Chemoprevention of Cancer and DNA Damage by Dietary Factors 2009.Pdf

Home , Health effects of wine

Chemoprevention of Cancer and DNA Damage by Dietary Factors

Edited by Siegfried Knasmu¨ller, David M. DeMarini, Ian Johnson, and Clarissa Gerha¨user Related Titles

Meyers, R. A. (ed.) Cancer From Mechanisms to Therapeutic Approaches

2007 ISBN: 978-3-527-31768-4

Brigelius-Flohé, R., Joost, H.-G. (eds.) Nutritional Genomics Impact on Health and Disease

2006 ISBN: 978-3-527-31294-8

Smolin, L. A., Grosvenor, M. B. Nutrition Science and Applications

2007 ISBN: 978-0-471-42085-9 zur Hausen, H. Infections Causing Human Cancer

2006 ISBN: 978-3-527-31056-2

Debatin, K.-M., Fulda, S. (eds.) Apoptosis and Cancer Therapy From Cutting-edge Science to Novel Therapeutic Concepts

2006 ISBN: 978-3-527-31237-5

Liebler, D. C., Petricoin, E. F., Liotta, L. A. (eds.) Proteomics in Cancer Research

2005 ISBN: 978-0-471-44476-3 Chemoprevention of Cancer and DNA Damage by Dietary Factors

Edited by Siegfried Knasmüller, David M. DeMarini, Ian Johnson, and Clarissa Gerhäuser The Editors All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and Prof. Dr. Siegfried Knasmüller publisher do not warrant the information contained Institute for Cancer Research in these books, including this book, to be free of Medical University Vienna errors. Readers are advised to keep in mind that Borschkegasse 8 a statements, data, illustrations, procedural details or 1090 Vienna other items may inadvertently be inaccurate. Austria Library of Congress Card No.: Dr. David M. DeMarini applied for US Environmental Protection Agency Environmental Carcinogenesis British Library Cataloguing-in-Publication Data Division A catalogue record for this book is available from the Research Triangle Park, NC 27711 British Library. USA Bibliographic information published by Prof. Ian Johnson the Deutsche Nationalbibliothek Norwich Research Park The Deutsche Nationalbibliothek lists this Institute of Food Research publication in the Deutsche Nationalbibliografie; Colney detailed bibliographic data are available on the Norwich, Norfolk NR4 7UA Internet at http://dnb.d-nb.de. United Kingdom # 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Dr. Clarissa Gerhäuser Weinheim German Cancer Research Centre (DKFZ) Toxicology and Cancer Risk Factors All rights reserved (including those of translation into Im Neuenheimer Feld 280 other languages). No part of this book may be 69120 Heidelberg reproduced in any form – by photoprinting, Germany microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Composition Thomson Digital, Noida, India Printing Strauss Gmbh, Mörlenbach Bookbinding Litges & Dopf GmbH, Heppenheim

Printed in the Federal Republic of Germany Printed on acid-free paper

ISBN: 978-3-527-32058-5 V

Contents

Preface XXVII

Foreword: Prevention of Cancer, and the Other Degenerative Diseases of Aging, Through Nutrition XXXI Bruce N. Ames and Joyce C. McCann

List of Contributors XXXIX

Part One General Principles 1

1 Molecular Mechanisms of Cancer Induction and Chemoprevention 3 Helmut Bartsch and Clarissa Gerhäuser 1.1 Cancer Chemoprevention 3 1.2 Molecular Mechanisms and Targets of Chemopreventive Agents 4 1.2.1 Carcinogen-Blocking Activities 5 1.2.2 Antimutagenic Effects and DNA Repair 5 1.2.3 Targeting Epigenetic Mechanisms 6 1.2.4 Radical-Scavenging and Antioxidant Effects 7 1.2.5 Anti-Inﬂammatory Mechanisms 8 1.2.6 Antitumor Promoting Activities 9 1.2.7 Antiproliferative Mechanisms 11 1.2.8 Induction of Apoptosis and Terminal Cell Differentiation 12 1.2.9 Inhibition of Angiogenesis (Angioprevention) 13 1.3 Perspectives 15 1.4 Conclusion 16 References 17

2 Types and Consequences of DNA Damage 21 Daniel T. Shaughnessy and David M. DeMarini 2.1 Introduction 21

Chemoprevention of Cancer and DNA Damage by Dietary Factors. Edited by Siegfried Knasmüller, David M. DeMarini, Ian Johnson, and Clarissa Gerhäuser Copyright 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32058-5 VI Contents

2.2 Types of DNA Damage 22 2.3 How to Detect DNA Damage Experimentally 24 2.4 DNA Damage Response 26 2.5 Types of DNA Repair 27 2.5.1 Direct DNA Repair 27 2.5.2 Base Excision Repair 27 2.5.3 Nucleotide Excision Repair 28 2.5.4 Mismatch Repair 29 2.5.5 Homologous and Nonhomologous Recombination for Repair of Double-Strand Breaks 29 2.5.6 DNA Damage Tolerance: SOS Repair and Trans-Lesion Synthesis 30 2.6 Types of Mutations 31 2.7 Assays to Detect Mutagens 31 2.8 Implications for Chemoprevention 32 References 32

3 Induction of DNA Damage and Cancer by Dietary Factors 35 Wolfram Parzefall, Nina Kager, and Siegfried Knasmüller 3.1 Introduction 35 3.2 Nitrosamines 35 3.3 Heterocyclic Aromatic Amines 37 3.4 Polycyclic Aromatic Hydrocarbons 40 3.5 Other Thermal Degradation Products 42 3.6 Mycotoxins 46 3.7 Carcinogens in Plant Foods 49 3.8 Food Additives and Pesticides/Herbicide Residues 49 3.9 Human Cancer Risks of Food Speciﬁc Carcinogens 50 References 52

4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview 57 Silvio De Flora, Carlo Bennicelli, Alessandra Battistella, and Maria Bagnasco 4.1 Antimutagenesis and Anticarcinogenesis 57 4.2 Strategies for Preventing Cancer and Other Mutation-Related Diseases 57 4.3 Classiﬁcation of Mechanisms of Chemopreventive Agents 59 4.4 Overview of Mechanisms of Inhibitors of Mutagenesis and Carcinogenesis 60 4.4.1 Extracellular Mechanisms 60 4.4.1.1 Inhibition of Uptake 60 4.4.1.2 Inhibition of Endogenous Formation 60 4.4.1.3 Complexation, Dilution, and Deactivation 61 4.4.2 Inhibition of Genotoxic Damage and Cancer Initiation 61 4.4.2.1 Modulation of Metabolism 61 Contents VII

4.4.2.2 Nucleophilicity 62 4.4.2.3 Antioxidant Mechanisms 62 4.4.2.4 Inhibition of Cell Replication 62 4.4.2.5 Maintenance of DNA Structure and Modulation of its Metabolism and Repair 63 4.4.2.6 Control of Gene Expression 63 4.4.3 Inhibition of Tumor Promotion 64 4.4.3.1 Anti-Inﬂammatory Activity 64 4.4.3.2 Signal Transduction Modulation 64 4.4.3.3 Protection of Gap Junctional Intercellular Communications (GJIC) 64 4.4.3.4 Induction of Cell Differentiation 65 4.4.3.5 Modulation of Apoptosis 65 4.4.4 Inhibition of Tumor Progression 65 4.4.4.1 Inhibition of Angiogenesis 65 4.4.4.2 Modulation of the Hormonal Status 66 4.4.4.3 Effects on the Immune System 66 4.4.5 Inhibition of Invasion and Metastasis 66 4.5 Concluding Remarks 67 References 68

5 Antioxidants and Cancer: Fact and Fiction 73 Andrew R. Collins and Alain Favier 5.1 Introduction 73 5.2 Fruits and Vegetables: the Evidence 73 5.3 Oxidative Damage and Antioxidants 74 5.4 Large-Scale Intervention Studies with Antioxidants 75 5.4.1 The Linxian Study 75 5.4.2 The Alpha-Tocopherol Beta-Carotene Trial in Finland 77 5.4.3 The Beta-Carotene and Retinol Efﬁciency Trial in the United States 77 5.4.4 The US Physicians Health Study 77 5.4.5 The MRC/BHF Heart Protection Study, United Kingdom 78 5.4.6 The SU.VI.MAX Study 78 5.4.7 Two Meta-Analyses 78 5.5 Using Surrogate Biomarkers for Disease Risk 79 5.5.1 Oxidation of Bases in DNA 79 5.5.2 Intervention Trials with Antioxidants, Using the Comet Assay to Assess DNA Damage 84 5.6 Nonantioxidant Effects of Antioxidants 87 5.7 Conclusions 87 References 88

6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer? 93 Hansruedi Glatt 6.1 Xenobiotics and Their Disposition – Basal Aspects 93 VIII Contents

6.2 Classification of Biotransformation Reactions and Enzymes 95 6.2.1 Conventional Phase 1/Phase 2 Concept 95 6.2.2 Toxicological Classification of Biotransformation Reactions 98 6.2.3 Chaos and Selection 99 6.2.4 Modified Phase 1/Phase 2 Terms Used in the Chemoprevention Area 100 6.2.5 Good and Bad Enzymes 101 6.3 Toxicokinetic Interactions Leading to Enhanced or Reduced Effects of Carcinogens 102 6.3.1 Individual Known Carcinogens 102 6.3.2 Wide Sets of Identified and Unidentified Carcinogens 103 6.4 Conclusions 106 References 107

7 Dietary Factors Regulate Metabolism of Carcinogens through Transcriptional Signaling Pathways 109 Soona Shin and Thomas W. Kensler 7.1 Introduction 109 7.2 Nrf2 110 7.2.1 Keap1-Mediated Regulation of Nrf2 112 7.2.2 Phosphorylation 113 7.2.2.1 Mitogen-Activated Protein Kinases 113 7.2.2.2 Protein Kinase C 114 7.2.2.3 CK2 114 7.3 Aryl Hydrocarbon Receptor 114 7.3.1 Regulation 115 7.4 Nuclear Receptors 116 7.4.1 Regulation of the CAR Pathway 117 7.4.2 Regulation of the PXR Pathway 117 7.5 Conclusions 118 References 118

8 Endocrine-Related Cancers and Phytochemicals 121 Johannes C. Huber and Johannes Ott 8.1 Introduction 121 8.2 Phytoestrogens and Endocrine-Related Cancers – Epidemiological Studies 122 8.2.1 Isoflavones and Breast Cancer 122 8.2.1.1 Isoflavones and the ‘‘Window of Opportunity’’ 122 8.2.1.2 Serum Isoflavones and Breast Cancer 122 8.2.2 Lignans and Breast Cancer 124 8.2.3 Phytoestrogens and Prostate Cancer 125 8.3 Mechanisms of Cancer Chemoprevention by Phytoestrogens 125 8.4 Estrogen Carcinogenesis 126 8.4.1 Estrogen Metabolites and Carcinogenesis 126 Contents IX

8.4.2 CYP1A1 129 8.4.2.1 Polymorphisms 129 8.4.2.2 Clinical Aspects of CYP1A1 Genotypes 130 8.4.2.3 Breast and CYP1A1 131 8.4.2.4 Pharmacogenomics and Phytochemicals 132 8.4.3 CYP1B1 133 8.4.3.1 Gene and Protein Structure of CYP1B1 133 8.4.3.2 Gene Regulation 133 8.4.3.3 Epigenetic Regulation 134 8.4.3.4 Polymorphisms 134 8.4.3.5 Breast Cancer 135 8.4.3.6 Endometrial Cancer 136 8.4.3.7 Pharmacogenomics and Phytochemicals 136 8.5 Conclusion 137 References 137

9 Inflammation-Induced Carcinogenesis and Chemoprevention 145 Hiroshi Ohshima, Susumu Tomono, Ying-Ling Lai, and Noriyuki Miyoshi 9.1 Introduction 145 9.2 Prevention of Inﬂammation-Associated Cancer by Avoidance of Causes of Tissue Damage 146 9.3 Chemoprevention by Modulating Inﬂammatory Processes 147 9.3.1 NF-kB 147 9.3.2 iNOS 148 9.3.3 COX-2 149 9.3.4 ROS-Generating Enzymes and Antioxidant Defense Mechanisms 149 9.4 Conclusion 150 References 151

10 DNA Methylation 153 Ian T. Johnson, Nigel J. Belshaw, and Giles O. Elliott 10.1 Introduction 153 10.2 Effects of Diet on DNA Methylation 155 10.3 Impact of Environment and Nutrition on the Human Epigenome 156 10.4 Modiﬁcation of DNA Methylation by Nutrients and Phytochemicals 157 10.4.1 Folates 157 10.4.2 Selenium 158 10.4.3 Polyphenols 159 10.4.4 Isothiocyanates 160 10.5 Conclusions 160 References 161 X Contents

11 Prevention of Angiogenesis and Metastasis 163 Tariq A. Bhat, Anil Mittal, and Rana P. Singh 11.1 Introduction 163 11.2 Angiogenesis 164 11.2.1 Angiogenesis Process 165 11.2.2 Tumor Angiogenesis 166 11.2.3 Angiopreventive Agents 166 11.2.4 Lymphangiogenesis 170 11.3 Metastasis 171 11.3.1 Basic Steps in Cancer Metastasis 172 11.3.2 Epithelial–Mesenchymal Transition in Metastasis 173 11.3.3 Invasion and Migration 174 11.3.4 Homing Mechanisms 175 11.3.5 Preventive Agents for Metastasis 177 11.4 Summary 177 References 178

12 Impact of Dietary Factors on the Immune System 183 Alexa L. Meyer 12.1 A Short Presentation of the Immune System 183 12.1.1 The First Line of Defense 183 12.1.2 Adaptive Immunity 185 12.1.3 The Immune System in Cancerogenesis 185 12.1.4 Cancer – A Serious Opponent 186 12.2 The Role of Nutrition in Immunity 186 12.2.1 Fat and Fatty Acids: Their Role in Inﬂammation and Beyond 186 12.2.2 Trace Elements 188 12.2.3 Vitamins 189 12.2.4 Nonnutritive Food Components 190 References 194

13 Epidemiological Studies 199 Anthony B. Miller 13.1 Introduction 199 13.2 Observational Epidemiology Studies: What Can We Learn From Them? 200 13.3 What Are We Trying To Do with Chemoprevention? 201 13.4 How Will We Know If We Are Successful? 201 13.5 The Example of Beta-Carotene 202 13.6 Folic Acid 204 13.7 Other Micronutrients 205 13.8 Green Tea and Other Agents 206 13.9 Conclusions 207 References 207 Contents XI

Part Two Experimental Models and Methods Used in Chemoprevention Studies 209

14 Methods Used for the Detection of Antimutagens: An Overview 211 Armen Nersesyan, Miroslav Misík,4 and Siegfried Knasmüller 14.1 Introduction 211 14.2 Mechanisms of DNA Protection 212 14.3 Methodological Aspects 212 14.3.1 End Points Used in Antigenotoxicity Studies 212 14.3.2 In Vitro Approaches 214 14.3.3 In Vitro Systems 215 14.3.4 Human Biomonitoring Studies 218 14.4 Limitations of the Predictive Value of Different Endpoints and Test Systems 219 14.5 Speciﬁcity of Protection 220 14.6 Dose–Effect Relationships 221 14.7 Future Trends 222 References 222

15 Methods to Determine Total Antioxidative Capacity and Oxidative DNA Damage 229 Karl-Heinz Wagner, Miroslav Misík,4 Armen Nersesyan, and Siegfried Knasmüller 15.1 Introduction 229 15.2 Methods 229 15.2.1 Trapping of Reactive Species 229 15.2.2 Approaches to Determine the Total Antioxidant Capacity 230 15.2.3 Free Radical Quenching Methods 231 15.2.4 Single-Electron Transfer Methods 234 15.3 Oxidation of Macromolecules 235 15.3.1 Biomarkers of Lipid Oxidation 235 15.3.2 Biomarkers of Protein Oxidation 238 15.4 Methods Used to Monitor Oxidative DNA Damage 239 15.4.1 In Vitro and In Vivo Approaches 240 15.5 Conclusions and Outlook 241 References 241

16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research 245 Wolfgang W. Huber and Michael Grusch 16.1 Introduction 245 16.2 Important Enzymes of Xenobiotic Metabolism in Cancer Chemoprevention Research 246 16.3 Measurement of Xenobiotic Metabolism: General Aspects 247 XII Contents

16.3.1 Determination of Enzyme Encoding genes (Investigation at DNA Level) 249 16.3.2 Determination of Enzyme Transcription (Investigation at RNA Level) 249 16.3.3 Determination of Enzyme Protein 250 16.3.3.1 Measurement of Enzyme Activity: General Aspects 252 16.3.3.2 Activity Measurements in Humans 255 16.4 Summary 255 References 256

17 Methods for the Analysis of DNA Methylation 263 Keith N. Rand and Peter L. Molloy 17.1 Introduction 263 17.2 Measurement of Global and Repeat Sequence Methylation 264 17.3 Measurement of Methylation of Individual Genes or Regions 267 17.3.1 Methylation Sensitive PCR 269 17.3.2 Non-Bisulﬁte-Based Assay 270 17.4 Scanning Genome-Wide for Changes in DNA Methylation 270 References 273

18 Methods Used to Study Alterations of Cell Signaling and Proliferation 277 Clarissa Gerhäuser 18.1 Introduction 277 18.2 Methods to Detect Alterations in Cell Signaling 278 18.2.1 Initializing Events 278 18.2.2 Signal Transduction to Intracellular Targets 280 18.2.2.1 Detection Using Phosphospeciﬁc Antibodies 280 18.2.2.2 Kinase Assay After Immunoprecipitation 281 18.2.3 Transcription Factor Activation 281 18.2.3.1 Transcription Factor–DNA Binding 281 18.2.3.2 Promoter Assays After Transfection with Reporter Gene Constructs 281 18.2.4 Gene Transcription and Translation 282 18.3 Methods to Measure Cell Proliferation 282 18.3.1 Microplate Screening Assays for Cytotoxicity 282 18.3.2 Cell Cycle Analysis [20] 282 18.3.3 Cell Death: Induction of Apoptosis 286 18.3.3.1 Methods to Detect Induction of Apoptosis 286 References 287

19 Methods for the Assessment of Antiangiogenic Activity 291 Clarissa Gerhäuser 19.1 Introduction 291 Contents XIII

19.2 Methods for Assaying Angiogenesis 291 19.2.1 In vitro Test Systems 292 19.2.1.1 Cell-Based Systems 292 19.2.1.2 Organ Culture Systems 295 19.2.2 In vivo Models 296 19.3 Conclusions 299 References 300

20 Nutrigenomics 303 Jan Frank and Gerald Rimbach 20.1 Deﬁning the ‘‘-omics’’ 303 20.2 Post-Transcriptional Gene Regulation by Small RNAs 305 20.3 Methods and Techniques Used in Nutrigenomic Research 306 20.3.1 Northern Blotting 306 20.3.2 Reverse Transcription Polymerase Chain Reaction 306 20.3.3 Microarrays 307 20.3.4 Microarray Data Normalization 310 20.3.4.1 Per Array Normalization 310 20.3.4.2 Per Gene Normalization 310 20.3.5 Microarray Data Analysis 311 20.3.5.1 Fold Change 311 20.3.5.2 Class Comparison, Class Discovery, and Class Prediction 313 20.4 Applications of Nutrigenomics 314 20.5 Proteomics 319 20.6 Metabolomics 320 20.7 Promoting Nutrigenomic Research 322 20.8 Summary 322 References 323

21 Preneoplastic Models and Carcinogenicity Studies with Rodents 335 Veronika A. Ehrlich and Siegfried Knasmüller 21.1 Introduction 335 21.2 Animal Models Based on the Use of Carcinogens 335 21.2.1 Use of Preneoplastic Lesions in Experimental Oncology 335 21.2.1.1 Altered Hepatic Foci (AHF) – Morphology and Phenotypes 337 21.2.1.1.1 Methodical Aspects 338 21.2.1.1.2 Initiators and Promoters of AHF 339 21.2.1.1.3 Mechanistic Aspects to the Action of Promoters 339 21.2.1.1.4 Inhibition of Foci Formation in the Liver 339 21.2.1.1.5 New Developments 340 21.2.1.2 Aberrant Crypts in the Colon 340 21.2.1.2.1 Morphology 340 21.2.1.2.2 Biochemical and Immunohistochemical Alterations of ACF 341 21.2.1.2.3 Genetic and Epigenetic Alterations 341 21.2.1.2.4 Methodological Aspects 341 XIV Contents

21.2.1.2.5 Use of the ACF Model for the Detection of Chemoprotective Compounds 343 21.2.1.2.6 New Developments 346 21.3 Genetically Engineered Rodent Models Used in Cancer Prevention Studies 346 þ 21.3.1 Intestinal Cancers in Transgenic ApcMin/ Mouse Model 347 21.3.2 Germline p53-Deﬁcient and p53 Mutated Animals 347 21.3.3 Rodent Models of Prostate Cancer 347 21.3.4 Other Transgenic Models 349 21.4 Xenograft Models 349 21.5 Conclusions 350 References 351

22 The Role of Nutrition in the Etiology of Human Cancer: Methodological Considerations Concerning Epidemiological Studies 357 Heiner Boeing 22.1 Introduction 357 22.2 Principles for Evaluating the Link between Nutrition and Cancer 358 22.2.1 Intervention Studies 358 22.2.2 Prospective Cohort Studies 359 22.2.3 Case-Control and Other Studies 359 22.3 Methodological Statistical Challenges Regarding Dietary Assessment 360 22.4 Complexity of Dietary Data and Their Analyses 361 22.5 Current Status of the Evidence 362 22.5.1 Fruit and Vegetables 362 22.5.2 Meat and Meat Products 363 22.5.3 Grains 364 22.5.4 Lipids (Fat) 364 22.5.5 Use of Alcoholic Beverages 364 22.6 Summary and Conclusion 365 References 365

Part Three Selected Chemoprotective Dietary Factors and Components 369

23 Carotenoids and Vitamin A 371 M. Cristina Polidori and Wilhelm Stahl 23.1 Introduction 371 23.2 Carotenoids – Biochemical Properties 374 23.3 b-Carotene – Cancer Prevention 376 23.4 Lycopene – Cancer Prevention 377 23.5 Other Carotenoids – Cancer Prevention 378 23.6 Retinoids – Biological Properties 378 Contents XV

23.7 Retinoids – Cancer Prevention 380 References 382

24 Selected Vitamins 385 24.1 Vitamin C 385 Pavel Kramata and Nanjoo Suh 24.1.1 Introduction 385 24.1.2 Physicochemical Properties 386 24.1.3 Bioavailability and Metabolism 387 24.1.4 Mechanism of Protection: In Vitro and In Vivo Studies 388 24.1.4.1 Vitamin C and Oxidative DNA Damage 389 24.1.4.2 Vitamin C, Cell Proliferation, Signal Transduction, and Apoptosis 389 24.1.5 Human Studies 390 24.1.5.1 Epidemiological Studies 390 24.1.5.2 Clinical Studies 391 24.1.6 Impact of Cooking and Food Processing on Protective Properties 392 24.2 Vitamin D 393 Heide S. Cross and Thomas Nittke 24.2.1 Introduction 393 24.2.1.1 How Much Vitamin D is Enough? 393 24.2.2 Vitamin D Synthesis 394

24.2.3 Bioavailability and Metabolism of 1,25-(OH)2-D3 394 24.2.4 Mechanisms of Protection 395 24.2.4.1 Vitamin D Signaling Pathways in Cancer 395 24.2.5 In Vivo Studies with 1,25-D3 for Tumor Prevention or Treatment 397 24.2.5.1 Treatment 397 24.2.5.2 Tumor Prevention 399 24.2.6 Impact of Processing on Vitamin D in Human Nutrition and Conclusion 402 24.3 Vitamin E 402 Hong Jin Lee and Nanjoo Suh 24.3.1 Introduction 402 24.3.2 Physicochemical Properties of Vitamin E, Chemical Structures, and Chemical Reactions 403 24.3.2.1 Structure of Vitamin E Components 403 24.3.2.2 Vitamin E in Human Diet 403 24.3.3 Bioavailability and Metabolism of Vitamin E 404 24.3.3.1 Bioavailability and Biopotency of Vitamin E 404 24.3.3.2 Metabolism of Vitamin E 404 24.3.4 In Vitro and Animal Studies of Vitamin E 406 24.3.4.1 Mechanism of Antioxidant Protection 406 24.3.4.2 Nonantioxidant Functions of Vitamin E 407 24.3.4.3 Anticancer Mechanisms of Vitamin E Action 407 XVI Contents

24.3.4.4 Vitamin E and Preclinical Studies 407 24.3.5 Vitamin E and Human Intervention Trials 408 24.3.6 Impact of Cooking, Processing, and Other Factors on Protective Properties of Vitamin E 409 References 409

25 Folate and Vitamins B2, B6, and B12 417 Philip Thomas and Michael Fenech 25.1 Introduction 417 25.2 Physicochemical and Transport Properties 418 25.3 Bioavailability and Metabolism of Active Compounds 420 25.3.1 Bioavailability 420 25.3.2 Metabolism 422 25.4 Mechanisms of Protection – In Vitro Studies 423 25.5 Results from Human Studies 424 25.6 Impact of Cooking, Processing, and Other Factors on Protective Properties 429 25.7 Conclusions 430 References 430

26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc 435 Dianne Ford and John Hesketh 26.1 Introduction 435 26.2 Selenium and Zinc in Food 435 26.3 Mechanisms of Chemoprevention by Selenium: Results of In Vitro and Animal Studies 436 26.4 Results from Human Studies of the Inﬂuence of Selenium Status on Cancer Risk 438 26.4.1 Genetic Inﬂuences on Selenium Metabolism and Disease Risk 440 26.5 Mechanisms of Chemoprevention by Zinc: Results of In Vitro and Animal Studies 441 26.5.1 Zinc as an Antioxidant 442 26.5.2 Effects of Zinc on Cell Proliferation and Apoptosis 443 26.5.3 Effects of Zinc on Cell Motility and Invasion 444 26.5.4 Effects of Zinc on Intermediary Metabolism 444 26.5.5 Intracellular Zinc Signaling in Cancer 444 26.5.6 Zinc Transporters and Cancer 445 26.5.7 Effects of Dietary Zinc Manipulation on Cancer Incidence and Progression in Animal Models 445 26.6 Human Studies of Links between Zinc, Carcinogenesis, and Tumor Progression 446 26.6.1 Associations Between Zinc Status and Cancer Incidence and Progression 446 Contents XVII

26.6.2 Effects of Dietary Zinc Manipulation on Cancer Incidence and Progression In Vivo 447 26.7 Conclusions 448 References 449

27 DNA Damage and Cancer Chemoprevention by Polyphenols 455 Ajaikumar B. Kunnumakkara, Preetha Anand, Kuzhuvelil B. Harikumar, and Bharat B. Aggarwal 27.1 Introduction 455 27.2 Physical–Chemical Properties of Polyphenols and Their Occurrence 455 27.3 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vitro Studies 460 27.3.1 Polyphenols Protect Against Chemical-Induced DNA Damage 460 27.3.2 Polyphenols Protect Against Radiation-Induced DNA Damage 464 27.4 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vivo Studies 465 27.4.1 Polyphenols Prevent Colon, Esophagus, and Gastric Cancer 465 27.4.2 Polyphenols Prevent Ascites Tumor 468 27.4.3 Polyphenols Inhibit Breast Cancer 468 27.4.4 Polyphenols Inhibit Liver Cancer 469 27.4.5 Polyphenols Inhibit Neuroblastoma and Glioma 470 27.4.6 Polyphenols Inhibit Leukemia 470 27.4.7 Polyphenols Inhibit Prostate Cancer 471 27.4.8 Polyphenols Inhibit Skin Cancer 472 27.4.9 Polyphenols Inhibit Lung Cancer 473 27.4.10 Polyphenols Inhibit Bladder Cancer 474 27.4.11 Polyphenols Inhibit Oral Cancer 474 27.5 Mechanisms of Chemoprevention by Phenolic Compounds: Results of Human Studies 475 27.6 Effects of Food Processing on Chemoprotective Properties of Polyphenols 476 27.7 Conclusion 476 References 477

28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients 483 Hye-Kyung Na, Joydeb Kumar Kundu, and Young-Joon Surh 28.1 Introduction 483 28.2 Antioxidant Effects 483 28.3 Anti-Inﬂammatory Effects 485 28.4 Anticarcinogenic Effects 488 28.4.1 Inhibition of Mouse Skin Carcinogenesis 488 28.4.2 Inhibition of Gastric Lesions and Tumorigenesis 489 28.4.3 Inhibition of Bladder Carcinogenesis 490 XVIII Contents

28.4.4 Inhibition of Tumor Cell Growth and Proliferation 490 28.4.5 Antimetastasis and Antiangiogenesis 492 28.4.6 Effects on Multidrug Resistance 492 28.5 Chemoprotective Effects 493 28.6 Conclusion 493 References 494

29 Tannins: Bioavailability and Mechanisms of Action 499 Fulgencio Saura-Calixto and Jara Pérez-Jiménez 29.1 Introduction 499 29.2 Physicochemical Properties 500 29.3 Bioavailability and Metabolism 501 29.3.1 Proanthocyanidins 501 29.3.2 Hydrolyzable Tannins 502 29.4 Mechanisms of Protection 503 29.5 Results of Human Studies 505 29.6 Impact of Cooking and Processing 506 References 506

30 Selected Flavonoids 509 30.1 Quercetin and other Flavonols 509 Loïc Le Marchand and Adrian A. Franke 30.1.1 Introduction 509 30.1.2 Physicochemical Properties of Active Compounds and their Occurrence 509 30.1.3 Biovailability and Metabolisms of Active Compounds 512 30.1.4 Mechanisms of Protection 514 30.1.5 Results of Human Studies 514 30.1.5.1 Cohort Studies 515 30.1.5.2 Case–Control Studies 515 30.1.6 Impact of Storage, Processing, and Cooking on Protective Properties 515 30.1.7 Research Needs 516 30.2 Anthocyanidins 516 Li-Shu Wang and Gary D. Stoner 30.2.1 Introduction 516 30.2.2 Physiochemical Properties of Anthocyanidins 517 30.2.3 Bioavailability and Metabolism of Anthocyanins 518 30.2.4 Mechanisms of Chemoprotection by Anthocyanidins and Anthocyanins 518 30.2.4.1 In Vitro Studies 518 30.2.4.2 Animal Studies 522 30.2.5 Human Studies 523 30.2.6 Impact of Processing on the Stability of Anthocyanins 524 30.2.7 Conclusions 524 Contents XIX

30.3 Proanthocyanidins 525 Clarissa Gerhäuser 30.3.1 Introduction 525 30.3.2 Physicochemical Properties and Occurrence 525 30.3.2.1 Physicochemical Properties 525 30.3.2.1.1 Interaction with Proteins 525 30.3.2.1.2 Antioxidant and Radical Scavenging Capacity 527 30.3.2.1.3 Complexation of Metal Ions 527 30.3.2.2 Occurrence 527 30.3.3 Bioavailability and Metabolisms [97–99,104–107] 528 30.3.4 Mechanisms of Protection: Results of In Vitro and Animal Studies 529 30.3.4.1 In vitro Antioxidant Activity 529 30.3.4.2 Potential Cancer Chemopreventive Activity in Cell Culture 529 30.3.4.3 Cancer Chemopreventive Activity in Animal Models 534 30.3.5 Results of Human Studies 534 30.3.5.1 Short-Term Intervention Studies 534 30.3.5.2 Epidemiological Studies 537 30.3.6 Impact of Cooking, Processing, and other Factors on Protective Properties 537 30.3.7 Conclusions 538 References 538

31 Phytoestrogens 547 31.1 Isoflavones: Sources, Intake, Fate in the Human Body, and Effects on Cancer 547 Alicja Mortensen, Sabine Kulling, Heidi Schwartz, and Gerhard Sontag 31.1.1 Introduction 547 31.1.2 Dietary Isoflavone Sources 547 31.1.3 Dietary Intake of Isoflavones 548 31.1.4 Absorption, Distribution, Metabolism, and Excretion of Isoflavones 549 31.1.4.1 Absorption 549 31.1.4.2 Metabolism 550 31.1.4.3 Distribution 551 31.1.4.4 Excretion 552 31.1.4.5 Pharmacokinetics 552 31.1.4.6 Factors Affecting the Pharmacokinetics of Isoflavones 552 31.1.5 Effects of Isoflavones on Breast, Prostate, and Intestinal Cancer 553 31.1.5.1 Breast Cancer 553 31.1.5.2 Prostate Cancer 554 31.1.5.3 Intestinal Cancer 554 31.2 Lignans 555 Eric Lainé, Christophe Hano, and Frédéric Lamblin 31.2.1 Introduction 555 31.2.2 Occurrence and Physicochemical Properties 556 XX Contents

31.2.2.1 Phytoestrogenic Nature 558 31.2.2.2 Antioxidant Properties 560 31.2.3 Bioavailability 561 31.2.4 Mechanisms of Protection: Results of In Vitro and Animal Studies 562 31.2.4.1 Inhibition of Tumorigenesis 562 31.2.4.2 Inhibition of Metastasis 562 31.2.4.3 Other Less Documented Mechanisms 562 31.2.5 Results of Human Studies 565 31.2.5.1 Breast Cancer 566 31.2.5.2 Prostate Cancer 567 31.2.5.3 Colon Cancer 567 31.2.6 Impact of Cooking, Processing, and Other Factors on Protective Properties 567 31.2.7 Conclusions 568 References 568

32 Chemopreventive Properties of Coffee and Its Constituents 579 Gernot Faustmann, Christophe Cavin, Armen Nersesyan, and Siegfried Knasmüller 32.1 Introduction 579 32.2 Bioactive Components in Coffee 580 32.3 Mechanisms of Chemoprevention 580 32.3.1 Protective Properties of Coffee 581 32.3.1.1 Antioxidant Effects 581 32.3.1.2 Induction of Detoxifying Enzymes 583 32.3.2 Protective Properties of Coffee Components 583 32.3.2.1 Antioxidant Effects 583 32.3.2.2 Induction of Detoxifying Enzymes 585 32.3.2.3 Protective Effects of Coffee Constituents Toward Genotoxic Carcinogens 585 32.3.2.4 Interaction of Coffee and Coffee Diterpenes with Cell Signaling Pathways 587 32.4 Coffee Consumption and Human Cancer Risks 588 32.5 Concluding Remarks 590 References 590

33 Tea and Its Constituents 595 33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis 595 Joshua D. Lambert and Chung S. Yang 33.1.1 Introduction 595 33.1.2 Bioavailability and Biotransformation of Tea Polyphenols 596 33.1.2.1 Biotransformation of Tea Polyphenols 596 33.1.2.2 Pharmacokinetics of Tea Polyphenols 598 33.1.3 In Vitro and Animal Studies of Cancer Prevention by Tea 599 Contents XXI

33.1.3.1 Antioxidative/Pro-Oxidative Activities 599 33.1.3.2 Effects on Carcinogen Metabolism 600 33.1.3.3 Prevention and Repair of DNA Damage 601 33.1.3.4 Prevention of Carcinogenesis and Potential Postinitiation Mechanisms 602 33.1.4 Studies on the Cancer-Preventive Activity of Tea in Humans 604 33.1.4.1 Antioxidative Activity 604 33.1.4.2 Effects on Carcinogen Metabolism 604 33.1.4.3 Prevention and Repair of DNA Damage 604 33.1.4.4 Epidemiological and Intervention Studies on Cancer Prevention by Tea 605 33.1.5 Impact of Cooking, Processing, and Other Factors on Protective Effects 607 33.1.6 Concluding Remarks 608 33.2 Black and Other Teas 609 Wentzel C.A. Gelderblom, Kareemah Gamieldien, and Elizabeth Joubert 33.2.1 Introduction 609 33.2.2 Physicochemical Properties of Active Compounds and their Occurrence 610 33.2.2.1 Black Tea 610 33.2.2.2 Rooibos Tea 610 33.2.2.3 Honeybush Tea 613 33.2.3 Bioavailability and Metabolism of Active Compounds 615 33.2.4 Mechanisms of Protection: Results of In Vitro and Animal Studies 616 33.2.4.1 Antimutagenic Properties 616 33.2.4.2 Antioxidant Properties 617 33.2.4.3 Studies in Animals 619 33.2.4.4 Cell Survival Parameters 620 33.2.5 Results of Human Studies 621 33.2.6 Impact of Heat and Processing on Protective Properties 622 33.2.6.1 Black Tea 622 33.2.6.2 Rooibos and Honeybush 622 33.2.7 Concluding Remarks 623 References 623

34 Protective Effects of Alcoholic Beverages and their Constituent 635 34.1 Wine 635 Philipp Saiko, Akos Szakmary, and Thomas Szekeres 34.1.1 Introduction 635 34.1.1.1 General Information and Historical Background 635 34.1.1.2 The Health Effects of Wine 636 34.1.1.3 Ingredients of Wine 636 34.1.2 Physicochemical Properties of Active Compounds, Occurrences, and Chemical Structures 637 XXII Contents

34.1.2.1 Resveratrol 637 34.1.2.1.1 History and Sources 637 34.1.2.1.2 French Paradox 638 34.1.2.1.3 Effects of Resveratrol 638 34.1.2.2 Piceatannol: A Naturally Occurring Resveratrol Metabolite 640 34.1.2.3 Gallic Acid 640 34.1.3 Bioavailability and Metabolism of Active Compounds 642 34.1.3.1 Bioavailability of Resveratrol 642 34.1.3.2 Metabolites of Resveratrol: Glucuronide and Sulfate Conjugates 642 34.1.3.3 Bioavailability of Resveratrol in Grape Juice Compared to Its Pure Aglycone 642 34.1.4 Mechanisms of Protection 643 34.1.4.1 Results of In Vitro Studies 643 34.1.4.2 Results of In Vivo Studies 643 34.1.5 Results of Human Studies 643 34.1.6 Conclusions 643 34.2 Beer 648 Metka Filipic, Janja Plazar, and Sakae Arimoto-Kobayashi 648 34.2.1 Introduction 648 34.2.2 Physicochemical Properties of Bioactive Compounds 649 34.2.2.1 Prenylated Flavonoids 649 34.2.2.2 Bitter Acids 650 34.2.2.3 Nitrogenous Compounds 651 34.2.3 Mechanisms of Chemoprevention: Results In Vitro and In Vivo 652 34.2.3.1 Prenylated Flavonoids 652 34.2.3.2 Bitter Acids 653 34.2.3.3 Nitrogenous Compounds and other Unidentiﬁed Components 653 34.2.4 Results of Human Studies 655 34.2.5 Effect of Beer Processing on Chemoprotective Properties 656 References 656

35 Sulfides in Allium Vegetables 663 Claus Jacob and Awais Anwar 35.1 Introduction 663 35.2 Physicochemical Properties 665 35.3 Bioavailability and Metabolisms of Active Compounds 667 35.3.1 Enzymatic Generation of Reactive Sulfur Species as Part of Binary Plant Defense Systems 667 35.3.2 Follow-On Products Formed by Degradation of Reactions with Biomolecules 669 35.3.3 Chemical Recycling of Allicin 670 Contents XXIII

35.4 Mechanisms of Protection 671 35.4.1 Sulfur-based Scavengers of Free Radicals and Other Oxidative Stressors 671 35.4.2 Prevention of Carcinogen Activation by Inhibition of Metabolic (Phase I) Enzymes 673 35.4.3 Induction of Phase II Enzymes 673 35.4.4 Sulfur Agents as HDAC Inhibitors 674 35.4.5 Interference with Cancer Promoting, Cell Signaling Pathways 674 35.4.6 Cell Cycle Arrest and Induction of Apoptosis 675 35.4.7 Sulfur Agents and Angiogenesis 676 35.4.8 Counteracting Multidrug Resistance 677 35.5 Results of Human Studies 677 35.6 Impact of Cooking, Processing, and Other Factors on Protective Properties 678 35.7 Outlook 680 35.8 Conclusion 681 References 681

36 Glucosinolates and Cruciferous Vegetables 685 L. Adele Boyd, Cris Gill, Tomas Borkowski, and Ian Rowland 36.1 Introduction 685 36.1.1 Cruciferous Vegetables and Their Components 685 36.1.2 Physicochemical Properties of Phytochemicals in Cruciferous Vegetables 685 36.2 Bioavailability and Metabolisms of Active Compounds 686 36.2.1 Gastric and Small-Intestinal Breakdown 689 36.2.2 Colonic Metabolism 689 36.2.3 Absorption from the Gut 689 36.2.4 Metabolism and Excretion of Hydrolysis Products 689 36.3 Mechanisms of Protection: Results of In Vitro and Animal Studies 690 36.3.1 Animal Studies 690 36.3.2 Effects of Vegetable Extracts and Components at the Cellular and Molecular Level 691 36.4 Human Studies 692 36.4.1 Epidemiology 692 36.4.2 Dietary Intervention Studies 692 36.4.2.1 Effects on Phase I and Phase II Enzyme Activities and Carcinogen Excretion in Humans 693 36.4.2.2 Antigenotoxic Effects of Cruciferous Vegetables 694 36.5 Impact of Cooking and Processing 695 36.5.1 Storage and Processing 695 36.5.2 Cooking 695 36.5.3 Consequences for Bioavailability 695 XXIV Contents

36.6 Conclusions 696 References 696

37 Chlorophyll 699 Hikoya Hayatsu, Tomoe Negishi, and Sakae Arimoto-Kobayashi 37.1 Introduction 699 37.2 Chemical Nature of Chlorophylls 699 37.3 Historical Background 701 37.4 Basic Studies on Antigenotoxic Activities of Chlorophylls and the Mechanism of the Actions 701 37.5 Chlorophyll–Carcinogen Complex Formation 702 37.6 Studies with Higher Organisms 704 37.7 Studies with Human Subjects 705 References 706

38 Dietary Fibers 709 Philip J. Harris and Lynnette R. Ferguson 38.1 Introduction 709 38.2 Physicochemical Properties of Active Compounds and Their Occurrence 711 38.3 Bioavailability and Metabolism of Active Compounds 712 38.4 Mechanisms of Chemoprevention: Results of In Vitro and Animal Studies 712 38.4.1 Ligniﬁed or Suberized Cell Walls 713 38.4.2 Cell Walls Containing Hydroxycinnamic Acids 714 38.5 Results of Human Studies 715 38.6 Impact of Cooking, Processing, and Other Factors on Protective Properties 716 38.7 Conclusions 716 References 717

39 Dietary Fiber Carbohydrates and their Fermentation Products 721 Lynnette R. Ferguson and Philip J. Harris 39.1 Introduction 721 39.2 Physicochemical Properties of Active Compounds and their Occurrence 721 39.3 Bioavailability and Metabolism of Active Compounds 724 39.4 Mechanisms of Chemoprevention: Results of In Vitro and Animal Studies 724 39.4.1 Hypotheses Suggesting that SCFAs are Protective 724 39.4.2 The Butyrate Hypothesis 724 39.4.3 Prebiotic Effects 725 39.4.4 Evidence from Animal Models 725 39.5 Results of Human Studies 726 Contents XXV

39.6 Impact of Cooking, Processing, and other Factors on Protective Properties 726 39.7 Conclusions 727 References 727

40 Lactobacilli and Fermented Foods 731 Sabine Fuchs, Reinhard Stidl, Verena Koller, Gerhard Sontag, Armen Nersesyan, and Siegfried Knasmüller 40.1 Introduction 731 40.2 Occurrence of Lactic Acid Bacteria 733 40.3 Detoxiﬁcation of Genotoxic Carcinogens by Lactic Acid Bacteria 733 40.4 Antioxidant Effects of Lactic Acid Bacteria 737 40.5 Effect of Lactic Acid Bacteria on the Immune Status 739 40.6 Results of Carcinogenicity Studies with Laboratory Rodents 740 40.7 Impact of Lactic Acid Bacteria on Cell Proliferation and Apoptosis 742 40.8 Results of Human Epidemiological Studies 742 40.9 Conclusions and Future Research 743 References 743

41 Fatty Acids and Cancer Prevention 749 Elizabeth K. Lund 749 41.1 Introduction 749 41.2 Fatty Acid Structure 750 41.3 Bioavailability 751 41.4 Epidemiology 752 41.5 Animal Models 753 41.6 In Vitro Studies 754 41.7 Mechanisms of Action 754 41.8 Conclusion 757 References 757

42 Protease Inhibitors 761 Ann R. Kennedy 42.1 Introduction 761 42.2 Physicochemical Properties of Active Compounds and Their Occurrence 761 42.3 Bioavailability and Metabolism of Active Compounds 762 42.4 Mechanisms of Protection and Results of In Vitro and Animal Studies 762 42.5 Results of Human Studies 764 42.6 Impact of Cooking Processing 766 References 766

Index 769 XXVII

Preface

The systematic search for antimutagenic and anticarcinogenic compounds in the human diet began as early as in the 1940s, and the new field of research was well underway in the 1950s. The first publications were concerned with the prevention of radiation-induced DNA damage and cancer by certain amino acids (such as cysteine), glutathione, and vitamins [1–4]. Subsequent investigations showed that some of these substances protected also against chemically induced effects [5–7]. By the early 1970s, a broad variety of different classes of dietary compounds with antimutagenic and anticarcinogenic properties were known (for review see Ref. [8]). Among the numerous early pioneers in this field, Tsuneo Kada and Lee Wattenberg deserve particular mention. Tsuneo Kada worked at the National Institute of Genetics in Mishima, Japan, and screened numerous plant extracts for their antimutagenic properties in bacterial test systems (for reviews see Refs [9–12]). Wattenberg and his colleagues from the University of Minnesota (USA) discovered the protective properties of cruciferous vegetables and glucosinolates, as well as many other dietary components, against chemically induced cancer in rodents and provided mechanistic explanations for these phenomena. When Silvio De Flora and Claes Ramel published the first systematic overview on different modes of prevention of DNA damage and cancer in 1988 [13], they described a broad variety of different mechanisms that had been discovered. The postulate of Doll and Peto that up to one-third of the cancer deaths in industrialized countries are due to dietary factors and may be prevented by developing adequate strategies [14] stimulated many researchers to work in this field. Numerous epidemiological investigations have confirmed their pioneering conclusion that the diet has a very strong impact on human cancer risks [15]. Recent advances in molecular biology have thrown up new important areas of research, including the investigation of the signaling pathways that activate transcription factors controlling the expression of proteins involved in the protection against DNA damage and cancer [16]. The development of high-throughput ‘‘omics’’ techniques has contributed substantially to the elucidation of these mechanisms and led to the discovery of new, hitherto unknown modes of action of dietary factors [17–21]. Also, the detection of the important role of epigenetic alterations in the induction of cancer, and the involvement of dietary factors in this process, and the discovery of the important effects of micronutrients on DNA stability and cancer

risks by Bruce Ames [22] and Michael Fenech [23] have extended older concepts that focused mainly on antioxidants and the direct inactivation of DNA reactive carcinogens by fruit and vegetable constituents. The editors of this book work in different areas of antimutagenesis and anticarcinogenesis and shared the opinion that it would be fruitful to produce a comprehensive volume that describes the different aspects and approaches currently being used to investigate and identify protective dietary factors. The book consists of three main parts. The first contains a description of the basic principles of DNA damage and cancer formation, as well as a number of chapters that describe mechanisms relevant to the development of chemoprevention strategies. These includethefunctionofoxidativedamageandhormonaleffects,theinvolvement of the immune system in tumor formation, and the role of drug metabolizing enzymes and micronutrients. The second part describes the different methods that are used to identify protective dietary compounds and investigate their modes of action. The authors felt that it was important not only to provide the readers with information required to establish the different experimental models in their laboratories but also to describe the limitations of the predictive value of different approaches in a critical way. This is of particular importance in view of the fact that authorities in the United States and in Europe have started to require that health claims on foods be justified by sound scientific evidence. This part of the book comprises chapters concerning methods for the detection of DNA damage, preneoplastic foci and animal cancer models, an overview of methods that are used to detect antioxidants, the investigation of signalling pathways, and the activation of transcription factors and DNA methylation. Further, contributions describe the use of high-throughput nutrigenomics techniques and measurements of drug metabolizing enzymes. The last part of the book contains chapters on selected dietary factors that have DNA and cancer-protective properties. The editors give a broad overview of physicochemical properties of active compounds and their occurrence, bioavailability and metabolisms of active compounds, mechanisms of protection with focus on results of in vitro and animal studies, results of human studies (if available), and the impact of processing on protective properties. The reader will find information concerning the effects of beverages (teas, coffee, wine, and beer), selected vitamins (A, B, C, D, and E) and other micronutrients, tannins and other polyphenolics, pigments, spice ingredients, dietary fiber, phytoestrogens, and lactic acid bacteria to name a few. The editors are very thankful to Mirolav Misík4 (Institute of Cancer Research, Medical University of Vienna) for his immense help in the compilation of this book, to Andreas Sendtko from Wiley International for his continuous support, and to Miel Delahaij who designed the cover. For most scientists, their main challenge and interest in their work is the search for new exciting results, and the realization of experiments aimed at verifying their hypotheses, rather than writing reviews. Therefore, the editors express their grati- tude to all colleagues who contributed to the success of this book, and hope it will stimulate others to work in the field of chemoprevention. We also hope it will be of interest and value to both health authorities and food industries and provide them Preface XXIX with new, relevant, and practical information on the prevention of DNA damage and cancer by dietary components.

S. Knasmüller D. DeMarini I. Johnson C. Gerhäuser References

1 Patt, H.M., Tyree, E.B., Straube, R.L. 11 Wattenberg, L.W. (1983) Inhibition of and Smith, D.E. (1949) Cysteine neoplasia by minor dietary constituents. protection against X irradiation. Science, Cancer Research, 43, 2448s–2453s. 110, 213–214. 12 Wattenberg, L.W. (1990) Inhibition of 2 Mikaelsen, K. (1952) The protective carcinogenesis by naturally-occurring effect of glutathione against radiation and synthetic compounds. Basic Life induced chromosome aberrations. Sciences, 52, 155–166. Science, 116, 172–174. 13 De Flora, S. and Ramel, C. (1988) 3 Mikaelsen, K. (1954) Protective Mechanisms of inhibitors of properties of cysteine, sodium muatgenesis and carcinogenesis. hyposulfite, and sodium cyanide against Classification and overview. Mutation radiation induced chromosome Research, 202, 285–306. aberrations. Proceedings of the National 14 Doll, R. and Peto, R. (1981) The causes Academy of Sciences of the United States of of cancer: quantitative estimates of America, 40, 171–178. avoidable risks of cancer in the United 4 Riley, H.P. (1952) Preliminary report on States today. Journal of the National the effect of certain chemicals on Cancer Institute, 66, 1191–1308. radiation damage to chromosomes. 15 World Cancer Research Fund/American Genetics, 37, 618–619. Institute for Cancer Research (2007) 5 Avanzi, S. (1957) Inibizione delleffetto Food, Nutrition, Physical Activity and the citologico dellacetato di Tallio per Prevention of Cancer: A Global Perspective, mezzo della cisteina. Caryologia, 10,96– AICR, Washington, DC. 101. 16 Osburn, W.O. and Kensler, T.W. (2008) 6 Avanzi, S. (1961) Chromosome breakage Nrf2 signaling: an adaptive response by pyrrolizidine alkaloids and pathway for protection against modification of the effect by cysteine. environmental toxic insults. Mutation Caryologia, 14, 251–261. Research, 659,31–39. 7 Moutschen, J. (1958) Cystamine as a 17 Zhang, X., Yap, Y., Wei, D., Chen, G. modifier of chromosome breaks caused and Chen, F. (2008) Novel omics by maleic hydrazide in broad bean. technologies in nutrition research. Radiation Research, 9, 188. Biotechnology Advances, 26, 169–176. 8 Gebhard, E. (1974) Antimutagens: 18 Rimbach, G., Fuchs, J. and Packer, L. data and problems. Humangenetik, (2005) Nutrigenomics, CRC Press/Taylor 24,1–32. and Francis, Boca Raton, FL. 9 Kada, T., Inoue, T., Morita, K. and 19 Milner, J.A. (2008) Nutrition and cancer: Namiki, M. (1986) Dietary desmutagens. essential elements for a roadmap. Progress in Clinical and Biological Cancer Letters, 269, 189–198. Research, 20, 6245–6253. 20 Narayanan, B.A. (2006) 10 Kada, T., Inoue, T. and Namiki, M. (1982) Chemopreventive agents alter global Environmental desmutagens and gene expression pattern: predicting antimutagens, in Environmental their mode of action and targets. Mutagenesis and Plant Biology (ed. E.J. Current Cancer Drug Targets, Klekowski), Praeger Scientific, New York. 6,711–727. XXX Preface

21 Choe, E. and Min, D.B. (2006) Proceedings of the National Academy of Chemistry and reactions of reactive Sciences of the United States of America, oxygen species in foods. Critical Reviews 103, 17589–17594. in Food Science and Nutrition, 46,1–22. 23 Fenech, M. (2002) Micronutrients and 22 Ames, B.N. (2006) Low micronutrient genomic stability: a new paradigm for intake may accelerate the degenerative recommended dietary allowances diseases of aging through allocation of (RDAs). Food and Chemical Toxicology, scarce micronutrients by triage. 40, 1113–1117. XXXI

Foreword: Prevention of Cancer, and the Other Degenerative Diseases of Aging, Through Nutrition Bruce N. Ames and Joyce C. McCann

I (B.N.A.) appreciate being asked to write about my work as a foreword for this admirable book on cancer prevention. Rather than talk about my past work, I will discuss my current vision, which I think will turn out to be my most important contribution to science.

The Triage Theory

The many degenerative diseases accompanying aging, such as cancer, immune dysfunction, cognitive decline, cardiovascular disease, and stroke, might be delayed by an inexpensive intervention. Ten years ago I proposed that the risk of cancer was increased by chronic, suboptimal consumption of micronutrients (approximately 40 essential minerals, vitamins, amino acids, and fatty acids) and that this should be easily remediable [1]. Since that time, the primary focus of my scientific work at the Nutrition and Metabolism Center at CHORI has been to assemble the investigative tools necessary and to test this hypothesis at the biochemical and molecular level and, most recently, in human trials. In 2006, I proposed the triage theory [2], which provides a unifying rationale for expanding my original proposal to include a causal link between micronutrient deficiencies and chronic disease in general. Armed with the novel analytic tools we have developed over the past 10 years and guided by the triage theory, I believe we are now in a position to make a major breakthrough that will change the way people think about nutrition and about the degenerative diseases of aging. Our hope is that this research will provide the justification for and impetus to the

Professor Ames is among the few hundred most- cited scientists in all ﬁelds, is a recipient of numerous awards, including the US National Medal of Science and the Japan Prize, and is a member of the US National Academy of Sciences and the Royal Swedish Academy of Sciences.

implementation of inexpensive public health programs that can significantly reduce chronic disease incidence and increase life span. Triage theory [2] posits that when supply of a micronutrient is inadequate, nature selects for a rebalancing of metabolism (e.g., by selection for different micronutrient binding constants) that ensures survival of the organism at the expense of less critical metabolism. I propose the consequences of this rebalancing include chronic diseases of aging. That nature may have developed such a system is logically consistent with the consensus that natural selection favors short-term survival for reproduction over long-term health. During evolution, micronutrient shortages were likely to be very common, for example, the 15 essential minerals are not distributed evenly on Earth; dietary sources and availability of micronutrients also fluctuated markedly [3]. The triage theory predicts that optimizing intake of approximately 40 essential micronutrients will reduce the risk of chronic diseases associated with aging and increase life span [2]. Micronutrients are remarkably inexpensive. Micronutrient intakes below recommended levels are unusually widespread not only in poor countries but also in US population across all segments of society, especially the poor, children, adolescents, the obese, and the elderly. High consumption of calorie- rich, micronutrient-poor unbalanced diets exacerbates the problem [2]. For example, about two-thirds of the US population have inadequate intakes of magnesium [2], almost all African-Americans are extremely low in vitamin D [4], and much of the population is low in a variety of other micronutrients, for example, omega-3 fatty acids, potassium, calcium, vitamin C, and vitamin E [2, 5]. There is little societal concern because no overt pathologies have been associated with marginal to moderate levels of deficiency. The triage theory predicts that the pathology is insidious, but we believe that it is measurable.

DNA Damage, Immune Dysfunction, and Mitochondrial Decay

We hypothesize that three insidious but measurable consequences of micronutrient triage are increased DNA damage (future cancer), immune dysfunction (future severe infection and inadequate response to vaccination), and mitochondrial decay (future cognitive dysfunction and accelerated brain aging). All three of these measurable consequences are known to increase with age. In addition, evidence from our own work and that of others, briefly reviewed here, indicates that sensitive assays targeted at these three end points have a high likelihood of detecting changes in individuals with modest micronutrient deficiencies. (a) DNA damage. Deficiency in each of the seven micronutrients (Mg, Fe, Zn, and vitamins B6, C, folic acid, and biotin) that we have so far examined results in increased DNA damage in humans, primary human cells in culture, or in rodents [2, 6–10]. Folate deficiency in human cells in culture was accompanied by cell-cycle arrest in the S-phase, apoptosis, and high uracil incorporation into DNA [11, 12]. Others have shown that DNA damage occurs in humans who are Other Effects of Deficiency: Vitamin K XXXIII

deficient in Fe, Zn, folate, and B12 or choline [2, 6, 13] and in rodents or human cell cultures deficient in Se, Cu, Ca, niacin, choline, pantothenate, and riboflavin [2]. Many of these and other micronutrient deficiencies, when studied epidemiologically, are associated with cancer [2, 6, 14–24]. A number of human intervention studies with micronutrients are associated with lowering of DNA damage or cancer [25–27]. (b) Mitochondrial oxidant release. A large literature provides evidence that mitochondrial decay occurs with age and results in increased production of oxidant byproducts of electron transport [2]. This decay appears to be a major contributor to both aging and its associated degenerative diseases, such as brain dysfunction, for example, complex I and Parkinsons disease; complex IV and Alzheimers disease [2]. In mice or human cells in culture, we found that deficiencies in Zn [10], Fe [8], biotin [7], or vitamin B6 resulted in increased mitochondrial oxidative decay [2]. In all four cases, the mechanism appears to involve inhibition of heme synthesis, which prevents complex IV formation [2, 7, 8]. (c) Immune dysfunction. Adaptive immune dysfunction occurs with aging [28] and with deficiencies of various micronutrients, for example, vitamins A, B6, B12, C, E, and folate [11] and Cu, Se, Fe, Zn, and tryptophan [29]. In addition, the pattern of increased proinflammatory cytokine production (IL-1, IL-6, TNF-a) and decreased IFN-g production during aging is also seen with deficiencies in vitamins A, D, E, Zn, and Fe in young individuals [28]. Vitamin D inadequacy has been associated with autoimmune diseases such as multiple sclerosis, type I diabetes, and other inflammatory diseases [30]. In addition to increased DNA damage [10] with zinc deficiency, another possible mechanism explaining its association with many cancers [19, 20, 22–24] is the demonstrated decline with zinc deficiency of the activity of natural killer (NK) cells, known to be cytotoxic to nascent tumor cells [31, 32].

Cellular Aging

Growth under modest deficiency of each of the three micronutrients so far examined [2], that is, vitamin B6, magnesium [9], and biotin [7], increases the rate of cellular senescence; folate [6] and vitamin B6 (Askree and Ames, unpublished) deficiencies result in slowing of the cell cycle; and telomere shortening has been observed with both Mg [9] and B6 deficiencies, the two micronutrients so far examined. We are currently investigating effects of other common micronutrient deficiencies (e.g., potassium and calcium) in cell culture.

Other Effects of Deficiency: Vitamin K

We expect that there will also be other adverse consequences of triage rebalancing that will be unique to different micronutrients and speciﬁc clusters of functions. We XXXIV Foreword

are just completing a comprehensive analysis of one micronutrient, vitamin K (vitK) (McCann and Ames, manuscript in preparation). Preliminary results indicate that the localization of vitK-dependent functions required for short-term survival in the liver, and other vitK-dependent functions in extrahepatic tissues, sets up a dichotomy that takes advantage of the preferential distribution of vitK to the liver to preserve critical functions when vitK availability is scarce. These results strongly support the central premise of the triage theory; a partial summary of the analysis follows. VitK [33] is a cofactor for a single enzyme required for the g-carboxylation of 14 different proteins, 10 with known functions. g-Carboxylation is required for these proteins to bind calcium, which is known to be required for protein function in almost all cases. Most of these 10 proteins are synthesized only in the liver and are required for coagulation. Only 3 of the 10 proteins (osteocalcin, matrix Gla protein, and Gas6) are not coagulation factors. In contrast to the coagulation factors, these three proteins are synthesized in extrahepatic tissues. We asked six questions in the analysis; results are briefly summarized. Question 1: Can the spectrum of functions requiring vitamin K be stratified according to their relative importance for short-term survival? Mouse knockout lethality was used to indicate essentiality. Knockouts for almost all of the vitamin K dependent proteins synthesized in the liver are unable to survive embryogenesis or the neonatal period; knockouts for the three extrahepatic proteins survive at least through weaning. Question 2: Is loss of any of these functions linked to age-associated chronic disease? Two of these knockout mutants have been studied extensively, and phenotypes are consistent with age-related disease (e.g., matrix Gla protein: early death from arterial calcification; osteocalcin: glucose dysregulation, weaker bones after ovariectomy). Question 3: Does the body use different mechanisms for managing the availability of vitamin K for different classes of functions? The major dietary source of vitK (vitK1) is preferentially directed to the liver [34]. Furthermore, it appears that vitK1 is mainly active in the liver, and another form of vitK that can be synthesized from vitK1 is mainly active in the g-carboxylation of extrahepatic proteins [35]. Question 4: Are functions required for short-term survival more resistant to vitamin K scarcity than less critical functions? Higher dietary intakes of vitK1 are required for g-carboxylation of the extrahepatic protein so far examined (osteocalcin) than are required for g-carboxylation of the only hepatic protein involved in coagulation so far examined [36]. Question 5: Is dietary inadequacy of vitamin K linked to age-associated chronic disease or risk markers? Dietary inadequacy of vitK1 is linked in some studies to several chronic diseases or conditions associated with aging, for example, arterial calcification and atherocsclerosis [37, 38], increased fracture risk [39], osteoarthritis [40], and insulin resistance [41]. Question 6: Is there a public health issue – that is, how widespread are vitamin K deficiencies in people at levels that could trigger loss of function and increase risk of age-associated chronic disease? Whether currently recommended intakes for vitK1 are sufficient for optimal carboxylation of the extrahepatic proteins has been questioned [42]. The average intakes of vitK1 in the United States [43] and the United Kingdom [44] are less than even the currently recommended intakes [45], which are based primarily on levels required for adequate coagulation function. Thus, a large percentage of the population may not to be receiving sufficient vitK1 for optimal g-carboxylation of proteins important for References XXXV long-term health; amounts of vitK1 in most vitamin pills are also quite low. Question 7: Do the answers to the above six questions collectively form a coherent mechanistic picture consistent with triage theory? The localization of the g-carboxylation of coagulation factors in the liver, and of other vitK-dependent proteins in extrahepatic tissues, sets up a dichotomy that takes advantage of the preferential distribution of vitK1 to the liver to preserve coagulation functions when vitK availability is scarce.

Conclusion

If the triage hypothesis is correct, we believe our work will demonstrate the critical importance of avoiding micronutrient malnutrition for a long and healthy life and change how people think about both nutrition and health. Most of the worlds population, including that of the United States, is inadequate in one or more micronutrients according to current intake recommendations. Yet, because there is no overt pathology associated with these levels of deficiency, there has been little public concern. Current recommendations do not take into account the insidious biochemical changes we are measuring. Thus, if our experiments are successful, they will indicate that current recommendations are too low for some micronutrients, meaning that even greater numbers of people are deficient. We think our work will demonstrate that insidious changes are indeed occurring at modest levels of deficiency and that these changes are likely to increase the risk of cancer, cardiovascular disease, and cognitive dysfunction. This result should have an enormous impact on public awareness, providing the impetus for real change in dietary practices that will slow the aging process and reduce chronic disease incidence. We hope to show that there is an inexpensive route to preventive medicine so that the steadily increasing costs of poor diets to the United Sates and the world can be slowed.

References

1 Ames, B.N. (1998) Micronutrients prevent 4 McCann, J.C. and Ames, B.N. (2008) Is cancer and delay aging. Toxicology Letters, there convincing biological or behavioral 102–103,5–18. evidence linking vitamin D deﬁciency to 2 Ames, B.N. (2006) A theory of evolutionary brain dysfunction? The FASEB Journal, 22, allocation of scarce micronutrients by 982–1001. enzyme triage: adequate micronutrient 5 Moshfegh, A., Goldman, J. and Cleveland, nutrition to delay the degenerative diseases L. (2005) What We Eat in America, of aging. Proceedings of the National NHANES 2001–2002: Usual Nutrient Academy of Sciences of the United States of Intakes from Food Compared to Dietary America, 103, 17589–17594. Reference Intakes, US Department of 3 Eaton, S.B. and Konner, M. (1985) Agriculture, Agricultural Research Paleolithic nutrition. A consideration of its Service. nature and current implications. The New 6 Courtemanche, C., Huang, A.C., Elson- England Journal of Medicine, 312, 283–289. Schwab, I., Kerry, N., Ng, B.Y. and Ames, XXXVI Foreword

B.N. (2004) Folate deficiency and ionizing in Biophysics and Molecular Biology, 92, radiation cause DNA breaks in primary 49–59. human lymphocytes: a comparison. The 15 Ishihara, J., Otani, T., Inoue, M., Iwasaki, FASEB Journal, 18, 209–211. M., Sasazuki, S. and Tsugane, S. (2007) 7 Atamna, H., Newberry, J., Erlitzki, E., Low intake of vitamin B-6 is associated Schultz, C.S. and Ames, B.N. (2007) Biotin with increased risk of colorectal cancer in deficiency inhibits heme synthesis and Japanese men. The Journal of Nutrition, impairs mitochondria in human lung 137, 1808–1814. fibroblasts. The Journal of Nutrition, 137, 16 Grau, M.V., Baron, J.A., Sandler, R.S., 1–6. Wallace, K., Haile, R.W., Church, T.R., 8 Walter, P.W., Knutson, M.D., Paler- Beck, G.J., Summers, R.W., Barry, E.L., Martinez, A., Lee, S., Xu, Y., Viteri, F.E. Cole, B.F., Snover, D.C., Rothstein, R. and and Ames, B.N. (2002) Iron deficiency Mandel, J.S. (2007) Prolonged effect of and iron excess damage mitochondria calcium supplementation on risk of and mitochondrial DNA in rats. colorectal adenomas in a randomized trial. Proceedings of the National Academy of Journal of the National Cancer Institute, 99, Sciences of the United States of America, 99, 129–136. 2264–2269. 17 Chavarro, J.E., Stampfer, M.J., Li, H., 9 Killilea, D.W. and Ames, B.N. (2008) Campos, H., Kurth, T. and Ma, J. (2007) A Magnesium deficiency accelerates cellular prospective study of polyunsaturated fatty senescence in cultured human fibroblasts. acid levels in blood and prostate cancer Proceedings of the National Academy of risk. Cancer Epidemiology, Biomarkers & Sciences of the United States of America, 105, Prevention, 16, 1364–1370. 5768–5773. 18 Dai, Q., Shrubsole, M.J., Ness, R.M., 10 Ho, E. and Ames, B.N. (2002) Low Schlundt, D., Cai, Q., Smalley, W.E., Li, M., intracellular zinc induces oxidative DNA Shyr, Y. and Zheng, W. (2007) The relation damage, disrupts p53, NFkB, and AP1 of magnesium and calcium intakes and a DNA-binding, and affects DNA repair in a genetic polymorphism in the magnesium rat glioma cell line. Proceedings of the transporter to colorectal neoplasia risk. The National Academy of Sciences of the United American Journal of Clinical Nutrition, 86, States of America, 99, 16770–16775. 743–751. 11 Courtemanche, C., Elson-Schwab, I., 19 Franklin, R.B. and Costello, L.C. (2007) Mashiyama, S.T., Kerry, N. and Ames, B.N. Zinc as an anti-tumor agent in prostate (2004) Folate deficiency inhibits the cancer and in other cancers. Archives þ proliferation of primary human CD8 T of Biochemistry and Biophysics, 463, lymphocytes in vitro. Journal of 211–217. Immunology, 173, 3186–3192. 20 Abnet, C.C., Lai, B., Qiao, Y.L., Vogt, S., 12 Mashiyama, S.T., Hansen, C.M., Roitman, Luo, X.M., Taylor, P.R., Dong, Z.W., Mark, E., Sarmiento, S., Leklem, J.E., Shultz, T.D. S.D. and Dawsey, S.M. (2005) Zinc and Ames, B.N. (2008) An assay for uracil concentration in esophageal biopsy in human DNA at baseline: effect of specimens measured by X-ray marginal vitamin B6 deficiency. Analytical fluorescence and esophageal cancer risk. Biochemistry, 372,21–31. Journal of the National Cancer Institute, 97, 13 Fenech, M. (2007) Nutrition and genome 301–306. health, in Nutrigenomics: Opportunities in 21 Fong, L.Y., Jiang, Y. and Farber, J.L. (2006) Asia, vol. 60 (eds E. Tai and P. Gillies), Zinc deficiency potentiates induction and S. Karger AG, Basel, pp. 49–65. progression of lingual and esophageal 14 Holick, M.F. (2006) Vitamin D: its role in tumors in p53-deficient mice. cancer prevention and treatment. Progress Carcinogenesis, 27, 1489–1496. References XXXVII

22 Jaiswal, A.S. and Narayan, S. (2004) Zinc evidence? The British Journal of Nutrition, stabilizes adenomatous polyposis coli 89, 552–572. (APC) protein levels and induces cell cycle 31 Moretta, L., Ciccone, E., Mingari, M.C., arrest in colon cancer cells. Journal of Biassoni, R. and Moretta, A. (1994) Cellular Biochemistry, 93, 345–357. Human natural killer cells: origin, 23 Prasad, A.S., Beck, F.W., Doerr, T.D., clonality, specificity, and receptors. Shamsa, F.H., Penny, H.S., Marks, S.C., Advances in Immunology, 55, 341–380. Kaplan, J., Kucuk, O. and Mathog, R.H. 32 Tapazoglou, E., Prasad, A.S., Hill, G., (1998) Nutritional and zinc status of head Brewer, G.J. and Kaplan, J. (1985) and neck cancer patients: an interpretive Decreased natural killer cell activity in review. Journal of the American College of patients with zinc deficiency with sickle Nutrition, 17, 409–418. cell disease. The Journal of Laboratory and 24 Liaw, K.Y., Lee, P.H., Wu, F.C., Tsai, J.S. Clinical Medicine, 105,19–22. and Lin-Shiau, S.Y. (1997) Zinc, copper, 33 Berkner, K.L. (2008) Vitamin K-dependent and superoxide dismutase in carboxylation. Vitamins and Hormones, 78, hepatocellular carcinoma. The American 131–156. Journal of Gastroenterology, 92, 2260–2263. 34 Schurgers, L.J. and Vermeer, C. (2002) 25 Fenech, M., Aitken, C. and Rinaldi, J. Differential lipoprotein transport (1998) Folate, vitamin B12, homocysteine pathways of K-vitamins in healthy subjects. status and DNA damage in young Biochimica et Biophysica Acta, 1570,27–32. Australian adults. Carcinogenesis, 19, 35 Cranenburg, E.C., Schurgers, L.J. and 1163–1171. Vermeer, C. (2007) Vitamin K: the 26 Goh, Y.I., Bollano, E., Einarson, T.R. and coagulation vitamin that became Koren, G. (2007) Prenatal multivitamin omnipotent. Thrombosis and Haemostasis, supplementation and rates of pediatric 98, 120–125. cancers: a meta-analysis. Clinical 36 Binkley, N.C., Krueger, D.C., Kawahara, Pharmacology and Therapeutics, 81, T.N., Engelke, J.A., Chappell, R.J. and 685–691. Suttie, J.W. (2002) A high phylloquinone 27 Ribeiro, M.L., Arcari, D.P., Squassoni, intake is required to achieve maximal A.C. and Pedrazzoli, J. Jr (2007) Effects of osteocalcin gamma-carboxylation. The multivitamin supplementation on DNA American Journal of Clinical Nutrition, 76, damage in lymphocytes from elderly 1055–1060. volunteers. Mechanisms of Ageing and 37 Berkner, K.L. and Runge, K.W. (2004) The Development, 128, 577–580. physiology of vitamin K nutriture and 28 DelaRosa, O., Pawelec, G., Peralbo, E., vitamin K-dependent protein function in Wikby, A., Mariani, E., Mocchegiani, E., atherosclerosis. Journal of Thrombosis and Tarazona,R.andSolana,R.(2006) Haemostasis, 2, 2118–2132. Immunological biomarkers of ageing in 38 Wallin, R., Schurgers, L. and Wajih, N. man: changes in both innate and (2008) Effects of the blood coagulation adaptive immunity are associated with vitamin K as an inhibitor of arterial health and longevity. Biogerontology, 7, calcification. Thrombosis Research, 122, 471–481. 411–417. 29 Wintergerst, E.S., Maggini, S. and Hornig, 39 Cockayne, S., Adamson, J., Lanham-New, D.H. (2007) Contribution of selected S., Shearer, M.J., Gilbody, S. and vitamins and trace elements to immune Torgerson, D.J. (2006) Vitamin K and the function. Annals of Nutrition and prevention of fractures: systematic review Metabolism, 51, 301–323. and meta-analysis of randomized 30 Zittermann, A. (2003) Vitamin D in controlled trials. Archives of Internal preventive medicine: are we ignoring the Medicine, 166, 1256–1261. XXXVIII Foreword

40 Neogi, T., Booth, S.L., Zhang, Y.Q., of vitamin K-1 (phylloquinone) in the Jacques, P.F., Terkeltaub, R., Aliabadi, P. American diet: data from the FDA Total and Felson, D.T. (2006) Low vitamin K Diet Study. Journal of the American Dietetic status is associated with osteoarthritis in Association, 96,149–154. the hand and knee. Arthritis and 44 Thane, C.W., Bolton-Smith, C. and Rheumatism, 54, 1255–1261. Coward, W.A. (2006) Comparative dietary 41 Yoshida, M., Booth, S.L., Meigs, J.B., intake and sources of phylloquinone Saltzman, E. and Jacques, P.F. (2008) (vitamin K1) among British adults in Phylloquinone intake, insulin sensitivity, 1986–7 and 2000–1. The British Journal of and glycemic status in men and women. Nutrition, 96, 1105–1115. The American Journal of Clinical Nutrition, 45 Institute of Medicine Food and Nutrition 88, 210–215. Board (2000) Dietary Reference Intakes for 42 Vermeer, C. and Hamulyak, K. (2004) Vitamin A, Vitamin K, Arsenic, Boron, Vitamin K: lessons from the past. Journal of Chromium, Copper, Iodine, Iron, Thrombosis and Haemostasis, 2, 2115–2117. Manganese, Molybdenum, Nickel, Silicon, 43 Booth, S.L., Pennington, J.A. and Sadowski, Vanadium, and Zinc, National Academy J.A. (1996) Food sources and dietary intakes Press, Washington, DC. XXXIX

List of Contributors

Bharat B. Aggarwal Maria Bagnasco The University of Texas M. D. Anderson University of Genoa Cancer Center Department of Health Sciences Department of Experimental Via A. Pastore 1 Therapeutics 16132 Genoa Cytokine Research Laboratory Italy 1515 Holcombe Boulevard, Box 143 Houston, TX 77030 Helmut Bartsch USA German Cancer Research Center Division of Epigenomics and Cancer Preetha Anand Risk Factors The University of Texas M. D. Anderson Im Neuenheimer Feld 280 Cancer Center 69120 Heidelberg Department of Experimental Germany Therapeutics Cytokine Research Laboratory Alessandra Battistella 1515 Holcombe Boulevard, Box 143 University of Genoa Houston, TX 77030 Department of Health Sciences USA Via A. Pastore 1 16132 Genoa Awais Anwar Italy Saarland State University School of Pharmacy Nigel J. Belshaw Division of Bioorganic Chemistry Institute of Food Research 66123 Saarbruecken Intestinal Biology and Health Germany Programme Norwich Research Park, Colney Sakae Arimoto-Kobayashi Norwich NR4 7UA Okayama University UK Faculty of Pharmaceutical Sciences Tsushima, Okayama 700-8530 Japan

Carlo Bennicelli Andrew R. Collins University of Genoa University of Oslo Department of Health Sciences Department of Nutrition Via A. Pastore 1 PB 1046 Blindern 16132 Genoa 0316 Oslo Italy Norway

Tariq A. Bhat Heide S. Cross Jawaharlal Nehru University Medical University of Vienna School of Life Sciences Department of Pathophysiology Cancer Biology Laboratory Waehringerguertel 18-20 New Delhi 110067 1090 Vienna India Austria

Heiner Boeing Silvio De Flora German Institute of Human Nutrition University of Genoa Department of Epidemiology Department of Health Sciences Potsdam-Rehbrücke Via A. Pastore 1 Arthur–Scheunert Allee 114–116 16132 Genoa 14558 Nuthetal Italy Germany David M. DeMarini Tomasz Borkowski U.S. Environmental Protection Agency University of Ulster Environmental Carcinogenesis Division School of Biomedical Sciences Research Triangle Park, NC 27711 Northern Ireland Centre for Food USA and Health Coleraine BT52 1SA Veronika A. Ehrlich UK Medical University of Vienna Department of Medicine I L. Adele Boyd Institute of Cancer Research Glanbia Nutritionals Borschkegasse 8a Glanbia Innovation Centre 1090 Vienna Leggetsrath Business Park Austria Kilkenny Ireland Giles O. Elliott Institute of Food Research Christophe Cavin Intestinal Biology and Health Nestlé Research Center Programme Quality and Safety Department Norwich Research Park, Colney P.O. Box 44, Vers-chez-les-Blanc Norwich NR4 7UA 1000 Lausanne 26 UK Switzerland List of Contributors XLI

Gernot Faustmann Jan Frank Medical University of Vienna Christian-Albrechts-University Department of Medicine I Institute of Human Nutrition Institute of Cancer Research and Food Science Borschkegasse 8a Hermann-Rodewald-Straße 6, 1090 Vienna Olshausenstrasse 40 Austria 24118 Kiel Germany Alain Favier Laboratoire Lésions des Acides Adrian A. Franke Nucléiques University of Hawaii CEA Cancer Research Center of Hawaii Grenoble 1236 Lauhala Street, Suite 407 France Honolulu, HI 96813 USA Michael Fenech CSIRO Human Nutrition Sabine Fuchs Gate 13, Kintore Avenue Medical University of Vienna Adelaide, BC 5000 Institute of Cancer Research Australia Department of Medicine I Borschkegasse 8a Lynnette R. Ferguson 1090 Vienna The University of Auckland Austria Faculty of Medical & Health Science Discipline of Nutrition Kareemah Gamieldien Private Bag 92019 Medical Research Council Auckland PROMEC Unit New Zealand P.O. Box 19070 Tygerberg 7505 Metka Filipic South Africa National Institute of Biology Department for Genetic Toxicology Wentzel C.A. Gelderblom and Cancer Biology Medical Research Council Ljubljana PROMEC Unit Slovenia P.O. Box 19070 Tygerberg 7505 Dianne Ford South Africa Newcastle University The Medical School Clarissa Gerhäuser Institute for Cell and Molecular German Cancer Research Center Biosciences and Human Nutrition Division of Epigenomics and Cancer Research Centre Risk Factors Framlington Place Im Neuenheimer Feld 280 Newcastle-upon-Tyne NE2 4HH 69120 Heidelberg UK Germany XLII List of Contributors

Cris Gill Philip J. Harris University of Ulster The University of Auckland School of Biomedical Sciences Faculty of Science Northern Ireland Centre for Food School of Biological Sciences and Health Private Bag 92019 Coleraine BT52 1SA Auckland UK New Zealand

Hansruedi Glatt Hikoya Hayatsu German Institute of Human Nutrition Okayama University (DIfE) Potsdam-Rehbruecke Faculty of Pharmaceutical Sciences Arthur-Scheunert Allee 114-116 Tsushima, Okayama 700-8530 14558 Nuthetal Japan Germany John Hesketh Michael Grusch Newcastle University Medical University of Vienna The Medical School Department of Medicine I Institute for Cell and Molecular Institute of Cancer Research Biosciences and Human Nutrition Borschkegasse 8A Research Centre 1090 Vienna Framlington Place Austria Newcastle-upon-Tyne NE2 4HH UK Christophe Hano Antenne Scientiﬁque Universitaire Johannes C. Huber de Chartres 21 Medical University Vienna Unité EA 1207 Biologie des Ligneux Department of Gynecologic et Grandes Cultures Endocrinology and Reproductive rue de Loigny la bataille Medicine 28 000 Chartres Waehringer Guertel 18-20 France 1090 Vienna Austria Kuzhuvelil B. Harikumar The University of Texas M. D. Anderson Wolfgang W. Huber Cancer Center Medical University of Vienna Department of Experimental Department of Medicine I Therapeutics Institute of Cancer Research Cytokine Research Laboratory Borschkegasse 8A 1515 Holcombe Boulevard, Box 143 1090 Vienna Houston, TX 77030 Austria USA List of Contributors XLIII

Claus Jacob Siegfried Knasmüller Saarland State University Medical University of Vienna School of Pharmacy Department of Medicine I Division of Bioorganic Chemistry Institute of Cancer Research 66123 Saarbruecken Borschkegasse 8a Germany 1090 Vienna Austria Ian T. Johnson Institute of Food Research Verena Koller Intestinal Biology and Health Medical University of Vienna Programme Department of Medicine I Norwich Research Park, Colney Institute of Cancer Research Norwich NR4 7UA Borschkegasse 8a UK 1090 Vienna Austria Elizabeth Joubert ARC Infruitec-Nietvoorbij Pavel Kramata Department of Food Sciences Rutgers, The State University Private Bag X5026 of New Jersey Stellenbosch 7599 Ernest Mario School of Pharmacy South Africa Department of Chemical Biology Nina Kager Susan Lehman Cullman Laboratory Medical University of Vienna for Cancer Research Department of Medicine I Piscataway, NJ 08854 Institute of Cancer Research USA Borschkegasse 8a 1090 Vienna Sabine Kulling Austria University of Potsdam Institute of Nutritional Science Ann R. Kennedy Arthur-Scheunert Allee 114-116 University of Pennsylvania School 14558 Nuthetal of Medicine Germany Department of Radiation Oncology 195 John Morgan Building, 3620 Joydeb Kumar Kundu Hamilton Walk Seoul National University Philadelphia, PA 19104-6072 College of Pharmacy USA National Research Laboratory of Molecular Carcinogenesis and Thomas W. Kensler Chemoprevention The Johns Hopkins University Seoul 151-742 Bloomberg School of Public Health South Korea Department of Environmental Health Sciences Baltimore, MD 21205 USA XLIV List of Contributors

Ajaikumar B. Kunnumakkara Hong Jin Lee The University of Texas M. D. Anderson Rutgers, The State University Cancer Center of New Jersey Department of Experimental Ernest Mario School of Pharmacy Therapeutics Department of Chemical Biology Cytokine Research Laboratory Susan Lehman Cullman Laboratory 1515 Holcombe Boulevard, Box 143 for Cancer Research Houston, TX 77030 Piscataway, NJ 08854 USA USA

Ying-Ling Lai Loïc Le Marchand University of Shizuoka University of Hawaii Graduate School of Nutritional Cancer Research Center of Hawaii and Environmental Sciences 1236 Lauhala Street, Suite 407 Global COE Program for Innovation Honolulu, HI 96813 in Human Health Sciences USA 52-1 Yada, Suruga-ku Shizuoka 422-8526 Elizabeth K. Lund Japan Institute of Food Research Norwich Research Park, Colney Eric Lainé Norwich NR4 7UA Antenne Scientiﬁque Universitaire UK de Chartres 21 Unité EA 1207 Biologie des Ligneux Alexa L. Meyer et Grandes Cultures University of Vienna rue de Loigny la bataille Institute of Nutritional Sciences 28 000 Chartres Althanstrasse 14 France 1090 Vienna Austria Joshua D. Lambert The Pennsylvania State University Anthony B. Miller Department of Food Science University of Toronto 332 Food Science Building Department of Public Health Sciences University Park, PA 16802 155 College Street USA Toronto, Ontario M5T 3M7 Canada Frédéric Lamblin Antenne Scientiﬁque Universitaire Miroslav Misík4 de Chartres 21 Medical University of Vienna Unité EA 1207 Biologie des Ligneux Department of Medicine I et Grandes Cultures Institute of Cancer Research rue de Loigny la bataille Borschkegasse 8a 28 000 Chartres 1090 Vienna France Austria List of Contributors XLV

Anil Mittal Tomoe Negishi Jawaharlal Nehru University Okayama University School of Life Sciences Faculty of Pharmaceutical Sciences Cancer Biology Laboratory Tsushima, Okayama 700-8530 New Delhi 110067 Japan India Armen Nersesyan Noriyuki Miyoshi Medical University of Vienna University of Shizuoka Department of Medicine I Graduate School of Nutritional Institute of Cancer Research and Environmental Sciences Borschkegasse 8a Global COE Program for Innovation 1090 Vienna in Human Health Sciences Austria 52-1 Yada, Suruga-ku Thomas Nittke Shizuoka 422-8526 Medical University of Vienna Japan Department of Pathophysiology Waehringer Guertel 18-20 Peter L. Molloy 1090 Vienna CSIRO Molecular and Health Austria Technologies Preventative Health Flagship Hiroshi Ohshima P.O. Box 184 University of Shizuoka North Ryde, NSW 1670 Graduate School of Nutritional Australia and Environmental Sciences Global COE Program for Innovation Alicja Mortensen in Human Health Sciences Technical University of Denmark 52-1 Yada, Suruga-ku National Food Institute (DTU) Shizuoka 422-8526 Department of Toxicology and Risk Japan Assessment Mørkhø Bygade 19 Johannes Ott 2860 Søborg Medical University Vienna Denmark DepartmentofGynecologicEndocrinology and Reproductive Medicine Hye-Kyung Na Waehringer Guertel 18-20 Seoul National University 1090 Vienna College of Pharmacy Austria National Research Laboratory of Molecular Carcinogenesis Wolfram Parzefall and Chemoprevention Medical University of Vienna Seoul 151-742 Department of Medicine I South Korea Institute of Cancer Research Borschkegasse 8a 1090 Vienna Austria XLVI List of Contributors

Jara Pérez-Jiménez Ian Rowland Department of Metabolism University of Reading and Nutrition Hugh Sinclair Unit for Human IF-CSIC Nutrition c/José Antonio Novais 10 Department of Food Biosciences 28040 Madrid Whiteknights Spain Reading RG6 6AP UK Janja Plazar National Institute of Biology Philipp Saiko Department for Genetic Toxicology Medical University of Vienna and Cancer Biology Clinical Institute of Medical Ljubljana and Chemical Laboratory Diagnostics Slovenia General Hospital of Vienna Waehringer Guertel 18-20 M. Cristina Polidori 1090 Vienna Heinrich-Heine University Düsseldorf Austria Institute of Biochemistry and Molecular Biology I Fulgencio Saura-Calixto PO Box 101007 Department of Metabolism 40001 Düsseldorf and Nutrition Germany IF-CSIC c/José Antonio Novais 10 Keith N. Rand 28040 Madrid CSIRO Molecular and Health Spain Technologies Preventative Health Flagship Heidi Schwartz P.O. Box 184 University of Vienna North Ryde, NSW 1670 Faculty of Chemistry Australia Department of Analytical and Food Chemistry Gerald Rimbach Waehringer Straße 38 Christian-Albrechts-University 1090 Vienna Institute of Human Nutrition Austria and Food Science Hermann-Rodewald-Str. 6, Daniel T. Shaughnessy Olshausenstrasse 40 National Institute of Environmental 24118 Kiel Health Sciences Germany Division of Extramural Research and Training, DHHS Research Triangle Park, NC 27709 USA List of Contributors XLVII

Soona Shin Gary D. Stoner The Johns Hopkins University The Ohio State University College School of Medicine of Medicine Department of Pharmacology Division of Hematology and Oncology and Molecular Sciences Department of Internal Medicine Baltimore, MD 21205 and Comprehensive Cancer Center USA 2001 Polaris PKWY Columbus, OH 43240 Rana P. Singh USA Jawaharlal Nehru University School of Life Sciences Nanjoo Suh Cancer Biology Laboratory Rutgers, The State University New Delhi 110067 of New Jersey India Ernest Mario School of Pharmacy Department of Chemical Biology Gerhard Sontag Susan Lehman Cullman Laboratory University of Vienna for Cancer Research Faculty of Chemistry Piscataway, NJ 08854 Department of Analytical USA and Food Chemistry Waehringer Str. 38 Young-Joon Surh 1090 Vienna Seoul National University Austria College of Pharmacy National Research Laboratory Wilhelm Stahl of Molecular Carcinogenesis Heinrich-Heine University Düsseldorf and Chemoprevention Institute of Biochemistry and Seoul 151-742 Molecular Biology I South Korea P.O. Box 101007 40001 Düsseldorf Akos Szakmary Germany Medical University of Vienna Department of Medicine I Reinhard Stidl Institute of Cancer Research Pouthongasse 26/17 Borschkegasse 8a 1150 Vienna 1090 Vienna Austria Austria

Thomas Szekeres Medical University of Vienna Clinical Institute of Medical and Chemical Laboratory Diagnostics General Hospital of Vienna Waehringer Guertel 18-20 1090 Vienna Austria XLVIII List of Contributors

Philip Thomas Li-Shu Wang CSIRO Human Nutrition The Ohio State University College Gate 13, Kintore Avenue of Medicine Adelaide, BC 5000 Division of Hematology and Oncology Australia Department of Internal Medicine and Comprehensive Cancer Center Susumu Tomono 2001 Polaris PKWY University of Shizuoka Columbus, OH 43240 Graduate School of Nutritional USA and Environmental Sciences Global COE Program for Innovation Chung S. Yang in Human Health Sciences Rutgers, The State University 52-1 Yada, Suruga-ku of New Jersey Shizuoka 422-8526 Ernest Mario School of Pharmacy Japan Department of Chemical Biology 164 Frelinghuysen Road Karl-Heinz Wagner Piscataway, NJ 08854-8020 University of Vienna USA Department of Nutritional Sciences Althanstrasse 14 1090 Vienna Austria Part One General Principles

1 Molecular Mechanisms of Cancer Induction and Chemoprevention Helmut Bartsch and Clarissa Gerh€auser

1.1 Cancer Chemoprevention

Advances in understanding the process of carcinogenesis at the cellular and molecular level [1] and progress in identifying the major causes of human cancer [2] have led to the development of cancer chemoprevention as a promising and relatively new prevention approach that includes prevention of recurrent cancer and second primary tumors. It is an adjunct to primary prevention, early detection, and cancer therapy. Chemoprevention is the process of inhibiting, delaying, or reversing carcinogenesis in the premalignant phase; it aims to halt or reverse the development and progression of precancerous cells to invasive cancer through the use of nontoxic nutrients, phytochemicals, and synthetic pharmacological agents. These can act during the time period from initiation to progression of tumors. Cancer chemoprevention has precedence in cardiology, in which cholesterol- lowering, antihypertensive, and antiplatelet agents are administered to prevent coronary heart disease in high-risk individuals. The concept of using chemopreventive agents to reduce cancer risk is based firmly on epidemiologic and experimental evidence indicating that specific agents can reduce carcinogenesis at various sites, including among others breast, colon/rectum, lung, prostate, and skin [3, 4]. Chemoprevention research has been stimulated by epidemiologic studies that have linked high consumption of fruit and vegetables to a reduced cancer risk [5]. This has provided initial leads for the identification of numerous naturally occurring (candidate) chemopreventive agents. Dietary components with potential cancer chemopreventive activity generally fall in six categories: besides vitamins, fiber, and minerals as nutrient components, human diet may contain up to 10 000 nonnutrient constituents belonging to the organosulfur compounds, polyphenols, and terpenoids [6, 7]. Chemopreventive agents suitable for the prevention of cancer in the general population should have high acceptance, low cost, oral consumability, high efficacy, no or low toxicity, and a known mechanism of action. When synthetic drugs are applied to high-risk groups, that is, those with precancerous lesions, a low but defined toxicity is often tolerated

in risk–beneﬁt evaluations. Promising chemopreventive agents currently investigated in preclinical and clinical studies and dealt with in this volume include naturally occurring anti-inﬂammatory agents, antiestrogens, micronutrients, phytochemicals, and some synthetic analogues.

1.2 Molecular Mechanisms and Targets of Chemopreventive Agents

Knowledge of molecular mechanisms is of importance not only for safe application of known agents but also for further development of novel potential cancer preventive agents [8–10]. The development of cancer is a multistage process that is broadly divided into initiation, promotion, and progression phases (Figure 1.1). Carcinogen- esis can be regarded as an accumulation of genetic and biochemical cell damage that offers many molecular targets for chemopreventive agents to prevent, inhibit, or slow the progression from early genetic lesions to invasive tumor development [11]. In the initiation phase, a carcinogen, either directly or after metabolic activation to a reactive molecule, interacts with intracellular macromolecules. This may cause DNA damage, which, if not repaired, can result in genetic and other cellular damage. Mutations eventually lead to an altered expression of oncogenes and tumor suppressor genes or continuous activation of protein kinases during the promotion phase, all of which ﬁnally result in modiﬁed cell structure, uncontrolled cell proliferation,

Figure 1.1 Multistage carcinogenesis and mechanisms relevant for cancer chemoprevention (HDACs, histone deacetylases). 1.2 Molecular Mechanisms and Targets of Chemopreventive Agents j5 tumor growth, and metastases. This cascade of events offers a variety of targets for chemopreventive intervention at every stage. As depicted in Figure 1.1, so far well- established chemopreventive mechanisms include modulation of drug metabolism, antimutagenic, antioxidant, radical-scavenging, anti-inﬂammatory, antitumor promoting, and antiproliferative activities as well as induction of terminal cell differentiation and apoptosis. Recently, antiangiogenic properties and potential to overcome epigenetic deregulation have been recognized as novel (adjunct) mechanisms of chemopreventive activity.

1.2.1 Carcinogen-Blocking Activities

Compounds effective at the initiation stage of carcinogenesis are termed as blocking agents, as they block carcinogen interaction with DNA. This is achieved inter alia by the inhibition of carcinogen uptake into the cell. For example, dietary fiber is believed to bind to and interact with carcinogens in the colonic lumen and therefore prevent damage to colon epithelial cells. The cancer preventive mechanism of calcium is partly attributed to the inactivation (inhibition of uptake) of procarcinogenic bile acids in the fecal matrix. Another important mechanism of blocking agents is the modulation of drug metabolism, often inhibition of metabolic activation of carcinogens by cytochrome P450 (phase 1) enzymes, and induction of drug detoxification (phase 2) enzymes to enhance excretion of procarcinogenic and reactive metabolites. Phase 1 enzymes metabolically activate some 1% of xenobiotics into electrophiles by addition of functional groups in an attempt to render these compounds more water soluble. As a protection against cellular damage, phase 2 enzymes such as glutathione- S-transferase, N-acetyl transferase, sulfotransferase, or UDP-glucuronosyl transferase conjugate the more reactive compounds to endogenous ligands and enhance their excretion. Because phase 1 enzymes often contribute to the activation of carcinogens while phase 2 enzymes are generally involved in their detoxication, inhibition of phase 1 enzymes concomitantly with induction of phase 2 enzymes is considered a basis for the efficacy of many chemopreventive compounds. Agents that selectively induce phase 2 enzymes are termed monofunctional inducers and include aryl/alkyl isothiocyanates present in cruciferous vegetables such as broccoli and cabbage. Furthermore, the organosulfur components diallylsulfide and diallyldisulfide from Allium species (garlic, onion) as well as catechols from green tea belong to this class. Bifunctional inducers such as indole-3-carbinol, which occurs in cruciferous vegetables as glucosinolate precursor, lead to a simultaneous induction of phase 1 and 2 enzyme activities.

1.2.2 Antimutagenic Effects and DNA Repair

Antimutagens can act to inhibit carcinogen uptake, modulate the activity of the cytochrome P450 enzymes, and block the activation of procarcinogens by a wide 6j 1 Molecular Mechanisms of Cancer Induction and Chemoprevention

variety of mechanisms. Antimutagens can also block carcinogen activation by inhibiting cyclooxygenases and lipoxygenases. Some antimutagens can enhance conjugation or binding of activated carcinogens to glutathione or other molecules to inactivate them and facilitate their removal. Other agents can block the binding of activated carcinogens to DNA, thus reducing the formation of DNA adducts. As an example, ellagic acid, a polyphenolic constituent of berries and nuts, has been shown to bind to and detoxify the diol epoxide of benzo[a]pyrene; by binding to DNA it has also been found to reduce the formation of O6-methylguanine by methylating carcinogens [12]. An additional antimutagenic mechanism, so far less explored, is to enhance DNA repair activity and ﬁdelity. Induction of poly(ADP-ribosyl)ation in human peripheral lymphocytes after treatment with ()-epigallocatechin gallate has been shown [13]. Also, intervention with kiwifruit for 6 weeks in healthy volunteers signiﬁcantly increased the ability of leukocytes to repair DNA breakage by free radicals [14].

1.2.3 Targeting Epigenetic Mechanisms

The term epigenetics is defined as heritable alterations in gene expression patterns that occur without DNA sequence changes. These heritable alterations are achieved by (i) methylation of cytosine bases in DNA and (ii) by post-translational histone modifications, such as acetylation, methylation, or phosphorylation. Epigenetic events occur very early during embryogenesis and throughout all stages of tumorigenesis. Hypermethylation of CpG-rich sequences in DNA of promoter regions is one of the most important epigenetic changes to occur in cancer cells, leading to transcriptional silencing of tumor suppressor and many other cancer relevant genes. Therefore, these changes are thought to be a key driving force in the development of cancer (reviewed by Hauser and Jung [15]). In contrast to tumor suppressor genes, which are irreversibly inactivated by genetic alterations, genes silenced by epigenetic modifications are still intact and can be reactivated by small molecules acting as modifiers of epigenetic mechanisms. Several natural products have recently been identified as inhibitors of DNA methyl transferases (DNMTs), the enzymes responsible for DNA methylation (Table 1.1). This is associated with the reactivation of methylation-silenced genes. Consequently, development of agents or food

Table 1.1 Naturally occurring inhibitors of DNA methyl transferases.

Category Compound Dietary source

Polyphenols Epigallocatechin gallate Green tea Caffeic acid Coffee, various fruits Chlorogenic acid Apple polyphenols Apples Genistein, daidzein Soy products Biochanin A Red clover 1.2 Molecular Mechanisms and Targets of Chemopreventive Agents j7 components that prevent or reverse the hypermethylation-induced inactivation of tumor suppressor genes is a new promising approach to cancer prevention. DNA forms a macromolecular complex with histone and nonhistone proteins designated as chromatin. Chromatin is responsible for the controlled storage of the genetic information within the nucleus. The structure of chromatin, which plays an important role in gene expression, is maintained by post-translational modifications of histones, such as acetylation, methylation, phosphorylation, sumoylation, and ubiquitination. As an example, histone acetylation results in an open and accessible chromatin and is associated with active gene transcription, whereas deacetylated, condensed chromatin mediates transcriptional repression. The steady state of reversible protein acetylation is maintained by a dynamic equilibrium between histone acetyl transferases (HATs) and histone deacetylases (HDACs). Inhibition of HDAC has been shown to affect the expression of genes that have an impact on apoptosis and cell cycle regulation, such as the cell cycle regulator p21, cyclins, apoptosis mediators, transcription factors, and retinoic acid receptors (RARs). Only few natural product inhibitors of HDACs have been described so far. For example, butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, inhibits HDAC activity at high mM concentrations and affects cellular differentiation, cell cycle arrest, apoptosis, invasion, and metastasis. Two sulfur- containing phytochemicals derived from garlic and broccoli, that is, diallyldisulfide (DADS) and sulforaphane, inhibit HDAC activity through their metabolites S-allylmercaptocysteine and sulforaphane cysteine. Interestingly, combination of sulforaphane and the DMNT-inhibiting isoflavone genistein enhanced the reactivation of methylation-silenced genes, implicating synergistic effects. In addition to these agents, natural product inhibitors of HATs and enzymes involved in additional histone modifications (ubiquitinylation, sumoylation, poly- ADP-ribosylation) have been described [15]. The impact of these epigenetic alterations in chemopreventive efficacy has yet to be demonstrated.

1.2.4 Radical-Scavenging and Antioxidant Effects

Insufficient oxygen consumption in mitochondria during fat metabolism (lipid peroxidation) might result in the production of reactive oxygen species (ROS). In a healthy organism, the formation of ROS is generally controlled by physiological antioxidant mechanisms, for example, intracellular glutathione and enzymatic mechanisms involving catalase, superoxide dismutase, and glutathione peroxidase. Manifestation of oxidative stress (immune diseases, chronic inflammation, and infection) disturbs the intracellular homeostasis of prooxidants and antioxidants. Overproduction of ROS results in the formation of DNA-reactive oxidation products including lipid peroxidation end products such as 4-hydroxy-2-nonenal and malondialdehyde, activation of carcinogens, and formation of promutagenic oxidized or modified DNA bases, and DNA strand breaks [16]. This can lead, if not repaired, to errors during DNA replication causing genetic alterations, induced transcription of protooncogenes and of ornithine decarboxylase, and increased risk for malignant 8j 1 Molecular Mechanisms of Cancer Induction and Chemoprevention

Table 1.2 Examples of naturally occurring antioxidants.

Category Compound Dietary source

Polyphenols Epigallocatechin gallate Green tea Quercetin Ubiquitous Ellagic acid Nuts, berries Curcumin Turmeric, curry Terpenoids b-Carotene Carrots Lycopene Tomatoes Vitamins Vitamin C (ascorbic acid) Citrus fruits Vitamin E (a-tocopherol) Oils of wheat germ, corn, soybean, and sunﬂower

transformation, all of which ultimately result in enhanced cell proliferation, tumor promotion, and progression. Radical scavengers and antioxidants inactivate ROS and are thought to restore the intracellular redox equilibrium. Some examples of dietary antioxidants are given in Table 1.2. The in vivo efﬁcacy of polyphenolic antioxidants has yet to be demonstrated, since bioavailability of these hydrophilic compounds is generally low, and it is questionable if protective intracellular or serum levels may be achieved. Supplementation with a-tocopherol (vitamin E), a lipophilic antioxidant, was shown to reduce prostate cancer incidence by 32% and overall mortality by 41% in heavy smokers [17]. However, in long-term large-scale chemoprevention trials, supplementation with lipophilic (synthetic) b-carotene, a component from orange- or red-colored vegetables such as carrots, failed to prevent lung cancer in male smokers [18]. In both trials, lung cancer incidence was elevated in smokers receiving high doses of b-carotene, possibly acting as prooxidant. This stresses the need for understanding the molecular mechanisms of chemopreventive agents so to avoid unwanted side effects.

1.2.5 Anti-Inflammatory Mechanisms

It is estimated that 19% of the global cancer burden is related to chronic inflammatory processes such as chronic gastritis, esophagitis, hepatitis, pancreatitis, ulcerative colitis, and Crohns disease. Hereby DNA damage from endogenous reactive species and excess production of nitric oxide (NO) and prostaglandins, that is, hormone-like endogenous mediators of inflammation, are thought to be among the causative factors of cellular injury and carcinogenesis. Cyclooxygenase (COX) 1 and 2 enzymes are involved in the biosynthesis of prostaglandins from arachidonic acid. Since prostaglandin levels are often elevated in tumor tissue, and COX-2 activity is induced in the premalignant stage of tumor development, anti-inflammatory agents such as inhibitors of the arachidonic acid cascade are promising chemopreventive agents. Nitric oxide is an important signaling molecule. It can physiologically act as a vasorelaxant, a modulator of neurotransmission, and is involved in the immune 1.2 Molecular Mechanisms and Targets of Chemopreventive Agents j9

Table 1.3 Examples of naturally occurring anti-inflammatory agents.

Category Compound Dietary source

Polyphenols Epigallocatechin gallate Green tea Quercetin Ubiquitous Curcumin Turmeric, curry Resveratrol Grapes, red wine Terpenoids b-Carotene Carrots Vitamins Vitamin E (a-tocopherol) Oils of wheat germ, corn, soybean, and sunflower Organosulfur compounds Diallylsulfide Garlic Diallyldisulfide

defense against pathogens and certain tumor cells. Chronic inflammation and persistent infections, however, result in the induction of the inducible form of NO synthase (iNOS) and an overproduction of NO. Long-term elevated levels of NO have been linked to early steps in carcinogenesis via nitrosative deamination of DNA bases and accumulation of reactive nitrogen and oxygen species, causing DNA adduct formation and other types of DNA damage [19, 20]. In Table 1.3, some examples of naturally occurring anti-inflammatory agents are summarized. Synthetic nonsteroidal anti-inflammatory drugs (NSAIDs) showing chemopreventive activity in animal models include sulindac, aspirin, piroxicam, and ibuprofen. Aspirin and sulindac are implicated as promising agents in human cancer chemoprevention and operate in part via inhibition of arachidonic acid metabolism via COX and lipoxygenase (LOX) pathways. Sulindac has been shown to lead to an almost total regression of colorectal adenomatous polyps in patients with FAP and Gardners syndrome. In a prospective mortality study, long-term aspirin use was found to reduce the relative risk of death from colon cancer.

1.2.6 Antitumor Promoting Activities

Tumor promoters such as the phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA) reversibly stimulate cell proliferation by signal transduction pathways. TPA application results in the activation of protein kinase C (PKC), which in turn is involved in the upregulation and activation of target proteins such as mitogen-activated protein (MAP) kinases (MAP kinase cascade) and ornithine decarboxylase (ODC). ODC catalyzes the decarboxylation of ornithine to putrescine, which is further converted to higher polyamines essential for duplication of DNA. Since this pathway is the only source of putrescine in mammalian cells, ODC is regarded as a key enzyme in polyamine biosynthesis. Although ODC and the resulting polyamines are essential for cellular proliferation, induction of ODC causes tumor promotion and cell transformation. Cultured tumor cells often contain high levels of ODC. Based on these characteristics, ODC is considered an attractive target in both chemoprevention 10j 1 Molecular Mechanisms of Cancer Induction and Chemoprevention

Table 1.4 Examples of inhibitors of ODC induction (anti-tumor promoters).

Category Compound Dietary source

Polyphenols Epigallocatechin gallate Green tea Quercetin Ubiquitous Curcumin Turmeric, curry Silymarin Milk thistle Terpenoids b-Carotene Carrots Lycopene Tomatoes Vitamins Vitamin A acid (retinoic acid) Vitamin E (a-tocopherol) Oils of wheat germ, corn, soybean, and sunﬂower Plant extracts Not identiﬁed Rosemary Ginger

and chemotherapy. Some examples of inhibitors of ODC induction are given in Table 1.4. Among the synthetic inhibitors of polyamine-related enzymes, 2-(diﬂuoromethyl) ornithine (DFMO) became the most well known [21]. ODC inactivation is associated with decreased transcription of the growth-related c-myc and c-fos genes. DFMO used as a single drug has only minor effects on tumor growth; however, in combination with other drugs, it prevents and inhibits the development of a variety of chemically induced cancers in animals with doses far lower than those administered for therapy. A recent study showing clinical prevention of recurrence of colorectal carcinomas by 70% by combined DFMO and sulindac doses supports the concept of combination chemoprevention as a valid strategy in human cancer prevention [22]. Hormones such as 17b-estradiol (E2) are regarded as endogenous tumor promoters. They interact with the estrogen receptors ERa and ERb, stimulate cell growth, and increase the risk for hormone-dependent tumor types such as breast and uterine cancer [23]. Recent reports indicate that E2 or its metabolites might also be involved in tumor initiation and act as mutagens that contribute to the formation of DNA adducts and chromosomal changes [24]. Release of these adducts from DNA and repair of the resulting damage may lead to DNA mutations that can initiate various types of hormone-related cancers. The promise of the antiestrogen tamoxifen is widely known based on its success in reducing the risk of breast cancer in women at high risk. The second-generation selective estrogen receptor modulator (SERM) raloxifene, already approved in the prevention of osteoporosis and without endometrial toxicity, is being compared with tamoxifen as a breast cancer chemopreventive in postmenopausal women. A third- generation SERM arzoxifene [25] with greater efﬁcacy than raloxifene in animal studies is being evaluated in a phase II study in patients scheduled for breast surgery and in high-risk subjects with multiple biomarker abnormalities [26]. Phytoestrogens are a broad group of plant-derived compounds that structurally resemble estrogens and bind to the estrogen receptor, mimicking the effects 1.2 Molecular Mechanisms and Targets of Chemopreventive Agents j11

Table 1.5 Major classes of phytoestrogens.

Category Compound Dietary source

Lignans Secoisolariciresinola Flaxseed, cereal, fruit, and vegetables Matairesinola Isoflavones Genistein, daidzein Soy products Biochanin A, formononetin Red clover Flavanones 8-Prenylnaringenin Hop Coumestans Coumestrol Bean shoots, alfalfa, clover, and sunflower seeds Stilbenes Resveratrol Grape skin, red wine aAfter metabolism to enterolactone and enterodiol. of estrogens. Major classes of phytoestrogens include the lignans, isoflavones, flavanones, coumestans, and stilbenes (Table 1.5). The last three groups are less abundant in the diet and less well studied. The influence of phytoestrogens on the prevention of hormone-related tumors is not conclusive [27, 28].

1.2.7 Antiproliferative Mechanisms

Accumulation of genetic damage and mutations during carcinogenesis can result in continuous activation of oncoproteins leading to uncontrolled cell proliferation and growth [1]. A potential mechanism in cancer prevention is therefore the inhibition of oncogene expression and activity (e.g., c-Ha-Ras, c-Myc). Uncontrolled cell proliferation often involves disorganization of cell cycle regulation and signal transduction pathways. A simpliﬁed model including receptor activation, signal transduction, and downstream effects is given in Figure 1.2. A ligand L (e.g., growth factors) interacts with its receptor R located in the cell membrane. The growth- stimulating signal is then transduced to the nucleus via phosphorylation/activation steps mediated by protein kinase cascades (e.g., PKC/Ras/Raf cascade; MAP kinase cascade), resulting in the activation or repression of gene transcription and consequently up- or downregulated protein expression. Deregulated protein expression can backregulate both signal transduction and transcription as well as inﬂuence intercellular communication with neighboring cells. Consequently, overexpression of hormone/growth factors and their receptors leads to a growth advantage of preneoplastic

Figure 1.2 Simplified model of signal transduction mechanisms leading to a possible growth advantage of preneoplastic cells. 12j 1 Molecular Mechanisms of Cancer Induction and Chemoprevention

Table 1.6 Natural products with antiproliferative activity.

Category Compound Dietary source

Polyphenols Epigallocatechin gallate Green tea Quercetin Ubiquitous Curcumin Turmeric, curry Genistein Soy product Terpenoids Limonene Essential oils from lavender Perillyl alcohol Mint, citrus fruits Vitamins Vitamin E Oils of wheat germ, corn, soybean, and sunﬂower

cells. Interactions with protein factors involved in signal transduction are mechanisms explored both in cancer therapy and in cancer prevention. Since tumor growth requires the formation of new blood vessels, inhibition of this process (antiangiogenesis) is also regarded as a feasible target (see Section 2.9). Some natural products with antiproliferative activity are summarized in Table 1.6.

1.2.8 Induction of Apoptosis and Terminal Cell Differentiation

The survival of multicellular organisms involves a balance between cell proliferation and cell death. Apoptosis (programmed cell death) is a genetically controlled response for cells to commit suicide. Apoptosis represents an innate cellular defense mechanism against carcinogen-induced cellular damage by inhibiting survival and growth of altered cells and by removing them at different stages of carcinogenesis. Accord- ingly, deregulation of apoptosis has been implicated in the onset and progression of cancer. Apoptosis can be initiated and inhibited by a variety of stimuli, both physiological and pathological. Examples of dietary components that have been demonstrated to induce apoptosis in experimental models in cultured cancer cells are summarized in Table 1.7. These compounds induce apoptosis by multiple different mechanisms. Efﬁcacy and selectivity in animal models and in humans still need further investigation. Another protective mechanism effective at late stages of carcinogenesis is the induction of terminal cell differentiation, that is, cell maturation and development to a normal, nonmalignant phenotype with a limited life span. The relation between cell proliferation, induction of cell differentiation, and

apoptosis is depicted in Figure 1.3. Cell cycle progression through the phases G1 (gap 1), S (synthesis, doubling of cell components), G2 (gap 2), and M (mitosis, cell division) is highly regulated by a series of proteins (cyclins, cyclin-dependent kinases). Changes in the expression of speciﬁc apoptosis-associated genes or excessive genetic damage stimulate cells to undergo apoptosis and elimination. In contrast, downregulation of genes that promote cell proliferation induces a cell

to differentiate, to escape cell cycle progression, and to enter G0 (resting) phase. 1.2 Molecular Mechanisms and Targets of Chemopreventive Agents j13

Table 1.7 Some phytochemicals as inducers of apoptosis.

Category Compound Dietary source

Polyphenols Epigallocatechin gallate Green tea Quercetin Ubiquitous Ellagic acid Nuts, berries Resveratrol Grapes, red wine Gingerol Ginger Curcumin Turmeric, curry Genistein Soy products Organosulfur Diallylsulﬁde, Garlic compounds diallyldisulﬁde, ajoene Phenethyl isothiocyanate Watercress, cruciferous vegetables Sulforaphane Broccoli, cruciferous vegetables Terpenoids Limonene Essential oils from lavender, mint, citrus fruits Perillyl alcohol Vitamins a-Tocopherol succinate Synthetic vitamin E derivative Vitamin A acid

Cell differentiation in experimental models can be induced by a variety of naturally occurring components summarized in Table 1.8; their preventive effectiveness in humans needs further evaluation.

1.2.9 Inhibition of Angiogenesis (Angioprevention)

Angiogenesis is deﬁned as the formation of new blood capillaries from pre-existing microvasculature by sprouting or intussusception (longitudinal division of existing vessels) and reﬁnes the primitive vasculogenic network into a complex vascular tree. The multistep organization of angiogenesis, which occurs mainly in the adult organism, is mediated by a sensitive balance of activators and inhibitors and includes (i) production of various growth factors, (ii) activation of endothelial cells

Figure 1.3 Relationship between cell proliferation, apoptosis, and terminal differentiation. 14j 1 Molecular Mechanisms of Cancer Induction and Chemoprevention

Table 1.8 Natural inducers of cell differentiation.

Category Compound Dietary source

Polyphenols Quercetin Ubiquitous Resveratrol Grapes, red wine Genistein Soy products Terpenoids Perillyl alcohol Essential oils from lavender, mint, citrus fruits Lycopene Tomatoes Vitamins Vitamin A acid, vitamin D, vitamin E Fermentation products Short-chain fatty acids, Fiber for example, butyrate Minerals Calcium Milk products

(ECs), (iii) production of lytic enzymes to digest the basement membrane and extracellular matrix, (iv) endothelial cell migration, proliferation, and tube formation, and (v) allowing cancer cells to invade and to form metastases (see Figure 1.4). Various signals have been identiﬁed to initiate angiogenesis by triggering the so-called angiogenic switch, where the regulatory balance is tipped in favor of capillary growth by increasing the expression of proangiogenic molecules. The potential to block tumor growth by inhibition of the neoangiogenic process represents a new approach to the treatment of solid tumors [29]. The high proliferation rate in the tumor deprived of proper vascularization would be balanced by cell

Figure 1.4 Individual steps in the process of angiogenesis involving endothelial cells and EC matrix (ECM) degradation. 1.3 Perspectives j15

Table 1.9 Examples of angiopreventive compounds [41].

Angiopreventive compounds Enzyme inhibited Other mechanisms of inhibition

Catechins (EGCG, Pro-MMP-2, MMP-1, MMP-2, # EC proliferation ECG) MMP-9, elastase, urokinase Curcumin MMP-9, COX-2, LOX, ODC Inhibits MMPs # EC proliferation, apoptosis Isoﬂavones MMP-2, MMP-8, MMP-9, # EC proliferation, block uPA (genistein, daidzein) elastase, tyrosine kinase EC migration and proliferation þ#EGF Resveratrol MMP-9, COX-1 and -2, TNF-a, Inhibits EC activation, FGF, and MAP kinase VEGF, wound healing

MMP, matrix metalloprotease; TNF-a, tumor necrosis factor a; uPA, urokinase type plasminogen activator (urokinase); EGF, epidermal growth factor; FGF, fibroblast growth factor; VEGF, vascular epithelial growth factor. death due to a lack of diffusion of nutrients and oxygen. Matrix metalloproteinases (MMPs), angiogenic growth factors, and their receptors are the main targets of an increasing number of clinical trials approved to test the tolerance and therapeutic efficacy of antiangiogenic agents, such as angiostatin, avastin, and endostatin. Also, most cancer chemopreventive agents show antiangiogenic properties when tested in vitro and in vivo angiogenesis models (Table 1.9). N-Acetyl-cysteine is able to reduce the invasive and metastatic potential of melanoma cells, and it inhibits endothelial cell invasion by directly inhibiting MMP activity. In addition, the chemopreventive catechins and flavonoids from green tea are potent inhibitors of MMP-2 and MMP-9. Inhibition of angiogenesis has also been demonstrated for NSAIDs as part of their chemopreventive activity. Thus, angiogenesis is a common and key target of most natural and synthetic chemopreventive agents, where they most likely suppress the angiogenic switch in (pre-)malignant tumor stages, a concept that was termed angioprevention [30, 31].

1.3 Perspectives

Current knowledge, still mostly derived from animal models, indicates that compounds with the most effective chemopreventive activity act via different multiple mechanisms. As indicated in Tables 1.1–1.9, a great variety of defined targets and mechanisms have already been characterized for the agents listed. Phytochemicals act by a plethora of antisurvival mechanisms, boost the hosts anti-inflammatory defense, and may sensitize malignant cells to cytotoxic agents and thus may also be beneficial to cancer patients when used as cochemotherapeutics [32]. A systematic and wider clinical application of this novel approach is now warranted. 16j 1 Molecular Mechanisms of Cancer Induction and Chemoprevention

Prevention of cancer through the activation of the immune system has been explored in recent years in preclinical systems, thanks to the availability of several new transgenic mouse models that closely mimic the natural history of human tumors. The most thoroughly investigated model of cancer immunoprevention is the mammary carcinoma of HER-2/neu transgenic mouse. For application in humans, a search for new human tumor antigens should be conducted among molecules that are directly involved in neoplastic transformation and are recognizable by the immune response. Novel tumor antigens fulfilling both conditions will be crucial for the development of cancer immunoprevention in man and will provide new targets also for cancer immunotherapy [33]. Well-designed intervention studies are necessary to assess the chemopreventive efficacy of agents in asymptomatic individuals as well as in high-risk groups. In such human trials, surrogate end point biomarkers should be integrated [34] to unravel mechanisms by which the chemopreventive agents act in the human body and to assess the predictive value of the marker. Such end point markers for clinical trials of chemopreventive agents include properties of epithelial precancer (IEN, intraepithelial neoplasia) that, for example, can be measured by computer-assisted image analysis [35]. Mechanisms relevant for chemoprevention can be used to establish a battery of test systems for the identification and development of novel chemopreventive agents [9, 36]. This concept has been successfully applied for the activity- guided isolation of chemoprotectants such as xanthohumol from hop [37]. Molecular biology studies have provided new information on novel promising targets, including regulatory molecules such as Nrf2 (nuclear factor erythroid- derived 2-related factor 2), epidermal growth factor receptor kinases, phosphatidylinositol 3-kinase, components of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway, nuclear factor-kB, cyclin D, angiogenic signaling pathways, matrix metalloproteases, DNA methyl transferases, and histone deacetylases. The development and/or identification of new agents for the control of these targets that are both safe and effective will determine the future progress in the cancer chemoprevention field. The abundance of flavonoids and related polyphenols as well as other structurally distinct phytochemicals in the plant kingdom and in our daily diet will likely lead to the identification of additional hitherto uncharacterized natural products with chemopreventive efficacy.

1.4 Conclusion

Cancer prevention including chemoprevention plays an integral and important part in cancer control. Several institutional bodies and the broad public should be aware of its importance and promote the development of scientifically based prevention programs. Many agents described in this treatise that prevent cancer, delay its onset, or revert premalignant conditions could have dramatic beneficial impacts on human health [38, 39]. Although there is an urgent need to develop such cancer References j17 chemopreventive agents, researchers in the field suspect that this area of scientific endeavor leads a Cinderella existence, both in terms of perception of importance and in terms of research funding, at least in Europe [40]. It is hoped that this encyclopedia will raise awareness among clinical and laboratory researchers of the importance of the development of novel, efficacious, and safe cancer preventatives and encourage young scientists to enter this field of research.

Acknowledgment

We thank Susanna Fuladdjusch for excellent secretarial help.

References

1 Hanahan, D. and Weinberg, R.A. (2000) International Journal of Cancer, 120, The hallmarks of cancer. Cell, 100, 451–458. 57–70. 8 Ren, S. and Lien, E.J. (1997) Natural 2 Stewart, B.W. and Kleihues, P. (eds) (2003) products and their derivatives as cancer World Cancer Report, IARC Press, Lyon. chemopreventive agents. Progress in Drug 3 Wattenberg, L.W. (1996) Chemoprevention Research, 48, 147–171. of cancer. Preventive Medicine, 25,44–45. 9 Steele, V.E. and Kelloff, G.J. (2005) 4 Kelloff, G.J., Lippman, S.M., Development of cancer chemopreventive Dannenberg, A.J., Sigman, C.C., drugs based on mechanistic approaches. Pearce, H.L., Reid, B.J., Szabo, E., Mutation Research, 591,16–23. Jordan, V.C. et al. (2006) Progress in 10 Liby, K.T., Yore, M.M. and Sporn, M.B. chemoprevention drug development: the (2007) Triterpenoids and rexinoids as promise of molecular biomarkers for multifunctional agents for the prevention prevention of intraepithelial neoplasia and and treatment of cancer. Nature Reviews. cancer – a plan to move forward. Clinical Cancer, 7, 357–369. Cancer Research, 12, 3661–3697. 11 De Flora, S. and Ferguson, L.R. (2005) 5 Potter, J.D. and Steinmetz, K. (1996) Overview of mechanisms of cancer Vegetables, fruit and phytoestrogens chemopreventive agents. Mutation as preventive agents, in Principles of Research, 591,8–15. Chemoprevention (eds B.W. Stewart, 12 Barch, D.H., Rundhaugen, L.M., D. McGregor and P. Kleihues), IARC Stoner, G.D., Pillay, N.S. and Rosche, W.A. Scientiﬁc Publications, No. 139, IARC (1996) Structure–function relationships of Press, Lyon, pp. 61–90. the dietary anticarcinogen ellagic acid. 6 Surh, Y.J. (2003) Cancer chemoprevention Carcinogenesis, 17, 265–269. with dietary phytochemicals. Nature 13 Bertram, B., Bollow, U., Reviews. Cancer, 3, 768–780. Rajaee-Behbahani, N., Burkle, A. and 7 Thomasset, S.C., Berry, D.P., Garcea, G., Schmezer, P. (2003) Induction of poly Marczylo, T., Steward, W.P. and (ADP-ribosyl)ation and DNA damage in Gescher, A.J. (2007) Dietary polyphenolic human peripheral lymphocytes phytochemicals: promising cancer after treatment with ()-epigallocatechin- chemopreventive agents in humans? gallate. Mutation Research, 534, A review of their clinical properties. 77–84. 18j 1 Molecular Mechanisms of Cancer Induction and Chemoprevention

14 Rush, E., Ferguson, L.R., Cumin, M., 22 Meyskens, F.L., Jr, McLaren, C.E., Thakur, V., Karunasinghe, N. and Plank, L. Pelot, D., Fujikawa-Brooks, S., (2006) Kiwifruit consumption reduces Carpenter, P.M., Hawk, E., Kelloff, G., DNA fragility: a randomized controlled Lawson, M.J. et al. (2008) pilot study in volunteers. Nutrition Difluoromethylornithine plus sulindac Research, 26, 197–201. for the prevention of sporadic colorectal 15 Hauser, A.-T. and Jung, M. (2008) adenomas: a randomized placebo- Targeting epigenetic mechanisms: controlled, double-blind trial. Cancer potential of natural products in cancer Prevention Research, 1,32–38. chemoprevention. Planta Medica, 74(13), 23 Yager, J.D. and Davidson, N.E. (2006) 1593–601. Estrogen carcinogenesis in breast cancer. 16 Nair, U., Bartsch, H. and Nair, J. (2007) The New England Journal of Medicine, Lipid peroxidation-induced DNA damage 354, 270–282. in cancer-prone inflammatory diseases: a 24 Cavalieri, E., Chakravarti, D., review of published adduct types and levels Guttenplan, J., Hart, E., Ingle, J., in humans. Free Radical Biology & Medicine, Jankowiak, R., Muti, P., Rogan, E. et al. 43, 1109–1120. (2006) Catechol estrogen quinones as 17 Heinonen, O.P., Albanes, D., Virtamo, J., initiators of breast and other human Taylor, P.R., Huttunen, J.K., cancers: implications for biomarkers of Hartman, A.M., Haapakoski, J., Malila, N. susceptibility and cancer prevention. et al. (1998) Prostate cancer and Biochimica et Biophysica Acta, 1766,63–78. supplementation with alpha-tocopherol 25 Sporn, M.B. (2004) Arzoxifene: a and beta-carotene: incidence and mortality promising new selective estrogen receptor in a controlled trial. Journal of the National modulator for clinical chemoprevention of Cancer Institute, 90, 440–446. breast cancer. Clinical Cancer Research, 10, 18 Omenn, G.S., Goodman, G.E., 5313–5315. Thornquist, M.D., Balmes, J., 26 Crowell, J.A. (2005) The chemopreventive Cullen, M.R., Glass, A., Keogh, J.P., agent development research program in Meyskens, F.L. et al. (1996) Effects of a the Division of Cancer Prevention of combination of beta carotene and vitamin the US National Cancer Institute: an A on lung cancer and cardiovascular overview. European Journal of Cancer, 41, disease. The New England Journal of 1889–1910. Medicine, 334, 1150–1155. 27 Rice, S. and Whitehead, S.A. (2006) 19 Bartsch, H. and Nair, J. (2006) Chronic Phytoestrogens and breast cancer: inflammation and oxidative stress in the promoters or protectors? Endocrine-Related genesis and perpetuation of cancer: role of Cancer, 13, 995–1015. lipid peroxidation, DNA damage, and 28 Oseni, T., Patel, R., Pyle, J. and Jordan, V.C. repair. Langenbecks Archives of Surgery, (2008) Selective estrogen receptor 391, 499–510. modulators and phytoestrogens. 20 Hussain, P.S. and Harris, C.C. (2007) Planta Medica, 74(13), 1656–1665. Inflammation and cancer: an ancient link 29 Albini, A. and Sporn, M.B. (2007) The with novel potentials. International Journal tumour microenvironment as a target for of Cancer, 121, 2373–2380. chemoprevention. Nature Reviews. Cancer, 21 Raul, F. (2007) Revival of 2- 7, 139–147. (difluoromethyl)ornithine (DFMO), 30 Tosetti, F., Ferrari, N., De Flora, S. and an inhibitor of polyamine biosynthesis, Albini, A. (2002) Angioprevention: as a cancer chemopreventive agent. angiogenesis is a common and key target Biochemical Society Transactions, 35, for cancer chemopreventive agents. 353–355. The FASEB Journal, 16,2–14. References j19

31 Noonan, D.M., Benelli, R. and Albini, A. potential cancer chemopreventive agents. (2007) Angiogenesis and cancer Mutation Research, 523–524, 163–172. prevention: a vision. Recent Results in 37 Gerhauser, C., Alt, A., Heiss, E., Cancer Research, 174, 219–224. Gamal-Eldeen, A., Klimo, K., Knauft, J., 32 DIncalci, M., Steward, W.P. and Neumann, I., Scherf, H.R. et al. (2002) Gescher, A.J. (2005) Use of cancer Cancer chemopreventive activity of chemopreventive phytochemicals as xanthohumol, a natural product derived antineoplastic agents. The Lancet Oncology, from hop. Molecular Cancer Therapeutics, 6, 899–904. 1, 959–969. 33 Lollini, P.L., Nicoletti, G., Landuzzi, L., 38 Schrijvers, D., Senn, H-.J., Mellstedt, H. De Giovanni, C. and Nanni, P. (2005) and Zakotnik, B. (eds) (2008) Handbook of New target antigens for cancer Cancer Prevention, European Society for immunoprevention. Current Cancer Drug Medical Oncology (ESMO). Targets, 5, 221–228. 39 Sporn, M.B. and Liby, K.T. (2005) Cancer 34 Miller, A.B., Bartsch, H., Boffetta, P., chemoprevention: scientific promise, Dragsted, L. and Vainio, H. (eds) (2001) clinical uncertainty. Nature Clinical Biomarkers in Cancer Chemoprevention, Practice Oncology, 2, 518–525. IARC Scientific Publications, No. 154, IARC 40 Gerhauser, C., Bartsch, H., Crowell, J., Press, Lyon. De Flora, S., DIncalci, M., Dittrich, C., 35 Kelloff, G.J., Sigman, C.C., Hawk, E.T., Frank, N., Mihich, E. et al. (2006) Johnson, K.M., Crowell, J.A. and Development of novel cancer Guyton, K.Z. (2001) Surrogate end-point chemopreventive agents in Europe: biomarkers in chemopreventive drug neglected Cinderella or rising development, in Biomarkers in Cancer phoenix? A critical commentary. ESF Chemoprevention (eds A.B. Miller, Workshop on Cancer Chemoprevention, H. Bartsch, P. Boffetta, L. Dragsted and DKFZ, Heidelberg, September 18–20, H. Vainio), IARC Scientific Publications, 2005. European Journal of Cancer, 42, No. 154, IARC Press, Lyon, pp. 13–26. 1338–1343. 36 Gerhauser, C., Klimo, K., Heiss, E., 41 Losso, J.N. (2003) Targeting excessive Neumann, I., Gamal-Eldeen, A., Knauft, J., angiogenesis with functional foods and Liu, G.Y., Sitthimonchai, S. et al. (2003) nutraceuticals. Trends in Food Science & Mechanism-based in vitro screening of Technology, 14, 455–468. j21

2 Types and Consequences of DNA Damage Daniel T. Shaughnessy and David M. DeMarini

2.1 Introduction

The integrity of DNA is essential for all cellular life known on Earth. However, DNA is constantly at risk of damage from spontaneous and exogenous processes. Consequently, an array of hundreds of proteins has evolved to repair and replicate DNA so that its integrity may be maintained despite a constant onslaught of damage. DNA damage elicits a response that includes (a) immediate restoration of the normal DNA structure via DNA repair through reversal or excision of the damage, (b) DNA damage tolerance by error-free or error-prone (mutagenic) mechanisms, or (c) cell death (apoptosis) (Table 2.1). If mutations result from DNA damage, then some mutations in somatic cells may lead to cancer or aging, whereas some mutations in germ cells may lead to heritable disease and contribute to evolution. Of course, not all mutations are harmful, and some (adaptive mutations) may provide enhanced survivability in certain environments or under certain conditions. Here we review brieﬂy the types of DNA damage, how the cell or an experimenter can detect that damage, the types of DNA repair, the types of DNA damage tolerance, and the types of mutations that can result from DNA damage. An important concept encompassing the above mechanisms is one we have called the mutagenesis paradigm, which involves the relationships between DNA damage, DNA repair, and mutation (Figure 2.1). Key to this concept is the distinction between DNA damage and mutation. DNA damage can be caused by spontaneous errors of nucleic acid metabolism or by endogenous or exogenous mutagens. Thus, mutagens do not make mutations directly. Instead, mutagens make DNA damage, which may involve a wide variety of lesions affecting either the phosphodiester backbone (the deoxyribose sugar moiety) or the nucleotides/bases. DNA damage generally does not change the sequence of nucleotides in DNA. Mutations, on the contrary, are changes in the sequence of nucleotides in DNA, and they result from the actions of the cell on the DNA damage. Thus, mutagens make DNA damage and cells make mutations; mutagenesis is a cellular process, requiring enzymes and, frequently, DNA replication.

Table 2.1 Biological responses to DNA damagea.

Reversal of base damage

Excision of damage, mispaired, or incorrect bases Base excision repair (BER) Nucleotide excision repair (NER) Transcription-coupled nucleotide excision repair (TC-NER) Alternative excision repair (AER) Mismatch repair (MMR)

Strand break repair Single-strand break repair (SSBR) Double-strand break repair (DSBR)

Tolerance of base damage Trans-lesion DNA synthesis (TLS) Postreplicative gap ﬁlling Replication fork progression

Cell cycle checkpoint activation Apoptosis

aFrom Ref. [1].

2.2 Types of DNA Damage

An assault on DNA from either endogenous or exogenous sources can result in a variety of types of damage (Figure 2.2). Errors during replication, incorporation of incorrect or damaged nucleotides, and chemical alterations of DNA bases in single-stranded and double-stranded DNA contribute to DNA damage from endogenous sources that threaten the cell with toxic or mutagenic consequences [1].

Figure 2.1 Mutagenesis paradigm. 2.2 Types of DNA Damage j23

Figure 2.2 Types of DNA damage.

One common error is the misincorporation of uracil (U), normally restricted to RNA, into DNA during replication. The resulting U:A base pair does not directly affect the coding properties of the DNA but, rather, the recognition and binding of regulatory proteins to DNA binding domains. The presence of U in DNA can also result from the spontaneous deamination of cytosine, a process that occurs 100–200 times/cell/day. A U:G mismatch that escapes repair, can result in C:G ! T:A transition mutations during replication [2]. In 50-CG-30 DNA sequences that comprise regulatory regions, cytosine is often methylated, and 5-methylcytosine is subject to spontaneous deamination to form thymine and, therefore, T:G mispairs. The base–sugar bonds in DNA are also subject to spontaneous (nonenzymatic) cleavage, resulting in the loss of DNA bases. Such damage, called abasic or apurinic/ apyrimidinic (AP) sites, can also lead to mutations if unrepaired [1]. 24j 2 Types and Consequences of DNA Damage

DNA in both the nucleus and mitochondria is subject to damage from reactive oxygen species (ROS) that are byproducts of normal aerobic metabolism [2, 3]. Damage from ROS can take the form of oxidized DNA bases, AP sites, or DNA strand breaks [1]. The most common oxidized base damage is 7,8-dihydro-8-oxoguanine (8-oxoG). This damage, which is present at orders of magnitude higher in mitochondria than in nuclear DNA, can induce mutations. 8-oxoG is unstable and can react with compounds such as peroxynitrate to produce even more mutagenic lesions. 8-oxoG can also arise in nucleotide pools and be incorporated during replication [1]. Other common base damages from ROS are thymine glycol and 4-hydroxy- 5-formamidopyrimidine (FaPy). ROS, and specifically oxygen radicals, also react with phospholipids in cellular membranes to produce the reactive aldehydes malondialdehyde and 4-hydroxynonenal, which can form cyclic-DNA adducts at high enough levels to pose mutagenic threats to the cell [1]. Numerous types of DNA damage result from exposure to both endogenous and exogenous alkylating agents. Methylating agents, for example, can react with DNA bases and even with the phosphodiester backbone. Nucleophilic sites, especially the N7 position of guanine, the O6 position of guanine, and the N3 position of adenine, are subject to attack from electrophilic alkylating agents [4]. The consequences of alkylation damage are toxicity, mutagenicity, and clastogenicity (DNA stand breakage and chromosomal damage). DNA can be aberrantly methylated by S-adenosylmethionine (SAM), an enzyme involved in the normal methylation of DNA. Other sources of endogenous alkylation are nitroso compounds, such as methylnitrosourea (MNU), which can be generated by the nitrosation of amino acids [5]. DNA alkylation can occur through exposure to numerous environmental compounds, including methylchloride, tobacco-specific nitrosamines, and dietary nitrosamines. Many cancer therapeutic drugs, such as temozolamide, are potent methylating agents [5]. Exposure to ionizing radiation (IR) from cosmic radiation or naturally occurring radon present in rocks and soil can lead to base damage and DNA strand breaks through the production of hydroxyl radicals from the effects of high-energy radiation on water [1]. Several types of DNA lesions result from exposure to ultraviolet (UV) radiation, including cyclobutane pyrimidine dimers and 6–4 photoproducts. Numerous environmental chemicals bind covalently with DNA to form adducts that affect transcription and replication and that can induce mutations. These include alkylating agents, aromatic amines, aflatoxins, heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins, and chemical agents that form cross-links within DNA or between DNA and proteins.

2.3 How to Detect DNA Damage Experimentally

One of the most common methods for detecting DNA damage in laboratory or population studies is the single cell gel electrophoresis (comet) assay. Developed by Östling and Johanson and modiﬁed by Singh and coworkers [6], the comet assay is a sensitive and relatively inexpensive method to monitor single- and double-strand 2.3 How to Detect DNA Damage Experimentally j25

DNA breaks (SSB and DSB, respectively) occurring at sites of DNA damage or at alkali-labile sites. Incubation with Escherichia coli Endonuclease III or FaPy glycosylase enzymes prior to unwinding and electrophoresis allows for incision and, therefore, detection of oxidatively modified DNA bases. The comet assay is widely used to monitor the influence of environmental and lifestyle exposures, including diet, exercise, and exposure to sunlight and air pollution, on DNA damage in leukocytes in human biomonitoring studies [7]. More recently, an immunofluores- cent probe for phosphorylated g-H2AX, which forms foci at sites surrounding DNA double-strand breaks, has been developed to monitor DNA damage in multiple cell types, including nucleated blood cells [8]. For detection of abasic (AP) sites, Nakamura et al. [9] have developed a sensitive assay that employs an aldehyde-reactive probe (ARP) that reacts with the aldehydic group of ring-opened AP sites. This method has been used to monitor endogenous levels of AP sites in multiple cell types, including liver, lung, and kidney, and in response to exposure to environmental chemicals [10]. Several approaches are used to detect covalently modified DNA (stable DNA adducts), including 32P-postlabeling, immunohistochemical methods using DNA-adduct-specific antibodies, HPLC followed by tandem mass spectroscopy, and accelerator mass spectrometry [11, 12]. The 32P-postlabeling assay, which relies on enzymatic digestion of DNA to nucleotides followed by addition of radiolabeled phosphate groups and chromatographic resolution of the adducted nucleotides, has been used in biomonitoring studies of occupational and environmental exposures to a variety of carcinogens [13]. Immunohistochemical methods rely on the use of antibodies that bind to specific DNA adducts. For example, an antibody specific to PAH–DNA adducts was used in a lung cancer study to investigate the relationship between lung cancer cases and adduct levels [12]. Different types of chromatography, for example, immunoaffinity, high-performance liquid chromatography (HPLC), or gas chromatography (GC), are used together with mass spectrometry methods to detect a wide range of DNA adducts resulting from exposures to carcinogens, such as aflatoxins, polycyclic aromatic hydrocarbons (PAHs), and dietary and tobacco-specific nitrosamines. Accelerator mass spectrometry (AMS) is a highly sensitive method of detecting adducts in vivo using radiolabeled (3Hor14C) compounds. For example, AMS was used to detect DNA adducts in patients undergoing colon cancer surgery. A radiolabeled version of the fried meat mutagen 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) was administered to patients prior to surgery and was found to have produced PhIP–DNA adducts in the human colon [14]. Ligation-mediated PCR (LM-PCR) is a method that has been developed to analyze DNA sequences in regions of DNA damage to compare sites of DNA damage to sites of mutation (determined by DNA sequencing after cells have replicated). In this assay, DNA is cleaved chemically or enzymatically at sites of DNA damage. For example, bacterial enzymes such as E. coli UvrABC endonuclease are used to make incisions in the vicinity of the damage resulting from exposure to UV light or to cross- linking agents such as cisplatinum. Gene-specific primers are then used in a nested PCR approach to amplify these DNA regions using labeled primers, followed by resolution of the fragments by gel electrophoresis [15]. 26j 2 Types and Consequences of DNA Damage

Among the measures of chromosomal damage from environmental chemicals, one of the most common is the cytokinesis-block micronucleus (CBMN) assay. Damage to DNA resulting in double-strand breaks, as well as aberrant chromosome segregation during mitosis, can result in either loss or ampliﬁcation of chromosomal regions. Micronuclei result from chromosomes or chromosomal fragments that lag behind at anaphase during nuclear division. In this assay, cytokinesis is inhibited by treatment with cytochalasin B, and damaged cells are scored for micronuclei in binucleated cells. The CBMN assay has been used in human studies to evaluate DNA damage in populations. For example, the CBMN assay was used to demonstrate that individuals who develop breast cancer, and their relatives, exhibited increased MN frequency in lymphocytes challenged with ionizing radiation [16].

2.4 DNA Damage Response

The DNA damage response consists of a set of molecular complexes that sense the various types of DNA damage described above and then signal the cell to respond in various ways to a particular type of damage. In essence, the signaling pathways result in (a) alterations in cell-cycle dynamics (checkpoints), (b) changes in the transcription of a variety of genes (transcriptional regulation), or (c) programmed cell death (apoptosis) [17]. A discussion of the features of the mammalian cell cycle is beyond the scope of this review. However, cell-cycle transitions from one stage to the next are regulated by a set of cyclin-dependent kinases (Cdk) that phosphorylate a variety of proteins involved in specific stages of the cell cycle. Cyclins are proteins whose concentrations vary according to specific stages of the cell cycle. DNA damage sensing mechanisms can initiate signaling that arrests the cell cycle so that DNA repair can occur before replication or cell division proceeds. In humans, some of the proteins associated with DNA damage checkpoints include ATM/ATR kinases (ATM and ATR), ATR- interacting proteins (ATRIP), RFC-like proteins (RAD17, RFC2 to RFC5), PCNA-like proteins (RAD, RAD1, HUS1), mediators (BRCA1, MDC1, 53BP1, Claspin), and effector kinases (CHK2 and CHK1). For example, the Mre11–Rad50–Nbs1 (MRN) mediator complex senses double- strand breaks (DSBs) and brings ATM (ataxia telangiectasia mutated) to the site of damage where ATM then autophosphorylates, effecting downstream signaling. Another kinase, ATR, is also associated with this process, and treatment of cells with ionizing radiation or UV light can result in ATM and ATR phosphorylating >700 proteins, especially those involved in DNA replication. Although a particular type of DNA damage may elicit a wide array of mediator and effector proteins associated with the DNA damage signaling response, only a subset of these may be needed to repair a specific lesion. The highly evolutionarily conserved complex cascade of cell signaling proteins that are called into action by DNA damage indicates how critical such signaling is for cell survival. Beyond controlling the cell cycle, the DNA damage signaling system also 2.5 Types of DNA Repair j27 regulates DNA repair and genomic stability, including pathways of more general importance to cell viability, such as insulin signaling, RNA splicing, spindle checkpoint, mitotic spindle and kinetochore proteins, tumor suppressor genes, and genes involved in chromatin remodeling. Epigenetic mechanisms are involved in DNA damage response [18] and play an important role in carcinogenesis [19]. Thus, the DNA damage response signaling pathways involve cellular functions far removed from just nucleic acid metabolism.

2.5 Types of DNA Repair

As shown in Table 2.1, DNA damage can be removed directly, reversing the base damage. Another approach involves excision of damaged or incorrect bases or nucleotides, exempliﬁed by base excision repair (BER) and nucleotide excision repair (NER). A variation on the latter is transcription-coupled repair (TC-NER). A simple mismatch (no damage, but the wrong base pairing), can be corrected by mismatch repair (MMR). When DNA damage results in breakage of the phosphodiester backbone, then single- or double-strand breaks can occur, and various mechanisms exist for repairing this type of damage. DNA damage tolerance involves a process whereby the DNA damage is not actually removed nor the damaged DNA repaired. Instead, replication past the damage or replication involving the undamaged strand occurs. The former can be an error-prone process, resulting in mutations, that is, a change in DNA sequence. As noted above, DNA damage can activate cell-cycle checkpoints and also apoptosis. Later, we review brieﬂy the types of DNA repair, which are discussed extensively elsewhere [1].

2.5.1 Direct DNA Repair

One type of DNA repair involves reversal of the damage. One classic example of this occurs in microbes and involves photolyases that remove UV photoproducts from DNA, leaving the DNA intact. Another example involves the direct removal of alkylation damage (e.g., a methyl group) from DNA by alkyltransferases, which are present in all cells. Both of these types of repair are referred to as direct repair because they remove only the damage itself and not any portion of the DNA.

2.5.2 Base Excision Repair

Another category of repair involves the removal of the damaged portion of DNA and possibly the surrounding area. The most parsimonious of these is BER, which involves the removal of the damaged base itself. It is generally used by the cell to remove nonbulky DNA adducts, alkyl groups, or oxidized, reduced, 28j 2 Types and Consequences of DNA Damage

or fragmented bases, or single-strand breaks. There are two general types: short- and long-patch BER. Short-patch BER dominates when BER is initiated by glycosylases; long-patch BER dominates when BER is initiated by the presence of apurinic/ apyrimidinic (AP) sites. In the case of short-patch BER, the process is initiated by DNA glycosylases that catalyze the hydrolysis of the N-glycosyl bonds (i.e., N-glycosidic bonds) linking the damaged or wrong base to the phosphodiester backbone. This can result in an abasic site, which is processed further by an AP endonuclease, which produces incisions or nicks in duplex DNA by hydrolysis of the phosphodiester bond immediately 50 to the AP site. Some DNA glycosylases have an AP lyase activity that cleaves the DNA 30 to the AP site. Hydrolysis of the phosphodiester bond 50 of the AP site produces a 50-terminal deoxyribose-phosphate that is removed by exonuclease. The missing base is replaced by DNA repair synthesis (by DNA polymerase polb), and the newly inserted base is joined covalently to the DNA by the ligase III X-ray repair cross-complementation (XRCC) group 1 complex. The opposite strand serves as the template for the insertion of the correct base to replace the damaged one. In the case of long-patch BER, the process is initiated by the presence of oxidized or reduced AP sites, 30-unsaturated aldehydes, or 30-phosphates because these types of damage are resistant to b-elimination by polb. This damage is processed further by the proliferating cell nuclear antigen (PCNA)-dependent, long-patch repair after the insertion of a nucleotide at the lesion site by polb. This process displaces the damaged strand,permitting DNA synthesisof an oligonucleotide (up to10 nucleotides) by pold or pole together with PCNA and replication factor C. The flap endonuclease 1 (FEN1) recognizes and cleaves off the damaged oligonucleotide flap structure, and ligase I catalyzes the final ligation step.

2.5.3 Nucleotide Excision Repair

As the name implies, NER (or global genomic NER) involves the removal of a stretch of nucleotides that includes the damaged nucleotide, and this occurs in transcriptionally silent regions of the DNA. Typical NER involves incision on both sides of the damaged nucleotide. A related process called alternative excision repair (AER) involves excision from only one side of the lesion. NER that occurs at the site of a stalled RNA polymerase II during transcription (i.e., in transcriptionally active regions of DNA) is called transcription-coupled nucleotide excision repair (TC-NER). In prokaryotic organisms, the process involves recognition and verification of the damage, followed by the bimodal incision of the strand containing the damage, excision of an oligonucleotide fragment, repair synthesis to fill the gap, and ending with ligation of the DNA fragment. In mammalian cells, global genomic NER generally involves, first, the recognition of the damage, which involves the binding of repair factors xeroderma pigmentosum group C and homologous recombinational repair group 23B (XPC-HR23B) to the lesion. Next, a preincision complex consisting of XPA and 2.5 Types of DNA Repair j29 replication factor A surrounds the lesion to verify it as a lesion as opposed to a natural variation in DNA structure. The recruitment to the lesion of the transcription initiation factor IIH (TFIIH) multiprotein complex, along with the helicase subunits of TFIIH, XPB, and XPD, results in the unwinding of the DNA at the site of the lesion. Next, a 30-incision is catalyzed by XPG, and a 50-incision is catalyzed by the excision repair cross-complementing 1 (CRCC1)-XPF complex. The resulting DNA gap is filled in by the PCNA-dependent polymerases pold and pole. Ligase 1 and associated proteins then ligate the fragment to the DNA. TC-NER occurs at transcriptionally active sites and is strand specific. It involves two different Cock- ayne syndrome complementation groups (A and B) that displace the stalled polymerase. Then TFIIH is recruited to the site, and the process proceeds in a manner similar to that of global NER.

2.5.4 Mismatch Repair

The MMR system corrects primarily replication errors (base–base or insertion– deletion mismatches) resulting from DNA polymerase errors. However, it can also repair alkylation damage, cisplatin-induced intrastrand cross-links, some large adducts (such as 2-aminoﬂuorene), and oxidative damage such as 8-oxoguanine. In humans, the mismatch recognition complex contains the proteins MSH2 and MSH6, which are homolgous to the bacterial protein coded by mutS. Once the heterodimer is bound to the mismatch, it undergoes a conformational change, and a second heterodimer (MUTLa) composed of mutL homologues MLH1 and PMS2 then binds to the MUTSa–DNA complex. Exonuclease I then excises the DNA strand containing the mispaired base, followed by the resynthesis of new DNA by pold.

2.5.5 Homologous and Nonhomologous Recombination for Repair of Double-Strand Breaks

Double-strand breaks can be repaired by either homologous or nonhomologous end- joining. Homologous recombination occurs during DNA replication (in S and G2 phases), whereas nonhomologous end-joining occurs during G0 and G1 phases. Homologous recombination uses the sister chromatid as the template for aligning the breaks in the proper fashion and is error-free, whereas nonhomologous end- joining (also called blunt-end repair) does not use sequence homology between the two breaks to ligate them and is, therefore, error-prone. Homologous recombination can be viewed as a type of DNA damage tolerance mechanism (discussed in the next section) because the DNA damage may not, in fact, be repaired and, instead, the cell simply uses an undamaged homologous template from which new DNA is to be made. Double-strand break repair (DSBR) by homologous recombination is catalyzed by the degradation of the DNA in the 50 to 30 direction by the MRE11–RAD50–NBS1 protein complex, generating 30 ssDNA, which is protected from degradation by 30j 2 Types and Consequences of DNA Damage

a heptameric ring complex of RAD52 proteins. A nucleoprotein ﬁlament consisting of RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, is then assembled by replication factor A, and RAD51 exchanges this single strand with the same sequence from the sister chromatid DNA. This dsDNA copy is used as the template to repair the lesion using the DNA synthesis machinery, and the resulting structures are resolved to generate the repaired DNA. Nonhomologous end-joining simply binds the ends of a double-strand break together without guidance from a template. The break is recognized by a heterodimer composed of the proteins KU70 and KU80, and this binding protects the DNA from digestion and associates the DNA with the DNA-dependent protein

kinase catalytic subunit DNA-PKcs. This complex activates XRCC4-ligase IV, which ligates the broken DNA fragments together after the MRE11–RAD50–NBS1 complex has processed the break. FEN11 and the protein Artemis also appear to be involved in this process.

2.5.6 DNA Damage Tolerance: SOS Repair and Trans-Lesion Synthesis

As noted previously, DNA damage tolerance involves replication of DNA past or around damage (using another strand for a template) without repairing or removing the damage. Thus, DNA damage tolerance mechanisms are not strictly DNA repair mechanisms because, in fact, the DNA damage is not actually repaired. Nonetheless, the consequence of DNA damage tolerance mechanisms can be a normal DNA sequence, such as that achieved by homologous recombination; however, it can also result in high probability of a mutation, such as that produced by the SOS response or certain types of trans-lesion synthesis. In recent years, the view has emerged that these latter mechanisms are the means by which cells make most mutations. SOS is the most well-studied error-prone mechanism, and in prokaryotes this process is initiated by various types of DNA lesions, especially strand breaks, which serve as a signal that initiates the induction of a cascade of gene products from >40 genes. The pKM101 plasmid in strains TA98 and TA100 of the Ames strains of Salmonella provides SOS repair. In essence, this process permits a DNA polymerase to replicate past the damaged template, but with a high probability of inserting an incorrect nucleotide. Thus, the cell likely survives, but in a mutant form. The alternative is cell death due to the inability of the standard DNA polymerase to bypass the damaged template. In eukaryotic cells, there does not appear to be a precise analogue to SOS repair; instead, error-prone repair or mutagenic processes appear to be constitutive and not induced by DNA damage. Among these, the polymerases among the so-called Y-family exhibit the ability to synthesize across lesions, lack exonucleolytic proofreading activities, and have high error rates even with intact templates [20]. The basis for their trans-lesion activity may be due to the fact that their active site is large enough to accommodate bulky DNA lesions. Current estimates are that humans have at least 15 DNA polymerases, with four among the Y-family, and 2.7 Assays to Detect Mutagens j31 at least half of all the human DNA polymerases are involved in trans-lesion synthesis or DNA repair. The human Y-family polymerases include polh, poli, polk, and REV1. Space does not permit a detailed discussion of trans-lesion synthesis, which is specialized for each Y-family polymerase. However, it is likely that trans-lesion synthesis accounts for the production of a signiﬁcant portion of the mutations initiated by DNA damage caused by an array of endogenous and exogenous mutagens.

2.6 Types of Mutations

As described at the beginning of this chapter, a mutation is a heritable change in the DNA sequence [21]. Mutations can be classiﬁed into three groups based solely on their location in the genome. A gene or point mutation is one that occurs within a gene and is typically a base substitution or a small (i.e., a few bases in length) deletion, duplication, insertion, or inversion of a set of nucleotides. A special subset of gene mutation is a frameshift mutation, which is the insertion or deletion of a small number of nucleotides that is not 3 or a multiple of 3. A chromosomal mutation is one that spans more than one gene and generally consists of large lesions, such as a large deletion, duplication, insertion, or inversion of a stretch of nucleotides. Translocations, that is, exchanges of genetic material between or within chromosomal regions, are an important type of chromosomal mutation. Finally, genomic mutation is the one class of mutation that does not exhibit a change in nucleotide sequence; instead, this is the increase or decrease in the number of chromosomes, for example, anueploidy.

2.7 Assays to Detect Mutagens

Over the past 30 years, a set of standardized assays has been incorporated into regulatory procedures for chemical and drug approval/safety evaluation, and we will review brieﬂy some of these. However, we note that there are many highly specialized systems for detecting mutations beyond those described here that are of great value for basic research. The standard battery of genetic toxicity assays used to detect the three classes of mutations includes microbial assays for gene mutation, mammalian cell assays for gene and chromosomal mutation, and in vivo assays for both gene and chromosomal mutation. Detailed discussions of these assays are available elsewhere, but collectively, these assays have a generalized ability to permit the recovery of a wide range of mutations and, thus, to detect a range of mutagens [22]. Some of the assays permit a detailed dissection and analysis of the types of mutations induced. Of all these assays, the Salmonella mutagenicity assay has been used the most for general screening to detect mutagens and antimutagens. The comet assay (described in Section 2.3) is the primary DNA damage assay used for antimutagenesis research. 32j 2 Types and Consequences of DNA Damage

2.8 Implications for Chemoprevention

Given the various types of DNA damage, the complex signaling pathways that identify the damage, and the various pathways by which the damage is processed (i.e., repaired, converted into a mutation, or is the trigger for cell death), antimutagens could conceivably alter many of these pathways to reduce mutagenesis. In addition, the wide array of mutation end points (gene, chromosomal, genomic), and assays (microbial, mammalian cells, in vivo) present a variety of opportunities to assess antimutagens and potential anticarcinogens. Critical to any such assessments is the need for all appropriate controls and survival measurements to assure that reductions in mutagenesis are not a consequence of reduction in survival. The following chapters describe a variety of studies that identify antimutagens and anticarcinogens and describe their possible mechanisms. Understanding the molecular basis by which cells deal with DNA damage provides many opportunities to explore antimutagenesis and anticarcinogenesis, and future studies will build on our increasing understanding of these processes.

Acknowledgments

We thank James Allen, Barbara Collins, and Leroy Worth for their helpful comments on this manuscript. We thank Sue Edelstein for Figure 2.2. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency and approved for publication. Approval does not signify that the contents reﬂect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. This work was supported in part by the Division of Extramural Research and Training of the NIH, National Institute of Environmental Health Sciences.

References

1 Friedberg, E.C., Walker, G.C., Siede, W., carcinogenesis. Annual Review of Wood, R.D., Schultz, R.A. and Pharmacology and Toxicology, 44, 239–267. Ellenberger, T. (2006) DNA Repair and 4 Wyatt, M.D. and Pittman, D.L. (2006) Mutagenesis, 2nd edn., ASM Press, Methylating agents and DNA repair Washington, DC. responses: methylated bases and sources 2 Barnes, D.E. and Lindahl, T. (2004) of strand breaks. Chemical Research in Repair and genetic consequences of Toxicology, 19, 1580–1594. endogenous DNA base damage in 5 Drablos, F., Feyzi, E., Aas, P.A., mammalian cells. Annual Review of Vaagbo, C.B., Kavli, B., Bratlie, M.S., Genetics, 38, 445–476. Pena-Diaz, J., Otterlei, M., Slupphaug, G. 3 Klaunig, J.E. and Kamendulis, L.M. (2004) and Krokan, H.E. (2004) Alkylation The role of oxidative stress in damage in DNA and RNA – repair References j33

mechanisms and medical significance. 13 Phillips, D.H. (1997) Detection of DNA DNA Repair, 3, 1389–1407. modifications by the 32P-postlabelling 6 Singh, N.P., McCoy, M.T., Tice, R.R. and assay. Mutation Research, 378,1–12. Schneider, E.L. (1988) A simple technique 14 Dingley, K.H., Curtis, K.D., Nowell, S., for quantitation of low levels of DNA Felton, J.S., Lang, N.P. and Turtletaub, damage in individual cells. Experimental K.W. (1999) DNA and protein adduct Cell Research, 175, 184–191. formation in the colon and blood after 7 Møller, P. (2006) The alkaline comet exposure to a dietary-relevant dose if 2- assay: towards validation in biomonitoring amino-1-methyl-6-phenylimidazo[4,5-b] of DNA damaging exposures. Basic & pyridine. Cancer Epidemiology, Biomarkers Clinical Pharmacology & Toxicology, & Prevention, 8, 507–512. 98, 336–345. 15 Besatarinia, A.H. and Pfiefer, G. (2008) 8 Ismail, I.S., Wadhra, T.I. and Investigating human cancer etiology by Hammarsten, O. (2007) An optimized DNA lesion footprinting and mutagenicity method for detecting gamma-H2AX in analysis. Carcinogenesis, 27, 1526–1537. blood cells reveals a significant 16 Fenech, M. (2002) Chromosomal interindividual variation in the gamma- biomarkers of genomic instability relevant H2AX response among humans. Nucleic to cancer. Drug Discovery Today, 7, Acids Research, 35, e36. 1128–1137. 9 Nakamura, J., Walker, V.E., Upton, P.B., 17 Harper, J.W. and Elledge, S.J. (2007) The Chiang, S.-Y., Kow, Y.W. and DNA damage response: ten years after. Swenberg, J.A. (1998) Highly sensitive Molecules and Cells, 28, 739–745. apurinic/apyrimidinic site assay can detect 18 Hassa, P.O. and Hottiger, M.O. (2005) An spontaneous and chemically induced epigenetic code for DNA damage repair depurinatin under physiological pathways? Biochemistry and Cell Biology, 83, conditions. Cancer Research, 58, 222–225. 270–285. 10 Rusyn, I., Asakura, S., Li, Y., Kosyk, O., 19 Grønbæk, K., Hother, C. and Jones, P.A. Koc, H., Nakamura, J., Upton, P.B. and (2007) Epigenetic changes in cancer. Swenberg, J.A. (2005) Effects of ethylene APMIS, 115, 1039–1059. oxide and ethylene inhalation on DNA 20 Nohmi, T. (2007) Novel DNA polymerases adducts, apurinic/apyrimidinic sites and and novel genotoxicity assays. Genes and expression of base excision DNA repair Environment, 29,75–88. genes in rat brain, spleen and liver. DNA 21 Preston, R.J. and Hoffmann, G.R. (2001) Repair, 4, 1099–1110. Genetic Toxicology, in Casarette and Doulls 11 Farmer, P.B. (2004) Exposure biomarkers Toxicology: The Basic Sciences of Poisons for the study of toxicological impact on (ed C.D. Klaassen), McGraw-Hill, carcinogenic processes. IARC Scientific New York, Chapter 9, pp. 321–350. Publications, 157,71–90. 22 Cimino, M.C. (2006) Comparative 12 Poirier, M.C., Santella, R.M. and overview of current international strategies Weston, A. (2000) Carcinogen and guidelines for genetic toxicology macromolecular adducts and their testing for regulatory purposes. measurement. Carcinogenesis, 21, Environmental and Molecular Mutagenesis, 353–359. 47, 362–390. j35

3 Induction of DNA Damage and Cancer by Dietary Factors Wolfram Parzefall, Nina Kager, and Siegfried Knasmuller€

3.1 Introduction

Over the last decades, strong efforts have been made to identify potential DNA- damaging and carcinogenic chemicals in human foods, and a number of different hazardous compounds were detected comprising nitrosamines (NAs), polycyclic aromatic hydrocarbons (PAHs), heterocyclic aromatic amines (HAs), and mycotoxins as well as thermal degradation products. Furthermore, attempts have been made to investigate potential adverse health effects caused by food additives and pesticide and herbicide residues. This chapter describes the formation and occurrence of different dietary carcinogens, their mode of action, and their potential health risks in humans. In many investigations with antimutagens and anticarcinogens, protective effects toward representatives of DNA-reactive carcinogens found in human foods were studied, and relevant information concerning this issue is included in the different sections.

3.2 Nitrosamines

The carcinogenic properties of NAs were discovered already in the 1950s, and their tumorigenic and DNA damaging effects were intensively studied by pioneers such as W. Lijinsky [1] and by scientists from the German Cancer Research Center in Heidelberg [2]. As of today several hundred structurally different compounds are known. NAs are nitroso derivatives of secondary amines and are either formed in cured meats or endogenously in the digestive tract. The low pH in the stomach favors the formation fromprecursors,that is, fromnitrate foundin vegetables(e.g., in red beet,spinach)and drinkingwaterandamines (whichare formedfromproteins).Nitratecanbereducedto nitrite during the digestive process, in particular by saliva. The most abundant

Scheme 3.1 Metabolic activation of NDEA. CYP2E1 hydroxylates the carbon atom a to the nitroso group. The resulting product splits into acetaldehyde and another unstable intermediate. The latter decomposes spontaneously and releases a nitrogen molecule, leaving behind a reactive alkylcarbonium ion as the ultimate electrophile responsible for adduct formation and toxicity.

carcinogenic NAs found in foods are N-nitroso-dimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), and N-nitroso-piperidine (NPIP). NAs are metabolically activated in the body, the most important pathway being hydroxylation of the carbon atom a to the nitroso group. The product is an unstable intermediate and splitting off a nitrogen molecule leads to a reactive alkylcarbenium ion that causes formation of protein and DNA adducts (Scheme 3.1). The carcinogenic properties of NAs have been studied intensively in experiments with laboratory rodents. The most important target organ of symmetric aliphatic NAs is the liver; other NAs cause tumors in different sites, for example, N-nitroso-bis-1, 2-oxopropylamine in the colon and N-nitroso-dibutylamine in the bladder. Asymmet- ricaliphaticNAscausecarcinomaspreferentiallyintheupperdigestivetract,primarily in the esophagus. Several findings support the assumption that NAs also cause DNA damage and cancer in humans. Therefore, IARC has classified the representatives NDMA and NDEA as Group 2A (probably carcinogenic to humans) and NPYR and NPIP as group 2B carcinogens (possibly carcinogenic to humans). In support to this classification are those of the tobacco-specific carcinogens N0-nitrosonornicotine (NNN) and 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) whose overall evaluation was upgraded by IARC from Group 2B to Group 1 (carcinogenic to humans). An example of the induction of cancer in humans by NAs is the high incidence of esophageal cancer in areas with high nitrate concentrations in water in China, which has been complemented by a more recent study showing a clear-cut association with standardized mortality from esophageal cancer in provinces of low and high total NA intake. In addition, in the province Guangzhou of South China, there is a high prevalence of nasal carcinomas that are possibly caused by consumption of salted dried fish, which contains in particular aliphatic NAs. However, NAs are 3.3 Heterocyclic Aromatic Amines j37 considered as only one chemical factor besides Epstein-Barr virus infection of nasal carcinoma. It is well documented that CYP2E1 that catalyzes the activation of NAs is induced by chronic ethanol consumption [3]. This phenomenon may at least in part explain the strong synergistic effects that alcoholism and smoking have on cancer incidence and cancer induction at specific sites, for example, in the larynx and in the esophagus, which was observed in several epidemiological studie. When NAs were given to rats over a lifetime in drinking water, the lowest observed effect levels of 10 mg/kg bw/d for NDEA for liver, urinary tract, and gastrointestinal tract tumors and of 200 mg/kg bw/d for NDELA for liver, GI tract, and hematopoietic/ lymphatic system tumors were determined. Human intake of exogenous NAs was estimated to be in the range of 36–140 mg/person/day. To this adds the endogenous formation estimated to be approximately 0.3–20 mg/person/day. In total, this might add up to 0.8–2.3 mg/kg/day for a person of 70 kg, which is only 10–25% of the carcinogenic dose in rats. Thus, the margin of exposure is extremely narrow (3.8–11). It is therefore justified to assume that NAs ingested with food or endogenously generated from precursors contribute to human cancer risks, and it is absolutely required to keep the intake of NAs and their precursors at levels as low as reasonably achievable (ALARA principle). Adequate measures to reduce human risks due to dietary exposure to NAs are the control of the nitrate and nitrite concentrations in drinking water and in specific vegetables as well as the reduced consumption of cured meat products. Furthermore, it has been shown in studies with laboratory rodents that dietary phenolics are potent inhibitors of the endogenous formation of NAs [4], and it was also demonstrated in intervention trials in humans that the excretion of the noncarcinogen N-nitroso- proline was significantly reduced by vitamin C. N-Nitroso-proline as a test substrate was formed in vivo from nitrate-containing red beet juice and the amino acid proline.

3.3 Heterocyclic Aromatic Amines

These compounds were detected by the Japanese researcher Takashi Sugimura in grilled ﬁsh in the early 1970s. At present, about 20 different HAs are known that belong to ﬁve groups, namely, imidazoquinolins (e.g., IQ, MeIQ), -quinoxalines (e.g., iQx, MeIQx), -pyridines (e.g., PhIP), aminocarbolines (e.g., AaC, MeAaC), and pyrido-indoles (Trp-P-1/2) as well as dipyrido-imidazoles (Glu-P-1/2). Scheme 3.2 shows structures of representatives of the different classes. J€agerstad et al. [5] demonstrated that HAs are formed during cooking of meats from amino acids, creatinine, and sugar. PhIP and AaC are the most abundant HAs formed in fried meat (hamburgers) and chicken; other compounds such as MeIQx and IQ were found at concentrations that are one or two orders of magnitude lower, and some of the dipyridoimidazoles have not yet been found in fried foods. Numerous studies have been carried out in which the concentrations of HAs were determined in different human foodstuffs (for details see Ref. [6]). 38j 3 Induction of DNA Damage and Cancer by Dietary Factors

Scheme 3.2 Chemical structures of some representative HAs.

HAs are metabolically activated via N-oxidation of the exocyclic amino group mainly by CYP1A2 in the liver and subsequently by O-acetylation that is catalyzed by N-acetyltransferases (NAT). In the case of PhIP (Scheme 3.3) and AaC, sulfotransferases (SULTs) play a key role in the further metabolic conversion to the DNA active metabolites. It has been shown in genotoxicity experiments that the reaction products of HAs cause DNA adduct formation; as a consequence, mutations are induced in specific oncogenes, which are considered to be responsible for their tumorigenic effects. It has been shown in many genotoxicity experiments that HAs are potent mutagens; for example, they induce mutations in bacterial assays already in the nanogram range per plate. In mammalian cells and in laboratory rodents, they are in general less active due to the higher activities of detoxifying enzymes. HAs cause induction of tumors in different organs of rodents, one of the main target tissues being the colon mucosa, and it has been postulated that cleavage of the b-glucuronidation products of HAs in the intestine by representatives of the intestinal microflora and subsequent acetylation by colon mucosa cells may account for this specific effect. In the case of PhIP, the induction of breast tumors, which was 3.3 Heterocyclic Aromatic Amines j39

Scheme 3.3 Metabolic activation of PhIP. The primary step of activation takes place mainly in the liver by N-oxidation of the exocyclic amino group by CYP1A2. Thereafter, O-acetylation is catalyzed by N-acetyltransferase2 (NAT2). The acetate ion as leavinggroupsplitsoffthatresultsinareactivenitreniumionasthe ultimate carcinogen. seen in rodent experiments, may be promoted due to its binding to the estrogen receptor [7]. The amounts of HAs required to induce tumors in laboratory rodents are in the range between 0.5 and 20 mg/kg bw/d (depending on the type of HA and species studied). Since the uptake levels in humans are substantially (up to three orders of magnitude) lower, it has been disputed if HAs contribute to cancer risks in man and it has been postulated that exposure to HAs via consumption of fried meats may explain the increasing incidence of colon cancer in rich (industrial) countries. Epidemiological studies are in many cases hampered by uncertainties of the exposure assessment, and in some well-designed investigations a clear-cut correlation between meat consumption, HA exposure, and the incidence of colorectal cancer was seen only in individuals with extremely high consumption levels. Some investigations suggested that individuals who have high CYP1A2 activities and polymorphisms in NAT2 gene (encoding for acetylation of the amines) and consume high levels of fried meat are at substantially increased risk of colon cancer [8]. These latter observations support the assumption that HAs cause colon cancer in humans. Numerous attempts have been made to develop strategies to protect humans against the adverse health effects of HAs (for an overview see Ref. [6]). They include reduction of the frying temperature, use of spicy marinades and polyphenol-rich oils during the preparation of meals, as well as consumption of speciﬁc foods that may interact with the metabolism of HAs. For example, it was shown in a human study 40j 3 Induction of DNA Damage and Cancer by Dietary Factors that the DNA-protective effects towards PhIP seen after consumption of Brussel sprouts are due to the reduction of the activity of SULT (Hoelzl, 2008 #86); another mode of protection seen with cruciferous vegetables is the induction of glucuronosyltransferase [9]. Also, lactic acid bacteria and dietary ﬁbers protect against HAs; in this case, direct binding mechanisms account apparently for the protective effects [10].

3.4 Polycyclic Aromatic Hydrocarbons

PAHs are products of incomplete combustion of organic materials that are found in all environmental compartments. Their chemical structures were resolved already in the 1930s. Since then about 500 chemically different PAHs have been identified. Human exposure to PAHs occurs via inhalation of particles and also by consumption of foods. In heavily contaminated industrial areas, the daily inhalative uptake by an individual may reach 100–900 ng of benzo[a]pyrene (B[a]P), and similar values (ranging 11–330 ng/day) were found after passive smoking. The corresponding levels for the main set of PAHs in dietary uptake are in the range of 200–400 ng/kg/day. A temporal decline of dietary intake has been noted. One main source may be the consumption of food and vegetables that are contaminated on their surface by PAH-containing particles, and the other is the consumption of barbequed meat in which PAHs are found in the outer crust. The most relevant compounds in foods are genotoxic and carcinogenic representatives such as benzo[a]anthracene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, and indeno[1,2,3-c,d]pyrene. Human cancer risk is commonly assessed on the basis of B[a]P as the lead compound of PAHs. Some authors have estimated the overall cancer risks under the assumption that carcinogenic PAHs other than B[a]P are formed in specific relations to the latter compound and for that purpose have applied PAH toxicity equivalency factors for exposure assessment. A more crude approach to account for other PAHs than B[a]P has been by multiplying B[a]P levels by a factor of 15 [11]. The pioneering work of the Millers who first discovered the protein binding properties of PAHs led to our knowledge on the reactivity of this class of compounds [12]. PAHs are metabolically activated to epoxides by cytochrome P-450 enzymes (in particular by CYP1A1). The main detoxification pathway is conjugation with glutathione by glutathione-S-transferases (GSTs). The ultimate DNA-reactive metabolites are bay region diol epoxides that form mainly adducts with the base guanosine and account for the mutagenic and carcinogenic properties of PAHs (Scheme 3.4). PAHs cause cancer in laboratory rodents, and certain model compounds such as B[a]P and 7,12-dimethylbenzanthracene (7,12-DMBA, CAS 57-97-6) have been used frequently in experimental oncology as model agents for stomach and breast cancer studies [13]. 3.4 Polycyclic Aromatic Hydrocarbons j41

Scheme 3.4 Metabolic activation of benzo(a)pyrene. AFB1 is attacked in the liver by several cytochrome P-450 isoforms, the major role is taken by CYP3A4 resulting in formation of an epoxide at the olefinic bond in 8,9 position. This is the ultimate reactive carcinogen that, after adducting DNA bases, transforms into a stable adduct by the opening of the pyrrol ring of the guanine base.

The development of biomarkers of PAH exposure has substantially contributed to the improvement of the risk assessment. The relation of environmental air pollution to PAHs with the associated biomarkers has been evaluated in a recent metastudy [14]. The urinary metabolite 1-hydroxypyrene and, to a lesser extent, PAH–DNA adducts in peripheral lymphocytes correlated well at the group level with exposure to B(a)P, even at low PAH exposures. Albumin or hemoglobin adducts did not well correlate with environmental exposure data, an observation that has been made earlier in occupational settings [15]. The dietary exposure of humans to PAHs can be reduced by use of low-temperature cooking and/or prevention of direct contact of meats with the ﬂames during barbequing. It has also been shown that compounds that induce GST activities protect from DNA damage and cancer. Already in the 1970s and 1980s, Wattenberg and coworkers identiﬁed a number of protective dietary constituents such as breakdown products of glucosinolates, constituents of allium vegetables, and coffee diterpenoids that protect against PAH-induced cancer formation in rats and mice [16], to name only a few. Recently, we reported the induction of GSTs in human lymphocytes by different dietary factors (e.g., by coffee consumption and gallic acid) and found that the induction of these enzymes led to protection against induction of DNA damage by B[a]P-7,8-dihydrodiol-9,10-epoxide, the ultimate metabolite of B(a)P [17]. 42j 3 Induction of DNA Damage and Cancer by Dietary Factors 3.5 Other Thermal Degradation Products

Over the last years, strong attempts have been made to identify thermal degradation products other than HAs in human foods. Typical examples are acrylamide (AA) and furans. AA has been found in specific foods at astonishingly high but varying levels (given as microgram AA/kg product), such as snacks (prezel sticks, salted biscuit, and cocktail snacks (1154 780), crisps, salted potato straws (766 559), french fries (424 498), and crisp bread (408 471) [18]. It is formed during the browning reaction (Maillard reaction) mainly from the amino acid asparagines and sugars such as glucose, fructose, lactose, and sucrose. Intake may vary widely between 0.3 and 2 mg/kg bw/day or may reach even 5 mg/kg bw/day at the 99th percentile in high consumers [19]. These are alarmingly high levels for a probable human carcinogen with a genotoxic mode of action. The primary effect is neurotoxicity and was detected after occupational exposure. AA is also toxic for reproduction and development in rats and mice and was found to be a germ cell mutagen. During metabolism, the reactive metabolite glycidamide is formed that is responsible for the genotoxicity and probably the carcinogenicity to several organs. However, epidemiological studies did not point to an association between either occupational or dietary exposure and an excess of cancer incidence so far; only a recent study has found an increased breast cancer risk (positive for estrogen receptors) in postmenopausal women after adjustment for smoking [20]. The innovative approach in this investigation was exposure monitoring by measuring hemoglobin adducts of AA and glycidamide and not by the usual assessment by food frequency questionnaires. It is worth noting that mammary tissue was also a target organ in rodent carcinogenicity studies. The authors of the aforementioned study suggest that AA itself may have a nongenotoxic mode of action to which a rodent study lends support by showing that the compound dose dependently induces DNA synthesis in three other hormone-dependent organs [21]. Health risk assessments of the general population are based on an average exposure to 0.4 mg/kg bw/day, which can increase in high consumers to 4 mg/kg bw/day. There appears to exist a sufficiently protective margin of exposure (MOE) of 200 for neurotoxicity in average consumers. However, the MOE for carcinogenic risks is approximately 300 and is considered to be not sufficiently protective. A similar conclusion is reached if a benchmark dose lower confidence limit (BMDL) for cancer is derived and divided by an uncertainty factor of 300 arriving at a tolerable daily intake of 1 mg/kg bw/day, which is in the range of average dose/consumption levels.Therefore,severalauthorsandauthoritieshaverecommendedexploringfurther measures to minimize acrylamide formation in food and to reduce human exposure. A multitude of other compounds are formed during food processing. Some are generated during the Maillard reaction, and their health effects are still unknown. A selection of a few representatives is given in Table 3.1, which provides information on the main endpoints of toxic effects. As can be seen, most compounds have a functional aldehyde group with the exception of furan that, however, can be Table 3.1 Some toxic compounds contaminating natural or home-made food.

Compound Occurrence Reactive metabolites Genotoxicity Carcinogenicity Other toxic effects

Furan In food that undergoes cis-2-butene-1,4-dial3 Depletion of ATP ! DNA HCC in male and ATP depletion heat treatment1 double strand breaks in female B6C3F1 mice hepatocyte suspension and male F344 rats5 culture4 CAS: 110-00-9 Naturally in certain woods Hepatocarcinogen in and during the combustion rodents6 of coals; found in engine exhausts, wood and tobacco O smoke2 Production by decarbonyla- Sufﬁcient evidence in tion of furfural. Use: produc- animal experiments tion of tetrahydrofuran, thiophene, and pyrrole Inadequate evidence in . te hra erdto Products Degradation Thermal Other 3.5 humans7

Furfural Furfural is a volatile Mostly negative in Inadequate evidence in Extensively absorbed component of a wide range of Salmonella typhimurium humans; limited evi- and eliminated after fruits and vegetables8 dence in experimental inhalation12 animals10 CAS: 98-01-1 Produced commercially by SCE in CHO cells and HCC in mice (gavage)11 Skin and mucous acid hydrolysis of pentosan human lymphocytes in vitro membrane O polysaccharides from non- irritation13 C food residues of food crops O H and wood wastes. Use:solvent in petroleum reﬁning, in production of phenolic resins9 CA in CHO, and V79 cells j 43 No SCE or CA in mouse bone marrow (Continued) 44

Table 3.1 (Continued) j nuto fDADmg n acrb itr Factors Dietary by Cancer and Damage DNA of Induction 3 Compound Occurrence Reactive metabolites Genotoxicity Carcinogenicity Other toxic effects

5-Hydroxy-methyl-- 5-Sulfooxymethylfurfural SMF interacts with DNA, HMF promotes growth of furfural (HMF) (SMF) RNA, and proteins ! colonic structural damage ! toxicity microadenomas17 CAS: 67-47-0 and mutagenicity15 OH O Product of Maillard 5-Chloromethylfurfural CMF is of higher mutagenic In colon dose-dependent reaction14 (CMF) is formed from potency in S. typhimurium induction of aberrant CH2 C O H HMF by allylic chlorination than SMF16 crypt foci18

Acrolein Formed from carbohydrates, Mother compound itself: Genotoxic in Drosophila21 Initiation of rat urinary amino acids, vegetable oils, acrolein bladder carcinogenesis24 and animal fats during heat- CAS: 107-02-8 O ing of foods; in tobacco H CCHC 2 smoke19; produced H commercially 2-year study in rats – no neoplastic response25 Used to produce acrylic acid Mutagenic in S. typhimurium Available data Strong irritant (starting material for acrylate and Escherichia coli22 and in inadequate for respiratory tract; polymers), to produce xeroderma pigmentosum evaluation of human cardiovascular system 23 26 27 DL-methionine, and as a ﬁbroblasts carcinogenicity toxicity LD50 : herbicide and slimicide20 7–46 mg/kg in rats

Acetaldehyde Metabolite of sugars and Mother compound itself: Cross-links with DNA Tumors of bronchi and Neurotoxic effects CAS: 75-07-0 ethanol; has been detected acetaldehyde in gastric mucosa and oral cavity " in chemical during embryonic O in plant extracts, tobacco colonic workers exposed to vari- development34 H3CC smoke, engine exhaust, mucosa cells29 ous aldehydes H ambient and indoor air, and in water28 Table 3.1 (Continued)

Compound Occurrence Reactive metabolites Genotoxicity Carcinogenicity Other toxic effects

DNA adducts of acetaldehyde Esophageal tumors: as- Teratogenic in in lymphocytes of alcohol sociated with genetically experimental abusers30 determined, high meta- animals35 bolic levels of acetaldehyde after drinking alcohol (IARC) Genotoxic in human lymphocytes31 Genotoxic in animal Inhalation: cytotoxic models32 and carcinogenic to nasal mucosa of rats33

Monochloro- Highest levels in Mutagenic in bacterial Oral chronic study in Decreased sperm propanediol36,37 chemically hydrolyzed vege- assays, but negative in rats: progressive motility and CAS: 96-24-2 table proteins the presence nephropathy, impaired fertility in of exogenous metabolic dose-dependent rats and other Cl Products Degradation Thermal Other 3.5 activation tubular hyperplasia mammals CH2 CH CH2 and adenoma Most likely in bread Also in mammalian Hyperplastic and neo- CNS lesions at OH OH and biscuits and in cells in vitro plastic lesions in testes, >25 mg/kg cooked meat or ﬁsh mammary gland, and pancreas Average daily intake No evidence of genotoxicity Malignant transforma- 2 lg per person in vivo tion of mouse ﬁbroblasts in vitro

Abbreviations: ATP, adenosine triphosphate; CA, chromosome aberrations; CHO, Chinese hamster ovary; CNS, central nervous system; HCC, hepatocellular carcinoma; – SCE, sister chromatid exchange. 1 37 References may be obtained from the authors. j 45 46j 3 Induction of DNA Damage and Cancer by Dietary Factors hydrolyzed to a dialdehyde. The availability and reactivity of the aldehyde groups appears to dictate the (geno)toxic effects. Another product formed during domestic cooking processes and also during some food manufacturing is 3-chloropropane-1,2- diol. This compound showed an apparent discrepancy between the genotoxicity results in vitro that were positive and the genotoxicity results in vivo that were negative. These ﬁndings may be explained by the fact that the main metabolic route in mammals is the formation of beta-chlorolactate and oxalic acid, while many bacteria metabolize 3-chloro-1,2-propanediol primarily via glycidol, the latter being a bacterial mutagen.

3.6 Mycotoxins

Mycotoxins are a chemically heterogeneous group of metabolites of moulds that are mostly formed in plant foods during cultivation and storage. ﬂ A atoxin B1 (AFB1) is probably the most potent genotoxic carcinogen in humans. It is formed by Aspergillus ﬂavus and A. parasiticus and found predominantly in foods such as oilseeds (e.g., peanuts, soybeans), cereals (e.g., maize, rice), spices (e.g., chili pepper), and tree nuts (e.g., almonds, pistachio) in countries with a hot and

humid climate. AFB1 is considered to account, in addition to viral infections, for the high prevalence of hepatocellular carcinoma (HCC) in countries of central Africa fl and China. The structurally related a atoxins G (AFG1, AFG2) are produced by A. parasiticus, which has a limited distribution. AFM1 is found in milk. The carcinogenicity of different aflatoxins has been evaluated by IARC [22, 23]. There is sufficient

evidence of carcinogenicity of AFB1,G1,andM1 to humans, limited evidence for AFB2, and inadequate evidence for AFG2, which all induce primarily liver tumors. AFB1 and other structurally related toxins are activated predominately in the liver by ﬁ CYP1A2 and 3A4, where CYP1A2 is active with high af nity at low AFB1 concentrations. In these reactions, an ultimate DNA-reactive metabolite, exo-AFB1-8,9-epoxide, is formed that binds to glutathione, serum albumin, and DNA. The binding to N7- guanine is accompanied by intercalation into the DNA molecule. Detoxiﬁcation of the reactive metabolites occurs mainly by GSTs (Scheme 3.5).

The genotoxic effects of AFB1 are well documented in experiments in vitro with bacterial and mammalian indicator cells and also in laboratory rodents. AFB1 is a potent liver carcinogen in rats, while only low or moderate effects were seen in mouse ﬁ strains that have higher GST activities. In humans, AFB1 exposure causes a speci c ﬁngerprint mutation (i.e., G ! Ttransversion) in codon 249 of the tumor suppressor gene p53. On the basis of this observation, it was possible to analyze DNA from HCC

and draw conclusions if they are due to exposure to AFB1. Indeed, such mutations were found in DNA from liver tumors collected in Africa and China but not in tissues

from HCC patients from Europe [24]. Exposure monitoring of AFB1 has been conducted by albumin adduct measurements and by the aﬂatoxin-speciﬁc urinary – adduct AFB1 N7-guanine. Ochratoxin A (OTA) is formed by Penicillium ochraceus and is found particularly in beans, coffee, grain, and pork products. It is believed to be the cause of Balkan 3.6 Mycotoxins j47

Scheme 3.5 Metabolic activation of aflatoxin B1. AFB1 is attacked in the liver by several cytochrome P-450 isoforms; the major role is taken by CYP3A4 resulting in an epoxide at the olefinic bond in 8,9 position. This is the ultimate reactive carcinogen that, after adducting DNA bases, transforms into a stable adduct by the opening of the pyrrol ring of the guanine base. endemic nephropathy (BEN), a chronic renal tubulointerstitial disease of previously unknown cause that often is accompanied by upper urinary tract urothelial cancer that was found in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia. The disease was ﬁrst described in the 1950s but appears to be decreasing since the 1990s [25]. OTA is mutagenic in certain in vitro tests with mammalian- and human- derived cells but not in bacteria, and it is still a matter of debate if it causes DNA adduct formation in humans [26]. OTA is carcinogenic in mouse and rat livers and kidneys, and two of its metabolites are immunosuppressive and may contribute to cancer etiology [27]. Recent advances in the understanding of endemic nephropathy now favor the causative role of aristolochic acid over the ubiquitous mycotoxin known as ochratoxin A. Speciﬁcally, (1) aristolactam–DNA adducts have been found in renal tissues and urothelial cancers of affected patients; (2) a signature mutation in the 48j 3 Induction of DNA Damage and Cancer by Dietary Factors p53 gene of upper urothelial cancers associated with this disease provided evidence of long-term exposure to aristolochic acid; (3) the renal pathophysiology and histopathology observed in endemic nephropathy most closely resemble those of aristolochic acid nephropathy [28]. A number of other mycotoxins may also cause human cancer; typical examples are

fumonisins (e.g., fumonisin B1), trichothecenes (nivalenol, deoxynivalenol), zearalenon, and other compounds such as patulin and citrinin. These toxins are found more frequently and at higher concentrations in human foods than aﬂatoxins. Results of in vitro mutagenicity tests indicate that some of these mycotoxins cause

DNA damage in mammalian/human cells at higher dose levels than AFB1, but results from animal studies are scarce and no ﬁrm conclusions concerning their potential mutagenic and carcinogenic risks can be drawn in general.

In the case of fumonisin B1, it has been shown, however, that it causes induction of liver cancer in rats after administration of relatively high doses [29], and it is notable that it has been postulated that the increased incidence of esophageal cancer in countries with high contamination levels of fusarium-infected foods (e.g., South Africa and China) may be due to exposure to these toxins [30]. The mechanism of carcinogenic action is possibly caused by both, genotoxic and

nongenotoxic mechanisms [31]. Although fumonisin B1 is not a bacterial mutagen [32], it causes (oxidative) DNA damage in mammalian cells and in rat kidney and is a clastogenic agent (induction of micronuclei and chromosomal aberrations in mammalian cells) [33]. The nongenotoxic mechanisms include complex alterations of cellular signaling pathways by disruption of lipid metabolism and changes in polyunsaturated fatty acids and phospholipid pools that eventually lead to an imbalance of both, apoptosis and proliferation rates [31]. Patulin is produced particularly by P. expansum, a fruit pathogen that causes apple rot. It is not a particularly potent toxin, but several studies have shown that it is genotoxic, and subcutaneous application in rats led to the formation of sarcomas. Although patulin appears not to be a strong genotoxin and carcinogen, exposure to this mycotoxin may contribute to human carcinogenic risk. Therefore, a WHO recommendation limits patulin in apple juice at a maximum concentration of 50 mg/l. The mycotoxin citrinin was originally isolated from P. citrinum but has also been found to be produced by some other fungi on human foods, in particular grain and cheese. Citrinin was nephrotoxic in all species tested, and since it causes nephropathy in livestock, it has been suspected as one of the causal agents of BEN. It was found recently that citrinin did not induce mutations in Salmonella tester strains but induced micronuclei with similar potency as OTA, although the mechanism is through aneuploidy [26]. Some other food components may protect from these fungal toxins: In a number of in vitro experiments and also in a few animal studies, it was demonstrated that lactic acid bacteria bind aﬂatoxins as well as other mycotoxins (OTA, citrinin, zearalenon) and it has been reported that consumption of fermented foods may protect humans

against their toxic effects. Also chlorophylls are apparently able to detoxify AFB1 by direct binding, and another mode of protection is the upregulation of GSTs that ﬁ inactivates DNA-reactive metabolites of AFB1 [34]. Such mechanism of detoxi cation 3.8 Food Additives and Pesticides/Herbicide Residues j49 is seen, for example, with breakdown products of glucosinolates from cruciferous vegetables and allium compounds. A synthetic and potent GST inducer is oltipraz, and intermittent results of human intervention trials in high AFB1 exposure areas in China were promising [35].

3.7 Carcinogens in Plant Foods

Plant-derived foods are generally regarded as healthy and safe. Therefore, cycasin and ptaquiloside were the only examples of plant-derived carcinogens for many years. Cycasin is found in the flower of cycad palms that was used to produce cakes and biscuits, while ptaquiloside is found in bracken fern sprouts consumed as vegetables in parts of Russia and China. In the early 1990s, B. Ames published a number of papers [36, 37] in which examples of plant-derived carcinogens were listed to which humans are exposed via the diet; they comprise constituents of mushrooms such as gyromitrin [38] and agaritin [39], alkylbenzenes (e.g., safrol, eugenol, methyleugenol) that are found in different spices such as nutmeg and basil, caffeic acid that is found in numerous vegetables and in coffee [40], and capsaicin the pungent principal of chilis. Ames emphasized that some of these compounds are equally potent in regard to DNA-damaging and carcinogenic properties as synthetic chemicals and that humans may be exposed to them even at much higher concentrations. Recent evaluation of the current state of knowledge by Ehrlich et al. [41] showed that the evidence for carcinogenic effects of the different plant-derived compounds is restricted in many cases to animal experiments that do not meet the current criteria of long-term carcinogenicity studies. In many of the trials only extremely high doses were tested, and it is unclear if the results obtained with rodents are relevant for humans. For example, it was claimed that coffee contains about 20 different chemicals that were shown to cause tumor formation in rodents [42], but no evidence of their potential carcinogenic effects was found in coffee drinkers in numerous epidemiologic studies (see Chapter 32). The weak positive relationship between coffee drinking and the occurrence of bladder and urinary tract cancer resided possibly on bias or confounding factors [43] and could in no way be confirmed for renal cancer in a large prospective study [44]. Some of the coffee constituents are even suggested to be protective [45], and coffee as a whole should be regarded as a potential effect modifier of carcinogenic exposures.

3.8 Food Additives and Pesticides/Herbicide Residues

Numerous investigations have been carried out in which the genotoxic and carcinogenic properties of food additives have been studied. The results are summarized in 50j 3 Induction of DNA Damage and Cancer by Dietary Factors various IARC publications [46–49]. Among the compounds suspected to possess carcinogenic properties are D-limonene and sodium saccharin. D-Limonene is present in citrus oils, and it was found to cause kidney cancer in male rats. Subsequent investigations showed that the induction of tumors is due to a mechanism specifically present in male rats. As the initial step in the carcinogenic process a chemical metabolite of D-limonene binds to a2m-globulin in the kidney. The presence of a2m-globulin is a prerequisite to the development of a renal syndrome, resulting in renal tubule protein overload, cytotoxicity, reparative cell proliferation, and eventually tumor development. This protein is present in the male rat only and is not found in humans, mice, and female rats. On the contrary, D-limonene was even found to suppress chemically induced mammary carcinogenesis. This anticarcinogenic effect is due to inhibition of post-translational farnesylation of the ras p21 protein that inhibits the activation of this oncogene and gives hope for improved chemotherapeutic regimens. In the case of sodium saccharin, it was shown that the formation of bladder tumors is also species specific. Due to the alkalinization of urine, a2m-globulin together with sodium saccharin and silicates precipitates in the urinary bladder of male rats causing chronic irritation, inflammation, and tumors. Compounds such as butter yellow were used for making margarine till 1937 and were found until the 1960s in consumer products such as shoe polish, pomaid, and grease. The compound was shown to be a potent liver carcinogen in rats and to bind covalently to liver proteins. 2-Furan-acetamide (AF-2)isa synthetic foodpreservativethatwas sold commercially in Japan since the early 1960s until it was withdrawn from the market in 1974 because of its genotoxicity and its carcinogenicity in rodents. IARC [47] evaluated the compound as showing sufficient evidence for carcinogenicity in experimental animals. Overall, it is unlikely that food additives that are presently used confer a relevant cancer risk on humans. On the contrary, it was postulated that antioxidants, which are widely used to increase the shelf life of foods, may in general reduce the genotoxic and carcinogenic effects of food contaminants discussed above. Pesticide and herbicide residues are detected in many fruits, vegetables, and grains with highly sensitive analytical techniques. Regulations in good agricultural practice give provisions that these residues occur below acute reference doses or no-observed- adverse-effect-levels (NOAELs). Although some of the pesticides in use are rodent carcinogens in lifetime high-dose experiments, the disparity between carcinogenic potency in rodents and human exposure becomes obvious since at realistic exposure human cancer risks can be calculated to be unmeasurably low, that is, less than one in a million of people exposed [50].

3.9 Human Cancer Risks of Food Specific Carcinogens

Based on the ﬁndings of Doll and Peto [51] who postulated that about one-thirds of all human cancer deaths in industrial countries are due to dietary factors (Table 3.2), 3.9 Human Cancer Risks of Food Specific Carcinogens j51 Table 3.2 Proportions of cancer deaths attributed to various factors [51].

Factor or class Range of acceptable of factors Best estimate (%) estimates (%) Data rating

Tobacco 30 25–40 Epidemiologically Alcohol 3 2–4 g Occupation 4 2–8 well documented Pollution 2 <1–5 Industrial products <1 <1–2 Uncertain Geophysical factorsa 32–4 g estimates Medicines and medical 1 0.5–3 procedures Infection 15 8–25b Diet 35 10–70 Reproductive and 71–13 Very uncertain sexual behavior g estimates Food additives <1 –5–2 Unknown ? ? aIncluding 1–2% cancer deaths due to UV sunlight. bDifferentiated for developed countries with 8% and 25% for developing countries.

Lutz and Schlatter published an interesting article in which they attempted to assess the contribution of different groups of dietary carcinogens to human cancer risks [52]. They attributed one-thirds of the annual cancer cases in Switzerland to different dietary factors and used available exposure data of different toxins and results of animal carcinogenicity studies. The results are listed in Figure 3.1. It can be seen that

Figure 3.1 Cancer risk estimates for dietary discussed in the this chapter are of little concern carcinogens (cancer cases per million lives). but may still be attributed to efforts of further This is a graphic representation of the major minimizing their impact on food-associated contributors to human cancer risk, regardless of cancer risks in humans. Data taken from their mechanism of action. The graph clearly Ref. [52]. illustrates that most of the food contaminants 52j 3 Induction of DNA Damage and Cancer by Dietary Factors adduct-forming chemicals account only for a relatively small fraction of the overall cancer cases, and the vast majority of cancer deaths could not be explained by exposure to dangerous chemicals but is apparently due to other factors such as obesity and low consumption of foods with DNA-stabilizing and protective properties. In conclusion, human diet contains numerous compounds with genotoxic and carcinogenic properties. However, only a few of them (e.g., AFB1 and aristolochic acid) are proven human carcinogens, and others are animal carcinogens but probably also constitute a potential human cancer risk. It is mandatory, therefore, to further assess (a) under what conditions these contaminants are formed, to reduce the exposure, and (b) to explore the mechanisms by which these food constituents act in experimental animals and humans, to improve risk characterization.Finally,itmustberecalledthatabalanceddietalsocontainsantioxidants and other plant-derived antimutagenic and anticarcinogenic substances that substantially may reduce the health hazards caused by the compounds described in this chapter.

References

1 Lijinsky, W. (1988) Intestinal cancer 6 Knasmuller,€ S., Holzl,€ C., Bichler, J., induced by N-nitroso compounds. Nersesyan, A. and Ehrlich, V. (2006) Toxicologic Pathology, 16, 198–204. Dietary Compounds Which Protect Against 2 Schm€ahl, D. (1987) Nitrosamine-induced Heterocyclic Amines, CRC Press, Boca carcinogenesis: experimental models for Raton, FL. human malignancies of multifactorial 7 Bennion, B.J., Cosman, M., Lightstone, origin, in Relevance of N-Nitroso F.C., Knize, M.G., Montgomery, J.L., Compounds To Human Cancer: Exposures Bennett, L.M., Felton, J.S. and Kulp, K.S. and Mechanisms (eds H. Bartsch, I.K. (2005) PhIP carcinogenicity in breast ONeill and R. Schulte-Hermann), IARC cancer: computational and experimental Scientiﬁc Publications, No. 84, IARC, evidence for competitive interactions with Lyon, pp. 239–245. human estrogen receptor. Chemical 3 Schwarz, M., Appel, K.E., Schrenk, D. Research in Toxicology, 18, 1528–1536. and Kunz, W. (1980) Effect of ethanol 8 Kadlubar, F., Kaderlik, R.K., Mulder, G.J., on microsomal metabolism of Lin, D., Butler, M.A., Teitel, C.H., dimethylnitrosamine. Journal of Cancer Minchin, R.F., Ilett, K.F., Friesen, M.D., Research and Clinical Oncology, 97, Bartsch, H. et al. (1995) Metabolic 233–240. activation and DNA adduct detection of 4 Wu, Y.N., Wang, H.Z., Li, J.S. and Han, C. PhIP in dogs, rats, and humans in (1993) The inhibitory effect of Chinese tea relation to urinary bladder and colon and its polyphenols on in vitro and in vivo carcinogenesis. Princess Takamatsu N-nitrosation. Biomedical and Symposia, 23, 207–213. Environmental Sciences, 6, 237–258. 9 Kassie, F., Uhl, M., Rabot, S., Grasl- 5 Jagerstad, M., Skog, K., Grivas, S. and Kraupp, B., Verkerk, R., Kundi, M., Olsson, K. (1991) Formation of Chabicovsky, M., Schulte-Hermann, R. heterocyclic amines using model systems. and Knasmuller,€ S. (2003) Mutation Research, 259, 219–233. Chemoprevention of 2-amino- References j53

3-methylimidazo[4,5-f]quinoline 16 Wattenberg, L.W. (1992) Inhibition of (IQ)-induced colonic and hepatic carcinogenesis by minor dietary preneoplastic lesions in the F344 rat by constituents. Cancer Research, 52, cruciferous vegetables administered 2085s–2091s. simultaneously with the carcinogen. 17 Steinkellner, H., Hoelzl, C., Uhl, M., Carcinogenesis, 24, 255–261. Cavin, C., Haidinger, G., Gsur, A., Schmid, 10 Stidl, R., Fuchs, S., Koller, V., Marian, B., R., Kundi, M., Bichler, J. and Knasmuller, Sontag, G., Ehrlich, V. and Knasmuller,€ S. S. (2005) Coffee consumption induces (2007) DNA-protective properties of lactic GSTP in plasma and protects lymphocytes acid bacteria, in The Activity of Natural against ( þ /)-anti-benzo[a]pyrene-7,8- Compounds in Disease Prevention and dihydrodiol-9,10-epoxide induced DNA- Therapy (eds Z. Dura ckova and D. damage: results of controlled human Slamenova), Slovak Academic Press, intervention trials. Mutation Research, 591, Bratislava, pp. 245–263. 264–275. 11 Hoelzl, C., Glatt, H., Meinl, W., Sontag, G., 18 Boon, P.E., de Mul, A., van der Voet, H., Haidinger, G., Kundi, M., Simic, T., van Donkersgoed, G., Brette, M. and van Chakraborty, A., Bichler, J., Ferk, F., Klaveren, J.D. (2005) Calculations of Angells, K., Nersesyan, A. and Knasmuller,€ dietary exposure to acrylamide. Mutation S. (2008) Consumption of Brussels sprouts Research, 580, 143–155. protects peripheral human lymphocytes 19 WHO/JECFA (2005) Summary and against 2-amino-1-methyl-6- conclusions of the sixty-fourth meeting of phenyllmidazo[4,5-b]pyridine (PhIP) and the Joint FAO/WHO Expert Committee on oxidative DNA-damage: results of a Food Additives (JECFA) pp. 7–17. controlled human intervention trial, 20 Olesen, P.T., Olsen, A., Frandsen, H., Molecular Nutrition and Food Research, 52, Frederiksen, K., Overvad, K. and 330–341. Tjonneland, A. (2008) Acrylamide 12 Miller, E.C. (1951) Studies on the exposure and incidence of breast cancer formation of protein-bound derivatives of among postmenopausal women in the 3,4-benzpyrene in the epidermal fraction Danish Diet, Cancer and Health Study. of mouse skin. Cancer Research, 11, International Journal of Cancer, 122, 100–108. 2094–2100. 13 Wattenberg, L.W. (1977) Inhibition of 21 Lafferty, J.S., Kamendulis, L.M., Kaster, J., carcinogenic effects of polycyclic Jiang, J. and Klaunig, J.E. (2004) hydrocarbons by benzyl isothiocyanate and Subchronic acrylamide treatment related compounds. Journal of the National induces a tissue-specific increase Cancer Institute, 58, 395–398. in DNA synthesis in the rat. Toxicology 14 Castano-Vinyals, G., DErrico, A., Malats, Letters, 154,95–103. N. and Kogevinas, M. (2004) Biomarkers of 22 IARC (1993) Aflatoxins, in Some exposure to polycyclic aromatic Naturally Occurring Substances: Food Items hydrocarbons from environmental air and Constituents, Heterocyclic Aromatic pollution. Occupational and Environmental Amines and Mycotoxins, WHO/IARC, Medicine, 61, e12. pp. 245–395. 15 Angerer, J., Mannschreck, C. and 23 IARC (2002) Aflatoxins, in Some Gundel, J. (1997) Biological monitoring Traditional Herbal Medicines, Some and biochemical effect monitoring of Mycotoxins, Naphthalene and Styrene, exposure to polycyclic aromatic IARC, pp. 171–300. hydrocarbons. International Archives 24 Pineau, P., Marchio, A., Battiston, C., of Occupational and Environmental Health Cordina, E., Russo, A., Terris, B., Qin, L.X., 70, 365–377. Turlin, B., Tang, Z.Y., Mazzaferro, V. and 54j 3 Induction of DNA Damage and Cancer by Dietary Factors

Dejean, A. (2008) Chromosome instability Mycotoxins, Naphthalene and Styrene, in human hepatocellular carcinoma IARC, pp. 301–366. depends on p53 status and aﬂatoxin 32 Ehrlich, V., Darroudi, F., Uhl, M., exposure. Mutation Research, 653,6–13. Steinkellner, H., Zsivkovits, M. and 25 Stefanovic, V. and Radovanovic, Z. (2008) Knasmueller, S. (2002) Fumonisin B(1) Balkan endemic nephropathy and is genotoxic in human derived associated urothelial cancer. Nature hepatoma (HepG2) cells. Mutagenesis, Clinical Practice, 5, 105–112. 17, 257–260. 26 Knasmuller,€ S., Cavin, C., Chakraborty, A., 33 Knasmuller,€ S., Bresgen, N., Kassie, F., Darroudi, F., Majer, B.J., Huber, W.W. and Mersch-Sundermann, V., Gelderblom, W., Ehrlich, V.A. (2004) Structurally related Zohrer, E. and Eckl, P.M. (1997) Genotoxic mycotoxins ochratoxin A, ochratoxin B, effects of three Fusarium mycotoxins, and citrinin differ in their genotoxic fumonisin B1, moniliformin and activities and in their mode of action in vomitoxin in bacteria and in primary human-derived liver (HepG2) cells: cultures of rat hepatocytes. Mutation implications for risk assessment. Nutrition Research, 391,39–48. and Cancer, 50, 190–197. 34 Steinkellner, H., Rabot, S., Freywald, C., 27 IARC (1993) Ochratoxin A, in Some Nobis, E., Scharf, G., Chabicovsky, M., Naturally Occurring Substances: Food Items Knasmuller,€ S. and Kassie, F. (2001) and Constituents, Heterocyclic Aromatic Effects of cruciferous vegetables and Amines and Mycotoxins, WHO/IARC, their constituents on drug metabolizing pp. 489–521. enzymes involved in the bioactivation 28 Grollman, A.P., Shibutani, S., Moriya, M., of DNA-reactive dietary carcinogens. Miller, F., Wu, L., Moll, U., Suzuki, N., Mutation Research, 480–481, 285–297. Fernandes, A., Rosenquist, T., Medverec, 35 Kensler, T.W.,Egner, P.A., Wang, J.B., Zhu, Z., Jakovina, K., Brdar, B., Slade, N., Y.R., Zhang, B.C., Lu, P.X., Chen, J.G., Turesky, R.J., Goodenough, A.K., Rieger, Qian, G.S., Kuang, S.Y., Jackson, P.E., R., Vukelic, M. and Jelakovic, B. 2007 Gange, S.J., Jacobson, L.P., Munoz, A. and Aristolochic acid and the etiology of Groopman, J.D. (2004) Chemoprevention endemic (Balkan) nephropathy. of hepatocellular carcinoma in aﬂatoxin Proceedings of the National Academy of endemic areas. Gastroenterology, 127, Sciences of the United States of America, 104, S310–S318. 12129–12134. 36 Ames, B.N. and Profet, M. (1992) Natures 29 Gelderblom, W.C., Snyman, S.D., Abel, S., pesticides. Natural Toxins, 1,2–3. Lebepe-Mazur, S., Smuts, C.M., Van der 37 Ames, B.N., Profet, M. and Gold, L.S. Westhuizen, L., Marasas, W.F.,Victor, T.C., (1990) Dietary pesticides (99.99% all Knasmuller, S. and Huber, W. (1996) natural). Proceedings of the National Hepatotoxicity and carcinogenicity of Academy of Sciences of the United States of the fumonisins in rats. A review America, 87, 7777–7781. regarding mechanistic implications for 38 IARC (1983) Gyromitrin (acetaldehyde establishing risk in humans. Advances formylmethylhydrazone), in IARC in Experimental Medicine and Biology, Monographs on the Evaluation of 392, 279–296. Carcinogenic Risks of Chemicals to Humans, 30 Marasas, W.F. (2001) Discovery and IARC, pp. 163–170. occurrence of the fumonisins: a historical 39 IARC (1983) Agaritine (L-glutamic acid-5- perspective. Environmental Health [2-(4-hydroxymethyl)-phenylhydrazide]), Perspectives, 109 (Suppl. 2), 239–243. in IARC Monographs on the Evaluation of 31 IARC (2002) Fumonisin B1, in Some Carcinogenic Risks of Chemicals to Humans, Traditional Herbal Medicines, Some IARC, pp. 63–69. References j55

40 WHO/IARC (1993) Caffeic acid, in IARC studies. International Journal of Cancer, Monographs on the Evaluation of 121, 2246–2253. Carcinogenic Risks to Humans, WHO/ 45 Huber, W.W. and Parzefall, W. (2005) IARC, pp. 115–134. Modiﬁcation of N-acetyltransferases and 41 Ehrlich, V., Nersesyan, A., Holzl,€ C., Ferk, glutathione S-transferases by coffee F., Bichler, J. and Knasmuller,€ S. (2006) components: possible relevance for cancer Kanzerogene und gentoxische risk. Methods in Enzymology, 401, 307–341. Pﬂanzeninhaltsstoffe, in Handbuch der 46 IARC (1983) Miscellaneous pesticides, in Lebensmitteltoxikologie (eds H. Dunkelberg, IARC Monographs on the Evaluation of T. Gebel and A. Hartwig), Wiley-VCH Carcinogenic Risks of Chemicals to Humans, Verlag GmbH, Weinheim, Germany, IARC, pp. 33–406. pp. 1915–1963. 47 IARC (1983) Some food additives, feed 42 Ames, B.N. and Gold, L.S. (1998) The additives and naturally occurring causes and prevention of cancer: the role substances, in IARC Monographs on the of environment. Biotherapy (Dordrecht, Evaluation of Carcinogenic Risks of Netherlands), 11, 205–220. Chemicals to Humans, IARC, pp. 33–291. 43 WHO/IARC (1991) Coffee, tea, mate, 48 IARC (1994) Acrolein, crotonaldehyde, methylxanthines and methylglyoxal, in furan, furfural, in Some Industrial IARC Monographs on the Evaluation of Chemicals, WHO/IARC, pp. 373–429. Carcinogenic Risks to Humans, IARC 49 WHO/IARC (1999) D-Limonene, IARC Working Group on the Evaluation of Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, 27 Carcinogenic Risks to Humans, Vol. 73, February to 6 March 1990, WHO/IARC, WHO/IARC, 307–327. pp. 41–197. 50 Gold, L.S., Stern, B.R., Slone, T.H., 44 Lee, J.E., Hunter, D.J., Spiegelman, D., Brown, J.P., Manley, N.B. and Ames, B.N. Adami, H.O., Bernstein, L., van den (1997) Pesticide residues in food: Brandt, P.A., Buring, J.E., Cho, E., English, investigation of disparities in cancer risk D., Folsom, A.R., Freudenheim, J.L., Gile, estimates. Cancer Letters, 117, 195–207. G.G., Giovannucci, E., Horn-Ross, P.L., 51 Doll, R. and Peto, R. (1981) The causes of Leitzmann, M., Marshall, J.R., Mannisto, cancer: quantitative estimates of avoidable S., McCullough, M.L., Miller, A.B., Parker, risks of cancer in the United States today. A.S., Pietinen, P., Rodriguez, C., Rohan, Journal of the National Cancer Institute, 66, T.E., Schatzkin, A., Schouten, L.J., Willett, 1191–1308. W.C., Wolk, A., Zhang, S.M. and Smith- 52 Lutz, W.K. and Schlatter, J. (1992) Warner, S.A. (2007) Intakes of coffee, tea, Chemical carcinogens and overnutrition milk, soda and juice and renal cell cancer in diet-related cancer. Carcinogenesis, 13, in a pooled analysis of 13 prospective 2211–2216. j57

4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview Silvio De Flora, Carlo Bennicelli, Alessandra Battistella, and Maria Bagnasco

4.1 Antimutagenesis and Anticarcinogenesis

Mutagenesis and carcinogenesis are two closely related processes, since carcinogenesis is initiated through a mutation in somatic cells, and several genetic and epigenetic events occur throughout the multiple steps leading to cancer. In addition, mutations in somatic cells have a broader signiﬁcance and impact on human health, since genomic and postgenomic alterations can be detected in a variety of pathological conditions and even in paraphysiological situations that characterize critical periods of life. Antimutagenesis and anticarcinogenesis aim at counteracting these noxious processes. For instance, the perinatal period is characterized by extensive nucleotide changes and alterations of multigene expression in the lung, and the thiol N-acetyl-L-cysteine (NAC), an analogue and precursor of reduced glutathione (GSH) working via a variety of mechanisms [1, 2], inhibits this physiological perturbation when given during pregnancy [3]. Alterations of mitochondrial DNA contribute to aging, and NAC attenuates this molecular damage [4]. Various chronic degenerative diseases may share common risk factors and common protective factors as well as common pathogenetic mechanisms, such as DNA damage, oxidative stress, and chronic inﬂammation [5]. For instance, formation of bulky DNA adducts and oxidative DNA damage may occur in arteries, thus contributing to the pathogenesis of atherosclerosis [6], and in cardiac myocytes, thus contributing to the pathogenesis of degenerative heart diseases [7]. Similar molecular alterations may be involved in the pathogenesis of chronic obstructive pulmonary diseases [7] and in glaucoma [8]. Thus, DNA represents a critical target in those diseases that are the leading causes of death in the population [9].

4.2 Strategies for Preventing Cancer and Other Mutation-Related Diseases

Generally speaking, diseases can be prevented at three operative levels, named primary prevention, secondary prevention, and tertiary prevention. There are some

uncertainties in the literature regarding the use of these terms and their application to cancer prevention. According to Last [10], primary prevention means preventing the occurrence of diseases, secondary prevention means early detection and intervention, preferably before the condition is clinically apparent, and has the aim of reversing, halting, or at least retarding the progress of a condition, and tertiary prevention means minimizing the effect of diseases ... by preventing complications and premature deteriorations. Therefore, primary prevention of cancer is addressed to apparently healthy individuals, irrespective of the fact that these individuals may be genetically more susceptible or heavily exposed to environmental/lifestyle carcinogens. Secondary prevention is addressed to cancer patients at a preclinical or early stage, and tertiary prevention is addressed to cancer patients after therapeutical intervention, with the goal of preventing local recurrences as well as invasion and distant metastases. Figure 4.1 shows an outline of the sequence of these prevention levels, as related to the growth of the neoplastic mass. A traditional approach in the prevention of cancer and other mutation-related diseases, based on risk assessment and risk management, consists in avoiding or minimizing exposures to recognized carcinogenic factors. Avoidance of exposure typically falls in a primary prevention setting. A complementary approach involves

Figure 4.1 Prevention strategies as related to growth patterns of the neoplastic mass and to the steps of the carcinogenesis process. The intervention points A, B, C, D, and E are discussed in the text (Sections 4.4.1–4.4.5, respectively). 4.3 Classification of Mechanisms of Chemopreventive Agents j59 the intake of protective factors modulating the host defense mechanisms by means of dietary and/or pharmacological agents. This approach is known as chemoprevention. Cancer chemoprevention was deﬁned as the administration of agents to prevent induction, to inhibit, or to delay the progression of cancer [11], or as the use of agents to slow the progression of, reverse, or inhibit carcinogenesis at a premalignant stage [12]. Therefore, as shown in Figure 4.1, chemoprevention has two distinctive targets, inhibition of carcinogenesis falling within primary prevention and reversal of carcinogenesis falling within secondary prevention. This distinction is not only academic but also bears practical relevance, since agents that work by inhibiting carcinogenesis are not necessarily effective in reverting carcinogenesis, and vice versa.

4.3 Classification of Mechanisms of Chemopreventive Agents

An optimal chemopreventive agent should possess some general requisites, including (a) efficacy, that is, the ability to lower the risk of developing cancer; (b) safety, that is, the lack of adverse effects (primum non nocere, meaning first, do not harm); (c) low cost, as related to benefits; and (d) practicality of use, regarding availability, stability, convenience of the administration route and schedule [13]. In addition, it is essential, for a potential use in humans, to understand their mechanism of action. Virtually, all multiple pathways leading to genotoxic damage and thereafter to mutation-related diseases can be modulated exogenously through dietary or pharmacological interventions. Several classifications of inhibitors of mutagenesis and carcinogenesis have been proposed during the last 30 years. A distinction between blocking agents, which prevent carcinogens from reacting with critical targets, and suppressing agents, which prevent the evolution of the neoplastic process, was made by Wattenberg [14]. According to Morse and Stoner [15], blocking agents inhibit tumor initiation, while suppressing agents inhibit promotion and progression. Kada et al. [16] distinguished desmutagens, which inactivate mutagens before they can attack DNA, from bioantimutagens, which interfere with fixation of DNA damage. Ramel et al. [17] classified stage-1 inhibitors that act extracellularly, and stage-2 inhibitors that act intracellularly. Other classifications, based on the concept that carcinogenesis can be viewed a continuum of mutagenic and mitogenic events [18], which are the targets for chemopreventive agents, were proposed by Kelloff et al. [19] and Steele and Kelloff [20]. In 1998, De Flora and Ramel [21] formulated a detailed classification of inhibitors of mutagenesis and carcinogenesis, which was periodically updated [22–26]. This classification follows the scholastic view recognizing the multistep nature of the carcinogenesis process. However, it is evident that certain mechanisms are reiterated several times along different phases of the process. As pointed out 20 years ago, the sequence of events ...should not be oversimplified by any rigid classification into rigid steps involved in the initiation–promotion–progression operative scheme [21]. 60j 4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview

4.4 Overview of Mechanisms of Inhibitors of Mutagenesis and Carcinogenesis

Taking into account these premises, we will now take an overview of the mechanisms by which chemopreventive agents can exert antimutagenic and anticarcinogenic effects. Due to space limitations and the availability of a huge literature, this overview will be rather schematic, and only a limited number of references will be provided. We refer to previews of review articles [21–26] for more analytical literature citations. Recent volumes covering in detail the properties of promising cancer chemopreventive agents are available [27].

4.4.1 Extracellular Mechanisms

4.4.1.1 Inhibition of Uptake The ﬁrst defense barriers against mutagens and carcinogens are in the extracellular environment. It is possible to hamper their penetration into the organism and their uptake by cells, and to favor their removal. Inhibition of penetration can be achieved by mechanical means, such as body shielding devices or washing and removal from the skin, oral mucosa and genital mucosa. The topical use of sunscreens provides protection against solar irradiation. According to the IARC evaluation [28], this reduces the risk of sunburns and probably prevents squamous-cell carcinoma of the skin. However, no conclusion can be drawn about prevention of basal-cell carcinoma and cutaneous melanoma, and the extension of intentional sun exposure in subjects using sunscreens may even increase the risk of cutaneous melanoma [28]. Inhibition of penetration can be achieved through pharmacological means, for instance, with drugs aimed at restoring impaired physiological mechanisms (e.g., the mucociliatory escalator), or through immunological means, for instance, with oral vaccines stimulating secretory IgA against carcinogens in the g.i. tract [21–23]. Cellular uptake can be attenuated by short-chain fatty acids (caproate, caprylate), putrescin iodide, aromatic amino acids, acylglycosylsterols, and dietary calcium [21, 23]. At the same time, it is possible to favor removal of carcinogenesis from the organism, for example, by means of dietary ﬁbers [21, 22]. Elimination of mutagens/carcinogens outside the cells can be achieved by means of multidrug resistance proteins, which belong to a series of ATP-binding cassette drug transporter proteins that extrude outside cells, not only cytostatic drugs but also, in general, excess levels of exogenous chemicals [29]. Another strategy is to favor the absorption of protective agents, for instance, by means of vitamin D3 and its analogues (deltanoids) [30].

4.4.1.2 Inhibition of Endogenous Formation Nitrosation is the main reaction leading to the endogenous formation of mutagens/ carcinogens in the organism. There is a variety of inhibitors of nitrosation, such as vitamins (ascorbic acid, a-tocopherol), sulfur compounds (bisulfite, cysteine, GSH, NAC, methionine, sulfamic acid), and phenols (catechol, cinnamic acid, chlorogenic acid, gallic acid, green tea catechins, hydroquinones, phenolic acids, pyrogallol, 4.4 Overview of Mechanisms of Inhibitors of Mutagenesis and Carcinogenesis j61 tannic acid, tannins, thymol, BHA, BHT) [31]. Moreover, several food extracts and beverages inhibit the nitrosation reaction, which attenuates the potential risks to the organism resulting from this process. Another approach for attenuating the endogenous formation of mutagens/carcinogens is targeted to the intestinal microbial flora, for instance, by means of fermented dairy products or by inhibiting conversion of lipids to diacetylglycerol (e.g., by calcium). In particular, due to the role of the intestinal microflora in colon carcinogenesis, probiotic ingredients may inhibit cancer development by competing with other components of the gut microflora, by generating antibacterial agents, and by producing lactic acid and other organic acids lowering the luminal pH [32].

4.4.1.3 Complexation, Dilution, and Deactivation These mechanisms can be accomplished either by physical or mechanical means (e.g., dietary ﬁbers, magnesium hydroxide, and maintenance of physiological pH in body ﬂuids) or by chemical reaction or enzyme-catalyzed reaction (e.g., calcium, oleic acid, C16–C24 unsaturated fatty acids, trapping agents, antioxidants, and vegetables with peroxidase or NADPH-oxidase activities) [21, 22].

4.4.2 Inhibition of Genotoxic Damage and Cancer Initiation

Once mutagens/carcinogens enter into cells, a variety of mechanisms tend to deactivate or block them. The selective efﬁciency of these mechanisms contributes to discriminate target cells from nontarget cells. It is noteworthy that stem cells are preferential targets for mutagens/carcinogens. We have shown that NAC is capable of inhibiting nucleotide alterations induced by 7,12-dimethylbenz(a)anthracene in transformed human mammary epithelial stem cells [33], which may provide a rationale for protecting the stem cells used for therapeutical purposes by means of chemopreventive agents. It has also been suggested that an alternative method of chemoprevention would be to reduce the stem cell pool [34].

4.4.2.1 Modulation of Metabolism An obvious target is to inhibit activation of promutagens/procarcinogens by Phase I enzymes that modify or create functional groups in the molecule. This property is shared by a variety of agents, mostly of natural origin, such as principles of cruciferous plants (phenols, arylalkyl isothiocyanates, indoles, and dithiolethiones), monocyclic monoterpenoids (limonene, menthol, and carveol), flavonoids, nicotinamide, short-chain saturated fatty acids (laurate), hemin, chlorophyllin, disulfiram, diethyldithiocarbamate, b-carotene, retinoids, and NADPH depletors (DHEA, fluasterone) [19, 22, 30, 35]. At the same time, it is feasible to induce Phase I detoxification mechanisms and Phase II conjugation pathways either via the antioxidant/electrophile response element (ARE/EpRE) or the redox- sensitive nuclear E2-related factor 2 (Nrf2) or to accelerate the decomposition of reactive metabolites. Examples of Phase II inducers are oltipraz and other dithiolethiones, natural and synthetic phenols and polyphenols, indoles, sulforaphane and 62j 4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview

0 other isothiocyanates, diterpene esters, riboflavin 5 -phosphate, S-allyl-L-cysteine, allylic sulfide, and butyrate [19, 22, 30, 36, 37]. A distinction has been made between monofunctional Phase II inducers and bifunctional Phase I/Phase II inducers via involvement of the Ah receptor and xenobiotic response element (XRE) [38]. This distinction, based on biochemical methods, has recently been confirmed by multigene expression analyses [39]. The simultaneous inhibition of metabolic activation and stimulation of detoxification would seem to be the optimal mechanistic approach. However, stimulation of metabolic activation, coordinated with detoxification and trapping of reactive metabolites, as produced by bifunctional inducers, is likely to be even more effective [25].

4.4.2.2 Nucleophilicity Due to the electrophilic nature of ultimate mutagens/carcinogens, it is important to trap them by either chemical reaction or enzyme-catalyzed conjugation. Examples of nucleophilic chemopreventive agents are NAC, GSH, cysteine and other thiols, sodium thiosulfate, diallyl sulﬁde, ergothioneine, polyphenols (ﬂavonoids, tannins, ellagic acid), creatinine, hemin, chlorophyllin, and magnesium ions [22, 40]. In addition to trapping the electrophilic metabolites, it is also possible to protect DNA nucleophilic sites by forming DNA adducts that mask binding sites, as it has been shown with ellagic acid, retinoids, and polyamines [21, 22].

4.4.2.3 Antioxidant Mechanisms Oxidative mechanisms contribute substantially to all steps of the carcinogenesis process and to the pathogenesis of several other mutation-related diseases. It is possible to counteract these mechanisms by scavenging reactive oxygen species and by interfering with biochemical pathways involved in oxidative stress. There are a large number and variety of antioxidants, working with different mechanisms. Some examples are provitamins and vitamins (b-carotene and other carotenoids, retinol, ascorbic acid, a-tocopherol), selenium, thioproline, ergothioneine, uric acid, diterpenes, polyphenols, resveratrol, xanthohumol, ﬂavonoids, NAC and other thiols, glycyrrhetinic acid, inducers of antioxidant enzymes (PSK-1, bismute nitrate),

inhibitors of prostaglandin E2 synthesis (eicosapentaenoic acid), nonsteroidal anti-inﬂammatory drugs (NSAIDs) acting as a speciﬁc or selective cyclooxygenase (COX-1 and COX-2) inhibitors (aspirin, sulindac, ibuprofen, indomethacin, piroxicam, celecoxib, etc.) [19, 22, 35].

4.4.2.4 Inhibition of Cell Replication Cell replication represents a crucial process throughout all stages of the carcinogenesis process, and proliferation is a sine qua non condicio for the development of the neoplastic mass. The same strategies that have been proposed as cell-cycle targeted therapies [41] could be used for cancer prevention, especially in a secondary prevention setting. Examples of typical chemopreventive agents endowed with antiproliferative properties are retinoids, glucocorticoids, isothiocyanates, deltanoids, NSAIDs, inhibitors of ornithine decarboxylase activity or of its induction, such as a-diﬂuoromethylornithine (DFMO), methylglyoxal bis(butylamidinohydrazone) 4.4 Overview of Mechanisms of Inhibitors of Mutagenesis and Carcinogenesis j63

(MGBB), inorganic and organic selenium compounds, calcium, ﬂuasterone, inositol hexaphosphate, and tamoxifen [19, 21, 22]. Inhibition of cell replication can be also achieved by promoting the proteasomal degradation of cyclins D1 and E [42].

4.4.2.5 Maintenance of DNA Structure and Modulation of its Metabolism and Repair Certain agents, such as cobaltous chloride, sodium arsenite, magnesium, ellagic acid, oltipraz, indole-3-carbinol, sulforaphane, and inhibitors of topoisomerases I (camptothecin) and II (genistein), have been shown to increase the ﬁdelity of DNA replication and repair [20–22, 30]. Other agents, such as cinnamaldehyde, coumarin, vanillin, umbelliferone, tannic acid, and poly(ADP-ribose) polymerase (PARP) protectors, stimulate DNA repair and/or reversion of DNA damage [21, 22]. Protease inhibitors and para-aminobenzoic acid (PABA) have been shown to inhibit error-prone DNA repair [22]. Folate, methionine, S-adenosyl-L-methionine (SAM), budesonide, and epigallocatechin gallate (EGCG) have been shown to correct hypomethylation. Folate plays an important role in DNA metabolism, being required for the synthesis of dTMP from dUMP [43], and its deﬁciency affects intrauterine growth retardation and weight at birth [44]. Other strategies involve blocking of telomerases or inhibition of their activity, for instance, by EGCG [45] or the epigenetic modulation of DNA, for example, via inhibition of histone deacetylase and modulation of DNA methylation [46].

4.4.2.6 Control of Gene Expression This is a delicate intervention point. In principle, as molecular indicators of safety, chemopreventive agents should not excessively alter per se the physiological baseline of gene expression, in the absence of noxious agents. At the same time, as molecular indicators of efficacy, they should be capable of attenuating the alterations induced by carcinogens. This concept has been evaluated in our laboratory by testing several chemopreventive agents, including oltipraz, phenethyl isothiocyanate (PEITC), 5,6-benzoflavone, indole-3-carbinol, NAC, and their combinations, by using cDNA microarrays for transcriptome analysis [39], antibody microarrays for proteome analysis [47], and micro-RNA microarrays for mirnome analysis (Izzotti et al.,in preparation). From a theoretical point of view, a variety of mechanisms could be exploited to control gene expression. However, one could argue whether they are genuine primary mechanisms or secondary effects. In addition to the aforementioned compounds, other examples of agents that have been shown to inhibit oncogene expression are SAM, retinoids, inhibitors of gene amplification (protease inhibitors), calcium, lovastatin, and limonene [22, 30]. The targeted inactivation of oncogenes, such as myc, ras, and abl, has proved to be successful in the therapy of cancer [48] and may be applied to secondary prevention as well. Oncogene sequences or activity can be inhibited by several means, for instance, by blocking target genes by hybridization of mRNA strands with antisense nucleotides [49], by using proteins that bind oncogene sequences, or by means of farnesyltransferase inhibitors, both synthetic and natural (limonene, perillyl alcohol), designed to prevent the isoprenylation and 64j 4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview

therefore the translocation of oncogenes (ras) to the plasma membrane [20, 50]. These agents have been successfully tested in animal models, also in combination with other drugs, such as the glucocorticoid budesonide [50]. Synthetic nucleotides can be used to control the expression of speciﬁc genes. They can be targeted either to mRNA, thereby inhibiting translation (the antisense strategy) or to speciﬁc DNA sequences where they inhibit translation (the antigene strategy) [51]. Oncogene products can be neutralized by either immunological or pharmacological means. Several strategies have been proposed for delivering deleted or mutated tumor suppressor genes, such as p53 [52] and fhit [53]. Micro-RNAs are further potential targets by using antisense oligonucleotide approaches for inhibiting their function and siRNA-like technologies for their replacement [54].

4.4.3 Inhibition of Tumor Promotion

As shown in Figure 4.1, the clonal expansion of initiated cells to form a benign tumor takes, in humans, years or more often decades. Certain crucial mechanisms involved in tumor initiation are reiterated in tumor promotion. Therefore, we refer to Section 4.4.2 regarding inhibition of DNA damage, inhibition of cell proliferation, scavenging of free radicals, and antioxidant activity.

4.4.3.1 Anti-Inflammatory Activity The last mechanism is interconnected with anti-inﬂammatory activity, for instance, via inhibition of NADPH oxidase (NOX) or of inducible nitric oxide synthase (iNOS) or COX, or lipooxygenase, thus interfering with arachidonic acid metabolism, or of signaling pathways, such as nuclear transcription factor (NF-kB), or via leukotriene receptor antagonism [55–57].

4.4.3.2 Signal Transduction Modulation Coordinately with anti-inﬂammatory activity, signal transduction modulation is also essential during tumor promotion, for instance, through inhibitors of protein kinases, such as mitogen-activated protein kinases (MAPKs), protein kinase C (PKC) and protein kinase B (PKB), transcription factors, such as NF-kB and activator protein-1 (AP-1), b-catenin-mediated signaling, and so on [58]. There is a huge literature on this subject, and the examples are very numerous, including, for instance, EGCG, NAC, genistein, capsaicin, curcumin, resveratrol, gingerol, anthocyanins, and so on [12, 19, 20, 24, 30, 56, 58–60]. Inhibition of proteases is one more approach to counteract tumor promotion [22, 30].

4.4.3.3 Protection of Gap Junctional Intercellular Communications (GJIC) Protection and integrity of gap junctional intercellular communications (GJIC), Cx43, and other connexins are crucial steps in the defense against the promotional growth of the neoplastic mass [34]. Typical examples are provided by b-carotene, canthax- anthine and other carotenoids, vitamin A and retinoids, green tea components, resveratrol, and caffeic acid phenethylene ester [19, 34]. 4.4 Overview of Mechanisms of Inhibitors of Mutagenesis and Carcinogenesis j65

4.4.3.4 Induction of Cell Differentiation Another strategy,viewing the tumors as caricatures of the process of tissue renewal [61], is induction of cell differentiation, for instance, by means of retinoids, deltanoids, calcium, genistein, glucocorticoids, sodium butyrate, hexamethylene bisacetamide, 1-b-D-arabinofuranosyl-cytosine, and 5-azacydine [20, 21, 24, 30, 61]. Due to the established role of vitamin D receptor (VDR) in cell proliferation and differentiation, VDR has been proposed as a target in cancer chemoprevention [62].

4.4.3.5 Modulation of Apoptosis This is an additional delicate intervention point. We recently reviewed almost 2000 studies, available up to late 2004, regarding modulation of apoptosis by 39 putative cancer chemopreventive agents [63]. The ability to stimulate apoptosis in various experimental test systems was shared by a number of agents, including provitamins, vitamins and their synthetic derivatives, such as b-carotene, lycopene, retinol (vitamin A), fenretinide (4-HPR), calciferol (vitamin D) and its derivatives; indoles, such as indole-3-carbinol and diindolylmethane; isothiocyanates, such as benzyl isothiocyanate (BITC) and PEITC; flavonoids, such as genistein and quercetin; alkaloids, such as berberine and capsaicin; phenols, such as curcumin and EGCG; xanthine derivatives, such as caffeine; thioethers, such as allyl sulfide; oligoelements and their derivatives, such as sodium selenite, other inorganic selenium compounds, and organoselenium compounds; glucocorticoids, such as dexamethasone and budesonide; NSAIDs, such as aspirin, sulindac sulfide, sulindac sulfone (exisulind), indomethacin, nimesulide, and celecoxib; selective estrogen modulators (SERMs), such as tamoxifen and raloxifene. On the contrary, the ability to inhibit apoptosis induced by a variety of stimuli was consistently reported with three agents only, including a-tocopherol (in 81 of 98 reviewed studies), ascorbic acid (119/173), and NAC (344/390). Inhibition of apoptosis by chemopreventive agents reflects their ability to counteract certain upstream signals, such as genotoxic damage, redox imbalances, and other forms of cellular stress that trigger apoptosis. Stimulation of apoptosis is a double-edged sword, since it is a protective mechanism in carcinogenesis but may contribute to the pathogenesis of other chronic degenerative diseases [63].

4.4.4 Inhibition of Tumor Progression

Tumor progression is the process that leads to the transformation of a benign tumor into cancer (Figure 4.1). During this relatively short-lasting stage, neoplastic cells acquire malignant properties. Previously discussed mechanisms, such as inhibition of genotoxic effects, antioxidant activity and scavenging of free radicals, inhibition of proteases, and signal transduction modulation, can also counteract the shift of a benign neoplastic mass to cancer.

4.4.4.1 Inhibition of Angiogenesis Neovascularization occurs at different stages of the carcinogenesis process [64] and plays an essential role in the evolution toward malignancy. Prevention of angiogenesis, 66j 4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview

for which the term angioprevention was coined [65], is thus quite an important strategy for both prevention and therapy of cancer. A number of agents, either of pharmacological or dietary origin, are targeted to block angiogenic factors, such as the family of vascular endothelial growth factors (VEGFs), playing an important role both in hemangiogenesis and lymphangiogenesis, basic ﬁbroblast-like growth factor (bFGF), transforming growth factor-b (TGF-b), interleukin-8 (IL-8), and so on. The list of inhibitors of angiogenesis includes, among others, angiostatin, endostatin, talidomide, linomide, retinol and synthetic retinoids such as 4-HPR, thiols such as NAC, ﬂavonoids such as genistein, green tea polyphenols such as EGCG, NSAIDs such as aspirin and celecoxib, protease inhibitors, calciferol, peroxisome proliferation-activated receptor (PPAR) g ligands, steroid antagonists, interferons, cytokines and chemokines, proline analogues, antibiotics, fumagillin, heparinoids, chemother- apeutics, inhibitors of metalloproteinases and of serinoproteinases, PKC inhibitors, and so on [24, 25, 64, 65]. Bevacizumab is an anti-VEGF monoclonal antibody that is now a standard agent in combination with cytotoxic therapy [66]. The largest class of drugs that block angiogenesis are the multitargeted tyrosine kinase inhibitors (TKIs) that target the VEGF receptor (VEGFR) [66].

4.4.4.2 Modulation of the Hormonal Status This approach provides an important strategy for the secondary prevention of hormone-dependent cancers, also including the prevention of second primary tumors, in both females and males. Examples are SERMs, such as tamoxifen, toremifene, raloxifene, arzoxifene, and so on, which are ligands of estrogen receptors (ERa,ERb), aromatase inhibitors, both steroidal (e.g., formestane, exemestane) and nonsteroidal (e.g., aminoglutethimide, fadrozole, anastrazole, letrozole, and vorozole), which block estrogen biosynthesis, selective androgen receptors antagonists (SARAs), such as bicalutamide and flutamide, soy isoflavones, such as genistein, inducers of estradiol 2-hydroxylation, such asindole-3-carbinol, oral contraceptives, 5a-reductase inhibitors, such as finasteride and epristeride, and dopamine antagonists [12, 19, 21, 24, 67–70].

4.4.4.3 Effects on the Immune System Due to the immunosuppressive tumor microenvironment, stimulation of the immune response can be also envisaged as a mechanism for inhibiting tumor progression. Apart from vaccination with tumor-speciﬁc antigens, which is a more suitable approach for therapeutical purposes, certain phytochemicals, vitamins and essential elements, such as polyphenols (e.g., EGCG, curcumin), isothiocyanates (e.g., PEITC), probiotics and prebiotics, lipotropes (choline, folate, methionine, and vitamin B12), a-tocopherol, retinoids, and selenium possess immunomodulating properties [19, 24, 71].

4.4.5 Inhibition of Invasion and Metastasis

Inhibition of invasion and metastasis falls within tertiary prevention and, in general, within the management of the oncologic patient after therapy and in parallel to 4.5 Concluding Remarks j67 rehabilitation. Therefore, in strict sense, it should be outside the boundaries of chemoprevention, which is usually applied at a premalignant stage [12]. Neverthe- less, several mechanisms that are potentially able to attenuate the risk of invasion and metastasis, such as signal transduction modulation, antioxidant activity, inhibition of cell proliferation, induction of cell differentiation, inhibition of neovascularization,oreffects on the immune system, are strictly interconnected with those that can be exploited in primary and/or secondary prevention of cancer, as previously discussed. For instance, immunity to p53 induced by an idiotypic network of anti-p53 antibodies may have protective effects toward tumor metastasis [73]. Other prevention strategies are speciﬁcally tailored to interfere with invasion and metastasis, taking into account the molecular mechanisms leading to these processes. Inhibitors of integrins are targeted to cell-adhesion molecules, with the goal of hampering the escape of transformed cells from the neoplastic mass [25, 65]. A variety of agents inhibit the proteases involved in degradation of the capillary basement membrane of hematic and lymphatic vessels and in modulating the interaction with the extracellular matrix. Among others, they include the family of tissue inhibitors of metalloproteinases (TIMPs), retinoids, protease inhibitors, cathepsin B, NAC, batimastat, marimastat, e-aminocaproic acid, polyphenols, eicosapentaenoic acid, and hydroxamic acid [19, 24, 25, 65, 72, 74]. In addition, an activation of antimetastasis genes could be achieved by enhancing the immunogenicity of tumor cells via insertion of genes encoding MHC-HLA and/or cytokines [75]. Dysfunctions in the tumor microenvironment are crucial in carcinogenesis. Therefore, several molecular targets for protecting the tumor microenvironment have been proposed, including for instance SERMs, targeted to estrogen receptors; curcumin, NAC, silibinin, xanthohumol, deguelin, EGCG, and resveratrol, targeted to Akt and NF-kB; sulforaphane and oltipraz, targeted to Nrf2-Keap1; rofexobib, celecoxib, and EGCG, targeted to COX-2; aspirin and other NSAIDs, targeted to both COX-1 and COX-2; sulforaphane, targeted to histone deacetylases; CDDO-imidazo- lide, targeted to the TGFb pathway; EGCG, resveratrol, apigenin, and sulforaphane, targeted to HIF1a; sulforaphane, EGCG, and fenretinide, targeted to VEGF [72].

4.5 Concluding Remarks

It should be noted that some of the mechanisms mentioned in this article are just of academic interest, at least for the time being. Moreover, several mechanisms are closely related and partially overlapping. It is difﬁcult to discriminate whether a given intervention point actually reﬂects a primary mechanism or a secondary mechanism or an effect. To give an example, an agent having antioxidant activity may modulate several end points, which should not be interpreted as primary mechanisms but rather as a consequence of an upstream modulation. It is also important to note that most inhibitors of mutagenesis and carcinogenesis work via multiple mechanisms of action and have pleiotropic properties, which sometimes include adverse effects. The predominance of protective effects versus adverse effects is variable depending 68j 4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview

on several factors, such as doses and administration routes, sequence of intake of mutagen/carcinogens and chemopreventive agents, biotransformation in the organism, individual susceptibility, and so on. The comprehension of mechanisms by which chemopreventive agents inhibit mutagenesis and carcinogenesis is essential also for establishing a rationale in the combination of different agents, an approach that mimicks the situation of complex mixtures, mainly of dietary origin. Like combined chemotherapy is the optimal choice for the treatment of cancer, cardiovascular diseases, and other major diseases, combined chemoprevention, based on mechanistic considerations, is a promise for the future in the area of chemoprevention.

Acknowledgments

We thank Dr Ilaria Righi for skillful assistance in preparation of the manuscript.

References

1 De Flora, S., Izzotti, A., DAgostini, F. 5 De Flora, S., Izzotti, A., Randerath, K., and Balansky, R.M. (2001) Mechanisms Randerath, E., Bartsch, H., Nair, J., of N-acetylcysteine in the prevention Balansky, R.M., van Schooten, F.J., of DNA damage and cancer, with Degan, P., Fronza, G., Walsh, D. and special reference to smoking-related Lewtas, J. (1996) DNA adducts in chronic end-points. Carcinogenesis, 22, 999–1013. degenerative diseases. Pathogenetic 2 De Flora, S., Izzotti, A., Albini, A., relevance and implications in preventive DAgostini, F., Bagnasco, M. and medicine. Mutation Research, 366,197–238. Balansky, R.M. (2004) Antigenotoxic 6 De Flora, S., Izzotti, A., Walsh, D., and cancer preventive mechanisms of Degan, P., Petrilli, G.L. and Lewtas, J. N-acetyl-L-cysteine, in Cancer (1997) Molecular epidemiology of Chemoprevention, Promising Cancer atherosclerosis. The FASEB Journal, 11, Chemopreventive Agents, Vol. 1 (eds G.J. 1021–1031. Kelloff, E.T. Hawk and C.C. Sigman), The 7 Izzotti, A., Balansky, R.M., Cartiglia, C., Humana Press Inc., Totowa, NJ, pp. 37–67. Camoirano, A., Longobardi, M. and 3 Izzotti, A., Balansky, R.M., Camoirano, A., De Flora, S. (2003) Genomic and Cartiglia, C., Longobardi, M., Tampa, E. transcriptional alterations in mouse fetus and De Flora, S. (2003) Birth related liver after transplacental exposure to genomic and transcriptional changes in cigarette smoke. The FASEB Journal, 17, mouse lung. Modulation by transplacental 1127–1129 (full text available online, April N-acetylcysteine. Mutation Research, 544, 22, 2003). 441–449. 8 Izzotti, A., Sacca, S.M., Cartiglia, C. and 4 Balansky, R.M., Izzotti, A., Scatolini, L., De Flora, S. (2003) Oxidative DNA DAgostini, F. and De Flora, S. (1996) damage in the eye of glaucoma patients. Induction by carcinogens and chemopre- The American Journal of Medicine, 114, vention by N-acetylcysteine of adducts to 638–646. mitochondrial DNA in rat organs. Cancer 9 De Flora, S., Quaglia, A., Bennicelli, C. and Research, 56,1642–1647. Vercelli, M. (2005) The epidemiological References j69

revolution of the 20th century. The FASEB neoplasia in humans with implications for Journal, 19, 892–897. cancer chemopreventive strategy. Cancer 10 Last, J.M. (1986) Scope and methods of Research, 52, 1651–1659. prevention, in Maxcy–Rosenau, Public 19 Kelloff, G.J., Boone, C.W., Steele, V.E., Health and Preventive Medicine (eds J.M. Crowell, J.A., Lubet, R.A., Greenwald, P., Last, J. Chin, J.E. Fielding, A.L. Frank, J.C. Hawk, E.T., Fay, J.R. and Sigman, C.C. Lashoff and R.B. Wallace), Appleton- (1996) Mechanistic considerations in the Century-Crofts, Norwalk, CT, pp. 3–7. evaluation of chemopreventive data, in 11 Sporn, M.B. and Newton, D.L. (1979) Principles of Chemoprevention Chemoprevention of cancer with (eds B.W. Stewart, D. McGregor and retinoids. Federation Proceedings, 38, P. Kleihues), IARC Sciences Publication, 2528–2534. No. 139, International Agency for 12 Kelloff, G.J. (2000) Perspectives on cancer Research on Cancer, Lyon, pp. 203–219. chemoprevention research and drug 20 Steele, V.E. and Kelloff, G.J. (2005) development. Advances in Cancer Research, Development of cancer chemopreventive 78, 199–334. drugs based on mechanistic approaches. 13 De Flora, S., Balansky, R., Scatolini, L., Mutation Research, 591,16–23. Di Marco, C., Gasparini, L., Orlando, M. 21 De Flora, S. and Ramel, C. (1988) and Izzotti, A. (1996) Adducts to nuclear Mechanisms of inhibitors DNA and mitochondrial DNA as of mutagenesis and carcinogenesis. biomarkers in chemoprevention, in Classiﬁcation and overview. Mutation Principles of Chemoprevention Research, 202, 285–306. (eds B.W. Stewart, D. McGregor 22 De Flora, S., Zanacchi, P., Izzotti, A. and P. Kleihues), IARC Sciences and Hayatsu, H. (1991) Mechanisms of Publication, No. 139, International food-borne inhibitors of genotoxicity Agency for Research on Cancer, Lyon, relevant to cancer prevention, in Mutagens pp. 291–301. in Food. Detection and Prevention 14 Wattenberg, L.W. (1981) Inhibitors of (ed. H. Hayatsu), CRC Press, Boca Raton, chemical carcinogens, in Cancer: FL, pp. 157–180. Achievements, Challenges and Prospects for 23 De Flora, S., Izzotti, A. and Bennicelli, C. the 1980s (eds J.H. Burchenal and (1993) Mechanisms of antimutagenesis H.F. Oettgen), Grune and Stratton, and anticarcinogenesis. Role in primary New York, NY, pp. 517–540. prevention, in Antimutagenesis and 15 Morse, M.A. and Stoner, G.D. (1993) Anticarcinogenesis Mechanisms III Cancer chemoprevention: principles and (eds G. Bronzetti, H. Hayatsu, S. prospects. Carcinogenesis, 14, 1737–1746. De Flora, M.D. Waters and D.M. Shankel), 16 Kada, T., Inoue, T. and Namiki, N. (1982) Plenum Press, New York, NY, pp. 1–16. Environmental desmutagens and 24 De Flora, S. (1998) Mechanisms of antimutagens, in Environmental inhibitors of mutagenesis and Mutagenesis and Plant Biology carcinogenesis. Mutation Research, 402, (ed. E.J. Klekowski), Praeger, New York, 151–158. NJ, pp. 137–151. 25 De Flora, S., Izzotti, A., DAgostini, F., 17 Ramel, C., Alekperov, U.K., Ames, B.N., Balansky, R.M., Noonan, D. and Albini, A. Kada, T. and Wattenberg, L.W. (1986) (2001) Multiple points of intervention in Inhibitors of mutagenesis and their the prevention of cancer and other relevance to carcinogenesis. Mutation mutation related diseases. Mutation Research, 168,47–65. Research, 480–481,9–22. 18 Boone, C.W., Kelloff, G.J. and Steele, V.E. 26 De Flora, S. and Ferguson, L.R. (2005) (1992) Natural history of intraepithelial Overview of mechanisms of cancer 70j 4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview

chemopreventive agents. Mutation Environmental and Molecular Mutagenesis, Research, 591,8–15. 151, 145–182. 27 Kellof, J., Hawk, E.T. and Sigman, C.C. 36 Pool-Zobel, B., Veeriah, S. and (eds) (2004) Promising Cancer Bohmer,€ F.-D. (2005) Modulation of Chemopreventive Agents, Vol. 1, Humana xenobiotic metabolising enzymes by Press, Totowa, NJ. anticarcinogen-focus on glutathione 28 IARC Handbooks of Cancer Prevention S-transferase and their role as targets of (2001) Sunscreens, International Agency for dietary chemoprevention in colorectal Research on Cancer, Vol. 5, World Health carcinogenesis. Mutation Research, 591, Organization, Lyon, France. 74–92. 29 Ferguson, L.R. and De Flora, S. (2005) 37 Yu, X. and Kensler, T. (2005) Nrf2 as a Multiple drug resistance, antimutagenesis target for cancer chemoprevention. and anticarcinogenesis. Mutation Research, Mutation Research, 591,93–102. 591,24–33. 38 Talalay, P., Fahey, J.W., Holtzclaw, W.D., 30 Greenwald, P., Kelloff, G.J., Boone, C.W. Prestera, T. and Zhang, Y. (1995) and McDonald, S.S. (1995) Genetic and Chemoprotection against cancer by cellular changes in colorectal cancer: phase 2 enzyme induction. Toxicology proposed targets of chemopreventive Letters, 82–83, 173–179. agents. Cancer Epidemiology, Biomarkers & 39 Izzotti, A., Bagnasco, M., Cartiglia, C., Prevention, 4, 691–702. Longobardi, M., Camoirano, A., Tampa, E., 31 Bartsch, H., Oshima, H. and Pignatelli, B. Lubet, R.A. and De Flora, S. (2005) (1988) Inhibitors of endogenous Modulation of multigene expression nitrosation. Mechanisms and implications and proteome proﬁles by in human cancer prevention. Mutation chemopreventive agents. Mutation Research, 202, 307–324. Research, 591, 212–223. 32 Commane, D., Hughes, R., Shortt, C. and 40 Arimoto, S., Kan-yama, K., Rai, H. and Rowland, I. (2005) The potential Hayatsu, H. (1995) Inhibitory effect of mechanisms involved in the anti- hemin, clorophyllin and related pyrrole carcinogenic action of probiotics. Mutation pigments on the mutagenicity of benzo[a] Research, 591, 276–289. pyrene and its metabolites. Mutation 33 De Flora, S., Scarfì, S., Izzotti, A., Research, 345, 127–135. DAgostini, F., Chang, C.-C., 41 Swanton, C. (2004) Cell-cycle targeted Bagnasco, M., De Flora, A. and Trosko, J.E. therapies. The Lancet Oncology, 5,27–36. (2006) Induction by 7,12-dimethylbenz(a) 42 Dragnev, K.H., Pitha-Rowe, I., Ma, Y., anthracene of molecular and biochemical Petty, W.J., Sekula, D., Murphy, B., alterations in transformed human Rendi, M., Suh, N., Desai, N.B., mammary epithelial stem cells, and Sporn, M.B., Freemantle, S.J. and protection by N-acetylcysteine. Dmitrovsky, E. (2004) Speciﬁc International Journal of Oncology, 29, chemopreventive agents trigger 521–529. proteasomal degradation of G1 cyclins: 34 Trosko, J.E., Chang, C.-C., Upham, B.L. and implications for combination therapy. Tai, M.-H. (2005) The role of human adult Clinical Cancer Research, 10, stem cells and cell–cell communication 2570–2577. in cancer chemoprevention and 43 Fenech, M. (2001) The role of folic acid and chemotherapy strategies. Mutation vitamin B12 in genomic stability of human Research, 591,187–197. cells. Mutation Research, 475,57–67. 35 Hartman, P.E. and Shankel, D.M. (1990) 44 Sram, R.J., Binkova, B., Lnenickova, Z., Antimutagens and anticarcinogens: a Solansky, I. and Dejmek, J. (2005) The survey of putative interceptor molecules. impact of plasma folate levels of mothers References j71

and newborns on intrauterine growth and cancer prevention. Mutation Research, retardation and birth weight. Mutation 591, 103–109. Research, 591, 302–310. 54 Esau, C.C. and Monia, B.P. (2007) 45 Naasani, I., Oh-Hashi, F., Oh-Hara, T., Therapeutic potential for microRNAs. Feng, W.Y., Johnston, J., Chan, K. and Advanced Drug Delivery Reviews, 59, Tsuruo, T. (2003) Blocking telomerase by 101–114. dietary polyphenols is a major mechanism 55 Chen, T., Nines, R.G., Peschke, S.M., for limiting the growth of human cancer Kresty, L.A. and Stoner, G.D. (2004) cells in vitro and in vivo. Cancer Research, Chemopreventive effects of a selective 63, 824–830. nitric oxide synthase inhibitor on 46 Belinsky, S.A., Klinge, D.M., Stidley, C.A., carcinogen-induced rat esophageal Issa, J.P., Herman, J.G., March, T.H. and tumorigenesis. Cancer Research, 64, Baylin, S.B. (2003) Inhibition of DNA 3714–3717. methylation and histone deacetylation 56 Oshima, H., Tazawa, H., Sylla, B.S. and prevents murine lung cancer. Cancer Sawa, T. (2005) Prevention of human Research, 63, 7089–7093. cancer by modulation of chronic 47 Izzotti, A., Bagnasco, M., Cartiglia, C., inflammatory processes. Mutation Longobardi, M., Balansky, R.M., Merello, Research, 591, 110–122. A., Lubet, R.A. and De Flora, S. (2005) 57 Steele, V.E., Holmes, C.A., Hawk, E.T., Chemoprevention of genome, Kopelovich, L., Lubet, R.A., Crowell, J.A., transcriptome, and proteome alterations Sigman, C.C. and Kelloff, G.J. (1999) induced by cigarette smoke in rat lung. Lipoxygenase inhibitors as potential European Journal of Cancer, 41, cancer chemopreventives. Cancer 1864–1874. Epidemiology, Biomarkers & Prevention, 8, 48 Felsher, D.W. (2003) Cancer revoked: 467–483. oncogenes as therapeutic targets. Nature 58 Kundu, J.K. and Surh, Y.-J.(2005) Breaking Reviews. Cancer, 3, 375–380. the relay in deregulated cellular signal 49 Jansen, B. and Zangemeister-Wittke, U. transduction as a rationale for (2002) Antisense therapy for cancer-the chemoprevention with anti–inflammatory time of truth. The Lancet Oncology, 3, phytochemicals. Mutation Research, 591, 672–683. 123–146. 50 Alyaqoub, F.S., Tao, L., Kramer, P.M., 59 Surh, Y.-J.(2003) Cancer chemoprevention Steele, V.E., Lubet, R.A., Gunning, W.T. with dietary phytochemicals. Nature and Pereira, M.A. (2007) Prevention of Reviews. Cancer, 3, 768–780. mouse lung tumors and modulation of 60 Weinstein, I.B. (1988) Strategies for DNA methylation by combined treatment inhibiting multistage carcinogenesis with budesonide and R115777 (Zarnestra based on signal transduction pathways. MT). Carcinogenesis, 28, 124–129. Mutation Research, 202, 413–420. 51 Helene, C. (1991) Rational design of 61 Pierce, G.B. and Speers, W.C. (1988) sequence-specific oncogene inhibitors Tumors as caricatures of the process of based on antisense and antigene tissue renewal: prospects for therapy by oligonucleotides. European Journal of directing differentiation. Cancer Research, Cancer, 27, 1466–1471. 48, 1996–2004. 52 Vogelstein, B. and Kinzler, K.W. (2001) 62 Thorne, J. and Campbell, M.J. (2008) The Achilles heel of cancer? Nature, 412, vitamin D receptor in cancer. The 865–866. Proceedings of the Nutrition Society, 67, 53 Zanesi, N., Pekarsky, Y. and Croce, C.M. 115–127. (2005) A mouse model of the fragile gene 63 DAgostini, F., Izzotti, A., Balansky, R.M., FHIT: from carcinogenesis to gene therapy Bennicelli, C. and De Flora, S. (2005) 72j 4 Mechanisms of Chemoprevention, Antimutagenesis, and Anticarcinogenesis: An Overview

Modulation of apoptosis by cancer. Clinical Cancer Research, 7, chemopreventive agents. Mutation 2620–2623. Research, 591, 173–186. 71 Ferguson, L.R. and Philpott, M. (2007) 64 Folkman, J. (2000) Incipient angiogenesis. Cancer prevention by dietary bioactive Journal of the National Cancer Institute, 92, components that target the immune 94–99. response. Current Cancer Drug Targets, 7, 65 Tosetti, F., Ferrari, N., De Flora, S. and 459–464. Albini, A. (2002) Angioprevention: 72 Albini, A. and Sporn, M.B. (2007) The angiogenesis is a common and key target tumour microenvironment as a target for for cancer chemopreventive agents. The chemoprevention. Nature Reviews. Cancer, FASEB Journal, 16,2–14. 7, 139–147. 66 Cabebe, E. and Wakelee, H. (2007) Role of 73 Erez-Alon, N., Herkel, J., Wolkowicz, R., anti-angiogenesis agents in treating Ruiz, P.J., Waisman, A., Rotter, V. and NSCLC: focus on bevacizumab abd Cohen, I.R. (1998) Immunity to p53 VEGFR tyrosine kinase inhibitor. Current induced by an idiotypic network of anti-p53 Treatment Options in Oncology, 8,15–27. antibodies: generation of sequence- 67 Lewis, J.S. and Jordan, V.C. (2005) specific anti-DNA antibodies and Selective estrogen modulators (SERMs): protection from tumor metastasis. Cancer mechanisms of anticarcinogenesis and Research, 58, 5447–5452. drug resistance. Mutation Research, 591, 74 Stetler-Stevenson, W.G., Hewitt, R. and 247–263. Corcoran, M. (1996) Matrix 68 Raghow, S., Kuliyev, E., Steakley, M., metalloproteinases and tumor invasion: Greenberg, N. and Steiner, M.S. (2000) from correlation and causality to the clinic. Efficacious chemoprevention of primary Seminars in Cancer Biology, 7, 147–154. prostate cancer by flutamide in an 75 Lollini, P.L., De Giovanni, C., Landuzzi, L., autochthonous transgenic model. Cancer Nicoletti, G., Frabetti, F., Cavallo, F., Research, 60, 4093–4097. Giovarelli, M., Forni, G., Modica, A., 69 Lu, L.-J.W., Anderson, K.E., Grady, J.J. and Modesti, A. et al. (1995) Transduction of Nagamani, M. (2000) Decreased ovarian genes coding for a histocompatibility hormones during a soya diet: implications (MHC) antigen and for its physiological for breast cancer prevention. Cancer inducer interferon-gamma in the same Research, 60, 4112–4121. cell: efficient MHC expression and 70 Buzdar, A. and Howell, A. (2001) Advances inhibition of tumor and metastasis in aromatase inhibition: clinical efficacy growth. Human Gene Therapy, 6, and tolerability in the treatment of breast 743–752. j73

5 Antioxidants and Cancer: Fact and Fiction Andrew R. Collins and Alain Favier

5.1 Introduction

It is a commonplace that fruits and vegetables are good for us – and that certain sectors of the population (such as children), or even certain nations, are reluctant to eat them. One of the most consistent and general findings to emerge from epidemiological studies of nutrition and human disease is that a high consumption of fruits and vegetables is associated with a lower incidence of chronic degenerative diseases, notably, vascular disease and various cancers. Many national and international health agencies are recommending that we eat five or more servings of these foods each day – a modest enough goal for affluent European and American middle classes, but probably unrealistic for the poorer members of society. In any case, the message is not entirely convincing, since we still do not know why fruits and vegetables protect us against cancer. In this chapter, we discuss the epidemiological evidence from case-control and prospective studies that fruits and vegetables are protective; the major hypothesis, popularly regarded as fact, that antioxidants in these foods are responsible for their health-giving properties; attempts to test this hypothesis by intervention studies; and possible alternative mechanisms by which fruits and vegetables might be protective.

5.2 Fruits and Vegetables: the Evidence

For many years, a considerable body of evidence from epidemiological studies of different kinds has suggested that the consumption of fruits and vegetables protects against various cancers. The most comprehensive survey is that published by the World Cancer Research Fund/American Institute for Cancer Research [1]. Since the ﬁrst survey 10 years ago [2], the evidence from cohort studies has weakened, so that now the evidence for protection is probable rather than convincing. Thus, fruits

and nonstarchy vegetables probably protect against cancers of the stomach, esophagus, larynx, pharynx, and mouth (and fruits also against cancer of the lung); allium vegetables probably protect against stomach cancer; and garlic in particular is probably protective against colorectal cancer.

5.3 Oxidative Damage and Antioxidants

It seems that all aerobic organisms are inevitably exposed to reactive forms of oxygen – and to the damage that the reactive oxygen species (ROS) can cause to biological molecules, including nucleic acids, lipids, and proteins. A small fraction of the oxygen passing through the respiratory chain in the mitochondria is released in . the form of the superoxide radical O2 . . eaq þ O2 ! O2 ðaddition of electrons to O2 during respirationÞ:

This is not particularly reactive, but it is converted by the enzyme superoxide

dismutase (SOD) to H2O2. SOD contains a transition metal at the active site. 2 þ . þ Cu þ O2 ! Cu þ O2

þ . þ 2 þ Cu þ O2 þ 2H ! Cu þ H2O2 ðreaction of superoxide dismutaseÞ

H2O2 in turn, via a reaction catalyzed by iron or copper, can lead to the production . of the hydroxyl radical OH.

2 þ . 3 þ H2O2 þ Fe ! OH þ Fe þ OH ðFenton reactionÞ: This is capable of oxidizing biological molecules including proteins, lipids, and nucleic acids. Most of this damage, however, is avoided; antioxidant defenses have evolved that remove the reactive species. SOD eliminates superoxide; catalase breaks

down H2O2; and the abundance of readily oxidized sulphydryl groups, particularly in the tripeptide glutathione (GSH), protects other molecules from damage. GSH is oxidized to dimer (GSSG) but is then regenerated by NADPH:

Inevitably, however, some oxidation of important biomolecules does occur, and it can be measured in normal healthy cells – most commonly, perhaps, as the oxidized base, 8-oxo-7,8-dihydroguanine (8-oxoGua) (see discussion below). 5.4 Large-Scale Intervention Studies with Antioxidants j75

In a series of papers published in the 1980s and 1990s, Ames and colleagues drew attention to the likely contribution of endogenous oxidation to human disease and aging [3,4]. DNA damage is the initiating event in carcinogenesis, and 8-oxoGua is a potentially mutagenic lesion, as it can mispair with adenine in replication. Ames et al. also stressed the likely importance of micronutrients such as vitamin C, vitamin E, carotenoids, and folic acid in protecting against damaging oxidation [4,5]. Evidence suggesting that carotenoids in particular may protect against cancer mounted. Peto et al. described a tendency in many epidemiological studies, for some human cancer risks to be inversely associated with blood retinol, and with blood or dietary b-carotene [6] and made a strong case for controlled intervention trials to determine whether b-carotene really does protect against cancer. How do we evaluate the antioxidant potential of different plant foods (or components thereof)? There are a variety of assays, based on radical quenching, chemical reducing activity, or protection of biological molecules (lipids or DNA) against oxidation. The ferric reducing activity of plasma (FRAP) assay, ﬁrst developed to assess antioxidant status in blood, has shown itself to be especially useful for comparing extracts of plant foods. According to the results obtained by Halvorsen et al. [7] in a survey of over a hundred fruits, berries, vegetables, pulses, nuts, grains, and so on, the FRAP values vary over orders of magnitude. Among the highest scoring foods are walnut, pomegranate, rose hip, bilberry, ginger, and capsicum. However, there is no guarantee that these foods will be effective in boosting antioxidant status in the body. Bioavailability is a crucial factor, as is the metabolism of the micronutrients once they have been taken up. Some micronutrients, such as vitamin C, are readily absorbed (and also rapidly cleared from the body). Flavonoids such as anthocyanidins are very poorly absorbed. Carotenoids, being lipid soluble, are absorbed to a greater extent from food if it is consumed with oil. Some ﬁber-rich vegetables may not release intracellular compounds before reaching the colon, and metabolism by gut bacteria may affect uptake, too.

5.4 Large-Scale Intervention Studies with Antioxidants

Table 5.1 summarizes the large-scale antioxidant trials carried out in the past two decades, in which clinical outcomes were assessed.

5.4.1 The Linxian Study

Linxian is a province of China with a high incidence of gastric cancer. A supplementation trial was carried out on almost 30 000 subjects aged 40–69 for a period of 5 years. Randomized into subgroups, they received a placebo, or various combinations of retinol, zinc, riboﬂavin, niacin, vitamin C, molybdenum, b-carotene, vitamin E, and selenium. Disease incidence was monitored, and there was a statistically Table 5.1 Summary of Large-Scale Antioxidant Intervention Trials.

Supplement Period of Trial Subjects (daily dose) intervention Outcome Reference 76 j nixdnsadCne:Fc n Fiction and Fact Cancer: and Antioxidants 5 Linxian, China 30 000, aged 40–69 Various combinations 5 years Significant decrease [8] of retinol (5000 IU), in cancer mortality riboflavin (3.2 mg), among those niacin (40 mg), receiving b-carotene, vitamin C (120 mg), vitamin E, and Se vitamin E (30 mg), b-carotene (15 mg), Zn (22.5 mg), Mo (30 mg), Se (50 mg) Alpha- tocopherol, 29 000 male smo- Four groups: a-to- Up to 8 years 18% increase (signif- [9] Beta- carotene kers, aged 50–69 copherol (50 mg); icant) in incidence of Cancer Prevention b-carotene (20 mg); lung cancer in those Study, Finland a-tocopherol þ taking b-carotene b-carotene; placebo CARET trial, USA 18 000 smokers, Two groups: b- 4 years (average) Significantly higher [11] former smokers, and carotene (30 mg) þ incidence of lung asbestos workers retinol (25 000 IU); cancer (þ28%) in placebo supplemented group Physicians 22 000 healthy male Two groups: b-caro- 12 years No difference in inci- [12] Health Study, USA physicians, 90% tene (25 mg); placebo dence of lung cancer nonsmokers or mortality between the two groups MRC/BHF Heart 20 000 patients with Two groups: vitamin 5 years No significant effects [13] Protection Study, UK coronary or other C (250 mg), vitamin E on cancer incidence occlusive arterial (600 mg), b-carotene or any other health disease, or diabetes (20 mg); placebo outcome measured SU.VI.MAX Study, France 13 000 men and Two groups: vitamin 7.5 years Significant reduction [14] women C (120 mg), vitamin E in cancer (31%) in (30 mg), b-carotene men receiving sup- (6 mg), Se (100 mg), plement; no effect in Zn (20 mg); placebo women 5.4 Large-Scale Intervention Studies with Antioxidants j77 significant, though modest, decrease in cancer mortality (and incidence) in the group receiving b-carotene, vitamin E, and selenium [8].

5.4.2 The Alpha-Tocopherol Beta-Carotene Trial in Finland

In this test of the effectiveness of a-tocopherol and b-carotene in preventing lung cancer, about 30 000 50–69-year-old male smokers in Finland were assigned to four groups and received b-carotene, vitamin E, b-carotene and vitamin E in combination, or a placebo. Supplementation lasted for up to 8 years. The main outcome of the study was a completely unexpected 18% increase in lung cancer incidence in those taking b-carotene, with or without vitamin E, relative to the placebo group [9]. There were no beneﬁcial effects of a-tocopherol or b-carotene on recurrence or progression of angina pectoris or subsequent major coronary events [10]. This result has so far not been satisfactorily explained, though it has been pointed out that the doses of antioxidants were high compared to a natural intake from food and were given in isolation, rather than in balance with other micronutrients; and that those diagnosed with lung cancer by the end of the study must have suffered the initial stages of carcinogenesis before the study started, and so this was not an ideal test of the cancer preventive ability of antioxidants.

5.4.3 The Beta-Carotene and Retinol Efficiency Trial in the United States

At about the same time as the trial in Finland, two studies were in progress in the United States. One was the Beta-Carotene and Retinol Efﬁciency Trial (CARET) trial involving 4060 men exposed to asbestos and over 14 000 smokers and ex-smokers (male and female), representing a group at high risk of lung cancer. They were supplemented with b-carotene plus retinyl palmitate, or with placebo. The study was stopped 21 months early when an interim analysis – brought forward a year in the light of the Alpha-Tocopherol Beta-Carotene (ATBC) results – showed accumulating evidence of a higher incidence of lung cancer in the active intervention group. The relative risk was 1.28, compared to that of the placebo group [11]. A similar caveat is attached to this study as to the ATBC study – that the subjects were already part way along the road to lung cancer. There is no way round this dilemma; because carcinogenesis is such a long process, very long-term supplementation trials, starting with young adults, or even children, would be necessary to check effects of antioxidants on initiation of the process.

5.4.4 The US Physicians Health Study

22 000 male physicians (mostly nonsmokers) were given b-carotene or placebo for 12.5 years: in contrast to the trials involving smokers, there were no signiﬁcant effects on disease risks, either beneﬁcial or harmful [12]. 78j 5 Antioxidants and Cancer: Fact and Fiction

5.4.5 The MRC/BHF Heart Protection Study, United Kingdom

In this more recent trial, 21 000 patients with cardiovascular disease or diabetes were given vitamin C, vitamin E, b-carotene, or placebo. Over a 5-year period, there was no signiﬁcant reduction in mortality from (or incidence of) any vascular disease, cancer, or other major outcome [13].

5.4.6 The SU.VI.MAX Study

This French trial was started in 1994 and has recruited 13 000 healthy subjects to receive a mixture of b-carotene, vitamin C, vitamin E, selenium, and zinc, or a placebo. The final results show a significant decrease in incidence of cancers overall in men but not in women [14]. No effect has been found on cardiovascular disease. The positive effect in men occurs only in those who start with a low biological status for vitamin C or b-carotene. The proposed explanation of the lack of effect in women is that their antioxidant intake (and concentration in the body) tends to be higher. There were interesting findings in relation to specific cancer types. Thus, supplementation of women – typically having an adequate dietary antioxidant intake – was associated with an increase in skin cancer incidence [15]. Men with normal levels of prostate-specific antigen (PSA) at the start show a significantly reduced prostate cancer incidence if supplemented, while those with an elevated blood PSA level have a higher incidence of prostate cancer (of borderline statistical significance) [16].

5.4.7 Two Meta-Analyses

By the early years of this century, a considerable number of intervention studies had taken place, varying in size, with different (combinations of)antioxidants, over a range ofdoses.Meta-analyses havebeenperformed,takingintoaccountfactorsinthedesign of each study to give appropriate weighting to the results. Nineteen clinical trials with vitamin E (with or without other micronutrients), involving almost 136 000 participants, were analyzed by Miller et al. [17]. They report a statistically significant positive associationbetweenthedoseofvitaminEandall-causemortality,theriskbeinggreater at doses above 150 IU/day; there was no significant decrease in risk at lower doses. Bjelakovic et al. [18] included trials with vitamin C, vitamin E, vitamin A, b-carotene, and selenium in their meta-analysis that covered nearly 233 000 subjects in 68 trials. Studies were assessed for their methodological quality: criteria included adequate randomization procedures, blinding, and follow-up. Well-designed and executed studies were regarded as having a low risk of bias; they accounted for over 180 000 subjects. They showed a significant increase in mortality in the groups taking supplements (though there was no effect of selenium or vitamin C). The studies defined as high bias-risk trials showed no significant effect of antioxidant supplements, good or bad. The message seems clear: antioxidants taken 5.5 Using Surrogate Biomarkers for Disease Risk j79 as supplements are not effective and may even be harmful. It is, however, important to stress the qualification of Bjelakovic et al. that because [they] examined only the influence of synthetic antioxidants, [these] findings should not be translated to potential effects of fruits and vegetables. In spite of the evidence from these trials, nutritional scientists, epidemiologists, and cancer specialists continue to believe that antioxidants must be beneficial – because a plausible mechanism exists, namely, the scavenging or quenching of reactive oxygen species. But it is generally realized that long-term supplementation trials with disease incidence as the outcome are no longer feasible, if only because of the ethical concerns, and what is needed is more information on the basic mechanisms of action of antioxidants in the body.

5.5 Using Surrogate Biomarkers for Disease Risk

If, instead of relying on actual occurrence of disease, we can identify markers of disease risk or early stages of the disease that are measurable in easily obtained samples (normally blood or urine), then we can carry out experimental interventions on small groups of volunteers over short periods and readily obtain quantitative results. This approach is obviously attractive – particularly, perhaps, because of the ease with which it yields numbers for feeding into statistical programs. But there are some important considerations: . The biomarkers should be biologically relevant; sufﬁciently variable from one person to the next, but not too variable at the intra-individual level; precise (with low experimental variability); and accurate (able to give an estimate close to the true value for whatever is being measured). . Normal epidemiological rules should be followed, such as including a placebo group and carrying out studies blind (where, ideally, neither the experimenter making the measurements nor the subjects know who is receiving which treatment). . To avoid obtaining false-negative results, it is important to estimate the power of a planned study to detect an expected difference, given the precision of the assay method, and the number of participants. . Results should be analyzed with the statistical rigor of conventional epidemiology.

5.5.1 Oxidation of Bases in DNA

When selecting a biomarker to examine effects of antioxidants on cancer risk, oxidized bases in DNA are an obvious choice, in view of the link between DNA damage and cancer, the ability of antioxidants – at least in vitro – to prevent DNA oxidation, and also because they are relatively easy to measure. They are not merely a 80j 5 Antioxidants and Cancer: Fact and Fiction

surrogate marker of cancer risk, however; they can be seen as an indicator of the overall redox state of the organism and can provide a useful independent marker in studies of other chronic diseases, for example, vascular disease or diabetes. Histori- cally, the method of choice for characterizing products of free radical attack on DNA was gas chromatography/mass spectrometry (GC–MS), since it allows the unambig- uous identification of these products as well as quantitative measurement. Thus, many oxidized species derived from purines and pyrimidines have been identified [19]. Accuracy of measurement is assured by including an internal standard labeled with a heavy isotope (so that it is separated by mass from the unlabeled entity). Generally, the DNA is chemically hydrolyzed to bases and then derivatized to give volatile products for the gas chromatography. Naturally, when interest shifted toward the background level of oxidation in DNA from cultured cells, peripheral white blood cells, tumor, or normal tissue, GC–MS was applied. High-performance liquid chromatography (HPLC) can also identify certain oxidation products [20]; nucleosides are detected with higher sensitivity than are bases, and so the DNA is hydrolyzed with a combination of enzymes that leaves sugars attached to bases. 8-Oxo-7,8-dihydro-20-deoxyguanosine (8-oxodGuo) is most commonly measured, using electrochemical detection (ECD), either coulometric or amperometric, the former being potentially more sensitive. Calibration is against a standard solution of the oxidized nucleoside. Recently, HPLC has been combined with tandem mass spectrometry (HPLC–MS/MS), giving the advantage of unequiv- ocal identification of oxidized nucleosides and the possible use of an internal heavy isotope-labeled standard [21]. A quite different approach has been adopted that uses lesion-specificrepair endonucleases from bacteria to cut the DNA at the sites of damage. Formamido- pyrimidine DNA glycosylase (FPG) is effective against 8-oxoGua (but it also detects formamidopyrimidines (FaPy) derived from damaged purines). The DNA strand breaks are measured using one of the three independent techniques: alkaline unwinding, alkaline elution, or the comet assay. The first of these depends on the unwinding of DNA strands from the starting point of a single-strand break (SSB); the extent of unwinding is assessed, after neutralization and shearing to small fragments, by hydroxyapatite chromatography that separates single- and double- stranded DNA fragments [22]. In alkaline elution, the ability of (denatured) DNA to pass through a filter is monitored; the shorter the DNA – that is, the more frequent the breaks – the faster it elutes (the usual analogy being with the escape of spaghetti from a collander) [23]. For the comet assay [24], cells are embedded in agarose on a microscope slide and lyzed; the DNA remains intact and essentially protein-free but still arranged as supercoiled loops. Breaks release the supercoiling. On electrophoresis (normally at high pH), the relaxed loops are able to escape and extend toward the anode, and images resembling comets are visualized by fluorescence microscopy. The percentage of DNA in the tail indicates the frequency of breaks. Adherents of each of these approaches have their own favorite ways of expressing results – for example, as fmol of 8-oxoGua per microgram of DNA, or 8-oxodGuo per 106 base pairs, or FPG-sensitive sites per 109 Da. Results with the different methods 5.5 Using Surrogate Biomarkers for Disease Risk j81

Table 5.2 A Selection of Typical Published Values for 8-OxoGua (or the Equivalent 8-Oxodguo) in Human Cells, Measured by Different Methods.

Method Material 8-OxoGua/106 Gua Reference

GC–MS Bronchial epithelial cells 575 [51] GC–MS Lymphoblastoid cell line 100 [52] HPLC–ECD Mononucleocytes 2.4 [53] HPLC–ECD Normal lymphocytes 4.2 [33] þ HPLC–ECD Normal CD4 T cells 40 [54] HPLC–ECD Mononucleocytes 4.9 [55] HPLC–ECD Hemodialysis patients lymphocytes 23 [56] HPLC–ECD Normal leukocytes 3.1 [57] HPLC–ECD Lymphocytes, bronchoscopy patients 8.4 [58] HPLC–ECD Leukocytes, healthy middle-aged 4.8 [59] subjects LC-MS Lung cancer cell lines 10 [60] LC-MS Prostate cancer cell lines 10 [61] FPG/comet assay Normal lymphocytes 0.34 [33] FPG/HPLC–ECD Cultured cells 0.3 [62] FPG/alkaline elution HeLa cells 0.4 [63] were seldom compared. However, by the mid-1990s, it became clear that there were serious discrepancies in estimates of background levels of oxidized bases in human cells. Measuring the same 36 human lymphocyte samples in one laboratory by HPLC–ECD and with the comet assay, for example, gave results that differed by an order of magnitude [25]. A far greater range of values is seen in the literature overall (Table 5.2). Expressing results in common units, 8-oxoGua (or 8-oxodGua) per 106 guanines, allows comparison with reported levels of other DNA adducts and gives a reasonable perspective on the likely biological significance of oxidative damage. Typically, GC–MS has given figures in the hundreds of guanines oxidized per million; HPLC estimates are around 3–40; and the enzymic approach finds less than 1. Though few recognized it at that time, it is now obvious that there is a tendency for further oxidation of DNA to occur during preparation of samples for chromatographic analysis. There are multiple steps of centrifugation, vortex mixing, enzymic hydrolysis, and – in the case of GC–MS – derivatization for a prolonged period at high temperature. A small amount of oxidation of Gua in terms of parts per million could completely mask a low background concentration. This possibility was critically examined by Ravanat et al. [26] who showed a clear relationship between time and temperature of derivatization and amount of 8-oxoGua measured by GC–MS. If unaltered Gua was removed before the derivatization step, the yield of 8-oxoGua was similar to that obtained by HPLC–ECD. Attempts have been made to limit the amount of oxidation by including antioxidants and chelating agents . (to remove transition metals that might catalyze OH production) in solutions used in sample preparation, and background levels of 8-oxoGua less than one per million have been reported [27]. 82j 5 Antioxidants and Cancer: Fact and Fiction

A meeting to discuss the problems in measuring oxidative DNA damage took place in Aberdeen in January 1997 [28]. The European Standards Committee on Oxidative DNA Damage (ESCODD) was formed after this meeting, with the aim of illuminating the problems by systematically comparing analytical methods in different laboratories, and we hoped to develop reliable, validated protocols that would be adopted as standard. Samples of 8-oxodGuo, oligonucleotides containing the oxidized base, calf thymus DNA with different amounts of 8-oxoGua, and HeLa cells with basal or artificially elevated levels of base oxidation were distributed to partners for analysis, so that we could compare techniques and test improved methods [29–33]. ESCODD operated for 6 years and was supported for the last 3 years by funding from the European Commission. It concluded . While GC–MS is invaluable for identifying oxidation products and can be used to measure high, experimentally induced levels of damage in DNA, it is not useful for estimating low basal levels in cellular DNA. Numerous published reports are based on GC–MS measurements grossly inflated by the artifact of oxidation during sample preparation. . HPLC–ECD is remarkably accurate when measuring relatively low levels of experimentally induced damage; results from seven out of eight laboratories measuring 8-oxodGuo in HeLa cells showed the same dose–response slope. However, in spite of the use of a standard protocol, there was a wide range of values when estimating background levels of damage, which must reflect different extents of adventitious oxidation occurring in different laboratories. . The wide range of HPLC-based estimates of background levels of damage was seen also in the final ESCODD trial, where partners analyzed lymphocyte DNA from groups of about 20 healthy volunteers recruited in their respective countries. Mean values ranged from 0.20 to 6.65 8-oxoGua per 106 guanines (median 4.24, mean 3.75, SD 2.37) (Figure 5.1) [33]. . The enzymic methods – using FPG to convert 8-oxoGua to DNA breaks – are less prone to spurious oxidation, as only very mild and brief sample processing is involved. In the same ESCODD exercise with lymphocytes, mean values across countries for the proportion of oxidized guanines ranged from 0.15 to 0.55 per 106 (median and mean 0.34; SD 0.15) (Figure 5.1) [33]. . On the basis of this analysis of 8-oxoGua in DNA of lymphocytes, we estimated the likely true background level of 8-oxoGua as between 0.3 and 4 per 106 Gua – the range between the median values (across countries) for the comet assay/FPG and HPLC, respectively. . HPLC–MS/MS, in principle a powerful technique since it can identify different oxidation products present at low concentrations, performed poorly in ESCODD trials and has yet to prove its potential. Much of the variability, in both HPLC and enzymic determinations, may be attributable to differences in practical skills between laboratories with the respective 5.5 Using Surrogate Biomarkers for Disease Risk j83

Figure 5.1 Mean concentrations of 8-oxodGuo (8-oxoGua) assessed by HPLC, or using the comet assay, in lymphocyte DNA from healthy young male subjects recruited in different countries (IT, Italy; BE, Belgium; SK, Slovakia; GB, Great Britain; DK, Denmark; SE, Sweden; PL, Poland; SP, Spain). Bars indicate SE of means. (Data from Ref. [33]).

assays. It seems to be the case that, as experience is gained in HPLC analysis, peaks of 8-oxodGuo become smaller, and there is a suspicion that, as we approach the true background level, it will become impossible to measure samples accurately without some prepurification, for example, using an immunoaffinity column to remove unmodified Gua. Even then, there will be a limitation – the amount of DNA available from a standard blood sample. 84j 5 Antioxidants and Cancer: Fact and Fiction

The enzymic methods have the advantage of requiring relatively little material (in the case of the comet assay, 30 ml of blood is sufficient for analysis). However, there are several potential problems with this approach: . FPG is not specific for 8-oxoGua; we must allow for the presence of some of the other substrates, FaPyAde and FaPyGua. . However, it is possible that FPG does not detect all potential FPG sites, if digestion conditions are not optimal, or if some lesions are inaccessible because of the conformation or sequence of DNA. . The enzymic methods depend on indirect calibration, normally using ionizing radiation to induce DNA breaks; the figure for breaks/Gy was established many years ago using very different irradiation conditions and analysis system, and its appropriateness is not certain. These problems are currently being addressed. Clearly, we have not yet established the definitive biomarker for oxidative DNA damage in human studies. While the comet assay with FPG may not be as precise as HPLC, it seems likely that it is more accurate, since it is affected less by artifact. And we have set a benchmark for assessing the credibility of published reports on background DNA oxidation: values of more than about five 8-oxoGua per 106 Gua should be regarded as dubious.

5.5.2 Intervention Trials with Antioxidants, Using the Comet Assay to Assess DNA Damage

The last section, we hope, justiﬁed the fact that we concentrate here on human trials employing the comet assay to measure DNA damage. There are various approaches: a single dose of antioxidant or antioxidant-rich food, or supplementation for a week or more; comparison of a supplemented group with a matched group receiving placebo, or a crossover trial in which all subjects receive placebo or supplement in different phases of the trial; measurement of endogenous damage, namely, strand breaks and oxidized bases (using lesion-speciﬁc endonucleases); measurement of

DNA breaks induced in lymphocytes treated ex vivo with H2O2 as an index of the resistance of the lymphocytes to oxidative attack. This area of research has been continually reviewed by Møller and Loft [34–36], and they conclude that, although there are problems in many reported trials with suboptimal study design, inappropriate subjects, or poor biomarker assays, there is a clear, statistically very significant tendency for supplementation to result in decreased damage. In view of the thorough treatment the topic has received, we will present here just a few examples, together with some comments. Generally, the frequency of endogenous strand breaks in lymphocyte DNA shows little or no change in supplementation with antioxidants. Strand breaks are not solely attributable to oxidative damage; they (or alkali-labile AP sites) may result from various kinds of damaging events occurring in the bases or in deoxyribose units of the DNA. They include transient intermediates in DNA repair pathways, particularly 5.5 Using Surrogate Biomarkers for Disease Risk j85 base excision repair (the nicked DNA present during nucleotide excision repair is rejoined very quickly in most cell types and is difficult to detect). So the lack of effect of antioxidants is not surprising. In contrast, the endogenous level of oxidized bases in lymphocytes is more readily affected by supplementation, though typically only after a relatively long period rather than a single dose. FPG sites or endonuclease III sites may be measured; if both, they can provide a useful independent confirmation of an effect. The first reported demonstration of a decrease in oxidized bases was in a placebo-controlled trial with 20 weeks of supplementation with a combination of vitamin C, vitamin E, and b-carotene. Subjects were healthy men in their 50s [37]. The decrease (in endonuclease III sites) was seen in both smokers and nonsmokers. In another trial, vitamin C was given to male smokers either in the form of ordinary capsules or in a slow release form: only in the latter group was a sustained decrease in FPG- and endonuclease III-sensitive sites seen [38]. Gallic acid as a supplement was found by Nersesyan et al. [39] to be more effective than vitamin C at decreasing the levels of endogenous base damage. It was soon realized – especially after the reports from the b-carotene intervention trials – that real foods, rich in certain or several antioxidants, might be more appropriate supplements than isolated compounds. A decrease in oxidized pyrimidines was seen during a 2-week supplementation with carotene-rich carrot juice [40], and we showed a similar effect after 4 weeks of daily consumption of 1 l of soy milk (rich in isoflavone phytoestrogens) [41]. Salvini et al. [42] in a crossover trial with high- or low-phenolic olive oil found a 30% decrease in oxidized purines with the high-phenolic, extra virgin olive oil. A single supplement, while not affecting endogenous damage, can have a marked effect on the resistance of lymphocyte DNA to oxidative attack in vitro, and this constitutes a useful, simple test for the ability of plant foods or their components to alter antioxidant status in humans. Lymphocytes taken after a large single dose of kiwifruit juice became resistant to H2O2-induced DNA breaks a few hours after the drink, following a peak concentration of vitamin C in the blood [43]. After a meal of fried onions, flavonoid glycosides were detected in the blood, and lymphocytes showed a delayed resistance to H2O2 [44]. Not surprisingly, this boost to antioxidant status is seen after longer term supplementation, too – for instance, in the 20-week vitamin C/vitamin E/b-carotene trial [37], or after 14 days of consumption of tomato puree representing 7 mg/day of lycopene [45]. Unusually, in this last study, lycopene was measured in both plasma and lymphocytes, and an inverse correlation was seen between the lycopene concentration and the level of oxidized pyrimidines. A study that delivered a particularly rich set of results was designed to test the notion that kiwifruit is a good source of antioxidants [46]. The study was designed as a crossover trial with three 3-week phases, representing different doses of kiwifruit – one per day, two per day, and three per day, separated by 2-week washout periods. The subjects (healthy young people) were divided into three groups, receiving the doses in different orders. Mean plasma vitamin C levels increased by up to 26%, depending on dose. Lymphocyte antioxidant status (resistance to H2O2-induced breaks) was enhanced – though, strangely, the greatest effect was seen with only one 86j 5 Antioxidants and Cancer: Fact and Fiction

Figure 5.2 Effects of kiwifruit supplementation on DNA damage (oxidized pyrimidines) and DNA repair (OGG1 activity in an in vitro assay) in lymphocytes from young healthy volunteers. Light gray, before supplementation; dark gray, after supplementation. Bars indicate SD. (Data from Ref. [46]).

kiwifruit a day. Endogenous DNA base oxidation (both purines and pyrimidines) was decreased by kiwifruit consumption (again with no sign of a dependence on kiwifruit dose) (Figure 5.2). The most exciting result (because unexpected) was a signiﬁcant increase in base excision repair capacity of lymphocyte extracts in an in vitro assay,with a substrate containing 8-oxoGua (i.e., measuring the activity of 8-oxoGua DNA glycosylase, OGG1) (Figure 5.2). The increase in repair enzyme activity was not accompanied by any detectable change in the levels of mRNA for OGG1, or for the enzyme AP endonuclease (APE1) that follows OGG1 in the pathway – consistent with the conclusion of Paz-Elizur et al. [47] that, in human lymphocyte samples, there is a poor correlation between mRNA levels and OGGactivity,measured by an in vitro assay with oligonucleotide containing 8-oxoGua as substrate. Factors other than regulation of transcription are apparently responsible for variations in enzyme activity. 5.7 Conclusions j87

We have recently found a similar stimulation of repair following dietary intervention with an antioxidant-rich diet in a group of subjects at risk of cardiovascular disease (Brevik et al., submitted). The same assay was used [48] to examine the effect of coenzyme Q10 (not a phytochemical but an antioxidant); a signiﬁcant stimulation of in vitro repair activity was seen.

5.6 Nonantioxidant Effects of Antioxidants

The value of dietary antioxidants per se has been brought into question by the clinical intervention studies discussed earlier. The smaller scale, biomarker-based studies have in many cases confirmed that antioxidants, or antioxidant-rich foods, can enhance the antioxidant status of blood and lymphocytes and decrease oxidative damage to DNA. But the biological significance of these effects remains in doubt; cellular antioxidant defenses are effective enough to prevent most of the potential damaging effects of free radicals in the body, and a certain amount of oxidative stress is presumably tolerated or is even necessary, for example, in the inflammatory response and in various cell signaling pathways. On the contrary, there are many other ways in which phytochemicals may have physiological effects, and in particular may modulate DNA damage, in addition to acting as antioxidants. They may, for instance, influence phase I and phase II xenobiotic metabolism, or antioxidant enzyme activities, either through gene expression or through direct effects on enzyme activity or stability. We are currently following up our finding of enhanced base excision repair in lymphocytes by studying individual phytochemicals and fruit extracts in cell culture model systems, in the belief that these nonantioxidant effects are likely to be at least as important in preventing the initiating event of carcinogenesis, that is, mutation arising from DNA damage. This reductionist approach is inevitable if we are to find out more about mechanisms of action. But in the end, we must address the complex issue of human variability. Genetic polymorphisms in genes related to cancer or its prevention are already known to influence the effects of dietary antioxidants; Dusinska et al. [49] found significant associations between smoking, GSTP1 genotype, plasma vitamin C, and purine oxidation in lymphocytes. Many hope that, with sufficient genetic information, it will be possible to provide a customized nutritional advice to individuals, aimed at maintaining health. Given the comparable complexities of foods and human genetics, this presents a major challenge.

5.7 Conclusions

This chapter focused on the links between fruits and vegetables – and the phytochemicals they contain – and the prevention of cancer. There is epidemiological evidence for protective effects, and there are similar inverse associations between 88j 5 Antioxidants and Cancer: Fact and Fiction

fruit and vegetable consumption and cardiovascular disease [50]. But we lack a full explanation of why these foods protect, and the public should not be misled into believing that antioxidants are the answer. In fact, there seems to be no advantage to healthy people in taking antioxidant supplements, and those at particular risk of cancer, including those with high PSA, heavy smokers, and asbestos workers, should avoid supplements. Meanwhile, while we wait for the results of research into the various other effects of phytochemicals in the body, the advice to consume ﬁve or more portions of fruit and vegetables per day is clearly very sensible.

References

1 WCRF/AICR (1997) Food, Nutrition and mineral combinations, cancer incidence, the Prevention of Cancer: A Global and disease-speciﬁc mortality in the Perspective, WCRF/AICR, Washington, general population. Journal of the National DC. Cancer Institute, 85, 1483–1492. 2 WCRF/AICR (2007) Food, Nutrition, 9 Alpha-Tocopherol Beta Carotene Cancer Physical Activity, and the Prevention of Prevention Study Group (1994) The effect Cancer, American Institute for Cancer of vitamin E and beta carotene on the Research, Washington, DC. incidence of lung cancer and other cancers 3 Ames, B.N. (1983) Dietary carcinogens in male smokers. New England Journal of and anticarcinogens. Science, 221, Medicine, 330, 1029–1035. 1256–1264. 10 Rapola, J.M., Virtamo, J., Ripatti, S., 4 Ames, B.N., Shigenaga, M.K. and Hagen, Haukka, J.K., Huttunen, J.K., Albanes, D., T.M.(1993) Oxidants, antioxidants, and the Taylor, P.R. and Heinonen, O.P. (1998) degenerative diseases of aging. Proceedings Effects of alpha tocopherol and beta of the National Academy of Sciences of the carotene supplements on symptoms, United States of America, 90, 7915–7922. progression, and prognosis of angina 5 Ames, B.N. (1998) Micronutrients prevent pectoris. Heart (British Cardiac Society), 79, cancer and delay aging. Toxicology Letters, 454–458. 102–103,5–18. 11 Omenn, G.S., Goodman, G.E., 6 Peto, R., Doll, R., Buckley, J.D. and Sporn, Thornquist, M.D., Balmes, J., Cullen, M.B. (1981) Can dietary beta-carotene M.R., Glass, A., Keogh, J.P., Meyskens, materially reduce human cancer rates? F.L. et al. (1996) Effects of a combination of Nature, 290, 201–208. beta carotene and vitamin A on lung 7 Halvorsen, B.L., Holte, K., Myhrstad, cancer and cardiovascular disease. M.C.W., Barikmo, I., Hvattum, E., New England Journal of Medicine, Remberg, S.F., Wold, A.-B., Haffner, K. 334, 1150–1155. et al. (2002) A systematic screening of total 12 Hennekens, C.H., Buring, J.E., Manson, J., antioxidants in dietary plants. The Journal Stampfer, M., Rosner, B., Cook, N.R., of Nutrition, 132, 461–471. Belanger, C., LaMotte, F. et al. (1996) Lack 8 Blot, W.J., Li, J.-Y., Taylor, P.R., Guo, W., of effect of long-term supplementation Dawsey, S., Wang, G.-Q., Yang, C.S., with beta carotene on the incidence of Zheng, S.-F. et al. (1993) Nutrition malignant neoplasms and cardiovascular intervention trials in Linxian, China: disease. New England Journal of Medicine, supplementation with speciﬁc vitamin/ 334, 1145–1149. References j89

13 Heart Protection Study Collaborative 20 Kasai, H., Crain, P.F., Kuchino, Y., Group (2002) MRC/BHF heart protection Nishimura, S., Ootsuyama, A. and study of antioxidant vitamin Tanooka, H. (1986) Formation of supplementation in 20536 high-risk 8-hydroxyguanine moiety in cellular individuals: a randomised placebo- DNA by agents producing oxygen radicals controlled trial. Lancet, 360,23–33. and evidence for its repair. Carcinogenesis, 14 Hercberg, S., Galan, P., Preziosi, P., 7, 1849–1851. Bertrais, S., Mennen, L., Malvy, D., 21 Cadet, J., Douki, T., Frelon, S., Sauvaigo, S., Roussel, A.M., Favier, A. and Briancon, S. Pouget, J.-P. and Ravanat, J.-L. (2002) (2004) The SU.VI.MAX Study: a Assessment of oxidative base damage to randomized, placebo-controlled trial of the isolated and cellular DNA by HPLC–MS/ health effects of antioxidant vitamins and MS measurement. Free Radical Biology & minerals. Archives of Internal Medicine, 164, Medicine, 33, 441–449. 2335–2342. 22 Ahnstrom,€ G. and Erixon, K. (1981) 15 Hercberg, S., Ezzedine, K., Guinot, C., Measurement of strand breaks by alkaline Preziosi, P., Galan, P., Bertrais, S., denaturation and hydroxyapatite Estaquio, C., Briancon, S., Favier, A., chromatography, in DNA Repair: A Latreille, J. and Malvy, D. (2007) Laboratory Manual of Research Antioxidant supplementation increases Procedures (eds E.C. Friedberg and the risk of skin cancers in women but not P.C. Hanawalt), Marcel Dekker, New York, in men. The Journal of Nutrition, 137, pp. 403–418. 2098–2105. 23 Kohn, K.W., Ewig, R.A.G., Erickson, L.C. 16 Meyer, F., Galan, P., Douville, P., Bairati, I., and Zwelling, L.A. (1981) Measurement of Kegle, P., Bertrais, S., Estaquio, C. and strand breaks and cross-links by alkaline Hercberg, S. (2005) Antioxidant vitamin elution, in DNA Repair: A Laboratory and mineral supplementation and prostate Manual of Research Procedures (eds E.C. cancer prevention in the SU.VI.MAX trial. Friedberg and P.C. Hanawalt), Marcel International Journal of Cancer, 116, Dekker, New York, pp. 379–401. 182–186. 24 Singh, N.P., McCoy, M.T., Tice, R.R. and 17 Miller, E.R. III, Pastor-Barriuso, R., Dalal, Schneider, E.L. (1988) A simple technique D., Riemersma, R.A., Appel, L.J. and for quantitation of low levels of DNA Guallar, E. (2005) Meta-analysis: high- damage in individual cells. Experimental dosage vitamin E supplementation may Cell Research, 175, 184–191. increase all-cause mortality. Annals of 25 Collins, A.R., Duthie, S.J., Fillion, L., Internal Medicine, 142,37–46. Gedik, C.M., Vaughan, N. and Wood, S.G. 18 Bjelakovic, G., Nikolova, D., Gluud, L.L., (1997) Oxidative DNA damage in human Simonetti, R.G. and Gluud, C. (2007) cells: the inﬂuence of antioxidants and Mortality in randomized trials of DNA repair. Biochemical Society antioxidant supplements for primary and Transactions, 25, 326–331. secondary prevention: systematic review 26 Ravanat, J.-L., Turesky, R.J., Gremaud, E., and meta-analysis. The Journal of the Trudel, L.J. and Stadler, R.H. (1995) American Medical Association, 297, Determination of 8-oxoguanine in DNA by 842–857. gas chromatography–mass spectrometry 19 Cadet, J., Berger, T., Douki, T. and and HPLC-electrochemical detection: Ravanat, J.-L. (1997) Oxidative damage to overestimation of the background level of DNA: formation, measurement and the oxidized base by the gas biological signiﬁcance. Reviews of chromatography–mass spectrometry Physiology, Biochemistry and Pharmacology, assay. Chemical Research in Toxicology, 8, 131,1–87. 1039–1045. 90j 5 Antioxidants and Cancer: Fact and Fiction

27 Hofer, T. and Moller,€ L. (2002) oxidatively damaged DNA. Free Radical Optimization of the workup procedure Biology & Medicine, 41, 388–415. for the analysis of 8-oxo-7,8-dihydro- 37 Duthie, S.J., Ma, A., Ross, M.A. and 20-guanosine with electrochemical Collins, A.R. (1996) Antioxidant detection. Chemical Research in Toxicology, supplementation decreases oxidative DNA 15, 426–432. damage in human lymphocytes. Cancer 28 Collins, A., Cadet, J., Epe, B. and Gedik, C. Research, 56, 1291–1295. (1997) Problems in the measurement of 38 Møller, P., Viscovich, M., Lykkesfeldt, J., 8-oxoguanine in human DNA. Report of a Loft, S., Jensen, A. and Poulsen, H.E. workshop, DNA Oxidation, held in (2004) Vitamin C supplementation Aberdeen, UK, January 19–21, 1997. decreases oxidative DNA damage in Carcinogenesis, 18, 1833–1836. mononuclear blood cells of smokers. 29 ESCODD (2002) Inter-laboratory European Journal of Nutrition, 43, 267–274. validation of procedures for measuring 39 Nersesyan, A., Hoelzl, C., Ferk, F., Misik, 8-oxo-7,8-dihydroguanine/8-oxo-7, M. and Knasmuller,€ S. Use of single cell gel 8-dihydro-20-deoxyguanosine in DNA. electrophoresis assays in dietary Free Radical Research, 36, 239–245. intervention trials, in The Comet Assay in 30 ESCODD (2000) Comparison of different Toxicology. C (eds A. Dhawan and D. methods of measuring 8-oxoguanine as a Anderson), Royal Society of Chemistry, marker of oxidative DNA damage. Free Cambridge, UK, in press. Radical Research, 32, 333–341. 40 Pool-Zobel, B.L., Bub, A., Muller, H., 31 ESCODD (2002) Comparative analysis of Wollowski, I. and Rechkemmer, G. (1997) baseline 8-oxo-7,8-dihydroguanine in Consumption of vegetables reduces mammalian cell DNA, by different genetic damage in humans: first results of methods in different laboratories: an a human intervention trial with approach to consensus. Carcinogenesis, 23, carotenoid-rich foods. Carcinogenesis, 18, 2129–2133. 1847–1850. 32 ESCODD (2003) Measurement of DNA 41 Mitchell, J.H. and Collins, A.R. (1999) oxidation in human cells by Effects of a soy milk supplement on plasma chromatographic and enzymic methods. cholesterol levels and oxidative DNA Free Radical Biology & Medicine, 34, damage in men – a pilot study. European 1089–1099. Journal of Nutrition, 38, 143–148. 33 ESCODD, Gedik, C.M. and Collins, A. 42 Salvini, S., Sera, F., Caruso, D., (2005) Establishing the background level of Giovannelli, L., Visioli, F., Saieva, C., base oxidation in human lymphocyte Masala, G., Ceroti, M. et al. (2006) Daily DNA: results of an interlaboratory consumption of a high-phenol extra-virgin validation study. The FASEB Journal, olive oil reduces oxidative DNA damage in 19,82–84. postmenopausal women. British Journal of 34 Møller, P. and Loft, S. (2002) Oxidative Nutrition, 95, 742–751. DNA damage in human white blood cells 43 Collins, B.H., Horska, A., Hotten, P.M., in dietary antioxidant intervention studies. Riddoch, C. and Collins, A.R. (2001) The American Journal of Clinical Nutrition, Kiwifruit protects against oxidative DNA 76, 303–310. damage in human cells and in vitro. 35 Møller, P. and Loft, S. (2004) Interventions Nutrition and Cancer, 39, 148–153. with antioxidants and nutrients in relation 44 Boyle, S.P., Dobson, V.L., Duthie, S.J., to oxidative DNA damage and repair. Kyle, J.A.M. and Collins, A.R. (2000) Mutation Research, 551,79–89. Absorption and DNA protective effects of 36 Møller, P. and Loft, S. (2006) Dietary flavonoid glycosides from an onion meal. antioxidants and beneficial effect on European Journal of Nutrition, 39, 213–223. References j91

45 Porrini, M. and Riso, P. (2000) Lymphocyte factors: effects of individual alcohol lycopene concentration and DNA sensitivity. Environmental Health protection from oxidative damage is Perspectives, 104, 1336–1338. increased in women after a short period of 54 Aukrust, P., Luna, L., Ueland, T., Johansen, tomato consumption. The Journal of R.F., Muller, F., Froland, S.S., Seeberg, Nutrition, 130, 189–192. E.C. and Bjoras, M. (2005) Impaired base 46 Collins, A.R., Harrington, V., Drew, J. and excision repair and accumulation of Melvin, R. (2003) Nutritional modulation oxidative base lesions in CD4 þ T cells of of DNA repair in a human intervention HIV-infected patients. Blood, 105, study. Carcinogenesis, 24, 511–515. 4730–4735. 47 Paz-Elizur, T., Elinger, D., Leitner-Dagan, 55 Breton, J., Sichel, F., Pottier, D. and Y., Blumenstein, S., Krupsky, M., Berrebi, Prevost, V. (2005) Measurement of 8-oxo- A., Schechtman, E. and Livneh, Z. (2007) 7,8-dihydro-20-deoxyguanosine in Development of an enzymatic DNA repair peripheral blood mononuclear cells: assay for molecular epidemiology studies: optimisation and application to samples distribution of OGG activity in healthy from a case-control study on cancers of the individuals. DNA Repair, 6,45–60. oesophagus and cardia. Free Radical 48 Tomasetti, M., Alleva, R., Borghi, B. and Research, 39,21–30. Collins, A.R. (2001) In vivo 56 Tarng, D.C., Liu, T.Y. and Huang, T.P. supplementation with coenzyme Q10 (2004) Protective effect of vitamin C on enhances the recovery of human 8-hydroxy-2[prime]-deoxyguanosine level lymphocytes from oxidative DNA damage. in peripheral blood lymphocytes of chronic The FASEB Journal, 15, 1425–1427. hemodialysis patients. Kidney 49 Dusinska, M., Ficek, A., Horska, A., International, 66, 820–831. Raslova, K., Petrovska, H., Vallova, B., 57 Yen, M.Y., Kao, S.H., Wang, A.G. and Wei, Drlickova, M., Wood, S.G. et al. (2001) Y.H. (2004) Increased 8-hydroxy- Glutathione S-transferase polymorphisms 20-deoxyguanosine in leukocyte DNA in inﬂuence the level of oxidative DNA Lebers hereditary optic neuropathy. damage and antioxidant protection in Investigative Ophthalmology & Visual humans. Mutation Research, 482,47–55. Science, 45, 1688–1691. 50 Ness, A.R. and Powles, J.W. (1997) Fruit 58 Harrison, K.L., Crosbie, P.A.J., Agius, and vegetables, and cardiovascular disease: R.M., Barber, P.V., Carus, M., Margison, a review. International Journal of G.P. and Povey, A.C. (2006) No association Epidemiology, 26,1–13. between N7-methyldeoxyguanosine and 51 Spencer, J.P.E., Jenner, A., Aruoma, O.I., 8-oxodeoxyguanosine levels in human Cross, C.E., Wu, R. and Halliwell, B. (1996) lymphocyte DNA. Mutation Research, Oxidative DNA damage in human 600, 125–130. respiratory tract epithelial cells. Time 59 Siomek, A., Gackowski, D., Rozalski, R., course in relation to DNA strand breakage. Dziaman, T., Szpila, A., Guz, J. and Biochemical and Biophysical Research Olinski, R. (2007) Higher leukocyte 8-oxo- Communications, 224,17–22. 7,8-dihydro-20-deoxyguanosine and lower 52 Jaruga, P. and Dizdaroglu, M. (1996) plasma ascorbate in aging humans? Repair of products of oxidative DNA base Antioxidants & Redox Signaling, 9, 143–150. damage in human cells. Nucleic Acids 60 Mambo, E., Chatterjee, A., de Souza-Pinto, Research, 24, 1389–1394. N.C., Mayard, S., Hogue, B.A., Hoque, 53 Nakajima, M., Takeuchi, T., Takeshita, T. M.O., Dizdaroglu, M., Bohr, V.A. and and Morimoto, K. (1996) Sidransky, D. (2005) Oxidized guanine 8-Hydroxydeoxyguanosine in human lesions and hOGG1 activity in lung cancer. leukocyte DNA and daily health practice Oncogene, 24, 4496–4508. 92j 5 Antioxidants and Cancer: Fact and Fiction

61 Trzeciak, A.R., Nyaga, S.G., Jaruga, P., method to estimate cellular oxidative Lohani, A., Dizdaroglu, M. and Evans, stress. Journal of Radiation Research, 45, M.K. (2004) Cellular repair of oxidatively 455–460. induced DNA base lesions is defective in 63 Hoffmann, S., Spitkovsky, D., Radicella, prostate cancer cell lines, PC-3 and J.P., Epe, B. and Wiesner, R.J. (2004) DU-145. Carcinogenesis, 25, 1359–1370. Reactive oxygen species derived from the 62 Orimo, H., Mie, N., Boiteux, S., Tokura, Y. mitochondrial respiratory chain are not and Kasai, H. (2005) Analysis of 8- responsible for the basal levels of oxidative hydroxyguanine (8-OH-Gua) released base modiﬁcations observed in nuclear from DNA by the formamidopyrimidine DNA of mammalian cells. Free Radical DNA glycosylase (Fpg) protein: a reliable Biology & Medicine, 36, 765–773. j93

6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer? Hansruedi Glatt

6.1 Xenobiotics and Their Disposition – Basal Aspects

Humansrequireacontinuoussupplyofwater,oxygen,andnutrients.Thesefactorsare not available in pure form in normal life, but are taken up from complex matrices containing numerous other constituents, which also may be absorbed. The term xenobiotic is used for all small organic chemicals that are neither nutrients nor their biotransformation products in a given organism. Sporadically, compounds of biologi- calorigin (e.g., phytochemicals) orpharmaceutical drugs areexemptedfrom theterm. Such a distinction is not meaningful, as numerous anthropogenic chemicals also occur naturally, and drugs are not only taken up for therapeutic purposes, but also unintentionally from contaminated water and food. More important, the organisms handle non-nutritive chemicals, whether natural or anthropogenic, in similar ways. Quantitatively, secondary metabolites of other organisms (plants, fungi, and bacteria) may constitute the major xenobiotics for humans. Another chief source is processing, in particular heating, of foodstuff, which initiates a plethora of chemical reactions. Many anthropologists place the advent of cooking at around 250 000 years ago,aminuteperiodcomparedtotheevolutionofthexenobiotic-metabolizingsystem. Forest fires, sunlight, diagenesis of sediments and lipid peroxidation are other natural factors generating chemical diversity in the human environment. The formation of some of these chemicals may be strongly enhanced by human activities. Drugs, food additives, residues of pesticides, and industrial chemicals are anthropogenic xenobiotics, which often receive much publicity. Most subjects may take up thousands of xenobiotics in quantities of >1 mg per day, and probably millions if one goes to the detection limit of actual analytical chemistry. The Greek word xenoV (foreign, foreigner) was often used with the connotation guest or friend. Indeed, some effects of xenobiotics are welcome and beneficial to health, whereas others may be adverse even at very low concentrations. And most xenobiotics will eventually disturb the finely tuned biological system when reaching sufficiently high levels. This implies the necessity to eliminate them. Excretion of

xenobiotics in terrestrial vertebrates primarily occurs via bile and urine, requiring sufficient hydrophilicity and restrictions in membrane penetration. Conversely, chemicals with the opposite properties (lipo- or amphiphilicity, facile membrane penetration) are those that readily enter the organism. This discrepancy between structural features favoring absorption and excretion can be largely bridged up by biotransformation reactions, which often are catalyzed by enzymes specifically created for this function. Structures and activities of xenobiotics areextremelydiverse.Therefore,theirhandlingisnotoptimizedtothesameextentasit is with endogenous metabolism. Interestingly, however, the xenobiotic-metabolizing systemispreparedfornewchemicalentities.Thus,mostanthropogenicxenobioticsare efficiently metabolized and eliminated. The same is true for heat-induced food constituents even in animals with no history of cooking. In return for this broad performance,ithastobeacceptedthatmetabolitesmaybeproducedthatinterferemore seriously with physiological functions than the parent xenobiotic. With regard to carcinogenesis, chemically reactive intermediates are particularly important. Anymetabolismrequiressomechemicalreactivity.Intheendogenousmetabolism, this reactivity is channeled by enzymes, coupling of sequential reactions and compart- mentalization, which largely leads to the avoidance of dangerous side reactions. This control has not reached the same perfection in xenobiotic metabolism. Xenobiotics are sometimes accidentally (parametabolically) metabolized by enzymes whose primary function lies in the endogenous metabolism. However, more importantareenzymeswithverybroadsubstratetolerancewithoutanyobviousfunction in the endogenous metabolism. The latter is indicated by viable individual- or species- related gene disruption as well as the lack of an aberrant phenotype in genetically engineered animal models in the absence of xenobiotic exposure. The number of these xenobiotic-metabolizing enzymes (XMEs) in the narrow sense may be approximately 100. Although the function of these enzymes is to eliminate the xenobiotics, some endogenous compounds (e.g., steroid and thyronine hormones) may also be metabolized, sometimes parametabolically (i.e., without any obvious function). Therefore, excessive expression of such enzymes, in the absence of a xenobiotic substrate, could leadtoendocrinedisturbance[1].Overall,theseparationofthesetwogroupsofenzymes (with primarily endogenous or xenobiotic-metabolizing function) is fluent. Exposure to xenobiotics may strongly vary in different situations. It is therefore not surprising that the expression of many XMEs is not constant, but regulated by the actual exposure. In general, a whole set of enzymes and other host factors handling a certain class of chemicals (e.g., aromatics, very small molecules or electrophiles) are abrogated. Typical regulation systems are presented in Chapter 7 of this monograph. The broad substrate tolerance of XMEs may lead to a competition among several substrates for the same enzyme, resulting in redirection of metabolic routes at complex exposures. Enzyme induction is another mechanism of interaction. Such interactions may lead to reduced or enhanced toxification of a chemical. Depending on the ecological niche, the kind and level of xenobiotic exposure may vary extremely between species. This is reflected in substantial species-dependent differences in xenobiotic metabolism. For example, humans and rats preferentially metabolize many phenols via sulfation at low exposure levels and via glucuronidation 6.2 Classification of Biotransformation Reactions and Enzymes j95 at high exposure levels. The major natural exposure to phenols occurs via phenolic plant metabolites. Carnivorous animals only experience low natural exposure to phenols; indeed, felines are largely deficient in enzymes catalyzing phenol glucuronidation, the high-capacity system [2]. Many XMEs are ambivalent – beneficial at many exposures, but disastrous at some other exposures. Most xenobiotics can be metabolized on various routes by different enzymes; thus, the individual XME is not vital. These aspects together with the varying exposure to xenobiotics have favored the development of species- and individual (genetic polymorphism)-dependent differences in xenobiotic metabolism.

6.2 Classification of Biotransformation Reactions and Enzymes

6.2.1 Conventional Phase 1/Phase 2 Concept

A scheme of common reactions in the biotransformation of xenobiotics is presented in Figure 6.1. In pharmacology and toxicology textbooks, these reactions are generally classified as follows: . Phase 1: Introduction, modification, or liberation of a functional group via oxidation, reduction, isomerization, or hydrolysis. . Phase 2: Conjugation reactions using a functional group present in the original xenobiotic or created in phase 1 metabolism. In the typical case, conjugation in phase 2 occurs with an anionic endogenous moiety, which normally enhances water solubility of the product, facilitating its excretion. However, water solubility is not sufficient for excretion. For example, direct renal elimination is negligible for ethanol – a compound showing the highest possible water solubility (miscible with water at any ratio). Ethanol is filtrated in the glomeruli, but then is reabsorbed together with water during the concentration of the urine in the tubule. Thus, more important than water solubility is a restriction in passive membrane penetration, which is achieved by the introduction of a negative charge. Transport of organic anions across membranes is mediated by special proteins using energy from ATP or electrochemical gradients as driving force. Distribution and substrate selectivity of these transporters allow for vectorial transport, a key factor for excretion. In cell culture, nearly every lipophilic compound is taken up into nearly every cell type. This observation suggests that passive uptake is possible, at least if sufficient time is available. However, it is now also evident that lipophilic compounds are subjected to various specific uptake and extrusion processes. Therefore, two other phases have been added to the absorption/elimination process of xenobiotics: . Phase 0: Absorption of the xenobiotic and counteracting processes; distribution of the xenobiotic and metabolic intermediates. 96j 6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer?

Figure 6.1 Scheme of common reactions in the the term phase 3 for the transport processes biotransformation of xenobiotics. Reactions in leading to excretion (block E). In the blocks A and B are often referred to as phase 1, chemoprevention area, block B is usually reactions in blocks C and D as phase 2. subsumed to phase 2, rather than phase 1 in the Sometimes a separate category (phase 3 traditional classification. reactions) is used for block D. Other authors use

. Phase 3: Excretion of terminal metabolites (involving vectorial distribution processes). Most reactions in biological systems are charge-driven addition and substitution reactions, involving a nucleophilic and an electrophilic reactant. This is true, in particular, for conjugation (phase 2) reactions and the formation of macromolecular adducts. Typical functional groups created in phase 1 metabolism clearly demonstrate either nucleophilic (e.g., hydroxyl, thiol, amino, and carboxyl groups) or electrophilic properties (e.g., epoxide and carbonyl groups). Enzymatic or spontaneous reactions can change these electronic characteristics (e.g., by oxidation of hydroxyl to carbonyl groups, isomerization or hydrolysis of arene oxides, or proton- ation of a hydroxyl group). With this complication, the charge-type of a functional group determines the further reactions it can undergo. Electrophilic, but not nucleophilic, metabolites are prone to form macromolecular adducts, as cellular macromolecules are rich in nucleophilic sites but largely deficient in electrophilic sites. Likewise, all conjugating enzymes require either an electrophilic or nucleophilic functional group (Figure 6.1). Glutathione transferases (GSTs) dominate the conjugation of electrophiles. Electrophilic intermediates (acyl-AMP and acyl- coenzyme A conjugates) also occur in the conjugation of carboxylic acids with amino acids. A larger diversity of conjugating enzymes utilize nucleophilic groups. The 6.2 Classification of Biotransformation Reactions and Enzymes j97 most important nucleophile-conjugating enzyme classes are the uridinedipho- sphate-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs). Other examples are the acetyl- and methyltransferases. This specificity of the conjugating enzymes implies, amongst other things, that UGTs cannot detoxify ultimate DNA- reactive genotoxicants nor compete with GSTs for the same substrate (unless this contains a nucleophilic center in addition to the electrophilic center). The phase 1/2 concept is teleological, based on certain ideas as to how excretable metabolites could be generated. The reality is more complex, as illustrated by the following examples: . Acetylation and methylation are conjugation reactions that do not introduce a negative charge into the molecule. These reactions decrease rather than increase water solubility. Hypothetical functions of the reactions involve a decrease in pharmacological or chemical activities. Conversely, a negative charge may already be introduced into a xenobiotic by a phase 1 reaction, for example, when aldehydes are oxidized to carboxylic acids. . Various metabolically formed sulfates and glucuronides can be hydrolyzed by sulfatases and b-glucuronidases, respectively [3]. As functional groups are un- masked, the reactions may be classified into phase 1. Likewise, hydroxylation may occuratothermolecularsitesevenafterconjugation.Inbothcases,phase1reactions occur after phase 2 reactions, in contrast to the sequence suggested by the names. . Some benzylic sulfates can be directly converted into the corresponding benzylic glutathione conjugates by GSTs via a substitution reaction [4]. Thus, typical phase 2 metabolites can be substrates for phase 2 enzymes. . Glutathione conjugates usually undergo further processing before excretion [5]. In general, they are converted into the corresponding cysteine conjugates by cleavage of glutamic acid and glycine, often followed by acetylation of the amino group of cysteine. The product is termed mercapturic acid. In other cases, b-lyases convert the cysteine conjugate to a thiol, which may be methylated by other enzymes. These reactions do not easily fit into the phase 1/2 classification, and are sometimes referred to as phase 3 of xenobiotic metabolism. Thus, the term phase 3 is used in different meanings, depending on authors and context. The phase 1/2 classification primarily relates to reactions rather than enzymes. Although it is common to refer to phase 1 and phase 2 enzymes in colloquial language, the match between enzyme and reaction class is not perfect. For example, some conjugating enzymes (e.g., acetyltransferases, SULTs) can use a conjugated xenobiotic as the donor substrate [6, 7]. In these cases, one xenobiotic (or xenobiotic metabolite) is conjugated (phase 2 reaction), while the other (the donor) is deconjugated (phase 1 reaction). Some glutathione and sulfo-conjugates are unstable and decompose spontaneously to regenerate the initial substrate or a positional or steric isomer of it [5, 6]. In the latter cases, the net effect produced by the conjugation is an isomerization. Other unstable sulfo-conjugates eliminate hydrogen sulfate, corresponding to a dehydration of the enzyme substrate [6]. In all these examples, so-called phase 2 enzymes mediate phase 1 reactions. 98j 6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer?

6.2.2 Toxicological Classification of Biotransformation Reactions

Withregardtocarcinogenesis,toxificationusuallyinvolvestheformationofelectrophilic intermediates capable of covalent binding to cellular macromolecules. Toxification may require one or several steps. Detoxification reactions may occur at different stages (Figure 6.2): we use the terms inactivation for the conversion of an active metabolite (ultimate toxicant) into an inactive metabolite, and sequestration for reactions that direct the xenobiotic into pathways avoiding the formation of the active metabolite. Nearly every type of functionalizing XME may be involved in initial steps of toxification of some carcinogens; an involvement of conjugating enzymes is possible, but less common. More restrictions apply for the final activation step, as the product has to be an electrophile. The most common types of electrophilic phase 1 metabolites are epoxides, free radicals, and carbonyl compounds (some aldehydes and quinones). They may be formed by cytochrome P-450 (CYP)-dependent monooxygenases, but not by flavine-dependent monooxygenases (FMOs), which oxidize heteroatoms (e.g., S, N, Se, and P) in their substrates. Alternatively, electrophilic phase 1 metabolites may be generated by lipoxygenases, myeloperoxidases, prostaglandin H synthases (also named cyclooxygenases, as both activities are contained in a single protein with two active centers) and other peroxidases. In addition, alcohol dehydrogenases and one-electron oxidoreductases (such as CYP reductase) are important in the formation of various aldehydes and free radicals, respectively. Among the conjugating enzymes, SULTs and acetyltransferases are particularly often involved in the formation of reactive metabolites, as acetate and sulfate are good leaving groups in certain chemical linkages (e.g., if the resulting cation is stabilized by resonance or inductive effects) [8, 9]. Sulfur mustards and episulfonium ions are reactive metabolites formed by GSTs from some haloalkanes [5]. Various acyl glucuronides are reactive, but have a strong preference for binding to proteins rather than nucleic acids [10].

Figure 6.2 Toxicological classification of predominating active metabolites in chemical biotransformation reactions. All types of XMEs carcinogenesis. Only a limited set of enzymes is may be involved in the sequestration-type of able to form or attack these metabolites and, detoxification reactions. Likewise, many enzyme thus, mediate final activation and inactivation, types may mediate initial steps of activation. respectively (see Section 6.2.2). Electrophilically reactive intermediates are the 6.2 Classification of Biotransformation Reactions and Enzymes j99

Many different enzymes can sequester progenotoxic compounds. In the early phase of metabolism, CYPs and FMOs may play major roles. After hydroxylation, UGTs, SULTs, and alcohol dehydrogenases may be particularly effective. The most common type of inactivation occurs via direct attack at the electrophilic group, which strongly restricts the enzyme types involved. GSTs show by far the broadest activity against the various types of electrophiles. In addition, specialized enzymes exist for common types of electrophiles: epoxide hydrolases for epoxides, quinone reductases for quinones, and aldehyde dehydrogenases as well as alcohol dehydrogenases (acting as aldehyde reductases) for aldehydes. In summary, reality differs from the widespread view that CYPs are the principal activatingenzymesandconjugationusuallyleadstodetoxification.Infact,toxificationby conjugating enzymes is not uncommon, and SULTs and acetyltransferases clearly represent high-risk enzymes similar to, or even more than, CYPs [8, 9]. This is highlighted by the history of chemoprevention. Early demonstrations of metabolism-based chemoprevention of tumorigenesis involved inhibition of SULTs by pentachlorophenol. This metabolic modulator effectively prevented the formation of DNA adducts and the induction of liver tumors by 10-hydroxysafrole, 2-acetylaminofluorene and its phase 1 metabolite, N-hydroxy-2-acetylaminofluorene, in mice and rats [11–13]. The wide use of liver postmitochondrial (S9) or microsomal systems in in vitro genotoxicity assays may have favored misconception on the role of phase 1 and phase 2 metabolism in toxification and detoxification [14]. S9 primarily represents a source of CYP activity, as only a single cofactor [NADP(H)] is normally supplemented. From negative and positive results in the absence versus presence of S9, respectively, it is often inferred that CYPs form the ultimate genotoxicant, ignoring that enzymes of the target cell may conduct the terminal activation. Supplementation of S9 with cofactors for conjugating enzymes often leads to decreased genotoxicity, an effect usually – but sometimes incorrectly – interpreted as detoxification. However, the major function of conjugation is the formation of metabolites that do not permeate the membranes. Therefore, reactive metabolites generated outside the target cell may not be detected due to the membrane barriers contained in standard test system, but may reduce the substrate concentration available for intracellular activating enzymes. In vivo, conjugation occurs within, but not outside, the cells. We have shown that the expression of conjugating enzymes, in particular of SULTs, within target cells leads to enhanced bioactivation of numerous progenotoxicants [8, 9]. These observations also point to the importance of the site of biotransformation. Formation of a reactive metabolite may be unproblematic if occurring in a cell rich in appropriate inactivating enzymes, or in terminally differentiated cells (e.g., mature enterocytes) if transfer to other cells is restricted. In both situations, toxification at uncritical sites may represent a sequestration for more sensible cells (e.g., stem cells).

6.2.3 Chaos and Selection

In general, the elimination of a xenobiotic does not occur as straight, as suggested by the phase 1/phase 2 model. A multitude of major and minor metabolites may be 100j 6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer?

excreted. Some of them may have undergone a long sequence of phase 1 and/or phase 2 and/or unclassified reactions. Potentially dangerous intermediates and cyclic reactions (e.g., oxidation/reduction or conjugation/deconjugation) may occur. Such chaotic traits are very prevalent. Chaotic processes combined with selection are a common mode of directional processes in physics, chemistry, biology, and social fields. The selection step in xenobiotic metabolism is the excretion, guided by active and passive distribution processes. A molecule of the xenobiotic is either excreted, evading any further metabolism, or persists in the organism. In general, it is only a question of time, until a persisting molecule is transformed by an actual XME or parametabolically by another host factor. The transformation may facilitate or impede excretion, again representing the selection step for the further fate. Compounds persisting for long periods in the organism may reach sufficiently high levels to activate transcription factors for XMEs often resulting in enhanced biotransformation. The occurrence of a multitude of different reactions on each level, and of two different major excretion routes (with urine and feces), is important to terminate futile cycling.

6.2.4 Modified Phase 1/Phase 2 Terms Used in the Chemoprevention Area

In the area of chemoprevention, the terms phase 1 and phase 2 are often used in a way that substantially differs from their conventional use in pharmacology, toxicology, and biochemistry. They are directly employed for classifying enzymes rather than reactions: . Phase I enzymes comprise a subset of the conventional phase 1 enzymes – those that are relatively more frequently involved in toxification reactions – primarily CYPs and peroxidases. . Phase II enzymes are characterized by a low risk of toxification and a frequent involvement in inactivation of reactive metabolites or sequestration of their metabolic precursors. A relatively large battery of such enzymes is controlled by the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), which can be activated by electrophilic or oxidative stress sensored by Kelch-like ECH- associated protein 1 (Keap1) (Chapter 7). This induction comprises several UGTs, GSTs,microsomal epoxide hydrolase, and NAD(P)H-quinone reductase 1 (NQO1). NQO1, which is often used as a marker for Nrf2-mediated gene activation, would be clearly classified into phase 1 in the conventional nomenclature. However, in concerted actions with UGTs or SULTs, it efficiently detoxifies many quinones. Due to this detoxifying role and the regulation by Nrf2, it is classified as a phase II enzyme by researchers in the chemoprevention area. It should be noted that Nrf2 does not only mediate the transcription of typical XMEs, but also of remotely associated and unassociated proteins, such as the catalytic subunit of g-glutamylcysteine synthetase (key enzyme in the synthesis of glutathione), selenium- dependent glutathione peroxidases (removing peroxides, which may be generated as a side product of xenobiotic metabolism), heme oxygenase (initiating the 6.2 Classification of Biotransformation Reactions and Enzymes j101

degradation of heme), transmembrane transporter proteins, and components of the proteasome. These proteins are also subsumed to varying extents under the term phase II enzymes by some authors. . Phase 2 enzymes (in the conventional nomenclature) that involve a relatively high risk for toxiﬁcation, such as SULTs and acetyltransferases, are largely ignored in the classiﬁcation used by many chemoprevention researchers. In an intervention study, a diet rich in Brussels sprout downregulated the level of SULT proteins in lymphocytes; such lymphocytes showed enhanced resistance to genotoxic effects induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [15], a food-borne carcinogen, whose terminal activation is mediated by SULTs [9, 16]. The underlying mechanism remains to be elucidated. Brussels sprout is a rich source of glucosinolates, which are potent activators of the Keap1/Nrf2 system. Possibly, enhanced proteasome activity may target some high-risk XME proteins.

6.2.5 Good and Bad Enzymes

The carcinogenicity of various compounds in animal models can be abolished or greatly diminished by genetic knockout or inhibition of individual XMEs (e.g., Refs [11–13, 17, 18]), implying that these enzymes are bad under these specific exposures. However, the adverse effects have to be overbalanced by positive effects in evolutionary eras, as knockout or knockdown can be easily achieved by numerous mutations. Other enzymes have shown protective effects in animal models or are associated with reduced cancer risks in epidemiological studies. For example, meta-analyses clearly indicate enhanced incidences of bladder tumors and leukemia [19] in GSTM1- deficient compared to proficient subjects (odds ratios 1.50 and 1.20, respectively). Possibly, this polymorphism also affects the incidence of lung tumors, with an odds ratio of 1.22 in all studies, but only 1.01 in the five largest studies (with the lowest risk for publication bias) reviewed in a recent meta-analysis [20], and with head and neck cancer (odds ratios 1.16) [19]. Similarly, GSTT1 deficiency appears to be associated with statistically significant increases in colorectal cancer, gastric cancer, head and neck cancer, leukemia, and lung cancer (odds ratios 1.37, 1.27, 1.08, 1.19, and 1.28, respectively). Upregulation of expression or amplification of genes could be easily achieved in evolution in the presence of selection pressure. Strangely, selection appears to occur in the opposite direction. The zero alleles of GSTM1 and GSTT1 has arisen by a deletion in the functional genes, implying that they are younger. In Caucasian populations, the frequencies of GSTM1 and GSTT1 zero alleles amount to nearly 70 and 45%, respectively [19]. These high frequencies make it likely that the deficiency brings benefits, which however have not yet been identified. The selection pressure of late-onset cancers may be low. However, reactive metabolites involved in late-onset cancers also induce other damage, leading to early-onset (e.g., transplacental) carcinogenesis and noncancerous diseases. This 102j 6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer?

could lead to selection pressures, which however have not been explored to my knowledge. Compared to the change in lifestyle, evolution is extremely slow. Therefore, the xenobiotic-metabolizing system may be far from optimally adjusted to current lifestyle and exposures, implying that its manipulation could be beneﬁcial. However, our knowledge on the dominating carcinogenic factors and mechanisms, and on other effects and functions of XMEs are too vague to design convincing health- promoting manipulations to the general population.

6.3 Toxicokinetic Interactions Leading to Enhanced or Reduced Effects of Carcinogens

6.3.1 Individual Known Carcinogens

XMEs and transport proteins can interact with many different xenobiotics. When bound to an enzyme, some xenobiotics are rapidly metabolized, others slowly or not at all.Atleastinthelattercases,processingofcompetingsubstratesisinhibited.Likewise, various xenobiotics can induce some XMEs. In typical cases, the induced enzymes can metabolize the inducing agent, but also many additional xenobiotics. Since biotrans- formationiscriticalfortheactionofmostchemicalcarcinogens,suchinteractionshave the potential for chemo-protection and -potentiation. This has been veriﬁed for many carcinogens in the literature. It is illustrated here by an example from our laboratory. 1-Methylpyrene is an abundant polycyclic aromatic hydrocarbon. It is genotoxic and carcinogenic in rodent models [21]. We have studied its biotransformation and the enzymes/transporters involved using in vitro and in vivo approaches. Benzylic hydroxylation followed by sulfation represents the activation pathway [9, 22]. Ring hydroxylation and further oxidation of the side chain to the carboxylic acid are prominent sequestration reactions [9, 22–24]. In the rat, activation primarily occurs in the liver. The active metabolite, 1-sulfooxymethylpyrene (1-SMP), is rapidly exported to the circulation, where it reversibly binds to serum albumin [25]; other transporters then mediate uptake into renal tubule cells. Based on this knowledge, potential interactions with other compounds were predicted. They were examined in the rat using the level of DNA adducts in different tissues as an end point. Results for liver and kidney are presented in Table 6.1. On every toxicokinetic level tested, we found interactions strongly affecting the adduct formation. Some interactions may have practical relevance. For example, administration of a moderately high level of ethanol (0.8 g/kg body mass) shortly before the proximate carcinogen, 1-hydroxy- methypyrene, enhanced the adduct formation up to 86-fold. Other modulators reduced the adduct formation or led to a shift in the target tissues. Thus, probenecid, an inhibitor of various transmembrane transporters, strongly enhanced the adduct formation in liver, but reduced the effect in kidney. Based on such knowledge, powerful chemopreventive strategies could be devised at exposures to 1-methylpyrene or, probably, any other well-investigated individual 6.3 Toxicokinetic Interactions Leading to Enhanced or Reduced Effects of Carcinogens j103

Table 6.1 Influence of metabolic modulators on the formation of DNA adducts in rats by 1-methylpyrene metabolites.a

Adduct level, as a multiple of control (without modulator) Test compound Modulator (dose per kg body mass) Liver Kidney

1-SMP Warfarin (250 mmol/kg): albumin ligand 0.11–0.25 — 1-HMP Probenecid (700 mmol/kg) inhibitor of 4.1–12.3 0.5–0.8 organic anion importers and exporters 1-HMP Ethanol (0.8 g/kg): competing substrate 17–18 50–86 for alcohol dehydrogenases and, indirectly, aldehyde dehydrogenases 1-HMP 4-Methylpyrazole (1000 mmol/kg): inhibitor 28–52 147–209 of alcohol dehydrogenases 1-HMP Disulfiram (250 mmol/kg): inhibitor of 3.8 — aldehyde dehydrogenases 1-HMP Pentachlorophenol (40 mmol/kg): inhibitor of 0.16–0.21 0.33 several SULTs and alcohol dehydrogenases 1-HMP Quercetin (40 mmol/kg): in vitro inhibitor of 1.0–1.4 1.0 many XMEs, including SULTs; rapidly conjugated in vivo aAnimals were intraperitoneally treated with 1-hydroxyoxymethylpyrene (1-HMP) or 1- sulfooxymethylpyrene (1-SMP) shortly after administration of the modulator. They were killed 30 min to 24 h afterward for the analysis of DNA adducts using 32P-postlabeling or mass spectrometric methods. Values are means from 3 to 10 modulator-treated animals versus a similar number of animals only treated with the corresponding vehicle. Ranges refer to different treatment periods or analytical methods. Data from Refs [23, 25] and manuscripts in preparation; –, not studied. carcinogen. However, their practical significance would be limited, as real life involves exposure to many different identified and unidentified carcinogens rather than an individual carcinogen. And when high exposure to a specific carcinogen is recognized, reduction of exposure rather chemoprevention, would nearly always be the preferred strategy. However, toxicokinetic knowledge could be used to avoid dramatic potentiating interactions.

6.3.2 Wide Sets of Identified and Unidentified Carcinogens

Adverse effects of protoxicants may be reduced by inhibition of toxifying enzymes. However,thisisonlythecasewhenanalternativedispositionmodeexists.Forexample, the ﬁrst activation step of 1-methylpyrene, the compound discussed in the preceding section, is side-chain hydroxylation catalyzed by CYPs. 1-Methylpyrene itself is too lipophilic to be directly excreted. The only other signiﬁcant initial biotransformation reaction is ring-oxidation, also mediated by CYPs. Therefore, one would have to selectively inhibit CYPs with side-chain regioselectivity and/or enhance CYP forms 104j 6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer?

withringregioselectivity,respectively.However,thetoxification/sequestrationrolesfor the individual CYPs will change with different carcinogens. General CYP inhibition would elevate the steady state of xenobiotics in the body, which may lead to enhanced interactions with many receptors. Furthermore, it could shift metabolism into parametabolic pathways, which involve a relatively high risk of formation of reactive intermediates(e.g.,freeradicalsformedbyperoxidases)andoccuratsiteslessprepared for reactive intermediates than, for example, hepatocytes are. Therefore, it is unlikely that manipulation of the CYP system offers feasible approaches for chemprevention at the complex exposures encountered by the general population. The next and final activation step of 1-methylpyrene involves sulfation of its benzylic alcohol. At this level, several alternative pathways exist. In the case of 1- methylpyrene, further oxidation of the side chain by alcohol and aldehyde dehydrogenases and ring-hydroxylation by CYPs dominate. In general, however, SULTs are primarily competed by UGTs. Although relatively many sulfo-conjugates are electrophilically reactive, I do not know about any example where a common substrate forms a reactive glucuronide. Carboxyl acids may form reactive glucuronides, but are no substrates of SULTs; therefore SULTinhibition would not enhance their activation. However, the potential chemopreventive potential of SULT inhibition is limited, as only a subset of all carcinogens is activated by these enzymes. Moreover, several SULTs are important in the regulation of the levels of various hormones, represent an important line of defense against food-borne hormones and neurotransmitters and, therefore, are discussed as potential targets (if inhibited) for endocrine disruption [26, 27]. Broad spectrum SULTforms, such as SULT1A1, do not appear to be critical for the regulation of endogenous hormones, but may be important for the disposition of phyto/xeno-oestrogens and also other deconjugated phenolic phytochemicals. In humans, but not rats, SULT1A1 is highly expressed in the gut. Expression of human SULT1A1 leads to the activation of numerous promutagens, including N-hydroxy-PhIP (and nearly 100 other compounds studied) [9]. Interestingly, this activation is completely inhibited even in the presence of low levels of dealcoholized (or regular) red wine, coffee, beetroot juice, and other plant preparations. So, it appears that SULTs have a higher affinity to many phytochemicals than to various progenotoxicants. We have created a mouse model, expressing human SULT1A1 in the gut (as in humans) and are exploring whether this expression leads to enhanced intestinal DNA damage after exposure to SULT1A1- dependent mutagens and whether this damage can be reduced by consumption of feed rich in secondary plant metabolites. In humans as well as in the recombinant mouse, SULT1A1 is also expressed in the tissues other than the gut. However, it is unlikely that secondary plant metabolites reach sufficient levels at these sites over long time periods to inhibit the enzyme. This is already indicated by results from conventional models. Although quercetin is a more potent inhibitor of various SULTs

(IC50 for human SULT1A1 0.07 mM [26]) than pentachlorophenol in vitro, only the latter compound suppressed the DNA adduct formation by 1-hydroxymethylypyrene in the rat in vivo (Table 6.1). In conclusion, it remains to be studied whether interaction of phytochemicals with SULTs can be used for chemoprevention in the gut; in other tissues the approach does not appear to be feasible. 6.3 Toxicokinetic Interactions Leading to Enhanced or Reduced Effects of Carcinogens j105

Similar to SULTs, acetyltransferases primarily are involved in the terminal activation and sequestration of protoxicants. Two xenobiotic-metabolizing acetyltransferases are found in humans, NAT1 and NAT2 (plus genetically unrelated enzymes that acetylate cysteine conjugates and specific endogenous substrates). NAT2 plays a much more prominentroleinthetoxificationoftheinvestigatedcompoundsthanNAT1[8].Genetic polymorphismshaveastrongimpactontheexpressionandactivityofthisenzyme.Slow and fast acetylators are present in Caucasian populations in an approximately 1 : 1 ratio. Epidemiological data suggest that slow acetylators have a moderately reduced risk for colon (odds ratio 0.92) and an elevated risk for bladder cancer (odds ratio 1.46) [19]. Therefore, one might speculate that tissue-selective inhibition of NAT2 in colon might be beneficial in fast acetylators, but it is not known as to how this could be reached. Next to reduced toxification, discussed in the preceding paragraphs, enhanced detoxification may be considered for chemoprevention. At exposure to numerous diverse protoxicants, one might strive to increase the levels or intrinsic activities of enzymes that are frequently involved in sequestration or inactivation reactions, but relatively rarely in toxification reaction. A battery of genes encoding such enzymes is controlled by Nrf2. A major activation mechanism involves chemical modification of Keap1, a binding partner of Nrf2 in the cytoplasm. DNA-reactive carcinogens are electrophiles and as such are able to react with the critical thiol groups of Keap1. However, under common human exposure conditions, the levels of the active metabolites may be too low for this effect. Moreover, not all electrophiles are equal. Hard electrophiles (e.g., epoxides and reactive esters) preferentially react with hard nucleophiles (e.g., amino and hydroxyl groups, present in large numbers in DNA), whereas soft electrophiles (e.g., polarized double bonds of quinones or a,b-unsaturated aldehydes) prefer soft nucleophiles (e.g., thiol groups present in glutathione, Keap1,andmanyotherproteins).Thus,variousactivatorsofKeap1mayprimarilyhave acytotoxic,ratherthangenotoxicpotential.However,theinduceddefensesystemdoes not inactivate only soft electrophiles but also hard ones and sequester their metabolic precursors. Indeed, activation of the Nrf2 has diminished the tumorigenesis induced by several carcinogens in animal models. Aparticular strong protection was observed fl in the rat against the hepatocarcinogenicity of a atoxin B1 [28]. An intervention study in a Chinese region with high aflatoxin exposure indicates that drugs and dietary factors activating Nrf2 can alter levels of urinary aflatoxin metabolites and nucleoside adducts, although the effects were not as strong as might have been hoped [29, 30]. It is still a long way to optimize chemopreventive treatments and to prove efficacy on actual fi fl tumorigenesis rather than biomarkers, speci cally against a atoxin B1 and generally against the whole set of carcinogenic factors in our environment. Efficacy may require continuous intervention starting at a young age. Thus, safety factors become important, too. Open questions are (a) Electrophiles that react with Keap1 will also react with numerous other proteins and, to varying extents, with other cellular structures. For example, after feeding broccoli to mice and rats, we could detect substantial levels of glucosinolates- derived DNA adducts in many tissues, and the corresponding glucosinolates were potent mutagens in bacteria and mammalian cell in culture (Baasanjav and Glatt, 106j 6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer?

in preparation). Under these conditions, the choice of the electrophile may become pivotal. Alternatively, one might prefer compounds that activate Nrf2 via a kinase-dependent pathway (Section 7.2.2) rather than chemical reactions at Keap1 thiol groups. (b) If long-term activation of Nrf2 is preventive against cancer (what has been demonstrated in special animal models, but not yet for the general population), one is tempted to ask the same questions as we have raised at the end of Section 6.2.5 for good enzymes: why has evolution not led to higher constitutive activity? Is there a price to pay in form of other, unfavorable health effects? (c) Another possible problem of the activation of the Nrf2 gene battery, like any manipulation of the xenobiotic-metabolizing system, is an altered bioavailability of some drugs or their active metabolites, requiring altered dosing. These drugs have to be identiﬁed. We have not yet addressed phase 0 of xenobiotic disposition as a possible target for chemoprevention. After oral administration of the food-derived carcinogens, [14C] PhIP plasma values were nearly twofold higher in mice with homozygous knockout of multidrug resistance protein 2 versus wild-type mice, demonstrating a role of this transmembrane transporter in restricting internal exposure to a carcinogen [31]. Concurrent oral administration of chlorophyll decreased the bioavailability of ﬂ a atoxin B1 by forming a complex resistant to absorption [32]. It is likely that these mechanisms are also operative with some other carcinogens, but these will only be subset of the numerous carcinogens that humans are exposed to.

6.4 Conclusions

Genetic and chemical modulation of the xenobiotic-metabolizing system has led to strong reductions and potentiations of effects of many individual carcinogens in animalmodels.Sucheffectscouldbeachievedbyalteredactivitiesoftoxifyingaswellas detoxifying enzymes forthecorrespondingcarcinogen. However,enzymes important in the activation of one carcinogen (in particular forms of CYPs, SULTs, and acetyltransferases) may play a prominent role in the sequestration of other toxicants. Therefore, this approach for chemoprevention is limited in a typical situation, when humansareexposedtoaplethoraofdifferenttoxicantsatlowlevelsratheradominating individual carcinogen. A number of other XMEs are frequently involved in the detoxification of many chemicals, but only relatively rarely in toxification reactions. Upregulation of such enzymes may be more promising for chemoprevention. However, in the absence of a specific exposure, any purposeful modification of the xenobiotic-metabolizing system is only sound, if one postulates that evolution has not managed to optimally adjust levels of individual enzymes to current lifestyle and exposures. This hypothesis is not unreasonable, as changes in lifestyle and exposures occurred extremely rapidly in human evolution and culture. However, it would be References j107 important to know the relative importance of various xenobiotics as carcinogens and toxicants to humans and their metabolic controls by XMEs. Although knowledge in this area is rapidly expanding, it is still far too limited to allow for a rational design for an improved xenometabolic system. In addition, more information on endogenous functions and parametabolic effects of XME is required. In experimental models, neonate animals are usually much more susceptible to chemical carcinogenesis than are adult animals. Therefore, if carcinogen metabolism is the target, a chemopreventive approach should start early and be continued over long periods of life. Thus, safety factors become important. For example, upregulation of the Nrf2 gene battery appears to be a potentially promising approach, at present. This upregulation is frequently mediated by electrophiles and oxidants, which react with Keap1, the sensor, but also with numerous other cellular macromolecules. However, formation of macromolecular adducts should always be regarded as a warning light in toxicology, requiring extensive safety testing. In conclusion, xenobiotic metabolism can be a promising target for chemoprevention when a limited number of identified xenobiotics represent the dominating carcinogens and cannot be sufficiently avoided – a rare setting. In the absence of the knowledge of the critical carcinogens, it is debatable whether one can expect net benefits resulting from modulation of the xenobiotic-metabolizing system.

References

1 Vansell, N.R. and Klaassen, C.D. (2001) Xenobiotics (ed. C. Ioannides), John Wiley Toxicology and Applied Pharmacology, 176, & Sons, Sussex, pp. 353–439. 187–194. 7 Levy, G.N. and Weber, W.W. (2002) in 2 Caldwell, J. (1982) Drug Metabolism Handbook of Enzyme Systems that Reviews, 13, 745–777. Metabolise Drugs and Other Xenobiotics (ed. 3 Kunert-Keil, C., Ritter, C.A., Kroemer, H.K. C. Ioannides), John Wiley & Sons, Sussex, and Sperker, B. (2002) in Handbook pp. 441–457. of Enzyme Systems that Metabolise 8 Glatt, H.R. and Meinl, W.(2005) Methods in Drugs and Other Xenobiotics (ed. C. Enzymology, 400, 230–249. Ioannides), John Wiley & Sons, Sussex, 9 Glatt, H.R. (2005) in Human Cytosolic pp. 521–554. Sulfotransferases (eds G.M. Paciﬁci and 4 Hiratsuka, A., Okada, T., Nishiyama, T., M.W.H. Coughtrie), Taylor & Francis, Fujikawa, M., Ogura, K., Okuda, H., London, pp. 281–306. Watabe, T. and Watabe, T. (1994) 10 Ritter, J.K. (2000) Chemico-Biological Biochemical and Biophysical Research Interactions, 129, 171–193. Communications, 202, 278–284. 11 Boberg, E.W., Miller, E.C., Miller, J.A., 5 Sheratt, P.J. and Hayes, S.J.D. (2002) in Poland, A. and Liem, A. (1983) Cancer Handbook of Enzyme Systems that Research, 43, 5163–5173. Metabolise Drugs and Other Xenobiotics (ed. 12 Ringer, D.P., Norton, T.R., Cox, B. and Howell, C. Ioannides), John Wiley & Sons, Sussex, B.A. (1988) Cancer Letters, 40,247–255. pp. 317–352. 13 Lai, C.-C., Miller, E.C., Miller, J.A. and 6 Glatt, H.R. (2002) in Handbook of Enzyme Liem, A. (1987) Carcinogenesis, 8, Systems that Metabolise Drugs and Other 471–478. 108j 6 Xenobiotic Metabolism: A Target for Nutritional Chemoprevention of Cancer?

14 Ku, W.W., Bigger, A., Brambilla, G., Glatt, 26 Waring, R.H., Ayers, S., Gescher, A.J., H.R., Gocke, E., Guzzie, P.J., Hakura, A., Glatt, H.R., Meinl, W., Jarratt, P., Kirk, C.J., Honma, M., Martus, H.J., Obach, R.S. and Pettitt, T., Rea, D. and Harris, R.M. (2008) Roberts, S. (2007) Mutation Research, 627, Journal of Steroid Biochemistry and 59–77. Molecular Biology, 108, 213–220. 15 Hoelzl, C., Glatt, H.R., Meinl, W., Sontag, 27 Kester, M.H., Bulduk, S., van Toor, H., G., Haidinger, G., Kundi, M., Simic, T., Tibboel, D., Meinl, W., Glatt, H.R., Falany, Chakraborty, A., Bichler, J., Ferk, F., C.N., Coughtrie, M.W., Schuur, A.G., Angelis, K., Nersesyan, A. and Knasmuller,€ Brouwer, A. and Visser, T.J. (2002) The S. (2008) Molecular Nutrition & Food Journal of Clinical Endocrinology and Research, 52, 330–341. Metabolism, 87, 1142–1150. 16 Muckel, E., Frandsen, H. and Glatt, H.R. 28 Yates, M.S., Kwak, M.K., Egner, P.A., (2002) Food and Chemical Toxicology, 40, Groopman, J.D., Bodreddigari, S., Sutter, 1063–1068. T.R., Baumgartner, K.J., Roebuck, B.D., 17 Buters, J.T.M., Sakai, S., Richter, T., Liby, K.T., Yore, M.M., Honda, T., Gribble, Pineau, T., Alexander, D.L., Savas, U., G.W.,Sporn, M.B. and Kensler, T.W.(2006) Doehmer, J., Ward, J.M., Jefcoate, C.R. and Cancer Research, 66, 2488–2494. Gonzalez, F.J. (1999) Proceedings of the 29 Wang, J.S., Shen, X., He, X., Zhu, Y.R., National Academy of Sciences of the United Zhang,B.C.,Wang,J.B.,Qian,G.S.,Kuang, States of America, 96, 1977–1982. S.Y., Zarba, A., Egner, P.A., Jacobson, L.P., 18 Miyata, M., Kudo, G., Lee, Y.H., Yang, T.J., Munoz, A., Helzlsouer, K.J., Groopman, Gelboin, H.V., Fernandez-Salguero, P., J.D. and Kensler, T.W. (1999) Journal of the Kimura, S. and Gonzalez, F.J. (1999) The National Cancer Institute, 91, 347–354. Journal of Biological Chemistry, 274, 30 Kensler,T.W.,Chen,J.G.,Egner,P.A.,Fahey, 23963–23968. J.W.,Jacobson,L.P.,Stephenson,K.K.,Ye,L., 19 Dong, L.M., Potter, J.D., White, E., Ulrich, Coady, J.L., Wang, J.B., Wu, Y., Sun, Y., C.M., Cardon, L.R. and Peters, U. (2008) Zhang, Q.N., Zhang, B.C., Zhu, Y.R., Qian, The Journal of the American Medical G.S., Carmella, S.G., Hecht, S.S., Benning, Association, 299, 2423–2436. L., Gange, S.J., Groopman, J.D. and Talalay, 20 Carlsten, C., Sagoo, G.S., Frodsham, A.J., P. (2005) Cancer Epidemiology, Biomarkers & Burke,W.and Higgins, J.P.(2008)American Prevention, 14,2605–2613. Journal of Epidemiology, 167,759–774. 31 Vlaming, M.L., Mohrmann, K., Wagenaar, 21 Rice, J.E., Rivenson, A., Braley, J. and E., de Waart, D.R., Elferink, R.P., Lagas, LaVoie, E.J. (1987) Journal of Toxicology and J.S., van Tellingen, O., Vainchtein, L.D., Environmental Health, 21, 525–532. Rosing, H., Beijnen, J.H., Schellens, J.H. 22 Engst, W., Landsiedel, R., Hermersdorfer,€ and Schinkel, A.H. (2006) The Journal of H., Doehmer, J. and Glatt, H.R. (1999) Pharmacology and Experimental Carcinogenesis, 20, 1777–1785. Therapeutics, 318, 319–327. 23 Ma, L., Kuhlow, A. and Glatt, H.R. (2002) 32 Simonich, M.T., Egner, P.A., Roebuck, PolycyclicAromaticCompounds,22,933–946. B.D., Orner, G.A., Jubert, C., Pereira, C., 24 Kollock, R., Frank, H., Seidel, A., Meinl, W. Groopman, J.D., Kensler, T.W., Dashwood, andGlatt,H.R.(2008)Toxicology,245,65–75. R.H., Williams, D.E. and Bailey, G.S. 25 Ma, L., Kuhlow, A. and Glatt, H.R. (2003) (2007) Carcinogenesis, 28, 1294–1302. Nova Acta Leopoldina NF87, 329, 265–272. j109

7 Dietary Factors Regulate Metabolism of Carcinogens through Transcriptional Signaling Pathways Soona Shin and Thomas W. Kensler

7.1 Introduction

Cells manifest many adaptive responses to environmental and endogenous stresses to promote their survival. In many cases, these responses include enhanced gene expression to expand cell capacity to detoxicate and/or eliminate xenobiotics including carcinogens. Cytochrome P-450s (CYPs) add or uncover a reactive functional group on which conjugative or nucleophilic trapping reactions can occur. Reactions performed by the multiple families of CYPs include oxidation, reduction, and hydrolysis. Metabolites formed by CYPs can sometimes be reactive, leading to damage of DNA and other biomolecules. Conjugating and other enzymes transform these toxic metabolites into nonreactive, more hydrophilic products that can be readily excreted. Examples of enzymes that belong to this category are UDP- glucuronosyl transferases (UGTs), glutathione S-transferases (GSTs), and NAD(P) H:quinone oxidoreductase 1 (NQO1). Modulation of the expression of these enzymes has been shown to dramatically modify pharmacological and toxicological responses to xenobiotics. Therefore, understanding how these enzymes are regulated is useful in terms of (i) providing molecular targets for screening of potential chemopreventive agents; (ii) identifying and evaluating food sources that modulate desired transcription factor targets; (iii) providing insight into downstream intermediate biomarkers of pharmacodynamic action of agent or dietary interventions before reaching the end point of cancer; and (iv) predicting pharmacokinetics, interindividual variations in response, and the side effects that might arise through activation of the target pathway. Multiple steps leading from a gene sequence in DNA to a functional protein are subject to regulation. However, the rate-limiting step is often the initiation of transcription that is regulated through transcription factors, DNA binding proteins that recognize cis-regulatory elements located in the upstream promoters of target genes. The function of transcription factors is affected by their subcellular localization, extent and type of post-translational modiﬁcation such as phosphorylation, acetylation or ubiquitinization, and consequently, stability. This chapter will

Figure 7.1 Transcription factors influencing the factors translocate to the nucleus where they expression of carcinogen-metabolizing induce gene expression affecting multiple enzymes. Activators induce expression of processes. Although processes are summarized enzymes involved in carcinogen metabolism as one arrow, each signaling pathway regulates through (1) direct binding to the transcription distinct patterns of responses (see text for factor (AhR, PXR, and CAR) or (2) indirect details). PXR in cytoplasm is shown by broken mechanisms involving modification of lines because it is a matter for debate whether cytoplasmic binding partners and/or PXR is located exclusively in the nucleus or not. coregulators (Nrf2 and CAR). Transcription

primarily focus on how dietary and other stimuli modulate the distribution and fate of transcription factors inﬂuencing the expression of carcinogen-metabolizing enzymes (summarized in Figure 7.1 and Table 7.1). As with the transcription factors, their transcriptional progeny can also be directly modulated in post-transcriptional ways, such as by direct phosphorylation of the enzymes. In addition, gene accessibility to transcription factors can be controlled by DNA methylation and other epigenetic means that silence regions of the genome. These regulatory mechanisms are also likely to be targets of dietary components.

7.2 Nrf2

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that belongs to the CapnCollar (CNC) subfamily of bZIP transcription factors. Nrf2 regulates constitutive and inducible expression of cytoprotective and antioxidant genes that catalyze the detoxication of xenobiotics through conjugation processes, inactivate Table 7.1 Summary of compounds that interact with signaling pathways.

Pathway Compounds Mode of action

Nrf2 3H-1,2-dithiole-3-thione Cruciferous vegetables Induce conformational changes in Keap1 that renders the Keap1 protein unable to repress Nrf2 signaling by reacting with sulfhydryl groups of Keap1 and/or by triggering kinase signaling cascades. Sulforaphane Cruciferous vegetables 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien- Synthetic analog of oleanolic acid 28-oyl]imidazole Butylated hydroxyanisole Antioxidant food preservative Curcumin Indian curry spice turmeric Triphlorethol-A Phlorotannin found in Ecklonia cava AhR 2,3,7,8-Tetrachlorodibenzo-p-dioxin, Environmental pollutants AhR ligands, induce dissociation of AhR from its polychlorinated biphenyls, benzo[a]pyrene cytoplasmic binding partners. Indole-3-carbinol Cruciferous vegetables Quercetin, epigallocatechin gallate Dietary ﬂavonols Act as antagonists against AhR signaling either by competing with TCDD for binding to the AhR or through an indirect mechanism involving Hsp90. CAR Phenobarbital, 6-(4-chlorophenyl)imidazo Induces translocation of CAR to the nucleus in [2,1-b][1,3]thiazole-5-carbaldehyde a PP2A-dependent way. CITCO and TCPOBOP O-(3,4-dichlorobenzyl)oxime, 1,4-bis are the only known ligands of CAR. [2-(3,5-dichloropyridyloxy)]benzene Wormwood Chinese herbal teas Yin Zhi Huang and Yin Chin Androstane steroidal compounds Bind to CAR, represses the constitutive activity. . Nrf2 7.2 PXR Sulforaphane Cruciferous vegetables Binds to PXR, inhibits recruitment of coactivator. Pregnenolone-16a-carbonitrile, rifampicin, PXR ligands, regulate interactions between PXR 5b-pregnane-3,20-dione, progesterone, and coregulators by protein phosphorylation. j

corticosterone, dexamethasone 111 Hyperforin St. Johns wort Kava kava, Wu Wei Zi, and Ging Cao Herbal remedies

See text for references. 112j 7 Dietary Factors Regulate Metabolism of Carcinogens through Transcriptional Signaling Pathways

reactive oxygen species, produce antioxidants, increase intracellular concentration of glutathione via synthesis or regeneration, remove unfolded or damaged proteins, and attenuate inﬂammation [1, 2]. Studies based on the analysis of Nrf2-disrupted mice support the importance of this pathway in its protection from exposure to environmental toxins, inﬂammatory stresses, neurodegeneration, asthma, and carcinogenesis [1]. As described below, the Nrf2 pathway can be modulated by dietary factors and pharmacological agents. This raises possibility for control and prevention of several chronic diseases through activation of Nrf2 signaling.

7.2.1 Keap1-Mediated Regulation of Nrf2

Nrf2, together with antioxidant response elements (AREs), and Keap1 are the three essential components of Nrf2 signaling. AREs are cis-elements found in promoters of target genes. Nrf2 heterodimerizes with small Maf proteins that collectively bind to AREs to up- or downregulate transcription of their target genes. Keap1, a repressor for Nrf2 action, retains Nrf2 in the cytoplasm and facilitates its degradation by ubiquitination and proteasomal hydrolysis. Nrf2 signaling responds to oxidative stresses and shear stresses, as well as to second messengers (nitric oxide, growth factors, oxidized lipids) [2]. The pathway can also be activated by pharmacological agents and natural compounds. These inducers facilitate nuclear translocation of Nrf2 and subsequent binding to AREs by changing conformation of Keap1 and/or triggering kinase signaling cascades (described in detail below). 3H-1,2-dithiole-3-thione (D3T) and sulforaphane have been isolated from cruciferous vegetables and are well-studied natural product activators of the Nrf2 signaling cascade. In addition, pharmacologic probes (1-[2-cyano-3-,12- dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), an extremely potent synthetic analog of oleanolic acid) and genetic approaches (siRNA against Keap1) are being used to better understand the full cytoprotective capacity of this pathway [1, 2]. Unlike other transcription factor pathways discussed in this chapter, inducers of the Nrf2 signaling cascade do not interact in a ligand–receptor mechanism, but rather have a common chemical feature: the ability to react with sulfhydryl groups of Keap1 [2]. Keap1 is an actin binding protein that forms a homodimer through its BTB domain (bric-a-brac, tramtrack, broad complex). This dimerization is required for the interaction with Nrf2. Keap1 serves as an adaptor molecule for cullin3-based E3 ligase. E3 ligase catalyzes ubiquitination of its substrate and targets the substrate to subsequent degradation. Under basal, unstressed conditions, Keap1 targets Nrf2 for degradation such that its half-life is 15–20 min and little accumulates in the nucleus. Binding of inducers to Keap1 leads to a conformational change that renders the Keap1 protein unable to repress Nrf2 signaling. Nrf2 then translocates to the nucleus and activates transcription of its target genes. It has been proposed recently that conformational changes in Keap1 lead to repression of Keap1-mediated degradation of Nrf2 rather than dissociation of Nrf2 from Keap1 [3]. Nrf2 can also regulate its own transcription in response to inducers by binding to AREs located in the promoter of the Nrf2 gene, thereby increasing the levels of this transcription factor [1]. 7.2 Nrf2 j113

Coactivators are the proteins that either direct activator recruitment of the transcriptional apparatus or remodel chromatin structure. Nrf2 cooperates with the small Maf proteins MafG and MafF for recognition of AREs. In addition, receptor- associated coactivator (RAC3) and steroid receptor coactivator-3 (SRC3) interact with the transactivation domain of Nrf2 to enhance transcriptional activity of the complex. CBP(CREB binding protein)/p300 interacts with coregulators as well as with the basal transcription machinery and is required for the activation of a number of transcription factors. CBP/p300 further enhances the transactivation activity mediated by RAC3/SRC3 [4]. Increased understanding of Keap1-dependent activation of Nrf2 signaling facilitated development of methods for screening of potential chemopreventive agents from natural compounds. On the basis of high-throughput mass spectrometry-based screening of modification of human Keap1, Liu et al. identified two chemopreventive compounds from extract of hops, xanthohumol, and xanthohumol D [5]. Using the Prochaska assay that monitors for increases in NQO1 activity, Talalay and colleagues first isolated and identified sulforaphane as an active inducer and chemopreventive agent from cruciferous vegetables such as broccoli [6]. This assay is now used in many laboratories for identifying bioactive food components with possible chemopreventive attributes.

7.2.2 Phosphorylation

Recent evidence suggests that a series of kinase pathways are involved in activation of Nrf2 signaling. Protein kinases are enzymes that catalyze transfer of the terminal phosphate group of ATP to serine, threonine, or tyrosine in proteins. This reaction can be reversed by dephosphorylation, which is catalyzed by phosphatases. Phos- phorylation affects signaling by either inducing a conformational change in the protein or creating a binding site that can be recognized by other proteins. Phos- phorylation of Nrf2 enables cross-talk with multiple signaling pathways and rapid adaptation to stresses. The following are only a few examples of kinases that are involved in the Nrf2 signaling cascade. A number of dietary compounds such as curcumin (an antioxidant found in the Indian curry spice turmeric), triphlorethol-A (phlorotannin found in Ecklonia cava), and sulforaphane modulate the fate of Nrf2 in a kinase-dependent manner [7–9], suggesting that kinase signaling can be used for development of cancer chemopreventive compounds. However, the network of responses resulting from interaction of Nrf2 with numerous kinase pathways remains to be determined.

7.2.2.1 Mitogen-Activated Protein Kinases The extracellular signal-regulated kinase (ERK) family, the c-Jun NH2-terminal kinase (JNK) family, and the p38 kinase family are the major constituents of mitogen-activated protein (MAP) kinases. Members of MAP kinases are important mediators of cellular responses, such as regulation of cell death or cell survival, cell cycle progression, and malignant transformation. In general, while the ERK pathway 114j 7 Dietary Factors Regulate Metabolism of Carcinogens through Transcriptional Signaling Pathways

responds to growth factors, p38 and JNK pathways are activated by growth and stress stimuli. It has been demonstrated that the ERK and JNK pathways play positive roles in mediating gene expression by the phenolic antioxidant butylated hydroxyanisole (BHA) as well as the nuclear translocation of Nrf2 in HepG2 cells [10]. In addition, triphlorethol-A-induced heme oxygenase-1 (HO-1) expression occurs via an ERK–Nrf2–ARE signaling pathway [8]. On the contrary, sulforaphane suppressed anisomycin (an antibiotic)-induced activation of p38 MAPK isoforms. Phosphorylation of Nrf2 protein by p38 enhanced the interaction between Nrf2 and Keap1 in vitro [9]. However, studies by other groups have shown that p38 plays a positive role in the induction of HO-1 and GCLM (the modiﬁer subunit of glutamate cysteine ligase) [7, 9].

7.2.2.2 Protein Kinase C Protein kinase C (PKC) subfamilies comprise at least 10 isoenzymes that are serine and threonine speciﬁc [7]. Among these isoenzymes, PKC delta regulates cell cycle and programmed cell death in various cell lines. For example, PKC delta mediates cytotoxicity induced by tumor necrosis factor (TNF) during inﬂammatory processes. It has been shown that phosphorylation of Ser40 of Nrf2 by PKC delta is necessary for Nrf2 release from Keap1 [1]. Furthermore, rottlerin (a PKC delta inhibitor) and PKC delta antisense oligonucleotides inhibited expression of Nrf2 target genes GCLM and HO-1 by curcumin in human monocytes [7].

7.2.2.3 CK2 The protein kinase CK2 (formerly casein kinase II) plays pleiotropic roles in signal transduction, gene expression, RNA synthesis, ubiquitination, and cell survival. It is activated when bound to calmodulin. Calmodulin is a calcium binding protein that undergoes a conformational change when activated. Two phosphorylated forms of human Nrf2 by CK2 have been identiﬁed: Nrf2-118 and Nrf2-98. They are increased in response to Nrf2 inducers. Phospho-118-Nrf2 does not bind to ARE and is more susceptible to degradation compared to phospho-98-Nrf2, suggesting that the latter has transcriptional activity. This ﬁnding implies that the sites and extent of phosphorylation differentially affect Nrf2 action [11].

7.3 Aryl Hydrocarbon Receptor

Aryl hydrocarbon receptor (AhR), a member of the basic helix–loop–helix/Per-Aryl hydrocarbon receptor nuclear translocator (Arnt)-Sim family, regulates transcription of CYP1As and UGTs in a ligand-dependent manner. Several AhR ligands are environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls, and benzo[a]pyrene. CYP1As including CYP1A1, 1A2, and 1B1 are enzymes that can biotransform endogenous and exogenous procarcinogens into toxic metabolites. Therefore, it has been widely accepted that the AhR pathway contributes to the bioactivation of carcinogens. Indeed, Ahr-null mice are more 7.3 Aryl Hydrocarbon Receptor j115 resistant to skin carcinogenesis induced by subcutaneous injection of benzo[a] pyrene compared to wild-type mice [12]. However, ﬁrst-pass metabolism through AhR–CYP1 signaling can also play a role in systemic elimination of carcinogens. For example, Cyp1a1-null mice are more susceptible to oral benzo[a]pyrene exposure than wild-type mice [13]. Furthermore, it has been demonstrated that AhR and Nrf2 upregulate each others expression, suggesting that these two pathways might cooperate for elimination of toxic xenobiotics [14].

7.3.1 Regulation

When bound to a ligand, AhR is released from its cytoplasmic binding partners the heat shock protein Hsp90, p23, and AhR-interacting protein (AIP). Upon dissociation, a nuclear localization signal (NLS) in the basic helix–loop–helix (bHLH) domain of AhR is exposed and AhR translocates into the nucleus. In addition, phosphorylation of the NLS by PKC negatively regulates nuclear translocation induced by binding of ligands. Following nuclear translocation, AhR dimerizes with its nuclear partner Arnt and recognizes the consensus sequence in its target genes, the xenobiotic- responsive element (XRE). Multiple XREs are found in the upstream enhancer region of CYP1A1; a GC box in the proximal promoter region is also required for transactivation [12]. Dietary flavonols quercetin and epigallocatechin gallate (EGCG, the most abundant catechin in green tea) act as antagonists against AhR signaling either by competing with TCDD for binding to the AhR or through an indirect mechanism involving Hsp90 [15, 16]. Green tea provides protection against polyaromatic hydrocarbon-induced cancers suggesting that inhibition of AhR signaling can be a target of chemoprevention. Therefore, AhR-based bioassays have been established as a tool for screening of inhibitors in food extracts. Among the herbs, the vegetables and the fruits, extracts of sage, green leafy ones such as spinach, and citrus were identified to contain inhibitors of AhR activation by TCDD [17]. Dietary agonists of AhR signaling have also been identified. Quercetin, as mentioned earlier, competes with TCDD and is a weak inducer of AhR signaling. Indole-3-carbinol (found in cruciferous plants including broccoli and brussels sprouts) is converted to an AhR agonist in the acidic environment in the stomach. The AhR pathway is a highly conserved pathway throughout evolution, and studies based on Ahr- null mice have shown that AhR plays a critical role in vascular development. Further- more, multiple candidates for endogenous ligands have been identified, implying the existence of physiological roles of AhR. These candidates compete with TCDD for binding to AhR and induce XRE-driven expression of CYP1A1. Examples are indigoid, equilenin (an equine estrogen), arachidonic acid metabolites, heme metabolites, and tryptophan metabolites. Taken together, these findings suggest that dietary agonists of AhR can be used to modulate physiological pathways [15]. It has been demonstrated that various cofactors are involved in the activation of AhR signaling. Remodeling of chromatin structure is an important aspect of transcriptional regulation by AhR. Brahma/SWI2-related gene 1 (Brg-1) protein 116j 7 Dietary Factors Regulate Metabolism of Carcinogens through Transcriptional Signaling Pathways

utilizes ATP to disrupt histone–DNA interactions within the CYP1A1 gene following treatment with TCDD. AhR/Arnt also work together with coactivator proteins including CBP/p300, SRC1, NCoA2, and p/CIP and the coactivator/corepressor protein RIP140. In addition, AhR interacts with the mediator/TRAP/DRIP/ARC multisubunit complex in a TCDD-dependent fashion [12]. The extent to which phytochemicals may influence these interactions is unknown. The AhR signaling pathway is regulated through a negative feedback loop. AhR repressor (AhRR) is one of the target genes of AhR and is exclusively located in the nucleus. AhRR can form a heterodimer with Arnt that leads to transcriptional repression. By using yeast two-hybrid screening method, ankyrin repeat protein 2 (ANKRA2) has been identified as a binding partner for AhRR [18]. ANKRA2 is a protein that interacts with histone deacetylases, enzymes that remove acetyl groups from tails of histones and render DNA bound by histones less accessible to transcription factors. AhR signaling can also be activated in an exogenous ligand-independent manner. For example, omeprazole (a proton pump inhibitor that suppresses production of gastric acid) induces transcription of CYP1A1 in an AhR-dependent manner, but it does not bind AhR directly. Disruption of cell–cell interaction and culturing cells under subconfluent conditions can also induce nuclear translocation of AhR from cytoplasm to nucleus and subsequent expression of CYP1A1 and CYP1B1 in the absence of exogenous ligand [12]. The mechanism and physiological relevance of ligand-independent activation need further investigation.

7.4 Nuclear Receptors

The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are transcription factors that belong to the nuclear receptor superfamily. Their roles as sensors for lipophilic xenobiotics have been established recently. PXR and CAR are the major regulators of CYP3A and CYP2B families, respectively. However, recent studies revealed that PXR regulates CYP2B and CAR regulates CYP3A in vivo. CYP2B and CYP3A metabolize a number of drugs used clinically; CYP3A4 alone is involved in the metabolism of more than 50% of pharmaceuticals [19]. Understanding PXR- and CAR-mediated regulation of these enzymes is critical for prediction of drug kinetics—namely, hepatic clearance of drugs, interindividual variation in drug response, and also drug–drug or drug–food interactions. For example, St. Johns wort, an herb that contains hyperforin (a potent inducer of human PXR), is a representative example for unwanted herb–drug interactions; interactions of St Johns wort with cyclosporin (an immunosuppressant drug) and indinavir (an antiviral drug) resulted in an acute heart transplant rejection and an increased HIV load, respectively, due to the reduction of plasma concentration of drugs [19, 20]. These nuclear receptors also regulate conjugating enzymes and transporters involved in drug or xenobiotic metabolism, such as UGT, sulfotransferase (SULT), GST, multidrug resistance protein (MRP), P-glycoprotein, and organic anion 7.4 Nuclear Receptors j117 transporter 2 [21]. In addition to their roles in regulating xenobiotic metabolism, nuclear receptors regulate conversion of cholesterol into bile acids and play a protective role against cholestasis. CYP7A, the rate-limiting enzyme in bile acid formation, is repressed by PXR. On the contrary, CAR upregulates CYP3A11, MRP3, and SULT2A1, enzymes involved in excretion of bile acids [20]. PXR and CAR share target genes, ligands, as well as coactivators. However, some activators induce downstream genes preferentially through CAR, while others do so through PXR [22].

7.4.1 Regulation of the CAR Pathway

Examples of chemicals that activate CAR signaling are phenobarbital, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime(CITCO), and 1,4-bis[2-(3,5- dichloropyridyloxy)]benzene (TCPOBOP) [20]. In addition, wormwood(componentofChineseherbalteasYinZhiHuangandYinChin)inducesCYP2B throughCAR.Ithasbeenshownthattheseherbalteasacceleratebilirubinclearanceina CAR-dependent manner [23]. Upon exposure to phenobarbital, CAR in the cytoplasmic compartment is released from chaperones, cytoplasmic CAR retention protein (CCRP) and Hsp90 and translocates to the nucleus. CAR forms a heterodimer with the retinoid X receptor (RXR) and binds to phenobarbital-responsive enhancer modules (PBREM) found in the promoter region of CYP2B. Among diverse chemicals that activate CAR signaling, TCPOBOP and CITCO are the only compounds reported to directly bind to CAR in mouse and human, respectively [20]. Additional mechanisms are involved in transcriptional upregulation of CYP2B by CAR in response to phenobarbital. Translocation of CAR to the nucleus depends on the activity of the protein phosphatase PP2A. In addition, glucocorticoid receptor-interacting protein 1 (GRIP1, coactivator of CAR) is reported to enhance nuclear translocation and transactivation by CAR. In the absence of exogenous compounds, steroid ligands bind to the receptor and render it transcriptionally inactive. This repression is reversed by treatment with activators [22]. However, the exact mechanism of activation of CAR signaling remains unknown.

7.4.2 Regulation of the PXR Pathway

Activators of PXR signaling are rifampicin, 5b-pregnane-3,20-dione, progesterone, corticosterone, hyperforin, and dexamethasone [20]. Herbal remedies such as kava kava, Wu Wei Zi, and Ging Cao also induce CYP3A4 through PXR [19]. Although it has been thought that PXR was a nuclear protein, in 2004 it was demonstrated that PXR is found in cytoplasm of mouse liver and that it translocates after interaction with its ligand, pregnendone-16a-carbonitrile (PCN) [20]. Under basal condition, PXR forms a complex with CCRP and Hsp90. Binding of ligand induces dissociation and translocation of PXR to the nucleus. In the nucleus, PXR forms a complex with RXR and binds to a xenobiotic-responsive enhancer module (XREM) in the promoter region of the 118j 7 Dietary Factors Regulate Metabolism of Carcinogens through Transcriptional Signaling Pathways

CYP3A gene. Multiple coactivators and corepressors are involved in the PXR pathway. Interactions between these coregulators with PXR are regulated by protein phosphorylation. Phosphorylation of PXR by protein kinase A (PKA) potentiates its interaction with coactivators. In contrast, PKC-mediated phosphorylation of PXR reduced transactivation and binding to a coactivator, steroid receptor coactivator-1 [21]. A number of assays have been developed for screening PXR ligands. Several drug–drug interactions could be attributed to activation of PXR and subsequent induction of CYP3A. Hyperforin and ritonavir (an antiviral drug) are examples of PXR ligands identiﬁed in this way [22]. Recently, sulforaphane has been identiﬁed as an antagonist of PXR, suggesting that diets can be a potential approach to reduce drug interactions [24].

7.5 Conclusions

This chapter has described how dietary factors interact with multiple transcriptional signaling pathways that regulate genes involved in the metabolism of carcinogens. It should be emphasized that this chapter has not reviewed all of the signaling pathways that transcriptionally regulate enzymes catalyzing the metabolism of carcinogens. Only pathways that are well known to respond to dietary factors and/or pharmacological agents are highlighted. Billions of dollars are spent per year on dietary supplements such as herbal medicines [19, 23]. These dietary supplements are often chosen based on their long- standing use in traditional medicines rather than critical scientific evaluation. Typically, their composition and formulation are not subject to strict standardization or regulation. However, recent mechanistic studies have identified transcriptional signaling pathways underlying the biochemical properties of some dietary factors. Information on the molecular targets of dietary factors is now available. This knowledge has offered tools for evaluating safety, efficacy, dosing strategy, and most importantly, methods for screening and identifying effective chemopreventive compounds out of complex dietary sources. There is no doubt that further mechanism-based studies need to be conducted in order to identify or develop more safe and potent chemopreventive compounds, supplements, and/or foods.

References

1 Kensler, T.W.,Wakabayashi, N. and Biswal, in cellular protective responses. Chemical S. (2007) Cell survival responses to Research in Toxicology, 18, 1779–1791. environmental stresses via the Keap1- 3 Kobayashi, A., Kang, M.I., Watai, Y., Tong, Nrf2-ARE pathway. Annual Review of K.I., Shibata, T., Uchida, K. and Pharmacology and Toxicology, 47,89–116. Yamamoto, M. (2006) Oxidative and 2 Dinkova-Kostova, A.T., Holtzclaw, W.D. electrophilic stresses activate Nrf2 through and Kensler, T.W. (2005) The role of Keap1 inhibition of ubiquitination activity of References j119

Keap1. Molecular and Cellular Biology, 26, coupled with ERK and JNK signaling 221–229. pathway in HepG2 cells. Molecular 4 Lin, W., Shen, G., Yuan, X., Jain, M.R., Yu, Carcinogenesis, 45, 841–850. S., Zhang, A., Chen, J.D. and Kong, A.N. 11 Pi, J., Bai, Y., Reece, J.M., Williams, J., Liu, (2006) Regulation of Nrf2 transactivation D., Freeman, M.L., Fahl, W.E., Shugar, D. domain activity by p160 RAC3/SRC3 and et al. (2007) Molecular mechanism of other nuclear co-regulators. Journal of human Nrf2 activation and degradation: Biochemistry and Molecular Biology, 39, role of sequential phosphorylation by 304–310. protein kinase CK2. Free Radical Biology & 5 Liu, G., Eggler, A.L., Dietz, B.M., Mesecar, Medicine, 42, 1797–1806. A.D., Bolton, J.L., Pezzuto, J.M. and van 12 Kawajiri, K. and Fujii-Kuriyama, Y. (2007) Breemen, R.B. (2005) Screening method Cytochrome P450 gene regulation and for the discovery of potential cancer physiological functions mediated by the chemoprevention agents based on mass aryl hydrocarbon receptor. Archives of spectrometric detection of alkylated Keap1. Biochemistry and Biophysics, 464, 207–212. Analytical Chemistry, 77, 6407–6414. 13 Uno, S., Dalton, T.P., Dragin, N., Curran, 6 Fahey, J.W., Dinkova-Kostova, A.T., C.P., Derkenne, S., Miller, M.L., Shertzer, Stephenson, K.K. and Talalay, P. (2004) H.G., Gonzalez, F.J. et al. (2006) Oral The Prochaska microtiter plate bioassay benzo[a]pyrene in Cyp1 knockout mouse for inducers of NQO1. Methods in lines: CYP1A1 important in detoxication, Enzymology, 382, 243–258. CYP1B1 metabolism required for immune 7 Rushworth, S.A., Ogborne, R.M., damage independent of total-body burden Charalambos, C.A. and OConnell, M.A. and clearance rate. Molecular (2006) Role of protein kinase C delta in Pharmacology, 69, 1103–1114. curcumin-induced antioxidant response 14 Shin, S., Wakabayashi, N., Misra, V., element-mediated gene expression in Biswal, S., Lee, G.H., Agoston, E.S., human monocytes. Biochemical and Yamamoto, M. and Kensler, T.W. (2007) Biophysical Research Communications, 341, NRF2 modulates aryl hydrocarbon 1007–1016. receptor signaling: inﬂuence on 8 Kang, K.A., Lee, K.H., Park, J.W., Lee, N.H., adipogenesis. Molecular and Cellular Na, H.K., Surh, Y.J., You, H.J. Chung, M.H. Biology, 27, 7188–7197. et al. (2007) Triphlorethol-A induces heme 15 Nguyen, L.P. and Bradﬁeld, C.A. (2007) oxygenase-1 via activation of ERK and The search for endogenous activators of NF-E2 related factor 2 transcription factor. the aryl hydrocarbon receptor. Chemical FEBS Letters, 581, 2000–2008. Research in Toxicology, 21, 102–116. 9 Keum, Y.S., Yu, S., Chang, P.P., Yuan, X., 16 Palermo, C.M., Westlake, C.A. and Kim, J.H., Xu, C., Han, J., Agarwal, A. et al. Gasiewicz, T.A. (2005) Epigallocatechin (2006) Mechanism of action of gallate inhibits aryl hydrocarbon receptor sulforaphane: inhibition of p38 mitogen- gene transcription through an indirect activated protein kinase isoforms mechanism involving binding to a 90 kDa contributing to the induction of heat shock protein. Biochemistry, 44, antioxidant response element-mediated 5041–5052. heme oxygenase-1 in human hepatoma 17 Amakura, Y., Tsutsumi, T., Nakamura, M., HepG2 cells. Cancer Research, 66, Kitagawa, H., Fujino, J., Sasaki, K., 8804–8813. Yoshida, T. and Toyoda, M. (2002) 10 Yuan, X., Xu, C., Pan, Z., Keum, Y.S., Kim, Preliminary screening of the inhibitory J.H., Shen, G., Yu, S., Oo, K.T. et al. (2006) effect of food extracts on activation of the Butylated hydroxyanisole regulates aryl hydrocarbon receptor induced by ARE-mediated gene expression via Nrf2 2,3,7,8-tetrachlorodibenzo-p-dioxin. 120j 7 Dietary Factors Regulate Metabolism of Carcinogens through Transcriptional Signaling Pathways

Biological & Pharmaceutical Bulletin, 25, Pregnane X receptor: promiscuous 272–274. regulator of detoxiﬁcation pathways. The 18 Oshima, M., Mimura, J., Yamamoto, M. International Journal of Biochemistry & Cell and Fujii-Kuriyama, Y. (2007) Molecular Biology, 39, 478–483. mechanism of transcriptional repression 22 Willson, T.M. and Kliewer, S.A. (2002) of AhR repressor involving ANKRA2, PXR, CAR and drug metabolism. Nature HDAC4, and HDAC5. Biochemical and Reviews Drug Discovery, 1, 259–266. Biophysical Research Communications, 364, 23 Lazar, M.A. (2004) East meets West: an 276–282. herbal tea ﬁnds a receptor. The Journal of 19 Tirona, R.G. and Bailey, D.G. (2006) Clinical Investigation, 113,23–25. Herbal product-drug interactions 24 Zhou, C., Poulton, E.J., Grun,€ F., mediated by induction. British Journal of Bammler, T.K., Blumberg, B., Clinical Pharmacology, 61, 677–681. Thummel, K.E. and Eaton, D.L. 20 Timsit, Y.E. and Negishi, M. (2007) CAR (2007) The dietary isothiocyanate and PXR: the xenobiotic-sensing sulforaphane is an antagonist of the receptors. Steroids, 72, 231–246. human steroid and xenobiotic nuclear 21 Matic, M., Mahns, A., Tsoli, M., Corradin, receptor. Molecular Pharmacology, 71, A., Polly, P. and Robertson, G.R. (2007) 220–229. j121

8 Endocrine-Related Cancers and Phytochemicals Johannes C. Huber and Johannes Ott

8.1 Introduction

The main task of sexual steroids is reproduction. Thus they exert angiogenic, mitotic, and reactive-oxygen-species (ROS) generating effects. Nidation and implantation of the early embryo are supported by free radicals resulting from the metabolization of estradiol to semiquinons and quinons. Angiogenesis is enhanced in the area of the inner genital tract after ovulation and is upregulated in the case of pregnancy. These effects are exerted by the ovarian hormones and their metabolites as well as by intensified mitosis, which is a precondition for the growing fetus [1]. These cellular events and molecular–biological signal transduction mechanisms are essential for mammalian reproduction. However, they exert unfavorable effects on endocrine related organs – first and foremost on the breasts. Thus, conversion of endogenous and exogenous hormones into metabolites exerting other than the reproductive effects is kind of an intelligent design after menopause. It has been estimated that more then two-thirds of human cancer could be prevented by appropriate lifestyle modifications. Although the exact percentage is uncertain, evidence from epidemiological, clinical, and laboratory studies indicate a link between cancer risk and nutritional factors. More than 250 population-based studies including case–control and cohort studies show that people who eat fruits or vegetables several times a day have approximately half the risk of developing cancer [2, 3]. Several nutritional and physiological models try to explain the beneficial effects of fruit and vegetables with most of the scientific research focusing on the phytochemical compounds. Phytochemicals are defined as bioactive nonnutrient compounds in fruits, vegetables, grains, plant foods that have been linked to reducing the major chronic diseases [4]. It is estimated that more than 5000 individual phytochemicals have been identified in fruits, vegetables, and grains, but a large percentage still remain unknown [4]. One interesting group of phytochemicals are phytoestrogens showing structural resemblance to mammalian ovarian hormones and thus exerting various effects on endocrine-related organs.

Phytoestrogens can be subdivided into two main groups: isoflavonoids and lignans [5]. They possibly modulate the effects of ovarian hormones. These compounds have been found to be abundant in plasma and urine of people living in areas with a low incidence of endocrine-related cancers [6]. Plant lignans and isoflavonoid glycosides are found in foods such as wholegrain rye bread, soya, and red clover. They are either hormone-like with inherent estrogenic activity or are converted by intestinal bacteria to weakly estrogenic compounds called bisphenols (or diphenols). These compounds influence several biological events including production, metabolism, and biological activities of the sex hormones, and they also affect, among others, many intracellular steroid-metabolizing enzymes [7]. This seems to result in an oncopreventive effect for endocrine-related cancers, which is supported by epidemiological and demographic studies. However, the preventive mechanisms, the clinical impact for the given patients, and the possible disadvantages of phytoestrogens are discussed controversially.

8.2 Phytoestrogens and Endocrine-Related Cancers – Epidemiological Studies

8.2.1 Isoflavones and Breast Cancer

A number of publications refer to the possible association between phytoestrogens or phytoestrogen-rich diets and breast cancer risk in women. There is some evidence that a diet containing soy bean products is chemoprotective. Some other studies were not able to demonstrate such a preventive effect of phytoestrogens [8–11]. Breast cancer, the most frequent and the most important endocrine-related malignancy, will be preferentially discussed in this review.

8.2.1.1 Isoflavones and the Window of Opportunity Summarizing the actual standard of knowledge, a window of opportunity for phytoestrogen consumption seems to exist: Soy consumption before puberty may have the same risk-lowering effect as does an early pregnancy [12]. These important experimental studies have now gained support from two other recent studies – one on animals [13] and one involving Japanese women [14] – in which high soy intake during adolescence was found to reduce the risk of breast cancer later in life. A Canadian study on 3000 cases and 3000 controls showed similar results: A diet rich in phytoestrogens before and during puberty was highly protective from breast cancer decades later [15].

8.2.1.2 Serum Isoflavones and Breast Cancer Variation in intestinal absorption of isoﬂavonoids seems to be an explanation for conﬂicting results in phytoestrogen prevention studies. Anestedcase–control study within the prospect cohort, one of the two Dutch cohorts participating in the European Prospective Investigation into Cancer and 8.2 Phytoestrogens and Endocrine-Related Cancers – Epidemiological Studies j123

Nutrition, investigated plasma phytoestrogens and subsequent breast cancer risk [16]. A total of 383 women (87 pre- or perimenopausal women and 296 postmenopausal women) who had developed breast cancer were selected as case subjects and were matched to 383 controls, on date of blood sampling. Plasma levels of isoflavones (daidzein, genistein, glycitein, O-desmethylangolensin, and equol) and lignans (enterodiol and enterolactone) were measured. Breast cancer odds ratios (OR) were calculated for tertiles of phytoestrogen plasma levels using conditional logistic regression analysis. For genistein, the risk estimate for the highest versus the lowest tertile was 0.68 (95% CI, 0.47–0.98). Similar protective effects, although not statistically significant, were seen for the other isoflavones. Results were the same in pre- or perimenopausal women and in postmenopausal women. Therefore, the authors concluded that high genistein circulating levels are associated with reduced breast cancer risk in the Dutch population. Anestedcase–control study from the Japan Public Health Center-based prospective study group evaluated the plasma isoflavone level and the subsequent risk of breast cancer among Japanese women [14]. A total of 24 226 women aged 40–69 years who responded to the baseline questionnaire and provided blood in 1990–1995 were observed until December 2002. During a mean followup of 10.6 years, 144 patients with newly diagnosed breast cancer were identified. Two matched controls for each patient were selected from the cohort. Isoflavone levels were assessed by plasma level and food frequency questionnaire, and the odds ratio of breast cancer according to isoflavone level was estimated using a conditional logistic regression model. The authors found a statistically significant inverse association between plasma genistein and risk of breast cancer, but no association for plasma daidzein. Adjusted odds ratios for the highest versus lowest quartile of plasma level were 0.34 for genistein (95% CI, 0.16–0.74; P for trend, 0.02) and 0.71 for daidzein (95% CI, 0.35–1.44; P for trend, 0.54). Median plasma genistein values in the control group were 31.9 ng/ml for the lowest and 353.9 ng/ml for the highest quartile groups. Regarding dietary intake of isoflavones, nonsignificant inverse associations were observed for both genistein and daidzein. These results confirmed the inverse association between genistein serum levels and the risk of breast cancer in contrast to dietary intake, which was the major parameter in many studies with conflicting results. However, conflicting data have been reported even in this scientific area: Another study including 333 women (aged 45–75 years) drawn from a large United Kingdom prospective study of diet and cancer, the European Prospective Investigation of Cancer and Nutrition-Norfolk study [17], and using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry methods incorporating triply [13] C-labeled standards measured seven phytoestrogens (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) in 114 spot urines and 97 available serum samples from women who developed breast cancer later. Results were compared with those from 219 urines and 187 serum samples from healthy controls matched by age and date of recruitment. Exposure to all isoflavones was significantly associated with increased breast cancer risk, so for equol 124j 8 Endocrine-Related Cancers and Phytochemicals

and daidzein. For a doubling of levels, odds ratios increased by 20–45% [log(2) odds ratio ¼ 1.34 (1.06–1.70; P ¼ 0.013) for urine equol, 1.46 (1.05–2.02; P ¼ 0.024) for serum equol, and 1.22 (1.01–1.48; P ¼ 0.044) for serum daidzein]. The use of single spot probes, which are not representative for an average level, has been mentioned as a limitation of this study.

8.2.2 Lignans and Breast Cancer

Lignans exist particularly in seeds like flaxseed, which is the richest source and additionally has high concentrations of alpha linolenic acid. The mammalian lignans enterodiol and enterolactone are formed from plant lignan glycoside precursors by the activity of the gut microflora in the proximal colon. Interestingly, in the human luteal phase a urinary peak excretion of endogenous enterolactone has been reported, indicating a bacterial enterlactone formation by estrogens in the bile reaching the colon during the enterohepatic circulation, which may be stimulated by luteal hormones [18]. In contrast to isoflavones, lignans do not have any detectable estrogenic activity in vivo and do not associate with estrogen receptors (ERs); lignans seem to be ligands for the nuclear type II estrogen binding sites, controlling histone-dependent gene modulation. This nuclear type II estrogen binding sites may mediate the anticancer action of lignans. Furthermore, enterolactone has a moderate activating effect on the pregnane X receptor, stimulating steroid metabolizing and xenobiotic detoxification enzymes [19]. Flaxseed is involved in insoluble fiber, and this may be another explanation on the oncopreventive effect of this compound by reducing the enterohepatic circulation of estrogens [20]. In a recent Finnish case–control study, 194 women with breast cancer and 208 community-based controls were investigated [21]. Their mean serum enterolactone concentrations were 20 nmol/l and 26 nmol/l for the breast cancer and control groups, respectively. An odds ratio of 0.38 was obtained in comparison with the highest and lowest quintiles of enterolactone concentrations adjusted for all known breast cancer risk factors. In another prospective study in Sweden [22] similar results were obtained: The high risk of breast cancer was associated with low plasma concentrations of enterolactone. However, an increased risk was found in the highest 12.5% of enterolactone concentrations. However, high values of plasma enterolactone above the 87.5th percentile were significantly associated with an increased breast cancer. On the basis of these findings, a plasma enterolactone concentration of about 30–70 nmol/l is probably protective against breast cancer, at least in Nordic countries with high whole-grain intake. This conclusion is in accordance with results from studies in men, which showed that this concentration of enterolactone protects against acute coronary events. However, in another prospective study in Dutch women, in which the ratios of enterolactone to creatinine were measured in overnight urine samples, women who had the highest enterolactone ratios showed a nonsignificant increase in breast 8.3 Mechanisms of Cancer Chemoprevention by Phytoestrogens j125 cancer risk [23]. In this study, enterolactone was only measured in 88 breast cancer cases that probably are too few in numbers to provide convincing evidence. At least 150–200 cases would be needed in this type of study as the results depend on two variables influenced by diet. Other recent studies suggest that enterolactone concentrations in plasma are more stable than urinary concentrations because of the extensive enterohepatic circulation of lignans.

8.2.3 Phytoestrogens and Prostate Cancer

The discovery of the estrogen receptor b (ERb) receptor in the prostate has led to an increase in research on phytoestrogens and prostate cancer, since ERb binds coumestrol and several other isoflavones with high affinity and may inhibit ER- mediated estrogen action through hetero-dimerization. A recent study [24] showed that administration of genistein to rats throughout their life led to downregulation of both estrogen receptors a and b in the prostate. This observation indicates that genistein can regulate steroid-receptor pathways and may prevent prostate cancer via this mechanism. Another study by the same group [25] showed that genistein inhibits the expression of the epidermal-growth-factor receptor and the ErbB2/Neu receptor in rat dorsolateral prostate; it could therefore block the growth-factor-mediated stimulation of cell proliferation. Interestingly, ERb expression is lost in malignant prostate cells in primary cultures and in prostate cancer tissues. There is fairly strong evidence to support the idea that a diet containing large amounts of soy and isoflavones protects against prostate cancer. However, there is less evidence to support the role of lignans, mainly because of the lack of data. Randomized studies assessing the effects of soy and rye-rich diets and isoflavone supplements in humans will begin in the near future. Randomized prospective studies investigating lignans are underway.

8.3 Mechanisms of Cancer Chemoprevention by Phytoestrogens

The potential beneficial effects of phytoestrogens on breast cancer may be mediated via different mechanisms. At the moment, a causal relation to disease prevention is hypothetical because it is unknown whether the effects of soy or unrefined cereal products are due to the content of phytoestrogens or to some other mechanism. Various isoforms of the estrogen receptor may be involved in these mechanisms such as ERb hetero-dimerization with ERa and a consequent reduction in estrogen effects. Isoflavones are natural agonists for ERb, antagonizing the strong ERa- mediated endocrine activities [26]. Other possible mechanisms include inhibition of tyrosine and other protein kinases, alteration of growth-factor activity, inhibition of angiogenesis, and inhibition of several enzymes including 3b-hydroxysteroid dehydrogenase, 17b-hydroxysteroid dehydrogenase type 1, 5a-reductase, phenol sulfa- tase, topoisomerase I and II, and aromatase. Chrysin, a compound of the passion fruit 126j 8 Endocrine-Related Cancers and Phytochemicals

exerts a strong aromatase inhibiting effect, which may provide interest for clinical use. Genistein, soy, and rye bran can also cause apoptosis in cancer cells both in vitro and in vivo [27]. Genistein stimulates several antioxidative enzymes, such as catalase, superoxide dismutase, glutathione peroxidise, and reductase, and it also induces tumor-cell differentiation. Genistein downregulates the epidermal-growth-factor receptor and ErbB2/Neu receptors in cancer cells may also inhibit tumor-cell invasion by inhibiting MMP9 (92 kDa type IV collagenase), and upregulates tissue inhibitor of metalloproteinases (TIMP1) and various trypsin inhibitors. Many in vitro results indicate the oncopreventive power of isoﬂavones, which must be conﬁrmed in DPC studies. Indole-3-carbinol (I3C) and genistein – and this is a further example – may act synergistically or supra-additively to stimulate BRCA1 and BRCA2 expression [28].

8.4 Estrogen Carcinogenesis

The mechanisms of carcinogenesis in the breast triggered by estrogen include the metabolism of estrogen to genotoxic, mutagenic metabolites, and the stimulation of tissue growth; these processes cause initiation, promotion, and progression of carcinogenesis. Estrogen metabolites, not ovarian 17b-estradiol, seem to be responsible for carcinogenesis [29]. These theories are supported by the ﬁndings of the WHI study: Although in the case of estrogen only arm breast cancer risk was reduced, the combined treatment with conjugated estrogen and medroxyprogesteron (MPA) was associated with a signiﬁcant higher risk [30]. MPA modulates estrogen metabolism. Thus it has been mentioned that combined use of gestagens and estrogens leads to estrogen hydroxylation to angiogenic 4- and 16-OH estrogen, both known to be metabolites with reproductive functions. Recently, various progesterone metabolites were investigated with regard to cancer risk. Similarities to estrogen metabolisms have been postulated: Progester- one metabolites with reproductive tasks are of higher risk for endocrine-related cancers [31]. However, this part of the review focuses on the well-established effects of estrogen metabolites, their association with carcinogenesis, and intervention strategies with phytochemicals.

8.4.1 Estrogen Metabolites and Carcinogenesis

Estrogen metabolism occurs in two steps, that is, phase I and phase II metabolism. Phase I metabolism in humans, hamsters, mice, and rats involves several cytochrome P-450 enzymes that catalyze the oxidative metabolism of estrone (E1) and estradiol (E2) predominantly to a 2-hydroxycatecholestrogen (cytochrome P-450 1A1, 1A2, and 3A) or to 4-hydroxycatecholestrogen (cytochrome P-450 1B1). In humans, 8.4 Estrogen Carcinogenesis j127 cytochrome P-450 1B1 is constitutively expressed in the breasts, ovaries, adrenal glands, and uterus, as well as in several other tissues. Cytochrome P-450 1B1 catalyzed products such as estrogen 3,4-quinoneform unstable adducts with adenine and guanine in DNA, leading to depurination and mutation in vitro and in vivo [29] (Scheme 8.1.) Phase II detoxication pathways – including sulfuration, methylation, and reaction with glutathione – protect breast tissue from damage caused by reactive metabolites of endogenous and exogenous chemicals [29]. To date, no studies have definitively demonstrated that estrogen metabolites contribute to human breast cancer, though two lines of evidence support this possibility. First, the hypothesis that potentially genotoxic estrogen metabolites contribute to human breast cancer depends on their capacity to form depurinated adducts on DNA and on their presence in breast tissue; in postmenopausal women, estrogen levels in breast tissue are 10–50 times the levels in blood [32]. Recently it was demonstrated that high levels of CYP1B1 expression and low levels of COMT expression in adjacent nontumor tissue were associated with a significantly increased breast cancer risk in a nonlinear manner [33]. Hydroxylation in position C2 forms stable DNA adducts without major mutage- nity, whereas hydroxylation in position C4 is responsible for the more dangerous depurinated adducts. Therefore, the balance between CYP1A and CYP1B activity defines the direction of estrogen hydroxylation and the formation of depurinated or stable adducts [34] (Scheme 8.2).

Scheme 8.1 Estrogen metabolism by phase I and II detoxification enzymes. 128j 8 Endocrine-Related Cancers and Phytochemicals

Scheme 8.2 Depurinated and stable adducts of estrogen metabolites.

The second line of evidence supporting the role of estrogen metabolites in human breast cancer comes from studies of associations of breast cancer risk and polymorphisms in genes encoding enzymes involved in estrogen synthesis and metabolism. The results of these studies have been inconsistent, probably because of the complexity of interactions between polymorphisms, environment, epigenetics, and the polymorphic structure of other genes. In order to understand the relations between breast cancer and estrogen metabolism, lifestyle, environmental risk factors, and the interaction between different estrogen metabolizing enzymes and polymorphisms should be considered. For example, Crooke et al. [35] assessed the association of combinations of 10 single- nucleotide polymorphisms in COMT, CYP-450 1A1, and 1B1 and glutathione- S-transferases M1 and T1 and the risk of breast cancer. They observed that an interaction between the low activity variant of COMT, two variants of CYP-450 1B1, and a variant of CYP-450 1A1 were significantly associated with an increased risk of breast cancer. However, these findings still require independent confirmation. The knowledge of the individual estrogen metabolism in an individual patient is not limited to the risk of breast cancer. Different estrogen metabolites have a different angiogenetic, angiostatic, mitotic, and apoptotic capacity and exhibit distinguished effects on neurotransmitters, mood, and heart muscle. These factors may provide useful information for an individual management of female health problems (Scheme 8.3). 8.4 Estrogen Carcinogenesis j129

Scheme 8.3 Estrogen metabolic pathway, the participating genes and their polymorphisms. (From Rebbeck et al. [36])

8.4.2 CYP1A1

CYP-450 1A1 catalyzes the 2-hydroxylation of estrone and estradiol into the cate- cholamines 2-hydroxy estrone (2-OHE1) and 2-hydroxy-estradiol (2-OHE2). These 2-hydroxy metabolites show reduced estrogenic effects and behave more like antiestrogens (in contrast to 4-OH and 16-OH metabolites). Besides, CYP1A1 also activates procarcinogens, such as polycyclic aromatic hydrocarbons (PAH) or heterocyclic aromatic amines (HA), present in tobacco smoke or broiled meat and plays a distinct role in the development of certain cancers, for example, lung and breast cancer [37]. CYP1 enzymes generally are responsible for metabolic activation of PAH and HCA heterocyclic amines. Thereby many PAHs, for example, benzo[a]pyrene (B[a]P), become activated to form harmful products that can covalently bind to nucleic acids and proteins to form adducts, then facilitating mutagenesis [36] (Scheme 8.4). Thus patients with higher CYP1A1 activity – induced or genetically determined – have predominantly 2-hydroxylated estrogen metabolites with lower estrogenic activity but are on higher risk for procarcinogen activation via tobacco smoke or broiled meat.

8.4.2.1 Polymorphisms As summarized by Chen et al. [39], T3801C, a substitution in the 30 noncoding region; A2455G, isoleucine change to valine at codon 462; T3205C, a transition mutation in the 30 noncoding region; C2453A, threonine to asparagine change at codon 461, are four polymorphisms that have been found and described in the CYP1A1 gene. Among them, C2453A is very rare and T3205C exists only in Africans or African- Americans, so most studies have focused on T3801C and A2455G. 130j 8 Endocrine-Related Cancers and Phytochemicals

Scheme 8.4 The metabolism of estradiol and estrone.

The one base substitution of thymine to cytosine in a noncoding region of the gene at position 3801 creates a MspI recognition site (CYP1A12A), which does not exist in the predominant genotype (T/T). A one base substitution adenine to thymine at position 2455 in the heme-binding region of exon 7 induces an amino acid change of isoleucine to valine at codon 462 (Ile462Val polymorphism). This polymorphism is usually linked with the MspI polymorphism (CYP1A12B). The presence of the Ile462Val polymorphism alone (CYP1A12C) is very rare in Caucasians. The above listed CYP1A1 polymorphisms have been shown not only to increase microsomal catalytic activity for metabolizing steroids but also for converting procarcinogens, including PAH and aromatic amines. The functional impact of polymorphisms found in CYP1A1 is still not clear. Cosma et al. [39] found signiﬁcantly elevated levels of inducible lymphocyte CYP1A1 enzyme activity in individuals carrying A2455G when compared to wild-type genotypic individuals. Crofts et al. [40] reported a threefold elevation in CYP1A1 enzymatic activity associated with A2455G G/G genotypes. The T3801C allele was also reported to encode an inducible form of CYP1A1.

8.4.2.2 Clinical Aspects of CYP1A1 Genotypes The CYP1A1 T/Twild type is present in about 81% of the population. A heterozygous genotype (CYP1A1 T/C) is present in about 18% of the population and is associated with an increased enzyme activity of CYP1A1. A homozygous genotype for the variant allele (C/C) has been found in about 1% of the population. The detected 8.4 Estrogen Carcinogenesis j131 genotype is associated with an increased enzyme activity. The positive effect of a highly active CYP1A1 enzyme, consisting in an increased formation of 2-OH estrogen metabolites, is overcome by its activation of procarcinogens and should be taken into account when one is consulting nicotine addicted patients. The CYP1A1 enzyme is also present in the lung and thus exposure to tobacco smoke must be avoided as well. In smokers higher levels of DNA adducts are found in breast tissue, therefore they are–with regard to the CYP1A1 polymorphism – at an increased risk of breast cancer in contrast to nonsmokers [41]. All CYP1A1 variants catalyze the formation of 2-, 4-, 6a-,and 15a-hydroxylated estrogen metabolites from E2 and E1, yet with varying catalytic efficiency and distinct region specificity. Although the variant CYP1A1.2 (Ile(462)Val) has a significantly higher catalytic activity for all hydroxylation sites and both substrates, it is the most pronounced for 2-hydroxylation. Catalytic efficiencies for the formation of the major metabolites, 2-OH-E2 and 2-OH-E1, by CYP1A1.2 were 5.7- and 12-fold higher, compared with the wild-type enzyme [42]. For female health, the reduced or enhanced phase I enzyme activity covers an important aspect. The faster working genotype eliminates estrogen quickly from the female organism; on the other side endocrine disrupters occupy the aryl receptor, inducing CYP1A1 activity. Aberrant estrogen metabolites, generated by this pathway may be responsible for an aberrant estrogen metabolism (Scheme 8.5).

8.4.2.3 Breast and CYP1A1 In a meta-analysis no signiﬁcant association between the CYP1A1 A2455G polymorphism and breast cancer risk in the worldwide population as well in allele as in genotype comparisons was found [39]. However, in a subgroup analysis, the G/G

Scheme 8.5 Ah receptors and CYP1A1. (From Bulun et al. [43]) 132j 8 Endocrine-Related Cancers and Phytochemicals

genotype tended to reduce the risk of breast cancer compared to the wild type (G/G versus G/A þ A/A or G/G versus A/A) for A2455G in east-Asians or in premenopausal women, though the number of studies was small. In the pathway of PAH metabolism, CYP1A1 tends to increase the risk of breast cancer by activating carcinogens, while in the pathway of estrogen metabolism, the CYP1A1 gene is inclined to reduce the risk. The G/G genotype of A2455G might function in estrogenic metabolism, which decreases the risk of breast cancer by influencing the level of estrogen or competing with the 16a-hydroxylation pathway. This effect was significant in premenopausal women but not in postmenopausal women who naturally showed significantly lower levels of estrogen. Consulting patients with a CYP1A1 A2455G G/G genotype must be emphasized about avoidance of carcinogens such as tobacco smoke. These data partly are in concordance with another meta-analysis published in the human genome epidemiology (HuGE) review [44]. In 17 studies, no consistent association between breast cancer and CYP1A1 genotype was found. No significant risk for the genotypes (1) 3801C/C (relative risk (RR) ¼ 0.97, 95% confidence interval (CI): 0.52, 1.80) or 3801T/C (RR ¼ 0.91, 95% CI: 0.70, 1.19) versus 3801T/T; (2) Val/Val (RR ¼ 1.04, 95% CI: 0.63, 1.74) or Ile/Val (RR ¼ 0.92, 95% CI: 0.76, 1.10) versus Ile/Ile; or (3) Asp/Asp (RR ¼ 0.95, 95% CI: 0.20, 4.49) or Thr/ Asp (RR ¼ 1.12, 95% CI: 0.87, 1.43) versus Thr/Thr (polymorphism Thr 461 Val) could be demonstrated. However, P-450 1A1 polymorphisms become clinically important in cigarette smoking females. Cigarette smoking increases breast cancer risk in women with CYP1A1 polymorphism. This is explained by the CYP1A1 activating effect of procarcinogens. According to the GAIL model, smoking should be avoided completely by females with a higher lifetime risk for breast cancer., Moreover, the knowledge about the CYP1A1 genotypes gives additional information about a higher risk constellation and can be used to persuade patients to avoid smoking.

8.4.2.4 Pharmacogenomics and Phytochemicals CYP1A1 enzyme activity in the liver may be increased by a diet rich in cruciferous vegetables (e.g., cabbage and broccoli), which contain I3C. I3C and its major in vivo product diindolymethane (DIM) are stimulating CYP1A1 activity in the liver, favoring a higher ratio of 2-OH/16-OH metabolites, which is regarded as a protective marker of breast cancer. In smokers, the higher CYP1A1 activity could be a disadvantage, which may also be induced by diet [45]. CYP1A1 is also involved in metabolizing caffeine to hypoxanthin and other metabolites. While the former metabolite reveals neuroprotective activities, the latter may be associated with geno- or germ-toxic side effects. This may be the same mechanism, as it is discussed for activating procancerogens by CYP1A1 in lung cancer patients. The clinical aspects of metabolization of different pharmacological drugs by CYP1A1 isoforms and the resulting medical impact in daily patient consultation are of special interest. Many drugs are known to be inactivated or converted by 8.4 Estrogen Carcinogenesis j133 the CYP1A1 system: analgesics (oxidation to benzoquinone), antiarrhythmics, anticoagulants (C6-hydroxylation), antineoplastics (oxidation), antiestrogens (N-demethylation), propanolol (N-dealkylation), caffeine (N3-demetylation), sexual hormones (C2-hydroxylation), muscle relaxants (C6-hydroxylation), and mycotoxins (epoxidation). Perforine stimulates CYP1A1 activity. Isoﬂavones have a suppressive effect on this enzyme, similar to retinoids, cholecalciferol, and hesperidine. Barbiturates enhance 2-hydroxylation of estrogens. The same effect can be observed in indole-3-carbinol and in epigallocatechin gallat. Data from several studies demonstrated that resveratrol inhibits aryl hydrocarbon- induced CYP1A1 activity in vitro by directly inhibiting CYP1A1/1A2 enzyme activity and by inhibiting the signal transduction pathway that upregulates the expression of carcinogen activating enzymes. These activities may be an important part of the chemopreventive activity of resveratrol in vivo [46].

8.4.3 CYP1B1

8.4.3.1 Gene and Protein Structure of CYP1B1 The CYP1B1 gene (Genbank accession no. U03688) consists of three exons and two introns located on chromosome 2p21 and spans 8.5 kb of genomic DNA (Genbank accession no. U56438). It encodes a 543 amino acid protein product that is found normally expressed in the nucleus of most cell types and exhibits cytoplasmic and nuclear localization in tubule cells of the kidney and secretory cells of breast tissue [47, 48].

8.4.3.2 Gene Regulation CYP1B1 is regulated by several key transcription factors, such as the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT) complex (AhR/ARNT), the Sp1 transcription factor, a cyclicAMP (cAMP)–response element–binding protein (CREB), and ER. Epigenetic factors, posttranscriptional modifications, and degradation pathways have recently been explored. Given that CYP1B1 is transcriptionally activated in several human cancers, it is considered as a potential target for anticancer therapy [49]. Recently, the expression of CYP1B1 and COMT in breast tissue and their associations with breast cancer risk were demonstrated. High levels of CYP1B1 expression and low levels of COMT expression in adjacent nontumor tissue were associated with a significantly increased breast cancer risk in a nonlinear manner. Odds ratios and 95% confidence intervals (in parentheses) for the midpoints of the first, second, fourth, and fifth quintiles of gene expression levels compared with the overall median levels in subjects with benign breast diseases were 0.21 (0.07–0.67), 0.81 (0.69–0.95), 1.20 (1.05–1.38), and 1.55 (1.12–2.15) for CYP1B1 and 1.72 (1.17–2.55), 1.19 (1.05–1.35), 0.83 (0.73–0.95), and 0.78 (0.65–0.93) for COMT, respectively [50]. 134j 8 Endocrine-Related Cancers and Phytochemicals

CYP1B1 is an inducible enzyme and is activated by polycyclic aromatic hydrocarbons and dioxin-like compounds [51, 52]. It is capable of activating a variety of carcinogens, such as arylamines; CYP1B1 encodes for an enzyme that catalyzes the formation of both 2- and 4-hydroxyestrone, with higher activity on the 4-hydroxyestrone compound [53, 54]. Levels of CYP1B1 gene expression in white blood cells have been investigated according to gender; results showed that females have a higher expression than males [55], though these data were not conﬁrmed by other studies [56, 57]. CYP1B1 gene expression during pregnancy has also been examined; it was found to be upregulated in the study of Stoilov et al. [58], whereas Cuthill et al. [59] did not conﬁrm these results.

8.4.3.3 Epigenetic Regulation Promoter methylation of CYP1B1 has been associated with a decreased activity of this gene [60]. CYP1B1 methylation takes place at multiple CpG sites within the CYP1B1 gene, some of which are contained within key promoter elements, such as DRE1, DRE2, DRE3, and Sp1 binding sites at 72 and 80 [61]. Methylation at these sites may decrease the accessibility of DNA-binding sites for proteins involved in AhR- mediated regulation [62] and may alter estrogen-mediated regulation of CYP1B1. DNA methylation of CYP1B1 has also been associated with survivalin breast cancer patients treated with tamoxifen [63].

8.4.3.4 Polymorphisms A summary of all known single nucleotide polymorphisms (SNP), including five different missense mutations and seven different common haplotypes, is provided on the Human Cytochrome P-450 Allele Nomenclature Committee home page [64]. Of these, five SNPs [C142G (R48G), G355T (A119S), C4326G (L432V), C4360G (A443G), and A4390G (N453S)] are known to result in amino acid substitutions. The C4326G transition (CYP1B13) leading to the corresponding amino acid transition [L432V (CYP1B1.3)] is associated with increased catalytic activity of the CYP1B1 enzyme in several studies [65–67]. A possible cause of this increase in the catalytic activity is changes in the tertiary (or quaternary) structure of the CYP1B1 protein, as the CYP1B1.3 polymorphism is located near a catalytically important heme-binding domain in CYP1B1 [68, 69]; furthermore, the CYP1B13 transition is also responsible for significant increases in AhR-mediated CYP1B1 gene expression during AhR-mediated signaling events [70, 71]. The Val432Leu polymorphism seems to have the largest impact on the catalytic properties of the enzyme; the Val432 allele displays threefold higher 4-hydroxylase activity than the Leu432 allele [72]. In vitro studies indicate that the CYP1B1 Val432Leu valine allele has higher 4-hydroxyestradiol:2-hydroxyestradiol and 4-hydroxyestrone:2-hydroxyestrone ratios than the leucine allele. Several other in vitro studies have also indicated that the leucine substitution may influence estrogen metabolism [73–76]. 4-Hydroxyestrogen has a higher angiogenetic and mitotic effects than 2-hydroxy-estrogens; this seems to be important for fetal implantation and pregnancy, but reveals probably disadvantages in the peri- and postmenopausal area. 8.4 Estrogen Carcinogenesis j135

8.4.3.5 Breast Cancer The association between the CYP1B1 Val432Leu polymorphism and breast cancer was assessed through a meta-analysis of all published case–control studies and a pooled analysis of both published and unpublished case–control studies from the Genetic Susceptibility to Environmental Carcinogens (GSEC) database [77]. Thirteen articles were included in the meta-analysis (14 331 subjects; 7514 cases, 6817 controls); nine data sets were included in the pooled analysis (6842 subjects; 3391 cases, 3451 controls). A summary meta- or pooled estimate of the association between the CYP1B1 Val432Leu polymorphism and breast cancer could not be calculated because of statistically signiﬁcant heterogeneity in the point estimates among studies. No association between the CYP1B1 Val432Leu polymorphism and breast cancer was observed in Asians (for Val/Val and Val/Leu combined, odds ratio ¼ 1.0, 95% CI: 0.8, 1.2). An inverse association was observed in populations of mixed/African origin (OR ¼ 0.8, 95% CI: 0.7, 0.9). The pooled analysis suggested a possible association in Caucasians (for Val/Val and Val/Leu combined, OR ¼ 1.5, 95% CI: 1.1, 2.1), with effect modiﬁcation across age categories [78]. Case-only and case–control studies have examined the gene–environment interaction and the interaction of CYP1B1 polymorphisms with other genetic and cellular factors. Such studies have found that smoking confers a further increase in breast cancer risk in individuals with the CYP1B13/3 genotype [79]. A prevailing hypothesis is that CYP1B1 is transcriptionally induced by polyaromatic hydrocarbons and other aromatic hydrocarbons (major constituents of cigarette smoke) resulting in increased levels of CYP1B1. Individuals carrying the hyperactive CYP1B13/3 genotype are more likely to metabolize polycyclic aromatic hydrocarbons and endogenous procarcinogens into toxic metabolites resulting in a further increase in exposure to carcinogens compared with nonsmokers [80]. Another association study demonstrated that women with the Leu/Leu genotype had a 2.3-fold (95% CI, 1.2–4.5) elevated risk of breast cancer – compared with those with the Val/Val genotype – after adjusting for potential confounding variables. The Val CYP1B1 allele increased the susceptibility to breast cancer in women who have been exposed to agricultural products used in farming (OR ¼ 2.18, 95% CI 1.10–4.32). These xenobiotics, mainly organochlorine hydrocarbons, are known to bind to the aromatic hydrocarbon receptor and to induce the expression of CYP1B1 enzyme. The excess risk for exposed women with a Val CYP1B1 homo/heterozygous genotype could result from a higher exposure to activated metabolites of pesticides or dioxin-like substances. Also, a higher induction of CYP1B1 enzyme by xenobiotics is thought to increase the level of genotoxic catechol–oestrogens among exposed women carrying the Val CYP1B1 allele. Results suggest that the Val CYP1B1 allele increases the susceptibility to breast cancer in women exposed to waste or agricultural pollutants [81]. As is the case for prostate cancer, DNA methylation may be an important determinant of CYP1B1 expression in breast and has been shown to predict the response to tamoxifen therapy [82]. A body mass index >24 kg/m2 has also been associated with an increased incidence of breast cancer in women carrying the CYP1B13 allele [83]. Interestingly, increased body mass index results in higher levels 136j 8 Endocrine-Related Cancers and Phytochemicals

of circulating endogenous estrogens, and this may be a contributing factor to breast cancer risk in obese women [84]. CYP-450 1B1 gene polymorphisms and postmenopausal breast cancer risk was investigated in hormone replacement therapy long time users. These data indicated that women who had used menopausal hormones for 4 years or longer and carried the CYP1B13/3 (432 Val/Val) genotype may be at an increased risk of breast cancer, OR 2.0 (95% CI 1.1–3.5), compared with long-term users without this genotype [85]. This may become important for an individualized hormone replacement therapy. A signiﬁcant association between the homozygous variant CYP1B1_1358_GG genotype and negative ERa status (P ¼ 0.005; OR 2.82, 95% CI: 1.37–5.82) with a signiﬁcant trend for CYP1B1_1358_A > Gand negative ERa status (P ¼ 0.003) as well as an association of CYP1B1_1358_GG and negative progesterone receptor (PR) status (P ¼ 0.015; OR 2.36, 95% CI: 1.18–4.70) and a P (trend) of 0.111 for CYP1B1_1358_A > G and negative PR status was observed. The CYP1B1_1358_GG genotype, known to encode higher CYP1B1 activity, is associated with ERa negativity [86].

8.4.3.6 Endometrial Cancer The highest levels of CYP1B1 are found in the endometrium [87]. Endometrial myoma tissue has significantly elevated 4-OHE2 levels compared with the surrounding normal myometrium, an effect which is abrogated by inhibition of CYP1B1 [88]. Furthermore, 4-OHE2 production has been shown to be responsible for endometrial carcinoma in mice [89]. These data suggest an important role for CYP1B1 in the induction of uterine cancers. Furthermore, a high-affinity, saturable, cytosolic- binding protein for 4-hydroxyestradiol may be a novel receptor mediating ERa- and ERb-independent effects of catechol estrogens [90]. Even so, the knowledge of the central role of estrogen in breast cancer has already led to the development of new preventive and therapeutic interventions that block receptor function or drastically reduce the levels of endogenous estrogen through the inhibition of its synthesis. The development of additional strategies on the basis of the inhibition of estrogen metabolism, inactivation of the reactive quinones, and specific inhibition of membrane estrogen receptor-activated second-messenger pathways will probably lead to the availability of additional effective intervention approaches.

8.4.3.7 Pharmacogenomics and Phytochemicals Grapefruit juice and also green tea are known to reduce CYP1B1 activity. This may become important for consultations of patients with CYP1B1 polymorphisms. Isoﬂavonoids are also selective substrates and inhibitors for CYP1A1 and CYP1B1 [91]. Methoxylated dietary ﬂavonoids, for example, DMF and 30,40-DMF, may be potent chemoprotectants by direct inhibition of CYP1B1/1A1 function and/ or their protein expression [92]. CYP-450 enzymes are also potential options for cancer therapy. The phytoestrogen resveratrol is a natural constituent of red wine and has known cancer preventive properties; this stilbene is metabolized by CYP1B1 to the References j137 anticancer agent piceatannol [93]. Coumarins are competitive inhibitors of cytochrome P-450 1B1, with equal potency for allelic variants.

8.5 Conclusion

Gene and enzyme modulation become a new aspect in pharmacological interventions. Phytochemicals are powerful instruments to stimulate gene and enzyme activity as they have a favorable effect on the aging organism.

References

1 Huber, J.C. (1998) Endokrine Gynakologie€ – 9 Saarinen, N.M., W€arri, A., Airio, M., Einfuhrung€ in die frauenspezifische Medizin, Smeds, A. and M€akel€a S. (2007) Role of Wilhelm Maudrich Verlag, Wien– dietary lignans in the reduction of breast Munchen€ –Bern. cancer risk. Molecular Nutrition & Food 2 Heber, D. (2004) Vegetables, fruits and Research, 51 (7), 857–866. phytoestrogens in the prevention of 10 Rice, S. and Whitehead, S.A. (2006) diseases. Journal of Postgraduate Medicine, Phytoestrogens and breast cancer – 50, 145–149. promoters or protectors? Endocrine-Related 3 Demark-Wahnefried, W., Rock, C.L., Cancer, 13 (4), 995–1015. Patrick, K. and Byers, T. (2008) Lifestyle 11 Mense, S.M., Hei, T.K., Ganju, R.K. and interventions to reduce cancer risk and Bhat, H.K. (2008) Phytoestrogens and improve outcomes. American Family breast cancer prevention: possible Physician, 77 (11), 1573–1578. mechanisms of action. Environmental 4 Liu, R.H. (2004) Potential synergy of Health Perspectives, 116 (4), 426–433. phytochemicals in cancer prevention: 12 Adlercreutz, H. (2002) Phytoestrogens and mechanism of action. The Journal of breast cancer. The Journal of Steroid Nutrition, 134 (12 Suppl), 3479S–3485S. Biochemistry and Molecular Biology, 5 Murkies, A.L., Wilcox, G. and Davis, S.R. 83 (1–5), 113–118. (1998) Clinical review 92: phytoestrogens. 13 Chiesa, G., Rigamonti, E., Lovati, M.R., The Journal of Clinical Endocrinology and Disconzi, E., Soldati, S., Sacco, M.G., Catò Metabolism, 83, 297–303. E.M., Patton, V., Scanziani, E., Vezzoni, P., 6 Adlercreutz, H. (1998) Epidemiology of Arnoldi, A., Locati, D. and Sirtori, C.R. phytoestrogens. Baillieres Clinical (2008) Reduced mammary tumor Endocrinology and Metabolism, 12 (4), progression in a transgenic mouse model 605–623. fed an isoflavone-poor soy protein 7 Ranjan, P., Dalela, D. and Sankhwar, S.N. concentrate. Molecular Nutrition & Food (2006) Diet and benign prostatic Research, 52 (10), 1121–1129. hyperplasia: implications for prevention. 14 Iwasaki, M., Inoue, M., Otani, T., Sasazuki, Urology, 68 (3), 470–476. S., Kurahashi, N., Miura, T., Yamamoto, S. 8 Duffy, C., Perez, K. and Partridge, A. and Tsugane, S.; Japan Public Health (2007) Implications of phytoestrogen Center-based prospective study group intake for breast cancer. CA – A Cancer (2008) Plasma isoflavone level and Journal for Clinicians, 57 (5), 260–277. subsequent risk of breast cancer among 138j 8 Endocrine-Related Cancers and Phytochemicals

Japanese women: a nested case–control (2001) Serum enterolactone and risk of study from the Japan Public Health breast cancer: a case–control study in Center-based prospective study group. eastern Finland. Cancer Epidemiology, Journal of Clinical Oncology, 26 (10), Biomarkers & Prevention, 10 (4), 339–344. 1677–1683. 22 Hulten, K., Winkvist, A., Lenner, P., 15 Thanos, J., Cotterchio, M., Boucher, B.A., Johansson, R., Adlercreutz, H. and Kreiger, N. and Thompson, L.U. (2006) Hallmans, G. (2002) An incident case- Adolescent dietary phytoestrogen intake referent study on plasma enterolactone and breast cancer risk (Canada). Cancer and breast cancer risk. European Journal of Causes & Control, 17 (10), 1253–1261. Nutrition, 41 (4), 168–176. 16 Verheus, M., van Gils, C.H., Keinan-Boker, 23 den Tonkelaar, I., Keinan-Boker, L., Veer, L., Grace, P.B., Bingham, S.A. and Peeters, P.V., Arts, C.J., Adlercreutz, H., Thijssen, P.H. (2007) Plasma phytoestrogens and J.H. and Peeters, P.H. (2001) Urinary subsequent breast cancer risk. Journal of phytoestrogens and postmenopausal Clinical Oncology, 25 (6), 648–655. breast cancer risk. Cancer Epidemiology, 17 Grace, P.B., Taylor, J.I., Low, Y.L., Luben, Biomarkers & Prevention, 10 (3), 223–228. R.N., Mulligan, A.A., Botting, N.P., 24 Fritz, W.A., Wang, J., Eltoum, I.E. and Dowsett, M., Welch, A.A., Khaw, K.T., Lamartiniere, C.A. (2002) Dietary Wareham, N.J., Day, N.E. and Bingham, genistein down-regulates androgen and S.A. (2004) Phytoestrogen concentrations estrogen receptor expression in the rat in serum and spot urine as biomarkers for prostate. Molecular and Cellular dietary phytoestrogen intake and their Endocrinology, 186 (1), 89–99. relation to breast cancer risk in European 25 Dalu, A., Haskell, J.F., Coward, L. and prospective investigation of cancer and Lamartiniere, C.A. (1998) Genistein, a nutrition-norfolk. Cancer Epidemiology, component of soy, inhibits the expression Biomarkers & Prevention, 13 (5), 698–708. of the EGF and ErbB2/Neu receptors in the 18 Liu, Z., Saarinen, N.M. and Thompson, rat dorsolateral prostate. Prostate, 37 (1), L.U. (2006) Sesamin is one of the major 36–43. precursors of mammalian lignans in 26 Hwang, C.S., Kwak, H.S., Lim, H.J., Lee, sesame seed (Sesamum indicum)as S.H., Kang, Y.S.,Choe, T.B., Hur, H.G. and observed in vitro and in rats. The Journal of Han, K.O. (2006) Isoﬂavone metabolites Nutrition, 136 (4), 906–912. and their in vitro dual functions: they can 19 Jacobs, M.N., Nolan, G.T. and Hood, S.R. act as an estrogenic agonist or antagonist (2005) Lignans, bacteriocides and depending on the estrogen concentration. organochlorine compounds activate the The Journal of Steroid Biochemistry and human pregnane X receptor (PXR). Molecular Biology, 101 (4–5), 246–253. Toxicology and Applied Pharmacology, 27 Limer, J.L. and Speirs, V. (2004) Phyto- 209 (2), 123–133. oestrogens and breast cancer 20 Adlercreutz, H., Hockerstedt,€ K., chemoprevention. Breast Cancer Research, Bannwart, C., Bloigu, S., H€am€al€ainen, E., 6 (3), 119–127. Fotsis, T. and Ollus, A. (1987) Effect of 28 Fan, S., Meng, Q., Auborn, K., Carter, T. dietary components, including lignans and and Rosen, E.M., (2006) BRCA1 and phytoestrogens, on enterohepatic BRCA2 as molecular targets for circulation and liver metabolism of phytochemicals indole-3-carbinol and estrogens and on sex hormone binding genistein in breast and prostate cancer globulin (SHBG). Journal of Steroid cells. British Journal of Cancer, 94 (3), Biochemistry, 27 (4–6), 1135–1144. 407–426. 21 Pietinen, P., Stumpf, K., M€annistoS.,€ 29 Yager, J.D. and Davidson, N.E. (2006) Kataja, V.,Uusitupa, M. and Adlercreutz, H. Estrogen carcinogenesis in breast cancer. References j139

The New England Journal of Medicine, 354 metabolism genotypes in postmenopausal (3), 270–282. breast cancer etiology. Cancer Epidemiology, 30 Stefanick, M.L., Anderson, G.L., Margolis, Biomarkers & Prevention, 16 (3), 444–450. K.L., Hendrix, S.L., Rodabough, R.J., 37 Cherala, Ganesh., Shapiro, Bernard H. and Paskett, E.D., Lane, D.S., Hubbell, F.A., Dmello, Anil P. (2007) Effect of perinatal Assaf, A.R., Sarto, G.E., Schenken, R.S., low protein diets on the ontogeny of select Yasmeen, S., Lessin, L. and Chlebowski, hepatic cytochrome p450 enzymes and R.T. (2006) WHI Investigators. Effects of cytochrome p450 reductase in the rat. Drug conjugated equine estrogens on breast Metabolism and Disposition: The Biological cancer and mammography screening in Fate of Chemicals, 35 (7), 1057–1063 postmenopausal women with [17392395 (P,S,E,B,D)]. hysterectomy. The Journal of the American 38 Chen, C., Huang, Y., Li, Y., Mao, Y.and Xie, Medical Association, 295 (14), 1647–1657. Y. (2007) Cytochrome P450 1A1 (CYP1A1) 31 Wiebe, J.P. (2006) Progesterone T3801C and A2455G polymorphisms in metabolites in breast cancer. Endocrine- breast cancer risk: a meta-analysis. Journal Related Cancer, 13 (3), 717–738. of Human Genetics, 52 (5), 423–435. Epub 32 van Landeghem, A.A., Poortman, J., 2007, April 11. Nabuurs, M. and Thijssen, J.H. (1985) 39 Cosma, G., Crofts, F., Taioli, E., Toniolo, P. Endogenous concentration and and Garte, S. (1993) Relationship subcellular distribution of estrogens in between genotype and function of the normal and malignant human breast human CYP1A1 gene. Journal of Toxicology tissue. Cancer Research, 45, 2900–2906. and Environmental Health, 40 (2–3), 33 Wen, W., Ren, Z., Shu, X.O., Cai, Q., Ye, C., 309–316. Gao, Y.T. and Zheng, W. (2007) Expression 40 Crofts, F., Taioli, E., Trachman, J., Cosma, of cytochrome P450 1B1 and catechol-O- G.N., Currie, D., Toniolo, P. and Garte, S.J. methyltransferase in breast tissue and (1994) Functional signiﬁcance of different their associations with breast cancer risk. human CYP1A1 genotypes. Carcinogenesis, Cancer Epidemiology, Biomarkers & 15 (12), 2961–2963. Prevention, 16 (5), 917–920. 41 Ishibe, N., Hankinson, S.E., Colditz, G.A., 34 Singh, S., Chakravarti, D., Edney, J.A., Spiegelman, D., Willett, W.C., Speizer, Hollins, R.R., Johnson, P.J., West, W.W., F.E., Kelsey, K.T. and Hunter, D.J. (1998) Higginbotham, S.M., Cavalieri, E.L. and Cigarette smoking, cytochrome P450 1A1 Rogan, E.G. (2005) Relative imbalances in polymorphisms, and breast cancer risk in the expression of estrogen-metabolizing the Nurses Health Study. Cancer Research, enzymes in the breast tissue of women 58 (4), 667–671. with breast carcinoma. Oncology Reports, 42 Kisselev, P., Schunck, W.H., Roots, I. and 14 (4), 1091–1096. Schwarz, D. (2005) Association of CYP1A1 35 Crooke, P.S., Ritchie, M.D., Hachey, D.L., polymorphisms with differential Dawling, S., Roodi, N. and Parl, F.F., metabolic activation of 17beta-estradiol (2006) Estrogens, enzyme variants, and and estrone. Cancer Research, 65 (7), breast cancer: a risk model. Cancer 2972–2978. Epidemiology, Biomarkers & Prevention, 15 43 Bulun, S.E., Zeitoun, K.M. and Kilic, G. (9), 1620–1629. (2000) Expression of dioxin-related 36 Rebbeck, T.R., Troxel, A.B., Walker, A.H., transactivating factors and target genes in Panossian, S., Gallagher, S., Shatalova, human eutopic endometrial and E.G., Blanchard, R., Norman, S., Bunin, endometriotic tissues. American Journal of G., DeMichele, A., Berlin, M., Schinnar, Obstetrics and Gynecology, 182 (4), 767–775. R., Berlin, J.A. and Strom, B.L. (2007) 44 Masson, L.F., Sharp, L., Cotton, S.C. and Pairwise combinations of estrogen Little, J. (2005) Cytochrome P-450 1A1 140j 8 Endocrine-Related Cancers and Phytochemicals

gene polymorphisms and risk of cytochromes P450 1A1 and P450 1B1 breast cancer: a HuGE review. allelic variants and other human American Journal of Epidemiology, 161 (10), cytochromes P450 in Salmonella 901–915. typhimurium NM2009. Drug Metabolism 45 Horn, T.L., Reichert, M.A., Bliss, R.L. and Disposition: The Biological Fate of and Malejka-Giganti, D. (2002) Chemicals, 29 (9), 1176–1182. Modulations of P450 mRNA in liver and 52 Paracchini, V., Raimondi, S., Gram, I.T., mammary gland and P450 activities Kang, D., Kocabas, N.A., Kristensen, V.N., and metabolism of estrogen in liver by Li, D., Parl, F.F., Rylander-Rudqvist, T., treatment of rats with indole- Soucek, P., Zheng, W., Wedren, S. and 3-carbinol. Biochemical Pharmacology, Taioli, E. (2007) Meta- and pooled analyses 64 (3), 393–404. of the cytochrome P-450 1B1 Val432Leu 46 Ciolino, H.P. and Yeh, G.C. (1999) polymorphism and breast cancer: a HuGE- Inhibition of aryl hydrocarbon-induced GSEC review. American Journal of cytochrome P-450 1A1 enzyme activity and Epidemiology, 165 (2), 115–125. CYP1A1 expression by resveratrol. 53 Shimada, T., Watanabe, J., Kawajiri, K., Molecular Pharmacology, 56 (4), 760–767. Sutter, T.R., Guengerich, F.P., Gillam, 47 Muskhelishvili, L., Thompson, P.A., E.M. and Inoue, K. (1999) Catalytic Kusewitt, D.F., Wang, C. and Kadlubar, properties of polymorphic human F.F. (2001) In situ hybridization and cytochrome P450 1B1 variants. immunohistochemical analysis of Carcinogenesis, 20 (8), 1607–1613. cytochrome P450 1B1 expression in 54 Hayes, C.L., Spink, D.C., Spink, B.C., Cao, human normal tissues. The Journal of J.Q., Walker, N.J. and Sutter, T.R. (1996) 17 Histochemistry and Cytochemistry, 49, Beta-estradiol hydroxylation catalyzed by 229–236. human cytochrome P450 1B1. Proceedings 48 Sissung, T.M., Price, D.K., Sparreboom, A. of the National Academy of Sciences of the and Figg, W.D. (2006) Pharmacogenetics UnitedStatesofAmerica,93(18),9776–9781. and regulation of human cytochrome P450 55 Finnstrom,€ N., Ask, B., Dahl, M.L., 1B1: implications in hormone-mediated Gadd, M. and Rane, A. (2002) Intra- tumor metabolism and a novel target for individual variation and sex differences therapeutic intervention. Molecular Cancer in gene expression of cytochromes Research, 4 (3), 135–150. P450 in circulating leukocytes. The 49 Rochat, B., Morsman, J.M., Murray, G.I., Pharmacogenomics Journal, 2 (2), Figg, W.D. and McLeod, H.L. (2001) 111–116. Human CYP1B1 and anticancer agent 56 Tuominen,R., Warholm, M., Moller,€ L. and metabolism: mechanism for tumor- Rannug, A. (2003) Constitutive CYP1B1 speciﬁc drug inactivation? The Journal of mRNA expression in human blood Pharmacology and Experimental mononuclear cells in relation to Therapeutics, 296, 537–541. gender, genotype, and environmental 50 Wen, W. (2007) Expression of factors. Environmental Research, 93 (2), cytochrome P450 1B1 and catechol- 138–148. O-methyltransferase in breast tissue and 57 Lemm, F., Wilhelm, M. and Roos, P.H. their associations with breast cancer risk. (2004) Occupational exposure to polycyclic Cancer Epidemiology, Biomarkers & aromatic hydrocarbons suppresses Prevention, 16 (5), 917–920. constitutive expression of CYP1B1 on 51 Shimada, T., Oda, Y., Gillam, E.M., the transcript level in human Guengerich, F.P. and Inoue, K. (2001) leukocytes. International Journal of Metabolic activation of polycyclic aromatic Hygiene and Environmental Health, hydrocarbons and other procarcinogens by 207 (4), 325–335. References j141

58 Stoilov, I., Akarsu, A.N., Alozie, I. et al. 67 Li, D.N., Seidel, A., Pritchard, M.P., Wolf, (1998) Sequence analysis and homology C.R. and Friedberg, T. (2000) modeling suggest that primary congenital Polymorphisms in P450 CYP1B1 glaucoma on 2p21 results from mutations affect the conversion of estradiol to the disrupting either the hinge region or the potentially carcinogenic metabolite conserved core structures of cytochrome 4-hydroxyestradiol. Pharmacogenetics, 10, P4501B1. American Journal of Human 343–353. Genetics, 62, 573–584. 68 Stoilov, I., Akarsu, A.N., Alozie, I. et al. 59 Cuthill, S., Poellinger, L. and Gustafsson, (1998) Sequence analysis and homology J.A. (1987) The receptor for 2,3,7,8- modeling suggest that primary congenital tetrachlorodibenzo-p-dioxin in the mouse glaucoma on 2p21 results from mutations hepatoma cell line Hepa 1c1c7. A disrupting either the hinge region or the comparison with the glucocorticoid conserved core structures of cytochrome receptor and the mouse and rat hepatic P4501B1. American Journal of Human dioxin receptors. The Journal of Biological Genetics, 62, 573–584. Chemistry, 262, 3477–3481. 69 Bailey, L.R., Roodi, N., Dupont, W.D. and 60 Widschwendter, M., Siegmund, K.D., Parl, F.F. (1998) Association of cytochrome Muller, H.M. et al. (2004) Association of P450 1B1 (CYP1B1) polymorphism with breastcancerDNAmethylationproﬁleswith steroid receptor status in breast cancer. hormone receptor status and response to Cancer Research, 58, 5038–5041. tamoxifen. Cancer Research, 64, 3807–3813. 70 Landi, M.T., Bergen, A.W., Baccarelli, A. 61 Tokizane, T., Shiina, H., Igawa, M. et al. et al. (2005) CYP1A1 and CYP1B1 (2005) Cytochrome P450 1B1 is genotypes, haplotypes, and TCDD- overexpressed and regulated by induced gene expression in subjects from hypomethylation in prostate cancer. Seveso, Italy. Toxicology, 207, 191–202. Clinical Cancer Research, 11, 5793–5801. 71 Poland, A., Glover, E. and Bradﬁeld, C.A. 62 Tokizane, T., Shiina, H., Igawa, M. et al. (1991) Characterization of polyclonal (2005) Cytochrome P450 1B1 is antibodies to the Ah receptor prepared by overexpressed and regulated by immunization with a synthetic peptide hypomethylation in prostate cancer. hapten. Molecular Pharmacology, 39,20–26. Clinical Cancer Research, 11, 5793–5801. 72 Henry, E.C., Rucci, G. and Gasiewicz, 63 Han, W., Kang, D., Park, I.A. et al. (2004) T.A. (1989) Characterization of multiple Associations between breast cancer forms of the Ah receptor: comparison of susceptibility gene polymorphisms and species and tissues. Biochemistry, 28, clinicopathological features. Clinical 6430–6440. Cancer Research, 10, 124–130. 73 Hatanaka, N., Yamazaki, H., Oda, Y., 64 Available at: http://www.imm.ki.se/ Guengerich, F.P., Nakajima, M. and Yokoi, CYPalleles/cyp1b1.htm. T. (2001) Metabolic activation of 65 Shimada, T., Watanabe, J., Kawajiri, K. et al. carcinogenic 1-nitropyrene by human (1999) Catalytic properties of polymorphic cytochrome P450 1B1 in Salmonella human cytochrome P450 1B1 variants. typhimurium strain expressing an Carcinogenesis, 20, 1607–1613. O-acetyltransferase in SOS/umu assay. 66 Hanna, I.H., Dawling, S., Roodi, N., Mutation Research, 497, 223–233. Guengerich, F.P. and Parl, F.F. (2000) 74 Henry, E.C., Rucci, G. and Gasiewicz, T.A. Cytochrome P450 1B1 (CYP1B1) (1989) Characterization of multiple forms pharmacogenetics: association of of the Ah receptor: comparison of species polymorphisms with functional and tissues. Biochemistry, 28, 6430–6440. differences in estrogen hydroxylation 75 Ikuta, T., Eguchi, H., Tachibana, T., activity. Cancer Research, 60, 3440–3444. Yoneda, Y. and Kawajiri, K. (1998) Nuclear 142j 8 Endocrine-Related Cancers and Phytochemicals

localization and export signals of the response to tamoxifen. Cancer Research, 64, human aryl hydrocarbon receptor. The 3807–3813. Journal of Biological Chemistry, 273, 83 Kocabas, N.A., Sardas, S., Cholerton, S., 2895–2904. Daly, A.K. and Karakaya, A.E. (2002) 76 Prokipcak, R.D. and Okey, A.B. (1988) Cytochrome P450 CYP1B1 and catechol Physicochemical characterization of the O-methyltransferase (COMT) nuclear form of Ah receptor from mouse genetic polymorphisms and breast hepatoma cells exposed in culture to cancer susceptibility in a Turkish 2,3,7,8-tetrachlorodibenzo-p-dioxin. population. Archives of Toxicology, 76, Archives of Biochemistry and Biophysics, 267, 643–649. 811–828. 84 Cook, L. (2003) Hormones, Genes, and 77 Available at: www.upci.upmc.edu/ Cancer, Oxford University Press, New York research/ccps/ccontrol/g_intro.html. (NY), pp. 371–379. 78 Paracchini, V., Raimondi, S., Gram, I.T., 85 Rylander-Rudqvist, T., Wedren, S., Kang, D., Kocabas, N.A., Kristensen, V.N., Granath, F., Humphreys, K., Ahlberg, S., Li, D., Parl, F.F., Rylander-Rudqvist, T., Weiderpass, E., Oscarson, M., Ingelman- Soucek, P., Zheng, W., Wedren, S. and Sundberg, M. and Persson, I. (2003) Taioli, E. (2007) Meta- and pooled analyses Cytochrome P450 1B1 gene of the cytochrome P-450 1B1 Val432Leu polymorphisms and postmenopausal polymorphism and breast cancer: a HuGE- breast cancer risk. Carcinogenesis, 24 (9), GSEC review. American Journal of 1533–1539. Epub 2003, July 4. Epidemiology, 165 (2), 115–125. Epub 2006, 86 Justenhoven, C., Pierl, C.B., Haas, S., October 19. Fischer, H.P., Baisch, C., Hamann, U., 79 Saintot, M., Malaveille, C., Hautefeuille, A. Harth, V., Pesch, B., Bruning,€ T., Vollmert, and Gerber, M. (2003) Interactions C., Illig, T., Dippon, J., Ko, Y.D. and between genetic polymorphism of Brauch, H. (2008) The CYP1B1_1358_GG cytochrome P450-1B1, sulfotransferase genotype is associated with estrogen 1A1, catecholo-methyltransferase and receptor-negative breast cancer. Breast tobacco exposure in breast cancer risk. Cancer Research and Treatment, 111 (1), International Journal of Cancer, 107, 171–177. 652–657. 87 Hayes, C.L., Spink, D.C., Spink, B.C., Cao, 80 Saintot, M., Malaveille, C., Hautefeuille, A. J.Q., Walker, N.J. and Sutter, T.R. (1996) and Gerber, M. (2003) Interactions 17h-Estradiol hydroxylation catalyzed by between genetic polymorphism of human cytochrome P450 1B1. Proceedings cytochrome P450-1B1, sulfotransferase of the National Academy of Sciences of the 1A1, catecholo-methyltransferase and United States of America, 93, 9776–9781. tobacco exposure in breast cancer risk. 88 Liehr, J.G., Ricci, M.J., Jefcoate, C.R., International Journal of Cancer, 107, Hannigan, E.V., Hokanson, J.A. and Zhu, 652–657. B.T. (1995) 4-Hydroxylation of estradiol by 81 Zheng, W., Xie, D.W., Jin, F., Cheng, J.R., human uterine myometrium and myoma Dai, Q., Wen, W.Q., Shu, X.O. and Gao, microsomes: implications for the Y.T. (2000) Genetic polymorphism of mechanism of uterine tumorigenesis. cytochrome P450-1B1 and risk of breast Proceedings of the National Academy of cancer. Cancer Epidemiology Markers and Sciences of the United States of America, 92, Prevention, 9, 147–150. 9220–9224. 82 Widschwendter, M., Siegmund, K.D., 89 Newbold, R.R. and Liehr, J.G. (2000) Muller, H.M. et al. (2004) Association of Induction of uterine adenocarcinoma in breast cancer DNA methylation proﬁles CD-1 mice by catechol estrogens. Cancer with hormone receptor status and Research, 60, 235–237. References j143

90 Raftogianis, R., Creveling, C., human oral epithelial cells: impact on Weinshilboum, R. and Weisz, J. (2000) DNA adduct formation and prevention by Estrogen metabolism by conjugation. polyphenols. Carcinogenesis, 26 (10), Journal of the National Cancer Institute. 1774–1781. Monographs, 27, 113–124. 93 Potter, G.A., Patterson, L.H., Wanogho, E., 91 Doostdar, H., Burke, M.D. and Mayer, R.T. Perry, P.J., Butler, P.C., Ijaz, T., Ruparelia, (2000) Bioﬂavonoids: selective substrates K.C., Lamb, J.H., Farmer, P.B., Stanley, and inhibitors for cytochrome P450 L.A. and Burke, M.D. (2002) The cancer CYP1A and CYP1B1. Toxicology, 144 (1–3), preventative agent resveratrol is converted 31–38. to the anticancer agent piceatannol by the 92 Wen, X. and Walle, T. (2005) Preferential cytochrome P450 enzyme CYP1B1. British induction of CYP1B1 by benzo[a]pyrene in Journal of Cancer, 86 (5), 774–778. j145

9 Inflammation-Induced Carcinogenesis and Chemoprevention Hiroshi Ohshima, Susumu Tomono, Ying-Ling Lai, and Noriyuki Miyoshi

9.1 Introduction

Chronic inflammation induced by biological, chemical, and physical factors has been associated with increased risk of human cancer at various sites. About 18% of 10 million new cancer cases in 2000 were attributable to inflammation associated with chronic infections caused by viruses, bacteria, and parasites [1]. This percentage increases further if chronic inflammatory diseases induced by chemical and physical factors (asbestos, cigarette smoke, gastric acid, hot beverages, etc.) or by unknown causes (ulcerative colitis, pancreatitis, etc.) are included. Inflammation facilitates the initiation of normal cells and their growth and progression to malignancy through production of proinflammatory cytokines and oxidants, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), and activation of signaling pathways involved in inflammation and carcinogenesis. Hanahan and Weinberg [2] recently proposed six major characteristics (self- sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis) that are required for a normal cell to become a tumor cell. These pathways could be disrupted by genetic alterations in genes involved in carcinogenesis (oncogenes and tumor suppressor genes) or by epigenetic processes such as gene methylation, post-translational modifications of proteins, and modification of gene expression patterns. Diverse proinflammatory cytokines, ROS, and RNS generated in inflamed tissues can cause genetic and epigenetic changes affecting these major pathways [3, 4] (Figure 9.1). Better understanding of the molecular mechanisms by which chronic inflammation increases cancer risk will lead to the development of new strategies for cancer prevention at many sites. In this chapter, we discuss possibilities for cancer prevention by modulating inflammatory processes and production of ROS and RNS in inflamed tissues with dietary factors.

Figure 9.1 A signaling pathway associated with inflammation- induced cancer. Possible chemopreventive agents are shown in shaded boxes.

9.2 Prevention of Inflammation-Associated Cancer by Avoidance of Causes of Tissue Damage

The first important point in controlling inflammation, thus in the prevention of inflammation-associated carcinogenesis, is avoidance of causes of tissue damage, namely, exposure to chemical and physical agents (asbestos, cigarette smoke, etc.) and infectious agents. Thus, modulation of lifestyle (by stopping smoking, etc.) and control of infection by improving sanitary conditions or by intervention with immunization or eradication may offer great potential for cancer prevention. Vaccination against hepatitis B virus has been shown to reduce the incidence of hepatocellular carcinoma [5]. The incidence of cervical cancer may be greatly reduced by vaccines against human papillomaviruses [6]. Eradication of Helicobacter pylori alone with omeprazole and antibiotics appears to have only a moderate effect on gastric cancer incidence worldwide [7]. However, H. pylori eradication reduced the prevalence of precancerous lesions [8] and also decreased the development of gastric cancer in H. pylori carriers without precancerous lesions, whereas it had no effect on 9.3 Chemoprevention by Modulating Inflammatory Processes j147 the incidence of gastric cancer in subjects with precancerous lesions [9]. As it has been reported that Lactobacillus acidophilus and Bifidobacterium-containing yogurt (AB-yogurt) improved the efficacy of H. pylori eradication by triple/quadruple therapy [10] and green tea extract suppressed H. pylori-induced gastritis in Mongolian gerbils [11], certain food components may be beneficial to prevent H. pylori-associated gastric cancer.

9.3 Chemoprevention by Modulating Inflammatory Processes

Various mechanisms have been proposed for the action of chemopreventive agents [4, 12, 13] (Figure 9.1). They include inhibition of signaling pathways (e.g., NF-kB), inhibition of oxidant-generating enzymes, such as NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS), and mediators of inﬂammation (e.g., cyclooxgenase (COX)-2), apoptosis induction, scavenging of ROS/RNS and antioxidants, and induction of xenobiotic-metabolizing enzymes (especially phase II enzyme induction).

9.3.1 NF-kB

Nuclear factor-kB (NF-kB) is a transcription factor, playing a crucial role in the regulation of inflammatory and immune responses and in carcinogenesis [14]. In unstimulated cells, NF-kB is sequestered and inactive in the cytoplasm through its association with inhibitory IkBs. Upon stimulation, IkBs are phosphorylated by IkB kinase (IKK) and degraded through the ubiquitin–proteasome-dependent pathway, leading to the release of NF-kB that enters the nucleus and activates genes important for immune and inflammatory responses, cell survival, and cell proliferation [14]. A variety of stimuli activate NF-kB, including inflammatory cytokines such as TNF-a,

ROS such as hydrogen peroxide (H2O2), infection with bacteria and viruses, various carcinogens and tumor promoters such as benzo[a]pyrene, the tobacco-specific nitrosamine N-nitrosonornicotine and phorbol myristate acetate, therapeutic agents such as taxol, apoptotic mediators such as anti-Fas, UV radiation, and many others [15]. NF-kB-regulated genes involved in carcinogenesis include c-Myc, epidermal growth factor receptor, cell adhesion molecules, iNOS, vascular endothelial cell growth factor, COX-2, Bcl-2, Bcl-xL, and others [16]. Thus, stimuli regulated by NF-kB during inflammation can be redirected as tumor growth signals. In addition, NF-kB has been found constitutively activated in many human tumor samples [15], supporting a crucial role of NF-kB in tumor development. Various chemopreventive agents (Scheme 9.1) can suppress NF-kB activation, including curcumin (a phytochemical component in turmeric), silymarin (a flavonoid from milk thistle, Silybum marianum L.), resveratrol (a grape phytoalexin), capsaicin, aspirin, celecoxib, retinoids, vitamin E, some plant polyphenols such as epigallocatechin gallate (EGCG, a major green tea polyphenol), and others [16]. 148j 9 Inflammation-Induced Carcinogenesis and Chemoprevention

Scheme 9.1 Chemical structures of dietary compounds with potential cancer chemopreventive activities.

However, inhibition of NF-kB activation has been reported to promote inﬂammation and carcinogenesis under certain circumstances [17].

9.3.2 iNOS

iNOS is induced in inﬂamed tissues at least partly via activation of NF-kB by cytokines such as TNF-a, IL-1b, and interferon-g. iNOS catalyzes the production of nitric oxide (NO) from L-arginine, and its expression has been reported in human cancer at a variety of sites, including the bladder, prostate, oral cavity, esophagus, stomach, colon, and breast [18]. Several recent reviews have discussed whether iNOS can be a target for cancer chemoprevention [4, 18, 19]. Various phytochemicals (Scheme 9.1) have been reported to suppress iNOS induction in cell culture systems, including curcumin, resveratrol, EGCG, genistein 9.3 Chemoprevention by Modulating Inflammatory Processes j149

(a soy isoflavone), allyl isothiocyanate (found in mustard oil), benzyl isothiocyanate (BITC, a cruciferous vegetable constituent), and zerumbone (a sesquiterpene from Zingiber zerumbet, a herbal plant that is also known as lempoyang) [19]. Effects of iNOS inhibitors on carcinogenesis have been examined in various animal models. Both inhibitory and enhancing effects have been reported, depending on the animal models used, the organ sites, the experimental protocols, and the type and specificity of iNOS inhibitors [18, 20]. Additional studies on the chemopreventive effects of iNOS inhibitors using more iNOS-selective inhibitors in site-specific and cell type- specific ways are needed.

9.3.3 COX-2

COX-1 and COX-2 catalyze the conversion of arachidonic acid to prostaglandins. COX-1 is a constitutive isoform that is present in most tissues and may carry out normal housekeeping functions. COX-2 is inducible, at least partly via activation of NF-kB, by chemical, physical, and biological stimuli such as cytokines, mitogens, growth factors, UV-light exposure, and tumor promoters and is overexpressed in numerous premalignant and malignant lesions, including those of the colorectum, breast, prostate and lung, and others. The role of COX-2 in carcinogenesis may include modulation of apoptosis and immunity, stimulation of angiogenesis, and promotion of tumor invasion [21]. Several nonsteroidal anti-inﬂammatory drugs (NSAIDs) such as aspirin, other traditional NSAIDs, and more recent COX-2 selective inhibitors (e.g., celecoxib) have been shown to be effective in decreasing risk of several types of cancer in epidemiological and clinical studies [22]. However, recent studies showed that long-term use of high doses of COX-2 selective inhibitors is associated with an increased cardiovascular toxicity. The mechanisms by which such nonsteroidal anti-inﬂammatory agents act to prevent cancer remain unclear. Both COX-dependent and COX-independent mechanisms have been proposed. Food ingredients that can suppress COX-1 and COX-2 include resveratrol, curcumin, green tea extract containing catechin and EGCG, genistein, BITC, zerumbone, galangin, luteolin, apigenin, 6-hydroxykaempferol, sasanquol, and wogonin [13, 19] (Scheme 9.1).

9.3.4 ROS-Generating Enzymes and Antioxidant Defense Mechanisms

In inﬂamed tissues, various ROS and RNS are generated by both enzymatic and nonenzymatic reactions. ROS and RNS can damage DNA, RNA, proteins, and lipids by oxidation, nitration, and halogenation reactions. The production of ROS and the defense against ROS-induced damage are regulated genetically and may be associated with individual susceptibility to inﬂammation-induced cancer. Several food ingredients and phytochemicals (BITC, silibinin, genistein, etc.) have been reported to inhibit the superoxide-generating enzyme, NADPH oxidase, in 150j 9 Inflammation-Induced Carcinogenesis and Chemoprevention

experimental systems [19] (Scheme 9.1). Dietary antioxidants such as tea polyphenols, curcumin, genistein, resveratrol, lycopene, pomegranate, and lupeol have been effective against cancers of the skin, prostate, breast, lung, and liver in experimental systems [23]. However, caution must be observed while using antioxidants and scavengers of ROS and RNS in humans, particularly as the mechanism of action and possible interactions with lifestyle factors such as smoking and alcohol drinking are poorly understood. Three large cancer chemoprevention trials conducted in the 1980s and 1990s using b-carotene as a chemopreventive agent showed that the incidence of lung cancer was increased significantly in subjects supplemented with b-carotene, especially those with smoking and alcohol drinking habits. The mechanisms underlying this increased risk remain unclear, but various possibilities have been proposed, including formation of the free radical b-carotene by cigarette smoke and propagation of free radical chain reactions [24]. Consumption of fruit and vegetables has been associated with reduced risks of cancers at various sites such as the mouth and pharynx, esophagus, stomach, colorectum, larynx, lung, ovary, bladder, and kidney. However, the mechanisms of cancer preventive effects of fruit and vegetables have not been established. Although the best, but still tentative, evidence is related to modulation of xenobiotic-metabolizing enzymes (especially phase II enzyme induction through activation of the Nrf-2 pathway), some other mechanisms may also be involved, including inhibition of endogenous formation of carcinogens, ROS- and RNS-induced DNA or tissue damage and lipid peroxidation, and modulation of apoptosis, inflammation, and cell signaling. Fruit and vegetables contain many nutrients and bioactive compounds, and they may act in concert to influence carcinogenesis [25].

9.4 Conclusion

Human cancer associated with chronic inflammation could be prevented by modulating inflammatory processes using anti-inflammatory drugs, dietary antioxidants, and other factors. However, further studies are clearly needed to identify problems associated with long-term use of drugs and food ingredients to inhibit certain signaling pathways, because many signaling pathways and molecules play dual roles in inflammation and carcinogenesis.

Acknowledgments

This work was supported in part by the Global COE program and by the Grant-in-Aid (18 509 001 to HO, 19 700 592 to NM) from the Ministry of Education, Science, Culture, and Sport, by the Grant-in-Aid for Cancer Research (19-19) from the Ministry of Health, Labor,and Welfare, Japan, and a grant on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation (KHC1023). References j151 References

1 Parkin, D.M. (2001) Global cancer 11 Matsubara, S., Shibata, H., Ishikawa, F., statistics in the year 2000. The Lancet Yokokura, T., Takahashi, M., Sugimura, T. Oncology, 2, 533–543. and Wakabayashi, K. (2003) Suppression 2 Hanahan, D. and Weinberg, R.A. (2000) of Helicobacter pylori-induced gastritis by The hallmarks of cancer. Cell, 100,57–70. green tea extract in Mongolian gerbils. 3 Ohshima, H., Tatemichi, M. and Sawa, T. Biochemical and Biophysical Research (2003) Chemical basis of inflammation- Communications, 310, 715–719. induced carcinogenesis. Archives of 12 Kelloff, G.J. et al. (2006) Progress in Biochemistry and Biophysics, 417,3–11. chemoprevention drug development: the 4 Ohshima, H., Tazawa, H., Sylla, B.S. and promise of molecular biomarkers for Sawa, T. (2005) Prevention of human prevention of intraepithelial neoplasia and cancer by modulation of chronic cancer – a plan to move forward. Clinical inflammatory processes. Mutation Cancer Research, 12, 3661–3697. Research, 591, 110–122. 13 Aggarwal, B.B. and Shishodia, S. (2006) 5 Chang, M.H. et al. (2000) Hepatitis B Molecular targets of dietary agents for vaccination and hepatocellular carcinoma prevention and therapy of cancer. rates in boys and girls. The Journal of the Biochemical Pharmacology, 71, 1397–1421. American Medical Association, 284, 14 Karin, M. (2006) Nuclear factor-kappaB in 3040–3042. cancer development and progression. 6 Garland, S.M. et al. (2007) Quadrivalent Nature, 441, 431–436. vaccine against human papillomavirus to 15 Garg, A. and Aggarwal, B.B. (2002) Nuclear prevent anogenital diseases. The New transcription factor-kappaB as a target for England Journal of Medicine, 356, cancer drug development. Leukemia, 16, 1928–1943. 1053–1068. 7 Moss, S.F. and Malfertheiner, P. (2007) 16 Bharti, A.C. and Aggarwal, B.B. (2002) Helicobacter and gastric malignancies. Nuclear factor-kappa B and cancer: its role Helicobacter, 12 (Suppl. 1), 23–30. in prevention and therapy. Biochemical 8 You, W.C. et al. (2006) Randomized Pharmacology, 64, 883–888. double-blind factorial trial of three 17 Shishodia, S. and Aggarwal, B.B. (2004) treatments to reduce the prevalence of Nuclear factor-kappaB: a friend or a foe in precancerous gastric lesions. Journal of the cancer? Biochemical Pharmacology, 68, National Cancer Institute, 98, 974–983. 1071–1080. 9 Wong, B.C. et al. (2004) Helicobacter pylori 18 Crowell, J.A., Steele, V.E., Sigman, C.C. eradication to prevent gastric cancer, in a and Fay, J.R. (2003) Is inducible nitric oxide high-risk region of China: a randomized synthase a target for chemoprevention? controlled trial. The Journal of the American Molecular Cancer Therapeutics, 2, 815–823. Medical Association 291, 187–194. 19 Murakami, A. and Ohigashi, H. (2007) 10 Sheu, B.S., Cheng, H.C., Kao, A.W., Wang, Targeting NOX, INOS and COX-2 in S.T., Yang, Y.J., Yang, H.B. and Wu, J.J. inflammatory cells: chemoprevention (2006) Pretreatment with Lactobacillus- using food phytochemicals. International and Bifidobacterium-containing yogurt Journal of Cancer, 121, 2357–2363. can improve the efficacy of quadruple 20 Lancaster, J.R. Jr and Xie, K. (2006) Tumors therapy in eradicating residual face NO problems? Cancer Research, 66, Helicobacter pylori infection after failed 6459–6462. triple therapy. The American Journal of 21 Zha, S., Yegnasubramanian, V., Nelson, Clinical Nutrition, 83, 864–869. W.G., Isaacs, W.B. and De Marzo A.M. 152j 9 Inflammation-Induced Carcinogenesis and Chemoprevention

(2004) Cyclooxygenases in cancer: Antioxidants & Redox Signaling, 10, progress and perspective. Cancer Letters, 475–510. 215,1–20. 24 Szabo, E. (2006) Selecting targets for 22 Ulrich, C.M., Bigler, J. and Potter, J.D. cancer prevention: where do we go from (2006) Non-steroidal anti-inﬂammatory here? Nature Reviews. Cancer, 6, 867–874. drugs for cancer prevention: promise, 25 IARC Working Group on the Evaluation perils and pharmacogenetics. Nature of Cancer-Preventive Strategies (2003) Reviews. Cancer, 6, 130–140. IARC Handbooks of Cancer Prevention, 23 Khan, N., Afaq, F. and Mukhtar, H. (2007) Volume 8: Fruits and Vegetables, Cancer chemoprevention through dietary International Agency for Research on antioxidants: progress and promise. Cancer, Lyon. j153

10 DNA Methylation Ian T. Johnson, Nigel J. Belshaw, and Giles O. Elliott

10.1 Introduction

Chromatin architecture is a major determinant of gene expression in higher organisms, and it is well established that many epigenetic signals are encoded by covalent modiﬁcations to DNA and the core histones [1]. One such epigenetic mark is methylation at the C5 position of cytosines lying 50 to guanosine in CpG dinucleotides. CpGs are distributed irregularly throughout the mammalian genome and tend to occur most abundantly in localized regions ranging from 0.5 to 4 kb in length called CpG islands (CGI), located mainly in the promoter regions of functional genes. Unmethylated CGIs are associated with open, transcriptionally active structure in the adjacent chromatin, whereas methylated CpGs recruit protein complexes that promote histone deacetylation, leading to chromatin compaction and silencing [2, 3]. This mechanism may have evolved as a means of silencing parasitic DNA elements, but in the complex genomes of higher organisms it also functions as a regulatory mechanism [4]. Recently, it has become clear that DNA methylation is susceptible to a host of poorly characterized environmental factors [5]. Tumorsexhibit a host of DNA methylation abnormalities, and since CGI methylation is transmitted through mitosis, aberrant epigenetic signals are ampliﬁed within proliferating tissues by clonal expansion, disrupting gene expression, and driving phenotypic changes in a similar way to mutation. Methylation of CpG dinucleotides is an active process maintained and regulated by two classes of DNA methyltransferase (DNMT) enzymes. DNA methyltransferases 3a and 3b (DNMT3a; DNMT3b) are essential for de novo methylation of DNA in the growing embryo, and inactivation of these enzymes arrests normal embryonic development [6]. DNA methyltransferase1 (DNMT1) is primarily a maintenance enzyme that methylates cytosines in CpG dinucleotides during DNA synthesis. It is essential for the accurate transmission of DNA methylation patterns through mitosis, and its deletion also causes embryonic lethality [7].

Figure 10.1 The diagram summarizes the methylated, including those associated with acquired abnormalities of DNA methylation that retrotransposons. Methylation of CpG islands occur in tumors and certain nonneoplastic leads to gene silencing and favors suppression of tissues (e.g., colorectal mucosa) in old age. In retrotransposition. In cancer, and in certain normal tissue, regions of the genome rich in aging cells, aberrant methylation of CpG islands cytosine–guanine dinucleotides (CpG islands) can cause silencing of normally expressed genes, located close to the promoter regions of actively whereas global demethylation of CpGs located expressed genes are largely unmethylated. elsewhere in the genome can activate Cytosines elsewhere in the genome tend to be retrotransposons and cause genomic instability.

DNA methylation patterns of neoplastic tissues typically differ markedly from those of normal tissue. In general, there is a reduction in methylation of cytosines remote from CGIs (DNA hypomethylation), coupled with hypermethylation of CGI located near or within the promoter regions of functional genes (Figure 10.1). It should be noted, however, that there appears to be no simple mechanistic link between these phenomena. Promoter hypermethylation occurs in virtually every type of human neoplasia, and the contribution of gene silencing caused by aberrant CGI methylation to the disruption of the normal phenotype is at least as great as that of somatic mutations. Genes in which inherited mutations are associated with familial cancer syndromes have in many cases been found to be susceptible also to methylation events that can contribute to carcinogenesis by providing a second hit. Moreover, it has also become clear that tumor-suppressor and DNA-repair genes not commonly mutated in tumors may frequently be silenced by CGI methylation [8]. Esteller et al. [9] reported that amongst a panel of 12 candidate genes, one or more was methylated in every one of the 15 different types of human tumor examined, but the methylation proﬁle differed markedly between different tumors. CGI methylation, and by implication gene silencing, affected virtually all of the main types of pathways contributing to the cancer phenotype, and often there was evidence for simultaneous methylation of genes associated with several different pathways in the same tumor. For example, p16INK4a, which is directly involved with regulation of the cell cycle, was methylated in virtually all the main types of tumor, but colorectal and gastric tumors were typically also methylated at p14ARF, MGMT, APC, and hMLH1. 10.2 Effects of Diet on DNA Methylation j155

The explosion of interest in CGI methylation in recent years has led to many new developments including a search for methylation biomarkers as a means of profiling tumors and tailoring strategies for chemotherapy, the development of therapeutic methods for the reversal of methylation and the re-expression of genes, and efforts to achieve a full analysis of the human epigenome. There is evidence that epigenetic changes are involved at all stages of the neoplastic sequence, but of particular interest is its contribution to the early stages of tumorigenesis. Feinberg et al. [10] have proposed that epigenetic changes, occurring in stem cells long before the emergence of discreet lesions, may act as epigenetic progenitors and as surrogates for the somatic mutations. Issa and colleagues [11] reported that the estrogen receptor gene ESR1 becomes methylated in the apparently normal flat mucosa of colorectal cancer patients. They went on to show that this also occurs in an age-dependent manner in cancer-free individuals, and speculated that ESR1 is itself a tumor- suppressor gene and that its age-dependent silencing may be a factor contributing to the increasing risk of colorectal cancer in the elderly. Although this role of ESR1 has not been confirmed, there is growing evidence that other genes that do have an established functional role in colorectal carcinogenesis also become methylated in the apparently normal mucosa of cancer patients [12]. The characterization of such field changes in the epithelia of the alimentary tract and other organs, their origins, and the effects on them of environmental factors, carcinogens, nutrients, and drugs are of growing interest for those concerned with strategies for cancer prevention.

10.2 Effects of Diet on DNA Methylation

The most significant feature of epigenetic marks is that unlike mutations they can, in principle, be modified so that abnormally silenced genes are reactivated. However, it is important to be aware that drug induced demethylation can also cause global demethylation as well as gene reactivation. It is also unclear whether some demethy- lating agents may modify the patterns of methylation of imprinted genes (loci methylated only at a single allele, inherited from one parent). Nevertheless, there is growing interest in the use of pharmaceutical intervention to modify epigenetic signals in established disease [13], and in the possibility that diet may exert an influence on the epigenetic status of tissues during the earliest stages of cancer development. Some of the best evidence that nutrients can modulate the epigenetic status of mammalian tissues comes from studies with mice carrying the agouti viable yellow gene. It is well established that mutations of the agouti locus cause a pleiotropic syndrome in which excessive amounts of yellow pigment are present in the fur. This physical abnormality is coupled with systemic effects including obesity, a noninsulin- dependent diabetic-like condition, and vulnerability to various types of cancer [14]. In the agouti viable yellow mouse (AIAP), the mutation consists of an intracisternal A particle (IAP). This is a retroviral DNA sequence that has become inserted close to the first coding exon of the agouti gene [15]. Expression of the gene varies with the 156j 10 DNA Methylation

methylation status of the 50 long terminal repeat within the inserted IAP. When this is methylated, the gene behaves like a wild-type allele and is expressed only in the hair follicle, but when it is unmethylated, gene expression becomes uncontrolled, causing the full agouti syndrome. Intermediate levels of methylation also occur, causing mottled fur and other variations on the agouti phenotype. The model thus provides a direct visual readout of the methylation status of the aberrant agouti gene. Wolff et al. [16] showed that by feeding pregnant mice on diets supplemented with high levels of folic acid and other methyl donors, it was possible to modify the expression of the agouti gene in their offspring. A higher proportion of offspring with wild-type coat color was obtained from the supplemented dams, and this was shown to be correlated with higher levels of DNA methylation within the IAP [17]. Cropley et al. [18] fed methyl-donor-supplemented diets to pregnant dams over a

period of 7 days during mid-gestation and then bred their offspring (F1) to observe any effects on the subsequent (F2) generation. The phenotypes in the F1 generation were ﬁ modi ed by supplementation, and the F2 generation also showed evidence of a shift in phenotype, which could only be attributed to the dietary supplementation of their grandmothers. This suggests that exposure to methyl donors in utero modiﬁes the epigenetic marks of the developing germ cells and that the signal is retained through gametogenesis, fertilization, and embryonic development of the next generation.

10.3 Impact of Environment and Nutrition on the Human Epigenome

Methylation of CGI increases with aging in many cell types and organisms, and this may profoundly modify their vulnerability to cancer [19]. To what extent do these processes occur in humans and are the sites or rates of DNA methylation modified by environmental factors, lifestyle, or health? Much remains to be done to address these issues but the mutability of the human epigenome appears to have been confirmed by recent studies using monozygotic (MZ) twins. Fraga et al. [20] showed that although patterns of DNA methylation across the genome were very similar in various tissues from young MZ twins, the patterns diverged in older twins, suggesting that exposure to differing environments may modify DNA methylation throughout life. However, to what extent these differences could be explained by passive intrinsic mechanisms, such as epigenetic drift, is not yet clear. Disease may also modify the epigenetic status of an individual in later life by accelerating CGI methylation in affected tissues. This mechanism is known to occur in the epithelial tissues of the alimentary tract affected by inflammatory disorders. For example, ulcerative colitis is associated with increased CGI methylation in the colon [21], perhaps as a direct consequence of elevated levels of the proinflammatory cytokine interleukin 6 (IL-6) [22]. It is interesting to speculate that a similar mechanism might operate systemically in individuals with chronic low-grade inflammation, such as occurs in obese patients, whose adipose tissue is a rich source of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-a) and IL-6 [23]. 10.4 Modification of DNA Methylation by Nutrients and Phytochemicals j157

10.4 Modification of DNA Methylation by Nutrients and Phytochemicals

10.4.1 Folates

The food-borne derivatives of folic acid collectively known as folates act as methyl donors in human metabolism and are of fundamental importance for normal DNA synthesis and repair. Conversion of deoxyuridylate to thymidylate requires the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate by the enzyme methyltetrahydrofolate reductase (MTHFR). Moreover, 5-methyltetrahydrofolate is itself essential for the conversion of homocysteine to methionine, the precursor of S-adenosylmethionine (SAM). This is the main methyl donor for intracellular methylation reactions, including that of cytosine. Low levels of 5-methyltetrahydrofolate could therefore, in principle, lead to low intracellular levels of SAM, elevated S-adenosylhomocysteine (SAH), and deﬁcient methylation of cytosine, both within CGI and elsewhere in the genome. A much simpliﬁed summary of methyl group metabolism is given in Figure 10.2. We

Figure 10.2 A highly simplified scheme for one-carbon metabolism, showing the central importance of S-adenosylmethionine (SAM) in relation to DNA methylation. Dietary folate restriction can lead to reduced levels of SAM and hence to aberrant methylation of DNA. THF, tetrahydrofolate; 5-MeTHF, 5-methyltetrahydrofolate; and 5,10-MeTHF, 5,10-methylenetetrahydrofolate. 158j 10 DNA Methylation

have previously observed that the experimental manipulation of dietary methyl donors modifies the epigenome of mice [17]. There is much current interest in the relationship between folate nutrition and the methylation status of the human genome. In vitro studies confirm that, at least in some cell types, DNA hypomethylation is associated with folate deficiency [24]. Rats maintained on diets deficient in methyl group donors (folate, methionine, choline, and vitamin B12) show significant genome-wide and gene-specific DNA hypomethylation, as well as gene-specific DNA hypermethylation, in the liver [25, 26], but in the colon, neither genome-wide nor gene-specific DNA methylation appears to be affected [27]. SAM:SAH ratios may be more resistant to perturbation by methyl donor deficiency in the colon mucosa compared to the liver, but severe and prolonged folate-deficiency has been shown to cause a decrease in colonic SAM:SAH without an effect on genome-wide or gene- specific DNA methylation [27]. In humans, there appears to be stronger evidence for effects of folate status on global DNA methylation than on the methylation status of particular CGIs. Pufulete et al. observed a positive correlation between folate status and colonic DNA methylation in individuals with [28] and without colorectal adenomas or cancers [29], and a similar correlation has also been reported for cervical tissue [30]. More recently, serum vitamin B12, but not folate status, was reported to be associated with CGI methylation of ESR1 in normal-appearing colonic mucosa [31]. A study investigating the impact of dietary folate and alcohol intake on CGI hypermethylation in colorectal tumors showed that methylation levels were higher in tumors from patients with low folate levels and/or high alcohol intake compared to those from patients with high folate levels and low alcohol intake, although the difference was not statistically significant [32]. In contrast, Slattery et al. [33] found no association between dietary folate, vitamins B6 and B12, methionine, or alcohol and the presence of the CGI methylator phenotype (CIMP), as defined by the presence of two or more methylated CGIs out of five, in colon tumors. In general, folate deficiency in humans has been found to be associated with global DNA hypomethylation [34] whereas supplementation has been associated with moderate increases in methylation [35]. However, there is growing concern that high levels of folate intake associated with fortification of cereals may lead to abnormalities of DNA methylation that could favor the development of colorectal carcinoma from adenomatous polyps [36]. Some support for this conjecture has recently been provided by studies describing what appears to be a protective effect of low folate status against colorectal cancer in a Swedish population [37] and a rise in the incidence of colorectal cancer following the introduction of mandatory supplementation of cereals with folic acid in both North America and Canada [38].

10.4.2 Selenium

High concentrations of selenium have been reported to exert inhibitory effects on DNMT1 activity in vitro and to decrease DNMT1 protein expression [39]. Although 10.4 Modification of DNA Methylation by Nutrients and Phytochemicals j159 contradictory results have also been obtained [40], selenium is regarded by some as another nutrient that may modify levels of DNA methylation. Davis et al. [41] showed that in rats fed selenium-deﬁcient diets, both liver and colon DNA were signiﬁcantly hypomethylated compared to those of rats fed diets supplemented with either selenite or selenomethionine. However, there is little evidence for similar effects on humans.

10.4.3 Polyphenols

Polyphenols are secondary plant metabolites that occur abundantly in the human diet. They are best known to food scientists as antioxidants, but they exert a variety of other biological activities including enzyme inhibition. Several studies have demonstrated the potent inhibition of DNMT activity by the polyphenol ()- epigallocatechin-3-gallate (EGCG) in vitro, with IC50 values ranging from 0.2 to 20 mM. Although EGCG is poorly absorbed in the human gut, such concentrations certainly occur at various sites in the lumen of the gastrointestinal tract, and may occur in plasma, and so the possibility that EGCG might reduce CGI methylation and cause reactivation of silenced genes in humans merits serious consideration [42]. EGCG is the major biologically active polyphenol present in green tea, a beverage widely consumed in parts of Asia, which appears to exert chemopreventative effects againstcancers of the alimentary tract and other sites [43]. Treatment of esophageal [44] and other cancer cells with EGCG in vitro leads to the reversal of gene-specific CGI methylation at several loci, and these changes are associated with re-expression of silenced genes. There is some evidence from observational studies in humans that a reduction in the intake of green tea leads to methylation of the CGI associated with the CDX2 gene [45], but further studies are needed to fully explore the role of green tea as a modifier of epigenetic signals. Although EGCG has been shown to be the most potent inhibitor of DNMTactivity, several other polyphenols exert similar, but weaker effects, including the tea polyphenols catechin, epicatechin, epigallocatechin, and epicatechin gallate and their metabolites, the coffee polyphenols caffeic acid and chlorogenic acid, and the flavonols quercetin, fisetin, and myricetin [42]. A recent study demonstrated the reactivation of silenced genes by demethylation of CpG islands in colorectal cancer cells by a reduction in DNMTprotein expression upon treatment with a polyphenolic extract from Annurca apples, which had chlorogenic acid, catechin, and epicatechin as its major constituents [46]. Epidemiologic and animal studies have suggested that phytoestrogens present in soy may be protective against some cancers, including those of the colon, breast, and prostate [47]. Several mechanisms have been proposed, including inhibition or reversal of epigenetic gene silencing. The major phytoestrogen present in soy is the polyphenolic isoflavone genistein, which has been shown to alter CpG island methylation patterns in mice [48]. In the agouti mouse model, maternal genistein supplementation was shown recently to increase methylation at the agouti locus 160j 10 DNA Methylation

reversing the agouti phenotype [49]. Genistein and the other isoﬂavones from soy, biochanin A and daidzein, have also been shown to inhibit DNMTactivity and reverse aberrant CGI methylation in esophageal and prostate cancer cell lines, leading to reactivation of gene expression. These effects were enhanced when combined with those of histone deacetylase (HDAC) inhibitors [50].

10.4.4 Isothiocyanates

Isothiocyanates are metabolites of glucosinolates, which are present only in cruciferous plants, such as brassicas, many of which are used as human food [51]. Like many other isothiocyanates, phenethyl isothiocyanate (PEITC), a metabolite of gluconasturtin from watercress, exhibits potent HDAC inhibitory activity and may reactivate gene expression by directly affecting chromatin remodeling [52]. Recently PEITC has been shown to exert a dual effect on prostate cancer cells in vitro [53], whereby GSTP1 is reactivated through effects both on HDAC activity and on CGI methylation.

10.5 Conclusions

The role of DNA methylation in the development of cancer is a major topic of research at present but most of this work remains focused on the biology of established tumors. Relatively little is known about the role of methylation in the earliest stages of neoplasia, where mechanisms of chemoprevention are most relevant. The possibility that nutrients and other biologically active food constituents can act as mediators of CGI methylation is intriguing. However, the absorption and bioavailability of biologically active secondary plant metabolites are often very low, and those that are absorbed are usually metabolized to forms with activity signiﬁcantly lower than their parent compounds. Further work to identify and evaluate any chemopreventive role for the numerous phytochemicals that may modify CGI methylation in humans is undoubtedly warranted. However, at present perhaps the most urgent questions relate to the possibility that excessive dietary exposure to folates may in some circumstances promote the development of cancer. Although low folate status has generally been regarded as a risk factor for colorectal cancer, the substantial rise in folic acid intake experienced by North American populations over the past decade may, some believe, lead to excessive levels of folate in a small proportion of the population, which could have had unintended consequences [38]. The many experimental studies on the impact of folate and other methyl group donors on the mammalian epigenome provide evidence for a complex pattern of effects that seems to depend on both organism and organ, which may also vary with the state of transformation of the cell type under investigation. Further research to explore the relevance of these phenomena to humans is urgently needed. References j161 References

1 Bird, A. (2002) Genes and Development, 16, 16 Wolff, G.L., Kodell, R.L., Moore, S.R. and 6–21. Cooney, C.A. (1998) FASEB Journal, 12, 2 Jones, P.L., Veenstra, G.J., Wade, P.A., 949–957. Vermaak, D., Kass, S.U., Landsberger, 17 Waterland, R.A. and Jirtle, R.L. (2003) N., Strouboulis, J. and Wolffe, A.P. Molecular and Cellular Biology, 23, (1998) Nature Genetics, 19, 187–191. 5293–5300. 3 Nan, X., Ng, H.H., Johnson, C.A., Laherty, 18 Cropley, J.E., Suter, C.M., Beckman, K.B. C.D., Turner, B.M., Eisenman, R.N. and and Martin, D.I. (2006) Proceedings of Bird, A. (1998) Nature, 393, 386–389. the National Academy of Sciences of the 4 Hendrich, B. and Tweedie, S. (2003) Trends United States of America, 103, in Genetics, 19, 269–277. 17308–17312. 5 Waterland, R.A. and Michels, K.B. (2007) 19 Liu, L., Wylie, R.C., Andrews, L.G. and Annual Review of Nutrition, 27, 363–388. Tollefsbol, T.O. (2003) Mechanisms of 6 Okano, M., Bell, D.W., Haber, D.A. and Li, Ageing and Development, 124, 989–998. E. (1999) Cell, 99, 247–257. 20 Fraga, M.F., Ballestar, E., Paz, M.F., 7 Lei, H., Oh, S.P., Okano, M., Juttermann, Ropero, S., Setien, F., Ballestar, M.L., R., Goss, K.A., Jaenisch, R. and Li, E. Heine-Suner, D., Cigudosa, J.C., Urioste, (1996) Development (Cambridge, England), M., Benitez, J., Boix-Chornet, M., Sanchez- 122, 3195–3205. Aguilera, A., Ling, C., Carlsson, E., 8 Esteller, M. (2006) British Journal of Cancer, Poulsen, P., Vaag, A., Stephan, Z., Spector, 94, 179–183. T.D., Wu, Y.Z., Plass, C. and Esteller, M. 9 Esteller, M., Corn, P.G., Baylin, S.B. and (2005) Proceedings of the National Academy Herman, J.G. (2001) Cancer Research, 61, of Sciences of the United States of America, 3225–3229. 102, 10604–10609. 10 Feinberg, A.P., Ohlsson, R. and 21 Issa, J.P., Ahuja, N., Toyota, M., Bronner, Henikoff, S. (2006) Nature Reviews M.P. and Brentnall, T.A. (2001) Cancer Genetics, 7,21–33. Research, 61, 3573–3577. 11 Issa, J.P., Ottaviano, Y.L., Celano, P., 22 Hodge, D.R., Peng, B., Cherry, J.C., Hurt, Hamilton, S.R., Davidson, N.E. and Baylin, E.M., Fox, S.D., Kelley, J.A., Munroe, D.J. S.B. (1994) Nature Genetics, 7, 536–540. and Farrar, W.L.(2005) Cancer Research, 65, 12 Shen, L., Kondo, Y., Rosner, G.L., Xiao, L., 4673–4682. Hernandez, N.S., Vilaythong, J., 23 Fantuzzi, G. (2005) The Journal of Allergy Houlihan, P.S., Krouse, R.S., Prasad, A.R., and Clinical Immunology, 115, 911–919, Einspahr, J.G., Buckmeier, J., Alberts, D.S., quiz 920. Hamilton, S.R. and Issa, J.P. (2005) Journal 24 Duthie, S.J., Grant, G. and Narayanan, S. of the National Cancer Institute, 97, (2000) British Journal of Cancer, 83, 1330–1338. 1532–1537. 13 Yoo, C.B. and Jones, P.A. (2006) Nature 25 James, S.J., Pogribny, I.P., Pogribna, M., Reviews. Drug Discovery, 5,37–50. Miller, B.J., Jernigan, S. and Melnyk, S. 14 Yen, T.T., Gill, A.M., Frigeri, L.G., Barsh, (2003) The Journal of Nutrition, 133, G.S. and Wolff, G.L. (1994) FASEB Journal, 3740S–3747S. 8, 479–488. 26 Pogribny, I.P. and James, S.J. (2002) Cancer 15 Michaud, E.J., van Vugt, M.J., Bultman, Letters, 187,69–75. S.J., Sweet, H.O., Davisson, M.T. and 27 Sohn, K.J., Stempak, J.M., Reid, S., Woychik, R.P. (1994) Genes and Shirwadkar, S., Mason, J.B. and Kim, Y.I., Development, 8, 1463–1472. (2003) Carcinogenesis, 24,81–90. 162j 10 DNA Methylation

28 Pufulete, M., Al-Ghnaniem, R., Rennie, 40 Ramachandran, K., Navarro, L., Gordian, J.A., Appleby, P., Harris, N., Gout, S., E., Das, P.M. and Singal, R. (2007) Emery, P.W. and Sanders, T.A. (2005) Anticancer Research, 27, 921–925. British Journal of Cancer, 92, 838–842. 41 Davis, C.D., Uthus, E.O. and Finley, J.W. 29 Pufulete, M., Al-Ghnaniem, R., Leather, (2000) The Journal of Nutrition, 130, A.J., Appleby, P., Gout, S., Terry, C., 2903–2909. Emery, P.W. and Sanders, T.A. (2003) 42 Fang, M., Chen, D. and Yang, C.S. (2007) Gastroenterology, 124, 1240–1248. The Journal of Nutrition, 137, 223S–228S. 30 Fowler, B.M., Giuliano, A.R., Piyathilake, 43 Yang, C.S., Maliakal, P. and Meng, X. C., Nour, M. and Hatch, K. (1998) Cancer (2002) Annual Review of Pharmacology and Epidemiology, Biomarkers & Prevention, 7, Toxicology, 42,25–54. 901–906. 44 Fang, M.Z., Wang, Y., Ai, N., Hou, Z., Sun, 31 Al-Ghnaniem, R., Peters, J., Foresti, R., Y., Lu, H., Welsh, W. and Yang, C.S. (2003) Heaton, N. and Pufulete, M. (2007) The Cancer Research, 63, 7563–7570. American Journal of Clinical Nutrition, 86, 45 Yuasa, Y., Nagasaki, H., Akiyama, Y., Sakai, 1064–1072. H., Nakajima, T., Ohkura, Y., Takizawa, T., 32 van Engeland, M., Weijenberg, M.P., Koike, M., Tani, M., Iwai, T., Sugihara, K., Roemen, G.M., Brink, M., de Bruine, Imai, K. and Nakachi, K. (2005) A.P., Goldbohm, R.A., van den Brandt, Carcinogenesis, 26, 193–200. P.A., Baylin, S.B., de Goeij, A.F. and 46 Fini, L., Selgrad, M., Fogliano, V., Graziani, Herman, J.G. (2003) Cancer Research, 63, G., Romano, M., Hotchkiss, E., Daoud, 3133–3137. Y.A., De Vol, E.B., Boland, C.R. and 33 Slattery, M.L., Curtin, K., Sweeney, C., Ricciardiello, L. (2007) The Journal of Levin, T.R., Potter, J., Wolff, R.K., Nutrition, 137, 2622–2628. Albertsen, H. and Samowitz, W.S. (2007) 47 Cross, H.S., Kallay, E., Lechner, D., International Journal of Cancer, 120, Gerdenitsch, W., Adlercreutz, H. and 656–663. Armbrecht, H.J. (2004) The Journal of 34 Rampersaud, G.C., Kauwell, G.P., Hutson, Nutrition, 134, 1207S–1212S. A.D., Cerda, J.J. and Bailey, L.B. (2000) The 48 Day, J.K., Bauer, A.M., DesBordes, C., American Journal of Clinical Nutrition, 72, Zhuang, Y., Kim, B.E., Newton, L.G., 998–1003. Nehra, V., Forsee, K.M., MacDonald, R.S., 35 Kim, Y.I., Baik, H.W., Fawaz, K., Knox, T., Besch-Williford, C., Huang, T.H. and Lee, Y.M.,Norton, R., Libby, E. and Mason, Lubahn, D.B. (2002) The Journal of J.B. (2001) The American Journal of Nutrition, 132, 2419S–2423S. Gastroenterology, 96, 184–195. 49 Dolinoy, D.C., Weidman, J.R., Waterland, 36 Kim, Y.I. (2006) Gut, 55, 1387–1389. R.A. and Jirtle, R.L. (2006) Environmental 37 Van Guelpen, B., Hultdin, J., Johansson, I., Health Perspectives, 114, 567–572. Hallmans, G., Stenling, R., Riboli, E., 50 Fang, M.Z., Chen, D., Sun, Y., Jin, Z., Winkvist, A. and Palmqvist, R. (2006) Gut, Christman, J.K. and Yang, C.S. (2005) 55, 1461–1466. Clinical Cancer Research, 11, 7033–7041. 38 Mason, J.B., Dickstein, A., Jacques, P.F., 51 Mithen,R.F.,Dekker,M.,Verkerk,R.,Rabot, Haggarty, P., Selhub, J., Dallal, G. and S. and Johnson, I.T. (2000) Journal of the Rosenberg, I.H. (2007) Cancer Science of Food and Agriculture, 80,967–984. Epidemiology, Biomarkers & Prevention, 16, 52 Dashwood, R.H. and Ho, E. (2007) 1325–1329. Seminars in Cancer Biology, 17, 363–369. 39 Fiala, E.S., Staretz, M.E., Pandya, G.A., El- 53 Wang, L.G., Beklemisheva, A., Liu, X.M., Bayoumy, K. and Hamilton, S.R. (1998) Ferrari, A.C., Feng, J. and Chiao, J.W. Carcinogenesis, 19, 597–604. (2007) Molecular Carcinogenesis, 46,24–31. j163

11 Prevention of Angiogenesis and Metastasis Tariq A. Bhat, Anil Mittal, and Rana P. Singh

11.1 Introduction

Cancer is a multistep process that leads to disregulated hyperproliferative disorder that involves transformation, apoptosis resistance, enhanced cell proliferation, invasion, angiogenesis, and metastasis. The most frequently occurring cancers are lung, prostate, breast, colorectal and stomach, and the four most deadly are lung, stomach, liver, and colorectal cancers in which angiogenesis and metastasis play critical roles [1]. Epidemiological studies suggest that with the increase in nonvegetarian dietary habits, a change to sedentary life style, and an exposure to large number of carcinogens, cancer incidences have also increased. Over 10 million new cases of cancer are being diagnosed worldwide, with about 6 million deaths every year [2]. Since 1990 until 2003, the cancer incidence and mortality have increased by about 22%. These data suggest for the urgent need of efficient diagnostic tools as well as preventive and therapeutic strategies for cancer control. In addition, the identification and targeting of critical steps in tumorigenesis constitute an important area that needs attention. In this regard, angiogenesis and metastasis play vital role in expansion of solid tumors and their dissemination to secondary organs. In clinical situation, solid metastatic tumors reduce the recovery potential of patients. The micrometastases are controlled by noninvasive treatments in which aggressive approaches based on high-dose chemotherapy or irradiation are common but only partially effective that exert severe side effects and develop drug resistance or radioresistance. In this regard, chemoprevention is an upcoming approach in which phytochemicals or synthetic agents reverse and/or suppress tumor growth and progression through various mechanisms. Many phytochemicals are included in our daily diet that show least or nontoxicity to normal cells. Therefore, it is likely that targeting tumor angiogenesis and metastasis by phytochemicals could be an effective strategy for cancer control. Specifically, the discovery of nontoxic phytochemicals could have greater practical significance compared to nonselective cytotoxic therapies to control

the tumor growth and metastasis by targeting angiogenesis, initiating apoptosis in tumors, and inhibiting invasive potential of tumor cells as well as tumor growth [3]. Therefore, it could be a rationale approach to examine nontoxic antitumor agents with antiangiogenic and antimetastatic activities for cancer control with reduced or no harmful effects. If we talk about the natural sources of drugs, currently more than 50% of drugs in clinical use have a natural-product origin, and about half of the worlds 25 best-selling pharmaceutical agents are derived from natural products [1]. Many countries, including the United States, Canada, India, China, Brazil, are developing botanical-drug research and testing because of the importance of phytochemicals to human life for their potential uses as preventive or therapeutic agents. In the United States, more than 10 countries have been on board for a national Cancer Institute project, Natural Inhibitor of Carcinogenesis, which has been examining over 5000 plant samples and has got approximately 250 active compounds for cancer chemoprevention [1]. Also, in December 2003, the European Parliament adopted new legislative policies to make it easier for traditional-medicine makers to demonstrate efficacy, within European Union member nations. Extensive research in last few years has revealed that treatment of cancer requires multitargeting including the suppression of multiple cell signaling pathways such as EGFR, IGF-1R, COX-2, iNOS, VEGF, HER2, and TNF. In this regard, many phytochemicals have been reported to hit multiple targets simultaneously. However, an upsurge is needed to examine their clinical efficacy and pharmacological safety. The current drugs used to treat most cancers are those that can block cell signaling, including growth factor signaling, prostaglandin production, inflammation, drug resistance, cell cycle progression, angiogenesis (e.g., VEGF), invasion (e.g., MMP), antiapoptosis, and cell proliferation [4]. In the following sections, we have provided an account of angiogenesis and metastasis, and their critical steps that could be targeted for prevention and intervention of tumorigenesis.

11.2 Angiogenesis

Angiogenesis, the growth of new blood vessels, is an important natural process that occurs in the body, in both physiological and pathological conditions. Angiogenesis occurs during the early embryo development, healing wounds, restoration of blood ﬂow to tissues after injury; and in females, during the monthly reproductive cycle to rebuild the uterus lining and during pregnancy to build the placenta. Angiogenesis is controlled through a series of on and off switches that are regulated by angiogenesis-stimulating factors and angiogenesis inhibitors, respectively. The balance between angiogenic factors and angiogenesis inhibitors determines the process of angiogenesis. The normal, healthy body maintains a perfect balance of angiogenesis modulators; however, in pathological conditions, the body may lose control over angiogenesis that results in excess or insufﬁcient growth of blood vessels. Excessive angiogenesis may occur in diseases such as cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, psoriasis, and many other 11.2 Angiogenesis j165

Figure 11.1 Critical role of angiogenesis in which also provide a route for metastatic tumor growth, progression, and metastasis. dissemination of cancer cells. The During cancer development, the angiogenesis angiopreventive strategies including the usage of process is initiated as early as dysplasia stage, nontoxic natural agents can prevent tumor and further growth and progression of solid growth, progression, and metastasis. tumors critically depend upon angiogenesis, similar conditions. In these conditions, new blood vessels feed diseased tissue and destroy normal tissue. Inadequate angiogenesis is accompanied by reduced blood vessels growth and circulation in conditions such as coronary artery disease, stroke, and delayed wound healing. It occurs when the tissue cannot produce adequate amounts of angiogenic growth factors, and thus therapeutic angiogenesis is aimed at stimulating new blood vessel growth to treat these conditions. In cancer, it allows tumors to expand as early as from dysplastic stage through promotion and progression, and cancer cells to escape into the circulation to form metastatic lesions in distant organs (Figure 11.1). Antiangiogenic interventions targeting new blood vessel growth are being developed to treat these conditions. Tumor angiogenesis, which is usually measured by the microvessel density, also has prognostic value for many cancers.

11.2.1 Angiogenesis Process

The angiogenesis process follows an orderly series of events outlined as follows: . Tissues produce and release angiogenic growth factors that diffuse into the nearby tissues. . These angiogenic growth factors recognize and bind to speciﬁc receptors located on the endothelial cells of nearby pre-existing blood vessels. . On binding to the receptors, growth factors activate endothelial cells and produce new molecules including enzymes such as matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). 166j 11 Prevention of Angiogenesis and Metastasis

. Enzymes dissolve tissue matrix and sheath-like covering surrounding the existing blood vessels. . The endothelial cells proliferate and migrate out in an orderly fashion through the dissolved portion of the tissue and existing vessels toward the injured/diseased tissue, such as tumor. . Adhesion molecules or integrins help to pull the new blood vessel sprouting forward. . Sprouting endothelial cells roll up to form a tubular structure to move through the tissue dissolved by MMPs and uPA. . Finally, newly formed blood vessel tubes are remodeled and stabilized by smooth muscle cells and pericytes into a matured vessel.

11.2.2 Tumor Angiogenesis

Tumor angiogenesis is the process of initiation and proliferation of blood vessels penetrating into the cancerous growths to supply nutrients and oxygen, and remove metabolic waste products from them simultaneously. The role of tumor angiogenesis in cancer progression was postulated about three decades ago, and now we have enough proof that it is essentially required for the solid tumor growth and metastasis [5]. The complex process of angiogenesis involves the cross communications of tumor cells, endothelial cells, phagocytes, and their secreted factors that are either promoters or inhibitors of angiogenesis [6]. Many proteins including VEGF and bFGF have been identiﬁed as potential angiogenic factors secreted by tumor cells to mediate angiogenesis to sustain tumor growth. VEGF signals through two tyrosine kinase receptors, VEGFR-1 and VEGFR-2 expressed predominantly on vascular endothelial cells. VEGFR-2 is the principal signaling receptor for vascular endothelial cells, whereas VEGFR-1 probably functions as a decoy receptor, serving to regulate the availability of VEGF in a given tissue [7]. Thus, VEGF receptor signaling constitutes an attractive target to inhibit tumor angiogenesis.

11.2.3 Angiopreventive Agents

Antiangiogenic strategies for cancer control and prevention have ushered in a new era of antiangiogenesis research resulting in clinical trials of many agents for their antiangiogenic efﬁcacy in different types of cancers [5, 8–12]. Many dietary and nondietary nontoxic phytochemicals have been found to target endothelial cell growth, proliferation, survival, and tube formation as well as tumor angiogenesis, and inhibit tumor or cancer cell growth in various in vitro and in vivo studies (Table 11.1; reviewed in Ref. [13]). Therefore, targeting of tumor angiogenesis by phytochemicals could be an effective strategy for cancer control. In this regard, many phytochemicals such as silibinin, quercetin, curcumin, and genistein target angiogenesis signaling and its components including VEGF, bFGF, MMPs, and uPA 11.2 Angiogenesis j167

Table 11.1 Phytochemicals with antiangiogenic and antimetastatic activities, and their sources and modes of action.

Phytochemicals Occurrence/source Mode of action

1 Genistein It is present in soybeans and Downregulates MMP-9 and soy products such as tofu and upregulates TIMP1; suppresses textured vegetable proteins endothelial cell proliferation, migration, and invasion; inhibits VEGF and COX-2 expression, and suppresses VEGF-induced tyrosine phosphorylation of receptor kinases; inhibits NF-kB activity and uPA production 2 Curcumin It is the principal curcuminoid Downregulates transcript levels of of the Indian curry spice VEGF and bFGF, and suppresses turmeric VEGF, MMP-2 and -9 expression, and activities of NF-kB, COX-2 and MAPK 3 Resveratrol/ It is a phytoalexin produced Inhibits capillary-like tube formation heyneol (tetramer naturally by several plants when by HUVEC and capillary of resveratrol) under attack by pathogens and is differentiation; suppresses VEGF sold as a nutritional supplement binding to HUVEC; inhibits derived primarily from Japanese NF-kB signaling knotweed, as well as chemically synthesized 4 Epigallocatechin- It is a type of catechin and is Suppresses ephrin-A1-mediated 3-gallate most abundant in green tea endothelial cell migration and tumor vasculature in HT-29 cells, and inhibits ERK1/2 activity as well as expression and secretion of VEGF. Inhibits MMP-2 and MMP-9 expression and activation in TRAMP model; inhibits COX-2 expression and activities of iNOS and NF-kB. Suppresses VEGF- induced tyrosine phosphorylation of Flk-1/KDR in endothelial cells 5 Epicatechin/ These are found in green tea Suppress in vitro and in vivo epigallocatechin/ and cocoa of the cocoa tree angiogenesis in various models epicatechin gallate 6 Flavopiridol It is derived from the plant Inhibits endothelial cell growth via alkaloid rohitukine isolated from suppression of hypoxia-induced the leaves and stems of Amoora expression of VEGF in human rohituka and later from neuroblastoma and monocytes Dysoxylum binectariferum both from India. Now, it is also synthesized chemically

(Continued) 168j 11 Prevention of Angiogenesis and Metastasis

Table 11.1 (Continued)

Phytochemicals Occurrence/source Mode of action

7 Torilin It is a sesquiterpene compound Suppresses neovascularization in purified from the fruits of Torilis bFGF-induced vessel formation in japonica mouse and chorioallantoic membrane in chick embryo models; downregulates hypoxia-induced expression of VEGF and IGF II in HepG2 human hepatoblastoma cells 8 Apigenin It is found in high amounts in Downregulates hypoxia-inducible parsley, thyme, and peppermint factor 1 (HIF-1) and VEGF as well as in a number of herbs, expression in human ovarian including chamomile herb, cancer cells; suppresses in vitro lemon balm herb, perilla, vervain angiogenesis herb, and yarrow 9 Quercetin It is a flavonoid (flavonol) found Inhibits MMP-2 and MMP-9 in citrus fruits and onion secretion from tumor cells; suppresses endothelial cell proliferation, migration, and tube formation 10 30-Hydroxyflavone/ These are found in citrus fruits Suppress in vivo angiogenesis 3040-dihydroxyfla- and grape fruit juice vone/2030- dihydroxyflavone 11 Fisetin/luteolin Fisetin is commonly found in Inhibits bFGF-induced corneal strawberries and other fruits and neovascularization vegetables. Luteolin is commonly found in leaves, but it is also present in rinds, barks, clover blossom, and ragweed pollen. It has also been isolated from Salvia tomentosa while its dietary sources include celery, green pepper, perilla, and camomile tea 12 Isoliquiritin It is derived from licorice root Inhibits in vitro tube formation by endothelial cells and in vivo angiogenesis 13 Magnosalin This natural product is isolated Suppresses in vivo angiogenesis from Flos magnoliae. Its synthetic derivative is 4-(3,4,5-trimethoxy- phenyl)-6-(2,4,5-trimethoxyphe- nyl)-2-diethylaminopyrimidine (TAS-202) 14 Sulfated beta (1 > 4) These are prepared from an Inhibits angiogenesis via interaction galacto- arabino-galacto-rhamno- with FGF-2 in chorioallantoic oligosaccharides galacturonan from Lupinus membrane assay polyphyllus Lindl 15 Philinopside A It is a sulfated saponin that is Suppresses proliferation, migration, isolated from the sea cucumber, and tube formation of HMECs; Pentacta quadrangulari inhibits receptor tyrosine kinases including VEGFR 11.2 Angiogenesis j169

Table 11.1 (Continued)

Phytochemicals Occurrence/source Mode of action

16 Silymarin It is a mixture of ﬂavonolignans Inhibits VEGF secretion in prostate extracted from the seeds of milk and breast cancer cells and tube thistle (Silybum marianum) formation by endothelial cells; suppresses RTK signaling and ERK1/2 activation in human epithelial carcinoma cells; inhibits MMP-2 expression and tube formation in HUVEC 17 Silibinin It is the major constituent of Inhibits growth, survival, invasion silymarin and is present in milk and migration, and tube formation thistle and artichoke by endothelial cells; inhibits ERK1/2, Akt, and NF-kB activation in HUVEC; inhibits MMP-2 expression; downregulates uPA in A549 lung cancer cells 18 Selenium Asparagus, garlic, mushrooms, Suppresses VEGF expression, nuts, wheat germ, seafood, and lowers microvessel density, and beef are the natural sources. It is collagenolytic activity of MMP-2 in also found in sulﬁde ores such as rat mammary carcinoma; induces pyrite apoptotic death of HUVEC and inhibits in vitro angiogenesis 19 Isomers of conju- It is a family of at least 13 isomers Abrogate angiogenesis in vitro and gated linoleic acid of linoleic acid found especially in vivo by suppressing the formation in meat and dairy products of microcapillary networks; lower derived from ruminants both serum and mammary gland VEGF concentration in breast cancer model 20 Phenethyl It is a constituent of many edible Inhibits ex vivo angiogenesis in CAM isothiocyanate cruciferous vegetables assay; reduces the survival and suppresses capillary-like tube formation and migration of HUVEC; downregulates VEGF secretion and VEGFR expression levels 21 Deguelin It is a derivative of rotenone and Abrogates endothelial cell growth, is extracted from several plant survival, migration, invasion and species belonging to legumes MMP production such as Lonchocarpus, Derris and Tephrosia 22 Vitamin K2 Vitamin K2 (menaquinone, Inhibits cell proliferation and tube menatetrenone) is normally formation by endothelial cells produced by bacteria in the intestines, but it is mostly found in various natural sources such as leafy green vegetables, particularly the dark green ones, such as spinach and kale, Brassica spp., and some fruits (Continued) 170j 11 Prevention of Angiogenesis and Metastasis

Table 11.1 (Continued)

Phytochemicals Occurrence/source Mode of action

23 Retenoic acid It is a derivative of vitamin A Downregulates production of IL-8 in found in carrot and spinach head and neck carcinoma; inhibits endothelial cells from responding to angiogenic growth factors 24 Ellagic acid It is a polyphenol antioxidant Downregulates VEGF- and found in numerous fruits and PDGF-induced signaling in vegetables including raspberries, endothelial cells and pericytes, strawberries, cranberries, wal- respectively nuts, pecans, pomegranates, and other plant foods 25 Peonidin 3-gluco- Black rice (Oryza sativa L. indica) Causes reduced expression of side and cyanidin MMP-9 and uPA 3-glucoside 26 Berberine It is an alkaloid found in plants Inhibits uPA expression and MMP-2 such as Berberis, goldenseal and hence, invasion in human lung (Hydrastis canadensis), and Coptis cancer cell line chinensis, usually in roots, rhizomes, stems, and bark 27 Cannabinoids These are terpenophenolic Inhibits cancer cell invasion via compounds present in increased expression of tissue cannabis (Cannabis sativa) inhibitor of matrix metalloproteinases-1

aMany plant extracts including that of green tea, soy, Viscum album coloratum, Livistona chinensis, Chrysobalanus icaco, Polypodium leucotomos, and Oriental herbal cocktail (ka-mi-kae-kyuk-tang) have been shown to inhibit various aspects of angiogenesis in experimental models.

(Table 11.1; [14–19]). Many plant extracts have also been reported to inhibit various attributes of angiogenesis (Table 11.1). Thus, the discovery of nontoxic angiopreventive agents, most likely from the natural sources, could have greater practical signiﬁcance compared to nonselective cytotoxic therapies to control the tumor growth and metastasis.

11.2.4 Lymphangiogenesis

Lymphangiogenesis plays an important physiological role in homeostasis, metabolism, and immunity. Lymphatic vessel formation process has also been implicated in a number of pathological conditions including metastasis, edema, rheumatoid arthritis, and impaired wound healing. The investigation of lymphatic system has received renewed interest, and speciﬁc lymphatic markers such as podoplanin, LYVE-1, PROX-1, desmoplakin, VEGF-C, VEGF-D, and their receptor VEGFR-3 have been discovered, which has provided an insight into the functional and molecular lymphatic biology [20, 21]. Instead of VEGFR-1 and -2, a third VEGF 11.3 Metastasis j171 receptor, VEGFR-3, and Fms-like tyrosine kinase 4 have been found to be predominantly expressed on lymphatic vessels during development [22]. Furthermore, instead of VEGF (or VEGF-A), it is VEGF-C and VEGF-D that bind to VEGFR-3 [22, 23]; VEGF-C and VEGF-D have been shown to induce tumor lymphangiogenesis and promote cancer metastasis in many studies [25–27]. These studies provided direct experimental evidence that tumors can activate tumor lymphangiogenesis, a process that has been questioned and neglected for a long time in the past. These studies also provide evidence that tumor angiogenesis can be divided into pathways that preferentially activate angiogenesis (driven by VEGF-A) and lymphangiogenesis (driven by VEGF-C and VEGF-D). Research on lymphatic metastasis and clinicopathological analyses of human cancer have recognized VEGF-C/VEGF-D/VEGFR-3 signaling as a promising target to prevent lymphangiogenesis and thereby to restrict metastatic spread of cancer [24, 28]. Lymphangiogenesis is also promoted by FGF-2, Ang-1, and in some cases by VEGF-A, apparently via upregulation of VEGF-C, VEGF-D [26, 29], or VEGFR-3 or via enhanced delivery of these growth factors by macrophages [30, 31]. Thus, targeting VEGF-C/VEGF-D/VEGFR-3 axis in cancer could also inhibit any contribution that FGF-2, Ang-1, or VEGF-A might make to tumor lymphangiogenesis. VEGF-C and VEGF-D can also promote tumor angiogenesis via activation of VEGFR-2 in the endothelial cells. Anti-VEGF therapy is shown to reduce lymphatic vessel density and expression of VEGFR-3 in breast tumor model [32]. mTOR is a signaling intermediate that regulates lymphangiogenesis as its inhibition by rapamycin has been reported to inhibit lymphangiogenesis [33]. Curcumin and glucocorticoids are shown to inhibit lymphangiogenesis [34, 35]. Also, the importance of VEGFR-3 in lymphangiogenesis has been established from the study in which anti-VEGR-3 antibody is found to inhibit lymph node as well as distant metastases, which was further augmented in combination with anti-VEGF-2 antibody [36]. Thus, the lymphangiogenesis signaling is a promising target to prevent cancer metastasis to distant sites.

11.3 Metastasis

One of the most important aspects of cancer progression and spread is metastasis, in which tumor cells acquire the ability to invade normal tissues and organs through tissue boundaries to form new cancerous lesions at sites distinct from the primary tumor [37]. The process through which cancer cells can spread within the body are varied, such as direct invasion of surrounding tissues, spread via the blood vascular system (hematogenous metastasis) and spread via the lymphatic system (lymphatic metastasis) [37, 38]. The molecular mechanisms involved in this process are not fully understood but those associated with cell–cell and cell–matrix adhesion, with the degradation of extracellular matrix (ECM), and with the initiation and maintenance of early growth at the new site are generally accepted to be very critical. Adhesion molecules (e.g., integrins and cadherins) play a major role in signaling from outside 172j 11 Prevention of Angiogenesis and Metastasis

to inside a cell, thereby controlling how a cell is able to sense and interact with its local environment [39]. Proteolytic enzymes and their inhibitors (e.g., MMPs and TIMPs) regulate the breakdown of extracellular matrix as well as the release of factors that can positively or negatively regulate the growth of cells [40]. It is not only the immediate cellular microenvironment but also the extended cellular microenvironment such as vascular insufﬁciency and hypoxia in the primary tumor that can modify cellular gene expression to enhance metastasis. Mechanisms of metastasis involve a complex array of genetic and epigenetic changes many of which are speciﬁc to both the types of tumor and the sites of metastasis.

11.3.1 Basic Steps in Cancer Metastasis

An account of the metastatic processes and associated molecular alterations has been given in Figure 11.2. Usually, cancer metastasis includes the following broad steps: . Invasion and inﬁltration of surrounding normal host tissue with penetration of small lymphatic or blood vessels.

Figure 11.2 Basic processes in the progression (left). Many molecular changes occur during of metastasis and associated molecular changes. each dynamic process of metastasis for the The epithelial–mesenchymal transition is an subsequent progression through the specific indication of epithelial tumors acquiring stages (right). There are many critical steps as migratory potential that is a prerequisite for depicted in the diagram that could be targeted for metastatic progression through various stages the prevention of metastasis. 11.3 Metastasis j173

. Release of cancer cells, either singly or in small clumps, into the general circulation. . Endurance in the circulation. . Arrest of these circulatory cancer cells in the capillary beds of distant organs. . Piercing-out of the lymphatic or blood vessel walls followed by survival and growth of the disseminated tumor cells at secondary site. Metastasis is usually an inefficient process since very few of the tumor cells that escape from a primary tumor ever form metastases. The metastatic process has long been formalized into a series of discrete stages perhaps best illustrated by the decathlon champion model introduced by Fidler [37]. These critical stages include escape from the primary tumor and intravasation into the blood vascular or lymphatic systems, survival in the circulation and avoidance of host defense mechanisms, arrest at a new site and extravasation into the tissue, and finally homing and growth at the new site. Tumor cells that form metastases must be capable of successfully prevailing through all stages that account for the inefficiency of the process. In solid epithelial tumors, epithelial–mesenchymal transition (EMT) is a process that facilitates the invasion and migration of metastasizing cancer cells. The accounts of these processes are discussed in the subsequent sections.

11.3.2 Epithelial–Mesenchymal Transition in Metastasis

Epithelial–mesenchymal transition is a well known phenomenon that is critically important during the embryonic development of multicellular organisms. During this process, polarized stationary epithelial cells are converted into motile mesenchymal cells that are loosely embedded in the ECM and can migrate during organ formation [41, 42]. Since primary solid tumor cells are tightly packed with each other as well as with normal cells, it is only after the detachment from the original place that they are able to metastasize. This process was not known for a long time; however, the extensive research in this area recognized the EMTas a critical mechanism to initiate tumor cell migration [43]. The tumors of epithelial origin are referred to as carcinomas while those of mesenchymal origin are referred to as sarcomas, and these are not interconverted except in certain cases known as sarcomatoid carcinomas. During the EMT, cells undergo the loss of epithelial markers, such as E-cadherin, and gain the expression of mesenchymal markers, such as N-cadherin and vimentin [44, 45]. The exact role of EMT in tumor progression is still debated; nevertheless, it could be particularly important in cancers with single-cell migration and early dissemination of tumor cells [41, 45]. Various carcinoma cell lines undergo a partial or complete EMT in vitro and many carcinomas show a diversity of phenotypes and malignant potential while losing most of their epithelial properties during progression, which is also taken as the criterion for the tumor grading (reviewed in Ref. [41]). There are parallel mechanisms of EMT in embryonic development and tumors; and several signaling pathways have been found to be common for both, development and tumor progression. There are many regulators of E-cadherin including Snail/Slug, SIP1, 174j 11 Prevention of Angiogenesis and Metastasis

E12/E47, TGF-beta, mesenchymal–epithelial transition (MET) factor, and a receptor tyrosine kinase that determine the EMT process (Figure 11.2). The correct understanding of the EMT process will lead to the recognition of promising targets for cancer control and prevention. This could be an important aspect of preventive control of metastasis, as micrometastases often develop after surgery, chemotherapy, and radiotherapy. Among the various targets to prevent EMT process are receptor tyrosine kinases, such as IGFR and ERB family, as well as nonreceptor kinases such as SRC. Trastuzumab (herceptin), a monoclonal antibody against ERB-B2 is an approved drug while many others are in late-stage clinical trials, such as Iressa, an inhibitor of EGFR, and SU5416, an inhibitor of VEGFR-2. Thus, inhibition of EMT or its reversal (MET) is an important aspect of slowing down the dedifferentiation and dissemination of tumor cells from the primary tumor.

11.3.3 Invasion and Migration

Tumors are disseminated to distant locations within the body through invasion and migration processes by which tumor cells migrate to new sites before homing them. This ability of a cancer cell to undergo migration and invasion that are similar to those that occur in normal, non-neoplastic cells during physiological processes, such as embryonic morphogenesis, wound healing, and immune cell trafficking, allows it to change position within the tissues and enter lymphatic and blood vessels for dissemination into the circulation, and it is then entrapped for metastatic growth in distant organs [46]. Invasion of cells of the surrounding tissue and destruction of normal tissue architecture are two important processes of malignant tumors. Two morphological patterns of tumor invasion can be distinguished, namely, single-cell and collective-cell invasion. The invasive potential and the invasion pattern of tumor cells are determined by various molecules including the changes in the expression and function of adhesive (e.g., cadherins and immunoglobulin domain-containing cell adhesion molecules) and regulatory proteins (e.g., Snail family members, SIP1, E12/E47, and transforming growth factor-beta). In this process, cells acquire a migratory, mesenchymal phenotype via downregulation of epithelial markers, such as E-cadherin, and upregulation of mesenchymal markers, such as N-cadherin and vimentin. The collective cell invasion and migration is less well understood since these cell sheets maintain the expression of epithelial adhesion structures and can invade the surrounding tissue and destroy the host organ. It has been demonstrated that podoplanin, a small mucin-like protein, mediates a pathway leading to collective cell migration and invasion in vivo and in vitro, although cells express E-cadherin even in advanced stages [47]. The migration process is very complex and involves many factors, structures, and interaction with extracellular matrix. The cell body modifies its shape and stiffness to interact with the surrounding tissue structures for efficient migration. For this, ECM provides the substrate and a barrier for the advancing cell body. Cell migration through tissues results from a continuous cycle of interdependent steps [48, 49]. First, the moving cell becomes polarized and elongates, followed by the pseudopod 11.3 Metastasis j175 formation via extension of the cells leading edge, which attaches to the ECM substrate. Subsequently, regions of the leading edge or the entire cell body contract, thereby generating traction force that leads to the gradual forward gliding of the cell body and its trailing edge. Cell protrusions that initiate ECM recognition and binding can be quite diverse in morphology and dynamics, and are termed as lamellipoda, filopoda, pseudopods, or invadopods [41, 50]. These cell protrusions contain fila- mentous actin, as well as varying sets of structural and signaling proteins, and lead to dynamic interactions with ECM substrates. Cell protrusions are the prerequisite for the onset and maintenance of cell motility in normal and cancer cells, which can be induced by chemokines and growth factors. Degradation of ECM is a prerequisite for the solid tumor cell migration and invasion, as it is the initial barrier for these processes to occur. The potential roles of proteolytic enzymes and their inhibitors in the metastatic process include the regulation of ECM degradation and release of factors influencing the growth of tumor cells. A fine balance between these enzymes and their inhibitors exists under normal condition, but it is often disturbed in the tumor environment. The breakdown of ECM and cellular adhesion contacts facilitates peritumor invasion, angiogenesis, intravasation, and extravasation, and also modulates the cellular signaling pathways in tumor cells. ECM degradation is mediated via members of proteinase family including the serine, aspartic acid, metallo- and cysteine proteinases. Among these, MMPs and the plasminogen activator family members are frequently and strongly implicated in cancer progression. MMPs have the capacity to degrade all components of the basement membranes and ECM [17]. Depending on the cell type and ECM substrate, focal contact assembly and migration can be regulated by different integrins, including a5b1, a6b1ora6b4 avb3, and a2b1 that bind fibronectin, laminin, fibronetin or vitronectin, and fibrillar collagen, respectively. Other, non- integrin receptors, such as CD44, discoidin receptors, CD26, immunoglobulin superfamily receptors, and surface proteoglycans also interact with ECM components and signal cell motility [49]. The tumor cells change their morphology to mimic endothelial cells during invasion process so as to get into the circulation through the blood capillary walls via intravasation process. This process is executed when tumor cells diapedese, which is regulated by MMPs and uPA. Within the circulation, these tumor cells employ methods to evade immune system in which they bypass recognition by NK-cells and CD4Th cells or expression of certain factors [51]. During circulation, they can undergo diapedesis to go out through the capillary walls using the same process, now called extravasation, into the distant site to home and grow there as metastases [51].

11.3.4 Homing Mechanisms

Usually, surviving the mechanical forces involved in tumor cell arrest and extravasation were considered to be significant barriers in the metastatic process and major sources of metastatic inefficiency. However, recent evidence has revealed that later postextravasational events may play a larger role in determining metastatic efficiency 176j 11 Prevention of Angiogenesis and Metastasis

than previously thought [52]. The development of intravital video microscopy (IVVM) has made it possible to visualize the arrest and extravasation of single cells in the lungs and livers of mice [53]. Through this technique, it has been shown that extravasation of tumor cells after arrest in the vasculature is possibly not inefficient, and in some cases is not even necessary for subsequent tumor growth [54]. Among the many factors affecting these processes are MMPs, TIMPs, and uPA that affect metastatic efficiency, in large part, by influencing the ability of cell to survive and grow in its new environment. At the new sites, tumor cells get arrested and must survive to form clinically relevant tumors. At these sites of arrest, resisting apoptosis is a critical event and a strong correlation has been observed between the metastatic efficiency of cell lines and their ability to resist apoptosis when challenged with a variety of stimuli in vitro [55]. The serine protease uPA is overexpressed and involved in the metastatic phenotype of many cancers [56]. uPA and its receptor uPAR have been studied in animal models and found to be correlated with the metastatic potential of cancers [57]. Mechanisms inducing apoptosis in metastasizing tumor cells vary in which several factors involved in receptor-mediated apoptotic pathways have been implicated. The well-characterized Fas/Fas ligand system, TNF-related apoptosis-inducing ligand (TRAIL), and DAP kinase constitute among important factors [58]. Studies suggest that defective Fas pathway signaling could be an important factor for encouraging metastasis at these secondary sites [59]. TRAIL knockout mice (as well as downregulation of DAP kinase by hypermethylation) has been shown to be more susceptible to both spontaneous and experimental metastasis using breast and renal cell carcinoma models [60]. Thus, to become apoptosis resistant at the site of arrest or after extravasation, tumor cells may acquire defects in these apoptosis-inducing signaling processes. Recent IVVM studies have demonstrated that a surprisingly large number of tumor cells remain viable as solitary cells for longer time after arrest and extravasation [61]. Once a tumor cell has become resident at a secondary site, it must divide and grow to form a secondary tumor. There are endogenous factors that regulate the dormancy of solitary tumor cells in the same way as the initiation of angiogenesis can be regulated by endogenous peptide fragments [62]. At secondary sites, these cells require an appropriate cellular microenvironment to proliferate. This is the essence of the seed-and-soil model of metastasis first postulated by Paget in the late nineteenth century. Many factors could be needed to support the growth of the arrested tumor cells. One class of molecules that may play a role in regulating the early growth of metastases is the chemokines. These soluble ligands and their receptors are involved in recognition and homing of leukocytes [63]. It has been demonstrated that breast cancer cell lines, as well as malignant tumors and their metastases, express the chemokine receptors CXC chemokine receptor-4 (CXCR4) and CCR7 at high levels, unlike normal breast epithelium [64]. The ligands for these receptors were predominantly expressed in the organs to which breast cancer most often metastasizes including lymph nodes, lung, liver, and bone marrow. At present, it is not very clear how these processes of EMT and tumor cell migration, invasion, and homing are mediated and regulated; however, it is conceivable that activation of 11.4 Summary j177 amoeboid-cell behavior could allow cancer cells to escape therapeutic agents that are designed to target integrins, proteases, and other related signaling pathway factors. A better understanding of these processes is, therefore, warranted.

11.3.5 Preventive Agents for Metastasis

Some natural agents have been investigated for their antimetastatic activity and potential molecular targets (Table11.1). For example, curcumin and epigallocatechin- 3-gallate have been reported to suppress MMP-2 and MMP-9 expression and cancer cell invasion [65, 66], while genistein suppresses MMP-9 and upregulates TIMP1 for its anti-invasive effect [17]. Quercetin and selenium inhibit MMP-2/9 secretion from tumor cells and genolytic activity of MMP-2, respectively, whereas silibinin and slymarin are shown to inhibit MMP-2 expression [67]. Prevention of cancer cells invasion via repression of MMPs and uPA expression has been observed using black rice anthocyanins (reviewed in Ref. [13]). Silibinin and berberine have been shown to suppress uPA expression in lung cancer cell line [19, 68]. It has been reported that inhibition of cancer cell invasion by cannabinoids occurs via increased expression of tissue inhibitor of matrix metalloproteinase-1 [69]. Stromal cell-derived factor-1 and its receptor CXCR4 are potential targets to prevent metastasis to distant site. Like hemopoietic cell homing, levels of stromal cell-derived factor-1 are high at sites of breast cancer metastasis including lymph node, lung, liver, and marrow. CXCR4 targeting using a synthetic polypeptide has been shown to inhibit breast cancer metastasis [70]. Immunotherapy plays an important role in the prevention of tumor cell homing and metastasis. Using anti-CD44 monoclonal antibody, inhibition of human melanoma growth and metastasis has been achieved in vivo. Also, alpha-D-glucan isolated from Tinospora cordifolia exerts immune-stimulating properties on tumor by activating NK cells [71]. Thus, there could be many critical promising targets to prevent or inhibit metastasis process by using natural or synthetic speciﬁc inhibitors.

11.4 Summary

Angiogenesis and metastasis constitute critical processes during solid tumor growth and progression. The studies so far have identiﬁed many vital molecules that mediate these processes and could be targeted in preventive strategies. There are many agents that have been found to modulate these targets in inhibiting tumor angiogenesis and metastasis as discussed above. However, more studies are needed to explore critical underlying mechanisms of angiogenesis, invasion, migration, homing, and growth of metastatic tumors for effective preventive measures. In addition, in this regard, studies with chemopreventive agents are also limited, which warrants an emphasis on discovering dietary or nondietary effective antiangiogenic and antimetastatic agents. Such strategies could form an integral part in cancer management. 178j 11 Prevention of Angiogenesis and Metastasis 11.5 Abbreviations

Akt protein kinase B Ang-1 angiopoietin 1 AP1 apoptosis protein-1 bFGF basic ﬁbroblast growth factor CAM cell adhesion molecule COX-2 cyclooxygenase 2 CXCR CXC chemokine receptor ECM extracellular matrix EGCG epigallocatechin-3-gallate EGF epidermal growth factor EGFR epidermal growth factor receptor EMT epithelial–mesenchymal transition FGF human ﬁbroblast growth factor FLK-1/KDR fetal liver kinase 1 FLT-1 fms-like tyrosine kinase 1 HER2 human epidermal growth factor receptor-2 HIF hypoxia-inducing factor HUVEC human umbilical vein endothelial cells IGF insulin-like growth factor IGFR insulin-like growth factor receptor IVVM intravital video microscopy LYVE-1 lymphatic vessel endothelial hyaluronan mTOR mammalian target of rapamycin MMP matrix metalloproteinase PROX-1 prospero-related homeobox gene 1 TRAIL tumor necrosis factor-related apoptosis-inducing ligand TGF transforming growth factor TIMP tissue inhibitor of metalloproteinases TNF tumor necrosis factor TRAMP transgenic adenocarcinoma of the mouse prostate uPA urokinase-type plasminogen activator uPAR urokinase-type plasminogen activator receptor VEGF vascular endothelial growth factor VEGFR VEGF receptor XIAP X-chromosome-linked inhibitor of apoptosis protein

References

1 Kong, J.M. and Liu, Y.(2006) New bioactive com/innovation/volumes/v6n2/ compounds from herbs: the active coverstory1.shtml. components of herbal plants possess 2 Frankish, H. (2003) 15 million new cancer properties that ﬁght disease. Innovation, cases per year by 2020, says WHO. The 6.(2). http://www.innovationmagazine. Lancet, 361, 1278. References j179

3 Singh, R.P. and Agarwal, R. (2006) metastasis associated properties in A431 Mechanisms of action of novel agents for cells overexpressing epidermal growth prostate cancer chemoprevention. factor receptor. British Journal of Endocrine-Related Cancer, 13, 751–778. Pharmacology, 128, 999–1010. 4 Aggarwal, B.B. and Shishodia, S. (2006) 16 Di, G.H., Li, H.C., Shen, Z.Z. and Shao, Molecular targets of dietary agents for Z.M. (2003) Analysis of anti-proliferation prevention and therapy of cancer. of curcumin on human breast cancer cells Biochemical Pharmacology, 71, 1397–1421. and its mechanism. Zhonghua Yi Xue Za 5 Folkman, J. (1971) Tumor angiogenesis: Zhi, 83, 1764–1768. therapeutic implications. The New England 17 Tosetti, F., Ferrari, N., Deﬂora, S. and Journal of Medicine, 285, 1182–1186. Albini, A. (2002) Angioprevention: 6 Ferrara, N. (2000) VEGF: an update on angiogenesis is a common key target for biological and therapeutic aspects. Current cancer chemopreventive agents. The Opinion in Biotechnology, 11, 617–624. FASEB Journal, 16,2–14. 7 Risau, W. (1997) Mechanisms of 18 Adhami, V.M., Siddiqui, I.A., Ahmad, N., angiogenesis. Nature, 386, 671–674. Gupta, S. and Mukhtar, H. (2004) Oral 8 Gimbrone, M.A. Jr, Leapman, S., Cotran, consumption of green tea polyphenols R. and Folkman, J. (1972) Tumor inhibits insulin-like growth factor-I- dormancy in vivo by prevention of induced signaling in an autochthonous neovascularization. The Journal of mouse model of prostate cancer. Cancer Experimental Medicine, 136, 261–276. Research, 64, 8715–8722. 9 Boehm, T., Folkman, J., Browder, T. and 19 Skogseth, H., Larsson, E. and Halgunset, J. OReally, M.S. (1997) Antiangiogenic (2005) Inhibitors of tyrosine kinase inhibit therapy of experimental cancer does not the production of urokinase plasminogen induce acquired drug resistance. Nature, activator in human prostatic cancer cells. 390, 404–407. Acta Pathologica et Microbiologica 10 Kerbel, R.S. (2000) Tumor angiogenesis: Scandinavica, 113, 332–339. past, present and the near future. 20 Zhang, B., Zhao, W.H., Zhou, W.Y., Yu, Carcinogenesis, 21, 505–515. W.S., Yu, J.M. and Li, S. (2007) Expression 11 Carmeliet, P. and Jain, R.K. (2000) of vascular endothelial growth factors-C Angiogenesis in cancer and other diseases. and -D correlate with evidence of Nature Medicine, 407, 249–257. lymphangiogenesis and angiogenesis in 12 Brekken, R.A., Li, C. and Kumar, S. (2002) pancreatic adenocarcinoma. Cancer Strategies for vascular targeting in tumors. Detection and Prevention, 31, 436–442. International Journal of Cancer, 100, 21 Stacker, S.A., Hughes, R.A. and Achen, 123–130. M.G. (2004) Molecular targeting of 13 Bhat, T.A. and Singh, R.P. (2008) Tumor lymphatics for therapy. Current angiogenesis: a potential target in cancer Pharmaceutical Design, 10,65–74. chemoprevention. Food and Chemical 22 Kaipanen, A., Kaipainen, A., Korhonen, J., Toxicology, 46, 1334–1345. Mustonen, T., van Hinsbergh, V.W., Fang, 14 Singh, R.P., Gu, M. and Agarwal, R. (2008) G.H., Dumont, D. and Breitman, M. Silibinin inhibits colorectal cancer growth (1995) Expression of the Fms-like tyrosine by inhibiting tumor cell proliferation and kinase 4 gene becomes restricted to angiogenesis. Cancer Research, 68, lymphatic endothelium during 2043–2050. development. Proceedings of the National 15 Huang, Y.T., Hwang, J.J., Lee, P.P., Ke, Academy of Sciences of the United States of F.C., Huang, J.H., Kandaswami, C., America, 92, 3566–3570. Middleton, E.J. Jr and Lee, M.T. (1999) 23 Joukov, V., Pajusola, K., Kaipainen, A., Effects of luteolin and quercetin, inhibitors Chilov, D., Lahtinen, I., Kukk, E., Saksela, of tyrosin kinase, on cell growth and O. and Kalkkinen, N. (1996) A novel 180j 11 Prevention of Angiogenesis and Metastasis

vascular endothelial growth factor, Moses, M.A. and Mulligan, R.C. (2004) VEGF-C, is a ligand for the Flt-4 (VEGFR- Dose-dependent response of FGF-2 for 3) and KDR (VEGFR-2) receptor tyrosine lymphangiogenesis. Proceedings of the kinases. The EMBO Journal, 15, 290–298. National Academy of Sciences of the United 24 M€akinen, T., Jussila, L., Veikkola, T., States of America, 101, 11658–11663. Karpanen, T., Kettunen, M.I., Pulkkanen, 31 Cursiefen, C., Chen, L., Borges, L.P., K.J., Kauppinen, R. and Jackson, D.G. Jackson, D., Cao, J., Radziejewski, C., (2001) Inhibition of lymphangiogenesis DAmore, P.A. and Dana, M.R. (2004) with resulting lymphedema in transgenic VEGF-A stimulates lymphangiogenesis mice expressing soluble VEGF receptor-3. and hemangiogenesis in inflammatory Nature Medicine, 7, 199–205. neovascularization via macrophage 25 Skobe, M., Hawighorst, T., Jackson, D.G., recruitment. The Journal of Clinical Prevo, R., Janes, L., Velasco, P., Riccardi, L. Investigation, 113, 1040–1050. and Alitalo, K. (2001) Induction of tumor 32 Whitehurst, B., Flister, M.J., Bagaitkar, J., lymphangiogenesis by vascular Volk, L., Bivens, C.M., Pickett, B. and endothelial growth factor-C promotes Castro-Rivera, E. (2007) Anti-VEGF-A breast cancer metastasis. Nature Medicine, therapy reduces lymphatic vessel density 7, 192–198. and expression of VEGFR-3 in an 26 Stacker, S.A., Caesar, C., Baldwin, M.E., orthotopic breast tumor model. Thornton, G.E., Williams, R.A., Prevo, R., International Journal of Cancer, 121, Jackson, D.G. and Nishikawa, S. (2001) 2181–2191. Vascular endothelial growth factor-D 33 Kobayashi, S., Kishimoto, T., Kamata, S., promotes the metastatic spread of tumors Otsuka, M., Miyazaki, M. and Ishikura, H. via the lymphatics. Nature Medicine, 7, (2007) Rapamycin, a specific inhibitor of 186–191. the mammalian target of rapamycin, 27 Mandriota, S.J., Jussila, L., Jeltsch, M., suppresses lymphangiogenesis and Compagni, A., Baetens, D., Prevo, R., lymphatic metastasis. Cancer Science, 98, Banerji, S. and Huarte, J. (2001) Vascular 726–733. endothelial growth factor-C-mediated 34 Matsuo, M., Sakurai, H., Koizumi, K. and lymphangiogenesis promotes tumor Saiki, I. (2007) Curcumin inhibits the metastasis. The EMBO Journal, 20, formation of capillary-like tubes by rat 672–682. lymphatic endothelial cells. Cancer Letters, 28 Kubo, H., Cao, R., Br€akenhielm, E., 251, 288–295. M€akinen, T., Cao, Y. and Alitalo, K. (2002) 35 Yano, A., Fujii, Y., Iwai, A., Kawakami, S., Blockade of vascular endothelial growth Kageyama, Y. and Kihara, K. (2006) factor receptor-3 signaling inhibits Glucocorticoids suppress tumor fibroblast growth factor-2-induced lymphangiogenesis of prostate cancer cells. lymphangiogenesis in mouse cornea. Clinical Cancer Research, 12,6012–6017. Proceedings of the National Academy of 36 Roberts, N., Kloos, B., Cassella, M., Sciences of the United States of America, 99, Podgrabinska, S., Persaud, K., Wu, Y., 8868–8873. Pytowski, B. and Skobe, M. (2006) 29 Tammela, T., Saaristo, A., Lohela, M., Inhibition of VEGFR-3 activation with the Morisada, T., Tornberg, J., Norrmen, C., antagonistic antibody more potently Oike, Y. and Pajusola, K. (2005) suppresses lymph node and distant Angiopoietin-1 promotes lymphatic metastases than inactivation of VEGFR-2. sprouting and hyperplasia. Blood, 10, Cancer Research, 66, 2650–2657. 4642–4648. 37 Fidler, I.J. (1990) Critical factors in the 30 Chang, L.K., Garcia-Cardena, G., Farnebo, biology of human cancer metastasis: F., Fannon, M., Chen, E.J., Butterfield, C., twenty-eighth G.H.A. Clowes Memorial References j181

Award lecture. Cancer Research, 50, 47 Wicki, A., Lehembre, F., Wick, N., 6130–6138. Hantusch, B., Kerjaschki, D. and 38 Sherwood, D.R. (2006) Cell invasion Christofori, G. (2006) Tumor invasion in through basement membranes: an anchor the absence of epithelial–mesenchymal of understanding. Trends in Cell Biology, 16, transition: podoplanin-mediated 250–256. remodeling of the actin cytoskeleton. 39 Juliano, R.L. (2002) Signal transduction by Cancer Cell, 9, 261–272. cell adhesion receptors and the 48 Friedl, P. and Brocker,€ E.B. (2000) The cytoskeleton: functions of integrins, biology of cell locomotion within three- cadherins, selectins, and dimensional extracellular matrix. Cellular immunoglobulin-superfamily members. and Molecular Life Sciences, 57,41–64. Annual Review of Pharmacology and 49 Friedl, P. and Wolf, K. (2003) Tumour-cell Toxicology, 42, 283–323. invasion and migration: diversity and 40 Imai, K., Hiramatsu, A., Fukushima, D., escape mechanisms. Nature Reviews. Pierschbacher, M.D. and Okada, Y. Cancer, 3, 362–374. (1997) Degradation of decorin by matrix 50 Adams, J.C. (2001) Cell-matrix contact metalloproteinases: identification of the structures. Cellular and Molecular Life cleavage sites, kinetic analyses and Sciences, 58, 371–392. transforming growth factor-beta1 51 Miles, F.L., Pruitt, F.L., van Golen, K.L. and release. The Biochemical Journal, 322, Cooper, C.R. (2008) Stepping out of the 809–814. flow: capillary extravasation in cancer 41 Thiery, J.P. (2002) Epithelial– metastasis. Clinical & Experimental mesenchymal transitions in tumour Metastasis, 25, 305–324. progression. Nature Reviews. Cancer, 2, 52 Cairns, R.A., Khokha, R. and Hill, R.P. 442–454. (2003) Molecular mechanisms of tumor 42 Thiery, J.P. (2003) Epithelial– invasion and metastasis: an integrated mesenchymal transitions in development view. Current Molecular Medicine, 3, and pathologies. Current Opinion in Cell 659–671. Biology, 15, 740–746. 53 Morris, V.L., Schmidt, E.E., Macdonald, 43 Birchmeier, C., Birchmeier, W. and Brand- I.C., Groom, A.C. and Chambers, A.F. Saberi, B. (1996) Epithelial–mesenchymal (1997) Sequential steps in hematogenous transitions in cancer progression. Acta metastasis of cancer cells studied by in vivo Anatomica, 156, 217–226. videomicroscopy. Invasion Metastasis, 17, 44 Lu, Z., Ghosh, S., Wang, Z. and Hunter, T. 281–296. (2003) Downregulation of caveolin-1 54 Al-Mehdi, A.B., Tozawa, K., Fisher, A.B., function by EGF leads to the loss of Shientad, L., Lee, A. and Muschel, R.J. E-cadherin, increased transcriptional (2000) Intravascular origin of metastasis activity of beta-catenin, and enhanced from the proliferation of endothelium- tumor cell invasion. Cancer Cell, 4, attached tumor cells: a new model for 499–515. metastasis. Nature Medicine, 6, 100–102. 45 Lee, J.M., Dedhar, S., Kalluri, R. and 55 Takasu, M., Tada, Y., Wang, J.O., Tagawa, Thompson, E.W. (2006) The epithelial– M. and Takenaga, K. (1999) Resistance to mesenchymal transition: new insights in apoptosis induced by microenvironmental signaling, development, and disease. The stresses is correlated with metastatic Journal of Cell Biology, 172, 973–981. potential in Lewis lung carcinoma. Clinical 46 Chambers, A.F., Groom, A.C. and & Experimental Metastasis, 17, 409–416. MacDonald, I.C. (2002) Dissemination 56 Helenius, M.A., Saramaki, O.R., Linja, and growth of cancer cells in metastatic M.J., Tammela, T.L. and Visakorpi, T. sites. Nature Reviews. Cancer, 2, 563–572. (2001) Amplification of urokinase gene in 182j 11 Prevention of Angiogenesis and Metastasis

prostate cancer. Cancer Research, 61, epigallocatechin-3-gallate suppresses rat 5340–5344. hepatic stellate cell invasion by 57 Binder, B.R., Mihaly, J. and Prager, G.W. inhibitionof MMP-2 expression and its (2007) uPAR–uPA–PAI–1 interactions and activation. Acta Pharmacologica Sinica, 27, signaling: a vascular biologists view. 1600–1607. Thrombosis and Haemostasis, 97, 336–342. 66 Hong, J.H., Ahn, K.S., Bae, E., Jeon, S.S. 58 Owen-Schaub, L.B., van Golen, K.L., Hill, and Choi, H.Y. (2006) The effects of L.L. and Price, J.E. (1998) Fas and Fas curcumin on the invasiveness of prostate ligand interactions suppress melanoma cancer in vitro and in vivo. Prostate Cancer lung metastasis. The Journal of and Prostatic Diseases, 9, 147–152. Experimental Medicine, 188, 1717–1723. 67 Vijayababu, M.R., Arunkumar, A., 59 French, L.A. and Tschopp, J. (2002) Kanagaraj, P., Venkataraman, P., Defective death receptor signaling as a Krishnamoorthy, G. and Arunakaran, J. cause of tumor immune escape. Seminars (2006) Quercetin downregulates matrix in Cancer Biology, 12,51–55. metalloproteinases 2 and 9 proteins 60 Cretney, E., Takeda, K., Yagita, H., expression in prostate cancer cells (PC-3). Glaccum, M., Peschon, J.J. and Smyth, Molecular and Cellular Biochemistry, 287, M.J. (2002) Increased susceptibility to 109–116. tumor initiation and metastasis in TNF- 68 Peng, P.L., Hsieh, Y.S., Wang, C.J., Hsu, related apoptosis-inducing ligand- J.L. and Chou, F.P. (2006) Inhibitory effect deficient mice. Journal of Immunology, 168, of berberine on the invasion of human 1356–1361. lung cancer cells via decreased 61 Naumov, G.N., Macdonald, I.C., productions of urokinase-plasminogen Chambers, A.F. and Groom, A.C. (2001) activator and matrix metalloproteinase-2. Solitary cancer cells as a possible source of Toxicology and Applied Pharmacology, 214, tumour dormancy? Seminars in Cancer 8–15. Biology, 11, 271–276. 69 Ramer, R. and Hinz, B. (2008) Inhibition of 62 Holmgren, L., OReilly, M.S. and Folkman, cancer cell invasion by cannabinoids via J. (1995) Dormancy of micrometastases: increased expression of tissue inhibitor balanced proliferation and apoptosis in the of matrix metalloproteinases-1. Journal presence of angiogenesis suppression. of the National Cancer Institute, 100, Nature Medicine, 1, 149–153. 59–69. 63 Campbell, J.J. and Butcher, E.C. (2000) 70 Liang, Z., Wu, T., Lou, H., Yu, X., Chemokines in tissue-specific and Taichman, R.S., Lau, S.K., Nie, S. and microenvironment-specific lymphocyte Umbreit, J. (2004) Inhibition of breast homing. Current Opinion in Immunology, cancer metastasis by selective synthetic 12, 336–341. polypeptide against CXCR4. Cancer 64 Muller, A., Homey, B., Soto, H., Ge, N., Research, 64, 4302–4308. Catron, D., Buchanan, M.E., McClanahan, 71 Nair, P.K., Rodriguez, S., Ramachandran, T. and Murphy, E. (2001) Involvement of R., Alamo, A., Melnick, S.J., Escalon, E., chemokine receptors in breast cancer Garcia, P.I. Jr and Wnuk, S.F. (2004) metastasis. Nature, 410,50–56. Immune stimulating properties of a novel 65 Zhen, M.C., Huang, X.H., Wang, Q., Sun, polysaccharide from the medicinal plant. K., Liu, Y.J., Li, W., Zhang, L.J. and Cao, Tinospora cordifolia. International L.Q. (2006) Green tea polyphenol Immunopharmacology, 4, 1645–1659. j183

12 Impact of Dietary Factors on the Immune System Alexa L. Meyer

Considering the constant threat of potentially harmful intruders from the environment, the importance of an adequate defense system is easily comprehensible. Especially in higher organisms, a sophisticated interplay of various cells, mediators, and other soluble factors provides an effective protection against a wide range of pathogens as well as worn-out and neoplastic body cells. Indeed, preventing tumor development is an important task of the immune system. Besides fighting virus infections that can cause cancer, the immune cells can actively recognize degenerated body cells and destroy them. However, more recent evidence has revealed that under certain circumstances, the immune system may also promote cancerogenesis. Manipulation of crucial events can therefore contribute to the reduction of cancer risk. Through its well-established influence on immune function, nutrition provides a significant means to this goal.

12.1 A Short Presentation of the Immune System

Although the multiple agents involved in the immune defense are interwoven, the system can be divided in categories to give a better overview (also see Figure 12.1).

12.1.1 The First Line of Defense

When a pathogen enters the body, it is confronted with cells and soluble factors of the natural or unspeciﬁc immune system. This branch is older and also found in lower animals. Many of its components are highly conserved in evolution. They are constitutively expressed and do not need a speciﬁc antigen to be activated. Indeed, they respond to certain common repetitive carbohydrate structures and lipopoly-

Figure 12.1 Overview of the human immune DC also present antigens from it to T cells system. A pathogen penetrating the body is of the specific immunity. These cells in first confronted to phagocytosing cells of turn enhance the function of macrophages, the innate immune system, macrophages or activate B lymphocytes to produce and dendritic cells that are both derived antibodies. Cytotoxic T (cT) cells can kill virus- from monocytes as well as neutrophil infected and tumor cells. This function is granulocytes. These cells try to destroy also accomplished by NK cells, a population the pathogen, but macrophages and of the innate immunity.

saccharides of the cell membranes of pathogens allowing them to discriminate foreign ones from their own. Upon recognition, cells and particles displaying these patterns are ingested by phagocytosing cells, macrophages, neutrophil granulocytes and dendritic cells (DC). This process is aided by specific plasma proteins known as the complement system that can also lyse certain pathogens [1]. Macrophages and neutrophils kill intruders by releasing reactive oxygen and nitrogen molecules in a reaction known as respiratory burst. In addition, they also induce inflammation by producing appropriate mediators, the proinflammatory cytokines and chemokines. In many a case, an infection can be warded off by innate mechanisms alone. However, a number of pathogens have evolved means to evade them, calling for a more effective defense. To this end, macrophages are able to process ingested structures and display peptide fragments on their surface. They share this property with another type of monocyte-derived innate immune cells, the dendritic cells. Both are the so-called professional antigen-presenting cells (APC) that are necessary for initiating a specific immune response [2]. 12.1 A Short Presentation of the Immune System j185

12.1.2 Adaptive Immunity

The main effectors of adaptive or specific immunity are the T and B lymphocytes. Each of them expresses a type of receptor on its surface that specifically binds a particular antigen. However, T lymphocytes only recognize their antigen if it is presented to them bound to a specific type of molecules encoded by a gene cluster termed the major histocompatibility complex (MHC) that exists in two classes. MHC type I molecules are found on all body cells containing a nucleus and serve to display predominantly intracellular antigens, own as well as foreign from viruses or other pathogens. In turn, MHC type II molecules binding exogenous antigens are only found on some cells like APC, for instance. Recognition of their specific antigen activates T lymphocytes and leads to their proliferation as, under normal conditions, the cells of the specific immune system are present in small numbers only. Once the pathogens are overcome, some lymphocytes develop into long-lived memory cells that allow a more rapid reaction if the same microorganism is encountered a second time [2]. An important fraction of T lymphocytes are the helper T (Th) cells characterized by the CD4 antigen on their surface and interacting with MHC II. They activate macrophages and B cells displaying their antigen. In the former, this leads to an enhanced fighting against the ingested pathogen and a cellular proinflammatory answer, while the latter transform into plasma cells producing antibodies against the fi speci c antigen. These branches are regulated by distinct types of Th cells, Th1 for the macrophages and Th2 for the B cells. Although other Th subtypes have been described, these two are generally regarded as major influencing factors directing the immune system toward a cellular or a humoral response. Both fractions do not differ morphologically but only in the cytokines they produce and which stimulate fi the target cells, mainly interferon (IFN)-g for Th1 and IL-4, IL-5, and less speci cally IL-10 for Th2 cells [3].

12.1.3 The Immune System in Cancerogenesis

A second type of T lymphocyte, the cytotoxic T lymphocyte (CTL), is a central player in anticancer immunity. CTL are characterized by the CD8 antigen on their surfaces and induce apoptosis or necrosis in virus-infected or degenerated cells. They react to typical peptides of cancer cells, the so-called tumor-associated antigens (TAA) displayed on MHC type I molecules, but to be fully activated they need the support of Th cells specific to the respective antigen [4–6]. The second kind of cytotoxic cells are the natural killer (NK) cells. These are lymphocytes as well, although belonging to the innate immune system. They also recognize body cells infected by intracellular pathogens like viruses, some parasites (Leishmania, Listeria) as well as tumor cells and kill them, but are not antigen-specific like CTL [7]. Thus, the immune system seems quite adapted to fighting tumors. Why does cancer develop nevertheless? 186j 12 Impact of Dietary Factors on the Immune System

12.1.4 Cancer – A Serious Opponent

A major problem is that TAA on the tumor cells neither are always recognized nor do activate the T cells, a phenomenon known as immunotolerance. Thus, presentation of the antigen plays a crucial role in tumor immunity. In humans, activated NK cells are capable of presenting antigens, but the most efficient among the APC are the dendritic cells. They up-take TAA from tumor cells killed by the NK cells. While the common way of presentation by APC is on MHC II molecules, DC are capable of cross-presentation, that is, displaying exogenous antigens on MHC I molecules. In this way, they can present the TAA to CTL as well. The DCs activity against the tumors, however, goes even farther inasmuch as they are able to kill tumors by themselves [8]. The immune system may, however, even promote cancerogenesis under certain circumstances. The involvement of inflammation in tumor development is widely acknowledged. The production of reactive oxygen species (ROS) can lead to DNA damage and mutagenic alterations. In addition, chemokines, mediators secreted in the course of inflammation recruit immune cells enable them to cross the blood vessel wall and penetrate the tissue. This property has been shown to be used by some tumors to help their invasion of other tissues during metastasis. Besides, some chemokines also promote angiogenesis and stimulate the growth of cancer cells [9]. Thus, influencing the immune system can provide a means to control cancer.

12.2 The Role of Nutrition in Immunity

Even from this scant overview of immune functions, it is easily comprehensible that a sufﬁcient supply of energy and nutrients is vital for the optimal function of this complex system. Beside proteins and amino acids needed to support the high turnover of immune cells and soluble parts, single nutrients but also nonnutritive food components have direct impacts on speciﬁc immune factors and reactions [10]. Some of the most relevant shall be presented in more detail in the following paragraphs.

12.2.1 Fat and Fatty Acids: Their Role in Inflammation and Beyond

Polyunsaturated fatty acids (PUFA) play a particular role in the regulation of inﬂammation being precursors of the eicosanoids, another group of mediators regulating immune but also other body functions. Two types of enzymes are responsible for the formation of these compounds: the cyclooxygenases (COX-1 and -2) and the lipoxygenases (LOX), with the former initiating the synthesis of the prostaglandins (PG) and thromboxanes (TX), summarized as prostanoids, the latter that of the leukotrienes (LT) [11]. The structure of the formed eicosanoids depends 12.2 The Role of Nutrition in Immunity j187

Scheme 12.1 Synthesis of prostaglandin (PG) synthesis. Fish oil is the best source for this PUFA E from polyunsaturated fatty acid (PUFA) and its consumption can influence the precursors. Arachidonic acid is the major membrane composition and thus the PG PUFA precursor in human cell membranes. production. As the ensuing PGs have different However, eicosapentaenoic acid is also a immunological effects, the immune response is substrate for the enzymes of eicosanoid modified. on the fatty acid they are derived from (compare Scheme 12.1). The two major substrates of COX and LOX are the n-6 fatty acid arachidonic acid (AA) and the n-3 fatty acid eicosapentaenoic acid (EPA). Both are cleaved from the phospholipids of cell membranes by phospholipase A2. In mammalian cells, however, AA is the most common substrate for COX and LOX due to its higher content in membranes and its greater affinity to the enzymes [12]. The membrane composition is influenced by fat intake and the typical Western diet is generally rich in n-6 fatty acids like, for instance, linoleic acid from which AA can be synthesized in the body through elongation and desaturation. In contrast, EPA is mainly contained in fatty fish and, to a lesser degree, in algae. The seeds and oils of flax, rape, walnut, and soy are sources of a-linolenic acid serving as a precursor to EPA although its conversion is rather inefficient. A higher intake of n-3 fatty acids, mostly in the form of fish oil, was shown to increase their content in the cell membranes and to competitively inhibit the formation of AA-derived eicosanoids and increase that of EPA-derived eicosanoids. 188j 12 Impact of Dietary Factors on the Immune System

Arachidonic acid gives rise to prostanoids of the 2-series and leukotrienes of the 4-series, EPA to prostanoids of the 3-series and leukotrienes of the 5-series. Eicosanoids exert a great variety of effects depending on the target tissues and the receptors through which their signal is transmitted [13]. They are mediators of immune functions and are particularly involved in the regulation of inflammation. Generally, those derived from AA are more potent stimulators of inflammation than those derived from EPA [14]. Accordingly, a higher intake of fish oil has been shown to mitigate excessive inflammation. This effect is not only mediated through modified eicosanoid synthesis. Indeed, n-3 fatty acids regulate peroxisome proliferator-activated receptors (PPAR). Upon binding of their ligands, these nuclear receptors act as transcription factors of target genes. Macrophages and dendritic cells express the g-isoform of this receptor type that inhibits inflammation and downregulates macrophage activity [15–17]. Incorporation of n-3 fatty acids from dietary fish oil in the membrane phospholipids has effects on cell functions by changing the composition of lipid rafts, structures mediating signal transduction. Thus, EPA caused the inhibition of T lymphocytes [15]. With regard to cytotoxicity, there is no clear evidence as to the effects of n-3 PUFA. On the one hand, inhibitory influences of fish oil on NK cell activity have been reported, on the other, in macrophages fish oil had no effect on the tumor cell killing compared to oils rich in n-6 fatty acids [18, 19]. Regardless of the fatty acid composition, a high fat diet is generally associated with impaired immunological functions including the ability of macrophages and NK cells to kill tumor cells [19, 20]. This effect may be partly mediated through the endocrine function of adipose tissue, as adiponectin was shown to downregulate IL-2-mediated NK cell activity [21].

12.2.2 Trace Elements

Trace elements are involved in immunological functions in several ways. First, they are cofactors of enzymes of cell replication like iron for ribonucleotide reductase or zinc for DNA- and RNA-polymerases. As such, they are crucial for the proliferation of lymphocytes during specific immune responses. Inadequate supply of iron or zinc therefore is associated with lower cell numbers, particularly of T lymphocytes. – Thymus atrophy was described [22 24]. The higher sensibility of Th1 cells adds to the impact of iron deficiency on cellular immunity [25]. Moreover, iron is also important for myeloperoxidase and the NADPH oxidase complex, catalyzing the production of ROS needed for the respiratory burst. This function is also impaired in iron deficiency despite undisturbed phagocytosis [22]. Besides its mentioned general involvement in cell replication, zinc is important for the development of T lymphocytes, as it is a cofactor of the stimulating nonapeptide thymulin, formerly called facteur thymique serique (FTS) secreted by the thymic epithelium. It is required for the differentiation of immature T cells. FTS is also an inducer of cytokine secretion in peripheral blood mononuclear cells and proliferation þ in CD8 T cells [26]. NK cell cytolytic activity is also depressed in zinc deficiency. 12.2 The Role of Nutrition in Immunity j189

In addition, the inhibitory receptor of NK cells depends on zinc, so that insufficient supply might lead to a dysregulation of NK function with unspecific cytotoxicity [26]. In contrast, cells differentiating in the bone marrow, the myeloid lines as well as the B lymphocytes, are not or only slightly affected by zinc deficiency. Monocytes and, to a lesser extent, granulocytes even showed increased proliferation under acute zinc deficiency [27]. A cofactor of the antioxidant enzyme glutathione peroxidase, selenium is involved in the protection of immune cells against the reactive molecules generated to kill pathogens. Accordingly, neutrophil granulocytes show a reduced capacity to kill ingested bacteria although phagocytosis as such is not affected [28]. Furthermore, selenium supplementation stimulates the expression of IL-2 receptors on T cells, thus enhancing their proliferation. A higher cytotoxicity of NK and cytotoxic T cells was observed in mice and humans supplemented with selenium [29]. A great part of these effects is related to the involvement of selenium in antioxidant processes [30]. This is supported by the observation that some viruses become more virulent in selenium-deficient hosts due to mutations in the viral genome that seem to be caused by a lower activity of glutathione peroxidase [31]. However, other mechanisms besides the regulation of oxidative status cannot be ruled out [29, 30].

12.2.3 Vitamins

Vitamins have very diverse functions in the body acting as cofactors of enzymes, being involved in metabolism, biosynthesis, and cell replication. Thus, they are also important for the immune system. The vitamins C (ascorbic acid) and E (tocopherols and tocotrienols) are among the most potent nonenzymatic antioxidants in the hydrophilic and lipophilic milieu, respectively. As mentioned before, the unspecific immune response relies partly on free radicals, reactive oxygen and nitrogen compounds that damage macromolecules like proteins, unsaturated fatty acids, and the DNA. The immune cells are themselves potential targets of these deleterious effects calling for an efficient protection. However, vitamins also exert direct effects on various immune functions. Discussing the actions of all the vitamins would go beyond the scope of this overview. Therefore, the focus shall be on the most relevant ones. Vitamin C or ascorbic acid is generally associated with the resistance against infections especially of the upper respiratory tract. While supplementation does not appear to have a significant preventive effect on colds and flues, the severity and duration of these infections can be slightly reduced [32, 33]. Vitamin C is one of the leading nonenzymatic antioxidants in the hydrophilic milieu and therefore plays a role in protecting the leukocytes from ROS generated during inflammation. Indeed, neutrophils and lymphocytes are rich in this vitamin, but their concentrations are rather unstable and decline during infections [34, 35]. Deficiency of ascorbic acid impairs the ability of neutrophils and monocytes to kill ingested pathogens. Their activity is enhanced by supplementation [36]. 190j 12 Impact of Dietary Factors on the Immune System

Although several other B-vitamins are relevant for the immune system, vitamin B12 (cobalamin) and folic acid are particularly worth mentioning because of their role in cell proliferation and nucleotide synthesis. These water soluble vitamins are cofactors in the metabolism of 1-carbon compounds (methyl-, methenyl-, methylene-, formyl-, and formimino-rests). Both act as transfer molecules taking these compounds over from various donators or from each other. Studies about the effects of these vitamins on immune functions are rather scarce, but there is evidence that insufﬁcient supply particularly affects cytotoxic T lymphocytes and NK cells, thus potentially impairing the defense against cancer [37–39]. Supplementation of folate has shown beneﬁcial effects on lymphocytes in the elderly [40]. Vitamin E, the common term used for the tocopherols and tocotrienols, is the lipophilic counterpart of vitamin C in its role as a major antioxidant, particularly in the cell membranes. It preserves their integrity and functionality and adequate cell signaling. Like ascorbic acid, it protects phagocytes. Oxidative stress has an inhibitory effect on the immune function, particularly on T lymphocytes and NK cells [41]. Vitamin E was repeatedly shown to improve the function and proliferation of these cells, especially in the elderly or subjects with a suppressed immune system [42–46]. Macrophages show an age-related increase in NO production leading to a higher COX activity. The ensuing enhanced production of PGE2 impairs Tcell proliferation and decreases their numbers. Vitamin E mitigates this effect by reducing NO levels probably through its antioxidant effects [44]. It must, however, be mentioned that many studies used very high doses of a-tocopherol that cannot be obtained through diet. Interestingly, it was not the highest dose of 800 mg/day that showed the greatest effect in a study in elderly subjects [42]. Vitamin A is the name given to retinol and related molecules. Its effects on the immune system have been known for long. It modulates monocyte and granulocyte differentiation and supplementation could increase respiratory burst and NK cell numbers [47]. Deeper insights into molecular regulation have permitted a new understanding of this relationship. Through the nuclear retinoic acid and retinoid X receptors (RAR and RXR, respectively), retinoic acid acts directly on target genes, thereby regulating cell proliferation and differentiation [48–50]. T lymphocytes are

particularly concerned. Retinoic acid stimulates the production of Th2 cytokines fi while suppressing the Th1 branch. On the contrary, vitamin A de ciency leads to a fl stronger Th1 and a weaker Th2 response with proin ammatory effects and impaired humoral immunity mirrored in lower antibody production after vaccine administration that can be reversed by supplementation [49]. The maturation of myeloid precursors to neutrophil granulocytes is stimulated by retinoic acid through RAR [48]. Vitamin A deficiency also impairs NK cell activity [51].

12.2.4 Nonnutritive Food Components

Foods can contain a number of components that have no nutritive value as such, supplying neither energy nor elements for biosynthesis processes, but having beneﬁcial effects on certain body functions including the immune defense. 12.2 The Role of Nutrition in Immunity j191

This means that they can also contribute to the fight against cancer [10]. Some of these compounds are already used as functional additives, and probiotic lactic acid bacteria are doubtlessly among the best known. Their effects on immune function are widely acknowledged and are supported by a number of scientific studies. Depending on the bacterial strain, a downregulation of inflammatory reactions was described, as well as stimulatory influences [52, 53]. However, an enhanced cellular immunity, innate and adaptive, has been observed in many studies with NK cells appearing as a particular target population [54–56]. In rodents, administration of lactic acid bacteria could reduce the development and growth of chemically induced tumors through enhancement of NK cell activity and cytokine production [57, 58]. A great variety of bioactive compounds are contained in plant foods and many of these, like carotenoids and flavonoids, have been found to influence the immune system in one way or the other. Many studies focused on b-carotene, one of the major carotenoids in human plasma [59]. Carotenoids are not essential for the organism, but due to their structural relationship to retinol, about 10% of the over 600 compounds described so far serve as provitamin A. Of these, b-carotene is the most effective and common. It was shown to stimulate the activity of NK cells and improve antigen presentation by monocytes. Increases in the numbers of these and T lymphocytes were also reported. These effects were obtained in cell cultures as well as in animals and humans [60, 61]. While especially b-carotene can be converted to vitamin A in the body, the observed immunological effects are to a major extent independent of its role as a provitamin. Indeed, other carotenoids lacking this activity were also effective. Differences seem to exist between the various carotenoids [60, 61]. Many studies in animals and humans used isolated carotenoids as supplements. However, foods rich in carotenoids can also stimulate immune functions like NK cell activity [62]. The immune stimulating effects of carotenoids have been associated with their antioxidant potential. While this may contribute to the improvement of immune functions, some effects were shown to be independent of it [62]. On the contrary, flavonoids have generally shown downregulatory effects on immunity. They suppress inflammation by interfering with eicosanoid synthesis, reducing generation of reactive oxygen molecules by neutrophils, and blocking the production of proinflammatory cytokines. Flavonoids inhibit COX and LOX activity as well as the transcription of the inducible COX-2 [63]. An inhibition of T cell proliferation and function, including T cell cytotoxicity was also shown. However, a stimulation of NK cell activity that was possibly mediated through an increased IFN release has been described [64, 65]. A stimulatory effect on lymphocytes and monocytes was also found in vitro for cocoa flavonoids. In this study, different effects were obtained with short chain and long chain flavonoid fractions and for different cell populations that may explain the variety in previous findings [66]. Furthermore, in vivo conditions may be different from the situation in vitro. Thus, consumption of purple sweet potato leaves, a food rich in flavonoids, also caused a higher natural cytotoxicity against tumor cells as well as an increased proliferation of T lymphocytes with a higher secretion of IL-2 and IL-4 [67]. 192j 12 Impact of Dietary Factors on the Immune System

The efﬁciency of vaccines and immunological cancer therapies can be optimized by the use of adjuvants, agents acting as enhancers. Two classes of secondary plant compounds have been successfully used to this end: the saponins and b-glucans. Saponins are glycosides of steroids or triterpenes. The combination of a polar and an apolar moiety is responsible for their surface activity and the formation of soapy foam when dissolved in water. Foods rich in saponins are pulses, spinach, garlic, and oats among others. Due to their chemical properties, they are hemolytic and have therefore often been considered as harmful. However, they also have a capacity to stimulate the immune system at low doses, leading to a higher antibody production and monocyte proliferation. In addition, they are potent inducers of þ CD4 helper T cells, leading to an increased production of Th1 and, to a lesser þ extent, Th2 cytokines, and of cytotoxic CD8 T lymphocytes (CTL). It was postulated that the effect on these latter is mediated by antigen presenting cells in which saponins facilitate antigen uptake generating pores in the cell membrane [68, 69]. b-Glucans are polysaccharides of b-linked glucose molecules. They are parts of the cell walls of some bacteria, fungi, and yeasts as well as some cereals like barley and oat, for instance. Structural differences exist between the b-glucans from various sources: in cereals, they contain mainly linear b 1,4-glucans interrupted by b 1,3 linkages and are water soluble. Several in vitro studies as well as in vivo trials

in animals showed a stimulation of Th1 immunity with a higher production of proinflammatory cytokines as well as an enhancement of oxidative burst and chemotaxis in macrophages and neutrophils, respectively. While the impact on cytokine production was less pronounced in humans, a better resistance to infections and decreased mortality have been reported in high-risk patients [70, 71]. Although nonnutritive food components may differ in the way they affect the immune system, their effects are mediated through the intestinal immune system. Indeed, the first contact with ingested compounds occurs in the gut and the importance of an effective defense system in this organ is evident considering the abundance of potential pathogens entering the body through this way. At the same time, the establishment of tolerance against innocuous food antigens and commensal gut microbiota is vital to avoiding hypersensitivity. Accordingly, the antigens reaching the gut undergo scrutiny. Intestinal epithelial cells (IEC) are able to act as nonprofessional antigen-presenting cells (APC), expressing MHC class II on their surface. However, antigen transport occurs mainly via specialized epithelial cells, the so-called M cells that are localized in the Peyers patches. These aggrega- tions of lymphoid tissue are the major site of encounter between antigens and intestinal immune cells, macrophages, dendritic cells, and lymphocytes. The ensuing activation of these cells and the production of cytokines can extend the local stimulus to a systemic immune response [72, 73]. In conclusion, a sufficient supply of vitamins and trace elements as well as macronutrients like fatty acids, for instance, is of central importance for a proper 12.2 The Role of Nutrition in Immunity j193

Table 12.1 Effects of selected nutrients and food components on immune functions that are effective against tumors.

Nutrient Status Cell type/function Effects References

Fat HI NK Activity # [18, 19] Fish oil, S NK Activity #? [17] n-3 FA S T lymphocytes Inhibition [14] S Eicosanoid synthesis Inﬂammation # [13, 14] Iron D T lymphocytes Numbers # [21, 22] Zinc D NK Cytotoxicity # [25] D T lymphocytes Proliferation # [25] D CTL Disturbed function [25] Selenium S CTL, NK Cytotoxicity " [28] Folate D NK Cytotoxicity # [37] D CTL Proliferation # [38] S CTL Proliferation " [39] Cobalamin D NK Activity # [36] D CTL Proliferation # [36] Vitamin E S NK, CTL Proliferation ", function " [41–45] Vitamin A D NK Activity # [50] S Th cells Shift toward Th2 [48] Probiotics S NK Cytotoxicity " [54–56, 58] b-Carotene S NK Cytotoxicity " [60, 61] Flavonoids S Eicosanoid synthesis Inﬂammation # [63] S NK Cytotoxicity " [64, 65, 67] Saponins S CTL Number, activity " [68, 69] b " -Glucans S Th1 lymphocytes Activity [70, 71]

D, deﬁciency; HI, high intake; S, supplementation.

immune function. Food components do not only supply energy and metabolites for biosynthesis processes, but can act as regulators and direct immune responses. Thus, anticancer immunity may also be inﬂuenced (see Table 12.1 for an overview). Evidence for immune stimulating effects of various nutrients that was obtained from numerous studies has often beguiled people into supplement- ing high doses of one or several compounds. This measure can, however, have negative consequences leading to a dysbalance of the immune system. Indeed, the various reactions have to be tightly regulated for an optimal protection against pathogens and the bodys own degenerated cells. A diverse, well-equili- brated diet is the best way to obtain a wide array of nutrients. Fruit, vegetables, whole grain cereals, milk products, and plant oils are good sources of essential micronutrients and fatty acids. Moderate meat consumption can improve the supply of iron, zinc, and some B vitamins, while a regular ﬁsh intake provides n-3 fatty acids. The relevance of natural foods is supported by the promising immune-regulatory effects of nonnutritive food components, many of which still await discovery. 194j 12 Impact of Dietary Factors on the Immune System

References

1 Revillard, J.P. (2001) Innate immunity. Warnock, M., Schmaier, A.H., European Journal of Dermatology, Yokoyama, C., Smyth, E.M., Wilson, S.J., 12, 224–227. FitzGerald, G.A., Garavito, R.M., Sui, D.X., 2 Janeway, C.A., Travers, P., Walport, M. and Regan, J.W. and Smith, W.L. (2007) Shlomchik, M.J. (2004) Immunobiology: Enzymes and receptors of prostaglandin The Immune System in Health and Disease, pathways with arachidonic acid-derived 6th edn, Garland Publishers, New York. versus eicosapentaenoid acid-derived 3 Kidd, P. (2003) Th1/Th2 balance: the substances and products. The Journal of hypothesis, its limitations, and Biological Chemistry, 282, 22254–22266. implications for health and disease. 13 Narumiya, S., Sugimoto, Y. and Alternative Medicine Review, 8, 223–246. Ushikubi, F. (1999) Prostanoid receptors: 4 Clarke, S.R.M. (2000) The critical role of structures, properties, and functions. CD40/CD40L in the CD4-dependent Physiological Reviews, 79, 1193–1226. generation of CD8 þ T cell immunity. 14 Calder, P.C. (2002) Dietary modification of Journal of Leukocyte Biology, 67, 607–614. inflammation with lipids. Proceedings of the 5 Dunn, G.P., Bruce, A.T., Ikeda, H., Nutrition Society, 61, 345–358. Old, L.J. and Schreiber, R.D. (2002) 15 Calder, P.C. (2003) n-3 polyunsaturated Cancer immunoediting: from fatty acids and inflammation: from immunosurveillance to tumor escape. molecular biology to the clinic. Lipids, Nature Immunology, 3, 991–998. 38, 343–352. 6 Andersen, M.H., Schrama, D., 16 Deckelbaum, R.J., Worgall, T.S. and Seo, T. Straten, P.T. and Becker, J.C. (2006) (2006) n-3 fatty acids and gene expression. Cytotoxic Tcells. The Journal of Investigative The American Journal of Clinical Nutrition, Dermatology, 126,32–41. 83, 1520S–1525S. 7 Moretta, L., Biassoni, R., Bottino, C., 17 Szanto, A. and Roszer, T. (2008) Nuclear Mingari, M.C. and Moretta, A. (2002) receptors in macrophages: a link between Natural killer cells: a mystery no more. metabolism and inflammation. FEBS Scandinavian Journal of Immunology, Letters, 582, 106–116. 55, 229–232. 18 Thies, F., Nebe-von-Caron, G., Powell, J.R., 8 Chan, C.W. and Housseau, F. (2008) The Yaqoob, P., Newsholme, E.A. and kiss of death by dendritic cells to cancer Calder, P.C. (2001) Dietary cells. Cell Death and Differentiation, supplementation with eicosapentaenoic 15,58–69. acid, but not with other long-chain n-3 or 9 Coussens, L.M. and Werb, Z. (2002) n-6 polyunsaturated fatty acids, decreases Inflammation and cancer. Nature, natural killer cell activity in healthy 420, 860–867. subjects aged >55 years. The American 10 Ferguson, L.R. and Philpott, M. (2007) Journal of Clinical Nutrition, 73, 539–548. Cancer prevention by dietary bioactive 19 Wallace, F.A., Neely, S.J., Miles, E.A. and components that target the immune Calder, P.C. (2000) Dietary fats affect response. Current Cancer Drug Targets, macrophage-mediated cytotoxicity towards 7, 459–464. tumour cells. Immunology and Cell Biology, 11 Funk, C.D. (2001) Prostaglandins and 78,40–48. leukotrienes: advances in eicosanoid 20 Barone, J., Hebert, J.R. and Reddy, M.M. biology. Science, 294, 1871–1875. (1989) Dietary fat and natural-killer-cell 12 Wada, M., DeLong, C.J., Hong, Y.H., activity. The American Journal of Clinical Rieke, C.J., Song, I., Sidhu, R.S., Yuan, C., Nutrition, 50, 861–867. References j195

21 Kim, K.-Y.,Kim, J.K., Han, S.H., Lim, J.-S., responses. Archivum Immunologiae et Kim, K.I., Cho, D.H., Lee, M.-S., Lee, J.-H., Therapiae Experimentalis 55, 289–297. Yoon, D.-J., Yoon, S.R., Chung, J.W., 31 Beck, M.A. (2001) Antioxidants and viral Choi, I., Kim, E. and Yang, Y. (2006) infections: host immune response and Adiponectin is a negative regulator of NK viral pathogenicity. Journal of the American cell cytotoxicity. Journal of Immunology, College of Nutrition, 20, 384S–388S. 176, 5958–5964. 32 Douglas, R.M., Hemil€a H., Chalker, E., 22 Ahluwalia, N., Sun, J., Krause, D., DSouza, R.R.D. and Treacy, B. (2004) Mastro, A. and Handte, G. (2004) Immune Vitamin C for preventing and treating the function is impaired in iron-deficient, common cold. Cochrane Database of homebound, older women. The American Systematic Reviews, 4, CD000980. Journal of Clinical Nutrition, 79, 516–521. 33 Wintergerst, E.S., Maggini, S. and 23 Mullick, S., Rusia, U., Sikka, M. and Hornig, D.H. (2007) Contribution of Faridi, M.A. (2006) Impact of iron selected vitamins and trace elements to deficiency anaemia on T lymphocytes & immune function. Annals of Nutrition and their subsets in children. The Indian Metabolism, 51, 301–323. Journal of Medical Research, 124, 647–654. 34 Washko, P., Rotrosen, D. and Levine, M. 24 Rink, L. and Gabriel, P. (2000) Zinc and the (1989) Ascorbic acid transport and immune system. Proceedings of the accumulation in human neutrophils. The Nutrition Society, 59, 541–552. Journal of Biological Chemistry, 25 Thorson, J.A., Smith, K.M., Gomez, F., 264, 18996–19002. Naumann, P.W. and Kemp, J.D. (1991) 35 Bergsten, P., Amitai, G., Kehrl, J., Role of iron on Tcell activation: TH1 clones Dhariwal, K.R., Klein, H.G. and Levine, M. differ from TH2 clones in their sensitivity (1990) Millimolar concentrations of to inhibition of DNA synthesis caused by ascorbic acid in purified human IgG Mabs against the transferrin receptor mononuclear leukocytes. Depletion and and the iron chelator deferoxamine. reaccumulation. The Journal of Biological Cellular Immunology, 134, 126–137. Chemistry, 265, 2584–2587. 26 Ibs, K.H. and Rink, L. (2003) Zinc-altered 36 Goldschmidt, M.C. (1991) Reduced immune function. The Journal of Nutrition, bactericidal activity in neutrophils from 133, 1452S–1456S. scorbutic animals and the effect of 27 King, L.E., Frentzel, J.W., Mann, J.J. and ascorbic acid on these target bacteria in vivo Fraker, P.J. (2005) Chronic zinc deficiency and in vitro. The American Journal of in mice disrupted Tcell lymphopoiesis and Clinical Nutrition, 54, 1214S–1220S. erythropoiesis while B cell lymphopoiesis 37 Tamura, J., Kubota, K., Murakami, H., and myelopoiesis were maintained. Sawamura, M., Matsushima, T., Journal of the American College of Nutrition, Tamura, T., Saitoh, T., Kurabayashi, H. and 24, 494–502. Naruse, T. (1999) Immunomodulation by 28 Arthur, J.R., McKenzie, R.C. and vitamin B12: augmentation of CD8 þ Beckett, G.J. (2003) Selenium and the T lymphocytes and natural killer cell immune system. The Journal of Nutrition, activity in vitamin B12-deficient patients by 133, 1457S–1459S. methyl-B12 treatment. Clinical and 29 Kiremidjian-Schumacher, L. and Roy, M. Experimental Immunology, 116,28–32. (1998) Selenium and immune function. 38 Kim, Y.I., Hayek, M., Mason, J.B. and Zeitschrift fur Ernahrungswiss, 37 (Suppl. 1), Meydani, S.N. (2002) Severe folate 50–56. deficiency impairs natural killer cell- 30 Hoffmann, P.R. (2007) Mechanisms by mediated cytotoxicity in rats. The Journal of which selenium influences immune Nutrition, 132, 1361–1367. 196j 12 Impact of Dietary Factors on the Immune System

39 Courtemanche, C., Elson-Schwab, I., Masucci, G., Malmberg, K.-J. and Mashiyama, S.T., Kerry, N. and Ames, B.N. Kiessling, R.V.R. (2007) A short-term (2004) Folate deficiency inhibits the dietary supplementation with high doses proliferation of primary human CD8 þ of vitamin E increases NK cell cytolytic T lymphocytes in vitro. Journal of activity in advanced colorectal cancer Immunology, 173, 3186–3192. patients. Cancer Immunology, 40 Field, C.J., Van Aerde, A., Drager, K.L., Immunotherapy, 56, 973–984. Goruk, S. and Basu, T.(2006) Dietary folate 47 Villamor, E. and Fawzi, W.W.(2005) Effects improves age-related decreases in of vitamin A supplementation on immune lymphocyte function. The Journal of responses and correlation with clinical Nutritional Biochemistry, 17,37–44. outcomes. Clinical Microbiology Reviews, 41 Betten, A., Dahlgren, C., Mellqvist, U.-H., 18, 446–464. Hermodsson, S. and Hellstrand, K. 48 Kastner, P. and Chan, S. (2001) Function (2004) Oxygen radical-induced natural of RARa during the maturation of killer cell dysfunction: role of neutrophils. Oncogene, 20, 7178–7185. myeloperoxidase and regulation by 49 Iwata, M., Eshima, Y. and Kagechika, H. serotionin. Journal of Leukocyte Biology, (2003) Retinoic acids exert direct effects on 75, 1111–1115. T cells to suppress Th1 development and 42 Meydani, S.N., Meydani, M., enhance Th2 development via retinoic acid Blumberg, J.B., Leka, L.S., Silber, G., receptors. International Immunology, Loszewski, R., Thompson, C., 15, 1017–1025. Pedrosa, M.C., Diamond, R.D. and 50 Dzhagalov, I., Chambon, P. and He, Y.W. Stollar, B.D. (1997) Vitamin E (2007) Regulation of CD8 þ T lymphocyte supplementation and in vivo immune effector function and macrophage response in healthy elderly subjects. A inflammatory cytokine production by randomized controlled trial. The Journal of retinoic acid receptor gamma. Journal of the American Medical Association, Immunology, 178, 2113–2121. 277, 1380–1386. 51 Dawson, H.D., Li, N.-Q., DeCicco, K.L., 43 Lee, C.-Y. and Wang, J.M.-F. (2000) Nibert, J.A. and Ross, A.C. (1999) Chronic Vitamin E supplementation improves cell- marginal vitamin A status reduces natural mediated immunity and oxidative stress of killer cell number and function in aging Asian men and women. The Journal of Lewis rats. The Journal of Nutrition, Nutrition, 130, 2932–2937. 129, 1510–1517. 44 Serafini, M. (2000) Dietary vitamin E and T 52 Ericksson, K.L. and Hubbard, N.E. (2000) cell-mediated function in the elderly: Probiotic immunomodulation in health effectiveness and mechanism of action. and disease. The Journal of Nutrition, International Journal of Developmental, 130, 403S–409S. Neuroscience, 18, 401–410. 53 Peña, J.A., Rogers, A.B., Ge, Z., Ng, V., 45 Malmberg, K.-J., Lenkei, T., Petersson, M., Li, S.Y., Fox, J.G. and Versalovic, J. (2005) Ohlum, T., Ichihara, F., Glimelius, B., Probiotic Lactobacillus spp. diminish Frodin,€ J.-E., Masucci, G. and Kiessling, R. Helicobacter hepaticus-induced (2002) A short-term dietary inflammatory bowel disease in supplementation of high doses of vitamin interleukin-10-deficient mice. Infection E increases T helper 1 cytokine production and Immunity, 73, 912–920. in patients with advanced colorectal cancer. 54 Nagao, F., Nakayama, M., Muto, T. and Clinical Cancer Research, 8, 1772–1778. Okumura, K. (2000) Effects of a fermented 46 Hanson, M.G., Özenci, V., Carlsten, M.C., milk drink containing Lactobacillus casei Glimelius, B.L., Frodin,€ J.-E.A., strain Shirota on the immune system in References j197

healthy human subjects. Bioscience, 63 Kim, H.P., Son, K.H., Chang, H.W. and Biotechnology, and Biochemistry, Kang, S.S. (2004) Anti-inflammatory plant 64, 2706–2708. flavonoids and cellular action 55 Parra, M.D., Martınez De Morentin, B.E., mechanisms. Journal of Pharmacological Cobo, J.M., Mateos, A. and Martınez, J.A. Sciences, 96, 229–245. (2004) Daily ingestion of fermented 64 Zhang, Y., Song, T.T., Cunnick, J.E., milk containing Lactobacillus casei Murphy, P.A. and Hendrich, S. (1999) DN114001 improves innate-defense Daidzein and genistein glucuronides capacity in healthy middle-aged people. in vitro are weakly estrogenic and Journal of Physiology and Biochemistry, activate human natural killer cells 60,85–92. at nutritionally relevant 56 Meyer, A.L., Micksche, M., Herbacek, I. concentrations. The Journal of Nutrition, and Elmadfa, I. (2006) Daily intake of 129, 399–405. probiotic as well as conventional yogurt 65 Middleton, E. Jr, Kandaswami, C. and has a stimulating effect on cellular Theoharides, T.C. (2000) The effects of immunity in young healthy women. plant flavonoids on mammalian cells: Annals of Nutrition and Metabolism, implications for inflammation, heart 50, 282–289. disease, and cancer. Pharmacological 57 Yasutake, N., Matsuzaki, T., Kimura, K., Reviews, 52, 673–751. Hashimoto, S., Yokokura, T. and 66 Kenny, T.P., Keen, C.L., Schmitz, H.H. Yoshikai, Y. (1999) The role of tumor and Gershwin, M.E. (2007) Immune necrosis factor (TNF)-a in antitumor effect effects of cocoa procyanidin oligomers of intrapleural injection of Lactobacillus on peripheral blood mononuclear cells. casei strain Shirota in mice. Medical Experimental Biology and Medicine, Microbiology and Immunology, 188,9–14. 232, 293–300. 58 Takagi, A., Matsuzaki, T., Sato, M., 67 Chen, C.M., Li, S.C., Lin, Y.L., Hsu, C.Y., Nomoto, K., Morotomi, M. and Shieh, M.J. and Liu, J.F. (2005) Yokokura, T. (2001) Enhancement of Consumption of purple sweet potato natural killer cytotoxicity delayed murine leaves modulates human immune carcinogenesis by a probiotic response: T-lymphocyte functions, microorganism. Carcinogenesis, lytic activity of natural killer 22, 599–605. cell and antibody production. 59 Bendich, A. and Olson, J.A. (1989) World Journal of Gastroenterology, Biological actions of carotenoids. The 11, 5777–5781. FASEB Journal, 3, 1927–1932. 68 Rajput, Z.I., Hu, S.H., Xiao, C.W. and 60 Hughes, D.A. (2001) Dietary carotenoids Arijo, A.G. (2007) Adjuvant effects of and human immune function. Nutrition, saponins on animal immune responses. 17, 823–827. Journal of Zhejiang University. B, 61 Chew, B.P. and Park, J.S. (2004) 8, 153–161. Carotenoid action on the immune 69 Sjolander,€ A., Cox, J.C. and Barr, I.G. response. The Journal of Nutrition, (1998) ISCOMs: an adjuvant with multiple 134, 257S–261S. functions. Journal of Leukocyte Biology, 62 Watzl, B., Bub, A., Briviba, K. and 64, 713–723. Rechkemmer, G. (2003) Supplementation 70 Akramiene, D., Kondrotas, A., of a low-carotenoid diet with tomato or Didziapetriene, J. and Kevelaitis, E. carrot juice modulates immune functions (2007) Effects of beta-glucans on the in healthy men. Annals of Nutrition and immune system. Medicina (Kaunas), Metabolism, 47, 255–261. 43, 597–606. 198j 12 Impact of Dietary Factors on the Immune System

71 Volman, J.J., Ramakers, J.D. and Plat, J. 73 Maldonado Galdeano, C., de Moreno de (2008) Dietary modulation of immune LeBlanc, A., Vinderola, G., Bibas function by b-glucans. Physiology & Bonet, M.E. and Perdigón, G. (2007) Behavior, 94, 276–284. Proposed model: Mechanism of 72 Heyman, M. (2001) How dietary antigens immunomodulation induced by probiotic access the mucosal immune system. bacteria. Clinical and Vaccine Immunology, Proceedings of the Nutrition Society, 14, 485–492. 60, 419–426. j199

13 Epidemiological Studies Anthony B. Miller

13.1 Introduction

Chemoprevention is a primary preventive intervention for cancer control, which has not come into practice as yet. Chemoprevention is based on two types of observations, from opposite sides of the cancer control spectrum. On the one side, the success of some forms of chemotherapy, or hormonotherapy, in prolonging the survival for some cancers. On the other side, in the case of tamoxifen, its success in the reduction of the occurrence of second primary cancers of the breast. By extrapolation, it is argued that an agent that is capable of treating advanced (symptomatic) cancer should also be capable of preventing the progression of precursors of the same cancer. Indeed, it is possible that similar agents would work as well, and potentially in lower, less toxic doses. The other epidemiology direction that has led to chemoprevention, and to the majority of the dietary factors considered in this volume, is the observation in several different types of epidemiologic studies that consumption of certain natural constituents of plants, and pharmacological agents, appears to be associated with a lower risk of some cancers. These observations, coupled with more basic science research, which seemed to indicate a mechanism of action for such agents in perturbing carcinogenicity, were sufﬁcient to initiate a series of investigations on chemoprevention. There are a number of parallels between chemoprevention and screening for cancer or its precursors. The success of such screening depends on an understanding of the natural history of precursors and a recognition that this natural history does not result in an inevitable progression of a detectable precursor to an invasive cancer. Rather, there may be different forms of a precursor that may not necessarily be on the same trajectory to cancer or that may or may not progress at the same rate or to the same extent. It is even possible in some instances for some of the early stages to be bypassed, or at least not be detectable, in an individual destined to develop cancer. So the demonstration that a screening test will detect a precursor, and that it can be ablated, does not necessarily mean that the individual has been beneﬁted.

In this chapter, I shall discuss some of the lessons learnt in the past two decades on chemoprevention, in relation to basic methodological principles as we understand them, and present some examples particularly relevant to the epidemiological underpinning of studies designed to evaluate the role of potential chemopreventive agents.

13.2 Observational Epidemiology Studies: What Can We Learn From Them?

There are two types of observational epidemiological studies that have informed chemoprevention, case–control studies and cohort (prospective) studies. Both of these studies suffer from a number of methodological difficulties, and in interpreting the findings from them we must always remember that they are observational, that is, the investigator observes but does not conduct an experiment; one can only take the findings as they relate to the subjects studied, and what they have done, or believe they have done. In population-based case–control studies of diet and cancer, a questionnaire is administered to an unselected series of newly diagnosed cancer cases of interest and a group of cancer-free controls selected to match the cancer cases by relevant characteristics including age and sex and representative of the population from which the cases are drawn. Hospital-based case–control studies are similar, though both cases and controls are derived from subjects in hospital, and they can hardly be regarded as representing a particular population. The methodological issues that affect such studies include the extent the subjects, both cases and controls, agree to participate in the study, the efficiency with which the data are collected, and the extent the subjects recall is influenced by their recent experience. The fact of diagnosis of a cancer in the cases, and in both groups, their recent dietary intake, has been shown in many studies to influence what they recall, even if the questionnaire is directed to their past experience. Bias (differential data between cases and controls) may be unavoid- able, therefore, and has led many to downgrade their interpretation of the findings from such studies. On the contrary, the ability to collect detailed data from cases and controls means that the information obtained may be more complete than in cohort studies, whereas in studies of rare conditions, case–control studies will be far more practical. In cohort studies, a group is identified, data collected from them, and then they are followed, usually for many years, to identify those who develop cancer. Cohort studies are rarely representative of a particular population, rather they tend to be convenience samples, that is, comprise groups selected because they are accessible in some way, for example, nurses, health professionals, participants in a screening programme. Because cohort studies are large (of the order of thousands or tens of thousands, rather than hundreds as in case–control studies), to permit sufficient numbers of cancers to develop, it is possible to collect rather limited data from each subject, with the dietary questionnaire usually being self-administered. Therefore, the precision with which dietary intake is measured is usually less than that for 13.4 How Will We Know If We Are Successful? j201 case–control studies. Although the major advantage is that the dietary data are collected before the diagnosis of cancer (thus avoiding the recall bias of case–control studies), cohort studies tend to suffer from major misclassification error, with a tendency to find null associations. Both types of studies collect data on foods consumed, and sometimes, dietary supplements. Thus, if a micronutrient is the subject of interest, such as in the study of a potential natural chemopreventive agent, estimates of its consumption have to be derived by running the data through diet data banks, often based on the US Department of Agriculture food tables or on national food tables of other countries, so that the possibility for misclassification error increases. Observational studies will therefore suffice to raise hypotheses, but it would be unwise to make policy decisions based upon such studies. Rather, it would be wise only to make policy decisions based upon the results of carefully designed experimental studies, as discussed below in terms of the example of beta-carotene.

13.3 What Are We Trying To Do with Chemoprevention?

The objective of primary prevention is to reduce the incidence of clinically significant cancers and consequently, reduce deaths from such cancers. Chemoprevention should have the same objective. The analogy with screening discussed in Section 13.1 should always be kept in mind. There is a distribution of cancers by severity, ranging from those that progress very slowly, if at all, to those that progress rapidly and soon metastasize and result in death. It is the rapidly progressive cancers that we should like most to prevent with chemoprevention. It could be argued that if the agent we use is capable of preventing the emergence of the cancers that are most easy to treat, and which only result in death under circumstances of major clinical delays or failure to treat, then we shall have achieved little. Indeed, offsetting any benefit from the prevention of easy-to-treat cancers will be the necessity for a large number of people to take the chemopreventive agent who in the event will not develop the relevant cancer and thus not benefit at all. Therefore, we should aim to prevent those cancers that are rapidly progressive, and that would otherwise result in death, by chemoprevention and select for our studies those people at highest risk of developing them. Unfortunately, there have been a number of instances where these desirable principles were ignored.

13.4 How Will We Know If We Are Successful?

All research involves comparison, and nearly all therapeutic agents for cancer have been introduced after evaluation by the most rigorous method known, the controlled clinical trial or the randomized clinical trial. Although observational epidemiology studies have resulted in the recognition of potential chemopreventive 202j 13 Epidemiological Studies

agents, beta-carotene being a prominent example, it was appreciated early in the development of chemoprevention that the same rigorous methodology should be used for new therapeutic agents. The difficulty related to the long time span anticipated from the time the chemopreventive agent was first used to the time when a detectable benefit would occur. This time period was likely to be much longer than was required for the evaluation of a new treatment for cancer. It is hardly surprising that investigators sought shortcuts and began to consider the use of intermediate end points, namely, precancerous (premalignant) lesions. The difficulty with this approach as already implied is that far more precancerous lesions can be detected than are required to account for the numbers of invasive cancers destined to occur in the future in the people studied. Furthermore, we have no means at present to determine whether a specific lesion is destined to progress, and if not amenable to treatment kill, or whether it will progress slowly to become a readily treatable cancer or – and this generally applies to the majority of such lesions – fail to progress and eventually regresses. Thus, there is a possibility that if we use precancerous lesions as an intermediate end point in a chemoprevention trial, we shall obtain the wrong answer. This means that we need to follow participants in our chemoprevention trial at least until we have been able to demonstrate a reduction in clinically significant invasive cancers and also make sure that we are able to demonstrate a subsequent reduction in site-specific mortality. However, even that is not sufficient, no chemopreventive agent with efficacy against cancer will be completely free of toxicity. Sometimes toxicity from a new agent may be demonstrated in the occurrence of unexpected events, even deaths. Thus, in our chemoprevention trial we should monitor for adverse events and record all causes of mortality. Only if we can demonstrate in our trial that the agent results in the reduction in the incidence of clinically significant invasive cancer and site-specific mortality, without an un- acceptable occurrence of unexpected toxicity and no increase in all-cause mortality, should we consider recommending the use of that agent for chemoprevention in the population. In effect, there are the same rigorous requirements for designing clinical trials for cancer chemoprevention as there are for trials evaluating cancer screening. These include the people to be recruited, randomization after informed consent, administration of the test (in this case chemopreventive agent – if possible double- blind), and follow-up for defined end points [1]. Space constraint does not permit discussion of these in detail here; instead, I shall use various examples to illustrate the principles involved.

13.5 The Example of Beta-Carotene

The story of beta-carotene is perhaps the greatest indictment against chemoprevention that we are ever likely to see. Observational epidemiology studies of diet and cancer appeared to justify the hope that beta-carotene would be an effective nontoxic chemopreventive agent [2]. Unfortunately, this optimism proved to be unfounded, 13.5 The Example of Beta-Carotene j203 and the cloud beta-carotene has placed over chemoprevention has not been lifted and perhaps never will. Research on the association of diet and cancer began with animal experimental studies, and then in the 1970s, a few research groups began to conduct case–control and eventually cohort studies to investigate the associations further. In general, it was found that for many cancers, plant foods were protective, a conclusion re- enforced by an authoritative review over a decade ago [3] and re-affirmed 10 years later [4]. Among the foods that seemed to be protective were various fruits and vegetables, including carrots. An early attempt in the analyses of these studies was to try and identify specific nutrients or micronutrients that could explain the protective effect, using some sort of food data bank. Early studies suggested that vitamin A was one of these protective agents, and with it we began to consider provitamin A, or beta-carotene, responsible for the orange color of carrots and several other vegetables and fruits. Beta-carotene, in contradistinction to vitamin A, was at least in the levels consumed in foods nontoxic. It was a short jump to make the assumption that beta-carotene in high dosage would be even more protective against cancer than physiological levels, and probably even at high dosage nontoxic, though it was known that there was a ceiling beyond which beta-carotene in the body was not converted to vitamin A, that is, the enzyme system responsible for this conversion had become saturated. The result was that people tended to become yellow after consuming high doses of beta-carotene because of the excess unconverted beta-carotene in blood. Although that should have been a warning, it did not take long to decide that a dose of up to 30 mg/day was potentially effective as chemoprevention. One of the cancers that appeared to be diet-associated was lung cancer, in part from the early vitamin A studies, succeeded by studies that covered a wider spectrum of foods and micronutrients. It was clear that diet-associations for lung cancer were less important than associations with tobacco use, and that cessation of smoking was likely to be a far more effective public health approach to control lung cancer than any dietary change or supplement use. However, once it was understood that cessation of smoking in those who had smoked for many years, especially to beyond the age of 40, did not result in lung cancer risk returning to normal, but that the risk acquired stayed the same, a potential diet-derived chemopreventive agent could have a major effect in reducing risk in those who had already acquired a substantial risk. Fortunately, those who believed this was likely were attuned to the necessity to establish the value of chemoprevention in a randomized trial setting and recognized that occurrence of lung cancer had to be the definitive end point. This led to the initiation of two major trials, both supported by the US National Cancer Institute, the Beta-Carotene And Retinol Efficacy Trial (CARET) in the United States [5] and the Alpha-Tocopherol And Beta-Carotene Trial (ATBC) in Finland [6]. Both trials used two agents in combination, beta-carotene and retinol in CARET, and alpha-tocopherol and beta-carotene in ATBC, but the common denominator was beta-carotene. Both recruited subjects believed to be at high risk of lung cancer, heavy smokers, and in the case of the CARET trial, asbestos-exposed persons also. The CARET trial recruited a vanguard group of subjects, in whom more intensive investigations into potential toxicity were 204j 13 Epidemiological Studies

conducted than the main group of subjects recruited subsequently. No major toxicity occurred in this group. I have described the sequence of events leading to the CARET trial DSMB being unblinded and the trial stopped after we learnt the unexpected results of the ATBC trial, a significant 16% increase of lung cancer in the intervention group after 8 years of follow-up [7]. The important point to note is that both CARET and ATBC trials experienced the same major adverse event, a statistically significant increase of lung cancer in the intervention arm: in CARET, a 28% increase in the intervention arm about 4 years after treatment had been initiated. This rapid effect suggested that beta-carotene was accelerating the progression of lung cancers already at a fairly advanced state of their preclinical course. Although this should have resulted eventually in the reduction in incidence of lung cancer in the intervention arms of both trials, there has so far been no reduction [8]. A particularly unfortunate aspect of the excess of lung cancer found in the CARET trial was that it affected asbestos workers. It also particularly affected continuing smokers, but seemed not to have an effect on nonsmokers. The other unexpected effect, seen in both trials, was an increase in cardiovascular disease mortality, although the relative risk of cardiovascular disease mortality decreased to 1.0 in CARET soon after the intervention stopped [8]. What seems to have gone wrong was an adverse effect of the loose beta- carotene that could not be metabolized; nonetheless, some potential mechanisms have been identified. The example of beta-carotene re-enforces two important principles: first, one cannot rely on observational studies alone to initiate chemoprevention, especially when based, as in the case of beta-carotene, on estimating the intake by converting data from questionnaires on intake of foods into estimated intake of micronutrients through computer data banks. Rather, it is essential to conduct a randomized trial with adequate follow-up. Second, it is essential to use the right end point in chemoprevention trials; in this example, both of the trials determine the incidence of and mortality from lung cancer, and mortality from other causes also. There were several other trials that used beta-carotene for chemoprevention that had lung cancer as a primary end point or were able to evaluate lung and other cancers as end points. A recent review has confirmed that none of the phase III trials of chemoprevention of lung cancer with the agents beta-carotene, retinol, 13-cis-retinoic acid, alpha-tocopherol, N-acetylcysteine (or acetylsalicylic acid) demonstrate beneficial, reproducible results [9].

13.6 Folic Acid

The hope that beta-carotene might be the only potential chemopreventive agent to show unexpected effects was dashed recently, when a trial was reported the effects of folic acid on colorectal adenomas [10]. Although a large, multicenter trial, it was planned from the outset to have colorectal adenomas as the end point, believed to be on the path to invasive colorectal cancer, yet suffering from the same disadvantages of 13.7 Other Micronutrients j205 precursor lesions already discussed. Those randomized in the trial were selected from those already treated for a colorectal adenoma, as there is a high risk of recurrence of adenoma and invasive cancer with follow-up. The end point was identiﬁed by colonoscopy, initially at 2 years after randomization, and then after another 2 years. No difference in adenoma frequency was noted at 2 years between the folate treated group and the placebo controls, or after another 2 years. However, the frequency of advanced adenomas was higher in the folate group. Folic acid was associated with higher risks of having three or more adenomas and of noncolorectal cancers. The important feature of this trial was that the follow-up was sufﬁciently prolonged to enable the adverse effect to be determined, even though its strict relevance to the occurrence of invasive cancer must remain uncertain. However, the wisdom of using presumed precursor lesions as the end point has to be questioned.

13.7 Other Micronutrients

Randomized trials have shown that supplementation with selenium or vitamin E is associated with a reduction of prostate cancer risk. Meyer et al. [11] assessed whether a supplementation with low doses of antioxidant vitamins and minerals could reduce the occurrence of prostate cancer and influence biochemical markers. This was an important departure from most other chemoprevention trials, which tended to use the maximum tolerated dose of the chosen agent. Their trial included 5141 men randomized to take either a placebo or a supplementation with nutritional doses of vitamin C, vitamin E, beta-carotene, selenium, and zinc daily for 8 years. In addition, the investigators included in their design biochemical markers of prostate cancer risk, such as prostate-specific antigen (PSA) and insulin-like growth factors (IGFs), which were measured on plasma samples collected at enrollment and at the end of follow-up from 3616 men. During the follow-up, 103 cases of prostate cancer were diagnosed. Overall, there was a moderate nonsignificant reduction in prostate cancer rate associated with the supplementation (hazard ratio ¼ 0.88; 95% CI ¼ 0.60–1.29). However, the stratification by risk built into the design showed that the effect differed significantly between men with normal baseline PSA (<3 mg/l) and those with elevated PSA (p ¼ 0.009). Among men with normal PSA, there was a statistically significant reduction in the rate of prostate cancer for those receiving the supplements (hazard ratio ¼ 0.52; 95% CI ¼ 0.29–0.92). In men with elevated PSA at baseline, the supplementation was associated with an increased incidence of prostate cancer of borderline statistical significance (hazard ratio ¼ 1.54; 95% CI ¼ 0.87–2.72). The findings supported the hypothesis that chemoprevention of prostate cancer can be achieved with nutritional doses of antioxidant vitamins and minerals, but only in those who presumably did not have a detectable cancer at baseline, as exhibited by a normal PSA level. One difficulty with this trial, however, is that it is quite unclear whether the cancers prevented would have resulted in death if chemoprevention had not been used. Therefore, it is to be hoped that the investigators will continue to 206j 13 Epidemiological Studies

follow the participants and report a mortality end point. There is a parallel here with another chemoprevention trial: that of tamoxifen in women selected at high risk of breast cancer. In this trial, a statistically significant reduction in breast cancer incidence was found, and the trial stopped to permit the use of the intervention in the placebo controls, and although the incidence reduction was confirmed after 7 years of follow-up, there was no reduction in breast cancer mortality [12]. Even when a cancer is relatively frequent, large numbers of individuals may have to be studied to obtain sufficient numbers of end points in a trial. This was seen in a trial of primary liver cancer chemoprevention in China. The effects of supplementation with four different combinations of vitamins and minerals on primary liver cancer mortality among 29 450 initially healthy adults from Linxian, China, were examined [13]. The investigators used a partial factorial design to evaluate the effect of four different vitamin–mineral combinations or a placebo daily for 5.25 years. The study outcome was primary liver cancer death, a total of 151 liver cancer deaths occurred during the analysis period. No statistically significant differences in liver cancer mortality were found comparing the presence and absence of any of the four intervention factors.

13.8 Green Tea and Other Agents

Head and neck cancer if diagnosed late may be very difficult to treat without incurring severe disability. This led to a number of investigators evaluating the use of synthetic derivatives of vitamin A, the retinoids, with little success. In a review, it was pointed out that some newer agents are being evaluated; however, high costs and side effects make these agents less attractive than phytochemical-containing foods such as green tea, pomegranate juice, and other natural compounds, since they cost less, are nontoxic, and are widely available [14]. While small trials have shown promising results using these agents, larger trials have yet to be conducted to establish their chemopreventive effects. Once again, it is critical that these are carried out to evaluate the impact on invasive cancers, not premalignant lesions. Although green tea, soy, and tomato-rich products (lycopene) have been associated with protection against prostate cancer, to date trials have not been reported using these agents [15]. However, prostate cancer may be particularly difficult to study unless care is taken to avoid making decisions on PSA-detected cancers, many of which are likely to have been overdiagnosed, that is, would not have become detectable in the absence of PSA screening in an individuals lifetime. The large prostate cancer prevention trial in the United States found a reduction in the overall incidence of prostate cancer in the intervention arm using finasteride, an inhibitor of steroid 5a-reductase, the enzyme that converts testosterone to the more potent androgen dihydrotestosterone, but found an increase of higher grade cancers among men randomized to that arm, as well as sexual side effects [16]. To date mortality has not been reported, but clearly is expected with some anxiety. In this trial, annual PSA screening tests, and biopsies in those men whose test were abnormal, were References j207 performed. Yet PSA screening has so far not been shown to reduce prostate cancer mortality. So the design of this large NCI-supported trial was faulty, as it relied upon PSA-detected cancers to provide the end point, and the reduction in prostate cancer incidence in the intervention arm was due to cancers with limited clinical significance. There was no reduction, even an increase, in the type of cancers likely to result in death from prostate cancer. Any trial of prostate cancer chemoprevention using nutritional agents must avoid this methodologic trap, an unlikely eventuality if the trial is performed in the United Sates, as there seems to be no understanding that screening tests have disadvantages and need to show they benefit those tested.

13.9 Conclusions

There are very substantial methodologic issues over studies of potential chemopreventive agents. Observational epidemiology (and experimental studies) may point to a potentially useful agent, but it must be followed by a properly designed randomized trial, with clinically signiﬁcant invasive cancer and cancer mortality as the end point. Deﬁnitive conclusions are not possible if precursor (premalignant) lesions are used as the end point.

References

1 Miller, A.B. (2006) Design of cancer Effects of a combination of beta carotene screening trials/randomized trials for and vitamin A on lung cancer and evaluation of cancer screening. World cardiovascular disease. The New England Journal of Surgery, 30, 1152–1162. Journal of Medicine, 334, 1150–1155. 2 Peto, R., Doll, R., Buckley, J.D. and Sporn, 6 The Alpha-Tocopherol, Beta-Carotene M.B. (1981) Can dietary beta-carotene Cancer Prevention Study Group (1994) materially reduce human cancer rates? The effect of vitamin E and beta carotene Nature, 290, 201–209. on the incidence of lung cancer and other 3 World Cancer Research Fund (1997) Food, cancers in male smokers. The New England Nutrition and the Prevention of Cancer: A Journal of Medicine, 330, 1029–1035. Global Perspective, WCRF/American 7 Miller, A.B., Buring, J. and Williams, O.D. Institute of Cancer, Research Washington, (2005) Stopping the carotene and retinol DC. efﬁcacy trial: the viewpoint of the safety and 4 World Cancer Research Fund/American endpoint monitoring committee, in Data Institute of Cancer Research (2007) Food, Monitoring in Clinical Trials (eds D.L. Nutrition, Physical Activity and the DeMets, C.D. Furberg and L.M. Friedman), Prevention of Cancer: A Global Perspective, Springer, New York, pp. 220–227. American Institute of Cancer Research, 8 Goodman, G.E., Thornquist, M.D., Washington, DC. Balmes, J., Cullen, M.R., Meyskens, F.L., 5 Omenn, G.S., Goodman, G.E., Thornquist, Omenn, G.S., Valanis, B. and Williams, M.D., Balmes, J., Cullen, M.R., Glass, A., J.H. (2004) The beta-carotene and retinol Keogh, J.P., Meyskens, F.L. et al. (1996) efﬁcacy trial: Incidence of lung cancer and 208j 13 Epidemiological Studies

cardiovascular disease mortality during 6 Robidoux, A., Bevers, T.B., Kavanah, M. year follow-up after stopping beta carotene et al. (2005) Tamoxifen for the prevention and retinol supplements. Journal of the of breast cancer: report of the National National Cancer Institute, 96, 1743–1750. Surgical Adjuvant Breast and Bowel 9 Gray, J., Mao, J.T., Szabo, E., Kelley, M., Project P-1 study. Journal of the National Kurie, J. and Bepler, G. (2007) American Cancer Institute, 97, 1652–1662. College of Chest Physicians. Lung cancer 13 Qu, C.X., Kamangar, F., Fan, J.H., Yu, B., chemoprevention: ACCP evidence-based Sun, X.D., Taylor, P.R., Chen, B.E., Abnet, clinical practice guidelines, 2nd edn. Chest, C.C.,et al. (2007) Chemoprevention of 132 (3 Suppl), 56S–68S. primary liver cancer: a randomized, 10 Cole, B.F., Baron, J.A., Sandler, R.S., Haile, double-blind trial in Linxian China. Journal R.W., Ahnen, D.J., Bresalier, R.S., of the National Cancer Institute, 99, McKeown-Eyssen, G., Summers, R.W. 1240–1247. et al. (2007) Folic acid for the prevention of 14 Klass, C.M. and Shin, D.M. (2007) Current colorectal adenomas, a randomized status and future perspectives of clinical trial. Journal of the American chemoprevention in head and neck cancer. Medical Association, 297, 2351–2359. Current Cancer Drug Targets, 7, 623–632. 11 Meyer, F., Galan, P., Douville, P., Bairati, I., 15 Fleshner, N. and Zlotta, A.R. (2007) Kegle, P., Bertrais, S., Estaquio, C. and Prostate cancer prevention: past, present, Hercberg, S. (2005) Antioxidant vitamin and future. Cancer, 110, 1889–1899. and mineral supplementation and prostate 16 Thompson, I.M., Goodman, P.J., Tangen, cancer prevention in the SU.VI. MAX trial. C.M., Lucia, M.S., Miller, G.J., Ford, L.G., International Journal of Cancer, 116, Lieber, M.M., Cespedes, R.D. et al. (2003) 182–186. The inﬂuence of ﬁnasteride on the 12 Fisher, B., Costantino, J.P., Wickerman, development of prostate cancer. New D.L., Cecchini, R.S., Cronin, W.M., England Journal of Medicine, 349, 215–224. Part Two Experimental Models and Methods Used in Chemoprevention Studies

14 Methods Used for the Detection of Antimutagens: An Overview Armen Nersesyan, Miroslav Mišík, and Siegfried Knasmuller€

14.1 Introduction

According to the current theory, mutations in genes (e.g., oncogenes and tumor- suppressor genes) play a key role in the etiology of cancer [1]. These alterations are usually somatic events, although germ-line mutations can predispose a person to heritable or familial cancer. DNA damage is also considered to accelerate aging processes and to be causally involved in degenerative diseases including Alzheimers, Parkinsons, and Huntigtons, and also in multiple sclerosis [2]; furthermore, damage to the genetic material in germ cells leads to heritable diseases and affects fertility [1–3]. To protect humans against DNA damage induced by environmental and dietary factors and its consequences, a broad variety of genotoxicity tests have been developed that are used for routine screening of chemicals and to investigate the molecular mechanisms leading to DNA damage and repair [4]. Numerous DNA- reactive carcinogens have been identified in the human diet. Typical examples are aflatoxins, heterocyclic aromatic amines (HAAs), polycyclic aromatic hydrocarbons (PAHs), nitrosamines, certain mycotoxins, and heavy metals, to name a few [3]. Apart from external DNA damage caused by chemical and physical factors, the integrity of the genetic material may be also affected by endogenous damage caused by reactive oxygen species (ROS) due to oxidative stress and by errors caused during replication and repair processes [3]. The search for dietary antimutagens (AMs) is based on the assumption that prevention of exogenous and endogenous DNA damage will have beneficial health effects such as protection against cancer and increase of life span [5]. The first dietary constituents that were found to elicit DNA-protective effects were vitamins (A, C, and E) and plant and spice extracts [6]. The identification of protective food constituents was also strongly stimulated by epidemiological studies, which indicated that the diet plays a major role in the etiology of human cancer. According to the estimates of Doll and Peto [7, 8],

approximately 30–35% of all human cancers are related to nutritional factors and might be prevented by dietary strategies.

14.2 Mechanisms of DNA Protection

De Flora and Ramel [9, 10] published schematic overviews on different mechanisms of antimutagens and anticarcinogens (see also Chapter 4). Many of the currently known antimutagens act simultaneously at different levels, and this feature makes the assessment of the relevance of a specific mode of action difficult in humans [5, 11]. Most of the compounds on which research has focused during the last years are secondary plant constituents and have no direct nutritional value. Typical examples are pungent spice ingredients, such as ginger and capsaicin, sulfides in Allium species, glucosinolates in cruciferous vegetables, and pigments such as chlorophylls and anthocyans [11]. For mammals and man, these plant constituents are xenobiotics, and as such, they are eliminated via reactions mediated by phase I and phase II enzymes. Phase I reactions are catalyzed by cytochrome P-450 enzymes and lead to the formation of electrophiles that undergo conjugation reactions triggered by phase II enzymes such as UDP-glucoronosyl-transferase (UGT), glutathion-S-transferase (GST), N-acetyltransferase (NAT), and sulfotransferase (SULT) [5, 12]. The detoxification products are excreted via the bile or the urine. Many genotoxic carcinogens such as aflatoxins, polycyclic aromatic hydrocarbons, and HAAs are biologically inactive per se and require conversion to DNA-reactive intermediates by phase I reactions; in some cases (e.g., HAAs), phase II enzymes (e.g., SULT and NAT) are also involved in the activation reactions [13]. Plant constituents may either inhibit the formation of DNA-reactive metabolites or act via the induction of detoxifying (phase II) enzymes (DEs). The molecular mechanisms upregulating the activity of xenobiotic drug metabolizing enzymes have been investigated in a number of studies (see, e.g., Refs [5, 11, 14, 15]).

14.3 Methodological Aspects

14.3.1 End Points Used in Antigenotoxicity Studies

Numerous experimental procedures have been developed that enable the detection of DNA alterations. These approaches can be grouped into three major categories, namely, (1) trials that detect primary damage, (2) indirect methods that detect phenomena associated with DNA alterations, in particular processes related to DNA repair processes, and (3) experiments that detect mutations in a strict sense (Figure 14.1). 14.3 Methodological Aspects j213

Figure 14.1 Schematic overview of different types of genotoxicity tests.

In addition, chemical methods have been developed that provide indirect evidence for potential DNA damaging or protective activities; for example, antioxidant effects can be monitored by measuring the lipid peroxidation and by measuring the formation of oxidized macromolecules (see also Chapter 15). The DNA reactivity of compounds can further be measured in vitro in experiments with individual DNA bases or with isolated DNA; also, enzyme measurements may provide information on processes that are associated with DNA protection [16]. The major advantage of methods that detect primary DNA damage is that no cell divisions are required [4]. Therefore, such measurements can be carried out with nondividing or slowly dividing cells in culture or with primary cells isolated from various inner organs. UDS experiments measure unscheduled DNA synthesis caused by repair processes and are based on the determination of incorporation of 3H-labeled thymidine into DNA using autoradiography [4]. This method has been largely replaced by single-cell gel electrophoresis (SCGE) or comet assays that are increasingly used in in vitro and in vivo studies with rodents and also in human intervention trials [17]. In this approach, the formation of comets formed due to migration of DNA in an electric ﬁeldisusedasameasureofDNAdamage.The procedure allows the detection of single- and double-strand breaks, apurinic sites, and conformational alterations of the DNA. Furthermore, lesion-speciﬁcenzymes 214j 14 Methods Used for the Detection of Antimutagens: An Overview

of microbial origin (endonuclease III and formamidopyrimidine glycosylase) can be used to monitor the endogenous formation of oxidized bases [17]. More recently, protocols were developed that enable to study modifications of repair processes and alterations of the sensitivity toward dietary carcinogens and DNA repair [18]. At present, attempts are being made to validate and standardize this relatively new technique that is also increasingly used in antimutagenicity studies. For a description of the guidelines see Tice et al. [19]; a review describing recent developments in regard to dietary human intervention trials can be found in Ref. [20]. In gene mutation assays with bacteria (e.g., in the Salmonella/microsome assay), þ induction of amino acid auxotrophy (e.g., his ! his ) or resistance toward antibiotics is used as an end point, while in experiments with mammalian cells, resistance toward antimetabolites that interfere with the synthesis of DNA bases is used for the detection of gene mutations (e.g., TGr, HPRT). In higher organisms (plants, insects, and mice), gene mutations can be detected by scoring of specific morphological alterations in somatic cells (e.g., eye color or wing spots in Drosophila, fur spots in mice, and stamen hair mutations in the plant Tradescantia) [21]. Chromosomal aberrations (CAs), both structural and numerical, can be detected microscopically in metaphase cells. By using fluorescence in situ hybridization (FISH), individual chromosomes or chromosomal regions can be distinguished, and alterations such as translocations that are missed with conventional techniques can be detected. Micronucleus (MN) formation results either from chromosome breakage (clastogenicity) or aneuploidy, and scoring of these extranuclear bodies is by far less time- consuming than conventional metaphase analyses of chromosomes [22, 23]. By using pancentromeric probes, it is possible to draw conclusions if MN are formed as a consequence of chromosomal breakage (clastogenicity) or aneuploidy.

14.3.2 In Vitro Approaches

Since the structure of the genetic material is similar in all organisms, a broad variety of indicator cells and organisms including prokaryotes, plants, and insects can be used in mutagenicity tests. The most commonly employed method for the detection of antimutagens are bacterial test systems, in particular the Salmonella/microsome assay, which was developed by Bruce Ames (for a description of the method see Ref. [24]). Bacterial experiments are easy to perform, and standardized protocols are available, their major advantage is that high cell densities, required for the detection of gene mutations, can be reached after short cultivation periods. A number of dietary compounds protect against heterocyclic aromatic amines, which are formed from amino acids in meats during cooking and are considered to play a role in the etiology of human colon cancer. The evaluation of the database shows that more than 600 complex mixtures and individual compounds were tested for protective properties in the past 30 years and that 90% of the results come from in vitro tests with bacterial and/or mammalian cells [25]. Also, dietary compounds 14.3 Methodological Aspects j215 that protect against other classes of genotoxic dietary carcinogens (aflatoxins, polycyclic aromatic amines, and nitrosamines) were mainly tested in these systems. One of the major disadvantages of antimutagenicity tests with microorganisms and also of experiments with conventional mammalian cells, which are used in routine genotoxicitytestingsuchasV-79andCHOcells,isthattheindicatorcellsdonotpossess drug metabolizing enzymes [26]. Therefore, exogenous enzyme homogenates from rodent livers and cofactors (e.g., S9 homogenate) are added to mimic the activation of procarcinogens. As a consequence, these systems do not reflect the induction of detoxifying phase IIenzymes and it is also unclear whether or not inhibition of phase I enzymes takes place intracellularly [27]. Figure 14.2 shows the differences between metabolic pathways and mechanisms of chemoprevention reflected in conventional cells (lacking drug metabolizing enzymes) and in primary cells. When the results of bacterial antimutagenicity tests and in vivo experiments with rodents are compared, it becomes apparent that many compounds acting as antimutagens in vitro are ineffective or even cause synergistic effects in the living animal [25]. On the basis of this observation, it can be assumed that only simple mechanisms such as direct binding effects are detected, while effects that involve alterations of the metabolism of genotoxic carcinogens are not adequately detected in conventional in vitro assays. A promising alternative to the use of conventional cell lines and bacteria are stable cell lines such as rat (e.g., H35, H4IIE) or human hepatoma cell lines (e.g., Hep3B and HepG2) that have retained the activities of enzymes usually lost during cultivation [28]. In experiments with these cells, no addition of liver enzymes is required for the activation of promutagens. The majority of data comes from results of experiments with HepG2 cells is currently available. Commonly used end points are MN formation and comet formation. It was shown that these cells detect the genotoxic effects of a broad variety of dietary carcinogens, including compounds such as safrol and HEMPA that give negative results in conventional in vitro tests but are mutagenic and carcinogenic in animal experiments [28, 29]. Several attempts have been made to use primary cells in genotoxicity and antimutagenicity studies. For example, Anderson and coworkers [30] used peripheral human lymphocytesandspermcellsincometassaystostudyprotectiveeffectsofflavonoidsand other compounds. However, the metabolic capacity of these cells is very low and many genotoxic agents do not give positive results unless exogenous enzyme homogenate is added. Furthermore, several attempts have been made to use primary liver cells. For example, Eckl and Raffelsberger [31] developed a protocol for the cultivation of primary rat liver cells that enables the detection of CA and MN. However, this procedure is not standardized and validated, and a major problem is that the activities of the drug metabolizing enzymes decline rapidly after the isolation of the cells [31].

14.3.3 In Vitro Systems

In 1970s and 1980s, the most frequently used end point in antimutagenicity studies with rodents was the induction of MN in bone marrow polychromatic erythrocytes 216j 14 Methods Used for the Detection of Antimutagens: An Overview

Figure 14.2 (a) and (b) Representation of enzymes (to which activation mix is added). mechanisms leading to the prevention of DNA Promutagens are activated by enzymes forming damage in indicator cells lacking xenobiotic drug ultimate DNA-reactive metabolites that enter the metabolizing enzymes and in cells possessing cells and bind to DNA. Antimutagens can phase I and phase II enzymes (e.g., primary inactivate the promutagens by direct binding (1), hepatocytes or HepG2 cells). (a) Shows the by inhibition of the activated enzymes (2), or by situation in experiments with cells lacking detoxification of the ultimate metabolite (3). The 14.3 Methodological Aspects j217 and CA experiments with bone marrow and peripheral blood cells [4]. These methods are sensitive towards radiation and directly acting compounds such as alkylating agents, but they are quite insensitive towards important classes of dietary procarcinogens such as nitrosamines and HAAs, as reactive metabolites do not reach the target cells [32]. Another disadvantage is that these models do not provide information on effects on target organs of tumor formation. Another approach that was developed in the 1960s is the host-mediated assay (HMAs) in which indicator organisms (mainly bacteria) were injected intraperitoneally into rodents treated by various genotoxic agents. Subsequently, the cells are recovered after short exposure periods (12 h) and plated on selective media for the analysis of DNA damage [33]. The subsequent development of transgenic animals enabled measurements in multiple organs and replaced largely the HMAs. The method is based on the construction of rodent strains in which bacterial genes (e.g., lacI and lacZ) were integrated into the host genomes. Subsequently, the animals were treated with test chemicals, DNA extracted from different tissues, and the target sequences isolated and transfected to bacteria for mutational analyses [34]. However, this approach is quite expensive and has been used only in a few protection studies. At present, one of the most widely used techniques for antigenotoxicity studies is the SCGE assay [27]. Different methods have been developed that enable the measurement of DNA damage in a broad variety of organs. Initially, intact cells were isolated with speciﬁc techniques after chemical exposure of animals (genotoxic agent with and/or without putative protective compounds). After determination of the viability, which is a critical parameter to obtain reliable results [35], the cells are transferred to agarose-coated slides, lyzed, and scored for induction of comets. Subsequently, Sasaki et al. [36] developed a fast protocol, in which the organs are homogenized and the nuclei isolated for comet analysis. As endpoints, different parameters, such as tail lengths, percentage of DNA in tail and, tail movement, are used in SCGE experiments. Guidelines that are also relevant for protection studies have been published by Tice et al. [19], Hartmann et al. [35], and Burlinson [37]. It is worth noting that in most antimutagenicity studies, livers and colons were used as target organs [38]. Typical examples for in vitro protection studies with SCGE technique are experiments with lactobacilli and constituents of vegetables [39, 40].

3 latter process may take place extracellularly or inhibit activating enzymes that are located in the intracellulary (3). Another important mode of target cells (2); furthermore, the DNA-reactive action is the induction of repair enzymes (4). metabolites may be inactivated in the cells (3). (b) Shows the situation in cells possessing phase Other modes of action are the induction of repair I and phase II enzymes (without activation mix). enzymes (4) or the induction of detoxifying Also, in this case antimutagens can inactivate enzymes (5). This latter process is regulated by mutagens extracellulary or intracellulary (1), or transcription factors. 218j 14 Methods Used for the Detection of Antimutagens: An Overview

14.3.4 Human Biomonitoring Studies

For many years, the most widely used technique to monitor human exposure to genotoxic carcinogens were the chromosomal analyses (CA) and micronucleus assay (MN) with peripheral lymphocytes [41, 42]. This methods was also widely used in occupational exposure studies and in experiments concerning the effects of lifestyle factors, and a number of studies were conducted in which potential protective effects of vitamin supplementation were investigated [43, 44]. Karyotype analysis of metaphase cells is a time-consuming procedure, and a possible alternative to this method is the MN assay with peripheral lymphocytes [23]. Since MN formation requires nuclear division, use of cytochalasin B, which leads to the formation of binucleated cells, enables to ensure that the nuclei that are evaluated have undergone nuclear division. A typical example of a protection study was published by Fenech [43] who showed that folate, in combination with vitamin B6 and B12 supplementation, leads to a decrease of the MN frequency in the Australian population. Recently, additional end points (nucleoplasmic bridges, nuclear buds, apoptosis, and necrosis) were included in the protocol and provide additional relevant information [41]. Nucleoplasmic bridges are assumed to occur when the centromeres of dicentric chromosomes are pulled to the opposite poles at anaphase and provide a measurement of chromosomal rearrangements; nuclear buds are thought to form as a consequence of gene amplification events [41]. MN can also be monitored in exfoliated cells from the oral and nasal cavity, bladder, cervix, and esophagus [45]. It has been stressed that the majority of human cancers (>90%) are of epithelial origin and that this simple method might have an equally or higher predictive value than studies with blood cells [46]. It is worth noting that with these cells, unlike in lymphocytes, the staining method plays an important role. Recently, we studied the MN frequencies in heavy smokers and found significant differences in results; that is, with DNA-specificstainsonlyreal MN frequencies are scored while with nonspecific ones, artifacts are recorded as MN [47]. A number of chemoprevention trials have been conducted, most of them with mouth mucosa cells. Increased MN rates by smoking, snuff, or betel chewing declined after the intervention with vitamin E, b-carotene, or vitamin A. A review of the results of trials with exfoliated cells is given in the articles of Majer et al. [45] and Holland et al. [48]. At present, the most widely, used approach for the detection of DNA-protective compounds in the diet are SCGE experiments with peripheral lymphocytes. Moller and Loft emphasized that the most adequate study designs are placebo-controlled parallel trials as seasonal effects can be avoided; however, crossover and sequential trials have the advantage that interindividual variations can be excluded [49]. The currently available database has been summarized and analyzed in recent reviews [20, 49, 50]. At present, results from 82 studies (with vitamin supplements, fruits, vegetables, and beverages such as green tea, coffee, and juices) are available. It is important to note that the results of intervention studies depend strongly on the use of controlled diets and that the number of subjects involved, as well as seasonal 14.4 Limitations of the Predictive Value of Different Endpoints and Test Systems j219 variations, gender, lifestyle, and age, plays a crucial role [20]. It was stressed that the inclusion of washout periods will strengthen the scientific value of such studies [20, 49]. Additional information concerning protective properties of foods and/or food components can be obtained by using lesion-specific enzymes (endo III and FPG) that allow the study of endogenous formation of oxidized purines and pyrimidines [17]. Recently, protocols have also been developed that enable to monitor protection against DNA damage caused by genotoxic carcinogens contained in the human diet such as HAAs, acrylamide, furan, mycotoxins, and glycidamide [20]. Bacterial mutagenicity tests have also been used to prove that dietary components alter the excretion of genotoxins. About 40 studies have been published showing that the mutagenicity of urine (which can be detected after consumption of fried meat containing HAAs) in an amine-sensitive S. typhimurium tester strain (YG1024) is altered by the consumption of dietary factors such as Brassica vegetables, black tea, or lactobacilli [51]. Interpretation of the biological consequences of alterations of urinary mutagenicity is problematic, as a decrease of HAA-induced mutagenicity does not necessarily indicate a protective effect, since it cannot be excluded that it is due to enhanced formation of electrophilic metabolites that bind to DNA [51]. The prevention of oxidative DNA damage can be also monitored by analyzing 8-hydroxy-20-deoxy-guanosine (8-OHdG) in urine, and this methodology has been used in a number of intervention trials [52]. It is also possible to measure the levels of the oxidized base in peripheral lymphocytes and sperm cells. Recent interlaboratory comparisons showed that the results of such experiments depend strongly on the experimental protocols and that oxidation processes in the samples have a strong impact on the outcome. At present, efforts are being made to develop reliable and standardized protocols [52, 53]. Enzyme measurements, for example, GST and UGT, can be carried out with samples collected before and after dietary interventions and provide valuable information on protective effects toward specific chemical carcinogens that are detoxified via these enzymes. For example, it was shown that coffee and Brassicas increase the activity of GST in humans and in another study, it was demonstrated that UGTactivity, which plays a crucial role in the detoxification of HAAs, can be induced by these vegetables (for review see Ref. [20]. Also, antioxidant enzymes such as superoxide dismutase or gluthathione peroxidase may be induced by dietary components (for review see Ref. [16]).

14.4 Limitations of the Predictive Value of Different Endpoints and Test Systems

In vitro models with indicator cells lacking activating/detoxifying enzymes reﬂect only simple protective mechanisms (direct binding of genotoxins and their DNA- reactive metabolites) but not protective modes of actions that are due to alterations of the metabolism of DNA-reactive carcinogens. A promising alternative is the use of primary cells or cell lines, which retained the activity of xenobiotic drug metabolizing enzymes in an inducible form [28]. However, a further limiting factor of all in vitro 220j 14 Methods Used for the Detection of Antimutagens: An Overview

approaches is that they do not reflect the uptake of dietary putative antimutagens via the intestinal tract and that effects of the intestinal microflora are not represented. Many dietary supplements, for example, chlorophylls, anthocyan, or curcumin, which are highly effective in in vitro tests, are poorly absorbed in the gastrointestinal tract, and they are unlikely to cause beneficial effects in inner organs. Therefore, more relevant information can be expected from the results of experiments with laboratory rodents. The development of experimental models with human flora- associated animals enables to mimic the impact of the intestinal human microflora [54]. Also, the use of artificial digestion systems that simulate the human gastrointestinal tract with the human intestinal microbial ecosystem may provide valuable information [55, 56]. As mentioned above, the SCGE assay is at present one of the most widely used methods to assess prevention of DNA damage in humans and in animals [20]. Recently, we showed in model experiments that the reduction of DNA migration by HAAs in rats by cruciferous vegetables is paralleled by a decrease of the formation of preneoplastic lesions in the liver and the colon [27]. This indicates that SCGE assays enable to draw conclusions on potential cancer-protective effects [20]. However, this may be not necessarily true for studies in which the impact of dietary factors on endogenous or ROS-induced damage is monitored. It is known that SCGE assays are extremely sensitive to oxidative damage, but these lesions may be repaired and have no significant consequences in terms of health risks [17, 57]. Collins et al. [58] investigated the levels of oxidized guanine (8-OHdG) in different European populations and found no significant associations between specific forms of cancer and oxidized DNA base levels in lymphocytes, whereas a significant association was seen with the incidence of coronary heart disease. However, a large number of data indicate that ROS are causally involved in the induction of initiated cells as well as in tumor promotion [16]. Since it is possible to establish direct links between certain endpoints such as MN and CA induction, DNA migration, and human health risks [59, 60] such as cancer and coronary heart disease, positive results of antigenotoxicity studies can be taken as an indication of protective effects. However, such claims should be verified in further prospective human studies or in laboratory experiments, in which prevention of cancer and other diseases is used as a firm endpoint [61].

14.5 Specificity of Protection

In many older antimutagenicity studies, genotoxic agents were used to which humans are not exposed under normal conditions, and no attempts were made to rule out the molecular mechanisms that account for the protective effects. The results of such experiments are mainly of academic interest. In the last years, the number of protection studies with representatives of compounds involved in the etiology of human cancer, such as nitrosamines, PAHs, HAAs, aﬂatoxins, heavy 14.6 Dose–Effect Relationships j221 metals, and ROS, increased, and strong efforts were made to clarify the underlying mechanisms [20]. It should be kept in mind that the protection spectrum of dietary constituents depends strongly on their mode of action. For example, pigments that act via direct binding inactivate only molecules with planar structures such as HAAs and PAHs, whereas compounds that induce GSTs are likely to have a broader protection spectrum, since these enzymes inactivate a broad variety of DNA-reactive electrophiles [14]. Inducers of speciﬁc DNA repair enzymes (e.g., those involved in excision repair) might be protective against a broad variety of DNA damaging molecules, but it is unclear if and to which extent dietary compounds that induce repair processes in bacteria are effective in humans [62].

14.6 Dose–Effect Relationships

In many laboratory experiments, high doses of genotoxic carcinogens were used, which exceed by far the realistic exposure conditions in humans. The use of such experimental conditions is often necessary, since otherwise no measurable effects can be induced. This is, for example, true for CA, MN, and SCGE experiments with nitrosamines, PAHs, and HAAs. Only in DNA adduct measurements with PAHs, low exposure levels are feasible. Also, concentrations of putative antimutagens, used in protection studies, were in many experiments unrealistically high. For example, the amounts of lactobacilli or isothiocyanates given in certain animal experiments can be achieved only after consumption of 10–20 l of yogurt per person per day or after consumption of extremely large amounts of Brassica vegetables (for details see Ref. [63]). In addition, coffee diterpenoids doses were unrealistically high in many animal experiments [64, 65]. The promising results that were obtained with certain compounds lead to the production of dietary supplements that contain active ingredients in much higher amounts than those in natural foods. For example, certain green tea preparations contain high EGCG levels, and it was postulated recently that these high doses might cause oxidative damage per se [66]. A number of studies indicate that many mutagens act biphasic; that is, they are protective at low dose levels only but cause DNA damage at higher concentrations. Typical examples are spice ingredients, caffeine, tannic acid, ﬂavonoids, and selenium [5]. A possible explanation for this phenomenon might be that these antimutagens are DNA reactive by themselves and induce a sort of adaptive response, similar to the one seen with ionising radiation which is due to induction of DNA repair enzymes and detoxifying enzymes [5]. The dose–effect relations of DNA protection are largely unknown, as in most experiments only few dose levels were tested. It may be tentatively assumed that linear dose–effect relations exist for compounds that directly bind to DNA-reactive 222j 14 Methods Used for the Detection of Antimutagens: An Overview

molecules, whereas for protective mechanisms caused by enzyme inhibition or induction, threshold doses are likely to exist [67].

14.7 Future Trends

A retrospective look shows that the quality of antimutagenicity studies has improved over the years. For the majority of the scientific community, it has become clear that the initial assumption that any method that can be used for the detection of mutagens can be used also for the detection of antimutagenicity is a false paradigm. As a consequence, attempts have been made (which are still continued) to develop in vivo tests with improved indicator cells that reflect a broad variety of protective mechanisms. Another important task concerns the development of human monitoring methods that provide information, if specific phenomena, which can be related on the basis of animal tests to prevention of the consequences of DNA damage (such as cancer), can also be seen in humans. Improvements of biochemical and chemical analytical methods will enable the measurement of enzymes related to DNA damage and of metabolites of carcinogens with known biological effects in humans [53]. The combination of SCGE experiments with -OMICs techniques, which have been developed in the past decade, and also with conventional biochemical methods, will provide mechanistic explanations for DNA-protective effects [16, 20]. A typical example is the interpretation of the protective effects seen after Brussels sprouts consumption toward PhIP-induced DNA damage by inhibition of sulfotransferases that are involved in the activation of this heterocyclic aromatic amine in humans and the explanation of the reduction of DNA migration due to the formation of oxidized bases by these vegetables by induction of antioxidant enzymes that was seen in a recent proteomics study [68, 69]. Also, microarray analyses have recently been conducted with peripheral lymphocytes that are used as target cells in most human antimutagenesis experiments [16]. Furthermore, OMICs-based techniques can also be used to elucidate if disease-related alterations of gene expression and protein patterns are reversed by compounds that elicited protective effects in DNA-protective studies and help draw conclusions on their chemoprevention activities.

References

1 Croce, C.M. (2008) Oncogenes and cancer. 3 Ames, B.N. (1983) Dietary carcinogens The New England Journal of Medicine, 358, and anticarcinogens. Oxygen radicals and 502–511. degenerative diseases. Science, 221, 2 Pacher, P., Beckman, J.S. and Liaudet, L. 1256–1264. (2007) Nitric oxide and peroxynitrite in 4 Kramer, P.J. (1998) Genetic toxicology. The health and disease. Physiological Reviews, Journal of Pharmacy and Pharmacology, 50, 87, 315–424. 395–405. References j223

5 Weisburger, J.H. (1999) Antimutagens, 16 Knasmueller, S., Nersesyan, A., Misik, M., anticarcinogens, and effective worldwide Gerner, C., Mikulits, W., Ehrlich, V., cancer prevention. Journal of Hoelzl, C., Szakmary, A. and Wagner, K.H. Environmental Pathology, Toxicology and (2008) Use of conventional and -omics Oncology, 18,85–93. based methods for health claims of dietary 6 Gebhart, E. (1974) Antimutagens: data and antioxidants: a critical overview. The British problems. Humangenetik, 24,1–32. Journal of Nutrition, 99 (E Suppl. 1), 7 Doll, R. and Peto, R. (1981) The causes of ES3–ES52. cancer: quantitative estimates of avoidable 17 Collins, A.R. (2004) The comet assay for risks of cancer in the United States today. DNA damage and repair: principles, Journal of the National Cancer Institute, 66, applications, and limitations. Molecular 1191–1308. Biotechnology, 26, 249–261. 8 Doll, R. (1992) The lessons of life: keynote 18 Hoelzl, C., Knasmueller, S., Mišík, M., address to the nutrition and cancer Collins, A., Dušínska, M. and Nersesyan, conference. Cancer Research, 52, A. (2008) Use of single cell gel 2024s–2029s. electrophoresis assays for the detection of 9 De Flora, S. (1998) Mechanisms of DNA-protective effects of dietary factors in inhibitors of mutagenesis and humans: recent results and trends. carcinogenesis. Mutation Research, 402, Mutation Research [Epub ahead in print]. 151–158. 19 Tice, R.R., Agurell, E., Anderson, D., 10 De Flora, S. and Ramel, C. (1990) Burlinson, B., Hartmann, A., Kobayashi, Classiﬁcation of mechanisms of inhibitors H., Miyamae, Y., Rojas, E., Ryu, J.C. and of mutagenesis and carcinogenesis. Basic Sasaki, Y.F. (2000) Single cell gel/comet Life Sciences, 52, 461–462. assay: guidelines for in vitro and in vivo 11 Ferguson, L.R., Philpott, M. and genetic toxicology testing. Environmental Karunasinghe, N. (2004) Dietary cancer and Molecular Mutagenesis, 35, 206–221. and prevention using antimutagens. 20 Nersesyan, A., Hoelzl, C., Ferk, F., Misik, Toxicology, 198, 147–159. M. and Knasmueller, S. Use of single cell 12 Lampe, J.W. (2007) Diet, genetic gel electrophoresis assays in dietary polymorphisms, detoxiﬁcation, and health intervention trials, in The Comet Assay in risks. Alternative Therapies in Health and Toxicology (eds A. Dhawan and D. Medicine, 13, S108–S111. Anderson), Royal Society of Chemistry, 13 Nagao, M. and Sugimura, T. (2000) Food Cambridge, UK (in press). Borne Carcinogens: Heterocyclic Amines, 21 Kilbey, B.J. (1984) Handbook of John Wiley & Sons, Inc., New York. Mutagenicity Test Procedures, Elsevier 14 Wattenberg, L.W. (1997) An overview of Science, Amsterdam. chemoprevention: current status and 22 Frank, D.W., Trzos, R.J. and Good, P.I. future prospects. Proceedings of the Society (1978) A comparison of two methods for for Experimental Biology and Medicine 216, evaluating drug-induced chromosome 133–141. alterations. Mutation Research, 56, 15 Kensler, T.W. and Shin, S. (2009) Dietary 311–317. factors regulate metabolism of 23 Fenech, M. (2002) Chromosomal carcinogens through transcriptional biomarkers of genomic instability relevant signaling pathways in chemoprevention of to cancer. Drug Discovery Today, 7, cancer and DNA damage by dietary 1128–1137. factors (eds S. Knasmueller, D.M. 24 Maron, D.M. and Ames, B.N. (1983) DeMarini, J.T. Johnson and C. Gerh€auser), Revised methods for the Salmonella Wiley-VCH Verlag GmbH & Co. KGaA, mutagenicity test. Mutation Research, 113, Weinheim, 109–120. 173–215. 224j 14 Methods Used for the Detection of Antimutagens: An Overview

25 Schwab, C.E., Huber, W.W., Parzefall, W., study of MeIQx in mice. Food and Chemical Hietsch, G., Kassie, F., Schulte-Hermann, Toxicology 34, 717–724. R. and Knasmueller, S. (2000) Search for 33 Knasmueller, S., Kerklaan, P. and Mohn, compounds that inhibit the genotoxic and G.R. (1986) Use of the DNA-repair host- carcinogenic effects of heterocyclic mediated assay for determining the organ aromatic amines. Critical Reviews in distribution of genotoxic factors in mice Toxicology, 30,1–69. treated orally with nitro-aromatic 26 Hewitt, N.J., de Kanter, R. and LeCluyse, compounds. Mutation Research, 164,9–17. E. (2007) Induction of drug metabolizing 34 Thybaud, V., Dean, S., Nohmi, T., de Boer, enzymes: a survey of in vitro J., Douglas, G.R., Glickman, B.W., methodologies and interpretations used Gorelick, N.J., Heddle, J.A., Heﬂich, R.H., in the pharmaceutical industry – do they Lambert, I., Martus, H.J., Mirsalis, J.C., comply with FDA recommendations? Suzuki, T. and Yajima, N. (2003) In vivo Chemico-Biological Interactions, 168, transgenic mutation assays. Mutation 51–65. Research, 540, 141–151. 27 Knasmueller, S., Steinkellner, H., Majer, 35 Hartmann, A., Agurell, E., Beevers, C., B.J., Nobis, E.C., Scharf, G. and Kassie, F. Brendler-Schwaab, S., Burlinson, B., Clay, (2002) Search for dietary antimutagens P., Collins, A., Smith, A., Speit, G., and anticarcinogens: methodological Thybaud, V. and Tice, R.R. (2003) aspects and extrapolation problems. Food Recommendations for conducting the and Chemical Toxicology 40, 1051–1062. in vivo alkaline Comet assay. 4th 28 Knasmueller, S., Parzefall, W., Sanyal, R., International Comet Assay Workshop. Ecker, S., Schwab, C., Uhl, M., Mersch- Mutagenesis, 18,45–51. Sundermann, V., Williamson, G., Hietsch, 36 Sasaki, Y.F., Tsuda, S., Izumiyama, F. and G., Langer, T., Darroudi, F. and Natarajan, Nishidate, E. (1997) Detection of A.T.(1998) Use of metabolically competent chemically induced DNA lesions in human hepatoma cells for the detection of multiple mouse organs (liver, lung, spleen, mutagens and antimutagens. Mutation kidney, and bone marrow) using the Research, 402, 185–202. alkaline single cell gel electrophoresis 29 Knasmueller, S., Mersch-Sundermann, V., (Comet) assay. Mutation Research, 388, Kevekordes, S., Darroudi, F., Huber, W.W., 33–44. Hoelzl, C., Bichler, J. and Majer, B.J. (2004) 37 Burlinson, B., Tice, R.R., Speit, G., Agurell, Use of human-derived liver cell lines for E., Brendler-Schwaab, S.Y., Collins, A.R., the detection of environmental and dietary Escobar, P., Honma, M., Kumaravel, T.S., genotoxicants: current state of knowledge. Nakajima, M., Sasaki, Y.F., Thybaud, V., Toxicology, 198, 315–328. Uno, Y., Vasquez, M. and Hartmann, A. 30 Anderson, D., Dobrzynska, M.M., (2007) Fourth International Workgroup on Basaran, N., Basaran, A. and Yu, T.W. Genotoxicity Testing: results of the in vivo (1998) Flavonoids modulate comet assay Comet assay workgroup. Mutation responses to food mutagens in human Research, 627,31–35. lymphocytes and sperm. Mutation 38 Singh, V., Belloir, C., Siess, M.H. and Le Research, 402, 269–277. Bon, A.M. (2006) Inhibition of carcinogen- 31 Eckl, P.M. and Raffelsberger, I. (1997) The induced DNA damage in rat liver and primary rat hepatocyte micronucleus colon by garlic powders with varying assay: general features. Mutation Research, alliin content. Nutrition and Cancer, 55, 392, 117–124. 178–184. 32 Breneman, J.W.,Briner, J.F., Ramsey, M.J., 39 Laky, B., Knasmueller, S., Gminski, R., Director, A. and Tucker, J.D. (1996) Mersch-Sundermann, V., Scharf, G., Cytogenetic results from a chronic feeding Verkerk, R., Freywald, C., Uhl, M. and References j225

Kassie, F. (2002) Protective effects of 47 Nersesyan, A., Kundi, M., Atefie, K., Brussels sprouts towards B[a]P-induced Schulte-Hermann, R. and Knasmuller, S. DNA damage: a model study with the (2006) Effect of staining procedures on the single-cell gel electrophoresis (SCGE)/ results of micronucleus assays with Hep G2 assay. Food and Chemical exfoliated oral mucosa cells. Cancer Toxicology, 40, 1077–1083. Epidemiology, Biomarkers & Prevention, 15, 40 Koller, V.J., Marian, B., Stidl, R., Nersesyan, 1835–1840. A., Winter, H., Simic, T., Sontag, G. and 48 Holland, N., Bolognesi, C., Kirsch-Volders, Knasmueller, S. (2008) Impact of lactic acid M., Bonassi, S., Zeiger, E., Knasmueller, S. bacteria on oxidative DNA damage in and Fenech, M. (2008) The micronucleus human derived colon cells. Food and assay in human buccal cells as a tool for Chemical Toxicology, 46,1221–1229. biomonitoring DNA damage: the HUMN 41 Fenech, M., Chang, W.P., Kirsch-Volders, project perspective on current status and M., Holland, N., Bonassi, S. and Zeiger, E. knowledge gaps. Mutation Research 659, (2003) HUMN project: detailed 93–108. description of the scoring criteria 49 Moller, P. and Loft, S. (2006) Dietary for the cytokinesis-block micronucleus antioxidants and beneficial effect on assay using isolated human oxidatively damaged DNA. Free Radical lymphocyte cultures. Mutation Research, Biology & Medicine, 41, 388–415. 534,65–75. 50 Hoelzl, C., Knasmueller, S., Mišík, M., 42 Natarajan, A.T. (2002) Chromosome Collins, A., Dušínska, M. and Nersesyan, aberrations: past, present and future. A. (2008) Use of single cell gel Mutation Research, 504,3–16. electrophoresis assays for the detection of 43 Fenech, M. (2002) Micronutrients and DNA-protective effects of dietary factors in genomic stability: a new paradigm for humans: recent results and trends. recommended dietary allowances (RDAs). Mutation Research (in press). Food and Chemical Toxicology, 40, 51 Steinkellner, H., Rabot, S., Freywald, C., 1113–1117. Nobis, E., Scharf, G., Chabicovsky, M., 44 Fenech, M., Baghurst, P., Luderer, W., Knasmueller, S. and Kassie, F. (2001) Turner, J., Record, S., Ceppi, M. and Effects of cruciferous vegetables and their Bonassi, S. (2005) Low intake of calcium, constituents on drug metabolizing folate, nicotinic acid, vitamin E, retinol, enzymes involved in the bioactivation of beta-carotene and high intake of DNA-reactive dietary carcinogens. pantothenic acid, biotin and riboflavin are Mutation Research, 480–481, 285–297. significantly associated with increased 52 Gedik, C.M. and Collins, A. (2005) genome instability – results from a dietary Establishing the background level of base intake and micronucleus index survey in oxidation in human lymphocyte DNA: South Australia. Carcinogenesis, 26, results of an interlaboratory validation 991–999. study. The FASEB Journal, 19,82–84. 45 Majer, B.J., Laky, B., Knasmueller, S. and 53 Knasmueller, S., Nersesyan, A., Mišík, M., Kassie, F. (2001) Use of the micronucleus Gerner, C., Mikulits, W., Ehrlich, V., assay with exfoliated epithelial cells as Hoelzl, C., Szakmary, A. and Wagner, K.- a biomarker for monitoring individuals H. Use of conventional and -OMICS based at elevated risk of genetic damage methods for health claims of dietary and in chemoprevention trials. antioxidants: a critical overview. The British Mutation Research, 489, 147–172. Journal of Nutrition, 99E Suppl 1:ES3–52. 46 Cairns,J.(1975)Mutationselectionandthe 54 Horie, H., Zeisig, M., Hirayama, K., natural history of cancer. Nature, 255, Midtvedt, T., Moller, L. and Rafter, J. (2003) 197–200. Probiotic mixture decreases DNA adduct 226j 14 Methods Used for the Detection of Antimutagens: An Overview

formation in colonic epithelium induced Zijno, A., Norppa, H. and Fenech, M. by the food mutagen 2-amino-9H-pyrido (2007) An increased micronucleus [2,3-b]indole in a human-flora associated frequency in peripheral blood lymphocytes mouse model. European Journal of Cancer predicts the risk of cancer in humans. Prevention, 12, 101–107. Carcinogenesis, 28, 625–631. 55 Blanquet, S., Zeijdner, E., Beyssac, E., 61 Soobrattee, M.A., Bahorun, T. and Meunier, J.P., Denis, S., Havenaar, R. and Aruoma, O.I. (2006) Chemopreventive Alric, M. (2004) A dynamic artificial actions of polyphenolic compounds in gastrointestinal system for studying the cancer. Biofactors, 27,19–35. behavior of orally administered drug 62 Ohta, T., Sutton, M.D., Guzzo, A., Cole, S., dosage forms under various physiological Ferentz, A.E. and Walker, G.C. (1999) conditions. Pharmaceutical Research, 21, Mutations affecting the ability of 585–591. theEscherichia coli UmuD0 protein to 56 Molly, K., Vande Woestyne, M. and participate in SOS mutagenesis. Journal of Verstraete, W. (1993) Development of a Bacteriology, 181, 177–185. 5-step multi-chamber reactor as a 63 Uhl, M., Kassie, F., Rabot, S., Grasl- simulation of the human intestinal Kraupp, B., Chakraborty, A., Laky, B., microbial ecosystem. Applied Microbiology Kundi, M. and Knasmuller, S. (2004) Effect and Biotechnology, 39, 254–258. of common Brassica vegetables (Brussels 57 Collins, A.R., Dusinska, M., Gedik, C.M. sprouts and red cabbage) on the and Stetina, R. (1996) Oxidative damage to development of preneoplastic lesions DNA: do we have a reliable biomarker? induced by 2-amino-3-methylimidazo Environmental Health Perspectives, 104 [4,5-f]quinoline (IQ) in liver and colon of (Suppl. 3), 465–469. Fischer 344 rats. Journal of Chromatography 58 Collins, A.R., Gedik, C.M., Olmedilla, B., B, 802, 225–230. Southon, S. and Bellizzi, M. (1998) 64 Huber, W.W., Scharf, G., Nagel, G., Oxidative DNA damage measured in Prustomersky, S., Schulte-Hermann, R. human lymphocytes: large differences and Kaina, B. (2003) Coffee and its between sexes and between countries, chemopreventive components kahweol and correlations with heart disease and cafestol increase the activity of O6- mortality rates. The FASEB Journal, 12, methylguanine-DNA methyltransferase in 1397–1400. rat liver: comparison with phase II 59 Bonassi, S., Hagmar, L., Stromberg, U., xenobiotic metabolism. Mutation Research, Montagud, A.H., Tinnerberg, H., Forni, 522,57–68. A., Heikkila, P., Wanders, S., Wilhardt, P., 65 Huber, W.W., Teitel, C.H., Coles, B.F., Hansteen, I.L., Knudsen, L.E. and Norppa, King, R.S., Wiese, F.W., Kaderlik, K.R., H. (2000) Chromosomal aberrations in Casciano, D.A., Shaddock, J.G., Mulder, lymphocytes predict human cancer G.J., Ilett, K.F. and Kadlubar, F.F. (2004) independently of exposure to carcinogens. Potential chemoprotective effects of the European Study Group on Cytogenetic coffee components kahweol and cafestol Biomarkers and Health. Cancer Research, palmitates via modification of hepatic 60, 1619–1625. N-acetyltransferase and glutathione 60 Bonassi, S., Znaor, A., Ceppi, M., Lando, S-transferase activities. Environmental and C., Chang, W.P., Holland, N., Kirsch- Molecular Mutagenesis, 44, 265–276. Volders, M., Zeiger, E., Ban, S., Barale, R., 66 Elbling, L., Weiss, R.M., Teufelhofer, O., Bigatti, M.P., Bolognesi, C., Cebulska- Uhl, M., Knasmueller, S., Schulte- Wasilewska, A., Fabianova, E., Fucic, A., Hermann, R., Berger, W.and Micksche, M. Hagmar, L., Joksic, G., Martelli, A., (2005) Green tea extract and Migliore, L., Mirkova, E., Scarfi, M.R., ()-epigallocatechin-3-gallate, the major References j227

tea catechin, exert oxidant but lack 1-methyl-6-phenylimidazo[4,5-b] antioxidant activities. FASEB, 19, 807–809. pyridine (PhIP) and oxidative 67 Verschoyle, R.D., Steward, W.P. and DNA-damage: results of a Gescher, A.J. (2007) Putative cancer controlled human intervention trial. chemopreventive agents of dietary origin: Molecular Nutrition and Food Research, 52, how safe are they? Nutrition and Cancer, 59, 330–341. 152–162. 69 Hoelzl, C., Lorenz, O., Haudek, V., 68 Hoelzl, C., Glatt, H., Meinl, W., Sontag, G., Gundacker, N., Knasmuller,€ S. and Haidinger, G., Kundi, M., Simic, T., Gerner, C. (2008) Proteome alterations Chakraborty, A., Bichler, J., Ferk, F., induced in human white blood cells by Angelis, K., Nersesyan, A. and consumption of Brussels sprouts: Knasmuller, S. (2008) Consumption results of a pilot intervention study. of Brussels sprouts protects peripheral Proteomics: Clinical Applications, 2, human lymphocytes against 2-amino- 108–117. j229

15 Methods to Determine Total Antioxidative Capacity and Oxidative DNA Damage Karl-Heinz Wagner, Miroslav Mišík, Armen Nersesyan, and Siegfried Knasmuller€

15.1 Introduction

Research on antioxidants has increased considerably during the past decades. One important issue in this respect is the determination of the real potential of a substance. The number of methods and end points to measure antioxidants increased in the same way (Figure 15.1). In this chapter, most of the commonly used methods to monitor the effects of antioxidants (antioxidative capacity, antioxidative potential) are described, thereby outlining the reaction mechanisms and major advantages and disadvantages of each method.

15.2 Methods

Methods used to measure oxidative stress can generally be divided into ﬁve main categories, namely, (1) physics-based approaches, (2) methods used for the determination of the antioxidant compounds (AOCs), (3) biochemical methods used to monitor the oxidation of macromolecules and their oxidation products, (4) approaches to the detection of ROS-induced DNA damage, and (5) methods used to measure antioxidant enzymes and transcriptional factors. This chapter concerns mainly the ﬁrst four, approaches that fall into the last category are described in other chapters of this book.

15.2.1 Trapping of Reactive Species

The only technique that can reveal free radicals directly and specify them is electron spin resonance (ESR), because it detects the presence of unpaired electrons.

Figure 15.1 Overview of different methodologies to determine oxidative stress.

However, ESR can be used to monitor only the fairly unreactive radicals, since reactive ones do not accumulate at levels high enough. One solution of this problem is to add traps or probes, which are agents that intercept reactive radicals reacting with them to form a stable radical that can be detected by ESR. Whole-body ESR techniques are being used with rodents. A wide range of traps are available for use in animals and cell culture systems such as N-tertbutyl-p-phenylnitrone, 5,5-dimethyl- 1-pyrroline N-oxide, 1,1,3-trimethyl-isoindole N-oxide, and 5,5-diethylcarbonyl- 1-pyrroline N-oxide [1–3]. A generally underestimated problem is that the reaction products giving an ESR signal can be rapidly removed in vivo and in cultured cells, both by enzymatic metabolism and by direct reduction by agents such as ascorbate [4].

15.2.2 Approaches to Determine the Total Antioxidant Capacity

Two main approaches have been developed to evaluate the antioxidant capacity in foods and human material. The ﬁrst measures the ability of a substance to transfer one electron to reduce compounds such as radicals, carbonyls, and metals. The most popular tests belonging to this category are the ferric iron reducing antioxidant parameter (FRAP), the Trolox equivalent antioxidant capacity (TEAC), and the diphenyl-1-picrylhydrazyl (DPPH) tests. Methods that fall into the second category are based on their ability to quench free radicals by hydrogen donation. The most 15.2 Methods j231 popular approaches at the moment are the oxygen radical absorbance capacity (ORAC) test, the total radical trapping antioxidant parameter (TRAP), the total oxidant scavenging capacity (TOSC) method (all measuring the effects in the hydrophilic compartment of the plasma), and the inhibition of linoleic acid and low-density lipoprotein (LDL) oxidation (Table 15.1).

15.2.3 Free Radical Quenching Methods

The ORAC assay measures the antioxidant inhibition of ROO-induced oxidations [5–7]. Therefore, it reflects the classical H donating ability of antioxidants in the hydrophilic compartment. The peroxyl radical reacts with a fluorescent probe, thereby forming a nonfluorescent product that can be quantitated by following the fluorescence over time. In earlier studies, b-phycoerythrin was used as the fluorescent agent emitting in the visible region (Exc 495 nm, Em 595 nm), but due to shortcomings and inconsistencies of the results, fluorescein or dichlorofluorescein are currently used, since they are less reactive and more stable. The antioxidative activity can be expressed as the lag time or the net integrated area under the fluorescence curve (AUC). ORAC values are reported as Trolox equivalents. Originally, the ORAC assay was limited to the measurement of hydrophilic chain breaking antioxidant capacity. Now a newer protocol is available, in which lipophilic and hydrophilic compounds are selectively separated by extraction, now also allows the quantification of lipophilic antioxidants using a mixture of acetone and water [8]. The advantage of the ORAC assay is that it can be automated. Convincing results have been obtained with 48- or 96-well plates coupled with a microplate reader. One important parameter is the temperature control (37 C), as small temperature differences decrease the reproducibility of the test. A principal drawback of the method is that the effect of oxidation of the photoreceptor of protein used does not necessarily reflect protection against oxidative damage of the protein itself. The TRAP assay is based on the use of 2,20-azobis(2,4-amidinopropane)dihydrochloride (AAPH), a hydrophilic azo-compound that generates peroxyl radicals [9]. AAPH decomposes at 37 C spontaneously with a known rate. Various substances such as 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), R-phycoerythrin, or 20-70-dichlorofluorescin have been used as oxidizable agents. The basic reactions of the procedure are similar to those of the ORAC assay. The . probe reacts with ROO radicals at low concentrations with a significant spectroscopic change in between the native and the oxidized sample, and no radical chain reaction beyond sample oxidation should occur. The antioxidant capacity is determined as the time required to consume all antioxidants by extension of the lag time for appearance of the oxidized probe when antioxidants are present. TRAP values are usually expressed as the lag time of the sample compared to the corresponding times for Trolox. The test is relatively complex to perform, requires experience, and is time-consuming. The use of the lag phase is based on the assumption that all antioxidants show a lag phase and that the lag phase corresponds to the antioxidative capacity. 232 j 5Mtost eemn oa nixdtv aaiyadOiaieDADamage DNA Oxidative and Capacity Antioxidative Total Determine to Methods 15

Table 15.1 Comparison of methods used to determine the total antioxidant capacity (TAC).

Test systems Principle End point Simplicity/throughput Biological relevance

The ORAC test H transfer Lag time area under the þþ/þþþ þþþ ﬂuorescence curve The TRAP H transfer Lag time / þþþ The total oxidant scavenging capacity, H transfer AUC DT50 / þþ CUPRAC CL/PCL Not known Lag time area under the þ/ þþ ﬂuorescence curve LDL oxidation H transfer Lag time Ultracentrifugation: / þþþ(in vitro) ELISA: þþ/þþ The Trolox equivalent Electron transfer D Optical density þþ/þþ antioxidant capacity The ferric iron reducing Electron transfer D Optical density þþþ/þþþ antioxidant parameter The CUPRAC Electron transfer D Optical density þþþ/þþþ The diphenyl-1-picrylhydrazyl test Electron transfer D Optical density þ/þ

þ , þþ, þþþ: positive; , , : negative. 15.2 Methods j233

One drawback of TRAP and ORAC assays is the interference of proteins that contribute up to 80% to the total antioxidant capacity [10]. Therefore, Trolox should be used as an internal standard and the samples must be either deproteinized or diluted prior to measurements. The TOSC assay was initially used for environment-related studies on marine organisms [11]. It is based on the inhibition of the radical-dependent formation of ethylene from ketomethiolbutyric acid by antioxidants. This procedure permits testing against peroxyl and hydroxyl radicals, as well as peroxynitrite. It can be conducted at physiological temperature and pH; nonlinear concentration-dependent activity variations can be examined easily and different types of antioxidant reactions (retardant or fast acting) can be distinguished. However, high-throughput analyses are not possible and multiple injections of each sample are required to observe ethylene formation. Further limitations are the multiple end points of calculated 20, fi 50, and 80% TOSC and the DT50 ( rst TOSC derivative of 50%). The chemiluminescence (CL) assay is a modification of the TRAP assay. Radical formation is followed by CL or photo-CL (PCL). CL is characterized by low emission intensity and by the fact that reactions with oxidants emit CL. The most widely used marker is luminol, but bioluminescent proteins such as pholasin are also becoming popular. The antioxidant capacity is the time of depressed light emission, which is measured at 10% recovery of light output. Recently, PCL was described, which is a commercial test system termed PHOTOCHEM for the determination of the integral antioxidative capacity towards . O2 [12]. In a strict sense, the method measures antiradical capacity. In contrast to many other assays used to determine AOC, this procedure requires no standardization of the pH and the temperature. However, to date, the system is marketed by only one company (Analytic Jena, Germany) and reagents for the hydrophilic and lipophilic assays are available from the manufacturer only; furthermore, a high throughput is not possible. Ascorbic acid is normally used for the determination of hydrophilic and Trolox for the lipophilic antioxidative capacity. Low-density lipoprotein oxidation is based on the autoxidation of linoleic acid or þ LDL that is induced in vitro mainly by Cu2 or some other azo-initiators [13]. LDL oxidation is of higher physiological relevance when tested under in vivo conditions and not ex vivo. The oxidation is monitored at 234 nm for conjugated dienes (CD) or by peroxide values for lipid hydroperoxides. LDL has to be freshly isolated from blood, which is a time- and material-consuming procedure and requires ultracentrifugation. During the preparation, low temperature and light protection are essential [14]. Furthermore, conjugated dienes can be formed in presence of polyunsaturated fatty acids. Recently, fluorescence and UV-based enzyme-linked immunosorbent assay (ELISA) assays with plasma became available, for which no complicated and time- consuming LDL isolation is needed. These methods can also be used for larger human trials. The AOC is determined in all these experiments either as AUC or as lag time until the antioxidants are consumed. An important modification was developed by Frankel and coworkers, who determined the secondary oxidation product hexanal from LDL [15]. Hexanal was chosen since it is the major oxidation product of n-6 fatty acids and is monitored with headspace gas chromatography (GC), the percentage inhibition 234j 15 Methods to Determine Total Antioxidative Capacity and Oxidative DNA Damage

of hexanal formation is used as a parameter for AOC. In many ex vivo studies, LDL was isolated, and subsequently, the substances were added and tested on their ability to delay oxidation. This scenario does not reﬂect in vivo conditions. Furthermore, not all oxidation inducers that are used ex vivo can be used for in vivo testing [16]. The crocin bleaching assay monitors the protection of AAPH-induced crocin bleaching by antioxidants [17]. Crocin is a mix of natural pigments and absorbs, similar to carotenoids, at 450 nm. Therefore, the interpretation of results can be complicated in experiments with food samples. Initially, the test was used for the analysis of plasma samples. One of its limitations is that crocin is not commercially available, but high-throughput with microplates is possible.

15.2.4 Single-Electron Transfer Methods

In these assays, the sample itself is an oxidant that abstracts an electron from the antioxidant, thereby causing color changes that are proportional to the AOC. When the change of absorbance is plotted against the antioxidant concentration, the slope of the curve reﬂects the total reducing capacity. In contrast to the methods described in the last chapter, no oxygen radical is present in the system; therefore, it can be assumed that the reducing capacity is equal to the antioxidant capacity. . The TEAC assay is a spectrophotometric test [18]. ABTS is oxidized by ROO to a green-blue radical cation. The ability of antioxidants to delay color formation is ex-

pressed relative to Trolox. Originally, the test used metmyoglobin and H2O2, and the ABTS radical was measured at 734 nm; meanwhile, various modifications have been developed. After generation of the ABTS radical, the sample to be tested is added, and subsequently, other chemicals such as manganese dioxide, ABAP, potassium persul- fate, or enzymes are used to generate the ABTS radical [1]. Temperatures higher than 37 C, which are not physiological, and different absorption maxima (415, 645, 734, or 815 nm) are frequently used. Depending on the protocol, the decrease or increase in ABTS radical absorbance in presence of the test sample or Trolox at a fixed time point is measured and the antioxidant capacity is calculated as Trolox equivalents. ABTS is not a physiological substance. It reacts fast with aqueous and organic solvents and substances with a low redox potential show a good response. Therefore, phenolic compounds or ascorbic acid react quite well with ABTS, whereas lipophilic compounds respond more weakly. The test is not restricted to a narrow pH range, but high hemoglobin concentration in the plasma may interfere with the measurements. The FRAP assay determines the reduction of 2,4,6-tripyridyl-s-triazine (TPTZ) in plasma to a colored product [19] and has also been adapted for food samples. Similar to the TEAC assay, compounds with a redox potential <0.7 Vare detected. FRAP is not able to monitor compounds that quench radicals such as proteins or thiol compounds such as glutathione (similar to all the reducing assays) and can therefore lead to an underestimation of the AOC. To maintain Fe solubility, the assay is conducted at acidic conditions (pH 3.6). Most redox reactions take place within a few minutes; therefore, both tests consider most of the substance effects (such as polyphenols) but not all, since some substances have longer reaction times [20]. This was recently 15.3 Oxidation of Macromolecules j235 shown for polyphenols such as caffeic, tannic, ferulic, or ascorbic acids, where the absorption steadily increased for hours [21]. Since the test reflects only the reducing potential and does not consider hydrogen transfer, it should be conducted only in combination with other methods to give a more complete picture. Similar to TEAC, it is a relatively easy test procedure, can be done manually or fully automated, and requires no expensive equipment. The copper reducing assay (CUPRAC, AOP-90) is a modification of the FRAP in þ þ which iron is replaced by Cu; Cu2 is reduced to Cu [22]. The assay is conducted at 490 nm with bathocuprine, or at 450 nm with neocuprine. Results are expressed as uric acid equivalents. Since copper has a lower redox potential than iron, but enhances the redox cycling potential, its pro-oxidative potential can be shown more sensitive [20]. Limitations regarding the underestimation of slower reactive molecules are similar to those of the other assays, but one of it advantages is that almost every antioxidant including thiols can be detected. The 2,2-diphenyl-1-picrylhydrazyl radical used in the DPPH assay is stable, deep purple, commercially available, and has not to be generated [23]. The loss of DPPH color at 515 nm after reaction with test compounds is measured either by the decrease of absorbance or by electron spin resonance. The concentration of a 50% decrease fi of the DPPH radical is de ned as EC50, and the time to reach it is TEC50. Since the DPPH assay uses a wavelength of 515 nm, it can interfere with substances with a similar spectrum, such as carotenoids. The test considers both electron transfer and hydrogen transfer reactions with the focus on the prior. Again, smaller molecules have better access to the radical site and contribute to a higher extent to the AOC. The test procedure itself is quite simple and fast and requires only a spectrophotometric device. For all the electron transfer tests, it can be assumed that they overestimate the effects of smaller molecules and hydrophilic substances such as ascorbic acid, uric acid, or polyphenols and reflect not always the situation in the organism. From the evaluation of the different assays, it becomes clear that no single test reflects the overall antioxidative capacity of antioxidants. Both hydrophilic and lipophilic activities must be considered, as well as hydrogen transfer and single- electron transfer mechanisms, and additional tests that reflect the inactivation of various reactive oxygen/nitrogen species are needed to fully estimate the AOC.

15.3 Oxidation of Macromolecules

Popular methods to determine lipid and protein oxidation and a short evaluation are summarized in Table 15.2.

15.3.1 Biomarkers of Lipid Oxidation

Cell membranes are highly susceptible to lipid oxidation due to their speciﬁc composition, which is characterized by high amounts of polyunsaturated fatty acids 236 j 5Mtost eemn oa nixdtv aaiyadOiaieDADamage DNA Oxidative and Capacity Antioxidative Total Determine to Methods 15

Table 15.2 Summary of main biomarkers for lipid and protein oxidation.

a Test Marker Methods Biological system Simplicity/throughput/importance

TBARS/MDA Lipid oxidation HPLC, GC–MS Plasma/serum, cells HPLC: þ/þ/ GC–MS: /þ/þ Conjugated dienes Lipid oxidation HPLC Foods, plasma, cells þ/þ/þ F2a-Isoprostanes Lipid oxidation GC–MS, ELISA Plasma/serum, urine GC–MS: //þ ELISA: /þ/þb Breath hydrocarbons Lipid oxidation GC Exhaled air þ//þ Aldehydes Lipid oxidation HPLC, GC–MS Plasma, LDL, urine HPLC: þ/þ/þ GC–MS: //þ Protein-bound carbonyls Protein oxidation Photometry, ELISA Plasma Photometry: þ/þ/ ELISA: þ/þ/ Advanced oxidation protein Protein oxidation ELISA Plasma, urine þ/þ/þ products a þ , positive; , negative. bMainly used for intervention studies, not for status analysis. 15.3 Oxidation of Macromolecules j237

(PUFAs). Lipid oxidation is a chain reaction and leads to structural and functional damage of membranes as well as to the formation of lipid hydroperoxides that are unstable and degrade to various secondary oxidation products. The formation of malondialdehyde (MDA) is the most widely used parameter of PUFA peroxidation among the thiobarbituric acid-reacting substances (TBARs) assay. Most protocols are based on reaction of MDA with thiobarbituric acid (TBA). One of the oldest and still most widely used methods is based on the precipitation of protein nearly at boiling conditions. The samples are heated for 1 h with TBA at low pH, and the pink chromogen formed absorbs at 532 nm. The sample preparation has been criticized since it is far from physiological conditions, and the free MDA in the original sample is not measured but the amount generated by decomposition of lipid peroxides during heating. Furthermore, it is known that other compounds such as sugars, amino acids, or bilirubin are also able to react with TBA [24]. The sensitivity of the test can be increased by combining it with high- performance liquid chromatography (HPLC) to separate such compounds before acidic heating [25]. In the last few years, several innovations have been introduced to improve the specificity of the test and to reduce known bias. In particular, the temperature at the deproteinization step has been reduced to physiological conditions. In addition, several methods have been developed that do not require derivatization or new derivatization agents such as 2,4-dinitrophenyl hydrazine or diaminonaphthalene [1]. Very recently, GC/MS-based methods have been applied that possess high sensitivity showing an overestimation of MDA levels. Although it is questionable whether MDA measurements are a reliable method for lipid oxidation, it is well documented by a large number of studies that increased levels are found in patients with ROS-related diseases [26]. Furthermore, using the method is more appropriate for an intervention design than for observation studies. The first step of PUFA oxidation is the conjugation of double bonds leading to the formation of conjugated dienes that absorb at around 234 nm. They can be absorbed by lipids and in plasma samples. The plasma preparation is more physiological and requires no heat treatment. In plasma samples, CDs are usually analyzed with HPLC–UV detection. Determination of the diene levels cannot be used alone to describe oxidative stress, but when measured with HPLC CD, it can support results obtained with other, more reliable parameters of oxidative stress. Although MDA and CD are both primary oxidation products, they can develop differently in the same sample due to different mechanisms of formation [27]. Isoprostanes are stable oxidation products from arachidonic acid, initially formed from phospholipids and released into circulation before the hydrolyzed form is excreted in urine. A large number of end products can be theoretically generated but interest has focused mainly on F2a-isoprostanes, the most promising marker for oxidative stress/injury being 8-prostaglandin F2a. At present, it is regarded as one of the most reliable markers of oxidative stress, although the presence of detectable concentrations of isoprostanes in biological fluids requires continuous lipid peroxidation. Several favorable characteristics make isoprostanes attractive as reliable markers for oxidative stress; they are specific oxidation products, stable, present in detectable quantities, increased strongly at in vivo oxidative stress, and their 238j 15 Methods to Determine Total Antioxidative Capacity and Oxidative DNA Damage

formation is modulated by antioxidants. Various approaches such as gas chromatography–mass spectrometry (GC–MS), GC–tandem MS, liquid chromatography–

tandem MS, and immunoassays are available for the detection of F2a-isoprostanes. The ﬁrst results were produced by using the MS technique, and various isoprostanes can be separated with this method. Recently, immunoassays have been developed that correlate quite well with the results obtained with GC–MS measurements in urine, but some discrepancies might occur in plasma when they were not tested on cross-reactivity with other prostaglandin metabolites [28]. Nevertheless, their use might be appropriate in intervention studies with various blood samplings of the same subject, thereby focusing not on the absolute levels but on the relative changes. The measurement of breath hydrocarbons is a noninvasive method that allows to determine LP through exhaled breath by measuring trace volatile hydrocarbons. Ethane formation results from n-3 oxidation, while pentane formation is caused by n-6 oxidation. Although the data reported on their consistency to describe lipid peroxidation are quite convincing, the limiting factor is their detection. They are mainly employed with GC–FID, but one concern is the background level in the breath since bacteria were shown to produce signiﬁcant amounts of these hydrocarbons in vivo. Furthermore, the separation of different hydrocarbons is not easy due to similar boiling points. Aldehydes represent stable products of PUFA oxidation. 4-Hydroxynonenal (4-HNE) and hexanal are mainly formed by n-6 fatty acid oxidation, while propanol and 4-hydroxyhexenal result from n-3 fatty acid oxidation [29]. High concentrations of 4-HNE have been shown to trigger well-known toxic pathways such as induction of caspases, the laddering of genomic DNA, and release of cytochrome C from mitochondria, which may lead to cell death. The most frequently used methods for the determination of aldehydes are GC–MS, GC-headspace, and HPLC. Also, polyclonal or monoclonal antibodies directed against 4-HNE protein conjugates are now frequently applied for 4-HNE measurements [30].

15.3.2 Biomarkers of Protein Oxidation

Markers of protein oxidation are less frequently used than lipid oxidation parameters (Table 15.2). They are mainly used in combination with lipid peroxidation markers, although their formation has been associated with several diseases [31]. Formation of protein-bound carbonyls is the most abundant end point used to monitor protein oxidation by a conventional colorimetric assay using 2,4-dinitro- phenylhdrazine. The test is easy to perform, but large quantities of solvents are required. Recently, an ELISA method has been developed and results have been found to correlate very well with the spectrophotometric method. Advanced oxidation protein products (AOPPs) are predominantly albumin and its aggregates which are damaged by oxidative stress. They contain abundantly dityr- osines that allow cross-linking, disulfide bridges, and carbonyl groups and are formed mainly by chlorinated oxidants such as hypochloric acid and chloramines resulting from myeloperoxidase activity [32]. AOPPs have several similar character- 15.4 Methods Used to Monitor Oxidative DNA Damage j239 istics as advanced glycation end products (AGEs)-modified proteins. Induction of proinflammatory activities, adhesive molecules, and cytokines is even more intense than that caused by AGEs. They are referred to as markers of oxidative stress as well as markers of neutrophil activation. Protein oxidation products mediated by chlorinated species generated by the enzyme myeloperoxidase were found in the extracellular matrix of human atherosclerotic plaques, and increased levels of advanced oxidation protein products were postulated to be an independent risk factor for coronary artery disease [33]. AOPPs are expressed as chloramine-T equivalents by measuring absorbance in acidic conditions at 340 nm in presence of potassium iodide. The test is easy to perform and can be carried out with microprobes.

15.4 Methods Used to Monitor Oxidative DNA Damage

A broad variety of mutagenicity tests which can be used to monitor the antioxidant effects of dietary factors are described in the chapter of Nersesyan et al. [34]. Some methods, which are in particular suitable to detect oxidative damage, are described below. The criticism concerning the sensitivity of conventional strains used in Salmonella/ microsome assay, for example, TA98 and TA100, leads to the construction of new sensitive strains TA102 (with the hisG428 ochre mutation on the multicopy plasmid pAQ1) and TA104 (without plasmids) that are highly responsive to ROS. One of the disadvantages of these strains is relatively high background frequencies. In single-cell gel electrophoresis (SCGE) assays, endogenous formation of oxidized purines and pyrimidines can be measured by lesion-speciﬁc enzymes endonuclease III (endo III) and formamidopyrimidine DNA glycosylase (FPG) [35]. Alterations of the sensitivity of the cells to the exogenous DNA damage can be monitored by the treatment of the cells with ROS-inducing chemicals (H2O2)or ionizing radiation. The latter approaches were used also in micronucleus (MN) assays with human lymphocytes. For example, Fenech and coworkers conducted an interesting study with red wine. In subsequent in vitro study, they tested individual constituents of wines for antioxidant properties [36]. An overview on human intervention trials in which SCGE technique was used can be seen in the reviews by Moller and Loft [37–39] and Hoelzl et al. [40]. The main oxidation product of DNA is 8-hydroxydeoxyguanosin (8-OHdG). The oxidation product can be measured easily in urine and lymphocytes. In some investigations, it was also measured in inner organs such as liver, lungs, and kidneys [41]. A detailed description of the methods can be found in the review by Knasmuller€ et al. [1]. The ﬁrst investigation was conducted in the late 1980s, and many protocols were developed based on gas chromatography coupled with mass spectrometry, liquid chromatography followed by gas chromatography coupled with mass spectrometry, high performance liquid chromatography, and enzyme-linked immunoassays [1]. The European Standards Committee on Oxidative DNA Damage (ESCODD) attempted to identify the reasons for large interlaboratory variations and 240j 15 Methods to Determine Total Antioxidative Capacity and Oxidative DNA Damage

developed standardized guidelines for their identiﬁcation [42]. Comparisons between classical results obtained with chemical analytical approaches and ELISA led to the conclusion that the latter method is quite unreliable [1].

15.4.1 In Vitro and In Vivo Approaches

Oxidative damage can be monitored in a variety of experimental settings. One of the classical approaches is the testing of a variety of potential antioxidants in models with subcellular fractions and/or intact cells. On the basis of the results, the most promising compounds can be selected for more expensive experiments, that is, studies with animals and humans. Figure 15.2 depicts the limitations and also the advantages of different models [1].

Figure 15.2 References j241

In vitro experiments do not provide information on the absorption of dietary compounds in gastrointestinal tract. It is unlikely that potent in vivo antioxidants such as curcumin, chlorophyllins, and anthocyanins are effective in inner organs as they are poorly absorbed. It is also worth noting in this context that stable human-derived cell lines that are widely used do possibly lack signaling pathways and transcription factors involved in antioxidant defense. For example, it is known that 50% of the tumors from which the cell lines are derived have mutated p53 [43]. Therefore, experiments with cell lines reﬂect direct scavenging of ROS but not necessarily ROS protection via cellular response systems, which may be even more relevant for protective effects in humans.

15.5 Conclusions and Outlook

The development of reliable methods and endpoints that can be used to detect oxidative damage is highly important in regard to investigating the consequences of host-mediated health effects, identifying individuals who are exposed to oxidative stress, and developing dietary strategies to improve the antioxidant status. As described above, a plethora of different methods have been developed that differ strongly in terms of reliability and cost. We have recently evaluated the usefulness of the different approaches that are currently used in regard to their potential use for justiﬁcation of health claims concerning the antioxidant properties of foods and concluded that biomarkers that are related to diseases and health risk may be more relevant than others [1]. Such relevant endpoints are, for example those indicative for oxidation of DNA, for example, SCGE assay, MN experiments, determination and measurement of oxidized DNA bases, determination of the excretion of isoprostanes that reﬂect lipid peroxidation due to COX2 and ROS, and measurements of LDL. As pointed out above, the overlap of different methods is quite poor, and the combination of the most relevant end points may be a suitable strategy in future investigations.

References

1 Knasmuller,€ S., Nersesyan, A., Mišık, M., pyrroline N-oxide (DECPO). Application to Gerner, C., Mikulits, W., Ehrlich, V., superoxide radical trapping. Tetrahedron Hoelzl, C., Szakmary, A. and Wagner, K.- Letters, 45, 149–152. H. (2008) Use of conventional and -omics 3 Khan, N., Wilmot, C.M., Rosen, G.M., based methods for health claims of dietary Demidenko, E., Sun, J., Joseph, J., OHara, antioxidants: a critical overview, The British J., Kalyanaraman, B. and Swartz, H.M. Journal of Nutrition, 99 (E-Suppl. 1), (2003) Spin traps: in vitro toxicity and ES3–ES52. stability of radical adducts. Free Radical 2 Karoui, H., Clement, J.-L., Rockenbauer, Biology & Medicine, 34, 1473–1481. A., Siri, D. and Tordo, P. (2004) Synthesis 4 Halliwell, B. and Whiteman, M. (2004) and structure of 5,5-diethoxycarbonyl-1- Measuring reactive species and oxidative 242j 15 Methods to Determine Total Antioxidative Capacity and Oxidative DNA Damage

damage in vivo and in cell culture: how initiated chemiluminescence of blood should you do it and what do the results plasma proteins to evaluate antioxidant mean? British Journal of Pharmacology, 142, homeostasis in humans. Redox Report, 6, 231–255. 43–48. 5 Ghiselli, A., Serafini, M., Maiani, G., 13 Esterbauer, H., Jurgens, G., Azzini, E. and Ferro-Luzzi, A. (1995) A Quehenberger, O. and Koller, E. (1987) fluorescence-based method for measuring Autoxidation of human low density total plasma antioxidant capability. Free lipoprotein: loss of polyunsaturated fatty Radical Biology & Medicine, 18,29–36. acids and vitamin E and generation of 6 Glazer, A.N. (1990) Phycoerythrin aldehydes. Journal of Lipid Research, 28, fluorescence-based assay for reactive 495–509. oxygen species. Methods in Enzymology, 14 Wagner, K.-H., Tomasch, R. and Elmadfa, 186, 161–168. I. (2001) Impact of diets containing corn oil 7 Cao, G., Alessio, H.M. and Cutler, R.G. or olive/sunflower oil mixture on the (1993) Oxygen-radical absorbance capacity human plasma and lipoprotein lipid assay for antioxidants. Free Radical Biology metabolism. European Journal of Nutrition, & Medicine, 14, 303–311. 40, 161–167. 8 Huang, D., Ou, B., Hampsch-Woodill, M., 15 Frankel, E.N., German, J.B. and Davis, P.A. Flanagan, J.A. and Prior, R.L. (2002) (1992) Headspace gas chromatography to High-throughput assay of oxygen radical determine human low density lipoprotein absorbance capacity (ORAC) using a oxidation. Lipids, 27, 1047–1051. multichannel liquid handling system 16 Frankel, E.N. and Meyer, A.S. (2000) The coupled with a microplate fluorescence problems of using one-dimensional reader in 96-well format. Journal of methods to evaluate multifunctional food Agricultural and Food Chemistry, 50, and biological antioxidants. Journal of the 4437–4444. Science of Food and Agriculture, 80, 9 Wayner, D.D.M., Burton, G.W., Ingold, 1925–1941. K.U. and Locke, S. (1985) Quantitative 17 Bors, W., Michel, C. and Saran, M. (1984) measurement of the total, peroxyl radical- Inhibition of the bleaching of the trapping antioxidant capability of human carotenoid crocin. A rapid test for blood plasma by controlled peroxidation. quantifying antioxidant activity. Biochimica The important contribution made by et Biophysica Acta, 796, 312–319. plasma proteins. FEBS Letters, 187,33–37. 18 Miller, N.J., Rice-Evans, C., Davies, M.J., 10 Ghiselli, A., Serafini, M., Natella, F. and Gopinathan, V. and Milner, A. (1993) A Scaccini, C. (2000) Total antioxidant novel method for measuring antioxidant capacity as a tool to assess redox status: capacity and its application to monitoring critical view and experimental data. Free the antioxidant status in premature Radical Biology & Medicine, 29, 1106–1114. neonates. Clinical Science, 84, 407–412. 11 Winston, G.W., Regoli, F., Dugas, A. Jr, 19 Benzie, I.F.F. and Strain, J.J. (1996) The Fong, J.H. and Blanchard, K.A. (1998) A ferric reducing ability of plasma (FRAP) as rapid gas chromatographic assay for a measure of antioxidant power: the determining oxyradical scavenging FRAP assay. Analytical Biochemistry, 239, capacity of antioxidants and biological 70–76. fluids. Free Radical Biology & Medicine, 24, 20 Prior, R.L., Wu, X. and Schaich, K. (2005) 480–493. Standardized methods for the 12 Popov, I., Volker,€ H. and Lewin, G. (2001) determination of antioxidant capacity and Photochemiluminescent detection of phenolics in foods and dietary antiradical activity. V. Application in supplements. Journal of Agricultural and combination with the hydrogen peroxide- Food Chemistry, 53, 4290–4302. References j243

21 Pulido, R., Bravo, L. and Saura-Calixto, F. related aldehydes. Free Radical Biology & (2000) Antioxidant activity of dietary Medicine, 11,81–128. polyphenols as determined by a modified 30 Sharma, R., Brown, D., Awasthi, S., Yang, ferric reducing/antioxidant power assay. Y., Sharma, A., Patrick, B., Saini, M.K., Journal of Agricultural and Food Chemistry, Singh, S.P., Zimniak, P., Singh, S.V. and 48, 3396–3402. Awasthi, Y.C. (2004) Transfection with 22 Apak, R., Guclu, K., Ozyurek, M. and 4-hydroxynonenal-metabolizing Karademir, S.E. (2004) Novel total glutathione S-transferase isozymes leads antioxidant capacity index for dietary to phenotypic transformation and polyphenols and vitamins C and E, using immortalization of adherent cells. their cupric ion reducing capability in the European Journal of Biochemistry/FEBS, presence of neocuproine: CUPRAC 271, 1690–1701. method. Journal of Agricultural and Food 31 Agarwal, R. (2004) Chronic kidney disease Chemistry, 52, 7970–7981. is associated with oxidative stress 23 Brand-Williams, W.C., Cuvelier, M.E. and independent of hypertension. Clinical Berset, C. (1995) Use of a free radical Nephrology, 61, 377–383. method to evaluate antioxidant activity. 32 Capeillere-Blandin, C., Gausson, V., Lebensmittel-Wissenschaft und-Technologie, Descamps-Latscha, B. and Witko-Sarsat, V. 28,25–30. (2004) Biochemical and spectro- 24 Meagher, E.A. and Fitzgerald, G.A. (2000) photometric significance of advanced Indices of lipid peroxidation in vivo: oxidized protein products. Biochimica et strengths and limitations. Free Radical Biophysica Acta, 1689,91–102. Biology & Medicine, 28, 1745–1750. 33 Kalousova, M., Zima, T., Tesar, V., 25 Wong, S.H.Y., Knight, J.A., Hopfer, S.M., Dusilova-Sulkova, S. and Skrha, J. (2005) Zaharia, O., Leach, C.N. Jr and Advanced glycoxidation end products in Sunderman, F.W. Jr (1987) Lipoperoxides chronic diseases: clinical chemistry and in plasma as measured by liquid- genetic background. Mutation Research, chromatographic separation of 579,37–46. malondialdehyde-thiobarbituric acid 34 Nersesyan, A., Misik, M. and Knasmuller,S.€ adduct. Clinical Chemistry, 33, 214–220. (2009) Experimental models for the 26 Del Rio, D., Stewart, A.J. and Pellegrini, N. detection of DNA protective effects, in (2005) A review of recent studies on Chemoprevention of Cancer and DNA malondialdehyde as toxic molecule and Damage by Dietary Factors (eds S. biological marker of oxidative stress. Knasmuller,€ D.M. DeMarini, I.T. Johnson Nutrition Metabolism and Cardiovascular and C., Gerh€auser), Wiley-VCH Verlag Diseases, 15, 316–328. GmbH & Co. KGaA, Weinheim, 211–227. 27 Konig, D., Wagner, K.H., Elmadfa, I. and 35 Collins, A.R., Dusinska, M., Gedik, C.M. Berg, A. (2001) Exercise and oxidative and Stetina, R. (1996) Oxidative damage to stress: significance of antioxidants with DNA: do we have a reliable biomarker? reference to inflammatory, muscular, and Environmental Health Perspectives, 104 systemic stress. Exercise Immunology (Suppl. 3), 465–469. Reviews, 7, 108–133. 36 Greenrod, W., Stockley, C.S., Burcham, P., 28 Young, I.S. (2005) Oxidative stress and Abbey, M. and Fenech, M. (2005) Moderate vascular disease: insights from isoprostane acute intake of de-alcoholized red wine, but measurement. Clinical Chemistry, 51, not alcohol, is protective against radiation- 14–15. induced DNA damage ex vivo: results of a 29 Esterbauer, H., Schaur, R.J. and Zollner, H. comparative in vivo intervention study in (1991) Chemistry and biochemistry of younger men. Mutation Research, 591, 4-hydroxynonenal, malonaldehyde and 290–301. 244j 15 Methods to Determine Total Antioxidative Capacity and Oxidative DNA Damage

37 Moller, P. and Loft, S. (2004) Interventions humans: recent results and trends. with antioxidants and nutrients in relation Mutation Research, in press. to oxidative DNA damage and repair. 41 Jin,X.,Chan,H.M.,Lok,E.,Kapal,K., Mutation Research, 551,79–89. Taylor,M.,Kubow,S.andMehta,R. 38 Moller, P. and Loft, S. (2006) Dietary (2008) Dietary fats modulate antioxidants and beneﬁcial effect on methylmercury-mediated systemic oxidatively damaged DNA. Free Radical oxidative stress and oxidative DNA Biology & Medicine, 41, 388–415. damage in rats. Food and Chemical 39 Moller, P. and Loft, S. (2002) Oxidative Toxicology, 46, 1706–1720. DNA damage in human white blood cells 42 Gedik,C.M.andCollins,A.(2005) in dietary antioxidant intervention studies. Establishing the background level of The American Journal of Clinical Nutrition, baseoxidationinhumanlymphocyte 76, 303–310. DNA: results of an interlaboratory 40 Hoelzl, C., Knasmuller,€ S., Misik, M., validation study. The FASEB Journal, Collins, A.R., Dusinska, M. and 19,82–84. Nersesyan, A. Use of single cell gel 43 Meulmeester, E. and Jochemsen, A.G. electrophoresis assays for the detection of (2008) p53: a guide to apoptosis. Current DNA-protective effects of dietary factors in Cancer Drug Targets, 8,87–97. j245

16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research Wolfgang W. Huber and Michael Grusch

16.1 Introduction

In the public, most discussion on the relationship between chemicals and disease, and on the relationship with cancer in particular, is focused on a procarcinogenic effect of substances, whereas prevention is more frequently being tied to their avoidance. However, the idea of exposure to chemicals as a means of reducing rather than enhancing carcinogenic potential has recently become more widespread and has developed into a target of increasing research [1–3]. Still, the term anticarcinogenic substances is often exclusively associated with chemotherapeutic drugs to which the patient is exposed after diagnosis of the disease. Indeed, there is at times an unclear distinction between the terms chemoprevention and chemotherapy due to the long-term character of the carcinogenic process in the human that may last for many years if not decades. Moreover, the carcinogenic process is subdivided into several phases during which susceptibility to anticancer strategies may change considerably and which may be described by the multistage model of carcinogenesis [4]: At first, the brief tumor initiation phase usually leads to irreversible genotoxic damage in a cell that results in greater susceptibility to the growth stimuli exerted in the subsequent tumor promotion phase. The latter, which covers the vast majority of the total duration of the carcinogenic process, leads to the formation of a benign tumor and is followed by the tumor progression phase in which malignancy is acquired, characterized by metastasis, low degree of tissue differentiation, and autonomous as well as infiltrative growth. Usually, cancer is not diagnosed until after the beginning of the progression phase so that chemoprevention, according to wide-spread interpretation, may also include the prevention of more advanced disease stages before diagnosis, thus comprising such action in all the three phases. Examples of chemoprevention strategies effective in latter phases would be growth receptor antagonism [5], induction of programmed cell death (apoptosis) [6], or starvation of the tumor by inhibition of the formation of tumor-specific blood vessels [7].

However, in the present chapter, we deal with an end point related to events that occur, in fact, even before tumor initiation, thus preceding any change to the target tissue. Before reaching this target tissue, toxicants and drugs are mostly subjected to the bodys multiple enzymes of xenobiotic metabolism resulting in metabolites that may differ considerably in effect from the respective parent compounds. Some of these metabolic steps may cause detoxification but others may also activate a compound and even be prerequisites for achieving the full toxic effect. Of course, these pathways are strongly substance dependent. Notably, the vast majority of the known genotoxic carcinogens must be enzymatically activated [8]. Therefore, a beneficial condition in xenobotic metabolism may be regarded as the first and therefore truly preventive line of defense against chemical carcinogenesis. The associated interests concern, on the one hand, the genetically determined precondition, particularly showing in human enzyme polymorphisms that appear to influence cancer incidence in carcinogen-exposed population groups [9, 10], and, on the other, it may concern modifications of enzyme status either by lifestyle factors or by explicit chemopreventive intervention [1–3, 11]. However, it also has to be kept in mind that detoxifying effects that would be beneficial in the tumor initiation phase may become undesirable during chemotherapy when toxicity to the tumor tissue is explicitly intended [12]. In dealing with the associated questions, tools are required to achieve a thorough characterization of the different aspects concerning the status of xenobiotic metabolism. The present chapter intends to give a brief overview of several of the most commonly applied methods that serve this purpose. The methods may be applied in human studies and/or in investigations using animal and in vitro models, either purely observational or upon active intervention.

16.2 Important Enzymes of Xenobiotic Metabolism in Cancer Chemoprevention Research

Conceivably, most cancer-associated research on xenobiotic metabolism is centered around enzymes that (a) metabolize relevant human carcinogens, (b) that may be beneficially modified by candidate chemoprotectants, and/or (c) that are associated with polymorphisms found to determine individual cancer risk. The overall goal of xenobiotic metabolism is to make chemicals more hydrophilic and to, thus, facilitate their excretion. The associated enzymes are subdivided into two groups described as phase I and phase II. Generally, the enzymes of phase I catalyze redox reactions and hydrolysis leading to the introduction of functional groups into the chemical. These steps tend to lead to carcinogen activation and the best-known representatives of phase I enzymes are found in the cytochrome P-450 (CYP) family. For example, CYP1A1, CYP1A2, and CYP2E1 activate polyaromatic hydrocarbons, aromatic amines, and alkylating compounds such as nitrosamines, respectively [8, 13]. More- over, several polymorphisms of CYP that may influence cancer risk have been described [8, 14, 15]. Nicotinamide quinone oxidoreductase 1 (NQO1) is an additional rather well-investigated and mostly protective enzyme that would belong to phase I due to its redox-related activity but has been attributed to phase II by several authors due to mechanistic considerations [16–18]. 16.3 Measurement of Xenobiotic Metabolism: General Aspects j247

Phase II enzymes cause the conjugation of xenobiotics that, in the vast majority of cases, will lead to carcinogen detoxification. Glutathione-S-transferases (GSTs), for which human polymorphisms are also known, may be regarded as the best investigated family of phase II enzymes, the most important members being termed as alpha (GSTA), mu (GSTM), pi (GSTP), and theta (GSTT) [19, 20]. Also, the associated cofactor glutathione plays an important role in the bodys antioxidant defense and the inducible limiting enzyme of glutathione synthesis, g-glutamylcysteine synthetase, represents a promising target of chemopreventive intervention as well [21–23]. Further phase II enzymes on which considerable research has hitherto been focused are N-acetyltransferases (NATs) and sulfotransferases (SULTs), with their ambivalent character between activation of some carcinogens and detoxification of others [10, 24, 25], and the predominantly detoxifying UDP-glucuronosyl transferase (UGT) [26–29]. For instance, much is known about the polymorphism of NAT indicating that rapid and slow acetylators are at a greater risk of developing colon and bladder cancer, respectively. This was explained by the activating capacity of NAT toward colon cancer causing heterocyclic amines, whereas NAT detoxifies aromatic amines associated with bladder cancer induction [10, 25, 30–33]. The status of several of these enzymes, which are often altered in combination, may depend on specific regulatory elements such as the pathway involving the antioxidant responsive element (ARE), Nrf2, and Keap1 [34]. This pathway is associated with the effect of so-called monofunctional inducers that cause increases in phase II but in not phase I metabolism, which would thus shift the general metabolic balance toward carcinogen detoxification and would therefore be interesting for chemoprotective strategies [17, 18, 22].

16.3 Measurement of Xenobiotic Metabolism: General Aspects

Like all other proteins, enzymes of xenobiotic metabolism are produced by translation from mRNAs that have previously been transcribed from genomic DNA. After possible further post-translational modification, the enzyme proteins exert the catalyzing activity that may detoxify as well as toxify many important carcinogens and other harmful compounds. Due to these various steps involved, enzymes of xenobiotic metabolism may be investigated essentially at four different levels: DNA, mRNA, protein, and activity. An overview of the associated end points is given in Figure 16.1. All these investigations provide information on different aspects of enzyme-related processes and thus cannot simply replace each other. It is therefore highly recommended to base ones knowledge on a particular enzyme on several, if not all, of these end points. Importantly, investigations of genotype, that is, at the DNA level, cover the hereditary preconditions of the enzymatic status of an individual, whereas the three other end points primarily supply phenotypical information on environmentally related modifications of this status, which may appear either as enzyme induction or as enzyme inhibition. Not all enzymes are susceptible to such modifications but, whenever such induction or inhibition occurs, it is absolutely required to distinguish 248j 16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research

Figure 16.1 End points in the investigation of transferase; NAT, N-acetyltransferse; PCR, xenobiotic metabolism in the course of events polymerase chain reaction; QRT-PCR, with examples of associated analysis methods. quantitative real-time RT-PCR; RT-PCR, reverse CDNB, 1-chloro-2,4-di-nitrobenzene; CYP, transcription PCR; SNP, single nucleotide cytochrome P-450; ELISA, enzyme-linked polymorphism. immunosorbent assay; GST, glutathione-S-

between the determined genotype and phenotype. Responsiveness to external stimuli plays a crucial role particularly for CYPs and GSTs, which are therefore among the most important and most investigated targets in chemoprevention studies and strategies. It should also be noted that novel data on formerly uninvestigated inducers or inhibitors may always change our knowledge, for instance, suggesting the responsiveness of an enzyme previously believed to be unmodiﬁable. An example would be the about 80% decrease in hepatic N-acetyltransferase activity that was recently observed in rats pretreated with the coffee components kahweol and cafestol [35, 36]. Of course, the choice of methodological approach would also strongly depend on the particular question to be elucidated. Some methods are more appropriate to obtain quick data on previously unknown substances or to be applied in screening and/or pilot studies while others are rather focused on the thorough elucidation of sophisticated mechanistic context. Some are well suited for human studies while others largely depend on animal or in vitro models, for example, due to inaccessible indicators in case of organ speciﬁcity of the effect. In the chapters that follow, several of the various possible approaches will be described in more detail. 16.3 Measurement of Xenobiotic Metabolism: General Aspects j249

16.3.1 Determination of Enzyme Encoding genes (Investigation at DNA Level)

As described above, enzyme investigations at DNA level are not capable of providing information on acquired modifications of enzyme status. However, they are useful in determining particularly those human enzymes for which genotype and phenotype areessentiallyidentical,thatis,investigations concerningenzymesthat,tothepresent knowledge, can neither be induced nor inhibited under more or less realistic in vivo conditions. For example, this concerns the so-called null variants where particular enzymes are altogether missing or lacking function. Due to the standardized character of most animal and in vitro models in this respect (except in the initial characterization of such models, for example, transgenic mice or particular cell lines), the classical indicationofDNA-levelinvestigationsofxenobioticenzymeswouldbetrue,genetically based human polymorphisms that may concern coding DNA sections as well as regulatory elements [20]. Consequently, such data will often accompany studies on cancer epidemiology. As an enormous advantage of this approach, the data can be obtainedinanyDNA-containingcellofaperson,therebyremainingindependentofthe often ethically and technically difficult acquisition of specific organ material. Methods commonly used in up-to-date studies carried out at DNA level include polymerase chain reaction (PCR), DNA sequencing, and array-based hybridization techniques [37], whereas the classical Southern blot [38, 39] is now more of historical significance. PCR is used for amplification of specific DNA regions with oligonucleotide primers. The sequence information necessary for primer design is available from publicly accessible databases such as those maintained by the National Center for Biotechnology Information (NCBI) in the United States or the European Molec- ular Biology Lab (EMBL). The amplified sequences can be scanned for polymorphisms by gel electrophoresis techniques [40], denaturing HPLC [41], or DNA sequencing. While sequencing is usually required for the identification of new polymorphisms, hybridization techniques in homogenous solution, such as the Taqman allelic discrimination assay [42], or array-based hybridization methods have been developed for the high-throughput detection of known polymorphisms. Micro- arraysarecreatedbyattachingthousandsofoligonucleotideprobestoasolidsurfacein an ordered fashion. Perfectly matched sequences hybridize more efficiently to the oligonucleotides on the array than sequences containing mismatches, and this can be exploited to screen multiple single nucleotide polymorphisms (SNPs), insertions, or deletions simultaneously [43]. While microarrays enable investigators to generate a vastamountofdatawithinashorttime,thecostsofsuchanalysesarealsoconsiderable.

16.3.2 Determination of Enzyme Transcription (Investigation at RNA Level)

Data on the formation of mRNAs transcripts in the nucleus are the earliest and therefore the most fundamental indicator of the occurrence of an enzyme modifying process, which underlines the relevance of this end point in mechanistic studies. Also, a change in mRNAs will usually precede the modiﬁcation of protein or activity 250j 16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research

levels so that further mechanistic conclusions may be drawn from the respective time courses of such events. The level of mRNAs may be influenced by transcription rate, mRNA processing, and transcript stability. However, mRNA determination does not cover any subsequent events that may further influence the effect of enzymes on the toxicants, that is, regulation at the translational and post-translational level. In contrast to DNA, RNAs are rather unstable molecules that may be degraded during experimentation by ubiquitous RNAses. Therefore, precautions have to be taken to work in an RNAse-free environment, for example, by explicit use of RNAse-free reagents. PCR has already been described as a crucial tool in DNA analysis and is also a standard method for detection of specific mRNA transcripts. However, when applied to mRNA analysis, the samples must first be reverse transcribed to obtain the corresponding cDNAs [44], and the method is therefore called reverse transcription PCR (RT-PCR). Amplification of a specific cDNA fragment corresponding to the mRNA under investigation is carried out with sequence-specific oligonucleotide primers and the amplification products are then resolved by gel electrophoresis. RT- PCR is an inexpensive method, only requiring a simple thermocycler that may presently be regarded as standard lab equipment. However, a disadvantage of this methodisthe semiquantitativecharacter ofits results. Anadvancement came with the development of quantitative real-time RT-PCR (QRT-PCR) where formation of the amplification product is monitored directly, yielding characteristic amplification plots that make a more accurate quantification possible [45, 46]. A typical example of an amplification plot in an mRNAexpression analysis is given in Figure 16.2. The greater accuracy of the results is in part counterbalanced by greater costs, particularly with regard to the necessary instrumentation, that is, a thermocycler suitable for online fluorescence detection. Despite that, QRT-PCR is increasingly replacing other standard methods for analysis of mRNA expression changes such as the northern blot [47] and RNAse protection assay [48], because it yields faster results, uses nonradioctive detection, and requires at least one order of magnitude less sample RNA. Similar as in DNA analysis, the simultaneous investigation of multiple enzyme mRNAs was made possible by the introduction of microarrays that may cover either the whole genome or specific enzyme families such as CYP [49]. Hybridization may also be used for histological localization of transcripts (in situ hybridization) [50]. However, this latter approach has to be regarded as more complicated than its protein-based equivalent (see later) and is therefore not as frequently used. As an alternative to mRNA quantification, transcriptional activity at genes of chemoprotective enzymes may also be investigated with transgenic animals or cells in which a reporter gene that visualizes transcription, for example, luciferase, is linked to a corresponding regulatory unit such as ARE [51, 52].

16.3.3 Determination of Enzyme Protein

Investigation of enzyme protein content will give further mechanistic information on the events and a comparison with mRNA data will help to distinguish between 16.3 Measurement of Xenobiotic Metabolism: General Aspects j251

Figure 16.2 Amplification plot from a QRT-PCR analysis. The PCR cycle number is plotted on the x-axis, the intensity of the fluorescent signal, which is generated by the amplification process, on the y-axis. Each curve represents one sample. The cycle number in which the fluorescence of each sample reaches a set threshold is called threshold cycle (Ct), and is proportional to the amount of mRNA in the original sample. transcriptional and translational (and possibly post-translational) effects. Most associated methodologies are based on the quality of proteins as typical antigens, that is, versatile immunological reactions may be used for their detection. These usually very sensitive methods involve one or two antibodies of which one is attached to a marker for visualization based on fluorescence, chemoluminescence, color, or radioactivity. A role in such reactions is often played by enzymes such as horseradish peroxidase or high-affinity complexes such as streptavidin–biotin. However, a disadvantage of such immune-based methods is the often-restricted availability of antibodies, the acquisition of which may either be costly or may depend on supply by specialized cooperation partners. Also, cross reactivity, that is, lack of specificity of the immune reactions may occasionally be a problem. Two commonly used methods in such investigations are the enzyme-linked immunosorbent assay (ELISA) [53, 54] and the Western blot technique [51, 55]. ELISA is based on antibodies attached to an enzyme that catalyzes a color reaction, usually in a microtiter plate, while, in Western blotting, separation through gel electrophoresis is followed by a transfer of the proteins to a blotting membrane on which the immune reactions are then carried out. Moreover, immunological detection of proteins is used in histology (immunohistochemistry), thus monitoring changes that are restricted to particular cells, for example, the pericentral cells of the liver acini, or cellular compartments, for example, cytoplasm. In cancer studies, such techniques are of 252j 16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research

particular relevance since certain enzymes may be preferentially expressed in preneoplastic cells. Clusters of such cells (foci) may serve as indicators of early carcinogenesis, probably related to their enzyme-related selection advantage. An example would be GSTP that is not present beyond traces in the unchanged liver and is therefore used as an important indicator in studies on hepatic preneoplasia [56, 57]. Not all methods that deal with xenobiotic metabolism are based on antibodies and/ or gel electrophoresis. For instance, a pattern of members of the GST family (alpha, mu, and pi, but not theta) may be separated and detected by a HPLC method that follows isolation out of biological material by a glutathione afﬁnity column [26, 58]. Perspectives for the future may be associated with techniques of proteomics with their capacity to analyze multiple proteins simultaneously. Such approaches may involve two-dimensional electrophoresis and subsequent HPLC-MS/MS [59, 60].

16.3.3.1 Measurement of Enzyme Activity: General Aspects Enzyme activity may be measured by a whole series of quick and inexpensive methods. Most approaches in this field are based on spectrophotometrical monitoring of either the appearance of a reaction product or the disappearance of a substrate or cosubstrate. Thus, the instruments required may be classified as standard equipment present in most labs. Because of all these advantages, several of these methods have developed into standard procedures at a worldwide level, so that there are now large databases available for comparison with any newly obtained results. Activity measurement is an appropriate approach in the investigation of xenobiotic metabolism whenever the economic acquisition of quick information is intended, or when an investigation involves a large number of samples, for example, in time- course, organ specificity, or dose–response studies. Although the required amount of material per sample may at times be larger than that in alternative approaches, activity measurement is an excellent means to obtain first information on the effects of unknown substances, as it would, for instance, be desirable in screening procedures. Of course, a major disadvantage of activity measurement is the lack of mechanistic information, so that further investigations are always required to decide whether an observed effect has occurred at the transcriptional, post-transcriptional, translational, or post-translational level. Also, the metabolism of a particular substrate is not always tied to a specific enzyme due to the potential involvement of alternative pathways, which may become particularly relevant under conditions of nonphysiological external modification. Therefore, downregulation of the synthesis of a particular carcinogen-activating enzyme protein must not necessarily result in reduced activation of the associated carcinogen and enhanced metabolism of a particular substrate does not unequivocally prove a true induction of the presumably responsible enzyme down to mRNA level. On the contrary, enzyme activity is what will ultimately activate or detoxify the carcinogen so that this end point is the one closest to the ultimate investigation target. Thus, data on altered activity alone may be of great relevance even in the absence of a clear explanation by molecular biology. It is conceivable that a critical role in activity-based methods must be attributed to the choice of model substrate. Of course, the most informative approaches would involve the investigation of activating and/or detoxifying reactions of the carcinogens 16.3 Measurement of Xenobiotic Metabolism: General Aspects j253 under investigation themselves. However, apart from potential problems associated with the handling of carcinogens, such reactions need not necessarily give measurable end points and/or previously published methods may not be available so that considerable time and resources would be required for their development. In case of multiple step metabolism of a carcinogen, the required substrate may be a metabolite rather than the parent compound and the latter may not be as easily obtained. For instance, the cooked food mutagen PhIP may be activated first through N-hydroxylation by CYP1A2 and, subsequently, by acetylation. Thus, to quantify the degree of this acetylation directly, it becomes inevitable to explicitly use NOH PhIP as the reaction substrate and, moreover, a radioactive marker rather than simple color was required to visualize the result [36, 61]. In such cases, the only solution would often be a complicated and time-consuming synthesis of the required substrate in ones own lab, which may even involve the development of the necessary synthesis procedures from scratch. Thus, despite their greater vicinity to the immediate goal of investigation, such specific approaches are often associated with the loss of many of the relevant advantages of activity measurements described above, such as low cost in time and money or large databases for comparison. As a viable alternative, it is recommended to use a series of model substrates for which a high degree of association with particular enzyme gene products has been established in numerous previous experiments. An overview of these assays is given in Table 16.1. Some of the substrates concerned are metabolized rather selectively by individual enzymes while others may provide a general overview of whole enzyme families. A good example of the latter is the metabolism of 1-chloro-2,4-dinitrobenzene (CDNB) that is today regarded as the most important indicator for general quantification of GSTs covering the important subforms alpha, mu, and pi (but not theta). Many studies that deal with CYP-related metabolism are based on the metabolic liberation of the compound resorufin from a series of resorufin derivatives that are individual substrates of specific CYP subforms. Moreover, CDNB- and resorufin-related assays, respectively, represent the two monitoring approaches involved in essentially all these simple spectrophotometric activity measurements: kinetics and end point determination. In principle, both method types involve a buffered reaction mixture containing the substrate, the cosubstrate, enzyme containing biological material, and, possibly, other factors that optimize the activity of the investigated enzyme. The applied biological material depends on the experimental model chosen (e.g., preparations from organs exposed in vivo or from cultured cells) and should be derived from the cellular fraction in which the enzyme of interest is contained (e.g., cytosol for GSTs and microsomes for CYP; see Table 16.1). The reaction is started and allowed to continue for a fixed amount of time during which the temperature (usually 37 or 25 C) has to be kept constant, for example, with the help of a water bath or in a thermostated cuvette. Enzyme activity is then determined by the speed of concentration change of a specific indicator substance while the reaction is linear. In kinetic studies, it is possible to monitor the indicator substance permanently during the reaction so that possible nonlinear sections of the curve may be excluded from calculation. However, in end point determinations, an additional step is required to visualize the indicator after the reaction. Thus, only the final 254j 16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research

Table 16.1 Model substrates for activity measurement of enzymes of xenobiotic metabolism.

Detection system, Primary target Cellular wavelength(s), end point, enzyme Substrate fraction or kinetics References

CYP1A1 Ethoxy-resorufin Microsomes Fluorescence (ex: 530 nm [48, 62] em: 585 nm), end point CYP1A2 Methoxy-resorufin Microsomes see above CYP2B1 Pentoxy-resorufin Microsomes see above CYP2B2 Benzyloxy-resorufin Microsomes see above CYP2E1 p-Nitrophenol Microsomes Visible (512 nm), end point [13, 48] NQO1 Menadione Cytosol Visible (550 nm), kinetics [63] Overall GST 1-Chloro-2,4-dinitro- Cytosol Visible (340 nm), kinetics [26, 64] (GSTA, GSTM, benzene (CDNB) GSTP) GSTM (mu) 1,2-Dichloro-4-nitro- Cytosol Visible (345 nm), kinetics [19, 64, 65] benzene (DCNB) GSTP (pi) 4-Vinylpyridine Cytosol UV (248 nm), kinetics [26, 66] GSTT (theta) 1,2-Epoxy-3-(p-nitro- Cytosol Visible (360 nm), kinetics [26, 67, 68] phenoxy)propane (EPNP) GSTO (omega) Hyxdroxyethyl disul- Cytosol Visible (340 nm), kinetics [69] fide (HEDS) GSTZ (zeta) Dichloroacetic acid Cytosol Visible (535 nm), end point [70] (DCA) NAT1 Sulfamethazine Cytosol HPLC, UV (254 nm), [71, 72] (SMZ) end point NAT2 p-Aminobenzoic acid Cytosol HPLC, UV (280 nm), [71, 73] (PABA) end point SULT1A1 2-Naphthol Cytosol Visible (405 nm), end point [48, 74] UGT1 p-Nitrophenol Microsomes Visible (400 nm), end point [26, 75] UGT2 [14C]- Microsomes Scintillation count, end point [26, 76] chloramphenicol

Unless indicated otherwise, analysis is carried out by means of a spectrophotometer. CYP, cytochrome P-450; GST, glutathione-S-transferase; NQO1, nicotinamide quinone oxidoreductase 1; SULT, sulfotransferase; UGT, UDP-glucuronosyl transferase.

amount of indicator can be quantiﬁed in each sample and linearity of the reaction can only be assumed. End point determinations would therefore usually require more preliminary work in the standardization of experimental conditions and leave a higher degree of uncertainty. Enzyme activity results are essentially always given in relation to protein, estimation of which may be quickly and easily achieved by the well-established Bradford/Biorad method [77]. Either, activity may simply be divided by the amount of protein in each reaction mixture or protein may be adjusted to equal concentrations before the reaction is started. The latter approach that adds a step (and possible error source) to the procedure would, on the other hand, exclude possible effects of protein inhibition that might become relevant when the differences in 16.4 Summary j255 protein between the samples become too large. However, dilution of high-activity samples is sometimes inevitable to still obtain a linear phase in the kinetic curve.

16.3.3.2 Activity Measurements in Humans Modiﬁcations of xenobiotic metabolism were shown to be strongly organ dependent, being often restricted to inner organs like the liver [26], that is, material not easily accessible in humans. Thus, there are limits to human in vivo studies aimed at the determination of effects on enzyme activity directly in the organs where they occur, for example, with regard to ethics or to the systematic shortcomings of biopsy samples. Enhancing effects on GST activity were occasionally observed in easily accessible blood or saliva [54, 78] but questions arose with regard to the origin of the monitored activity, particularly given the fact that an increase in plasma GSTmay also occur as a consequence of liver cell degradation [79, 80]. However, in the frame of an alternative approach, volunteers may be given model substrates representative of particular enzymes and overall metabolic capacity may be monitored through analysis of blood and urine concentrations of speciﬁc metabolites, resulting from in vivo metabolism of the parent compound anywhere in the body. For example, caffeine is used as a model substrate to monitor both the activity of CYP1A2 and NAT [81, 82]. Of course, in case of multistep or multiple-pathway metabolism, this approach cannot unequivocally distinguish, for example, between increased formation and reduced degradation of a particular metabolite. An additional alternative means of using human material in the investigation of xenobiotic metabolism would be to employ metabolically competent cell lines of human origin such as the liver- derived HepG2 that may provide at least some qualitative information with regard to the in vivo situation [83, 84].

16.4 Summary

The effect of genotoxic carcinogens is strongly influenced by the enzymes of xenobiotic metabolism. This concerns metabolic activation, which is mostly even a prerequisite for the genotoxic effect, as well as detoxification. Therefore, enzymatic status, either genetically predetermined or as a result of environmental modifications, may be decisive for individual susceptibility to carcinogens. In conclusion, knowledge on xenobiotic metabolism would be of utmost relevance with regard to health recommendations and in the development of chemopreventive intervention strategies. Importantly, the acquisition of such knowledge strongly depends on the choice of appropriate tools of which an overview is given in this chapter. Investiga- tions can be carried out at the level of DNA, mRNA, protein, and enzyme activity, each giving individual information on particular aspects of xenobiotic metabolism. The pattern reaches from the genetically determined precondition to the ultimate action at the metabolized chemical possibly influenced by acquired modifications. In the end, chemopreventive action should be based on wide knowledge derived from several if not all of these end points. 256j 16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research References

1 Gerhauser, C. (2005) Beer constituents 9 Coles, B., Nowell, S.A., MacLeod, S.L., as potential cancer chemopreventive Sweeney, C., Lang, N.P. and Kadlubar, F.F. agents. European Journal of Cancer, 41, (2001) The role of human glutathione 1941–1954. S-transferases (hGSTs) in the 2 Huber, W.W. and Parzefall, W. (2005) detoxification of the food-derived Modification of N-acetyltransferases and carcinogen metabolite N-acetoxy-PhIP, glutathione S-transferases by coffee and the effect of a polymorphism in components: possible relevance for hGSTA1 on colorectal cancer risk. cancer risk. Methods in Enzymology, 401, Mutation Research, 482,3–10. 307–341. 10 Hein, D.W., Doll, M.A., Fretland, A.J., Leff, 3 Schwab, C.E., Huber, W.W., Parzefall, W., M.A., Webb, S.J., Xiao, G.H., Hietsch, G., Kassie, F., Schulte Hermann, Devanaboyina, U.S., Nangju, N.A. and R. and Knasmuller, S. (2000) Search for Feng, Y. (2000) Molecular genetics and compounds that inhibit the genotoxic and epidemiology of the NAT1 and NAT2 carcinogenic effects of heterocyclic acetylation polymorphisms. Cancer aromatic amines. Critical Reviews in Epidemiology, Biomarkers & Prevention, 9, Toxicology, 30,1–69. 29–42. 4 Schulte-Hermann, R., Marian, B. and 11 Sweeney, C., Coles, B.F., Nowell, S., Lang, Bursch, W. (1999) Tumor promotion, in N.P. and Kadlubar, F.F. (2002) Novel Toxicology (eds H. Marquardt, S., Sch€afer, markers of susceptibility to carcinogens in R. McClellan and F. Welsch), Academic diet: associations with colorectal cancer. Press. Toxicology, 181–182,83–87. 5 Mullauer, L., Grasl-Kraupp, B., Bursch, W. 12 Coles, B.F. and Kadlubar, F.F. (2003) and Schulte-Hermann, R. (1996) Detoxification of electrophilic compounds Transforming growth factor beta by glutathione S-transferase catalysis: 1-induced cell death in preneoplastic foci determinants of individual response to of rat liver and sensitization by the chemical carcinogens and antiestrogen tamoxifen. Hepatology, 23, chemotherapeutic drugs? Biofactors, 17, 840–847. 115–130. 6 Grasl-Kraupp, B., Bursch, W., Ruttkay- 13 Sohn, O.S., Fiala, E.S., Requeijo, S.P., Nedecky, B., Wagner, A., Lauer, B. and Weisburger, J.H. and Gonzalez, F.J. (2001) Schulte-Hermann, R. (1994) Food Differential effects of CYP2E1 status on restriction eliminates preneoplastic cells the metabolic activation of the colon through apoptosis and antagonizes carcinogens azoxymethane and carcinogenesis in rat liver. Proceedings methylazoxymethanol. Cancer Research, of the National Academy of Sciences 61, 8435–8440. of the United States of America, 91, 14 Guengerich, F.P. (2008) Cytochrome p450 9995–9999. and chemical toxicology. Chemical Research 7 Atiqur Rahman, M. and Toi, M. (2003) in Toxicology, 21,70–83. Anti-angiogenic therapy in breast cancer. 15 Nebert, D.W. and Dalton, T.P. (2006) The Biomedicine & Pharmacotherapy, 57, role of cytochrome P450 enzymes in 463–470. endogenous signalling pathways and 8 Ingelman-Sundberg, M. (2004) Human environmental carcinogenesis. Nature drug metabolising cytochrome P450 Reviews. Cancer, 6, 947–960. enzymes: properties and polymorphisms. 16 Talalay, P. and Dinkova-Kostova, A.T. Naunyn-Schmiedebergs Archives of (2004) Role of nicotinamide quinone Pharmacology, 369,89–104. oxidoreductase 1 (NQO1) in protection References j257

against toxicity of electrophiles and cancer susceptibility of NAT2 slow reactive oxygen intermediates. Methods in acetylators towards aromatic amines: a Enzymology, 382, 355–364. review considering ethnic differences. 17 Cuendet, M., Oteham, C.P., Moon, R.C. Toxicology Letters, 128, 229–241. and Pezzuto, J.M. (2006) Quinone 26 Huber, W.W., Prustomersky, S., Delbanco, reductase induction as a biomarker for E.H., Uhl, M., Scharf, G., Turesky, R.J., cancer chemoprevention. Journal of Thier, R. and Schulte-Hermann, R. (2002) Natural Products, 69, 460–463. Enhancement of the chemoprotective 18 Jaiswal, A.K. (1994) Jun and Fos regulation enzymes glucuronosyl transferase and of NAD(P)H: quinone oxidoreductase glutathione transferase by the coffee gene expression. Pharmacogenetics, 4, components Kahweol and Cafestol in 1–10. specific organs of the rat. Archives of 19 Hayes, J.D., Flanagan, J.U. and Jowsey, I.R. Toxicology, 76, 209–217. (2005) Glutathione transferases. Annual 27 King, C.D., Rios, G.R., Green, M.D. Review of Pharmacology and Toxicology, 45, and Tephly, T.R. (2000) UDP- 51–88. glucuronosyltransferases. Current Drug 20 Coles, B.F. and Kadlubar, F.F. (2005) Metabolism, 1, 143–161. Human alpha class glutathione S- 28 Nowell, S.A., Massengill, J.S., Williams, S., transferases: genetic polymorphism, Radominska Pandya, A., Tephly, T.R., expression, and susceptibility to disease. Cheng, Z., Strassburg, C.P., Tukey, R.H., Methods in Enzymology, 401,9–42. MacLeod, S.L., Lang, N.P. and Kadlubar, 21 Huber, W.W., Scharf, G., Rossmanith, W., F.F. (1999) Glucuronidation of Prustomersky, S., Grasl-Kraupp, B., Peter, 2-hydroxyamino-1-methyl-6- B., Turesky, R.J. and Schulte-Hermann, R. phenylimidazo[4,5-b]pyridine (2002) The coffee components Kahweol by human microsomal UDP- and Cafestol induce g-glutamylcysteine glucuronosyltransferases: synthetase, the rate limiting enzyme of identification of specific UGT1A family chemoprotective glutathione synthesis, in isoforms involved. Carcinogenesis, 20, several organs of the rat. Archives of 1107–1114. Toxicology, 75, 685–694. 29 Kensler, T.W. (1997) Chemoprevention 22 Hayes, J.D. and McMahon, M. (2001) by inducers of carcinogen detoxication Molecular basis for the contribution of the enzymes. Environmental Health antioxidant responsive element to cancer Perspectives, 105 (Suppl. 4), 965–970. chemoprevention. Cancer Letters, 174, 30 Hein, D.W. (2006) N-Acetyltransferase 2 103–113. genetic polymorphism: effects of 23 Lu, S.C. (1999) Regulation of hepatic carcinogen and haplotype on urinary glutathione synthesis: current concepts bladder cancer risk. Oncogene, 25, and controversies. FASEB Journal, 13, 1649–1658. 1169–1183. 31 Kadlubar, F.F. and Badawi, A.F. (1995) 24 Nowell, S., Ambrosone, C.B., Ozawa, S., Genetic susceptibility and MacLeod, S.L., Mrackova, G., Williams, S., carcinogen–DNA adduct formation in Plaxco, J., Kadlubar, F.F. and Lang, N.P. human urinary bladder carcinogenesis. (2000) Relationship of phenol Toxicology Letters, 82–83, 627–632. sulfotransferase activity (SULT1A1) 32 Lang, N.P., Butler, M.A., Massengill, J., genotype to sulfotransferase phenotype in Lawson, M., Stotts, R.C., Hauer Jensen, M. platelet cytosol. Pharmacogenetics, 10, and Kadlubar, F.F. (1994) Rapid metabolic 789–797. phenotypes for acetyltransferase and 25 Golka, K., Prior, V., Blaszkewicz, M. and cytochrome P4501A2 and putative Bolt, H.M. (2002) The enhanced bladder exposure to food-borne heterocyclic 258j 16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research

amines increase the risk for colorectal 40 Godde, R., Akkad, D.A., Arning, L., cancer or polyps. Cancer Epidemiology, Dekomien, G., Herchenbach, J., Biomarkers & Prevention, 3, 675–682. Kunstmann, E., Meins, M., Wieczorek, S., 33 Minchin, R.F., Kadlubar, F.F. and Ilett, Epplen, J.T. and Hoffjan, S. (2006) K.F. (1993) Role of acetylation in colorectal Electrophoresis of DNA in human genetic cancer. Mutation Research, 290,35–42. diagnostics – state-of-the-art, alternatives 34 Dinkova-Kostova, A.T., Liby, K.T., and future prospects. Electrophoresis, 27, Stephenson, K.K., Holtzclaw, W.D., Gao, 939–946. X., Suh, N., Williams, C., Risingsong, R., 41 ODonovan, M.C., Oefner, P.J., Roberts, Honda, T., Gribble, G.W., Sporn, M.B. and S.C., Austin, J., Hoogendoorn, B., Guy, C., Talalay, P. (2005) Extremely potent Speight, G., Upadhyaya, M., Sommer, S.S. triterpenoid inducers of the phase 2 and McGuffin, P. (1998) Blind analysis of response: correlations of protection denaturing high-performance liquid against oxidant and inflammatory stress. chromatography as a tool for mutation Proceedings of the National Academy of detection. Genomics, 52,44–49. Sciences of the United States of America, 102, 42 Livak, K.J., Flood, S.J., Marmaro, J., Giusti, 4584–4589. W. and Deetz, K. (1995) Oligonucleotides 35 Lampe, J.W., King, I.B., Li, S., Grate, M.T., with fluorescent dyes at opposite ends Barale, K.V., Chen, C., Feng, Z. and Potter, provide a quenched probe system useful J.D. (2000) Brassica vegetables increase for detecting PCR product and nucleic and apiaceous vegetables decrease acid hybridization. PCR Methods and cytochrome P450 1A2 activity in humans: Applications, 4, 357–362. changes in caffeine metabolite ratios in 43 Wang, D.G., Fan, J.B., Siao, C.J., Berno, A., response to controlled vegetable diets. Young, P., Sapolsky, R., Ghandour, G., Carcinogenesis, 21, 1157–1162. Perkins, N., Winchester, E., Spencer, J., 36 Huber, W.W., Teitel, C.H., Coles, B.F., Kruglyak, L., Stein, L., Hsie, L., Topaloglou, King, R.S., Wiese, F.W., Kaderlik, K.R., T., Hubbell, E., Robinson, E., Mittmann, Casciano, D.A., Shaddock, J.G., Mulder, M., Morris, M.S., Shen, N., Kilburn, D., G.J., Ilett, K.F. and Kadlubar, F.F. (2004) Rioux, J., Nusbaum, C., Rozen, S., Potential chemoprotective effects of the Hudson, T.J., Lipshutz, R., Chee, M. coffee components kahweol and cafestol and Lander, E.S. (1998) Large-scale palmitates via modification of hepatic identification, mapping, and genotyping of N-acetyltransferase and glutathione single-nucleotide polymorphisms in the S-transferase activities. Environmental and human genome. Science, 280 1077–1082. Molecular Mutagenesis, 44, 265–276. 44 Gerard, G.F., Fox, D.K., Nathan, M. and 37 Shi, M.M. (2001) Enabling large-scale DAlessio, J.M. (1997) Reverse pharmacogenetic studies by high- transcriptase. The use of cloned Moloney throughput mutation detection and murine leukemia virus, reverse genotyping technologies. Clinical transcriptase to synthesize DNA from Chemistry, 47, 164–172. RNA. Molecular Biotechnology, 8,61–77. 38 Brown, T. (2001) Southern blotting. 45 Grusch, M., Drucker, C., Peter- Current Protocols in Protein Science, Vorosmarty, B., Erlach, N., Lackner, A., Appendix 4, Appendix 4G. Losert, A., Macheiner, D., Schneider, W.J., 39 Meijerman, I., Sanderson, L.M., Smits, Hermann, M., Groome, N.P., Parzefall, P.H., Beijnen, J.H. and Schellens, J.H. W., Berger, W., Grasl-Kraupp, B. and (2007) Pharmacogenetic screening Schulte-Hermann, R. (2006) Deregulation of the gene deletion and duplications of of the activin/follistatin system in CYP2D6. Drug Metabolism Reviews, 39, hepatocarcinogenesis. Journal of 45–60. Hepatology, 45, 673–680. References j259

46 Ginzinger, D.G. (2002) Gene 53 Snawder, J.E. and Lipscomb, J.C. (2000) quantification using real-time quantitative Interindividual variance of cytochrome PCR: an emerging technology hits the P450 forms in human hepatic mainstream. Experimental Hematology, 30, microsomes: correlation of individual 503–512. forms with xenobiotic metabolism and 47 Grusch, M., Rosenberger, G., Fuhrmann, implications in risk assessment. G., Braun, K., Titscher, B., Szekeres, T., Regulatory Toxicology and Pharmacology, Fritzer-Skekeres, M., Oberhuber, G., 32, 200–209. Krohn, K., Hengstschlaeger, M., Krupitza, 54 Steinkellner, H., Hoelzl, C., Uhl, M., G. and Jayaram, H.N. (1999) Benzamide Cavin, C., Haidinger, G., Gsur, A., Schmid, riboside induces apoptosis independent of R., Kundi, M., Bichler, J. and Knasmuller, Cdc25A expression in human ovarian S. (2005) Coffee consumption induces carcinoma N.1 cells. Cell Death and GSTP in plasma and protects lymphocytes Differentiation, 6, 736–744. against ( þ /)-anti-benzo[a]pyrene-7,8- 48 Huber, W.W., Rossmanith, W., Grusch, M., dihydrodiol-9,10-epoxide induced DNA- Haslinger, E., Prustomersky, S., Peter- damage: results of controlled human Vorosmarty,B., Parzefall, W.,Scharf, G. and intervention trials. Mutation Research, 591, Schulte-Hermann, R. (2008) Effects of 264–275. coffeeanditschemopreventivecomponents 55 Sy, S.K., Ciaccia, A., Li, W., Roberts, E.A., kahweol and cafestol on cytochrome P450 Okey, A., Kalow, W. and Tang, B.K. (2002) and sulfotransferase in rat liver. Food and Modeling of human hepatic CYP3A4 Chemical Toxicology, 46,1230–1238. enzyme kinetics, protein, and mRNA 49 Chin, K.V. and Kong, A.N. (2002) indicates deviation from log-normal Application of DNA microarrays in distribution in CYP3A4 gene expression. pharmacogenomics and toxicogenomics. European Journal of Clinical Pharmacology, Pharmaceutical Research, 19, 1773–1778. 58, 357–365. 50 Vejda, S., Cranfield, M., Peter, B., Mellor, 56 Drucker, C., Parzefall, W., Teufelhofer, O., S.L., Groome, N., Schulte-Hermann, R. Grusch, M., Ellinger, A., Schulte- and Rossmanith, W.(2002) Expression and Hermann, R. and Grasl-Kraupp, B. (2006) dimerization of the rat activin subunits bC Non-parenchymal liver cells support the and bE: evidence for the formation of novel growth advantage in the first stages of activin dimers. Journal of Molecular hepatocarcinogenesis. Carcinogenesis, 27, Endocrinology, 28, 137–148. 152–161. 51 Higgins, L.G., Cavin, C., Itoh, K., 57 Schausberger, E., Hufnagl, K., Parzefall, Yamamoto, M. and Hayes, J.D. (2008) W., Gerner, C., Kandioler-Eckersberger, D., Induction of cancer chemopreventive Wrba, F., Klimpfinger, M., Schulte- enzymes by coffee is mediated by Hermann, R. and Grasl-Kraupp, B. (2004) transcription factor Nrf2. Evidence that the Inherent growth advantage of (pre) coffee-specific diterpenes cafestol and malignant hepatocytes associated with kahweol confer protection against nuclear translocation of pro-transforming acrolein. Toxicology and Applied growth factor alpha. British Journal of Pharmacology, 226, 328–337. Cancer, 91, 1955–1963. 52 Wang, X.J., Hayes, J.D. and Wolf, C.R. 58 Ostlund Farrants, A.-K., Meyer, D.J., Coles, (2006) Generation of a stable antioxidant B., Southan, C., Aitken, A., Johnson, P.J. response element-driven reporter gene cell and Ketterer, B. (1987) The separation of line and its use to show redox-dependent glutathione transferase subunits by using activation of nrf2 by cancer reverse-phase high-pressure liquid chemotherapeutic agents. Cancer Research, chromatography. The Biochemical Journal, 66, 10983–10994. 245, 423–428. 260j 16 Measurement of Enzymes of Xenobiotic Metabolism in Chemoprevention Research

59 Teufelhofer, O., Parzefall, W., Elbling, L., alpha, beta-unsaturated aldehyde products Kainzbauer, E., Grasl-Kraupp, B., of radical reactions and lipid peroxidation Zielinski, C., Schulte-Hermann, R. and by human glutathione transferases. Gerner, C. (2006) Divide and conquer: rat Proceedings of the National Academy of liver tissue proteomics based on the Sciences of the United States of America, 91, analysis of purified constituents. 1480–1484. Electrophoresis, 27, 4112–4120. 67 Fjellstedt, T.A., Allen, R.H., Duncan, B.K. 60 Wang, Y., Al-Gazzar, A., Seibert, C., Sharif, and Jakoby, W.B. (1973) Enzymatic A., Lane, C. and Griffiths, W.J. (2006) conjugation of epoxides with glutathione. Proteomic analysis of cytochromes P450: a The Journal of Biological Chemistry, 248, mass spectrometry approach. Biochemical 3702–3707. Society Transactions, 34, 1246–1251. 68 Thier, R., Wiebel, F.A., Schulz, T.G., 61 Turesky, R.J., Lang, N.P., Butler, M.A., Hinke, A., Bruning, T. and Bolt, H.M. Teitel, C.H. and Kadlubar, F.F. (1991) (1998) Comparison of GST theta activity in Metabolic activation of carcinogenic liver and kidney of four species. Archives of heterocyclic aromatic amines by human Toxicology Supplement, 20, 471–474. liver and colon. Carcinogenesis, 12, 69 Girardini, J., Amirante, A., Zemzoumi, K. 1839–1845. and Serra, E. (2002) Characterization of an 62 Burke, M.D. and Mayer, R.T. (1974) omega-class glutathione S-transferase Ethoxyresorufin: direct fluorimetric from Schistosoma mansoni with assay of a microsomal O-dealkylation glutaredoxin-like dehydroascorbate which is preferentially inducible by reductase and thiol transferase activities. 3-methylcholanthrene. Drug Metabolism European Journal of Biochemistry, 269, and Disposition, 2, 583–588. 5512–5521. 63 Sanchez, R.I., Mesia-Vela, S. and 70 Tong, Z., Board, P.G. and Anders, M.W. Kauffman, F.C. (2003) Induction of NAD (1998) Glutathione transferase zeta (P)H quinone oxidoreductase and catalyses the oxygenation of the carcinogen glutathione S-transferase activities in dichloroacetic acid to glyoxylic acid. The livers of female August-Copenhagen Irish Biochemical Journal, 331 (Pt 2), 371–374. rats treated chronically with estradiol: 71 Ilett, K.F., Ingram, D.M., Carpenter, D.S., comparison with the Sprague–Dawley rat. Teitel, C.H., Lang, N.P., Kadlubar, F.F. and The Journal of Steroid Biochemistry and Minchin, R.F. (1994) Expression of Molecular Biology, 87, 199–206. monomorphic and polymorphic 64 Habig, W.H., Pabst, M.J. and Jakoby, W.B. N-acetyltransferases in human colon. (1974) Glutathione S-transferases. The Biochemical Pharmacology, 47, 914–917. first enzymatic step in, mercapturic acid 72 Reeves, P.T., Minchin, R.F. and Ilett, K.F. formation. Journal of Biological Chemistry, (1988) Induction of sulfamethazine 249, 7130–7139. acetylation by hydrocortisone in the rabbit. 65 Manson, M.M., Ball, H.W., Barrett, M.C., Drug Metabolism and Disposition, 16, Clark, H.L., Judah, D.J., Williamson, G. 110–115. and Neal, G.E. (1997) Mechanism of action 73 Chan, K., Miners, J.O. and Birkett, D.J. of dietary chemoprotective agents in rat (1988) Direct and simultaneous high- liver: induction of phase I and II drug performance liquid chromatographic metabolizing enzymes and aflatoxin B1 assay for the determination of metabolism. Carcinogenesis, 18, p-aminobenzoic acid and its conjugates 1729–1738. in human urine. Journal of 66 Berhane, K., Widersten, M., Engstrom, A., Chromatography, 426, 103–109. Kozarich, J.W. and Mannervik, B. (1994) 74 Frame, L.T.,Ozawa, S., Nowell, S.A., Chou, Detoxication of base propenals and other H.C., DeLongchamp, R.R., Doerge, D.R., References j261

Lang, N.P. and Kadlubar, F.F. (2000) A 80 Mazur, W., Gonciarz, M., Kajdy, M., simple colorimetric assay for phenotyping Mazurek, U., Jurzak, M., Wilczok, T. and the major human thermostable phenol Gonciarz, Z. (2003) Blood serum sulfotransferase (SULT1A1) using platelet glutathione alpha s-transferase (alpha cytosols. Drug Metabolism and Disposition, GST) activity during antiviral therapy 28, 1063–1068. in patients with chronic hepatitis C. 75 Kaderlik, K.R., Mulder, G.J., Turesky, R.J., Medical Science Monitor, 9 (Suppl. 3), Lang, N.P., Teitel,C.H., Chiarelli, M.P. and 44–48. Kadlubar, F.F. (1994) Glucuronidation of 81 Le Marchand, L., Franke, A.A., Custer, L., N-hydroxy heterocyclic amines by human Wilkens, L.R. and Cooney, R.V. (1997) and rat liver microsomes. Carcinogenesis, Lifestyle and nutritional correlates of 15, 1695–1701. cytochrome CYP1A2 activity: inverse 76 Roland, N., Rabot, S. and Nugon-Baudon, associations with plasma lutein and alpha- L. (1996) Modulation of the biological tocopherol. Pharmacogenetics, 7,11–19. effects of glucosinolates by inulin and 82 Le Marchand, L., Sivaraman, L., Franke, oat fibre in gnotobiotic rats inoculated A.A., Custer, L.J., Wilkens, L.R., Lau, A.F. with a human whole faecal flora. Food and Cooney, R.V. (1996) Predictors of and Chemical Toxicology, 34, 671–677. N-acetyltransferase activity: should 77 Bradford, M.A. (1976) A rapid sensitive caffeine phenotyping and NAT2 method for the quantification of genotyping be used interchangeably in microgram quantities of protein utilizing epidemiological studies? Cancer the principle of protein binding. Analytical Epidemiology, Biomarkers & Prevention, 5, Biochemistry, 72, 248–254. 449–455. 78 Sreerama, L., Hedge, M.W. and Sladek, 83 Knasmuller, S., Mersch-Sundermann, N.E. (1995) Identification of a class 3 V., Kevekordes, S., Darroudi, F., Huber, aldehyde dehydrogenase in human saliva W.W., Hoelzl, C., Bichler, J. and Majer, and increased levels of this enzyme, B.J. (2004) Use of human-derived liver glutathione S-transferases, and cell lines for the detection of DT-diaphorase in the saliva of subjects environmental and dietary genotoxicants: who continually ingest large quantities of current state of knowledge. Toxicology, coffee or broccoli. Clinical Cancer Research, 198, 315–328. 1, 1153–1163. 84 Scharf, G., Prustomersky, S., Knasmuller, 79 Giannini, E., Risso, D., Ceppa, P., Botta, F., S., Schulte-Hermann, R. and Huber, W.W. Chiarbonello, B., Fasoli, A., Malfatti, F., (2003) Enhancement of glutathione and Romagnoli, P., Lantieri, P.B. and Testa, R. g-glutamylcysteine synthetase, the rate (2000) Utility of alpha-glutathione S- limiting enzyme of glutathione synthesis, transferase assessment in chronic by chemoprotective plant-derived food and hepatitis C patients with near normal beverage components in the human alanine aminotransferase levels. Clinical hepatoma cell line HepG2. Nutrition and Biochemistry, 33, 297–301. Cancer, 45,74–83. j263

17 Methods for the Analysis of DNA Methylation Keith N. Rand and Peter L. Molloy

17.1 Introduction

Among many methods that can be used to determine or estimate methylation levels, we concentrate in this short review on those methods that have become reasonably popular, or those that in our opinion are particularly worthy of consideration for use in the field of chemoprevention of cancer. We consider methods that determine the overall level of methylation of the genome, either in total or by assaying specific classes of repeat sequences, methods that can be used to interrogate the methylation status of individual genes, and finally consider emerging methods that can be used to profile DNA methylation across the genome and so be used to identify specific changes associated with an intervention. Comprehensive reviews with references to a wide range of methods are already available and recommended for further reading. The review by Havlis and Trbusek [1] contains a table that compares sensitivity, amount of DNA needed, DNA form, repeatability, limitations, and major advantages, while that of Fraga and Esteller [2] is particularly good in its descriptions of HPLC, HPCE, and in situ methods. A more recent extensive review of methods was published in 2006 [3]. For the wide range of methods that depend upon bisulfite conversion, the review by Clark et al. [4] has full details on the bisulfite conversion protocol and a troubleshooting guide. Later reviews cover quantitative PCR methods as applied to methylation studies [5], and a well-written comprehensive review clearly explains the principles of the different methods [6]. Useful reviews covering approaches to genome-wide DNA methylation analysis are those of Ushijima [7], Zilberman and Henikoff [8], and Beck and Rakyan [9]. In addition to considering whether the effect of dietary or other factors on chemoprevention of cancer and DNA damage is mediated through epigenetic mechanisms, it is important to consider what should be measured. In the case of the human genome, pie graphs that allow comparison of the composition of the genome and the CpG distribution have been constructed [10]. The major repetitive

elements are SINEs (mostly Alu elements) containing 27% of the CpGs of the whole genome, LINEs 12%, and LTRs 7%. Other important repeats are Alpha satellite and Classical Satellite, containing 2.8 and 4.2% of CpGs, respectively. Because repeat sequences are also normally heavily methylated, an even higher percentage of total methylated CpGs are present in these repeats. Researchers using animal models must of course bear in mind the differences in the proportions of the different parts of the particular animals genome and their CpG content. In cancer, although a reduction in overall methylation level is often observed, there is also a redistribution of methylation so that some parts of the genome such as CpG islands and sometimes regions on a megabase scale show large increases in methylation. Most of the loss of methylation in DNA of cancer cells involves repeat sequences, but normally methylated CpG-rich regions near some genes also become demethylated in cancer. The methylome thus comprises a number of different components, and it is yet to be understood how these may be differentially impacted by dietary and other factors.

17.2 Measurement of Global and Repeat Sequence Methylation

Since a number of well-known dietary factors, including folate and vitamin B12, are critically involved in one carbon metabolism and the supply of methyl groups through the donor S-adenosyl methionine, it is to be expected that levels of dietary supply might impact on the total level of 5-methyl cytosine (5meC) in the genome. Measurement of total 5meC content can thus provide an indication of the potential for an epigenetic impact. Even for dietary impacts that might be expected to be uniform across an animal effects have been seen to be significantly organ and target specific [11]. It is also important to bear in mind that measurement of total methylation levels largely reflects the averaged methylation of different repeat sequence families in the genome. There is evidence from the selective effects of mutations in ICF syndrome (mutated DNMT3B) as well as differential hypomethylation of repeat families that have been seen in some cancers that there is a level of differential and independent control over the methylation of different repeat sequence families. Targeted assays are expected to be valuable in studying chromosomal instability in cancer that has been associated with hypomethylation of satellite and pericentromeric repeats such as Sat2 and Sat3 [12] and that contain only a small proportion of CpG sites. Likewise the demethylation of promoter-containing inter- spersed repeats such as LINE-1 and Alu elements may impact cancer development differently – for example, through transcriptional interference – and again targeted assays could provide useful insights into the chain of events leading to cancer initiation and development. In Table 17.1 we consider assays that measure both overall levels of genomic methylation as well as assays for specific repeat sequence classes. The most direct and comprehensive means of determining the genomic content of 5meC involve digestion of DNA to nucleotides or bases and their separation using HPLC, HPCE, or thin layer chromatography to quantify relative levels. These methods 17.2 Measurement of Global and Repeat Sequence Methylation j265

Table 17.1 Global and repeat sequence DNA methylation analysis.

Measures DNA required Comments References

HPLC Ratio of 5meC 1–5 mg Enzymatic digestion to [36] to C þ 5meC nucleotides followed by HPLC HPLC/tandem Ratio of 5meC 1 mg Acid hydrolysis to free [13] mass to C þ 5meC bases, followed by HPLC spectrometry and mass spectrometry. RNA removal is critical HPCE Ratio of 5meC 0.5–1 mg Acid hydrolysis to free [37] to C þ 5meC bases, followed by HPCE Thin layer Ratio of 5meC <1 mg Requires 32P labeling [38, 39] chromatography to C þ 5meC 5meC Amount of 2 mg Immunoassay for 5meC [14] Immunoassay 5meC in DNA in denatured DNA fixed to membrane. Accurate DNA quantification is critical Methyl acceptor Unmethylated 500 ng Use of dam methylase [40] assay CpG sites control allows better standardization. Not suitable for use with damaged DNA Cytosine exten- Ratio of C to 1 mg or less Use of HpaII and [16] sion assay 5meC at BssH11, respectively, restriction sites biases toward assessment of repeat sequences and CpG islands LUMA Ratio of C to 100–500 ng Pyrosequencing-based [17] 5meC at version of cytosine restriction sites extension assay; suitable for high throughput Southern blot Length 100–500 ng Qualitative or semiquan- [12] distribution of titative estimation of restriction methylation levels fragments Repeat Sequence 5meC level at 50 ng bisulfite- Proportion of C versus T [19, 41] PCR specific sites treated DNA at target CpG sites within repeat measured using either sequence COBRA assay or amplicons pyrosequencing

tend to be time consuming and laborious, and traditional HPLC analysis requires amounts of DNA that may be limiting in some circumstances. A recent publication describes an approach coupling acid hydrolysis of DNA to release bases followed by HPLC and mass spectrometry detection that provides an efﬁcient and reasonably high throughput means of quantiﬁcation. The method was applied to a study of the 266j 17 Methods for the Analysis of DNA Methylation

effect of MTHFR C677T genotype on age-related changes in DNA methylation [13]. An anti-5meC DNA immunoassay provides an alternate approach that has been applied to a rat model of chemoprevention of colon cancer by celoxicob [14]. The choice of method will depend on the availability of necessary equipment for each method, and thin layer chromatography separation provides a reasonable low-tech option. A number of methods have been developed as surrogates for the measurement of genomic methylation levels. The CpG specific DNA methyltransferase Sss1 can be used to measure the available unmethylated CpG sites in the genome, and the use of this approach is made more accurate by using Dam methylase (GATC sites) to normalize the amount of methylatable DNA. This method has been used for example in the studies on folic acid supplements and prevention of colorectal cancer [15]. It is important to ensure the quality of input DNA as it has been reported that damaged DNA is not good substrate and can give inaccurate but reproducible results [2]. Methylation at a subset of CpG sites across the genome can be determined following digestion of DNA with the methylation sensitive and insensitive isoschi- zomers HpaII and MspI that cut at CCGG sites. Incorporation of radioactive dCTP provides a measurement of unmethylated sites (cut by HpaII) compared with total sites (cut by MspI). BssHII can be used to bias measurement toward CpG islands, where its cut site CGCGCG is enriched [16]. A development of the end-filling approach, the LUMA assay, is configured for high-throughput analysis but requires specialized equipment. It is based on pyrosequencing technology, and the frequency of HpaII cut sites is normalized in the same reaction to the frequency of EcoRI sites [17]. Measurement of methylation levels of specific repeat sequence families has historically relied on digestion with methylation sensitive restriction enzymes followed by Southern blotting and hybridization with specific repeat sequence probes. Though somewhat laborious, this method allows a reasonable qualitative estimation of loss of methylation, provided the initial DNA is of good quality. Methods for PCR amplification of repeat sequences following bisulfite treatment of genomic DNA have begun to emerge. Because of the heterogeneous nature of repeat sequences, much of which has arisen through mutation of CpG sites to TpG or CpA, design considerations are important for their proper application and interpretation. Reaction of single stranded DNA with sodium bisulfite results in the deamination of cytosine to form uracil, while 5meC is unreactive. Thus, after amplification, the state of a methylatable CpG site will be read as CpG or TpG depending on whether the cytosine was methylated or not respectively in the original DNA. In designing primers for amplifying repeat sequences from bisulfite-treated DNA, it is important to avoid covering CpG sites as this can lead to biased priming and it is also important to validate that amplification from unmethylated and methylated template DNA is equivalent as sequence-dependent amplification bias is not uncommon (see reference [4] for discussion). In quantifying methylation at CpG sites within amplicons, it is important to take into account that while C at a CpG site corresponds to a methylated cytosine in the original DNA, a T may represent either an unmethylated cytosine or a sequence variation, that is, a TpG site (or arise 17.3 Measurement of Methylation of Individual Genes or Regions j267 from other variations such as unmethylatable CpA site). The level of background mutation at a CpG site can be determined from analysis of DNA fully methylated in vitro using Sss1 methylase. PCR-based assays for human LINE-1 and Alu elements that use COBRA or pyrosequencing to quantify repeat sequence hypomethylation have been developed by Yang et al. [18]. A similar assay for rat LINE-1 sequences [19] was used to study DNA hypomethylation in choline-induced liver cancer. The use of PCR-based assays for hypomethylation of different repeat sequence families has the potential to provide a higher resolution analysis using lower amounts of input DNA, and we have been developing such assays targeted to different repeat families (Rand, Molloy et al., in preparation).

17.3 Measurement of Methylation of Individual Genes or Regions

Changes in the methylation state of multiple genes is now a well-recognized hallmark of cancer development [20]. Hypermethylation of promoters of many genes in cancer is accompanied by their transcriptional silencing. Among these, the most extensively studied are those classified as CpG islands (6.8% of CpGs are found in CpG islands in humans). Hypomethylation of a number of gene promoters that are normally developmentally or tissue-specifically regulated is also common in cancer. The majority of methods used for analyzing the methylation state of individual genes are based on bisulfite conversion of DNA followed by PCR amplification. The review by Clark et al. [4] describes a range of different assays that are used to measure methylation of bisulfite-treated DNA along with the discussion of potential problems and approaches to overcome them. In Table 17.2 we present a selection of those assays most readily applicable to studies of diet/chemoprevention in cancer development. For measurement of levels of DNA methylation in the range 5–100%, PCR primers should be designed to amplify bisulfite-converted DNA independent of its methylation state. This involves designing primers to regions that lack methylatable CpG sites, but that contain a number of Cs so that primers will be specific for the products of conversion, where Cs are replaced by Us in the target sequence. A critical part of assay development, to ensure that subsequent quantification of methylation is accurate, is to establish that the amplification efficiency of methylated and unmethylated templates is equal. Here we recommend the use of whole genome amplified DNA (using commercial kits based on Phi29 DNA polymerase) as an unmethylated control and DNA methylated in vitro using Sss1 methylase (also commercially available) as methylated control template. The most detailed profile of methylation within an amplicon can be derived by cloning and sequencing individual molecules. This can identify heterogeneity of methylation patterns and different classes of molecules (e.g., unmethylated and highly methylated) such as might be seen on imprinted genes. Chhibber and Schroeder [21] have proposed that single molecule amplification after bisulfite treatment followed by ultradeep sequencing (using barcoding to allow multiple 268j 17 Methods for the Analysis of DNA Methylation

Table 17.2 Methylation analysis of specific genes following bisulfite treatment of DNA.

Measures Comments References

Clonal Position of 5-meCs in Provides most detailed anal- [4] sequencing individual molecules ysis, showing nature of heterogeneity Direct PCR prod- Average level of 5-meC Semiquantitative for methyl- [4, 42] uct sequencing at individual positions ation levels above 10% Pyrosequencing Average level of 5-meC High throughput and [43] at individual positions quantitative. Requires specialized equipment (commercial services available) massCLEAVE Quantification of meth- Amplicon is copied to RNA [44] (www. ylation at individual and from T7 RNA polymerase sequenom.com) groups of CpG sites promoter incorporated in primer. Base-specific cleavage produces fragments that are analyzed by mass spectrometry Melting profile Melting temperature of Simple to perform: depends [22] analysis amplicon on number of CpG sites. Can distinguish fully methylated and unmethylated as well as amplicons showing intermediate methylation ERMA Level of CpG sites in Application of methyl accep- [45] amplicon tance assay to individual amplicons MANIC Cytosine level in top Biotin-primer-based capture [46] strand of top strand after incorporation of fluorescent dCTP COBRA Fraction of DNA cut at Applicable for subset of sites [23, 47] a specific site where methylation (i.e., presence of CpG versus TpG) in amplicon results in the presence or absence of a restriction site. Improved quantification using capillary electrophoresis SNuPE Methylation at single Selective extension of primer [48, 49] CpG sites at site within amplicon, depending on presence of C or T. Can be adapted to high throughput systems Methylation Fraction of DNA ampli- Primers designed to depend [4, 25] specific PCR fied by methylation specifically on the methyla- specific primers tion state of specific CpGs (either methylated – MSP, or unmethylated – USP) 17.3 Measurement of Methylation of Individual Genes or Regions j269 sample analysis) will provide a high-throughput approach to give much information on methylation haplotypes the actual pattern of methylated CpGs in each molecule. Although providing the most information, the required sequencing of multiple clones is labor intensive, and direct Sanger sequencing or pyrosequencing of amplicons can give average methylation levels at individual CpG sites within an amplicon. Pyrosequencing provides the best quantification, though read lengths are limited to about 100 bases. The mass spectrometry-based massCLEAVE method of Sequenom also provides quantitative data on multiple CpG sites or groups of sites within an amplicon and can be used for high-throughput analysis. A rapid way to assess the proportion of methylated and unmethylated alleles in a population and to identify the presence of intermediate levels of methylation is to conduct a melting profile analysis of the PCR product. This can be done on Real Time PCR machines using the fluorescent dyes such as SybrGreen or Syto9 [22]. Two methods that allow quantification of the overall level of methylation in an amplicon are the ERMA and MANIC assays. The ERMA assay uses Sss1 methylase to methylate all CpG sites (that is originally methylated in genome) in the amplicon using tritiated S-adenosyl methionine and standardizes this against methylation by Dam methylase of GATC sites incorporated in the primers. The MANIC method measures incorporation of fluorescently labeled cytosine into the top stand of an amplicon that is primed using a biotinylated primer. Both methods are relatively straightforward and useful for following methylation levels where the pattern of methylation is heterogeneous. The most widely used method for following methylation at individual sites in an amplicon is COBRA that relies on sequence differences between methylated and unmethylated DNA following bisulfite treatment that give rise to differences in restriction sites. Gel-based analysis can be used to quantify the levels of cut and uncut DNA and infer the methylation level at a site. Care needs to be taken to ensure complete digestion. Also, the narrow dynamic range of ethidium bromide-stained gels can lead to underestimation of strong signals and variable background fluorescence can effect quantification. These problems can be avoided by using capillary-electrophoresis-based analysis [23]. The use of primers internal to amplicon adjacent to a CpG site allows methylation at any CpG site to be quantified through the dependence of extension by a single nucleotide on the presence of a C or T (MS-SNuPE) and can be adapted to high-throughput platforms.

17.3.1 Methylation Sensitive PCR

Many epigenetic changes induced by dietary or environmental factors are likely to occur only in a fraction of cells and not be visible using methods discussed above where the DNA is ampliﬁed in an unbiased manner. Initiating events in cancer development, such as methylation of tumor suppressor gene promoters, may be present only in a small fraction of cells, but it is important to be able to monitor their presence. For example, methylation of multiple tumor-related genes in relation to alcohol and folate intake was studied in colon cancer using methylation sensitive PCR 270j 17 Methods for the Analysis of DNA Methylation

(MSP) [24]. In MSP primers are designed to regions containing methylatable CpG sites, either matching the MSP or the unmethylated (USP) – MSP primers can be used for selective amplification of as little as one methylated molecule among 10 000 unmethylated molecules [25]. For quantification, MSP assays can be monitored in real time using fluorescent dyes or using specific probes to sequences within the amplicon. Standard curves for different fractions of methylated and unmethylated DNA should be constructed and a methylation-independent control PCR run to quantify the level of amplifiable DNA in the reaction. It is important to monitor for artefacts that may arise, for example, through incomplete C to U conversion in the bisulfite reaction; potential pitfalls in application of MSP are discussed in Clark et al. [4].

17.3.2 Non-Bisulfite-Based Assay

An alternate assay approach that allows simultaneous assessment of multiple CpG sites, either within the same or different genes, is the methylation-specific multiple probe ligation assay (MS-MLPA). This assay relies on the lack of digestion at specific sites by methylation sensitive enzymes to leave intact DNA strands that act as templates for ligation of oligonucleotides and subsequent multiplexed PCR amplification [26]. Though requiring an initial investment of effort in developing assays, this assay requires relatively low levels of DNA, does not require pretreatment with bisulfite, and provides a quantitative output. MS-MLPA was used to quantify methylation of 24 tumor suppressor genes in response to different antimetabolites [27]. A variation of MS-MLPA has used bisulfite-treated DNA [28]. A more highly multiplexed system [29] that uses bisulfite-treated DNA and a similar principle to MS-MLPA to interrogate approximately 1500 individual CpG sites is commercially available from Illumina. Sites in this GoldenGate assay are targeted to cancer-related CpG islands, and custom design is also available.

17.4 Scanning Genome-Wide for Changes in DNA Methylation

The assessment of the effects of nutrition and environmental factors on specific gene methylation has largely relied on a candidate gene approach, where the target genes were chosen because of their known or suspected relationship with the metabolic processes or disease under study. The emergence of powerful new technologies for scanning across the genome to identify changes in the state of methylation of single- copy genes offers the prospect of opening up to scrutiny the whole methylome. Although too costly for routine analysis, these technologies will allow an unbiased search for epigenetic impacts, allowing subsequent development of specific targeted assays. A range of methods have been developed that utilize methylation sensitive enzymes to scan for differences in methylation profiles; although incomplete in coverage and often technically challenging, these have been used extensively to 17.4 Scanning Genome-Wide for Changes in DNA Methylation j271 identify methylation changes between normal and cancer tissue (reviewed in [7]). Recent approaches to genome-wide analysis of methylation patterns are well covered in Ref. [8], which contains a useful table comparing platforms for large-scale analysis of DNA methylation and in [9] that discusses global methylation profiling, especially array-based methods and next-generation sequencing. Fan et al. [30] provide a very good review of principles for interpretation of genome-wide studies (although not focusing on methylation). In the discussion below we will focus on the developing new approaches that provide most comprehensive genome coverage and are best supported on commercial platforms. The Illumina Infinium DNA Methylation BeadChip is the only genome-wide array system that uses bisulfite-treated DNA for interrogation of methylation status. It uses arrayed oligonucleotides to interrogate approximately 27 000 CpG sites covering more than 14 000 well-annotated genes and microRNA promoters. The alternate approach is to fractionate the methylated or unmethylated compartment of the genome before analysis using platforms discussed below. Three basic approaches have been used to separate out the methylated or unmethylated fraction of the genome: 1. Shearing DNA to a size range of 400–1000 bp followed by the capture of the methylated fraction using antibodies to 5meC termed MeDIP [31] or using methylated DNA-binding proteins such as the MBD2/MBD3L1 complex that has a high affinity for methylated DNA, termed MIRA [32]. For microarray analysis hybridization of the captured methylated fraction is compared with that of the input, unfractionnated DNA to identify enriched regions. Although possibly being subject to effects of CpG density, the MeDIP procedure has become the most widely used. A Bayesian statistical approach has been developed for analysis of MeDIP microarray hybridization data [33]. 2. A number of variations rely on digestion of DNA with methylation sensitive enzymes. An example of these is the HELP Assay that uses comparative hybridization of the HpaII (methylation-sensitive) and MspI (methylation-insensitive) digestion products of one DNA specimen [34]. 3. Digestion of DNA with the methylation-dependent enzyme McrBC that digests methylated DNA with a low level of sequence specificity. Cutting occurs between two sites of sequence purine-5meC-G spaced 55–1000 base pairs apart. A most recent development of this approach in which hybridization of total DNA is compared with that of the high molecular weight (>1000 bp) unmethylated fraction remaining after McrBC digestion and which is aided by improved bioinformatic tools is the CHARM assay [35]. A number of commercial platforms are available for analysis and these are summarized in Table 17.3. Affymetrix, Agilgent, and Nimblegen all produce oligonucleotide arrays that are suitable for hybridization of DNA fractionated using one of the above three methods and providing different coverage of either promoters or CpG islands. The repertoire of arrays is still evolving, and ideal coverage would at least include all known promoters and CpG islands. Such a custom array (Nimblegen) was used for analysis of DNA fractionated using the 272 j

Table 17.3 Commercially available tools for genome-wide or custom methylation studies. Methylation DNA of Analysis the for Methods 17

Manufacturer/technology Applications/coverage Species Comments, examples of use References

Affymetrix/arrays 25–28 000 promoters Human, mouse 25 mers oligonucleotides. Shorter probe [50] (http://www.affymetrix.com/index. (includes 60% CpG islands) length than other platforms balanced by affx) higher probe density, Coverage from 7toþ2.5 kb Agilent/arrays (http://www.chem. CpG island or promoter ar- Human, mouse, 60 mers, average of 6–10 probes per CpG [51] agilent.com/scripts/PHome.asp rays 15–28 000 regions custom island 5toþ2.5 kb on promoter arrays Nimblegen/arrays http://www. Human, mouse, and rat Human, mouse, 50–75 mers 2.5 kb covered in single [31, 52] nimblegen.com/) promoter arrays, rat, custom array or 6 kb in 2 array set. Human 15 400–18 000 promoters promoter plus CpG island array available. Have been used with MeDip, MIRA, CHARM Illumina/bead arrays Infinium Human Methyla- Human 27 K CpG sites per sample, covering [53] (http://www.illumina.com/) tion27 BeadChip more than 14 000 genes, single CpG resolution. Bisulfite modification then whole genome amplification GoldenGate Cancer set or custom BeadArray, or for higher throughput, [29] VeraCode (which needs BeadXpress Reader) Illumina Solexa/sequencing High-throughput sequencing Not applicable Bisulfite conversion, then attached to [54] (http://www.illumina.com/sequenc- surface and amplified in situ. Sequenced ing/Technology.ilmn) by synthesis using mixture of four fluorescently labeled reversible chain terminators 454 Life Sciences/sequencing High-throughput sequencing Not applicable Bisulfite conversion, then amplification [55–57] (http://www.454.com/) on beads in aqueous-oil emulsion and or Roche website pyrosequencing

Links are only to the main page for each company, as these are less likely to change over time. A search can be made from the main page. References j273

CHARM protocol [35]. A key consideration in utilizing genome-wide hybridization to identify methylation differences is the availability of good bioinformatic support. Different groups have used probe-by-probe comparisons or averaged across a sliding window to smooth out inherent noise in the signals. The next phase of technology development that will provide even more detailed insight into changes in methylation state will come from application of genome-wide sequencing technologies, using either bisulﬁte-treated DNA or DNA fractionated as for array hybridization. Some early examples are included in Table 17.3. However, there are still signiﬁcant technical challenges as well as cost constraints before such approaches enter widespread use.

References

1 Havlis, J. and Trbusek, M. (2002) 8 Zilberman, D. and Henikoff, S. (2007) 5-Methylcytosine as a marker for the Genome-wide analysis of DNA monitoring of DNA methylation. Journal of methylation patterns. Development, Chromatography B-Analytical Technologies 134, 3959–3965. in the Biomedical and Life Sciences, 9 Beck, S. and Rakyan, V.K. (2008) The 781, 373–392. methylome: approaches for global DNA 2 Fraga, M.E. and Esteller, M. (2002) DNA methylation profiling. Trends in Genetics, methylation: a profile of methods and 24, 231–237. applications. Biotechniques, 33, 632. 10 Rollins, R.A., Haghighi, F., Edwards, J.R., 3 Brena, R.M., Huang, T.H.M. and Plass, C. Das, R., Zhang, M.Q., Ju, J.Y. and (2006) Quantitative assessment of DNA Bestor, T.H. (2006) Large-scale structure methylation: potential applications for of genomic methylation patterns. Genome disease diagnosis, classification, and Research, 16, 157–163. prognosis in clinical settings. Journal of 11 Pogribny, I.P., James, S.J., Jernigan, S. and Molecular Medicine, 84, 365–377. Pogribna, M. (2004) Genomic 4 Clark, S.J., Statham, A., Stirzaker, C., hypomethylation is specific for Molloy, P.L. and Frommer, M. (2006) DNA preneoplastic liver in folate/methyl methylation: bisulphite modification and deficient rats and does not occur in analysis. Nature Protocols, 1, 2353–2364. non-target tissues. Mutation Research – 5 Sulewska, A., Niklinska, W., Fundamental and Molecular Mechanisms Kozlowski, M., Minarowski, L., of Mutagenesis, 548,53–59. Niklinski, J., Dabrowska, K. and 12 Ehrlich, M., Hopkins, N.E., Jinag, G.C., Chyczewski, L. (2007) Detection of DNA Dome, J.S., Yu, M.C., Woods, C.B., methylation in eucaryotic cells. Folia Tomlinson, G.E., Chintagumpala, M. et al. Histochemica et Cytobiologica, 45, 315–324. (2003) Satellite DNA hypomethylation in 6 Moskalyov, E.A., Eprintsev, A.T. and karyotyped Wilms tumors. Cancer Genetics Hoheisel, J.D. (2007) DNA methylation and Cytogenetics, 141,97–105. profiling in cancer: from single 13 Kok, R.M., Smith, D.E.C., Barto, R., nucleotides towards the methylome. Spijkerman, A.M.W., Teerlink, T., Molecular Biology, 41, 723–736. Gellekink, H.J., Jakobs, C. and 7 Ushijima, T. (2005) Innovation – detection Smulders, Y.M. (2007) Global DNA and interpretation of altered methylation methylation measured by liquid patterns in cancer cells. Nature Reviews. chromatography-tandem mass Cancer, 5, 223–231. spectrometry: analytical technique, 274j 17 Methods for the Analysis of DNA Methylation

reference values and determinants in 22 Wojdacz, T.K. and Dobrovic, A. (2007) healthy subjects. Clinical Chemistry and Methylation-sensitive high resolution Laboratory Medicine, 45, 903–911. melting (MS-HRM): a new approach for 14 Pereira, M.A., Tao, L., Wang, W., Li, Y., sensitive and high-throughput assessment of Umar, A., Steele, V.E. and Lubet, R.A. methylation. Nucleic Acids Research, 35,e41. (2004) Modulation by celecoxib and 23 Brena, R.M., Auer, H., Kornacker, K., difluoromethylornithine of the Hackanson, B., Raval, A., Byrd, J.C. and methylation of DNA and the estrogen Plass, C. (2006) Accurate quantification receptor-alpha gene in rat colon tumors. of DNA methylation using combined Carcinogenesis, 25, 1917–1923. bisulfite restriction analysis coupled with 15 Mathers, J.C. (2005) Reversal of DNA the Agilent 2100 Bioanalyzer platform. hypomethylation by folic acid Nucleic Acids Research, 34, e17. supplements: possible role in colorectal 24 van Engeland, M., Weijenberg, M.P., cancer prevention. Gut, 54, 579–581. Roemen, G., Brink, M., de Bruine, A.P., 16 Pogribny, I.P., Yi, P. and James, S.J. (1999) Goldbohm, R.A., van den Brandt, P.A., A new method to rapidly assess differences Baylin, S.B. et al. (2003) Effects of dietary in genome-wide and CpG island folate and alcohol intake on promoter methylation in normal and neoplastic methylation in sporadic colorectal cancer: DNA. FASEB Journal, 13, A1550–A2550. The Netherlands Cohort Study on Diet and 17 Karimi, M., Johansson, S., Stach, D., Cancer. Cancer Research, 63, 3133–3137. Corcoran, M., Grander, D., Schalling, M., 25 Herman, J.G., Graff, J.R., Myohanen, S., Bakalkin, G., Lyko, F. et al. (2006) Nelkin, B.D. and Baylin, S.B. (1996) LUMA (LUminometric Methylation Methylation-specific PCR: a novel PCR Assay) – a high throughput method to the assay for methylation status of CpG analysis of genomic DNA methylation. islands. Proceedings of the National Academy Experimental Cell Research, 312, of Sciences of the United States of America, 1989–1995. 93, 9821–9826. 18 Yang, I., Park, I.Y.,Jang, S.M., Shi, L.H., Ku, 26 Nygren, A.O.H., Ameziane, N., H.K. and Park, S.R. (2006) Rapid Duarte, H.M.B., Vijzelaar, R., Waisfisz, Q., quantification of DNA methylation through Hess, C.J., Schouten, J.P. and Errami, A. dNMP analysis following bisulfite–PCR. (2005) Methylation-specific MLPA Nucleic Acids Research, 34, e61. (MS-MLPA): simultaneous detection of 19 Asada, K., Kotake, Y., Asada, R., CpG methylation and copy number Saunders, D., Broyles, R.H., Towner, R.A., changes of up to 40 sequences. Nucleic Fukui, H. and Floyd, R.A. (2006) LINE-1 Acids Research, 33, e128. hypomethylation in a choline-deficiency- 27 Sasaki, S., Kobunai, T., Kitayama, J. and induced liver cancer in rats: dependence Nagawa, H. (2008) DNA methylation and on feeding period. Journal of Biomedicine sensitivity to antimetabolites in cancer cell and Biotechnology, 2006, 17142. lines. Oncology Reports, 19, 407–412. 20 Feinberg, A.P., Ohlsson, R. and 28 Dahl, C. and Guldberg, P. (2007) A ligation Henikoff, S. (2006) The epigenetic assay for multiplex analysis of CpG progenitor origin of human cancer. methylation using bisulfite-treated DNA. Nature Reviews Genetics, 7,21–33. Nucleic Acids Research, 35, e144. 21 Chhibber, A. and Schroeder, B.G. (2008) 29 Bibikova, M., Lin, Z.W., Zhou, L.X., Single-molecule polymerase chain Chudin, E., Garcia, E.W., Wu, B., reaction reduces bias: application to DNA Doucet, D., Thomas, N.J. et al. (2006) methylation analysis by bisulfite High-throughput DNA methylation sequencing. Analytical Biochemistry, profiling using universal bead arrays. 377,46–54. Genome Research, 16, 383–393. References j275

30 Fan, J.B., Chee, M.S. and Gunderson, K.L. epigenetic method for the diagnosis and (2006) Highly parallel genomic assays. classification of brain tumors. Molecular Nature Reviews Genetics, 7, 632–644. Cancer Research, 2, 196–202. 31 Weber, M., Davies, J.J., Wittig, D., 39 Barciszewska, M.Z., Barciszewska, A.M. Oakeley, E.J., Haase, M., Lam, W.L. and and Rattan, S.I.S. (2007) TLC-based Schubeler, D. (2005) Chromosome-wide detection of methylated cytosine: and promoter-specific analyses identify application to aging epigenetics. sites of differential DNA methylation in Biogerontology, 8, 673–678. normal and transformed human cells. 40 DeSmet, C., DeBacker, O., Faraoni, I., Nature Genetics, 37, 853–862. Lurquin, C., Brasseur, F. and Boon, T. 32 Rauch, T., Li, H.W., Wu, X.W. and (1996) The activation of human gene Pfeifer, G.P. (2006) MIRA-assisted MAGE-1 in tumor cells is correlated with microarray analysis, a new technology for genome-wide demethylation. Proceedings the determination of DNA methylation of the National Academy of Sciences of the patterns, identifies frequent methylation United States of America, 93, 7149–7153. of homeodomain-containing genes in lung 41 Yang, A.S., Estecio, M.R.H., Doshi, K., cancer cells. Cancer Research, 66, Kondo, Y., Tajara, E.H. and Issa, J.P.J. 7939–7947. (2004) A simple method for estimating 33 Down, T.A., Rakyan, V.K., Turner, D.J., global DNA methylation using bisulfite Flicek, P., Li, H., Kulesha, E., Gr€af, S., PCR of repetitive DNA elements. Nucleic Johnson, N. et al. (2008) A Bayesian Acids Research, 32, e38. deconvolution strategy for 42 Lewin, J., Schmitt, A.O., Adorjan, P., immunoprecipitation-based DNA Hildmann, T. and Piepenbrock, C. (2004) methylome analysis. Nature Biotechnology, Quantitative DNA methylation analysis 26, 779–785. based on four-dye trace data from direct 34 Khulan, B., Thompson, R.F., Ye, K., sequencing of PCR amplificates. Fazzari, M.J., Suzuki, M., Stasiek, E., Bioinformatics, 20, 3005–3012. Figueroa, M.E., Glass, J.L. et al. (2006) 43 Tost, J. and Gut, I.G. (2007) DNA Comparative isoschizomer profiling of methylation analysis by pyrosequencing. cytosine methylation: the HELP assay. Nature Protocols, 2, 2265–2275. Genome Research, 16, 1046–1055. 44 Coolen, M.W., Statham, A.L., 35 Irizarry, R.A., Ladd-Acosta, C., Gardiner-Garden, M. and Clark, S.J. (2007) Carvalho, B., Wu, H., Brandenburg, S.A., Genomic profiling of CpG methylation Jeddeloh, J.A., Wen, B. and Feinberg, A.P. and allelic specificity using quantitative (2008) Comprehensive high-throughput high-throughput mass spectrometry: arrays for relative methylation (CHARM). critical evaluation and improvements. Genome Research, 18, 780–790. Nucleic Acids Research, 35, e19. 36 Ramsahoye, B.H. (2002) Measurement 45 Galm, O., Rountree, M.R., Bachman, K.E., of genome wide DNA methylation by Jair, K.W., Baylin, S.B. and Herman, J.G. reversed-phase high-performance liquid (2002) Enzymatic regional methylation chromatography. Methods, 27, 156–161. assay: a novel method to quantify regional 37 Paz, M.F., Fraga, M.F., Avila, S., CpG methylation density. Genome Guo, M.Z., Pollan, M., Herman, J.G. and Research, 12, 153–157. Esteller, M. (2003) A systematic profile of 46 Yamamoto, T., Nagasaka, T., Notohara, K., DNA methylation in human cancer cell Sasamoto, H., Murakami, J., Tanaka, N. lines. Cancer Research, 63, 1114–1121. and Matsubara, N. (2004) Methylation 38 Zukiel, R., Nowak, S., Barciszewska, A.M., assay by nucleotide incorporation: a Gawronska, I., Keith, G. and quantitative assay for regional CpG Barciszewska, M.Z. (2004) A simple methylation density. Biotechniques, 36, 846. 276j 17 Methods for the Analysis of DNA Methylation

47 Xiong, Z.G. and Laird, P.W. (1997) 52 Rauch, T. and Pfeifer, G.P. (2005) COBRA: a sensitive and quantitative DNA Methylated-CpG island recovery assay: a methylation assay. Nucleic Acids Research, new technique for the rapid detection of 25, 2532–2534. methylated-CpG islands in cancer. 48 Gonzalgo, M.L. and Jones, P.A. (1997) Laboratory Investigation, 85, 1172–1180. Rapid quantitation of methylation 53 Steemers, F.J., Chang, W.H., Lee, G., differences at specific sites using Barker, D.L., Shen, R. and Gunderson, K.L. methylation-sensitive single nucleotide (2006) Whole-genome genotyping with the primer extension (MS-SNuPE). Nucleic single-base extension assay. Nature Acids Research, 25, 2529–2531. Methods, 3,31–33. 49 Kaminsky, Z.A., Assadzadeh, A.A., 54 Bentley, D.R. (2006) Whole-genome Flanagan, J. and Petronis, A. (2005) Single re-sequencing. Current Opinion in Genetics nucleotide extension technology for & Development, 16, 545–552. quantitative site-specific-evaluation of 55 Margulies, M., Egholm, M., Altman, W.E., C-met/C in GC-rich regions. Nucleic Acids Attiya, S., Bader, J.S., Bemben, L.A., Research, 33, e95. Berka, J., Braverman, M.S. et al. (2005) 50 Schumacher, A., Kapranov, P., Genome sequencing in microfabricated Kaminsky, Z., Flanagan, J., high-density picolitre reactors. Nature, Assadzadeh, A., Yau, P., Virtanen, C., 437, 376–380. Winegarden, N. et al. (2006) Microarray- 56 Meyer, M., Stenzel, U. and Hofreiter, M. based DNA methylation profiling: (2008) Parallel tagged sequencing on the technology and applications. Nucleic Acids 454 platform. Nature Protocols, 3, 267–278. Research, 34, 528–542. 57 Wheeler, D.A., Srinivasan, M., 51 Wolber, P.K., Collins, P.J., Lucas, A.B., Egholm, M., Shen, Y., Chen, L., De Witte, A. and Shannon, K.W. (2006) McGuire, A., He, W.,Chen, Y.J. et al. (2008) DNA Microarrays Part a: Array Platforms The complete genome of an individual by and Wet-Bench Protocols, Academic Press, massively parallel DNA sequencing. vol. 410, pp. 28–57. Nature, 452, 872–875. j277

18 Methods Used to Study Alterations of Cell Signaling and Proliferation Clarissa Gerh€auser

18.1 Introduction

Cell signaling is a term used to describe a complex interactive system of signals that act to regulate or mediate a cellular response [1]. Growth factor signals are propagated from the cell surface through the action of transmembrane receptors to intracellular effectors that control critical functions in human cancer cells, such as cell differentiation, cell growth, angiogenesis, and inhibition of cell death and apoptosis [2]. Enzymes termed protein kinases couple phosphate groups to tyrosine, serine, or threonine residues in specific amino acid sequence motifs of target proteins. This process can be reversed by protein phosphatases. Many different types of protein kinases and phosphatases have been identified that recognize specific motifs and proteins. This variety determines specificity. The importance of phosphorylation to cellular regulation is underscored by the fact that cells have over a thousand different protein kinases [3]. These include transmembrane receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR)) or cytoplasmic kinases (e.g., phosphatidylinositol-3-kinase (PI3K)). Through site-specific regulation of phosphorylation, cells can rapidly switch on or modulate many major signaling and metabolic pathways. Phosphorylation regulates enzyme activities and protein binding and activates cascades of protein phosphorylation in signal transduction pathways (e.g., mitogen-activated protein kinase (MAPK) pathway). In cancer cells, these signaling pathways are often altered and result in a phenotype characterized by uncontrolled growth and increased capability to invade surrounding tissue. Altera- tions in receptor tyrosine kinase pathways have been implicated in oncogenic activation, mitogenic stimulation, and tumor angiogenesis. Therefore, these kinases and their downstream signaling pathways represent attractive targets for cancer prevention and therapy [2]. This chapter will summarize methodology to investigate key signal transduction pathways that are altered in cancer cells, using epidermal growth factor (EGF) signaling as an example. Downstream propagation of activated EGFR involves the Ras/MAPK-dependent pathway, the PI3K/AKT-dependent

pathway [2], and signaling through STATs (signal transducers and activators of transcription) [4] (summarized in Figure 18.1). In addition, test systems to measure cell proliferation, cell cycle progression, and cell death by apoptosis are presented.

18.2 Methods to Detect Alterations in Cell Signaling

One of the best investigated receptor protein tyrosine kinase families is the HER/ErbB family [5]. HER receptors and their ligands are frequently overexpressed in many tumor types. As an example, HER-2/neu is ampliﬁed in a subset of breast tumors, and overexpression is associated with adverse prognosis [2]. ErbB-1, better known as the EGFR, is a 170 kDa transmembrane protein with an extracellular ligand binding domain and a cytosolic domain with protein tyrosine kinase (PTK) activity. The ligand binding domain is able to bind several distinct ligands including the EGF.

18.2.1 Initializing Events

Initial steps of EFG signaling include ligand binding, receptor dimerization, and receptor activation by autophosphorylation (Figure 18.1). Ligand binding assays can be performed with intact cells, crude membrane preparations, detergent-solubilized receptor preparations [6], or receptor preparations affinity purified with monoclonal antibodies [7]. The cell line A-431 derived from a human epidermoid carcinoma of the vulva has been proven particularly useful for EGFR preparations due to its high content of 2.5 106 EGF receptors/ cell [6], but EGFR expression is generally high in most cell lines. Routinely, EGF labeled with 125I has been used for binding studies. For competition assays, labeled EGF is incubated in the presence of increasing concentration of unlabeled growth factor. Similarly, compounds or extract can be added simultaneously to investigate inhibition of ligand binding. Quantification is achieved by scintillation counting [6]. More recent reports describe the use of fluorescent rhodamine-labeled EGF with fluorescence correlation spectroscopy [8], or europium-labeled EGF with time- resolved fluorescence determination [9]. Receptor dimerization can be detected with purified EGFR, membrane preparations, or intact cells. Activated, dimerized receptors are cross-linked by addition of reagents such as l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) or bis (sulfosuccinimidyl)suberate (BS3). Dimers are detected at a molecular weight of 340 kDa by immunoblotting with EGFR antibodies. Traditional methods to detect phosphorylation and to measure protein kinase (PK) activity are mainly based on the use of 32P-labeled adenosine triphosphate (ATP). Membrane preparations of A-431 cells are incubated with [g-32P]ATP and EGF to stimulate autophosphorylation in the presence or absence of an inhibitor. After incubation, reaction mixtures are spotted on filter paper, and unbound ATP Figure 18.1 Scheme of epidermal growth factor This reaction is reversed by the PIP3 phosphatase signaling (modified from Refs [4, 5, 12, 27, 28]). PTEN (phosphatase and tensin homologue), Binding of the ligand EGF to its receptor causes which has been recognized as a tumor receptor dimerization. This results in activation suppressor. PIP3 functions as a second of the receptors protein tyrosine kinase domain messenger to recruit AKT and other cytoplasmic through autophosphorylation. (a) Ras/MAPK kinases with the so-called PH (pleckstrin pathway (right): The phosphotyrosine binding homologue) domains to the inner surface of the domain of the scaffold protein Shc binds to the cell membrane. AKT is activated by a dual activated receptor, resulting in phosphorylation regulatory mechanism that requires both of Shc at two tyrosine residues. This promotes translocation to the plasma membrane and binding of the adaptor protein Grb2 and phosphorylation through 3-phosphoinositide- membrane recruitment of the guanine dependent protein kinases. Phosphorylation by nucleotide releasing factor Sos (son of AKT leads to activation or inhibition of several sevenless) to the activated receptor dimer [28]. target proteins, resulting in cell growth, survival, Sos then activates Ras (reversed by the GTPase and proliferation through various activating protein (GAP)) leading to the mechanisms [27]. Dephosphorylation through activation of Raf. Raf subsequently the general phosphatase PPA2 inactivates phosphorylates MEK1/2 (a MAP kinase) that AKT [12]. (c) STAT signaling (middle): STATs activates, respectively, the MAP kinases ERK1 (signal transducers and activators of and ERK2. This pathway results in the activation transcription) are a family of proteins that act of a series of transcription factors including Elk, downstream of tyrosine kinase receptors enhanced cell proliferation, and promotion of including EGFR. STATs are activated cell survival [13]. (b) PI3K/AKT pathway (left): through phosphorylation. Activated STATs Grb2 is also bound to the docking protein Gab1. form dimers, translocate to the nucleus, and Recruitment of Gab1 leads to tyrosine initiate target gene transcription. STAT3 and phosphorylation at multiple sites of Gab1. This STAT5 stimulate cell cycle progression, allows binding and activation of the p85 angiogenesis, and inhibition of apoptosis and regulatory subunit of PI3K, leading to the are therefore considered oncogenes. Conversely, stimulation of PI3K. The p110 subunit of PI3K STAT1 target genes are involved in cell cycle catalyses the conversion of PIP2 arrest and apoptosis (not shown) and have (phosphatidylinositol 4,5-bisphosphate) to PIP3 properties consistent with a tumor suppressor (phosphatidylinositol 3,4,5-trisphosphate). gene [29]. 280j 18 Methods Used to Study Alterations of Cell Signaling and Proliferation

is removed by washing. Receptor autophosphorylation is detected by scintillation counting. Alternatively, reaction mixtures can be submitted to gel electrophoresis followed by autoradiography [10, 11]. Applications of these methods are, however, hampered by generation of radioactive waste and short half-life of 32P-labeled ATP. Alternatively, EGFR autophosphorylation can be detected using phosphorylation- speciﬁc antibodies in Western blotting.

18.2.2 Signal Transduction to Intracellular Targets

After activation of the membrane receptor, the extracellular signal is transmitted to intracellular targets by a network of interacting proteins (Figure 18.1). These proteins act as signaling cascades, such as the MAPK cascade, or the PI3K/AKT pathway. Signal transmission in the MAPK pathway is initiated by activation of a small G protein such as Ras and followed by the sequential activation of cytosolic protein kinases, which activate the kinase at the next level until the ﬁnal regulatory molecule is activated (Raf ¼ MAP3K, MEK1/2 ¼ MAPKK, ERK1/2 ¼ MAPK). In the PI3K/AKT pathway, inactive AKT is targeted to the plasma membrane by the PI3K product PIP3 and activated by dual phosphorylation via phosphoinositide-dependent kinases (PDKs) [12]. AKT has multiple downstream targets that are either activated or inhibited by phosphorylation and are involved in glucose metabolism, apoptosis, proliferation, angiogenesis, and more. Kinase cascade activation can be measured by immunoblotting using phosphospeciﬁc antibodies for phosphorylated substrates, or at the activity level using kinase assays.

18.2.2.1 Detection Using Phosphospecific Antibodies Proteins of interest are mostly cytosolic proteins; therefore, care has to be taken that cell membranes are properly disrupted during lysate preparation. Also, protein degradation and dephosphorylation should be prevented by use of specific inhibitors for proteases and phosphatases. Activation or inhibition of activation of the MAPK cascade is sensitively analyzed by detection of ERK1/2 (also known as p42/p44 MAPK) phosphorylation. Protein lysates of serum-starved cells treated with inhibitors and stimulated with EGF are separated by SDS polyacrylamide electrophoresis. ERK1/2 phosphorylation is detected by immunoblotting with antiphospho antibodies [13]. Due to an increase in molecular weight, phosphorylated bands migrate slightly slower in the electrophoretic field. Under optimized conditions, staining with ERK1/2 antibodies (not phosphospecific) will detect four bands after EGF stimulation, p42/p44 as well as phospho-p42/phospho-p44. Blots can be scanned for quantification, and phosphorylation signals are normalized to the expression of the nonphosphorylated protein. A similar protocol can be used to detect AKT activation [12]. Alternatively, phosphorylated ERK1/2 can be quantified by phosphospecific ELISAs (enzyme-linked immunosorbent assays) that are commercially available. AKT activation involves translocation to the plasma membrane, which can be investigated by immunoblotting after cell fractionation into plasma membrane and cytosolic fractions [12]. 18.2 Methods to Detect Alterations in Cell Signaling j281

18.2.2.2 Kinase Assay After Immunoprecipitation Immunoprecipitation of a kinase of interest with bead-immobilized antibodies enhances specificity and is useful when phosphospecific antibodies are not available. The enriched protein kinase preparation is incubated with a reaction mixture containing [g-32P]ATP, myelin basic protein as a general phosphorylation substrate or other appropriate specific phosphorylation substrates, and potential inhibitors. Phosphorylation is quantified by autoradiography after gel electrophoresis, extensive washing to remove unbound [g-32P]ATP, and gel drying. Alternatively, aliquots can be spotted on phosphocellulose paper squares, which are immediately washed in phosphoric acid to remove excess radioactivity. Phosphorylation is quantified by scintillation counting [13].

18.2.3 Transcription Factor Activation

Often, the ﬁnal regulatory molecule of a signal transduction pathway is a transcription factor that is activated by phosphorylation to bind to a target sequence or regulatory element in the promoter region of responsive genes. Alternatively, a transcription factor such as NF-kB is liberated from an inhibitory protein that is marked for degradation by phosphorylation. Consequently, transcription of these genes is enhanced and may result in elevated protein expression (Figure 18.1). Methods have been developed to analyze transcription factor–DNA binding. In addition, activation of transcription factors can be measured in promoter assays after transfection with reporter constructs.

18.2.3.1 Transcription Factor–DNA Binding The most widely used method is the electrophoretic mobility shift assay (EMSA), also known as band shift, gel shift, or gel retardation assay. The assay is based on the fact that binding of protein to a DNA fragment results in the reduction of its electrophoretic mobility in nondenaturing polyacrylamide gels. Short 32P-labeled oligonucleotides or DNA fragments are incubated with nuclear extracts, separated by electrophoresis, dried, and analyzed by autoradiography. Binding specificity is confirmed by an addition of a large excess of mutant or unspecific oligonucleotide. Concomitant incubation with an antibody directed against the transcription factor will further shift the complex to a larger molecular weight (super shift) [14]. Recently, transcription factor profiling assays based on an ELISA format have been developed (e.g., BD Mercury Trans Factor kits, Active Motives TransAM kits). They are rapid, as sensitive as classical EMSA assays, and allow colorimetric detection and simultaneous analysis of up to 96 samples [1].

18.2.3.2 Promoter Assays After Transfection with Reporter Gene Constructs To quantify transcription factor or promoter activation, cells are transfected with reporter gene constructs that induce visually identiﬁable characteristics. In these constructs, ﬂuorescent or luminescent proteins, for example, the luciferase gene, are put under the control of the target promoter or a regulatory element (transcription 282j 18 Methods Used to Study Alterations of Cell Signaling and Proliferation

factor binding site) and the activity of the reporter gene product is quantitatively measured as an indication of transcription factor activation [15]. Incubation of transfected cells with test compounds in the absence or presence of an inducer will allow detection of transcription factor activation or inhibition of activation.

18.2.4 Gene Transcription and Translation

As a result of a signal transduction cascade, the transcription of target genes is stimulated and may result in elevated protein expression. Enhanced mRNA expression can be detected by Northern blotting techniques [16] or quantiﬁed by (real-time) RT-PCR (reverse-transcribed polymerase chain reaction) experiments [17]. In addition, protein expression of the target gene can be analyzed by immunoblotting.

18.3 Methods to Measure Cell Proliferation

The survival of multicellular organisms involves a balance between cell proliferation and cell death. As outlined above, overexpression of hormone/growth factors and their receptors might present a growth advantage to preneoplastic cells. Also, continuous accumulation of genetic damage and mutations during carcinogenesis can result in uncontrolled cell proliferation and cell growth.

18.3.1 Microplate Screening Assays for Cytotoxicity

Methods for determining cell proliferation also allow detection of cytotoxicity induced by treatment with inhibitors of cell signaling or cell growth. Cytotoxicity describes the effects of interventions on cell viability, cell growth, or metabolic functions. Simple screening methods used to indicate cytotoxicity fall into these three categories, as summarized in Table 18.1. When determining cytotoxic activity of test compounds, it is important to consider kinetic and dose–response effects. Also, cell numbers used for incubation may inﬂuence the detection and results of cytotoxicity evaluation and should be optimized accordingly [18, 19]. Information on the mode of activity can be achieved with mode-speciﬁc methods. These include effects on cell cycle progression measured by cell cycle analyses, or by determining induction of cell death by detection of apoptosis, for example, by caspase-3/7 activity assays (Table 18.1). Further details and additional methods are described below.

18.3.2 Cell Cycle Analysis [20]

Cell cycle analysis with ﬂow cytometry after staining of DNA with DNA ﬂuoro- chromes allows to position cells in their respective phases of the cell cycle according Table 18.1 Summary of in vitro methods for detecting cytotoxicity (from Refs [18, 19]).

Assay Principle Instrumentation required Reference

Altered cell viability Trypan blue dye uptake Vital dye that only enters dead cells Phase contrast inverted microscope [18] – Basic protocol 1 with damaged cell membrane. Live cells exclude the dye. Percentage of viable cells of total cells is determined by cell counting.

Lactate dehydrogenase (LDH)a[ Indicator of increased membrane Microtiter plate spectrophotometer [18] – Basic protocol 2 [19] leakage (commercial kits available) permeability associated with cell Decrease of absorption at 340 nm death when LDH activity is detected (modiﬁcations with colorimetric in cell culture supernatants. detection available)

Neutral red dye retention Vital dye that is taken up and retained Microtiter plate spectrophotometer [18] – Basic protocol 3

in viable cells. Absorption at 550 nm (ﬂuorescence Proliferation Cell Measure to Methods 18.3 reading possible at excitation (Ex) 488 nm, emission (Em) 540 nm)

Propidium iodide (PI) or ﬂuorescent PI is excluded from living cells but Fluorescence microtiter plate reader [18] – Basic protocol 4 marker attachment to double enters dead cells and binds to DNA. Ex 530 nm, Em 645 nm stranded nucleic acids Calcein-AM is membrane Live/dead assay Fluorescence microtiter plate reader [18] – Alternate protocol 6 permeable and cleaved by intact cells to calcein. Ethidium homodimer is Simultaneous detection of live cells: membrane impermeable and only Ex 488 nm, Em 530 nm dead cells: enters nonviable cells, where it labels Ex 530 nm, Em 585 nm nucleic acids (optimization required). j 283 (Continued) 284 j 8MtosUe oSuyAtrtoso elSgaigadProliferation and Signaling Cell of Alterations Study to Used Methods 18 Table 18.1 (Continued)

Assay Principle Instrumentation required Reference

ATP quantiﬁcation ATP as a viability marker is Luminometer, CCD camera [19] measured via a luciferase-catalyzed (ﬂuorescence microtiter plate photon-emitting reaction. reader)

Effects on cell growth and proliferation Cell counting Done along with trypan blue uptake Phase contrast inverted microscope [18] – Basic protocol 1 for viability.

Kenacid blue (KB) dye binding or Staining of membrane proteins after Microtiter plate spectrophotometer [18] – Basic protocol 5 [30] ﬁ Sulforhodamine B (SRB) staining cell xation. Dead (unattached) cells KB: Absorption at 577 (or 600) nm are removed by washing. Solubilized SRB: Absorption at 515 (or 490) nm dye is proportional to live cell count. Comparison with untreated control required.

[3H]Thymidine uptake [3H]Thymidine incorporates into Liquid scintillation counter [18] – Basic protocol 6 nucleic acids of proliferating cells. Toxicants decrease the rate of cell division (complex method, but sensitive).

Cell cycle analysis with PI PI binds to DNA. The distribution of Flow cytometer [18] – Basic protocol 7 a cell population in the cell cycle phases is based on DNA contents (G0/G1: 2n, S: >2n,G2/M: 4n) Caspase-3/7 activity assays An indicator-labeled substrate Fluorescence microtiter plate reader [19] (commercial kits available) peptide is cleaved and generates Ex 485 nm, Em 527 nm a ﬂuorescent or luminescent signal. Luminometer

Effects on cellular metabolic processes MTT[b reduction MTT is an indicator of mitochondrial Microtiter plate spectrophotometer [18] – Basic protocol 8 function in live cells. It is reduced Absorption at 550–570 nm to insoluble formazan crystals (time intensive).

Alamar blue (resazurin) reduction Water soluble, nontoxic redox Microtiter plate spectrophotometer [18] – Basic protocol 9 [19] indicator, no extraction or ﬁxation Absorption at 570 or 600 nm required. Cultures can be monitored Fluorescence microtiter plate reader repeatedly. Ex 530 nm, Em 590 nm 83Mtost esr elProliferation Cell Measure to Methods 18.3

Fluorescent dye loss Membrane-permeant ﬂuorescent Fluorescence microtiter plate reader [18] – Basic protocol 10 c dyes such as CFDA-AM[ are Ex 585 nm, Em 530 nm retained in intact cells after esterase- induced cleavage. Leakage of CFDA indicates cell damage. aAbbreviations: Em, emission; Ex, excitation; KB, kenacid blue; LDH, lactate dehydrogenase; PI, propidium iodide; SRB, sulforhodamine B. bMTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. cCFDA-AM: acetoxymethyl ester of 5-carboxyﬂuorescein diacetate. j 285 286j 18 Methods Used to Study Alterations of Cell Signaling and Proliferation

to their DNA content, that is, G0/G1, S, and G2/M. Various techniques for measuring DNA content have been developed. They differ in the method used for cell permea- bilization (detergent versus prefixation with alcohols) and composition of the staining solution [20]. An advantage of the fixation methods is the possibility of storage for extended periods. Consequently, cells can be treated with compounds and fixed independent of the timing of their analysis. Propidium iodide is the most widely used DNA fluorochrome. Since PI binds to both DNA and RNA, staining solutions contain RNase A to degrade RNA. DNA content measurements can also be used to detect apoptotic cells. DNA of apoptotic cells is fractionated by apoptosis-associated endonucleases. After extraction of these short DNA fragments, apoptotic cells can fi therefore be identi ed as cells with a fractional sub-G1 DNA content [21].

18.3.3 Cell Death: Induction of Apoptosis

Apoptosis (programmed cell death) is a genetically controlled response of cells to commit suicide. Apoptosis represents an innate cellular defense mechanism against carcinogen-induced cellular damage by inhibiting survival and growth of altered cells and removing them at different stages of carcinogenesis. Accordingly, deregulation of apoptosis has been implicated in the onset and progression of cancer. Apoptotic cells are characterized by speciﬁc morphological and biochemical changes. The morphological features of apoptosis consist of chromatin condensation, cell shrink- age, and membrane blebbing, which can be clearly observed by light microscopy. The biochemical changes during apoptosis induction include (i) the appearance of phosphatidylserine on the cell membrane surface, (ii) increased mitochondrial membrane permeability, (iii) activation of caspases, (iv) DNA fragmentation, and (v) protein cleavage at speciﬁc locations [21, 22].

18.3.3.1 Methods to Detect Induction of Apoptosis On the basis of these characteristics of apoptotic cells, multiple methods have been described to detect the induction of apoptosis by dietary or therapeutic agents [23]. Basically, all methods have some limitations and are not completely specific and quantitative [21, 22, 24]. 1. Phosphatidylserine is normally situated on the inner surface of the cytoplasmic membrane. During apoptosis, phosphatidylserine is translocated to the outer surface. Annexin V, a highly conserved 35 kDa protein, has a high affinity to phosphatidylserine and can be used, as a fluorescent conjugate, to detect phosphatidylserine externalization by flow cytometry or fluorescent microscopy. 2. Breakdown of the mitochondrial transmembrane potential can be measured by fluorochromes that are sensitive to the electrochemical potential within the mitochondria, such as JC-1. These changes can be detected by flow cytometry or fluorescent microscopy [25]. 3. The caspases are a group of aspartic acid-specific cysteine proteases that are activated during apoptosis. These unique proteases, which are synthesized as References j287

inactive proforms, are involved in the initiation and execution of apoptosis once activated by proteolytic cleavage. Caspase assays are based on the measurement of caspase processing to an active enzyme (detected by Western blotting) and of their proteolytic activity using suitable peptide substrates that yield fluorescent or colored products after cleavage [26]. A number of commercial kits and reagents are available to assess apoptosis based on caspase function. 4. During the execution phase of apoptosis, nucleases are activated that cleave DNA into 180–200 bp increments. This DNA fragmentation (DNA laddering) can be detected by gel electrophoresis. At the single-cell level, the TUNEL (TdT-mediated dUTP nick-end labeling) assay measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP(a) at 30-OH DNA ends using the enzyme terminal deoxynucleotidyl transferase. The fluorescein-12-dUTP- labeled DNA can then be visualized directly by fluorescence microscopy or quantified by flow cytometry. Alternatively, biotinylated nucleotides can be incorporated and detected via streptavidin-coupled horseradish peroxidase with suitable colorimetric substrates. DNA fragmentation is also detectable by flow fi cytometry after PI staining of xed cells via the occurrence of a sub-G1 peak in cell cycle analyses as described above. 5. Poly(ADP-ribose)polymerase (PARP) is a zinc-dependent eukaryotic DNA binding protein that specifically recognizes DNA strand breaks produced during apoptosis induction. The 113 kDa protein serves as a substrate for effector caspases and is cleaved during apoptosis into 89 and 24 kDa fragments, which can be detected by Western blotting as specific markers of apoptosis. For more in-depth mechanistic evaluation of apoptosis induction, changes in the expression and phosphorylation of pro- and antiapoptotic proteins can be detected by Western blotting. Some relevant mechanisms observed during the induction of apoptosis by dietary factors have been summarized by Khan et al. [23].

References

1 Davis, M.A. (2004) Making mechanistic John Wiley and Sons, Inc., New York, connections between cell signaling pp. 14.10.11–14.10.12. pathways and pathological endpoints. 4 Silva, C.M. (2004) Role of STATs Toxicologic Pathology, 32 (Suppl. 1), as downstream signal transducers 131–135. in Src family kinase-mediated 2 Bianco, R., Melisi, D., Ciardiello, F. and tumorigenesis. Oncogene, 23, Tortora, G. (2006) Key cancer cell signal 8017–8023. transduction pathways as therapeutic 5 Yarden, Y. and Sliwkowski, M.X. (2001) targets. European Journal of Cancer, Untangling the ErbB signalling network. 42, 290–294. Nature Reviews Molecular Cell Biology, 3 Yamada, K.M. (2008) Signal transduction, 2, 127–137. in Current Protocols in Cell Biology (eds J.S. 6 Carpenter, G. (1985) Binding assays for Bonifacino, M. Dasso, J.B. Harford, J. epidermal growth factor. Methods in Lippincott-Schwartz, and K.M. Yamada,), Enzymology, 109, 101–110. 288j 18 Methods Used to Study Alterations of Cell Signaling and Proliferation

7 Hsuan, J. and Yaish, P. (1991) Southern blots. Methods in Enzymology, Affinity purification of active epidermal 152, 572–581. growth-factor receptor using 17 Lutfalla, G. and Uze, G. (2006) Performing monoclonal-antibodies. Methods in quantitative reverse-transcribed Enzymology, 200,378–388. polymerase chain reaction experiments. 8 Pramanik, A. and Rigler, R. (2001) Methods in Enzymology, 410, 386–400. Ligand–receptor interactions in the 18 Ehrich, M. and Sharova, L. (2000) In vitro membrane of cultured cells monitored by methods for detecting cytotoxicity, in fluorescence correlation spectroscopy. Current Protocols in Toxicology (eds J.A. Bus, Biological Chemistry, 382, 371–378. L.G. Costa, E. Hodgson, D.A. Lawrence, 9 Mazor, O., Hillairet de Boisferon, M., and D.J. Reed), John Wiley and Sons, Inc., Lombet, A., Gruaz-Guyon, A., Gayer, B., New York, pp. 2.6.1–2.6.27. Skrzydelsky, D., Kohen, F., Forgez, P. et al. 19 Riss, T.L. and Moravec, R.A. (2004) Use of (2002) Europium-labeled epidermal multiple assay endpoints to investigate the growth factor and neurotensin: novel effects of incubation time, dose of toxin, probes for receptor-binding studies. and plating density in cell-based Analytical Biochemistry, 301,75–81. cytotoxicity assays. Assay and Drug 10 Cohen, S. (1983) Purification of the Development Technologies, 2,51–62. receptor for epidermal growth-factor from 20 Darzynkiewicz, Z. and Juan, G. (1997) a-431 cells – its function as a tyrosyl kinase. DNA content measurement for DNA Methods in Enzymology, 99, 379–387. ploidy and cell cycle analysis, in Current 11 Karaszkiewicz, J.W. and Henrich, C.J. Protocols in Cytometry (eds J.P. Robinson, (1997) Using protein kinase and protein Z. Darzynkiewicz, R. Hoffmann, J.P. phosphatase inhibitors to dissect protein Nolan, Orfao F A., P.S. Rabinovitch and S. phosphorylation pathways, in Current Watkins), John Wiley and Sons, Inc., Protocols in Immunology (eds J.E. Coligan, New York, pp. 7.5.1–7.5.24. B.E. Bierer, D.H. Margulies, E.M. Shevach, 21 Pozarowski, P., Grabarek, J., and W.Strober), John Wiley and Sons, Inc., Darzynkiewicz, Z. (2003) Flow cytomentry New York. pp. 11.17.11–11.17.20. of apoptosis, in Current Protocols in 12 Pankov, R. (2004) Determination of Akt/ Cytometry (eds J.P. Robinson, Z. PKB signaling, in Current Protocols in Cell Darzynkiewicz, R. Hoffmann, J.P. Nolan, Biology (eds J.S. Bonifacino, M. Dasso, J.B. A. Orfao, P.S. Rabinovitch and S. Watkins), Harford, J. Lippincott-Schwartz and K.M. John Wiley and Sons, Inc., New York, Yamada), John Wiley and Sons, Inc., pp. 7.19.11–17.19.33. New York, pp. 14.16.11–14.16.12. 22 Hall, P.A. (1999) Assessing apoptosis: a 13 Shaul, Y.and Seger, R. (2005) The detection critical survey. Endocrine-Related Cancer, of MAPK signaling, in Current Protocols in 6,3–8. Cell Biology (eds J.S. Bonifacino, M. Dasso, 23 Khan, N., Afaq, F. and Mukhtar, H. (2007) J.B. Harford, J. Lippincott-Schwartz and Apoptosis by dietary factors: the suicide K.M. Yamada), John Wiley and Sons, Inc., solution for delaying cancer growth. New York, pp. 14.13.11–14.13.34. Carcinogenesis, 28, 233–239. 14 Kerr, L.D. (1995) Electrophoretic mobility 24 Sgonc, R. and Gruber, J. (1998) Apoptosis shift assay. Oncogene Techniques, detection: an overview. Experimental 254, 619–632. Gerontology, 33, 525–533. 15 Brasier, A.R. and Ron, D. (1992) Luciferase 25 Lugli, E., Troiano, L., Cossarizza, A. (2007) reporter gene assay in mammalian cells. Polychromatic analysis of mitochondrial Methods in Enzymology, 216, 386–397. membrane potential using JC-1, in Current 16 Wahl, G.M., Meinkoth, J.L. and Protocols in Cytometry (eds J.P. Robinson, Kimmel, A.R. (1987) Northern and Z. Darzynkiewicz, R. Hoffmann, J.P. References j289

Nolan, A. Orfao, P.S. Rabinovitch and 28 Henson, E.S. and Gibson, S.B. (2006) S. Watkins), John Wiley and Sons, Inc., Surviving cell death through epidermal New York, pp. 7.32.31–37.32.15. growth factor (EGF) signal transduction 26 Kaufmann, S.H., Kofttke, T.J.,Martins, M., pathways: implications for cancer therapy. Henzing, A.J. and Earnshaw, W.C. (2001) Cellular Signalling, 18, 2089–2097. Analysis of caspase activation during 29 Quesnelle, K.M., Boehm, A.L. and apoptosis, in Current Protocols in Cell Grandis, J.R. (2007) STAT-mediated EGFR Biology (eds J.S. Bonifacino, M. Bonifacino, signaling in cancer. Journal of Cellular J.B. Harford, J. Lippincott-Schwartz and Biochemistry, 102, 311–319. K.M. Yamada), John Wiley and Sons, Inc., 30 Skehan, P., Storeng, R., Scudiero, D., New York, pp. 18.12.11–18.12.29. Monks, A., McMahon, J., Vistica, D., 27 Vivanco, I. and Sawyers, C.L. (2002) The Warren, J.T., Bokesch, H. et al. (1990) phosphatidylinositol 3-Kinase AKT New colorimetric cytotoxicity assay for pathway in human cancer. Nature Reviews anticancer-drug screening. Journal of the Cancer, 2, 489–501. National Cancer Institute, 82, 1107–1112. j291

19 Methods for the Assessment of Antiangiogenic Activity Clarissa Gerh€auser

19.1 Introduction

Angiogenesis is the formation of new blood vessels from already established vasculature and plays an essential role in tumor growth and survival. Without adequate blood supply, a tumor cannot grow beyond a critical size of 1–2mm2 due to lack of oxygen and nutrients [1]. The process of angiogenesis is regulated by growth factors and includes multiple steps, as outlined in Figure 19.1. Endothelial cells are activated by angiogenic growth factors released from the tumor (a). The basement membrane of the blood vessels is degraded by proteinases such as collagenases and plasminogen activator (b). Endothelial cells start to proliferate and migrate toward the angiogenic stimulus. This step also includes the recruitment of matrix metalloproteinases (MMPs) for extracellular matrix remodeling (c). Then, a new basement membrane is formed around the immature blood vessels, and the ends of two outgrowing blood vessels merge to form a lumen (d). This process offers multiple targets for antiangiogenic agents, such as (i) production of growth factors, (ii) activation of endothelial cells, (iii) production of lytic enzymes to digest the basement membrane and extracellular matrix, and (iv) endothelial cell migration, proliferation, and tube formation.

19.2 Methods for Assaying Angiogenesis

Several recent reviews have critically summarized and evaluated current in vitro and in vivo test systems for antiangiogenic research [2–8]. Until now, no single established test model for antiangiogenic research fulﬁlls the needs for a large-scale quantitative screening assay.

Figure 19.1 Steps involved in angiogenic development. The process of angiogenesis is comprised of (a) release of angiogenic factors; (b) degradation of the basement membrane and endothelial cell proliferation; (c) migration and tube formation (differentiation); and (d) lumen formation and stabilization of the new vessel with a basement membrane and pericytes (derived from [15]).

19.2.1 In vitro Test Systems

19.2.1.1 Cell-Based Systems Cell-based systems have been developed using human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMEC), and bovine aortic endothelial cells (BAEC). They cover a limited range of inhibitory mechanisms depending on the test setup and are very useful for mechanistic elucidations, but do not provide the full spectrum of potential targets. Assays that are easily quantifiable include cell proliferation assays, cell motility assays (phagokinetic track, in vitro wounding), chemotaxis (trans-membrane) assays and tube-formation assays ([4], summary in Table 19.1). Inhibition of cell proliferation can be measured by MTTreduction, [3H]thymidine incorporation, or BrdU-labeling as end points. Todetermine specificity, the inhibitory potential of test compounds should be compared in a series of different cell types, such as fibroblasts and cells of epithelial origin. Cell migration is easily measured in the wound healing assay. A confluent monolayer of endothelial cells is scraped with a pipette tip to create a cell-free area [7]. Digital images are taken directly after wounding and after an additional 12–18 h incubation period in the presence of inhibitory compounds. The area 19.2 Methods for Assaying Angiogenesis j293

Table 19.1 Summary of in vitro angiogenesis assays [7].

Assay Advantages Disadvantages

Cell proliferation MTT reduction 1. Measures cell viability 1. Cells not necessarily proliferating 2. No measure of drug toxicity [3H]thymidine 1. Measures DNA replication 1. Uses radiation incorporation 2. No measure of drug toxicity BrdU-labeling 1. Measures DNA replication 1. No measure of drug toxicity (no radiation) Cell-cycle analysis 1. Measures apoptosis and 1. Cells have to be in suspension for therefore toxicity of drug analysis 2. Measures DNA replication 3. Measures percentage of cells proliferating

Cell migration Wound healing 1. Measures rate of endothelial cell 1. Quantification is somewhat (migration) migration arbitrary 2. Technical problems in achieving identical conditions of confluence Phagokinetic track 1. Measures total cell movement 1. Only a small number of cells (cell motility) 2. Measures directional effects of studied drug 2. Unnatural substrate to migrate on Boyden chamber 1. Measures migration in response 1. Technically difficult to set up (chemotaxis) to a gradient 2. Problems in maintaining trans 2. Extremely sensitive to small filter gradients changes in concentration 3. Difficult to obtain accurate cell counts 4. Time consuming to analyze

Cell differentiation Tube formation on 1. Endothelial cells pushed down 1. Lumen formation is under debate Matrigel differentiation pathway 2. Nonendothelial cells also form (matrix assay) 2. Formation of tube-like structures tubes 3. Quick 3. Homogeneous pattern of tubule lengths 3D gel assay 1. More closely mimics the in vivo 1. Long time period situation 2. Problems of quantifying a 3D 2. Tubules form in all three structure dimensions 1. Long time period Coculture 1. Tubules form lumen 2. Undeﬁned interactions between 2. More heterogeneous pattern of endothelial and other cells tubule lengths 3. Closer to in vivo situation

(Continued) 294j 19 Methods for the Assessment of Antiangiogenic Activity

Table 19.1 (Continued)

Assay Advantages Disadvantages

Organ culture assays 1 Rat aortic ring All: All: 2 Chick aortic arch 1. Mimic the in vivo situation 1. Difﬁcult to quantify 3 Porcine aortic artery 2. Include surrounding cells and 2. Growth requirements differ matrix between explant and cell out-growth 4 Placental vein disk 3. Endothelial cells are not prolif- 3. Time consuming erating at start of assay. 4. Nonhuman (except placental vein disk assay)

covered with migrated cells after incubation is compared by image analysis with that of an untreated control sample. In the phagokinetic track assay, cells are cultured on colloidal-gold-plated coverslips or on beads attached to the bottom of 96-well plates. Cell motility can be measured by observing the track left by the cells [7]. In a process called chemotaxis, endothelial cells move along a gradient of angiogenic factors. This property is measured in modified Boyden chambers. A Boyden chamber is composed of two medium-filled compartments separated by a microporous membrane, which may be coated with collagen to promote cell adhesion. In general, endothelial cells are placed in the upper compartment and are allowed to migrate through the pores of the membrane into the lower compartment, in which chemotactic agents (fibronectin, conditioned medium) are present. Migrating cells appearing on the underside of the filter are quantified by counting [9]. The experimental setup can be modified to measure the invasive potential of cancer cells (cell invasion). In this case, the membrane is coated with Matrigel as an extracellular matrix, and cancer cells are stimulated to migrate toward a chemotactic agent [10]. Matrigel is a mixture of extracellular and basement membrane proteins derived from the mouse Engelbreth–Holm–Swarm sarcoma [7]. Cell differentiation: Culture of endothelial cells on Matrigel in the matrix assay allows in vitro investigation of endothelial cell attachment, migration, and differentiation into tubules within 4–24 h. Tubule structures are evaluated from microscopic pictures. Since Matrigel is expensive, and only a part of the network can be analyzed in one image when cells are cultured in 12- to 96-well plates, downscaling of the assay to a 384- and 1536-well format has been described [7]. In the 3D gel assay, endothelial cells are cultured between two layers of Matrigel over prolonged periods of time (>12 days), which allows upward branching to a 3D network of tubules. This assay is difficult to analyze and quantify [7]. Coculture assays of endothelial cells and stromal cells, for example, fibroblasts, for 12–14 days mimic the in vivo situation more closely and allow formation of tubules containing a lumen [7]. Mechanistic investigations: Endothelial cells can be stimulated by hypoxic conditions to express angiogenesis-related proteins, such as hypoxia-inducible factor-1a (HIF-1a), the proto-oncoprotein c-MYC, vascular endothelial growth factor (VEGF), 19.2 Methods for Assaying Angiogenesis j295 as well as the basement membrane modulators including matrix metalloprotease MMP-2 and its endogenous inhibitor TIMP-2. Expression of these factors can be investigatedatthetranscriptionallevelbyRT-PCR(reversetranscriptase polymerasechain reaction)[11].In addition, the activity ofMMP-2 and TIMP-2 secreted to the cell culture medium can be analyzed by zymography. MMPs are reversibly inhibited by exposure to the detergent SDS (sodium dodecylsulfate) generally used in gel electrophoresis, and can be reactivated when SDS is removed. Separation of cell culture supernatants in polyacrylamide gels polymerized in the presence of digestible proteins, such as gelatin, allows easy detection of MMP activity after renaturation and staining with Coomassie blue, indicated by a clear zone of lytic activity inabluebackground[12].Additionalmolecularpathwaysastargetsofchemopreventive agents in the prevention of angiogenesis (angioprevention) have been summarized in Ref. [13]. One problem associated with all these cell-based in vitro assays arises from the fact that endothelial cells are normally in a quiescent state. Prolonged culture may induce alterations in activation state, karyotype, expression of cell surface antigens, and growth properties [4]. Therefore, these assays may only have limited value as models for in vivo reactions. Another problem, which is only recently being recognized, is the fact that all endothelial cells are not alike. Large vessel-derived endothelial cells such as HUVECs structurally differ from endothelial cells of microvascular origin, such as HMECs [7]. Also, the response to growth factors and inhibitors may vary with the species (human versus murine) and the organ source of these endothelial cells [4, 7]. As a result, in vitro assays should be performed using endothelial cells from more than one source, or more importantly, should be followed up with one or more in vivo assay of angiogenesis.

19.2.1.2 Organ Culture Systems Organ culture systems represent an intermediate between cell-based in vitro assays and animal models. A summary of most commonly used models is given in Table 19.1. Disks or segments of rat aorta, porcine carotid artery, or human placental veins are cultured in a matrix such as ﬁbrin over a period of 10–14 days, and endothelial microvessel growth is monitored by digital imaging [7]. These assays are quite useful, but have limitations in that vessel outgrowth is initiated from large vessels instead of microvessels. Another limitation is the fact that angiogenic factors that are released by tumor cells and the tumor cells themselves are not present in the organ culture system [4]. Also, a large variation in microvessel growth has been observed between individual donor animals, human placentas, or even between vessels from the same donor. This requires careful comparison with untreated controls and positive control substances when the effects of potential antiangiogenic compounds are investigated, and repeated analysis with fragments from several donors is necessary [14]. A modiﬁcation is the chicken aortic arch assay based on the culture of embryonic arch endothelial cells that are more similar to microvascular endothelial cells, but unlike the in vivo situation, undergo proliferation before explanation. This assay avoids the use of laboratory animals and is rapid with only 1–3 days of culture [5]. 296j 19 Methods for the Assessment of Antiangiogenic Activity

19.2.2 In vivo Models

In vivo animal models arethe ultimate choice of investigating the effects of angiogenesis- influencing factors. However, these assays are expensive, tedious, and time consuming. Adetailed description of most currently utilized models is given in [6–8]. Advantages and disadvantages associated with these models are summarized in Table 19.2. . Microcirculatory preparations in chamber models allow continuous, noninvasive in situ observation of new vessels with light microscopy. Most commonly used models include the rabbit ear chamber first described in 1924, the dorsal skinfold chamber developed in the 1940s, and the cranial window [3]. Cells, tissue, or sponges are placed inside a transparent chamber, and physiological effects, such as blood flow, can be measured. Angiogenesis can be quantified by intravital microscopy or by immunostaining of excised tissue. For implanting xenografts, immu- nocompromised animals can be used [6]. . The chick chorioallantoic membrane assay utilizes fertilized chicken eggs. This assay was introduced to angiogenesis research in 1974 by Folkmann and his group. The chorioallantoic membrane (CAM) is highly vascularized and serves the embryo as an initial respiratory system. The assay is carried out in ovo, after removing a part of the egg shell, or in vitro [8]. Test substances are placed on the membrane in polymer pellets, which may hinder their release depending on solubility. . The corneal angiogenesis assay was also first described by the Folkmann group in 1974. Apocket is made in the cornea, and test tumors or tissues, when introduced into this pocket, elicit the growth of new vessels. This assay is still considered as one of the best in vivo assays, since the cornea itself is avascular. Thus, any vessels seen in the cornea after stimulation by angiogenesis-inducing tissues or factors are new vessels. Test inhibitors can be administered orally or systemically. The vascular response can be monitored by direct observation throughout the course of the experiment [5]. . Vascularization into polymer matrix implants is studies in the sponge implant and the Matrigel plug assay. These assays are less difficult as the corneal angiogenesis assay. A polymer matrix containing cells and/or angiogenic factors is subcutaneously implanted. Vascularization into the implant is assessed by immunohistochemical detection of CD31, a surface marker of endothelial cells, or by measuring the blood/hemoglobin content. Comparison of different studies is complicated by differences in sponge size, shape, and composition [7]. . Tumor models allow the analysis of antiangiogenic drugs, since tumors require new blood vessels to grow beyond a certain size. Vessels in tumors can be detected by immunohistochemical staining as described above [7]. . The hollow fiber assay was introduced in 1995 by the NCI in the drug development programme to identify potential drug candidates. Tumor cells are implanted into animals inside biocompatible polyvinylidene fluoride fibers. Extensive vascular networks to the hollow fibers have been observed after extended postimplantation 19.2 Methods for Assaying Angiogenesis j297

Table 19.2 Summary of in vivo angiogenesis assays (modified from [6–8]).

a Assay

Advantages Disadvantages References

Chamber models: rabbit ear chamber; dorsal skinfold chamber (rats, mice); cranial window chamber 1. Noninvasive monitoring 1. Invasive: chamber placement [6, 7] 2. 3D vessel growth can be followed requires surgery over a relatively long period 2. Technically difficult 3. Long-term observation minimizes 3. Expensive when done in rabbits number of mice used 4. Nonspecificinflammatory response 4. Dorsal skinfold assay less expensive 5. Can get surgery associated than rabbit ear chamber assay angiogenesis 6. Skinfold chamber poorer optical quality

Chick chorioallantoic membrane (CAM) assay 1. Technically simple 1. Nonmammalian [6, 7, 16] 2. Inexpensive 2. Embryonic 3. Suitable for large-scale screening 3. Very sensitive to increases in oxygen 4. Permits noninvasive observations tension 5. Suitable for mammalian xenografts 4. Due to the pre-existing vascular network, visualization of new capillaries can be difficult 5. Immune response can mask new vasculature 6. Long-term observation restricted 7. Nonspecificinflammatory reactions common 8. Drugs that require metabolic activation cannot be assessed

Corneal angiogenesis assay 1. Reliable 1. Expensive [6–8, 17] 2. Immunologically privileged site 2. Technically difficult 3. Absence of pre-existing vessels 3. Ethically questionable 4. Permits noninvasive and long-term 4. Avascular structure, atypical for monitoring angiogenesis in vivo 5. Procedure technically demanding in murine eye 6. Nonspecificinflammatory response with some compounds

Sponge/matrix implant 1. Inexpensive 1. Nonspeciﬁc immune responses may [6–8, 18] lead to an angiogenic response (Continued) 298j 19 Methods for the Assessment of Antiangiogenic Activity

Table 19.2 (Continued)

a Assay

Advantages Disadvantages References

2. Technically simple, reproducible 2. Sponge composition varies, making 3. Well-tolerated procedure inter experimental comparisons 4. May replicate hypoxic tumor difﬁcult environment 3. Not a typical site for angiogenesis 5. Time course of response can be in vivo recorded 4. Variable retention of test compounds within implant 5. The subcutaneous tissue is not a highly relevant site for tumor growth 6. Animals have to be kept singly

Matrigel plug assay 1. Nonartificial, providing a more 1. Expensive [7, 8] natural environment for angiogenesis 2. Analysis is time consuming. 2. Technically simple 3. Matrigel is not chemically defined 3. Suitable for large-scale screening 4. Difficult to make plugs unifore in 3D-shape 5. Analysis in plugs time consuming 6. The subcutaneous tissue is not a highly relevant site for tumor growth

Tumor models: syngeneic, orthotopic, or as xenografts 1. Can follow pharmacokinetics of drug 1. Tumor environment depends on [7] as well as antiangiogenic effects tumor growth site (orthotopic versus 2. Long-term studies possible subcutaneous) 2. Real-time studies not possible

Hollow-fiber assays 1. More suitable than surface assays for 1. Nonspecificinflammatory response [6] tumor angiogenesis 2. Not a typical site for pathological 2. Permits long-term observation angiogenesis in vivo 3. Well-tolerated procedure 3. Not adapted for noninvasive monitoring 4. Yet to be validated

Angiomouse 1. Visualization is noninvasive 1. Sensitivity can be limited by [7, 8, 19] 2. Allows for real-time imaging of quenching due to surrounding tissue, angiogenesis especially skin 2. Hypoxia can decrease green ﬂuorescent protein (GFP) gene expression and hence, the degree of ﬂuorescence 19.3 Conclusions j299

Table 19.2 (Continued)

a Assay

Advantages Disadvantages References

Zebraﬁsh 1. Intact whole animal 1. Does not indicate exact point in [7, 8] 2. Technically simple angiogenic cascade speciﬁcally 3. Allows gene analysis of vessel disrupted development 2. Expensive to maintain in breeding 4. Relatively fast assay (6–12 h) condition 5. Fully quantitative 3. Does not distinguish between 6. Large numbers of animals available cytotoxic effects and genuine inhibition for statistical analyses 4. Nonmammalian 7. Suitable for large-scale screening 5. Embryonic aAlso see Ref. [3] for a comparison and evaluation of potential stimuli, ability to quantify vessels, invasiveness, ease of experiment, time required for preparation, possibility for long-term observation and costs.

times in vivo, but the model is not fully developed and validated for quantitative analyses yet [6]. . The Angiomouse is a system in which green fluorescent protein (GFP) is used indirectly to image tumor angiogenesis. Tumor cells expressing green fluorescent protein are injected into mice. New blood vessels formed by the host vasculature are not fluorescent and are consequently imaged as well-defined, dark networks against the bright green background. This is a noninvasive method and allows real-time imaging of angiogenesis [7]. . Zebrafish assay: The zebrafish (Danio rerio) is a small tropical freshwater fish, which is used increasingly in studies on angiogenesis. Angiogenesis is measured either by microangiography or by endogenous alkaline phosphatase staining of blood vessels. Transgenic zebrafish lines with fluorescent GFP-labeled blood vessels have also been developed, which greatly simplify the imaging of vessels [7]. The model has several advantages as summarized in Table 19.2.

19.3 Conclusions

Although the in vitro tests provide an initial step for evaluation of the effects of a test compound, can be carried out rapidly, and are easily quantiﬁed, their value for the identiﬁcation of effective antiangiogenic compounds may be limited. Organ cultures such as the aortic ring assay appear to provide some useful links between in vitro and in vivo conditions and allow interactions between the endothelial cells 300j 19 Methods for the Assessment of Antiangiogenic Activity

and their surrounding environment (smooth muscle cells, pericytes, basement membrane, and matrix). Nevertheless, results form in vitro assays have to be validated by a variety of in vivo assays. A single in vivo model is not sufficient to investigate angiogenesis, since there are variations within different species used, specific microenvironments, organ sites, and the manner of administration of the test substances. In vivo assays are time consuming, expensive, prone to high variability, and often difficult to quantify, but are absolutely necessary to obtain meaningful results [7].

References

1 Carmeliet, P. and Jain, R.K. (2000) J.M. and McEwan, R.N. (1987) A rapid Angiogenesis in cancer and other diseases. in vitro assay for quantitating the invasive Nature, 407, 249–257. potential of tumor cells. Cancer Research, 2 Auerbach, R., Auerbach, W. and 47, 3239–3245. Polakowski, I. (1991) Assays for 11 Bertl, E., Bartsch, H. and Gerhauser, C. angiogenesis: a review. Pharmacology & (2006) Inhibition of angiogenesis and Therapeutics, 51,1–11. endothelial cell functions are novel 3 Jain, R.K., Schlenger, K., Hockel, M. and sulforaphane-mediated mechanisms in Yuan, F. (1997) Quantitative angiogenesis chemoprevention. Molecular Cancer assays: progress and problems. Nature Therapeutics, 5, 575–585. Medicine, 3, 1203–1208. 12 Birkedal-Hansen, H., Yamada, S., 4 Auerbach, R., Akhtar, N., Lewis, R.L. and Windsor, J., Poulsen, A.H., Lyons, G., Shinners, B.L. (2000) Angiogenesis assays: Stetler-Stevenson, W. and Birkedal- problems and pitfalls. Cancer Metastasis Hansen, B. (2003) Matrix Reviews, 19, 167–172. metalloproteinases. Current 5 Auerbach, R., Lewis, R., Shinners, B., Protocols in Cell Biology, 18 [Chapter 10, Kubai, L. and Akhtar, N. (2003) Unit 10]. Angiogenesis assays: a critical overview. 13 Albini, A., Noonan, D.M. and Ferrari, N. Clinical Chemistry, 49,32–40. (2007) Molecular pathways for cancer 6 Hasan, J., Shnyder, S.D., Bibby, M., angioprevention. Clinical Cancer Research, Double, J.A., Bicknel, R. and Jayson, G.C. 13, 4320–4325. (2004) Quantitative angiogenesis assays 14 Bertl, E., Klimo, K., Heiss, E., Klenke, F., in vivo – a review. Angiogenesis, 7,1–16. Peschke, P., Becker, H., Eicher, T., 7 Staton, C.A., Stribbling, S.M., Tazzyman, Herhaus, C. et al. (2004) Identiﬁcation of S., Hughes, R., Brown, N.J. and Lewis, C.E. novel inhibitors of angiogenesis using a (2004) Current methods for assaying human in vitro anti-angiogenesis assay. angiogenesis in vitro and in vivo. International Journal for Cancer Prevention, International Journal of Experimental 1,47–61. Pathology, 85, 233–248. 15 Wernert, N., Stanjek, A., Kiriakidis, S., 8 Norrby, K. (2006) In vivo models of Hugel, A., Jha, H.C., Mazitschek, R. and angiogenesis. Journal of Cellular and Giannis, A. (1999) Inhibition of Molecular Medicine, 10, 588–612. angiogenesis in vivo by ets-1 antisense 9 Chen, H.C. (2005) Boyden chamber assay. oligonucleotides-inhibition of Ets-1 Methods in Molecular Biology, 294,15–22. transcription factor expression by the 10 Albini, A., Iwamoto, Y., Kleinman, H.K., antibiotic fumagillin. Angewandte Chemie, Martin, G.R., Aaronson, S.A., Kozlowski, 38, 3228–3231. References j301

16 Ponce, M.L. and Kleinmann, H.K. (2003) 18 Akhtar, N., Dickerson, E.B. and Auerbach, The chick chorioallantoic membrane as an R. (2002) The sponge/Matrigel invivoangiogenesismodel.CurrentProtocols angiogenesis assay. Angiogenesis, 5,75–80. in Cell Biology, 15 [Chapter 19, Unit 19]. 19 Hoffman, R. (2002) Green ﬂuorescent 17 Campochiaro,P.A.andHackett,S.F.(2003) protein imaging of tumour growth, Ocular neovascularization: a valuable metastasis, and angiogenesis in mouse model system. Oncogene, 22, 6537–6548. models. The Lancet Oncology, 3, 546–556. j303

20 Nutrigenomics Jan Frank and Gerald Rimbach

20.1 Defining the -omics

The revolutionary progress in recombinant DNA technology culminated in the sequencing of the entire human genome in 2001 [1, 2] and, subsequently, the genomes of the mouse [3, 4] and rat [5]. This knowledge now enables the study of the complex interplay of the diet and/or specific nutrients with the genetic makeup and health of individuals as well as entire populations. Rapid advancement in the development of high-throughput biomolecular techniques and their frequent application in system-wide experimental approaches have led to a noticeable propagation of the so-called -omics (e.g., genomics, transcriptomics, proteomics, metabolomics, etc.; see Box 20.1). Nutritionists are now increasingly using these state-of-the- art technologies to study the molecular basis of the health effects of specific components of the diet. The term nutrigenomics – short for nutritional genomics – refers to the study of the impact of specific nutrients, dietary components, or entire diets on gene expression. It is not to be confused with nutrigenetics, which investigates how genetic variability affects the bodys response to dietary components. Hence, nutrigenomics and nutrigenetics are closely related disciplines but approach the interplay of diet and genes from opposing starting points. Nutrigenomics is an upcoming discipline that uses high-throughput molecular biology techniques such as transcriptomics, proteomics, and metabolomics (Figure 20.1). The term genomics refers to the study of all nucleotide sequences in chromosomes – the genome – of an organism. It can be divided further into structural genomics (DNA sequence analysis and mapping of the genome of an organism) and functional genomics ((system-wide) experimental approaches to study gene function). The term transcriptomics is used to describe methods that measure the relative amounts of mRNA to determine patterns and levels of gene expression and their regulation. Proteomics is the study of the expression of the

Box 20.1 Terminology used in nutrigenomics

Genome The entire set of nucleotide sequences contained in the chromosomes of an organism Genomics The study of the genome Transcriptomics The study of the mRNA molecules (formed) within a biological system (cell, tissue, organism) Proteome All proteins (formed) within a biological system Proteomics Large-scale analysis of all proteins and peptides within a biological system Metabolite A substance formed during chemical processes within a biological system Metabolome All metabolites formed in a simple biological system (e.g., cell) Metabonome All metabolites formed in a complex biological system (e.g., organ or organism) Metabolomics Large-scale analysis of all metabolites (formed) within a biological system Lipidome All lipophilic substances (formed) within a biological system Lipidomics Large-scale analysis of all lipophilic substances within a biological system Glycome All sugars (formed) within a biological system Glycomics Large-scale analysis of all sugars within a biological system

complete set of proteins expressed in a cell, tissue, or organism and, accordingly, metabolomics investigates the proﬁle and functions of all metabolites generated in a simple (e.g., cell) or complex (e.g., entire organ or organism) system. Some scientists distinguish between these two approaches and refer to the study of metabolite formation in a simple system as metabolomics and in a complex system as metabonomics. The vast amount of data generated with such system- wide approaches requires the application of advanced bioinformatics tools to manage comprehensive data handling. The ﬁrst book on nutrigenomics has recently been published, covering a wide variety of aspects related to the effects of nutrients on gene expression and their role in health and disease. For further information on nutrigenomics, the interested reader is referred to this comprehensive book [6]. 20.2 Post-Transcriptional Gene Regulation by Small RNAs j305

Figure 20.1 Transcriptomics, proteomics, and metabolomics as analytical tools in nutrition research.

20.2 Post-Transcriptional Gene Regulation by Small RNAs

In the course of gene transcription and translation, the organism uses a number of RNAs that do not code for a peptide but serve other molecular functions. Some of these noncoding RNAs, such as ribosomal and transfer RNA, facilitate the ribosomal assembly of peptides and have been known for decades. Others, such as small interfering RNA (siRNA) and microRNA (miRNA), have been discovered relatively recently and effect post-transcriptional gene-silencing by a process termed RNA interference (RNAi, reviewed in Ref. [7]). siRNA and miRNA are both short (19–25 nucleotides (nt)) and double-stranded RNA species. Their major distinguishing features are their origin (exogenous versus endogenous) and the gene-silencing pathways toward which they direct their respective target mRNAs. miRNAs are encoded in the genome and transcribed by an RNApolymerase into long (from hundreds to thousands of nt) primary miRNAs, which are cleaved by the ribonuclease (RNase) Drosha and its cofactor Pasha into stem-loop-structured precursor miRNAs (pre-miRNAs; 70 nt). These pre-miRNAs are exported from the nucleus into the cytoplasm by Exportin 5 where they are further processed by the RNase Dicer into small (22 nt) miRNAs. These mature miRNAs are unwound and one strand (the guide strand) is loaded into the RNA-induced silencing complex (RISC), a multiprotein complex containing both known (including argonaute and Dicer) and as yet unidentiﬁed proteins, while the other strand (passenger strand) is probably degraded. Several of these miRNA-loaded RISC complexes bind to target mRNA, inhibiting its translation, and directing it toward processing bodies (P-bodies) for storage or degradation. The exact fate of the target mRNA is determined by the degree of base complementarity between the miRNA and mRNA. Perfect complementarity triggers the siRNA pathway leading to complete 306j 20 Nutrigenomics

degradation of the target mRNA involving the RNase in RISC, a phenomenon mainly observed in plants and to a much lesser extent in animals. Imperfect base complementarity activates the miRNA pathway resulting in the inhibition of gene translational (at the initiation or elongation step) and/or partial RNA decomposition (through destabilization due to poly(A)-shortening of the transcript) [7, 8]. It has been calculated that each miRNA binds on average 100 different target mRNAs, allowing post-transcriptional silencing of many different genes by a single miRNA. In fact, it was computed that approximately 30% of the genes of the vertebrate genome are regulated by miRNA [9]. RNAi has become a valuable experimental tool to study gene function and is increasingly investigated for its therapeutic value. For further reading, see the excellent reviews authored by Cowland et al. [8] and Rana [7].

20.3 Methods and Techniques Used in Nutrigenomic Research

20.3.1 Northern Blotting

For a long time, the expression of individual genes has been determined by quantification of RNA with Northern blotting. Briefly, this technique requires the isolation of RNA (usually total RNA or mRNA, although Northern blotting can also be used for miRNA quantification) from a sample, separation of the transcripts (according to size) by gel-electrophoresis, and their visualization with specific dyes (most commonly ethidium bromide). The RNA bands are then transferred (blotted) and fixed to a nitrocellulose or nylon membrane and hybridized with sequence- specific probes (short segments of complementary DNA (cDNA) or RNA) for the gene(s) in question. To allow quantitative measurements of the gene transcripts, the probes are either labeled with (i) radioactive phosphorous (32Por33P) or (ii) with an antigen or hapten, which itself binds an antibody-linked enzyme (e.g., horseradish peroxidase or alkaline phosphatase). Quantification is achieved by (i) the determination of radioactivity or (ii) by the measurement of chemiluminiscence or fluorescence of the product of the enzymatic reaction after addition of the substrate for the respective antibody-bound enzyme.

20.3.2 Reverse Transcription Polymerase Chain Reaction

The classical approach of Northern blotting has been partly replaced by more sensitive techniques such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). RT-PCR is an in vitro method that produces cDNA copies of selected stretches of (single-stranded) mRNA isolated from a sample by incubation of the sample RNA with an excess of viral reverse transcriptase, sequence-speciﬁc or random oligonucleotide primers, and deoxyribonucleoside triphosphates (dNTPs). 20.3 Methods and Techniques Used in Nutrigenomic Research j307

The resulting cDNA is then amplified in a thermal cycler in the presence of a heat- stable DNA polymerase (isolated from a thermophilic organism, most commonly Thermophilus aquaticus (Taq) but heat-stable DNA polymerases from other organisms have been used too), dNTPs, two sequence-specific primers (one for each complementary strand of the investigated gene, typically 20 bases long), reaction buffer, and magnesium. The reaction mixture is heated to 95 C, a temperature at which the double-stranded cDNA denatures to single strands, and then cooled to 55 C to allow annealing of the primers to the complementary DNA strands. The temperature is then raised to 72 C, the optimum temperature for the heat-stable DNA polymerase, which synthesizes complementary DNA along the annealed strand using the appropriate dNTPs. The newly synthesized double-stranded cDNA is then denatured to single strands by heating to 95 C and serves as a template for DNA synthesis during the following temperature cycle. Usually, around 25–40 temperature cycles are performed to (almost exponentially) amplify the target DNA. Theoretically, n cycles result in 2n copies of the target sequence; therefore, extremely small amounts of sample RNA are sufficient for analysis by RT-PCR. Quantification of the generated DNA is achieved by using DNA-binding dyes, whose fluorescence can be measured in real time during the amplification cycles (hence real-time RT-PCR), using fluorescence spectroscopy. The concentration of the mRNA, and accordingly the level of gene expression, can be deduced from the number of cycles necessary to exceed a preset fluorescence threshold (e.g., 10 times the SD of the baseline signal) [10]. A modification of this general real-time RT-PCR protocol, featuring improved sensitivity without the need for prior RNA purification, has been developed by Chen and colleagues [11] to allow the direct quantification of miRNA. They used a two-step PCR protocol employing stem-loop primers for the reverse transcription of miRNA into stem-loop cDNA in the first step and conventional linear primers for the quantitative real-time RT-PCR in the second step. The utilization of stem-loop primers permitted the discrimination of mature miRNAs differing in only one nucleotide and very high sensitivity in samples containing as little as 25 pg total RNA [11]. For more detailed information on Northern blotting and PCR, the reader is referred to the excellent original and review articles available in the literature (e.g., Refs [10, 12–14]).

20.3.3 Microarrays

Northern blotting and RT-PCR have their limitations, as they can only analyze gene expression for a limited number of candidate genes at a time. The development of DNA microarray technology has rendered it possible to determine the expression of thousands of genes, or even entire genomes, simultaneously. The operating principle of DNA microarrays relies on the base pairing of oligonucleotides within a sample with known immobilized oligonucleotides (capture probes) on a support material (e.g., glass slides). The use of representative oligonucleotides rather than the entire nucleotide sequence of a given gene allows a higher density (larger 308j 20 Nutrigenomics

number) of probes and a smaller overall size of the microarray. A typical DNA microarray experiment follows a characteristic series of steps: (1) RNA extraction from a sample; (2) reverse transcription of the RNA to obtain cDNA and (2a) labeling of the cDNA with specific dye(s) (usually fluorophores, such as Cyanine 3 and 5), or (2b) reverse transcription of the cDNA to obtain cRNA and labeling of the cRNA; (3) hybridization of the labeled cDNA or cRNA onto the microarray under defined conditions (e.g., time, temperature, etc.); (4) washing of the slides to remove nonhybridized labeled oligonucleotides; (5) signal detection (e.g., using an appropriate scanning device); and (6) data analysis (Figure 20.2). Modifications of this general approach were adopted by Affymetrix for the development of their GeneChip arrays. On Affymetrix GeneChip arrays, several different oligonucleotide probes are photolitographically spotted onto quartz wafers for each gene. Furthermore, for each nucleotide sequence (probe) designed to match a specific target sequence, a second, almost identical, probe is synthesized, which differs in only one base in the center of the nucleotide sequence. This allows the quantification and subtraction of nonspecific cross-hybridization (see Section 20.3.4), and the combination of these two approaches guarantees microarrays that produce data of very high quality. Because whole-genome microarray experiments produce an enormous amount of data not all of which may be of interest to a particular researcher, focused arrays have been made (commercially) available and are being increasingly used. Pathway- focused microarrays, for example, are used for the expression quantification of all or the most important genes relevant to a certain biological process (e.g., apoptosis) or disease (e.g., cancer). The major biological processes currently studied using DNA arrays in the context of cancer nutrigenomics are summarized in Table 20.1. Some

Figure 20.2 Schematic representation of the analytical steps involved in a gene chip experiment. 20.3 Methods and Techniques Used in Nutrigenomic Research j309

Table 20.1 Biological processes studied through the application of DNA microarray technology in the context of cancer nutrigenomics.

Biological processes represented on DNA microarrays

Angiogenesis Cellular antioxidant defense Cell cycle Cell mobility Cell surface receptor-linked signal transduction Acute phase response DNA damage response DNA metabolism DNA packaging DNA recombination DNA repair DNA replication and chromosome cycle G1/S and G2/M transition of mitotic cell cycle Histogenesis and organogenesis Mitosis Nucleo, nucleoside, nucleotide, and nucleic acid metabolism Oncogenesis Transcription regulation and transcription DNA-dependent transcription

companies also offer custom-made arrays providing researchers with the possibility to design arrays entirely according to their personal research needs. Tissue-specific microarrays represent another more focused array platform that can be used for the quantification of all genes expressed in a certain tissue or organ. These focused microarrays offer faster and easier data processing due to the smaller number of data points produced and can be much cheaper compared to whole-genome microarrays. Microarrays can also be used for the simultaneous quantification of a large number of small RNAs. However, to do so certain methodological problems need to be addressed to ensure meaningful data are obtained [15]. For example, the melting temperatures (Tm) of miRNA can vary from 55 to 74 C and thus hybridization of the microarray at a fixed temperature will result in lower signals for capture probes with a fi Tm lower than, and reduced speci city for probes with a Tm higher, than the used hybridization temperature. This problem can be overcome by using, for example, fi locked nucleic acid (LNA)-modi ed capture probes with more uniform Tm [16]. LNA are synthetic RNA nucleotide analogues whose sugar (ribose) rings have been modified by the introduction of a methylene bridge between the 20-oxygen and the 40-carbon, thus reducing the conformational flexibility of the ribose and increasing the structural organization of the phosphate backbone [17]. Replacing selected fi nucleotides of the capture probe with LNA, the Tm of each individual LNA-modi ed oligonucleotide capture probe can be adjusted depending on its length, the number of LNA incorporated, and the nature of the complementary strand (DNA or RNA). 310j 20 Nutrigenomics

Introducing LNA into the capture probes of their microarray platform (miChip), Castoldi et al. [16] were able to adjust all capture probes to a uniform hybridization temperature (72 C) and to sensitively and accurately quantify mature miRNA differing in as little as one nucleotide without prior puriﬁcation of the RNA. For a more detailed discussion of the application of microarray technology for miRNA proﬁling and associated pitfalls see Ref. [15]. Since it is beyond the scope of this chapter to describe in detail all the available variants of microarray technology, the interested reader is referred to the comprehensive book edited by Schena [18], who has been a pioneer in the development of DNA microarray technology, and to the excellent reviews by Elliott et al. [19], Liu- Stratton et al. [20], and Spielbauer and Stahl [21] for a more detailed description of microarray technology and its numerous applications in nutrition and medical research.

20.3.4 Microarray Data Normalization

A single microarray experiment results in an enormous amount of gene expression data; therefore, this data should be scrutinized for evidently ﬂawed results. The remaining data points need to be normalized (to correct for differences in the background signal, dye intensity, array-to-array variations, etc.) before comparative analyses of the obtained data can be accomplished. In commercial microarrays, some normalization occurs as part of the experimental procedure [22].

20.3.4.1 Per Array Normalization Per array normalizations are used to control for variations in signal intensity due to inconsistencies in sample preparation, washing and/or the production of the microarray. Commonly, all individual measurements are divided by a constant value such as the median of all intensity values. This global normalization procedure, however, is not suitable for experiments where more than 50% of the genes represented on the array are likely to be regulated in the same direction (up- or downregulated, respectively), as it may mask these observations. Normalization to a positive control gene is an alternative per array normalization procedure that uses positive controls from a housekeeping gene or another genome for normalization. Similar to the above, all intensity values are divided by the median intensity value of all positive controls represented on the array. As a third alternative, speciﬁc algorithms are used by some commercial microarray technologies to derive a constant value by which all signal intensities then are divided [22].

20.3.4.2 Per Gene Normalization Per gene normalization may be used for normalization of a gene across all samples within an experiment. If additional arrays/samples are included in the analysis, normalization needs to be recalculated using the complete new set of data. This kind of normalization can be useful to relate the signal intensity for a speciﬁc gene on the array to the signal intensity at baseline (without treatment) in a time–course 20.3 Methods and Techniques Used in Nutrigenomic Research j311 experiment; thus, the signal strength of a certain gene in a sample is divided by the signal strength of the same gene at baseline/without treatment. The most common approach for per gene normalization is the division by the median of all signal intensities for that particular gene across all samples within an experiment. This allows the direct comparison of all gene expression levels of a gene within the experiment, but care should be taken to standardize all microarray procedures as small differences in microarray and sample handling may result in gene expression levels falsely classiﬁed as differentially expressed [22].

20.3.5 Microarray Data Analysis

The huge number of data points generated during microarray experiments requires careful selection and application of mathematical and statistical procedures in order to detect and compare changes in gene expression. Data processing with sophisticated bioinformatics tools and the identification of (truly and not arbitrarily) differentially expressed genes in response to a specific nutrient or diet certainly pose the greatest challenges in the application of microarray technology. Because of the complexity of the data generated and the plethora of factors that may affect the outcome of microarray experiments (such as differences in experimental design, for example, treatment and time of sampling, RNA extraction protocols, hybridization conditions, or normalization procedures, to name a few), the Microarray Gene Expression Database Society (MGED), a grass-roots movement, has established itself in an attempt to develop standards for data handling [23]. An additional problem with microarray experiments that makes the comparison of gene expression data generated in different laboratories difficult is the output of the data in a number of formats and units. Because DNA microarrays are a comparatively new tool for the quantification of gene expression, there has been a lack of standards to follow to obtain meaningful presentation of data. Such standardization would allow microarray data to be easily interpreted, experiments to be reproduced, and would assist in the establishment of gene expression databases. In an effort to create such guidelines, the MGED group has formulated the minimum information about a microarray experiment (MIAME) that should be recorded and reported when referring to microarray data [23]. The MGED consortium identified six crucial parts of a microarray experiment that should be carefully described using controlled vocabularies: (1) experimental design; (2) array design; (3) samples; (4) hybridization; (5) measurements; and (6) normalization controls (see Box 20.2; [23]). Adherence to these guidelines will make the comparison of data obtained in different laboratories and across different experiments easier.

20.3.5.1 Fold Change Following normalization, the microarray data (usually given as spot intensities or ratios) are converted into numerical values. The ﬁrst step in data analysis, then, is to deﬁne a threshold level for the acceptance of a gene as expressed (present) or not 312j 20 Nutrigenomics

Box 20.2 MIAME Standards

Part 1: Experimental Design Description of the experiment, contact details of the author and/submitter, title. Information given in this section should enable the user to reconstruct the experimental design Part 2: Array Design Systemic description of all arrays used in the experiment, including the physical design and the genes represented. Detailed specification of (i) array as a whole (e.g., glass support); (ii) each type of element or spot (e.g., synthesized oligonucleotides); and (iii) properties of each element (e.g., DNA sequence) Part 3: Samples Description of the labeled nucleic acids used for hybridization. Detailed information on source of sample (such as organism or cell type), treatment(s) applied, extraction and labeling of the nucleic acids Part 4: Hybridization Description of Hybridization procedure (hybridization solution, blocking agent, washing procedure, quantity of labeled target used, hybridization time, volume, temperature, and apparatus) Part 5: Measurements Description of the experimental results by providing images of the original array scans, the quantification matrices based on image analysis and the final gene expression matrix after normalization. The reported information should facilitate the comprehension of the image analysis performed and the underlying methodology (e.g., calculations) Part 6: Normalization Controls Specification of normalization strategy (e.g., housekeeping genes), normalization and quality control algorithms, identities and location of the array elements used as controls, and hybridization extract preparation Source: [23].

expressed (absent) based on experimental conditions and background signal intensity. As mentioned earlier, data analysis is the most sophisticated part of a microarray experiment, although the questions commonly asked (e.g., Which gene(s) are differentially expressed and by how much?) appear to be fairly simple. One of the more basic approaches to answer these questions is the determination of fold changes in gene expression after careful normalization of the data (see above). A fold change of 2 (for gene expression in one treatment group or sample compared to a differently treated one) is usually considered of sufficient magnitude to alter biological processes and therefore frequently employed for the identification of genes that are differentially expressed. However, mRNA concentrations of genes that are expressed at very low levels (and should therefore be regarded as absent) will easily be changed twofold without significantly affecting biological processes and should therefore be excluded 20.3 Methods and Techniques Used in Nutrigenomic Research j313 from the analyses. It may, thus, sometimes be necessary to set the fold change threshold to a higher value than 2. Initial comparison of gene expression levels in a sample with a control sample will give a list of up- or downregulated candidate genes. At least three independent replicates of such an experiment should be performed to obtain meaningful data and reduce the number and increase the accuracy of the identified candidate genes. Analysis of gene expression in replicates will then commonly give a shortened list of candidate genes, because not all genes identified as up- or downregulated in one sample will be present in a replicate sample. Increasing the number of replicates will further reduce the background noise and the number of falsely identified regulated genes. The thus derived list of differentially regulated genes can then be used for statistical analysis.

20.3.5.2 Class Comparison, Class Discovery, and Class Prediction According to their primary aims, most microarray experiments fall into one of the three general categories: class comparison, class discovery, or class prediction. The purpose of class comparison experiments is to determine whether or not the gene expression profile (mean expression level) in one group (class) differs from that in another group and which genes are differentially expressed. The majority of nutrigenomic studies can be assigned to the category of class comparison experiments. Class discovery experiments are performed to retrospectively divide samples/ treatments into groups with (a certain degree of) homogeneity in gene expression (extent and/or direction) within the group, but distinct differences between the groups. Class prediction experiments derive multivariate mathematical models from a subset of all samples to predict the class affiliation of a gene or sample and may be used to, for example, predict if a sample was taken from a healthy or neoplastic tissue based on its gene expression profile. However, class prediction experiments are not common in nutrigenomic research. Suggested reading regarding microarray data analyses: Tefferi et al. [24], McShane et al. [25], and Mills [22]. For the analysis of microarray data, a number of computer programs supporting all major operating systems are freely available for download and installation on a personal computer (e.g., dChip). Internet-based programs that can be accessed and operated with the help of a Web browser and commercial software packages are also available. Two major types of microarray analysis software are available: specific and comprehensive programs, respectively. Specific programs are specialized tools that carry out only one or a few different analyses, such as a statistical test or a clustering algorithm. A list of such specific programs can be viewed at: http://ihome.cuhk. edu.hk/b400559/arraysoft_mining_specific.html. Comprehensive analysis software can be used to perform a larger number of different analyses and commonly aims at providing a single programme for all (or most) required analyses. Other bioinformatics resources, such as Genbank, Unigene, OMIM (all accessible via http://www.ncbi.nlm.nih.gov), SOURCE (http://source.stanford.edu), Medminer (http://discover.nci.nih.gov/textmining/main.jsp), DRAGON (http://pevsnerlab. kennedykrieger.org/dragon.htm), KEGG GENES (http://www.genome.jp/kegg/ genes.html), Onto-Express (http://vortex.cs.wayne.edu/ontoexpress/), and many more, are available that can aid in the interpretation of microarray data. 314j 20 Nutrigenomics

20.4 Applications of Nutrigenomics

The more complex the experimental model, the more complicated it gets to control all variables that may affect gene expression. Therefore, because of the simplicity to modulate only a single parameter (such as a nutrient), the majority of nutrigenomic experiments are performed in cell culture models. Both primary and immortalized cells are frequently employed to investigate the role of nutrients in the development or prevention of cancer. Using cells in isolation from other naturally adjacent cell types, however, limits the biological significance of these experiments. Therefore, animal models, where experimental parameters are relatively easy to control too, are often employed to study nutrient–gene interactions. In animal studies, however, gene expression in response toa dietary treatment isoften monitored usingpooled samples andatasinglepointintime.Becausegeneexpressioncanchangesignificantlywithina relativelyshorttime,itshouldideallybeprofiledrepeatedlyoveralongerperiodandfor each animal individually. Furthermore, changes in gene expression should be tested for their ability to alter the corresponding biological end points to establish their biological relevance [26]. Human studies of nutrient–gene interactions are less common because of the major difficulties in standardizing experimental conditions andtheproblemsinassigningobservedgene-regulatoryeffectstoaparticularnutrient with the required certainty. The innovative tools of nutrigenomics, especially gene microarraytechnology,maybeusedtodevelopnovelapproachestostudythebiological functions of nutrients and dietary compounds. DNA microarrays, for example, allow the genome-wide scanning for effects of nutrients without the necessity to limit ones attention to one or more specific target gene(s), thus reducing the risk of failure to recognize a biological effect. In microarray studies, sample size has a significant effect onhowconfidentlygenescanbedeclareddifferentiallyexpressed[27].Duetofinancial limitations, however, microarray experiments in humans are often statistically under- powered. Overall, the level of confidence one can assign to the results from a human microarraystudyhighlydependsontheskilloftheexperimenter,therobustness ofthe experimental design, and most importantly on the statistical power [28]. Other problematic issues concerning the gene array data, especially in human studies, may be the reporting of results, the experimental reproducibility, and the interpretation of the biologically relevant information [29]. Furthermore, gene expression microarray experiments produce lists of differentially regulated genes whose biological functions are often unknown, which again complicates data interpretation. However, if experiments are planned and conducted carefully, nutrigenomics may help in clarifying the molecular functions of dietary factors. Nutrigenomics may also serve to characterize the carcinogenic and anticarcinogenic properties of dietary compounds or to identify and establish new biomarkers. Table 20.2 summarizes nutrigenomic studies using high-throughput microarray technology to study carcinogenesis and its prevention by dietary factors including fat and fatty acids, vitamins and provitamins, phytochemicals, minerals and trace elements, and mycotoxins. Another novel application of microarray technology could be the study of the bioavailability and/or bioactivity/ biopotency of nutrients. Ascan bederived from the above, thepotential applications of Table 20.2 Studies using high-throughput nutrigenomic techniques/gene expression arrays to study carcinogenesis and its prevention by dietary compounds.

Number of Test substance Cell line or tissue Species genes monitored References

Dietary fat and fatty acids Butyrate Primary human colonocytes, LT-97, and HT-29 Human >14 000 [37] colon cancer cells High-fat diet Colon Rat 8799 [38] n-3-, n-6-, n-9-PUFA Colonocytes Rat 9028 [39] n-6-PUFA Mammary adenocarcinoma Rat 5184 [40]

Vitamins and provitamins b-Carotene H661 lung cancer and BEAS-2B nonmalignant Human 3156 [41] bronchoepithelial cells Folic acid HCT-116, Caco-2, HT-29, and LS513 colon Human 2 · 96 [42] cancer cells Folic acid, vitamin B12 HT-29 colon cancer cells Human 1026 [43] Nutrigenomics of Applications 20.4 Retinyl acetate Skin Rat 8740 [44] / Retinoic acid F9 Wt teratocarcinoma cells, F9 RARb2 cells Mouse Not speciﬁed [45] Retinoic acid BEAS-2B and BEAS-2B-R1 bronchial epithelial Human 11 069 [46] cells Retinoic acid BEAS-2B and BEAS-2BNNK bronchial Human 11 069 [47] epithelial cells Retinoic acid, ascorbic acid MCF-7 breast cancer cells Human 1152 [48] 1a,25-Dihydroxyvitamin D(3) SW480-ADH colon cancer cells Human >12 665 [49] (Continued) j 315 316 j 0Nutrigenomics 20

Table 20.2 (Continued)

Number of Test substance Cell line or tissue Species genes monitored References

1a,25-Dihydroxyvitamin D(3) LNCaP prostate cancer cells Human 3000 [50] 1a,25-Dihydroxyvitamin D(3) MCF-7 and MDA-MB-231 breast cancer cells Human 2000 [51] 1a,25-Dihydroxyvitamin D(3) Primary and cultured (ALVA-31) prostate cancer cells Human >20 000 [52] 1a,25-Dihydroxyvitamin D(3) LNCaP prostate cancer cells Human 23 000 [53] 1a,25-Dihydroxyvitamin D(3) Primary cultures of normal and malignant human Human >20 000 [54] prostatic epithelial cells 1a,25-Dihydroxyvitamin D(3) LNCaP prostate cancer cells Human 5600 [55] 1a,25-Dihydroxyvitamin D(3) OVCAR3 ovary cancer cells Human 14 500 [56] 1a,25-Dihydroxyvitamin D(3) MCF-7 breast cancer cells Human 14 500 [57] 1a,25-Dihydroxyvitamin D(3) Duodenal mucosa cells Rat 8799 [58] 1a,25-Dihydroxyvitamin D(3) Primary keratinocytes Human 2135 [59] Menadione MCF-7 breast cancer cells Human 17 000 [60] RRR-a-tocopheryl acetate Liver Rat >7000 [26] RRR-a-tocopheryl acetate Testes Rat >7000 [61] RRR-a-tocopheryl acetate, all HepG2 liver carcinoma cells and rat liver Human and rat 14 500 [62] rac-a-tocopheryl acetate RRR-a-tocopheryl succinate PC-3 prostate cancer cells Human 267 [63] Tocotrienols MCF-7 and MDA-MB-231 breast cancer cells Human 1200 [64, 65] Tocotrienols Mammary tumors from MCF-7 implanted mice Mouse 1176 [64, 65] Vitamin E, lycopene Prostate carcinoma from MatLyLu Dunning Rat 7000 [66] prostate cancer cell-injected rats Phytochemicals Ellagic acid, resveratrol LNCaP prostate cancer cells Human 2400 [67] Ergosterol peroxide HT-29 colon cancer cells Human 8500 [68] Flavonoids HT-29 colon cancer cells Human 96 [69] Flavone HT-29 colon cancer cells Human 9850 [70] Flavone MDA-MB-468, MDA-MB-361, and BT20 breast Human 24 650 [71] cancer cells Flavopiridol Primary T cells and monocytes Human 5032 [72] Biochanin A LNCaP prostate cancer cells Human 2000 [73] Biochanin A Primary hepatocytes, Human 96 Equol, daidzein LNCaP prostate cancer cells Human 14 500 [74] Genistein TCCSUP bladder tumor cells Human 884 [75] Genistein LNCaP prostate cancer cells Human >557 [76] Genistein PC3 cells and bone and subcutaneous tumors Human and mouse 14 500 [77] from PC3 cells-implanted mice Indole-3-carbinole, 3, 30- PC-3 prostate cancer cells Human 22 125 [78] diindolylmethane Indole-3-carbinole MCF-7 breast cancer cells Human 960 [79] 0 Indole-3-carbinole, 3, 3 - Liver Rainbow trout >8000 [80] Nutrigenomics of Applications 20.4 diindolylmethane (Oncorhynchus mykiss) 3,30-Diindolylmethane MDA-MB-231 breast cancer cells Human 22 215 [81] 3,30-Diindolylmethane MCF-7 breast cancer cells Human >20 000 [82] Isoliquiritigenin A549 lung cancer cells Human 111 [83] Isochaihulactone A549 lung cancer cells Human >38 500 [84] Lycopene MCF-7, MDA-MB-231, and MCF-10a breast Human 202 [85] cancer cells Lycopene MCF-7, MDA-MB-231, and MCF-10a breast Human 25 000 [86] cancer cells (Continued) j 317 318 j 0Nutrigenomics 20

Table 20.2 (Continued)

Number of Test substance Cell line or tissue Species genes monitored References

Quercetin CO115 colon adenocarcinoma cells Human 38 500 [87] Sulforaphane Small intestine Mouse 6000 [88] Sulforaphane Caco-2 colon cancer cells Human 14 500 [89] Sulforaphane Liver Mouse >34 000 [90] Taxifolin HCT116 colon cancer cells Human 3096 [91] Vanillin, cinnamaldehyde HCT116 colon cancer cells Human 14 500 [92]

Minerals and trace elements Calcium, heme Colon mucosa Rat 365 [93] Heme Colon mucosa Rat 365 [94] Selenium Intestine Mouse 6347 [95] Selenium-methylselenocysteine Mammary tumors from Mouse 1176 [96] TM6-cell-implanted mice Zinc IMR-90 lung ﬁbroblast Human 704 [97]

Mycotoxins Aﬂatoxin-B1 Liver Rainbow trout >1400 [98] Ochratoxin A Kidney Rat 696 [99] Ochratoxin A Liver and kidney Rat 7000 [100] Ochratoxin A HK-2 kidney cells Human 14 500 [101] 20.5 Proteomics j319 nutrigenomics and particularly transcriptomics in the ﬁeld of cancer and chemoprevention research are manifold.

20.5 Proteomics

Changes in mRNA concentrations and, hence, in gene expression alone do not necessarily result in differences in the concentration and/or activity of the target protein(s). The effects of nutrients or other dietary compounds of interest on the transcriptome should, therefore, always be confirmed by quantitative analysis of the respective protein (e.g., by Western blotting). In this regard, high-throughput proteomic approaches are becoming increasingly popular in nutrition research because they bypass the step of gene expression and facilitate direct assessment of the effects on functional proteins. Proteomic studies generally address one of the three main topics: protein expression, structure, or function [30]. For the analysis of the proteome, proteins are extracted from a sample (e.g., cell or tissue homogenate) and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE; according to charge in the first and according to size in the second dimension) [31]. Very complex protein mixtures (e.g., containing highly hydrophobic membrane proteins) may not be easily resolved by 2D-PAGE, and further sample cleanup or prefractionation (e.g., according to cellular compartment or solubility) may be required. The separated proteins are visualized by staining with silver nitrate, Coomassie Brilliant Blue, fluorescent, or other appropriate dyes. The resulting images of the protein spots on the gels are digitalized and can then be compared visually or, more accurately, with advanced computer programs to identify differences in the protein contents and compositions of the samples. Thus, detected proteins of interest can be extracted from the gels for further characterization and identification. To this end, the separated proteins may be blotted onto membranes and hybridized with specific, labeled antibodies. Alternatively, spots may be excised and subjected to Edman sequencing to obtain the amino acid sequence(s), which can then be compared with entries in public databases [32]. To this end, a number of search engines, such as Mascot, PEAKS, OMSSA, and SEQUEST, to name a few, are available on the Internet and can be used to match protein or peptide sequences from analyzed samples to known protein sequences deposited in these databases. One disadvantage with the above-mentioned techniques is that each spot needs to be transferred and analyzed individually, thus making these approaches relatively time consuming. Therefore, advanced techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) coupled with high- performance liquid chromatography (HPLC) have become powerful tools for high- throughput proteomic analyses [33]. The most commonly used approach for proteomic studies is schematically given in Figure 20.3. Because of its large-scale approach, proteomics has many potential applications in nutrition and cancer research including the identification of protein profiles indicative of a certain disease (diagnostic biomarkers) (Box 20.3). 320j 20 Nutrigenomics

Figure 20.3 Schematic representation of the most widely used approach and the involved techniques in proteomics research.

20.6 Metabolomics

Just as changes in mRNA concentrations may not always result in changes in cellular protein levels, alterations in protein levels do not necessarily bring about changes in protein activity. The metabolome (Box 20.1), the full set of endogenous and exogenous metabolites in a biological system (such as a cell, organ, organism, or biological ﬂuid), however, can be regarded as the end point of gene expression. Thus, changes in the concentrations of metabolites may be better suited to describe the biochemical state of a biological system and may, consequently, be a better measure of gene function than the transcriptome and proteome.

Box 20.3 Potential applications of proteomics in nutrition and cancer research . Identification of (cancer) biomarkers . Comparison of protein profiles in healthy and transformed cells and tissues for diagnostic purpose . Discovery of signal transduction pathways involved in carcinogenesis and their modulation by dietary factors . Analysis of proteins relevant to cell proliferation, differentiation, and cell death . Confirmation of differently expressed genes (end point measurement) 20.6 Metabolomics j321

A major problem associated with metabolomic research is the great diversity of metabolites (which may comprise lipids, proteins, and carbohydrates; see Box 20.1) and their diverse and often opposing physical and chemical properties (e.g., solubility in extraction solvents) and, consequently, the limitations of analytical methods to accurately quantify all metabolites in a given system. Transcriptomics and proteomics, on the other hand, only have to handle one class of compounds each (nucleic acids and proteins, respectively) with relatively similar chemical and physical properties. Mass spectrometry (coupled with liquid or gas chromatography systems) and nuclear magnetic resonance spectroscopy are the two most frequently employed techniques to determine metabolite spectra in biological systems. Less often-employed techniques used for the separation and detection of the metabolome include capillary electrophoresis and electrochemical detection (coupled with HPLC), respectively (Table 20.3). The large number of metabolites in a biological system and the resulting high number of signals in a metabolite spectrum (up to several thousand signals), make data handling particularly challenging and the use of highly sophisticated statistical and bioinformatics tools necessary. During metabolomic studies, experimental conditions (e.g., dietary intake) need to be tightly controlled, to minimize extrinsic factors that may affect metabolite spectra [34, 35]. Once metabolite profiles have been determined and/or individual metabolites have been isolated and quantified, these data can be compared using freely available electronic databases such as the Human Metabolome Database (HMDB; http://www.hmdb.ca/). The HMDB contains detailed information on three kinds of data, namely (i) chemical, (ii) clinical, and (iii) molecular biology/biochemistry data, regarding low molecular weight metabolites present in human tissues and body fluids. The database currently holds entries of 2500 hydrophilic and lipophilic metabolites present at low (<1 nM) to high (>1 mM) concentrations in humans and 5500 protein and DNA sequences linked to these metabolite entries. Approxi- mately half the information contained in about 90 data fields for each entry is devoted to chemical and clinical data and the other half to enzymatic and/or biochemical data. Much of the information is hyperlinked to other online databases (KEGG, PubChem, MetaCyc, ChEBI, PDB, Swiss-Prot, and GenBank) and applets for structure and pathway viewing. The HMDB is complemented by the integration of the databases DrugBank (containing information on 1500 drugs) and FooDB (containing information on 3500 food components and food additives) [36].

Table 20.3 The most common separation and detection techniques used in metabolomic research.

Separation methods Detection methods

Gas chromatography Mass spectrometry High-performance liquid chromatography Nuclear magnetic resonance spectroscopy Capillary electrophoresis Electrochemical detection 322j 20 Nutrigenomics

20.7 Promoting Nutrigenomic Research

A number of organizations and societies have been founded to promote nutrigenomic research. The European Nutrigenomics Organization (NuGO), for example, is a network of excellence funded by the European Commissions Research Directorate General that aims at promoting nutrigenomic research by, among other things, training European scientists in the use of nutrigenomic technologies, developing and integrating genomic technologies for the beneﬁtofnutritional science, and providing a platform for exchange of nutrigenomic expertise on the Internet and in the laboratories of the participating partners (universities, research organizations, and small- to medium-sized businesses). The Nutrigenomics Soci- ety is a closely associated and interlinked organization cooperating with NuGO to foster collaborations between nutrigenomic researchers, enhance the quality of all types of nutrigenomic experiments, and provide and improve educational materials (http://www.nugo.org).

20.8 Summary

Despite being a very powerful tool for the discovery of nutrient–gene interactions, the application of global (e.g., genome-wide) nutrigenomic approaches also poses a philosophical problem, as it is not clear how they fit accepted scientific method. More often than not, in nutrigenomic studies the differential expression of a specific gene facilitated by a dietary factor was not detected while testing a specific hypothesis, but rather through screening of a large number of genes without a specific hypothesis in mind. On the other hand, the broader hypothesis that a certain dietary factor affects gene expression is so nearly unfalsifiable as to be valueless. Therefore, one has to ask oneself, if such post hoc justifications are of value. Could similarly plausible justifications be contrived for a gene randomly selected from the genome? Or should high-throughput nutrigenomic techniques be regarded as a method of generating hypotheses, requiring further biomolecular methods for hypothesis testing? The use of nutrigenomic methods is increasing rapidly and, as databases fill up, these questions will require more urgent consideration. The application of microarray technology to investigate the role of specific nutrients or diets in the maintenance of health and prevention of disease is becoming increasingly popular. However, because of the many factors that may influence the performance and outcome of microarray experiments, and the enormous amount of data that can be generated, it is essential to assure the comparability and reproducibility of results obtained. In this regard, the formulation of the MIAME standards has been a significant milestone for this still young discipline, and the adherence to the MIAME conventions should ensure the meaningful interpretation of microarray data. Because changes in transcriptional activity may not necessarily result in altered levels of the gene product or its biological activity, microarray data should always be References j323 supported by the measurements of protein concentrations and the assessment of specific functional end points (e.g., enzyme activity, metabolite production). If performed, reported, and interpreted with the appropriate care, nutrigenomics has great potential to elucidate the role of dietary factors in the promotion of health and the prevention of disease including cancer.

Acknowledgments

Dr. Frank was supported by a grant (FR 2478/1-1) from the German Research Foundation (Deutsche Forschungsgemeinschaft). The Institute of Human Nutrition and Food Science at the Christian-Albrechts-University, Kiel, Germany, is an associated member of the NuGO network.

References

1 McPherson, J.D., Marra, M., Hillier, L., X.N., Fujiyama, A., Hattori, M., Toyoda, Waterston, R.H., Chinwalla, A., Wallis, J., A., Yada, T., Park, H.S., Sakaki, Y., Sekhon, M., Wylie, K., Mardis, E.R., Shimizu, N., Asakawa, S., Kawasaki, K., Wilson, R.K., Fulton, R., Kucaba, T.A., Sasaki, T., Shintani, A., Shimizu, A., Wagner-McPherson, C., Barbazuk, W.B., Shibuya, K., Kudoh, J., Minoshima, S., Gregory, S.G., Humphray, S.J., French, L., Ramser, J., Seranski, P., Hoff, C., Evans, R.S., Bethel, G., Whittaker, A., Poustka, A., Reinhardt, R. and Lehrach, Holden, J.L., McCann, O.T., Dunham, A., H. (2001) A physical map of the human Soderlund, C., Scott, C.E., Bentley, D.R., genome. Nature, 409 (6822) 934–941. Schuler, G., Chen, H.C., Jang, W., 2 Venter, J.C., Adams, M.D., Myers, E.W., Green, E.D., Idol, J.R., Maduro, V.V., Li, P.W., Mural, R.J., Sutton, G.G., Smith, Montgomery, K.T., Lee, E., Miller, A., H.O., Yandell, M., Evans, C.A., Holt, R.A., Emerling, S., Kucherlapati, Gibbs, R., Gocayne, J.D., Amanatides, P., Ballew, Scherer, S., Gorrell, J.H., Sodergren, E., R.M., Huson, D.H., Wortman, J.R., Clerc-Blankenburg, K., Tabor, P., Naylor, Zhang, Q., Kodira, C.D., Zheng, X.H., S., Garcia, D., de Jong, P.J., Catanese, J.J., Chen, L., Skupski, M., Subramanian, G., Nowak, N., Osoegawa, K., Qin, S., Rowen, Thomas, P.D., Zhang, J., Gabor Miklos, L., Madan, A., Dors, M., Hood, L., Trask, G.L., Nelson, C., Broder, S., Clark, A.G., B., Friedman, C., Massa, H., Cheung, Nadeau, J., McKusick, V.A., Zinder, N., V.G., Kirsch, I.R., Reid, T., Yonescu, R., Levine, A.J., Roberts, R.J., Simon, M., Weissenbach, J., Bruls, T., Heilig, R., Slayman, C., Hunkapiller, M., Bolanos, Branscomb, E., Olsen, A., Doggett, N., R., Delcher, A., Dew, I., Fasulo, D., Cheng, J.F., Hawkins, T., Myers, R.M., Flanigan, M., Florea, L., Halpern, A., Shang, J., Ramirez, L., Schmutz, J., Hannenhalli, S., Kravitz, S., Levy, S., Velasquez, O., Dixon, K., Stone, N.E., Cox, Mobarry, C., Reinert, K., Remington, K., D.R., Haussler, D., Kent, W.J., Furey, T., Abu-Threideh, J., Beasley, E., Biddick, K., Rogic, S., Kennedy, S., Jones, S., Bonazzi, V., Brandon, R., Cargill, M., Rosenthal, A., Wen, G., Schilhabel, M., Chandramouliswaran, I., Charlab, R., Gloeckner, G., Nyakatura, G., Siebert, R., Chaturvedi, K., Deng, Z., Di Francesco, Schlegelberger, B., Korenberg, J., Chen, V., Dunn, P., Eilbeck, K., Evangelista, C., 324j 20 Nutrigenomics

Gabrielian, A.E., Gan, W., Ge, W., Gorokhov, M., Graham, K., Gropman, B., Gong, F., Gu, Z., Guan, P., Heiman, T.J., Harris, M., Heil, J., Henderson, S., Higgins, M.E., Ji, R.R., Ke, Z., Ketchum, Hoover, J., Jennings, D., Jordan, C., K.A., Lai, Z., Lei, Y., Li, Z., Li, J., Liang, Y., Jordan, J., Kasha, J., Kagan, L., Kraft, C., Lin, X., Lu, F., Merkulov, G.V., Milshina, Levitsky, A., Lewis, M., Liu, X., Lopez, J., N., Moore, H.M., Naik, A.K., Narayan, Ma, D., Majoros, W., McDaniel, J., V.A., Neelam, B., Nusskern, D., Rusch, Murphy, S., Newman, M., Nguyen, T., D.B., Salzberg, S., Shao, W., Shue, B., Nguyen, N., Nodell, M., Pan, S., Peck, J., Sun, J., Wang, Z., Wang, A., Wang, X., Peterson, M., Rowe, W., Sanders, R., Wang, J., Wei, M., Wides, R., Xiao, C., Scott, J., Simpson, M., Smith, T., Sprague, Yan, C., Yao, A., Ye, J., Zhan, M., Zhang, A., Stockwell, T., Turner, R., Venter, E., W., Zhang, H., Zhao, Q., Zheng, L., Wang, M., Wen, M., Wu, D., Wu, M., Xia, Zhong, F., Zhong, W., Zhu, S., Zhao, S., A., Zandieh, A. and Zhu, X. (2001) The Gilbert, D., Baumhueter, S., Spier, G., sequence of the human genome. Science, Carter, C., Cravchik, A., Woodage, T., Ali, 291 (5507) 1304–1351. F., An, H., Awe, A., Baldwin, D., Baden, 3 Okazaki, Y., Furuno, M., Kasukawa, T., H., Barnstead, M., Barrow, I., Beeson, K., Adachi, J., Bono, H., Kondo, S., Nikaido, Busam, D., Carver, A., Center, A., Cheng, I., Osato, N., Saito, R., Suzuki, H., M.L., Curry, L., Danaher, S., Davenport, Yamanaka, I., Kiyosawa, H., Yagi, K., L., Desilets, R., Dietz, S., Dodson, K., Tomaru, Y., Hasegawa, Y., Nogami, A., Doup, L., Ferriera, S., Garg, N., Schonbach, C., Gojobori, T., Baldarelli, R., Gluecksmann, A., Hart, B., Haynes, J., Hill, D.P., Bult, C., Hume, D.A., Haynes, C., Heiner, C., Hladun, S., Quackenbush, J., Schriml, L.M., Kanapin, Hostin, D., Houck, J., Howland, T., A., Matsuda, H., Batalov, S., Beisel, K.W., Ibegwam, C., Johnson, J., Kalush, F., Blake, J.A., Bradt, D., Brusic, V., Chothia, Kline, L., Koduru, S., Love, A., Mann, F., C., Corbani, L.E., Cousins, S., Dalla, E., May, D., McCawley, S., McIntosh, T., Dragani, T.A., Fletcher, C.F., Forrest, A., McMullen, I., Moy, M., Moy, L., Murphy, Frazer, K.S., Gaasterland, T., Gariboldi, B., Nelson, K., Pfannkoch, C., Pratts, E., M., Gissi, C., Godzik, A., Gough, J., Puri, V., Qureshi, H., Reardon, M., Grimmond, S., Gustincich, S., Hirokawa, Rodriguez, R., Rogers, Y.H.,Romblad, D., N., Jackson, I.J., Jarvis, E.D., Kanai, A., Ruhfel, B., Scott, R., Sitter, C., Smallwood, Kawaji, H., Kawasawa, Y., Kedzierski, M., Stewart, E., Strong, R., Suh, E., R.M., King, B.L., Konagaya, A., Thomas, R., Tint, N.N., Tse, S., Vech, C., Kurochkin, I.V., Lee, Y., Lenhard, B., Wang, G., Wetter, J., Williams, S., Lyons, P.A., Maglott, D.R., Maltais, L., Williams, M., Windsor, S., Winn-Deen, Marchionni, L., McKenzie, L., Miki, H., E., Wolfe, K., Zaveri, J., Zaveri, K., Abril, Nagashima, T., Numata, K., Okido, T., J.F., Guigo, R., Campbell, M.J., Sjolander, Pavan, W.J., Pertea, G., Pesole, G., K.V., Karlak, B., Kejariwal, A., Mi, H., Petrovsky, N., Pillai, R., Pontius, J.U., Qi, Lazareva, B., Hatton, T., Narechania, A., D., Ramachandran, S., Ravasi, T., Reed, Diemer, K., Muruganujan, A., Guo, N., J.C., Reed, D.J., Reid, J., Ring, B.Z., Sato, S., Bafna, V., Istrail, S., Lippert, R., Ringwald, M., Sandelin, A., Schneider, C., Schwartz, R., Walenz, B., Yooseph, S., Semple, C.A., Setou, M., Shimada, K., Allen, D., Basu, A., Baxendale, J., Blick, L., Sultana, R., Takenaka, Y., Taylor, M.S., Caminha, M., Carnes-Stine, J., Caulk, P., Teasdale, R.D., Tomita, M., Verardo, R., Chiang, Y.H., Coyne, M., Dahlke, C., Wagner, L., Wahlestedt, C., Wang, Y., Mays, A., Dombroski, M., Donnelly, M., Watanabe, Y., Wells, C., Wilming, L.G., Ely, D., Esparham, S., Fosler, C., Gire, H., Wynshaw-Boris, A., Yanagisawa, M., Glanowski, S., Glasser, K., Glodek, A., Yang, I., Yang, L., Yuan, Z., Zavolan, M., References j325

Zhu, Y., Zimmer, A., Carninci, P., R.S., Kulbokas, E.J., Kulp, D., Landers, T., Hayatsu, N., Hirozane-Kishikawa, T., Leger, J.P., Leonard, S., Letunic, I., Levine, Konno, H., Nakamura, M., Sakazume, N., R., Li, J., Li, M., Lloyd, C., Lucas, S., Ma, B., Sato, K., Shiraki, T., Waki, K., Kawai, J., Maglott, D.R., Mardis, E.R., Matthews, L., Aizawa, K., Arakawa, T., Fukuda, S., Hara, Mauceli, E., Mayer, J.H., McCarthy, M., A., Hashizume, W., Imotani, K., Ishii, Y., McCombie, W.R., McLaren, S., McLay, K., Itoh, M., Kagawa, I., Miyazaki, A., Sakai, McPherson, J.D., Meldrim, J., Meredith, K., Sasaki, D., Shibata, K., Shinagawa, A., B., Mesirov, J.P., Miller, W., Miner, T.L., Yasunishi, A., Yoshino, M., Waterston, R., Mongin, E., Montgomery, K.T., Morgan, Lander, E.S., Rogers, J., Birney, E. and M., Mott, R., Mullikin, J.C., Muzny, D.M., Hayashizaki, Y. (2002) Analysis of the Nash, W.E., Nelson, J.O., Nhan, M.N., mouse transcriptome based on functional Nicol, R., Ning, Z., Nusbaum, C., annotation of 60,770 full-length cDNAs. OConnor, M.J., Okazaki, Y., Oliver, K., Nature, 420 (6915), 563–573. Overton-Larty, E., Pachter, L., Parra, G., 4 Waterston, R.H., Lindblad-Toh, K., Pepin, K.H., Peterson, J., Pevzner, P., Birney, E., Rogers, J., Abril, J.F., Agarwal, Plumb, R., Pohl, C.S., Poliakov, A., Ponce, P., Agarwala, R., Ainscough, R., T.C., Ponting, C.P., Potter, S., Quail, M., Alexandersson, M., An, P., Antonarakis, Reymond, A., Roe, B.A., Roskin, K.M., S.E., Attwood, J., Baertsch, R., Bailey, J., Rubin, E.M., Rust, A.G., Santos, R., Barlow, K., Beck, S., Berry, E., Birren, B., Sapojnikov, V., Schultz, B., Schultz, J., Bloom, T., Bork, P., Botcherby, M., Bray, Schwartz, M.S., Schwartz, S., Scott, C., N., Brent, M.R., Brown, D.G., Brown, Seaman, S., Searle, S., Sharpe, T., S.D., Bult, C., Burton, J., Butler, J., Sheridan, A., Shownkeen, R., Sims, S., Campbell, R.D., Carninci, P., Cawley, S., Singer, J.B., Slater, G., Smit, A., Smith, Chiaromonte, F., Chinwalla, A.T., D.R., Spencer, B., Stabenau, A., Stange- Church, D.M., Clamp, M., Clee, C., Thomann, N., Sugnet, C., Suyama, M., Collins, F.S., Cook, L.L., Copley, R.R., Tesler, G., Thompson, J., Torrents, D., Coulson, A., Couronne, O., Cuff, J., Trevaskis, E., Tromp, J., Ucla, C., Ureta- Curwen, V., Cutts, T., Daly, M., David, R., Vidal, A., Vinson, J.P., Von Davies, J., Delehaunty, K.D., Deri, J., Niederhausern, A.C., Wade, C.M., Wall, Dermitzakis, E.T., Dewey, C., Dickens, M., Weber, R.J., Weiss, R.B., Wendl, M.C., N.J., Diekhans, M., Dodge, S., Dubchak, West, A.P., Wetterstrand, K., Wheeler, R., I., Dunn, D.M., Eddy, S.R., Elnitski, L., Whelan, S., Wierzbowski, J., Willey, D., Emes, R.D., Eswara, P., Eyras, E., Williams, S., Wilson, R.K., Winter, E., Felsenfeld, A., Fewell, G.A., Flicek, P., Worley, K.C., Wyman, D., Yang, S., Foley, K., Frankel, W.N., Fulton, L.A., Yang, S.P., Zdobnov, E.M., Zody, M.C. Fulton, R.S., Furey, T.S., Gage, D., Gibbs, and Lander, E.S. (2002) Initial R.A., Glusman, G., Gnerre, S., Goldman, sequencing and comparative analysis of N., Goodstadt, L., Grafham, D., Graves, the mouse genome. Nature, 420 (6915), T.A., Green, E.D., Gregory, S., Guigo, R., 520–562. Guyer, M., Hardison, R.C., Haussler, D., 5 Gibbs, R.A., Weinstock, G.M., Metzker, Hayashizaki, Y., Hillier, L.W., Hinrichs, M.L., Muzny, D.M., Sodergren, E.J., A., Hlavina, W., Holzer, T., Hsu, F., Hua, Scherer, S., Scott, G., Steffen, D., Worley, A., Hubbard, T., Hunt, A., Jackson, I., K.C., Burch, P.E., Okwuonu, G., Hines, Jaffe, D.B., Johnson, L.S., Jones, M., S., Lewis, L., DeRamo, C., Delgado, O., Jones, T.A., Joy, A., Kamal, M., Karlsson, Dugan-Rocha, S., Miner, G., Morgan, M., E.K., Karolchik, D., Kasprzyk, A., Kawai, Hawes, A., Gill, R., Celera, Holt, R.A., J., Keibler, E., Kells, C., Kent, W.J., Kirby, Adams, M.D., Amanatides, P.G., Baden- A., Kolbe, D.L., Korf, I., Kucherlapati, Tillson, H., Barnstead, M., Chin, S., 326j 20 Nutrigenomics

Evans, C.A., Ferriera, S., Fosler, C., Caccamo, M., Clamp, M., Clarke, L., Glodek, A., Gu, Z., Jennings, D., Kraft, Curwen, V., Durbin, R., Eyras, E., C.L., Nguyen, T., Pfannkoch, C.M., Sitter, Searle, S.M., Cooper, G.M., Batzoglou, S., C., Sutton, G.G., Venter, J.C., Woodage, Brudno, M., Sidow, A., Stone, E.A., T., Smith, D., Lee, H.M., Gustafson, E., Venter, J.C., Payseur, B.A., Bourque, G., Cahill, P., Kana, A., Doucette-Stamm, L., Lopez-Otin, C., Puente, X.S., Chakrabarti, Weinstock, K., Fechtel, K., Weiss, R.B., K., Chatterji, S., Dewey, C., Pachter, L., Dunn, D.M., Green, E.D., Blakesley, R.W., Bray, N., Yap, V.B., Caspi, A., Tesler, G., Bouffard, G.G., De Jong, P.J., Osoegawa, Pevzner, P.A., Haussler, D., Roskin, K.M., K., Zhu, B., Marra, M., Schein, J., Bosdet, Baertsch, R., Clawson, H., Furey, T.S., I., Fjell, C., Jones, S., Krzywinski, M., Hinrichs, A.S., Karolchik, D., Kent, W.J., Mathewson, C., Siddiqui, A., Wye, N., Rosenbloom, K.R., Trumbower, H., McPherson, J., Zhao, S., Fraser, C.M., Weirauch, M., Cooper, D.N., Stenson, Shetty, J., Shatsman, S., Geer, K., Chen, Y., P.D., Ma, B., Brent, M., Arumugam, M., Abramzon, S., Nierman, W.C., Havlak, Shteynberg, D., Copley, R.R., Taylor, M.S., P.H., Chen, R., Durbin, K.J., Egan, A., Riethman, H., Mudunuri, U., Peterson, Ren, Y., Song, X.Z., Li, B., Liu, Y., Qin, X., J., Guyer, M., Felsenfeld, A., Old, S., Cawley, S., Worley, K.C., Cooney, A.J., Mockrin, S. and Collins, F. (2004) DSouza, L.M., Martin, K., Wu, J.Q., Genome sequence of the Brown Norway Gonzalez-Garay, M.L., Jackson, A.R., rat yields insights into mammalian Kalafus, K.J., McLeod, M.P., evolution. Nature, 428 (6982), 493–521. Milosavljevic, A., Virk, D., Volkov, A., 6 Rimbach, G., Fuchs, J. and Packer, L. (eds) Wheeler, D.A., Zhang, Z., Bailey, J.A., (2005) Nutrigenomics, Oxidative Stress and Eichler, E.E., Tuzun, E., Birney, E., Disease Series, CRC Press, Boca Raton, FL. Mongin, E., Ureta-Vidal, A., Woodwark, 7 Rana, T.M. (2007) Illuminating the C., Zdobnov, E., Bork, P., Suyama, M., silence: understanding the structure and Torrents, D., Alexandersson, M., Trask, function of small RNAs. Nature Reviews B.J., Young, J.M., Huang, H., Wang, H., Molecular Cell Biology, 8 (1), 23–36. Xing, H., Daniels, S., Gietzen, D., 8 Cowland, J.B., Hother, C. and Gronbaek, Schmidt, J., Stevens, K., Vitt, U., K. (2007) MicroRNAs and cancer. Acta Wingrove, J., Camara, F., Mar Alba, M., Pathologica, Microbiologica et Abril, J.F., Guigo, R., Smit, A., Dubchak, Immunologica, 115 (10), 1090–1106. I., Rubin, E.M., Couronne, O., Poliakov, 9 Lewis, B.P., Burge, C.B. and Bartel, D.P. A., Hubner, N., Ganten, D., Goesele, C., (2005) Conserved seed pairing, often Hummel, O., Kreitler, T.,Lee, Y.A.,Monti, ﬂanked by adenosines, indicates that J., Schulz, H., Zimdahl, H., thousands of human genes are Himmelbauer, H., Lehrach, H., Jacob, microRNA targets. Cell, 120 (1), 15–20. H.J., Bromberg, S., Gullings-Handley, J., 10 Heid, C.A., Stevens, J., Livak, K.J. and Jensen-Seaman, M.I., Kwitek, A.E., Lazar, Williams, P.M. (1996) Real time J., Pasko, D., Tonellato, P.J., Twigger, S., quantitative PCR. Genome Research, 6 Ponting, C.P., Duarte, J.M., Rice, S., (10), 986–994. Goodstadt, L., Beatson, S.A., Emes, R.D., 11 Chen, C., Ridzon, D.A., Broomer, A.J., Winter, E.E., Webber, C., Brandt, P., Zhou, Z., Lee, D.H., Nguyen, J.T., Nyakatura, G., Adetobi, M., Chiaromonte, Barbisin, M., Xu, N.L., Mahuvakar, V.R., F., Elnitski, L., Eswara, P., Hardison, R.C., Andersen, M.R., Lao, K.Q., Livak, K.J. and Hou, M., Kolbe, D., Makova, K., Miller, Guegler, K.J. (2005) Real-time W., Nekrutenko, A., Riemer, C., Schwartz, quantiﬁcation of microRNAs by stem- S., Taylor, J., Yang, S., Zhang, Y., loop RT-PCR. Nucleic Acids Research, 33 Lindpaintner, K., Andrews, T.D., (20), e179. References j327

12 Clarke, A.M., Mapstone, N.P. and Quirke, C., Aach, J., Ansorge, W., Ball, C.A., P. (1992) Molecular biology made easy. Causton, H.C., Gaasterland, T., The polymerase chain reaction. The Glenisson, P., Holstege, F.C., Kim, I.F., Histochemical Journal, 24 (12), 913–926. Markowitz, V., Matese, J.C., Parkinson, 13 Bustin, S.A. (2000) Absolute H., Robinson, A., Sarkans, U., Schulze- quantification of mRNA using real-time Kremer, S., Stewart, J., Taylor, R., Vilo, J. reverse transcription polymerase chain and Vingron, M. (2001) Minimum reaction assays. Journal of Molecular information about a microarray Endocrinology, 25 (2), 169–193. experiment (MIAME): toward standards 14 Roth, C.M. (2002) Quantifying gene for microarray data. Nature Genetics, 29 expression. Current Issues in Molecular (4), 365–371. Biology, 4 (3), 93–100. 24 Tefferi, A., Bolander, M.E., Ansell, S.M., 15 Yin, J.Q., Zhao, R.C. and Morris, K.V. Wieben, E.D. and Spelsberg, T.C. (2002) (2008) Profiling microRNA expression Primer on medical genomics. Part III: with microarrays. Trends in Biotechnology, Microarray experiments and data 26 (2), 70–76. analysis. Mayo Clinic Proceedings, 77 (9), 16 Castoldi, M., Schmidt, S., Benes, V., 927–940. Noerholm, M., Kulozik, A.E., Hentze, 25 McShane, L.M., Shih, J.H. and M.W. and Muckenthaler, M.U. (2006) A Michalowska, A.M. (2003) Statistical sensitive array for microRNA expression issues in the design and analysis of gene profiling (miChip) based on locked expression microarray studies of animal nucleic acids (LNA). RNA, 12 (5), models. Journal of Mammary Gland 913–920. Biology and Neoplasia, 8 (3), 359–374. 17 Braasch, D.A. and Corey, D.R. (2001) 26 Barella, L., Muller, P.Y., Schlachter, M., Locked nucleic acid (LNA): fine-tuning Hunziker, W., Stocklin, E., Spitzer, V., the recognition of DNA and RNA. Meier, N., de Pascual-Teresa, S., Chemistry & Biology, 8 (1), 1–7. Minihane, A.M. and Rimbach, G. (2004) 18 Schena, M. (ed.) (1999) DNA Microarrays: Identification of hepatic molecular A Practical Approach, Practical Approach mechanisms of action of alpha-tocopherol Series, Oxford University Press, New York. using global gene expression profile 19 Elliott, R.N., Bacon, J.R. and Bao, Y.(2004) analysis in rats. Biochimica et Biophysica Nutritional genomics, in Phytochemicals Acta, 1689 (1), 66–74. in Health and Disease (eds Y. Bao and R. 27 Page, G.P., Edwards, J.W., Barnes, S., Fenwick), Marcel Dekker, Inc., New York, Weindruch, R. and Allison, D.B. (2003) A pp. 1–23. design and statistical perspective on 20 Liu-Stratton, Y., Roy, S. and Sen, C.K. microarray gene expression studies in (2004) DNA microarray technology in nutrition: the need for playful creativity nutraceutical and food safety. Toxicology and scientific hard-mindedness. Letters, 150 (1), 29–42. Nutrition, 19 (11–12), 997–1000. 21 Spielbauer, B. and Stahl, F. (2005) Impact 28 Rensink, W.A. and Hazen, S.P. (2006) of microarray technology in nutrition and Statistical issues in microarray data food research. Molecular Nutrition and analysis. Methods in Molecular Biology, Food Research, 49 (10), 908–917. 323, 359–366. 22 Mills, K. (2005) Analysis of microarray 29 Walker, M.S. and Hughes, T.A. (2008) data, in Nutrigenomics, vol. 17 (eds G. Messenger RNA expression profiling Rimbach, J. Fuchs and L. Packer), CRC using DNA microarray technology: Press, Boca Raton, FL, USA, pp. 43–66. diagnostic tool, scientific analysis or un- 23 Brazma, A., Hingamp, P., Quackenbush, interpretable data? International Journal of J., Sherlock, G., Spellman, P., Stoeckert, Molecular Medicine, 21 (1), 13–17. 328j 20 Nutrigenomics

30 Kussmann, M., Raymond, F. and Affolter, primary, adenoma and tumor human M. (2006) OMICS-driven biomarker colon cells by favourably modulating discovery in nutrition and health. Journal expression of glutathione S-transferases of Biotechnology, 124 (4), 758–787. genes, an approach in nutrigenomics. 31 Gorg, A., Obermaier, C., Boguth, G., Carcinogenesis, 26 (6), 1064–1076. Harder, A., Scheibe, B., Wildgruber, R. 38 Fujiwara, K., Ochiai, M., Ubagai, T., and Weiss, W. (2000) The current state of Ohki, M., Ohta, T., Nagao, M., Sugimura, two-dimensional electrophoresis with T. and Nakagama, H. (2003) Differential immobilized pH gradients. gene expression profiles in colon Electrophoresis, 21 (6), 1037–1053. epithelium of two rat strains with distinct 32 Gevaert, K. and Vandekerckhove, J. (2000) susceptibility to colon carcinogenesis Protein identification methods in after exposure to PhIP in combination proteomics. Electrophoresis, 21 (6), with dietary high fat. Cancer Science, 94 1145–1154. (8), 672–678. 33 Daniel, H. (2002) Genomics and 39 Davidson, L.A., Nguyen, D.V., Hokanson, proteomics: importance for the future of R.M., Callaway, E.S., Isett, R.B., Turner, nutrition research. The British Journal of N.D., Dougherty, E.R., Wang, N., Lupton, Nutrition, 87 (Suppl 2), S305–S311. J.R., Carroll, R.J. and Chapkin, R.S. (2004) 34 Whitfield, P.D., German, A.J. and Noble, Chemopreventive n-3 polyunsaturated P.J. (2004) Metabolomics: an emerging fatty acids reprogram genetic signatures post-genomic tool for nutrition. The during colon cancer initiation and British Journal of Nutrition, 92 (4), progression in the rat. Cancer Research, 64 549–555. (18), 6797–6804. 35 Gibney, M.J., Walsh, M., Brennan, L., 40 Escrich, E., Moral, R., Garcia, G., Costa, I., Roche, H.M., German, B. and van Sanchez, J.A. and Solanas, M. (2004) Ommen, B. (2005) Metabolomics in Identification of novel differentially human nutrition: opportunities and expressed genes by the effect of a high-fat challenges. The American Journal of n-6 diet in experimental breast cancer. Clinical Nutrition, 82 (3), 497–503. Molecular Carcinogenesis, 40 (2), 73–78. 36 Wishart, D.S., Tzur, D., Knox, C., Eisner, 41 Keijer, J., Bunschoten, A., Palou, A. and R., Guo, A.C., Young, N., Cheng, D., Franssen-van Hal, N.L. (2005) Beta- Jewell, K., Arndt, D., Sawhney, S., Fung, carotene and the application of C., Nikolai, L., Lewis, M., Coutouly, M.A., transcriptomics in risk–benefit evaluation Forsythe, I., Tang, P., Shrivastava, S., of natural dietary components. Biochimica Jeroncic, K., Stothard, P., Amegbey, G., et Biophysica Acta, 1740 (2), 139–146. Block, D., Hau, D.D., Wagner, J., Miniaci, 42 Novakovic, P., Stempak, J.M., Sohn, K.J. J., Clements, M., Gebremedhin, M., Guo, and Kim, Y.I. (2006) Effects of folate N., Zhang, Y., Duggan, G.E., Macinnis, deficiency on gene expression in the G.D., Weljie, A.M., Dowlatabadi, R., apoptosis and cancer pathways in colon Bamforth, F., Clive, D., Greiner, R., Li, L., cancer cells. Carcinogenesis, 27 (5), Marrie, T., Sykes, B.D., Vogel, H.J. and 916–924. Querengesser, L. (2007) HMDB: the 43 Pellis, L., Dommels, Y., Venema, D., Human Metabolome Database. Nucleic Polanen, A.V., Lips, E., Baykus, H., Kok, Acids Research, 35 (Database issue), F., Kampman, E. and Keijer, J. (2008) D521–D526. High folic acid increases cell turnover and 37 Pool-Zobel, B.L., Selvaraju, V., Sauer, J., lowers differentiation and iron content in Kautenburger, T., Kiefer, J., Richter, K.K., human HT29 colon cancer cells. The Soom, M. and Wolfl, S. (2005) Butyrate British Journal of Nutrition, 99 (4), may enhance toxicological defence in 703–708. References j329

44 Burns, F.J., Chen, S., Xu, G., Wu, F. and 51 Swami, S., Raghavachari, N., Muller, Tang, M.S. (2002) The action of a dietary U.R., Bao, Y.P. and Feldman, D. (2003) retinoid on gene expression and cancer Vitamin D growth inhibition of breast induction in electron-irradiated rat skin. cancer cells: gene expression patterns Journal of Radiation Research, 43 (Suppl), assessed by cDNA microarray. Breast S229–S232. Cancer Research and Treatment, 80 (1), 45 Zhuang, Y., Faria, T.N., Chambon, P. and 49–62. Gudas, L.J. (2003) Identification and 52 Guzey, M., Luo, J. and Getzenberg, R.H. characterization of retinoic acid receptor (2004) Vitamin D3 modulated gene beta2 target genes in F9 teratocarcinoma expression patterns in human primary cells. Molecular Cancer Research, 1 (8), normal and cancer prostate cells. Journal 619–630. of Cellular Biochemistry, 93 (2), 271–285. 46 Ma, Y., Koza-Taylor, P.H., DiMattia, D.A., 53 Krishnan, A.V., Shinghal, R., Hames, L., Fu, H., Dragnev, K.H., Turi, T., Raghavachari, N., Brooks, J.D., Peehl, Beebe, J.S., Freemantle, S.J. and D.M. and Feldman, D. (2004) Analysis of Dmitrovsky, E. (2003) Microarray analysis vitamin D-regulated gene expression in uncovers retinoid targets in human LNCaP human prostate cancer cells using bronchial epithelial cells. Oncogene, 22 cDNA microarrays. Prostate, 59 (3), (31), 4924–4932. 243–251. 47 Pitha-Rowe, I., Petty, W.J., Feng, Q., Koza- 54 Peehl, D.M., Shinghal, R., Nonn, L., Seto, Taylor, P.H., Dimattia, D.A., Pinder, L., E., Krishnan, A.V., Brooks, J.D. and Dragnev, K.H., Memoli, N., Memoli, V., Feldman, D. (2004) Molecular activity of Turi, T., Beebe, J., Kitareewan, S. and 1,25-dihydroxyvitamin D3 in primary Dmitrovsky, E. (2004) Microarray cultures of human prostatic epithelial analyses uncover UBE1L as a candidate cells revealed by cDNA microarray target gene for lung cancer analysis. The Journal of Steroid chemoprevention. Cancer Research, 64 Biochemistry and, Molecular Biology 92 (3), (21), 8109–8115. 131–141. 48 Kim, K.N., Pie, J.E., Park, J.H., Park, Y.H., 55 Ikezoe, T., Gery, S., Yin, D., OKelly, J., Kim, H.W. and Kim, M.K. (2006) Binderup, L., Lemp, N., Taguchi, H. and Retinoic acid and ascorbic acid act Koeffler, H.P. (2005) CCAAT/enhancer- synergistically in inhibiting human binding protein delta: a molecular target breast cancer cell proliferation. The of 1,25-dihydroxyvitamin D3 in androgen- Journal of Nutritional Biochemistry, 17 (7), responsive prostate cancer LNCaP cells. 454–462. Cancer Research, 65 (11), 4762–4768. 49 Palmer, H.G., Sanchez-Carbayo, M., 56 Zhang, X., Li, P., Bao, J., Nicosia, S.V., Ordonez-Moran, P., Larriba, M.J., Wang, H., Enkemann, S.A. and Bai, W. Cordon-Cardo, C. and Munoz, A. (2003) (2005) Suppression of death receptor- Genetic signatures of differentiation mediated apoptosis by 1,25- induced by 1alpha,25-dihydroxyvitamin dihydroxyvitamin D3 revealed by D3 in human colon cancer cells. Cancer microarray analysis. The Journal of Research, 63 (22), 7799–7806. Biological Chemistry, 280 (42), 50 Qiao, S., Pennanen, P., Nazarova, N., Lou, 35458–35468. Y.R.and Tuohimaa,P. (2003) Inhibition of 57 Towsend, K., Trevino, V., Falciani, F., fatty acid synthase expression by Stewart, P.M., Hewison, M. and 1alpha,25-dihydroxyvitamin D3 in Campbell, M.J. (2006) Identification of prostate cancer cells. The Journal of Steroid VDR-responsive gene signatures in breast Biochemistry and Molecular Biology cancer cells. Oncology, 71 (1–2), 85 (1), 1–8. 111–123. 330j 20 Nutrigenomics

58 Kutuzova, G.D. and DeLuca, H.F. (2007) Tocotrienol-rich fraction from palm oil 1,25-Dihydroxyvitamin D3 regulates affects gene expression in tumors genes responsible for detoxification in resulting from MCF-7 cell inoculation in intestine. Toxicology and Applied athymic mice. Lipids, 39 (5), 459–467. Pharmacology, 218 (1), 37–44. 66 Siler, U., Barella, L., Spitzer, V., Schnorr, 59 Moll, P.R., Sander, V.,Frischauf, A.M. and J., Lein, M., Goralczyk, R. and Wertz, K. Richter, K. (2007) Expression profiling of (2004) Lycopene and vitamin E interfere vitamin D treated primary human with autocrine/paracrine loops in the keratinocytes. Journal of Cellular Dunning prostate cancer model. The Biochemistry, 100 (3), 574–592. FASEB Journal, 18 (9), 1019–1021. 60 Chuang, Y.Y., Chen, Y., Gadisetti, 67 Narayanan, B.A., Narayanan, N.K., Chandramouli, V.R., Cook, J.A., Coffin, Stoner, G.D. and Bullock, B.P. (2002) D., Tsai, M.H., DeGraff, W., Yan, H., Interactive gene expression pattern in Zhao, S., Russo, A., Liu, E.T.and Mitchell, prostate cancer cells exposed to phenolic J.B. (2002). Gene expression after antioxidants. Life Sciences, 70 (15), treatment with hydrogen peroxide, 1821–1839. menadione, or t-butyl hydroperoxide in 68 Kobori, M., Yoshida, M., Ohnishi- breast cancer cells. Cancer Research, 62 Kameyama, M. and Shinmoto, H. (2007) (21), 6246–6254. Ergosterol peroxide from an edible 61 Rota, C., Barella, L., Minihane, A.M., mushroom suppresses inflammatory Stocklin, E. and Rimbach, G. (2004) responses in RAW264.7 macrophages Dietary alpha-tocopherol affects and growth of HT29 colon differential gene expression in rat testes. adenocarcinoma cells. British Journal of IUBMB Life, 56 (5), 277–280. Pharmacology, 150 (2), 209–219. 62 Hundhausen, C., Frank, J., Rimbach, G., 69 Veeriah, S., Kautenburger, T., Stoecklin, E., Muller, P.Y. and Barella, L. Habermann, N., Sauer, J., Dietrich, H., (2006) Effect of vitamin E on cytochrome Will, F. and Pool-Zobel, B.L. (2006) Apple P450 mRNA levels in cultured flavonoids inhibit growth of HT29 human hepatocytes (HepG2) and in rat liver. colon cancer cells and modulate Cancer Genomics & Proteomics, 3 (3–4), expression of genes involved in the 183–190. biotransformation of xenobiotics. 63 Malafa, M.P., Fokum, F.D., Andoh, J., Molecular Carcinogenesis, 45 (3), 164–174. Neitzel, L.T., Bandyopadhyay, S., Zhan, 70 Herzog, A., Kindermann, B., Doring, F., R., Iiizumi, M., Furuta, E., Horvath, E. Daniel, H. and Wenzel, U. (2004) and Watabe, K. (2006) Vitamin E Pleiotropic molecular effects of the pro- succinate suppresses prostate tumor apoptotic dietary constituent flavone in growth by inducing apoptosis. human colon cancer cells identified by International Journal of Cancer, 118 (10), protein and mRNA expression profiling. 2441–2447. Proteomics, 4 (8), 2455–2464. 64 Nesaretnam, K., Ambra, R., Selvaduray, 71 Ullmannova, V. and Popescu, N.C. (2007) K.R., Radhakrishnan, A., Canali, R. and Inhibition of cell proliferation, induction Virgili, F. (2004) Tocotrienol-rich fraction of apoptosis, reactivation of DLC1, and from palm oil and gene expression in modulation of other gene expression by human breast cancer cells. Annals of the dietary flavone in breast cancer cell lines. New York Academy of Sciences 1031, Cancer Detection and Prevention, 31 (2), 143–157. 110–118. 65 Nesaretnam, K., Ambra, R., Selvaduray, 72 Lam, L.T., Pickeral, O.K., Peng, A.C., K.R., Radhakrishnan, A., Reimann, K., Rosenwald, A., Hurt, E.M., Giltnane, Razak, G. and Virgili, F. (2004) J.M., Averett, L.M., Zhao, H., Davis, R.E., References j331

Sathyamoorthy, M., Wahl, L.M., Harris, determined by cDNA microarray analysis. E.D., Mikovits, J.A., Monks, A.P., The Journal of Nutrition, 133 (4), Hollingshead, M.G., Sausville, E.A. and 1011–1019. Staudt, L.M. (2001) Genomic-scale 79 Chatterji, U., Riby, J.E., Taniguchi, T., measurement of mRNA turnover and the Bjeldanes, E.L., Bjeldanes, L.F. and mechanisms of action of the anti-cancer Firestone, G.L. (2004) Indole-3-carbinol drug flavopiridol. Genome Biology, 2 (10), stimulates transcription of the interferon RESEARCH0041. gamma receptor 1 gene and augments 73 Rice, L., Samedi, V.G., Medrano, T.A., interferon responsiveness in human Sweeney, C.A., Baker, H.V., Stenstrom, breast cancer cells. Carcinogenesis, 25 (7), A., Furman, J. and Shiverick, K.T. (2002) 1119–1128. Mechanisms of the growth inhibitory 80 Tilton, S.C., Givan, S.A., Pereira, C.B., effects of the isoflavonoid biochanin A on Bailey, G.S. and Williams, D.E. (2006) LNCaP cells and xenografts. Prostate, 52 Toxicogenomic profiling of the hepatic (3), 201–212. tumor promoters indole-3-carbinol, 74 Takahashi, Y., Lavigne, J.A., Hursting, 17beta-estradiol and beta-naphthoflavone S.D., Chandramouli, G.V., Perkins, S.N., in rainbow trout. Toxicological Sciences, 90 Kim, Y.S.and Wang, T.T.(2006) Molecular (1), 61–72. signatures of soy-derived phytochemicals 81 Rahman, K.W., Li, Y., Wang, Z., Sarkar, in androgen-responsive prostate cancer S.H. and Sarkar, F.H. (2006) Gene cells: a comparison study using DNA expression profiling revealed survivin as a microarray. Molecular Carcinogenesis, 45 target of 3,30-diindolylmethane-induced (12), 943–956. cell growth inhibition and apoptosis in 75 Chen, C.C., Shieh, B., Jin, Y.T., Liau, Y.E., breast cancer cells. Cancer Research, 66 (9), Huang, C.H., Liou, J.T.,Wu, L.W.,Huang, 4952–4960. W., Young, K.C., Lai, M.D., Liu, H.S. and 82 Mulvey, L., Chandrasekaran, A., Liu, K., Li, C. (2001) Microarray profiling of gene Lombardi, S., Wang, X.P., Auborn, K.J. expression patterns in bladder tumor cells and Goodwin, L. (2007) Interplay of treated with genistein. Journal of genes regulated by estrogen and Biomedical Science, 8 (2), 214–222. diindolylmethane in breast cancer 76 Suzuki, K., Koike, H., Matsui, H., Ono, Y., cell lines. Molecular Medicine, 13 (1–2), Hasumi, M., Nakazato, H., Okugi, H., 69–78. Sekine, Y., Oki, K., Ito, K., Yamamoto, T., 83 Ii, T., Satomi, Y., Katoh, D., Shimada, J., Fukabori, Y., Kurokawa, K. and Yamanaka, Baba, M., Okuyama, T., Nishino, H. and H. (2002) Genistein, a soy isoflavone, Kitamura, N. (2004) Induction of cell cycle induces glutathione peroxidase in the arrest and p21(CIP1/WAF1) expression human prostate cancer cell lines LNCaP in human lung cancer cells by and PC-3. International Journal of Cancer, isoliquiritigenin. Cancer Letters, 207 (1), 99 (6), 846–852. 27–35. 77 Li, Y., Che, M., Bhagat, S., Ellis, K.L., 84 Chen, Y.L., Lin, P.C., Chen, S.P., Lin, Kucuk, O., Doerge, D.R., Abrams, J., C.C., Tsai, N.M., Cheng, Y.L., Chang, Cher, M.L. and Sarkar, F.H. (2004) W.L., Lin, S.Z. and Harn, H.J. (2007) Regulation of gene expression and Activation of nonsteroidal anti- inhibition of experimental prostate cancer inflammatory drug-activated gene-1 via bone metastasis by dietary genistein. extracellular signal-regulated kinase 1/2 Neoplasia, 6 (4), 354–363. mitogen-activated protein kinase revealed 78 Li, Y., Li, X. and Sarkar, F.H. (2003) Gene a isochaihulactone-triggered apoptotic expression profiles of I3C- and DIM- pathway in human lung cancer A549 cells. treated PC3 human prostate cancer cells The Journal of Pharmacology and 332j 20 Nutrigenomics

Experimental Therapeutics 323 (2), chemopreventive effect of taxifolin is 746–756. exerted through ARE-dependent gene 85 Chalabi, N., Delort, L., Le Corre, L., Satih, regulation. Biological & Pharmaceutical S., Bignon, Y.J. and Bernard-Gallon, D. Bulletin, 30 (6), 1074–1079. (2006) Gene signature of breast cancer 92 King, A.A., Shaughnessy, D.T., Mure, K., cell lines treated with lycopene. Leszczynska, J., Ward, W.O., Umbach, Pharmacogenomics, 7 (5), 663–672. D.M., Xu, Z., Ducharme, D., Taylor, J.A., 86 Chalabi, N., Satih, S., Delort, L., Bignon, Demarini, D.M. and Klein, C.B. (2007) Y.J. and Bernard-Gallon, D.J. (2007) Antimutagenicity of cinnamaldehyde and Expression profiling by whole-genome vanillin in human cells: global gene microarray hybridization reveals expression and possible role of DNA differential gene expression in breast damage and repair. Mutation Research, cancer cell lines after lycopene exposure. 616 (1–2), 60–69. Biochimica et Biophysica Acta, 1769 (2), 93 van der Meer-van Kraaij, C., Kramer, E., 124–130. Jonker-Termont, D., Katan, M.B., van der 87 Murtaza, I., Marra, G., Schlapbach, R., Meer, R. and Keijer, J. (2005) Differential Patrignani, A., Kunzli, M., Wagner, U., gene expression in rat colon by dietary Sabates, J. and Dutt, A. (2006) A heme and calcium. Carcinogenesis, 26 (1), preliminary investigation demonstrating 73–79. the effect of quercetin on the expression 94 van der Meer-van Kraaij, C., van Lieshout, of genes related to cell-cycle arrest, E.M., Kramer, E., van der Meer, R. and apoptosis and xenobiotic metabolism in Keijer, J. (2003) Mucosal pentraxin (Mptx), human CO115 colon-adenocarcinoma a novel rat gene 10-fold down-regulated in cells using DNA microarray. colon by dietary heme. The FASEB Biotechnology and Applied Biochemistry, 45 Journal, 17 (10), 1277–1285. (Pt 1), 29–36. 95 Rao, L., Puschner, B. and Prolla, T.A. 88 Thimmulappa, R.K., Mai, K.H., Srisuma, (2001) Gene expression profiling of low S., Kensler, T.W., Yamamoto, M. and selenium status in the mouse intestine: Biswal, S. (2002) Identification of Nrf2- transcriptional activation of genes linked regulated genes induced by the to DNA damage, cell cycle control and chemopreventive agent sulforaphane by oxidative stress. The Journal of Nutrition, oligonucleotide microarray. Cancer 131 (12), 3175–3181. Research, 62 (18), 5196–5203. 96 Unni, E., Kittrell, F.S., Singh, U. and 89 Traka, M., Gasper, A.V., Smith, J.A., Sinha, R. (2004) Osteopontin is a potential Hawkey, C.J., Bao, Y. and Mithen, R.F. target gene in mouse mammary cancer (2005) Transcriptome analysis of human chemoprevention by Se- colon Caco-2 cells exposed to methylselenocysteine. Breast Cancer sulforaphane. The Journal of Nutrition, 135 Research, 6 (5), R586–R592. (8), 1865–1872. 97 Ho, E., Courtemanche, C. and Ames, B.N. 90 Hu, R., Xu, C., Shen, G., Jain, M.R., Khor, (2003) Zinc deficiency induces oxidative T.O., Gopalkrishnan, A., Lin, W., Reddy, DNA damage and increases p53. B., Chan, J.Y.and Kong, A.N. (2006) Gene expression in human lung fibroblasts. The expression profiles induced by cancer Journal of Nutrition, 133 (8), 2543–2548. chemopreventive isothiocyanate 98 Tilton, S.C., Gerwick, L.G., Hendricks, sulforaphane in the liver of C57BL/6J J.D., Rosato, C.S., Corley-Smith, G., mice and C57BL/6J/Nrf2 (/) mice. Givan, S.A., Bailey, G.S., Bayne, C.J. and Cancer Letters, 243 (2), 170–192. Williams, D.E. (2005) Use of a rainbow 91 Lee, S.B., Cha, K.H., Selenge, D., trout oligonucleotide microarray to Solongo, A. and Nho, C.W. (2007) The determine transcriptional patterns in References j333

aﬂatoxin B1-induced hepatocellular Mantle, P., Cavin, C. and Schilter, B. carcinoma compared to adjacent liver. (2006) A toxicogenomics approach to Toxicological Sciences, 88 (2), 319–330. identify new plausible epigenetic 99 Luhe,€ A., Hildebrand, H., Bach, U., mechanisms of ochratoxin a Dingermann, T. and Ahr, H.J. (2003) A carcinogenicity in rat. Toxicological new approach to studying ochratoxin A Sciences, 89 (1), 120–134. (OTA)-induced nephrotoxicity: expression 101 Arbillaga, L., Azqueta, A., van Delft, J.H. proﬁling in vivo and in vitro employing and Lopez de Cerain, A. (2007) In vitro cDNA microarrays. Toxicological Sciences, gene expression data supporting a DNA 73 (2), 315–328. non-reactive genotoxic mechanism for 100 Marin-Kuan, M., Nestler, S., Verguet, C., ochratoxin A. Toxicology and Applied Bezencon, C., Piguet, D., Mansourian, R., Pharmacology, 220 (2), 216–224. Holzwarth, J., Grigorov, M., Delatour, T., j335

21 Preneoplastic Models and Carcinogenicity Studies with Rodents Veronika A. Ehrlich and Siegfried Knasmuller€

21.1 Introduction

Animal models provide an intact biological system for evaluating the efﬁcacy of cancer-preventive strategies. Rodents have been particularly useful for in vivo studies concerning the development of cancer in different organs. Furthermore, they provide valuable tools to study chemopreventive activity of various synthetic and natural compounds. These models can be broadly divided into genetic cancer models and models in which carcinogenesis is induced chemically. This chapter gives a short overview on the different approaches with emphasis on those that are frequently used for the investigation of chemopreventive food components. Section c21.2 describes rodent models for the identiﬁcation of protective agents, which are based on the use of standard chemical carcinogens. In Section c21.3, genetically engineered rodent models are discussed and Section c21.4 focuses on xenografts used in dietary chemoprevention research.

21.2 Animal Models Based on the Use of Carcinogens

The major goal in the development of carcinogen-induced tumor models was the rapid generation of neoplasia. Most protocols involve high-dose regimens of a single genotoxic carcinogen, such as dimethylbenzanthracene (DMBA) or azoxymethane (AOM). Table21.1 lists examples that are based on the use of chemical carcinogens for induction of tumors (or preneoplastic lesions) in different organs and the application of chemopreventive agents to interfere with the process of carcinogenesis.

21.2.1 Use of Preneoplastic Lesions in Experimental Oncology

Preneoplastic lesions in liver and colon have been used in experimental cancer research for more than 30 years.

Table 21.1 Examples for chemoprevention studies with rodents based on the use of chemical carcinogens for tumor initiation.

Examples of chemoprevention a b c Animals Carcinogen studies

A/J mice (spontane- Initiation with tobacco-related Green tea, black tea, EGCG, thea- ous lung tumors); t.o. carcinogens (NNK, B[a]P, flavins: # lung tumor incidence and lung NDMA) volume (reviewed in Ref. [6]) Hairless HOS:HR-1 DMBA-initiated and UVB- Beetroot (Beta vulgaris) extract: mice; t.o. skin promoted or NOR-1-initiated # incidence of skin tumors [3] and TPA-promoted skin carcinogenesis Swiss albino mice; t.o. DMBA as initiator and TPA as Resveratrol: # DMBA þTPA-in- skin promoter of skin tumors duced tumor incidence [7] Sprague–Dawley rats; NMU-induced mammary Soy protein isolate: # NMU-induced t.o. breast tumors tumor incidence [9] ICR mice; t.o. liver DEN-induced and TPA-promot- Beetroot (Beta vulgaris) extract: ed liver tumors # incidence and multiplicity of liver tumors [3] F344 rats; t.o. prostate DMAB- and PhIP-induced non- Isoflavones (genistein, daidzin, invasive carcinomas in the ven- mixture) and lycopene: # DMAB tral prostate, DMAB þ testoster- (testosterone)-induced tumors; one treatment also induces in- lycopene: $ PhIP-induced carcino- vasive cancers outside the mas (for review see Ref. [4]) prostate ACI rats; t.o. stomach MNNG-induced gastric prolifer- Fermented brown rice: # incidence ative lesions and multiplicity of gastric proliferative lesions [10] Sprague–Dawley rats; DMH-induced colorectal Polymeric black tea polyphenols: # t.o. colon carcinogenesis tumor volume and multiplicity [8] Mice, F344 rats; t.o. DMH- and NMU-initiated colon b-Carotene: # colon tumor incidence colon carcinogenesis (nonsignificantly); reviewed in Ref. [1] Wistar rats; t.o. BBN-initiated bladder Dietary ginger extract: # multiplicity bladder carcinogenesis of urothelial lesions (hyperplasia and neoplasia) [2] SPFalbino Wistar rats Preneoplastic acinar lesions in- Dietary antioxidants (b-carotene, t.o. pancreas duced in rat pancreas by vitamin C, and selenium): # pan- azaserine creatic tumors when given in combination [5]

at.o., target organ. bB[a]P, benzo[a]pyrene; BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; DEN, N-nitrosodiethylamine; DMAB, 3,20-dimethyl-4-aminobiphenyl; DMBA, 7,12-dimethylbenz[a]anthracene, DMH, 1,2- dimethylhydrazine; EGCG, epigallocatechin-3-gallate; ENNG, N-ethyl-N0-nitro-N-nitroguanidine; MNNG, N-methyl-N0-nitro-N-nitroguanidine; NDMA, N-nitrosodimethylamine; NMU, N-methylnitrosourea; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NOR-1, ()-(E)-4-methyl-2-[(E)- hydroxyamino]-5-nitro-6-methoxy-3-hexanamide; TPA, 12-O-tetradecanoylphorbol-13-acetate; PhIP, 2-amino-1-methyl-6-phenylimidiazo[4,5-b]pyridine. c# Decrease; $ no alteration. 21.2 Animal Models Based on the Use of Carcinogens j337

Cancer formation by chemical compounds is a slow process due to a stepwise, sequential transformation of normal into malignant cells via intermediate cell populations. Multistage carcinogenesis involves at least three distinct stages, namely (i) initiation (DNA damage by a genotoxic carcinogen), (ii) subsequent promotion (preferential growth of initiated cells, promoted by nongenotoxic agents) to an identiﬁable lesion (e.g., an enzyme-altered focus), and (iii) progression (transition to a malignant phenotype and genotype) [11, 12]. For a detailed description of the multistage process of carcinogenesis see Chapter 1. Precancerous lesions are considered to be derived from initiated cells by clonal expansion and consist of morphologically and/or functionally altered cell populations. These surrogate end points for tumor formation can be detected after relatively short time periods (i.e., after a few weeks) and only a small number of animals are needed (usually 8–10 per experimental group). Preneoplastic lesions have been identiﬁed in a number of organs, for example, in the skin (epidermal dysplasia and hyperplasia, epithelial papilloma), lung (alveolar and focal hyperplasia, nodular lesions), pancreas (atypical acinar foci), kidney (tubules with irregular epithelium), mammary gland (hyperplastic alveolar nodules), and also in liver (altered hepatic foci) and colon (aberrant crypt foci, ACF); for overview see Ref. [13]. However, preneoplastic models other than aberrant crypt foci in the colon or altered hepatic foci have rarely been used in chemoprevention studies. Therefore, the following sections focus on preneoplastic lesions in liver (Section 21.2.1.1) and colon (Section 21.2.1.2).

21.2.1.1 Altered Hepatic Foci (AHF) – Morphology and Phenotypes For almost four decades, AHF have been known as the earliest emerging distinct phenotypic parenchymal changes indicating hepatocarcinogenesis in rats and mice. These lesions have been discussed as potential end points in carcinogenicity testing since the early 1980s [14]. It is well documented that AHF increase in number and size with continued exposure to both genotoxic and nongenotoxic carcinogens [14, 15]. Some of the phenotypical abnormalities of AHF are stable; however, under specific conditions, some phenotypical characteristics are lost (phenotypic reversion). Bannasch and coworkers [14] state that neither there is any hepatocarcinogenic agent that does not elicit AHFnor is there any model of hepatocarcinogenesis without formation of these lesions prior to the manifestation of benign or malignant hepatocellular neoplasms. In the early years, a classification system was developed, which was based on the staining behavior of AHF cells, and defined clear, acidophilic, intermediate, tigroid, basophilic, and also mixed cell types [14, 15]. In subsequent years, it was shown that the expression of a variety of enzymes differs between AHF and the surrounding tissue. Based on these observations, histochemical methods were developed that enable the detection of enzymatically altered AHF mainly in rat liver (for review see Refs [13, 16]). At present, the most widely used end point is the expression of the þ placental form of glutathione-S-transferase (GSTp ), which can be detected by þ immunohistochemistry. The majority of all foci stain positive for GSTp [16]; a representative focus is shown in Figure 21.1. Another frequently used marker is g-glutamyl-transpeptidase. 338j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

þ Figure 21.1 This figure depicts a GSTp focus in the liver of a male rat, which was subjected to a single dose of a genotoxic agent (N-nitrosomorpholine, 250 mg/kg body weight) and subsequent administration of a promoting agent (phenobarbital, daily dose 50 mg/kg body weight) for 5 months.

21.2.1.1.1 Methodical Aspects AHF can be used to detect tumor initiating (Figure 21.2a) and promoting properties (Figure 21.2b) of chemicals. To distinguish between these features, the animals are treated with the compounds according to different schedules [17].

Figure 21.2 Different treatment schedules for agent or another form of enhancement such as the detection of initiating and promoting partial hepatectomy. (b) The DNA-reactive carcinogens in experiments with AHF as carcinogen is administered for 8–12 weeks biological end point. (a) The potential initiator is followed by the exposure to the potential administered once or continuously for up to promoting agent. 18 weeks. This can be followed by a promoting 21.2 Animal Models Based on the Use of Carcinogens j339

21.2.1.1.2 Initiators and Promoters of AHF Numerous synthetic and natural compounds have been identiﬁed that initiate and/or promote the formation of AHF [17]. Typical examples for initiators are nitrosamines (which are the most frequently used ﬂ carcinogens in mechanistic studies), urethane, a atoxin B1 (AFB1), and heterocyclic aromatic amines (HAAs) [18]. Also polycyclic aromatic hydrocarbons such as benzo- [a]pyrene (B[a]P) cause formation of AHF in rats, although the liver is not a target organ for tumor induction of this compound. A wide spectrum of agents has been tested for enhancement of foci in rodent liver in the various models in use. Typical examples of promoters are phenobarbital, steroid hormones, such as ethinyl estradiol and testosterone, hypolipidemic drugs, and polychlorinated biphenyls (for review see Ref. [17]). Promotion processes may also occur spontaneously, for example, by endogenous hormones or dietary factors (e.g., calorie intake). A very interesting observation was made in experiments with rats in which the dietary restriction to 40% of control levels reduced the number and volume of AHF by 85% within 3 months (lowered DNA replication and increased apoptosis were observed by the authors). When treated with a tumor promoter (nafenopin) after food restriction, only half as many hepatocellular adenomas and carcinomas were found as compared to animals fed ad libitum throughout their lifetime. The authors concluded that restricted calorie intake preferentially enhances apoptosis of preneoplastic cells [19].

21.2.1.1.3 Mechanistic Aspects to the Action of Promoters A number of studies have been conducted in which the ratio between cell division and programmed cell death during development of liver cancer was investigated. It was shown that the cell division rates are increased in AHF compared to normal tissue; in adenomas and carcinomas even higher division rates were observed. Also the death rates (apoptosis) increased gradually from normal to preneoplastic to adenoma and carcinoma tissue [20]. Further studies showed that the prenoplastic tissue is more susceptible to stimulation of cell replication and cell death [21, 22] and that tumor promoters evidently act as survival factors by inhibiting apoptosis in preneoplastic liver cells, thereby stimulating growth of preneoplastic lesions. Interestingly, withdrawal of tumor promoter led to excessive elimination of preneoplastic lesions, whereas normal tissue was less affected [21].

21.2.1.1.4 Inhibition of Foci Formation in the Liver Numerous investigations have been conducted to identify compounds that prevent the formation of hepatic foci. These agents were protective either at the initiation level (i.e., when administered before and/or simultaneously with the genotoxic carcinogen) or at the promotion level (after carcinogen treatment). Examples for anti-initiators are food constituents such as alliin or chlorophyll and chlorophyllin (see Table 21.4), which reduce DNA damage caused by AFB1 [23, 24] and butylated hydroxytoluene, which inhibited the foci formation caused by 2-acetyl-aminoﬂuorene [25]. Also glucosinolates, components of cruciferous vegetables, were found protective toward AFB1. Cruciferous plants themselves inhibited foci formation induced by the heterocyclic aromatic amine IQ [26, 27]. 340j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

A number of compounds were identified that prevent foci promotion when administered after treatment with the initiating carcinogen. For example, acetaminophen and aminophenol were protective against development of foci that had been induced by a nitrosamine in the liver [17]. Flavanone, a nonpolar flavonoid, administered by dietary route, acted as anti-initiator and antipromotor by reducing þ significantly the formation of placental GSTp foci during the initiation period

(with AFB1) and their growth [28]. Table 21.4 (Section 21.2.1.2) lists some further examples for chemoprevention studies in rodents using the AHF model. Mechanistical aspects regarding the molecular mechanisms of antimutagenesis and anticarcinogenesis by dietary factors are discussed in Chapter 5.

21.2.1.1.5 New Developments Grasl-Kraupp and coworkers [29] developed an ex vivo cell culture model for initiated rat hepatocytes. Following treatment of rats with a nitrosamine (N-nitrosomorpholine), hepatocytes were isolated after 22 days (maxi- þ mal occurrence of GSTp cells) and cultivated in vitro. Then the cells were treated either with the mitogen cyproterone acetate or with transforming growth factor þ (TGF-b) for 1–3 days. In culture (ex vivo), the rate of DNA replication of GSTp cells þ was compared to that of normal hepatocytes. It was found that GSTp cells show an inherent growth advantage and a preferential response toward the effects of TGF-b and cyproterone acetate, as in the in vivo situation. Based on these results, the authors stress that this ex vivo system may provide a useful tool to elucidate biological and molecular changes during the initiation of carcinogenesis [29].

21.2.1.2 Aberrant Crypts in the Colon

21.2.1.2.1 Morphology The luminal surface of the large intestine is lined by a simple columnar epithelial layer. Its surface is folded into a number of deep cavities called crypts of Lieberkuhn€ [30]. Between, and linking the crypts, is a flat mucosal surface. Colonic epithelium is a self-renewing tissue with high turnover rates that requires tight control of cell proliferation and apoptosis. Accumulation of genetic and epigenetic alterations (e.g., apoptosis, cellular differentiation, shifting of the proliferative zone) leads to the development of an aberrant crypt pattern. Aberrant crypt foci were first described in 1987 by Bird who discovered that the treatment of rats with colon-specific carcinogens causes the formation of ACF, which can be visualized in methylene blue-stained whole mount preparations [31]. ACF are defined as single or multiple crypts that (i) have altered luminal openings, (ii) exhibit thickened epithelia, and (iii) are larger than adjacent normal crypts [31]. They consist of altered cells, which exhibit cytoplasmic basophilia, a high nuclear to cytoplasmic ratio, prominent nucleoli, loss of globlet (mucus excreting) cells, loss of polarity, and increased proliferative activity in the upper part of the crypt; for review see Ref. [32]. These preneoplastic lesions are generally divided into three groups, namely dysplastic, nondysplastic (atypical), and mixed type (for details see Ref. [33]). In ACF without dysplasia, the crypts are enlarged and have slightly enhanced nuclei, no mucin depletion and crypt cells staining positive for PCNA (proliferating cell nuclear antigen) and Ki-67 related antigen (proliferation markers) remain in the lower part 21.2 Animal Models Based on the Use of Carcinogens j341 of the crypts. In ACF with dysplasia, crypts are more elongated, and the nuclei enlarged. The major site of positive staining cells of PCNA and Ki-67 is extended to the upper part of the crypts. Mixed-type ACF show combination of the features of pure adenomatous pattern (with dysplasia) and hyperplastic characteristics. Figure 21.3a and b depicts typical aberrant crypts, which are abnormally large, darkly stained, and slightly elevated. Dysplastic crypts with a slit-shaped luminal opening are shown in Figure 21.3a; Figure 21.3b depicts nondysplastic crypts with a larger pericryptical zone (i.e., area between bordering crypts). In humans, ACF were first described in 1991 by Roncucci et al. and Pretlow et al. [34, 35]. They resemble those seen in rodents induced by carcinogens and some data support the assumption that they are precursors of colorectal tumors (for details see Refs [33, 36]).

21.2.1.2.2 Biochemical and Immunohistochemical Alterations of ACF Previous studies explored phenotype alterations of ACF by means of biochemical and immunohistochemical methods. A number of such alterations are typical for ACF, for example, altered enzyme activities (such as cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), and hexosaminidase) and altered patterns of cell adhesion molecules (such as epithelial and placental cadherin, as well as the carcinoembryonic antigen (CEA) or proteins, which are used as markers for cell proliferation (such as Ki-67 and PCNA). The most important features are listed in Table 21.2; for review see Ref. [33].

21.2.1.2.3 Genetic and Epigenetic Alterations Different genetic alterations have been identified in ACF in humans and also in chemically induced ACF in rats; a detailed overview is given by Mori et al. [36] and Cheng and Lai [33]. Many genes, which are considered to be involved in colon carcinogenesis (e.g., oncogenes, such as K-ras, tumor suppressor genes, such as Apc and FHIT, and mismatch repair genes, such as hMSH2), were found to be altered in ACF. Furthermore, involvement of epigenetic alterations, such as CpG island methylation and microsatellite instability, has been observed in ACFas well as in the formation and progression of colorectal carcinomas. This supports the assumption that they (ACF or a specific subpopulation) indeed represent preneoplastic lesions. Table 21.3 lists up different alterations that were identified in ACF. Inactivation of the Apc (adenomatous polyposis) gene can be found in colorectal tumors in humans and to a much lesser extent also in human ACF. The Apc protein destabilizes oncogenic b-catenin and therefore intact Apc acts as a tumor suppressor gene. When Apc or b-catenin genes are mutated, as occurs in the majority of colorectal cancer, b-catenin cannot be degraded but accumulates, associates with the transcription factor Tcf4, translocates to the nucleus where it activates Wnt target proliferative genes and oncogenes (c-myc, cyclin D1 and c-jun), and transformation further proceeds to adenomas and adenocarcinomas.

21.2.1.2.4 Methodological Aspects As in AHF experiments, ACF studies allow to discriminatebetween initiating and promoting compounds. The treatment schedule is similar to that used for the detection of liver foci, but different model chemicals are used. 342j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

Figure 21.3 Colonic mucosal surface stained with methylene blue (100). Part (a) depicts an aberrant crypt focus with a high level of dysplasia, which is microscopically elevated with an elongated slit-shaped luminal opening (arrow). Part (b) shows a nondysplastic crypt with round dilated lumen (arrow).

Only a few compounds have been detected, which are initiators of colon cancer and aberrant crypts. The most frequently used agents are DMH and its metabolite AOM [38]. AOM treatment leads to DNA methylation and increases proliferation and mutations of colonic epithelial cells. AOM-induced colon tumors in rodents share many histopathological characteristics with human tumors. 21.2 Animal Models Based on the Use of Carcinogens j343

Table 21.2 Biochemical and immunohistochemical alterations of ACF.a

End point Comment

Hexosaminidase # Activity in more than 95% of foci and tumors in rats, might be a marker for an early events in colon carcinogenesis (gene closely located to Apc gene), not a marker for human ACF Carcinoembryonic antigen " CEA level (¼ intracellular adhesion molecule), detected in a (CEA) high proportion of human ACF, expression of CEA was related to the sizes of the foci but not to the presence or degree of dysplasia P-cadherin (P-c); E-cadherin P-c and E-c ¼ cell adhesion molecules; P-c is aberrantly (E-c) expressed in ACFprior to and independent of E-c and b-catenin b-Catenin Transcriptional activator of oncogenes; " nuclear expression in rat and human ACF (see also Section 21.3.1) Cyclooxygenase 2 (COX-2) Overexpression in ACF, adenomas, and carcinomas of AOM- induced rats (may contribute to ACF growth and sequential tumor growth) Inducible nitric oxide iNOS expression " in dysplastic but not in hyperplastic ACF synthase (iNOS) (in AOM-induced rat colon) Cell proliferation markers Several studies in rodent and human ACF show positive Ki-67, PNCA, and P16INK4a staining of PCNA and Ki-67, suggesting an altered pattern of cell proliferation þ þ GSTp GSTp expression " in rat and human ACF and in colorectal carcinomas might be associated in humans with K-ras expression Changes in mucin Abnormal expression of gastric mucin seen in rat and human production aFor review see Refs [32, 36, 37].

Also heterocyclic aromatic amine induce formation of ACF. HAAs are formed during cooking of meats; they cause cancer in the colon of rodents and in other organs as well [39], and evidence is accumulating that HAAs are involved in the etiology of colon cancer in humans [40]. Other agents that cause ACF are N-methyl-N-nitrosourea [41] and 3,2-dimethyl- 4-aminobiphenyl (DMABP), but these compounds were hardly ever used in mechanistic and chemoprevention studies [1, 42]. Furthermore, the ACF model was intensely used in studies aimed at detecting dietary factors, which cause tumor promotion in the colon (e.g., reﬁned sugars, thermolyzed sucrose, thermolyzed protein, fat, and hemin) [43–45].

21.2.1.2.5 Use of the ACF Model for the Detection of Chemoprotective Compounds Numerous studies have been conducted aimed at identifying compounds that are protective toward colon cancer with the ACF model. Corpet and Tache reviewed the potency of chemopreventive agents in the AOM rat model. They found in total 137 articles and results for about 186 complex mixtures and individual compounds are available (the data can be downloaded from http://www.inra.fr/reseau-nacre/ sci-memb/corpet/indexan.html). The establishment of a ranking order of protective potency showed that the most potent were polyethylene glycol, pluronic 344j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

Table 21.3 Epigenetic and genetic alterations in ACF.a

b Alteration Remarks

K-ras mutation K-ras activation was identiﬁed in ACF from rats in several studies; also found in humans Apc mutation Apc inactivation found in human ACF (4%), K-ras activation is more common at the ACF stage; Apc mutation rate is much higher in CRC and adenomas hMSH2 mutation Mismatch repair gene alteration found in ACF isolated from mouse colons CpG island methylation Hypermethylation of CpG islands leads to suppression of transcription of tumor suppressor genes and repair genes; found in 53% of ACF of humans with sporadic CRC but only in 11% of FAP patients Microsatellite instability Detected in animal and in human ACF Fragile histidine triad Lost in CRC (40%) – only few ACF isolated from colon cancer (FHIT), candidate tumor patients showed reduced expression; the reduced expression of suppressor gene FHIT was correlated with the degree of dysplasia

aFor review see Refs [32, 36, 37]. bCRC, colorectal carcinoma; FAP, familial adenomatous polyposis.

F68 (a polyethylene glycol-like block polymer), perilla oil containing b-carotene, and indole-3-carbinol (for details see Ref. [42]). In addition, many other dietary constituents were found protective, for example, vitamins (1a-hydroxy-D5, fluorinated analogue of vitamin D3, vitamin A, and retinoids), lactobacilli in fermented foods (e.g., Bifidobacterium animalis, Bifidobacterium longum,andStreptococcus thermophilus), different glucosinolates in Brassica vegetables (e.g., garden cress, broccoli), carotenoids, and fibers (e.g., inulin, wheat bran, xylooligosaccharide, fructooligo- saccharide, resistant starch, and beet fiber) to name only a few [42]. In most of the studies, DMH or AOM were used to cause foci formation and the putative protective compounds were added either before or after administration of the carcinogen. The prevention during the foci initiation phase might be due to either inactivation of DNA-reactive molecules, inhibition of metabolic activation, or induction of DNA-repair processes [46] and is compound specific. Since humans are not exposed to DMH and its metabolite AOM, chemoprotective effects seen against DMH-induced foci cannot be extrapolated to the human situation. However, it is assumed that the further development of preneoplastic cells (promotion, progression) is triggered by molecular processes that are independent of the chemical carcinogen used [47]. Therefore, antipromoting effects seen in the AOM/DMH–ACF model might be considered relevant for humans. Furthermore, HAAs were used in a number of chemoprevention studies in which inhibition of ACF formation was used as an end point (for review see Refs [48, 49]), and a number of dietary components such as fibers, chlorophyllins, Brassica vegetables, and lactobacilli were found protective. In this context, it is interesting that epidemiological studies indicate that consumption of these factors is also 21.2 Animal Models Based on the Use of Carcinogens j345 inversely related to the incidence of colon cancer in humans. One of the problems of the use of HAAs in ACF studies is that the foci yield is relatively low, even when the animals are treated with high doses (up to 100 mg/day for several days) when compared to levels of human HAA consumption (meat/fish eaters: micrograms per day). The foci frequency could be substantially increased by feeding the animals a high-fat and fiber-free diet, which facilitates the detection of putative protective effects [50]. In contrast to AOM or DMH, it is not possible to induce ACF with a single dose of HAA; therefore, it is not possible to distinguish clearly between anti-initiating and antipromoting effects in these experiments. The review by Mori et al. [36] gives a comprehensive overview on studies of chemoprevention of colorectal cancer using the ACF model. Further recent examples for dietary factors used in this model (or in the AHF model) are listed in Table 21.4.

Table 21.4 Examples of chemoprevention studies in rodents using AHF or ACF.

a Compound Result/mechanism References

Vitamin D (1a,25- # formation of AOM-induced ACF by facilitating [51, 52]

OH2D3)(1a-OHD5) degradation of b-catenin and decrease in c-myc expression in rats o-3 fatty acids # of colon carcinogenesis in AOM-initiated rats; [51, 54] anti-inﬂammatory activities (inhibition of IL-1 and TNF-a synthesis) Inulin-type fructans # AOM- or DMH-induced ACF; anti-inﬂammatory [51, 53] activities in rats and mice Dried onion # number and size of AOM- and IQ-induced ACF [56] during initiation and promotion in type 2 diabetic mice (db/db) Diet supplemented with # AOM initiated ACF and BCAC in type 2 diabetic [55] citrus unshiu (satsuma) mice (db/db) Green tea polyphenols # AOM-induced ACF by # nuclear expression of [57] b-catenin and cyclin D1 and " of apoptosis in F344 rats Diet supplemented with # DMH-induced ACF and MCF in Wistar rats [58] fresh cruciferous vegetables Cruciferous vegetables Brussels sprouts: # frequency of IQ-induced ACF [26] þ (Brussels sprouts, red and GSTp hepatic foci; red cabbage: # size of IQ- þ cabbage juice) induced GSTp hepatic foci in F344 rats þ Chlorophyllin and # AFB1-induced GSTp hepatic foci and ACF in [24] chlorophyll F344 rats

Garlic powder with high # AFB1-induced and 2-AAF (and partial hepatec- [23] þ levels of alliin tomy)-promoted GSTp hepatic foci; induction of

enzymes involved in AFB1 detoxification (in rats) a# " $ fl Decrease, increase, no alteration; ACF, aberrant crypt foci; AFB1,a atoxin B1; 2-AAF, 2-acetylaminofluorene; AHF, altered hepatic foci; AOM, azoxymethane; BCACs, b-catenin accumulating crypts, GSTp, placental form of glutathione-S-transferase; IQ, 2-amino- 3-methylimidazo[4,5-f]quinoline; MCF, mucin-depleted foci. 346j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

21.2.1.2.6 New Developments Although numerous studies show that ACF have been used extensively for the identification of dietary factors enhancing or reducing the risk for colorectal cancer, some studies suggest that misleading results may be obtained with certain compounds [59]. For example, it is well documented that cholic acid, a primary bile acid, is a strong tumor promoter in the colon, whereas it significantly decreases the number of ACF [60, 61]. A similar contradiction was seen with the xenoestrogen genistein [62–64]. It was repeatedly postulated by Japanese groups that altered crypts with accumulation of b-catenin in the cytoplasm and/or nucleus (b-catenin accumulating crypts, BCAC) are more reliable biomarkers for colon cancer development; for review see Ref. [36]. It was shown that cholic acid increases the frequency of AOM- induced BCAC in rats. In a critical comment by Pretlow and Bird [65], it is stated that BCAC in fact represent specific dysplastic ACF. In a subsequent paper by Hao et al. [66], human ACF were analyzed for b-catenin expression and in approximately 56% of dysplastic ACF, b-catenin was increased, whereas in ACF with atypia, b-catenin in the cytoplasm was only seen in 2% of total ACF. As mentioned above, Magnuson and coworkers [60] also found that the number of ACF at early time points did not predict tumor incidence in rats treated with cholic acid. Therefore, the authors suggest that aberrant crypt multiplicity (ACM ¼ ACF with multiple aberrant crypts) should be measured in future studies, since ACM correlate particularly well with the incidence of colorectal adenomas and carcinomas in AOM-induced rat models [67]. Another potential short-term end point for colon cancer might be mucin- depleted foci (MDF ¼ crypt foci with absent or scant mucous production). In AOM-treated rats, such foci could be visualized with high-iron diamine Albicon blue [68]. Their frequency was lower than that of ACF and they were histological more dysplastic than non-MDF. In a recent article, it was shown that the number of MDFfoci declined in AOM-treated rats or after piroxicam (a colon cancer inhibiting drug) administration, whereas their frequency increased after treatment with cholic acid [68].

21.3 Genetically Engineered Rodent Models Used in Cancer Prevention Studies

The most sophisticated models of human cancer currently available are based on the use of genetically engineered animals to mimic pathophysiological and molecular features of human malignancies [69]. These approaches have greatly facilitated efforts in understanding tumor biology and identifying the role of speciﬁc genes in carcinogenesis as well as in normal development. The elucidation of molecular sites of action (targets) for dietary components is fundamental to the development of effective prevention strategies and approaches. Some of the most frequently used models are described brieﬂyinthefollowing section. 21.3 Genetically Engineered Rodent Models Used in Cancer Prevention Studies j347

21.3.1 þ Intestinal Cancers in Transgenic ApcMin/ Mouse Model

þ The ApcMin/ mouse is one of the main animal models used to study the effect of dietary agents on colorectal cancer. The animals contain a germline inactivation of the Apc gene and are strongly predisposed to develop intestinal tumors at relatively young age. As mentioned earlier (see Section 21.2.1.2.3), the Apc protein destabilizes oncogenic b-catenin and therefore intact Apc acts as a tumor suppressor gene. This model is considered to reflect the situation of patients with familial adenomatous polyposis or of individuals carrying the first Apc mutation in somatic cells who are predisposed for developing sporadic colon cancer. The major drawback of this model is that tumors occur predominantly in the small intestine (which is not a target tissue in humans) and not in the colon [53]. Some examples of chemoprevention studies þ using the ApcMin/ mouse model are listed in Table 21.5; for review see also Ref. [70]. Corpet and Pierre [38] studied the correlation between the results of chemoprevention studies using ACF as an end point and data from experiments with the þ ApcMin/ mouse model. Comparisons of the efficacy of protective agents in the þ ApcMin/ mouse and in the ACF rat model indicated a significant correlation (p < 0.001).

21.3.2 Germline p53-Deficient and p53 Mutated Animals

Mutations of the p53 tumor suppressor gene are the most frequently observed genetic lesions in human cancer; that is, over 50% of all human tumors examined to date have identiﬁable p53 gene point mutations or deletions [71]. Heterozygous p53 knockout (p53 þ /) mice have some analogy to humans susceptible to heritable forms of cancer due to decreased p53 dosage (such as individuals with Li–Fraumeni syndrome). Hursting et al. [70] evaluated the ability of several dietary interventions to offset the increased susceptibility of homozygous p53/ to spontaneous tumorigenesis. Also p53 þ / Wnt-1 transgenic models, dominant negative p53 mutant mice, as well as tissue-speciﬁc inactivation of the p53 gene have been used in rodent models for chemoprevention studies. Some results of experiments with dietary factors are listed in the review by Hursting et al. [70].

21.3.3 Rodent Models of Prostate Cancer

Numerous transgenic and gene knockout rodent models of prostate cancer have been generated that develop lesions ranging from prostatic intraepithelial neoplasia to invasive disease. They are dependent on molecular mechanisms already implicated in human prostate carcinogenesis (for review of mouse models see Ref. [72]) and can broadly be divided in two categories, namely (1) models in which viral oncogenes are expressed in prostate tissues (TRAMP/TRAP and Lady 12T-10 model) and (2) models 348j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

Table 21.5 Examples of genetically engineered animal models used for the identification of chemopreventive substances.

Examples for chemopreven- a b c Animal model Principle tion studies

Apc (adenomatous polyposis Contains a nonsense muta- Inulin-type fructans, wheat coli gene)Min mouse; t.o. tion at the murine Apc gene, bran [79], sulforaphane [80], colon which is a tumor suppressor green tea extract and poly- gene phenols (EGCG, PPE; for review see Ref. [6]: # of intestinal tumors TRAMP (transgenic adeno- Prostate specific expression Green tea polyphenols, carcinoma of mouse pros- of viral oncogenes (SV40 genistein: inhibition of tate) model; t.o. prostate early genes) prostate carcinogenesis; reviewed in Ref. [72] TRAP (transgenic rats with SV40 large T antigen under g-Tocopherol, resveratrol: adenocarcinomas of the probasin promoter control, # tumors in the prostate; for prostate) model; t.o. prostate allowing prostate-specific review see Ref. [4] gene expression; develop prostate adenocarcinomas at mostly 100% incidence Lady 12T-10 transgenic Prostate specific expression Antioxidants: # tumors in the model; t.o. prostate of a viral oncogene (long prostate; reviewed in Ref. [72] probasin promoter-driven large T-antigen)

Nkx3.1;Pten mutant mice; Combinatorial loss of Vitamin D (1a,25-OH2D3): t.o. prostate function of the tumor # prostate intraepithelial suppressor genes Nkx3.1 and neoplasia [77] Pten c-Ha-ras proto-oncogene Animals carrying the human D-Limonene: # MNU-in- transgenic rats; t.o. breast proto-oncogene H-ras 128 are duced breast tumor develop- highly susceptible to MNU ment [76]; soy isoﬂavones: induced mammary # premalignant mammary carcinogenesis lesions [78] p53þ/ Wnt-1 transgenic Animals deﬁcient in the Dietary soy and calorie mice; t.o. breast tumor suppressor gene p53, restriction: delayed tumor highly susceptible to mam- development [81] mary carcinogenesis Apcþ/ Msh2/ mice; t.o. Murine model of intestinal Dietary folate: # small small intestine, colon tumorigenesis, which carries intestinal and colorectal a heterozygous mutation in tumorigenesis, only if the Apc gene and a null provided before the mutation in the Msh2 (DNA establishment of neoplastic mismatch repair) gene foci [75]

at.o., target organ. bMNU, N-methyl-N-nitrosourea. c# Decrease; EGCG, epigallocatechin gallate; PPE, polyphenon E (standardized green tea polyphenol preparation). 21.4 Xenograft Models j349 with genetic modiﬁcations of pathways implicated in human prostate development (PTEN model). Some examples of their use in chemoprevention studies are listed in Table 21.5.

21.3.4 Other Transgenic Models

In the recent years, numerous models have been developed in which cancer-related genes are overexpressed or inactivated. Many of these transgenic and knockout strains are catalogued in online databases such as the induced mutation registry database and mouse genome informatics site (http://www.informatics.jax.org) maintained by the Jackson Laboratory or the NCI-sponsored mouse models of human cancer consortia (for details see http://emice.nci.nih.gov). SV40 large T antigen and other transgenic models for prostate cancer, mice with alterations in the ErbB/Ras signaling pathway, HPV-16 transgenic mice, as well as animals with alterations in genes involved in metabolism, immunocompetence, and epigenetic regulation have been utilized in cancer prevention studies. Furthermore, a number of models for animals that are deficient in DNA repair have been developed. Loss of function of the base excision repair gene Myh þ enhances tumorigenesis in the colon of ApcMin/ mice [73]. Mutations in the Atm gene, which is involved in DNA double-strand break repair and cell cycle checkpoint results in genetic instability, increased oxidative stress, and cancer. Several studies were conducted with Atm-deficient mice to examine the role of antioxidants in cancer prevention [74]. For example, Song et al. [75] reported that dietary folate supplementation significantly protected against small intestinal and colorectal tumorigenesis when it was administered to mice deficient in Msh2 (DNA mismatch repair gene) and Atm. However, the authors state that the timing of folate intervention is critical in providing an effective and safe chemopreventive effect.

21.4 Xenograft Models

This approach is based on the implantation of tumor cells (harvested from a cancer patient or grown in vitro) into animals. Xenografts are widely used in preclinical in vivo studies of antineoplastic drugs and furthermore in chemoprevention trials. Surgical orthotopic implantation of intact fragments of human cancer (transplanted into the corresponding organ of immunodeﬁcient rodents) is employed for modeling colon, breast, prostate, and liver cancer. In some cases, the animals are additionally treated with tumor promoters. Since the importance of the tumor microenvironment has become more evident, it has been suggested by Frese and Tuveson [69] that the xenograft model is more appropriately termed animal culture [69]. To give some examples, Xu et al. [82] observed inhibition of hepatocellular carcinoma formation after injection of human hepatoma cells (HepG2) 350j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

subcutaneously in mice and intragastrical administration of selenium-enriched green tea extract compared to the control group. Furthermore, Yuan et al. [83] described inhibition of liver and pulmonary metastases of orthotopic colon cancer (HT-29 carcinoma cells implanted in colons of nude mice) after EGCG administration. In another study, sulforaphane suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice [84].

21.5 Conclusions

The use of laboratory animals has been criticized repeatedly for ethical reasons, but they still provide more valuable information as in vitro approaches, as they reflect absorption, distribution, as well as metabolism of the tested substances. In earlier evaluations, the shortcomings and pitfalls of currently used in vitro models have been emphasized; for example, see Ref. [85] The importance of animal studies was also stressed by Corpet and Pierre, who conducted a meta-analysis of selected chemopreventive agents (aspirin, b-carotene, calcium, and wheat bran studies) to find out if rodent models of colon carcinogenesis are good predictors of chemopreventive efficacy in humans [1]. The carcinogen-induced rat studies matched human trials (controlled intervention studies of adenoma recurrence) for aspirin, calcium, and b-carotene and were comparable for wheat bran. The authors concluded that the rodent models are useful and important tools to study mechanisms of carcinogenesis and chemoprevention and to screen for cancer- protective agents. It should be taken into account that predictions are not accurate for all agents. Furthermore, in animal models based on high-dose exposure to genotoxic carcinogens, such as DMBA or AOM/DMH derivatives, tumors develop through a multistep process similar to that observed in human carcinogenesis (i.e., in the colon). Moreover, the easy identification of preneoplastic lesions (such as AHF, ACF, or MDF) in short-term studies makes these models useful tools for screening the potential chemopreventive efficacy of dietary substances. However, experimental limitations of animal models should always be taken into account; for example, high dosages of genotoxic carcinogens induce large-scale genetic damage in a random fashion and most of the carcinogens in use have no apparent etiology in human cancer. Furthermore, the interpretation of the activity of preventive compounds in these models can often be confounded by the metabolic activation or detoxification of the high-dose carcinogen that has been used for initiation [70]. Therefore, it might be difficult to extrapolate data from animal experiments to the human situation. Although there are some limitations in genetically engineered rodent models available for cancer prevention studies, animals with specific (and human-like) genetic susceptibilities for cancer provide powerful new tools for testing interventions and/or specific compounds that may inhibit the process of carcinogenesis in humans. References j351

Acknowledgments

We thank Bettina Grasl-Kraupp and Rolf Schulte-Hermann for their valuable and helpful comments on this manuscript.

References

1 Corpet, D.E. and Pierre, F. (2005) How 7 Kalra, N., Roy, P., Prasad, S. and Shukla, Y. good are rodent models of carcinogenesis (2008) Resveratrol induces apoptosis in predicting efﬁcacy in humans? A involving mitochondrial pathways in systematic review and meta-analysis of mouse skin tumorigenesis. Life Sciences, colon chemoprevention in rats, mice and 82, 348–358. men. European Journal of Cancer, 41, 8 Patel, R., Ingle, A. and Maru, G.B. (2008) 1911–1922. Polymeric black tea polyphenols inhibit 2 Ihlaseh, S.M., de Oliveira, M.L., Teran, E., 1,2-dimethylhydrazine induced colorectal de Camargo, J.L. and Barbisan, L.F. (2006) carcinogenesis by inhibiting cell Chemopreventive property of dietary proliferation via Wnt/beta-catenin ginger in rat urinary bladder chemical pathway. Toxicology and Applied carcinogenesis. World Journal of Urology, Pharmacology, 227, 136–146. 24, 591–596. 9 Simmen, R.C., Eason, R.R., Till, S.R., 3 Kapadia, G.J., Azuine, M.A., Sridhar, R., Chatman, L. Jr, Velarde, M.C., Geng, Y., Okuda, Y., Tsuruta, A., Ichiishi, E., Korourian, S. and Badger, T.M. (2005) Mukainake, T., Takasaki, M., Konoshima, Inhibition of NMU-induced mammary T., Nishino, H. and Tokuda, H. (2003) tumorigenesis by dietary soy. Cancer Chemoprevention of DMBA-induced Letters, 224,45–52. UV-B promoted, NOR-1-induced TPA 10 Tomita, H., Kuno, T., Yamada, Y., Oyama, promoted skin carcinogenesis, and T., Asano, N., Miyazaki, Y., Baba, S., DEN-induced phenobarbital Taguchi, A., Hara, A., Iwasaki, T., promoted liver tumors in mice by extract of Kobayashi, H. and Mori, H. (2008) beetroot. Pharmacological Research, 47, Preventive effect of fermented brown rice 141–148. and rice bran on N-methyl-N0-nitro- 4 Shirai, T. (2008) Signiﬁcance of N-nitrosoguanidine-induced gastric chemoprevention for prostate cancer carcinogenesis in rats. Oncology Reports, development: experimental in vivo 19,11–15. approaches to chemoprevention. Pathology 11 Femia, A.P. and Caderni, G. (2008) Rodent International, 58,1–16. models of colon carcinogenesis for the 5 Woutersen, R.A., Appel, M.J. and Van study of chemopreventive activity of Garderen-Hoetmer, A. (1999) Modulation natural products. Planta Medica, 74, of pancreatic carcinogenesis by 1602–1607. antioxidants. Food and Chemical Toxicology, 12 Pitot, H.C. and Dragan, Y.(1994) Chemical 37, 981–984. induction of hepatic neoplasia, in The 6 Yang, C.S., Lambert, J.D., Ju, J., Lu, G. and Liver: Biology and Pathobiology, 3rd edn Sang, S. (2007) Tea and cancer prevention: (eds I.M. Arias, J.L. Boyer, N. Fausto, molecular mechanisms and human W.B. Jakoby, D.A. Schachter and D.A. relevance. Toxicology and Applied Shafritz), Raven Press, New York, Pharmacology, 224, 265–273. pp. 1467–1495. 352j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

13 Williams, G.M. (1999) Chemically and tumor regression. Hepatology, 25, induced preneoplastic lesions in rodents 906–912. as indicators of carcinogenic activity. 22 Grasl-Kraupp, B., Luebeck, G., Wagner, A., IARC Scientific Publications, Low-Baselli, A., de Gunst, M., Waldhor, T., 146, 185–202. Moolgavkar, S. and Schulte-Hermann, R. 14 Bannasch, P., Haertel, T. and Su, Q. (2003) (2000) Quantitative analysis of tumor Significance of hepatic preneoplasia in risk initiation in rat liver: role of cell replication identification and early detection of and cell death (apoptosis). Carcinogenesis, neoplasia. Toxicologic Pathology, 31, 21, 1411–1421. 134–139. 23 Berges, R., Siess, M.H., Arnault, I., Auger, 15 Bannasch, P., Nehrbass, D. and Kopp- J., Kahane, R., Pinnert, M.F., Vernevaut, Schneider, A. (2001) Significance of M.F. and le Bon, A.M. (2004) Comparison hepatic preneoplasia for cancer of the chemopreventive efficacies of garlic chemoprevention. IARC Scientific powders with different alliin contents Publications, 154, 223–240. against aflatoxin B1 carcinogenicity in rats. 16 Pitot, H.C. (1990) Altered hepatic foci: Carcinogenesis, 25, 1953–1959. their role in murine hepatocarcinogenesis. 24 Simonich, M.T., Egner, P.A., Roebuck, Annual Review of Pharmacology and B.D., Orner, G.A., Jubert, C., Pereira, C., Toxicology, 30, 465–500. Groopman, J.D., Kensler, T.W., Dashwood, 17 Williams, G.M. (1989) The significance of R.H., Williams, D.E. and Bailey, G.S. chemically-induced hepatocellular altered (2007) Natural chlorophyll inhibits foci in rat liver and application to aflatoxin B1-induced multi-organ carcinogen detection. Toxicologic Pathology, carcinogenesis in the rat. Carcinogenesis, 17, 663–672; discussion 673–664. 28, 1294–1302. 18 Sakai, H., Tsukamoto, T., Yamamoto, M., 25 Maeura, Y., Weisburger, J.H. and Williams, Kobayashi, K., Yuasa, H., Imai, T., Yanai,T., G.M. (1984) Dose-dependent reduction of Masegi, T. and Tatematsu, M. (2002) N-2-fluorenylacetamide-induced liver Distinction of carcinogens from mutagens cancer and enhancement of bladder cancer by induction of liver cell foci in a model for in rats by butylated hydroxytoluene. Cancer detection of initiation activity. Cancer Research, 44, 1604–1610. Letters, 188,33–38. 26 Kassie, F., Uhl, M., Rabot, S., Grasl- 19 Grasl-Kraupp, B., Bursch, W., Ruttkay- Kraupp, B., Verkerk, R., Kundi, M., Nedecky, B., Wagner, A., Lauer, B. and Chabicovsky, M., Schulte-Hermann, R. Schulte-Hermann, R. (1994) Food and Knasmuller, S. (2003) restriction eliminates preneoplastic cells Chemoprevention of 2-amino- through apoptosis and antagonizes 3-methylimidazo[4,5-f ]quinoline (IQ)- carcinogenesis in rat liver. Proceedings of the induced colonic and hepatic preneoplastic National Academy of Sciences of the United lesions in the F344 rat by cruciferous States of America, 91, 9995–9999. vegetables administered simultaneously 20 Schulte-Hermann, R., Bursch, W., Low- with the carcinogen. Carcinogenesis, 24, Baselli, A., Wagner, A. and Grasl-Kraupp, 255–261. B. (1997) Apoptosis in the liver and its role 27 Uhl, M., Kassie, F., Rabot, S., Grasl- in hepatocarcinogenesis. Cell Biology and Kraupp, B., Chakraborty, A., Laky, B., Toxicology, 13, 339–348. Kundi, M. and Knasmuller, S. (2004) Effect 21 Grasl-Kraupp, B., Ruttkay-Nedecky, B., of common Brassica vegetables (Brussels Mullauer, L., Taper, H., Huber, W., Bursch, sprouts and red cabbage) on the W. and Schulte-Hermann, R. (1997) development of preneoplastic lesions Inherent increase of apoptosis in liver induced by 2-amino-3-methylimidazo tumors: implications for carcinogenesis [4,5-f]quinoline (IQ) in liver and colon of References j353

Fischer 344 rats. Journal of Chromatography 37 Ehrlich, V.E., Huber, W., Grasl-Kraupp, B., B, 802, 225–230. Nersesyan, A. and Knasmuller,€ S. (2004) 28 Siess, M.H., Le Bon, A.M., Canivenc- Use of preneoplastic lesions in colon and Lavier, M.C. and Suschetet, M. (2000) liver in experimental oncology. Radiology Mechanisms involved in the and Oncology, 38, 205–216. chemoprevention of flavonoids. Biofactors, 38 Corpet, D.E. and Pierre, F. (2003) Point: 12, 193–199. from animal models to prevention of colon 29 Low-Baselli, A., Hufnagl, K., Parzefall, W., cancer. Systematic review of Schulte-Hermann, R. and Grasl-Kraupp, chemoprevention in min mice and, B. (2000) Initiated rat hepatocytes in choice of the model system. Cancer primary culture: a novel tool to study Epidemiology, Biomarkers & Prevention, 12, alterations in growth control during the 391–400. first stage of carcinogenesis. 39 Ohgaki, H. (2000) Carcinogenicity in Carcinogenesis, 21,79–86. animals and specific organs – rodents, in 30 Potten, C.S. (1992) The significance of Food Borne Carcinogens: Heterocyclic spontaneous and induced apoptosis in the Amines (eds M. Nagao and T. Sugimura), gastrointestinal tract of mice. Cancer John Wiley & Sons, Ltd, Chichester, Metastasis Reviews, 11, 179–195. pp. 197–228. 31 Bird, R.P. (1987) Observation and 40 Augustsson, K. and Steineck, G. (2000) quantification of aberrant crypts in the Cancer risk based on epidemiological murine colon treated with a colon studies, in Food Borne Carcinogens: carcinogen: preliminary findings. Cancer Heterocyclic Amines (eds M. Nagao and T. Letters, 37, 147–151. Sugimura), John Wiley & Sons, Ltd, 32 Alrawi, S.J., Schiff, M., Carroll, R.E., Chichester, pp. 332–348. Dayton, M., Gibbs, J.F., Kulavlat, M., Tan, 41 Intiyot, Y., Kinouchi, T., Kataoka, K., D., Berman, K., Stoler, D.L. and Anderson, Arimochi, H., Kuwahara, T., G.R. (2006) Aberrant crypt foci. Anticancer Vinitketkumnuen, U. and Ohnishi, Y. Research, 26, 107–119. (2002) Antimutagenicity of Murdannia 33 Cheng, L. and Lai, M.D. (2003) Aberrant loriformis in the Salmonella mutation assay crypt foci as microscopic precursors of and its inhibitory effects on azoxymethane- colorectal cancer. World Journal of induced DNA methylation and aberrant Gastroenterology, 9, 2642–2649. crypt focus formation in male F344 rats. 34 Roncucci, L., Stamp, D., Medline, A., The Journal of Medical Investigation, 49, Cullen, J.B. and Bruce, W.R. (1991) 25–34. Identification and quantification of 42 Corpet, D.E. and Tache, S. (2002) Most aberrant crypt foci and microadenomas in effective colon cancer chemopreventive the human colon. Human Pathology, 22, agents in rats: a systematic review of 287–294. aberrant crypt foci and tumor data, ranked 35 Pretlow, T.P., Barrow, B.J., Ashton, W.S., by potency. Nutrition and Cancer, 43,1–21. ORiordan, M.A., Pretlow, T.G., Jurcisek, 43 Corpet, D.E., Stamp, D., Medline, A., J.A. and Stellato, T.A. (1991) Aberrant Minkin, S., Archer, M.C. and Bruce, W.R. crypts: putative preneoplastic foci in (1990) Promotion of colonic human colonic mucosa. Cancer Research, microadenoma growth in mice and rats fed 51, 1564–1567. cooked sugar or cooked casein and fat. 36 Mori, H., Yamada, Y., Kuno, T. and Hirose, Cancer Research, 50, 6955–6958. Y. (2004) Aberrant crypt foci and beta- 44 Poulsen, M., Molck, A.M., Thorup, I., catenin accumulated crypts: significance Breinholt, V. and Meyer, O. (2001) The and roles for colorectal carcinogenesis. influence of simple sugars and starch Mutation Research, 566, 191–208. given during pre- or post-initiation on 354j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

aberrant crypt foci in rat colon. Cancer Steroid Biochemistry and Molecular Biology, Letters, 167, 135–143. 97, 129–136. 45 Zhang, X.M., Chan, C.C., Stamp, D., 53 Pool-Zobel, B.L. (2005) Inulin-type Minkin, S., Archer, M.C. and Bruce, W.R. fructans and reduction in colon cancer (1993) Initiation and promotion of colonic risk: review of experimental and human aberrant crypt foci in rats by data. The British Journal of Nutrition, 93 5-hydroxymethyl-2-furaldehyde in (Suppl. 1), S73–S90. thermolyzed sucrose. Carcinogenesis, 14, 54 Reddy, B.S. (2004) Studies with the 773–775. azoxymethane-rat preclinical model for 46 De Flora, S. and Ramel, C. (1990) assessing colon tumor development and Classiﬁcation of mechanisms of inhibitors chemoprevention. Environmental and of mutagenesis and carcinogenesis. Basic Molecular Mutagenesis, 44,26–35. Life Sciences, 52, 461–462. 55 Suzuki, R., Kohno, H., Yasui, Y., Hata, K., 47 Vogelstein, B., Fearon, E.R., Hamilton, Sugie, S., Miyamoto, S., Sugawara, K., S.R., Kern, S.E., Preisinger, A.C., Leppert, Sumida, T., Hirose, Y.and Tanaka,T.(2007) M., Nakamura, Y., White, R., Smits, A.M. Diet supplemented with citrus unshiu and Bos, J.L. (1988) Genetic alterations segment membrane suppresses during colorectal-tumor development. The chemically induced colonic preneoplastic New England Journal of Medicine, 319, lesions and fatty liver in male db/db mice. 525–532. International Journal of Cancer, 120, 48 Dashwood, R.H. (2002) Modulation of 252–258. heterocyclic amine-induced mutagenicity 56 Tache, S., Ladam, A. and Corpet, D.E. and carcinogenicity: an A-to-Z guide to (2007) Chemoprevention of aberrant chemopreventive agents, promoters, and crypt foci in the colon of rats by dietary transgenic models. Mutation Research, 511, onion. European Journal of Cancer, 43, 89–112. 454–458. 49 Schwab, C.E., Huber, W.W., Parzefall, W., 57 Xiao, H., Hao, X., Simi, B., Ju, J., Jiang, H., Hietsch, G., Kassie, F., Schulte-Hermann, Reddy, B.S. and Yang, C.S. (2008) Green R. and Knasmuller, S. (2000) Search for tea polyphenols inhibit colorectal aberrant compounds that inhibit the genotoxic and crypt foci (ACF) formation and prevent carcinogenic effects of heterocyclic oncogenic changes in dysplastic ACF in aromatic amines. Critical Reviews in azoxymethane-treated F344 rats. Toxicology, 30,1–69. Carcinogenesis, 29, 113–119. 50 Kassie, F., Sundermann, V.M., 58 Arikawa, A.Y. and Gallaher, D.D. (2008) Edenharder, R., Platt, K.L., Darroudi, F., Cruciferous vegetables reduce Lhoste, E., Humbolt, C., Muckel, E., Uhl, morphological markers of colon cancer M., Kundi, M. and Knasmuller, S. (2003) risk in dimethylhydrazine-treated rats. The Development and application of test Journal of Nutrition, 138, 526–532. methods for the detection of dietary 59 Hirose, Y., Kuno, T., Yamada, Y., Sakata, K., constituents which protect against Katayama, M., Yoshida, K., Qiao, Z., Hata, heterocyclic aromatic amines. Mutation K., Yoshimi, N. and Mori, H. (2003) Research, 523–524, 183–192. Azoxymethane-induced beta-catenin- 51 Kim, Y.S. and Milner, J.A. (2007) Dietary accumulated crypts in colonic mucosa of modulation of colon cancer risk. The rodents as an intermediate biomarker for Journal of Nutrition, 137, 2576S–2579S. colon carcinogenesis. Carcinogenesis, 24, 52 Murillo, G. and Mehta, R.G. (2005) 107–111. Chemoprevention of chemically-induced 60 Magnuson, B.A., Carr, I. and Bird, R.P. mammary and colon carcinogenesis by (1993) Ability of aberrant crypt foci 1alpha-hydroxyvitamin D5. The Journal of characteristics to predict colonic tumor References j355

incidence in rats fed cholic acid. Cancer end-point marker for colorectal cancer in Research, 53, 4499–4504. azoxymethane-induced rats. 61 Magnuson, B.A. and Bird, R.P. (1993) Carcinogenesis, 20, 2267–2272. Reduction of aberrant crypt foci induced in 68 Femia, A.P., Dolara, P. and Caderni, G. rat colon with azoxymethane or (2004) Mucin-depleted foci (MDF) in the methylnitrosourea by feeding cholic acid. colon of rats treated with azoxymethane Cancer Letters, 68,15–23. (AOM) are useful biomarkers for colon 62 Steele, V.E., Pereira, M.A., Sigman, C.C. carcinogenesis. Carcinogenesis, 25, and Kelloff, G.J. (1995) Cancer 277–281. chemoprevention agent development 69 Frese, K.K. and Tuveson, D.A. (2007) strategies for genistein. The Journal of Maximizing mouse cancer models. Nature Nutrition, 125, 713S–716S. Reviews, 7, 645–658. 63 Pereira, M.A., Barnes, L.H., Rassman, V.L., 70 Hursting, S.D., Nunez, N.P., Patel, A.C., Kelloff, G.V. and Steele, V.E. (1994) Use of Perkins, S.N., Lubet, R.A. and Barrett, J.C. azoxymethane-induced foci of aberrant (2005) The utility of genetically altered crypts in rat colon to identify potential mouse models for nutrition and cancer cancer chemopreventive agents. chemoprevention research. Mutation Carcinogenesis, 15, 1049–1054. Research, 576,80–92. 64 Rao, C.V., Wang, C.X., Simi, B., Lubet, R., 71 Hollstein, M., Sidransky, D., Vogelstein, B. Kelloff, G., Steele, V. and Reddy, B.S. and Harris, C.C. (1991) p53 mutations in (1997) Enhancement of experimental human cancers. Science, 253,49–53. colon cancer by genistein. Cancer Research, 72 Klein, R.D. (2005) The use of genetically 57, 3717–3722. engineered mouse models of prostate 65 Pretlow, T.P. and Bird, R.P. (2001) cancer for nutrition and cancer Correspondence re: Y. Yamada et al., chemoprevention research. Mutation Frequent beta-catenin gene mutations and Research, 576, 111–119. accumulations of the protein in the 73 Sieber, O.M., Howarth, K.M., Thirlwell, C., putative preneoplastic lesions lacking Rowan, A., Mandir, N., Goodlad, R.A., macroscopic aberrant crypt foci Gilkar, A., Spencer-Dene, B., Stamp, G., appearance, in rat colon carcinogenesis. Johnson, V., Silver, A., Yang, H., Miller, Cancer Res., 60: 3323–3327, 2000; and J.H., Ilyas, M. and Tomlinson, I.P. (2004) Sequential analysis of morphological and Myh deﬁciency enhances intestinal biological properties of beta-catenin- tumorigenesis in multiple intestinal þ accumulated crypts, provable neoplasia (ApcMin/ ) mice. Cancer premalignant lesions independent of Research, 64, 8876–8881. aberrant crypt foci in rat colon 74 Reliene, R., Fleming, S.M., Chesselet, carcinogenesis. Cancer Res., 61: M.F. and Schiestl, R.H. (2008) Effects of 1874–1878, 2001. Cancer Research, 61, antioxidants on cancer prevention and 7699–7701. neuromotor performance in Atm deﬁcient 66 Hao, X.P., Pretlow, T.G., Rao, J.S. and mice. Food and Chemical Toxicology, 46, Pretlow, T.P. (2001) Beta-catenin 1371–1377. expression is altered in human colonic 75 Song, J., Sohn, K.J., Medline, A., Ash, C., aberrant crypt foci. Cancer Research, 61, Gallinger, S. and Kim, Y.I. (2000) 8085–8088. Chemopreventive effects of dietary folate 67 Rijken, P.J., Timmer, W.G., van de Kooij, on intestinal polyps in Apc þ /Msh2/ A.J., van Benschop, I.M., Wiseman, S.A., mice. Cancer Research, 60, 3191–3199. Meijers, M. and Tijburg, L.B. (1999) Effect 76 Asamoto, M., Ota, T., Toriyama-Baba, H., of vegetable and carotenoid consumption Hokaiwado, N., Naito, A. and Tsuda, H. on aberrant crypt multiplicity, a surrogate (2002) Mammary carcinomas induced in 356j 21 Preneoplastic Models and Carcinogenicity Studies with Rodents

human c-Ha-ras proto-oncogene compounds sulforaphane and transgenic rats are estrogen-independent, dibenzoylmethane alone and in Min/ þ but responsive to D-limonene treatment. combination in Apc mouse. Cancer Japanese Journal of Cancer Research, 93, Research, 67, 9937–9944. 32–35. 81 Hursting, S.D., Perkins, S.N., Phang, J.M. 77 Banach-Petrosky, W., Ouyang, X., Gao, H., and Barrett, J.C. (2001) Diet and cancer Nader, K., Ji, Y., Suh, N., DiPaola, R.S. and prevention studies in p53-deﬁcient mice. Abate-Shen, C. (2006) Vitamin D inhibits The Journal of Nutrition, 131, 3092S–3094S. the formation of prostatic intraepithelial 82 Xu, J., Yang, F., An, X. and Hu, Q. (2007) neoplasia in Nkx3.1;Pten mutant mice. Anticarcinogenic activity of selenium- Clinical Cancer Research, 12, 5895–5901. enriched green tea extracts in vivo. Journal 78 Matsuoka, Y., Fukamachi, K., Hamaguchi, of Agricultural and Food Chemistry, 55, T., Toriyama-Baba, H., Kawaguchi, H., 5349–5353. Kusunoki, M., Yoshida, H. and Tsuda, H. 83 Yuan, J.H., Li, Y.Q. and Yang, X.Y. (2007) (2003) Rapid emergence of mammary Inhibition of epigallocatechin gallate on preneoplastic and malignant lesions in orthotopic colon cancer by upregulating human c-Ha-ras proto-oncogene the Nrf2-UGT1A signal pathway in nude transgenic rats: possible application for mice. Pharmacology, 80, 269–278. screening of chemopreventive agents. 84 Myzak, M.C., Tong, P., Dashwood, W.M., Toxicologic Pathology, 31, 632–637. Dashwood, R.H. and Ho, E. (2007) 79 Pool-Zobel, B.L. and Sauer, J. (2007) Sulforaphane retards the growth of human Overview of experimental data on PC-3 xenografts and inhibits HDAC reduction of colorectal cancer risk by activity in human subjects. Experimental inulin-type fructans. The Journal of Biology and Medicine, 232, 227–234. Nutrition, 137, 2580S–2584S. 85 Knasmuller, S., Steinkellner, H., Majer, 80 Shen, G., Khor, T.O., Hu, R., Yu, S., Nair, B.J., Nobis, E.C., Scharf, G. and Kassie, F. S., Ho, C.T., Reddy, B.S., Huang, M.T., (2002) Search for dietary antimutagens Newmark, H.L. and Kong, A.N. (2007) and anticarcinogens: methodological Chemoprevention of familial aspects and extrapolation problems. Food adenomatous polyposis by natural dietary and Chemical Toxicology, 40, 1051–1062. j357

22 The Role of Nutrition in the Etiology of Human Cancer: Methodological Considerations Concerning Epidemiological Studies Heiner Boeing

22.1 Introduction

Among the many factors potentially defining the risk of a population to experience the various forms of cancer is nutrition. Although the age structure of a population is probably the most important factor defining the number of cancer cases in a population, it also becomes obvious over the last decades that lifestyles also play an important role. Besides smoking, nutrition, obesity, and physical activity had been identified as major components influencing cancer occurrence in a population and individual cancer risk. Over the decades, there had been the estimate that about 30% of the avoidable cancer occurrence is due to diet [1]. Experimental research, which is the focus of this book, is often driven by more interest in contributing to the overall knowledge about the mechanism implicated in carcinogenesis and less on public health goals in lowering disease occurrence. Although such a position is well justified by the individual perspective, research programs are more and more focusing on the translational aspect of study findings. Thus, the experimental research approach might receive considerable public health attention if integrated into the concept of generating biological and mechanistic support for a risk or preventive effect of a nutritional factor proposed to change risk on population level due to epidemiological associations. Nowadays, the evaluation of factors potentially changing the risk in a population is performed under strict conditions following the evidence-based approach in clinical medicine [2]. This evidence-based nutritional prevention approach uses primarily human epidemiological studies including intervention trials and is taking the biological mechanisms as required supportive element if a high level of evidence is assigned [3]. In this chapter, the principles and current status of the evidence-based nutritional prevention approach is described, and how far this approach could help in selecting experimental research areas that have a high potential for contributing to translate their findings into public health actions.

Chemoprevention of Cancer and DNA Damage by Dietary Factors. Edited by Siegfried Knasmüller, David M. DeMarini, Ian Johnson, and Clarissa Gerhäuser Copyright Ó 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32058-5 358j 22 The Role of Nutrition in the Etiology of Human Cancer

22.2 Principles for Evaluating the Link between Nutrition and Cancer

Experimental studies are strong in identifying causal links. However, they have the disadvantage that their variation is limited to the experimental setting and that it remains unclear how the observed experimental variation can be set in the context of the variation seen in the very complex situation when linking individual behavior with cancer risk. The latter type of analysis is the subject of epidemiology. In epidemiology, observed variations in study populations are used to identify associations between nutritional factors and cancer end points. However, these links are often provided with uncertainty regarding the causal role. Thus, most schemas of generating evidence focus on the consistency of epidemiological results from properly conducted studies and require also well-founded biological mechanisms [4]. This portfolio-like approach is different from approaches that focus primarily only on intervention studies and clinical trials [5]. However, the portfolio approach also takes the epidemiological studies as the primary source of evidence and is following a hierarchy of study designs to provide evidence for a causal link. In the case of link between nutrition and cancer, the portfolio approach might be more adequate than the intervention study design approach since each study design has its speciﬁc strengths and weaknesses in this research ﬁeld.

22.2.1 Intervention Studies

The intervention approach applies an experimental design and requires the random allocation of humans to a treatment and control group that differs only in the intervention. If successfully and adequately conducted, the intervention approach is considered to give proof of causal relationships and also to provide quantitative estimates for successful prevention. However, in the case of cancer as end point, due to the low incidence, the intervention studies need to cover several years and has to include several ten thousands of study subjects. This study design has been successfully applied in studies that tested nutrients and other compounds applicable as supplements [6]. However, this approach has become more difﬁcult to conduct if behavioral change is the target of the intervention [7]. Major issues are the adherence to the intervention and the assessment of compliance. Particularly challenging is the assessment of diet unbiased by the intervention [8]. A proper and unbiased dietary assessment seems particularly important since behavioral change is usually associated with many other changes including compensatory dietary changes due to the intervention. Most intervention designs favor an isocaloric situation across the groups with no change in weight. If change in weight occurs in one of the study arms of the intervention study, the interpretation of study results becomes more complex for all those end points in which diet and anthropometric indices play a role. Further issues that need to be considered in the case of cancer are potentially long-lasting induction periods, stage of carcinogenesis in which the intervention is effective, and effective dose. Recent examples of long-lasting and 22.2 Principles for Evaluating the Link between Nutrition and Cancer j359 large-scale intervention studies do not motivate to favor this approach as a major principle to obtain evidence for a link between nutrition and cancer.

22.2.2 Prospective Cohort Studies

Prospective cohort studies do not intervene but observe study populations regarding the relation of their modifiable (diet, smoking, physical activity) and nonmodifiable (age, sex, genetic background) factors with incidence or mortality, subsequent to the initial assessment of individual characteristics. Modern cohort studies also built up large-scale biobanks with biological material obtained at recruitment for further use during the course of the study [9]. Prospective cohort studies can include several hundred thousands of subjects, and some of them already last over decades with repeated measurements of lifestyle factors and collection of blood or other body fluids [10]. The primary aim of cohort studies is to investigate incidence during follow-up and to establish risk models explaining the event of disease occurrence by characteristics of the individuals having this event compared to those not having this event during observation time. When biological measurements are included, the preferred study design is the nested case-control or case-cohort study [11]. Both the study designs take the prospective nature of the study into account and are more efficient in using the case information under the light of often costly laboratory measurements.

22.2.3 Case-Control and Other Studies

The design of a case-control study is based on the Bayesian theorem and reﬂects that the odds of ratios of exposed to unexposed among cases and controls is equivalent to the ratios of diseased and nondiseased subjects in exposed and unexposed subjects. The latter situation is found in cohort studies. Thus, by assessing the odds ratio of exposed to unexposed subjects in cases and controls, the relative risk can be estimated. This statistical relationship allows the conduct of cost-effective studies since cases continuously arise from the general population and this population also provides the controls. However, issues such as the selection of controls from the source population including comparable participation rates to the cases and the retrospective nature of the exposure assessment limit the use of this design for studies regarding nutrition and cancer [12]. These issues have been critically discussed during past years, and it was concluded that due to the probability of selection and information, bias case-control studies are not the preferred study design for the relationship between nutrition and cancer. As described above, for the evaluation of the nutrition and cancer relationship, all epidemiological study designs can contribute evidence despite the well-recognized overall advantage of the intervention approach. This conclusion also calls for a systematic collection of all epidemiological study results available for the research question. The World Cancer Research Fund [13], a charity organization based in 360j 22 The Role of Nutrition in the Etiology of Human Cancer

London, has devoted most of its activities during the past years to this type of systematic collection of study results on nutrition and cancer relationship coupled with a framework for evaluating the evidence based on this result collection [13]. Other institutions, such as the German Nutrition Society, are also pursuing similar approaches to establish evidence in the area of nutrition and cancer within the framework of their activities for disease prevention through nutrition [3]. The high number of prospective studies in the area of nutrition and cancer established in the 1990s with the collection of biological material can effectively be used for generating further evidence of causality if intervention studies are subsequently planned using the same intermediate markers as being evaluated in the observational studies [14]. This concept requires a number of well-designed small intervention studies with intermediate markers that are much easier to conduct than large-scale intervention studies with cancer end points. Such concepts might also fit better to the resources and qualification of experimental groups working in the field of nutrition and cancer. However, such concept will only work effectively if a close interaction between experimental and epidemiological groups exists, particularly with respect to the development and use of intermediate biomarkers. The establishment of the level of evidence does not give quantitative figures regarding the effect. This type of information is provided by meta-analyses using the published information about relative risk in a standardized way. Having provided the initial data for their specific analyses, sometimes the groups conducting these meta- analyses have to contact the researcher [15]. Also, pooling of study results via standardized table information or individual data sets has become a practice [16]. For the future, many of the current research questions will be investigated by pooling the available empirical data on a worldwide scale. A particular issue of the systematic evaluation of the evidence and investigation of the quantitative effects is the heterogeneity across study results. Consistency of study results has been assumed as a requirement for a high level of evidence. In reality, not all studies obtain similar effect measures and quantitative relative risk estimates. Therefore, meta-analytical program packages provide statistical parameters regarding heterogeneity of the study results. Often, research into a question indicates statistical significant heterogeneity across studies. In this case, an overall relative risk estimate across all studies is not justified. Sometimes, further evaluation can identify the reason of heterogeneity, which could be the study design, region, or gender, for example.

22.3 Methodological Statistical Challenges Regarding Dietary Assessment

An important research area in the ﬁeld of nutrition and cancer is the assessment of diet. The major issue is the measurement error that describes the degree of wrongly reported dietary intake compared to the truth. If a measurement error exists at a larger extent, the estimates of relative risk are biased and do not apply to the underlying population [17]. To estimate the measurement error of an assessment, 22.4 Complexity of Dietary Data and Their Analyses j361 instrument techniques need to be available that estimate dietary intake in an unbiased way. Such techniques are available but only for energy by using the doubly labeled water technique and for nitrogen by using nitrogen excretion in urine [18]. For most other dietary factors, the estimate of intake is valid only on a relative scale. Particularly difﬁcult seems to be the estimate of the proportion of fat in the diet. This is due to the fact that a good biomarker for fat intake is missing and that the estimate of the composition of diet regarding the energy providing nutrients is distorted by anthropometric indices [19]. Thus, the validity of dietary information is often investigated by comparing the proposed assessment instrument with more advanced instruments. In general, short-term methods such as the recording of diet over several days or the repeated report of dietary intake during the past day are thought to provide a more valid quantitative estimate of dietary intake than methods that require the assessment of past intake by averaging the frequency and portion size over a certain time period. The latter approach is called the food frequency method and has gained prominence by being the standard assessment instrument in large-scale prospective studies. Validation studies usually compare the food frequency instrument with other short-term methods and biomarker studies. Statistical methods have been published that use the results of validation studies to correct the estimate of the relative risk of the measurement error inherent in the basic dietary assessment [20]. The correction of the relative risk of measurement error could also been done by the calibration approach [21]. Calibration means that a more sophisticated dietary assessment instrument is applied in a subgroup of a cohort and that the food frequency data are regressed to the consumption values of the calibration instrument. Nowadays, statistical methods have been developed that combine short-term and long-term dietary information and obtain the best estimate for a subject from the dietary information being assembled for this individual [22,23]. Further progress in dietary assessment is assumed by the use of new technologies. Already available methods can be adopted for use in a new context, for example, as web-based tool, or new techniques can be introduced such as recording purchases by bar code reading or describing meals by photos made through mobile phones.

22.4 Complexity of Dietary Data and Their Analyses

The experimental scientiﬁc focus is usually directed to the compounds in foods and the understanding of how these compounds alter the relation between nutrition and cancer. Estimates of consumption of these compounds in human populations have to be derived from food intake data by using food tables. In statistical terms, the relation of compounds in a food is taken as given, and variation in consumption data on compounds across individuals is the result of the variation in foods with different ﬁxed compound composition. Compound variation across individuals is therefore the result of a complex process that has not been investigated so much in the past. Results from epidemiological studies with respect to the effect of a single nutritional compound on disease risk, even if derived from complex statistical models with 362j 22 The Role of Nutrition in the Etiology of Human Cancer

adjustment for other dietary compounds, are therefore not easy to interpret according to causality. Food pattern analysis is considered as a research tool to unravel the complex relationships between food intake and the effect of compounds in foods on disease risk and has been increasingly applied in nutritional epidemiology during the past years [24]. One other approach to consider the complexity of dietary data is the use of isocaloric statistical modeling that is associated with the term exchange models. Isocaloric statistical modeling means to model the respective disease risk associated with a change in a compound under the assumption of a constant energy supply [25]. This assumption can be realized only if other energy providing components are exchanged in an isocaloric manner. In epidemiological exchange models, the relative risk is assessed for situations in which various energy providing compounds are exchanged with each other. This concept is an extension of the concept of energy adjustment. The latter concept means that only the part of the variation of a nutrient is considered in the risk model that is not due to variation in energy intake.

22.5 Current Status of the Evidence

Despite the increasing number of study results emerging from the many prospective studies in the fieldofnutritionandcancer,itwillbecomeincreasinglydifficult to completely change the conclusions and levels of evidence being proposed so far by expert panels on research questions for which many study results are already available. Thus, the evaluation of the evidence by the second expert panel of the Ref. [13] has been tabulated for five dietary variables that are considered to be one of the most important dietary factors with impact on cancer occurrence (Table 22.1).

22.5.1 Fruit and Vegetables

In the early 1990s, the available study results, mostly case-control studies, were nearly consistent in the observation that cancer cases report less intake of fruit and vegetables prior to clinical cancer occurrence than controls [26]. These study results were taken as breakthrough regarding the role of diet in cancer prevention and led to the 5-A-day campaign as public private partnership between National Cancer Insti- tute of the United States and the food industry and retail companies. Since then, especially data from cohort studies investigating cancer incidence in conjunction with intake of fruit and vegetables could not always conﬁrm the role of this food item regarding reducing the risk of cancer. Thus, the initial assignment of the level of evidence as convincing such as by the ﬁrst panel of the WCRF [27] was changed to probable by an expert panel of the International Agency for Research on Cancer (IARC) in 2003 [28]. Although nowadays the evidence is still rated as probable (Table 22.1) for some cancer sites, the quantitative effect itself on reduction of cancer 22.5 Current Status of the Evidence j363

Table 22.1 Level of evidence for five of the most important dietary factors with implication to cancer risk of 19 cancer sites according to the second expert panel of the WCRF 2007 [13].

Fruit/ Meat and Cancer sites vegetables meat products Grains Lipids Alcohol

Mouth/pharynx, larynx, !! ~~~ Esophagus !! ~ ! ~~~ Stomach !! ~ Colon ! ~~~ !! ~ (animal) ~~~ Rectum ! ~~~ !! ~~~ Lung !!/!~ ~ Bladder Kidney ^^ Pancreas !/ ~ Liver !/ ~~ Gallbladder Skin Breast ~ (postmenopausal) ~~~ Ovary /! Uterus /!~ Cervix Prostate ~

!!, probable evidence for a decrease in risk with increasing consumption; !, limited/insufﬁcient evidence for a decrease in risk with increasing consumption; ~~~, convincing evidence for an increase in risk with increasing consumption; ~~, probable evidence for an increase in risk with increasing consumption; ~, limited/insufﬁcient evidence for an increase in risk with increasing consumption; ^^, probable evidence of no effect.

risk by the increasing intake of fruit and vegetables by three servings is seen today more in the range of 5–10% instead of the previously 23%.

22.5.2 Meat and Meat Products

Recent prospective studies and subsequent meta-analyses highlighted that the intake of meat and, to a larger extent, meat products originating from pork, beef, and lamb increases the risk of colorectal cancer. In the recent evaluation of the second WCRF panel [13], meat and meat products were given a high level of evidence to increase cancer risk because of the consistency of the ﬁndings. Old data from case-control studies as well as more recent results from prospective studies have also extended the spectrum of cancer sites in which this food group might play a role at the last expert evaluation. The primary focus regarding the biological meaning of these ﬁndings refers more to the role of haem iron as source for nitrosation and formation of N-nitroso compounds and less to the methods of cooking and food preparation of meat and meat products. For the latter practices, convincing epidemiological data 364j 22 The Role of Nutrition in the Etiology of Human Cancer

are still missing and research into biomarkers for preparation and cooking practices suitable to be applied in epidemiological studies is ongoing.

22.5.3 Grains

Dietary fiber as previously neglected component of the diet has been proposed by Burkitt in 1972 to reduce the risk of colorectal cancer [29]. This hypothesis was investigated since then with varying success. Although early case-control studies provided support for this hypothesis, prospective data that had been published at the beginning of this millennium were mostly negative in assigning this dietary factor a role in colorectal carcinogenesis. Also, intervention studies using intermediate markers of risk such as polyp recurrence were mostly negative. Support for a role of fiber particularly from grains for this cancer site came recently from the two largest prospective studies by number of participants. There is further evidence emerging from the prospective cohort studies that high intake of fiber from grains is associated with reduced risk of cancers of the upper gastrointestinal tract including the stomach.

22.5.4 Lipids (Fat)

Lipids, also termed fat, have been proposed to be the driving force for increased cancer risk due to the ecological studies linking mortality rates with population ﬁgures of fat use in the 1970s, particularly for breast and corpus uteri cancer [30]. This initial hypothesis has not been conﬁrmed in most of the individual-based studies linking individual fat consumption with cancer incidence. However, recently, data emerged from prospective studies that measurement error and a low discrimination ability of some dietary assessment instrument regarding high and low fat consumer might be the reason for not having seen increased risk of postmenopausal cancer with increasing fat consumption. The WCRF panel also pointed out that fat consumption might also increase the risk of lung cancer. However, the data so far are not consistent, and it should be considered that the number of smokers is high among subjects with lung cancer. Smokers might have a special diet, for example, low in fruit and vegetables and high in fat.

22.5.5 Use of Alcoholic Beverages

Alcohol has been rated by WHO as the substance for which convincing (sufficient) evidence exists that its use increases cancer risk. This organization has identified five cancer sites that have been linked with the use of alcohol: mouth, pharynx, larynx, esophagus, liver, and female breast. Recently, colorectal cancer was added to this list [31]. A similar list of cancer sites being convincingly linked with the use of alcohol has been identified by the WCRF panel (Table 22.1). In contrast to cardiovascular diseases, a decrease in risk with moderate consumption compared to low or no consumption of alcohol has not been proposed by the expert panel. Occasionally, References j365 cancer studies also observe the lowest risk in those subjects with moderate consumption. Such observations should be interpreted with caution because the use of alcoholic beverages is linked with other lifestyle factors and lifetime moderate alcohol use might reflect moderation in other lifestyle aspects.

22.6 Summary and Conclusion

The generation of evidence is a systematic process done by collecting and evaluating all epidemiological studies conducted on the research question followed by a careful evaluation of the biological mechanisms and results from experimental research. Each epidemiological study contributes to the evidence by its study design and the study result. Currently, there seems the most promising concept for generating evidence that uses large-scale cohort studies by unraveling associations including biomarker and intermediate end points and subsequently testing these associations in small-scale intervention studies for causality by using these biomarkers and intermediate end points taking the pros and cons of each study design in the area of nutrition and cancer into account. In this process, it is helpful that most of the study results are nowadays given in quantitative terms with a point estimate of relative risk including conﬁdence intervals that are suitable for quantitative meta-analyses. Quantitative meta-analyses will allow using the epidemiological data generated on the worldwide scale in a better manner. In this context, it is evident that the next generations of study results contributing to the question of nutrition and cancer have to consider the most recent methodological concepts. Major principles of these concepts have been outlined in this chapter. These include the better estimate of dietary intake by new statistical concepts and the inclusion of biomarkers, the use of advanced statistical modeling including isocaloric exchange models, and the application of food pattern analyses instead of single food group analyses to consider the complexity of dietary intake in relation to disease etiology. Having these advancements in mind, the interaction between epidemiology and experimental research might foster the translation of basic research ﬁndings into public health practice.

References

1 WHO (2003) The World Cancer Report, ernahrungsmitbedingter€ Krankheiten, 2006 2003 edn, IARC Press, Lyon. edn, DGE, Bonn. 2 Kroke, A., Boeing, H., Rossnagel, K. and 4 Wiseman, M. (2004) Observational versus Willich, S.N. (2004) History of the concept randomised trial evidence, Lancet 364, of levels of evidence and their current 755–756, author reply 756–757. status in relation to primary prevention 5 Sackett, D.L., Rosenberg, W.M., through lifestyle interventions. Public Haynes, R.B. and Richardson, W.S. (1996) Health Nutrition, 7, 279–284. Evidence based medicine: what it is and 3 DGE (2006). Evidenzbasierte Leitlinie: what it isnt. British Medical Journal, Fettkonsum und Pravention€ ausgewahlter€ 312,71–72. 366j 22 The Role of Nutrition in the Etiology of Human Cancer

6 Albanes, D., Heinonen, O.P., Huttunen, and Schulze, M.B. (2008) Plasma fetuin-A J.K., Taylor, P.R., Virtamo, J., Edwards, levels and the risk of type 2 diabetes. B.K., Haapakoski, J., Rautalahti, M., Diabetes, 57 (10), 2762–2767. Hartman, A.M. and Palmgren, J. (1995) 12 Giovannucci, E., Stampfer, M.J., Colditz, Effects of alpha-tocopherol and beta- G.A., Manson, J.E., Rosner, B.A., carotene supplements on cancer incidence Longnecker, M., Speizer, F.E. and Willett, in the alpha-tocopherol beta-carotene W.C. (1993) A comparison of prospective cancer prevention study. The American and retrospective assessments of diet in Journal of Clinical Nutrition, 62, the study of breast cancer. American 1427S–1430S. Journal of Epidemiology, 137, 502–511. 7 Chlebowski, R.T.and Grosvenor, M. (1994) 13 WCRF and AICR (2007) Food, Nutrition, The scope of nutrition intervention trials Physical Activity, and the Prevention of with cancer-related endpoints. Cancer, 74, Cancer: a Global Perspective, 2007 edn, 2734–2738. AICR, Washington, DC. 8 Neuhouser, M.L., Tinker, L., Shaw, P.A., 14 Michels, K.B. and Willett, W.C. (2008) The Schoeller, D., Bingham, S.A., Horn, L.V., Womens Health Initiative Randomized Beresford, S.A., Caan, B., Thomson, C., Controlled Dietary Modiﬁcation Trial: a Satterﬁeld, S., Kuller, L., Heiss, G., Smit, post-mortem. Breast Cancer Research and E., Sarto, G., Ockene, J., Stefanick, M.L., Treatment. Mar 30, Epub ahead of print. Assaf, A., Runswick, S. and Prentice, R.L. 15 Schulze, M., Schulz, M., Heidemann, C., (2008) Use of recovery biomarkers to Schienkiewitz, A., Hoffmann, K. and calibrate nutrient consumption self- Boeing, H. (2007) Fiber and magnesium reports in the Womens Health Initiative. intake and incidence of type 2 diabetes. American Journal of Epidemiology, 167, Archives of Internal Medicine, 167, 956–965. 1247–1259. 16 Koushik, A., Hunter, D.J., Spiegelman, D., 9 Riboli, E., Hunt, K.J., Slimani, N., Ferrari, Beeson, W.L., van den Brandt, P.A., Buring, P., Norat, T.,Fahey, M., Charrondiere, U.R., J.E., Calle, E.E., Cho, E., Fraser, G.E., Hemon, B., Casagrande, C., Vignat, J., Freudenheim, J.L., Fuchs, C.S., Overvad, K., Tjonneland, A., Clavel- Giovannucci, E.L., Goldbohm, R.A., Chapelon, F., Thiebaut, A., Wahrendorf, J., Harnack, L., Jacobs, D.R. Jr, Kato, I., Krogh, Boeing, H., Trichopoulos, D., V., Larsson, S.C., Leitzmann, M.F., Trichopoulou, A., Vineis, P., Palli, D., Marshall, J.R., McCullough, M.L., Miller, Bueno-De-Mesquita, H.B., Peeters, P.H., A.B., Pietinen, P., Rohan, T.E., Schatzkin, Lund, E., Engeset, D., Gonzalez, C.A., A., Sieri, S., Virtanen, M.J., Wolk, A., Barricarte, A., Berglund, G., Hallmans, G., Zeleniuch-Jacquotte, A., Zhang, S.M. and Day, N.E., Key,T.J., Kaaks, R. and Saracci, R. Smith-Warner, S.A. (2007) Fruits, (2002) European Prospective Investigation vegetables, and colon cancer risk in a pooled into Cancer and Nutrition (EPIC): study analysis of 14 cohort studies. Journal of the populations and data collection. Public National Cancer Institute, 99,1471–1483. Health Nutrition, 5,1113–1124. 17 Kristal, A.R. and Potter, J.D. (2006) Not the 10 Carandang, R., Seshadri, S., Beiser, A., time to abandon the food frequency Kelly-Hayes, M., Kase, C.S., Kannel, W.B. questionnaire: counterpoint. Cancer and Wolf, P.A. (2006) Trends in incidence, Epidemiology, Biomarkers & Prevention: A lifetime risk, severity, and 30-day mortality Publication of the American Association for of stroke over the past 50 years. The Journal Cancer Research, Cosponsored by the of the American Medical Association, 296, American Society of Preventive Oncology, 15, 2939–2946. 1759–1760. 11 Stefan, N., Fritsche, A., Weikert, C., 18 Kroke, A., Klipstein-Grobusch, K., Voss, S., Boeing, H., Joost, H.-G., H€aring, H.-U. Moseneder, J., Thielecke, F., Noack, R. and References j367

Boeing, H. (1999) Validation of a self- validation for use as a covariate in a model administered food-frequency to estimate usual food intake. Journal of the questionnaire administered in the American Dietetic Association, 106, European Prospective Investigation into 1556–1563. Cancer and Nutrition (EPIC) Study: 24 Schulze, M.B., Hoffmann, K., Kroke, A. comparison of energy, protein, and and Boeing, H. (2003) An approach to macronutrient intakes estimated with the construct simpliﬁed measures of dietary doubly labeled water, urinary nitrogen, and patterns from exploratory factor analysis. repeated 24-h dietary recall methods. The The British Journal of Nutrition, 89, American Journal of Clinical Nutrition, 70, 409–419. 439–447. 25 Schulze, M.B., Schulz, M., Heidemann, 19 Voss, S., Kroke, A., Klipstein-Grobusch, K. C., Schienkiewitz, A., Hoffmann, K. and and Boeing, H. (1998) Is macronutrient Boeing, H. (2008) Carbohydrate intake and composition of dietary intake data affected incidence of type 2 diabetes in the by underreporting? Results from the EPIC- European Prospective Investigation into Potsdam Study. European Prospective Cancer and Nutrition (EPIC)-Potsdam Investigation into Cancer and Nutrition. Study. The British Journal of Nutrition, 99, European Journal of Clinical Nutrition, 52, 1107–1116. 119–126. 26 Block, G., Patterson, B. and Subar, A. 20 Rosner, B.A., Michels, K.B., Chen, Y.H. (1992) Fruit, vegetables, and cancer and Day, N.E. (2008) Measurement error prevention: a review of the epidemiological correction for nutritional exposures with evidence. Nutrition and Cancer, 18,1–29. correlated measurement error: Use of the 27 WCRF, and AICR (1997) Food, Nutrition method of triads in a longitudinal setting. and the Prevention of Cancer: A Global Statistics in Medicine, 27, 3466–3489. Perspective, 1997 edn, AICR, Washington, 21 Ferrari, P., Day, N.E., Boshuizen, H.C., DC. Roddam, A., Hoffmann, K., Thiebaut, A., 28 IARC (2003) Handbooks of Cancer Pera, G., Overvad, K., Lund, E., Prevention No 8: Fruit and vegetables, 2003 Trichopoulou, A., Tumino, R., Gullberg, edn, IARC Press, Lyon. B., Norat, T., Slimani, N., Kaaks, R. and 29 Burkitt, D.P., Walker, A.R. and Painter, Riboli, E. (2008) The evaluation of the diet/ N.S. (1972) Effect of dietary ﬁbre on disease relation in the EPIC study: stools and the transit-times, and its role considerations for the calibration and the in the causation of disease. Lancet, 2, disease models. International Journal of 1408–1412. Epidemiology, 37 (2), 368–378. 30 Armstrong, B. and Doll, R. (1975) 22 Haubrock, J., Harttig, U., Volatier, J.L., Environmental factors and cancer Dekkers, A. and Boeing, H. (2008) incidence and mortality in different Estimating usual dietary intake countries, with special reference to dietary distributions by using the multiple-source practices. International Journal of Cancer, method (personal communication). 15, 617–631. 23 Subar, A.F., Dood, K.W., Guenther, P.M., 31 Boffetta, P., Hashibe, M., La Vecchia, C., Kipnis, V., Midthune, D., McDowell, I., Zatonski, W. and Rehm, J. (2006) The Tooze, J.A., Freedman, L.S. and Krebs- burden of cancer attributable to alcohol Smith, S.M. (2006) The food propensity drinking. International Journal of Cancer, questionnaire: concept, development, and 119, 884–887. Part Three Selected Chemoprotective Dietary Factors and Components

23 Carotenoids and Vitamin A M. Cristina Polidori and Wilhelm Stahl

23.1 Introduction

A great deal of attention has been focused in the recent past on the mechanisms underlying the beneficial effects of fruits and vegetables in the chemoprevention of several kinds of cancer [1]. Fruits and vegetables and their dietary constituents affect the immune system, hormonal status, metabolism of carcinogens, and gene expression. However, a large body of evidence shows that several constitutive compounds of fruits and vegetables also inhibit and reverse the damaging effects of oxidation reactions associated with the process of carcinogenesis. Aerobic metabolism and endogenous defense reactions are associated with the formation of reactive oxygen and nitrogen species, for example, hydroxyl radical, superoxide anion radical, hydrogen peroxide, singlet oxygen, nitric oxide, or peroxynitrite. These reactive intermediates are capable of damaging biologically relevant molecules such as DNA, proteins, lipids, or carbohydrates, processes that are thought to be involved in the pathobiochemistry of several diseases. Depending on the nature of the reactive oxygen species (ROS), several forms of DNA damage occur, including modification and loss of bases, strand breaks, or DNA–protein cross-linking. Among base modification the formation of 8-hydroxy-2-deoxyguanosine (8-OHdG) is a major reaction, and the compound has been detected following ROS exposure in vitro and in vivo. Although 8-OHdG is efficiently removed by excision repair, it may be promutagenic leading to Gto T transversions. Such mutations have been determined in genes whose modified gene products play a role in the initiation, development, and progression of cancer. There is increasing evidence that ROS are also involved in signal transduction pathways. Their signaling properties are at least partly due to interactions with redox-sensitive proteins including tyrosine kinases or redox-sensitive transcriptory proteins like the Nrf2–KEAP1 system. Although the implication of ROS-dependent signaling is only beginning to be mapped, it is likely that such interactions are relevant with respect to the regulation of growth and differentiation and thus to cancer development and control. Different lines of biological defense against oxidative damage have been developed, which can be categorized into

Scheme 23.1 Chemical structure of all-trans retinol.

Scheme 23.2 Chemical structure of retinoic acid.

prevention, interception, and repair. Interception is the domain of antioxidants and antioxidant vitamins, and micronutrients belong to the most important nonenzymatic antioxidant systems in the human organism. Important micronutrients are the carotenoids that comprise a group of natural pigments commonly occurring in fruit and vegetables consumed by humans. Carotenoids with cyclic b-ionone end groups are precursors of vitamin A. The term vitamin A (all-trans retinol, Scheme 23.1) is often used as a general term for all compounds that exhibit the biological activity of retinol, while the term retinoid refers to both naturally occurring and synthetic compounds bearing a structural resemblance to all-trans retinol. An important biologically active form of vitamin A is retinoic acid (Scheme 23.2). The best studied provitamin A carotenoid is b-carotene. Within the body, b-carotene can undergo cleavage to retinal which is then reduced to give rise to two molecules of retinol. To date, a wide range of carotenoids have been isolated and quantified from fruits and vegetables, and up to 50 carotenoids are known as being available from the diet and absorbed and metabolized by the human body. The carotenoids detected in mg–ng/g quantities in human tissues, however, are substantially less and are shown in Scheme 23.3 (b-carotene, a-carotene, canthaxanthin, b-cryptoxanthin, zeaxanthin, and lutein). Carotenoids have antioxidant properties, scavenge ROS, activate signaling pathways leading to antioxidant response activation, and upregulate the expression of detoxifying enzymes. In other words, they exert beneficial effects by counteracting one of the major endogenous causes of cancer, that is, oxidative (DNA) damage. The antioxidant activity of retinol and its derivatives is moderate; however, the compound plays a major role in cellular signaling, for example, as a ligand of a family of nuclear receptors involved in the regulation of gene expression. In vitro cell culture studies, studies on animal models, and different types of human studies all support the idea that carotenoids and vitamin A play a role in the prevention of cancer. The purpose of this chapter is to review the major findings resulting from the studies on carotenoids found in the human diet and body according to their preventive effects against certain types of cancer. Human trials on the preventive effects of selected carotenoids and 23.1 Introduction j373

Scheme 23.3 Chemical structures of b-carotene, a-carotene, canthaxanthin, b-cryptoxanthin, zeaxanthin, and lutein. vitamin A will be classified as primary prevention trials if including general or healthy populations, whereas trials including participants with specific cancer will be classified as secondary prevention. Tertiary prevention (treatment) trials will be introduced when appropriate on the basis of relevant results published in the literature. As such, 374j 23 Carotenoids and Vitamin A

particular attention will be given to b-carotene and lung cancer, lycopene and prostate cancer, retinoids and cervical cancer, and retinoids and skin cancer, gastric cancer, and leukemia. Cancers that have been studied in relation to carotenoid- and vitamin A- mediated prevention include those of mouth/pharynx/larynx, lung, esophagus, prostate, and skin.

23.2 Carotenoids – Biochemical Properties

Carotenoids are among the most widespread natural colorants, and more than 600 different compounds have been identified until know, with b-carotene as the most prominent. They are synthesized by plants, bacteria, fungi, and algae and are responsible for the bright colors of various fruits and vegetables and the coloration of the leaves in autumn when the covering chlorophyll is lost. The importance of carotenoids in nature is reflected by various biological functions including provitamin A activity, antioxidant properties, and accessory functions in the light harvesting system of plants. Most carotenoids contain an extended system of conjugated double bonds, which is responsible for their color. This core of a carotenoid is substituted with different end groups including b-ionone rings as in the case of b-carotene. According to the number of double bonds, several cis/trans (E/Z) configurations are possible for a molecule. Carotenoids tend to isomerize and form a mixture of mono- and poly-cis isomers in addition to the all-trans form. In most cases the all-trans form is predominant in nature. On the basis of their composition, carotenoids can be divided in two classes: carotenes that contain only carbon and hydrogen and oxocarotenoids (xanthophylls) that carry at least one oxygen atom. From a biochemical point of view, carotenoids are grouped as provitamin A and nonprovitamin A compounds. The provitamin A carotenoids, b-carotene, a-carotene, and cryptoxanthin may serve as precursors of retinol and are capable of preventing classical vitamin A deficiency diseases. fi 1 Carotenoids ef ciently scavenge singlet molecular oxygen ( O2) and peroxyl radicals. Singlet oxygen quenching by carotenoids occurs via physical or chemical 1 quenching. Physical quenching involves the transfer of excitation energy from O2 to the carotenoid, resulting in ground state oxygen and an excited triplet state carotenoid. The energy is dissipated between the excited carotenoid and the surrounding solvent to yield the ground state carotenoid and thermal energy. In the process of physical quenching, the carotenoid remains intact and can undergo further cycles of singlet oxygen quenching. The rate constants for the reaction of carotenoids with singlet oxygen are in the range of 109 M 1 s 1, that is, near diffusion control. fi 1 b-Carotene and other carotenoids are the most ef cient natural O2-quenchers; their quenching activity is closely related to the number of conjugated double bonds present in the molecule. 23.2 Carotenoids – Biochemical Properties j375

Chemical quenching contributes less than 0.05% to the overall quenching of 1 fi O2 but is responsible for the nal decomposition of carotenoids, a process also known as photobleaching. b-Carotene and other carotenoids efficiently scavenge peroxyl radicals, especially at low oxygen tension. It should be noted that carotenoids may also act as pro-oxidants under specific conditions. Such properties have been discussed in context with adverse effects observed upon b-carotene supplementation at high levels. Apart from their antioxidant activity, several other biological properties have been assigned to carotenoids including impact on cellular signaling [2]. One pathway of intercellular signaling is provided by cell-to-cell channels called gap junctions. The influence of carotenoids on gap junctional communication does not correlate with their antioxidant activity. Among the carotenoids, b-carotene and canthaxanthin belong to the most effective stimulators of gap junctional communication. In cell culture, carotenoids reversibly inhibit the progression of carcinogen-initiated fibroblasts to the transformed state. This inhibitory effect has been found to be related to an increased gap junctional communication induced by these compounds. Carotenoids influence gap junctional intercellular communication correlated with an increased formation of connexin protein. The levels of other proteins involved in the control of cell proliferation and growth factor signaling are also modulated at the expression level, suggesting direct effects of carotenoids on transcription. There is substantial evidence for an impact of carotenoids on various transcription systems, such as the retinoid receptors (RAR, RXR) activator protein-1 (AP-1), peroxisome proliferator-activated receptors (PPAR), xenobiotic receptors, and antioxidant response element (ARE). Such regulatory properties are related to the anticancer activity of carotenoids, and the modulation of a network of transcription systems may provide the molecular basis for the synergistic anticancer effects [3]. The importance of b-carotene as a precursor of vitamin A has been established since many years, and the reader is referred to the extended literature. It should be noted that recent studies provide evidence that b-carotene has a higher affinity to the cleaving enzyme compared to other provitamin A compounds and appears to be the superior source for vitamin A. As mentioned above, fruits and vegetables are the major dietary sources of carotenoids. Although b-carotene and lutein are found in many different kinds of fruits and vegetables, only a few products contain other important carotenoids such as lycopene; 90% of dietary lycopene in the United States is derived from tomatoes and tomato products. An important source for b-carotene is carrot, but spinach, broccoli, or green and red peppers also contribute to human supply. Fruits contain considerable amounts of the provitamin A carotenoids b-carotene and b-cryptoxanthin, and it has been suggested that they are among the most important sources for vitamin A in developing countries. Carotenoids are also found in seafood including lobster and salmon. Commercially, astaxanthin is applied as a feed additive to obtain the typical color of salmon flesh. Further, large amounts of carotenoids, especially b-carotene, are chemically synthesized for use in the food or feed industry. Absorption of carotenoids from dietary sources follows the lipid pathway. In the small intestine, ingested carotenoids are incorporated into micelles. Micelles are 376j 23 Carotenoids and Vitamin A

formed from dietary lipids and bile acids that facilitate absorption of carotenoids into the intestinal mucosa cell. Thus, the intestinal uptake of these compounds is improved by the coingestion of oil, margarine, or butter. The intact carotenoids are incorporated into chylomicrons that are released into the lymphatic system. In blood plasma, carotenoids appear initially in the chylomicron and VLDL fraction, whereas their levels in other lipoproteins such as LDL and HDL rise at later time points with peak levels at 24–48 h. The major vehicle of hydrocarbon carotenoids is the LDL, while the more polar oxocarotenoids are found in LDL and HDL. Carotenoids accumulate in human tissues. Interindividual differences in carotenoid levels were reported, but high levels of lycopene and b-carotene were always found in liver, adrenals, and testes, whereas other tissues such as lung and kidney contained less.

23.3 b-Carotene – Cancer Prevention

The measure of carotenoids most widely used in studies on cancer is the b-carotene concentration in serum or plasma, whose range in cohort studies is 17–141 mg/dl (0.32–2.63 mmol/l) in healthy humans [4]. The concentrations of carotenoids in blood and tissue are markers of exposure to carotenoids in the diet. Another way of estimating carotenoid exposure is obviously the quantitative measurement of dietary intake, though the reliability of the latter is largely dependent upon data collection method and is still a matter of debate. Problems in estimating exposure by means of measurement of carotenoid plasma levels include (a) the inadequacy of circulating levels to represent tissue concentrations, (b) the ability of plasma carotenoid levels to reflect recent dietary intake rather than exposure over decades, (c) individual differences in the metabolism and kinetics of carotenoids, and (d) carotenoid alterations as a consequence of blood sample handling and storage. Additional factors influencing serum carotenoid concentrations and presumably also those of tissues include dietary intake of carotenoids, fat content of the diet, acidic fiber content of the diet, smoking habit, alcohol consumption, and food processing. The strength of the association between carotenoid exposure and cancer risk may depend on the balance between the amount of carotenoids available and the extent of the exposure to other risk factors, such as cigarette smoking. Various foods, nutrients, and dietary patterns may also confound the relationship between carotenoid measures and disease. Intake of carotenoids, for example, is strongly associated with the intake of several potentially protective nutrients, such as fiber, vitamin E, vitamin C, and flavonoids. This implies that carotenoid levels may simply be markers of fruit and vegetable intake. This issue also hinders clear-cut conclusions on early and more recent observational data, suggesting cancer preventive effects of b-carotene against lung (20–50% lower risk for the highest b-carotene intake or plasma concentration), oral and pharyngeal cancers [5], the incidence of which tended to be inversely related to b-carotene (or provitamin A carotenoids) intake or blood concentrations (reviewed in Ref. [4]). According to the results of cohort studies and case-control studies, 23.4 Lycopene – Cancer Prevention j377 investigating dietary and serum b-carotene against esophageal cancer, foods containing b-carotene seem to be protective [5]. Using the primary data from 11 cohort studies carried out in North America and Europe, M€annisto€ and coworkers [6] analyzed the association between carotenoid intakes estimated from food frequency questionnaires and the incidence of colorectal cancer during 6–20 years of follow-up of healthy subjects. The calculation of multivariate relative risks of over 7800 incident cases of colorectal cancer diagnosed among over 700 000 participants showed no association of intakes of specific carotenoids with colorectal cancer risk. Comparison of the highest quintile of intake with the lowest showed relative risks ranging from 0.92 for lutein plus zeaxanthin to 1.04 for lycopene [6]. Recent prospective studies and meta-analyses show that the association between prediagnostic serum carotenoids including b-carotene and prostate cancer risk is absent [7]. High serum b-carotene concentrations appeared to be associated with increased risk of aggressive, clinically relevant prostate cancer [7]. Finally, the overview of the results from cohort and case- control studies addressing the role of b-carotene against skin cancers reveals scarce evidence that foods containing b-carotene protect against the risk of nonmelanoma skin cancers [5]. No clinical trial of b-carotene as a single agent has shown a risk reduction of any cancer type, despite evidence indeed being available for the increase in the risk of lung cancer among smokers and asbestos workers receiving b-carotene supplements at high doses (resulting in 10–15-fold blood concentrations higher than normal) [8, 9]. Of the five randomized controlled trials and one cohort study investigating b-carotene supplements and lung cancer, four studies showed increased risk with b-carotene intervention (reviewed in Ref. [5]). A pooled analysis of three of the four trials showed a relative risk of 1.10 for supplemented versus nonsupplemented subjects [5]. A review of the three randomized controlled trials and two cohort studies on the effects of b-carotene supplementation against prostate cancer shows no evidence of either protection or increased risk (reviewed in Ref. [5]).

23.4 Lycopene – Cancer Prevention

Lycopene, a carotenoid consumed primarily from tomatoes, watermelons, and few other plant-based foods, is a promising chemopreventive agents in the diet. Lycopene is the most potent carotenoid antioxidant, has an antiproliferative effect, reduces plasma low-density lipoprotein cholesterol, improves immune function, and reduces inﬂammation. Lycopene is the major carotenoid found in the human prostate gland and is not a provitamin A carotenoid but yet possesses a powerful antioxidant activity as a singlet oxygen quencher [10]. Prostate cancer is second only to lung cancer as the cause of cancer-related deaths in American men and is responsible for over 29 000 deaths per year. There is a paucity of reliable data on DNA damage in prostate tissue, despite the evidence suggesting that a single serving of tomatoes or tomato products ingested daily may contribute to protect from DNA damage [11]. As DNA damage seems to be involved in the 378j 23 Carotenoids and Vitamin A

pathogenesis of prostate cancer, the regular ingestion of tomatoes or tomato products, however, might prevent the disease. Epidemiological studies have shown an inverse association between dietary intake of tomatoes, tomato products, lycopene, and prostate cancer [12]. On the contrary, lycopene and other carotenoids have been recently shown to be unrelated to prostate cancer [7], suggesting that lycopene or tomato-based regimens may not be effective for prostate cancer prevention. In addition, the FDA recently published a drastic report on the absence of credible scientiﬁc evidence to support an association between lycopene intake and a reduced risk of prostate, lung, colorectal, gastric, breast, ovarian, endometrial, or pancreatic cancer as well as for an association between tomato consumption and a reduced risk of lung, colorectal, breast, cervical, or endometrial cancer [13]. As far as lycopene and prostate cancer are concerned, this picture, however, could be much different when looking at cancer patients. Small clinical studies with cancer patients, in fact, showed that increased lycopene consumption before prostatectomy inhibits the progression of the disease, also lowering prostate-speciﬁc antigen (PSA) levels as well as DNA damage in leukocytes and prostate cells (for review, see Ref. [14]).

23.5 Other Carotenoids – Cancer Prevention

Cohort studies and case-control studies on total serum carotenoids and total dietary carotenoids, respectively, as well as case-control studies on provitamin A carotenoids, serum and dietary a-carotene, investigated their role in the prevention of mouth/ pharynx/larynx cancers (reviewed in Ref. [5]). Overall, there is consistent evidence of a protection of the above-cited compounds against oral cancers, with a dose–response relationship [5]. Cohort and case-control studies investigating total dietary carotenoids and serum or plasma carotenoids as well as dietary and serum b-cryptoxanthin in the prevention of lung cancer provide substantial evidence of a protection, with a clear dose–response relationship, while according to the results of cohort and case- control studies on dietary and serum total carotenoids against esophageal cancer, there is not sufﬁcient evidence in favor of a protective effect. Recent prospective studies and meta-analyses show that the association between prediagnostic serum carotenoids (lycopene, a-carotene, b-carotene, b-cryptoxanthin, lutein, and zeaxanthin) and prostate cancer risk is absent [7, 13].

23.6 Retinoids – Biological Properties

The term vitamin A comprises all C-20-b-ionone derivatives that exhibit a biological activity such as that of all-trans retinol. Biologically important oxidation products of retinol are retinal and retinoic acid that occur in several isomeric forms such as 11-cis retinal, 9-cis, or all-trans-retinoic acid. Vitamin A and its derivatives are essential to 23.6 Retinoids – Biological Properties j379 processes such as vision, immune function, reproduction and maintenance of epithelial tissue, or differentiation. Deficiency is associated with night blindness, loss of vision, growth retardation, fetal reabsorption, and immunodeficiency. Retinol itself is rarely found in foods. The major dietary sources of vitamin A are provitamin A carotenoids (see above) and retinyl esters, which are present in liver, milk, and various meat products. Retinol is absorbed by the intestinal mucosa cells and bound to the cytosolic retinol binding protein II (CRBP II). CRBP II apparently facilitates intestinal vitamin A trafficking and metabolism. After esterification with long-chain fatty acids, retinyl esters are packaged into chylomicrons, transported to the liver, and stored there in the form of fatty acid esters. The stellate cells of the liver are the main storage site of retinyl esters in the body. Vitamin A can be remobilized upon cleavage of retynil esters and is bound to retinol binding protein (RBP), the specific transport protein present in plasma. Each mole of retinol binds equimolar with RBP to form holo-RBP that further binds to a molecule of transthyretin. The retinol–RBP–TTR complex is not filtered by the glomerulus and circulates in the plasma. Tissues are then able to take the retinol up as needed via cellular retinoid binding protein. The plasma levels of retinol, respectively, holo-RBP are homeostatically controlled, and plasma concentration of retinol decrease significantly only when the liver reserves are almost depleted. Most of the cellular activities of vitamin A important for the control of proliferation are mediated by its oxidation products all-trans-retinoic acid and various cis-retinoic acid isomers. Retinoic acid is believed to be synthesized by the cells, as a needed process, which involves intracellular proteins that regulate the amount of retinoic acid produced. Specific proteins also help to determine the intracellular usage and trafficking of the compound within the cell. In the nucleus, retinoid signaling is transduced by two groups of nuclear receptors, the retinoic acid receptor (RAR) family and the retinoid X receptor (RXR) family. Each family, RAR and RXR, includes three isotypes designated as a, b, and g that are encoded by three different genes. The ligand-dependent receptors form various dimers, and the ligand–receptor complexes act as inducible transcription factors selectively binding to retinoic acid response elements (RAREs) located in the promoter regions of target genes. Natural ligand of the RAR receptor family is all-trans-retinoic acid. Both, RARs and RXRs, are activated by 9-cis retinoic acid, though it has recently been debated whether this compound is a natural ligand. The retinoic acid receptors function either directly on retinoid response elements or indirectly by modifying the responses of other transcription factors, and it has been shown that they are involved in the regulation of the expression of several hundred genes. However, the role of all-trans retinoic may extend beyond the regulation of gene transcription because a large number of noncoding RNAs are also modulated by retinoic acid. In addition, extranuclear mechanisms of action of retinoids are operative. It should be noted that synthetic derivatives of vitamin A exhibit a variety of biological effects that may have implications in cancer chemoprevention and therapy. On the basis of their structure, they act either agonistic or antagonistic compared to the natural congeners. 380j 23 Carotenoids and Vitamin A

23.7 Retinoids – Cancer Prevention

In a recent meta-analysis of eight prospective studies on over 430 000 healthy subjects followed up for 6–16 years, no association was found between vitamin A intake from food only and the risk of lung cancer (multivariate relative risk ¼ 0.96) [15]. Two randomized controlled trials investigated retinol supplements and skin cancer in selected participants at risk of developing nonmelanoma skin cancer, showing relative risks of 1.10 for basal cell carcinoma and 0.93 for squamous cell carcinoma (reviewed in Ref. [5]). In the b-Carotene and Retinol Efficacy Trial (CARET), 25.000 IU of retinyl palmitate was given in combination with 30 mg b-carotene, and, as mentioned above, a statistically significant increased risk of all lung cancers in the treated subjects was shown [9]. In another trial exploring without patient randomization the effects of retinol or b-carotene supplements against lung cancer in asbestos-exposed subjects, the adjusted effect estimate was of 0.67 in supplemented subjects compared to nonsupplemented subjects [16]. The amount of retinoic acids in the diet is very small, in the range 10–100 mg/day. All-trans-retinoic acid is derived from retinol by oxidation of the C-15 alcohol group to a carboxylic acid. Because of its acidic nature, it is more water soluble than retinol or retinal but still poorly so. The concentration of all-trans-retinoic acid in the plasma of fasting individuals is 4–14 nmol/l. Most tissues of the body contain all-trans-retinoic acid at concentrations of 40–6000 pmol/g wet weight. The concentration of all-trans-retinoic acid is <1% that of all-trans retinol in human plasma and <5% that of total vitamin A in the tissues of healthy animals and humans, and all-trans-retinoic acid is present only in traces in plants. Therefore, all-trans-retinoic acid is a very minor constituent of the diet. Some foods, such as dairy products, sugar, and comestible oils, have been fortified with vitamin A but not with all-trans- retinoic acid, which is not available as a dietary supplement (unlike vitamin A and carotenoids) [17]. Because all-trans-retinoic acid is rapidly metabolized in the body and is not stored in the liver or other organs, it does not accumulate over time. Recent studies suggest that in both subjects without complaints of uterine cervical lesions and the HPV-6/11, 16/18 infected cases of low- grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and invasive cancers, there is a high negative association between serum all-trans-retinoic acid levels and different grades of cervical lesions. Both retinoids and carotenoids inhibit experimentally induced carcinogenesis in the postinitiation phase, consistent with an inability to arrest the progression of preneoplastic cells to fully transform cells. In both cases, activity is tightly correlated with the ability of retinoids and carotenoids to upregulate expression of connexin 43 and thus increases gap junctional communication. This action is consistent with studies of human neoplasia and preneoplasia, in which downregulated expression of connexin 43 is an early event. Retinoids act via interaction with retinoic acid nuclear receptors, whereas there is evidence that carotenoids may activate PPAR receptors. According to the mechanisms of action of retinoids cited above, the latter have been shown to be potent modulators of epithelial cell growth and differentiation and 23.7 Retinoids – Cancer Prevention j381 active indeed against carcinogenesis in animal models and humans with tumoral lesions [18]. The amount of all-trans-retinoic acid ingested in the diet, as mentioned above, poses neither a benefit nor a risk, and the exposure to all-trans-retinoic acid is limited to the oral or topical treatment of clinical disorders. The maximum oral dose is 2 mg/kg body weight per day. At these doses, administration of all-trans-retinoic acid (for the treatment of leukemia, for instance) is embryotoxic and teratogenic [17]. The adverse effects shown by oral supplementation of all-trans-retinoic acid at low doses (20–30 mg/day) include dry oral and nasal mucous membranes [17]. Invasive cervical carcinoma is preceded by a precancerous phase, cervical intraepithelial neoplasia (CIN), which can be detected on cervical smears and confirmed by colposcopy and biopsy. Moderate and severe intraepithelial neoplasia (CIN2 and CIN3) are treated mainly with surgery to prevent progression to invasive carcinoma, while nonsurgical methods of preventing the progression or inducing regression of CIN are urgently needed. Randomized controlled trials (RCTs) and non-RCTs comparing the efficacy of four different retinoids [N-(4-hydroxyphenyl)retinamide (fenretinide), 9-cis-retinoic acid, all-trans-retinoic acid 13-cis-retinoic acid (isotretinoin)] for treating CIN in women show that the retinoids studied are not effective at causing regression of CIN3 but may have some effect on CIN2, while the data on CIN1 is inadequate. On the basis of this meta-analysis, in addition, the retinoids studied are reasonably well tolerated but are not effective at preventing progression of CIN of any grade. In the attempt of identifying the association of serum all-trans- retinoic acid levels with the proliferation status in terms of proliferating cell nuclear antigen expression as it is an antiproliferation agent and with the grades of cervical lesions, a highly significant association was observed for the proliferating cell nuclear antigen expression with the grades of cervical lesions. Acute promyelocytic leukemia (APL), characterized by a translocation between the promyelocytic leukemia gene (PML) on chromosome 15 and the retinoic acid receptor-a gene on chromosome 17, has become a model for targeted treatment of cancer. Differentiation therapy with all-trans-retinoic acid in combination with chemotherapy has significantly improved survival in patients with APL, both in children and adults. The first use of chemotherapy was unsuccessful due to lack of supportive care and cytotoxic agent-related exacerbated coagulopathy. The first breakthrough came from the use of anthracyclines that improved the complete remission rate, though the 5-year overall survival could be attained only in a small proportion of patients. Inducing the differentiation of APL cells rather than killing them by use of all-trans-retinoic acid in treating APL resulted in terminal differentiation of APL cells and a 90–95% complete remission rate of patients, thereby turning differentiation therapy in cancer treatment. The combination of all-trans-retinoic acid with chemotherapy further improved the 5-year overall survival especially after the introduction of arsenic trioxide. Importantly, both all-trans-retinoic acid and arsenic trioxide trigger catabolism of the PML–RAR-a fusion protein, which is the key player in APL leukemogenesis. Hence, in treating APL both all-trans-retinoic acid and arsenic trioxide represent paradigms for molecularly targeted therapy. All-trans- retinoic acid has also been used successfully in differentiated therapy of skin cancer, Kaposis sarcoma, and cutaneous T-cell lymphoma, but its usefulness is limited by 382j 23 Carotenoids and Vitamin A

the rapid emergence of acquired all-trans-retinoic acid resistance involving multi- factoral mechanisms. A key mechanism of resistance involves all-trans-retinoic acid-induced catabolism of all-trans-retinoic acid. Novel strategies to overcome this important limitation of exogenous all-trans-retinoic acid therapy involve the inhibition of the cytochrome P-450-dependent all-trans-retinoic acid 4-hydroxylase enzymes responsible for all-trans-retinoic acid metabolism. These inhibitors are also referred to as retinoic acid metabolism blocking agents. Fenretinide, mentioned above as an agent preventing CIN progression, is an aminophenol-containing synthetic retinoid derivative of all-trans-retinoic acid. Clini- cal studies of fenretinide have shown side effects consisting of night blindness and ocular toxicity, but a new molecule, p-dodecylaminophenol, has been produced to maintain the anticancer activity without side effects. p-Dodecylaminophenol has been shown to inhibit cell growth in human prostate cancer cell lines progressively from low to high concentration in a dose-dependent manner,both the i.v.andi.p. administration suppressed tumor growth in mice in vivo, and showed no effects on blood retinol concentrations, in contrast to reductions after fenretinide administration. Finally, studies of isotretinoin, etretinate, and acitretin have been shown to be effective chemotherapeutic agents in studies of patients with xeroderma pigmentosum, the nevoid basal cell carcinoma syndrome. Patients actively developing large numbers of new skin cancers may also beneﬁt from this approach. Although isotretinoin and acitretin share overlapping toxicities, there are differences that may affect drug choice. Because low doses may be effective, there are advantages to beginning the treatment at a low dose, and subsequently, increasing the dose if necessary, based on patient response. Due to the fetotoxicity and teratogenicity mentioned above, laboratory monitoring including pregnancy testing should be performed before and during treatment. Long-term toxicity, primarily involving the skeletal system, can be monitored with imaging studies.

References

1 Linseisen, J., Rohrmann, S., Miller, A.B., Carotenoids and transcriptions. Bueno-de-Mesquita, H.B., Buchner,€ F.L., Archives of Biochemistry and Biophysics, Vineis, P., Agudo, A., Gram, I.T. et al. 430,89–96. (2007) Fruit and vegetable consumption 4 International Agency for Research on and lung cancer risk: updated information Cancer (1998) IARC Handbooks of Cancer from the European Prospective Prevention; Carotenoids, Vol. 2, Oxford Investigation into Cancer and Nutrition University Press, Oxford, UK. (EPIC). International Journal of Cancer, 121, 5 World Cancer Research Fund 1103–1114. International (2007) Food, Nutrition, 2 Stahl, W., Ale-Agha, N. and Polidori, M.C. Physical Activity and the Prevention of (2002) Non-antioxidants properties of Cancer: A Global Perspective, American carotenoids. Biological Chemistry, 383, Institute of Cancer Research, 553–558. Washington, DC. 3 Sharoni, Y., Danilenko, M., Dubi, N., 6 M€annisto,€ S., Yaun, S.S., Hunter, D.J., Ben-Dor, A. and Levy, J. (2004) Spiegelman, D., Adami, H.O., Albanes, D., References j383

van den Brandt, P.A., Buring, J.E. et al. of prostate cancer: do we have the evidence (2007) Dietary carotenoids and risk of from intervention studies? Current colorectal cancer in a pooled analysis of 11 Opinion in Clinical Nutrition and Metabolic cohort studies. American Journal of Care, 9, 722–727. Epidemiology, 16, 246–255. 12 Giovanucci, E. (1999) Tomatoes, tomato- 7 Peters, U., Leitzmann, M.F., Chatterjee, based products, lycopene, and cancer: N., Wang, Y., Albanes, D., Gelmann, E.P., review of the epidemiologic literature. Friesen, M.D., Riboli, E. and Hayes, R.B. Journal of the National Cancer Institute, (2007) Serum lycopene, other carotenoids, 17, 317–331. and prostate cancer risk: a nested case- 13 Kavanaugh, C.J., Trumbo, P.R. and control study in the prostate, lung, Ellwood, K.C. (2007) The U.S. Food and colorectal, and ovarian cancer screening Drug Administrations evidence-based trial. Cancer Epidemiology, Biomarkers & review for qualiﬁed health claims: Prevention, 16, 962–968. tomatoes, lycopene, and cancer. Journal of 8 Virtamo, J., Pietinen, P., Huttunen, J.K., theNationalCancer Institute, 99,1074–1085. Korhonen, P., Malila, N., Virtanen, M.J., 14 Rao V. (ed.) (2006) Tomatoes, Lycopene and Albanes, D., Taylor, P.R., Albert, P. and Human Health; Preventing Chronic ATBC Study Group (2003) Incidence of Diseases, Caledonian Science Press. cancer and mortality following alpha- 15 Cho, E., Hunter, D.J., Spiegelman, D., tocopherol and beta-carotene Albanes, D., Beeson, W.L., van den Brandt, supplementation: a postintervention P.A., Colditz, G.A., Feskanich, D., Folsom, follow-up. Journal of the American Medical A.R. et al. (2006) Intakes of vitamins Association, 23, 476–485. A, C and E and folate and multivitamins 9 Goodman, G.E., Thornquist, M.D., and lung cancer: a pooled analysis of 8 Balmes, J., Cullen, M.R., Meyskens, F.L., prospective studies. International Journal of Jr, Omenn, G.S., Valanis, B. and Williams, Cancer, 118, 970–978. J.H., Jr (2004) The Beta-Carotene and 16 Musk, A.W., de Klerk, N.H., Ambrosini, Retinol Efﬁcacy Trial: incidence of lung G.L., Eccles, J.L., Hansen, J., Olsen, N.J., cancer and cardiovascular disease Watts, V.L., Lund, H.G., Pang, S.C., Beilby, mortiality during 6-year follow-up after J. and Hobbs, M.S. (1998) Vitamin A and stopping beta-carotene and retinol cancer prevention I: observations in supplements. Journal of the National workers previously exposed to asbestos at Cancer Institute, 96, 1743–1750. Wittenoom, Western Australia. 10 Di Mascio, P., Devasagayam, T.P., Kaiser, International Journal of Cancer, 75,355–361. S. and Sies, H. (1990) Carotenoids, 17 IARC (1999) IARC Handbooks of Cancer tocopherols and thiols as biological singlet Prevention; Retinoids, Vol. 4, Oxford molecular oxygen quenchers. Biochemical University Press, Oxford, UK. Society Transactions, 18, 1054–1056. 18 Fields, A.L., Soprano, D.R. and Soprano, 11 Ellinger, S., Ellinger, J. and Stehle, P. K.J. (2007) Retinoids in biological control (2006) Tomatoes, tomato products and and cancer. Journal of Cellular Biochemistry, lycopene in the prevention and treatment 102, 886–898. j385

24 Selected Vitamins

24.1 Vitamin C Pavel Kramata and Nanjoo Suh

24.1.1 Introduction

Vitamin C is an essential nutrient synthesized by plants and most animals but not by humans, primates, guinea pigs, and several other organisms. Insufficient dietary intake of vitamin C results in scurvy or scorbutus, a Latin term used in the scientific name of vitamin C, ascorbic acid. The symptoms of scurvy include purple spots on the skin, bleeding from mucous membranes, spongy gums, and the loss of teeth, and if not treated, the disease is fatal. The most common victims of scurvy were sailors and soldiers of the past who did not have access to fresh fruits and vegetables for long periods of time, usually more than a month. The disease was successfully treated by many native cultures, but it was first proven to be preventable by citrus fruit in 1747. The antiscorbutic principle was identified and named ascorbic acid in 1932 by Albert Szent-Gyorgyi who won the Nobel Prize for medicine in 1937. The compound was chemically synthesized in 1934. The minimum dose required for the prevention of scurvy is approximately 10 mg/ day. Although fruits and vegetables are the most abundant source of vitamin C (Table 24.1), the animal liver and brain also contain this vitamin in significant amounts. The Food and Nutrition Board of the United States National Academy of Sciences recommends a vitamin C daily allowance of 75 mg for women and 90 mg for men with additional 35 mg for smokers. The upper intake level has been set to 2000 mg, but many individuals take regularly much higher daily doses from dietary supplements. The best-known advocate of large doses of vitamin C for the prevention of many diseases including cancer was Linus Pauling, the Nobel Prize double laureate, chemist, and peace activist.

Table 24.1 Vitamin C content in fruits and vegetables.

Vitamin C Vitamin C a a Fruit (mg/kg fresh wet wt) Vegetable (mg/kg fresh wet wt)

Apple (green) 60 Broccoli 870 Banana 110 Cabbage 490 Grapefruit 360 Carrot 60 Grape (green) 30 Cauliﬂower 430 Kiwi 590 Celery 80 Lemon 580 Garlic 170 Mango 370 Lettuce (iceberg) 30 Orange 540 Onion 50 Pear 60 Potato 70 Pineapple 120 Spring onion 260 Plum 40 Tomato 170 Strawberry 770 Turnip (green) 170

Data taken from Ref. [38]. aAscorbic acid and dehydroascorbic acid.

24.1.2 Physicochemical Properties

Vitamin C (L-ascorbic acid, Scheme 24.1) is a white crystalline solid highly soluble in water (33 g/100 ml at 20 C). Under physiological conditions, except in the gastric

juice, vitamin C is present as a monoanion (ascorbate) with a pKa value of 4.25.

Scheme 24.1 Oxidation of ascorbate by two successive, reversible one-electron oxidation steps and irreversible degradation of dehydroascorbate. (Adapted from Ref. [2].) 24.1 Vitamin C j387

Ascorbate can function as a reducing agent donating one or two electrons, forming ascorbyl free radical and dehydroascorbate, respectively (Scheme 24.1). Both the products can be recycled back to ascorbate. If not reduced, dehydroascorbate is hydrolyzed irreversibly to 2,3-diketogulonate (Scheme 24.1) [1]. Ascorbate is an essential cofactor of enzymes participating in diverse metabolic pathways, including the synthesis of collagen, carnitine, and several hormones (epinephrine, norepinephrine, and neuropeptides), and in the reduction of iron, copper, and other metals [1]. The symptoms of scurvy result from a decreased activity of these enzymes. Besides its role in enzymatic reactions, ascorbate acts as a potent antioxidant in nonenzymatic reactions in biological ﬂuids scavenging reactive oxygen and nitrogen species that include free radicals (hydroxyl, peroxyl, superoxide anion, and nitrogen dioxide), antioxidant-derived radicals (a-tocopheroxyl radical), nonradical species (hypochlorous acid, ozone, singlet oxygen, nitroxide, and peroxynitrite), and others [2]. Since ascorbate is sufﬁciently reactive and the ascorbyl free radical is relatively stable, the radical replaces highly reactive and potentially harmful species in the reduction process. This is considered critical for the prevention of many human degenerative diseases including cancer [2]. In the presence of metal ions, vitamin C can act as a pro-oxidant in vitro, inducing the formation of reactive hydroxyl radicals from hydrogen peroxide or lipid hydroperoxides:

þ þ þ ascorbate þ Fe3 ! ascorbyl radical. þ Fe2 þ H

2 þ . 3 þ H2O2 þ Fe ! HO þ Fe þ HO

Although the low availability of catalytic metal ions sequestered by various metal binding proteins questions the relevance of the reaction and the role of vitamin C as pro-oxidant in vivo [2], transition metal ion-independent decomposition of lipid hydroperoxide mediated by vitamin C in vitro has been reported [3].

24.1.3 Bioavailability and Metabolism

Vitamin C ingested predominantly as ascorbic acid is absorbed from food by the epithelial cells of the small intestine into the surrounding blood capillaries. Circulating ascorbate is filtered in the kidney and partially reabsorbed into the blood stream through renal epithelial cells. The difference between the amount of ascorbate filtered and the amount reabsorbed constitutes renal excretion. Bioavail- ability of orally administered vitamin C is thus determined by the difference between intestinal absorption and renal excretion and depends on the dose [4]. Adrenal and pituitary glands accumulate the greatest quantity of ascorbate (30–50 mg/100 g) followed by liver, spleen, eye lens, pancreas, kidney, and brain (10–30 mg/100 g). The loss of ascorbic acid from a theoretical total body pool has been estimated at 2.6–4.1% per day, but the loss in specific tissues may vary [5]. 388j 24 Selected Vitamins

The mean blood plasma concentrations of ascorbate in humans from 30, 100, and 200 mg oral daily doses of vitamin C at steady state were 9, 56, and 75 mM, respectively, revealing sigmoidal relationship with a steep portion of the curve between 30 and 100 mg doses. Ascorbate begins to appear in urine at daily doses above 100 mg and most of the absorbed ascorbate is excreted in urine at the 500 mg dose. Decreased bioavailability keeps plasma levels below 100 mM even at a 1000 mg oral dose [6]. In contrast, vitamin C can reach as much as 70-fold higher plasma levels when administered intravenously [7]. Intravenous administration of very high doses of vitamin C (up to 60 g) has been used for cancer treatment [8]. Ascorbate and the oxidized form, dehydroascorbate, are transported across cellular membranes by two different systems [9]. Ascorbate is transported into the cell by sodium-dependent vitamin C transporters SVCT1 and SVCT2 whereas dehydroascorbate is transported by glucose transporters GLUT1, GLUT3, and GLUT4. The SVCT transporters have a higher afﬁnity for ascorbate than the GLUT transporters have for dehydroascorbate. SVCT1 is predominantly expressed in epithelial cells, including those of the intestine, kidney, and liver, and mediates high-capacity transport of ascorbate. SVCT2 is expressed in more specialized cells of the brain, eye, and placenta and has been found to maintain the intracellular level of ascorbate and provide protection against oxidative stress. Both transporter isoforms are subject to downregulation by ascorbate, which limits bioavailability of vitamin C from high oral doses. In addition, SVCT1 expression declines with age and may explain why elderly individuals require higher levels of dietary vitamin C than younger individuals to reach comparable concentrations of ascorbate in serum [4]. Transport of both ascorbate and dehydroascorbate contributes to ascorbate intracellular accumulation. Ascorbate recycling, a model developed with the use of isolated human neutrophils, explains intracellular regeneration of ascorbate from dehydroascorbate under conditions of increased oxidative stress [5]. In the extracellular space under oxidizing conditions, ascorbate is converted to dehydroascorbate, which is rapidly transported into cells by the glucose transporters and reduced back to ascorbate. The intracellular reduction is mediated by glutathione and thioredoxin/ glutaredoxin family of enzymes using NADPH as a source of reducing equivalents. The reduction produces an outside-to-inside dehydroascorbate gradient and is likely a driving force for transport of dehydroascorbate across cell membranes. Since the reduction is fast and the product exits cells at relatively slow rate, ascorbate can reach high (millimolar) intracellular concentrations. The dehydroascorbate transport is competitively inhibited by glucose; hyperglycemia may interfere with the process of ascorbate recycling in some cells and lead to a higher exposure to oxidative stress [5].

24.1.4 Mechanism of Protection: In Vitro and In Vivo Studies

Development of cancer is believed to result from changes in the sequence of DNA – mutations – that affect genes critical for the regulation of cell proliferation and death. DNA mutations are generated by incorrect repair or bypassing of DNA lesions formed by genotoxic agents. These agents are either exogenous environmental toxins 24.1 Vitamin C j389 or endogenous damaging molecules produced during normal cellular metabolism or pathological processes such as inﬂammation [10]. Although both groups of agents contribute to DNA damage in humans, it has been estimated that oxidative stress – a sustained attack of reactive oxygen species (ROS) on DNA – causes 104–105 hits per cell per day [2]. In addition, reactive molecules have the capacity to also modify lipids and proteins and change their biological functions that may lead to pathological conditions. Oxidative stress occurs in the cell when more free radicals and active intermediates are formed than eliminated by cellular redox homeostasis systems. Restoring the balance by decreasing the level of ROS with dietary components – antioxidants – is one of the most widely explored strategies in cancer prevention. As an antioxidant, vitamin C can diminish DNA, lipid, and protein oxidative damage by reducing the total levels of radicals and reactive species. Although a direct oxidative damage to DNA, both chromosomal and mitochondrial, is considered a major contributing factor in carcinogenesis, products of lipid and protein oxidation may also form DNA adducts or have an indirect effect on DNA mutagenesis by affecting the ﬁdelity of DNA replication and DNA repair [11]. Effects of vitamin C on biomarkers of DNA oxidation are discussed in more detail.

24.1.4.1 Vitamin C and Oxidative DNA Damage Oxidative damage to DNA affects predominantly the guanine base and induces the formation of mutagenic 8-oxo-7,8-dehydroguanine (8-oxoGua). The level of 8-oxoGua or its nucleoside derivative, 8-oxo-7,8-dehydro-20-deoxyguanosine (8-oxodG), is the most commonly measured biomarker of oxidative damage [12]; however, other DNA lesions are also formed [13]. The effect of vitamin C supplementation on biomarkers of oxidative DNA damage in humans has been measured in more than 20 studies (reviewed in Ref. [14]). Most studies assessed levels of 8-oxoGua, 8-oxodG, or strand breaks in DNA of white blood cells isolated from subjects exposed to oxidative stress (caused by smoking, environmental smoke) and treated with vitamin C or placebo. Alternatively, white blood cells collected from vitamin C-treated subjects were challenged with oxidative stress ex vivo. Most studies in humans have demonstrated an inverse correlation between plasma concentrations of vitamin C and levels of oxidized DNA base lesions. Results from some experiments showed protection of white blood cells against ex vivo oxidative insult, suggesting that vitamin C increases cellular antioxidant status. In contrast, there was no evidence that vitamin C can alter the levels of DNA strand breakage, but it is important to note that DNA breaks are not a speciﬁc biomarker of oxidative damage. Several studies found that a vitamin C-mediated decrease of oxidized DNA base lesions is linked to an increase of 8-oxoGua or 8-oxodG levels in plasma or urine. These results suggested that vitamin C does not block oxidative DNA damage but rather stimulates DNA repair and possibly acts as a pro-oxidant [14]. The pro-oxidant hypothesis in humans is controversial and should be supported by additional experimentation.

24.1.4.2 Vitamin C, Cell Proliferation, Signal Transduction, and Apoptosis In cell culture models, vitamin C induces cytotoxicity associated with auto-oxidation and production of the reactive hydroxyl radical from H2O2 in the presence of 390j 24 Selected Vitamins

transition metal ions in the medium [14]. Since the availability of catalytic metal ions is likely limited in vivo, these results, indicating a pro-oxidant activity of vitamin C, may not be physiologically relevant. However, it has been reported that high concentrations of vitamin C (0.3–20 mM) kill tumor but not normal cells in vitro

via ascorbate-mediated extracellular generation of H2O2 in the absence of transition metal ions, suggesting that H2O2 may itself be the toxic agent [4]. Although the selective sensitivity of tumor cells to H2O2 is not clear, the observation provides a potential mechanism of action for the antitumor effect of intravenously administered

high-dose vitamin C and implicates it as a vehicle for H2O2 delivery in cancer treatment [4]. Vitamin C modulates gene expression by affecting levels of reactive oxygen species that act as subcellular messengers in gene regulatory and signal transduction pathways. Vitamin C has been shown to inhibit the activation of transcription factors NF-kB and AP-1, stimulate the expression of a mismatch repair protein MLH1 and apoptosis-inducing proteins p53 and p73, and sensitize cells to apoptotic cell death induced by DNA-damaging agents such as UV light, cisplatin, or etoposide [14]. The effects of vitamin C on gene expression have also been demonstrated by measuring numerous differentiation markers, mainly in cells of mesenchymal origin and stem cells, and at least some appear to be speciﬁcally induced by ascorbate rather than by auto-oxidation products of vitamin C in vitro [14].

24.1.5 Human Studies

24.1.5.1 Epidemiological Studies A comprehensive research report of the World Cancer Research Fund on nutrition and cancer recently concluded that diets made up from fruits and nonstarchy vegetables probably decrease risk of development of some human cancers, particularly in the mouth, pharynx, larynx, esophagus, stomach, lung, pancreas, and colon, and that foods containing vitamin C have a preventive effect on the development of esophageal cancer [15]. Support for the conclusion about foods containing vitamin C and esophageal cancer was based on the analysis of results of 1 cohort, 19 case– control, and 3 ecological studies [15]. The importance of vitamin C in cancer prevention associated with diets is difficult to assess since fruits and vegetables also contain many other potentially protective antioxidants such as carotenoids, phenols, and flavonoids as well as other bioactive phytochemicals. A high level of vitamin C in blood may indicate a better protection of the organism from oxidative damage; alternatively, it may only serve as indicator of high consumption of fruits and vegetables and thus a generally healthier lifestyle with no protective function of the vitamin itself. Inverse correlation between levels of vitamin C in blood and overall mortality has been reported, but for cancer-related mortality, this trend was observed only among men [16, 17]. Numerous epidemiological studies have attempted to resolve this controversy by examining effects of vitamin C dietary supplements on cancer development in observational case–control and prospective cohort studies. In general, studies on 24.1 Vitamin C j391 cancers whose prevention has been positively associated with consumption of fruits and vegetables also tend to show positive results with vitamin C dietary supplements [18–22]. Results of observational cohort and case–control studies should be interpreted with caution since well-known limitations (reliance on questionnaires and their analysis, influence of many potentially confounding factors and behavior patterns linked to the use of vitamin supplements) may significantly affect outcomes of these studies. Intervention clinical trials are considered a more accurate and reliable measure of effects of micronutrients in cancer prevention, and their results provide the most convincing evidence regarding dietary supplements and cancer risk.

24.1.5.2 Clinical Studies Several large, randomized, placebo-controlled, double-blind clinical trials have been designed to assess efficacy of dietary supplements that include vitamin C for treatment and prevention of cancer, mainly cancer-related mortality, development of cancer or precancerous lesions, and progression of existing cancers. The largest randomized clinical trial reporting cancer-related mortality in conjunction with vitamin C dietary intervention to date, the Linxian Nutrition Interven- tion Trial, included 29 594 healthy adults living in an area with one of the worlds highest rates of esophageal and gastric cancers. A vitamin C (120 mg) and molybdenum supplement alone, or in combination with other vitamins, was taken by the study subjects daily for 5.25 years. The trial did not find a significant difference in cancer-related mortality between any of the treatment groups and a control group (reviewed in Ref. [23]). A risk of development of cancer in subjects receiving vitamin C supplements was assessed in several smaller trials. In a subgroup of the large prevention trial, a 6-year prospective study of 3318 adults with esophagus and/or gastric dysplasias, subjects received a daily supplement containing 180 mg of vitamin C along with other vitamins or placebo; no significant benefit of the vitamin treatment was observed. In another trial, progression of precancerous gastric lesions was followed for 3 years among 1980 subjects at high risk of gastric cancer receiving a daily dietary supplement of vitamin C (600 mg), vitamin E, and b-carotene or placebo in Venezuela; no statistically significant difference in cancer progression rate was observed between vitamin and placebo groups [24]. Another recent study found no effect of a 7.3-year twice-daily treatment with vitamin C (250 mg), vitamin E, and selenium for the prevention of precancerous gastric lesions that included 2956 adults [25]. Combina- tions of vitamin C (1000 or 400 mg daily) and vitamin E were tested for the prevention of colorectal adenoma, a precursor of invasive cancer, in two separate 4- and 2-year trials that included 751 and 137 adults, respectively; neither study reported results indicating efficacy of the treatments (reviewed in Ref. [23]). The French study SU.VI. MAX followed 12 741 adults for 7.5 years and reported a reduced rate of prostate cancer by 48% in men, with normal levels of prostate-specific antigen, and overall reduction of cancer incidence by 31% in men but not in women taking a combination of vitamin C (120 mg), vitamin E, b-carotene, selenium, and zinc (reviewed in Ref. [26]). The Physicians Health Study II, an ongoing large trial that has enrolled 392j 24 Selected Vitamins

15 000 male US physicians, is investigating effects of daily vitamin C (500 mg), vitamin E, b-carotene, and multivitamin supplementation, alone or in combination, on the risk of development of cancer. The study will be completed in 2008 [27]. Progression of existing cancers was monitored in a small clinical trial of 65 patients with previously treated transitional cell carcinoma of the bladder. The study found a 50% decrease in development of new tumors in patients treated for 10 months with high doses of vitamin C (2000 mg) and other vitamins compared to that in patients treated with the recommended daily allowance of these vitamins (reviewed in Ref. [23]). Two earlier small trials reported no effect of high doses of vitamin C (10 000 mg) for tumor progression in 123 and 100 patients with advanced colorectal, gastrointestinal, and lung cancers regardless of prior chemotherapy (reviewed in Ref. [28]). It is likely that inhibition of progression of existing tumors in cancer patients with altered drug metabolism and history of various cancer therapies requires a very different intervention than primary prevention of carcinogenesis in healthy subjects. In addition, therapeutic approach may require high levels of vitamin C in plasma that are impossible to reach with dietary supplementation. A nondietary approach to treating advanced cancers with intravenous applications of large doses of vitamin C (>10 000 mg) that could reach high plasma levels (70-fold higher than attainable by oral administration [7]) has been practiced [8] and recently showed some success [29]. A phase I study of high-dose intravenous vitamin C treatment in terminally ill patients with solid tumors is in progress [30]. The clinical trials appear to demonstrate only a marginal or no effect of supplemental vitamin C for cancer prevention. Although the results are disappointing, it would be premature to dismiss vitamin C as a human cancer-preventive agent. Only few well-designed studies have been performed to date; the size, design, and duration of the trial, unknown confounders, selection of population, compliance of the study subjects, supplement doses and combinations, and the selection of tested outcomes are only examples of factors that may negatively influence the study results [23, 28]. Large and sufficiently long clinical trials based on a systematic small-scale approach that would identify optimal combinations of the study parameters for preventing specific cancers should be performed before a positive role of vitamin C in the prevention of human cancer is ruled out.

24.1.6 Impact of Cooking and Food Processing on Protective Properties

Vitamin C is stable during storage of uncut fruits and vegetables [31] but very unstable during food processing; the extent of the loss depends on temperature and time of the treatment [32]. Pasteurization at 85 C for 8 min was found to decrease vitamin C content of strawberry juice by 30% compared to unpasteurized juice [33]. Leaching contributes to losses of vitamin C; cooking of broccoli in water significantly decreases the level of vitamin C whereas steam cooking has no influence [34]. Short-term deep fat frying or stir frying preserves vitamin C content better than pressure cooking, boiling, microwave cooking, or baking [32]. Dehydration is another process that dramatically reduces vitamin C content; prunes had 65 or 76% less vitamin C than 24.2 Vitamin D j393 plums after they were dried at 60 or 85 C [35]. It is worth noting, however, that drying significantly increases the overall antioxidant capacity of fruits and vegetables [36]. Vitamin C in food can be destroyed during chilling, chilled storage, portioning and distribution, reheating, and hot-holding, and the loss depends on the type of the food and specific parameters of the process [37]. The loss of vitamin C in intact vegetables after cooking is lower compared to the loss in mashed, chopped, or shredded products and increases with increasing time and temperature of storage. Reheating and hot-holding of food are major causes of deterioration of nutritional quality. In general, the greater the time needed for reheating and hot-holding, the greater the loss of vitamin C [37].

24.1.7 Acknowledgment

This work was supported by NIH Grants to N.S.

24.2 Vitamin D Heide S. Cross and Thomas Nittke

24.2.1 Introduction

24.2.1.1 How Much Vitamin D is Enough? Although vitamin D is not a true nutrient for most mammals, since it is primarily synthesized in skin cells in the presence of adequate sunlight providing UVB, vitamin D deficiency, and insufficiency, has become a well-recognized problem worldwide. In areas where sun exposure is prevented by sunscreen use, is limited above latitudes of 34 north or south or during winters, in populations with traditional clothing that covers most of the skin, and especially in migrants with darkly pigmented skin that move from southern to northern countries, lack of vitamin D precursor is widely evident. Also, in the elderly due to decreased capacity of synthesis in the skin in addition to little outdoors activity, low vitamin D levels are prevalent. Food sources are poor (primarily in fatty fish or fortified foods) and intake recommendations are too low since these are based on information gathered around lifestyle including outdoors activities that have ceased to exist in the general population. While, in humans, circulating vitamin D levels of 20–29 nmol/l serum will indeed prevent rickets, levels closer to 75–80 nmol/l are needed to support other physiological functions of vitamin D [39], especially those involved in limiting growth of cells and increasing differentiation. These functions will be subsequently described in detail. While vitamin D deficiency has been known to be causative in bone diseases, a recently recognized association of vitamin D insufficiency with risk of several types of cancer has drawn considerable attention. Vitamin D insufficiency may annually account for several thousand premature deaths from, for instance, colon cancer [40], 394j 24 Selected Vitamins

mammary cancer [41], and prostate cancer [42], to name only a few of the most frequent nonfamilial malignancies prevalent in Western industrialized countries. Dietary factors have long been recognized to modulate a variety of molecular targets not only for prevention of cancer but also for therapy, whereas the vitamin D connection has remained obscure until lately. Also, controversy was remarkable, although epidemiological data clearly identiﬁed low levels of serum 25-hydroxyvitamin D3 (25-D3), the immediate precursor of the active vitamin D metabolite, as an indicator of enhanced incidence of different malignancies. Clinical observations even suggested that optimal serum levels of 25-D3 at diagnosis improved disease-free survival time. While recommended exposure time of face and hands to the sun during summer is only 10–15 min to accumulate enough vitamin D precursor per day [43], this was reinterpreted by dermatologists to potentially cause a melanoma epidemic.

24.2.2 Vitamin D Synthesis

Vitamin D3 is synthesized from the sterol 7-dehydrocholesterol by UV irradiation in the upper skin layer and by heat in the lower layer. Transport of vitamin D3 from the skin to fatty tissue for storage or to the liver is carried out by the vitamin D binding protein (DBP). Activation of vitamin D3 occurs in the liver mainly by 25-hydroxylation by the mitochondrial cytochrome P-450-based enzyme CYP27A1, although microsomal cytochrome P-450 enzymes might also be involved [44]. The product of this hydroxylation is 25-D3, the major circulating form. 25-D3 binds with high afﬁnity to DBP and the circulating metabolite is thus protected from degradation. It is present in human plasma at concentrations in the range of 10–80 ng/ml (25–200 nmol/l), which is representative of the vitamin D status in vivo. In a recent paper, this wide range of human plasma concentration of 25-D3 was associated with a variety of human diseases, not only malignancies but also such diverse illnesses as diabetes, immune diseases, and infections [45].

24.2.3

Bioavailability and Metabolism of 1,25-(OH)2-D3

25-D3 is converted to the active steroid hormone 1,25-dihydroxyvitamin D3 (1,25-

(OH)2-D3; 1,25-D3) by 1a-hydroxylation. This occurs, to a major part, in proximal tubule cells of the kidney by another cytochrome P-450 enzyme, CYP27B1.While 25-D3 is present in human serum at nanomolar concentrations, normal 1,25-D3 levels are 1000-fold lower. Patients with chronic kidney disease exhibit reduced 1,25-D3 serum levels as well as frank rickets or osteomalacia since the endocrine function of the steroid hormone for calcium/phosphate homeostasis can no longer be maintained. As part of the calcium homeostatic loop, CYP27B1 is strongly downregulated by 1,25-D3 and upregulated by parathyroid hormone (PTH). In a negative feedback loop, vitamin D, when sufficiently available, destructs itself: another P-450 enzyme, CYP24A1, 24-hydroxylates and thereby inactivates 25-D3 24.2 Vitamin D j395 as well as 1,25-D3, the latter at a 10-fold higher efficiency. The renal enzyme CYP24A1 is expressed at high constitutive levels, but it is also present in extrarenal cells representing a variety of normal vitamin D target organs, such as intestinal and bone cells or keratinocytes. However, it was suggested that CYP24A1 is undetectable in vitamin D target cells unless exposed to 1,25-D3 by a vitamin D receptor (VDR)- mediated mechanism. Recently, it was demonstrated [46] that this is apparently not the case in human colon: during tumorigenesis expression of CYP24A1 mRNA is increased. Interestingly, in addition to renal cells there is widespread potential for extrarenal synthesis of 1,25-D3, since positivity for CYP27B1 was demonstrated by Zehnder et al. [47] in many types of cells (see also Figure 24.1). With colon cell hyperproliferation during premalignancy, expression of the synthesizing hydroxylase CYP27B1 is increased, as well as that of the VDR. Only during outright human malignancy in high-grade colon tumors, expression is again repressed, in contrast to that of CYP24A1 [48, 49]. These experimental data gave rise to the hypothesis that extra- renally synthesized 1,25-D3 could be a physiological mechanism that normally exists to prevent cancerous growth. This protection is somehow eliminated by unknown counteractive mechanisms that could include promoter methylation of CYP27B1 and promoter demethylation of CYP24A1 during tumor progression. An actual mechanistic link between 25-D3 serum concentrations and cancer incidence was not apparent yet. Only when the actual synthesis and degradation of 1,25-D3 in a variety of cancer cells, such as those derived from colon, breast, and prostate, was demonstrated by high-pressure liquid chromatography (HPLC) (see Ref. [50]), it became accepted that this steroid hormone could play alternative roles in human physiology due to localized tissue accumulation. It also solved the riddle why the incidence of malignancies rises with vitamin D insufficiency, at 25-D3 levels below 80 nmol/l serum, as an efficient extrarenal 1,25-D3 synthesis completely depends on high serum levels of the precursor. It should be considered as well that although mineral homeostasis is regulated by the picomolar serum concentrations of 1,25-D3, growth inhibition of cancer cells generally occurs only at a 1000-fold higher nanomolar concentration that could be reached due to localized CYP27B1 activity in tissues. A negative correlation of serum 1,25-D3 with tumor incidence has never been detected, not even at the highest physiological concentrations, whereas there is a clear negative correlation with 25-D3 levels. In addition, a recent human pilot study by Holt et al. [51] demonstrated, for the first time, that rectal crypt proliferation correlated inversely with serum 25-D3 levels.

24.2.4 Mechanisms of Protection

24.2.4.1 Vitamin D Signaling Pathways in Cancer Besides its classical role in mediating calcium and phosphate homeostasis, 1,25-D3 (see Figure 24.1) has nonclassical roles that include cell differentiation and antiproliferative activity, a positive effect on apoptosis and negative action on angiogenesis. 1,25-D3 exerts transcriptional activation and repression of target genes 396j 24 Selected Vitamins

Figure 24.1 Extrarenal and renal synthesis of 1.25-D3.

by binding to VDR. Studies on knockout mice deﬁcient in VDR and in key components of the vitamin D metabolic pathway demonstrated transcriptional effects in many target genes (for review see Ref. [52]). Although knockout clearly resulted in bone-related diseases, alopecia, infertility, growth retardation, and so on, none of the in vivo models deﬁcient in parts of the vitamin D system developed tumors spontaneously; however, incidence was more rapid, subsequently enhanced, when a carcinogen was added or when VDR knockout was coupled to tumor suppressor gene deactivation [53]. Only in the colon did VDR knockout in mice lead to hyperproliferation and an increasing level of a DNA damage marker [54]. 24.2 Vitamin D j397

VDR target gene expression is modulated through synergistic 1,25-D3 binding and dimerization with the retinoic X-receptor. This induces phosphorylation and conformational change of the VDR followed by the release of corepressors (nuclear receptor corepressors, NCoRs; silencing mediator for retinoid and thyroid hormone receptors, SMRT; histone deacetylase, HDAC) and repositioning of the VDR activation function 2 domains. The complex then binds within the promoter region to vitamin D response elements (VDREs) that are typically composed of two hexameric sequences organized as repeats interspaced by varying numbers of nucleotides. This initiates the recruitment of coactivators as prerequisite for the full transcriptional activation. Steroid receptor coactivators (SRCs), nuclear coactivator-62 kDa– Ski-interacting protein (NCoA62-SKIP), histone acetyltransferases (HATs), CREB binding protein (CBP)–p300, and polybromo- and SWI-2-related gene-1-associated factor (PBAF-SNF) acetylate histones and thereby mediates the open chromatin state. This facilitates the binding of the activation function 2 (AF2) of the nuclear receptor heterodimer to the vitamin D receptor-interacting protein 205 (DRIP205) and further to the mediator complex containing other DRIPs that bridges the nuclear receptor–coactivator complex with the basal transcription apparatus for transcription initiation (see Ref. [55]). Epigenetic events may play a major role in regulating the extrarenal vitamin D system: VDR-mediated signaling can be repressed by such mechanisms in prostate and mammary cancer cells. Kim et al. [56] demonstrated that 1,25-D3 itself could induce DNA methylation, thereby suppressing the CYP27B1 gene. Khorchide et al. [57] were able to show that in normal human prostate cells the CYP24A1 promoter was methylated and expression was suppressed, whereas this was not the case in highly malignant prostate cells that had high 1,25-D3 catabolizing activity. In kidney cells, induction of the CYP24A1 gene by 1,25-D3 is by far the strongest. In nonrenal cells, besides regulation of vitamin D hydroxylases, vitamin D exerts direct and indirect effects. For instance, mediators of cell cycle disruption such as certain cyclins and cyclin-dependent kinase inhibitors (p21, p27) are transcriptionally directly regulated by 1,25-D3/VDR. However, many cell cycle-regulating genes that are affected by 1,25-D3 do not contain VDREs and these are modulated due to cross- talk with other pathways affected by 1,25-D3: the downregulation of the epidermal growth factor receptor (EGFR) by 1,25-D3 is a good example of this mechanism. In Table 24.2, major target pathways of 1,25-D3 in relation to the cell cycle, apoptosis, the Wnt pathway (as part of epithelial–mesenchymal transition during progressing malignancy of epithelial cells), and differentiation are described.

24.2.5 In Vivo Studies with 1,25-D3 for Tumor Prevention or Treatment

24.2.5.1 Treatment While it is clear that the active vitamin D metabolite 1,25-D3 has to be supplied to patients at nanomolar doses to be effective for tumor treatment, and this will surely result in hypercalcemia, this may not be the case in animals – and a signiﬁcant inhibition of metastasis is observed in prostate and lung murine models treated with 1,25-D3 without adverse side effects (see Ref. [58]). Similar results were achieved with 398j 24 Selected Vitamins

Table 24.2 Major cancer-related signaling pathways targeted by 1,25-D3.

Anticancer effect Signaling event

Antiproliferative Induction of p27, p21, p15, GADD45a expression Activation of TGFb signaling Inhibition of Wnt-b-catenin-TCF4 pathway and telomerase Repression of SKP2, MYC, and EGF receptor expression Differentiation Induction of E-cadherin expression Apoptosis Induction of BAX, BAK Activation of IGF1R-PI3K-Akt pathway Downregulation of Bcl2 and Bcl-X(L)

These data obtained mainly by in vitro experiments demonstrate the antitumorigenic potential of 1,25-D3 exerted via direct (transcriptional activation by 1,25-D3 bound to VDR) and indirect actions (genes in question are not known to contain a VDRE). (For more details see Ref. [52].)

diverse cancer xenografts in rodents (see also Table 24.3). However, for clinical use of 1,25-D3 in tumor therapy, it will be essential to initially deﬁne a safe and effective clinical treatment regimen. For instance, employing a high dose, intermittent 1,25- D3 regimen could potentially result in antitumor activity with a lower toxicity proﬁle. Toavoid the problem of hypercalcemia in patients, a variety of analogues of 1,25-D3 were developed. The goal was either to obtain substances that are more effective than 1,25-D3, as antimitotic agents to be able to reduce the concentration applied, or to

Table 24.3 Activity of 1,25-D3 in animal tumor models.

Site of cancer/ animal model Treatment Anticancer effect Reference

Colon Apcmin 1,25-D3 Decrease in polyp number and [97] tumor load (sum of all polyp area) Breast MCF-7/VEGF and 1,25-D3 Antiangiogenesis; [98] MDA-435S involvement in endothelial xenografts cell apoptosis Prostate PC3 xenografts 1,25-D3 þ Antiproliferative Ahmed et al. mitoxantrone/ (2002) dexamethason Nkx3.1; Pten 1,25-D3 Delayed incidence of prostate [99] (mouse) Intraepithelial neoplasia Mat-Lylu (rat) 1,25-D3 Reduction of lung metastases [58]

Note: For brevity, animal models of pancreatic cancer, squamous cell carcinoma, and ovarian cancer were not described. Studies carried out with vitamin D analogues were excluded. MCF-7/VEGF, MCF-7 breast cancer cells transfected with vascular endothelial growth factor (VEGF). 24.2 Vitamin D j399 have analogues with a lower hypercalcemic potential. This can be obtained by modifying, for instance, the side-chain structures around the C-24 position. Numer- ous analogues have been synthesized so far, but the one effective for several types of tumors has not been obtained yet. Another potentially more successful approach could be combination therapy. 1,25-D3 acts synergistically with some chemotherapeutic agents. It potentiates the activity of platinum analogues, taxanes, and DNA-intercalating agents. Optimal enhancement is seen when 1,25-D3 is administered before or simultaneously with chemotherapy, never when afterward [59]. Such increased antitumor effects with 1,25-D3 combination therapy offers opportunities for clinical use of the steroid hormone across several tumor types, where modest effects are observed with chemotherapy alone (for more details, see Table 24.4).

24.2.5.2 Tumor Prevention Several meta-analyses and prospective studies (see Ref. [60]) indicate that vitamin D can be considered a tumor progression-preventive steroid hormone. Increasing the efﬁciency of the vitamin D system in a targeted manner could conceivably prevent either premalignancies or their progression to clinically manifest primary tumors. This may be achieved by nutritional factors such as calcium, soy, and folate, especially in the colon. A high percentage of an apparently healthy population is deﬁcient both in vitamin D and in calcium. Nutritional calcium intake is inversely associated with colorectal cancer incidence. Risk reductions are in the range of 15–40% for the highest versus the lowest intake categories. There is increasing evidence that calcium and vitamin D act largely together in controlling colon epithelial cell proliferation. In the Polyp Prevention Study Group Trial, supplementation of calcium with vitamin D during follow-up was inversely associated with adenoma recurrence, and even more so with multiple recurrences [61]. An interaction between nutritional calcium and vitamin D in protection against colorectal cancer may be due to the ability of luminal calcium to suppress degradation of 1,25-D3 synthesized in colonocytes – CYP24A1 expression was doubled in mice fed low dietary calcium, and the expression of the cyclin-dependent kinase inhibitor p21 was reduced by 50%. This could lead to a reduced colonic accumulation of the antimitotic prodifferentiating 1,25-D3 and hyperproliferation [62]. Reduced incidence not only of hormone-related cancers such as mammary and prostate tumors but also of colon tumors has been linked to the consumption of a typical Asian diet containing soy. Soy and red clover are prominent sources of phytoestrogens that bind preferentially to estrogen receptor (ER)-b. The presence of this receptor is associated with a normal prostate and mammary gland, whereas it is diminished during tumor progression. ER-b is prominently expressed in normal colon mucosa. When mice are fed a diet containing soy, expression of the synthesizing vitamin D hydroxylase CYP27B1 is enhanced and that of the catabolic hydroxylase CYP24A1 is decreased [63]. Methylation/demethylation processes in promoter sequences of vitamin D hydroxylases may lead to reduced, subsequently, enhanced expression of these enzymes, and folate is a well-known physiological methyl donor. This could provide a link to the 400 j 4Slce Vitamins Selected 24 Table 24.4 Clinical trials with vitamin D.

Dosing schedule (vitamin D and Cancer type Study (patients) Treatment Response 1,25-D3) Reference

Prevention Colon polyp Phase II/III Vitamin D þ calcium Detection of new adenomas on follow-up Daily 1000 IU a prevention (2500) colonoscopy 3 or 5 years after study entry vitamin D Phase II (88) Vitamin D þ calcium Monitoring biomarkers of risk for colon Daily 800 IU vitamin D a cancer in patients at high risk High-grade PIN Phase II (50) Calcitriol MTD and PSA end point, follow-up 2 years Daily p.o. a

Intervention Prostate cancer Phase II (34) 1,25-D3 þ dexamethasone þ 13 HRPC patients had a >50% reduction Daily p.o. [100] carboplatin in PSA Phase II (43) 1,25-D3 þ 19% patients had PSA decline of 50%, Intermittent thrice [101] dexamethasone (AIPC) persisting for 28 days weekly p.o. Phase II (24) 1,25-D3 þ docetaxel þ 60 mg 1,25-D3; 55% chemotherapy naïve High-dose pulse p.o. [102] estramustine patients had 50% reduction in PSA Phase II (80) 1,25-D3 þ dexamethasone Objective: tumor vessel density, PIN, Twice weekly p.o. a (before prostatectomy) apoptosis and cell cycle markers, PSA, VDR, and MTD Phase II (48) 1,25-D3 (DN-101) þ Objective: MTD and PSA end point High-dose pulse p.o. a mitoxantrone þ prednisone Phase II (30) 1,25-D3 þ naproxen Objective: dose escalation, MTD Unspecified a Phase II (250) 1,25-D3 (DN-101) þ Weekly 45 g 1,25-D3 (DN-101); 58% of Weekly p.o. [103] docetaxel DN-101 treated patients had a >50% decline in PSA. Associated with improved survival Phase III (40) Vitamin D3 þ soy (Novasoy Objective: surveillance for prostate cancer. Weekly p.o. a tablets) Weekly 45 mg 1,25-D3 (DN-101) Phase III (900) 1,25-D3 (DN-101) þ Objective: survival in metastatic AIPC Weekly p.o. a docetaxel Colon cancer Unspecified (20) Vitamin D3 þ calcium Objective: safety, proliferative labeling index Unspecified a carbonate in patients with resected colon cancer, and effects on biological markers

Note: Clinical trials carried out exclusively with vitamin D analogues, and studies regarding only dose and toxicity relationship are excluded. AIPC, androgen-independent prostate cancer; HRPC, hormone refractory prostate cancer; MTD, maximum tolerated dose; p.o., oral administration; PIN, prostatic intraepithelial neoplasia; PSA, prostate speciﬁc antigen. aOngoing studies in the United Sates (see http://www.clinicaltrials.gov). 42VtmnD Vitamin 24.2 j 401 402j 24 Selected Vitamins

observation that reduced intake of nutritional folate present in leafy green vegetables is associated with enhanced incidence of colon cancer [63].

24.2.6 Impact of Processing on Vitamin D in Human Nutrition and Conclusion

Duetothewidespreadfearofsundamagetotheskin,aswellasthefactthateffectiveness of solar UVB radiation in vitamin D synthesis depends on latitude, season, time of day, and skin pigmentation, it has been previously suggested that the major source of vitamin D should come from dietary ingredients and not sun exposure. Fish is an excellent source of vitamin D, especially oily fish including salmon and mackerel. It is interesting to note that farmedsalmon hadapproximately 25% of the vitamin D content wild salmon had. Even within species, vitamin D content in fish varied widely [64]. Little is known about the effect of various cooking conditions on vitamin D content in fish. However, it can be assumed that, due to the stability of a steroid molecular structure, the precursor should be quite stable. In a Norwegian study on the vitamin D status and the impact of three cooked fish meals containing cod liver, it was demonstrated that, after consumption, the amount of vitamin D had increased 54-fold from the previous low basal level [65]. While cod liver or cod liver oil is probably not a preferred mode of vitamin D consumption in many populations, it does indicate the potency even after the cooking process. Preservation of the molecular structure is most probably not true for the active metabolite 1,25-D3 synthesized by hydroxylation reactions. This, however, is of little relevance for the efficacy of 1,25-D3 in cancer prevention, since the active substance is synthesized in human body from the precursor as described above. Taken together, 1,25-D3 has prominent antiproliferative, antiangiogenic, and prodifferentiating effects in a wide range of tumor cells. These effects are mediated through perturbation of several important cellular signaling pathways. For clinical trials, however, the problem of administering side effect-free doses remains. However, dietary modulation of extrarenal 1,25-D3 synthesis in organs potentially prone to tumor incidence could, by locally enhancing the concentration of 1,25-D3 in these tissues, lead to an enhanced apoptotic and antimitotic activity, thereby preventing tumor cell growth.

24.3 Vitamin E Hong Jin Lee and Nanjoo Suh

24.3.1 Introduction

Vitamin E was discovered after ﬁnding that a puriﬁed diet containing casein, cornstarch, lard, butterfat, salt, and adequate doses of vitamin A, vitamin B, and 24.3 Vitamin E j403 vitamin C caused sterility in rats [66]. Fertility was restored by the administration of lettuce leaves containing an unknown nutrient that was called vitamin E [67]. In 1936, vitamin E was crystallized and characterized by Evans et al. who named it tocopherol after Greek words tocos and phero, which mean childbirth and to bring, respectively [67]. In 1964, a group of compounds related to vitamin E harboring three double bonds in the long side chain was discovered and named tocotrienols [68]. Since then, the term vitamin E has been used for a group of naturally occurring tocopherols (a, b, g, d) and their corresponding tocotrienols (a, b, g, d) [69]. The fat-soluble vitamin E scavenges free radicals such as ROS and thus prevents the oxidation of polyunsaturated fatty acids (PUFAs) in cellular membranes and lipoproteins [69, 70]. The free radicals generated in the biological redox system and/ or by an exposure to different external factors such as UV light, pollutants, and ionizing radiation can cause reversible or irreversible damage to DNA and other biological molecules [69] associated with a number of pathological conditions including cancer [71], cardiovascular diseases, and diabetes. In consequence, vitamin E has been investigated in many studies as a candidate for preventing numerous human diseases. In this section, we will focus on activities of vitamin E related to the prevention and treatment of cancer.

24.3.2 Physicochemical Properties of Vitamin E, Chemical Structures, and Chemical Reactions

24.3.2.1 Structure of Vitamin E Components The a-, b-, g-, d-isomers of tocopherols and tocotrienols are distinguished by a different number and position of methyl substituents linked to the chromanol ring (Scheme 24.2). Tocopherols with a saturated phytyl side chain have three 0 0 chiral centers at C2, C 4,andC 8 indicating that there are eight possible stereoisomers in each tocopherol. However, all naturally occurring tocopherols have an RRR conﬁguration. Tocotrienols have an unsaturated isoprenoid side chain and one center of asymmetry at the C2 position. Commercially available forms of vitamin E may consist of (i) a mixture of naturally occurring tocopherols and tocotrienols, (ii) RRR-a-tocopherol (also known as D-a-tocopherol), (iii) synthetic a-tocopherol including stereoisomers (also known as all rac-a-tocopherol or DL-a-tocopherol), and (iv) acetate or succinate a-tocopherol derivatives that prevent the hydroxyl residue at C6 position from oxidation when exposed to air [72, 73].

24.3.2.2 Vitamin E in Human Diet The most signiﬁcant sources of vitamin E are oils and fats. Vegetable oils are the most abundant source because all natural forms of vitamin E are derived from plants. The content of vitamin E in food analyzed mainly by gas chromatography or high- performance liquid chromatography, and published from 1970 to 2002, has been 404j 24 Selected Vitamins

Scheme 24.2 Vitamin E comprises eight natural occurring forms, four tocopherols and four tocotrienols. These forms are different in number and position of methyl groups on the chromanol ring and the presence and absence of unsaturated bonds in the phytyl tail.

reviewed by Eitenmiller and Lee [74]. Table 24.5 shows the average concentration of tocopherols and tocotrienols in representative foods [74, 75].

24.3.3 Bioavailability and Metabolism of Vitamin E

24.3.3.1 Bioavailability and Biopotency of Vitamin E Vitamin E activity is presented in international units (IUs); one IU is equivalent to one milligram of all-rac-a-tocopheryl acetate [76]. The currently accepted relative biopotency of vitamin E isoforms equals 1.49 IU of a-tocopherol, 1.36 IU of a-tocopherol acetate, 0.75 IU of b-tocopherol, 0.15 IU of g-tocopherol, 0.02 IU of d-tocopherol, 0.45 IU of a-tocotrienol, and 0.08 IU of b-tocotrienol to 1.00 IU of all- rac-a-tocopheryl acetate and is based on the fetal resorption in rats [76]. The Panel on Antioxidant Nutrients and Related Compounds concluded that a-tocopherol is two times more active than all-rac-a-tocopherol, on the basis of the results from studies analyzing the ratio of a-tocopherol acetate to all-rac-a-tocopherol acetate in

plasma using deuterium-labeled d3-RRR-a-tocopherol acetate and d6-all-rac-a- tocopherol acetate [76].

24.3.3.2 Metabolism of Vitamin E All forms of vitamin E are absorbed in the small intestine by passive diffusion in enterocytes, are incorporated into chylomicrons, and then are secreted into the lymphatic system. However, absorption in the digestive tract does not determine the absorption efﬁciency of different forms of vitamin E [70, 76]. In blood, lipoprotein lipase hydrolyzes the chylomicrons and provides fatty acid and vitamin E to tissues. 24.3 Vitamin E j405

Table 24.5 Vitamin E content of representative dietary components.

Tocopherol content (mg/100 g) Tocotrienol content (mg/100 g)

Dietary component ab gdabgd

Almonda 31.8 0.2 1.3 Trace 0.2 — Trace — Asparagusb 1.14 0.03 0.14 ————— Avocadoesb 2.66 0.08 0.39 ————— Blackberriesb 1.43 0.04 1.42 0.85 ———— Blueberriesb 0.58 — 0.38 0.02 —— 0.08 — Bread (wheat)a 0.3 0.2 0.8 0.3 0.1 0.5 —— Broccolib 1.44 Trace 0.31 — Trace ——— Cabbageb 0.05 ——— 0.33 — 0.32 — Canola and 21.9 Trace 37.6 1.3 Trace — Trace — rapeseed oila Carrotsb 0.86 0.01 —————— Caster oila 1.2 1.8 28.7 38.5 — Trace —— Corn oila 18.6 1.1 67.9 3.1 2.9 — 3.3 — Cottonseed oila 40.4 0.4 33.2 0.1 Trace Trace Trace — Cranberriesb 1.23 Trace 0.04 ———0.33 — Eggsa 1.9 0.1 0.6 Trace 0.3 ——— Fish (ﬂounder)a 0.5 ——————— Green olivesb 3.81 ——————— Hazel nuta 21.7 0.5 3.2 0.1 ———— Kiwib 0.70 Trace 0.02 0.01 ———— Larda 1.0 — 0.1 — Trace ——— Lettuceb 0.18 — 0.31 0.01 0.01 ——— Olive oila 13.5 0.1 1.0 Trace ———— Palm oila 14.8 Trace 0.2 Trace 12.2 1.0 21.5 3.7 Parsleyb 0.75 — 0.53 ————— Peanut oila 17.9 0.3 14.6 2.3 Trace Trace Trace — Pistachioa 3.0 0.1 37.6 0.5 0.5 — 2.1 — Plumsb 0.28 — 0.05 — 0.02 — 0.22 — Raspberriesb 0.85 0.09 1.39 1.15 —— Soybean oila 8.2 1.0 69.3 28.1 Trace Trace Trace — Spinachb 1.96 — 0.21 ————— Sunﬂower oila 55.4 1.1 5.8 0.5 Trace Trace Trace — Tomatoesb 0.53 Trace 0.14 Trace Trace ——— Walnuta 1.3 Trace 22.6 2.1 ———— Wheat germ oila 149.2 57.8 22.4 2.5 2.3 5.4 0.2 —

—: Not detectable. aFrom Ref. [74]. bFrom Ref. [75]: the values of vitamin E are from raw and unprocessed vegetables and fruits.

The chylomicron remnants are recognized and taken up by the liver, where a-tocopherol transfer protein (a-TTP) preferentially binds a-tocopherol and prevents it from lysosomal degradation [70, 77]. Furthermore, a-TTP transfers a-tocopherol to the synthesis site of very low-density lipoproteins (VLDLs) and returns a-tocopherol to the circulating lipoproteins [70]. When the plasma level of vitamins E is exceeded, 406j 24 Selected Vitamins

vitamins are metabolized and secreted into urine or bile after initial w-oxidation and several steps of b-oxidation. The product of a-tocopherol oxidation is a-CEHC (2,5,7,8-tetramethyl-2-(20-carboxyethyl)-6-hydroxychroman) detectable in plasma, urine, and bile [70].

24.3.4 In Vitro and Animal Studies of Vitamin E

24.3.4.1 Mechanism of Antioxidant Protection Various components of vitamin E such as a-tocopherol and g-tocopherol play an essential role in cellular defense mechanism against various oxidants [69]. They act as scavenger of a peroxyl radical, terminating the chain oxidation of PUFAs [70]. As illustrated in Scheme 24.3, in the absence of tocopherols, the peroxyl radical (ROO.) reacts with PUFAs, and further auto-oxidation of PUFAs occurs in the membrane or lipoproteins. Tocopherol (TOH) donates hydrogen atom to the peroxyl radical faster than does the target lipid (RH), scavenges the radical, and terminates the chain reaction [69, 70]. The antioxidant activities of different vitamin E isoforms decrease in the order a-tocopherol >b-tocopherol ﬃ g-tocopherol >d-tocopherol when measured in vitro, showing a correlation between efﬁciency and methyl substitution in chromanol ring [78]. However, g-tocopherol is a much more effective antioxidant

Scheme 24.3 Scavenging reaction of peroxy radical (ROO.) and terminates chain tocopherols. Lipid peroxy radical (ROO.), acting reaction by donating hydrogen atom to peroxy as a chain carrier, oxidizes the target lipid (RH) by radical faster than target lipid (RH). taking hydrogen atom from target lipid (RH). Tocopheroxyl radical (TO.) is more stable than Lipid radical (R.) can react with molecular free radicals and can be reduced to tocopherol oxygen and supplies lipid peroxy radical (ROO.) (TOH) by ascorbic acid, glutathione, and for further lipid oxidation. In the presence of coenzyme Q. vitamin E, tocopherol (TOH) scavenges lipid 24.3 Vitamin E j407 against reactive nitrogen species (RNS) than a-tocopherol as it forms 5-nitro- g-tocopherol, a reaction not feasible with a-tocopherol [79]. The tocopheroxyl radical is less reactive than peroxyl radical and can be converted to the parent tocopherol by other H-atom donors such as vitamin C, glutathione, and coenzyme Q (Scheme 24.3) [69, 70]. It has been demonstrated that vitamin C supplementation can normalize the level of vitamin E in smokers, suggesting an interaction between vitamin E and vitamin C [70].

24.3.4.2 Nonantioxidant Functions of Vitamin E Protein kinase C (PKC) is one of the key signaling molecules involved in the regulation of cell proliferation and differentiation [80]. Inhibition of PKC by a-tocopherol resulting in inhibition of platelet aggregation and reduction of nitric oxide and superoxide production has been demonstrated in many types of cells [80]. The effect is likely related to the activation of protein phosphatase 2A by a-tocopherol that dephosphorylates and inactivates PKC, rather than to a tocopherol–protein interaction [80]. However, it has been proposed that the role of a-tocopherol in PKC- bound speciﬁc signaling stems from an antioxidant effect on the membrane, based on the observation that oxidants and antioxidants induce opposite PKC-linked responses [78].

24.3.4.3 Anticancer Mechanisms of Vitamin E Action Mechanisms of vitamin E anticancer activity have been investigated for many years [73, 81, 82] and can be summarized as follows: (a) induction of apoptosis (via TGF-b, Fas, and Caspase-3, -4, -7, -8, and -9 activation, ROS generation, and GADD45b and Bax translocation); (b) blockage of survival and cell proliferation and induction of cell cycle arrest (via induction of peroxisome proliferator-activated receptor-g (PPAR-g), inhibition of estrogen receptor and androgen receptor (AR), inhibition of cyclin D1 and cyclin E, and induction of p21 and p27); (c) blockage of metastasis (via inhibition of MMP-9, VEGFrelease and mRNA expression); and (d) anti-inﬂammatory activity via inhibition of cyclooxygenase-2 (COX-2) (more speciﬁc with g-tocopherol) [73, 81, 83].

24.3.4.4 Vitamin E and Preclinical Studies Results from preclinical studies with RRR-a-tocopherol acetate or all-rac-a-tocopherol in breast, prostate, or colon cancer models are not consistent [73]. A majority of studies investigating the protective effects of certain types of vitamin E in animal models of mammary cancer prevention show little or no effect [82]. However, g-tocopherol, the most common form of vitamin E in US diet and the second most common form in human tissues, has demonstrated anti-inﬂammatory and antitumor activity in many animal models of colon, breast, and prostate cancers [84, 85]. In addition to studies with tocopherols, tocotrienols have shown strong antioxidant activity as well as anticancer properties, although the literature on tocotrienols and cancer is limited [86]. More animal studies may be necessary to determine whether individual isoforms of tocopherols or tocotrienols have different anticancer activities in preclinical studies. 408j 24 Selected Vitamins

24.3.5 Vitamin E and Human Intervention Trials

Epidemiologic evidence supporting a link between vitamin E and cancer is limited, and the results of a few completed studies with vitamin E are not consistent [72]. Numerous human intervention studies with vitamin E have been reported, but we will discuss results from only a few key vitamin E clinical trials. From 1986 to 1991, individuals of ages 40–69 from Linxian, China, were recruited for testing the effect of daily vitamin and mineral supplementation for mortality and cancer incidence [87]. Out of 29 584 adults, approximately 15 000 who received daily vitamin and mineral supplementation of b-carotene (15 mg b-carotene daily), vitamin E (30 mg a-tocopherol daily), and selenium (50 mg daily) for 5.2 years had lower total mortality, suggesting that vitamin and mineral supplementation of the diet including vitamin E may effect a reduction in cancer risk in this population [87]. From 1985 to 1993, a randomized, double-blind, placebo-controlled alpha-tocopherol, beta-carotene (ATBC) lung cancer prevention study examined the effect of a-tocopherol (50 mg/ day) and b-carotene (20 mg/day) daily supplementation on the incidence of lung cancer and possibly other cancers [88]. The ATBC cancer prevention study found that male smokers receiving 50 mg/day of synthetic DL-a-tocopherol acetate had 32% lower prostate cancer incidence and 41% reduction in prostate cancer deaths compared to a control group [89]. After the ATBC cancer prevention study, two large randomized trials assessing the effect of vitamin E supplementation on cancer incidence were conducted: the Heart Outcomes Prevention Evaluation (HOPE) trial [90] and the Womens Health Study (WHS) trial [91]. In the randomized, double-blind, placebo-controlled international HOPE trial conducted for 4.5 years between 1993 and 1999, testing RRR-a-tocopherol acetate (400 IU/day) supplementation, no significant association between cancer incidence and vitamin E supplementation was found [90]. The large WHS trial that used 600 IU of natural-source vitamin E (RRR-a-tocopherol acetate) taken by study subjects every other day for more than 10 years also provided no signs of overall benefit with regard to the prevention of major adverse cardiovascular events or cancer and did not affect total mortality but decreased cardiovascular mortality in healthy women [91]. An ongoing clinical study of vitamin E, the NCI-funded Selenium and Vitamin E Cancer Prevention Trial (SELECT) of 32 500 men will determine the effectiveness of either selenium (200 mg/day L-selenomethionine) and/or vitamin E (400 IU/day all- rac-a-tocopheryl acetate) at reducing prostate cancer incidence and mortality [92]. This 12-year study is scheduled to finish by 2013 [92]. The outcomes from several vitamin E human clinical studies do not consistently show a beneficial effect of the long-term supplementation with RRR-a-tocopherol or synthetic all-rac-a-tocopheryl acetate for the prevention of cancers, including that of the breast, prostate, and colon [73, 90, 91, 93]. It has been suggested that dietary habits, type and dose of vitamin E supplementation, and the lifestyle of the patients may contribute to the study outcomes [73]. More studies are needed to determine the optimum dosage, the bioavailability, and, in particular, the use of various vitamin E isoforms. References j409

24.3.6 Impact of Cooking, Processing, and Other Factors on Protective Properties of Vitamin E

The amount of vitamin E in fruits, vegetables, nuts, beans, and various oils is affected by species, variety, maturity, growing condition such as weather, sunlight, and season, and harvest [75]. Variation in the level of vitamin E in food can be related to processing procedures, storage time, and conditions [75]. Chun et al. reported vitamin E values for fruit, vegetables, and several processed products representing key foods in US diet [75]. Among vitamin E components, a-tocopherol was found in highest quantity in most fruits and vegetables. Alpha- and g-tocotrienols were detectable in several fruits and vegetables but at levels mostly lower than 0.1 mg/100 g. Fruits, vegetables, and their processed products provide a good source of vitamin E to the US consumer because of the quantity consumed [75], but many studies have shown that processing causes significant losses of vitamin E [94]. Vitamin E values in unprocessed foods such as vegetables and fish, commercially prepared foods such as breakfast cereals, and canned food items have been reported [95]. Although fat-soluble vitamin E is not as severely affected by processing as water-soluble vitamins, vitamin E can be inactivated during processing and losses in vitamin E content seem to be closely associated with lipid degradation [95]. According to the same report, cooking temperature, time, and exposure to light and oxidative conditions can affect vitamin E degradation. In the grain group, corn is a significant source of vitamin E, but tocopherols can be easily destroyed in cooking processes during the production of tortillas. Soybeans, nuts, and vegetable oils represent a good source of vitamin E, and g-tocopherol is predominant in legumes and fairly resistant to cooking losses. The level of g-tocopherol is thus higher than that of the a-tocopherol in diets that consist largely of legumes [95]. Since the high intake of soybean and corn oils in American diet contributes to the consumption of large amounts of g-tocopherol [96], more studies may be needed to understand the importance of g-tocopherol and tocotrienols to the prevention and treatment of cancer.

Acknowledgment

This work was supported by NIH grants to N.S.

References

References to Section 24.1

1 Padayatty, S.J., Katz, A., Wang, Y., Eck, P., 2 Carr, A. and Frei, B. (1999) Does vitamin C Kwon, O., Lee, J.H., Chen, S., Corpe, C., act as a pro-oxidant under physiological Dutta, A., Dutta, S.K. and Levine, M. (2003) conditions? The FASEB Journal, 13, Vitamin C as an antioxidant: evaluation of 1007–1024. its role in disease prevention. Journal of the 3 Lee, S.H., Oe, T. and Blair, I.A. (2001) American College of Nutrition, 22,18–35. Vitamin C-induced decomposition of lipid 410j 24 Selected Vitamins

hydroperoxides to endogenous antioxidant? A review of novel actions and genotoxins. Science, 292, 2083–2086. reactions of vitamin C. Free Radical 4 Li, Y. and Schellhorn, H.E. (2007) New Research, 39, 671–686. developments and novel therapeutic 15 World Cancer Research Fund/American perspectives for vitamin C. The Journal of Institute for Cancer Research (2007) Nutrition, 137, 2171–2184. Food, Nutrition, Physical Activity, and the 5 Rumsey, S.C. and Levine, M. (1998) Prevention of Cancer: A Global, Perspective, Absorption, transport, and disposition of AICR, Washington, DC. ascorbic acid in humans. The Journal of 16 Khaw, K.T., Bingham, S., Welch, A., Nutritional Biochemistry, 9, 116–130. Luben, R., Wareham, N., Oakes, S. and 6 Padayatty, S.J. and Levine, M. (2001) New Day, N. (2001) Relation between plasma insights into the physiology and ascorbic acid and mortality in men and pharmacology of vitamin C. Canadian women in EPIC-Norfolk prospective Medical Association Journal, 164, 353–355. study: a prospective population study. 7 Padayatty, S.J., Sun, H., Wang, Y., European prospective investigation into Riordan, H.D., Hewitt, S.M., Katz, A., cancerandnutrition.Lancet,357,657–663. Wesley, R.A. and Levine, M. (2004) 17 Loria, C.M., Klag, M.J., Caulﬁeld, L.E. Vitamin C pharmacokinetics: implications and Whelton, P.K. (2000) Vitamin C for oral and intravenous use. Annals of status and mortality in US adults. The Internal Medicine, 140, 533–537. American Journal of Clinical Nutrition, 8 Block, K.I. and Mead, M.N. (2003) 72, 139–145. Vitamin C in alternative cancer treatment: 18 Kubo, A. and Corley, D.A. (2007) historical background. Integrative Cancer Meta-analysis of antioxidant intake and Therapies, 2, 147–154. the risk of esophageal and gastric cardia 9 Wilson, J.X. (2005) Regulation of vitamin adenocarcinoma. The American Journal of C transport. Annual Review of Nutrition, 25, Gastroenterology, 102, 2323–2330. 105–125. 19 Tsugane, S. and Sasazuki, S. (2007) Diet 10 Federico, A., Morgillo, F., Tuccillo, C., and the risk of gastric cancer: review of Ciardiello, F. and Loguercio, C. (2007) epidemiological evidence. Gastric Cancer, Chronicinﬂammationandoxidativestress 10,75–83. in human carcinogenesis. International 20 Howe, G.R. and Burch, J.D. (1996) Journal of Cancer, 121,2381–2386. Nutrition and pancreatic cancer. Cancer 11 Klaunig, J.E. and Kamendulis, L.M. Causes & Control, 7,69–82. (2004) The role of oxidative stress in 21 Patterson, R.E., White, E., Kristal, A.R., carcinogenesis. Annual Review of Neuhouser, M.L., Potter, J.D. (1997) Pharmacology and Toxicology, 44, 239–267. Vitamin supplements and cancer risk: 12 Collins, A.R., Cadet, J., Moller, L., the epidemiologic evidence. Cancer Poulsen, H.E. and Vina, J. (2004) Are we Causes & Control, 8, 786–802. sure we know how to measure 8-oxo-7,8- 22 Ruano-Ravina, A., Figueiras, A., dihydroguanine in DNA from human Freire-Garabal, M. and Barros-Dios, J.M. cells? Archives of Biochemistry and (2006) Antioxidant vitamins and risk of Biophysics, 423,57–65. lung cancer. Current Pharmaceutical 13 Cadet, J., Douki, T., Gasparutto, D. and Design, 12, 599–613. Ravanat, J.L. (2003) Oxidative damage to 23 Coulter, I.D., Hardy, M.L., Morton, S.C., DNA: formation, measurement and Hilton, L.G., Tu, W., Valentine, D. and biochemical features. Mutation Research, Shekelle, P.G. (2006) Antioxidants 531,5–23. vitamin Cand vitamin E for the prevention 14 Duarte, T.L. and Lunec, J. (2005) Review: and treatment of cancer. Journal of General when is an antioxidant not an Internal Medicine, 21,735–744. References j411

24 Plummer, M., Vivas, J., Lopez, G., Levine, M. (2006) Intravenously Bravo, J.C., Peraza, S., Carillo, E., Cano, E., administered vitamin C as cancer Castro, D., Andrade, O., Sanchez, V., therapy: three cases. Canadian Medical Garcia, R., Buiatti, E., Aebischer, C., Association Journal, 174, 937–942. Franceschi, S., Oliver, W. and Munoz, N. 30 Stephenson, C.M., Levin, R.D. and (2007) Chemoprevention of precancerous Lis, C.G. (2007) Phase 1 trial of high-dose gastric lesions with antioxidant vitamin intravenous vitamin C treatment for supplementation: a randomized trial in a patients with cancer. Journal of the high-risk population. Journal of the American Osteopathic Association, 107, National Cancer Institute, 99,137–146. 212–213. 25 You, W.C., Brown, L.M., Zhang, L., Li, 31 Kevers, C., Falkowski, M., Tabart, J., J.Y., Jin, M.L., Chang, Y.S., Ma, J.L., Pan, Defraigne, J.O., Dommes, J. and K.F., Liu, W.D., Hu, Y., Crystal-Mansour, Pincemail, J. (2007) Evolution of S., Pee, D., Blot, W.J., Fraumeni, J.F. Jr, antioxidant capacity during storage of Xu, G.W. and Gail, M.H. (2006) selected fruits and vegetables. Journal of Randomized double-blind factorial trial Agricultural and Food Chemistry, 55, of three treatments to reduce the 8596–8603. prevalence of precancerous gastric 32 Fillion, L. and Henry, C.J. (1998) Nutrient lesions. Journal of the National Cancer losses and gains during frying: a review. Institute, 98, 974–983. International Journal of Food Sciences and 26 Huang, H.Y., Caballero, B., Chang, S., Nutrition, 49, 157–168. Alberg, A.J., Semba, R.D., Schneyer, C.R., 33 Klopotek, Y., Otto, K. and Bohm, V. (2005) Wilson, R.F., Cheng, T.Y., Vassy, J., Processing strawberries to different Prokopowicz, G., Barnes, G.J.J. 2nd and products alters contents of vitamin C, Bass, E.B. (2006) The efﬁcacy and safety total phenolics, total anthocyanins, and of multivitamin and mineral supplement antioxidant capacity. Journal of use to prevent cancer and chronic disease Agricultural and Food Chemistry, 53, in adults: a systematic review for a 5640–5646. National Institutes of Health State-of-the- 34 Gliszczynska-Swiglo, A., Ciska, E., Science Conference. Annals of Internal Pawlak-Lemanska, K., Chmielewski, J., Medicine, 145, 372–385. Borkowski, T. and Tyrakowska, B. (2006) 27 Christen, W.G., Gaziano, J.M. and Changes in the content of health- Hennekens, C.H. (2000) Design of promoting compounds and antioxidant Physicians Health Study II: a activity of broccoli after domestic randomized trial of beta-carotene, processing. Food Additives and vitamins E and C, and multivitamins, in Contaminants, 23, 1088–1098. prevention of cancer, cardiovascular 35 Piga, A., Del Caro, A. and Corda, G. disease, and eye disease, and review of (2003) From plums to prunes: results of completed trials. Annals of inﬂuence of drying parameters on Epidemiology, 10, 125–134. polyphenols and antioxidant activity. 28 Davies, A.A., Davey Smith, G., Journal of Agricultural and Food Harbord, R., Bekkering, G.E., Sterne, Chemistry, 51, 3675–3681. J.A., Beynon, R. and Thomas, S. (2006) 36 Vinson, J.A., Zubik, L., Bose, P., Nutritional interventions and outcome in Samman, N. and Proch, J. (2005) Dried patients with cancer or preinvasive fruits: excellent in vitro and in vivo lesions: systematic review. Journal of the antioxidants. Journal of the American National Cancer Institute, 98, 961–973. College of Nutrition, 24,44–50. 29 Padayatty, S.J., Riordan, H.D., 37 Williams, P.G. (1996) Vitamin retention Hewitt, S.M., Katz, A., Hoffer, L.J. and in cook/chill and cook/hot-hold hospital 412j 24 Selected Vitamins

food-services. Journal of the American 47 Zehnder, D. et al. (2001) Extrarenal Dietetic Association, 96, 490–498. expression of 25-hydroxyvitamin d(3)-1 38 Holland, A.A., Welch, A.A., Unwin, I.D., alpha-hydroxylase. The Journal of Clinical Buss, D.H., Paul, A.A. and Southgate, Endocrinology and Metabolism, 86 (2), D.A.T. (1991) McCance and Widdowsons 888–894. The Composition of Foods, 5th edn, The 48 Bises, G. et al. (2004) 25-hydroxyvitamin Royal Society of Chemistry, Ministry of D3-1alpha-hydroxylase expression in Agriculture, Fisheries, and Food, London. normal and malignant human colon. The Journal of Histochemistry and Cytochemistry, 52 (7), 985–989. References to Section 24.2 49 Cross, H.S. et al. (2001) 25-Hydroxyvitamin D(3)-1alpha- 39 Calvo, M.S., Whiting, S.J. and Barton, hydroxylase and vitamin D receptor gene C.N. (2005) Vitamin D intake: a global expression in human colonic mucosa is perspective of current status. The Journal elevated during early cancerogenesis. of Nutrition, 135 (2), 310–316. Steroids, 66 (3–5), 287–292. 40 Garland, C.F. and Garland, F.C. (1980) 50 Cross, H.S. et al. (1997) Vitamin D Do sunlight and vitamin D reduce metabolism in human colon the likelihood of colon cancer? adenocarcinoma-derived Caco-2 cells: International Journal of Epidemiology, expression of 25-hydroxyvitamin 9 (3), 227–231. D3-1alpha-hydroxylase activity and 41 Garland, F.C. et al. (1990) Geographic regulation of side-chain metabolism. The variation in breast cancer mortality in the Journal of Steroid Biochemistry and United States: a hypothesis involving Molecular Biology, 62 (1), 21–28. exposure to solar radiation. Preventive 51 Holt, P.R. et al. (2002) Colonic epithelial Medicine, 19 (6), 614–622. cell proliferation decreases with 42 Schwartz, G.G. and Hulka, B.S. (1990) Is increasing levels of serum 25-hydroxy vitamin D deﬁciency a risk factor for vitamin D. Cancer Epidemiology, prostate cancer? (Hypothesis). Anticancer Biomarkers & Prevention, 11 (1), 113–119. Research, 10 (5A), 1307–1311. 52 Deeb, K.K., Trump, D.L. and Johnson, 43 Holick, M.F. (1989) Vitamin D: C.S. (2007) Vitamin D signalling biosynthesis, metabolism, and mode of pathways in cancer: potential for action, in Endocrinology (ed. L.J. De Groot anticancer therapeutics. Nature Reviews. et al.), Grune & Stratton, New York, pp. Cancer, 7 (9), 684–700. 902–926. 53 Zinser, G.M., Sundberg, J.P. and Welsh,

44 Prosser, D.E. and Jones, G. (2004) J. (2002) Vitamin D3 receptor ablation Enzymes involved in the activation and sensitizes skin to chemically induced inactivation of vitamin D. Trends in tumorigenesis. Carcinogenesis, 23 (12), Biochemical Sciences, 29 (12), 664–673. 2103–2109. 45 Heaney, R.P. (2003) Long-latency 54 Kallay, E. et al. (2001) Characterization of deﬁciency disease: insights from calcium a vitamin D receptor knockout mouse as a and vitamin D. The American Journal of model of colorectal hyperproliferation Clinical Nutrition, 78 (5), 912–919. and DNA damage. Carcinogenesis, 22 (9), 46 Cross, H.S. et al. (2005) The vitamin D 1429–1435. endocrine system of the gut: its 55 Haussler, M.R. et al. (1998) The nuclear possible role in colorectal cancer vitamin D receptor: biological and prevention. The Journal of Steroid molecular regulatory properties revealed. Biochemistry and Molecular Biology, 97 Journal of Bone and Mineral Research, 13 (1–2), 121–128. (3), 325–349. References j413

56 Kim, M.S. et al. (2007) 1alpha, Steroid Biochemistry and Molecular Biology, 25(OH)2D3-induced DNA methylation, 103 (3–5), 642–644. suppresses the human CYP27B1 gene. 65 Brustad, M. et al. (2004) Vitamin D Molecular and Cellular Endocrinology, status in a rural population of 265–266, 168–173. northern Norway with high fish liver 57 Khorchide, M., Lechner, D. and consumption. Public Health Nutrition, Cross, H.S. (2005) Epigenetic regulation 7 (6), 783–789. of vitamin D hydroxylase expression and activity in normal and malignant human prostate cells. The Journal of References to Section 24.3 Steroid Biochemistry and Molecular Biology, 93 (2–5), 167–172. 66 Evans, H.M. and Bishop, K.S. (1922) On 58 Getzenberg, R.H. et al. (1997) Vitamin D the existence of a hitherto unrecognized inhibition of prostate adenocarcinoma dietary factor essential for reproduction. growth and metastasis in the Dunning rat Science, 56, 650–651. prostate model system. Urology, 50 (6), 67 Wolf, G. (2005) The discovery of the 999–1006. antioxidant function of vitamin E: the 59 Hershberger, P.A. et al. (2002) Cisplatin contribution of Henry A. Mattill. Journal potentiates 1,25-dihydroxyvitamin D3- of Nutrition, 135, 363–366. induced apoptosis in association with 68 Pennock, J.F., Hemming, F.W. and increased mitogen-activated protein Kerr, J.D. (1964) A reassessment of kinase kinase kinase 1 (MEKK-1) tocopherol in chemistry. Biochemical and expression. Molecular Cancer Biophysical Research Communications, Therapeutics, 1 (10), 821–829. 17, 542–548. 60 Giovannucci, E. et al. (2006) Prospective 69 Wang, X. and Quinn, P.J. (1999) Vitamin study of predictors of vitamin D status E and its function in membranes. Progress and cancer incidence and mortality in in Lipid Research, 38, 309–336. men. Journal of the National Cancer 70 Traber, M.G. (2007) Vitamin E regulatory Institute, 98 (7), 451–459. mechanisms. Annual Review of Nutrition, 61 Grau, M.V. et al. (2003) Vitamin D, 27, 347–362. calcium supplementation, and colorectal 71 Rodrigo, R., Guichard, C. and Charles, R. adenomas: results of a randomized trial. (2007) Clinical pharmacology and Journal of the National Cancer Institute, 95 therapeutic use of antioxidant vitamins. (23), 1765–1771. Fundamental & Clinical Pharmacology, 21, 62 Kallay, E. et al. (2005) Colon-specific 111–127. regulation of vitamin D hydroxylases: a 72 Brigelius-Flohe, R., Kelly, F.J., Salonen, possible approach for tumor prevention. J.T., Neuzil, J., Zingg, J.M. and Azzi, A. Carcinogenesis, 26 (9), 1581–1589. (2002) The European perspective on 63 Cross, H.S. (2007) Extrarenal vitamin D vitamin E: current knowledge and future hydroxylase expression and activity in research. The American Journal of Clinical normal and malignant cells: Nutrition, 76, 703–716. modification of expression by 73 Kline, K., Lawson, K.A., Yu, W. and epigenetic mechanisms and dietary Sanders,B.G.(2007)VitaminE andcancer. substances. Nutrition Reviews, 65 (8 Pt 2), Vitamins and hormones, 76,435–461. S108–S112. 74 Eitenmiller, R.R. and Lee, J. (2004) 64 Lu, Z. et al. (2007) An evaluation of the Vitamin E: Food Chemistry, Composition, vitamin D3 content in fish: is the vitamin and Analysis, Marcel Dekker, New York. D content adequate to satisfy the dietary 75 Chun, J., Lee, J., Ye, L., Exler, J. and requirement for vitamin D? The Journal of Eitenmiller, R.R. (2006) Tocopherol and 414j 24 Selected Vitamins

tocotrienol contents of raw and processed 84 Jiang, Q., Christen, S., Shigenaga, M.K. fruits and vegetables in the United States and Ames, B.N. (2001) Gamma- diet. Journal of Food Composition and tocopherol, the major form of vitamin E Analysis, 19, 196–204. in the US diet, deserves more attention. 76 Hoppe, P.P. and Krennrich, G. (2000) The American Journal of Clinical Nutrition, Bioavailability and potency of natural- 74, 714–722. source and all-racemic alpha-tocopherol 85 Dietrich, M., Traber, M.G., Jacques, P.F., in the human: a dispute. European Journal Cross, C.E., Hu, Y. and Block, G. (2006) of Nutrition, 39, 183–193. Does gamma-tocopherol play a role in the 77 Mustacich, D.J., Bruno, R.S. and primary prevention of heart disease and Traber, M.G. (2007) Vitamin E. Vitamins cancer? A review. Journal of the American and Hormones, 76,1–21. College of Nutrition, 25, 292–299. 78 Traber, M.G. and Atkinson, J. (2007) 86 Sen, C.K., Khanna, S. and Roy, S. (2007) Vitamin E, antioxidant and nothing Tocotrienols in health and disease: the more. Free Radical Biology & Medicine, 43, other half of the natural vitamin E family. 4–15. Molecular Aspects of Medicine, 28, 79 Hensley, K., Benaksas, E.J., Bolli, R., 692–728. Comp, P., Grammas, P., Hamdheydari, 87 Blot, W.J., Li, J.Y., Taylor, P.R., Guo, W., L., Mou, S., Pye, Q.N., Stoddard, M.F., Dawsey, S., Wang, G.Q., Yang, C.S., Wallis, G., Williamson, K.S., West, M., Zheng,S.F.,Gail,M.,Li,G.Y.et al. Wechter, W.J. and Floyd, R.A. (2004) (1993) Nutrition intervention trials in New perspectives on vitamin E: Linxian, China: supplementation with gamma-tocopherol and specific vitamin/mineral carboxyelthylhydroxychroman combinations, cancer incidence, and metabolites in biology and medicine. Free disease-specific mortality in the general Radical Biology & Medicine, 36,1–15. population. Journal of the National 80 Azzi, A., Ricciarelli, R. and Zingg, J.M. Cancer Institute, 85, 1483–1492. (2002) Non-antioxidant molecular 88 The ATBC Cancer Prevention Study functions of alpha-tocopherol (vitamin Group (1994) The alpha-tocopherol, beta- E). FEBS Letters, 519,8–10. carotene lung cancer prevention study: 81 Stone, W.L.,Krishnan, K., Campbell, S.E., design, methods, participant Qui, M., Whaley, S.G. and Yang,H. (2004) characteristics, and compliance. Annals of Tocopherols and the treatment of colon Epidemiology, 4,1–10. cancer. Annals of the New York Academy of 89 Heinonen, O.P., Albanes, D., Virtamo, Sciences, 1031, 223–233. J., Taylor, P.R., Huttunen, J.K., 82 Kline, K., Lawson, K.A., Yu, W. and Hartman, A.M., Haapakoski, J., Malila, Sanders, B.G. (2003) Vitamin E and N., Rautalahti, M., Ripatti, S., Maenpaa, breast cancer prevention: current status H., Teerenhovi, L., Koss, L., Virolainen, and future potential. Journal of Mammary M. and Edwards, B.K. (1998) Prostate Gland Biology and Neoplasia, 8,91–102. cancer and supplementation with alpha- 83 Jiang, Q., Elson-Schwab, I., tocopherol and beta-carotene: incidence Courtemanche, C. and Ames, B.N. (2000) and mortality in a controlled trial. Gamma-tocopherol and its major Journal of the National Cancer Institute, metabolite, in contrast to alpha- 90, 440–446. tocopherol, inhibit cyclooxygenase 90 Lonn, E., Bosch, J., Yusuf, S., Sheridan, activity in macrophages and epithelial P., Pogue, J., Arnold, J.M., Ross, C., cells. Proceedings of the National Academy Arnold, A., Sleight, P., Probstfield, J. and of Sciences of the United States of America, Dagenais, G.R. (2005) Effects of long- 97, 11494–11499. term vitamin E supplementation on References j415

cardiovascular events and cancer: a metabolism. FASEB Journal, 13, randomized controlled trial. Journal of the 1145–1155. American Medical Association, 293, 97 Huerta, S., Irwin, R.W., Heber, D., Go, 1338–1347. V.L., Koeffler, H.P., Uskokovic, M.R. and 91 Lee, I.M., Cook, N.R., Gaziano, J.M., Harris, D.M. (2002) 1alpha,25-(OH)(2)-D Gordon, D., Ridker, P.M., Manson, J.E., (3) and its synthetic analogue decrease Hennekens, C.H. and Buring, J.E. (2005) tumor load in the Apc(min) Mouse. Vitamin E in the primary prevention of Cancer Research, 62, 741–746. cardiovascular disease and cancer: the 98 Mantell, D.J., Owens, P.E., Bundred, N.J., Womens Health Study: a randomized Mawer, E.B. and Canfield, A.E. (2000) controlled trial. Journal of the American Alpha,25-dihydroxyvitamin D(3) inhibits Medical Association, 294,56–65. angiogenesis in vitro and in vivo. 92 Lippman, S.M., Goodman, P.J., Circulation Research, 87, 214–220. Klein, E.A., Parnes, H.L., Thompson, 99 Banach-Petrosky, W., Ouyang, X., Gao, I.M. Jr, Kristal, A.R., Santella, R.M., H., Nader, K., Ji, Y.,Suh, N., DiPaola, R.S. Probstfield, J.L., Moinpour, C.M., and Abate-Shen, C. (2006) Vitamin D Albanes, D., Taylor, P.R., Minasian, inhibits the formation of prostatic L.M.,Hoque,A.,Thomas,S.M., intraepithelial neoplasia in Nkx3.1;Pten Crowley, J.J., Gaziano, J.M., Stanford, mutant mice. Clinical Cancer Research, 12, J.L., Cook, E.D., Fleshner, N.E., Lieber, 5895–5901. M.M., Walther, P.J., Khuri, F.R., Karp, 100 Flaig, T.W., Barqawi, A., Miller, G., Kane, D.D., Schwartz, G.G., Ford, L.G. and M., Zeng, C., Crawford, E.D. and Glode, Coltman, C.A. Jr (2005) Designing the L.M. (2006) A phase II trial of Selenium and Vitamin E Cancer dexamethasone, vitamin D, and Prevention Trial (SELECT). Journal of the carboplatin in patients with hormone- National Cancer Institute, 97,94–102. refractory prostate cancer. Cancer, 107, 93 Patterson, R.E., White, E., Kristal, A.R., 266–274. Neuhouser, M.L. and Potter, J.D. (1997) 101 Trump, D.L., Potter, D.M., Muindi, J., Vitamin supplements and cancer risk: Brufsky, A. and Johnson, C.S. (2006) the epidemiologic evidence. Cancer Phase II trial of high-dose, intermittent Causes & Control, 8, 786–802. calcitriol (1,25 dihydroxyvitamin D3) and 94 Gliszczynska-Swiglo, A., Ciska, E., dexamethasone in androgen-independent Pawlak-Lemanska, K., Chmielewski, J., prostate cancer. Cancer, 106,2136–2142. Borkowski, T. and Tyrakowska, B. (2006) 102 Tiffany, N.M., Ryan, C.W., Garzotto, M., Changes in the content of health- Wersinger, E.M. and Beer, T.M. (2005) promoting compounds and antioxidant High dose pulse calcitriol, docetaxel and activity of broccoli after domestic estramustine for androgen independent processing. Food Additives and prostate cancer: a phase I/II study. Journal Contaminants, 23, 1088–1098. of Urology, 174, 888–892. 95 Wyatt, C.J., Carballido, S.P. and 103 Beer, T.M., Higano, C.S., Saleh, M., Mendez, R.O. (1998) Alpha- and gamma- Dreicer, R., Hudes, G., Picus, J., Rarick, tocopherol content of selected foods in M., Fehrenbacher, L. and Hannah, A.L. the Mexican diet: effect of cooking losses. (2007) Phase II study of KOS-862 in Journal of Agricultural and Food Chemistry, patients with metastatic androgen 46, 4657–4661. independent prostate cancer previously 96 Brigelius-Flohe, R. and Traber, M.G. treated with docetaxel. Invest New Drugs, (1999) Vitamin E: function and 25, 565–570. j417

25 Folate and Vitamins B2, B6, and B12 Philip Thomas and Michael Fenech

25.1 Introduction

The B vitamins are a group of eight water-soluble vitamins that play crucial roles in cellular metabolism. Deficiency in some of these micronutrients can lead to genome and epigenome damage resulting in chromosome loss and/or breakage [1, 2]. Here, we focus on four members of this group, mainly folate and vitamins B2, B6, and B12, their metabolic relationships within the folate/methionine pathway and the potential influences on genomic instability events. Folate (vitamin B9) is essential for the synthesis of DNA and the maintenance and regulation of cell division and is found in food sources such as leafy green vegetables, dry beans, peas, lentils, fortified cereals, liver, and yeast [3]. Folate was first discovered in 1931 by Dr Lucy Wills, while working with anemic pregnant women in Bombay in India [4]. She found that the symptoms of anemia could be alleviated by treating patients with yeast. It was supposed that yeast contained an unknown component that could be potentially important in the large-scale treatment of anemia [4]. The subsequently named Wills factor was eventually isolated from spinach leaves in 1941 and renamed as folic acid. Folate derives its name from the Latin word folium meaning leaf [4]. Folate deficiency has been associated with increased risk for a number of cancers, neural tube defects, and neurodegenerative disorders such as Alzheimers disease (AD) [5–7]. Riboflavin (vitamin B2) was initially isolated as a yellow-green pigment from milk in 1879 and given the name lactoflavin (lacto, for milk and flavin for its yellow coloration) [8]. Later, two independent research groups headed by Richard Kuhn in Germany and Paul Karrer in Switzerland were able to isolate and crystallize a compound identical to lactoflavin, which was now more commonly referred to as riboflavin as it contained the sugar ribose. Riboflavin is the central component of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which participate in a wide range of redox reactions that are critical to the function of aerobic cells. It plays a key role in energy metabolism and in the effective regulation of fats, carbohydrates, and proteins [8]. B2 is a photosensitive vitamin present in a

wide variety of foods such as milk, cheese, leafy green vegetables, liver, almonds, and yeast. Riboflavin deficiency is classically associated with the oral–ocular–genital syndrome, angular cheilitis, photophobia, and scrotal dermatitis, and reduces the efficiency of enzymes required in the folate/methionine pathway such as methylenetetrahydrofolate reductase (MTHFR) for which it is an active cofactor [8, 9]. Vitamin B6 has been shown to be important for normal cognitive function and in lowering the risk of cardiovascular disease [1, 10]. Vitamin B6 consists of three water- soluble pyrimidine derivatives, pyridoxine, pyridoxal, and pyridoxamine, together with their phosphate esters and was first defined in 1934 by Paul Gyorgy. All the three forms are precursors to the metabolically active form of vitamin B6 pyridoxal- 50-phosphate (P5P) [11]. The B6 vitamin pyrimidine derivatives are first oxidized to pyridoxal and then phosphorylated in the liver to P5P. P5P is the main circulatory form of B6 and is considered an accurate measure of individual B6 status [12]. P5P plays a vital role as a cofactor in a large number of essential enzymatic reactions including protein metabolism, niacin production, and neurotransmitter function [11]. Vitamin B6 is photosensitive and can be found in significant quantities in bananas, nuts and seeds, liver, poultry, most fish, wheat germ, and yeast. Vitamin B12 has a colorful history involving three Nobel prizes resulting from its discovery, isolation, and the resultant chemical structure. Until the 1920s, pernicious anemia, a blood disorder involving the failure of red blood cells to develop, usually resulted in death [4]. Two physicians, George Minot and William Murphy, followed up on George Whipples studies that liver could improve the formation of red corpuscles in anemic dogs. They began feeding their patients large amounts of liver and, in 1926, were able to announce that a daily diet of about a pound of liver could indeed control the deadly anemia. The active liver component so effective in the treatment of anemia was to become known as vitamin B12 [4]. In 1956, Dorothy Hodgkin completed studies on the chemical structure of vitamin B12 using X-ray crystallography and received the Nobel Prize in chemistry in 1964 for her efforts. Robert Woodward finally synthesized Vitamin B12 in 1971. Food sources rich in vitamin B12 are eggs, meat, poultry, shellfish, milk, and milk products. Vitamin B12 functions as a cofactor for methionine synthase that transfers a methyl group from folate to homocysteine to synthesize methionine and yield tetrahydrofolate, which is ultimately involved in the DNA synthesis and the formation of red blood cells [2], Vitamin B12 is also vitally important for the normal functioning of the brain, because of its role in maintaining the myelin sheath surrounding nerve cells [1].

25.2 Physicochemical and Transport Properties

In 1943, the chemical structure of folate was revealed and in its synthesized pure crystalline form was widely used to treat several types of anemia (Scheme 25.1). These synthesized group of compounds were made up of heterocyclic compounds based on the 4-[(pteridin-6-methyl)amino]benzoic acid skeleton (PABA) that was associated with varying numbers of glutamate residues [4]. Natural folates that are 25.2 Physicochemical and Transport Properties j419

Scheme 25.1 Showing chemical structure of (a) folic acid, (b) riboflavin (vitamin B2), (c) B12, and (d) B6. readily found in leafy green vegetables, liver, and yeast were found to differ structurally from these synthetic forms. Primarily, polyglutamated compounds are formed through the conjugation of additional glutamate groups as well as various di- or tetrahydro forms resulting from the reduction of the pteridine rings [4]. Folic acid is now considered to denote the compound in the oxidized or synthetic form, whereas folate is used to describe the reduced or natural form. Humans do not generate folate endogenously as they are not capable of synthesizing PABA and thus need to maintain cellular folate levels through an adequate diet or supplementation [4]. The conversion of dietary folate to an active intracellular coenzyme requires many physiological and biochemical processes. For folate, a conjugase enzyme is required to deconjugate polyglutamated folate in the small intestine; receptors are required for active uptake into the intestinal brush border epithelium, carried to the liver by the hepatoportal circulation where monoglutamated folate is reduced and methylated to form 5-methyltetrahydrofolate that is exported into the blood, and it is then taken up by a receptor/pinocytosis mechanism where it is subsequently stored in cells in the polyglutamated form by the activity of folyl-gamma-polyglutamate synthetase [2]. The capacity of 5-methyltetrahydrofolate to donate its methyl group for the regeneration of methionine from homocysteine (Hcy) depends on the activity of methionine 420j 25 Folate and Vitamins B2, B6, and B12

synthase [2]. On the other hand, the activity of thymidylate synthase and MTHFR determines the probability of 5,10-methylenetetrahydrofolate donating its methyl group for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). Defects in any one or more of the key enzymes and uptake proteins for folate conversion can limit the bioavailability of intracellular folate necessary for essential metabolic reactions [2]. Free riboflavin (7,8-dimethyl-10-ribityl-isoalloxazine), which is present in milk and eggs, consists of an isoalloxazine ring that is bound to a ribitol side chain (Scheme 25.1) [8]. Most riboflavins are present in the form of the derivatives FAD or FMN that occur in a noncovalently bound form to enzymes that allow this form of riboflavin to be readily available for absorption [8]. For dietary riboflavin to be absorbed, riboflavin in the free form needs to be hydrolyzed by enterocyte phosphatases to form FMN that is further converted to FAD. Free riboflavin is transported into the plasma bound to immunoglobulins or protein albumins [8]. P5P is one of natures most versatile cofactors, playing a major role in the metabolism of different amino acids that are found in numerous pathways, ranging from the interconversion of a amino acids to the synthesis of antibiotic compounds [13]. The structural features of the pyridoxal molecule essential for the catalysis of these nonenzymatic reactions are the formyl group, the phenolic group, and the heterocyclic ring with nitrogen arranged in the 4, 3, and 1 positions [11]. This allows this versatile molecule to be directly involved in reactions involving transamination of amino acids to keto acids to be used as metabolic fuel, decarboxylation of amino acids to yield important hormones and neurotransmitters, as well as having a role as a cofactor for enzymes in the transulfuration pathway such as cystathionine b-synthase required for antioxidant production [13]. Pyridoxine and its derivatives are absorbed by simple diffusion in the upper small intestine and transported to the liver to be transformed into P5P, which is then exported out of the liver bound to an albumin carrier. B12 is the most chemically complex of all the vitamins (Scheme 25.1). The structure of B12 is based on a corrin structure similar to the porphyrin ring found in hemoglobin, chlorophyll, and cytochrome [14]. The central metal ion is cobalt where four of the six coordination sites are provided by the corrin ring and a fifth by a dimethylbenzimidazole group [14]. The sixth coordination site within the molecule can be variable with one of the four ions being present including a cyano (CN), a 0 hydroxyl group ( OH), a methyl ( CH3), or a 5 -deoxyadenosyl group [14].

25.3 Bioavailability and Metabolism of Active Compounds

25.3.1 Bioavailability

Bioavailability can best be defined as the proportion of a nutrient ingested that can be made available for metabolic processes and/or ultimate storage. The bioavailability 25.3 Bioavailability and Metabolism of Active Compounds j421 of micronutrients can be influenced by a multitude of different factors. In the case of folate, bioavailability is governed by the degree of intestinal absorption [15]. Most naturally occurring dietary folate exists in a polyglutamyl form that needs to be deconjugated by the pH-dependent (pH 6.5–7.0) enzyme folylpoly g-glutamyl carboxypeptidase (FGCP) [15]. This enzyme requires zinc as an active cofactor and is associated with the jejunal brush border membrane cleaving glutamate moieties from the folate molecule. Folate is therefore transported and subsequently absorbed in the intestine in the monoglutamate form. Reduction in the effectiveness of this enzymatic cleavage could impair bioavailability of folate resulting from decreased absorption from polyglutamate sources [15]. Certain genetic polymorphisms have been suggested to influence folate bioavailability. Polymorphisms within the glutamate carboxypeptidase II gene such as H475Y have been shown to reduce the activity of the FGCP enzyme resulting in impaired intestinal folate absorption and subsequent bioavailability [16]. The primary route for membrane transport of reduced folates into mammalian cells and tissues is regulated by the reduced folate carrier system. Polymorphisms within the reduced folate carrier such as the A80G have been shown to influence the absorption of folate resulting in lowered folate bioavailability [17]. It has also been noted that alterations in pH within the upper small intestine resulting from physiological conditions or medications could impair folate absorption [18]. Theupperileumisthemainsiteforriboflavin absorption and depends on an active transport system rather than passive diffusion. The bioavailability of riboflavin from various food sources has not been well characterized, but it has been estimated that at least 95% of food flavin can be absorbed per meal [19, 20]. It has been shown that dietary fiber can enhance the absorption of riboflavin and therefore increases its bioavailability by slowing down the movement of chyme in the intestine, increasing the interaction time between the riboflavin and the absorption sites [21]. B6 vitamers occur in various forms in both animal and plant food sources. The forms present in plant sources consist mainly of pyridoxine and the phosphorylated forms that have reduced bioavailability. Further to this, a large proportion of plant origin B6 is glucosylated resulting in a reduction in vitamin B6 bioavailability [22]. Vitamin B6 present in food from animal sources consists mainly of pyridoxamine and pyridoxal that exhibit high availability, up to 100% in tuna, while in foods from plant sources it is considerably lower ranging 20–40% [23]. Vegetarians therefore are at particular risk for low vitamin B6 intake. The conversion of dietary vitamin B12 to intracellular active coenzyme requires many physiological processes involving a number of intermediate steps. For vitamin B12, at least five different peptides (R binder, intrinsic factor, ileal receptors, transcobalamin I, and transcobalamin II) are required to deliver vitamin B12 from the gut to the tissues, and a further four enzymes (cblF, cblC/D, microsomal reductase, and cblE/G) are necessary to convert vitamin B12 to the appropriate reduced state to function as a coenzyme with methionine synthase [2]. Defects in any one or more of the key enzymes and uptake proteins can limit the bioavailability of vitamin B12 resulting in a state of cellular deficiency, regardless of adequate dietary intake [2, 24]. 422j 25 Folate and Vitamins B2, B6, and B12

In the dietary intakes reference based on populations in the United States, it is assumed that 50% of dietary vitamin B12 is absorbed by healthy adults [25].

25.3.2 Metabolism

Folate and vitamins B2, B6, and B12 play an important role in DNA metabolism and maintenance (Figure 25.1) [1, 2]. Folate is required for the synthesis of dTMP from dUMP [4]. Under conditions of folate deﬁciency, dUMP accumulates and as a result uracil is incorporated into DNA instead of thymine [26]. There is good evidence to suggest that excessive incorporation of uracil in DNA not only leads to point mutations but may also result in the generation of single- and double-stranded DNA breaks, chromosome breakage, and micronucleus formation [27, 28]. It has been proposed that this mechanism is the likely cause of increased colon cancer risk associated with low-folate intake [1, 6]. Some evidence suggests that both vitamin B12 and vitamin B6 may similarly cause high uracil incorporation by restricting synthesis of the folate form required for dTMP synthesis (i.e., 5,10-methylenetetrahydrofolate), resulting in increased chromosome breakage and genome instability [1, 7]. Deﬁciency of folate, B6, and B12 mimic radiation exposure causing single- and double-strand breaks that are a strong predicative risk factor for certain cancers [3]. Folate and vitamin B12 are required for the synthesis of methionine and S-adenosyl methionine (SAM), the common methyl donor required for the maintenance of methylation

Figure 25.1 Metabolism of folic acid. SAM, deoxythymidine monophosphate; Cob(I), S-adenosyl methionine; MTRR, methionine reduced form of vitamin B12; Cob(III), oxidized synthase reductase; MTR, methionine synthase; form of vitamin B12. Adapted from Wagner C. SHMT, serine hydroxymethyltransferase; THF, (1995) Biochemical role of folate in cellular tetrahyrdofolate; DHF, dihydrofolate; MTHFR, metabolism. In: Folate in Health and Disease methylenetetrahydrofolate reductase; dUMP, (ed. L.B. Bailey), Marcel Dekker, New York, p. 26. deoxyuridine monophosphate; dTMP, 25.4 Mechanisms of Protection – In Vitro Studies j423 patterns in DNA that determine gene expression and DNA conformation [7, 29]. When the concentration of vitamin B12 and methionine is low, both SAM synthesis and methylation of DNA are reduced, inhibition by SAM of MTHFR is minimized resulting in the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, thus favoring an increase in the dUMP pool and the uracil incorporation into DNA [2]. Deficiencies in folate, vitamin B6, and B12 can therefore lead to (a) an elevated DNA damage rate and altered methylation of DNA, both of which are important risk factors for cancer [27, 28], (b) an increased level in Hcy status, an important risk factor for cardiovascular disease, neural tube defects, and neurodegenerative disorders [3–5], (c) deficiencies in vitamin B2 and B6 that act as cofactors for MTHFR and cystathionine b synthase, respectively, may lead to elevated levels of Hcy and deficiencies in the availability of glutathione, a natural antioxidant. These same metabolic deviations may also play an important role in some developmental and neurological abnormalities [27, 28]. The blood levels of folate and vitamin B12 required to prevent clinical outcomes such as anemia or hyperhomocysteinemia are properly defined. However, it is still uncertain whether these levels as well as those of vitamin B2 and B6 are adequate to minimize chromosome damage rates, optimize DNA methylation status, and maintain genome stability. Evidence is provided from in vitro studies with human cells and in vivo cross-sectional and intervention studies in humans to identify the role these B group vitamins play in genome stability and the associated disease risk.

25.4 Mechanisms of Protection – In Vitro Studies

It has been shown that fragile sites in chromosomes are expressed when human lymphocytes are cultured in the absence of folic acid and thymidine in culture medium [2, 30]. Furthermore, under these conditions chromosome breakage and micronucleus (MN) expression are increased simultaneously suggesting a similar mechanism underlying the expression of fragile sites and chromosome breakage [2, 30]. Reidys experiments showed that lymphocytes cultured in folic acid-deficient medium exhibit increased levels of excision repair during G2 because the cytosine arabinoside-induced chromosome aberration level (which is indicative of excision repair activity) was more than doubled by this treatment and further enhanced by the addition of deoxyuridine [2]. The treatment of human lymphoid cells in culture with methotrexate results in a large increase in the dUTP/dTTP ratio and a much increased incorporation rate of uracil in DNA [31]. The connection between uracil incorporation and the generation of DNA strand breaks was confirmed in more recent studies using the single-cell gel electrophoresis method; in this method, the extent of uracil incorporation was measured by treating the nuclei with uracil DNA glycosylase followed by the measurement of resulting DNA breaks [31]. Vitamin B12 deficiency was found to decrease genomic methylation by 35% and increase uracil incorporation by 105% in rat colonic tissue after a 10-week trial. The B12-deficient diet was not severe enough to cause anemia but still created aberrations in base 424j 25 Folate and Vitamins B2, B6, and B12

substitution and methylation of colonic DNA, potentially contributing to increased carcinogenic risk [32]. Folate deﬁciency has also been shown to induce aneuploidy in human lymphocytes for chromosomes 17 and 21, which is observed in breast cancer and leukemia. Increased risk for these cancers has been associated with folate deﬁciency, which may result in demethylation of centromeric repeat sequences, inducing abnormal chromosomal distribution during nuclear division [33].

25.5 Results from Human Studies

The early evidence of chromosome damage in human cells in vivo from folate and vitamin B12 deficiency was first obtained from studies linking the expression of Howell–Jolly bodies in erythrocytes with megaloblastic anemia [2]. Howell–Jolly bodies are whole chromosomes or chromosome fragments that lag behind at anaphase during the production and maturation of the red blood cell and in fact they are the same as micronuclei, the alternative and the most commonly used term for this chromosome damage biomarker. Micronucleated erythrocytes in humans are most readily observed in splenectomized subjects because the spleen actively filters micronucleated erythrocytes from the blood [2, 34]. A case study of a 30-year-old male with Crohns disease with a very high level of micronuclei in erythrocytes (67/1000 cells) showed that this was associated with a low serum folate (1.9 ng/ml; normal range >2.5 ng/ml) and a low red cell folate (70 ng/ml; normal range >225 ng/ml). Micronucleus frequency was reduced to 12/1000 cells, serum folate increased to >20 ng/ml, red cell folate increased to 1089 ng/ml after 25 days with a daily oral dose of 25 mg folinic acid [34]. One of the main observations of this study was that minimum spontaneous MN frequencies were observed only when serum folate levels exceeded 15–20 ng/ml that was higher than the values accepted as normal by clinicians for prevention of anemia (i.e., 6–15 ng/ml) [2]. A small cross-sectional study (N ¼ 22) on the influence of blood micronutrients on micronucleus frequency in erythrocytes of splenectomized subjects preselected because they had among the highest or lowest micronucleus frequencies from a larger population (N ¼ 122) showed that elevated micronucleus index was strongly associated with low levels of serum folate (<4 ng/ml) and low levels of plasma B12 (<200 pg/ml) [2]. Blood samples from the same cohort of individuals analyzed in the studies of splenectomized individuals [2, 34] were also analyzed for uracil content in lymphocytes [27, 28]. Uracil levels in DNA were 70-fold higher in individuals with serum folate <4 ng/ml relative to individuals with serum folate >4 ng/ml. Uracil levels in DNA were rapidly reduced (within 3 days) after daily supplementation with 5 mg folic acid in both the deficient (<4 ng/ml serum folate) and non- deficient groups (>4 ng/ml serum folate). These changes were accompanied by corresponding reductions in erythrocyte micronucleus frequency but over a longer time frame [27, 28]. 25.5 Results from Human Studies j425

A small number of case studies link vitamin B12 deficiency with increased levels of chromosome aberrations [2]. Of 10 patients with pernicious anemia (which is a manifestation of vitamin B12 deficiency), 3 had elevated chromosome aberrations, and 8 had increased levels of micronucleus frequency in bone marrow preparations [35]. A female infant with transcobalamin II (the transporter for vitamin B12 in plasma) deficiency showed elevated levels of aneuploidy (hypodiploidy in approximately 30% of the cells) and increased chromosomal breakage in the bone marrow with reduction in hypodiploidy to 10% of cells after 5 months treatment with folate and vitamin B12 supplements [2]. Combined deficiency in folic acid and vitamin B12 was associated with (a) transient 7q- in one patient and (b) in a series of patients produced a persistent abnormal deoxyuridine suppression test result (which is indicative of inadequate capacity to generate dTMP) and increased frequency of chromosomes showing despiralization and chromosomal breaks [2]. The latter studies showed that it took up to 84 days after supplementation with folic acid and vitamin B12 before the deoxyuridine suppression and the chromosomal morphology tests returned to normal. With regard to the question of chromosome despiralization, it is important to note that the DNA methylation inhibitor, 5-azacytidine, induces distinct undercondensation of the heterochromatin regions of chromosomes 1, 9, 15, 16, and Y and the specific loss of these chromosomes as micronuclei in human lymphocytes in vitro [36]. Similarly the immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which is caused by mutation in the DNA methyl transferase gene, is characterized by the despiralization of heterochromatin of chromosomes 1, 9, and 16 and loss of these chromosomes into micronuclei and nuclear blebs [2]. A series of studies have been performed to investigate the interrelationship between DNA damage in somatic cells and blood status for folate, vitamin B12, and homocysteine. As a marker of chromosome damage, the cytokinesis-block micronucleus method was used in lymphocytes, which has been shown in numerous studies to be a reliable and sensitive biomarker of chromosome breakage and chromosome loss that occurs spontaneously or as a result of elevated exposure to genotoxins [2, 5]. Preliminary studies comparing DNA damage rate and micronutrient status in vegetarians and nonvegetarians had indicated that there was a significant negative correlation between micronucleus frequency in lymphocytes and plasma vitamin B12 status in young men [37]. Theprevalenceoffolatedeficiency, vitamin B12 deficiency, and hyperhomocysteinemia was investigated in 64 healthy men aged between 50 and 70 years, and the relationship of these micronutrients with the micronucleus frequency in cytokinesis-blocked lymphocytes was determined [38]. Of the men, 23% had serum folate concentration less than 6.8 nmol/l, 16% had red blood cell folate concentration less than 317 nmol/l, 4.7% were vitamin B12 deficient (<150 pmol/l), and 37% had plasma homocysteine levels greater than 10 mmol/l. In total, 56% of the apparently healthy men had nonoptimal values for folate, vitamin B12, or homocysteine. The micronucleus index of these men (mean SEM, 19.2 1.1, P ¼ 0.02, N 34) was significantly elevated when compared to that of men who had higher 426j 25 Folate and Vitamins B2, B6, and B12

concentrations of folate and vitamin B12 and lower plasma homocysteine (16.3 1.3, N ¼ 30). Interestingly, the micronucleus index in men with normal concentrations of folate and vitamin B12 but homocysteine levels greater than 10 mmol/l (19.4 1.7, P < 0.05, N ¼ 15) was also significantly higher when compared to those with normal folate, vitamin B12, and homocysteine less than 10 mmol/l. Micronucleus index and plasma homocysteine were also significantly (P ¼ 0.0086) and positively correlated (r ¼ 0.415) in those subjects who were not deficient in folate or vitamin B12. The micronucleus index was not significantly correlatedwithfolateindices,buttherewasasignificant (P ¼ 0.013) negative correlation with serum vitamin B12 (r ¼0.315). It was apparent that elevated homocysteine status, in the absence of vitamin deficiency, and low, but not deficient, vitamin B12 status are important risk factors for increased chromosome damage in lymphocytes [38]. Across-sectionalstudy(N ¼ 49 males, 57 females) and a randomized double- blind placebo-controlled dietary intervention study were performed (N ¼ 31, 32 per group) to determine the effect of folate and vitamin B12 on DNA damage (micronucleus formation and DNA methylation) and plasma homocysteine (Hcy) in young Australian adults aged 18–32 years [2, 24]. None of the volunteers was folate deficient (i.e., red cell folate <136 nmol/l), and only 4.4% (all females) were vitamin B12 deficient (i.e., serum B12 < 150 pmol/l). The cross-sectional study showed that (a) the frequency of micronucleated (MNed) cells was positively correlated with plasma Hcy in males (R ¼ 0.293, P < 0.05) and (b) in females MNed cell frequency was negatively correlated with serum B12 (R ¼0.359, P < 0.01), but (c) there was no significant correlation between micronucleus index and red cell folate status. The results also showed that the level of unmethylated CpG (DNA) (measured using the Sss1 methylase method) was not significantly related to vitamin B12 or folate status. The dietary intervention involved supplementation with 700 mgfolic acid and 7 mg vitamin B12 in wheat bran cereal for 3 months followed by 2000 mg folic acid and 20 mg vitamin B12 via tablets for a further 3 months. In the supplemented group, MNed cell frequency was significantly reduced during the intervention by 25.4% in those subjects with initial MNed cell frequency in the high 50%, but there was no change in those subjects in the low 50th percentile for initial MNed cell frequency. The reduction in MNed cell frequency was significantly correlated with serum B12 (R ¼0.49, P < 0.0005) and plasma Hcy (R ¼ 0.39, P < 0.006) but was not significantly related to red cell folate. DNA methylation status was not altered in the supplemented group. The greatest decrease in plasma Hcy (by 37%) during the intervention was observed in those subjects in the supplemented group with initial plasma Hcy in the high 50% and correlated significantly with increases in red cell folate (R ¼0.64, P < 0.0001), but not with serum B12. The results from this study suggest that (a) MNed cell frequency is minimized when plasma Hcy is below 7.5 mmol/l and serum B12 is above 300 pmol/l, and (b) dietary supplement intake of 700 mg folic acid and 7 mg vitamin B12 is sufficient to minimize MNed cell frequency and plasma Hcy in young adults. Thus, it appears that elevated plasma Hcy, a risk factor for cardiovascular disease, may also be a risk factor for chromosome damage. 25.5 Results from Human Studies j427

Other studies have shown that global DNA methylation in lymphocytes or colonic tissue is influenced by the extent of folate intake. The depletion–repletion study performed by Jacob et al. [2] with postmenopausal women in a metabolic unit showed more than 100% increase in DNA hypomethylation after 9 weeks on low folate (56–111 mg/day) and a subsequent increase in DNA methylation after a further 3 weeks on a high folate diet (286–516 mg/day). Fowler et al. [39] and Cravo and colleagues [40] showed, using the Sss1 methylase assay, that cervical and gastric/ colonic/rectal epithelium DNA methylation is significantly correlated to serum and tissue folate concentration, respectively. Furthermore, it was shown that intrinsic methylation of DNA was lower in the normal colorectal mucosa of adenoma and carcinoma patients; however, supplementation with 10 mg folic acid/day for 6 months increased methylation 15-fold (P < 0.0002) and 3 months after cessation of therapy methylation decreased fourfold [2]. Vitamin B6 intake has been found to be inversely associated with increased risk for a number of cancers including prostate, lung, colorectal, and breast cancer as well as cardiovascular diseases [1, 6, 7]. Supplementation appears to reduce the risk, but it has been suggested that it may be necessary to take levels above the RDA (>1.3 mg/ day) to obtain maximum effectiveness for risk management. Diets low in vitamin B6 have also been associated with brain dysfunction and neurologic conditions, such as depression and Parkinsons disease [3, 41]. B6 is required for the neurotransmitter synthesis of serotonin and dopamine, which is essential for normal nerve cell communication [42]. There is not enough evidence to indicate whether B6 deficiency affects genome stability, even though this is plausible given its role as a cofactor for serine hydroxymethyl transferase in the folate/methionine cycle (Figure 25.1). Deficiencies in folate, B2, B6, and B12 can cause accumulation of Hcy, a risk factor for vascular diseases such as microangiopathy and macroangiopathy [3, 8]. Hcy is metabolized through two major routes, the B6-dependent transulfuration pathway and the folate-, B12- and B2-dependent remethylation to methionine. Recent studies have shown that Hcy accumulation resulting from low intake of these B vitamins contribute to AD by exposing neurons to increased levels of DNA damage leading to eventual cell death [3, 5]. Nutritional supplementation of both B6 and magnesium has also been claimed to alleviate the symptoms of autism resulting in an improvement in verbal IQ [43]. It has been shown in riboflavin-deficient rats exposed to hepatocarcinogens that there is an increase in DNA strand breakage and in the induction of repair enzymes (poly(ADP-ribose) polymerase, DNA polymerase beta, and DNA ligase) that contribute to the incidence of malignant transformations. Both the effects were found to be reversed to near normal levels upon supplementation (1 mg/100 g diet), suggesting a chemopreventative role for this vitamin [8]. Riboflavin deficiency has also been implicated as a risk factor for cervical dysplasia in a cross-study of 257 cases showing an increased risk when riboflavin intake falls below 1.2 mg/day [8]. Both in vitro and in vivo studies suggest that high riboflavin intake in a low-folate background could increase genome instability in lymphocytes [9, 44]. The interactive relationships between folate, MTHFR genotype, and its cofactor riboflavin are complex and have been shown to affect a number of biomarkers of 428j 25 Folate and Vitamins B2, B6, and B12

Figure 25.2 Mechanistic framework explaining (iv) initiation of cancer caused by CpG the interrelationship between MTHFR genotype, hypomethylation and aneuploidy; and riboflavin (R), and folic acid (F) with respect to (v) initiation of cancer caused by increased BFB (i) CpG methylation and uracil in DNA; cycles, MNi (originating from acentric (ii) aneuploidy and micronuclei (MNi) chromosome fragments), NBUDs, and NPBs. originating from chromosome loss events; For brevity, other carcinogenic mechanisms (iii) MNi (originating from acentric chromosome induced by altered genome methylation, such as fragments), nuclear buds (NBUDs), silencing of tumor suppressor genes and/or nucleoplasmic bridges (NPBs), and activation of oncogenes, are not included in the breakage–fusion–bridge (BFB) cycles; diagram.

genomic instability. A flow diagram of these relationships and potential outcomes are highlighted in Figure 25.2. The framework predicts that (1) as folate concentration increases, genomic instability events arising from aneuploidy and breakage–fusion–bridge cycles (BFB) are minimized, (2) genome hypomethylation and aneuploidy are minimized during high MTHFR activity, (3) the risk of BFB cycles is increased under high-riboflavin and low-folate conditions, (4) the risk of genome hypomethylation and aneuploidy are maximized under both low-riboflavin and low-folate conditions, (5) reduced MTHFR activity may decrease MNi frequency caused by uracil incorporation and subsequent chromosome breakage but may increase MNi originating from chromosome loss or gain resulting from DNA hypomethylation. These complex relationships may go some way to explain why the MTHFR C677T polymorphism may influence the relative risk for certain clinical outcomes. The induction of chromosome breakage and BFB cycles caused by uracil incorporation into the DNA may be more relevant to conditions such as leukemia or lymphoma, whereas failure to sustain adequate SAM synthesis may be more relevant to cancers caused by DNA hypomethylation or aneuploidy-driven developmental defects such as Down syndrome [9]. 25.6 Impact of Cooking, Processing, and Other Factors on Protective Properties j429

25.6 Impact of Cooking, Processing, and Other Factors on Protective Properties

Nutritional content of various food types in relation to vitamin stability can be severely compromised as a result of methods used in food preparation, processing, and storage. Folate losses resulting from such processes are the result of a combination of thermal degradation and leaching of the vitamin into the cooking water [45]. Between 55 and 70% of folate has been shown to be retained in meat and poultry that has been boiled, stewed, or braised, while roasting or baking resulted in a retention rate of between 60 and 95% [46]. Fish prepared by poaching and/or steaming showed a folate retention rate of between 70 and 100%, while oven-baked fish showed folate losses of between 10 and 20% [46]. The effect of boiling on the folate retention rate in spinach and broccoli was found to be 49% and 44%, respectively, while steaming was found to have no effect on folate composition or the degree of availability [47]. Analysis of beef liver and strawberries before and after a 7-month freezing period showed no significant change in folate content [48]. Although the water-soluble vitamins are sensitive to heat treatments, riboflavin is unusual in that it is sensitive to the effects of both alkali and light but stable to heat and the oxidative effects of air. Riboflavin losses occur in food processing mainly as a result of leaching [49]. It has been shown that blanching of lima beans and peas resulted in a 30% reduction in riboflavin content, while cooked chicken retained significant amounts of riboflavin (up to 94%) [50, 51]. Riboflavin content following g irradiation of beef, lamb, pork, and turkey and grass prawns showed no change in the riboflavin content following post-irradiation cooking [52, 53]. The storage stability of riboflavin can be adequately maintained in dry products if protected from light [54]. Vitamin B6 is unstable under conditions of prolonged heat treatment common to both food preparation and processing. The stability of B6 depends on the vitamer content of the food, as pyridoxine has been shown to be more heat tolerant than both pyridoxal and pyridoxamine [55]. It has been shown that water blanching lima beans resulted in a vitamin B6 loss of between 19 and 24%, while steam blanching lead to a loss of only 15–17% [56]. Richardson et al. determined the vitamin B6 content of six foods that had been frozen, canned using conventional heat treatment, and irradiated using gamma rays. The B6 activity was found to be reduced after 9 months of storage at 20 C in the frozen foods and at ambient temperatures for the canned and irradiated foods. At each time point analyzed, the B6 activity of the heat-treated and irradiated food was between 40 and 60% the activity found in frozen foods [57]. Cooking methodology has been found to have a profound effect on B6 content [58]. Stewed or braised beef was found to have a reduced B6 content of 56 and 58%, respectively, with a similar reduction of 58 and 45% found under similar cooking conditions using pork. Part of the loss is attributed to the release of meat juice. If meat juice is included, a total B6 retention rate of 80 and 63% was found for beef and a 68 and 62% retention rate for pork [58]. Boiled vegetables have been shown to have reduced total vitamin B6 rates between 16 and 61%, whereas steaming resulted in higher B6 retention with the losses ranging between 10 and 24% [58]. 430j 25 Folate and Vitamins B2, B6, and B12

It has been shown that appreciable amounts of vitamin B12 are lost during milk processing. As a result of boiling milk for 5 min, a 30% reduction in B12 content was observed, whereas continual boiling for 30 min led to a 50% total reduction [59]. It has also been shown that milk pasteurization can lead to a 10% reduction in B12, whereas a 50% loss was evident after a 5 min microwave treatment [59]. Storage can also affect the B12 content of milk. Exposure of pasteurized milk to fluorescent light for 24 h showed a significant decrease in B12 concentration but was unduly affected following refrigeration for 9 days at 4 C in the dark under normal domestic conditions [60]. Further studies have shown that roasted meat had a B12 reduction of 33% while scrambled eggs, boiled eggs, andfried eggs varied in their B12bioavailability averaging 3.7%, 8.9%, and 9.2%, respectively [25]. The cooking of fish through various methods including boiling, frying, and steaming was found to reduce the B12 content to varying degrees ranging from 2–14% [61].

25.7 Conclusions

In conclusion, folate and vitamins B2, B6, and B12 play important roles in DNA synthesis and are essential for normal metabolic homeostasis. Deﬁciencies in these micronutrients can lead to genomic instability that can increase the risk of developmental and degenerative diseases. It may be possible that the above RDI intakes of these vitamins may be required in some individuals, as increasing evidence gathers for common single-nucleotide polymorphisms that alter signiﬁcantly the activity of proteins required for the absorption, transport, and metabolism of these vitamins. It is therefore anticipated that optimizing intake of folate and relevant B vitamins on an individual basis may be essential to maintain a healthy genomic and metabolic balance potentially leading to reduced risks for certain cancers and other related genome instability disorders.

References

1 Ames, B.N. (2001) DNA damage from 4 Hoffbrand, A.V. and Weir, D.G. (2001) micronutrient deﬁciencies is likely to be a The history of folic acid. British Journal of major cause of cancer. Mutation Research/ Haematology, 113, 579–589 Fundamental and Molecular Mechanisms of 5 Thomas, P. and Fenech, M. (2007) A Mutagenesis, 475,7–20. review of genome mutation and 2 Fenech, M. (2001) The role of folic acid Alzheimers disease. Mutagenesis, 22, and Vitamin B12 in genomic stability 15–33. of human cells. Mutation Research, 475, 6 Zhang, S.M., Moore, S.C., Lin, J., Cook, 57–67. N.R., Manson, J.E., Lee, I.M. and 3 Ames, B.N. (2004) A role for supplements Buring, J.E. (2006) Folate, vitamin B6, in optimizing health: the metabolic tune- multivitamin supplements, and colorectal up. Archives of Biochemistry and Biophysics, cancer risk in women. American Journal of 423, 227–234. Epidemiology, 163, 108–115. References j431

7 Zhang, S.M., Willett, W.C., Selhub, J., non-homocysteine related mechanism. Hunter, D.J., Giovannucci, E.L., Holmes, Life Sciences, 77, 2735–2742. M.D., Colditz, G.A. and Hankinson, S.E. 18 Gregory, J.F., III (2001) Case study: folate (2003) Plasma folate, vitamin B6, vitamin bioavailability. The Journal of Nutrition, B12, homocysteine, and risk of breast 131, 1376S–1382S. cancer. Journal of the National Cancer 19 Zempleni, J., Galloway, J.R. and Institute, 95, 373–380. McCormick, D.B. (1996) 8 Powers, H.J. (2003) Riboflavin (vitamin Pharmacokinetics of orally and B-2) and health. The American Journal of intravenously administered riboflavin in Clinical Nutrition, 77, 1352–1360. healthy humans. The American Journal of 9 Kimura, M., Umegaki, K., Higuchi, M., Clinical Nutrition, 63,54–66. Thomas, P. and Fenech, M. (2004) 20 Baker, C.J. (1997) Bioavailability of Methylenetetrahydrofolate reductase riboflavin. European Journal of Clinical C677T polymorphism, folic acid and Nutrition, 51, S38–S42. riboflavin are important determinants of 21 Roe, D.A., Wrick, K., Mclain, D. and Van genome stability in cultured human Soest, P. (1978) Effects of dietary fibre lymphocytes. The Journal of Nutrition, 134, sources on riboflavin absorption. 48–56. Federation Proceedings, 37, 756 (abstract). 10 Calvaresi, E. and Bryan, J. (2001) B 22 Reynolds, R.D. (1988) Bioavailability of Vitamins cognition and aging: a review. vitamin B-6 from plant foods. The The Journals of Gerontology, 56, P327–339. American Journal of Clinical Nutrition, 48, 11 Schneider, G., Kack, H. and Lindqvist, Y. 863–867. (2000) The manifold of vitamin B6 23 Roth-Maier, D.A., Kettler, S.I. and dependent enzymes. Structure, 8,R1–R6. Kirchgessner, M. (2002) Availability of 12 Leklem, J.E. (1990) Vitamin B6: a status vitamin B6 from different food sources. report. The Journal of Nutrition, 120, International Journal of Food Sciences and 1503–1507. Nutrition, 53, 171–179. 13 Metzler, D.E., Ikawa, M. and Snell, S.S. 24 Fenech, M., Aitken, C. and Rinaldi, J. (1953) The general mechanism for (1998) Folate, vitamin B12, homocysteine vitamin B6-catalysed reactions. Journal status and DNA damage in young of the American Chemical Society, 76, Australian adults. Carcinogenesis, 19, 648–652. 1163–1171. 14 Battersby, A.R. (1994) How nature builds 25 Watanabe, F. (2007) Vitamin B12 sources the pigments of life: the conquest of and bioavailability. Experimental Biology vitamin B12. Science, 264, 1551–1557. and Medicine, 232, 1266–1274. 15 McNulty, H. and Pentieva, K. (2004) Folate 26 Eto, I. and Krumdieck, C.L. (1986) Role of bioavailability. Proceedings of the Nutrition vitamin B12 and folate deficiencies in Society, 63, 529–536. carcinogenesis. Advances in Experimental 16 Devlin, A.M., Ling, E.H., Peerson, J.M., Medicine and Biology, 206, 313–330. Fernando, S., Clarke, R., Smith, A.D. and 27 Blount, B.C. and Ames, B.N. (1995) DNA Halsted, C.H. (2000) Glutamate damage in folate deficiency. Baillieres carboxypeptidase II: a polymorphism Clinical Haematology, 8, 461–478. associated with lower levels of serum folate 28 Blount, B.C., Mack, M.M., Wehr, C.M., and hyperhomocysteinemia. Human MacGregor, J.T., Hiatt, R.A., Wang, G., Molecular Genetics, 9, 2837–2844. Wickramasinghe, S.N., Everson, R.B. and 17 Yates, Z. and Lucock, M. (2005) G80A Ames, B.N. (1997) Folate deficiency causes reduced folate carrier SNP modulates uracil misincorporation into human DNA cellular uptake of folate and affords and chromosome breakage: implications protection against thrombosis via a for cancer and neuronal damage. 432j 25 Folate and Vitamins B2, B6, and B12

Proceedings of the National Academy of 37 Fenech, M. and Rinaldi, J. (1994) The Sciences of the United States of America, 94, relationship between micronuclei in 3290–3295. human lymphocytes and plasma levels of 29 Zingg, J.M. and Jones, P.A. (1997) Genetic vitamin C, vitamin E, vitamin B12 and folic and epigenetic aspects of DNA acid. Carcinogenesis, 15, 1405–1411. methylation on genome expression, 38 Fenech, M.F., Dreosti, I.E. and Rinaldi, evolution, mutation and carcinogenesis. J.R. (1997) Folate, vitamin B12, Carcinogenesis, 18, 869–882. homocysteine status and chromosome 30 Sutherland, G.R. (1979) Heritable fragile damage rate in lymphocytes of older men. sites on human chromosomes I. Factors Carcinogenesis, 18, 1329–1336. affecting expression in lymphocyte 39 Fowler, B.M., Giuliano, A.R., Piyathilake, culture. American Journal of Human C., Nour, M. and Hatch, K. (1998) Genetics, 31, 125–135. Hypomethylation in cervical tissue: is 31 Duthie, S.J. and McMillan, P. (1997) Uracil there a correlation with folate, status? misincorporation in human DNA detected Cancer Epidemiology, Biomarkers & using single cell gel electrophoresis. Prevention, 7, 901–906. Carcinogenesis, 18, 1709–1714. 40 Cravo, M., Fidalgo, P., Pereira, A.D., 32 Choi, S.-W., Friso, S., Ghandour, H., Gouveia-Oliveira, A., Chaves, P., Selhub, Bagley, P.J., Selhub, J. and Mason, J.B. J., Mason, J.B., Mira, F.C. and Leitao, C.N. (2004) Vitamin B-12 deficiency induces (1994) DNA methylation as an anomalies of base substitution and intermediate biomarker in colorectal methylation in the DNA of rat colonic cancer: modulation by folic acid epithelium. The Journal of Nutrition, 134, supplementation. European Journal of 750–755. Cancer Prevention, 3, 473–479. 33 Wang, X., Thomas, P., Xue, J. and Fenech, 41 Bernstein, A.L. (1990) Vitamin B6 in M. (2004) Folate deficiency induces clinical neurology. Annals of the New York aneuploidy in human lymphocytes in Academy of Sciences, 585, 250–260. vitro-evidence using cytokinesis-blocked 42 Leklem, J.L. (1999) Vitamin B6, in Modern cells and probes specific for chromosomes Nutrition in Health and Disease (eds M.E. 17 and 21. Mutation Research, 551, Shils, J.A. Olsen, M. Shike and A.C. Ross), 167–180. Baltimore, Williams and Wilkins, pp. 34 Everson, R.B., Wehr, C.M., Erexson, G.L. 413–421. and MacGregor, J.T. (1988) Association of 43 Angley, M., Semple, S., Hewton, C., marginal folate depletion with increased Paterson, F. and Mckinnon, R. (2007) human chromosomal damage in vivo: Children and autism-part 2-management demonstration by analysis of with complimentary medicines and micronucleated erythrocytes. Journal of the dietary interventions. Aust Fam Physician, National Cancer Institute, 80, 525–529. 36, 827–830. 35 Jensen, M.K. (1977) Cytogenetic findings in 44 Fenech, M., Baghurst, P., Luderer, W., pernicious anaemia. Comparison between Turner, J., Record, S., Ceppi, M. and results obtained with chromosome studies Bonassi, S. (2005) Low intake of calcium, and, the micronucleus test. Mutation folate, nicotinic acid, vitamin E, retinol, Research, 45,249–252. {beta}-carotene and high intake of 36 Guttenbach, M. and Schmid, M. (1994) pantothenic acid, biotin and riboflavin are Exclusion of specific human significantly associated with increased chromosomes into micronuclei by genome instability – results from a dietary 5-azacytidine treatment of lymphocyte intake and micronucleus index survey in cultures. Experimental Cell Research, 211, South Australia. Carcinogenesis, 26, 127–132. 991–999. References j433

45 Eitenmiller, R.R. and Landen, W.O. (1999) 54 Borenstein, B. (1971) Rationale and Folate, CRC Press, Boca Raton, FL. technology of food fortification with 46 Bergstrom, L. (1994) National food vitamins, minerals and amino acids. Food administration, Uppsala, Sweden. Technology, 2, 171–186. 47 McKillop, D.J., Pentieva, K., Daly, D., 55 Gregory, J.F. and Hiner, M.E. (1983) McPartlin, J.M., Hughes, J., Strain, J.J., Thermal stability of vitamin B6 Scott, J.M. and McNulty, H. (2002) The compounds in liquid model food systems. effect of different cooking methods on Journal of Food Science, 48, 1323–1327. folate retention in various foods that are 56 Raab, C.A., Luh, B.S. and Schweigert, B.S. amongst the major contributors to folate (1973) Effects of heat processing on the intake in the UK diet. The British Journal of retention of vitamin B6 in lima beans. Nutrition, 88, 681–688. Journal of Food Science, 38, 544–545. 48 Vahteristo, L.T., Lehikoinen, K.E., 57 Richardson, L.R., Wilkes, S. and Ritchey, Ollilainen, V., Koivistoinen, P.E. and S.J. (1961) Comparative vitamin B6 activity Varro, P. (1998) Oven-baking and Frozen of frozen, irradiated and heat-processed Storage Affect Folate Vitamer Retention. foods. The Journal of Nutrition, 73,363–368. Lebensmittel-Wissenschaft und Technologie, 58 Bognar, A. (1993) Studies on the influence 31, 329–333. of cooking on the vitamin B6 content of 49 Berlitz, H.D. and Grosch, W. (1999) food. Bioavailability 93. Nutritional, Vitamins, Springer-Verlag, Germany. chemical and food processing implications 50 Guerrant, N.B. and OHara, M.B. (1953) of nutrient availability, conference Vitamin retentions in peas and lima beans proceedings, part 2, May 9–12. (ed. U. after blanching, processing in tin and in Schlemmer) Bundesforschunganstalt Fur glass, after storage and after cooking. Food Ernahrung, Ettlingen, pp. 346. Technology, 7, 473–477. 59 Watanabe, F., Abe, K., Fujita, T., Goto, M., 51 Al Khalifa, A.S. and Dawood, A. (1993) Hiemori, M. and Nakano, Y. (1998) Effects Effects of cooking methods on thiamin and of microwave heating on the loss of vitamin riboflavin contents of chicken meat. Food B(12) in foods. JournalofAgriculturaland Chemistry, 48,69–74. Food Chemistry, 46, 206–210. 52 Fox, J.B., Lakritz, L., Hampson, J., 60 Andersson, I. and Oste, R. (1994) Richardson, F., Ward, K. and Thayer, D.W. Nutritional quality of pasteurized milk. (1995) Gamma irradiation effects on Vitamin B12, folate and ascorbic acid thiamin and riboflavin in beef, lamb, pork, content during storage. International Dairy and turkey. Journal of Food Science, 60, Journal, 4, 161–172. 596–598. 61 Nishioka F M., kanosue, F., Miyamoto, E. 53 Lee, K.F. and Hau, L.B. (1996) Effect of and Watanabe, F. (2006) Characterization gamma irradiation and post irradiation of vitamin B12 in skipjack meats and loss cooking on riboflavin, riboflavin and of the vitamin from the fish meats by niacin contents of grass prawns (Penaeus various cooking conditions. Vitamins, 80, Monodon). Food Chemistry, 55, 379–382. 507–511. j435

26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc Dianne Ford and John Hesketh

26.1 Introduction

Increased intake of a number of essential dietary micronutrients has been linked with cancer prevention and many of these are the focus of other chapters in this book. The current chapter focuses on the essential micronutrients selenium and zinc since intakeofboththesenutrientsisreportedtobemarginalinmanypartsoftheworld[1,2] and, although they have a number of separate and different biological activities consistent with cancer chemoprevention, antioxidant activity and effects on cell cycle progression and apoptosis are likely components of the anticancer activity of both these micronutrients. The following sections describe, separately, the biological activities of selenium and zinc at the cellular level that are consistent with anticancer activity.Ouraimistosummarizeandevaluateevidencerelatingdirectlytothepremise that dietary sufﬁciency of both micronutrients is important in cancer prevention.

26.2 Selenium and Zinc in Food

Dietary selenium mostly occurs as selenomethionine, selenocysteine, or selenium methylselenocysteine, all of which are metabolized to selenide. Selenide is central to selenium metabolism (see Figure 26.1). It is the starting point for synthesis of the amino acid selenocysteine that is incorporated into 25 selenoproteins where it takes part in a range of oxidoreductase reactions [3, 4]. Selenocysteine is incorporated into the selenoproteins during their synthesis by a unique mechanism that uses a speciﬁc tRNA that recognizes the UGA codon; recoding of UGA for selenocysteine-tRNA requires a speciﬁc structure within the 30 untranslated region of the selenoprotein mRNA (selenocysteine insertion sequence, SECIS) and a number of proteins that bind to this structure [3]. Alternatively, selenide can be converted into a series of selenometabolites (methyselenol, dimethyselenide) by methylation reactions. There is active debate over whether the chemopreventive effects of increased selenium

Figure 26.1 Se metabolism. Dietary Se is recoding of the UGA by a specific structure commonly found as the amino acids (SECIS: selenocysteine insertion sequence) in selenomethionine and selenocysteine or as the 30 untranslated region of the selenoprotein Se-methylselenocysteine. In the body, these can mRNA. Alternatively, Se-methylselenocysteine be converted to selenide that is used to can be metabolized to methylselenol that is synthesize the selenoproteins (selenocysteine- though to be biologically active, for example, containing proteins) by a mechanism involving a inducing apoptosis. Active forms of selenium are specific tRNA recognized by UGA and involving in bold boxes.

intake are through the increased activity of selenoproteins or the effects of selenometabolites, particularly those generated through methylation of selenide [4]. Foods based on animal proteins, in particular meat but also eggs and diary products, provide zinc in its most readily bioavailable form. Chelation of zinc by dietary phytates in plant-based sources reduces zinc absorption and contributes to zinc deﬁciency in developing countries, but there is currently no evidence for adverse health effects resulting from reduced zinc absorption from varied vegetarian diets in developed countries [5].

26.3 Mechanisms of Chemoprevention by Selenium: Results of In Vitro and Animal Studies

Selenium is incorporated into selenoproteins, some of which play important roles in protecting cells from oxidative damage [1, 3, 4, 6]; since such damaging events have been suggested to initiate tumorigenic events, there is a physiological rationale for selenium having a chemopreventive effect. A large number of animal studies have investigated the effect of increased selenium intake on induction of tumors and the majority of studies show a signiﬁcantly lower tumor incidence [4, 7]. 26.3 Mechanisms of Chemoprevention by Selenium: Results of In Vitro and Animal Studies j437

Selenoproteins include the well-characterized glutathione peroxidases (GPx) GPx1, 2 and 4, all of which reduce peroxides and hydroperoxides [6] and so protect cells from oxidative damage by preventing generation of high concentrations of reactive oxygen species. In addition, thioredoxin reductases (also selenoproteins) regulate cell redox state. Recent structural analysis of the novel selenoproteins Sel W, M, L, T, H, and the 15 kDa selenoprotein shows that they contain thioredoxin redox folds, and both selenoproteins M and S have redox functions [8, 9]. Such redox and cell protective functions could be a basis for the chemopreventive effects of Se. However, the selenoproteins may act through other mechanisms and recently GPx2 has not only been shown to inhibit invasion and migration of tumor cells but also to be a target for the Wnt signaling pathway [10]. Selenometabolites such as methylselenol have been reported to induce phase II conjugating enzymes, lower DNA adduct formation and damage and so promote cell cycle arrest and apoptosis [4]. Selenomethionine has also been reported to activate p53 [11]. There is strong evidence that high concentrations of both selenomethionine and selenometabolites in cell culture induce apoptosis [4]. Consistent with such effects are recent observations from microarray studies that show that, in transformed prostate and breast cancer cell lines, selenomethionine and methyselenol alter expression of genes in pathways that regulate cell cycle progression, cell proliferation and apoptosis [12]. The mechanism by which selenium modulates apoptosis and cell cycle control is not known but such actions could play a key role in the cancer preventative effects of selenium. Possible mechanisms include inhibition of ROS formation by the selenoproteins GPx1 and 4, so lowering activation of NF-kB, inhibition of 5-lipopxygenase by GPx4 altering the pattern of arachidonic acid metabolites and leukotrienes and so modulating cell proliferation and tumor survival, regulation of protein kinase C or regulation of other redox functions such as those of the 15 kDa selenoprotein that is a major selenoprotein in the prostate [4]. A major argument for suggesting that the selenometabolites, rather than selenoproteins, are active in the mechanism of chemoprevention by selenium is that the selenium supplementation of 200 mg/day found to lower cancer risk in a US population would probably give an overall daily selenium intake of over 250 mg/day, considerably higher than the approximately 150 mg/day found to be sufﬁcient to optimize plasma glutathione peroxidase activity and selenoprotein P levels [13]. However, we do not know what daily intake is necessary to optimize target tissue (e.g., prostate, colon) selenoprotein activities so it remains possible that higher intakes protect against tumorigenesis by modulating selenoprotein activities in key target tissues. Further evidence that the selenoproteins are indeed important in chemoprevention is now emerging from studies of transgenic mice; for example, mice lacking the selenocysteine-tRNA gene show altered p53 expression [14] and mice that develop prostate cancer because of targeted SV40 T antigen expression show increased tumorigenesis when a second transgene is introduced that modiﬁes the selenocysteine tRNA and causes depletion of a range of selenoproteins including GPx1–3 and selenoprotein P [15]. 438j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

26.4 Results from Human Studies of the Influence of Selenium Status on Cancer Risk

In addition to animal studies, there is evidence from epidemiology that increased selenium intake reduces cancer risk [4] and data from a major supplementation trial suggest increased selenium intake lowers risk of some cancers, although the outcomes of other clinical trials have been varied. Cancer incidence in different populations worldwide is associated with selenium intake, with populations that have a low selenium intake showing a greater rate of mortality from cancers (see Ref. [1]). Recently, two prospective studies have examined cancer mortality in relation to selenium status as assessed by blood selenium. A trial involving middle-aged individuals in France showed that individuals with the lowest plasma selenium had the highest cancer mortality rate over the subsequent 10 years [16]. Similarly, in the US Womens Health and Aging Study individuals with the highest blood selenium and carotenoid concentrations were at lowest risk of cancer mortality [17]. Whilst such correlations between selenium intake or selenium status and cancer mortality are intriguing and suggest a potential link between selenium intake/metabolism and cancer risk they could reflect many other causative mechanisms. Although limited in the extent to which they can be interpreted, these studies are important in providing an impetus for alternatively designed studies of selenium intake and cancer risk. To date, the single most important study of selenium intake and cancer risk has been the Nutritional Prevention of Cancer (NPC) trial carried out in the US [18, 19]. This was a supplementation trail with 200 mg/day (or placebo) and involved more than 1000 subjects who had a clinical record of nonmelanoma skin cancer. After 6.5 years, those individuals receiving the selenium supplementation showed significantly lower total cancer incidence with lower incidence of prostate, lung, and colorectal cancers (CRC). After a further 2-year follow-up period, the significant effect on total cancer incidence remained as did the protective effect of selenium against prostate cancer. Subgroup analysis of this trial indicates that both the initial and follow-up data provide evidence for a protective effect of selenium against cancer incidence in men and in former smokers. For both total cancer incidence, as well as prostate and colorectal cancer incidence, the effect of selenium supplementation was the greatest in those individuals, whose plasma selenium was below 106 mg/l, corresponding to a level of selenium status comparable to most European populations. Results from four other supplementation trials suggest increased selenium intake lowers cancer incidence. Supplementation with either 30–50 mg/day for 8 years or 200 mg/day for 2 years [20] led to lower incidence of liver cancer (50% fall in the 8-year trial) and lower hepatitis. Such effects of increased selenium intake may not be applicable to other cancers because of the importance of viral hepatitis in the etiology of liver cancer and the known influence of selenium on viral infection [1, 21]. In addition to the NPC trial, there have been a large number of cohort and case–control studies that have examined the possible link between selenium intake or selenium status and incidence of specific cancers. The results from such studies have been contradictory and variable [1, 22]. For prostate cancer, although a number of small studies have failed to show any association of selenium status and disease risk, 26.4 Results from Human Studies of the Influence of Selenium Status on Cancer Risk j439 there have been six nested case–control or case–control studies that involve more than 300 participants, and five of these report a significant inverse relationship between selenium status (assessed by plasma, serum, or toenail selenium) and prostate cancer risk; one of the recent studies [23] failed to find an overall relationship between serum selenium and prostate cancer risk but interestingly higher selenium status was associated with lower disease risk in either men who were smokers or men who had a high vitamin E intake or took a multivitamin supplement. This apparent association in men who also had increased intake of other micronutrients/vitamins is also reflected in the outcome of a recent trail in which men who were supplemented with selenium and multivitamins had a lower risk of prostate cancer compared to men who had received the placebo [24]. Thus, overall the evidence at present suggests that increased selenium intake may indeed lower theriskofprostatecancerinmen,particularlyifcombinedwithincreased multivitamin intake. This assessment is in line with the recent WCRF (World Cancer Research Fund) statement that increased selenium intake probably protects from prostate cancer. A clearer picture should emerge from the ongoing long-term SELECT selenium supplementation trial. The evidence for an association of selenium status and the risk of other cancers is less convincing, although WCRF state that for both lung and colorectal cancer there is limited evidence that increased selenium intake lowers risk of these diseases. This is especially true of breast cancer for which large case–control studies show no association of disease risk with the selenium status assessed by plasma or toenail selenium [22]. The situation is different for both lung cancer and colorectal cancer where the majority of relatively small studies have produced conflicting data but where recent meta-analyses suggest that there is an inverse relationship between selenium status and incidence of these two cancers. The first suggestion that increased selenium intake might protect from CRC came from the NPC trial where selenium supplementation with 200 mg/day was found to lead to a lower incidence of CRC at the first analysis after approximately 6 years. This is supported by results from three small studies but several other medium-sized case–control studies and one large study failed to show any relationship between CRC incidence and selenium status [22]. However, a recent meta-analysis combining three studies (the Wheat Bran Fiber Trial, Polyp Prevention Trial, and Polyp Prevention Study) has examined the occurrence of a new adenoma in relation to blood selenium in over 1700 patients who had recently had an colonic adenoma removed [25] and the data showed that individuals in the highest quartile in terms of blood selenium concentration had significantly lower odds of developing a new adenoma. Results from a relatively small trial in Italy are consistent with these effects of selenium in that a 5-year supplementation with 200 mg/day selenium together with zinc and vitamins A, C, and E led to a lower rate of recurrence of colonic adenomas. Again nested case–control studies of selenium status in relation to lung cancer risk have produced variable results [22]. Two medium-large size studies, one in the US and the other in The Netherlands, report a lower risk of lung cancer in individuals with higher selenium status as estimated from toenail selenium, but another very large cohort study in the US failed to show such a protective effect. A meta-analysis of 440j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

16 studies [26] suggests that increased selenium intake is associated with a lower risk of lung cancer; interestingly, subgroup analysis showed that the effects were only statistically significant in groups of lower selenium status (serum selenium less than 100 mg/l). Results from the NPC trial initially showed a lower incidence of lung caner after 10 years of selenium supplementation [27] but after a further 3 years there was no longer a statistically significant effect. Considering the overall picture from these studies, one is left with two impres- sions: the potential lowering of disease risk by increased selenium intake and the variation in observed effects between the studies. The basis of the variation is not clear but there are several possibilities: the length of the follow-up period, the population age, the gender and lifestyle characteristics (e.g., if they are smokers), the possibility that the effects of selenium depends on other nutrient intake, and finally the possibility that there is genetic variation between individuals in their selenium metabolism and response to dietary selenium. To date, it has not been possible to dissect out a particular factor or factors responsible for the variation but several studies point to dietary and lifestyle factors influencing the effect of selenium status on cancer risk. In the NPC trail, the significant effect of selenium supplementation on prostate cancer and CRC was only statistically significant in the lowest quartile of the participants stratified on the basis of initial plasma selenium status [18, 19]. Compila- tion of data from several case control and observational studies suggests that the inverse association of prostate cancer risk and selenium status is seen in populations in which selenium status in the higher range [23]. Similarly, a meta-analysis from the three studies of CRC [25] also indicates that the effect of selenium on cancer risk was seen at the higher range of selenium intake. These observations may be compatible if a threshold level of selenium status is required to obtain a protective effect. Data from a lung cancer meta-analysis study [26] are also consistent with such a view. However, a case–control study carried out in a region of Spain of low selenium intake showed a relationship between plasma selenium and CRC incidence, although the study was small. A recent nested case–control indicates that other dietary and lifestyle factors may also modulate the influence of selenium intake on disease susceptibility [23]. Although, overall, the study failed to show any significant association between serum selenium concentration and prostate cancer risk, an interaction between selenium and vitamin E intake was found in that serum selenium concentration was significantly associated with lower disease risk in men who also had a high vitamin E intake. Similarly, high serum selenium concentration was associated with lower disease risk in men who smoked. Interestingly, results from two supplementation trials indicate that increased selenium intake (50 mg/day) together with additional vitamin E and b-carotene lowered death rates from lung and esophageal cancer [28].

26.4.1 Genetic Influences on Selenium Metabolism and Disease Risk

A small number of single nucleotide polymorphisms (SNPs) have been identified as being functional in the genes encoding selenoproteins. AT/Cvariation in the protein 26.5 Mechanisms of Chemoprevention by Zinc: Results of In Vitro and Animal Studies j441 coding region of the GPx1 gene (rs1050450) leads to an amino acid change at codon 198 that causes functional changes with the Leu variant of the protein having lower activity. The Leu allele has been reported to show increased association with lung, breast, and bladder cancer [12] but the association with higher risk of breast cancer has not been confirmed in some studies. Of course, the disease consequences of this SNP might only be evident when combined with other SNPs or diet and in this regard it is interesting to note that the impact of the Leu allele on susceptibility to either bladder or breast cancer has been reported to be influenced by a second SNP – one within the gene that codes for another antioxidant defense protein, manganese superoxide dismutase. Furthermore, a stronger association between alcohol intake and smoking with lung cancer risk has been reported in the homozygous Leu carriers than in carriers of the other variants [29]. An SNP (T/C variant) within the region of the GPx4 gene corresponding to the 30 UTR at position 718 (rs713041) has been found to have functional consequences in terms of expression of GPx4 and other selenoproteins [12]. Reporter gene studies have shown that the C variant produces a higher level of reporter activity and in human volunteers it was found that the SNP led to differences in responses of GPx4, GPx1, and GPx3 protein expression or activity to Se supplementation or withdrawal [30]. Interestingly, a small association study indicated that the T variant was associated with a lower risk of colon cancer [31]. Two polymorphic variants, a C/T substitution at position 811 (rs5845) and a G/A at position 1125 (rs5859), have been identified in the region of Sep15 gene that corresponds to the 30 UTR of the mRNA [32]; reporter gene experiments have indicated that the combination of C811 and T1125 may affect SECIS function, suggesting that these two SNPs would affect selenium incorporation during Sep15 synthesis. Furthermore, malignant mesothelioma cells expressing the A variant at position 1125 were less responsive to growth inhibition by increased selenium supply. There are two reports of an association of the combined variants with breast or lung cancer [33, 34]. Although limited in number and size of the populations analyzed, the above SNP- disease association studies indicate that genetic variation within selenoprotein genes may affect susceptibility to a number of cancers. More large studies are needed to substantiate this premise but at present the findings are consistent with both a chemopreventive effect of selenium and the view that this effect occurs through the selenoproteins.

26.5 Mechanisms of Chemoprevention by Zinc: Results of In Vitro and Animal Studies

Zinc is essential to a wide spectrum of cellular processes, highlighted by the recent estimate that 3–10% of all human genes account for zinc-associated proteins [35], so is a compelling candidate for playing multifaceted roles in tumorigenesis and cancer progression and, therefore, in cancer prevention or treatment. The premise is supported by various lines of evidence concerning both functional properties of zinc consistent with anticancer actions and observations relating directly to anticancer 442j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

effects of zinc. This evidence includes effects of zinc in cell culture models consistent with anticancer activity, associations of zinc status with cancer susceptibility/occurrence, and efficacy of zinc supplementation, mostly in animal models but, inconsis- tently and to a limited degree, also in humans, to reduce cancer incidence or progression. Key aspects of this evidence relating to the proposition that zinc may be an important dietary factor in cancer prevention are summarized below. Zinc has antioxidant properties that may be important in protecting the normal cell from events leading to cancer, consistent with observations that zinc deficiency is associated with increased oxidative stress [36, 37]. The antioxidant properties of zinc will be considered below. Franklin and Costello [38] categorize the cellular actions of zinc relevant to antitumor action as effects in tumor cells on (1) growth and proliferation; (2) motility and invasion, and (3) intermediary metabolism. This categorization provides a convenient framework for considering many of the cellular actions of zinc consistent with a potential role in cancer treatment, and will be adopted also for the purpose of this summary. The role of zinc as an intracellular signal is a cross-cutting aspect of zinc cell biology, germane to all four categories of zinc action, so will be considered in the specific context of potential actions relevant to cancer. Also, it is becoming apparent that changes in intracellular zinc content, accompanied by changes in zinc transporter expression, are associated with malignancy, specifically in cancers of the prostate and breast. Thus, the following summary will also include evidence relating to this aspect of cellular zinc metabolism in the context of cancer. Whilst most of the evidence considered below demonstrates functions of zinc consistent with a chemopreventive action, it should be noted that increased zinc levels within cancer cells are not unequivocally associated with cancer-protective effects. For example, the increased intracellular concentration of zinc measured in a Tamoxifen-resistant breast cancer cell line, compared with the parent MCF-7 cell line [39], reveals a role for zinc that may increase, rather than reduce, cancer progression. Such examples serve to highlight the diversity and complexity of cellular responses to zinc that may be relevant in the context of cancer.

26.5.1 Zinc as an Antioxidant

Zinc may reduce cellular oxidative stress, and so protect against DNA damage, through a number of mechanisms including induction of zinc-responsive genes coding for proteins with antioxidant properties, antagonism of redox-active transition metals, and sulfydryl stabilization. With respect to zinc-mediated induction of antioxidant genes, increased expression of the small, cysteine-rich, intracellular zinc-binding protein metallothionein is probably the most important quantitatively, since the family of genes coding for this protein are exquisitely sensitive to cellular zinc status and the protein is a potent scavenger of the hydroxyl radical [40, 41]. Increased zinc availability substantially increases the transcription of the metallothionein genes through the binding of the zinc-dependent transcription factor MTF1 to multiple copies of the metal response element (MRE) sequence in the 26.5 Mechanisms of Chemoprevention by Zinc: Results of In Vitro and Animal Studies j443

Scheme 26.1 The generation of hydroxyl radicals through þ þ oxidation of Fe2 and Cu . Donation of a single electron to hydrogen peroxide by these redox-active transition metal cations generates the highly reactive hydroxyl radical. This reaction is þ þ prevented by the substitution of Fe2 or Cu by the redox-inert þ Zn2 cation. promoter regions [42]. A substantial oxidant-protective effect of zinc mediated through competition with redox-active transition metals, speciﬁcally iron and copper, þ is likely to be through substitution at binding sites in proteins in which Fe2 and þ Cu can donate electrons to hydrogen peroxide via Fenton reactions, to generate the highly reactive hydroxyl radical [37] (Scheme 26.1). The enzyme d-aminolevulinate dehydratase, which is inactivated by oxygen as a result of thiol-oxidation leading to disulﬁde formation, has been studied extensively in the context of zinc-induced sulfydryl stabilization, providing good evidence for an antioxidant action of zinc mediated thought this process [43]. Other proteins apparently protected by zinc through such a mechanism include tubulin, alanyl tRNA synthetase, and protein farnesyltransferase [43].

26.5.2 Effects of Zinc on Cell Proliferation and Apoptosis

Proapoptotic effects of zinc on a variety of cell types, including various tumor cell lines, have been observed and the mechanisms underpinning this effect are being elucidated. This function of zinc has been studied particularly extensively in prostate tumor cells, driven by the strong interest in the relationship between zinc and prostate cancer stemming from the fact that the prostate accumulates unusually high levels of zinc. This accumulation of zinc is important for the ability of the prostate to secrete citrate into prostatic ﬂuid. Also, prostate carcinogenesis involves the loss of a zinc-accumulating cellular phenotype. Zinc-induced apoptosis in malignant prostate cells is reported to be through a dual action to increase the expression of the Bax gene through a zinc-dependent transcription factor (distinct from MTF1, for which no binding sites exist in the promoter region) and also to promote mitochondrial insertion and subsequent oligomerization of Bax, associated with the production of pores through which cytochrome c release occurs [38]. Consistent with induction by the zinc of Bax-mediated apoptosis in prostate tumor cells, it has been reported that growth of PC-3 prostate xenograft tumors in mice was inhibited by zinc treatment and was accompanied by increased Bax expression and apoptosis [38]. Evidence for proapoptotic effects of zinc in other cancer cell types includes induction of apoptosis by zinc accumulation in choriocarcinoma cells [44] and in human epithelial ovarian cancer cells, the latter accompanied by a measured increase in Bax expression [45]; increased Bax expression accompanied by apoptosis and reduced tumor growth in a rat model of N-nitrosomethylbenzylamine (NMBA)-induced 444j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

esophageal cancer on dietary supplementation of zinc-deﬁcient animals [46]; zinc- induced apoptosis through both caspase-dependent and -independent pathways in HEP-2 cancer cells [47].

26.5.3 Effects of Zinc on Cell Motility and Invasion

A decrease in cell motility associated with increased cellular zinc levels in malignant cells has been proposed as a mechanism through which zinc may modulate cancer progression [38]. For example, increased zinc levels suppressed the ability of LNCaP cells to invade Matrigel [48] and zinc supplementation of prostate cancer cell lines reduced expression of intercellular adhesion molecule-1 and reduced cell invasiveness and adhesion [49], all consistent with an ability of zinc to reduce host tissue invasion and metastasis by cancer cells. In contrast, an increased intracellular zinc concentration associated with acquisition of a Tamoxifen resistant phenotype was associated with increased growth and invasion of MCF-7 breast cancer cells [50], highlighting the potential cell speciﬁcity of the effect of zinc on motility and invasion and, hence, the need to consider potential cell-type-speciﬁc effects of zinc in cancer chemoprevention.

26.5.4 Effects of Zinc on Intermediary Metabolism

Zinc is pivotal in maintaining the unique metabolic proﬁle of prostate epithelial cells, which allows the secretion of high concentrations of citrate into the prostatic ﬂuid by inhibiting mitochondrial aconitase activity to spare this intermediary metabolite [38]. Respiration is further inhibited in prostate cells through the inhibition of terminal oxidation in the mitochondria by the elevated intracellular zinc content. Malignant transformation of prostate epithelial cells involves loss of their zinc-accumulating capability and the concomitant switch to citrate oxidation, combined with the release of the inhibitory effect on terminal oxidation and a concomitant increase in the rate of oxidative respiration. Franklin and Costello proffer the view that the metabolic effects of zinc are incompatible with malignant transformation, given that the metabolic decrease in zinc and citrate is an early event in the development of malignancy [38].

26.5.5 Intracellular Zinc Signaling in Cancer

The view that zinc should be viewed as an intracellular signal, akin to calcium, is gaining increasing acceptance. The intracellular free zinc content is negligible and, in response to extracellular signals such as redox stress or cytokines, zinc is released from protein binding sites, notably metallothionein, or is transported into and within the cell as a result of modulated activity of zinc transporter proteins. Zinc can then bind to and activate zinc-dependent transcription factors or, alternatively, interact 26.5 Mechanisms of Chemoprevention by Zinc: Results of In Vitro and Animal Studies j445 directly with components of intracellular signaling pathways. Reports of zinc- modulation of intracellular signaling pathways are numerous and the effects diverse in nature, with multiple potential effects that may impinge upon tumorigenesis. Modulation by zinc of NF-kB activity in tumor cells may be of central importance in the cell-signaling actions of zinc germane to carcinogenesis. NF-kB plays a central role in the cellular response to oxidative stress and in immune function, both of which may play important roles in cancer preventive actions of zinc [51]. Even with respect to this speciﬁc example, however, effects of zinc appear inconsistent and cell- type speciﬁc. For example, zinc inhibited TNF-a induced activation of NF-kB in PC-3 and DU-145 prostate cancer cells [49] but increased NF-kB activity in both immortalized embryonic hippocampal H19-7 cells [52] and in HUT-78 human malignant lymphoblastoid cells [53] through increased phosphorylation, and thus dissociation, of the NF-kB inhibitory protein IkB-a.

26.5.6 Zinc Transporters and Cancer

The zinc transporter LIV-1 (ZIP6) is regulated by estrogen and was identified as a candidate gene for the metastatic spread of ER positive breast cancer to the lymph nodes by virtue of an association between LIV-1 expression and lymph node involvement in the disease [54]. LIV-1 expression was associated with grade in patients with primary breast cancer, being most highly expressed in low histological grade tumors [39], in agreement with a potential role for the functional activity of this transporter contributing toward breast cancer phenotype (or alternatively with the expression being associated with the coexpression of another functionally relevant maker). This same analysis identified a positive association between the expression of the zinc transporter HKE4 (ZIP7) and the well-known proliferation marker Ki67. Direct evidence for a link between HKE4 function and breast cancer phenotype is provided by the reported observation that siRNA knockdown of this transporter in a Tamoxifen-resistant cell line model prevented an observed zinc-induced activation of signaling pathways that may be involved in the aggressive phenotype [39]. As noted above, malignant transformation of prostate epithelial cells involves loss of the unique zinc-accumulating phenotype of this cell type. Evidence that the zinc transporter Zip1 mediates zinc uptake into prostate cells [55] coupled with the observation that Zip1 expression was reduced in zinc-depleted malignant prostate glands, compared to adjacent, nonmalignant, zinc-accumulating tissue [56], identifies a pivotal role for this zinc transporter in prostate cancer.

26.5.7 Effects of Dietary Zinc Manipulation on Cancer Incidence and Progression in Animal Models

A variety of studies in rodent models demonstrate an effect of zinc deﬁciency to increase susceptibility to tumor development when exposed to carcinogenic compounds or an apparent effect of dietary zinc to reduce carcinogenesis and/or cancer 446j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

progression. For example, zinc deﬁciency in mice increased the incidence of lingual and esophageal carcinogenesis induced by 4-nitroquinoline 1-oxide [57] and in both rats and mice increased esophageal cell proliferation and the incidence of NMBA- induced esophageal tumors [46, 58]. In rats, incidence of NMBA-induced esophageal tumorigenesis in zinc-deﬁcient animals was reduced by dietary zinc supplementation [46] and zinc supplementation of mice with developing PC3 prostate cell xenograft tumors reduced tumor growth [38].

26.6 Human Studies of Links between Zinc, Carcinogenesis, and Tumor Progression

26.6.1 Associations Between Zinc Status and Cancer Incidence and Progression

Interpretation of the results of studies reporting associations between poor zinc status and increased risk or progression of cancer is, of course, compromised by the possibility that poor zinc status may be associated with other relevant aspects of suboptimal nutrition that are not identified and, therefore, not controlled. Also, it is generally not possible in such studies to exclude the possibility that a measured reduction in zinc status in cancer patients may be a consequence, rather than a cause, of cancer development. Nonetheless, and particularly in view of the properties of zinc consistent with pleiotropic roles in cancer prevention, such studies are important and merit consideration. Selected examples are summarized below. A number of studies support the premise that reduced zinc status increases tumor incidence or progression. For example, a study of patients with newly-diagnosed squamous cell carcinoma of the oral cavity, oropharynx, larynx, and hypopharynx identified a correlation between pretreatment zinc status, assayed by the measurement of zinc in plasma, lymphocytes and granulocytes, and clinical outcome [59]. Specifically, tumor size and the stage of disease at diagnosis was correlated with zinc status, independent of a measure of prognostic nutritional status based on measures of serum albumin and tranferrin, triceps fold thickness, and cutaneous hypersensitivity to test antigens [60] and unrelated to smoking and alcohol intake. Furthermore, adequate nutritional status, including zinc status, was associated with an increased disease-free interval. Consistent with an apparent effect of poor zinc status on the development, rather than progression, of head and neck cancer, a prospective study in a Chinese cohort showed a strong association between esophageal biopsy tissue zinc concentration and reduced risk of developing esophageal squamous cell carcinoma [61]. An association between zinc status and cancer was also indicated in a study of patients with hepatocellular carcinoma, who had significantly lower plasma zinc concentration compared with healthy controls [62]. A further link between zinc status and cancer was indicated in this study by the observation that tissue zinc concentration was reduced significantly in the cancerous tissue compared with the surrounding normal tissue. Also in support of an interaction between reduced zinc status and cancer, significantly reduced serum zinc concentrations were measured in patients 26.6 Human Studies of Links between Zinc, Carcinogenesis, and Tumor Progression j447 with cancer of the digestive tract (colorectal carcinoma and esophageal carcinoma) compared with healthy controls [63]. In contrast to reports of positive associations between zinc status and reduced cancer incidence, a case–control study based on a population in Utah, which examined the association of dietary factors with prostate cancer, identified no association between cancer incidence and dietary intake of zinc assessed on the basis of a quantitative food frequency questionnaire [64]. In agreement with these findings, a study of similar design carried out in Serbia also found no link between dietary zinc intake and the incidence of prostate cancer [65]. It is notable that the studies cited above that identify no apparent link between zinc status and cancer were based on measurement of dietary zinc intake, as opposed to measurement of tissue zinc concentrations, highlighting the possibility that dietary evaluation may be an inappropriate tool in this context and/or that influences of zinc status on cancer are manifest only when perturbations are of a sufficient magnitude to result in measured changes in tissue zinc concentrations.

26.6.2 Effects of Dietary Zinc Manipulation on Cancer Incidence and Progression In Vivo

As discussed above, at least a component of the anticancer action of zinc may be attributable to antioxidant effects. Evidence that zinc can have a biologically significant antioxidant effect in human subjects in vivo is provided by the results of the large- scale (3643 participant; 11 centers) Age-Related Eye Disease Study. This study detected a significant reduction in the risk of developing advanced age-related macular degeneration in a higher risk group of the cohort studied given a daily 80 mg zinc supplement over a 6.3 year follow-up period [51]. Human studies on the potential protective effects of zinc on cancer development/ progression have focused largely on prostate cancer and to date have yielded conflicting results. For example, self-reported use of zinc supplements was associated with reduced risk of prostate cancer, and showed a dose–response effect, in a population-based case–control study conducted in Washington, consistent with a protective effect of zinc on the development of prostate cancer although, as the authors note, there remains a need to investigate the role on this association of other lifestyle variables associated with supplement use [66]. In contrast with these findings, and again with the caveat that other lifestyle factors associated with the use of zinc supplements could not be excluded, intake of supplemental zinc at doses exceeding 100 mg/day or over a period of 10 or more years was associated with increased risk of prostate cancer in 46 974 US men (Health Professionals Follow-up Study) followed over 14 years [67]. A third study investigating a potential association between the use of nutrient supplements and the development of prostate cancer involved self-reported use in Swedish patients (n ¼ 1499) and controls (n ¼ 1130) [68]. This case–control study identified no difference in the prevalence of zinc supplement use between patients and controls and no statistically significant difference in prostate cancer risk between supplement users and nonusers stratified by frequency, duration, or cumulative exposure of zinc supplementation. Reasons for the lack of an 448j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

apparently consistent effect of zinc supplementation on prostate cancer incidence may include variability in dose and/or bioavailability of the zinc formulas administered, duration of supplementation, and interactions with other dietary components. In relation to the potential efﬁcacy of zinc supplementation in the treatment of other forms of cancer, a preliminary study in 60 patients undergoing chemotherapy for colorectal or esophageal carcinoma found that fewer (30%) patients who received a daily combined supplement of zinc (21 mg/day) and selenium over 60 days experienced a decline in parameters indicative of nutritional status that was observed in 80% of patents who did not receive the supplement, indicating that supplementation with micronutrients, including zinc, may improve the clinical course of patients with intestinal cancers [63].

26.7 Conclusions

In summary, there is strong evidence that increased selenium intake lowers tumor incidence in a number of animal models of carcinogenesis [4, 7]. In humans, the evidence of increased selenium intake lowering cancer incidence is not unanimous but there is good evidence linking selenium and prevention of prostate cancer and limited evidence in lowering lung and colon cancer risk. More important, several studies indicate that the lower cancer risk associated with lower selenium intake is observed in conjunction with the other factors – genetic variation in enzymes involved in cell protection from oxidative damage (e.g., superoxide dismutase), or altered intake of the other micronutrients such as vitamin E and carotenoids. Thus, increased selenium intake may be particularly chemopreventive in conjunction with other dietary changes. Crucially, there is still debate over the level of dietary selenium required to lower the risk of cancer. Although a recent study [69] indicates that the overall optimal selenium intake is 130 mg/day, this is related to overall mortality rather than cancer prevention. There is a need to carry out major trials to determine the level of dietary selenium that is chemopreventive and in this regard the SELECT study should provide very useful information. However, it will also be important to assess as to how this level is modulated by other dietary constituents and genetic factors. Although selenium has been strongly implicated in protecting cells from oxidative damage and modulation of cell cycle control/apoptosis, the mechanism of chemoprevention by increased selenium intake is still unknown. In particular, there is active debate over whether these effects occur through the selenoproteins or through selenometabolites such as methyselenol. However, the use of transgenic animal models and results from pilot disease association studies of several SNPs suggest that indeed the selenoproteins may be the mechanistic basis of the lower disease risk and the chemopreventive effects of increased selenium intake. The evidence summarized above provides clear potential links between dietary zinc and cancer prevention. In particular, zinc has pleitotropic cellular actions consistent with an effect to reduce cancer incidence and progression, but these References j449 appear to include effects specific to particular cell types. In addition, studies in both rodent models and inconsistent and limited evidence in human subjects suggests that zinc plays a role in cancer chemoprevention. However, current evidence is far from sufficient to conclude that zinc obtained from the diet or through the use of supplements has an important role to play in cancer chemoprevention. Effects of dietary zinc are likely to be specific to cancer type and may be modified by other factors such as interaction with other dietary components and host genotype, highlighting the potential need to consider interindividual cancer susceptibility with respect to a potential modulating role of dietary zinc. Hence the results of rigorous, controlled, intervention trials in humans are required to guide dietary recommendations concerning cancer chemoprevention by zinc. As reviewed above, many studies have now provided evidence at the cellular level that increased selenium and zinc may have chemopreventive effects. However, key questions still remain to be answered. It will be important in the future to determine whether the actions of selenium are through the selenoproteins, low molecular weight selenocompounds, or both. Similarly, the pleiotropic involvement of zinc in normal cellular function makes it a challenge to establish unequivocally the primary mechanisms through which anticancer effects are mediated, so further research is required in this area. Interestingly, the antioxidant effects of zinc and selenium may be linked through the low molecular weight, cysteine-rich protein metallothionein, which binds 7 zinc ions with sulfydryl ligands from its 20 cysteines. Release of zinc on oxidation of metallothionein disulfide pairs by low molecular weight selenium compounds or by selenoenzymes, including glutathione peroxidase, is believed to comprise a component of the antioxidant effect of selenium [41]. Besides such mechanistic studies, large supplementation trials need to be carried out to obtain definitive evidence as to whether increased intake of selenium or zinc lowers the incidence of cancers. Several studies suggest that zinc or selenium intake may lower cancer incidence when increased together with changes in other dietary antioxidants and micronutrients. So, increased zinc and selenium intake should not been studied in isolation but in relation to overall dietary antioxidants. In addition, genetic factors may influence the efficacy of increased intake. Trials carried out to determine zinc or selenium chemopreventive effects need to take such genetic and multinutrient factors into account.

References

1 Rayman, M.P. (2005) Selenium national food supplies: methodology and in cancer prevention: a review of the regional estimates. Public Health Nutrition, evidence and mechanism of action. 8, 812–819. Proceedings of the Nutrition Society, 3 Hatﬁeld, D.L. and Gladyshev, V.N. (2002) 64, 527–542. How selenium has altered our 2 Wuehler, S.E., Peerson, J.M. and Brown, understanding of the genetic code. K.H. (2005) Use of national food balance Molecular and Cellular Biology, 22, data to estimate the adequacy of zinc in 3565–3576. 450j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

4 Whanger, P.D. (2004) Selenium and its Sec gene (Trsp) in mouse mammary relationship to cancer: an update. The epithelium. Molecular and Cellular Biology, British Journal of Nutrition, 91,11–28. 23, 1477–1488. 5 Hunt, J.R. (2003) Bioavailability of iron, 15 Diwadkar-Navsariwala, V., Prins, G.S., zinc, and other trace minerals from Swanson, S.M., Birch, L.A., Ray, V.H., vegetarian diets. The American Journal of Hedayat, S., Lantvit, D.L. and Diamond, Clinical Nutrition, 78, 633S–639S. A.M. (2006) Selenoprotein deﬁciency 6 Brigelius-Flohe, R. (2006) Glutathione accelerates prostate carcinogenesis in a peroxidases and redox-regulated transgenic model. Proceedings of the transcription factors. Biological Chemistry, National Academy of Sciences of the United 387, 1329–1335. States of America, 103, 8179–8184. 7 Combs, G.F. Jr and Gray, W.P. (1998) 16 Akbaraly, N.T.,Arnaud, J., Hininger-Favier, Chemopreventive agents: selenium. I., Gourlet, V., Roussel, A.M. and Berr, C. Pharmacology & Therapeutics, 79, 179–192. (2005) Selenium and mortality in the 8 Ferguson, A.D., Labunskyy, V.M., elderly: results from the EVA study. Clinical Fomenko, D.E., Arac, D., Chelliah, Y., Chemistry, 51, 2117–2123. Amezcua, C.A., Rizo, J., Gladyshev, V.N. 17 Ray, A.L., Semba, R.D., Walston, J., and Deisenhofer, J. (2006) NMR structures Ferrucci, L., Cappola, A.R., Ricks, M.O., of the selenoproteins Sep15 and SelM Xue, Q.L. and Fried, L.P. (2006) Low serum reveal redox activity of a new thioredoxin- selenium and total carotenoids predict like family. The Journal of Biological mortality among older women living in the Chemistry, 281, 3536–3543. community: the womens health and aging 9 Dikiy, A. et al. (2007) SelT, SelW, SelH, and studies. The Journal of Nutrition, 136, Rdx12: genomics and molecular insights 172–176. into the functions of selenoproteins of a 18 Clark, L.C. et al. (1996) Effects of selenium novel thioredoxin-like family. Biochemistry, supplementation for cancer prevention in 46, 6871–6882. patients with carcinoma of the skin A 10 Kipp, A., Banning, A. and Brigelius-Flohe, randomized controlled trial. Nutritional R. (2007) Activation of the glutathione Prevention of Cancer Study Group. Journal peroxidase 2 (GPx2) promoter by beta- of American Medical Association, 276, catenin. Biological Chemistry, 388, 1957–1963. 1027–1033. 19 Dufﬁeld-Lillico, A.J., Reid, M.E., Turnbull, 11 Seo, Y.R., Kelley, M.R. and Smith, M.L. B.W., Combs, G.F. Jr, Slate, E.H., (2002) Selenomethionine regulation of Fischbach, L.A., Marshall, J.R. and Clark, p53 by a ref1-dependent redox L.C. (2002) Baseline characteristics and the mechanism. Proceedings of the National effect of selenium supplementation on Academy of Sciences of the United States of cancer incidence in a randomized clinical America, 99, 14548–14553. trial: a summary report of the Nutritional 12 Hesketh, J. (2008) Nutrigenomics and Prevention of Cancer Trial. Cancer selenium: gene expression patterns, Epidemiology, Biomarkers & Prevention, 11, physiological targets and genetics. Annual 630–639. Review of Nutrition, 28, 157–177. 20 Yu, S.Y., Zhu, Y.J. and Li, W.G. (1997) 13 Xia, Y., Hill, K.E., Byrne, D.W., Xu, J. and Protective role of selenium against Burk, R.F. (2005) Effectiveness of hepatitis B virus and primary liver cancer selenium supplements in a low-selenium in Qidong. Biological Trace Element area of China. The American Journal of Research, 56, 117–124. Clinical Nutrition, 81, 829–834. 21 Broome, C.S., McArdle, F., Kyle, J.A., 14 Kumaraswamy, E. et al. (2003) Selective Andrews, F., Lowe, N.M., Hart, C.A., removal of the selenocysteine tRNA [Ser] Arthur, J.R. and Jackson, M.J. (2004) An References j451

increase in selenium intake improves consumption, and risk for lung cancer. immune function and poliovirus handling Cancer Letters, 247, 293–300. in adults with marginal selenium status. 30 Meplan, C., Crosley, L., Nicol, F., Horgan, The American Journal of Clinical Nutrition, G., Mathers, J. and Hesketh, J. (2008) 80, 154–162. Functional effects of a common single 22 Navarro Silvera, S.A. and Rohan, T.E. nucleotide polymorphism (GPX4C718T) (2007) Trace elements and cancer risk: a in the glutathione peroxidase 4 gene: review of the epidemiologic evidence. interaction with gender. American Journal Cancer Causes & Control, 18,7–27. of Clinical Nutrition, 87, 1019–1027. 23 Peters, U. et al. (2007) Serum selenium and 31 Bermano, G., Pagmanditis, V., Hooloway, risk of prostate cancer: a nested V., Kadri, N., Mowatt, N. and Hesketh, J. case–control study. The American Journal of (2007) Evidence that a polymorphism Clinical Nutrition, 85, 209–217. within the 30 UTR of glutathione 24 Meyer, F., Galan, P., Douville, P., Bairati, I., peroxidase 4 is functional and is associated Kegle, P., Bertrais, S., Estaquio, C. and with susceptibility to colorectal cancer. Hercberg, S. (2005) Antioxidant vitamin Genes and Nutrition, 2, 227–232. and mineral supplementation and prostate 32 Hu, Y.J. et al. (2001) Distribution and cancer prevention in the SU.VI.MAX trial. functional consequences of nucleotide International Journal of Cancer, 116, polymorphisms in the 30-untranslated 182–186. region of the human Sep15 gene. Cancer 25 Jacobs, E.T. et al. (2004) Selenium and Research, 61, 2307–2310. colorectal adenoma: results of a pooled 33 Hu, Y.J.and Diamond, A.M. (2003) Role of analysis. Journal of the National Cancer glutathione peroxidase 1 in breast cancer: Institute, 96, 1669–1675. loss of heterozygosity and allelic 26 Zhuo, H., Smith, A.H. and Steinmaus, C. differences in the response to selenium. (2004) Selenium and lung cancer: a Cancer Research, 63, 3347–3351. quantitative analysis of heterogeneity in 34 Jablonska, E., Gromadzinska, J., Sobala, the current epidemiological literature. W., Reszka, E. and Wasowicz, W. (2008) Cancer Epidemiology, Biomarkers & Lung cancer risk associated with selenium Prevention, 13, 771–778. status is modified in smoking individuals 27 Reid, M.E., Duffield-Lillico, A.J., Garland, by Sep15 polymorphism. European Journal L., Turnbull, B.W., Clark, L.C. and of Nutrition, 47,47–54. Marshall, J.R. (2002) Selenium 35 Andreini, C., Banci, L., Bertini, I. and supplementation and lung cancer Rosato, A. (2006) Counting the zinc- incidence: an update of the nutritional proteins encoded in the human genome. prevention of cancer trial. Cancer Journal of Proteome Research, 5, 196–201. Epidemiology, Biomarkers & Prevention, 11, 36 Prasad, A.S. (2008) Clinical, 1285–1291. immunological, anti-inflammatory and 28 Blot, W.J. (1997) Vitamin/mineral antioxidant roles of zinc. Experimental supplementation and cancer risk: Gerontology, 43, 370–377. international chemoprevention trials. 37 Ho, E. (2004) Zinc deficiency DNA damage Proceedings of the Society for and cancer risk. The Journal of Nutritional Experimental Biology and Medicine, Biochemistry, 15, 572–578. 216, 291–296. 38 Franklin, R.B. and Costello, L.C. (2007) 29 Raaschou-Nielsen, O., Sorensen, M., Zinc as an anti-tumor agent in prostate Hansen, R.D., Frederiksen, K., cancer and in other cancers. Archives of Tjonneland, A., Overvad, K. and Vogel, U. Biochemistry and Biophysics, 463, 211–217. (2007) GPX1 Pro198Leu polymorphism, 39 Taylor, K.M., Morgan, H.E., Smart, K., interactions with smoking and alcohol Zahari, N.M., Pumford, S., Ellis, I.O., 452j 26 Micronutrients and Susceptibility to Cancer: Focus on Selenium and Zinc

Robertson, J.F. and Nicholson, R.I. (2007) implications for prostate cancer The emerging role of the LIV-1 subfamily progression. Carcinogenesis, 27, of zinc transporters in breast cancer. 1980–1990. Molecular Medicine, 13, 396–406. 50 Hiscox, S., Morgan, L., Green, T.P., 40 Sato, M. and Bremner, I. (1993) Oxygen Barrow, D., Gee, J. and Nicholson, R.I. free radicals and metallothionein. Free (2006) Elevated Src activity promotes Radical Biology & Medicine, 14, 325–337. cellular invasion and motility in tamoxifen 41 Maret, W. (2003) Cellular zinc and redox resistant breast cancer cells. Breast Cancer states converge in the metallothionein/ Research and Treatment, 97, 263–274. thionein pair. The Journal of Nutrition, 133, 51 Prasad, A.S. and Kucuk, O. (2002) Zinc in 1460S–1462S. cancer prevention. Cancer Metastasis 42 Andrews, G.K. (2001) Cellular zinc Reviews, 21, 291–295. sensors: MTF-1 regulation of gene 52 Min, Y.K., Park, J.H., Chong, S.A., Kim, expression. Biometals, 14, 223–237. Y.S., Ahn, Y.S., Seo, J.T., Bae, Y.S. and 43 Powell, S.R. (2000) The antioxidant Chung, K.C. (2003) Pyrrolidine properties of zinc. The Journal of Nutrition, dithiocarbamate-induced neuronal cell 130, 1447S–1454S. death is mediated by Akt, casein kinase 2, 44 Bae, S.N., Kim, J., Lee, Y.S.,Kim, J.D., Kim, c-Jun N-terminal kinase, and IkappaB M.Y. and Park, L.O. (2007) Cytotoxic effect kinase in embryonic hippocampal of zinc-citrate compound on progenitor cells. Journal of Neuroscience choriocarcinoma cell lines. Placenta, 28, Research, 71, 689–700. 22–30. 53 Bao, B., Prasad, A.S., Beck, F.W. and 45 Bae, S.N., Lee, Y.S., Kim, M.Y., Kim, J.D. Sarkar, F.H. (2007) Zinc up-regulates and Park, L.O. (2006) Antiproliferative and NF-kappaB activation via phosphorylation apoptotic effects of zinc-citrate compound of IkappaB in HUT-78 (Th0) cells. (CIZAR(R)) on human epithelial ovarian FEBS Letters, 581, 4507–4511. cancer cell line, OVCAR-3. Gynecologic 54 Manning, D.L. et al. (1994) Oestrogen- Oncology, 103, 127–136. regulated genes in breast cancer: 46 Fong, L.Y., Nguyen, V.T. and Farber, J.L. association of pLIV1 with lymph node (2001) Esophageal cancer prevention in involvement. European Journal of Cancer, zinc-deficient rats: rapid induction of 30, 675–678. apoptosis by replenishing zinc. Journal of 55 Franklin, R.B., Ma, J., Zou, J., Guan, Z., the National Cancer Institute, 93, Kukoyi, B.I., Feng, P. and Costello, L.C. 1525–1533. (2003) Human ZIP1 is a major zinc uptake 47 Rudolf, E., Rudolf, K. and Cervinka, M. transporter for the accumulation of zinc in (2005) Zinc-induced apoptosis in HEP-2 prostate cells. Journal of Inorganic cancer cells: the role of oxidative stress and Biochemistry, 96, 435–442. mitochondria. Biofactors, 23, 107–120. 56 Franklin, R.B., Feng, P., Milon, B., 48 Ishii, K., Otsuka, T., Iguchi, K., Usui, S., Desouki, M.M., Singh, K.K., Kajdacsy- Yamamoto, H., Sugimura, Y., Yoshikawa, Balla, A., Bagasra, O. and Costello, L.C. K., Hayward, S.W. and Hirano, K. (2004) (2005) hZIP1 zinc uptake transporter Evidence that the prostate-specific antigen down regulation and zinc depletion þ (PSA)/Zn2 axis may play a role in human in prostate cancer. Molecular Cancer, prostate cancer cell invasion. Cancer 4, 32. Letters, 207,79–87. 57 Fong, L.Y., Jiang, Y. and Farber, J.L. (2006) 49 Uzzo, R.G., Crispen, P.L., Golovine, K., Zinc deficiency potentiates induction and Makhov, P., Horwitz, E.M. and Kolenko, progression of lingual and esophageal V.M. (2006) Diverse effects of zinc on NF- tumors in p53-deficient mice. kappaB and AP-1 transcription factors: Carcinogenesis, 27, 1489–1496. References j453

58 Fong, L.Y. and Magee, P.N. (1999) Dietary European Journal of Clinical Nutrition, 55, zinc deﬁciency enhances esophageal cell 293–297. proliferation and N-nitrosomethyl- 64 West, D.W., Slattery, M.L., Robison, L.M., benzylamine (NMBA)-induced esophageal French, T.K. and Mahoney, A.W. (1991) tumor incidence in C57BL/6 mouse. Adult dietary intake and prostate Cancer Letters, 143,63–69. cancer risk in Utah: a case–control 59 Prasad, A.S., Beck, F.W., Doerr, T.D., study with special emphasis on Shamsa, F.H., Penny, H.S., Marks, S.C., aggressive tumors. Cancer Causes & Kaplan, J., Kucuk, O. and Mathog, R.H. Control, 2,85–94. (1998) Nutritional and zinc status of head 65 Vlajinac, H.D., Marinkovic, J.M., Ilic, M.D. and neck cancer patients: an interpretive and Kocev, N.I. (1997) Diet and prostate review. Journal of the American College of cancer: a case–control study. European Nutrition, 17, 409–418. Journal of Cancer, 33, 101–107. 60 Buzby, G.P., Mullen, J.L., Matthews, D.C., 66 Kristal, A.R., Stanford, J.L., Cohen, J.H., Hobbs, C.L. and Rosato, E.F. (1980) Wicklund, K. and Patterson, R.E. (1999) Prognostic nutritional index in Vitamin and mineral supplement use is gastrointestinal surgery. American Journal associated with reduced risk of prostate of Surgery, 139, 160–167. cancer. Cancer Epidemiology, Biomarkers & 61 Abnet, C.C., Lai, B., Qiao, Y.L., Vogt, S., Prevention, 8, 887–892. Luo, X.M., Taylor, P.R., Dong, Z.W., Mark, 67 Leitzmann, M.F., Stampfer, M.J., Wu, K., S.D. and Dawsey, S.M. (2005) Zinc Colditz, G.A., Willett, W.C. and concentration in esophageal biopsy Giovannucci, E.L. (2003) Zinc supplement specimens measured by X-ray use and risk of prostate cancer. Journal of ﬂuorescence and esophageal cancer risk. the National Cancer Institute, 95, Journal of the National Cancer Institute, 97, 1004–1007. 301–306. 68 Chang, E.T., Hedelin, M., Adami, H.O., 62 Liaw, K.Y., Lee, P.H., Wu, F.C., Tsai, J.S. Gronberg, H. and Balter, K.A. (2004) Re: and Lin-Shiau, S.Y. (1997) Zinc, copper, Zinc supplement use and risk of and superoxide dismutase in prostate cancer. Journal of the National hepatocellular carcinoma. The American Cancer Institute, 96, 1108; author reply Journal of Gastroenterology, 92, 2260–2263. 1108–1109. 63 Federico, A., Iodice, P., Federico, P., Del 69 Bleys, J., Navas-Acien, A. and Guallar, E. Rio, A., Mellone, M.C., Catalano, G. and (2008) Serum selenium levels and all- Federico, P. (2001) Effects of selenium and cause, cancer, and cardiovascular mortality zinc supplementation on nutritional status among US adults. Archives of Internal in patients with cancer of digestive tract. Medicine, 168, 404–410. j455

27 DNA Damage and Cancer Chemoprevention by Polyphenols Ajaikumar B. Kunnumakkara, Preetha Anand, Kuzhuvelil B. Harikumar, and Bharat B. Aggarwal

27.1 Introduction

Our knowledge about what cancer is, what causes cancer, how to prevent cancer, and the cell signaling pathways involved in tumorigenesis has increased signiﬁcantly within the last half a century. This increased knowledge has created some dilemmas and paradoxes. For instance, generally, it is now believed that the majority of cancers are preventable, yet predictions indicate that more than 1 million Americans will be diagnosed with cancer in 2008, numbers higher than ever before. General consensus is that less than 90% of cancer-related deaths are due to metastasis of the disease, yet most drugs in the market focus on apoptosis of the primary tumor. Lifestyle is linked up to 90% of cancers. Carcinogens exist in our environment, food and drinks, and surroundings. Carcinogens can exert their effect through the induction of mutations, deoxyribonucleic acid (DNA) double-strand breaks, and DNA adduct formations (Figure 27.1). Studies also indicate that the polyphenolic phytochemicals have great potential to prevent and treat cancer. These phytochemicals include the phenolic acids, anthocyanins, catechins, stilbenes, resveratrol, ellagic acid, and several other ﬂavonoids [1]. Studies show that these phytochemicals inhibit the proliferation of cancer cells in vitro and in vivo and can prevent the carcinogenesis induced by chemicals and radiation. The studies also suggest that polyphenolic compounds inhibit the DNA damage induced by different carcinogens and thereby prevent carcinogenesis [2, 3]. This review focuses on the potential of selected polyphenols such as resveratrol, ellagic acid, silymarin, and tea polyphenols to suppress the DNA damage induced by different chemicals and to prevent cancer.

27.2 Physical–Chemical Properties of Polyphenols and Their Occurrence

Resveratrol (3,40,5-trihydroxy stilbene) is a phytoalexin that belongs to the stilbene class of compounds (Figure 27.3). It is an off-white powder (extracted by methanol)

Figure 27.1 Role of DNA damage in tumorigenesis induced by carcinogens and infections.

with a melting point of 253–255 C and molecular weight of 228.25. Resveratrol exists in both cis and trans forms, and the latter is the more stable form. The word resveratrol is derived from a Latin word res meaning which comes from, from the plant name veratrum, from the suffix ol indicating the presence of an alcohol moiety in the structure. Takaoka first isolated the compound in 1940 from the roots of the white hellebore (Veratrum grandiflorum O. Loes); Nonomura et al. isolated it in 1963 from the roots of a traditional Japanese and Chinese plant (Polygonum cuspidatum). So far, resveratrol has been reported to exist in more than 70 plant species. Resveratrol was reported to be in peanuts (Arachis hypogaea) and different types of berries (Vaccinium). Fresh grape skin contains about 50–100 mg of resveratrol per gram wet weight, thereby contributing to a relatively high concentration of resveratrol in red wine and grape juice. A series of epidemiological studies has proven an inverse relationship between consumption of red wine and incidence of cardiovascular diseases; the phenomenon is termed the French Paradox. Several studies have shown that the cardiovascular effect of red wine is mainly due to its resveratrol content (Figure 27.2) [4]. Ellagic acid is another antimutagenic and anticarcinogenic compound and is principally obtained from plants such as oak (Quercus) [5, 6], sweetgum (Liquidambar styraciflua), linden (Tilia), aile (Alnus), eucalyptus species, chestnut (Castanea dentata), pomegranate, guava, strawberry, raspberry, blackberry, pistachio, mango, hazelnut, walnut, pepper, plum, apricot, peach, black raisin, red raisin, currant, tea, grape, wines, and aged brandies in oak casks (Figure 27.3). Ellagic acid is a yellow crystalline powder slightly soluble in water, alcohol, and ether and soluble in alkalies. 27.2 Physical–Chemical Properties of Polyphenols and Their Occurrence j457

Figure 27.2 Sources and structure of resveratrol.

Melting point is 350 C. Studies suggest that ellagic acid decreases the abnormal cell growth in the human colon, prevents the development of cells infected with human papillomavirus, which is related to cervical cancer, and promotes the apoptotic growth of cancerous cells of the prostate. The apoptotic process incited by this antioxidant (ellagitannin) also has beneficial effects on cancers of the breast, lung, esophagus, and skin. Carcinogenesis inhibition by ellagic acid has been demonstrated in animals with tumors of the esophagus, tongue, lung, colon, liver, and skin. Also, ellagic acid is a pharmacologically active compound, and it has been found to control hemorrhages in animals and humans by activating the Hageman factor. Figure 27.3 shows the structure of ellagic acid and its sources. Silymarin consists of a family of flavonoids (silybin, isosilybin, silychristin, silydianin, and taxifoline) commonly found in the dried fruit of the milk thistle plant, Silybum marianum (Figure 27.4). The molecular weight is 482.4 (silibinin) and melting point 158 C. It is anhydrous in nature, insoluble in water, and readily soluble in organic solvents. Research within the last decade has shown that silymarin can suppress the proliferation of a variety of tumor cells (e.g., prostate, breast, ovary, colon, lung, bladder); this is accomplished through cell cycle arrest at the G1/S-phase, induction of CDK inhibitors (such as p15, p21, and p27), downregulation of antiapoptotic gene products (e.g., Bcl-2 and Bcl-xL), inhibition of cell survival kinases (AKT, PKC, and MAPK), and inhibition of inflammatory transcription factors 458j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

Figure 27.3 Sources and structure of ellagic acid.

(e.g., nuclear factor-kappa B (NF-kB)). The studies also showed that silymarin can also downregulate gene products involved in the proliferation of tumor cells (cyclin D1, epidermal growth factor receptor, cyclooxygenase-2 (COX-2), TGF-beta), invasion (MMP-9), angiogenesis (VEGF), and metastasis (adhesion molecules). The anti- inﬂammatory effects of silymarin are mediated through suppression of NF-kB- regulated gene products, including COX-2, liquid oxygen, inducible nitric oxide synthase, tumor necrosis factor, and interleukin (IL)-1. Numerous studies in animals showed that silymarin acts as a chemopreventive agent against a variety of carcinogens/tumor promoters, including UV light, 7,12-dimethylbenz(a)anthracene (DMBA), phorbol 12-myristate 13-acetate, and others. Silymarin has also been shown to sensitize tumors to chemotherapeutic agents through downregulation of the multidrug resistent protein (MDR) protein and other mechanisms. It binds to both estrogen and androgen receptors, and downregulates prostate-speciﬁc antigen (PSA). In addition to its chemopreventive effects, silymarin exhibits antitumor activity against human tumors (e.g., prostate and ovary) in rodents. Various clinical trials have indicated that silymarin is bioavailable and pharmacologically safe. Here, we describe the chemopreventive effects of silymarin and its protection against the DNA damage induced by different agents. Figure 27.4 shows the structure of the silymarin components and their sources. 27.2 Physical–Chemical Properties of Polyphenols and Their Occurrence j459

Figure 27.4 Sources and structure of silymarin and its analogues.

Next to water, tea (Camellia sinensis) is the most commonly consumed beverage worldwide because of its characteristic aroma, flavor, and health benefits. Beverage- grade tea is manufactured from the leaves and buds of the plant C. sinensis and is commercially available in three forms: green, black, and oolong tea. The catechins present in green tea are commonly called polyphenols and are flavanols in nature. The major catechins found in green tea are ()-epicatechin, ()-epigallocatechin, ()-epicatechin-3-gallate, and ()-epigallocatechin-3-gallate (EGCG). The chemical structures of these polyphenols are shown in Figure 27.5. These polyphenols are antioxidant and anti-inflammatory in nature and have been shown to possess anticarcinogenic activity in several in vitro and in vivo systems. Of these major polyphenols, EGCG has been shown to be the major and most effective chemopreventive agent and has been extensively studied (e.g., its use in skin photoprotective activity) in several disease models. It is a white powder, soluble in water at least to 5 mg/ml, giving a clear solution. EGCG should be stable in solution at 2–8 C at neutral to slight acidic pH [7]. 460j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

Figure 27.5 Sources and structure of tea polyphenols.

27.3 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vitro Studies

In the past decade, research has shown that phenolic compounds may prevent cancer through inhibition of the DNA damage induced by different agents including

hydrogen peroxide (H2O2), potassium bromate (KBrO3), tobacco smoke condensate, – and benzo(a)pyrene (C20H12). Tables 27.1 27.4, summarizes the protective effect of phenolic compounds on DNA damage induced by different chemicals and radiation. A brief description of the effect of phenolic compounds on DNA damage induced by different agents is given below.

27.3.1 Polyphenols Protect Against Chemicals-Induced DNA Damage

The research over the past few years showed that polyphenols are potent agents

against H2O2, HQ, BQ, BT, vitamin C, and bleomycin-induced DNA damage. The studies showed that resveratrol inhibited KBrO3-induced DNA damage. KBrO3 was 27.3 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vitro Studies j461

Table 27.1 Resveratrol inhibits DNA damage induced by activity of different agents.

3þ Cr and H2O2-induced DNA damage in calf thymus DNA 3þ Cr and H2O2-induced DNA damage in calf thymus DNA H2O2-induced DNA damage in rat pheochromocytoma cells H2O2 and HQ-induced strand breaks in phi X-174 plasmid DNA H2O2-induced DNA damage in CHO cells H2O2-induced DNA damage in PBL H2O2-induced DNA damage in human lymphocytes H2O2-induced DNA damage in mononuclear cells of umbilical blood H2O2-induced mutation by Ames test H2O2-induced DNA damage in C6 glioma cells TAR and H2O2-induced DNA damage in rat ﬁbroblasts and mouse mammary epithelial cells KBrO3-induced DNA damage in the kidney of rats Cisplatin and mitomycin C-induced DNA damage in human PBMC Cisplatin and selenium–cisplatin conjugate ([NH(3)][2]Pt[SeO(3)], Se-Pt) in human blood platelets, lymphocytes UVA-induced DNA damage in HaCaT human keratinocytes UV-induced DNA damage in cultured arteries Oxidative DNA damage in vascular smooth muscle cells from stroke-prone hypertensive rats B[a]P-induced DNA damage in mouse sperm B[a]P-induced DNA damage in mice lung B[a]P-DNA adduct formation in human bronchial epithelial cells B[a]P and DMBA-induced DNA damage in mouse epidermis B[a]P-induced DNA damage in human oral epithelial cells N-Hydroxy-PhIP-induced DNA adduct formation in human mammary epithelial cells Nitrobenzene-induced DNA damage in mice Fluorophore A2E-induced DNA damage DMBA-induced DNA adducts in human PMN IQ-induced DNA damage in male F344 rats ROS-induced DNA damage TCDD-induced DNA damage in human mammary epithelial cells MNNG-induced DNA damage in Chinese hamster lung ﬁbroblasts Estrogen-induced DNA damage in breast cells 5-Aminolevulinic acid-induced DNA damage in vitro Mutagenicity in plasmid SK(þ) DNA Peroxynitrite-induced DNA damage in vitro

DNA damage caused by the photovoltaic effect of nano-TiO2 DNA damage in experimental diabetic neuropathy 3-NBA-induced DNA mutation in human lung epithelial A549 cells b-Amyloid-induced oxidative stress in PC12 cells Ethanol-induced oxidative DNA damage in mouse brain cells Depurinating estrogen–DNA adduct formation in calf thymus DNA

Abbreviations: B[a]P, benzo[a]pyrene; IQ, 2-amino-3-methylimidazo[4,5-f ]quinoline; MNNG, N-methyl-N0-nitro-N-nitrosoguanidine; 3-NBA, 3-nitrobenzanthrone; PBL, human peripheral blood lymphocytes; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PMN, human polymorphonuclear neutrophils; TAR, tobacco smoke condensate; ROS, reactive oxygen species; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; UV, ultraviolet. Selected references: [8–14]. 462j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

Table 27.2 Ellagic acid inhibits DNA damage induced by activity of different agents.

þ Cu2 -mediated oxidative DNA lesion

H2O2-induced mutation H2O2-induced DNA SSBs in cells of the digestive gland of mussels H2O2-and bleomycin-induced DNA damage in CHO cells AFB1 mutagenesis in strain TA 100 of Salmonella typhimurium Covalent binding of NBMA metabolites to DNA in cultured rat esophagus DMBA–DNA binding and DMBA metabolism in secondary cultures of mammary epithelial cells MNU-induced DNA adduct formation Aroclor 1254-induced DNA adduct formation DBP-induced DNA adduct formation DBP-induced DNA adduct formation in the human breast cells MCF-7 Endrin-induced DNA single-strand breaks Pentachlorophenol-induced oxidative DNA damage in mouse liver Cyclophosphamide, MNNG, N-nitroso-N-ethylurea, mitomycin C, and URE-induced genotoxicity TCDD-induced DNA SSB 2-Aminoﬂuorene-DNA adduct formation in human bladder cancer cells Radiation-induced DNA strand breaks produced in rat lymphocytes Gamma radiation-induced DNA damage

ﬂ Abbreviations: AFB1,a atoxin B1; DBP, dibenzo[a,l]pyrene; DMBA, 7,12-dimethylbenz(a) anthracene; H2O2, hydrogen peroxide; MNU, methylnitrosourea; 3-NBA, 3-nitrobenzanthrone; SSB, single-strand break; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; URE, urethane. Selected references: [15–18].

given to rats treated previously with resveratrol, and oxidative damage to kidney DNA was estimated after 6 h by measuring 8-oxo-7,8-dihydro-20-deoxyguanosine referred to as deoxyguanosine (dG). Levels of dG in the renal genomic DNA signiﬁcantly

increased by more than 100% after the KBrO3 treatment, and resveratrol abolished completely the KBrO3-induced DNA damage. The studies showed that resveratrol,

Table 27.3 Silymarin inhibits DNA damage induced by different agents activity.

H2O2-induced DNA damage in HUVECs H2O2-induced DNA damage in human lymphocytes (no effect) – H2O2 BLM-induced DNA damage HQ-, BQ-, BT-, H2O2-, BLM-, and Vit C-induced DNA damage Trp- and IQ-induced DNA damage in human blood and sperm samples Acetaminophen-induced DNA strand breaks Isoproterenol-induced DNA damage in rat cardiac myocytes N-nitrosodiethylamine-induced DNA adduct formation UV-induced DNA damage in SKH-1 hairless mice UVB-induced thymine dimer-positive cells UVB-induced DNA damage in human epidermal keratinocytes UVA-induced damage to human HaCaT keratinocytes

Abbreviations: BLM, bleomycin; BQ, benzoquinone; BT, benzenetriol; HQ, hydroquinone; HUVEC, human umbilical vein endothelial cell;IQ, 2-amino-3-methylimidazo(4,5-f ) quinoline; Trp,3-amino-1- methyl-5H-pyrido(4,3-b)indole; Vit C, sodium ascorbate; UV, ultraviolet. Selected references: [19–22]. 27.3 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vitro Studies j463

Table 27.4 Tea polyphenols inhibits DNA damage induced by different agents activity.

Benzo[a]pyrene-induced DNA damage

Aﬂatoxin B1-induced DNA damage 2-Aminoﬂuorene-induced DNA damage

H2O2-induced DNA damage tert-Butyl hydroperoxide-induced DNA damage PhIP-induced DNA damage 1,2-Dimethylhydrazine-induced DNA damage in rat colonic mucosa UVA-induced DNA damage

Abbreviations: H2O2, hydrogen peroxide; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; UV, ultraviolet. Selected references [23–30].

ellagic acid, silymarin, and tea polyphenols inhibited H2O2-induced DNA damage in different systems including human lymhocytes, in human umbilical vein cells, in rat pheochromocytoma (PC12) cells, rat ﬁbroblasts, and mouse mammary epithelial cells; phi X-174 plasmid DNA; CHO cells; human peripheral blood lymphocytes; mononuclear cells of umbilical blood, C6 glioma cells and in cells from the mussel (Unio tumidus) digestive gland [15, 20, 23, 31]. The studies also showed that polyphenols have high protective activity against tobacco smoke condensate and benzo[a]pyrene-induced DNA damage. The treatment of resveratrol inhibited tobacco smoke condensate-induced DNA damage in rat ﬁbroblasts and mouse mammary epithelial cells.

C20H12, an aryl hydrocarbon receptor ligand present in cigarette smoke and car exhaust, is thought to negatively affect male reproduction and cause stomach and lung cancer. The studies showed that resveratrol inhibits damage caused by

C20H12-induced benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) DNA adducts in all components of the seminiferous tubules, in mouse lungs, in human bronchial epithelial cells, mouse epidermis, and in human oral epithelial cells [11]. ﬂ The studies also showed that ellagic acid inhibited a atoxin B1 (AFB1) mutagenesis in Salmonella typhimurium and DNA damage in cultured rat and human tracheo- bronchial tissues. At a dose of 1000 mg/plate, ellagic acid inhibited the mutation frequency by more than 90%. Ellagic acid was also tested for its ability to inhibit the DNA binding and adduct formation of AFB1 in cultured explants of rat trachea and human tracheobronchus. Ellagic acid caused a dose-dependent inhibition in the covalent binding of AFB1 to the DNA of both the rat trachea (9–57% inhibition) and human tracheobronchus (24–79% inhibition). Green tea polyphenols also inhibited AFB1-induced DNA damage. Cisplatin and mytomycin C are cytotoxic anticancer agents that are known to cause DNA damage in normal cells. The studies showed that reveratrol inhibited cisplatin and selenium–cisplatin conjugate ([NH(3)][2]Pt[SeO(3)], Se-Pt)-induced DNA damage in human blood platelets and lymphocytes. More recent studies showed that resveratrol inhibited the cisplatin- and mitomycin C-induced DNA damage in human peripheral blood lymphocytes. Mizutani et al. showed that the administration of resveratrol to male and female stroke-prone spontaneously 464j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

hypertensive rats inhibited the level of 8-hydroxydeoxyguanosine in the urine. In an earlier study, Mizutani et al. [10] showed that resveratrol inhibited the oxidative DNA damage in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. These polyphenols also inhibit N-hydroxy-PhIP-induced DNA adduct formation in human mammary epithelial cells; nitrobenzene-induced DNA damage in mice; ﬂuorophore A2E-induced DNA damage; DMBA-induced DNA adducts in human polymorphonuclear neutrophils; quinoline (IQ)-induced DNA damage in male F344 rats; 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced DNA damage in human mammary epithelial cells; N-methyl-N-nitro-N-nitrosoguanidine-induced DNA damage in Chinese hamster lung ﬁbroblast cells, estrogen-induced DNA damage in breast cells; 5-aminolevulinic acid-induced DNA damage in vitro,peroxynitrite- induced DNA damage in vitro; DNA damage caused by the photovoltaic effect of nano-TiO(2); DNA damage in experimental diabetic neuropathy; 3-nitrobenza- throne-induced DNA mutation in human lung epithelial A549 cells; b-amyloid- induced DNA damage in PC12 cells caused by vitisin A or heyneanol A [13]; ethanol-induced oxidative DNA damage in mouse brain cells; depurinating estrogen-DNA adduct formation in calf thymus DNA; inhibited N-nitrosobenzylmethylamine metabolism and DNA binding in cultured rat esophagus; DMBA- and N-methyl-N-nitrosourea-induced DNA damage; benzo[a]pyrene-induced DNA damage, endrin- and pentachlorophenol-induced DNA damage; acetaminophen-, isoproterenol-, N-nitrosodiethylamine-, Trp-, and IQ-induced DNA damage [22]; and 1,2-dimethylhydrazine-induced oxidative DNA damage in rat colon mucosa [26].

27.3.2 Polyphenols Protect Against Radiation-Induced DNA Damage

Ungvari et al. [9] showed that resveratrol inhibits the DNA damage induced by UV radiation in cultured arteries. Another study showed that resveratrol enhances UVA-induced DNA damage in the human keratinocyte cell line. Thresiamma et al. [17] showed that ellagic acid inhibited radiation-induced genotoxicity in mice. Whole-body radiation in mice produced micronuclei and chromosomal aberrations that were found to be signiﬁcantly inhibited by oral administration of ellagic acid (200 mmol/kg). Moreover, administration of ellagic acid inhibited the DNA strand breaks produced in rat lymphocytes upon irradiation. Nemavarkar et al. [18] showed that ellagic acid protected normal yeast cells from gamma radiation-induced damage. Using rad52 mutants that lack a recombinant DNA repair pathway, protection was brought about solely by reducing DNA damage rather than by interfering with DNA repair. Gu et al. [14] showed that silymarin inhibited ultraviolet light B (UVB)-induced thymine dimer-positive cells. Silymarin also inhibits UVB-induced DNA damage in human epidermal keratinocytes. The studies also showed that green tea polyphenols also have great potential in the prevention of radiation-induced DNA damage [27–30, 32]. 27.4 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vivo Studies j465

27.4 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vivo Studies

Numerous studies showed the ability of polyphenols in the prevention and treatment of cancer. A brief description of the ability of polyphenols against the prevention and treatment of cancer in animals is given in the following section. Tables 27.5–27.8 summarizes the chemopreventive activity of polyphenols in animals.

27.4.1 Polyphenols Prevent Colon, Esophagus, and Gastric Cancer

Colorectal cancer, also called colon cancer or large bowel cancer, includes cancerous growths in the colon, rectum, and appendix. It is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. In the United States it is estimated that 148 810 new cases and 49 960 deaths will occur due to colon cancer in 2008. The studies over the past few years showed that

Table 27.5 Chemopreventive activity of resveratrol against different cancer.

Model Rodents Doses/Route

Ascites Mice 1 mg/kg/i.p. Ascites Mice 2.5, 5, 10, 20 mg/kg/i.p. Breast cancer Mice 100, 200 ppm/diet Breast cancer Mice 25 mg/kg/i.p. Breast cancer Mice 1 mg/l/d.w. (Her-2) Breast cancer Rats 10 ppm/diet Breast cancer Mice (Include dose/route) Colon cancer Mice 0.05%, 0.2%/diet Colon cancer Rats 8 mg/kg/p.o. (DMH) Colon cancer Rats 200 mg/kg/d.w. (AOM) Colon cancer Mice 0.1%/d.w. (Min mouse) Esophageal cancer Rats 1, 2 mg/kg/p.o. Gastric cancer Mice 500, 1000, 1500 mg/kg/i.t. Glioma Rats 10, 40 mg/kg/i.p. Hepatoma Rats 10, 50 ppm/diet Liver cancer Mice 10, 15 mg/kg/i.p. Liver cancer Mice 10, 15 mg/kg/i.p. Leukemia Mice 12.5, 25, 50 mg/kg/i.g. Lung cancer Mice 20 mg/kg/i.p Neuroblastoma Mice 2, 10, 50 mg/kg/p.o. Prostate cancer Mice 625 mg/kg/route? Melanoma Mice 10 mmol/topical Skin cancer Mice 85 nM/topical Skin cancer Mice 1, 5, 10, 25 mmol/topical

Selected reference: [4]. 466j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

Table 27.6 Chemopreventive activity of ellagic acid against different cancers.

Model Rodents Dose/Route

Esophageal cancer Rats 4 g/kg/diet Esophageal cancer Rats 4 g/kg/diet Intestinal cancer Mice 1565 mg/kg/diet Liver cancer Rats 0.005%/diet Liver cancer Rats 400 ppm/diet Lung cancer Mice 12 mg/ml/d.w. Lung cancer Mice 0.2, 2 mmol/diet Lung cancer Mice 50 mmol/kg/i.v. Lung adenoma Mice 100 mg/kg/diet Lung metastasis Mice 200 nmol/kg/i.v. Mammary tumor Rats 0.1%/d.w. Mammary tumor Rats 0.1%/diet Multiorgan carcinogenesis Rats 1%/diet Multiorgan carcinogenesis Fish 1000, 2000 ppm/diet Skin cancer Mice 1500 nmol/topical Skin cancer Mice 3 mg/l/d.w. Tongue cancer Rats 400 ppm/diet

Selected references: [33–39].

Table 27.7 Chemopreventive activity of silymarin against different cancers.

Cancer Rodent Dose/route

Bladder cancer Mice 200 mg/kg Bladder cancer Mice 500 ppm/diet Breast cancer Mice 414 mmol/l/kg,/p.o. Breast cancer Mice 0.3%/diet Breast cancer Rats 0.03, 0.1, 0.3, or 1%/diet Colon cancer Rats 5000, 10 000 ppm/diet Liver cancer Rats 1000 ppm/diet Lung cancer Mice 0.033, 0.10, 0.33, 1.0%/diet Lung cancer Mice 0.05, 0.1%/diet Lung cancer Mice 200 mg/kg/p.o. Oral tongue cancer Rats 100, 500 ppm/diet Prostate cancer Mice 0.1, 0.5, 1%/diet Prostate cancer Mice 0.05, 0.1%/diet Prostate cancer Rats 0.05%, 0.1%/diet Prostate cancer Rats 100, 500 ppm/diet Skin cancer Mice 9 mg/topical Skin cancer Mice 1%/diet Skin cancer Mice 0.5, 1 mg/cm2/topical Skin cancer Mice 0.5%/diet Skin cancer Mice 3, 6, 12 mg/topical

Selected references: [40–47]. 27.4 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vivo Studies j467

Table 27.8 Chemopreventive activity of green tea polyphenols against different cancers.

Cancer Rodent Dose/Route

Bladder cancer Mice 100 or 250 mg/kg Breast cancer Rats — Colon cancer Rats 0.05, 0.01, or 0.002% Colon cancer Mice 0.4%/diet Colon cancer Rats 1800–3600 ppm Colon cancer Rats 50 mg/kg Colon cancer Rats 2% (w/v) Colon cancer Rats 0.12 or 0.24% Colon cancer Mice 5 mg in 0.2 ml, oral Gastric cancer Rats 0.5–1.0%, drinking water Liver cancer Rats 0.1% in drinking water Lung cancer Mice 5 mg in 0.2 ml oral Lung cancer Mice — Lung cancer Mice 0.5% drinking water Prostate cancer Mice 0.1% in drinking water Prostate cancer Mice — Prostate cancer Mice 0.06% in drinking water Oral cancer Rats 200 mg/kg Skin cancer Mice — Skin cancer Mice 0.1%, in drinking water Skin cancer Mice — Skin cancer Mice 6 mg/animal/topical Skin cancer Mice 6 mg/animal/topical Skin cancer Mice —

Selected references: [48–55]. plant polyphenols have great potential in the prevention of gastrointestinal tract cancers. The studies showed that resveratrol, silymarin, and tea polyphenols inhibit azoxymethane-induced colon carcinogenesis in animals [42, 56–60]. Moreover, studies also showed the protective activity of resveratrol and its synthetic analogue 3,4,5,40-tetramethoxystilbene inhibited adenoma development in Apc(Min þ ) mice. Several other studies showed that resveratrol and tea polyphenols inhibited 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis animals. Administra- tion of tea polyphenols also inhibited N-methyl-N-nitrosourea-induced colorectal cancer in rats and both diethylnitrosamine (DEN)- and benzo(a)pyrene (BP)-induced forestomach neoplasia in A/J mice [50]. Esophageal cancer is malignancy of the esophagus. It is estimated that in the United States 16 470 new cases and 14 280 deaths will occur due to esophageal cancer in 2008. The studies showed that resveratrol inhibits N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 male rats [4]. The higher expression of COX-1, the upregulated COX-2 expression, and the increased levels of PGE(2) synthesis were all signiﬁcantly decreased by administering resveratrol. Administration of ellagic acid also inhibited NMBA-induced esophageal cancer in rats and in Apc-mutated Min mice [33]. 468j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

Stomach or gastric cancer can develop in any part of the stomach and may spread throughout the stomach and to other organs, particularly the esophagus and the small intestine, and is the fourth most common cancer worldwide. In the United States, it is estimated that 21 500 new cases and 10 880 will occur due to gastric cancer in 2008. Zhou et al. studied the effect of resveratrol in a nude mouse model of gastric cancer established by injecting human primary gastric cancer cells into subcutaneous tissue [4]. Resveratrol (500 mg/kg, 1000 mg/kg, and 1500 mg/kg) was directly injected beside the tumor six times every 2 days. The results showed that resveratrol inhibited the development of gastric cancer in mice. Administration of tea polyphenols also inhibited N-methyl-N0-nitro-N-nitrosoguanidine (MNNG)- induced gastric cancer in rats [51].

27.4.2 Polyphenols Prevent Ascites Tumor

Polyphenols are potent therapeutic agents against ascites tumor. The studies showed that resveratrol administration to rats inoculated with a fast growing tumor (the Yoshida AH-130 ascites hepatoma) caused a significant decrease (25%) in the tumor cell content. The flow cytometric analysis of the tumor cell population revealed the existence of an aneuploid peak (representing 28% of total), which suggests that resveratrol causes apoptosis in the tumor cell population; this likely results in the decreased cell number. In 2003, Liu et al. showed that resveratrol could inhibit the growth of H22 tumor in Balb/c mice. This study showed that the antitumor effect of resveratrol might be related to directly inhibiting the growth of H22 cells and indirectly inhibiting its potential effect on nonspecific host immunomodulatory activity [61].

27.4.3 Polyphenols Inhibit Breast Cancer

Worldwide, breast cancer is the second most common type of cancer after lung cancer (10.4% of all cancer incidence, both sexes counted). In the United States, it is estimated that 184 450 new cases and 40 930 deaths will occur in 2008. Chemopre- vention of polyphenols has emerged as a cost-effective approach to control the incidence of breast cancer. The studies from our group and others showed that resveratrol inhibited the formation of DMBA-induced mammary tumors in female Sprague Dawley rats. The analysis of the tumor tissues showed that resveratrol suppresses DMBA-induced mammary carcinogenesis through the downregulation of NF-kB, COX-2, and matrix metalloprotease-9 expression. Moreover, resveratrol is also capable of delaying the development and reducing the metastasizing capacity of spontaneous mammary tumors in HER-2/neu transgenic mice. The chemopreventive effect of resveratrol might be related to the downregulation of HER-2/neu expression and the induction of apoptosis in tumor cells. The studies showed that resveratrol significantly lowered tumor growth, decreased angiogenesis, and increased the apoptotic index in ER (estrogen receptor) a- and ERbþ MDA-MB-231 27.4 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vivo Studies j469 tumors in nude mice compared to controls. More recent studies showed that vaticanol C (Vat-C, a novel resveratrol tetramer) inhibited mouse metastatic mammary cancers carrying mutations in p53 that produce a metastatic spectrum similar to that seen in human breast cancers. The studies also showed that ellagic acid inhibited 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female F344 rats [37, 62]. The efficacy of a complex of silybin with phosphatidylcholine on the development of spontaneously appearing mammary tumors in HER-2/neu transgenic mice has also been evaluated. This complex was effective in delaying the onset of spontaneous mammary tumors and reducing the size of the primary mammary tumor and the size and number of lung metastases. The complex was also effective in downregulating HER-2/neu gene expression. The chemopreventive efficacy of black tea polyphenols (Polyphenon-B) during the preinitiation phase of DMBA-induced mammary carcinogenesis was also studied [49]. Intragastric administration of DMBA-induced adenocarcinomas and the dietary administration of Polyphenon-B effectively suppressed the incidence of mammary tumors as evidenced by modulation of xenobiotic metabolizing enzymes and oxidant–antioxidant status, inhibition of cell proliferation and angiogenesis, and induction of apoptosis [49].

27.4.4 Polyphenols Inhibit Liver Cancer

Hepatocellular carcinoma (HCC) is the third deadliest and fifth most common human cancer worldwide. In the United States, it is estimated that 21 370 new cases will be diagnosed with liver cancer and 18 410 deaths will occur in 2008. The studies showed that plant polyphenols have great potential in the prevention of HCC. Studies showed that dietary resveratrol could inhibit the proliferation and metastasis of tumors and hyperlipidemia in Donryu rats subcutaneously implanted with an ascites hepatoma cell line, AH109A [4, 63]. With the addition of 10 or 50 ppm of resveratrol to the diet of hepatoma-bearing rats for 20 days, solid tumor growth and metastasis tended to be suppressed dose-dependently. Resveratrol dose dependently suppressed both the serum triglyceride and very low-density lipoprotein þ low-density lipoprotein (VLDL LDL) cholesterol levels. These results suggested that dietary resveratrol has hypolipidemic effects with a tendency for antitumor growth and antimetastatic effects in hepatoma-bearing rats. In another study, the treatment of hepatoma22 (H22) tumor- bearing mice with 10 or 15 mg/kg resveratrol for 10 days inhibited the growth of murine transplantable liver cancer by 36.3 or 49.3%, respectively [4]. This study also showed that the underlying antitumor mechanism of resveratrol might involve the inhibition of the cell cycle progression by decreasing the expression of the cyclinB1 and p34cdc2 proteins. Resveratrol in combination with 5-fluorouracil (5FU) also inhibited the growth of murine H22 after the mice bearing H22 tumors were treated with 10 or 15 mg/kg resveratrol for 10 days. Ellagic acid also inhibited hepatocarcinogenesis induced by N-2-fluorenylacetamide (FAA) in male ACI/N rats. Exposure to FAA alone induced a substantial number 470j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

of altered foci, and at the end of experiment (week 36), the incidence of hepatocellular neoplasms was 100%. In the group receiving ellagic acid together with FAA, the number of altered foci was decreased at all time points and at termination of the study; the final incidence of hepatocellular neoplasms (30%) was also reduced. Another study showed that dietary administration of ellagic acid (0.005%) to rats significantly reduced the number of gamma-glutamyl transpeptidase-positive foci fl induced by a atoxin B1 (AFB1); such foci are considered to be precursor of hepatocellular neoplasms. Silymarin was also found to be effective in suppressing the hepatocarcinogenesisinducedbyN-nitrosodiethylamine(NDEA)inrats[22].Adminis- tration of silymarin reduces the number of liver tumor nodules as well as lipid peroxidation; it also elevated the antioxidant defense system in NDEA-treated rats [43]. The studies with tea polyphenols showed that these compounds inhibited NDEA-induced precancerous liver lesions in rats. Tea polyphenols (0.1%) were given to Wistar rats in drinking water during the 8 weeks of the experiment. The results also showed that GST-Pi mRNA and protein expression increased significantly in the

NDEA-CCl4-PH-treated group, which is consistent with the changing of GST- Pi-positive foci. Tea pigments and tea polyphenols had an inhibitory effect on the

overexpression of GST-Pi mRNA and protein in NDEA-CCl4-PH-treated rats. These results suggest that tea pigments and tea polyphenols are effective in preventing the occurrence and progression of precancerous liver lesions in rats [52].

27.4.5 Polyphenols Inhibit Neuroblastoma and Glioma

Neuroblastoma is the most common extracranial solid cancer in childhood and the most common cancer in infancy, with an annual incidence of about 650 new cases per year in the United States. The studies showed that resveratrol inhibited the outgrowth of tumors by as much as 80% in mouse xenograft models of human neuroblastoma [64]. The bioavailability of the resveratrol in serum was in the low micromolar range (2–10 mmol/l), and no accumulation was observed in tumor tissue. When resveratrol was given as peritumor injection, bioavailability was increased and rapid tumor regression occurred. Glioma is a cancer of the brain that begins in glial cells (cells that surround and support nerve cells). Approximately 10 000 Americans have been diagnosed with malignant glioma each year. The studies showed that resveratrol caused signiﬁcant glioma cell cytotoxicity and apoptosis, exerted antitumor effects on the subcutaneous and intracerebral gliomas, and inhibited angiogenesis in rat RT-2 subcutaneous gliomas [4].

27.4.6 Polyphenols Inhibit Leukemia

Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation of blood cells, usually white blood cells (leukocytes). As of 2008, it is estimated that each year approximately 44 270 individuals will be 27.4 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vivo Studies j471 diagnosed with leukemia in the United States and 21 710 individuals will die of the disease. The studies showed the antitumor and immunomodulatory effects of resveratrol on mouse lymphocytic leukemia cells L1210 in vivo [4]. Resveratrol exerted a dose-related regulatory effect on both innate and speciﬁc immune functions in L1210-bearing mice. A normalization of CD4/CD8 ratios in these tumors was noted after the treatment with resveratrol, as were enhanced of lymphocyte proliferation, NK cell activity, and anti-SRBC titers. Moreover, resveratrol suppressed interleukin-6 cellular content and release and mRNA expression in L1210-induced tumors in mice.

27.4.7 Polyphenols Inhibit Prostate Cancer

Cancer of the prostate gland is the most common invasive malignancy and a major cause of cancer-related deaths in male population in the United States (with an estimate of 186 320 new cases and 28 660 deaths in 2008) and is typically detected in the elderly population with a relatively slower rate of growth and progression. Many dietary phytochemicals have shown promising chemopreventive and therapeutic effects, at least in preclinical models of prostate cancer. Dietary administration of resveratrol significantly reduced the incidence of poorly differentiated prostatic adenocarcinoma by one-eighth of the untreated group in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice [4]. Moreover, this phytochemical also inhibited growth of advanced human prostate carcinoma xenografts in nude mice and inhibits 3,20-dimethyl-4-aminobiphenyl-induced prostate carcinogenesis in male F344 rats [46]. Oral infusion of a polyphenolic fraction isolated from green tea (GTP) also has great potential in the prevention and treatment of prostate cancer. At a human achievable dose (equivalent to six cups of green tea per day), it significantly inhibits prostate cancer development and increases survival in TRAMP mice. green tea polyphenols (GTP) (0.1% w/v) provided as the sole source of drinking fluid to TRAMP mice from 8 to 32 weeks of age resulted in (i) significant delay in primary tumor incidence and tumor burden, (ii) significant decrease in prostate (64%) and genitourinary (GU) (72%) weight, (iii) significant inhibition in serum insulin-like growth factor-1 (IGF-1) and restoration of insulin-like growth factor binding protein-3 (IGF BP-3) levels, and (iv) marked reduction in the protein expression of proliferating cell nuclear antigen (PCNA) in the prostate compared with water-fed TRAMP mice. In this study, GTP infusion resulted in almost complete inhibition of distant site metastases. Another study on the same mouse model showed that continuous GTP infusion for 32 weeks resulted in substantial reduction in tumor growth and the expression of NF-kB, IKKa,IKKb, RANK, NIK, and STAT-3 in dorsolateral prostate of TRAMP mice. The study also showed that EGCG inhibited early but not late-stage prostate cancer in TRAMP mice [54]. In another study, the chemopreventive effects of GTP, water extract of black tea, and their major constituents epigallocatechin-3-gallate and theaflavins were compared in athymic nude mice implanted with androgen- sensitive human prostate cancer CWR22Rnu1 cells. The results showed that the 472j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

treatment with all the tea ingredients resulted in (i) signiﬁcant inhibition in growth of implanted prostate tumors, (ii) reduction in the level of serum prostate speciﬁc antigen, (iii) induction of apoptosis accompanied with upregulation in Bax and decrease in Bcl-2 proteins, and (iv) decrease in the levels of VEGF protein.

27.4.8 Polyphenols Inhibit Skin Cancer

Skin cancer is a malignant growth on the skin that develops in the epidermis (the outermost layer of skin). It is estimated that more than 1 000 000 new cases of this cancer will be diagnosed and 1000 deaths will occur in the United States in 2008. Numerous studies provided the ability of polyphenols to inhibit the initiation, promotion, and progression of skin cancer. Resveratrol is one of the main polyphenols that has a great potential in the prevention and therapy of skin cancer. It inhibited DMBA-induced skin cancer in mice. This phytochemical also showed chemoprotective capabilities against DMBA-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted mouse skin two-stage carcinogenesis models. Another study conducted by Reagan-Shaw et al. [4] showed that resveratrol inhibited UVB-induced skin cancers in mice by modulating the expression and function of cell cycle regulatory proteins cyclin-D1 and -D2, cyclin-dependent kinase (CDK)-2, -4, and -6, and WAF1/p21. Ellagic acid also proved as a chemopreventive agent against

skin cancer by its ability to inhibit the formation C20H12 and 3-methylcholanthrene- induced skin tumorigenesis [39] in mice. Ellagic acid inhibited lung metastasis of B16F10 melanoma cells in mice [36]. Several reports have described the efficacy of silymarin in preventing the initiation and progression of skin cancer. This phytochemical reduced the incidence, multiplicity, and volume of the tumors in Sencar mouse skin [47] and decreases cell proliferation and increases apoptosis in skin tumor. Silybin was found to suppress the UVB-induced photocarcinogenesis in SKH1 hairless mice. Topical or dietary application of silybin decreased the onset of tumor appearance, multiplicity, and volume of skin cancer by downregulating inflammatory and angiogenic markers such as HIF-1a, STAT3, NF-kB, COX-2, and inducible nitric oxide synthase. Silymarin also inhibited the UVB-induced immunosuppression in mice through the downregulation of IL-12. Several studies evaluated the chemopreventive activity of GTP against skin tumorigenicity induced by different chemicals and radiation. In the two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene as the initiating agent followed by twice weekly applications of TPA as tumor promoter, topical application of GTP on female SENCAR mice afforded significant protection against skin tumorigenicity. This study also showed that oral feeding of GTP in drinking water to female SENCAR mice also protected against skin tumorigenesis in DMBA– TPA-treated animals. GTP when administered topically or orally significantly inhibited polycyclic aromatic hydrocarbons (PAHs)–DNA adduct formation in epidermis after topical application of [3H]benzo[a]pyrene or [3H]DMBA. In another study, topical application of GTP (6 mg/animal) concurrently with each application of 27.4 Mechanisms of Chemoprevention by Phenolic Compounds: Results of In Vivo Studies j473 either TPA (3.2 nmol) or mezerein (3.2 nmol) in stage I or stage II of the murine skin tumor promotion, respectively, resulted in significant protection against skin papilloma formation in terms of both tumor multiplicity (42–50%) and tumor growth (43–54%)andinhibitedpapillomastosquamouscellcarcinomas(SCCs). In order to identify which of the specific epicatechin derivatives present in GTP is responsible for these inhibitory effects, green tea phytochemicals were evaluated for their inhibitory effects against TPA-caused induction of epidermal ODC activity in mice. Among these, ()epigallocatechin-3-gallate, which was the major constituent present in GTP by weight, exerted the maximum inhibition against TPA-caused induction of epidermal ODC activity when compared with several other naturally occurring polyphenols [65]. GTP was also evaluated for their ability to inhibit UVB radiation-induced skin carcinogenesis in animals. Chronic oral feeding of GTP (0.1%, w/v) in drinking water resulted in significantly lower tumor yield and extended TDT50 as compared to animals receiving normal drinking water. Topical application of GTP before UVB irradiation also afforded protection against photocarcinogenesis; however, the protective response was lower than that observed by oral feeding of GTP in drinking water [66], and this protection is mediated through (a) the induction of immunoregulatory cytokine IL-12, (b) IL-12-dependent DNA repair following nucleotide excision repair mechanism, (c) the inhibition of UV-induced immunosuppression through IL-12-dependent DNA repair, (d) the inhibition of angiogenic factors, and (e) the stimulation of cytotoxic T cells in a tumor microenvironment.

27.4.9 Polyphenols Inhibit Lung Cancer

Lung cancer is the predominant cause of cancer mortality in developed countries with a 5-year survival of less than 10%. It is estimated that more than 215 020 new cases of lung cancer will be diagnosed and 161 840 deaths will occur in the United States, in 2008. The studies showed that resveratrol, at doses of 2.5 and 10 mg/kg, significantly reduced the tumor volume (42%), tumor weight (44%), and metastasis of the lung (56%) in mice bearing highly metastatic Lewis lung carcinoma (LLC) tumors. These studies also suggest that the antitumor and antimetastatic activities of resveratrol might be due to the inhibition of DNA synthesis in LLC cells. However, two other studies showed that resveratrol has no effect in the inhibition of lung cancer in animals [67]. Ellagic acid is another important polyphenol that has been reported to have chemopreventive activity against lung cancer. The studies showed that ellagic acid inhibited benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone- induced pulmonary adenoma formation and N-nitrosodiethylamine-induced lung tumorigenesis in mice. Ellagic acid was able to significantly reduce lung tumor incidence to 20.0% from the control value of 72.2%; it increases the glutathione levels in the lungs [68]. Another study showed the effect of ellagic acid on 4-NQO-induced tongue carcinogenesis and the number and area of silver-stained nucleolar organizer region proteins (AgNORs) in male F344 rats. Feeding ellagic acid to the rats significantly reduced the incidence of tongue neoplasms (squamous cell papilloma 474j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

and carcinoma) and preneoplastic lesions (hyperplasia and dysplasia) and the number and area of AgNORs per nucleus [35]. Dietary administration of silybin was found to reduce the incidence, multiplicity, and angiogenesis of lung tumors induced by urethane [44]. It was also found to inhibit the growth of human nonsmall cell lung carcinoma xenografts in nu/nu mice. However, Yan et al. [69] showed that dietary

administration of silybin did not inhibit C20H12-induced pulmonary adenoma in mice. The administration of GTP has a great potential in inhibiting lung cancer in animals. Oral intubation of GTP at a dose of 5 mg in 0.2 ml water 30 min prior to challenge with carcinogen afforded signiﬁcant protection against both diethylnitrosamine (DEN)- and benzo(a)pyrene-induced forestomach and lung tumorigenesis in A/J mice [50]. In another study, the inhibitory effects of Polyphenon E (a standardized green tea polyphenol preparation containing 65% ()-epigallocatechin-3-gallate) and caffeine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor progression from adenoma to adenocarcinoma were evaluated. Histopatho- logic analysis indicated that Polyphenon E administration signiﬁcantly reduced the incidence (by 52%) and multiplicity (by 63%) of lung adenocarcinoma. It also inhibited cell proliferation in adenocarcinomas, enhanced apoptosis in adenocarcinomas and adenomas, and lowered levels of c-Jun and extracellular signal-regulated kinase (Erk) 1/2 phosphorylation. The administration of black tea polyphenols also inhibited the formation of lung cancer in animals. The studies showed that black tea phenols inhibited pathogenesis of pulmonary tumors induced by a tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) [70, 71], and benzo(a) pyrene (B[a]P)-induced lung carcinogenesis in mice [72].

27.4.10 Polyphenols Inhibit Bladder Cancer

Bladder cancer the fourth most common cancer in men and eighth most common cancer in women, represents an important health problem. In the United States, estimated 68 810 new cases of bladder cancer will be diagnosed and approximately 14 100 deaths will be attributed to bladder cancer in 2008. The studies showed that siliymarin inhibited bladder cancer induced by tobacco smoke carcinogen N-butyl- N- (4-hydroxybutyl) nitrosamine in ICR mice [40]. Polyphenon E was also examined for its chemopreventive efﬁcacy against 4-hydroxybutyl(butyl)nitrosamine-induced urinary bladder cancer. The administration of Polyphenon E (100 or 250 mg/kg body weight/day) caused a dose-dependent decrease in palpable urinary bladder tumors (low dose, 14 of 34; high dose, 6 of 35; controls, 20 of 34) in rats [48].

27.4.11 Polyphenols Inhibit Oral Cancer

Oral cancer is any cancerous tissue growth located in the mouth. In 2008, in the United States alone, about 34 000 individuals will be diagnosed with oral cancer. Several studies showed that polyphenols inhibit chemically induced oral cancer 27.5 Mechanisms of Chemoprevention by Phenolic Compounds: Results of Human Studies j475 in animals. Feeding ellagic acid to rats signiﬁcantly reduced the incidence of 4-NQO- induced tongue carcinogenesis (squamous cell papilloma and carcinoma) and preneoplastic lesions in male F344 rats [35, 45]. Moreover, silymarin elevated the activity of phase II enzymes and decreased the polyamine content and prostaglandin levels in tongue mucosa [45]. The studies also showed that administration of green tea polyphenols inhibited 4-NQO-induced oral cancer. On supplementation of GTPs for 30 days (200 mg/kg) for the oral cancer-induced rats, there was a moderate increase in the levels of GSH, PSH, and total thiols and a decrease in the levels of GSSG, conjugated dienes, and the activity of GGT. Thus, GTP reduces the oxidant production thereby maintaining the endogenous low molecular weight cellular thiols in oral cancer-induced rats.

27.5 Mechanisms of Chemoprevention by Phenolic Compounds: Results of Human Studies

Human clinical studies have demonstrated that polyphenols have significant cancer chemopreventive and therapeutic effects. The efficacy of silymarin is being evaluated in cancer patients either alone or in combination with other chemotherapeutic agents. Silipide, a silibinin formulation, was given orally to patients with colorectal adenocarcinoma at doses of 360, 720, or 1440 mg daily for 7 days and high levels of silibinin were achieved in colorectal mucosa of the patients [73]. Silibinin phytosome (SiliphosR), a commercial preparation of silibinin, at a dose of 13 g, divided in three daily doses, appears to be well tolerated in patients with advanced prostate cancer. Silymarin was used (along with soy, lycopene, and antioxidants) in a phase III clinical trial to delay prostate-specific antigen progression after prostatectomy and radiotherapy in prostate cancer patients. Silymarin is also tried against promyelocytic leukemia. The investigators administered 800 mg/day silymarin during the patients methotrexate and 6-mercaptopurine chemotherapy. During the 4 months of treatment with silymarin, the patient had normal liver transaminase levels and there was no further interruption of therapy. In a nonrandomized study, patients with brain metastases receiving stereotactic radiotherapy with omega-3 fatty acids and silymarin had longer survival times and a decreased number of radionecroses [73]. The studies suggest that the use of ellagic acid reduces chemotherapy-induced toxicity, particularly neutropenia, in hormone refractory prostate cancer (HRCP) patients [74]. The studies also showed that green tea polyphenols have great potential in the prevention and treatment of cancer. It is reported that green tea consumption reduced the risk of prostate cancer [75]. In Japan, Public Health Center-based Prospective Study of green tea showed that consumption of green tea was associated with a dose-dependent decrease in the risk of advanced prostate cancer. Green tea catechins in capsules given to patients with high-grade prostate intraepithelial neoplasia (PIN) demonstrated cancer preventive activity by inhibiting the conversion of high-grade PIN lesions to cancer [75]. In another case-control study, conducted 476j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

in southeast China in 2004–2005 on 1009 female patients, green tea consumption was found to be associated with a reduced risk of developed breast cancer. A recent meta-analysis of 13 papers on breast cancer showed that the combined results of green tea consumption from four studies showed a reduced risk of breast cancer for highest versus lowest intake groups. Daily consumption of green tea in a prospective cohort study with over 8000 individuals resulted in delayed cancer onset, and a follow- up study of breast cancer patients found that stages I and II breast cancer patients experienced a lower recurrence rate and longer disease-free period. Tea was found to be more protective against breast cancers in individuals with low catechol- O-methyltransferase activity alleles compared to those with high activity alleles in one study, but such an association was not found in another. A study in Shanghai demonstrated that daily consumption of the equivalent of two or three cups of green tea reduced the risk of esophageal cancer among nonsmokers/nondrinkers of alcoholic beverages. EGCG delivered in the form of capsule (200 mg orally for 12 weeks) has been reported to be effective in patients with human papilloma virus (HPV) infected cervical lesions. A prospective cohort study over 10 years in Japan showed that the daily consumption of green tea delayed the onset of cancer in both smokers and nonsmokers [75].

27.6 Effects of Food Processing on Chemoprotective Properties of Polyphenols

Numerous studies showed that food processing may alter the content of polyphenols and their ability to act as antioxidants and cancer chemopreventive agents. Cooking or heat processing of berries can lead to the degradation of resveratrol [76]. Storage was found to increase the content of free ellagic acid because of ellagitannin hydrolysis [77]. The studies also showed that red wines fermented on-hull had higher initial concentrations of antioxidant polyphenolics compared to a corresponding hot- pressed juice, but changes in antioxidant capacity during storage were more affected by time than by storage temperature despite lower concentrations of ellagic acid present at 37 C compared to 20 C [78]. Experimental evidence indicates that the composition of tea polyphenols undergo major changes during fermentation when converted from white tea to green tea and ﬁnally to black tea [79]. The processing to black tea involves withering, rolling, fermentation, drying, and sieving. The superoxide dismutase activity increased at the withering stage and declined during processing [80]. Thus, food processing could modulate the content of polyphenols and its chemopreventive activities.

27.7 Conclusion

In conclusion, various studies demonstrate that dietary polyphenols can suppress carcinogen-induced DNA damage and thus prevent cancer. Epidemiological studies References j477 also suggest that fruits and vegetables can prevent cancer. The results from nutritional intervention studies in humans, however, are not equivocal. Although these food- derived phytochemicals have proven to be nontoxic in vivo systems, more human trials are needed to recognize their true potential.

Acknowledgment

We would like to acknowledge Kristi M. Speights and Michael Worley for carefully editing this manuscript. Due to lack of space, we have not included all the references in this chapter.

References

1 Thomasset, S.C., Berry, D.P., Garcea, G., 7 Katiyar, S.K. and Elmets, C.A. (2001) Green Marczylo, T., Steward, W.P. and tea polyphenolic antioxidants and skin Gescher, A.J. (2007) Dietary polyphenolic photoprotection (review). International phytochemicals – promising cancer Journal of Oncology, 18, 1307–1313. chemopreventive agents in humans? 8 Lopez-Burillo, S., Tan, D.X., Mayo, J.C., A review of their clinical properties. Sainz, R.M., Manchester, L.C. and International Journal of Cancer, 120, Reiter, R.J. (2003) Melatonin, xanthurenic 451–458. acid, resveratrol, EGCG, vitamin C and 2 Soobrattee, M.A., Bahorun, T. and alpha-lipoic acid differentially reduce Aruoma, O.I. (2006) Chemopreventive oxidative DNA damage induced by Fenton actions of polyphenolic compounds in reagents: a study of their individual and cancer. Biofactors, 27,19–35. synergistic actions. Journal of Pineal 3 Lee, K.W. and Lee, H.J. (2006) The roles of Research, 34, 269–277. polyphenols in cancer chemoprevention. 9 Ungvari, Z., Orosz, Z., Rivera, A., Biofactors, 26, 105–121. Labinskyy, N., Xiangmin, Z., Olson, S., 4 Harikumar, K.B. and Aggarwal, B.B. Podlutsky, A. and Csiszar, A. (2007) (2008) Resveratrol: a multitargeted agent Resveratrol increases vascular oxidative for age-associated chronic diseases, stress resistance. American Journal of Cell cycle, 7, 1020–1035. Physiology, 292, H2417–H2424. 5 Doussot, F., De Jeso, B., Quideau, S. and 10 Mizutani, K., Ikeda, K., Nishikata, T. and Pardon, P. (2002) Extractives content in Yamori, Y.(2000) Phytoestrogens attenuate cooperage oak wood during natural oxidative DNA damage in vascular smooth seasoning and toasting; inﬂuence of muscle cells from stroke-prone tree species, geographic location, spontaneously hypertensive rats. Journal of and single-tree effects. Journal of Hypertension, 18, 1833–1840. Agricultural and Food Chemistry, 50, 11 Wen, X. and Walle, T. (2005) Preferential 5955–5961. induction of CYP1B1 by benzo[a]pyrene in 6 Lei, Z., Jervis, J. and Helm, R.F. (2001) Use human oral epithelial cells: impact on of methanolysis for the determination of DNA adduct formation and prevention total ellagic and gallic acid contents by polyphenols. Carcinogenesis, 26, of wood and food products. Journal of 1774–1781. Agricultural and Food Chemistry, 49, 12 Kumar, A., Kaundal, R.K., Iyer, S. and 1165–1168. Sharma, S.S. (2007) Effects of resveratrol 478j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

on nerve functions, oxidative stress and 20 Yu, T.W. and Anderson, D. (1997) Reactive DNA fragmentation in experimental oxygen species-induced DNA damage and diabetic neuropathy. Life Sciences, 80, its modification: a chemical investigation. 1236–1244. Mutation Research, 379, 201–210. 13 Jang, M.H., Piao, X.L., Kim, H.Y.,Cho, E.J., 21 Lewerenz, V., Hanelt, S., Nastevska, C., Baek, S.H., Kwon, S.W. and Park, J.H. El-Bahay, C., Rohrdanz, E. and Kahl, R. (2007) Resveratrol oligomers from Vitis (2003) Antioxidants protect primary rat amurensis attenuate beta-amyloid-induced hepatocyte cultures against oxidative stress in PC12 cells. Biological & acetaminophen-induced DNA strand Pharmaceutical Bulletin, 30, 1130–1134. breaks but not against acetaminophen- 14 Guo, L., Wang, L.H., Sun, B., Yang, J.Y., induced cytotoxicity. Toxicology, 191, Zhao, Y.Q., Dong, Y.X., Spranger, M.I. and 179–187. Wu, C.F. (2007) Direct in vivo evidence of 22 Ramakrishnan, G., Augustine, T.A., protective effects of grape seed Jagan, S., Vinodhkumar, R. and Devaki, T. procyanidin fractions and other (2007) Effect of silymarin on antioxidants against ethanol-induced N-nitrosodiethylamine induced oxidative DNA damage in mouse brain hepatocarcinogenesis in rats. Experimental cells. Journal of Agricultural and Food Oncology, 29,39–44. Chemistry, 55, 5881–5891. 23 Feng, Q., Torii, Y., Uchida, K., 15 Labieniec, M. and Gabryelak, T. (2006) Nakamura, Y., Hara, Y. and Osawa, T. Study of interactions between phenolic (2002) Black tea polyphenols, theaflavins, compounds and H2O2 or Cu(II) ions in prevent cellular DNA damage by inhibiting B14 Chinese hamster cells. Cell Biology oxidative stress and suppressing International, 30, 761–768. cytochrome P450 1A1 in cell cultures. 16 Smith, W.A., Freeman, J.W. and Gupta, Journal of Agricultural and Food Chemistry, R.C. (2001) Effect of chemopreventive 50, 213–220. agents on DNA adduction induced by the 24 Lin, D., Kadlubar, F.F. and Chen, J. (1998) potent mammary carcinogen dibenzo[a,l] Direct detoxification of N-acetoxy-PhIP by pyrene in the human breast cells MCF-7. tea polyphenols: a possible mechanism for Mutation Research, 480–481,97–108. chemoprevention against PhIP-DNA 17 Thresiamma, K.C., George, J. and adduct formation in vivo. Zhonghua Yu Kuttan, R. (1998) Protective effect of Fang Yi Xue Za Zhi [Chinese Journal of curcumin, ellagic acid and bixin on Preventive Medicine] 32, 265–269. radiation induced genotoxicity. Journal of 25 Lin, D.X., Thompson, P.A., Teitel, C., Experimental & Clinical Cancer Research, Chen, J.S. and Kadlubar, F.F. (2003) Direct 17, 431–434. reduction of N-acetoxy-PhIP by tea 18 Nemavarkar, P., Chourasia, B.K. and polyphenols: a possible mechanism for Pasupathy, K. (2004) Evaluation of chemoprevention against PhIP-DNA radioprotective action of compounds using adduct formation. Mutation Research, Saccharomyces cerevisiae. Journal of 523–524, 193–200. Environmental Pathology, Toxicology and 26 Lodovici, M., Casalini, C., De Filippo, C., Oncology, 23, 145–151. Copeland, E., Xu, X., Clifford, M. and 19 Anderson, D., Yu, T.W., Phillips, B.J. and Dolara, P. (2000) Inhibition of Schmezer, P. (1994) The effect of various 1,2-dimethylhydrazine-induced oxidative antioxidants and other modifying agents DNA damage in rat colon mucosa by black on oxygen-radical-generated DNA tea complex polyphenols. Food and damage in human lymphocytes in the Chemical Toxicology, 38, 1085–1088. COMET assay. Mutation Research, 307, 27 Zhao, J.F., Zhang, Y.J.,Jin, X.H., Athar, M., 261–271. Santella, R.M., Bickers, D.R. and References j479

Wang, Z.Y. (1999) Green tea protects 35 Tanaka, T., Kojima, T., Kawamori, T., against psoralen plus ultraviolet A-induced Wang, A., Suzui, M., Okamoto, K. and photochemical damage to skin. The Journal Mori, H. (1993) Inhibition of of Investigative Dermatology, 113, 4-nitroquinoline-1-oxide-induced rat 1070–1075. tongue carcinogenesis by the naturally 28 Katiyar, S.K., Perez, A. and Mukhtar, H. occurring plant phenolics caffeic, ellagic, (2000) Green tea polyphenol treatment to chlorogenic and ferulic acids. human skin prevents formation of Carcinogenesis, 14, 1321–1325. ultraviolet light B-induced pyrimidine 36 Menon, L.G., Kuttan, R. and Kuttan, G. dimers in DNA. Clinical Cancer Research, 6, (1995) Inhibition of lung metastasis in 3864–3869. mice induced by B16F10 melanoma cells 29 Huang, C.C., Wu, W.B., Fang, J.Y., by polyphenolic compounds. Cancer Chiang, H.S., Chen, S.K., Chen, B.H., Letters, 95, 221–225. Chen, Y.T. and Hung, C.F. (2007) 37 Hirose, M., Akagi, K., Hasegawa, R., ()-Epicatechin-3-gallate, a green tea Yaono, M., Satoh, T., Hara, Y., polyphenol is a potent agent against Wakabayashi, K. and Ito, N. (1995) UVB-induced damage in HaCaT Chemoprevention of 2-amino-1-methyl- keratinocytes. Molecules, 12, 1845–1858. 6-phenylimidazo[4,5-b]-pyridine (PhIP)- 30 Parshad, R., Sanford, K.K., Price, F.M., induced mammary gland carcinogenesis Steele, V.E., Tarone, R.E., Kelloff, G.J. and by antioxidants in F344 female rats. Boone, C.W. (1998) Protective action of Carcinogenesis, 16, 217–221. plant polyphenols on radiation-induced 38 Harttig, U., Hendricks, J.D., Stoner, G.D. chromatid breaks in cultured human cells. and Bailey, G.S. (1996) Organ speciﬁc, Anticancer Research, 18, 3263–3266. protocol dependent modulation of 31 Leanderson, P., Faresjo, A.O. and 7,12-dimethylbenz[a]anthracene Tagesson, C. (1997) Green tea polyphenols carcinogenesis in rainbow trout inhibit oxidant-induced DNA strand (Oncorhynchus mykiss) by dietary ellagic breakage in cultured lung cells. Free acid. Carcinogenesis, 17, 2403–2409. Radical Biology & Medicine, 23, 235–242. 39 Mukhtar, H., Das, M. and Bickers, D.R. 32 Morley, N., Clifford, T., Salter, L., (1986) Inhibition of Campbell, S., Gould, D. and Curnow, A. 3-methylcholanthrene-induced skin (2005) The green tea polyphenol tumorigenicity in BALB/c mice by chronic ()-epigallocatechin gallate and green tea oral feeding of trace amounts of ellagic acid can protect human cellular DNA from in drinking water. Cancer Research, 46, ultraviolet and visible radiation-induced 2262–2265. damage. Photodermatology 40 Vinh, P.Q., Sugie, S., Tanaka, T., Hara, A., Photoimmunology & Photomedicine, Yamada, Y., Katayama, M., Deguchi, T. and 21,15–22. Mori, H. (2002) Chemopreventive effects 33 Daniel, E.M. and Stoner, G.D. (1991) The of a ﬂavonoid antioxidant silymarin on effects of ellagic acid and 13-cis-retinoic N-butyl-N-(4-hydroxybutyl)nitrosamine- acid on N-nitrosobenzylmethylamine- induced urinary bladder carcinogenesis in induced esophageal tumorigenesis in rats. male ICR mice. Japanese Journal of Cancer Cancer Letters, 56, 117–124. Research: Gann, 93,42–49. 34 Paivarinta, E., Pajari, A.M., Torronen, R. 41 Malewicz, B., Wang, Z., Jiang, C., Guo, J., and Mutanen, M. (2006) Ellagic acid and Cleary, M.P., Grande, J.P. and Lu, J. (2006) natural sources of ellagitannins as possible Enhancement of mammary chemopreventive agents against intestinal carcinogenesis in two rodent models tumorigenesis in the Min mouse. Nutrition by silymarin dietary supplements. and Cancer, 54,79–83. Carcinogenesis, 27, 1739–1747. 480j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

42 Volate, S.R., Davenport, D.M., Muga, S.J. carcinogenesis by black tea polyphenols: and Wargovich, M.J. (2005) Modulation of modulation of xenobiotic-metabolizing aberrant crypt foci and apoptosis by dietary enzymes, oxidative stress, cell herbal supplements (quercetin, curcumin, proliferation, apoptosis, and angiogenesis. silymarin, ginseng and rutin). Molecular Carcinogenesis, 46, 797–806. Carcinogenesis, 26, 1450–1456. 50 Katiyar, S.K., Agarwal, R. and Mukhtar, H. 43 Ramakrishnan, G., Raghavendran, H.R., (1993) Protective effects of green tea Vinodhkumar, R. and Devaki, T. (2006) polyphenols administered by oral Suppression of N-nitrosodiethylamine intubation against chemical carcinogen- induced hepatocarcinogenesis by induced forestomach and pulmonary silymarin in rats. Chemico-Biological neoplasia in A/J mice. Cancer Letters, Interactions, 161, 104–114. 73, 167–172. 44 Singh, R.P., Deep, G., Chittezhath, M., 51 Mei, Y., Wei, D. and Liu, J. (2005) Kaur, M., Dwyer-Nield, L.D., Modulation effect of tea polyphenol toward Malkinson, A.M. and Agarwal, R. (2006) N-methyl-N0-nitro-N-nitrosoguanidine- Effect of silibinin on the growth and induced precancerous gastric lesion in progression of primary lung tumors in rats. The Journal of Nutritional Biochemistry, mice. Journal of the National Cancer 16, 172–177. Institute, 98, 846–855. 52 Gong, Y., Han, C. and Chen, J. (2000) Effect 45 Yanaida, Y., Kohno, H., Yoshida, K., of tea polyphenols and tea pigments on the Hirose, Y., Yamada, Y., Mori, H. and inhibition of precancerous liver lesions in Tanaka, T. (2002) Dietary silymarin rats. Nutrition and Cancer, 38,81–86. suppresses 4-nitroquinoline 1-oxide- 53 Lu, G., Liao, J., Yang, G., Reuhl, K.R., induced tongue carcinogenesis in male Hao, X. and Yang, C.S. (2006) Inhibition of F344 rats. Carcinogenesis, 23, 787–794. adenoma progression to adenocarcinoma 46 Kohno, H., Suzuki, R., Sugie, S., Tsuda, H. in a 4-(methylnitrosamino)-1-(3-pyridyl)- and Tanaka, T. (2005) Dietary 1-butanone-induced lung tumorigenesis supplementation with silymarin inhibits model in A/J mice by tea polyphenols and 3,20-dimethyl-4-aminobiphenyl-induced caffeine. Cancer Research, 66, 11494–11501. prostate carcinogenesis in male F344 rats. 54 Harper, C.E., Patel, B.B., Wang, J., Clinical Cancer Research, 11, 4962–4967. Eltoum, I.A. and Lamartiniere, C.A. (2007) 47 Lahiri-Chatterjee, M., Katiyar, S.K., Epigallocatechin-3-Gallate suppresses Mohan, R.R. and Agarwal, R. (1999) early stage, but not late stage prostate A ﬂavonoid antioxidant, silymarin, affords cancer in TRAMP mice: mechanisms of exceptionally high protection against action. Prostate, 67, 1576–1589. tumor promotion in the SENCAR mouse 55 Javed, S., Mehrotra, N.K. and Shukla, Y. skin tumorigenesis model. Cancer (1998) Chemopreventive effects of black Research, 59, 622–632. tea polyphenols in mouse skin model of 48 Lubet, R.A., Yang, C.S., Lee, M.J., Hara, Y., carcinogenesis. Biomedical and Kapetanovic, I.M., Crowell, J.A., Environmental Sciences, 11, 307–313. Steele, V.E.,Juliana, M.M. and Grubbs, C.J. 56 Tessitore, L., Davit, A., Sarotto, I. and (2007) Preventive effects of polyphenon E Caderni, G. (2000) Resveratrol depresses on urinary bladder and mammary cancers the growth of colorectal aberrant crypt foci in rats and correlations with serum and by affecting bax and p21(CIP) expression. urine levels of tea polyphenols. Molecular Carcinogenesis, 21, 1619–1622. Cancer Therapeutics, 6, 2022–2028. 57 Weisburger, J.H., Rivenson, A., Aliaga, C., 49 Kumaraguruparan, R., Seshagiri, P.B., Reinhardt, J., Kelloff, G.J., Boone, C.W., Hara, Y. and Nagini, S. (2007) Steele, V.E., Balentine, D.A., Pittman, B. Chemoprevention of rat mammary and Zang, E. (1998) Effect of tea extracts, References j481

polyphenols, and epigallocatechin gallate mitochondria. Clinical Cancer Research, on azoxymethane-induced colon cancer. 13, 5162–5169. Proceedings of the Society for Experimental 65 Agarwal, R., Katiyar, S.K., Zaidi, S.I. and Biology and Medicine, 217, 104–108. Mukhtar, H. (1992) Inhibition of skin 58 Luceri, C., Caderni, G., Sanna, A. and tumor promoter-caused induction of Dolara, P. (2002) Red wine and black tea epidermal ornithine decarboxylase in polyphenols modulate the expression of SENCAR mice by polyphenolic fraction cycloxygenase-2, inducible nitric oxide isolated from green tea and its individual synthase and glutathione-related enzymes epicatechin derivatives. Cancer Research, in azoxymethane-induced f344 rat colon 52, 3582–3588. tumors. The Journal of Nutrition, 132, 66 Wang, Z.Y., Agarwal, R., Bickers, D.R. and 1376–1379. Mukhtar, H. (1991) Protection against 59 Sengupta, A., Ghosh, S. and Das, S. (2003) ultraviolet B radiation-induced Tea can protect against aberrant crypt foci photocarcinogenesis in hairless mice by formation during azoxymethane induced green tea polyphenols. Carcinogenesis, 12, rat colon carcinogenesis. Journal of 1527–1530. Experimental & Clinical Cancer Research, 67 Berge, G., Ovrebo, S., Botnen, I.V., 22, 185–191. Hewer, A., Phillips, D.H., Haugen, A. and 60 Xiao, H., Hao, X., Simi, B., Ju, J., Jiang, H., Mollerup, S. (2004) Resveratrol inhibits Reddy, B.S. and Yang, C.S. (2008) Green benzo[a]pyrene-DNA adduct formation in tea polyphenols inhibit colorectal aberrant human bronchial epithelial cells. British crypt foci (ACF) formation and prevent Journal of Cancer, 91, 333–338. oncogenic changes in dysplastic ACF in 68 Khanduja, K.L., Gandhi, R.K., Pathania, V. azoxymethane-treated F344 rats. and Syal, N. (1999) Prevention of Carcinogenesis, 29, 113–119. N-nitrosodiethylamine-induced lung 61 Liu, H.S., Pan, C.E., Yang,W.and Liu, X.M. tumorigenesis by ellagic acid and (2003) Antitumor and quercetin in mice. Food and Chemical immunomodulatory activity of resveratrol Toxicology, 37, 313–318. on experimentally implanted tumor of 69 Yan, Y., Wang, Y., Tan, Q., Lubet, R.A. and H22 in Balb/c mice. World Journal of You, M. (2005) Efﬁcacy of deguelin and Gastroenterology, 9, 1474–1476. silibinin on benzo(a)pyrene-induced lung 62 Hirose, M., Nishikawa, A., Shibutani, M., tumorigenesis in A/J mice. Neoplasia, 7, Imai, T. and Shirai, T. (2002) 1053–1057. Chemoprevention of heterocyclic amine- 70 Yang, G., Wang, Z.Y., Kim, S., Liao, J., induced mammary carcinogenesis in rats. Seril, D.N., Chen, X., Smith, T.J. and Environmental and Molecular Mutagenesis, Yang, C.S. (1997) Characterization of early 39, 271–278. pulmonary hyperproliferation and tumor 63 Miura, D., Miura, Y. and Yagasaki, K. progression and their inhibition by (2003) Hypolipidemic action of dietary black tea in a 4-(methylnitrosamino)- resveratrol, a phytoalexin in grapes and red 1-(3-pyridyl)-1-butanone-induced lung wine, in hepatoma-bearing rats. Life tumorigenesis model with A/J mice. Sciences, 73, 1393–1400. Cancer Research, 57, 1889–1894. 64 van Ginkel, P.R., Sareen, D., 71 Yang, G.Y., Liu, Z., Seril, D.N., Liao, J., Subramanian, L., Walker, Q., Ding, W., Kim, S., Bondoc, F. and Darjatmoko, S.R., Lindstrom, M.J., Yang, C.S. (1997) Black tea constituents, Kulkarni, A., Albert, D.M. and Polans, A.S. theaﬂavins, inhibit 4-(methylnitrosamino)- (2007) Resveratrol inhibits tumor growth 1-(3-pyridyl)-1-butanone (NNK)-induced of human neuroblastoma and mediates lung tumorigenesis in A/J mice. apoptosis by directly targeting Carcinogenesis, 18,2361–2365. 482j 27 DNA Damage and Cancer Chemoprevention by Polyphenols

72 Banerjee, S., Manna, S., Saha, P., blueberries and bilberries. Journal of Panda, C.K. and Das, S. (2005) Black tea Agricultural and Food Chemistry, 51, polyphenols suppress cell proliferation 5867–5870. and induce apoptosis during benzo(a) 77 Zafrilla, P., Ferreres, F. and pyrene-induced lung carcinogenesis. Tomas-Barberan, F.A. (2001) Effect of European Journal of Cancer Prevention, processing and storage on the antioxidant 14, 215–221. ellagic acid derivatives and ﬂavonoids of 73 Ramasamy, K. and Agarwal, R. (2008) red raspberry (Rubus idaeus) jams. Journal Multitargeted therapy of cancer by of Agricultural and Food Chemistry, 49, silymarin. Cancer Letters, 269, 352–362. 3651–3655. 74 Falsaperla, M., Morgia, G., Tartarone, A., 78 Talcott, S.T. and Lee, J.H. (2002) Ellagic Ardito, R. and Romano, G. (2005) Support acid and ﬂavonoid antioxidant content of ellagic acid therapy in patients with muscadine wine and juice. Journal of hormone refractory prostate cancer Agricultural and Food Chemistry, 50, (HRPC) on standard chemotherapy using 3186–3192. vinorelbine and estramustine phosphate. 79 Katiyar, S., Elmets, C.A. and Katiyar, S.K. European Urology, 47, 449–454, discussion (2007) Green tea and skin cancer: 454–445. photoimmunology, angiogenesis and 75 Khan, N. and Mukhtar, H. (2008) DNA repair. The Journal of Nutritional Multitargeted therapy of cancer by green Biochemistry, 18, 287–296. tea polyphenols. Cancer Letters, 269, 80 Palavan-Unsal, N., Arisan, E.D. and 269–280. Terzioglu, S. (2007) Polyamines in tea 76 Lyons, M.M., Yu, C., Toma, R.B., Cho, S.Y., processing. International Journal of Food Reiboldt, W., Lee, J. and van Breemen, R.B. Sciences and Nutrition, 58, 304–311. (2003) Resveratrol in raw and baked j483

28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients Hye-Kyung Na, Joydeb Kumar Kundu, and Young-Joon Surh

28.1 Introduction

Ginger has a long history of medicinal use dating back 2500 years. The rhizome of Zingiber officinale, one of the most widely used species of the ginger family, has been used not only as a common condiment for various foods and beverages but also as a useful component of oriental herbal medicine. The flavoring ingredients of ginger reside in its volatile oil (oleoresin). The major pungent ingredients present in ginger include 6-gingerol [1-(40-hydroxy-30-methoxyphenyl)-5-hydroxy- 3-decanone] and its dehydrated product 6-shogaol [1-(4-hydroxy-3-methoxyphenyl)- 4-decen-3-one]. Other pungent ingredients include zingerone and paradol. Some pungent ingredients derived from ginger possess strong antioxidative and anti- inflammatory properties [1]. This chapter highlights the cancer chemopreventive and chemotherapeutic potential of antioxidative and anti-inflammatory substances present in the rhizome of ginger, with special focus on pungent ingredients of the species Zingiber officinale Roscoe.

28.2 Antioxidant Effects

6-Gingerol, a major pungent ingredient of ginger (Z. ofﬁcinale Roscoe, Zingiber- þ aceae) decreased peroxidation of phospholipid liposomes in the presence of Fe3 and ascorbate, but zingerone had only a weak inhibitory effect [2]. 6-Gingerol also inhibited xanthine oxidase activity [3] and protected against cisplatin-induced renal damage by restoring or potentiating the cellular antioxidant defense [4]. 6-Paradol, a minor pungent phenolic substance found in ginger and other Zingiber- aceae plants including Grains of Paradise (Aframomum melegueta Roscoe), also has the same vanilloid structure found in gingerol and shogaol (Scheme 28.1). 6-Paradol and its nonpungent synthetic analogue, 6-dehydroparadol, protected against

Scheme 28.1 Chemical structures of representative pungent ingredients found in ginger (Zingiber officinale Roscoe, Zingiberaceae).

oxidative base modiﬁcation (8-hydroxy-deoxyguanosine formation) in calf thymus

DNA exposed to H2O2 followed by UV irradiation [5]. Both 6-gingerol and 6-paradol suppressed the phorbol ester-induced superoxide production in differentiated human promyelocytic leukemia (HL-60) cells [6]. Topical application of 6-paradol and its derivatives inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced

H2O2 production and myeloperoxidase activity in the dorsal skin of mice [5]. The antioxidative effects of 6-gingerol were verified by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 20,70-dichlorodihydrofluorescein diacetate (DCFH-DA) assays, and the compound was shown to protect HL-60 cells from oxidative stress [7]. Likewise, pretreatment with 6-gingerol reduced UVB-induced intracellular ROS accumulation, which was accompanied by activation of caspase-3, -8, and -9, and Fas expression in immortalized human keratinocytes HaCaT [8]. Two novel glucosides of 6-gingerdiol, namely 1-(4-O-beta-D-glucopyranosyl- 3-methoxyphenyl)-3,5-dihydroxydecane and 5-O-beta-D-glucopyranosyl-3-hydroxy- 1-(4-hydroxy-3-methoxyphenyl)decane, were isolated from fresh ginger (Zingiber officinale Roscoe) [9]. Antioxidative activities of these gingerdiol glucosides were investigated using a linoleic acid model system and by their DPPH radical scavenging ability. Although the former compound had no substantial antioxidant activity, the latter glucoside elicited as strong activity as the aglycon, 6-gingerdiol, in both measurements [9]. 28.3 Anti-Inflammatory Effects j485

The structure–activity relationship of gingerol and related compounds isolated from rhizomes of ginger was assessed. Gingerol-related compounds substituted with an alkyl group bearing the 10-, 12-, or 14-carbon chain length were isolated and their antioxidant activities were evaluated by three different methods. The results suggested that the substituents on the alkyl chain might contribute to both radical scavenging effect and the inhibitory effect on autoxidation of oils, while the inhibitory effect against the peroxidation of liposome was somewhat influenced by the alkyl chain length [10]. Reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO–), can also cause DNA damage and influence signal transduction, thereby contributing to carcinogenic processes. 6-Gingerol was shown to be a good scavenger of peroxyl radicals generated by pulse radiolysis [2]. The compound significantly inhibited the expression of inducible NO synthase (iNOS) and production of NO in lipopolysaccharide (LPS)-stimulated J774.1 murine macrophages [11]. Moreover, 6-gingerol effectively suppressed peroxynitrite-induced oxidation of DCF-DA, oxidative single-strand DNA breaks, and protein nitrosylation [2]. Therefore, 6-gingerol is likely to be a potent inhibitor of NO synthesis and also an effective protector against RNS-mediated damage. Reaction of 6-gingerol with peroxynitrite gave rise to the formation of a symmetrical dimer of 6-gingerol covalently linking each other at the aromatic ring. This dimer appeared to be generated from a phenoxyl radical intermediate produced as a consequence of one-electron oxidation of 6-gingerd by peroxynitrite-derived radicals [12]. The redox-sensitive transcription factor nuclear factor E2-related factor 2 (Nrf2) plays a pivotal role in regulating the expression of a wide array of antioxidant/phase-2 detoxifying enzymes. Under basal conditions, the interaction of the cytosolic protein Keap1 (Kelch-like ECH-associated protein 1) with Nrf2 results in a low level of expression of cytoprotective genes whose promoter regions contain the antioxidant response element (ARE) [13]. 10-Shogaol has been shown to alkylate the cysteine 151 residue of recombinant human Keap1, which is proposed to stimulate nuclear accumulation of Nrf2 and subsequent upregulation of ARE-mediated antioxidant/ detoxifying enzyme expression [14].

28.3 Anti-Inflammatory Effects

Ginger is described in Ayurvedic and Tibb systems of medicine to be useful in treating inflammation and rheumatism. Thus, ginger has a broad spectrum of anti- inflammatory actions and has been used as an ethnobotanical or alternative medicine for the management of inflammatory conditions including rheumatic disorders. One of the distinct features of inflammation is elevated oxygenation of arachidonic acid, which is mainly catalyzed by cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), resulting in the production of prostaglandins and leukotrienes, respectively. Prosta- glandin E2 (PGE2) and leukotriene 4 as representative products of COX and 5-LOX, respectively, are considered to be important mediators of inflammation. Treatment of 486j 28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients

patients suffering from rheumatoid arthritis, osteoarthritis, and musculoskeletal disorders with powdered ginger revealed that more than three-quarters of arthritis patients experienced, to varying degrees, relief in pain and swelling [15]. None of the patients reported adverse effects of ginger consumption for the period of 3 months to 2.5 years [15]. At least one of the mechanisms by which ginger shows its ameliorative effects is attributable to the inhibition of prostaglandin and leukotriene biosynthesis through suppression of COX and 5-LOX, respectively [15, 16]. This pharmacological property distinguishes ginger from nonsteroidal anti-inﬂammatory drugs (NSAIDs) because the dual inhibitors of COX and LOX may have a better therapeutic proﬁle but lesser side effects than NSAIDs [16]. A series of structurally related natural pungent products including capsaicin, gingerol, and gingerdione among others were found to be potent inhibitors of 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) biosynthesis in intact human leukocytes [17]. Several compounds within this series were also found to

inhibit PGE2 formation, with gingerdione being the most potent. Members of the vanilloid family of pungent compounds can suppress arachidonic acid metabolism by acting as dual inhibitors of COX and 5-LOX, which could account, in part, for their anti-inﬂammatory and analgesic properties [17]. A later study conﬁrmed the presence of gingerols as potent inhibitors of COX and 5-LOX in the rhizomes of ginger [18]. Ginger extract as well as the COX-2 selective inhibitor celecoxib attenuated the LPS-

induced rat trachea hyper-reactivity and reduced the serum levels of PGE2 and thromboxane. The myeloperoxidase activity and the number of inﬂammatory cells in LPS-stimulated rat bronchoalveolar lavage were also reduced by the same treatment. Incubation with hydroalcoholic extract of ginger or celecoxib reduced the release of

PGE2 and thromboxane from LPS-simulated rat lung parenchyma. These results suggest that ginger attenuates rat lung inﬂammation, at least partly, by suppressing COX [19]. Seventeen pungent oleoresin principles of ginger and synthetic analogues were evaluated for inhibition of COX-2 enzyme activity in an intact cell assay model using cultured human airways epithelial A549 cells stimulated with interleukin (IL)-1b. 8-Paradol and 8-shogaol, as well as two synthetic analogues, 3-hydroxy-1-(4-hydroxy- 3-methoxyphenyl)decane and 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane, showed strong inhibitory effects on IL-1b-induced COX-2 enzyme activity [20]. The structure–activity relationship analysis of these phenolic compounds revealed that three important structural features, that is, (i) lipophilicity of the alkyl side chain, (ii) substitution pattern of hydroxy and carbonyl groups on the side chain, and (iii) substitution pattern of hydroxy and methoxy groups on the aromatic moiety, are linked to their inhibitory effects on COX-2 enzyme activity. Partially puriﬁed fractions from the dichloromethane extracts of organically grown samples of fresh [21] or processed dry [22] ginger were analyzed by gas chromatography-mass spectrometry. Most of the fractions containing gingerols

and/or gingerol derivatives showed marked inhibition of LPS-induced PGE2 production in human leukemia cells [21, 22]. Differentiated U937 cells were treated with LPS for 24 h in the presence or absence of organic extracts of ginger or standardized fractions containing 6-, 8-, 10-gingerol or 6-shogaol [23]. Although extracts containing 28.3 Anti-Inflammatory Effects j487 gingerols were capable of inhibiting LPS-induced COX-2 expression, shogaol-rich fractions had no effect on COX-2 expression [23]. Aktan et al. examined the inhibitory effects of a stable 6-gingerol metabolite, RAC-6-dihydroparadol (6-DHP), and a closely related gingerol analogue, RAC-2- hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one [a capsaicin/gingerol (Cap- sarol) analogue referred to as ZTX42], on the expression and activity of iNOS and NO production in LPS-stimulated RAW 264.7 murine macrophages. Both ZTX42 and 6-DHP significantly inhibited LPS-induced NO production [24]. Although both compounds partially inhibited the catalytic activity of iNOS, their inhibitory effect was predominantly due to attenuation of iNOS protein production. This occurred at the transcriptional level, since the above compounds abrogated LPS-induced IkBa degradation and the subsequent nuclear translocation of the NF-kB, a key transcription factor involved in regulating expression of many proinflammatory genes, including COX-2 and iNOS. Ginger extract inhibited the production of proinflammatory cytokines, such as IL-12, tumor necrosis factor-alpha (TNF-a) and IL-1b, and proinflammatory chemokines (RANTES and MCP-1) in LPS-stimulated murine peritoneal macrophages [25]. As one of the active ingredients responsible for anti-inflammatory properties of ginger, 6-gingerol inhibited the production of proinflammatory cytokines from LPS-stimulated macrophages [26]. Mitogen-activated protein kinase phosphatase-5 (MKP5) has been shown to play a role in mediating anti-inflammatory activities of some chemopreventive phytochemicals [27]. MKP5 overexpression decreased cytokine-induced NF-kB activation, COX-2 expression, and cytokine production in normal human primary prostatic epithelial cells, corroborating potent anti-inflammatory activity of MKP5 [27]. 6-Gingerol upregulated MKP5 in normal prostate epithelial cells. Moreover, prostate cancer cell lines (DU 145, PC-3, LNCaP, and LAPC-4) retained the ability to upregulate MKP5 following 6-gingerol exposure, suggesting the potential of this phytochemical in prostate cancer treatment. The skin permeation and the topical anti-inflammatory properties of ginger extracts were investigated. A commercial ginger dry extract and a dry extract enriched with gingerols exerted anti-inflammatory activity by inhibiting croton oil-induced ear edema in mice [28]. Topical application of 6-gingerol significantly suppressed the TPA-induced inflammation as demonstrated by amelioration of mouse ear edema [29]. Likewise, topically applied 6-paradol and its derivatives inhibited TPA-induced ear edema in mice [5]. Vascular permeability has been used as an indirect measurement of inflammation induced by irritants or tumor promoters. In parallel with protection against TPA-induced inflammation, 6-gingerol reduced the vascular permeability of the epidermis of mice treated with TPA. Intraperitoneal administration of 6-gingerol (50–100 mg/kg body weight) also produced an inhibition of paw edema induced by carrageenan [30]. The chemopreventive property of 6-gingerol has been attributed to its strong anti- inflammatory activity. Thus, topical application of 6-gingerol inhibited the tumor promoter-induced expression of COX-2 in mouse skin, and this was achieved through inactivation of NF-kB [31]. Topically applied 6-gingerol also abrogated 488j 28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients

UVB-induced expression of COX-2 and its mRNA transcript as well as nuclear translocation of NF-kB [8]. The same study also revealed the inhibitory effects of 6-gingerol on UVB-induced ROS accumulation, IkBa degradation, NF-kB activation, and COX-2 expression in an immortalized human keratinocyte cell line (HaCaT) [8].

28.4 Anticarcinogenic Effects

The anticancer properties of ginger are attributed to the presence of certain pungent vanilloids, namely, 6-gingerol, 6-shogaol, 6-paradol, zingerone, and so on. A number of mechanisms that may be involved in the chemopreventive and chemotherapeutic effects of ginger and its components have been reported [1, 32] and updated below (also schematically represented in Figure 28.1).

28.4.1 Inhibition of Mouse Skin Carcinogenesis

Topical application of the ethanol extract of ginger (Zingiber ofﬁcinale Roscoe, Zingiberaceae) strongly inhibited skin carcinogenesis in SENCAR mice initiated

Figure 28.1 Chemopreventive and chemotherapeutic potential of ginger. 28.4 Anticarcinogenic Effects j489 by 7,12-dimethylbenz[a]anthracene and promoted with TPA [33]. 6-Gingerol inhibited TPA-promoted skin tumorigenesis and ornithine decarboxylase (ODC) activity, a biochemical hallmark of tumor promotion, in female ICR mice [29]. Like 6-gingerol, 6-paradol inhibited tumor promotion, TNF-a production, and the ODC activity induced by TPA in mouse skin [6]. 6-Paradol and its nonpungent analogue, 6-dehydroparadol, also protected against TPA-induced mouse skin tumor promotion [5]. Both compounds and some of their analogues, when topically applied, reduced H2O2 production, myeloperoxidase activity, and the ODC activity in mouse skin treated with TPA [5].

28.4.2 Inhibition of Gastric Lesions and Tumorigenesis

Ginger root has been used traditionally for the treatment of gastrointestinal ailments and is also reported to have gastroprotective activity in animal models. The orally administered acetone extract of ginger (1000 mg/kg) and the same amounts (100 mg/ kg) of 6-gingerol or zingiberene, a main terpenoid from ginger extract, significantly inhibited HCl/ethanol-induced gastric lesions [34]. A new antiulcer principle named 6-gingesulfonic acid was isolated from ginger, which was more potent than 6-gingerol and 6-shogaol [35, 36]. 6-Gingerol shares the structural similarity with capsaicin, a principal pungent ingredient of hot chili pepper, and hence acts as a vanilloid-receptor agonist. In an attempt to elucidate the role of vanilloid receptor type 1 (VR1) in gastric mucosal defense mechanisms, Horie et al. determined the effects of capsaicin and 6-gingerol [37]. Intragastric administration of capsaicin, a prototypic VR1 agonist, resulted in substantial protection against HCl-induced gastric lesions, which was attenuated by the VR1 antagonists, such as ruthenium red and capsazepine. Likewise, 6-gingerol significantly inhibited the formation of HCl-induced gastric lesions, and its gastroprotective effect was blocked by treatment with ruthenium red. The results of this study suggest that 6-gingerol and capsaicin exert protective effects against acid- induced gastric lesions through interaction with VR1 [37]. Gingerol given in the diet at a concentration of 0.02% significantly decreased the number of intestinal tumors formed in the azoxymethane-treated male F344 rats [38]. Oral administration of ginger to male Wistar rats protected against the 1,2-dimethylhydrazine-induced colon carcinogenesis, which appears to be associated with potentiation of cellular antioxidant defense capacity and attenuation of oxidative stress [39]. Helicobacter pylori is one of the etiological factors for dyspepsia, peptic ulcer disease, and the development of gastric and colon cancer. A fractionated methanol extract of the dried powdered ginger rhizome containing gingerols inhi- þ bited the growth of 19 strains of H. pylori, including 5 CagA strains [40], suggesting the chemopreventive potential of ginger, especially gingerols, in H. pylori-induced gastric and colon carcinogenesis. Combination of extracts from ginger and the antibiotic clarithromycin had synergistic or additive effects on the suppression of growth of H. pylori [41]. 490j 28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients

28.4.3 Inhibition of Bladder Carcinogenesis

Male Wistar rats fed 1% ginger in a basal diet developed signiﬁcantly lower number of urothelial lesions (hyperplasia and neoplasia) induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water [42].

28.4.4 Inhibition of Tumor Cell Growth and Proliferation

6-Gingerol induced apoptosis in human promyelocytic leukemia (HL-60) cells [43]. The ability of 6-gingerol to induce apoptosis in HL-60 cells was prevented by catalase, suggesting that 6-gingerol-induced cell death could be mediated by ROS [7]. Shogaol and some structurally related compounds isolated from rhizomes of Chinese ginger also induced apoptosis of HL-60 cells [44]. Activator protein 1 (AP-1) has a critical role in tumor promotion. Epidermal growth factor (EGF) induces cell transformation and AP-1 activity. 6-Gingerol and 6-paradol caused inhibition of EGF-induced transformation of cultured mouse epidermal JB6 cells, but their underlying mechanisms are apparently different [45]. While apoptosis appeared to account for the blockade of cell transformation by 6- paradol, 6-gingerol is likely to exert its antitumorigenic effects through inhibition of AP-1 activation in EGF-stimulated JB-6 cells. Transforming growth factor-beta1 (TGF-b1) promotes tumor progression in the late stages of carcinogenesis, through induction of epithelial-mesenchymal transition. Inhibition of the AP-1 transcriptional complex by 6-gingerol blocked TGF-b1-induced epithelial–mesenchymal transition in immortalized human skin keratinocyte cell line transfected with mutant c-Ha-ras [46]. Treatment of two human pancreatic cancer cell lines, one expressing wild-type (wt) p53 and the other expressing mutated p53, with 6-gingerol resulted in the inhibition of cell growth through the cell cycle arrest at G1 phase in both cell lines [47]. In this study, 6-gingerol decreased both cyclin A and cyclin-dependent kinase (Cdk) expression. These events led to reduced phosphorylation of retinoblastoma protein (Rb) and the subsequent blockade of S phase entry. 6-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild-type p53-expressing cells, and this was related to the increased phosphorylation of Akt/protein kinase B. These results suggest that 6-gingerol can circumvent the resistance of mutant p53-expressing cells toward chemotherapy by inducing apoptotic cell death, while it exerts cytostatic effects on wild-type p53-expressing cells by inducing growth arrest [47]. The modulatory effects of 6-gingerol on testosterone-induced alterations on apoptosis related proteins were examined in both androgen-sensitive human prostate cancer (LNCaP) cells and in ventral prostate of Swiss albino mice in vivo. 6-Gingerol treatment induced apoptosis in LNCaP cells [48]. The compound also restored the levels of p53 in mouse prostate and elevated the expression of Bax 28.4 Anticarcinogenic Effects j491 and activated caspase-9 and caspase-3 which were reduced by testosterone in both LNCaP cells and in murine prostate. In addition, 6-gingerol attenuated testosterone-induced expression of Bcl-2 and survivin in both LNCaP cells and mouse prostate in vivo [48]. Cyclin D1 is a proto-oncogene that is overexpressed in many cancers and plays a role in cell proliferation through activation by b-catenin signaling. NSAID-activated gene-1 (NAG-1) is a cytokine associated with proapoptotic and antitumorigenic properties. 6-Gingerol treatment suppressed cell proliferation and induced apoptosis and G1 cell cycle arrest in human colorectal cancer cells [49]. According to this study, the induction of apoptosis by 6-gingerol was associated with a decrease in cyclin D1 resulting from reduced nuclear translocation of b-catenin and proteoly- sis of cyclin D1 and an increase in NAG1 expression. 6-Gingerol facilitated TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis by increasing caspase-3/7 activation, while the compound alone affected viability only slightly in gastric cancer cells [50]. Cotreatment with TRAIL and 6-gingerol signiﬁcantly reduced the growth of human gastric cancer (HGC) cells xenografted in athymic nude mice. 6-Gingerol was shown to downregulate the expression of cIAP1, which suppresses caspase-3/7 activity, by inhibiting TRAIL-induced NF-kB activation. Unlike 6-gingerol, 6-shogaol alone reduced the viability of gastric cancer cells by damaging microtubules and induced mitotic arrest. Moreover, intraperitoneal administration of 6-shogaol alone attenuated the growth of HGC cells tumor xenograft in nude mice [50]. In another study, 6-shogaol exhibited the most potent cytotoxicity against human nonsmall cell lung adenocarcinoma (A549), ovarian cancer (SK-OV-3), melanoma (SK-MEL-2), and colon cancer (HCT15) cells [51]. 6-Shogaol also inhibited proliferation of two transgenic mouse ovarian cancer cell lines, C1 (genotype: p53 / ,c-myc, þ þ K-ras) and C2 (genotype: p53 / ,c-myc, Akt) [51]. 6-Shogaol effectively induced apoptosis of Mahlavu cells, a poorly differentiated and p53-mutated human hepatoma cell line with high expression of multidrug resistance gene-1 (MDR-1) and Bcl-2, via a caspase-dependent mechanism [52]. The 6-shogaol-induced apoptosis of these cells involved an initial overproduction of ROS followed by a severe depletion of intracellular glutathione (GSH) contents. N-Acetylcysteine (NAC), an antioxidant and a precursor of GSH biosynthesis, almost completely protected against apoptotic cell death induced by 6-shogaol. Similarly, exogenously added GSH could also provide protection with an equal efﬁcacy [52]. 6-Shogaol, not 6-gingerol, inhibited the growth of human colorectal carcinoma COLO 205 cells and induced apoptosis through modulation of mitochondrial functions regulated by ROS [53]. ROS generation occurred in the early stages of 6-shogaol-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. Upregulation of Bax, Fas, and FasL and downregulation of Bcl-2 and Bcl-XL were observed in 6-shogaol-treated COLO 205 cells. NAC, not other antioxidants, suppressed 6-shogaol-induced apoptosis. The expression of growth arrest and DNA damag-inducible protein 153 (GADD153) and its 492j 28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients

mRNA transcript and protein was markedly induced in response to 6-shogaol [53]. 6-Paradol, like 6-gingerol, induced apoptosis when treated to HL-60 cells [43]. 6-Paradol and other structurally related derivatives, 10-paradol, 3-dehydroparadol, 6-dehydroparadol, and 10-dehydroparadol, with the exception of 3-paradol induced apoptosis through a caspase-3-dependent mechanism in an oral squamous carcinoma KB cell line [54].

28.4.5 Antimetastasis and Antiangiogenesis

6-Gingerol inhibited both the vascular endothelial growth factor (VEGF)- and basic ﬁbroblast growth factor (bFGF)-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF [55]. Moreover, intraperitoneal administration of 6-gingerol to mice receiving i.v. injection of B16F10 melanoma cells reduced the number of lung metastasis [55]. NF-kB can be constitutively activated in ovarian cancer cells and may contribute to increased transcription and translation of angiogenic factors. Treatment of cultured ovarian cancer cells with ginger extracts induced profound growth inhibition in A2780, SKOV3, and ES-2 cells [56]. 6-Shogaol was found to be the most active of the individual ginger components tested. Ginger extracts treatment resulted in the inhibition of NF-kB activation as well as diminished secretion of VEGF and IL-8 in ES-2 cells. The effects of 6-gingerol on adhesion, invasion, motility, activity, and the production of matrix metalloproteinase (MMP)-2 or -9 were investigated in human breast cancer (MDA-MB-231) cells. Treatment of MDA-MB-231 cells with increasing concentrations of 6-gingerol led to a concentration-dependent decrease in cell migration and motility, which was accompanied by reduced activities of MMP-2 or MMP-9 [57]. Expression of MMP-2 and MMP-9 mRNA was also decreased by 6-gingerol treatment [57].

28.4.6 Effects on Multidrug Resistance

P-glycoprotein (P-gp), a product of the MDR1 gene, is the one of the efﬂux ATP binding cassette family transporters. P-gp is overexpressed frequently in tumor cells resistant to multiple anticancer agents. 6-Gingerol inhibited the efﬂux of P-gp substrates and reversed the multidrug resistance in KB-C2 cells that express P-gp [58]. It is likely that 6-gingerol has an inhibitory effect on P-gp and may increase the bioavailability of the anticancer drugs. Likewise, 6-gingerol inhibited P-gp-mediated [3H]digoxin transport in porcine kidney epithelial cells transfected with MDR1 gene [59]. 28.6 Conclusion j493

28.5 Chemoprotective Effects

Several lines of evidences suggest that oxidative stress is implicated in the nephrotoxicity of a widely used synthetic anticancer drug cisplatin. The ethanol extract of Z. ofﬁcinale alone and in combination with vitamin E partially ameliorated cisplatin- induced renal injury [4]. This protection was mediated either by preventing the cisplatin-induced decline of the renal antioxidant defense capacity or by their direct free radical scavenging activity.

28.6 Conclusion

The rhizome of ginger (Zingiber officinale Roscoe, Zingiberaceae) has long been used as a common spice as well as an important component of oriental herbal medicine. Some pungent ingredients of ginger, such as 6-gingerol, 6-shogaol, and 6-paradol, have potent anti-inflammatory and antioxidative properties, which may contribute to its chemopreventive and chemotherapeutic potential. Recent studies have revealed the critical molecules involved in cellular redox and inflammatory signaling.

Figure 28.2 Intracellular signaling pathways as targets for anticarcinogenic actions of ginger ingredients. 494j 28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients

These include the transcription factors, Nrf2, NF-kB, and AP-1 as well as their regulators. Activation of Nrf2 can induce transcription of a battery of antioxidant and other cytoprotective genes, while NF-kB and AP-1 are responsible for upregulation of a wide array of proinflammatory genes that encode COX-2, iNOS, and cytokines. Ginger and its pharmacologically active ingredients, by targeting some of these signaling molecules, can exert antioxidant and anti-inflammatory as well as anticarcinogenic effects (Figure 28.2). Well-designed intervention trials should be needed to verify chemopreventive or chemotherapeutic activity of ginger and its defined ingredients in humans.

Acknowledgments

This work was supported by research grants from the BioGreen 21 Program (no. 20070301-034-042) awarded to Y.-J. Surh and MOEHRD, Basic Research Promotion Fund (KRF-2006-E00002) from Korea Research Foundation, Republic of Korea (to H.-K. Na).

References

1 Surh, Y.-J., Lee, E. and Lee, J.M. (1998) Anti-tumor-promoting activities Chemoprotective properties of some of selected pungent phenolic substances pungent ingredients present in red pepper present in ginger. Journal of and ginger. Mutation Research, 402, Environmental Pathology, Toxicology 259–267. and Oncology, 18, 131–139. 2 Aeschbach, R., Loliger, J., Scott, B.C., 7 Wang, C.C., Chen, L.G., Lee, L.T. and Murcia, A., Butler, J., Halliwell, B. and Yang, L.L. (2003) Effects of 6-gingerol, Aruoma, O.I. (1994) Antioxidant actions of an antioxidant from ginger, on inducing thymol, carvacrol, 6-gingerol, zingerone apoptosis in human leukemic HL-60 cells. and hydroxytyrosol. Food and Chemical In Vivo, 17, 641–645. Toxicology, 32,31–36. 8 Kim, J.K., Kim, Y., Na, K.M., Surh, Y.J. and 3 Chang, W.S., Chang, Y.H., Lu, F.J. and Kim, T.Y. (2007) [6]-Gingerol prevents Chiang, H.C. (1994) Inhibitory effects of UVB-induced ROS production and COX-2 phenolics on xanthine oxidase. Anticancer expression in vitro and in vivo. Free Radical Research, 14, 501–506. Research, 41, 603–614. 4 Kuhad, A., Tirkey, N., Pilkhwal, S. and 9 Sekiwa, Y., Kubota, K. and Kobayashi, A. Chopra, K. (2006) 6-Gingerol prevents (2000) Isolation of novel glucosides related cisplatin-induced acute renal failure in to gingerdiol from ginger and their rats. Biofactors, 26, 189–200. antioxidative activities. Journal of 5 Chung, W.Y., Jung, Y.J.,Surh, Y.J.,Lee, S.S. Agricultural and Food Chemistry, and Park, K.K. (2001) Antioxidative and 48, 373–377. antitumor promoting effects of [6]-paradol 10 Masuda, Y., Kikuzaki, H., Hisamoto, M. and its homologs. Mutation Research, and Nakatani, N. (2004) Antioxidant 496, 199–206. properties of gingerol related 6 Surh, Y.-J., Park, K.K., Chun, K.S., Lee, compounds from ginger. Biofactors, L.J., Lee, E. and Lee, S.S. (1999) 21, 293–296. References j495

11 Ippoushi, K., Azuma, K., Ito, H., Horie, H. and lung inflammation. Prostaglandins, and Higashio, H. (2003) [6]-Gingerol Leukotrienes, and Essential Fatty Acids, inhibits nitric oxide synthesis in activated 77, 129–138. J774.1 mouse macrophages and prevents 20 Tjendraputra, E., Tran, V.H., peroxynitrite-induced oxidation and Liu-Brennan, D., Roufogalis, B.D. and nitration reactions. Life Sciences, 73, Duke, C.C. (2001) Effect of ginger 3427–3437. constituents and synthetic analogues 12 Ippoushi, K., Ito, H., Horie, H. and on cyclooxygenase-2 enzyme in Azuma, K. (2005) Mechanism of inhibition intact cells. Bioorganic Chemistry, of peroxynitrite-induced oxidation and 29, 156–163. nitration by [6]-gingerol. Planta Medica, 21 Jolad, S.D., Lantz, R.C., Solyom, A.M., 71, 563–566. Chen, G.J., Bates, R.B. and 13 Surh, Y.J. (2003) Cancer chemoprevention Timmermann, B.N. (2004) Fresh with dietary phytochemicals. Nature organically grown ginger (Zingiber Reviews Cancer, 3, 768–780. officinale): composition and effects on 14 Luo, Y., Eggler, A.L., Liu, D., Liu, G., LPS-induced PGE2 production. Mesecar, A.D. and van Breemen, R.B. Phytochemistry, 65, 1937–1954. (2007) Sites of alkylation of human Keap1 22 Jolad, S.D., Lantz, R.C., Chen, G.J., by natural chemoprevention agents. Bates, R.B. and Timmermann, B.N. Journal of the American Society for Mass (2005) Commercially processed dry Spectrometry, 18, 2226–2232. ginger (Zingiber officinale): composition 15 Srivastava, K.C. and Mustafa, T. (1992) and effects on LPS-stimulated PGE2 Ginger (Zingiber officinale) in rheumatism production. Phytochemistry, 66, and musculoskeletal disorders. Medical 1614–1635. Hypotheses, 39, 342–348. 23 Lantz, R.C., Chen, G.J., Sarihan, M., 16 Grzanna, R., Lindmark, L. and Solyom, A.M., Jolad, S.D. and Frondoza, C.G., (2005) Ginger – an herbal Timmermann, B.N. (2007) The effect medicinal product with broad anti- of extracts from ginger rhizome on inflammatory actions. Journal of Medicinal inflammatory mediator production. Food, 8, 125–132. Phytomedicine: International Journal of 17 Flynn, D.L., Rafferty, M.F. and Phytotherapy and, Phytopharmacology, Boctor, A.M., (1986) Inhibition of human 14, 123–128. neutrophil 5-lipoxygenase activity by 24 Aktan, F., Henness, S., Tran, V.H., gingerdione, shogaol, capsaicin and Duke, C.C., Roufogalis, B.D. and related pungent compounds. Ammit, A.J. (2006) Gingerol metabolite Prostaglandins, Leukotrienes, and Medicine, and a synthetic analogue Capsarol inhibit 24, 195–198. macrophage NF-kappaB-mediated iNOS 18 Kiuchi, F., Iwakami, S., Shibuya, M., gene expression and enzyme activity. Hanaoka, F. and Sankawa, U. (1992) Planta Medica, 72, 727–734. Inhibition of prostaglandin and 25 Tripathi, S., Bruch, D. and Kittur, D.S., leukotriene biosynthesis by gingerols (2008) Ginger extract inhibits LPS induced and diarylheptanoids. Chemical & macrophage activation and function. BMC Pharmaceutical Bulletin, 40, 387–391. Complementary and Alternative Medicine, 19 Aimbire, F., Penna, S.C., 8,1. Rodrigues M., Rodrigues, K.C., Lopes- 26 Tripathi, S., Maier, K.G., Bruch, D. and Martins, R.A. and Sertie, J.A. (2007) Kittur, D.S. (2007) Effect of 6-gingerol Effect of hydroalcoholic extract of on pro-inflammatory cytokine production Zingiber officinalis rhizomes on and costimulatory molecule expression LPS-induced rat airway hyperreactivity in murine peritoneal macrophages. 496j 28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients

The Journal of Surgical Research, 35 Yamahara, J., Hatakeyama, S., Taniguchi, 138, 209–213. K., Kawamura, M. and Yoshikawa, M. 27 Nonn, L., Duong, D. and Peehl, D.M. (1992) Stomachic principles in ginger. II. (2007) Chemopreventive anti- Pungent and anti-ulcer effects of low polar inflammatory activities of curcumin and constituents isolated from ginger, the other phytochemicals mediated by MAP dried rhizoma of Zingiber officinale Roscoe kinase phosphatase-5 in prostate cells. cultivated in Taiwan. The absolute Carcinogenesis, 28, 1188–1196. stereostructure of a new diarylheptanoid. 28 Minghetti, P., Sosa, S., Cilurzo, F., Yakugaku Zasshi, 112, 645–655. Casiraghi, A., Alberti, E., Tubaro, A., 36 Yoshikawa, M., Yamaguchi, S., Kunimi, K., Loggia, R.D. and Montanari, L. (2007) Matsuda, H., Okuno, Y., Yamahara, J. and Evaluation of the topical anti-inflammatory Murakami, N. (1994) Stomachic principles activity of ginger dry extracts from in ginger. III. An anti-ulcer principle, solutions and plasters. Planta Medica, 6-gingesulfonic acid, and three 73, 1525–1530. monoacyldigalactosylglycerols, 29 Park, K.K., Chun, K.S., Lee, J.M., Lee, S.S. gingerglycolipids A, B, and C, from and Surh, Y.J. (1998) Inhibitory effects of Zingiberis Rhizoma originating in Taiwan. [6]-gingerol, a major pungent principle of Chemical & Pharmaceutical Bulletin, 42, ginger, on phorbol ester-induced 1226–1230. inflammation, epidermal ornithine 37 Horie, S., Yamamoto, H., Michael, G.J., decarboxylase activity and skin tumor Uchida, M., Belai, A., Watanabe, K., promotion in ICR mice. Cancer Letters, Priestley, J.V. and Murayama, T. (2004) 129, 139–144. Protective role of vanilloid receptor type 1 30 Young, H.Y.,Luo, Y.L.,Cheng, H.Y.,Hsieh, in HCl-induced gastric mucosal lesions in W.C., Liao, J.C. and Peng, W.H. (2005) rats. Scandinavian Journal of Analgesic and anti-inflammatory activities Gastroenterology, 39, 303–312. of [6]-gingerol. Journal of 38 Yoshimi, N., Wang, A., Morishita, Y., Ethnopharmacology, 96, 207–210. Tanaka, T., Sugie, S., Kawai, K., Yamahara, 31 Kim, S.O., Kundu, J.K., Shin, Y.K., Park, J. and Mori, H. (1992) Modifying effects of J.H., Cho, M.H., Kim, T.Y. and Surh, Y.J. fungal and herb metabolites on (2005) [6]-Gingerol inhibits COX-2 azoxymethane-induced intestinal expression by blocking the activation of carcinogenesis in rats. Japanese Journal of p38 MAP kinase and NF-kappaB in Cancer Research, 83, 1273–1278. phorbol ester-stimulated mouse skin. 39 Manju, V. and Nalini, N. (2005) Oncogene, 24, 2558–2567. Chemopreventive efficacy of ginger, a 32 Shukla, Y. and Singh, M. (2007) Cancer naturally occurring anticarcinogen during preventive properties of ginger: a brief the initiation, post-initiation stages of 1,2 review. Food and Chemical Toxicology, dimethylhydrazine-induced colon cancer. 45, 683–690. Clinica Chimica Acta, 358,60–67. 33 Katiyar, S.K., Agarwal, R. and Mukhtar, H. 40 Mahady, G.B., Pendland, S.L., Yun, G.S., (1996) Inhibition of tumor promotion in Lu, Z.Z. and Stoia, A. (2003) Ginger SENCAR mouse skin by ethanol extract of (Zingiber officinale Roscoe) and the Zingiber officinale rhizome. Cancer gingerols inhibit the growth of Cag A þ Research, 56, 1023–1030. strains of Helicobacter pylori. Anticancer 34 Yamahara, J., Mochizuki, M., Rong, H.Q., Research, 23, 3699–3702. Matsuda, H. and Fujimura, H. (1988) The 41 Nostro, A., Cellini, L., Di Bartolomeo, S., anti-ulcer effect in rats of ginger Cannatelli, M.A., Di Campli, E., Procopio, constituents. Journal of Ethnopharmacology, F., Grande, R., Marzio, L. and Alonzo, V. 23, 299–304. (2006) Effects of combining extracts References j497

(from propolis or Zingiber officinale) with 50 Ishiguro, K., Ando, T., Maeda, O., Ohmiya, clarithromycin on Helicobacter pylori. N., Niwa, Y., Kadomatsu, K. and Goto, H. Phytotherapy Research, 20, 187–190. (2007) Ginger ingredients reduce viability 42 Ihlaseh, S.M., de Oliveira, M.L., Teran, E., of gastric cancer cells via distinct de Camargo, J.L. and Barbisan, L.F. (2006) mechanisms. Biochemical and Biophysical Chemopreventive property of dietary Research Communications, 362, 218–223. ginger in rat urinary bladder chemical 51 Kim, J.S., Lee, S.I., Park, H.W., Yang, J.H., carcinogenesis. World Journal of Urology, Shin, T.Y., Kim, Y.C.,Baek, N.I., Kim, S.H., 24, 591–596. Choi, S.U., Kwon, B.M., Leem, K.H., Jung, 43 Lee, E. and Surh, Y.-J. (1998) Induction of M.Y. and Kim, D.K. (2008) Cytotoxic apoptosis in HL-60 cells by pungent components from the dried rhizomes of vanilloids, [6]-gingerol and [6]-paradol. Zingiber officinale Roscoe. Archives of Cancer Letters, 134, 163–168. Pharmacal Research, 31, 415–418. 44 Wei, Q.Y., Ma, J.P., Cai, Y.J., Yang, L. and 52 Chen, C.Y., Liu, T.Z., Liu, Y.W., Tseng, Liu, Z.L. (2005) Cytotoxic and apoptotic W.C., Liu, R.H., Lu, F.J., Lin, Y.S., Kuo, activities of diarylheptanoids and gingerol- S.H. and Chen, C.H. (2007) 6-shogaol related compounds from the rhizome of (alkanone from ginger) induces apoptotic Chinese ginger. Journal of cell death of human hepatoma p53 mutant Ethnopharmacology, 102, 177–184. Mahlavu subline via an oxidative stress- 45 Bode, A.M., Ma, W.Y.,Surh, Y.-J.and Dong, mediated caspase-dependent mechanism. Z. (2001) Inhibition of epidermal growth Journal of Agricultural and Food Chemistry, factor-induced cell transformation and 55, 948–954. activator protein 1 activation by 53 Pan, M.H., Hsieh, M.C., Kuo, J.M., Lai, [6]-gingerol. Cancer Research, 61, 850–853. C.S., Wu, H., Sang, S. and Ho, C.T. (2008) 46 Davies, M., Robinson, M., Smith, E., 6-Shogaol induces apoptosis in human Huntley, S., Prime, S. and Paterson, I. colorectal carcinoma cells via ROS (2005) Induction of an epithelial to production, caspase activation, and GADD mesenchymal transition in human 153 expression. Molecular Nutrition & Food immortal and malignant keratinocytes by Research, 52, 527–537. TGF-beta1 involves MAPK, Smad and 54 Keum, Y.S., Kim, J., Lee, K.H., Park, K.K., AP-1 signalling pathways. Journal of Surh, Y.-J., Lee, J.M., Lee, S.S., Yoon, J.H., Cellular Biochemistry, 95, 918–931. Joo, S.Y., Cha, I.H. and Yook, J.I. (2002) 47 Park, Y.J., Wen, J., Bang, S., Park, S.W. and Induction of apoptosis and caspase-3 Song, S.Y. (2006) [6]-Gingerol induces cell activation by chemopreventive [6]-paradol cycle arrest and cell death of mutant and structurally related compounds in KB p53-expressing pancreatic cancer cells. cells. Cancer Letters, 177,41–47. Yonsei Medical Journal, 47, 688–697. 55 Kim, E.C., Min, J.K., Kim, T.Y., Lee, S.J., 48 Shukla, Y., Prasad, S., Tripathi, C., Singh, Yang, H.O., Han, S., Kim, Y.M. and Kwon, M., George, J. and Kalra, N., (2007) In vitro Y.G. (2005) [6]-Gingerol, a pungent and in vivo modulation of testosterone ingredient of ginger, inhibits angiogenesis mediated alterations in apoptosis related in vitro and in vivo. Biochemical and proteins by [6]-gingerol. Molecular Biophysical Research Communications, Nutrition & Food Research, 51, 1492–1502. 335, 300–308. 49 Lee, S.H., Cekanova, M. and Baek, S.J. 56 Rhode, J., Fogoros, S., Zick, S., Wahl, H., (2008) Multiple mechanisms are involved Griffith, K.A., Huang, J. and Liu, J.R., in 6-gingerol-induced cell growth arrest (2007) Ginger inhibits cell growth and and apoptosis in human colorectal cancer modulates angiogenic factors in ovarian cells. Molecular Carcinogenesis, 47, cancer cells. BMC Complementary and 197–208. Alternative Medicine, 7, 44. 498j 28 Antioxidant, Anti-inflammatory, and Anticarcinogenic Effects of Ginger and Its Ingredients

57 Lee, H.S., Seo, E.Y., Kang, N.E. and on P-glycoprotein function. Biochemical Kim, W.K. (2008) [6]-Gingerol and Biophysical Research Communications, inhibits metastasis of MDA-MB-231 327, 866–870. human breast cancer cells. The 59 Zhang, W. and Lim, L.Y. (2008) Effects of Journal of Nutritional Biochemistry, 19, spice constituents on P-glycoprotein- 313–319. mediated transport and CYP3A4-mediated 58 Nabekura, T., Kamiyama, S. and Kitagawa, metabolism in vitro. Drug Metabolism and S. (2005) Effects of dietary Disposition: The Biological Fate of Chemicals, chemopreventive phytochemicals 36, 1283–1290. j499

29 Tannins: Bioavailability and Mechanisms of Action Fulgencio Saura-Calixto and Jara Perez-Jimenez

29.1 Introduction

Tannins are a major group of oligomeric and polymeric dietary polyphenols made up of ﬂavanol monomers (proanthocyanidins or condensed tannins) and polyesters of a sugar moiety and gallic acid or ellagic acid (hydrolyzable tannins). Over the last century, studies on tannins have moved from industrial applications (leather production) and organoleptic properties in foods to the present day research into biological properties associated with disease prevention. Physiological effects of dietary tannins will depend on their release from the food matrix and their absorption and fermentation in the gastrointestinal tract. The action of digestive enzymes may release a fraction of food tannins, which may be partially absorbed through the small intestine mucosa. Unabsorbed tannins and tannins associated with cell walls reach the colon, where they may be fermented by bacterial microﬂora, releasing some tannins and yielding different metabolites. Tannins absorbed in both the small and the large intestine may exert systemic effects. Several studies performed through cell culture assays and animal experiments have suggested that tannins may provide some protection against cancer. However, there have not yet been any clinical trials in humans. Knowledge of the intake of tannins and other polyphenols in common diets may be useful to elucidate their relative contribution to the health effects associated with the intake of antioxidants, especially in relation to gastrointestinal cancer. Note that most data on tannin intake take into account only low molecular weight food tannins extracted with aqueous organic solvents, ignoring the biological activity of a substantial part of nonextractable tannins that are subjected to colonic fermentation.

29.2 Physicochemical Properties

Hydrolyzable tannins are polyesters of a sugar moiety (or other nonaromatic polyhydroxy compounds) and gallic acid (gallotannins) or ellagic acid (ellagitannins) (Scheme 29.1). These compounds undergo hydrolytic cleavage to the respective sugar and acid moiety upon treatment with diluted acids. Condensed tannins or proanthocyanidins are polyhydroxyflavanol oligomers or polymers. They consist of flavan-3-ol or flavan-3,4-diol monomers that are usually linked by carbon–carbon bonds in the 4 ! 6orthe4! 8 position (B-type proantho- caynidins). B-type proanthocyanidins can be classified according to the hydroxylation pattern(s) of the chain extender unit(s), and the most commons in plant foods are procyanidins, prodelphinidins, and propelargonidins. The name proanthocyanidins is derived from the fact that when treated with acid, they react to form insoluble phlobaphenes and red-colored anthocyanidin solutions (Scheme 29.2). Tannins – especially proanthocyanidins – have the ability to complex strongly with carbohydrates and proteins. Tannins are sequestered into pores in the polysaccharide structure, an association that is influenced by molecular size, conformational mobility andshape,andwatersolubility.Whentanninsinteractwithproteins,theresultisusually a precipitationofthecomplex,butundercertainconditionssomesolublecomplexesare formed. Tannin–protein interactions are influenced by characteristics of the protein (size,aminoacidcomposition,pI,etc.),characteristicsofthetannin(size,structure),and conditions of the reaction (pH, temperature, solvent, and time) [1]. The major dietary sources of proanthocyanidins are fruits (particularly berries), legumes, nuts and some cereals (sorghum, barley), wine, beer, and other beverages. In the case of hydrolyzable tannins, ellagitannins have been reported, among others, in almost all varieties of berries, nuts (pecans, walnuts, brazil nuts, peanuts, and cashews), blue plum, pomegranate (fruit and juice), red apples, navel oranges, pink and white grapefruit, tangerine, tangelo, peach, brown and green pear, white and red grapes, oak-aged wines, and kiwi as well as beer.

Scheme 29.1 Structure of an ellagitannin (left) and a gallotannin (right). 29.3 Bioavailability and Metabolism j501

Scheme 29.2 Hydrolysis of a proanthocyanidins in an acidic medium.

29.3 Bioavailability and Metabolism

Very little is known about the metabolic fate and bioavailability of tannins. To exert their biological properties, tannins should be available to some extent in the target tissue. Therefore, the biological properties of tannins may depend on their absorption in the gut and their bioavailability. It must be remembered that molecules from tannins appearing in blood or excreted in urine can be very different from that when ingested.

29.3.1 Proanthocyanidins

It is not yet clearly established whether proanthocyanidins are depolymerized in the stomach or not, and it has been suggested that food bolus may buffer the acid secretion in the stomach, preventing acidic hydrolysis of proanthocyanidins [2, 3]. 502j 29 Tannins: Bioavailability and Mechanisms of Action

In small intestine digestion, the main factor that affects the fate of metabolization of proanthocyanidins is their degree of polymerization. It has been observed that the procyanidin dimers B2 and B5 are hydrolyzed to epicatechin in isolated rat small intestine. Also, it has been reported that orally administered procyanidin B2 and B3 dimers were absorbed and excreted by rats and that dimers and trimers from catechin have similar permeability coefficients to that of catechin and close to that of mannitol, a marker of paracellular transport [4]. However, absorption is much lower or entirely absent in the case of higher oligomers, and especially of polymers, which may reach the colon nearly intact [5]. During small intestine digestion, high molecular weight proanthocyanidins may form less digestible complexes with protein, starch, and digestive enzymes. Another important site where tannins become available in the gastrointestinal tract is the large intestine. Most of the ingested proanthocyanidins reach the colon, where they become fermentable substrate for bacterial microflora along with other nondigestible constituents. The abundant microflora in the colon plays a critical role in the metabolism of tannins. After microbial enzyme metabolism of any tannin that reaches the colon, there are two possible routes available, breakdown of the original tannin structure into absorbable metabolites or a breakdown into nonabsorbable metabolites (probably mid-molecular weight tannins); these remain in the colonic lumen, where they may counteract the effects of dietary pro-oxidants in the colon produced during colonic bacterial metabolism. Several authors have found that proanthocyanidins are metabolized to a large extent by gut microflora, the main metabolites produced being phenlyacetic, phe- nylpropionic, and phenylbutyric acids [4, 5]. The higher the degree of polymerization is, the lower the degree of metabolization will be [5]. As to the possibility of depolymerization of proanthocyanidins in the colon to their monomeric units, to our knowledge no intestinal bacteria so far described are capable of performing this transformation.

29.3.2 Hydrolyzable Tannins

Few studies have evaluated the rate of hydrolysis of hydrolyzable tannins into monomers (ellagic acid or gallic acid) during enzymatic digestion in the stomach and small intestine. The possibility of gastric absorption of ellagic acid has recently been reported in pigs [6], but results of absorption in the small intestine are contradictory [7, 8]. One possible explanation for this discrepancy is the poor water solubility of ellagic acid, which would result in low concentrations of free ellagic acid in plasma, and another is the fact that ellagic acid binds irreversibly to cellular DNA and proteins, which may also account for its limited transcellular absorption and its ability to form poorly soluble complexes with calcium and magnesium ions in the intestine [9]. Following absorption, ellagic acid undergoes conjugation, and conjugated forms with methyl, glucuronyl, and sulfate groups have been found in plasma and excreted in urine in humans. 29.4 Mechanisms of Protection j503

Two different systems for gastric absorption of gallic acid have been described, a rapid permeation system for intact gallic acid and a slow permeation system for conjugated derivatives [10], while in the small intestine, gallic acid is absorbed, peaking after about 1 h, and is metabolized to other compounds, of which the most abundant in humans is 4-O-methylgallic acid [11]. When hydrolyzable tannins reach the colon, they are subjected to the action of certain lactobacilli with distinct tannase activity, which hydrolyzes hexahydroxy- diphenoyl groups in ellagitannins and galloyl groups in gallotannins [12]. A pathway has been proposed for the degradation of ellagitannin by human microbiota via hydrolysis to ellagic acid and its microbial transformation to urolithin A and urolithin B, which are detected in plasma as glucuronides after absorption [13]. A recent study of the complete metabolism of ellagitannins in pig extensively reported conjugated metabolites in bile, which would imply very active enterohepatic circulation of these compounds [6].

29.4 Mechanisms of Protection

Over the last decade, studies on cell cultures and on experimental animals treated with tannins have shown that these compounds may exert several beneﬁcial effects. It is believed that tannins may exert their biological effects in two different ways: (1) as an unabsorbable, complex structure with binding properties that may produce local effects in the gastrointestinal tract (antioxidant, radical scavenging, antimicrobial, antiviral, and antinutrient effects) or (2) as absorbable tannins (probably low molecular weight) and absorbable metabolites from colonic fermentation of tannins that may produce systemic effects in various organs. Several studies with different cell cultures have reported chemopreventive effects of tannins on breast, oral, prostate, stomach, and skin cancer [14–18]. Most of these studies have tested the molecules of tannins as present in foods, assuming that these polyphenols are absorbed to reach these target tissues to exert the potential anticarcinogenic effect. However, this approach does not take into account the bioavailability of tannins referred above, as a result of which the amount of the tannins as present in the food matrix that would reach the different organs would be negligible and would not even remotely approach the concentration used in the referred studies. It suggests that the original tannins should only be used in cell cultures when studying colorectal cancer, while for other kinds of cancers their corresponding metabolites should be tested. In the case of colorectal cancer, several studies (with cell cultures and experimental animals) have concluded that tannins – both hydrolyzable tannins and proanthocyanidins – may produce a chemopreventive effect for colorectal cancer through various mechanisms: 1. Induction of apoptosis through, among other processes, activation of initiator caspasa-9, and effector caspase-3 [19]. 504j 29 Tannins: Bioavailability and Mechanisms of Action

2. Inhibition of the inflammation signaling pathway that increases the risk of colon cancer by inhibiting expression of the enzyme COX-2 (cyclooxygenase- 2) [20]. 3. Reduction of the levels of MMP-2 and MMP-9 secreted to the extracellular medium [21]. 4. Generation of an antioxidant environment in the colon. Since overproduction of free radicals and hence of oxidative stress has been linked to cancer, antioxidants may help to prevent the onset of this disease. Note, in this connection, that tannins are able to inhibit lipid peroxidation and lipoxygenases in vitro,andtheyarealsoabletoscavengeradicalssuchashydroxyl, superoxide, and peroxyl that are known to be important in cellular pro-oxidant states. 5. Inhibition of DNA oxidative damage, another effect associated with the antioxidant properties of tannins [22]. 6. Stimulation of the growth of beneficial colonic bacteria, such as Lactobacillus and Bifidobacterium spp., and inhibition of the growth of nondesirable bacteria, such as Clostridium [23]. Interestingly, the observed effects did not take place in normal cells. As to the products that have been tested, the studies with hydrolyzable tannins focus mainly on punicalagin, present in pomegranate, and ellagic acid, while the studies on proanthocyanidin properties used extracts obtained from grape, wine, or apple. However, these works only consider low molecular weight proanthocyanidins, with a mean degree of polymerization between three and eight units. This means that the possible chemopreventive effects of high molecular weight proanthocyanidins, which remain in the residues of the common aqueous–organic extractions performed, have not been systematically considered. However, supplementation to rats with a grape product rich in high molecular weight proanthocyanidins (about 20%) revealed a capacity to inhibit proliferation in the colonic epithelium by reducing the total number of crypts per milliliter and producing shorter crypts [24]. In this study, besides high concentrations of high molecular weight proanthocyanidins, the product tested contained numerous low molecular weight proanthocyanidins and other polyphenols. This suggests that mixtures of polyphenols may enhance the health beneficial effects of a single polyphenol or tannin through synergistic action. Similarly, it has been observed that pomegranate juice (rich in polyphenols, including hydrolyzable tannins) induced apoptosis on HT-29 cells, when concentrations of the hydrolyzable tannin punicalagin or total tannins comparable to the concentrations present in pomegranate juice had no effect [19]. Therefore, the possible chemopreventive effect should be evaluated in the context of the overall intake of antioxidants and other chemopreventive compounds in adiet. 29.5 Results of Human Studies j505

29.5 Results of Human Studies

Most intervention studies on the relationship between antioxidants and chronic disease, including gastrointestinal cancer, have tested specific antioxidants (mainly vitamin C, vitamin E, and b-carotene) or specific foods. A more comprehensive view of this field may be gained by examining the antioxidant capacity of whole diets rather than single antioxidants or foods. In this way, epidemiological studies have suggested that dietary antioxidants may play a role in the prevention of a number of diseases [25]. Considering the overall intake in a common diet in the approach to tannins may be useful for epidemiological studies and for the design of intervention studies. A number of authors have estimated the daily intake of tannins based on food composition and consumption survey data. The mean proanthocyanidin intake in the United States, according to the USDA (United States Department of Agriculture) food composition database, is estimated at 53.6 mg/day/person. Recently, Saura-Calixto et al. [26] determined and estimated the intake of highly polymerized proanthocyanidins in the Spanish diet at 450 mg/person/day. The reason why the intake data reported in the United States are low is that USDA composition data do no not take into account dietary intake of high molecular weight proanthocyanidins. There are no data concerning ellagitannins and gallotannins intake in the literature. Saura-Calixto et al. [26] estimated the intake of hydrolyzable polyphenols (polyphenols associated with high molecular weight compounds that includes hydrolyzable tannins and other phenolic acids) in the Spanish population at around 1250 mg/person/day. Nevertheless, these data may, respectively, underestimate and overestimate the hydrolyzable tannin intake. In the case of the Spanish diet, a total intake of polyphenols (2.8 g/p/day) has been reported, including extractable polyphenols (phenolic acids, flavonoids, and low molecular weight proanthocyanidins), high molecular weight proanthocyanidins, and hydrolyzable phenolics. It is estimated that hydrolyzable phenolics and high molecular weight proanthocyanidins represent about 50% of the total intake of polyphenols. Clinical trials have failed to show any benefit from antioxidant supplementation in the prevention of gastrointestinal cancer [27]. The reported clinical trials only analyzed isolated antioxidants (e.g., 15–50 mg b-carotene/day, 120–2000 mg vitamin C/day, 30–600 mg vitamin E/day), but the potential effect and the synergistic action of dietary polyphenols that reach the colonic lumen (estimated at 2.5 g/day) [26] are ignored. Note that tannins and other polyphenols, unlike vitamins, remain nearly intact until they reach the colon and are degraded there, releasing absorbable and nonabsorbable metabolites that may play a chemopreventive role. Further studies would be necessary to elucidate such potential action of dietary polyphenols in the colonic lumen. 506j 29 Tannins: Bioavailability and Mechanisms of Action

29.6 Impact of Cooking and Processing

There are only a very limited number of references to the influence of storage and processing on stability of tannins. It has been reported that ellagitannins were reduced by up to 40% after 9 months storage at 20 C [28] and that the amount of ellagic acid in frozen-stored raspberries decreased significantly (14–21%) in the course of 1 year [29]. Proanthocyanidins may be degraded during the drying process or polymerized to more highly polymerized compounds, which can render the extraction and quantification of proanthocyanidins in dried foods incomplete. Similarly, various processing procedures entailing the use of high temperatures have been found to reduce proanthocyanidin content. For example, this has been reported after cooking of faba beans, processing of sorghum bran into cookies and bread, thermal processing, and canning of peaches or heating of grapes [30–33].

References

1 Dey, P.M. and Harborne, J.B. (1989) 6 Espın, J.C., Gonzalez-Barrio, R., Cerda, B., Methods in Plant Biochemistry Volume 1. López-Bote, C., Rey, A.I. and Tomas- Plant Phenolics, Academic Press, San Barberan, F.A. (2007) Iberian pig as a Diego. model to clarify obscure points in the 2 Spencer, J.P., Chaudry, F., Pannala, A.S., bioavailability and metabolism of Srai, S.K., Debnam, E. and Rice-Evans, C. ellagitannins in humans. Journal of (2000) Decomposition of cocoa Agricultural and Food Chemistry, 55, procyanidins in the gastric milieu. 10476–10485. Biochemical and Biophysical Research 7 Smart, R.C., Huang, M.T., Chang, R.L., Communications, 272, 236–241. Sayer, J.M., Jerina, D.M. and Conney, D.H. 3 Rios, L.Y., Bennett, R.N., Lazarus, S.A., (1986) Disposition of the naturally Remesy, C., Scalbert, A. and Williamson, occurring antimutagenic plant phenol, G. (2002) Cocoa procyanidins are stable ellagic acid, and its synthetic derivatives, during gastric transit in humans. The 3-O-decylellagic acid and 3,3-di-O- American Journal of Clinical Nutrition, 76, methylellagic acid in mice. Carcinogenesis, 1106–1110. 7, 1663–1667. 4 Deprez, S., Mila, I., Huneau, J.F., Tome,D. 8 Seeram, N.P., Henning, S.M., Zhang, Y., and Scalbert, A. (2001) Transport of Suchard, M., Li, Z. and Heber, D. (2006) proanthocyanidin dimer, trimer, and Pomegranate Juice ellagitannin polymer across monolayers of human metabolites are present in human plasma intestinal epithelial Caco-2 cells. and some persist in urine for up to 48 Antioxidants & Redox Signaling, 3, hours. The Journal of Nutrition, 136, 957–967. 2481–2485. 5 Gonthier, M.-P., Donovan, J.L., Texier, O., 9 Whitley, A.C., Stoner, G.D., Felgines, C., Remesy, C. and Scalbert, A. Darby, M.W. and Walle, T. (2003) (2003) Metabolism of dietary procyanidins Intestinal epithelial cell accumulation in rats. Free Radical Biology and Medicine, of the cancer preventive polyphenol 35, 837–844. ellagic acid extensive binding to protein References j507

and DNA. Biochemical Pharmacology, 66, growth of human breast cancer cells. 907–915. Molecular Cancer Therapeutics, 4, 537–546. 10 Konishi, Y., Zhao, Z. and Shimizu, M. 17 Shoji, T., Masumoto, S., Moriichi, N., (2006) Phenolic acids are absorbed from Kobori, M., Kanda, T., Shinmoto, H. and the rat stomach with different absorption Tsushida, T. (2005) Procyanidin trimers to rates. Journal of Agricultural and Food pentamers fractionated from apple inhibit Chemistry, 54, 7539–7543. melanogenesis in B16 mouse melanoma 11 Shahrzad, S., Aoyagi, K., Winter, A., cells. Journal of Agricultural and Food Koyama, A. and Bitsch, I. (2001) Chemistry, 53, 6105–6111. Pharmacokinetics of gallic acid and its 18 Miura, T., Chiba, M., Kasai, K., Nozaka, H., relative bioavailability from tea in healthy Nakamura, T., Shoji, T., Kanda, T., Ohtake, humans. The Journal of Nutrition, 131, Y. and Sako, T. (2008) Apple procyanidins 1207–1210. induce tumor cells apoptosis through 12 Osawa, R., Kuroiso, K., Goto, S. and mitochondrial pathway activation of Shimizu, A. (2000) Isolation of tannin- caspase-3. Carcinogenesis, 29, 585–593. degrading lactobacilli from humans and 19 Seeram, N.P., Adams, L.S., Henning, fermented foods. Applied and S.M., Nio, Y., Zhang, Y., Nair, M.G. and Environmental Microbiology, 66, Heber, D. (2005) In vitro antiproliferative, 3093–3097. apoptotic and antioxidant activities of 13 Cerda B., Periago, P., Espın, J.C. and punicalagin, ellagic acid and a total Tomas-Barberan, F.A. (2005) Metabolism pomegranate tannin extract are enhanced of antioxidant and chemopreventive in combination with other polyphenols as ellagitannins from strawberries, found in pomegranate juice. The Journal of raspberries, walnuts, and oak-aged wine in Nutritional Biochemistry, 16, 360–367. humans: identification of biomarkers and 20 Adams, L.S., Seeram, N.P., Aggarwal, individual variability. Journal of B.B., Takada, Y., Sand, D. and Heber, D. Agricultural and Food Chemistry, 53, (2006) Pomegranate juice, total 227–235. pomegranate ellagitannins and 14 Agarwal, C., Veluri, R., Kaur, M., Chou, punicalagin suppress inflammatory cell S.C., Thompson, J.A. and Agarwal, R. signaling in colon cancer cells. Journal of (2007) Fractionation of high molecular Agricultural and Food Chemistry, 54, weight tannins in grape seed extract and 980–995. identification of procyanidin B2-3,30-di- 21 Bawadi, H.A., Bansode, R.R., Trappey, A., O-gallate as a major active constituent II, Truax, R.E. and Losso, J.N. (2005) causing growth inhibition and apoptotic Inhibition of Caco-2 colon, MCF-7 and death of DU145 human prostate Hs578 breast and DU 145 prostatic cancer carcinoma cells. Carcinogenesis, 28, cell proliferation by water-soluble black 1478–1484. bean condensed tannins. Cancer Letters, 15 Hibashami, H., Shokji, T., Shibuya, I., 218, 153–162. Higo, K. and Kanda, T. (2004) Induction of 22 Giovannelli, L., Testa, G., De Filippo, apoptosis by three types of procyanidins C., Cheynier, V.,Clifford, M.N. and Dolara, isolated from apple (Rosaceae Malus P. (2000) Effect of complex polyphenols pumila) in human stomach cancer KATO and tannins from red wine on DNA III cells. International Journal of Molecular oxidative damage of rat colon mucosa Medicine, 13, 795–799. in vivo. European Journal of Nutrition, 39, 16 Ramljak, D., Romanczyk, L.J., Methen, Y., 207–212. Barlow, L.J., Thompson, N. et al. (2005) 23 Dolara, P., Luceri, C., De Filippo, C., Pentameric procyanidins from Femia, A.P., Giovannelli, L., Caderni, G., Theobroma cacao selectively inhibits Cecchini, C., Silvi, S., Orpianesi, C. and 508j 29 Tannins: Bioavailability and Mechanisms of Action

Cresci, A. (2005) Red wine polyphenols 29 De Ancos, B., Iban~cez, E., Reglero, G. influence carcinogenesis, intestinal and Cano, M.P. (2000) Frozen storage microflora, oxidative damage and gene effects on anthocyanins and volatile expression profiles of colonic mucosa in compounds of raspberry fruit. Journal of F344 rats. Mutation Research, 591, Agricultural and Food Chemistry, 48, 237–246. 873–879. 24 López-Oliva, M.E., Agis-Torres, A., 30 Sharma, A. and Sehgal, S. (1992) Effect of Garcıa-Palencia, P., Goñi, I. and Muñoz- domestic processing, cooking and Martınez, E. (2006) Induction of germination on the trypsin inhibitor epithelial hypoplasia in cecal and distal activity and tannin content of faba bean colonic mucosa by grape antioxidant (Vicia faba). Plant Foods for Human dietary fiber. Nutrition Research, 26, Nutrition, 42, 127–133. 651–658. 31 Larrauri, J.A., Rupérez, P. and Saura- 25 Arts, I.C. and Hollman, P.C. (2005) Calixto, F. (1997) Effect of drying Polyphenols and disease risk in temperature on the stability of epidemiological studies. The American polyphenols and antioxidant activity Journal of Clinical Nutrition, 81, 317s–325s. of red grape pomace peels. Journal of 26 Saura-Calixto, F., Serrano, J. and Goñi, I. Agricultural and Food Chemistry, 45, (2007) Intake and bioaccessibility of total 1390–1393. polyphenols in a whole diet. Food 32 Awika, J.M., Dykes, L., Gu, L., Rooney, L.W. Chemistry, 101, 492–501. and Prior, R.L. (2003) Processing of 27 Bjelakovic, G., Nikolova, D., Simoneti, R. sorghum (Sorghum bicolor) and sorghum and Gloud, C. (2004) Antioxidant products alters procyanidin oligomer and supplements for prevention of polymer distribution and content. Journal gastrointestinal cancer: a systematic of Agricultural and Food Chemistry, 51, review. Lancet, 364, 1219–1228. 5516–5521. 28 H€akkinen, S., K€arenlampi, S., Mykk€anen, 33 Hong, Y.J.,Barrett, D.M. and Mitchell, A.E. H., Heinonen, M. and Torr€ onen,€ R. (2000) (2004) Liquid chromatography/mass Ellagic acid-content in berries: influence of spectrometry investigation of the impact of domestic processing and storage. thermal processing and storage on peach European Food Research and Technology, procyanidins. Journal of Agricultural and 212,75–80. Food Chemistry, 52, 2366–2371. j509

30 Selected Flavonoids

30.1 Quercetin and other Flavonols Loïc Le Marchand and Adrian A. Franke

30.1.1 Introduction

The health benefits of plant-based diets have received much research attention. Among the many phytochemicals with potential cancer-protective properties, flavonoids are among the most common and most widely distributed in the human diet. Among the subclasses of flavonoids, flavonols are the most abundant in foods, with kaempferol and myricetin and, especially, quercetin being the predominant flavonols consumed. This chapter reviews the main physical and chemical properties of these main flavonols, their occurrence in the diet, their uptake and biovailability, and the mechanisms of action and epidemiological evidence related to their potential protective effect against cancer.

30.1.2 Physicochemical Properties of Active Compounds and their Occurrence

Flavonols (officially called 3-hydroxy-2-phenylchromen-4-one) are characterized by a benzopyrone system (rings A and C) that contains a phenolic constituent (B-ring) at position 2 and a hydroxyl group at carbon-3 (Scheme 30.1). They are typically hydroxylated at positions 5 and 7 as a result of their biosynthesis involving one phenylpropane(cinnamateviatheshikimatepathway)andthreeacetateunits(derived from malonate). The three-dimensional representation of quercetin (Scheme 30.2) illustrates the almost planar configuration of this class of flavonoids that explains their abilitytointercalatewithDNA[1,2].Kaempferolandmyricetinhaveonefewerandone additional hydroxyl group, respectively, in the B-ring (Scheme 30.1). A total of 393 differentstructureshavebeenreportedforflavonols[3].However,weonlyreferhereto the most commonly occurring aglycones listed in Scheme 30.1.

Scheme 30.1 Structures of flavonols.

The one physical property that distinguishes the flavonol aglycones the most from other flavonoids is their UV absorption peak in the 350–370 nm range (which occurs in addition to that at about 260–280 nm typical of most flavonoids) (Table 30.1). These absorbance wavelengths protect plants from UVdamage [4] and give flavonols a slight yellow color (hence their name, which is derived from flavus, the Latin for yellow). Characteristically, this absorbance level can be changed by the traditional shift reagents [5]. Another characteristic of flavonols is that they show few but characteristic proton magnetic resonance signals that are shifted downfield due to the aromatic nature of all protons. These signals appear as 2 Hz-doublets for A-ring protons and as (double)-doublets for B-ring protons depending on the substitution at that ring (Table 30.1). Also, another characteristic of flavonols is their reaction with complex- ing agents that produces fluorescent colors under 366 nm light [5, 6]. In nature, flavonols are produced by almost all orders of the plant kingdom. With few exceptions, they occur in plant tissue as glycosides, most frequently bound with sugars to the hydroxyls at positions 3 and/or 7 and/or to the OH groups in the B-ring. This renders these molecules water soluble. However, if the glycoside moiety is acylated, they can become lipophilic, especially when esterified with aromatic acids, such as cinnamates [6]. These aromatic esters are particularly protective against UV damage because of their additional absorbing properties. Over 2000 glycosides (including those for flavones) have been reported so far [7].

Scheme 30.2 Three-dimensional structure of quercetin. 30.1 Quercetin and other Flavonols j511

Table 30.1 Physical and other characteristic properties of quercetin.

Molecular formula C15H10O7 Molecular weight (g/mol) 302.2357 Nuclide mass (g/mol) 302.0427 Melting point of dihydrate Anhydrous at 95–97 C; decays at 314 C (yellow needles from dil. alcohol) Solubilitya (not soluble in water) 1 g/290 ml abs. alcohol at 20 C (1 g/23 ml when boiling); in acetic acid; alkaline solutions (yellow) b UV in MeOH (lmax in nm) 255, 269 (sh), 299 (sh), 370 l e c UV in 96% EtOH ( max in nm; [ ]) 373 [20 892] d HNMR signals (ppm in acetone-d6 or DMSO-d6) 6.27 (H-6; d2), 6.47 (H-8; d2), 7.82 (H-20; d2), 6.97 (H-50; d8), 7.72 (H-60; dd2,8) e LD50 orally in mice 160 mg/kg l e ﬁ max, absorbance maximum; nm, nanometer; , molar extinction coef cient; sh, shoulder; d6, deuterated six times; d2, doublet 2 Hz; d8, doublet 8 Hz; dd 2,8, double-doublet 2 Hz and 8 Hz; dil., dilute; abs., absolute; LD50, dose at which 50% of population dies; UV, ultra violet absorbance; HNMR, proton magnetic resonance; ppm, parts per million; DMSO, dimethyl sulfoxide. aMerck Index, 10th edn, 1983. bSee Ref. [5]. cCRC Handbook of Chemistry and Physics, 1970. dRef. [6, 18]. eSullivan et al. (1951) Proceedings of Society for Experimental Biology and Medicine, 77, 269.

In the diet, flavonols are one of the most common flavonoids, both in terms of distribution and of concentration in foods. Their levels rival those of anthocyanins observed in blue-purple plant parts and of catechins in cocoa or tea [8, 9]. Interest has focused on the distribution of flavonols in plants for chemotaxonomy [10–13] and, more recently, on their reported biological activities and health benefits [14]. Quer- cetin, the most common flavonol, may constitute up to 91% of the flavonol content of a flavonol-rich diet [15, 16]. Food levels of flavonols, and other flavonoids, were recently compiled from 199 peer-reviewed publications by the US Department of Agriculture (USDA) in a database available online [8], a useful resource not only because it lists typical concentration ranges but also because it gives a confidence rating for the data [17]. The USDA database will continuously be updated with new data, such as those recently published for onions [18]. Flavonol levels vary widely in foods depending on various factors (genetics, soil, climate, UV radiation, and others). Very high quercetin levels are reported for capers (2384 mg/kg), lovage (1700 mg/kg), and most importantly, due to their high intake, for onions (up to 2000 mg/kg). However, the reported quercetin levels in onions also show much variation, with levels as low as a few milligrams per kilogram [18]. Within foods, quercetin concentrations are higher in the outermost parts than in the inner parts (reviewed in [15]), due to the lower water content and higher UVexposure of the outer part. Wide variation in flavonol levels is also observed for tomatoes, with concentrations 79% higher in organically grown than conventionally grown tomatoes [19]. The average dietary intake of flavones and flavonols (and their main food sources) was estimated 512j 30 Selected Flavonoids

to be 20–24 mg in the United States (tea 26%, onions 24%, apples 8%), 23 mg in the Netherlands (tea 48%, onions 29%, apples 7%), 26 mg in Wales/UK (tea 82%, onions 10%), 4 mg in Finland (apples and onions), 5 mg in Spain (tea 26%, onions 23%, apples 8%), and 16 mg in Japan (onions 46%, molokheya 10%, apples 7%, green tea 5%) [9]. Usually, at least 70–80% of these amounts are flavonols, as illustrated by an Italian study that reported a mean intake of 22.4 13.0 mg for flavonols and 0.5 0.3 mg for flavones [20].

30.1.3 Biovailability and Metabolisms of Active Compounds

After much debate, it is now agreed that flavonol glycosides as consumed are not transported as such into the blood stream but exist in the circulation as glucuronide and/or sulfate conjugates [21–23]. Consequently, they need to be hydrolyzed to aglycones as a prerequisite for absorption through the gut mucosa, and three major routes of absorption have been described (Figure 30.1): (a) hydrolysis by the gut bacteria followed by passive diffusion through enterocytes; (b) hydrolysis through lactate phloridzin hydrolase (LPH), an enzyme bound to the brush border membrane, followed by passive diffusion through the enterocyte; and (c) transport of the intact glycoside through the apical membrane of the enterocyte via sodium- dependent glucose transporter 1 (SGLT1), followed by hydrolysis via cytosolic beta- glucosidase (CBG). In addition, it has been noted that hydrolysis occurs as early as in the oral cavity [24]. Typical liver conjugation reactions by UGT, SULT, and COMT enzymes (Figure 30.1) could theoretically also take place on the basolateral side of the enterocyte. Although the gut flora can hydrolyze all flavonol glycosides (pathway a), pathway b is thought to be important for mono/diglycosides and pathway c for quercetin-40-O-glucoside [23, 25]. A combination of these uptake pathways may be relevant to many flavonol glycosides. However, for rutin, one of the most commonly occurring glycosides, and for other biosides (defined by two sugars attached to each other then bound to flavonols), pathway a applies exclusively. This absorption pathway is characterized by the longer time needed to reach maximum plasma levels, indicating that it is occurring in the distal part of the small intestine, compared to the two other pathways that take place in the proximal part. Uptake via pathway c can be interrupted by efflux of the absorbed glucoside to the lumen via multidrug resistance- associated protein 2 (MDRP2) [22, 23]. With the increasing concentration of bacteria encountered during progression through the gut, flavonols are not only hydrolyzed but also extensively degraded [26, 27] (ring A- and B-related fragments in the lower part of Figure 30.1). Some of these degradation products are absorbed from the gut, probably by passive diffusion, and may exert beneficial biological effects. For example, dihydroxyphenyl acetic acid (DHPAA) has been shown to reduce the proliferation of prostate and colon cancer cells [28]. These metabolic products were found in urine at concentrations up to 50% relative to dose [29] and mostly as glucuronates [30]. It has been recommended that urinary levels of some of these degradation products (hydroxyphenyl acetic acid, 3-methoxy-4-hydroxy-phenyl acetic acid, and, particularly, DHPAA) be used as 30.1 Quercetin and other Flavonols j513

Figure 30.1 Absortion, transport, and catecholamine orthomethyl transferase; MRP2/ metabolism of ingested quercetin glycosides.Q- 3, multidrug resistance-associated protein 2/3; 3-Glu-Rha, quercetin-3-O-rhamnosyl(1–6) UGT, UDP-glucuronyltransferase; SULT, 0 0 glucoside (rutin); Q-3,4 -Glu2, quercetin-3,4 -O- sulfotransferases; PHL, phloroglucinol; DHPPA, diglucoside; Q-40-Glu, quercetin-40-O-glucoside; 3,4-dihydroxyphenyl propionic acid; 3,4-DHPAA, QMGs, quercetin monoglucosides; Q, quercetin; dihydroxyphenyl acetic acid

Q-Glur, quercetin-O-glucuronides; Q-SO4, (homoprotocatechuic acid); HPPA, 3- quercetin sulfates; Q-30-OMe, 30-O-methylethers hydroxyphenyl propionic acid; HPAA, 3- of quercetin (isorhamnetin) or its conjugates; hydroxyphenyl acetic acid; MHPAA, 3-methoxy- LPH, lactate phloridzin hydrolase; SGLT1, 4-hydroxyphenyl acetic acid (homovanillic acid); sodium-dependent glucose transporter 1; CBG, boxed molecules are predominant metabolites cytosolic beta-glucosidase; COMT, found in plasma and urine after flavonol intake. markers of flavonol intake [31]. However, these catabolic metabolites are not specifictoflavonols since they could originate from several other flavonoids or other natural products, or they could be ingested in the diet [28, 32]. Therefore, when estimating dietary flavonol exposure, it is preferable to measure the parent flavonol in plasma or urine [33], although their urinary recovery can be as little as 0.5–1% relative to dose [23, 25, 34]. Controlled feeding trials with intake determined by food analyses [16, 34, 35], as well as cross-sectional studies with intake estimated from a questionnaire and food composition data [33, 36], showed that urinary and/or plasma flavonols reflect dietary intake despite a large intraindividual variation of up to 91%. 514j 30 Selected Flavonoids

30.1.4 Mechanisms of Protection

Quercetin and other related flavonols have been shown to inhibit carcinogen-induced tumors in rodents. The evidence to date suggests that this protective effect may result from their actions on a number of important mechanisms involved in carcinogenesis. Like other flavonoids, flavonols have been shown to be highly effective scavengers of most types of oxidizing molecules, including singlet oxygen and various free radicals [37] that are involved in DNA damage and tumor promotion [38]. Flavonols may also exert a beneficial effect through their impact on bioactivation of carcinogens. Many chemical carcinogens require transformation by xenobiotics-metabolizing enzymes into a more reactive form to bind to DNA and possibly cause a mutation. If not repaired, this mutation may initiate or promote the carcinogenesis process. Quercetin and kaempferol have been shown to inhibit cytochrome P-450 enzymes of the CYP1A family [39]. These enzymes are known to bioactivate polycyclic aromatic hydrocarbons, a class of human carcinogens. Quercetin has also been shown to inhibit CYP3A4 and to contribute to the suppressive effect of grapefruit on this enzyme [39, 40]. CYP3A4 is the most abundant p450 enzyme in the liver and metabolizes a number of carcinogens and medications. Quercetin and kaempferol have also been shown to inhibit sulfotransferase 1A1, possibly representing an important mechanism for the chemoprevention of sulfation-induced carcinogenesis [39]. Glucuronidation, catalyzed by the UDP-glucuronyltransferase (UGT) family of enzymes, is a major detoxifying pathway for endogenous steroids, drugs, and carcinogens, and UGTs were induced by quercetin, kaempferol, and other selected flavonoids in a prostate tumor cell line [41]. Quercetin has also been shown to have anti-inflammatory properties by inhibiting cycloxygenase-2 (COX-2) and inducible nitric oxide synthase [42, 43] Chronic inflammation is thought to play an important role in a number of cancers and COX-2 inhibitors are being studied as chemopreventative agents against colorectal cancer. In estrogen-dependent tumor cell lines or animal models, quercetin has been shown to have antiproliferative activity that has been related to its antiestrogenic effect [44]. In other in vitro models, flavonols have been shown to affect cell-signaling and cell cycle regulation. Quercetin was reported to inhibit lung cancer cell proliferation by activating the extracellular signal-regulated kinase (ERK) and triggering apoptosis [45] to inhibit protein tyrosine kinase that is also involved in cell proliferation [46], or to cause cell cycle arrest and apoptosis by a p53-dependent mechanism [47]. Finally, quercetin has been shown to downregulate the levels of oncogenic Ras in cancer cells [48].

30.1.5 Results of Human Studies

Human data on the potential cancer beneﬁts of ﬂavonols are mostly derived from epidemiological studies, either prospective or retrospective in design. 30.1 Quercetin and other Flavonols j515

30.1.5.1 Cohort Studies In a prospective study of 10 054 Finnish men and women followed for 30 years, men withahighquercetinorappleintakehadalowerincidenceofallcancersandlung cancer; similarly, men with a high myricetin intake had a lower incidence of prostate cancer [49]. No association was found with cancers of the stomach, colorectum, and breast. A prospective study involving 120 850 men and women in the Netherlands found no association between flavonol intake and cancers of the stomach, colon, or lungs during 4.3 years of follow-up [50]. Similarly, flavonols, including quercetin, kaempferol, and myricetin, were not associated with breast cancer in the Nurses Health Study (NHS) -II [51] or with colorectal cancer in the NHS-I and Health Professional Follow-up Study [52]. However, intake of kaempferol and its main food sources, tea and broccoli, was inversely associated with the risk of ovarian cancer in NHS-I [53]. In the Multiethnic Cohort Study, intakes of total flavonols, kaempferol, quercetin, and myricetin were found to be associated with a reduced risk of pancreatic cancer [54]. These associations were particularly marked among smokers.

30.1.5.2 Case–Control Studies Acase–control study in Hawaii reported an inverse association between lung cancer and intakes of onion, apple, and quercetin [55]. A case–control study of breast cancer in Greece also reported a nonstatistically significant inverse association with kaempferol intake [56], whereas a study in Western New York reported an inverse association between quercetin intake and prostate cancer [57]. Intakes of quercetin and kaempferol were also reported to be inversely associated with stomach cancer in a Spanish study [58]. Similarly, a study in Scotland found an inverse association between intakes of flavonols and quercetin and colorectal cancer [59]. Finally, an Italian study reported an inverse association of flavonols intake with kidney cancer [20]. In summary, human studies are still few. They suggest a possible protective effect of flavonol against cancers at various sites (lung, prostate, ovarian, and pancreas). However,this evidence remains limited and is based solely on dietary intake estimates.

30.1.6 Impact of Storage, Processing, and Cooking on Protective Properties

Food processing is expected to have little impact on the biological effects of flavonols, limited to a lowering of their concentrations in foods and, in some instances, a modification of their conjugation pattern. This may lead to a lower flavonol intake and to changes in pharmacokinetics after absorption. Cooking has been shown to result in a decrease in flavonol content in foods, as illustrated in tomatoes by a 30–60% decrease in quercetin concentration observed with boiling [15, 60, 61]. Similarly, boiling onions led to a 20–25% loss in quercetin content through leaching into the cooking water, without degradation; a similar loss was observed after frying but, in this case, with degradation [62, 63]. Finally, blanching has been reported to decrease the total flavonol content of sweet-potato leaves by 56% [64]. It has also been shown that storage of onions at ambient 516j 30 Selected Flavonoids

temperature after homogenization changes the conjugation pattern, without altering total quercetin levels. In contrast, total quercetin concentration in onions decreased 36% within the ﬁrst 2 weeks of curing after harvest, with little further loss in the following 6 months of home-like storage conditions [63]. In our experience, neither freeze-drying nor storage of the unprocessed food at 80 C for 16 months led to a signiﬁcant loss of quercetin levels [15].

30.1.7 Research Needs

As mentioned above, the epidemiological data available to date rely exclusively on dietary questionnaire information. The evidence for a cancer-protective effect of flavonols in humans would be much strengthened if similar results were obtained using biomarkers to assess usual intake. Only limited research has been conducted on potential biomakers of flavonol intake. However, for isoflavones, it has been shown that urinary excretion is a good marker for both circulating levels and dietary intake [65–67], suggesting that similar markers could be developed for flavonols. Such markers should also include the main catabolic metabolites since they appear to also have interesting beneficial properties. In humans, glucuronide and sulfate conjugates are the predominant circulating forms for flavonols. However, only the bioactivity of the unconjugated (free) forms of flavonols has been studied and the assumption has been that the conjugates are not reactive. Therefore, there is a need to determine the bioactivity of glucuronide and sulfate flavonol conjugates and to assess whether deconjugation mechanisms of glucuronides and sulfates may reactivate flavonol to its free form. Finally, additional small-scale human studies need to be conducted to further assess the effects of specific flavonols and flavonol-rich foods on specific cancer- related pathways, such as carcinogen biotransformation, cell proliferation, apoptosis, and inflammation. In conclusion, the human evidence for a cancer-protective effect of flavonols is still limited but clearly warrants additional research both in the laboratory and in epidemiologic studies. The evidence, although promising, needs strengthening before large-scale chemoprevention trials are undertaken or before any specific health recommendations can be made to the public.

30.2 Anthocyanidins Li-Shu Wang and Gary D. Stoner

30.2.1 Introduction

The anthocyanidins occur ubiquitously in the plant kingdom and belong to the flavonoid group of plant polyphenols. Anthocyanidins are natures colors as they 30.2 Anthocyanidins j517 confer the bright red, blue, and purple colors on fruits and vegetables, and the autumn foliage of deciduous trees. Of potential importance to human health is the relatively high concentration of anthocyanidins in the diet. The daily intake of anthocyanidins in US diet is estimated to be as much as 180–215 mg or about 10-fold higher than that of other flavonoids such as quercetin, genistein, and apigenin [68]. Among the most abundant commercial sources of anthocyanidins are berries; for example, blueberries, blackberries, black currants, elderberries, grapes, and strawberries [69]. The anthocyanidin content of the berry increases with its maturation stage, and is highest in ripe berries. In general, the darker the berry, the higher its anthocyanidin content and the higher its antioxidant potential. Genetics has a major effect on anthocyanidin content in berries since different cultivars of a given berry type can vary significantly in anthocyanidin content [70]. Epidemiologic studies suggest that the consumption of anthocyanidins lowers the risk of cardiovascular disease, arthritis, diabetes, and cancer due, at least in part, to their antioxidant and anti-inflammatory activities [71]. In a recent review, we summarized studies on cancer preventative activities of the anthocyanins, including results from laboratory studies as well as data from human epidemiological studies [72]. This section of the chapter includes a discussion of both anthocyanidins and anthocyanins, because investigations have been conducted with pure anthocyanidins even though anthocyanins are the forms of these molecules found in nature. Laboratory studies have provided clues on molecular mechanism(s) by which anthocyanidins/anthocyanins inhibit carcinogenesis, but there is still much to be learned. Moreover, the relevance of the in vitro studies with anthocyanidins/ anthocyanins to the in vivo situation needs to be confirmed because the concentrations of anthocyanidins/anthocyanins used in in vitro studies are usually much higher than can be achieved in vivo.

30.2.2 Physiochemical Properties of Anthocyanidins

The basic structure of the anthocyanidins is shown in Scheme 30.3. The six most prevalent anthocyanidins in nature are pelargonidin, cyanidin, peonidin, delphinidin, malvidin, and petunidin. Individual berry types usually contain several different anthocyanidins; however, they tend to be enriched in one type or another. For example, blueberries are enriched in delphinidins, black raspberries in cyanidins, and strawberries in pelargonidins. As stated above, in nature, different anthocyanidins are commonly bound to sugars such as glucose, galactose, rhamnose, xylose, or arabinose; the resulting molecules are termed anthocyanins [72]. Sugar components of anthocyanins are usually conjugated to the anthocyanidin structure via the C3 hydroxyl group in ring C. Several hundred anthocyanins are known varying in the basic anthocyanidin skeleton and the position and extent to which the glycosides are attached to the skeleton [72]. In contrast to other ﬂavonoids, anthocyanins carry a positive charge in acidic solution. They are water soluble, and depending upon pH and the presence of chelating metal ions, they are intensely colored in blue, purple, or red. 518j 30 Selected Flavonoids

Scheme 30.3 Chemical structure of anthocyanidins [73].

30.2.3 Bioavailability and Metabolism of Anthocyanins

The bioavailability, pharmacokinetics of distribution, and metabolism of anthocyanins in animals and in humans have been summarized in a recent review [73]. In both animals and humans, anthocyanins are absorbed as intact glycosides rather than their component anthocyanidins, and their absorption and elimination is rapid. In general, the efﬁciency of their absorption is poor, often lower than 1% of the administered dose. Interestingly, the principal metabolites of anthocyanins in urine are methylated derivatives, only very small amounts of glucuronide or sulfate conjugates are found.

30.2.4 Mechanisms of Chemoprotection by Anthocyanidins and Anthocyanins

30.2.4.1 In Vitro Studies Most in vitro studies of the anticarcinogenic effects of anthocyanidins/anthocyanins have been conducted with human tumor cell lines. These studies are summarized in detail in a recent review article [72] and the reader is referred to this article for more detailed information. In vitro studies have used either pure anthocyanidins/anthocyanins for study, or they have employed extract fractions from various natural sources of these compounds. As illustrated in Table 30.2, Table 30.2 Anticarcinogenic effects of anthocyanidins/anthocyanins and anthocyanin-rich extracts in vitro.

Anthocyanidin/ Anticancer effect anthocyanin source Concentration Cells References

Antioxidation " ORAC value Six types of berry extracts 50 mg/ml Human endothelial cells [74] # ROS Concord grape extract 10–20 mg/ml Normal human breast cells [75]

Phase II enzyme activation " Glutathione-related enzymes Concord grape extract 10–20 mg/ml Normal human breast cells [75] " NAD(P)H:quinone reductase

Anticell proliferation # Cell proliferation Five anthocyanidins 12.5–200 mg/ml Human stomach, colon, [76] and ﬁve anthocyanins breast, and lung cancer cells

Induction of apoptosis " Caspase-3 and PARP activity Cyanidin 3-glucoside 10–100 mM Human breast, stomach, [77] and peonidin 3-glucoside liver, cervical, and lung cancer cells " Cytochrome c release Delphinidin 30–180 mM Human prostate cancer cells [78] " Caspases-3, -6, -8, and -9 02Anthocyanidins 30.2 Induction of differentiation " Activation of transglutaminase Black raspberry extract 100 mg/ml Human oral SCC cells [79] enzymes involved in keratin production " Granulocyte differentiation Cyanidin-3-O-beta- 25–225 mg/ml Human leukemic cells [80] glucopyranoside

(Continued) j 519 520

Table 30.2 (Continued) j 0Slce Flavonoids Selected 30 Anthocyanidin/ Anticancer effect anthocyanin source Concentration Cells References

Anti-inﬂammation # COX-1 and COX-2 Cyanidin-3-O-glucoside 12.5–200 mg/ml Human breast, colon, [81] stomach, and lung cancer cells # NF-kB activation Pomegranate extract 10–40 mg/ml Normal human [82] epidermal keratinocytes

Antiangiogenesis # VEGF Black raspberry extract 1–100 mg/ml Human oral SCC cells [79, 83] and mouse epidermal cells # Matrigel endothelial Six anthocyanidins and 1–25 mM Human umbilical [84] cell tube formation one anthocyanin vein endothelial cells

Anti-invasiveness # MMP-9 and uPA Extract from black rice, 50–200 mg/ml Human liver and cervical cancer cells [85] peonidin 3-glucoside, and cyanidin 3-glucoside (extract), 25–100 mM (anthocyanin) # Cell invasion and motility (Matrigel)

ORAC, oxygen-radical absorbing capacity; AP-1, activator protein 1; PARP, poly(ADP-ribose)polymerase; COX, cyclooxygenase; NF-kB, nuclear factor kappa B; PGE2, prostaglandin E2; VEGF, vascular endothelial growth factor; SCC, squamous cell carcinoma; MMP, matrix metalloproteinases; uPA, urokinase plasminogen activator. 30.2 Anthocyanidins j521

Figure 30.2 Schematic illustration of known molecular mechanisms that may be involved in the chemopreventive mechanisms of anthocyanidin/anthocyanin [72]. These compounds stimulate apoptosis and inhibit P13K, ERKs/p38k, and I8Ba pathways. anthocyanidins/anthocyanins and different extracts enriched in these compounds elicit multiple anticarcinogenic effects in vitro including antioxidation [74, 75], activation of phase II metabolizing enzymes [75], inhibition of cell proliferation [76], induction of apoptosis [77, 78] and cell differentiation [79, 80], and anti-inﬂammato- ry [81, 82], antiangiogenic [79, 83, 84], and anti-invasive [85] effects. The expression levels of relevant genes associated with these cellular processes are also inﬂuenced by these compounds/mixtures (see Table 30.2 and Figure 30.2). Anticarcinogenic effects of anthocyanidins are related to their structure and are due principally to the presence of hydroxyl groups in position 3 of ring C and in the 30,40,and50 positions of ring B in the molecule. For example, anticancer effects of delphinidin, with three hydroxyl groups on the B-ring, are superior to those of malvidin and peonidin that have only one hydroxyl group on the B-ring (Scheme 30.3). Anthocyanidins appear to be more potent inhibitors of carcinogenesis in vitro than anthocyanins [76], suggesting that sugars attached to the anthocyanidin nucleus interfere with its anticarcinogenic effects. Several investigations have compared the antiproliferative effects of anthocyanidins/ anthocyanins on normal versus cancer cells in vitro and found that these compounds selectively inhibit the growth of cancer cells [86, 87]. The mechanism(s) for this selectivity are unknown. 522j 30 Selected Flavonoids

The concentrations of anthocyanins/anthocyanidins or extract fractions required to elicit anticarcinogenic effects in vitro are much higher (10–200 mM) than the levels of these compounds observed in the blood of animals administered anthocyanin-containing diets or juices (2–25 nM). This may be due, in part, to the instability of anthocyanins at physiological pH. The half-life of most anthocyanins in culture medium at pH 7.0 is less than 5 h [88]. Results of in vitro studies with anthocyanidins are also difﬁcult to extrapolate to the in vivo situation because anthocyanidins are unstable in vivo and are generally not found in plasma, in urine, or in tissues [89]. Thus, perhaps the most appropriate use of the in vitro data in Table 30.2 is that of identifying possible mechanistic biomarkers for clinical investigations with anthocyanins or anthocyanin-containing foodstuffs.

30.2.4.2 Animal Studies Anthocyanidins/anthocyanins and fruit extracts enriched in these compounds have been shown to inhibit the development of multiple cancer types in carcinogen-treated animals and in animals with a hereditary predisposition to cancer. Table 30.3 presents results of some of the published studies; the reader is referred to a recent review article [72] for a more extensive discussion of the available data. In a rat model of squamous cell carcinoma of the esophagus, a diet containing an anthocyanin-rich extract from black raspberries was shown to reduce the number of N- nitrosomethylbenzylamine (NMBA)-induced esophageal papillomas [90]. This reduction was associated with the ability of the extract to inhibit cell proliferation, inﬂammation, and angiogenesis and to stimulate apoptosis. Lala et al. [91], using the azoxymethane (AOM)-induced colon cancer model in rats, showed that an anthocyanin-rich extract from bilberry, chokeberry, and grape administered in the diet signiﬁcantly reduced AOM-induced aberrant crypt foci. This reduction was associated with decreased cell proliferation and COX-2 gene expression. In the mouse skin cancer model, delphinidin and pomegranate extracts enriched in anthocyanins and tannins were shown to inhibit UVB- or TPA (12-O-tetradecanoylphorbol-13-acetate)-induced skin cancer development when applied topically to the skin. Delphinidin inhibited UVB-mediated DNA damage and pomegranate extracts modulated the mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-k B) pathways [82, 92]. Using the nude mouse xenotransplant model, cyanidin-3- glucoside [93] and anthocyanins from black rice [77] were shown to inhibit the development of tumors produced by subcutaneous injection of lung tumor cells. The mechanism of this inhibition was not investigated. In the rat mammary carcinogenesis model, diets containing 5% blueberry or black raspberry powders reduced the proliferation index in mammary tissues from estrogen-treated animals compared to control rats [94]. Finally, a juice prepared from blueberries, red grapes, raspberries, and elderberries reduced the size and cyclin D1 expression of tumors in nude mice produced after subcutaneous injection of human prostate cancer cells [95]. The positive inhibitory effects of these anthocyanin-rich preparations in the gastrointestinal (GI) tract and on the skin are probably mediated by the localized absorption of anthocyanins into the target tissues. However, observations that anthocyanins also protect against cancers of the lung, breast, and prostate following 30.2 Anthocyanidins j523

Table 30.3 Anticarcinogenic effects of anthocyanidins/ anthocyanins and anthocyanin-rich extracts in vivo.

Anthocyanidin/ Cancer types anthocyanin source Concentration Mechanisms References

Esophagus Anthocyanin-rich extract 3.5 mmol/g diet # Cell proliferation [90] from black raspberries " Apoptosis # Inﬂammation # Angiogenesis

Colon Anthocyanin-rich extract 3.85 g/kg diet # Cell proliferation [91] from bilberry, chokeberry, and grape # COX-2 expression

Skin Delphinidin 1 mg/topical Inhibition of [82] application UVB-mediated apoptosis and DNA damage Anthocyanin- and 2 mg/topical Modulation of MAPK [92] hydrolysable-tannin-rich application and NF-kB pathways pomegranate extracts

Lung Cyanidin-3-glucoside 9.5 mg/kg i.p. ND [93] injection Anthocyanins from 0.5% (wt/wt) by ND [77] black rice oral gavage

Breast Blackberries and blueberries 5% in diet # Cell proliferation [94] Prostate Fruit juice from blueberries, 10% in drinking # Cell proliferation [95] red grapes, raspberries, water and elderberries

MAPK, mitogen-activated protein kinases; i.p., intraperitoneal; ND: not determined. oral administration suggest that these compounds are absorbed in sufﬁcient quantities to be protective in internal organs.

30.2.5 Human Studies

Epidemiological studies in humans regarding the effects of anthocyanidins on cancer risk have been summarized [72]. These studies have not provided convincing evidence of the anticancer effects of anthocyanidins. For example, in Italy, studies of the effects of dietary anthocyanidins on the risk of oral, pharyngeal, and prostate cancers failed to demonstrate protective effects, and supplementation of anthocyanins in the diet of cancer patients receiving chemotherapy did not result in increased inhibition of tumor development compared to chemotherapy alone. 524j 30 Selected Flavonoids

Although epidemiological studies have not shown that anthocyanin intake reduces cancer risk in humans, they suggest that anthocyanin intake may reduce certain parameters of oxidative stress. For example, a study in Germany showed that individuals who consumed an anthocyanin/polyphenolic-rich fruit juice had reduced oxidative DNA damage and a signiﬁcant increase in reduced glutathione compared to controls [96]. In an investigation involving Barretts esophagus patients, the oral administration of freeze-dried black raspberry powder (which contains about 5–7% anthocyanins) in a slurry of water daily for 6 months reduced levels of 8-epi- prostaglandin F2a (8-Iso-PGF2) and 8-OHdG in urine [72].

30.2.6 Impact of Processing on the Stability of Anthocyanins

Anthocyanins can be destroyed during the processing of fruits and vegetables [69]. Temperature has been reported to produce a logarithmic destruction of anthocyanin pigments with time of heating at a constant temperature. Light also has deleterious effects on anthocyanin stability, and light exposure of anthocyanin-containing beverages should be avoided. Interestingly, adding sugar to anthocyanin-containing beverages stabilizes the compounds because it inhibits the action of degradative enzymes that remove the glucosides and interferes with condensation reactions.

30.2.7 Conclusions

Anthocyanidins/anthocyanins have been shown to exhibit anticarcinogenic activity against multiple cancer cell types in vitro and tumor types in vivo. Potential cancer chemopreventiveactivitiesofanthocyaninsrevealedfrominvitrostudiesincluderadical scavenging activity, stimulation of phase II detoxifying enzymes, reduced cell proliferation, inﬂammation, angiogenesis, and invasiveness, and induction of apoptosis and differentiation.Anthocyanins modulatethe expressionand activation of multiple genes associated with these cellular functions including genes involved in PI3K/Akt, ERK, JNK, and MAPK pathways (Figure 30.2). In vivo studies have shown that dietary anthocyanins inhibit cancers of the gastrointestinal tract, and topically applied anthocyanins inhibit skin cancer, likely due to localized absorption of the compounds. Pharmacokineticdataindicatethattheabsorptionofanthocyaninsintothebloodstream of rodents (and humans) is minimal; however, dietary administration of these compounds appears to inhibit breast cancer in rats and tumors produced in nude mice by xenotransplantationof lungandprostatecancercells.Thus,inthese model systems,the systemicdeliveryofabsorbedanthocyaninsappearstobesufﬁcienttoconferprotection. Although experimental studies have clearly demonstrated the anticancer activity of anthocyanins, epidemiological studies have not revealed protective effects in humans. This may be due to complexities associated with accurately determining anthocyanin consumption in human populations relative to other dietary factors. Future studies aimed at enhancing the absorption of anthocyanins and/or their metabolites may be necessary for their optimal use in the chemoprevention of human cancer. 30.3 Proanthocyanidins j525

30.3 Proanthocyanidins Clarissa Gerh€auser

30.3.1 Introduction

Proanthocyanidins (PAs), also known as condensed tannins, represent the second most abundant class of plant polyphenols after lignin [97]. PAs are widely distributed in plants and plant-derived food, especially in fruit, legume seeds, cereal grains, and a variety of beverages including wine, beer, tea, cocoa, and cider [98]. PAs in food affect parameters such as astringency, bitterness, sourness, sweetness, salivary viscosity, aroma, and color formation [99]. They are of interest in nutrition and medicine because of their potent antioxidant capacity and possible protective effects on human health in reducing the risk of chronic diseases such as cardiovascular diseases and cancers (reviewed in Refs [98, 99]). The name proanthocyanidins is based on the formation of colored anthocyanidins upon heating in acidic solution. Synonyms for proanthocyanidins used in the literature are condensed tannins, vegetable tannins, flavans, flavolans (2–10 catechin units), polyflavans, catechins, macromolecular phenolic substances, leucoanthocyanidins, condensed proanthocyanidins, polymeric proanthocyanidins, oligomeric proanthocyanidins, procyanidins, procyanidolic oligomers, plant polyphenols, pycnogenols, flava- nolicoligomers,flavanotannins,andcondensedflavonoid-tanningsubstances[99,100].

30.3.2 Physicochemical Properties and Occurrence

PAs are di-, tri-, and oligomeric condensation products of flavan-3-ol monomers (Scheme 30.4). Dimeric PAs of the so-called B-type have one 4 ! 6or4! 8 interflavan linkage, whereas A-type PAs are more rigid and the monomer subunits are further linked by an unusual second ether linkage. C-type PAs are trimers composed of three flavan-3-ol subunits with 4 ! 6or4! 8 bonds [98]. Proantho- cyanidins are divided into 15 subclasses, depending on their monomer composition [99, 101]. Only three classes appear to be prominent in human nutrition (listed in Ref. [102]): procyanidins, composed of catechin and epicatechin monomers; prodelphinidins, additionally consisting of gallocatechin and epigallocatechin subunits; and propelargonidins, which are (epi)afzelechin polymers ([101]; summary in Ref. [99]). Generally, soluble PA oligomers possess an average molecular weight of 1000–6000, but values up to 20 000 have also been reported [99]. PAs generally possess 12–16 phenolic OH-groups and 5–7 aromatic rings per 1000 units of relative molecular mass.

30.3.2.1 Physicochemical Properties

30.3.2.1.1 Interaction with Proteins Tannins are generally capable to form unspe- ciﬁc complexes with proteins and to hide leather. 526j 30 Selected Flavonoids

Scheme 30.4 Structures of proanthocyanidin dimers and trimers of the A-, B-, and C-type. Some typical proanthocyanidins are procyanidin A1 [epicatechin-(4b ! 8, 2b ! O ! 7)-catechin], procyanidin B1 [epicatechin-(4b ! 8)-catechin], and procyanidin b ! b ! C1[epicatechin-(4 8)-epicatechin-(4 8)-epicatechin].

Astringency associated with (unripe) fruit, beverages (such as wine, cider, tea, and beer) and bitterness of chocolate, results from the interaction of PAs with salivary proline-rich proteins [99]. This characteristic property also provides protection against herbivores and pathogens (bacteria, fungi, yeast, and viruses) [98]. Reaction with proteins, which is directly related to the degree of oligomerization, is also important for wine making [100]. PA-protein interaction is a dynamic, generally reversible surface phenomenon driven by hydrophobic effects, which is reinforced by the establishment of hydrogen 30.3 Proanthocyanidins j527 bonds. Phenolic hydroxyl groups of PAs serve as proton donors and carbonyl groups of the peptide bonds as proton acceptors. Proline-rich proteins (such as collagen, gelatine, and salivary proteins) were found to have highest binding affinity through hydrophobic interaction. Also, carbohydrate residues in salivary glycoproteins may enhance affinity and specificity of the interaction. Binding is also influenced by the molecular weight, the degree of galloylation, and the pattern of hydroxylation. Larger PAs with a degree of polymerization (DP) of more than 3 and the presence of galloyl groups increase the affinity for proteins. Unspecific complexation of mucosal proteins may also account for the antidiarrhoeal effect of tannins. Interaction of PAs with digestive enzymes or dietary proteins may lower net protein utilization and results in reduced weight gain. This is partly counteracted by enhanced secretion of digestive enzymes and especially salivary proteins as a defense mechanism (reviewed in Ref. [98]).

30.3.2.1.2 Antioxidant and Radical Scavenging Capacity PAs like all polyphenols are able to scavenge reactive oxygen species (ROS), including singlet oxygen as well as superoxide anion-, hydroxyl-, peroxyl-, and nitric oxide radicals, through electron- donating properties and generation of relatively stable phenoxyl radicals, which are generally less harmful than the initial radical species. PAs formed from (epi) gallocatechin units with 30,40,50-trihydroxyphenyl B-rings (prodelphinidins) are more efﬁcient radical scavengers than PAs with o-dihydroxyphenyl-substituted B-rings (procyanidins). A-type PAs with two bonds between monomer units are less potent antioxidants than B-type PAs. No clear conclusion could be drawn whether the degree of polymerization has an effect on radical-scavenging activity or not [98].

30.3.2.1.3 Complexation of Metal Ions PAs with o-dihydroxyphenyl groups in the B-ring (e.g., procyanidins) very effectively complex Fe(III) as well as Al(III) and Cu (II). This has biological consequences as PAs have been shown to lower bioavailability of nonheme dietary iron through the gut barrier [98]. On the other hand, ﬂavo- noid–metal complexes apparently are more effective in scavenging superoxide anion radicals than the parent ﬂavonoids. Also, reduction of free iron and copper levels may lower the risk of hydroxyl radical formation through Fenton or Haber–Weiss reactions [99].

30.3.2.2 Occurrence PAs are widespread in plants and occur in fruits, berries, bark, leaves, seeds, nuts, and cereal grains. Quantitative data based on a systematic determination of PA mono- to decamerswithnormalphaseHPLCbyGuetal.[97,103]becameavailableonlyrecently and have been summarized in a comprehensive database [102]. As indicated in Figure30.3,mostimportantdietarysourcesofPAsarefruitsandberries(chokeberries (Aroniamelanocarpa),cranberries,blueberries, apple),nuts,grains, andspicessuch as cinnamon and cocoa (dark chocolate), whereas their content in vegetables is generally low [97, 103]. Cultivar and genetic background are major factors affecting PA proﬁle and content. For example, PA content in wild lowbush blueberries is about twofold higherthanincultivated highbushblueberries. MostfoodsourcescontainB-typePAs. 528j 30 Selected Flavonoids

Figure 30.3 Proanthocyanidin content of fruits (left), nuts, cereal and pulses (middle), spices, and supplements (right); adapted from Ref. [103]. Sorghum is a type of grass, which is an important food crop in less-developed regions of the world.

Only cranberry, peanut, plum, avocado, and curry were found to contain A-type PAs, and some novel structures were found in cinnamon [97, 99].

30.3.3 Bioavailability and Metabolisms [97–99,104–107]

It has been estimated that daily PA consumption varies from 10 to 500 mg, depending on dietary habits, age, and gender [99]. Gu et al. [103] reported an average PA intake in the United States of about 60 mg/day/person. About 74% of the ingested PAs possess aDP> 3 [103]. Of all ﬂavonoids, PAs appear to be the least well absorbed. They are relatively stable in the gastric juice [106, 107]. Bioavailability of small PAs up to trimers has 30.3 Proanthocyanidins j529 been reported [97]. Whereas sulfated metabolites and procyanidin dimers have been detected in urine, only catechin glucuronides and methylated glucuronide metabolites were measurable in plasma, kidney, and liver [99]. PAs with a DP > 3 are generally poorly bioavailable due to their molecular size, low permeability through paracellular absorption, and their likely complexation with luminal and mucosal proteins [99]. In vitro incubations with human colonic microﬂora suggest that PAs will be catabolized into low-molecular-weight phenolic acids such as mono- and dihydroxy-phenylacetic acid, hydroxyphenylpropionic acid, and phe- nylvaleric acid when they reach the colon. These phenolic acids will then be absorbed, further metabolized by conjugation, and excreted in urine [106]. They may exert bioactivities such as antioxidant activity depending on their substitution pattern. It has been observed that PA intake alters fecal bacterial composition in the rat gastrointestinal tract. A transient shift toward tannin-resistant Gram-negative Enterobacteria and Bacteroides species after 3 weeks of 0.7 and 2% PA diets has been reported [97].

30.3.4 Mechanisms of Protection: Results of In Vitro and Animal Studies

30.3.4.1 In vitro Antioxidant Activity As outlined above, PAs possess potent antioxidant activity, based on several mechanisms including direct radical-scavenging effects, chelation of transition metals, and inhibition of enzymes through protein interactions. A summary of in vitro antioxidant effects of PAs from various sources is given in Aron and Kennedy [99]. Antioxidant capacity in vivo will depend on factors such as bioavailability, absorption through the gut barrier, and metabolism. Antioxidant effects may be relevant locally in the gut, or systemically after absorption of small PAs or PA metabolites. Any conjugation of the phenolic hydroxyl groups with a sulfate, glucuronide, or methyl group will lower reducing capacity [104].

30.3.4.2 Potential Cancer Chemopreventive Activity in Cell Culture Potential cancer chemopreventive activities, which were detected in vitro in cell culture experiments, of a series of PA-enriched plant extracts derived from grape seed, cocoa, pine bark, hazel bark, cranberries, blueberries, apple, and Japanese quince are summarized in Table 30.4. PA-enriched extracts were shown to exert potential cancer-protective effects via antigenotoxic activity, modulation of enzyme activities related to detoxiﬁcation, hormone metabolism, inﬂammation, oxidation and reduction, regulation of cell cycle progression and signal transduction pathways of cell growth and proliferation, induction of apoptosis, stimulation of immune functions and DNA repair, suppression of oncogenes and tumor formation, and antiangiogenic mechanisms [99, 105, 108–111]. It is worth noting that a cocoa PA pentamer selectively inhibited the growth of breast cancer cell lines, whereas normal mammary cells were more resistant. Similar effects were reported for a cocoa PA extract on prostate (cancer) cell lines 530 j 0Slce Flavonoids Selected 30 Table 30.4 Overview of potential anticancer effects of proanthocyanidin-enriched preparations in vitro in cell culture.

Source Species, organ (cell lines) Effects Author, year References

Grape seedsa (Vitis vinifera, Vitaceae) GSPE Human oral keratinocytes Protection from tobacco-induced oxidative Bagchi, 1999 [108] stress GSPE Human umbilical vein endothelial cells TNF-a-induced VCAM expression # Sen, 2001 [105] (HUVEC) GSPE Human umbilical vein endothelial cells Proliferation #, DNA-synthesis #, apopto- Agarwal, 2004 [110] (HUVEC) sis ", antiangiogenic effects: MMP-2 secretion #, capillary tube formation on matrigel # Proanthocyanidin extract Human breast cancer (MCF-7), lung Cytotoxicity, Bcl-2 ", c-Myc #, growth en- Ye, 1999 [108, 110] (IH636) cancer (A-427), gastric adenocarcinoma hancement of normal gastric mucosa cells (CRL 1739) GSPE Chang liver cells, established from Protection from chemotherapy-induced Joshi, 1999, 2000 [99, 108] nonmalignant human tissue hepatotoxicity, regulation of expression of apoptosis-related proteins p53, Bcl-2, c-Myc GSPE Rat hepatoma (FaO) Antigenotoxic, prevention of H2O2-in- Llópiz, 2004 [99] duced DNA damage GSPE Mouse hepatoma (Hepa1c1c7) Antioxidant, antiproliferative activity Matito, 2003 [99] GSPE Rat colon cancer (RCN-9) Caspase 3 activity ", apoptosis " Nomoto, 2004 [99] OPC Human colorectal cancer (SNU-C4) Bax ", Bcl-2 mRNA #, caspase 3 level and Kim, 2005 [111] activity ", apoptosis " Procyanidin dimers B1, B2 Human colon cancer (HT-29) No effect on AP-1 reporter activity Jeong, 2004 [99] GSPE Human prostate cancer (DU145) G1 cell cycle arrest ", apoptosis ", dissi- Agarwal, 2000, 2002 [99] pation of mitochondrial membrane potential GSPE Human prostate cancer (DU145) Inhibition of NF-kB, apoptosis " Dhanalakshmi, 2003 [99] GSPE Human prostate cancer (LNCaP) ATM kinase ", Chk2 ", anoikis and Kaur, 2006 [99] apoptosis " via ROS production GSPE Human prostate carcinoma (DU145) MAPK and NF-kB signaling #, MMP-2 and Vayalil, 2004 [110] -9 expression # 0 Procyanidin B2-3,3 -di-O- Human prostate carcinoma (DU145) Proliferation #, apoptosis " (galloyl groups Agarwal, 2007 [121] gallate are important for antiproliferative activity) GSPE Human breast cancer (MCF-7) Protection from peroxyl radicals, Faria, 2006 [99] antiproliferative Gravinol (GSPE) Mouse mammary carcinoma (4T1) human Proliferation #, apoptosis ", Bax/Bcl-2 ", Mantena, 2006 [110] breast cancer (MDA-MB-468, MCF-7) caspase 3 activity " Gravinol (GSPE) Human epidermoid carcinoma (A431), NF-kB/p65 and IKKa expression #, MAPK Meeran and Katiyar, 2008 [110] human keratinocytes signaling #, levels of proinﬂammatory proteins # GSPE Human colon cancer (CaCo2), normal Proliferation #, apoptosis " in cancer cells Engelbrecht, 2007 [122] colon cells (NCM460) but not in normal cells. PI3-kinase/PKB survival-related pathway #

Cocoa (Theobroma cacao L., Malvaceae) B and C type procyanidins Phosphatidyl cholin liposomes Lipid peroxidation #, antioxidant protec- Verstraeten, 2005 [99] tion of cell membranes Procyanidin pentamers Human aortic endothelial cells ErbB-2 expression #, angiogenesis # Kenny, 2004 [99] Procyanidin pentamer Various human breast cancer and normal Antiproliferative, depolymerization of mi- Ramljak, 2005 [99] mammary cell lines tochondrial membranes, G1-modulatory proteins CdC-2 #, p53 #, normal cell lines Proanthocyanidins 30.3 are more resistant Human prostate cancer (DU145, 22Rv1) Growth inhibition, but not of normal Jourdain, 2006 [99] and normal cells (RWEP-1) prostate cells

Pine bark (Pinus sylvestris L., Pinaceae) Pycnogenol endothelial cells (ECV 304) Protection from oxidative stress by reactive Virgili, 1998 [99]

nitrogen species j (Continued) 531 532

Table 30.4 (Continued) j 0Slce Flavonoids Selected 30

Source Species, organ (cell lines) Effects Author, year References

Pycnogenol Human keratinocytes (HaCaT), murine NF-kB-dependent gene expression # Packer, 1999 [99] macrophages (Raw264.7) Procyanidin dimers B1,B2 Murine macrophages (Raw264.7) Little effect on NF-kB-dependent gene Park, 2000 [99, 111] expression Procyanidin trimer C2, Murine macrophages (Raw264.7) NF-kB dependent gene expression " Park, 2000 [99, 111] pycnogenol Procyanidin dimers B1,B2 Murine macrophages (Raw264.7) No effect on NF-kB-dependent gene Jeong, 2004 [99] expression Gallate-free procyanidins Antioxidant activity, radical scavengers Tourino, 2005 [99]

Hazel bark (Hamamelis virginiana, Hamamelidaceae) Galloylated procyanidin Human keratinocyte cultures Proliferation " Deters, 2001 [109] fractions 1% Procyanidins in topic Human skin Prevention of SDS-induced skin irritation Deters, 2001 [109] formulation Polymeric procyanidin frac- Human hepatoma cells (HepG2) B(a)P-induced DNA damage # by scav- Dauer, 2003 [109] tions (DP 16–28) enging of B(a)P metabolites or ROS formed during activation

American cranberry (Vaccinium macrocarpon, Ericaceae) OPC Mouse keratinocytes (308) TPA-induced ODC activity #, antioxidant Kandil, 2002 [111] activity

Blueberry (Vaccinium myrtillus, V. angustifolium, Ericaceae) Human keratinocytes (HaCaT) VEGF expression #, angiogenesis # Roy, 2002 [99] Procyanidins with mDP 5.65 Human prostate cancer, mouse liver Antiproliferative activity Schmidt, 2004 [99] cancer cells Human prostate cancer (LNCaP, DU145) Antiproliferative activity Schmidt, 2006 [99] Blueberry extract Human prostate cancer (DU145) MMP-2 and -9 expression # Matchett, 2005, 2006 [99, 110]

Apple (Malus · domestica, Rosaceae) B- and C-type procyanidins Human stomach cancer (KATO III) DNA fragmentation, apoptosis " via ROS Hibasami, 2004 [99] formation Procyanidin-enriched fraction Human colon cancer (SW620) Antiproliferative activity, PKC #, ERK ", Gosse, 2005, 2006 [99] ODC #, polyamine synthesis #, apoptosis " ACT Mouse melanoma (B16), mouse Proliferation #, apoptosis ", mitochondrial Miura, 2008 [123] mammary tumor cells (BALB-MC.E12) membrane permeability ", cytochrome c release ", caspase-3 and -9 activation (especially by PA pentamer and higher degree fractions)

Japanese Quince fruit (Chaenomeles japonica, Rosaceae) Procyanidin extract (trimer to Human peripheral blood mononuclear MMP-2 and -9 gelatinase activity # Strek, 2007 [110] oligomer) cells (PBMC), human leukemia (HL60)

Abbreviations: ACT, apple-condensed tannins consisting of PAs (80%) and monomers, ﬂavonoids and other polyphenols (20%) [123]; ATM, ataxia telangiectasia mutated; GSPE, grape seed proanthocyanidin-rich extract; IKK, I-kB (inhibitor of NF-kB) kinase; MAPK, mitogen-activated protein kinase; (m)DP, (mean) degree of polymerization; MMP, matrix metalloproteases; NF-kB, nuclear factor kappa B; ODC, ornithine decarboxylase; OPC, oligomeric procyanidins; PKC, protein kinase C; Pycnogenol, standardized pine bark water extract, consisting of procyanidin monomers and oligomers as well as phenolic acids as minor constituents [113]; ROS, reactive oxygen species; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoyl-13-acetate; SDS, sodium dodecyl sulfate; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; " activation, induction, upregulation; # inhibition, reduction, downregulation. 03Proanthocyanidins 30.3 j 533 534j 30 Selected Flavonoids

(summarized in Ref. [99]). Also, galloylated PA fractions from hazel bark (Hamamelis virginiana) induced proliferation of cultured human keratinocytes.

30.3.4.3 Cancer Chemopreventive Activity in Animal Models Cancer chemopreventive activities of PAs derived from grape seeds, cocoa, apple, and pine bark in animal models are summarized in Table 30.5 [97, 99, 105, 108, 110]. Earlier studies on grape seed proanthocyanidin-rich extract (GSPE) mainly reported prevention of chemically or UV-induced skin carcinogenesis after topical application or administration in the diet. In recent years, PA extracts have also been tested in chemically induced breast, colon, lung, and pancreatic carcinogenesis models, in various xenograft experiments with human or murine cancer cells injected or transplanted subcutaneously before PA intervention, as well as in a transgenic model for prostate cancer. Interestingly, the preventive effect of GSPE on 7,12-dimethylbenzo [a]anthracene (DMBA)-induced mammary carcinogenesis in the rat depended on the choice of rodent diet. A PA-enriched fraction derived from apple at very low concentrations (0.01% in drinking water) potently inhibited azoxymethane-induced cancer precursor lesions in rat colon (aberrant crypt foci) by 50%. Potent inhibition in the same model was also observed with GSPE (0.1–1% in the diet). Growth of cancer cells in xenograft models was consistently reduced by GSPE, and these effects were associated with induction of apoptosis. In the transgenic TRAMP model for prostate cancer, GSPE increased the number of preneoplastic prostate intraepithelial neoplasia (PIN), but reduced the number of prostate adenocarcinoma. This may indicate that the progression of PIN to adenocarcinoma is inhibited. Overall, these data indicate that although bioavailability of oligomeric PAs with a DP > 3 is limited, dietary intervention with PA extracts results in a signiﬁcant reduction of tumor growth, not only in colon but also in various other organs.

30.3.5 Results of Human Studies

Investigations on potential cancer preventive effects of PAs in humans are limited, whereas numerous studies on vascular- and cardioprotective effects, mainly by cocoa, red wine, pine bark, and grape seed extracts, as well as antimicrobial and urinary health effects, especially by cranberry juice, have been conducted. For an overview of these studies see Refs [97–99, 105, 107, 109, 111–115].

30.3.5.1 Short-Term Intervention Studies With respect to skin cancer prevention by PAs, similar to studies in animal models, topical application of 1% GSPE cream to human volunteers 30 min prior to UVA and UVB radiation slightly increased sun protection of the skin. This was attributed to radical-scavenging mechanisms of the PA extract [116]. In a double-blind crossover study by Record et al. [117] designed to measure fecal water antioxidant capacity and free radical production, 18 volunteers consumed high- and low-procyanidin chocolate for 4 weeks, separated by a 4-week washout period. Both interventions had no effect on direct antioxidant activity of fecal water, although Table 30.5 Overview of potential anticancer effects of proanthocyanidin-enriched preparations in vivo in animal models.

Source Species, organ Effects Author, year References

Grape seeds (Vitis vinifera, Vitaceae) Mouse Protection of TPA-induced ROS production in pe- Bagchi, 1998 [108] ripheral macrophages, lipid peroxidation #, and DNA fragmentation # in brain and liver Mouse Cytoprotection from drug-induced toxicity, Cyp2E1 # Bagchi, 2002 [99] Mouse Prevention of nicotine-DNA adducts Cheng, 2003 [99] GSPE topical Mouse skin TPA-induced ODC activity #, PKC activity #, DMBA/ Bomser, 1999, 2000; Zhao, [99, 105] TPA-induced skin tumorigenesis # 1999 GSPE, 0.2 or 0.5% in SKH-1 hairless mouse Prevention of UVB-induced photocarcinogenesis Mittal, 2003 [97, 110] AIN-76 diet (incidence, multiplicity and size), transformation to carcinoma #, lipid peroxidation #, tissue fat # GSPE, 0.2 or 0.5% in SKH-1 hairless mouse skin UVB-induced immune suppression (IL-10) #, IL-12 Sharma and Katiyar, 2006; [99, 110] AIN-76A diet ", reduced UVB-induced depletion of antioxidant Sharma, 2007 enzymes, inhibition of UVB-induced H2O2 and NO production, lipid peroxidation #, protein oxidation #, MAPK and NF-kB signaling # GSPE 0.1–1% in AIN- Rat, AOM-induced aberrant crypt foci 72–88% reduction in ACF, ODC activity # in distal Singletary and Meline, 2001 [97, 110] 93G diet (ACF) in colon colon GSPE 0.1–1% in AIN- Rat, DMBA-induced mammary No effect Singletary and Meline, 2001 [97, 110] 03Proanthocyanidins 30.3 93G diet tumors GSPE 5% in Teklad 4% Rat, DMBA-induced mammary 44% reduction in tumor numbers, no effect when Kim, 2004 [97] rodent diet tumors fed at 1. 5 or 5% in AIN-76A diet Procyanidin dimers Aromatase-transfected MCF-7 breast Hormone-dependent tumor growth #, aromatase Eng, 2003; Kijima, 2006 [99] cancer xenograft model expression and activity # Sarcoma 180 xenograft model Enhancement of doxorubicin toxicity, immune Zhang, 2005 [99] modulation

GSPE 0.2%, 0.5% Balb/c mice, 4T1 mouse mammary 30 and 52% reduction in tumor volume, apoptosis ", Mantena, 2006 [110] j 535 tumor cells transplanted s.c. lung metastases #, survival " (Continued) 536 Table 30.5 (Continued) j 0Slce Flavonoids Selected 30 Source Species, organ Effects Author, year References

GSPE 200 mg/kg b.w. by Human colorectal cancer (HT29) Proliferation #, apoptosis and PARP cleavage ", p21 Kaur, 2006 [110] gavage xenograft expression GSPE 200 mg/kg b.w. by Mouse prostate (TRAMP) 46% reduction in weight of genitourinary tract or- Raina, 2007 [110] gavage gans, PIN ", adenocarcinoma #, expression of PCNA, cyclins, and CDKs # GSPE 50 or 100 mg/kg Human epidermoid carcinoma (A431) Tumor growth #, mRNA expression of PCNA, cyclin Meeran and Katiyar 2008 [110] b.w. by gavage xenograft D1 #, NF-kB activity #

Cocoa (Theobroma cacao L., Malvaceae) CLPr (0.25% in oriental Rat PhiP-induced pancreatic carcinogenesis (initiation Yamagishi, 2002 [105] CRF-1 basal diet) stage) #, no effect on mammary cancer CLPr (0.25% in oriental Rat Lung carcinogenesis # in multiorgan carcinogenesis Yamagishi, 2003 [97] CRF-1 basal diet) model, no effect on small intestine, colon, kidney

Apple (Malus domestica, Rosaceae) Procyanidin-enriched Rats, AOM-induced ACF Colon cancer prevention: 50% ACF reduction Gosse, 2005 [99, 124] fraction (0.01% in drinking water) ACT, 1% in drinking Mice, B16 mouse melanoma cells Enhanced survival of B16-transplanted mice, >50% Miura, 2008 [123] water transplanted s.c. reduction in tumor volume, apoptosis " (TUNEL staining)

Pine bark (Pinus sylvestris L., Pinaceae) Trimer > dimer > monomer Mouse skin TPA-induced ODC activity # Gali, 1994 [99]

Abbreviations: ACF, aberrant crypt foci; ACT, apple condensed tannins – 80% PAs, 20% monomers, ﬂavonoids, and other polyphenols [123]; AOM, azoxymethane; CDK, cyclin-dependent kinase; CLPr, cacao liquor proanthocyanidins; DMBA, 7,12-demethylbenzo(a)anthracene; GSPE, grape seed proanthocyanidin-rich extract; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor kappa B; NO, nitric oxide; ODC, ornithine decarboxylase; PARP, poly(ADP-ribose)polymerase; PCNA, proliferating cell nuclear antigen; PhiP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PIN, prostate intraepithelial neoplasia; PKC, protein kinase C; ROS, reactive oxygen species; TPA, 12-O-tetradecanoyl-13-acetate; TUNEL, TdT-mediated dUTP-biotin nick end labeling; " activation, induction, upregulation; # inhibition, reduction, downregulation. 30.3 Proanthocyanidins j537 they lowered free radical production to a similar extent. These data demonstrated that factors in chocolate other than ﬂavan-3-ols and procyanidins may be responsible for the observed effects [117].

30.3.5.2 Epidemiological Studies Evaluation of associations between PA consumption and cancer incidence in epidemiological studies has been hampered by a lack of quantitative data on dietary levels of PAs and PA consumption. Only recently, Theodoratou and colleagues compared the intake of six major classes of flavonoids including PAs with the risk of colorectal cancer in a large prospective case–control study in Scotland with 1456 incident cases and 1456 population-based controls [118]. Overall, PA uptake of more than 45 mg/day (derived mainly from tea, apple, and red wine consumption) was associated with a statistically significant 22% reduction in colorectal cancer risk (odds ratio 0.78; 95% confidence interval: 0.63–0.96, P for trend 0.031). Cutlerandcoworkersinvestigateddietaryflavonoidintakeandriskoflung,colorectal, breast, pancreatic, and upper aerodigestive cancers among postmenopausal women in the Iowa Womens Health Study, a large prospective cohort study with 34 708 participants. Cancer occurrence was followed for 18 years from 1986 to 2004. PA intake was estimated from the USDA database [102]. After adjustment for multiple variables, lung cancerincidencewas25%lowerforthose20%of womenwiththehighestconsumption of PA compared to those women with lowest consumption (hazard ratio ¼ 0.75; 95% confidence interval: 0.57–0.97). These effects were even stronger in past and current smokers with highest PAuptake (34% reduction in lung cancer risk). None of the other tumor types was influenced by PA intake in this study [119].

30.3.6 Impact of Cooking, Processing, and other Factors on Protective Properties

Several reports indicate that PA content is influenced by food preparation and processing. PAs usually are concentrated in the peel of fruits or the bran of grains. Industrial food processing methods such as fruit peeling, dehulling of seeds, nuts and almonds, decortication and bolting of cereals, grinding, juice filtration, and berry maceration can decrease total PA content [106]. PAs are high in fresh fruit such as plums and grapes, but undetectable in prune (dried plum) and raisins (dried grape). This indicates that PAs are degraded during drying [97]. Also, long-term storage of apple juice at room temperature for 9 months resulted in a total loss of PA content [106]. Natural cloudy apple juice contains about 2.5-fold more PAs than processed clear apple juice. This was associated with a significant higher antioxidant capacity of cloudy apple juice than of clear juice [120]. Cooking also significantly influences PA levels in food. Simmering of Pinto beans in waterfor2 hstronglyreducedthePAlevels,especiallyoligo-andpolymers(Figure30.3). PAcontentsofbabyfoodspreparedfrom100%fruitcontainedlowerPAconcentrations than fresh fruit, and especially levels of oligomeric PAs were reduced [97]. As indicated above, cocoa and dark chocolate contain significant levels of PAs. Interestingly, PA content of cocoa is dramatically altered by processing. A method 538j 30 Selected Flavonoids

called Dutch processing, which comprises the alkalinization of the cleaned centers (nibs) of cocoa beans, was introduced about 200 years ago as a method to reduce bitterness in cocoa. Consequently, the PA content of milk chocolate, which is typically produced from a Dutch processed cocoa, is often much lower than PA amounts in dark chocolate, in which Dutch processed powder is used less frequently. Also, PA levels in defatted cocoa beans decline with fermentation of beans and with the temperature used for roasting of underfermented nibs [107].

30.3.7 Conclusions

PAs represent an abundant class of plant polyphenols. Information on dietary uptake has been limited due to a lack of quantitative data; therefore, their cancer preventive effects in humans may have been underestimated so far. PAs with a DP > 3 are most likely not bioavailable and will reach the colon, where they may exert a local biological effect prior to degradation by the gut microﬂora. Nevertheless, in animal models, dietary intervention with PA extracts results in a signiﬁcant reduction of tumor growth in various organs beside the colon.

References

References to Section 30.1

1 Sun, H., Tang, Y., Xiang, J., Xu, G., Zhang, 5 Markham, K.R. (1982) Techniques of Y., Zhang, H. and Xu, L. (2006) Flavonoid Identification, Academic Press, Spectroscopic studies of the interaction New York. between quercetin and G-quadruplex 6 Franke, A.A. and Markham, K.R. (1989) DNA. Bioorganic & Medicinal Chemistry Quercetin-3-O-a-[2-p-hydroxybenzoyl-4-p- Letters, 16, 3586–3589. coumaroylrhamnopyraranoside], an 2 Ferguson, L.R. and Denny, W.A. (2007) aglycone-like flavonol from Librocedrus Genotoxicity of non-covalent interactions: bidwillii. Phytochemistry, 28, 3566–3568. DNA intercalators. Mutation Research, 623, 7 Williams, C. (2006) Flavone and flavonol 14–23. O-glycosides, in Flavonoids: Chemistry, 3 Valant-Vetshera, K. and Wollenweber, E. Biochemistry, and Applications (eds O. (2006) Flavones and flavonols, in Andersen and K. Markham), Taylor and Flavonoids: Chemistry, Biochemistry, Francis Group, LLC, Boca Raton, FL, and Applications (eds O. Andersen and pp. 749–856. K. Markham), Taylor and Francis 8 US Department of Agriculture (2007) Group, LLC, Boca Raton, FL, pp. 617–748. USDA Database for the Flavonoid Content 4 Gould, K. and Lister, C. (2006) Flavonoid of Selected Foods, Release 2.1. functionsin plants, inFlavonoids: Chemistry, 9 Kyle, J. and Duthie, G. (2006) Flavonoids in Biochemistry, and Applications (eds O. food, in Flavonoids: Chemistry, Biochemistry, Andersen and K. Markham), Taylor and and Applications (eds O. Andersen and K. Francis Group, LLC, Boca Raton, FL, Markham), Taylorand Francis Group, LLC, pp. 397–442. Boca Raton, FL, pp. 219–262. References j539

10 Andersen, O. and Markham, K. (2006) Montella, M., Ramazzotti, V., Franceschi, Flavonoids: Chemistry, Biochemistry and S. and LaVecchia, C. (2007) Flavonoids and Applications, Taylor and Francis Group, the risk of renal cell carcinoma. Cancer LLC, Boca Raton, FL. Epidemiology, Biomarkers & Prevention, 16, 11 Harborne, J. and Mabry, T. (1982) The 98–101. Flavonoids: Advances in Research, Chapman 21 Hollman, P.C.H. (2004) Absorption, and Hall, New York. bioavailability, and metabolism of 12 Harborne, J., Mabry, T. and Mabry, H. flavonoids. Pharmaceutical Biology, 42, (1975) The Flavonoids: Part 1, Chapters 1 to 74–83. 11, Academic Press, New York. 22 Walle, T. (2004) Absorption and 13 Harborne, J.B. and Williams, C.A. (2000) metabolism of flavonoids. Free Radical Advances in flavonoid research since 1992. Biology & Medicine, 36, 829–837. Phytochemistry, 55, 481–504. 23 Manach, C. and Donovan, J.L. (2004) 14 Clifford, M. and Brown, J. (2006) Dietary Pharmacokinetics and metabolism of flavonoids and health: broadening the dietary flavonoids in humans. Free Radical perspective, in Flavonoids: Chemistry, Research, 38, 771–785. Biochemistry, and Applications (eds O. 24 Walle, T., Browning, A.M., Steed, L.L., Andersen and K. Markham), Taylor and Reed, S.G. and Walle, U.K. (2005) Francis Group, LLC, Boca Raton, FL, Flavonoid glucosides are hydrolyzed and pp. 319–370. thus activated in the oral cavity in humans. 15 Franke, A.A., Custer, L.J., Arakaki, C. and The Journal of Nutrition, 135,48–52. Murphy, S. (2004) Vitamin C and flavonoid 25 Manach, C., Williamson, G., Morand, C., levels of fruits and vegetables consumed in Scalbert, A. and Remesy, C. (2005) Hawaii. Journal of Food Composition and Bioavailability and bioefficacy of Analysis, 17,1–35. polyphenols in humans. I. Review of 97 16 Noroozi, M., Burns, J., Crozier, A., Kelly, bioavailability studies. The American I.E. and Lean, M.E. (2000) Prediction of Journal of Clinical Nutrition, 81, dietary flavonol consumption from fasting 230S–242S. plasma concentration or urinary excretion. 26 Booth, A.N., Murray, C.W., Jones, F.T. and European Journal of Clinical Nutrition, 54, DeEds, F. (1956) The metabolic fate of 143–149. rutin and quercetin in the animal body. The 17 Holden, J., Bhagwat, S. and Patterson, K. Journal of Biological Chemistry, 223, (2002) Development of a multi-nutrient 251–257. data quality evaluation system. Journal of 27 Schneider, H., Schwiertz, A., Collins, M.D. Food Composition and Analysis, 15,339–526. and Blaut, M. (1999) Anaerobic 18 Slimestad, R., Fossen, T. and Vagen, I.M. transformation of quercetin-3-glucoside by (2007) Onions: a source of unique dietary bacteria from the human intestinal tract. flavonoids. Journal of Agricultural and Food Archives of Microbiology, 171,81–91. Chemistry, 55, 10067–10080. 28 Gao, K., Xu, A., Krul, C., Venema, K., Liu, 19 Mitchell, A.E., Hong, Y.J., Koh, E., Barrett, Y., Niu, Y., Lu, J., Bensoussan, L., Seeram, D.M., Bryant, D.E., Denison, R.F. and N.P., Heber, D. and Henning, S.M. (2006) Kaffka, S. (2007) Ten-year comparison of Of the major phenolic acids formed during the influence of organic and conventional human microbial fermentation of tea, crop management practices on the content citrus, and soy flavonoid supplements, of flavonoids in tomatoes. Journal of only 3,4-dihydroxyphenylacetic acid has Agricultural and Food Chemistry, 55, antiproliferative activity. The Journal of 6154–6159. Nutrition, 136,52–57. 20 Bosetti, C., Rossi, M., McLaughlin, J.K., 29 Sawai, Y., Kohsaka, K., Nishiyama, Y. and Negri, E., Talamini, R., Lagiou, P., Ando, K. (1987) Serum concentrations of 540j 30 Selected Flavonoids

rutoside metabolites after oral 37 Le Marchand, L. (2002) Cancer preventive administration of a rutoside formulation to effects of flavonoids: a review. Biomedicine humans. Arzneimittel-Forschung, 37, & Pharmacotherapy, 56, 296–301. 729–732. 38 Cerutti, P.A. (1985) Prooxidant states and 30 Rechner, A.R., Kuhnle, G., Bremner, P., tumor promotion. Science, 227, 375–381. Hubbard, G.P., Moore, K.P. and Rice- 39 Moon, Y.J., Wang, X. and Morris, M.E. Evans, C.A. (2002) The metabolic fate (2006) Dietary flavonoids: effects on of dietary polyphenols in humans. Free xenobiotic and carcinogen metabolism. Radical Biology & Medicine, 33, Toxicology In Vitro, 20, 187–210. 220–235. 40 Fuhr, U. and Kummert, A. (1995) The fate 31 Gross, M., Pfeiffer, M., Martini, M., of naringenin in humans: a key to Campbell, D., Slavin, J. and Potter, J. (1996) grapefruit juice–drug interactions? The quantitation of metabolites of Clinical Pharmacology and Therapeutics, 58, quercetin flavonols in human urine. 365–373. Cancer Epidemiology, Biomarkers & 41 Sun, X.Y., Plouzek, C.A., Henry, J.P., Prevention, 5, 711–720. Wang, T.T. and Phang, J.M. (1998) 32 Rechner, A.R., Smith, M.A., Kuhnle, G., Increased UDP-glucuronosyltransferase Gibson, G.R., Debnam, E.S., Srai, S.K., activity and decreased prostate specific Moore, K.P. and Rice-Evans, C.A. (2004) antigen production by biochanin A in Colonic metabolism of dietary prostate cancer cells. Cancer Research, 58, polyphenols: influence of structure on 2379–2384. microbial fermentation products. Free 42 Mutoh, M., Takahashi, M., Fukuda, K., Radical Biology & Medicine, 36, 212–225. Komatsu, H., Enya, T., Matsushima- 33 Mennen, L.I., Sapinho, D., Ito, H., Hibiya, Y., Mutoh, H., Sugimura, T. and Bertrais, S., Galan, P., Hercberg, S. and Wakabayashi, K. (2000) Suppression by Scalbert, A. (2006) Urinary flavonoids and flavonoids of cyclooxygenase-2 promoter- phenolic acids as biomarkers of intake for dependent transcriptional activity in colon polyphenol-rich foods. The British Journal cancer cells: structure–activity of Nutrition, 96, 191–198. relationship. Japanese Journal of Cancer 34 de Vries, J.H.M., Hollman, P.C., Research, 91, 686–691. Meyboom, S., Buysman, M.N.C.P., Zock, 43 Raso, G.M., Meli, R., Di Carlo, G., Pacilio, P.L., van Staveren, W.A. and Katan, M.B. M. and Di Carlo, R. (2001) Inhibition of (1998) Plasma concentrations and urinary inducible nitric oxide synthase and excretion of the antioxidant flavonols cyclooxygenase-2 expression by flavonoids quercetin and kaempferol as biomarkers in macrophage J774A.1. Life Sciences, 68, for dietary intake. The American Journal of 921–931. Clinical Nutrition, 68,60–65. 44 Miodini, P., Fioravanti, L., Di Fronzo, G. 35 Franke, A.A., Custer, L.J., Wilkens, L.R., Le and Cappelletti, V. (1999) The two phyto- Marchand, L., Nomura, A.M.Y.,Goodman, oestrogens genistein and quercetin exert M.T., and Kolonel, L.N. (2002) Liquid different effects on oestrogen receptor chromatographic analysis of dietary function. British Journal of Cancer, 80, phytoestrogens from human urine and 1150–1155. blood. Journal of Chromatography B, 777, 45 Hung, H. (2007) Dietary quercetin 43–57. inhibits proliferation of lung 36 Radtke, J., Linseisen, J. and Wolfram, G. carcinoma cells. Forum of Nutrition, 60, (2002) Fasting plasma concentrations of 146–157. selected flavonoids as markers of their 46 So, F.V., Guthrie, N., Chambers, A.F., ordinary dietary intake. European Journal of Moussa, M. and Carroll, K.K. (1996) Nutrition, 41, 203–209. Inhibition of human breast cancer cell References j541

proliferation and delay of mammary multiethnic cohort study. American Journal tumorigenesis by flavonoids and of Epidemiology, 166, 924–931. citrus juices. Nutrition and Cancer, 26, 55 Le Marchand, L., Murphy, S.P., Hankin, 167–181. J.H., Wilkens, L.R. and Kolonel, L.N. 47 Plaumann, B., Fritsche, M., Rimpler, H., (2000) Intake of flavonoids and lung Brandner, G. and Hess, R.D. (1996) cancer. Journal of the National Cancer Flavonoids activate wild-type p53. Institute, 92, 154–160. Oncogene, 13, 1605–1614. 56 Peterson, J., Lagiou, P., Samoli, E., Lagiou, 48 Psahoulia, F.H., Moumtzi, S., Roberts, A., Katsouyanni, K., La Vecchia, C., Dwyer, M.L., Sasazuki, T., Shirasawa, S. and J. and Trichopoulos, D. (2003) Flavonoid Pintzas, A. (2007) Quercetin mediates intake and breast cancer risk: a preferential degradation of oncogenic Ras case–control study in Greece. British and causes autophagy in Ha-RAS- Journal of Cancer, 89, 1255–1259. transformed human colon cells. 57 McCann, S.E., Ambrosone, C.B., Moysich, Carcinogenesis, 28, 1021–1031. K.B., Brasure, J., Marshall, J.R., 49 Knekt, P., Kumpulainen, J., Jarvinen, R., Freudenheim, J.L., Wilkinson, G.S. and Rissanen, H., Heliovaara, M., Reunanen, Graham, S. (2005) Intakes of selected A., Hakulinen, T. and Aromaa, A. (2002) nutrients, foods, and phytochemicals and Flavonoid intake and risk of chronic prostate cancer risk in western New York. diseases. The American Journal of Clinical Nutrition and Cancer, 53,33–41. Nutrition, 76, 560–568. 58 Garcia-Closas, R., Gonzalez, C.A., Agudo, 50 Goldbohm, R.A., vant Veer, P., van den A. and Riboli, E. (1999) Intake of specific Brandt, P.A., vant Hof, M.A., Brants, carotenoids and flavonoids and the risk of H.A., Sturmans, F. and Hermus, R.J. gastric cancer in Spain. Cancer Causes & (1995) Reproducibility of a food frequency Control, 10,71–75. questionnaire and stability of dietary 59 Theodoratou, E., Kyle, J., Cetnarskyj, R., habits determined from five annually Farrington, S.M., Tenesa, A., Barnetson, repeated measurements. European Journal R., Porteous, M., Dunlop, M. and of Clinical Nutrition, 49, 420–429. Campbell, H. (2007) Dietary flavonoids 51 Adebamowo, C.A., Cho, E., Sampson, L., and the risk of colorectal cancer. Cancer Katan, M.B., Spiegelman, D., Willett, W.C. Epidemiology, Biomarkers & Prevention, 16, and Holmes, M.D. (2005) Dietary flavonols 684–693. and flavonol-rich foods intake and the risk 60 Gennaro, L., Leonardi, C., Esposito, F., of breast cancer. International Journal of Salucci, M., Maiani, G., Quaglia, G. and Cancer, 114, 628–633. Fogliano, V. (2002) Flavonoid and 52 Lin, J., Zhang, S.M., Wu, K., Willett, W.C., carbohydrate contents in Tropea red Fuchs, C.S. and Giovannucci, E. (2006) onions: effects of homelike peeling and Flavonoid intake and colorectal cancer risk storage. Journal of Agricultural and Food in men and women. American Journal of Chemistry, 50, 1904–1910. Epidemiology, 164, 644–651. 61 Crozier, A., Lean, M.E.J., McDonald, M.S. 53 Gates, M.A., Tworoger, S.S., Hecht, J.L., De and Black, C. (1997) Quantitative analysis Vivo, I., Rosner, B. and Hankinson, S.E. of the flavonoid content of commercial (2007) A prospective study of dietary tomatoes, onion, lettuce, and celery. flavonoid intake and incidence of epithelial Journal of Agricultural and Food Chemistry, ovarian cancer. International Journal of 45, 590–595. Cancer, 121, 2225–2232. 62 Makris, D.P. and Rossiter, J.T. (2001) 54 Nothlings, U., Murphy, S.P., Wilkens, L.R., Domestic processing of onion bulbs Henderson, B.E. and Kolonel, L.N. (2007) (Allium cepa) and asparagus spears Flavonols and pancreatic cancer risk: the (Asparagus officinalis): effect on flavonol 542j 30 Selected Flavonoids

content and antioxidant status. Journal of 70 Wang, S.Y. and Lin, H.S. (2000) Agricultural and Food Chemistry, 49, Antioxidant activity in fruits and leaves of 3216–3222. blackberry, raspberry, and strawberry 63 Price, K.R., Bacon, J.R. and Rhodes, M.J.C. varies with cultivar and developmental (1997) Effect of storage and domestic stage. Journal of Agricultural and Food processing on the content and Chemistry, 48, 140–146. composition of flavonol glucosides in 71 Harborne F J.B. and Grayer F R.J. (1994) onion (Allium cepa). Journal of Agricultural The anthocyanins, in The Flavonoids: and Food Chemistry, 45, 938–942. Advances in Research Since 1986 (ed. J.B. 64 Chu, Y.-H., Chang, C.-L. and Hsu, H.-F. Harborne), Chapman and Hall, London, (2000) Flavonoid content of several pp. 1–20. vegetables and their antioxidant activity. 72 Wang, L.-S. and Stoner, G.D. (2008) Journal of the Science of Food and Anthocyanins and their role in cancer Agriculture, 80, 561–566. prevention. Cancer Letters, 269 (2), 65 Franke, A.A., Custer, L.J. and Hundahl, 281–290. S.A. (2004) Determinants for urinary and 73 Prior, R.L. and Wu, X. (2006) plasma isoflavones in humans after soy Anthocyanins: structural characteristics intake. Nutrition and Cancer, 50 (2), that result in unique metabolic patterns 141–154. and biological activities. Free Radical 66 Maskarinec, G., Singh, S., Meng, L. and Research, 40, 1014–1028. Franke, A.A. (1998) Dietary soy intake and 74 Bagchi, D., Sen, C.K., Bagchi, M. and urinary isoflavone excretion among Atalay, M. (2004) Anti-angiogenic, women from a multiethnic population. antioxidant, and anti-carcinogenic Cancer Epidemiology, Biomarkers & properties of a novel anthocyanin-rich Prevention, 7, 613–619. berry extract formula. Biochemistry, 69, 67 Seow, A., Shi, C.-Y., Franke, A.A., Hankin, 75–80. J., Lee, H.-P. and Yu, M.C. (1998) 75 Singletary, K.W., Jung, K.J. and Giusti, M. Isoflavonoid levels in spot urine are (2007) Anthocyanin-rich grape extract associated with frequency of dietary soy blocks breast cell DNA damage Journal of intake in a population-based sample of Medicinal Food, 10, 244–251. middle-aged and older Chinese in 76 Zhang, Y., Vareed, S.K. and Nair, M.G. Singapore. Cancer Epidemiology, (2005) Human tumor cell growth Biomarkers & Prevention, 7, 135–140. inhibition by nontoxic anthocyanidins, the pigments in fruits and vegetables. Life Sciences, 76, 1465–1472. References to Section 30.2 77 Chen, P.N., Chu, S.C., Chiou, H.L., Chiang, C.L., Yang, S.F. and Hsieh, Y.S. 68 Hertog, M.G., Hollman, P.C., Katan, M.B. (2005) Cyanidin 3-glucoside and peonidin and Kromhout, D. (1993) Intake of 3-glucoside inhibit tumor cell growth and potentially anticarcinogenic flavonoids induce apoptosis in vitro and suppress and their determinants in adults in the tumor growth in vivo. Nutrition and Cancer, Netherlands. Nutrition and Cancer, 20, 53, 232–243. 21–29. 78 Mukhtar, H., Adhami, V.M., Afaq, F., 69 Delgado-Vargas, F., Jimenez, A.R. and Saleem, M., Sarfaraz, S. and Khan, N. Paredes-López, O. (2000) Natural (2008) Delphinidin induces apoptosis and pigments: carotenoids, anthocyanins, and growth inhibition of prostate cancer PC-3 betalains: characteristics, biosynthesis, cells via inhibition of Notch-1 and NF-kB/ processing, and stability. Critical Reviews in PI3K pathways. Proceedings of the Annual Food Science and Nutrition, 40, 173–289. Meeting of the American Association for References j543

Cancer Research, Los Angeles, CA, USA, (2006) Black rice anthocyanins inhibit vol. 48, #375. cancer cell invasion via repression of 79 Rodrigo, K.A., Rawal, Y., Renner, R.J., MMPs and u-PA expression. Chemico- Schwartz, S.J., Tian, Q., Larsen, R.E. and Biological Interactions, 163, 218–229. Mallery, S.R. (2006) Suppression of the 86 Hakimuddin, F., Paliyath, G. and tumorigenic phenotype in human oral Meckling, K. (2004) Selective cytotoxicity of squamouscellcarcinomacellsbyanethanol a red grape wine ﬂavonoid fraction against extract derived from freeze-dried black MCF-7 cells. Breast Cancer Research and raspberries.Nutrition andCancer,54,58–68. Treatment, 85,65–79. 80 Fimognari, C., Berti, F., Nusse,€ M., 87 Galvano, F., La Fauci, L., Lazzarino, G., Cantelli-Forti, G. and Hrelia, P. (2004) Fogliano, V., Ritieni, A., Ciappellano, S., Induction of apoptosis in two human Battistini, N.C., Tavazzi, B. and Galvano, leukemia cell lines as well as G. (2004) Cyanidins: metabolism and differentiation in human promyelocytic biological properties. The Journal of cells by cyanidin-3-O-beta- Nutritional Biochemistry, 15,2–11. glucopyranoside. Biochemical 88 Duthie, S.J., Jenkinson, A.M., Crozier, A., Pharmacology, 67, 2047–2056. Mullen, W., Pirie, L., Kyle, J., Yap, L.S., 81 Reddy, M.K., Alexander-Lindo, R.L. and Christen, P. and Duthie, G.G. (2006) The Nair, M.G. (2005) Relative inhibition of effects of cranberry juice consumption on lipid peroxidation, cyclooxygenase antioxidant status and biomarkers relating enzymes, and human tumor cell to heart disease and cancer in healthy proliferation by natural food colors. Journal human volunteers. European Journal of of Agricultural and Food Chemistry, 53, Nutrition, 45, 113–122. 9268–9273. 89 Francis, F.J. (1989) Food colorants: 82 Afaq, F., Malik, A., Syed, D., Maes, D., anthocyanins. Critical Reviews in Food Matsui, M.S. and Mukhtar, H. (2005) Science and Nutrition, 28, 273–314. Pomegranate fruit extract modulates UV- 90 Wang, L.S., Hecht, S., Carmella, S., Rocha, B-mediated phosphorylation of mitogen- C., Zikri, N., Henry, C., Larue, B., activated protein kinases and activation of McIntyre, C., Lechner, J.F. and Stoner, nuclear factor kappa B in normal human G.D. (2008) Prevention of rat esophageal epidermal keratinocytes. Photochemistry squamous cell cancer using extracts from and Photobiology, 81,38–45. freeze-dried black raspberries. 83 Huang, C., Li, J., Song, L., Zhang, D., Tong, Proceedings of the Annual Meeting of the Q., Ding, M., Bowman, L., Aziz, R. and American Association for Cancer Stoner, G.D. (2006) Black raspberry Research, Los Angeles, CA, USA, vol. 49, extracts inhibit benzo(a)pyrene diol- #3822. epoxide-induced, activator protein 1 91 Lala, G., Malik, M., Zhao, C., He, J., Kwon, activation and VEGF transcription by Y., Giusti, M.M. and Magnuson, B.A. targeting the phosphotidylinositol 3- (2006) Anthocyanin-rich extracts inhibit kinase/Akt pathway. Cancer Research, 66, multiple biomarkers of colon cancer in 581–587. rats. Nutrition and Cancer, 54,84–93. 84 Favot, L., Martin, S., Keravis, T., 92 Afaq, F., Saleem, M., Kueger, C.G., Reed, Andriantsitohaina, R. and Lugnier, C. J.D. and Mukhtar, H. (2005) Anthocyanin- (2003) Involvement of cyclin-dependent and hydrolyzable tannin-rich pathway in the inhibitory effect of pomegranate fruit extract modulates delphinidin on angiogenesis. MAPK and NF-kappaB pathways and Cardiovascular Research, 59, 479–487. inhibits skin tumorigenesis in CD-1 85 Chen, P.N., Kuo, W.H., Chiang, C.L., mice. International Journal of Cancer, 113, Chiou, H.L., Hsieh, Y.S. and Chu, S.C. 423–433. 544j 30 Selected Flavonoids

93 Ding, M., Feng, R., Wang, S.Y., Bowman, 100 Khanbabaee K. and van Ree T. (2001) L., Lu, Y., Qian, Y., Castranova, V., Jiang, Tannins: classification and definition. B.H. and Shi, X. (2006) Cyanidin-3- Natural Product Reports 18, 641–649. glucoside, a natural product derived from 101 Beecher, G.R. (2003) Overview of dietary blackberry, exhibits chemopreventive flavonoids: nomenclature, occurrence and chemotherapeutic activity. The and intake. The Journal of Nutrition, 133, Journal of Biological Chemistry, 281, 3248S–3254S. 17359–17368. 102 Nutrient Data Laboratory (2004) USDA 94 Ravoori, S., Vadhanam, M.V., Kausar, H., Database for the Proanthocyanidin Content Moktar, A., Vuppalapati, P.K., Schultz, D.J. of Selected Foods, US Department of and Gupta, R.C. (2008) Effect of select Agriculture, http://www.nal.usda.gov/ berries on estrogen-induced mammary fnic/foodcomp/Data/PA/PA.pdf. tissue proliferation. Proceedings of the 103 Gu, L.W., Kelm, M.A., Hammerstone, Annual Meeting of the American J.F., Beecher, G., Holden, J., Haytowitz, Association for Cancer Research, Los D., Gebhardt, S. and Prior, R.L. (2004) Angeles, CA, USA, vol. 49: #3821. Concentrations of proanthocyanidins in 95 Singh, J., Yao, M., Jardine, G. and Dong, common foods and estimations of Q. (2008) Retardation of prostate cancer normal consumption. The Journal of growth in vitro and in vivo by a dietary Nutrition, 134, 613–617. phytochemical cocktail. Proceedings of 104 Scalbert, A., Deprez, S., Mila, I., Albrecht, the Annual Meeting of the American A.M., Huneau, J.F. and Rabot, S. (2000) Association for Cancer Research, Los Proanthocyanidins and human health: Angeles, CA, USA, vol. 49: #5463. systemic effects and local effects in the 96 Rossi, M., Garavello, W., Talamini, R., gut. Biofactors, 13, 115–120. Negri, E., Bosetti, C., Dal Maso, L., Lagiou, 105 Cos, P., De Bruyne, T., Hermans, N., P., Tavani, A. et al. (2007) Flavonoids and Apers, S., Berghe, D.V. and Vlietinck, A.J. the risk of oral and pharyngeal cancer: a (2004) Proanthocyanidins in health care: case–control study from Italy. Cancer current and new trends. Current Epidemiology, Biomarkers & Prevention, 16, Medicinal Chemistry, 11, 1345–1359. 1621–1625. 106 Manach, C., Scalbert, A., Morand, C., Remesy, C. and Jimenez, L. (2004) Polyphenols: food sources and References to Section 30.3 bioavailability. The American Journal of Clinical Nutrition, 79, 727–747. 97 Prior, R.L. and Gu, L.W. (2005) Occurrence 107 Hackman, R., Polagruto, J., Zhu, Q., Sun, and biological significance of B., Fujii, H. and Keen, C. (2008) Flavanols: proanthocyanidins in the American diet. digestion, absorption and bioactivity. Phytochemistry, 66, 2264–2280. Photochemistry Reviews, 7,195–208. 98 Santos-Buelga, C. and Scalbert, A. (2000) 108 Bagchi, D., Bagchi, M., Stohs, S.J., Ray, Proanthocyanidins and tannin-like S.D., Sen, C.K. and Preuss, H.G. (2002) compounds: nature, occurrence, dietary Cellular protection with intake and effects on nutrition and health. proanthocyanidins derived from grape Journal of the Science of Food and seeds. Advances in Optical Biopsy and Agriculture, 80, 1094–1117. Optical Mammography, 957, 260–270. 99 Aron, P.M. and Kennedy, J.A. (2008) 109 Dixon, R.A., Xie, D.Y. and Sharma, S.B. Flavan-3-ols: nature, occurrence and (2005) Proanthocyanidins: a final frontier biological activity. Molecular Nutrition & in flavonoid research? New Phytologist, Food Research, 52,79–104. 165,9–28. References j545

110 Nandakumar, V., Singh, T. and Katiyar, colorectal cancer. Cancer Epidemiology, S.K. (2008) Multi-targeted prevention and Biomarkers & Prevention, 16, 684–693. therapy of cancer by proanthocyanidins. 119 Cutler, G.J., Nettleton, J.A., Ross, J.A., Cancer Letters, 269 (2), 378–387. Harnack, L.J., Jacobs, D.R. Jr, Scrafford, 111 Koleckar, V., Kubikova, K., Rehakova, Z., C.G., Barraj, L.M., Mink, P.J. et al. (2008) Kuca, K., Jun, D., Jahodar, L. and Opletal, Dietary flavonoid intake and risk of L. (2008) Condensed and hydrolysable cancer in postmenopausal women: the tannins as antioxidants influencing the Iowa Womens Health Study. health. Mini Reviews in Medicinal International Journal of Cancer, 123, Chemistry, 8, 436–447. 664–671. 112 Scalbert, A. (1991) Antimicrobial 120 Oszmianski, J., Wolniak, M., Wojdylo, A. properties of tannins. Phytochemistry, 30, and Wawer, I. (2007) Comparative study 3875–3883. of polyphenolic content and antiradical 113 Packer, L., Rimbach, G. and Virgili, F. activity of cloudy and clear apple juices. (1999) Antioxidant activity and biologic Journal of the Science of Food and properties of a procyanidin-rich extract Agriculture, 87, 573–579. from pine (Pinus maritima) bark, 121 Agarwal, C., Veluri, R., Kaur, M., Chou, pycnogenol. Free Radical Biology and S.C., Thompson, J.A. and Agarwal, R. Medicine, 27, 704–724. (2007) Fractionation of high molecular 114 Rasmussen, S.E., Frederiksen, H., weight tannins in grape seed extract and Struntze Krogholm, K. and Poulsen, L. identification of procyanidin B2-3,30-di- (2005) Dietary proanthocyanidins: O-gallate as a major active constituent occurrence, dietary intake, bioavailability, causing growth inhibition and apoptotic and protection against cardiovascular death of DU145 human prostate disease. Molecular Nutrition & Food carcinoma cells. Carcinogenesis, 28, Research, 49, 159–174. 1478–1484. 115 Williamson, G. and Manach, C. (2005) 122 Engelbrecht, A.M., Mattheyse, M., Ellis, Bioavailability and bioefficacy of B., Loos, B., Thomas, M., Smith, R., polyphenols in humans. II. Review of 93 Peters, S., Smith, C. et al. (2007) intervention studies The American Journal Proanthocyanidin from grape seeds of Clinical Nutrition, 81,243S–255S. inactivates the PI3-kinase/PKB pathway 116 Bagchi, D., Bagchi, M., Stohs, S.J., Das, and induces apoptosis in a colon cancer D.K., Ray, S.D., Kuszynski, C.A., Joshi, cell line. Cancer Letters, 258, 144–153. S.S. and Pruess, H.G. (2000) Free radicals 123 Miura, T., Chiba, M., Kasai, K., Nozaka, and grape seed proanthocyanidin extract: H., Nakamura, T., Shoji, T., Kanda, T., importance in human health and disease Ohtake, Y. et al. (2008) Apple procyanidins prevention. Toxicology, 148, 187–197. induce tumor cell apoptosis through 117 Record, I.R., McInerney, J.K., Noakes, M. mitochondrial pathway activation of and Bird, A.R. (2003) Chocolate caspase-3. Carcinogenesis, 29, 585–593. consumption, fecal water antioxidant 124 Gosse, F., Guyot, S., Roussi, S., Lobstein, activity, and hydroxyl radical production. A., Fischer, B., Seiler, N. and Raul, F. Nutrition and Cancer, 47, 131–135. (2005) Chemopreventive properties of 118 Theodoratou, E., Kyle, J., Cetnarskyj, R., apple procyanidins on human colon Farrington, S.M., Tenesa, A., Barnetson, cancer-derived metastatic SW620 cells R., Porteous, M., Dunlop, M. et al. (2007) and in a rat model of colon carcinogenesis. Dietary flavonoids and the risk of Carcinogenesis, 26,1291–1295. j547

31 Phytoestrogens

31.1 Isoflavones: Sources, Intake, Fate in the Human Body, and Effects on Cancer Alicja Mortensen, Sabine Kulling, Heidi Schwartz, and Gerhard Sontag

31.1.1 Introduction

Over the last two decades the biological effects of the soy isoflavones daidzein, genistein, and glycitein on human health have been extensively investigated. Development of analytical methods made the determination of isoflavone contents in foods and dietary supplements and the creation of several isoflavone databases possible, thereby allowing an estimation of the daily intake of isoflavones in different populations or in their subgroups. Epidemiological studies and investigation of the metabolism, kinetics, and effects in in vivo and in vitro systems yielded a wealth of in part consistent, in part contradictory evidence of potential beneficial health effects. In this section, typical dietary isoflavone sources are listed, the intake in different populations is discussed, information on absorption, distribution, metabolism, excretion, and pharmacokinetics is presented and finally a short insight into the current knowledge of isoflavone effects on breast, prostate, and intestinal cancers is given.

31.1.2 Dietary Isoflavone Sources

The main food sources of isoflavones are traditional soy foods such as tofu or soy milk, new-generation soy products such as soy desserts or soy burgers and commonly consumed foodstuffs in the production of which soy flour or soy protein isolates are used [1–5]. Isoflavone concentrations in the former two food groups cover a wide range (Table 31.1), which is due to the high natural variability of the isoflavone content in soybeans and due to the variability introduced by processing [6, 7].

Table 31.1 Contents of the isoflavones daidzein, genistein and glycitein in soy products.

Product Total isoflavones (mg/100 g)

Soy ﬂour 60–265 Tofu 5.1–64 Soy milk 1.3–21 Miso 23–126 Natto 20–124 Tempeh 6.9–63 Soy sauces 0.1–23 Soy burgers 0.1–26 Soy yogurt 1.6–12 Soy milk drinks 1.0–11 Soy cheeses 2.3–33

References to the data sources are summarized in Ref. [8].

Commonly and frequently consumed foodstuffs containing variable, but generally low, concentrations of total isoflavones (usually <2 mg/100 g) are bakery products such as bread and muffins, meat products such as sausages, and canned food items such as tuna or meatless chili [3, 9, 10]. Nonsoy foods containing low concentrations of isoflavones are other legumes, cereals (high microgram/kilogram range), fruits, vegetables, and nuts (low-to-medium microgram/kilogram range) [4, 11–14]. Infant formulas, however, may contain high concentrations of total isoflavones (up to 30 mg/100 g in instant products) [5, 15]. Highest isoflavone concentrations are found in nutritional supplements that may consist of up to 20% isoflavones [16]. Isoflavone supplements are commonly manufactured from extracts of soy and red clover and in some cases also from kudzu root, adding more isoflavones (formononetin and biochanin A from red clover, puerarin from kudzu). In unprocessed soybeans and red clover, isoflavones occur as malonylglucosides, glucosides, and, to a lesser degree, as free aglucones. Processing such as heat treatment or fermentation causes changes in the isoflavone conjugation pattern resulting in increased contents of acetylglucosides, glucosides, and aglucones [17]. The structures of conjugated and free isoflavones are given in Scheme 31.1.

31.1.3 Dietary Intake of Isoflavones

Recent estimates indicate isoflavone intakes of 15–50 mg (expressed as aglycone equivalents) per day in Asian countries. The mean daily isoflavone intake among older Japanese adults ranges from 25 to 50 mg. Intake in Korea, Hong Kong, and Singapore is lower than in Japan, and significant regional intake differences exist for China [18]. Isoflavone intake in Western countries does not usually exceed 1–3 mg/ day since soy is not commonly consumed in Western diets. However, certain subgroups of the Western population have a higher intake of isoflavones: vegetarians 31.1 Isoflavones: Sources, Intake, Fate in the Human Body, and Effects on Cancer j549

Scheme 31.1 Structures of isoflavones. and soy consumers (3–12 mg/day), soy formula-fed infants (approximately 40 mg/day), and consumers of soy or red clover-based dietary supplements (20–150 mg/day) [8, 19]. Up to now data on isoﬂavone intakes in Eastern Europe do not exist.

31.1.4 Absorption, Distribution, Metabolism, and Excretion of Isoflavones

The absorption, distribution, metabolism, and excretion (ADME) as well as the bioavailability of isoﬂavones have not yet been fully elucidated. Most of the information is available for isoﬂavones daidzein and genistein and to a lesser extent for glycitein.

31.1.4.1 Absorption After ingestion, conjugated isoflavones (malonyl- and acetylglucosides as well as glucosides) are hydrolyzed to their aglycones by lactase-phlorizin-hydrolase (LPH) in the apical membrane of the lumen of the small intestine and/or by bacterial intestinal glucosidases or glucuronidases of the human microbiota [20, 21]. Deglycosylation releases the aglycone into the lumen, where the latter might be absorbed into the enterocyte by passive diffusion [22, 23]. The involvement of transporters for aglycone absorption in the intestine still remains an open question. Alternatively, the active transport of flavonoid glucosides, for example, quercetin glucosides, by the sodium-dependent glucose cotransporter (SGLT1), and the subsequent cleavage of glucosides by a cytosolic b-glucosidase (CBG) has been discussed [24]. However, to date there is little evidence that isoflavone glucosides interact with SGLT1 [25, 26]. Moreover, a recent publication completely rejected the transport of flavonoids via SGLT1 [27]. 550j 31 Phytoestrogens

31.1.4.2 Metabolism Intestinal bacteria play an essential role in isoflavone metabolism. For daidzein as well as genistein the intestinal metabolism has been studied extensively in vitro and in vivo [28–34]. It has been reported that daidzein is converted by the gut microflora to the isoflavanone dihydrodaidzein (DHD), which can be further metabolized to both the isoflavane equol and to 50-methyldeoxybenzoin O-desmethylangolensin (O-DMA) (see Scheme 31.2). Only 30% of the adult Western population and 50–60% of the adult Asians as well as those of vegetarians excrete equol in urine after consuming soy foods [35]. The equol producer is defined on the basis of urinary and serum equol concentrations [36]. Unlike other isoflavones, equol has a chiral center and can therefore exist in two distinct optically active isomeric forms, R- and S-equol. However, the enantiomer produced by the human microbiota from daidzein is known to be S-()-equol [37]. Genistein, analogous to daidzein, is first reduced by gut bacteria to dihydrogenistein (DHG), followed by a cleavage of the C-ring to form 60-hydroxy-O-DMA (60OH-O-DMA). The corresponding equol derivative 5-OH-equol has not yet been identified. On the other hand, 60OH-O-DMA can be further degraded by the colonic microflora to yield 4-hydroxyphenyl-2-propionic acid. Decarboxylation can then lead to the putative metabolic end product 4-ethylphenol. Until now, 4-hydroxyphenyl-2-

Scheme 31.2 Metabolism of daidzein by the human gut microbiota (main metabolism route) and by cytochrome P-450 enzymes (minor metabolism route). 31.1 Isoflavones: Sources, Intake, Fate in the Human Body, and Effects on Cancer j551 propionic acid has been identified only in rat urine and in in vitro incubations with human microflora [32, 33]. Little is known about the bacterial metabolism of glycitein, the third soy isoflavone. Recently, several reduced as well as reduced and demethylated metabolites of glycitein were identified in vitro and in vivo, including 6-OH-daidzein, dihydro- glycitein, 6-OH-DHD, 6-methoxy-equol, 6-OH-equol, 50-OH-O-DMA, and 50-methoxy-O-DMA [34, 38, 39]. The 40-methyl ethers of daidzein and genistein – the red clover isoflavones formononetin and biochanin A, respectively – are rapidly demethylated in vitro and in vivo resulting in high plasma concentrations of daidzein and genistein [40–42]. This is in contrast to glycitein, the O-methylether of 6-OH- daidzein, which seems to be a rather stable molecule [39]. Upon absorption, isoflavones are in part metabolized by cytochrome P-450 enzymes (P-450) in the liver. Several mono- and dihydroxylated daidzein and genistein metabolites – predominantly in the C-6, -8, and -30 positions – have been identified in vitro [43, 44] and in human urine [34, 45, 46]. P-450 1A1, 1A2, 1B1, 2E1, and 3A4 have been determined to be involved in the phase I metabolism of genistein and daidzein [45]. Recently, the oxidative metabolism of glycitein has also been assessed. Several mono- and dihydroxylated metabolites as well as demethylated derivatives have been reported in vitro and in vivo [34, 45, 46]. However, compared to the metabolism by the action of the gut microbiota the oxidative metabolism of isoflavones represents only a side pathway. Once absorbed, isoflavones are efficiently conjugated either with glucuronic acid or, to a lesser extent, with sulfate. In addition, some sulfoglucuronides, diglucur- onides, and disulfates may also be formed. Conjugation takes place either in the liver or in the intestinal epithelium [47, 48]. As a consequence, isoflavones are present in the circulation in predominantly conjugated forms. In human urine, 1–3% of genistein is found in free form, 62–64% as monoglucuronide, 13–19% as diglucur- onide, 6–12% as sulfoglucuronide, 2–3% as monosulfate, and 3–6% as disulfate [49]. The isoenzymes that efficiently conjugate isoflavones are the UDP-glucuronosyl transferases (UGTs) 1A1, 1A6, 1A8, 1A9, and 1A10 as well as sulfotransferase enzymes (SULT) 1A1, 1A2, 1A3, 1B1, 1E1, 1C2, 2B1a, and 2B1b. Conjugation takes place predominantly at the hydroxyl group at C-7 where the native isoflavone glucosides generally have their glucose residue (see Scheme 31.1) [48–51].

31.1.4.3 Distribution It has been shown that isoflavones and their metabolites are widely distributed within body fluids, but definitive tissue distribution studies have not been performed in humans so far. It is not known whether the measured plasma concentrations and the distribution of metabolites in the plasma are also representative for individual organs, particularly for potential target organs such as breast, prostate, and thyroid. In this context, only few data are available. The concentrations of genistein and daidzein in the breast tissue of premenopausal women were comparable to those found in plasma; however, the equol concentrations were higher [52, 53]. The prostate fluid of men was found to contain higher concentrations of isoflavones than found in plasma [54, 55]. In humans, the concentrations of unconjugated isoflavones in the 552j 31 Phytoestrogens

plasma were relatively low. Data comparing the conjugated and unconjugated isoﬂavone fractions in human tissue are not available. However, in rats it was shown that tissues contain a much higher fraction of aglycon than does plasma [33]. For instance, the fraction of free genistein was 49% in the mammary gland tissue of the females, and even as much as 80 or 100% in ovaries and uterus, respectively, compared to less than 5% in the plasma.

31.1.4.4 Excretion The main route of excretion of isoﬂavones is via the kidney. Faecal excretion appears to be minimal, but very few human studies have investigated this issue in detail. Human trials have indicated the faecal excretion of daidzein and genistein lies within the range of 1–4% and urinary excretion between 5 and 35% of the ingested dose [56, 57].

31.1.4.5 Pharmacokinetics Isoflavones are readily absorbed from the gastrointestinal tract. In general, maximum plasma levels were achieved between 5 and 9 h after ingestion. Very often, plasma concentration–time curves exhibit two peaks: one early peak 1–2 h after isoflavone intake and the highest peak between 5 and 9 h. This biphasic shape is assumed to reflect the enterohepatic recycling as well as absorption occurring successively in the small intestine and in the colon. Elimination of isoflavones is quite slow, with half-life values of 6–8 h. A dose of 50 mg of either daidzein or genistein, yields a peak plasma concentration of 2 mmol/l. Isoflavones are therefore the most well-absorbed flavonoids [58]. Pharmacological doses of isoflavones lead to very high plasma levels; for example, doses of 4 and 8 mg of genistein per kilogram body weight caused peak plasma concentrations up to 9.5 and 17.9 mmol/l, respectively [59].

31.1.4.6 Factors Affecting the Pharmacokinetics of Isoflavones Human ADME of isoflavones are subject to considerable interindividual variation. For example, a 12- and 15-fold interindividual variation for genistein and daidzein excretion, respectively, and a 600-fold interindividual variation for the equol excretion were reported after soy consumption. The high variation in metabolism is likely to be largely a consequence of interindividual differences in the gut microflora [60, 61]. Furthermore, a variety of factors associated with food processing, food composition, and food texture have been proposed to influence the pharmacokinetics and the metabolism of isoflavones: Considerable evidence is available that isolated isoflavone-7-O-b-glucosides have a higher bioavailability than their corresponding aglycones when ingested as purified single compounds [48]. However, in a complex food matrix such as soy milk, isoflavone aglycones show a comparable or higher bioavailability than their corresponding malonyl-glucosides [62, 63]. Also, the food texture and the food composition seem to have a substantial impact. A liquid texture, such as soy milk, yields faster absorption rates and higher peak plasma concentrations compared to a food item such as tofu or tempeh with a solid texture [64]. There is some evidence that a diet rich in indigestible carbohydrates promotes the growth or 31.1 Isoflavones: Sources, Intake, Fate in the Human Body, and Effects on Cancer j553 activity of the bacteria responsible for equol production in the colon [65]. Further studies are required to determine the relative importance of these different factors that potentially affect the bioavailability of isoflavones. Such data are important to define optimal levels for isoflavone intake on the basis of a benefit–risk assessment.

31.1.5 Effects of Isoflavones on Breast, Prostate, and Intestinal Cancer

The historically low rates of breast and prostate cancers in Asian countries [66] have contributed to the scientific interest in the chemoprotective effects of soy. Although several compounds have been considered putative anticarcinogens in soybeans [67], the potential beneficial effects of soy isoflavones on cancer have been extensively studied over the past two decades (for reviews see Refs [8, 68–72] and references cited therein). The protective effects of isoflavones on cancer may be due to their estrogenic activity, which may contribute to lowering the circulating levels of unconjugated sex hormones [73], due to their antioxidant activity, which may prevent free radical damage to the cells genetic material, thus preventing mutations and the initiation of carcinogenesis [74], due to promotion of apoptosis [75] and inhibition of angiogenesis [76], and, in case of early exposure to isoflavones, due to a preadolescent development of mammary tissue to make it more resistant to carcinogens, slowing cancer promotion [77].

31.1.5.1 Breast Cancer In vitro studies of the effects of soy isoflavones on the proliferation of breast cancer cells indicate that physiologically relevant concentrations (1 mmol/l) stimulate the proliferation of the estrogen receptor (ER) positive (ER þ ) MCF-7 breast cancer cell line, by an ER-dependent mechanism. At higher concentrations (>10 mmol/l), inhibitory effects have been recorded for genistein that were attributed to an estrogen-independent mechanism of action. Furthermore, isoflavones have been shown to inhibit the process of tumor invasion in vitro, though significant effects are apparent only at relatively high isoflavone concentrations (10–75 mmol/l). Other possible mechanisms of action of the soy isoflavones in breast cancer include inhibition of angiogenesis, inhibition of enzymes involved in estrogen biosynthesis and metabolism, and antioxidant effects. Genistein has also been shown to upregulate the phase II detoxification enzymes in breast cancer cells and in nonneoplastic mammary cells, thus conferring protection against genotoxic effects (for review and further references see Refs [8, 78]). Although several animal studies have confirmed cancer-preventing effects of a soy- based diet, investigations in laboratory animals treated with isoflavones demonstrate increased incidence of mammary tumors, or indicate that isoflavones can promote breast cancer [8, 78]. The time of exposure to soy isoflavones seems to be critical for theireffectsonbreastcancerinlaboratoryanimals;forexample,pre-pubertalexposure to genistein and daidzein results in a cancer-protective effect, whereas in utero and adult exposure has not been found to protect against breast cancer. In some studies, in utero exposure to genistein has even been reported to promote breast cancer 554j 31 Phytoestrogens

development [8, 79]. The reported carcinogenic effect of isoflavones in laboratory animals should be further elucidated [80]. It is currently unclear how this finding relates to breast cancer risk in Western women who start using isoflavone-rich supplements around and after menopause, and to breast cancer survivors. The evidence that soy intake by humans reduces breast cancer is equivocal [70, 78]. The existing hypothesis is based on studies of Japanese immigrants, on animal studies, and on epidemiological studies involving soy. A recent review applying case–control studies on the association of soybean intake and breast cancer risk in Asian women consistently suggests an inverse association for both pre- and postmenopausal breast cancer. However, too few randomized control trials are available to reach conclusions on the effects of isoflavones on breast cancer in Western women [79]. A recent meta-analysis of 18 epidemiological studies revealed a small inverse association between soy intake and breast cancer in both Western and Asian women [81].

31.1.5.2 Prostate Cancer A variety of in vitro studies have demonstrated that isoflavones can inhibit growth of human prostate cancer cell lines, and cell lines derived from normal human prostatic epithelium. Animal studies indicate that genistein and soy isoflavone mixtures may protect against prostate cancer. In addition, genistein has been observed to enhance prostate cancer treatment, to inhibit metastasis, and to improve survival. However, data from both epidemiological and intervention studies are inconsistent with regard to soy consumption, isoflavone exposure, and prostate cancer risk (for reviews see Refs [8, 72, 77, 78]).

31.1.5.3 Intestinal Cancer Although colorectal cancer (CRC) is not considered hormone dependent, the human intestines are known to contain ERs [82], and there are a variety of mechanisms by which estrogens may decrease CRC risk [71]. As soy isoflavones are phytoestrogens, a considerable attention has been given to soy and isoflavones as potential dietary modulators of CRC risk. Furthermore, the following observations implicate the possible protective action of soy on CRC: (a) vegetarian diets, which often include soy, appear to be associated with a lower risk of CRC; (b) Asian populations, which consume large amounts of soy products, have a low incidence of CRC; (c) the incidence of CRC is increasing in Japan as the diet becomes increasingly Westernized [67]; and (d) soy contains a number of substances with potential anticancer activity [8, 67]. A limited number of animal studies have investigated the effects of soy isoflavones on intestinal cancer. Isolated genistein has been observed both to enhance and to inhibit colon cancer whereas exposure to soy products containing varying amounts of isoflavones had either no effect or a protective effect against colon cancer. Both decrease in precancerous colonic lesions and decrease in tumor incidence have been detected (for reviews see Refs [8, 71, 72, 83]). However, observations that genistein increased chemically induced colon cancer [84] and precancerous colonic lesions [85] in laboratory animals indicate the possibility that isoflavones consumed as pure compounds may exert adverse effects [83]. 31.2 Lignans j555

Epidemiological studies exploring the association between the soy intake and the CRCrisk have produced mixed results [71, 86, 87]. If soy does reduce the CRCrisk, it is not clear whether isoﬂavones are responsible for the protective effects. At this point, the evidence is too limited to draw conclusions about the impact of soy or isoﬂavones on colon cancer risk.

31.2 Lignans Eric Laine, Christophe Hano, and Frederic Lamblin

31.2.1 Introduction

Lignans are diphenolic compounds resulting from the dimerization of two monolignols (Scheme 31.3) that are also used by plants to synthesize lignin, the well-known polymer deposited in plant vessels conducting water and mineral salts from the roots.

Scheme 31.3 Biosynthetic pathway leading to secoisolariciresinol, the main lignan accumulated in flaxseed (in its diglucosylated form SDG) and matairesinol. DP, dirigent protein; PLR, pinoresinol-lariciresinol reductase; SD, secoisolariciresinol dehydrogenase. 556j 31 Phytoestrogens

Despite their quite simple structure, lignans are very varied, they differ by the type of linkage between the two monomers and by additional modifications that occur after dimerization (Scheme 31.3). Nevertheless, the role of lignans in planta remains unclear. Some of them have been demonstrated to be toxic for animals, or antibacterial and antifungal, and they belong to secondary metabolites; these features lead to the hypothesis of their involvement in plant defense, but experimental evidences are generally lacking to prove this assessment. Moreover, like many phenolic compounds, ligans also exhibit antioxidant activity (demonstrated in vitro). Thus, they could possibly act to prevent free-radical-induced damages, especially in the case of seeds with a high unsaturated fatty acid content such as flax. In 1982, Adlercreutz [88] of Finland proposed the hypothesis that lignans could have preventive effects against breast cancer. Some lignan (e.g., secoisolariciresinol)- derived metabolites display specific structural features that allow them to closely resemble estrogens and that give them the ability to bind to estrogen receptors or to interfere with estrogen metabolism. They are thus considered as phyto-SERMs (selective estrogen receptor modulators) and are also referred to as phytoestrogens. They will be reviewed here.

31.2.2 Occurrence and Physicochemical Properties

Lignans are widespread in seed plants. They vary in their localization and in their concentrations in the plant. Some plants such as sesame or flax accumulate lignans in seeds whereas other plants show high contents in woody part of the shoots or in roots or rhizomes. Among the phytoestrogenic lignans available in diet secoisolariciresinol, matairesinol, 7-hydroxymatairesinol and sesamin are worth mentioning (Scheme 31.4). Their precursors such as pinoresinol and lariciresinol (Scheme 31.3) are likely transformed to the same compound in the intestine (see Section 31.2.3) and thus can be included in the total available phytoestrogenic lignans [89]. Heartwood of trees can also provide high quantities of lignans but is not part of the diet. The content in phytoestrogenic lignans ranges from trace to more than 1% of dry weight as found in flaxseeds. Many data on the lignan content of various plant foods are now available. However, significant differences in lignan contents can be observed when different published studies are compared. Indeed, for the same species the estimated content may notably vary depending on analytical method, cultivar, growth conditions, or also date of harvest. The extraction method is of great importance, not only because it directly influences lignan yield but also because it allows selective recovery of different compounds. The number of detected compounds will vary from one study to another. As an example, destructive methods under severe conditions will not allow the detection of the major compound 7-hydroxymatairesinol in cereals [90]. Therefore, the data presented in Table 31.2 (adapted from recently published studies [90, 91] focusing on at least four major lignans) give indicative values establishing a list of the main dietary sources of plant lignans. 12Lignans 31.2

Scheme 31.4 Chemical structures of four major dietary lignans from plants (a) and the mammalian lignans enterodiol and enterolactone (b) obtained after metabolization by the gut microflora (arrows). Note that pinoresinol and lariciresinol (precursors of j secoisolariciresinol) can also be converted into enterodiol and enterolactone. 557 558j 31 Phytoestrogens

Table 31.2 Lignan content of various seeds, nuts, vegetables, fruits, and beverages.

Seco Matai Pino 7-OH matai Lari Sesamin

Flaxseedsa 165 759 529 871 35 1780 nd Sesame seedsa 240 1137 47 136 7209 13 060 62 724 Rye brana 462 729 1547 1017 1503 nd Wheat brana 868 410 138 2787 672 nd Oat brana 90 440 567 712 766 nd Cashew nutsa 316 55 19 31 307 nd Almondsa 159 24 208 27 233 82 Brassica vegetablesb 19 12 1691 599 Apricotb 31 0 314 105 Strawberryb 5 0 212 117 Coffeeb 9.2 nd 1.3 9.1 Tea (Earl Grey)b 6.2 1.5 27 28.9 Beer (lager)b 1 nd 21.7 9.0 Red wine, Franceb 47.5 5.9 9.5 16.1

seco: secoisolariciresinol; matai: matairesinol; pino: pinoresinol; 7-OH matai: 7-hydroxymatairesinol; lari: lariciresinol. nd: not detected; blanks indicate that the compound was not taken into account in the study. aIn mg/100 g (Smeds et al. [90]). bIn mg/100 g fresh edible weight (Milder et al. [91]).

Flaxseed is by far the richest source of dietary phytoestrogenic lignans, mainly secoisolariciresinol, accumulated in the seed coat in its diglucosylated form (secoisolariciresinol diglucoside, SDG). Lignans can also be found in other oilseeds (sesame, sunﬂower, and pumpkin), in cereal brans (rye, wheat), and in nuts (cashew nuts, Brazil nuts, almonds, peanuts). Fruits (including berries) and vegetables also contain nonnegligible amounts of lignans, especially Brassica vegetables (mainly pinoresinol and lariciresinol). Despite their relatively low lignan content, alcoholic and nonalcoholic beverages (including fruit juices) contribute to 37% of daily intake in Dutch men and women [92].

31.2.2.1 Phytoestrogenic Nature Phytoestrogenic lignans, after converting into enterolignans (enterodiol or enterolactone, Scheme 31.4), can bind to estrogen receptor (Figure 31.1). This selective estrogen receptor modulator nature is due to their structural similarities, peculiarly

Figure 31.1 Mechanisms leading to the humans. Estrogen signaling is usually associated modulation of estrogen response by mammalian with gene activation (genomic effect): after lignans. (a) Biological actions of estrogens estrogen (E) binding, ERs dimerize and bind to mediated by estrogen binding to one of the two ERE (estrogen responsive element) sequences specific receptors (ERa and ERb). These localized in the promoter region of target genes. receptors belong to the nuclear receptor family The subsequent transcriptional activation of and are encoded by two distinct genes in ER-dependent genes triggers different cellular 31.2 Lignans j559

responses leading either to cell proliferation or or as antagonist (hindering the estradiol binding sexual differentiation. Estrogens also exert to the ER and subsequent signaling pathway). nongenomic actions associated with The response will vary according to the organs, the activation of various protein-kinase cascades. tissues, or cells that can differ by the type of ER (b) Structural similarities (phenyl and hydroxyl (SERM affinity will differ according to the ER groups) between enterodiol and estrogen type) and by the types of coactivators and estradiol allowing mammalian lignans to bind to corepressors recruited in estrogen signaling. The estrogen receptors. Mammalian lignans act as estrogen response can be modulated by the SERM. (c) Mechanisms of interaction of recruitment of other transcription coactivators mammalian lignans (ML) with the estrogen and/or corepressors than those recruited after signaling system. ML can bind to estrogen estradiol binding. Mammalian lignans can also receptors and modulate the estrogen response. act by modulating estrogen biosynthesis This response will depend upon the types of ML (moderate aromatase inhibiting activity), and estrogen and their respective activation (increasing sulfatation leading to concentrations.Depending not only on its nature inactive form of estrogen), and bioavailability but also on the ratio of natural estrogen (increasing plasma SHBG levels resulting in (estradiol) and ML concentration, the latter can lower estrogen uptake). act as agonist (mimicking a binding of estradiol) 560j 31 Phytoestrogens

hydroxyl groups and aromatic ring structure (Figure 31.1b). Depending not only on its nature but also on the ratio of natural estrogen (estradiol) and phytoestrogen concentration, the phytoestrogen can act as agonist (mimicking binding of estradiol) or as antagonist (hindering the estradiol binding to the ER). Even when acting as an agonist, the resulting effect can be different from the estradiol effect because the transcription coactivators and/or coinhibitors recruited by the ER–phytoestrogen complex can differ from those recruited after estradiol binding (Figure 31.1c). For example, the effect on prepubescent or postmenopausal females can be specific, due to their low endogenous estrogen levels. Their action can also vary according to the organ or even the cell type because binding of phytoestrogen can be easier with ERb compared to ERa as it has been observed with isoflavone derivatives [93]. Moreover, coactivators and coinhibitors can differ between cell types, thus mediating different physiological processes. For these reasons, a phyto-SERM can have mimicking or opposite effect compared to natural endogenous estradiol depending on the cell type considered (e.g., in bones). Even when acting as agonist, it is generally accepted that phytoestrogenic lignans (e.g., secoisolariciresinol, the main flax lignan) are not as proestrogenic as isoflavones. No detrimental effect has been observed so far on mammal reproduction as it can occur with strong phytoestrogens such as isoflavones found in Fabaceae (soybean, red clover). Flax could thus provide mammalian lignans (ML) that are less proestrogenic than isoflavones but retain some of the benefits of phytoestrogens. Phytoestrogens do not act solely via their binding to ER; they also modulate or interfere with a number of metabolic steps involved in the synthesis or conversion of endogenous sex hormones. For example, it has been shown that enterolactone, a mammalian lignan, inhibits the aromatase (whose function is to aromatize andro- gens thus producing estrogens, Figure 31.1c) [94], including in MCF-7 breast cancer cells [95] where it has been shown to reduce the estradiol level. Phytoestrogens can also regulate the levels of active and inactive (sulfated) forms of estradiol and free or bound estradiol (to SHBG (sex hormone binding globulin) [96, 97] (Figure 31.1c). Both interactions affect the levels of active hormone; some of these interactions have been evidenced with lignans. It can be noted that some lignans are also able to bind to other sex hormone receptors.

31.2.2.2 Antioxidant Properties Kitts et al. [98] have studied antioxidant properties of SDG and enterolignans; they evidenced an inhibition of DNA degradation. Prasad [99] showed in 1997 that SDG had antioxidant effect similar to vitamin E and that the aglycone form (secoisolariciresinol) and enterolactone were about five times more efficient. Lipid peroxidation is playing a role in carcinogenesis and every compound that is able to prevent it is potentially chemopreventive against cancer. This feature could explain at least partly the chemoprotective effect observed against colon cancer [100]. Rajesha et al. [101] observed that supplementation of flaxseed in the diet of rats receiving a free-radical-

generating toxin (CCl4) allowed to maintain or enhance the hepatic enzyme activities 31.2 Lignans j561 involved in free radical scavenging such as peroxidase, catalase, and superoxide dismutase; these activities were dramatically reduced in the control.

31.2.3 Bioavailability

Secoisolariciresinol diglucoside, discovered in 1956, is the most abundant and best studied phytoestrogenic flax lignan, and we will therefore focus on it. Its content in flaxseed can exceed 1.5% of DWand represent more than 5% of the DW of the seed coat. SDG, ester-linked to hydroxy-methyl-glutaric acid (HMGA), forms the backbone of a lignan macromolecule (SDG–HMG complex) that also contains hydroxycinnamic acid glucosides. A much less studied lignan, matairesinol, is also present in flaxseed although in much lower concentration. Hydroxymatairesinol, close to the latter, is also found in a number of foodstuffs derived from whole-grain cereals. When ingested, these three lignans are not always directly bioavailable. Glitso et al. [102], when conducting experiments on pig feed with rye, observed that most of the lignans present in the ileum were still conjugated. For example, SDG has first to be released from the HMG complex. It is then deglycosylated and eventually converted by human intestinal microbiota (among which Clostridium coccoides, Eubacterium rectale, Lactonifactor longoviformis) in enterolactone and enterodiol, the so-called enterolignans or mammalian lignans; enterodiol is also eventually converted into enterolactone [103, 104] (Scheme 31.4). For this reason, it is generally considered that SDG and matairesinol effects result from the enterolactone presence in blood. This explains why most of the clinical experimentations aimed at linking lignan intake and cancer occurrence include quantification of enterolactone in blood and urine to estimate lignan intake and metabolization. Nevertheless, direct transfer of secoisolariciresinol and matairesinol in bloodstream through gut also does exist, although it has long been neglected [105]; this could also produce specific effects in cell metabolism, especially for matairesinol. One should also keep in mind that microbiota of the intestine is prone to wide variation among the population due to different diet, antibiotic intake, and so on. Lampe et al. [106] concluded from their study that controlled feeding studies have demonstrated dose-dependent urinary lignan excretion in response to flaxseed consumption (main source of SDG); however, even in the context of controlled studies, there are substantial interindividual variations in plasma concentrations and urinary excretion of enterolignans. Kuijsten et al. [107] have performed a monitoring of blood and urine concentration in mammalian lignans after ingestion of purified SDG. Their results confirm previous observation by other authors: enterolignans appeared in plasma 8–10 h after ingestion of the purified SDG. Enterolactone reached its maximum about 20 h after ingestion. The mean elimination half-life of enterolactone was about 13 h. Within 3 days, up to 40% of the ingested SDGwas excreted as enterolignans via urine, with the majority (58%) as enterolactone [107]. The mean rates of blood enterolactone 562j 31 Phytoestrogens

in omnivorous humans in various countries range from 4 to 30 nmol/l [108], with the mean value at 20 nmol/l in the population analyzed by this Finnish team. A recent survey conducted on a large European population sample gives about 10 nmol/l for enterolactone plus enterodiol in plasma [109].

31.2.4 Mechanisms of Protection: Results of In Vitro and Animal Studies

Most of the work is conducted either in vivo with mice or rats, and deals with mammary or prostate tumors (induced by DMBA or grafted), or in vitro with model cancer cell lines. Most studies yielded results in favor of a preventive effect of phytoestrogenic lignans. A selection of published data is presented in Table 31.3 (for more detailed reviews see also Refs [110–112] on breast cancer).

31.2.4.1 Inhibition of Tumorigenesis Jenab and Thomson [119] related in 1996 an inhibition of colon cancer when feeding rats to either flaxseed or defatted flaxseed meal. They observed that total urinary lignan excretion was negatively correlated with the total number of aberrant crypts and the total number of aberrant crypt foci in the distal colon. There were no significant differences between the flaxseed and the corresponding defatted flaxseed groups. It was concluded that flaxseed has a colon cancer protective effect, that it is due, in part, to secoisolariciresinol. In 2002, Saarinen et al. demonstrated, using rats, inhibition of mammary carcinomas by enterolactone (summarized in Ref. [112]). Bylund et al. [118] published in 2005, the results of work conducted on mice bearing prostatic tumors and found an inhibitory effect of hydroxymatairesinol on tumor development. Similarly, Demark- Wahnefried et al. [123, 124], in a pilot study of dietary fat restriction and flaxseed supplementation in men with prostate cancer, observed a significant reduction of proliferation of benign prostatic epithelium, as well as increased apoptosis in the prostatic tissue.

31.2.4.2 Inhibition of Metastasis The ability to inhibit metastasis multiplication was estimated in vitro in a cell invasion assay by Chen and Thompson [114] and Magee et al. [115]. Although both groups used the same experimental system and design, the group headed by Magee observed no effect whereas the group headed by Thompson observed inhibition. In 1999, Li et al. (summarized in Ref. [111]) published the results of injection of lignans on model mice with melanoma metastases: mice that received the highest doses of SDG had less metastasis.

31.2.4.3 Other Less Documented Mechanisms It is widely thought that therapeutic and preventive effects of SDG in cancer prevention results from its phytoestrogenic nature. Nevertheless, lignans found in various plant families have been shown to possess interesting properties that can contribute to cancer prevention in in vitro or animal model studies. Table 31.3 Effects of purified lignans or lignan-rich diets on cancer models in vivo (experimental animals) and in vitro (cancer cell lines).

Cancer model/animal Type of cancer Compound/source or cell line Effects Author, year Reference

Breast/in vivo Sesamin DMBA-induced SD rats Reduced number of tumors Hirose, 1992 [112] Breast/in vivo SDG DMBA-induced SD rats Reduced number of tumors Thompson, 1996 [112] Breast/in vivo SDG DMBA-induced SD rats Reduced tumor volume, number and Thompson, 1996 [113] incidence of new tumors Breast/in vivo SDG N-Methyl-N-nitrosourea No effect of flaxseed diet on tumor size, Rickard, 1999 [112] (MNU)-induced mammary multiplicity, or incidence tumor in SD rats Dose-dependent effect of SDG on tumor multiplicity: low SDG level promoted and high SDG level reduced tumor multiplicity Breast/in vivo 7-Hydroxymatairesinol DMBA-induced SD rats Reduced tumor volume and growth Saarinen, 2001 [112] Breast/in vivo flaxseed diet Athymic Ncr nu/nu mice Reduced tumor growth and metastasis, Dabrosin, 2002 [111] bearing orthotopic MDA- decreased levels of VEGF (vascular en- MB-435 tumors dothelial growth factor) Breast/in vivo Enterolactone DMBA-induced SD rats Reduced growth of tumors and reduced Saarinen, 2002 [112] volume of newly established tumors Breast/in vitro Enterodiol, enterolactone ER-human breast cancer cell Inhibition of cell adhesion, invasion, and Chen, 2003 [114] lines, MDA-MB-435 and migration, the steps involved in the MDA-MB-231 metastasis cascade Breast/in vitro Secoisolariciresinol, Breast cancer cell line MDA- No antimetastasic effect; only secoiso- Magee, 2004 [115] matairesinol, enterodiol, MB-231 lariciresinol and enterodiol induced a Lignans 31.2 enterolactone significant decrease in cell invasion Breast/in vivo SDG and flaxseed diet Athymic Ncr nu/nu mice Reduced incidence of metastasis, no Chen, J, 2006 [112] bearing orthotopic MDA- significant difference in tumor

MB-435 tumors recurrence j 563 (Continued) Table 31.3 (Continued)

Cancer model/animal Type of cancer Compound/source or cell line Effects Author, year Reference 564 j

Breast/in vivo Flaxseed diet, enterodiol MCF-7 breast cancer xeno- Inhibition of estradiol-induced growth, Bergman [116] Phytoestrogens 31 or enterolactone grafts in ovariectomized angiogenesis, and secretion of VEGF Jungestrom,€ 2007 mice Breast/in vivo Lariciresinol DMBA-induced SD rats, Inhibition of tumor growth and Saarinen, 2008 [117] MCF-7 breast cancer xeno- angiogenesis in both models increased grafts in athymic nude mice apoptosis in MCF-7 xenografts Prostate/in vivo Rye bran diet LNCaP Human prostate Reduced and delayed growth, increased Bylund, 2000 [111] adenocarcinoma xenografts tumor cell apoptosis in nude mice Prostate/in vivo 7-Hydroxymatairesinol LNCaP Human prostate Tumor growth inhibition, reduced cell Bylund, 2005 [118] adenocarcinoma xenografts proliferation, increased apoptosis in nude mice Prostate/in vivo Flaxseed diet Transgenic adenocarcinoma Tumor growth inhibition, reduced cell Lin, 2002 [111] mouse prostate (TRAMP) proliferation, increased apoptosis model Colon/in vivo Flaxseed diet Azoxymethane-induced tu- Reduced number of tumors Jenab, 1996 [119] mors in SD rats Colon/in vivo Rye bran diet Azoxymethane-induced tu- Reduced number of tumors Davies, 1999 [111] mors in rats Colon/in vivo and Enterolactone Colo201 human cells trans- Inhibition of tumor growth and cell Danbara, 2005 [120] in vitro planted in athymic mice growth, increased apoptosis Colo201 human cell line Cell growth inhibition, increased apoptosis Colon/in vitro Enterodiol, enterolactone Human colonic cancer Se- and time-dependent decreases in cell Qu, 2005 [121] SW480 numbers; increased apoptosis Melanoma/in vivo SDG B16BL6 murine melanoma Decreased pulmonary metastasis of Li, 1999 [111] cells in C57BL/6 mice melanoma cells and growth inhibition of metastatic tumors Liver/in vivo and 7-Hydroxymatairesinol, AH109 hepatoma cells Inhibition of proliferation and invasion Miura, 2007 [122] in vitro enterolactone AH109A hepatoma in rats Reduced tumor growth and metastasis

DMBA: 7,12-dimethylbenz[a]anthracene; SD: Sprague–Dawley. 31.2 Lignans j565

Amongst these features, one can cite antiangiogenesis properties, that is, inhibition of blood vessel formation [116, 117] or the stimulation of breast cell differentiation that could prevent breast cancer and has been observed in young female rats [125], resulting eventually in less tumorigenesis [126]. Also to be noted, some enterolignans and lignans have been shown to exert a proapoptotic effect on tumor cell lines [120]. Antiproliferative effects have also been suggested as some of the lignans have already been showed to have these properties, but these features have not been found so far for the phytoestrogenic lignans such as secoisolaraiciresinol.

31.2.5 Results of Human Studies

The teams headed by Adlercreutz (in Finland) and Thompson (in Canada) were the first to study the preventive effects of phytoestrogenic lignans found in flaxseed. Since then, numerous epidemiological as well as prospective studies have been published; but summarizing their results is difficult because some of them are contradictory [110–112]. Most studies deal with breast, prostate, and colon cancers. They were conducted mainly by Finnish and Canadian teams and have generated quite contradictory results: some of them found a correlation between lignan intake, enterolactone concentration in blood and urine, and the rate of cancer, and some of them did not. Nevertheless, other teams, or even the same, have evidenced benefits of lignans in in vitro or in vivo studies. The main sources of data are epidemiological studies in which lignan intake estimation is based on food frequency questionnaires and lignan content data of foodstuffs. Generally, this is substantiated by blood (circulating) and urine (excreted) enterolactone quantification. A drawback of this method, compared to supplementation experiments, is that the population quartile with the highest lignan intake is often more vegetarian, as lignans are mainly found in whole seeds and berries, and as a consequence consumes probably less animal-derived food. One should thus be cautious when interpreting results. Nevertheless, these observations allow driving hypothesis before eventually launching supplementation experiments. Flaxseed is generally used for this purpose due to its very high SDG content and its ease of use both in human and animal experiments. Unfortunately, purified lignans are so far unavailable or too costly and thus can be used only when conducting experiments with small animals such as mice, except in experiments designed to follow the enterolactone contents after a single ingestion. However, supplementation with whole flaxseed, often used in small mammal experiments, has a drawback of delivering omega-3 fatty acids (a-linolenic acid is the main fatty acid in flaxseed oil) that can also play a role in some of the observed benefits, especially in mammary tumorigenesis [113]; this is nevertheless the case for more studies dealing with cardiac diseases (that are also prevented by SDG). To overcome this, it would be better to compare the effect of two flax cultivars (one being rich in omega-3 fatty acid and poor in lignan content, whereas the other showing 566j 31 Phytoestrogens

opposite features) as it has been made in studies dealing with stress [127] and blood pressure, or to use only ﬂaxseed hull supplements that contain no oil. In all cases, the mechanisms involved in preventive effects are still to be elucidated because hypotheses are numerous, among which the antiestrogenic effect is more recognized, but it cannot be ruled out that a number of mechanisms contribute to the beneﬁcial effect. Main results are described below. For more details, see also Adlercreutz [110] and Saarinen et al. [112] who have recently published reviews on these experiments.

31.2.5.1 Breast Cancer Ingram et al. [128], Pietinen et al. [129], and Olsen et al. [130] have presented results in favor of a preventive effect. Nevertheless, the recent papers by Kilkkinen et al. [131] or Zeleniuch-Jacquotte et al. [132, 133] report no correlation between enterolactone blood concentrations and breast cancer incidence. However, one can note that the cohorts studied did not include an identified subpopulation that consumed flaxseed. Thus, even the mean enterolactone rates in the highest quartile (54 nmol/l for Pietinen et al. [129], who observed a benefit, and 45 nmol/l for Kilkkinen [131] and Zeleniuch-Jacquotte [132, 133], who observed no benefit) are far below those that can be found in people who consume either flaxseed (only 0.6% of people) or lignan supplementation. For the latter, the rates are between 65 and 167 nmol/l for enterolactone supplementation alone and up to 270 nmol/l when combined with enterodiol, for a daily intake of about 30 g flaxseed [107]. This could explain some of the discrepancies observed among published results. More recent studies tend to identify and separate better-defined groups, and thus allow to evidence correlations that were found nonsignificant in previous studies because the differences were minimized by the size and heterogeneity of the groups. In these studies, the benefitof dietary lignan became more evident. For example, Olsen et al. in 2004 [130] and Mc Cann et al. in 2006 [134] demonstrated a preventive effect of lignan intake, but it was restricted to breast cancer of ER-negative type (which presents the worst prognosis). Such quite surprising results suggest that lignans, even when they are phytoestrogenic, could also act via mechanisms other than those involving ER. On the contrary, Touillaud et al. [135] following a French postmenopausal cohort study showed a significant protective effect of lignan intake that was limited to ER þ tumors. But recently, breast cancer risk was investigated in relation to the estimated lignan intake among a cohort study of more than 50 000 postmenopausal women [136]. Overall, a significant 17% risk reduction for breast cancer in the high-lignan quartile was observed, but no heterogeneity across ER subtypes. Recent studies exploring the association between plasma phytoestrogen levels and breast cancer risk led to contradictory conclusions. In a population-based case–control study in Germany, Piller et al. [137] observed that premenopausal breast cancer risk decreased with increasing plasma enterolactone concentrations. These results gave support to the potential role of mammalian lignans for breast cancer prevention among premenopausal women in Western populations. However, in a nested case–control study within one of the two Dutch cohorts participating in the 31.2 Lignans j567

European Prospective Investigation into Cancer and Nutrition, Verheus et al. [138] observed no effect of lignans (enterodiol and enterolactone) on breast cancer risk.

31.2.5.2 Prostate Cancer A Finnish team studying the correlation between enterolactone level in blood and prostate cancer found no correlation [139]. Stattin et al., in 2004, had similar results [140] although the same team produced results on mice showing beneﬁts of lignans [118].

31.2.5.3 Colon Cancer In a case–control study, Kuijsten et al. [141] observed a substantial reduction in colorectal adenoma risk among subjects with high plasma concentrations of enterolignans, but the same team published in 2008 [142] the results of a cohort study (35 000 participants followed for 7 years) that did not support the hypothesis that high plasma enterodiol or enterolactone concentrations could be associated with reduced risk of colorectal cancer.

31.2.6 Impact of Cooking, Processing, and Other Factors on Protective Properties

Data concerning the content of plant lignans (mammalian lignan precursors) in baked or other processed foods such as dairy products and edible oils tend to prove their relatively high stability. Flaxseed (whole seed or meal) can be incorporated in various processed foods (multigrain breads, muffins, cookies, and other baked goods) to improve our dietary consumption of mammalian lignan precursors. The stability of SDG has been investigated in these processed foods [143, 144]. It appears that the major part of the theoretical yield of SDG can be recovered, indicating that it withstands the normal temperature of the baking process. Kuijsten et al. [145] observed that the consumption of crushed or milled flaxseed substantially increased the bioconversion rate of lignans into enterolignans in humans compared to whole seed consumption (1.4 and 3.2 increase in plasma enterolignan concentrations after consumption of crushed and ground flaxseed, respectively). Incorporation of rye bread in the diet of pigs increased their level of circulating enterolactone [146]. These results demonstrate that lignans from baked foods can also be metabolized into mammalian lignan by the gut microflora. Hyv€arinen et al. [147] demonstrated that SDG added to milk prior to high temperature pasteurization, fermentation, and milk renneting was recovered in dairy products such as yogurt, cheese, and whey-based drinks and remained stable over a 6-month storage period. In vegetables, an average 25% decrease in total lignan content was observed after boiling or frying, indicating the relatively high stability of the compounds in cooking temperatures [91]. Edible roasted sesame seed oil contains lignans such as sesamol, sesamin, and sesamolin. Secosiolariciresinol and matairesinol can also be recovered from roasted nuts and seeds but the yield may depend upon roasting temperature. Pinoresinol can 568j 31 Phytoestrogens

be found as the major compound in cold-pressed extra-virgin olive oil. Dietary commercialized flaxseed oil contains very low quantities of lignans. The major part ofSDGthataccumulatesintheseedcoatisretainedintheflax cake during the pressing process. This finding has stimulated the development of processes for the production of flax hull-enriched fractions with elevated levels of SDG for use as a functional food.

31.2.7 Conclusions

Although in vivo and in vitro data are globally in favor of a chemoprotective effect of lignans against tumor growth, multiplicity and metastasis, epidemiological studies are much less conclusive. This discrepancy could be due to the difference of concentration of circulating lignans between epidemiological studies and in vivo experiments. In the latter, supplementations with flaxseed, cereal bran, or lignans were performed, thus allowing higher enterolactone concentrations compared to those in the population (even the highest quartile) involved in epidemiological studies. The availability of purified lignans at reasonable cost would allow better- defined supplementation experiments. At present, lignan extraction and purification methods still need improvement. The mechanisms by which phytoestrogenic lignans prevent cancers still remain unclear. These compounds may act via their phytoestrogenic properties (interaction with the estrogen signaling pathway) and also via free radical scavenging, preventing the onset of tumors, or also via other means such as antiangiogenesis or apoptosis induction, preventing the cancer development. The elucidation of these mechanisms requires further investigation.

References 3 Ritchie, M.R. (2008) (Phytoestrogen database) Phyto-oestrogen database. http:// medicine.st-andrews.ac.uk/research/docs/ References to Section 31.1 ritchie/. Accessed on 3.3.2008. 4 Thompson, L.U., Boucher, B.A., Liu, Z., 1 Franke, A.A., Hankin, J.H., Yu, M.C., Cotterchio, M. and Kreiger, N. (2006) Maskarinec, G., Low, S.-H. and Custer, L.J. Phytoestrogen content of foods consumed (1999) Isoflavone levels in soy foods in Canada, including isoflavones, lignans, consumed by multiethnic populations in and coumestan. Nutrition and Cancer, 54, Singapore and Hawaii. Journal of 184–201. Agricultural and Food Chemistry, 47, 5 US Department of Agriculture, A. R. S . 977–986. (2002) USDA–Iowa State University Data- 2 Hutabarat, L.S., Greenfield, H. and base on the Isoflavone Content of Foods. Mulholland, M. (2001) Isoflavones and Release 1.3.2002, Nutrient Data Laboratory coumestrol in soybeans and soybean Web site http://www.nal.usda.gov/fnic/ products from Australia and Indonesia. foodcomp/Data/isoflav/isoflav.html. Journal of Food Composition and Analysis, 6 Lee, S.J., Ahn, J.K., Kim, S.H., Kim, J.T., 14,43–58. Han, S.J., Jung, M.Y. and Chung, I.M. References j569

(2003) Variation in isoflavone of soybean nutritional and health aspects in humans. cultivars with location and storage The Journal of Nutritional Biochemistry, 9, duration. Journal of Agricultural and Food 193–200. Chemistry, 51, 3382–3389. 15 Murphy, P.A., Song, T., Buseman, G. 7 Setchell, K.D. and Cole, S.J. (2003) and Barua, K. (1997) Isoflavones in Variations in isoflavone levels in soy foods soy-based infant formulas. Journal of and soy protein isolates and issues related Agricultural and Food Chemistry, 45, to isoflavone databases and food labeling. 4635–4638. Journal of Agricultural and Food Chemistry, 16 Nurmi, T., Mazur, W., Heinonen, S., 51, 4146–4155. Kokkonen, J. and Adlercreutz, H. (2002) 8 Mortensen, A., Kulling, S.E., Schwartz, H., Isoflavone content of the soy based Rowland, I., Ruefer, C., Rimbach, G., supplements. Journal of Pharmaceutical Cassidy, A., Magee, P., Millar, J., Hall, W.L., and Biomedical Analysis, 28,1–11. Kramer Birkved, F., Sorensen, I.K. and 17 Coward, L., Smith, M., Kirk, M. and Sontag, G. (2009) Analytical and Barnes, S. (1998) Chemical modification of compositional aspects of isoflavones in isoflavones in soyfoods during cooking food and their biological effects. Journal of and processing. The American Journal of Molecular Nutrition and Food Research (in Clinical Nutrition, 68, 1486S–1491S. press). 18 Messina, M., Nagata, C. and Wu, A.H. 9 Horn-Ross, P.L., Barnes, S., Lee, M., (2006) Estimated Asian adult soy protein Coward, L., Mandel, J.E., Koo, J., John, and isoflavone intake. Nutrition and E.M. and Smith, M. (2000) Assessing Cancer, 55,1–12. phytoestrogen exposure in epidemiologic 19 Bakker, M.I. (2004) Dietary intake of studies: development of a database (United phytoestrogens, RIVM report 320103002. States). Cancer Causes & Control, 11, http://www.rivm.nl/bibliotheek/ 289–298. rapporten/320103002.pdf. 10 Pillow, P.C., Duphorne, C.M., Chang, S., 20 Day, A.J., DuPont, M.S., Ridley, S., Rhodes, Contois, J.H., Strom, S.S., Spitz, M.R. and M., Rhodes, M.J., Morgan, M.R. and Hursting, S.D. (1999) Development of a Williamson, G. (1998) Deglycosylation of database for assessing dietary flavonoid and isoflavonoid glycosides by phytoestrogen intake. Nutrition and human small intestine and liver beta- Cancer, 33,3–19. glucosidase activity. FEBS Letters, 436, 11 Liggins, J., Bluck, L.J., Runswick, S., 71–75. Atkinson, C., Coward, W.A. and Bingham, 21 Day, A.J., Cañada, F.J., Diaz, J.C., Kroon, S.A. (2000) Daidzein and genistein P.A., Mclauchlan, R., Faulds, C.B., Plumb, contents of vegetables. The British Journal G.W., Morgan, M.R. and Williamson, G. of Nutrition, 84, 717–725. (2000) Dietary flavonoid and isoflavone 12 Liggins, J., Bluck, L.J., Runswick, S., glycosides are hydrolysed by the lactase site Atkinson, C., Coward, W.A. and Bingham, of lactase phlorizin hydrolase. FEBS S.A. (2000) Daidzein and genistein content Letters, 468, 166–170. of fruits and nuts. The Journal of Nutritional 22 Setchell, K.D.R., Brown, N.M., Zimmer- Biochemistry, 11, 326–331. Nechemias, L., Brashear, W.T., Wolfe, B.E., 13 Liggins, J., Mulligan, A., Runswick, S. and Kirschner, A.S. and Heubi, J.E. (2002) Bingham, S.A. (2002) Daidzein and Evidence for lack of absorption of soy genistein content of cereals. Journal of isoflavone glycosides in humans, Clinical Nutrition, 56, 961–966. supporting the crucial role of intestinal 14 Mazur, W.M., Duke, J.A., Wahala, K., metabolism for bioavailability. The Rasku, S. and Adlercreutz, H. (1998) American Journal of Clinical Nutrition, 76, Isoflavonoids and lignans in legumes: 447–453. 570j 31 Phytoestrogens

23 Rice-Evans, C. (2004) Flavonoids and metabolites dihydrodaidzein, isoflavones: absorption, metabolism, and dihydrogenistein, 60-OH-O-dma, and bioactivity. Free Radical Biology & Medicine, cis-4-OH-equol in human urine by gas 36, 827–828. chromatography–mass spectroscopy 24 Walgren, R.A., Lin, J.T., Kinne, R.K. and using authentic reference compounds. Walle, T. (2000) Cellular uptake of dietary Analytical Biochemistry, 274, 211–219. flavonoid quercetin 40-beta-glucoside by 32 Coldham, N.G., Howells, L.C., Santi, A., sodium-dependent glucose transporter Montesissa, C., Langlais, C., King, L.J., SGLT1 The Journal of Pharmacology and Macpherson, D.D. and Sauer, M.J. (1999) Experimental Therapeutics, 294, 837–843. Biotransformation of genistein in the rat: 25 Oitate, M., Nakaki, R., Koyabu, N., elucidation of metabolite structure by Takanaga, H., Matsuo, H., Ohtani, H. and product ion mass fragmentology. The Sawada, Y. (2001) Transcellular transport Journal of Steroid Biochemistry and of genistein, a soybean-derived isoflavone, Molecular Biology, 70, 169–184. across human colon carcinoma cell line 33 Coldham, N.G., Darby, C., Hows, M., King, (Caco-2). Biopharmaceutics & Drug L.J., Zhang, A.Q. and Sauer, M.J. (2002) Disposition, 22,23–29. Comparative metabolism of genistein by 26 Murota, K., Shimizu, S., Miyamoto, S., human and rat gut microflora: detection Izumi, T., Obata, A., Kikuchi, M. and and identification of the end-products of Terao, J. (2002) Unique uptake and metabolism. Xenobiotica, 32,45–62. transport of isoflavone aglycones by 34 Heinonen, S.M., Hoikkala, A., Wahala, K. human intestinal Caco-2 cells: comparison and Adlercreutz, H. (2003) Metabolism of of isoflavonoids and flavonoids. The the soy isoflavones daidzein, genistein and Journal of Nutrition, 132, 1956–1961. glycitein in human subjects. Identification 27 Kottra, G. and Daniel, H. (2007) Flavonoid of new metabolites having an intact glycosides are not transported by the isoflavonoid skeleton. The Journal of Steroid human Na þ /glucose transporter when Biochemistry and Molecular Biology, 87, expressed in Xenopus laevis oocytes, but 285–299. effectively inhibit electrogenic glucose 35 Rowland, I.R., Wiseman, H., Sanders, uptake. The Journal of Pharmacology and T.A., Adlercreutz, H. and Bowey, E.A. Experimental Therapeutics, 322, 829–835 (2000) Interindividual variation in 28 Kelly, G.E., Nelson, C., Waring, M.A., metabolism of soy isoflavones and lignans: Joannou, G.E. and Reeder, A.Y. (1993) influence of habitual diet on equol Metabolites of dietary (soya) isoflavones in production by the gut microflora. Nutrition human urine. Clinica Chimica Acta, 223, and Cancer, 36,27–32. 9–22. 36 Setchell, K.D. and Cole, S.J. (2006) Method 29 Chang, Y.C. and Nair, M.G. (1995) of defining equol-producer status and its Metabolism of daidzein and genistein by frequency among vegetarians. The Journal intestinal bacteria. Journal of Natural of Nutrition, 136, 2188–2193. Products, 58, 1892–1896. 37 Setchell, K.D., Clerici, C., Lephart, E.D., 30 Joannou, G.E., Kelly, G.E., Reeder, A.Y., Cole, S.J., Heenan, C., Castellani, D., Waring, M. and Nelson, C. (1995) A Wolfe, B.E., Nechemias-Zimmer, L., urinary profile study of dietary Brown, N.M., Lund, T.D., Handa, R.J. and phytoestrogens. The identification and Heubi, J.E. (2005) S-Equol, a potent ligand mode of metabolism of new isoflavonoids. for estrogen receptor beta, is the exclusive The Journal of Steroid Biochemistry and enantiomeric form of the soy isoflavone Molecular Biology, 54, 167–184. metabolite produced by human intestinal 31 Heinonen, S., Wahala, K. and Adlercreutz, bacterial flora. The American Journal of H. (1999) Identification of isoflavone Clinical Nutrition, 81, 1072–1079. References j571

38 Simons, A.L., Renouf, M., Hendrich, S. soybean isoflavone daidzein in its aglycone and Murphy, P.A. (2005) Metabolism of and glucoside form: a randomized, double- glycitein (7,40-dihydroxy-6-methoxy- blind, cross-over study. The American isoflavone) by human gut microflora. Journal of Clinical Nutrition, 87 (5), Journal of Agricultural and Food Chemistry, 1314–1323. 53, 8519–8525. 47 Sfakianos, J., Coward, L., Kirk, M. and 39 Ruefer, C.E., Maul, R., Donauer, E., Fabian, Barnes, S. (1997) Intestinal uptake and E.J. and Kulling, S.E. (2007) In vitro and in biliary excretion of the isoflavone genistein vivo metabolism of the soy isoflavone in rats. The Journal of Nutrition, 127, glycitein. Molecular Nutrition & Food 1260–1268. Research, 51, 813–823. 48 Doerge, D.R., Chang, H.C., Churchwell, 40 Setchell, K.D., Brown, N.M., Desai, P., M.I. and Holder, C.L. (2000) Analysis of Zimmer-Nechemias, L., Wolfe, B.E., soy isoflavone conjugation in vitro and in Brashear, W.T., Kirschner, A.S., Cassidy, A. human blood using liquid and Heubi, J.E. (2001) Bioavailability of chromatography–mass spectrometry. pure isoflavones in healthy humans and Drug Metabolism and Disposition, 28, analysis of commercial soy isoflavone 298–307. supplements. The Journal of Nutrition, 131, 49 Adlercreutz, H., van der Wildt, J., Kinzel, 1362S–1375S. J., Attalla, H., Wahala, K., Makela, T., Hase, 41 Kulling, S.E. and Metzler, M. (2002) T. and Fotsis, T. (1995) Lignan and Oxidative metabolism and genotoxic isoflavonoid conjugates in human urine. potential of major isoflavone The Journal of Steroid Biochemistry and phytoestrogens. Journal of Chromatography Molecular Biology, 52,97–103. B, 777, 211–218. 50 Nakano, H., Ogura, K., Takahashi, E., 42 Hur, H. and Rafii, F. (2000) Harada, T., Nishiyama, T., Muro, K., Biotransformation of the isoflavonoids Hiratsuka,A.,Kadota,S.andWatabe, biochanin A, formononetin, and glycitein T. (2004) Regioselective monosulfation by Eubacterium limosum. FEMS and disulfation of the phytoestrogens Microbiology Letters, 192,21–25. daidzein and genistein by human 43 Kulling, S.E., Honig, D.M., Simat, T.J. and liver sulfotransferases. Drug Metzler, M. (2000) Oxidative in vitro Metabolism and Pharmacokinetics, 19, metabolism of the soy phytoestrogens 216–226. daidzein and genistein. Journal of 51 Clarke, D.B., Lloyd, A.S., Botting, N.P., Agricultural and Food Chemistry, 48, Oldfield, M.F., Needs, P.W. and Wiseman, 4963–4972. H. (2002) Measurement of intact sulfate 44 Kulling, S.E., Honig, D.M. and Metzler, M. and glucuronide phytoestrogen conjugates (2001) Oxidative metabolism of the soy in human urine using isotope dilution isoflavones daidzein and genistein in liquid chromatography–tandem mass 13 humans in vitro and in vivo. Journal of spectrometry with [ C3]isoflavone Agricultural and Food Chemistry, 49, internal standards. Analytical Biochemistry, 3024–3033. 309, 158–172. 45 Roberts-Kirchhoff, E.S., Crowley, J.R., 52 Hargreaves, D.F., Potten, C.S., Harding, Hollenberg, P.F. and Kim, H. (1999) C., Shaw, L.E., Morton, M.S., Roberts, S.A., Metabolism of genistein by rat and human Howell, A. and Bundred, N.J. (1999) cytochrome P450s. Chemical Research in Two-week dietary soy supplementation has Toxicology, 12, 610–616. an estrogenic effect on normal 46 Ruefer, C.E., Bub, A., Moseneder,€ J., premenopausal breast. The Journal of Winterhalter, P., Stuertz, M. and Kulling, Clinical Endocrinology and Metabolism, 84, S.E. (2008) Pharmacokinetics of the 4017–4024. 572j 31 Phytoestrogens

53 Maubach, J., Bracke, M.E., Heyerick, A., American Journal of Clinical Nutrition, 75, Depypere, H.T., Serreyn, R.F., Mareel, 126–136. M.M. and De Keukeleire, D. (2003) 60 Karr, S.C., Lampe, J.W., Hutchins, A.M. Quantitation of soy-derived and Slavin, J.L. (1997) Urinary isoflavonoid phytoestrogens in human breast tissue and excretion in humans is dose dependent at biological fluids by high-performance low to moderate levels of soy-protein liquid chromatography. Journal of consumption. The American Journal of Chromatography B, 784, 137–144. Clinical Nutrition, 66,46–51. 54 Morton, M.S., Chan, P.S., Cheng, C., 61 Rowland, I., Wiseman, H., Sanders, T., Blacklock, N., Matos-Ferreira, A., Adlercreutz, H. and Bowey, E. (1999) Abranches-Monteiro, L., Correia, R., Metabolism of oestrogens and Lloyd, S. and Griffiths, K. (1997) Lignans phytoestrogens: role of the gut microflora. and isoflavonoids in plasma and prostatic Biochemical Society Transactions, 27, fluid in men: samples from Portugal, 304–308. Hong Kong, and the United Kingdom. 62 Tsangalis, D., Wilcox, G., Shah, N.P. and Prostate, 32, 122–128. Stojanovska, L. (2005) Bioavailability of 55 Hedlund, T.E.,Maroni, P.D., Ferucci, P.G., isoflavone phytoestrogens in Dayton, R., Barnes, S., Jones, K., Moore, postmenopausal women consuming soya R., Ogden, L.G., Wahala, K., Sackett, H.M. milk fermented with probiotic and Gray, K.J. (2005) Long-term dietary bifidobacteria. The British Journal of habits affect soy isoflavone metabolism Nutrition, 93, 867–877. and accumulation in prostatic fluid in 63 Kano, M., Takayanagi, T., Harada, T., Caucasian men. The Journal of Nutrition, Sawada, Y. and Ishikawa, F. (2006) 135, 1400–1406. Bioavailability of isoflavones after ingestion 56 Xu, X., Harris, K.S., Wang, H.J., Murphy, of soy beverages in healthy adults. The P.A. and Hendrich, S. (1995) Journal of Nutrition, 136,2291–2296. Bioavailability of soybean isoflavones 64 Cassidy, A., Brown, J.E., Hawdon, A., depends upon gut microflora in women. Faughnan, S., King, L.J., Millward, J., The Journal of Nutrition, 125, 2307–2315. Zimmer-Nechemias, L., Wolfe, B. and 57 Tew, B.Y.,Xu, X., Wang, H.J., Murphy, P.A. Setchell, K.D. (2006) Factors affecting the and Hendrich, S. (1996) A diet high in bioavailability of soy isoflavones in wheat fiber decreases the bioavailability of humans after ingestion of physiologically soybean isoflavones in a single meal fed to relevant levels from different soy foods. women. The Journal of Nutrition, 126, The Journal of Nutrition, 136,45–51. 871–877. 65 Cassidy, A. (2006) Factors affecting the 58 Manach, C., Williamson, G., Morand, C., bioavailability of soy isoflavones in Scalbert, A. and Remesy, C. (2005) humans. Journal of AOAC International, Bioavailability and bioefficacy of 89, 1182–1188. polyphenols in humans. I. Review of 97 66 Pisani, P., Parkin, D.M., Bray, F. and bioavailability studies. The American Ferlay, J. (1999) Estimates of the worldwide Journal of Clinical Nutrition, 81 (Suppl), mortality from 25 cancers in 1990. 230–242. International Journal of Cancer, 83,18–29. 59 Busby, M.G., Jeffcoat, A.R., Bloedon, L.T., 67 Messina, M. and Barnes, S. (1991) The role Koch, M.A., Black, T., Dix, K.J., Heizer, of soy products in reducing risk of cancer. W.D., Thomas, B.F., Hill, J.M., Crowell, Journal of the National Cancer Institute, 83, J.A. and Zeisel, S.H. (2002) Clinical 541–546. characteristics and pharmacokinetics of 68 Arliss, R.M. and Biermann, C.A. (2002) Do purified soy isoflavones: single-dose soy isoflavones lower cholesterol, inhibit administration to healthy men. The atherosclerosis, and play role in cancer References j573

prevention? Holistic Nursing Practice, 17, action: current evidence for a role in breast 40–48. and prostate cancer. The British Journal of 69 Cassidy, A., Albertazzi, P., Nielsen, I.L., Nutrition, 91, 513–531. Hall, W., Williamson, G., Tetens,I., Atkins, 79 Lamartiniere, C.A. (2002) Timing of S., Cross, H., Manios, Y., Wolk, A., Steiner, exposure and mammary cancer risk. C. and Branca, F. (2006) Critical review of Journal of Mammary Gland Biology and health effects of soyabean phytoestrogens Neoplasia, 7,67–76. in post-menopausal women. Proceedings of 80 DFG_Senate_Commission_on_ the Nutrition Society, 65,1–17. Food_Safety (2007) Isoflavones as 70 Messina, M., Kucuk, O. and Lampe, J.W. phytoestrogens in food supplements and (2006) An overview of the health effects of dietary foods for special medical purposes. isoflavones with an emphasis on prostate http://www.dfg.de/aktuelles_presse/ cancer risk and prostate-specific antigen reden_stellungnahmen/2007/download/ levels. Journal of AOAC International, 89, sklm_isoflavones_phytoestrogene.pdf. 1121–1134. Accessed on 3.3.2008. 71 Messina, M.J. (2002) Soy foods and soy 81 Trock, B., Hilikivi-Clarke, L. and Clarke, R. isoflavones and menopausal health. (2006) Meta-analysis of soy intake and Nutrition in Clinical Care, 5, 272–282. breast cancer risk. Journal of the National 72 Sarkar, F.H. and Li, Y. (2003) Soy Cancer Institute, 98, 459–471. isoflavones and cancer prevention. Cancer 82 Enmark, E. and Gustafsson, J.A. (1999) Investigation, 21, 744–757. Oestrogen receptors: an overview. Journal 73 Cornwell, T., Cohick, W. and Raskin, I. of Internal Medicine, 246, 133–138. (2004) Dietary phytoestrogens and health. 83 Bennik, M.R. and Rondini, E.A. (2002) Phytochemistry, 65, 995–1016. Phytoestrogens, estrogens and risk of 74 Messina, M., Persky, V., Setchell, K.D.R. colon cancer, in Phytoestrogens and Health and Barnes, S. (1994) Soy intake and cancer (eds G.S. Gilani and J.J.B. Anderson), risk: a review of the in vitro and in vivo data. AOCS Press, Champaign, IL, USA, pp. Nutrition and Cancer, 21,113–131. 427–439. 75 Balabhadrapathruni, S., Thomas, T.J., 84 Rao, C.V., Wang, C.X., Simi, B., Lubet, R., Yurkow, E.J., Amenta, P.S. and Thomas, T. Kelloff, G., Steele, V. and Reddy, B.S. (2000) Effects of genistein and structurally (1997) Enhancement of experimental related phytoestrogens on cell cycle colon cancer by genistein. Cancer Research, kinetics and apoptosis in MDA-MB-468 57, 3717–3722. human breast cancer cells. Oncology 85 Gee, J.M., Noteborn, H.P., Polley, A.C. and Reports, 7,3–12. Johnson, I.T. (2000) Increased induction of 76 Fotsis, T., Pepper, M., Adlercreutz, H. and aberrant crypt foci by 1,2-dimethyl- Hase, T. (1995) Genistein, a dietary hydrazine in rats fed diets containing ingested isoflavonoid inhibits cell purified genistein or genistein-rich proliferation and in vitro angiogenesis. The soya protein. Carcinogenesis, 21, Journal of Nutrition, 125, 790S–797S. 2255–2259. 77 Lamartiniere, C.A., Cotroneo, M.S., Fritz, 86 Messina, M. and Bennink, M. (1998) W.A., Wang, J., Mentor-Marcel, R. and Soyfoods, isoflavones and risk of colon Elgavish, A. (2002) Genistein cancer; a review of the in vitro and in vivo chemoprevention: timing and evidence. Ballieres Clinical Endocrinology mechanisms of action in murine and Metabolism, 10, 707–728. mammary and prostate. The Journal of 87 Spector, D., Anthony, M., Alexander, D. Nutrition, 132, 552S–558S. and Arab, L. (2003) Soy consumption and 78 Magee, P.J. and Rowland, I.R. (2004) colorectal cancer. Nutrition and Cancer, 47, Phyto-oestrogens, their mechanism of 1–12. 574j 31 Phytoestrogens

References to Section 31.2 Inhibition of human aromatase by mammalian lignans and isoflavonoid 88 Adlercreutz, H., Fotsis, T., Heikkinen, R., phytoestrogens. The Journal of Steroid Dwyer, J.T., Woods, M., Goldin, B.R. and Biochemistry and Molecular Biology, 44, Gorbach, S.L. (1982) Excretion of the 147–153. lignans enterolactone and enterodiol and 95 Brooks, J.D. and Thompson, L.U. (2005) of equol in omnivorous and vegetarian Mammalian lignans and genistein postmenopausal women and in women decrease the activities of aromatase and with breast cancer. Lancet, 8311, 17beta-hydroxysteroid dehydrogenase in 1295–1299. MCF-7 cells. The Journal of Steroid 89 Heinonen, S., Nurmi, T., Liukkonen, K., Biochemistry and Molecular Biology, 94, Poutanen, K., W€ah€al€a, K., Deyama, T., 461–467. Sansei Nishibe, S. et al. (2001) In vitro 96 Martin, M.E., Haourigui, M., Pelissero, C., metabolism of plant lignans: new Benassayag, C. and Nunez, E.A. (1996) precursors of mammalian lignans Interactions between phytoestrogens and enterolactone and enterodiol. Journal of human sex steroid binding protein. Life Agricultural and Food Chemistry, 49, Sciences, 58, 429–436. 3178–3186. 97 Schottner, M. and Spiteller, G. (1998) 90 Smeds, A.I., Eklund, P.C., Sjoholm,€ R.E., Lignans interfering with 5alpha- Willfor,€ S.M., Nishibe, S., Deyama, T. dihydrotestosterone binding to human sex and Holmbom, B.R. (2007) hormone-binding globulin. Journal of Quantification of a broad spectrum of Natural Products, 61, 119–121. lignans in cereals, oilseeds, and nuts. 98 Kitts, D.D., Yuan, Y.V., Wijewickreme, Journal of Agricultural and Food Chemistry, A.N. and Thompson, L.U. (1999) 55, 1337–1346. Antioxidant activity of the flaxseed lignan 91 Milder, I.E., Arts, I.C., van de Putte, B., secoisolariciresinol diglycoside and its Venema, D.P. and Hollman, P.C. (2005) mammalian lignan metabolites enterodiol Lignan contents of Dutch plant foods: a and enterolactone. Molecular and Cellular database including lariciresinol, Biochemistry, 202,91–100. pinoresinol, secoisolariciresinol and 99 Prasad, K. (1997) Hydroxyl radical- matairesinol. The British Journal of scavenging property of secoisolariciresinol Nutrition, 93, 393–402. diglucoside isolated from flax-seed. 92 Milder, I.E., Feskens, E.J., Arts, I.C., Bueno Molecular and Cellular Biochemistry, 168, de Mesquita, H.B., Hollman, P.C. and 117–123. Kromhout, D. (2005) Intake of the plant 100 Pool-Zobel, B.L., Adlercreutz, H., Glei, lignans secoisolariciresinol, matairesinol, M., Liegibel, U.M., Sittlingon, J., lariciresinol, and pinoresinol in Dutch Rowland, I., Wahala, K. and Rechkemmer, men and women. The Journal of Nutrition, G. (2000) Isoflavonoids and lignans have 135, 1202–1207. different potentials to modulate oxidative 93 Mueller, S.O., Simon, S., Chae, K., Metzler, genetic damage in human colon cells. M. and Korach, K.S. (2004) Phytoestrogens Carcinogenesis, 21, 1247–1252. and their human metabolites show distinct 101 Rajesha, J., Murthy, K.N., Kumar, M.K., agonistic and antagonistic properties on Madhusudhan, B. and Ravishankar, G.A. estrogen receptor alpha (ERalpha) and (2006) Antioxidant potentials of flaxseed ERbeta in human cells. Toxicological by in vivo model. Journal of Agricultural Sciences, 80,14–25. and Food Chemistry, 31, 3794–3799. 94 Adlercreutz, H., Bannwart, C., W€ah€al€a, K., 102 Glitsø, L.V., Mazur, W.M., Adlercreutz, Makela, T., Brunow, G., Hase, T., H., W€ah€al€a, K., M€akel€a, T., Sandstrom,€ B., Arosemena, P.J., Kellis, J.T. Jr, et al. (1993) Bach Knudsen, K.E. (2000) Intestinal References j575

metabolism of rye lignans in pigs. 111 Wescott, N.D. and Muir, A.D. (2003) Flax The British Journal of Nutrition, 84 seed in disease prevention and health 429–437. promotion. Phytochemistry Reviews, 2, 103 Setchell, K.D., Lawson, A.M., Borriello, 257–288. S.P., Harkness, R., Gordon, H., Morgan, 112 Saarinen, N.M., W€arri, A., Airio, M., D.M., Kirk, D.N. et al. (1981) Lignan Smeds, A. and M€akel€a, S. (2007) Role of formation in man: microbial involvement dietary lignans in the reduction of breast and possible roles in relation to cancer. cancer risk. Molecular Nutrition & Food Lancet, 8236,4–7. Research, 51, 857–866. 104 Clavel, T., Lippman, R., Gavini, F., Dore, J. 113 Thompson, L.U., Rickard, S.E., and Blaut, M. (2007) Clostridium Orcheson, L.J. and Seidl, M.M. (1996) saccharogumia sp. nov. and Lactonifactor Flaxseed and its lignan and oil longoviformis gen. nov., sp. nov., two novel components reduce mammary tumor human faecal bacteria involved in the growth at a late stage of carcinogenesis. conversion of the dietary phytoestrogen Carcinogenesis, 17, 1373–1376. secoisolariciresinol diglucoside. 114 Chen, J. and Thompson, L.U. (2003) Systematic and Applied Microbiology, 30, Lignans and tamoxifen, alone or in 16–26. combination, reduce human breast 105 Muir, A.D. (2006) Flax lignans analytical cancer cell adhesion, invasion, and methods and how they inﬂuence our migration in vitro. Breast Cancer Research understanding of biological activity. and Treatment, 80, 163–170. Journal of AOAC International, 89, 115 Magee, P.J., McGlynn, H. and Rowland, 1147–1157. I.R. (2004) Differential effects of 106 Lampe, J.W., Atkinson, C. and Hullar, isoﬂavones and lignans on invasiveness M.A. (2006) Assessing exposure to of MDA-MB-231 breast cancer cells lignans and their metabolites in humans. in vitro. Cancer Letters, 20,35–41. Journal of AOAC International, 89, 116 Bergman Jungestrom,€ M., Thompson, 1174–1181. L.U. and Dabrosin, C. (2007) Flaxseed 107 Kuijsten, A., Arts, I.C., Vree, T.B. and and its lignans inhibit estradiolinduced Hollman, P.C. (2005) Pharmacokinetics growth, angiogenesis, and secretion of of enterolignans in healthy men and vascular endothelial growth factor in women consuming a single dose of human breast cancer xenografts in vivo. secoisolariciresinol diglucoside. The Clinical Cancer Research, 13, 1061–1067. Journal of Nutrition, 135, 795–801. 117 Saarinen, N.M., W€arri, A., Dings, R.P., 108 Whitten, P.L. and Patisaul, H.B. (2001) Airio, M., Smeds, A.I. and M€akel€a, S. Cross-species and interassay (2008) Dietary lariciresinol attenuates comparisons of phytoestrogen action. mammary tumor growth and reduces Environmental Health Perspectives, 109, blood vessel density in human MCF-7 5–20. breast cancer xenografts and carcinogen- 109 Peeters, P.H., Slimani, N., van der induced mammary tumors in rats. Schouw, Y.T., Grace, P.B., Navarro, C., International Journal of Cancer, 123, Tjonneland, A., Olsen, A., Clavel- 1196–1204. Chapelon, F. et al. (2007) Variations in 118 Bylund, A., Saarinen, N., Zhang, J.X., plasma phytoestrogen concentrations in Bergh, A., Widmark, A., Johansson, A., European adults. The Journal of Nutrition, Lundin, E., Adlercreutz, H. et al. (2005) 137, 1294–1300. Anticancer effects of a plant lignan 110 Adlercreutz, H. (2007) Lignans and 7-hydroxymatairesinol on a prostate human health. Critical Reviews in Clinical cancer model in vivo. Experimental Biology Laboratory Sciences, 44, 483–525. and Medicine, 230, 217–223. 576j 31 Phytoestrogens

119 Jenab, M. and Thompson, L.U. (1996) 126 Chen, J., Tan, K.P., Ward, W.E. and The influence of flaxseed and lignans Thompson, L.U. (2003) Exposure to on colon carcinogenesis and flaxseed or its purified lignan during P-glucuronidase activity. Carcinogenesis, suckling inhibits chemically induced rat 17, 1343–1348. mammary tumorigenesis. Experimental 120 Danbara, N., Yuri, T., Tsujita-Kyutoku, M., Biology and Medicine, 228, 951–958. Tsukamoto, R., Uehara, N. and Tsubura, 127 Spence, J.D., Thornton, T., Muir, A.D. A. (2005) Enterolactone induces and Westcott, N.D. (2003) The effect of apoptosis and inhibits growth of Colo 201 flax seed cultivars with differing content human colon cancer cells both in vitro and of alpha-linolenic acid and lignans on in vivo. Anticancer Research, 25, responses to mental stress. Journal of the 2269–2276. American College of Nutrition, 22, 121 Qu, H., Madl, R.L., Takemoto, D.J., 494–501. Baybutt, R.C. and Wang, W. (2005) 128 Ingram, D., Sanders, K., Kolybaba, M. Lignans are involved in the antitumor and Lopez, D. (1997) Case control study of activity of wheat bran in colon cancer phyto-oestrogens and breast cancer. The SW480 cells. The Journal of Nutrition, 135, Lancet, 350, 990–994. 598–602. 129 Pietinen, P., Stumpf, K., Mannisto, S., 122 Miura, D., Saarinen, N.M., Miura, Y., Kataja, V., Uusitupa, M. and Adlercreutz, Santti, R. and Yagasaki, K. (2007) H. (2001) Serum enterolactone and Hydroxymatairesinol and its mammalian risk of breast cancer: a case–control metabolite enterolactone reduce the study in eastern Finland. Cancer growth and metastasis of subcutaneous Epidemiology, Biomarkers & Prevention, 10, AH109A hepatomas in rats. Nutrition and 339–344. Cancer, 58,49–59. 130 Olsen, A., Knudsen, K.E., Thomsen, B.L., 123 Demark-Wahnefried, W., Price, D.T., Loft, S., Stripp, C., Overvad, K., Moller, S. Polascik, T.J.,Robertson, C.N., Anderson, and Tjonneland, A. (2004) Plasma E.E., Paulson, D.F., Walther, P.J., enterolactone and breast cancer Gannon, M. et al. (2001) Pilot study of incidence by estrogen receptor status. dietary fat restriction and flaxseed Cancer Epidemiology, Biomarkers & supplementation in men with prostate Prevention, 13, 2084–2089. cancer before surgery: exploring the 131 Kilkkinen, A., Virtamo, J., Vartiainen, E., effects on hormonal levels, prostate- Sankila, R., Virtanen, M.J., Adlercreutz, specific antigen, and histopathologic H. and Pietinen, P. (2004) Serum features. Urology, 58,47–52. enterolactone concentration is not 124 Demark-Wahnefried, W., Robertson, associated with breast cancer risk in a C.N., Walther, P.J., Polascik, T.J., nested case–control study. International Paulson, D.F. and Vollmer, R.T. (2004) Journal of Cancer, 108, 277–280. Pilot study to explore effects of low-fat, 132 Zeleniuch-Jacquotte, A., Adlercreutz, H., flaxseed-supplemented diet on Shore, R.E., Koenig, K.L., Kato, I., Arslan, proliferation of benign prostatic A.A. and Toniolo, P. (2004) epithelium and prostate-specific Circulating enterolactone and risk antigen. Urology, 63,900–904. of breast cancer: a prospective study in 125 Tan, K.P., Chen, J., Ward, W.E. and New York. British Journal of Cancer, Thompson, L.U. (2004) Mammary gland 5,99–105. morphogenesis is enhanced by exposure 133 Zeleniuch-Jacquotte, A., Lundin, E., to flaxseed or its major lignan during Micheli, A., Koenig, K.L., Lenner, P., suckling in rats. Experimental Biology and Muti, P., Shore, R.E., Johansson, I. et al. Medicine, 229, 147–157. (2006) Circulating enterolactone and References j577

risk of endometrial cancer. 141 Kuijsten, A., Arts, I.C., Hollman, P.C., International Journal of Cancer, vant Veer, P. and Kampman, E. (2006) 15 (119), 2376–2381. Plasma enterolignans are associated with 134 McCann, S.E., Kulkarni, S., Trevisan, M., lower colorectal adenoma risk. Cancer Vito, D., Nie, J., Edge, S.B., Muti, P. and Epidemiology, Biomarkers & Prevention, 15, Freudenheim, J.L. (2006) Dietary lignan 1132–1136. intakes and risk of breast cancer by tumor 142 Kuijsten, A., Hollman, P.C., Boshuizen, estrogen receptor status I. Breast Cancer H.C., Buijsman, M.N., vant Veer, P., Kok, Research and Treatment, 99, 309–311. F.J., Arts, I.C. and Bueno-de-Mesquita, 135 Touillaud, M.S., Thiebaut, A.C., Fournier, H.B. (2008) Plasma enterolignan A., Niravong, M., Boutron-Ruault, M.C. concentrations and colorectal cancer risk and Clavel-Chapelon, F. (2007) Dietary in a nested case–control study. American lignan intake and postmenopausal breast Journal of Epidemiology, 167, 734–742. cancer risk by estrogen and progesterone 143 Muir, A.D. and Westcott, N.D. (2000) receptor status. Journal of the National Quantitation of the lignan Cancer Institute, 99, 475–486. secoisolariciresinol diglucoside in baked 136 Suzuki, R., Rylander-Rudqvist, T., Saji, S., goods containing flax seed or flax meal. Bergkvist, L., Adlercreutz, H. and Wolk, Journal of Agricultural and Food Chemistry, A. (2008) Dietary lignans and 48, 4048–4052. postmenopausal breast cancer risk by 144 Hyv€arinen, H.K., Pihlava, J.M., oestrogen receptor status: a prospective Hiidenhovi, J.A., Hietaniemi, V., cohort study of Swedish women. British Korhonen, H.J. and Ryh€anen, E.L. (2006) Journal of Cancer, 12, 636–640. Effect of processing and storage on the 137 Piller, R., Chang-Claude, J. and Linseisen, stability of flaxseed lignan added to J. (2006) Plasma enterolactone and bakery products. Journal of Agricultural genistein and the risk of premenopausal and Food Chemistry, 54,48–53. breast cancer. European Journal of Cancer 145 Kuijsten, A., Arts, I.C., vant Veer, P. and Prevention, 3, 225–232. Hollman, P.C. (2005) The relative 138 Verheus,M.,vanGils,C.H.,Keinan-Boker, bioavailability of enterolignans in L., Grace, P.B., Bingham, S.A.and Peeters, humans is enhanced by milling and P.H. (2007) Plasma phytoestrogens and crushing of flaxseed. The Journal of subsequent breast cancer risk. Journal of Nutrition, 135, 2812–2816. Clinical Oncology, 25,648–655. 146 Bach Knudsen, K.E., Serena, A., Kjaer, 139 Kilkkinen, A., Virtamo, J., Virtanen, M.J., A.K., Tetens, I., Heinonen, S.M., Nurmi, Adlercreutz, H., Albanes, D. and T.and Adlercreutz, H. (2003) Rye bread in Pietinen, P. (2003) Serum enterolactone the diet of pigs enhances the formation of concentration is not associated with enterolactone and increases its levels in prostate cancer risk in a nested plasma, urine and feces. The Journal of case–control study. Cancer Epidemiology, Nutrition, 133, 1368–1375. Biomarkers & Prevention, 12,1209–1212. 147 Hyv€arinen, H.K., Pihlava, J.M., 140 Stattin, P., Bylund, A., Biessy, C., Kaaks, Hiidenhovi, J.A., Hietaniemi, V., R., Hallmans, G. and Adlercreutz, H. Korhonen, H.J. and Ryh€anen, E.L. (2006) (2004) Prospective study of plasma Effect of processing and storage on the enterolactone and prostate cancer risk stability of flaxseed lignan added to dairy (Sweden). Cancer Causes & Control, 15, products. Journal of Agricultural and Food 1095–1102. Chemistry, 54, 8788–8792. j579

32 Chemopreventive Properties of Coffee and Its Constituents Gernot Faustmann, Christophe Cavin, Armen Nersesyan, and Siegfried Knasmuller€

32.1 Introduction

Coffee is named after the province Kaffa in todays Ethiopia. More than 1000 years ago, the first coffee plants were cultivated in Yemen; Venetian traders brought coffee to Europe in 1615 and the first coffee houses opened up in cities such as Vienna, London, and Venice at the end of the 17th century. Today, coffee is one of the most widely consumed beverages worldwide. The global average per capita consumption is approximately 1 kg of green bean equivalent per year. The highest amounts are consumed by North Americans (>4 kg) and Europeans (4 kg); the heaviest drinkers are at present the Finns with an annual average intake of 11 kg [1]. The impact of coffee on human health has been investigated intensively but most of the earlier studies have focused on potential adverse effects. However, in the last decades, some emerging evidence suggests the possibility for protective health effects. Consumption of coffee has been associated with reduced risk of several diseases including the prevention of DNA damage [2], hepatitis and certain cancers [3], neurodegenerative diseases such as Parkinsons and Alzheimers diseases [4, 5], and diabetes type II [6]. While the protective effects against DNA damage and cancer may be due to protection against reactive oxygen species (ROS) and induction of detoxifying enzymes, the inverse association between coffee consumption and incidence of diabetes has been attributed to reduced postprandial uptake of glucose, inhibition of hepatic glucose-6-phosphatase, reduction of body weight, and increased magnesium uptake. In the case of the neurological disorders, the impact of caffeine on the dopaminergic system may be responsible for protective effects [7]. Possible adverse effects, which have been claimed in some studies include miscarriage, reflux, gastric irritations, and cardiovascular diseases. However, evidence for these negative effects is, in general, controversial and was found only in heavy consumers (and may be due to confounding factors such as smoking, and

dietary habits such as low consumption of fruit and vegetables, low physical exercise, etc.). Epidemiological studies addressing the impacts of coffee intake on classic health outcomes such as cancer, cardiovascular disease, osteoporosis, and developmental effects have been largely inconsistent [7, 8]. Overall, no signiﬁcant trend associating moderate coffee consumption with potential adverse effect has emerged from the large epidemiological database on coffee and health.

32.2 Bioactive Components in Coffee

Coffee contains a broad variety of different chemicals (for review, see Ref. [9]). It has been estimated that 1400 compounds are contained in the volatile fraction that accounts for the specific flavor. The chemical structures of the most intensely studied nonvolatile components are shown in Scheme 32.1. The molecular configurations of the melanoidins, which are formed during the roasting process, are largely unknown. The type of coffee plants used, the production processes, and also the brewing methods have a strong impact on the concentrations of different bioactive components (see Table 32.1). During roasting at temperatures between 180 and 240 C for 8–15 min, the concentration of chlorogenic acids (CGAs), the most abundant hydroxycinnamic acids (HCAs) in coffee, declines as a function of time and temperature, while niacin and N-methylpyridinium (from trigonelline) and melanoidins (complex of sugars/amino acids, chlorogenic acids generated by Maillard reaction) are formed. Paper filtration leads to an almost complete removal of the diterpenes cafestol and kahweol (C þ K) and the concentrations of caffeine can be modified by different technologies. Instant coffees contain similar concentrations of C þ K and caffeine as paper filtered brews. In general, the most common preparation method is paper filtration. Italians prefer espresso and mocha and in Scandinavia, Turkey, Greece, and also in former Yugoslavia, unfiltered coffees are widely consumed. The use of different production and brewing methods offers the possibility to design specific coffees with increased health effects. However, the development of successful strategies depends on the knowledge concerning the molecular mechanisms that account for the health effects.

32.3 Mechanisms of Chemoprevention

Two main modes of action are of particular interest in regard to the DNA and cancer protective effects of coffee and its components, namely, inhibition of oxidative damage by ROS and alterations of the activities of drug metabolizing enzymes involved in the activation and detoxiﬁcation of DNA reactive carcinogens. 32.3 Mechanisms of Chemoprevention j581

Scheme 32.1 Structures of bioactive constituents of coffee.

32.3.1 Protective Properties of Coffee

32.3.1.1 Antioxidant Effects The antioxidant (AO) effects of coffee have been studied in a number of in vitro and animal experiments and more recently also in humans. Under in vitro conditions, genotoxic effects were observed with relatively high coffee concentrations in bacterial mutagenicity tests and also in studies with mammalian and human cells. These effects could be explained by the pro-oxidative activities of phenolic constituents at high concentration that result in the formation of 582j 32 Chemopreventive Properties of Coffee and Its Constituents

Table 32.1 Concentration of selected bioactive constituents in coffee.

Content in green beans (% of dry matter)

Effect of Content in coffee Compound Arabica Robusta roasting brew (mg/100 ml)

Caffeine 1.2 2.2 $ 1–80a Chlorogenic acids 6.5 10 ## 25–75 Cafestol and kahweol 1.3–1.9 0.2–1.5 # 0.03–17b Melanoidins 0.0 0.0 """ 25c

aDecaffeinated coffee (1–2 mg/100 ml), drip (paper) ﬁltered coffee (50–80 mg/100 ml). bScandinavian style (10–17 mg/100 ml) > espresso (0.3–3 mg/100 ml) > drip (paper) ﬁltered (0.03–0.4 mg/100 ml). cUnit ¼ % of coffee brew dry matter.

hydrogen peroxide. However, when coffee was tested at lower (physiologically more relevant) concentrations,consistentlyprotectiveeffects againstROS-mediateddamage were observed and it was postulated that the adverse effects do not reflect the physiological situation in vivo animal studies [10]. The latter assumption is also strengthened by the fact that no evidence for genotoxic effects was seen. Data on AO effects from studies with animal models are rather scarce. No effects were seen regarding alterations of isoprostane levels, which reflect lipid peroxidation, but an increase of 8-hydroxy-20-deoxyguanosine (8-OHdG) excretion was found in rats after administration of relatively high doses of coffee (0.62% in the diet) by Sakamoto et al. [11]; in another feeding study with 4.5% coffee beverage, the total antioxidant capacity (TAC) of plasma was clearly enhanced [12]. Asignificantreductionoftheformationofoxidizedpurinesandpyrimidinesandalso

decreased sensitivity toward ROS (H2O2)-induced DNA migration in peripheral lymphocytes was seen in a small human intervention study after consumption of 600 ml of coffee for 5 days by Bichler and coworkers [2]. In the same trial, also a 38% increase of superoxide dismutase (SOD) activity was observed in plasma, while the activity of glutathione peroxidase (GPx) was not affected. The only human study in which an adverse effect of coffee was found was the questionnaire-based investigation of Giovannelli and coworkers [13]; they reported a direct association between coffee drinkingandDNAmigrationduetotheformationofformamidopyrimidineglycosylase (FPG)-sensitive lesions in single-cell gel electrophoresis (SCGE) experiments. In the same study, also an association of DNA damage and the plasma level of lycopene was reported,whileininterventiontrialsprotectiveeffectsofthiscarotenoidwerefound[14]. The evidence for AO effects of coffee is also supported by the observation that plasma glutathione (GSH) levels were found to be increased (16%) in Italian coffee drinkers [15]. It is likely that this effect is due to the induction of g-glutamylcysteine synthetase (g-GCS) by diterpenes, which was observed in experiments with rats by Huber et al. [16]. It is known that GSH is not only a substrate of glutathione S-transferases (GSTs), which catalyze the enzymatic inactivation of speciﬁc ROS, but also a potent antioxidant per se. 32.3 Mechanisms of Chemoprevention j583

A number of in vitro and animal studies were conducted to ﬁnd out as to which coffee types have the highest AO properties, but the results are rather controversial. Richelle et al. [17] found in LDL oxidation assays with human plasma that the AO activity of green Robusta beans is twice as high as that of green Arabica beans but the differences disappeared after roasting. Decaffeination and/or addition of milk had no impact on the ROS protective properties in this study. According to Moreira et al. [18], chlorogenic acids, but not caffeine account for the AO properties of coffee, which were shown to be higher in ground than in instant coffees. On the contrary, several studies indicate that the ROS scavenging effects increase during the roasting process, which leads to a decline of CGAs; for example, an enhancement of AO activity was reported by Del Castillo et al. [19] and also by Nicoli et al. [20]. These ﬁndings can be taken as an indication that Maillard products may account for the AO effects of coffee. Under in vitro conditions, the maximal AO effect was observed after medium roasting, suggesting that both CGA and melanoidins act together.

32.3.1.2 Induction of Detoxifying Enzymes The impact of coffee on the activities of drug metabolizing enzymes has been studied intensely in animal experiments by Huber and coworkers [21, 22]. Briefly, they found induction of phase I (such as CYP1A1 and CYP1A2) and also of phase II enzymes, in particular of UDP-glucuronosyl-transferase (UDPGT) and GST. The latter observations are in agreement with animal studies with unroasted beans [23]. The induction of GST may explain the protective effect of coffee against aflatoxin B1-induced DNA damage which was seen in in vitro studies and in ex vivo experiments with hepatocytes and the prevention of induction of preneoplastic hepatic lesions in rats by this mycotoxin. Also the protection against polycyclic aromatic hydrocarbons (PAHs), which was already observed in the 1970s by Wattenberg and coworkers in carcinogenicity studies with rodents and later also in genotoxicity experiments with human derived liver cells may be due to induction of this enzyme system [24, 25]. Also in human studies evidence for induction of GSTafter consumption of coffee was obtained. For example, Steinkellner et al. [26] found a weak increase of the overall GST activity (8%) and of GST-p (threefold) in plasma, while the activity of GST-a was not affected after consumption of 1 l unfiltered coffee/day for a period of 5 days. The authors postulated that the inhibition of DNA damage by benzo[a]pyrene (B[a]P) in lymphocytes, which they found in subsequent single cell gel electrophoresis experiments, is due to the induction of GST.

32.3.2 Protective Properties of Coffee Components

32.3.2.1 Antioxidant Effects Examples for results of in vitro studies concerning the ROS protective properties of individual coffee components are listed in Table 32.2. 584j 32 Chemopreventive Properties of Coffee and Its Constituents

Table 32.2 Antioxidant properties of individual coffee components in vitro.

Test compound Results and comments

# 1 Caffeine Formation of OH and O2; protection against oxidative damage of calf thymus DNA and of radical induced migration in human lymphocytes Hydroxycinnamic acids Numerous investigations showed inactivation of peroxides

CGAs, CA, FA, and p-CMA (t-BOOH, H2O2), scavenging of DPPH radicals, inhibition of LP, and reduction of radical induced DNA damage in bacterial and mammalian cells; the antioxidant activity of CGA and CA was higher than that of Trolox Cafestol and kahweol No antioxidant effects were found in in vitro experiments with human lymphocytes [2] Maillard products Most studies were conducted with model mixtures, only in melanoidins one, melanoidins from coffees with different degrees of roasting were tested, antiradical effects were found to # with roasting time but prevention of LAperoxidation " with roasting Guaiacol and derivatives Fat-soluble phenolic compounds were found to possess high antioxidant activity in MDA experiments Trigonelline and niacin Were found to possess lower antioxidant properties than CGA and caffeine (measurement of deoxyribose degradation) Caffeoyltryptophan and Caused similar effects as CA and CGA (DPPH radical protocatechuic acid scavenging) Pyrolysis products of caffeic Different components were found to be protective at low doses acid (Stadler et al., 1996) Heterocyclic compounds Strongest effects were seen with 2-acetylpyrrole and 1-methylpyrrole (measurement of conversion of hexanal to hexanoic acid)

Abbreviations: CA, caffeic acid; CGA, chlorogenic acid; DPPH, diphenyl-1-picrylhydrazyl; FA, ferulic 1 . acid; LA, linoleic acid; LP, lipid peroxidation; MDA, malondialdehyde; O2, singlet oxygen; OH , hydroxylradical; p-CMA, p-coumaric acid; t-BOOH, t-butylhydroperoxide.

Only few investigations concerning the antioxidant properties of individual coffee constituents in animals have been published. Caffeine feeding to rats increased the survival of the animals after ionizing radiation but the total peroxyl radical trapping antioxidant activity (TRAP) and thiobarbituric acid reactive substances (TBARS) were not signiﬁcantly altered in plasma in a further study. In another experiment, a signiﬁcant reduction of chromosomal aberrations was observed after treatment of mice with caffeine [27]. þ When rats were fed with chlorogenic acid (CA), they became resistant to Cu2 - induced oxidation of lipoproteins in plasma. The latter acid and CGA prevented also the induction of catalase (CAT), GPx, and GSH reductase by the oxidant paraquat in the livers of rats but did not affect the TBARS levels and SOD activity in the liver [28]. On the other hand, an increase of 8-OHdG excretion in the urine of rats was found with CA (145 mg/kg body weight) by Zhizhina and Blyukhterova [29]. It is notable that the amount of acid given in the latter study is equivalent to the consumption of more than 100 cups of coffee per person, therefore these results are probably not relevant for humans. 32.3 Mechanisms of Chemoprevention j585

In C57Bl/KsJ-db/db mice, caffeic acid significantly increased superoxide dismutase, CAT, and GPx activities and their respective mRNA levels, while lowering H2O2 and thiobarbituric acid reactive substances levels, in the erythrocytes and liver (0.02% caffeic acid in the diet for 5 weeks) [30]. In rats treated with streptozotocin, the supplementation of ferulic acid resulted in a decrease in the TBARS, hydroperoxides and an increase in GSH compared to the control. Ferulic acid also resulted in increased activities of SOD, CAT, and GPx [31]. These results tend to show that the administration of caffeic and ferulic acids helps in enhancing the AO capacity of caffeic acid in an in vivo model of oxidative stress [32]. Dietary supplementation of caffeic acid (0.2 and 0.8%) in rats resulted in a significant increase of a-tocopherol both in plasma and lipoprotein. The plasma AO capacity was increased compared to the control and lipoproteins from caffeic acid-fed rats were more resistant than þ control to Cu2 -catalyzed oxidation [33]. These data show that caffeic and ferulic acids act as AO and help in enhancing the AO capacities in vivo. The effects of caffeic acid on oxidative stress are studies in rats submitted to oxidative stress by iron overload. Male Wistar rats were fed semisynthetic diets containing iron (0.2% of the diet) with and without caffeic acid (0.65% of the diet) for 4 weeks. Caffeic acid prevented the pro-oxidant effects of high iron doses and also reduced iron-induced hypercholesterolemia [32]. The AO of coffee components can be not assessed adequately in in vitro models, which do not reflect alterations of signaling pathways that activate genes related to antioxidant defense mechanisms [34]. In this context, it is notable that N- methylpyridinium, a degradation product of trigonelline, failed to show antioxidant properties in vitro, but when it was administered to rats it caused a strong increase of the TAC of plasma. These effects were also paralleled by a moderate induction of UDPGT and GST in the liver [12].

32.3.2.2 Induction of Detoxifying Enzymes Different coffee components have been shown to either induce detoxifying phase II enzymes and/or to activate enzymes that catalyze the activation of genotoxic carcinogens. Examples of the effects of different bioactive compounds contained in coffee on drug metabolizing enzymes are listed in Table 32.3.

32.3.2.3 Protective Effects of Coffee Constituents Toward Genotoxic Carcinogens The alterations of drug metabolizing enzymes by different coffee components may be causally related to their antimutagenic and anticarcinogenic properties that were seen in a number of studies. The effects of caffeine that were found in animal studies have been reviewed by IARC [9]; in more recent studies, signiﬁcant protective effects toward the induction of breast cancer by 2-amino-1-methyl-6-phenyl-imidazo-[4,5-b]pyridine (PhIP) have been found in experiments with rats [35], which were explained by reduced enzymatic formation of DNA reactive PhIP metabolites. In this context, it is notable that also in a human study a signiﬁcant inverse association of coffee intake with breast cancer incidence was observed only among postmenopausal women [36]. Overall, the results of a 22-year follow-up study suggest a weak inverse association 586j 32 Chemopreventive Properties of Coffee and Its Constituents

Table 32.3 Effects of coffee components on drug metabolizing and DNA repair enzymes.

Test compound Results and comments

Caffeine In vitro Twofold " of CYP1A activity in R hepatocytes with 1.0 mM; evidence of DNA repair inhibition at 0.06–0.3 mM In vivo Twofold " of hepatic CYP1A2 activity in R (0.04% in food); fourfold " of hepatic CYP1A1 and CYP1A2 100 mg/kg b.w. was fed to R; no effects on GSTactivity in R and M; trend toward inhibition of NAT in colons of R but not in other organs; GSH depletion by 40% in a further study with R

Hydroxycinnamic acids In vitro # Of NAT in the gastrointestinal microﬂora with 400 mM of CGA, CA, and FA In vitro Threefold " of GST in liver of R by 0.5% CA in the diet; FA (100 mg/kg body weight) caused a 27% " of hepatic GSTand " of QR in liver and colons of R; GST " in intestines of M by 50–80% with CGA and CA (0.2% in diet); twofold " of GST in the epidermis of M after topical application of CGA; 14% # of hepatic GST activity in R after a SD of 500 mg/kg b.w. CA

Cafestol and kahweol In vitro " Of GST activity in human derived cell lines (HepG2, THLE, Beas 2B); 18% # of SULT subtypes, 40% " of GST and 20% " of UDPGT when HepG2 were exposed to 0.92 mg/ml [25]; 50% " of g-GCS in HepG2 when exposed to 62 mM; for more examples see [37, 38]) In vitro # Of different hepatic CYP450s with 0.2% in the diet in R, SULT # in the same study by 25% (Huber et al., 2007); 13% # of hepatic NATactivity in R (0.02% in the diet) (Huber, unpublished); 0.2% in the diet resulted in a fourfold " of GSTactivity, a threefold " of GSH (" of g-GCS) and a ninefold " of UDPGT in the liver; signiﬁcant " of hepatic activity of O6-MGMTwith 0.04% C þ K in the diet [65]

Melanoidins No conclusive data

N-Methylpyridinium In vitro >20% " of GST activity in CaCo2 cells (0.025 g/100 ml) In vitro GST " in R to a lesser extent than in vitro; 65% " of UDPGTwhen 22 mg/kg b.w. were fed to R [12]

For details see Huber et al. (2005, 2008) [21, 22] and IARC [9]. Abbreviations: b.w., body weight; CA, caffeic acid; C þ K, cafestol and kahweol; CGA, chlorogenic acid; FA, ferulic acid; g-GCS, g-glutamylcysteine synthetase; GSH, glutathione; GST, glutathione S-transferase; M, mice; NAT, N-acetyl transferase; QR, quinone reductase; R, rats, SULT; sulfotransferase, O6-MGMT; O6-methyl-guanine-DNA-methyltransferase.

between caffeine-containing beverages and the risk of postmenopausal breast cancer [36]. However, it is also notable that in a number of animal studies, caffeine was found to increase tumor formation by speciﬁc carcinogens such as 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline-1-oxide (4HAQO), and B(a)P [9]. 32.3 Mechanisms of Chemoprevention j587

The most intensely studied coffee components in regard to chemoprevention of DNA damage and cancer are C þ K. For example, Cavin et al. [37, 38] showed that feeding of the diterpenes to rats protects hepatocytes against DNA damage by AFB1; also, in human epithelial and mammalian cell lines protection toward this mycotoxin was observed. The diterpenes (C þ K) are also apparently protective against PAHs; for example, reduction of B(a)P-induced DNA damage was found in experiments with a human bronchial cell line. Protection against this compound was also seen in carcinogenicity studies with rats [24] and in hamsters oral administration of the diterpenes decreased the number of 7,12-dimethylbenz[a]anthracene (DMBA)-induced buccal pouch tumors. Enzyme measurements indicate that the chemopreventive properties toward AFB1 and PAHs are apparently due to induction of GSTs, which play a key role in the detoxification of these carcinogens. Huber et al. [39] compared in a comprehensive study the protective effects of a number of different dietary antimutagens in regard to prevention of formation of PhIP–DNA adducts in rat colons and found the strongest reduction with C þ K. Subsequent studies indicated that the prevention of heterocyclic amine-induced damage is due to induction of detoxifying enzymes and/or prevention of activation by CYP isozymes and/or NAT. However, these experiments were carried out with relatively high doses of the diterpenes (0.1% of the diet), which are not relevant for humans and may cause hypercholesterolemia. It is notable that the latter animal and human experiments with coffee did not yield evidence of protection toward heterocyclic amines [14]. Coffee itself causes, in contrast to the diterpenes, a pronounced induction of phase I enzymes (in particular CYP1A2) and this may explain why no effects were seen in the later studies. Also, HCAs were tested in a number of carcinogenicity studies with animals and in some of them protective properties were discovered (for review, see Ref. [21]). A study performed by Matsunaga and coworkers [40] demonstrated that supplementation with CGA significantly reduced multiplicity of colon tumors and labeling indices of epithelial cells from non-neoplastic mucosa compared to the control group in a rat model of azoximethane-induced colon carcinogenesis.The same group investigated also the effect of CGA on N-methyl-nitrosourea-induced glandular stomach carcinogenesis in rats. They could demonstrate that the incidence of adenomatous hyperplasia and the proliferation index of non-neoplastic epithelial cells were significantly lower as in control rats by exposure in the postinitiation phase upon CGA supplementation, indicating CGA might be used for the prevention of human stomach cancer [41]. CGA was also shown to be effective in diminishing tongue carcinogenesis. Feeding of CGA significantly reduced the incidence of tongue neoplasms (squamous cell papilloma and carcinoma) and of preneoplastic lesions (hyperplasia and dysplasia) when administered to rats concurrently with the carcinogen 4-nitroquinoline-1-oxide [42].

32.3.2.4 Interaction of Coffee and Coffee Diterpenes with Cell Signaling Pathways Two recent investigations shed light on the molecular mechanisms that account for the induction of xenobiotic drug metabolizing enzymes of coffee. Cavin and coworkers reported in 2008 [43] that coffee induces under in vitro conditions the 588j 32 Chemopreventive Properties of Coffee and Its Constituents

activity of the transcription factor Nrf2 in rat and human hepatocytes and explained the induction of different enzymes by this phenomenon. Subsequently, Higgins þ þ et al. [44] compared the induction of various protective enzymes in nrf2 / and knockout nrf2 / mice. They found more pronounced upregulation of mRNA transcription of genes encoding for NAD(P)H:quinone oxidoreductase 1, GST-a, þ þ UDPGT 1A6, and the glutamate cysteine ligase catalytic subunit in nrf2 / animals and concluded that their activation is regulated by the transcription factor. This is an interesting observation, as a large number of processes, which convey protection toward carcinogens and oxidative damage are controlled by Nrf2 and recent studies showed that potent chemopreventive dietary components, for example, sulforaphane and epigallocatechin gallate, act via Nrf2 induction [45, 46]. The results of additional in vitro studies indicate that the diterpenes C þ K may account partly for the Nrf2 inducing effects of coffee.

32.4 Coffee Consumption and Human Cancer Risks

Data on associations between coffee consumption and the incidence of specific forms of cancer in humans are summarized in Table 32.4. An article that was published in 1981 by MacMahon et al. [47] reported on a significant association between coffee intake and the incidence of pancreas cancer. Subsequently, numerous follow-up studies were conducted and the results were strongly controversial. The findings were evaluated in 1991 by IARC [9] that concluded that the results do not indicate an increased risk. In six out of seven large cohort studies, which were all conducted after 1991, no indication for increased risk was observed and La Vecchia and Tavani [3] concluded in a recent review that it is unlikely that a causal relationship between coffee intake and the incidence of pancreatic cancer exists. Moreover, also Hart et al. [48] analyzed all available publications (till April 2007) and emphasized that the findings are not indicative of an association. In the case of bladder cancer, consistently moderate positive associations were found in a number of studies, but also in this case it is possible that the results are biased by confounding factors (in particular by smoking) [3]. It is notable in this context that no urinary bladder tissue proliferation was observed in rats after repeated supplementation of the drinking water with coffee for a period of up to 6 weeks [49]. Table 32.4 also shows that the results of some studies suggest that coffee consumption is inversely related to the incidence of gastrointestinal cancer. The results obtained in studies concerning the incidence of hepatocellular carcinomas (HCC) are even more promising. In this context, it is notable that a number of animal studies indicate that ROS play a key role in the etiology of colon cancer and HCC (for details, see Refs [50–53]) and it may be tentatively assumed that the antioxidant effects of coffee account for its anticarcinogenic potential. It is also well documented that the incidence of liver cirrhosis is substantially lower in coffee consumers [54, 55] and it is known that this inflammatory disease, which is characterized by oxidative damage, correlates strongly with the incidence of HCC [56]. 32.4 Coffee Consumption and Human Cancer Risks j589

Table 32.4 Association between coffee consumption and cancer risks in different organs.

Cancer site Evidence References

Liver 8 CCSs, 9 CHSs; in all studies dose-dependent risk #; [57, 58] (hepatocellular effects were seen at low intake levels (1 cup/day); risks # carcinoma) 20–50% at intake levels 1 cup/day in prospective studies; recent MA (4 CHS and 5 CCS/4 CHS and 6 CCS) indicate # risk of 43% (95% CI, 51–33) by of 2 cups/day and 41% # (95% CI, 51–28) in drinkers compared to nondrinkers; effects more pronounced in alcohol consumers and subjects with a history of liver disease Gastrointestinal Evidence for inverse relationship between coffee and [59, 60] tract cancer of the large bowel; MA found a pooled RR of 0.97 (95% CI, 0.73–1.29) for 5 CHSs, the combined RR of 12 CCSs was 0.72 (95% CI, 0.61–0.84); later CHSs suggest stronger effects in , than < and higher protection in colon than in rectum; decaffeinated coffee may be more beneficial; most CCSs found inverse association with colon cancer, but there is inconsistency for rectal cancer Upper digestive No association with cancer of the oral cavity, pharynx, and [9, 61] tract esophagus; after 1991, 5 CCSs and 1 CHS failed to find an association; one CCS found a risk # of 40% for oral/ pharyngeal and esophageal cancer (>3 versus 1 cup(s)/ day); a Swedish CHS found " risk of stomach cancer by 22% (increment of 1 cup/day); pooled results of 23 CCSs and CHSs are largely reassuring against " risk for stomach cancer Pancreas 21 CCSs, in 10 a moderate positive association was found; [9, 62] most latter studies failed to find associations; a Japanese CCS found a U-shaped relation (lowest risk among occa- sional consumers); if any, a strong association can be excluded Bladder 22 CCSs and 2 CHSs, risk " in 16 CCSs and in 1 CHS; MA [9, 63, 64] (34 CCSs and 3 CHSs) found risk " by 20% (coffee drinkers versus abstainers), more pronounced in < than in , and similar for regular and decaffeinated coffee; pooled results of 10 European CCS indicate an " risk in nonsmokers when coffee is consumed heavily (10 cups/day: 80% risk "); later CCS show controversial results; a strong association can be excluded

Abbreviations: CCS, case-control study; CHS, cohort study; MA, meta-analysis.

In this context, it is notable that recent animal experiments showed that the supplementation of drinking water with instant coffee prevented the induction of cirrhosis by CCl4 in rats and it was found in the same study that clinical markers, such as alanine transaminase (ALT), aspartate transaminase (AST), and g-glutamyl transferase (GGT), are strongly reduced by coffee supplementation and also by coffee-speciﬁc diterpenes. Also, in human studies, the evidence for reduction of ALT, 590j 32 Chemopreventive Properties of Coffee and Its Constituents

AST, and GGT levels was seen in coffee drinkers, in particular, those who are heavy alcohol consumers.

32.5 Concluding Remarks

Taken together, the evaluation of the current state of knowledge on DNA and cancer protective properties of coffee shows that it is a highly promising beverage having beneficial effects on humans. On the basis of recent studies, evidence is accumulating that its consumption prevents oxidative DNA damage as well as the incidence of cancers in different organs. These findings are of particular relevance in regard to the fact that coffee is consumed in large quantities all over the world. As mentioned above, the identification of specific protective compounds and the characterization of their molecular mechanisms may pave the way for the development of brands with improved antioxidant and anticarcinogenic properties.

References

1 (2003) Earth Trends. Agriculture and food Crehuet Navajas, R. (2007) Alzheimers database. World Resources Institute disease and coffee: a quantitative review. (WRI), http://earthtrends.wri.org/ Neurological Research, 29,91–95. searchable_db/index.php?theme=8. 6 vanDam,R.M.andHu,F.B.(2005) 2 Bichler, J., Cavin, C., Simic, T., Coffee consumption and risk of type 2 Chakraborty, A., Ferk, F., Hoelzl, C., diabetes: a systematic review. The Journal Schulte-Hermann, R., Kundi, M., of the American Medical Association, 294, Haidinger, G., Angelis, K. and Knasmuller, 97–104. S. (2007) Coffee consumption protects 7 Schilter, B., Holzhauser, D., Cavin, C. human lymphocytes against oxidative and (2001) Health beneﬁts of coffee. 19th 3-amino-1-methyl-5H-pyrido[4,3-b]indole International Conference on Coffee acetate (Trp-P-2) induced DNA-damage: Science, ASIC, Trieste, Italy. results of an experimental study with 8 Higdon, J.V. and Frei, B. (2006) Coffee and human volunteers. Food and Chemical health: a review of recent human research. Toxicology, 45, 1428–1436. Critical Reviews in Food Science and 3 La Vecchia, C. and Tavani, A. (2007) Nutrition, 46, 101–123. Coffee and cancer risk: an update. 9 IARC (1991) Coffee, tea, mate, European Journal of Cancer Prevention, 16, methylxanthines and methylglyoxal, in 385–389. IARC Monographs on the Evaluation of the 4 Hernan, M.A., Takkouche, B., Caamano- Carcinogenic Risks to Humans, World Isorna, F. and Gestal-Otero, J.J. (2002) A Health Organization, Lyon, pp. 41–206. meta-analysis of coffee drinking, 10 Stadler, R.H., Turesky, R.J., Muller, O., cigarette smoking, and the risk of Markovic, J. and Leong-Morgenthaler, Parkinsons disease. Annals of Neurology, P.M. (1994) The inhibitory effects of coffee 52,276–284. on radical-mediated oxidation and 5 Barranco Quintana, J.L., Allam, M.F., mutagenicity. Mutation Research, 308, Serrano Del Castillo, A. and Fernandez- 177–190. References j591

11 Sakamoto, W., Isomura, H., Fujie, K., Agricultural and Food Chemistry, 49, Nishihira, J., Ozaki, M. and Yukawa, S. 3438–3442. (2003) Coffee increases levels of urinary 18 Moreira, D.P., Monteiro, M.C., Ribeiro- 8-hydroxydeoxyguanosine in rats. Alves, M., Donangelo, C.M. and Trugo, Toxicology, 183, 255–263. L.C. (2005) Contribution of chlorogenic 12 Somoza, V., Lindenmeier, M., Wenzel, E., acids to the iron-reducing activity of coffee Frank, O., Erbersdobler, H.F. and beverages. Journal of Agricultural and Food Hofmann, T. (2003) Activity-guided Chemistry, 53, 1399–1402. identiﬁcation of a chemopreventive 19 del Castillo, M.D., Ames, J.M. and Gordon, compound in coffee beverage using in vitro M.H. (2002) Effect of roasting on the and in vivo techniques. Journal of antioxidant activity of coffee brews. Journal Agricultural and Food Chemistry, 51, of Agricultural and Food Chemistry, 50, 6861–6869. 3698–3703. 13 Giovannelli, L., Saieva, C., Masala, G., 20 Nicoli, M.C., Anese, M., Parpinel, M.T., Testa, G., Salvini, S., Pitozzi, V., Riboli, E., Franceschi, S. and Lerici, C.R. (1997) Loss Dolara, P. and Palli, D. (2002) Nutritional and/or formation of antioxidants during and lifestyle determinants of DNA food processing and storage. Cancer Letters, oxidative damage: a study in a 114,71–74. Mediterranean population. Carcinogenesis, 21 Huber, W.W. and Parzefall, W. (2005) 23, 1483–1489. Modiﬁcation of N-acetyltransferases and 14 Nersesyan, A., Hoelzl, C., Ferk, F., Misik, glutathione S-transferases by coffee M. and Knasmuller,€ S. (2009) Use of single components: possible relevance for cancer cell gel electrophoresis assays in dietary risk. Methods in Enzymology, 401, 307–341. intervention trials, in The Comet Assay in 22 Huber, W.W.,Rossmanith, W., Grusch, M., Toxicology, (eds A. Dhawan and D. Haslinger, E., Prustomersky, S., Peter- Anderson), Royal Society of Chemistry, Vorosmarty, B., Parzefall, W., Scharf, G. Cambridge, UK. and Schulte-Hermann, R. (2008) Effects of 15 Esposito, F., Morisco, F., Verde, V., Ritieni, coffee and its chemopreventive A., Alezio, A., Caporaso, N. and Fogliano, components kahweol and cafestol on V. (2003) Moderate coffee consumption cytochrome P450 and sulfotransferase increases plasma glutathione but not in rat liver. Food and Chemical Toxicology, homocysteine in healthy subjects. 46, 1230–1238. Alimentary Pharmacology & Therapeutics, 23 Lam, L.K., Sparnins, V.L. and Wattenberg, 17, 595–601. L.W. (1987) Effects of derivatives of 16 Huber, W.W., Scharf, G., Rossmanith, W., kahweol and cafestol on the activity of Prustomersky, S., Grasl-Kraupp, B., glutathione S-transferase in mice. Journal Peter, B., Turesky, R.J. and Schulte- of Medicinal Chemistry, 30, 1399–1403. Hermann, R. (2002) The coffee 24 Wattenberg, L.W. (1983) Inhibition of components kahweol and cafestol induce neoplasia by minor dietary constituents. gamma-glutamylcysteine synthetase, the Cancer Research, 43, 2448s–2453s. rate limiting enzyme of chemoprotective 25 Majer, B.J., Hofer, E., Cavin, C., Lhoste, glutathione synthesis, in several organs E.,Uhl,M.,Glatt,H.R.,Meinl,W.and of the rat. Archives of Toxicology, 75, Knasmuller, S. (2005) Coffee diterpenes 685–694. prevent the genotoxic effects of 2-amino- 17 Richelle, M., Tavazzi, I. and Offord, E. 1-methyl-6-phenylimidazo[4,5-b]pyridine (2001) Comparison of the antioxidant (PhIP) and N-nitrosodimethylamine in a activity of commonly consumed human derived liver cell line (HepG2). polyphenolic beverages (coffee, cocoa, and Food and Chemical Toxicology, 43, tea) prepared per cup serving. Journal of 433–441. 592j 32 Chemopreventive Properties of Coffee and Its Constituents

26 Steinkellner, H., Hoelzl, C., Uhl, M., 34 Knasmuller,€ S., Nersesyan, A., Mišık, M., Cavin, C., Haidinger, G., Gsur, A., Schmid, Gerner, C., Mikulits, W., Ehrlich, V., R., Kundi, M., Bichler, J. and Knasmuller, Hoelzl, C., Szakmary, A. and Wagner, K.- S. (2005) Coffee consumption induces H. (2008) Use of conventional and -OMICS GSTP in plasma and protects lymphocytes based methods for health claims of dietary against ( þ /)-anti-benzo[a]pyrene-7,8- antioxidants: a critical overview. The British dihydrodiol-9,10-epoxide induced DNA- Journal of Nutrition, 99 (Suppl. 1), S3–S52. damage: results of controlled human 35 Hirose, M., Nishikawa, A., Shibutani, M., intervention trials. Mutation Research, 591, Imai, T. and Shirai, T. (2002) 264–275. Chemoprevention of heterocyclic amine- 27 Farooqi, Z. and Kesavan, P.C. (1992) induced mammary carcinogenesis in rats. Radioprotection by caffeine pre- and post- Environmental and Molecular Mutagenesis, treatment in the bone marrow 39, 271–278. chromosomes of mice given whole-body 36 Ganmaa, D., Willett, W.C., Li, T.Y., gamma-irradiation. Mutation Research, Feskanich, D., van Dam, R.M., Lopez- 269, 225–230. Garcia, E., Hunter, D.J. and Holmes, M.D. 28 Tsuchiya, T., Suzuki, O. and Igarashi, K. (2008) Coffee, tea, caffeine and risk of (1996) Protective effects of chlorogenic breast cancer: a 22-year follow-up. acid on paraquat-induced oxidative stress International Journal of Cancer, 122, in rats, Bioscience, Biotechnology, and 2071–2076. Biochemistry, 60, 765–768. 37 Cavin, C., Mace, K., Offord, E.A. and 29 Zhizhina, G.P. and Blyukhterova, N.V. Schilter, B. (2001) Protective effects of (1997) Effect of metal ions and xenobiotics coffee diterpenes against aﬂatoxin B1- on endogenous oxidation of DNA. induced genotoxicity: mechanisms in rat Biochemistry, 62,88–94. and human cells. Food and Chemical 30 Jung, U.J., Lee, M.K., Park, Y.B.,Jeon, S.M. Toxicology, 39, 549–556. and Choi, M.S. (2006) Antihyperglycemic 38 Cavin, C., Bezencon, C., Guignard, G. and and antioxidant properties of caffeic acid in Schilter, B. (2003) Coffee diterpenes db/db mice. The Journal of Pharmacology prevent benzo[a]pyrene genotoxicity in rat and Experimental Therapeutics, 318, and human culture systems. Biochemical 476–483. and Biophysical Research Communications, 31 Balasubashini, M.S., Rukkumani, R., 306, 488–495. Viswanathan, P. and Menon, V.P. (2004) 39 Huber, W.W., McDaniel, L.P., Kaderlik, Ferulic acid alleviates lipid peroxidation in K.R., Teitel, C.H., Lang, N.P. and Kadlubar, diabetic rats. Phytotherapy Research, 18, F.F. (1997) Chemoprotection against the 310–314. formation of colon DNA adducts from 32 Lafay, S., Gueux, E., Rayssiguier, Y.,Mazur, the food-borne carcinogen 2-amino-1- A., Remesy, C. and Scalbert, A. (2005) methyl-6-phenylimidazo[4,5-b]pyridine Caffeic acid inhibits oxidative Stress and (PhIP) in the rat. Mutation Research, 376, reduces hypercholesterolemia induced by 115–122. iron overload in rats. International Journal 40 Matsunaga, K., Katayama, M., Sakata, K., for Vitamin and Nutrition Research. 75, Kuno, T., Yoshida, K., Yamada, Y., Hirose, Y., 119–125. Yoshimi, N. and Mori, H. (2002) Inhibitory 33 Nardini, M., Natella, F., Gentili, V., Di effectsofchlorogenicacidonazoxymethane- Felice, M. and Scaccini, C. (1997) Effect of induced colon carcinogenesis in male F344 caffeic acid dietary supplementation on the rats. Asian Paciﬁc Journal of Cancer antioxidant defense system in rat: an in vivo Prevention, 3,163–166. study. Archives of Biochemistry and 41 Shimizu, M., Yoshimi, N., Yamada, Y., Biophysics, 342, 157–160. Matsunaga, K., Kawabata, K., Hara, A., References j593

Moriwaki, H. and Mori, H. (1999) Gastroenterology and Hepatology, 6, Suppressive effects of chlorogenic acid on 275–282. N-methyl-N-nitrosourea-induced 49 Lina, B.A., Rutten, A.A. and Woutersen, glandular stomach carcinogenesis in male R.A. (1993) Effect of coffee drinking on cell F344 rats. The Journal of Toxicological proliferation in rat urinary bladder Sciences, 24, 433–439. epithelium. Food and Chemical Toxicology, 42 Tanaka, T., Kojima, T., Kawamori, T., 31, 947–951. Wang, A., Suzui, M., Okamoto, K. and 50 Bartsch, H., Nair, J. and Owen, R.W. (2002) Mori, H. (1993) Inhibition of Exocyclic DNA adducts as oxidative stress 4?-nitroquinoline-1-oxide-induced rat markers in colon carcinogenesis: potential tongue carcinogenesis by the naturally role of lipid peroxidation, dietary fat and occurring plant phenolics caffeic, ellagic, antioxidants. Biological Chemistry, 383, chlorogenic and ferulic acids. 915–921. Carcinogenesis, 14, 1321–1325. 51 Bruce, W.R., Giacca, A. and Medline, A. 43 Cavin, C., Marin-Kuan, M., Langouet, S., (2000) Possible mechanisms relating diet Bezencon, C., Guignard, G., Verguet, C., andriskofcoloncancer.CancerEpidemiology, Piguet, D., Holzhauser, D., Cornaz, R. and Biomarkers & Prevention, 9,1271–1279. Schilter, B. (2008) Induction of Nrf2- 52 Teufelhofer, O., Parzefall, W., Kainzbauer, mediated cellular defenses and alteration E., Ferk, F., Freiler, C., Knasmuller, S., of phase I activities as mechanisms of Elbling, L., Thurman, R. and Schulte- chemoprotective effects of coffee in the Hermann, R. (2005) Superoxide liver. Food and Chemical Toxicology, 46, generation from Kupffer cells contributes 1239–1248. to hepatocarcinogenesis: studies on 44 Higgins, L.G., Cavin, C., Itoh, K., NADPH oxidase knockout mice. Yamamoto, M. and Hayes, J.D. (2008) Carcinogenesis, 26, 319–329. Induction of cancer chemopreventive 53 Hussain, S.P., Hofseth, L.J. and Harris, enzymes by coffee is mediated by C.C. (2003) Radical causes of cancer. transcription factor Nrf2. Evidence that the Nature Reviews, 3, 276–285. coffee-speciﬁc diterpenes cafestol and 54 La Vecchia, C. (2005) Coffee, liver kahweol confer protection against enzymes, cirrhosis and liver cancer. acrolein. Toxicology and Applied Journal of Hepatology, 42, 444–446. Pharmacology, 226, 328–337. 55 Cadden, I.S., Partovi, N. and Yoshida, E.M. 45 Juge, N., Mithen, R.F. and Traka, M. (2007) (2007) Review article: possible beneﬁcial Molecular basis for chemoprevention by effects of coffee on liver disease and sulforaphane: a comprehensive review. function. Alimentary Pharmacology & Cellular and Molecular Life Sciences, 64, Therapeutics, 26,1–8. 1105–1127. 56 Vali, L., Hahn, O., Kupcsulik, P., Drahos, 46 Na, H.K. and Surh, Y.J. (2008) Modulation A., Sarvary, E., Szentmihalyi, K., Pallai, Z., of Nrf2-mediated antioxidant and Kurucz, T., Sipos, P. and Blazovics, A. detoxifying enzyme induction by the green (2008) Oxidative stress with altered tea polyphenol EGCG. Food and Chemical element content and decreased ATP level Toxicology, 46, 1271–1278. of erythrocytes in hepatocellular 47 MacMahon, B., Yen, S., Trichopoulos, D., carcinoma and colorectal liver metastases. Warren, K. and Nardi, G. (1981) Coffee and European Journal of Gastroenterology & cancer of the pancreas. The New England Hepatology, 20, 393–398. Journal of Medicine, 304, 630–633. 57 Larsson, S.C. and Wolk, A. (2007) Coffee 48 Hart, A.R., Kennedy, H. and Harvey, I. consumption and risk of liver cancer: a (2008) Pancreatic cancer: a review of the meta-analysis. Gastroenterology, 132, evidence on causation. Clinical 1740–1745. 594j 32 Chemopreventive Properties of Coffee and Its Constituents

58 Bravi,F.,Bosetti,C.,Tavani,A.,Bagnardi, 63 Zeegers, M.P., Tan, F.E., Goldbohm, R.A. V.,Gallus,S.,Negri,E.,Franceschi,S. and van den Brandt, P.A. (2001) Are coffee and La Vecchia, C. (2007) Coffee drinking and tea consumption associated with and hepatocellular carcinoma risk: urinary tract cancer risk? A systematic ameta-analysis.Hepatology, 46, review and meta-analysis. International 430–435. Journal of Epidemiology, 30, 353–362. 59 Giovannucci, E. (1998) Meta-analysis of 64 Sala, M., Cordier, S., Chang-Claude, J., coffee consumption and risk of colorectal Donato, F., Escolar-Pujolar, A., Fernandez, cancer. American Journal of Epidemiology, F., Gonzalez, C.A., Greiser, E., Jockel, 147, 1043–1052. K.H., Lynge, E., Mannetje, A., Pohlabeln, 60 Tavani,A. and La Vecchia, C. (2004) Coffee, H., Porru, S., Serra, C., Tzonou, A., Vineis, decaffeinated coffee, tea and cancer of the P., Wahrendorf, J., Boffetta, P. and colon and rectum: a review of Kogevina, M. (2000) Coffee consumption epidemiological studies, 1990–2003. and bladder cancer in nonsmokers: a Cancer Causes & Control, 15, 743–757. pooled analysis of case-control studies in 61 Botelho, F., Lunet, N. and Barros, H. (2006) European countries. Cancer Causes & Coffee and gastric cancer: systematic Control, 11, 925–931. review and meta-analysis. Cadernos de 65 Huber, W.W., Scharf, G., Nagel, G., saude publica, 22, 889–900. Prustomersky, S., Schulte-Hermann, R., 62 Michaud, D.S., Giovannucci, E., Willett, Kaina, B., (2003) Coffee and its W.C., Colditz, G.A. and Fuchs, C.S. (2001) chemopreventive components Kahweol Coffee and alcohol consumption and the and Cafestol increase the activity of O6- risk of pancreatic cancer in two methylguanine-DNA methyltransferase prospective United States cohorts. Cancer in rat liver-comparison with phase II Epidemiology, Biomarkers & Prevention, 10, xenobiotic metabolism. Mutation Research, 429–437. 522,57–68. j595

33 Tea and Its Constituents

33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis Joshua D. Lambert and Chung S. Yang

33.1.1 Introduction

Tea (Camellia sinensis, Theaceae) is a beverage with worldwide popularity second only to water [1]. Tea is consumed in one of the three major forms: green tea, black tea, or oolong tea [2, 3]. These types of tea differ with regard to production methods and chemical composition. Green tea, which accounts for 20% of world consumption, is prepared by pan frying the tea leaves to inactivate polyphenol oxidase. This process preserves the characteristic tea catechins (Scheme 33.1). By contrast, black tea, representing 80% of world tea consumption, is prepared by crushing the tea leaves and allowing an oxidative process to occur commonly known as fermentation. This process results in the oxidation of the tea catechins to form theaflavins (Scheme 33.NaN), as well as, the oligomeric polyphenolic compounds called thearubigins. Oolong tea is a partially oxidized product that retains higher levels of catechins and contains newly formed polyphenolic oligomers. A typical cup of brewed green tea contains, by dry weight, 30–40% catechins including epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3- gallate (EGCG). By contrast, a typical cup of black tea contains 3–10% catechins, 2–6% theaflavins, and >20% thearubigins [2]. Both tea and tea constituents have been extensively studied for their potential as cancer-preventive agents. In this chapter, we review the effect of tea and tea constituents on oxidative stress, carcinogen metabolism, and DNA damage as possible mechanisms of the chemopreventive activity of tea. We also discuss some of the key studies in animal models of carcinogenesis and briefly review some of the proposed postinitiation mechanisms for the cancer-preventive effects of tea. We hope to point out some of the outstanding questions regarding cancer prevention by tea and stimulate research in this area.

Scheme 33.1 Structures of the major tea polyphenols.

33.1.2 Bioavailability and Biotransformation of Tea Polyphenols

33.1.2.1 Biotransformation of Tea Polyphenols We and others have extensively studied the biotransformation of the green tea polyphenols [3–6] and found that these compounds undergo extensive biotransformation and may be subject to active efﬂux. Tea catechins are subject to methylation, glucuronidation, sulfation, and ring-ﬁssion metabolism. EGC is readily methylated by catechol-O-methyltransferase (COMT) to form 40-O-methyl-()-EGC and EGCG is methylated to form 400-O-methyl-()-EGCG and 40,400-O-dimethyl-()-EGCG [7]. Rat liver cytosol shows higher COMT activity toward EGCG and EGC than does

human or mouse liver cytosol. In addition, the Km and Vmax values are higher for EGC than for EGCG. At low concentrations of EGCG, the dimethylated compound is the major product, whereas at high EGCG concentrations, monomethylated EGCG metabolites are formed. Studies of EGCG and EGC glucuronidation reveal that EGCG-400-O-glucuronide is the major metabolite formed by humans, mice, and rats [8]. Mouse small intestinal fi microsomes have the greatest catalytic ef ciency (Vmax/Km) for glucuronidation followed, in decreasing order, by mouse liver, human liver, rat liver, and rat small intestine. Human UGT1A1, 1A8, and 1A9 have high glucuronidation activity toward EGCG, with the intestinal-specific UGT1A8 being the most efficient. EGC-30-O-glucuronide is the major product formed by microsomes from mice, rats, and humans with the liver microsomes having a higher efficiency than intestinal microsomes. On the basis of these studies, it appears that mice are more similar to humans in terms of enzymatic ability to glucuronidate tea catechins than are rats. Vaidyanathan et al. have shown that EC undergoes sulfation catalyzed by human and rat intestinal and liver enzymes in cytosol, with the human liver being the most efficient [9]. Sulfotransferase (SULT)1A1 is largely responsible for this 33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis j597 activity in the liver; however, both SULT1A1 and SULT1A3 are active in the human intestine. EGCG is also sulfated by human, mouse, rat liver cytosol, although the structures of the metabolites remain to be determined [10]. Rat has the greatest activity followed by mouse and humans. Our recent results from data-dependent tandem mass spectrometric analysis of mouse urine samples after intraperitoneal or intragastric administration of EGCG have shown that methylated EGCG (or glucuronidated or sulfated EGCG) can be further glucuronidated and/or sulfated (or methylated) to form related mixed EGCG metabolites (Sang, unpublished data). At toxic doses, EGCG can form two cysteine adducts in vivo, EGCG-200-cysteine and EGCG-20-cysteine [11]. These metabolites can be detected in urine following administration of 200–400 mg/kg, i.p. or 1500 mg/kg, i.g. EGCG. We hypothesize that these metabolites form as a result of oxidation of EGCG to a quinone or semiquinone that then reacts with the sulfhydryl group of cysteine. It is possible that similar metabolites will be formed by reaction with glutathione and N-acetylcysteine, but these metabolites remain to be discovered. Active efflux has been shown to limit the bioavailability and cellular accumulation of many compounds. The multidrug resistance-associated proteins (MRP) are ATP- dependent efflux transporters that are expressed in many tissues and are overexpressed in many human tumor types. These proteins may play a role in limiting the bioavailability of tea catechins. We have reported that indomethacin (MRP inhibitor) increases the intracellular accumulation of EGCG, EGCG 400-O-methyl-EGCG, or 40,400-di-O-methyl-EGCG by 10-, 11-, or 3-fold in Madin-Darby canine kidney (MDCKII) cells overexpressing MRP-1 [12]. Similarly, treatment of MRP-2 overexpressing MDCKII cells with MK-571 (an MRP-2 inhibitor) results in 10-, 15-, or 12-fold increase in the intracellular levels of EGCG, 400-O-methyl-EGCG, and 40,400-di-O-methyl-EGCG, respectively. Vaidyanathan and Walle have shown that treatment of Caco-2 cells with MK-571 enhances the apical-to-basolateral movement of EC and ECG [13, 14]. MK-571 also reduced the efflux of EC-sulfates from the cytosol to the apical well, suggesting that the EC-sulfates are also substrates for MRP-2 [13]. Although we have previously suggested that the uptake of EGCG into HT-29 cells takes place predominantly by passive diffusion, our recent results have suggested that carrier-mediated transport may also play a role (unpublished results). Others have demonstrated that ECG is a substrate for the monocarboxylate transporter (MCT) and that inhibition of this transporter by benzoic acid or phloretin significantly reduced uptake by Caco-2 cells [14]. In addition to phase II metabolites, our laboratory has identified several ring fission products of tea catechins in human urine and plasma after oral ingestion of decaffeinated green tea [15]. The compounds 5-(30,40,50-trihydroxyphenyl)-g- valerolactone (M4), 5-(30,40-dihydroxyphenyl)-g-valerolactone (M6), and 5-(30,50- dihydroxyphenyl)-g-valerolactone (M60) are believed to be derived from microbial metabolism in the colon. Indeed, anaerobic fermentation of EGC, EC, and ECG with human fecal microflora has been shown to result in the production of M4, M6, and M60 [16]. 598j 33 Tea and Its Constituents

The combined effects of MRP-1, MRP-2, and MCT on the bioavailability of tea polyphenols remain to be determined in vivo. MRP-2, with its localization on the apical membrane of the small intestine, likely acts to limit EGCG bioavailability by actively exporting EGCG in the enterocyte back into the intestinal lumen either before or after being methylated by cytosolic COMT or glucuronidated by UGT. The remaining fraction of EGCG would then be absorbed into the portal circulation, enter the liver, and could subsequently be effluxed by MRP-2 located on the canalicular membrane of the hepatocytes. In contrast, MRP-1 is located on the basolateral membrane of enterocytes, hepatocytes, and other tissues. Substrates of this pump are effluxed from the interior of the cells into the blood stream or interstitial space. The role of MRP-1 would be expected to increase the bioavailability of EGCG in vivo. The influence of MRP-1 and -2 on the bioavailability of EGCG in vivo, however, is likely to depend on the tissue distribution of each efflux protein. It has been reported that the expression level of MRP-2 is 10-fold higher than that of MRP-1 in human jejunum [17]. Efflux of EGCG by MRP-2 may be predominant in the intestine, resulting in a decrease of bioavailability.

33.1.2.2 Pharmacokinetics of Tea Polyphenols Pharmacokinetic studies of tea catechins have been conducted in rats, mice, and humans [18]. Following i.g. administration of decaffeinated green tea (200 mg/kg) to rats, plasma levels of EGCG, EGC, and EC were ﬁt to a two-compartment model with elimination half-lives of 165, 66, and 67 min, respectively. The absolute bioavailability of EGCG, EGC, and EC after i.g. administration of decaffeinated green tea is 0.1, 14, and 31%, respectively. By comparison, the absolute bioavailability of EGCG in mice following i.g. administration of 75 mg/kg EGCG is 26.5%. Concentrations of EGCG in the small intestine and colon are 45 and 7.9 nmol/g, respectively, following i.g. administration. The levels in other tissues are less than 0.1 nmol/g. Although more than 50% of plasma EGCG is present as glucuronide, EGCG is present mainly in the free form in tissues [19]. Administration of 50–2000 mg/kg, i.g. EGCG to mice resulted in a linear increase in the plasma, liver, and prostate. In contrast, the levels of EGCG in the small intestine and colon plateaued at 500 mg/kg, i.g. [20]. These results suggest that small intestinal and colonic tissues become saturated with EGCG, resulting in a plateau of the levels in these tissues. Recently, Chu et al. [21] reported the pharmacokinetics of orally administered green tea catechins in the plasma and fetuses of pregnant rats. Following treatment with 55 mg/kg, i.g. green tea extract, EC had the highest, and GCG had

the lowest, Cmax in the maternal plasma at 9.8 and 0.04 mM, respectively. These levels were 10 and 50–100 times higher than those in the placenta and the fetus, respectively. Treating rats with a green tea polyphenol preparation (0.6% wt/v) in the drinking fluid has been shown to result in increasing plasma levels over a 14-day period with levels of EGC and EC being higher than those of EGCG [22]. Plasma levels then decrease over the subsequent 14 days suggesting an adaptive effect. When the same polyphenol preparation was given to mice, the EGCG levels in the 33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis j599 plasma, lung, and liver were much higher than in rats. These levels of EGCG in the mouse peaked on day 4 and then decreased to less than 20% of the peak values by day 10 [22]. Several studies of the systemic bioavailability of orally administered green tea and catechins in human volunteers have been conducted. We have shown that oral administration of 20 mg green tea solids/kg body weight results in Cmax in the plasma for EGC, EC, and EGCG of 223, 124, and 77.9 ng/ml, respectively [23]. Plasma EC and EGC are present mainly in glucuronidated and sulfated form whereas 77% of the EGCG is in free form. EGC but not EC is also methylated (to 40-O-methyl-EGC) in humans. EGCG has also been found as a methylated metabolite. The maximum plasma concentration of 40,400-di-O-methyl-EGCG is 20% of that of EGCG but the cumulative excretion of 40,400-di-O-methyl-EGCG in urine is 10-fold higher (140 mg) than that of EGCG (16 mg) over 24 h [24]. In addition to methylated and conjugated metabolites, the ring-fission metabolites M4, M6, and M60 have been detected in urine at 8, 4, and 8 mM, respectively following ingestion of 200 mg EGCG [15, 24]. Chow et al. [25] have demonstrated that after treating human volunteers with green tea polyphenol with a dosing schedule of 800 mg once daily for 4 weeks, there was an increase in the area under the plasma EGCG concentration–time curve from 95.6 to 145.6 min/(mg min). No significant changes were observed in the pharmacokinetics of EGCG after repeated green tea polyphenol treatment with a regimen of 400 mg twice daily. Similarly, there was no significant change in the area under the curve for EGC or EC.

33.1.3 In Vitro and Animal Studies of Cancer Prevention by Tea

33.1.3.1 Antioxidative/Pro-Oxidative Activities Tea polyphenols have strong antioxidant properties due to the radical scavenging and metal chelating abilities of dihydroxyl- and trihydroxyl-substituted aromatic rings. These compounds have been shown to scavenge a number of different reactive oxygen species (ROS) in vitro (reviewed in Refs [1, 26]). An increasing number of studies have also demonstrated these antioxidative effects in vivo, although the effects are not as strong. Senthil Kumaran et al. [27] have demonstrated that supplementation with EGCG can reduce age-related increases in oxidative stress in rats. For example, treating 24- month-old rats with 100 mg/kg, i.g. EGCG decreased the levels of lipid peroxidation and protein carbonylation in the liver by 50 and 39%, respectively. The hepatic levels of both antioxidants (e.g., reduced glutathione) as well as antioxidant enzymes (e.g., superoxide dismutase (SOD)) were increased by EGCG treatment. Similar effects were also observed in skeletal muscle. In a second study, the same research group found that treating 24-month-old rats for 30 days with 2 mg/kg, i.g. EGCG reduced the levels of protein carbonyls and malonyldialdehyde, increased levels of ascorbic acid, a-tocopherol, and reduced glutathione in the brain [28]. In both of these studies, no effects were observed in young rats, suggesting that EGCG offered no protective effect in the absence of increased oxidative stress. This, as well as the type of tea 600j 33 Tea and Its Constituents

studied and the dose used, may explain why some other studies have observed no signiﬁcant effect of tea or tea polyphenols on antioxidant status [1, 29]. Our laboratory and others have suggested a role for pro-oxidative effects of tea components. EGCG, as well as the other catechins, are unstable under cell culture conditions and subject to oxidation and polymerization, with concurrent

formation of superoxide anion and H2O2 [30, 31]. Inclusion of SOD and catalase, which stabilize the EGCG and prevent formation of reactive oxygen species, in cell culture also ablates a number of the biological effects attributed to EGCG. For example, exogenous catalase completely inhibited EGCG-induced apoptosis in H661 human lung cancer cells [32]. EGCG-mediated induction of apoptosis and increased gene expression in the transforming growth factor (TGF)b pathway in transformed bronchial epithelial cells were ablated by inclusion of exogenous catalase, but EGCG-mediated decreases of genes expression in the bone morphoge- netic protein (BMP) pathway were not affected [33]. These results suggest a role

for H2O2 in the effects on TGFb but not in the effects on BMP. Exogenous SOD prevented EGCG-induced decreases of epidermal growth factor receptor protein level and phosphorylation, but not cell growth inhibition, in human esophageal cancer cells [34]. The importance of antioxidative and pro-oxidative activities in vivo remains to be determined. It is possible that EGCG, and other catechins, work by directly scavenging free radicals and chelating free transition metals and thereby reducing oxidative stress. It is also possible that in the absence of pre-existing oxidative stress, tea polyphenols generate a low level of reactive oxygen species that stimulate upregulation of the endogenous antioxidant systems by mechanisms such as the antioxidant response element/Nrf2 signaling pathway. Indeed, Yuan et al. [35] have recently reported that treating colon cancer xenograft-bearing nude mice with dietary EGCG caused dose-dependent upregulation of Nrf2 expression in orthotopically implanted colon tumors. Shen et al. [36] reported similar results in nontumor-bearing C57bl/6J mice.

33.1.3.2 Effects on Carcinogen Metabolism Studies have suggested that tea polyphenols can inhibit the expression of carcinogen activating enzymes such as cytochromes P-450 (CYP) and increase the levels of enzymes that detoxify carcinogens. The changes in the plasma and tissue levels of carcinogen metabolites are likely due to changes in the expression of the metabolizing enzymes rather than direct affects on enzyme activity. The following are several examples of modulation of carcinogen metabolism by tea preparations. Krishnan et al. [37] have shown that pretreatment of Swiss mice with 1% black tea polyphenols blunted the increase in the expression of CYP1A1 and 1A2 induced by a single oral dose of benzo[a]pyrene (B[a]P, mg/mouse). This is likely due to interference at the aromatic hydrocarbon receptor level. Western blot analysis showed that B[a]P-induced expression of these two isoforms was decreased by 55–58% and 79–86% by black tea polyphenols in the liver and the lung, respectively. Similar decreases in the microsomal activity of CYP1A1 and CYP1A2 were observed. 33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis j601

Intragastric treatment of rats with theaflavins (20 mg/kg) for 4 weeks reduced CYP1A1 activity in the intestine but not in the liver [38]. This discrepancy may be due to the limited systemic bioavailability of theaflavins. Theaflavins also decreased the protein levels of CYP2E1 in intestinal microsomes in the same study. Long-term treatment of rats with green or black tea resulted in an increased expression of hepatic CYP1A1 and 1A2 in the absence of carcinogen treatment [39, 40]. Studies at our laboratory have suggested that induction of CYP1A1 and 1A2 activity is likely due to the caffeine [41]. Studies in animal models also support the idea that tea treatment can induce Phase II enzymes. Treatment of piglets with 0.2% green tea extract (45% EGCG) for 3 weeks increased the rate of formation of glutathione conjugated aflatoxin

(AF)B1 by small intestinal microsomes in ex vivo studies from 11.0 pmol/mg protein/min to 16.0 pmol/mg protein/min [42]. Maliakal et al. [43] have reported that treatment of female Wistar rats with 2% green tea solution for 4 weeks was shown to increase cytosolic GST activity in the liver. However, studies after that, in which Wistar rats were given 833 mg/kg, i.g. tea polyphenols once daily for 6 months, showed no effect on hepatic GST activity [44]. Oral gavage treatment with EGCG (200 mg/kg) has been shown to upregulate gene expression of g-glutamyl- transferase, glutamate cysteine ligase, and hemeoxygenase 1 in C57bl/6J mice [36]. This effect appeared to take place via the Nrf2-antioxidant response element pathway. Pretreating rats with 2% green tea as the sole source of drinking ﬂuid for 6 weeks prior to a single dose of 2-amino-3-methylimididazo[4,5-f]quinoline (IQ) was found to increase excretion of glucuronide and sulfate metabolites of IQ in urine compared to rats pretreated with water [45]. Others, however, have suggested that caffeine may be the major tea component responsible for this activity [46].

33.1.3.3 Prevention and Repair of DNA Damage Prevention of DNA damage and repair of such damage represent potential mechanisms for the prevention of carcinogenesis. Tea and tea components have been shown to inhibit carcinogen-induced DNA damage in a number of cell-line studies. For example, cotreatment of human leukocytes with EGCG (2 mM) and bleomycin (20 mg/ml) resulted in a 50% decrease in bleomycin-induced DNA damage compared to treatment with bleomycin alone [47]. Yen et al. [48] have reported green tea, black tea, and Oolong tea extract to protect dose-dependently Chang liver cells from B[a]P-induced DNA damage. Pure EGC, EGCG, and theaﬂavins (10–50 mM) dose-dependently protected cells against B[a]P-induced DNA damage. At higher concentrations, EGC, EGCG, and theaﬂavins induced DNA damage by pro-oxidative mechanisms. Studies in animal models have been consistent with results in cell-line studies. Jiang et al. [49] have reported that pretreatment of C57bl/6 Big Blue lacI transgenic mice with 2% green tea prior to a single dose of B[a]P resulted in a 54% decrease in characteristic GC to TA transversions in the liver compared to water-treated controls. In contrast, green tea administration did not inhibit DNA adduct formation in the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis 602j 33 Tea and Its Constituents

model in A/J mice even though tea did reduce tumorigenesis [50]. Xu et al. [51] have, however, shown that green tea can prevent the formation of 8-oxo-2-deoxyguanosine in the same model. These results suggest that the ability of tea to prevent DNA adduct formation and oxidative DNA damage contribute to its cancer-preventive activity. C3(1) SV40 T,t antigen transgenic multiple mammary adenocarcinoma (TAg) mice spontaneously develop mammary adenocarcinoma. Treating mice with 0.05% green tea catechins (60% EGCG) or black tea theaflavins as the sole source of drinking fluid for 18 weeks decreased the levels of 3-(2-deoxy-D-erythro-pentofuranosyl) pyrimido[1,2-]purin-10(3H)one, a malonyldialdehyde-DNA adduct, in the tumors by 78 and 63%, respectively [52]. These decreases were related to increased survival and decreased mammary tumor volume compared to water-treated controls (data discussed below). In another series of studies, Lin et al. [53] reported that pretreating rats with 3% green tea extract as the sole source of drinking fluid for 10 days reduced 2-amino-3-methylimididazo[4,5-f ]quinoline (PhIP) DNA adduct formation. PhIP- DNA adducts were reduced by 50–63% in colon, heart, lung, and liver by green tea treatment. Based on cell-free studies, the authors suggest that this effect is due to the inhibition of PhIP–DNA binding, but the concentrations of tea catechins used in these mechanistic studies were quite high (0.2–1 mM) and the results remain to be demonstrated in vivo. An interesting recent study has suggested that EGCG can prevent photocarcinogenesis via an IL-12-dependent DNA repair pathway. Meeran et al. [54] have reported that application of 1 mg/cm2 EGCG to the backs of wild-type (C3H/HeN) mice inhibited UVB-induced photocarcinogenesis, but no inhibition was observed in IL-12 / littermates. The authors found that this phenomenon was related to decreased repair of pyrimidine dimers in the skin of IL-12 / mice compared to wild-type littermates.

33.1.3.4 Prevention of Carcinogenesis and Potential Postinitiation Mechanisms Cancer prevention by tea and tea components has been studied in many different animal models of carcinogenesis (reviewed in Refs [1, 55]). Tea and tea constituents inhibit the development of oral, esophageal, forestomach, stomach, intestinal, colon, liver, prostate, skin, and breast cancers in animal models. Although most studies have focused on the activity of tea polyphenols, several studies have reported the importance of caffeine in cancer prevention (reviewed in Ref. [55]). Several recent studies highlight the inhibitory activity of tea preparations in animal models of tumorigenesis, and begin to provide some mechanistic data related to the inhibitory effect. Kaur et al. [52] have recently reported that treating TAg mice, which spontaneously develop mammary tumors, with 0.05% green tea catechins or 0.05% black tea theaﬂavins for 25 weeks resulted in an increase in the mean survival time (151 and 154 days, respectively, compared to 144 days for control). Treatment with green tea catechins and black tea theaﬂavins also reduced tumor load by 25 and 39%, respectively. Inhibition of tumorigenesis was associated with increased apoptosis as

well as decreased levels of the oxidative DNA adduct M1dG (discussed above) 33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis j603 compared to water-treated controls. The authors propose that the antioxidative activity of tea polyphenols may play a role in the prevention of mammary carcinogenesis; they also point out that tea polyphenols likely work through multiple mechanisms. Our laboratory has extensively studied the antitumorigenic activities of tea poly- þ phenols in the APCmin/ mouse model of intestinal tumorigenesis. We have reported that EGCG as the sole source of drinking fluid dose-dependently (0.02–0.32%, wt/v) inhibited small intestinal tumorigenesis in this model [56]. Inhibition of tumor multiplicity was associated with increased expression of E-cadherin and decreased levels of nuclear b-catenin, c-Myc, phospho-Akt, and phospho-extracellular regulated kinase (Erk) 1/2. We compared the effectiveness of EGCG as pure compound with a defined catechin mixture, Polyphenon E (PPE), containing 65% EGCG [57]. Total tumor multiplicity was decreased by both dietary PPE (0.12%) or the corresponding amount of dietary EGCG (0.08%). Although there was a trend suggesting the PPE was more effective than EGCG at reducing total tumor multiplicity (70 vesrus 51% decrease), the difference was not statistically significant. Further studies are required to more fully elucidate whether PPE or another green tea catechin preparation is more þ effective at inhibiting tumorigenesis in the APCmin/ mouse than EGCG; such studies should also examine the nature of the interaction between the catechins if one exists. We and others have examined the effect of PPE and EGCG on the development of aberrant crypt foci (ACF) and adenomas in carcinogen-treated rats. Treating rats with PPE (0.12–0.24% in the diet) for 8 weeks following injection with azoxymethane (AOM) dose-dependently decreased the total number of ACF per rat by 16.3 and 36.9% at 0.12 and 0.24% PPE, respectively [58]. Decreases in ACF multiplicity were associated with decreased nuclear b-catenin, cyclin D1, and retinoid X receptor a staining. Carter et al. [59] examined the effect of EGCG on PhIP-induced ACF in the rat when EGCG was given during the postinitiation phase of the carcinogenic process. Treatment with EGCG for 15 weeks reduced PhIP-induced ACF by 71% compared to water-treated controls. This decrease in ACF was associated with a 40% decrease in bromodeoxyuridine labeling in the crypt, suggesting that EGCG is inhibiting aberrant cell proliferation. The underlying mechanisms remain unclear. Lu and colleagues have demonstrated the importance of caffeine in the cancer-preventive effects of tea in the UVB-induced complete skin carcinogenesis model [60]. Topical application of caffeine inhibited tumor formation and induced apoptosis in tumors. Irradiation of SKH-1 hairless mice for 20 weeks results in the formation of cellular patches in the epidermis that are immunoreactive to antibodies that recognize mutated p53 [61]. Discontinuation of UVB-treatment results in a disappearance of these patches over time. Lu et al. [61] have also shown that treating mice with orally administered EGCG or topical caffeine as the sole source of drinking fluid, following discontinuation of UVB-treatment, enhanced the disappearance of these p53 mutant patches. 604j 33 Tea and Its Constituents

33.1.4 Studies on the Cancer-Preventive Activity of Tea in Humans

33.1.4.1 Antioxidative Activity Supplementation of healthy human volunteers with catechins (500 mg/day) for 4 weeks resulted in an 18% decrease in plasma oxidized low-density lipoprotein (LDL) compared to controls [62]. Similarly, Hsu et al. [63] have reported that supplementation of hemodialysis patients with 455 mg/day green tea catechins for 3 months decreased plasma hydrogen peroxide, C-reactive protein, and several proinﬂammatory cytokines compared to placebo-treated controls. Tea catechins also blunted dialysis-induced increases in several inﬂammatory markers including plasma levels of Fas ligand and interleukin (IL)-6 soluble receptor.

33.1.4.2 Effects on Carcinogen Metabolism Studies in humans have not yielded strong results regarding the modulation of CYP expression and activity by tea and tea polyphenols. For example, Chow et al. [64] demonstrated that a 4-week treatment of human volunteers with 800 mg/day PPE (65% EGCG) did not signiﬁcantly alter the activity of CYP1A2, CYP2D6, or CYP2C9 based on the pharmacokinetics of selected probe compounds. Similarly, treating healthy volunteers with 844 mg decaffeinated green tea extract (59% EGCG) for 14 days did not signiﬁcantly affect CYP3A4 or CYP2D6 activity [65]. These results raise questions about the relevance to humans of the observed CYP-modulating effect in animal studies. By contrast, there is evidence from human intervention studies to suggest that green tea preparations can modulate phase II metabolism. Treating human volunteers for 4 weeks with 800 mg/day PPE increased glutathione-S-transferase (GST-p) activity in blood lymphocytes [66]. This effect was greatest in individuals with the lowest tertile baseline GST-p activity (80% increase compared to baseline). Other studies have demonstrated that such increases in the activity and expression of phase II metabolizing enzymes may be related to the anticarcinogenic activity of tea polyphenols. For example, a recent intervention study in China showed that a 3-month treatment with 500 or 1000 mg/day green tea polyphenols increased urinary

excretion of the mercapturic acid conjugates of AFB1 (AFB1-NAC) by 10-fold and 8.4-fold compared to baseline [67].

33.1.4.3 Prevention and Repair of DNA Damage In a pilot study by Schwartz et al. [68], heavy smokers and nonsmokers were treated with green tea (400–500 mg green tea powder per cup) ﬁve times per day for 4 weeks. The authors found that the levels of B[a]P-deoxyguanosine (B[a]P-dG) adducts were higher in smokers than in nonsmokers (two to fourfold elevation), but tea-treatment caused a 50% decrease in B[a]P-dG adducts by the end of the experiment. Tea treatment for 4 weeks also reduced the number of 8-OHdG positive cells in smokers to 50% of the pretreatment levels. Other researchers have reported that supplementation with green tea or green tea preparations can reduce biomarkers of oxidative DNA damage. For example, 33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis j605

Hakim et al. [69] found in a randomized, controlled intervention study that supplementation of heavy smokers (>10 cigarettes per day) with 4 cups of decaffeinated green tea (73.5 mg catechins per cup) per day for 4 months reduced urinary 8-OHdG levels by 31% compared to control. A recent study in China found that green tea polyphenols modulated the formation of aﬂatoxin-albumin (AfA) adducts in the serum [67]. Treatment AfA-seropositive individuals with 500 or 1000 mg green tea polyphenols for 3 months resulted in a dose- and time-dependent decrease in the serum levels of AfA. Similarly, green tea polyphenol treatment was also found to reduce urinary levels of 8-OHdG in a population at high-risk of liver cancer (i.e., seropositive for AfA) [67]. Supplementation with 500 or 1000 mg/day green tea polyphenols for 3 months resulted in a dose-dependent decrease in urinary 8-OHdG levels compared to placebo.

33.1.4.4 Epidemiological and Intervention Studies on Cancer Prevention by Tea Many case–control studies have shown that subjects who consumed large amounts of tea had lower cancer risk; in particular, risk of gastric and esophageal cancers was lower in green tea consumers in Japan and China. Gao et al. [70] reported in 1994 that green tea consumption was associated with a reduced risk of esophageal cancer. From the Shanghai Cancer Registry, 1016 eligible cases of esophageal cancer were matched with controls, and patient interviews were conducted. After adjustment for known confounders, a protective effect was observed in women. For women consuming 1–14.9 g of dry green tea leaves per month (one cup of tea typically contains 2 g tea leaves), the odds ratio (OR) was 0.77 (95% CI: 0.39–1.53) whereas for those consuming 15 g tea per month, the OR was 0.34 (95% CI: 0.17–0.69). Among men, the OR was 0.80, but not statistically significant. In another study, green tea drinking was found to be inversely associated with risk of stomach cancer, with ORs of 0.77 (95% CI: 0.52–1.13) among female heavy tea drinkers and 0.76 (CI: 55–1.27) among male heavy tea drinkers [71]. In a more recent study, we and our collaborators investigated the association between prediagnostic urinary tea polyphenols, their metabolites, and the risk of developing gastric and esophageal cancers. Using a nested case–control design, we compared 190 cases of gastric cancer and 46 cases of esophageal cancer with 772 control subjects from the Shanghai Cohort [72]. Urinary EGC positivity showed a statistically significant inverse association with gastric cancer (OR ¼ 0.52, 95% CI ¼ 0.28–0.97) after adjustment for confounders. The protective effect was primarily seen among subjects with below population median levels of serum carotenoids. Similar tea polyphenol–cancer risk associations were observed for combined risk of gastric and esophageal cancers [73]. Tea consumption has also been associated with the reduced risk of other types of cancer. For example, a population-based case–control study of women of Asian descent living in Los Angeles found that green tea drinkers had a significantly reduced risk of breast cancer (OR ¼ 0.71 and 0.53 for consumption of 0–85.7 ml and >85.7 ml of tea per day, respectively) [74]. Among women who carried at least one low-activity COMT allele, the effect was even stronger, and black tea was also found 606j 33 Tea and Its Constituents

to reduce risk [75]. The effect of black tea was not observed in the original study [74]. The authors concluded that individuals with a low-activity COMT allele have a reduced risk of breast cancer because they metabolize tea polyphenols less efficiently and, therefore, had prolonged exposure to the active parent compound. The Japanese Public Health Center-based Prospective Study (JPHC) reported an association between green tea consumption and decreased risk of advanced prostate cancer [76]. In a cohort of 49 920 men, a dose-dependent decrease in the risk of advanced prostate cancer was observed (p for trend ¼ 0.01). There was, however, no association between tea consumption and risk of localized prostate cancer. Not all epidemiological studies have observed an inverse relationship between tea consumption and cancer risk. A review of 21 epidemiological investigations of gastrointestinal cancer or precancerous lesions suggested a protective effect of green tea on adenomatous polyps and chronic atrophic gastritis formation, but there was no clear evidence for the prevention of stomach and intestinal cancer [77]. Similarly, two prospective studies in Japan found no association between green tea intake and decreased breast cancer risk [78]. In the Ohsaki National Health Insurance Cohort Study in Japan, after an 11-year follow-up, although green tea consumption was associated with decreased mortality due to cardiovascular diseases, it had no association with cancer deaths [79]. A possible reason for inconsistent results of epidemiological studies of tea and cancer prevention could be differences in study design. Case–control studies are considered less powerful than prospective studies because of the many confounding factors associated with the case–control design. As discussed by Tsubono et al. and others [80], individuals with a stomach problem, which predisposes the individual to risk for gastric cancer, may refrain from drinking green tea because of stomach irritation. Similarly, the population selected may play a critical role in demonstrating the effect of tea consumption. As discussed above, a greater protective effect of tea against breast cancer was observed in women with at least one low-activity allele of COMT. In collaboration with Dr. Mimi C. Yu, we have also demonstrated the importance of nutritional status [73]. In that study, the protective effect against colon cancer was more clearly seen in subjects with lower serum levels of carotenoids. The quantity and quality of the tea consumed and accurate determination of tea consumption may also affect the outcome of epidemiological studies. Reliance on questions of number of cups of tea consumed per day represents a potential weakness in many epidemiological studies. In future studies, more precise quantitative and qualitative information of tea consumption should be a goal in the study design. To report in grams of tea consumed per month is a good approach, especially when loose tea leaves are consumed and the habit of making tea is known. Likewise objective measurements of exposure biomarkers, such as the catechins and their metabolites, in urine or plasma could be very useful and should be further utilized where appropriate. A key to accurately assessing the efficacy of tea and tea polyphenols for the prevention of cancer will be carefully designed intervention studies. To be most 33.1 Green Tea and Its Constituents: Protection Against DNA Damage and Carcinogenesis j607 efficient and effective, such studies will likely need to focus on the progression of existing precancerous lesions to cancer, or the recurrence of cancer in people who have had therapy for a primary tumor. A recent double-blind study by Bettuzzi et al. followed 200 individuals with high-grade prostate intraepithelial neoplasia (PIN) receiving either 600 mg of green tea catechins daily or placebo (100 individuals in each group) for 12 months. Only 3% of the patients in the catechin treatment group developed prostate cancer, whereas the rate of cancer development on the placebo group was 30% [81]. No adverse effect was associated with the treatment. These results are very exciting, and the impact would be tremen- dous if the results could be reproduced in similar trials with larger number of subjects. Previously, a phase I study of green tea extract in patients with advanced lung cancer found that a dose of up to 3 g/m2/day for 16 weeks was well tolerated but produced no objective response in tumor volume [82]. Although the results of this study were negative, the study size was small (n ¼ 17) and the disease stage was likely inappropriate since most dietary components lack sufficient potency to affect late- stage cancer of any type. More interesting data are likely to be generated from an ongoing Phase II chemoprevention trial currently being conducted by a consortium of cancer centers and universities in Canada and the United States in former heavy smokers using PPE.

33.1.5 Impact of Cooking, Processing, and Other Factors on Protective Effects

We and others have shown that although tea polyphenols are stable under acidic and anaerobic conditions, at neutral or basic pH, the compounds undergo oxidation with the formation of higher molecular weight compounds as well as reactive oxygen species including H2O2 [30, 31]. Tea catechins are also subject to epimerization [30]. The stability of tea catechins depends on concentration with higher concentration preparations of tea catechins being relatively more stable than lower concentration preparations [30]. For example, we have observed that the t1/2 of EGCG at 37 C in deionized water is 30 or 150 min when the starting concentration of EGCG is 20 or 96 mM, respectively [30, 31]. Sang et al., and others, have reported that tea catechins are more stable in deionized water than in tap water [30]. This is likely due to the higher content of transition metals, such as iron and copper, in tap water, which promotes oxidation of tea catechins via a Fenton- type reaction. Based on the fact that tea catechins bind strongly to proteins, it has been hypothesized that addition of milk to tea may interfere with the bioavailability and biological activities of tea polyphenols. Although this has been shown to occur in vitro, it has not been consistently observed in vivo [144]. For example, Kyle et al. have shown that addition of milk to black tea did not alter black tea-mediated changes in total plasma antioxidant content or total plasma phenol content [145]. Another study found similar results with regard to plasma total antioxidant content, but it did observe that milk reduced plasma catechins levels [146]. Further studies are needed to assess the 608j 33 Tea and Its Constituents

impact of other ingredients and dietary components on the biological activity of tea and tea polyphenols.

33.1.6 Concluding Remarks

In the present review, we have discussed some of the laboratory and human studies demonstrating the antioxidant activity of tea, as well as its activity of preventing DNA damage, inhibiting carcinogen activation, and inducing carcinogen detoxification. These activities represent possible mechanisms by which tea and tea components could prevent carcinogenesis. We have reviewed some of the recent studies demonstrating the cancer-preventive activity of tea in animal models of carcinogenesis, epidemiological studies, and human intervention studies. It is likely that more than one mechanism is involved in the cancer-preventive activity of tea and, given that tea has been shown to inhibit carcinogenesis at all stages (i.e., initiation, promotion, and progression), both pre- and postinitiation preventive activities are likely to be important. The effective concentrations of tea polyphenols needed to demonstrate these activities in vivo compared to blood and tissue concentrations due to daily tea consumption are a critical issue when we assess the cancer prevention effects due to tea consumption. For example, some of the studies have used concentrations of 1–2.5% tea polyphenols as the sole source of drinking fluid. If we calculate the EGCG based on the assumptions that fluid consumption is 3 ml/day for mice, the average weight of mice is 20 g, and that EGCG represents 25% of the dry weight of the extract, this represents a daily EGCG dose of 375–938 mg/kg. Based on allometric scaling, this corresponds to a dose in humans of 1.2–3 g/day or the equivalent of 6–15 cups of green tea per day (assuming 200 mg EGCG per cup) [83]. In studies using green tea catechin preparations, the equivalent doses will be even higher. While such doses are achievable in populations that consume large amount of green tea, they are likely higher than the average consumption of green tea, especially in Europe and the United States. The available epidemiological studies on this topic have been inconsistent [1, 4, 5]. Further mechanistic studies in vivo are needed to clarify which of the mechanisms are key to cancer prevention and which may be irrelevant because they are carcinogen-specific or are artifacts of cell culture studies. Further, to really establish the cancer preventive usefulness of tea and tea constituents, intervention studies in at-risk human populations are needed. The recent study by Bettuzzi et al. [81] is a good precedent. Although the study population was quite small (n ¼ 30 per group), the results are very exciting. Future studies with larger populations are warranted. Finally, the emerging evidence that high concentrations of tea polyphenols have pro-oxidative activity could be problematic and may indicate the potential for adverse effects. Indeed, although tea has a long history of safe use as a beverage, there are a number of recent case reports and animal studies suggesting that high 33.2 Black and Other Teas j609 doses of dietary supplements containing green tea and high doses of green tea polyphenols can cause hepatotoxicity as well as gastrointestinal toxicity [84–86]. Careful monitoring of these potential adverse effects are needed in future human studies, and the risks should be weighed against the cancer-preventive benefits of tea and tea compounds.

Acknowledgments

Supported by NIH grants CA125780 (to JDL) and CA88961 (to CSY), and American Institute for Cancer Research Grant 05A047 (to JDL).

33.2 Black and Other Teas Wentzel C.A. Gelderblom, Kareemah Gamieldien, and Elizabeth Joubert

33.2.1 Introduction

Black tea is one of the most consumed beverages after water, and it is estimated that black tea constitutes 80% of the dried tea manufactured despite the fact that green tea has been the primary focus as a health beverage [87]. The South African herbal teas, rooibos (Aspalathus linearis), and honeybush (Cyclopia spp.) are consumed on a regular basis locally, while their popularity is rapidly increasing worldwide, with Germany having become the major export market. Both these plants came to the attention of Thurnberg, a botanist, during the 1700s. Medicinal properties were reported for C. genistoides in 1830, but rooibos, as a health beverage, attracted attention only in 1968 for its calming effect on colicky babies [88]. The major emphasis regarding the health properties of these teas has been on the presence of polyphenolic constituents, and more specifically their antioxidant properties. The possible protective role against the adverse effects of reactive oxygen species in cells and their association with many chronic diseases, such as cardiovascular disease and cancer, and aging has been the topic of many investigations. These include investigations on the chemopreventive properties of flavonoids that cover several biological aspects involving many research disciplines. Numerous studies have been conducted on the chemical properties, bioavailability, and biotransformation of flavonoids in different cellular systems in vitro and in vivo using animal models. Information about the bioactivity derived from these studies form the basis in formulating possible mechanisms involved in their potential chemopreventive properties in humans. This section will review the cancer-protective properties of black tea and the lesser known South African herbal teas, rooibos and honeybush. Aspects of their antioxidant, antimutagenic, and anticarcinogenic properties will be emphasized with reference to their possible role in cancer prevention in humans. 610j 33 Tea and Its Constituents

33.2.2 Physicochemical Properties of Active Compounds and their Occurrence

33.2.2.1 Black Tea The most significant group of bioactives of black tea is its phenolic constituents, especially the catechins and their oxidation products. The fermentation of tea leaves, a polyphenol oxidase-catalyzed oxidation followed by chemical oxidation and condensation reactions, leads to a substantial reduction (about 85%) in the catechin content. The major oxidation–condensation products are theaflavins, thearubigins (Table 33.1) [89], and theasinensins [90]. No quantitative data are available for the latter compounds. Theaflavins, bright orange-red dimers with a benzotropolone functionality, form through oxidative coupling of the pyrogallol and catechol B rings of two catechin molecules. The main theaflavins are theaflavin, theaflavin-3-gallate, theaflavin-30- gallate, and theaflavin-3,30-digallate [89]. Other minor compounds are the stereoisomers of theaflavin, isotheaflavins, neotheaflavins and related compounds, that is, theaflavic acids formed through oxidative coupling of the B-ring of the catechins with gallic acid, and theoflagallins, products of gallic acid and gallocatechin quinones [91]. During tea fermentation, unstable theasinensin quinones, with the B-rings of two catechins connected by a CC bond, accumulate, which are converted to theasinensins (colorless) during heating and drying of the leaves. The major theasinensins of black tea are theasinensins A and D, isomeric dimers of () epigallocatechin-3- gallate differ in configuration of the biphenyl bonds [90]. Thearubigins, an acidic, water-soluble, and the predominant polyphenol fraction of black tea (about 60% of the solids) is believed to be derived largely from ()-epigallocatechin and EGCG, via the intermediate quinones, theaflavins, other benzotropolones, and theasinensins [92]. It is a heterogeneous mixture of compounds that is still ill-defined but comprising amongst other proanthocyanidin type polymers, theafulvins and oolongtheanins [89]. Research is in progress to identify new thearubigin type compounds. Other monomeric compounds of interest in black tea due to their antioxidant properties and content are flavonols. Black tea contains a number of quercetin, kaempferol, and myricetin glycosides, making up 2–3% while caffeine constitutes up to 3–6% of the water-soluble extract [89].

33.2.2.2 Rooibos Tea Rooibos tea, a caffeine-free herbal tea, has a unique phenolic composition [88, 93] in that it contains two novel compounds, aspalathin, a C-linked glucoside dihydrochalcone, and aspalalinin, a cyclic dihydrochalcone. Aspalathin is the major monomeric flavonoid of unfermented rooibos and together with its corresponding flavones, orientin and isoorientin, consists some of the major flavonoids of fermented rooibos (Table 33.2). Other C-glucosides are the dihydrochalcone, nothofagin and its corresponding flavones, vitexin and isovitexin. The fermentation process, an enzymatically initiated oxidation process, followed by chemical oxidation, is accompanied by a decrease in the dihydrochalcones. Approximately, 7% of the dihydrochalcones originally present in the 33.2 Black and Other Teas j611

Table 33.1 Structures and content of catechin precursors and their oxidation products, theaflavins and thearubigins, in black tea [89].

Compound type and structure Compound Substitution Contenta

Catechins R1 R2 ECb HH ECG Gallate H 16–53c EGC H OH EGCG Gallate OH

Theaﬂavins R1 R2 TFd HH OH R2 TF3G Gallate H 11–32e O 0 TF3 G H Gallate OH TFDG Gallate Gallate HO O OH

O HO O OH O

OH R1

Oligomeric proanthocyanidin type R1 R2 thearubigins (n ¼ 2–5) OH Hor Hor OH Gallate OH

HO O R1 O

R R2 n

Other thearubigins (Oolongtheanin) 121f OH O-gallate OH

HO O OH HO OH O O OH HO O amg/150 ml serving containing 525 mg solids. bEC: ()-epicatechin; ECG: ()-epicatechin gallate; EGC: ()-epigallocatechin; EGCG: ()- epigallocatechin gallate. cTotal catechin content. dTF, theaflavin; TF3G, theaflavin-3-gallate; TF30G, theaflavin-30-gallate; TFDG, theaflavin-digallate. eTotal theaflavin content. fTotal thearubigin content. 612j 33 Tea and Its Constituents

Table 33.2 Content of major monomeric phenolic compounds per serving of unfermented and fermented rooibos (data adapted from Ref. [143]).

Compound type and structure Compound Substitution Ua Fa

Dihydrochalcones R OH Aspalathin OH 20 1.7 Nothofagin H 0.8 0.3b HO OH OH R O R1 R2 R3 HO OH HO OH O

Flavones Orientin C-Glucosyl H OH 1.7 2.1 Isoorientin H C-Glucosyl OH 2.0 2.6 OH Vitexin C-Glucosyl H H 0.2 0.3 R1 Isovitexin H C-Glucosyl H <0.1 0.7 HO O R3 R R2 OH O

Flavonols Isoquercitrin O-Glucosyl 0.9c <0.1c Rutin O-Rutinosyl TPPd 105 90 OH

HO O OH

O OH O R aU, unfermented; F, fermented; mg/150 ml serving containing 300 mg solids. bData adapted from Ref. [88]. cSum of isoquercitrin and rutin content. dTotal polyphenol content – mg gallic acid equivalents/150 ml serving.

unfermented tea is retained after fermentation. The ratio of dihydrochalcones to total ethyl acetate soluble polyphenols also decrease during processing, either due to its conversion to other ethyl acetate soluble polyphenols or its conversion to polymeric compounds, not soluble in ethyl acetate. The presence of flavanones dihydroorientin, dihydroisoorientin, and hemiphlorin in fermented rooibos suggests oxidative conversion of the dihydrochalcones via flavanones to their 33.2 Black and Other Teas j613 corresponding flavones during fermentation. The presence of the novel compound, 5,7-dihydroxy-6-C-b-D-glucopyranosylchromone also suggests oxidative degradation of dihydroisoorientin during fermentation. Other flavonoids isolated from rooibos include ( þ )-catechin, chrysoeriol, luteolin and its 7-O-linked glucoside, and quercetin O-linked glycosides such as quercetin-3-robinobioside, hyperoside, isoquercitrin, and rutin. The presence of ligans, that is, vladinol and secoisolariciresinol, and the coumarin esculetin was demonstrated in fermented rooibos [93]. Several benzoic and cinnamic acids have been isolated from fermented rooibos [88, 93]. Fermentation of rooibos is accompanied by the development of a red-brown color, contributed to the formation of polymeric substances. However, there is very little information about the polymeric fraction of rooibos [88]. The water extract of fermented rooibos contains as much as 50% complex tannin-like substances, while a methanol extract of unfermented rooibos contains approximately 14% tannin. Tannin, isolated from unfermented rooibos, is an irregular heteropolymer of the procyanidin type with ( þ )-catechin and ()-epicatechin as chain-extending units and ( þ )-catechin as terminal flavanol. Low concentrations of procyanidin B3 and, bis-fisetinidol-(4b,6:4b,8)-catechin, a trimer, are present in fermented rooibos.

33.2.2.3 Honeybush Tea Cyclopia species is also distinct because it is caffeine-free. The species differ in their phenolic fingerprint, but in all cases the major monomeric compounds are the xanthone C-glucoside, mangiferin, and the flavanone rutinoside, hesperidin [94]. Quantitative analysis of water extracts of the four most commercially relevant species, that is, C. intermedia, C. subternata, C. genistoides, and C. sessiliflora, revealed that in addition to mangiferin and hesperidin, isomangiferin and eriocitrin are also present in substantial quantities in some species. High-temperature fermentation (>60 C), an essential step in the manufacture of traditional honeybush tea, greatly reduces the content of total polyphenol and individual compounds of the hot water soluble solids (Table 33.3). In-depth investigation of the phenolic composition of Cyclopia spp. is limited to C. intermedia and C. subternata [94]. In addition to mangiferin and hesperidin, C. intermedia was found to contain, among others, the xanthone, isomangiferin, the flavanones hesperetin, naringenin, prunin, eriodictyol, and eriodictyol glycosides, the flavones luteolin and diosmetin, the flavonols kaempferol glucosides including C-6 and C-8 glucosides and a glycosylated 30,40-methylenedioxyflavonol, the isoflavones formononetin, afrormosin, pseudobaptigen, fujikinetin, calycosin, wistin, and a diglycosylated isoflavone, and the coumestans medicagol, flemichapparin, and sophoracoumestan. C. subternata, differing greatly from C. intermedia in phenolic composition, afforded in addition to mangiferin, isomangiferin and hesperidin compounds such as the flavones 5-deoxyluteolin, luteolin, and scolymoside, the flavanones hesperetin, narirutin, and eriocitrin, a flavan glucoside, a C-6 glucosylk- aempferol, the isoflavone orobol, and the flavanol epigallocatechin 3-O-gallate. 614 j 3TaadIsConstituents Its and Tea 33 Table 33.3 Contenta of major monomeric phenolic compounds per serving of unfermented and fermented Cyclopia species (data adapted from Ref. [94]).

Substitution C. intermedia C. subternata C. genistoides C. sessiliflora Compound type and structure Compound Ub Fc UF U F U F

Xanthones R1 R2 R Mangiferin C-Glucosyl H 7.5 0.6 3.6 0.2 30.1 12.9 13.2 0.6 2 Isomangiferin H C-Glucosyl nd nd 0.5 nd 5.2 2.8 2.7 nd HO OOH

R1 R2 R1 OH OH O

Flavanones O Hesperidin O-Rutinosyl CH3 3.4 1.4 1.3 0.8 2.8 1.4 1.5 1.4 R2 Eriocitrin O-Rutinosyl H 0.5 nd 1.4 0.8 nd nd 1.0 0.4 R O 1 OH TPPc 91 49 97 53 86 66 88 51

OH O amg/150 ml serving containing 300 mg solids. bU, unfermented; F, fermented. cTotal polyphenol content – g gallic acid equivalents/150 ml serving. 33.2 Black and Other Teas j615

No coumestans were isolated from C. subternata. No information is as yet available on the polymeric fraction of honeybush tea, except that it contains proanthocyanindin type structures.

33.2.3 Bioavailability and Metabolism of Active Compounds

The major pathways of polyphenol biotransformation involving glucuronidation, sulfation, and O-methylation mainly occur in the small intestine and the liver while the degradation products are produced by the gastrointestinal microflora. Numerous studies regarding the bioavailability of the monomeric catechins have been conducted, while the black tea theaflavins and to a lesser extent the thearubigins have received less attention [95, 96]. The bioavailability of the theaflavins and thearubigins are extremely low compared to the green tea catechins, as compounds with a molecular weight more than 500, containing 5 or more hydrogen-bond donors and/or 10 or more hydrogen-bond acceptors, are poorly absorbed. Degradation products of the theaflavins by colonic bacteria, similar to catechins and/or catechin breakdown metabolites, could yield smaller degradation products with less hydrogen bonds, making them more bioavailable. Despite the low bioavailability, reabsorption of conjugates entering the enterohepatic circulation would result in the accumulation of polyphenols and their phase II conjugates reaching levels that could exhibit pharmacological effects. The metabolic fate of caffeine differs between rodents and humans although up to 60–80% is metabolized and excreted in urine. In humans, caffeine is effectively metabolized in the liver to paraxanthine, theobromine, and theophylline that constitute 80, 11, and 4%, respectively. These metabolites exhibited properties similar to caffeine except for theobromine that exhibited a weaker activity regarding autoimmune and inflammatory disorders [97]. At present, very little is known about the bioavailability and metabolism of the major polyphenolic rooibos constituents, that is, aspalathin, nothofagin, orientin, and isoorientin. A recent study showed that aspalathin is converted to its corresponding flavanones, that is, eriodictyol C-6 and C-8 glucosides, and flavones, orientin and isoorientin, under physiologically relevant conditions (37 C in a phosphate buffer of pH 7.4) within 6 h [98]. The pharmocokinetics and tissue distribution of orientin was recently determined in rat tissues but no metabolites were reported [99]. Numerous studies have been conducted on the flavonols, quercetin and luteolin, but they occur at very low levels in rooibos. Although fewer studies have been performed with honeybush tea compared to rooibos, more studies on the biological properties of the major constituents, the xanthone, mangiferin, and the flavavones, hesperidin and narirutin have been conducted, as they are common constituents of citrus. Three polyhydroxy and one polyhydroxy methoxy metabolites were observed in the urine of rats [100]. Hesperidin and narirutin are rhamnoglycosides that are absorbed from the gut as aglycones. Very little is known about their metabolism although the presence of monoglucuronides of hesperetin, the aglycone of hesperidin, has been reported. Some phenolic acids resulting from human 616j 33 Tea and Its Constituents

microbial metabolism of naringenin and hesperetin have been detected in rat urine [95].

33.2.4 Mechanisms of Protection: Results of In Vitro and Animal Studies

When considering the chemopreventive properties of teas and their polyphenolic compounds, the antimutagenic and antioxidant properties and the disruption of the cell cycle events relating to their antiproliferative and proapoptotic events are of importance and need to be evaluated. Investigations into the modulating effects of teas in preventing the induction and/or promotion of early preneoplastic lesions in experimental cancer models have been reported.

33.2.4.1 Antimutagenic Properties Black tea and the catechin oxidation products, theaflavins and thearubigins, exhibit antimutagenic properties against different classes of carcinogens, including aromatic and heterocyclic amines, polycyclic aromatic hydrocarbons, n-nitrosamines, and aflatoxins using various tester strains of Salmonella typhimurium and Escherichia coli mutagenicity assays. Numerous studies have been described on the protective effects of black tea, theaflavins, and thearubigins against carcinogen, oxidative DNA damage, and DNA adduct formation [101]. Both mutagenic and antimutagenic effects of caffeine have been reported. The mutagenic effects are mostly ascribed to the interaction of caffeine with mutagenic agents and/or chemicals [102]. However, no mutagenic effects were noticed in vivo. Black tea, but not decaffeinated black tea, decreases the urinary excretion of mutagens and promutagens of rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline, by interfering with their bioactivation and metabolism due to the presence of caffeine [103]. It was suggested that, due to the poor bioavailability of the catechin condensation products, caffeine and its microbial metabolites should also be taken into account when considering the protective effects of black tea against mutagenesis. Studies on the protective effect against DNA damage and antimutagenic effects of the South African herbal teas, rooibos and honeybush (C. intermedia), have recently been reviewed [104, 105]. Fermented rooibos protects against benzo[a]pyrene- induced chromosomal aberrations and genotoxicity of 2-acetylaminofluorene (2-AAF) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP). Depending on the carcinogen used, the antimutagenic effects of rooibos and some of the unfermented honeybush species compared favorably with black tea using the Salmonella typhimurium mutagenicity assay [106]. Of the major rooibos flavonoids, aspalathin, nothofagin, orientin, and isoorientin exhibited moderate activities while luteolin, a minor constituent, exhibited properties similar to EGCG, the major green tea flavonoid [107]. The antimutagenic properties of hesperidin and mangiferin were fl similar, while hesperetin exhibited comparable properties to EGCGwhen a atoxin B1 was used as mutagen. Against 2-AAF-induced mutagenesis, mangiferin exhibits the highest antimutagenic properties with hesperetin having only weak protective properties. Hesperidin, however, stimulates the mutagenic response of 2-AAF. 33.2 Black and Other Teas j617

33.2.4.2 Antioxidant Properties The antioxidant properties of polyphenolic plant constituents and their protective role against excessive oxidative damage and disease development in humans have been the focus of many studies. Black tea and the theaflavins have been reported to scavenge the synthetic ABTSradical cation and to protect LDL against oxidation [108]. Theaflavin digallate was the most effective, followed by the two theaflavin gallates with theaflavin exhibiting the weakest activity. Black tea and the theaflavins not only scavenged ROS but also prevented superoxide production by inhibiting xanthine oxidase in human promyelocytic leukemia (HL-60) cells [109]. Nitric oxide synthase was also inhibited by theaflavin digallate in murine macrophages [110]. Inhibition of these events plays an important role in the inhibition of ROS formation and anti-inflammatory responses of black tea and the subsequent inhibition of PMA- induced cancer promotion in mouse skin. Black tea, and specifically theaflavin, was found to be more effective than green tea and its individual catechin constituents in abrogating the production of nitric oxide and superoxide anion in macrophages [87]. The contribution of caffeine to the antioxidant activity of black tea has also been evaluated in Jurkat T cells. Fe-induced oxidative damage was decreased, which was associated with a decrease in the glutathione peroxidase activity indicating antioxidant properties for caffeine [111]. Comparative studies of water-soluble solids of different teas showed that their relative order of efficacy in in vitro antioxidant assays depends on the specific assay, as well as the composition of the samples used. The latter emphasizes the effect of variation in chemical composition, introduced by varieties or species, genotypes, environmental factors, and processing of the plant material. In general, however, certain trends in relative order of activity are obvious (Table 33.4), most notably that green tea > black tea, black tea > oolong tea (mostly), unfermented rooibos > fermented rooibos, and for the same species unfermented honeybush > fermented honeybush. In some cases, unfermented rooibos is more effective than black tea. Oxidative changes taking place during fermentation tend to decrease the antioxidant activity of the water-soluble extract. In the FRAP assay, unfermented C. intermedia, C. subternata, and C. sessiliflora had higher activity than fermented rooibos, while unfermented C. sessiliflora offered the same protection against microsomal lipid peroxidation as fermented rooibos. None of the honeybush species was as effective as those of unfermented rooibos and C. sinensis teas in the in vitro antioxidant assays. Studies on the antioxidant properties of the major flavonoid of rooibos, aspalathin, . showed that it exhibits similar activity to quercetin in scavenging O2 , but it was more effective than its corresponding flavones, orientin and isoorientin [112]. Of the major honeybush polyphenols, mangiferin exhibited the highest antioxidant capacity in the ABTS assay compared to hesperidin and minor constituents, hesperetin, narirutin, eriodictyol, and naringenin [94]. In the FRAP assay, mangiferin exhibited similar activity than hesperidin, hesperetin, and eriodictyol with eriocitrin exhibiting the highest activity. In contrast, mangiferin exhibited a weaker protective activity compared to aglycones luteolin, hesperetin, and eriodictyol in the lipid peroxidation assay using rat liver microsomes, presumably due to its high hydrophilicity as a result 618 j 3TaadIsConstituents Its and Tea 33

Table 33.4 Relative order of activitya of Camellia sinensis, rooibos, and honeybush teas in different antioxidant assays.

Sample set Assay Rooibos and C. sinensis teas Rooibos and honeybush species 1 DPPH scavenging Green > rooibos (U) > rooibos (F) > rooibos (SF) > black > oolong þ 2ABTSscavenging Green > black > rooibos (U) oolong > rooibos (F) Rooibos (U, F) > honeybush species (U, F); C gen (U) C sess (U) C sub (U) C int (U) > C gen (F) > C int (F) C sess (F) C sub (F) 2 FRAP Green > rooibos (U) > black > rooibos (F) oolong Rooibos (U) > honeybush species (U); rooibos (F) C gen (U); C int (U) C sub (U) C sess (U) > C gen (U) > C gen (F) > C sub (F) ¼ C sess (F) C int (F) 3O2 scavenging C sess (U) > C sub (U) C sess (F) C gen (U) > C gen (F) > C sub (F) C mac (u) C int (U) > C int (F) C mac (F) 1 b-Carotene bleaching method Green > black > oolong > rooibos (F) > (coupled linoleic acid oxidation) rooibos (U) > rooibos (SF) 2 Microsomal lipid peroxidation Green > rooibos (U) black oolong > rooibos (F) C sess (U) rooibos (F); C sess (U) > C int (U) C sub (U) C gen (F) C gen (U) > C sub (F) C int (F) C sess (F) aData as referenced in Ref. [105]; C gen: C. genistoides; C sess: C. sessiliflora; C sub: C. subternata; C int: C. intermedia; C mac: C. maculata; U, unfermented; F, fermented. 33.2 Black and Other Teas j619 of the presence of the sugar residue. The inhibition of lipid peroxidation was ascribed to the Fe chelating and free radical scavenging properties of the catechol and oxo- hydroxyl structural arrangements. A recent study showed that aspalathin and enriched polyphenolic extracts from rooibos exhibit pro-oxidant effects in vitro [113]. Similar findings were reported for theaflavin digallate [114].

33.2.4.3 Studies in Animals Black tea and the theaflavins have been shown to reduce the induction of early preneoplastic lesion in different organs including the lung, colon, skin, stomach, esophagus, liver, and buccal pouch in rats and mice [96]. Black tea inhibits the progression of NNK-induced lung adenoma into adenocarcinoma by suppressing the cell proliferation rate. The preventive properties of theaflavin on the DMBA-induced buccal pouch carcinogenesis counter the increase of NF-kB, mutant p53, and Bcl-2, while the proapopototic proteins Bax and caspase-3 are upregulated [115]. In the liver, black tea polyphenols decrease the number and size of preneoplastic lesion by upregulating the expression of P21 while inhibiting the expression of cyclin D1 and CDK4 [116]. Inhibition of oxidative cell injury has been suggested as a possible mechanism of chemoprevention by black tea. In the prostate, the physiological levels of androgen shift the pro-oxidant–antioxidant balance toward oxidative stress that could result in the deregulation of cell growth affecting cancer initiation and promotion. Protection of testosterone-induced oxidative stress in rats by theaflavins and thearubigins was associated with a significant reduction in lipid peroxidation and decreased activities of superoxide dismutase, catalase, and glutathione reductase [117]. A redox reaction also seems to be involved in the inhibition of DNA adduct formation and colon carcinogenesis of the ultimate carcinogen N-acetoxy- PhIP in the presence of black tea polyphenols [118]. The induction of oxidative stress by theaflavin digallate, associated with the depletion of glutathione, was suggested to play a determinant role in the induction of apoptosis in squamous human cancer cells compared to normal fibroblast [114]. Caffeine inhibits carcinogenesis in different animal models (see Ref. [119] for references), although an enhancing effect was reported for heterocyclic amines of colon carcinogenesis presumably due to the induction of CYP1A2 [120]. At present, no study has shown caffeine to be carcinogenic in mice and rats up to levels of 230 and 391 mg/kg per day [121]. However, the chemopreventive properties of caffeine are similar to the black tea catechins, including antioxidant and antimutagenic properties, inhibition of signal transduction related to cell proliferation and the induction of apoptosis. Theafluvin and theaflavin fail to affect the activities of the hepatic phase I and phase II liver enzymes in rats while both the polyphenolic fractions decrease the CYP1A1 and CYP3A apoprotein levels in the intestine. The phase II enzymes were also not affected in the intestine, although the theafulvins decrease the activity of the UDP-glucuronosyl transferase I family substrate, 1-napthol [122]. In contrast to these findings, black tea was found to be more effective than green tea in stimulating phase II enzymes in the liver while decaffeinated black tea had no effect. The involvement of flavanols and caffeine in mediating these effects was questioned while a specific role of theaflavins and thearubigens was suggested [123]. 620j 33 Tea and Its Constituents

However, a recent study showed that caffeine activates hepatic glucuronosyl transferase as well as CYP1A2 [124]. Feeding studies in rats showed that fermented and unfermented rooibos and honeybush (C. intermedia) signiﬁcantly increased the activities of the phase II enzyme, glutathione-S-transferase a, while unfermented rooibos and honeybush enhanced the activity of UDP-glucuronosyl transferase. Both fermented and unfermented herbal teas markedly signiﬁcantly increased and decreased the GSH and GSSG levels, respectively, resulting in an increase in the GSH:GSSG ratio [104, 105].

No effect was noticed on the liver ORAC. The mutagenicity of 2-AAF and AFB1 was signiﬁcantly reduced by liver cytosolic preparations of rats fed fermented and unfermented herbal teas. Although no effect on the induction of cytochrome P-450 was observed, liver microsomal preparations of the rats fed the unfermented

herbal teas and fermented rooibos reduced the activation of AFB1 to its reactive mutagenic metabolite. The data indicated that components of rooibos and honeybush tea significantly modulate the drug metabolizing enzymes associated with carcinogen metabolism as well as the oxidative status in the liver. Black tea failed to affect any of the above parameters but significantly reduced the GSSG and ORAC levels in the liver. Treatment of rats with rooibos tea inhibited the expression of CYP2C11 in the liver [125] while another study showed that rooibos increased the activity and expression of CYP3A in rat intestine [126] suggesting that some drug/ herb interactions are likely to occur. An organic solvent extract of fermented and unfermented rooibos and honeybush (C. intermedia) protects against TPA tumor promotion in mouse skin [127]. No direct relationship between the antioxidant and the protection against tumor promotion appears to exist as unfermented honeybush, exhibiting a weaker protection against microsomal lipid peroxidation than unfermented rooibos, showed a higher protective effect against tumor promotion in mouse skin. The relative concentrations of flavonoid subgroups seem to be important as green tea and unfermented honeybush, having the highest flavanol/proanthocyanidin content, exhibited the highest protection. However, mangiferin and hesperidin, the major honeybush polyphenols, exhibit antioxidant and anti-inflammatory effects and are thus likely to be involved in the antitumor promoting effects of honeybush. A recent study showed that mangiferin reduces the TPA-induced ROS production and DNA fragmentation in peritoneal macrophages [128]. The relative contribution of different flavonoid subgroups and/or nonflavonoids still needs to be elucidated.

33.2.4.4 Cell Survival Parameters Studiesindifferentcellculturesweremainlyconductedwiththeaflavinandreportedon the antiproliferation and proapoptotic effects in different carcinoma cells including skin,colon,breast,lung,andprostate.Theseeventsareassociatedwiththeupregulation ofproapoptoticproteins,Baxandcaspase-9,downregulationofNF-kB,andinhibitionof the cyclooxygenase-2. Theaflavin was also shown to exhibit antiproliferative effects in different cancer cells, by inhibiting cyclin kinase p21 and cyclin B expression [129]. Other studies reported the inhibition of c-jun phosphorylation transcription activity of AP-1 and the downregulation of MAPK pathways. Caffeine also showed various effects 33.2 Black and Other Teas j621 on the cell cycle causing growth arrest in cancer cells. Studies in mouse epidermal cells showed that the caffeine-induced inhibition of cell proliferation and induction of apoptosis are associated with the prevention of retinoblastoma phosphorylation by disrupting the growth signal-induced activation of CDK4 [130]. Limited studies were conducted with rooibos related to cell growth parameters. Rooibos was shown to inhibit cyclooxygenase-2 (COX-2) expression in human breast epithelial cells, presumably via modulation of NF-kB binding to DNA [131]. Thea- flavin monogallate also inhibited the expression of COX-2 in transformed cells and not in their normal counterparts at both mRNA and protein levels [129]. The modulation of COX-2 expression was suggested to play an important role in chemopreventive properties of rooibos and black tea.

33.2.5 Results of Human Studies

Epidemiological studies suggest an inverse association between tea consumption and development of cardiovascular disease and cancer. Black tea consumption increased the total catechin concentration and antioxidant capacity of the plasma, although many contradictory findings have been reported, some of which could be ascribed to the addition of milk [132]. With respect to cancer chemoprevention, the effect of black tea consumption on the development of different cancers in humans has been reviewed [96, 133–135]. Although many studies in experimental animals provided evidence of the anticancer properties of black tea, the evidence between tea consumption and prevention of human cancer development yielded less clear conclusions. Some of these include a prospective cohort study in the Netherlands showing no protective effect against breast, colorectal, and stomach cancers, while a case–control study on ovarian cancer in China did not find any conclusive results with black tea compared to green tea. A prospective cohort study in the United States showed that black tea reduces colon cancer although a meta-analysis of studies conducted in North America, Asia, and Europe did not find sufficient evidence to suggest that black tea is a potential chemopreventive agent against colorectal cancer. Notwith- standing the moderate protection in some studies, the role of black tea consumption in the reduction of colon, lung, stomach, esophagus, kidney, and breast cancers in humans remains inconclusive. Inconsistent findings regarding tea consumption and esophageal cancer can also be related to the temperature of tea preparation. It is also known that different risk factors are involved when considering cancer development in populations of different countries, which can selectively affect the preventive effects of black tea. Inconsistency in the outcome between the studies could also be related to many factors including diet, smoking and drinking, age and the bioavailability of the tea polyphenols that will depend on an individuals metabolism and gut microflora. These different parameters may significantly affect the uptake and availability of biologically active constituents while microbial metabolites might also play an important role in health benefits attributed to tea consumption [96]. 622j 33 Tea and Its Constituents

No studies investigating the chemopreventive properties of rooibos and honeybush teas have been conducted in humans.

33.2.6 Impact of Heat and Processing on Protective Properties

33.2.6.1 Black Tea Tea catechins and theaflavins are susceptible to further changes during heat processing and even preparation of the infusion. Factors that play a role are pH of the solution, temperature, and heating time. Catechins were found to be highly stable under acidic conditions (pH < 4), but their stability decreases with increasing pH between 4 and 8, while rapid degradation occurs in alkaline solutions (pH > 8). High temperatures during production of tea beverages result in epimerization of the catechins. EGCG is changed to gallocatechin gallate (GCG) when autoclaved at 121 C for 20 min [136]. The large amounts of GCG found in certain ready-to-drink teas were attributed to epimerization of EGCG. Epimerization was found to take place more easily in tap water than purified water. Variation between the pH and mineral ion complexity of these two types of water were implicated [137]. Epimerization does not affect in vitro antioxidant activity in a liposome system, but GCG was found to be slightly more effective than its epimer EGCG, which was attributed to the change in stereochemistry [138]. However, epicatechins have significantly lower redox potentials than the catechins [89]. Degradation was found to follow epimerization [136]. The rate of degradation of the catechins differs, with EGCG and EGC more susceptible to degradation than ECG and EC [139]. Degradation of theaflavins is also enhanced by increased pH and temperature and they are less stable than the catechins. Different theaflavins have varying stability, with theaflavin and theaflavin-3-gallate the least stable, either in boiling water or in alkaline solution [139]. In aqueous solution (pH 7.3), theaflavin will gradually oxidize, yielding bistheaflavinB andtheanaphtoquinone[140].Both catechinsand theaflavinshavepoor long-termstability insolutionwith approximately 50% degraded within the firstmonth of storage at room temperature [139]. Pasteurization and storage of tea-containing beverages, accompanied by slow chemical oxidation, resulted in a reduction in the oxygen scavenging ability but an increase in the radical scavenging ability of the beverages[141].Thelatteroutcomewasunexpectedandwasspeculatedtohaveresulted from the formation of macromolecular compounds (more stable, polymerized phenolic compounds) with greater radical scavenging power than that of the original polyphenols. The greater radical scavenging power of these polymeric compounds was hypothesized to be the result of increased resonance delocalization of radicals.

33.2.6.2 Rooibos and Honeybush Infusions of rooibos and honeybush have traditionally been prepared by prolonged heating at low heat in favor of a strong brew. This practice became largely obsolete with the availability of tea bags on the market that rapidly release the ﬂavor components. However, extraction for manufacture of rooibos and honeybush water-soluble powders for use by the food industry exposes the extracts to high heat References j623

(90 C) in excess of 30 min. It is not known to what extent heating would affect the composition and thus bioactivity of these herbal teas. Heating of rooibos extract increased its ability to protect against lipid peroxidation in the Rancimat method but not in the b-carotene bleaching method [105]. Since slow conversion of aspalathin to the ﬂavanones (S)- and (R)-eriodictyol-6-C-b-D-glucopyranoside occurs at 30 C in the presence of oxygen [142], it is likely that some aspalathin will degrade during extract manufacture when higher temperature prevails, as well as during storage of a product such as rooibos iced tea.

33.2.7 Concluding Remarks

Black and/or the herbal teas play an important role in the daily intake of health- promoting flavonoids, which include caffeine when considering the quantities of black tea consumed. Discrepancies in their biological effects and variations in research findings and/or outcomes in different in vitro test systems and in vivo studies in animals and humans highlight the inherent problems of using these natural products. As some of the reactive biological constituents are not known, many still have to be identified making the standardization of the teas impossible. The composition of a cup of tea is determined by many factors such as variety, climate, cultivation, and manufacturing conditions. Further variations are introduced by the type of packaging (tea bag) and conditions such as agitation, ratio of leaf to water, and heating time when preparing the infusion. The chemopreventive properties of green tea seem to outperform black tea in both animal and human studies although there are many exceptions. Fermentation of the herbal teas also resulted in a significant decrease in the major polyphenolic constituents that coincide with a reduction of the antimutagenic and antioxidant properties. In mouse skin, however, unfermented honeybush exhibits a higher protective effect than unfermented rooibos despite its lower antioxidant and antimutagenic properties implying that specific tissue/tea interactions as a result of compositional differences are of importance. Other reasons for the variation in results have recently been highlighted [131] and included aspects regarding differences in diets, study designs, and protocols, routes of administration, and the stability of tea constituents in solution and in the diet. The relationship between the tissue levels and the chemopreventive properties in a specific organ also has to be considered.

References

References to Section 33.1

1 Yang, C.S., Maliakal, P. and Meng, X. 2 Balentine, D.A., Wiseman, S.A. and (2002) Inhibition of carcinogenesis by tea. Bouwens, L.C. (1997) The chemistry of Annual Review of Pharmacology and tea ﬂavonoids. Critical Reviews in Food Toxicology, 42,25–54. Science and Nutrition, 37, 693–704. 624j 33 Tea and Its Constituents

3 Lambert, J.D. and Yang, C.S. (2003) 11 Sang, S., Lambert, J.D., Hong, J., Tian, S., Cancer chemopreventive activity and Lee, M.J., Stark, R.E., Ho, C.T. and bioavailability of tea and tea polyphenols. Yang, C.S. (2005) Synthesis and structure Mutation Research, 523–524, 201–208. identification of thiol conjugates of 4 Li, C., Meng, X., Winnik, B., Lee, M.J., ()-epigallocatechin gallate and their Lu, H., Sheng, S., Buckley, B. and urinary levels in mice. Chemical Research Yang, C.S. (2001) Analysis of urinary in Toxicology, 18, 1762–1769. metabolites of tea catechins by liquid 12 Hong, J., Lambert, J.D., Lee, S.H., chromatography/electrospray ionization Sinko, P.J. and Yang, C.S. (2003) mass spectrometry. Chemical Research in Involvement of multidrug resistance- Toxicology, 14, 702–707. associated proteins in regulating cellular 5 Kohri,T.,Nanjo,F.,Suzuki,M.,Seto,R., levels of ()-epigallocatechin-3-gallate Matsumoto, N., Yamakawa, M., Hojo, H., and its methyl metabolites. Biochemical Hara, Y., Desai, D., Amin, S., Conaway, and Biophysical Research Communications, C.C. and Chung, F.L. (2001) Synthesis of 310, 222–227. ()-[4-3H]epigallocatechin gallate and its 13 Vaidyanathan, J.B. and Walle, T. (2001) metabolic fate in rats after intravenous Transport and metabolism of the tea administration. JournalofAgriculturaland flavonoid ()-epicatechin by the human Food Chemistry, 49,1042–1048. intestinal cell line Caco-2. Pharmaceutical 6 Hu, M., Chen, J. and Lin, H. (2003) Research, 18, 1420–1425. Metabolism of flavonoids via enteric 14 Vaidyanathan, J.B. and Walle, T. (2003) recycling: mechanistic studies of Cellular uptake and efflux of the tea disposition of apigenin in the Caco-2 cell flavonoid ()epicatechin-3-gallate in the culture model. The Journal of human intestinal cell line Caco-2. The Pharmacology and Experimental Journal of Pharmacology and Experimental Therapeutics, 307, 314–321. Therapeutics, 307, 745–752. 7 Lu, H., Meng, X. and Yang, C.S. (2003) 15 Li, C., Lee, M.J., Sheng, S., Meng, X., Enzymology of methylation of tea Prabhu, S., Winnik, B., Huang, B., catechins and inhibition of catechol- Chung, J.Y., Yan, S., Ho, C.T. and O-methyltransferase by Yang, C.S. (2000) Structural identification ()-epigallocatechin gallate. Drug of two metabolites of catechins and their Metabolism and Disposition, 31, 572–579. kinetics in human urine and blood after 8 Lu, H., Meng, X., Li, C., Sang, S., tea ingestion. Chemical Research in Patten, C., Sheng, S., Hong, J., Bai, N., Toxicology, 13, 177–184. Winnik, B., Ho, C.T.and Yang, C.S. (2003) 16 Meselhy, M.R., Nakamura, N. and Glucuronides of tea catechins: Hattori, M. (1997) Biotransformation of enzymology of biosynthesis and ()-epicatechin 3-O-gallate by human biological activities. Drug Metabolism and intestinal bacteria. Chemical & Disposition, 31, 452–461. Pharmaceutical Bulletin, 45, 888–893. 9 Vaidyanathan, J.B. and Walle, T. (2002) 17 Taipalensuu, J., Tornblom, H., Glucuronidation and sulfation of the tea Lindberg, G., Einarsson, C., Sjoqvist, F., flavonoid ()-epicatechin by the human Melhus, H., Garberg, P., Sjostrom, B., and rat enzymes. Drug Metabolism and Lundgren, B. and Artursson, P. (2001) Disposition, 30, 897–903. Correlation of gene expression of ten 10 Lu, H. (2002) Mechanistic Studies on the drug efflux proteins of the ATP-binding phase II metabolism and absorption of tea cassette transporter family in normal catechins, in Toxicology, Rutgers, The human jejunum and in human State University of New Jersey, New intestinal epithelial Caco-2 cell Brunswick, p. 160. monolayers. The Journal of Pharmacology References j625

and Experimental Therapeutics, 299, Chemical Research in Toxicology, 15, 164–170. 1042–1050. 18 Chen, L., Lee, M.J., Li, H. and Yang, C.S. 25 Chow, H.H., Cai, Y., Hakim, I.A., (1997) Absorption, distribution, Crowell, J.A., Shahi, F., Brooks, C.A., elimination of tea polyphenols in rats. Dorr, R.T., Hara, Y. and Alberts, D.S. Drug Metabolism and Disposition, 25, (2003) Pharmacokinetics and safety of 1045–1050. green tea polyphenols after multiple- 19 Lambert, J.D., Lee, M.J., Lu, H., Meng, X., dose administration of epigallocatechin Hong, J.J., Seril, D.N., Sturgill, M.G. and gallateandpolyphenonEinhealthy Yang, C.S. (2003) Epigallocatechin-3- individuals. Clinical Cancer Research, 9, gallate is absorbed but extensively 3312–3319. glucuronidated following oral 26 Higdon, J.V. and Frei, B. (2003) Tea administration to mice. The Journal of catechins and polyphenols: health effects, Nutrition, 133, 4172–4177. metabolism, and antioxidant functions. 20 Lambert, J.D., Lee, M.J., Diamond, L., Critical Reviews in Food Science and Ju, J., Hong, J., Bose, M., Newmark, H.L. Nutrition, 43,89–143. and Yang, C.S. (2006) Dose-dependent 27 Senthil Kumaran, V., Arulmathi, K., levels of epigallocatechin-3-gallate in Srividhya, R. and Kalaiselvi, P. (2008) human colon cancer cells and mouse Repletion of antioxidant status by EGCG plasma and tissues. Drug Metabolism and and retardation of oxidative damage Disposition, 34,8–11. induced macromolecular anomalies in 21 Chu, K.O., Wang, C.C., Chu, C.Y., aged rats. Experimental Gerontology, 43, Chan, K.P., Rogers, M.S., Choy, K.W. and 176–183. Pang, C.P. (2006) Pharmacokinetic 28 Srividhya, R., Jyothilakshmi, V., studies of green tea catechins in maternal Arulmathi, K., Senthilkumaran, V. and plasma and fetuses in rats. Journal of Kalaiselvi, P. (2008) Attenuation of Pharmaceutical Sciences, 95, 1372–1381. senescence-induced oxidative 22 Kim, S., Lee, M.J., Hong, J., Li, C., exacerbations in aged rat brain by Smith, T.J., Yang, G.Y., Seril, D.N. and ()-epigallocatechin-3-gallate. Yang, C.S. (2000) Plasma and tissue levels International Journal of Developmental of tea catechins in rats and mice during Neuroscience, 26, 217–223. chronic consumption of green tea 29 Rietveld, A. and Wiseman, S. (2003) polyphenols. Nutrition and Cancer, 37, Antioxidant effects of tea: evidence from 41–48. human clinical trials. The Journal of 23 Lee, M.J., Maliakal, P., Chen, L., Meng, X., Nutrition, 133, 3285S–3292S. Bondoc, F.Y., Prabhu, S., Lambert, G., 30 Sang, S., Lee, M.J., Hou, Z., Ho, C.T. and Mohr, S. and Yang, C.S. (2002) Yang, C.S. (2005) Stability of tea Pharmacokinetics of tea catechins after polyphenol ()-epigallocatechin-3-gallate ingestion of green tea and and formation of dimers and epimers ()-epigallocatechin-3-gallate by humans: under common experimental conditions. formation of different metabolites and Journal of Agricultural and Food Chemistry, individual variability. Cancer Epidemiology, 53, 9478–9484. Biomarkers & Prevention, 11, 1025–1032. 31 Akagawa, M., Shigemitsu, T. and 24 Meng, X., Sang, S., Zhu, N., Lu, H., Suyama, K. (2003) Production of Sheng, S., Lee, M.J., Ho, C.T. and hydrogen peroxide by polyphenols and Yang, C.S. (2002) Identiﬁcation and polyphenol-rich beverages under quasi- characterization of methylated and physiological conditions. Bioscience, ring-ﬁssion metabolites of tea catechins Biotechnology, and Biochemistry, 67, formed in humans, mice, and rats. 2632–2640. 626j 33 Tea and Its Constituents

32 Yang, G.Y., Liao, J., Kim, K., Yurkow, E.J. 39 Sohn, O.S., Surace, A., Fiala, E.S., and Yang,C.S. (1998) Inhibition of growth Richie, J.P. Jr, Colosimo, S., Zang, E. and and induction of apoptosis in human Weisburger, J.H. (1994) Effects of green cancer cell lines by tea polyphenols. and black tea on hepatic xenobiotic Carcinogenesis, 19, 611–616. metabolizing systems in the male F344 33 Vittal, R., Selvanayagam, Z.E., Sun, Y., rat. Xenobiotica, 24, 119–127. Hong, J., Liu, F., Chin, K.V. and Yang, 40 Xu, M., Bailey, A.C., Hernaez, J.F., C.S. (2004) Gene expression changes Taoka, C.R., Schut, H.A. and induced by green tea polyphenol Dashwood, R.H. (1996) Protection by ()-epigallocatechin-3-gallate in human green tea, black tea, and indole-3-carbinol bronchial epithelial 21BES cells against 2-amino-3-methylimidazo[4,5-f ] analyzed by DNA microarray. Molecular quinoline-induced DNA adducts and Cancer Therapeutics, 3, 1091–1099. colonic aberrant crypts in the F344 rat. 34 Hou, Z., Sang, S., You, H., Lee, M.J., Carcinogenesis, 17, 1429–1434. Hong, J., Chin, K.V. and Yang, C.S. 41 Chen, L., Bondoc, F.Y., Lee, M.J., (2005) Mechanism of action of Hussin, A.H., Thomas, P.E. and ()-epigallocatechin-3-gallate: Yang, C.S. (1996) Caffeine induces auto-oxidation-dependent inactivation of cytochrome P4501A2: induction of epidermal growth factor receptor and CYP1A2 by tea in rats. Drug Metabolism direct effects on growth inhibition in and Disposition, 24, 529–533. human esophageal cancer KYSE 150 cells. 42 Tulayakul, P., Dong, K.S., Li, J.Y., Cancer Research, 65, 8049–8056. Manabe, N. and Kumagai, S. (2007) The 35 Yuan, J.H., Li, Y.Q. and Yang, X.Y. (2007) effect of feeding piglets with the diet Inhibition of epigallocatechin gallate on containing green tea extracts or coumarin orthotopic colon cancer by upregulating on in vitro metabolism of aflatoxin B1 by the Nrf2-UGT1A signal pathway in nude their tissues. Toxicon, 50, 339–348. mice. Pharmacology, 80, 269–278. 43 Maliakal, P.P., Coville, P.F. and 36 Shen, G., Xu, C., Hu, R., Jain, M.R., Wanwimolruk, S. (2001) Tea Nair, S., Lin, W., Yang, C.S., Chan, J.Y.and consumption modulates hepatic drug Kong, A.N. (2005) Comparison of metabolizing enzymes in Wistar rats. The ()-epigallocatechin-3-gallate elicited Journal of Pharmacy and Pharmacology, liver and small intestine gene expression 53, 569–577. profiles between C57BL/6J mice and 44 Liu, T.T., Liang, N.S., Li, Y., Yang,F., Lu, Y., C57BL/6J/Nrf2 (/) mice. Meng, Z.Q. and Zhang, L.S. (2003) Effects Pharmaceutical Research, 22, 1805–1820. of long-term tea polyphenols 37 Krishnan, R., Raghunathan, R. and consumption on hepatic microsomal Maru, G.B. (2005) Effect of polymeric drug-metabolizing enzymes and liver black tea polyphenols on benzo(a)pyrene function in Wistar rats. World Journal of [B(a)P]-induced cytochrome P4501A1 Gastroenterology, 9, 2742–2744. and 1A2 in mice. Xenobiotica, 35, 45 Embola, C.W., Weisburger, J.H. and 671–682. Weisburger, M.C. (2001) Urinary 38 Catterall, F., McArdle, N.J., Mitchell, L., excretion of N-OH-2-amino-3- Papayanni, A., Clifford, M.N. and methylimidazo[4,5-f]quinoline-N- Ioannides, C. (2003) Hepatic and glucuronide in F344 rats is enhanced by intestinal cytochrome P450 and green tea. Carcinogenesis, conjugase activities in rats treated with 22, 1095–1098. black tea theafulvins and theaflavins. 46 McArdle, N.J., Clifford, M.N. and Food and Chemical Toxicology, Ioannides, C. (1999) Consumption of tea 41, 1141–1147. modulates the urinary excretion of References j627

mutagens in rats treated with IQ. Role of adduct formation. Mutation Research, caffeine. Mutation Research, 441,191–203. 523–524, 193–200. 47 Glei, M. and Pool-Zobel, B.L. (2006) The 54 Meeran, S.M., Mantena, S.K., main catechin of green tea, Elmets, C.A. and Katiyar, S.K. (2006) ()-epigallocatechin-3-gallate (EGCG), ()-Epigallocatechin-3-gallate prevents reduces bleomycin-induced DNA damage photocarcinogenesis in mice through in human leucocytes. Toxicology In Vitro, interleukin-12-dependent DNA repair. 20, 295–300 Cancer Research, 66, 5512–5520. 48 Yen, G.C., Ju, J.W. and Wu, C.H. (2004) 55 Lambert, J.D., Hong, J., Yang,G.Y.,Liao, J. Modulation of tea and tea polyphenols on and Yang, C.S. (2005) Inhibition of benzo(a)pyrene-induced DNA damage in carcinogenesis by polyphenols: evidence Chang liver cells. Free Radical Research, from laboratory investigations. The 38, 193–200. American Journal of Clinical Nutrition, 49 Jiang, T., Glickman, B.W.and de Boer, J.G. 81, 284S–291S. (2001) Protective effect of green tea 56 Ju, J., Hong, J., Zhou, J.N., Pan, Z., against benzo[a]pyrene-induced Bose, M., Liao, J., Yang, G.Y., Liu, Y.Y., mutations in the liver of Big Blue Hou, Z., Lin, Y., Ma, J., Shih, W.J., transgenic mice. Mutation Research, Carothers, A.M. and Yang, C.S. (2005) 480–481, 147–151. Inhibition of intestinal tumorigenesis in 50 Shi, S.T., Wang, Z.Y., Smith, T.J., Apc min/ þ mice by ()-epigallocatechin- Hong, J.Y., Chen, W.F., Ho, C.T. and 3-gallate, the major catechin in green tea. Yang, C.S. (1994) Effects of green tea Cancer Research, 65, 10623–10631. and black tea on 4-(methylnitrosamino)-1- 57 Hao, X., Bose, M., Lambert, J.D., Ju, J., (3-pyridyl)-1-butanone bioactivation, Lu, G., Lee, M.J., Park, S., Husain, A., DNA methylation, and lung Wang, S., Sun, Y. and Yang, C.S. (2007) tumorigenesis in A/J mice. Cancer Inhibition of intestinal tumorigenesis in Research, 54, 4641–4647. Apc(min/ þ ) mice by green tea 51 Xu, Y., Ho, C.T., Amin, S.G., Han, C. and polyphenols (polyphenon E) and Chung, F.L. (1992) Inhibition of tobacco- individual catechins. Nutrition and speciﬁc nitrosamine-induced lung Cancer, 59,62–69. tumorigenesis in A/J mice by green tea 58 Xiao, H., Hao, X., Simi, B., Ju, J., Jiang, H., and its major polyphenol as antioxidants. Reddy, B.S. and Yang, C.S. (2008) Green Cancer Research, 52, 3875–3879. tea polyphenols inhibit colorectal 52 Kaur,S.,Greaves,P.,Cooke,D.N., aberrant crypt foci (ACF) formation and Edwards, R., Steward, W.P., prevent oncogenic changes in dysplastic Gescher, A.J. and Marczylo, T.H. (2007) ACF in azoxymethane-treated F344 rats. Breast cancer prevention by green tea Carcinogenesis, 29, 113–119. catechins and black tea theaﬂavins in 59 Carter, O., Dashwood, R.H., Wang, R., the C3(1) SV40 T,t antigen transgenic Dashwood, W.M., Orner, G.A., mouse model is accompanied by Fischer, K.A., Lohr, C.V., Pereira, C.B., increasedapoptosisandadecreasein Bailey, G.S. and Williams, D.E. (2007) oxidative DNA adducts. Journal of Comparison of white tea, green tea, AgriculturalandFoodChemistry, epigallocatechin-3-gallate, and caffeine as 55, 3378–3385. inhibitors of PhIP-induced colonic 53 Lin, D.X., Thompson, P.A., Teitel, C., aberrant crypts. Nutrition and Cancer, Chen, J.S. and Kadlubar, F.F. (2003) 58,60–65. Direct reduction of N-acetoxy-PhIP by tea 60 Lou, Y.R., Lu, Y.P., Xie, J.G., Huang, M.T. polyphenols: a possible mechanism for and Conney, A.H. (1999) Effects of oral chemoprevention against PhIP-DNA administration of tea, decaffeinated tea, 628j 33 Tea and Its Constituents

and caffeine on the formation and growth human glutathione s-transferases by of tumors in high-risk SKH-1 mice polyphenone intervention. Cancer previously treated with ultraviolet B light. Epidemiology, Biomarkers & Prevention, Nutrition and Cancer, 33, 146–153. 16, 1662–1666. 61 Lu, Y.P., Lou, Y.R., Liao, J., Xie, J.G., 67 Tang, L., Tang, M., Xu, L., Luo, H., Peng, Q.Y., Yang, C.S. and Conney, A.H. Huang, T., Yu, J., Zhang, L., Gao, W., (2005) Administration of green tea or Cox, S.B. and Wang, J.S. (2008) caffeine enhances the disappearance of Modulation of aflatoxin biomarkers in UVB-induced patches of mutant p53 human blood and urine by green tea positive epidermal cells in SKH-1 mice. polyphenols intervention. Carcinogenesis, Carcinogenesis, 26, 1465–1472. 29, 411–417. 62 Inami, S., Takano, M., Yamamoto, M., 68 Schwartz, J.L., Baker, V., Larios, E. and Murakami, D., Tajika, K., Yodogawa, K., Chung, F.L. (2005) Molecular and cellular Yokoyama, S., Ohno, N., Ohba, T., effects of green tea on oral cells of Sano, J., Ibuki, C., Seino, Y. and smokers: a pilot study. Molecular Nutrition Mizuno, K. (2007) Tea catechin & Food Research, 49,43–51. consumption reduces circulating 69 Hakim, I.A., Harris, R.B., Brown, S., oxidized low-density lipoprotein. Chow, H.H., Wiseman, S., Agarwal, S. International Heart Journal, 48, 725–732. and Talbot, W. (2003) Effect of increased 63 Hsu, S.P., Wu, M.S., Yang, C.C., tea consumption on oxidative DNA Huang, K.C., Liou, S.Y., Hsu, S.M. and damage among smokers: a randomized Chien, C.T. (2007) Chronic green tea controlled study. The Journal of Nutrition, extract supplementation reduces 133, 3303S–3309S. hemodialysis-enhanced production of 70 Gao, Y.T., McLaughlin, J.K., Blot, W.J., hydrogen peroxide and hypochlorous Ji, B.T., Dai, Q. and Fraumeni, J.F. Jr acid, atherosclerotic factors, and (1994) Reduced risk of esophageal cancer proinflammatory cytokines. The American associated with green tea consumption. Journal of Clinical Nutrition, 86, Journal of the National Cancer Institute, 1539–1547. 86, 855–858. 64 Chow, H.H., Hakim, I.A., Vining, D.R., 71 Ji, B.T., Chow, W.H., Yang, G., Crowell, J.A., Cordova, C.A., Chew, W.M., McLaughlin, J.K., Gao, R.N., Zheng, W., Xu, M.J., Hsu, C.H., Ranger-Moore, J. and Shu, X.O., Jin, F., Fraumeni, J.F. Jr, Alberts, D.S. (2006) Effects of repeated and Gao, Y.T. (1996) The influence green tea catechin administration on of cigarette smoking, alcohol, and human cytochrome P450 activity. Cancer green tea consumption on the risk of Epidemiology, Biomarkers & Prevention, carcinoma of the cardia and distal 15, 2473–2476. stomach in Shanghai, China. Cancer, 65 Donovan, J.L., Chavin, K.D., Devane, C.L., 77, 2449–2457. Taylor, R.M., Wang, J.S., Ruan, Y. and 72 Lee, M.J., Wang, Z.Y., Li, H., Chen, L., Markowitz, J.S. (2004) Green tea Sun, Y., Gobbo, S., Balentine, D.A. and (Camellia sinensis) extract does not alter Yang, C.S. (1995) Analysis of plasma cytochrome p450 3A4 or 2D6 activity in and urinary tea polyphenols in healthy volunteers. Drug Metabolism and human subjects. Cancer Epidemiology, Disposition, 32, 906–908. Biomarkers & Prevention, 4, 393–399. 66 Chow, H.H., Hakim, I.A., Vining, D.R., 73 Sun, C.L., Yuan, J.M., Lee, M.J., Crowell, J.A., Tome, M.E., Ranger- Yang, C.S., Gao, Y.T., Ross, R.K. and Moore, J., Cordova, C.A., Yu, M.C. (2002) Urinary tea polyphenols Mikhael, D.M., Briehl, M.M. and in relation to gastric and esophageal Alberts, D.S. (2007) Modulation of cancers: a prospective study of men in References j629

Shanghai, China. Carcinogenesis, 23, volunteers with high-grade prostate 1497–1503. intraepithelial neoplasia: a preliminary 74 Wu, A.H., Yu, M.C., Tseng, C.C., report from a one-year proof-of-principle Hankin, J. and Pike, M.C. (2003) Green study. Cancer Research, 66, 1234–1240. tea and risk of breast cancer in Asian 82 Laurie, S.A., Miller, V.A., Grant, S.C., Americans. International Journal of Kris, M.G. and Ng, K.K. (2005) Phase I Cancer, 106, 574–579. study of green tea extract in patients with 75 Wu, A.H., Tseng, C.C., Van Den Berg, D. advanced lung cancer. Cancer Chemo- and Yu, M.C. (2003) Tea intake, COMT therapy and Pharmacology, 55,33–38. genotype, and breast cancer in Asian- 83 Schneider, K., Oltmanns, J. and American women. Cancer Research, Hassauer, M. (2004) Allometric principles 63, 7526–7529. for interspecies extrapolation in 76 Kurahashi, N., Sasazuki, S., Iwasaki, M., toxicological risk assessment: empirical Inoue, M. and Tsugane, S. (2008) Green investigations. Regulatory Toxicology and tea consumption and prostate cancer risk Pharmacology, 39, 334–347. in Japanese men: a prospective study. 84 Galati, G., Lin, A., Sultan, A.M. and American Journal of Epidemiology, 167, OBrien, P.J. (2006) Cellular and in vivo 71–77. hepatotoxicity caused by green tea 77 Borrelli, F., Capasso, R., Russo, A. and phenolic acids and catechins. Free Radical Ernst, E. (2004) Systematic review: green Biology & Medicine, 40, 570–580. tea and gastrointestinal cancer risk. 85 Bonkovsky, H.L. (2006) Hepatotoxicity Alimentary Pharmacology & Therapeutics, associated with supplements containing 19, 497–510. Chinese green tea (Camellia sinensis). 78 Suzuki, Y., Tsubono, Y., Nakaya, N., Annals of Internal Medicine, 144,68–71. Koizumi, Y. and Tsuji, I. (2004) Green tea 86 Isbrucker, R.A., Edwards, J.A., Wolz, E., and the risk of breast cancer: pooled Davidovich, A. and Bausch, J. (2006) analysis of two prospective studies in Safety studies on epigallocatechin gallate Japan. British Journal of Cancer, (EGCG) preparations. Part 2: dermal, 90, 1361–1363. acute and short-term toxicity studies. Food 79 Kuriyama, S., Shimazu, T., Ohmori, K., and Chemical Toxicology, 44, 636–650. Kikuchi, N., Nakaya, N., Nishino, Y., Tsubono, Y. and Tsuji, I. (2006) Green tea References to Section 33.2 consumption and mortality due to cardiovascular disease, cancer, and all 87 Sarkar,A.andBhaduri,A.(2001)Black causes in Japan: the Ohsaki study. The tea is a powerful chemopreventor of Journal of the American Medical reactive oxygen and nitrogen species: Association, 296, 1255–1265. comparison with its individual catechin 80 Tsubono, Y., Nishino, Y., Komatsu, S., constituents and green tea. Biochemical Hsieh, C.C., Kanemura, S., Tsuji, I., and Biophysical Research Nakatsuka, H., Fukao, A., Satoh, H. Communications, 284, 173–178. and Hisamichi, S. (2001) Green tea 88 Joubert, E. and Schulz, H. (2006) and the risk of gastric cancer in Japan. Production and quality aspects of rooibos The New England Journal of Medicine, tea and related products. A review. Journal 344, 632–636. of Applied Botany and Food Quality, 81 Bettuzzi, S., Brausi, M., Rizzi, F., 80, 138–144. Castagnetti, G., Peracchia, G. and 89 Balentine, D.A., Wiseman, S.A. and Corti, A. (2006) Chemoprevention of Bouwens, L.C.M. (1997) The chemistry of human prostate cancer by oral tea ﬂavonoids. Critical Reviews in Food administration of green tea catechins in Science and Nutrition, 37, 693–704. 630j 33 Tea and Its Constituents

90 Tanaka, T., Watarumi, S., Matsuo, Y., Pharmacokinetics and tissue distribution Kamei, M. and Kouno, I. (2003) study of orientin in rat by liquid Production of theasinensins A and D, chromatography. Journal of epigallocatechin gallate dimers of black Pharmaceutical and Biomedical Analysis, tea, by oxidation–reduction dismutation 47, 429–434. of dehydrotheasinensin A. Tetrahedron, 100 Wang, H., Ye, G., Ma, C.-H., Tang, Y.-H., 59, 7939–7947. Fan, M.-S., Li, Z.-X. and Huang, C.-G. 91 Robertson, A. (1992) The chemistry and (2007) Identiﬁcation and determination biochemistry of black tea production: the of four metabolites of mangiferin in rat non-volatiles, in Tea: Cultivation to urine. Journal of Pharmaceutical and Consumption (eds K.C. Willson and M.N. Biomedical Analysis, 45, 793–798. Clifford), Chapman & Hall, London. 101 Gupta, S., Saha, B. and Giri, A.K. (2002) 92 Haslam, E. (2003) Thoughts on Comparative antimutagenic and thearubigins. Phytochemistry, 64,61–73. anticlastogenic effects of green and black 93 Shimamura, N., Miyase, T., Umehara, K., tea: a review. Mutation Research, 512, Warashina, T. and Fujii, S. (2006) 37–65. Phytoestrogens from Aspalathus linearis. 102 Nehlig, A. and Debry, G. (1994) Potential Biological & Pharmaceutical Bulletin, genotoxic, mutagenic and antimutagenic 29, 1271–1274. effects of coffee. Mutation Research, 94 Joubert, E., Richards, E.S., Van der 317, 145–162. Merwe, J.D., De Beer, D., Manley, M. and 103 McArdle, N.J., Clifford, M.N. and Gelderblom, W.C. (2008) Effect of species Ioannides, C. (1999) Consumption of tea variation and processing on phenolic modulates the urinary excretion of composition and in vitro antioxidant mutagens in rats treated with IQ. Role of activity of aqueous extracts of Cyclopia spp. caffeine Mutation Research, 44, 191–203. (honeybush tea). Journalof Agriculturaland 104 Mckay, D.L. and Blumberg, J.B. (2006) A Food Chemistry, 56,954–963. review of the bioactivity of South African 95 Manach, C., Williamson, G., Morand, C., herbal teas: rooibos (Aspalathus linearis) Scalbert, A. and Remesy, C. (2005) and honeybush (Cyclopia inntermedia). Bioavailability and bioefﬁcacy of Phytotherapy Research, 21,1–16. polyphenols in humans. Review of 97 105 Joubert, E., Gelderblom, W.C.A., Louw, A. bioavailability studies. The American and De Beer, D. (2008) South African Journal of Clinical Nutrition, 81, herbal teas: Aspalathus linearis, Cyclopia 230s–242s. spp. and Athrixia phylicoides: a review. 96 Lambert, J.D. and Yang, C.S. (2003) Journal of Ethnopharmacology, 119, Cancer chemopreventive activity and 376–412. bioavailability of tea and tea 106 Van der Merwe, J.D., Joubert, E., polyphenols. Mutation Research, Richards, E.S., Manley, M., 523–524, 201–208. Snijman, P.W., Marnewick, J.L. and 97 Horrigan, L.A., Kelly, J.P. and Connor, T.J. Gelderblom, W.C.A.(2006) A comparative (2006) Immunomodulatory effects of study on the antimutagenic properties of caffeine: friend or foe? Pharmacology & aqueous extracts of Aspalathus linearis Therapeutics, 111, 877–892. (rooibos), different Cyclopia spp. 98 Krafczyk, N. and Glomb, M.A. (2008) (honeybush) and Camellia sinensis teas. Characterization of phenolic compounds Mutation Research, 611,42–53. in rooibos tea. Journal of Agricultural and 107 Snijman, P.W., Swanevelder, S., Food Chemistry, 56, 3368–3376. Joubert, E., Green, I.R. and 99 Li, D., Wang, Q., Yuan, Z., Zhong, L., Gelderblom, W.C.A. (2007) The Xu, L., Cui, Y. and Duan, K. (2008) antimutagenic activity of the major References j631

flavonoids of rooibos (Aspalathus linearis): and apoptosis. Toxicology In Vitro, 22, some dose–response effects on mutagen 598–609. activation–flavonoid interactions. 115 Mohan, K.V.P.C., Devaraj, H., Hara, Y. Mutation Research, 631, 111–123. and Nagini, S. (2005) Antiproliferative 108 Miller, N.J., Castelluccio, C., Tijburg, L. and apoptosis inducing effect of and Rice-Evans, C. (1996) The antioxidant lactoferrin and black tea polyphenol properties of theaflavins and their gallate combination on hamster buccal pouch esters-radical scavengers or metal carcinogenesis. Clinical Biochemistry, chelators? FEBS Letters, 392,40–44. 38, 879–886. 109 Lin, J.-K., Chen, P.-C., Ho, C.-T. and Lin- 116 Jia, X., Han, C. and Chen, J. (2002) Effects Shiau, S.-Y. (2000) Inhibition of xanthine of tea on preneoplastic lesions and cell oxidase and suppression of intracellular cycle regulators in rat liver. Cancer reactive oxygen species in HL-60 cells by Epidemiology, Biomarkers & Prevention, theaflavin-3,30-digallate, () epigallo- 11, 1663–1667. catechin-3-gallate, and propyl gallate. 117 Siddiqui, I.A., Raisuddin, S. and Journal of Agricultural and Food Chemistry, Shukla, Y. (2005) Protective effects of 48, 2736–2743. black tea on testosterone induced 110 Lin, Y.L., Tsai, S.H., Lin-Shiau, S.Y., oxidative damage in prostate. Cancer Ho, C.T. and Lin, J.K. (1999) Theaflavin-3, Letters, 227, 125–132. 30-digallate from black tea blocks the nitric 118 Lin, D.-X., Thompson, P.A., Teitel, C., oxide synthase by down-regulating the Chen, J.-S. and Kadlubar, F.F. (2003) activation of NF-kB in macrophages. Direct reduction of N-acetoxy-PhIP by tea European Journal of Pharmacology, polyphenols: a possible mechanism for 367, 379–388. chemoprevention against PhIP–DNA 111 Erba,D.,Riso,P.,Foti,P.,Criscuoli,F. adduct formation. Mutation Research, and Testtolin, G. (2003) Black tea extract 523–524, 193–200. supplementation decreases oxidative 119 Lu, G., Liao, J., Yang, G., Reuhl, K.R., damage in Jurkat T cells. Archives of Hao, X. and Yang, C.S. (2006) Inhibition Biochemistry and Biophysics, of adenoma progression to 416, 196–201. adenocarcinoma in a 4- 112 Joubert, E., Winterton, P., Britz, T.J. and (methylnitrosamino)-1-(3-pyridyl)-1- Ferreira, D. (2004) Superoxide anion and butanone-induced lung tumorigenesis a,a-diphenyl-b-picrylhydrazyl radical model in A/J mice by tea polyphenols and scavenging capacity of rooibos caffeine. Cancer Research, 66, (Aspalathus linearis) aqueous extracts, 11494–11501. crude phenolic fractions, tannin and 120 Tsuda, H., Sekine, K., Uehara, N., flavonoids. Food Research International, Takasuka, N., Moore, M.A., Konno, Y., 37, 133–138. Nakashita, K. and Degawa, M. (1999) 113 Joubert, E., Winterton, P., Britz, T.J. and Heterocyclic amine mixtures Gelderblom, W.C.A. (2005) Antioxidant carcinogenesis and its enhancement by and pro-oxidant activities of aqueous caffeine in F344 rats. Cancer Letters, extracts and crude polyphenolic fractions 143, 229–234. of tooibos (Aspalathus linearis). Journal of 121 Nawrot, P., Jordan, S., Eastwood, J., Agricultural and Food Chemistry, 53, Rotstein, J., Hugenholtz, A. and 10260–10267. Feeley, M. (2003) Effects of caffeine on 114 Schuck, A.G., Ausubel, M.B., human health. Food Additives and Zuckerbraun, H.L. and Babich, H. (2008) Contaminants, 20,1–30. Theaflavin-3,30-digallate, a component of 122 Catterall, F. McArdle, N.J. Mitchell, L. black tea: an inducer of oxidative stress Papayanni, M.N. Clifford, M.N. 632j 33 Tea and Its Constituents

Ioannides, C. 2003 Hepatic and intestinal 129 Hou, Z., Lambert, J.D., Chin, K.-V. and cytochrome P450 and conjugase activities Yang, C.S.(2004) Effects of tea polyphenols in rats treated with black continuous on signal transduction pathways related to ingestion of herbal teas on intestinal cancer chemoprevention. Mutation CYP3A in the rat Journal of Research, 555,3–19. Pharmacological Sciences, 103, 214–221. 130 Hashimoto, T., He, Z., Ma, W.-Y., 123 Bu-Abbas, A., Clifford, M.N., Walker, R. Schmid, P.C., Bode, A.M., Yang, C.S. and and Ioannides, C. (1998) Contribution of Dong, Z. (2004) Caffeine inhibits cell caffeine and ﬂavanols in the induction of proliferation by G0/G1 arrest in JB6 cells. hepatic phase II activities by green tea. Cancer Research, 64, 3344–3349. Food and Chemical Toxicology, 36, 131 Na, H.K., Mossanda, K.S., Lee, J.Y. and 617–621. Surh, Y.J. (2004) Inhibition of phorbol 124 Nikaidou, S., Ishizuka, M., Maeda, Y., ester-induced COX-2 expression by some Hara, Y., Kazusaka, A. and Fujita, S. (2005) edible African plants. Biofactors, Effect of green tea extracts, caffeine, and 21, 149–153. catechins on hepatic drug metabolising 132 Langley-Evans, S.C. (2000) Consumption enzyme activities and the mutagenic of black tea elicits an increase in plasma transformation of carcinogens. The antioxidant potential in humans. Japanese Journal of Veterinary Research, International Journal of Food Sciences and 52, 185–192. Nutrition, 51, 309–315. 125 Jang, E.-H., Park, Y.-C. and Chung, W.-G. 133 Shukla, Y. (2007) Tea and cancer (2004) Effects of dietary supplements on chemoprevention. Asian Paciﬁc Journal of the induction and inhibition of Cancer Prevention, 8, 155–166. cytochrome P450s protein expression in 134 Yang, C.S., Lambert, J.D., Ju, J., Lu, G. and rats. Food and Chemical Toxicology, Sang, S. (2007) Tea and cancer prevention: 42, 1749–1756. molecular mechanisms and human 126 Matsuda, K., Nishimura, Y., Kurata, N., relevance. Toxicology and Applied Iwase, M. and Yasuhara, H. (2007) Pharmacology, 224, 265–273. Effects of continuous ingestion of herbal 135 Ju, J.J., Lambert, G.L. and Yang, C.S. teas on intestinal CYP3A in the rat. (2007) Inhibition of carcinogenesis by tea Journal of Pharmacological Sciences, constituents. Seminars in Cancer Biology, 103, 214–221. 17, 395–402. 127 Marnewick, J.L., Joubert, E., Joseph, S., 136 Chen, Z.Y., Zhu, Q.Y., Tsang, D. and Swanevelder, S., Swart, P. and Huang, Y. (2001) Degradation of green Gelderblom, W.C.A. (2005) Inhibition of tea catechins in tea drinks. Journal of tumour promotion in mouse skin by Agricultural and Food Chemistry, 49, extracts of rooibos (Aspalathus linearis) 477–482. and honeybush (Cyclopia intermedia), 137 Wang, H. and Helliwell, K. (2000) unique South African herbal teas. Cancer Epimerisation of catechins in green Letters, 224, 193–202. tea infusions. Food Chemistry, 70,337–344. 128 Sanchez, G.M., Re, L., Giuliani, A., 138 Seeram, N.P. and Nair, M.G. (2002) Núñez-Selles, A.J., Davidson, G.P. and Inhibition of lipid peroxidation and León-Fernandez, O.S. (2000) Protective structure–activity-related studies of effects of Mangifera indica L. extract, dietary constituents anthocyanins, mangiferin and selected antioxidants anthocyanidins and catechins. Journal of against TPA-induced biomolecules Agricultural and Food Chemistry, oxidation and peritoneal macrophage 50, 5308–5312. activation in mice. Pharmacological 139 Su, Y.L., Leung, L.K., Huang, Y. and Research, 42, 565–573. Chen, Z.Y. (2003) Stability of tea References j633

theaflavins and catechins. Food Chemistry, Salmonella mutagenicity assay: possible 83, 189–195. mechanisms involved. MSc thesis, 140 Tanaka, T., Inoue, K., Betsumiya, Y., Stellenbosch University, Stellenbosch, Mine, C. and Kouno, I. (2001) Two types South Africa. of oxidative dimerization of black tea 144 van het Hof, K.H., Kivits, G.A., polyphenol theaflavin. Journal of Weststrate, J.A. and Tijburg, L. B. (1998) Agricultural and Food Chemistry, Bioavailability of catechins from tea: the 49, 5785–5789. effect of milk. European Journal of Clinical 141 Manzocco, L., Anese, M. and Nicoli, C. Nutritions, 52, 356–359; Siebert, K.J., (1998) Antioxidant properties of tea Troukhanova, N.V. and Lynn, P.Y. (1996) extracts as affected by processing. Nature of polyphenol—protein Lebensmittel-Wissenschaft & Technologie, interactions. Journal of Agricultural and 31, 694–698. Food Chemistry, 44,80–85. 142 Marais, C., Janse van Rensburg, W., 145 Kyle, J.A., Morrice, P.C., McNeill, G. and Ferreira, D. and Steenkamp, J.A. (2000) Duthie, G.G. (2007) Effects of infusion (S)- and (R)-Eriodictyol-6-C-b-D- time and addition of milk on content and glucopyranoside, novel keys to the absorption of polyphenols from black tea. fermentation of rooibos (Aspalathus Journal of Agricultural and Food Chemistry, linearis). Phytochemistry, 55,43–49. 55, 4889–4894. 143 Van der Merwe, J.D. (2005) A comparative 146 Reddy, V.C., Vidya Sagar, G.V., study on protection of Cyclopia spp. Sreeramulu, D., Venu, L. and Raghunath, (honeybush), Aspalathus linearis (rooibos) M. (2007) Addition of milk does not alter and Camellia sinensis teas against aflatoxin the antioxidant activity of black tea. Annals B1 induced mutagenesis in the of Nutrition and Metabolism, 49, 189–195. j635

34 Protective Effects of Alcoholic Beverages and their Constituent

34.1 Wine Philipp Saiko, Akos Szakmary, and Thomas Szekeres

34.1.1 Introduction

34.1.1.1 General Information and Historical Background Wine is an alcoholic beverage made from fermentation of fruit or grape juice. A wine may consist of a single type of grape or may contain a blend of different types of grapes. The natural chemical balance of grapes is such that they can ferment without the addition of sugars, acids, enzymes, or other nutrients. Although other fruits such as apples and berries can also be fermented, the resulting wines are normally named after the fruit from which they are produced (e.g., apple wine) and are generically known as fruit or country wine. Others, such as barley wine and rice wine (e.g., sake), are made from starch-based materials and resemble beer more than wine, while ginger wine is fortified with brandy. In these cases, the use of the term wine is a reference to the higher alcohol content rather than to the production process. Law in many jurisdictions protects the commercial use of the English word wine and its equivalent in other languages. Wine is produced by fermenting crushed grapes using various types of yeasts that consume sugars added to grapes and convert them into alcohol. Various varieties of grapes and strains of yeast are used depending on the types of wine produced. The scientific history of wine stretches back much longer than its first written account in the Bible, with the earliest evidence dating around 5000 BC. A pottery jar recovered in present-day Iran provides the earliest chemical evidence so far discovered [1]. Wine was identified by the presence of calcium salts of tartaric acid, only present in large amounts in grapes and in the resins of terebinth trees [2]. This resin was widely used in ancient times as an additive to wine to inhibit bacterial growth. Though wild grape trunks have been found to originate as far back as the eighth millennium, this archaeological discovery marks the earliest scientific record of

fermented wine as part of human culture. From the ﬁfth millennium BC, wine spread from its postulated origins in the Southern Caucasus to Palestine, Syria, Egypt, and Mesopotamia and subsequently to the Mediterranean [2], but after the fall of the Roman Empire wine making declined. During the Dark Ages, wine making was kept alive mainly through the efforts of Christian monasteries. As the Church extended their monasteries, they began to develop some of the ﬁnest vineyards in Europe. In the Medieval period, wine was still considered a staple of everyday diet, because most of Europe lacked reliable sources of drinking water. However, in the following centuries wine had to face the rival of a clean and readily available supply of drinking water, and was no longer needed as a major part of the daily diet.

34.1.1.2 The Health Effects of Wine In 1992, Renaud and de Lorgeril observed a lower mortality rate in coronary heart disease in France compared to other North European countries and the United States, despiteasimilarintakeofhighlevelsofsaturatedfat,identicalsmokinghabits,andlack ofexercise[3].Thisobservationwasbroughttotheattentionofthemedicalcommunity and the lay public, and became known as the French Paradox. The authors explained theparadoxintermsoftheconsumptionoftheso-calledMediterranean diet,withan abundance of vegetables, fruits, olive oil, and – especially – red wine. They suggested thisdifferencecouldbeattributedtothehighconsumptionof(red)winesbytheFrench population. In the United States, a boom in red wine consumption was initiated in the 1990s by 60 Minutes, and other news reports on the French Paradox. Population studies have observed a J-curve association between wine consumption and the risk of heart disease. This means that abstainers and heavy drinkers have an elevated risk, whilst moderate drinkers have a lower risk. They also found that moderate consumption of other alcoholic beverages might be cardioprotective, although the association is considerably stronger for wine.

34.1.1.3 Ingredients of Wine Wine is a rich source of biologically active phytochemicals, chemicals found in plants. Phytochemicals are divided into distinct subgroups according to their structure and function in the plant. The polyphenols are one of the most prominent groups in disease prevention. Currently, more than 8000 of them have been identified, which are ubiquitous in plant-borne foods. Polyphenols are not only associated with color and with sensory properties but also linked to the health benefits ascribed to fruits, vegetables, and wine. The grape polyphenols may be classified into the following three groups: 1. Nonflavonoids, derived from hydroxycinnamic acids (paracoumaric acid, caffeic acid, chlorogenic acid, and ferulic acid) and hydroxybenzoic acids (gallic acid, protecatechouric acid, and vanillic acid). 2. Flavonoids, comprising the largest class (several thousand) of phenolic compounds including flavonols (e.g., querecetin and myricetin), isoflavonols, flavanones, flavanals (e.g., catechin, epicatechin, and procyanidin), and anthocyanins (e.g., delphinidin, cyanidin, and malvidin). 34.1 Wine j637

3. Stilbenes, constituting a relatively small group of phenolic compounds that are usually synthesized in plants in response to stress conditions. Their structures contain two benzene rings connected by a methylene bridge. Resveratrol (3,40,5- trihydroxy-trans-stilbene; RV) is the most extensively studied stilbene derivative. Most flavonoids occurring in plants are conjugated to sugar-moieties, pectins, and organic acids, or are polymerized with other flavonoids to polymers. Products of many fruits and vegetables, such as strawberries, blueberries, green and black tea, tomatoes, yellow onions, soy, and chocolate, contain considerable amounts of polyphenols. Red grapes and wine of the Vitis vinifera varieties are especially rich in these polyphenols, and more than 500 phenolic compounds have been recognized in wine thus far [2]. Evidence from laboratory studies suggests that red wine may possess superior health benefits including the prevention of cancer because it contains more polyphenols than white wine, which is due to the production process (see below). Red wines obtained by traditional maceration can have a polyphenol content of more than 3 g/l. RV is thought to be at least partly responsible for the health benefits of red wine, since it has been shown to exert a range of both cardioprotective and chemoprotective mechanisms, thus being the most prominent of these polyphenols. Red wine contains much greater amounts of RV than white wine does, since RV is concentrated in the grape skins and seeds, and the manufacturing procedure of red wine includes prolonged contact of grape juice with these parts. Plant polyphenols are recognized for their antioxidative activities, thereby protecting cells from oxidative damage caused by free radicals. Electron acceptors such as molecular oxygen react easily with them to become reactive oxygen species (ROS). Polyphenols scavenge free radicals, thus breaking the free radical chain reaction of lipid peroxidation, which has been implicated in the development of cancer. However, it is inherently difficult to evaluate the beneficial effects of specific polyphenolic antioxidants, since a large number of individual compounds may occur in a single food. For example, over 60 different chemically distinct flavonoids are known to occur in a given red wine. Numerous scientific studies have been conducted to attempt to arrive at one consistent index for food antioxidant power. Since it has been proven that the dietary intake of compounds exerting antioxidant activity is of great medical value, a number of chemical, biological, and electrochemical methods have been proposed to evaluate the antioxidant potential of naturally occurring agents such as RV.

34.1.2 Physicochemical Properties of Active Compounds, Occurrences, and Chemical Structures

34.1.2.1 Resveratrol

34.1.2.1.1 History and Sources Resveratrol (3,40,5-trihydroxy-trans-stilbene; Scheme 34.1) was ﬁrst isolated in 1940 as an ingredient of the roots of white hellebore (Veratrum grandiﬂorum O. Loes) and has since been found in a wide variety of about 70 plant species, including grapes, mulberries, and peanuts [4]. RV is a polyphenol and 638j 34 Protective Effects of Alcoholic Beverages and their Constituent

Scheme 34.1 Chemical structure of resveratrol.

has been classified as a phytoalexin for being synthesized in spermatophytes in response to injury, UV irradiation, and fungal attack. RV was identified in 1963 as the active constituent of the dried roots of Polygonum cuspidatum, also called Ko-J O-kon in Japan, and used in traditional Asian medicine against suppurative dermatitis, gonor- rhea, favus, and hyperlipemia [5]. RV was first detected in grapevines (Vitis vinifera)in 1976 and then in wine in 1992 [6]. In grapes, especially when infected with Botrytis cinerea, RV is exclusively synthesized in the leaf epidermis and in grape skins, but not in the flesh. Fresh grape skins contain 50–100 mg RV per gram, corresponding to 5–10% of their biomass [7]. Since grape skins are not fermented in the production process of white wines, only red wines contain considerable amounts of RV. Its concentrations measured in a sampling of red wine varieties ranged from 2 to 40 mM [7].

34.1.2.1.2 French Paradox Epidemiological studies have revealed an inverse correlation between the red wine consumption and the incidence of cardiovascular disease, a phenomenon commonly known as the French Paradox, that is, the fact that the incidence of heart infarction in France is about 40% lower than in the rest of Europe, despite a diet being traditionally rich in saturated fat [3]. This led to the suggestion that RV might be the active principal of red wine. Indeed, RV protects the cardiovascular system by a large number of mechanisms including defense against ischemic- reperfusion injury, promotion of vasorelaxation, protection and maintenance of intact endothelium, antiatherosclerotic properties, inhibition of low-density lipoprotein oxidation, suppression of platelet aggregation, and estrogen-like actions [8, 9].

34.1.2.1.3 Effects of Resveratrol Besides its effects on the cardiovascular system, RV exhibits a remarkable inhibitory potential in various stages of tumor development [9]. The antitumor activity of RV was first revealed by its ability to reduce the incidence of carcinogen-induced development of cancers in experimental animals [10]. Subse- quently, RV has been shown to exert numerous effects that may block tumor development at several discrete stages during the multigenic process of carcinogenesis, involving interactions between RV and manifold targets [11]. These targets include kinases [12], steroid hormone receptors [13], ROS [14], ribonucleotide reductase (RR) [15], and DNA polymerases [16]. RV causes an arrest at the S/G2 phase transition of the cell cycle [17] and is capable of inducing differentiation and apoptosis in a multitude of human tumor cell lines. RV has also been identified as an effective inhibitor of RR [15, 18]. RR catalyzes the rate-limiting step of de novo DNA 34.1 Wine j639 synthesis, namely, the reduction of ribonucleotides into the corresponding deoxyribonucleoside triphosphates (dNTPs). The importance of all these targets for cancer development is well known and therefore RV can beneficially contribute to cancer prevention. As tissue inflammation provokes tumor promotion, anti-inflammatory agents are viewed as a valuable chemopreventive modality against this mechanism of carcinogenesis [14]. RV has been shown to exert substantial antiphlogistic activity in an in vivo rat model [19]. The key molecular targets implicated herein are cyclooxygenases (COX-1 and COX-2). COX-1 and COX-2 are, respectively, constitutive and inducible enzymes that catalyze the production of proinflammatory prostaglandins from arachidonic acid [14]. Prostaglandins stimulate tumor growth by acting on cell proliferation, angiogenesis, and immunosuppression. RV effects against cellular COX activity involve its direct inhibitory action against COX-1 and COX-2 and its suppression of transcriptional COX-2 upregulation [20]. As prostaglandins not only stimulate tumor cell growth but also suppress immune surveillance, COX enzymes are likely important targets of the cancer preventive activity of RV [20]. Arachidonic acid is also metabolized via lipoxygenase (LOX) to produce hydroperoxyeicosate- traenoic acids (HPETEs) or leukotrienes. Arachidonic acid metabolites derived from LOX pathways play an important role in growth-related signal transduction, implying that intervention through these pathways could be useful for attenuating cancer progression. RV inhibits LOX and COX in K562 myelogenous leukemia cells [21]. LOX-derived metabolites have an (indirect) influence on the development as well as the progression of human cancers [22]. Figure 34.1 displays the effects of RV on arachidonic acid metabolism. Furthermore, RV induces a multitude of effects that depend on the cell type (e.g., NF-kB modulation in cancer cells versus neural cells), cellular condition (normal, stressed, or malignant) and concentration (proliferative versus growth arrest) and can have opposing activities. The final readout depends on the balance of these partially opposing effects. Single alterations in cell physiology, signaling, and

Figure 34.1 Effects of resveratrol on arachidonic acid metabolism. 640j 34 Protective Effects of Alcoholic Beverages and their Constituent

metabolism result often in a cascade of changes that cannot always be restored by reversion of the single original change. RV targets whole pathways and sets of intracellular events rather than a single enzyme and therefore offers a less specific but more gentle (fewer side effects) and possibly more effective strategy for therapy to restore homeostasis. Therefore, a keen interest in RV has emerged due to its evident value as a cancer preventive and cardioprotective dietary substance. RVmay provide an alternative (and early)intervention approachthatcould prevent/delaydisease onset, emendthecourse of disease, and/or prevent further damage. Since the identification of resveratrols health benefits are largely owed to its high abundance in certain plants and foods, the discovery of further naturally occurring stilbenes, as well as, chemically modified analogues that are superior to RV in their cancer chemopreventive properties may be expected. This overview of molecular targets implicated in cancer antagonism by RV underscores the complexity underlying biological responses to this drug, which is probably common to many other molecules generated for self-defense. The efficacy of RV against distinct mechanisms of disease development is an indicator of its potential value for the prevention of various human diseases. Subsequently, the search and identification of more effective preventive agents among stilbene natural products is warranted. Table 34.1 briefly summarizes cancer-related targets and biological effects of RV.

34.1.2.2 Piceatannol: A Naturally Occurring Resveratrol Metabolite In contrast to the detailed knowledge of RV activities in biological systems, little is knownabouttheeffects ofotherpolyhydroxylatedstilbenes. RVundergoescytochrome P-450 catalyzed hydroxylation to piceatannol (3,30,40,5-tetrahydroxy-trans-stilbene; PCA; Scheme 34.2) and two other unidentiﬁed mono- and dihydroxy-RV analogues. This demonstratesthat a natural dietarycancer preventativeagent can be convertedto a compound with known chemopreventive and anticancer activity by enzyme CYP1B1, which is overexpressed in a wide variety of human tumors. Importantly, these ﬁndings give insight into the functional role of cytochrome P-450 enzyme CYP1B1 and support the hypothesis that CYP1B1 might be serving as a growth suppressor enzyme in tumors [23].ComparablewithRV,PCAdisplayscytotoxicactivityinacuteleukemiaand lymphoma cells and exerts antiproliferative activity in colon cancer cells [24].

34.1.2.3 Gallic Acid Gallic acid (3,4,5-trihydroxybenzoic acid; GA; Scheme 34.3) is found in gallnuts, sumac, tea leaves, oak bark, grapes, various herbs, and in red and white wines. In particular, red wine has a high content of this phenolic acid. GA can be present as free molecule or as part of the tannin molecule (gallotannin). It was recently shown that GA antagonizes P-selectin-mediated platelet leucocyte interactions [25] and could be jointly responsible for the beneﬁcial effects of red wine and the French Paradox. Other beneﬁcial effects might be the antidiabetic and antiangiogenic effects of GA þ containing fruit extracts [26] and the induction of Ca2 -dependent apoptosis in leukemia cells [27]. Altogether, GA was described as an excellent free radical 34.1 Wine j641

Table 34.1 Cancer-related targets and biological effects of resveratrol.

Class of targets Molecular targets Biological effects

Direct radical scavenging ROS DNA stability, lipidoxidation, apoptosis/cell survival Antioxidant/phase II SOD, catalase, GR DNA stability, lipidoxidation, enzymes apoptosis/cell survival Heme oxidase 1 (HO-1) Radical scavenging, anti-inflammatory, antiapoptotic GST, NQO1, ARE/EpRE activation, UDP-glucuronyl transferase ERb-activated Arachidonic acid related COX-1 þ 2 (COX-2 via NF-kB) Anti-inflammatory, antitumor promoting Estrogen related Selective estrogen receptor Superagonist, agonist, modulation (SERM) antagonist CYP 1A1, CYP 1B1 Inhibition of estrogen-metabolizing phase I enzymes Modulation of signaling Raf, Src, MAPK, PKD, Cell growth arrest, cell death, kinases PKCd differentiation Modulation of global gene p300 (acetylase), SIRT1 Cell survival, apoptosis delay, expression through (deacetylase) inflammatory response chromatin remodeling Transcription factors p53/p21 Cell cycle arrest, apoptosis IkB kinase/NF-kB Cell survival AP1 Tumor growth promoter Other cell cycle related Ribonucleotide reductase, DNA synthesis, cell cycle replicative DNA polymerases arrest Survivin Cell survival, apoptosis Cell death related TRAIL/DR4 þ 5 Apoptosis Fas/CD95 Apoptosis Mitochondria dependent, Apoptosis cytochrome c, Apaf-1 PI3K/Akt, ERa dependent Cell survival Ceramide Apoptosis

Scheme 34.2 Chemical structure of piceatannol. 642j 34 Protective Effects of Alcoholic Beverages and their Constituent

Scheme 34.3 Chemical structure of gallic acid.

scavenger and as inducer of differentiation as well as programmed cell death in numerous tumor cell lines and might play an important role in the prevention of malignant transformation and cancer development.

34.1.3 Bioavailability and Metabolism of Active Compounds

34.1.3.1 Bioavailability of Resveratrol Several in vivo studies in animals and humans demonstrated a very low intestinal uptake of RV leading to trace amounts in the bloodstream based on extensive metabolism in the gut and liver. Rapid metabolism is also the main reason for the short initial half-life of the primary molecule (8–14 min) [28]. The bulk of an intravenous dose of RV is converted to sulfate conjugates within 30 min in humans. A detailed analysis of plasma metabolites after oral dosing was not possible; however, both sulfate and glucuronide conjugates were detected [29].

34.1.3.2 Metabolites of Resveratrol: Glucuronide and Sulfate Conjugates Although modifications such as glucuronidation and sulfation typically reduce the cell permeability of drugs and aid in their excretion, the undeniable in vivo efficacy of administered RV, despite its low bioavailability, has led to the suggestion that its metabolites are likely to be the active principal. However, resveratrol-3-sulfate fails to inhibit CYPs [30] and there is currently no evidence that any metabolite is able to permeate the plasma membrane. Recently, the absorptive efficiency of three polyphenolic constituents (trans- resveratrol, þ -catechin, and quercetin) was investigated after oral application to healthy human subjects in three different media (white wine, grape juice, and vegetable juice/homogenate) [31]. All compounds were present in serum and urine predominantly as glucuronide and sulfate conjugates, reaching peak concentrations in the former around 30 min after consumption. The absorption of these three polyphenols was broadly equivalent in aqueous and alcoholic matrices; however, their peak plasma concentrations reached only 10–40 nM, whereas in vitro biologic activities have been studied at 5–100 mM [31].

34.1.3.3 Bioavailability of Resveratrol in Grape Juice Compared to Its Pure Aglycone In grape juice, the level of free RV is rather low, and cis- and trans-Piceid (RV-3-O-b- D-glucoside; Polydatin) are the major RV derivatives. This suggests a lower bioavailability of RV glycosides in grape juice in comparison to its pure aglycone form in 34.1 Wine j643 wine [32]. These findings were confirmed by another study reporting that RV concentrations in Italian red wine ranged from 8.6 to 24.8 mM, whereas the RV concentration in grape juice was only 1.6 mM [33]. Given that in vivo concentrations of individual metabolites can be much higher than those of the native compound, further studies are needed to determine (1) whether the metabolites represent inactivated forms of the drug, (2) act as a pool from which free RV can be released in various tissues, or (3) are themselves active in promoting many of the health benefits attributed to RV.

34.1.4 Mechanisms of Protection

34.1.4.1 Results of In Vitro Studies A large number of resveratrols beneﬁcial health effects, such as anticancer, antiviral, neuroprotective, antiaging, and anti-inﬂammatory effects, have been reported in vitro. Table 34.2 summarizes a variety of resveratrols anticancer activities.

34.1.4.2 Results of In Vivo Studies As the mechanisms of resveratrols broad cancer chemopreventive effects are not completely understood, continued efforts are needed, especially well-designed preclinical studies in animal models that closely mimic/represent human disease, to establish the usefulness of RV as cancer chemopreventive agent. Table 34.3 gives an overview of such preclinical studies including the observed effects.

34.1.5 Results of Human Studies

Overall, in vivo studies in rodents clearly show great promise for RV in the prevention and treatment of cancers. However, only a few phase I clinical trials are currently underway for oral RV in humans at doses up to 7.5 g per day. A National Cancer Institute-sponsored study has been completed recently [34], suggesting that consumption of RV does not cause serious adverse events. RV and six metabolites were recovered from plasma and urine, among them two monoglucuronides and RV-3- sulfate. The area under the plasma concentration curve (AUC) values for these metabolites were up to 23 times greater than those of RV [34]. Cancer chemopreventive effects of RV in cells in vitro require levels of at least 5 mM/l, intimating that consumption of high-dose RV might be insufﬁcient to elicit systemic levels synony- mous with cancer chemopreventive efﬁcacy. However, the high systemic levels of RV metabolites clearly suggest that they might be the active principals of the parent drug.

34.1.6 Conclusions

Chemopreventive agents such as RV might be used not only to prevent but also to treat cancer since the molecular targets are similar. Due of their pharmacological 644 j 4Poetv fet fAchlcBvrgsadterConstituent their and Beverages Alcoholic of Effects Protective 34 Table 34.2 Results of in vitro studies.

a Mechanism Assay system lM

Estrogenic/antiestrogenic and scavenging properties MCF-7 and MVLN breast cancer cells 0.1–25 Inhibition of aryl hydrocarbon-induced cytochrome P-450 1A1 HepG2 hepatoma cells and MCF-7 breast cancer cells 0.5–5 enzyme activity and CYP1A1 expression Modulation of the catalytic activity and mRNAexpression of the Cultured MCF-7 human breast carcinoma cells 1–20 procarcinogen-activating human cytochrome P-450 1B1 Inhibition of cell growth, G1-phase arrest, and induction of Human A431 epidermoid carcinoma cells 1–50 apoptosis Inhibition of COX-2 transcription and activity in phorbol ester- Human mammary 184B5/HER epithelial cells (and prema- 2.5–40 treated human mammary epithelial cells lignant MSK Leuk1 oral epithelial cells) Suppression of TNF-induced activation of nuclear transcrip- U937 myeloid leukemia cells, Jurkat lymphoid, and HeLa and 5 tion factor NF-kB, activator protein-1, and apoptosis H4 epithelial cells Inhibition of the binding of labeled estradiol to the estrogen MCF-7 breast cancer cells 10 receptor Stimulation of the proliferation of estrogen-dependent breast T47D breast cancer cells 10 cancer cells Growth inhibition, induction of apoptosis by downregulation EC-9706 esophageal cancer cells 10 of Bcl-2 and upregulation of bax Modulation of ERE-luciferase activity and estrogen-inducible MCF-7, T47D, LY2, and S30 breast cancer cells 10–15 protein expression Inhibition of the clonal growth of tumor cells 32Dp210, HL-60, U937, and L1210 leukemia cells 10–80 Depletion of intracellular dCTP, dTTP, dATP, and dGTP pools HL-60 leukemia cells 12.5 (inhibition of ribonucleotide reductase) Inhibition of 14C-cytidine incorporation into DNA (inhibition HL-60 leukemia cells 12.5 of ribonucleotide reductase) Growth inhibition, accumulation of cells at the S/G2 phase Caco-2 colon cancer cells 25 transition of the cell cycle, and signiﬁcant decrease of ornithine decarboxylase (ODC) activity Cell division cycle arrest at S/G2-phase transition HL-60 leukemia cells 30 Increase in the content of cyclins A and B1 as well as cyclin- SW480 colon cancer cells 30 dependent kinases Cdk1 and Cdk2, and promotion of Cdk1 phosphorylation Increase in the expression and kinase activities of positive MCF-7 breast cancer cells <50 G1/S and G2/M regulators, resulting in cell cycle blockade at the S-phase and apoptosis induction Inhibition of the expression and function of the androgen LNCaP prostate cancer cells 50–100 receptor Decrease of the expression and kinase activities of positive MDA-MB-231 breast cancer cells <200 G1/S and G2/M cell cycle regulators and inhibition of ribonucleotide reductase activity a Lowest concentration at which reproducible changes have been observed, or IC50 or EC50, if provided. 41Wine 34.1 j 645 646 j 4Poetv fet fAchlcBvrgsadterConstituent their and Beverages Alcoholic of Effects Protective 34 Table 34.3 Results of in vivo studies.

a Species Assay system/results Daily dose

Male BALB/c mice Inhibition of the growth of murine hepatocellular H22 carcinoma cells implanted in mice: RV 5–15 mg/kg (abd.) induced an S-phase arrest of tumor cells, and inhibition of tumor growth was further enhanced in combination with 5-FU Female Sprague–Dawley rats Inhibition of mammary carcinogenesis induced by 7,12-dimethylbenz(a)anthracene: RV had no 10 ppm (diet) effect on body weight gain and tumor volume but produced striking reductions in the incidence and multiplicity of tumors, and extended the latency period of tumor development Male F344 rats Inhibition of azoxymethane-induced colon carcinogenesis: RV significantly reduced the number 200 mg/kg (d.w.) and multiplicity of isolated ACF (aberrant crypt foci) and abolished large ACF Apc(Minþ) mice Prevention of the formation of colon tumors and reduction of the formation of small intestinal 0.01% (d.w.) tumors by 70%: RV downregulated genes that are directly involved in cell cycle progression or cell proliferation (cyclins D1 and D2, DP-1 transcription factor, and Y-box binding protein) HER-2/neu transgenic mice Inhibition of the development of spontaneous mammary tumors: RV supplementation delayed the 0.2 mg/kg (d.w.) development of spontaneous mammary tumors, reduced the mean number and size of mammary tumors, and diminished the number of lung metastases Female Sprague–Dawley rats Delay of MNU-induced mammary tumorigenesis: RVcaused a 28-day increase in tumor latency and 100 mg/kg (gav.) reduced the multiplicity of tumors from 6.0 in the control group to 3.9 in the treatment group Female athymic nude mice Inhibition of mammary carcinogenesis induced by MDA-MB-231 breast adenocarcinoma cells: RV 25 mg/kg (i.p.) caused significantly lower tumor growth, decreased angiogenesis, and increased the apoptotic index in ERa– ERbþ MDA–MB-231 tumors Syngeneic A/J mice Inhibition of subcutaneous neuroblastomas in mice: RV suppressed the growth rate, resulting in 40 mg/kg (i.p.) 70% long-term survival Fischer 344 rats Lower tumor growth rate, longer animal survival time, and higher animal survival rate; immu- 40 mg/kg (i.p.) nohistochemical analyses showed that the s.c. gliomas from RV-treated rats had fewer microvessel densities than did control rats Female C57BL/6 strain mice Significant reduction of tumor volume, tumor weight, and metastasis to the lung in mice bearing 2.5–10 mg/kg highly metastatic Lewis lung carcinoma (LLC) tumors: RV dose-dependently inhibited angiogenesis (i.p.) and had no effect on either initial or final body weight in LLC-bearing mice compared to that in normal mice Female syngeneic C57BL/6N Growth inhibition of B16-BL6 melanoma cells in vivo: Treatment with RV significantly delayed 50 mg/kg (i.p.) mice tumor growth, without mortality or body weight changes, but did not reduce the number of lung metastases after i.v. injection of B16-BL6 cells Male Wistar rats RVadministration to rats inoculated with the fast growing Yoshida AH-130 ascites hepatoma caused 1 mg/kg (i.p.) a 25% decrease of tumor cells, associated with an increase of cells in the G2/M phase of the cell cycle. Male F344 rats Inhibition of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis: RV 1–2 mg/kg (i.p. or significantly reduced number and size of NMBA-induced tumors p.o.) Male Wistar rats Inhibition of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis: RV markedly reduced 8 mg/kg (p.o.) tumor incidence, the degree of histological lesions, and the size of tumors. C57Bl6/J mice Treatment with RV significantly inhibited the growth of murine T241 fibrosarcoma in mice and 1 mg/kg (p.o.) suppressed FGF-2- and VEGF-stimulated angiogenesis Nude mice Inhibition of implanted human primary gastric carcinoma cells: RV could significantly inhibit 500–1500 mg/kg carcinoma growth when it was injected near the carcinoma and induced implanted tumor cells to (s.c.) undergo apoptosis Female SKH-1 hairless mice Topical application of skin with RV (both pre- and post-treatment) resulted in a highly significant 25–50 mM (top.) inhibition in tumor incidence and delayed onset of tumorigenesis abd: abdominal injection; d.w.: drinking water; gav: gavage; i.p.: intraperitoneal; p.o.: per os; s.c.: subcutaneous injection; top.: topical application. a Lowest concentration at which reproducible changes have been observed, or IC50 or EC50, if provided. 41Wine 34.1 j 647 648j 34 Protective Effects of Alcoholic Beverages and their Constituent

safety, RV and its metabolites/analogues may be applied in combination with other chemotherapeutic agents to enhance their efficacy thus minimizing chemotherapy- induced toxicity. By inhibition of COX and cytochrome P-450 enzymes and by induction of quinone reductase (QR), RV can simultaneously inhibit promutagen bioactivation, stimulate carcinogen detoxification, and protect the organism against the adverse effects of diverse environmental toxins. Furthermore, RV inhibits prostaglandins, nitric oxide (NO) formation, and the generation of ROS, therefore preventing their stimulative effect on tumor development. Besides acting as a chemopreventive agent, several in vitro, ex vivo, and in vivo experiments have shown that RV suppresses the growth of various cancer cell lines by inhibiting DNA polymerases, RR, and by inducing cell cycle arrest or apoptosis. Although the most exciting in vivo data relate to its cancer chemopreventive and chemotherapeutic activity, some studies also demonstrate beneficial effects on cardiovascular, neurological, and hepatic systems. RV is commonly referred to as a dirty molecule, meaning that it seems to interact with many different proteins, including cyclooxygenases, ribonucleotide reductase, and DNA polymerases. Thus, its activity cannot be resumed in a unique mechanism of action but likely results from various complementary actions of different biochemical pathways. However, the question of whether RV itself can accumulate to bioactive levels in target organs remains to be addressed. Opposing results and controversies involving the available data are leading to the suggestion that RV metabolites might be the active principal, and call for additional experiments. Long-term in vivo epidemiological studies are highly warranted to determine the preventive and therapeutic efficacy of dietary or supplemented RV on tumor development and progression.

34.2 Beer Metka Filipic, Janja Plazar, and Sakae Arimoto-Kobayashi

34.2.1 Introduction

Beer is the world oldest alcoholic beverage and has been a valuable constituent of human diet in many cultures. Historians believe that ancient Mesopotamians and Sumerians were brewing beer as early as 10 000 years ago. Beer remained popular in ancient Greece and Rome till wine overtook it and beer became known as a barbarian drink. In Europe in the Middle Age, monks reﬁned the process of beer making and introduced the use of hops as ﬂavoring and preservative. The most important milestone in beer history is Louis Pasteurs discovery of the yeasts role in fermentation in 1857, which founded the basis of modern brewery. Today brewing industry is a huge global business, and beer is one of the most commonly consumed alcoholic beverages. Beer is a generic name that covers a vast range of types and brands. It is produced from barley, water, and hops, with the addition of yeast for fermentation. 34.2 Beer j649

Different conditions during brewing, such as the variety of barley and malting procedure, temperature and pH during mashing, the variety of hops added during wort-boiling, and temperature during yeast fermentation deﬁne the type and quality of beer.

34.2.2 Physicochemical Properties of Bioactive Compounds

Beer contains many components with antimutagenic and anticarcinogenic properties, which have been found in experiments both in vitro and in vivo [35–37]. These include polyphenolic compounds, B vitamins (B6, B12, and folic acid), dietary fibers, and nitrogenous compounds [38–41]. The content of polyphenolic compounds in beer is around 500 mg/l; 70–80% originate from malt, while 20–30% of the polyphenols derive from the dried female inflorescences (hops or hop cones) of the hop plant (Humulus lupulus L., Cannabinacecae) [42]. The polyphenolic compounds in beer that may have cancer-preventive activity include phenolic acids (caffeic, ferulic acid, vanilin), flavonoles (quercetin, rutin, and kaempferol), isoflavonoids (formo- noneitin, daidzein, genistein, and biochanin A), catechins (( þ )-catechin, catechin gallate, and epicatechin gallate), prenylated flavonoids (xanthohumol, XN; isoxanthohumol, IXN; 8-prenylnaringenin, 8-PN; and 6-prenylnaringenin), and bitter acids (humulone, lupulone). A comprehensive overview of the occurrence of phenolic compounds with cancer-preventive properties in beer has been recently published by Gerh€auser [38]. Several bioactive compounds with cancer-preventive properties, such as certain polyphenols, B vitamins, dietary fibers that are not beer specific are described elsewhere in this book. This section provides an overview of the chemistry and the role of beer-specific compounds (prenylated flavonoids, bitter acids, and specific nitrogen compounds) in cancer prevention.

34.2.2.1 Prenylated Flavonoids Hops are a rich source of prenylated flavonoids, which are secreted along with bitter acids and essential oils as part of the hop resin (lupulin) by glandular trichomes found on the adaxial surfaces of cone bracts of the inflorescence. These compounds are also found in trichomes on the underside of young leaves [43]. Prenylated flavonoids that have been isolated from hop resin are mostly chalcones and flavanones (Scheme 34.4) [43].

Scheme 34.4 General chemical structure of the flavonoids: (a) chalcone and (b) flavanone. 650j 34 Protective Effects of Alcoholic Beverages and their Constituent

Scheme 34.5 Conversion of XN into 8PN. Acidic environment catalyzes the isomerization of XN to IXN, which is then converted into 8PN through metabolism by the cytochrome P-450 enzyme system.

Chalcones (e.g., xanthohumol) are flavonoids with an open C-ring, in which the two aromatic rings are joined by a three-carbon a, b-unsaturated carbonyl group. Flavanones (e.g., isoxanthohumol, 8-prenylnaringenin) have a closed C-ring and lack the double bond in the 2, 3-position of the C-ring in the flavane nucleus. Chalcones are easily converted to flavanones, either chemically in acidic conditions or enzymatically by chalcone isomerase (Scheme 34.5) [44]. The most important prenylated flavonoid in hop resin is the prenylated chalcone xanthohumol (Scheme 34.5) (30-[3,3-dimethyl allyl]-2,4,40-trihydroxy-60-methoxychal- cone, XN), representing 80–90% of the flavonoids in hops (0.1–1% of the hops dry weight) [43].

34.2.2.2 Bitter Acids For brewing the most important hop compounds are hop bitter acids (prenylated acylphloroglucinols): a-acids or humulones (1) and b-acids or lupulones (2) (Scheme 34.6). The two series comprise three constituents differing in the nature

Scheme 34.6 Structures of a- and b-acids. Each group comprises three compounds differing in the nature of the side chain, which is derived from the hydrophobic amino acids: leucine, valine, and isoleucine. 34.2 Beer j651

Scheme 34.7 Isomerization of humulones to isohumulones. of the side chain, which is derived from the hydrophobic amino acids, leucine, valine, and isoleucine (Scheme 34.7). They represent up to 25% dry weight of hop cones and have strong bacteriostatic activity. Hop acids occur as pale-yellowish solids in the pure state, are weak acids, exhibit very poor solubility in water, and have almost no bitter taste [42]. During brewing, hops are boiled with wort, which leads to isomerization of humulones to cis/trans pairs of isohumulones (Scheme 34.7), and impart their bitter taste to beer and stabilize the beer foam. Isohumulones represent one of the most abundant classes of polyphenolics in beer, while lupulones are extremely sensitive to oxidation and do not survive the brewing process [42].

34.2.2.3 Nitrogenous Compounds Polypeptides are important factors keeping the fine foam of beer. Six to nine percent of the nonvolatile components in beer are nitrogenous compounds including peptides, nucleic acids, and amines. Pseudouridine (5-ribosyluracil; Scheme 34.8) is a ubiquitous structural constituent of RNA. In beer, it has been identified as an antimutagenic substance toward alkylating agents: N-methyl-N0-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) [41]. The amount of pseudouridine in beer was estimated at about 4.0 mg/l beer. Glycine betaine (Scheme 34.8) is distributed widely in animals, plants, and microorganisms. Its principal physiological role is as an osmolyte and methyl donor in transmethylation reactions. In beer, glycine betaine has been identified as an antimutgenic component toward fish mutagen 2-chloro-4-methylthiobutanoic acid (CMBA) [40].

Scheme 34.8 Chemical structure of pseudouridine and glycine betaine. 652j 34 Protective Effects of Alcoholic Beverages and their Constituent

34.2.3 Mechanisms of Chemoprevention: Results In Vitro and In Vivo

Activity-guided fractionation of German-style pilsener led to the isolation of more than 30 compounds [39]. Most of the isolated compounds were then tested for their potential cancer-chemopreventive activities by measuring their (i) antioxidant effects, for example, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals; (ii) modulation of carcinogen metabolism by reduction of activating phase I cytochrome P-450 1A (CYP1A) enzyme activity and induction of detoxifying phase II NAD(P)H:QR enzyme activity; and (iii) anti-inflammatory mechanisms through inhibition of inducible nitric oxide synthase (iNOS) induction and inhibition of COX-1 activity. The results showed that various structural classes of compounds had quite distinct profiles of activity. Benzoic and cinnamic acid derivatives, proanthocyanidins, and flavanones exerted radical-scavenging activity. Catechins were also good COX-1 inhibitors, but had only marginal effects on CYP1A and QR activity. The most complex activities are exerted by the prenylated chalcone xanthohumol and (prenylated) flavanones isoxanthohumol, naringenin, 6- and 8-prenylnaringenin, and 6-geranylnaringeni, which are very potent inhibitors of CYP1A activity, enhance QR activity as an indication for elevated detoxification, and possess anti-inflammatory potential.

34.2.3.1 Prenylated Flavonoids Miranda et al. [36] were the first to show antimutagenic effects of XN, IXN, and 8PN. In a bacterial test system with Salmonella typhimurium, all three prenyalted flavonoids, at micromolar concentrations, inhibited the mutagenicity of the heterocyclic aromatic amine (HC) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), which was activated by of cDNA-expressed human CYP1A2. It has been suggested that the underlying mechanism is the inhibition of metabolic activation as seen in direct enzymatic experiments in which XN, IXN, and 8PN strongly inhibited activity of cDNA-expressed CYP1A1, CYP1B1, and CYP1A2 [45]. In metabolically active human hepatoma HepG2 cells and in precision-cut rat liver slices XN completely prevented formation of IQ and benzo(a)pyrene-induced DNA damage at concentrations as low as 10 nM [46, 47]. Interestingly, while XN inhibited CYP1A activity when measured in microsomes from precision-cut rat liver slices, no inhibition was detected when measured in whole tissue [47]. XN showed protective effects also against tert-butyl hydroperoxide-induced oxidative DNA damage in HepG2 cells and in precision-cut rat liver slices [46, 47] and against menandion- induced DNA damage in murine hepatoma Hepa1c1c7 cells [48]. However, protection against oxidative DNA damage was less pronounced than the one against indirect acting carcinogens. All three studies indicated that the protection against oxidative damage was most probably mediated by induction of a cellular defense system. In murine hepatoma Hepa1c1c7 cells, XN (and to lesser extent also IXN and 8PN) induced quinone reductase by the activation of transcription factors interacting with the antioxidant responsive element (ARE) [38, 48]. XN and to lesser extent also IXN were shown to be able to scavenge reactive oxygen species and inhibit superoxide radical and nitric oxide production [38]. Thus, these results indicate that XN may 34.2 Beer j653 prevent initiation of carcinogenesis by inhibiting phase I enzymes, induction of Phase II enzymes, reactive oxygen radical scavenging, and inhibition of radical and nitric oxide production. XN was also found to cause cytotoxic and antiproliferative effects in different types of cancer cells in vitro [38]. The experiments showed that XN exhibited anti- inflammatory activity by inhibiting endogenous prostaglandin synthesis through inhibition of cyclogenases COX-1 and COX-2, and at nanomolar concentrations prevented the formation of carcinogen (dimethylbenz(a)anthracene, DMBA)-induced preneoplastic lesions in mammary gland tissue. These results show that XN exhibits anti-inflammatory and antiproliferative effects at the promotional stage of carcinogenesis and prevents the formation of carcinogen-induced preneoplastic lesions (Figure 34.2). Recently, 8PN gained considerable attention as it has been identified as one of the most potent known phytoestrogens [38]. It inhibited proliferation and/or induced apoptosis in different types of cancer cells in vitro [49–51], and it as been suggested that the underlying mechanism might be inhibition of aromatase activity and thus inhibition of estrogen synthesis [52]. 8PN has also been shown to inhibit angiogenesis in vitro and in vivo [38]. The content of 8PN in hops and beer is relatively low; however, in human intestine, XN and IXN are rapidly metabolized to 8PN, and it has been estimated that even after moderate consumption of beer the exposure values might fall within the range of human biological activity [53].

34.2.3.2 Bitter Acids Despite the fact that bitter acids, particularly isohumulone, are the most abundant polyphenols in beer, studies concerning their potential cancer-preventive properties are rather scarce. Humulones and lupulones have been shown to have radical- scavenging activity and to inhibit lipid peroxidation. In addition, humulone has been shown to have anti-inﬂammatory, antitumor-promoting, and antiangiogenic activity. For all these phenomena, the authors suggested to be partly due to the inhibition of the overexpression of COX-2 [38]. Humulone and a mixture of hop bitter acids induced apoptosis in human leukaemia HL-60 cells, and molecular and gene expression analysis indicated that bitter acids of hop induce apoptosis via mitochondrial- and receptor-mediated pathways in HL-60 cells [38]. Two recent reports suggest that isohumulones also may have cancer-preventive potential.Yajimaetal.[54]showedthatisohumulonesactivateperoxisomeproliferator- activated receptors (PPARs) a and g. This report is interesting in view of the potential roleofPPARsandtheirligandsincancerprevention,andalsoasacausalfactorincolon carcinogenesis [38]. In another study, Nozawa et al. [55] reported that feeding of rats with isohumulones suppressed the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in colon, and the results suggested that the chemopreventive effect was mediated by the inhibition of prostaglandin 2 production.

34.2.3.3 Nitrogenous Compounds and other Unidentified Components In our early studies, we [35] found that beer suppress mutagenicity of direct-acting mutagens:preactivatedheterocyclicaminesandN-methyl-N0-nitro-N-nitrosoguanidine 654j 34 Protective Effects of Alcoholic Beverages and their Constituent

Figure 34.2 Mechanisms of cancer preventive the promotion and progression stages, activities of XN. At the initiation stage of cancer XN is an effective anti-inflammatory agent that development, XN prevents DNA damage by inhibits endogenous prostaglandin (PGE) suppressing metabolic activation of synthesis through the suppression of COX-2 procarcinogens via inhibition of CYP enzymes. It activity, thus preventing PGE-mediated also prevents ROS-induced DNA damage by its promotion of cell growth and initiation of antioxidative and radical-scavenging activity and angiogenesis. Antiangiogenic activity may be by inducing quinine reductase activity. XN exerted also through inhibition of nitric oxide as . . inhibits the production of NO by excessive and prolonged NO generation suppressing the expression of iNOS, thus promotes the production of vascular endothelial suppressing reactive nitrogen species (RNS) growth factor (VEGF), a known inducer of formation, which can induce DNA damage. At angiogenesis.

in S. typhimurium mutagenicity assays. It also suppressed MNNG-induced formation of O6-methylguanine in bacterial DNA. Pseudouridine has been identified as one of the active compounds against MNNG-induced mutations [41]. One possibility of the mechanism of antimutagenicity of pseudouridine is entrapment of methyl-radical derived from MNNG. Glycine betaine has been shown to suppress the mutagenicity of salted fish mutagen CMBA [40], which reacts with 20-deoxyguanosine producing stable N7-guanine adducts [56]. Heterocyclic amines, including 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 3-amino-1-methyl-5H-pyrido[4.3-b]indole (Trp-P-2), are formed when muscle meats 34.2 Beer j655 are cooked at high temperatures and which may pose a cancer risk. In vivo studies with rats and mice showed that beer protects against HC-induced DNA adducts and preneoplastic lesions in multiple organs. For example freeze-dried beer given to mice with drinking water, reduced the formation of MeIQx–DNA adducts in liver and lungs, Trp-P-2–DNA adducts in liver, and PhIP–DNA adducts in colon and lungs [57, 58]. Feeding of freeze-dried beer in the diet to rats significantly lowered the incidence and multiplicity of mammary tumors, and the number of ACF in colon [37, 59]. It has been also shown that beer components have at least one target associated with the observed antimutagenicity, namely, the suppression of the activity of CYP enzymes involved in activation of heterocyclic amines [58]. Furthermore, consumption of beer has been shown to have radioprotective effects. Monobe et al. [60] reported that consumption of beer reduced chromosome aberrations in human lymphocytes that were subsequently irradiated with X-rays or charged carbon ions in vitro. Pseudouridine, which has been identified as an antimutagen against alkylation-induced mutagenicity, also reduced chromosome aberrations in irradiated human lymphocytes [61].

34.2.4 Results of Human Studies

Consumption of beer is generally associated with alcohol exposure as alcohol-free beers currently account for only 2–3% of the beer market. There is an increasing evidence that low alcohol consumption (10–15 g alcohol/day, equivalent to about one glass (0.3 l) of beer per day) reduces mortality in comparison to nondrinking and heavy drinking [62]. However, there is also convincing evidence that high alcohol consumption is related to carcinogenesis [63]. Several epidemiological studies showed positive associations between beer consumption and the development of different types of cancer [64]. However, these studies are difﬁcult to interpret due to numerous confounding factors related to lifestyle and dietary habits. On the contrary, a Canadian study involving 1253 subjects (617 cases and 636 controls) indicated that beer consumption (10 g alcohol/day) signiﬁcantly decreased the risk of prostate cancer [65]. Also, after a 12-year follow-up in a large prospective cohort study of Finish male smokers (alpha-tocopherol, beta-carotene cancer prevention study) a decreased relative risk for renal cell cancer was reported both for the highest alcohol intake category and for the highest beer intake category [66]. However, due to the obvious positive association between high alcohol consumption and other types of cancer, these data should be interpreted with caution. A recent human study, focused on nutritional properties of beer, showed that beer consumption increases the plasma antioxidant capacity [67]. It has been shown that phenolic acids from beer are readily absorbed from the gastrointestinal tract and are present in blood after being largely metabolized to form glucuronide and sulfate conjugates [68]. In another study, increase of plasma antioxidant activity together with improved plasma lipid levels and anticoagulant activities was observed in hyperho- lesterolaemic volunteers after supplementation of their diet with 330 ml beer per day for 30 consecutive days [69]. 656j 34 Protective Effects of Alcoholic Beverages and their Constituent

Beer also contains signiﬁcant amounts of several B vitamins, including B6, B12 and folic acid, which play important role in cancer prevention. It has been estimated that moderate consumption of beer can cover up to one half of the recommended daily intake of these vitamins [70].

34.2.5 Effect of Beer Processing on Chemoprotective Properties

Although XN is the major prenylated flavonoid in hops and potentially the most effective cancer chemopreventive agent in beer, it is generally a minor flavonoid in beer due to the isomerization of chalcones into flavanones during the beer-brewing process (Scheme 34.5; [44]. In beer, the content of IXN is about 10- to 20-fold higher than the content of XN, depending on the type of beer. In addition to the isomerization process, the amount of XN in beer is further largely reduced during brewing due to incomplete extraction and adsorption to insoluble malt proteins and yeast cells during the fermentation process. Subsequently, the levels of XN in beer are slightly increased again due to the second, late addition of hops to the boiling wort. Gerh€auser et al. [38] measured XN concentration in a series of German beers by solid-phase extraction and high-pressure liquid chromatography. The levels of XN were 0.08 0.03 mg/l (0.23 0.08 mM), while the concentration of IXN in Pilsner beer reached 1.6 mg/l (mean concentrations in normal brews 0.74 0.5 mg/l). They concluded that the amounts of prenylated flavonoids in a maximally recommended daily amount of beer (up to 0.5 l) are not sufficient to achieve the chemopreventive activities of the studied flavonoids. Recently, XN-enriched beer has been produced with the Xan-technology [71]. However, it has not yet been introduced as a commercially available beer. Despite the fact that concentrations of individual compounds with cancer- protective activities in beer are low, the in vitro and in vivo studies showed that whole beer exerts protective effects particularly against genotoxicity and carcinogenic potential of indirect-acting carcinogens including HC, to which humans are unavoidably exposed (with the exception of strict vegetarians). The results suggest that active component(s) not yet identified are responsible for the protective effects and/or these effects are a result of combined action of the complex mixture of multiple, biologically active components of beer.

References

References to Section 34.1

1 McGovern, P.E., Glusker, D.L., Exner, L.J. disease prevention. Journal of Clinical and Voigt, M.M. (1996) Neolithic resinated Laboratory Analysis, 11, 287–313. wine. Nature, 381, 480–481. 3 Renaud, S. and de Lorgeril, M. (1992) 2 Soleas, G.J., Diamandis, E.P. and Wine, alcohol, platelets, and the French Goldberg, D.M. (1997) Wine as a biological paradox for coronary heart disease. Lancet, ﬂuid: history, production, and role in 339, 1523–1526. References j657

4 Baur, J.A. and Sinclair, D.A. (2006) H.H., Farnsworth, N.R., Kinghorn, A.D., Therapeutic potential of resveratrol: the Mehta, R.G., Moon, R.C. and Pezzuto, J.M. in vivo evidence. Nature Reviews. Drug (1997) Cancer chemopreventive activity of Discovery, 5, 493–506. resveratrol, a natural product derived from 5 Nonomura, S., Kanagawa, H. and grapes. Science, 275, 218–220. Makimoto, A. (1963) Chemical 15 Fontecave, M., Lepoivre, M., Elleingand, constituents of polygonaceous plants. I. E., Gerez, C. and Guittet, O. (1998) Studies on the components of Ko-J O-Kon Resveratrol, a remarkable inhibitor of (Polygonum cuspidatum Sieb. Et Zucc.). ribonucleotide reductase. FEBS Letters, Yakugaku Zasshi, 83, 988–990. 421, 277–279. 6 Siemann, E.H. and Creasy, L.L. (1992) 16 Locatelli, G.A., Savio, M., Forti, L., Concentration of phytoalexin resveratrol in Shevelev, I., Ramadan, K., Stivala, L.A., wine. American Journal of Epidemiology, 43, Vannini, V., Hubscher, U., Spadari, S. 49–52. and Maga, G. (2005) Inhibition of 7 Gusman, J., Malonne, H. and Atassi, G. mammalian DNA polymerases by (2001) A reappraisal of the potential resveratrol: mechanism and structural chemopreventive and chemotherapeutic determinants. The Biochemical Journal, properties of resveratrol. Carcinogenesis, 389, 259–268. 22, 1111–1117. 17 Ragione, F.D., Cucciolla, V., Borriello, A., 8 Hao, H.D. and He, L.R. (2004) Pietra, V.D., Racioppi, L., Soldati, G., Mechanisms of cardiovascular protection Manna, C., Galletti, P. and Zappia, V. by resveratrol. Journal of Medicinal Food, 7, (1998) Resveratrol arrests the cell division 290–298. cycle at S/G2 phase transition. Biochemical 9 Saiko, P., Szakmary, A., Jaeger, W. and and Biophysical Research Communications, Szekeres, T. (2008) Resveratrol and its 250,53–58. analogs: defense against cancer, coronary 18 Horvath, Z., Saiko, P., Illmer, C., disease and neurodegenerative maladies or Madlener, S., Hoechtl, T., Bauer, W., Erker, just a fad? Mutation Research, 658,68–94. T., Jaeger, W., Fritzer-Szekeres, M. and 10 Dong, Z. (2003) Molecular mechanism of Szekeres, T. (2005) Synergistic action of the chemopreventive effect of resveratrol. resveratrol, an ingredient of wine, with Mutation Research, 523–524, 145–150. Ara-C and tiazofurin in HL-60 human 11 Aggarwal, B.B. and Shishodia, S. (2006) promyelocytic leukemia cells. Molecular targets of dietary agents for Experimental Hematology, 33, 329–335. prevention and therapy of cancer. 19 Stewart, J.R., Ward, N.E., Ioannides, C.G. Biochemical Pharmacology, 71, 1397–1421. and OBrian, C.A. (1999) Resveratrol 12 Stewart, J.R., Christman, K.L. and OBrian, preferentially inhibits protein kinase C.A. (2000) Effects of resveratrol on the C-catalyzed phosphorylation of a cofactor- autophosphorylation of phorbol ester- independent, arginine-rich protein responsive protein kinases: inhibition of substrate by a novel mechanism. protein kinase D but not protein kinase C Biochemistry, 38, 13244–13251. isozyme autophosphorylation. Biochemical 20 Subbaramaiah, K., Chung, W.J., Pharmacology, 60, 1355–1359. Michaluart,P.,Telang,N.,Tanabe,T., 13 Bhat, K.P., Lantvit, D., Christov, K., Mehta, Inoue, H., Jang, M., Pezzuto, J.M. and R.G., Moon, R.C. and Pezzuto, J.M. (2001) Dannenberg, A.J. (1998) Resveratrol Estrogenic and antiestrogenic properties inhibits cyclooxygenase-2 transcription of resveratrol in mammary tumor models. and activity in phorbol ester-treated Cancer Research, 61, 7456–7463. human mammary epithelial cells. The 14 Jang, M., Cai, L., Udeani, G.O., Slowing, Journal of Biological Chemistry, 273, K.V., Thomas, C.F., Beecher, C.W., Fong, 21875–21882. 658j 34 Protective Effects of Alcoholic Beverages and their Constituent

21 MacCarrone, M., Lorenzon, T., Guerrieri, model. The Journal of Pharmacology and P. and Agro, A.F. (1999) Resveratrol Experimental Therapeutics, 302, 369–373. prevents apoptosis in K562 cells by 29 Walle, T., Hsieh, F., DeLegge, M.H., Oatis, inhibiting lipoxygenase and J.E. Jr and Walle, U.K. (2004) High cyclooxygenase activity. European Journal of absorption but very low bioavailability of Biochemistry/FEBS, 265,27–34. oral resveratrol in humans. Drug 22 Steele, V.E., Holmes, C.A., Hawk, E.T., Metabolism and Disposition, 32, 1377–1382. Kopelovich, L., Lubet, R.A., Crowell, J.A., 30 Yu, C., Shin, Y.G., Kosmeder, J.W., Sigman, C.C. and Kelloff, G.J. (2000) Pezzuto, J.M. and van Breemen, R.B. Potential use of lipoxygenase inhibitors for (2003) Liquid chromatography/tandem cancer chemoprevention. Expert Opinion mass spectrometric determination of on Investigational Drugs, 9, 2121–2138. inhibition of human cytochrome P450 23 Potter, G.A., Patterson, L.H., Wanogho, E., isozymes by resveratrol and resveratrol- Perry, P.J., Butler, P.C., Ijaz, T., Ruparelia, 3-sulfate. Rapid Communications in Mass K.C., Lamb, J.H., Farmer, P.B., Stanley, Spectrometry, 17, 307–313. L.A. and Burke, M.D. (2002) The cancer 31 Goldberg, D.M., Yan, J. and Soleas, G.J. preventative agent resveratrol is converted (2003) Absorption of three wine-related to the anticancer agent piceatannol by the polyphenols in three different matrices by cytochrome P450 enzyme CYP1B1. British healthy subjects. Clinical Biochemistry, 36, Journal of Cancer, 86, 774–778. 79–87. 24 Wolter, F., Clausnitzer, A., Akoglu, B. and 32 Meng, X., Maliakal, P., Lu, H., Lee, M.J. Stein, J. (2002) Piceatannol, a natural and Yang, C.S. (2004) Urinary and plasma analog of resveratrol, inhibits progression levels of resveratrol and quercetin in through the S phase of the cell cycle in humans, mice, and rats after ingestion of colorectal cancer cell lines. The Journal of pure compounds and grape juice. Journal Nutrition, 132, 298–302. of Agricultural and Food Chemistry, 52, 25 Appeldoorn, C.C., Bonnefoy, A., Lutters, 935–942. B.C., Daenens, K., van Berkel, T.J., 33 Wang, Y., Catana, F., Yang, Y., Roderick, R. Hoylaerts, M.F. and Biessen, E.A. (2005) and van Breemen, R.B. (2002) An LC-MS Gallic acid antagonizes P-selectin- method for analyzing total resveratrol in mediated platelet–leukocyte interactions: grape juice, cranberry juice, and in wine. implications for the French paradox. Journal of Agricultural and Food Chemistry, Circulation, 111, 106–112. 50, 431–435. 26 Liu, Z., Schwimer, J., Liu, D., Greenway, 34 Boocock, D.J., Faust, G.E., Patel, K.R., F.L., Anthony, C.T. and Woltering, E.A. Schinas, A.M., Brown, V.A., Ducharme, (2005) Black raspberry extract and M.P., Booth, T.D., Crowell, J.A., Perloff, fractions contain angiogenesis inhibitors. M., Gescher, A.J., Steward, W.P. and Journal of Agricultural and Food Chemistry, Brenner, D.E. (2007) Phase I dose 53, 3909–3915. escalation pharmacokinetic study in 27 Isuzugawa, K., Inoue, M. and Ogihara, Y. healthy volunteers of resveratrol, a þ (2001) Ca2 -dependent caspase activation potential cancer chemopreventive agent. by gallic acid derivatives. Biological & Cancer Epidemiology, Biomarkers & Pharmaceutical Bulletin, 24, 844–847. Prevention, 16, 1246–1252. 28 Marier, J.F., Vachon, P., Gritsas, A., Zhang, J., Moreau, J.P. and Ducharme, M.P. (2002) Metabolism and disposition of References to Section 34.2 resveratrol in rats: extent of absorption, glucuronidation, and enterohepatic 35 Arimoto-Kobayashi, S., Sugiyama, C., recirculation evidenced by a linked-rat Harada, N., Takeuchi, M., Takemura, M. References j659

and Hayatsu, H. (1999) Inhibitory effects prenylflavonoids from hops and beer: to of beer and other alcoholic beverages on your good health! Phytochemistry, 65, mutagenesis and DNA adduct formation 1317–1330. induced by several carcinogens. Journal of 44 Nikolic, D., Li, Y., Chadwick, L.R., Pauli, Agricultural and Food Chemistry, 47, G. F. and van Breemen, R. B. (2005) 221–230. Metabolism of xanthohumol and 36 Miranda, C.L., Yang, Y.H., Henderson, isoxanthohumol prenylated flavonoids M.C., Stevens, J.F., Santana-Rios, G., from hops (Humulus lupulus L.), by human Deinzer, M.L. and Buhler, D.R. (2000) liver microsomes. Journal of Mass Prenylflavonoids from hops inhibit the Spectrometry, 40, 289–299. metabolic activation of the carcinogenic 45 Henderson, M.C., Miranda, C.L., Stevens, heterocyclic amine 2-amino-3- J.F., Deinzer, M.L. and Buhler, D.R. (2000) methylimidazo[4,5-f]quinoline, mediated In vitro inhibition of human P450 enzymes by cDNA-expressed human CYP1A2. Drug by prenylated flavonoids from hops, Metabolism and Disposition, 28, 1297–1302. Humulus lupulus. Xenobiotica, 30, 235–251. 37 Nozawa, H., Tazumi, K., Sato, K., Yoshida, 46 Plazar, J., Zegura, B., Lah, T.T. and Filipic, A., Takata, J., Arimoto-Kobayashi, S. and M. (2007) Protective effects of Kondo, K. (2004) Inhibitory effects of beer xanthohumol against the genotoxicity of on heterocyclic amine-induced benzo(a)pyrene (BaP), 2-amino-3- mutagenesis and PhIP-induced aberrant methylimidazo[4,5-f]quinoline (IQ) and crypt foci in rat colon. Mutation Research, tert-butyl hydroperoxide (t-BOOH) in 559, 177–187. HepG2 human hepatoma cells. Mutation 38 Gerhauser, C. (2005) Beer constituents as Research, 632,1–8. potential cancer chemopreventive agents. 47 Plazar, J., Filipic, M. and Groothuis, European Journal of Cancer, 41, 1941–1954. G.M.M. (2008) Antigenotoxic effect of 39 Gerhauser, C., Alt, A.P., Klimo, K., Knauft, xanthohumol in rat liver slices. Toxicology K., Frank, N. and Becker, H. (2002) In Vitro, 22, 318–327. Isolation and potential cancer 48 Dietz, B.M., Kang, Y.H., Liu, G., Eggler, chemopreventive activities of phenolic A.L., Yao, P., Chadwick, L.R., Pauli, G.F., compounds of beer. Phytochemistry Review, Farnsworth, N.R., Mesecar, A.D., van 1, 369–377. Breemen, R. B. and Bolton, J.L. (2005) 40 Kimura, S., Hayatsu, H. and Arimoto- Xanthohumol isolated from Humulus Kobayashi, S. (1999) Glycine betaine in lupulus inhibits menadione-induced DNA beer as an antimutagenic substance damage through induction of quinone against 2-chloro-4-methylthiobutanoic reductase. Chemical Research in Toxicology, acid, the sanma-fish mutagen. Mutation 18, 1296–1305. Research, 439, 267–276. 49 Brunelli, E., Minassi, A., Appendino, G. 41 Yoshikawa, T., Kimura, S., Hatano, T., and Moro, L. (2007) 8-Prenylnaringenin Okamoto, K., Hayatsu, H. and Arimoto- inhibits estrogen receptor-alpha mediated Kobayashi, S. (2002) Pseudouridine, an cell growth and induces apoptosis in antimutagenic substance in beer towards MCF-7 breast cancer cells. Journal of N-methyl-N0-nitro-N-nitrosoguanidine Steroid Biochemistry and Molecular Biology, (MNNG). Food and Chemical Toxicology, 40, 107, 140–148. 1165–1170. 50 Delmulle, L., Bellahcene, A., Dhooge, W., 42 De Keukeleire, D. (2000) Fundamentals of Comhaire, F., Roelens, F., Huvaere, K., beer and hop chemistry. Quimica Nova, 23, Heyerick, A., Castronovo, V. and De 108–112. Keukeleire, D. (2006) Anti-proliferative 43 Stevens, J.F. and Page, J.E. (2004) properties of prenylated flavonoids from Xanthohumol and related hops (Humulus lupulus L.) in human 660j 34 Protective Effects of Alcoholic Beverages and their Constituent

prostate cancer cell lines. Phytomedicine, 57 Arimoto-Kobayashi, S., Ishida, R., Nakai, 13, 732–734. Y., Idei, C., Takata, J., Takahashi, E., 51 Diller, R.A., Riepl, H.M., Rose, O., Frias, Okamoto, K., Negishi, T. and Konuma, T. C., Henze, G. and Prokop, A. (2007) Ability (2006) Inhibitory effects of beer on of prenylflavanones present in hops to mutation in the Ames test and DNA adduct induce apoptosis in a human Burkitt formation in mouse organs induced by lymphoma cell line. Planta Medica, 73, 2-amino-1-methyl-6-phenylimidazo[4,5-b] 755–761. pyridine (PhIP). Biological & 52 Monteiro, R., Faria, A., Azevedo, I. and Pharmaceutical Bulletin, 29,67–70. Calhau, C. (2007) Modulation of breast 58 Arimoto-Kobayashi, S., Takata, J., cancer cell survival by aromatase Nakandakari, N., Fujioka, R., Okamoto, K. inhibiting hop (Humulus lupulus L.) and Konuma, T.(2005) Inhibitory effects of flavonoids. Journal of Steroid Biochemistry heterocyclic amine-induced DNA adduct and Molecular Biology, 105, 124–130. formation in mouse liver and lungs by 53 Possemiers, S., Bolca, S., Grootaert, C., beer. Journal of Agricultural and Food Heyerick, A., Decroos, K., Dhooge, W., De Chemistry, 53, 812–815. Keukeleire, D., Rabot, S., Verstraete, W. 59 Nozawa, H., Nakao, W., Takata, J., and Van de Wiele, T. (2006) The Arimoto-Kobayashi, S. and Kondo, K. prenylflavonoid isoxanthohumol from (2006) Inhibition of PhIP-induced hops (Humulus lupulus L.) is activated mammary carcinogenesis in female rats by into the potent phytoestrogen ingestion of freeze-dried beer. Cancer 8-prenylnaringenin in vitro and in the Letters, 235, 121–129. human intestine. Journal of Nutrition, 136, 60 Monobe, M. and Ando, K. (2002) Drinking 1862–1867. beer reduces radiation-induced 54 Yajima, H., Ikeshima, E., Shiraki, M., chromosome aberrations in human Kanaya, T., Fujiwara, D., Odai, H., lymphocytes. Journal of Radiation Research, Tsuboyama-Kasaoka, N., Ezaki, O., 43, 237–245. Oikawa, S., and Kondo, K. (2004) 61 Monobe, M., Arimoto-Kobayashi, S. and Isohumulones, bitter acids derived from Ando, K. (2003) Beta-pseudouridine, a hops, activate both peroxisome beer component, reduces radiation- proliferator-activated receptor alpha and induced chromosome aberrations in gamma and reduce insulin resistance. human lymphocytes. Mutation Research, Journal of Biological Chemistry, 279, 538,93–99. 33456–33462. 62 Thun, M.J., Peto, R., Lopez, A.D., Monaco, 55 Nozawa, H., Nakao, W., Zhao, F. and J.H., Henley, S.J., Heath, C.W. and Doll, R. Kondo, K. (2005) Dietary supplement of (1997) Alcohol consumption and mortality isohumulones inhibits the formation of among middle-aged and elderly US adults. aberrant crypt foci with a concomitant New England Journal of Medicine, 337, decrease in prostaglandin E2 level in rat 1705–1714. colon. Molecular Nutrition & Food Research, 63 WCRF/AICR (1997) in Food, Nutrition and 49, 772–778. the Prevention of Cancer: A Global 56 Kimura, S., Nakayama, M., Hatano, T., Perspective, World Cancer Research Fund/ Segawa, A., Watanabe, T., Hayatsu, H. and American Institute for Cancer Research, Arimoto-Kobayashi, S. (2005) Washington, DC. Characterization of adducts formed in the 64 Ruf, J.C. (2003) Overview of reaction of 2-chloro-4-methylthiobutanoic epidemiological studies on wine, health acid with 20-deoxyguanosine. Chemical and mortality. Drugs Under Experimental Research in Toxicology, 18, 1755–1761. and Clinical Research, 29, 173–179. References j661

65 Jain, M.G., Hislop, G.T., Howe, G.R., 68 Nardini, M., Natella, F., Scaccini, C. and Burch, J.D. and Ghadirian, P. (1998) Ghiselli, A. (2006) Phenolic acids Alcohol and other beverage use and from beer are absorbed and extensively prostate cancer risk among Canadian men. metabolized in humans. Journal International Journal of Cancer, 78, of Nutritional Biochemistry, 17,14–22. 707–711. 69 Gorinstein, S., Caspi, A., Libman, I., 66 Mahabir, S., Leitzmann, M.F., Leontowicz, H., Leontowicz, M., Tashma, Virtanen, M.J., Virtamo, J., Pietinen, P., Z., Katrich, E., Jastrzebski, Z. and Albanes, D. and Taylor, P.R. (2005) Trakhtenberg, S. (2007) Bioactivity of beer Prospective study of alcohol drinking and and its inﬂuence on human metabolism. renal cell cancer risk in a cohort of International Journal of Food Sciences and Finnish male smokers. Cancer Nutrition, 58,94–107. Epidemiology Biomarkers & Prevention, 14, 70 Bamforth, C.W. (2002) Nutritional aspects 170–175. of beer: a review. Nutrition Research, 22, 67 Ghiselli, A., Natella, F., Guidi, A., 227–237. Montanari, L., Fantozzi, P. and Scaccini, C. 71 Wunderlich, S., Zurcher, A. and Back, W. (2000) Beer increases plasma antioxidant (2005) Enrichment of xanthohumol in the capacity in humans. Journal of Nutritional brewing process. Molecular Nutrition & Biochemistry, 11,76–80. Food Research, 49, 874–881. j663

35 Sulfides in Allium Vegetables Claus Jacob and Awais Anwar

35.1 Introduction

Garlic, onions, and related plants of the genus Allium have been part of folk medicine for many centuries all over the world [1, 2]. Although the precise health benefits of such vegetables and their products are still a matter of debate, recent research has provided a number of new insights that deepen our understanding of Allium plants and derived products, such as oils and powders of garlic and onion [3]. Numerous biologically active ingredients, including allicin and polysulfides, have been identified and their interactions with biomolecules, cells, and organisms described. In many cases, there seems to be a broad-range antimicrobial activity associated with sulfur-containing compounds derived from the Allium species, which manifests itself in diverse antibacterial, antifungal, and pesticidal activities [4]. These activities seem to be the result of an intrinsic cytotoxicity of various (sulfur) compounds present in these plants. In garlic, these compounds include allicin, diallyldisulfide (DADS), diallyltrisulfide (DATS), diallyltetrasulfide (DATTS), and ajoene, to name just a few (see Scheme 35.1 for chemical structures). These compounds are currently of great interest in medicine and agriculture as possible natural antibiotics, fungi- cides, and green pesticides.1) Equally interesting are literature reports describing the potential anticancer activity of garlic, onions, and their respective ingredients. Such reports are not new, and we can trace the first serious, scientific claims of garlic as an anticancer agent back to the 1980s [5].2) A review by Eric Block, published in 1992, provides a comprehensive overview of the chemical and biochemical knowledge in Allium research up to that date, which is still largely valid today [1]. Nonetheless, the last 5–10 years have

1) Most of the issues surrounding the antimi- dients besides sulfur agents, such as selenium crobial activity of garlic compounds have re- compounds and allixin. cently been reviewed and will not be discussed 2) This Chinese study linking the consumption here further. It should also be pointed out that of garlic with a reduced risk of stomach cancer garlic contains a range of other active ingre- is often seen as a ﬁrst key epidemiological report in this area.

Scheme 35.1 A selection of the sulfur chemistry (5), and, quite frequently, AM (6). Apart from found in garlic. This chemistry is initiated by the such reactions, allicin also decomposes upon enzymatic conversion of alliin (1) to allicin (3), aging or heating. These decomposition which is triggered by physical damage to the processes are chemically far from trivial and garlic clove (e.g. chopping, crushing) or result in a wide range of sulfur compounds, microbial attack. This conversion is catalyzed by including DAS (7), DADS (8), DATS (9), DATTS the C-S-lyase enzyme alliinase and proceeds via (10), 3-vinyl-3,4-dihydro-1,2-dithiin (11), 2-vinyl- allyl sulfenic acid (2). Allicin reacts readily with 2,4-dihydro-1,3-dithiin (12), and ajoene (13) (the thiol-containing biomolecules, forming a range latter occurs as E-ajoene and Z-ajoene, both are of follow-on products, such as SAC (4), SAMC biologically active, and E-ajoene is shown here). 35.2 Physicochemical Properties j665 witnessed the emergence of a signiﬁcant body of new evidence regarding the chemical and biochemical properties of natural sulfur compounds, which also includes their possible roles in cancer prevention and therapy. Some of the more striking aspects of chemoprevention have recently formed the basis of several excellent reviews, such as the ones by Amagase, Davis, Dashwood, Mersch-Sunder- mann, Shukla, Singh, Surh, and their respective colleagues [3, 6–11]. As part of this chapter, we will consider a range of garlic- and onion-derived substances capable of preventing the formation of cancer cells, preventing cancer cell proliferation, and killing cancer cells (e.g., by induction of apoptosis).

35.2 Physicochemical Properties

Before we start, we need to briefly consider the biological chemistry of sulfur agents, which is highly complicated and diverse, and often, but not exclusively, related to the redox activity of sulfur. A number of recent reviews and perspectives have been published that deal with these issues in considerable detail [12–15]. It is therefore sufficient here to highlight a few key aspects. First of all, the element sulfur is a true redox chameleon that occurs in around 10 different formal oxidation states in vivo, ranging from 2 in thiols and some sulfides to þ 6 in sulfate, including a few fractional oxidation states (see Table 35.1). This inherent flexibility in oxidation states is reflected by sulfurs ability to participate in a range of mechanistically different redox processes that include one- and two-electron transfer reactions, radical reactions, atom transfer reactions, and various exchange reactions, such as thiol/disulfide exchanges. The resulting network of reactive sulfur species (RSS) able to exist in vivo is extensive and embraces not only some of the better known sulfur species, such as thiols, disulfides, and sulfate, but also lesser known, often quite reactive sulfur species, such as thiyl radicals, sulfenic, sulfinic, and sulfonic acids, thiosulfinates and thiosulfonates, sulfoxides and sulfones, S-nitrosothiols, polysulfides, perthiols, perthiyl radicals, hydropolysulfides, and isothiocyanates, as well as certain inorganic fi fi sul de anions, such as hydrogen sul de (H2S, which is in equilibrium with HS at pH 7.4) [13, 16]. Some of these RSS are summarized in Table 35.1, which is, of course, far from complete.

3 Further products, such as diallylsulfoxide and unknown formation pathway. Garlic is also rich in diallylsulfone have also been found (structures a number of compounds that are biologically not shown). Full reduction of sulfur, for instance active yet do not contain sulfur, such as allixin via exchange reactions or thermal degradation, (16). Please note that this selection of results in the liberation of the cell signaling compounds provides only a fraction of the sulfur molecule H2S, which at pH 7.4 is in equilibrium compounds present in garlic, onions, and related with the HS anion (14). Recently, Allium species, which have been selected due to thiacremonone (15) has been found in garlic as their suspected roles in cancer yet another sulfur agent and with a hitherto chemoprevention. 666j 35 Sulfides in Allium Vegetables

Table 35.1 Brief overview of reactive sulfur species (RSS), including chemical formulae, sulfur oxidation states, and in vivo occurrence of the relevant RSS.

General Sulfur chemical oxidation Sulfur species formula state (R ¼þ1) Reactivity Occurrence (examples only)

Hydrogen sulfide H2S 2 Reducing, metal Inorganic sulfur agent, for binding example, released from DATS Thiol RSH 2 Reducing, metal Cysteine in proteins, binding enzymes, GSH Sulfide (organic) RSR 2 Weakly reducing As methionine in proteins and enzymes Disulfide radical RSSR 1.5 Strongly reducing Formed from RS and GSH anion one electron during OS donor Thiyl radical RS 1 Reducing, Formed by oxidation of dimerization RSH, for example, in bacterial RNRase Perthiol RSSH 1 Strongly reducing Reduced form of a polysulfide, such as DATS Disulfide RSSR 1 Weakly oxidizing, In many proteins and occasionally enzymes reducing Polysulfide RSxR 1, Weakly oxidizing Natural products, for

(x 3) (0)x2,1 example, DATS, DATTS, varacin, lenthionine Sulfenic acid RSOH 0 Reducing, Formed from RSH during oxidizing, and OS, found in Prx enzymes dimerization Sulfoxide RS(O)R 0 Weakly oxidizing Alliin, also in (accidentally) or reducing oxidized methionine Thiosulfinate RS(O)SR þ1,1 Effective oxida- Natural products (e.g., tion of thiols allicin), found in Prx Sulfinic acid RS(O)OH þ2 Usually inert, Overoxidized form of reduction by sulfur, found in Prx sulfiredoxin

Sulfone RS(O)2R þ2 Weakly oxidizing In some natural products and (accidentally) oxidized methionine þ Thiosulfonate RS(O)2SR 3, 3 Effective oxida- In some natural products, tion of thiols otherwise unclear, possibly formed during OS

Sulfonic acid RS(O)2OH þ4 Redox inert End product of sulfur (in absence of oxidation, found in taurine enzymes) 2 þ Sulfate SO4 6 Redox inert in Inorganic sulfur agent, for absence of example, in heparin sulfate enzymes

Abbreviation: RNRase, ribonucleotide reductase; Prx, human peroxiredoxin enzymes. 35.3 Bioavailability and Metabolisms of Active Compounds j667

As far as our discussion of chemopreventive agents is concerned, one must bear in mind that the underlying chemistry of sulfur compounds is complicated and differs from compound to compound. Thiols, for instance, are often highly reducing and exhibit almost ideal antioxidant, radical, and peroxide scavenging properties. In contrast, thiosulfinates, such as the garlic compound diallyl thiosulfinate (allicin) and thiosulfonates, are rather oxidizing and able to modify most protein-based cysteine thiols within seconds. Other chemotypes, such as di- and polysulfides, are less reactive or exhibit an unusual chemistry rich in secondary, follow-on reactions. Therefore, it is often difficult to classify a given sulfur agent as pro- or antioxidant, as beneficial or as detrimental to cells. Such properties are relative and depend on the particular (biochemical) context under which the compounds are studied. This may also explain why different studies using the same sulfur compound occasionally arrive at different or even apparently contradictory results or interpretations. Such discrepancies may arise when the same compound is applied within different contexts, at different concentrations, or in different cell lines.

35.3 Bioavailability and Metabolisms of Active Compounds

35.3.1 Enzymatic Generation of Reactive Sulfur Species as Part of Binary Plant Defense Systems

A range of RSS relevant in the context of chemoprevention, such as allicin and diallylpolysulfides, are not simply synthesized and stored in the plant. In garlic, allicin is synthesized in the clove on demand, that is, when the clove is physically injured or attacked by microbes [17]. The chemistry behind allicin synthesis is rather intriguing. The cysteine derivative g-glutamyl-S-alk(en)yl-L-cysteine is hydrolyzed and oxidized to yield alliin (Scheme 35.1). Alliin, which is present in the cytosol, is a comparatively chemically unreactive sulfoxide, although some reports claim that it may inhibit angiogenesis in HUVEC cells in vitro [18]. Alliin is converted to the highly aggressive thiosulfinate allicin by the C-S-lyase enzyme, alliinase, which is normally stored separately from alliin in vacuoles, and only encounters its substrate when the garlic clove is damaged. Allicin, which is then formed within seconds, reacts readily with a wide range of thiol-based targets, such as cysteine residues in peptides, proteins, and enzymes [14]. A similar binary substrate/enzyme system able to generate an aggressive sulfur species on demand also exists in onions (Scheme 35.2). Here, most of the chemistry of biologically active species is associated with the formation and subsequent reaction pathways of the lachrymatory-factor (LF), although a garlic-like thiosulfinate chemistry also exists. The LF is formed from isoalliin in the presence of the (onion) enzymes alliinase and lachrymatory-factor synthase (LFS) [19]. The LF is chemically unstable and readily dimerizes or hydrolyses to yield a wide range of rather 668j 35 Sulfides in Allium Vegetables

Scheme 35.2 Aspects of sulfur chemistry found dimer of the LF, trans-3,4-diethyl-1,2-dithietane- in onions. Unlike in garlic with its characteristic 1,1,-dioxide (21), differs dramatically from the allyl-(2-propenyl) chemistry, onions contain dimer of (18)or(2) and is given as just one compounds with a 1-propenyl group. Although example to illustrate the rather unusual there are some similarities, such as the chemistry based on the LF. The LF is also formation of an allicin-like thiosulfinate (19) sensitive to hydrolysis, which results in from the alliin-like precursor isoalliin (trans-1- numerous follow-on products, possibly

propenyl-L-cysteine sulfoxide, PRENCSO) (17) including H2S, and may result in the formation of via 1-propenyl sulfenic acid (18), there are also 1,2,4-trithiolanes (22)(cis- and trans-forms, distinct differences due to a different reactivity of stereochemistry not shown). In onions, the the conjugated double bonds. As a result, (18) chemistry of the LF somewhat overshadows the is converted to the lachrymatory factor (20)by typical thiosulfinate chemistry of (18), which is the enzyme LF synthase. This reaction is not shown on the left (represented by compound possible for allyl sulfenic acid (2). The LF is highly (19)). This chemistry in part resembles the reactive and endows onions with an extensive follow-on chemistry of allicin and may include and complicated sulfur chemistry, which is quite methyl-, ethyl-, and propyl-based mono-, di-, and distinct from the one of garlic, yet equally polysulfides. interesting from a biological perspective. The

interesting follow-on products, including trans-3,4-diethyl-1,2-dithietane-1,1,-diox-

ide, 1,2,4-trithiolanes, and, probably, H2S [1]. Perhaps surprisingly, such binary substrate/enzyme systems to generate RSS on demand are fairly common in plants and fungi, where they mostly serve to ﬁght off 35.3 Bioavailability and Metabolisms of Active Compounds j669 invading organisms or potential consumers. For instance, a substrate/enzyme combination is also responsible for the synthesis of lenthionine in Shiitake mushrooms. Similarly, the formation of reactive isothiocyanates from fairly unreactive glucosinolates in plants of the family Crucifereae is controlled by the myrosinase enzymes [4]. Most of the agents formed in this context, such as LF, allicin, and isothiocyanates, are either too unstable chemically or too reactive to survive for long in the presence of other biomolecules, especially the ones containing thiol groups. Allicin, for instance, is sensitive to elevated temperatures and will be destroyed by cooking or decompose after a couple of days at room temperature (see Scheme 35.1 and Section 35.6). Alternatively, it may react with thiols present in small molecules, peptides, proteins, or enzymes to form a range of follow-on products, such as S-allylmercaptocysteine (SAMC) [1]. As a consequence, a human diet rich in garlic and onions will not automatically also be rich in allicin, LF, or similarly aggressive sulfur agents. A recent review by Amagase entitled Clarifying the Real Bioactive Constituents of Garlic has underlined the importance of avoiding any such misunderstandings, for instance, when dealing with aged garlic extracts (AGE) [6].3)

35.3.2 Follow-On Products Formed by Degradation of Reactions with Biomolecules

Among the various follow-on products4) of allicin, we can distinguish between compounds formed by decomposition of allicin in the absence of other reaction partners and reaction products of allicin with other biomolecules, most notably thiols. The first group of compounds includes the various diallylsulfides, such as diallylsulfide (DAS), DADS, DATS, and DATTS, as well as a range of higher sulfides, such as diallylpenta-, hexa-, and heptasulfide, whose presence and biological roles are still less clear. In addition, dithiins and the interesting disulfide ajoene are also formed as part of – chemically rather complicated – allicin decomposition pathways. In onion, major follow-on products of the LF reaction pathway are trans- 3,4-diethyl-1,2-dithietane-1,1,-dioxide, 1,2,4-trithiolanes, and various methyl-, ethyl-, and propyl-containing sulfides (Scheme 35.2). The precise composition of such preparations varies widely and depends critically on the manufacturing process. Apart from chemical decomposition, we need to account for various reactions of allicin with thiol-containing biomolecules (Scheme 35.1). The reaction of allicin with

3) AGE is not a pure compound or a precise systems therefore points toward the involve- mixture of pure compounds and is therefore ment of follow-on products, such as DAS, poorly defined chemically. As a consequence, DADS, ajoene, and polysulfides. its use in many biochemical and biological 4) In this chapter, we will use the expression assays is limited (and the same applies to garlic follow-on products rather than secondary extracts and oils). Nonetheless, the activity of products to avoid any possible confusion with AGE in a particular test system often provides a secondary metabolites, which also fre- fi rst lead for more in-depth investigations. quently occur in plants. Unlike the follow-on fi Interestingly, AGE does not contain signi cant products, the latter are not formed by amounts of allicin, and its activity in test chemical decomposition or degradation. 670j 35 Sulfides in Allium Vegetables

cysteine results in agents such as S-allylcysteine (SAC) and SAMC, both of which possess interesting chemopreventive properties. In the human body, allicin is also likely to be sequestered by reduced glutathione (GSH), leading to the formation of S-allylmercaptoglutathione.

A very recent report by Benavides et al. has also provided initial evidence for H2S release from various di- and polysulfides found in garlic (Scheme 35.1) [16]. Such a fi connection between garlic and polysul des on the one hand and H2S and H2S signaling on the other hand has been proposed earlier as part of the chemistry of fi polysul des, which may be possible under physiological conditions [14]. H2S formation is apparently the result of an exhaustive reduction of polysulfides by GSH, especially in the case of DATS, DATTS, and higher sulfides. It may also occur in the case of DADS. The various sulfides, disulfides, and polysulfides are chemically rather stable and considerably less reactive when compared with allicin. Together with the reaction products of allicin, they are therefore found in most garlic products and must be considered as the likely contenders for chemopreventive activity, although a major role for allicin and related thiosulfinates should not be ruled out up front. Interestingly, polysulfides are also fairly stable metabolically, and plasma concentrations of DATS as high as 31 mM have been achieved in rats following injection of this compound [20]. Furthermore, allicin – as well as most of the sulfides mentioned in this section – is fairly lipophilic; such compounds are able to cross cell membranes and enter cells with comparable ease. This may explain their diverse and often rather high biological activity, which has been observed for such sulfur compounds in vitro and in animal models.

35.3.3 Chemical Recycling of Allicin

The loss of allicin from garlic due to decomposition or chemical reactions has frequently raised the question if aged garlic extract, powders, or oils are indeed still biologically active and beneficial to human health. The presence of various biologically active follow-on agents other than allicin may point toward such a longer term activity of cooked or processed garlic (see Sections 35.5 and 35.6). In addition, it has been reported that allicin is in part being recycled from its own decomposition product DADS by cytochrome P-450 and flavin-containing mono-oxygenase enzymes that are present in many human cells – especially in certain cancer cells [21]. Is it therefore possible that certain cancer cells convert DADS to allicin and hence commit suicide, while normal cells are not affected? After all, oxidative activation of reactive (sulfur) species, such as thiosulfinates, has been discussed before in the context of oxidative stress (OS), and allicin is indeed synthesized chemically by

oxidation of DADS in the presence of H2O2 [17]. The notion of allicin formation in situ is particularly intriguing, since it is supported by two possible reaction pathways, both of which are relevant in the context of human cells. First, enzymatic conversion of DADS to allicin by oxidase enzymes may preferably occur in cancer cells, which often exhibit higher levels of such enzymes when compared with healthy cells. To a certain 35.4 Mechanisms of Protection j671 degree, such oxidative activation would therefore be cancer cell speciﬁc and may allow DADS (and other active ingredients of Allium vegetables) to act as selective agents. Second, there is some evidence of a redox interplay between DADS on the one side and DATS and DATTS on the other. Recent reports by Munday and colleagues have pointed toward the ability of DATS and DATTS to (catalytically) . – – generate superoxide (O2 ) and therefore also H2O2 in cells in the presence of dioxygen and GSH [22]. Since DADS, DATS, and DATTS are usually jointly present in garlic products and consumed together, H2O2 generation by DATS and DATTS may result in allicin formation (from DADS) and therefore trigger the chemopreventive, that is, cytotoxic effects associated with this thiosulﬁnate. Such hypotheses and their implications for chemoprevention obviously still need to be explored further.

35.4 Mechanisms of Protection

There are various mechanisms by which chemopreventive agents may interfere with cancer cells at the initiation, promotion, and progression stages of cancer [11, 23]. A rough and simplified classification of different biochemical modes of action is provided in Figure 35.1.5) Many sulfur compounds found in garlic, onion, and related plants, in one way or another, seem to exhibit one or several of these chemopreventive activities. It should be mentioned from the outset that this section will primarily focus on sulfur compounds found in garlic, since most of the literature on Allium vegetables is concerned with this plant. In certain instances, such as for the mono-, di-, and polysulfides, similar considerations apply to the sulfur analogues found in onions, although the methyl-, ethyl-, and propyl-containing agents present in onions are generally somewhat less active than the corresponding allyl-containing agents found in garlic. Unfortunately, many aspects of the sulfur chemistry of onion are also still only poorly understood, partially because the sulfur compounds found in onions are often unstable or difficult to obtain and handle in practice.

35.4.1 Sulfur-based Scavengers of Free Radicals and Other Oxidative Stressors

Antioxidants play a major role at the initiation stage of cancer development. It is well known that free radicals and related reactive oxygen species (ROS) cause DNA mutations, some of which lead to the formation of cancer. A number of garlic-derived products and compounds, such as AGE, garlic and onion oils, allyl mercaptan (AM),

5) This classiﬁcation is not complete or mutually exclusive. It is only used to disentangle the many compounds and their associated activities – and to enable a better overview of the ﬁeld. 672j 35 Sulfides in Allium Vegetables

Figure 35.1 A schematic overview of the the prevention of metastasis formation (both of different cancer chemopreventive activities which are related to the inhibition of cancer cell discussed as part of this chapter. The scheme proliferation), or the ability of DAS and DATS to illustrates key chemopreventive modes of action, counteract MDR, are not listed explicitly but are the corresponding cellular targets or events, and discussed in the text. Alliin is shown here a selected list of natural sulfur agents from Allium because of recent evidence that it inhibits vegetables that are associated with a particular angiogenesis. Please note that this figure does mode of action. It should be noted that the not provide a complete overview of all possible classification used here is only meant to provide chemopreventive mechanisms, nor does it a rough and simplified guide to the different contain an exhaustive overview of all sulfur biochemical events, compounds, and chemical agents and their respective activities (for transformations associated with them. Other instance, little is known to date about the activity activities, such as inhibition of angiogenesis and of the chemically less accessible DATTS).

DAS, DADS, DATS, SAC, SAMC, and ajoene, have been considered as possible antioxidants that protect DNA, proteins, enzymes, and lipids from oxidation and peroxidation [3, 10, 11]. Among them, the thiol compound AM is perhaps the most obvious scavenger, since it is able to react directly with a wide range of radicals and electrophilic compounds. The antioxidant chemistry behind the other compounds is less obvious, although, in principle, all of them may react with and hence sequester 35.4 Mechanisms of Protection j673

ROS or form AM in the presence of GSH. Some of the sulfur compounds can also react directly with other carcinogens, such as aromatic nitrocompounds, quinolines, and quinones.

35.4.2 Prevention of Carcinogen Activation by Inhibition of Metabolic (Phase I) Enzymes

Another possibility of interfering directly with carcinogens involves the inhibition of cytochrome P-450 enzymes [3, 24]. In essence, inhibition of these and related metabolic enzymes prevents metabolism and possible oxidative activation of carcinogenic agents. Diallylsulfoxide and diallylsulfone, for instance, both inhibit the activity of cytochrome P-450 2B1, and similar ﬁndings have been made for DAS and DADS [25]. Likewise, DAS and ajoene are able to prevent the chemical ﬂ activation of carcinogenic agents such as a atoxin B1 and its metabolic products via phase I enzyme inhibition [26]. Such effects are not clear-cut, however, and DAS, DADS, and DATS may also act as inducers – not inhibitors – of cytochrome P-450 enzymes, as observed for cytochrome P-450 1A1, 2B1, and 3A1 in rat liver [27].

35.4.3 Induction of Phase II Enzymes

In many instances, natural sulfur compounds associated with sequestration of carcinogens act via induction of detoxifying phase II enzymes, such as glutathione-S-transferase (GST), microsomal epoxide hydrolase (mEH), quinone reductase (QR), and UDP-glucuronosyltransferase (UGT) [10]. The chain of cellular events leading from sulfur agents to such phase II detoxifying agents is quite complex. DAS, DADS, DATS, and dithiins are among the more prominent phase II enzyme inducers [28, 29]. At the molecular level, these sulfur compounds seem to interact with the Nrf2–Keap1 complex, most probably as oxidants. Keap1 contains many redox-sensitive cysteine residues and upon oxidation releases Nrf2. The latter enters the nucleus, where it binds to the antioxidant responsive element (ARE) that controls the expression of a range of phase II enzymes [11]. Other reports claim that AGE may stimulate the expression of not only phase II enzymes but also various antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). For instance, an increased GPx activity has beenobservedinthelungsofmice exposedtoDATS.Here,theunderlyingbiochemical mechanisms are less obvious.6) DAS and DATS can also increase the levels of GSH in the liver,lung, and/or forestomach, an effect only observed for the allylsulﬁdes of garlic and not for the propylsulﬁdes of onions. It is possible that the chemistry of the allyl moiety plays a decisive – or even the decisive – role in this particular context [10].

6) Interestingly, some reports claim that seleni- many selenium compounds increase the ac- um compounds – and not sulfur agents – are tivity of GPx and are also toxic against cancer among the major chemopreventive constitu- cells. ents of (selenium-enriched) garlic. Indeed, 674j 35 Sulfides in Allium Vegetables

35.4.4 Sulfur Agents as HDAC Inhibitors

In many types of cancer, cellular levels of proteins and enzymes are altered due to epigenetic changes. These changes occur without modifications in the DNA encoding for these proteins, but, for instance, due to changes in transcription factor activity. Agents able to redepress silenced but intact genes may return some normality to the cancer cell – for instance, by promoting differentiation, slowing proliferation, or inducing apoptosis. Todate, anumberofcompoundshavebeenidentifiedthatredepressbeneficial,yet silenced, genes. Among these agents, the histone deacetylase (HDAC) inhibitors occupy a prominent role [8, 30]. HDAC inhibitors increase the level of histone acetylation and therefore tend to enhance gene expression, including the expression of DNA binding proteins and transcription factors. Ultimately, such processes may result in a redepression of silenced genes and subsequently differentiation or slowed proliferationof cancer cellsoreven cancer cell death.Within this context,a recent study has described a possible correlation between AM and DADS on the one hand and reduced HDAC activity, histone (H3 and H4) hyperacetylation, p21 expression, and inhibition of (human colon) tumor cell proliferation on the other hand [31]. Further- more, sulfur agents able to increase histone acetylation levels are DADS (in Caco-2 and HT-29 colon tumor cells, K562 human leukemia cells, and DS19 mouse erythroleu- kemia cells), DATS (in PC-3 prostate tumor cells), SAC and allicin (in DS19 cells), and SAMC (in DS19 cells, Caco-2 human colon, and T47D human breast cancer cells) [10]. The precise chain of cellular events leading from these sulfur compounds to HDAC inhibition, the subsequent expression of certain proteins and apparent chemoprevention, possibly via G2/M phase cell cycle arrest in the cancer cells, is still a matter of future investigation. At the molecular level, it is known that many HDAC enzymes contain an active-site zinc ion. The latter may be a prime target for sulfur compounds, which are known to interact very strongly with zinc ions in various proteins and enzymes, where they behave as adventitious ligands to the metal ion, occupy the substrate binding sites, and hence inhibit the metalloenzyme in question [8]. Judging by the chemistry of many HDAC inhibitors, such as hydroxamic acid derivatives, which in essence are ligands for zinc ions, AM, DADS, and related compounds may indeed form a new class of potent (but probably unspecific) HDAC inhibitors.7)

35.4.5 Interference with Cancer Promoting, Cell Signaling Pathways

To a certain extent, the events surrounding redepression of beneﬁcial enzymes are mirrored by the inhibition of the bad pathways, which stimulate the proliferation of

7) Unlike thiols, disulfides are only poor ligands for zinc ions. Therefore DADS – and possibly polysulfides – may not be active on their own but may become activated by reduction to AM, for instance, in the presence of GSH. 35.4 Mechanisms of Protection j675 malignant cells and protect them against apoptosis. Such pathways are often highly complex and involve a wide range of different proteins and enzymes. A recent review, by Dorai and Aggarwal, summarizes the different cellular signaling pathways affected by chemopreventive agents, including sulfur compounds from Allium vegetables [23]. One prominent signaling pathway responsible for cancer cell proliferation is controlled by proteins such as NF-kB and AP1, which in turn are affected by various other signaling proteins, such as NF-kB-inducing kinase (NIK) and mitogen-activated protein (MAP) kinases or the AKT signaling pathway. Several studies have described the role of garlic in inhibiting the activation of NF-kB. A report by Borek describes the activity of AGE as an NF-kB inhibitor [32]. Among the pure sulfur compounds, there are various reports linking diallylsulfides to a suppressed NF-kB activation. For instance, DADS and DATS reduce lipopolysaccharide-induced NF-kB activation (and nitric oxide production) in macrophages [33]. Within this context, a recently discovered, novel sulfur compound isolated from garlic, called thiacremonone (see Scheme 35.1), seems to provide effective cancer chemoprevention by modulating NF-kB and the tumor necrosis factor-a (TNF-a) [34]. Nonetheless, the relationship between natural sulfur compounds, NF-kB, and cell proliferation is not straightforward. Ajoene, for instance, appears to induce apoptosis in human promyeloleukemic cells by a mechanism that generates ROS and actually activates NF-kB [35].

35.4.6 Cell Cycle Arrest and Induction of Apoptosis

Apart from modulating NF-kB activity, sulfur compounds are known to interfere with a range of other cellular pathways. For instance, DADS inhibits hepatic and tumoral HMG-CoA reductase, and this inhibition has been associated with the suppressed growth of H-ras oncogene transformed tumor xenografts [36]. Perhaps even more interesting, however, are events that not only slow down cancer cell proliferation, but also kill cancer cells. In this context, DATS and DATTS have received considerable attention. A series of independent studies published during the last 5 years have found activity against MGC803 and SGC7901 human gastric cancer cell lines [37], HCT-15 and DLD-1 colon cancer cell lines [38], Caco-2 and HT-29 colon carcinoma cancer cells [39], and PC-3 and DU145 prostate cancer cell lines [40], to name just a few. Singh et al. also noted a cancer cell selective activity of DATS against PC-3 cells, while normal prostate cells (Pr-EC) were largely unaffected. The possibility that a chemically simple molecule such as DATS may distinguish between cancer cells and normal cells is intriguing, and this has led to an intensive search for the biochemical causes of this effect [41]. Among them, oxidative modification of cysteine residues in proteins, such as b-tubulin, via a thiol/trisulfide exchange mechanism involving DATS is a possibility, although DATS is not a particularly strong oxidant [38]. Interestingly, it appears that fi . polysul des such as DATS are also able to (catalytically) generate O2 in the presence of dioxygen and GSH and hence raise intracellular levels of OS, which may push cancer cells already high in ROS over a critical ROS threshold [14, 22]. In this context, 676j 35 Sulfides in Allium Vegetables

Singh and colleagues have suggested that DATS may actually trigger the degradation of ferritin, which is associated with iron ion release, subsequent ROS generation, G2/ M cycle arrest, and apoptosis, a chain of events that may in part be under the control of

the c-Jun NH2-terminal kinase (JNK) signaling axis [10]. Apart from DATS, a mitotic arrest has also been observed for DAS, DADS, SAMC, and ajoene, although the underlying (bio)chemistry is less apparent (yet may still be associated with ROS formation). Interestingly, cell cycle arrests are not limited to the G2/M phase: DADS may also cause arrest in the S phase, while DATS can cause arrest in the G1 phase via a decrease in cyclin D1 and an increase in p27 in human gastric cancer cell line BGC823 [10]. The ability of sulfur compounds to kill cancer cells is not solely linked to mitotic arrest via ROS formation. Induction of apoptosis by changing the ratio of Bcl-2 to Bax provides another entry point that leads to apoptosis via the caspase pathway [10]. In this context, DATS, ajoene, and, to a lower extent, DADS and DAS have been associated with a decrease in Bcl-2 levels, which triggers the intrinsic pathway of cell death. The biochemistry surrounding apoptosis in cancer cells is, of course, quite complex, and other pathways, such as the Akt–Bad pathway (in the case of DATS) and

calcium-dependent pathways leading to increased levels of H2O2 and caspase-3 activity (in the case of DADS) have also been implicated. The role of p53 in sulfur compound-induced cell death is still not fully understood.8) Finally, the possibility of ﬁ H2S release from polysul des, which has been postulated for DATS and DATTS (and also DADS), may associate these agents with a host of different cell signaling pathways [16]. If, and to which extent, this may also include signaling pathways associated with cancer promotion or progression is still unclear and needs to be investigated further.

35.4.7 Sulfur Agents and Angiogenesis

Another rather interesting aspect of sulfur-based chemoprevention is the inhibition of angiogenesis and metastasis formation [10]. Within this context, several studies have shown that AGE and a range of active ingredients, such as DATS, DADS, DAS, ajoene, and SAMC, can inhibit cell proliferation and hence angiogenesis in various models, such as in a colorectal cancer cell line model (HT-29, SW480, SW620) and human umbilical vein endothelial cells (HUVECs). Among the sulfur agents discussed so far, DATS seems to be the most active in the HUVEC model, and its activity seems to be associated with caspase-3 activity, PARP cleavage, and apoptotic cell death. Apart from inducing cell death, AGE also attenuated cell migration in rat

8) For more details on sulfur compounds from of the rather complicated relationship between garlic and their role in apoptosis of cancer sulfur compounds from Allium vegetables, cells, see Herman-Antosiewicz et al. [10]. Two cancer chemoprevention in different cancer recent reviews, one by Mersch-Sundermann cells (cell lines), and the possible modes of and colleagues [9] and the other by Shukla and action [13]. Kalra [3], provide further, more general details 35.5 Results of Human Studies j677 sarcoma cells, pointing toward several activities of AGE that together seem to immobilize and kill cancer cells and also prevent angiogenesis and possibly the formation of metastases. The ability of natural sulfur agents to prevent metastasis formation has also be demonstrated for SAMC, which in mice reduced the number of lung metastasis caused by implanted PC-3 cells by an astonishing 85.5% and completely prevented the formation of adrenal gland metastases [42]. Aspects surrounding these antiangiogenic and antimetastatic activities are summarized in a recent review by Singh et al. [10].

35.4.8 Counteracting Multidrug Resistance

Finally, a number of studies have linked organic sulfur compounds from garlic to the modulation of multidrug resistance (MDR) in human cancer cells: MDR poses a serious problem in chemotherapy, since it protects certain cancer cells from the toxic effects of anticancer drugs. The mechanism of protection frequently involves an overexpression of the drug efﬂux protein P-glycoprotein. Studies involving K562 leukemia cells and human MDR carcinoma KB-C2 cells indicate that DAS and DATS may prevent resistance against anticancer drugs, such as vinblastine (DAS in K562 leukemic cells) and daunorubicin (DAS, DATS in KB-C2 cells). In both instances, modulation of MDR by the sulfur agents seems to be the result of an interference with P-glycoprotein overexpression [3].

35.5 Results of Human Studies

Human studies involving organic sulfur agents derived from Allium vegetables can be divided into three main groups. There are sporadic reports of classical trials that involve the use of pure and synthetic agents and placebo controls. Such reports are rare, however, and their outcomes are often inconclusive and perhaps even questionable. An intervention study involving synthetic DATS and selenium has been conducted in China [43]. It points toward reduced morbidity rates from malignant tumors in the group of participants taking DATS and selenium when compared to the placebo control group. Most studies involving human subjects have not been conducted with synthetic sulfur agents, though. Some studies have used food supplements, such as garlic and onion powders, as part of a controlled diet, while others have followed an epidemiological approach, relying on the natural food habits of different population groups. Many of these studies have already been reviewed [44, 45]. As part of this chapter, we will limit ourselves to highlighting a few key findings. Intervention studies in Japan with AGE and patients suffering from colorectal cancer indicate that garlic is able to decrease the risk of new adenomas and the size of adenomas in these patients significantly. Similarly, a cohort study in the Netherlands seems to confirm a decreased risk for stomach, colorectal, breast, and lung cancers 678j 35 Sulfides in Allium Vegetables

due to the regular intake of onion, leek, and garlic supplements [3]. The findings of such cohort studies with controlled diets have been confirmed by epidemiological surveys [46]. The latter frequently link a diet rich in Allium, most notably garlic, to a decreased risk of cancer, especially stomach cancer. As mentioned above, such studies date back to the 1980s, yet have often been marred by statistically insignificant or even conflicting results. In a few cases, the consumption of onions has even been associated with a slightly increased risk of cancer (rectum carcinoma), although this increase was not statistically significant [47]. Overall, there seems to be a substantial body of evidence pointing toward a decreased risk of cancer in populations with an Allium-rich diet, which may be most pronounced in, but not limited to, incidences of stomach cancer. There may be a couple of explanations for these findings, including an antibacterial activity of many sulfur compounds against Helicobacter pylori, or the chemical reactivity of many sulfur compounds, which allows them to scavenge various carcinogens [46]. Future studies, either with cohorts and controlled diets, or by using an epidemiological approach, may provide a clearer indication of the impact sulfur agents from Allium vegetables have on human health. Such studies may be complemented by a thorough meta-analysis of already available data and possibly more reliable trials with pure sulfur agents, such as allicin, DAS, DADS, DATS, DATTS, SAC, SAMC, and ajoene. Finally, it must be pointed out that the health benefits of such sulfur compounds may be not only limited to cancer chemoprevention but may also include other aspects, such as lowering cholesterol and, of course, antimicrobial activity. In turn, not all health benefits ascribed to garlic, onions, and related vegetables may necessarily be due to the presence of sulfur agents, but could also result from the various other ingredients, such as allixin, trace elements, and various phenolic compounds [6].

35.6 Impact of Cooking, Processing, and Other Factors on Protective Properties

Processing of garlic, onions, and related species has already been discussed above as part of the synthetic pathways leading to allicin in garlic, the LF in onions, and a host of follow-on products. Interestingly, many of the active ingredients contained in garlic cloves, onion bulbs, and the like are not destroyed by chopping, heating, or cooking, as is the case with most other vegetables and fruits [48]. Quite on the contrary, many of the more interesting ingredients of processed garlic and onions are actually formed as a direct result of processing, including allicin, the LF and their respective follow-on products, such as DATS, DATTS, ajoene, and the various other mono-, di-, and polysulfides. In fact, alliin and isoalliin alone are rather unreactive chemically and have a limited biological activity. Since studies have shown that in the human body alliin is most probably not processed to allicin or any of the follow-on compounds discussed here, alliinase conversion is crucial for the appearance of most 35.6 Impact of Cooking, Processing, and Other Factors on Protective Properties j679 of the chemopreventive and antimicrobial activities associated with garlic (and probably onions), their products, and compounds. As far as heating, cooking, or microwaving is concerned, one would therefore expect little loss of (chemopreventive) activity, assuming that the alliin-to-allicin conversion catalyzed by alliinase has already taken place and the follow-on products of garlic compensate for the loss of allicin. Indeed, this seems to be the case: Although heating and microwaving both decrease the chemopreventive activity of freshly crushed garlic due to inhibition of alliinase, they have little effect on this activity once allicin has been formed in the crushed garlic sample [49–51]. This retention of activity fi has been con rmed for the scavenging of superoxide and hydroxyl radicals, H2O2 and peroxynitrite [50, 51]. As mentioned above, such effects may to some extent be due to the presence of AM, which is formed as part of allicin decomposition. The resistance of chemopreventive properties against heating has also been confirmed in a DNA-based assay, where microwaved, oven-heated, and untreated garlic samples were compared: Once the alliin-to-allicin conversion had taken place, neither heating nor microwaving had any notable effects on the ability of crushed garlic to inhibit the binding of the mammary carcinogen 7,12-dimethyl- benzene(a)anthracene (DMBA) metabolites to rat mammary epithelial cell DNA [49]. It is therefore safe to claim that garlic, unlike many other fruits and vegetables, retains considerable activity even after cooking, assuming allicin is formed first. The follow-on products of allicin may differ in heated and nonheated samples. Higher polysulfides, for instance, are unlikely to survive heat treatment, which may result in an increased presence of DADS and DATS. Nonetheless, even fried garlic will still contain a cocktail of powerful chemopreventive agents, such as

DAS, DADS, allylmercaptan, and H2S. Less is known about the (follow-on) chemistry of sulfur agents in onions, yet it is likely that similar considerations will apply. This leads us to perhaps the most important question of all, that is, if it is healthier to consume raw or cooked garlic. In essence, this is a question of chemistry. Swallowing a whole clove of raw garlic may ultimately provide the human body with alliin, while eating raw, but freshly crushed garlic, will deliver a fair amount of allicin. The latter is quite stable chemically under acidic conditions and will survive well in stomachs acidic environment, and yet will react with other biomolecules to form disulfides. Cooked garlic, on the other hand, is rich in DATS and a couple of other diallylsulfides, which not only may last longer than allicin, but may also, at least in part, be converted to other sulfur agents in the body, such as AM and SAMC. Since virtually all of these sulfur agents exhibit a certain biological activity, neither chopping, heating, nor cooking garlic will abolish eventual health benefits credited to garlic and its sulfur compounds, although these processes may well alter them. As before, comparable considerations also apply to onions, although the matter is somewhat more complicated in this case because of the involvement of two enzymes, that is, alliinase and LFsynthase. In the end, it is also not advisable to swallow a whole onion. 680j 35 Sulfides in Allium Vegetables

35.7 Outlook

The previous sections have taken a closer look at garlic, its active sulfur ingredients, and their possible roles in the various facets of cancer chemoprevention. Overall, sulfur-containing agents seem to be associated with a variety of chemopreventive actions, ranging from simple sequestration of carcinogens to the control of complex cellular signaling pathways. In some cases, the different modes of action seem to be associated with chemically distinct sulfur compounds, while in other instances, several chemopreventive modes of action seem to converge on just one molecule. In general, chemoprevention and health benefits seem to dominate the field of garlic compounds. Nonetheless, there are also some reports that point toward a rather sinister role of such sulfur compounds, for instance, in generating ROS, OS, and stimulating NF-kB activity. Among the compounds discussed above, DAS has been associated with an enhanced hepatocarcinogenesis when applied subsequently to carcinogen treatment (while DADS inhibited carcinogenesis) [52]; DAS, DATS, and a few other alkylsulfides have been associated with the enhancement of diethylnitrosamine-induced GST-positive foci in rat liver [53]; ajoene has been associated with the formation of ROS, which may either lead to the formation of cancer cells by DNA modification or kill cancer cells as postulated for DATS and varacin, which appear to act via a radical formation mechanism [14, 35].9) Nonetheless, plants of the genus Allium, their products, and chemical compounds contained therein seem to provide numerous health benefits that are only in part affected by food processing, such as cooking. While many of these benefits are now well documented in the literature, and are also slowly finding their way into applications among the wider public, there are still many questions remaining. While it has not been possible to summarize all possible chemopreventive actions of natural sulfur compounds as part of this chapter, the literature provided should allow access to the wider, and more in-depth, discussion of the matters and also to the open questions still surrounding Allium vegetables, natural sulfur compounds, and cancer chemopreventive activities associated with them. In this context, we should emphasize that Allium does not simply equal garlic and that the focus of this chapter on the latter is due to the fact that the Allium literature is overwhelmingly dominated by garlic. Nonetheless, onions, and also the less studied plants, such as leeks and chives, provide an equally interesting set of compounds, many of which may turn out to possess chemopreventive properties. Other edible sources of sulfur compounds, such as asparagus, which appears to contain the cyclic trisulfide 1,2,3,-trithiane-5-carboxylic acid, and Petai (Parkia Speciosa), which is a source of numerous sulfides and polysulfides, have hardly been considered yet.

9) Varacin is a cyclic pentasulﬁde from Lissocli- num vareau whose cytotoxic biological activity and underlying biochemical modes of action seem to be associated with the formation of r O2 radicals [41]. References j681

35.8 Conclusion

Garlic, onions, leek, and related Allium vegetables have been used as a source of medicines in many cultures and throughout history. These plants contain a wide range of sulfur agents with distinct chemical reactivity, biochemical proﬁles, and associated biological activities against micro-organisms and cancer. As we have seen, allicin and its follow-on products are among the numerous sulfur compounds found in these plants. These RSS exhibit a wide range of cancer chemopreventive activities, such as simple chemical antioxidant activities, induction of phase II enzymes, modulation of cellular signaling pathways, interference with histone deacetylation, and induction of apoptosis. Although the precise underlying chemistry and mode(s) of biochemical action of the compounds are often unknown, redox reactions and binding to metalloproteins, both hallmarks of sulfur chemistry, seem to explain some of the activities observed in cell culture, animals, and humans. The latter link the consumption of Allium plants to a decreased risk of cancer, in particular, stomach cancer. This apparent chemopreventive activity in humans is combined with an extraordinary resistance of many sulfur agents against food processing, including heating and cooking, and a high bioavailability. These properties clearly distinguish sulfur compounds from most other chemopreventive plant products and ensure a prominent role among the various natural preventive and therapeutic agents. It is therefore safe to assume that garlic, onions, and the like will continue to provide ample opportunity for multidisciplinary research and product development in the future as well as tasty – and probably healthy – dishes for people not afraid of the odor associated with them [54, 55].

Acknowledgments

TheauthorswouldliketothankTorstenBurkholz(SaarlandStateUniversity)forhelpful discussions and referencing. This work has been supported ﬁnancially by the Saarland State University, the German Academic Exchange Service (DAAD, grant number D/07/09986), and the Landesforschungsfoerderprogramm of the State of Saarland.

References

1 Block, E. (1992) The organosulfur 3 Shukla, Y. and Kalra, N. (2007) Cancer chemistry of the genus Allium – chemoprevention with garlic and implications for the organic chemistry of its constituents. Cancer Letters, 247,167–181. sulfur. Angewandte Chemie, 31, 4 Jacob, C. (2006) A scent of therapy: 1135–1178. pharmacological implications of natural 2 Rivlin, R.S. (2001) Historical perspective products containing redox-active sulfur on the use of garlic. Journal of Nutrition, atoms. Natural Product Reports, 23, 131, 951s–954s. 851–863. 682j 35 Sulfides in Allium Vegetables

5 Mei, X., Wang, M., Xu, H.X., Pan, X.Y., Proceedings of the National Academy of Gao, C.Y., Han, N. and Fu, M.Y. (1982) Sciences of the United States of America, 104, Garlic and gastric cancer – the effect of 17977–17982. garlic on nitrile and nitrate in gastric juice. 17 Jacob, C. and Anwar, A. (2008) The Acta Nutrimenta Sinca, 4,53–56. chemistry behind redox regulation with a 6 Amagase, H. (2006) Clarifying the real focus on sulfur redox systems. Physiologia bioactive constituents of garlic. Journal of Plantarum, 133, 469–480. Nutrition, 136, 716s–725s. 18 Mousa, A.S. and Mousa, S.A. (2005) Anti- 7 Davis, C.D. (2007) Nutritional angiogenesis efficacy of the garlic interactions: credentialing of molecular ingredient alliin and antioxidants: role of targets for cancer prevention. Experimental nitric oxide and p53. Nutrition and Cancer, Biology and Medicine, 232, 176–183. 53, 104–110. 8 Dashwood, R.H. and Ho, E. (2007) Dietary 19 Luthra, P.M. and Luthra, R. (2002) histone deacetylase inhibitors: from cells Propanthial S-oxide synthase: potential to mice to man. Seminars in Cancer Biology, target to develop flavoursome, 17, 363–369. nonlachrymatory user-friendly onions. 9 Wu, X., Kassie, F. and Mersch- Current Science, 83, 1439–1440. Sundermann, V. (2005) Induction of 20 Sun, X., Guo, T., He, J., Zhao, M.H., Yan, apoptosis in tumor cells by naturally M., Cui, F.D. and Deng, Y.H. (2006) occurring sulfur-containing compounds. Determination of the concentration of Mutation Research, 589,81–102. diallyl trisulfide in rat whole blood using 10 Herman-Antosiewicz, A., Powolny, A.A. gas chromatography with electron-capture and Singh, S.V. (2007) Molecular targets of detection and identification of its major cancer chemoprevention by garlic-derived metabolite with gas chromatography mass organosulfides. Acta Pharmacologica spectrometry. Yakugaku Zasshi-Journal of Sinica, 28, 1355–1364. the Pharmaceutical Society of Japan, 126, 11 Surh, Y.J. (2003) Cancer chemoprevention 521–527. with dietary phytochemicals. Nature 21 Germain, E., Auger, J., Ginies, C., Siess, Reviews Cancer, 3, 768–780. M.H. and Teyssier, C. (2002) In vivo 12 Steudel, R. (2002) The chemistry of metabolism of diallyl disulfide in the rat: organic polysulfanes R-S-n-R (n > 2). identification of two new metabolites. Chemical Reviews, 102, 3905–3945. Xenobiotica, 32, 1127–1138. 13 Jacob, C., Giles, G.I., Giles, N.M. and Sies, 22 Munday, R., Munday, J. and Munday, C.M. H. (2003) Angewandte Chemie, (2003) Comparative effects of mono-, di-, International Edition, 42, 4742–4758. tri-, and tetrasulfides derived from plants 14 Munchberg,€ U., Anwar, A., Mecklenburg, of the allium family: redox cycling in vitro S. and Jacob, C. (2007) Polysulfides as and hemolytic activity and phase 2 enzyme biologically active ingredients of garlic. induction in vivo. Free Radical Biology & Organic & Biomolecular Chemistry, 5, Medicine, 34, 1200–1211. 1505–1518. 23 Dorai, T. and Aggarwal, B.B. (2004) Role of 15 Giles, G.I. and Jacob, C. (2002) Reactive chemopreventive agents in cancer therapy. sulfur species: an emerging concept in Cancer Letters, 215, 129–140. oxidative stress. Biological Chemistry, 383, 24 Reicks, M.M. and Crankshaw, D.L. (1996) 375–388. Modulation of rat hepatic cytochrome 16 Benavides, G.A., Squadrito, G.L., Mills, P-450 activity by garlic organosulfur R.W., Patel, H.D., Isbell, T.S., Patel, R.P., compounds. Nutrition and Cancer, 25, Darley-Usmar, V.M., Doeller, J.E. and 241–248. Kraus, D.W. (2007) Hydrogen sulfide 25 Brady, J.F., Ishizaki, H., Fukuto, J.M., Lin, mediates the vasoactivity of garlic. M.C., Fadel, A., Gapac, J.M. and Yang, C.S. References j683

(1991) Inhibition of cytochrome-P-450 2e1 macrophages. Journal of Agricultural by diallyl sulfide and its metabolites. and Food Chemistry, 54, 3472–3478. Chemical Research in Toxicology, 4, 34 Ban, J.O., Yuk, D.Y., Woo, K.S., Kim, T.M., 642–647. Lee, U.S., Jeong, H.S., Kim, D.J., Chung, 26 Tadi, P.P., Lau, B.H.S., Teel, R.W. and Y.B.,Hwang, B.Y.,Oh, K.W. and Hong, J.T. Herrmann, C.E. (1991) Binding of (2007) Inhibition of cell growth and aflatoxin B1 to DNA inhibited by ajoene induction of apoptosis via inactivation of and diallyl sulfide. Anticancer Research, 11, NF-kappa B by a sulfur compound isolated 2037–2041. from garlic in human colon cancer cells. 27 Wu, C.C., Sheen, L.Y., Chen, H.W., Kuo, Journal of Pharmacological Sciences, 104, W.W., Tsai, S.J. and Lii, C.K. (2002) 374–383. Differential effects of garlic oil and its three 35 Dirsch, V.M., Gerbes, A.L. and Vollmar, major organosulfur components on the A.M. (1998) Ajoene, a compound of hepatic detoxification system in rats. garlic, induces apoptosis in human Journal of Agricultural and Food Chemistry, promyeloleukemic cells, accompanied 50, 378–383. by generation of reactive oxygen 28 Guyonnet, D., Siess, M.H., Le Bon, A.M. species and activation of nuclear factor and Suschetet, M. (1999) Modulation of kB. Molecular Pharmacology, 53, phase II enzymes by organosulfur 402–407. compounds from allium vegetables in rat 36 Singh, S.V. (2001) Impact of garlic tissues. Toxicology and Applied organosulfides on p21(H-ras) processing. Pharmacology, 154,50–58. The Journal of Nutrition, 131, 1046S–1048S. 29 Kensler, T.W.,Curphey, T.J.,Maxiutenko, Y. 37 Ha, M.W., Ma, R., Shun, L.P., Gong, Y.H. and Roebuck, B.D. (2000) and Yuan, Y. (2005) Effects of allitridi on Chemoprotection by organosulfur cell cycle arrest of human gastric cancer inducers of phase 2 enzymes: cells. World Journal of Gastroenterology, 11, dithiolethiones and dithiins. Drug 5433–5437. Metabolism and Drug Interactions, 17,3–22. 38 Hosono, T., Fukao, T., Ogihara, J., Ito, Y., 30 de Ruijter, A.J., van Gennip, A.H., Caron, Shiba, H., Seki, T. and Ariga, T. (2005) H.N., Kemp, S. and van Kuilenburg, A.B. Diallyl trisulfide suppresses the (2003) Histone deacetylases (HDACs): proliferation and induces apoptosis of characterization of the classical HDAC human colon cancer cells through family. The Biochemical Journal, 370, oxidative modification of beta-tubulin. The 737–749. Journal of Biological Chemistry, 280, 31 Druesne, N., Pagniez, A., Mayeur, C., 41487–41493. Thomas, M., Cherbuy, C., Duee, P.H., 39 Jakubikova, J. and Sedlak, J. (2006) Garlic- Martel, P. and Chaumontet, C. (2004) derived organosulfides induce cytotoxicity, Diallyl disulfide (DADS) increases histone apoptosis, cell cycle arrest and oxidative acetylation and p21(waf1/cip1) expression stress in human colon carcinoma cell lines. in human colon tumor cell lines. Neoplasma, 53, 191–199. Carcinogenesis, 25, 1227–1236. 40 Xiao, D., Herman-Antosiewicz, A., 32 Borek, C. (2001) Antioxidant health effects Antosiewicz, J., Xiao, H., Brisson, M., of aged garlic extract. The Journal of Lazo, J.S. and Singh, S.V. (2005) Diallyl Nutrition, 131, 1010S–1015S. trisulfide-induced G(2)-M phase cell cycle 33 Liu, K.L., Chen, H.W.,Wang, R.Y.,Lei, Y.P., arrest in human prostate cancer cells is Sheen, L.Y. and Lii, C.K. (2006) DATS caused by reactive oxygen species- reduces LPS-induced iNOS expression, dependent destruction and NO production, oxidative stress, and hyperphosphorylation of Cdc25C. NF-kappaB activation in RAW 264.7 Oncogene, 24, 6256–6268. 684j 35 Sulfides in Allium Vegetables

41 Xiao, D., Lew, K.L., Kim, Y.A., Zeng, Y., radical-scavenging property of garlic. Hahm, E.R., Dhir, R. and Singh, S.V. Molecular and Cellular Biochemistry, 154, (2006) Diallyl trisulfide suppresses growth 55–63. of PC-3 human prostate cancer xenograft 49 Song, K. and Milner, J.A. (2001) The in vivo in association with Bax and Bak influence of heating on the anticancer induction. Clinical Cancer Research, 12, properties of garlic. The Journal of 6836–6843. Nutrition, 131, 1054S–1057S. 42 Howard, E.W., Ling, M.T., Chua, C.W., 50 Pedraza-Chaverri, J., Medina-Campos, Cheung, H.W., Wang, X. and Wong, Y.C. O.N., Avila-Lombardo, R., Zuniga-Bustos, (2007) Garlic-derived S-allylmercapto A.B. and Orozco-Ibarra, M. (2006) Reactive cysteine is a novel in vivo antimetastatic oxygen species scavenging capacity of agent for androgen-independent prostate different cooked garlic preparations. Life cancer. Clinical Cancer Research, 13, Sciences, 78, 761–770. 1847–1856. 51 Pedraza-Chaverri, J., Medina-Campos, 43 Li, H., Li, H.Q., Wang, Y., Xu, H.X., Fan, O.N. and Segoviano-Murillo, S. (2007) W.T., Wang, M.L., Sun, P.H. and Xie, X.Y. Effect of heating on peroxynitrite (2004) An intervention study to prevent scavenging capacity of garlic. Food and gastric cancer by micro-selenium and large Chemical Toxicology, 45, 622–627. dose of allitridum. Chinese Medical Journal, 52 Hirose, M., Imaida, K., Tamano, S. and 117, 1155–1160. Ito, N. (1994) Cancer chemoprevention by 44 Amagase, H., Petesch, B.L., Matsuura, H., antioxidants. Food Phytochemicals for Kasuga, S. and Itakura, Y. (2001) Intake of Cancer Prevention Ii, 547, 122–132. garlic and its bioactive components. 53 Takada,N.,Matsuda,T.,Otoshi,T., Journal of Nutrition, 131, 955s–962. Yano, Y., Otani, S., Hasegawa, T., Nakae, 45 Shukla, Y. and Pal, S.K. (2004) Dietary D.,Konishi,Y.andFukushima,S. cancer chemoprevention: an overview. (1994) Enhancement by organosulfur International Journal of Human Genetics, 4, compounds from garlic and onions 265–276. of, diethylnitrosamine-induced 46 Bianchini, F. and Vainio, H. (2001) Allium glutathione-S-transferase positive foci in vegetables and organosulfur compounds: the rat-liver. Cancer Research, 54, do they help prevent cancer? 2895–2899. Environmental Health Perspectives, 109, 54 Anwar, A., Burkholz, T., Scherer, C., 893–902. Abbas, M., Lehr, C.M., Diederich, M. and 47 Dorant, E., vandenBrandt, P. and Jacob, C. (2008) Naturally occurring Goldbohm, R.A. (1996) A prospective reactive sulfur species, their implications cohort study on the relationship between for cancer therapy and possible modes of onion and leek consumption, garlic biochemical action. Journal of Sulfur supplement use and the risk of colorectal Chemistry, 29, 251–268. carcinoma in The Netherlands. 55 Jacob, C. and Anwar, A. (2008) Perspective Carcinogenesis, 17, 477–484. on recent developments on sulfur 48 Prasad, K., Laxdal, V.A., Yu, M. and containing agents and hydrogen sulfide Raney, B.L. (1996) Evaluation of hydroxyl signaling. Planta Medica, 74, 1580–1592. j685

36 Glucosinolates and Cruciferous Vegetables L. Adele Boyd, Cris Gill, Tomas Borkowski, and Ian Rowland

36.1 Introduction

36.1.1 Cruciferous Vegetables and Their Components

The family Cruciferae comprises over 350 genera and 3000 species. The primary vegetable crucifers are those of the species Brassica oleracea L., which are the most widely consumed and include a large number of vegetables such as red cabbage (var. rubra); white cabbage (var. alba);cauliﬂower (var. botrytis); savoy cabbage (var. sabauda); Brussels sprouts (var. gammier); broccoli (var. italica); watercress (Rorippa nasturtium-aquaticum); radish (Raphanus sativa L); and mustard greens (B. juncea L., B. nigra L., or B. rapa L.). Such vegetables of the Cruciferae family are widely recognized as sources of valuable micro- and macronutrients as well as a wide range of phytochemicals that may confer a chemoprotective effect against cancer [1].

36.1.2 Physicochemical Properties of Phytochemicals in Cruciferous Vegetables

The most well-studied phytochemicals within cruciferous vegetables (CVs are the glucosinolates (GLS), a class of sulfur- and nitrogen-containing glycosides present in high amounts exclusively within the Cruciferae. The glucosinolates are hydrolyzed by plant myrosinase on injury to the plant, or by intestinal microﬂora, to form isothiocyanates (ITCs), indoles, nitriles, and thiocyanates (Scheme 36.1), which appear to be the bioactive agents [2, 3]. Cruciferae also contain other biologically active compounds including retinol, ascorbic acid, a-tocopherol, lutein, b-carotene, folate, and a range of phenolic compounds [4] (see Table 36.1).

Scheme 36.1 Chemical structures of glucosinolates and their breakdown products following enzymatic hydrolysis by myrosinase.

36.2 Bioavailability and Metabolisms of Active Compounds

The bioavailability of GLS and their bioactive derivatives is highly variable, as it is affected by a wide range of factors that have an impact on the release from food matrix, absorption, distribution, metabolism, and excretion. The initial hydrolysis of GLS is catalyzed by myrosinase, which is released from its cellular compartment during plant tissue disruption by chopping and during chewing. Myrosinase cleaves the glucosidic bond in the glucosinolate, giving rise to glucose and an unstable thiohydroxamate-O-sulfonate. The spontaneous rearrangement of this sulfonate molecule creates a wide range of possible products including ITCs, nitriles and elemental sulfur, thiocyanates, epithionitriles, oxazolidine-2-thiones, or indolyl compounds (Scheme 36.1), the overall proﬁle of which depends on the type of side þ chain, reaction conditions (e.g., presence of Fe2 ions and pH), and myrosinase activity [5]. At neutral pH, ITCs are usually formed, while under acidic or alkaline conditions, nitriles are the major product. Epithionitriles are formed from GLS with unsaturated side chains. It should be noted that processing conditions of vegetables can have a major impact on these processes and alter the proﬁle of products. In particular, inactivation of myrosinase by cooking has a marked effect on absorption (see the following sections). Table 36.1 Structures and sources of dietary glucosinolates and isothiocyanates.

Chemical structure Glucosinolate (R-GLS) Isothiocyanate (R-N¼C¼S) Plant source

a CH2¼CHCH2 2-Propenyl (sinigrin) 2-Propenyl (allyl) Brussels sprouts, cauliﬂower, kale, broccoli, cabbage, horseradish, mustard, radish a CH2¼CHCH2CH2 3-Butenyl (gluconapin) 3-Butenyl Brussels sprouts, kale, rape, Chinese cabbage, turnip greens, green cabbage a CH2¼CHCH2CH2CH2 4-Pentenyl (glucobrassicanapin) 4-Pentenyl Kale, Chinese cabbage, pak-choi, collards, turnip

a Compounds Active of Metabolisms and Bioavailability 36.2 CH2¼CHCH(OH)CH2 2-Hydroxy-3-butenyl (progoitrin) 2-Hydroxy-3-butenyl Brussels sprouts, kale, broccoli, red cabbage, swede SHCH2CH2CH2CH2 4-Mercaptobutyl 4-Mercaptobutyl Rockets, rapeseed CH3SCH2CH2CH2 3-Methylthiopropyl (glucoibervirin) 3-Methylthiopropyl Cabbages, cauliflower CH3SCH2CH2CH2CH2 4-Methylthiobutyl (glucoerucin) 4-Methylthiobutyl Rockets, broccoli, kohlrabi CH3SCH2CH2CH2CH2CH2 5-Methylthiopentyl (glucoberteroin) 5-Methylthiopentyl Cauliflower, cabbage a CH3SCH¼CHCH2CH2 4-Methyltio-3-butenyl (glucoraphasatin) 4-Methyltio-3-butenyl Radish, broccoli, rapeseed, turnip, horseradish CH3 Methyl (glucocapparin) Methyl Cabbage, horseradish, radish CH3CH2CH(CH3) 1-Methylpropyl (glucocochlearin) 1-Methylpropyl Rapeseed, mustard CH3CH(CH3) 1-Methlethyl (glucoputranjivin) 1-Methlethyl Radish, horseradish, mustard b CH2¼CHCH2CH(OH)CH2 2-hydroxy-4-pentenyl (gluconapoleiferin) 2-hydroxy-4-pentenyl Turnip, rapeseed, broccoli b HOCH2CH(CH3) 1-Methyl-2-hydroxyethyl (glucosisymbrin) 1-Methyl-2-hydroxyethyl Radish, mustard CH3SOCH2CH2CH2CH2 4-Methylsulfinylbutyl (glucoraphanin) 4-Methylsulfinylbutyl Broccoli, cabbage, brussels sprouts, (sulforaphane) cauliflower, kale CH3SOCH2CH2CH2CH2CH2 5-Methylsulfinylpentyl (glucoalyssin) 5-Methylsulfinylpentyl Brussels sprouts, broccoli, red cabbage CH3SO[CH2]7 7-Methylsulfinylheptyl (glucoibarin) 7-Methylsulfinylheptyl Watercress CH3SO[CH2]8 8-Methylsulfinyloctyl 8-Methylsulfinyloctyl Watercress

a j CH3SOCH¼CHCH2CH2 4-Methylsulﬁnyl-3-butenyl (glucoraphenin) 4-Methylsulﬁnyl-3-butenyl Radish, broccoli 687 CH3SO2CH2CH2CH2CH2 4-Methylsulphonylbutyl (glucoerysolin) 4-Methylsulphonylbutyl Rapeseed, cabbage (Continued) Table 36.1 (Continued) 688

¼ ¼ j

Chemical structure Glucosinolate (R-GLS) Isothiocyanate (R-N C S) Plant source Vegetables Cruciferous and Glucosinolates 36

C6H5CH2 Benzyl (glucotropaeolin) Benzyl Horseradish, garden cress C6H5CH2CH2 2-Phenethyl (gluconasturtiin) 2-Phenethyl Watercress, radish, turnip p-HOC6H4CH2 4-Hydroxybenzyl (glucosinalbin) 4-Hydroxybenzyl Radish, rapeseed, mustard

Indole-3-methyl (glucobrassicin) Indole-3-methylc Broccoli, brussels sprouts, cauliﬂower, kale, cabbage

4-Methoxy-3-indolylmethyl 4-Methoxy-3-indolylmethylc Cauliﬂower, brussels sprouts, cabbage, (4-methoxyglucobrassicin) broccoli, kohlrabi, swede, radish

4-Hydroxy-3-indolylmethyl 4-Hydroxy-3-indolylmethylc Cauliﬂower, brussels sprouts, cabbage, (4-hydroxglucobrassicin) swede, radish, horseradish

1-Methoxy-3-indolylmethyl 1-Methoxy-3-indolylmethylc Broccoli, brussels sprouts, cauliﬂower, (neoglucobrassicin) cabbage, swede aGlucosinolate with side-chain double bond in the presence of an epithiospeciﬁer protein the ITC may rearrange to produce an epithionitrile. bb-Hydroxy-ITCs are unstable and spontaneously cyclize to oxazolidine-2-thiones (e.g., 5-vinyloxazolidine-2-thione ! goitrin). cIndole glucosinolates break down to unstable indole ITCs that undergo lysis to form corresponding alcohol, such as indole-3-carbinol. The alcohol can condense into di-, tri-, or tetramers. In the presence of ascorbic acid, ascorbigen and thiocyanate are formed. As with methionine-derived glucosinolates, indolyl-3-acetonitrile can be created instead of an unstable ITC. 36.2 Bioavailability and Metabolisms of Active Compounds j689

36.2.1 Gastric and Small-Intestinal Breakdown

Intact GLSs are relatively stable to gastric and small-intestinal conditions, and losses of less than 30% due to hydrolysis have been reported, although binding and adsorption to protein in meals can cause additional losses [6]. Isothiocyanates formed in the food by myrosinase action or during chewing are much less stable than glucosinolates and readily form condensation products and bind to proteins.

36.2.2 Colonic Metabolism

It has been estimated that about 60% of intact GLS from food reaches the colon where it can be hydrolyzed by the resident microﬂora to ITCs to varying extents [6].

36.2.3 Absorption from the Gut

Although there is no conclusive evidence for absorption of intact GLS from the human gut, hydrolysis products appear to be rapidly absorbed, with one study reporting a peak plasma concentration 1 h after ingestion of ITCs [7]. Subsequently, the level declined by ﬁrst-order kinetics with a t½ of 1.77 h, reﬂecting a rapid distribution or metabolism. There is very limited information on the absorption of nitriles and indoles derived from GLS.

36.2.4 Metabolism and Excretion of Hydrolysis Products

Glucosinolate-derived bioactive products undergo phase I and phase II xenobiotic metabolism, mainly in the liver and small intestine, and the corresponding metabolites, rather than the parent compounds, are likely to reach the target tissues in the body, in bioavailable and bioactive form. The metabolism of the isothiocyanates occurs primarily via the mercapturic acid pathway, where the glutathione-conjugated ITCs are modiﬁed by a series of enzymatic reactions to produce N-acetylcysteine dithiocarbamates and excreted in the urine. A number of studies have reported the increased presence of these dithiocarbamates in urine after consumption of cruciferous vegetables in humans, and as such it has been used as an uptake/compliance marker [8]. Organic nitriles are usually detoxiﬁed by sulfur transferases into the less toxic thiocyanate and corresponding aldehyde. Indole-3-carbinol (I3C) is metabolized by mixed function oxidase and alcohol dehydrogenase systems to yield various products, including indole-3-carboxaldehyde, 5-hydroxyindolecarboxaldehyde, and the corresponding carboxylicacid. In mammalian 690j 36 Glucosinolates and Cruciferous Vegetables

cells in culture, substantial amounts of diindolylmethane have been found to accumulate in the nucleus. In general, the major metabolic pathway of structurally different glucosinolate degradation products is conjugation to GSH, then N-acetylation, and subsequent excretion as the corresponding mercapturate.

36.3 Mechanisms of Protection: Results of In Vitro and Animal Studies

36.3.1 Animal Studies

The main biological effects observed in animals after exposure to CVor purified ITCs are changes in enzyme activities, decreased levels of DNA damage, and reductions in colonic aberrant crypt foci (ACF) formation. Aberrant crypt foci, thought to be the earliest morphological changes to occur during colonic mucosal neoplasia, have been observed in the human colon and in rats and mice treated with carcinogens and have been used as an surrogate marker for colon cancer for assessing activity of chemoprotective agents [9]. Garden cress, Brussel sprouts, and red cabbage have been demonstrated to reduce ACF formation in rats induced by the heterocyclic amine; 2-amino-3-methyl-imidazo[4,5-f ]quinoline (IQ); and the DNA-alkylating agents 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM) [10–12]. In addition to these findings, garden cress and its glucosinolate derivatives glucotrapaeolin and benzyl isothiocyanate (BITC) have been shown to reduce IQ-induced genotoxicity almost completely. In an attempt to identify the mechanism behind this antigenotoxic effect, detoxification enzyme activities have also been assessed. Hepatic UDPGT-2 was significantly induced in response to garden cress juice and its constituents suggesting a possible role in the antigenotoxic effects [10]. ACF formation was reduced when rodents were fed vegetable extracts during and after carcinogen exposure, but the reduction appears to be significantly higher in animals fed the extract prior to and/or during carcinogen treatment. These findings support the role of CVs at both the initiation and the postinitiation stages of carcinogenesis. Studies have been carried out to investigate the ability of phenylethylisothiocyanate (PEITC) to protect against ACF formation and DNA adducts, induced by a range of carcinogens [13]. A significant reduction in AOM-induced ACF numbers and in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)–DNA adducts was observed. It has also been shown that PEITC induces Glutathione-S-transferase (GST) activity and GSH content in the colon [14], which could at least in part be responsible for the chemoprotective effects exerted in the digestive tract of rats. Asignificant reduction in levels of PhIP- and IQ-induced DNA adducts in response to preinitiation, postinitiation, and continuous exposure of I3C has been observed. In addition, I3C has also been shown to reduce ACFformation and tumor induction at both the initiation and postinitiation stages of IQ and AOM-induced carcinogenesis [9]. 36.3 Mechanisms of Protection: Results of In Vitro and Animal Studies j691

36.3.2 Effects of Vegetable Extracts and Components at the Cellular and Molecular Level

In vitro studies have focused on a number of end points including modulation of phase I and phase II enzymes and DNA damage, together with proliferation and apoptosis. Several ITCs present in CV,namely, sulforaphane (SFN), BITC, and PEITC have all been shown to act as substrates for four GSTs, namely, A1-1, M1-1, M4-4, and P1- 1 [15]. SFN has been demonstrated to induce phase II enzyme activity and to inhibit benzo(a)pyrene (B[a]P) and H2O2-induced DNA damage in colonic LS-174 cells. The ultimate chemopreventive effects of CVs probably involve complex cellular interactions. In addition to their effects on metabolic enzymes, ITCs have been shown to inhibit cell proliferation by either inducing apoptosis or cell-cycle arrest in colon cancer cell cultures. They also appear to be more toxic toward transformed/malignant cells than normal cells of the colon, suggesting ITCs to be promising new agents in cancer therapy through their selective effects on cancer cells [16]. Apoptosis induction has been observed in many colon cell lines in response to a number of ITCs and indoles including PEITC, BITC, SFN, I3C, and DIM [16–19]. Found in high quantities in Brussel sprouts, AITC has also been shown to cause HT29 cells to become blocked in mitosis; however, the morphological features of these cells showed no sign of apoptosis [19]. Apoptosis, first indicated by the induction of the mitogen-activated protein kinase (MAPK) and jun N-terminal kinase (JNK), leads to mitochondrial cytochrome c release, followed by caspase 3 and 9 activation, before nucleus condensation and DNA fragmentation. These effects have been observed in both HT29 and HCT116 cells [17, 18]. It has been observed that ITCs initiate both P53-dependent and P53-independent apoptosis in colon cells [18]. These effects are of particular interest as most cancer cells exhibiting a mutation, deletion, or inactivation of p53 are resistant to chemopreventive agents. In addition to the induction of caspases 7 and 9, demonstrating the involvement of the mitochondrial pathway, a time- and dose-dependent change in both protein expression of antiapoptotic Bc1-XL and proapoptotic Bax and Bak proteins have been observed after exposure to PEITC, SFN, DIM, and I3C [18]. The glucosinolate hydrolysis products, BITC, PEITC, and N-methoxyindole-3- fi carbinol (NI3C) have been shown to signi cantly increase cell numbers in the G2/M phase of the cell cycle, as well as cause an increase in phosphorylation of the G2/M phase checkpoint enforcer Chk2, p21, and p27 expression [20, 21]. There is recent evidence from human lung cancer cells treated with PEITC, BITC, and SFN that tubulin may be a major binding target for ITC, and that this can lead to apoptosis and mitotic arrest [22]. The ITCs were found to disrupt microtubule polymerization in vitro and in vivo. In conclusion, studies in animal models and in vitro suggest that CVs, containing high levels of ITCs and indoles, may protect against cancer of the colon, partly by modulating detoxification enzymes, resulting in the prevention of initiation/DNA damage, and partly by modulating postinitiation events, in particular, inhibition of 692j 36 Glucosinolates and Cruciferous Vegetables

proliferation and induction of apoptosis. It would appear that chemoprotection by ITCs and indoles occurs via different mechanisms.

36.4 Human Studies

36.4.1 Epidemiology

The majority of studies have focused on the relationship between CVs, glucosinolates and their metabolites, and colorectal cancer, although it should be noted that there is some epidemiological evidence of a protective effect of CVs on other cancer sites, for example, breast [3], bladder [23], prostate [24], and lung [25]. The majority of epidemiological studies, usually case–control studies, evaluating the association between fruit and vegetable consumption and colon cancer risk have reported inverse associations although some recent prospective studies have weakened the association [26]. The association between vegetables and colon cancer appears to be stronger for the dark-green vegetables [27], and among the subgroups of these vegetables, CVs have shown strong negative associations between consumption and colon cancer risk in both sexes [28]. The epidemiological studies evaluating the relationship between CV consumption and colon cancer risk predominantly indicate protective effects. For example, a reduction in the risk that was consistent for both sexes and for both the colon and the rectum (statistically signiﬁcant for colon cancer in females and for rectal cancer in males) was seen with cruciferous vegetable consumption in sample populations of Majorca and northeastern Italy [29, 30]. More recently, epidemiological studies have focused on the relationship between glucosinolate exposure and cancer risk. Moy et al. [31] investigated the association between urinary total ITC level and CRC risk in 225 incident cases of colorectal cancer and 1119 matched controls as part of a cohort of 18 244 men in Shanghai, with 16 years of follow-up. High levels of urinary total ITCs were associated with a reduced risk of colorectal cancer, 5 years after baseline measurements of ITCs. The inverse association became stronger as the duration of follow-up increased, and after 10 years, the ORs for the second, third, and fourth quartiles of urinary ITCs were 0.61, 0.51, and 0.46, respectively, compared to that for the ﬁrst quartile (P for trend ¼ 0.006). The study suggests that dietary ITCs may exert tumor-inhibitory effects, especially during earlier stages of carcinogenesis.

36.4.2 Dietary Intervention Studies

The difﬁculties of assessing the protective effects of dietary regimen, foods, and food constituents on cancer risk in humans have been discussed by Gill and Rowland [32]. As cancer is an impractical end point due to ethical considerations, cost, and duration, studies have focused on intermediate end points (biomarkers). It should 36.4 Human Studies j693 be noted that in contrast to markers for cardiovascular risk, such as serum cholesterol, cancer biomarkers have not been fully validated. Three main end points have been utilized during these studies: phase I and phase II enzyme activities, carcinogen excretion, and antioxidant effects including decreased oxidative DNA damage. Despite their lack of strict validation as cancer biomarkers, these end points have strong mechanistic links with cancer risk.

36.4.2.1 Effects on Phase I and Phase II Enzyme Activities and Carcinogen Excretion in Humans The potential of CVs to induce both phase I and phase II enzymes during carcinogen metabolism in humans has been demonstrated in many studies [33–36]. Cruciferous vegetable consumption has been shown to result in the induction of CYP1A2 activities, which are known to mediate metabolism of certain carcinogens such as PhIP and 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx), which are found in cooked meat and fish. For example, dietary supplementation with CV (subjects ingested 250 g each of Brussels sprouts and broccoli per day) was reported to significantly increase the urinary excretion of N(2)-hydroxy-PhIP-N(2)-glucuronide after the consumption of cooked meat to approximately 130% of levels observed when subjects avoided CV. There was a concomitant increase in hepatic CYP1A2, as demonstrated by changes in saliva caffeine kinetics [36]. In a similar study by the same group [35], CV consumption decreased the urinary excretion of unmetabo- lized MeIQx and PhIP by 21–23% after a cooked meat meal. In addition, there was an increase in urinary mutagenicity during the cruciferous-vegetable phase, although no increase in PhIP–DNA adducts in lymphocytes (an indicator of PhIP activation to a genotoxin) was seen. It seems likely that the increased mutagenicity was due to the breakdown of the conjugated carcinogen metabolites in the mutagenicity assay [35]. Glutathione-S-transferases are members of a large multigene family responsible for the elimination of activated carcinogens from the body by producing highly polar molecules that are readily excreted. GSTs catalyze this detoxification through the conjugation of carcinogens with glutathione (GSH). Low GST activity has been correlated with a higher tumor incidence in the colonic mucosa [37]. Studies in humans have demonstrated that CV consumption can increase GST levels and activity in addition to glutathione levels in lymphocytes. Brassica vegetables (radish, cauliflower, broccoli, and cabbage) have been demonstrated to induce GSTm activity in peripheral lymphocytes [34]. Furthermore, after subjects consumed Brussel sprouts, levels of GSTa and GSTp were increased in plasma and in rectal cells, respectively [38]. The GSTM1 gene deletion is the best-studied polymorphism of GSTand occurs in up to 50% of populations depending on their ethnic background [39]. It has been shown that CVs exert protective effects on colon cancer risk in individuals with a specific genotype; however, these effects depend on a variety of other variables, such as smoking and age [40]. The findings of these studies suggest that the positive genotypes are desirable due to an increase in detoxification of carcinogens in these individuals [41]; however, in those individuals with the null genotypes, ITCs may act 694j 36 Glucosinolates and Cruciferous Vegetables

longer, due to slower excretion, and exert their effects in a way other than detoxiﬁca- tion [15, 42]. In addition to effects on dietary carcinogens, CVs have also been demonstrated to alter the metabolism of cigarette smoke carcinogens, such as the nitrosamine 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in humans. After the consumption of watercress for 2 days, a signiﬁcant increase in urinary levels of NNAL and NNAL-glucuronide was observed [43], suggesting that watercress induces UDP- glucuronosyltransferase activity in humans. In addition, a correlation between the levels of these metabolites and PEITC intake, as measured by total urinary PEITC–- NAC, was observed. These results support the hypothesis that PEITC may inhibit oxidative metabolism of NNK in humans.

36.4.2.2 Antigenotoxic Effects of Cruciferous Vegetables Measuring oxidative DNA damage in human lymphocytes gives an indication of the integrated rate of DNA damage in the body and is suggested to be a potential biomarker for cancer risk. An inverse, though insignificant, association between CV intake (assessed by validated dietary questionnaires) and oxidative DNA damage has been reported [44]. A number of intervention trials in healthy subjects have been conducted involving consumption of Brussels sprouts, watercress, or a sprouting vegetable mixture. DNA damage was assessed by the single-cell gel electrophoresis assay (Comet assay) or by measuring 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodG) excretion (an indicator of oxidative DNA damage). Verhagen et al. [45] investigated the effect on 8-oxodG excretion of consumption of 300 g of cooked Brussels sprouts per day for 3 weeks by five volunteers. Control subjects consumed 300 g of glucosinolate- free vegetables. In the sprouts group, the levels of 8-oxodG were significantly decreased (by 28%) during the intervention period. More recently, these results were confirmed and extended by Hoelzl et al. [46], who investigated the effect of the same dose of Brussel sprouts in eight subjects. Sprout consumption was associated with a significant decrease (97%) in PhIP-induced DNA damage in lymphocytes assessed by the Comet assay. There was no protection against damage induced by another cooked food mutagen, 3-amino-1-methyl-5H-pyrido[4,3- b]-indole (Trp-P-2). There was evidence that the protective effect may be due to the inhibition of sulfotransferase 1A1, which plays a key role in the activation of PhIP. In addition, a decrease of the endogenous formation of oxidized bases was observed, and DNA damage caused by hydrogen peroxide was lower (39%) after the intervention. In a study of the effect of consumption of watercress (85 g/day for 6 weeks) on DNA damage in lymphocytes assessed by the Comet assay, Gill et al. [47] reported a reduction in basal DNA damage (by 17%, in basal plus oxidative purine DNA damage (by 24%)), and in DNA damage induced by ex vivo challenge with hydrogen peroxide (by 9.4%). The beneficial changes seen after watercress intervention were greater and more significant in smokers than in nonsmokers. A good correlation has been observed between the DNA damage occurring in colonocytes and the levels observed in lymphocytes of subjects participating in supplementation studies [48]. Therefore, effects observed in peripheral lymphocytes should be consistent with site-specific effects, such as those seen in the colon. 36.5 Impact of Cooking and Processing j695

36.5 Impact of Cooking and Processing

Storage and industrial and domestic processing of CVs can cause marked changes in glucosinolate levels, but the effects depend on the type of cruciferous vegetable and processing (e.g., temperature, ﬁne chopping versus coarse chopping), as well as on the type of glucosinolate.

36.5.1 Storage and Processing

Storage of broccoli at room temperature markedly decreases levels of the main GLS glucoraphanin and glucobrassicin (50–80% in 5 days), although at 4 C the losses are not as great [49]. Interestingly, indole glucosinolates in broccoli actually increased on storage at 10 C. The impact of various processing methods on GLS levels has been extensively studied, but the results are inconsistent. In general, physical disruption of plants, for example, by chopping, blending, and freezing or thawing, allows mixing of glucosinolates and myrosinase with the formation of isothiocyanates and other breakdown products. In general, this leads to a marked decrease in glucosinolate content by up to 75% [50], although Verkerk et al. [51] reported 15-fold increases in glucosinolate content after chopping and storage of white cabbage, possibly as a response mimicking pest damage. Canning results in substantial thermal degradation of glucosinolates, with losses up to 70%.

36.5.2 Cooking

When cruciferous vegetables are boiled, glucosinolate levels decline signiﬁcantly (up to 75%) with the degree of losses depending on the type of vegetable, time of cooking, amount of water, and glucosinolate type [50]. Microwave cooking also resulted in considerable losses, but steaming and stir-frying retained high levels of the compounds, probably because of reduced leaching of the water-soluble compounds and inactivation of myrosinase in the case of frying [50].

36.5.3 Consequences for Bioavailability

Rungapamestry et al. [52] have investigated the implications of inactivation of myrosinase by cooking for bioavailability of glucosinolates. In human feeding trials, hydrolysis of glucosinolates and absorption of isothiocyanates were more after ingestion of uncooked CVs than after consumption of cooked plants in which myrosinase was inactivated. It appears that cooking affects the site of release of breakdown products of glucosinolates: after consumption of raw vegetables, ITCs are 696j 36 Glucosinolates and Cruciferous Vegetables

produced during chewing and are absorbed mainly in the upper gastrointestinal tract, whereas after consumption of cooked plants, the glucosinolates remain intact until they reach the colon where they are hydrolyzed by the resident microﬂora.

36.6 Conclusions

Results of human, animal, and in vitro studies have provided considerable evidence that CVs and their constituents have the potential to reduce colon cancer risk partly by modulating detoxiﬁcation enzymes, resulting in prevention of initiation/DNA damage, and partly by modulating postinitiation events, in particular, inhibition of proliferation and induction of apoptosis. It has been demonstrated from these studies that the protective effects of these vegetables do, in part, come from their glucosinolate, ITC, and indole content, although it is likely that other components, especially antioxidants such as carotenoids and vitamin C, also play a role.

References

1 Vegetable Crucifers: Status Report. 9 Wargovich, M.J., Chen, C.D., Available at www.arsgrin.gov/npgs/ Jimenez, A., Steele, V.E., Velasco, M., cgc_reports/crucifer1201.htm. Stephen, L.C., Price, R., Gray, K. 2 Keum, Y.S., Jeong, W.S. and Kong, A.N. and Kelloff, G.J. (1996) Cancer (2004) Mutation Research, 555, 191–202. Epidemiology, Biomarkers & Prevention, 3 Ambrosone, C.B., McCann, S.E., 5, 355–360. Freudenheim, J.L., Marshall, J.R., Zhang, 10 Kassie, F., Rabot, S., Uhl, M., Huber, W., Y. and Shields, P.G. (2004) The Journal of Qin, H.M., Helma, C., Schutte-Hermann, Nutrition, 134, 1134–1138. R. and Knassmuller, S. (2002) 4 ONeill, M.E., Carroll, Y., Corridan, B., Carcinogenesis, 23, 1155–1161. Olmedilla, B., Granado, F., Blanco, I., Van 11 Uhl, M., Kassie, F., Rabot, S., den Berg, H., Hininger, I., Rousell, A.M., Grasl-Kraupp, B., Chakraborty, A., Chopra, M., Southon, S. and Thurnham, Laky,B.,Kundi,M.andKnassmuller,S. D.I. (2001) The British Journal of Nutrition, (2004) Journal of Chromatography, 802, 85, 499–507. 225–230. 5 Ludikhuyze, L.L. and Hendrickx, M. (2000) 12 Smith, T.K., Mithen, R. and Johnson, I.T. Journal of Food Protection, 63, 400–403. (2003) Carcinogenesis, 24, 491–495. 6 Maskell, I. and Smithard, R. (1994) The 13 Knasmuller, S., Friesen, M.D., Holme, British Journal of Nutrition, 72, 455–466. J.A., Alexander, J., Sanyal, R., Kassie, F. 7 Ye, L., Dinkova-Kostova, A.T., Wade, K.L., and Bartsch, H. (1996) Mutation Research, Zhang, Y., Shapiro, T.A. and Talalay, P. 350,93–102. (2002) Clinica Chimica Acta, 316,43–53. 14 Van Lieshout, E.M.M., Peters, W.H.M. and 8 Shapiro, T.A., Fahey, J.W., Dinkova- Jansen, J.B.M.J. (1996) Carcinogenesis, 17, Kostova, A.T., Holtzclaw, W.D., 1439–1445. Stephenson, K.K., Wade, K.L., Ye, L. and 15 Kolm, R.H., Danielson, U.H., Zhang, Y., Talalay, P. (2006) Nutrition and Cancer, 55, Talalay, P. and Mannervik, B. (1995) The 53–62. Biochemical Journal, 311, 453–459. References j697

16 Gamet-Payrastrel, L., Lumeau, S., Gasc, N., 29 Benito, E., Obrador, A., Stiggelbout, A., Cassar, G., Rollin, P. and Tulliez, J. (1998) Bosch, F.X., Mulet, M., Munoz, N. and Anti-Cancer Drugs, 9, 141–148. Kaldor, J. (1990) International Journal of 17 Hu, R., Kim, B.R., Chen, C., Hebbar, V.and Cancer, 45,69–76. Kong, A.N.T. (2003) Carcinogenesis, 24, 30 Bidoli, E., Franceschi, S., Talamini, R., 1361–1367. Barra, S. and La Vecchia, C. (1992) 18 Pappa, G., Lichtenburg, M., Iori, R., International Journal of Cancer, 50, 223–229. Barillari, J., Bartsch, H. and Gerhauser, C. 31 Moy, K.A., Yuan, J.M., Chung, F.L., Van (2006) Mutation Research, 599,76–87. Den Berg, D., Wang, R., Gao, Y.T. and Yu, 19 Smith, T.K., Lund, E.K., Clarke, R.G., M.C. (2008) Cancer Epidemiology, Bennett, R.N. and Johnson, I.T. (2005) Biomarkers & Prevention, 17, 1354–1359. Journal of Agricultural and Food Chemistry, 32 Gill, C.I.R. and Rowland, I.R. (2002) The 53, 3895–3901. British Journal of Nutrition, 88, S73–S77. 20 Visanji, J.M., Duthie, S.J., Pirie, L., 33 Kall, M.A., Vang, O. and Clausen, J. (1996) Thompson, D.G. and Padﬁeld, P. J. (2004) Carcinogenesis, 17, 793–799. Journal of Nutrition, 134, 3121–3126. 34 Lampe, J.W., King, I.B., Li, S., Grate, M.T., 21 Neave, A.S., Sarup, S.M., Seidelin, M., Barale, K.V., Chen, C., Feng, Z. and Potter, Duus, F. and Vang, O. (2004) Toxicological J.D. (2000) Carcinogenesis, 21, 1157–1162. Sciences, 83, 126–135. 35 Murray, S., Lake, B.G., Gray, S., Edwards, 22 Mi, L., Xiao, Z., Hood, B.L., A.J., Springall, C., Bowey, E.A., Dakshanamurthy, S., Wang, X., Govind, S., Williamson, G., Boobis, A.R. and Conrads, T.P., Veenstra, T.D. and Chung, Gooderham, N.J. (2001) Carcinogenesis, 22, F.L. (2008) The Journal of Biological 1423–1420. Chemistry, 283 (32), 22136–22146. 36 Walters, D.G., Young, P.J., Angus, L., 23 Tang, L., Zirpoli, G.R., Guru, K., Moysich, Knize, M.G., Boobis, A.R., Gooderham, K.B., Zhang, Y., Ambrosone, C.B. and N.J. and Lake, B.G. (2004) Carcinogenesis, McCann, S.E. (2008) Cancer Epidemiology, 25, 1659–1669. Biomarkers & Prevention, 17, 938–944. 37 Grubben, M.J., Nagengast, F.M., Katan, 24 Kirsh, V.A., Peters, U., Mayne, S.T., Subar, M.B. and Peters, W.H.(2001) Scandinavian A.F., Chatterjee, N., Johnson, C.C. and Journal of Gastroenterology. Supplement, Hayes, R.B. (2007) Journal of the National 234,68–76. Cancer Institute, 99, 1200–1209. 38 Nijhoff, W.A., Grubben, M.J., Nagengast, 25 London, S.J., Yuan, J.-M., Chung, F.-L., F.M., Jansen, J.B., Verhagen, H., Van Gao, Y.-T., Coetzee, G.A., Ross, R.K. and Poppel, G. and Peters, W.H. (1995) Yu, M.C. (2000) Lancet, 356, 724–729. Carcinogenesis, 16, 2125–2128. 26 WCRF (2007) Food, Nutrition Physical 39 Seidgard, J., Varachek, W.R., Pero, R.W. Activity and the Prevention of Cancer: A and Pearson, W.R. (1988) Proceedings of the Global Perspective, World Cancer Research National Academy of Sciences of the United Fund/American Institute for Cancer States of America, 85, 7293–7297. Research. 40 Turner, C.M., Smith, G., Sachse, C., 27 Satia-Abouta, J., Galanko, J.A., Martin, Lightfoot, T., Garner, R.C., Wolf, C.R., C.F., Ammerman, A. and Sandler, R.S. Forman, D., Bishop, D.T. and Barrett, J.H. (2004) International Journal of Cancer, 109, (2004) International Journal of Cancer, 112, 728–736. 259–264. 28 Voorrips, L.E., Goldbohm, R.A., Van 41 Talalay, P. and Fahey, J.W. (2001) Journal of Poppel, G., Sturmans, F., Hermus, R.J.J. Nutrition, 131, 3027S–3033S. and Van den Brandt, P.A. (2000) 42 Seow, A., Yuan, J.M., Sun, C.L., Van Den American Journal of Epidemiology, 152, Berg, D., Lee, H.P. and Yu, M.C. (2002) 1081–1092. Carcinogenesis, 23, 2055–2061. 698j 36 Glucosinolates and Cruciferous Vegetables

43 Hecht, S.S., Carmella, S.G. and Bradbury, I. and Rowland, I.R. (2007) The Murphy, S.E. (1999) Cancer Epidemiology, American Journal of Clinical Nutrition, 85, Biomarkers & Prevention, 8, 907–913. 504–510. 44 Giovanelli, L., Saieva, C., Masala, G., Testa, 48 Pool-Zobel, B.L., Domacher, I., Lambertz, G., Salvini, S., Pitoxxi, V., Riboli, E., Dolara, R., Knoll, M. and Seitz, H.K. (2004) P. and Palli, D. (2002) Carcinogenesis, 23, Mutation Research, 551, 127–34. 1483–1489. 49 Rodrigues, A.S. and Rosa, E.A.S. (1999) 45 Verhagen, H., Poulsen, H.E., Loft, S., van Journal of the Science of Food and Poppel, G., Willems, M.I.and van Bladeren, Agriculture, 79, 1028–1032. P.J. (1995) Carcinogenesis, 16,969–970. 50 Song, L. and Thornalley, P.J. (2007) Food 46 Hoelzl, C., Glatt, H., Meinl, W., Sontag, G., and Chemical Toxicology, 45, 216–224. Haidinger, G., Kundi, M., Simic, T., 51 Verkerk, R., Dekker, M. and Jongen, Chakraborty, A., Bichler, J., Ferk, F., W.M.F. (2001) Journal of the Science of Food Angelis, K., Nersesyan, A. and Knasmuller,€ Agriculture, 81, 953–958. S. (2008) Molecular Nutrition & Food 52 Rungapamestry, V.,Duncan, A.J., Fuller, Z. Research, 52, 330–341. and Ratcliffe, B. (2007) Proceedings of the 47 Gill, C.I.R., Haldar, S., Boyd, A., Bennett, Nutrition Society, 66,69–81. R., Whiteﬁeld, J., Butler, M., Pearson, J., j699

37 Chlorophyll Hikoya Hayatsu, Tomoe Negishi, and Sakae Arimoto-Kobayashi

37.1 Introduction

Humans eat green plants throughout their lives. While chlorophylls do not constitute major nutrients in the diet, green vegetables are regarded as an important item of food that can protect humans against cancer [1]. During the past decades, scientiﬁc research has been performed to ﬁnd clues if these pigments can have preventive effects against cancer and other health deterioration. As a result, these studies have reached the point to show that the green pigment can trap certain kinds of carcinogens, thereby promoting their excretion from the human body. In this chapter, recent progress is described in the light of the information that has been accumulated by the decades-long research efforts from many laboratories worldwide.

37.2 Chemical Nature of Chlorophylls

A shown in Scheme 37.1, chlorophylls have the tetrapyrrole macrocyclic structure. The structure is different from ordinary porphyrins (Scheme 37.2) in that one pyrrole ring has a dihydro structure and that it has a fifth ring with five carbon atoms as the ring constituting elements. Chlorophylls have Mg as the central metal and bear a long-chain aliphatic alcohol esterified to a carboxylate on a side chain. Chlorophylls are unstable toward acids, bases, light, and oxidation. Nearly 100 chlorophylls with different chemical structures are known. In vegetables, the two main chlorophylls, a and b, are present in a ratio of 3 : 1. Chlorophylls a and b are water insoluble. Chlorophylls absorb light intensely: chlorophylls a and b have absorption bands around 440 and 660 nm with molar extinction coefficients (e) of about 100 000. Isolation of reasonably pure chlorophylls from natural sources required a complicated solvent extraction procedure back in the 1960s [3]. Even now, solvent extractions followed by a combination of several column chromatographic procedures are

Scheme 37.1 Structures of chlorophyll and chlorophyllin.

standard for the preparation of individual chlorophylls [2]. There is, however, a recent development by the use of counter current chromatography (CCC). Thus, Jubert and Bailey obtained 300 mg of chlorophyll a and 100 mg of chlorophyll b, both virtually 100% pure, from 450 g fresh spinach by CCC [4].

Scheme 37.2 Structures of protoporphyrin and hemin. 37.4 Basic Studies on Antigenotoxic Activities of Chlorophylls and the Mechanism j701

For various practical purposes, chlorophyllin is used, a stable, water-soluble derivative of chlorophyll. It is produced by hydrolyzing chlorophylls and replacing the central metal magnesium with other metals such as copper and iron (Scheme 37.1). As a sodium salt of the carboxylate residues in the structure, chlorophyllin is easily soluble in water, and is stable toward heat and light. It absorbs visible light strongly: an aqueous solution of chlorophyllin at a concentration of 0.01% is deeply green. Puriﬁed chlorophyllin is not easily obtainable. Commercial chlorophyllin samples contain its derivatives and inorganic salts as contaminants. A full discussion on this issue can be found in the article of Dushwood [5].

37.3 Historical Background

The beneﬁcial effects of chlorophyllin have been recognized almost a century ago, and sodium copper chlorophyllin is being used as medicine for tissue healing and for deodorizing exhalation, and as food additive for coloration. The annual production of chlorophyllin in Japan was 40–50 tons for the year 1992. Orally taken chlorophyllin is known to be safe and ingested chlorophyllin is almost entirely excreted in the feces. A comprehensive review on its safety and fate in the human body has been published by Harrison and coauthors [6]. Chlorophylls, on the contrary, are undoubtedly nontoxic to humans at doses taken from diet, but their behavior within the human body has not been studied intensely. Obviously, the scanty availability and the unstable nature of pure chlorophyll samples have precluded extensive scientiﬁc research. For the purpose of photodynamic therapy of cancer, chlorophyllin derivatives are studied for their possible use as sensitizers, because they can be excited by light to form cytotoxic singlet oxygen. Chlorophylls, on the contrary, are considered to be unsuitable as medicine, for they are too unstable [2].

37.4 Basic Studies on Antigenotoxic Activities of Chlorophylls and the Mechanism of the Actions

In our own laboratory, we started search on antigenotoxic biological compounds in the late 1970s. The experimental methodology we used was the bacterial mutagenicity assays. The first interesting finding [7] was the inhibitory actions of porphyrin compounds, hemin, protoporphyrin (Scheme 37.2), and chlorophyllin, against the mutagenicity of a potent mutagen, Trp-P-1, one of the food pyrolysate heterocyclic amines [8]. It was noted that the þ His mutation of Salmonella typhimurium TA98 in the presence of S9 mix was 50% suppressed by hemin with its amount only 1.8 equivalent to that of the mutagen. Suppression at 100% was observed with 50 M equivalent. Hemin showed similar strong inhibitory activities against polycyclic fl aromatic hydrocarbons and a atoxin B1 [9]. Also, chlorophyllin was antimutagenic in 702j 37 Chlorophyll

Scheme 37.3 A model for a complex between Trp-P-1 and chlorophyllin.

these tests, although the inhibition was somewhat weaker than hemin. By inspecting the chemical structures of the mutagens and comparing them with those of the inhibitors, we concluded that the inhibition could be a result of affinity between the planar surfaces of the mutagen and the porphyrins. Based on several simple experiments, in which we observed precipitate formation in water from hemin and the mutagen, and also noticed a nonadditive nature of their UVabsorption spectra on mixing the two compounds, we postulated that either hemin or protoporphyrin can form a complex with Trp-P-1. This assumption was verified in subsequent experiments (Scheme 37.3). Chlorophylls and chlorophyllin have been investigated for their inhibitory effects against genotoxicity in general. Their inhibitions were in many cases quite strong: as exemplified in Figure 37.1. Generally, polycyclic genotoxicants having planar molecular surfaces are subject to the inhibition by chlorophyllin. Some genotoxicants that do not have such planar structures may, in some studies, be categorized in the inhibition susceptible group, but in these cases, the inhibitions are often not strong. Several reviews have been published on antimutagenic effects of chlorophyllins (for review, see Refs [10–14]). The majority of these studies were conducted at the cellular level. Studies using higher organisms, which have opened up the prospect for human trials, are discussed below (chapter 37.6).

37.5 Chlorophyll–Carcinogen Complex Formation

We observed that the absorption spectrum of a one-to-one mixture of chlorophyll and Trp-P-2 in phosphate buffer at pH 7.4 was different from the estimated sum of the spectra for individual components [15] (Figure 37.2a). With chlorophyllin, in place of chlorophyll, the nonadditive character of the mixture spectrum was more pronounced (Figure 37.2b). This observation constitutes the evidence for complex 37.5 Chlorophyll–Carcinogen Complex Formation j703

Figure 37.1 Inhibition of the mutagenicity of Trp-P-2(NHOH) in Salmonella by chlorophyll and chlorophyllin. Trp-P-2(NHOH), 0.05 nmol, was assayed on S. typhimurium TA98, without metabolic activation, in the presence or absence of the pigments. þ 100% mutagenicity corresponds to 2310 His revertants per plate. The data represent values obtained after subtracting the solvent-control numbers (30 5 revertants per plate).

Figure 37.2 Absorption spectra of Trp-P-2-chlorophyll (or chlorophyllin) mixtures and difference spectra against the calculated sum of spectra for individual components. The solutions contained Trp-P-2 and/or pigment (20 mM each) in 0.1 M sodium phosphate buffer at pH 7.4. (a) —, chlorophyll þ Trp-P-2; - - -, Trp-P-2; ––, chlorophyll; (b) — chlorophyllin þ Trp-P-2; - - -, Trp-P-2; ––, chlorophyllin. 704j 37 Chlorophyll

formation between these planar molecules, chlorophyll and Trp-P-2. Further evidence came from experiments using solid-supported chlorophyllin; that is, we produced chlorophyllin fixed on Sepharose [16] and chitosan [17] and found that carcinogens bearing planar molecular surfaces were adsorbed strongly to the solid- supported chlorophyllin, whereas carcinogens that do not have such structures were not significantly absorbed. Dashwood and his colleagues conducted extensive research on the complex formation of chlorophylls with carcinogens. Based on experimental and theoretical studies, they developed three-dimensional models for complexes of chlorophyllin fl with a atoxin B1 [18] and heterocyclic amines [19]. In this context, it is notable that phthalocyanine, a structure analogue of porphyrin, forms one-to-one complexes with polycyclic planar compounds, and the blue rayon in which copper phthalocyanine trisulfonate is covalently linked as ligand has been used for extracting polycyclic carcinogens from the environment [20].

37.6 Studies with Higher Organisms

As the principal mechanism of the preventive action is the trapping of carcinogens by complex formation, it can be expected that experimental cancers and DNA damage in higher organisms caused by oral administration of carcinogens would be inhibited by chlorophylls. Since chlorophylls are largely unabsorbed through the digestive tract, it can be anticipated that carcinogens in their chlorophyll-complexed forms would be excreted as such without much chance to be absorbed into the organism. We used Drosophila wing spot tests to investigate the mutagenic effects of orally administered agents. By growing larvae with feed containing a carcinogen, it is possible to detect the agents ability to cause mutations in the adult fly wing hairs. We were able to show that hair mutations caused by Trp-P-2 were almost completely eradicated by simultaneous administration of a mix chlorophyll (chlorophylls a and b, 7 : 3) [15]. Also, chlorophyllin caused a strong inhibitory effect. In further studies, chlorophyllin was shown to protect against many other carcinogens that have planar fl molecular structures, for example, a atoxin B1 and benzo(a)pyrene. In addition, Sepharose-supported chlorophyllin was also effective in suppressing mutation induction by these agents, indicating that the mechanism operating in these experiments is the complex formation [21]. Experiments using rodents to explain the chemopreventive actions of chlorophylls have been reported from many laboratories. We reported that the genotoxic actions of Trp-P-2 in mice was suppressed; that is, DNA adduct formation in multiple organs was inhibited up to 90% by coadministration of chlorophyllin-chitosan [22]. Further- more, studies of Hasegawa et al. [23] and of Xu and Dashwood [24] showed that heterocyclic amine-induced carcinogenesis of rat internal organs was effectively inhibited by coadministration of chlorophyllin. A recent study from Baileys labora- fl tory showed that a atoxin B1-induced multiorgan carcinogenesis in rats was strongly 37.7 Studies with Human Subjects j705 inhibited by coadministered chlorophylls isolated from spinach [25]. It was shown that, when chlorophylls were simultaneously given, the aflatoxin excretion into feces increased more than fourfold over that of the control. Furthermore, by using the fluorescence quenching technique, these researchers established the nature of the fl complex as a one-to-one adduct between chlorophyll and a atoxin B1. Among the other rodent trials concerning the preventive effect of chlorophyllin [26–29], several studies on skin tumorigenesis are worthy of note. While an investigation with mice showed protection by topically applied chlorophyllin against 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin papilloma [26], work from another laboratory indicated protection of orally administered chlorophyllin in rats against skin tumor induction by topically applied benzo(a)pyrene [27]. In the latter case, mechanisms other than trapping of carcinogen by chlorophyllin must be operating. In fact, there have been indications that chlorophylls affect the metabolisms of carcinogens, possibly through indirect mechanisms [30, 31]. The most comprehensive as well as venturing trial concerning cancer chemoprevention by chlorophylls is that performed by Bailey and his colleagues with rainbow trouts. Their use of the fish for the assay produced the first demonstration of the anticancer effect of chlorophyllin in vertebrates in mid-1990s [32]. The decades- long exploration has culminated in the recently published fruitful results [33, 34]. Using dibenzo(a,l)pyrene (DBP) as carcinogen, with more than 12 000 rainbow trout, the researchers showed that (1) increasing chlorophyllin coadministration doses produced successive inhibition in liver and stomach DBP–DNA adduct levels at several DBP doses administered; (2) tumor inhibition that resulted from 30–68% was predictable from the adduct response; and (3) animal responses with high doses of DBP were complicated, but indicated critical importance of establishing carcinogen end point dose–response relationships in chemopreventive studies [33]. Using the same carcinogen, DBP, these scientists studied subsequently the protective properties of natural chlorophyll in rainbow trout [34]. They used a 3 : 1 mix of chlorophylls a and b isolated from spinach by the CCC technology [4]. One group of 140 trout was fed with a diet containing DBP (224 ppm) alone, and the second group received a diet containing DBP plus 1000–6000 ppm chlorophyll, for 4 weeks. DBP induced high tumor incidences in stomach (56%) and liver (51%), while cofeeding of chlorophyll resulted in significantly decreased incidences (stomach to 29% with 2000 ppm chlorophyll, 23% with 4000 ppm, and 19% with 6000 ppm; liver to 21% with 2000 ppm, 28% with 4000 ppm, and 26% with 6000 ppm). In addition, an in vitro examination showed that the DBP molecule can form a tight complex with two molecules of chlorophyll, which explains inhibition of the carcinogen uptake into the liver.

37.7 Studies with Human Subjects

In 1997, a large-scale human trial was launched by a joint team of the United States and the Peoples Republic of China [35]. Residents of Qidong, near Shanghai, 706j 37 Chlorophyll

have been exposed to aflatoxins unavoidably through their diet, and the risk for hepatocellular carcinoma is very high in this area. A total of 180 healthy residents participated in the study. They were given tablets containing 100 mg copper chlorophyllin, which were prepared in the United States, and consumed one tablet 20 min before meal, three times/day, for duration of 4 months. Fifty percent of the people for the study took placebo tablets that did not contain chlorophyllin. The chlorophyllin used contained Cu chlorin e4, its ethyl ester, and e6 as major fl components. The end point examined was the urinary content of a atoxin B1-N7- guanine, which is a biomarker of the biologically effective dose of aflatoxin. Analyses were conducted for 169 samples of 12 h urine collected on the 12th week fl of the intervention. The results showed that the levels of a atoxin B1-N7-guanine among the adduct-positive urines (60% of samples from either of the test and control subjects) decreased significantly by the chlorophyllin uptake. The research team, led by Kensler, concluded that prophylactic interventions with chlorophyllin or supplementation of diets with food rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers. A notable observation in this monumental human trial was that many of the participants blood sera turned green. The researchers identified these colors as chlorin e4 ethyl ester and chlorin e4, which were ingredients in the tablets taken by the participants. This finding suggested, according to the report, that the mechanism of chlorophyllin chemoprevention may involve the presence of these components in the serum [36]. The next obvious problem concerning the question was whether natural chlorophylls were eventually effective as chlorophyllin in humans. Bailey announced at a conference in December 2007 that in three individuals who consumed chlorophyll fl pills together with a tiny amount of radiolabeled a atoxin B1 the urinary excretion of aflatoxin was significantly reduced [37]. A follow-up of the work is expected to verify the results, leading to a considerable impact on the human society.

References

1 World Cancer Research Fund/American 4 Jubert, C. and Bailey, G. (2007) Isolation of Institute for Cancer Research (2007) Food, chlorophylls a and b from spinach by Nutrition, Physical Activity and the counter-current chromatography. Journal Prevention of Cancer: A Global Perspective, of Chromatography A, 1140,95–100. AICR, Washington, DC. 5 Dashwood, R.H. (1997) The importance of 2 Grimm, B., Porra, R.J., Rudiger, W. and using pure chemicals in (anti) Scheer, H. (2006) Chlorophylls and mutagenicity studies: chlorophyllin as Bacteriochlorophylls: Biochemistry, a case in point. Mutation Research, 381, Biophysics, Functions and Applications, 283–286. Springer, Dordrecht, The Netherlands. 6 Harrisson, J.W.E., Levin, S.E. and 3 Vernon, L.P. and Seely, G.R. (1966) Trabin, B. (1954) The safety and fate of The Chlorophylls, Academic Press, potassium sodium copper chlorophyllin New York. and other copper compounds. Journal of References j707

the American Pharmaceutical Association, 17 Arimoto-Kobayashi, S., Harada, N., 43, 722–737. Tokunaga, R., Odo, J. and Hayatsu, H. 7 Arimoto, S., Ohara, Y., Namba, T., (1997) Adsorption of mutagens to Negishi, T. and Hayatsu, H. (1980) chlorophyllin-chitosan, an insoluble Inhibition of the mutagenicity of amino form of chlorophyllin. Mutation Research, acid pyrolysis products by hemin and other 381, 243–249. biological pyrrole pigments. Biochemical 18 Breinholt, V., Schimerlik, M., and Biophysical Research Communications, Dashwood, R. and Bailey, G. (1995) 29, 662–668. Mechanisms of chlorophyllin 8 Nagao, M. and Sugimura, T. (2000) Food anticarcinogenesis against aflatoxin B1: Borne Carcinogens: Heterocyclic Amines, complex formation with the carcinogen. John Wiley & Sons Ltd, Chichester, UK. Chemical Research in Toxicology, 8, 506–514. 9 Arimoto, S., Negishi, T. and Hayatsu, H. 19 Dashwood, R., Yamane, S. and Larsen, R. (1980) Inhibitory effect of hemin on the (1996) Study of the forces of stabilizing mutagenic activities of carcinogens. complexes between chlorophylls and Cancer Letters, 11,29–33. heterocyclic amine mutagens. 10 Hayatsu, H., Arimoto, S. and Negishi, T. Environmental and Molecular Mutagenesis, (1988) Dietary inhibitors of mutagenesis 27, 211–218. and carcinogenesis. Mutation Research, 20 Hayatsu, H. (1992) Cellulose bearing 202, 429–446. covalently linked copper phthalocyanine 11 Waters, M.D., Stack, H.F., Jackson, M.A., trisulfonate as an adsorbent selective for Brockman, H.E. and De Flora, S. (1996) polycyclic compounds and its use in Activity profiles of antimutagens: in vitro studies of environmental mutagens and and in vivo data. Mutation Research, 350, carcinogens. Journal of Chromatography, 109–129. 597,37–56. 12 Ferguson, L.R., Philpott, M. and 21 Negishi, T., Nakano, H., Kitamura, A., Karunashighe, N. (2004) Dietary cancer Itome, C., Shiotani, T. and Hayatsu, H. and prevention using antimutagens. (1994) Inhibitory activity of chlorophyllin Toxicology, 198, 147–159. on the genotoxicity of carcinogens in 13 Odin, A.P. (1997) Antimutagenicity of the Drosophila. Cancer Letters, 83, 157–164. porphyrins and non-enzyme porphyrin- 22 Anzai, N., Taniyama, T., Nakandakari, N., containing proteins. Mutation Research, Sugiyama, C., Negishi, T., Hayatsu, H. and 387,55–68. Negishi, K. (2001) Inhibition of DNA 14 Bulmer, A.C., Ried, K., Blanchfield, J.T. adduct formation and mutagenic action of and Wagner, K.-H. (2008) The anti- 3-amino-1-methyl-5H-pyrido[4,3-b]indole mutagenic properties of bile pigments. by chlorophyllin-chitosan in rpsL Mutation Research, 658,28–41. transgenic mice. Japanese Journal of Cancer 15 Negishi, T., Arimoto, S., Nishizaki, C. and Research, 92, 848–853. Hayatsu, H. (1989) Inhibitory effect of 23 Hasegawa, R., Hirose, M., Kato, T., chlorophyll on the genotoxicity of Hagiwara, A., Boonyaphiphat, P., 3-amino--methyl-5H-pyrido[4,3-b]indole Nagao, M., Ito, N. and Shirai, T. (1995) (Trp-P-2). Carcinogenesis, 10, 145–149. Inhibitory effect of chlorophyllin on 16 Arimoto, S., Fukuoka, S., Itome, PhIP-induced mammary carcinogenesis C.,Nakano,H.,Rai,H.andHayatsu, in female F344 rats. Carcinogenesis, H. (1993) Binding of polycyclic 16, 2243–2246. planar mutagens to chlorophyllin 24 Xu, M. and Dashwood, R.H. (1999) resulting in inhibition of the mutagenic Chemoprevention studies of heterocyclic activity. Mutation Research, 287, amine-induced colon carcinogenesis. 293–305. Cancer Letters, 143, 179–183. 708j 37 Chlorophyll

25 Simonich, M.T., Egner, P.A., 32 Breinholt, V., Hendricks, J., Pereira, C., Roebuck, B.D.R., Orner, G.A., Jubert, C., Arbogast, D. and Bailey, G. (1995) Dietary Pereira, C., Groopman, J.D., Kensler, T.W., chlorophyllin is a potent inhibitor of Dashwood, R.H., Williams, D.E. and aflatoxin B1, hepatocarcinogenesis in Bailey, G.S. (2007) Natural chlorophyll rainbow trout. Cancer Research, 55,57–62. inhibits aflatoxin B1-induced multi-organ 33 Pratt, M.M., Reddy, A.P., Hendricks, J.D., carcinogenesis in the rat. Carcinogenesis, Pereira, C., Kensler, T.W. and Bailey, G.S. 28, 1294–1302. (2007) The importance of carcinogen dose 26 Singh, A., Singh, S.P. and Bamezai, R. in chemoprevention studies: quantitative (1996) Modulatory influence of interrelationships between, dibenzo[a,l] chlorophyllin on the mouse skin pyrene dose, chlorophyllin dose, target papillomagenesis and xenobiotic organ DNA adduct biomarkers and final detoxication system. Carcinogenesis, tumor outcome. Carcinogenesis, 28, 17, 1459–1463. 611–624. 27 Park, K.K. and Surh, Y.J. (1996) 34 Simonich, M.T., McQuistan, T., Jubert, C., Chemopreventive activity of chlorophyllin Pereira, C., Hendricks, J.D., against mouse skin carcinogenesis by Schimerlik, M., Zhu, B., Dashwood, R.H., benzo[a]pyrene and benzo[a]pyrene-7,8- Williams, D.E. and Bailey, G.S. (2008) dihydrodiol-9,10-epoxide. Cancer Letters, Low-dose dietary chlorophyll inhibits 102, 143–149. multi-organ carcinogenesis in the rainbow 28 Sugie, S., Okamoto, K., Makita, H., trout. Food and Chemical Toxicology, 46, Ohnishi, M., Kawamori, T., Watanabe, T., 1014–1024. Tanaka, T. and Mori, H. (1996) Inhibitory 35 Egner, P.A., Wang, J.-B., Zhu, Y.-R., effect of chlorophyllin on diethyl- Zhang, B.-C., Wu, Y., Zhang, Q.-N., nitrosamine and phenobarbital-induced Qian, G.-S., Kuang, S.-Y., Gange, S.J., hepatocarcinogenesis in male F344 rats. Jacobson, L.P., Helzlsouer, K.J., Japanese Journal of Cancer Research, 87, Bailey, G.S., Groopman, J.D. and 1045–1051. Kensler, T.W. (2001) Chlorophyllin 29 Xu, M., Orner, G.A., Bailey, G.S., intervention reduces aflatoxin-DNA Stoner, G.D., Horio, D.T. and adducts in individuals at high risk Dashwood, R.H. (2001) Post-initiation for liver cancer. Proceedings of the effects of chlorophyllin and indole-3- National Academy of Sciences of carbinol in rats given 1,2-dimethyl the United States of America, 98, hydrazine or 2-amino-3-methylimidazo 14601–14606. [4,5-f ]quinoline. Carcinogenesis, 22, 36 Egner, P.A., Stansbury, K.H., Snyder, E.P., 309–314. Rogers, M.E., Hintz, P.A. and Kensler, T.W. 30 Fahey, J.W., Stephenson, K.K., (2000) Identification and characterization Dinkovakostova, A.T., Egner, P.A., of chlorin e4 ethyl ester in sera of Kensler, T.W. and Talalay, P. (2005) individuals participating in the Chlorophyll, chlorophyllin and related chlorophyllin chemoprevention trial. tetrapyrroles are significant inducers of Chemical Research in Toxicology, 13, mammalian phase 2 cytoprotective genes. 900–906. Carcinogenesis, 26, 1247–1255. 37 Bailey, G.S. (2007) Cancer 31 Yun, C.H., Jeong, H.G., Jhoun, J.W. and chemoprevention by chlorophylls: from Guengerich, F.P. (1995) Non-specific animal models to humans. The 9th inhibition of cytochrome P450 activities by International Conference on Mechanisms chlorophyllin, in human and rat liver of Antimutagenesis and microsomes. Carcinogenesis, 16, Anticarcinogenesis, Dec. 1–5, Jeju Island, 1437–1440. Korea, Abstracts, p. 53. j709

38 Dietary Fibers Philip J. Harris and Lynnette R. Ferguson

38.1 Introduction

There is a widespread belief that high intakes of dietary fiber (DF) are likely to benefit health, including protection against diseases such as colorectal cancer (CRC). A considerable number of reviews have focused on the arguments as to whether or not DF has been definitively proved to have the necessary beneficial properties [1–4]. A key consideration in such arguments is that DF is not a single entity, but rather a heterogeneous group of materials. Moreover, since DF was originally defined by Trowell [5] as the skeletal remains of plant cells that are resistant to hydrolysis by the enzymes of man, that is, plant cell walls, the materials included in definitions of DF have increased. Most recent definitions include nonstarch polysaccharides (NSPs) in addition to those in plant cell walls, resistant starches (RS), and nondigestible oligosaccharides (NDOs). Some of these recent definitions also include physiological criteria, for example, the definition adopted by the American Association of Cereal Chemists [6, 7]. A similar definition has also been proposed by Codex Alimentarius (a joint FAO/WHO organization) at the 27th session of the Codex Committee on the Nutrition and Foods for Special Uses [8, 9], as follows: Dietary fiber means carbohydrate polymers with a degree of polymerization (DP) not lower than 3 which are neither digested nor absorbed in the small intestine. A degree of polymerization not lower than 3 is intended to exclude mono- and disaccharides. It is not intended to reflect the average DP of a mixture. Dietary fiber consists of one or more of edible carbohydrate polymers naturally occurring in the food as consumed; carbohydrate polymers, which have been obtained from food raw material by physical, enzymatic, or chemical means; and synthetic carbohydrate polymers. Dietary fiber generally has properties such as decrease intestinal transit time and increasing stools bulk; fermentable by colonic microflora; reduce blood total and/or LDL cholesterol levels; and reduce postprandial blood glucose and/or insulin levels. With the exception of nondigestible edible carbohydrate polymers naturally occurring in foods as consumed where a declaration or claim is made with respect to dietary fiber,

a physiological effect should be scientifically demonstrated by clinical studies and other studies as appropriate. The establishment of criteria to quantify physiological effects is left to national authorities. However, the following alternative definition has been proposed by the Joint FAO/WHO Expert Consultation on Carbohydrates in Human Nutrition [10]: Dietary fiber consists of intrinsic plant cell wall polysaccharides. This definition is narrower even than that originally proposed by Trowell [5] in that it includes only the cell wall polysaccharides and not the whole plant cell walls. The intention is to determine dietary fiber as NSP by an enzymatic–chemical method [11, 12] rather than by enzymatic–gravimetric methods. Much of the historic literature makes the implicit assumption that the NSP component is critical to cancer protection. Although polysaccharides are the most abundant component of the cell walls of food plants, other components, particularly phenolic components such as lignin and suberin, which usually occur in only small amounts, may have health benefits, yet they will not be determined. However, many of these components will be quantified using enzymatic–gravimetric methods A footnote to the proposed Codex definition adds When derived from a plant origin, dietary fiber may include fractions of lignin and/or other compounds when associated with polysaccharides in the plant cell walls and if these compounds are quantified by the AOAC gravimetric analytical method for dietary fiber analysis. Fractions of lignin and the other compounds (proteic fractions, phenolic compounds, waxes, saponins, phytates, cutin, phytosterols, etc.) intimately associated with plant polysaccharides are often extracted with the polysaccharides in the AOAC 991.43 method. These substances are included in the definition of fiber insofar as they are actually associated with the poly- or oligosaccharidic fraction of fiber. However, when extracted or even reintroduced into a food containing nondigestible polysaccharides, they cannot be defined as dietary fiber. When combined with polysaccharides, these associated substances may provide additional beneficial effects. Other problems are also associated with the narrower definition of dietary fiber and with using enzymatic–chemical methods for quantification. First, it ignores the indigestible carbohydrate components that have been included in the definitions of dietary fiber such as that proposed by Codex. Second, enzymatic–gravimetric methods are currently used in almost every country in the world. Third, enzymatic–chemical methods cannot distinguish between intrinsic plant cell wall polysaccharides and extracted plant cell wall polysaccharides or other NSPs added to the food. An exception would be where the added NSP has a distinctive monosaccharide composition, for example, glucomannans. The proposed Codex definition recognizes the potential health significance of materials other than NSP that are not hydrolyzed within the human digestive tract. For this reason, the footnote to the definition includes associated materials that are estimated by the AOAC 991.43 total DF method. Although such considerations may appear pedantic, the definition of DF is important, since it determines the range of chemical and structural types to be considered for chemopreventive properties. An accompanying review [13] considers the question as to whether dietary-fiber carbohydrates may prevent CRC. This review focuses on whole plant cell walls, which even today make up most of the DF in Western diets. In particular, we consider 38.2 Physicochemical Properties of Active Compounds and Their Occurrence j711 evidence for a likely role in CRC protection of phenolic components in food plant cell walls.

38.2 Physicochemical Properties of Active Compounds and Their Occurrence

Food plants are composed of a variety of different cell types, but the most abundant are the parenchyma cells. The walls of this cell type are usually thin and composed mostly of NSPs consisting of cellulose and other polysaccharides (noncellulosic polysaccharides) [13]. In most fruits and vegetables, the noncellulosic polysaccharides contain mostly pectic polysaccharides (pectins), with the smaller portions of xyloglucans. However, the cell walls of cereal grains and of certain fruits and vegetables (commelinid monocotyledons), such as pineapple, contain large proportions of heteroxylans. The cell walls of cereal grains also contain (1 ! 3), (1 ! 4)-b-glucans, which are particularly abundant in oats and barley [14]. The heteroxylans have covalently attached to them the phenolic compound ferulic acid (Scheme 38.1), which is a hydroxycinnamic acid. Ferulic acid also occurs covalently attached to pectic polysaccharides in the parenchyma cell walls of those vegetables botanically classified as members of the order Caryophyllales, including beetroot, spinach, and silverbeet. In addition to ferulic acid, other hydroxycinnamic acids occur covalently linked to heteroxylans and, in the Caryophyllales, to pectic polysaccharides [14] (Scheme 38.2). These hydroxycinnamic acids include dimers of ferulic acid (dehydrodiferulic acids) and small proportions of p-coumaric acid (Scheme 38.1). Food plants also contain cell types, usually in small proportions, with walls that contain the polymeric phenolic component lignin. Lignin is covalently linked to the noncellulosic polysaccharides and is a complex, hydrophobic polymer formed by the oxidative polymerization of mostly three types of hydroxycinnamyl alcohol precursors (monolignols) [15]. Cells with lignified walls occur, for example, in certain fruits, such as pears, in the outer cell layers (pericarp) of wheat bran, and in mature asparagus spears. The outer cell layers of wheat bran, which have lignified

Scheme 38.1 Structures of (a) p-coumaric acid (R ¼ H) and ferulic ¼ acid (R OCH3) and (b) an example of a dehydrodiferulic acid (the 5-50 dimer). 712j 38 Dietary Fibers

Scheme 38.2 Structures of cell wall chains of the pectic polysaccharide polysaccharides showing the points of rhamnogalacturonan I [39] and which in plants of attachment of ferulic acid. (a) The heteroxylan the order Caryophyllales have ferulic acid glucuronoarabinoxylan, which occurs in the cell attached to them. Dimers of ferulic acid walls of cereals and other commelinid (dehydrodiferulic acids) and the small amounts monocotyledons; (b and c) arabinans and of p-coumaric acid are attached to these galactans, respectively, that occur as side polysaccharides in the same way as ferulic acid.

walls, have been separated on a large scale from the underlying cell types using a PeriTech debranning system [16]. Suberin, another hydrophobic, polymeric phenolic component occurs covalently linked to noncellulosic polysaccharides in the walls of cork cells that comprise the skins of tubers such as potato. Suberin has a complex structure with a unique polyphenolic domain containing hydroxycinnamates and a polyaliphatic domain, together with associated waxes [17, 18].

38.3 Bioavailability and Metabolism of Active Compounds

Components of dietary ﬁber generally become bioavailable after digestion and fermentation by colonic bacteria. They are not further metabolized after this time.

38.4 Mechanisms of Chemoprevention: Results of In Vitro and Animal Studies

Mechanisms that have been associated with CRC protection by plant cell walls fall into two classes: those involving intact, undigestible cell walls and those involving 38.4 Mechanisms of Chemoprevention: Results of In Vitro and Animal Studies j713 the digestion and fermentation of digestible cell walls such as the walls of most parenchyma cells in food plants [3]. Polymeric phenolic components of plant cell walls have been implicated in mechanisms involving intact, undigestible cell walls. The presence of lignin or suberin in a plant cell wall restricts or completely prevents the digestion and fermentation of the polysaccharides [3]. However, the presence of hydroxycinnamic acids covalently attached to the polysaccharides in parenchyma cell walls does not prevent the digestion of the polysaccharides and the hydroxycinnamic acids are released through digestion and fermentation. It is these compounds that the evidence suggests are implicated in CRC protection.

38.4.1 Lignified or Suberized Cell Walls

The question as to whether particular DFs, or sources of DF, are chemopreventive must consider possible mechanisms related to the multistep development of CRC [19, 20]. The evolution of CRC is defined by the genetic and epigenetic changes, and much of the disease initiation and behavior relates to the accumulation of small mutations. Although some of these may be inherited, others may result from exposure of the gastrointestinal tract, over many years, to dietary mutagens [21]. If dietary mutagens and other types of carcinogens are important, then mechanisms that reduce exposure to such compounds are likely to be protective. Some of the earliest epidemiological studies linked low fecal weights to an increased risk of cancer of the colon [22]. Lignified cell walls or suberized cell walls, which are largely unaltered during their passage through the gut, increase fecal bulk and decrease transit time [3]. This is directly beneficial in bulking and thus effectively diluting carcinogens, as well as reducing the potential contact time for carcinogen exposure of the gastrointestinal tract, by moving the excreta more rapidly through the colon. In vitro studies have shown that isolated plant cell walls (DF) can bind carcinogens [23, 24] and this will further decrease the likelihood of carcinogen absorption in vivo. We compared the abilities of several plant cell wall preparations to adsorb in vitro six known dietary carcinogens all of which were heterocyclic aromatic amines (HAAs). Although a cell wall preparation containing mainly the walls of parenchyma cells adsorbed thecarcinogens poorly,preparations from potato skinsandfrom commercial cork,whichcontainsuberin,adsorbedthemwell.Preparations containinglignifiedcell walls, such as from wheat bran, adsorbed the carcinogens to an intermediate extent [23]. The hydrophobic nature of suberin and lignin is considered to increase the ability of plant cell walls to bind hydrophobic carcinogens. Cereals have been considered as a good source of DF, and most nutritional guidelines would support their consumption. However, it is important to recognize that brans of different cereal species have different adsorptive abilities. For example, Harris et al. [24] compared the abilities of brans from five different cereals to adsorb a known environmental mutagen, 1,8- dinitropyrene (DNP). These brans were obtained from known cultivars using defined milling conditions, and were chemically characterized. The ranking order (most effective to least effective) was as follows: rice, wheat, maize, barley, and oats. 714j 38 Dietary Fibers

As indicated above, our in vitro studies shown that suberized cell walls had the best adsorptive abilities. We therefore tested in Fischer-344 rats the chemopreventive properties of cell wall preparations from potato skins and commercial cork (both at 5%) in a standard animal diet. Both types of suberized cell walls had the ability to protect against aberrant crypts induced by a known dietary carcinogen (the heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) [25]. Wheat bran, which contains lignified cell walls, has also been shown to protect against CRC. A series of studies have related physiological effects of wheat bran-containing diets to CRC protection in animal models. A diet containing 10% wheat bran was shown to protect against the formation of aberrant crypts caused by IQ in Fischer-344 rats [26]. Aberrant crypts were used as a surrogate marker of CRC. This was followed by a mechanistic study in which it was found that the wheat-bran diet significantly modified the absorption, metabolism, and excretion of 14C-labeled IQ administered by gavage [27]. For a short time (up to 2 h) after gavage of a low dose of IQ (1 mg/kg), there were higher concentrations of radioactivity in the plasma of rats fed with wheat bran compared with that of the controls, but lower concentrations thereafter. At all sample times, Kestell and coworkers [27] found lower concentrations of radioactivity in the plasma of rats fed with wheat bran compared with the plasma of control rats following a high dose of IQ (50 mg/kg). The wheat-bran diet significantly retarded the metabolism of IQ in the plasma, at either dose. There were also differences in the nature and concentrations of IQ metabolites in the urine of rats fed with the wheat-bran diet compared with the rats fed with the control diet.

38.4.2 Cell Walls Containing Hydroxycinnamic Acids

Hydroxycinnamic acids, particularly ferulic acid, which occur covalently linked to the wall polysaccharides in the parenchyma cells of certain food plant species (see above), are released into the human colon by microbial hydroxycinnamoyl esterases at the same time that the cell wall polysaccharides are being digested and fermented [28]. The abilities of ferulic, p-coumaric and 5,5-dehydrodiferulic acids to protect against different types of mutation in a bacterial (Salmonella typhimurium) model have been compared [29]. All showed antimutagenic properties, as did the related compound curcumin, and also an extract containing hydroxycinnamic acids obtained by the saponiﬁcation of the cell walls of wheat coleoptiles, a source of parenchyma cells. Three known mutagens, representing different mechanisms of action (bleomycin, hydrogen peroxide, and IQ) were used to chemically induce reversion mutation. Both pure hydroxycinnamic acids and the extract from the cell walls showed ability to reduce mutations by all three mutagens, albeit in bacterial cells. Further studies showed that ferulic and p-coumaric acids could protect against both oxidative stress and genotoxicity in cultured mammalian cells [28]. Both acids also reduced the activity of the xenobiotic metabolizing enzyme cytochrome P-450 1A and downregulated the expression of the cyclooxygenase-2 enzyme. The related compound curcumin is a known anticarcinogen, and at equitoxic doses, the activities of the plant cell wall hydroxycinnamic acids were equal to or superior to those of curcumin. 38.5 Results of Human Studies j715

Thus, providing the plant cell wall hydroxycinnamic acids that can reach effective levels in the colon, they could provide an important mechanism by which DFs in the form of plant cell walls of certain food plants protect against CRC.

38.5 Results of Human Studies

The most compelling observational evidence for relating a dietary intake to disease risk comes from cohort studies in which diet is measured at several time points prior to disease development. Several major cohort studies have related high DF intake to reduced CRC risk, but not all have reached the same conclusion. Some important examples are given below. A multiethnic cohort study [30] involving a total of 85 903 men and 105 108 women, who had completed a quantitative food frequency questionnaire in 1993–1996, indicated an inverse association between DF intake and CRC risk in men, but several confounding factors prevented the data reaching statistical significance for the inverse association in women. The European Prospective Investigation into Cancer and Nutrition (EPIC) cohort study [31] prospectively related DF intake and CRC incidence in 519 978 individuals aged 25–70 years, recruited from 10 European countries. DF in foods was inversely related to the incidence of large bowel cancer (adjusted relative risk 0.75 [95% CI 0.59–0.95] for the highest versus the lowest quintile of intake). The authors suggested that, in populations with low average intake of DF, an approximate doubling of total DF intake from foods could reduce the risk of colorectal cancer by 40%. The USA-based Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial [32] was a randomized controlled trial that estimated DF intake using a 137-item food frequency questionnaire, and related this intake to the incidence of adenoma as assessed by sigmoidoscopy. The authors considered dietary factors for 33 971 participants without polyps, with 3591 cases who had at least one histologically verified adenoma in the distal large bowel. A high intake of DF, particularly from grains and cereals, and fruits, was associated with a decreased risk of adenoma of the distal large bowel. In the Japan Collaborative Cohort Study [33], high DF intake was related to decreased risk of CRC. This prospective study considered food frequency questionnaire data on 43 115 men and women (combined), 40–79 years of age. The study showed a statistically significant trend that related increased DF intake to reduced CRC risk, especially colon cancer. However, Michels et al. [34] challenged the positive conclusions about the effects of DF reached by studies such as those described above. Michels et al. [34] used a different method of analysis in their studies of 76 947 women (in the Nurses Health Study) and 47 279 men (the Health Professionals Follow-up Study). They suggested that there is considerable confounding by other dietary and lifestyle factors and could not agree that high DF intake was protective. They suggested that it may instead be a marker for other dietary and lifestyle habits. This analytical 716j 38 Dietary Fibers

approach was used and the same conclusion reached in a review by Park et al. [35] (from the same research group). Reanalysis of data was also done by Bingham et al. [36]. These authors reanalyzed the EPIC trial data, including the new data from CRC cases that had developed since the initial publications. They questioned whether the observed association of reduced CRC risk with increased DF from foods could be confounded by folate intake. They confirmed a statistically significant inverse association for colon cancer, especially the left-sided colon cancer (P < 0.001). However, when all possible confounders were taken into account, the association between DF and rectal cancer failed to reach statistical significance. Only limited numbers of prospective randomized human clinical trials of DF intake that predict CRC risk have been done. Those available have generally measured adenoma rather than cancer per se. Asano and Mcleod [37] performed a Cochrane collaboration on five such trials that they considered to have valid trial designs. However, since they combined five studies that used very different types of DF, the validity of this analysis may be questioned. Bresalier [38] identified reasons why potential chemopreventive agents, selected on good scientific grounds, may give apparently negative answers in prospective randomized human clinical trials. He pointed to the need to consider what point the agent is being administered relative to the multistep nature of carcinogenesis, the baseline levels of disease risk in the population under study, and the duration of study. He also emphasized the complexity of dietary interactions and the need to consider dose–r- esponse effects. All these factors could be important in terms of DF and CRC risk.

38.6 Impact of Cooking, Processing, and Other Factors on Protective Properties

There are little available data on the impact of cooking on the properties of DF. However, processing could potentially be beneﬁcial. Most plant-based foods contain a mixture of cell types, and processing may provide a good means of separating these to optimize certain properties. For example, we have previously pointed out that wheat bran is a good source of DF, but contains a number of different cell types. Peritech technology permitted fractionation of hard, Australian wheat to produce an aleurone- rich and a pericarp-rich fraction [36]. Rats were fed diets containing 10% of either of these fractions, and cell walls isolated from the feces. While the aleurone walls were partially degraded, there was no evidence of degradation of the pericarp walls. We suspect that either fraction may protect against cancer, but by a different mechanism.

38.7 Conclusions

In the last 30 years, the definition of DF has been incrementally expanded. With the early definitions, increased consumption of DF could only be achieved by increasing References j717 the intake of whole grains, brans, or other food plant material that would increase the intake of plant cell walls containing significant amounts of phenolic components. Our ongoing studies have led us to conclude that suberized and lignified cell walls, especially suberized, may play an important role in the protection against Western diseases, including CRC. Failure to distinguish suberized and lignified plant cell walls from other sources of NSPs may provide a major limitation in current assessments of the role of DF in preventing colorectal cancer in humans. We express a serious concern that some of the new DF definitions make it possible to increase DF without consuming any such materials, potentially with negative connotations for cancer risk in human populations [3].

References

1 Harris, P.J. and Ferguson, L.R. (1993) 10 Mann, J., Cummings, J.H., Englyst, H.N., Dietary fibre: its composition and role in Key, T., Liu, S., Riccardi, G., protection against colorectal cancer. Summerbell, C., Uauy, R., van Dam, R.M., Mutation Research, 290,97–110. Venn, B., Vorster, H.H. and Wiseman, M. 2 Harris, P.J. and Ferguson, L.R. (1999) (2007) FAO/WHO scientific update Dietary fibres may protect or enhance on carbohydrates in human nutrition: carcinogenesis. Mutation Research, conclusions. European Journal of Clinical 443,95–110. Nutrition, 61 (Suppl. 1), S132–S137. 3 Ferguson, L.R., Chavan, R.R. and 11 Englyst, H.N., Quigley, M.E. and Harris, P.J. (2001) Changing concepts Hudson, G.J. (1994) Determination of of dietary fiber: implications for dietary fibre as non-starch polysaccharides carcinogenesis. Nutrition and Cancer, with gas-liquid chromatographic, high- 39, 155–169. performance liquid chromatographic 4 Ferguson, L.R. and Harris, P.J. (2003) or spectrophotometric measurement The dietary fibre debate: more food for of constituent sugars. Analyst, thought. Lancet, 361, 1487–1488. 119, 1497–1509. 5 Trowell, H. (1972) Crude fibre, dietary 12 Englyst, K.N., Liu, S. and Englyst, H.N. fibre and atherosclerosis. Atherosclerosis, (2007) Nutritional characterization and 16, 138–140. measurement of dietary carbohydrates. 6 De Vries, J.W. (2001) The Definition European Journal of Clinical Nutrition, of Dietary Fiber. Cereal Foods World, 61 (Suppl. 1), S19–S39. 46, 112–126. 13 Ferguson, L.R. and Harris, P.J. (2008) 7 De Vries, J.W. (2003) On defining dietary Dietary-fibre carbohydrates and their fibre. Proceedings of the Nutrition Society, fermentation products: do they protect 62,37–43. against colorectal cancer? in 8 Codex Committee on Nutrition and Foods Chemoprevention of Cancer and DNA for Special Dietary Uses (2005) 27th Damage by Dietary Factors session, Bonn, Germany. (edsS.Knasmuller,I.Johnson,D.DeMarini 9 McCleary, B.V. (2007) An integrated and C. Gerhauser), Wiley International. procedure for the measurement of total 14 Harris, P.J. and Smith, B.G. (2006) Plant dietary fibre (including resistant starch), cell walls and cell-wall polysaccharides: non-digestible oligosaccharides and structures, properties and uses in food available carbohydrates. Analytical and products. International Journal of Food Bioanalytical Chemistry, 389, 291–308. Science and Technology, 41, 129–143. 718j 38 Dietary Fibers

15 Ralph, J., Lundquist, K., Brunow, G., Lu, F., 25 Ferguson, L.R. and Harris, P.J. (1998) Kim, H., Schatz, P.F., Marita, J.M., Suberized plant cell walls suppress Hatfield, R.D., Ralph, S.A., formation of heterocyclic amine-induced Christensen, J.H. and Boerjan, W. aberrant crypts in a rat model. Chemico- (2004) Lignins: natural polymers from Biological Interactions, 114, 191–209. oxidative coupling of 4-hydroxyphenyl- 26 Ferguson, L.R. and Harris, P.J. (1996) propanoids. Phytochemistry Reviews, Studies on the role of specific dietary fibres 3,29–60. in protection against colorectal cancer. 16 Harris, P.J. (2005) Diversity in plant Mutation Research, 350, 173–184. cell walls, in Plant Diversity and Evolution: 27 Kestell, P., Zhao, L., Zhu, S., Harris, P.J. Genotypic and Phenotypic Variation in and Ferguson, L.R. (1999) Studies on the Higher Plants (ed. R.J. Henry), CAB mechanism of cancer protection by wheat International Publishing, Wallingford, bran: effects on the absorption, Oxon. metabolism and excretion of the food 17 Bernards, M.A. (2002) Demystifying carcinogen 2-amino-3-methylimidazo suberin. Canadian Journal of Botany, [4,5-f]quinoline (IQ). Carcinogenesis, 80, 227–240. 20, 2253–2260. 18 Bernards, M.A., Summerhurst, K.K. and 28 Ferguson, L.R., Zhu, S.T. and Harris, P.J. Razem, F.A. (2004) Oxidases, peroxidases (2005) Antioxidant and antigenotoxic and hydrogen peroxide: the suberin effects of plant cell wall hydroxycinnamic connection. Phytochemistry Reviews, acids in cultured HT-29 cells. Molecular 3, 112–126. Nutrition & Food Research, 49, 585–593. 19 Bodmer, W.F. (2006) Cancer genetics: 29 Ferguson, L.R., Lim, I.F., Pearson, A.E., colorectal cancer as a model. Journal of Ralph, J. and Harris, P.J. (2003) Bacterial Human Genetics, 51, 391–396. antimutagenesis by hydroxycinnamic 20 Jass, J.R. (2007) Classification of colorectal acids from plant cell walls. Mutation cancer based on correlation of clinical, Research, 542,49–58. morphological and molecular features. 30 Nomura, A.M., Hankin, J.H., Henderson, Histopathology, 50, 113–130. B.E., Wilkens, L.R., Murphy, S.P., Pike, 21 Ferguson, L.R. and Philpott, M. (2008) M.C., Le Marchand, L., Stram, D.O., Nutrition and Mutagenesis. Annual Review Monroe, K.R. and Kolonel, L.N. (2007) of Nutrition, 28, 313–329. Dietary fiber and colorectal cancer risk: 22 Burkitt, D.P., Walker, A.R. and Painter, the multiethnic cohort study. Cancer N.S. (1972) Effect of dietary fibre on stools Causes & Control, 18, 753–764. and the transit-times, and its role in the 31 Bingham, S.A., Day, N.E., Luben, R., causation of disease. Lancet, 2, Ferrari, P., Slimani, N., Norat, T., 1408–1412. Clavel-Chapelon, F., Kesse, E., Nieters, A., 23 Harris, P.J., Triggs, C.M., Roberton, A.M., Boeing, H., Tjonneland, A., Overvad, K., Watson, M.E. and Ferguson, L.R. (1996) Martinez, C., Dorronsoro, M., The adsorption of heterocyclic aromatic Gonzalez, C.A., Key, T.J., Trichopoulou, A., amines by model dietary fibres with Naska, A., Vineis, P., Tumino, R., contrasting compositions. Chemico- Krogh, V., Bueno-de-Mesquita, H.B., Biological Interactions, 100,13–25. Peeters, P.H., Berglund, G., Hallmans, G., 24 Harris, P.J., Sasidharan, V.K., Lund, E., Skeie, G., Kaaks, R., Riboli, E. and Roberton, A.M., Triggs, C.M., European Prospective Investigation into Blakeney, A.B. and Ferguson, L.R. (1998) Cancer and Nutrition (2003) Dietary fibre Adsorption of a hydrophobic mutagen to in food and protection against colorectal cereal brans and cereal bran dietary fibres. cancer in the European Prospective Mutation Research, 412, 323–331. Investigation into Cancer and Nutrition References j719

(EPIC): an observational study. Lancet, Miller, A.B., Pietinen, P., Rohan, T.E., 361, 1496–1501. Schatzkin, A., Willett, W.C., Wolk, A., 32 Peters, U., Sinha, R., Chatterjee, N., Zeleniuch-Jacquotte, A., Zhang, S.M. and Subar, A.F., Ziegler, R.G., Kulldorff, M., Smith-Warner, S.A. (2005) Dietary fiber Bresalier, R., Weissfeld, J.L., Flood, A., intake and risk of colorectal cancer: a Schatzkin, A., Hayes, R.B. and pooled analysis of prospective cohort Prostate, Lung, Colorectal, and Ovarian studies. Journal of the American Medical Cancer Screening Trial Project Team Association, 294 2849–2857. (2003) Dietary fibre and colorectal 36 Bingham, S.A., Norat, T., Moskal, A., adenoma in a colorectal cancer early Ferrari, P., Slimani, N., Clavel-Chapelon, detection programme. Lancet, 361, F., Kesse, E., Nieters, A., Boeing, H., 1491–1495. Tjonneland, A., Overvad, K., Martinez, C., 33 Wakai, K., Date, C., Fukui, M., Dorronsoro, M., Gonzalez, C.A., Tamakoshi, K., Watanabe, Y., Ardanaz, E., Navarro, C., Quiros, J.R., Hayakawa, N., Kojima, M., Kawado, M., Key, T.J., Day, N.E., Trichopoulou, A., Suzuki, K., Hashimoto, S., Tokudome, S., Naska, A., Krogh, V., Tumino, R., Palli, D., Ozasa, K., Suzuki, S., Toyoshima, H., Panico, S., Vineis, P., Bueno-de-Mesquita, Ito, Y., Tamakoshi, A. and Group, J.S. H.B., Ocke, M.C., Peeters, P.H., (2007) Dietary fiber and risk of colorectal Berglund, G., Hallmans, G., Lund, E., cancer in the Japan collaborative cohort Skeie, G., Kaaks, R. and Riboli, E. (2005) study. Cancer Epidemiology, Biomarkers & Is the association with fiber from foods in Prevention, 16, 668–675. colorectal cancer confounded by folate 34 Michels, K.B., Fuchs, C.S., intake? Cancer Epidemiology, Biomarkers & Giovannucci, E., Colditz, G.A., Prevention, 14 1552–1556. Hunter, D.J., Stampfer, M.J. and 37 Asano, T. and McLeod, R.S. (2002) Willett, W.C. (2005) Fiber intake and Dietary fibre for the prevention of incidence of colorectal cancer among colorectal adenomas and carcinomas. 76,947 women and 47,279 men. Cancer Cochrane Database of Systematic Reviews, Epidemiology, Biomarkers & Prevention, CD003430. 14, 842–849. 38 Bresalier, R.S. (2008) Chemoprevention of 35 Park, Y., Hunter, D.J., Spiegelman, D., colorectal cancer: why all the confusion? Bergkvist, L., Berrino, F., van den Brandt, Current Opinion Gastro, 24,48–50. P.A., Buring, J.E., Colditz, G.A., 39 Harris, P.J., Chavan, R.R. and Freudenheim, J.L., Fuchs, C.S., Ferguson, L.R. (2008) Production and Giovannucci, E., Goldbohm, R.A., characterisation of two wheat-bran Graham, S., Harnack, L., Hartman, A.M., fractions: an aleurone-rich and a pericarp- Jacobs, D.R. Jr, Kato, I., Krogh, V., rich fraction. Molecular Nutrition & Food Leitzmann, M.F., McCullough, M.L., Research, 49, 536–545. j721

39 Dietary Fiber Carbohydrates and their Fermentation Products Lynnette R. Ferguson and Philip J. Harris

39.1 Introduction

Despite the long-held belief that dietary fibers (DFs) protect against colorectal cancer (CRC), the topic has more recently become controversial [1]. An important consideration in such discussions is the definition of DF. Initially, the term was applied to only plant cell walls in food, but the meaning has since been expanded to include all nonstarch polysaccharides (NSPs), resistant starches (RSs), and nondigestible oligosaccharides (NDOs). The definition of DF is discussed in an accompanying review [2]. In this chapter, we focus on whether the dietary fiber carbohydrates, including all NSPs, RSs, and NDOs, are likely to play a role in cancer prevention. All these dietary fiber carbohydrates escape digestion in the small intestine and act as substrates for bacterial growth and metabolism in the cecum and colon where they are partly or completely digested and fermented in the large bowel, resulting in the production of short-chain fatty acids (SCFAs), succinate, hydrogen, carbon dioxide, and methane. A number of these products have been ascribed cancer preventive properties, with varying degrees of evidence. The products have a variety of effects, including the acidification of the lumen contents, and the modification of xenobiotic metabolizing enzyme (XME) activities [3]. A number of authors [4] have focused attention on the production of SCFAs, especially butyrate. Another area of increasing interest is the prebiotic properties of dietary fiber carbohydrates to modify the composition of the colonic microbiota, as reviewed by Lim et al. [5].

39.2 Physicochemical Properties of Active Compounds and their Occurrence

Dietary ﬁber carbohydrates include the polysaccharides in intact plant cell walls, NSPs used as food additives (such as gelling agents, thickeners, stabilizers, and emulsiﬁers), RSs, and NDOs. The polysaccharides in intact plant cell walls are usually

the most abundant dietary fiber carbohydrates. Although plant cell walls vary greatly in their compositions depending on cell type and plant species, they all have a similar basic construction [6]. They have a fibrillar phase of cellulose microfibrils embedded in a matrix usually composed mostly of noncellulosic polysaccharides with a variety of different structures (Scheme 39.1). In the majority of fruits and vegetables (eudicotyledons and noncommelinid monocotyledons), most of the noncellulosic polysaccharides are typically pectins, with smaller proportions of xyloglucans (Scheme 39.1). However, in some fruits and vegetables (commelinid monocotyledons), for example, pineapples, heteroxylans (glucuronoarabinoxylans) are the major noncellulosic polysaccharides (Scheme 39.1). In cereal grains, the noncellulosic polysaccharides are predominantly heteroxylans (arabinoxylans) in wheat and (1 ! 3), (1 ! 4)-b-glucans in oats and barley (Scheme 39.1).

Scheme 39.1 Structures of common plant cell heteroxylan found in the primary cell walls of wall polysaccharides. (a) Cellulose; (b) and (c) commelinid fruits and vegetables (the homogalacturonan and rhamnogalacturonan arabinoxylans in cereal cell walls have a similar 1 (RG-1), the most abundant types of structure but contain no, or only small polysaccharides in pectin; (d) xyloglucan; proportions of, glucuronic acid); (f) (1 ! 3), (e) glucuronoarabinoxylan (GAX), the type of (1 ! 4)-b-glucan. 39.2 Physicochemical Properties of Active Compounds and their Occurrence j723

NSPs used as food additives are obtained from a variety of sources. They include polysaccharides obtained from plant cell walls, for example, cellulose from wood pulp and cotton linters (the short fibers remaining on cotton seeds after the long fibers have been removed), pectin from lemons, limes, and apples, and a number of polysaccharides, for example, galactomannans (such as guar gum) and xyloglucans, from the thick cell walls of a number of leguminous seeds [7]. They also include gums secreted by plants (exudate gums), such as gum arabic, and mucilages secreted by the outer layer (epidermis) of certain seeds, such as psyllium (ispaghula) from Plantago ovata. They are also obtained from the cell walls of seaweeds, for example, agar and carrageenans, and are synthesized by microorganisms, such as xanthan and gellan gums. Except for cellulose, all these NSPs are water soluble and have been referred to as soluble fiber polysaccharides. RS is the sum of starch and products of starch degradation not absorbed in the small intestine of healthy individuals. Four forms of RS (RS1, 2, 3 and 4) are now often recognized [8, 9]. The first form, RS1, occurs where starch granules are physically entrapped within plant cells and are inaccessible to a-amylases; it is present in seeds and partly milled cereal grains. RS2 is represented by native (raw) starch granules that are highly resistant to a-amylases because of their crystallinity. Such starch granules occur in only some food plants, for example, potatoes, green banana fruit, and high-amylose cultivars of maize. The third form, RS3, is retro- graded starch formed when gelatinized starch, formed by heating starch granules, is cooled. This form occurs for example in cooled cooked potatoes and in bread. A forth form, RS4, is now commonly recognized and comprises chemically modified starches (etherized, esterified, or cross-linked) often used in processed foods. Each of these forms of RS has different characteristics and is likely to have different physiological effects in the colon. NDOs are resistant to digestion in the human small intestine, but are degraded by bacteria in the colon. NDOs are all water soluble and include fructans, comprising inulin and fructooligosaccharides (FOSs) (Scheme 39.2), which occur in a variety of food plants such as onions and garlic, and are extracted commercially from chicory roots and Jerusalem artichoke tubers for use in the food industry [10]. NDOs also include the a-galactosides raffinose, stachyose, verbascose, and ajugose (Scheme 39.2) that occur in the seeds of many leguminose plants [11], as well as synthetic molecules such as polydextrose [12].

Scheme 39.2 Structures of common NDOs. (a) and (b) Isokestose and nystose, two fructooligosaccharides of the inulin type; (c) and (d) raffinose and stachyose, two a-galactosides. 724j 39 Dietary Fiber Carbohydrates and their Fermentation Products

39.3 Bioavailability and Metabolism of Active Compounds

Components of dietary ﬁber carbohydrates generally become bioavailable after digestion and fermentation by colonic bacteria. They are not further metabolized after this time.

39.4 Mechanisms of Chemoprevention: Results of In Vitro and Animal Studies

39.4.1 Hypotheses Suggesting that SCFAs are Protective

Wong et al. [13] reviewed the role in colonic health of fermentation and the main SCFAs produced by this (acetate, propionate, and butyrate). Each of the three is utilized differently: acetate enters the bloodstream and is metabolized by peripheral tissues, most of the propionate is taken up by the liver, and butyrate is the major energy source for colonocytes. The rate of production of the total SCFA and the relative amounts of the different acids depend on the particular type or source of NSP, RS, or NDO, on the colonic microbial species and their numbers, and on gut transit time. For example, one of the reasons that RS have been suggested as chemopreventive is that, compared to NSPs, their digestion and fermentation result in a higher molar proportion of butyrate. Ferguson et al. [14] replaced normal maize starch in the diet of rats with three preparations of RS2 (native potato starch, native high-amylose maize starch, and an a-amylase-treated native high-amylose maize starch). In addition to quantifying the production of SCFA in the cecum, they estimated the effects on fecal bulking and transit time, both of which have also been suggested as protecting against colorectal cancer. Although all the RS preparations had major effects on the fecal weight and on the weight of the cecum, they only slightly shortened the transit times. All increased the amount of starch reaching the cecum and SCFAproduction in the order (lowest to highest effect) high-amylose maize starch, a-amylase-treated high-amylose maize starch, and native potato starch. Native potato starch, but not high-amylose maize starch, enhanced the proportion of butyrate. Thus, even though these preparations are all classiﬁed as RS2, there were marked differences among them in relation to effects suggested as having signiﬁcance for colorectal cancer prevention.

39.4.2 The Butyrate Hypothesis

Of the SCFAs, butyrate is recognized as the main energy source for colonocytes. Many in vitro studies have shown various effects of butyrate that have been suggested as being consistent with chemoprevention [13]. For example, butyrate stimulates cell differentiation, cell cycle arrest, and apoptosis and has also been suggested as having 39.4 Mechanisms of Chemoprevention: Results of In Vitro and Animal Studies j725 epigenetic effects through inhibition of the histone deacetylase enzyme. Butyrate also decreases the transformation of the primary into secondary bile acids. Repeatedly, statements of the following sort appear in the literature: Butyrate is a key bioactive product of dietary ﬁber fermentation thought to play a key role in cancer prevention. One contributory mechanism in this role is the regulation of apoptosis by butyrate [15]. These authors go on to describe a novel mechanism that may explain how butyrate can induce apoptosis. Perhaps, what is more controversial is whether apoptosis is actually a chemoprevention mechanism. Although apoptosis is unquestionably a mechanism of tumor cell kill, DAgnostini et al. [16] review much evidence suggesting that apoptosis is not a good mechanism of chemoprevention. The butyrate hypothesis may thus be a good example of how mechanistic studies using in vitro models often fail to reﬂect the multidimensional nature of the gut.

39.4.3 Prebiotic Effects

Prebiotics have been defined as food ingredients that are largely undegraded in the small bowel and can beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria [17]. A number of NSP, RS, and NDO have been claimed to have this effect. For example, FOS has been reported to reduce colonic inflammation significantly and increase the numbers of Lactobacillus and Bifidobacterium cells and species [18, 19]. Used in combination with Lactobacillus paracasei, FOS has been reported to increase the numbers of Lactobacillus and Bifidobacterium cells and species in the colon, while reducing the numbers of pathogenic Clostridium and Enterobacterium [20]. However, materials usually regarded as prebiotics have also been shown to have negative effects. For example, at high doses, FOS ingestion increases intraluminal osmolarity, leading to diarrhoea as a common side effect [21]. Lim et al. [5] point to a concern that the evidence for sustainable human health benefits of prebiotics is missing at present.

39.4.4 Evidence from Animal Models

We have previously referred to studies in rodent models using chemical induction of colon cancer that show variable effects of different DFs or DF sources [8, 22]. In general, these studies showed protection against CRC with DF sources such as wheat or rye bran where the plant cell walls (DF) contain phenolic compounds, whereas other NSPs, including pectin, carageenan, agar, psyllium, and guar gum, enhanced rather than reduced tumor formation. Both the NDO inulin and the NSP pectin þ increased the number of intestinal polyps in ApcMin/ mice [23, 24], whereas rye bran reduced the polyp number in the same model [25]. If rodents are fed an elemental diet for a long time, their colon starts to atrophy [26]. Most studies on NSP, RS, or NDO have related high levels of fermentation with increased cell proliferation, and it has been suggested that this may prevent colonic atrophy and be an important function of SCFAs [4]. Where the evidence is available, 726j 39 Dietary Fiber Carbohydrates and their Fermentation Products

as in Mandir et al. [27], the data support the conclusion that increased digestion and fermentation increases the probability of increased cell division. However, this effect may be more likely to enhance rather than reduce carcinogenesis. Mandir et al. [27] þ used an ApcMin/ mouse model of intestinal cancer and directly examined the ability of different fermentable DF preparations to stimulate colonic cell division, in comparison with their effects on CRC. Although a slight amount of atrophy (as seen in the mice fed on control diets lacking DF) did not enhance carcinogenesis or appear detrimental to the health of the rodents, those DF preparations that stimulated cell division also substantially increased the incidence of intestinal carcinogenesis. This is also consistent with the epidemiologic data [28]. However, we note that in humans colonic atrophy may be a problem only in rare situations, for example, in subjects with inﬂammatory bowel diseases on an elemental diet [29].

39.5 Results of Human Studies

Whether or not epidemiologic studies show that DF is protective against CRC depends not only upon the evidence (which is generally positive), but also upon the method of statistical analysis. Thus, both the EPIC study of cancer risk factors across Europe and a large meta-analysis of a number of studies initially showed that DF was strongly protective [30, 31]. However, further analyses, which were claimed necessary because of possible confounding by other dietary risk factors, led to a loss of statistical significance [31, 32]. It should be noted, however, that this conclusion has been challenged [33]. Where there is likely to be confounding by other risk factors, the only way to establish proof is a randomized clinical trial. A Cochrane review [34] found that only five trials reached their standards for analysis. Of these, three used some type of wheat bran-based supplement, one used counseling to increase DF intake, and only one tested an NSP source in the form of 3.5 g per day of psyllium husk as a DF intervention [35]. All five studies identified by Asano and McLeod [34] gave data on the recurrence of at least one adenoma, but only one of these studies showed statistically significant differences in adenoma recurrence between the intervention and control arms of the study. The intervention using psyllium husk significantly increased, rather than decreased, the risk of adenoma recurrence. It should be noted, however, that an adenoma is a biomarker of cancer but is not itself formally classified as cancer.

39.6 Impact of Cooking, Processing, and other Factors on Protective Properties

It is well recognized that different food processing technologies may maintain, enhance, or destroy the concentrations of NSP, RS, or NDO in a raw material or food [36]. There is also increasing evidence that the presence of other food components may modify the action of NSP, RS, or NDO and their fermentation References j727 in the colon [37]. Morita and coworkers [37] reviewed the way in which proteins and peptides can modulate the digestion and fermentation of NSP or RS. Rats fed on a diet containing 20% high-amylose corn starch and with casein as the sole protein source produced very much higher amounts of cecal SCFA compared to rats fed on a diet containing normal cornstarch. When the casein was substituted with rice or potato protein, cecal succinate production decreased and cecal butyrate increased. The authors suggested that different protein sources may play a role in correcting an imbalance in the ratio of carbohydrate and nitrogen as fermentative substrates for cecal bacteria. In additional, they may promote butyrate production.

39.7 Conclusions

Many of the publications claiming beneficial effects of the dietary fiber carbohydrates NSP, RS, or NDO use in vitro models that fail to duplicate the complexity of the human colon. We suggest that the only way to answer some of the current questions is to establish double-blind, placebo-controlled clinical trials to examine systematically specific types of dietary fiber carbohydrates.

References

1 Lawlor, D.A. and Ness, A.R. (2003) nonstarch polysaccharides. Physiological Commentary: the rough world of Reviews, 81, 1031–1064. nutritional epidemiology: does dietary 5 Lim, C.C., Ferguson, L.R. and Tannock, fibre prevent large bowel cancer? G.W. (2005) Dietary fibres as prebiotics: [comment]. International Journal of implications for colorectal cancer. Epidemiology, 32, 239–243. Molecular Nutrition & Food Research, 2 Harris, P.J. and Ferguson, L.R. (2008) 49, 609–619. Dietary fibres, in Chemoprevention of 6 Harris, P.J. (2006) Primary and secondary Cancer and DNA Damage by Dietary Factors plant cell walls: a comparative overview. (eds S. Knasmuller,€ I. Johnson, D. De New Zealand Journal of Forestry Science, Marini and C. Gerhauser), Wiley-VCH 36,36–53. Verlag GmbH, Weinheim. 7 Harris, P.J. and Smith, B.G. (2006) Plant 3 Kestell, P., Zhao, L., Zhu, S., Harris, P.J. cell walls and cell-wall polysaccharides: and Ferguson, L.R. (1999) Studies on the structures, properties and uses in food mechanism of cancer protection by wheat products. International Journal of Food bran: effects on the absorption, Science and Technology, 41, 129–143. metabolism and excretion of the food 8 Ferguson, L.R., Chavan, R.R. and Harris, carcinogen 2-amino-3-methylimidazo P.J. (2001) Changing concepts of dietary [4,5-f]quinoline (IQ). Carcinogenesis, fiber: implications for carcinogenesis. 20, 2253–2260. Nutrition and Cancer, 39, 155–169. 4 Topping, D.L. and Clifton, P.M. (2001) 9 Englyst, H.N., Kingman, S.M. and Short-chain fatty acids and human colonic Cummings, J.H. (1992) Classification and function: roles of resistant starch and measurement of nutritionally important 728j 39 Dietary Fiber Carbohydrates and their Fermentation Products

starch fractions. European Journal of of fructooligosaccharides on intestinal Clinical Nutrition, 46 (Suppl. 2), S33–S50. flora and human health. Bifidobacteria 10 Flamm, G., Glinsmann, W., Kritchevsky, Microflora, 5,37–50. D., Prosky, L. and Roberfroid, M. (2001) 20 Bomba, A., Nemcova, R., Gancarcikova, S., Inulin and oligofructose as dietary fiber: a Herich, R., Guba, P. and Mudronova, D. review of the evidence. Critical Reviews in (2002) Improvement of the probiotic effect Food Science and Nutrition, 41, 353–362. of micro-organisms by their combination 11 Martinez-Villaluenga, C., Frias, J. and with maltodextrins, fructo-oligosaccharides Vidal-Valverde, C. (2008) Alpha- and polyunsaturated fatty acids. The British galactosides: antinutritional factors or Journal of Nutrition, 88 (Suppl. 1), S95–S99. functional ingredients? Critical Reviews in 21 Lara-Villoslada, F., de Haro, O., Camuesco, Food Science and Nutrition, 48, 301–316. D., Comalada, M., Velasco, J., Zarzuelo, A., 12 Craig, S.A.S., Holden, J.F., Troup, J.P., Xaus, J. and Galvez, J. (2006) Short-chain Auerbach, M.H. and Frier, H.I. (1998) fructooligosaccharides, in spite of being Polydextrose as soluble fiber: physiological fermented in the upper part of the large and analytical aspects. Cereal Foods World, intestine, have anti-inflammatory activity 43, 370–376. in the TNBS model of colitis. European 13 Wong, J.M., de Souza, R., Kendall, C.W., Journal of Nutrition, 45, 418–425. Emam, A. and Jenkins, D.J. (2006) Colonic 22 Harris, P.J. and Ferguson, L.R. (1999) health: fermentation and short chain fatty Dietary fibres may protect or enhance acids. Journal of Clinical Gastroenterology, carcinogenesis. Mutation Research, 443, 40, 235–243. 95–110. 14 Ferguson, L.R., Tasman-Jones, C., Englyst, 23 Pajari, A.M., Rajakangas, J., Paivarinta, E., H. and Harris, P.J. (2000) Comparative Kosma, V.M., Rafter, J. and Mutanen, M. effects of three resistant starch (2003) Promotion of intestinal tumor preparations on transit time and short- formation by inulin is associated with an chain fatty acid production in rats. accumulation of cytosolic beta-catenin in Nutrition and Cancer, 36, 230–237. Min mice. International Journal of Cancer, 15 Chirakkal, H., Leech, S.H., Brookes, K.E., 106, 653–660. Prais, A.L., Waby, J.S. and Corfe, B.M. 24 Rajakangas, J., Pajari, A.M., Misikangas, (2006) Upregulation of BAK by butyrate in M. and Mutanen, M. (2006) Nuclear factor the colon is associated with increased Sp3 kappaB is downregulated and correlates binding. Oncogene, 25, 7192–7200. with p53 in the Min mouse mucosa during 16 DAgostini, F., Izzotti, A., Balansky, R.M., an accelerated tumor growth. International Bennicelli, C. and De Flora, S. (2005) Journal of Cancer, 118, 279–283. Modulation of apoptosis by cancer 25 Mutanen, M., Pajari, A.M. and Oikarinen, chemopreventive agents. Mutation S.I. (2000) Beef induces and rye bran Research, 591, 173–186. prevents the formation of intestinal polyps 17 Schrezenmeir, J. and de Vrese, M. (2001) in Apc(Min) mice: relation to beta-catenin Probiotics, prebiotics, and synbiotics: and PKC isozymes. Carcinogenesis, approaching a definition. The American 21, 1167–1173. Journal of Clinical Nutrition, 73, 26 Janne, P., Carpentier, Y. and Willems, G. 361S–364S. (1977) Colonic mucosal atrophy induced 18 Marteau, P. and Boutron-Ruault, M.C. by a liquid elemental diet in rats. (2002) Nutritional advantages of probiotics The American Journal of Digestive Diseases, and prebiotics. The British Journal of 22, 808–812. Nutrition, 87 (Suppl. 2), S153–S157. 27 Mandir, N., Englyst, H. and Goodlad, R.A. 19 Hidaka, H., Eida, T., Takizawa, T., (2008) Resistant carbohydrates stimulate Tokunaga, T. and Tashiro, Y. (1986) Effects cell proliferation and crypt fission in wild- References j729

type mice and in the Apc mouse model of Schatzkin, A., Willett, W.C., Wolk, A., intestinal cancer, association with Zeleniuch-Jacquotte, A., Zhang, S.M. and enhanced polyp development. The British Smith-Warner, S.A. (2005) Dietary fiber Journal of Nutrition, 100, 711–721. intake and risk of colorectal cancer: a 28 Preston-Martin, S., Pike, M.C., Ross, R.K., pooled analysis of prospective cohort Jones, P.A. and Henderson, B.E. (1990) studies. Journal of American Medical Increased cell division as a cause of human Association, 294 2849–2857. cancer. Cancer Research, 50, 7415–7421. 32 Michels, K.B., Fuchs, C.S., Giovannucci, 29 Ferguson, L.R., Philpott, M. and Dryland, E., Colditz, G.A., Hunter, D.J., Stampfer, P. (2007) Nutrigenomics in the whole- M.J. and Willett, W.C. (2005) Fiber intake genome scanning era: Crohns disease as and incidence of colorectal cancer among example. Cellular and Molecular Life 76,947 women and 47,279 men. Cancer Sciences, 64, 3105–3118. Epidemiology, Biomarkers & Prevention, 30 Bingham, S.A., Day, N.E., Luben, R., 14, 842–849. Ferrari, P., Slimani, N., Norat, T., Clavel- 33 Bingham, S. (2006) The fibre-folate debate Chapelon, F., Kesse, E., Nieters, A., in colorectal cancer. Proceedings of the Boeing, H., Tjonneland, A., Overvad, K., Nutrition Society, 65,19–23. Martinez, C., Dorronsoro, M., Gonzalez, 34 Asano, T.and McLeod, R.S. (2002) Dietary C.A., Key, T.J., Trichopoulou, A., Naska, A., fibre for the prevention of colorectal Vineis, P., Tumino, R., Krogh, V., Bueno- adenomas and carcinomas. Cochrane de-Mesquita, H.B., Peeters, P.H., Database of Systematic Reviews, Berglund, G., Hallmans, G., Lund, E., CD003430. Skeie, G., Kaaks, R., Riboli, E. and 35 Bonithon-Kopp, C., Kronborg, O., Giacosa, European Prospective Investigation into A., Rath, U. and Faivre, J. (2000) Calcium Cancer, and Nutrition. (2003) Dietary fibre and fibre supplementation in prevention in food and protection against colorectal of colorectal, adenoma recurrence: a cancer in the European Prospective randomised intervention trial. European Investigation into Cancer and Nutrition Cancer Prevention Organisation Study (EPIC): an observational study. Lancet, Group. Lancet, 356, 1300–1306. 361 1496–1501. 36 Morell, M.K., Konik-Rose, C., Ahmed, R., 31 Park, Y., Hunter, D.J., Spiegelman, D., Li, Z. and Rahman, S. (2004) Synthesis of Bergkvist, L., Berrino, F., van den Brandt, resistant starches in plants. Journal of P.A., Buring, J.E., Colditz, G.A., AOAC International, 87, 740–748. Freudenheim, J.L., Fuchs, C.S., 37 Morita, T., Kasaoka, S. and Kiriyama, S. Giovannucci, E., Goldbohm, R.A., (2004) Physiological functions of resistant Graham, S., Harnack, L., Hartman, A.M., proteins: proteins and peptides regulating Jacobs, D.R. Jr, Kato, I., Krogh, V., large bowel fermentation of indigestible Leitzmann, M.F., McCullough, M.L., polysaccharide. Journal of AOAC Miller, A.B., Pietinen, P., Rohan, T.E., International, 87, 792–796. j731

40 Lactobacilli and Fermented Foods Sabine Fuchs, Reinhard Stidl, Verena Koller, Gerhard Sontag, Armen Nersesyan, and Siegfried Knasmuller€

40.1 Introduction

Lactic acid bacteria (LAB) have been used since approximately 15 000 years to convert fresh milk into products with an extended endurance by early civilizations such as Sumerians, Babylonians, and Egyptians. The production of yogurt is postulated to originate from nomadic tribes in the Middle East and it is mentioned in early Persian scripts that the fecundity and longevity of Abraham was due to the consumption of yogurt. In the beginning of the 20th century, the Nobel laureate Ilya Metchnikov emphasized the beneficial health effects of consumption of fermented foods. Numerous positive health effects have been attributed to fermented foods containing LAB in the last decades (for review, see [1]); they include the improvement of the immune status and of allergic diseases, prevention of inflammations (e.g., gastroenteritis, Crohns disease, ulcerative colitis), viral and Helicobacter pylori infections and of travellers disease (diarrhea due to infections with Escherichia coli strains) as well as protection against DNA damage and cancer. The present chapter focuses on the latter issues and describes the mechanisms that account for the chemopreventive properties (Figure 40.1) as well as the current evidence for associations between the consumption of fermented foods and the reduction of the incidence of different forms of cancer in humans. Inthelastyears,alargenumberofnewstrainshavebeenisolated,which may be used for the production of fermented foods and possibly protect humans against oxidative and chemically induced DNA damage and malignant diseases. To identify cultures with optimized chemopreventive properties, the molecular mechanisms that account for these beneficial health effects have to be identified.

Figure 40.1 Schematic illustration of mechanisms by which LAB prevent DNA damage and tumor formation. 40.3 Detoxification of Genotoxic Carcinogens by Lactic Acid Bacteria j733

40.2 Occurrence of Lactic Acid Bacteria

The term probiotics has been defined by the Food and Agricultural Organization as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host and comprise different types of microorganisms including lactic acid bacteria which ferment the sugar lactose to lactic acid as well as yeasts. LAB comprises a number of different genera such as lactobacilli, bifidobacteria, and enterococci. Recently, new strains have been isolated from the human microflora and new methods have been developed, which are based on DNA- sequencing that enable a better characterization of the different species [2, 3]. To extend the life span of probiotics in the gut and improve their colonization, compounds such as oligofructose and inulin (prebiotics) that are metabolized by LAB, are added to milk products. Probioticsareusedfortheproductionofavarietyoffermentedfoodsincludingyogurt (usually containing two or more different strains), cheese, and fermented vegetables including sauerkraut and kimchi. The most widely used strains for yogurt production are Lactobacillus (L.) casei, L. acidophilus, and L. bulgaricus, more recently also Bifido- bacteria strains are used. The different species and subspecies differ substantially in regard to their shelf life and also in their survival during their passage in the gastrointestinal (GI) tract. Bifidobacteria are in general more sensitive toward oxygen than lactobacilli and it is technologically more difficult to cultivate them. High-quality yogurts contain up to 108 viable LAB/ml; however, in some of the marketed products, the titers are 2 to 3 orders of magnitude lower and the number of viable cells decline as a function of the storage time. LAB are also part of the intestinal microflora. In humans, more than 400 different bacterial species have been identified in the gut and it has been estimated that up to 2 kg of the intestinal content of the GI-tract (1014 cells) are bacteria. The density of – LAB found in the human gut is in the range between 106 and 109 g 1. It is notable that LAB are found in higher quantities in the intestinal contents of vegetarians than in meat eaters and it has been stressed that the lower incidence of colon cancer found in vegetarians may be causally related to this phenomenon (see below). Many of the strains for which beneficial health effects have been made, for example, L. casei Shirota or L. rhamnosus GG, have been isolated from the microflora of humans and it has been claimed that the survival of the strains of human origin in the GI-tract is increased.

40.3 Detoxification of Genotoxic Carcinogens by Lactic Acid Bacteria

A number of studies showed that LAB are able to detoxify the representatives of different classes of genotoxic carcinogens. Examples for the results of such studies are listed in Table 40.1. 734 j 0Lcoail n emne Foods Fermented and Lactobacilli 40 Table 40.1 Detoxification of DNA-reactive chemicals by lactic acid bacteriaa.

Chemicals Occurrence/relevance Effects of lactic acid bacteria

Heterocyclic aromatic amines IQ, PhIP, AaC, DiMeIQx, MeIQx, IQx Thermal degradation products formed during Direct binding to cell walls of LAB; effects were seen frying of meats from amino acids, glucose, and in vitro and in animals and humans, also short-chain kreatinin; cause colon and other forms of cancer in fatty acids formed by LAB may play an important animals; PhIP and AaC are the most abundant HAs role; binding effects are strain and compound in fried meats speciﬁc

Mycotoxins

Aflatoxin B1 (AFB1) Formed by Aspergillus flavus, the strongest known Numerous studies reported binding of AFB1 by human hepatocarcinogen LAB. The inactivation of the other toxins was described in only few investigations; the detoxification of AFB1, DON, ZEA, and FB1 was observed also with dead cells (and is probably due to binding); PAT and OTA were detoxified only with living cells (mechanism unclear) Ochratoxin A (OTA) Formed by Aspergillus ochraceus – cancer formation in kidney and bladder, genotoxin Patulin (PAT) Found in fruits, genotoxic in mammalian cells, carcinogenic effects not known Deoxynivalenol (DON) Fusarium toxins, DON may cause esophageal Nivalenol (NIV) cancer in humans, genotoxic in human cells Zearalenone (ZEA) Fumonisin B1 (FB1) Induces liver cancer in rats, may cause esophageal cancer in humans Nitro compounds 2-Nitrofluorene (2NF) Combustion product in diesel emissions, genotoxic Reduction of mutagenic effects of these compounds and carcinogenic in rats in Salmonella/microsome assays, DNA damage by MNNG was reduced in colon cells of rats in vivo 03Dtxfcto fGntxcCrioesb atcAi Bacteria Acid Lactic by Carcinogens Genotoxic of Detoxification 40.3 N-Methyl-N0-nitro-N-nitrosoguanidine (MNNG) Synthetic model carcinogen 4-Nitro-O-phenylene-diamine Found in low concentrations in ambient air; component of hair dyes; carcinogenic in rats and mice; causes DNA damage and mutations in bacteria 4-Nitroquinoline-1-oxide (4NQO) Model genotoxin

Miscellaneous Dimethylhydrazine (DMH) Used as a model compound for the induction of Prevention of DMH-induced DNA damage and colon tumors ACF-formation in colons of rats Benzo(a)pyrene (B(a)P) Model compound for polycyclic aromatic B(a)P was used in vitro with LAB, its mutagenicity hydrocarbons (PAH), genotoxic and carcinogenic was decreased by LAB in bacterial tests but no for humans and animals effects were seen in rats (in vivo)

AaC, 2-amino-9H-pyrido[2,3-b]indole; DiMeIQx, 2-amino-3,4,8-trimethyl-imidazo[4,5-f ]quinoxaline; HA, heterocyclic aromatic amines; IQ, 2-amino-3-methyl-imidazo- [4,5-f ]quinoline; MeIQx, 2-amino-3,8-dimethyl-imidazo[4,5-f ]quinoxaline; PhIP, 2-amino-1-methyl-6-phenyl-imidazo[4,5-f ]pyridine. aSee Ref. [4]. j 735 736j 40 Lactobacilli and Fermented Foods

An important group of food borne carcinogens inactivated by LAB are heterocyclic aromatic amines (HAs). The protective properties of LAB toward these food borne carcinogens were demonstrated mainly in in vitro experiments (i.e., in chemical analytical studies and in bacterial mutagenicity tests (for review, see [5]). Recently, Stidl et al. [6] conducted a comprehensive study with representatives of eight different LAB species, from each 12 strains were tested. The highest removal rates (monitored in HPLC-CEAD measurements) were seen with representatives of the L. helveticus and Streptococcus (S.) thermophilus groups while other species (e.g., L. kefir, L. plantarum) were by far less effective. The ranking order of the binding capacity toward different HAs declined in the order AaC > DiMeIQx >MeIQx >IQx > PhIP. Apart from in vitro experiments, animal and human studies have also been conducted. For example, Zsivkovits et al. [7] showed that specific LAB strains protect rats against IQ (2-amino-3-methyl-imidazo[4,5-f]quinoline)-induced DNA damage in liver and colon. The effects were time- and dose-dependent (maximal protection after 4 h) and were seen under conditions that are relevant for humans. Tavan et al. [8] conducted a similar study and observed reduction of DNA migration in single cell gel electrophoresis (SCGE) experiments; furthermore, they also found prevention of HA-induced preneoplastic lesions (aberrant crypt foci, ACF) in the colon with fermented milk. In this context, it is notable that the inoculation of microfloras from meat consumers and vegetarians into gnotobiotic (germ-free) rats showed that the extent of HA-induced damage in colon cells is substantially reduced in the latter group [9]. Also, the results of chemical analytical studies support the assumption of protective effects of LAB in animals and humans. For example, it was found that the uptake of radiolabeled 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) [10] is reduced by coadministration of LAB in rats. Also in humans, the consumption of LAB led to a reduction of the urinary and fecal excretion of HAs [8, 11]. Another important group of chemicals that is detoxified by LAB are mycotoxins. fl Several in vitro studies showed that a atoxin B1 (AFB1), which is considered to be the most potent human carcinogen and accounts for the high prevalence for liver cancer in Central Africa and China, is inactivated by the bacteria [12, 13]. However, evidence of the effects in vivo is confined to one study in which it was shown that the absorption of the toxin in the GI-tract of chickens is reduced by LAB and experiments concerning the prevention of hepatic cancer and/or DNA damage in animals are lacking. Also with Fusarium toxins (e.g., deoxynivalenol, DON and nivalenol, NIV), it has been found that LAB remove these compounds from liquid media but no data from animal and human studies are available at present. All these effects appear to be due to the binding of the chemicals to the cell walls of the bacteria as they were seen with living and with heat inactivated cells. Another mode of action may account for the decrease of the mutagenic effects of the mycotoxins patulin (PAT) and ochratoxin A (OTA) in human-derived cells, which was described by Fuchs and coworkers [14]. They found that these toxins are eliminated by living but not by heat inactivated bacteria; the most potent removal of PAT was observed with a Bifidobacterium (B.) animalis strain (VM12) while OTA was most efficiently detoxified by a L. acidophilus strain (VM20). 40.4 Antioxidant Effects of Lactic Acid Bacteria j737

Among the compounds listed in Table 40.1, only 2-nitrofluorene (2NF) is relevant as a risk factor for humans. The other nitro compounds and also dimethylhydrazine (DMH) are synthetic chemicals that do not occur constitute health hazards for humans. B(a)P is an important carcinogen of human relevance that is contained in foods and tobacco smoke, but protective effects of LAB that were seen under in vitro conditions in bacterial tests could not be confirmed in subsequent animal studies [15]. Another important mechanism for prevention of DNA damage in the GI-tract is the reduction of nitrate by nitratereductase that has been found in the strains used for yogurt production; since nitrate is required for the formation of nitrosamines, a decrease of the nitrate concentrations will reduce the endogenous formation of these carcinogens [16]. It has also been claimed that representatives of the intestinal microflora (i.e., Clostridia and Bacteroides strains) reduce the reactivation of HA-glucuronides in the colon by b-glucuronidase [17]; but no results from animal and human studies are available, which confirm this hypothesis. Several studies have been published that concern the protective effects of LAB toward DNA damage caused by fecal water (FW), an aqueous fraction that is prepared from human feces by high-speed centrifugation. Several in vitro experiments indicate that bacteria (in particular, Bifidobacteria and L. plantarum) protect human-derived colon cells against FW-induced DNA damage. Also in animal and human studies, evidence of protective effects of LAB was obtained. For example, Klinder et al. [18] reported that feeding of LAB to rats reduced the genotoxic effect of FW samples prepared from their feces and the same observation was made by Oberreuther- Moschner [19] in a human intervention trial with a L. acidophilus and a B. longum strain. In both studies, reduction of the genotoxicity of FW was monitored in SCGE tests with HT 29 cells. One of the problems of the interpretation of these findings is due to the fact that the compounds that account for the DNA-damaging properties of FWare not known and it is unclear at present if the genotoxic properties of FWsamples seen in vitro are causally related to colon cancer risks. Klinder and coworkers [18] compared the genotoxic effect of FW samples of normal rats and of animals with AOM-induced tumors; as they found higher activities in samples from tumor-bearing animals, they postulated that FW-genotoxicity is an indicator for colon tumor risks. Also the mode of action by which LAB detoxify FW is not known at present but it is notable that significant protective effects were only seen with viable bacteria [15].

40.4 Antioxidant Effects of Lactic Acid Bacteria

A number of investigations indicate that LAB protect against reactive oxygen species (ROS) that are considered to play a key role in the etiology of colon cancer [20, 21] and other malignant diseases [22, 23]. Table 40.2 summarizes the results of biochemical and genotoxicity studies in which protective effects against ROS were observed either with intact cells or with 738j 40 Lactobacilli and Fermented Foods

Table 40.2 Examples of studies concerning the antioxidative effects of LABa.

LAB strains Results

In vitro experiments

L. plantarum Scavenging of O2 by Mn(II) L. fermentum E-3, L. fermentum E-8 Prevention of lipid peroxidation by high levels of glutathione, expression of Mn-SOD and secretion

of H2O2 L. rhamnosus GG, L. rhamnosus Scavenging of superoxide anion radicals and peroxyl Lc 705, L. acidophilus LA, L. paracasei radicals by L. rhamnosus GG YEC, Biﬁdobacterium Bb12, Escherichia coli L. delbrueckii ssp. lactis, L. delbrueckii Protection of proteins against free radical oxidation, ssp. bulgaricus, L. acidophilus, L. casei highest protective effects with L. delbrueckii ssp. bulgaricus Y-23 55 Strains from 16 different Inhibition of DNA migration induced in human colon

LAB species cells by plumbagin and H2O2; strongest effects seen with S. thermophilus, no correlation with SOD

Animal experiments L. lactis NZ9800; L. plantarum LAB strains with SOD activity protected rats against NCIM8826; both wild type and chemically (TNBS) induced colitis, while SOD deﬁcient recombinant with SOD activity strains were ineffective; also with the enzyme itself positive effects were seen B. biﬁdum strain Yakult Prevention of lipid peroxidation of colon mucosa cells of mice induced by iron overload leading to the release

of H2O2 Lactobacillus sp. SBT 2028; Cell-free extract of Lactobacillus sp. SBT 2028, a strain L. rhamnosus SBT 2257 with high antioxidant activity reduced hemolysis in rats caused by vitamin E deﬁciency

Human studies L. fermentum ME-3 Improvement of the TAA, TAS, and GSH in the blood of healthy volunteers by L. fermentum L. fermentum ME-3, L. buchneri S-15, Consumption of fermented goat milk improved L. plantarum LB-4 resistance of the lipoprotein fraction to oxidation, lowered levels of peroxidized lipoproteins, oxidized LDL, enhanced the GSH redox ratio, isoprostanes, and TAA

GSH, reduced glutathione; Mn, manganese; LDL, low density lipoprotein; SOD, superoxide dismutase; TAA, total antioxidative activity; TAS, total antioxidant status. aSee Ref. [25].

cell-free extracts in vitro and also studies with animals and humans. Recent findings of Koller et al. [24] showed that specific LAB strains, in particular representatives of the species S. thermophilus protect human-derived colon cells (HT 29) against ROS- induced DNA migration. However, it is notable that some of the strains they tested caused severe DNA migration per se and the authors postulated that these effects may fi be due to formation of H2O2 that has been found earlier with speci c LAB strains. 40.5 Effect of Lactic Acid Bacteria on the Immune Status j739

The antioxidant defense system of LAB has been studied intensely over the last decades and several protective mechanisms have been discovered. ROS may be inactivated either by direct scavenging or via enzymatic reaction. The most important direct scavengers are antioxidants such as NADPH/NADH and glutathione that are present in some strains in high concentrations. Following oxidation, GSH is reduced by GSH-reductase that uses NADPH as a source of reducing power [26]. The most important enzymatic oxidative stress resistance mechanism in LAB is performed by a coupled NADH-oxidase/peroxidase system. Intracellular O2 is þ eliminated by NADH-oxidase activity that uses oxygen to oxidize NADH to NAD giving rise to formation of H2O2 that is subsequently inactivated by NADH-peroxidase. In strains lacking this latter enzyme, the hydrogen peroxide can be detoxified by other enzymes such as catalase, GSH-peroxidase or pyruvate that reacts none- nzymatically with H2O2 to form H2O and acetate [27]. Different types of catalase have been identified in LAB. Apart from the true catalases, which are only active in the presence of hematin, also nonheme pseudocatalases were found in a number of genera [28, 29]. However, some Lactobacillus and Streptococcus species are completely devoid of these enzymes [30]. Another important enzyme system that decreases the steady state level of reactive . oxygen are superoxide dismutases (SOD) that convert O2 into H2O2. Most LAB possess these enzymes, their classification depends on their cofactors (mostly Mn and Fe) and MnSOD is by far the most abundant form [30]. Not all LAB possess SOD [31] and strong differences in the activity of these enzymes were found in comparative screening studies. For some L. plantarum strains, it was shown that they compensate the lack of SOD activity by an alternative nonenzymatic defense system that involves accumulation of high-intracellular concentrations of scavenging manganese ions [32–34]. It is notable that Koller et al. [24] found high SOD levels only in the representatives of the S. thermophilus group but no activities in some Lactobacillus species such as L. kefir, B. breve, and L. casei. While in a number of studies with specific strains possessing SOD, protective effects were seen in inflammation models with animals, no correlation between the prevention of ROS-induced DNA damage and SOD activity of LAB strains was seen in human colon cells. Also, reports in which protective effects of specific LAB strains against ulcerative colitis and Crohns disease were found support the assumption of ROS protective properties of LAB as it is known that oxidative damage is involved in IBD.

40.5 Effect of Lactic Acid Bacteria on the Immune Status

Apart from the direct and enzymatic inactivation of ROS, LAB interact also with cell signaling pathways and activate transcription factors in humans that are involved in the inﬂammations and release of oxygen species [35, 36]. It has been shown 740j 40 Lactobacilli and Fermented Foods

in a number of in vitro and in vivo studies that the bacteria cause a downregulation of inflammatory cytokines (e.g., TNF-a, IL-6, and IL-12) [35, 36]; recently, such effects were also found in human trials [37]. The anti-inflammatory effects of LAB involve inhibition of the NF-kB pathway, probably through stabilization of I-kBa [38, 39]. Downregulation of this transcription factor was seen, for example, in models in which reactions were induced by pathogenic bacteria or lipopolysaccharides and it was shown that the effects in the hosts are mediated by specific pattern recognition receptors for microbial products; in particular, the toll-like receptor group (TLRs) plays an important role [40, 41]. In addition to the interactions with signaling pathways involved in inflammatory responses, it is also known that the bacteria affect other functions of the immune response (for review, see [36]). For example, it was found in a human trial that probiotic colonization produced a balanced T-helper cell response (Th1 ¼ Th2 ¼ Th3/Th1) preventing the imbalance is associated with different clinical diseases and it was also shown in humans that yogurt consumption increases the natural cytotoxicity against K562 erythroleucemic cells [37]. Also, results of animal studies indicate that the phagocytotic activity in macrophages is substantially increased by LAB (for review, see [36]). In this context, it is notable that also prebiotics (oligofructose and inulin) that are used for yogurt production have positive effects on the immune system; an excellent review on this topic has been published recently by Seifert and Watzl [42].

40.6 Results of Carcinogenicity Studies with Laboratory Rodents

In most animal studies, the impact of LAB on colon carcinogenesis has been investigated with model compounds such as DMH and azoxymethane (AOM). As end points, either formation of colon tumors or, more frequently, ACF were used. In some of these trials with LAB strains, substantial protective effects were found but some of the investigations also yielded negative results [36, 43]. The extrapolation of the results of such studies to humans is in general, problematic as the chemical carcinogens used do not play a role in the etiology of human colon cancer. Since the animals were treated in almost all trials with AOM or DMH after LAB administration, the protective effects may be due to direct inactivation of the chemicals and not to inhibition of formation and/or clonal expansion of cancer cells. More relevant are ﬁndings that showed that the bacteria are also protective toward colon, mammary, and liver carcinogenesis caused by HAs that are considered to play a role in the etiology of human cancer [44]. LAB are obviously also able to reduce tumor growth. For example, it was reported that Biﬁdobacteria cause regression of sarcomas in mice [45] and with L. casei Shirota protective effects against transplantable tumors were found. Furthermore, it was reported that the same strains inhibit metastasis in guinea pigs after injection of Table 40.3 Epidemiological studies concerning associations between the consumption of LAB-containing foods and specific forms of human cancera.

Study-type Country/year Results

Case–control studies 1010 breast cancer cases; 1950 controls France/1986 Reduced risk by yogurt (RR ¼ 0.8; CI 0.6–1.0) 152 proximal colon cancer cases; 201 distal USA/1988 Reduced risk by cultured milk (OR ¼ 0.65; CI 0.41–1.01) colon cancer cases; 618 controls 133 breast cancer cases; 289 controls Netherlands/1989 Reduced risk of by fermented milk (OR ¼ 0.63; CI 0.41–0.96) 746 colon cancer cases; 746 controls USA/1992 Yogurt was signiﬁcantly protective against colon cancer (RR ¼ 0.83; CI 0.70–0.98) 06Rslso acngnct tde ihLbrtr Rodents Laboratory with Studies Carcinogenicity of Results 40.6 154 small adenoma cases; 208 large France/1996 Yogurt was found to be protective against large adenomas (OR ¼ 0.5; CI 0.3–0.9) adenoma cases; 426 polyp free cases; 171colon cancer cases; 309 controls 180 bladder cancer cases; 445 controls Japan/2002 Habitual intake of LAB reduced the risk of bladder cancer (OR ¼ 0.46; CI 0.27–0.79)

Prospective studies 331 men and 350 women cases of USA/1994 Milk consumption and intake of total fermented dairy products not related to colorectal adenomas; 9159 men and adenoma risk (RR ¼ 0.85; CI 0.43–1.70) 8585 women controls 215 incident colon cancer and 111 rectal Netherlands/1994 No signiﬁcant protective effect against colon cancer with fermented milk cancer cases within 120 852 persons (RR ¼ 0.89; CI 0.60–1.33) 203 incident colon cancer cases within USA/1996 No association between colon cancer and intake of fermented dairy 47 935 persons products (RR ¼ 0.84; CI 0.54–1.29)

Double-blind intervention study 138 Patients with bladder carcinomas Japan/1995 Prevention of cancer recurrence by L. casei preparation caused a signiﬁcant prophylactic effect compared to a placebo in two of three groups (p ¼ 0.01) a

See Ref. [4]. j 741 742j 40 Lactobacilli and Fermented Foods

cancer cells and similar observations were also made in experiments with mice (for review, see [35]).

40.7 Impact of Lactic Acid Bacteria on Cell Proliferation and Apoptosis

Apart from inactivation of DNA-reactive carcinogens and/or ROS and modulation of the immune system, also other properties of LAB may be involved in their cancer protective effects. It has been shown in a number of in vitro studies with different cancer cell lines originating from the GI-tract (e.g., IC-6, HT29, HGC-27) that the bacteria cause a decrease in the cell proliferation [46–49]. These observations were also confirmed in animal experiments (i.e., in AOM models with rats) [50] and in a recent human study with colorectal cancer (CRC) (n ¼ 37) and polypectomized (n ¼ 43) patients by Rafter and coworkers [51]. They reported that the consumption of synbiotic food (i.e., a combination of inulin with L. rhamnosus GG and B. lactis (Bb12) causes a significant reduction of the genotoxicity of FW samples collected from the patients. One of the mechanisms that has been claimed to account for the antimutagenic properties of LAB is the alteration of the formation of secondary bile acids that cause a proliferative stimulus on colon mucosa cells and are considered to be a risk factor for CRC [52–55]. Results of human intervention trials suggested that probiotic diets increase the dehydroxylation of primary bile acids; furthermore, it was found in an artificial human GI-ecosystem that the bacteria cause precipitation of deconjugated bile acids [56–58]. However, also adverse effects were seen, for example, in a study with rats an increase of secondary bile acids was reported after feeding an L. acidopilus strain [59] and more experiments are required to draw firm conclusions. Another mode of action that may play a role is the formation of short-chain fatty acids (SCFAs). It is known from animal experiments that prebiotics increase the levels of SCFA (in particular, of butyrate) that are known to slow down the division rates of colon cells [50, 52, 53]. In some studies also, the impact of LAB on apoptosis rates was monitored in colon cells and the induction of this form of programmed cell death (which is characteristic of the tumor cells) was seen in vitro and also in animal experiments [47, 50]. Also, SCFA may be involved in this phenomenon.

40.8 Results of Human Epidemiological Studies

The results obtained in human epidemiological studies concerning the intake of LAB in fermented foods and the incidence of different forms of cancer are controversial (Table 40.3). While in some case–control studies, the evidence for an inverse association of consumption of yogurt and the incidence of speciﬁc forms of cancer References j743

(in particular, of colorectal tumors) was seen, no indication for such effects was found in large prospective studies.

40.9 Conclusions and Future Research

One of the reasons for the conflicting results obtained in the human studies may be due to the fact that they do not take into consideration the quality of the fermented products (i.e., the concentrations of viable LAB/ml) and the type of strains used for their production. It is known that the concentration of viable bacteria varies considerably in yogurts (between 105 and 108 cells/ml; the latter concentrations are only found in high-quality products) and that the number of viable cells declines substantially as a function of the storage time of the products. As described above, it was found in the few available comprehensive studies that the inactivation of genotoxic carcinogens and also the ROS protective properties of LAB are highly strain and species specific. In the case of the improvement of the immune functions, it has been claimed repeatedly by dietary industries that strains isolated from the human gut are more effective compared to naturally occurring strains but recent investigations showed that also conventional products cause comparable effects and more experimental data are required to enable to draw firm conclusions. Overall, the results obtained with LAB so far are promising and show that fermented foods containing strains with beneficial properties may contribute substantially to the prevention of DNA damage and cancer in humans provided that it is possible to develop strategies on the basis of sound scientific data.

References

1 Ouwehand, A.C., Salminen, S. and Therapy (eds Z. Durackova and S. Isolauri, E. (2002) Probiotics: an overview Knasmueller), Slovak Academic Press, of beneficial effects. Antonie van Bratislava, pp. 253–271. Leeuwenhoek, 82, 279–289. 5 Knasmueller, S., Steinkellner, H., 2 Kaouther, B.A., Vaughan, E.E. and deVos, Hirschl, A.M., Rabot, S., Nobis, E.C. and W.M. (2007) Advancedmoleculartoolsforthe Kassie, F. (2001) Impact of bacteria in dairy identification of lactic acid bacteria. The products and of the intestinal microflora Journal of Nutrition, 137,741–747. on the genotoxic and carcinogenic 3 Pfeiler, E.A. and Klaenhammer, T.R.(2007) effects of heterocyclic aromatic amines. The genomics of lactic acid bacteria. Mutation Research, 480–481, 129–138. Trends in Microbiology, 15, 546–553. 6 Stidl, R., Sontag, G., Koller, V. and 4 Stidl, R., Fuchs, S., Koller, V., Marian, B., Knasmueller, S. (2008) Binding of Sontag, G., Ehrlich, V. and Knasmueller, S. heterocyclic aromatic amines by lactic acid (2007) DNA-protective properties of lactic bacteria: results of a comprehensive acid bacteria, in The Activity of Natural screening trial. Molecular Nutrition & Food Compounds in Diseases Prevention and Research, 52, 322–329. 744j 40 Lactobacilli and Fermented Foods

7 Zsivkovits, M., Fekadu, K., Sontag, G., GG reduces aflatoxin B1 transport, Nabinger, U., Huber, W.W., Kundi, M., metabolism, and toxicity in Caco-2 Cells. Chakraborty, A., Foissy, H. and Applied and Environmental Microbiology, Knasmuller, S. (2003) Prevention of 73, 3958–3964. heterocyclic amine-induced DNA damage 14 Fuchs, S., Sontag, G., Stidl, R., Ehrlich, V., in colon and liver of rats by different Kundi, M. and Knasmuller,€ S. (2008) lactobacillus strains. Carcinogenesis, Detoxification of patulin and ochratoxin A, 24, 1913–1918. two abundant mycotoxins, by lactic acid 8 Tavan, E., Cayuela, C., Antoine, J.M., bacteria. Food and Chemical Toxicology, Trugnan, G., Chaugier, C. and Cassand, P. 46, 1398–1407. (2002) Effects of dairy products on 15 Burns, A.J. and Rowland, I.R. (2004) heterocyclic aromatic amine-induced rat Antigenotoxicity of probiotics and colon carcinogenesis. Carcinogenesis, prebiotics on faecal water-induced DNA 23, 477–483. damage in human colon adenocarcinoma 9 Humblot, C., Lhoste, E., Knasmuller,€ S., cells. Mutation Research, 551, 233–243. Gloux, K., Bruneau, A., Bensaada, M., 16 Fernandes, C.F., Shahani, K.M. and Durao, J., Rabot, S., Andrieux, C. and Amer, M.A. (1987) Therapeutic role of Kassie, F. (2005) Protective effencts dietary lactobacilli and lactobacillic of Brussel sprouts, oligosaccharides fermented dairy products. Microbiological and fermented milk towards 2-amino-3- Reviews, 46, 343–356. methylimidazo[4,5-f ]quinoline (IQ)- 17 Johannson, G., Holmen, A., Persson, L., induced genotoxicity in the human flora Hogstedt, B., Wassen, C., Ottova, L. and associated F344 rat: role of xenobiotic Gustafsson, J.A. (1998) Dietary influence metabolising enzymes and intestinal on some proposal risk factors for colon microflora. Journal of Chromatography B, cancer-fecal and urinary mutagenic activity 802, 231–237. and the activity of some intestinal bacterial 10 Zhang, X.B. and Ohta, Y. (1993) enzymes. Cancer Detection and Prevention, Microorganisms in the gastrointestinal 21, 258–266. tract of the rat prevent absorption of the 18 Klinder, A., Forster, A., Caderni, G., mutagen-carcinogen 3-amino-1,4- Femia, A.P. and Pool-Zobel, B.L. (2004) dimethyl-5H-pyrido[4,3-b]indole. Fecal water genotoxicity is predictive of Canadian Journal of Microbiology, tumor-preventive activities by inulin-like 39, 841–845. oligofructoses, probiotics (Lactobacillus 11 Lidbeck, A., Övervik, E., Rafter, J., rhamnosus and Bifidobacterium lactis), Nard, C.E. and Gustaffson, J.A. (1992) and their synbiotic combination. Nutrition Effects of Lactobacillus acidophilus and Cancer, 49, 144–155. supplements on mutagen excretion in 19 Oberreuther-Moschner, D.L., Jahreis, G., feces and urine of humans. Microbial Rechkemmer, G. and Pool-Zobel, B.L. Ecology in Health and Disease, 5,59–67. (2004) Dietary intervention with the 12 IARC (1993) Monographs on the Evaluation probiotics Lactobacillus acidophilus 145 of Carcinogenic Risks to Humans. Some and Bifidobacterium longum 913 modulates Naturally Occuring Substances: Food Items the potential of human faecal water to and Constituents, Heterocyclic Aromatic induce damage in HT29clone19A cells. Amines and Mycotoxins, vol. 56 The British Journal of Nutrition, 91, International Agency for Research on 925–932. Cancer, Lyon. 20 Bartsch, H., Nair, J. and Owen, R.W. (2002) 13 Gratz, S., Wu, Q.K., El-Nezami, H., Exocyclic DNA adducts as oxidative stress Juvonen, R.O., Mykkanen, H. and Turner, markers in colon carcinogenesis: potential P.C. (2007) Lactobacillus rhamnosus strain role of lipid peroxidation, dietary fat and References j745

antioxidants. Biological Chemistry, 383, Environmental Microbiology, 64, 915–921. 1359–1365. 21 Bruce, W.R., Giacca, A. and Medline, A. 29 Johnston, M.A. and Delwiche, E.A. (1965) (2000) Possible mechanisms relating diet Distribution and characteristics of the and risk of colon cancer. Cancer catalases of lactobacillaceae. Journal of Epidemiology, Biomarkers & Prevention, 9, Bacteriology, 90, 347–351. 1271–1279. 30 Kullisaar, T., Zilmer, M., Mikelsaar, M., 22 Skrzycki, M., Scibior, D., Podsiad, M. and Vihalemm, T., Annuk, H., Kairane, C. and Czeczot, H. (2008) Activity and protein Kilk, A. (2002) Two antioxidative level of CuZnSOD and MnSOD in benign Lactobacilli strains as promising and malignant liver tumors. Clinical probiotics. International Journal of Food Biochemistry, 41,91–96. Microbiology, 72, 215–224. 23 Bartsch, H. and Nair, J. (2006) Chronic 31 Lin, M.Y. and Yen, C.L. (1999) inﬂammation and oxidative stress in the Antioxidative ability of lactic acid bacteria. genesis and perpetuation of cancer: role of Journal of Agricultural and Food Chemistry, lipid peroxidation, DNA damage and 47, 1460–1466. repair. Langenbecks Archives of Surgery/ 32 Archibald, F. (1986) Manganese: its Deutsche Gesellschaft fur Chirurgie, 391, acquisition by and function in the lactic 499–510. acid bacteria. Critical Reviews in 24 Koller, V.J., Marian, B., Stidl, R., Microbiology, 13,63–109. Nersesyan, A., Winter, H., Simic, T., 33 Archibald, F.S. and Fridovich, I. (1981) Sontag, G. and Knasmuller,€ S. (2008) Manganese and defenses against oxygen Impact of lactic acid bacteria on oxidative toxicity in Lactobacillus plantarum. Journal DNA damage in human derived colon of Bacteriology, 146, 928–936. cells. Food and Chemical Toxicology, 46, 34 Archibald, F.S. and Fridovich, I. (1981) 1221–1229. Manganese, superoxide dismutase and 25 Koller, V.J. (2006) Investigations oxygen tolerance in some lactic acid concerning the protective effects of bacteria. Journal of Bacteriology, 146, probiotic lactic acid bacteria towards 928–936. reactive oxygen species. Master Thesis. 35 Matsuzaki, T. (1998) Immunomodulation Institute of Cancer Research, Department by treatment with Lactobacillus casei strain of Medicine I, Medical University of Shirota. International Journal of Food Vienna, Vienna Austria, pp. 17–18. Microbiology, 41, 133–140. 26 Cabiscol, E., Tamarit, J. and Ros, J. (2000) 36 Meydani, S.N. and Ha, W.K. (2000) Oxidative stress in bacteria and protein Immunologic effects of yogurt. The damage by reactive oxygen species. American Journal of Clinical Nutrition, 71, International Microbiology, 3,3–8. 861–872. 27 van Niel, E.W., Hofvendahl, K. and Hahn- 37 Meyer, A.L., Elmadfa, I., Micksche, M. and Hagerdal, B. (2002) Formation and Herbacek, I. (2007) Immunonutrition: conversion of oxygen metabolites by feeding the bodys defence, in The Activity Lactococcus lactis subsp. lactis ATCC 19435 of Natural Compounds in Diseases under different growth conditions. Applied Prevention and Therapy (eds Z. Durackova and Environmental Microbiology, 68, and S. Knasmuller),€ Slovak Academic 4350–4356. Press, Bratislava, pp. 273–296. 28 Hertel, C., Schmidt, G., Fischer, M., 38 Tien, M.T., Girardin, S.E., Regnault, B., Oellers, K. and Hammes, W.P. (1998) Le Bourhis, L., Dillies, M.A., Coppee, J.Y., Oxygen-dependent regulation of the Bourdet-Sicard, R., Sansonetti, P.J. and expression of the catalase gene katA of Pedron, T. (2006) Anti-inﬂammatory effect Lactobacillus sakei LTH677. Applied and of Lactobacillus casei on Shigella-infected 746j 40 Lactobacilli and Fermented Foods

human intestinal epithelial cells. Journal of proliferation and oxidative stress in vitro. Immunology, 176, 1228–1237. Letters in Applied Microbiology, 42, 452–458. 39 Menard, S., Candalh, C., Bambou, J.C., 48 Russo, F., Orlando, A., Linsalata, M., Terpend, K., Cerf-Bensussan, N. and Cavallini, A. and Messa, C. (2007) Effects of Heyman, M. (2004) Lactic acid bacteria Lactobacillus rhamnosus GG on the cell secrete metabolites retaining antigrowth and polyamine metabolism in inflammatory properties after intestinal HGC-27 human gastric cancer cells. transport. Gut, 53, 821–828. Nutrition and Cancer, 59, 106–114. 40 Tohno, M., Kitazawa, H., Shimosato, T., 49 Baricault, L., Denariaz, G., Houri, J.J., Matsumoto, M., Katoh, S., Kawai, Y. and Bouley, C., Sapin, C. and Trugnan, G. Saito, T. (2005) A swine toll-like receptor (1995) Use of HT-29, a cultured human 2-expressing transfectant as a potential colon cancer cell line, to study the effect of primary screening system for fermented milks on colon cancer cell immunobiotic microorganisms. FEMS growth and differentiation. Carcinogenesis, Immunology and Medical Microbiology, 16, 245–252. 44, 283–288. 50 Le Leu, R.K., Brown, I.L., Hu, Y.,Bird, A.R., 41 Kelly, D. and Conway, S. (2005) Bacterial Jackson, M., Esterman, A. and Young, G.P. modulation of mucosal innate immunity. (2005) A synbiotic combination of resistant Molecular Immunology, 42, 895–901. starch and Bifidobacteria lactis facilitates 42 Seifert, S. and Watzl, B. (2007) Inulin and apoptotic deletion of carcinogen-damaged oligofructose: review of experimental data cells in rat colon. The Journal of Nutrition, on immune modulation. The Journal of 135, 996–1001. Nutrition, 137, 2563–2567. 51 Rafter, J., Bennett, M., Caderni, G., 43 Bolognani, F., Rumney, C.J., Pool- Clune, Y., Hughes, R., Karlsson, P.C., Zobel, B.L. and Rowland, I.R. (2001) Effect Klinder, A., ORiordan, M., of lactobacilli, bifidobacteria and inulin on OSullivan, G.C., Pool-Zobel, B., the formation of aberrant crypt foci in rats. Rechkemmer, G., Roller, M., Rowland, I., European Journal of Nutrition, 40, 293–300. Salvadori, M., Thijs, H., Van Loo, J., 44 Reddy, B.S. and Rivenson, A. (1993) Watzl, B. and Collins, J.K. (2007) Dietary Inhibitory effect of Bifidobacterium longum synbiotics reduce cancer risk factors in on colon, mammary, and liver polypectomized and colon cancer patients. carcinogenesis induced by 2-amino-3- The American Journal of Clinical Nutrition, methylimidazo[4,5-f ]quinoline, a food 85, 488–496. mutagen. Cancer Research, 53, 3914–3918. 52 Hague, A. and Paraskeva, C. (1995) The 45 Hitchins, A.D. and McDonough, F.E. short-chain fatty acid butyrate induces (1989) Prophylactic and therapeutic apoptosis in colorectal tumour cell lines. aspects of fermented milk. The American European Journal of Cancer Prevention, Journal of Clinical Nutrition, 49, 675–684. 4, 359–364. 46 Thoreux, K., Senegas-Balas, F., Bernard- 53 Hague, A., Elder, D.J., Hicks, D.J. and Perrone, F., Giannarelli, S., Denariaz, G., Paraskeva, C. (1995) Apoptosis in Bouley, C. and Balas, D. (1996) Modulation colorectal tumour cells: induction by the of proliferation, second messenger levels, short chain fatty acids butyrate, propionate and morphotype expression of the rat and acetate and by the bile salt intestinal epithelial cell line IEC-6 by deoxycholate. International Journal of fermented milk. Journal of Dairy Science, Cancer, 60, 400–406. 79,33–43. 54 Marchetti, C., Migliorati, G., Moraca, R., 47 Choi, S.S., Kim, Y., Han, K.S., You, S., Riccardi, C., Nicoletti, I., Fabiani, R., Oh, S. and Kim, S.H. (2006) Effects of Mastrandrea, V. and Morozzi, G. (1997) Lactobacillus strains on cancer cell Deoxycholic acid and SCFA-induced References j747

apoptosis in the human tumor cell-line during diet supplementation with HT-29 and possible mechanisms. Cancer Lactobacillus rhamnosus GG. Minerva Letters, 114,97–99. Gastroenterol Dietol, 48,45–49. 55 Reddy, B.S. (1998) Prevention of colon 58 De Boever, P., Wouters, R., Verschaeve, L., cancer by pre- and probiotics: evidence Berckmans, P., Schoeters, G. and from laboratory studies. The British Journal Verstraete, W. (2000) Protective effect of of Nutrition, 80, S219–223. the bile salt hydrolase-active Lactobacillus 56 Park, Y.H., Kim, J.G., Shim, Y.W., reuteri against bile salt cytotoxicity. Applied Kim, S.H. and Whang, K.Y.(2007) Effect of Microbiology and Biotechnology, 53, dietary inclusion of Lactobacillus 709–714. acidophilus ATCC43121 on cholesterol 59 Casiraghi, M.C., Canzi, E., Zanchi, R., metabolism in rats. Journal of Microbiology Donati, E. and Villa, L. (2007) Effects of and Biotechnology, 17, 655–662. a synbiotic milk product on human 57 Mirasoli, M., Roda, A., Montagnani, M., intestinal ecosystem. Journal of Applied Azzaroli, F. and Roda, E. (2002) Cholic acid Microbiology, 103, 499–506. metabolism in human fecal cultures j749

41 Fatty Acids and Cancer Prevention Elizabeth K. Lund

41.1 Introduction

The potential importance of dietary fat in the process of carcinogenesis has been recognized since the 1930s [1]. Historically, a high fat intake has been considered to be a potential risk factor for a number of cancers [2], in particular breast cancer, but the evidence supporting such a premise has weakened over the recent years [3], as more sophisticated epidemiological methods have systematically excluded related confounding factors such as obesity or meat intake. This early focus on total fat intake has meant that the attributes of individual types of fat have been largely ignored until recent decades. However, Hugh Sinclair had already suggested that ﬁsh oils might be important in protecting against cancer as early as 1956 [4] and, over 30 years ago, Pearce and Dayton [5] highlighted a concern that in a cohort of over 400 elderly men put on a diet designed to reduce cardiovascular disease, that is, one containing in the region of 40% of the fat as polyunsaturated fatty acids (PUFAs), cancer incidence was approximately double that in the control group after 8 years. In a preceding paper relating to the same study, the fat source was reported to be predominantly corn oil, which would have led to a diet high in omega-6 fatty acids. The consequence of the lack of awareness as to the importance of the type of PUFAs in the diet is that the quality of data collected historically in ecological, case–control, and cohort studies has been compromised, with food frequency questionnaires often only having two or three questions on relevant food stuffs such as seafood or types of cooking fats and in many cases the use of supplements has not been reported. Furthermore, the quality of data on the different fatty acids in the food compositional databases has been sparse and when it is listed can only be an estimate based on a wide variety of sources of the relevant foods. The body is also exposed to short chain fatty acids such as acetate, butyrate, and propionate formed as a result of bacterial fermentation of nonabsorbed carbohydrate in the colonic lumen, or present in fermented foods; however, these are outside the

scope of this chapter where I have chosen to focus simply on the longer chain fatty acids derived from the diet and, in particular, the polyunsaturated fatty acids.

41.2 Fatty Acid Structure

We consume fatty acids in our diet derived from both plant and animal sources. All sources of fats contain a mixture of saturated, monounsaturated, and polyunsaturated fatty acids with the hard fats such as lard and butter being predominantly composed of saturated fats while oils contain a much higher percentage of unsaturated fatty acids, either monounsaturated as in olive oil or polyunsaturated as in maize oil. Fatty acids are consumed largely as triglycerides, three fatty acids esterified with a glycerol backbone, which are derived from the fat stores of both plants and animals. We also consume a wide range of phospholipids derived from cell membranes. The free fatty acid is removed from the glycerol or phosphate group during digestion, before absorption and re-esterification in the intestinal mucosa. Fatty acids are in many cases described by a nonsystematic name, often related to the food they were first derived from as well as by a systematic name. To avoid confusion, it is normal to use an abbreviated symbol describing the number of carbon atoms in the chain followed by the degree of saturation, such that octade-

canoic acid (C18H36O2) is more frequently referred to as stearic acid (C18 : 0) while octadec-9-enoic acid (C18H34O2) is called oleic acid (C18 : 1) and has 1 double bond in the 9th position from the carboxyl terminal (Scheme 41.1). Oleic acid is a monounsaturated fatty acid (MUFA) while those with more than one double bond are referred to as PUFAs. The vast majority of dietary fatty acids have an even number of carbon atoms in the chain and most PUFAs are found in the cis form, the trans form being produced during the processing of oils to form fats with a higher melting point. The trans fatty acids have a more linear structure and so pack more closely together than those in the cis form. In contrast to their systematic names, in biology the position of the double bond is counted from the methyl end of the fatty acid as this better links those fatty acids with related functions, metabolism, and activities. Thus, we have the n-6 or omega-6 fatty acids such as linoleic acid (LA), C18 : 2 n-6 and gamma-linolenic acid (GLA), C18 : 3 n-6, or the omega 3 fatty acids such as alpha-linolenic acid (ALA), C18 : 3 n-3. One group of oils that has received a great deal of attention in relation to a wide range of chronic diseases are the fish oils: eicosapentaenoic acid (EPA), C20 : 5 n-3 and docosahexaenoic acid (DHA), C22 : 6 n-3. These fatty acids are not actually produced by fish but are derived from the phytoplankton found in cold water regions. There is limited, but strengthening, evidence that these may help to reduce the risk of some cancers as well as their better-recognized roles in the prevention of inflammatory diseases and cardiovascular disease. Finally, conjugated linoleic acid (CLA) has received some attention as potentially protective. Conjugated fatty acids are found naturally in meat and dairy products and are distinguished by having at least two double bonds adjacent to each other. 41.3 Bioavailability j751

Scheme 41.1 Fatty acid structures demon- active fatty acids but drawn in linear form. strating the naming of fatty acids as used in The position of the first double bond from the biology. Part (a) uses the C18 series of fatty acids methyl end is also important in the formation of to demonstrate the impact of additional double a wide range of metabolites such as eicosanoids, bonds on the linearity of the fatty acids, which in docosanoids, and resolvins involved in cell turn impacts on membrane fluidity. Part (b) signaling. shows the structures of the most biologically

41.3 Bioavailability

Absorption of fatty acids from the intestinal lumen relies not only on the effective emulsiﬁcation of foods and hydrolysis of lipids but also on successful transfer across the apical membrane. Transport into the enterocyte is believed to occur by a combination of diffusion and a saturable protein-mediated transport, but the relative 752j 41 Fatty Acids and Cancer Prevention

importance of each route is not yet established [6]. We do, however, know that effective transport of very long chain PUFAs depends on the presence of intestinal fatty acid transport proteins such as I-FATPand FABPpm (plasma membrane fatty acid binding protein), andthat polymorphisms in their structure may lead to malabsorption of these fatty acids [7]. Whether these deal with n-3 and n-6 fatty acids differently does not appear to have been investigated, although FABPs in the brain are known to have a greater affinity for the n-3 than n-6 PUFAs [8]. In the enterocyte, the fatty acids are re- esterified into triglycerides and incorporated into chylomicrons before being exported from the enterocyte in chlomicrons. Once in the body, fatty acids are distributed through out the body predominantly in lipoprotein particles before a reversal of the uptake process occurs to release the fatty acid into the cell. Within the cell, PUFAs are incorporated into the membrane-associated phospholipid fraction with subsequent potential influence on formation of different classes of eicosanoids, docosanoids, and resolvins, membrane fluidity and cell signaling. Following supplementation, individual fatty acid concentrations in the plasma can reach as high as 400 uM, albeit much of this will be esterified and associated with lipoproteins [9].

41.4 Epidemiology

The epidemiological evidence for a protective effect of n-3 fatty acids is subject to the well-recognized limitations of all such studies as well as those more specific to this group, outlined above. Two recent reviews of this area from The World Cancer Research Fund – WCRF/AICR [10] and by Maclean et al. [11] suggest that there is no substantial evidence for a protective effect of these PUFAs from ecological studies, which is observational rather than intervention studies. The review by Maclean et al., that is observational rather than focuses on 19 cohort studies covering 11 different categories: lung, breast, aerodigestive, prostate, skin, bladder, colorectal, lymphoma, ovarian, pancreatic, and stomach. She did not, however, included the European Prospective Investigation into Cancer and Nutrition Study (EPIC) [12] in her report as well as a number of other recent studies. The WCRF/AICR report reviewed the literature for 18 sites and reported on fish intake in 15 of these and referred to the omega PUFAs for 9 sites (data on CD associated with the report). The degree to which PUFA intake was broken down to n-3 versus n-6 or the constituent fatty acids is very variable between studies, for example, ALA is usually included in total n-3 fatty acids although it is probably not biologically active and is poorly converted to EPA and particularly DHA. Although there were a significant number of studies looking at colorectal and breast cancer, the conclusions of the panel were that there was no association between breast cancer and fish or n-3 fatty acid intake but for colorectal cancertheysuggestthereislimitedevidencefora protectiveeffectof fish.Nobenefitof fish consumption or n-3 fatty acids is reported in relation to breast cancer, even in the EPIC study where there was a wide range of fish consumption across the populations [13]. For all the other cancers examined, both the amount and quality of the data available meant no conclusions could be drawn. 41.5 Animal Models j753

In the most recent analysis of the colorectal cancer data in relation to fish intake [14], Geelen et al. conclude that there is a slight protective effect of fish consumption particularly on women and on populations with a wider range of intake. This effect is more significant for recent studies where the quality of dietary assessment methods has improved. This meta-analysis gives greater weight to the EPIC study that assesses the impact of fish intake over a much wider range of populations and shows a very clear dose–response effect in relation to intake and risk. Those in the population eating fish at least twice a week were shown to have a 40% reduction in risk, with this level of intake counteracting increased risk associated with red meat consumption. Whether the effects of fish are related to the n-3 fatty acid content is unclear because of the poor quality of the data available for assessing intake. However, previous human intervention studies with n-3 fatty acids, using biomarkers of risk such as crypt cell proliferation and apoptosis [15,16], suggest these lipids are beneficial to intestinal health. There is no epidemiological evidence of a protective effect of conjugated fatty acids in the diet.

41.5 Animal Models

While epidemiological studies have provided only limited evidence that the pattern of dietary fatty acids may influence cancer risk, animal studies are more convincing, albeit often with rather extreme differences in dietary composition far in excess of anything realistically achievable for most people. However, the disease models themselves are usually more aggressive than that found for most human cancers and so these studies can provide useful data. Colorectal cancer studies using chemical carcinogens, subcutaneous injection of HT29 cells, or the use of the APCmin mouse model, all suggest the consumption of fish oil containing EPA and DHA compared to corn oil (rich in LA C18 : 2 n-6) reduces tumor number and/or size and that this is associated with reduced cell proliferation and increased apoptotic cell death [17–19]. A protective effect of a-linolenic acid (C18 : 3 n-3) has been reported in some cases but the effect is less consistent across studies. Animal-based studies have examined the effects of different PUFAs not only on tumor number but also on markers of increased risk of cancer, that is increased cell proliferation and apoptosis and the numbers and size of premalignant lesions such as aberrant crypt foci (ACF). These studies suggest that marine n-3 fatty acids can both stimulate apoptosis and also suppress mitosis while LA has the opposing effect. There is also evident that the naturally occurring isomers of CLAs reduce the tumor number in the APCmin mouse model but that the commercially available isoforms have the opposite affect [20] but that all forms may help to reduce metastasis [21]. The literature related to prevention of breast cancer using animal models is similar to that for colorectal cancer models [22] and recent studies have also suggested a protective effect of the fish oil derived fatty acids in relation to prostate cancer in both genetically susceptible Pten knockout mice and in animals with implanted 754j 41 Fatty Acids and Cancer Prevention

tumor cells [23,24]. Studies investigating the effects of different PUFAs on animal models of other cancers are relatively scarce. For example, we have shown that n-3 fatty acids might be able to modify the risk of esophageal adenocarcinoma [25]. In the case of hepatic cancer, it has been shown that n-3 fatty acids reduce metastasis of an implanted tumor cell line and the subsequent proliferation by reducing the adhesion of the cells to endothelial cells [26], although there is conﬂicting evidence as to whether these fatty acids prevent or promote colon cancer metastasis in the liver [27,28]. Additionally, it has been shown that n-6 fatty acids increase metastasis of breast cells to the lung in a dose-dependent manner [29], while gamma linolenic acid has been suggested as a preventative treatment in bladder cancer [30].

41.6 In Vitro Studies

A wide range of cell lines have been used to assess the impact of fatty acids on cell proliferation andapoptosis. Cell culturestudies provideconsistent datato suggestthat PUFAs reduce cell number in many cancer cell lines with DHA and EPA having the most impact, while ALA, GLA, and AA may have the intermediate effects and LA has little or no effect on cell number at physiological concentrations. Such studies also provide useful insight into mechanisms of action but should always be interpreted with caution, as doses and delivery methods may not always be very physiological.

41.7 Mechanisms of Action

The key factors in limiting tumor initiation and early progression are mitosis and apoptosis. n-3 PUFAs, especially EPA and DHA are affective in reducing the cell number in several cancer cell lines. Such a reduction in cell number can be achieved by either reduced cell division or increased apoptosis or a combination. In the colorectal cancer cell line, HT-29, both effects can be observed [31]. Similarly, in rat models of colorectal cancer it has been shown that dietary fish oil both increases apoptosis and reduces mitosis [32,33], the effect on apoptosis apparently being the better predictor of tumor outcome [34]. We have shown similar effects in the terminal end bud of the rat mammary gland in healthy adult rats fed fish oil (17% energy as fat) [35], while Olivo and Hilakivi-Clarke [36] reported similar results in low fat diets (16% energy from fat) containing fish oil rather than corn oil, fed prepuberty, but this response was reversed in rats on a high fat diet (39% energy as fat). Similarly, fish oil has also been reported to inhibit carcinogenesis in the prostate gland as compared to corn oil [23,24]. n-3 PUFAs may not only reduce tumor progression through effects on mitosis and apoptosis but they can also reduce angiogenesis compared to n-6 fatty acids [37]. How then can these biologically active lipids have such diverse effects on a wide range of risk factors? Many potential mechanisms have been proposed, all of which 41.7 Mechanisms of Action j755

Figure 41.1 A summary of the several routes by themselves but also to intracellular lymphocytes, which polyunsaturated fatty acids may act on lamina propria cells, macrophages, and dendritic colorectal epithelial cell survival by altering cells will potentially also affect survival of any apoptosis, cell proliferation and cell differentia- damaged cells by for example impacting on Fas tion. Although focused on colorectal cancer signaling. Modulation of cell signaling also these mechanisms are likely to be generic for affects expression of genes associated with many cell types liable to develop into tumors. metastasis and angiogenesis. Changes not only to the epithelial cells

probably interact with each other. These have recently been very thoroughly reviewed by Calviello et al. [38,39] and are summarized in Figure 41.1. First, we know that the long chain PUFAs, AA, and EPA are substrates for the cyclooxygenases (COX) and lipoxygenases (LOX) that catalyze the first step in the formation of a large class of more or less proinflammatory signaling molecules: the prostaglandins, thromboxanes, and leukotrienes. The prostaglandins formed from AA tend to be very inflammatory whilst those from EPA may show little or no inflammatory activity. The two types of fatty acids compete for cyclooxygenase and lipoxygenase such that the ratio of fatty acids in the diet will impact on the pattern of eicosanoid production in different tissues [40] as well as that of docosanoid and resolving production [41]. However, fatty acids not only impact on prostanoid synthesis by this direct effect but they have also been shown to modify expression of COX-2 at the mRNA and protein level in both cell lines and human tissue [42–44]. The impact of different PUFAs on inflammation and COX-2 expression is of considerable interest as cancer development is more common in people with certain inflammatory conditions and COX-2 expression is elevated in many cancers. Furthermore, the use of COX inhibitors shows their considerable potential as chemotherapeutic agents [45]. Intriguingly, COX-2 expression has also been linked to changes in b-catenin signaling such that a downregulation of PGE-2 production 756j 41 Fatty Acids and Cancer Prevention

arising as a result of n-3 PUFA exposure would induce b-catenin degradation. It is hypothesized that this would lead to a reduction in expression of survivin, a gene controlled by the b-catenin signaling pathway, and thus an increase in apoptosis. b-Catenin also controls the expression of other cancer-related proteins such as PPAR-d, MMP-7, and VEGF, all previously shown to be modulated by n-3 PUFAs [38,46,47]. Furthermore, it is widely reported that COX-2 protein inhibits apoptosis, so that a reduction in COX-2 induced by n-3 fatty acids would again be predicted to increase cancer cell death. Alterations in the expression of a wide range of other proteins associated with apoptosis, cell cycling, and differentiation, which are induced by n-3 PUFAs as opposed to n-6 PUFAs, have been reported in the literature. These include the BCL family of proteins, p21Cip/Waf1, cyclins (E, D, A), cyclin-dependent kinase inhibitors (p27, p57, p19), c-MYC, and nSREBP. Additionally, other proteins related to angiogenesis and metastasis such as E-cadherin, CAM-1 and MMP-2 and -9 are all downregulated by n-3 PUFAs. Inducible nitric oxide synthase (iNOS) is also reduced in response to these fatty acids, an effect which would be predicted to reduce the metastatic potential of cancer cells [39]. It is now also apparent that n-3 PUFAs might also be able to increase DNA repair at least in breast cancer [48]. It is, as yet, unclear as to how all these changes in gene signaling interrelate or are initiated; however, it is likely that changes in membrane fluidity and thus receptor- mediated signaling are involved as well as the changes mediated through the eicosanoid pathway discussed above. Additionally, we know that fatty acids can act as ligands for the family of peroxisome proliferator-activated receptors (PPARs) that are themselves activated by prostaglandins [49]. PPARs control expression of a number of proteins associated with cell cycle, apoptosis, and differentiation as well as those related to lipid metabolism and drug metabolizing enzymes. In turn, their activity is regulated by phosphorylation, mediated through a diverse range of signaling molecules including ERK, JNK, MAPK, TGF-b, PKA, and PKC [50] many of which have been linked to EPA- or DHA-mediated changes in cell function in obesity and inflammation. However, reporter assays suggest this mechanism cannot fully explain the differences in effect between n-3 and n-6 series fatty acids. However, differences in response may be due to these two lipid groups differentially binding to the obligate dimer RXR [18]. Another important aspect of the PUFAs is the number of double bonds and thus their propensity to become oxidized. It is hypothesized that the presence of high levels of PUFAs in the cell and mitochondrial membrane makes cells more vulnerable to oxidative stress and this in turn reduces glutathione levels and induces apoptosis. We have shown that the addition of lipid-soluble antioxidants inhibits the apoptosis induced by fish oils in vitro and that the reduction of glutathione increases apoptosis in the DMH rat model of colorectal cancer [51]. Changes in intracellular redox state are known to modify cell-signaling pathways, modifying MAP kinases, NF-kB, and AP-1, as well as an increase in apoptosis being observed in a more oxidized environment [52]. All these effects show clear lipid peroxidation dependency and thus may provide an explanation as to why the use of antioxidant vitamins in human intervention studies has proven to be counterproductive [53]. However, both References j757 n-6 and n-3 fatty acids are liable to peroxidation and so any opposing effects of these two groups of lipids can only partially be explained by such a mechanism.

41.8 Conclusion

There is increasing epidemiological evidence that consumption of ﬁsh oils may be protective against cancer, particularly of the colon. There is a considerable body of evidence from animal studies to support this hypothesis and a wide range of interrelated potential mechanisms to explain such observations. It would seem wise to encourage populations to increase their intake of n-3 fatty acids and perhaps reduce intake of n-6 fatty acids based on current evidence.

References

1 Watson, A. and Mellanby, E. (1930) Tar 8 Hanhoff, T., Lucke, C. et al. (2002) Insights cancer in mice. II. The condition of skin into binding of fatty acids by fatty acid when modified by external treatment or binding proteins. Molecular and Cellular diet, as a factor in influencing this Biochemistry, 239 (1–2), 45–54. cancerous reaction. British Journal of 9 Marangoni, F., Angeli, M.T. et al. (1993) Experimental Pathology, 11, 311–322. Changes of n-3 and n-6 fatty acids in 2 Hill, M.J. (1987) Dietary fat and human plasma and circulating cells of normal cancer (review). Anticancer Research, subjects, after prolonged administration of 7 (3 Pt A), 281–292. 20:5 (EPA) and 22:6 (DHA) ethyl esters and 3 Rothstein, W.G. (2006) Dietary fat, prolonged washout. Biochimica et coronary heart disease, and cancer: Biophysica Acta, 1210 (1), 55–62. a historical review. Preventive Medicine, 10 WCRF/AICR (2007) Food, Nutrition, 43 (5), 356–360. Physical Activity, and the Prevention of 4 Sinclair, H.M. (1956) Deficiency of Cancer: A Global Perspective, World Cancer essential fatty acids and atherosclerosis, Research Fund/American Institute for etcetera. Lancet, 270 (6916), 381–383. Cancer Research. 5 Pearce, M.L. and Dayton, S. (1971) 11 Maclean, C.H., Newberry, S.J. et al. (2005) Incidence of cancer in men on a diet high Effects of omega-3 fatty acids on cancer. in polyunsaturated fat. Lancet, 1 (7697), Evidence Report/Technology Assessment 464–467. (Summary), No. 113, pp. 1–4. 6 Porter, C.J., Trevaskis, N.L. et al. (2007) 12 Norat, T., Bingham, S. et al. (2005) Meat, Lipids and lipid-based formulations: fish, and colorectal cancer risk: the optimizing the oral delivery of lipophilic European Prospective Investigation into drugs. Nature Reviews. Drug Discovery, 6 (3), Cancer and Nutrition. Journal of the 231–248. National Cancer Institute, 97 (12), 906–916. 7 Stremmel, W. (1988) Uptake of fatty acids 13 Engeset, D., Alsaker, E. et al. (2006) Fish by jejunal mucosal cells is mediated by consumption and breast cancer risk. The a fatty acid binding membrane protein. European Prospective Investigation into The Journal of Clinical Investigation, 82 (6), Cancer and Nutrition (EPIC). International 2001–2010. Journal of Cancer, 119 (1), 175–182. 758j 41 Fatty Acids and Cancer Prevention

14 Geelen, A., Schouten, J.M. et al. (2007) Fish model simulating radical prostatectomy. consumption, n-3 fatty acids, and Neoplasia, 8 (2), 112–124. colorectal cancer: a meta-analysis of 24 Berquin, I.M., Min, Y. et al. (2007) prospective cohort studies. American Modulation of prostate cancer genetic risk Journal of Epidemiology, 166 (10), by omega-3 and omega-6 fatty acids. 1116–1125. The Journal of Clinical Investigation, 117 (7), 15 Anti, M., Marra, G. et al. (1997) Modulating 1866–1875. effect of omega-3 fatty acids on the 25 Lee, G., Mehta, S. et al. (2005) Impact of proliferative pattern of human colorectal aspirin and fish oil on development of mucosa. Advances in Experimental Medicine esophageal adenocarcinoma in a rat and Biology, 400, 605–610. surgical model assessed microscopically 16 Courtney, E.D., Matthews, S. et al. (2007) and by gene expression, Cpg island Eicosapentaenoic acid (EPA) reduces crypt methylation and Western blot. cell proliferation and increases apoptosis Gastroenterology, 128 (Suppl. 2) (4), in normal colonic mucosa in subjects with A22–A23. a history of colorectal adenomas. 26 Iwamoto, S., Senzaki, H. et al. (1998) International Journal of Colorectal Disease, Effects of fatty acids on liver metastasis of 22 (7), 765–776. ACL-15 rat colon cancer cells. Nutrition and 17 Roynette, C.E., Calder, P.C. et al. (2004) n-3 Cancer, 31 (2), 143–150. polyunsaturated fatty acids and colon 27 Griffini, P., Fehres, O. et al. (1998) Dietary cancer prevention. Clinical Nutrition, omega-3 polyunsaturated fatty acids 23 (2), 139–151. promote colon carcinoma metastasis in rat 18 Lund, E. (2006) Dietary fatty acids and liver. Cancer Research, 58 (15), 3312–3319. colon cancer. Scandinavian Journal of Food 28 Gutt, C.N., Brinkmann, L. et al. (2007) and Nutrition, 50 (1), 39–44. Dietary omega-3-polyunsaturated fatty 19 Johnson, I.T. and Lund, E.K. (2007) Review acids prevent the development of article: nutrition, obesity and colorectal metastases of colon carcinoma in rat liver. cancer. Alimentary Pharmacology & European Journal of Nutrition, 46 (5), Therapeutics, 26 (2), 161–181. 279–285. 20 Mandir, N. and Goodlad, R.A. (2008) 29 Rose, D.P., Hatala, M.A. et al. (1993) Effect Conjugated linoleic acids differentially of diets containing different levels of alter polyp number and diameter in the linoleic acid on human breast cancer Apc(min/ þ ) mouse model of intestinal growth and lung metastasis in nude mice. cancer. Cell Proliferation, 41 (2), 279–291. Cancer Research, 53 (19), 4686–4690. 21 Soel, S.M., Choi, O.S. et al. (2007) 30 Solomon, L.Z., Jennings, A.M. et al. (1998) Influence of conjugated linoleic acid Is gamma-linolenic acid an effective isomers on the metastasis of colon cancer intravesical agent for superficial bladder cells in vitro and in vivo. The Journal of cancer? In vitro cytotoxicity and in vivo Nutritional Biochemistry, 18 (10), 650–657. tolerance studies. Urological Research, 22 Yee, L.D., Young, D.C. et al. (2005) Dietary 26 (1), 11–15. (n-3) polyunsaturated fatty acids inhibit 31 Clarke, R.G., Lund, E.K. et al. (1999) Effect HER-2/neu-induced breast cancer in mice of eicosapentaenoic acid on the independently of the PPARgamma ligand proliferation and incidence of apoptosis rosiglitazone. The Journal of Nutrition, in the colorectal cell line HT29. Lipids, 135 (5), 983–988. 34 (12), 1287–1295. 23 Kelavkar, U.P., Hutzley, J. et al. (2006) 32 Chang, W.L., Chapkin, R.S. et al. (1998) Prostate tumor growth and recurrence can Fish oil blocks azoxymethane-induced rat be modulated by the omega-6:omega-3 colon tumorigenesis by increasing cell ratio in diet: athymic mouse xenograft differentiation and apoptosis rather than References j759

decreasing cell proliferation. The Journal of docosahexaenoic acid in human colon Nutrition, 128 (3), 491–497. cancer cells. Cancer Research, 63 (5), 33 Latham, P., Lund, E.K. et al. (1999) Dietary 972–979. n-3 PUFA increases the apoptotic response 41 Calder, P.C. (2007) Immunomodulation to 1,2-dimethylhydrazine, reduces mitosis by omega-3 fatty acids. Prostaglandins, and suppresses the induction of Leukotrienes, and Essential Fatty Acids, carcinogenesis in the rat colon. 77 (5–6), 327–335. Carcinogenesis, 20 (4), 645–650. 42 Llor, X., Pons, E. et al. (2003) The effects of 34 Chang, W.C., Chapkin, R.S. et al. (1997) fish oil, olive oil, oleic acid and linoleic acid Predictive value of proliferation, on colorectal neoplastic processes. Clinical differentiation and apoptosis as Nutrition, 22 (1), 71–79. intermediate markers for colon 43 Lee, G., Lund, E. et al. (2004) Suppression tumorigenesis. Carcinogenesis, 18 (4), of COX-2 expression in human 721–730. adenocarcinoma cells (OE33) cultured 35 Kramer, F., Lund, E.K. et al. (2003) with eicosapentaenoic acid (EPA). Gut, A comparison of the effect of soy 53 (100 Suppl. 3), A26–A126. isoflavonoids and fish oil on cell 44 Mehta, S., Boddy, A. et al. (2008) Effect of proliferation, apoptosis and expression n-3 polyunsaturated fatty acids on Barretts of oestrogen receptor and in the epithelium in the human lower mammary gland and colon of the rat. esophagus. American Journal of Clinical Proceedings of the Nutrition Society, Nutrition, 87 (4), 949–956. 61, 148. 45 Goossens, L., Pommery, N. et al. (2007) 36 Olivo, S.E. and Hilakivi-Clarke, L. (2005) COX-2/5-LOX dual acting anti- Opposing effects of prepubertal low- and inflammatory drugs in cancer high-fat n-3 polyunsaturated fatty acid chemotherapy. Current Topics in diets on rat mammary tumorigenesis. Medicinal Chemistry, 7 (3), 283–296. Carcinogenesis, 26 (9), 1563–1572. 46 Bassaganya-Riera, J. and Hontecillas, R. 37 Szymczak, M., Murray, M. et al. (2008) (2006) CLA and n-3 PUFA differentially Modulation of angiogenesis by omega-3 modulate clinical activity and colonic polyunsaturated fatty acids is mediated by PPAR-responsive gene expression in a pig cyclooxygenases. Blood, 111 (7), model of experimental IBD. Clinical 3514–3521. Nutrition, 25 (3), 454–465. 38 Calviello, G., Resci, F. et al. (2007) 47 Kobayashi, N., Barnard, R.J. et al. (2006) Docosahexaenoic acid induces Effect of altering dietary omega-6/omega-3 proteasome-dependent degradation of fatty acid ratios on prostate beta-catenin, down-regulation of survivin cancer membrane composition, and apoptosis in human colorectal cancer cyclooxygenase-2, and prostaglandin E2. cells not expressing COX-2. Carcinogenesis, Clinical Cancer Research, 12 (15), 28 (6), 1202–1209. 4662–4670. 39 Calviello, G., Serini, S. et al. (2007) n-3 48 Hilakivi-Clarke, L., Olivo, S.E. et al. (2005) Polyunsaturated fatty acids and the Mechanisms mediating the effects of prevention of colorectal cancer: prepubertal (n-3) polyunsaturated fatty molecular mechanisms involved. acid diet on breast cancer risk in rats. Current Medicinal Chemistry, 14 (29), The Journal of Nutrition, 135 (Suppl. 12), 3059–3069. 2946S–2952S. 40 Narayanan, B.A., Narayanan, N.K. et al. 49 Stoll, B.A. (2002) n-3 fatty acids and lipid (2003) Modulation of inducible nitric oxide peroxidation in breast cancer inhibition. synthase and related proinflammatory The British Journal of Nutrition, 87 (3), genes by the omega-3 fatty acid 193–198. 760j 41 Fatty Acids and Cancer Prevention

50 Burns, K.A. and Vanden Heuvel, J.P. 52 Jackson, M.J., Papa, S. et al. (2002) (2007) Modulation of PPAR activity via Antioxidants, reactive oxygen and nitrogen phosphorylation. Biochimica et Biophysica species, gene induction and mitochondrial Acta, 1771 (8), 952–960. function. Molecular Aspects of Medicine, 51 Latham, P., Lund, E.K. et al. (2001) Effects 23 (1–3), 209–285. of cellular redox balance on induction of 53 Russell, R.M. (2004) The enigma of beta- apoptosis by eicosapentaenoic acid in carotene in carcinogenesis: what can be HT29 colorectal adenocarcinoma cells and learned from animal studies. The Journal of rat colon in vivo. Gut, 49 (1), 97–105. Nutrition, 134 (1), 262S–268S. j761

42 Protease Inhibitors Ann R. Kennedy

42.1 Introduction

Protease inhibitors are a well-established class of cancer chemopreventive agents, and many different types of protease inhibitors exhibit cancer chemopreventive effects [1–6]. The protease inhibitor that has been evaluated the most extensively as a cancer chemopreventive agent is the soybean-derived protease inhibitor, the Bowman–Birk inhibitor (BBI), which has been evaluated in human trials in the form of BBI Concentrate (BBIC) [1–4]. BBIC is an extract of soybeans enriched in BBI, which has the ability to prevent carcinogenesis in many different in vitro and in vivo animal carcinogenesis assay systems [1–4]. BBIC achieved Investigational New Drug (IND) status with the Food and Drug Administration (FDA) in the United States in 1992, and numerous human trials have been performed [7]. BBI/BBIC also has anti-inflammatory activity in animal assay systems (reviewed in Refs. [1, 3]). It is expected that BBI is responsible for both the anticarcinogenic and anti-inflammatory activities of BBIC. While the mechanisms for the anticarcinogenic effects of BBI are unknown, several different mechanisms are possible. The most likely explanation for the anti-inflammatory effects of BBI is the direct and potent inhibitory effects of BBI on specific proteases playing major roles in inflammation [3].

42.2 Physicochemical Properties of Active Compounds and Their Occurrence

Protease inhibitors are widely distributed in both the plant and animal kingdoms. They are found in many common foods, including legumes, cereals, oilseeds, nuts, fruits, vegetables, eggs, potatoes, and other dairy and animal products. Many vegetables contain protease inhibitors that are likely to be cancer chemopreventive agents [1]. Soybeans are unusually rich in protease inhibitor activity, and several

different protease inhibitors are known to be present in soybeans. It is believed that the protease inhibitor activity involved in both anticarcinogenic and anti-inﬂamma- tory effects is the chymotrypsin inhibitor (C.I.) activity [1–4, 8]. In soybeans, the protease inhibitor with C.I. activity is BBI [1–4, 8]. The structure of BBI has been determined by Odani and Ikenaka [9] and is discussed in reference [3].

42.3 Bioavailability and Metabolism of Active Compounds

It is known from animal studies that, when ingested as part of the diet, BBI reaches the intestines in an intact form, is taken up into the bloodstream and distributed to organs throughout the body [3]. Information about the absorption, distribution, and excretion of BBI comes primarily from animal studies utilizing radiolabeled BBI. Studies indicate that approximately half of the BBI administered orally is excreted in the feces in an unaltered form, while the rest enters the intestinal epithelial cells or crosses the intestinal lumen via a paracellular mechanism. At 3 h after an oral 125I-BBI dose, BBI is widely distributed in the body and is present in an active form in all major internal organs (with only very low levels in the brain). It is known that some of the BBI excreted into the urine still possesses protease inhibitor activity. When 125I-BBI is administered to animals by oral gavage, the calculated serum half-

life (T1/2) is 10 h in both rats and hamsters [3]. While the amounts of BBI reaching organs outside the gastrointestinal tract following oral administration in cancer prevention studies are relatively low, the concentrations of BBI reaching internal organs, such as the liver, breasts, prostate, lung, and so on, are expected to be sufﬁcient on a cellular/molar basis to have cancer chemopreventive activity in those organs [3, 10].

42.4 Mechanisms of Protection and Results of In Vitro and Animal Studies

BBI is known to inhibit the proteolytic activity of several well-characterized proteases, including trypsin, chymotrypsin, cathepsin G, elastase, and chymase (reviewed in Refs [1, 3]). Despite the ability to inhibit the activity of these proteases, BBI does not have detectable effects, at the doses studied in animals, on the biological functions in which these proteases are known to play important roles [1, 3]. Its ability to prevent cancer is thought to be due to its ability to make various endpoints altered by carcinogen exposure return to a normal state, as reviewed elsewhere [1–4, 8]). BBI has the ability to return the levels of expression of a number of different biologically active substances whose levels have been altered by exposure to external stimuli (e.g., carcinogens) to levels that are approximately normal for the system being examined. The ability of BBI to regulate biologic phenomena may be due to a number of different effects, some of which are as follows. BBI has some antioxidant activity, the ability to modify arachidonic acid metabolism, and the capability to alter patterns 42.4 Mechanisms of Protection and Results of In Vitro and Animal Studies j763 of gene expression, as reviewed in several references [1–4, 8]. BBI is known to be highly anti-inflammatory, presumably due to its ability to affect the activity of one or more proteases playing important roles in inflammatory processes (e.g., cathepsin G, elastase, and chymase) and its ability to affect the production of free radicals (reviewed in Refs. [1–4]). The ability of BBI to serve as an anti-inflammatory agent does not appear to affect the normal functioning of the immune system [3]. It has been reported that BBI stimulates DNA repair mechanisms in normal tissues, such as nucleotide excision repair and nonhomologous end joining, which can explain its ability to serve as a radioprotective agent [11, 12]. The radioprotective effect of BBI depends on the presence of a functional wild-type TP53. Since many tumors have lost TP53 function during their development, it has been suggested that BBI could be used as a radioprotective agent during radiotherapy of tumors characterized by a mutant TP53 [11, 12]. BBI could have other beneficial uses during or after radiation therapy as well [3]. As an example, it is likely that it could prevent second malignancies from developing as a result of radiation therapy, as it is known to prevent radiation-induced carcinogenesis in vivo (e.g., Ref. [13]), as illustrated in Figure 42.1.

Figure 42.1 Inhibition of iron ion or proton killed). Exposure to proton or iron ion radiation radiation-induced malignant lymphoma or rare significantly increased the incidence rates of tumors in mice by Bowman–Birk Inhibitor malignant lymphoma (p < 0.001) and rare Concentrate (BBIC). Male CBA/JCR HSD mice tumors (p < 0.02), and dietary supplementation were irradiated at the NASA Space Radiation with BBIC, at 1% (w/w) of the animal diet, Laboratory (Brookhaven National Laboratory, reduced the incidence rates of malignant Upton, NY) with 0.5 Gy iron ions (1 GeV/n) or lymphoma and rare tumors in the irradiated 3 Gy protons (1 GeV/n) and followed for animals to levels that were not significantly approximately 2 years (when all remaining higher than those in the Sham Radiation Control animals in the various treatment groups were groups (p 0.5). Data are from Ref. [13]. 764j 42 Protease Inhibitors

The most biologically important cancer preventive activity of BBI may not be related to beneﬁcial effects on DNA repair activities, however, as it can be given to cells and animals at long periods after carcinogen exposure and still have cancer preventive activity [1, 3]. For anticarcinogenic activities, it is widely assumed that the crucial time for DNA repair processes to play a beneﬁcial role is during carcinogen exposure.

42.5 Results of Human Studies

A phase I human trial utilizing a single exposure to BBIC began in patients with oral leukoplakia in 1992. In this study, a total of 24 patients were treated with BBIC at doses from 25–800 C.I. units and studied over a 1-month period. No toxicity or adverse side effects from BBIC were observed [7, 14]. Multiple dosing of patients with oral leukoplakia utilizing doses of BBIC from 100–1066 C.I. units per day for a total period of 6 months occurred as part of a phase IIa trial [15, 16]. BBIC was administered to patients as an oral rinse that was swallowed in this oral cancer prevention trial utilizing patients with oral leukoplakia. A total of 32 patients received BBIC treatment in this trial. BBIC was well tolerated with no evidence of laboratory, symptom or clinical side effects [7, 15, 16]. As part of the phase IIa trial, it was observed that BBIC treatment led to a dose-dependent reduction in lesion size in the patients with oral leukoplakia [7, 15, 16]. Effects on the expression levels of certain types of proteolytic activities and the neu oncogene (used as the surrogate endpoint biomarkers for the trial) were also observed [7, 15–17]. Aphase IIb/III trial of BBIC in patients with oral leukoplakia is currently being performed and is expected to be completed within the next few years. This phase IIb trial is a randomized, double- blind, placebo-controlled trial involving patient treatment for 6 months. Patients in remission at 6 months continue the drug therapy up to a maximum time of 1.5 year on BBIC or placebo tablets. Another phase I human trial utilized a tablet formulation of BBIC in patients with benign prostatic hyperplasia (BPH) [7, 18]. At doses of 100, 200, 400, and 800 C.I. units/day, BBIC tablets (or placebo tablets) were administered to patients for 6 months in this trial; a total of 19 patients were enrolled at doses up to 800 C.I. units per day. For each BBIC dose group, there were three patients taking BBIC and one taking the placebo formulation; an additional three patients were added to the 800 C.I. units/ day dosage group. Thus, 15 of the patients were treated with doses of BBIC. This was a double-blind, randomized, phase I trial of BBIC in BPH patients. As part of this trial, there was no dose-limiting toxicity of BBIC observed. Other findings from the trial are as follows. There was a statistically significant decrease in serum PSA levels in all BBIC-treated patients. Some BBIC-treated patients exhibited a relatively large reduction in serum PSA levels, ranging up to a 43% reduction. There was also a statistically significant decrease in serum triglyceride levels and a decrease in prostate volume in the treated patients. The scores recorded in response to a urinary symptom questionnaire indicated improved urinary activities in the BBIC-treated patients; 42.5 Results of Human Studies j765 however, the control subjects exhibited similar improvements in urinary activities during the course of the trial. The data obtained in this trial, particularly the data suggesting that BBIC treatment may lead to reduced serum PSA levels and reduced prostate volumes, suggest that BBIC could be useful for prostate cancer chemoprevention. A new prostate cancer prevention program utilizing BBIC has begun at the University of Pennsylvania. Another area of BBIC human trials involved patients with ulcerative colitis (left- sided disease or pancolitis), who received BBIC or placebo tablets over a 3-month treatment period [19]. This was a randomized, double-blind, placebo-controlled trial: 50% of the patients received placebo tablets and 50% received BBIC tablets at a dosage of 800 C.I. units per day. A total of 14 patients were treated with BBIC in this trial. The primary goals of the trial were to assess the safety of the drug, BBIC, in patients with ulcerative colitis and assess the effect of BBIC on serum inflammation markers and tissue protease levels, which would hopefully serve as disease markers for future trials. No serious adverse events occurred during the trial. Some nausea and disease symptom exacerbation was observed, but these occurred in equal frequency in the active drug group and the placebo group [19]. The trial results suggest a potential benefit over placebo for both achieving clinical response and induction of remission in patients with active ulcerative colitis without apparent toxicity [19]. One patient with metastatic prostate cancer was treated with BBIC tablets for 6 months at a dosage of 800 C.I. units per day. There were no adverse events reported for this patient, and the patient tolerated the BBIC tablets without any difficulties [7]. Serum PSA levels were maintained at approximately a constant level during the BBIC therapy period, but rose to a much higher level following BBIC treatment. The results of PSA measurements for this patient with metastatic prostate cancer suggest that BBIC may have had a growth inhibitory effect on the prostate cancer cells during the treatment period and suggest the possible use of BBIC in prostate cancer patients [7]. Several other BBIC trials are ongoing and the results are not yet available. The BBIC studies performed thus far have utilized BBIC at doses of up to 1066 C.I. units per day for as long as 1.5 years of treatment. It is expected that well over 100 patients have been treated with BBIC thus far (the exact number is unknown, as many of the patients treated with BBIC are part of ongoing current placebo-controlled trials, and the exact number of patients who are receiving or have received BBIC treatment cannot be determined). Thus far, there have been a total of two serious adverse events reported to the US FDA, both of which were considered unrelated to the drug treatment. While some side effects have been reported by patients, it is not clear whether the observed adverse events are drug related in most cases. At this point in the human drug experience with BBIC, some gas or bloating can be considered an expected side effect of drug treatment. This is a common side effect associated with consumption of soybeans and other types of beans. This side effect is expected to last only for approximately a 1-week period after the beginning of high levels of soybean/bean consumption. This side effect has been relatively rare in the BBIC trials performed thus far. This is presumably because BBIC is essentially a protein extract of soybeans, and flatulence or bloating from bean products is due to difficulties in the digestion of the soybean carbohydrates. 766j 42 Protease Inhibitors

42.6 Impact of Cooking Processing

The members of the Bowman–Birk family of protease inhibitors are stable through normal cooking processing [1, 3]. They can, however, be destroyed by autoclaving. Autoclaved BBIC has served as a control substance in numerous BBIC animal studies [1, 3].

References

1 Kennedy, A.R. (1993) Overview: Inhibitor Concentrate. Nutrition and anticarcinogenic activity of protease Cancer, 19, 281–302. inhibitors, in Protease Inhibitors as Cancer 9 Odani, S. and Ikenaka, T.J. (1973) Scission Chemopreventive Agents (eds W. Troll and of soybean Bowman-Birk proteinase A.R. Kennedy), Plenum Publishing inhibitor into two small fragments having Corporation, New York, pp. 9–64. either trypsin or chymotrypsin inhibitor 2 Kennedy, A.R. (1993) In vitro studies of activity. Journal of Biochemistry, 74, anticarcinogenic protease inhibitors, in 857–860. Protease Inhibitors as Cancer 10 Kennedy, A.R. (1995) The evidence for Chemopreventive Agents (eds W. Troll and soybean products as cancer preventive A.R. Kennedy), Plenum Publishing agents. The Journal of Nutrition, 125, Corporation, pp. New York, 65–91. 733s–743s. 3 Kennedy, A.R. (1998) Chemopreventive 11 Dittman, K.H., Mayer, C. and Rodemann, agents: protease inhibitors. Pharmacology H.P. (2003) Radioprotection of normal & Therapeutics, 78, 167–209. tissue to improve radiotherapy: the effect of 4 Kennedy, A.R. (1994) Prevention of the Bowman Birk protease inhibitor. carcinogenesis by protease inhibitors. Current Medicinal Chemistry, 3 (5), Cancer Research, 54 (7 Suppl.), 360–363. 1999s–2005s. 12 Dittmann, K., Toulany, M., Classen, J., 5 Troll, W., Frenkel, K. and Wiesner, R. Heinrich, V., Milas, L. and Rodemann, (1984) Protease inhibitors as H.P. (2005) Selective radioprotection of anticarcinogens. Journal of the National normal tissues by Bowman-Birk Cancer Institute, 73, 1245–1250. proteinase inhibitor (BBI) in mice. 6 Troll, W., Wiesner, R. and Frenkel, K. Strahlentherapie und Onkologie: Organ der (1987) Anticarcinogenic action of protease Deutschen Rontgengesellschaft, 181 (3), inhibitors. Advances in Cancer Research, 49, 191–196. 265–283. 13 Kennedy, A.R., Davis, J.G., Carlton, W. and 7 Kennedy, A.R. (2005) The status of human Ware, J.H. (2008) Effects of dietary trials utilizing Bowman-Birk Inhibitor antioxidant supplementation on the Concentrate from soybeans, in Soy in development of malignant lymphoma and Health and Disease Prevention (ed. M. other neoplastic lesions in mice exposed to Sugano), CRC Press, Boca Raton, FL, pp. proton or iron ion radiation. Radiation 207–223, Chapter 12. Research, 169, 615–625. 8 Kennedy, A.R., Szuhaj, B.F., Newberne, 14 Armstrong, W.B., Kennedy, A.R., Wan, P.M. and Billings, P.C. (1993) Preparation X.S., Atiba, J., McLaren, C.E. and and production of a cancer Meyskens, F.L. Jr (2000) Single dose chemopreventive agent, Bowman-Birk administration of Bowman-Birk Inhibitor References j767

Concentrate (BBIC) in patients with oral and neu oncogene expression in patients leukoplakia. Cancer Epidemiology, with oral leukoplakia treated with the Biomarkers and Prevention, 9,43–47. Bowman-Birk inhibitor. Cancer 15 Armstrong, W.B., Kennedy, A.R., Wan, Epidemiology, Biomarkers and Prevention, X.S., Taylor, T.H., Nguyen, Q.A., Jensen, J., 8, 601–608. Thompson, W., Lagerberg, W. and 18 Malkowicz, S.B., McKenna, W.G., Vaughn, Meyskens, F.L. Jr (2000) Clinical D.J., Wan, X.S., Propert, K.J., Rockwell, K., modulation of oral leukoplakia and Marks, S.H.F., Wein, A.J. and Kennedy, protease activity by Bowman-Birk A.R. (2001) Effects of Bowman-Birk Inhibitor Concentrate in a Phase IIa inhibitor concentrate in patients with Chemoprevention Trial. Clinical Cancer benign prostatic hyperplasia. The Prostate, Research, 6, 4684–4691. 48,16–28. 16 Meyskens, F.L. Jr (2001) Development of 19 Lichtenstein, G.R., Deren, J.J., Katz, S., Bowman-Birk Inhibitor for Lewis, J.D., Kennedy, A.R. and chemoprevention of oral head and neck Ware, J.H. (2008) Bowman-Birk cancer. Annals of the New York Academy of Inhibitor Concentrate (BBIC): Sciences, 952, 116–123. a novel therapeutic agent for patients 17 Wan, X.S., Meyskens, F.L. Jr, Armstrong, with active ulcerative colitis. Digestive W.B. and Kennedy, A.R. (1999) Diseases and Sciences, 53 (1), Relationship between a protease activity 175–180. j769

Index

a agouti viable yellow mouse (AIAP) 155 aberrant crypt foci (ACF) 337, 340, 341, 343, alanine transaminase (ALT) 589 347, 603, 736 alcohol dehydrogenases 98 – biochemical/immunohistochemical alcohol-free beers 655 alterations 341, 343 alcoholic active compounds 642 – development 603 – bioavailability 642 – formation 344, 690 – metabolism 642 – genetic/epigenetic alterations 341 alcoholic beverage 635, 648 aberrant crypt foci causing agents 343 – beer 648 – 3,2-dimethyl-4-aminobiphenyl (DMABP) aldehyde dehydrogenases 99 343 aldehyde-reactive probe (ARP) 25 – N-methyl-N-nitrosourea 343 alkylation-induced mutagenicity 655 2-acetylaminoﬂuorene (2-AAF) 616 alkylbenzenes 49 – induced mutagenesis 616 – eugenol 49 acetylsalicylic acid 204 – safrol 49 acrylic acid 44 all-trans-retinoic acid 380, 381 activating/detoxifying enzymes 219 all-trans retinol 372 active steroid hormones 394 – chemical structure 372 – bioavailability 394 Allium species 5, 212 – 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3; – garlic 5 1,25-D3) 394 – onion 5 – metabolism 394 alpha-linolenic acid (ALA) 750, 753 acute promyelocytic leukemia (APL) 381 alpha-tocopherol and beta-carotene trial – cells 381 (ATBC) 203 adaptive immunity 185 altered hepatic foci (AHF) 337–339 adenosine triphosphate (ATP) 278 – cells 337 – binding cassette drug transporter proteins – foci formation inhibition 339 60 – methodological aspects 338, 341 adhesion molecules 171 – model 345 – cadherins 171 – morphology 337 – integrins 171 – phenotypes 337 advanced glycation end products (AGEs) – promoters action 339 239 Alzheimers disease 417 advanced oxidation protein products amino acid sequence search engines 319 (AOPPs) 238 – Mascot 319 aﬂatoxin B1 (AFB1) 706, 736 – OMSSA 319 – N7-guanine 706 – PEAKS 319

– SEQUEST 319 – angiostatin 15 2-amino-1-methyl-6-phenylimidazo [4,5-b] – antimetastatic activities 164 pyridine (PhIP) 616 antigenotoxicity 212, 220 2-amino-3-methyl-imidazo[4,5-f ]quinoline – coronary heart disease 220 (IQ) 690, 714 – DNA alterations 212 – 14C-labeled 714 – end points 212 – DNA adducts 690 antigen-presenting cells (APC) 184, 192 – genotoxicity 690 antimutagenicity 220 a-amylase-treated high-amylose maize – research 31 starch 724 – specificity of protection 220 androgen-sensitive human prostate cancer antimutagenicity tests 215 (LNCaP) cells 490 – bacterial 215 angiogenesis 291, 295 – disadvantages 215 – assays 291, 293, 297 antioxidant assays 618 – cell-based systems 292 – order of activity 618 – development steps 292 antioxidant compounds (AOCs) 229 – growth factors 15, 166 antioxidant effect(s) 239, 407, 447, 483, 529 – inhibition 65, 164 – mutagenicity tests 239 – organ culture systems 295 antioxidant enzymes 126, 189 – prevention 295 – glutathione peroxidase 189 – stimulating factors 164 antioxidant responsive element (ARE) 247, angiomouse 299 652 – green fluorescent protein (GFP) 299 antioxidant(s) 234, 390, 600 angiopreventive agents 166 – carotenoids 390 animal culture, see xenograft model – defense protein 441 animal models 295, 335, 372, 725, 753–754 – flavonoids 390 – cancer chemopreventive activity 534 – genes 442 – cell-based in vitro assays 295 – phenols 390 – in vivo models 296 – polyphenolics 476 animal tumor models 398 – protection mechanisms 62, 149, 406 anthocyanidins 516, 517 – rich food 84 – bioavailability 518 antioxidative/anti-inflammatory – chemical structure of 518 substances 483 – chemoprotection mechanisms 518 antiproliferative mechanisms 11, 640 – metabolism of 518 antitumor activity 407 – physiochemical properties of 517 AOAC gravimetric analytical method 710 anthocyanins 517, 519 APCmin mouse model 726, 753 – anticarcinogenic effects 519–520, 523 apoptosis 12 – cancer preventative activities of 517 – associated genes 12 – chemoprotection mechanisms 518 apoptosis detection methods 286 – containing foodstuffs 522 – DNA fragmentation 287 – elicit anticarcinogenic effects 522 – mitochondrial transmembrane – epidemiological studies in humans 523 apoptosis-inducing proteins 390 – fruits/vegetables processing 524 – p53 390 – in vitro studies 518 – p73 390 – rich extracts 519, 523 apoptotic cell death 753 – schematic illustration of molecular apoptotic mediators 147 mechanisms 521 apurinic/apyrimidinic (AP) sites 23 anti-inflammatory drugs 149 arachidonic acid metabolism. 639, 762 anti-VEGF therapy 171 – shematic presentation 639 antiangiogenic activity 291 Aronia melanocarpa 527 – matrix metalloproteinases (MMPs) 291 artificial human GI-ecosystem 742 – new blood vessels formation 291 aryl hydrocarbon receptor (AhR) 114 antiangiogenic agents 15 – ligands 111 Index j771

– nuclear translocator 133 – health properties 609 – signaling pathway 114, 116 – impact of heat/processing 622 ascorbate 387, 388 – physicochemical properties 610 – blood plasma concentrations 388 – polyphenols 619 – function 387 – polyphenols in liver/lung 600 – role 387 bovine aortic endothelial cells (BAEC) 292 ascorbinic acid 189 Bowman–Birk inhibitor (BBI) 761–764 ascorbyl free radical 387 – anti-inflammatory activity 761 aspartate transaminase (AST) 589 – cancer preventive activity 764 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic Bowman–Birk inhibitor concentrate acid (ABTS) 231 (BBIC) 761, 763, 764 – assay 617 – anti-inflammatory activity 761 2,20-azobis(2,4-amidinopropane) – human trials 765 dihydrochloride (AAPH) 231 – treated patients 764 azoxymethane (AOM) 740 – treatment 764, 765 – colon carcinogenesis 646 Bradford/Biorad method 254 – tumors 342, 737 Brassica vegetables 221 – models 742 BrdU-labeling 292 breakage-fusion-bridge cycles 428 b breast cancer 206, 553–554, 566–567 bacterial microflora 502 – plasma enterolactone 566 bacterial mutagenicity tests 219 brewing industry 648 Balkan endemic nephropathy 47 butyrate hypothesis 724–725 Barretts esophagus patients 524 Bayesian theorem 359 c benchmark dose lower confidence limit CA assay, see Karyotype analysis (BMDL) 42 Caco-2 cells 597 benign prostatic hyperplasia (BPH) 764 – treatment 597 benzo [a]pyrene (B[a]P) 41, 339 caffeic acid 585 – metabolic activation 41 – in vivo model of oxidative stress 585 benzoic acid skeleton 418 caffeine 603, 619 benzyl isothiocyanate (BITC) 690 – importance 603 Bifidobacteria 733, 740 – role 619 Bifidobacterium animalis strain 736 caffeine feeding 584 – VM12 736 – rats 584 Bifidobacterium cells 725 cancer-preventive activity 604 bifunctional inducers 5 – antioxidative activity 604 – indole-3-carbinol 5 – effects on carcinogen metabolism 604 bioactive compounds 191, 649, 685 – repair of DNA damage 604 – physicochemical properties 649 – tea consumption 605 – polycyclic aromatic hydrocarbons 514 – tea in humans 604 bioinformatics resources 313 cancer-related proteins 756 – DRAGON 313 – MMP-7 756 – Genbank 313 – PPAR-d 756 – KEGG GENES 313 – VEGF 756 – Medminer 313 cancer chemoprevention 3, 202, 246, 455 – OMIM 313 – chemopreventive agents 3 – Onto-Express 313 – definition 3 – SOURCE 313 – molecular mechanisms 4 – Unigene 313 – xenobiotic metabolism 246 bioluminescent proteins 233 cancer chemopreventive agents 3, 761 bis(sulfosuccinimidyl)suberate (BS3) 278 – protease inhibitors 761 black tea 595, 609, 621, 622 cancer models 563 – cancer-protective properties 609 – in vivo/in vitro 563 772j Index

– genetically engineered rodent models cell proliferation measurement methods 12, 346 13, 282 – preventative agent 640 – cell cycle analysis 282 – studies 346–349 – cell death 286 b-carotene 202, 203, 374–376 – microplate screening assays for – antioxidant activity 375 cytotoxicity 282 – cancer prevention 376 cell protrusions 175 – concentration 376 cell-signaling pathways 277, 278, 587, 756 – in blood 203 – coffee diterpenes 587 – nontoxic chemopreventive agent 202 – EFG signaling 278 – vitamin A 203 – electrophoretic mobility shift assay b-carotene and retinol efficacy trial (EMSA) 281 (CARET) 77, 203, 380 – gene constructs 281 b-carotene bleaching method 623 – gene transcription 282 case control studies 359–360 – immunoprecipitation of kinase 281 b-catenin signaling pathway 755, 756 – methods to detect alterations 278 carcinogen metabolism 600, 620 – signal transduction 280 – cytochromes P-450 (CYP) 600 – transcription factor activation 281 – effects 600 – using phosphospecific antibodies 280 cardiovascular disease 78 cellular adhesion 175 carotenoids 371, 372, 374, 375, 378 cellular antioxidant defense capacity 489 – antioxidant properties 372 cellular immunity 188 – biochemical properties 374–376 cellular oxidative stress 442 – cancer prevention 378 cellular zinc levels 444 – classes 374 cervical intraepithelial neoplasia (CIN) 381 – gap junctional intercellular – CIN2 381 communication 375 – CIN3 381 – introduction 371–374 – progression 382 case-control studies 362, 363 chamber models 296 catechin breakdown metabolites 615 – microcirculatory preparations 296 catechin oxidation products 616 chemiluminescence (CL) assay 233 catechin precursors 611 chemokine receptors 176 – structures and content of 611 chemoprevention mechanisms 199, 713, 724 catechol-O-methyltransferase (COMT) 596 – beta-carotene 202 cell-adhesion molecules 67 – effects 159, 504 – epithelial cadherin 341 – in vitro/animal studies 713, 724 – placental cadherin 341 – lignified/suberized cell walls 713 cell-cell interaction 116, 147, 341 – lung cancer 204 cell cycle 282, 397, 437, 469, 616, 691, 724 – objective of 201 – analysis 282 – tamoxifen 206 – arrest 724 – toxicity 202 – events 616 chemotaxis 294 – phases 691 chemotherapeutic drugs 245, 382 – progression 437, 469 chick chorioallantoic membrane (CAM) – regulating genes 397 assay 169, 296 cell death, see apoptosis chlorin e4 706 cell maturation 12 1-chloro-2,4-dinitrobenzene (CDNB) 253 cell-matrix adhesion 171 chlorogenic acid 159 cell migration 292 chlorophyll 699–701 – wound healing 292 – antigenotoxic activities 701 cell motility 294 – carcinogen complex formation 702–704 cell proliferating markers 341 – chemical nature 699 – Ki-67 341 – historical background 701 – PCNA 341 – introduction 699 Index j773

– mechanism of the actions 701 – risk 715, 716 – structures 700 Comet assay 694 – types 699 complementary DNA (cDNA) 306–308 chlorophyllin 700, 701, 704, 706 condensate-induced DNA damage 463 – sepharose-supported 704 conjugated linoleic acid (CLA) 750 – solid-supported 704 conjugating enzymes 97, 99, 116 – structures 700 – transporters 116 – use 701 conjugation reactions 95 chromatography separation 266 constitutive androstane receptor (CAR) 116 chromosomal aberration (CAs) analyses 214, – pathway 117 230 – retention protein 117 chromosome breakage 26, 425 copper reducing assay 235 – biomarker 425 corneal angiogenesis assay 296 – rates 423 coronary artery disease 165 chymotrypsin inhibitor (C.I.) activity 762 CpG distribution 263 cisplatin-induced renal injury 483, 493 – animals genome 264 Codex Committee 709 – methylation level 264 Codex deﬁnition 710 CpG island methylation patterns 159 coffee 579 CpG-rich sequences 6 – antioxidant (AO) effects 581, 583, 585 crocin bleaching 234 – antioxidant properties 584 – antioxidants 234 – bioactive components in 580 Crohns disease 739 – cancer protective effects of 580 cruciferous vegetables 5, 40, 132, 169, 685, – cell signaling pathways 587 692, 694, 695 – constituents 585 – antigenotoxic effects 694 – DNA damage 579 – bioavailability consequences 695 – epidemiological studies 580 – broccoli 5, 132 – hydroxycinnamic acids (HCAs) 580 – cabbage 5, 132 – mechanisms of chemoprevention 580 – components 685 – on human health 579 – cooking 695 – protective properties 583, 585 – dietary intervention studies 692 – structures of bioactive chemicals 581 – human studies 692 – total antioxidant capacity (TAC) of – physicochemical properties 685 plasma 582 – storage/processing 695 coffee consumption 588 cruciferous vegetables active compounds 686 – bladder cancer 588 – bioavailability 686 – cancer risks in different organs 589 – metabolisms 686 – human cancer risks 588 crypt cells 340 cohort studies 359, 365 – proliferation 753 colon aberrant crypts 340 cyclic-DNA adducts 24 – morphology 340 cyclin-dependent kinases (Cdk) 26 colon cancer 269, 567, 740 – inhibitor 399 – methylated sequence 269 cyclobutane pyrimidine dimers 24 – risk 692 cyclooxygenases (COX) 621, 639, 714, 755, colon cancer model 522 756 – azoxymethane (AOM) 522 – COX-2 expression 621, 755, 756 colonic atrophy 725 – COX-2 protein 756 colorectal cancer (CRC) 554, 709, 712–714, – enzymes 639, 714 721, 742 – inhibitors 755 – cell line 754 Cyclopia species 613, 614 – data 753 – monomeric phenolic compounds – evolution 713 content 614 – models 753 cytochrome P-450 (CYP) family 109, 246, – protection 712 394, 514, 620 774j Index

cytokinesis-block micronucleus (CBMN) dihydroxyphenyl acetic acid (DHPAA) 512 assay 26 1,25-dihydroxyvitamin D3 (1,25-D3) cytosine-guanine dinucleotides 154 394–399, 401, 402 cytosolic b-glucosidase (CBG) 512, 549 – combination therapy 399 cytosolic retinol binding protein II 7,12-dimethylbenzo[a]anthracene (CRBP II) 379 (DMBA) 534, 705 – role 379 – induced buccal pouch carcinogenesis 619 cytotoxicity detection 283 – mammary tumors 468 – in vitro methods 283–285 – skin papilloma 705 – metabolism 462 d dimethylhydrazine (DMH) 737 Dam methylase 266 – foci 344 DBP-DNA adduct levels 705 – rat model 756 deoxyribonucleoside triphosphates diphenyl-1-picrylhydrazyl (DPPH) tests 230 (dNTPs) 306 disregulated hyperproliferative disorder 163 deoxythymidine monophosphate DNA 6, 16, 24, 28–30, 35, 38, 40, 47, 49, 80, (dTMP) 420 84, 86, 105, 109, 213, 217, 220, 307, 311, 314, – synthesis 422 339, 344, 349, 388, 389, 422, 425, 427, 504, deoxyuridine monophosphate 420 620, 648, 742 detoxication pathways 127 – active metabolites 38 detoxifying effects 246 – alkylation 24 detoxifying enzymes 583, 585 – base endogenous oxidation 86 – coffee components 585 – base lesions 389 – induction 583, 585 – binding dyes 307 dibenzo(a,l)pyrene (DBP) 705 – binding proteins 109 dietary antimutagens (AMs) 211 – extraction 217 dietary antioxidants 87, 449 – fragments 28, 30, 620 dietary assessment 360–361 – glycosylase 80 – methodological statistical challenges – lesions 24 360 – ligase 427 – potential importance 749 – metabolism 422 dietary fiber (DF) 709, 710, 713, 715, 716, 721 – methyl transferase gene 6, 16, 425 – constituents 710 – migration 213, 220 – definition 710, 721 – oxidation 84 – human clinical trials 716 – oxidative damage 504 – introduction 709–711 – protective effects 40 – preparations 726 – reactive carcinogens 35, 105, 742 dietary fiber active compounds 711, 712, 721, – reactive metabolite(s) 47, 48 724 – repair processes 344 – bioavailability 712, 724 – replication 7, 340 – metabolism 712, 724 – sequence(s) 23, 30, 733 – occurrence 711, 721–724 – strand(s) 80, 423, 427 – physicochemical properties 711, 721–724 – synthesis 28, 43, 418, 473 dietary fiber carbohydrates 721 DNA-adducts 10, 25, 27, 36, 62, 81, 102, 103, – fermentation products 721 105, 455, 655 dietary fiber protective properties 716, 726 – formation 48, 616, 619, 704 – impact of cooking/processing 716, 726 – HC-induced 655 dietary fiber fermentation 725 – specific antibodies 25 – role 725 DNA-alkylating agents 690 2-(difluoromethyl)ornithine (DFMO) 10, 62 DNA damage 21, 22, 24, 25, 27, 29, 31, 32, 41, dihydrogenistein (DHG) 550 48, 59, 79, 86, 104, 211, 219, 220, 377, 389, 5,7-dihydroxy-6-C-b-D- 425, 427, 455, 458, 460, 463, 464, 485, 601, glucopyranosylchromone 613 604, 616, 652, 690, 691, 694, 731, 743 dihydroxylated metabolites 551 – alkylating agents 24 Index j775

DNA damage checkpoints 26 – migration 14, 167 – ATM/ATR kinases 26 Engelbreth–Holm–Swarm sarcoma cell – ATR-interacting proteins 26 294 – RFC-like proteins 26 enhanced cell proliferation 163 DNA-damaging agents 390 enterodiol 558–559 DNA fluorochrome 286 enterolactone 558–559 – propidium iodide 286 – 1,8-dinitropyrene (DNP) 713 DNA hypomethylation 158, 267, 427, 428 enzyme-linked immunosorbent assays – epithelium 427 (ELISAs) 251, 280 DNA methylation 110, 135, 153, 156, 159, enzyme induction 94 263, 270, 342, 423, 425–427 enzyme inhibition 222 – BeadChip 271 enzyme measurements 219 – genome scanning 270 enzyme methyltetrahydrofolate – global/repeat sequence 265 reductase 157 – inhibitor 425 enzyme myeloperoxidase 239 – mass spectrometry detection 265 enzyme protein 250 – measurement 264 – determination 250 – PCR methods 263 enzyme transcription 249 DNA methyltransferase (DNMT) 153, 159 epidemiology 199 – activity 159 – chemoprevention 199 – enzymes 153 – data 516 – protein expression 159 – rules 79 DNA microarray technology 307, 309, 310 – studies 163 DNA protection mechanisms 212, 221 epidermal growth factor (EGF) 15, 490 – dose-effect relations of 221 – induced transformation 490 DNA repair mechanisms 5, 6, 27, 84, – signaling 277 763, 764 epidermal growth factor receptor – types 27–31 (EGFR) 125, 277, 397 DNA repair enzymes 221, 586 epidermal growth factor signaling 277, 279 docosahexaenoic acid (DHA) 750 – binding of ligand 279 dose-response effect 753 epigallocatechin (EGC) 595 dose-response relationship 378 epigallocatechin gallate (EGCG) 63, 600, double-strand breaks 29 616, 622 – repair 29 – epimer 622 drug approval/safety evaluation 31 epigenetics 6 drug-food interactions 116 – defination 6 drug metabolizing 586 – factors 133 – coffee components effects 586 – modifications 6 Dutch processing 538 – processes 145 – signals 155 e epithelial–mesenchymal transition (EMT) Edman sequencing 319 process 173, 174 eicosanoid synthesis 191 epithelial precancer 16 eicosapentaenoic acid (EPA) 750 ErbB/Ras signaling pathway 349 electron spin resonance (ESR) 229, 230 error-prone repair/mutagenic processes – whole-body 230 30 electrophilic metabolites 219 Escherichia coli 616 – formation 219 esophageal cancer 36, 467, 754 electrophoretic mobility shift assay estrogen 126–128, 131, 132, 558, 653 (EMSA), 281 – biological actions 558 ellagic acid 456–458, 463, 472, 473, 476 – metabolism 126, 132, 136 – structure 458 – metabolites 126–128, 131 endothelial cells 166, 294 – synthesis inhibition 653 – hypoxia-inducible factor-1a (HIF-1a) 294 estrogen receptor (ER) 124, 125, 399, 553 776j Index

– b receptor 125 fibroblast growth factor 492 – selective estrogen receptor 641 flavin adenine dinucleotide (FAD) 417 ethane formation 238 flavine-dependent monooxygenases European Commissions Research Directorate (FMOs) 98 General 322 flavonols 509 European Molecular Biology Lab (EMBL) – against cancers 515 249 – biological effects of 515 European Nutrigenomics Organization – cancer-protective effect of 516 (NuGO) 322 – case-control study 515 European Prospective Investigation Into – Cohort studies 515 Cancer And Nutrition (EPIC) 715, 716, – cooking 515 726, 752, 753 – dietary intake of 511 – study 726, 752, 753 – food processing 515 – trial data 716 – health benefits of plant-based diets 509 European Standards Committee on Oxidative – human data 514 DNA Damage (ESCODD) 82, 239 – nurses health study 515 evidence-based nutritional prevention – phenolic constituent (B-ring) 509 approach 357 – physicochemical properties 509 ex vivo system 340 – plasma/urine 513 excessive genetic damage 12 – protection mechanisms 514 exocyclic amino group 38 – structures of 510 experimental oncology 335 fluorescence in situ hybridization 214 – preneoplastic lesions use 335 folate/methionine pathway 418 extracellular matrix (ECM) 171 – methyle-netetrahydrofolate reductase – degradation 14, 175 (MTHFR) 418 – substrates 175 folic acid 204 extracranial solid cancer 470 – disadvantages of 204 – effects of 204 f – noncolorectal cancers 205 facal water 737 folk medicine 663 facteur thymique serique (FTS) 188 – garlic 663 farnesyltransferase inhibitors 63 – onions 663 Fas/Fas ligand system 176 food additives 50 fat 749 – genotoxic/carcino genic properties 50 fat-soluble vitamin E 409 – residues 50 – free radicals 403 Food and Agricultural Organization 733 – ROS 403 Food and Drug Administration (FDA) 761 fatty acid 187, 749, 751, 752, 753, 754, 755 Food and Nutrition Board of the United – bioavailability 751–752 States 385 – composition 188 food frequency instrument 361 – epidemiology 752–753 – calibration means 361 – impact 754 – method 361 – mechanisms 754 food matrix 499 – structure 750, 751 – lignan content data of 565 – types 755 formamidopyrimidine DNA glycosylase fermented food 731 (FPG) 239 – introduction 731 frameshift mutation 31 ferric iron reducing antioxidant parameter free radical quenching methods 231 (FRAP) 230 freeze-dried beer 655 – assay 617 French Paradox 456, 636, 638, 640 – test 230 fructooligosaccharides (FOSs) 723 – values 75 fusarium toxins 736 ferulic acid 711 – deoxynivalenol (DON) 736 FGCP enzyme 421 – nivalenol (NIV) 736 Index j777 g glucocorticoid budesonide 64 g-glutamyl-transferase 589, 601 Glucosinolate-derived bioactive products 689 – gene expression of 601 – xenobiotic metabolism 689 gallocatechin gallate 622 glucosinolates (GLS) 685, 686, 689 gamma-linolenic acid (GLA) 750 – bioavailability 686 gap junctional intercellular – colonic metabolism 689 communication 64, 375 – gastric/small-intestinal breakdown 689 garlic 664 – gut absorption 689 – chopping 664 glucosinolates 685, 686 – crushing 664 – chemical structures 686 garlic compound 663, 667 glucuronide conjugates 642 – diallyl thiosulfinate (allicin) and 667 glutathione-S-transferases 40, 128, 212, 247, gas chromatography-mass spectrometry 690, 693 (GC-MS) 80, 238, 239 glutathione conjugates 97 gastric lesions 489 – ITCs 689 – inhibition 489 glutathione peroxidise 126, 582 gastrointestinal tract 503, 733 glutathione transferases 96 gel electrophoresis 280, 295 grape polyphenols 636 gene array 314 grapefruit juice 136 gene chip experiment 308 grape seed proanthocyanidin-rich extract – schematic representation 308 (GSPE) 534 gene expression 156, 322, 423, 763 green Robusta beans 583 – analysis 313 – AO activity of 583 – arrays 315 green tea 206, 595, 623 – data 310 – chemopreventive properties 623 – profile 313 – treated with green tea 604 gene mutation assays 214 green tea polyphenols 596, 605 – bacteria 214 – aflatoxin-albumin 605 – detection of 214 – biotransformation of 596 gene polymorphisms 136 green vegetables 699 genetic polymorphisms 87, 421 genome 271 h – methylated/unmethylated fraction 271 HCl-induced gastric lesions 489 genome-wide/custom methylation 272 HDAC inhibitory activity 160 – commercially available tools 272 Heart Outcomes Prevention Evaluation genomic methylation 266, 423 (HOPE) 408 genomic/postgenomic alterations 57 helicobacter pylori 146, 489 genomics 303 hepatic N-acetyltransferase activity 248 – definition 303 hepatic cancer 754 – functional genomics 303 hepatocellular carcinoma 46, 469, 588, 706 – structural genomics 303 herb-drug interactions 116 genotoxic carcinogens 212, 215, 219, 221, hetero-cyclic ring 420 246, 350, 585 heterocyclic aromatic amines (HAAs) 35, 37, – AOM/DMH derivatives 350 211,214, 339, 713, 736 – DMBA 350 – dietary compounds 214 – dose-effect relationships 221 high-throughput nutrigenomic techniques – high-dose exposure 350 315 – metabolism of 215 high-performance liquid chromatography genotoxicity tests 213, 215 (HPLC) 25, 80, 237, 319, 395, 656 – schematic overview of 213 high-throughput microarray technology 314 German Nutrition Society 360 high-throughput molecular biology GI-tract 737, 742 techniques 303 – DNA damage 737 – metabolomics 303 glioma cell cytotoxicity 470 – proteomics 303 778j Index

– transcriptomics 303 – oxidized levels 220 histone acetyltransferases (HATs) 397 hydroxyl radical 389 hollow fiber assay 296 hypomethylation 267 homocysteine 426 – gene promoters 267 homozygous genotype 130 – PCR-based assays 267 homozygous variant 136 hypoxia-inducible factor 294 honeybush tea 613–615 hormone-dependent cancers 66 i hormone-related cancers 10 immune cells 189 hormone refractory prostate cancer immune factors 186 (HRCP) 475 immune system 16, 66, 175, 183, 184, 185, hospital-based case-control studies 200 186, 189 human atherosclerotic plaques 239 – effects 66 human cancer 349 – overview 184 – NCI-sponsored mouse models 349 immunoassays 238 human cancer etiology 357 immunohistochemical analysis 646 – nutrition role 357 in vitro antioxidant assays 617 human drug experience 765 in vitro cell culture studies 372 human enzyme polymorphisms 246 in vitro studies 754 human epidemiological studies 742 in vivo angiogenesis assays 297–299 – results 742 inducible nitric oxide synthase 756 human gut microbiota 550 – inhibitors 149 – daidzein metabolism 550 inducing agent 102 human hepatoma cells 349 inflammation-associated cancer 146 – HepG2 349 – prevention 146 human lung cancer cells 691 inflammation-induced cancer 149 human lymphoid cells 423 inflammation recruit immune cells 186 human metabolome database 321 inflammation signaling pathway 504 – data information 321 inflammatory cytokines 740 human microvascular endothelial cells – IL-12 740 (HMEC) 292 – IL-6 740 human pancreatic cancer cell lines 490 inflammatory human papillomaviruses 146 – angiogenic markers 472 human tumor antigens 16 – immune responses 147 human umbilical vein endothelial cells inflammatory reactions 191 (HUVEC) 292 – downregulation 191 humans carcinogen excretion 693 inorganic/organic selenium compounds 63 – enzyme activities 693 insulin-like growth factors (IGFs) 205 hydrocarbon receptor ligand 463 – binding protein 471 hydrogen transfer reactions 235 intermediary metabolism 444 hydrolysis products 689 – zinc effects 444 – excretion 689 internet-based programs 313 – metabolism 689 intervention studies 358–359 hydrolyzable tannins 500, 502 intestinal epithelial cells 192 hydrophilic compounds 8 intracellular coenzyme 419, 421 hydrophobic amino acids 651 intracellular glutathione 491 hydroxyapatite chromatography 80 intracellular pathogens 185 hydroxycinnamic acids 714, 715 – viruses 185 – cell walls containing 714 intracellular redox equilibrium 8 – plant cell wall 715 intracellular signaling pathways 445 hydroxycinnamyl alcohol precursors 711 investigational new drug 761 8-hydroxydeoxyguanosin (8-OHdG) 219, ionizing radiation 24 239, 371, 484, 582 iso-thiocyanates 685, 686 – DNA 239 – level 692 Index j779 isoflavanone dihydrodaidzein (DHD) 550 Lewis lung carcinoma (LLC) 647 isoflavones 547 Li–Fraumeni syndrome 347 – absorption 549 lignans 555 – antioxidant effects 553 – antioxidant activity 556 – dietary intake of 548 – bioavailability 561 – food sources of 547 – chemical structures of 557 – gastrointestinal tract 552 – content of seeds 558 – glucuronic acid 551 – diphenolic compounds 555 – in soy products 548 – glycoside precursors 124 – intestinal cancer 554–555 – in seed plants 556 – metabolism 550 – physicochemical properties 556 – nonsoy foods 548 – phytoestrogenic nature 558 – O-desmethylangolensin (O-DMA) 550 – preventive effects of breast cancer 556 – prostate cancer 554 – receptor complexes 379 – red clover 548 Linxian nutrition intervention trial 391 – structures 549 lipid oxidation 235 – UDP-glucuronosyl transferases – biomarkers for 236 (UGTs) 551 – cell membranes 235 isoprostanes 237 – parameters 238 – detectable concentrations of 237 lipid peroxidation 237 isothiocyanates 689 lipid peroxy radical 406 lipophilic activities 235 j – hydrogen transfer 235 Japanese public health center-based – single electron transfer mechanisms 235 prospective study (JPHC) 606 lipopolysaccharide jun N-terminal kinase (JNK) 691 – simulated rat lung parenchyma 486 Jurkat T cells 617 – stimulated murine peritoneal macrophages 487 k lipoproteins 376 K562 erythroleucemic cells 740 – HDL 376 kaempferol 514 – LDL 376 Karyotype analysis 218 lipoxygenase (LOX) 9, 755 – pathways 9 l – derived metabolites 639 L. acidophilus strain 736, 742 liver cancer 267 – VM20 736 locked nucleic acid (LNA)-modified capture lactase-phlorizin-hydrolase (LPH) 549 probes 309 lactic acid bacteria (LAB) 731, 733, 734, 737, long-chain aliphatic alcohol 699 739, 742, 743 long-patch BER 28 – antimutagenic properties 742 low-density lipoprotein (LDL) 405, 469 – antioxidant defense system 739 – oxidation 230, 231 – antioxidant effects 737–739 low-phenolic olive oil 85 – apoptosis 742 lung cancer 203 – cell proliferation 742 – ATBC 204 – DNA damage mechanisms 732 – beta-carotene 204 – DNA-reactive chemicals detoxification 734 – CARET 204 – effect on immune status 739 – smoking 203 – epidemiological studies 741 – tobacco 203 – carcinogen detoxification 733–737 lung cancer murine models 397 – introduction 731 lung tumors 474 – occurrence 733 lycopene 206, 377 – ROS protective properties 743 – cancer prevention 377 – strains 739 lymphatic metastasis 171 lactobacillus species 739 – clinicopathological analysis 171 780j Index

lymphocytes 220 – tumor suppressor genes 270 – antioxidant 85 MGED consortium 311 – oxidized DNA 220 – microarray experiment 311 lymphoid tissue 192 MIAME standards 312, 322 lytic enzymes 14 microarray analysis software 313 – production of 14 – comprehensive programs 313 – specific programs 313 m microarray data 313 macromolecules 235 – analysis 311 – oxidation 235 – folding 311–313 macrophages fish oil 188 – normalization 310 Madin–Darby canine kidney cells 597 – procedure 310 magnesium ions 502 microarray experiments 312, 313 Maillard reaction 42 – comparison 313 major histocompatibility complex – discovery 313 (MHC) 185 – prediction 313 malignant lymphoma cells 15, 763 microarray gene expression Database Society malondialdehyde 237 (MGED) 311 – formation 237 microarray platform 310 mammalian tissues 155 – miChip 310 mammary carcinogenesis 646 microarray technology 322 – inhibition 646 – application 322 manganese superoxide dismutase 441 micronucleus 426 MANIC method 269 – formation 214 MAP kinase cascade 11 micronutrient 201, 205 mass spectrometry 321, 597 microsomal reductase 421 – analysis 597 miRNA pathway 306 – mouse urine samples 597 mismatch repair genes 29, 341 matrix-assisted laser desorption/ionization – hMSH2 341 time-of-flight mass spectrometry 319 mismatch repair protein 390 matrix metalloproteinases (MMPs) 15, 165 – MLH1 390 McrBC digestion 271 mismatch repair (MMR) system 22, 29 MDR protein 458 mitochondrial DNA 57 Mediterranean diet 636 – alterations 57 membrane fatty acid binding protein 752 mitogen-activated protein 9, 691 menopausal hormones 136 – kinases 9, 64, 113, 691 meta-analytical program packages 360 – pathway 277 metabolomic research 321 MNNG-induced mutations 654 – detection methods 321 MNU-induced mammary tumorigenesis 646 – problem 321 monocarboxylate transporter (MCT) 597 – separation methods 321 monocyte-derived innate immune cells 184 metabonomics system 304 monoglutamated folate 419 metal ion 420 monomeric phenolic compounds 612 metal response element (MRE) sequence 442 – content 612 metastasis 562 monounsaturated fatty acid (MUFA) 750 – inhibition 562 – oleic acid 750 – process 177 mouse mammary epithelial cells 463 methionine synthase 418 mouth/pharynx/larynx cancers 378 methyl donors 156 mRNA concentrations 312, 319, 320 methylation 267 mRNA expression analysis 250 – analysis 268 mRNA transcription 588 – measurement 267 – genes encoding 588 – specific genes 6, 268 MTHFR genotype 427 – specific multiple probe ligation assay 270 mucin depleted foci 346 Index j781 multidrug resistance-associated proteins nondigestible oligosaccharides (NDOs) 709, (MRP) 116, 512, 597 721, 723, 725 multiprotein complex 305 – structures 723 – argonaute 305 nonenzymatic antioxidants 189 – Dicer 305 nongenotoxic mechanisms 48 multistage carcinogenesis 337 nonnutritive food components 192, 193 – mechanisms 4 nonstarch polysaccharides (NSPs) 709, 721 – stages 337 nonsteroidal aminoglutethimide 66 mutation-related diseases 58, 59 nonsteroidal antiinflammatory drugs 9 mycotoxins patulin (PAT) 736 nonvegetarian dietary habits 163 northern blotting 306, 307 n – limitations 307 N-acetyl-L-cysteine (NAC) 15, 57 NPC trial 439, 440 N-acetyltransferase (NAT) 212, 247 nuclear factor erythroid-2-related factor 2 100 N-hydroxy-2-acetylaminofluorene 99 nuclear localization signal (NLS) 115 N-hydroxy-PhIP-induced DNA adduct 464 nuclear magnetic resonance (NMR) N-methoxyindole-3-carbinol 691 spectroscopy 230 N-methyl-N-nitrosourea-induced DNA nuclear receptors 116 damage 464, 651 nuclear retinoic acid 190 N-nitrosodiethylamine-induced lung nucleic acid metabolism 21 tumorigenesis 473 nucleophilic chemopreventive agents 62 N-nitrosomethylbenzylamine (NMBA) 443, – NAC 62 522, 647 nucleoplasmic bridges 218 – induced esophageal tumorigenesis 467 nucleotide excision repair 28 – inhibition 647 nutrient-gene interactions 314, 322 NADPH oxidase 64, 147 nutrigenetics 303 – complex 188 nutrigenomic research 306–314 National Center for Biotechnology Information – data analysis 311 (NCBI) 249 – data normalization 310 natural killer (NK) cells 185 – microarrays 307–310 – inhibitor 164 – northern blotting 306 natural-product origin 164 – reverse transcription polymerase chain NCI-funded selenium 408 reaction 306–307 neurodegenerative disorders 417 nutrigenomics 304 – Alzheimers disease 417 – definition 303 neurotoxic effects 44 – terminology 304 neutrophil granulocytes 184 nutrigenomics applications 314–319 nicotinamide quinone oxidoreductase 246 o nitrate-containing red beet juice 37 O-linked glycosides 613 nitrenium ion 39 – hyperoside 613 nitrogenous compounds 651 – isoquercitrin 613 – amines 651 – rutin 613 – nucleic acids 651 observational epidemiology 200 nitrosation inhibitors 60 oligomeric/polymeric dietary – vitamins 60 polyphenols 499 NK cells 188 omega-6 fatty acids 750 NMBA-induced esophageal – linoleic acid 750 tumorigenesis 446 oncogene sequences 63, 211 NNK-induced lung adenoma 619 oolong tea 595 noncellulosic polysaccharides 711, 722 ornithine decarboxylase 7, 62 noncoding RNAs 305 ovarian hormones 121 – ribosomal RNA 305 oxalic acid 46 – transfer RNA 305 oxidase enzymes 670 782j Index

oxidative damage 81, 239, 240 – effects of isoflavones 553 – in vivo approaches 240 – lignans 562 – monitoring methods 239 – resveratrol 136 oxidative metabolism 126 PKC-linked responses 407 oxidative polymerization 711 PKC-mediated phosphorylation 118 oxidative stress 190, 230 plant-derived carcinogens 49 – categories 229 plant defense systems 667 – measurement 229 – content 567 – methodologies 230 plant polyphenols 147 oxygen radical absorbance capacity – epigallocatechin gallate 147 (ORAC) 230 plant polyphenols 516 plasma antioxidant capacity 655 p plasma carotenoid levels 376 p-coumaric acid 711 plasma genistein 123 – structures 711 platelet aggregation 407 p53 tumor suppressor gene 347 polyamine-related enzymes 10 PAH-DNA adducts 41 – synthetic inhibitors 10 PAH-induced cancer formation 41 polyaromatic hydrocarbons 135 PAH metabolism 132 polycyclic aromatic hydrocarbons (PAHs) 24, parathyroid hormone (PTH) 394 40, 211, 339, 472, 583, 701 pathogenetic mechanisms 57 polycyclic carcinogens 704 – DNA damage 57 polycyclic genotoxicants 702 – oxidative stress 57 polycyclic planar compounds 704 PCNA-dependent polymerases 29 polyglutamated compounds 419 pentachlorophenol-induced oxidative DNA Polygonum cuspidatum 638 damage 462 polyhydroxyflavanol oligomers 500 PeriTech debranning system 712 polymerase chain reaction (PCR), 249 peroxisome proliferator-activated receptors – DNA analysis 250 (PPARs) 188, 756 – fluorescent dyes 269 pesticide/herbicide residues 50 – methylation sensitive 269 Peyers patches 192 – mRNA analysis 250 phenethyl isothiocyanate 160 polyphenolic rooibos constituents 615 phenobarbital-responsive enhancer modules – bioavailability 615 (PBREM) 117 – metabolism 615 phenolic acids 455, 655 polyphenol-rich oils 39 phenolic antioxidant 114 Polypodium leucotomos 170 phenylethylisothiocyanate (PEITC) 690 polyunsaturated fatty acids (PUFA) 186, 403, pholasin 233 749, 750, 755 phosphatidylinositol-3-kinase 277 – routes 755 phosphoinositide-dependent kinases – type 749 (PDKs) 280 porphyrin ring 420 phospholipid liposomes 483 portfolio approach 358 – peroxidation 483 post-translational histone modifications 6, photobleaching process 375 109, 145 physical factors 145 – acetylation 6 – asbestos 145 – epigenetic events 6 – gastric acid 145 – methylation 6 phytochemicals 636 – phosphorylation 6 phytoestrogens 11, 547, 560 PPAR receptors 380 – absorption, distribution, metabolism, and prebiotic effects 725 excretion (ADME) 549 – definition 725 – dietary intake of isoflavones 548 precancerous gastric lesions 391 – dietary isoflavone sources 547 prediagnostic serum carotenoids 378 – diets 122 pregnane X receptor 124 Index j783 premalignant lesions 753 protease inhibitors 761, 762, 766 – identification 350 – active compounds 761, 762 proangiogenic molecules 14 – bioavailability 762 – expression 14 – Bowman–Birk family 766 proanthocyanidin dimers 526 – cooking processing impact 766 – structures 526 – metabolism 762 – proanthocyanidin-enriched preparations – occurrence 761 in vitro in cell culture 530–533 – physicochemical properties 761 – proanthocyanidin-enriched preparations protease inhibitors protection in vivo in animal models 535–536 mechanisms 762 Proanthocyanidins 525 – in vitro/animal studies results 762 – bioavailability of 528 protection spectrum 221 – cancer chemopreventive activities of 534 protein-bound carbonyls 238 – cardiovascular diseases 525 – formation 238 – degree of polymerization (DP) 527 protein kinase 113, 278, 407 – di-, tri-, and oligomeric condensation – subfamilies 114 525 – PKC/Ras/Raf cascade 11 – epidemiological Studies 537 protein oxidation 235, 238 – food preparation 537 – biomarkers for 236, 238 – in fruits 527 protein tyrosine kinase (PTK) 278 – in plants 527 proteomics 319–320 – in vitro antioxidant activity 529 – potential applications 320 – physicochemical properties 525 – schematic representation 320 – potential anticancer effects 535 proton radiation-induced malignant – protein interaction 526 lymphoma 763 – scavenge reactive oxygen species – inhibition 763 (ROS) 527 protoporphyrin 700 – skin cancer prevention 534 – structures 700 pro-oxidative activities 600 provitamin A 203 – importance of 600 – carotenoid 372, 377 proapopototic proteins Bax 619 PSA levels 764, 765 proapoptotic effects 443 public health goals 357 probiotics 733 PUFA peroxidation 237 – definition 733 PUFA precursor 187 procarcinogenic effect 245 PUFAs 752, 754, 756 procyanidin dimers 529 – aspect 756 – catechin glucuronides 529 pulmonary diseases 57 – methylated glucuronide metabolites pungent compounds 486 529 PXR Pathway 117 procyanidins 525 – regulation 117 programmed cell death 114 pyrosequencing technology 266 proinflammatory cytokines 145, 191, 487 pyrrole ring 699 – IL-12 487 proliferating cell nuclear antigen (PCNA) 28, q 340 QRT-PCR analysis 251 promyelocytic leukemia gene (PML) 381 – amplification plot 251 propelargonidins 525 Quercetin 509 prostate-specific antigen (PSA) 78, 205 – absortion, transport, and metabolism – levels 378 of 513 prostate cancer 135, 438, 567 – characteristic properties of 511 – rodent models 347 – extracellular signal-regulated kinase prostate cells 378 (ERK) 514 prostate intraepithelial neoplasia (PIN) 534, – protective mechanisms 514 607 – three-dimensional structure of 510 784j Index

r – chemoprevention studies 336 radiation-induced carcinogenesis 763 – preneoplastic models 335 radical scavenging capacity 8, 527 rooibos tea 610, 613, 616, 622 – antioxidant 527 – antimutagenic effects 616 radioprotective agent 763 – unfermented/fermented 610, 612, 613 randomized controlled trials (RCTs) 381 Ras/MAPK pathway 279 s reactive nitrogen species (RNS) 145, 485 S-adenosyl methionine 157, 422 reactive oxygen species (ROS) 24, 74, 112, salmonella/microsome assay 214 121, 145, 186, 211, 371, 389, 617, 637, 737 Salvia tomentosa 168 – accumulation 484 scavenging reaction 406 – dependent signaling 371 secoisolariciresinol 555, 565 – induced damage 220 – biosynthetic pathway 555 – inducing chemicals 239 secoisolariciresinol dehydrogenase 555 reactive sulfur species, overview 666 secoisolariciresinol diglucoside (SDG) 560 real-time reverse transcription-polymerase – antioxidant properties 560 chain reaction (real-time RT-PCR) 306, 307 – flax cake 568 receptor-mediated pathways 653 – hydroxy-methyl-glutaric acid (HMGA) 561 – apoptotic pathways 176 – preventive effects 562 recombinant DNA technology 303 – seed coat 568 red grapes 637 – sex hormone binding globulin 560 red wines 638 selective androgen receptors antagonists 66 redox-active transition metal cations 443 selective estrogen modulators (SERMs) 10, 65 redox-sensitive transcription factor 485 – arzoxifene 10 redox-sensitive transcriptory proteins 371 selenium 435 redox cycling potential 235 – deficient diets 159 redox reactions 417 – deficient hosts 189 resistant starche 709, 721 – metabolism 435, 440 resveratrol – vitamin E 205 – chemopreventive activity 465 selenocysteine-tRNA gene 437 – dose 469 selenometabolites 437 – effects 638 – methylselenol 437 retinoic acid(s) 372, 380 serum b-cryptoxanthin 378 – chemical structure 372 sham radiation control groups 763 retinoic acid receptor (RAR) family 379 short-chain fatty acids (SCFAs) 721, 727, 742, retinoic acid response elements (RAREs) 749 379 – acetate 749 retinoid X receptor (RXR) family 190, 379 – butyrate 749 retinoids 378, 380 – formation 742 – biological properties 378 – hypotheses 724 – cancer prevention 380 – production 724 retinol binding protein (RBP) 379 – propionate 749 retroviral DNA sequence 155 signal transduction 114, 280 reverse transcription polymerase chain – mechanisms 11 reaction (RT-PCR) 230, 306–307 – modulation 64, 67 – limitations 307 – pathway(s) 11, 133 rheumatoid arthritis 170 – intracellular targets 280 ribonucleotide reductase 188, 638 signaling molecules 407 RNA-induced silencing complex (RISC) signaling pathways 164 305 single-cell gel electrophoresis (SCGE) RNA interference process (RNAi ) 305 assays 213, 239, 582 RNA polymerase 305 – comet assay 694 rodent – DNA migration 736 – carcinogenicity studies 335 single-electron transfer methods 234 Index j785 single nucleotide polymorphisms – antioxidant properties 617–619 (SNPs) 134, 249, 440 – beverage-grade 459 singlet molecular oxygen 374 – bioavailability 615 sister chromatid exchange (SCE) 230 – cancer-preventive effects of 603 small interfering RNA (siRNA) 305 – chemopreventive properties 616 – post-transcriptional gene regulation 305 – components 600 smokers 604 – containing beverages 622 sodium-dependent glucose transporter 1 – human studies results 621 (SGLT1) 512 – impact of cooking 607 sodium-dependent vitamin C – metabolism 615 transporters 388 – pharmacokinetic studies of 598 – SVCT1 388 – pro-oxidative effects of 600 – SVCT2 388 – proteins 607 sodium dodecylsulfate (SDS) 295 – storage 622 solar irradiation 60 – types of 595 Sorghum 528 tea polyphenols 596, 598, 603 starch-based materials 635 – antitumorigenic activities of 603 stereotactic radiotherapy 475 – bioavailability 596 steroid receptor coactivators (SRCs) 397 – biotransformation of 596 steroid-receptor pathways 125 – pharmacokinetics of 598 stromal cell-derived factor-1 177 – structures of 596 sulfotransferase enzymes 212, 247, 694, 551 tert-butyl hydroperoxide-induced oxidative – distribution 551 DNA damage 652 – excretion 552 therapeutic agents, ascites tumor 468 – inhibition 104 thermal degradation products 42 – pharmacokinetics 552 thermophilus aquaticus (Taq) 307 sulfur agents 665 thiobarbituric acid (TBA) 237 sulfur-containing compounds 663, 667 – reacting substances assay 237, 584 superoxide dismutase (SOD) activity 126, – malondialdehyde 230 582, 739 tissue-specific microarrays 309 synthetic ABTS radical cation 617 TNF-related apoptosis 491 synthetic derivatives 65 tobacco-specific nitrosamines 25 – b-carotene 65 tocopherols scavenging reaction 406 – lycopene 65 toll-like receptor group (TLRs), role 740 total antioxidant capacity (TAC) 232 t – approaches 230 T-cell lymphoma 381 – determination methods comparison 232 T-helper cell 740 – TRAP/ORAC assays 233 tamoxifen 199 total oxidant scavenging capacity (TOSC) – resistant breast cancer cell line 442 method 230, 231 – resistant cell line model 445 total peroxyl radical trapping antioxidant – resistant phenotype 444 activity 584 tannins 499, 500 total reactive antioxidant potential – bioavailability 499 (TRAP) 230 – hydrolyzable 500 toxifying enzymes 103 taqman allelic discrimination assay 249 TPA-induced tartaric acid 635 – inflammation 487 TdT-mediated dUTP nick-end labeling – ROS production 620 (TUNEL) 287 transcription systems 100, 110, 375, 390 tea 595, 603, 616, 617, 619, 621, 622 – AP-1 390 – active compounds 615 – NF-kB 390 – animal protection mechanisms 616 – pathways 112 – animals studies 619 – peroxisome proliferator-activated receptors – antimutagenic properties 616 (PPAR) 375 786j Index

– retinoid receptors (RAR, RXR) activator US department of agriculture (USDA) 511 protein-1 (AP-1) 375 – in onions 511 – xenobiotic receptors 375 transcriptomics, definition 303 v transforming growth factor -b (TGF-b) 66, vanilloid receptor 489 340 vascular diseases 427 transgenic animals 217 – macroangiopathy 427 – development of 217 vascular endothelial growth factor (VEGF) 294 þ transgenic ApcMin/ mouse model 347 – induced tyrosine phosphorylation 167 – intestinal cancers 347 – signals 166 transitional cell carcinoma 392 vitamin A 371, 372, 373, 378, 379, 380 translesion synthesis 30 – introduction 371–374 transmembrane transporters 102 – role 372 transport proteins 102 – synthetic derivatives 379 transulfuration pathway 420 vitamin B12 264 – cystathionine b-synthase 420 vitamin C 385, 386, 387, 389, 390, 392 trapping antioxidant parameter (TRAP) 231 – antitumor effect 390 travellers disease 731 – apoptosis 389 trolox equivalent antioxidant capacity – bioavailability 387–388 (TEAC) 230 – cell proliferation 389 tube-formation assays 292 – content 386, 392 tumor cells 175, 176, 186 – dietary intervention 391 – associated antigens 185 – metabolism 387–388 – cell growth/proliferation inhibition 490 – oxidative DNA damage 389 – specific antigens 66 – physicochemical properties 386–387 – suppressor genes 211 – pro-oxidant activity 390 tumor prevention/treatment 397 – protection mechanism 388 – in vivo studies 397 – signal transduction 389 tumor progression 173 – supplements 391 tumor promoters 9 vitamin C protection mechanism 388 – phorbol ester 9 – impact of food processing 392 tumor suppressor genes 341 – in vitro/in vivo studies 388–389 – Apc 341 – studies of 390, 391 – FHIT 341 vitamin D 393, 395, 396, 397, 399, 400, 402 two-dimensional polyacrylamide gel – clinical trials 400 electrophoresis (2D-PAGE) 319 – content 402 – deficiency 393 u – hydroxylase 399 UDP-glucoronosyl-transferase 212, 247, 583 – introduction 393–394 – family substrate 514, 619 – metabolic pathway 396 UGA codon 435 – metabolite 394 ultraviolet light – negative feedback loop 394 – absorption spectra 702 – precursor 394 – induced immunosuppression 472 – processing impact 402 – irradiation 638 – synthesis 402 – radiation-induced skin carcinogenesis 473 – target organs 395 uric acid 235 – VDR-mediated signaling 397 urine 219, 512 vitamin D binding protein (DBP) 394 – metabolic products 512 vitamin D receptor (VDR), 65, 396 – mutagenicity of 219 – interacting protein 397 urothelial cancers 47 – mediated mechanism 395 urothelial lesions 490 – mediated signaling 397 USA-based prostate, lung, colorectal, and – target gene expression 397 ovarian (PLCO) cancer screening trial 715 vitamin D response elements (VDREs) 397 Index j787 vitamin E 402, 403, 404 – in vitro tests 220 – bioavailability 404 xenobiotic enzymes 249 – cancer prevention trial 408 – DNA-level investigations 249 – components 403 xenobiotic metabolism 87, 94, 100, 107, 116, – dietary components 405 246, 247, 254 – human diet 403 – activity measurement of enzymes 254 – introduction 402–403 – DNA 247 – isoforms 406 – end points investigation 248 – metabolism 404 – enzymes 246 – structure 403 – goal of 246 – humans activity measurements 255 w – measurement of 247 water-soluble vitamins 417, 429 – modifications of 255 – metabolism 417 – mRNAs 247 web-based tool 361 xenobiotic metabolizing enzyme (XME) 721 Western blotting 319 – cytochrome P450 714 white blood cells 470 xenobiotics disposition 93 whole-genome microarray experiments 308 xenograft models 349–350 wine 636 – growth of cancer cells 534 – health effects 636 xenograft tumors 443 world cancer research fund (WCRF) 359, 390, xeroderma pigmentosum 28, 382 752 – panel 363, 364 y – statement 439 yogurt production 733 x z xanthine derivatives 65 zebrafish assay 299 – caffeine 65 zinc-binding protein metallothionein 442 xenobiotic zinc-deficient animals 446 – detoxification enzymes 124 zinc-dependent transcription factor 442, 443, – metabolizing acetyltransferases 105 444 – metabolizing enzymes 94, 150 zinc-induced apoptosis 443 – metabolizing system 106 zinc accumulation 443 xenobiotic-responsive enhancer module zinc manipulation 445 (XREM) 117 – effects 445 xenobiotic response element (XRE) 62 zinc metabolism 442 xenobiotic drug metabolizing enzymes 212, zinc supplements 447 219 zinc transporter proteins 444, 445 – activity of 219 zymography 295