Interrelations Between Essential Metal Ions and Human Diseases Interrelations Between Essential Metal Ions and Human Diseases Metal Ions in Life Sciences Volume 13

Metal Ions in Life Sciences 13

Astrid Sigel Helmut Sigel Roland K.O. Sigel Editors Interrelations between Essential Metal Ions and Human Diseases Interrelations between Essential Metal Ions and Human Diseases Metal Ions in Life Sciences Volume 13

Series Editors: Astrid Sigel, Helmut Sigel, and Roland K.O. Sigel

For further volumes: http://www.springer.com/series/8385 and http://www.mils-series.com Astrid Sigel • Helmut Sigel • Roland K.O. Sigel Editors

Interrelations between Essential Metal Ions and Human Diseases Editors Astrid Sigel Helmut Sigel Department of Chemistry Department of Chemistry Inorganic Chemistry Inorganic Chemistry University of Basel University of Basel Spitalstrasse 51 Spitalstrasse 51 CH-4056 Basel CH-4056 Basel Switzerland Switzerland [email protected] [email protected]

Roland K.O. Sigel Institute of Inorganic Chemistry University of Zürich Winterthurerstrasse 190 CH-8057 Zürich Switzerland [email protected]

ISSN 1559-0836 ISSN 1868-0402 (electronic) ISBN 978-94-007-7499-5 ISBN 978-94-007-7500-8 (eBook) DOI 10.1007/978-94-007-7500-8 Springer Dordrecht Heidelberg New York London

Library of Congress Control Number: 2014931237

© Springer Science+Business Media Dordrecht 2013 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com) Historical Development and Perspectives of the Series

Metal Ions in Life Sciences*

It is an old wisdom that metals are indispensable for life. Indeed, several of them, like sodium, potassium, and calcium, are easily discovered in living matter. However, the role of metals and their impact on life remained largely hidden until inorganic chemistry and coordination chemistry experienced a pronounced revival in the 1950s. The experimental and theoretical tools created in this period and their application to biochemical problems led to the development of the ﬁ eld or discipline now known as Bioinorganic Chemistry , Inorganic Biochemistry, or more recently also often addressed as Biological Inorganic Chemistry . By 1970 Bioinorganic Chemistry was established and further promoted by the book series Metal Ions in Biological Systems founded in 1973 (edited by H.S., who was soon joined by A.S.) and published by Marcel Dekker, Inc., New York, for more than 30 years. After this company ceased to be a family endeavor and its acquisition by another company, we decided, after having edited 44 volumes of the MIBS series (the last two together with R.K.O.S.) to launch a new and broader minded series to cover today’s needs in the Life Sciences. Therefore, the Sigels new series is entitled

Metal Ions in Life Sciences.

After publication of the ﬁ rst four volumes (2006–2008) with John Wiley & Sons, Ltd., Chichester, UK, and the next ﬁ ve volumes (2009–2011) with the Royal Society of Chemistry, Cambridge, UK, we are happy to join forces now in this still new endeavor with Springer Science & Business Media B.V., Dordrecht, The Netherlands; a most experienced Publisher in the Sciences .

v vi Historical Development and Perspectives of the Series

The development of Biological Inorganic Chemistry during the past 40 years was and still is driven by several factors; among these are (i) the attempts to reveal the interplay between metal ions and peptides, nucleotides, hormones or vitamins, etc., (ii) the efforts regarding the understanding of accumulation, transport, metabolism and toxicity of metal ions, (iii) the development and application of metal-based drugs, (iv) biomimetic syntheses with the aim to understand biological processes as well as to create effi cient catalysts, (v) the determination of high-resolution structures of proteins, nucleic acids, and other biomolecules, (vi) the utilization of powerful spectroscopic tools allowing studies of structures and dynamics, and (vii), more recently, the widespread use of macromolecular engineering to create new biologically relevant structures at will. All this and more is and will be refl ected in the volumes of the series Metal Ions in Life Sciences . The importance of metal ions to the vital functions of living organisms, hence, to their health and well-being, is nowadays well accepted. However, in spite of all the progress made, we are still only at the brink of understanding these processes. Therefore, the series Metal Ions in Life Sciences will endeavor to link coordination chemistry and biochemistry in their widest sense. Despite the evident expectation that a great deal of future outstanding discoveries will be made in the interdisciplin- ary areas of science, there are still “language” barriers between the historically separate spheres of chemistry, biology, medicine, and physics. Thus, it is one of the aims of this series to catalyze mutual “understanding”. It is our hope that Metal Ions in Life Sciences proves a stimulus for new activities in the fascinating “fi eld” of Biological Inorganic Chemistry. If so, it will well serve its purpose and be a rewarding result for the efforts spent by the authors.

Astrid Sigel and Helmut Sigel Department of Chemistry, Inorganic Chemistry, University of Basel, CH-4056 Basel, Switzerland

Roland K.O. Sigel Institute of Inorganic Chemistry, University of Zürich, CH-8057 Zürich, Switzerland

October 2005, October 2008, and August 2011 Preface to Volume 13

Interrelations Between Essential Metal Ions and Human Diseases

Most of the 13 metals and 3 metalloids and their ions, which are covered in this volume, have been proven to be essential for humans. Indeed, it is an old wisdom that metal ions are indispensable for life. The main group metals, i.e., sodium, potassium, magnesium, and calcium, belong to the so-called bulk elements, and they occur in humans (70 kg) between about 20 g (Mg) and 1000 g (Ca) [H. Sigel, A. Sigel, H. G. Seiler, in Handbook on Metals in Clinical and Analytical Chemistr y, Eds H. G. Seiler, A. Sigel, H. Sigel, Dekker, New York, 1994, pp. 1–12]. The remaining 9 metals are transition elements, including zinc, and they all occur at trace levels, though iron and zinc dominate in humans with about 4 and 2.5 g, respectively. All the other metals, as well as the three metalloids (silicon, arsenic, selenium), occur only at ultra-trace levels, e.g., manganese and cobalt with about 12 and 1 mg, respectively. They comprise the essential elements manganese, cobalt, copper, molybdenum, and selenium; chromium, vanadium, nickel, silicon, and arsenic have been proposed as being essential in the second half of the last century. However, it turned out that their essentiality is difﬁ cult to establish because, if at all, they are certainly needed only in ultra-trace amounts, and because of their prevalence in the environment from natural and anthropomorphic sources, it has been difﬁ cult to prove whether or not there is a requirement for them, though the likelihood for vanadium and silicon as being essential appears to be high. The introductory Chapter 1 presents an overview of the topic, metal ions and infectious diseases, as seen from the clinic. The dilemma is that next to the bulk elements, also the trace elements are required by both, humans and bacterial pathogens. Since these metal ions are both necessary for life, but toxic in excess, metal homeostasis is tightly controlled by both bacteria and humans. Thus, pathogens utilize a variety of strategies to sense, acquire, store, and export metal ions in/from the vertebrate host.

vii viii Preface to Volume 13

The bulk elements sodium, potassium, magnesium, and calcium are dealt with in Chapters 2 to 4. All these elements are essential for human health and the chapters summarize their basic physiological actions. For example, a proper cellular Mg 2+ homeostasis is in all instances compulsory; defi ciency or overload gives rise to diseases, and these are described. Interestingly, evolution has thoroughly exploited the chemical properties of Ca2+ , i.e., its fast ligand-exchange rate and its reversible binding to sites with an irregular geometry, and selected it as a carrier of cellular signals. The next chapters focus on the roles of the transition elements beginning in Chapter 5 with vanadium : Since vanadate can be considered a close blueprint of phosphate with respect to its built-up, it likely takes over a regulatory function in metabolic processes depending on phosphate; e.g., phosphatases can be inhibited and kinases activated, but its essentiality for humans has not been proven. Yet in 1982/83 the discovery of vanadate-dependent bromoperoxidase in the marine mac- roalga Ascophyllum nodosum established that some forms of life need it. At common concentrations it is non-toxic for humans and this opens up a wide playground for pharmacological applications. Similarly, is chromium essential, pharmacologically relevant or toxic? At present chromium cannot be considered as an essential element because (i) nutritional data demonstrating chromium defi ciency and improvement in symptoms from chromium supplementation are lacking, and (ii) no biomolecules have convincingly been demonstrated to bind chromium and to have an essential function in the body. Manganese , covered in Chapter 7 , is important for human health. Though it is absolutely necessary for development, metabolism, and the antioxidant system, excessive exposure or intake may lead to manganism, a neurodegenerative disorder that causes dopaminergic neuronal death and parkinson-like symptoms. The effects of iron defi ciency or overload are covered in great detail in Chapter 8. Iron is a redox-active metal which is abundant in the Earth’s crust. It has played a key role in the evolution of living systems and as such it is an essential element in a wide range of biological phenomena, being critical for the function of an enormous array of enzymes, energy transduction mechanisms, and oxygen carriers. Since the redox nature of iron renders the metal toxic in excess, all biological organisms carefully control iron levels. For example, low body iron levels are related to anemia, whereas systemic iron overload results from, e.g., hyperabsorption, and can be treated by iron-chelation therapy. Furthermore, iron chelators have been widely investigated for the treatment of cancer, tuberculosis, and malaria. Cobalt and its role in human health and disease is primarily defi ned by the functioning of cobalamin (vitamin B12 ); it is dealt with in Chapter 9. Cobalamin acts in humans as a cofactor for methylmalonyl-coenzyme A mutase and methionine synthase, both enzymes being important for health. Especially the dysfunction of methionine synthase causes disruption of many cellular processes and leads to disease. In contrast, so far no nickel -containing enzyme or cofactor is known in higher animals. However, nickel has been included in the group of “possibly essential elements” for animals and humans already in the 1970s and its importance for plants, bacteria, archaea, and unicellular eukaryotes is well documented. In this context Preface to Volume 13 ix

Helicobacter pylori, a gram-negative bacterium, may be mentioned. This pathogen colonizes the human gut, giving rise to acute and chronic gastric pathologies, including peptic ulcer, and possibly also to gastric carcinomas and lymphomas. The toxic effects of nickel can produce serious respiratory, cardiovascular, and kidney diseases; they also alter the immune response giving rise to dermatitis, etc. Copper , the metal of Chapter 11 , represents in humans the 3rd most abundant transition metal; it is essential but it can also harm cells due to its potential to catalyze the generation of toxic reactive oxygen species. Therefore, the transport of copper and the cellular copper content are tightly regulated. Nutritional copper defi - ciency gives rise to anemia, to neuropathies, to impaired immune responses, etc. Genetic copper defi ciency leads to Menkes disease and distal hereditory peripheral neuropathy. Genetic copper overload causes Wilson’s disease and infantile cirrhosis. Ingestion of high doses of copper gives rise to nausea, vomiting, headache, diarrhea, hemolytic anemia, gastrointestinal hemorrhage, liver as well as kidney failure and fi nally death may occur. Furthermore, alterations of copper homeostasis have been associated with neurodegenerative diseases such as prion diseases, Alzheimer’s disease, Parkinson’s disease or Huntington’s disease, etc., but the exact role of copper in these important neurological disorders remains unclear. Zinc is dealt with in Chapter 12 : The total amount of zinc in a human (70 kg) is 2 to 3 g, i.e., there is nearly as much zinc as there is iron. Also the cellular Zn 2+ concentrations are rather high, that is, nearly as high as those of major metabolites like ATP. The vast knowledge of the physiological functions of zinc in at least 3000 proteins and the recent recognition of fundamental regulatory functions of Zn2+ ions released from cells or within cells links this nutritionally essential metal ion to numerous human diseases. It is not only the right amount of zinc in the diet that maintains health, at least as important is the proper functioning of the dozens of proteins that control cellular zinc homeostasis and regulate its intracellular traffi c. Zinc and its role in organ pathophysiology as well as in genetic, metabolic, chronic, and infectious diseases are covered. The essential trace element molybdenum , treated in Chapter 13, plays a crucial role in human health and disease. Remarkably, it is the only metal of the 2nd transition row (4d) of the periodic table with a biological role for humans. Four mammalian Mo-dependent enzymes are known, all of them harboring a pterin-based molybdenum cofactor (Moco) in their active site. In the focus are the individual pathways and the clinical and cellular consequences of their dysfunction. In all these enzymes molybdenum catalyzes oxygen transfer reactions from or to substrates using water as oxygen donor or acceptor, whereby it shuttles between the oxidation states +IV and +VI. Especially important are the functions and defi ciencies of xanthine dehydrogenase and sulfi te oxidase. The underlying molecular basis of Moco defi ciency, possible treatment options, and links to other diseases including neuropsychiatric disorders are discussed. The metalloid silicon is the second most abundant element in the Earth’s crust behind oxygen and has many industrial applications including its use as an additive in the feed and beverage industry. Chapter 14 discusses the possible biological potential of the metalloid, which is bioavailable as orthosilicic acid, and its potential x Preface to Volume 13 benefi cial effects on human health. Asbestos, its fi brous crystalline form, is a health hazard promoting asbestosis and leading to signifi cant impairment of lung function and an increased cancer risk. Specifi c biochemical or physiological functions of silicon, if any, are largely unknown, although generally thought to exist. Can the toxic metalloid arsenic sustain life? Clearly, the biochemical and physiological properties of arsenic are invariably linked with the toxicity of this element. The aim of Chapter 15 is (i) to summarize the evidence for benefi cial or sustaining roles of arsenic in living organisms, including its substitution for phosphorus, and (ii) to summarize its Janus-faced role in both causing and treating human disease. Arsenic oxide, deadly at high doses, is also an approved and effective drug for the treatment of acute promyelocytic leukemia. The well known toxicity of this element and its ability to cause diseases, including cancer of the skin, lung, bladder, liver, and kidney, make it a health hazard. So far it has not been recognized as being essential for humans because it has been diffi cult to establish whether or not there is a requirement for arsenic at ultra-trace levels considering its prevalence in the environment from natural and anthropomorphic sources. In contrast, selenium is established as an essential micronutrient for mammals, but it is also proven to be toxic in excess, leading to selenosis. Selenium exerts its biological functions through selenoproteins which contain selenocysteine. In fact, 25 selenoproteins are encoded in the human genome; most of their known functions are involved in redox systems and signaling pathways. Overall, this volume offers a wealth of information about human health and the interrelations between essential, or possibly essential, metals or metalloids.

Astrid Sigel Helmut Sigel Roland K.O. Sigel Contents

Historical Development and Perspectives of the Series ...... v

Preface to Volume 13 ...... vii

Contributors to Volume 13 ...... xvii

Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series ...... xxi

Contents of Volumes in the Metal Ions in Life Sciences Series ...... xxiii

1 Metal Ions and Infectious Diseases. An Overview from the Clinic ...... 1 Peggy L. Carver Abstract ...... 2 1 Introduction ...... 3 2 Iron ...... 5 3 Zinc ...... 10 4 Selenium ...... 14 5 Copper ...... 18 6 Chromium ...... 19 7 Manganese ...... 20 8 Summary and Future Developments ...... 22 References ...... 23 2 Sodium and Potassium in Health and Disease ...... 29 Hana R. Pohl , John S. Wheeler , and H. Edward Murray Abstract ...... 30 1 Introduction ...... 30 2 Physiology of Sodium and Potassium in Humans ...... 32 3 Pathology Associated with Sodium Levels ...... 38

xi xii Contents

4 Pathology Associated with Potassium Levels ...... 41 5 Conclusion ...... 45 References ...... 46 3 Magnesium in Health and Disease...... 49 Andrea M. P. Romani Abstract ...... 50 1 Introduction ...... 50 2 Cellular Magnesium Homeostasis ...... 54 3 Magnesium in Disease ...... 55 4 Conclusions ...... 73 References ...... 75 4 Calcium in Health and Disease ...... 81 Marisa Brini , Denis Ottolini , Tito Calì , and Ernesto Carafoli Abstract ...... 82 1 Introduction ...... 83 2 General Properties of Calcium as a Signaling Agent ...... 88 3 Intracellular Calcium Handling ...... 93 4 Calcium as a Regulator of Biological Processes ...... 100 5 The Ambivalence of the Calcium Signal: Defects of Calcium Regulation and Disease ...... 116 6 Conclusions ...... 126 References ...... 130 5 Vanadium. Its Role for Humans ...... 139 Dieter Rehder Abstract ...... 139 1 Introduction ...... 140 2 Distribution and Cycling of Vanadium ...... 142 3 The Aqueous Chemistry of Vanadium and the Vanadate- Phosphate Antagonism ...... 147 4 The Medicinal Potential of Vanadium...... 152 5 Concluding Remarks and Prospects ...... 164 References ...... 167

6 Chromium: Is It Essential, Pharmacologically Relevant, or Toxic? ...... 171 John B. Vincent Abstract ...... 172 1 Introduction ...... 172 2 Is Chromium Essential? ...... 173 3 Is Chromium Pharmacologically Relevant?...... 180 4 Is Chromium Toxic? ...... 191 5 Concluding Remarks and Future Direction ...... 192 References ...... 194 Contents xiii

7 Manganese in Health and Disease ...... 199 Daiana Silva Avila , Robson Luiz Puntel , and Michael Aschner Abstract ...... 200 1 Introduction ...... 200 2 Manganese Transport ...... 206 3 Manganism. A Neurodegenerative Disease ...... 210 4 Symptoms and Sensitive Populations ...... 211 5 Manganism versus Parkinson’s Disease ...... 211 6 Manganese in the Etiology of Other Neurodegenerative Disorders . 212 7 Molecular Mechanisms of Toxicity ...... 214 8 Genetic Susceptibility ...... 216 9 Treatment ...... 217 10 General Conclusions ...... 218 References ...... 220 8 Iron: Effect of Overload and Defi ciency ...... 229 Robert C. Hider and Xiaole Kong Abstract ...... 230 1 Introduction ...... 231 2 Iron Defi ciency and Anemia ...... 248 3 Systemic Iron Overload ...... 255 4 Iron-Selective Chelators with Therapeutic Potential ...... 266 5 Neuropathology and Iron ...... 277 6 The Role of Iron Chelation in Cancer Therapy ...... 281 7 Iron and Infection ...... 282 8 Overview and Future Developments ...... 284 References ...... 286 9 Cobalt: Its Role in Health and Disease ...... 295 Kazuhiro Yamada Abstract ...... 296 1 Introduction ...... 296 2 Cobalamin, Vitamin B12 ...... 297 3 Vitamin B12 Defi ciency and Disease...... 310 4 Non-corrinoid Cobalt ...... 314 5 Implications and Future Development ...... 315 References ...... 317 10 Nickel and Human Health ...... 321 Barbara Zambelli and Stefano Ciurli Abstract ...... 322 1 Introduction: The Double Face of Nickel in Biological Systems ...... 322 2 Nickel Hazard for Human Health ...... 324 3 Nickel-Dependent Infectious Diseases ...... 336 xiv Contents

4 Nickel Essentiality in Animals and Humans ...... 348 5 Conclusions and Outlook ...... 350 References ...... 352 11 Copper: Effects of Defi ciency and Overload ...... 359 Ivo Scheiber , Ralf Dringen , and Julian F. B. Mercer Abstract ...... 360 1 Introduction ...... 360 2 Copper Biochemistry and Homeostasis ...... 361 3 Copper Defi ciency Disorders ...... 371 4 Copper Overload Disorders ...... 375 5 Neuropathology and Copper ...... 376 6 Overview and Future Developments ...... 380 References ...... 381 12 Zinc and Human Disease ...... 389 Wolfgang Maret Abstract ...... 390 1 Introduction ...... 390 2 Zinc Biochemistry ...... 391 3 Zinc in Organ Pathophysiology ...... 398 4 Zinc in Disease ...... 404 5 General Conclusions ...... 407 References ...... 409 13 Molybdenum in Human Health and Disease ...... 415 Guenter Schwarz and Abdel A. Belaidi Abstract ...... 416 1 Introduction ...... 417 2 Defi ciencies in Molybdenum Enzymes ...... 419 3 Molybdenum Cofactor Defi ciencies ...... 426 4 Association of Molybdenum with Other Disorders ...... 440 5 Concluding Remarks and Future Developments ...... 442 References ...... 444 14 Silicon: The Health Benefi ts of a Metalloid ...... 451 Keith R. Martin Abstract ...... 452 1 Introduction ...... 452 2 Silicon Biochemistry ...... 453 3 Silicon and Its Potential Health Benefi ts ...... 457 4 Toxicology of Silicon and Silica ...... 463 5 Potential Medicinal Uses of Silicon and Silicates ...... 467 6 Summary and Future Directions ...... 468 References ...... 469 Contents xv

15 Arsenic. Can This Toxic Metalloid Sustain Life? ...... 475 Dean E. Wilcox Abstract ...... 476 1 Introduction ...... 476 2 Toxicity ...... 481 3 Sustaining Roles...... 484 4 Beneﬁ cial Uses ...... 490 5 Summary ...... 492 References ...... 494 16 Selenium. Role of the Essential Metalloid in Health ...... 499 Suguru Kurokawa and Marla J. Berry Abstract ...... 500 1 Introduction ...... 501 2 Selenium in Biomolecules ...... 502 3 Function of Selenoproteins ...... 509 4 Selenium and Disease ...... 516 5 Health Beneﬁ ts of Selenium in Humans ...... 520 6 General Conclusions ...... 525 References ...... 527

Index ...... 535

Contributors to Volume 13

Numbers in parentheses indicate the pages on which the authors’ contributions begin. Michael Aschner Department of Pediatrics and Pharmacology , The Kennedy Center for Research on Human Development and The Molecular Toxicology Center , Nashville , TN 37232-0414 , USA, [email protected] (199) Daiana Silva Avila Biochemistry Graduation Program , Universidade Federal do Pampa , Uruguaiana , Rio Grande do Sul , Brazil, [email protected] (199) Abdel A. Belaidi Institute of Biochemistry, Department of Chemistry, Center for Molecular Medicine , University of Cologne , Zuelpicher Str. 47, D-50674 Köln , Germany (415) Marla J. Berry Department of Cell & Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu , HI 96813 , USA, [email protected] (499) Marisa Brini Department of Biology , University of Padova, Via U. Bassi 58/B, I-35131 Padova, Italy, [email protected] (81) Tito Calì Department of Biology , University of Padova, Via U. Bassi 58/B, I-35131 Padova, Italy (81) Ernesto Carafoli Venetian Institute of Molecular Medicine (VIMM), Via G. Orus 2, I-35129 Padova, Italy, [email protected] (81) Peggy L. Carver University of Michigan College of Pharmacy, Department of Clinical, Social, and Administrative Sciences, 428 Church St., Ann Arbor, MI 48109-1065 , USA, [email protected] (1) Stefano Ciurli Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology , University of Bologna, I-40127 Bologna, Italy, ste- [email protected] (321) Ralf Dringen Centre for Biomolecular Interactions Bremen , University of Bremen, D-28334 Bremen , Germany (359)

xvii xviii Contributors to Volume 13

Robert C. Hider Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, Stamford Street, London SE1 9NH, UK, [email protected] (229) Xiaole Kong Institute of Pharmaceutical Science , King’s College London, Franklin- Wilkins Building, Stamford Street, London SE1 9NH, UK, [email protected] (229) Suguru Kurokawa Department of Cell & Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu , HI 96813 , USA, [email protected] (499) Wolfgang Maret King’s College London, School of Medicine, Diabetes and Nutritional Sciences Division, Metal Metabolism Group, Franklin Wilkins Bldg, 150 Stamford St. , London SE1 9NH , UK, [email protected] (389) Keith R. Martin School of Nutrition and Health Promotion, Healthy Lifestyles Research Center , Arizona State University, 500 North 3rd Street , Phoenix , AZ 85004 , USA, [email protected] (451) Julian F. B. Mercer Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Melbourne Campus at Burwood, VIC 3125 , Australia, [email protected] (359) H. Edward Murray Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, 1600 Clifton Road, Mailstop F-57 , Atlanta , GA 30333 , USA (29) Denis Ottolini Department of Biology , University of Padova, Via U. Bassi 58/B, I-35131 Padova, Italy (81) Hana R. Pohl Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, 1600 Clifton Road, Mailstop F-57, Atlanta , GA 30333 , USA, [email protected] (29) Robson Luiz Puntel Biochemistry Graduation Program , Universidade Federal do Pampa, Uruguaiana , Rio Grande do Sul , Brazil, [email protected] (199) Dieter Rehder Chemistry Department, University of Hamburg, D-20146 Hamburg , Germany, [email protected] (139) Andrea M. P. Romani Department of Physiology and Biophysics, School of Medicine , Case Western Reserve University, 10900 Euclid Avenue , Cleveland , OH 44106-4970 , USA , [email protected] (49) Ivo Scheiber Department of Parasitology, Faculty of Science , Charles University , Prague , Czech Republic (359) Guenter Schwarz Institute of Biochemistry, Department of Chemistry, Center for Molecular Medicine , University of Cologne, Zuelpicher Str. 47, D-50674 Köln , Germany, [email protected] (415) Contributors to Volume 13 xix

John B. Vincent Department of Chemistry , The University of Alabama , Tuscaloosa , AL 35487-0336 , USA, [email protected] (171) John S. Wheeler Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, 1600 Clifton Road, Mailstop F-57 , Atlanta , GA 30333 , USA (29) Dean E. Wilcox Department of Chemistry , Dartmouth College , Hanover , NH 03755 , USA, [email protected] (475) Kazuhiro Yamada Department of Biochemistry , Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road , Bethesda , MD 20814 , USA, [email protected] (295) Barbara Zambelli Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology , University of Bologna, I-40127 Bologna , Italy, [email protected] (321)

Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series edited by the SIGELs and published by Dekker/Taylor & Francis (1973–2005)

Volume 1: Simple Complexes Volume 2: Mixed-Ligand Complexes Volume 3: High Molecular Complexes Volume 4: Metal Ions as Probes Volume 5: Reactivity of Coordination Compounds Volume 6: Biological Action of Metal Ions Volume 7: Iron in Model and Natural Compounds Volume 8: Nucleotides and Derivatives: Their Ligating Ambivalency Volume 9: Amino Acids and Derivatives as Ambivalent Ligands Volume 10: Carcinogenicity and Metal Ions Volume 11: Metal Complexes as Anticancer Agents Volume 12: Properties of Copper Volume 13: Copper Proteins Volume 14: Inorganic Drugs in Deﬁ ciency and Disease Volume 15: Zinc and Its Role in Biology and Nutrition Volume 16: Methods Involving Metal Ions and Complexes in Clinical Chemistry Volume 17: Calcium and Its Role in Biology Volume 18: Circulation of Metals in the Environment Volume 19: Antibiotics and Their Complexes Volume 20: Concepts on Metal Ion Toxicity Volume 21: Applications of Nuclear Magnetic Resonance to Paramagnetic Species Volume 22: ENDOR, EPR, and Electron Spin Echo for Probing Coordination Spheres Volume 23: Nickel and Its Role in Biology Volume 24: Aluminum and Its Role in Biology Volume 25: Interrelations Among Metal Ions, Enzymes, and Gene Expression Volume 26: Compendium on Magnesium and Its Role in Biology, Nutrition, and Physiology Volume 27: Electron Transfer Reactions in Metalloproteins

xxi xxii Titles of Volumes 1–44 in the Metal Ions in Biological Systems Series

Volume 28: Degradation of Environmental Pollutants by Microorganisms and Their Metalloenzymes Volume 29: Biological Properties of Metal Alkyl Derivatives Volume 30: Metalloenzymes Involving Amino Acid-Residue and Related Radicals Volume 31: Vanadium and Its Role for Life Volume 32: Interactions of Metal Ions with Nucleotides, Nucleic Acids, and Their Constituents Volume 33: Probing Nucleic Acids by Metal Ion Complexes of Small Molecules Volume 34: Mercury and Its Effects on Environment and Biology Volume 35: Iron Transport and Storage in Microorganisms, Plants, and Animals Volume 36: Interrelations Between Free Radicals and Metal Ions in Life Processes Volume 37: Manganese and Its Role in Biological Processes Volume 38: Probing of Proteins by Metal Ions and Their Low-Molecular- Weight Complexes Volume 39: Molybdenum and Tungsten. Their Roles in Biological Processes Volume 40: The Lanthanides and Their Interrelations with Biosystems Volume 41: Metal Ions and Their Complexes in Medication Volume 42: Metal Complexes in Tumor Diagnosis and as Anticancer Agents Volume 43: Biogeochemical Cycles of Elements Volume 44: Biogeochemistry, Availability, and Transport of Metals in the Environment Contents of Volumes in the Metal Ions in Life Sciences Series edited by the SIGELs

Volumes 1–4 published by John Wiley & Sons, Ltd., Chichester, UK (2006–2008) < http://www.Wiley.com/go/mils >

Volume 5–9 by the Royal Society of Chemistry, Cambridge, UK (2009–2011) < http://www.rsc.org/shop/metalionsinlifesciences > and from Volume 10 on by Springer Science & Business Media BV, Dordrecht, The Netherlands (since 2012) < http://www.mils-series.com >

Volume 1 Neurodegenerative Diseases and Metal Ions

1 The Role of Metal Ions in Neurology. An Introduction Dorothea Strozyk and Ashley I. Bush 2 Protein Folding, Misfolding, and Disease Jennifer C. Lee, Judy E. Kim, Ekaterina V. Pletneva, Jasmin Faraone-Mennella, Harry B. Gray, and Jay R. Winkler 3 Metal Ion Binding Properties of Proteins Related to Neurodegeneration Henryk Kozlowski, Marek Luczkowski, Daniela Valensin, and Gianni Valensin 4 Metallic Prions: Mining the Core of Transmissible Spongiform Encephalopathies David R. Brown 5 The Role of Metal Ions in the Amyloid Precursor Protein and in Alzheimer ’ s Disease Thomas A. Bayer and Gerd Multhaup

xxiii xxiv Contents of Volumes in the Metal Ions in Life Sciences Series

6 The Role of Iron in the Pathogenesis of Parkinson ’ s Disease Manfred Gerlach, Kay L. Double, Mario E. Götz, Moussa B.H. Youdim, and Peter Riederer 7 In Vivo Assessment of Iron in Huntington ’ s Disease and Other Age-Related Neurodegenerative Brain Diseases George Bartzokis, Po H. Lu, Todd A. Tishler, and Susan Perlman 8 Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis Lisa J. Whitson and P. John Hart 9 The Malfunctioning of Copper Transport in Wilson and Menkes Diseases Bibudhendra Sarkar 10 Iron and Its Role in Neurodegenerative Diseases Roberta J. Ward and Robert R. Crichton 11 The Chemical Interplay between Catecholamines and Metal Ions in Neurological Diseases Wolfgang Linert, Guy N.L. Jameson, Reginald F. Jameson, and Kurt A. Jellinger 12 Zinc Metalloneurochemistry: Physiology, Pathology, and Probes Christopher J. Chang and Stephen J. Lippard 13 The Role of Aluminum in Neurotoxic and Neurodegenerative Processes Tamás Kiss, Krisztina Gajda-Schrantz, and Paolo F. Zatta 14 Neurotoxicity of Cadmium, Lead, and Mercury Hana R. Pohl, Henry G. Abadin, and John F. Risher 15 Neurodegerative Diseases and Metal Ions. A Concluding Overview Dorothea Strozyk and Ashley I. Bush

Subject Index

Volume 2 Nickel and Its Surprising Impact in Nature

1 Biogeochemistry of Nickel and Its Release into the Environment Tiina M. Nieminen, Liisa Ukonmaanaho, Nicole Rausch, and William Shotyk 2 Nickel in the Environment and Its Role in the Metabolism of Plants and Cyanobacteria Hendrik Küpper and Peter M.H. Kroneck 3 Nickel Ion Complexes of Amino Acids and Peptides Teresa Kowalik- Jankowska, Henryk Kozlowski, Etelka Farkas, and Imre Sóvágó Contents of Volumes in the Metal Ions in Life Sciences Series xxv

4 Complex Formation of Nickel(II) and Related Metal Ions with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids Roland K.O. Sigel and Helmut Sigel 5 Synthetic Models for the Active Sites of Nickel-Containing Enzymes Jarl Ivar van der Vlugt and Franc Meyer 6 Urease: Recent Insights in the Role of Nickel Stefano Ciurli 7 Nickel Iron Hydrogenases Wolfgang Lubitz, Maurice van Gastel, and Wolfgang Gärtner 8 Methyl-Coenzyme M Reductase and Its Nickel Corphin

Coenzyme F 430 in Methanogenic Archaea Bernhard Jaun and Rudolf K. Thauer 9 Acetyl-Coenzyme A Synthases and Nickel-Containing Carbon Monoxide Dehydrogenases Paul A. Lindahl and David E. Graham 10 Nickel Superoxide Dismutase Peter A. Bryngelson and Michael J. Maroney 11 Biochemistry of the Nickel-Dependent Glyoxylase I Enzymes Nicole Sukdeo, Elisabeth Daub, and John F. Honek 12 Nickel in Acireductone Dioxygenase Thomas C. Pochapsky, Tingting Ju, Marina Dang, Rachel Beaulieu, Gina Pagani, and Bo OuYang 13 The Nickel-Regulated Peptidyl-Prolyl cis/trans Isomerase SlyD Frank Erdmann and Gunter Fischer 14 Chaperones of Nickel Metabolism Soledad Quiroz, Jong K. Kim, Scott B. Mulrooney, and Robert P. Hausinger 15 The Role of Nickel in Environmental Adaptation of the Gastric Pathogen Helicobacter pylori Florian D. Ernst, Arnoud H.M. van Vliet, Manfred Kist, Johannes G. Kusters, and Stefan Bereswill 16 Nickel-Dependent Gene Expression Konstantin Salnikow and Kazimierz S. Kasprzak 17 Nickel Toxicity and Carcinogenesis Kazimierz S. Kasprzak and Konstantin Salnikow

Subject Index xxvi Contents of Volumes in the Metal Ions in Life Sciences Series

Volume 3 The Ubiquitous Roles of Cytochrome P450 Proteins

1 Diversities and Similarities of P450 Systems: An Introduction Mary A. Schuler and Stephen G. Sligar 2 Structural and Functional Mimics of Cytochromes P450 Wolf-D. Woggon 3 Structures of P450 Proteins and Their Molecular Phylogeny Thomas L. Poulos and Yergalem T. Meharenna 4 Aquatic P450 Species Mark J. Snyder 5 The Electrochemistry of Cytochrome P450 Alan M. Bond, Barry D. Fleming, and Lisandra L. Martin 6 P450 Electron Transfer Reactions Andrew K. Udit, Stephen M. Contakes, and Harry B. Gray 7 Leakage in Cytochrome P450 Reactions in Relation to Protein Structural Properties Christiane Jung 8 Cytochromes P450. Structural Basis for Binding and Catalysis Konstanze von König and Ilme Schlichting 9 Beyond Heme-Thiolate Interactions: Roles of the Secondary Coordination Sphere in P450 Systems Yi Lu and Thomas D. Pﬁ ster 10 Interactions of Cytochrome P450 with Nitric Oxide and Related Ligands Andrew W. Munro, Kirsty J. McLean, and Hazel M. Girvan 11 Cytochrome P450-Catalyzed Hydroxylations and Epoxidations Roshan Perera, Shengxi Jin, Masanori Sono, and John H. Dawson 12 Cytochrome P450 and Steroid Hormone Biosynthesis Rita Bernhardt and Michael R. Waterman 13 Carbon-Carbon Bond Cleavage by P450 Systems James J. De Voss and Max J. Cryle 14 Design and Engineering of Cytochrome P450 Systems Stephen G. Bell, Nicola Hoskins, Christopher J.C. Whitehouse, and Luet L. Wong 15 Chemical Defense and Exploitation. Biotransformation of Xenobiotics by Cytochrome P450 Enzymes Elizabeth M.J. Gillam and Dominic J.B. Hunter Contents of Volumes in the Metal Ions in Life Sciences Series xxvii

16 Drug Metabolism as Catalyzed by Human Cytochrome P450 Systems F. Peter Guengerich 17 Cytochrome P450 Enzymes: Observations from the Clinic Peggy L. Carver

Subject Index

Volume 4 Biomineralization. From Nature to Application

1 Crystals and Life: An Introduction Arthur Veis 2 What Genes and Genomes Tell Us about Calcium Carbonate Biomineralization Fred H. Wilt and Christopher E. Killian 3 The Role of Enzymes in Biomineralization Processes Ingrid M. Weiss and Frédéric Marin 4 Metal–Bacteria Interactions at Both the Planktonic Cell and Bioﬁ lm Levels Ryan C. Hunter and Terry J. Beveridge 5 Biomineralization of Calcium Carbonate. The Interplay with Biosubstrates Amir Berman 6 Sulfate-Containing Biominerals Fabienne Bosselmann and Matthias Epple 7 Oxalate Biominerals Enrique J. Baran and Paula V. Monje 8 Molecular Processes of Biosiliciﬁ cation in Diatoms Aubrey K. Davis and Mark Hildebrand 9 Heavy Metals in the Jaws of Invertebrates Helga C. Lichtenegger, Henrik Birkedal, and J. Herbert Waite 10 Ferritin. Biomineralization of Iron Elizabeth C. Theil, Xiaofeng S. Liu, and Manolis Matzapetakis 11 Magnetism and Molecular Biology of Magnetic Iron Minerals in Bacteria Richard B. Frankel, Sabrina Schübbe, and Dennis A. Bazylinski 12 Biominerals. Recorders of the Past? Danielle Fortin, Sean R. Langley, and Susan Glasauer 13 Dynamics of Biomineralization and Biodemineralization Lijun Wang and George H. Nancollas xxviii Contents of Volumes in the Metal Ions in Life Sciences Series

14 Mechanism of Mineralization of Collagen-Based Connective Tissues Adele L. Boskey 15 Mammalian Enamel Formation Janet Moradian-Oldak and Michael L. Paine 16 Mechanical Design of Biomineralized Tissues. Bone and Other Hierarchical Materials Peter Fratzl 17 Bioinspired Growth of Mineralized Tissue Darilis Suárez-González and William L. Murphy 18 Polymer-Controlled Biomimetic Mineralization of Novel Inorganic Materials Helmut Cölfen and Markus Antonietti

Subject Index

Volume 5 Metallothioneins and Related Chelators

1 Metallothioneins. Historical Development and Overview Monica Nordberg and Gunnar F. Nordberg 2 Regulation of Metallothionein Gene Expression Kuppusamy Balamurugan and Walter Schaffner 3 Bacterial Metallothioneins Claudia A. Blindauer 4 Metallothioneins in Yeast and Fungi Benedikt Dolderer, Hans-Jürgen Hartmann, and Ulrich Weser 5 Metallothioneins in Plants Eva Freisinger 6 Metallothioneins in Diptera Silvia Atrian 7 Earthworm and Nematode Metallothioneins Stephen R. Stürzenbaum 8 Metallothioneins in Aquatic Organisms: Fish, Crustaceans, Molluscs, and Echinoderms Laura Vergani 9 Metal Detoxiﬁ cation in Freshwater Animals. Roles of Metallothioneins Peter G.C. Campbell and Landis Hare Contents of Volumes in the Metal Ions in Life Sciences Series xxix

10 Structure and Function of Vertebrate Metallothioneins Juan Hidalgo, Roger Chung, Milena Penkowa, and Milan Vašák 11 Metallothionein-3, Zinc, and Copper in the Central Nervous System Milan Vašák and Gabriele Meloni 12 Metallothionein Toxicology: Metal Ion Trafﬁ cking and Cellular Protection David H. Petering, Susan Krezoski, and Niloofar M. Tabatabai 13 Metallothionein in Inorganic Carcinogenesis Michael P. Waalkes and Jie Liu 14 Thioredoxins and Glutaredoxins. Functions and Metal Ion Interactions Christopher Horst Lillig and Carsten Berndt 15 Metal Ion-Binding Properties of Phytochelatins and Related Ligands Aurélie Devez, Eric Achterberg, and Martha Gledhill

Subject Index

Volume 6 Metal-Carbon Bonds in Enzymes and Cofactors

1 Organometallic Chemistry of B 12 Coenzymes Bernhard Kräutler 2 Cobalamin- and Corrinoid-Dependent Enzymes Rowena G. Matthews 3 Nickel-Alkyl Bond Formation in the Active Site of Methyl-Coenzyme M Reductase Bernhard Jaun and Rudolf K. Thauer 4 Nickel-Carbon Bonds in Acetyl-Coenzyme A Synthases/Carbon Monoxide Dehydrogenases Paul A. Lindahl 5 Structure and Function of [NiFe]-Hydrogenases Juan C. Fontecilla-Camps 6 Carbon Monoxide and Cyanide Ligands in the Active Site of [FeFe]-Hydrogenases John W. Peters 7 Carbon Monoxide as Intrinsic Ligand to Iron in the Active Site of [Fe]-Hydrogenase Seigo Shima, Rudolf K. Thauer, and Ulrich Ermler xxx Contents of Volumes in the Metal Ions in Life Sciences Series

8 The Dual Role of Heme as Cofactor and Substrate in the Biosynthesis of Carbon Monoxide Mario Rivera and Juan C. Rodriguez 9 Copper-Carbon Bonds in Mechanistic and Structural Probing of Proteins as well as in Situations where Copper Is a Catalytic or Receptor Site Heather R. Lucas and Kenneth D. Karlin 10 Interaction of Cyanide with Enzymes Containing Vanadium and Manganese, Non-Heme Iron, and Zinc Martha E. Sosa-Torres and Peter M.H. Kroneck 11 The Reaction Mechanism of the Molybdenum Hydroxylase Xanthine Oxidoreductase: Evidence against the Formation of Intermediates Having Metal-Carbon Bonds Russ Hille 12 Computational Studies of Bioorganometallic Enzymes and Cofactors Matthew D. Liptak, Katherine M. Van Heuvelen, and Thomas C. Brunold

Subject Index

Author Index of MIBS -1 to MIBS -44 and MILS -1 to MILS - 6

Volume 7 Organometallics in Environment and Toxicology

1 Roles of Organometal(loid) Compounds in Environmental Cycles John S. Thayer 2 Analysis of Organometal(loid) Compounds in Environmental and Biological Samples Christopher F. Harrington, Daniel S. Vidler, and Richard O. Jenkins 3 Evidence for Organometallic Intermediates in Bacterial Methane

Formation Involving the Nickel Coenzyme F 430 Mishtu Dey, Xianghui Li, Yuzhen Zhou, and Stephen W. Ragsdale 4 Organotins. Formation, Use, Speciation, and Toxicology Tamas Gajda and Attila Jancsó 5 Alkyllead Compounds and Their Environmental Toxicology Henry G. Abadin and Hana R. Pohl 6 Organoarsenicals: Distribution and Transformation in the Environment Kenneth J. Reimer, Iris Koch, and William R. Cullen 7 Organoarsenicals. Uptake, Metabolism, and Toxicity Elke Dopp, Andrew D. Kligerman, and Roland A. Diaz-Bone Contents of Volumes in the Metal Ions in Life Sciences Series xxxi

8 Alkyl Derivatives of Antimony in the Environment Montserrat Filella 9 Alkyl Derivatives of Bismuth in Environmental and Biological Media Montserrat Filella 10 Formation, Occurrence and Signiﬁ cance of Organoselenium and Organotellurium Compounds in the Environment Dirk Wallschläger and Jörg Feldmann 11 Organomercurials. Their Formation and Pathways in the Environment Holger Hintelmann 12 Toxicology of Alkylmercury Compounds Michael Aschner, Natalia Onishchenko, and Sandra Ceccatelli 13 Environmental Bioindication, Biomonitoring, and Bioremediation of Organometal(loid)s John S. Thayer 14 Methylated Metal(loid) Species in Humans Alfred V. Hirner and Albert W. Rettenmeier

Subject Index

Volume 8 Metal Ions in Toxicology: Effects, Interactions, Interdependencies

1 Understanding Combined Effects for Metal Co-Exposure in Ecotoxicology Rolf Altenburger 2 Human Risk Assessment of Heavy Metals: Principles and Applications Jean- Lou C.M. Dorne, George E.N. Kass, Luisa R. Bordajandi, Billy Amzal, Ulla Bertelsen, Anna F. Castoldi, Claudia Heppner, Mari Eskola, Stefan Fabiansson, Pietro Ferrari, Elena Scaravelli, Eugenia Dogliotti, Peter Fuerst, Alan R. Boobis, and Philippe Verger 3 Mixtures and Their Risk Assessment in Toxicology Moiz M. Mumtaz, Hugh Hansen, and Hana R. Pohl 4 Metal Ions Affecting the Pulmonary and Cardiovascular Systems Massimo Corradi and Antonio Mutti 5 Metal Ions Affecting the Gastrointestinal System Including the Liver Declan P. Naughton, Tamás Nepusz, and Andrea Petroczi 6 Metal Ions Affecting the Kidney Bruce A. Fowler xxxii Contents of Volumes in the Metal Ions in Life Sciences Series

7 Metal Ions Affecting the Hematological System Nickolette Roney, Henry G. Abadin, Bruce Fowler, and Hana R. Pohl 8 Metal Ions Affecting the Immune System Irina Lehmann, Ulrich Sack, and Jörg Lehmann 9 Metal Ions Affecting the Skin and Eyes Alan B.G. Lansdown 10 Metal Ions Affecting the Neurological System Hana R. Pohl, Nickolette Roney, and Henry G. Abadin 11 Metal Ions Affecting Reproduction and Development Pietro Apostoli and Simona Catalani 12 Are Cadmium and Other Heavy Metal Compounds Acting as Endocrine Disrupters? Andreas Kortenkamp 13 Genotoxicity of Metal Ions: Chemical Insights Woijciech Bal, Anna Maria Protas, and Kazimierz S. Kasprzak 14 Metal Ions in Human Cancer Development Erik J. Tokar, Lamia Benbrahim-Tallaa, and Michael P. Waalkes

Subject Index

Volume 9 Structural and Catalytic Roles of Metal Ions in RNA

1 Metal Ion Binding to RNA Pascal Aufﬁ nger, Neena Grover, and Eric Westhof 2 Methods to Detect and Characterize Metal Ion Binding Sites in RNA Michèle C. Erat and Roland K.O. Sigel, 3 Importance of Diffuse Metal Ion Binding to RNA Zhi-Jie Tan and Shi-Jie Chen 4 RNA Quadruplexes Kangkan Halder and Jörg S. Hartig 5 The Roles of Metal Ions in Regulation by Riboswitches Adrian Ferré-D’Amaré and Wade C. Winkler 6 Metal Ions: Supporting Actors in the Playbook of Small Ribozymes Alexander E. Johnson-Buck, Sarah E. McDowell, and Nils G. Walter 7 Multiple Roles of Metal Ions in Large Ribozymes Daniela Donghi and Joachim Schnabl Contents of Volumes in the Metal Ions in Life Sciences Series xxxiii

8 The Spliceosome and Its Metal Ions Samuel E. Butcher 9 The Ribosome: A Molecular Machine Powered by RNA Krista Trappl and Norbert Polacek 10 Metal Ion Requirements in Artiﬁ cial Ribozymes that Catalyze Aminoacylations and Redox Reactions Hiroaki Suga, Kazuki Futai, and Koichiro Jin 11 Metal Ion Binding and Function in Natural and Artiﬁ cial Small RNA Enzymes from a Structural Perspective Joseph E. Wedekind 12 Binding of Kinetically Inert Metal Ions to RNA: The Case of Platinum(II) Erich G. Chapman, Alethia A. Hostetter, Maire F. Osborn, Amanda L. Miller, and Victoria J. DeRose

Subject Index

Volume 10 Interplay between Metal Ions and Nucleic Acids

1 Characterization of Metal Ion-Nucleic Acid Interactions in Solution Maria Pechlaner and Roland K.O. Sigel 2 Nucleic Acid-Metal Ion Interactions in the Solid State Katsuyuki Aoki and Kazutaka Murayama 3 Metal Ion-Promoted Conformational Changes of Oligonucleotides Bernhard Spingler 4 G-Quadruplexes and Metal Ions Nancy H. Campbell and Stephen Neidle 5 Metal Ion-Mediated DNA-Protein Interactions Barbara Zambelli, Francesco Musiani, and Stefano Ciurli 6 Spectroscopic Investigations of Lanthanide Ion Binding to Nucleic Acids Janet R. Morrow and Christopher M. Andolina 7 Oxidative DNA Damage Mediated by Transition Metal Ions and Their Complexes Geneviève Pratviel 8 Metal Ion-Dependent DNAzymes and Their Applications as Biosensors Tian Lan and Yi Lu xxxiv Contents of Volumes in the Metal Ions in Life Sciences Series

9 Enantioselective Catalysis at the DNA Scaffold Almudena García-Fernández and Gerard Roelfes 10 Alternative DNA Base Pairing through Metal Coordination Guido H. Clever and Mitsuhiko Shionoya 11 Metal-Mediated Base Pairs in Nucleic Acids with Purine- and Pyrimidine-Derived Nucleosides Dominik A. Megger, Nicole Megger, and Jens Müller 12 Metal Complex Derivatives of Peptide Nucleic Acids Roland Krämer and Andrij Mokhir

Subject Index

Volume 11 Cadmium: From Toxicity to Essentiality

1 The Bioinorganic Chemistry of Cadmium in the Context of Its Toxicity Wolfgang Maret and Jean-Marc Moulis 2 Biogeochemistry of Cadmium and Its Release to the Environment Jay T. Cullen and Maria T. Maldonado 3 Speciation of Cadmium in the Environment Francesco Crea, Claudia Foti, Demetrio Milea, and Silvio Sammartano 4 Determination of Cadmium in Biological Samples Katrin Klotz, Wobbeke Weistenhöfer, and Hans Drexler 5 Imaging and Sensing of Cadmium in Cells Masayasu Taki 6 Use of 113 Cd NMR to Probe the Native Metal Binding Sites in Metalloproteins: An Overview Ian M. Armitage, Torbjörn Drakenberg, and Brian Reilly 7 Solid State Structures of Cadmium Complexes with Relevance for Biological Systems Rosa Carballo, Alfonso Castiñeiras, Alicia Domínguez-Martín, Isabel García Santos, and Juan Niclós-Gutierrez 8 Complex Formation of Cadmium(II) with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids Roland K.O. Sigel, Miriam Skilandat, Astrid Sigel, Bert P. Operschall, and Helmut Sigel 9 Cadmium(II) Complexes of Amino Acids and Peptides Imre Sóvágó and Katalin Várnagy Contents of Volumes in the Metal Ions in Life Sciences Series xxxv

10 Natural and Artiﬁ cial Proteins Containing Cadmium Anna F. Peacock and Vincent L. Pecoraro 11 Cadmium in Metallothioneins Eva Freisinger and Milan Vašák 12 Cadmium-Accumulating Plants Hendrik Küpper and Barbara Leitenmaier 13 Cadmium Toxicity in Plants Elisa Andresen and Hendrik Küpper 14 Toxicology of Cadmium and Its Damage to Mammalian Organs Frank Thévenod and Wing-Kee Lee 15 Cadmium and Cancer Andrea Hartwig 16 Cadmium in Marine Phytoplankton Yan Xu and François M.M. Morel

Subject Index

Volume 12 Metallomics and the Cell Guest Editor: Lucia Banci

1 Metallomics and the Cell: Some Deﬁ nitions and General Comments Lucia Banci and Ivano Bertini 2 Technologies for Detecting Metals in Single Cells James E. Penner-Hahn 3 Sodium/Potassium Homeostasis in the Cell Michael J.V. Clausen and Hanna Poulsen 4 Magnesium Homeostasis in Mammalian Cells Andrea M.P. Romani 5 Intracellular Calcium Homeostasis and Signaling Marisa Brini, Tito Calì, Denis Ottolini, and Ernesto Carafoli 6 Manganese Homeostasis and Transport Jerome Roth, Silvia Ponzoni, and Michael Aschner 7 Control of Iron Metabolism in Bacteria Simon Andrews, Ian Norton, Arvindkumar S. Salunkhe, Helen Goodluck, Wafaa S.M. Aly, Hanna Mourad- Agha, and Pierre Cornelis 8 The Iron Metallome in Eukaryotic Organisms Adrienne C. Dlouhy and Caryn E. Outten xxxvi Contents of Volumes in the Metal Ions in Life Sciences Series

9 Heme Uptake and Metabolism in Bacteria David R. Benson and Mario Rivera 10 Cobalt and Corrinoid Transport and Biochemistry Valentin Cracan and Ruma Banerjee 11 Nickel Metallomics: General Themes Guiding Nickel Homeostasis Andrew M. Sydor and Deborah B. Zamble 12 The Copper Metallome in Prokaryotic Cells Christopher Rensing and Sylvia Franke McDevitt 13 The Copper Metallome in Eukaryotic Cells Katherine E. Vest, Hayaa F. Hashemi, and Paul A. Cobine 14 Zinc and the Zinc Proteome Wolfgang Maret 15 Metabolism of Molybdenum Ralf R. Mendel 16 Comparative Genomics Analysis of the Metallomes Vadim N. Gladyshev and Yan Zhang

Subject Index

Volume 13 Interrelations between Essential Metal Ions and Human Diseases (this book)

Volume 14 The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment (in preparation) Guest Editors: Peter M.H. Kroneck and Martha E. Sosa-Torres

1 The Early Earth Atmosphere and Early Life Catalysts Sandra I. Ramírez Jiménez 2 Living on Acetylene, a Primordial Energy Source (Acetylene Hydratase) Felix ten Brink 3 Carbon Monoxide, Toxic Gas and Fuel for Anaerobes and Aerobes: Carbon Monoxide Dehydrogenases Jae-Hun Jeoung, Jochen Fesseler, Sebastian Götzl, and Holger Dobbek 4 Investigations of the Efﬁ cient Electrocatalytic

Interconversions of CO 2 and CO by Nickel-Containing Carbon Monoxide Dehydrogenases Vincent Wang, Stephen W. Ragsdale, and Fraser A. Armstrong 5 Nature ’ s Toolbox to Handle Dihydrogen (Hydrogenases) Alison Parkin Contents of Volumes in the Metal Ions in Life Sciences Series xxxvii

6 The Making of the Greenhouse Gas Methane (Methanogenesis) Dariusz A. Sliwa and Stephen W. Ragsdale 7 Light-Dependent Production of Dioxygen (Photosynthesis) Vittal Yachandra and Junko Yano 8 Production of Dioxygen in the Dark (Dismutases of Oxyanions) Jennifer DuBois 9 Transition Metal Complexes and Activation of Dioxygen (Model Compounds, Catalysis) Gereon M. Yee and William B. Tolman 10 Respiratory Conservation of Energy with Dioxygen (Respiratory Chain/Cytochrome c Oxidase) Shinya Yoshikawa 11 Methane Monooxygenase: Breaking up Methane with Iron and Copper Matthew H. Sazinsky and Stephen J. Lippard

12 Cleaving the N,N Triple Bond: The Transformation of N 2 to NH 3 (Nitrogenase) Chi Chung Lee, Yilin Hu, and Markus W. Ribbe 13 The Production of Ammonia by Multiheme Cytochromes c Jörg Simon and Peter M.H. Kroneck 14 No Laughing Matter: The Making of the Greenhouse

Gas Dinitrogen Monoxide (N 2 O Reductase) Oliver Einsle 15 Hydrogen Sulﬁ de: A Toxic Gas Produced by Dissimilatory Sulfate Reduction and Consumed by Microbial Oxidation Larry L. Barton, Marie- Laure Fardeau, and Guy Fauque 16 Anaerobic Oxidation of Methane and Ammonia Mike S.M. Jetten 17 Transformation of Dimethylsulfoxide Ulrike Kappler

Subject Index

Comments and suggestions with regard to contents, topics, and the like for future volumes of the series are welcome. Chapter 1 Metal Ions and Infectious Diseases. An Overview from the Clinic

Peggy L. Carver

Contents ABSTRACT ...... 2 1 INTRODUCTION ...... 3 1.1 Role of Antioxidants ...... 3 1.2 Host Defense Responses to Infection ...... 3 1.3 Alterations in Serum Levels of Trace Elements ...... 4 1.4 Nutritional Immunity ...... 4 1.5 Natural Resistance-Associated Macrophage Protein (Nramp) ...... 5 1.6 Calprotectin ...... 5 2 IRON ...... 5 2.1 Human Pharmacology and Pharmacokinetics ...... 5 2.2 The Complex Defense-Counter Defense System in the Battle for Iron...... 6 2.3 Role of Iron in Infectious Diseases ...... 6 2.3.1 Dialysis Patients ...... 7 2.3.2 Malaria ...... 7 2.3.3 Human Immunodeﬁ ciency Virus ...... 8 2.3.4 Diabetes ...... 8 2.3.5 Iron Overload ...... 8 2.3.6 Role of Iron Chelators in Infection ...... 9 3 ZINC ...... 10 3.1 Human Pharmacology and Pharmacokinetics ...... 10 3.1.1 Zn-Metallothionein (Zn-MT) ...... 11 3.1.2 Zn-Metallo β-Lactamases ...... 11 3.2 Role of Zinc in Infectious Diseases ...... 11 3.2.1 Cystic Fibrosis ...... 11 3.2.2 Prevention of Childhood Diarrhea and Respiratory Tract Infections ...... 12 3.2.3 The Common Cold ...... 12

P. L. Carver (*) University of Michigan College of Pharmacy, Department of Clinical, Social, and Administrative Sciences , 428 Church St. , Ann Arbor , MI 48109-1065 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 1 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_1, © Springer Science+Business Media Dordrecht 2013 2 Carver

3.2.4 Prevention or Treatment of Malaria ...... 13 3.2.5 Burn Patients ...... 13 3.2.6 Wound Healing ...... 13 3.2.7 Critically Ill Patients ...... 13 3.2.8 Sickle Cell Disease ...... 14 4 SELENIUM ...... 14 4.1 Human Pharmacology and Pharmacokinetics ...... 14 4.2 Role of Selenium in Infectious Diseases ...... 15 4.2.1 Human Immunodeﬁ ciency Virus ...... 15 4.2.2 Intensive Care Unit Sepsis ...... 16 4.2.3 Role of Selenium in Other Infections ...... 17 5 COPPER ...... 18 5.1 Human Pharmacology and Pharmacokinetics ...... 18 5.2 Role of Copper in Infectious Diseases ...... 19 5.2.1 Copper/Zinc Ratio ...... 19 6 CHROMIUM ...... 19 6.1 Human Pharmacology and Pharmacokinetics ...... 19 6.2 Role of Chromium in Infectious Diseases ...... 20 7 MANGANESE ...... 20 7.1 Human Pharmacology and Pharmacokinetics ...... 20 7.2 Role of Manganese in Infectious Diseases ...... 21 7.2.1 Arginase ...... 21 7.2.2 Manganese Superoxide Dismutase ...... 22 8 SUMMARY AND FUTURE DEVELOPMENTS ...... 22 ABBREVIATIONS ...... 22 ACKNOWLEDGMENT ...... 23 REFERENCES ...... 23

Abstract Trace elements (TEs) are required by both humans and bacterial pathogens. Although metal ion homeostasis is tightly controlled in humans, growing evidence suggests that pathogens utilize a variety of means designed to circumvent the sequestration of TEs. Colonizing pathogenic microorganisms employ a variety of strategies to sense, acquire, store, and export metal ions in the vertebrate host which include the biosynthesis and utilization of siderophores, and the expression of high-affi nity metal-ion transporters. For iron, selenium, and zinc, signifi cant correlations have been shown between TE levels in plasma, serum, or tissues, and the prevention or treatment of a variety of infectious diseases; fewer such data exist for copper, chromium, or manganese. TEs are often employed as antioxidants, and as supplements in patients with TE-defi cient states. The role of TE supplementation in humans as antioxidants remains controversial, but has demonstrated signifi cant benefi t in the role of selenium for patients with sepsis, and of zinc for the prevention of several infectious diseases.

Keywords burns • chromium • copper • critically ill • Cu/Zn ratio • cystic ﬁ brosis • diarrhea • human immunodeﬁ ciency virus (HIV) • infectious diseases • intensive care unit • iron • malaria • manganese • mycobacterium • pneumonia • selenium • sepsis • supplementation • trace elements • zinc

Please cite as: Met. Ions Life Sci. 13 (2013) 1–28 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 3

1 Introduction

Trace elements (TEs) are often deﬁ ned as minerals that are required by adult humans in amounts between 1 to 100 mg/day. Nutrition is a two-edged sword when dealing with the treatment or prevention of infectious diseases, since bacteria, like humans, have a need for TEs [1 ]. Since metal ions are both necessary for life, but toxic in excess, metal homeostasis is tightly controlled by both bacteria and humans. When infecting humans, bacteria must acquire nutrients required for survival from the host environment. Colonizing pathogenic microorganisms employ a variety of strategies to sense, acquire, store, and export metal ions, which include the biosynthesis and utilization of siderophores, and the expression of high-afﬁ nity metal-ion transporters [2 ]. In order to control the availability of metals while restricting access by bacteria, humans have developed a variety of immune strategies.

1.1 Role of Antioxidants

Much of the research on TEs and infection has evaluated the response of the host to the onset of infection, particularly in critically ill patients, including those with trauma or severe burns. Any injured patient will develop an acute-phase response and a systemic infl ammatory response syndrome (SIRS) with the production of numerous mediators, including cytokines, which modulate the metabolic response. Oxidative stress is defi ned as a state in which the level of toxic reactive oxygen intermediates overcomes the endogenous antioxidant defenses of the host, resulting in damage to DNA, RNA, proteins, carbohydrates, and unsaturated fatty acids of the cell membrane. In critically ill patients, hyperinfl ammation, cellular immune dysfunction, and oxidative stress, combined with pathophysiologic events leading to mitochondrial dysfunction and SIRS, can result in multiple organ dysfunction and high rates of mortality. Manzanares et al. [ 3 ] recently performed a meta analysis of the outcomes of 21 randomized clinical trials in patients who received antioxidant micronutrients versus placebo. The use of antioxidants was associated with a signifi cant reduction in overall mortality.

1.2 Host Defense Responses to Infection

In the human antioxidant defense armamentarium, a variety of cytosolic, mitochondrial, and plasma antioxidants serve to protect tissues from the accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can lead to target end organ dysfunction and death. In addition to nonenzymatic endogenous antioxidant defense mechanisms (e.g., uric acid, glutathione, bilirubin, thiols, albumin, and nutrition factors, including vitamins and phenols), enzymatic defense mechanisms such as catalase (Cu, Fe), copper-zinc superoxide dismutase (Cu/Zn SOD), manganese 4 Carver superoxide dismutase (Mn-SOD) and glutathione peroxidase (GPx), are responsible for neutralizing ROS and RNS [3 , 4 ]. ROS activate the nuclear transcription factor NF kappa beta (NFκB). Activation of NFκB is modulated by Se, Zn, and vitamins C and E. Cu is also part of ferroxidases such as ceruloplasmin. The SODs initiate the antioxidant process, transforming the superoxide anion into hydrogen peroxide, which is further metabolized, ﬁ rst by catalase, then by the different GPxs [5 ]. Host defense responses to infection include the release of cytokines, including tumor necrosis factor (TNF), interferon alpha (IFNα ), and interleukins (IL) [1 , 6 ]. Cytokine-mediated anorexia results in reduced nutrient intake and sequestration of critical nutrients such as iron, copper, and zinc [1 ]. During SIRS, low plasma levels of endogenous TEs are observed, secondary to escape of TEs from the interstitial compartment due to capillary leakage, hemodilution, and hemodialysis or continuous renal replacement therapies [3 , 7 – 9 ]. Critically ill, burn, and trauma patients are characterized by an increased free radical production, which is proportional to the severity of the injury [5 ]. The most severe depletions of antioxidants occur in the most critically ill patients.

1.3 Alterations in Serum Levels of Trace Elements

Relationships between TE doses and serum TE concentrations vary for each TE and in varying underlying clinical conditions. SIRS is characterized by decreased serum levels of Fe, Se, and Zn, along with increased levels of Cu [5 , 10 , 11 ]. In patients with major burns, however, Cu defi ciency is observed. A recent study in clinically stable patients undergoing long-term administration of parenteral nutrition demonstrated a signifi cant dose-response relationship between weekly TE doses and serum TE concentrations for Zn, Cr, and Mn, but not for Se, Cu, or Fe [12 ]. Serum levels of Cu, Zn, Se and Fe in 44 patients with tuberculosis (TB) were compared to a control group of healthy individuals, at baseline and at the end of an intensive phase of anti-TB chemotherapy. Concentrations of Zn, Se, and Fe were signifi cantly lower (P < 0.05) while that of Cu and the Cu/Zn ratio signifi cantly higher (P < 0.05) in TB patients versus controls. Further, TB patients with human immunodefi ciency virus (HIV) coinfection had signifi cantly lower serum Zn and Se concentrations, and signifi cantly higher Cu/Zn ratios compared to those in TB patients without HIV coinfection (P < 0.05). Serum Cu concentration and Cu/Zn ratios declined signifi cantly after anti-TB chemotherapy, irrespective of HIV serostatus (P < 0.05) [13 ].

1.4 Nutritional Immunity

“Nutritional immunity” is a term used to describe the starvation of pathogens by the host for the vital metal ions Fe, Zn, and Mn. All bacterial pathogens must have mechanisms to circumvent nutritional immunity, but complex host defense mechanisms 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 5 to counteract these bacterial counter-defense mechanisms have also evolved [14 ]. For example, Borrelia burgdorferi, the causative agent of Lyme disease, and the only pathogen known to be an exception to the obligatory requirement for host Fe, circumvents host-mediated Fe sequestration by substituting Mn2+ in place of Fe2+ and thus does not require Fe 2+ to infect the host. In response, vertebrates encode additional mechanisms to restrict Mn2+ availability [15 ].

1.5 Natural Resistance-Associated Macrophage Protein (Nramp)

The Nramp family constitutes a large class of metal-ion membrane transporters, localized either at the cell surface or in intracellular vesicles, which translocate a wide range of divalent metal substrates, including Mn, Fe, Co, Cu, Zn, and Cd. The ﬁ rst of these to be mechanistically studied was mammalian DMT1 (divalent metal transporter) [16 ].

1.6 Calprotectin

Calprotectin (CP) is a metal chelating molecule that acts as a Mn scavenger in the context of Staphylococcus aureus infection. Although the antibacterial and antifungal properties of calprotectin were ﬁ rst attributed to its ability to sequester Zn, more recent studies demonstrate that calprotectin-dependent depletion of Mn also occurs in abscesses caused by S. aureus [2 ]. CP binds Mn2+ and Zn2+ with high afﬁ nity and essentially “starves” bacteria of these essential nutrients [17 ].

2 Iron

2.1 Human Pharmacology and Pharmacokinetics

In humans, a complex system of transporters regulates Fe homeostasis, which is maintained through careful coordination of duodenal absorption and recycling of Fe stores (see Chapter 8). The role of Fe in infectious diseases has been intensively studied. Since Fe serves as an important cofactor for enzymes, and is involved in many basic cellular functions and metabolic pathways of bacteria and fungi, they have developed sophisticated mechanisms for its acquisition. 6 Carver

2.2 The Complex Defense-Counter Defense System in the Battle for Iron

Human hosts tightly regulate Fe levels, sequestering this nutrient intracellularly as a mechanism to prevent bacterial growth. Since Fe 3+ is almost insoluble, nearly two- thirds of the Fe within vertebrates is complexed to the porphyrin heme in hemoglobin. Extracellular Fe is rapidly removed by transferrin and lactoferrin, proteins with a high affi nity for Fe. Infl ammation and febrile conditions can increase ferritin and lactoferrin synthesis. Hemopexin is a heme-scavenging protein found in serum which binds heme with high affi nity. Infection and infl ammation alter Fe homeostasis through immune-mediated mechanisms that further restrict the supply of readily available Fe. Iron administration alone does not appear to cause bacterial growth; however, once the transferrin saturation exceeds a critical threshold, free Fe becomes available for bacterial utilization. In addition, bacteria and fungi have evolved complex strategies to acquire Fe from vertebrate hosts. Pathogens employ one or more Fe transport mechanisms, depending on the type of Fe found in the host, while the host counters by increasing synthesis of Fe binding proteins such as transferrin and lactoferrin. The resulting Fe starvation of pathogen limits its growth, allowing the host time to eradicate the infection via immune-related mechanisms [18 ]. Two main mechanisms for obtaining host Fe include the development of receptors that can bind transferrin, lactoferrin, or hemoglobin, or the production of siderophores [19 ]. Siderophores are low-molecular-weight Fe chelators secreted by bacteria and fungi, that compete with transferrin for available Fe. Siderophores bind Fe3+ with an affi nity stronger than that of transferrin or lactoferrin. Energy-dependent transport of siderophores across the outer membranes of bacteria is mediated by TonB-dependent receptors. To counteract siderophores, vertebrates produce neutrophil gelatinase-associated lipocalin (NGAL; siderocalin), which binds and seques- ters siderophores [14 ]. Staphylococcus aureus uses non-siderophore mechanisms to acquire Fe from hemoglobin. By secreting hemolytic toxin, S. aureus lyses erythrocytes to release hemoglobin, which binds to a surface receptor on the bacteria. Fe is transported as heme into the bacterial cell for use as a nutrient.

2.3 Role of Iron in Infectious Diseases

In vitro evidence and animal studies suggest that increased Fe availability promotes bacterial growth and virulence. The risk of increased infections with administration of intravenous (IV) Fe has also been supported in limited animal studies. For example, in a murine model of E. coli sepsis, administration of IV Fe sucrose was associated with a mortality rate of nearly 60% when septic mice were also administered Fe, as compared to a mortality rate of 0% in mice with sepsis alone, or in those 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 7 administered Fe alone [20 ]. The relationship between Fe and infection has been investigated in human patient populations infected with malaria and in those at high risk for infection.

2.3.1 Dialysis Patients

Renal failure resulting in hemodialysis is an independent risk factor for infection in hemodialysis patients. Teehan and colleagues [21 ] evaluated Fe storage levels in hemodialysis patients receiving IV iron and found that patients with replete Fe indices were at increased risk for bacteremia compared with patients having deﬁ cient iron stores.

2.3.2 Malaria

Controversy continues over whether the benefi t of Fe supplementation in Fe-defi cient individuals outweighs the potential risk of malaria, and whether Fe supplementation should be restricted to Fe-defi cient or anemic patients. In a recent study in 785 Tanzanian children living in an area of intense malaria transmission, the presence of naturally occurring Fe defi ciency signifi cantly decreased the odds of parasitemia, severe malaria, and malaria-associated mortality [22 ]. However, international guidelines support Fe supplementation in children under 2 years of age in areas with a high prevalence of anemia. Iannotti et al. [23 ] reviewed 26 randomized controlled trials of preventive, oral Fe supplementation in young children (<5 years) and found confl icting data regarding the benefi ts and adverse effects of Fe supplementation on malaria. In several smaller studies in Gambia, Nepal, and Tanzania, Fe supplementation was associated with a signifi cant increase in fever-associated severe malaria, a 16% greater risk of adverse events due to malaria [ 24 – 26 ]. However, in several studies [27 – 30], including two large (832 and 25,490 children) studies in Tanzania and Nepal [ 29 , 30 ], no signifi cant differences in the rate of parasitemia, parasite density, or frequency of malaria were observed. They concluded that additional research is needed in populations affected by HIV and tuberculosis, and that Fe supplementation in preventive programs may need to be targeted. However, it is also important to note that nearly all trials of Fe supplementation took place in conjunction with some form of malaria control (e.g., bed nets, malaria prophylaxis), and some trials took place in area of lower malaria transmission [31 – 36 ]. Pregnant women, like children, are among the populations in greatest need of Fe supplementation while also being at greatest risk of malaria [31 ]. As gestation proceeds, latent malarial protozoa become active, especially in the Fe-rich placenta [ 37 ]. Although some studies have reported that Fe defi ciency is associated with a decreased prevalence and severity of malaria in pregnant women, [ 38 , 39 ], and that Fe supplementation in pregnant women increases the risk of malaria, these effects are likely diminished by factors such as host immunity, host Fe status, and effective malaria surveillance and control. A recent meta analysis of 23 studies conducted in 8 Carver countries having some risk of malaria concluded that there is no evidence that Fe supplementation increases placental malaria. However, only 2 of the 23 studies reported malaria outcomes [40 ], and the authors qualifi ed their fi ndings by noting that there was a signifi cantly increased risk of malaria associated with Fe supplementation in areas without adequate malaria surveillance and treatment programs. The interaction between Fe level, Fe supplementation and susceptibility to maternal and childhood malaria remains a concern, in particular in areas without adequate malaria surveillance and treatment programs [31 , 35 , 41]. Several ongoing studies are currently comparing the risk of malaria in Fe-supplemented versus non-supplemented pregnant women [31 ].

2.3.3 Human Immunodeﬁ ciency Virus

During chronic diseases, such as acquired immunodeﬁ ciency disease, Fe is rapidly sequestered by macrophages, causing a condition known as anemia of chronic disease, in which total body stores of Fe range from normal to increased, but are sequestered and unavailable even to the host, causing an anemia which functions as a defense against infection [42 ].

2.3.4 Diabetes

Patients with diabetic ketoacidosis appear uniquely susceptible to infections caused by Rhizopus species, lending support to the role of Fe uptake in the pathogenesis of this infection. In the presence of low pH and high glucose, phagocytes are dysfunctional, with impaired chemotaxis and defective intracellular oxidative and non- oxidative killing [43 ]. Patients with diabetic ketoacidosis have elevated serum levels of free Fe, and acidic (7.3–6.88) pHs [44 , 45 ]. In vitro , R. oryzae grows profusely at acidic conditions upon addition of exogenous Fe, but only at pHs ≤7.4.

2.3.5 Iron Overload

In vertebrates, administration of large doses of Fe, or the presence of medical conditions such as thalassemia, solid organ or hematopoietic stem cell transplantation in which Fe overload is present, are all strong risk factors for the development of bacterial and fungal infections. In patients with thalassemia, the need for frequent transfusions often results in Fe overload, and infections continue to be among the major (12–46%) causes of mortality. The bacteria isolated most frequently include Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Streptococcus pneumoniae, Salmonella typhi, Yersinia enterocolitica and other Gram-negative bacteria [46 ]. Fe overload is common in patients undergoing hematopoietic stem cell transplantation (HSCT), particularly in patients with myelodysplastic syndromes, in part 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 9 due to frequent administration of red blood cell transfusions prior to HSCT, adding exogenous Fe loading at a rate of 200–250 mg per unit of red blood cells. In these patients, hepatic Fe concentrations often approach levels observed in hereditary hemochromatosis. In HSCT recipients, elevated ferritin, hepatic Fe, hepcidin, and Fe bone marrow stores have all been shown to correlate with a markedly increased risk for the development of invasive fungal infections, including those caused by Aspergillus, Candida, Cryptococcus, Histoplasma, Paracoccidioides, Pneumocystis, Pythium, Rhizopus, Trichosporon , and Mucor [47 – 58 ]. Similarly, in patients undergoing liver transplantation, elevated serum Fe and hepatic Fe overload is associated with decreased long term survival, regardless of whether the patient had hereditary hemochromatosis [59 , 60]. In 153 patients undergoing liver transplantation, 31 invasive fungal infections were observed in 28 patients, of which 21 (68%) were caused by Candida , 7 (23%) by Aspergillus , 2 (6%) by Cryptococcus , and 1 (3%) by Saccharomyces . Stainable Fe in the hepatic explant was found in 48 patients (31%) and was strongly and independently associated with the development of post transplantation fungal infections [ 59 ].

2.3.6 Role of Iron Chelators in Infection

The predisposition to Mucorales (zygomycoses), Yersinia , and Vibrio vulniﬁ cus infections in patients treated with deferoxamine is now known to be due to the formation of feroxamine, a deferoxamine-Fe chelate which acts as a siderophore, delivering Fe to the pathogen. Deferoxamine strips ferric Fe from transferrin and attaches itself on the mold through an inducible receptor, and the Fe is transported intracellularly by an active reduction of the ferric form into the more soluble ferrous form [ 61 – 65 ]. In contrast, in a mouse model of diabetic ketoacidosis, mice are protected from R. oryzae infection by administration of the Fe chelators deferiprone and deferasirox, which do not act as siderophores [66 – 68 ]. Unfortunately, not all Mucorales have the same susceptibility to effective Fe chelators. For example, Cunninghamella bertholletiae and Mucor species display higher deferasirox minimal inhibitory and fungicidal concentrations than do Rhizopus species. The possible utility of deferasirox as an adjunctive therapy for mucormycosis has been evaluated in small studies, with mixed results. In an open label study of deferasirox in combination with antifungal therapy, seven of eight patients survived [69 ]. A recent multicenter, placebo-controlled, double-blinded clinical trial assessed the potential role of administration of deferasirox, in combination therapy with antifungal agents, in the treatment of 14 patients with mucormycosis. Patients with mucormycosis treated with deferasirox had a higher mortality rate at 90 days than in those who received placebo (82% versus 22%, respectively), possibly because more patients in the deferasirox group had hematologic malignancy, neutropenia, and/or pulmonary involvement. Further study is necessary to determine the possible beneﬁ ts or harms of deferasirox [70 , 71 ]. 10 Carver

3 Zinc

3.1 Human Pharmacology and Pharmacokinetics

Zinc affects multiple aspects of the immune system, and the response to infection, and is required for normal development and function of cells mediating innate immunity, neutrophils and natural killer cells, as well as macrophages. Zinc acts as a cofactor for more than 3000 metalloenzymes and proteins (Chapter 12 ), including Cu-Zn superoxide dismutase and metallothionein, as well as a large family of Zn proteins involved in gene transcription (such as the Zn fi nger proteins) which are important in maintenance of the immune system or in the prevention of infectious diseases [72 – 74 ]. Zn circulates at a concentration of 70 to 120 μg/dL, with 60 percent loosely bound to albumin and 30 percent tightly bound to macroglobulin. The primary stores of Zn include the liver and kidney; mostly intracellularly bound to metalloproteins. Zn is actively absorbed throughout the small intestine, mainly in the duodenum and jejunum via an intricate homeostatic mechanism which is regulated by metallothionein. Typically, Zn absorption is 20 to 40% bioavailable but metallothionein found in the gut enterocyte binds Cu more avidly than Zn. Zn homeostasis (see also Chapter 12) is probably maintained by a combination of changes in fractional absorption and endogenous fecal Zn excretion. Zn is transported bound to albumin, and taken up by peripheral tissues and by the liver where it may be stored as metallothionein. Zn excretion is primarily via the gastrointestinal tract, although up to 10 percent of the circulating Zn is also excreted through urine; urinary excretion typically ranges from 0.5 to 0.8 mg/day [72 – 74 ]. Because most Zn is bound to albumin, measured Zn levels may be reduced in patients with hypoalbuminemia; however, the correlation between Zn and albumin levels is weak, and plasma levels are loosely correlated with Zn stores. Thus, plasma levels do not reliably identify individuals with Zn defi ciency. A recent (2012) meta analysis of studies correlating dietary Zn intake and serum or plasma levels of Zn in healthy adults reported that for every doubling of Zn dose, the plasma concentration changes by 6% [75 ]. Although plasma levels correlate with doses, and are generally a good index of Zn status in healthy individuals, these levels are depressed during infl ammatory disease states [12 ]. In healthy individuals, plasma, urinary, and hair Zn are reliable biomarkers of Zn status [76 ]. Zn defi ciency is associated with impaired phagocytic function, lymphocyte depletion, decreased immunoglobulin production, a reduction in the T4+/T8+ ratio, and decreased interleukin-2 production [77 – 79 ]. Mild Zn defi ciency is common, especially in developing countries, because the diet is relatively low in Zn and contains signifi cant amounts of plant or vegetable phytates found in cereal proteins, which reduce Zn absorption. Zn absorption may also be impaired in patients with severe liver disease, although levels increase within one week following liver transplantation [80 , 81 ]. Zn defi ciency is also associated with pancreatic disease or insuffi ciency, since pancreatic enzymes, while necessary for release of dietary Zn, also contain Zn-complexing ligands. Rarely, Zn defi ciency can occur 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 11 due to malabsorption of Zn in patients with an autosomal recessive disease caused by mutations in the SLC39A4 gene on chromosome 8q24.3, which encodes ZIP4, a Zn transporter in the gastrointestinal tract, resulting in acrodermatitis enteropathica [74 , 82 ]. Recent studies suggest that Zn defi ciency can result in a signifi cant increase in the incidence of diarrhea and upper respiratory tract infections, as well as morbidity and mortality from these infections. Diabetics (both type 1 and type 2) can exhibit hyperzincuria due to alterations of Zn metabolism, which may have a role in the immune dysfunction associated with diabetes mellitus [83 ]. While Zn supplementation in diabetic patients may improve immune function, it increases the HbA1c levels and leads to worsening glucose intolerance [84 ].

3.1.1 Zn-Metallothionein (Zn-MT)

Physiologically, sepsis increases Zn-MT production, likely mediated by the up- regulation of the metallothionein gene by interleukin-1, leading to Zn sequestration in the intestinal and liver cells. The physiologic beneﬁ t of Zn as a negative acute- phase reactant may be to minimize Zn availability for bacterial use in its own DNA replication [72 ].

3.1.2 Zn-Metallo β-Lactamases

Recent studies in multidrug-resistant Acinetobacter have highlighted the role of Zn in Zn-metallo β-lactamases. “Starving” Acinetobacter baumannii (with Zn chelators) restored susceptibility of the organism, highlighting the possibility of Zn limitation strategies as a possible mechanism to combat carbapenem resistance in Acinetobacter [ 85 ].

3.2 Role of Zinc in Infectious Diseases

In recent years, much interest has been generated by the possibility that subclinical Zn defi ciency may signifi cantly increase the incidence of, and morbidity and mortality from, diarrhea and upper respiratory tract infections. Several studies have now demonstrated that Zn supplementation of select high risk populations can have substantial health benefi ts.

3.2.1 Cystic Fibrosis

Low plasma Zn concentrations have been reported in approximately 30% of young infants with cystic fi brosis identifi ed by newborn screening. Since fecal Zn losses correlated with fecal fat excretion, investigators have suggested that cystic fi brosis interferes with enterohepatic recycling of Zn [86 ]. 12 Carver

The existence of positive correlations between Zn and IL-2, and between Zn or active thymulin and natural killer (NK) cell activity suggest a close link among Zn failure, impaired IL-2 activity, low thymulin level, and reduced NK activity in cystic fi brosis patients with both normal and growth retardation. Although the role of NK cells is unknown in cystic fi brosis, Zn supplementation has been suggested as a means to induce a complete saturation of thymulin molecules and correct cellular immune defects [87 ]. Abdulhamid et al. [88 ] performed a double blind placebo controlled study investigating the effect of oral supplementation of elemental Zn (30 mg daily for 1 year) on the rate of respiratory tract infections, use of antibiotics and plasma cytokines in 26 children with cystic fi brosis. Zn supplementation reduced the number of days of oral antibiotics used to treat respiratory tract infections, and was marginally effective in reducing percentage increase in plasma IL-6 and IL-8 while increasing the percentage change in ex vivo generation of IL-2 in isolated mononuclear cells; this effect was greater in patients who exhibited low plasma Zn at baseline than those who had plasma Zn levels identical to normal subjects. The authors concluded that a higher daily Zn dose may be needed to decrease respiratory tract infections and modify immune responses [88 ]. A later small, retrospective study of cystic fi brosis patients with normal serum Zn evaluated a higher dosage of Zn (5 mg/kg Zn sulfate daily to a maximum of 150 mg daily). They reported that Zn supplementation resulted in a signifi cant decrease in the number of infections [89 ].

3.2.2 Prevention of Childhood Diarrhea and Respiratory Tract Infections

Several meta analyses [90 – 92] have concluded that preventive Zn supplementation (with doses ranging from 15 to 140 mg/week of elemental Zn) for ≥3 months to Zn-deﬁ cient children 2–5 years old in developing countries reduces the frequency and severity of diarrhea by 13 percent, and the incidence of clinically conﬁ rmed pneumonia by about 20 percent.

3.2.2.1 Treatment of Childhood Diarrhea

A recent meta analysis of 24 randomized trials concluded that in children >6 months old from populations in which Zn defi ciency is common, evidence suggests that oral Zn supplementation reduces the severity and duration of acute diarrhea [91 , 93 , 94 ]. Zn supplementation is probably helpful for treatment of acute diarrhea even in populations without Zn defi ciency, perhaps because Zn has specifi c local inhibitory effects on some enteric pathogens and toxins.

3.2.3 The Common Cold

A large number of studies have evaluated the effect of Zn lozenges on the duration or severity of common cold symptoms. A recent Cochrane analysis of 13 therapeutic trials and two preventive trials concluded that 7 days of Zn treatment is associated 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 13 with a signiﬁ cant reduction in the duration and severity of common cold symptoms, and a decreased rate of developing a cold. However, adverse events including bad taste and nausea are higher in patients taking Zn [95 ]. Some investigators feel that if utilized, Zn lozenges must have a minimal daily dose of elemental Zn of at least 75 mg, and be started within 24h of the onset of the common cold [96 ].

3.2.4 Prevention or Treatment of Malaria

Trials examining whether Zn supplementation reduces morbidity or mortality from childhood malaria have had confl icting results. Several small trials from Asia and Africa showed that supplementation reduced clinic visits or mortality due to malaria [97 ], while several others reported non-signifi cant reductions of morbidity or clinic visits due to malaria [92 , 98 , 99 ]. Zn supplementation does not appear to have a benefi cial effect when used as an adjunct to treatment of the disease. Although Zn levels tend to decrease by ~70% during the acute phase response in children with Falciparum malaria, a large placebo-controlled trial of Zn supplementation in children, performed in a malaria- endemic area of Africa, also failed to show a signifi cant effect of Zn supplementation on overall mortality or malaria-related mortality [99 – 102 ].

3.2.5 Burn Patients

In patients with major burns, IV administration of 2.9 μmol Se, 40.4 μmol Cu, and 406 μmol Zn daily for 3 weeks results in improved wound healing, and a 65% reduction in the rate of nosocomial pneumonia [103 , 104]. Current guidelines from the European Society for Clinical Nutrition and Metabolism recommend enteral supplementation of Se, Cu, and Zn at a “higher than standard doses” after burn injury, based upon the above studies [105 ].

3.2.6 Wound Healing

Application of topical Zn oxide application to post-surgical wounds can signiﬁ - cantly decrease the occurrence of Staphylococcus aureus , and the need for postoperative antibiotics [106 ]. In burn patients, IV Zn supplementation (in combination with Se and Cu) was associated with increased skin concentrations of Zn and Se, improved wound healing, and a reduction in pulmonary infections [5 , 107 – 109 ].

3.2.7 Critically Ill Patients

In 1996, Berger et al. [110 ] reported that in 11 intensive care unit (ICU) patients, Zn levels were decreased in the fi rst week of ICU stay. In patients on home total parenteral nutrition (TPN) with a diagnosis of catheter sepsis or pancreatitis, 14 Carver administration of 30 mg IV Zn daily for 3 days resulted in a signifi cantly higher febrile response, as evidenced by increased temperature in the Zn versus a control (placebo) group [111 ]. Heyland and colleagues [112 ] conducted a systematic review of Zn supplementation in critically ill patients, including 4 randomized controlled trials, where IV administration of Zn showed nonsignifi cant trends toward reductions in mortality and ICU length of stay. They concluded that evidence to support the use of IV Zn is lacking, as the 6 available studies were too small to detect even a moderate effect, and that current recommendations for high dose Zn supplementation in critically ill patients should be revised. However, considering the importance of Zn in free radical scavenging, anabolism, and immunity, large rigorously designed randomized trials are warranted, to evaluate the effects of Zn supplementation in severe septic patients [113 ].

3.2.8 Sickle Cell Disease

Gupta and Chaubey conducted a double-blind, placebo-controlled, randomized controlled trial of 130 sickle cell patients in India. Zn (200 mg orally 3 times daily) or placebo was administered for 1.5 years. There was a signifi cant reduction of the mean number of infective episodes in the Zn-supplemented group [114 ]. Prasad et al. [115 ] evaluated Zn supplementation in 32 patients with sickle cell disease who were divided into 3 groups, based upon their level of granulocytic or lymphocytic Zn levels (mild, moderate defi ciency, or no Zn defi ciency). Subjects were administered Zn acetate (50 to 75 mg of elemental Zn orally daily) for 2 or 3 years, or placebo, in the mild, moderate defi ciency, or no Zn defi ciency groups, respectively. Prolonged Zn supplementation resulted in increased, lymphocyte and granulocyte Zn, and IL-2 production, and a decreased number of documented bacte- riologically positive infections. In a later study by the same investigator, Bao, Prasad and colleagues [116 ] conducted a double-blind, randomized, placebo- controlled trial to further evaluate the role of Zn supplementation (25 mg orally 3 times daily for 3 months) on the incidence of infections, oxidative stress, and biomarkers for chronic infl ammation in patients with sickle cell disease. The Zn-supplemented group experienced a decreased incidence of infections, and signifi cant decreases in lipopolysaccharide-induced TNF-α and IL-1 β mRNAs, and TNF-induced NFκB- DNA binding in mononuclear cells compared with the placebo group.

4 Selenium

4.1 Human Pharmacology and Pharmacokinetics

Serum Se levels vary widely in different parts of the world, as does the ingestion of dietary Se. As Se has a narrow therapeutic range, the optimal range of dietary intake of Se is narrow; potentially toxic intakes are closer to recommended dietary intakes 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 15 than for other dietary trace minerals (see also Chapter 16). While Se status can be assessed by determining the Se concentration of whole blood, plasma, serum, or erythrocytes, plasma or serum levels are the most commonly used and are reasonably accurate biomarkers of Se status, responding to short-term changes in intake [117 – 119 ]. While Se supplementation may be benefi cial in individuals with low levels of Se, it is potentially toxic if administered to those who already have normal or high levels. People whose serum or plasma Se concentration is ≥122 μg/L should not supplement with Se [120 ]. Currently, Se is the most intensively studied TE with respect to the treatment or prevention of a variety of infections in humans. Se defi ciency (serum or plasma Se ≤85 μg/L) has been linked to the incidence, virulence, or disease progression of viral infections and has correlated with several infectious diseases, including HIV, sepsis or pneumonia in ICU or burn patients, and prostatitis. In healthy subjects, Se can be found in plasma associated with selenoprotein- P (52%), glutathione peroxidase (39%), albumin (9%), and free Se (<1%) [ 121, 122]. Among the >30 selenoproteins which have been identifi ed, 4 forms of glutathione peroxidase have been shown to be important in antioxidant defense [120 ], while selenoprotein-P appears to play a role in protection versus infection [123 ]. Cu, Mn, Zn, Fe, and Se are required for the activity of SOD, catalase, and glutathione peroxidase, respectively [3 ]. Se is found in relatively high amounts in the liver, spleen, and lymph nodes, which are involved in hematopoietic and immune function potential. Se is incorporated into at least 25 selenoproteins and thus is a constituent of multiple antioxidant defense systems [ 124]. In mice, selenoprotein-P appears to provide protection against the parasitic infection trypanosomiasis [123 ]. Impaired cell-mediated immunity has been demonstrated when tissue stores of Se are depleted. Natural killer cell activity is enhanced when Se is supplemented in the diet of Se-depleted individuals [ 125 ].

4.2 Role of Selenium in Infectious Diseases

4.2.1 Human Immunodeﬁ ciency Virus

HIV replication is inhibited by Se [126 , 127 ], and a number of studies have shown a linear relationship between Se defi ciency (usually defi ned as a serum level ≤85 μg/L) and a reduction in CD4 cell counts in HIV-infected patients. In HIV-infected individuals, several studies [126 ,128 – 131] have associated low (not necessarily defi cient) serum Se levels with lower CD4 counts, increased viral load, rapid progression of HIV, and higher mortality. When serum Se levels are controlled for serum albumin, or for an acute phase response (defi ned as an α 1 -acid glycoprotein level ≥100 mg/dL or a C-reactive protein ≥1 mg/dL), these associations disappear [132 ], which may be attributable to the lowering of blood Se concentration by the acute-phase response in individuals with more advanced HIV-1 infection. 16 Carver

4.2.1.1 Selenium Supplementation in HIV

Two randomized controlled trials have shown apparent benefi t from Se supplementation in HIV-infected patients [133 , 134]. Burbano et al. [133 ] conducted a randomized, placebo-controlled study evaluating the administration of Se to HIV-infected individuals in the US who were not Se-defi cient (with Se defi ciency defi ned as a serum Se >85 μg/L). Se supplementation (200 μg orally daily) resulted in a decreased rate of hospital admissions (P = 0.02) and in the percentage of hospital admissions due to infection (P = 0.01). Similarly, Hurwitz et al. [134 ], conducted a double-blind, randomized, placebo-controlled trial in the US in 174 HIV-infected subjects with Se levels >75 μg/L. Administration of Se 200 μg orally daily for 18 months signifi cantly increased serum Se concentrations (∆ = 32.2 ± 24.5 versus 0.5 ± 8.8 μg/L; P < 0.001) in adherent subjects. Higher serum Se levels predicted decreased HIV viral load (P < 0.02) and increased CD4 count (P < 0.04) even after covariance for demographic factors, antiretroviral therapy regimen and adherence, HIV-disease stage and duration, and hepatitis-C virus co-infection. Moreover, Se-treated subjects in whom serum Se levels changed ≤26.1 μg/L displayed elevations in viral load and, as a result, decreases in CD4 count. However, others have criticized the method by which the data were analyzed and the relevance of the differences recorded in CD4+ cell count and viral load [135 ]. By contrast, Kupka et al. [135 ] reported that Se supplementation (200 μg of Se daily, as selenomethionine during the antenatal and post-partum periods) had no effect on HIV-1 viral load or CD4+ cell count in 913 HIV-infected Tanzanian pregnant women in whom use of antiretroviral therapy was uncommon, although it reduced the risk of mortality in children older than 6 weeks. However, the Se status of these subjects was unknown. In a case-control study of 259 HIV-infected drug users, 47 (18.1%) patients whose plasma Se level was < 135 μg/L had a three-fold higher risk of developing mycobacterial disease (primarily tuberculosis), than did those with higher plasma Se levels.

4.2.2 Intensive Care Unit Sepsis

As noted above, studies have consistently demonstrated decreased plasma Se concentrations in critically ill patients, especially those with septic shock. In ICU patients with severe septic shock, there is a 40% decrease in plasma Se concentrations [ 136 , 137 ] and in selenoprotein-P, following admission to the ICU. ICU mortality is strongly associated with the minimum Se concentration [137 ]. Se has emerged as the most important antioxidant micronutrient in the critically ill, particularly in burn and trauma patients [5 ]. Intravenous selenite has a biphasic action: ﬁ rstly as a prooxidant and, after incorporation into selenoenzymes, as an antioxidant [138 ]. Since glutathione peroxidase, the body’s most important antioxidant enzyme, is directly dependent on Se, strategies focused on replacement of Se have been evaluated as a means to reduce infections, the length of hospital stay, and mortality. 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 17

The role of supplemental Se (given as sodium selenite) has been evaluated in a number of studies, with confl icting outcomes. The 2004 and the updated 2008 Cochrane data base reviews [4 ] of 7 trials ([139 – 145 ]) of supplemental IV sodium selenite concluded that there was limited evidence to recommend supplementation of critically ill patients with Se, and that additional trials of adequate size and appropriate methodology were required in order to overcome the defects of previous studies. Since 2007, several larger clinical trials of Se supplementation [146 , 147 ] have been published, with confl icting outcomes. The studies have varied in patient populations, method of Se administration (as a bolus followed by continuous infusion versus continuous infusion), and duration of therapy [4 , 139 , 144, 146 , 147 ]. Few trials reported on outcomes other than mortality and there were insuffi cient data to examine the effect of methodological superiority or dose of Se on the outcomes. However, two recent meta analyses [3 , 148] evaluated the results of 21 randomized clinical trials of antioxidant micronutrients, including Se, in critically ill patients. They concluded that bolus administration of Se followed by transient prooxidant effect of an IV bolus followed by the antioxidant effect of continuous infusion seems effi ca- cious and well tolerated and was associated with a signifi cant reduction in mortality and in the duration of mechanical ventilation, with a trend towards a reduction in infections. Reductions in the risk of mortality were highest in those patients with a higher risk of death. Trials using loading doses, high doses (>500 μg daily) and a longer duration of Se therapy appear to be associated with lower mortality.

4.2.3 Role of Selenium in Other Infections

In a small study in adult subjects in the United Kingdom, Broome et al. [149 ] demonstrated a functional outcome of Se supplementation (50 or 100 μg Se orally per day for 15 weeks as sodium selenite) on the immune system of subjects with fairly low (<1.2 μmol/L) Se status. When patients were challenged with an oral, live, attenuated poliovirus, Se-supplemented patients demonstrated an augmented cellular immune response as evidenced by an increased production of IFN and other cytokines, an earlier peak T cell proliferation, and an increase in T helper cells. In addition, they cleared the virus more rapidly than did those given placebo [149 ]. Most studies in surgical patients report only postoperative or post-ICU admission Se levels. However, Stoppe et al. [124 ] recently reported pre- and postoperative Se, Zn, and Cu levels in patients undergoing elective cardiac surgery. Fifty of 60 patients (83.3%) exhibited Se defi ciency prior to surgery, and all patients demonstrated signifi cant decreases in Se levels postoperatively, as compared to baseline (pre-operative) levels. The intra-operative decrease in TE concentrations was most pronounced in patients who developed multi-organ failure post-operatively [ 124]. As discussed above (under Zinc, Section 3.2.5 ) administration of Cu, Zn, and Se supplements for 3 weeks has been found to decrease the incidence of pneumonia following severe burns [103 – 105 , 108 ]. In cystic fi brosis patients, there is evidence both for and against antioxidant supplementation with vitamins E and C, β-carotene, and Se. However, a recent meta analysis concluded that antioxidant supplementation 18 Carver in cystic fi brosis is not yet recommended beyond routine care, because while levels of antioxidants in the blood improved, there was no improvement in lung function, and quality of life decreased in groups taking supplements [150 ].

5 Copper

5.1 Human Pharmacology and Pharmacokinetics

Among the biologically important Cu-containing enzymes which have been described, Cu/Zn SOD (a component of antioxidant defense) and ceruloplasmin play important roles in the development of infections (see also Chapter 11 , in general). Cu is absorbed in the proximal small intestine and stomach, with absorption occurring by a saturable active transport process at lower levels of dietary Cu and by passive diffusion at high levels of dietary Cu [151 , 152]. The Menkes P-type ATPase (ATP7A) is responsible for Cu traffi cking to the secretory pathway for effl ux from enterocytes and other cells. Absorbed Cu is loosely bound to plasma albumin and amino acids in the portal blood and taken to the liver, where most of it is taken up on the fi rst pass and incorporated into ceruloplasmin, a Cu-containing protein which transports Cu from the liver to peripheral tissues. Ceruloplasmin binds to its receptors on the cell surface; Cu is then released from its binding protein and enters the cell [151 , 152]. Cu homeostasis is largely regulated by excretion of Cu into the gastrointestinal tract via the bile; ~50% of Cu is excreted in the bile while the remaining half is excreted through other gastrointestinal secretions. Metallothionein, synthesized in the liver, may act as a Cu storage protein. Acquired Cu defi ciency is rare, but has been well documented in humans. Hematologic features of Cu defi ciency include anemia (usually microcytic) and neutropenia, which can be mistaken for Fe defi ciency anemia. In this setting, administration of Fe supplements can worsen Cu defi ciency because excess Fe competes with Cu and decreases net Cu absorption. Similarly, because Cu and Zn are com- petitively absorbed from the jejunum via metallothionein, high doses of Zn (>150 mg/day) can result in Cu defi ciency in normal individuals. Excessive Zn ingestion can occur due to prolonged use of oral Zn supplements for the treatment of common colds, administration of parenteral Zn in patients on chronic hemodialysis, or occa- sionally when trace elements in TPN are withheld in patients with cholestasis. Patients with major burns are unique for having Cu defi ciency, as compared to trauma patients with the systemic infl ammatory response syndrome, in whom serum levels of Cu are increased, and Fe, Se, and Zn decreased [5 ]. Ceruloplasmin (like ferritin) is an acute phase reactant, and serum Cu and ceruloplasmin levels are increased in adult patients with infl ammatory processes, pregnancy, coronary artery disease, cirrhosis, diabetes, malignancies, and renal failure [ 153 ]. Confl icting data have been reported in children; although Teslariu and Nechifor reported decreased serum levels of Cu and Zn in otherwise healthy 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 19 children with acute urinary tract infections [ 154], Wang et al. reported no correlation between Cu levels and severity of illness scores in children admitted to an ICU [ 155 ]. Ceruloplasmin has an independent role in Fe metabolism, in which it serves as a plasma ferroxidase, converting Fe to a valence that can be bound by plasma transferrin. Metallothionein, synthesized in the liver, may act as a Cu storage protein.

5.2 Role of Copper in Infectious Diseases

To date, studies examining the relationship between Cu levels and the development of infections have found no correlation with the development of infectious diseases; however, in several patient populations, correlations have been found between an increased Cu/Zn plasma ratio and decreases in immune-related markers or responses to infection. In addition, as noted above, administration of Cu, Zn, and Se supplements for 3 weeks have been found to decrease pneumonia following severe burns [103 – 105 , 108 ].

5.2.1 Copper/Zinc Ratio

In patients undergoing peritoneal dialysis, Guo et al. noted the Cu/Zn ratio was strongly correlated with nutritional abnormalities, oxidative stress, infl ammation, and immune dysfunction, including a negative correlation of the Cu/Zn ratio with the percentages of B- and T-lymphocyte subsets and the ratio of CD4/CD8 antigens [ 156 ]. As noted earlier, TB patients with HIV coinfection demonstrated signifi cantly higher Cu/Zn ratios compared to those in TB patients without HIV coinfection (P < 0.05) [13 ]. Similarly, children with chronic hepatitis B infection had signifi cantly lower plasma levels of Mn, Se, Zn (but not Cu), and signifi cantly higher Cu/Zn ratios prior to interferon therapy (P < 0.001) as compared to a control group [6 ].

6 Chromium

6.1 Human Pharmacology and Pharmacokinetics

Chromium is absorbed predominantly in the small intestine and is transported in the circulation bound to albumin and transferrin [157 ]. The total body Cr concentration is the main homeostatic control of its gut absorption. Dietary bioavailability of Cr is very low and almost all of the ingested Cr is excreted via feces [158 , 159 ]. Cr absorption is enhanced in the setting of Zn and Fe defi ciency, suggesting that these minerals compete for intestinal absorption [157 ]. Patients receiving parenteral nutrition with usually prescribed doses of Cr can have abnormally elevated serum and urine 20 Carver concentrations in part attributable to contamination of amino acid products, especially in patients with renal dysfunction [160 ]. Although there appears to be a signifi cant dose-response relationship between Cr doses and serum Cr concentrations, serum Cr equilibrates slowly with tissue stores [12 ]. Cr is excreted mainly through the urine; however, some Cr is excreted in the feces through bile and small intestinal losses. Urinary losses increase with metabolic stress, trauma, and ascorbic acid defi cits. Cr defi cits induce glucose intolerance, and glucose intolerance can further drive these urinary losses of Cr (see also Chapter 6 , in general).

6.2 Role of Chromium in Infectious Diseases

Chromium is a cofactor for insulin function that enhances insulin effects to improve glucose metabolism through the glucose tolerance factor [160 ]. While diabetics, particularly those with altered glucose levels, are known to have an increased prevalence of infectious diseases, thus far no studies have evaluated the role of Cr as a risk factor for infectious diseases.

7 Manganese

7.1 Human Pharmacology and Pharmacokinetics

Manganese is a component of metalloenzymes such as manganese superoxide dismutase, arginase, glutamate synthetase, and pyruvate carboxylase and is associated with oxidative phosphorylation and mucopolysaccharide metabolism (see also Chapter 7). An average adult has 10–12 mg Mn incorporated into the active center of various metalloenzymes [161 ]. Particular interest has been paid to Mn-SOD, which is located primarily in mitochondria, which are important for detoxifying the superoxide radical to hydrogen peroxide [12 ]. Mn is excreted mainly from the bile, and thus can accumulate in patients with cholestasis. A number of proteins involved in Mn transport have been identifi ed including the putative uptake proteins divalent metal transporter-1 (DMT1), transferrin receptor (TfR) and ATP13A2 (also known as PARK9), as well as the effl ux protein Fpn. Previously, the only protein known to be operant in cellular Mn export was the Fe-regulating transporter, Fpn [162 ]. Mn absorption, transport, and excretion are tightly regulated because Mn is both essential at low dose and toxic at higher doses. While Mn is transported by simple diffusion in the large intestine, Mn is absorbed by active transport in the small intestine [163 ]. Absorption, effl ux, and distribution of Mn appear to be inversely related to stored Fe, with Fe defi ciency facilitating Mn absorption. Only about 5% of dietary Mn appears to be absorbed; however, absorption is greater in neonates and children 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 21 than in adults, and in females than in males. Fe defi ciency increases the absorption, effl ux, and distribution of orally administered Mn into the body, and in delivery to the brain possibly via Nramp [161 ,162 , 164 ]. Once absorbed, Mn is transported to the liver where ~80% of plasma Mn is bound to β1-globulin, a small fraction is bound to transferrin, an Fe-binding protein. Mn in the liver is conjugated with bile and >90% of Mn is excreted by secretion into the intestine via the hepatobiliary system, where a small fraction is reabsorbed and the remainder is excreted in the feces. Decreased elimination of Mn in patients with poor biliary excretion (e.g., neonates and adults with cholestasis) may result in increased delivery of Mn to the brain and other tissues, increasing the potential for toxicity [161 ]. In vivo experiments in mice and rats have defi ned the range (1–3.5%) of GI absorption of Mn [161 ]. While Mn is transported by simple diffusion in the large intestine, Mn is absorbed by active transport in the small intestine. Mn excretion into bile is likely active as well because it depends on concentration gradients. A plethora of plasma proteins or ligands have been implicated as specifi c Mn carrier proteins, including transglutaminase, β1-globulin, albumin, and transferrin. In fact, approximately 80% of plasma Mn is bound to β1-globulin. a small fraction of plasma Mn is bound to transferrin, while approximately 80% of plasma Mn is associated with albumin and β1-globulin. Despite the demonstration that Mn preferentially binds to albumin in the plasma of both rabbits and humans, emerging evidence has provided evidence for weaker binding of Mn to albumin compared to Cd and Zn [163 ]. Because 60–80% of Mn is contained in red blood cells, erythrocyte or whole- blood Mn concentrations appear to be the most accurate and reproducible parameter [ 163 ]. Several investigators [12 , 165 ] have demonstrated a correlation between Mn supplementation and serum concentrations and in long term (up to 20 years) patients receiving parenteral nutrition, while Siepler et al. did not [166 ].

7.2 Role of Manganese in Infectious Diseases

Many organisms can compensate for the loss of antioxidant enzymes by the formation of catalytic Mn-antioxidants during periods of Mn abundance. It has been proposed that cells utilize these Mn-antioxidant complexes as a “backup” for Cu/Zn SOD1: when Mn is abundant, surplus intracellular Mn 2+ forms antioxidant complexes and when Mn is limited, cells rely on the high efﬁ ciency of SODs [167 ].

7.2.1 Arginase

The Mn-containing enzyme arginase down-regulates nitric oxide (NO) production by competing with nitric oxide synthase (NOS) for arginine. NO is important in host immune defense, since it is utilized by the immune system to generate peroxynitrite 22 Carver which kills bacteria; however, septic shock is associated with an overproduction of NO [168 ]. In humans, plasma arginase is elevated (and levels of arginine usually reduced) in a variety of conditions, including sickle cell disease, oxidative stress, malaria, and cystic ﬁ brosis. Clinically, the ratio of plasma arginine/(plasma ornithine + citrulline), which has been termed the ‘global arginine bioavailability ratio’ (GABR) has proven more useful as a biomarker in some disorders than have plasma concentrations of arginine alone. For example, a low GABR represent an independent risk factor for morbidity and mortality in sickle cell patients [169 ]. In animal studies, Mn-SOD activity in the heart, and arginase activity in the liver, were lower in piglets fed a low Mn diet, and the relative arginase activity increased with enhanced dietary Mn and correlated with Mn concentrations in the liver [170 ].

7.2.2 Manganese Superoxide Dismutase

Following burns, trauma, and surgery, despite no changes in Mn serum concentrations, Mn (but not Cu or Zn) concentrations are increased within burn scars, emphasizing the importance of Mn-SOD, a mitochondrial antioxidant defense, in wound healing [ 5 ]. Despite the signiﬁ cant and evolving role of Mn in pathogens, to date, no published studies have correlated Mn plasma or serum levels with the prevention or treatment of infectious diseases, or addressed the role of supplementation or chelation of Mn in humans.

8 Summary and Future Developments

The role of trace elements in infectious diseases is complex. Signiﬁ cant correlations have been demonstrated for Fe, Se, and Zn and infections; fewer data exist for Cu, Cr, or Mn. However, as the synergistic role of these TEs is further elucidated, investigators, clinicians, and most importantly, patients, will beneﬁ t from a more complete understanding of this complex biological system and the prevention or treatment of infectious diseases.

Abbreviations

Cp calprotectin Cu/Zn SOD copper-zinc superoxide dismutase DMT1 divalent metal transporter Fpn ferroportin GABR global arginine bioavailability ratio 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 23

GI gastrointestinal GPx glutathione peroxidase HIV human immunodeﬁ ciency virus HSCT hematopoietic stem cell transplantation ICU intensive care unit IFNα interferon α IL interleukin IV intravenous Mn manganese Mn-SOD manganese superoxide dismutase N F κB NF kappa beta NGAL neutrophil gelatinase-associated lipocalin; also known as siderocalin NK cell natural killer cell NO nitric oxide NOS nitric oxide synthase Nramp natural resistance-associated macrophage protein RNS reactive nitrogen species ROS reactive oxygen species SIRS systemic inﬂ ammatory response syndrome SOD superoxide dismutase TB tuberculosis TEs trace elements TfR transferrin receptor TNF tumor necrosis factor TPN total parenteral nutrition Zn-MT Zn-metallothionein

Acknowledgment I would like to thank Vincent Pecoraro for his invaluable comments, suggestions, and editing of this manuscript.

References

1. H. Donabedian, Nutr. J. 2006 , 5 , 21. 2. J. A. Hayden, M. B. Brophy, L. S. Cunden, E. M. Nolan, J. Am. Chem. Soc. 2013 , 135, 775–787. 3. W. Manzanares, R. Dhaliwal, X. Jiang, L. Murch, D. K. Heyland, Crit. Care 2012 , 16 , R66. 4. A. Avenell, D. W. Noble, J. Barr, T. Engelhardt, Cochrane Database Syst. Rev. 2004 , CD003703. DOI: 10.1002/14651858.CD003703.pub2 . 5. M. M. Berger, Nutr. Clin. Pract. 2006 , 21 , 438–449. 6. N. Balamtekin, A. E. Kurekci, A. Atay, S. Kalman, V. Okutan, E. Gokcay, A. Aydin, K. Sener, M. Safali, O. Ozcan, Biol. Trace Elem. Res. 2010 , 135 , 153–161. 7. R. Chiolero, M. M. Berger, Contrib. Nephrol. 2007 , 156 , 267–274. 8. M. Zappitelli, M. Juarez, L. Castillo, J. Coss-Bu, S. L. Goldstein, Intensive Care Med. 2009 , 35 , 698–706. 9. D. Rucker, R. Thadhani, M. Tonelli, Semin. Dial. 2010 , 23 , 389–395. 10. G. M. Giammanco, G. Pizzo, S. Pecorella, S. Distefano, V. Pecoraro, M. E. Milici, Oral Microbiol. Immunol. 2002 , 17 , 89–94. 24 Carver

11. M. Geoghegan, D. McAuley, S. Eaton, J. Powell-Tuck, Curr. Opin. Crit. Care 2006 , 12 , 136–141. 12. I. F. Btaiche, P. L. Carver, K. B. Welch, J. Parenter. Enteral. Nutr. 2011 , 35 , 736–747. 13. A. Kassu, T. Yabutani, Z. H. Mahmud, A. Mohammad, N. Nguyen, B. T. Huong, G. Hailemariam, E. Diro, B. Ayele, Y. Wondmikun, J. Motonaka, F. Ota, Eur. J. Clin. Nutr. 2006, 60 , 580–586. 14. M. I. Hood, E. P. Skaar, Nat. Rev. Microbiol. 2012 , 10 , 525–537. 15. J. E. Posey, F. C. Gherardini, Science 2000 , 288 , 1651–1653. 16. L. T. Jensen, M. C. Carroll, M. D. Hall, C. J. Harvey, S. E. Beese, V. C. Culotta, Mol. Biol. Cell 2009 , 20 , 2810–2819. 17. S. M. Damo, T. E. Kehl-Fie, N. Sugitani, M. E. Holt, S. Rathi, W. J. Murphy, Y. Zhang, C. Betz, L. Hench, G. Fritz, E. P. Skaar, W. J. Chazin, Proc. Natl. Acad. Sci. USA 2013 , 110 , 3842–3846. 18. V. Braun, K. Hantke, Curr. Opin. Chem. Biol. 2011 , 15 , 328–334. 19. E. D. Weinberg, Biochim. Biophys. Acta 2009 , 1790 , 600–605. 20. R. A. Zager, A. C. Johnson, S. Y. Hanson, Kidney Int. 2004 , 65 , 2108–2112. 21. G. S. Teehan, D. Bahdouch, R. Ruthazer, V. S. Balakrishnan, D. R. Snydman, B. L. Jaber, Clin. Infect. Dis. 2004 , 38 , 1090–1094. 22. M. Gwamaka, J. D. Kurtis, B. E. Sorensen, S. Holte, R. Morrison, T. K. Mutabingwa, M. Fried, P. E. Duffy, Clin. Infect. Dis. 2012 , 54 , 1137–1144. 23. L. L. Iannotti, J. M. Tielsch, M. M. Black, R. E. Black, Am. J. Clin. Nutr. 2006 , 84 , 1261–1276. 24. S. Sazawal, R. E. Black, M. Ramsan, H. M. Chwaya, R. J. Stoltzfus, A. Dutta, U. Dhingra, I. Kabole, S. Deb, M. K. Othman, F. M. Kabole, Lancet 2006 , 367 , 133–143. 25. A. W. Smith, R. G. Hendrickse, C. Harrison, R. J. Hayes, B. M. Greenwood, Ann. Trop. Paediatr. 1989 , 9 , 17–23. 26. J. M. Tielsch, S. K. Khatry, R. J. Stoltzfus, J. Katz, S. C. LeClerq, R. Adhikari, L. C. Mullany, S. Shresta, R. E. Black, Lancet 2006 , 367 , 144–152. 27. J. Berger, J. L. Dyck, P. Galan, A. Aplogan, D. Schneider, P. Traissac, S. Hercberg, Eur. J. Clin. Nutr. 2000 , 54 , 29–35. 28. J. P. Chippaux, D. Schneider, A. Aplogan, J. L. Dyck, J. Berger, Bull. Soc. Pathol. Exot. 1991 , 84 , 54–62. 29. C. Menendez, E. Kahigwa, R. Hirt, P. Vounatsou, J. J. Aponte, F. Font, C. J. Acosta, D. M. Schellenberg, C. M. Galindo, J. Kimario, H. Urassa, B. Brabin, T. A. Smith, A. Y. Kitua, M. Tanner, P. L. Alonso, Lancet 1997 , 350 , 844–850. 30. J. van den Hombergh, E. Dalderop, Y. Smit, J. Trop. Pediatr. 1996 , 42 , 220–227. 31. N. Spottiswoode, M. Fried, H. Drakesmith, P. E. Duffy, Adv. Nutr. 2012 , 3 , 570–578. 32. M. R. Desai, R. Dhar, D. H. Rosen, S. K. Kariuki, Y. P. Shi, P. A. Kager, F. O. Ter Kuile, J. Nutr. 2004 , 134 , 1167–1174. 33. M. R. Desai, J. V. Mei, S. K. Kariuki, K. A. Wannemuehler, P. A. Phillips-Howard, B. L. Nahlen, P. A. Kager, J. M. Vulule, F. O. ter Kuile, J. Infect. Dis. 2003 , 187 , 658–666. 34. H. Z. Ouedraogo, M. Dramaix-Wilmet, A. N. Zeba, P. Hennart, P. Donnen, Trop. Med. Int. Health 2008 , 13 , 1257–1266. 35. D. E. Roth, R. E. Black, J. U. Ojukwu, J. U. Okebe, D. Yahav, M. Paul, Evidence-Based Child Health: A Cochrane Review Journal 2010 , 5 , 1186–1188. 36. S. A. Richard, N. Zavaleta, L. E. Caulﬁ eld, R. E. Black, R. S. Witzig, A. H. Shankar, Am. J. Trop. Med. Hyg. 2006 , 75 , 126–132. 37. E. D. Weinberg, J. Moon, Drug Metab. Rev. 2009 , 41 , 644–662. 38. E. R. Kabyemela, M. Fried, J. D. Kurtis, T. K. Mutabingwa, P. E. Duffy, J. Infect. Dis. 2008 , 198 , 163–166. 39. M. Nacher, R. McGready, K. Stepniewska, T. Cho, S. Looareesuwan, N. J. White, F. Nosten, Trans Roy. Soc. Trop. Med. Hyg. 2003 , 97 , 273–276. 40. J. P. Pena-Rosas, L. M. De-Regil, T. Dowswell, F. E. Viteri, Cochrane Database Syst. Rev. 2012 , 12 , CD004736. DOI: 10.1002/14651858.CD004736.pub4 . 41. H. Ekvall, Z. Premji, A. Bjorkman, Trop. Med. Int. Health 2000 , 5 , 696–705. 42. C. Y. Lan, G. Rodarte, L. A. Murillo, T. Jones, R. W. Davis, J. Dungan, G. Newport, N. Agabian, Mol. Microbiol. 2004 , 53 , 1451–1469. 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 25

43. R. Y. Chinn, R. D. Diamond, Infect. Immun. 1982 , 38 , 1123–1129. 44. W. M. Artis, J. A. Fountain, H. K. Delcher, H. E. Jones, Diabetes 1982 , 31 , 1109–1114. 45. A. S. Ibrahim, B. Spellberg, T. J. Walsh, D. P. Kontoyiannis, Clin. Infect. Dis. 2012 , 54 Suppl. 1 , S16–22. 46. G. Rahav, V. Volach, M. Shapiro, D. Rund, E. A. Rachmilewitz, A. Goldfarb, Br. J. Haematol. 2006 , 133 , 667–674. 47. A. Busca, M. Falda, P. Manzini, S. D'Antico, A. Valfre, F. Locatelli, R. Calabrese, A. Chiappella, S. D'Ardia, F. Longo, A. Piga, Biol. Blood Marrow Transplant 2010 , 16 , 115–122. 48. V. Pullarkat, S. Blanchard, B. Tegtmeier, A. Dagis, K. Patane, J. Ito, S. J. Forman, Bone Marrow Transplant 2008 , 42 , 799–805. 49. M. H. Miceli, L. Dong, M. L. Grazziutti, A. Fassas, R. Thertulien, F. Van Rhee, B. Barlogie, E. J. Anaissie, Bone Marrow Transplant 2006 , 37 , 857–864. 50. J. Maertens, H. Demuynck, E. K. Verbeken, P. Zachee, G. E. Verhoef, P. Vandenberghe, M. A. Boogaerts, Bone Marrow Transplant 1999 , 24 , 307–312. 51. A. Altes, A. F. Remacha, P. Sarda, F. J. Sancho, A. Sureda, R. Martino, J. Briones, S. Brunet, C. Canals, J. Sierra, Bone Marrow Transplant 2004 , 34 , 505–509. 52. S. I. Strasser, K. V. Kowdley, G. E. Sale, G. B. McDonald, Bone Marrow Transplant 1998 , 22 , 167–173. 53. C. Garcia-Vidal, A. Upton, K. A. Kirby, K. A. Marr, Clin. Infect. Dis. 2008 , 47 , 1041–1050. 54. J. Kanda, C. Mizumoto, H. Kawabata, T. Ichinohe, H. Tsuchida, N. Tomosugi, K. Matsuo, K. Yamashita, T. Kondo, T. Ishikawa, T. Uchiyama, Biol. Blood Marrow Transplant 2009 , 15 , 956–962. 55. D. P. Kontoyiannis, G. Chamilos, R. E. Lewis, S. Giralt, J. Cortes, Raad, II, J. T. Manning, X. Han, Cancer 2007 , 110 , 1303–1306. 56. A. Mahindra, R. Sobecks, L. Rybicki, B. Pohlman, R. Dean, S. Andresen, M. Kalaycio, J. Sweetenham, B. Bolwell, E. Copelan, Bone Marrow Transplant 2009 , 44 , 767–768. 57. E. Ozyilmaz, M. Aydogdu, G. Sucak, S. Z. Aki, Z. N. Ozkurt, Z. A. Yegin, N. Kokturk, Bone Marrow Transplant 2010 , 45 , 1528–1533. 58. T. Tachibana, M. Tanaka, H. Takasaki, A. Numata, S. Ito, R. Watanabe, R. Hyo, R. Ohshima, M. Hagihara, R. Sakai, S. Fujisawa, N. Tomita, H. Fujita, A. Maruta, Y. Ishigatsubo, H. Kanamori, Int. J. Hematol. 2011, 93 , 368–374. 59. J. Alexander, A. P. Limaye, C. W. Ko, M. P. Bronner, K. V. Kowdley, Liver Transpl. 2006 , 12 , 1799–1804. 60. J. K. Chow, B. G. Werner, R. Ruthazer, D. R. Snydman, Clin. Infect. Dis. 2010 , 51 , e16–23. 61. J. R. Boelaert, M. de Locht, J. Van Cutsem, V. Kerrels, B. Cantinieaux, A. Verdonck, H. W. Van Landuyt, Y. J. Schneider, J. Clin. Invest. 1993 , 91 , 1979–1986. 62. J. R. Boelaert, A. Z. Fenves, J. W. Coburn, Am. J. Kidney Dis. 1991 , 18 , 660–667. 63. J. R. Boelaert, G. F. van Roost, P. L. Vergauwe, J. J. Verbanck, C. de Vroey, M. F. Segaert, Clin. Nephrol. 1988 , 29 , 261–266. 64. J. R. Boelaert, P. L. Vergauwe, J. M. Vandepitte, Ann. Intern. Med. 1987 , 107 , 782–783. 65. M. H. Branton, S. C. Johnson, J. D. Brooke, J. A. Hasbargen, Rev. Infect. Dis. 1991 , 13 , 19–21. 66. A. S. Ibrahim, J. E. Edwards, Jr., Y. Fu, B. Spellberg, J. Antimicrob. Chemother. 2006 , 58 , 1070–1073. 67. A. S. Ibrahim, T. Gebermariam, Y. Fu, L. Lin, M. I. Husseiny, S. W. French, J. Schwartz, C. D. Skory, J. E. Edwards, Jr., B. J. Spellberg, J. Clin. Invest. 2007 , 117 , 2649–2657. 68. A. S. Ibrahim, T. Gebremariam, G. Luo, Y. Fu, S. W. French, J. E. Edwards, Jr., B. Spellberg, Antimicrob. Agents Chemother. 2011 , 55 , 1768–1770. 69. B. Spellberg, D. Andes, M. Perez, A. Anglim, H. Bonilla, G. E. Mathisen, T. J. Walsh, A. S. Ibrahim, Antimicrob. Agents Chemother . 2009 , 53 , 3122–3125. 70. B. Spellberg, A. S. Ibrahim, P. V. Chin-Hong, D. P. Kontoyiannis, M. I. Morris, J. R. Perfect, D. Fredricks, E. P. Brass, J. Antimicrob. Chemother. 2012 , 67 , 715–722. 71. R. Soman, N. Gupta, A. Shetty, C. Rodrigues, J. Antimicrob. Chemother. 2012 , 67 , 783–784. 72. S. C. Burjonrappa, M. Miller, J. Pediatr. Surg. 2012 , 47 , 760–771. 73. W. Maret, Adv. Nutr. 2013 , 4 , 82–91. 74. A. S. Prasad, J. Trace Elem. Med. Biol. 2012 , 26 , 66–69. 26 Carver

75. N. M. Lowe, M. W. Medina, A. L. Stammers, S. Patel, O. W. Souverein, C. Dullemeijer, L. Serra-Majem, M. Nissensohn, V. Hall Moran, Br. J. Nutr. 2012 , 108 , 1962–1971. 76. N. M. Lowe, K. Fekete, T. Decsi, Am. J. Clin. Nutr. 2009 , 89 , 2040S–2051S. 77. A. H. Shankar, A. S. Prasad, Am. J. Clin. Nutr. 1998 , 68 , 447S–463S. 78. A. S. Prasad, Mol. Cell Biochem. 1998 , 188 , 63–69. 79. A. S. Prasad, S. Meftah, J. Abdallah, J. Kaplan, G. J. Brewer, J. F. Bach, M. Dardenne, J. Clin. Invest . 1988 , 82 , 1202–1210. 80. M. R. Narkewicz, N. Krebs, F. Karrer, K. Orban-Eller, R. J. Sokol, Hepatology 1999 , 29 , 830–833. 81. M. D. Pescovitz, P. L. Mehta, R. M. Jindal, M. L. Milgrom, S. B. Leapman, R. S. Filo, Clin. Transplant 1996 , 10 , 256–260. 82. M. R. Bleackley, R. T. Macgillivray, Biometals 2011 , 24 , 785–809. 83. R. M. Walter, Jr., J. Y. Uriu-Hare, K. L. Olin, M. H. Oster, B. D. Anawalt, J. W. Critchﬁ eld, C. L. Keen, Diabetes Care 1991 , 14 , 1050–1056. 84. J. J. Cunningham, A. Fu, P. L. Mearkle, R. G. Brown, Metabolism 1994 , 43 , 1558–1562. 85. M. I. Hood, B. L. Mortensen, J. L. Moore, Y. Zhang, T. E. Kehl-Fie, N. Sugitani, W. J. Chazin, R. M. Caprioli, E. P. Skaar, PLoS Pathog. 2012 , 8 , e1003068. 86. N. F. Krebs, J. E. Westcott, T. D. Arnold, B. M. Kluger, F. J. Accurso, L. V. Miller, K. M. Hambidge, Pediatr. Res. 2000 , 48 , 256–261. 87. E. Mocchegiani, M. Provinciali, G. Di Stefano, A. Nobilini, G. Caramia, L. Santarelli, A. Tibaldi, N. Fabris, Clin. Immunol. Immunopathol. 1995 , 75 , 214–224. 88. I. Abdulhamid, F. W. Beck, S. Millard, X. Chen, A. Prasad, Pediatr. Pulmonol. 2008 , 43 , 281–287. 89. S. Van Biervliet, S. Vande Velde, J. P. Van Biervliet, E. Robberecht, Ann. Nutr. Metab. 2008 , 52 , 152–156. 90. R. Aggarwal, J. Sentz, M. A. Miller, Pediatrics 2007 , 119 , 1120–1130. 91. Z. A. Bhutta, S. M. Bird, R. E. Black, K. H. Brown, J. M. Gardner, A. Hidayat, F. Khatun, R. Martorell, N. X. Ninh, M. E. Penny, J. L. Rosado, S. K. Roy, M. Ruel, S. Sazawal, A. Shankar, Am. J. Clin. Nutr. 2000 , 72 , 1516–1522. 92. M. Y. Yakoob, E. Theodoratou, A. Jabeen, A. Imdad, T. P. Eisele, J. Ferguson, A. Jhass, I. Rudan, H. Campbell, R. E. Black, Z. A. Bhutta, BMC Public Health 2011 , 11 Suppl. 3 , S23. 93. M. Lukacik, R. L. Thomas, J. V. Aranda, Pediatrics 2008 , 121 , 326–336. 94. M. Lazzerini, L. Ronfani, Cochrane Database Syst. Rev. 2012 , 6 , CD005436. 95. M. Singh, R. R. Das, Cochrane Database Syst. Rev. 2011 , CD001364. 96. A. S. Prasad, Curr. Opin. Clin. Nutr. Metab. Care 2009 , 12 , 646–652. 97. A. H. Shankar, B. Genton, M. Baisor, J. Paino, S. Tamja, T. Adiguma, L. Wu, L. Rare, D. Bannon, J. M. Tielsch, K. P. West, Jr., M. P. Alpers, Am. J. Trop. Med. Hyg. 2000 , 62 , 663–669. 98. C. J. Bates, P. H. Evans, M. Dardenne, A. Prentice, P. G. Lunn, C. A. Northrop-Clewes, S. Hoare, T. J. Cole, S. J. Horan, S. C. Longman, D. Stirling, P. J. Aggett, Br. J. Nutr. 1993 , 69 , 243–255. 99. J. Veenemans, P. Milligan, A. M. Prentice, L. R. Schouten, N. Inja, A. C. van der Heijden, L. C. de Boer, E. J. Jansen, A. E. Koopmans, W. T. Enthoven, R. J. Kraaijenhagen, A. Y. Demir, D. R. Uges, E. V. Mbugi, H. F. Savelkoul, H. Verhoef, PLoS Med. 2011 , 8 , e1001125. 100. S. Sazawal, R. E. Black, M. Ramsan, H. M. Chwaya, A. Dutta, U. Dhingra, R. J. Stoltzfus, M. K. Othman, F. M. Kabole, Lancet 2007 , 369 , 927–934. 101. C. Duggan, W. B. MacLeod, N. F. Krebs, J. L. Westcott, W. W. Fawzi, Z. G. Premji, V. Mwanakasale, J. L. Simon, K. Yeboah-Antwi, D. H. Hamer, J. Nutr. 2005 , 135 , 802–807. 102. Z. A. P. S. Group, Am. J. Clin. Nutr. 2002 , 76 , 805–812. 103. M. M. Berger, A. Shenkin, J. Trace Elem. Med. Biol. 2007 , 21 Suppl. 1 , 44–48. 104. M. M. Berger, F. Spertini, A. Shenkin, C. Wardle, L. Wiesner, C. Schindler, R. L. Chiolero, Am. J. Clin. Nutr. 1998 , 68 , 365–371. 105. K. G. Kreymann, M. M. Berger, N. E. Deutz, M. Hiesmayr, P. Jolliet, G. Kazandjiev, G. Nitenberg, G. van den Berghe, J. Wernerman, Dgem, C. Ebner, W. Hartl, C. Heymann, C. Spies, Espen, Clin. Nutr. 2006 , 25 , 210–223. 1 Metal Ions and Infectious Diseases. An Overview from the Clinic 27

106. M. S. Agren, U. Ostenfeld, F. Kallehave, Y. Gong, K. Raffn, M. E. Crawford, K. Kiss, A. Friis-Moller, C. Gluud, L. N. Jorgensen, Wound Repair Regen. 2006 , 14 , 526–535. 107. M. M. Berger, P. Eggimann, D. K. Heyland, R. L. Chiolero, J. P. Revelly, A. Day, W. Raffoul, A. Shenkin, Crit. Care 2006 , 10 , R153. 108. M. M. Berger, M. Baines, W. Raffoul, M. Benathan, R. L. Chiolero, C. Reeves, J. P. Revelly, M. C. Cayeux, I. Senechaud, A. Shenkin, Am. J. Clin. Nutr. 2007 , 85 , 1293–1300. 109. M. M. Berger, C. Binnert, R. L. Chiolero, W. Taylor, W. Raffoul, M. C. Cayeux, M. Benathan, A. Shenkin, L. Tappy, Am. J. Clin. Nutr. 2007 , 85 , 1301–1306. 110. M. M. Berger, C. Cavadini, R. Chiolero, H. Dirren, J. Trauma 1996 , 40 , 103–109. 111. C. L. Braunschweig, M. Sowers, D. S. Kovacevich, G. M. Hill, D. A. August, J. Nutr. 1997 , 127 , 70–74. 112. D. K. Heyland, N. Jones, N. Z. Cvijanovich, H. Wong, J. Parenter. Enteral. Nutr. 2008 , 32 , 509–519. 113. S. Rinaldi, F. Landucci, A. R. De Gaudio, Curr. Drug Targets 2009 , 10 , 872–880. 114. V. L. Gupta, B. S. Chaubey, J Assoc Physicians India 1995 , 43 , 467–469. 115. A. S. Prasad, F. W. Beck, J. Kaplan, P. H. Chandrasekar, J. Ortega, J. T. Fitzgerald, P. Swerdlow, Am. J. Hematol. 1999 , 61 , 194–202. 116. B. Bao, A. S. Prasad, F. W. Beck, D. Snell, A. Suneja, F. H. Sarkar, N. Doshi, J. T. Fitzgerald, P. Swerdlow, Transl. Res. 2008 , 152 , 67–80. 117. G. Hardy, I. Hardy, W. Manzanares, Nutr. Clin. Pract. 2012 , 27 , 21–33. 118. K. Ashton, L. Hooper, L. J. Harvey, R. Hurst, A. Casgrain, S. J. Fairweather-Tait, Am. J. Clin. Nutr. 2009 , 89 , 2025S–2039S. 119. K. Venardos, K. Ashton, J. Headrick, A. Perkins, Mol. Cell Biochem. 2005 , 270 , 131–138. 120. M. P. Rayman, Lancet 2012 , 379 , 1256–1268. 121. X. Forceville, J. Trace Elem. Med. Biol. 2007 , 21 Suppl. 1 , 62–65. 122. X. Forceville, V. Mostert, A. Pierantoni, D. Vitoux, P. Le Toumelin, E. Plouvier, M. Dehoux, F. Thuillier, A. Combes, Eur. Surg. Res. 2009 , 43 , 338–347. 123. R. F. Burk, K. E. Hill, Biochim. Biophys. Acta 2009 , 1790 , 1441–1447. 124. C. Stoppe, G. Schalte, R. Rossaint, M. Coburn, B. Graf, J. Spillner, G. Marx, S. Rex, Crit. Care Med. 2011 , 39 , 1879–1885. 125. L. Kiremidjian-Schumacher, M. Roy, H. I. Wishe, M. W. Cohen, G. Stotzky, Biol. Trace Elem. Res. 1994 , 41 , 115–127. 126. M. P. Look, J. K. Rockstroh, G. S. Rao, K. A. Kreuzer, U. Spengler, T. Sauerbruch, Biol. Trace Elem. Res. 1997 , 56 , 31–41. 127. J. E. Spallholz, L. M. Boylan, H. S. Larsen, Ann. NY Acad. Sci. 1990 , 587 , 123–139. 128. A. Campa, G. Shor-Posner, F. Indacochea, G. Zhang, H. Lai, D. Asthana, G. B. Scott, M. K. Baum, J. Acquir. Immune Deﬁ c. Syndr. Hum. Retrovirol. 1999 , 20 , 508–513. 129. M. K. Baum, G. Shor-Posner, S. Lai, G. Zhang, H. Lai, M. A. Fletcher, H. Sauberlich, J. B. Page, J. Acquir. Immune Deﬁ c. Syndr. Hum. Retrovirol. 1997 , 15 , 370–374. 130. R. Kupka, G. I. Msamanga, D. Spiegelman, S. Morris, F. Mugusi, D. J. Hunter, W. W. Fawzi, J. Nutr. 2004 , 134 , 2556–2560. 131. A. Cirelli, M. Ciardi, C. de Simone, F. Sorice, R. Giordano, L. Ciaralli, S. Costantini, Clin. Biochem. 1991 , 24 , 211–214. 132. P. K. Drain, J. M. Baeten, J. Overbaugh, M. H. Wener, D. D. Bankson, L. Lavreys, K. Mandaliya, J. O. Ndinya-Achola, R. S. McClelland, BMC Infect. Dis. 2006 , 6 , 85. 133. X. Burbano, M. J. Miguez-Burbano, K. McCollister, G. Zhang, A. Rodriguez, P. Ruiz, R. Lecusay, G. Shor-Posner, HIV Clin. Trials 2002 , 3 , 483–491. 134. B. E. Hurwitz, J. R. Klaus, M. M. Llabre, A. Gonzalez, P. J. Lawrence, K. J. Maher, J. M. Greeson, M. K. Baum, G. Shor-Posner, J. S. Skyler, N. Schneiderman, Arch. Intern. Med. 2007 , 167 , 148–154. 135. R. Kupka, F. Mugusi, S. Aboud, G. I. Msamanga, J. L. Finkelstein, D. Spiegelman, W. W. Fawzi, Am. J. Clin. Nutr. 2008 , 87 , 1802–1808. 136. X. Forceville, D. Vitoux, R. Gauzit, A. Combes, P. Lahilaire, P. Chappuis, Crit. Care Med. 1998 , 26 , 1536–1544. 28 Carver

137. Y. Sakr, K. Reinhart, F. Bloos, G. Marx, S. Russwurm, M. Bauer, F. Brunkhorst, Br. J. Anaesth. 2007 , 98 , 775–784. 138. W. Manzanares, A. Biestro, M. H. Torre, F. Galusso, G. Facchin, G. Hardy, Intensive Care Med . 2011 , 37 , 1120–1127. 139. M. W. Angstwurm, L. Engelmann, T. Zimmermann, C. Lehmann, C. H. Spes, P. Abel, R. Strauss, A. Meier-Hellmann, R. Insel, J. Radke, J. Schuttler, R. Gartner, Crit. Care Med. 2007 , 35 , 118–126. 140. M. W. Angstwurm, J. Schottdorf, J. Schopohl, R. Gaertner, Crit. Care Med. 1999 , 27 , 1807–1813. 141. M. M. Berger, M. J. Reymond, A. Shenkin, F. Rey, C. Wardle, C. Cayeux, C. Schindler, R. L. Chiolero, Intensive Care Med. 2001 , 27 , 91–100. 142. B. Kuklinski, M. Buchner, R. Schweder, R. Nagel, Z. Gesamte Inn. Med. 1991, 46 , 145–149. 143. D. Lindner, J. Lindner, G. Baumann, H. Dawczynski, K. Bauch, Med. Klin. (Munich) 2004 , 99 , 708–712. 144. V. Mishra, M. Baines, S. E. Perry, P. J. McLaughlin, J. Carson, R. Wenstone, A. Shenkin, Clin. Nutr. 2007 , 26 , 41–50. 145. T. Zimmermann, S. Albrecht, H. Kuhne, U. Vogelsang, R. Grutzmann, S. Kopprasch, Med. Klin. (Munich) 1997 , 92 Suppl. 3 , 3–4. 146. X. Forceville, B. Laviolle, D. Annane, D. Vitoux, G. Bleichner, J. M. Korach, E. Cantais, H. Georges, J. L. Soubirou, A. Combes, E. Bellissant, Crit. Care 2007 , 11 , R73. 147. P. J. Andrews, A. Avenell, D. W. Noble, M. K. Campbell, C. G. Battison, B. L. Croal, W. G. Simpson, J. Norrie, L. D. Vale, J. Cook, R. de Verteuil, A. C. Milne, G. Trials Management, Trials 2007 , 8 , 25. 148. T. S. Huang, Y. C. Shyu, H. Y. Chen, L. M. Lin, C. Y. Lo, S. S. Yuan, P. J. Chen, PLoS One 2013 , 8 , e54431. 149. C. S. Broome, F. McArdle, J. A. Kyle, F. Andrews, N. M. Lowe, C. A. Hart, J. R. Arthur, M. J. Jackson, Am. J. Clin. Nutr. 2004 , 80 , 154–162. 150. L. Shamseer, D. Adams, N. Brown, J. A. Johnson, S. Vohra, Cochrane Database Syst. Rev. 2010 , CD007020. 151. R. A. Wapnir, Am. J. Clin. Nutr. 1998 , 67 , 1054S–1060S. 152. K. E. Mason, J. Nutr. 1979 , 109 , 1979–2066. 153. G. S. Sacks, Nutr. Clin. Pract. 2003 , 18 , 352. 154. O. Teslariu, M. Nechifor, Rev. Med. Chir. Soc. Med. Nat. Iasi. 2012 , 116 , 883–887. 155. G. Wang, X. Feng, X. Yu, X. Xu, D. Wang, H. Yang, X. Shi, Biol. Trace Elem. Res. 2013 , 152, 300–304. 156. C. H. Guo, P. C. Chen, M. S. Yeh, D. Y. Hsiung, C. L. Wang, Clin. Biochem. 2011, 44, 275–280. 157. E. G. Offenbacher, F. X. Pi-Sunyer, B. J. Stoecker, Chromium , Eds B. L. O'Dell, R. A. Sunde, Marcel Dekker, New York, 1997. 158. E. G. Offenbacher, H. Spencer, H. J. Dowling, F. X. Pi-Sunyer, Am. J. Clin. Nutr. 1986 , 44, 77–82. 159. W. Mertz, J. Nutr. 1993 , 123 , 626–633. 160. J. I. Boullata, J. Infus. Nurs. 2013 , 36 , 16–23. 161. G. Hardy, Gastroenterology 2009 , 137 , S29–35. 162. M. R. Dewitt, P. Chen, M. Aschner, Biochem. Biophys. Res. Commun. 2013 . 163. A. B. Bowman, G. F. Kwakye, E. H. Hernandez, M. Aschner, J. Trace Elem. Med. Biol. 2011 , 25 , 191–203. 164. A. W. Dobson, K. M. Erikson, M. Aschner, Ann. N. Y. Acad. Sci. 2004 , 1012 , 115–128. 165. Y. Takagi, A. Okada, K. Sando, M. Wasa, H. Yoshida, N. Hirabuki, J. Parenter. Enteral. Nutr. 2001 , 25 , 87–92. 166. J. K. Siepler, R. A. Nishikawa, T. Diamantidis, R. Okamoto, Nutr. Clin. Pract. 2003 , 18 , 370–373. 167. V. C. Culotta, M. J. Daly, Antioxid. Redox Signal 2013 . 168. M. Mori, T. Gotoh, J. Nutr. 2004 , 134 , 2820S–2825S; discussion 2853S. 169. S. M. Morris, Jr., Curr. Opin. Clin. Nutr. Metab. Care 2012 , 15 , 64–70. 170. J. Pallauf, C. Kauer, E. Most, S. D. Habicht, J. Moch, J. Anim. Physiol. Anim. Nutr. (Berl) 2012 , 96 , 993–1002. Chapter 2 Sodium and Potassium in Health and Disease

Hana R. Pohl, John S. Wheeler, and H. Edward Murray

Contents ABSTRACT ...... 30 1 INTRODUCTION ...... 30 2 PHYSIOLOGY OF SODIUM AND POTASSIUM IN HUMANS ...... 32 2.1 Action of Sodium and Potassium on Membranes ...... 32 2.1.1 Nervous System ...... 32 2.1.2 Muscular System ...... 33 2.2 Homeostasis of Sodium and Potassium ...... 33 2.2.1 Absorption and Distribution of Potassium ...... 34 2.2.2 Absorption and Distribution of Sodium ...... 34 2.2.3 Potassium Excretion and Secretion in the Kidneys ...... 34 2.2.4 Sodium Excretion and Secretion in the Kidneys ...... 35 2.3 Mechanism of Other Physiological Systems Inﬂ uencing Sodium and Potassium Homeostasis ...... 36 2.3.1 Potassium ...... 36 2.3.2 Sodium ...... 37 3 PATHOLOGY ASSOCIATED WITH SODIUM LEVELS ...... 38 3.1 Hyponatremia...... 38 3.2 Hypernatremia ...... 40 4 PATHOLOGY ASSOCIATED WITH POTASSIUM LEVELS ...... 41 4.1 Hypokalemia ...... 41 4.2 Hyperkalemia ...... 43 5 CONCLUSION ...... 45 ABBREVIATIONS ...... 45 REFERENCES ...... 46

H. R. Pohl (*) • J. S. Wheeler • H. E. Murray Agency for Toxic Substances and Disease Registry (ATSDR), US Department of Health and Human Services, 1600 Clifton Road, Mailstop F-57 , Atlanta , GA 30333 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 29 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_2, © Springer Science+Business Media Dordrecht 2013 30 Pohl, Wheeler, and Murray

Abstract Sodium and potassium are essential for human health. They are important ions in the body and are associated with many physiologic and pathophysiologic processes. The chapter summarizes the basic physiologic actions of sodium and potassium on membranes of the neurologic and muscular systems. It provides information regarding the kinetics, i.e., absorption, distribution, and excretion of these ions and their movement between the intracellular and extracellular compartments. It also explains the physiologic systems that can inﬂ uence proper homeostasis between sodium and potassium. Concentrations of sodium in the blood that exceed or do not reach the normal value range are called hypernatremia or hyponatremia, respectively. Similarly, the clinicians recognize hyperkalemia and hypokalemia. Pathologies associated with these states are described and examples of some of the diseases are presented here.

Keywords homeostasis • hyperkalemia • hypernatremia • hypokalemia • hypona tremia • potassium • sodium

Please cite as: Met. Ions Life Sci. 13 (2013) 29–47

1 Introduction

This chapter provides an overview of sodium and potassium and their importance in human physiology and pathology. Sodium and potassium are essential in maintaining cellular homeostasis. Most metabolic processes are dependent on or affected by these electrolytes. Among the functions of these electrolytes are maintenance of osmotic pressure and water distribution in various body ﬂ uid compartments, maintenance of proper pH, regulation of the proper function of the heart and other muscles, involvement in oxidation-reduction (electron transport) reactions, and participation in catalysis as cofactors for enzymes. Dietary requirements for sodium and potassium vary widely, but generally, daily intake should be only in small amounts [1 ]. Normal plasma levels for sodium in adults range from 136 to 146 mEq/L, and this balance is normally maintained by an average dietary intake of 90 to 250 mEq per day. Sodium excretion tends to reﬂ ect sodium intake, and on an average diet, urine sodium excretion will range between 80 and 180 mEq per day. Potassium is essential for the proper function of all cells, tissues, and organs in the human body. It is also crucial to heart function and plays a key role in skeletal and smooth muscle contraction, making it important for normal digestive and muscular function. Normal plasma levels for potassium in adults range from 3.5 to 5.0 mEq/L, and this balance is usually maintained in adults on an average dietary intake of 80 to 200 mEq per day. It is noted that the normal intake, minimal need, and maximum tolerance for potassium is almost the same as that for sodium. 2 Sodium and Potassium in Health and Disease 31

Sodium ions are the major cations of extracellular fl uid, whereas, potassium ions are the major cations of the intracellular fl uid [2 ]. To maintain internal fl uid and electrolyte balance, water, sodium, and potassium are in constant movement between the intracellular and extracellular body compartments. Potassium and sodium ions are particularly important in the renal regulation of acid-base balance because hydrogen ions are substituted for sodium and potassium ions in the renal tubule. Potassium plays a key role in that potassium bicarbonate is the primary intracellular inorganic buffer. Potassium enters the cell more readily than sodium and initiates the brief sodium-potassium exchange across the cell membranes. In the nerve cells, this sodium-potassium fl ux generates the electrical potential that aids the conduction of nerve impulses. When potassium leaves the cell, it changes the membrane potential and allows the nerve impulse to progress. This electrical potential gradient, created by the “sodium-potassium pump”, helps generate muscle contractions and regulates the heartbeat. Discovery of the sodium-potassium pump in the 1950s by a Danish scientist, Jens Christian Skou, marked an important step forward in our understanding of how ions enter and leave cells. This physiologic function is of particular signifi cance for excitable cells such as nerve cells, which depend on this pump for responding to stimuli and transmitting impulses [3 ]. Cellular uptake of potassium is regulated by the sodium-potassium pump, while movement of potassium out of the cell is governed by passive forces (cell membrane permeability and chemical and electrical gradients to the potassium ions). Another of the pump’s most important functions is preventing the swelling of cells. If sodium is not pumped out, water accumulates within the cell causing it to swell and ultimately burst. Abnormal levels of these electrolytes may result in a variety of pathological disorders [ 2 ]. For example, too high a concentration of sodium, a condition called hypernatremia, leads to edema (swelling of tissues due to excess fl uid retention) thirst, and lessened urine production. Hyponatremia is a low level of serum sodium and is usually characterized by headache, confusion, seizures, muscle spasms, nausea, and vomiting. Too much potassium, called hyperkalemia, characterized by irritability, nausea, decreased urine production, and cardiac arrest. Fatigue is the most common symptom of chronic potassium defi ciency. Early symptoms include muscle weakness, slow refl exes, and dry skin or acne; these initial problems may progress to nervous disorders, insomnia, slow or irregular heartbeat, and loss of gastrointestinal tone. A sudden loss of potassium may lead to cardiac arrhythmia. Low potassium may impair glucose metabolism and lead to elevated blood sugar. In more severe potassium defi ciency, there can be serious muscle weakness, bone fragility, central nervous system changes, decreased heart rate, and even death. Potassium is very important in cellular biochemical reactions and energy metabolism; it participates in the synthesis of proteins from amino acids in the cell. Potassium also functions in carbohydrate metabolism; it is active in glycogen and glucose metabolism, converting glucose to glycogen that can be stored in the liver for future energy. Potassium is important for normal growth and for building muscle. 32 Pohl, Wheeler, and Murray

Though sodium is readily conserved by the body, there is no effective method for potassium conservation. Even when a potassium shortage exists, the kidneys continue to excrete it. Since the human body relies on potassium balance for a regularly contracting heart and a healthy nervous system, it is essential to strive for this electrolyte’s balance. The renin-angiotensin-aldosterone system and vasopressin levels play an important role in regulating the electrolyte levels in the body. Pathological states of the system can be accompanied by imbalances of potassium and sodium levels. A complex interplay of physiological control systems maintains ﬂ uid, sodium, and potassium homeostasis. When this interplay of physiological systems is disrupted, or when homeostatic mechanisms can no longer maintain intracellular, extracellular or interstitial ﬂ uid, an imbalance of sodium and potassium will occur. The following discussion will address some of the complexities of the physiology and pathology involved with sodium and potassium interactions.

2 Physiology of Sodium and Potassium in Humans

2.1 Action of Sodium and Potassium on Membranes

2.1.1 Nervous System

One of the major roles of potassium/sodium balance in the body is that of the nerve impulse. A differential in sodium and potassium concentration forms a polarity across the nerve membrane that when stimulated (electrical, chemical, mechanical, or thermal) leads to depolarization and propagation of the nerve impulse along the cell membrane [4 ]. In the nerve cell, active sodium-potassium pumps create this differential by pumping two K+ atoms into the cell for every three Na+ atoms pumped out of the cell. Active pumping, along with negatively charged ions of other molecules inside the cell, leads to a voltage potential across the cell membrane. The resulting voltage is approximately –70mV [5 ]. Following membrane stimulation the membrane becomes permeable to Na+ ions, allowing Na + inside the cell, thus eliminating the electrical potential across the membrane (depolarization). Depolarization propagates in all directions from the initial point. For a very brief time, the membrane is unable to depolarize again and remains unresponsive. It is the nature of this delicate balance of sodium and potassium across the neuronal membrane that leads to diseases and physiological imbalances which result in a number of different neurological problems. Chemicals such as the organochlorine DDT gain their physiological disrupting power by interfering with the sodium channel across the axonal membrane, thus leading to variety of toxic effects, including lethality [6 ]. 2 Sodium and Potassium in Health and Disease 33

2.1.2 Muscular System

Similar in function to the membrane of neurons is the membrane function of the muscle fi ber [2 ]. The muscle fi ber, when stimulated by acetylcholine, depolarizes, propagating the depolarization into the deeper muscle through the transverse tubules, leading to the release of calcium ion followed by a contraction of the myofi brils of the muscle and thus movement of the muscle. Na+ and K+ play a key role in the depolarization of the muscle cell membrane. Polarization, as with the neuron, requires an active ion pump and energy in the form of ATP to create an ion gradient across the cell’s membrane. As with neurons, the muscle cell membrane becomes impervious to Na+ while Na+ ions are actively pumped out of the cell and K+ ions into the cell; however, some K+ diffuse back out at a slower rate than Na + is pumped out. This ion gradient, along with anions of many organic compounds and proteins inside the cell, create a voltage across the cell membrane. When the membrane is stimulated (electrically or mechanically, but usually chemically with acetylcholine), the membrane becomes permeable to sodium and voltage suddenly drops, thereby depolarizing the membrane. The depolarization propagates in all directions, moving into the muscle through transverse tubes, leading to Ca2+ release and the subsequent contraction of muscle. Diseases and xenobiotics can interfere with many steps along this complicated process of muscle cell depolarization and contraction. Interference can occur at the cell membrane, with Na+ /K+ balance, with Ca2+ infl ux, and with many other pathways. Many toxins and therapeutic agents work by inhibiting cell depolarization and repolarization.

2.2 Homeostasis of Sodium and Potassium

Homeostasis of Na+ and K+ is critical to life, especially extracellular K+ levels. A number of homeostatic mechanisms keep Na+ and K+ regulated. Normal extracellular and intracellular Na + and K+ are [7 ]: intracellular K+ 140 mEq/L extracellular K+ 5 mEq/L intracellular Na+ 12 mEq/L extracellular Na+ 140 mEq/L Intracellular K+ levels can be affected by insulin, aldosterone, β-adrenergic stimulation, acid base abnormalities, cell lysis, and strenuous exercise [ 8 ]. While short- term regulation involves cellular redistribution, long-term regulation involves renal excretion and reabsorption. 34 Pohl, Wheeler, and Murray

2.2.1 Absorption and Distribution of Potassium

The recommended intake of potassium for adolescents and adults is 4700 mg/day [9 ]. Following ingestion, K + is rapidly absorbed by active uptake in the mucosal lining of the intestine. This rapid uptake could lead to severe K+ imbalance if it was not for the rapid absorption of K+ into cells (see Section 4.2 ). Ninetyeight percent of gastrointestinally absorbed K + is stored in cells, with 2% being found extracellularly [8 ]. Even though cellular storage allows for the rapid regulation of extracellular K + , long-term regulation is carried out in the kidney.

2.2.2 Absorption and Distribution of Sodium

The average daily intake of sodium for males over 20 in the United States is 4,243 mg/day. For women it is 2,980 mg/day [10 ]. The Food and Drug Administration recommends that daily intake not exceed 2,300 mg/day for healthy individuals and no more than 1,500 mg/day for sensitive individuals (hypertensive, blacks, middle- aged, and older) [11 ]. Sodium is rapidly and actively taken up by the mucosal lining of the gastrointestinal (GI) tract [10 ]. Unlike K+ , however, it is not rapidly sequestered into the cells. Only around 10% of Na+ body burdens are found in the cells, 40% remains in extracellular ﬂ uid [ 4 ]. Na + is excreted through urine, feces, perspiration, and tears. It is also secreted back into the intestines at the rate of 25 grams per day. To remain in homeostasis, the intestines must absorb 25–35 of sodium every day [8 ]. This amount plus the amount of Na+ lost from other routes (urine and perspiration) needs to be reabsorbed every day for Na + homeostasis to occur. It is easy to see why diseases such as diarrhea and intestinal inﬂ uenza can easily upset the Na+ maintenance in the body and quickly lead to life threatening situations.

2.2.3 Potassium Excretion and Secretion in the Kidneys

A small percentage of excess K+ is excreted in the feces, while the bulk of K + excretion occurs in the urine following fi ltration, reabsorption, and secretion in the kidneys (see Figure 1 ). The kidney fi lters around 800 mg of K + per day of which approximately 65% and 27% is reabsorbed in the proximal tubule and loop of Henle, respectively [8 ]. These percentages remain fairly constant from day to day and do not signifi cantly regulate daily variations from changes in diet and absorption. The work of regulating daily variations occurs mainly in the secretion of K+ in the distal tubules and cortical collecting tubules [11 ]. 2 Sodium and Potassium in Health and Disease 35

Figure 1 Nephron. Image used with permission of the Regents of the University of Michigan. http://www.med.umich.edu/ lrc/secondlook/ .

Under normal potassium intake the amount of absorption exceeds what the body needs, and secretion into the distal tubules and cortical collecting tubules eliminates the excess through excretion in the urine. Under extreme K+ deﬁ ciencies reabsorption in the distal tubules can actually exceed secretion and thus conserve K+ .

2.2.4 Sodium Excretion and Secretion in the Kidneys

Some sodium is lost in feces and sweat, but as was seen with potassium, the majority of sodium regulation in the body occurs in the kidney (see Figure 1 ). In the kidney, sodium ions (approximately 70%) are reabsorbed into the proximal tubules and loop of Henle after ﬁ ltration through the glomerulus [4 ]. However, unlike K+ , the driving force of 36 Pohl, Wheeler, and Murray

Na+ homeostasis is the glomerular fi ltration rate and tubule reabsorption. By the time the fi ltrate reaches the distal tubules almost all the Na+ has been reabsorbed. As the fi ltrate formed at the glomerulus passes through the proximal tubules, loop of Henle, and distal tubules, the solution undergoes several transformations in tonicity that allows (along with active Na+ uptake throughout the loop) for reabsorption of water and Na+ . The ascending limb is impermeable to water yet still actively secretes Na + causing the interstitial space around the ascending limb to become hypertonic. Since the interstitial space around the ascending limb is immediately adjacent to the descending limb, it creates an osmotic gradient between fl uid inside the descending limb and the interstitial fl uid. This gradient drives the removal of water from the descending limb, thereby increasing the fl uid tonicity (forming a hypertonic solution). As the fl uid makes its way out of the descending limb into the ascending limb the tubule becomes impermeable to water, yet Na + continues to be actively pumped out. This results in a hypotonic fl uid low in Na+ that leaves the ascending loop of Henle. Following the reabsorption of Na + in the ascending loop of Henle, Na + reabsorption continues in the distal tubules. It is in this region of the kidney where water retention occurs. The pituitary gland, in response to decreased water concentration in the blood, releases stored antidiuretic hormone (ADH) into the circulatory system. ADH causes the epithelial cells of the distal convoluted tubules to become more permeable to water, thus concentrating urine and saving water during times of water stress. Na + homeostasis is critical to life and thus requires the amount of sodium intake to equal the amount of Na+ excretion. There are numerous feedback loops and hormonal controls in play to regulate Na+ excretion such as blood pressure (pressure natriuresis and diuresis), blood volume, antidiuretic hormone, angiotensin II, arterial baroreceptor, low pressure stretch receptors refl exes, aldosterone, and natriuretic peptide. Regardless of the mechanism (complex or simple), all these feedbacks work by altering either glomerular fi ltration rates or by Na+ reabsorption. Xenobiotics, disease, or even fever can cause any of these mechanisms to alter Na+ balance. It is therefore necessary to have a complex system of redundancy and rapid response to maintain critical Na+ balance.

2.3 Mechanism of Other Physiological Systems Inﬂ uencing Sodium and Potassium Homeostasis

2.3.1 Potassium

Aldosterone : See the discussion of aldosterone’s effects on Na + below. Aldosterone increases the Na+ /K + ATPase pump as Na+ is conserved, K+ is secreted into the urine.

β -adrenergic stimulation: Activation of β2 -adrenergic receptors by stimulants such + as epinephrine causes K to move into cells. Drugs that block β2 receptors can prevent the uptake of K+ into cells. Acid-base abnormalities: The activity of the sodium-potassium ATPase pump is inhibited in the presence of increased hydrogen ion concentration. Therefore 2 Sodium and Potassium in Health and Disease 37 disease or physiological states that affect acid-base balance can affect K + homeostasis as well [8 ]. Cell lysis: Necrosis or major cell death can lead to the release of intracellular K+ causing a disruption in K+ homeostasis. Strenuous exercise : Muscle cells release K+ during long-duration exercise. Usually this is not a problem except in individuals that may already be sensitive to K + disturbances (diabetics, people taking beta blockers).

2.3.2 Sodium

Pressure natriuresis and diuresis : Blood pressure drives both urinary volume and the amount of Na + fi ltered into the proximal tubule. While increases and decreases in natriuresis pressure can help regulate Na + homeostasis when such pressure changes occur as a result of disease (e.g., hypertension) or other causes, the increase or decrease in pressure can cause imbalances in sodium. Blood volume: Changes in blood volume quickly lead to changes in cardiac output and blood pressure. As discussed above, blood pressure changes can lead to changes in Na+ excretion. Antidiuretic hormone : As previously discussed (see sodium excretion and secretion in the kidney), the pituitary gland, in response to decreased water concentration in the blood, releases stored antidiuretic hormone into the circulatory system. ADH causes the epithelial cells of the distal convoluted tubules to become more permeable to water, thus concentrating urine and saving water during times of water stress. Angiotensin II: Decreased levels of angiotensin II result in decreased reabsorption of Na + in the renal tubules. Thus decreases in angiotensin II are seen following increases in sodium intake. Angiotensin II works by modifying the natriuresis pressure mechanism, decreasing angiotensin II and increasing pressure when sodium needs to be excreted [12 ]. It also indirectly stimulates aldosterone secretion and constricts efferent arterioles. Angiotensin II is decreased by inhibiting renin, an angiotensin II precursor. In some individuals, this renin-angiotensin system (RAS) does not operate as effi ciently, and greater increases in arteriole pressure are needed to excrete sodium. This may lead to hypertension in some individuals [ 8 ]. Arterial baroreceptor and low pressure stretch receptors refl exes : Sympathetic activity can constrict renal arterioles, increase tubular reabsorption, and stimulate renin release, all leading to increased retention of sodium. This type of refl ex is likely to occur from decreased blood volume, as in following a large hemorrhage. Aldosterone : Na + absorption in the kidney (the ascending limb of the loop of Henle, the distal convoluted tubules, and collecting ducts) is greatly infl uenced by the amount of aldosterone excreted by the adrenal cortex [4 ]. When Na+ levels drop, the adrenal cortex secretes aldosterone, which results in an increase in the active reabsorption of Na+ . 38 Pohl, Wheeler, and Murray

3 Pathology Associated with Sodium Levels

3.1 Hyponatremia

Hyponatremia represents a decrease in the serum sodium concentration below the lower end of the normal range (136 mEq/L) [13 ]. Clinical signs and symptoms associated with hyponatremia include hypotension, and decreased extracellular fl uid osmolarity resulting in intracellular fl uid increase [14 ]. Hyponatremia is the most common electrolyte disorder. In one study, the prevalence of hyponatremia was 28% in acute hospital care patients at the time of admission and 21% in ambulatory patients [15 ]. The risk factors for hyponatremia include use of diuretics, liver failure, heart failure, myocardial infarction, and endocrine changes which are mostly found in older patients. Hyponatremia is associated with various conditions that can be grouped into dilutional disorders (characterized by water intake in excess of output; the condition implies impaired water excretion) and depletional disorders (caused by sodium depletion in excess of water depletion or replacement of fl uid losses with water alone). See Table 1 for pathologic states associated with hyponatremia.

Table 1 Pathology associated with hyponatremia a . Causes of hyponatremia Associated diseases Water intake higher than output; always impaired Primary: chronic renal failure, acute renal water excretion (dilutional disorders) failure (recovery phase), SIADH Neuroendocrine: adrenal and pituitary insuffi ciency With edema: congestive heart failure, hepatic cirrhosis, toxemia in pregnancy Osmotic: severe hyperglycemia Diuretics: thiazides Sodium depletion higher than water depletion or Severe diarrhea, vomiting, blood loss, replacement of fl uid losses with water alone excessive sweating (depletional disorders = extrarenal losses) a All tables were modifi ed from Chandrasoma and Taylor [14 ] and Merck [13 ].

Dilutional disorders include primary causes such as renal failure and the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Other causes of dilutional disorders include neuroendocrine dysfunction (adrenal and pituitary insuffi ciency), diseases linked to sodium retention and edema (congestive heart failure, cirrhosis, nephrotic syndrome), osmotic hyponatremia (severe hyperglycemia in diabetes), and drug-induced disorders (mercurial diuretics, chlorothiazide diuretics). Hyponatremia with hypotonicity can also be induced by diets with high water and low salt intake or by excessive beer drinking. SIADH is an example of a dilutional disorder. The syndrome was fi rst described almost 50 years ago [16 ]. The diagnostic criteria include hyponatremia with 2 Sodium and Potassium in Health and Disease 39 hypotonicity of plasma, high urine osmolarity relative to plasma, increased renal sodium excretion, absence of edema, and normal renal and adrenal function. SIADH explains about 60% of all types of chronic hyponatremia and is the most common type of hyponatremia in hospitalized patients [17 ]. SIADH is associated with 4 major etiologies: nonmalignant pulmonary diseases, neoplasms with ectopic hormone production, neurologic disorders, and use of several pharmaceutics [18 ]. SIADH is linked to euvolemic hyponatremia described as an increase in total water with normal or near normal sodium levels. It is associated with inappropriate secretion of arginine vasopressin (AVP), the hormone that regulates excretion of water by kidneys. Excessive release of AVP unrelated to plasma osmolarity occurs in about 40% of patients with SIADH. In the case of impaired glomerular fi ltration rate (renal failure) hyponatremia is caused by inadequate glomerular fi ltration of water (i.e., the body cannot get rid of water taken in). However, this usually happens when the fi ltration rate is substantially reduced to about 20–30% of the normal rate [14 ]. The common ground of diseases such as congestive heart failure, cirrhosis, and nephrotic syndrome is the edematous state . Hyponatremic patients with these diseases have abnormal renal retention of sodium resulting in extracellular fl uid volume overload and edema. They also have retention of water causing hyponatremia with hypotonicity. Drugs such as thiazide diuretics are an important cause of hyponatremia especially in elderly women. The mechanism of action is inhibition of Na+ -Cl– symport (co-transporter) located in the cortical part of the ascending loop of Henle and the distal convoluted tubules of the kidneys resulting in the failure of these ions to reabsorb [19 , 20 ]. Thiazides also increase calcium reabsorption in the distal tubule. Complications of thiazide therapy are hyponatremia, hypokalemia, hypercalcemia, hyperglycemia, and hyperlipidemia. Hyponatremia can also be induced by loop diuretics (e.g., furosemide, bumetanide). These diuretics block the Na+ -K+ -2Cl– symport which facilitates ion movement from the tubular lumen into the tubular cells in the ascending part of the loop of Henle [20 ]. The mechanism of action of the loop diuretics lays in competing for the Cl– binding sites of the symport. This may lead to natriuresis and hyponatremia, hypokalemia, hypomagnesemia, and dehydration. Genetic mutation of the Na+ -K+ -2Cl– symport encoding gene may lead to impaired function of the symport; the clinical presentation is severe volume depletion, hypokalemia, and metabolic alkalosis with increased prenatal mortality. The disease is called type I Bartter’s syndrome [21 ]. A special case of hyponatremia is with hypertonicity. It was described in patients with uncontrolled diabetes mellitus with severe hyperglycemia [14 ]. The increased glucose concentration causes water to move from the intracellular to the extracellular compartment resulting in decreased sodium concentration (i.e., dilutional state). Depletional disorders include severe diarrhea, vomiting, blood loss, and excessive sweating accompanied by large oral intake of water. Diarrhea is an example of a condition linked to depletional hyponatremia. The most common causes of diarrhea are bacterial enterotoxins (e.g., Vibrio cholerae ), bacterial invasion of gastric mucosa (e.g., some Shigella, Salmonella), and enteroviruses. 40 Pohl, Wheeler, and Murray

Diarrhea is also associated with hypokalemia and metabolic acidosis. The condition may become severe and lead to mortality, especially in susceptible populations such as the elderly, those debilitated by other diseases, and the very young. In a retrospective study in Nepal, 5 children died out of 57 who were admitted to the hospital with diarrhea [ 22 ]. Most patients (70%) were younger than 2 years. Electrolyte disturbance was reported in 46 (80%) patients, and acid-base disturbance was reported in all tested. Hyponatremia was present in 56% of patients and was either isolated (26%) or associated with hypokalemia (26%). Hypokalemia was found in 46% of patients and was isolated in 14%. In a two year prospective study in Nigeria, 191 children under 15 years of age were admitted to the hospital with diarrhea and protein energy malnutrition [23 ]. The most often observed disturbance was metabolic acidosis that was reported in 108 (56.3%) of patients. Hypokalemia was found in 45 (23.4%) and hyponatremia in 25 (13%) of patients. Clinical risk factors contributing to mortality in children hospitalized for diarrhea were studied in Turkey [24 ]. In a cohort of 400 children, 27 (6.75%) died. Signiﬁ cant factors contributing to fatalities included severe malnutrition, co-existent sepsis, hypogly- cemia, hypoalbuminemia, Shigella infection, hyponatremia (p = 0.016), hypokalemia (p = 0.00041) and metabolic acidosis (p = 0.0069).

3.2 Hypernatremia

Hypernatremia represents an elevation in the serum sodium concentration above the higher end of the normal range (145 mEq/L) [13 ]. Clinical signs and symptoms associated with hypernatremia include hypertension, increased extracellular fl uid volume, and increased extracellular fl uid osmolarity resulting in intracellular fl uid loss [ 14]. See Table 2 for pathologic states associated with hypernatremia. Hypernatremia is not as common as hyponatremia. It is associated with abnormal renal excretion of water with inadequate water intake disorders such as in pituitary ADH defi ciency (central diabetes insipidus) and nephrotic syndrome (nephrotic diabetes insipidus), in which kidneys are ADH unresponsive, or with osmotic diuresis such as severe glycosuria and manitol diuresis. Other diseases and states that may be accompanied by hypernatremia are chronic renal failure, recovery phase of acute renal failure, hypocalcemia, hypokalemia, and sickle cell anemia.

Table 2 Pathology associated with hypernatremia. Causes of hypernatremia Associated diseases Abnormal renal excretion of water with Diabetes insipidus, renal failure, inadequate intake loop diuretics Water depletion with normal renal water Excessive sweating; conservation diarrhea (children) Excessive intake of sodium with limited Poisoning water intake 2 Sodium and Potassium in Health and Disease 41

Another mechanism of hypernatremia is water depletion with normal renal conservation of water but inadequate intake of water; causes include excessive sweating and diarrhea (pronounced in children). For example, hypernatremia was reported in 6 children (3.1%) with severe diarrhea in a cohort of 191 (see Section 3.1 .) [23 ]. However, hyponatremia was far more frequent. i.e., in 13% of the cohort.

4 Pathology Associated with Potassium Levels

4.1 Hypokalemia

Hypokalemia represents the low potassium levels. In adults, potassium blood levels drop below 3.5 mEq/L, which is the lower range of normal values. Clinical signs and symptoms associated with hypokalemia include neuromuscular (weakness, paralysis, fasciculation and tetany), gastrointestinal (ileus, nausea, vomiting, abdominal distention), and renal effects (polyuria) [14 ]. Cardiac effects present themselves as dysrhythmias and conduction defects. ECG manifestations include decreased amplitude and broadening of the T waves, prominent U waves, ST segment depression, increased QRS duration, and increase in P wave amplitude and duration. The changes may lead to atrioventricular block and cardiac arrest [ 25 – 27]. With hypokalemia, cardiac arrest occurs during systole [ 28]. See Table 3 for pathologic states associated with hypokalemia.

Table 3 Pathology associated with hypokalemia. Causes of hypokalemia Associated diseases Increased extrarenal losses Severe diarrhea, laxative abuse, vomiting, excessive sweating, villous adenoma Increased renal losses With metabolic acidosis: renal tubular acidosis, diabetic ketoacidosis With metabolic alkalosis: diuretics, post hypercapnea, mineralocorticoid excess syndrome, Bartter’s syndrome With no speciﬁ c acid-base disorder: acute renal failure (recovery phase), post obstructive diuresis, osmotic diuresis, saline intake Potassium shifts into cells Alkalemia, β-adrenergic activity, familial hypokalemic (redistribution) periodic paralysis, theophylline toxicity

Potassium homeostasis depends on external balance (i.e., dietary intake and absorption versus excretion) and internal balance (i.e., the distribution of potassium between intracellular and extracellular ﬂ uids [14 ]). External losses include those through the gastrointestinal tract (e.g., diarrhea, villous adenoma of recto-sigmoid colon, inadequate intake) or through the skin 42 Pohl, Wheeler, and Murray

(e.g., profuse sweating). Urine potassium is usually <20 mEq/24 hours. In external losses through the kidneys, urine potassium is usually >20 mEq/24 hours. Eating disorders and starvation: Anorexia nervosa and bulimia are psychologi- cal eating disorders. Medical consequences of these eating disorders include heart damage, failure of the endocrine system, perforation of the stomach or esophagus, aspiration of vomit, erosions of teeth enamel, and depression [ 29 ]. Death by starvation has been reported in up to 24% of the patients with anorexia. Biochemical changes are also pronounced [30 ]. Hypokalemia is the most common electrolyte disturbance. It is often refl ected by changes on the electrocardiograms. Metabolic alkalosis is found in patients who vomit or abuse diuretics, whereas acidosis is found in those abusing laxatives. In laxative abuse, potassium is lost directly from the intestines. In contrast, the loss of potassium in those who vomit is largely due to metabolic alkalosis, which is secondary to loss of hydrogen ions in the vomitus. This results in increased availability of bicarbonate from blood and increased renal excretion of potassium [31 ]. Hypokalemic nephropathy is also associated with laxative abuse. Severe chronic hypokalemia in these patients was found to result in a progressive decrease in renal function and histological changes suggestive of chronic glomerular damage. Chronic tubulo-interstitial nephropathy has been also reported [32 , 33 ]. Hypokalemia is also associated with starvation related to other causes. For example, hypokalemia was reported in malnourished children on poor protein-calorie diets all over the world. In these children, decrease in total body potassium was correlated with decreased muscle potassium established by analysis of biopsy samples [34 – 36 ]. This result correlated with loss of total muscle mass. In contrast, muscle water was increased. Wasting is one aspect of the muscle loss; however, a contributing factor may be a decreased muscle build-up. Several laboratory studies showed the importance of potassium in protein synthesis. A study in young chicken demonstrated that there was a signifi cant decrease in the incorporation of injected L-leucine-1-14 C into skeletal muscle of chicken fed a potassium-defi cient diet [37 ]. Similarly, when rats were maintained on a potassium-defi cient diet, the animals stopped growing within a few days and the incorporation of [ 3 H]leucine into skeletal muscle protein in vivo was reduced by 28–38% [38 ]. Related to the above topic is the refeeding syndrome. It illustrates the metabolic and clinical changes in the body that occur in the process of aggressive nutritional rehabilitation of starved patients. The most important manifestation is hypophosphatemia [39 ]. Hypokalemia, hypomagnesemia, hyperglycemia, fl uid overload, and thiamine defi ciency may also be present. During starvation, potassium is depleted in the cells. During refeeding, increased insulin secretion promotes cellular uptake of potassium, resulting in hypokalemia. The outcome is an imbalance of electrochemical potential on membranes leading to cardiac arrhythmias and arrest. Neuromuscular dysfunction is also observed. The refeeding syndrome was reported in up to 25% of adults with cancer. Causes for potassium renal losses are complex [26 , 27 ]. Contributing clinical factors are increased mineralocorticoid-receptor stimulation (primary hyper-reninism distinguished by increased renin and aldosterone levels that cannot be suppressed 2 Sodium and Potassium in Health and Disease 43 by saline); primary aldosteronism (e.g., Conn syndrome); a primary increase in the effectiveness and/or amount of non-aldosterone mineralocorticoid-receptor agonist (e.g., Cushing syndrome, congenital adrenal hyperplasia); and increased distal sodium delivery and/or non-reabsorbable ions in the distal nephron (e.g., magnesium defi cit, Bartter syndrome) [27 ]. Clinical data indicate that renal losses of potassium are often related to adverse effects to therapy (e.g., penicillin, gentamicin, cisplatin, diuretics). For example, hypokalemia was reported in 10% to 40% of patients on thiazide diuretics [ 40 ]. The mechanism includes increased exchange of Na+ for K + and increased production of aldosterone as a response to diuretic hypovolemia [19 ]. It is well established that acid-base imbalance and electrolyte disorders are associated with diabetes . Recent reports indicate that low potassium is a possible risk factor for developing type 2 diabetes [41 ]. Redistribution losses are the consequence of potassium shifts into cells from the extracellular fl uids. By this mechanism, hypokalemia is present in respiratory alkalosis, increased β2 -adrenergic activity, theophylline toxicity, and in familial hypokalemic periodic paralysis.

Stimulation of β2 -adrenergic receptors redistributes potassium into cells by increasing the activity of sodium-potassium ATPase. States of increased sympathetic responsiveness can be observed in myocardial infarction, delirium tremens, or major head trauma. These states are also associated with shifts in potassium levels. Hypokalemia is common in congestive heart failure due to a defect in sodium- potassium ATPase activity and intracellular transfer of potassium caused by oxidative stress and neurohormonal activation [42 ]. Hypokalemia in the presence of congestive heart failure may lead to serious outcomes [ 43 ]. These include impaired diuresis because of decreased natriuresis and lack of suppression of renin secretion, reduced myocardial performance, and elevated risk for ventricular arrhythmia and sudden death. Recent studies indicated that heart failure itself may stimulate metabolic changes such as insulin resistance [44 ]. These in turn may worsen the primary condition. A study in hospitalized patients with heart failure and a depressed left ventricular ejection fraction reported 30-day and 1-year mortality as 7.1% and 25.5%, respectively [45 ]. Impaired renal function is a major factor that inﬂ uences the prognosis of patients with heart failure [46 ].

4.2 Hyperkalemia

In adults, hyperkalemia refers to blood values of potassium >5 mEq/L. Clinical manifestations of hyperkalemia include neuromuscular effects (weakness, ascending paralysis, and respiratory failure) and ECG changes (peaked T waves, ﬂ attened P waves, widened QRS complex) [14 ]. The changes in heart conductivity can lead to sinus arrest, ventricular tachycardia, and ﬁ brillation at >10 mEq/L [25 ]. With hyperkalemia, cardiac arrest occurs during diastole [28 ]. See Table 4 for pathologic states associated with hyperkalemia. 44 Pohl, Wheeler, and Murray

Table 4 Pathology associated with hyperkalemia. Causes of hyperkalemia Associated diseases Decreased excretion Renal failure (acute and chronic), severe oliguria due to severe dehydration or shock Endocrine dysfunction Adrenocortical insufﬁ ciency, hyporeninemic-hypoaldosteronism Potassium shifts out of cells Acidosis, hypertonic states, massive release in burns, (redistribution) rhabdomyolysis or crush injury, or severe infection

Hyperkalemia is less common than hypokalemia. However, it still affects about 8% of patients in US hospitals [ 25]. There are two major mechanisms for hyperkalemia development. Redistribution hyperkalemia is caused by potassium shifting from the intracellular space into the extracellular space, thus raising serum potassium concentration. Potassium is forced out of cells in exchange for hydrogen ion in both metabolic and respiratory acidosis. Similarly, potassium leaks out of cells in hypertonic states, in burns and injuries, and in massive digitalis overdose. Hyperkalemia secondary to impaired potassium excretion is the major cause of this electrolyte disorder. It may be due to aldosterone defi ciency (e.g., primary adrenal failure, Addison’s disease) or tubular unresponsiveness to aldosterone (e.g., chronic renal diseases, some pharmaceuticals). Hyperaldosteronism is a disease caused by an excess production of adrenal hormone aldosterone. This hormone is responsible for sodium and potassium balance, which then directly controls water balance to maintain appropriate blood pressure and blood volume. With adrenal insuffi ciency, there is inappropriate sodium excretion. When adrenal aldosterone production is increased (as in shock, heart failure, or cirrhosis) sodium excretion is decreased. People with a defi ciency of aldosterone, especially found in association with cortisol defi ciency in Addison’s disease, have low blood volume and therefore low blood pressure, low sodium and high potassium. Just the opposite is seen in hyperaldosteronism. There are several drugs that affect the renin-angiotensin-aldosterone system and thus may impact potassium levels. A review of studies that administered angiotensin- converting enzyme inhibitors, angiotensin receptor blockers, aldosterone receptor antagonists, and direct renin inhibitors alone or in combination to patients with hypertension, heart failure, or chronic kidney disease revealed that the risk of hyperkalemia on monotherapy of hypertension is low (≤2%) but increases to about 5% in combination therapy [47 ]. Increased incidence was also observed in patients with heart failure or chronic kidney disease (5% to 10%). The syndrome of hyporeninemic hypoaldosteronism (SHH) that also belongs to this category is associated with several renal diseases. SHH includes low plasma renin activity, low plasma aldosterone, and hyperkalemia. The syndrome is also common in patients with diabetes mellitus. In a study of 210 outpatient diabetics, metabolic alkalosis was the most common acid-base imbalance [48 ]. The most common electrolyte disorders were hypernatremia in patients with serum creatinine <1.2 mg/dL, and hyponatremia and hyperkalemia in patients with higher creatinine levels (>3.1 mg/dL). 2 Sodium and Potassium in Health and Disease 45

Renal diseases with changes in urine output are another obvious reason for potassium disbalance. Patients with acute renal failure present with anorexia, nausea, vomiting, lethargy, and increased blood pressure [28 ]. The onset of oliguria is sudden; proteinuria and hematuria are common. There is a progressive increase in serum urea nitrogen, creatinine, potassium, phosphate, and sulfate. In contrast, serum sodium, calcium, and bicarbonate are decreased. The etiology for inducing acute renal failure is numerous and the disease is classically divided into pre-renal, renal (intrinsic), and post-renal failure. Multiple animal models have been developed to induce acute renal failure by different mechanisms [ 49]. These laboratory studies contribute to a better understanding of the disease. In chronic kidney disease, the changes develop at a slower rate. Therefore, the organism has time to compensate for partial loss of function. For example, uremia and azotemia occur only when renal failure is advanced; usually when the creatinine clearance decreases to about 30–40% of normal [14 ]. The inability to concentrate urine, resulting in polyuria, is one of the early signs of chronic kidney failure. Metabolic acidosis is caused by the failure of hydrogen ion excretion. Hyperkalemia is one of the later signs of the disease; so is the development of secondary hyperparathyroidism and renal osteodystrophy. When pre-dialysis mortality was studied in a large cohort of men (N = 1,227), both hypo- and hyperkalemia were linked to mortality in white patients [50 ]. Black patients seemed to better toler- ate hyperkalemia than whites. Hypokalemia was associated with faster chronic kidney disease progression in both races.

5 Conclusion

Sodium and potassium are essential to life. These ions are involved in many physiological processes, and their imbalance may impair proper function in various organs and/or entire systems in the body. It is beyond the scope of this chapter to describe in detail all the diseases. The interested reader is encouraged to ﬁ nd more information in the medical texts and scientiﬁ c papers cited here.

Abbreviations

ADH antidiuretic hormone ATP adenosine 5′-triphosphate AVP arginine vasopressin DDT dichlorodiphenyltrichloroethane ECG electrocardiogram GI gastrointestinal RAS renin-angiotensin system SHH syndrome of hyporeninemic hypoaldosteronism SIADH syndrome of inappropriate antidiuretic hormone 46 Pohl, Wheeler, and Murray

References

1. N. W. Tietz, Fundamentals of Clinical Chemistry, 3rd ed, W. B. Saunders, Philadelphia, 1987, 1010 p. 2. W. Ganong, Review of Medical Physiology , Lang Medical Books, Norwalk, Connecticut, 1991. 3. J. C. Skou, Biochim. Biophys. Acta 1957 , 23 , 394–401. 4. J. W. Hole Jr., Human Anatomy and Physiology, Wm. C. Brown Company Publishers, Dubuque, Iowa, 1978, 814. 5. M. W. Barnett, P.M. Larkman, Pract. Neurol . 2007 , 7 , 192–197. 6. H. P. Vijverberg, J.M. van der Zalm, J. van der Bercken, Nature 1982 , 295 , 601–603. 7. J. L. Lewis, III., Water and Sodium Balance, in The Merk Manual for Health Care Professionals , Eds R. S. Porter, J. L. Kaplan, Merck Sharp & Dohme Corp., Whitehouse Station, NJ, 2012, 1. http://www.merckmanuals.com/professional/endocrine_and_metabolic_disorders/fluid_ metabolism/water_and_sodium_balance.html . 8. A. C. Guyton, J. E. Hall, Textbook of Medical Physiology, 10th ed, Saunders, Philadelphia, xxxii, 2000, 1064 p. 9. H. Karppanen, P. Karppanen, E. Mervaala, J. Hum. Hypertens. 2005, 19 , S10–S19. 10. L. R. Johnson, Gastrointestinal Physiology , 7th ed, The Mosby physiology monograph series, Mosby Elsevier, Philadelphia, 2007, xii, 160 p. 11. G. Giebisch, G. Malnic, R. W. Berliner, Control of Renal Potassium Excretion , in The Kidney , Eds B. Brenner, F. Rector, W. B. Saunders Co., Philadelphia, 1996, p. 371–407. 12. S. D. Crowley, S. B. Gurley, M. J. Herrera, P. Ruiz, R. Grifﬁ ths, A. P. Kumar, H. S. Kim, O. Smithies, T. H. Le, and T. M. Coffman, Proc. Natl. Acad. Sci. USA 2006 , 103 , 17985–17990. 13. R. Berkow, A. J. Fletcher, Eds, The Merk Manual of Diagnosis and Therapy, 16th ed. Merk Research Laboratories, Rahway, NJ, 1992, 14. P. T. Chandrasoma, C. R. Taylor, Concise Pathology, Appleton and Lange Publishers, Norwalk, Connecticut, 1991. 15. R. C. Hawkins, Clin. Chim. Acta 2003 , 337 , 169–172. 16. F. C. Bartter, W. B. Schwartz, Am. J. Med. 1967 , 42 , 790–806. 17. P. H. Baylis, Int. J. Biochem. Cell Biol. 2003 , 35 , 1495–1499. 18. H. M. Siragy, Endocr. Pract. 2006 , 12 , 446–457. 19. J. K. Hix, S. Silver, R.H. Sterns, Semin. Nephrol. 2011 , 31 , 553–566. 20. D. Wile, Ann. Clin. Biochem. 2012 , 49 , 419–431. 21. M. Carmosino, G. Procino, M. Svelto, Biol. Cell 2012 , 104 , 201–212. 22. G. S. Shah, B. K. Das, S. Kumar, M. K. Singh, G. P. Bhandari, Kathmandu Univ. Med. J . (KUMJ ) 2007 , 5 , 60–62. 23. F. A. Odey, I. S. Etuk, M. H. Etukudoh, M. M. Meremikwu, Niger Postgrad. Med. J . 2010 , 17 , 19–22. 24. G. Uysal, A. Sokmen, S. Vidinlisan, Indian J. Pediatr. 2000 , 67 , 329–333. 25. N. El-Sherif, G. Turitto, Cardiol. J . 2011 , 18 , 233–245. 26. J. Pepin, C. Shields, Emerg. Med. Pract . 2012 , 14 , 1–17; quiz 17–18. 27. R. J. Unwin, F. C. Luft, D. G. Shirley, Nat. Rev. Nephrol. 2011 , 7 , 75–84. 28. M. Krupp, Fluid and Electrolyte Disorders, in Current Medical Diagnosis and Treatment , Eds S. A. Schroeder, M. A. Krupp, L. M. Tierney, Jr., S. J. McPhee, Appleton and Lange Publishers, Norwalk, Connecticut, 1990, p. 593–594. 29. N. W. Brown, South Med. J. 1985 , 78 , 403–405. 30. A. P. Winston, Ann. Clin. Biochem. 2012 , 49 , 132–143. 31. J. J. Weinstein, N. Engl. J. Med. 2003 , 349 , 2363–2364; author reply 2363–2364. 32. E. M. Abdel-Rahman, A.V. Moorthy, Clin. Nephrol. 1997 , 47 , 106–11. 33. Y. Arimura, H. Tanaka, T. Yoshida, M. Shinozaki, T. Yanagida, T. Ando, H. Hirakata, M. Fujishima, Nephrol. Dial. Transplant 1999 , 14 , 957–959. 34. B. L. Nichols, G. A. Alleyne, D. J. Barnes, C. D. Hazlewood, J. Pediatr. 1969 , 74 , 49–57. 2 Sodium and Potassium in Health and Disease 47

35. G. A. Alleyne, D. J. Millward, G. H. Scullard, J. Pediatr . 1970 , 76 , 75–81. 36. B. L. Nichols, J. Alvarado, C. F. Hazlewood, F. Viteri, J. Pediatr . 1972 , 80 , 319–330. 37. K. E. Rinehart, W. R. Featherston, J. C. Rogler, J. Nutr. 1968 , 95 , 627–632. 38. I. Dorup, T. Clausen, Br. J. Nutr. 1989 , 62 , 269–284. 39. J. Fuentebella, J. A. Kerner, Pediatr. Clin. North Am . 2009 , 56 , 1201–1210. 40. M. Schulman, R. G. Narins, Am. J. Cardiol. 1990 , 65 , 4E–9E; discussion 22E–23E. 41. R. Chatterjee, H. C. Yeh, D. Edelman, F. Brancati, Expert Rev. Endocrinol. Metab . 2011 , 6 , 665–672. 42. A. Bielecka-Dabrowa, D. P. Mikhailidis, L. Jones, J. Rysz, W. S. Aronow, M. Banach, Int. J. Cardiol . 2012 , 158 , 12–17. 43. S. G. Coca, M. A. Perazella, G. K. Buller, Am. J. Kidney Dis. 2005 , 45 , 233–247. 44. H. Ashraﬁ an, M. P. Frenneaux, L. H. Opie, Circulation 2007 , 116 , 434–448. 45. R. S. Bhatia, J. V. Tu, D. S. Lee, P. C. Austin, J. Fang, A. Haouzi, Y. Gong, P. P. Liu, N. Engl. J. Med. 2006 , 355 , 260–269. 46. C. C. Lang, D. M. Mancini, Heart 2007 , 93 , 665–671. 47. M. R. Weir, M. Rolfe, Clin. J. Am. Soc. Nephrol . 2010 , 5 , 531–548. 48. N. Sotirakopoulos, I. Kalogiannidou, M. Tersi, K. Armentzioiou, D. Sivridis, K. Mavromatidis, Saudi J. Kidney Dis. Transpl . 2012 , 23 , 58–62. 49. A. P. Singh, A. Junemann, A. Muthuraman, A. S. Jaggi, N. Singh, K. Grover, R. Dhawan, Pharmacol. Rep. 2012 , 64 , 31–44. 50. J. Hayes, K. Kalantar-Zadeh, J. L. Lu, S. Turban, J. E. Anderson, C. P. Kovesdy, Nephron Clin. Pract . 2012 , 120 , c8–16. Chapter 3 Magnesium in Health and Disease

Andrea M. P. Romani

Contents ABSTRACT ...... 50 1 INTRODUCTION ...... 50 1.1 Distribution of Magnesium in the Human Body...... 50 1.2 Intestinal Magnesium Absorption and Release into the Blood...... 51 1.2.1 Apical Side ...... 51 1.2.2 Cellular Transport ...... 52 1.2.3 Basolateral Side ...... 53 1.3 Renal Magnesium Handling and Reabsorption ...... 53 2 CELLULAR MAGNESIUM HOMEOSTASIS ...... 54 2.1 Cellular Magnesium Transport Mechanisms ...... 55 2.2 Regulation of Magnesium Transport ...... 55 3 MAGNESIUM IN DISEASE ...... 55 3.1 Hypermagnesemia ...... 56 3.1.1 Hypermagnesemia in Renal Failure ...... 57 3.2 Hypomagnesemia...... 57 3.2.1 Cardiovascular Pathologies ...... 59 3.2.2 Hyperaldosteronism ...... 62 3.2.3 Diabetes...... 62 3.2.4 Metabolic Syndrome ...... 64 3.2.5 Alcoholism ...... 65 3.2.6 Inﬂ ammation ...... 66 3.2.7 Renal Pathologies ...... 67 3.2.8 Magnesium and Tumors ...... 69 3.2.9 Magnesium and Prenatal Pathologies ...... 70

A. M. P. Romani (*) Department of Physiology and Biophysics, School of Medicine , Case Western Reserve University , 10900 Euclid Avenue , Cleveland , OH 44106-4970 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 49 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_3, © Springer Science+Business Media Dordrecht 2013 50 Romani

3.3 Pharmacological Agents Causing Hypomagnesemia ...... 71 3.3.1 Proton Pump Inhibitors ...... 72 3.3.2 Anti-epidermal Growth Factor Receptor Antibodies ...... 72 4 CONCLUSIONS ...... 73 ABBREVIATIONS ...... 74 ACKNOWLEDGEMENTS ...... 75 REFERENCES ...... 75

Abstract Mammalian cells tightly regulate cellular Mg2+ content through a variety of transport and buffering mechanisms under the control of various hormones and cellular second messengers. The effect of these hormones and agents results in dynamic changes in the total content of Mg2+ being transported across the cell membrane and redistributed within cellular compartments. The importance of maintaining proper cellular Mg2+ content optimal for the activity of various cellular enzymes and metabolic cycles is underscored by the evidence that several diseases are characterized by a loss of Mg2+ within speciﬁ c tissues as a result of defective transport, hormonal stimulation, or metabolic impairment. This chapter will review the key mechanisms regulating cellular Mg2+ homeostasis and their impairments under the most common diseases associated with Mg2+ loss or deﬁ ciency.

Keywords alcoholism • cancer • cell cycle • diabetes • homeostasis • hormones • hypertension • inﬂ ammation • insulin • metastases • Mg2+ • Mg2+ transport

Please cite as: Met. Ions Life Sci. 13 (2013) 49–79

1 Introduction

Magnesium is the 4th most abundant element in the human body and the 2nd most abundant cation within human cells after potassium. The human body contains about 760 mg of magnesium at birth. This amount increases to 5 g at age 4–5 months and to 25 g at adulthood [1 ]. How this increase is regulated or stimulated is presently unknown. In the following lines we will provide a general picture of how Mg2+ is absorbed at the intestinal level, and the role of the kidney in controlling urinary Mg2+ loss.

1.1 Distribution of Magnesium in the Human Body

Approximately 60% of whole body magnesium is found in bones, 30% to 40% in skeletal muscles and soft tissues, and 1% in the extracellular fl uid [2 ]. In bones, magnesium is mainly distributed along the Havers’s channels, where it contributes to form hydroxyapatite crystals [3 ]. In net terms, this magnesium accounts to about 1% of bone ash [2 ,3 ]. At an early stage, most of this magnesium can readily exchange 3 Magnesium in Health and Disease 51 with serum, representing an optimal store to compensate occasional dietary defi ciency. As age progresses the proportion of readily exchangeable magnesium in the bone decreases signifi cantly. In individuals consuming magnesium-enriched diet a positive association between bone mineral density and magnesium content within the erythrocytes has been reported [4 ]. Not much is known about the role of magnesium within skeletal muscles other than that it controls ATP content and utilization for contraction purposes and reticular Ca2+ uptake and release [5 ]. In soft tissue, magnesium acts as a cofactor of many enzymes involved in energy metabolism, protein synthesis, and RNA and DNA synthesis [ 6]. It also plays a major role in the maintenance of the electrical potential of nervous tissue and cell membranes.

1.2 Intestinal Magnesium Absorption and Release into the Blood

Diet and water are the main sources of magnesium intake. The recommended daily dose of Mg 2+ is ~300 mg for men and 250 mg for women [ 7] unless pregnant, in which case an increase to ~350 mg is suggested. These doses correspond to the amount of Mg2+ eliminated daily through the urinary and digestive systems [7 ]. Dietary Mg2+ is absorbed at the apical side of intestinal epithelial cells and transported throughout the cell to be released into the blood at the basolateral side of the cell [7 ].

1.2.1 Apical Side

Limited information is available about the modality by which Mg2+ is absorbed from the intestinal lumen. The operation of specifi c and saturable Mg2+ accumulation mechanisms has been observed in brush border cells of the ileum [8 ] but also duodenum and jejunum [9 , 10 ]. More recently, attention has been paid to the distribution and operation of apical Mg2+ channels, namely TRPM6 [11 ] and TRPM7 [12 ]. They are members of the melastatin subfamily of t ransient r eceptor p otential (TRP) channels [13 ]. TRPM6 is specifi cally located in the colon and in the distal convolute tubule of the nephron [11 ]. TRPM7 is ubiquitously expressed in the majority of mammalian cells [71 ], including the various segments of the small intestine [14 ]. The specifi c modalities of operation and regulation of these channels have been amply discussed in several reviews [15 – 18 ], and will not be discussed here. For the purpose of this review, we will only mention that both TRPM6 and TRPM7 operate as tetramers, and present a α-kinase domain at their C-terminus, which phosphorylates serine and threonine residues within an α-helix structure [19 – 21 ]. Presently, only annexin I [ 22 ], myosin IIA heavy chain [23 ,24 ], and calpain [25 ] have been clearly identifi ed as phosphorylation substrates for the TRPM7 kinase domain while the best known target for TRPM6 kinase domain is TRPM7 itself [26 ]. 52 Romani

Aside from the speciﬁ c location, the most striking difference between the two channels is that TRPM6 but not TRPM7 expression and activity are modulated by diet and estrogens. Estrogens (17β-estradiol) markedly upregulate TRPM6 mRNA in both colon and kidney while having no effect on TRPM7 mRNA [ 27 , 28 ]. In the absence of estrogens, the r epressor of e strogen receptor a ctivity (REA) binds to the 6th, 7th and 8th β-sheets of TRPM6 kinase domain in a phosphorylation-dependent manner and inhibits TRPM6 activity [27 ]. Short-term estrogen administration dis- sociates the binding between REA and TRPM6, resulting in increased channel activity [27 ]. Dietary Mg 2+ restriction increases TRPM6 mRNA expression both in the kidney and the colon [28 , 29 ], whereas Mg2+ enriched diet upregulates TRPM6 mRNA expression only in the colon, increasing intestinal absorption [28 ]. In contrast, neither dietary Mg2+ manipulation affects TRPM7 mRNA expression in the two organs [28 , 29 ]. Thus, evidence is there that genetic factors and variation in dietary Mg 2+ content control TRPM6 expression and activity in the large intestine to favor Mg 2+ absorption, while renal Mg2+ resorption only occurs following dietary Mg2+ restriction [28 , 29 ]. TRPM6 channel activity is also modulated by RACK1 (r eceptor for a ctivated protein k inase C ), which binds directly to the α-kinase domain of TRPM6, and possibly TRPM7 due to the high homology (>84%) between the two kinase domains [30 ]. RACK1 binds the same β-sheets involved in REA regulation [27 ], and inhibits the channel activity of TRPM6 and TRPM7. Activation of protein kinase C (PKC), the natural ligand for RACK1, completely prevents the inhibitory effect of RACK1 on TRPM6 channel activity [30 ], suggesting a competition of PKC for TRPM6 towards RACK1.

1.2.2 Cellular Transport

At front of a total cellular Mg2+ concentration of 15 to 20 mM [ 31 – 33 ], cytoplasmic free Mg2+ concentration accounts for ~0.5 to 1 mM [31 – 33], suggesting that as Mg2+ enters the cell, it is rapidly buffered by ATP, phosphonucleotides, and proteins. It is hypothesized that cytoplasmic proteins can bind Mg2+ and contribute to its buffering within this cellular compartment while transporting it to the basolateral side for dismissal into the circulation. Aside from calmodulin [34 ], troponin C [ 35 ], parvalbumin [36 ], and S100 protein [37 ], for which a Mg2+ binding consensus sequence has been reported, the number or nature of Mg2+ binding proteins remains elusive. Parvalbumin and calbindin-D28k , two proteins abundantly present within intestinal and renal cells, have been indicated as transcellular transporters of the Mg2+ accumulated at the apical domain of the cell, accelerating its delivery rate to the basolateral domain for dismissal. In intestinal cells, these proteins contribute to Ca2+ binding and transport upon vitamin D stimulation. Whether these proteins operate in a similar manner for Mg 2+ in the intestinal epithelium is presently unde- ﬁ ned. The physiological relevance of Mg2+ binding by cytoplasmic proteins and their ability to transport Mg 2+ from the apical to the basolateral side of intestinal and renal cells is highly questioned since parvalbumin null mice do not exhibit hypomagnesemia or signiﬁ cant changes in tissue Mg2+ handling and homeostasis [38 ]. 3 Magnesium in Health and Disease 53

A variable percentage of the Mg2+ present in the diet is not absorbed and is eliminated through the intestine. This percentage varies based upon the diet composition and the complex form in which magnesium is present in the diet and its solubility. Magnesium sulfate, magnesium hydroxide, magnesium chloride, magnesium oxide, magnesium oxalate, magnesium gluconate, and magnesium citrate are among the most common forms of magnesium salts present in the diet, or available in multi-vitamin and multi-mineral dietary supplements [ 39]. Each of these compounds is characterized by different solubility and intestinal absorption rate, varying from very little solubility for magnesium oxide to good solubility for magnesium citrate [ 39 ].

1.2.3 Basolateral Side

Once delivered to the basolateral domain of the intestinal cell, Mg2+ is extruded into the bloodstream through a Na+ -dependent Mg2+ extrusion mechanism termed Na+ / Mg2+ exchanger. The ﬁ rst evidence for the operation of such a mechanism was provided by Gunther, Vormann and Forster in 1984 [40 ]. In two sequential studies [40 , 41 ], these authors detailed how this Na+ /Mg2+ exchanger operates, and its inhibition by amiloride. Several other groups have conﬁ rmed the presence and operation of this extrusion mechanism in various mammalian cell types (see [42 ] for a list). The current consensus is that this Mg2+ extrusion mechanism becomes active upon phosphorylation by cAMP, and operates as an antiporter, strictly requiring a physiological concentration of extracellular Na+ to be fully operative [43 ]. Under conditions in which a less than optimal concentration of extracellular Na + is available, Mg 2+ can be extruded from the cell into the bloodstream through the operation of a subsidiary, and not fully characterized Na+ -independent Mg2+ extrusion mechanism [ 44 ], which appears to utilize both anions and cations to promote Mg 2+ transport (reviewed in [16 ]).

1.3 Renal Magnesium Handling and Reabsorption

Upon dismissal into the blood stream, about one third of serum Mg2+ circulates bound to proteins (mainly albumin), or in a complex with anions [45 ], whereas the remaining two thirds (~0.7 mM) is in the free form. This serum Mg2+ concentration is in equilibrium with the concentration in the extracellular space, and both are just slightly higher than the free [Mg2+ ] in the cell cytoplasm. As a result of this distribution, the majority of mammalian cells is at, or near a zero trans condition in terms of magnesium concentration across the cell membrane. Serum magnesium undergoes renal glomerular ﬁ ltration as other serum cations. Approximately 25%–30% of ﬁ ltered Mg2+ is reabsorbed in the proximal tubule, and ~65% is reabsorbed in the thick ascending limb of the Henle’s loop [46 ]. It is in this segment of the nephron that various hormones (vasopressin, PTH, etc.) and drugs 54 Romani

(cyclosporine, cisplatin, gentamycin, etc.) operate to increase or decrease Mg2+ reabsorption [46 ]. The increase in reabsorption occurs through paracellular and transcellular Mg2+ transport mechanisms: passive paracellular transport via claudins favors bulk Mg2+ absorption while active transcellular transport mechanisms mediate the ﬁ ne-tuning of Mg 2+ absorption. Claudin 16 (originally known as paracellin-1 [47 ]) and claudin 19 [48 ] form the tight junction component through which bulk Mg 2+ absorption occurs. This pathway is controlled by the CaSR (calcium sensing receptor [49 ]) via negative feedback of the PKA/cAMP signaling [50 ], but it is also inﬂ uenced by the proper expression of the EGF receptor (EGFR), with defects in either signaling pathway resulting in increased Mg2+ loss in the urine [51 , 52]. The involvement of the CaSR in Mg2+ reabsorption is also supported by the evidence that this sensor in the basolateral membrane of renal epithelial cells is activated by equivalent concentrations of Ca2+ and Mg 2+ [ 53 ]. Activation of the CaSR inhibits apical K + channels and Na+ /K + /Cl – co-transporter activity [54 ], overall reducing lumen positive potential and paracellular transport of divalent cations. The hormone-mediated increase in Mg 2+ reabsorption and cellular Mg 2+ content ultimately results in an enhanced operation rate of the basolateral, cAMP-modulated Na+ /Mg 2+ exchanger previously described. In contrast, exposure to cyclosporine, gentamycin, cisplatin, or other drugs will decrease the activity of paracellular and transcellular Mg2+ transport mechanisms, ultimately reducing the operation of the Na+ /Mg 2+ exchanger. The residual 5%–10% Mg 2+ reabsorption occurs in the distal convolute tubule [ 46 ]. It is in this segment of the nephron that TRPM6 would play a major role in enhancing Mg2+ accumulation from the lumen into the cells and ultimately into the bloodstream, and where major changes in TRPM6 expression have been observed as a result of variations in dietary Mg 2+ content [ 28 ,29 ]. Interestingly, EGF also controls TRPM6 expression through Erk1/2 and AP-1 [54 , 55 ]. Because of the key role of the renal apparatus in controlling Mg 2+ reabsorption, it is not surprising that several genetic and iatrogenic diseases primarily impair renal Mg2+ reabsorption (see below).

2 Cellular Magnesium Homeostasis

Total cellular Mg2+ content ranges between 15 to 18 mM, well below the concentration predicted by the Nernst equation (~55 mM), whereas cytosolic free Mg 2+ concentration (0.5–0.8 mM) is slightly below the concentration present in the extracellular environment [56 , 57 ]. Within the cell, Mg 2+ is distributed within cytoplasm and cellular organelles. In the cytoplasm, more than 95% of Mg 2+ located therein is in the form of a complex with ATP and phosphonucleotides [58 , 59 ]. As for the organelles, Mg2+ is abundantly localized within nucleus, mitochondria, and endoplasmic reticulum [56 ], in which it regulates the activity of numerous enzymes, channels, and genes, directly and indirectly controlling metabolic and bioenergetics processes [56 ]. This well deﬁ ned distribution points to a tightly regulated cellular Mg2+ homeostasis through a combination of transport and chelating mechanisms. 3 Magnesium in Health and Disease 55

2.1 Cellular Magnesium Transport Mechanisms

Our current understanding of Mg2+ transport across the cell membrane indicates that Mg 2+ exits the cell via an exchange mechanism, tentatively identiﬁ ed as Na+ /Mg2+ exchanger [60 , 61] based upon its strict functional dependence on physiological extracellular Na+ concentration [56 , 60], and via an alternative pathway termed Na+ - independent extrusion mechanism, which appears to utilize different cations and anions in the process (reviewed in [56 ] and [62 ]). Entry of Mg2+ into the cell occurs through channels or electrogenic transporters. Several Mg2+ entry mechanisms have been identiﬁ ed. Yet, it remains still unclear to which extent these mechanisms cooperate in mediating Mg2+ entry, or whether Mg2+ accumulation primarily occurs through a pre-dominant mechanism, perhaps different in diverse cells.

2.2 Regulation of Magnesium Transport

Several exhaustive review articles have addressed the specifi c modality of operation and regulation of the Mg 2+ transport mechanisms [56 , 62 – 66 ], and we refer to those reviews for further information. For the purpose of this chapter, we will only mention that Mg2+ entry and extrusion is under hormonal control. Hormones that increase cellular cAMP level (e.g., catecholamine, glucagon, PGE2, etc.) all promote Mg2+ extrusion primarily via the Na + /Mg2+ exchanger [16 ]. Conversely, hormones (insulin, vasopressin, etc.) that decrease/prevent cAMP production or activate protein kinase C signaling, all favor Mg 2+ accumulation primarily via TRPM6 or TRPM7 [16 ]. In the case of Mg2+ entry, the involvement of Erk1/2 and associated signaling components has been observed or postulated [67 , 68 ]. Both Mg2+ extrusion and Mg2+ accumulation are quantitatively and timely limited processes [69 , 70], implying the movement of Mg2+ from and to specifi c cellular compartments. Cytoplasm is but one of the cellular compartments involved in Mg2+ transport out of the cell or into the cells [ 71 , 72 ], other compartments being mitochondria and the endo-sarco-plasmic reticulum [71 , 72 ]. The mechanisms involved in Mg 2+ transport in and out of these compartments, however, are not yet fully elucidated. In the case of the cytoplasm, evidence is there that pathological conditions that decrease cellular ATP content through dysmetabolic processes [73 – 75 ] ultimately cause cellular Mg2+ loss or defi ciency.

3 Magnesium in Disease

Both hyper- and hypomagnesemia occur in human patients. Hypermagnesemia is less common than hypomagnesemia and is mostly iatrogenic (i.e., medically induced) in nature. Hypomagnesemia, on the other hand, can result from different 56 Romani causes but because in its initial phase it is associated with vague and non-specifi c symptoms, it often goes undetected in the large population until an individual checks in a hospital or a medical facility for another pathological condition. This association raises the question as to which extent hypomagnesemia is connected in a cause-effect relation to the concurrent disease. The following pages will address the medical concept and the main pathological causes of hyper- and hypo-magnesemia. In addition, because several of the most common human pathologies frequently present hypomagnesemia as an associate condition, efforts will be made to provide a better understanding of the possible cause-effect relation between hypomagnesemia or magnesium defi ciency and the onset of a specifi c disease and/or its main complications.

3.1 Hypermagnesemia

Hypermagnesemia is defi ned as an abnormally elevated Mg 2+ level in the blood [ 76]. Usually, it is the result of an excess of magnesium in the body. Whole body Mg 2+ homeostasis is the result of equilibrium between absorption (intestine), storage (bones), and excretion (kidneys). Hence, hypermagnesemia is usually the result of diseases in any of these compartments. Because the kidneys are very effective at excreting excess Mg2+ , hypermagnesemia occurs rarely and is mostly observed when renal creatinine clearance falls below 30 mL urine per minute. Thus, hypermagnesemia develops almost exclusively in patients with kidney failure who are given magnesium salts or Mg2+ -containing drugs such as laxatives or antacids, and it is usually concurrent with hypocalcemia and/or hyperkalemia [ 76]. Main symptoms are: impaired breathing, hypotension, decreased or absent deep tendon refl exes, muscle weakness, arrhythmia, and bradycardia as a result of abnormal electrical conduction at the nervous, muscular, and cardiac level. High serum Mg2+ concentrations are associated with nausea and vomiting, in the attempt to renormalize the electrolyte level. Because some of these symptoms occur following hypocalcemia and/or hyperkalemia onset, it is diffi cult to determine to which extent either of these two conditions contributes to the appearance of the symptoms. Symptoms usually worsen based upon the serum Mg2+ concentration: hyporefl exia is present at serum Mg2+ concentrations ≥4.0 mEq/L, prolonged atrioventricular conduction occurs at a concentration ≥5.0 mEq/L while heart block and cardiac arrest occur at concentrations between ≥10 and 13 mEq/L. One condition in which high levels of serum Mg2+ are usually attained as a result of therapeutic approach is pre-eclampsia. In this clinical condition, prevention of pre-eclampic uterine contractions usually requires concentrations between 4.0 and 7.0 mEq/L (≥2.5 and 4.5 mM, respectively) [ 77 ]. Serum Mg 2+ concentrations at which maternal toxicity but also neonate depression, hypotonia, and low Apgar scores are observed are usually in excess of 7.0 mEq/L [77 ]. Loss of patella refl ex occurs between 7.0 and 10 mEq/L; respiratory depression between 10.0 and 13.0 3 Magnesium in Health and Disease 57 mEq/L: altered atrioventricular conduction and heart block at 15.0 and 25.0 mEq/L, and cardiac arrest at >25mEq/L.

3.1.1 Hypermagnesemia in Renal Failure

Clinical evaluation of a cohort of patients on hemodialysis indicates that serum Mg2+ concentration lower than 2.77 mg/dL (1.14 mmol/L) is a signifi cant predictor for increased all-cause mortality. This mean serum Mg 2+ concentration would be considered indicative of mild hypermagnesemia in the healthy population. Hence, it appears that a serum Mg2+ concentration higher than normal in hemodialysis patients will be largely asymptomatic because of better survival of these patients under hemodialysis conditions [78 ]. Consistent with this interpretation, serum Mg2+ levels lower than 1.14 mmol/L appear to be signifi cantly associated with the presence of vascular calcifi cations of the hand arteries in the absence of other concauses. Overall, these results suggest that higher than normal serum Mg 2+ concentrations may play an important protective role in the development of vascular calcifi cation in hemodialysis patients [78 ]. Results from a longitudinal study in end-stage renal disease patients suggest that hypermagnesemia may retard the development of arterial calcifi cations in this pathological state [79 ]. Signifi cantly lower values of carotid intima-media thickness and aortic pulse wave velocity values, two surrogate markers for vascular calcifi cation, have been observed in patients affected by chronic kidney disease (CKD) and presenting high serum magnesium levels (0.90–1.32 mmol/L, or 2.18–3.21 mg/dL), indicating a lower arteriosclerotic burden associated with a lower risk of cardiovascular events and mortality [80 ]. Consequently, CKD patients with mildly elevated serum Mg2+ levels could have a survival advantage over those with lower magnesium levels [80 ]. Aside from renal insuffi ciency/failure, conditions predisposing to hypermagnesemia have been identifi ed with hemolysis, lithium intoxication, adrenal insuffi - ciency, and hyperparathyroidism. Yet, a full understanding of the mechanisms whereby these clinical conditions predispose to hypermagnesemia is yet lacking.

3.2 Hypomagnesemia

Hypomagnesemia (or hypomagnesaemia) is an electrolyte disturbance characterized by an abnormally low level of magnesium in the blood [81 ]. Normal serum Mg2+ levels in humans ranges between 1.5–2.5 mg/dL (or 1.0–1.2mmol/L) [ 45 ]. When the serum Mg2+ level is lower than 0.7 mmol/L, we refer to the condition as hypomagnesemia. This term strictly refers to the Mg2+ level in the serum, and is not and should not be equated to magnesium defi ciency, although the two conditions can be related in specifi c patients. Magnesium defi ciency refers to an intake of dietary magnesium below minimal levels, and can result in numerous symptoms 58 Romani and diseases. The majority of the symptoms and conditions can generally be remedied by increasing Mg2+ in the diet or via oral supplements. In the most severe cases, intravenous Mg2+ supplementation is necessary to rapidly restore Mg2+ level within tissues and serum. Although hypomagnesemia is usually indicative of a systemic magnesium defi cit, hypomagnesemia can be present without Mg2+ defi ciency, and vice versa . Hence, three distinct conditions can be observed: (a) Hypomagnesemia without magnesium defi ciency (b) Hypomagnesemia with magnesium defi ciency (c) Magnesium defi ciency without hypomagnesemia Hypomagnesemia may result from a number of conditions including inadequate intake of magnesium, chronic diarrhea, malabsorption, chronic stress, alcoholism, and (ab)use of medications such as diuretics or antacids of the proton pump inhibitor family (e.g., omeprazole and similar). The most common signs and symptoms of hypomagnesemia are: muscle weakness, muscle cramps, cardiac arrhythmia, increased irritability of the nervous system, with tremors, athetosis, jerking, nystagmus, and extensor plantar refl ex. Additionally, confusion, disorientation, hallucination, depression, epileptic fi ts, hypertension, tachycardia, and tetany may be present in a signifi cant percentage of cases. Usually, symptoms are bland or not existent when hypomagnesemia is between 0.5 and 0.7 mmol/L, to become more apparent and severe when magnesemia falls below 0.5 mmol/L [82 ]. Magnesium defi ciency is not uncommon in hospitalized patients. Ten to twenty percent of all hospitalized patients and 60–65% of patient in intensive care units (ICU) have hypomagnesemia. The condition is usually under-diagnosed because (i) testing for serum magnesium levels is not routine, and (ii) not always lower cellular Mg2+ content correlates with low serum Mg 2+ level. Low levels of Mg 2+ in blood may be the result of low Mg2+ content in the diet, defective Mg2+ absorption in the intestines, or increased Mg2+ excretion by the kidneys. Magnesium defi ciency and hypomagnesemia is often observed in acute myocardial infarction, usually within the fi rst 48 hours after a heart attack, or as the result of drug and medication intake, or gastrointestinal and renal causes.

Drugs : Alcohol intake is one of the primary causes of hypomagnesemia. Hypo- magnesemia occurs in 30% of patients with alcohol abuse and 85% with delirium tremens, due to malnutrition, chronic diarrhea, and direct effect of alcohol on liver, muscle tissues, and neurons. Alcohol also stimulates renal Mg2+ excretion, which is also increased because of ketoacidosis, hypophosphatemia, and hyperaldosteronism resulting from liver disease. Hypomagnesemia is also observed in severe cases of thiamine deﬁ ciency because magnesium is required to transform thiamine into thiamine pyrophosphate.

2+ Medications : Loop and thiazide diuretics; antibiotics that block Mg resorption in the loop of Henle (i.e., aminoglycoside, gentamicin, tobramycin, amphotericin, pentamidine, viomycin); proton pump inhibitors (i.e., omeprazole); digitalis; catecholamine and adrenergic agonist; cisplatin and cyclosporin, which both stimulate renal excretion; and insufﬁ ciency in selenium, vitamin D, and vitamin B6. 3 Magnesium in Health and Disease 59

Gastrointestinal causes : The distal portion of the digestive tract secretes high levels of magnesium. Thus, hypomagnesemia can occur as the result of secretory diarrhea in Crohn’s disease, ulcerative colitis, Whipple’s disease, and celiac sprue. Magnesium loss also occur in cases of malabsorption and acute pancreatitis.

Renal causes : Renal magnesium loss is observed in Gitelman/Bartter’s syndrome, postobstructive diuresis, diuretic phase of acute tubular necrosis, and in kidney transplant. Massive urinary Mg2+ loss is observed in ~40% of diabetic patients, most likely as the result of glycosuria and ketoaciduria. The following pages present an overview of the most common pathologies associated with low Mg2+ content within tissues or in the circulation. Where possible, an indication of the role of hypomagnesemia or low cellular Mg2+ content for the onset of the main pathology or its complications will be provided.

3.2.1 Cardiovascular Pathologies

Reduced serum Mg2+ content has often been observed in several cardiac and cardiovascular pathologies including acute myocardial infarction, speciﬁ c forms of arrhythmias, and hypertension. Because the association is usually observed a poste- riori, at the time the patient seeks medical attention for the concurrent cardiovascular pathology, it is difﬁ cult to determine whether reduced Mg2+ content in the blood, and perhaps within the affected tissue, is an epiphenomenon or has any causal connection with the onset of the disease or its manifestation.

3.2.1.1 Cardiac Arrhythmias

The effects of low Mg2+ levels on cardiac rhythm have been studied for more than 70 years. Magnesium plays an essential role in maintaining normal cardiac electro- physiology, mostly by acting as a natural Ca2+ channel blocker or as an antagonist for Na +, thus limiting the cellular content of this cation. Consequently, it is hypothesized that inadequate serum and tissue Mg2+ concentrations contribute to the onset of various cardiac arrhythmias. Among these, we can list ventricular tachycardia (VT), ventricular fi brillation (VF), long QT and torsades de pointes, as well as atrial and ventricular extra systoles or premature beats, all conditions predisposing to sudden cardiac death. Magnesium defi cit is also observed in the setting of congestive heart failure (CHF), which affects more than 5 million people just in the US, and in the setting of hypertension, which affects more than 30 million Americans. In the case of CHF, approximately 600,000–700,000 new patients are diagnosed with the disease every year. These patients have a very high propensity for ventricular arrhythmias, which represent one of the prominent causes of death in the group, and are frequently linked to hypomagnesemia. In these patients, Mg 2+ defi ciency may result from elevated circulating levels of catecholamines, aldosterone, and vasopressin, and from increased urinary Mg2+ excretion consequent to diuretic and digoxin therapy. With the exception of spironolactone and other diuretics that spare 60 Romani potassium and magnesium, the treatment with diuretics of the thiazide family (the most commonly used) increases urinary Mg2+ excretion by a minimum of 25% to as much as 400% above basal level. This increased loss of Mg 2+ affects the response to digitalis therapy in CHF patients, who may eventually necessitate a dose that is twice the amount administered to CHF patients with normal serum Mg2+ level to control cardiac performance and rhythm. The concomitant administration of magnesium instead can reduce the dose of digitalis required to control the disease, therefore decreasing the risk of toxicity. Several forms of arrhythmias including ventricular tachyarrhythmias and torsades de pointes are attenuated or sedated with Mg2+ replacement or Mg2+ boluses [83 ]. Examination of the effects of pharmacological i.v. doses of Mg2+ on heart rate and rhythm suggests an inverse relationship between sudden death from arrhythmias and serum Mg2+ levels, prompting the idea that patients with low Mg2+ levels may require Mg2+ administration either orally or parenterally. Candidates for i.v. Mg 2+ treatment include patients that respond less than optimally to conventional antiarrhythmic therapy. The notion that arrhythmias precipitated by digitalis can be effectively reversed by injections of magnesium dates back to 1930 [ 84], and has been confi rmed by several other studies thereafter (reviewed in [85 ]). Similarly, several studies have found that the use of oral Mg2+ may decrease the risk of arrhythmias associated with long QT syndrome [86 ], coronary artery disease [87 ], and mitralic valve replacement [88 ]. Interestingly, patients that received an oral combination of magnesium and potassium supplementation presented signifi cant increase in the serum concentration of both cations. Hence, it would appear that due to the simplicity, cost-effectiveness, and safety of magnesium salts, such a supplementation could be a fi rst-line option for treating patients with frequent but not life- threatening ventricular tachyarrhythmias. Several trials have attempted to delineate the usefulness of Mg 2+ supplementation in other cardiovascular diseases including myocardial infarction and coronary diseases [87 ]. The results of the studies, however, have been inconsistent and inconclusive [89 ], not fully supporting the imple- mentation of Mg2+ treatment for these diseases. Whether this lack of results depends on the severity of the condition, the bioavailability of Mg2+ , or the possibility that for certain diseases Mg2+ supplementation is more preventive than curative, still remains to be elucidated.

3.2.1.2 Hypertension

Several epidemiological studies have highlighted an inverse relationship between serum Mg2+ level and hypertension, with higher blood pressure values being observed in the presence of lower Mg2+ levels [90 ]. Because serum Mg2+ level represents <1% of total Mg 2+ content, from this relationship it has been inferred that Mg 2+ is low not only in the circulation but also within tissues. In good agreement with this hypothesis, several authors have observed that hypertensive patients have reduced Mg 2+ content within erythrocytes [91 , 92 ], platelets [93 ], and other cell types. In the majority of these studies, the decrease in cellular Mg2+ content was associated with an increase in cellular Na+ and/or Ca 2+. In keeping with these results, using an in vitro system 3 Magnesium in Health and Disease 61

Altura and coworkers demonstrated that low Mg2+ causes vasoconstriction of blood vessels while increasing external Mg2+ content dilates the vessels and blocks vasoconstriction induced by epinephrine or other agents [94 ]. Further, Resnick and his group observed intracellular Mg2+ defi cit in hypertensive patients [95 ], and noted that blood pressure was inversely related to basal fasting cellular free Mg2+ level irrespective of whether the patients were hypertensive or normotensive. These results paralleled a report from a Swiss group showing that blood pressure is directly proportional to cellular free Ca2+ [96 ], along the lines that Ca2+ and Mg2+ have opposite effects within tissues and that their tissue contents are inversely related. Despite the consis- tency of these reports, evidence for a direct and clear effect of Mg2+ supplementation on blood pressure has remained largely inconclusive. Recently, Kass et al. [97 ] undertook an extensive reviewing of this topic by screening 141 relevant articles in the literature. By applying several stringent criteria, the authors narrowed down the pertinent articles to 22, which were used for their meta-analysis. These articles provided information from 12 different countries around the world, and from studies with both parallel (13) and cross-over (10) design. Although not all individual trials showed signifi cant reduction in blood pressure, the combination of all trials indicated a signifi cant decrease in both systolic (3–4 mmHg) and diastolic (2–3 mmHg) blood pressure, the effect becoming more evident in trials of cross-over design and with an intake >370 mg/day [97 ]. In addition, Mg2+ appeared to have a more pronounced blood pressure lowering effect when it was administered with normal to high potassium intake and with low sodium intake [97 ]. A similar benefi cial effect has been observed when Mg2+ has been administered with taurine, and attributed to the ability of these two agents to reduce intracellular Ca2+ and Na + levels [ 98 ]. Whatever the mechanism, patients with higher 24 h urinary levels of Mg 2+ /creatinine and taurine/creatinine presented signifi cantly lower incidence of cardiovascular risks including cerebrovascular accidents, coronary heart disease, congestive heart failure, and myocardial infarction [98 ]. Presently, not a single comprehensive hypothesis on how Mg2+ exerts its antihypertensive effect is available. An in-depth reviewing of the possible modalities of Mg 2+ action in the fi eld suggests that Mg2+ can exert its effect through different mechanisms. In addition to the mentioned ability of Mg2+ to operate as a natural Ca2+ -channel blocker, which explains the observed antithesis of free Ca2+ and Mg 2+ levels within cells and their direct and inverse relationship, respectively, with hypertension, several other possibilities are at hand. Cellular Mg2+ level has been reported 2+ 2+ to inversely relate to IP3 -mediated mobilization of reticular Ca and Ca -ATPase activity [ 99 ], and to reactive oxygen species formation [100 ], the enhancement of both processes being implicated with increased vascular tone and hypertrophic vascular remodeling. Additional effects observed at the vasculature level include increased production of nitric oxide [101 ] and prostacyclins [102 ], which promote endothelium-dependent and endothelium-independent vascular relaxation. Other possible mechanisms of action for Mg2+ include antiinfl ammatory and antioxidative effects, modulation of cell growth [103 ], and reduction of circulating LDL levels and cholesterol delivery to endothelial cells [104 ]. All these mechanisms can have direct implications for atherosclerosis onset and progression and for maintenance of proper vascular structure and function. 62 Romani

Yet, comparison of serum Mg2+ , vascular dysfunction, hypertension and atherosclerosis has failed to support a direct association among these parameters. Thus, low serum Mg 2+ level is not currently considered a risk factor for the development of these conditions [105 ]. In fact, several studies have reported no differences in serum Mg2+ level in hypertensive patients (reviewed in [106 ]). Thus, evidence is there that not all hypertensive patients are hypomagnesemic, and not all Mg2+ -defi cient patients are hypertensive. Some hypertensive subgroups, however, consistently present altered Mg2+ homeostasis. These subgroups include African-Americans or individuals of African descent, elderly patients, or patients with malignant hypertension, metabolic syndrome, or obesity [107 ]. While emphasis has been placed in identifying genetic causes of hypertension, limited information is available as to whether genetic alterations of Mg 2+ homeostasis and transport play a signifi cant role in hypertension onset and progression in the indicated subgroups. In this respect, the group of Touyz has proposed that dysregulation or alteration of the Mg2+ entry channel TRPM7 may play an important role in abnormal cellular Mg 2+ homeostasis in hypertension [107 ]. This group, in fact, observed an altered magnesium infl ux in vascular smooth muscle cells in SHR rats, associated with down-regulation of vascular TRPM7 [107 ] as well as cardiovascular and renal remodeling, fi brosis, and infl ammation associated with down-regulation of renal TRPM7 following infusion of aldosterone in mice [108 ]. Noteworthy, several of these responses were ameliorated by dietary Mg2+ supplementation [109 ]. The studies of Touyz’s group have predominantly been carried out in animals. Yet, the obtained results forebode the likelihood that similar changes and alterations also occur in the human setting.

3.2.2 Hyperaldosteronism

The results discussed at the end of the previous paragraph shed some light on the clinical observation that hyperaldosteronism is one of the main endocrinopathies associated with hypomagnesemia [ 10 ]. The disease is characterized by urinary Mg 2+ loss and low level of circulating Mg 2+, but the mechanisms behind these events are not fully elucidated. One possibility is that the Mg 2+ loss is due to the elevated Na + retention resulting from aldosterone hypersecretion, which exchanges with cellular Mg2+ perhaps through the Na + /Mg2+ exchanger, triggering a signiﬁ cant loss of cellular Mg2+ in various tissues. At the same time, it is conceivable that urinary Mg2+ loss depends on a defect in expression or activity of the ubiquitous Mg2+ entry channels TRPM7 but also TRPM6, which is deputed to speciﬁ cally reabsorb Mg2+ in the distal convolute tubule of the nephron. This possibility is supported by the data from Touyz’s group [107 ] mentioned above.

3.2.3 Diabetes

Diabetes is one of the best known diseases that induces Mg2+ loss in both animals and humans. Despite the large body of evidence in the medical literature, the majority of the reports are correlative at best. 3 Magnesium in Health and Disease 63

Both type-1 (T1DM) and type-2 diabetes (T2DM) are characterized by hypomagnesemia, hypermagnesuria, and lower Mg2+ level within tissues. Because T2DM presents the majority of all the diabetic cases diagnosed every year in the human population, more attention has been paid to this condition, in the attempt to determine whether the Mg2+ loss observed in the disease is a predisposing condition or an epiphenomenon. Insulin has long been recognized as one of the hormones playing a major role in regulating cellular Mg2+ homeostasis. Experimental and clinical evidence indicates that insulin increases cellular Mg2+ content although the mechanism of action is not completely clear. One possibility evidenced by Romero and collaborators in erythrocytes is that insulin can directly modulate the Na+ /Mg2+ exchanger [110 ]. Alternatively, insulin could increase cellular Mg2+ indirectly by enhancing cellular K + content while decreasing cellular Na+ content [ 111]. It is currently unclear whether insulin has a direct effect on the expression and activity of TRPM6 and TRPM7. Recent epidemiological studies, however, indicate the occurrence of defective mutations in the intestinal expression and activity of TRPM6 involved in dietary Mg2+ absorption [112 ] in a cohort of diabetic women. Magnesium accumulation directly correlates with the rates of glucose accumulation within tissues following insulin administration. This has been observed in liver cells [113 ], cardiac myocytes [114 ], and β-cells [115 ]. Moreover, experiments conducted in our laboratory have consistently indicated that the decrease in tissue Mg2+ content observed in T1DM animals correlates directly with the level of K+ , but inversely with the level of Na+ and Ca2+ within liver, skeletal muscle, and heart [75 ], i.e., tissues directly involved in controlling glycemia. In addition, the extrusion rate of Mg2+ from diabetic hepatocytes [75 ] and cardiac myocytes [ 116] is dramatically enhanced both in intact cells and in purifi ed plasma membrane [117 ], and is renormalized by the exogenous insulin administration [116 ], or artifi cial increase of glucose and glycogen within plasma membrane vesicles [117 ]. Diabetic animals also exhibit a marked increase in urinary Mg2+ loss [ 75 ], mimicking what has been reported to occur in diabetic patients. Whether this effect depends on the absence of an insulin stimulatory effect on renal Mg2+ reabsorption is presently undefi ned. The interplay between insulin and Mg2+ is reciprocal in that Mg2+ -defi cient animals present reduced levels of insulin receptor phosphorylation [118 ], at least in skeletal muscles, with consequent reduction in muscle glucose accumulation. Determination of cellular [Mg2+ ]i under similar experimental conditions indicates the reduction of cytosolic Mg2+ from physiological ≥0.7 mM [ 45] to half those values [119 ], thus affecting several Mg2+ -dependent enzymes requiring phosphorylation. In addition, these conditions can set the basis for reduced insulin-stimulated cellular metabolism, and predispose to insulin resistance. Conversely, Mg 2+ addition can restore several of these dysmetabolic conditions, if not all. In particular, Mg2+ intake appears to be directly and signifi cantly associated with insulin sensitivity in a threshold fashion [120 ]. Overall, these results strongly suggest that cellular Mg 2+ defi ciency can actually be the cause rather than the result of insulin resistance. 64 Romani

In keeping with the dependence of Mg2+ accumulation on an effective glucose uptake, hypomagnesemia appears to be prevalent in individuals with poor glycemic control [120 ], and actually relate inversely to the effectiveness of the metabolic control and glycated hemoglobin (HbA1C) levels [120 ]. On the other hand, several treatments for T2DM appear to increase cellular Mg2+ levels. Oral antidiabetic drugs such as metformin or pioglitazone increase Mg2+ levels within hepatocytes [121 ] or adipocytes [122 ] as well as circulating Mg2+ levels.

3.2.3.1 Diabetes Complications

Mg2+ deﬁ ciency has also been implicated as a predisposing factor to the onset and development of diabetic complications, along the lines of what has already been indicated for hypertension. Inﬂ ammation, atherosclerosis, oxidative stress, i.e., the main functional and metabolic changes observed in hypertensive patients, also play an essential role in the progression of diabetic cardiomyopathy, nephropathy, neuropathy, and retinopathy. All these complications as well as diabetic hyperalgesia are attenuated to a varying extent by magnesium supplementation [123 ].

3.2.4 Metabolic Syndrome

About 20 years ago, Resnick formulated the ‘ionic hypothesis’ for hypertension and other metabolic disorders. Based on his hypothesis, hypertension, insulin resistance, and type 2 diabetes are associated with an increase in intracellular Ca 2+ and a decrease in intracellular Mg2+ [ 124 ]. Yet, the exact mechanisms behind the onset of the metabolic syndrome are not completely defi ned. Most of the patients are of middle age, sedentary, and with varying degrees of obesity, mostly central obesity, and insulin resistance. Stress is considered a contributing factor. The most important factors implicated in the disease onset are weight gain, genetics, endocrine disorders (e.g., polycystic ovary syndrome in women of reproductive age), aging, and sedentary lifestyle, (i.e., low physical activity and excess caloric intake). The exact sequence of events is also not clear. Is it obesity or insulin resistance that causes the metabolic syndrome? Or, does the metabolic syndrome cause obesity and insulin resistance? Or, are these three conditions an expression of a more far-reaching metabolic and hormonal derangement? To complicate the issue, several infl ammatory markers including C-reactive protein, interleukin 6 (IL-6), and tumor necrosis factor α (TNFα) are usually increased in these patients [125 ]. Irrespective of whether the metabolic syndrome (aka syndrome X) is cause or consequence of insulin resistance and obesity, all three conditions are associated with a deranged cellular and serum Mg2+ homeostasis. Due to the limited number of studies on the topic, it is unclear whether Mg2+ defi ciency predisposes to the disease or it is the results of the incurrent dysmetabolism and/or insulin resistance (see previous paragraph). 3 Magnesium in Health and Disease 65

3.2.5 Alcoholism

Alcoholism is another of the most common human pathologies associates with Mg 2+ defi ciency. Experimental evidence indicates that in the case of alcoholism, it is the alcohol consumption that induces Mg 2+ loss from tissues, and ultimately with the urine. This pattern has been observed in both human patients and in animal models. At the liver level, ethanol administration induces a marked decrease in cytoplasmic ATP content through the change in pyridine nucleotides associated with ethanol metabolism by the alcohol dehydrogenase [126 ]. This effect occurs in a time- and dose-dependent manner [ 126]. The observed decrease in ATP content removes an essential complexing agent from the cell cytoplasm that ultimately results in an increase in cellular free [Mg 2+] and a detectable Mg 2+ extrusion from the hepatocyte via the Na+ /Mg2+ exchanger [126 ]. Inhibiting the exchanger or the alcohol dehydrogenase prevents Mg 2+ loss [126 ]. Similar effects have been observed following acute [126 ] and chronic [127 ] ethanol administration, as well as addition of repeated doses of alcohol with a small interval in between [ 128 ]. More importantly, acute and chronic ethanol administrations exert an inhibitory effect on the protein kinase C signaling involved in favoring Mg 2+ accumulation [129 ]. As a result of this inhibition, Mg2+ cannot be effectively accumulated within the cell until ethanol has been removed from the system and protein kinase C can properly translocate to the cell membrane [129 ]. This inhibitory effect lasts for more than 1 hour following an acute ethanol administration [129 ], and for almost two weeks in a chronic ethanol model [ 129 ]. Under both acute and chronic conditions, Mg2+ is lost from the cytoplasm as well as mitochondria and endoplasmic reticulum [130 ]. Magnesium losses qualitatively similar to those observed in the liver have been reported to occur in other tissues including skeletal muscles [131 ], vascular smooth muscle cells [132 ], and neurons [ 133], and they have been associated with the high incidence of vasospasm and stroke plaguing chronic alcoholics, and possibly delirium tremens insurgence [ 134]. In the case of the skeletal muscles, alcohol acceler- ates protein catabolism and muscle atrophy but it is presently unclear whether Mg 2+ loss plays any role in the process (reviewed in [135 ]). Magnesium defi cit appears to play a signifi cant role in modulating the infl ammatory response induced by EtOH. Physiological Mg2+ levels inhibit the release of proinfl ammatory cytokines while promoting production and release of antiinfl ammatory cytokines [ 136 ]. Because infl ammation plays a key role in the onset of ste- atohepatitis and its progression towards alcohol liver disease (ALD) [137 ], it can be easily hypothesized that cellular and systemic Mg2+ defi cit modulates the immune response of liver resident macrophages (Kupffer), and circulating monocytes, and lymphocytes, with major consequences for liver function and cyto-architectonics. Lastly, physiological Mg2+ levels have been related to proper cell cycle progression [103 ]. Hence, it is possible that lower than normal cellular Mg2+ levels affect hepatocyte regeneration following alcohol-induced liver apoptosis [137 ]. While alcohol administration promotes Mg2+ wasting, Mg2+ supplementation ameliorates several neuronal, muscular, and hepatic biochemical functions. Because 66 Romani of the variety of symptoms and functions ameliorated by Mg2+ supplementation, it would appear that Mg 2+ acts as a coenzyme in biochemical reactions but also as a regulator of cellular signaling pathways such as adenylyl cyclase [138 ], protein kinase C [139 ], and Erk1/2 MAPKs [140 ]. In the case of the heart, for example, dietary Mg 2+ supplementation ameliorates the myocardial dysfunction induced by acute or chronic ethanol administration, and renormalizes heart size as well as iso- metric force and isotonic shortening [141 ]. The mechanism(s) behind these effects are not elucidated. Magnesium is considered to act as a natural Ca2+ -channel blocker. Thus, it is possible that the changes in force development and cell shortening depend on the restoration of a normal cellular Ca 2+ level that directly impacts the contractile myofi laments. Less clear is whether the renormalization of heart size depends on the restoration of normal cellular Ca2+ levels or, alternatively, on direct effects of Mg2+ on protein synthesis and mRNA translation.

3.2.6 Inﬂ ammation

As indicated in the previous paragraph, seminal work by different laboratories supports the notion that an increase in systemic infl ammation is associated with Mg 2+ defi cit. This response is characterized by increased serum levels of TNFα and infl ammatory cytokines [136 ] while the production and release of antiinfl ammatory cytokines is reduced [136 ]. Currently, two main mechanisms are invoked to explain the increase in infl ammatory cytokines in the case of Mg2+ defi ciency: (i) the Ca2+ - channel blocking effect of Mg2+ is attenuated, resulting in increased Ca 2+ entry within the immune-competent cells, with enhanced cell priming towards an infl ammatory phenotype/response, and/or (ii) the reactive oxygen species production, which is increased under Mg2+ defi ciency conditions through non fully-elucidated mechanism(s), promotes membrane oxidation and activation of NFκB [142 ]. Irrespective of whether these two mechanisms cooperate or act independently to increase the infl ammatory response, the net result in an increased production of infl ammatory cytokines that can be detected in the circulation. In addition, Mg2+ - defi cient animals are more susceptible to septic shock [143 ]. Administration of lipopolysaccharide (LPS) results in a mortality rate in excess of 70% within 3 hours in these animals as compared to no lethal effect in control animals [143 ]. Magnesium supplementation prior to LPS administration signifi cantly increases the survival rate of the animals. In agreement with the above observation, Altura and collaborators reported that Mg 2+ defi ciency results in an increased production of specifi c cytokines [144 ] via de novo synthesis of ceramide in vascular smooth muscle cells [ 145], and that inhibition of ceramide synthesis attenuates NFκB activation and cytokines production [145 ]. Recently, in collaboration with Dr. Bernstein’s laboratory, we have provided evidence that monocytes from women undergoing preterm labor synthesize elevated levels of pro-infl ammatory cytokines, and that pharmacological doses of MgSO4 , commonly used as a tocolytic agent to stop preterm labor, completely block cytokines production [146 ,147 ]. 3 Magnesium in Health and Disease 67

All together, these results indicate that optimal levels of Mg2+ are key to modulate systemic and local inﬂ ammation by regulating inﬂ ammatory cytokine production and release.

3.2.7 Renal Pathologies

The kidney plays an important role in controlling human body Mg2+ content through reabsorption in the Henle’s loop and the distal convolute tubule. Thus, it is not surprising that several renal diseases impact the ability of the organ to reabsorb Mg 2+ to a signiﬁ cant extent, causing Mg2+ wasting and Mg 2+ deﬁ cit. In the following paragraphs, the most common renal Mg 2+ handling diseases and the predominant location within the nephron will be commented. We refer to several recent reviews for a more in-depth description of the causes and complications [148 , 149 ].

3.2.7.1 Bartter’s Syndrome

Bartter’s syndrome is characterized by Na+ and Cl – wasting, hypokalemia, metabolic alkalosis, and increased production of renin and aldosterone [150 ]. The disease affects the thick ascending limb (TAL) of the Henle’s loop, and is the result of autosomal recessive mutations of various genes involved in Na+ and Cl– transport including that encoding for the Na + /K+ /Cl– co-transporter (NKCC2 or SLC12A1). The disease is associated with hypermagnesuria and hypomagnesemia in approximately 50% of the cases. The precise explanation for this variability is not clear, especially if we consider that all the various forms of Bartter’s syndrome are characterized by inhibition of ion transport in the TAL and by a varying level of dissipation of the electrochemical gradient that drives the reabsorption of divalent cations such as Ca2+ and Mg2+ . Consequently, it is difﬁ cult to determine the electrochemical gradient responsible for Mg2+ but also Ca2+ reabsorption in the TAL since the associated polyuria will determine volume depletion and changes in tubular and systemic ionic concentrations. Moreover, patients presenting speciﬁ c mutations of the chloride channel (CLC-Kb) located in the basolateral domain of the distal convolute tubule (DCT) present a combined phenotype of Bartter’s syndrome plus Gitelman’s syndrome, and more persistent hypomagnesemia [151 ]. The reason as to why mutations in the CLC-Kb channel in this segment distal to the TAL are associated with such a phenotype is not completely understood.

3.2.7.2 Gitelman’s Syndrome

Gitelman’s syndrome is a salt wasting condition characterized by metabolic and ionic conditions reminiscent of the Bartter’s syndrome. Also in this condition the production of renin and aldosterone are increased but to a lower extent than in the 68 Romani

Bartter’s syndrome. The disease affects specifi cally SLC12A3 isoform, located in the DCT. The syndrome can be mimicked by the chronic administration of thiazide diuretics, which specifi cally block the SLC12A3 encoded transporter NCC [152 ]. The patients affected by this syndrome present consistently hypomagnesemia, and a decreased expression of the Mg2+ selective channel TRPM6 in the DCT has been indicated as the most likely reason for the Mg2+ defi cit. However, the mechanism(s) responsible for the reduced TRPM6 expression remain(s) speculative at the moment. Aldosterone has been indicated as a possible culprit of the reduced channel expression, as the secretion of this hormone is increased in Gitelman’s syndrome, and the treatment with spironolactone, which antagonizes aldosterone, ameliorates urinary Mg 2+ loss, and increases serum Mg2+ level.

3.2.7.3 Defects in Claudin Expression

Defects in paracellin-1 (a.k.a. claudin 16, encoded by CLDN16 ) result in an autosomal recessive disorder termed familial hypomagnesemia with hypercalciuria and nephrocalcinosis, or FHHNC [47 ]. These defects consist in single amino acid mutation of this protein, which forms tight junction in the TAL and the DCT. Based upon the single amino acid mutation, a more or less severe phenotype is observed, with a variable degree of urinary Mg2+ and Ca2+ loss. These mutations affect the regulation of the tight junction by cAMP and consequently the reabsoprtion of Mg2+ and Ca2+ by cAMP-mediated hormones like parathyroid hormone. Claudin 19 contributes to form tight junction in the TAL and DCT by forming a heterotetramer with claudin 16 [48 ]. Consequently, single amino acid mutations in claudin 19 sequence have also been associated with Mg2+ wasting and hypomagnesemia [48 ].

3.2.7.4 Defects in TRPM6 Expression

As indicated in Sections 2 and 3 , TRPM6 constitutes the Mg2+ entry mechanism of choice in the DCT and in the distal portion of the intestine in which it promotes Mg2+ reabsorption and absorption, respectively. Autosomal recessive mutations in this gene product are cause of hypomagnesemia with secondary hypocalcemia (HSH), with consequent increase in neuromuscular excitability, muscle spasm, tetany and convulsions [ 11 ]. Interestingly, supplementation with high doses of Mg2+ is sufﬁ cient to renormalize calcemia while serum Mg 2+ levels remain suboptimal and indicate a defective intestinal absorption of Mg2+ via TRPM6. It has to be noted that urinary Mg2+ wasting may not be notice- able in most patients under day-to-day conditions, becoming more detectable following the supplementation with high doses of Mg 2+ especially if administered intravenously [ 51 ]. 3 Magnesium in Health and Disease 69

3.2.7.5 Defects in Epidermal Growth Factor Signaling

Hypomagnesemia associated with psychomotor and mental retardation, and epileptic seizures has been observed in patients with mutation in the EGF (epidermal growth factor) gene [52 ]. Because of these mutations, the patients present a defective secretion of EGF in the interstitium and a limited or absent activation of the EGF receptor in the DCT [52 ]. Subsequent studies have indicated that EGF-receptor activation promote Mg 2+ entry through TRPM6 [ 54] by enhancing the channel expression in the cell membrane from the endosomal compartment. As described more in details in the following Section 3.3.2 , this EGF/EGFR/TRPM6 pathway becomes inactivated by speciﬁ c monoclonal antibodies used therapeutically to treat metastatic forms of colon cancer.

3.2.8 Magnesium and Tumors

Magnesium plays an essential role in numerous cell functions including progression through the cell cycle [103 ]. Since the 1970s, the group of Rubin has advocated an essential role of Mg2+ as a regulator of cell proliferation and protein synthesis irrespective of the cell type [ 153 , 154 ]. Results from this laboratory and from the groups of Touyz, Maier, and Wolf have provided signifi cant lines of evidence that low levels of cellular Mg2+ impact the ability of the cells to properly synthesize proteins and to progress through the various mitotic steps [103 , 142 , 154 , 155]. Conversely, an increase in cellular Mg 2+ correlated well with DNA and protein synthesis, and with tissue growth, while quiescent tissues present lower levels of Mg2+ . Because of this role of Mg2+ in cell growth, the attention of several researchers has obviously focused on the possible involvement of Mg2+ in tumor development and metastatization. The results obtained so far support an intriguing scenario, with very clear and interesting distinction. At variance of normal cells, which depend on Mg2+ content and availability for proper growth, tumor cells are essentially independent on magnesium availability and stop growing only when extra-cellular Mg2+ is reduced to a very low level (e.g., 0.2 mM or less [155 ]). Under these conditions, p21, p27, and p53 cell cycle regulatory proteins become up-regulated [155 ] or activated [ 156] while the cell cycle promoting proteins cyclin D and cyclin E, and several cyclins-dependent kinases become down-regulated [155 ]. These effects are associated to changes in the level of several MAP kinases including Erk1/2 and p38 [155 ]. Associated cDNA studies have indicated that more than 30 genes are affected by up-and-down changes in Mg 2+ content [ 157 ]. Many of these genes control cell proliferation while other control cell-matrix interaction (e.g., integrin), or antioxidant defenses (e.g., glutathione S-transferase). The latter gene is of particular relevance because it not only contributes to the antioxidant defenses of the cell but also regulates cell proliferation and differentiation. Consistent with this widespread inhibitory role of a low Mg 2+ level on cell growth, mice exposed to a Mg2+ -defi cient diet and grafted for various solid 70 Romani tumors exhibited a reduced tumor growth compared to mice maintained on a normal Mg2+ diet [158 ]. This observation strikes an interesting correlation with the clinical evidence that colon cancer patients treated with monoclonal antibodies anti-EGF receptor presents a reduction in the primary tumor size and in metastases while presenting hypomagnesemia and increased urinary Mg2+ wasting [159 ] (see also Section 3.3.2 ). Because of the role of Mg 2+ as chemo-attractant for endothelial cells [160 ], low Mg 2+ levels negatively affect endothelial cell proliferation [161 ]. In line with this observation, Mg2+ -defi cient mice presented less developed and vascularized tumors [158 ]. The mechanism responsible for the reduced vascularization and angiogenesis is not clear. In part, it can be attributed to the reduced ability of endothelial cells to proliferate and migrate. In part, however, it may depend on the accelerated senescence associated with growth arrest which the endothelial cells and the tumor cells experience [161 ]. At the same time, Mg 2+ defi ciency is associated with a higher level of basal infl ammation, and the higher level of proinfl ammatory cytokines may certainly play a role in limiting angiogenesis and accelerating the deterioration of endothelial cells. The protective or at least limiting effect of low Mg 2+ levels for the growth of primary tumors and the associated angiogenesis process does not apply to the metastatization process. Based upon the reduction in cell growth, a lower number of metastases would be expected to be present in Mg2+ -defi cient mice carrying solid tumors, and many of these metastases would be of smaller size. In fact, quite the opposite, as a larger number of lung metastases have been observed in Mg2+ - defi cient mice [ 158]. This could be explained by the observed overexpression of metalloproteinases and other proteinases and the increased expression of vascular cell adhesion molecules (VCAMs) within the primary tumor cells [162 ]. On the other hand, Mg2+ is essential for the proper activity of NM23-H1, an 8 member genes family with very well established antimetastatic activity [163 ]. It is therefore possible that in a low Mg2+ environment this gene complex does not operate effectively in controlling and suppressing the diffusion of metastasis. Taken together, the effect of Mg2+ defi cit or defi ciency appears to span to both sides of the aisle. On one side, Mg2+ defi cit limits tumor growth and proliferation and tumor-related angiogenesis. On the other side, it promotes tissue invasion and metastatization. In the middle, we have multiple effects of up- and down-regulation of a variety of enzymes, molecules, and infl ammation agents that contribute to the fi nal outcome in this complex scenario.

3.2.9 Magnesium and Prenatal Pathologies

Magnesium has been widely used for more than 60 years in the US in maternal/ perinatal settings [164 ]. Its use has been mainly in two areas: (i) preterm labor, and (ii) preeclampsia. 2+ The rationale behind the use of Mg (mostly MgSO4 ) in preterm labor is that it decreases muscle contractility by limiting Ca2+ accumulation within the muscle cell. The utilization as a tocolytic has been widespread although the mechanism of action 3 Magnesium in Health and Disease 71 is not completely understood and some studies have indicated a limited or nihil beneﬁ cial effect as tocolytic [165 ].

As for preeclampsia, MgSO4 is commonly used for seizure prophylaxis. In this 2+ case, MgSO4 has to be administered at doses that rapidly increase serum Mg level to 2.5 mM or higher [166 ]. Its anticonvulsant effect can be attributed to slowing down neuromuscular conduction, depression of vasomotor center, and blockade of peripheral neuromuscular transmission [166 ]. One of the main complications of pre-term labor is the occurrence of cerebral palsy in premature infants born as early as 27 weeks of gestations or earlier [167 ]. The disease is ~80-fold higher in preterm delivered babies than in babies delivered at term, and is characterized by permanent abnormal gross and ﬁ ne motor functioning. The main cause has been attributed to disturbances in the developing fetal or infant brain [167 ]. Several clinical observations in the 1990s indicated that newborns of very low birth weight exposed to

MgSO 4 while in utero, mostly as a tocolytic for preterm labor or as prevention for eclamptic seizures, presented a much lower incidence of cerebral palsy than newborns of similar birth weight not exposed to the agent [168 ]. The suggestion that

MgSO 4 could act as a neuroprotective agent for at risk newborn was the object of several other studies many of which although not all conﬁ rmed the notion. Because of this inconsistency, it was not until 2009 that the role of MgSO4 as a neuroprotective agent for at risk newborn was ultimately conﬁ rmed [ 169]. This led the American College of Obstetrics and Gynecology (ACOG) to issue a committee opinion on the use of MgSO4 for neonatal neuroprotection, and to establish clear guidelines for the dosage and modality of administration [170 ].

While the benefi cial effect of MgSO4 treatment in at-risk perinatal conditions appears to be fi nally accepted, the mechanism(s) behind this effect is not completely elucidated. Recent studies by Bernstein’s and our laboratories [146 ,147 ] provide compelling evidence that Mg2+ may act as an antiinfl ammatory agent on maternal and perhaps neonatal monocytes, reducing the synthesis and production of circulating proinfl ammatory cytokines including TNFα and IL-1 among others by modulating NFκB signaling. These results are well in line with the reports by the Altura group discussed earlier [144 ], coincidentally and independently published at the same time as ours [146 ]. Interestingly, high levels of infl ammatory cytokines have been reported to be present in the cerebral fl uid of newborns with cerebral palsy [167 ], thus providing a far reaching relevance to our reports and to those by the Altura group. Because of obvious ethical restrictions, however, it is currently undefi ned whether the infl ammatory cytokines present in the fl uids of the newborns are maternal in origin or they are generated endogenously by the newborn’s immune system in response to proinfl ammatory stimuli released by the mother.

3.3 Pharmacological Agents Causing Hypomagnesemia

Increasing evidence in the literature indicates that proton pump inhibitors and anti- EGFR antibodies have become the two fast rising groups of pharmacological agents inducing hypomagnesemia and magnesium waste in patients. 72 Romani

3.3.1 Proton Pump Inhibitors

The ﬁ rst observation of hypomagnesemia associated with the use of a proton pump inhibitor (PPI) dates back to 2006. Since then, the incidence of episodes has progressively increased due to the wide utilization of prescription PPI products (in excess of 70 million in 2009 [ 171]) and availability of several PPI products over the counter. The increasing trend of episodes prompted the FDA to issue a warning in 2011 to the public indicating that prescription PPI may cause severe hypomagnesemia when taken for prolonged periods of time (1 year or longer) [172 ]. Recent reviewing of the data in the literature indicates that hypomagnesemia: (i) can be severe (as low as 0.35 mM [173 ], as compared to the normal lower limit of 0.76 mM [ 45]); (ii) it is often (>60%) associated with hypokalemia [173 ] and hypocalcemia, the latter springing from altered parathyroid hormone release; (iii) it regresses rapidly upon discontinuation of PPIs [173 ]; but (iv) it reoccurs just as rapidly following PPIs re-introduction in the therapy [173 ]. Presently, the mechanism responsible for the onset of hypomagnesemia is not fully elucidated. Modeling of intestinal Mg2+ absorption under conditions resembling the decrease in intestinal pH elicited by the proton pump inhibitor esomeprazole suggests a reduced intestinal absorption of Mg2+ in the distal portion of the intestine [171 ]. This inhibition is likely to be the result of a proton neutralizing effect on carboxyl side chains of glutamic acid and aspartic acid residues deemed essential for Mg2+ binding and conduction in the pore-forming region of TRPM7 and TRPM6 [171 ]. Frequent but small doses of Mg2+ supplementation appear to be beneﬁ cial in preserving proper circulating Mg2+ levels following PPI-induced hypomagnesemia [174 ]. Because of the association between low Mg2+ levels and diseases like diabetes, osteoporosis, hypertension, arrhythmias, and congestive heart failure, patients taking PPI for extended periods of time should be advised to consistently monitor their serum Mg2+ level and perhaps discontinue at time the use of the proton pump inhibitor.

3.3.2 Anti-epidermal Growth Factor Receptor Antibodies

Anti-EGF receptor antibodies represent the second class of pharmaceutical agents consistently associated with an increased incidence of hypomagnesemia and magnesium waste. These agents are commonly used for the treatment of various forms of cancer, including cancer of the colon, ovary, lung, prostate, and kidney among others. Persistent and/or abnormal activation of the EGF receptor has been observed in all these neoplastic conditions. Following engagement by its ligand, the EGF receptor becomes activated and recruits several signaling pathways including MEK, ERK, PI-3-K, STATS, and PLC-γ [ 175 ], which are potent oncogenic regulators of tumor cell growth, invasion, angiogenesis, and metastasis. On the other hand, activation of the EGF receptor results in the upregulation of TRPM6 via Erk1/2 [54 ], thus promoting physiological intestinal absorption and renal reabsorption of Mg 2+ . Administration of anti-EGFR antibodies will then inhibit the activation of the 3 Magnesium in Health and Disease 73 oncogenic regulators mentioned above but also Mg 2+ absorption and reabsorption, inducing hypomagnesemia. As the use of these antineoplastic agents has increased, so has the occurrence of hypomagnesemia increased [159 ]. As in the case of the proton pump inhibitors, hypomagnesemia is associated with hypocalcemia and hypokalemia [159 ]. Comparison among the different forms of anti-EGFR antibodies indicates that pni- tumumab, a fully human monoclonal antibody used for metastatic colon cancer, presents the highest incidence of hypomagnesemia, often as severe as 0.9 mg/dL, or half the physiological level [176 ]. The occurring hypomagnesemia does not appear to lead to major complications other than neuroexcitability and neuromuscular spasms [176 ]. Nevertheless, an aggressive treatment in patients with severe hypomagnesemia is recommended, with very high doses of magnesium (up to10 g) being needed to achieve a clinically signiﬁ cant reversal of the symptoms. Weekly Mg2+ administration is usually inadequate as serum Mg2+ return to low baseline level within 3–4 days. In several patients daily to twice-a-week doses of intravenous Mg2+ as high as 6–10 g/dose have been required, and some of these cases have registered a continuous or worsening hypomagnesemia despite the treatment [ 177 ]. The most likely explanation for such a negative outcome is that as long as the EGF receptor in the kidney and intestine is inhibited, renal Mg2+ wasting and ineffective intestinal Mg2+ absorption will persist or worsen. Moreover, because the inhibition of the EGF receptor is rapid and long-lasting during anti-EGFR therapy, the effectiveness of intravenous Mg 2+ supplementation is largely diminished. Also, patients treated with these antineoplastic agents show a marked hypoalbuminemia [176 ]. The causes for this effect are unknown. Nevertheless, a reduced level of circulating albumin has a two-fold effect in promoting hypomagnesemia and other ionic alterations: (i) it exacerbates the loss of Ca2+ and Mg2+ as lower amounts of these cations are protein- complexed, and (ii) it increases indirectly the doses of anti- EGFR antibodies present in the circulation, thus promoting more pronounced and long lasting effects of the antineoplastic agent on the EGFR, and consequently on Mg2+ homeostasis.

4 Conclusions

Due to space constrains, and the complexity of the ﬁ eld, we have only tapped on the main pathologies and iatrogenic conditions associated with hypomagnesemia and altered cellular Mg2+ homeostasis, and attempted to provide the reader with a framework to appreciate the perhaps incomplete intricacies of Mg2+ homeostasis and its regulation, as well as its physio-pathological implications. Each pathological condition mentioned here has been the topic of several ad hoc reviews in recent years, which are cited in the preceding sections; we refer the interested reader to them for a more in-depth evaluation. As our understanding of the regulation of Mg2+ homeostasis progresses, we are conﬁ dent that new tools will become available to properly address the key physiological role Mg2+ plays inside the cell and in the whole human body. 74 Romani

Abbreviations

ACOG American College of Obstetrics and Gynecology ALD alcohol liver disease AP-1 activator protein-1 ATP adenosine 5′-triphosphate cAMP 3′,5′-cyclic adenosine monophosphate CaSR calcium sensing receptor CHF chronic heart failure CHF congestive heart failure CKD chronic kidney disease CVD cardiovascular disease DCT distal convolute tubule EGF epidermal growth factor EGFR epidermal growth factor receptor Erk extracellular signal regulated kinases FDA Food and Drug Administration FHHNC familial hypomagnesemia with hypercalciuria and nephrocalcinosis HbA1c glycated hemoglobin A1c HSH hypomagnesemia with secondary hypocalcemia i.v. intravenous ICU intensive care unit IL interleukin IP3 inositol triphosphate LPS lipopolysaccharide MAPKs mitogen activated protein kinases N F κB nuclear factor kappa-light-chain-enhancer of activated B cells NKCC 2 Na+ /K+ /Cl– cotransporter PKA protein kinase A PKC protein kinase C PPI proton pump inhibitor PTH parathyroid hormone QT interval between Q and T wave in electro cardiogram. RACK1 receptor for activated protein kinase 1 REA repressor of estrogen receptor activity SLC12A1 solute transporter class 12 isoform A1 SLC12A3 solute transporter class 12 isoform A3 T1DM type 1 diabetes mellitus T2DM type 2 diabetes mellitus TAL thick ascending limb TNFα tumor necrosis factor α TRMP6 transient receptor potential melastatin subfamily isoform 6 TRP transient receptor potential TRPM7 transient receptor potential melastatin subfamily isoform 7 3 Magnesium in Health and Disease 75

VCAM vascular cell adhesion molecule VF ventricular ﬁ brillation VT ventricular tachycardia

Acknowledgements This work was supported by NIAAA-11593 and in part by NIH-HL090969.

References

1. E. M. Widdowson, R. A. McCance, C. M. Spray, Clin. Sci. 1951 , 10 , 113–125. 2. H. A. Schroeder, A. P. Nason, I. H. Tipton, J. Chronic Dis. 1969 , 21 , 815–841. 3. P. O. Webster, Am J. Clin. Nutr. 1987 , 45 , 1305–1312. 4. K. K. Rude, M. Olerich, Osteoporos. Int. 1996 , 6 , 453–46 5. P. Volpe, B. H. Alderson-Lang, G. A. Nickols, Am. J. Physiol. 1990 , 258 , C1077–C108 6. J. A. Cowan, BioMetals 2002, 15 , 225–235. 7. M. E. Shils, Magnesium , in Modern Nutrition in Health and Disease, Eds M. E. Shils, J. A. Olson, M. Shike, A. C. Ross, 9th edn, Lippincott, Williams, and Wilkins, 1995 , pp. 169–192. 8. A. E. Gomez-Ayala, F. Lisbona, I. Lopez-Aliaga, I. Pallares, M. Barrionuevo, S. Hartiti, M. C. Rodriguez-Matas, M. S. Campos, Lab. Anim. 1998, 32, 72–79. 9. M. Baillien, H. Wang, M. Cogneau, Magnesium 1995 , 8 , 315–329. 10. M. Baillien, M. Cogneau, Magnesium 1995 , 8 , 331–339. 11. K. P. Schlingmann, S. Weber, M. Peters, N. L. Niemann, H. Vitzthum, K. Klingel, M. Kratz, E. Haddad, E. Ristoff, D. Dinour, M. Syrrou, S. Nielsen, M. Sassen, S. Waldegger, H. W. Seyberth, M. Konrad, Nat. Genet. 2002 , 31 , 166–170. 12. M. J. Nadler, M. C. Hermosura, K. Inabe, A. L. Perraud, Q. Zhu, A. J. Stokes, T. Kurosaki, J. P. Kinet, R. Penner, A. M. Scharenberg, A. Fleig, Nature 2001 , 411 , 590–595. 13. B. Nilius, T. Voets, Pﬂ ugers Arch.-Eur. J. Physiol. 2005 , 451 , 1–10. 14. B. J. Kim, K. J. Park, H. W. Kim, S. Choio, J. Y. Jun, I. Y. Chang, J.-H. Jeon, I. So, S. J. Kim, World J. Gastroenterol. 2009 , 14 , 5799–5804. 15. C. Schmitz, F. Deason, A.-L. Perraud, Magnes. Res. 2007 , 20 , 6–18. 16. A. Romani, Cellular Magnesium Homeostasis in Mammalian Cells, in Metallomics and the Cell , Vol 12 of Metal Ions in Life Sciences , Volume Ed L. Banci, Series Eds A. Sigel, H. Sigel, R. K. O. Sigel, Springer, Dordrecht, 2013, pp. 69–118. 17. G. A. Quamme, Am. J. Physiol. 2010 , 298 , 407–429. 18. V. Chubanov, T. Gudermann, K. P. Schlingmann, Pﬂ ugers Arch. 2005 , 451 , 228–234. 19. A. G. Ryazanov, FEBS Lett. 2002 , 514 , 26–29. 20. L. V. Ryazanova, K. S. Pavur, A. N. Petrov, M. V. Dorovkov, A. G. Ryazanov, Mol. Biol. (Mosk) 2001 , 35 , 321–332. 21. C. Schmitz, A.-L. Perraud, C. O. Johnson, K. Inabe, M. K. Smith, R. Penner, T. Kurosaki, A. Fleig, A. M. Scharenberg, Cell 2003 , 114 , 191–200. 22. M. V. Dorovkov, A. G. Ryazanov, J. Biol. Chem. 2004 , 279 , 50643–50646. 23. K. Clark, J. Middelbeek, N. A. Morrice, C. G. Figdor, E. Lasonder, F. N. van Leeuwen, PLoS One 2008 , 3 , e1876 (1–10). 24. K. Clark, J. Middelbeek, E. Lasonder, N. G. Dulyaninova, N. A. Morrice, A. G. Ryazanov, A. R. Bresnick, C. G. Figdor, F. N. van Leeuwen, J. Mol. Biol. 2008 , 378 , 790–803. 25. L.-T. Su, M. A. Agapito, M. Li, W. Simpson, A. Huttenlocher, R. Habas, L. Yue, L. W. Runnels, J. Biol. Chem. 2006 , 281 , 11260–11270. 26. C. Schmitz, M. V. Dorovkov, X. Zhao, B. J. Davenport, A. G. Ryazanov, A.-L. Perraud, J. Biol. Chem. 2005 , 280 , 37763–37771. 27. G. Cao, J. van der Wijst, A. van der Kemp, F. van Zeeland, R. J. Bindels, J. G. Hoenderop, J. Biol. Chem. 2009 , 284 , 14788–14795. 76 Romani

28. W. M. Groenestege, J. G. Hoenderop, L. van den Heuvel, N. Knoers, R. J. Bindels, J. Am. Soc. Nephrol. 2006 , 17 , 1035–1043. 29. L. J. Rondón, W. M. Groenestege, Y. Rayssiguier, A. Mazur, Am. J. Physiol. 2008 , 294 , R2001–R2007. 30. G. Cao, S. Thébault, J. van der Wijst, A. van der Kemp, E. Lasonder, R. J. Bindels, J. G. Hoenderop, Curr. Biol. 2008 , 18 , 168–176. 31. A. Romani, A. Scarpa, Arch. Biochem. Biophys. 1992 , 298 , 1–12. 32. F. I. Wolf, A. Torsello, S. Fasanella, A. Cittadini, Mol. Asp. Med. 2003 , 24 , 11–26. 33. F. I. Wolf, A. Cittadini, Mol. Aspects. Med. 2003 , 24 , 3–9. 34. S. Oki, M. Ikura, M. Zhang, Biochemistry 1997 , 36 , 4309–4316. 35. S. Wang, S. E. George, J. P. Davis, J. D. Johnson, Biochemistry 1998 , 37 , 14539–14544. 36. D. Allouche, J. Parello, Y. H. Sanejouand, J. Mol. Biol. 1999 , 285 , 857–873. 37. Y. Ogoma, H. Kobayashi, T. Fujii, Y. Kondo, A. Hachimori, T. Shimizu, M. Hatano, Int. J. Biol. Macromol. 1992 , 14 , 279–286. 38. H. Belge, P. Gailly, B. Schwaller, J. Loffi ng, H. Debaix, E. Riveira-Munoz, R. Beauwens, J. P. Devogelaer, J. G. Hoenderop, R. J. Bindels, O. Devuyst, Proc. Natl. Acad. Sci. USA 2007 , 104 , 14849–14854. 39. Ods.od.nih.gov. 2009-07-13, http://ods.od.nih.gov/factsheets/Magnesium-HealthProfessional/ 40. T. Gunther, J. Vormann, R. Forster, Biochem. Biophys. Res. Commun. 1984 , 119 , 124–131. 41. T. Gunther, J. Vormann, Biochem. Biophys. Res. Commun. 1985 , 130 , 540–545. 42. A. Romani, A. Scarpa, Front. Biosci. 2000 , 5 , D720–D734. 43. T. Gunther, Magnes. Res. 2007 , 20 , 89–99. 44. T. Gunther, Miner. Electrolyte Metab. 1993, 19 , 259–265. 45. Geigy Scientifi c Tables, Ed C. Lentner, Ciba-Geigy, 1984, Basel, Switzerland. 46. L. J. Dai, G. Ritchie, D. Kerstan, H. S. Kang, D. E. Cole, G. A. Quamme, Physiol Rev . 2001 , 81 , 51–84. 47. D. B. Simon, Y. Lu, K. A. Choate, H. Velazquez, E. Al-Sabban, M. Praga, G. Casari, A. Bettinelli, G. Colussi, J. Rodrigues-Soriano, D. McCredie, D. Milford, S. Sanjad, R. P. Lifton, Science , 1999 , 285 , 1103–1106. 48. J. Hou, A. Renigunta, A. S. Gomes, M. Hou, D. L. Paul, S. Waldegger, D. A. Goodenough, Proc. Natl. Acad. Sci. USA 2009 , 106 , 15350–15355. 49. M. A. Khan, A. D. Conigrave, Br. J. Pharmacol . 2010 , 159 , 1039–1050. 50. D. Muller, P. J. Kausalya, F. Claverie-Martin, I. C. Meij, P. Eggert, V. Garcia-Nieto, W. Hunziker, Am. J. Hum. Genet . 2003 , 73 , 1293–1301. 51. R. Y. Walder, D. Landau, P. Meyer, H. Shalev, M. Tsolia, Z. Borochowitz, M. B. Boettger, G. E. Beck, R. K. Englehardt, R. Carmi, V. C. Sheffi eld, Nat. Genet. 2002 , 31, 171–174. 52. W. M. Groenestege, S. Thebault, J. van der Wijst, D. van den Berg, R. Janssen, S. Tejpar, L. P. van den Heuvel, E. van Cutsem, J. G. Hoenderop, N. V. Knoers, R. J. Bindels, J. Clin. Invest. 2007 , 117 , 2260–2267. 53. B. W. Bapty, L. J. Dai, G. Ritchie, F. Jirik, L. Canaff, G. N. Hendy, G. A. Quamme, Kidney Intern. 1998 , 53 , 583–592. 54. A. Ikari, C. Okude, H. Sawada, Y. Yamazaki, J. Sugatani, M. Miwa, Biochem. Biophys. Res. Commun. 2008, 369 , 1129–1133. 55. A. Ikari, A. Sanada, C. Okude, H. Sawada, Y. Yamazaki, J. Sugatani, M. Miwa, J. Cell. Physiol. 2010, 222 , 481–487. 56. A. Romani, Arch. Biochem Biophys, 2011 , 512 , 1–23. 57. P. W. Flatman, J. Membr. Biol. 1984 , 80 , 1–14. 58. A. Scarpa, F. J. Brinley, Fed. Proc. 1981 , 40 , 2646–2652. 59. D. Lüthi, D. Günzel, J. A. McGuigan, Exp. Physiol . 1999 , 84 , 231–252. 60. T. Gunther, Magnesium 1986 , 5 , 53–59. 61. M. Kolisek, A. Nestler, J. Vormann, M. Schweigel-Röntgen, Am. J. Physiol . 2012 , 302 , C318–C326. 62. A. Romani, in Physiology of Magnesium Homeostasis , Eds R. Vink, M. Nechifor, Univ. of Adelaide Publishing Co., Adelaide, Australia, 2011, pp. 13–58. 3 Magnesium in Health and Disease 77

63. R. T. Alexander, J. G. Hoenderop, R. J. Bindels, J. Am. Soc. Nephrol. 2008 , 19 , 1451–1458. 64. R. M. Touyz, Y. He, A. C. I. Montezano, G. Yao, V. Chubanov, T. Gudemann, G. E. Callera, Am. J. Physiol. 2006 , 290 , R73–R78. 65. A. Yogi, G. E. Callera, T. T. Antunes, R. C. Tostes, R. M. Touyz, Magnes. Res. 2010 , 23 , S207–S215. 66. G. A. Quamme, Curr. Opin. Gastroenterol . 2008 , 24 , 230–235. 67. L. M. Torres, C. Cefaratti, B. Perry, A. Romani, Mol. Cell. Biochem. 2006 , 288 , 191–199. 68. S. J. Kim, H. S. Kang, M. S. Kang, X. Yu, S. Y. Park, I. S. Kim, N. S. Kim, S. Z. Kim, Y. G. Kwak, J. S. Kim, Biochem. Biophys. Res. Commun. 2005 , 333 , 1132–1138. 69. A. Romani, A. Scarpa, Nature 1990 , 346 , 841–844. 70. A. Romani, A. Scarpa, FEBS Lett. 1990 , 269 , 37–40. 71. A. M. Romani, Recent Pat. Biotechnol . 2012 , 6 , 212–222. 72. T. E. Fagan, A. Romani, Am. J. Physiol. 2000 , 279 , G943–G950. 73. V. Gaussin, P. Gailly, J.-M. Gillis, L. Hue, Biochem J. 1997 , 326 , 823–827. 74. P. Dalal, A. Romani, Metabolism 2010 , 59 , 1663–1671. 75. T. E. Fagan, C. Cefaratti, A. Romani, Am. J. Physiol. 2004 , 286 , E184–E193. 76. W. Jahnen-Dechent, M. Ketteler, Clin. Kidney J. 2012 , 5 , i3–i14. 77. J. F. Lu, C. H. Nightingale, Clin. Pharmacokinet . 2000 , 38 , 305–314. 78. E. Ishimura, S. Okuno, T. Yamakawa, Magnes. Res. 2007 , 20 , 237–244. 79. E. Ishimura, S. Okuno, K. Kitatani, Clin. Nephrol. 2007 , 68 , 222–227. 80. S. Salem, H. Bruck, F. H. Bahlmann, Am. J. Nephrol. 2012 , 35 , 31–39. 81. R. Whang, E. M. Hampton, D. D. Whang, Ann. Pharmacother. 1994 , 28 , 220–226. 82. S. M. al-Ghamdi, E. C. Cameron, R. A. Sutton, Am. J. Kidney Dis. 1994 , 24 , 737–752. 83. K. Hoshino, K. Ogawa, T. Hishitani, T. Isobe, Y Eto, J. Am. Coll. Nutr. 2004 , 23 , 497S–500S. 84. S. Young, E. M. L. Goh, U. H. McKillop, C. F. Stanford, D. P. Nicholls, E. R. Trimble, Br. J. Clin. Pharmac. 1991 , 32 , 717–721. 85. M. Zehender, T. Meinertz, T. Faber, J. Am. Coll. Cardiol . 1997 , 29 , 1028–1034. 86. K. Hoshino, K. Ogawa, T. Hishitani, T. Isobe, Y. Etoh, Pediatr. Int. 2006 , 48 , 112–117. 87. M. Seelig, Am. J. Cardiol. 1989 , 63 , 4G– 21G. 88. B. Lichodziejewska, J. Kłos, J. Rezler, K. Grudzka, M. Dłuzniewska, A Budaj, L. Ceremuzynski, Am. J. Cardiol. 1997 , 79 , 768–772. 89. J. Shepherd, J. Jones, G. K. Frampton, L. Tanajewski, D. Turner, A. Price, Health Technol. Assess. 2008, 12 (28), iii-iv, ix-95. 90. M. Houston, J. Clin Hypert. 2011 , 13 , 843–847. 91. R. M. Touyz, F. J. Milne, H. C. Seftel, S. G. Reinach, S. Afr. Med. J. 1987 , 72 , 377–381. 92. J. T. Wright, Jr, M. Rahman, A. Scarpa, M. Fatholahi, V. Grifﬁ n, R. Jean-Baptiste, M. Islam, M. Eissa, S. White, J. G. Douglas, Hypertension 2003 , 42 , 1087–1092. 93. R. M. Touyz, F. J. Milne, S. G. Reinach, Clin. Exp. Hypertens. A 1992, 14 , 1189–209. 94. B. M. Altura, B. T. Altura, A. Carella, P. D. Turlapaty, Artery 1981 , 9 , 212–231. 95. L. Resnick, Prog. Cardiovasc. Dis. 1999 , 42 , 1–22. 96. T. F. Lüscher, B. Waeber, J. Cardiovasc. Pharmacol. 1991 , 18 (Suppl. 3), S1–S3. 97. L. Kass, J. Weekes, L. Carpenter, Eur. J. Clin. Nutr. 2012 , 66 , 411–418. 98. W. Abebe, M. S. Mozaffari, Am. J. Cardiovasc. Dis . 2011 , 1 , 293–311. 99. F. Nakamura, R. D. Minshall, G. C. Le Breton, S. F. Rabito, Hypertension 1996 , 28 , 444–449. 100. K. Satake, J. D. Lee, H. Shimizu, H. Uzui, Y. Mitsuke, H. Yue, T. Ueda, Magnes. Res. 2004 , 17 , 20–27. 101. R. Landau, J. A. Scott, R. M. Smiley, Brit. J. Obstetr. Gynaecol. 2004 , 111 , 446–451. 102. N. Soltani, M. Keshavarz, H. Sohanaki, S, Zahedi Asl, A. R. Dehapour, Eur. J. Pharmacol. 2005 , 508 , 177–181. 103. R. M. Touyz, G. Yao, J. Cell Physiol. 2003 , 197 , 326–335. 104. Y. Rayssiguier, E. Gueux, D. Weiser, J. Nutr. 1981 , 111 , 1876–1883. 105. N. M. Kaplan, Am. Fam. Physician 1998 , 58 , 1323–1330. 106. F. R. Beyer, H. O. Dickinson, D. J. Nicolson, G. A. Ford, J. Mason, Cochran Database Syst. Rev . 2006 , 3 , CD004805. 78 Romani

107. R. M. Touyz, Am. J. Physiol . 2008 , 294 , H1103–H1118. 108. A. Yogi, G. E. Callera, S. E. O'Connor, Y. He, J. W. Correa, R. C. Tostes, A. Mazur, R. M. Touyz, J. Hypertens. 2011 , 29 , 1400–1410. 109. R. Horton, E. G. Biglieri, J. Clin. Endocrinol. Metab . 1962 , 22 , 1187–1192. 110. A. Ferreira, A. Rivera, J. R. Romero, J. Cell Physiol. 2004 , 199 , 434–440. 111. M. D. Resh, R. A. Nemenoff, G. Guidotti, J. Biol. Chem . 1980 , 255 , 10938–10945. 112. Y. Song, Y. H. Hsu, T. Niu, J. E. Manson, J. E. Buring, S. Liu, BMC Med. Genet . 2009 , 10:4; DOI: 10.1186/1471-2350-10-4 . 113. L. M. Torres, J. Youngner, A. Romani, Am. J. Physiol. 2005 , 288 , G195–G206. 114. A. Romani, V. Matthews, A. Scarpa, Circ. Res. 2000 , 86 , 326–333. 115. J. C. Henquin, T. Tamagawa, M. Nenquin, M. Cogneau, Nature 1983 , 301 , 73–74. 116. G. Reed, C. Cefaratti, L. N. Berti-Mattera, A. Romani, J. Cell. Biochem. 2008 , 104, 1034–1053. 117. C. Cefaratti, A. Romani, Metabolism 2003 , 52 , 1464–1470. 118. A. Suarez, N. Pulido, A. Casla, B. Casanova, F. J. Arrieta, A. Rovira, Diabetologia 1995 , 38 , 1262–1270. 119. L. M. Resnick. B. T. Altura, R. K. Gupta, J. H. Laragh, M. H. Alderman, B. M. Altura, Diabetologia 1993 , 36 , 767–770. 120. M. Barbagallo, L. J. Dominguez, Arch. Biochem. Biophys. 2007 , 40–47. 121. J. Nadler, S. Scott, Biochem. Biophys, Res. Commun. 1994 202 , 416–421. 122. S. A. Ewis, M. S. Abdel-Rahman, J. Appl. Toxicol. 1995 , 15 , 387–390. 123. F. Guerrero-Romero, M. Rodriguez-Moran, Arch. Med. Res . 2005 , 250–257. 124. L. M. Resnick, Am. J. Hypertens. 1993, 6 , 123S–134S. 125. D. Vykoukal, M. G. Davies, J. Vascular Surgery 2011 , 54 , 819–831. 126. P. A. Tessman, A. Romani, Am J. Phyisol. 1998 , 275 , G1106–1116. 127. A. Young, C. Cefaratti, A. Romani, Am. J. Physiol . 2003 , 284 , G57–G67. 128. A. Young, L.N. Berti-Mattera, A. Romani, Alcohol. Clin. Exp. Res . 2007 , 31 , 1240–1251. 129. L. M. Torres, C. Cefaratti, L. Berti-Mattera, A. Romani, Am. J. Physiol . 2009 , 297 , G621–G631. 130. L. M. Torres, B. Konopnika, L. N. Berti-Mattera, C. Liedtke, A. Romani, Alcohol. Clin. Exp. Res. 2010 , 34 , 1659–1669. 131. E. Gonzales-Reimers, A. Conde-Martel, F. Santolaria-Fernandez, A. Martinez-Riera, F. Rodriguez-Moreno, T. González-Hernández, V. V. Castro-Aleman, Alcohol 1993, 28 , 311–318. 132. Z.-W. Yang, J. Wang, T. Zheng, B. T. Altura, B. M. Altura, Alcohol 2001 , 24 , 145–153. 133. A. N. Babu, T. P. Cheng, A. Zhang, B. T. Altura, B. M. Altura, Brain Res. Bull. 1999 , 50 , 59–62. 134. B. M. Altura, B. T. Altura, Alcohol 1999 , 19 , 119–130. 135. A. Romani, Magnes. Res. 2008 , 21 , 197–204. 136. A. Mazur, J. A. Maier, E. Rock, E. Gueux, W. Nowacki, Y. Rayssiguier, Arch. Biochem. Biophys. 2007 , 458 , 48–56. 137. M. Adachi, D. A. Brenner, Dig. Dis. 2005 , 23 , 255–263. 138. M. E. Maguire, Trends Pharmacol. Sci. 1984 , 5 , 73–77. 139. Y. Konno, S. Ohno, Y. Akita, H. Kawasaki, K. Suzuki, J. Biochem. 1989 , 106 , 673–678. 140. W. F. Waas, K. N. Dalby, Biochemistry 2003 , 42 , 2960–2970. 141. R. A. Brown, M. Crawford, M. Natavio, P. Petrowski, J. Ren, Alcohol. Clin. Exp. Res . 1998 , 22 , 2062–2072. 142. S. Ferrè, E. Baldoli, M. Leidi, J. A. Maier, Biochim. Biophys. Acta 2010 , 1802 , 952–958. 143. C. Malpuech-Brugere, W. Nowacki, E. Rock, E. Gueux, A. Mazur, Y. Rayssiguier, Biochim. Biophys. Acta 1999 , 1453 , 35–40. 144. N. C. Shah, J. P. Liu, J. Iqbal, M. Hussain, X.-C. Jiang, Z. Li, Y. Li, T. Zheng, W. Li, A. C. Sica, J. L. Perez-Albela, B. T. Altura, B. M. Altura, Int. J. Clin. Exp. Med. 2011 , 4 , 103–118. 145. B. M. Altura, N. C. Shah, G. Shah, A. Zhang, W. Li, T. Zheng, J. L. Perez-Albela, B. T. Altura, Am. J. Phyisol. 2012 , 302 , H319–H332. 146. J. Sugimoto, A. M. Romani, A. M. Valentin-Torres, A. A. Luciano, C. M. Ramirez Kitchen, N. Funderburg, S. Mesiano, H. B. Bernstein, J. Immunol. 2012, 188 , 6338–6346. 3 Magnesium in Health and Disease 79

147. H. Suzuki-Kakisaka, J. Sugimoto, M. Tetarbe, A. M. Romani, C. M. Ramirez Kitchen, H. B. Bernstein, Am. J. Reprod. Immunol. 2013 , 70, 213–220. 148. H. Dimke, G. J. Hoenderop, R. J. Bindels, Clin. Sci . 2010 , 118 , 1–18. 149. J. M. Topf, P. T. Murray, Rev. Endocr. Metab. Disord . 2003 , 4 , 195–206. 150. F. C. Bartter, P. Pronove, J.R. Gill, Jr., M. R. C. Maccardle, Am. J. Med. 1962 , 33 , 811–828. 151. N. Jeck., M. Konrad, M. Peters, S. Weber, K. E. Bonzel, H. W. Seyberth, Pediatr. Res. 2000 , 48 , 754–758. 152. H. J. Gitelman, J. B. Graham, L. G. Welt, Trans. Assoc. Am. Physicians 1966 , 79 , 221–235. 153. H. Rubin, Proc. Natl. Acad. Sci. USA 1975 72 , 3551–3555. 154. H. Rubin, Arch Biochem. Biophys . 2007 , 458 , 16–23. 155. A. Sgambato, F. I. Wolf, B. Faraglia, A. Cittadini, J. Cell Physiol. 1999 , 180 , 245–254. 156. A. Ikari, H. Sawada, A. Sanada, C. Tonegawa, Y. Yamazaki, J. Sutanagi, J. Cell Biochem . 2011 , 112 , 3563–3572. 157. F. I. Wolf, V. Trapani, M. Simonacci, A. Boninsegna, A. Mazur, J. A. M. Maier, Nutr. Cancer 2009 , 61 , 1131–1136. 158. A. Nasulewicz, J. Wietrzyk, F. I. Wolf, S. Dzimira, J. Madej, J. A. Maier, Biochim. Biophys. Acta 2004 , 1739 , 26–32. 159. S. Muallem, O. W. Moe, J. Clin. Invest . 2007 , 117 , 2086–2089. 160. K. A. Lapidos, E. C. Woodhouse, E. C. Kohn, L. Masiero, Angiogenesis 2001 , 4 , 21–28. 161. J. A. M. Maier, C. Malpuech-Brugere, W. Zimowska, Y. Rayssiguier, A. Mazur, Biochim. Biophys. Acta 2004 , 1689 , 13–24. 162. S. Ferrè, A. Mazur, J. A. M. Maier, Magnes. Res . 2007 , 20 , 66–71. 163. W. Ma, J. Chen, X. Xue, Z. Wang, H. Liu, T. Wang, Biochem. Biophys. Res. Commun . 2008 , 371 , 425–430. 164. P. Scheans, Neonatal Network 2012 , 31 , 121–124. 165. D. A. Grimes, K. Nanda, Obstet. Gynecol . 2006 , 108 , 986–989. 166. P. G. Pryde, R. Mittendorf, Obstet. Gynecol . 2009 , 114 , 669–673. 167. Center for Disease Control and Prevention. Cerebral Palsy: http://www.cdc.gov/ncbddd/cp/ index.html 168. K. B. Nelson, J. K. Grether, Pediatrics 1995 , 95 , 263–269. 169. L. W. Doyle, C. A. Crowther, P. Middleton, S. Marret, Cochrane Database Syst. Rev . 2009 , 1 , CD004661. 170. American College of Obstetricians and Gynecologists Committee on Obstetric Practice, Obstet. Gynecol . 2010 , 115 , 669–671. 171. J. P. F. Bai, E. Hausman, R. Lionberger, X. Zhang, Mol. Pharmac . 2012 , 9 , 3495–3505. 172. http://www.fda.gov/drugs/drugsafety/ucm245011.htm 173. M. W. Hess, J. G. Hoenderop, R. J. M. Biondels, J. P. H. Drenth, Aliment. Pharmacol. Therap . 2012 , 36 , 405–413. 174. T. Cundy, A. Dissanayake, Clin. Endocrinol. (Oxford) 2008 , 69 , 338–341. 175. I. Vivancoa, K. Mellinghoff, Curr. Op. Oncol. 2010 , 22 , 573–578. 176. P. Maliakal, A. Ledford, Exp. Therap. Med . 2010 , 1 , 307–311. 177. D. Schrag, K. Y. Chung, C. Flombaum, L. Saltz, J. Natl. Cancer Inst . 2005 , 97 , 1221–1224. Chapter 4 Calcium in Health and Disease

Marisa Brini , Denis Ottolini , Tito Calì , and Ernesto Carafoli

Contents ABSTRACT ...... 82 1 INTRODUCTION ...... 83 1.1 Calcium in Nature and in Living Organisms ...... 83 1.2 Regulation of Calcium in Biological Fluids ...... 84 1.3 Calcium in the Mineralized Compartment of the Organisms ...... 85 2 GENERAL PROPERTIES OF CALCIUM AS A SIGNALING AGENT ...... 88 3 INTRACELLULAR CALCIUM HANDLING ...... 93 3.1 Transport of Calcium Across Membrane Boundaries ...... 93 3.2 Spatiotemporal Dynamics of the Calcium Signal ...... 94 3.3 Regulation of the Calcium Signal by the Cell Organelles ...... 97 4 CALCIUM AS A REGULATOR OF BIOLOGICAL PROCESSES ...... 100 4.1 Gene Transcription ...... 100 4.2 Intracellular Proteolysis ...... 101 4.3 Protein Phosphorylation and Dephosphorylation ...... 103 4.4 Calcium and Bioenergetics ...... 106 4.5 Muscle Contraction ...... 108 4.6 Secretion ...... 110 4.7 Calcium in the Beginning of Cell Life ...... 112 4.8 Apoptotic Cell Death and Autophagy ...... 113

M. Brini (*) • D. Ottolini • T. Calì Department of Biology , University of Padova , Via U. Bassi 58/B , I-35131 Padova , Italy e-mail: [email protected] E. Carafoli (*) Venetian Institute of Molecular Medicine (VIMM) , Via G. Orus 2 , I-35129 Padova , Italy e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 81 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_4, © Springer Science+Business Media Dordrecht 2013 82 Brini, Ottolini, Calì, and Carafoli

5 THE AMBIVALENCE OF THE CALCIUM SIGNAL: DEFECTS OF CALCIUM REGULATION AND DISEASE ...... 116 5.1 Neuronal Diseases...... 116 5.1.1 Ataxia ...... 116 5.1.2 Migraine ...... 118 5.2 Neurodegenerative Diseases ...... 118 5.2.1 Parkinson’s Disease ...... 119 5.2.2 Alzheimer’s Disease ...... 120 5.2.3 Huntington’s Disease ...... 120 5.2.4 Amyotrophic Lateral Sclerosis ...... 121 5.3 Genetic Hearing Loss...... 122 5.4 Cardiac Diseases (Cardiomyopathies) ...... 123 5.5 Skeletal Muscle Diseases ...... 124 5.5.1 Malignant Hyperthermia ...... 124 5.5.2 Central Core Disease ...... 125 5.5.3 Brody’s Disease ...... 125 5.5.4 Duchenne Muscular Dystrophy ...... 125 6 CONCLUSIONS ...... 126 ABBREVIATIONS ...... 127 ACKNOWLEDGMENTS ...... 129 REFERENCES ...... 130

Abstract Evolution has exploited the chemical properties of Ca2+ , which facilitate its reversible binding to the sites of irregular geometry offered by biological macromolecules, to select it as a carrier of cellular signals. A number of proteins bind Ca2+ to speciﬁ c sites: those intrinsic to membranes play the most important role in the spatial and temporal regulation of the concentration and movements of Ca2+ inside cells. Those which are soluble, or organized in non-membranous structures, also decode the Ca2+ message to be then transmitted to the targets of its regulation. Since Ca2+ controls the most important processes in the life of cells, it must be very carefully controlled within the cytoplasm, where most of the targets of its signaling function reside. Membrane channels (in the plasma membrane and in the organelles) mediate the entrance of Ca2+ into the cytoplasm, ATPases, exchangers, and the mitochondrial Ca 2+ uptake system remove Ca 2+ from it. The concentration of Ca 2+ in the external spaces, which is controlled essentially by its dynamic exchanges in the bone system, is much higher than inside cells, and can, under conditions of pathology, generate a situation of dangerous internal Ca2+ overload. When massive and persistent, the Ca2+ overload culminates in the death of the cell. Subtle conditions of cellular Ca2+ dyshomeostasis that affect individual systems that control Ca 2+, generate cell disease phenotypes that are particularly severe in tissues in which the signaling function of Ca2+ has special importance, e.g., the nervous system.

Keywords bones • calcium binding proteins • calcium regulated functions • calcium signaling • calcium transporters • cardiomyopathies • muscle diseases • neurodegenerative diseases • teeth

Please cite as: Met. Ions Life Sci. 13 (2013) 81–137 4 Calcium in Health and Disease 83

1 Introduction

1.1 Calcium in Nature and in Living Organisms

Calcium is the third most abundant metal in nature: it follows aluminium, which is by far the most abundant, and sodium, and it is followed by magnesium. Na, Ca, Mg, and Fe are found in nature in comparable abundance, each of them making up about 3% of the earth’s crust [ 1 , 2 ]. However, in the earth’s mantle, i.e., the layer immediately underneath the crust, Mg2+ is instead much more abundant than Ca2+ . Ca2+ is found in rocks, soil, and waters: in the sea its concentration is about 10 mM (however, in sea water Mg2+ is about fi ve fold more abundant than Ca2+ ). In nature, Ca2+ is present in salts of various compositions. In living organisms these salts have long been known to be essential in the formation of skeletal structures: in higher organisms, Ca2+ phosphate is the major salt of bones and teeth, whereas in lower organisms other salts, e.g., Ca2+ carbonates and sulfates, are the major contributors to skeletal or other structural components. In plants, Ca2+ oxalate precipitates are found, and Ca2+ picolinate is abundant in spore-forming microorganisms [3 , 4 ]. In animal organisms there is a large difference between the concentration of Ca 2+ in the body fl uids and extracellular spaces and that within cells: this difference is the basis for the signaling role of Ca2+ that will be discussed below. In man, the concentration of Ca2+ in plasma is between 2.1 and 2.6 mM [5 ] and is in the same mM range in most extracellular spaces, including the lymph, which is considered equivalent to the extracellular spaces. However, there are signifi cant exceptions, a prominent one being for instance the endolymph of the inner ear, where the concentration of extracellular Ca2+ is in the low μM range. An important problem is the relationship between total and free (ionized) Ca 2+ , which may vary from fl uid to fl uid and, in any case, is not easy to determine. Ca2+ exists in at least three basic forms: ionized, complexed to organic compounds, and bound (precipitated) in the inorganic salts mentioned above. An equilibrium exists between these forms, which is regulated by hormones (see below) and diet, and of course by the rules of chemistry. For instance, in blood plasma (where most Ca2+ of the blood is found) Ca 2+ is divided roughly equally between the ionized and complexed forms. By contrast, in milk, which contains about 30 mM total Ca2+ , about 2 mM is free Ca2+ , about 20 mM is associated with casein micelles, and about 8 mM is Ca 2+ bound to phosphate (Ca2+ -hydrogen phosphate) and citrate. The cerebrospinal fl uid is also worth mentioning, because of its unusually large percentage of ionized Ca2+ : 1.1 mM ionized versus 1.4 mM total (i.e., about 80% is ionized). Especially large differences between free and total Ca2+ are found in the intracellular ambient, but the matter of the intracellular space, where not only Ca2+ binding ligands but also organellar transport and storage are active in determining the ratio between free (ionized) and bound Ca2+ , has special complexities. It will be discussed in more detail later on. 84 Brini, Ottolini, Calì, and Carafoli

1.2 Regulation of Calcium in Biological Fluids

The concentration of Ca2+ in the blood of mammals (including humans), on which the concentration of Ca2+ in the extracellular spaces and, eventually, inside cells depends, is regulated by three hormones: parathyroid hormone, calcitonin, and the active form of vitamin D (1,25-OH vitamin D3, calcitriol). The major function of parathyroid hormone is to increase Ca2+ in the blood: in the absence of parathyroid hormone plasma Ca 2+ may decrease by up to 50%, whereas an excess of parathyroid hormone results in hypercalcemia. The hormone maintains the blood plasma Ca2+ concentration by acting on the bones, the kidney, and the intestine. In bones, it regulates the dynamic equilibrium between Ca2+ adsorbed to the surface of bones 2+ [in the form of Ca10 (PO4 )6 (OH)2 ] and Ca in plasma by promoting its outfl ow from the bone. In the absence of the hormone, the reverse process is favored, leading to a lowering of blood Ca 2+. In the kidneys, parathyroid hormone decreases the excretion of Ca 2+ essentially by promoting its reabsorption from the glomerular fi ltrate, but also, indirectly, by promoting the production of calcitriol through the action of renal 25-hydroxyvitamin D1 α-hydroxylase. The resulting calcitriol then increases the intestinal absorption of Ca2+ (see below). The stimulation of the renal hydroxylase is the indirect mechanism by which parathyroid hormone promotes the absorption of Ca2+ . Calcitonin lowers plasma Ca 2+ by inhibiting osteoclasts’ motility and spreading. Osteoclasts are the target cells of the hormone: when incubated with calcitonin, they rapidly loose the ruffl ed borders typical of resorbing bone, decreasing the fl ow of Ca2+ from bones to blood (osteoblasts and osteoclasts will be discussed in detail in Section 1.3 ). Calcitonin decreases the intestinal absorption of Ca 2+ , and inhibits its reabsorption by kidney tubules. The level of calcitonin in blood increases when plasma Ca2+ rises, and drops when it decreases and its secretion by the producing cells (the C cells of the thyroid) has been proposed to be stimulated by the gastrointestinal hormones. The active forms of the vitamin D endocrine system are the derivatives 1,25(OH)2D, calcitriol, which is the major biologically active form, and 24,24(OH)2D3: the latter is produced in the kidney by the activation of the 24-hydroxylase by calcitriol, and has a role in bone development and parathyroid function. Calcitriol increases bone mineralization by increasing the absorption of Ca2+ in the intestine, i.e., it makes more of it available to osteoblasts. It also stimulates the proliferation of the osteoblasts. The increased absorption of Ca2+ in the intestine is due to the promotion of the expression of the Ca2+ -binding protein calbindin, an EF-hand protein of which two forms exist: calbindin D-28k, which has 6 EF-hand Ca2+ -binding motifs (of which, however, only four are operationally active), and calbindin D-9k, which has two EF-hand motifs. Calbindin D-9k is closely related to the S-100 Ca2+ -binding proteins, and is considered to be a member of their family. Calbindin D-28k, instead, has only minimal sequence homology to calbindin D-9k and to the S-100 proteins, and is closely related to calretinin, which also has 6 EF-hand motifs. Calcitriol stimulates the synthesis of calbindins by acting on a specifi c nuclear receptor (the VDR receptor) that recognizes a 4 Calcium in Health and Disease 85 specifi c response element in the promoter of the gene. Calbindin D-9k is very abundant in the intestinal mucosa, and present in smaller amounts in other tissues. Its concentration in the intestine parallels the rate of Ca2+ absorption. Calbindin D-28k is present in brain and numerous other tissues, including avian intestine. However, it is not expressed in mammalian intestine, and its expression in the brain is not vitamin D-dependent. The absorption of Ca2+ in the intestine occurs by a saturable active transport process dependent on vitamin D, and by a vitamin D-independent passive paracellular process (it has been claimed that also the passive paracellular absorption is stimulated by vitamin D). The saturable transport process consists of the penetration of Ca2+ into the mucosal cell through luminal Ca2+ channels, of its buffering by calbindin D-9k (calbindin D-28k as well in birds) which increases the rate of its trans- cytosolic diffusion to the basolateral membrane, and by its ejection to the extracellular fl uid of the lamina propria by the basolateral plasma membrane Ca2+ ATPase. Calcitriol stimulates the expression of the Ca2+ entry channels, of calbindin D-9k, and of the Ca2+ -exporting plasma membrane pumps.

1.3 Calcium in the Mineralized Compartment of the Organisms

About 1.5 billion years ago, a large transfer of geologic minerals (including CaCO 3 ) occurred into the oceans due to the violent moves of tectonic plates. The natural selection forced the living organisms of the sea to develop more protective body parts (such as shells or scales) to cope with the new mineral-rich environment. The evolution of exoskeletons increased enormously the pace of animal evolution but limitations such as small body size, lack of surface sensory organs and reduced movement/locomotion inspired a new evolutionary step culminating with the dislocation of mineralized skeleton from the outside to the inside of animal bodies, as well as with the replacement of the Ca2+ carbonate, used to build marine exoskeletons, with the chemically more stable 2+ 2+ Ca phosphate Ca3 (PO4 )2 in the form of Ca hydroxyapatite Ca5 (PO4 )3 (OH) (usually written Ca10 (PO 4 ) 6 OH2 ) [6 , 7 ]. The development of endoskeletons (bones and teeth) gave vertebrates improved mobility and mechanical competence. It also provided them with a ready source of key inorganic ions like Ca2+ , Mg2+ , and phosphate. The earliest mineralized structures in the vertebrate lineage were tooth-like structures in the mouth or in the skin arranged to form a protective shield, while the ﬁ rst endoskeleton appeared as cartilagineous and gradually evolved through the process of endochondral ossiﬁ cation [8 ]. Mineralized tissues are composite structures consisting of an inorganic mineral phase, an organic phase, and cells. The crystals in bone have a length of ~20–50 nm and a width of 12–20 nm, depending on age and species; in dentin they are of similar size, but enamel crystals are ~10 times larger [9 , 10]. Bone apatite nanocrystals exhibit a variety of substitutions and vacancies that make the Ca/P molar ratio distinct from the stoichiometric hydroxyapatite ratio of 1.67 [11 ]. 86 Brini, Ottolini, Calì, and Carafoli

The cell-produced organic matrices speed up biomineralization and, as crystals grow, the association with proteins, such as osteocalcin, also regulates bone remodeling, maintaining the hydroxyapatite bone mineral in a dynamic state. Dentin mineral is remodeled to a much lesser extent, although the roots are remodeled as a response to disease, and the remodeling is under cellular control. Enamel mineral is not remodeled, but the enamel matrix is degraded as mineralization takes place. With time, depending on tissue site and animal diet, bone and dentin mineral pro- 2 − gresses from a poorly crystalline apatite with high HPO4 content and a low level of crystallinity to a mineral with somewhat higher crystallinity, lower acid phosphate content, and a more organized structure, albeit with more carbonate substitutions [12 ,13 ]. Bone and dentin consist of apatite crystals deposited in an oriented fashion on a collagen scaffold. Type I collagen is predominant and is associated with bone, dentin, cementum, skin, ligaments, and tendons. Collagen is an insoluble fi brous protein consisting of three polypeptide chains wound into a repeating triple- helical fi bril [14 ]. The fi brils line up head-to-tail to form repeating arrays on which the mineral particles align with their long axes parallel to the fi bril axis. The apatite crystals deposit fi rst within the holes and then spread throughout the matrix [ 15 ], the initial mineralization occurring at the cellular level. Osteoblasts and odontoblasts control the production and mineralization of the extracellular collagen protein matrix in bone and teeth, osteoclasts instead remove bone mineral and bone matrix. Thus, bone cells regulate the formation and resorption of bone, which is a key step in regulating body Ca2+ (body Mg2+ and phosphate as well). In the bone formation phase, clusters of osteoblasts on the bone surface produce the bone matrix constituents by rapidly depositing collagen on which, after maturation of osteoid matrix, mineralization occurs. At the end of the matrix-secreting period, osteoblasts entrapped in the new bone matrix differentiate into osteocytes, characterized by long cell processes rich in microfi laments that form a network permeating the entire bone matrix during its formation and before its calcifi cation. The exact function of osteocytes is still unclear but they possibly respond to bone tissue strain and enhance bone remodeling activity by recruiting osteoclasts to sites where bone remodeling is required [ 16]. As mentioned above, osteoclasts are responsible for bone resorption. They lower the pH within the bone-resorbing compartment to as low as 4.5 thanks to the action of their plasma membrane H+ pump, which helps mobilize bone mineral [17 ] as they secrete lysosomal enzymes like acid phosphatase, cathepsin K, matrix metalloproteinase 9, and gelatinase [18 ] that digest the organic matrix. During resorption, mature osteoclasts absorb vast amounts of Ca2+ and their survival is ensured via the Na+ /Ca2+ exchangers, which extrude Ca2+ into the extracellular space preventing the development of deleterious Ca 2+ overload in their cytoplasm [19 , 20 ]. Interestingly, recent evidence has shown that isoforms 1 and 4 of the plasma membrane Ca2+ ATPases also mediate Ca2+ extrusion from mature osteoclasts contributing to their differentiation and survival [21 ]. The function of osteoblasts and osteoclasts is regulated locally by cytokines and by systemic hormones (see Section 1.2 ) ([ 22 , 23 ] and references therein). Vitamin D maintains general Ca2+ homeostasis (see above) by acting on kidneys and small intestine but also directly 4 Calcium in Health and Disease 87 on bones, where it increases osteoclastic resorption [24 ]. The parathyroid (PTH) hormone also stimulates bone resorption (but also stimulates bone formation when administered intermittently). The secretion of PTH is controlled by a negative feedback of Ca 2+ on the parathyroid gland cells through the plasma membrane Ca2+ sensor [25 ]. The end result is a constant Ca 2+ concentration in the extracellular fl uid due to the maintenance of the appropriate concentration of PTH in body fl uids [25 ]. Biomineralization is thus a dynamic process characterized by the constant equilibrium between crystal deposition and removal. The chemistry of bone hydroxy- 2+ − apatite requires that bone formation includes a supply of Ca and H 2 PO4 and some way to dispose of 1.4 H + per each Ca 2+ equivalent deposited. The source of Ca 2+ is obviously the extracellular fl uid, but the metal must be taken up by osteoblasts and the mechanism of this process is still poorly understood. Calbindin [ 26] has been suggested to have a role but calbindin-negative osteoblasts still transport Ca 2+ at a normal rate [27 ]. As for phosphate, osteoblasts express high levels of alkaline phosphatase (ALP), that cleaves the pyrophosphate produced within the osteoclasts [ 28 , 29 ] and is likely to have a role. An additional transport mechanism that removes H + is required for the rapid deposition of hydroxyapatite. High concentrations of phosphate and Ca2+ at neutral pH will form an initial precipitate, but mineral formation becomes limited as the pH falls below 5.6 [ 30]. A sodium-hydrogen exchange appears to be the system that holds the pH at slightly elevated levels to permit mineral deposition [ 31 ]. The biggest obstacle in the understanding of the molecular process of bone formation is the mechanism by which osteocytes cause the precipitation of Ca2+ phosphate. The solubility limit of Ca2+ phosphate in human bone is approximately 7 × 10–5 M at physiological pH and temperature. The concentration of free Ca 2+ in the –3 3 − plasma and extracellular fl uids (ECF) is higher than 10 M, that of PO4 ion, mostly 2 − in the form of the HPO4 , is very low in plasma. Under these conditions, when plasma or ECF come in contact with bone surfaces, plasma Ca2+ is supersaturated with respect to bone. Nevertheless, the free Ca2+ level in the ECF and plasma is still maintained at approximately 10 –3 M, without continuous deposition of hydroxyapatite crystals. Two main theories have been proposed: the fi rst relies on an organic or inorganic precursor seeding that directs the formation of apatite from soluble inorganic ions by the action of noncollagenous proteins, such as the SIBLING family of proteins [32 ], that would act at the surface of bones and teeth to modify the solubility of hydroxyapatite, thus promoting or inhibiting mineralization [ 33, 34]. The second theory is based on the formation of matrix vesicles (MV) as the initial site of primary nucleation in the mineralization of calcifi ed cartilage, bone, and dentin. MVs are extracellular vesicles of about 20–200 nm in diameter derived from the plasma membrane of mineral forming cells (chondrocytes, osteoblasts, and odontoblasts) [35 ] by a budding process. They are enriched in numerous proteins, among them annexins (A2, A5, and A6) [36 –38 ] and in phosphatidylserine, which facilitate Ca2+ -dependent annexin binding and enable annexins to form Ca2+ channels (see above) [39 , 40 ]. The MVs would initiate intravesicular mineral formation either by regulating the ratio of Pi to pyrophosphate (PPi) and by serving as nucleation sites for apatite deposition. Thus, mineralization would begin with the formation of hydroxyapatite crystals 88 Brini, Ottolini, Calì, and Carafoli within the MVs and proceed with the propagation of hydroxyapatite through the membrane into the extracellular matrix [32 ]. The hydroxyapatite would then be propagated in clusters around MVs and fi ll the space between collagen fi brils. PPi, which inhibits the formation of hydroxyapatite [41 ] formed by nucleotide pyrophosphatase/ phosphodiesterase 1 (NPP1) from nucleotide triphosphates, but also by ankylosis progressive homolog (ANKH, a homolog of the mouse progressive ankylosis (ank) gene product), would be hydrolyzed by ALP [42 ]. Other sites of intracellular mineral deposition are the mitochondria. Their interplay with Ca2+ will be discussed in Section 3 . For the discussion of their possible role in the biomineralization process it will be suffi cient to mention that in addition to Ca 2+ they accumulate inorganic phosphate [43 ], precipitating hydroxyapatite in the alkaline environment of their matrix. The precipitates are frequently seen as electron-dense granules within mitochondria that have accumulated massive amounts of Ca2+ and phosphate [44 ]. They are also observed within the mitochondria of cells that experience conditions of pathological cytosolic Ca 2+ overload and within the mitochondrial profi les of cells normally exposed to high Ca2+ traffi c in the cytosol, e.g., those of mineralized tissues [45 ]. The granules have been isolated and found to contain, in addition to hydroxyapatite, a number of organic components. Surprisingly, however, even under conditions of high saturation of Ca2+ and phosphate found in the mitochondrial matrix, they remain amorphous [46 ]. These amorphous granules have been suggested to be involved in the process of biological mineralization (it may be signifi cant that osteoclasts are rich in mitochondria) [45 ]: they could in other words be a means to store high concentrations of Ca2+ and phosphate in a non-crystalline and more readily available form. A hypothesis for their possible role has been proposed by Lehninger [46 ] who postulated that the amorphous granules would be somehow stabilized as micropackets by biological factor(s) and transported to mineralization sites where they would form apatitic bone mineral. Phosphocitrate (PC), which has been identifi ed in mammalian mitochondria, may be one factor involved in the process of granule stabilization [47 ] and in the prevention of Ca2+ phosphate precipitation in cells, or cellular compartments, that maintain a high concentration of Ca2+ and phosphate (e.g., the mitochondria).

2 General Properties of Calcium as a Signaling Agent

Na + and Ca2+ are the major cationic components of extracellular spaces, whereas K+ , Mg2+ (and Zn 2+ ) are the major intracellular metals. Within cells, nearly all of the K + and about 75% of the Na + is free, whereas a much higher proportion of the other three metals is present in bound forms. This is particularly true for Ca2+ , the ionized concentration of which within cells is a negligible fraction of its total concentration (see above). However, the issue of total and ionized Ca2+ inside cells is complex. The usual measurements of total cell Ca2+ yield values in the 1 to 10 mM range. These values cover ionized Ca2+ , and Ca2+ bound to the usual inorganic ligands and small molecular weight organic molecules, and to specifi c binding proteins. They cover 4 Calcium in Health and Disease 89 also, and especially, Ca 2+ sequestered within organelles like the endo(sarco)plasmic reticulum, the Golgi system and, under special conditions (see below), the mitochondria. The ionized Ca2+ in the cytosol, which is the cell compartment where most of the targets of its signaling function reside, is on the order of 100 nM. Clearly, this concentration is much lower than in all other districts of the organism, where the ionized/free ratio is solely determined by the action of “inert” Ca 2+ ligands. Within cells the control of Ca 2+ concentration is a dynamic operation: inert ligands like small molecular weight components and binding proteins do have a role in it, as they have in all other parts of the organism. But they could not possibly lower free Ca2+ in the ambient to values in the nM range. These extremely low concentrations are only achieved thanks to the concerted operation of membrane transporting systems (pumps, exchangers, and channels): their activity maintains (cytosolic) Ca2+ at the nM level, which is demanded by its signaling function. Transporters that exchange Ca2+ across the membrane barriers separating the intracellular organelles from the cytosol generate Ca2+ stores in the former that ensure the availability of the adequate supply of Ca2+ to the cytosol. As mentioned, the main Ca2+ -storing organelles are the endo(sarco)plasmic reticulum, the Golgi system and, under special conditions, the mitochondria. Very large amounts of Ca2+ are contained in the reticulum, in which the ratio of ionized versus bound Ca2+ can be considered similar to that of the extracellular spaces, yielding free Ca2+ concentrations in the mM range. Very likely, a similar situation also prevails in the Golgi system. Under conditions of normal cell life, mitochondria are not a quantitatively signifi cant Ca2+ store. Their matrix could be considered similar to the cytosol, with free Ca2+ concentrations in the nM range. Mitochondria, however, can accumulate very large amounts of Ca 2+ under the conditions of cytosolic Ca 2+ overload frequently occurring in pathology. They do so because they take up inorganic phosphate together with Ca 2+ (see above), precipitating amorphous hydroxyapatite in the matrix. Under these conditions, mitochondria become very large Ca2+ stores, but nearly all the Ca2+ they contain is unavailable for rapid exchanges with the cytosol. This massive accumulation of Ca2+ by mitochondria is an important defense device: it enables cells to clear out excess Ca 2+ from the cytosol, giving them the time to survive cytosolic Ca2+ storms. Irrespective of the difference in the various parts of the organism, the difference between free and bound Ca2+ in cells is determined by the unusual propensity of the metal to be ligated, which in turn refl ects its peculiar coordination chemistry. According to the rules of coordination chemistry the interaction of metal ions with coordinating ligands is determined by valency, which determines the charge of the metal, the ionic radius, the polarizability, i.e., the ease with which the electron cloud of the metal is distorted by external electrical forces, the hydration energy, which expresses the ease with which the attached water molecules are stripped off the metal, and the radius of the hydrated ion, which determines the charge density. The combination of these properties explains why Ca2+ is so easily complexed, and why it can fi t optimally in binding (coordination) sites of irregular geometry, such as those offered by biological molecules like proteins. One can for instance compare Ca 2+ to the other important Group 2 metal, Mg2+ (Table 1 ). 90 Brini, Ottolini, Calì, and Carafoli

Table 1 Some properties of un-hydrated and hydrated Ca2+ and Mg2+ . Ionic radius Polarizability Hydration energy Hydrated ions 24 3 Å α 0 × 10 cm kcal/g ion Å Ca 2+ 0.99 0.531 410 4.5 Mg 2+ 0.65 0.012 495 5.9

The smaller size of divalent Mg2+ and its very low polarizability value do not permit much ﬂ exibility in the geometry of the coordinating site (the ligands, as in the case of Ca2+ , are usually oxygens) which tends to be a more or less perfect octa- hedron: perfect octahedral cavities, naturally, do not easily come about in biological macromolecules. The properties of Ca2+ , by contrast, are compatible with coordinating sites of irregular geometry, as one expects to ﬁ nd in biological macromolecules. Synthetic low-molecular-weight compounds offer a visual representation of the differences in the coordinating demands of Ca2+ and Mg 2+ (Figure 1 ) [ 48]. The distance between the metal and the ligating oxygen atoms may vary by as much as 0.52 Å in the case of Ca2+ , but by only 0.12 Å in the case of Mg2+ .

Figure 1 Hypothetic comparison of the binding of Ca 2+ and Mg 2+ to an EF-hand protein motif. The chemical properties of the two ions described in Table 1 determine the higher ability of Ca2+ to ﬁ t into binding sites of irregular geometry.

In tissues and fl uids of animal organisms Ca2+ is ligated by a number of inorganic and organic low-molecular-weight molecules. Normally, this type of binding occurs with low affi nity. High affi nity (and specifi c) Ca 2+ binding, such as necessary for the 4 Calcium in Health and Disease 91 regulation of its signaling function demands coordinating sites of higher complexity, such as those offered by proteins. Evolution has developed several such motifs, some of them are present in hundreds of proteins: the regulation of the signaling function of Ca 2+ relies most frequently on three of them: the EF hand helix-loop- helix motif, the C2 motif, and the annexin Ca2+ -binding fold. The EF hand motif is found in a large number of protein families (about 66 sub-families are known [49 ]). The typical EF hand structure (Figure 2 ) consists of two α-helical domains inter- ● ● ● ● rupted by a loop usually containing 12 residues with the pattern X Y Z -Y - ●● ● X -Z; X,Y,Z,-X,-Y,-Z are the residues that coordinate Ca2+ , and the dots ( ) are residues not involved in the coordination: the residue that follows Z is a central invariant Gly. At positions X and Y the side chains of Asp or Asn contribute coordinating oxygens, whereas Asp, Asn, or Ser are found at position Z. A peptide carbonyl oxygen coordinates Ca2+ at position -Y, and a water oxygen usually coordinates Ca2+ at position -X. An Asp or a Glu are conserved residues at position -Z. EF hand motifs usually occur in pairs, but proteins have also been found that contain only one EF hand motif or an odd number of EF hand motifs: for instance, the C-terminal domain of the large subunit of calpain (see below) contains 5. However, this penta- EF hand domain forms a heterodimer with the small subunit of calpain that also contains 5 EF hand motifs.

Figure 2 Top: 3D structure of the calmodulin (CaM) (PDB fi le 3CLN) EF-hand domain (top left), of the synaptotagmin I (PDB fi le 1TJX) C2b motif (top middle), and of the full-length annexin A1 (PDB fi le 1MCX) (top right, repeats 1 to 4 are shown in red, yellow, purple, and green, respectively). The calcium ions are depicted as orange spheres and the residues involved in its coordination are shown as sticks. Bottom from left to right: typical EF-hand of CaM, synaptotagmin I C2 motif, and annexin I AB, AB’, and DE calcium-binding sites are shown with the coordinating residues (sticks). Calcium ions are green and water molecules cyan, distances in Å are shown as yellow dotted lines. 92 Brini, Ottolini, Calì, and Carafoli

The C2 domain, initially identifi ed in a Ca 2+ -dependent protein kinase, protein kinase C, is a 130 residues domain now found in numerous proteins involved in cell signaling and other important intracellular processes, e.g., membrane traffi cking [50 ]. The domain forms an eight-stranded antiparallel β-sandwich consisting of a pair of four-stranded β-sheets. Ca2+ is coordinated, in a depression formed at the edge of the β-sandwich by loops connecting β-sheets 2–3 and 6–7 (Figure 1 ), by carbonyl oxygens, mono- and bidentate Asp side chain oxygens, and a water oxygen. Annexins are a broad family of Ca2+ -dependent phospholipid-binding proteins that are widely distributed in eukaryotic cells [ 51 ]. They are intracellular, but some (e.g., annexins A1, A2, A5) are found outside of cells. They are involved in diverse biological processes, among them the inhibition of phospholipase, traffi cking of membrane vesicles (endo-exocytosis), Ca2+ channel formation, anchoring of other proteins to the cell membrane. Outside cells, annexins play roles in the mechanism of blood coagulation and in fi brinolysis, and are important actors in the antiinfl ammatory responses. All annexins are composed of a divergent N-terminal domain (the “head” region) and a conserved C-terminal domain (the “core” domain) [52 ]. The core domain contains 4 (8 in annexin A6) repeats with 5 α-helices A–E. Helices A, B, D, and E form a coiled-coil structure with the shape of a curved disk with loops connecting helices A and B, and D and E on the convex side of the disk. The loops harbor the Ca2+ -binding sites, whereas the N-terminal domain of the molecule is on the concave side of the disk, from which it is expelled upon binding of Ca2+ . Annexins contain 3 types of Ca 2+ binding sites: type II (AB loop), type III (DE loop), and the AB’ site (Figure 2 ). In site II 3 backbone carbonyl oxygens coordinate Ca2+ in a conserved sequence (M,L)-K-G-(A,L)-G-T. The side chain of an acidic residue 39 amino acids downstream the conserved sequence, coordinates Ca 2+ in a bidentate fashion, and two more coordinating oxygens are contributed by water molecules. In site III the coordination sphere comprises 2 backbone carbonyl oxygens from the DE loop which is not part of the conserved sequence. A bidentate acidic residue close in sequence and 3 water molecules complete the coordination sphere. The coordination sphere of the AB’ site comprises a backbone carbonyl oxygen downstream of the conserved sequence for the AB loop, the bidentate side chain of an acidic residue close by, and 5 water oxygens: the coordination in site AB’ is thus 8, rather than 7 as in sites II and III. Numerous X-ray structures of annexins are now available. Many contain empty or partially occupied Ca2+ -binding sites: annexin A1 has 6 to 8 Ca2+ ions bound, annexin A2 and A3 have 5, annexin A5 up to 10. The affi nity of annexins for Ca2+ is rather low, compared, for instance, to that of EF hands: this could be due to the large number of water oxygens involved in the coordination of Ca2+ . The ease with which Ca2+ can be ligated in sites of great geometric variability explains its evolutionary choice as a cellular signaling agent. As is self-evident, signaling agents must be bound reversibly by diverse molecules that control their concentration in the vicinity of the targets of their messenger function. The coordination chemistry properties discussed above permit the control to occur optimally for the case of Ca2+ : they would, by contrast, not permit it for that of Mg 2+ . There is one additional dividend to the evolutionary choice of Ca2+ as a signaling agent. As is self-evident, to prevent prohibitive energetic costs to modulate their concentration, signaling agents must be maintained within cells at very low free concentrations: 4 Calcium in Health and Disease 93 this is easily achieved in the case of Ca2+ , thanks to the ease with which it can be complexed. The resulting sub-μM concentration of cytosolic free Ca2+ prevents the precipitation of Ca 2+-phosphate salts, which would otherwise inevitably occur if both Ca2+ and phosphate were in the mM range. This has made possible the use of phosphate as a universal energy currency.

3 Intracellular Calcium Handling

3.1 Transport of Calcium Across Membrane Boundaries

The proteins containing the Ca2+ -binding motifs described in Section 2 are soluble, or associated to non-membranous structures. Their role in the regulation of cell Ca2+ is certainly important, but is quantitatively limited, as cell physiology may demand the (temporary) ligation of amounts of Ca2+ that could overcome their total Ca2+ - binding capacity. Cells, however, also contain a wealth of proteinaceous Ca2+ - binding systems that are intrinsic to membranes: they play the major role in the control of cell Ca 2+ , as they do not only bind Ca2+ , but also move it back and forth across the plasma membrane and the membranes of the organelles. They can “buffer” it even if present in the membranes in minute amounts. The systems that mediate the traffi c of Ca2+ across membranes are channels, ATPases (routinely termed pumps), exchangers (normally Na+ /Ca2+ -exchangers), and a specifi c mitochondrial electrophoretic transport system (the Ca2+ uniporter). These systems have been described in numerous detailed reviews, including one we have published in the preceding issue of the present series [ 53]. They will thus only be described very briefl y to facilitate the understanding of the discussions in the following Sections. The systems that mediate the entry of Ca2+ into the cytosol from the external spaces, or the lumen of the organelles, are homo- or hetero-polymeric protein complexes that can be gated, (i) by voltage across the plasma membrane, (ii) by the binding of specifi c external ligands to their extracellular portions (e.g., neurotransmitters in neurons), or, (iii) by the binding of ligands generated by stimulatory agonists in the cytosol. These ligands (second messengers) act on channels in the membrane of the endo(sarco)plasmic reticulum (ER, SR) and the Golgi system

(e.g., InsP3 ) and possibly on that of the acidic organelles. A fourth type of channels, which have been defi ned molecularly and functionally only recently, are the so called store-operated plasma membrane channels, that are activated by the empty- ing of the Ca2+ store in the ER [54 ]. They will have to be discussed in some more detail later on, since they are especially relevant to the content of this contribution. The Ca 2+ ATPases are located in the plasma membrane (the PMCA pump) and in the membrane of the ER, SR, and the Golgi system [55 ]. Their mechanism of transport is now understood in atomic detail thanks to the solution of the crystal structure of the sarcoplasmic reticulum pump (the SERCA pump). They are high Ca2+ affi nity transporters, i.e., they interact effi ciently with Ca2+ even in the sub-μM concentrations of the cytosol, and are regulated by a number of mechanisms, from phosphorylation processes that could be direct or mediated by 94 Brini, Ottolini, Calì, and Carafoli

accessory proteins, to the interaction with regulatory proteins, e.g., CaM in the case of the PMCA pump. The Na+ /Ca2+ -exchangers are present in the plasma membrane and in the inner membrane of mitochondria. They are lower Ca2+ affi nity systems, which transport bulk amounts of Ca 2+ , e.g., across the plasma membrane whenever the physiological need arises to rapidly extrude large amounts of Ca 2+ , e.g, in heart myocytes at the end of the contraction phase. Mitochondria take up Ca2+ by an electrophoretic system that responds to the membrane potential which is negative inside and maintained across the inner membrane by the operation of the respiratory chain. Its molecular identity has been clarifi ed only recently [56 ,57 ]: the system has very low affi nity for Ca 2+ , yet, it works effi ciently in the intracellular environment. This apparent paradox will be discussed in more detail later on, as it is central to the subject matter of this contribution. The acidic compartment of the cell has also recently been claimed to contain Ca2+ uptake and release systems (see for instance [53 ] for a recent review in which this aspect is mentioned). The Ca2+ release system from its organelles has been claimed to by a special channel type, the two-pore channel (TPC). The matter of the acidic compartment in the control of cell Ca2+ is a controversial issue, and will be discussed in more detail in Section 3.2 .

3.2 Spatiotemporal Dynamics of the Calcium Signal

Changes in the intracellular Ca2+ concentration regulate numerous important biological processes, ranging from cell origin to cell death. The versatility of Ca 2+ as an intracellular messenger depends on the tight control of its spatial and temporal distribution. The spatio-temporal pattern of Ca2+ signals is shaped by a sophisticated machinery that regulates precisely its amplitude and duration in a site-specifi c manner. The components of this machinery which include membrane transport systems and binding proteins have been succinctly described in Sections 2 and 3.1 . Their concerted operation generates cellular Ca2+ microdomains which have a distinct physical localization and specifi c functional signifi cance. The participation of intracellular organelles in the regulation of Ca2+ signaling has been introduced in Section 3.1 , and will be discussed in more detail later on. Excitable cells, i.e., neurons and muscle cells, are particularly responsive to the spatio-temporal regulation of the Ca2+ signal, as they are essential in the tuning of special processes such as contraction, secretion, synaptic transmission, etc. Not surprisingly, their dysregulation generates major human pathologies, e.g., cardiac and neurodegenerative diseases. Temporally, Ca2+ signals occur in two main forms: waves and oscillations, each of them generated by the specifi c activation of groups of membrane channels that are differently distributed throughout the cell. Propagating Ca 2+ waves originate mainly from the release of Ca 2+ from internal stores through the opening of intracellular channels, the most important being those in the InsP 3R and RyR. Polycistin-2 and TPC have also recently been claimed to release Ca2+ from the ER and the acidic Ca2+ 4 Calcium in Health and Disease 95 stores (lysosomes and/or endosomes), respectively, thus activating neighboring 2+ InsP3 R and RyR channels which are sensitive to Ca itself (see below). On the TPC channels a controversy has recently arisen, as it has been claimed that they are Na+ - selective, rather than Ca2+ -selective, and that the main cation they release from the endosomes/lysosomes is Na+ and not Ca2+ [ 58 ]. According to general consensus a transient increase of cytosolic Ca2+ may propagate as a wave when the initial localized increase triggers a regenerative process, known as Ca2+ -induced Ca2+ release (CICR), originally described by Ford and Podolski and by Endo and coworkers in 1970 [ 59 , 60 ]. The process, fi rst described in skeletal muscles, is now known to take place also in other cell types, e.g., cardiac myocytes and neurons. The opening of specifi c membrane channels generates two main types of intracellular waves. The most common ones are those mediated by the opening of

InsP 3 Rs. They were fi rst reported in the eggs of a medaka fi sh during fertilization [ 61], and were then found in different egg species, including those of frogs and rodents. Recently, it has emerged that they may serve specifi c functions also in other cell types, e.g., neurons. The main factors determining the generation of the wave format are the site and magnitude of InsP 3 generation. Localized events of InsP 3 R opening, known as “puffs”, are responsible for a local Ca 2+ release that may act as trigger to generate a global Ca2+ wave. The rate of intracellular Ca 2+ wave propaga- 2+ tion is a complex function of InsP 3 and Ca diffusion, sequential CICR via sensitized InsP3 Rs (and RyRs), and the distribution and composition of the ion channel/ receptor clusters. The other Ca2+ wave type, which is mediated by the regenerative activation of RyRs is rare. It has been observed in cardiac myocytes, and it is not clear whether it occurs under normal physiological conditions. Spontaneous localized Ca 2+ release events, often called ‘sparks’, result from the opening of clusters of RyRs in the sarcoplasmic reticulum by local CICR, and have been suggested to cause the large regenerative Ca2+ release that controls contraction. The generation of this type of wave depends on the sensitivity and the distribution of the RyR channels, and Ca2+ entry through voltage-gated plasma membrane Ca2+ channels modulate their frequency in myocytes, thus attributing a role to extracellular Ca2+ as well [62 ]. An important question is the propagation of Ca2+ waves among cells, which has a role in the physiology and pathology of different tissues [63 ]. Emerging evidence has shown that cell communication through gap junctions, and the diffusion of second messengers like InsP3 , are basic to the propagation of the intercellular waves by paracrine signaling between adjacent cells. ATP appears to be the most common paracrine messenger, but the mechanism of its release is still obscure. The signaling cascade starts from the release of ATP in the extracellular ambient and its diffusion to the plasma membrane purinergic receptors. Two type of purinergic receptors are activated by the extracellularly released ATP: the ionotropic receptors that are ligand-gated ion channels, (i.e., those of the P2X family) and the metabotropic receptors (i.e., those of the P2Y family) that are coupled to the generation of InsP3 . The generation of the intercellular Ca2+ waves appears to depend on the stimulation 2+ of metabotropic receptors and to the release of Ca from the ER by InsP3 . External ATP is the major extracellular messenger utilized by many cell types, but other 96 Brini, Ottolini, Calì, and Carafoli molecules, such as glutamate in astrocytes and neurons [64 , 65] and Ca2+ itself by acting on the membrane Ca2+ sensor receptor [66 ] may act as paracrine messengers. Thus, the waves do not only transfer information from one side of the cell to the other, they also propagate the Ca2+ signal among adjacent cells. The transfer of information through Ca2+ waves is not exclusively determined by its diffusion, as the cytoplasm contains an elevated concentration of Ca2+ binding proteins and other molecules that hinder its diffusion. In addition, free Ca2+ is captured by the Ca2+ transport systems that either eject it or sequester it in the organelles. They also hinder the propagation of its message, thus making the waves a sophisticated tunable way to transmit the message. The generation of Ca2+ hot spots at the mouth of the Ca2+ release/infl ux channels activates sensors with different affi nities for Ca2+ . This is the concept of Ca2+ microdomains that has forcefully emerged in the last 15–20 years. Among these domains, those generated by the juxtaposition of ER mitochondrial membranes (the so-called mitochondria-associated ER membranes, MAMs), and those generated by the close contacts between the plasma membrane (PM) and the ER [where the ORAI1/STIM1 complexes are formed (see Section 3.3 )] are the most important. Both will be discussed in more detail in the next section. Whereas waves shape the spatial regulation of the Ca2+ signal, its temporal regulation is determined by oscillations. The rhythmic changes of the plasma membrane potential in the heart, or the burst of action potential in neurons have long been known to produce fl uctuations in cytosolic Ca2+ . The seminal observations by Cobbold and coworkers in the mid-1980s [ 67] have then shown that Ca 2+ oscillations may also occur in non-excitable cells, e.g., during fertilization of oocytes and in hormone-stimulated hepatocytes. By general assumption, the oscillatory behavior has a physiological advantage over the sustained elevation of Ca2+ as the latter could be deleterious to the cell. Ca2+ -dependent processes that require activation by high Ca2+ are satisfi ed by the oscillatory regime that prevents persistent Ca2+ overload. Oscillations also avoid long-lasting receptor desensitization. In the oscillatory regime, the concentration of Ca2+ would be permitted to periodically exceed the threshold for enzyme activation, but its sustained global level would remain below the threshold. Usually, Ca2+ oscillations are characterized by a constant amplitude and a variable frequency, which ranges from 5 to 60 seconds depending to the cell type, and on the nature and the strength of the stimulus that initiates them. In excitable cells such as neurons, heart, and neuroendocrine cells, the transient [Ca 2+] elevation is due to Ca 2+ entry through voltage-operated (VOCCs) or receptor operated Ca 2+ channels (ROCCs) activated by neurotransmitters. In non-excitable cells, instead, the main mechanism of the oscillatory Ca 2+ elevation is through the activation of plasma membrane receptors coupled to G proteins and the generation of InsP 3 . Two possible mechanisms have been proposed for the generation of the InsP 3 generated oscillatory signals: either an oscillatory production of InsP 3 or the oscillatory inactivation of InsP3 receptors. Both mechanisms appear to operate in different cell types, the common denominator being the positive and negative feedback by Ca2+ on the release system. For example, in hormone-stimulated hepatocytes and in pancreatic acinar cells oscillations are driven by the cycling of the InsP 3 channels between a fully open and a largely closed state, rather than by 4 Calcium in Health and Disease 97

oscillations in InsP3 levels. But in kidney epithelial cells spatiotemporal changes in 2+ the concentration of InsP 3 appear instead to be synchronous with Ca oscillations. Other molecules, i.e., cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), have also been shown to mediate the mobilization of Ca2+ from internal stores. An intriguing aspect that has recently emerged is that the inﬂ ux of Ca 2+ across the plasma membrane also plays a critical role in delivering the oscillatory signal to the correct cellular locus. As ER Ca 2+ stores empty, Ca2+ enters through store- operated channels (SOCCs). As will be discussed in the next section, these channels are controlled by STIM proteins, which are sensors of ER luminal Ca 2+ levels. The extra-reticular portion of the STIM1 protein has been shown to move cyclically in and out of the ER plasma membrane junctions during each Ca 2+ oscillatory spike, thus reversibly activating ORAI1 channels and Ca2+ entry.

3.3 Regulation of the Calcium Signal by the Cell Organelles

Intracellular organelles have a dual role in Ca2+ signaling: they are both the target of regulation and its effectors. Specifi c organellar function are Ca2+ regulated processes, but at the same time organelles play an essential role in the defi nition of the spatiotemporal characteristics of the Ca2+ signal. On this, mitochondria absolve the main role. They have received increasing attention in the last few years since their coupling with ER and the plasma membrane is the essential feature in the process of local regulation of Ca2+ signaling. The mitochondrial inner membrane contains a specifi c Ca2+ transport machinery composed by an uptake uniporter (MCU [ 68 ]) which is composed by a tetrameric pore forming subunit plus two regulatory components, MICU1, MICU2, and MCUR1 [69 – 71 ], and by a Na+ /Ca2+ extrusion system [72 ], which operates in most cell types (in some cells mitochondria operate instead of a H+ /Ca2+ exchanger). The uptake of Ca2+ uses as driving force the electrochemical gradient generated across the inner mitochondrial membrane (IMM) by the chemiosmotic operation of the respiratory chain. It also depends critically on microdomains of high Ca2+ concentration generated by the opening of Ca 2+ channels in the neighboring ER that satisfy the low Ca2+ affi nity of the uniporter, thus permitting the accumulation of Ca2+ into the matrix. The outer mitochondrial membrane (OMM) has been traditionally considered freely permeable to Ca2+ , thus excluding it from a specifi c regulatory role in the handling of Ca2+ . However, more recent evidence has shown that its VDAC channels favor the Ca 2+ transfer from the ER to mitochondria thanks to their coupling between the InsP3 R and MCU [73 ]. Thus, OMM proteins would also have a role in the handling of Ca2+ by mitochondria. As mentioned, the concept of microdomains and of signal compartmentalization has recently received wide attention, and general consensus now supports the notion that, in many cases, these microdomains have a specifi c physical organization and biochemical properties. This is the case of the previously mentioned MAMs, which are specialized regions where ER and mitochondria become tethered by specifi c proteins that maintain their distance in the range of 10–30 nm [74 – 76 ]. MAMs have 98 Brini, Ottolini, Calì, and Carafoli been identifi ed in the 1990s [77 , 78 ], but only recently a signaling role has been attributed to them. They are involved in several important cellular functions, ranging from Ca2+ signaling, lipid biosynthesis, mitochondrial division, dynamics regulation of ER and mitochondria membranes [79 ]. The physical link between ER and mitochondria depend on mitofusin 2, which is partitioned between ER and mitochondria [80 ] and which is crucial for the transfer of Ca2+ from the former to the latter. This transfer is guaranteed by the chaperone Grp75-mediated interaction between the mitochondrial outer membrane voltage-dependent anion-channel pro- 2+ tein 1 (VDAC1) and the InsP3 R [73 ]. The transfer of Ca is not only important as a response to cell stimulation, it is constitutively critical to proper cell bioenergetics, as documented by bioenergetics defects, and by increase in autophagy observed in

InsP3 silenced cells [81 ]. Another important example of the compartmentalization of the Ca2+ signal is the Ca2+ infl ux from the extracellular ambient in response to the depletion of intracellular stores. The plasma membrane store-operated Ca2+ entry (SOCE) is a widespread and conserved Ca2+ infl ux pathway, that mediates Ca2+ infl ux following the loss of Ca2+ from the ER. Its gating is regulated by mitochondria: by buffering the Ca2+ released from the ER and that entering through store-operated Ca2+ channels (SOCCs), they reduce the Ca2+ -dependent inactivation of the latter, increasing the extent of store depletion and the activation of SOCCs. The mechanism by which the decrease of Ca2+ concentration in the ER initiates the SOCE process has now been clarifi ed. When the ER luminal Ca2+ decreases, Ca2+ is released by the N-terminal low-affi nity EF hand of the single pass STIM protein in the ER lumen, causing the association of the C-terminal portion of STIM molecules to form clusters that make contacts with the plasma membrane, originating the so-called “puncta” structures. The targets of the STIM1 clusters are ORAI1 proteins, that are the pore-forming subunits of the SOCCs. The STIM1 proteins recruit ORAI1 channels and gate their pore opening. Other organelles such as the Golgi apparatus, the acidic compartment of the cell, and the nucleus are also involved in the dynamical shaping of the Ca2+ signal. The Golgi apparatus is equipped with its own Ca2+ transporters and Ca 2+ bind- 2+ ing proteins, thus making it an ER-like Ca store. InsP3 R and, especially in neurons and cardiac myocytes, RyR channels mediate the Ca2+ release from the Golgi vesicles. The resident Ca2+ ATPase SPCA, and a SERCA pump are responsible for Ca2+ reuptake in their lumen. The relative contribution of these different transporters varies with the cell type: interestingly, it has recently emerged that their differential distribution on the Golgi membranes generates heterogeneity in the Golgi intraluminal Ca 2+ concentration. At variance with the other Ca 2+ -ATPases, the SPCA pump also mediates Mn2+ transport. The transport of Mn2+ from the cytosol to the lumen of Golgi has an important detoxifying role, but is also essential to the function of the resident Golgi enzymes involved in the process of protein glycosylation [82 , 83 ]. Whereas the Golgi apparatus is generally recognized as a releasable Ca2+ reservoir, the role of the acidic organelles as Ca2+ stores is instead still controversial. 4 Calcium in Health and Disease 99

Two pore Ca2+ channels have been identifi ed in the membranes of endosomes and lysosomes and have been proposed to be gated by the second messenger NAADP (see above), triggering Ca2+ release from them [84 ]. The primary structure of TPC contains two six-transmembrane domain repeats, unlike all other Na+ and Ca2+ channels that contain four. They have been fi rst identifi ed in sea urchin eggs [ 85 ] and then also in mammalian cells [86 ] and have received considerable attention as they have been proposed to regulate global Ca2+ through the crosstalk with both 2+ intracellular Ca channels in other membrane systems, i.e., the InsP 3 R and the RyR channels, and the plasma membrane Ca2+ channels. Ca2+ released from acidic stores has been proposed to be promoted by NAADP, to trigger further Ca2+ release via a CICR mechanism and to modify plasma membrane excitability by modulating the Ca2+ release from endosomes or lysosomes specifi cally positioned beneath the plasma membrane. However, some aspects of the process by which lysosomes, endosomes, and acidic organelles handle Ca2+ are still unclear, beginning with the mechanism by which they accumulate Ca 2+ in their lumen. The driving force has been claimed to be a proton gradient generated by the vacuolar H+ -ATPase [87 ], but the precise details of the Ca2+ uptake mechanism are still elusive. Very recently, the Ca2+ releasing function of the TPCs in the acidic organelles has been questioned, as the direct recording of TPC currents in endolysosomes has shown that it is carried + 2+ by Na rather than Ca , and is activated by PI(3,5)P2 , an endolysosome-specifi c phosphoinositide, and not by NAADP. Na+ would thus be the principal cation in the lysosome, casting doubts on this compartment as a Ca2+ store [58 ]. As for the nucleus, the matter of Ca2+ permeability of the nuclear envelope is still an open issue. Alternative proposals suggest that the pores of the nuclear envelope exist in freely permeable or gated states depending on physiological conditions and demands. Numerous experiments with fl uorescent Ca2+ dyes but also with selectively targeted recombinant probes have shown that the kinetics of cytosolic and nuclear Ca2+ increases induced by cell stimulation were temporally nearly indistinguishable, suggesting that the envelope does not represent a barrier to the free diffusion of Ca2+ . Other experiments, however, have found that the Ca2+ signals evoked by the stimulation of cells were temporally delayed in the nucleus. A conciliatory view could propose that nuclear pores may be either passively permeable to Ca2+ , or restrict its passage depending on different cell types or metabolic condition. Earlier 2+ work had shown that the nuclear envelope contains InsP 3 Rs and RyRs and a Ca pump identical to that of the ER. Most enzymes of the phosphoinositide cycle have also been found in the nuclear envelope, suggesting an independent Ca 2+ regulation in the nucleus. Clearly, the problem is to understand how plasma membrane agonists that activate the phosphatidylinositol cycle would be coupled to the process occurring at the nuclear envelope. The fi nding that the nuclear envelope folds inside the nucleoplasm forming invaginations suggests that this structural arrangement may facilitate the agonist-induced delivery of Ca 2+ to selective sub-compartments of the nucleoplasm. Irrespective of the mechanism that governs the nuclear envelope permeability to Ca2+ , a specifi c nuclear function, gene transcription, is selectively regulated by Ca 2+ . 100 Brini, Ottolini, Calì, and Carafoli

4 Calcium as a Regulator of Biological Processes

4.1 Gene Transcription

The ability of Ca2+ to infl uence the expression of genes only became known at the end of the last century, but rapidly developed into one of the most important Ca 2+ signaling areas. Since the Ca2+ signals, including those in the nucleus, generally occur in the form of rapid transients, most of the work on their effects has focused on immediate early genes, i.e., on genes, which generally code for short lived transcription factors without the intermediation of de novo protein synthesis. The expression of late response genes occurs with much slower kinetics, and is instead dependent on de novo protein synthesis. Late response genes are frequently activated by transcription factors that are the products of immediate early genes, thus, they may also be under the control of (nuclear) Ca2+ . The fi rst clear evidence that nuclear Ca2+ had a role in gene regulation was perhaps the demonstration [88 ] that a non-diffusible Ca 2+ chelator, microinjected into the nucleus of AtT20 cells, attenuated the nucleoplasmic Ca2+ transients induced by the activation of voltage-gated plasma membrane Ca2+ channels, and simultaneously blocked gene expression mediated by the transcription factor cAMP responsive element-binding protein (CREB). The cytosolic Ca 2+ transients and the transcriptional activation mediated by the serum response DNA regulatory element (SRE), which is known to be a target of the Ca2+ sensitive extracellular signal-regulated kinases- microtubule-associated protein (ERK-MAP) kinase, which is activated by Ca2+ in the cytoplasm, were instead unaffected. In addition to targeting CREB [89 ], nuclear Ca2+ also stimulates the CREB co-activator CBP [90 ] (CREB binding protein), which, however, also binds to other DNA binding proteins, transmitting the Ca 2+ message to a number of other transcription factors. CREB-CBP-driven transcription is driven by the phosphorylation of CREB by nuclear CaMK IV [91 ], activated in turn by the increase of nuclear Ca2+ (other kinases may also phosphorylate CREB). The kinase phosphorylates CREB on Ser133, promoting its interaction with CBP, however, for transcription to start, CBP itself must also be phosphorylated by CaMK IV. CaMK IV has also recently been found to regulate the process of alternative splicing of the primary transcripts of numerous genes (see Section 4.3 ). Gene transcription, however, can also be regulated by the Ca2+ binding EF hand protein downstream regulatory element antagonist modulator (DREAM) [92 , 93 ], a multifunctional protein that also has roles outside the nucleus. At low levels of nuclear Ca2+ DREAM interacts with downstream responsive element (DRE) sites present in the promoter of a many genes, repressing transcription. As nuclear Ca2+ increases, DREAM binds it, leaving the DRE sites and allowing transcription to initiate. DREAM was initially found to regulate the transcription of the dynorphin gene, but is now known to regulate a number of other genes, including some that code for Ca2+ -regulating proteins, e.g., one of the Na+ /Ca2+ -exchangers [94 ], and one subunit of the L-type Ca2+ channels [95 ]. Another mechanism by which Ca2+ can infl uence the transcription of genes involves the translocation of transcription factors from the cytoplasm to the nucleus. One well 4 Calcium in Health and Disease 101 understood case is that of nuclear factor of activated T cells (NFAT), a transcription factor that is transported into the nucleus by calcineurin after the latter has dephosphorylated it in response to the increase of cytoplasmic Ca2+ [96 ] (discussed in more detail in Section 4.3). Another case is that of the nucleo-cytoplasmic shuttling of the Forkhead transcription factor FoxO3a, which has a role in cell death processes. FoxO3a is translocated to the nucleus in response to several cell-death promoting stimuli, causing transcriptional activation of cell death-inducing genes. In hippocampal neurons the activation of extrasynaptic NMDA receptors [97 ] causes the translocation of FoxO3a to the nucleus. The activation of synaptic NMDA receptors, instead, inhibits the translocation, protecting the neurons from death-inducing signals. The protection process involves CaMK IV, stimulated by nuclear Ca2+ transients induced by the activation of synaptic NMDA receptors. The protection process is likely to be related to a gene transcription process, which would modulate the translocation of the transcription factor; i.e., its release from the DNA and its export from the nucleus. Ca2+ can regulate gene transcription by still another mechanism, which involves a dual function of the L-type Ca2+ entry channels. As just mentioned, it regulates gene transcription by promoting nuclear Ca2+ transients. However, a C-terminal fragment of the pore-forming subunit of the channel which is located in the nucleus is produced in neurons [98 ] of developing and adult brains. Its production and nuclear localization are developmentally regulated, and the infl ux of Ca 2+ through the L-type channel themselves, or through NMDA receptors, causes its export from the nucleus. Within the nucleus, the fragment affects the transcription of a number of genes, including some that are involved in the regulation of Ca2+ .

4.2 Intracellular Proteolysis

The Ca2+ -dependent intracellular cysteine proteases now known as calpains were discovered in 1964 [99 ]. They are regulatory, rather than strictly degradative, enzymes, i.e., they catalyze the limited proteolysis of proteins involved in numerous cell functions, irreversibly modulating them [ 100 ]. They are considered to be cytoplasmic enzymes, but recent research has shown that they also exist in mitochondria, where they are claimed to cleave a number of substrates, including apoptosis-inducing factors like the AIF. Calpains do not have a strict substrate recognition sequence. The tertiary structure, rather than primary structure elements, of the attacked proteins appears to determine their substrate preference. In small peptides, calpains have specifi city for sites in which a small hydrophobic amino acid is in position P2, and a large hydrophobic amino acid in position P1 [101 ]. The calpain family now comprises numerous isoforms (15 in the human genome), some of which have multiple spliced variants. Some isoforms are ubiquitous, i.e., calpain 1 (u-calpain), calpain 2 (m-M calpain), and calpain 10, others are tissue-specifi c, e.g., calpain 3 (also termed p94) for muscle, calpain 8 (also termed nCl-2) for stomach, calpain 9 (also termed nCl-4) for the digestive tract. Ubiquitous calpains play roles in all cells: their physiological functions are still incompletely understood, but their involvement in the regulation of processes like cell differentiation, cell cycle 102 Brini, Ottolini, Calì, and Carafoli regulation, cell death, embryonic development, and a number of nervous tissue functions is fairly well documented. Defects of ubiquitous calpains may be lethal, e.g., the knocking out of either the calpain 1 or calpain 2 genes, but may also be compatible with life; positional cloning has identifi ed variants of the calpain 10 gene that are associated with increased susceptibility to type 2 diabetes in American and European populations [102 ]. By contrast, ubiquitous calpains tend to become hyperactivated in disease conditions, in which a dysfunction of Ca2+ homeostasis creates a condition of cellular Ca2+ overload, e.g., cardiomyopathies, muscular dystrophies, neuronal excitotoxicity, Alzheimer disease, cataract formation; permanent (i.e., not regulated) calpain activation in these conditions promotes the unregulated cleavage of target, but also non-target, protein substrates, leading to irreversible cell damage. Defects of tissue- specifi c calpains may generate tissue-specifi c disease phenotypes: limb-girdle muscular dystrophy type 2A is caused by mutations in the calpain 3 gene [ 103], and the digestive tract-specifi c calpain 9 is down-regulated in gastric cancer cell lines. Its depletion in fi broblasts using antisense RNA was found to be tumorigenic, suggesting that in these systems calpain 9 acts as a tumor suppressor. The calpain superfamily is divided into the typical and atypical families depending on the domain structure. Typical calpains are heterodimers of a large (about 80 kDa) catalytic subunit, and a small (28 kDa) regulatory subunit. The most extensively investigated typical calpains are calpain 1 and calpain 2. They differ in Ca 2+ sensitivity, calpain 1 being optimally stimulated by μM Ca2+ , and calpain 2 by mM Ca2+ . The catalytic subunit can be divided into four domains. The N-terminal domain I becomes autocatalytically hydrolyzed, leading to the activation of the enzyme and to the detachment of the regulatory subunit from the heterodimer. Domain II (divided in subdomains IIa and IIb) is the catalytic core of the enzyme. It contains the catalytic triad (Cys-His-Asn) typical of cysteine proteases. Domain III contains a C2 Ca2+ -binding domain typical of other Ca2+ -binding proteins. It is the linker between the catalytic core of the protein and its Ca2+ -binding domain. The C-terminal domain IV has homology to CaM, and contains 5 EF hand Ca2+ binding motifs. The fi fth one is not operational, but interacts with the analogous EF hand domain of the regulatory subunit, forming the heterodimer. The small regulatory subunit is composed of domain V and domain VI. Domain V has a high Gly content: its hydrophobicity suggests that it may be involved in the binding of calpain to the plasma membrane. A proline-rich stretch separates it from domain VI, which is homologous to domain IV of the catalytic subunit. It thus has 5 EF hand Ca 2+ binding motifs, of which the fi fth does not bind Ca2+ , but mediates the interaction with domain IV of the large subunit. Several partial and complete crystal structures of calpains are now available: they have confi rmed the predictions from sequence studies. They have shown that subdomain IIa, which contains the catalytic Cys, and subdomain IIb, which contains the catalytic His and Asn, are held apart in the absence of Ca2+ , disrupting the catalytic triad and keeping the enzyme inactive. The binding of Ca2+ to domain IV, but possibly to domain III as well, induces a conformational change that moves sub-domain IIa closer to sub-domain IIb, reconstituting the catalytic triad, and activating protease 4 Calcium in Health and Disease 103 activity. Two non-EF hand Ca2+ binding domains that have recently been identifi ed [104 ] in sub-domains IIa and IIb are also involved in the conformational change. The reason for the difference in Ca2+ sensitivity between calpain 1 and calpain 2 has never been satisfactorily explained, and has implications for the physiological role of calpain 2 (but for that of calpain 1 as well, since the concentration of Ca 2+ necessary to activate it is 10- to 100-fold higher than that known to prevail in normal cytoplasms). A number of factors that could somehow lower the concentration of Ca2+ necessary to activate these calpains have been proposed, but none has been convincingly validated. The general idea has thus gained consensus that calpains would only become activated when the concentration of Ca2+ in the cytosol increases abnormally, as frequently occurring in several disease conditions (see above). The idea would also make sense if one considers that the effect of calpains on target proteins, be it an activation or an inhibition, is in any case irreversible, i.e., hardly compatible with a physiological regulatory role. Related to this point is the matter of autoproteolysis as a mechanism for calpain activation. Work on calpain 1 and 2 has shown that their incubation with Ca2+ induces the rapid autoproteolysis of both the large and the small subunits, reducing substantially the Ca2+ requirement for their activation. This has led to the proposal that calpains would be pro-enzymes activated by autoproteolysis. However, other studies have shown that both the intact and the cleaved forms of the enzyme are capable of cleaving substrates, and the crystal structure of calpain 2 has indeed confi rmed that the autoproteolysis removes an N-terminal fragment from the large subunit that does not block the catalytic site. Atypical calpains do not contain the small regulatory subunit, and some do not even contain the penta EF hand domain 4. Their Ca2+ sensitivity is thus an open problem, although they may contain the Ca 2+-binding sites outside domain IV. For instance, in ubiquitous calpain 10 domain IV is replaced by a domain structurally related to domain III (which is also found in two other atypical calpains, calpain 5 and 6). In addition to the absence of domain IV, calpain 10 does not contain the Ca 2+ -binding motifs in domain II (which are instead present in atypical calpain 5), and its Ca2+ -dependent activation mechanism is thus unclear. Calpastatin [105 ] is a natural protein inhibitor of calpains. It has 4 repeated, poorly homologous inhibitory domains of about 140 amino acids (domains I, II, III, and IV) and an N-terminal domain L that has no inhibitory activity. Three subdomains have been identifi ed in each inhibitory domain: sub-domain A binds to domain IV of calpain, sub-domain B, which has little inhibitory activity by itself, is essential for calpastatin activity, and sub-domain C binds to domain VI in the small regulatory subunit of calpain.

4.3 Protein Phosphorylation and Dephosphorylation

Ca2+ has an important role in the phosphorylation and dephosphorylation of proteins, which is a major mechanism for the regulation of metabolism. A large number of enzymes functioning in diverse metabolic pathways are phosphorylated on 104 Brini, Ottolini, Calì, and Carafoli serine or threonine residues by Ca 2+-dependent kinases, and are dephosphorylated by calcineurin, the only Ca2+ -dependent protein phosphatase so far known. Most of the Ca2+ -dependent protein kinases are CaM-stimulated enzymes, but other kinases, the most prominent example being protein kinase C, decode the Ca2+ signal without the intermediation of CaM. The protein kinase C family contains several members, some of which are not Ca2+ -regulated. Five or six of them are binding Ca2+ to a C2 domain normally located in the N-terminal moiety of the protein. Binding of Ca2+ to the C2 domain helps targeting the kinase to the plasma membrane, where it can effectively search for its membrane embedded activatory ligand, diacylglycerol. The CaM-dependent kinases (CaMKs) phosphorylate serine or threonine residues in specifi c consensus sequences of the substrates, although their substrate specifi cities can overlap. The catalytic domain of most of them contains acidic residues that interact with the basic residues in the consensus sequences. CaMKs can be divided into narrow-specifi city and broad-specifi city CaMKs [106 –108 ]. The former comprise myosin light chain kinase (MLCK), phosphorylase kinase (PhK), and eukaryotic elongation factor 2 kinase (eEF-2K, also called CaMK III). MLCK has only the regulatory light chain of myosin as substrate. Apart from the CaM- binding domain, it contains an AID domain (atypical interacting domain; a domain of unknown function found in many proteins, which is part of a broader consensus sequence termed octicosapeptide). MLCK is normally activated by Ca2+ release from the sarcoplasmic or endoplasmic reticulum, and the phosphorylation of myosin initiates contraction of smooth muscle and potentiates that of skeletal muscle. PhK phosphorylates and activates glycogen phosphorylase, accelerating glycogen degradation: it is present in many tissues, but is particularly abundant in liver and muscle. It is a holoenzyme of 4 catalytic γ-subunits, complexed to 4 each regulatory α-, β-, and δ-subunits. The δ-subunit, actually, is CaM, which remains unusually associated to the holoenzyme even in the absence of Ca2+ . PhK is activated by the binding of Ca 2+ to the δ-subunits, and the activation is potentiated by the phosphorylation of the α- and β-subunits by protein kinase A. eEF-2K phosphorylates elongation factor 2, promoting ribosomal translocation along mRNA during translation: the phosphorylation inactivated eEF-2. The catalytic center of eEF-2k bears no homology to those of the other CaMKs. Its regulation is complex, and entails binding of CaM, autophosphorylation, and phosphorylation by a variety of kinases. The broad specifi city CaMKs comprise CaMK I, CaMK II, CaMK IV, and CaM- dependent kinase kinase (CaMKK). CaMK I, a protein of about 40kDa, is abundant in brain, liver, and intestine. The binding of Ca2+ /CaM activates the enzyme by disrupting the inhibitory interaction of an AID with the ATP-binding pocket. CaMKK phosphorylates CaMK I, activating it up to 20-fold. CaMK I phosphorylates a number of proteins, including brain proteins, but its physiological role remains elusive. CaMK II is a ubiquitous kinase that has been involved in the regulation of a large number of processes [109 ]. It is the product of 4 separate genes, the isoforms having propensity to form a holoenzyme structure thanks to the presence of a C-terminal association domain. The holoenzyme forms a double hexameric ring-shaped struc- 4 Calcium in Health and Disease 105 ture that facilitates regulatory properties. When subunits in the holoenzyme bind Ca2+ /CaM, they become activated, and trans-phosphorylate adjacent subunits at Thr-286 increasing their affi nity for CaM and making them Ca 2+ -independent. Since the number of subunits in the holoenzyme that become active and autophosphory- lated at Thr-286 depends on the Ca2+ concentration, CaMK II is able to “decode” the frequency and the amplitude of the Ca2+ oscillations. This ability to “prolong” the signaling of Ca2+ after its transient has abated has been exploited to involve CaMK II in processes like learning and memory. CaMK II is present in the cytosol and is also associated with organelles, in line with the large number of proteins it phosphorylates. One variant of CaMK II contains a nuclear localization sequence and has been shown to regulate gene transcription, at least in cardiac myocytes. CaMK II plays an important role in the regulation of synaptic transmission: indeed, several neuronal proteins are phosphorylated by CaMK II, among them the NMDA and AMPA glutamate receptors. CaMK IV has a more restricted tissue distribution: it is expressed abundantly in neuronal cells, in T-cells, and in the testis. It is activated by the binding of CaM, and further activated by phosphorylation by CaMKK: unusually, the phosphorylation by CaMKK renders CaMK IV Ca2+ -independent. CaMK IV has a nuclear localization sequence and has a prominent role in the nucleus, where it phosphorylates numerous transcription factors. It also phosphorylates the heterogeneous nuclear ribonucleoprotein (hnRNP) L which is the transactive factor that then interacts with the CA (cysteine-adenosine) repeat in the CAMK IV-responsive RNA elements (CaRRE) of numerous genes to regulate the splicing process of their primary transcripts [110 , 111]. Interestingly, one of these genes encodes a plasma membrane Ca2+ pump. CaMKK is similar in the organization of domains and function to the other CaMKs, however, it also has distinctive features: it does not contain the acidic residue that is used by other CaMKs to recognize basic residues next to the phosphorylated Ser or Thr, but contains instead an Arg- and Pro-rich insert that is important in the phosphorylation of CaMK I and IV. Unusually, both CaMKK and its substrates (CaMK I and CaMK IV) must bind CaM for phosphorylation to occur. The only Ca 2+ -dependent protein phosphatase so far known is calcineurin (Cn), a dimer of a 58–64 kDa catalytic subunit (CnA), and a tightly bound 19 kDa, Ca2+ - binding regulatory subunit (CnB), which is a canonical EF hand protein with 4 Ca2+ - binding motifs [112 ]. It is the product of three human genes, and is expressed in most tissues. However, it is particularly abundant in the brain (hence, its name), where isoform α predominates: it represents about 1% of the total brain protein. In brain, Cn triggers a phosphatase cascade that opposes the stimulatory effects of PKA and CaMKs: it has been implicated in a large series of brain processes, from the expression and activity of ion channels, to the release of neurotransmitters, to the recycling of synaptic vesicles. The catalytic domain of the phosphatase is located in the N-terminal moiety of CnA, and is followed by a domain that binds CnB and by two further domains, one that binds CaM and one that acts as an autoinhibitory sequence. Importantly, in the absence of CaM calcineurin is inactive, and is thus peculiarly under dual Ca 2+ regulation, by CaM and by its own “calmodulin”, i.e., the CaM- like 19 kDa subunit. 106 Brini, Ottolini, Calì, and Carafoli

This multiple Ca2+ regulation mechanism is reminiscent of that of calpain (see above), which, however, instead of exogenous CaM, has, its own “calmodulin” incorporated in its sequence (the penta EF hand domain IV). Cn is a phosphatase with a binuclear iron-zinc binuclear active center, which has a dual substrate speci- ﬁ city: it dephosphorylates both phosphoseryl/threonyl and tyrosyl substrates. It prefers substrates in which a basic residue lies at the N-terminal side of the phosphorylated amino acid, and no acidic residue lies at its C-terminal side. CnB is tightly bound to CnA even in the absence of Ca 2+ , and is required for phosphatase activity. The binding of CaM to CnA displaces the autoinhibitory domain, expos- ing the catalytic center and activating phosphatase activity: however, prolonged exposure of the active site facilitates the oxidation of catalytic Fe 2+, inactivating the enzyme. Paradoxically, then, CaM can be either activating or inhibiting depending on the duration of the Ca 2+ signal. A 240 kDa protein termed Cain/Cabin 1 has been described as an endogenous inhibitor of calcineurin. Cn is also inhibited by fungal immunosuppressive compounds (FK-506, cyclosporin), which bind to their respective immunophilins and then bind to calcineurin. The immunosuppressive drugs are used to prevent organ rejection after transplant operations and in the treatment of autoimmune diseases. Cn plays an important role in the regulation of gene expression: this has been established mostly through studies of T cell activation: the liberation of Ca2+ in the cytosol by the activation of InsP3 -linked receptors activates calcineurin to dephos- phorylate the transcription factor NFAT, which exposes its nuclear localization sequence and translocates it to the nucleus together with calcineurin. NFAT dephosphorylation also increases its DNA binding and transcriptional activity. The export of NFAT from the nucleus depends on its rephosphorylation by GSK 3, and on calcineurin inactivation upon Ca2+ removal/decrease [96 ].

4.4 Calcium and Bioenergetics

Shortly after initial fi ndings on the transport of Ca 2+ in mitochondria, it was discovered [113 , 114] that three enzymes of the citric acid cycle of the mitochondrial matrix (the pyruvate, the α-ketoglutarate, and the NADH-dependent isocitrate dehy- 2+ drogenases) are activated by Ca in the micromolar range (K m 1 to 50 μM) [ 115 ]. Thus, it became evident that the mitochondrial Ca2+ transport process had an essential role in the regulation of ATP production and in maintaining the proper bioenergetics balance of the cells: a problem, in those early days, was the low affi nity of the mitochondrial Ca2+ uptake by uniporter, which in principle would not have permitted mitochondria to effi ciently take up Ca2+ in the physiological ambient of the cytosol. As discussed above, the problem was solved decades later by the demonstration that the release of Ca2+ from the ER exposed neighboring mitochondria to a Ca2+ concentration high enough to overcome the low affi nity of the uniporter. The machinery for energy production by mitochondria is the electron transport chain (ETC) of the inner mitochondrial membrane, which is composed of fi ve 4 Calcium in Health and Disease 107 multiprotein complexes. Three of them (I, III, IV) pump protons (H+ ) across the inner membrane, ejecting them from mitochondrial matrix, thus establishing the electrochemical gradient which is used by complex V (the ATP synthase) to produce ATP. Reducing equivalents from the citric acid cycle are transported by the respiratory chain from NADH and FADH2 , to oxygen which is converted to H2 O. The negative- inside electrochemical gradient generated by their travel down to O 2 is not only used to synthetize ATP. It is also used to drive the transfer of Ca2+ across the inner membrane into the mitochondrial matrix. Since the entry of Ca2+ dissipates the membrane potential, it temporarily abolishes the synthesis of ATP. However, the entry of Ca2+ into the matrix stimulates the activity of the citric acid cycle, thus enhancing the delivery of reducing equivalents to the respiratory chain. The mechanisms of the regulation of the three citric acid cycle enzymes by transient Ca2+ increases in the matrix are different [ 115 ]. The pyruvate dehydrogenase complex represents “the point of no return” in carbohydrate metabolism. The complex is therefore subject to stringent regulation and its activity is directly inhibited by the end product acetylCoA/CoA and NADH/NAD+ ratios, but also, more importantly, by reversible phosphorylation by highly specifi c kinases and phosphatases in the mitochondrial matrix. The phosphorylated pyruvate dehydrogenase is activated by the Ca2+ -dependent dephosphorylation by the pyruvate dehydrogenase phosphatase. Two phosphatase isoforms are present in mammalian mitochondria, PDP1 and PDP2, each containing a Mg2+ -dependent catalytic subunit, designated as PDP1c and PDP2c, of which only the fi rst is activated by Ca 2+ . Ca2+ regulation of pyruvate dehydrogenase may thus vary according to the distribution of the two isoforms in different tissues, or physiological situations, e.g., the nutrition status [ 116 , 117 ]. Mammalian NAD-isocitrate dehydrogenase consists of three subunits associated to form an octamer. It has complex regulatory properties: it is inhibited by increasing ATP/ADP and NADH/NAD+ ratios (a property shared with the pyruvate dehydrogenase system and oxoglutarate dehydrogenase). Ca2+ causes a marked decrease 2+ in the K m of the dehydrogenase, the Ca sensitivity of which is infl uenced by the ATP/ADP ratio (it becomes more sensitive to Ca2+ at lower ratios). Two Ca2+ ions are bound per dehydrogenase octamer, to motifs different from the canonical Ca 2+ - binding motifs discussed above. Oxoglutarate dehydrogenase is a multienzyme complex that has similarities to pyruvate dehydrogenase. It is also end-product inhibited by increases in the succinyl CoA/CoA and NADH/NAD+ ratios. However, unlike pyruvate dehydrogenase, it is not regulated by reversible phosphorylation. Ca2+ acts directly on the enzyme markedly decreasing its K m. The Km is also decreased by the decrease in the ATP/ ADP ratio. As in the case of the NAD-isocitrate dehydrogenase, decreases in this ratio also markedly increase the sensitivity of the enzyme to Ca2+ . Between 2.5 and 5 Ca 2+ are bound to each oxoglutarate dehydrogenase complex. As in the case of the NAD-isocytrate dehydrogenase, canonical Ca2+ -binding sites have been found in the subunits of the complex. The possibility to directly monitor mitochondrial Ca2+ transients generated by cell stimulation has permitted to analyze in detail the activation of the three matrix enzymes by Ca2+ . Thus, it has been shown that the Ca 2+ transients monitored 108 Brini, Ottolini, Calì, and Carafoli directly within the mitochondria paralleled the increase of NADH [118 ] and of ATP production [119 ]. The entity of the increase was proportional to the amplitude of the matrix Ca2+ transients. Interestingly, imaging studies on single cells have shown that oscillations of cytosolic Ca2+ were transmitted to mitochondria resulting in the sustained activation of the matrix enzymes, thus extending the NADH increase for times longer than those of the Ca2+ transient [120 ]. In addition to the three citric acid cycle enzymes, a number of other potential mitochondrial targets of Ca 2+ regulation have also been proposed that may directly or indirectly infl uence respiration and hence, ATP synthesis. For instance, the mitochondrial F1F0 ATPase itself may be activated by μM concentrations of Ca2+ ions by a mechanism involving the release of a small inhibitory protein [121 ]. Ca2+ can also improve energy metabolism by favoring the transport of the NADH equivalents produced in the cytosol during the glycolysis to the mitochondrial matrix. The transport is mediated by two shuttle mechanisms: the glycerol phosphate shuttle and the malate-aspartate shuttle, both of which can be activated by extramitochondrial Ca2+ . The FAD-glycerol phosphate dehydrogenase is located on the cytoplasmic surface of the inner membrane: together with the cytoplasmic NAD-glycerol phosphate dehydrogenase it forms the glycerol phosphate shuttle. The aspartate/glutamate carrier (AGC1, or aralar), is a component of the malate- aspartate shuttle [122 , 123 ]. The proteins of both shuttle pathways contain EF hand Ca2+ -binding sites that face the intermembrane space and are sensitive to Ca 2+ increases occurring in the proximity of mitochondria.

4.5 Muscle Contraction

The history of Ca 2+ as intracellular messenger actually initiated with studies of heart muscle contraction. It is traced back to 1883, when S. Ringer discovered that Ca2+ was essential for cardiac contractility [124 ]. It took a long time to realize that Ca2+ acts as a messenger not only in the contraction of heart, but also in that of skeletal muscles. The concept of “excitation-contraction coupling” (ECC) was eventually established, i.e., the concept of a mechanism that links electrical phenomena occurring at the plasma membrane with the activation of contractile proteins [125 ]. The mechanism by which muscle contraction is regulated by Ca2+ is now well understood, and will thus only be described very succinctly, to focus on its different molecular details in skeletal, cardiac, and smooth muscles. Contractile proteins include myosin, actin, tropomyosin, and troponin, which are organized into functional units (the sarcomere). Myosin thick fi laments are surrounded by actin polymers thin fi laments organized in a hexagonal array together with tropomyosin and troponin. Troponin is distributed along the entire length of the thin fi lament at inter- vals of about 40 nm. The periodicity is determined by the arrangement of tropomyosin molecules which fi t in the grooves of the double stranded actin fi laments. The myosin and actin fi laments slide along each other utilizing energy from ATP hydrolysis, thus shortening the sarcomere unit in the contraction process. 4 Calcium in Health and Disease 109

Tropomyosin and troponin confer Ca2+ sensitivity to it, troponin being the Ca2+ sensor that allows contraction to occur. It is a complex of troponin C, I and T. Troponin I is the subunit that inhibits the ATPase activity of the actin-myosin complex, troponin T promotes the binding with myosin and regulates the interaction between the troponin components, and troponin C binds Ca2+ to four EF hand motifs. The mechanism of the regulation by Ca2+ is similar in skeletal and cardiac muscles, but differs in smooth muscle, where, instead of the troponin-tropomyosin complex, a Ca2+ CaM-dependent myosin light chain kinase operates. In skeletal muscles, ECC occurs by mechanical coupling involving the interaction between L-type channels in specialized structures of the plasma membrane, the T tubules. They are formed by PM invaginations that establish physical contact with specialized portions of the SR (the terminal cisternae) permitting the coupling between the voltage gated L-type Ca2+ channels with the RyR channels in the SR. The plasma membrane depolarization is sensed by the L-type channels in the T-tubules and directly transmitted to the RyR. Several proteins participate in the junction between PM and SR [126 , 127], among them the transmembrane proteins triadin and junctin that mediate the contact between RyRs and the SR protein calsequestrin, which senses the luminal Ca 2+ concentration in the SR, and transmits the information to RyR via triadin. The contraction of skeletal muscles depends on the Ca2+ release from the SR store by RyRs, the relaxation phase is instead mediated by Ca 2+ reuptake in the SR by isoform 1 of the SERCA pump. In the cardiac muscle, instead, even if the release from SR is the triggering event for contraction, Ca 2+ entry from L-type channels is required to induce the CICR mechanism and thus the opening of RyRs. The depolarizing action potential originating from the sino-atrial node induces contraction starting from the right atrium forcing blood into the ventricles. When the action potential travels across the heart the membrane of cardiac myocytes becomes depolarized causing the opening of L-type voltage-gated channels and the infl ux of Ca2+ into a restricted region between the plasma membrane and the membrane of SR (the junctional zone or dyadic cleft). This Ca2+ infl ux is not suffi cient per se to activate contraction, but it induces the opening of a clusters of RyRs located in the SR membrane opposed to the PM and thus activates the CICR mechanism and the consequent mobilization of Ca2+ from SR. The diffusion of Ca2+ from the junctional zone then generates a global Ca2+ increase that activates the contractile machinery [128 ]. As in skeletal muscle, after Ca2+ has activated the contractile proteins it is rapidly extruded from the cytosol to permit the next action potential to trigger a new contraction. In cardiac myocytes the main systems that remove Ca 2+ from the cytosol are the plasma membrane Na+ /Ca2+ exchanger and isoform 2 of the SERCA pump of the SR. The relative contribution of these systems differs according to the species. The PMCA pumps of the plasma membrane do not have a quantitatively signifi cant role in the extrusion of Ca2+ from the cardiomyocyte but can regulate contraction in a subtler way, linked to the modulation of the NO synthase [129 ]. The ECC in smooth muscles differs from that of skeletal and cardiac muscles [ 130 ]. Smooth muscle cells form a layer that wraps up hollow organs such as blood vessels, intestine, bladder, airways, uterus etc. Their contractile properties are 110 Brini, Ottolini, Calì, and Carafoli functionally important in these organs, as they permit dynamic changes in their luminal volume that may regulate the movements of the content of the organs, as in the case of peristalsis or urine expulsion. Uterine smooth muscle exerts a dual action: it relaxes during gestation to accommodate fetal growth, and contracts during parturition. Similarly, the contractility of smooth muscle in the vessel wall controls blood pressure and fl ow to the body. In most cases the changes in plasma membrane depolarization driven by action potentials are suffi cient to promote Ca2+ infl ux into the myocytes through the voltage- gated channels that directly activate contraction. In arterial smooth muscle cells, where the membrane potential across the plasma membrane is about −50/–40 mV, oscillations of approximately 10 mV above or below these values trigger changes in global Ca2+ that engage the contractile apparatus and cause maximal dilatation or constriction. Contraction of smooth muscle cells occurs even in the absence of extracellular 2+ Ca , when driven by agonists that trigger the opening of the intracellular InsP 3 R and RyR channels and the activation of a CICR mechanism. This is the case of the urinary bladder, the gastrointestinal trait, and the airways, but also of arteries where Ca2+ sparks have been detected. It is also the case of smooth muscle cells from the 2+ 2+ colon or the portal vein, where the InsP3 -mediated Ca release originates a Ca wave [131 ].

4.6 Secretion

Ca2+ is especially important in the fusion of the secretory vesicles with the plasma membrane [132 ], but it also has a role in the process of vesicle maturation [ 133 ]. Conceptually, the release of the vesicle content can be divided into four steps: vesicle docking, vesicle priming, Ca2+ triggering, and the vesicle fusion reaction itself. Two secretion processes are especially well characterized: the synaptic transmission in neurons and the insulin secretion in pancreatic β cells. Synaptic and endocrine exocytosis use the same Ca 2+ -triggering mechanisms, but differ in the mechanism by which the vesicles are docked and prepared for fusion (i.e., primed). Vesicle exocytosis is managed by the SNARE (soluble N-ethylmaleimide- sensitive factor attachment protein receptor) fusion machinery [134 , 135 ]. The complex includes a number of proteins having different localization and roles: synaptobrevin-2 (syb2) on the vesicle, syntaxin-1 (syx1), and SNAP-25 on the plasma membrane interact with each other to form a very stable bundle of four coiled α-helices. Accessory factors, including complexins, Munc13, Munc18, and synaptotagmins also participate in the assembling of the complex (for reviews see, e.g., [136 –138 ]). Synaptotagmins are single pass transmembrane proteins that bind Ca2+ with relatively low affi nity (K d > 10 μM) at two C2 domains in their C-terminal portion (these domains are not functional in all synaptotagmin isoforms). Interestingly, membrane phospholipids also participate in Ca2+ binding to C2 domains, which, due to their different affi nity for Ca 2+, could operate cooperatively to regulate both 4 Calcium in Health and Disease 111 constitutive secretion, triggered by spontaneous fl uctuation in cytosolic Ca2+ and thus occurring in resting condition, and evoke exocytosis triggered by massive Ca2+ entry promoted by the action potential. Synaptotagmin I (Syt1) is the Ca 2+ sensor in many Ca 2+ -sensitive processes of neurotransmission; it binds both syntaxin and SNAP-25 and transduces the Ca 2+ signal into a nanomechanical activation of the membrane fusion machinery, thus causing vesicle fusion. Complexin, which is another Ca2+ -binding protein, activates SNARE-complexes before synaptotagmin action, and clamps fusion by preventing complete SNARE-complex assembly until Ca2+ binds to synaptotagmin [139 ]. The molecular details of the fusion process have been investigated in depth, and the data obtained by studying secretion events in reconstituted membranes have shown that Ca 2+ entry through the VOCCs is necessary to recruit vesicles at the plasma membrane rather than to trigger their fusion. According to this model docked vesicles are tethered to the membrane through a non-primed or primed excitosome complex. Synaptotagmin 1 is a member of the complex that can be in either a Ca 2+-bound or a Ca 2+-unbound form, differentiating releasable pools. Only docked vesicles primed by Ca 2+ binding to synaptotagmin would be released following the depolarization signal. Ca 2+ binding to the sensor in the transmembrane sector of the voltage-gated channel induces conformational changes to the channel transmitted through the interaction between the intracellular loop connecting transmembrane domain II and III of the VOCCs and the TM domain of synaptotagmin. Thus, according to this view, synaptotagmin acts as a priming protein rather than as a fusion inducing protein, and the sensors for fusion are instead the VOCCs channels themselves. This model, named excitosome model, adequately explains secretion events occurring in the sub-millisecond timescale, which especially characterizes synaptic transmission, satisfying the time requirements of the of excitation-secretion coupling process [140 ]. As for endocrine secretion, the role of Ca2+ in regulating insulin secretion is of special interest as it has been proposed to be linked to the generation of local Ca 2+ microdomains beneath the plasma membrane would differentially control the release of different pools of vesicles. Nutrient-induced increases in intracellular free Ca2+ concentrations are the key trigger for insulin release from pancreatic islet β-cells [141 ]. In healthy β-cells, the uptake of glucose by a facilitated glucose transporter and glycolytic and mitochondrial metabolism increases the ATP concentration. The increased ATP/ADP ratio promotes the closure of the ATP-sensitive K+ 2+ (KATP ) channels, plasma membrane depolarization, and Ca infl ux through voltage- gated channels: ultimately, it promotes the fusion of insulin containing large dense core vesicles (LDCVs) with the plasma membrane. The subsequent opening of voltage-gated K+ channels repolarizes the cell to terminate exocytosis. Convincing evidence shows that in β-cells L-type (voltage-gated) Ca 2+ channels are most active close to sites where LDCVs are clustered [142 ] and it has also been proposed that these channels may physically interact with them [ 143 ]. In addition, it has been suggested that Ca2+ release from the ER or even from the LDCVs by CICR or NAADP-gated channels, may locally contribute to the increase of the Ca 2+ levels close to the vesicle surface. 112 Brini, Ottolini, Calì, and Carafoli

4.7 Calcium in the Beginning of Cell Life

The initiation of embryo development depends on the intracellular Ca2+ increase in the egg during fertilization. In most species, including mammals, Ca2+ is also responsible for inducing changes in oocytes that make them mature for fertilization. In particular, the resumption and progression of the meiosis, which is a step necessary to activate oocytes to initiate embryo development, is directly associated with an activating Ca2+ signal generated during sperm-oocyte fusion [ 144 , 145 ]. This general mechanism, however, differs among species with respect to the spatiotemporal confi guration of the Ca2+ signal. In some species, e.g., in sea urchins, starfi sh, frogs, and fi sh, a single Ca 2+ transient in the oocyte is suffi cient for the activation, in others, e.g., ascidia and mammals, an oscillatory pathway is instead required. The molecular machinery of the signaling cascade also differs among species, being essentially dependent on the activation of the phosphoinositide pathway in sea urchins, starfi sh, and frogs, and on the release of a protein moiety containing a molecule named sperm factor (SF) in mammals. This factor has been molecularly identifi ed only in mammals and found to be a sperm-specifi c isoform of phospholipase, PLCζ [ 146 ]. Thus, if in one case the generation of the diffusible second messenger InsP3 is driven by the activation of a Src-family kinase and PLCγ, in mammals the sperms directly delivers PLCζ, which hydrolyzes membrane PIP 2 to InsP3 and diacylglycerol (DAG). The genera- 2+ tion of InsP3 induces the release of Ca from the intracellular stores (cADPR/ryano- 2+ dine receptor channels may contribute to the Ca mobilization). The role of InsP3 in the globalization of the Ca2+ signal is now generally accepted. However, recent work has demonstrated that the initial liberation of Ca 2+ is promoted in the cortex of the egg by NAADP acting on special stores, that would then promote the liberation 2+ of Ca from the InsP3 stores via a CICR process [147 ]. The NAADP sensitive stores which initiate the liberation of Ca2+ have been proposed to be the acidic organelles [84 ] which, as has been mentioned above, have been recently questioned as Ca 2+ stores [58 ]. It has also been proposed that the initial increase of Ca2+ in the cortex of starfi sh eggs could be promoted by NAADP acting on a novel plasma membrane channel [148 ]. Irrespective of the mechanism by which the elevation of intracellular Ca2+ at fertilization is produced and shaped, its immediate consequence is the exocytosis of cortical granules (CG) which modifi es the component of zona pellucida to prevent polyspermy and ensures the formation of the diploid zygote. The actin cytoskeleton is prominently involved in the reorganization of the cortical domain of the cell that promotes these processes [ 149]. The release from meiosis arrest is mediated by a Ca2+ -CaM dependent protein kinase II degradation of cyclin B [ 150 ]. Interestingly, it has been shown that each sperm-induced Ca2+ increase is accompanied by a parallel increase in CaMKII activity [151 ]. Considering that the elevation of Ca2+ occurs in a relatively rapid temporal sequence, e.g., minutes, oscillations may extend the effect of Ca 2+ for hours after sperm fusion, and may thus be advantageous for other processes in mammals. 4 Calcium in Health and Disease 113

Interestingly, it has been shown that the recruitment of maternal mRNA for new protein synthesis occurs during the oscillation, and is proportional to the magnitude of Ca 2+ stimulation. Pulsatile activation of CaMKII appears to underline enhanced gene expression [152 ], however, it has also been shown that cell cycle progression is required to recruit mRNA, implying that the process is not under exclusive Ca 2+ control [153 ].

4.8 Apoptotic Cell Death and Autophagy

As has now become clear Ca2+ does not only regulate biological processes necessary to cell survival and wellness: it also participates in processes that may culminate in cell death, e.g., apoptosis and autophagy. However, it must be understood that these processes are not just ways for the cells to die: they also represent a sophisticated mechanism for cell quality control and rescue, which are necessary to the harmoni- ous development of the organism. Apoptosis is necessary to normal cell homeostasis as it eliminates cells that are damaged or unnecessary. Autophagy is the general term used to defi ne a cellular process responsible for the delivery of proteins or organelles to lysosomes. Apoptosis occurs through two conventional pathways: (i) the extrinsic pathway which is typically initiated by death receptors acting on the plasma membrane and the activation of the death-inducing caspase cascade [154 ], and (ii) the intrinsic pathway, which acts through the permeabilization of the mitochondrial outer membrane that releases cytochrome c and induces caspase activation [155 ]. The second pathway is regulated by Ca2+ , and will thus be discussed here. Ca2+ -mediated apoptosis can be triggered by physiological signals, but multiple cytotoxic agents also lead to it by disrupting Ca2+ homeostasis: the inhibitor of SERCA pumps thapsigargin and the alkaloid staurosporine are the best known. They have been used to dis- sect the molecular details of the pathway and have established that the Bcl-2 family of proteins is important to it. Bcl-2 is overexpressed in a number of cancer cells as it promotes their survival [ 156 ]. That Ca2+ was involved in the Bcl-2-linked apoptosis process was indicated by the fi nding that the protein controlled the Ca2+ signal, and by work showing that Bcl-2, which is not only localized in the cytoplasm and in the nuclear envelope, but also associates with the ER and mitochondrial membranes, 2+ regulates the InsP3 -mediated Ca release [157 ]. The fi rst evidence that Bcl-2 overexpression directly affected Ca2+ homeostasis came from work on hematopoietic cells, where its overexpression prevented the reduction of the cytosolic free Ca2+ induced by the withdrawal of interleukin-3, at the same time protecting the cells from apoptosis [158 ]. It was later shown that the overexpression of Bcl-2 reduced the Ca2+ content of the ER and Golgi lumina, and reduced the Ca2+ release following 2+ InsP3 stimulation [ 159 , 160]. As a result, it also prevented the mitochondrial Ca overload and the cell death it would induce. Different mechanisms have been proposed for the control of ER luminal Ca 2+ by Bcl-2, and it has been concluded that it could be linked to differences in Bcl-2 114 Brini, Ottolini, Calì, and Carafoli

expression and localization in various cells, or to the isoform of InsP3 receptor expressed in them. Initial studies showed that Bcl-2 increased the ER Ca2+ leak by increasing membrane permeability [159 , 160 ], but other studies showed instead that Bcl-2 depleted Ca2+ pools by interacting with the SERCA pump [ 161 ], and still other studies proposed that Bcl-2 interacted directly with the InsP 3 receptor, either stimulating or inhibiting it [162 , 163 ]. A more recent study even suggested that Bcl-2 inhibited Ca2+ entry by downregulating L-type channels, thus eventually preventing mitochondrial Ca2+ uptake [164 ]. In addition to Bcl-2 (and Bcl-xL, another antiapoptotic member of the family), also the proapoptotic members of the Bcl-2 family Bax and Bak regulate Ca 2+ homeostasis. Their knockdown reduces the Ca2+ content of the ER, whereas Bax overexpression increases it [165 , 166]. Accordingly, Bax/Bak MEF double knockout cells were found to be resistant to apoptotic stimuli [167 ]. As for autophagy, it is classifi ed in three main classes: microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy. They differ in the way by which cellular organelles and proteins are phagocytosed and delivered to lysosomes, so that their constituents are recycled to satisfy the energy demands during metabolic stress. Microautophagy implies a direct delivery of a small portion of cytoplasm by invagination and fi ssion of the lysosomal membrane. CMA is a very selective process that relies on the help of chaperones that recognize specifi c sequence motifs in proteins, e.g., the KFERQ pentapeptide, and of the lysosome- associated membrane protein type 2A (LAMP-2A) to target unfolded or aggregated proteins to the lysosomes. Macroautophagy is characterized by the formation of double membranous vesicles called autophagosomes. Under non-stress conditions, low levels of autophagy guarantee normal cellular homeostasis by removing dysfunctional proteins or even organelles: an intensively studied autophagy specializa- tion is the removal of dysfunctional mitochondria, a process named mitophagy [ 168 , 169 ]. Autophagy must thus be considered a positive process: not surprisingly, therefore, its impairment or alteration are often involved in pathologies like cancer and neurodegenerative disorders [170 ]. More than 30 atg genes have been identifi ed that regulate macroautophagy, and in mammals the serine/threonine kinase mTOR has a central role in the process. mTOR controls cell growth and metabolism in response to nutrients, growth factors, ATP, and stress. Emerging evidence indicates that, as in the case of apoptosis, intracellular Ca2+ regulates autophagy, even if the exact role of Ca 2+ remains still ambiguous. Numerous reports suggest an inhibitory role, others a stimulatory role

[171 , 172]. The inhibitory role has been linked to InsP3 R activity since the silencing of the receptor by siRNA or its inhibition by xestospongin or Li+ induced autophagy

[ 81 , 173 ]. An intriguing explanation for the control of autophagy by the InsP 3 R pro- poses that it could act as a scaffold and, by simultaneously binding beclin 1 (a protein required for autophagy) and Bcl-2, could participate in the formation of antiautophagic Bcl-2/beclin 1 complexes, which would sequester beclin 1 and prevent the activation of autophagy [174 ]. By converse, the activating role of Ca2+ has been associated to agents that induce its increase in cytosolic concentration, e.g., the SERCA pump inhibitor thapsigargin 4 Calcium in Health and Disease 115

Figure 3 The cartoon illustrates the most important players in the control of cellular Ca2+ homeostasis and in the systems that decode its function. The free cytosolic Ca 2+ concentration is maintained in the nM range by buffering proteins and by the action of pumps and other transporters on the plasma membrane or in the membrane of the organelles (ER and Golgi). When the cell is stimulated, channels in the plasma membrane and in the organelles are opened, generating the immediate increase of cytosolic Ca2+ which is then returned to the basal concentration by the system above. The precise control of Ca 2+ homeostasis is fundamental for important activities of the cell, e.g., gene transcription in the nucleus, energy transformation in mitochondria, exocytosis (secretion) mechanisms, muscle contraction (T-tubules). The values of Ca2+ concentration indicated in the cartoon are only representative and may vary depending on the cell type. The insets surrounded by black lines represent specifi c compartments or organelles. The fi lling color indicates the concentration of Ca 2+ in the compartment/organelle (e.g., dark blue: high Ca2+ concentration, light blue: low Ca2+ concentration). All symbols and acronyms in the cartoon are explained and described in the text. Some clarifi cations: in the lysosomes the putative system that accumulates Ca 2+ in the lumen of the organelle is indicated with a question mark, as it has not been characterized. In the ER/SR numerous Ca2+ -buffer proteins are present, however only calsequestrin (Calseq) is indicated. Inside the vesicles , the little green shapes with spikes represent the secreted molecules. The arrows indi- 2+ 2+ cate the direction of Ca ion fl uxes. CaR, IP3 R, STIM, Mfn2, and NCX refer to Ca sensors, + 2+ InsP3 R, STIM1, mitofusin 2, and Na /Ca exchanger, respectively, in the text.

or the Ca2+ ionophore ionomycin. Increased cytosolic Ca2+ activates autophagy via a signaling pathway involving the CaMKK, the ΑMP-activated protein kinase (AMPK), and mTOR. An independent pathway in which the elevation in cytosolic Ca2+ activates autophagy in AMPK-knock out fi broblasts has also been proposed [175 ]. While the unambiguous correlation between cytosolic Ca2+ levels and autophagy activity is 116 Brini, Ottolini, Calì, and Carafoli diffi cult to demonstrate, a clear correlation has been established with the state of fi lling of the ER Ca2+ store. This underlines the close relationship between the autophagic and apoptotic pathways, since the latter is also strictly related to the concentration of luminal ER Ca2+ and to the amount that can be released by the ER. Briefl y, Bcl-2 acts a suppressor of both Ca 2+-dependent apoptotic and autophagic processes. In the autophagy pathway, the proposal that Bcl-2 acts by binding beclin 1, and also by lowering ER Ca 2+ concentration has been convincingly documented [ 176 – 178 ]. It has also been shown that treatments which enhance autophagy, e.g., that with the mTOR inhibitor rapamycin, or the deprivation of nutrients, also remodeled the intracellular Ca2+ signaling machinery [179 , 180 ]. The comprehensive cartoon of Figure 3 summarizes visually the information provided up to this point on the systems for the control of cell Ca 2+, and on the processes regulated by its signal.

5 The Ambivalence of the Calcium Signal: Defects of Calcium Regulation and Disease

As has been made clear in the preceding section, the Ca2+ message is vital to the correct functioning of most cell processes. It has also been made clear that Ca2+ within cells must be controlled with utmost precision, even when the aim of the Ca2+ signal is to terminate cells in the apoptotic process. Conditions may arise, however, in which the control of cell Ca2+ fails, in which cases cells predictably develop various forms of pathological dysfunctions. Conditions in which the lack of control of cell Ca2+ is massive and global terminate rapidly cell life: these are the cases of toxic cell death induced by massive conditions of Ca 2+ overload. However, subtler Ca2+ defects that affect single systems for the control of Ca 2+ permit cell life to continue, albeit with various degrees of discomfort. A number of these conditions are genetic, and their study has even contributed to the understanding of the mechanisms for the control of Ca2+ and the decoding of its message. A brief description of these conditions will be offered in the next sections, which will cover only the most important (and interesting) among them.

5.1 Neuronal Diseases

5.1.1 Ataxia

Ataxias are neurological disorders characterized by lack of coordination in voluntary movements. There are 3 types of ataxia: cerebellar, sensory, and vestibular. The last two types involve problems in the dorsal spinal cord (due to impaired proprioception) and in the vestibular system, respectively: they represent a minor percentage of all 4 Calcium in Health and Disease 117 ataxias. The cerebellar ataxias, and more precisely the spinocerebellar type (SCA), are the most intensively studied: Ca2+ frequently has been involved in their pathogenesis. They are caused by defects in more than 30 genes [181 ]. Their correlation to the impairment of Ca2+ homeostasis is obvious in patients which present mutations in proteins involved in Ca2+ regulation. Mutations in CACNA1A, a subunit of CAV2.1 voltage-dependent P/Q-type Ca2+ channels, which are expressed abundantly in cerebellar Purkinje cells indeed induce SCA6 [182 ]: the most frequent mutation is the expansion of CAG repeats, that specify glutamines (more than 19Q in SCA6) at the C-terminal of the subunit. SCA6 is thus one of the poly- glutamine (poly-Q) neurological diseases. The N-terminal portion of the protein containing the long poly-Q chain speciﬁ ed by the CAG repeats is processed by proteases, producing a (presumably toxic) fragment that can aggregate or translocate to other cell compartments. The Ca2+ channel that generates the poly-Q fragment seems not to be damaged [ 183 ], and its mutant subunit can aggregate even if not cleaved by the protease [184 ]. Other point mutations in the CACNA1A gene induce different ataxic phenotypes, e.g., episodic ataxia type 2 and early-onset cerebellar atrophy [ 185]. Partial deletions in the gene of InsP3 R1, instead, cause SCA15, SCA16, and SCA29 [ 186, 187] and a marked downregulation of the

InsP 3 R has been found in several SCA15 patients [181 ]. Alterations of Ca2+ inﬂ ux into neurons due to glutamate excitotoxicity can be involved in the pathogenesis of SCA5, a condition caused by a mutation in the SPTBN2 gene (β-III spectrin): the mutant protein becomes unable to stabilize the glutamate transporter EEAT4 in the plasma membrane of Purkinje cells, predisposing them to excitotoxicity [188 ]. Also SCA1, SCA2, and SCA3 are poly-glutamine diseases: in these cases the mutation affects ataxin-1, -2, and -3, respectively [181 ]. The major function of ataxin-1 is the regulation of the transcription of several genes [189 ]. Ataxin-2 is instead probably involved in the control of mRNA regulation [ 190 ] and ataxin-3 is a potent transcriptional repressor with deubiquitinating activity [191 ]. Mouse ataxin-1 mutants have altered levels of several proteins involved in Ca2+ homeostasis, most of them with Ca2+ buffering function and located in the ER

[192 ]. Evidence has been provided that the InsP3 R interacts with mutant forms of ataxin-1, ataxin-2, and ataxin-3 [191 , 192 ]: accordingly, mice with InsP3 R deletions display an ataxic phenotype [193 ]. Mitochondrial Ca2+ handling has also been studied in a SCA28 disease model: the ablation of the AFG3L2 gene, which encodes a mitochondrial protease mutated in this form of ataxia, causes impaired mitochondrial Ca2+ uptake and respiratory chain dysfunction. The impaired mitochondrial Ca 2+ uptake is due to the increased organelle fragmentation and to the loss of ER-mitochondria connections [194 ]. Ca 2+ pumps have also been involved in ataxias. A defect of one of the PMCA pump isoforms, PMCA3, has recently been discovered in a case of X-linked human cerebellar ataxia. The defect impairs the ability of the pump to properly eject Ca 2+ from cells overexpressing it [195 ]. Genetic defects of a special PMCA2 isoform which cause deafness (see Section 5.3 ) also causes equilibrium defects. The ataxic “Wriggle Sagami” mouse model, in which a genetically defective PMCA2 has been detected, displays impaired development of Purkinje cells dendrites and synaptic connections [196 ]. 118 Brini, Ottolini, Calì, and Carafoli

As repeatedly mentioned above, the role of the release of Ca 2+ from lysosomes through a two-pore channel (TPC) is controversial. Emerging evidence has nevertheless shown that in Niemann-Pick disease type C, a metabolic neurodegenerative disease causing ataxia with selective loss of Purkinje neurons, there is an alteration of lysosome-related Ca2+ signaling [197 ].

5.1.2 Migraine

Migraine is a disorder characterized by headache in combination with nausea, photophobia, phonophobia, and vomiting. The headache, which is unilateral and pulsating, is generally preceded by an “aura”, a neurological symptom characterized by a visual (or other sensory) disturbance [198 ]. Migraine has generally a polygenic and multifactorial inheritance, but some monogenic types also exist. Among them the best studied is the rare autosomal dominant familial hemiplegic migraine (FHM) [199 ]. Three genes have been linked to the disease (see [200 ] for a brief review): CACNA1A , that encodes the pore-forming subunit of the voltage-gated CAV2.1 P/Q-type Ca2+ channel [201 ], ATP1A2, that encodes the α-2 subunit of the Na + /K+ pump, and SCN1A, that encodes the neuronal voltage-gated sodium channel. A novel mutation has recently also been found in the SLC4A4 gene, encoding + the Na /HCO3 transporter. CAV2.1 channels are involved in the initiation of action potential-evoked neurotransmitter release and are expressed in the presyn- aptic terminals and the somato-dendritic membrane of spinal cord and brain neurons [202 ] (their mutations have also been linked to SCA6 and episodic ataxia type 2, see above). Two mice models that recapitulate FHM have contributed signiﬁ - cantly to the study of the human disease. A number of reports have described a gain-of- function of mutant CAV2.1 channels with increased open probability and activation at lower voltage [203 ]. Other reports have described alterations of the inactivation of CAV2.1 due to the increasing dissociation of regulatory G proteins [203 ].

5.2 Neurodegenerative Diseases

The evidence that connects Ca2+ dyshomeostasis with neurodegenerative diseases like Parkinson’s (PD), Alzheimer’s (AD), Huntington’s diseases (HD), and amyotrophic lateral sclerosis (ALS) is now abundant [204 ]. All these diseases are characterized by the loss of speciﬁ c neurons somehow linked to the deposition of abnormal proteinaceous aggregates. Ca2+ dysregulation and alteration of Ca 2+ signaling have also been detected in animal and cellular model of all these diseases, suggesting that they could have a role in the apoptotic death of neurons [205 ]. Multiple mitochondrial defects are also present. Mitochondria move along axons and supply the energy necessary to the pumps for the extrusion of Ca 2+ (and other) ions. Neurons are uniquely exposed to glutamate excitotoxicity, which can lead to massive Ca 2+ 4 Calcium in Health and Disease 119 inﬂ ux and to the activation of detrimental enzymes like phospholipases, endonucleases, and proteases (e.g., calpains). As discussed in Section 3.2 , mitochondria can temporarily counteract the effects of cytosolic Ca2+ overload by buffering the excessive level of Ca2+ in the cytosol.

5.2.1 Parkinson’s Disease

The progressive death of dopaminergic neurons (DN) of the substantia nigra pars compacta containing proteinaceous aggregates of α-syn called Lewy bodies is the hallmark of Parkinson’s disease. Only about 5% of PD cases are genetic, with mutations in several proteins, among them α-synuclein (α-syn), parkin, DJ-1, PINK1, and LRRK2. All these proteins are somehow involved in the functions of mitochondria [205 ], suggesting the possible involvement of the organelles in the etiology of PD. The ﬁ rst indication came from the discovery that the ingestion of 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP), a poison of complex I of the respiratory chain, or the exposure to pesticides like rotenone and paraquat (which also inhibit complex I), induced a phenotype that recapitulated almost all the aspects of idiopathic PD. In addition to the toxicity of α-syn aggregates and mitochondria damage, oxidative stress and Ca2+ homeostasis impairment have also come into focus as possible factors in the molecular etiology of PD [206 ]. The role of Ca2+ dyshomeostasis is supported by a number of observations [207 ]: DNs are peculiarly susceptible to “Ca2+ insult” since they use L-type Ca2+ channels for their normal pacemaking activity, instead of the Na+ channels used by other types of neurons. They are constitutively exposed to the risk of Ca 2+ overload, and it has indeed been shown that those DNs that express high levels of Ca 2+ -buffering proteins (e.g., calbindin D28K, calretinin, and parvalbumin) are protected from degeneration [208 ]. Another interesting characteristic of DNs is the lower total mitochondrial mass with respect to other cells, which decreases their Ca2+ -buffering power [208 ]. As for the oxidative damage, it could be linked to the dyshomeostasis of Ca2+ , as the Ca2+ defect could be responsible for the excessive production of ROS. A protein called DJ-1, which is involved in the defense of cells against oxidative stress [209 ], counteracts the ROS produced during the partial uncoupling of mitochondria in the course of pacemaking activity, which is a mechanism used by DNs to limit the uptake of Ca2+ [ 210 ]. Thus, the DJ-1 KO-DNs have enhanced vulnerability to Ca2+ -induced ROS production. Evidence for the participation of DJ-1 in Ca2+ homeostasis is limited, but our laboratory has recently shown that the protein increases the ER-mitochondria connection, and thus the correct Ca2+ transfer between the two organelles [211 ]. Interestingly, this effect is shared with α-syn, the other protein that is frequently mutated in PD, and with parkin [212 – 214 ]. Abundant information is also available on the toxic effect of α-syn aggregates (or mutants) on Ca2+ homeostasis [ 204, 206]. As for PINK1, it has been claimed to control the mitochondrial Na + /Ca2+ exchanger, its deletion predisposing cells to mitochondrial Ca2+ overload [ 215 ]. It has also been claimed that PINK1 deletion directly impairs mitochondrial Ca 2+ accumulation [216 ]. 120 Brini, Ottolini, Calì, and Carafoli

5.2.2 Alzheimer’s Disease

Alzheimer’s disease, which is the most common form of dementia, represents one of the most important pathologies in developed countries [217 ]. Although ageing is acknowledged as a primary risk factor, the cause of the disease is still obscure. The extracellular space of cerebral cortex in AD patients presents aggregates, called “plaques”, of β-amyloid (Abeta), a 29–43 amino acid peptide, originated by the cleavage of a large transmembrane protein (the amyloid precursor protein (APP)), by the β and γ secretases. Proteinaceous aggregates of the phosphorylated tau protein, called “neurofi brillary tangles”, are also present within neurons. 95% of AD cases are sporadic [208 ], but familial forms of the disease occur in patients with mutations in APP, and in the ER proteins presenilin 1 and 2 (PS1 and PS2, respectively), which are the catalytic core of γ-secretase. Ca2+ dyshomeostasis is increasingly recognized as an important factor in the etiology of AD. Elevated levels of intracellular Ca 2+ have been observed in the areas of brain affected by AD pathology, with stimulation of several Ca 2+ activated enzymes, phosphorylation of tau, and processing of APP to Abeta [ 208]. The latter peptide may then initiate a vicious circle in which its oligomers would form pores in the cell membranes that potentiate Ca2+ entry and increase cytosolic Ca2+ . However, Abeta has also been claimed to impair glutamatergic signaling, by somehow reducing the number of NMDA receptors and thus the infl ux of Ca2+ into the neurons. Interestingly, the intracellular portion of APP, that is released after secretase cleavage, modulates Ca2+ effl ux from ER, thus also altering intracellular Ca2+ homeostasis [208 ]. Mutant forms of PS1 and PS2 also modify InsP 3R and RyR activity, thus altering ER Ca 2+ release. PS1 has been claimed to form Ca 2+ -conducting pores in the ER membrane. Its mutation reduced ER Ca2+ leak and thus enhanced the ER Ca2+ levels. Mutant PS were also shown to increase the expression and the sensitvity of ER Ca2+ release channels, thus promoting exaggerated Ca2+ release after stimulation. However, the pore-forming ability of PS is controversial, as other reports have not confi rmed it and failed to measure enhanced ER Ca 2+ levels in cells overexpressing mutant PSs [ 218]. PSs also regulate the activity of other Ca 2+ -related proteins as sorcin, calmyrin, and calsenilin/DREAM [219 ], and modify the activation of store-operated Ca2+ channels, as they alter the expression of the STIM protein [ 218 ]. Very recently, some works describe a direct role of PSs in the regulation of MAM activities, e.g., Ca2+ transfer from ER to mitochondria [213 ].

5.2.3 Huntington’s Disease

Huntington’s disease is a purely genetic disease, caused by a mutation in the ﬁ rst exon of the gene that encodes the ubiquitous protein huntingtin (Htt). The mutation increases the length of the poly-glutamine tract normally present in the N-terminus portion of the protein and eventually causes the death of neurons of brain regions involved in motor circuit (neostriatum). HD is thus one of the poly- glutamine diseases. The number of CAG repeats determines the age of onset of 4 Calcium in Health and Disease 121

HD and its severity (35Qs being the upper limit for a normal life). Htt is a 348 kDa protein of unclear function, however, it has been proposed to be involved in important processes like gene transcription, apoptosis, and organelle regulation [204 ]. Its abnormal poly-Q tract is cleaved by proteases (caspase 6), and the fragment has the propensity to aggregate to form ﬁ brils or oligomers, which have been variously proposed to be toxic or even protective to cells [ 204 , 220 ]. The idea is now gaining momentum that the toxic species is the Htt monomer, which could explain why the larger aggregates, which may “sequester” the monomer, could be protective. Ample evidence suggests an action of Htt on Ca2+ signaling [221 ], and a disruption of mitochondrial Ca2+ homeostasis in HD cells has indeed been documented by different groups (it has even been proposed that Htt can interact directly with mitochondria [220 ]). The Htt fragment can migrate to the cell nucleus, where it would be involved in the regulation of the expression of gene, including that of the InsP3 R [220 ]. However, Htt has also been shown to directly interact with 2+ 2+ InsP 3R and to regulate ER Ca release [204 ]. Augmented Ca leak from RyR, and subsequent cell death, has also been observed in neurons expressing mutant Htt [ 222 ]. Work in our laboratory has shown that the Ca2+ dyshomeostasis condition is associated to a severe damage in mitochondrial dynamics [223 ]. HD neurons display elevated expression of metabotropic glutamate receptors, which can activate

InsP 3 signaling. The NMDA glutamate receptor appears to be hypersensitized by mutant Htt with subsequent increased Ca2+ inﬂ ux in the neurons [208 ]. A recent report has linked extracellular Ca2+ and glutamate toxicity, by showing that a novel compound protects mutant Htt expressing neuronal cells from apoptosis by TRPC1- mediated SOCE inhibition [224 ].

5.2.4 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis is caused by the loss of motor neurons in the motor cortex and the spinal cord [225 ]. The molecular/cellular phenotype is characterized by oxidative stress, organelle dysfunctions, and Ca2+ imbalance [226 ]. About 5–10% of ALS cases are familiar, and 20% of them present a mutation in the gene that encodes superoxide dismutase 1 (SOD1) [227 ]. The remaining genetic cases are caused by mutations of numerous other genes, e.g., TDP-43 (TAR DNA binding protein 43), VAPB (vesicle-associated membrane protein-associated protein B/C) and FUS (fused in sarcoma). As in PD, HD, and AD, ALS neurons also present proteinaceous inclusions in the soma and in the axon which are composed by ubiquitin and the proteins cited above. Most ALS research is now concentrated on the mutations of SOD1: since the protein is critical in the defense against oxidative stress, the notion that oxidative stress is at the basis of ALS cellular phenotype has traditionally occupied the central stage. However, a number of aspects of the cellular phenotype of ALS are not solely explained by oxidative stress: they could instead be explained by Ca2+ signaling dysfunctions. Motor neurons are normally exposed to numerous and rapid Ca 2+ 122 Brini, Ottolini, Calì, and Carafoli transients, which are necessary for their physiological rhythmic activity. As a result, they have lower Ca2+ buffering capacity than other neurons [208 ]. This exposes them to the risk of Ca 2+ overload, which is exacerbated by their higher number of AMPARs [204 ]. Interestingly, the Ca 2+ -permeability of AMPARs is augmented by the mutations of SOD1, and the Arg-Gln substitution in the GluR2 subunit, which blocks Ca2+ inﬂ ux, is defective in ALS patients. Surprisingly, however, even if glutamate excitotoxicity explains several aspects of the ALS phenotype, the deletion of the glutamate transporter in astrocytes (that induces an increased concentration of the neurotransmitter around the neurons) has no deleterious effects on motor neurons [204 ]. Ca 2+-buffering defects have been found in the mitochondria of synapses of mutant SOD1 mice, suggesting the possibility that impairment of mitochondrial Ca 2+ handling is important in the pathogenesis of ALS. Interestingly, recent studies have indeed shown that VAPB (one of the proteins mutated in genetic forms of ALS) has a role in mitochondrial dynamics and in the ER-mitochondria Ca2+ transfer (as other proteins do in AD and PD (see above)) [204 , 213 , 228 ].

5.3 Genetic Hearing Loss

Hearing depends on the conversion of the sound waves transmitted through the endolymph of the inner ear into signals that are transduced by the hair cells of the Corti organ through the mechanoelectric transduction (MET) process. The sound waves defl ect the stereociliar bundle that protrude from the hair cells, inducing the opening of the MET channels that mediate the penetration of K + and Ca 2+ into the stereociliar cytoplasm: only about 0.2% of the total MET current is carried by Ca2+ , that must be exported back to the endolymph by a special variant of isoform 2 of the plasma membrane Ca2+ pump: a splicing insert in the fi rst cytosolic loop directs the variant to the apical portion of the hair cell [ 229 ], and a second insert in its C-terminal cytosolic CaM-binding domain truncates it about 50 residues short of the normal C-terminus (PMCA2wa ). PMCA2 is unique among the isoforms of the PMCA pump because of its ability to function very effectively even in the absence of the natural activator CaM, and the doubly spliced variant wa has lower Ca2+ -pumping activity than the full length, unspliced isoform. These special characteristics of the PMCA pump have evidently been evolutionarily adjusted to satisfy the requirements of the Ca2+ balance between the stereociliar cytoplasm and the endolymph: an extracellular fl uid in which the uniquely low Ca2+ concentration (see above) must be constantly maintained at its very low μM level. This demands a PMCA variant that is able to pump Ca2+ with limited effi ciency, protected by the oscillations in the natural activator CaM that would instead greatly infl uence the activity of all other PMCA pump isoforms. In the endolymph, Ca 2+ binds reversibly to a single pass stereociliar EF hand Ca2+ -binding protein, cadherin 23, which, together with protocadherin 15, forms the tips links that organize the stereociliar bundle to promote its defl ection. The balance 4 Calcium in Health and Disease 123 of Ca2+ between the stereocilia and the endolymph is vital to the correct operation of the stereocilia bundle, and must be maintained with the exquisite precision that is necessary for the correct functioning of the MET process: it is thus not surprising that mutations of the stereociliar PMCA pump (and/or of the Ca2+ binding ability of cadherin 23) should have been found to generate a deafness phenotype. Such phenotypes have been described in mice and also in humans [230 – 232]: the defects of the PMCA pump are invariably characterized by an effi ciency of Ca2+ pumping that is lower than that of the wt PMCA2 wa pump (which, as mentioned, is lower than that of the full length unspliced PMCA2 pump): the pump defect has been analyzed molecularly in both mice and humans, and found to predominantly affect the long term, unstimulated basal ability of the pump to export Ca2+ to the endolymph rather that the burst of pump activity in response to the arrival of a large Ca 2+ load. Depending on the functional severity of the pump mutations, the deafness phenotype may or may not demand a concomitant loss of function mutation of cadherin 23.

5.4 Cardiac Diseases (Cardiomyopathies)

Ca2+ links the electrical signals at the heart sarcolemma with the contraction of the myocytes [233 ]. The concerted operation of the proteins/systems involved in the myocyte contraction process allows heart to function normally. Its disruption leads to diverse disease phenotypes, which are generally classifi ed into four categories: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM) and arrhythmogenic right ventricular dysplasia (ARVD) (reviewed in [ 234]). A large number of mutations in the genes of Ca 2+ -dependent contractile proteins have been identifi ed in HCM, DCM, and RCM (reviewed in [ 235]), but none of the two disease-causing gene defects identifi ed in familial forms of ARVD are related to them. HCM- and RCM-causing mutations increase the Ca2+ sensitivity of cardiac muscle contraction because they impair the interaction of TnT with TnI, whereas DCM-causing mutations decrease it due to an increased affi nity of TnT for tropomyosin. HCM-causing mutations in myosin and myosin-binding protein C have also been described that lead to increased Ca 2+ sensitivity of cardiac myofi laments. The diastolic and systolic dysfunction observed in HCM and DCM, respectively, fi ts well with the increase and decrease in the myofi lament Ca2+ sensitivity and might lead to increase and decrease of the ventricular wall stress. Genetic defects of cardiac ryanodine receptor (RyR2) have instead been identifi ed in ARVD [ 236]. The cardiac RyR associates with four molecules of FKBP12.6 (the immunophilin mentioned above that binds the immunosuppressive agent FK506). Four mutations map in the cytosolic portion of the receptor, two of them clustering in the central FKBP12.6- interacting domain. The mutations cause hypersensitivity of the receptor to activate levels of Ca2+ , and lead to abnormal excitation-contraction coupling and arrhythmias (they eventually also trigger apoptosis and/or necrosis of the cardiomyocytes). 124 Brini, Ottolini, Calì, and Carafoli

Impairments in Ca2+ cycling are also considered early signs for the adaptive hypertrophic response upon heart damage or increased volume load [ 237]. During this adaptation, a number of changes associated with the process of Ca2+ handling have been observed: the activity of the SERCA pump increases, thus resulting in augmented SR store loading. With the progression of the disease, failing hearts exhibit increased sensitivity of RyRs to activation by luminal Ca2+ . The potentiated spontaneous Ca2+ release [ 238 ] would contribute to the decreased SR Ca 2+ content. The expression level of 2+ another important Ca homeostasis actor, i.e., the InsP3 R, is also critical for the main- 2+ tenance of rhythmic heart Ca signals. InsP3 Rs are typically 50-fold less abundant in healthy cardiomyocytes than RyRs [239 ], but their expression increases signiﬁ cantly during hypertrophy and heart failure [240 ]. The location of InsP 3R next to the RyRs might be important in the process of CICR and thus in EC-coupling.

5.5 Skeletal Muscle Diseases

Malignant hyperthermia, central core disease, and Brody’s disease are three Ca 2+ - related pathologies that affect skeletal muscles. Another muscular disease (Duchenne muscular dystrophy) also involves Ca2+ control deﬁ ciencies, but the central genetic defect of the disease affects a protein that is not directly Ca2+ -related.

5.5.1 Malignant Hyperthermia

Malignant hyperthermia (MH) is a disorder in which the exposure to volatile anes- thetics and muscle relaxants (e.g., succinylcholine) causes a dysregulation (increase) of myoplasmic Ca 2+ that produces hypercontraction of muscles in susceptible patients. The condition occurs with a frequency of 1:3000 cases, with a mortality rate which originally was 70–80%, but is now less than 5% [241 ] thanks to the discovery of the positive therapeutic effects of dantrolene, a drug that inhibits the release of Ca2+ through the RyR [242 ]. During the MH episode the release of Ca2+ from the SR, probably activated by the anesthetic drugs, becomes uncontrolled, overwhelming the Ca2+ homeostatic capacity of myocytes, and resulting in a sustained muscle contracture that consumes completely the cellular ATP and dramatically increases heat production. The death of myocytes is eventually caused by the loss of membrane integrity with leakage of muscle cell contents [243 ]. The principal cause of the susceptibility are mutations in the RyR1 gene [244 ], but mutations in the gene that encodes the α1 subunit of the L-type voltage Ca2+ channel (CACNA1S [245 ]) have also been detected. The current view on the molecular etiology of the disease claims that the mutations predispose the RyR1 to become more readily opened. An alternative proposal involves instead the participation of ECCE (excitation-coupled Ca 2+ entry) [246 ] and does not consider the increased leakage of Ca2+ from the SR [247 ]. 4 Calcium in Health and Disease 125

5.5.2 Central Core Disease

Central core disease (CCD), a congenital myopathy that induces weakness of the muscles of lower extremities, is also caused by mutations of the RyR1 [248 ]. The disease is characterized by a particular amorphous area (called “core”), frequently located in the center of the fi bers of muscle, in which no mitochondria are found [249 ]. Lack of glycogen granules and myofi brillar disorganization has also been described [250 ]. The mechanisms by which the mutations cause the CCD molecular phenotype, and why different mutations in the same protein can generate two pathologies as different as MH and CCD is still obscure. However, four types of channel defects have been recognized: (i) mutations that cause hyper-sensitization to electrical and pharmacological stimuli (as in MH); (ii) mutations that result in leaky channels and in depletion of Ca2+ from SR; (iii) mutations that cause excitation–contraction uncoupling (“E-C hypothesis”), with lack of ability of the Cav1.1 channel to activate the initial release of Ca2+ from the SR; (iv) mutations that cause a decrease of RyR1 channels expression in SR membranes [251 ]. The trigger event of the myopathy seems to be the inability of myocytes to achieve a cytosolic Ca 2+ concentration suffi cient to induce the contraction of muscle fi bers [244 ].

5.5.3 Brody’s Disease

Brody’s disease (BD) is a rare early-onset myopathy caused by mutations in the ATP2A1 gene that encodes isoform 1 of the SERCA pump. The disease is characterized by muscle stiffness: the contraction phase is normal, but the time of relaxation, after vigorous exercise or rapid movement, increases signifi cantly and slight atrophy in type 2 fi bers is usually present [ 252]. The pathophysiology of BD is now clarifi ed: defective SERCA activity causes a slow and diffi cult relaxation, since the reuptake of the Ca2+ released from the SR is impaired. Some patients show decreased activity of the pump, without mutations in its gene: these cases are classifi ed as “Brody syndromes” [253 ]. Two drugs are available for the therapy of the disease: dantrolene, which inhibits the release of Ca2+ from the SR (see above), and verapamil, a blocker of the L-type channels, and thus of the membrane depolarization that triggers the initial SR-Ca 2+ release. However, they only partially cure the symptoms [254 ].

5.5.4 Duchenne Muscular Dystrophy

Mutations in dystrophin, a protein encoded by a very large and complex gene, cause Duchenne muscular dystrophy (DMD), an invariably fatal X-linked disease that causes progressive muscle weakness with subsequent muscular degeneration. Dystrophin is part of a large complex of at least 10 proteins [255 ] and forms a transmembrane bridge between intracellular actin and the extracellular matrix. 126 Brini, Ottolini, Calì, and Carafoli

The dystrophin gene is susceptible to deletions, splicing errors and frame shifts which decrease or abolish its expression and disrupt the complex causing plasma membrane instability [256 ]. In parallel with these dystrophin defects, the Ca 2+ content of DMD myocytes has been found to be extremely elevated, suggesting the involvement of Ca2+ in the pathophysiology of the disease [257 ,258 ]. Normal muscles that undergo intensive and stressing exercises can experience pain, swelling, and inﬂ ammation, all effects that appear to be correlated to excessive Ca2+ entry into the myocytes [259 ]. In DMD muscles that are predisposed to be weak and fragile, the situation is more dramatic [ 260]. Plasma membrane ruptures conclusively allow the entering of excessive Ca2+ in the myocyte cytosol, but it has also been proposed that stretch-activated channels that normally allow the entering of Ca2+ are more active in DMD muscles [257 ]. The condition of Ca2+ overload, which is likely to be exacerbated by the defective Ca2+ -buffering capacity documented in DMD [261 ], would activate detrimental proteases, e.g., calpains, that induce myonecrosis [262 ]. Another important effect of the Ca2+ imbalance in the myocytes is the increased production of ROS. Recent evidence has linked the mechanical stress-mediated entering of Ca2+ into DMD myocytes to the potentiation of ROS production [263 ]. DMD, however, also affects the heart: abundant data in literature have discussed the relationship between Ca2+ imbalance and heart function in DMD hearts [264 ]. DMD patients generally display dilated cardiomyopathy and heart failure, which could probably be due to the altered handling of Ca2+ by SR [ 265]. However, the proposal is debated: no differences in heart SR Ca2+ content have been found in some reports [ 266 ], whereas other reports have instead shown increased SR Ca2+ level [267 ].

6 Conclusions

The Ca2+ signal controls the most important processes which shape cell life, from its origin at fertilization to its end in the process of programmed death. Ca2+ must thus be very precisely controlled in the cell ambient: to this aim evolution has developed numerous means from specifi c binding proteins to systems that transport Ca2+ across membrane boundaries. They maintain cell Ca2+ at a basal (very low) set point, that is selectively and transiently increased according to the demands of the targets of its message. Cells are sealed to external Ca 2+ by the plasma membrane barrier, that only admits the passage of Ca2+ in a carefully controlled way from the virtually unlimited source in the external spaces. The fact that the concentration of Ca2+ is much higher in the external spaces than inside cells is a dynamically favorable situation, as it ensures that even slight increases of the permeability of the plasma membrane, such as those produced by the opening of specifi c channels, promptly generate signifi - cant swings of Ca2+ within the cytosol. The main reservoir of Ca2+ in the organism is the bone compartment, in which dynamic exchanges reversibly regulate Ca2+ in the circulating fl uids and in the extracellular spaces of the tissues. The dynamics of Ca2+ exchanges in bones is 4 Calcium in Health and Disease 127 controlled by hormones, as is the absorption of Ca 2+ in the intestine and its excretion and resorption in the kidneys. One distinctive feature of the Ca2+ signal is its ambivalence: the correct functioning of cell life demands its absolute spatial and temporal control within the cell ambient. Should this control become defective, various degrees of cell dyscomfort will ensue, that in extreme cases culminate in cell death.

Abbreviations

AD Alzheimer’s disease AGC aspartate/glutamate carrier AID atypical interacting domain AIF apoptosis-inducing factor ALP alkaline phosphatase ALS amyotrophic lateral sclerosis AMP adenosine 5′-monophosphate AMPA 2-amino-3-hydroxyl-5-ethyl-4-isoxazolepropionic acid AMPAR 2-amino-3-hydroxyl-5-ethyl-4-isoxazolepropionic acid receptor AMPK AMP-activated protein kinase ANKH ankylosis progressive homolog APP amyloid precursor protein ARVD arrhythmogenic right ventricular dysplasia ATP adenosine 5′-triphosphate BD Brody’s disease cADPR cyclic adenosine diphosphate ribose CaM calmodulin CaMK calmodulin-dependent kinase CaMKK calmodulin-dependent kinase kinase cAMP cyclic adenosine 5′-monophosphate CaRRE CaMKIV-responsive RNA elements CBP CREB-binding protein CCD central core disease CG cortical granules CICR Ca2+ -induced Ca2+ release CMA chaperone-mediated autophagy Cn calcineurin CoA coenzyme A CREB cAMP responsive element-binding protein DAG diacylglycerol DCM dilated cardiomyopathy DMD Duchenne muscular dystrophy DN dopaminergic neuron DRE downstream responsive element DREAM downstream regulatory element antagonist modulator 128 Brini, Ottolini, Calì, and Carafoli

ECC excitation-contraction coupling ECCE excitation-coupled Ca2+ entry ECF extracellular ﬂ uid eEF-2K eukaryotic elongation factor 2 kinase = CaMKIII ER endoplasmic reticulum ERK-MAP extracellular signal-regulated kinases-microtubule-associated protein ETC electron transport chain

FADH2 ﬂ avin adenine dinucleotide (reduced) FHM familial hemiplegic migraine FKBP FK506 binding protein FUS fused in sarcoma GSK3 glycogen synthase kinase 3 HCM hypertrophic cardiomyopathy HD Huntington’s disease Htt huntingtin hnRNP heterogeneous nuclear ribonucleoprotein IMM inner mitochondrial membrane

InsP 3 inositol 1,4,5-trisphosphate InsP 3 R inositol 1,4,5-trisphosphate receptor KO-DNs knock out dopaminergic neurons LAMP-2A lysosome-associated membrane protein type 2A LDCV large dense core vesicle MAM mitochondria-associated endoplasmic reticulum membrane MCU mitochondrial Ca2+ uniporter MET mechanoelectric transduction MH malignant hyperthermia MICU1 mitochondrial calcium uptake 1 MLCK myosin light chain kinase MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mTOR mammalian target of rapamycin MV matrix vesicle NAADP nicotinic acid adenine dinucleotide phosphate NAD nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide (reduced) NFAT nuclear factor of activated T cells NMDA N-methyl D-aspartate NPP1 nucleotide pyrophosphatase/phosphodiesterase 1 OMM outer mitochondrial membrane ORAI1 calcium release-activated calcium channel protein 1 P2X, P2Y purinergic receptors type X and type Y PC phosphocitrate PD Parkinson’s disease PDP pyruvate dehydrogenase phosphatase PhK phosphorylase kinase Pi inorganic phosphate 4 Calcium in Health and Disease 129

PI(3,5)P2 phosphatidylinositol 3,5-bisphosphate PIP2 phosphatidylinositol 4,5-bisphosphate PKA protein kinase A PL phospholipase PM plasma membrane PMCA plasma membrane Ca2+ -ATPase poly-Q poly-glutamine PPi inorganic diphosphate = pyrophosphate PS presenilin PTH parathyroid hormone RCC restrictive cardiomyopathy ROCC receptor-operated Ca2+ channels ROS reactive oxygen species RyR ryanodine receptor SCA spinocerebellar ataxia SERCA sarco/endoplasmic reticulum Ca2+ -ATPase SF sperm factor siRNA small interference ribonucleic acid SNARE soluble N-ethylmaleimide sensitive factor attachment protein receptor SOCCs store-operated Ca2+ channels SOCE store-operated Ca2+ entry SOD superoxide dismutase SPCA secretory pathway Ca2+ ATPase SR sarcoplasmic reticulum SRE serum response DNA regulatory element STIM sensors stromal interaction molecule Syt1 synaptotagmin I TAR transactivation response element TDP-43 TAR DNA-binding protein 43 TM transmembrane Tn troponin TPC two-pore channel TRP transient receptor potential channel VAPB vesicle-associated membrane-associated protein B/C VDAC1 voltage-dependent anion channel 1 VDR voltage-dependent receptor VOCCs voltage-operated Ca2+ channels

Acknowledgments The original work by the authors has been supported over the years by grants from the Italian Ministry of University and Research (FIRB2001 to E.C., PRIN 2003, 2005 and 2008 to M.B), the Telethon Foundation (Project GGP04169 to M.B.), the FP6 program of the European Union (FP6 Network of Excellence NeuroNe, LSH-2003-2.1.3-3 to E.C. and Integrated Project Eurohear to E.C.), the Human Frontier Science Program Organization to E.C., to ERANet-Neuron (nEUROsyn), and CARIPARO Foundation to E.C, the Italian National Research Council (CNR) and by a grant from the University of Padova (Progetto di Ateneo 2008 CPDA082825) to M.B. 130 Brini, Ottolini, Calì, and Carafoli

References

1. W. S. Fyfe, Geochemistry , Clarendon Press, Oxford, 1974. 2. F. H. Day, The Chemical Elements in Nature , George Harrup, London, 1963. 3. A. Kornberg, J. A. Spudich, D. L. Nelson, M. P. Deutscher, Annu. Rev. Biochem. 1975 , 37 , 51–78. 4. H. O. Halverson, Soc. Gen. Micro. Symp. 1963 , 13 , 343–368. 5. J. S. Woodhead, in Biochemical Aspects of Human Disease, Ed R. S. Elkeles, Blackwell, Oxford, 1982. 6. J. A. Ruben, A. F. Bennett, Nature 1981 , 291 , 411–413. 7. S. Omelon, J. Georgiou, Z. J. Henneman, L. M. Wise, B. Sukhu, T. Hunt, C. Wynnyckyj, D. Holmyard, R. Bielecki, M. D. Grynpas, PLoS One 2009 , 4 , e5634. 8. S. Mundlos, B. R. Olsen, FASEB J. 1997 , 11 , 227–233. 9. J. Kirkham, S. J. Brookes, R. C. Shore, W. A. Bonass, D. A. Smith, M. L. Wallwork, C. Robinson, Connect. Tissue Res. 1998 , 38 , 91–100; discussion 139–145. 10. W. Tong, M. J. Glimcher, J. L. Katz, L. Kuhn, S. J. Eppell, Calcif. Tissue Int. 2003 , 72 , 592–598. 11. A. L. Boskey, Curr. Osteoporos. Rep. 2006 , 4 , 71–75. 12. K. Verdelis, L. Lukashova, M. Yamauchi, P. Atsawasuwan, J. T. Wright, M. G. Peterson, D. Jha, A. L. Boskey, Eur. J. Oral. Sci. 2007 , 115 , 296–302. 13. A. Carden, M. D. Morris, J. Biomed. Opt. 2000 , 5 , 259–268. 14. G. N. Ramachandran, C. M. Venkatachalam, Biochim. Biophys. Acta 1966 , 120 , 457–458. 15. A. J. Hodge, Connect. Tissue Res. 1989 , 21 , 137–147. 16. L. E. Lanyon, Calcif. Tissue Int. 1993 , 53 Suppl. 1 , S102–106; discussion S106–107. 17. I. A. Silver, R. J. Murrills, D. J. Etherington, Exp. Cell. Res. 1988 , 175 , 266–276. 18. J. M. Delaisse, T. L. Andersen, M. T. Engsig, K. Henriksen, T. Troen, L. Blavier, Microsc. Res. Tech. 2003 , 61 , 504–513. 19. B. S. Moonga, R. Davidson, L. Sun, O. A. Adebanjo, J. Moser, M. Abedin, N. Zaidi, C. L. Huang, M. Zaidi, Biochem. Biophys. Res. Commun. 2001 , 283 , 770–775. 20. J. P. Li, H. Kajiya, F. Okamoto, A. Nakao, T. Iwamoto, K. Okabe, Endocrinology 2007 , 148 , 2116–2125. 21. H. J. Kim, V. Prasad, S. W. Hyung, Z. H. Lee, S. W. Lee, A. Bhargava, D. Pearce, Y. Lee, H. H. Kim, J. Cell. Biol. 2012 , 199 , 1145–1158. 22. D. J. Hadjidakis, I. I. Androulakis, Ann. N. Y. Acad. Sci. 2006 , 1092 , 385–396. 23. J. Kular, J. Tickner, S. M. Chim, J. Xu, Clin. Biochem. 2012 , 45 , 863–873. 24. H. F. DeLuca, Am. J. Clin. Nutr. 2004 , 80 , 1689S–1696S. 25. N. Chattopadhyay, E. M. Brown, Mol. Genet. Metab. 2006 , 89 , 189–202. 26. N. Balmain, D. Hotton, P. Cuisinier-Gleizes, H. Mathieu, J. Bone. Miner. Res. 1989 , 4 , 565–575. 27. C. I. Turnbull, K. Looi, J. E. Mangum, M. Meyer, R. J. Sayer, M. J. Hubbard, J. Biol. Chem. 2004 , 279 , 55850–55854. 28. J. Lotz, D. Steeger, G. Hafner, W. Ehrenthal, J. Heine, W. Prellwitz, Calcif. Tissue Int. 1995 , 57 , 253–257. 29. L. Hessle, K. A. Johnson, H. C. Anderson, S. Narisawa, A. Sali, J. W. Goding, R. Terkeltaub, J. L. Millan, Proc. Natl. Acad. Sci. USA 2002 , 99 , 9445–9449. 30. B. Strates, W. F. Neuman, Proc. Soc. Exp. Biol. Med. 1958 , 97 , 688–691. 31. C. R. Redhead, J. Physiol. 1988 , 401 , 455–468. 32. H. P. Wiesmann, U. Meyer, U. Plate, H. J. Hohling, Int. Rev. Cytol. 2005 , 242 , 121–156. 33. C. Qin, O. Baba, W. T. Butler, Crit. Rev. Oral. Biol. Med. 2004 , 15 , 126–136. 34. B. Huang, Y. Sun, I. Maciejewska, D. Qin, T. Peng, B. McIntyre, J. Wygant, W. T. Butler, C. Qin, Eur. J. Oral Sci. 2008 , 116 , 104–112. 35. H. C. Anderson, Clin. Orthop. Relat. Res. 1995 , 266–280. 36. M. Balcerzak, E. Hamade, L. Zhang, S. Pikula, G. Azzar, J. Radisson, J. Bandorowicz-Pikula, R. Buchet, Acta Biochim. Pol. 2003 , 50 , 1019–1038. 4 Calcium in Health and Disease 131

37. T. Kirsch, Curr. Opin. Rheumatol. 2006 , 18 , 174–180. 38. N. N. Nahar, L. R. Missana, R. Garimella, S. E. Tague, H. C. Anderson, J. Bone Miner. Metab. 2008 , 26 , 514–519. 39. T. Kirsch, G. Harrison, E. E. Golub, H. D. Nah, J. Biol. Chem. 2000 , 275 , 35577–35583. 40. T. Kirsch, H. D. Nah, D. R. Demuth, G. Harrison, E. E. Golub, S. L. Adams, M. Paciﬁ ci, Biochemistry 1997 , 36 , 3359–3367. 41. W. N. Addison, F. Azari, E. S. Sorensen, M. T. Kaartinen, M. D. McKee, J. Biol. Chem. 2007 , 282 , 15872–15883. 42. R. A. Terkeltaub, Am. J. Physiol. Cell. Physiol. 2001 , 281 , C1–C11. 43. A. L. Lehninger, C. S. Rossi, J. W. Greenawalt, Biochem. Biophys. Res. Commun. 1963 , 10 , 444–448. 44. J. W. Greenawalt, C. S. Rossi, A. L. Lehninger, J. Cell. Biol. 1964 , 23 , 21–38. 45. F. Gonzales, M. J. Karnovsky, J. Biophys. Biochem. Cytol. 1961 , 9 , 299–316. 46. A. L. Lehninger, Biochem. J. 1970 , 119 , 129–138. 47. W. P. Tew, C. D. Malis, J. E. Howard, A. L. Lehninger, Proc. Natl. Acad. Sci. USA 1981 , 78 , 5528–5532. 48. E. Carafoli, J. T. Penniston, Sci. Am. 1985 , 253 , 70–78. 49. N. Nakayama, H. Kawasaki, R. Kretsinger, Topics Biol. Inorg. Chem. 2000 , 3 , 29–58. 50. E. A. Nalefski, J. J. Falke, Protein Sci. 1996 , 5 , 2375–2390. 51. P. Raynal, H. B. Pollard, Biochim. Biophys. Acta 1994 , 1197 , 63–93. 52. A. Rosengarth, H. Luecke, J. Mol. Biol. 2003 , 326 , 1317–1325. 53. M. Brini, T. Cali, D. Ottolini, E. Carafoli, Met. Ions Life Sci. 2013 , 12 , 119–168. 54. R. S. Lewis, Cold Spring Harb. Perspect. Biol. 2011 , 3 . 55. M. Brini, E. Carafoli, Physiol. Rev. 2009 , 89 , 1341–1378. 56. J. M. Baughman, F. Perocchi, H. S. Girgis, M. Plovanich, C. A. Belcher-Timme, Y. Sancak, X. R. Bao, L. Strittmatter, O. Goldberger, R. L. Bogorad, V. Koteliansky, V. K. Mootha, Nature 2011 , 476 , 341–345. 57. D. De Stefani, A. Raffaello, E. Teardo, I. Szabo, R. Rizzuto, Nature 2011 , 476 , 336–340. 58. X. Wang, X. Zhang, X. P. Dong, M. Samie, X. Li, X. Cheng, A. Goschka, D. Shen, Y. Zhou, J. Harlow, M. X. Zhu, D. E. Clapham, D. Ren, H. Xu, Cell 2012 , 151 , 372–383. 59. L. E. Ford, R. J. Podolsky, Science 1970 , 167 , 58–59. 60. M. Endo, M. Tanaka, Y. Ogawa, Nature 1970 , 228 , 34–36. 61. J. C. Gilkey, L. F. Jaffe, E. B. Ridgway, G. T. Reynolds, J. Cell. Biol. 1978 , 76 , 448–466. 62. H. Cheng, W. J. Lederer, Physiol. Rev. 2008 , 88 , 1491–1545. 63. L. Leybaert, M. J. Sanderson, Physiol. Rev. 2012 , 92 , 1359–1392. 64. V. Parpura, T. A. Basarsky, F. Liu, K. Jeftinija, S. Jeftinija, P. G. Haydon, Nature 1994 , 369 , 744–747. 65. B. Innocenti, V. Parpura, P. G. Haydon, J. Neurosci. 2000 , 20 , 1800–1808. 66. A. M. Hofer, S. Curci, M. A. Doble, E. M. Brown, D. I. Soybel, Nat. Cell Biol. 2000 , 2 , 392–398. 67. N. M. Woods, K. S. Cuthbertson, P. H. Cobbold, Nature 1986 , 319 , 600–602. 68. M. Patron, A. Raffaello, V. Granatiero, A. Tosatto, G. Merli, D. De Stefani, L. Wright, G. Pallafacchina, A. Terrin, C. Mammucari, R. Rizzuto, J. Biol. Chem. 2013 , 288 , 10750–10758. 69. F. Perocchi, V. M. Gohil, H. S. Girgis, X. R. Bao, J. E. McCombs, A. E. Palmer, V. K. Mootha, Nature 2010, 467 , 291–296. 70. M. Plovanich, R. L. Bogorad, Y. Sancak, K. J. Kamer, L. Strittmatter, A. A. Li, H. S. Girgis, S. Kuchimanchi, J. De Groot, L. Speciner, N. Taneja, J. Oshea, V. Koteliansky, V. K. Mootha, PLoS One 2013 , 8 , e55785. 71. K. Mallilankaraman, C. Cardenas, P. J. Doonan, H. C. Chandramoorthy, K. M. Irrinki, T. Golenar, G. Csordas, P. Madireddi, J. Yang, M. Muller, R. Miller, J. E. Kolesar, J. Molgo, B. Kaufman, G. Hajnoczky, J. K. Foskett, M. Madesh, Nat. Cell Biol. 2012 , 14 , 1336–1343. 72. L. Boyman, G. S. Williams, D. Khananshvili, I. Sekler, W. J. Lederer, J. Mol. Cell. Cardiol. 2013 , 59, 205–213. 73. G. Szabadkai, K. Bianchi, P. Varnai, D. De Stefani, M. R. Wieckowski, D. Cavagna, A. I. Nagy, T. Balla, R. Rizzuto, J. Cell Biol. 2006 , 175 , 901–911. 132 Brini, Ottolini, Calì, and Carafoli

74. R. Rizzuto, P. Pinton, W. Carrington, F. S. Fay, K. E. Fogarty, L. M. Lifshitz, R. A. Tuft, T. Pozzan, Science 1998, 280, 1763–1766. 75. G. Csordas, C. Renken, P. Varnai, L. Walter, D. Weaver, K. F. Buttle, T. Balla, C. A. Mannella, G. Hajnoczky, J. Cell Biol. 2006 , 174 , 915–921. 76. J. R. Friedman, L. L. Lackner, M. West, J. R. DiBenedetto, J. Nunnari, G. K. Voeltz, Science 2011 , 334 , 358–362. 77. J. E. Vance, J. Biol. Chem. 1990 , 265 , 7248–7256. 78. A. E. Rusinol, Z. Cui, M. H. Chen, J. E. Vance, J. Biol. Chem. 1994 , 269 , 27494–27502. 79. A. A. Rowland, G. K. Voeltz, Nat. Rev. Mol. Cell Biol. 2012 , 13 , 607–625. 80. O. M. de Brito, L. Scorrano, Nature 2008 , 456 , 605–610. 81. C. Cardenas, R. A. Miller, I. Smith, T. Bui, J. Molgo, M. Muller, H. Vais, K. H. Cheung, J. Yang, I. Parker, C. B. Thompson, M. J. Birnbaum, K. R. Hallows, J. K. Foskett, Cell 2010 , 142 , 270–283. 82. P. Pizzo, V. Lissandron, P. Capitanio, T. Pozzan, Cell Calcium 2011 , 50 , 184–192. 83. I. Vandecaetsbeek, P. Vangheluwe, L. Raeymaekers, F. Wuytack, J. Vanoevelen, Cold Spring Harb. Perspect. Biol. 2011 , 3 . 84. A. Galione, Cold Spring Harb. Perspect. Biol. 2011 , 3 , a004036. 85. D. L. Clapper, T. F. Walseth, P. J. Dargie, H. C. Lee, J. Biol. Chem. 1987 , 262 , 9561–9568. 86. J. M. Cancela, G. C. Churchill, A. Galione, Nature 1999 , 398 , 74–76. 87. S. Patel, R. Docampo, Trends Cell Biol. 2010 , 20 , 277–286. 88. G. E. Hardingham, S. Chawla, C. M. Johnson, H. Bading, Nature 1997 , 385 , 260–265. 89. G. E. Hardingham, F. J. Arnold, H. Bading, Nat. Neurosci. 2001 , 4 , 261–267. 90. S. Chawla, G. E. Hardingham, D. R. Quinn, H. Bading, Science 1998 , 281 , 1505–1509. 91. B. Mayr, M. Montminy, Nat. Rev. Mol. Cell. Biol. 2001 , 2 , 599–609. 92. A. M. Carrion, W. A. Link, F. Ledo, B. Mellstrom, J. R. Naranjo, Nature 1999 , 398 , 80–84. 93. B. Mellstrom, J. R. Naranjo, Semin. Cell Dev. Biol. 2001 , 12 , 59–63. 94. R. Gomez-Villafuertes, B. Torres, J. Barrio, M. Savignac, N. Gabellini, F. Rizzato, B. Pintado, A. Gutierrez-Adan, B. Mellstrom, E. Carafoli, J. R. Naranjo, J. Neurosci. 2005 , 25 , 10822–10830. 95. J. J. Ronkainen, S. L. Hanninen, T. Korhonen, J. T. Koivumaki, R. Skoumal, S. Rautio, V. P. Ronkainen, P. Tavi, J. Physiol. 2011 , 589 , 2669–2686. 96. P. G. Hogan, L. Chen, J. Nardone, A. Rao, Genes Dev. 2003 , 17 , 2205–2232. 97. O. Dick, H. Bading, J. Biol. Chem. 2010 , 285 , 19354–19361. 98. N. Gomez-Ospina, F. Tsuruta, O. Barreto-Chang, L. Hu, R. Dolmetsch, Cell 2006 , 127 , 591–606. 99. G. Guroff, J. Biol. Chem. 1964 , 239 , 149–155. 100. I. Bertipaglia, E. Carafoli, Subcell. Biochem. 2007 , 45 , 29–53. 101. D. Cuerrier, T. Moldoveanu, P. L. Davies, J. Biol. Chem. 2005 , 280 , 40632–40641. 102. Y. Horikawa, N. Oda, N. J. Cox, X. Li, M. Orho-Melander, M. Hara, Y. Hinokio, T. H. Lindner, H. Mashima, P. E. Schwarz, L. del Bosque-Plata, Y. Oda, I. Yoshiuchi, S. Colilla, K. S. Polonsky, S. Wei, P. Concannon, N. Iwasaki, J. Schulze, L. J. Baier, C. Bogardus, L. Groop, E. Boerwinkle, C. L. Hanis, G. I. Bell, Nat. Genet. 2000 , 26 , 163–175. 103. I. Richard, O. Broux, V. Allamand, F. Fougerousse, N. Chiannilkulchai, N. Bourg, L. Brenguier, C. Devaud, P. Pasturaud, C. Roudaut, D. Hillaire, M.-R. Passos-Bueno, M. Zatz, J. A. Tischﬁ eld, M. Fardeau, C. E. Jackson, D. Cohen, J. S. Beckmann, Cell 1995 , 81 , 27–40. 104. T. Moldoveanu, C. M. Hosﬁ eld, D. Lim, J. S. Elce, Z. Jia, P. L. Davies, Cell 2002 , 108 , 649–660. 105. A. Okitani, D. Goll, M. Stromer, R. Robson, Federation Proc. 1976 , 35 , 1746. 106. S. S. Hook, A. R. Means, Annu. Rev. Pharmacol. Toxicol. 2001 , 41 , 471–505. 107. G. A. Wayman, H. Tokumitsu, M. A. Davare, T. R. Soderling, Cell Calcium 2011 , 50 , 1–8. 108. J. Robinson, R. J. Colbran, in Encyclopedia of Biological Chemistry , Ed W. L. D. Lane, Elsevier, 2004, Vol. 1, pp. 281–286. 109. A. Hudmon, H. Schulman, Biochem. J. 2002 , 364 , 593–611. 110. J. A. Lee, Y. Xing, D. Nguyen, J. Xie, C. J. Lee, D. L. Black, PLoS Biol 2007 , 5 , e40. 4 Calcium in Health and Disease 133

111. J. Yu, Y. Hai, G. Liu, T. Fang, S. K. Kung, J. Xie, J. Biol. Chem. 2009 , 284 , 1505–1513. 112. J. Aramburu, A. Rao, C. B. Klee, Curr. Top. Cell Regul. 2000 , 36 , 237–295. 113. F. D. Vasington, J. V. Murphy, J. Biol. Chem. 1962 , 237 , 2670–2677. 114. H. F. Deluca, G. W. Engstrom, Proc. Natl. Acad. Sci. USA 1961 , 47 , 1744–1750. 115. R. M. Denton, Biochim. Biophys. Acta 2009 , 1787 , 1309–1316. 116. T. Karpova, S. Danchuk, E. Kolobova, K. M. Popov, Biochim. Biophys. Acta 2003 , 1652 , 126–135. 117. P. J. Leblanc, M. Mulligan, A. Antolic, L. Macpherson, J. G. Inglis, D. Martin, B. D. Roy, S. J. Peters, Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008 , 295 , R1224–1230. 118. R. Rizzuto, C. Bastianutto, M. Brini, M. Murgia, T. Pozzan, J. Cell Biol. 1994 , 126 , 1183–1194. 119. L. S. Jouaville, P. Pinton, C. Bastianutto, G. A. Rutter, R. Rizzuto, Proc. Natl. Acad. Sci. USA 1999 , 96 , 13807–13812. 120. G. Hajnoczky, L. D. Robb-Gaspers, M. B. Seitz, A. P. Thomas, Cell 1995 , 82 , 415–424. 121. P. R. Territo, V. K. Mootha, S. A. French, R. S. Balaban, Am. J. Physiol. Cell Physiol. 2000 , 278 , C423–435. 122. A. del Arco, J. Satrustegui, J. Biol. Chem. 1998 , 273 , 23327–23334. 123. F. M. Lasorsa, P. Pinton, L. Palmieri, G. Fiermonte, R. Rizzuto, F. Palmieri, J. Biol. Chem. 2003 , 278 , 38686–38692. 124. S. Ringer, J. Physiol. 1883 , 4 , 29–42 23. 125. S. Ebashi, M. Endo, I. Ohtsuki, in Calcium as a Cellular Regulator, Ed E. C. C. Klee, Oxford University Press, New York, Oxford, 1999, pp. 579–595. 126. E. Rios, G. Brum, Nature 1987 , 325 , 717–720. 127. B. A. Block, T. Imagawa, K. P. Campbell, C. Franzini-Armstrong, J. Cell Biol. 1988 , 107 , 2587–2600. 128. C. J. Fearnley, H. L. Roderick, M. D. Bootman, Cold Spring Harb. Perspect. Biol. 2011 , 3 , a004242. 129. E. J. Cartwright, D. Oceandy, L. Neyses, Pﬂ ügers Arch. 2009 , 457 , 665–671. 130. D. C. Hill-Eubanks, M. E. Werner, T. J. Heppner, M. T. Nelson, Cold Spring Harb. Perspect. Biol. 2011 , 3 , a004549. 131. M. Iino, H. Kasai, T. Yamazawa, EMBO J. 1994 , 13 , 5026–5031. 132. B. Katz, R. Miledi, J. Physiol. 1967 , 189 , 535–544. 133. T. Voets, Neuron 2000 , 28 , 537–545. 134. R. A. Easom, Semin. Cell Dev. Biol. 2000 , 11 , 253–266. 135. J. Lang, Eur. J. Biochem. 1999 , 259 , 3–17. 136. J. Malsam, S. Kreye, T. H. Sollner, Cell. Mol. Life Sci. 2008 , 65 , 2814–2832. 137. J. B. Sorensen, Annu. Rev. Cell Dev. Biol. 2009 , 25 , 513–537. 138. S. M. Wojcik, N. Brose, Neuron 2007 , 55 , 11–24. 139. Z. P. Pang, T. C. Sudhof, Curr. Opin. Cell Biol. 2010 , 22 , 496–505. 140. D. Atlas, Annu. Rev. Biochem. 2013 , 82, 607–635. 141. G. A. Rutter, T. Tsuboi, M. A. Ravier, Cell Calcium 2006 , 40 , 539–551. 142. L. Olivos Ore, A. R. Artalejo, Trends Neurosci. 2004 , 27 , 113–115. 143. O. Wiser, M. Trus, A. Hernandez, E. Renstrom, S. Barg, P. Rorsman, D. Atlas, Proc. Natl. Acad. Sci. USA 1999 , 96 , 248–253. 144. S. A. Stricker, Dev. Biol. 1999 , 211 , 157–176. 145. M. Whitaker, Physiol. Rev. 2006 , 86 , 25–88. 146. C. M. Saunders, M. G. Larman, J. Parrington, L. J. Cox, J. Royse, L. M. Blayney, K. Swann, F. A. Lai, Development 2002 , 129 , 3533–3544. 147. T. P. Collins, R. Bayliss, G. C. Churchill, A. Galione, D. A. Terrar, Cell Calcium 2011 , 50 , 449–458. 148. F. Moccia, D. Lim, G. A. Nusco, E. Ercolano, L. Santella, FASEB J. 2003 , 17 , 1907–1909. 149. L. Santella, A. Puppo, J. T. Chun, Int. J. Dev. Biol. 2008 , 52 , 571–584. 150. T. Lorca, F. H. Cruzalegui, D. Fesquet, J. C. Cavadore, J. Mery, A. Means, M. Doree, Nature 1993 , 366 , 270–273. 134 Brini, Ottolini, Calì, and Carafoli

151. S. Markoulaki, S. Matson, A. L. Abbott, T. Ducibella, Dev. Biol. 2003 , 258 , 464–474. 152. T. Ducibella, R. M. Schultz, J. P. Ozil, Semin. Cell Dev. Biol. 2006 , 17 , 324–332. 153. J. Backs, P. Stein, T. Backs, F. E. Duncan, C. E. Grueter, J. McAnally, X. Qi, R. M. Schultz, E. N. Olson, Proc. Natl. Acad. Sci. USA 2010 , 107 , 81–86. 154. A. Ashkenazi, Nat. Rev. Cancer 2002 , 2 , 420–430. 155. R. J. Youle, A. Strasser, Nat. Rev. Mol. Cell Biol. 2008 , 9 , 47–59. 156. S. Cory, J. M. Adams, Nat. Rev. Cancer 2002 , 2 , 647–656. 157. C. W. Distelhorst, M. D. Bootman, Cell Calcium 2011 , 50 , 234–241. 158. G. Baffy, T. Miyashita, J. R. Williamson, J. C. Reed, J. Biol. Chem . 1993 , 268 , 6511–6519. 159. P. Pinton, D. Ferrari, P. Magalhaes, K. Schulze-Osthoff, F. Di Virgilio, T. Pozzan, R. Rizzuto, J. Cell Biol. 2000 , 148 , 857–862. 160. R. Foyouzi-Youssefi , S. Arnaudeau, C. Borner, W. L. Kelley, J. Tschopp, D. P. Lew, N. Demaurex, K. H. Krause, Proc. Natl. Acad. Sci. U S A 2000 , 97 , 5723–5728. 161. E. S. Dremina, V. S. Sharov, K. Kumar, A. Zaidi, E. K. Michaelis, C. Schoneich, Biochem. J. 2004 , 383 , 361–370. 162. R. Chen, I. Valencia, F. Zhong, K. S. McColl, H. L. Roderick, M. D. Bootman, M. J. Berridge, S. J. Conway, A. B. Holmes, G. A. Mignery, P. Velez, C. W. Distelhorst, J. Cell Biol. 2004 , 166 , 193–203. 163. C. White, C. Li, J. Yang, N. B. Petrenko, M. Madesh, C. B. Thompson, J. K. Foskett, Nat. Cell Biol. 2005 , 7 , 1021–1028. 164. N. Diaz-Prieto, I. Herrera-Peco, A. M. de Diego, A. Ruiz-Nuno, S. Gallego-Sandin, M. G. Lopez, A. G. Garcia, M. F. Cano-Abad, Cell Calcium 2008 , 44 , 339–352. 165. N. N. Danial, S. J. Korsmeyer, Cell 2004 , 116 , 205–219. 166. M. Chami, A. Prandini, M. Campanella, P. Pinton, G. Szabadkai, J. C. Reed, R. Rizzuto, J. Biol. Chem. 2004 , 279 , 54581–54589. 167. L. Scorrano, S. A. Oakes, J. T. Opferman, E. H. Cheng, M. D. Sorcinelli, T. Pozzan, S. J. Korsmeyer, Science 2003 , 300 , 135–139. 168. R. J. Youle, D. P. Narendra, Nat. Rev. Mol. Cell Biol. 2011 , 12 , 9–14. 169. G. Ashrafi , T. L. Schwarz, Cell Death Differ. 2013 , 20 , 31–42. 170. B. Ravikumar, S. Sarkar, J. E. Davies, M. Futter, M. Garcia-Arencibia, Z. W. Green- Thompson, M. Jimenez-Sanchez, V. I. Korolchuk, M. Lichtenberg, S. Luo, D. C. Massey, F. M. Menzies, K. Moreau, U. Narayanan, M. Renna, F. H. Siddiqi, B. R. Underwood, A. R. Winslow, D. C. Rubinsztein, Physiol. Rev. 2010 , 90 , 1383–1435. 171. J. P. Decuypere, G. Bultynck, J. B. Parys, Cell Calcium 2011 , 50 , 242–250. 172. J. M. Vicencio, S. Lavandero, G. Szabadkai, Cell Calcium 2010 , 47 , 112–121. 173. S. Sarkar, D. C. Rubinsztein, Autophagy 2006 , 2 , 132–134. 174. J. M. Vicencio, C. Ortiz, A. Criollo, A. W. Jones, O. Kepp, L. Galluzzi, N. Joza, I. Vitale, E. Morselli, M. Tailler, M. Castedo, M. C. Maiuri, J. Molgo, G. Szabadkai, S. Lavandero, G. Kroemer, Cell Death Differ. 2009 , 16 , 1006–1017. 175. A. Grotemeier, S. Alers, S. G. Pfi sterer, F. Paasch, M. Daubrawa, A. Dieterle, B. Viollet, S. Wesselborg, T. Proikas-Cezanne, B. Stork, Cell Signal. 2010 , 22 , 914–925. 176. M. Hoyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I. S. Mathiasen, M. Jaattela, Mol. Cell 2007 , 25 , 193–205. 177. N. R. Brady, A. Hamacher-Brady, H. Yuan, R. A. Gottlieb, FEBS J. 2007 , 274 , 3184–3197. 178. S. Swerdlow, C. W. Distelhorst, Dev. Cell 2007 , 12 , 178–179. 179. J. P. Decuypere, K. Welkenhuyzen, T. Luyten, R. Ponsaerts, M. Dewaele, J. Molgo, P. Agostinis, L. Missiaen, H. De Smedt, J. B. Parys, G. Bultynck, Autophagy 2011 , 7 , 1472–1489. 180. J. P. Decuypere, D. Kindt, T. Luyten, K. Welkenhuyzen, L. Missiaen, H. De Smedt, G. Bultynck, J. B. Parys, PLoS One 2013 , 8 , e61020. 181. A. Matilla-Duenas, I. Sanchez, M. Corral-Juan, A. Davalos, R. Alvarez, P. Latorre, Cerebellum 2010, 9 , 148–166. 182. K. Ishikawa, H. Fujigasaki, H. Saegusa, K. Ohwada, T. Fujita, H. Iwamoto, Y. Komatsuzaki, S. Toru, H. Toriyama, M. Watanabe, N. Ohkoshi, S. Shoji, I. Kanazawa, T. Tanabe, H. Mizusawa, Hum. Mol. Genet. 1999 , 8 , 1185–1193. 4 Calcium in Health and Disease 135

183. H. Saegusa, M. Wakamori, Y. Matsuda, J. Wang, Y. Mori, S. Zong, T. Tanabe, Mol. Cell. Neurosci. 2007 , 34 , 261–270. 184. K. Watase, C. F. Barrett, T. Miyazaki, T. Ishiguro, K. Ishikawa, Y. Hu, T. Unno, Y. Sun, S. Kasai, M. Watanabe, C. M. Gomez, H. Mizusawa, R. W. Tsien, H. Y. Zoghbi, Proc. Natl. Acad. Sci. USA 2008 , 105 , 11987–11992. 185. S. Naik, K. Pohl, M. Malik, A. Siddiqui, D. Josifova, Pediatr. Neurol. 2011 , 45 , 328–330. 186. E. Storey, R. J. Gardner, M. A. Knight, M. L. Kennerson, R. R. Tuck, S. M. Forrest, G. A. Nicholson, Neurology 2001 , 57 , 1913–1915. 187. E. Storey, R. J. Gardner, Handb. Clin. Neurol. 2012 , 103 , 561–565. 188. Y. Ikeda, K. A. Dick, M. R. Weatherspoon, D. Gincel, K. R. Armbrust, J. C. Dalton, G. Stevanin, A. Durr, C. Zuhlke, K. Burk, H. B. Clark, A. Brice, J. D. Rothstein, L. J. Schut, J. W. Day, L. P. Ranum, Nat. Genet. 2006 , 38 , 184–190. 189. H. T. Orr, J. Cell. Biol. 2012 , 197 , 167–177. 190. Y. Jin, H. Suzuki, S. Maegawa, H. Endo, S. Sugano, K. Hashimoto, K. Yasuda, K. Inoue, EMBO J. 2003 , 22 , 905–912. 191. I. Bezprozvanny, Neurochem. Res. 2011 , 36 , 1186–1197. 192. A. Kasumu, I. Bezprozvanny, Cerebellum 2012 , 11 , 630–639. 193. M. Matsumoto, T. Nakagawa, T. Inoue, E. Nagata, K. Tanaka, H. Takano, O. Minowa, J. Kuno, S. Sakakibara, M. Yamada, H. Yoneshima, A. Miyawaki, Y. Fukuuchi, T. Furuichi, H. Okano, K. Mikoshiba, T. Noda, Nature 1996 , 379 , 168–171. 194. F. Maltecca, D. De Stefani, L. Cassina, F. Consolato, M. Wasilewski, L. Scorrano, R. Rizzuto, G. Casari, Hum. Mol. Genet. 2012 , 21 , 3858–3870. 195. G. Zanni, T. Cali, V. M. Kalscheuer, D. Ottolini, S. Barresi, N. Lebrun, L. Montecchi-Palazzi, H. Hu, J. Chelly, E. Bertini, M. Brini, E. Carafoli, Proc. Natl. Acad. Sci. USA 2012 , 109 , 14514–14519. 196. M. Brini, T. Cali, D. Ottolini, E. Carafoli, FEBS J. 2013 , 280, 5385–5397. 197. E. Lloyd-Evans, F. M. Platt, Cell Calcium 2011 , 50 , 200–205. 198. B. de Vries, R. R. Frants, M. D. Ferrari, A. M. van den Maagdenberg, Hum. Genet. 2009 , 126 , 115–132. 199. M. B. Russell, A. Ducros, Lancet Neurol. 2011 , 10 , 457–470. 200. O. D. Uchitel, J. Physiol. 2012 , 590 , 1–2. 201. R. A. Ophoff, G. M. Terwindt, M. N. Vergouwe, R. van Eijk, P. J. Oefner, S. M. Hoffman, J. E. Lamerdin, H. W. Mohrenweiser, D. E. Bulman, M. Ferrari, J. Haan, D. Lindhout, G. J. van Ommen, M. H. Hofker, M. D. Ferrari, R. R. Frants, Cell 1996 , 87 , 543–552. 202. D. Pietrobon, Curr. Opin. Neurobiol. 2005 , 15 , 257–265. 203. D. Pietrobon, Biochim. Biophys. Acta 2012 . 204. T. Calì, D. Ottolini, M. Brini, Cell Calcium 2012 , 52 , 73–85. 205. T. Calì, D. Ottolini, M. Brini, Biofactors 2011 , 37 , 228–240. 206. D. Ottolini, T. Calì, M. Brini, Res. Rep. Biochem. 2013 , 3 , 55–70. 207. D. J. Surmeier, P. T. Schumacker, J. Biol. Chem. 2013 , 288 , 10736–10741. 208. U. Wojda, E. Salinska, J. Kuznicki, IUBMB Life 2008 , 60 , 575–590. 209. M. R. Cookson, Cold Spring Harb. Perspect. Med. 2012 , 2 , a009415. 210. J. N. Guzman, J. Sanchez–Padilla, D. Wokosin, J. Kondapalli, E. Ilijic, P. T. Schumacker, D. J. Surmeier, Nature 2010 , 468 , 696–700. 211. D. Ottolini, T. Calì, A. Negro, M. Brini, Hum. Mol. Genet. 2013 , 22 , 2152–2168. 212. T. Calì, D. Ottolini, A. Negro, M. Brini, J. Biol. Chem. 2012 , 287 , 17914–17929. 213. T. Calì, D. Ottolini, M. Brini, DNA Cell Biol. 2013 , 32 , 140–146. 214. T. Calì, D. Ottolini, A. Negro, M. Brini, Biochim. Biophys. Acta 2013 , 1832 , 495–508. 215. S. Gandhi, A. Wood-Kaczmar, Z. Yao, H. Plun-Favreau, E. Deas, K. Klupsch, J. Downward, D. S. Latchman, S. J. Tabrizi, N. W. Wood, M. R. Duchen, A. Y. Abramov, Mol. Cell 2009 , 33 , 627–638. 216. B. Heeman, C. Van den Haute, S. A. Aelvoet, F. Valsecchi, R. J. Rodenburg, V. Reumers, Z. Debyser, G. Callewaert, W. J. Koopman, P. H. Willems, V. Baekelandt, J. Cell Sci. 2011 , 124 , 1115–1125. 136 Brini, Ottolini, Calì, and Carafoli

217. R. Brookmeyer, E. Johnson, K. Ziegler-Graham, H. M. Arrighi, Alzheimers Dement. 2007 , 3 , 186–191. 218. K. Honarnejad, J. Herms, Int. J. Biochem. Cell Biol. 2012 , 44 , 1983–1986. 219. L. Fedrizzi, E. Carafoli, Biofactors 2011 , 37 , 189–196. 220. M. Giacomello, R. Hudec, R. Lopreiato, Biofactors 2011 , 37 , 206–218. 221. M. Giacomello, J. C. Oliveros, J. R. Naranjo, E. Carafoli, Prion 2013 , 7 , 76–84. 222. M. Suzuki, Y. Nagai, K. Wada, T. Koike, Biochem. Biophys. Res. Commun. 2012 , 429 , 18–23. 223. V. Costa, M. Giacomello, R. Hudec, R. Lopreiato, G. Ermak, D. Lim, W. Malorni, K. J. Davies, E. Carafoli, L. Scorrano, EMBO Mol. Med. 2010 , 2 , 490–503. 224. J. Wu, H. P. Shih, V. Vigont, L. Hrdlicka, L. Diggins, C. Singh, M. Mahoney, R. Chesworth, G. Shapiro, O. Zimina, X. Chen, Q. Wu, L. Glushankova, M. Ahlijanian, G. Koenig, G. N. Mozhayeva, E. Kaznacheyeva, I. Bezprozvanny, Chem. Biol. 2011 , 18 , 777–793. 225. L. M. Duffy, A. L. Chapman, P. J. Shaw, A. J. Grierson, Neuropathol. Appl. Neurobiol. 2011 , 37 , 336–352. 226. T. A. Gennarelli, D. I. Graham, Semin. Clin. Neuropsychiatry 1998 , 3 , 160–175. 227. S. Battistini, C. Ricci, E. M. Lotti, M. Benigni, S. Gagliardi, R. Zucco, M. Bondavalli, N. Marcello, M. Ceroni, C. Cereda, J. Neurol. Sci. 2010 , 293 , 112–115. 228. J. Grosskreutz, L. Van Den Bosch, B. U. Keller, Cell Calcium 2010 , 47 , 165–174. 229. G. Antalffy, A. S. Mauer, K. Paszty, L. Hegedus, R. Padanyi, A. Enyedi, E. E. Strehler, Cell Calcium 2012 , 51 , 171–178. 230. M. Giacomello, A. De Mario, S. Primerano, M. Brini, E. Carafoli, Int. J. Biochem. Cell Biol. 2012 , 44 , 679–683. 231. J. M. Schultz, Y. Yang, A. J. Caride, A. G. Filoteo, A. R. Penheiter, A. Lagziel, R. J. Morell, S. A. Mohiddin, L. Fananapazir, A. C. Madeo, J. T. Penniston, A. J. Grifﬁ th, N. Engl. J. Med. 2005 , 352 , 1557–1564. 232. R. Ficarella, F. Di Leva, M. Bortolozzi, S. Ortolano, F. Donaudy, M. Petrillo, S. Melchionda, A. Lelli, T. Domi, L. Fedrizzi, D. Lim, G. E. Shull, P. Gasparini, M. Brini, F. Mammano, E. Carafoli, Proc. Natl. Acad. Sci. USA 2007 , 104 , 1516–1521. 233. D. M. Bers, Annu. Rev. Physiol. 2008 , 70 , 23–49. 234. D. Fatkin, R. M. Graham, Physiol. Rev. 2002 , 82 , 945–980. 235. S. Morimoto, Cardiovasc. Res. 2008 , 77 , 659–666. 236. N. Tiso, D. A. Stephan, A. Nava, A. Bagattin, J. M. Devaney, F. Stanchi, G. Larderet, B. Brahmbhatt, K. Brown, B. Bauce, M. Muriago, C. Basso, G. Thiene, G. A. Danieli, A. Rampazzo, Hum. Mol. Genet. 2001 , 10 , 189–194. 237. J. Heineke, J. D. Molkentin, Nat. Rev. Mol. Cell Biol. 2006 , 7 , 589–600. 238. Z. Kubalova, D. Terentyev, S. Viatchenko-Karpinski, Y. Nishijima, I. Gyorke, R. Terentyeva, D. N. da Cunha, A. Sridhar, D. S. Feldman, R. L. Hamlin, C. A. Carnes, S. Gyorke, Proc. Natl. Acad. Sci. USA 2005 , 102 , 14104–14109. 239. J. Kockskamper, A. V. Zima, H. L. Roderick, B. Pieske, L. A. Blatter, M. D. Bootman, J. Mol. Cell Cardiol. 2008 , 45 , 128–147. 240. D. Harzheim, A. Talasila, M. Movassagh, R. S. Foo, N. Figg, M. D. Bootman, H. L. Roderick, Channels (Austin) 2010 , 4 , 67–71. 241. D. C. Kim, Korean J. Anesthesiol. 2012 , 63 , 391–401. 242. T. Krause, M. U. Gerbershagen, M. Fiege, R. Weisshorn, F. Wappler, Anaesthesia 2004 , 59 , 364–373. 243. H. Rosenberg, M. Davis, D. James, N. Pollock, K. Stowell, Orphanet J. Rare Dis. 2007, 2, 21. 244. D. H. Maclennan, E. Zvaritch, Biochim. Biophys. Acta 2011 , 1813 , 948–964. 245. N. Monnier, V. Procaccio, P. Stieglitz, J. Lunardi, Am. J. Hum. Genet. 1997 , 60 , 1316–1325. 246. R. A. Bannister, I. N. Pessah, K. G. Beam, J. Gen. Physiol. 2009 , 133 , 79–91. 247. A. M. Hurne, J. J. O’Brien, D. Wingrove, G. Cherednichenko, P. D. Allen, K. G. Beam, I. N. Pessah, J. Biol. Chem. 2005 , 280 , 36994–37004. 248. W. J. Durham, X. H. Wehrens, S. Sood, S. L. Hamilton, Subcell. Biochem. 2007 , 45 , 273–321. 4 Calcium in Health and Disease 137

249. S. Treves, A. A. Anderson, S. Ducreux, A. Divet, C. Bleunven, C. Grasso, S. Paesante, F. Zorzato, Neuromuscul. Disord. 2005 , 15 , 577–587. 250. A. D. Lyfenko, S. A. Goonasekera, R. T. Dirksen, Biochem. Biophys. Res. Commun. 2004 , 322 , 1256–1266. 251. S. Treves, H. Jungbluth, F. Muntoni, F. Zorzato, Curr. Opin. Pharmacol. 2008 , 8 , 319–326. 252. A. A. Benders, J. H. Veerkamp, A. Oosterhof, P. J. Jongen, R. J. Bindels, L. M. Smit, H. F. Busch, R. A. Wevers, J. Clin. Invest. 1994 , 94 , 741–748. 253. N. C. Voermans, A. E. Laan, A. Oosterhof, T. H. van Kuppevelt, G. Drost, M. Lammens, E. J. Kamsteeg, C. Scotton, F. Gualandi, V. Guglielmi, L. van den Heuvel, G. Vattemi, B. G. van Engelen, Neuromuscul. Disord. 2012 , 22 , 944–954. 254. A. Hovnanian, Subcell. Biochem. 2007 , 45 , 337–363. 255. P. L. McNeil, R. A. Steinhardt, Annu. Rev. Cell Dev. Biol. 2003 , 19 , 697–731. 256. D. G. Allen, N. P. Whitehead, E. W. Yeung, J. Physiol. 2005 , 567 , 723–735. 257. D. G. Allen, O. L. Gervasio, E. W. Yeung, N. P. Whitehead, Can. J. Physiol. Pharmacol. 2010 , 88 , 83–91. 258. F. W. Hopf, P. R. Turner, R. A. Steinhardt, Subcell. Biochem. 2007 , 45 , 429–464. 259. G. L. Warren, C. P. Ingalls, D. A. Lowe, R. B. Armstrong, Exerc. Sport Sci. Rev. 2001 , 29 , 82–87. 260. B. J. Petrof, J. B. Shrager, H. H. Stedman, A. M. Kelly, H. L. Sweeney, Proc. Natl. Acad. Sci. USA 1993 , 90 , 3710–3714. 261. A. Pertille, C. L. de Carvalho, C. Y. Matsumura, H. S. Neto, M. J. Marques, Int. J. Exp. Pathol. 2010 , 91 , 63–71. 262. J. G. Tidball, M. J. Spencer, Int. J. Biochem. Cell Biol. 2000 , 32 , 1–5. 263. R. J. Khairallah, G. Shi, F. Sbrana, B. L. Prosser, C. Borroto, M. J. Mazaitis, E. P. Hoffman, A. Mahurkar, F. Sachs, Y. Sun, Y. W. Chen, R. Raiteri, W. J. Lederer, S. G. Dorsey, C. W. Ward, Sci. Signal 2012 , 5 , ra56. 264. T. R. Shannon, Am. J. Physiol. Heart Circ. Physiol. 2009 , 297 , H1965–1966. 265. J. Finsterer, C. Stollberger, Cardiology 2003 , 99 , 1–19. 266. B. Fraysse, A. Liantonio, M. Cetrone, R. Burdi, S. Pierno, A. Frigeri, M. Pisoni, C. Camerino, A. De Luca, Neurobiol. Dis. 2004 , 17 , 144–154. 267. V. Robert, M. L. Massimino, V. Tosello, R. Marsault, M. Cantini, V. Sorrentino, T. Pozzan, J. Biol. Chem. 2001 , 276 , 4647–4651. Chapter 5 Vanadium. Its Role for Humans

Dieter Rehder

Contents aBSTRACT...... 139 1 Introduction...... 140 2 Distribution and Cycling of Vanadium...... 142 2.1 Vanadium in Nature...... 142 2.2 Pharmacokinetics and Pharmacodynamics...... 144 3 The Aqueous Chemistry of Vanadium and the Vanadate-P hosphate Antagonism...... 147 4 The Medicinal Potential of Vanadium...... 152 4.1 Diabetes Mellitus...... 152 4.2 Activity in Health Hazards Other than Diabetes...... 156 4.2.1 Treatment of Cancer...... 156 4.2.2 Cardiovascular Effects; Bacterial and Viral Diseases...... 159 4.2.3 Diseases Caused by Parasites...... 162 5 Concluding Remarks and Prospects...... 164 Abbreviations...... 166 References...... 167

Abstract Vanadium is the 21st most abundant element in the Earth’s crust and the 2nd-to-most abundant transition metal in sea water. The element is ubiquitous also in freshwater and nutrients. The average body load of a human individual amounts to 1 mg. The omnipresence of vanadium hampers checks directed towards its essentiality. However, since vanadate can be considered a close blueprint of phosphate with respect to its built-up, vanadate likely takes over a regulatory function in metabolic processes depending on phosphate. At common concentrations, vanadium is non-toxic. The main source for potentially toxic effects caused by vanadium is exposure to high loads of vanadium oxides in the breathing air of vanadium processing industrial

D. Rehder (*) Chemistry Department, University of Hamburg, D-20146 Hamburg, Germany e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 139 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_5, © Springer Science+Business Media Dordrecht 2013 140 Rehder enterprises. Vanadium can enter the body via the lungs or, more commonly, the stomach. Most of the dietary vanadium is excreted. The amount of vanadium resorbed in the gastrointestinal tract is a function of its oxidation state (VV or VIV) and the coordination environment. Vanadium compounds that enter the blood stream are subjected to speciation. The predominant vanadium species in blood are vanadate and vanadyl bound to transferrin. From the blood stream, vanadium becomes distributed to the body tissues and bones. Bones act as storage pool for vanadate. The aqueous chemistry of vanadium(V) at concentration <10 μM is dominated by vanadate. At higher concentrations, oligovanadates come in, decavanadate in particular, which is thermodynamically stable in the pH range 2.3–6.3, and can further be stabilized at higher pH by interaction with proteins. The similarity between vanadate and phosphate accounts for the interplay between vanadate and phosphate-dependent enzymes: phosphatases can be inhibited, kinases activated. As far as medicinal applications of vanadium compounds are concerned, vanadium’s mode of action appears to be related to the phosphate- vanadate antagonism, to the direct interaction of vanadium compounds or fragments thereof with DNA, and to vanadium’s contribution to a balanced tissue level of reactive oxygen species. So far vanadium compounds have not yet found approval for medicinal applications. The antidiabetic (insulin-enhancing) effect, however, of a singular vanadium complex, bis(ethylmaltolato)oxidovanadium(IV) (BEOV), has revealed encouraging results in phase IIa clinical tests. In addition, in vitro studies with cell cultures and parasites, as well as in vivo studies with animals, have revealed a broad potential spectrum for the application of vanadium coordination compounds in the treatment of cardiac and neuronal disorders, malignant tumors, viral and bacterial infections (such as influenza, HIV, and tuberculosis), and tropical diseases caused by parasites, e.g., Chagas’ disease, leishmaniasis, and amoebiasis.

Keywords antiparasitic vanadium compounds • antiviral potential • cardiovascular effects • essentiality of vanadium • insulin-enhancing action • vanadate-phosphate antagonism

Please cite as: Met. Ions Life Sci. 13 (2013) 139–169

1 Introduction

Vanadium is a versatile and omnipresent element that can attain the oxidation states –III to +V. Low-valent vanadium is stabilized by strongly π-accepting ligands, carbon monoxide in particular, high valent vanadium by σ and π donors represented by hard, oxygen and nitrogen functional ligands. Soft ligands such as thio-functional ones are predominantly found in vanadium compounds with vanadium in intermediate oxidation states. Vanadium nitrogenase is an example for a naturally occurring vanadium compound where vanadium switches in-between the oxidation states +II 5 Vanadium. Its Role for Humans 141 and +IV: In vanadium nitrogenase from nitrogen fixing bacteria such asAzotobacter , vanadium – an integral constituent of the Fe7VS9 M-cluster – is coordinated to three sulfides, a histidine-N, and two oxygen functions of homocitrate. Vanadium(III) coordinated to water molecules is present in the vanadocytes of sea squirts. The +IV and +V oxidation states, which are the by far predominating ones in physiologically IV 2+ V 3+ V + relevant vanadium systems, typically contain the V O , V O or V O2 ‘core’, although there are exceptions. An example for a ‘bare’ vanadium(IV) complex is the naturally occurring amavadin, where V4+ is coordinated to two tetradentate N-oxyimino-2,2′-dipropionate ligands. Amavadin is found in mushrooms belonging to the genus Amanita, such as the fly agaric. Theoxido vanadium(V) core is present in vanadate-dependent haloperoxidases from, inter alia, marine algae, with vanadate − H2VO4 coordinatively linked to an active center histidine-N. To date, vanadate-dependent haloperoxidases and vanadium nitrogenases have remained the only identified naturally occurring vanadium-based enzymes. Whether or not vanadium is an essential element for evolutionary younger organisms, including vertebrates, remains to be verified. A functional role of simple vanadium compounds (vanadate in particular) in vertebrates, and hence also in humans, is likely, an assumption which is based on the similarity between vanadate and phosphate. In this context, the vanadate-dependent haloperoxidases are of particular interest since they mimic, or model, enzymes involved in phosphate metabolism, where the protein binding domain for phosphate is blocked by vanadate. The competitive behavior of vanadate with respect to phosphate is likely also the clue for the insulin-mimetic/insulin-enhancing potential of vanadium compounds, and hence the surge in the design of antidiabetic vanadium complexes during the past two decades. These auspicious developments have also initiated research towards the design of biologically active vanadium complexes in the search of pharmacological control of cancer, cardiovascular imbalances, and diseases caused by viruses, bacteria, amoebae, and flagellate protozoan parasites. In several cases, ligands have been employed that relate to original pharmacologically applied drugs, with the aim to increase the efficacy of the drug, and to widen the spectrum of therapeutic use by exploiting the cooperative effect of the metal and the ligand. The research into these medicinal applications includes functional alternatives to the phosphate-vanadate antagonism, e.g., the direct interaction of the vanadium compound with the DNA of a tumor cell or the pathogen. The broad medicinal potential of vanadium will be extemporized in Section 4 of this chapter. Section 2.1 is dedicated to vanadium’s distribution and speciation in nature, and hence, its availability, potential inalienability, and occasional toxicity for humans. Section 2.2 addresses resorption, speciation in the blood stream and in the cytosol, tissue distribution, and excretion of vanadium within the human body, and hence, vanadium’s gateways commonly referred to as pharmacokinetics and pharmacodynamics. Given the importance of the similarities (and, to some extent, also the dissimilarities) between vanadate and phosphate for the potentiality of vanadium in toxic as well as in beneficial issues (such as regulatory function and medicinal applications), an extra section, Section 3, is devoted to the vanadate- phosphate antagonism. 142 Rehder

2 Distribution and Cycling of Vanadium

2.1 Vanadium in Nature

The Earth’s crust, including the aqua- and atmosphere, contains ≈130 ppm (by mass) vanadium, making vanadium the 21st most abundant element in the outer- most sphere of our home planet. The abundance of vanadium in the Earth’s crust thus exceeds its occurrence in the Universe (≈1 ppm) and in the Sun (≈ 0.4 ppm) by about two orders of magnitude [1]. Volcanic areas with basaltic layers are particularly rich in vanadium, as are hard coal (up to 0.34%) and some oil shales and crude oil. Venezuelan crude oil can contain up to 0.12% V, mostly in the form of oxidovanadium(IV) porphyrins (Figure 1a). VO2+ ions are strongly complexed by porphyrins and related chelators, and the enrichment of crude oil with vanadium is due to its extraction from vanadium-bearing rock by porphyrins present in oil that had passed through rocky layers. Natural release of vanadium mainly goes back to weathering of vanadium-containing rock and the erosion of soil. Vanadium concentrations in seawater and freshwater are around 30 nM. At the prevailing oxic conditions and about neutral to slightly alkaline pH, soluble − + vanadate(V), H2VO4 , is the dominant species. The high Na concentration in seawa- + − ter implies that ion pairs Na [H2VO4] are formed. Under non-oxic conditions, sparingly soluble oxidovanadium(IV) hydroxide ‘VO(OH)2’ is generated, transported in water in the form of colloids and absorbed to floating particulate sediment, or solubilized by complexing ligands. Vanadium is the second-to-most abundant transition metal in seawater, out- 2 − classed only by molybdenum (MoO4 , c = 100 nM). The nanomolar concentration excludes the formation of vanadates of higher nuclearity otherwise typical for vanadates (see below). Vanadate concentrations in potable water are around 10 nM. In volcanic areas with basalt layers, the concentration of vanadium in underground water, and consequently also in drinking water, can rise to 2.5 μM [2]. Where drinking

a b C2H5 CH 3 CH3 H3C 2- C2H5 H3C N O N O N O O V O O V N N O O H C CH 3 3 O O O N (H2C)2 CH3 CO2H H3C

Figure 1 (a) The porphinogenic vanadium compound oxidovanadium(IV)-deoxiphylloerythrin from crude oil. (b) Amavadin, present in the fly agaric Amanita( muscaria), is a non-oxido vanadium compound containing the ligand (S,S)-N-oxyimino-2,2′-diisopropionate(3–). 5 Vanadium. Its Role for Humans 143 water is supplied through lead water pipe systems, vanadate is removed through the formation of deposits of sparingly soluble vanadinite, PbCl2⋅3Pb3[VO4]2. A decrease of pH and an increase of the phosphate concentration (e.g., by addition of phosphate- based corrosion inhibitors to drinking water) can re-mobilize vanadate (eqns 1 and 2) and thus eventually increase vanadate concentrations beyond a tolerable level [3].

++2 −− PbCl23⋅31Pb VO4 ++21HH 06Pb 24VO + 2Cl (1) []2 2 PbCl ⋅33Pb VO ++HPOP−+HH→⋅bClP32bPOV↓+ O− (2) 23[]4 2 4 23[]4 2 24 Vanadinite is a common mineral containing vanadium in the oxidation state +V. Vanadium’s first discovery by the Spanish mineralogist Manuel del Rio yF ernández in1801 goes back to this mineral. Vanadium-based minerals are otherwise comparatively rare, i.e., most of the vanadium in the Earth’s crust is dissipated in other minerals, rocks, and sediments. Examples for defined minerals with vanadium in oxidation states other than +V are minasragrite, VOSO4⋅5H2O, and patronite, V(S2)2, with vanadium(IV), and karelianite, V2O3, with vanadium(III). Note that, under oxic conditions, only the oxidation states +V and, to some extent, also +IV are stable. Vanadium(II) has been found in forsterite (Mg2SiO4) and enstatite 2+ (MgSiO3) in chondrules of meteorites such as the Vigarano meteorite [4]. Here, V can partially occupy Mg2+ and Ca2+ sites. High contents of vanadium are found in various sea squirts (ascidians), in fan worms, and in Amanita mushrooms such as the fly agaric [5]. In ascidians, vanadium concentrations in specialized blood cells, where the predominant form of III 2+ vanadium is [V (H2O)5HSO4] , can go up to 0.3 M. The concentration of vanadium in the fly agaric exceeds that in other plants by a factor of 102. In the fly agaric, also known as toad stool, vanadium is present as amavadin, a low molecular mass non- oxido VIV complex (Figure 1b). Vanadium contents in food average 30 μg kg–1, the daily intake via food and beverages is 10 μg to 2 mg, only a minute proportion of which becomes resorbed. The body pool of an average human being (70 kg body mass) amounts to ca. 1 mg V, the average blood plasma concentration to 45 nM. Oral intake of vanadium is somewhat increased for sportsmen and bodybuilders resorting to preparations containing VOSO4. This ‘vanadyl fuel’ allegedly helps increasing the muscle mass. Since almost all of the vanadium is excreted in the form of insoluble VO(OH)2 prior to resorption, potential harm due to vanadium overload is not likely to occur. Still another – and more critical – source of vanadium intake is breathable air in urban and industrialized areas. Vanadium, in the form of vanadium(IV) and vanadium(V) oxides, VOx, is present in air in particulate form or absorbed to tiny dust particles and aerosols, and thus enters the lungs and the pulmonary system, from where it becomes distributed in the body after solubilization. The main natural sources for vanadium loads in the atmosphere are continental dust, marine aerosols, and volcanic emissions [6]. In rural areas, the concentration of vanadium oxides is in the range of 50 ng m–3; pollution can go up to >103 ng m–3 in urban settings, and in industrial areas in particular [7], where combustion of petroleum and oil are the main 144 Rehder

contributors to aerial VOx. Potential toxic effects of vanadium overloads [8], in particular irritations of the respiratory tract in workers exposed to high loads of vanadium oxides at their working place, will briefly be addressed in the next section.

2.2 Pharmacokinetics and Pharmacodynamics

As noted, critical exposure to vanadium compounds, vanadium oxides in particular, is confined to inhalation in the frame of occupational exposure, including mining and milling of vanadium ores, metallurgical processing involving ferrovanadin, production of catalysts, batteries and glass melt additives based on vanadium oxides, and cleaning of oil-fired boilers. Inhaled vanadium oxides cause rhinitis, irritations of the respiratory tract and – commonly transient – pulmonary malfunctions such as bronchitis, pneumonia, and asthma. Whether or not vanadium oxides can promote lung cancer has yet to be shown. In any case, the maximum allowable concentration of V2O5 at the working place, the MAC value, has been set by the World Health Organisation to 0.05 mg m–3 (40-h week, 8-h time-weighted average). Vanadium oxides are readily absorbed in the lungs and enter the blood stream − after solubilization in the form of vanadate, H2VO4 . Skin does not appear to allow for an appreciable import of vanadium. The main ‘natural’ source for the body’s vanadium supply thus is dietary uptake, a comparatively ineffective process because up to 99% of the dietary vanadium is usually excreted with the feces. The main routes of vanadium uptake and distribution in the body are sketched in Figure 2. − Dietary forms of vanadium are either vanadate, H2VO4 , present mainly in drinking water, and oxidovanadium(IV) compounds {VOL}, where {VOL} represents any ligand-stabilized VO2+. Free VO2+ is, as noted, essentially unavailable, since it forms sparingly soluble oxidovanadium hydroxide, allowing for nanomolar concentration 2+ – − of ‘free’ VO (actually [VO(OH)3] ) at the best. H2VO4 is more easily taken up in the gastrointestinal tract. However, vanadate(V) is partially reduced in the stomach and precipitated in the form of VO(OH)2 in the slightly alkaline medium of the intestines. Vanadium can also enter the blood stream by injection or infusion, either intentionally when injected intravenously (or intraperitoneally), or accidentally when present as a ‘contaminant’ in infusion solutions [9]. Vanadium compounds ending up in the blood stream either after resorption in the gastrointestinal tract, or via the lungs, or by infusion/injection, are subjected to redox interconversion between VV and VIV, depending on the oxygen tension and the presence of redox-active agents. The main transporter for both anionic vanadate(V), cationic VO2+, and neutral or charged {LVIVO} is transferrin [10]. Transferrin (Tf) forms binary complexes {VO2+-Tf} and ternary complexes {VOL-Tf} and {VOL′-Tf}, where L is a ligand originally coordinated to VO2+, and L′ a low molecular mass (lmm) ligand provided by blood serum, such as lactate [11], the serum lmm compound with the highest concentration, i.e., 1.5 mM. The VO2+ ion binds into the same protein pocket as Fe3+, and hence to two tyrosinates, an aspartate, and the Nε of a histidine, plus a synergistic carbonate (Figure 3a). With increasing blood 5 Vanadium. Its Role for Humans 145

Respiratory tract Lungs V2O5, VO2, V2O3 Bone

Dietary vanadium 2+ Blood plasma Heart, Kidney, VO H2VO4 H2VO4 Liver, Spleen L eL VO2+-Tf {LVO} {L/L'VO}-Tf Brain, Muscle, Adipose tissue Feces Infusion; Injection VO(OH)2 H2VO4 , {LVO} Urinary excretion H2VO4 Urinary excretion H2VO4

Figure 2 uptake, distribution and excretion of vanadium compounds. Uptake routes are indicated by broad arrows, excretion routes by broken arrows, and distribution routes by standard arrows and equilibrium arrows, respectively. Main vanadium compounds are indicated. Abbreviations: Tf = transferrin, L is any ligand provided by the nutritional matrix or in a medicinally applied vanadium compound, L′ is a low molecular mass ligand present in blood serum, and {L/L′VO} is the abbreviation for a VO2+ complex with L and/or L′.

a Arg NH + NH2 H2N Carbonate O H O O C b N Asp O O O O N His V O O V N O Tyr O O HN O His HO O Tyr

Figure 3 Likely binding modes of VO2+ in (a) the ternary VO2+-transferrin complex [12b], and (b) in the ternary complexes LVO2+-albumin or LVO2+-immunoglobulin (L = ethylmaltol), coordinating through a histidine [12a]. In (a), the Tyr trans to the oxido ligands binds just weakly. serum concentrations of vanadium, high-molecular mass transporters other than Tf come in, namely serum albumin (Ab) and immunoglobulin G (Ig) [12]. These proteins preferentially form ternary complexes, {VOL-Ab} and {VOL-Ig}. As shown in Figure 3b, the protein binds to the VOL moiety via a histidine residue. Plasma vanadium contents decline in three phases: The first phase is a rapid decline with a half-life t1/2 of 1 hour, followed by a second intermediate decline 146 Rehder

(t1/2 ≈ 26 hours) and a third slow decline with t1/2 ≈ 10 days. Vanadium contents in blood are thus reduced to about 30% within the first 24 hours 9[ ]. Clearing occurs directly via urinary excretion, and after distribution over tissues of the inner compartment (heart, liver, kidney, spleen), the outer compartment (brain, muscle, adipose tissue), and bones. About 50% of the vanadium is recovered in urine after 12 days. The residence time of vanadium in bones, where it replaces phosphorus in hydroxyapatite, Ca5(PO4)3OH, is ca. 1 month [13], corresponding to a half-life of 4–5 days. There are several alternative routes by which vanadium compounds can be transported from the blood plasma into blood and tissue cells. Vanadate is essentially − present, at pH ≈ 7, in the form of dihydrogenvanadate, H2VO4 (the pKa is 8.2), and 2 − may use phosphate and sulfate channels: Vanadate and phosphate HPO4 / − H2PO4 (pKa = 7.2) are structurally very similar (see also the next section). Vanadate and oxidovanadium(IV) bound to transferrin can enter the intracellular space by endocytosis – analogously to Fe3+, the main target ion for transferrin. An additional conceivable path – for a stable vanadium coordination compound with a sufficiently lipophilic coordination sphere – is diffusion across the cell membrane. The feasibil- ity of this latter route of entry has been demonstrated for the uptake of the complex

[VO(pyridinone)2H2O] by erythrocytes [14]. The low absorption rate of dietary vanadium and the rather efficient desorption of excess vanadium that has entered the blood and body tissues diminish toxic effects that contemporarily can emerge, such as irritations of the conjunctivae and the respiratory system on exposure to vanadium oxides in the breathing air (see above), or (mild) gastrointestinal and renal problems in the course of medicinal applications of vanadium compounds. The no-effect level has been set to a daily intake of 10 mg V per kg body mass. The respective limit values for intravenous application is 7 mg kg–1, for breathing air 35 mg m–3. Acute poisoning in animals fed an about tenfold excess of vanadium compounds causes paralysis, convulsion, and eventually death [5,15]. Vanadium compounds are considered potentially genotoxic and thus mutagenic, teratogenic, and ‘suspected carcinogenic’. Classification as a carcinogen is based on the fact that vanadium induces the formation of tumor-associated antigens, and that it can directly and indirectly damage DNA and affect DNA repair [1b,16]. ‘Indirectly’ here refers to the potentiality of VO2+ to effect the formation of reactive oxygen species (ROS) such as the OH radical in a Fenton-like reaction (eqn. 3a), and superoxide when directly interacting with O2 (eqn. 3b). VO2++HO HHVO3+ OO• H (3a) ++22 →+2 + VO2+−OH34OHVO • OH−+ (3b) ++22→+24 2 + Superoxide in turn can cause the release of iron from the iron storage protein ferritin [17] and thus contributes to the disruption of iron homeostasis. In rat models, vanadium provokes neuro-toxicological effects in the brain, such as demyelination, i.e., damage of the myelin sheet of neurons. Myelin is a lipid-rich membrane of the nerves, and vanadium apparently promotes its peroxidative destruction [8]. 5 Vanadium. Its Role for Humans 147

While carcinogenic properties of vanadium compounds via ROS formation and interference with phosphokinases [18] are well in the realm of possibility, it should also be noted that vanadium compounds damage DNA in tumor cells more effectively than in healthy cells. As elaborated in Section 4.2.1, many vanadium compounds have an antitumor activity. Vanadium species can also annihilate reactive oxygen species. This is demonstrated by the sequence in equation (4) for the oxidation of peroxide to superoxide and further to O2 [19].

2+ + VOIV  +→OV2−− VOO2  + e–   2  ()2  + 2+ VOVVOV2−  →  OO• −  + e−  ()2   ()2  2+ 2+ VOV•OV−  →  IV OO + (4)  ()2    2 In reference to Paracelsus, who noted that “All substances are poisons; it is solely the dose that differentiates between a poison and a remedy” (“Alle Ding’ sind Gift, und nichts ohn’ Gift; allein die Dosis macht, daß ein Ding kein Gift ist”), there is so far no solid basis for categorizing vanadium compounds as harmful when administered in sensible amounts. Rather, as discussed in more detail in the oncoming sections, vanadium is likely an essential element in as far as vanadate can interfere with phosphatases, phosphorylases, and kinases and, more generally, is involved in regulating the phosphate metabolism and phosphate-dependent energetic processes. In addition, the participation of VIV and VV in levelling ROS suggests that vanadium can be beneficial in the treatment of several diseases and malfunctions related to ROS imbalances. Generally, vanadium(V), in particular when present as vanadate, is more toxic than vanadium(IV). As noted, VO2+ either forms a sparingly soluble hydroxide or is ‘masked’, through coordination, by a variety of physiologically available ligand systems. Biological detoxification of vanadate occurs via integration into the hydroxyapatite structure of the bones (vide supra) and by reduction to vanadium(IV) [20]. Glutathione, ascorbate, NADH, and NADPH are examples for agents that can reduce − 2+ vanadate. In ascidians, reduction equivalents for the reduction of H2VO4 to VO are supposedly delivered by NADPH (generated in the pentose phosphate pathway) via the redox couple 2GSH ⇌ GSSG + 2H+ + 2e–, where GSH and GSSG are the reduced and oxidized forms of glutathione, respectively [21]. Vanadium(III) plays, if any, a minor role only, since vanadium(IV) is not easily reduced to vanadium(III) at physiological conditions – and if so, rapidly re-oxidized to vanadium(IV).

3 The Aqueous Chemistry of Vanadium and the Vanadate-Phosphate Antagonism

At strongly acidic conditions (pH <2), cationic octahedral aqua complexes of VIII, IV 2+ V + 3+ 2+ V O , and V O2 can be present in aqueous media, i.e., [V(H2O)6] , [VO(H2O)5] , + 3+ and [VO2(H2O)4] . While [V(H2O)6] has Oh symmetry, the structures of the aqua 148 Rehder

III 3+ IV 2+ V + [V (H2O)6] [V O(H2O)5] [V O2(H2O)4]

OH 2 O O H2O OH2 V H2O OH2 V H O OH V H2O O 2 2 H O OH 2 2 H2O OH OH2 2 OH2 OH2

cis trans d(V-OH2): 1.99 Å d(V=O): 1.6; d(V-OH2 ): 2.0; d(V-OH2 ): 2.2 Å

Figure 4 Cationic aqua complexes of vanadium(III, IV, and V) that can exist in strongly acidic aqueous media. For structure data see ref. [22].

2+ + complexes of VO and VO2 are somewhat distorted due to the trans influence exerted by the doubly bonded oxido ligand(s), giving rise to comparatively weakly bonded water trans to the oxo group(s). In addition, there are distortions in the octahedral arrangement as shown in Figure 4, reducing the local symmetry to C4v in 2+ + [VO(H2O)5] and C2v in [VO2(H2O)4] [22] (for the respective distances d(V-O) see Figure 4). Strongly acidic conditions are provided in the stomach, but otherwise physiologically irrelevant – with the exception of the vanadium sequestering blood 3+ 2+ cells of ascidians, where V mainly exists in the form of [V(H2O)5HSO4] . In the presence of a ligand L, partial or complete replacement of H2O provides stability of the resulting complex(es) also in the less acidic, neutral, and slightly alkaline regimes. If L is a bidentate, singly negatively charged ligand, such as lactate, III 2+ the composition of the resulting mono-ligand complexes is [V (H2O)4L] , IV + V [V O(H2O)3L] , and [V O2(H2O)2L]. Depending on the pH, mixed aqua-hydroxido III + IV complexes can form, such as [V (OH)(H2O)3L] , [V O(OH)(H2O)2L], and V – [V O2(OH)(H2O)L] . Further, monooxidovanadium(V) complexes come in, for V 2+ V + instance [V O(H2O)3L] and [V O(OH)(H2O)2L] . As the denticity and charge of the ligands increases – an example is citrate [23] – the diversity of potential vanadium species present around pH 7 rises substantially. Vanadium(IV) and vanadium(V) are easily interconverted by physiologically rel- + + 2+ evant redox agents such as NAD /NADH, NADP /NADPH, FAD /FADH2, glutathione, and ascorbate. Further, VIV and VV redox-interact with reactive oxygen − 2+ species. The redox potential for the couple H2VO4 /VO (eqn. 5) at pH 7 is −0.34 V, which compares to −0.32 V for the couple NAD+/NADH.

– HHVO ++43++eH– VO2 + O (5) []24  2 The most prominent inorganic vanadium species present at micromolar concentra- − tions and pH 7 is dihydrogenvanadate, H2VO4 : At the physiological ionic strength of I ≈ 0.15 M, the pKa for the protonation/deprotonation equilibrium (eqn. 6), is 8.2 [24].

––2 HHVO HVO + + (6) []24 []4 5 Vanadium. Its Role for Humans 149

O O O 2 O 3 Va O HO Va O V O H O O HO H O H H O O O Vb OH Vb Vc O O O 2- O Vb Vc O Vb H2VO4 H2V2O7 O O O O O O O Va O Va OH 5 4 H O O 3- H3V10O28

4- 5- V4O12 V5O15

Figure 5 Vanadate(V) species that can be present in aqueous media, depending on concentration, pH, and stabilization by electrostatic interaction with, e.g., proteins. The protonation grade of decavanadate shown here corresponds to that at pH ≈ 2.7.

With increasing concentration, condensed vanadate species form. In the slightly acidic to alkaline regime (ca. pH 5–9), these are predominantly divanadate 2– 4– 5– [H2V2O7] , cyclic tetravanadate [V4O12] , and cyclic pentavanadate [V5O15] . In (6–n)– the pH range 2.3–6.3, decavanadate [HnV10O28] (n = 3–0) comes in. For the structural formulae of these oligovanadates see Figure 5. Commonly, physiological vanadate concentrations at subtoxic levels are too low to allow for the formation of oligovanadates. Locally, however, concentration enhancement or template-directed nucleation [25] may occur, and the oligovanadate(s) then formed can interact with proteins and DNA. An example is the incorporation of tetravanadate into Cu,Zn superoxide dismutase [26a], and the promotion of the hydrolytic cleavage of the ester bond in a phosphodiester RNA model by tetravanadate [26b]. Decavanadate is thermodynamically unstable at pH values exceeding 6.3; its decay into vanadates of lower nuclearity (including monovanadate) is, however, kinetically hampered, allowing for half-lives at pH 7, and even somewhat above pH 7, of several hours. Additional stabilization takes place by close interaction with proteins such as tubulins [27] and myosin [28] as well as with mitochondria [29]. Tubulins are proteins that assemble to structural elements (termed microtubules) of cytoskeletons. Myosin is involved in muscle contraction. Myosin binds decavanadate with high affinity, forming a myosin-MgATP-decavanadate complex that blocks the contractile cycle. Decavanadate that targets mitochondrial proteins inhibits mitochondrial oxygen consumption. Stabilization of decavanadate can also be achieved by its incorporation into tiny water pools of intracellular compartments – as suggested by model experiments with reverse micelles with nanoscopically confined water in the micelles’ cavities [30]. Vanadate and phosphate are structural analogues, and vanadate is only slightly larger than phosphate: The geometrical diameters are 6.2 Å for vanadate and 5.8 Å for phosphate, the volumes of the circumscribing spheres amount to 125 Å3 for vanadate and 102 Å3 for phosphate. These values are based on the following 150 Rehder

Asp O Glu Arg O- HN Arg NH2 Gly, Ile O HN Arg O N H + NH + HN H N NH H 2 O + NH H 2 - H2N 2 O HN O O +H N NH N HO V O- 2 2 O O NH HO V O N H N O HO V His HN 2 O OH NH HO Ser S His Arg Ser Cys

Asp O O O HO O HO HO R O R H R H OH OH R O O O O O H O O P OH H H O O P O O P OH O P OH O OH H N OH N N HN N HN HN His HN

Figure 6 Top: The active sites of vanadate-substituted (and thus inhibited) rat acid phosphatase (left) [32a], the Cys215Ser mutant of protein tyrosine phosphatase 1B (center) [32b], and bovine phosphotyrosyl phosphatase (right) [32c]. Bottom: The mechanism of phosphate ester hydrolysis as catalyzed by a phosphatase. The transition state {} is ‘fixed’ as vanadate becomes coordinated into the active center. structure parameters: r(O2–) = 1.36 Å, d(V-O) = 1.72 Å, d(P-O) = 1.54 Å; V-O and P-O distances for tetrahedral coordination geometry, where r(VV) = 0.36 and r(PV) = 0.17 Å. From a geometrical point of view, the two anions are thus essentially indistinguishable, making vanadate an efficient competitor for phosphate in binding sites commonly targeted by phosphate. There are, however, also substantial differences: At pH ≈ 7 and physiological ionic strength, vanadate is almost exclusively − present in the form of dihydrogenvanadate, H2VO4 , while phosphate exists in 2 − approximately equal amounts of mono- and dihydrogenphosphate, HPO4 and − H2PO4 . The higher average charge enables phosphate to interact more efficiently than vanadate with dipoles (such as water) and anions (e.g., anionic amino acid residues in protein matrices). On the other hand, phosphorus can attain the coordination number 5 in transitional states only, while the d-block element vanadium easily extends its coordination number by forming stable penta- and hexa-coordinate complexes. Hence, once incorporated in lieu of phosphate into the active site of a phosphate-dependent enzyme, this enzyme is commonly deactivated with respect to its original function. Naturally occurring enzymes relying on vanadate in a penta-coordinate trigonal- bipyramidal environment are the haloperoxidases in fungi, lichen, marine algae [5,31a], and Streptomyces [31b]; an example for a vanadate-inhibited phosphate- dependent enzyme is the vanadate variant of rat acid phosphatase [32] (Figure 6, top left), with the same first coordination sphere environment for vanadium as in 5 Vanadium. Its Role for Humans 151

O Base O

3' O O O O O O O O O O P O Base V P O Base V V O V O OH HO O HO O O O 5' O O O O H2O

O O O HO Base O + P O V V O HO OH O O O O

Figure 7 Proposed mechanism for the pyrovanadolysis of the DNA primer [36]: Divanadate attacks the primer at the 3′ position, a process which affords mediation by Mn2+. The transiently formed 3′-divanadophospho-nucleotide is hydrolytically split into divanadate and the phosphonu- cleotide to which DNA polymerase falls back.

vanadate-dependent haloperoxidases. Interestingly, vanadate-inhibited phosphatases do have some haloperoxidase activity [33], while vanadate-dependent haloperoxidases can exhibit phosphatase activity [34]. In the lower part of Figure 6, the mechanistic sequence of the phosphoester hydrolysis as catalyzed by phosphatases is illustrated. Apart from vanadate, many complexes of VIII, VIV, and VV with organic ligands that are related to or mimic physiologically available ligands, effectively inhibit phosphatases [35], likely after off-dissociation of all or part of the ligands and thus formation of vanadate or {VOxL} intermediates with an ‘open’ coordination site capable of interacting with, and thus blocking, the active site of the enzyme. Vanadates can also activate biochemical processes. An example for the activation of an otherwise phosphate-dependent process is the linkage of divanadate 2 − (‘pyrovanadate’, H2V2O7 ) to the 3′-end of the primer nucleotide sequence of DNA [DNA primers serve as a starting point for DNA replication (and hence DNA synthesis) through DNA polymerase]. This activates degradation (‘pyrovanadolysis’) of the primer, making available nucleotides for DNA replication by DNA polymerase [36]. The proposed mechanism for pyrovanadolysis is shown in Figure 7. Activation of kinases will be addressed in the context of the antidiabetic action of vanadium compounds in Section 4.1. 3– In solutions containing vanadate and phosphate, phosphovanadates, [HVPO7] 2– and [H2VPO7] , are present at pH 7 (the pKa is 7.2 at physiological ionic strength) [37] (eqn. 7). This mixed anhydride is labile; the formation constant at pH 7 is about 20 M–1, as compared to the formation constant of 350 M–1 for divanadate. Phosphovanadate hence is less stable against hydrolysis than divanadate by an order 2– of magnitude, but more stable than diphosphate, [H2P2O7] , by six orders of magnitude. Phosphate can also form mixed species with VO2+ [38]: The dominant species 152 Rehder

O 2 O O 3 O HO V O O V O P OH HO V R O O O O O O O 2 3 HVO3(O2) HVPO6(O2) HVO3(OR)

Figure 8 examples for physiologically potentially relevant peroxidovanadates, peroxidovanado- phosphates, and vanadate esters (from left to right).

in the slightly acidic regime is ‘VO(HPO4)’, where hydrogenphosphate is a ligand for VO2+ rather than a counter-ion. Stabilization against hydrolysis in the neutral to slightly alkaline range is again achieved by nutritionally or physiologically available organic ligands.

––2 2– HHVO + HPOV+ + HHPO + O (7) []24 []4  []27 2 In view of the omnipresence of hydrogen peroxide which, at low concentration levels, is an important signalling and regulatory molecule in many biological processes, the potential formation of peroxidovanadates [39] and peroxidovanadophos- 2– 3– phates [40] such as HVO3(O2) and HVPO6(O2) is also to be considered, as is the generation of vanadate esters according to equation (8). The monoesters of vanadate and divanadate – analogues of the physiologically important phosphate and diphosphate esters – are, however, hydrolytically labile. Formation constants in the case of aliphatic residues R are in the order of 0.1 M–1, for aromatic residues (phenyl esters) around 1 M–1 [41]. Figure 8 contains peroxidovanadates and vanadate esters of potential importance.

– – HH24VO +→ROHH VO32OR  + O (8) []  ()

4 The Medicinal Potential of Vanadium

4.1 Diabetes Mellitus

Worldwide, about 10% of the population are suffering from diabetes mellitus – knowingly and unknowingly. Approximately 90% of all diabetic cases are ascribed to type 2 diabetes, the remaining 10% to type 1 diabetes [42a]. Type 1 diabetes (“juvenile diabetes”) goes along with absent or only residual insulin supply by the β cells in the Langerhans islets of the pancreas, commonly caused by degeneration of the β cells in the frame of autoimmune reactions, or by accidental dysfunction or loss of the pancreas. Type 2 diabetes (“adult onset diabetes”) is related, in its initial stage, to insufficient response of the cellular insulin receptors to insulin. In its 5 Vanadium. Its Role for Humans 153 advanced stage, a feed-back mechanism comes in, provoking β cell failure caused by de-differentiation of the β cells [43]. Type 2 diabetes typically concerns people beyond the age of 50; its onset can be kept in check by physical exercise and reasonable nutritional behavior. However, diabetes type 2 is nowadays increasingly diagnosed also with young people and even children; this juvenile onset type 2 diabetes appears to be correlated to obesity [42b]. Intact β cells produce proinsulin, a peptide hormone consisting of an A-chain with 21 amino acids (aa), a B-chain (30 aa), and a C-chain (31 aa) (for the role of the C peptide see [86]), connecting the A- and B-chains. The C-chain is detached in the final step of insulin synthesis; in genuine insulin, the A- and B-chains are linked through two cystines. Insulin is stored as a C3-symmetric hexamer, with the monomers linked through histidines via zinc ions [44]. The discharge of the active, monomeric form of insulin is initiated by elevated blood glucose levels. Insulin targets the cellular insulin receptor, triggering a complex mechanism by which glucose becomes internalized into the cytosol, followed by glucose metabolism. Further, insulin is involved in the inhibition of gluconeogenesis (the synthesis of glucose from smaller building blocks, for example amino acids), and in glycogen- esis (the synthesis of glycogen). Insulin is thus strongly involved in glucose homeostasis. In addition, insulin stimulates lipogenesis and inhibits lipolysis, and thus prevents ketoacidosis caused by the accumulation of ketonic bodies such as acetyl acetic acid in the blood. Ketonic bodies are causative for the severe disease patterns accompanied with progressive states of diabetes, such as retinopathy and dying off of limbs. Many inorganic (vanadate, vanadyl sulfate, peroxidovanadates) and organic vanadium compounds have been tested positive with respect to effectuating cellular glucose uptake and controlling free fatty acid levels. A selection of vanadium complexes of the general composition {VOL}, where L represents an organic ligand (system) in the coordination sphere, is provided in Figure 9; test conditions and results are summarized in Table 1 [45–52]. The compounds have been successfully tested in vitro (i.e., with cell cultures) and/or in vivo with diabetic rats or mice and, in the case of the maltolato complex 1b, with human individuals. Complex 1b (bis(ethylmaltolato)oxidovanadium(IV), BEOV), has passed clinical trials phase I and IIa with type 2 diabetic volunteers, essentially with encouraging response [13]. The ligand L in {VOL} largely influences the efficacy of a vanadium compound by steering resorption, transport, and stability of the complex, and thus the availability of the actual antidiabetic species, i.e., vanadate, at the locus operandi. Commonly, vanadium complexes are clearly more effective than inorganic vanadium compounds, underlining the advantageous bioavailability and pharmaceutical efficacy of organic vanadium compounds [13]. Where inorganic vanadium com- − pounds, vanadate (H2VO4 ) in particular, induce hypoglycemic effects, such as tea/ vanadate decoctions (last entry in Table 1) [52], this effect is likely due to the inter- mittent formation of a coordination compound with tea ingredients. Normal insulin supply and insulin ‘sensing’ provided, insulin docks to the car- boxyterminal segment of the extracellular α-subunits, IRα, of the trans-membrane insulin receptor (a tyrosine kinase), de-repressing the tyrosine kinase activity of the 154 Rehder

R1 O R3 O O O O V R2 O V O O O O O N O R2 3 1 R R X = H: 2a; O 1 2 3 X X = OH: 2b R = CH3; R , R = H: Maltol; 1a 1 2 3 R = C2H5; R , R = H: Ethyl maltol; 1b 1 2 3 R = C5H11; R = CH3; R = OCH3: Allixin; 1c O H3C O O CH3 O V O O OH O O 2 O O CH3 H3C OH2 CH3 V H2O O N N O V 5 O O H C N 3 O O O O 3 4 O H2O O V O O O O O 8 O S Poly- O OH2 N N V V CH3 glutamate O S O N OH2 (CH2)3 6 O 7 [Co] [Vitamin B12]

Figure 9 A selection of vanadium coordination compounds with antidiabetic in vitro and/or in vivo potential. Compounds 2 and 8 contain VV, the other complexes VIV. See Table 1 and text for details and references.

intracellular β subunit, IRβ. In such a way, phosphorylation of the tyrosine residues of IRβ is promoted and the cytosolic signal cascade initiated, ending up in the activation of the glucose transporter (GLUT4). Once activated, GLUT 4 becomes translocated to the cell membrane where it picks up glucose for delivery into, and metabolism within, the cytosol. The basic signal transduction is illustrated in Figure 10. Among the various interposed and branching steps [45], insulin receptor substrates (IRS), phosphatidylinositol 3-kinase (PI3K, also known as Akt), and protein kinase B (PKB), have been considered. IRS are cytosolic proteins with tyrosine residues, and PKBs are kinases which have available serine and/or tyrosine residues for phosphorylation. PKB directly targets the glucose transporter. In the case of missing insulin supply (type 1 diabetes), or insufficient receptivity of the insulin receptor for insulin (type 2 diabetes), IRβ is dephosphorylated through the action of a protein tyrosine phosphatase (PTPase, PTP-1B), annulling signalling for glucose intake by GLUT4 and thus provoking hyperglycemia. A possible mechanism of action of antidiabetic vanadium compounds in stimulating cellular glucose uptake in the case of hyperglycemia is included in Figure 10. Accordingly, any vanadium compound {VOL} will be broken down in the extra- and/or intracellular space to generate vanadate. As noted in 5 Vanadium. Its Role for Humans 155 [ 50 ] Ref. [ 13 ] [ 52 ] [ 46 ] [ 46 ] [ 51 ] [ 45 ] [ 47 ] [ 47 ] [ 48 ] [ 49 ] mice. y and hyperleptinemia glycogen synthase kinase, improvement of gene expression of gene expression glycogen synthase kinase, improvement levels Alleviation of hyperglycemia, hypercholesterolemia, hypercholesterolemia, of hyperglycemia, Alleviation Antidiabetic effect (for abbreviations cf. text and F igure 10 ) cf. text (for abbreviations Antidiabetic effect Reduction in blood glucose and glycosylated hemoglobin Lowering of blood glucose Lowering Lowering of blood glucose, phosphorylation Akt and Lowering of blood glucose Lowering and hyperlipidemia of hyperglycemia Alleviation of blood glucose Lowering Stimulation of glucose uptake and metabolism Stimulation of glucose uptake Inhibition of lipolysis Phosphorylation of protein kinase B Phosphorylation of IR β and IRS; glycogen accumulation tea decoction albumin, 6 albumin, 7 Complex no. Complex (see F igure 9 ) 1b 1c 2a 2a, 2b 8 vanadate/black 3 3 4 5 + serum per os Mode of application per os per os intraperitoneal per os injection tail vein per os in vitro in vitro in vitro in vitro c A selection of vanadium coordination compounds with antidiabetic potential. A selection of vanadium mice derive from cross-breading of female glucose-intolerant KK (Kyoji Kando)-mice with male obese A from cross-breading of female glucose-intolerant KK (Kyoji mice derive y mice y mice rats rats rats rats a a a a a transformed mice fibroblasts b STZ stands for streptozotocin, a naturally occurring glucosamine derivative that destroys the β cells in Langerhans islets. that destroys STZ stands for streptozotocin, a naturally occurring glucosamine derivative SV (for Simian virus) 3T3 fibroblasts are pseudo-adipocytes. KK-A STZ Table 1 Table Target a b c Human individuals STZ STZ SV STZ STZ Rat adipocytes Rat adipocytes Differentiated 3T3-L1 mouse adipocytes Differentiated KK-A 156 Rehder

{VOL} H2O

O H2VO4 IR Glucose α α extra

β β PKB intra PI3K O {VOL} OPO3H OPO3H H2O OPO3H IRS GLUT4 H2VO4

OPO3H PTP Pyruvate Glycogen

OVO3H

Figure 10 Signal cascade for the internalization of glucose by the glucose transporter GLUT4, as triggered by the phosphorylated insulin receptor (IR). In the absence of insulin (diabetes 1) or insufficient insulin response (diabetes 2), protein tyrosine phosphatase 1B (PTP) dephosphorylates the IR, and the glucose intake is annulled. Vanadate can block PTP and thus restore the signalling path. Several of the steps of the signal cascade are shown: IRS = insulin receptor substrate, PI3K = phosphatidylinositol 3-kinase (which activates protein kinase B, also known as Akt), PKB = protein kinase B.

Section 3, vanadate is an efficient phosphatase inhibitor. Consequently, inhibition of PTP-1B by vanadate [53] prevents dephosphorylation of IRβ and thus restores the signalling path.

4.2 Activity in Health Hazards Other than Diabetes

4.2.1 Treatment of Cancer

Several animal and in vitro studies have revealed the virtue of inorganic and, more pronounced, organic vanadium compounds in reducing or preventing neoplasia (the malignant proliferation of cells), and thus tumors, including cancer and its metastatic potential, in various target tissues (see [54] and [55] for comprehensive reviews on cancer prevention and treatment with vanadium compounds). Selected examples are collated in Figure 11, and specific test results are provided in Table 2 [56–59]. The vanadocene derivative 10 is a recent advancement of the more basic vana- 5 docene (η -C5H5)2VCl2 (= Cp2VCl2; Cp = cyclopentadienyl), introduced a quarter of a century ago by Köpf and Köpf-Maier, who demonstrated that the compound effectively degenerates and kills Ehrlich ascites tumor cells [60]. Ehrlich ascites are derived from breast tumors of female mice. The o-phenanthroline complex 11, dubbed “metvan”, stands for another group of closely related ‘classical’ vanadium coordination compounds that turned out to be particularly operant against various 5 Vanadium. Its Role for Humans 157

O C2H5 O O O O O OCH3 N O HO V V O Methoxybenzyl- H C N O O O Cl 3 pentadienyl H O NH O V cyclo Ethylcarboxy-4- NH Cl CH N pyrimidinone O N 3 O CH O C2H5 3 OCH 9a 9b 10 3 C2H5

H C C H 3 O 2 5 Ethylidenehydrazine- O O N carbothioamide N OSO3 V Salicylidene- V NH N tryptophan N S N O N CH N HN 3 H3C CH3 O N S o-Phenanthroline Thiazole 11 CH3 12

Figure 11 Vanadium(V) and vanadium(IV) coordination compounds with anticancer potential. Simplistic names of the ligands are provided. 9b is a hydrolysis product of 9a. For details of the mode of action see Table 2 and text.

Table 2 Selected vanadium coordination compounds with antitumor activity. Complex Target; no. (see mode of application Figure 11) Effect Remarks Ref. HeLaa tumor cells 9a Sufficiently more Species present [56] toxic (IC50 = 44 μM) at pH 7: 9a – towards tumor than (= [VO2L2] ), 9b – towards non-tumor (= [VO2(L)OH] ), cells vanadates Human kidney cancer 10 IC50 = 0.55 μM [57] cells (CAKI-1) CAKI-1 mice; 10 Loss of body weight, Maximum tolerable [57] intraperitoneal deadly toxic in some dose: 20 mg kg–1 cases; reduced tumor d–1 growth Leukemia and myeloma 11 Effective at nM and Effective [58] cells, cells derived low μM conc. Cell also against from breast and apoptosis associated cisplatin-resistant testicular cancer with the generation ovarian cancer of ROS and depletion of GSH Colon cancer cells 12 Decrease of cell viability; More efficient than [59] IC50 = 48 μM cisplatin a HeLa tumor cells derive from cervical cancer (the cervix is the lower part of the uterus, connecting to the vagina). 158 Rehder cancer cell lines, including cisplatin-resistant ovarian and testicular cancer [58]. The oxidovanadium(V) pyrimidinone complex 9 and the oxidovanadium(IV) complex 12 are examples for more recent developments in the search of vanadium- based anticancer compounds. The operating mode of anticancer vanadium compounds is still elusive. Modes of action that have been proposed include (i) Inhibition of protein tyrosine phosphatases and activation of protein kinases; (ii) Activation of tyrosine phosphorylases, with concomitant activation of signal transduction pathways, followed by apoptosis and/or activation of tumor suppressor genes; (iii) Cleavage of, or intercalation into, DNA, resulting in cell cycle arrest; (iv) enhanced formation of reactive oxygen species (ROS); (v) Down-regulation of ferritin expression and disruption of ferritin with concomitant, iron-induced mediation of ROS. As in the case of antidiabetic vanadium species, the ligand system mediates the resorption and pharmacokinetics of the anticancer compound. In many cases, the active species again is likely vanadate, formed by (partial) degradation of the original drug: Speciation studies of the anionic pyrimidinone (L) complex, – [VO2L2] (9a), have demonstrated that at pH 7 the complex 9a coexists with the – hydrolysis products, [VO2(OH)L] (9b), and mono-, di-, and tetravanadate [56]. Intervention of vanadate with phosphatases, phosphorylases, and kinases will alter and eventually disrupt or enforce signalling paths involved in the regulation of the proliferation of malignant cells. Inorganic vanadate generated under physiological conditions from, for example, [VO(maltol)2] (1a in Figure 9) has been shown to discriminate between hepatocytes and hepatoma cells in as far as vanadate significantly increases the generation of ROS (superoxide and hydrogen peroxide), and concomitantly causes cell cycle arrest in hepatoma but not in normal liver cells (hepatocytes) [61]. Similarly, the formation of ROS by Fe2+ – after vanadium-induced disintegration of ferritin – may be responsible for the vulnerability of astrocytoma cells, while astrocytes (cells of the brain and spinal chord) remain unaffected [62]. Vanadocenes, Cp′2VCl2 (Cp′ stands for substituted Cp), such as complex 10 in Figure 11 will become partially hydrolyzed under physiological conditions to form x+ [Cp′2V(H2O)x(OH)2–x] (x = 0, 1, 2), and hence in a fashion comparable to the 2 + hydrolysis of cisplatin, cis-[(NH3)2PtCl2]. The {Pt(NH3)2 } moiety of cisplatin can directly interact with DNA by coordinating to the N-bases of DNA. The harder 2+ 4+ 2+ (with respect to Pt ) V in the {Cp′2V } moiety supposedly prefers coordination to oxo functionalities of the phosphoester linkages (Figure 12a). Alternatively, Cp′2V(OH)2 can interact with the phosphates via hydrogen bonds. In any case, the resulting ‘kink’ in the DNA will counteract DNA’s replication. Compounds such as metvan (11 in Figure 11), having an aromatic system strongly coordinated to vanadium, may interact with DNA, and thus deactivate DNA and cell proliferation, via intercalation (π−π interaction) (Figure 12b). 5 Vanadium. Its Role for Humans 159

a NH2 b HN N O O N H2N Guanosine Adenine N O N O P HN O O N O O O O P R N O O O O X P O O N V O H N V X O 2 O N O N NH P O Cytosine O R O N O O N Thymidine H O

Figure 12 Possible interactions of stable (fragments of) vanadium compounds with DNA. 2+ (a) The Cp2V moiety of vanadocene (e.g., compound 10) binds to two adjacent phosphates. (b) The o-phenanthroline unit of compound 11 intercalates in-between two nucleobases. X can be OH or H2O. The π stacking is indicated by broken lines.

4.2.2 Cardiovascular Effects; Bacterial and Viral Diseases

Interference of vanadium with protein tyrosine phosphatase (PTPase), in particular the inhibition of PTPase discussed in the context of the insulin-enhancing properties of vanadium compounds in the preceding section, appears to be a major mode of action also in beneficial effects for the vascular system in general, and vanadium’s cardio-protective effects in particular. The inhibition of PTPase by coordination of vanadate to the active site cysteine, as well as oxidative inhibition (cysteine → cystine) by vanadium-induced production of peroxide, result in an up-regulation of protein kinase B (Akt). Akt plays an important role in the regulation of cardiac hypertrophy and angiogenesis (the formation of new blood vessels from preformed ones), and thus in the prevention of, and recovery after, myocardial infarction. The up-regulation of Akt in turn enhances the expression of epithelial nitrogen oxide synthase (NO synthase) and thus the release of the vasodilator NO [63]. The maltolato complex 1a in Figure 9 [64], the pyridinethiolato complex 13, and the picolinato-bis(peroxido)vanadium(V) complex 14 in Figure 13 [65] perform significant cardio-protection in test animals (Table 3) [63–71]. Several studies on vanadium’s potentiality in deactivating viral and bacterial infections have appeared during the last decade, and selected compounds and results are provided in Figure 13 and Table 3. The vanadium-substituted polyoxidotungstate IV V 11– [(V O)2(V O)(SbW9O33)2] exhibits antiviral activity against viruses causing influenza and Dengue fever, and is also active against HIV-1 (human immune deficiency virus) and SARS (severe acute respiratory syndrome)in vitro [67a]. The cluster contains a linear {O=VO4}3 core (with the vanadium centers in a tetragonal-pyramidal 160 Rehder

O O S O N V N O S O N V O O 13 O O O 14 R

N(C2H5)2 NHO HN N O N O V R V S NH NH N N N Cl Cl O N HN V R R 15 16 NH OHN H O OH O O V N O O V N H3C N S N (His496) NH 17 18 CH3

Figure 13 Vanadium complexes with cardio-protective effects (13 and 14) or operant against viral (15, 16, and 17) and bacterial infections (17 and 18). The cut-out 17 is the active center of vanadate-dependent haloperoxidase, e.g., from Corallina inaequalis (see text and Table 3).

environment), sandwiched by two {SbW9O33} halves in which the tungsten ions are octahedrally coordinated [67b]. At physiological conditions, this so-called ‘Keggin sandwich’ likely decomposes into the genuinely active species vanadate(V) and tungstate(VI). Anti-HIV activity has also been reported for the physiologically stable, water-soluble oxidovanadium(IV)-porphyrin 15 [68]. Water solubility of 15 is provided by the aminosulfonyl substituent at the porphyrin skeleton. The complex inhibits HIV-1 replication in virus infected Hut/CCR5 cells, possibly by targeting and deactivating the viral reverse transcriptase [Hut/CCR5 cells are derived from Hut78 cells, a human T cell line. Hut/CCR5 cells express the chemokine receptor CCR5, a protein on the surface of T lymphocytes (a group of white blood cells)]. The oxidovanadium(IV) xylyl-bicyclam complex 16 is a further example for an agent that is highly effective against strains of HIV-1 and HIV-2 [69], with IC50 values of 0.1–0.3 μM (IC50 values denote the concentration of a drug or prodrug at which the life function of 50% of the viable cells is inhibited). A possible mode of action is through binding of the complex to the chemokine receptor type 4 (CXCR-4), a receptor protein that HIV can use to infect T lymphocytes. Docking of compound 16 to CXCR-4 can occur via direct coordination of the metal center to aspartate and glutamate residues of the protein, or via weak interaction of the oxidovanadium moiety with tryptophan residues, and/or by hydrophobic interaction between tryptophan residues and the bicyclam rings. Antiviral and antibacterial activity has also been noted for vanadate-dependent haloperoxidase isolated from the marine alga Corallina inaequalis, and its alkalophilic mutant Pro395Asp/Leu241Val/Thr343Ala in particular [70]. The enzyme contains 5 Vanadium. Its Role for Humans 161

Table 3 Vanadium coordination compounds in the treatment of cardiovascular diseases, viral, and bacterial infections. Complex Target; no. (see mode of application Figure 13) Effect Mode of action Ref. Female rats 13 Amelioration Activation of Akt [63] of myocardial signalling → injuries expression of NO synthase Rats; intravenously 1a Limitation of Inhibition of tyrosine [64] reperfusion phosphatase injury Rats; orally 14 Neuro-protection Enhancement of [65] downstream Akt Rat cardiac myocites 14 Amelioration of Increase of levels of [66] cardio- NO synthase dysfunction Influenza virus, Dengue see text Antiviral activity (not reported) [67] fever virus, HIV-1, SARS; in vitro HIV-1 15 Inhibition of HIV-1 Inhibitory activity [68] replication in towards HIV-1 host cells reverse transcriptase HIV-1 and HIV-2 strains 16 Anti-HIV activity Binding to the [69] receptor CXCR-4b Herpes simplex, 17a Antiviral and Formation of HOBr [70] 1 Coxsackievirus B4, antimicrobial and O2 Staphylococcus aureus, activity Pseudomonas aeruginosa; – pH 8, H2O2 + Br Mycobacterium tuberculosis 18 Growth inhibition (not reported) [71] a The active center of the Pro395Asp/Leu241Val/Thr343Ala mutant of the vanadate-dependent chloroperoxidase from the alga Corallina inaequalis. b See text for details.

− vanadate (H2VO4 ) coordinated to the Nε of a histidine residue in the active center (17 in Figure 13). The native peroxidase catalyzes the oxidation of halides, for example, bromide to hypobromous acid, HOBr (eq. 9a), and – in a side-reaction – 1 generates singlet oxygen O2 (eq. 9b), commonly at slightly acidic conditions. 1 The mutant is active at pH ~ 8, allowing for the disinfection, through HOBr and O2, of medicinal equipment under particularly mild and thus moderate conditions. Strong microbial abatement was observed for both enveloped (Herpes simplex) and non- enveloped viruses (Coxsackievirus B4), as well as for Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa). Br – HO HH+ HOBr O (9a) ++22 →+2 HOBrBHO 1OHr– OH+ (9b) +→22 22++ + 162 Rehder

Antibacterial action has also been reported for complex 18 in Figure 13. The VV IV thiosemicarbazone (tsc) complex [VO2(tsc)] and its V precursor [VO(acac)tsc] (acac = acetylacetonate(1–)) inhibit the pathogen of tuberculosis, Mycobacterium tuberculosis [71]. Minimal inhibitory concentrations (MIC values) of these vanadium complexes are lower than for the free tsc ligand, supporting a role of the vanadium center in these antituberculosis drugs.

4.2.3 Diseases Caused by Parasites

Vanadium coordination compounds have also been shown to exhibit potential in the abatement of epidemic diseases caused by parasites (amoebae and flagellates) predominantly in tropical and subtropical countries. Examples are amoebiasis, Chagas’ disease (American trypanosomiasis), and leishmaniasis. As in the case of antiviral and antibacterial vanadium compounds, none of these compounds has so far achieved the status of clinical tests, and studies have thus been restricted to in vitro tests with cultures of the parasites. In many cases, these studies reveal antiparasitic properties of the vanadium species, which are about comparable to or even more effective than established medications. In this section, selected examples of antiparasitic vanadium complexes are briefly described. For a recent overview see also the review by D. Gambino [72]. Respective vanadium compounds are illustrated in Figure 14; selected results are summarized in Table 4 [73–77].

O O 2 NH2 Salicyl- V O N O semicarbazone O N N O V N 5-Methyl- O N O salicyl H3CO 2-Furoyl- 2 N o-Phenan- hydrazide OH throline 19 20

HO Galactose 2+ O OH VO (LH-1)2 HO HL = O +H N Galactomannan 3 NH2 O O Glutaminate HO OH O O (Gln) O V O O O O O Mannose O O OH (Gln) 21 22

Figure 14 Complexes and formulations that have been shown to exert antiparasitic potential against parasites causing amoebiasis (19), Chagas’ disease (20), and leishmaniasis (20, 21, 22). For details see text and Table 4. 5 Vanadium. Its Role for Humans 163

Table 4 Vanadium compounds that kill parasites causing (tropical) diseases. Complex no. (see Target Figure 13) Effect Remarks Ref. Tropozoites of 19 Amoebocidal More effective than [73] Entamoeba Metronidazole; slightly histolytica toxic against HeLa cells (in vitro) Epimastigotes of 20 Trypanocidal Causes conformational [75] Trypanosomas changes in supercoiled cruzi (in vitro) plasmid DNA Promastigotes and 20 Leishmanicidal Cytotoxic against leukemia [74] amastigotes HL-60 cells of Leishmania (in vitro) Promastigotes and 21 Leishmanicidal on Diminishes also superoxide [76] amastigotes amastigotes; growth production by macrophages of Leishmania inhibition of (in vitro) promastigotes Leishmania-infected 22 Reduction in parasite Similar effects are brought [77] 2– mice (in vivo) burden; expansion about by [HVO2(O2)2)] of antileishmanial T-cells

According to the World Health Organisation, about 50 million people are affected worldwide by amoebiasis, with infections clearly cumulating in tropical countries. The etiologic agent of the disease is the amoeba Entamoeba histolytica, present in contaminated food and water in particular in areas of low sanitary and hygiene standards. Transmission occurs mainly by the fecal-oral route and through direct contact. 90% of the infected people are asymptomatic. For the remaining 10% the symptoms include diarrhea and, in more serious cases, dysentery (an inflammatory disorder mainly of the colon) with mucus and blood, the latter stemming from amoebae that succeed to overcome the epithelium of the intestines and thus travel to other organs, the liver in particular, where they cause deadly abscesses. The death toll amounts to ca. 70,000 per year. The hydrazone complex 19 in Figure 14 is an example for an efficient amoebocidal (pro)drug [73]. With an IC50 = 0.36 μM, the compound is more efficient than the standard drug Metronidazole (IC50 = 1.89 μM), and somewhat more toxic than Metronidazole against human cervical HeLa cancer cell lines. Chagas’ disease is caused by the flagellate protozoan parasiteTrypanosomas cruzi, transmitted by the feces of “kissing bugs”, blood-sucking bugs belonging to the subfamily Triatominae. About 10 million people are infected, with ca. 10,000 deaths per year. The disease is mainly distributed in Latin America, but also increasingly spreading into North America and Europe. Primary symptoms are skin lesions and swelling of the eye lids; secondary symptoms include digestive and neurological alteration, and cardiac disorder up to heart failure. The oxidovanadium complex 164 Rehder

20 [74] contains a salicylidene semicarbazone ligand and hence a ligand that has also been shown to convey anticancer (compound 12) and antiamoebiasis activity (19). The additional ligand in 20 is phenanthroline, capable of intercalating DNA (Figure 12b). The metabolic pathways of parasites such as Trypanosoma and Leishmania are similar to those in tumor cells, suggesting a similar mode of action of anti-parasitic and anti-tumor drugs. Complexes such as compound 20, which are about as trypanocidal against epimastigotes (a developmental state of the parasite in the bug) of T. cruzi as the reference drug Nifurtimox, are in fact cytotoxic against leukemia cells [74] and cause conformational changes in plasmid DNA [75]. Complex 20 is also active on the promastigote and amastigote forms (the flagellate and non-flagellate stages, respectively) of Leishmania parasites, responsible for the tropical and subtropical disease leishmaniasis. The vectors for this disease, which is associated with malnutrition and weakness of the immune system, are sandflies of the subfamily Phlebotomina. About 12 million people are affected worldwide. The most serious form of this disease is visceral leishmaniasis, which goes along with high fever, weight loss, swelling of the spleen and liver, and anemia. Visceral leishmaniasis is mainly distributed in Brazil, India, Bangladesh, and Sudan, and accounts for 60,000 deaths each year. Oxidovanadium complexes of galactomannan (21 in Figure 14) have been shown to be leishmanicidal on amastigotes of L. amazonensis, and to inhibit the growth of the promastigotes of this parasite [76]. Galactomannan is a polysaccharide with a mannose backbone and galactose side groups, isolated from the lichen Ramalina celastri, which is abundant in South Brazil. Antileishmanial effects have 2– further been noted, with infected mice, for bis(peroxido)vanadate, [HVO2(O2)2] , 2– and combinations of glutaminate and [HVO2(O2)2] (see 22 in Figure 14 for a tentative formulation of a formula unit) [77]. Finally, decavanadate deters the growth of the leishmania parasite, likely by interaction of decavanadate’s hydrolysis – product [H2VO4] with phosphatases [78], hence a mechanistic aspect which is again reminiscent of the antidiabetic, insulin-enhancing action of vanadate and (hydrolytically labile) vanadium complexes. For the molecular built-up of decavanadate see Figure 5.

5 Concluding Remarks and Prospects

The element vanadium was discovered in 1801 by Andrés Manuel del Rio y Fernández in vanadinite from the district Zimapán in Mexico [5]. It was rediscovered by Nils Gabriel Sefström in iron ore from the Taberg in Småland (Sweden) in 1830 by treating a “black powder obtained from the manufacturing of bar iron”, and in 1869/70 Sir Henry Enfield Roscoe in England was the first one who succeeded to isolate (an impure form of) metallic vanadium by reduction of VCl2 with H2 [79,87], nowadays widely employed in particularly tough and hard chromium- vanadium-iron alloys. Along with metallic vanadium, vanadium oxides (“VOx”) are 5 Vanadium. Its Role for Humans 165 in use as catalysts in oxidation reactions (an example is the production of sulfuric acid from SO2), as an UV-absorbing additive in glass ware, and in the form of lithium and silver vanadates in lithium batteries [80]. The first indication for the presence of vanadium in a living organism, i.e., the sugar beet, goes back to 1888 [81]; the essentiality of vanadium for some life forms became established in 1982/83 by the discovery of vanadate-dependent bromoperoxidase in the marine macro-alga Ascophyllum nodosum [82]. Toxic effects of vanadium were first reported by John Priestley [15], who applied large doses of sodium vanadate to animals, tediously describing their ailment and final death. Exploratory medicinal applications of vanadium, again in the form of vanadate, were carried out in Lyon (France) back in 1897–98 [83] on probands with health problems as diverse as anemia, tuberculosis, rheumatism, neurasthenia, and diabetes where, in the latter case, some decrease of blood glucose was observed. The prospective use of vanadium compounds in the treatment of diabetes thus has a long-standing tradition. The presently most advanced study of vanadium’s potential in the treatment of human diabetics are clinical tests phase IIa, carried out in 2007/8 with bis(ethylmaltolato)oxidovanadium(IV), BEOV [13], a compound developed in the group of Chris Orvig in Vancouver, Canada. The successful application of vanadium compounds in low doses with diabetic animals (streptozotozin-induced rats in particular) and with type 2 diabetic humans has initiated research into medicinally active vanadium compounds for other medicinal applications, among these tropical and subtropical diseases caused by infectious parasites responsible for leishmaniasis, Chagas’ disease, and amoebiasis, just as diseases caused by viruses (HIV, influenza), and bacteria (tuberculosis). In all cases, in vitro tests with cell cultures or cultured parasites, as well as sporadic in vivo tests with infected test animals, have provided results that should encourage further investigations into the field of vanadium-based medications.G iven that classical pharmaceuticals tend to have, sometimes severe, side effects, and parasites can develop resistance, the development of potential (pro-)drugs based on the essentially non-toxic element vanadium certainly is a future challenge. The possibly most important aspect in choosing vanadium as the central metal in these coordination compounds is their (partial) rebuilt to vanadate(V) and oxidovanadium(IV) in the physiological broth. Vanadate is an antagonist of phosphate – as demonstrated for the first time by Cantley in 1977 for the inhibition of the Na,K-ATPase, the sodium-potassium pump [84]. Based on the interference of vanadate with metabolic processes depending on phosphate in general, and on phosphate-regulated enzymatic activity in particular, vanadate has also been introduced into the amelioration of cardiac dysfunctions, and in fighting malignant tumors. The latter application, first investigated systematically by Köpf and Köpf-Maier [60], is based, at least in part, on the direct interaction of stable vanadium-ligand fragments with the DNA of cancer cells – and is thus reminiscent of the action of the well established anticancer drug cisplatin. Stable fragments in vanadium compounds are represented by the cyclopentadienide ligands in vanadocenes, and by N-functional, oligodentate aromatic ligands such as ortho-phenanthroline and derivatives thereof. 166 Rehder

Last but not least, yet another functional aspect of vanadium compounds is the ability of VO2+ to interfere – in a Fenton-like reaction – with the tissue concentrations of reactive oxygen species [85].

Abbreviations

Ab albumin acac acetonylacetonate(1–) Akt protein kinase ATPase adenosine triphosphate cleaving enzyme BEOV bis(ethylmaltolato)oxidovanadium CAKI human renal carcinoma cell line CCR5 chemokine receptor of T lymphocytes Cp cyclopentadienide CXCR-4 chemokine receptor type 4 FAD flavin adenine dinucleotide (oxidized form)

FADH2 flavin adenine dinucleotide (reduced form) GLUT glucose transporter GSH glutathione GSSG oxidized form of glutathione HeLa cells cervical cancer cell line derived from Henrietta Lacks HIV human immune deficiency virus

IC50 50% inhibitory concentration; the values denote the concentration of a drug or prodrug at which the life function of 50% of the viable cells are inhibited Ig immunoglobulin IR insulin receptor IRS insulin receptor substrate lmm low molecular mass (ligand) MAC maximum allowable concentration at the working place MIC minimal inhibitory concentration NAD nicotinamide adenine dinucleotide (oxidized form) NADH nicotinamide adenine dinucleotide (reduced form) NADP nicotinamide adenine dinucleotide phosphate (oxidized form) NADPH nicotinamide adenine dinucleotide phosphate (reduced form) PIK phosphatidyl-inositol kinase PKB protein kinase B PTPase protein tyrosine phosphatase ROS reactive oxygen species SARS severe acute respiratory syndrome STZ streptozotocin SV Simian virus Tf transferrin tsc thiosemicarbazone 5 Vanadium. Its Role for Humans 167

References

1. (a) D. Rehder, Future Med. Chem. 2012, 4, 1823–1837. (b) D. Rehder, in Detoxification of Heavy Metals, Eds I. Sherameti, A. Varma, Soil Biology 2011, 30, ch. 11. 2. T. Shimizu, K. Ichikawa, M. Masayuki, Y. Shijo, Bunseki Kagaku 1989, 38, 201–203. 3. T. L. Gerke, K. G. Scheckel, M. R Schock, Environ. Sci. Technol. 2009, 43, 4412–4418. 4. g. Giuli, G. Pratesi, S. G. Eeckhout, E. Paris, 68th Ann. Meteoritic. Soc. Meeting, Zurich 2006, p. 5148. 5. D. Rehder, Bioinorganic Vanadium Chemistry, John Wiley & Sons, Chichester, 2008, pp 87–155. 6. D. G. Barcelaux, J. Toxicol. Clinic. Toxicol. 1999, 37, 265–278. 7. M. D. Cohen, ACS Symposium Series 2007, 974, 217–239. (b) T. Scior, A. Guevara-García, P. Bernard, Q.-T. Do, D. Domeyer, S. Laufer, Mini Rev. Med. Chem. 2005, 5, 995–1008. 8. J. O. Olopade, J. R. Condor, Curr. Topics Toxicol. 2011, 7, 33–39. 9. G. Heinemann, B. Fichti, W. Vogt, Clin. Pharmacol. 2003, 55, 241–245. 10. A. Gorzsás, I. Andersson, L. Pettersson, Eur. J. Inorg. Chem. 2006, 3559–3565. 11. D. Sanna, P. Buglyó, G. Micera, E. Garribba, J. Biol. Inorg. Chem. 2010, 15, 825–839. 12. (a) D. Sanna, L. Bíro, P. Buglyó, G. Micera, E. Garribba, J. Inorg. Biochem. 2012, 115, 87–99. (b) G. C. Justino, E. Garribba, S. Mehtab, J. Costa Pessoa, J. Biol. Inorg. Chem. 2013, 18, 803–813. 13. K. H. Thomson, J. Lichter, C. LeBel, M. C. Scaife, J. H. McNeill, C. Orvig, J. Inorg. Biochem. 2009, 103, 554–558. 14. T. C. Delgado, A. I. Tomaz, I. Corriera, J. Costa Pessoa, J. G. Jones, C. F. G. C. Geraldes, M. M. C. A. Castro, J. Inorg. Biochem. 2005, 99, 2328–2339. 15. J. Priestley, Phil. Trans. Roy. Soc. London 1876, 166, 495–556. 16. V. A. Ehrlich, A. K. Nersesyan, K. Alefie, C. Hoelzl, F. Ferk, J. Bichler, E. Valic, A. Schaffer, R. Schulte-Herrmann, M. Fenech, K.-H. Wagner, S. Knasmüller, Environ. Health Perspect. 2008, 116, 1689–1693. 17. H. P. Monteiro, C. C. Winterburn, A. Stern, Free Radic. Res. Commun. 1991, 12–13, 125–129. 18. N. Gao, M. Ding, J. Z. Zheng, Z. Zhang, S. S. Leonard, K. J. Liu, X. Shi, B.-H. Jiang, J. Biol. Chem. 2002, 277, 31963–31971. 19. H. Kelm, H.-J. Krüger, Angew. Chem. Int. Ed. 2001, 40, 2344–2348. 20. E. J. Baran, Chem. Biodivers. 2008, 5, 1475–1484. 21. (a) J. R. Treberg, J. E. Stacey, W. R. Driedzic, Comp. Biochem. Physiol. B 2012, 161, 323–330. (b) H. Michibata,T. Ueki, Biomol. Concepts 2010, 1, 97–107. 22. J. Krakowiak, D. Lundberg, I. Persson, Inorg. Chem. 2012, 51, 9598–9609. 23. M. Kaliva, E. Kyriakakis, A. Salifoglou, Inorg. Chem. 2002, 41, 7015–7023. 24. H. Schmidt, I. Andersson, D. Rehder, L. Pettersson, Chem. Eur. J. 2001, 7, 251–257. 25. J. Schemberg, K. Schneider, U. Demmer, E. Warkentin, A. Müller, U. Ermler, Angew. Chem. Int. Ed. 2007, 46, 2409–2413. 26. (a) L. Wittenkeller, A. Abraha, R. Ramasamy, D. Mota de Freitas, L. A. Theisen, D. C. Crans, J. Am. Chem. Soc. 1991, 113, 7872–7881. (b) N. Steens, A. M. Ramadan, T. N. Parac-Vogt, Chem. Commun. 2009, 965–967. 27. S. Lobert, N. Isern, B. S. Hennington, J. J. Correia, Biochemistry 1994, 33, 6244–6252. 28. M. Aureliano, Dalton Trans. 2009, 9093–9100. 29. S. S. Soares, C. Gutiérrez-Merino, M. Aureliano, J. Inorg. Biochem. 2007, 101, 789–796. 30. (a) N. E. Levinger, L. C. Rubenstrunk, B. Baruah, D. C. Crans, J. Am. Chem. Soc., 2011, 133, 7205–7214. (b) D. C. Crans, N. E. Levinger, Acc. Chem. Res., 2012, 45, 1637–1645. 31. (a) M. Weyand, H.-J. Hecht, M. Kieß, M.-F. Liaud, H. Vilter, D. Schomburg, J. Mol. Biol. 1999, 293, 595–611. (b) L. Kaysser, P. Bernhardt, S.-J. Nam, S. Loesgen, J. G. Ruby, P. Skewes-Cox, P. R. Jensen, W. Fenical, B. S. Moore, J. Am. Chem. Soc. 2012, 134, 11988–11991. 32. (a) Y. Lindquist, G. Schneider, P. Vihko, Eur. J. Biochem. 1994, 221, 139–142. (b) M. Z. Mehdi, A. K. Srivastava, Arch. Biochem. Biophys. 2005, 440, 158–164. (c) M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 1997, 36, 15–23. 168 Rehder

33. N. Tanaka, V. Dumay, Q. Liao, A. J. Lange, R. Wever, Eur. J. Biochem. 2002, 269, 2162–2167. 34. R. Renirie, W. Hemrika, R. Wever, J. Biol. Chem. 2000, 16, 11650–11667. 35. C. C. McLauchlan, J. D. Hooker, M. A. Jones, Z. Dymon, E. A. Backhus, B. A. Greiner, N. A. Dorner, M. A. Youkhana, L. M. Manus, J. Inorg. Biochem. 2010, 104, 274–281. 36. B. Akabayov, A. W. Kulczyk, S. R. Akabayov, C. Theile, L. W. McLaughlin, B. Beauchamp, A. M. van Oijen, C. C. Richardson, J. Biol. Chem., 2011, 286, 29146–29157. 37. M. J. Gresser, A. S. Tracey, K. M. Parkinson, J. Am. Chem. Soc. 1986, 108, 6229–62343. 38. T. Kiss, E. Kiss, G. Micera, D. Sanna, Inorg. Chim. Acta 1998, 283, 202–210. 39. I. Andersson, A. Gorzsás, C. Kerezsi, I. Tóth, L. Pettersson, Dalton Trans. 2005, 3658–3666. 40. I. Andersson, S. Angus-Dunge, O. W. Howarth, L. Pettersson, J. Inorg. Biochem. 2000, 80, 51–58. 41. (a) A. S. Tracey, M. J. Gresser, Proc. Natl. Acad. Sci. USA 1986, 83, 609–613. (b) A. S. Tracey, M. J. Gresser, Can. J. Chem. 1988, 66, 2570–2574. 42. (a) T. Scully, Nature 2012, 485, S2–S3. (b) L. Chen, D. J. Magliano, P. L. Zimmer, Nat. Rev. Endocrinol. 2012, 8, 228–236. 43. C. Talchai, S. Xuan, H. V. Lin, L. Sussel, D. Accili, Cell 2012, 150, 1223–1234. 44. M. F. Dunn, Biometals 2005, 18, 295–303. 45. M. Hiromura, Y. Adachi, M. Machida, M. Hattori, H. Sakurai, Metallomics 2009, 1, 92–100. 46. G. R. Willsky, L.-H. Chi, M. Godzala III, P. J. Kostyniak, J. J. Smee, A. M. Trujillo, J. A. Alfano, W. Ding, Z. Hu, D. C. Crans, Coord. Chem. Rev. 2011, 255, 2258–2269. 47. J. Gätjens, B. Meier, Y. Adachi, H. Sakurai, D. Rehder, Eur. J. Inorg. Chem. 2006, 3575–3585. 48. J. Nilsson, A. A. Shteinman, E. Degerman, E. A. Enyedy, T. Kiss, U. Behrens, D. Rehder, E. Nordlander, J. Inorg. Biochem. 2011, 105, 1795–1800. 49. H. Ou, L. Yan, D. Mustafi, M. W. Makinen, M. J. Brady, J. Biol. Inorg. Chem. 2005, 10, 874–886. 50. S. Karmaker, T. K. Saha, Y. Yoshikawa, H. Sakurai, ChemMedChem 2007, 2, 1607–1612. 51. R. Mukherjee, E. G. Donnay, M. A. Radomski, C. Miller, D. A. Redfern, A. Gericke, D. S. Damron, N. E. Brasch, Chem. Commun. 2008, 3783–3785. 52. T. A. Clark, C. E. Heylinger, M. Kopilas, A. L. Edel, A. Junaid, F. Aguilar, D. D. Smyth, J. A. Thliveris, M. Merchant, H. K. Kim, G. N. Pierce, Metabolism 2012, 61, 742–753. 53. K. G. Peters, M. G. Davis, B. W. Howard, M. Pokross, V. Rastogi, C. Diven, K. D. Greis, E. Eby-Wilkens, M. Maier, A. Evdokimov, S. Soper, F. Genbauffe, J. Inorg. Biochem. 2003, 96. 321–330. 54. A. Bishayee, A. Waghray, M. P. Patel, M. Chatterjee, Cancer Lett. 2010, 294, 1–12. 55. M. M. Evangelou, Oncol. Hematol. 2002, 42, 249–265. 56. H. Faneca, V. A. Figueiredo, I. Tomaz, G. Gonçalves, F. Avecilla, M. C. Pedroso de Lima, C. F. G. C. Geraldes, J. Costa Pessoa, M. M. C. A. Castro, J. Inorg. Biochem. 2009, 103, 601–608. 57. I. Fichtner, J. Claffey, A. Deally, B. Gleeson, M. Hogan, M. R. Markelova, H. Müller-Bunz, H. Weber, M. Tacke, J. Organomet. Chem. 2010, 695, 1175–1181. 58. O. J. D’Cruz, F. M. Uckun, Expert Opin. Investig. Drugs 2002, 11, 1829–1836. 59. N. A. Lewis, F. Liu, L. Seymour, A. Magnuse, T. R. Erves, J. Faye Arca, F. A. Beckford, R. Venkatraman, A. Gonzáles-Sarrías, F. R. Fronczkek, D. G. VanDerveer, N. P. Seeram, A. Liu, W. L. Jarrett, A. A. Holder, Eur. J. Inorg. Chem. 2012, 664–677. 60. P. Köpf-Maier, H. Köpf, Drugs Future 1986, 11, 297–319. 61. (a) Q. Wang, T.-T. Liu, Y. Fu, K. Wang, X.-G. Yang, J. Biol. Inorg. Chem. 2010, 15, 1087– 1097. (b) T.-T. Liu, Y.-J. Liu, Q. Wang, X.-G. Yang, K. Wang, J. Biol. Inorg. Chem. 2012, 17, 311–320. 62. J. O. Olopade, A. B. Madhan Kumar, A. Das, X. Liu, B. Todorich, J. L. Liang, B. Slagle-Webb, J. R. Connor, Proc. Am. Ass. Cancer Res. 2009, 50, 1344 (#5575). 63. (a) Md. S. Bhuiyan, K. Fukunaga, J. Pharmacol. Sci. 2009, 110, 1–13. (b) K. Fukunaga, Yakugaku Zasshi 2012, 132, 279–284. 64. D. A. Liem, C. C. Gho, B. C. Gho, S. Kazim, O. C. Manintveld, P. D. Verdouw, D. J. Duncker, J. Pharmacol. Exp. Therapeut. 2004, 309, 1256–1262. 5 Vanadium. Its Role for Humans 169

65. C. L. Walker, M. J. Walker, N.-K. Liu, E. C. Risberg, X. Gao, J. Chen, X,-M. Xu, PLoS ONE 2012, 7, e30012. 66. K. T. Keyes, J. Xu, B. Long, C. Zhang, Z. Hu, Y. Ye, Am. J. Phys. Heart Circ. Physiol. 2010, 298, H1198–H1208. 67. (a) S. Shigeta, S. Mori, T. Yamase, N. Yamamoto, N. Yamamoto, Biomed. Pharmacother. 2006, 60, 211–219. (b) T. Yamase, E. Ishikawa, K. Fukaya, H. Nojiri, T. Taniguchi, T. Atake, Inorg. Chem. 2004, 43, 8150–8157. 68. S.-Y. Wong, R. W.-Y. Sun, N. P.-Y. Chung, C.-L. Lin, C.-M. Che, Chem. Commun. 2005, 3544–3546. 69. A. Ross, D. C. Soares, D. Covelli, C. Pannecouque, L. Budd, A. Collins, N. Robertson, S. Parsons, E. De Clercq, P. Kennepohl, P. J. Sadler, Inorg. Chem. 2010, 49, 1122–1132. 70. R. Renirie, A. Dewilde, C. Pierlot, R. Wever, D. Hober, J.-M. Aubry, J. Appl. Microbiol. 2008, 105, 264–270. 71. P. I. da S. Maia, F. R. Pavan, C. Q. F. Leite, S. S. Lemos, G. F. de Sousa, A. A. Batista, O. R. Nascimento, J. Ellena, E. E. Castellano, E. Niquet, V. M. Deflon, Polyhedron 2009, 28, 398–406. 72. D. Gambino, Coord. Chem. Rev. 2011, 255, 2193–2203. 73. M. R. Maurya, A. A. Khan, A. Azam, S. Ranjan, N. Mondal, A. Kumar, F. Avecilla, J. Costa Pessoa, Dalton Trans. 2010, 39, 1345–1360. 74. J. Benítez, L. Becco, I. Correira, S. M. Leal, H. Guiset, J. Costa Pessoa, J. Lorenzo, S. Tanco, P. Escobar, V. Moreno, B. Garat, D. Gambino, J. Inorg. Biochem. 2011, 105, 303–312. 75. J. Benítez, L. Guggeri, I. Tomaz, G. Arrambide, M. Navarro, J. Costa Pessoa, B. Garat, D. Gambino, J. Inorg. Biochem. 2009, 103, 609–616. 76. G. R. Noleto, A. L. R. Mercê, M. Iacomini, P. A. J. Gorin, V. T. Soccol, M. B. M. Oloveira, Molec. Cellul. Biochem. 2002, 233, 73–83. 77. A. K. Haldar, S. Banerjee, K. Naskar, D. Kalita, N. S. Islam, S. Roy, Experim. Parasitol. 2009, 122, 145–154. 78. T. L. Turner, V. H. Nguyen, C. C. McLauchlan, Z. Dymon, B. M. Dorsey, J. D. Hooker, M. A. Jones, J. Inorg. Biochem. 2012, 108, 96–104. 79. H. E. Roscoe, Phil. Trans. Roy. Soc. A 1870, 160, 317–331. 80. Y. J. Kim, A. C. Marschilok, K. J. Takeuchi, E. S. Takeuchi, J. Power Sources 2011, 196, 6781–6787. 81. E. O. von Lippmann, Ber. Dtsch. Chem. Ges. 1888, 21, 3492–3493. 82. H. Vilter, K.-W. Glombitza, A. Grawe, Bot. Mar. 1983, 26, 331–340. 83. B. Lyonnet, X. Martz, E. Martin, La Presse Médicale 1899, 32, 191–192. 84. L. C. Cantley, Jr., L. Josephson, R. Warner, M. Yanagisawa, C. Lechene, G. Guidotti, J. Biol. Chem. 1977, 252, 7421–7423. 85. D. C. Crans, A. M. Trujillo, P. S. Pharazyn, M. D. Cohen, Coord. Chem. Rev. 2011, 255, 2178–2192. 86. L. Nordquist, M. Johansson, Vasc. Health Risk Manag. 2008, 4, 1283–1288. 87. H. E. Roscoe, Philos. Mag. 1870, 39, 146–150. Chapter 6 Chromium: Is It Essential, Pharmacologically Relevant, or Toxic?

John B. Vincent

Contents ABSTRACT ...... 172 1 INTRODUCTION ...... 172 2 IS CHROMIUM ESSENTIAL? ...... 173 2.1 Current Opinions ...... 173 2.2 Evidence ...... 173 2.2.1 “Low Chromium” Rodent Diets ...... 173 2.2.2 Absorption and Transport ...... 176 2.2.3 Total Parenteral Nutrition ...... 179 3 IS CHROMIUM PHARMACOLOGICALLY RELEVANT? ...... 180 3.1 Rodent Disease Model Studies...... 180 3.2 Clinical Studies ...... 182 3.3 Proposed Mechanisms of Action ...... 186 3.3.1 Insulin Signaling ...... 186 3.3.2 Cholesterol and Fatty Acid Metabolism...... 189 3.3.3 Inﬂ ammation and Oxidative Stress ...... 190 4 IS CHROMIUM TOXIC? ...... 191 4.1 Chromate ...... 191 4.2 Chromium Picolinate and Other Cr(III) Complexes ...... 191 5 CONCLUDING REMARKS AND FUTURE DIRECTION ...... 192 ABBREVIATIONS AND DEFINITIONS ...... 193 ACKNOWLEDGMENT ...... 194 REFERENCES ...... 194

J. B. Vincent (*) Department of Chemistry , University of Alabama , Tuscaloosa , AL 35487-0336 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 171 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_6, © Springer Science+Business Media Dordrecht 2013 172 Vincent

Abstract Over fi fty years ago, the element chromium (as the trivalent ion) was proposed to be an essential element for mammals with a role in maintaining proper carbohydrate and lipid metabolism. Evidence for an essential role came from dietary studies with rodents, studies on the effects of chromium on subjects on total parenteral nutrition, and studies of the absorption and transport of chromium. Over the next several decades, chromium-containing nutritional supplements became so popular for weight loss and muscle development that sales were second only to calcium among mineral supplements. However, the failure to identify the responsible biomolecules(s) that bind chromium(III) and their mode of action, particularly a postulated species named glucose tolerance factor or GTF, resulted in the status of chromium being questioned in recent years, such that the question of its being essential needs to be formally readdressed. At the same time as chromium(III)’s popularity as a nutritional supplement was growing, concerns over its safety appeared. While chromium has been conclusively shown not to have benefi cial effects on body mass or composition and should be removed from the list of essential trace elements, chromium(III) compounds are generally nontoxic and have benefi cial pharmacological effects in rodents models of insulin insensitivity, although human studies have not conclusively shown any benefi cial effects. Mechanisms have been proposed for these pharmacological effects, but all suffer from a lack of consistent supporting evidence.

Keywords chromium • insulin sensitivity • insulin signaling • rats • type 2 diabetes

Please cite as: Met Ions Life Sci. 13 (2013) 171–198

1 Introduction

Recently, a paradigm shift has occurred in terms of the status of chromium. While fi rst proposed to be an essential element in the late 1950s and accepted as a trace element in the 1980s, scientifi c studies have continued to fail to produce convincing evidence of this status. In the 1990s, statements to justify the status of chromium despite the results of studies such as “Chromium is a nutrient and not a drug, and it will therefore benefi t only those who are defi cient or marginally defi cient in Cr” [1 ] were common in review articles [ 1 –3 ]. Recent studies have led to a reinterpretation of the status of chromium. The status of chromium as an essential element is no longer supported by experimental data. In fact, chromium is now best understood as a therapeutic agent. However, the potential benefi ts of the use of chromium as a therapeutic agent are uncertain, and its mechanism of action in increasing insulin sensitivity and possibly infl uencing lipid metabolism at a molecular level is poorly understood. This review will examine the data on which chromium was proposed to be an essential element and describe the problems with this interpretation, discuss the evidence for a therapeutic role for chromium in animal models of diabetes and insulin resistance, and evaluate the potential toxicity as chromium(III) complexes when used at pharmacologically relevant doses. 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 173

2 Is Chromium Essential?

2.1 Current Opinions

Chromium reduces body fat, causes weight loss, causes weight loss without exercise, causes long-term or permanent weight loss, increases lean body mass or builds muscle, increases human metabolism, and controls appetite or craving for sugar, while 90% of US adults do not consume diets with suffi cient chromium to support normal insulin function, resulting in increased risk of obesity, heart disease, elevated blood fat, high blood pressure, diabetes, or some other adverse effect on health. Any or all of the above representations may come to mind when thinking about chromium and its relationship to human nutrition. Most people think of chromium in terms of weight loss and lean muscle mass development as a result of nutraceutical product marketing. However, the Federal Trade Commission (FTC) of the United States ordered entities associated with the nutritional supplement chromium picolinate to stop making each of the above representations in 1997 because of the lack of “competent and reliable scientifi c evidence” [4 ]. Overwhelming scientifi c evidence currently indicates that chromium does not affect body mass and body composition of healthy individuals and that chromium nutritional defi ciency is rare (if it exists at all) [ 5 ]. Yet, although the ruling by the FTC is over 15 years old, such representations can still be found in the popular media. In the United States, the National Research Council of the National Academies of Science recognized chromium as an essential trace element in 1980 and reviewed this position in 1989 and 2002 [6 – 8]. However, in 1980 and 1988, chromium was determined to have an estimated safe and adequate daily dietary intake (ESADDI) of 50–200 μg, while in 2002 this was changed to an adequate intake (AI) of 30 μg. The Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP) [ 9 ] in 2009 determined that chromium defi ciency in farm animals had never conclusively been observed such that ‘no evidence of the essentiality of Cr(III) as a trace element in animal nutrition’ exists. As discussed in Section 2.2 , the status of chromium is at best uncertain currently, and the element should probably be removed from the list of essential trace elements.

2.2 Evidence

2.2.1 “Low Chromium” Rodent Diets

Over fi fty years ago, Cr was suggested to be an essential trace element in the mammalian diet. In this work reported by Mertz and Schwarz [ 10 ], previously considered the pioneering work in the fi eld, rats were fed a torula yeast-based diet, which compromised the health of the rats. The rats developed necrotic liver degeneration and apparently impaired glucose tolerance in response to an intravenous 174 Vincent glucose load [10 ]. Selenium was discovered to reverse the liver disorder but not the glucose intolerance; as a result, the authors proposed a new dietary requirement, coined glucose tolerance factor (GTF) was absent from the torula yeast-based diet and responsible for the glucose intolerance [11 ]. In an effort to identify the missing dietary component, a variety of chemicals and some foods were added to the diet. Most notably, inorganic compounds containing over 40 different elements (200– 500 mg element/kg body mass) could not restore glucose tolerance, while several inorganic Cr(III) complexes (200 mg Cr/kg body mass) restored glucose tolerance [ 12 ]. Brewer’s yeast and acid-hydrolyzed porcine kidney powder were identifi ed as natural sources of the missing dietary component and were found to contain appreciable quantities of Cr [ 12]. When given by stomach tube (500–1000 mg/kg body mass), brewer’s yeast, porcine kidney powder, and concentrates made from them restored proper glucose metabolism in rats on the torula yeast-based diet [ 12 ]. Consequently, Mertz and Schwarz proposed the active ingredient of GTF was Cr3+ , making Cr an essential trace element for the mammalian diet [12 ]. As this became the primary evidence for an essential role for Cr, one must ask what exactly this work established? The Cr content of the regular laboratory rat diet and of the torula yeast-based diet have not been determined; thus, the rats were not shown to actually receive a diet lacking or defi cient in Cr; the studies only indicated that adding Cr to the diet could lead to potential effects on apparent glucose intolerance. Subsequently, the Cr content of torula yeast has been determined, but the Cr content has been found to range signifi cantly in value [ 13, 14]; the content probably varies based on the growth conditions. As a result, the content of the original diet simply cannot be established. In addition, the rats were housed in wire mesh cages, possibly with stainless steel components (as the metal composition of the wire was not reported), allowing the rats to obtain Cr by chewing on these components. Thus, the actual Cr intake of the rats in these studies is impossible to gauge. As subsequent studies have shown that rat in metal free cages on purifi ed diets fail to develop Cr defi ciency, these studies fail to establish that the animals developed a Cr defi ciency. The results do raise another possible explanation, one that was not originally considered – the Cr added to the torula yeast-based diet was having a pharmacological or therapeutic effect and not correcting a nutritional defi ciency. The magnitude of the doses of Cr utilized in these studies need to be put into perspective. An American consuming a nutritionist-designed diet [15 ] or self-selected diet [16 ] consumes about 30 μg of Cr daily. This value, 30 μg Cr/day, is the value set as the adequate intake (AI) by the Food and Nutrition Board of the Institute of Medicine of the National Academy of Sciences (USA) [8 ]; as defi ned, the AI indicates that >98% of the population receiving this quantity of an item display no health problems from defi ciency. Given the average body mass of a human, 65 kg, gives an adequate Cr intake of less than 0.5 μg Cr/kg per day. Rats on the torula yeast-based diet that was supplemented with Cr compounds received at least 400 times this quantity, a supra-nutritional dose. These comparisons, of course, make the assumption that the biochemistry of Cr is similar in rodents and primates. 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 175

Attempts have been made to establish the nutritional status of Cr using nutritionally compromised diets supplemented with Cr, most notably in the 1990s [17 – 19 ]; the rationale behind these diets was that stresses that increase urinary Cr loss could potentially lead over time to chromium defi ciency. However, these studies suffer from some of the same fl aws in assumptions as the initial studies. Rats were provided a high-sugar or high-fat diet (supposedly a “low-Cr” diet with ca. 30 μg Cr/kg diet) with additional mineral stresses for 24 weeks, resulting in compromised lipid and carbohydrate metabolism in the rats. The addition of 5 ppm Cr to the drinking water of rats on the stressed diets led to plasma insulin levels tending to be higher in intravenous glucose tolerance tests after 24 weeks on the diet [ 17 ]. Unfortunately, the Cr intake compared to the Cr loss in the rats was not determined so that whether the rats were maintaining a Cr balance cannot be established. However, as described in Section 2.2.2 , the amount of urinary Cr loss is directly dependent on the amount of Cr intake so that the rats should not have developed a Cr defi ciency. Consequently, the results should be interpreted in terms of supplemental Cr having a benefi cial effect on diet-induced insulin resistance, a pharmacological rather than nutritional effect. An analysis of the actual Cr content of the diet is in order. A male Wistar rat (as used in Refs [ 17 – 19]) on average in a subchromic study consumes 20 g of food a day and has an average body mass of 217 g [ 20 ]. Twenty grams of food containing 33 μg Cr/kg food provides 0.66 μg Cr. Thus, 0.66 μg Cr/d for a 217 g rat is 3.0 μg Cr/kg body mass per day, six times what a human intakes. Thus, the “low-Cr” diet was not defi cient, unless rats require more than six times the Cr dose that humans do. In contrast, a male Wistar rat on average drinks 147 mL of water [20 ]. This volume of water supplemented with 5 ppm Cr provides 735 μg Cr daily or 3.39 mg/kg body mass. This is approximately 100 times the adequate intake of an American male (35 μg Cr/day) [8 ]. Again, indicating the lowering of plasma-insulin levels by addition of Cr can only be considered a pharmacological effect. Finally, a most recent study appears to have unambiguously demonstrated that Cr has a pharmacological rather than a nutritional effect in mammals [21 ]. Whether Cr is an essential element was examined for the fi rst time in carefully controlled metal- free conditions using a series of purifi ed diets containing various Cr contents. Male lean Zucker rats were housed in specially designed metal-free cages for six months and fed the AIN-93G diet with no added Cr in the mineral mix component of the diet (containing 16 μg Cr/kg diet), the standard AIN-93G diet (containing added 1,000 μg Cr/kg), the standard AIN-93G diet supplemented with 200 μg Cr/kg, or the standard AIN-93G diet supplemented with 1,000 μg Cr/kg. The Cr content of the diet had no effect on the body mass or food intake. Similarly, the Cr content of the diet had no effect on the glucose levels in glucose tolerance or insulin tolerance tests. However, a distinct and statistically signifi cant trend toward lower insulin levels under the curve after a glucose challenge was observed with increasing Cr content in the diet; rats on the supplemented AIN-93G diets had signifi cantly lower areas (P <0.05) than rats on the low-Cr diet. The study revealed that a diet with as little Cr as reasonably possible had no effect on body composition, glucose metabolism, or insulin sensitivity compared with a “Cr-suffi cient” diet; however, pharmacological 176 Vincent quantities of Cr had a concentration-dependent effect on lowering insulin levels in glucose tolerance tests, indicating that Cr may have a pharmacological effect increasing insulin sensitivity in healthy rats [21 ]. In summary, a complete paradigm shift has occurred in the fi eld of Cr nutrition, where for four decades, Cr had been considered to have only a nutritional, not a pharmacological effect. Now Cr is realized to have a pharmacological effect rather than a nutritional one. Nutritional studies cannot be used to determine whether Cr is an essential element. Studies using as little Cr as possible in the diet have failed to establish any signs of Cr defi ciency. Without conclusive positive evidence, Cr cannot be considered an essential trace element. A demonstration that Cr could potentially be an essential element will probably require the isolation of a biomolecule that is essential to some critical biological process and requires Cr to perform its essential function. As described below (Section 3.3 ), this has not occurred.

2.2.2 Absorption and Transport

Cr is absorbed by passive diffusion when intaken orally. This has been convincingly demonstrated by a double perfusion technique using segments of the small intestine of rats; these studies revealed that over a 100-fold range of [Cr3 O(propionate)6 + (H2 O)3 ] (Cr3) concentrations (10–1000 ppb), chromium absorption was a nonsatu- rable process [22 ]. Additionally, studies following the fate of orally administered 51 Cr have not observed a change in % Cr absorption over a range of intakes [23 , 24 ].

Most recently, rats gavaged with a dose of CrCl 3 absorbed approximately 0.2% of the Cr over a 2000-fold range of doses (0.01–20 mg Cr) [ 24 ]. Another interesting conclusion that can be drawn from the intestinal perfusate studies is that Cr appears to be actively transported out of the intestinal cells, as approximately 94% of the Cr entering the cells was cleared from the cells (leaving only approximately 6% behind to be stored). However, no transporter is known for Cr3+ . This suggests the possibility that Cr3+ may be bound to some chelating ligand and actively transported in this form; this is an area requiring further research. Changes in diet could affect the amount of Cr absorption and potentially affect the mechanism, although changes in mechanism have not been demonstrated. For example, the presence of added amino acids, phytate (high levels), ascorbic acid, and oxalate, but not low levels of phytate, in the diet reportedly altered the extent of Cr uptake (reviewed in [25 ]), although the changes (while statistically signifi cant in some cases) were relatively small in a small percentage of absorption. Once in the bloodstream, Cr 3+ binds almost exclusively to the Fe-transport protein transferrin. The association of transferrin and Cr has been reviewed previously [ 26 ]. Cr-loaded transferrin has been demonstrated to transport Cr in vivo [ 27 ,28 ]. Injection of 51Cr-transferrin into rats resulted in incorporation of 51 Cr into tissues. The transport of Fe into tissue by endocytosis of transferrin has been found to be insulin sensitive, as the transport of Cr; injection of labeled transferrin and insulin resulted in a several fold increase in urinary Cr [28 ]. Thus, transferrin, in an insulin- dependent fashion, can transfer Cr to tissues from which it is excreted 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 177 in the urine. The binding of Cr to transferrin is quite tight, although the apparent binding constants for the two metal binding sites differ by approximately 105 [ 29 ]; the in vitro binding of Cr3+ from inorganic salts has been shown to be quite slow [ 29 ], although these studies were performed in the presence of ambient bicarbonate concentrations. This also suggests that Cr may be carried to transferrin as a chelate complex. However, recent studies in the author’s laboratory reveal that at the bicarbonate concentration of human blood (~20 mM) the binding of Cr3+ is quite rapid (B. Liu, G. Deng, K. Wu, and J. B. Vincent, unpublished results). Once Cr is brought into the cell by endocytosis, it must leave the endosome to enter the cell cytosol. As Cr3+ is not readily reduced by any biological reducing agents, so that it can be transported by divalent metal ion transporters (in a fashion similar to Fe), it must be transported by another mechanism; this is another area requiring further research [30 ]. A human study of chromium absorption as a function of Cr intake has often been cited as evidence of an essential role for Cr; however, this single study requires reproduction. Anderson and Koslovsky have reported an inverse relationship between dietary chromium intake and degree of absorption observed in human studies [ 31 ]. The data suggest that absorption of Cr varies approximately from 0.5 to 2.0% for Cr intakes of ~15–50 μg per day. This diffi cult to perform study is far from defi nitive; for example, a distinct difference is found if the data are separated into male and female subjects. For males, no statistical variation occurs for chromium absorption as a function of intake, while an apparent inverse trend is observed for the female subjects. However, these data are in striking contrast to this same lab’s studies reported two years earlier [32 ]. Chromium absorption was determined to be ~0.4% for free-living individuals; when Cr intake was increased by over fourfold, urinary chromium excretion increased over fourfold while maintaining ~0.4% absorption of chromium for both males and females. The difference between the two studies lies in the range of Cr intakes of ~15–50 μg per day for the former and ~60–260 μg per day for the latter, suggesting that an inverse relationship between Cr intake and absorption, if it exists, exists only at the lowest portion of the range of intakes. The former study requires a careful examination in terms of statistical analysis and propagation of error, in addition to reproduction, before this study can be used as evidence for an essential role for Cr in humans (or female humans). Cr concentrations in the human urine and blood serum are proportional to Cr intake [32 , 33], while human urine Cr concentrations do not correlate with serum glucose, insulin, or lipid parameters or with age or body mass [32 ]. Additionally, in rats, Cr concentrations in the liver and kidney correlate with Cr intake [34 ]. Urinary Cr loss is increased in type 2 diabetic subjects [35 , 36], raising the question of whether the increased Cr loss could result in a conditional Cr defi ciency; however, studies with model diabetic rats (alloxan-treated rats [37 ] and Zucker diabetic fatty rats [ 38]) have shown that the increases in urinary Cr excretion are the result of increases in Cr absorption (perhaps simply as a result of increased water consumption). Thus, urinary Cr loss is controlled by absorption of Cr, and Cr apparently is not a conditionally essential element. 178 Vincent

An increase in urinary Cr excretion has been reported for human subjects on self-selected diets in response to a glucose challenge, while no effect was observed for individuals taking a Cr supplement (200 μg Cr as CrCl3 for 3 months) [33 ]. Urinary Cr loss after a glucose challenge was found not to be predictable and suggested to not refl ect Cr status [33 ]. Yet, the extent of movement of chromium to the urine in response to a glucose challenge did change, from an increase at normal Cr intake to no increase when supplemented with Cr (the inverse of the expected observation). Also in this study, the Cr intake of the individuals in the study was not established. The results from humans on self-selected diets are consistent with studies of urinary Cr loss in subjects on diets supplemented with a variety of varying carbohydrates [39 ]. The greater the increase in the amount of insulin in the blood in response to the various carbohydrates, the more Cr was lost in the urine [ 39 ]. Thus, Cr appears to be mobilized in response to insulin, rather than directly to glucose or other carbohydrates. A range of responses to the carbohydrates was noted. Some of the subjects who in response to the diets had the highest circulating blood insulin levels had decreased abilities to mobilize Cr for excretion in the urine (within 90 min); thus, a group of subjects with decreased carbohydrate tolerance appeared to have decreased urinary Cr loss [ 39]. The Cr content of the self-selected diets of individuals in the study was not determined, and the subjects do not appear to have been questioned about whether they were consuming any Cr-containing supplements [39 , 40 ]. Urinary Cr excretion after a glucose tolerance test does not differ between control men or hyperinsulinemic men or differ between men on diets with differing high amylase cornstarch contents [41 ]. Eight of 10 healthy individuals have been found to have increased urinary Cr loss (ng Cr/min) for 4 hours after an oral glucose tolerance test compared to the 4 hours before the test such that the mean Cr loss was signifi cantly greater after the test than before, while no mean effect was observed for 13 diabetic subjects [42 ]. Finally, Morris and coworkers conducting hyperinsulinemic euglycemic clamp studies have shown that increases in blood insulin levels, not specifi cally blood glucose levels, are responsible for a decrease in plasma Cr and an accompanying increase in urinary Cr loss [43 ], consistent with their earlier studies demonstrating increased urinary Cr loss after an oral glucose challenge [44 ]. Thus, humans appear to increase urinary Cr loss in response to an increase in blood insulin concentrations (whether from a carbohydrate or insulin challenge) although the magnitude of the change appears to be quite variable, including some individuals who may not respond potentially as a result of decreased glucose tolerance. This increase apparently results from the increased movement of Cr bound to transferrin as noted above. Rats have been conclusively shown to increase Cr excretion in response to an insulin or glucose challenge [27 , 28 , 45]. If Cr were essential and had a role under physiological conditions in insulin sensitivity, this increase in urinary Cr loss in response to insulin could potentially serve as a biomarker for Cr. However, studies on rats on the purifi ed diets containing as low as possible to very high Cr contents (described in ref. [21 ]) show that the increase in rate of urinary Cr loss does not correlate with Cr intake, even at the lowest Cr content [46 ]. At the highest Cr intake and thus highest background rate urinary Cr loss, insulin did not stimulate an 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 179 increase in rate of Cr loss [ 46 ]. These results are very similar to those described above in humans [33 ]; thus, insulin-stimulated Cr loss is not a biomarker for Cr status, and the movement of Cr in response to insulin does not provide evidence for its being essential. One cannot help but notice that Cr appears to be set up in terms of transport to play a role in glucose metabolism. In the bloodstream, Cr binds tightly to one site of transferrin. While transferrin is kept only 30% saturated with iron and has similar binding constants for both Fe3+ binding sites, Cr3+ binds more rapidly and more tightly to the site that Fe3+ binds to more slowly; thus, transferrins appear primed to carry Cr in addition to Fe. As transferrin movement is insulin-sensitive, Cr bound to transferrin is delivered to tissues in an insulin-sensitive fashion; this transport of Cr is primarily to the skeletal muscle [ 27 , 28], where most glucose is metabolized in response to insulin. Cr is then rapidly removed from these tissues.

2.2.3 Total Parenteral Nutrition

Starting in the late 1970s, studies of patients on total parenteral nutrition (TPN) have been used to support the proposal that chromium is an essential element [47 – 50 ]. This stems from patients on TPN who developed impaired glucose utilization or glucose intolerance and neuropathy or encephalopathy [ 47 , 48 ,51 – 55 ]. The symptoms were reversed by chromium infusion and not by other treatments, including insulin administration alone. While limited to less than ten individual cases, these studies have been interpreted as providing evidence of clinical symptoms associated with chromium defi ciency that can be reversed by supplementation. Another patient on TPN who developed symptoms of adult-onset diabetes and hyperlipidemia but died had low tissue chromium levels [56 ]. Additionally, the effects of chromium supplementation on fi ve patients on TPN requiring a substantial amount of exogenous insulin have been examined. Three subjects displayed no benefi cial response while two showed a possible benefi cial response to chromium supplementation [ 57]. Subjects received TPN containing 10 μg Cr/day followed by supplementation with an additional 40 μg Cr/day for 3 days and then restoration of the normal TPN. Curiously the development of symptoms that were reversible by chromium supplementation does not correlate with serum chromium levels [49 ], indicating that either serum chromium levels are not an indicator of chromium defi ciency or that another factor is in operation. Additionally, these incidences of diagnosed potential chromium defi ciency have been questioned recently as they lack consistent relationships between the chromium in the TPN, time on TPN before symptoms appear, serum chromium levels and symptoms [58 ]. The most notable features of these studies are the quantities of Cr administered. In the cases where apparent defi ciencies were reported, the TPN solutions provided 2–240 μg Cr/day. For comparison, all the Cr in the TPN is introduced into the bloodstream, while only 0.5% of Cr in the regular diet is absorbed into the bloodstream. Thus, 30 μg of Cr in a typical daily diet presents only ~0.15 μg Cr to the bloodstream. 180 Vincent

The TPN solutions are consequently providing 13–67 times the required amount of chromium; thus, based on these data, the TPN solutions cannot be considered Cr-deﬁ cient . Subjects were, in turn, treated with 40–250 μg Cr/day added to the TPN solution to alleviate their conditions, clearly pharmacological doses, as the largest dose provided 1.7×103 times more chromium than a standard diet. Consequently, the results with the insulin-resistant TPN patients can only be considered as providing evidence for a pharmacological role of chromium. The data are not relevant for examining whether chromium is an essential element. Not surprisingly, as TPN provides ten or more micrograms of chromium per day, TPN patients are accumulating chromium in their tissues [59 , 60 ]. Calls are appear- ing for the re-examination of the chromium levels in TPN solutions in terms of a need to reduce recommended levels [61 ]. In summary, evidence to designate chromium an essential element does not exist. While the possibility always exists that evidence could surface in the future to support a biological role for chromium, such assumptions cannot be taken into current considerations. The next review of the status of chromium by the Committee on the Scientiﬁ c Evaluation of Dietary Reference Intakes of the National Academies of Science (USA) must seriously consider revising its status.

3 Is Chromium Pharmacologically Relevant?

3.1 Rodent Disease Model Studies

Several rat models of type 2 diabetes have been utilized to examine the effects of Cr(III) administration [5 ]. Three models have symptoms arising from mutations of the leptin receptor: the JCR:LA-cp, Zucker obese and Zucker diabetic fatty rats. Leptin is a hormone produced by adipocytes that signals the brain that the appetite should be suppressed. Consequently, as the leptin signaling system is blocked at the receptor, the JCR:LA-cp and Zucker obese rats become markedly obese and insulin- resistant and possess somewhat elevated blood glucose levels and elevated levels of blood insulin, triglycerides, and cholesterols. The Zucker diabetic fatty (ZDF) rats have an additional, uncharacterized mutation that results in these rats developing symptoms very comparable to type 2 diabetes in humans, including elevated blood glucose levels, in addition to the high triglycerides and cholesterol levels. In contrast to the obese models, the ZDF rats have smaller body masses than healthy Zucker rats. Some general statements for studies of Cr(III) complex administration using these three models can be made. When Cr is administered at a young age, it has no effect on body mass and food intake [ 62 – 69]. Cr administration generally appears to have no effect on fasting blood glucose levels but to lower glycated hemoglobin levels. (This might be explained by the data of Vincent and coworkers [66 ], which show that while glucose levels tend to be lower in Cr-treated animals at several instances during the administration period, that this effect is not signifi cant; however, glycated hemoglobin levels, which serve as a window to the average 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 181 exposure of red blood cells to glucose over 60–90 days, refl ect a benefi cial effect on blood glucose over this time.) Cr appears generally to be benefi cial to lipid metabolism, lowering total cholesterol levels; however, effects on other lipid variables are inconsistent. Thus, in these rat models of diabetes and obesity-related insulin resistance, Cr appears to have benefi cial effects on insulin resistance, marginally benefi cial effects on blood glucose, and benefi cial effects on the grossly elevated plasma lipid levels. Unfortunately, only a tiny percentage of human type 2 diabetes cases are the result of mutations in leptin or its receptor. The Goto–Kakizaki rat is a non-obese model of type 2 diabetes; the origins of the diabetes at a molecular level are not known. Two studies have examined the effects of [Cr(pic)3 ] (1–100 mg/kg daily) for either 4 or 32 weeks [70 , 71 ]. Unfortunately the reports do not indicate whether the dose is of Cr as the compound or of the compound (in which case ~12.5% of the dose would be Cr). No effects were observed on body mass, fasting blood glucose or insulin levels, or glucose or insulin areas under the curve in a glucose tolerance test. For this model, Cr(III) appears to have no appreciable effect. The chemical streptozotocin when administered intravenously or intraperitoneally relatively selectively kills the beta cells of the pancreas, destroying nearly all the body’s ability to produce insulin. Thus, rats treated with the chemical serve as an excellent model of type 1 diabetes (not type 2 diabetes). To generate a better model for type 2 diabetes studies, the addition of a high-fat diet has been utilized in addition to the chemical treatments or streptozotocin has been given to newborn rats, rather than adults. Four studies have examined the effects of Cr supplementation on these type 2 models where they found lower fasting glucose, total cholesterol, and triglycerides concentrations [72 – 75]. Studies using just streptozotocin have given inconsistent results but are also very different in design from one another making interpretation diffi cult [ 5 ]. In summary, the results of the studies with rats undergoing modifi ed streptozotocin treatments (lower fasting glucose but not insulin and effects on lipids) are different from those of the Goto–Kazizaki rats (no effects) that are in turn different from the results from the leptin-receptor mutation models (lower fasting insulin but not glucose and effects on lipids). No great dependence appears on dose (when the doses are supranutritional), length of time of Cr administration or form of Cr. The origin of the diabetes appears to make a signifi cant difference on the potential benefi ts of Cr administration. Mouse models of diabetes with mutations to the genes for leptin, the ob/ob mouse, and leptin receptor, the db/db mouse, have also been studied in terms of effects of Cr(III) administration. Both these models display obvious obesity. Unfortunately, not all the studies have used well-defi ned forms of Cr. The results of these studies have been confl icting in terms of fasting blood glucose and cholesterol concentrations, although glucose and insulin levels in glucose tolerance tests consistently tend to be lower [76 – 84 ] (reviewed in [5 ]). Thus, with one exception, Cr(III) treatment of rat and mouse models of type 2 diabetes have had benefi cial effects, although the effects differ from one model to the other. These differences in the models may be signifi cant to the results observed in human clinical trials. 182 Vincent

3.2 Clinical Studies

While human studies of the effects of chromium supplementation have failed to observe effects in healthy subjects, clinical trials of the effects of chromium supplements on type 2 diabetic subjects have failed to generate consistent results. A recent review that included only studies that were placebo-controlled and used a chemically well-defi ned form of Cr identifi ed 19 studies that met the criteria [ 5 ]. (Not including well-defi ned studies eliminates Cr sources such as “Cr-enhanced yeast”). Nine of the 19 reports reported no effects from supplementation; another may or may not have seen signifi cant changes depending on how the statistical analysis is performed. Studies using 150–1000 μg Cr daily for 6 weeks to 16 months have reported no effects from Cr, while studies using 200–1000 μg for 10 days to 6 months reported benefi cial effects. Studies using over 100 subjects, that should have more power to distinguish potential differences, reported no effects in one case and benefi cial effects from supplementation in the others. Several studies are quite small, lacking the statistical power to potentially observe effects. Similarly, no pattern was identifi ed in terms of benefi cial effects on particular symptoms from Cr supplementation. Fourteen studies examined fasting blood glucose levels. Five reported that levels dropped with supplementation while nine observed no effect. Four studies observed no effect on fasting insulin levels while levels were lower in three studies. Triglyceride levels were unaffected in four studies and lower in two studies. Glycated hemoglobin levels were reported to be lower in four studies, but no change was reported in fi ve studies. Effects on cholesterol levels were slightly more consistent. Seven studies reported no lowering of total cholesterol while three noted decreases. For HDL, six studies reported no effect, while a single study reported an increase in levels; for LDL, fi ve studies reported no effects, while only a single study reported a decrease. In response to some type of a glucose challenge, four studies observed no effects on glucose levels while three saw positive effects; in terms of insulin response, one study had mixed results depending on the time interval that Cr was administered, while another reported positive effects. Behavior of the blood variables across the studies was simply found to be too inconsistent to draw any fi rm conclusions. This inconsistent behavior existed whether these studies are broken down by the compound used, the amount of Cr, the number of subjects, or the length of the study [5 ]. Two thorough meta-analyses of the effects of Cr supplements on type 2 diabetic subjects have been reported. Althius et al. [85 ] in 2002 performed a meta-analysis on studies under a contract from the Offi ce of Dietary Supplements of the National Institutes of Health (USA). Using their criterion for inclusion (trials containing a Cr treatment group and a control), the authors identifi ed only four studies of subjects with type 2 diabetes for analysis. The combined data from the studies, except those from a study by Anderson and coworkers [86 ], showed no effect from chromium on glucose or insulin concentrations. Thus, they concluded that the data on diabetics were inconclusive. The authors also examined the effects of Cr supplements on healthy subjects or subjects with impaired glucose tolerance (but not type 2 diabetes) 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 183 in 14 trials including 425 subjects; no associations between Cr administration and glucose or insulin concentrations were found. Another meta-analysis was reported by Balk et al. in 2007 [87 ], the most thorough meta-analysis on Cr supplementation in terms of blood variables reported to that date. Forty-one randomized controlled trials were identifi ed that examined the effects of chromium supplementation on glucose metabolism and lipids concentrations in ≥10 non-pregnant adults (i.e., healthy and diabetic subjects) for ≥3 weeks. However, almost half were determined to be of poor quality. Nine studies were funded by the food or supplement industry, 18 were by non-industry sources, and 14 did not indicate the funding source. Ten studies used Brewer’s yeast, 15 studies used

CrCl3 , 5 studies used Cr nicotinate and 15 studies used [Cr(pic)3 ]; some studies compared multiple sources of Cr. No benefi t from Cr supplementation was identifi ed for healthy individuals [87 ]. Eighteen studies were identifi ed that examined type 2 diabetic subjects. Cr supplementation was found to statistically improve glycemic control in type 2 diabetics. The effects were fairly small but signifi cant overall. When broken down by Cr source, the effects were small but signifi cant for subjects on yeast and [Cr(pic)3 ] but not CrCl 3 . Most signifi cantly, the authors determined the results were not defi nitive because of the poor quality and heterogeneity of the studies. Overall Cr did not affect lipid levels, while [Cr(pic)3 ] lowered glycated hemoglobin levels. However, lower glycated hemoglobin levels were only observed in 3 interventions out of 14, two of which came from a single, large study (that of Anderson and coworkers [86 ]). Amongst fasting glucose studies, a trend was observed that industry-sponsored studies were more likely to observe benefi cial effects. The authors also expressed concerns that the Brewer’s yeast results suggested that another component in the yeast may be having an effect because effects were observed at lower doses of Cr. As a bottom line the authors concluded that Cr supplementation ‘may have a modest effect’ on glucose metabolism in type 2 diabetics but that ‘the large heterogeneity and the overall poor quality limit the strength of our conclusions’ and that more randomized trials are required [ 87]. The study was supported by a contract from the Agency for Healthcare Research and Quality (US Department of Health and Human Services). Three studies meeting the appropriate criteria have appeared since the Balk et al. meta-analysis. These are a small study by Lai [88 ] with Cr yeast with a 10 subject treatment and 10 subject control that observed small effects on plasma glucose, insulin, and glycated hemoglobin; a study with Cr yeast utilizing 57 subjects by Kleefstra et al. [89 ] that observed no effects; and a study by Cefalu et al . [ 90 ] with

93 subjects that observed no effects with 1000 μg Cr daily as [Cr(pic)3 ]. These studies, because of the participant size of the last two, would have signifi cantly affected the results of the meta-analysis if they could have been included, making any effect of Cr on fasting glucose in type 2 diabetics even more questionable. One must also note that any meta-analysis is likely to be biased toward the positive as studies with negative results tend to be published less frequently than positive reports. Basically, the results come down to the following: (i) clinical trials on Cr(III) complex supplementation for healthy subjects observe no effects from treatment, (ii) clinical studies on Cr(III) complex supplementation are equivocal for type 2 diabetic 184 Vincent subjects, and (iii) the results of the trials with diabetic subjects are basically only considered equivocal, rather than without observable effect, because of the results of the single large, well-designed study by Anderson and coworkers [86 ]. This study is unique in being the only study using subjects from China and needs to be independently repeated. In a review in 1998, Anderson [91 ] split studies on Cr supplementation of type 2 diabetics into two groups: subjects receiving ≤200 μg Cr daily and subjects receiving >200 μg Cr daily. Using all the studies identifi ed with diabetic subjects to that date, Anderson suggested that >200 μg Cr were required for diabetic subjects to generate an observable effect. The effect appeared to be largest for [Cr(pic)3 ] where this apparent effect was the result of only the single study by Anderson and coworkers [86 ]). Subsequently, this requirement has commonly been cited. However, studies since 1998 have failed to follow the trend identifi ed by Anderson. Cefalu and coworkers [90 , 92] in a preliminary and then in a subsequent report potentially may have found a relationship that might explain the different results between populations in the various studies. In a double-blind, placebo-controlled study, 93 subjects with a fasting plasma glucose level of at least 6.94 mmol L–1 received 1000 μg Cr daily as [Cr(pic)3 ] or placebo for 24 weeks [90 ]. Comparison of the treatment and control groups found no effects on body mass, percentage body fat, free fat mass, or abdominal fat deposits, fasting glucose, glycated hemoglobin, or insulin sensitivity. Yet, effects were observed when the Cr-receiving subjects at the end of the study were divided into responders (≥10% increase in insulin sensitivity from baseline) and non-responders. At baseline, responders had lower insulin sensitivity and higher fasting glucose and glycated hemoglobin levels than non- responders. Thus, Cefalu and coworkers might potentially have identifi ed predictors for type 2 diabetic subjects that might preferentially respond to Cr treatment. These results will need to be carefully tested in additional studies where the ‘responder’ group is identifi ed before the Cr administration to establish whether a subsequent difference is actually manifested. According to the American Diabetes Association in its 2010 Clinical Practices Recommendations, ‘Benefi t from chromium supplementation in people with diabetes or obesity has not been conclusively demonstrated and therefore cannot be recommended’ [93 ]. The American Diabetes Association dropped any mention of chromium in its 2011, 2012, and 2013 recommendations. In December 2003, Nutrition 21, the major supplier of chromium picolinate, petitioned the United States Food and Drug Administration (FDA) for eight qualifi ed health claims: 1. Chromium picolinate may reduce the risk of insulin resistance. 2. Chromium picolinate may reduce the risk of cardiovascular disease when caused by insulin resistance. 3. Chromium picolinate may reduce abnormally elevated blood sugar levels. 4. Chromium picolinate may reduce the risk of cardiovascular disease when caused by abnormally elevated blood sugar levels. 5. Chromium picolinate may reduce the risk of type 2 diabetes. 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 185

6. Chromium picolinate may reduce the risk of cardiovascular disease when caused by type 2 diabetes. 7. Chromium picolinate may reduce the risk of retinopathy when caused by abnormally high blood sugar levels. 8. Chromium picolinate may reduce the risk of kidney disease when caused by abnormally high blood sugar levels [94 ] . After extensive review, the FDA issued a letter of enforcement discretion allowing only one (No. 5) qualifi ed health claim for the labeling of dietary supplements [94 , 95 ]: ‘One small study suggests that chromium picolinate may reduce the risk of type 2 diabetes. FDA concludes that the existence of such a relationship between chromium picolinate and either insulin resistance or type 2 diabetes is highly uncertain.’ The small study was performed by Cefalu et al. [96 ]. This study was a placebo- controlled, double-blind trial examining 1000 μg/day of Cr as [Cr(pic) 3 ] on 29 obese subjects with a family history of type 2 diabetes; while no effects of the supplement were found on body mass or body fat composition or distribution, a signifi cant increase in insulin sensitivity was observed after four and eight months of supplementation. This raises the question of why the discrepancy between human and rodent studies exists. Rodent studies observing benefi cial effects generally provided rats between 80 and 1000 μg Cr/kg body mass daily. Based on mass, this would correspond to 5.2 to 65 mg Cr daily for an average 65 kg human. Even when corrected for the increased metabolic rate of rats compared to humans, this range corresponds to ~1 to 13 mg of Cr daily. Thus, human clinical trials may have only started to approach the dose necessary to see a benefi cial effect in humans. The amount of Cr used in clinical trials needs to be increased before ruling out that Cr has no effect on type 2 diabetic subjects. However, one cannot rule out that something is unique about rodents that allows Cr to have benefi cial effects. Unfortunately, studies of Cr supplementation on farm animals are also equivocal and often use doses in proportion to body mass even smaller than those used in human clinical trials [5 , 97 ]. Recently, Vincent [5 ] has proposed that in order to defi nitely determine whether Cr supplementation has an effect on diabetics, human clinical trials should: (1) be performed with suffi cient power to be able to realistically observe effects, on subjects whose baseline characteristics are well established, and for periods of time of at least 4–6 months. Knowing baseline characteristics is particularly important, given the possibility at the current dosages that only subjects with the highest degrees of insulin resistance may be responsive to Cr. (2) be performed with larger doses of Cr(III). Studies using JCR:LA-cp or ZDF rats utilized 80–1000 μg Cr/kg daily corresponding to approximately 5.2–65 mg daily for a human (based on body mass). If corrected for the increased metabolic rate of rats, this still correspond to ~1–13 mg daily. Studies are needed using 5–7 mg Cr(III) daily for 4–6 months or longer. (3) be carefully monitored for any deleterious effects, especially when using the higher doses of Cr(III). 186 Vincent

3.3 Proposed Mechanisms of Action

3.3.1 Insulin Signaling

When many bioinorganic chemists or nutritionists think of a biological form of chromium, glucose tolerance factor (or GTF) may be their fi rst thought. As has been reviewed many times recently [5 , 25 , 98], the studies postulating the existence of GTF are fl awed, and the material isolated from Brewer’s yeast and also called GTF is an artifact of its isolation. The term GTF should be removed from the lexicon of the chemistry and nutrition communities. What then can be said about the action of chromium at a molecular level? Given that Cr(III) appears to have pharmacological effects in increasing insulin sensitivity and altering lipid metabolism in rodent diabetes models, Cr must interact directly with some biomolecules(s) to generate these effects. To begin to elucidate how Cr can affect insulin sensitivity at a molecular level, the effects of Cr on cultured mammalian cells have been probed. However, research results are contradictory such that the state of the fi eld is not immediately clear (reviewed in [98 ]). Using the lesson learned from toxicology studies of [Cr(pic)3 ] (see Section 4.2 ), some of the discrepancies might be explained based on the stability of the Cr(III) complexes and what form of Cr(III) is actually being presented to the cells (and whether this form is biologically relevant); yet, this does not aid in elucidating the site of action of Cr. Most of these studies have used adipocytes (or preadipocytes) or skeletal muscles, cells known to incorporate Fe via endocytosis of transferrin. These cells should, thus, intake Cr via transferrin endocytosis. Given that Cr-loaded transferrin can be readily prepared, the physiologically relevant form of Cr, i.e., Cr transferrin, should be used in cell culture studies to deliver Cr to the cells. One result is nearly uniform across cell culture studies utilizing skeletal muscle, adipocytes, or adipocyte-like cells – Cr enhances glucose uptake and metabolism in a fashion dependent on insulin (see, for example, [99 ]). Numerous pathways by which a Cr biomolecule could manifest itself in these effects have been proposed. However, research results in in vitro and in vivo systems are contradictory, such that the state of the fi eld is not immediately clear (Table 1 ). Attention has been focused on two sites of action in the insulin signaling cascade as the potential sites of Cr action, insulin receptor (IR) and Akt. The most thorough studies observing increased IR signaling from Cr(III) treatment were reported by Brautigan and coworkers [100 ]. Preincubation of Chinese hamster ovary (CHO) cells overexpressing IR with [Cr(pic)3 ], Cr histidine (actually a complex mixture of numerous Cr-histidine complexes), or [Cr3 O(propionate) 6 + (H2 O)3 ] (Cr3) activated IR tyrosine kinase activity in the cells at low doses of insulin. While the concentration dependence was only examined for Cr histidine, the effect was concentration-dependent. Neither insulin binding to the cells nor IR number was affected. Additionally, the addition of Cr did not inhibit dephosphorylation of the IR by endogenous phosphatases or added PTP1B (phosphotyrosine phosphatase 1B). Also, Cr apparently did not alter redox regulation of PTP1B (i.e., by trapping 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 187

Table 1 Selected studies of effects of chromium administration on insulin signaling pathway. a Cell or organism Chromium compound Effect Refs.

Skeletal muscle CrCl3 , [Cr(pic)3 ], Up-regulation of insulin receptor [ 152 ] Cr peptide complexes mRNA levels

Insulin-resistant [Cr(pic)3 ] No effect on insulin receptor [ 120 ] 3T3-L1 and Akt mRNA levels adipocytes

Chinese hamster [Cr(pic)3 ], Cr3, Activated IR kinase activity [100 ] ovary cells Cr histidine

JCR:LA rat [Cr(pic)3 ] Increased insulin receptor, IRS-1, [ 63 ] and Akt phosphorylation and increased PI3K activity

3T3-L1 Cr(D-phe)3 Increased phosphorylation of Akt [ 153 ] adipocytes but not insulin receptor

3T3-L1 [Cr(pic)3 ] No effect on phosphorylation of [ 113 , 115 ] adipocytes insulin receptor, IRS-1, or Akt KK/HIJ mice Milk powder enriched Increased IRS-1 tyrosine phospho- [154 ] skeletal with trivalent Cr rylation, increased Akt activity, muscle and decreased IRS-1 serine-307 phosphorylation 3T3-L1 Cr histidine Increased insulin-stimulated glucose [ 101 ] adipocytes uptake and insulin-stimulated tyrosine phosphorylation of IR C2C12 skeletal Cr oligo- mannuronate Enhanced phosphorylation of IR, [ 155 ] muscle cells PI3K, and Akt and AMPK a Table adapted from [5 ].

the oxidized inactive form or by preventing its reduction and reactivation). CrCl3 and Cr histidine were found not to activate the kinase activity of a recombinant fragment of IR. The authors concluded that Cr inside the cell modiﬁ ed the receptor in some manner, activating its kinase activity [100 ]. Subsequently, Brautigan, et al. [101 ] demonstrated that Cr histidine stimulated tyrosine phosphorylation of IR in 3T3-L1 adipocytes in the presence of insulin but not of MAPK (mitogen-activated protein kinase) or 4E-BP1, markers for activation of transcription and translation, respectively, in the presence of insulin; glucose uptake in the presence of insulin was also stimulated by Cr histidine. The effects of Cr histidine were also examined in competition with those of vanadate [101 ]; the results were interpreted in terms of Cr having an action involving IR activation and potentially in another action beyond IR activation that increases GLUT4 transport.

Sreejayan and coworkers [81 ] using Cr(D-phenylalaninate)3 (Cr(D-phe)3 ) have generated evidence for an association between Cr and Akt. Cr(D-phe)3 (5 or 25 μM for 10 days) was found to increase insulin-stimulated glucose uptake by cultured mouse 3T3-adipocytes. Treatment of the cells with 5 μM Cr for 0.5 to 4 hours or 0.1 to 100 μM Cr for 2 hours did not increase insulin-stimulated phosphorylation of IR (Tyr1146) signiﬁ cantly, while under similar conditions insulin-stimulated Akt phosphorylation (Thr308) was increased signiﬁ cantly. 188 Vincent

To reconcile the heterogeneous results in the studies with cultured cells (Table 1 ), the complexes need to be studied under uniform conditions – the same cells treated in the same manner for the same period of time with the same Cr complex at the same concentrations. Additionally the Cr compounds need to be examined over a range of concentrations over varying periods of time with each of the cell types. The stability of the Cr complexes in the culture media needs to be established. Only in this manner will the actual Cr species in contact with the cells be established. Similarly, the distribution, concentration, and form of the Cr in the cells needs to be determined. Control experiments using just the ligands need to be performed to determine if any effects arise from just the ligands. Without this type of comprehensive treatment, progress in interpreting the body of cell culture experiments is going to be diffi cult if not impossible as has already been found in toxicology studies (see Section 4.2 ). Studies would probably be best performed if Cr-transferrin, the form of Cr by which the metal is delivered to cells, were utilized. One specifi c biomolecule has been proposed as the biologically active chromium- binding molecule. This is the only biomolecule other than transferrin known to bind Cr in vivo, low-molecular-weight Cr-binding substance (LMWCr or chromodulin). This molecule occurs in the tissue, the bloodstream, and the urine and appears to bind Cr in the tissues for its elimination from the body via the urine. The history of studies of this molecule has been exhaustively reviewed [5 , 102 ] and is beyond the coverage of this review. The inability of the organic portion of this Cr-peptide complex to be characterized generated signifi cant controversy, as the situation bore similarity to the previous inability to characterize the organic component of GTF [103 ]. Another important concern is that a Cr-loading procedure is necessary in the purifi cation of LMWCr, so that the peptide could be followed (by its Cr content) through the isolation procedure; thus, the animal providing the tissue or body fl uid is usually administered a Cr(III) or Cr(VI) source or such a source is added to the 2 − tissue homogenate or fl uid [5 , 102]. Rupture of CrO 4 -treated mammalian cultured cells resulted in Cr being bound to a low-molecular-weight species with spectroscopic properties similar to LMWCr [104 ]. This was interpreted in terms of LMWCr being an artifact generated during isolation; however, the unnatural method of pre- 2 − senting CrO4 in high concentration to cultured cells also suffers from the types of problems discussed above when using cultured cells. Thus, this study only shows that apoLMWCr can potentially bind Cr in a cell extract and potentially bind Cr tight enough to remove it from other biomolecules, consistent with the results of the isolation procedures of LMWCr described above. The Cr environment of LMWCr has been characterized by a variety of techniques including paramagnetic NMR, EPR, X-ray absorbance, and variable temperature magnetic susceptibility [105 , 106 ]. The peptide component has recently been sequenced by mass spectrometry [107 ]; the sequence begins with four glutamate residues whose cyclizing blocked attempts at Edman degradation sequencing. The peptide binds four chromic ions with identical binding constants and cooperativity as apoLMWCr (within experimental error) [107 ]. LMWCr has been found to stimulate insulin-dependent glucose incorporation and metabolism in isolated rat adipocytes [ 99 , 104] and in vitro to stimulate 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 189

(or perhaps retard the deactivation of) the kinase activity of the insulin-activated insulin receptor [108 , 109 ]. A mechanism for LMWCr in amplifying insulin signaling has been proposed [110 , 111]. This proposal was put forward when Cr was thought to be essential; the mechanism needs to be altered, so that it would be in vogue under conditions of Cr supplementation, so that abnormally high concentrations of holoLMWCr are generated. In this mechanism, apoLMWCr is stored in insulin-sensitive cells. Responses to increases in blood insulin concentrations result in activation of the insulin- signaling cascade: insulin binds to its receptor bringing about a conformational change that results in the autophosphorylation of tyrosine residues on the internal side of the receptor, transforming the receptor into an active tyrosine kinase and transmitting the signal from insulin into the cell. In response to this signaling, transferrin moves from the bloodstream into cells, carrying in part Cr3þ into the cells. The Cr fl ux results in loading of LMWCr with Cr. The holoLMWCr then binds to the insulin receptor, presumably assisting to maintain the receptor in its active conformation and amplifying insulin signaling. This mechanism requires demonstration that it can (or cannot be) active in vivo to verify (or refute); clear demonstration that the IR is directly involved in increasing insulin sensitivity by Cr would support this mechanism. As Cr is probably not an essential element, LMWCr could be part of a Cr detoxifi cation system as suggested by Yamamoto, Wada, and Ono [ 112 ]; Cr supplementation, which leads to increased Cr concentrations in the body, could lead to increased concentrations of holoLMWCr, capable in turn of affecting insulin signaling. Studies need to determine the origin of LMWCr, i.e., what protein is it made from and what enzymes are involved? Is the holoLMWCr biologically active at physiological levels (suggesting a potential biological role for Cr) or is it signifi cantly active only when Cr concentrations are high? Does LMWCr interact with the IR in vivo , or does it manifest its effects elsewhere?

3.3.2 Cholesterol and Fatty Acid Metabolism

Elmendorf and coworkers have examined the effects of CrCl3 and [Cr(pic)3 ] on 3T3- L1 adipocytes [113 – 117 ] (however see [118 , 119 ]). In their ﬁ rst report [113 ], CrCl3 and [Cr(pic)3 ] were shown to increase GLUT4 transport to the plasma membrane in the presence of insulin. Cr treatment did not affect IR, insulin receptor substrate-1 (IRS-1), PI3K, or Akt regulation but decreased plasma membrane cholesterol.

Subsequently, the effects of [Cr(pic)3 ] were shown to be dependent on the glucose concentration of the media with the effects being observed at 25 mM, but not 5.5 mM [114 ]. [Cr(pic)3 ] activated AMPK (AMP-activated protein kinase) and improved defects in cholesterol transporter ABCA1 trafﬁ cking and cholesterol accrual in the high glucose treated cells [117 ].These researchers have postulated that Cr manifested its effects via affecting the cholesterol homeostasis and the membrane ﬂ uidity

[ 113 –117 ]. Yao and coworkers [ 120 ,121 ] determined that [Cr(pic) 3 ] increased glucose uptake and metabolism and GLUT4 transport in 3T3-L1 adipocytes; the effects 190 Vincent were independent of insulin. Cr (60 nM) had no effect on IR or Akt phosphorylation but was found to activate MAPK independent of its effect on GLUT4 translocation. They also looked at the effects of Cr at both 25 and 5.5 mM glucose in their studies described above; similar results were observed at both glucose concentrations in contrast to Elmendorf and coworkers.

The use of exclusively [Cr(pic)3 ] in some of the studies examining membrane properties generates some questions that may be related to differing results between cell studies. While not particularly lipophilic, despite being neutral in charge [122 ], the compound still appears to be able to partition to a signiﬁ cant degree to cell membranes. This membrane incorporation of [Cr(pic)3 ] results, for example, in increased membrane permeability [123 ]. Thus, some of the observations related to cholesterol homeostasis may be speciﬁ cally related to the use of [Cr(pic)3 ], its lipophilicity, and its stability in cell culture media. Notable in this regard is a recent report showing that [Cr(pic)3 ] associates with the lipid interface in reverse micelle model membranes and that a similar association could explain the increased association of the insulin receptor, phosphorylated IRS-1, and phosphorylated Akt in detergent- resistant membrane microdomains [124 ].

3.3.3 Inﬂ ammation and Oxidative Stress

Jain and Kannan have shown that monocytes exposed to high glucose concentrations have lower levels of the cytokine TNF-α (tumor necrosis factor-α) in the presence of 100 μM CrCl3 for 24 hours at 37°C [125 ]. Treatment with CrCl3 also inhibited stimulation of TNF-α secretion in these cells by 50 μM H2 O2 . Lipid peroxidation and protein oxidation in the presence of H2 O2 was also inhibited by CrCl3 . As increased TNF-α secretion may be associated with insulin resistance, Jain has proposed in an interview that increased insulin sensitivity arising from Cr administration may be mediated by lowering of TNF-α levels [126 ]. In a follow-up study,

CrCl3 in combination with estrogen lowered lipid peroxidation in high glucose- treated monocytes [127 ]. The combination was also found to decrease interleukin-6 (IL-6) secretion. Cr was proposed to potentiate the effects of estrogen [127 ]. Curiously, another group has shown that Cr(III) treatment (350–500 ppm) results in increased TNF-α production by macrophages (in the absence of high glucose concentrations) [128 ]. This activation by chromium (CrCl 3 ) may be regulated by tyrosine kinases [129 ]. The results in the presence of high glucose could also point to an association between reactive oxygen species and chromium, but these studies must be considered extremely preliminary. Additionally, the fate of Cr in these cell culture studies needs to be examined. Subsequent studies in Zucker diabetic fatty [65 ] and streptozotocin-induced diabetic rats [130 ] have found that Cr(III) administration can lower blood levels of TNF-α, IL-6, and C-reactive protein, although differences appeared to be observed depending on choice of Cr(III) complex administered. Cr(III) administration has also been reported to lower blood levels of TNF-α in a clinical trial of type 2 diabetic subjects, although again differences appeared to be observed depending on choice of Cr(III) complex administered [131 ]. 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 191

4 Is Chromium Toxic?

4.1 Chromate

Lay and coworkers [132 , 133 ] have proposed that chromate generated enzymatically (i.e., from hydrogen peroxide or other species generated by enzymes) from Cr(III) in the body could act as a phosphotyrosine phosphatase (PTP) inhibitor, in a similar manner to vanadate, and that the site of action of Cr is at the PTPs. The proposal that chromate could be involved in chromium action in vivo is based on the ability of hydrogen peroxide to oxidize Cr(III) compounds to chromate, suggesting the apparent beneﬁ cial effects of Cr actually stem from side effects of its toxicity [133 ]. To demonstrate this, Lay and coworkers exposed chromium picolinate, CrCl3 and the basic chromium carboxylate cation Cr3 to 0.10–1 mM hydrogen peroxide for 1–6 h in 0.10 M HEPES buffer at pH 7.4. This resulted in the formation of chromate in efﬁ ciencies of from 1% ([Cr(pic)3 ] for 6 h with 1 mM H2 O2 ) to 33% (the cation for 6 h with 1 mM H 2 O2 ). The cation could also be oxidized with hypochloride or glucose oxidase or xanthine oxidase (enzymes that produce H2 O2 ). However, when one considers the amount of Cr humans consume from their diet and from nutritional supplements and the low % absorption and that cell concentrations of peroxide are 10 –7 to 10–8 M while numerous reductants (such as ~5 mM ascorbate) are present, the probability that cell concentrations of chromate could even approach the K i of chromate for phosphatases is negligibly small [5 ]. Similarly, toxicity from chromate at these concentrations is unlikely. Given the enormous doses of Cr(III) complexes shown to have no detrimental effects (see Section 4.2 ), this proposed mechanism of toxicity from chromate generated from Cr(III) sources can be ignored.

4.2 Chromium Picolinate and Other Cr(III) Complexes

The potential toxicity of Cr picolinate, [Cr(pic)3 ], the most popular form of Cr supplement over the last two decades, has been an area of intense debate, but consensus has probably recently been reached (for recent reviews see [5 , 134 , 135 ]). In mammalian cell culture studies and mammalian studies in which the complex is given intravenously [5 , 134 ], [Cr(pic)3 ] is clearly toxic and mutagenic, unlike other commercial forms of Cr(III) supplements. The ﬁ rst study to raise concerns about potential toxic effects, by Stearns and coworkers [136 ], demonstrated, using CHO cells, that [Cr(pic)3 ] as a solid suspension in acetone or the mother liquor from the synthesis of [Cr(pic)3 ] (before the compound precipitates from solution) caused chromosomal aberrations. Subsequent studies have shown that the complex gives rise to a variety of types of oxidative damage and is clastogenic [ 137 – 143]. This led, for example, in fruit ﬂ ies (Drosophila ) to dominant female sterility, appreciable delays in development of larvae and adults, and lower success rates in pupation and eclosion; the Cr dosage in these studies was approximately equivalent to a 192 Vincent human consuming one 200 μg Cr-containing supplement a day [144 ]. The ability of

[Cr(pic)3 ] to generate chromosomal aberrations in polytene chromosomes of the salivary glands of Drosophila larvae was also examined; in the [Cr(pic)3 ]-treated group, 53% of the identifi ed chromosomal arms were positively identifi ed as containing one or more aberrations, while no aberrations were observed for the identifi ed chromosomal arms of the control group [ 145]. No effects on Drosophila were observed for other Cr(III) compounds examined [144 ,145 ]. However, when given orally to mammals, [Cr(pic)3 ] does not appear to be toxic nor appear to be a mutagen or carcinogen. An NIH-commissioned study of the effects of up to 5% of the diet (by mass) of male and female rats and mice for up to 2 years found no harmful effects on female rats or mice or male mice and at most ambiguous data for one type of carcinogenicity in male rats (along with no changes in body mass in either sex of rats or mice)

[ 146]. Despite numerous claims that [Cr(pic) 3 ] is absorbed better than inorganic forms of Cr used to model dietary Cr, CrCl3 , Cr nicotinate (the second most popular form of Cr sold as a nutritional supplement), and [Cr(pic) 3 ] are absorbed to a similar degree in rats [24 , 147 ,148 ]. Only 1% of absorbed Cr from the supplement is found in the bloodstream as [Cr(pic)3 ], suggesting that little of the intact molecule is absorbed [149 ]. When ingested, the complex probably hydrolyzes near the stomach lining, releasing the Cr, which is subsequently absorbed. The picolinate ligands also alter the redox properties of the Cr center such that it is more susceptible to undergoing redox chemistry in the body than hexaaqua Cr(III) [150 ,151 ]. The hydrolysis of the complex is probably fortuitous, releasing the Cr before the intact [Cr(pic) 3 ] complex can be absorbed to an appreciable level and potentially enter into redox chemistry, in contrast to the cell studies where the very stable, neutral complex could be absorbed intact. The message of these confl icting results is that applying solutions of Cr(III) compounds to cultured cells in general does not present Cr(III) to the cells in a comparable fashion to that in which Cr(III) is presented to cells in the body; the difference may be crucial to the results and interpretation of the study. In summary, Cr(III) supplementation appears to be safe at levels currently used in nutritional supplements and in pharmacology studies, in line with assessments by the Food and Drug Administration (USA) and European Food Safety Authority. However, as no benefi t has been demonstrated for Cr supplementation of healthy individuals, any potential risk from supplementation would appear to outweigh potential benefi ts at the current time.

5 Concluding Remarks and Future Direction

At present Cr cannot be considered as an essential element as (i) nutritional data demonstrating Cr defi ciency and improvement in symptoms from Cr supplementation are lacking and (ii) no biomolecules have convincingly been demonstrated to bind Cr and have an essential function in the body. No benefi cial effects have 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 193 convincingly been demonstrated from Cr supplementation by healthy humans. Cr(III) supplementation appears to be safe at levels currently used in nutritional supplements and in pharmacology studies. While studies with rodent models repro- ducibly demonstrate benefi cial effects from Cr supplementation at pharmacological doses, the scientifi c literature for clinical trials in diabetic humans lacks consistent and reproducible outcomes. Future clinical studies need to be more carefully designed including the utilization of an appropriate number of subjects and appropriate amount of administered Cr, the use of well characterized Cr(III) compounds, and the examination of whether particular subgroups of type 2 diabetic subjects are likely to benefi t from chromium supplementation. Further studies are required to investigate the mechanism and mode of action of Cr(III) at the molecular level in enhancing insulin sensitivity and potentially improving cholesterol metabolism.

Abbreviations and Deﬁ nitions

AI adequate intake AMPK AMP-activated protein kinase CHO Chinese hamster ovary + Cr3 [Cr3 O(propionate)6 (H2 O)3 ] Cr(D-phe)3 Cr(D-phenylalaninate)3 [Cr(pic)3 ] chromium picolinate 4E-BP1 4E-binding protein-1 ESADDI estimated safe and adequate daily dietary intake FDA Food and Drug Administration FEEDAP Panel on Additives and Products or Substances Used in Animal Feed FTC Federal Trade Commission GLUT4 glucose transporter type 4 GTF glucose tolerance factor HDL high density lipoprotein HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IL-6 interleukin-6 IR insulin receptor IRS-1 insulin receptor substrate-1 LDL low density lipoprotein LMWCr low-molecular-weight chromium-binding substance MAPK mitogen-activated protein kinase PI3K phosphatidylinositol 3-kinase PTP phosphotyrosine phosphatase PTP1B phosphotyrosine phosphatase 1B TNF-α tumor necrosis factor-α TPN total parenteral nutrition ZDF Zucker diabetic fatty (rats) 194 Vincent

Acknowledgment The author wishes to thank the USDA for supporting his recent research on the nutritional biochemistry of chromium (National Research Initiative Grant 2009-35200- 05200 from the USDA Cooperative State, Research, Educational, and Extension Service).

References

1. R. A. Anderson, J. Am. Coll. Nutr. 1998 , 17 , 548–555. 2. R. A. Anderson, Regul. Toxicol. Pharmacol. 1997 , 26 , S35–S41. 3. R. A. Anderson, Biol. Trace Elem. Res. 1992 , 32 , 19–24. 4. Federal Trade Commission, Docket No. C-3758 Decision and Order, 1997, http://www.ftc. gov/os/1997/07/nutritid.pdf (accessed 14 December 2012). 5. J. B. Vincent, The Bioinorganic Chemistry of Chromium, John Wiley and Sons, Chichester, UK, 2013. 6. National Research Council, Recommended Dietary Allowances, 9th Ed. Report of the Committee on Dietary Allowances, Division of Biological Sciences, Assembly of Life Science, Food and Nutrition Board, Commission on Life Science, National Research Council , National Academy Press, Washington, D.C., 1980. 7. National Research Council, Recommended Dietary Allowances, 10th Ed. Subcommittee on the tenth edition of the RDAs, Food and Nutrition Board, Commission on Life Sciences, National Research Council , National Academy Press, Washington, D.C., 1989. 8. National Research Council, Dietary Reference Intakes for Vitamin A, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. A Report of the Panel of Micronutrients, the Subcommittee on Upper Levels of Nutrients and Interpretations and Uses of Dietary Reference Intakes, and the Standing Committee on the Scientifi c Evaluation of Dietary Reference Intakes , National Academies of Sciences, Washington, D.C., 2002. 9. Panel on Additives and Products or Substances Used in Animal Feed, EFSA J. 2009 , 1043 , 1–69. 10. W. Mertz, K. Schwarz, Arch. Biochem. Biophys. 1955 , 58 , 504–506. 11. K. Schwarz, W. Mertz, Arch. Biochem. Biophys. 1957 , 72 , 515–518. 12. K. Schwarz, W. Mertz, Arch. Biochem. Biophys. 1959 , 85 , 292–295. 13. P. R. Shepherd, C. Elwood, P. D. Buckley, L. F. Blackwell, Biol. Trace Elem. Res. 1992 , 32 , 109–113. 14. R. A. Anderson, J. H. Brantner, M. M. Polansky, J. Agric. Food Chem. 1978 , 26 , 1219–1221. 15. R. A. Anderson, A. S. Kozlovsky, Am. J. Clin. Nutr. 1985 , 41 , 117–121. 16. R. A. Anderson, N. A. Bryden, M. M. Polansky, J. Am. Diet. Assoc. 1993 , 93 , 462–464. 17. J. S. Stiffl er, J. S. Law, M. M. Polansky, S. J. Bhathena, R. A. Anderson, Metabolism 1995 , 44 , 1314–1320. 18. J. S. Striffl er, M. M. Polansky, R. A. Anderson, Metabolism 1998 , 47 , 396–400. 19. J. S. Striffl er, M. M. Polansky, R. A. Anderson, Metabolism 1999 , 48 , 1063–1068. 20. U. S. Environmental Protection Agency, Recommendations for and Documentation of Biological Values for Use in Risk Assessment, U. S. Environmental Protection Agency, Washington, D.C., EPA/600/6–87/008 (NTIS PB88179874), 1988. 21. K. R. Di Bona, S. Love, N. R. Rhodes, D. McAdory, S. H. Sinha, N. Kern, J. Kent, J. Strickland, A. Wilson, J. Beaird, J. Ramage, J. F. Rasco, J. B. Vincent, J. Biol. Inorg. Chem. 2011, 16 , 381–390. 22. H. J. Dowling, E. G. Offenbacher, F. X. Pi-Sunyer, J. Nutr. 1989 , 119 , 1138–1145. 23. W. Mertz, E. E. Roginski, R. C. Reba, Am. J. Physiol. 1965 , 209 , 489–494. 24. K. Kottwitz, N. Lachinsky, R. Fischer, P. Nielsen, Biometals 2009 , 22 , 289–295. 25. J. B. Vincent, Polyhedron 2001 , 20 , 1–26. 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 195

26. J. B. Vincent, M. J. Hatﬁ eld, Nutr. Abstr. Rev., Ser. A 2004 , 74 , 15N–23N. 27. B. J. Clodfelder, J. B. Vincent, J. Biol. Inorg. Chem. 2005 , 10 , 383–393. 28. B. J. Clodfelder, R. G. Upchurch, J. B. Vincent, J. Inorg. Biochem. 2004 , 98 , 522–533. 29. Y. Sun, J. Ramirez, S. A. Woski, J. B. Vincent, J. Biol. Inorg. Chem. 2000 , 5 , 129–136. 30. N. R. Rhodes, P. A. LeBlanc, J. F. Rasco, J. B. Vincent, Biol. Trace Elem. Res. 2012 , 148 , 409–414. 31. R. A. Anderson, A. S. Kozlovsky, J. Clin. Nutr. 1985 , 41 , 1177–1183. 32. R. A. Anderson, M. M. Polansky, N. A. Bryden, K. Y. Patterson, C. Veillon, W. H. Glinsmann, J. Nutr. 1983 , 113 , 276–281. 33. R. A. Anderson, M. M. Polansky, N. A. Bryden, E. E. Roginski, K. Y. Patterson, C. Veillon, W. Glinsmann, Am. J. Clin. Nutr. 1982 , 36 , 1184–1193. 34. R. A. Anderson, N. A. Bryden, M. M. Polansky, J. Am. Coll. Nutr. 1997 , 16 , 1184–1193. 35. B. W. Morris, S. MacNeil, C. A. Hardisty, S. Heller, C. Burgin, T. A. Gray, J. Trace Elem., Med. Biol. 1999 , 13 , 57–61. 36. B. W. Morris, G. J. Kemp, C. A. Hardisty, Clin. Chem. 1985 , 31 , 334–335. 37. W. Feng, W. Ding, Q. Qian, Z. Chai, Biol. Trace Elem. Res. 1998 , 63 , 129–138. 38. N. R. Rhodes, D. McAdory, S. Love, K. R. Di Bona, Y. Chen, K. Ansorge, J. Hira, N. Kern, J. Kent, P. Lara, J. F. Rasco, J. B. Vincent, J. Inorg. Biochem. 2010, 104, 790–797. 39. R. A. Anderson, N. A. Bryden, M. M. Polansky, S. Reiser, Am. J. Clin. Nutr. 1990 , 51 , 864–868. 40. S. Reiser, A. S. Powell, C.-Y. Yang, J. J. Canary, Am. J. Clin. Nutr. 1987 , 45 , 580–587. 41. K. M. Behall, J. C. Howe, R. A. Anderson, J. Nutr. 2002 , 132 , 1886–1891. 42. C. T. Gurson, G. Saner G, Am. J. Clin. Nutr. 1978 , 31 , 1158–1161. 43. B. W. Morris, S. Mac Neil, K. Stanley, T. T. A. Gray, R. Fraser, J. Endocrin. 1993 , 39 , 339–345. 44. B. W. Morris, A. Blumsohn, S. Neil, T. A. Gray, Am. J. Clin. Nutr. 1992 , 55 , 989–991. 45. B. J. Clodfelder, J. Emamaullee, D. D. D. Hepburn, N. E. Chakov, H. S. Nettles, J. B. Vincent, J. Biol. Inorg. Chem. 2001 , 6 , 608–617. 46. S. T. Love, K. R. D Bona, S. H. Halda, D. McAdory, B. R. Skinner, J. F. Rasco, J. B. Vincent, Biol. Trace Elem. Res. 2013 , in press. 47. K. N. Jeejeebhoy, R. C. Chu, E. B. Marliss, G. R. Greenberg, A. Bryuce-Robertson, Am. J. Clin. Nutr. 1977 , 30 , 531–538. 48. H. Freund, S. Atamian, J. E. Fischer, JAMA 1979 , 241 , 496–498. 49. K. N. Jeejeebhoy, Nutr. Rev. 1999 , 57 , 329–335. 50. K. N. Jeejeebhoy, J. Trace Elem. Exp. Med. 1999 , 12 , 95–89. 51. R. B. Brown, S. Forloines-Lynn, R. E. Cross, W. D. Heizer, Dig. Dis. Sci. 1986 , 31 , 661–664. 52. A. H. Verhage, W. K. Cheong, K. N. Jeejeebhoy, J. Parent. Enteral Nutr. 1996 , 20 , 123–127. 53. M. Via, C. Scurlock, J. Raikhelkar, G. Di Louzzo, J. I. Mechanick, Nutr. Clin. Prac. 2008 , 23 , 325–328. 54. T. C. Drake, K. D. Rudser, E. R. Seaquist, A. Saeed, Endocrin. Pract. 2012 , 18 , 394–398. 55. O. J. Phung, R. A. Quercia, K. Keating, W. L. Baker, J. L. Bell, C. M. White, C. I. Coleman, Am. J. Health-Syst. Pharm. 2010 , 67 , 535–541. 56. R. E. Scully, E. J. Mark, W. F. Mcneely, B. U. McNeely, New England J. Med . 1990 , 322 , 829–841. 57. O. Wongseelashote, M. A. Daly, E. H. Frankel, Nutrition 2004 , 20 , 318–320. 58. D. M. Stearns, Biofactors 2000 , 11 , 149–162. 59. L. Howard, C. Ashley, D. Lyon, A. Shenkin, J. Parenter. Enteral Nutr. 2007 , 31 , 388–396. 60. A. Moukarzel, Gastroenterology 2009 , 137 (Suppl. 5), S18–S28. 61. I. F. Braiche, P. L. Carver, K. B. Welch, J. Parenter. Enteral Nutr. 2011 , 35 , 736–747. 62. S. D. Proctor, S. E. Kelly, K. L. Stanhope, P. J. Havel, J. C. Russell, Diabetes Obes. Metab. 2007 , 9 , 87–95. 63. Z. Q. Wang, X. H. Zhang, J. C. Russell, M. Hulver, W. T. Cefalu, J. Nutr. 2006 , 136 , 415–420. 196 Vincent

64. W. T. Cefalu, Z. Q. Wang, X. H. Zhang, J. C. Russell, J. Nutr. 2002 , 132 , 1107–1114. 65. S. K. Jain, J. L. Croad, T. Velusamy, J. L. Rains, R. Bull, Mol. Nutr. Food Res. 2010 , 54 , 1–10. 66. B. J. Clodfelder, B. M. Gullick, H. C. Lukaski, Y. Neggers, J. B. Vincent, J. Biol. Inor. Chem. 2005 , 10 , 119–130. 67. N. Talpur, B. W. Echard, T. Yasmin, D. Bagchi, H. G. Preuss, Mol. Cell. Biochem. 2003 , 252 , 369–377. 68. M. S. Mozaffari, R. Abdelsayed, J. Y. Liu, H. Wimborne, A. El-Remessy, A. El-Marakby, Nutr. Metab. 2009 , 6 , 51. 69. Y. Sun, B. J. Clodfelder, A. A. Shute, T. Irvin, J. B. Vincent, J. Biol. Inorg. Chem. 2002 , 7 , 852–862. 70. A. Abdourahman, J. G. Edwards, IUBMB Life 2008 , 60 , 541–548. 71. D. S. Kim, T. W. Kim, J. S. Kang, J. Trace Elem. Med. Biol. 2004 , 17 , 243–247. 72. A. Dogukan, N. Sahin, M. Tuzcu, V. Juturu, C. Orhan, M. Onderci, J. Komoroski, K. Sahin, Biol. Trace Elem. Res. 2009 , 131 , 124–132. 73. K. Sahin, M. Onderci, M. Tuzcu, B. Ustandag, G. Cikim, I. H. Ozercan, V. Sriramoju, V. Juturu, J. R. Komorowski, Metabolism 2007 , 56 , 1233–1240. 74. A. Shinde Urmila, G. Sharma, J. Xu Yan, S. Dhalla Naranjan, K. Goyal Ramesh, Exp. Clin. Endocrinol. Diabetes 2004 , 112 , 248–252. 75. U. A. Shinde, R. K. Goyal, J. Cell. Mol. Med. 2003 , 7 , 322–329. 76. F. R. Flatt, L. Juntti-Breggren, P. O. Breggren, B. J. Gould, S. K. Swanston-Flatt, Diabetes Res. 1989 , 10 , 147–151. 77. R. W. Tuman, R. J. Doisy, Diabetes 1977 , 26 , 820–826. 78. G. L. Mannering, J. A. Shoeman, D. W. Shoeman, Biochem. Biophys. Res. Commun. 1994 , 200 , 1455–1462. 79. C. Rink, S. Roy, S. Khanna, T. Rink, D. Bagchi, C. K. Sen, Physiol. Genomics 2007 , 27 , 370–379. 80. C. D. Seaborn, B. J. Stoecker, J. Nutr. 1989 , 119 , 1444–1451. 81. X. Yang, K. Palanichamy, A. C. Ontko, M. N. Rao, C. X. Fang, J. Ren, N. Sreejayan, FEBS Lett. 2005 , 28 , 1458–1464. 82. X. Yang, S. Y. Li, F. Dong, J. Ren, N. Sreejayan, J. Inorg. Biochem. 2006 , 100 , 1187–1193. 83. F. Dong, X. Yang, N. Sreejayan, J. Ren, Obesity 2007 , 15 , 2699–2711. 84. N. Sreejayan, F. Dong, M. R. Kandadi, X. Yang, J. Ren, Obesity 2008 , 16 , 1331–1337. 85. M. D. Althius, N. E. Jordan, E. A. Ludington, J. T. Wittes, Am. J. Clin. Nutr. 2002 , 76 , 148–155. 86. R. A. Anderson, N. Cheng, N. A. Bryden, M. M. Polansky, N. Cheng, J. Chi, J. Feng, Diabetes 1997 , 46 , 1786–1791. 87. E. Balk, A. Tatsioni, A. Lichenstein, J. Lau, A. G. Pittas, Diabetes Care 2007 , 30 , 2154–2163. 88. Lai, M.-H. J. Clin. Biochem. Nutr. 2008 , 43 , 191–198 89. N. Kleefstra, R. O. B. Gans, S. T. Houweling, S. J. L. Bakker, S. Verhoeven, R. O. B. Gans, B. Meyboom-de Jng, H. J. G. Bilo, Diabetes Care 2007 , 30 , 1092–1096. 90. W. T. Cefalu, J. Rood, P. Pinsonat, J. Qin, O. Sereda, L. Levitan, R. A. Anderson, X. H. Zhang, X. H. Zhang, J. M. Martin, Z. Q. Wang, B. Newcomer, Metabolism 2010 , 59 , 755–762. 91. R. A. Anderson, J. Am. Coll. Nutr. 1988 , 17 , 548–555. 92. Z. Q. Wang, J. Qin, J. Martin, X. H. Zhang, O. Sereda, R. A. Anderson, P. Pinsonat, W. T. Cefalu, Metabolism , 2007 , 56 , 1652–1655. 93. American Diabetes Association, Diabetes Care 2010 , 23 (Suppl. 1), S11–S61. 94. Food and Drug Administration, Qualified Health Claims: Letter of Enforcement Discretion – Chromium Picolinate and Insulin Resistance (Docket No. 2004Q-0144), 2005. http://www.fda.gov/food/ingredientspackaginglabeling/labelingnutrition/ucm073017.htm (accessed 3 April 2010). 95. P. R. Trumbo, K. C. Elwood, Nutr. Rev ., 2006, 64 , 357–363 96. W. T. Cefalu, A. D. Bell-Farrow, J. Stegner, Z. Q. Wang, T. King, T. Morgan, J. G. Terry, J. Trace Elem. Exp. Med . 1999 , 12 , 71–83. 6 Is Chromium Essential, Pharmacologically Relevant or Toxic? 197

97. M. D. Lindemann, in The Nutritional Biochemistry of Chromium , Ed J. B. Vincent, Elsevier, Amsterdam, 2007, pp. 85–118. 98. J. B. Vincent, S. T. Love, Chem. Biodivers. 2012 , 9 , 1923–1941. 99. A. Yamamoto, O. Wada, H. Suzuki, J. Nutr. 1988 , 118 , 39–45. 100. H. Wang, A. Kruszewski, D. L. Brautigan, Biochemistry 2005 , 44 , 8167–8175. 101. D. L. Brautigan, A. Kruszewski, H. Wang, Biochem. Biophys. Res. Commun. 2006 , 347 , 769–773. 102. J. B. Vincent, R. Bennett, in The Nutritional Biochemistry of Chromium, Ed J. B. Vincent, Elsevier, Amsterdam, 2007, pp. 139–160. 103. A. Levina, R. Codd, C. T. Dillion, P. A. Lay, Prog. Inorg. Chem. 2003 , 51 , 145–644. 104. A. Levina, H. H. Harris, P. A. Lay, J. Am. Chem. Soc. 2007 , 129 , 1065–1075. 105. C. M. Davis, J. B. Vincent, Arch. Biochem. Biophys. 1997 , 339 , 335–343. 106. L. Jacquamet, Y. Sun, J. Hatﬁ eld, W. Gu, S. P. Cramer, M. W. Crowder, G. A. Lorigan, J. B. Vincent, J.-M. Latour, J. Am. Chem. Soc. 2003 , 125 , 774–780. 107. Y. Chen, H. M. Watson, J. Gao, S. H. Sinha, C. Cassady, J. B. Vincent, J. Nutr. 2011 , 141 , 1225–1232. 108. C. M. Davis, J. B. Vincent, Biochemistry 1997 , 36 , 4382–.4385. 109. C. M. Davis, A. C. Royer, J. B. Vincent, Inorg. Chem. 1997 , 36 , 5316–5320. 110. J. B. Vincent, Acc. Chem. Res. 2000 , 33 , 503–510. 111. J. B. Vincent, J. Am. Coll. Nutr. 1999 , 18 , 6–12. 112. A. Yamamoto, O. Wada, T. Ono, Eur. J. Biochem. 1987 , 165 , 627–631. 113. G. Chen, P. Liu, G. R. Pattar, L. Tackett, P. Bhonagiri, A. B. Strawbridge, J. S. Elmendorf, Mol. Endocrinol. 2006 , 20 , 857–870. 114. G. R. Pattar, L. Tackett, P. Liu, J. S. Elmendorf, Mutat. Res. 2006 , 610 , 93–100. 115. E. M. Horvath, L. Tackett, A. M. McCarthy, P. Raman, J. T. Brozincki, J. S. Elmendorf, Mol. Endocrinol. 2008 , 22 , 937–950. 116. E. M. Horvath, L. Tackett, J. S. Elmendorf, Biochem. Biophys. Res. Commun. 2008 , 372 , 639–643. 117. W. Sealls, B. A. Penque, J. S. Elmendorf, Arterioscler. Thromb. Vasc. Biol. 2011 , 31 , 1139–1140. 118. E. M. Horvath, L. Tackett, A. M. McCarthy, P. Raman, J. T. Brozincki, J. S. Elmendorf, Mol. Endrocrin . 2010 , 24 , 1308. 119. E. M. Horvath, L. Tackett, J. S. Elmendorf, Biochem. Biophys. Res. Commun. 2010 , 401 , 319. 120. Y. Wang, M. Yao, J. Nutr. Biochem. 2009 , 20 , 982–991. 121. Y.-Q. Wang, Y. Dong, M.-H. Yao, Clin. Exp. Pharmacol. Physiol. 2009 , 36 , 843–849. 122. K. F. Kingry, A. C. Royer, J. B. Vincent, J. Inorg. Biochem. 1998 , 72 , 79–88. 123. G.W. Evans, T. D. Bowman, J. Inorg. Biochem. 1992 , 46 , 243–250. 124. A. Al-Qatati, P. W. Winter, A. L. Wolf-Ringwall, P. B. Chatterjee, A. K. Van Orden, D. C. Crans, D. A. Roess, B. G. Barisas, Cell Biochem. Biophys. 2012 , 62 , 441–450. 125. S. K. Jain, K. Kannan, Biochem. Biophys. Res. Commun. 2001 , 289 , 687–691. 126. T. Kordella, Diabetes Forecast 2004 , 57 , 83–85. 127. S. K. Jain, K. Rogier, L. Prouty, S. K. Jain, Free Radic. Biol. Med. 2004 , 37 , 1730–1735. 128. I. Catelas, A. Petit, D. J. Zukor, J. Antoniou, O. L. Huk, Biomaterials 2002 , 24 , 383–391. 129. L. Luo, A. Petit, J. Antoniou, D. J. Zukor, O. L. Huk, R. C. W. Liu, F. M. Winnik, F. Mwale, Biomaterials 2006 , 27 3351–3360. 130. S. K. Jain, J. L. Rains, J. L. Croad, Free Radic. Biol. Med. 2007 , 43 , 1124–1131. 131. S. K. Jain, G. Kahlon, L. Morehead, R. Dhawan, B. Lieblong, T. Stapleton, G. Caldito, R. Hoeldtke, S. N. Levine, P. F. Bass, III, Mol. Nutr. Food Res. 2012 , 56 , 1333–1341. 132. I. Mulkani, A. Levina, P. A. Lay, Angew. Chem. Int. Ed. 2004 , 43 , 4504–4507. 133. A. Levina, P. A. Lay, Coord. Chem. Rev. 2005 , 249 , 281–298. 134. J. B. Vincent, Dalton Trans. 2010 , 39 , 3787–3794. 135. D. Stallings, J. B. Vincent, Topics Nutraceutical Res. 2006 , 4 , 89–112. 136. D. M. Stearns, J. P. Wise Sr., S. R. Patierno, K. E. Wetterhahn, FASEB J. 1995 , 9 , 1643–1648. 137. D. M. Stearns, S. M. Silveira, K. K. Wolf, A. M. Lake, Mutat. Res. 2002 , 513 , 135–142. 198 Vincent

138. K. R. Manygoats, M. Yazzie, D. M. Stearns, J. Biol. Inorg. Chem. 2002 , 7 , 791–798. 139. P. Whitaker, R. H. San, J. J. Clarke, H. E. Seifried, V. C. Dunkel, Food Chem. Toxicol. 2005 , 43 , 1619–1625. 140. D. Bagchi, M. Bagchi, J. Balmoori, X. Ye, S. J. Stohs, Res. Commun. Mol. Pathol. Pharmacol. 1997 , 97 , 335–346. 141. D. Bagchi, S. J. Stohs, B. W. Downs, M. Bagchi, H. G. Preuss, Toxicology 2002 , 180 , 5–22. 142. V. H. Coryell, D. M. Stearns, Mutat. Res. 2006 , 610 , 114–223. 143. M. Jana, A. Rajaram, R. Rajaram, Toxicol. Appl. Pharmacol. 2009 , 237 , 331–344. 144. D. D. D. Hepburn, J. Xiao, S. Bindom, J. B. Vincent, J. O'Donnell, Proc. Natl. Acad. Sci. USA 2003 , 100 , 3766–3771. 145. D. M. Stallings, D. D. D. Hepburn, M. Hannah, J. B. Vincent, J. O'Donnell, Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2006 , 610 , 101–113. 146. M. D. Stout, A. Nyska, B. J. Collins, K. L. Witt, G. E. Kissling, D. E. Malarkey, M. J. Hooth, Food Chem. Toxicol. 2009 , 47 , 729–733. 147. K. L. Olin, D. M. Stearns, W. H. Armstrong, C. L. Keen, Trace Elem. Electrolytes 1994 , 11 , 182–186. 148. R. A. Anderson, N. A. Bryden, M. M. Polansky, K. Gautschi, J. Trace Elem. Exp. Med. 1996 , 9 , 11–25. 149. Research Triangle Institute, Project Report, [ 14 C]Chromium Picolinate Monohydrate: Disposition and Metabolism in Rats and Mice, submitted to National Institutes of Environmental Health Sciences , 2002 . 150. J. K. Speetjens, R. A. Collins, J. B. Vincent, S. A. Woski, Chem. Res. Toxicol. 1999 , 12 , 483–487. 151. S. Chaudhary, J. Pinkston, M. M. Rabile, J. D. Van Horn, J. Inorg. Biochem. 2005, 99, 787–794. 152. W. Qiao, Z. Peng, Z. Wang, J. Wei, A. Zhou, Biol. Trace Elem. Res. 2009 , 131 , 133–142. 153. F. Dong, M. R. Kandadi, J. Ren, N. Sreejayan, J. Nutr. 2008 , 138 , 1846–1851. 154. W. Y. Chen, C. J. Chen, C. H. Liu, F. C. Mao, Diabetes Obes. Metab. 2009 , 11 , 293–303. 155. C. Hao, J. Hao, W. Wang, Z. Han, G. Li, L. Zhang, X. Zhao, G. Yu, PLoS One 2011 , 6 , e24598. Chapter 7 Manganese in Health and Disease

Daiana Silva Avila , Robson Luiz Puntel , and Michael Aschner

Contents ABSTRACT ...... 200 1 INTRODUCTION ...... 200 1.1 Manganese Essentiality ...... 201 1.2 Manganese Pharmacokinetics ...... 202 1.3 Manganese Biochemistry and Physiology ...... 204 2 MANGANESE TRANSPORT ...... 206 2.1 Manganese Uptake in Relation to Oxidative State ...... 207 2.2 Cellular Manganese Uptake ...... 208 2.3 Cellular Manganese Efﬂ ux ...... 209 3 MANGANISM. A NEURODEGENERATIVE DISEASE ...... 210 4 SYMPTOMS AND SENSITIVE POPULATIONS ...... 211 5 MANGANISM VERSUS PARKINSON’S DISEASE ...... 211 6 MANGANESE IN THE ETIOLOGY OF OTHER NEURODEGENERATIVE DISORDERS ...... 212 6.1 Manganese and Amyotrophic Lateral Sclerosis ...... 212 6.2 Manganese and Alzheimer’s Disease ...... 213 6.3 Manganese and Huntington’s Disease ...... 213 7 MOLECULAR MECHANISMS OF TOXICITY ...... 214 7.1 Dopamine Oxidation ...... 214 7.2 Mitochondrial Dysfunction ...... 215 7.3 Astrocytosis ...... 215 8 GENETIC SUSCEPTIBILITY ...... 216 9 TREATMENT ...... 217

D. S. Avila • R. L. Puntel Biochemistry Graduation Program , Universidade Federal do Pampa , Uruguaiana, Rio Grande do Sul , Brazil e-mail: [email protected]; [email protected] M. Aschner (*) Department of Pediatrics and Pharmacology , The Kennedy Center for Research on Human Development and The Molecular Toxicology Center , Nashville , TN 37232 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 199 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_7, © Springer Science+Business Media Dordrecht 2013 200 Avila, Puntel, and Aschner

10 GENERAL CONCLUSIONS ...... 218 ABBREVIATIONS ...... 219 ACKNOWLEDGMENTS ...... 220 REFERENCES ...... 220

Abstract Manganese is an important metal for human health, being absolutely necessary for development, metabolism, and the antioxidant system. Nevertheless, excessive exposure or intake may lead to a condition known as manganism, a neurodegenerative disorder that causes dopaminergic neuronal death and parkinsonian- like symptoms. Hence, Mn has a paradoxal effect in animals, a Janus-faced metal. Extensive work has been carried out to understand Mn-induced neurotoxicity and to ﬁ nd an effective treatment. This review focuses on the requirement for Mn in human health as well as the diseases associated with excessive exposure to this metal.

Keywords dopamine • essentiality • manganese • manganese enzymes • manganism • mitochondria • neurodegenerative diseases • parkinson’s disease- related genes • treatment

Please cite as: Met. Ions Life Sci. 13 (2013) 199–227

1 Introduction

Manganese, a group 7 metal in the periodic table, is the twelfth most abundant element in the earth’s crust. It exists in a number of chemical and physical forms in the atmosphere’s particulate matter and in water [ 1]. Mn does not occur naturally in a pure state, and is found as both inorganic and organic compounds, the inorganic form being the most common. Because the Mn outer electron shell can donate up to 7 electrons, it can occur in 11 different oxidation states, varying from −3 to +7 [2 ]. In living tissue, Mn has been found as Mn 2+ , Mn 3+ , and possibly as Mn 4+ , while Mn5+ , Mn 6+ , Mn 7+, and other complexes of Mn at lower oxidation states, are not observed in biological materials [3 , 4 ]. The versatile chemical properties of Mn have enabled its industrial usage in iron and steel production, manufacture of dry cell batteries, production of potassium permanganate and other chemicals, as oxidant in the production of hydroquinone, manufacture of glass and ceramics, textile bleaching, as an oxidizing agent for electrode coating in welding rods, adhesives, paint, matches and ﬁ reworks, and tanning of leather. Organic compounds of Mn are also present in fuel additive, methylcyclopentadienyl manganese tricarbonyl (MMT) as well as in several fungicides. Moreover, considering that Mn is a paramagnetic metal, namely that it has unpaired electrons in its outer d shell, it can also be detected with magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) [1 , 5]. These techniques allow for the tracking of Mn dynamics repeatedly in the same subject in vivo [1 , 6]. Mn can also interact with 7 Manganese in Health and Disease 201

fl uorophore fura-2, by quenching and increasing its fl uorescence, representing a new methodological approach for in vitro kinetic studies. Thus, given its ubiquitous nature and widespread use in both industrial and non-industrial processes, several health organizations have expressed concern about the potential health effects of occupational/environmental Mn exposure. Mn is an essential element for humans, animals, and plants; it is required for growth, development, and maintenance of health. Routes of Mn exposure are mainly through dietary intake, dermal absorption, and inhalation. Accordingly, the primary source of Mn intoxication in humans is due to occupational exposure as in miners, smelters, welders, and workers in dry-cell battery factories [ 7 – 10 ], in which signifi cant neurological dysfunction has been associated with Mn exposure [11 ,12 ]. Indeed, epidemiological studies of industrial workers have suggested a relationship between elevated environmental Mn exposure and an increased risk for parkinsonian disturbances [13 – 17], an association that has also been supported by numerous laboratory studies [18 – 24]. While the exact mechanisms underlying the neurotoxic effects of Mn remain unclear, these studies collectively suggest that elevated environmental exposures to Mn may be suffi cient to exacerbate the emergence of neurological diseases [23 , 24 ]. Thus, in the next sections of this chapter we will discuss some details concerning Mn in health and disease.

1.1 Manganese Essentiality

Mn is an essential nutrient necessary for a variety of metabolic functions including those involved in normal human development, activation of certain metalloenzymes, energy metabolism, immunological system function, nervous system function, reproductive hormone function, and in antioxidant enzymes that protect cells from damage due to free radicals [25 , 26]. Mn also plays an essential role in regulation of cellular energy, bone and connective tissue growth, and blood clotting. Mn is an important cofactor for a variety of enzymes, including those involved in neurotransmitter synthesis and metabolism [27 ]. Indeed, in the mammalian brain, small amounts of Mn are required for brain development, cellular homeostasis, and for the activity of multiple enzymes [28 – 30 ]. Additionally, Mn is believed to be involved in the stellate process production in astrocytes, as well as in the metabolism of brain glutamate to glutamine, a step carried out by glutamine synthetase (GS). Taking into account the variety of enzymatic processes which require Mn, an inadequate daily supply of the metal is associated with a variety of health repercussions, ranging from generalized growth impairment, birth defects, reduced fertility, and impaired bone formation, to altered metabolisms of lipids, proteins, and carbohydrates [31 , 32 ]. However, few occurrences of Mn defi ciencies have been reported in humans, with symptoms including dermatitis, slowed growth of hair and nails, decreased serum cholesterol levels, decreased levels of clotting proteins, increased serum calcium and phosphorus concentrations, and increased alkaline phosphatase activity [25 ,33 ,34 ]. In addition, several human diseases have been reported to be 202 Avila, Puntel, and Aschner associated to low blood Mn concentrations, including epilepsy, Mseleni disease, Down’s syndrome, osteoporosis, and Perthest disease [35 ], nevertheless, the role of Mn defi ciency in these diseases remains unclear. In general, highly severe defi ciencies in dietary Mn supply are necessary to observe clinical symptoms [36 , 37 ]. The U.S. Food and Drug Administration (U.S. FDA) suggests a Reference Daily Intake (RDI) for Mn at 2 mg/day for adults (Federal Register 2007, 72 FR 62149), although there is no consensus regarding the safe and adequate levels of this nutrient for various age groups. This recommended dosage is based upon the U.S. National Research Council’s (NRC), which established estimated safe and adequate dietary intake (ESADDI) of 2–5 mg/day for adults [ 38]. Additionally, it is known that Mn essentiality in humans varies depending of the life-stage and of the sex [39 ]. Accordingly, it is suggested by the National Academy of Sciences (NAAS) that an adequate intake of Mn is 2.3 mg/day for adult men and 1.8 mg/day for adult women [39 ,40 ]. The difference is accounted for by differential Mn absorption in men versus women [41 ]; it has been attributed to lower serum ferritin concentrations in men as compared to women [39 , 41 ]. Lactating or pregnant women are also thought to have increased Mn requirement [39 ]. Moreover, life-stages are also known to infl uence dietary Mn requirements. Accordingly, in newborns (less than six months of age) adequate Mn intake is defi ned as 3 μg/day; at seven to twelve months of age, adequate Mn intake increases to 600 μg/day [39 ]. In children one to three years of age, adequate Mn intake approximates 1.2 mg/day, and in children four to eight years of age, the adequate Mn intake increases to 1.5 mg/day.

1.2 Manganese Pharmacokinetics

Intricate regulation of Mn absorption and tissue specifi c accumulation is crucial for the proper regulation of the activity of Mn-dependent enzymes. Thus, understanding Mn’s essentiality and toxicity in the brain requires knowledge of its regulation in the periphery. Three major factors have been postulated to modulate plasma Mn levels. First, given that the main source of Mn is diet, tight regulation of gastrointestinal absorption of Mn is crucial. Second, following Mn absorption and a concomitant increase in plasma Mn levels, transport of Mn to target organs, including the liver, is necessary to prevent Mn-induced toxicity in the periphery. Finally, Mn must be eliminated from the plasma via shuttling to bile [42 ]. Thus, homeostatic controls tightly restrict Mn absorption and regulate Mn excretion to maintain stable tissue levels despite fl uctuations in daily Mn dietary exposure. However, exposure to high Mn concentrations, as might occur in occupational settings, may overwhelm homeostatic controls and results in elevations in tissue Mn concentrations. Accordingly, both pulmonary uptake and particulate transport via the olfactory bulb [ 42 , 43 ] can lead to deposition of Mn within the striatum and cerebellum and infl ammation of the nasal epithelium [44 ]. It is generally accepted that Fe has a strong infl uence on Mn homeostasis, since both metals share binding and uptake via the transferrin (Tf) transporter and the 7 Manganese in Health and Disease 203 divalent metal transporter-1 (DMT-1) (see more details in Section 2 of this chapter). It is known that Mn ions (Mn3+ ) bind at the same location as ferric ions (Fe3+ ) on the large glycoprotein molecule mucin, which is known to stabilize the ions, preventing precipitation in the lumen of the gastrointestinal tract [ 45]. Moreover, both metals are known to have an affi nity for the intercellular metal binding molecule mobilferrin [46 ]. Absorption of metal ions into enterocytes is known to take place via transmembrane transporters. Thus, during Fe defi ciency the number of transporters in enterocyte membranes is increased in order to maximize Fe absorption [47 ]. This will inevitably result in increased Mn absorption, particularly in the absence of Fe. Indeed, in rodent models, Fe defi ciency is associated with increased Mn absorption across the gastrointestinal tract, as well as increased Mn deposition in the brain [ 48 , 49 ]. Moreover, the absorption of Mn by the gastrointestinal tract is highly dependent upon the quantity of ingested Mn and net accumulated levels in the plasma. While Mn is transported by simple diffusion in the large intestine, Mn is absorbed by active transport in the small intestine [42 ]. In contrast, Mn excretion into bile is driven by concentration gradients leading to its fl ow from liver to bile [50 ]. About 3–5% of dietary Mn is absorbed in the gastrointestinal tract as Mn 2+ and Mn 4+ [29 ]. Mn2+ is oxidized to Mn3+ by liver and plasma ceruloplasmin and transported through the blood [51 , 52]. Mn tends to form tight complexes with other ligands [4 ]. Accordingly, a variety of plasma proteins or ligands have been implicated as specifi c Mn carrier proteins, including transglutaminase, beta-globulin, albumin, and Tf [ 53 , 54 ]. As a result, its free plasma and tissue concentrations tend to be extremely low [55 ]. Intracellular Mn2+ is sequestered in the mitochondria of the brain and liver via the Ca2+ uniporter [ 56 , 57 ]. Thus, mitochondria are the primary pool of intracellular Mn; however, nuclei have also been implied (remains debatable) to preferentially accumulate this metal [21 ,58 , 59 ]. In addition, it was recently shown that Mn2+ may induce fragmentation of the Golgi apparatus, indicating a specifi c role of this compartment in maintaining Mn homeostasis [60 ]. The Golgi harbors the Ca2+ /Mn2+ - ATPases of the secretory pathway (SPCAs) [61 ], which possesses a high-affi nity Mn2+ transport capacity [62 ]. This is also supported by in vivo studies reporting that brain areas with high SPCA expression also show enhanced Mn 2+ accumulation upon continuous systemic MnCl2 infusion in mice [63 ], and by the observation that a gain-of-function mutation in SPCA, which specifi cally enhances Golgi Mn2+ transport, improves survival of Mn 2+-exposed cells [64 ]. Thus, failure of effi cient Mn2+ detoxifi cation by saturating the SPCA-mediated removal via the Golgi may result in enhanced Mn2+ accumulation in the mitochondria, thereby causing mitochondrial impairment [60 ]. Mn enters the brain from the blood either across the cerebral capillaries and/or the cerebrospinal fl uid (CSF). At normal plasma concentrations, Mn appears to enter into the CNS primarily across the capillary endothelium, whereas at high plasma concentrations, transport across the choroid plexus appears to predominate [65 , 66 ], consistent with observations on the rapid appearance and persistent elevation 204 Avila, Puntel, and Aschner of Mn in this organ [67 ]. Indeed, radioactive Mn injected into the blood stream is concentrated in the choroid plexus within 1 hour after injection. Three days post- injection it is localized at the dentate gyrus and CA3 of the hippocampus [68 ]. The concentration of Mn in the brain varies across brain regions. The highest Mn levels are found in the globus pallidus in humans and in the hypothalamus in rats [28 ,69 –75 ]. Spectroscopy in rats has demonstrated that mitochondria in the basal ganglia accumulate the highest amount of Mn [76 , 77]. Differential metal transporter expression patterns and diffusion constants for Mn in various brain regions must explain, at least in part, the asymmetry in Mn accumulation across brain regions [ 78 ]. The preferential accumulation of Mn in basal ganglia is often associated with a clinical disorder referred to as manganism, which is characterized by a set of extrapyramidal symptoms resembling idiopathic Parkinson’s disease (IPD) (see more details in Section 3 of this chapter). However, further characterization of the absorption and elimination kinetics, as well as Mn uptake and export pathways is necessary to better understand the basis of differential Mn accumulation across different brain regions. The physiological half-life of Mn in the adult rat and primate brain is approximately 51 to 74 days [52 , 55 , 73 , 79 ]. The main excretion mechanism for Mn depends on normal liver function. Indeed, blood Mn concentrations are increased during the active phase of acute hepatitis as well as in post hepatic cirrhosis, and a signifi cant correlation exists between blood Mn and the activities of liver enzymes in patients with hepatitis and cirrhosis [80 , 81]. Corroborating these observations, MRI has consistently shown signal hyperintensity in the globus pallidus in cirrhotic patients [ 82 ]. Furthermore, direct measurements in pallidal samples obtained from the autopsies of cirrhotic patients revealed several-fold increases in Mn concentrations, and histopathologic evaluations showed Alzheimer’s type II astrocytosis [83 ]. The disorder is characterized as hepatic encephalopathy, and it is associated with cognitive, psychiatric, and motor impairments, all of which are known to be also associated with manganism [84 ]. From physiologically based pharmacokinetic models, it has been proposed that 54 Mn clearance from the body follows biphasic elimination, with a short “fast” elimination phase (with half-times of around a few days) followed by a longer “slow” elimination phase. This elimination behavior was consistently observed with all exposure routes. Thus, the availability of tracer studies for multiple exposure routes permitted a comparison of dose route differences in elimination. Accordingly, the faster clearance in monkeys and humans occurred from oral exposure, whereas the slowest clearance occurred following intravenous (iv ) administration [85 ].

1.3 Manganese Biochemistry and Physiology

As pointed out above, Mn is an essential nutrient necessary for a variety of functions. Accordingly, it acts as an activator of the gluconeogenic enzymes pyruvate carboxylase and isocitrate dehydrogenase, is involved in protecting mitochondrial membranes 7 Manganese in Health and Disease 205 through superoxide dismutase, and it activates glycosyl transferase, which is involved in mucopolysaccharide synthesis [86 ], just to name a few. Mn it is also a cofactor for many other enzymes, such as transferases, hydrolases, lyases, and integrins. Nevertheless, additional investigation is needed to identify the complete set of Mn-dependent enzymes in mammalian systems as for many of these activities, Mn is not the only metal that can act as a cofactor; iron, magnesium or copper are readily able to substitute it. In addition, the majority of Mn-dependent enzymes are found in bacteria and plants and only a few have been systematically studied in mammalians. Although Cu and Mg can substitute for Mn as a cofactor for some enzymes, there is a subset of enzymes with roles in neuron and/or glial that are strictly dependent upon the presence of Mn. These discrete Mn-binding proteins (manganoproteins) include glutamine synthetase, superoxide dismutase 2 (SOD2), arginase, pyruvate decarboxylase, and serine/threonine phosphatase [87 – 89 ]. Glutamine synthetase is the most abundant manganoprotein; it is predominantly expressed in astrocytes, where it converts glutamate to glutamine. Because GS contains four Mn ions per octamer [90 ], Mn has been proposed to regulate GS activity. In fact, insuffi cient Mn increases glutamate traffi cking, glutamatergic signaling, and excitotoxicity [91 ]. Furthermore, it has been proposed that the increased susceptibility to seizures observed in individuals with Mn defi ciency may be due to diminished GS levels and/or activity [92 ]. Arginase regulates elimination of ammonia from the body by converting L-arginine, synthesized from ammonia, to L-ornithine and urea as part of the urea cycle. Moreover, in the brain, L-arginine is converted to nitric oxide by neuronal nitric oxide synthetase. Proper regulation of arginase promotes neuronal survival by impairing nitric oxide signaling [93 , 94 ]. Pyruvate carboxylase is an essential enzyme required for glucose metabolism that interacts with Mn to generate oxaloacetate, a precursor of the tricarboxylic acid (TCA) cycle [95 ]. Interestingly, in the brain, pyruvate carboxylase is predominantly expressed in astrocytes [58 , 96]. Protein phosphatase-1 is essential for glycogen metabolism, cell progression, regulation synthesis, and release of neurotrophins, which promote neuronal survival and synaptic membrane receptors and channels [97 ]. Finally, SOD2 is a mitochondrial enzyme that detoxifi es superoxide anions through the formation of hydrogen peroxide. Although the concentration of Mn within neurons is low (<10 −5 M), their high mitochondrial energy demands is correlated with a propensity for increased SOD2 in neurons compared to glia [28 , 42 ]. Furthermore, loss of SOD2 activity increases the susceptibility to mitochondrial inhibitor induced toxicity and causes oxidative stress [98 ]. Thus, Mn defi ciencies, although not frequently reported in humans, may result in several biochemical and structural defects [35 , 99 , 100]. Accordingly, taking into account the Mn-dependent enzymatic processes, it is clear that inadequate daily supply of this metal may be associated with a variety of health repercussions [31 , 32 ]. In contrast, excessive levels of Mn can accumulate in the brain and lead to neurotoxicity. Because the area of the CNS comprising the basal ganglia is very complex and its function is dependent upon the precise function and balance of several 206 Avila, Puntel, and Aschner neurotransmitters, it is not surprising that symptoms of manganism often overlap with IPD. Indeed, evidence exists to support that persistent exposure to Mn may predispose an individual to acquire dystonic movements associated with PD (more details concerning Mn toxicity are provided in Section 3 of this chapter).

2 Manganese Transport

Considering the delicate relationship between essentiality and toxicity, Mn homeostasis is vital for the optimal functioning of any organism. Although some research has focused on mechanisms associated with the transport of Mn across the blood- brain barrier (BBB), the exact identity of the carrier(s) involved in Mn traffi cking into the brain remains somewhat controversial. Over the past decades, active transport [65 ] and facilitated diffusion [51 , 66] mechanisms have been described. More recently, Mn transport has been ascribed to high affi nity metal transporters of Ca and Fe. Several of these transporters include DMT-1, which belongs to the family of natural resistance-associated macrophage protein (NRAMP) [ 47 , 101]; ZIP8, a member of the solute carrier-39 [102 ]; Tf receptor (TfR) [51 , 103], which is known to be responsible for Fe3+ uptake; voltage-regulated [104 ] and store-operated Ca2+ channels [105 ]; and the ionotropic glutamate receptor Ca2+ channels [106 ]. It was also observed that distribution after intravenous injection of 54 Mn is readily transported into the brain as the free ion [ 66]. Moreover, studies have also demonstrated that a Mn-citrate complex could be transported either by a monocarboxylate transporter (MCT) [107 ], organic anion transporter polypeptide (OATP) or ATP-binding cassette (ABC) superfamilies [108 ]. Leak-pathways for Mn also likely exist, especially in areas lacking intact BBB, namely the circumventricular organs [109 ] (Figure 1 ). While the relative contribution of each of these transporters and/or mechanism(s) remains unknown, it is likely that optimal tissue Mn concentrations are maintained by the involvement of all of them. As pointed out above, in plasma more than 80% of Mn2+ is bound to 2+ α2 - macroglobulin and albumin. However, these complexes (Mn -albumin or α 2 -macroglobulin) may not take part in the transport across BBB (see Figure 1 ). TRPM7, which is ubiquitously expressed in vertebrates and functions as an active Ca2+ -selective transporter and a serine/threonine protein kinase, is a putative Mn transporter. The kinase activity is important for its metal transport function. Specifi cally, the transporter operates by regulating intracellular Ca 2+ levels and Mg 2+ homeostasis through the creation of an inward current, thus contributing to the establishment of a cellular membrane potential. Physiological levels of Mg 2+ and Ca2+ are necessary for maintaining the permeability of TRPM7 to Mn2+ , Co 2+ , and Ni2+ [ 110]. Finally, the homomeric purinoreceptors, including P2X and P2Y, have been invoked to transport Mn. These receptors are ATP-dependent and ubiquitously expressed on endothelial cells [111 – 113]. Nevertheless, purinoreceptors have a relatively lower affi nity for Mn than for the other divalent metals they transport (Ca > Mg > Ba > Mn) [110 ]. 7 Manganese in Health and Disease 207

Mn transport into CNS Blood –Brain Barrier

Blood compartment Brain Parenchyma

? ZIP8 /ZIP14 Mn2+ Tf Mn3+ Citrate Mn2+ Free Mn2+ Leak pathways DMT-1 Mn2+ Mn2+ -albumin

Physiological Mn plasma levels

Figure 1 Mechanism of Mn transport across the BBB under physiological Mn exposure levels. Transporters and relevant manganese oxidation states associated with Mn transport are demonstrated. Mn bound to albumin is excluded from passing the BBB given its size. Arrow size depicts the relative importance of each of the transporters in this process, bolder arrows representing more prominent transport mechanisms. Please refer to the discussion for additional details. Since it has yet to be determined whether ZIP8 functions to transport Mn across the BBB, the process has been annotated with a question mark.

Although non-protein-bound Mn enters the brain more rapidly than Tf-bound Mn [65 ,66 ], the question remains as to which form represents the predominant mechanism of transport in situ . Analyses of transport mechanisms based on tracer techniques employing bolus injections of Mn into the circulation cannot be easily interpreted. Thus, while tracer studies represent a sensitive technique for quantifying Mn transport, it must be noted that the information derived from such studies does not necessarily reﬂ ect the chemically active or functional forms in which Mn exists and is transported in vivo. This is due to the saturation of blood ligands for Mn and the likelihood that Mn in the free form exists at concentrations in excess to those expected under physiological conditions. Thus, transport kinetics under such conditions may not necessarily mimic physiological conditions. In the next subsections, we brieﬂ y discuss some aspects related to Mn transport in biological systems.

2.1 Manganese Uptake in Relation to Oxidative State

Mn can cross the BBB and blood-cerebrospinal ﬂ uid barrier (BCB) through several carriers (see Figure 1 ) and in different oxidation states [42 ]. Indeed, emerging reports have indicated that Mn2+ can be transported via DMT-1, the divalent metal/ 208 Avila, Puntel, and Aschner bicarbonate ion symporters ZIP8 and ZIP14, various calcium channels, the solute carrier-39 (SLC39) family of zinc transporters, park9/ATP13A2, and the Mg transporter hip14. Accordingly, DMT-1 belongs to a large family of metal transporters, which are responsible for the transport of divalent metals ions, including Mn, Fe, Cu, and Cd [114 ]. Thus, DMT-1 is involved in Mn accumulation in the brain. DMT-1 works as hydrogen ion symporter, transporting one hydrogen ion and one divalent cation in the same direction. This protein is responsible also for exporting the Mn2+ , which is released into the cytoplasm [115 ]. Alternatively, Mn 2+ ions may be directly transported from the blood stream by crossing the cellular membrane through voltage-regulated or glutamate-activated ionic channels, which are usually responsible for the transport of Ca2+ into the cell [116 ]. Finally, emerging experimental data has indicated that huntingtin-interacting protein 14 and 14L (Hip14, Hip14L) mediates transport of Mn2+ and other divalent metals (Mg2+ , Sr 2+ , Ni 2+ , Ca 2+ , Ba2+ , Zn 2+ ) across cell membranes [ 117 , 118]. Alternatively, Mn 3+ entry via the TfR, which mediates Fe 3+ uptake, is also considered [88 ].

2.2 Cellular Manganese Uptake

A critical regulator of brain Mn levels is the DMT-1 (also referred to as the DCT, or divalent cation transporter) which is known to shuttle both Mn2+ and Fe 2+ ions, as well as other divalent metals. This transporter belongs to the NRAMP gene family [47 , 101 ]. Disruption of the orthologous DMT-1 gene in the rat or mouse, results in signifi cantly lower tissue levels and uptake of Mn and Fe in the brain [ 119 –121 ]. Consistent with a role for DMT-1 in brain Mn uptake, nasal Mn absorption is also signifi cantly attenuated in b/b rats, and olfactory DMT-1 protein levels are signifi - cantly elevated in Fe-defi cient rats [122 ]. Notably, a recent study has shown that DMT-1 contributes to neurodegeneration in an experimental model of PD [123 ]. These authors observed increased expression of a specifi c DMT-1 isoform (DMT-1/Nramp2/Slc11a2) in the substantia nigra of PD patients. Moreover, the authors also showed that the administration of 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, a dopaminergic toxin used in experimental models of Parkinson’s disease) increased DMT-1 expression in the ventral mesencephalon of mice, which was concomitant with Fe accumulation, oxidative stress, and dopaminergic cell loss [123 ]. Additionally, DMT-1-mediated metal transport across rat brain endothelial cells in culture has been reported to be pH-, temperature-, and Fe-dependent [110 , 124 , 125 ]. The TfR is the major cellular receptor for Tf-bound Fe, but because Tf can also bind trivalent Mn, TfR can also mediate Mn3+ transport by endocytosis. Mn 3+ internalized through the endocytic pathway must be released from Tf and reduced to Mn 2+, which is transported through DMT-1 to the cytosol. The TfR is an active transporter that is pH- and Fe-dependent [110 ]. Both in vivo and in vitro studies have reported that Mn is effi ciently transported via the TfR. For example, a spontaneous mutation in a murine gene linked to the TfR, referred to as hypotransferrinemic, 7 Manganese in Health and Disease 209 results in a drastic serum TfR defi ciency, impairs Mn transport, and disrupts Fe deposition [126 , 127 ]. Additional cellular Mn transporters include the Mn-citrate transporters and the Mn-bicarbonate symporters. Indeed, a small fraction of Mn has been found in the plasma as citrate complex. Crossgrove and Yokel have demonstrated that a Mn-citrate tridentate complex with a non-coordinated central carboxylate moiety is a probable substrate for the anion transporter or a monocarboxylate transporter [ 107 ]. Moreover, the members of the organic anion transporter polypeptide or ATP- binding cassette superfamilies may transport Mn-citrate complexes [108 ]. The Mn-bicarbonate symporters, ZIP8 and ZIP14, have also been identifi ed as members of the solute carrier-39, and are expressed on brain capillaries [102 ], although it has yet to be determined whether these proteins are functional at the BBB [ 1 ,128 ]. − Nevertheless, these symporters utilize a HCO3 gradient as the driving force for Mn uptake across the plasma membrane. Gitler and colleagues have recently reported that the park9 gene responsible for early-onset Parkinsonism also transports Mn [118 ]. The park9 gene encodes a putative P-type transmembrane ATPase (ATP13A2) protein. Although the exact function of park9 is unknown, it is generally thought to be a shuttle for cations, including Mn across the cell membrane. Biochemical studies have demonstrated that the highest and lowest park9 mRNA levels are localized within the substantia nigra and cerebellum, respectively [129 ]. Mn transport via voltage-regulated channels [104 ], store-operated channels [105 ], ionotropic glutamate receptor channels [106 ] (all Ca2+ channels), and choline transporters [130 ] has also been described. Another possible mechanism for Mn transport involves dopamine transporters (DAT). It is believed that DAT facilitates Mn transport into dopaminergic striatal neurons and that Mn accumulates in the globus pallidus via axonal transport [ 131 , 132]. As a result, blockage of the DAT in the striatum would attenuate Mn accumulation in striatal neurons and would cause decreased Mn concentrations in the globus pallidus [132 ] (see Figure 2 ). Nevertheless, while the tissue specifi c expression of each of the aforementioned Mn transporters is yet to be determined, it is likely that optimal tissue Mn levels are maintained through the involvement of all the above and likely other unknown Mn transporters.

2.3 Cellular Manganese Efﬂ ux

Little information exists on the putative extracellular transport mechanisms of Mn. In the intestine ferroportin-1 (Fpn) is located at the basal membrane and exports Fe to the circulation [133 , 134]. Yin and colleagues [135 ] have implicated Fpn as a Mn exporter. Using an inducible HEK293T cell model, they showed that Fpn expression reduced Mn-induced toxicity and decreased Mn accumulation. In addition, Mn was also able to increase Fpn levels [ 136]. From these studies, it was concluded that Fpn participates in Mn efﬂ ux (see Figure 2 ). In accordance to previous data, it was recently shown that Mn is a substrate for Fpn, and that this export process is inhibited 210 Avila, Puntel, and Aschner

Mn2+

Mn2+ Mn2+ Mn2+

Mitochondria TfR Golgi Nucleus HIP Mn3+

DAT 14

Mn 4

2+ Mn Mn2+ Mitochondria Ca2+ channels

DMT-1 ZIP 4 / ZIP 8 Citrate Mn2+ Mn2+ Mn2+

2+ - Mn / HCO 3

Figure 2 Identifi ed and putative Mn transporters. These illustrated Mn transporters have been demonstrated to facilitate Mn traffi cking (uptake, storage, effl ux) between the extra- and intracellular milieu. Each of these transporter proteins has also been implicated in the transport of other metals. by a low extracellular pH and by incubation in a high K+ medium, indicating the involvement of transmembrane ion gradients in Fpn-mediated Mn transport [137 ]. Interestingly, Fpn is expressed in tissues involved in both Fe and Mn homeostasis, including the developing and mature reticuloendothelial system, the duodenum, liver, and the pregnant uterus [138 , 139]. Fpn has also been identifi ed in cells of the central nervous system including those of the BBB, choroid plexus, neurons, oligodendrocytes, astrocytes, and retina [ 133 , 140 ]. Nevertheless, it has yet to be determined whether Mn shares Fe exporters, such as Fpn, in this process. Moreover, the role of Fpn in Mn homeostasis remains to be elucidated in each of the neural cell types.

3 Manganism. A Neurodegenerative Disease

Manganism (also referred to as locura manganica) has been known for 150 years and it is caused by the preferential accumulation of Mn in brain areas rich in dopaminergic (DAergic) neurons (caudate nucleus, putamen, globus pallidus, substantia nigra, and subthalamic nuclei) [79 , 141 , 142]. Mn can readily oxidize catecholamines, 7 Manganese in Health and Disease 211 including dopamine (DA), altering homeostasis in these areas [70 ]. Perturbation in striatal DA levels likely explains the biphasic syndrome experienced by patients with manganism. An initial phase of increased DA production is associated with psychotic episodes commonly observed in psychiatric patients [143 ]. As Mn poisoning progresses, catecholamine levels decrease, most likely due to the loss of nigrostriatal DAergic neurons and, consequently, the parkinsonian-like symptoms ensue [1 , 13 , 70]. Hence, in early stages of manganism, upon cessation of Mn exposure, the symptoms might be reversed, whilst in patients with motor disturbances, manganism is irreversible [144 ].

4 Symptoms and Sensitive Populations

The initial stages of manganism are characterized by psychiatric symptoms, including emotional liability, mania, compulsive or aggressive behavior, irritability, reduced response speed, hallucinations, feeding and sex disturbances, intellectual deﬁ cits, humor changes, sex dysfunctions, as well as mild motor impairment. In established manganism cases, the classic extrapyramidal symptoms (motor symptoms) become prominent and include mask-like face, limb rigidity, mild tremors, gait disturbance, cock-like walk, slurred speech, excessive salivation and sweating, and a disturbance of balance, all of which are also observed in IPD [144 – 147 ]. Considering the routes and sources of exposure, affected populations commonly include welders, miners, and people that work in a Mn-polluted environment [147 – 149 ]; infants fed with Mn-containing formulas [109 ]; patients with hepatic encephalopathy [150 , 151], and subjects fed with parenteral nutrition [152 , 153]. In addition, subjects that have genetic pre-disposition have been recently considered as sensitive populations, as described in Section 8 .

5 Manganism versus Parkinson’s Disease

Idiopathic Parkinson’s disease is a progressive neurodegenerative disorder with a slow onset, and compared with the familial forms of the disease, it is associated with advanced age (>55 years of age). The four cardinal signs of IPD are tremor at rest, bradykinesia (hypokinesia and akinesia), rigidity and postural instability [ 154 –156 ]. The disease is characterized by loss of neurons [154 ] in the substantia nigra pars compacta (SNpc) and decreased DA levels in the caudate and putamen [155 ]. Excessive brain Mn levels can also represent a risk factor for IPD [ 16 , 145 , 147 – 149 ]. Case-controlled studies have revealed a strong correlation between Mn-exposed populations and increased susceptibility to PD [16 ,145 , 147 –149 ]. Manganism and PD share in their etiology common cellular mechanisms such as preferential accumulation of Mn within mitochondria resulting in oxidative stress [21 , 157] and selective DAergic neurotoxicity [ 1 , 158 , 159]. Parkinsonism in welders 212 Avila, Puntel, and Aschner

versus non-welders is clinically distinguishable only by the age of onset (46 versus 63 years, respectively) [149 , 160 ]. Furthermore, the prevalence of PD is higher among welders versus age-standardized individuals in the general population [ 149 , 160]. However, direct evidence that Mn exposure in welders is responsible for this increased prevalence has not been reported yet. Manganism commonly occurs in response to acute Mn exposures, whereas PD likely reﬂ ects long-term exposure to relatively low Mn exposures [ 145 , 158 ]. Manganism features less frequent kinetic tremor, or no tremor versus patients with PD [129 ,147 – 149 ]. Acute high Mn exposures also lead to dystonias and a “cock- walk” with symptoms becoming progressive and irreversible [147 ,149 ]. In addition to affecting the basal ganglia, manganism is also known to affect other brain regions, including the cortex and hypothalamus and at the morphological level leading to neuronal loss and reactive gliosis in the globus pallidus and substantia nigra pars reticulata (SNpr) in the absence of Lewy bodies, which are a hallmark of PD [ 147, 149, 161, 162]. Furthermore, in manganism, damage to the striatum (caudate nucleus and putamen) and subthalamic nucleus may occur, while the SNpc is generally spared whilst PD is predominantly characterized by neuronal loss in the SNpc [161 ]. PD and manganism are analogous in several mechanistic ways. Mn-induced neurotoxicity involves mitochondrial dysfunction, increase in endoplasmic reticulum stress factors and oxidative stress, as also observed in PD patients studied post-mortem [ 145 ,148 , 158 , 161]. Mn can cause the increase in ﬁ bril formation by α-synuclein, thus inducing neuronal death. The α-synuclein aggregates, named Lewy bodies, are one of the hallmarks of PD. Furthermore, there are several genetic factors associating both disorders, which will be described in detail below (Section 8 ).

6 Manganese in the Etiology of Other Neurodegenerative Disorders

6.1 Manganese and Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons (MNs) in the spinal cord and the cortex [163 ]. ALS patients gradually loose MNs, and muscles weaken, ultimately leading to respiratory distress and death [163 ]. A small percentage of ALS cases are hereditary, due to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Expression of these mutant forms of SOD1 in animal models leads to ALS-like phenotypes [164 ]. Oxidative stress is proposed to be a central mechanism leading to Mn cell loss in ALS, other contributing mechanisms include excitotoxicity, astrocytosis, mitochondrial impairment, calcium/magnesium imbalance, and Mn toxicity [165 –167 ]. Mn-SOD (SOD2), in contrast to its orthologue, SOD1, does not contribute to the genetic predisposition to ALS [168 ], but it may slow down ALS-like syndrome progression in mice, and its activity has been shown to be reduced in the serum and the CSF of ALS patients [ 168 ]. 7 Manganese in Health and Disease 213

Mn levels were found to be lower in blood cells, but were signiﬁ cantly increased in the sera of ALS patients [169 , 170 ], supporting a role for Mn-mediated neurotoxicity in ALS.

6.2 Manganese and Alzheimer’s Disease

Heavy metals, especially the essential metal Zn and the non-essential metal Al, have been shown to play a role in amyloid-beta (Abeta) aggregation and toxicity, both of which are characteristics of Alzheimer’s disease (AD) [171 ]. In a recent study, Abeta precursor-like protein 1 (APLP1) was found to be the most up-regulated gene in the frontal cortex of monkeys (Cynomologous macaques) chronically exposed to Mn. This result was associated with cortical Mn accumulation [172 ], cortical neuron degeneration, and apoptotic marker expression [172 ], consistent with previous reports of cognitive impairment in those animals. Conversely, over-expression of Abeta in mice led to Mn accumulation in the brain, suggesting that Abeta could play a role in Mn homeostasis and toxicity [173 ]. Similar to ALS, despite Mn accumulation, SOD2 antioxidant activity is reduced in AD, likely contributing to oxidative stress [174 ]. Furthermore, Mn can also produce alterations related to AD without the senile plaque formation. In cases of chronic Mn exposure, neuronal degeneration in the globus pallidus is associated with the development of Alzheimer’s type II astrocytosis, in which cells typically exhibit enlarged, pale nuclei, margination of chromatin and, often, prominent nucleoli [175 ].

6.3 Manganese and Huntington’s Disease

Huntington’s disease (HD) is a progressive neurodegenerative disease with a prevalence of 5 in 10,000 people worldwide. HD is characterized by motor impairment, cognitive deterioration, emotional disturbance, and psychiatric defi cits, caused by expansion in the glutamine-encoding triplet repeat by mutation in the HTT gene [175 ]. Environmental factors have also been suggested to contribute to the residual variation in age of onset, perhaps even more so than genetic factors [ 176 ]. In this context, metals such as Mn may be involved in modulating and interacting with HD. A study by Williams et al. [ 177] described a novel gene-environment interaction between expression of mutant HTT and Mn. Specifi cally, acute Mn exposure of cultured striatal cells unexpectedly decreased the vulnerability of mutant expressing cells (ST HdhQ111/Q111 ) to Mn cytotoxicity compared to wild-type (STHdh Q7/Q7 ) [177 ]. Furthermore, total intracellular Mn levels following Mn exposure in STHdh Q7 /Q 7 and STHdh Q111 /Q111 cells were signifi cantly lower in mutant than wild-type cells [ 177 , 178]. Moreover, the mutant HTT–Mn interaction was corroborated in vivo using the YAC128Q mouse model of HD; these mice accumulated less Mn in the striatum than wild-type animals following subcutaneous Mn injections [179 ]. 214 Avila, Puntel, and Aschner

7 Molecular Mechanisms of Toxicity

The cellular, intracellular, and molecular mechanism(s) underlying Mn neurotoxicity are incompletely understood [21 ,109 , 141 , 142 , 158 , 161 ]. Nevertheless, it has been demonstrated that Mn affects numerous biological activities, dependent upon levels and routes of exposure, dosage, age of the exposed individual, and exposure duration [6 , 180 , 181]. Mn is known to induce increased oxidative stress, a well- established molecular mechanism of Mn-induced toxicity. Below, we discuss the main mechanisms that are believed to mediate Mn-induced neurodegeneration.

7.1 Dopamine Oxidation

Dopamine (DA) is one of the most abundant catecholamines within the brain, controlling locomotion, emotion, and neuroendocrine system. Chronic exposure to Mn has been shown to cause the degeneration of nigrostriatal DAergic neurons leading to symptoms that resemble PD [13 ,148 , 182]. However, Mn’s effects are dependent upon the experimental conditions, form of Mn (oxidation state), route of administration, and exposure duration [ 1 , 6 ,142 , 147 , 183]. Postnatal Mn exposure causes a decline in pre-synaptic DAergic functioning, reduced DA transporter expression, and DA uptake in the striatum, and a long-lasting decrease in DA efﬂ ux [ 184, 185 ]. Conversely, in adult animal models, exposure to Mn inhibits DA neurotransmission and depletes striatal DA [29 , 144 , 183 , 186 ,187 ], thereby resulting in motor deﬁ cits [161 ]. Although it is generally accepted that free radicals play a key role in mediating Mn-induced DAergic neurodegeneration [188 , 189 ], the precise mechanism of Mn-induced neurotoxicity remains unknown. One hypothesis invokes the ability of Mn to enhance reactive oxygen species (ROS) generation, thus forming quinines [82 , 190 , 191 ]. Indeed, the Mn-catalyzed autoxidation of DA involves redox cycling of Mn2+ and Mn3+ in a reaction that generates ROS and DA-o -quinone, thereby leading to oxidative damage [82 , 190 – 192]. Thus, elevated rate of autoxidation of cytoplasmic DA induced by Mn may contribute to DAergic cell death secondary to the formation of cytotoxic quinones and ROS [190 , 191 ]. Mn-induced DA oxidation is a complex process involving several steps in which semiquinone, aminochrome intermediates, L-cysteine or copper and NADH are implicated [158 , 182 , 193]. Mechanisms underlying semiquinone and aminochrome- induced damage in the Mn-induced neurodegenerative process likely include: (i) NADH or NADPH depletion; (ii) inactivation of enzymes by oxidizing thiol groups or essential amino acids; (iii) formation of ROS; and (iv) lipid peroxidation. It is noteworthy that neither Mn2+ nor Mn 3+ can generate hydroxyl radicals from hydrogen peroxide and/or superoxide via Fenton-type or Haber-Weiss-type reactions, while Mn2+ can scavenge and detoxify superoxide radicals [3 , 188 ]. 7 Manganese in Health and Disease 215

7.2 Mitochondrial Dysfunction

As mentioned in Section 1.2 , intracellular Mn preferentially accumulates in the mitochondria, mainly as Mn2+ via the Ca2+ uniporter [21 ,157 ,194 – 196 ]. Elevated intramitochondrial Mn interferes with oxidative respiration, leading to excessive production of ROS, and consequently, mitochondrial dysfunction [21 ,157 ,194 –196 ]. The ability of Mn to enhance oxidative stress is due to the transition of its oxidative state +2 to +3, which increases its pro-oxidant capacity [192 ]. Superoxide produced in the mitochondrial electron transport chain may catalyze this transition through a set of reactions similar to those mediated by SOD and thus lead to the increased oxidant capacity of the metal [195 ]. Since Mn3+ has greater pro-oxidant potential than Mn2+ , its production in the mitochondria may accentuate oxidative damage [197 ]. Moreover, Mn can directly impair mitochondrial function by inhibiting the mitochondrial electron transfer chain [21 , 88 , 198 ], resulting in a reduced ATP production, · − 3+ increased leakage of electrons, and increased O2 production [199 ]. Although, Mn is more potent at inhibiting complex I [3 , 197 ], Mn2+ is the predominant species within cells and is largely bound to ATP [196 , 197]. Notably, Mn in biological media in any of the oxidation states will spontaneously generate Mn3+ . Interestingly, even trace amounts of Mn3+ can cause formation of ROS [200 ]. The involvement of ROS in Mn-induced mitochondrial dysfunction is also supported by observations on the efﬁ cacy of antioxidants in attenuating its effects [201 ]. Mn also interferes with Ca 2+ homeostasis in mitochondria by inhibiting its efﬂ ux [202 ,203 ]. Oxidative stress generated by high Mn concentrations leads to the induction and opening of the mitochondrial permeability transition (MPT) pore, a Ca2+ - dependent process, resulting in increased solubility to protons, ions, and solutes, loss of the mitochondrial inner membrane potential (Δψm), impairment of oxidative phosphorylation, and ATP synthesis and mitochondrial swelling [ 202 ,204 ,205 ]. Indeed, Mn has been shown to decrease Δψm in a concentration-dependent manner, indicating that this Ca2+ -dependent process likely mediates Mn neurotoxicity [204 ,206 , 207 ]. Apoptotic mechanisms secondary to changes in mitochondrial function have also been implicated in Mn-induced neurotoxicity. Ca2+ -induced MPT opening leads to the activation of the Bcl-2 family of proteins, especially Bax/Bak, culminating with the release of cytochrome c (Cyt c ) [208 , 209]. Cyt c activates, via ERK (extracellular signal-regulated kinases), the cysteine protease caspase-3, which mediates apoptosis, chromatin condensation and DNA fragmentation [210 ]. Consistent with these observations, Mn exposure has been shown to lead to ERK and caspase-3 activation in astrocytes [204 ]. Furthermore, DNA strand breakage at low Mn levels was reported, thereby reinforcing the mitochondrial role in mediating its neurotoxicity [211 ].

7.3 Astrocytosis

Astrocytes make up approximately 50% of the human brain volume [ 212] and assume many critical pathophysiological roles essential for normal neuronal activity, including glutamate uptake, glutamine release, K+ and H+ buffering, volume regulation and 216 Avila, Puntel, and Aschner membrane–membrane mediated trophic cell signaling [92 , 180 ]. Unlike neurons, astrocytes concentrate Mn to levels at least 50-fold higher than the culture media, thus functioning as the major homeostatic regulators and storage site for Mn [213 , 214]. Primate models of Mn toxicity have shown astrocytic pathological alterations (Alzheimer type II) [17 , 22 , 151 ] and exposure of cultured astrocytes to patho- physiologically relevant concentrations of Mn led to a dose- and time- dependent cell swelling, which appears to be a consequence of oxidative stress and changes in MPT [215 ]. Increased accumulation of Mn in astrocytes has also been shown to alter glutamate homeostasis and elicit excitatory neurotoxicity [27 ]. For example, Mn decreases astrocytic glutamate uptake [180 , 181 , 216] and reduces the expression of the astrocytic glutamate:aspartate transporter (GLAST) [27 , 217 , 218 ], leading to increased extracellular glutamate levels. Additionally, expression of glutamine transporters was downregulated in Mn-exposed cultured astrocytes [219 ], contributing to the disruption of the glutamate-glutamine cycling in the brain. The inhibition of the Na + /K+ -ATPase by reactive oxygen species also likely contributes to Mn-induced astrocytic dysfunction [220 ]. Mn increases the uptake of the amino acid L-arginine, a substrate for the inducible form of nitric oxide synthase (iNOS), which can lead to ROS production as a consequence of nitric oxide production [ 221 , 222 ]. ROS have also been shown to directly interfere with glutamate uptake [223 ], possibly via oxidation of thiol groups in the transporter protein [220 ]. Thus, Mn accumulation in astrocytes has the potential to lead to oxidative damage in these cells as well as adverse effects on glutamate clearance from the extracellular space.

8 Genetic Susceptibility

Recently, the association of mutations of PD-related genes and manganism has been reported. DJ-1 (Park7), together with parkin (Park2) and Pink1 (Park6), form an E3 ubiquitin-ligase complex that is involved in α-synuclein degradation [224 ]. The physiological functions of these proteins involve protection against oxidative stress. Both mitochondrial dysfunction and oxidative stress can modulate the ubiquitin- proteasome pathway and have been implicated as causative factors for the abnormal accumulation of proteins in familial forms of PD [145 ]. Recessive inheritance of PARK 2 mutations may also cause increased environmental sensitivity to Mn exposure, as observed by Aboud et al. [225 ]. Using human induced pluripotent stem cells (hIPCs) derived from a subject at genetic risk by PARK 2 mutation, the authors found signiﬁ cant high reactive oxygen species levels and increased mitochondrial fragmentation after Mn exposure in vitro [ 225 ]. Mutations in parkin are associated with early onset of PD, associated with DAergic neurodegeneration, however, absent Lewy bodies formation [145 ]. The parkin gene encodes an E3 ubiquitin ligase, which has cytoprotective properties. Transient transfection with the parkin gene in SH-SY5Y cells inhibits Mn-induced cell death [226 ]. Exposure to welding fumes containing Mn led to decreased Park2 7 Manganese in Health and Disease 217 protein levels in DAergic brain areas in rodents [227 ]. Loss of function and/or decreased expression of parkin has been associated with overexpression of DMT-1 and linked to PD [ 123 , 227], as well as manganism [228 ]. Conversely, increased Parkin expression levels have been shown to attenuate Mn-induced neurotoxicity, likely by reducing its transport [226 , 228 ]. DJ-1 was also decreased in striatum of rats exposed chronically to welding fumes [ 227]. Mutations in dj-1 account for 1–2% of early-onset cases of PD [229 ]. The protein encoded by this gene is expressed in the brain, including neurons within the SNPc and striatum, areas primarily affected in PD [230 ]. DJ-1 expression has been localized to the matrix and intermembrane space of mitochondria [231 ] and it is thought to function as an antioxidant protein [232 ]. Dj-1 -knockout mice exhibit increased mitochondrial free radical formation and inactivation of enzymes [232 ]. Leucine-rich repeat kinase 2 (LRRK2) or PARK8 is a cytoplasmic enzyme present in DAergic neurons. Mutations in this gene causing increased kinase activity lead to typical features of PD [233 ]. Kinases require the formation of an ATP- divalent metal cation complex, and Mg 2+ typically participates in this catalysis. Recent studies in G2019S cells, where LRRK2 is mutated and shows increased enzyme activity, have demonstrated that Mn2+ can displace Mg2+ at the active site and increase the catalytic rate of the enzyme [234 ]. Because this mutation is present in 22–41% of PD cases, changes in the enzyme activity caused by Mn may result in a gain-of-function type mechanism of toxicity, leading to decreased cell survival [234 ]. Furthermore, mutations in a putative Mn exporter gene SLC30A10 have been recently described [235 ]. These mutations are associated with marked motor impairment, including a Parkinsonian-like syndrome. This inherited autosomal recessive mutation leads to hypermanganesemia, dystonia, polycythemia, and hepatic cirrhosis [236 ]. The hypermanganesemia associated with SLC30A10 mutation is extreme, with patients having whole blood Mn levels of 1200–6400 nmol/L, compared with normal whole blood Mn (<320 nmol/L) [236 ].

9 Treatment

Even though manganism has been studied for several years, treatment approaches are still controversial. Some authors have shown that levodopa, a standard treatment for PD, decreased Mn symptoms; however most of the studies do not indicate this drug as effi cient [ 148 , 149]. Hence, other drugs have been tested in non-human animals and some of these treatments may be used in future clinical trials. Antiinfl ammatory agents, such as indomethacin and para-aminosalicilic acid, reduced Mn-induced increase in oxidative stress (isoprostanes) and neuroinfl ammation (prostaglandin E2) [237 – 240 ]. Notably, indomethacin protected against progressive spine degeneration and dendritic damage in striatal medium spiny neurons of mice exposed to Mn [239 , 240 ]. This protection is probably mediated by the transcription factor NF-κB [ 241 ]. Using transgenic mice expressing a transcription 218 Avila, Puntel, and Aschner

factor fused to a green ﬂ uorescent protein (GFP), Moreno and coworkers showed that Mn exposure increased NF-κB reporter activity and nitric oxide synthase 2 (NOS2) expression in both microglia and astrocytes, and that these effects were prevented by supplementation with steroid 17β-estradiol [241 ]. Estrogen also decreased neuronal protein nitration in treated mice and inhibited apoptosis in striatal neurons co-cultured with Mn-treated astrocytes in vitro [241 ] . Furthermore, tamoxifen, an estrogen-related compound, effectively reversed glutamate transport inhibition in a Mn-induced model of glutamatergic deregulation, suggesting a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis [242 ]. In agreement with this study, Xu et al. showed that the pretreatment of rats with the NMDA ( N -methyl- D -aspartate) antagonist MK801 protected neurons from Mn-induced glutamate excitotoxicity [243 ]. Synthetic and natural antioxidants have been tested in Mn-induced neurotoxicity models. The chain breaking antioxidant Trolox concomitantly administered to Mn in developing pups showed neuroprotective effects by decreasing apoptotic activity, isoprostane levels and p38 phosphorylation [244 ]. Ebselen and diethyl-2-phenyl- 2-tellurophenyl vinyl phosphonate are organochalcogens that also reversed the neurotoxic effects of this metal. Natural products such as Melissa ofﬁ cinalis extract and silymarin reduced oxidative stress associated to Mn exposure [245 ,246 ]. Several studies have addressed genetic factors that mediate Mn toxicity. Streifel and coworkers used mice lacking NOS, postulating that they would be protected from the neurotoxic effects of Mn [247 ]. They found that loss of NOS2 reduced NO-induced peroxynitrite formation, thus attenuating Mn-related peroxynitrite adduct formation in the striatal-pallidum and substantia nigra pars reticulata. These mice showed attenuated alterations in neurobehavioral function and neurochemistry in vivo and loss of NOS2 also prevented astrocyte-mediated neuronal apoptosis in vitro [247 ]. Expression of parkin, an E3 ubiquitin ligase also linked to PD, protects against Mn toxicity, as observed in SH-SY5Y cells [248 ]. Conversely, deletion of parkin leads to an increase in DMT-1 levels, thus causing increase in Mn uptake [ 248 ]. Furthermore, it was reported for yeast that expression of PARK9, a gene linked to PD, protected cells from Mn toxicity [118 ]. In C. elegans, Benedetto et al. observed that Mn-induced DAergic neurotoxicity requires the NADPH dual-oxidase BLI-3, suggesting that in vivo BLI-3 activity promotes the conversion of extracellular DA into toxic reactive species, which, in turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress and cell degeneration [248 ]. BLI-3 knockout or inhibition may represent a novel strategy for mitigating Mn neurotoxicity.

10 General Conclusions

Because of its paradoxal effects on human health, Mn exposure or intake has been studied for quite some time. Several mechanisms have been proposed for manganism, such as (i) dopamine oxidation; (ii) glial toxicity, particularly in astrocytes; 7 Manganese in Health and Disease 219

(iii) oxidative stress; (iv) mitochondrial dysfunction; (v) alteration in the expression of PD-related genes. However, there are many questions that have yet to be clariﬁ ed. Treatment approaches have also been investigated, focusing on the mechanisms that were described in this chapter. Remarkably, although Mn intake is necessary for the normal functioning of the organism, it is necessary to regulate its environmental and occupational exposures, as once excessive exposure occurred, it may lead to neurological dysfunction for which effective treatment has yet to be developed.

Abbreviations

ABC ATP-binding cassette Abeta amyloid β AD Alzheimer’s Disease ALS amyotrophic lateral sclerosis APLP amyloid β precursor-like protein ATP adenosine 5′-triphosphate BBB blood-brain barrier BCB blood-cerebrospinal fl uid barrier CA3 cornu Ammonis, region 3 CNS central nervous system CSF cerebrospinal fl uid Cyt c cytochrome c DA dopamine DAT dopamine transporter type DMT divalent metal transporter ERK extracellular signal-regulated kinase ESADDI estimated safe and adequate dietary intake FDA Food and Drug Administration Fpn ferroportin GFD green fl uorescent protein GLAST glutamate:aspartate transporter GS glutamine synthetase HD Huntington’s Disease Hip huntingtin-interacting protein hIPCs human induced pluripotent stem cells HTT huntingtin iNOS nitric oxide synthase (inducible form) IPD idiopatic Parkinson’s disease LRRK2 leucine-rich repeat kinase 2 MCT monocarboxylate transporter MMT methylcyclopentadienyl manganese tricarbonyl MNs motor neurons MPT mitochondrial transition pore 220 Avila, Puntel, and Aschner

MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MRI magnetic resonance imaging NAAS National Academy of Sciences NADH nicotinamide adenine dinucleotide reduced NADPH nicotinamide adenine dinucleotide phosphate reduced NMDA N -methyl- D -aspartate NOS nitric oxide synthase NRAMP natural resistance-associated macrophage protein NRC National Research Council OATP organic anion transporter polypeptide PARK Parkinson protein PD Parkinson’s disease PET positron emission tomography RDI reference daily intake ROS reactive oxygen species SLC39 solute carrier-39 SNpc substantia nigra pars compacta SNpr substantia nigra pars reticulata SOD superoxide dismutase SPCA Ca2+ /Mn2+ ATPase of the secretory pathway SPECT single-photon emission computed tomography TCA tricarboxylic acid Tf transferrin TfR transferrin receptor TRPM7 transient receptor potential cation channel, subfamily M, member 7

Acknowledgments M.A. acknowledges funding by R01ES10563 and P30ES00267; D.S.A. acknowledges support by FAPERGS (ARD11/1673-7) and CNPq (Universal 476471/2011-7).

References

1. M. Aschner, T. R. Guilarte, J. S. Schneider, W. Zheng, Toxicol. Appl. Pharmacol . 2007 , 221 , 131–147. 2. USEPA, Health Assessment Document for Manganese, EPA 600-83-013F, U.S.E.P. Agency, Editor, 1984, Washington, DC. 3. F. S. Archibald, C. Tyree, Arch. Biochem. Biophys . 1987 , 256 , 638–650. 4. C. L. Keen, in Proceedings of the Workshop on the Bioavailability and Oral Toxicity of Manganese, Ed S. Velazquez, US EPA, Environmental Criteria and Assessment Ofﬁ ce, 1995 , 3–11. 5. T. Inoue, T. Majid, R. G. Pautler, Rev. Neurosci . 2011 , 22 , 675–694. 6. M. C. Newland, Neurotoxicology 1999 , 20 , 415–432. 7. R. M. Bowler, S. Gysens, E. Diamond, S. Nakagawa, M. Drezgic, H. A. Roels, Neurotoxicology 2006 , 27 , 315–326. 8. Y. M. Jiang, X. A. Mo, F. Q. Du, X. Fu, X. Y. Zhu, H. Y. Gao, J. L. Xie, F. L. Liao, E. Pira, W. Zheng, J. Occup. Environ. Med . 2006 , 48 , 644–649. 9. J. E. Myers, M. L. Thompson, S. Ramushu, T. Young, M. F. Jeebhay, L. London, E. Esswein, K. Renton, A. Spies, A. Boulle, I. Naik, A. Iregren, D. J. Rees, Neurotoxicology 2003 , 24 , 885–894. 7 Manganese in Health and Disease 221

10. K. Ono, K. Komai, M. Yamada, J. Neurol. Sci . 2002 , 199 , 93–96. 11. D. G. Cook, S. Fahn, K. A. Brait, Arch. Neurol . 1974 , 30 , 59–64. 12. H. Roels, R. Lauwerys, J. P. Buchet, P. Genet, M. J. Sarhan, I. Hanotiau, M. de Fays, A. Bernard, D. Stanescu, Am. J. Ind. Med . 1987 , 11 , 307–327. 13. A. Barbeau, Neurotoxicology 1984 , 5 , 13–35. 14. F. M. Corrigan, L. Murray, C. L. Wyatt, R. F. Shore, Exp. Neurol . 1998 , 150 , 339–342. 15. J. M. Gorell, C. C. Johnson, B. A. Rybicki, E. L. Peterson, G. X. Kortsha, G. G. Brown, R. J. Richardson, Neurology 1997 , 48 , 650–658. 16. B. A. Rybicki, C. C. Johnson, J. Uman, J. M. Gorell, Mov. Disord . 1993 , 8 , 87–92. 17. M. Yamada, S. Ohno, I. Okayasu, R. Okeda, S. Hatakeyama, H. Watanabe, K. Ushio, H. Tsukagoshi, Acta Neuropathol . 1986 , 70 , 273–278. 18. R. Lucchini, L. Selis, D. Folli, P. Apostoli, A. Mutti, O. Vanoni, A. Iregren, L. Alessio, Scand. J. Work Environ. Health 1995 , 21 , 143–149. 19. H. A. Roels, P. Ghyselen, J. P. Buchet, E. Ceulemans, R. R. Lauwerys, Br. J. Ind. Med . 1992 , 49 , 25–34. 20. R. Lucchini, P. Apostoli, C. Perrone, D. Placidi, E. Albini, P. Migliorati, D. Mergler, M. P. Sassine, S. Palmi, L. Alessio, Neurotoxicology 1999 , 20 , 287–297. 21. C. E. Gavin, K. K. Gunter, T. E. Gunter, Neurotoxicology 1999 , 20 , 445–453. 22. C. W. Olanow, P. F. Good, H. Shinotoh, K. A. Hewitt, F. Vingerhoets, B. J. Snow, M. F. Beal, D. B. Calne, D. P. Perl, Neurology 1996 , 46 , 492–498. 23. R. H. Gwiazda, D. Lee, J. Sheridan, D. R. Smith, Neurotoxicology 2002 , 23 , 69–76. 24. R. Witholt, R. H. Gwiazda, D. R. Smith, Neurotoxicol. Teratol . 2000 , 22 , 851–861. 25. ATSDR (Agency of Toxic Substances and Disease Registry), Toxicological Proﬁ le for Manganese, U.S. Department of Health and Human Services Public Health Service, 2000. 26. IOM, Dietary Reference Intakes: Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc, Ed Institute of Medicine, National Academy Press, Washington DC, 2011. 27. K. M. Erikson, M. Aschner, Neurochem. Int . 2003 , 43 , 475–480. 28. J. R. Prohaska, Physiol. Rev . 1987 , 67 , 858–901. 29. D. G. Barceloux, J. Toxicol. Clin. Toxicol . 1999 , 37 , 293–307. 30. V. C. Culotta, M. Yang, T.V. O’Halloran, Biochim. Biophys. Acta 2006 , 1763 , 747–758. 31. C. L. Keen, J. L. Ensunsa, M. H. Watson, D. L. Baly, S. M. Donovan, M. H. Monaco, M. S. Clegg, Neurotoxicology 1999 , 20 , 213–223. 32. J. W. Finley, J. G. Penland, R. E. Pettit, C. D. Davis, J. Nutr . 2003 , 133 , 2849–2856. 33. C. L. Keen, S. Zidenberg-Cherr, Manganese Toxicity in Humans and Experimental Animals , in Manganese in Health and Disease, Ed D. J. Klimis-Tavantzis, CRC Press., Boca Raton, 1994, pp. 193–205. 34. J. W. Finley, C. D. Davis, Biofactors 1999 , 10 , 15–24. 35. J. A. Pennington, S. A. Schoen, Int. J. Vitam. Nutr. Res . 1996 , 66 , 350–362. 36. J. L. Greger, J. Nutr . 1998 , 128 , 368S–371S. 37. NAS, Panel on Micronutrients . 2001 , available at www.nap.edu/books/0309072794/html/ . 38. S. C. Sistrunk, M. K. Ross, N. M. Filipov, Environ. Toxicol. Pharmacol . 2007 , 23 , 286–296. 39. J. W. Finley, P. E. Johnson, L. K. Johnson, Am. J. Clin. Nutr . 1994 , 60 , 949–955. 40. M. Aschner, J. L. Aschner, Neurosci. Biobehav. Rev . 1991 , 15 , 333–340. 41. K. Thompson, R. M. Molina, T. Donaghey, J. E. Schwob, J. D. Brain, M. Wessling-Resnick, FASEB J . 2007 , 21 , 223–230. 42. D. C. Dorman, B. E. McManus, C. U. Parkinson, C. A. Manuel, A. M. McElveen, J. I. Everitt, Inhal. Toxicol . 2004 , 16 , 481–488. 43. J. J. Powell, R. Jugdaohsingh, R. P. Thompson, Proc. Nutr. Soc . 1999 , 58 , 147–153. 44. M. E. Conrad, J. N. Umbreit, E. G. Moore, C. R. Rodning, Blood 1992 , 79 , 244–247. 45. H. Gunshin, B. Mackenzie, U.V. Berger, Y. Gunshin, M.F. Romero, W.F. Boron, S. Nussberger, J. L. Gollan, M. A. Hediger, Nature 1997 , 388 , 482–488. 46. S. J. Garcia, K. Gellein, T. Syversen, M. Aschner, Toxicol. Sci . 2006 , 92 , 516–525. 47. V. A. Fitsanakis, N. Zhang, J. G. Anderson, K. M. Erikson, M. J. Avison, J. C. Gore, M. Aschner, Toxicol. Sci . 2008 , 103 , 116–124. 222 Avila, Puntel, and Aschner

48. C. D. Klaassen, Toxicol. Appl. Pharmacol . 1974 , 29 , 458–468. 49. M. Aschner, M. Gannon, Brain Res. Bull . 1994 , 33 , 345–349. 50. A. Takeda, J. Sawashita, S. Okada, Brain Res . 1995 , 695 , 53–58. 51. A. C. Foradori, A. Bertinchamps, J. M. Gulibon, G. C. Cotzias, J. Gen. Physiol . 1967 , 50 , 2255–2266. 52. G. C. Cotzias, A. Bertinchamps, Clinical Experiences with Manganese, in Metal Binding in Medicine, Eds M. J. Seven, L. A. Johnson, Lippincott Co., Philadelphia, 1960, pp. 50–57. 53. G. C. Cotzias, K. Horiuchi, S. Fuenzalida, I. Mena, Neurology 1968 , 18 , 376–382. 54. T. E. Gunter, J. S. Puskin, Biophys. J . 1972 , 12 , 625–635. 55. J. J. Liccione, M. D. Maines, J. Pharmacol. Exp. Ther . 1988 , 247 , 156–161. 56. C. Zwingmann, D. Leibfritz, A. S. Hazell, J. Cereb. Blood Flow Metab . 2003 , 23 , 756–771. 57. K. Kalia, W. Jiang, W. Zheng, Neurotoxicology . 2008 , 29 , 466–470. 58. M. R. Sepulveda, F. Wuytack, A. M. Mata, J. Neurochem . 2012 , 123 , 824–836. 59. P. Vangheluwe, M. R. Sepulveda, L. Missiaen, L. Raeymaekers, F. Wuytack, J. Vanoevelen, Chem. Rev . 2009 , 109 , 4733–4759. 60. V. K. Ton, D. Mandal, C. Vahadji, R. Rao, J. Biol. Chem . 2002 , 277 , 6422–6427. 61. M. R. Sepulveda, T. Dresselaers, P. Vangheluwe, W. Everaerts, U. Himmelreich, A. M. Mata, F. Wuytack, Contrast Media Mol. Imaging . 2012 , 7 , 426–434. 62. S. Mukhopadhyay, A. D. Linstedt, Proc. Natl. Acad. Sci. USA 2011 , 108 , 858–863. 63. V. A. Murphy, K. C. Wadhwani, Q. R. Smith, S. I. Rapoport, J. Neurochem. 1991, 57, 948–954. 64. O. Rabin, L. Hegedus, J. M. Bourre, Q. R. Smith, J. Neurochem . 1993 , 61 , 509–517. 65. R. E. London, G. Toney, S. A. Gabel, A. Funk, Brain Res. Bull . 1989 , 23 , 229–235. 66. A. Takeda, T. Akiyama, J. Sawashita, S. Okada, Brain Res . 1994 , 640 , 341–344. 67. M. Yukawa, K. Amano, M. Suzuki-Yasumoto, M. Terai, Arch. Environ. Health 1980 , 35 , 36–44. 68. E. Bonilla, E. Salazar, J. J. Villasmil, R. Villalobos, Neurochem. Res . 1982 , 7 , 221–227. 69. E. D. Bird, A. H. Anton, B. Bullock, Neurotoxicology 1984 , 5 , 59–65. 70. V. J. Bush, T. P. Moyer, K. P. Batts, J. E. Parisi, Clin. Chem . 1995 , 41 , 284–294. 71. D. C. Dorman, M. F. Struve, R. A. James, B. E. McManus, M. W. Marshall, B. A. Wong, Toxicol. Sci . 2001 , 60 , 242–251. 72. A. St-Pierre, L. Normandin, G. Carrier, G. Kennedy, R. Butterworth, J. Zayed, Inhal. Toxicol . 2001 , 13 , 623–632. 73. A. Tracqui, J. Tayot, P. Kintz, G. Alves, M. A. Bosque, P. Mangin, Forensic Sci. Int . 1995 , 76 , 199–203. 74. M. Morello, A. Canini, P. Mattioli, R. P. Sorge, A. Alimonti, B. Bocca, G. Forte, A. Martorana, G. Bernardi, G. Sancesario, Neurotoxicology 2008 , 29 , 60–72. 75. J. J. Liccione, M. D. Maines, J. Pharmacol. Exp. Ther . 1989 , 248 , 222–228. 76. A. Nong, J. G. Teeguarden, H. J. Clewell, 3rd, D. C. Dorman, M. E. Andersen, J. Toxicol. Environ. Health A 2008 , 71 , 413–426. 77. M. C. Newland, C. Cox, R. Hamada, G. Oberdorster, B. Weiss, Fundam. Appl. Toxicol . 1987 , 9 , 314–328. 78. J. Versieck, F. Barbier, A. Speecke, J. Hoste, Clin. Chem . 1974 , 20 , 1141–1145. 79. L. Spahr, R. F. Butterworth, S. Fontaine, L. Bui, G. Therrien, P. C. Milette, L. H. Lebrun, J. Zayed, A. Leblanc, G. Pomier-Layrargues, Hepatology 1996 , 24 , 1116–1120. 80. R. F. Butterworth, L. Spahr, S. Fontaine, G. P. Layrargues, Metab. Brain Dis. 1995, 10, 259–267. 81. K. Weissenborn, C. Ehrenheim, A. Hori, S. Kubicka, M. P. Manns, Metab. Brain Dis . 1995 , 10 , 219–231. 82. A. S. Hazell, R. F. Butterworth, Proc. Soc. Exp. Biol. Med . 1999 , 222 , 99–112. 83. J. D. Schroeter, A. Nong, M. Yoon, M. D. Taylor, D. C. Dorman, M. E. Andersen, H. J. Clewell, 3rd, Toxicol. Sci . 2011 , 120 , 481–498. 84. S. H. Zlotkin, S. Atkinson, G. Lockitch, Clin. Perinatol . 1995 , 22 , 223–240. 85. A. B. Bowman, K. M. Erikson, M. Aschner, Manganese – The Two Faces of Essentiality and Neurotoxicity, in Metals and Neurodegeneration, Ed S. Huang, Research Signpost, Kerala, India, 2010. 86. A. Takeda, Brain Res. Brain Res. Rev . 2003 , 41 , 79–87. 7 Manganese in Health and Disease 223

87. D. W. Christianson, Prog. Biophys. Mol. Biol . 1997 , 67 , 217–252. 88. F. C. Wedler, R. B. Denman, Curr. Top. Cell Regul . 1984 , 24 , 153–169. 89. P. K. Maciejewski, D. L. Rothman, Neurochem. Int . 2008 , 52 , 809–825. 90. T. Eid, A. Williamson, T. S. Lee, O. A. Petroff, N. C. de Lanerolle, Epilepsia 2008 , 49 Suppl. 2 , 42–52. 91. D. E. Ash, J. D. Cox, D. W. Christianson, Met. Ions Biol. Syst . 2000 , 37 , 407–428. 92. A. G. Estevez, M. A. Sahawneh, P. S. Lange, N. Bae, M. Egea, R. R. Ratan, J. Neurosci . 2006 , 26 , 8512–8516. 93. A. S. Mildvan, M. C. Scrutton, M. F. Utter, J. Biol. Chem . 1966 , 241 , 3488–3498. 94. A. C. Yu, J. Drejer, L. Hertz, A. Schousboe, J. Neurochem . 1983 , 41 , 1484–1487. 95. N. M. Fong, T. C. Jensen, A. S. Shah, N. N. Parekh, A. R. Saltiel, M. J. Brady, J. Biol. Chem . 2000 , 275 , 35034–35039. 96. O. A. Andreassen, A. Dedeoglu, A. Friedlich, K. L. Ferrante, D. Hughes, C. Szabo, M. F. Beal, Exp. Neurol . 2001 , 168 , 419–424. 97. L. S. Hurley, C. L. Keen, Manganese , in Trace Elements in Human Health and Animal Nutrition , Vol. 1, 5th edn., Ed W. Mertz, Academic Press, San Diego, CA, 1987, pp. 185–222. 98. C. Keen, Nutritional and Toxicological Aspects of Manganese Intakes: An Overview , in Risk Assessment of Essential Elements, Eds W. Mertz, C. O. Abernathy, S. S. Olin, ILSI Press, Washington DC, 1994, pp. 221–235. 99. J. H. Freeland-Graves, C. Llanes, Models to Study Manganese Deﬁ ciency, in Manganese in Health and Disease , Ed D. J. Klimis-Tavantzis, CRC Press, Boca Raton, 1994. 100. M. D. Garrick, K. G. Dolan, C. Horbinski, A. J. Ghio, D. Higgins, M. Porubcin, E. G. Moore, L. N. Hainsworth, J. N. Umbreit, M. E. Conrad, L. Feng, A. Lis, J. A. Roth, S. Singleton, L. M. Garrick, BioMetals 2003 , 16 , 41–54. 101. L. He, K. Girijashanker, T. P. Dalton, J. Reed, H. Li, M. Soleimani, D. W. Nebert, Mol. Pharmacol . 2006 , 70 , 171–180. 102. L. Davidsson, B. Lonnerdal, B. Sandstrom, C. Kunz, C. L. Keen, J. Nutr . 1989 , 119 , 1461–1464. 103. C. M. Lucaciu, C. Dragu, L. Copaescu, V. V. Morariu, Biochim. Biophys. Acta 1997, 1328, 90–98. 104. A. Riccio, C. Mattei, R. E. Kelsell, A. D. Medhurst, A. R. Calver, A. D. Randall, J. B. Davis, C. D. Benham, M. N. Pangalos, J. Biol. Chem . 2002 , 277 , 12302–12309. 105. S. S. Kannurpatti, P. G. Joshi, N. B. Joshi, Neurochem. Res . 2000 , 25 , 1527–1536. 106. J. S. Crossgrove, R. A. Yokel, Neurotoxicology 2004 , 25 , 451–460. 107. J. S. Crossgrove, D. D. Allen, B. L. Bukaveckas, S. S. Rhineheimer, R. A. Yokel, Neurotoxicology 2003 , 24 , 3–13. 108. K. M. Erikson, K. Thompson, J. Aschner, M. Aschner, Pharmacol. Ther . 2007 , 113 , 369–377. 109. V. A. Fitsanakis, G. Piccola, A. P. Marreilha dos Santos, J. L. Aschner, M. Aschner, Hum. Exp. Toxicol . 2007 , 26 , 295–302. 110. Z. Xiang, G. Burnstock, Neuroreport 2005 , 16 , 903–907. 111. R. A. North, Physiol. Rev . 2002 , 82 , 1013–1067. 112. N. Tanaka, K. Kawasaki, N. Nejime, Y. Kubota, K. Nakamura, M. Kunitomo, K. Takahashi, M. Hashimoto, K. Shinozuka, J. Pharmacol. Sci . 2004 , 95 , 174–180. 113. C. Marzano, M. Pellei, F. Tisato, C. Santini, Anticancer Agents Med. Chem . 2009 , 9 , 185–211. 114. A. Sacher, A. Cohen, N. Nelson, J. Exp. Biol . 2001 , 204 , 1053–1061. 115. J. S. Crossgrove, R. A. Yokel, Neurotoxicology 2005 , 26 , 297–307. 116. A. Goytain, R. M. Hines, G. A. Quamme, J. Biol. Chem . 2008 , 283 , 33365–33374. 117. A. D. Gitler, A. Chesi, M. L. Geddie, K. E. Strathearn, S. Hamamichi, K. J. Hill, K. A. Caldwell, G. A. Caldwell, A. A. Cooper, J. C. Rochet, S. Lindquist, Nat. Genet . 2009 , 41 , 308–315. 118. A. C. Chua, E. H. Morgan, J. Comp. Physiol. B 1997 , 167 , 361–369. 119. M. D. Fleming, C. C. Trenor, 3rd, M. A. Su, D. Foernzler, D. R. Beier, W. F. Dietrich, N. C. Andrews, Nat. Genet . 1997 , 16 , 383–386. 120. M. D. Fleming, M. A. Romano, M. A. Su, L. M. Garrick, M. D. Garrick, N. C. Andrews, Proc. Natl. Acad. Sci. USA 1998 , 95 , 1148–1153. 224 Avila, Puntel, and Aschner

121. K. Thompson, R. M. Molina, T. Donaghey, J. D. Brain, M. Wessling-Resnick, Am. J. Physiol. Gastrointest. Liver Physiol . 2007 , 293 , G640–644. 122. J. Salazar, N. Mena, S. Hunot, A. Prigent, D. Alvarez-Fischer, M. Arredondo, C. Duyckaerts, V. Sazdovitch, L. Zhao, L. M. Garrick, M. T. Nunez, M. D. Garrick, R. Raisman-Vozari, E. C. Hirsch, Proc. Natl. Acad. Sci. USA 2008 , 105 , 18578–18583. 123. V. A. Fitsanakis, G. Piccola, J. L. Aschner, M. Aschner, Neurotoxicology 2006 , 27 , 60–70. 124. V. A. Fitsanakis, G. Piccola, J. L. Aschner, M. Aschner, J. Neurosci. Res . 2005 , 81 , 235–243. 125. T. K. Dickinson, A. G. Devenyi, J. R. Connor, J. Lab. Clin. Med . 1996 , 128 , 270–278. 126. C. C. Trenor, 3rd, D. R. Campagna, V. M. Sellers, N. C. Andrews, M. D. Fleming, Blood 2000 , 96 , 1113–1118. 127. S. Potocki, M. Rowinska-Zyrek, D. Witkowska, M. Pyrkosz, A. Szebesczyk, K. Krzywoszynska, H. Kozlowski, Curr. Med. Chem . 2012 , 19 , 2738–2759. 128. A. Ramirez, A. Heimbach, J. Grundemann, B. Stiller, D. Hampshire, L. P. Cid, I. Goebel, A. F. Mubaidin, A. L. Wriekat, J. Roeper, A. Al-Din, A. M. Hillmer, M. Karsak, B. Liss, C. G. Woods, M. I. Behrens, C. Kubisch, Nat. Genet . 2006 , 38 , 1184–1191. 129. P. R. Lockman, K. E. Roder, D. D. Allen, J. Neurochem . 2001 , 79 , 588–594. 130. W. N. Sloot, J. B. Gramsbergen, Brain Res . 1994 , 657 , 124–132. 131. J. G. Anderson, P. T. Cooney, K. M. Erikson, Environ. Toxicol. Pharmacol. 2007, 23, 179–184. 132. L. J. Wu, A. G. Leenders, S. Cooperman, E. Meyron-Holtz, S. Smith, W. Land, R. Y. Tsai, U. V. Berger, Z. H. Sheng, T. A. Rouault, Brain Res . 2004 , 1001 , 108–117. 133. M. D. Garrick, Genes Nutr . 2011 , 6 , 45–54. 134. Z. Yin, H. Jiang, E. S. Lee, M. Ni, K. M. Erikson, D. Milatovic, A. B. Bowman, M. Aschner, J. Neurochem . 2010 , 112 , 1190–1198. 135. M. B. Troadec, D. M. Ward, E. Lo, J. Kaplan, I. De Domenico, Blood 2010 , 116 , 4657–4664. 136. M. S. Madejczyk, N. Ballatori, Biochim. Biophys. Acta 2012 , 1818 , 651–657. 137. S. Abboud, D. J. Haile, J. Biol. Chem . 2000 , 275 , 19906–19912. 138. A. S. Zhang, S. Xiong, H. Tsukamoto, C. A. Enns, Blood 2004 , 103 , 1509–1514. 139. P. Hahn, T. Dentchev, Y. Qian, T. Rouault, Z. L. Harris, J. L. Dunaief, Mol. Vis . 2004 , 10 , 598–607. 140. M. Aschner, D. C. Dorman, Toxicol. Rev . 2006 , 25 , 147–154. 141. M. Aschner, K. M. Erikson, D. C. Dorman, Crit. Rev. Toxicol . 2005 , 35 , 1–32. 142. G. Gianutsos, M. T. Murray, Neurotoxicology 1982 , 3 , 75–81. 143. D. B. Calne, N. S. Chu, C. C. Huang, C. S. Lu, W. Olanow, Neurology 1994 , 44 , 1583–1586. 144. A. Benedetto, C. Au, M. Aschner, Chem. Rev . 2009 , 109 , 4862–4884. 145. A. B. Bowman, G. F. Kwakye, E. Herrero Hernandez, M. Aschner, J. Trace Elem. Med. Biol . 2011 , 191–203. 146. C. W. Olanow, Ann. N. Y. Acad. Sci . 2004 , 1012 , 209–223. 147. R. G. Lucchini, E. Albini, L. Benedetti, S. Borghesi, R. Coccaglio, E. C. Malara, G. Parrinello, S. Garattini, S. Resola, L. Alessio, Am. J. Ind. Med . 2007 , 50 , 788–800. 148. B. A. Racette, L. McGee-Minnich, S. M. Moerlein, J. W. Mink, T. O. Videen, J. S. Perlmutter, Neurology 2001 , 56 , 8–13. 149. J. A. Chalela, L. Bonillha, R. Neyens, A. Hays, Neurocrit. Care 2011 , 14 , 456–458. 150. A. Pentschew, F. F. Ebner, R. M. Kovatch, J. Neuropathol. Exp. Neurol . 1963 , 22 , 488–499. 151. D. B. Bertinet, M. Tinivella, F. A. Balzola, A. de Francesco, O. Davini, L. Rizzo, P. Massarenti, M. A. Leonardi, F. Balzola, J. Parenter. Enteral Nutr . 2000 , 24 , 223–227. 152. K. M. Hambidge, R. J. Sokol, S. J. Fidanza, M. A. Goodall, J. Parenter. Enteral Nutr . 1989 , 13 , 168–171. 153. J. Jankovic, J. Neurol. Neurosurg. Psychiatry 2008 , 79 , 368–376. 154. A. J. Lees, J. Hardy, T. Revesz, Lancet 2009 , 373 , 2055–2066. 155. E. Tolosa, G. Wenning, W. Poewe, Lancet Neurol . 2006 , 5 , 75–86. 156. C. E. Gavin, K. K. Gunter, T. E. Gunter, Toxicol. Appl. Pharmacol . 1992 , 115 , 1–5. 157. M. Aschner, K. M. Erikson, E. Herrero Hernandez, R. Tjalkens, Neuromolecular Med . 2009 , 11 , 252–266. 7 Manganese in Health and Disease 225

158. D. L. Stredrick, A. H. Stokes, T. J. Worst, W. M. Freeman, E. A. Johnson, L. H. Lash, M. Aschner, K. E. Vrana, Neurotoxicology 2004 , 25 , 543–553. 159. B. A. Racette, J. A. Antenor, L. McGee-Minnich, S. M. Moerlein, T. O. Videen, V. Kotagal, J. S. Perlmutter, Mov. Disord. 2005, 20 , 492–496. 160. T. R. Guilarte, Environ. Health Perspect . 2010 , 118 , 1071–1080. 161. S. von Campenhausen, B. Bornschein, R. Wick, K. Botzel, C. Sampaio, W. Poewe, W. Oertel, U. Siebert, K. Berger, R. Dodel, Eur. Neuropsychopharmacol . 2005 , 15 , 473–490. 162. A. Eisen, S. Kim, B. Pant, Muscle Nerve 1992 , 15 , 219–224. 163. E. P. Pioro, H. Mitsumoto, Clin. Neurosci . 1995 , 3 , 375–385. 164. S. H. Appel, D. Beers, L. Siklos, J. I. Engelhardt, D. R. Mosier, Amyotroph. Lateral Scler. Other Motor Neuron. Disord . 2001 , 2 Suppl. 1 , S47–54. 165. Y. Kawahara, S. Kwak, Amyotroph. Lateral Scler. Other Motor Neuron. Disord . 2005 , 6 , 131–144. 166. Y. Li, J. C. Copin, L. F. Reola, B. Calagui, G. T. Gobbel, S. F. Chen, S. Sato, C. J. Epstein, P. H. Chan, Brain Res . 1998 , 814 , 164–170. 167. J. Tomkins, S. J. Banner, C. J. McDermott, P. J. Shaw, Neuroreport 2001 , 12 , 2319–2322. 168. E. Kapaki, C. Zournas, G. Kanias, T. Zambelis, A. Kakami, C. Papageorgiou, J. Neurol. Sci . 1997 , 147 , 171–175. 169. H. Nagata, S. Miyata, S. Nakamura, M. Kameyama, Y. Katsui, J. Neurol. Sci . 1985 , 67 , 173–178. 170. A. Gaeta, R. C. Hider, Br. J. Pharmacol . 2005 , 146 , 1041–1059. 171. L. A. Hazelwood, R. B. Free, D. M. Cabrera, M. Skinbjerg, D. R. Sibley, J. Biol. Chem . 2008 , 283 , 36441–36453. 172. C. J. Maynard, R. Cappai, I. Volitakis, R. A. Cherny, A. R. White, K. Beyreuther, C. L. Masters, A. I. Bush, Q. X. Li, J. Biol. Chem . 2002 , 277 , 44670–44676. 173. L. Esposito, J. Raber, L. Kekonius, F. Yan, G. Q. Yu, N. Bien-Ly, J. Puolivali, K. Scearce- Levie, E. Masliah, L. Mucke, J. Neurosci . 2006 , 26 , 5167–5179. 174. G. P. Bates, Nat. Rev. Genet . 2005 , 6 , 766–773. 175. N. S. Wexler, J. Lorimer, J. Porter, F. Gomez, C. Moskowitz, E. Shackell, K. Marder, G. Penchaszadeh, S. A. Roberts, J. Gayan, D. Brocklebank, S. S. Cherny, L. R. Cardon, J. Gray, S. R. Dlouhy, S. Wiktorski, M. E. Hodes, P. M. Conneally, J. B. Penney, J. Gusella, J. H. Cha, M. Irizarry, D. Rosas, S. Hersch, Z. Hollingsworth, M. MacDonald, A. B. Young, J. M. Andresen, D. E. Housman, M. M. De Young, E. Bonilla, T. Stillings, A. Negrette, S. R. Snodgrass, M. D. Martinez-Jaurrieta, M. A. Ramos-Arroyo, J. Bickham, J. S. Ramos, F. Marshall, I. Shoulson, G. J. Rey, A. Feigin, N. Arnheim, A. Acevedo-Cruz, L. Acosta, J. Alvir, K. Fischbeck, L. M. Thompson, A. Young, L. Dure, C. J. O’Brien, J. Paulsen, A. Brickman, D. Krch, S. Peery, P. Hogarth, D. S. Higgins, Jr., B. Landwehrmeyer, Proc. Natl. Acad. Sci. USA 2004 , 101 , 3498–3503. 176. B. B. Williams, D. Li, M. Wegrzynowicz, B. K. Vadodaria, J. G. Anderson, G. F. Kwakye, M. Aschner, K. M. Erikson, A. B. Bowman, J. Neurochem . 2010 , 112 , 227–237. 177. B. B. Williams, G. F. Kwakye, M. Wegrzynowicz, D. Li, M. Aschner, K. M. Erikson, A. B. Bowman, Toxicol. Sci . 2010 , 117 , 169–179. 178. M. Wegrzynowicz, H. K. Holt, D. B. Friedman, A. B. Bowman, J. Proteome Res . 2012 , 11 , 1118–1132. 179. M. Aschner, Environ. Health Perspect . 2000 , 108 Suppl. 3 , 429–432. 180. L. Normandin, A. S. Hazell, Metab. Brain Dis . 2002 , 17 , 375–387. 181. A. W. Dobson, K. M. Erikson, M. Aschner, Ann. N. Y. Acad. Sci . 2004 , 1012 , 115–128. 182. P. K. Pal, A. Samii, D. B. Calne, Neurotoxicology 1999 , 20 , 227–238. 183. C.C. Huang, Y.H. Weng, C.S. Lu, N.S. Chu, T.C. Yen, J. Neurol . 2003 , 250 , 1335–1339. 184. S. A. McDougall, C. M. Reichel, C. M. Farley, M. M. Flesher, T. Der-Ghazarian, A. M. Cortez, J. J. Wacan, C. E. Martinez, F. A. Varela, A. E. Butt, C. A. Crawford, Neuroscience 2008 , 154 , 848–860. 185. C. M. Reichel, J. J. Wacan, C. M. Farley, B. J. Stanley, C. A. Crawford, S. A. McDougall, Neurotoxicol. Teratol . 2006 , 28 , 323–332. 226 Avila, Puntel, and Aschner

186. A. McNeill, D. Birchall, S. J. Hayﬂ ick, A. Gregory, J. F. Schenk, E. A. Zimmerman, H. Shang, H. Miyajima, P. F. Chinnery, Neurology 2008 , 70 , 1614–1619. 187. J. Donaldson, D. McGregor, F. LaBella, Can. J. Physiol. Pharmacol . 1982 , 60 , 1398–1405. 188. K. M. Erikson, D. C. Dorman, L. H. Lash, M. Aschner, Toxicol. Sci . 2007 , 97 , 459–466. 189. J. Donaldson, F. S. LaBella, D. Gesser, Neurotoxicology 1981 , 2 , 53–64. 190. R. Graumann, I. Paris, P. Martinez-Alvarado, P. Rumanque, C. Perez-Pastene, S. P. Cardenas, P. Marin, F. Diaz-Grez, R. Caviedes, P. Caviedes, J. Segura-Aguilar, Pol. J. Pharmacol . 2002 , 54 , 573–579. 191. S. H. Reaney, D. R. Smith, Toxicol. Appl. Pharmacol . 2005 , 205 , 271–281. 192. J. Segura-Aguilar, C. Lind, Chem. Biol. Interact . 1989 , 72 , 309–324. 193. B. Chance, J. Biol. Chem . 1965 , 240 , 2729–2748. 194. T. E. Gunter, C. E. Gavin, M. Aschner, K. K. Gunter, Neurotoxicology 2006 , 27 , 765–776. 195. T. E. Gunter, D. R. Pfeiffer, Am. J. Physiol . 1990 , 258 , C755–786. 196. S. F. Ali, H. M. Duhart, G. D. Newport, G. W. Lipe, W. Slikker, Jr., Neurodegeneration 1995 , 4 , 329–334. 197. S. Zhang, J. Fu, Z. Zhou, Toxicol. In Vitro 2004 , 18 , 71–77. 198. H. R. Scholte, J. Bioenerg. Biomembr . 1988 , 20 , 161–191. 199. D. HaMai, A. Campbell, S. C. Bondy, Free Radic. Biol. Med . 2001 , 31 , 763–768. 200. C. J. Chen, S. L. Liao, Exp. Neurol . 2002 , 175 , 216–225. 201. C. E. Gavin, K. K. Gunter, T. E. Gunter, Biochem. J . 1990 , 266 , 329–334. 202. F. Spadoni, A. Stefani, M. Morello, F. Lavaroni, P. Giacomini, G. Sancesario, Exp. Brain Res . 2000 , 135 , 544–551. 203. Z. Yin, J. L. Aschner, A. P. dos Santos, M. Aschner, Brain Res . 2008 , 1203 , 1–11. 204. M. Zoratti, I. Szabo, Biochim. Biophys. Acta 1995 , 1241 , 139–176. 205. K. V. Rao, M. D. Norenberg, J. Biol. Chem . 2004 , 279 , 32333–32338. 206. D. Milatovic, Z. Yin, R. C. Gupta, M. Sidoryk, J. Albrecht, J. L. Aschner, M. Aschner, Toxicol. Sci . 2007 , 98 , 198–205. 207. D. R. Green, J. C. Reed, Science 1998 , 281 , 1309–1312. 208. S. J. Korsmeyer, M. C. Wei, M. Saito, S. Weiler, K. J. Oh, P. H. Schlesinger, Cell Death Differ . 2000 , 7 , 1166–1173. 209. P. X. Petit, S. A. Susin, N. Zamzami, B. Mignotte, G. Kroemer, FEBS Lett . 1996 , 396 , 7–13. 210. E. A. Malecki, Brain Res. Bull . 2001 , 55 , 225–228. 211. M. K. Chen, J. S. Lee, J. L. McGlothan, E. Furukawa, R. J. Adams, M. Alexander, D. F. Wong, T. R. Guilarte, Neurotoxicology 2006 , 27 , 229–236. 212. M. Aschner, M. Gannon, H. K. Kimelberg, J. Neurochem . 1992, 58 , 730–735. 213. M. Aschner, K. E. Vrana, W. Zheng, Neurotoxicology 1999 , 20 , 173–180. 214. K. V. Rama Rao, P. V. Reddy, A. S. Hazell, M. D. Norenberg, Neurotoxicology 2007 , 28 , 807–812. 215. A. S. Hazell, M. D. Norenberg, Neurochem. Res . 1997 , 22 , 1443–1447. 216. K. Erikson, M. Aschner, Neurotoxicology 2002 , 23 , 595–602. 217. K. M. Erikson, R. L. Suber, M. Aschner, Neurotoxicology 2002 , 23 , 281–288. 218. M. Sidoryk-Wegrzynowicz, E. Lee, J. Albrecht, M. Aschner, J. Neurochem . 2009 , 110 , 822–830. 219. T. D. Hexum, R. Fried, Neurochem. Res . 1979 , 4 , 73–82. 220. A. S. Hazell, M. D. Norenberg, Neurochem. Res . 1998 , 23 , 869–873. 221. A. S. Hazell, Neurochem. Int . 2002 , 41 , 271–277. 222. A. Volterra, Ren. Physiol. Biochem . 1994 , 17 , 165–167. 223. W. Zhou, J. B. Milder, C. R. Freed, J. Biol. Chem . 2008 , 283 , 9863–9870. 224. A. A. Aboud, A. M. Tidball, K. K. Kumar, M. D. Neely, K. C. Ess, K. M. Erikson, A. B. Bowman, Neurotoxicology 2012 , 33 , 1443–1449. 225. Y. Higashi, M. Asanuma, I. Miyazaki, N. Hattori, Y. Mizuno, N. Ogawa, J. Neurochem . 2004 , 89 , 1490–1497. 226. K. Sriram, G. X. Lin, A. M. Jefferson, J. R. Roberts, O. Wirth, Y. Hayashi, K. M. Krajnak, J. M. Soukup, A. J. Ghio, S. H. Reynolds, V. Castranova, A. E. Munson, J. M. Antonini, FASEB J . 2010 , 24 , 4989–5002. 7 Manganese in Health and Disease 227

227. J. A. Roth, S. Singleton, J. Feng, M. Garrick, P. N. Paradkar, J. Neurochem . 2010 , 113 , 454–464. 228. S. Hague, E. Rogaeva, D. Hernandez, C. Gulick, A. Singleton, M. Hanson, J. Johnson, R. Weiser, M. Gallardo, B. Ravina, K. Gwinn-Hardy, A. Crawley, P. H. St George-Hyslop, A. E. Lang, P. Heutink, V. Bonifati, J. Hardy, Ann. Neurol . 2003 , 54 , 271–274. 229. J. A. Olzmann, J. R. Bordelon, E. C. Muly, H. D. Rees, A. I. Levey, L. Li, L. S. Chin, J. Comp. Neurol . 2007 , 500 , 585–599. 230. R. M. Canet-Aviles, M. A. Wilson, D. W. Miller, R. Ahmad, C. McLendon, S. Bandyopadhyay, M. J. Baptista, D. Ringe, G. A. Petsko, M. R. Cookson, Proc. Natl. Acad. Sci. USA 2004 , 101 , 9103–9108. 231. T. Taira, Y. Saito, T. Niki, S. M. Iguchi-Ariga, K. Takahashi, H. Ariga, EMBO Rep . 2004 , 5 , 213–218. 232. C. Paisan-Ruiz, A. E. Lang, T. Kawarai, C. Sato, S. Salehi-Rad, G. K. Fisman, T. Al-Khairallah, P. St George-Hyslop, A. Singleton, E. Rogaeva, Neurology 2005 , 65 , 696–700. 233. J. P. Covy, B. I. Giasson, J. Neurochem . 2010 , 115 , 36–46. 234. M. R. Dewitt, P. Chen, M. Aschner, Biochem. Biophys. Res. Commun . 2013 , 432 , 1–4. 235. K. Tuschl, P. T. Clayton, S. M. Gospe, Jr., S. Gulab, S. Ibrahim, P. Singhi, R. Aulakh, R. T. Ribeiro, O. G. Barsottini, M. S. Zaki, M. L. Del Rosario, S. Dyack, V. Price, A. Rideout, K. Gordon, R. A. Wevers, W. K. Chong, P. B. Mills, Am. J. Hum. Genet . 2012 , 90 , 457–466. 236. A. P. Santos, R. L. Lucas, V. Andrade, M. L. Mateus, D. Milatovic, M. Aschner, M. C. Batoreu, Toxicol. Appl. Pharmacol . 2012 , 258 , 394–402. 237. D. Santos, D. Milatovic, V. Andrade, M. C. Batoreu, M. Aschner, A. P. Marreilha dos Santos, Toxicology 2012 , 292 , 90–98. 238. D. Milatovic, T. J. Montine, M. Aschner, Methods Mol. Biol . 2011 , 758 , 195–204. 239. D. Milatovic, R. C. Gupta, Y. Yu, S. Zaja-Milatovic, M. Aschner, Toxicol. Appl. Pharmacol . 2011 , 256 , 219–226. 240. J. A. Moreno, K. M. Streifel, K. A. Sullivan, W. H. Hanneman, R. B. Tjalkens, Toxicol. Sci . 2011 , 122 , 121–133. 241. E. Lee, M. Sidoryk-Wegrzynowicz, Z. Yin, A. Webb, D. S. Son, M. Aschner, Glia 2012 , 60 , 1024–1036. 242. Z. Xu, K. Jia, B. Xu, A. He, J. Li, Y. Deng, F. Zhang, Toxicol. Ind. Health 2010 , 26 , 55–60. 243. F. M. Cordova, A. S. Aguiar, Jr., T. V. Peres, M. W. Lopes, F. M. Goncalves, D. Z. Pedro, S. C. Lopes, C. Pilati, R. D. Prediger, M. Farina, K. M. Erikson, M. Aschner, R. B. Leal, Arch. Toxicol . 2013 , 92 , 638–644. 244. Y. Chtourou, K. Trabelsi, H. Fetoui, G. Mkannez, H. Kallel, N. Zeghal, Neurochem. Res . 2011 , 36 , 1546–1557. 245. E. N. Martins, N. T. Pessano, L. Leal, D. H. Roos, V. Folmer, G. O. Puntel, J. B. Rocha, M. Aschner, D. S. Avila, R. L. Puntel, Brain Res. Bull . 2012 , 87 , 74–79. 246. K. M. Streifel, J. A. Moreno, W. H. Hanneman, M. E. Legare, R. B. Tjalkens, Toxicol. Sci . 2012 , 126 , 183–192. 247. J. A. Roth, Neuromolecular Med . 2009 , 11 , 281–296. 248. A. Benedetto, C. Au, D. S. Avila, D. Milatovic, M. Aschner, PLoS Genet . 2010 , 6 , e1001084. Chapter 8 Iron: Effect of Overload and Deficiency

Robert C. Hider and Xiaole Kong

Contents ABSTRACT...... 230 1 Introduction...... 231 1.1 Aqueous Iron Solution Chemistry...... 231 1.2 Iron-Dependent Proteins. The Nature of the Iron Binding Sites...... 235 1.2.1 Heme-Containing Proteins...... 235 1.2.2 Iron-Sulfur Proteins...... 236 1.2.3 non-heme, Non-sulfur, Iron-Dependent Enzymes...... 237 1.2.4 Transport and Iron Storage Proteins...... 238 1.3 Iron Transport...... 240 1.3.1 Cellular Iron Transport...... 241 1.3.2 Regulation of Iron Metabolism...... 245 1.4 Iron Physiology...... 246 1.4.1 The Role of Hepcidin...... 247 2 Iron Deficiency and Anemia...... 248 2.1 Iron Requirements of Man...... 248 2.2 The Influence of Anemia on Human Physiology...... 250 2.3 Dietary Sources of Iron...... 252 2.4 Iron Fortification...... 253 2.5 Oral Iron Supplementation...... 253 2.6 Anemia of Chronic Disease...... 254 3 Systemic Iron Overload...... 255 3.1 non-transferrin Bound Iron...... 256 3.2 Hereditary Hemochromatosis...... 256 3.2.1 HFE Hemochromatosis...... 257 3.2.2 Juvenile Hemochromatosis...... 257 3.2.3 ferroportin Disease...... 257 3.2.4 Treatment by Iron Chelation...... 258

R.C. Hider (*) • X. Kong Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, Stamford Street, London, SE1 9NH, UK e-mail: [email protected]; [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 229 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_8, © Springer Science+Business Media Dordrecht 2013 230 Hider and Kong

3.3 Transfusional Siderosis...... 258 3.3.1 The Hemoglobinopathies...... 258 3.3.2 Myelodysplastic Syndrome...... 262 3.4 Hereditary Disorders of Mitochondrial Iron Overload...... 263 3.4.1 Sideroblastic Anemia...... 263 3.4.2 friedreich’s Ataxia...... 263 3.4.3 Glutaredoxin-5 Deficiency...... 264 3.5 Animal Models of Iron Overload...... 264 3.6 Genetic Screening for Thalassemia...... 264 4 Iron-Selective Chelators with Therapeutic Potential...... 266 4.1 Design Features...... 266 4.1.1 Metal Selectivity...... 266 4.1.2 Ligand Selection...... 267 4.1.3 Hexadentate, Tridentate, and Bidentate Iron(III) Complexes...... 267 4.1.4 Critical Features for Clinical Application: Molecular Size and Hydrophobicity...... 270 4.1.5 Toxicity of Chelators and Their Iron Complexes...... 270 4.2 Orally Active Iron Chelators in Current Use...... 272 4.2.1 Tridentate Chelators...... 272 4.2.2 Bidentate Chelators...... 274 4.2.3 use of Iron Chelators to Treat Diseases Other than Thalassemia...... 276 5 nEuropathology and Iron...... 277 5.1 Alzheimer’s Disease...... 278 5.2 Parkinson’s Disease...... 279 5.3 Pantothenate Kinase-2 Deficiency...... 279 5.4 Macular Degeneration...... 279 5.5 Potential of Iron Chelators for the Treatment of Neurodegeneration...... 280 6 The Role of Iron Chelation in Cancer Therapy...... 281 7 Iron and Infection...... 282 7.1 Tuberculosis...... 283 7.2 Malaria...... 283 8 Overview and Future Developments...... 284 Abbreviations...... 285 References...... 286

Abstract Iron is a redox active metal which is abundant in the Earth’s crust. It has played a key role in the evolution of living systems and as such is an essential element in a wide range of biological phenomena, being critical for the function of an enormous array of enzymes, energy transduction mechanisms, and oxygen carriers. The redox nature of iron renders the metal toxic in excess and consequently all biological organisms carefully control iron levels. In this overview the mechanisms adopted by man to control body iron levels are described. Low body iron levels are related to anemia which can be treated by various forms of iron fortification and supplementation. Elevated iron levels can occur systemi- cally or locally, each giving rise to specific symptoms. Systemic iron overload results from either the hyperabsorption of iron or regular blood transfusion and can be treated by the use of a selection of iron chelating molecules. The symptoms of many forms of neurodegeneration are associated with elevated levels of iron in certain regions of the brain and iron chelation therapy is beginning to find an application in the treatment of such diseases. Iron chelators have also been widely 8 Iron: Effect of Deficiency and Overload 231 investigated for the treatment of cancer, tuberculosis, and malaria. In these latter studies, selective removal of iron from key enzymes or iron binding proteins is sought. Sufficient selectivity between the invading organism and the host has yet to be established for such chelators to find application in the clinic. Iron chelation for systemic iron overload and iron supplementation therapy for the treatment of various forms of anemia are now established procedures in clinical medicine. Chelation therapy may find an important role in the treatment of various neurodegenerative diseases in the near future.

Keywords anemia • iron • iron chelators • iron overload • iron toxicity • neurodegeneration

Please cite as: Met. Ions Life Sci. 13 (2013) 229–294

1 Introduction

1.1 Aqueous Iron Solution Chemistry

Iron, element 26 in the periodic table, is the fourth most abundant element in the Earth’s crust. It has the maximum value of nuclear binding energy of all elements and so tends to accumulate as a result of star based fusion reactions. It is 1000 times more abundant than both copper and zinc. Its position in the middle of the first transition metal row of the periodic table leads to incompletely filled d orbitals and thus the possibility of various oxidation states, the most common being (II), d6, (III), d5, and (IV), d4. Iron(IV) is highly reactive and is restricted to intermediates formed during enzyme catalytic cycles, whereas iron(II) and iron(III) are widely distributed in solution, in the solid phase, and bound to proteins. When iron salts (both iron(II) and iron(III)) are dissolved in water, they undergo hydrolysis (equation 1), the process effectively being the deprotonation of coordinated water molecules.

3+ 2+ OH2 OH2 H O H O 2 OH2 2 OH FeIII FeIII + H+ H O H O 2 OH2 2 OH2 (1) OH2 OH2

With iron(II) (ferrous iron), the hydrolysis of micromolar solutions tends to take place at pH values greater than 7.0 (Figure 1). In contrast, with iron(III) (ferric iron), the charge density on the metal ion is much higher, thereby polarizing the water 232 Hider and Kong

Figure 1 Iron(III) and iron(II) speciation plots; [iron]total, 1 μM; KH2O solubility products, iron(III): 2.79 × 10–39 M4, iron(II): 4.87 × 10–17 M3.

molecule more effectively than iron(II). As a result, dissociation of the proton from hexa-aqua iron(III) (1) occurs more easily, hydrolysis initiating at much lower pH values, typically pH 2.0 (Figure 1). Whereas it is possible to prepare a 10 μM iron(II) sulfate solution at pH 7.0, the maximum concentration of iron(III) sulfate at this pH values will be less than 10–18M. Hydroxide ions are also able to crosslink hydrated iron(III) species forming dimers and oligomeric anions (2, 3) [1], finally generating an insoluble polymer of ferrihydrate [2]. The condensation reaction occurs rapidly at

neutral pH values, the initial phase of oligomer formation producing species between 16 and 30 iron atoms. In the presence of suitable ligands, for instance HEIDI (4) and citrate (5), these complexes can be stabilized [3]. As a direct consequence of this high affinity for oxygen donors, iron(III) must be coordinated by ligands in order to maintain appreciable aqueous solubility at most physiological pH values. 8 Iron: Effect of Deficiency and Overload 233

CO2H CO2H HO

N CO2H HO2C HO CO2H

The two most common geometries for iron(II) and iron(III) complexes are octahedral for 6-coordinate complexes (1) and tetrahedral for 4-coordinate complexes (6). Octahedral stereochemistry is more frequently observed. As iron(III) possesses a smaller ionic radii than iron(II) (0.65 and 0.78 Å, respectively) and a higher net charge (3+ and 2+), the charge density on the iron(III) ion is much greater than that on the iron(II) ion (0.59 versus 0.27 eÅ–2). This large difference leads to a markedly different ligand selectivity. Iron(III) favors coordination by charged oxygen atoms such as hydroxide, phenoxide, phosphate, and carboxylates, whereas iron(II) favors interaction with aromatic amines, imidazoles, and sulfhydryl groups (see Table 1 [4] for a direct comparison of affinity constants with a range of ligands). As a result of this difference in ligand selectivity, the addition of complexing agents has a major influence on the FeIII/FeII reduction potential. tris-Phenanthroline 3+ iron(II) is exceptionally stable, shifting the reduction potential between Fe(Phen)3 2+ and Fe(Phen)3 in favor of the iron(II) complex (E0 = +1.1 volt). In contrast, desferrioxamine-B (7), an iron(III)-selective siderophore binds iron(III) much more

HO O H S N N O Fe S S CH3 S O N + H3N HN O HO O N OH 6 7 tightly than iron(II) resulting with a reduction potential of −0.45 volt. The range of reduction potentials for iron complexes spans the range +1.1 v to −0.8 v and it is significant to note that a large proportion of this range is covered by iron-proteins (+0.4 v to −0.5 v) [5]. The redox activity of the FeIII/FeII pair can, depending on the coordination of the metal, reduce molecular oxygen to form the superoxide anion (equation 2) and reduce hydrogen peroxide to the hydroxyl radical (equation 3), the latter equation corresponding to the Fenton reaction. As superoxide can be further

FeII OOFeIII ·− (2) ++22 FeIII HO FeIII OH− OH· (3) ++22 + 234 Hider and Kong

Table 1 Stability constants for iron complexes. Iron(III) Iron(II) III II Ligand log Keq N pFe log Keq N pFe Catecholate O 43.8 3 15.5 13.5 2 6.0

O Phenanthroline 14.1 3 <14.6 21.0 3 11.5

N N

Oxalate − O O 18.5 3 <14.6 5.2 2 6.0

O O − Acetohydroxamate O 28.3 3 <14.6 8.5 2 7.6

O N H Citric acid (5) 18.2 2 16.7 4.4 1 6.1 Nitrilotriacetate + 24.0 2 18.1 12.8 2 7.1 O C H CO3 3 N

CO3

EDTA O2C O C 25.1 1 23.5 14.3 1 12.4 + 2 NH + NH CO2 CO2 Desferrioxamine-B (7) 30.5 1 26.6 9.5 1 6.0 Histidine O 4.7 1 <14.6 10.4 2 6.0

N O + NH HN 3 The affinity constants refer to the cumulative constants in the following equation: KK12K3 Fe ++LLFeLFeL23+ L  FeL . pFe is defined as concentration of uncoordinated iron –6 –5 III when [Fe]total = 10 M, [Ligand]total = 10 M at pH 7.4. Data taken from [4]. For pFe , a value of II <14.6 indicates that the ligand has a lower affinity than H2O at pH 7.4. For pFe , a value of 6.0 indicates that the ligand does not bind iron(II) at pH 7.4. N: maximum number of ligands in complex converted to the highly damaging hydroxyl radical, rapid generation of these species is damaging to cells [6]. If the reduction potential of an iron complex is either high (> +0.2 v) or low (< −0.2 v) then redox cycling does not occur readily because, under the former condition iron(II) is preferentially stabilized and under 8 Iron: Effect of Deficiency and Overload 235 the latter, iron(III) is preferentially stabilized. However with a ligand possessing a reduction potential close to zero, for instance EDTA (Eo = +0.12 v) both the iron(III) and iron(II) complexes are relatively stable and so the coordinated iron can readily redox cycle between the two oxidation states. As with ligand selectivity, the kinetic lability of a metal complex is heavily dependent on the charge density of the cation, the higher the value, the slower the rate of exchange. One method adopted to compare the lability of a series of metal ions is to monitor the rate of exchange of water molecules on the corresponding aqueous ions (equation 4). For ions such as Ca2+, which have a low charge density,

Fe()HO3+ + HO*(Fe HO)*HO + HO (4) 26 22 52()2 the water-exchange rates approach the diffusion limit (k = 5 × 108 s–1); this renders Ca2+ ideal for a rapid signalling role in cells. The value for iron(II) is 4.4 × 106 s–1 and for iron(III) it is 1.6 × 102 s–1, iron(III) exchanging much more slowly than iron(II). Clearly, iron(II) is kinetically labile and is able to exchange rapidly between binding sites. In contrast iron(III) is much less labile, the rate of exchange between multidentate ligands being particularly slow; for instance the half-life of the donation of iron(III) from FeIII∙EDTA (1mM) to desferrioxamine-B (7, 1 mM) is 42 h [5].

1.2 Iron-Dependent Proteins. The Nature of the Iron Binding Sites

Man has over 500 iron-containing metalloproteins [2].

1.2.1 Heme-Containing Proteins

In this protein class, iron is bound to a porphyrin molecule by the 4 aromatic nitrogen atoms in octahedral fashion, the axial ligands are typically provided by the protein, but can also provide a binding site for gaseous molecules such as dioxygen (Figure 2). There are three main classes of heme proteins: oxygen carriers, as typified by hemoglobin and myoglobin; activators of molecular oxygen, as typified by peroxidases, catalases, and cytochrome P450s. Peroxidases and catalases oxidize a range of compounds using H2O2 as a substrate; cytochrome P450s hydroxylate a wide range of xenobiotic and endogenous substrates, including steroids, using oxygen as a substrate. Electron transport proteins, as typified by cytochromes a, b, and c, are components of the respiratory chain, they accept electrons from reduced donor molecules, transferring them to appropriate acceptors, thereby linking substrate oxidation to cytochrome c oxidase [2]. 236 Hider and Kong

Figure 2 Heme center in hemoglobin.

Figure 3 2Fe-2S and 4Fe-4S clusters: S; Fe.

1.2.2 Iron-Sulfur Proteins

In this protein class, iron is bound to sulfur in tetrahedral fashion. Two predominate core structures are found in man, 2Fe-2S clusters and 4Fe-4S clusters (Figure 3). Many of the proteins that contain these clusters are involved in electron transfer. Thus ferredoxins are located in electron transfer chains and can also act as donors to enzymes where they exchange electrons between redox centers which are physically separated. The valence state of 2Fe-2S clusters oscillates between 2+ and 3+ and with 4Fe-4S clusters, between 1+ and 2+. The activity of these proteins is not limited to one-electron transfer and iron-sulfur clusters are found in dehydratases and S-adenosylmethionine-dependent enzymes [2]. 8 Iron: Effect of Deficiency and Overload 237

1.2.3 Non-heme, Non-sulfur, Iron-Dependent Enzymes

These enzymes fall into two broad categories, those containing either mononuclear or dinuclear iron. A large number of mononuclear non-heme iron enzymes are involved with the activation of dioxygen to catalyze the hydroxylation of a wide range of substrates. With the α-oxoglutarate-dependent enzymes, iron(II) is bound to the enzyme via two histidines and an aspartate residue (Figure 4) and is involved with the hydroxylation of amino acids and the demethylation of histones [6]. Pterin-dependent hydroxylases fall into a related enzyme group and these are responsible for the hydroxylation of aromatic amino acids [6]. The dinuclear iron proteins typically contain a four helix bundle protein fold surrounding a (μ-carboxylato)diiron core. The two iron atoms are typically separated by less than 0.4 nm and have one or more bridging carboxylate ligands, together with bridging oxo or hydroxo ligands [7]. The diiron center is bound to the protein via imidazole and carboxylate functions (Figure 5). Such centers are found in the H chains of ferritin, where iron(II) is oxidized to iron(III) and deposited in a core of ferrihydrite and in ribonucleotide reductase where the diiron center generates a tyrosyl radical which is utilized in the conversion of ribosides to deoxyribosides [7].

Figure 4 Proposed mechanism of Fe(II)- and 2-oxoglutarate-dependent dioxygenase-catalyzed reactions. In the example of asparaginyl hydroxylation, the reaction proceeds through a radical mechanism involving an iron-oxo intermediate, which adopts both iron(III) and iron(IV) oxidation states during the process. The decarboxylation of 2-oxoglutarate occurs simultaneously. Reproduced with permission from [6]; copyright 2007, Nature Publishing Group. 238 Hider and Kong

Figure 5 The metal binding sites of cytoplasmic and nuclear iron(II)-dependent enzymes. (a) ribonucleotide reductase; (b) ferroxidase center on the H-ferritin subunit.

1.2.4 Transport and Iron Storage Proteins

In view of the potential redox activity of many simple iron complexes, the levels of such labile iron forms are maintained at carefully controlled limits, both extracellularly and intracellularly. In the extracellular compartments iron is almost entirely transported between cells, tightly bound to transferrin. Within cells, ferritin acts as an iron sink and is in equilibrium with the labile iron(II) pool, which depending on the cell type, is limited to the range 0.5–2 μM [8]. Serum transferrin is a globular protein which possesses two high affinity iron(III) binding sites (Figure 6a) [9]. Each site consists of two tyrosines, one histidine, one aspartate, provided by the protein, together with a synergistically bound carbonate anion. These ligands create an octahedral coordination sphere (Figure 6b) with a high selectivity for iron(III) and an affinity of 10–20 M–1 (conditional stability constant at pH 7.4) [9]. Serum transferrin is typically circulating in the blood at a level of 35 μM, with between 25 and 35% of the iron binding sites occupied. Under such conditions the levels of the so called non-transferrin bound iron are vanishingly small. Transferrin delivers iron to cells which express transferrin receptors [10,11]. Ferritin is a protein composed of 24 homologous subunits, designed to create a large aqueous enclosure for the storage of iron atoms. Each ferritin molecule can accommodate up to 4500 iron atoms [12] (Figure 7). Ferritin is the major iron storage protein for all mammalian tissues and consists of a mixture of two subunits referred to as L- and H-subunits. In general, L-rich ferritins are characteristic of 8 Iron: Effect of Deficiency and Overload 239

Figure 6 (a) Human serum transferrin [9]; (b) coordination sphere of ferric ion in the N-lobe binding site of human serum transferrin [11]. Reproduced with permission from [25]; copyright 2012, Elsevier.

organs storing iron for long periods (e.g., liver) and these molecules tend to possess a high iron content. H-rich ferritins, which are characteristic of the heart, have a major role in iron detoxification, they possess a ferroxidase site (see Figure 5b) and tend to contain a lower iron content. Iron(II) enters the various pores of the oligomeric structure (Figure 7b) and is rapidly oxidized to iron(III) either by the growing core of ferrihydrite or at the ferroxidase site of the H-subunit [13]. Ferritin molecules are constantly being synthesized and removed to lysosomes, where they are degraded, thereby releasing the entrapped iron [14]. This iron enters the cytosolic labile iron pool under the tight control of iron(II) transport proteins, which are located in the lysosomal membrane. 240 Hider and Kong

Figure 7 The three-dimensional structure of the ferritin subunit and 24-mer. (a) 24-meric human H-chain ferritin molecule viewed down the four-fold symmetry axis (PDB 1FHA). (b) The crystal growth mechanism of core formation in ferritin [13]. Reproduced with permission from [13]; copyright 1981, Elsevier.

1.3 Iron Transport

In mammalian cells there is a labile iron pool which supplies iron to the mitochondrion for incorporation into heme and iron-sulfur cluster proteins (Figure 8). It is also the source of iron for many cytoplasmic iron-dependent enzymes. As non- coordinated iron salts can catalyze the formation of toxic oxygen-containing radicals, the levels of this labile iron pool must be tightly controlled. Cells must be able to sense iron levels and regulate homeostasis in such a manner as to maintain non- toxic levels. The concentration of this iron pool is determined by the rates of iron uptake, utilization for incorporation into iron proteins, storage in ferritin and iron export from the cell (Figure 8). The concentration of the pool falls into the 0.5–2 μM range and a likely structure is iron(II) bound to a single molecule of glutathione (8) [15]. This structure ensures protection against autoxidation of iron(II) and offers a mechanism for the introduction of iron into protein-bound iron sulfur clusters via glutaredoxins [8]. The selection of iron(II) in preference to iron(III) for this role is logical in view of the kinetic lability of iron(II) being 5 × 104 times higher than that of iron(III). 8 Iron: Effect of Deficiency and Overload 241

Figure 8 Cytoplasmic iron fluxes. DMT1, divalent metal transporter-1; IRP, iron responsive protein. FBXL5, F-box and leucine-rich repeat protein 5.

O H O N N H − O O− O + S H3N O H2O OH2 II Fe H2O OH2

OH2

1.3.1 Cellular Iron Transport

Within the circulation, transferrin-bound iron is the principle iron source for all mammalian tissues; although under iron overload conditions, transferrin becomes saturated and non-transferrin bound iron appears in the serum and is taken up by cardiac and endocrine tissue by uncharacterized transporters. 242 Hider and Kong

1.3.1.1 Transport of Iron-Loaded Transferrin

There are at least three classes of transferrin receptors in mammalian cells; TfR1, which is expressed on many cell types, TfR2, which is restricted to hepatocytes and erythroid cells, and a third type, only located in epithelial kidney cells. The basic mechanism of iron transport is the same for each class. Fe2-transferrin-TfR complexes bind to clathrin-coated pits which are internalized into endosomes. The endosome lumen is acidified to pH 5.5, whereupon both transferrin and TfR undergo conformation changes to release transferrin-bound iron. A ferrireductase reduces the released iron(III) to iron(II), which is a substrate for the divalent metal-ion transporter 1 (DMT1) (Figure 9). Both apo transferrin and TfR are recycled to the cell surface. It has been estimated that a transferrin molecule experiences over 200 such cycles during its life time [10].

1.3.1.2 Absorption of Dietary Iron

Inorganic iron present in the diet is predominantly iron(III) and this is reduced on the surface of duodenal enterocytes by the ferrireductase DCYTB (Figure 10). The resulting iron(II) enters the cell through the divalent metal transporter

Figure 9 Macrophage iron fluxes. 8 Iron: Effect of Deficiency and Overload 243

Figure 10 Duodenal enterocyte located on intestinal villae. Associated with the unidirectional movement of iron. which is proton-coupled [16]. DMT1 has been shown to transport other divalent metals, such as Zn(II), Mn(II), and Co(II), but the physiological relevance of this property is unclear. Heme iron is transported from the lumen of the duodenum by the transport protein HCP1 [17]. Once absorbed, heme is degraded by heme oxygenase to form iron(II) (Figure 10). Thus both DMT1 and HCP1 supply iron to the labile iron pool (possibly as FeII. Glutathione, 8). 244 Hider and Kong

1.3.1.3 Mitochondrial Iron Transport

Mitochondria are major sites of iron consumption in mammalian cells as they accommodate the enzymes involved in heme and iron-sulfur cluster synthesis. The uptake of iron is dependent on membrane potential and facilitated by the transport proteins mitoferrin-1 and mitoferrin-2 [18]. Mitoferrin-2 is widely distributed in different tissues, whereas mitoferrin-1 is restricted to erythroid cells. Ferrochelatase, the terminal enzyme in heme synthesis receives iron(II) directly from the inner mitochondrial membrane and has been demonstrated to form a oligomeric complex with mitoferrin-1 [19]. It has been suggested that frataxin shuttles iron(II) from the same membrane, directing iron to enzymes involved in iron sulfur cluster synthesis.

1.3.1.4 Ferroportin-Mediated Iron Efflux

Duodenal enterocytes, macrophages, hepatocytes, and CNS cells release iron in a controlled manner by use of the iron efflux transporter ferroportin (FPN1). This transporter was independently discovered by three groups and was originally termed Ireg1 [20], ferroportin [21], and MTP [22]. The term ferroportin has been widely adopted. The gene encodes a highly hydrophobic membrane protein with 10–12 transmembrane spanning domains, which bears little sequence identity with any other transporter family. Indeed, ferroportin is specific for iron(II) and is the only iron exporter identified to date. Ferroportin is located on the basolateral side of duodenal enterocytes and in the cytoplasmic membranes of both macrophages and hepatocytes (Figure 10). For efficient transfer of freshly effluxed iron(II) to apo transferrin, ferroportin is frequently closely coupled with a multi-copper ferroxidase, for instance, hephaestin or ceruloplasmin, whereupon the iron(II) is rapidly oxidized to iron(III), without the generation of oxygen-containing free radicals.

1.3.1.5 Iron Metabolism Facilitated by the Macrophage

Macrophages acquire iron via the transferrin receptor, hemoglobin, via erythrophagocytosis and the haptoglobin receptor and heme, sequestered by hemopexin via the lipoprotein receptor, CD91 (Figure 9). Changes in the surface glycoproteins of red blood cells take place as they age. These modified proteins are recognized by receptors on the macrophage surface and trigger phagocytosis. Haptoglobin and hemopexin scavenge hemoglobin and heme respectively. Both are released in the circulation as a result of hemolysis (Figure 9) [23]. On entry to the cytoplasm, the heme moiety is degraded by heme oxidase and the resulting iron(II) enters the labile iron pool. On exposure to the lumen of a phagolysosome, hemoglobin is degraded, releasing heme which is able to penetrate the membrane also gaining access to heme oxidase. Iron(II) is exported from the macrophage via ferroportin and the effluxed iron(II) is oxidized to iron(III) by ceruloplasmin. 8 Iron: Effect of Deficiency and Overload 245

1.3.2 Regulation of Iron Metabolism

Iron homeostasis is regulated by balancing iron uptake with intracellular storage and use. In mammalian cells, this is largely achieved at the level of protein synthesis. Regulatory sequences in mRNA molecules are located in the noncoding regions situated at 5’ and 3’ extremities of the coding regions. Location in the 5’ region is usually associated with initiation of translation, whereas location in the 3’ region is associated with enhanced mRNA stability [24]. The regulatory sequences relating to iron metabolism are termed iron responsive elements (IRE) and their structural features are well established [25]. Iron responsive proteins (IRP) bind to IREs and thereby control the translation of the related mRNA. An IRE consists of an upper stem of five base-paired residues that assume a helical structure which is separated from a lower stem of base-paired structures by an unpaired cytosine, thereby creating a bulge in the structure (Figure 11). At the top of the structure is a six membered loop which includes the sequence AGU.

Figure 11 The iron responsive element is an RNA stem-loop structure. The upper and lower stems are composed of base-pairs which are held in a helical conformation. In the six-membered loop AsafNAsafdasdasdasd, the C at position 1 of the loop forms a base-pair with the G at position 5. This C-G base-pair structures the loop, allowing the A, G, and U residues to form a region that can form multiple hydrogen bonds with proteins. The upper and lower stems are separated by an unpaired “bulge” C that confers flexibility on the structure by interrupting the helix. Reproduced with permission from [25]; copyright 1997, Elsevier. 246 Hider and Kong

Figure 12 Mechanism of iron-regulatory protein 1 (IRP1) in the translational regulation of (a) transferrin receptor and (b) ferritin.

Table 2 Known genes with IRE regions. Gene name IRE position Function H and L Ferritin 5’UTR Iron storage Erythroid δ-amino-levulinate synthase 5’UTR Erythroid heme synthesis Succinate dehydrogenase 5’UTR TCA cycle m-Aconitase 5’UTR TCA cycle Ferroportin 5’UTR Iron efflux DMT1 3’UTR Iron uptake Transferrin receptor 3’UTR Iron uptake Cell cycle control gene (CDC14A) 3’UTR Cell cycle

These residues constitute the major recognition feature for IRPs, both the bulged C and the AGU region fit into binding pockets of IRP molecules. The affinity constant for this interaction falls in the range 10–30 pM [2,26]. In man there are two IPRs and when iron levels are low they both bind to mRNA. When iron levels are adequate, IRP1 acquires a [4Fe-4S] cluster converting it to the enzyme aconitase (Figure 8) and IRP2 is degraded by proteasomes. The binding of a IPR to the transferrin receptor mRNA enhances the stability of RNA and hence increases protein synthesis (Figure 12a). This in turn promotes receptor mediated iron uptake. The effect on ferritin mRNA is to reduce protein synthesis (Figure 12b), thereby increasing the availability of intracellular iron. A number of proteins with a central role in iron metabolism are controlled in analogous fashion (Table 2).

1.4 Iron Physiology

While the body levels of other dietary metals can be regulated by excretion in the feces and urine, humans do not possess the ability to remove excess iron from the 8 Iron: Effect of Deficiency and Overload 247

Figure 13 Iron homeostasis in a normal adult man. Loss is uncontrolled (epithelial desquamation and in women, menstruation).

body. Consequently, several proteins have evolved which regulate iron homeostasis, their location being primarily in duodenal endothelial cells, hepatocytes and reticuloendothelial macrophages. It is essential for these cells to function in concert. Regulation of total body iron is achieved by feedback mechanisms that operate on iron absorption. Approximately 4 mg of iron circulates bound to transferrin (this is about 0.1% of total body iron). Seventy five percent of body iron is in the form of heme proteins, mainly hemoglobin, the remainder being located in cells either in non-heme enzymes or ferritin. Although only 1–2 mg of iron is absorbed each day by normal individuals, 20 mg is transferred daily to the bone marrow for erythropoiesis and is efficiently recycled by macrophages (Figure 13). There is no active excretion mechanism for iron; loss is uncontrolled and results mainly from bleeding and epithelial desquamation. It has been estimated that the average iron atom survives in a human for approximately ten years.

1.4.1 The Role of Hepcidin

Hepcidin is a 25 residue peptide containing four disulfide bridges (Figure 14). It has an amphipathic nature and possesses a net positive charge. The structure is highly conserved within mammals and fish [27]. Removal of the N-terminal pentapeptide leads to loss of activity. Hepcidin regulates iron export from a range of cells by 248 Hider and Kong binding to ferroportin, causing internalization and degradation of the iron-efflux protein. The mechanism of this internalization is similar to that of other receptors and involves hepcidin-induced phosphorylation and subsequent ubiquitination of ferroportin. Thus, under conditions of high hepcidin serum levels, ferroportin located in the duodenal epithelial cells and macrophages will be internalized and such cells are unable to efflux iron. Under conditions of low hepcidin serum levels, the opposite holds and thus, high iron fluxes enter the serum originating from both duodenal epithelial cells and macrophages.

Figure 14 Human hepcidin.

Hepcidin is produced primarily in the hepatocyte, the synthesis being regulated by both the iron and the inflammatory status of the organism. High concentrations of fully saturated transferrin trigger the involvement of bone morphogenetic protein (BMP), which in turn facilitates the phosphorylation of the transcription factor SMAD4. SMAD4 controls hepcidin synthesis. Hepcidin synthesis is also regulated by erythropoietic signals such as GDF15. In addition, hepcidin synthesis is regulated by inflammatory stimuli, thus, interleukin 6 activates the JAK/STAT signaling pathway which again stimulates the hepcidin promoter [28]. Thus, under conditions where man is replete in iron (high saturation of transferrin), hepcidin synthesis is increased and there is an overall retention of iron by the duodenum, hepatocyte, and macrophage. The converse also holds, thus conditions, where a low transferrin saturation exists, will lead to the inhibition of hepcidin production and a concomitant enhanced release of iron from the same group of cells. Significantly, elevation of interleukin 6 occurs when man is infected by microorganisms and parasites. Such an action triggers hepcidin synthesis, reducing the extracellular availability of iron and thereby enhancing the host defense [29].

2 Iron Deficiency and Anemia

2.1 Iron Requirements of Man

Healthy term infants with a normal birth weight are born with high hemoglobin levels and sufficient iron stores to support growth for the first six months of life [30]. Babies are born with body iron loads, typically in the region of 80 mg/kg; the corresponding value for adult men is 55 mg/kg. Thus, the low iron content in human milk does not present a problem to the suckling infant and renders infection to be less of a problem. 8 Iron: Effect of Deficiency and Overload 249

Figure 15 Daily iron requirements. Information taken from [31].

Preterm and low birth weight infants exhaust their iron stores at an earlier age than normal children and thus, there is a greater chance of anemia with such children. The transfer of iron from maternal blood to the fetus occurs mainly in the third trimester (Figure 15). Thus premature infants are born with lower iron stores. At six months, solids and cereals should be given to babies, with the goal of beginning to provide appreciable levels of iron in the diet. Iron requirement for the rest of life remains close to 1 mg/day (Figure 15) [31], only exceeding this requirement during the adolescent growth spurt, with menstruating females and during pregnancy. Iron requirements increase dramatically during the second and third trimesters of pregnancy, reaching a requirement of 5–6 mg/day. Iron requirement during lactation does not increase. It is estimated that most pregnant women in developing countries and between 30 and 40% of pregnant women in developed countries are iron-deficient [32]. Blood donations (500 mL/year) require an additional 0.6–0.7 mg iron per day, a significant addition to the normal adult requirement of 1.1 mg/day. In developing countries intestinal parasitic infections can cause appreciable blood loss which in turn leads to increased iron requirements. A list of causes of iron deficiency is presented in Table 3. 250 Hider and Kong

Table 3 Causes of iron Inadequate dietary iron intake deficiency. Single food diets in infancy Dieting, fasting, and malnutrition Diet with high content of inhibitors of iron absorption Antacid therapy Increased iron requirements Rapid growth during adolescence Menstruation Pregnancy Erythropoietin therapy Trauma Increased iron loss Bleeding from gastrointestinal and genitourinary tracts Intravascular hemolysis Parasitic infestations Intense exercise Blood donation Decreased absorption of iron Diseases of the stomach and small intestine Celiac disease Infection by Helicobacter pylori Crohn’s disease Genetic defects Mutation of DMT1 gene Mutation of glutaredoxin-5 gene

2.2 The Influence of Anemia on Human Physiology

After birth, the erythron has the priority for transferrin-bound iron as compared to other tissues. In general, erythrocyte production is unperturbed until the body iron stores are depleted. When the stores become limiting, the saturation of transferrin decreases and patients then show signs of iron-deficient erythropoiesis; protoporphyrin and zinc protoporphyrin appear in the erythrocytes and these changes are followed by the onset of microcytosis. Iron deficiency is known to be associated with decreased physical activity as demonstrated by exercise tolerance and work performance [33]. These effects have been thoroughly investigated in rodent models. As hemoglobin levels decrease, so do those of myoglobin and the cytochromes. Thus, there is diminished oxygen transport in the blood and diminished oxygen diffusion in muscle. There is also a decrease in the mitochondrial iron-sulfur content which is associated with a decrease of mitochondrial oxidative capacity. These changes have a major influence on physical activity; the Harvard Stop Test score registers impaired performance even with mild anemia (Figure 16a) [34], as does the worktime in workers performing a sub maximal exercise test (Figure 16b) [35]. 8 Iron: Effect of Deficiency and Overload 251

Figure 16 (a) Harvard Step Test score among a group of Guatemalan agricultural laborers [34]. (b) Work time in a group of Indonesia agricultural workers performing a submaximal exercise test as a function of severity of anemia [35].

The adverse influence of anemia on mental behavior is more difficult to demonstrate, but as 25% of the body oxygen is utilized by the brain, adverse effects can be anticipated. Iron is required for the myelination of the spinal cord, for the synthesis of chemical transmitters, particularly dopamine and serotonin and for mitochondrial function. The influence of anemia on mental development has been reviewed and details from 18 studies described [36]. Taken together, they support the 252 Hider and Kong hypothesis that iron deficiency is associated with developmental delay. With longer term intervention studies, again there is evidence of a causal link between iron deficiency anemia and poor developmental performance [36].

2.3 Dietary Sources of Iron

There are two major classes of iron in the diet, heme iron and non-heme iron. The former is derived from hemoglobin, myoglobin, and cytochromes and is found in animal sources such as red meat and seafood. Non-heme iron is derived mainly from plant sources such as lentils, beans, rice and cereals (Table 4). The absorption of heme iron is relatively efficient, ranging from 15 to 35%; whereas the absorption of non-heme iron is less efficient (2–20%) and can be inhibited by the presence of other components in the diet. Heme iron, which is strongly chelated by protoporphyrin IX, is absorbed by the HCP-1 transporter (Section 1.3.1.2), and is not subject to competition from other iron-binding compounds which are present in the lumen of the gut. In contrast, non-heme iron is absorbed in the iron(II) state by DMT-1 (Section 1.3.1.2) and so is susceptible to the presence of iron chelating ligands in the gut lumen. Compounds such as phytates, polyphenols, and tannins, which are widely distributed in plant foods, are capable binding to both iron(II) and iron(III), rendering the iron non-bioavailable [37]. When such compounds bind iron(II), they autoxidize the metal to iron(III). Phytate is present in legumes, rice, and cereals. Tannic acid is present in tea and coffee. Their presence can inflict a powerful inhibitory influence

Table 4 Iron content in food. Fe (mg per serving) Food Heme Non-Heme % of daily requirement (male) Liver sausage 85 g 9.4 – 52 Beef steak 85 g 3.2 – 18 Turkey 85 g 2.3 – 13 Chicken breast 85 g 1.1 – 6 Halibut 85 g 0.9 – 5 Iron fortified cereal 236 mL – 18 100 Soybean 236 mL – 8.8 48 Lentils 236 mL – 6.6 36 Spinach 236 mL – 6.4 35 White rice 236 mL – 3.2 18 Raisins 236 mL – 2.7 15 Egg 1 – 1 6 Brown bread 1 slice – 0.9 5 Taken from the US Department of Agriculture database. 236 mL ≡ 1 standard cup 8 Iron: Effect of Deficiency and Overload 253 of non-heme iron absorption. Conversely, the presence of vitamin C (ascorbic acid) enhances the absorption of non-heme iron, the reduced form of the vitamin reducing iron(III) to the more bioavailable iron(II) (equation 5).

O HO O HO O O 3+ +Fe + Fe2+ +H+ HO OH HO O (5) O O AscH Asc A typical iron intake for an adult male is 16–18mg/day, whereas for women it is lower, typically 12 mg/day. Thus, for most situations there should be sufficient iron in the diet, although the consumption of energy-restricted diets or those rich in poorly bioavailable iron can contribute to inadequate iron absorption. Selected food sources and their iron contents are presented in Table 4.

2.4 Iron Fortification

Iron fortification of commonly used foods is a practical and cost-effective strategy to improve the iron nutrition of a large population. The concept has been widely adopted in developed countries over the past 50 years, typically with the iron fortification of breakfast cereals. The efficacy of iron fortification in the developing countries has not been so successful and improved guidelines and materials are required. The following iron preparations have been used; ferrous sulfate (promotes oxidation of fats and so leads to rancidity), ferrous fumarate (likely to be similar to ferrous sulfate), elemental iron (finely divided metallic iron), ferric pyrophosphate and ferric EDTA. The bioavailability of the ferric salts and elemental iron is lower than that of ferrous sulfate. The World Health Organization (WHO) has issued recommendations for a range of iron compounds to be used and the level to be adopted in developing countries. Good efficiency has been reported from South Africa, Morocco, Vietnam, Chile, Thailand, and Kenya [32]. In principle, adverse effects are possible with respect to iron overloading diseases and in regions with a high prevalence of malaria, however, this is less likely to occur with iron fortification when compared with iron supplementation (Section 2.5).

2.5 Oral Iron Supplementation

Another approach to control iron deficiency is iron supplementation, which unlike fortification delivers a relatively large dose of iron in the absence of food. The majority of patients with iron deficiency anemia respond to oral iron therapy. A wide range of iron preparations are available for oral application, the majority being iron(II) by 254 Hider and Kong virtue of its greater bioavailability. Thus ferrous sulfate, ferrous fumarate, and ferrous gluconate are widely marketed, indeed ferrous sulfate was introduced as a therapy for anemia by Blaud in 1832 [38]. By virtue of its low cost and good bioavailability ferrous sulfate is still widely used for oral therapy, however, its use is associated with a range of gastrointestinal side effects that frequently lead to poor compliance. There are “slow release” formulations available which can reduce side effects, but typically they also reduce iron absorption when compared to the parent compound. Another problem with iron(II) salts in oral formulations is that they can inhibit the absorption of a wide range of other therapeutics, including antibiotics, L-dopa, and thyroxine [39]. Up to 40% of patients have symptoms associated with the oral administration of iron(II) salts. These symptoms can be avoided by utilizing iron(III) complexes, although the bioavailability of simpler iron(III) salts is lower than the analogous iron(II) salts due to the extreme low solubility of iron(III) hydroxide (Section 1.1). However, if the iron(III) complex is sufficiently stable and water-soluble, then the bioavailability can reach that achieved by ferrous sulfate. Iron(III) maltol [40] and iron(III) polymaltose [41] are two such compounds reported to possess excellent bioavailability, although there is controversy as to the effectiveness of such compounds [42]. Presumably the iron complexes are reduced by DCYTB, thereby generating iron(II) which is the main substrate for DMT1 (Section 1.3.1.2).

2.6 Anemia of Chronic Disease

The anemia of chronic disease is an acquired disorder of iron homeostasis which may be associated with infection, malignancy, organ failure or trauma (Table 5). The anemia is typically mild to moderate and the erythrocyte size being close to

Table 5 Incidence of anemia in various disease groups. Disorder Percentage of cases with anemia Infection Tuberculosis 20 HIV 10–35 Trauma Heart transplant 70 Intensive care 40 Autoimmune Rheumatoid arthritis 15 Inflammatory bowel disease 10–70 Cancer Palliative care 40–75 Chronic kidney disease 40 Heart failure 30 Aging (>65 years) 10–20 Data adopted from [43]. 8 Iron: Effect of Deficiency and Overload 255 normal. Macrophages, which normally recycle iron (Section 1.3.1.5), retain it and intestinal iron absorption is reduced under these pathological conditions [43]. An association with proinflammatory cytokines was suspected for many years and it has recently been established that this modified cellular behavior is associated with elevated levels of hepcidin (Section 1.4.1) [44,45]. Expression of hepcidin that is inappropriately high for the normal body iron status results in interruption of intestinal iron absorption and macrophage iron recycling. Thus, there is less iron available for erythropoiesis. IL-6 is the cytokine that induces hepcidin expression [45] and its levels have been shown to correlate with the severity of anemia in rheumatoid arthritis, cancer, and aging [43]. Treatment options for patients with anemia of chronic disease depends on the severity of the anemia and since the anemia is derived from the innate immune response to infection, there is little clinical support for the use of exogenous iron or erythroid-stimulating agents, such as erythropoietin. In principle it is possible to mobilize macrophage iron by chelation and to redistribute it to the transferrin iron pool [46,47], however, to date no such compounds have been introduced into the clinic. In contrast to the above general statements, in the specific cases of autoimmune disorders and end-stage renal disease, iron treatment in conjunction with erythroid- stimulating agents can be effective [48,49]. With the use of erythropoietin, the rate of iron mobilization from stores is unable to match the iron requirements of the expanding red cell mass and consequently iron supplementation is required. In many patients oral supplements will not provide an optimal supply of iron and such patients are frequently intolerant to oral iron therapy, in such cases intravenous iron preparations can be introduced. These preparations consist of a carbohydrate ligand and either ferric hydroxide or ferric oxide cores. They are designed to be non-immunogenic and to be efficiently removed from the circulation by macrophages, which catabolize the complex and render the iron available for apo transferrin. Iron dextrin was the first such preparation to be used clinically, but it has been associated with a wide range of side effects. However, there are now much improved formulations available [50,51]. An advantage of some of these preparations is that doses up to 1 g of iron can be administered in a single infusion [51].

3 Systemic Iron Overload

Iron overload can be present in one of two general forms. Firstly, in situations where erythropoiesis is normal but the plasma iron level exceeds the iron binding capacity of transferrin (e.g., hereditary hemochromatosis); the resulting non-transferrin bound iron (NTBI) is deposited in parenchymal cells of the liver, heart, and endocrine tissues [52]. The second type results from increased catabolism of erythrocytes (e.g., transfusional iron overload). In this situation iron initially accumulates in reticuloendothelial macrophages, but subsequently spills over into the NTBI pool and parenchymal cells. Parenchymal iron deposition leads to organ damage. 256 Hider and Kong

3.1 Non-transferrin Bound Iron

When transferrin is fully saturated, any iron entering the blood forms part of the NTBI pool. NTBI, unlike transferrin, lacks an address system and tends to be absorbed by highly vascular tissue, such as the heart and endocrine organs. As iron accumulates in these organs, redox cycling leads to the generation of toxic oxygen radicals. Transferrin also contributes to defence against infection by depriving microorganisms of an iron supply [29]. Conversly, the presence of NTBI presents a weakness, rendering the host more susceptible towards infection. There are many small molecules in the blood which are capable of binding iron(III), the principle oxidation state of iron in the serum. These include acetate, phosphate, and citrate. Of these, citrate has the highest affinity for iron(III) [53] and there are two dominating complexes under the conditions provided by serum, [Fe(citrate)2] (9) and [Fe3(citrate)3] (10) [53,54]. At an iron concentration of 1 μM, complex (9) dominates at physiological citrate levels (typically 100 μM), whereas at a level of 10 μM iron the oligomeric iron citrate (10) begins to dominate [53]. Another important iron binding component of the serum is albumin, which is present at a relatively high concentration (600 μM) [55,56]. Which of these three forms of NTBI gains facile entry into parenchymal cells has not been established.

O −O −O O O O O O O O O O O O Fe O O Fe O O O Fe O O O O O Fe O O − O O − O − O O O O O O

9 10

3.2 Hereditary Hemochromatosis

The term hereditary hemochromatosis covers a heterogeneous group of iron overload disorders (Table 6). They are linked to gene mutations associated with hepcidin, leading to low levels of hepcidin. This in turn leads to the hyperabsorption of dietary iron, up to 8–10 mg per day. Over a period of time transferrin saturation gradually increases from the normal value of 30% to 100%, which is associated with the appearance of non-transferrin bound iron and the associated inappropriate tissue distribution of iron [57]. In hereditary hemochromatosis, hepatic iron overload can lead to fibrosis, cirrhosis, and carcinoma of the liver [58] and to non-hepatic symptoms including cardiomyopathy, diabetes, hypogonadism, and arthritis [59]. In contrast, the CNS does not develop iron overload. 8 Iron: Effect of Deficiency and Overload 257

Table 6 Hereditary iron overload disorders. Type Gene Onset Clinical expression HFE hemochromatosis 1 HFE Late Hepatic Juvenile hemochromatosis 2A HFE2 Early Cardiac and endocrine Juvenile hemochromatosis 2B HAMP Early Cardiac and endocrine TfR2 hemochromatosis 3 TFR2 Late Hepatic Ferroportin disease 4 SLC40AI Late Hepatic

The above symptoms tend to develop late in life, but the disease can be managed by phlebotomy (each 500 mL of blood contains 250 mg of iron), the earlier the diagnosis the better. Indeed, early diagnosed patients experience a normal life span. In addition to phlebotomy patients should avoid iron supplementation and limit consumption of red meat and alcohol.

3.2.1 HFE Hemochromatosis

HFE hemochromatosis is the most frequent form of hemochromatosis [60] and there is a particularly high prevalence among North European caucasians (1 in 200). The discovery of HFE was the first cloning success to contribute to our understanding of iron metabolism [61]. This important finding was rendered possible by the observation of Simon et al. that the hemochromatosis phenotype is frequently associated with a particular human leukocyte antigen haplotype [62]. The HFE protein is an atypical membrane-bound MHC class 1 molecule which interacts with β2-microglobulin; its link with iron transport remains to be established. Many patients bear a mutant HFE, with a C282Y substitution and HFEC282Y fails to incorporate in the plasma membrane [63]. The frequency of HFEC282Y homozygosity is 1:200, although the clinical penetrance of the disease is lower [58,64]. Additional HFE mutations are also associated with hemochromatosis [65]. Significantly ablation of HFE promotes a hemochromatotic phenotype in mice [66].

3.2.2 Juvenile Hemochromatosis

Juvenile hemochromatosis unlike HFE hemochromatosis is a relatively rare disease mainly located in Southern Europe [67]. These patients rapidly accumulate iron and are more likely to present with cardiomyopathy and endocrinopathy than with liver disease (Table 6). This pathology is linked to the mutation of the gene responsible for hemojuvelin (a bone morphogenetic protein receptor operating upstream of the hepcidin pathway), which leads to the expression of extremely low levels of hepcidin. A small subset of these patients possesses a mutation to the HAMP gene, which encodes for hepcidin. Low hepcidin leads to enhanced iron absorption (Section 1.4.1).

3.2.3 Ferroportin Disease

Ferroportin disease is characterized by moderate to severe iron overload. It is more frequent than Types 2 and 3 hemochromatosis and is caused by mutations to the 258 Hider and Kong ferroportin gene. Affected individuals express high hepcidin levels and the disease displays phenotypic heterogeneity [68].

3.2.4 Treatment by Iron Chelation

Phlebotomy is an extremely efficient method of removing iron. Treatment of severely iron-loaded hemochromatosis patients typically involves weekly phlebotomy; which can lead to removal of up to 1g of iron every month. Iron chelation therapy cannot achieve such high excretion rates but does have the advantage of reducing NTBI levels (vide infra). Patients suffering from juvenile hemochromatosis may rapidly develop cardiac complications and such people can benefit from a combination of phlebotomy and chelation therapy. In principle, orally active iron chelators could find application in the treatment of hemochromatosis, and deferasirox (vide infra) has been reported to show good efficacy [69].

3.3 Transfusional Siderosis

The management of anemias associated with ineffective erythropoiesis (for instance thalassemia, sickle cell disease, hemolytic anemia, and myelodysplastic syndromes) requires frequent blood transfusion. Blood contains plenty of iron (0.5 mg mL–1) and this accumulates in the tissues as man does not actively excrete iron (Section 1.4). In addition, ineffective erythropoiesis inhibits hepcidin expression which leads to the stimulation of dietary iron absorption. Overall this pathology leads to transfusional siderosis, the formation of non-transferrin bound iron and the inappropriate iron loading of liver, cardiac, and endocrine tissue [70].

3.3.1 The Hemoglobinopathies

Thalassemia and sickle cell anemia are the most common worldwide monogenic diseases. They occur at their highest frequency in countries of the developing world and it has been estimated that there are 250 million carriers. 300,000 children are born each year with major hemoglobin disorders [71]. The high incidence of these disorders reflects their association with malaria resistance which has developed over thousands of years [72]. More than 3,000 million people live in malarious areas and currently the disease is responsible for at least two million deaths each year. Any protective mechanism against this dominating infection that develops within the native population will be gradually amplified and this has been the situation with thalassemia and sickle cell anemia where a range of mutations have gradually accumulated within the population. The correspondence between the distribution of malaria in the Old World before major control programs were established and the distribution of thalassemia is striking (Figure 17) [73]. The cellular mechanisms related to this type of protection are complex but increasingly well understood [73,74]. 8 Iron: Effect of Deficiency and Overload 259

Figure 17 (a) Distribution of malaria in the Old World before major control programs were established. (b) Distribution of α- and β-thalassemia [73]. 260 Hider and Kong

Figure 18 Changes of human hemoglobin with development. There is a switch from hemoglobin-F to adult hemoglobin-A together with a small amount of hemoglobin-A2 in the immediate post natal period [75]. Hemoglobins; F = α2γ2; A = α2β2; A2 = α2δ2. Reproduced with permission from [75]; copyright 2001, Blackwell Science Ltd.

Table 7 Annual births with Thalassemia dominated symptoms major hemoglobin disorders. Hb Bart’s hydrops (α°/α°) 5,000 Hb H disease (α°/α+) 10,000 β-Thalassemia major (β°) 23,000 Hb E/β-Thalassemia 19,000 Total 57,000 Sickle cell anemia dominated symptoms SS disease 217,000 S/β-Thalassemia 11,000 HbC/S 55,000 Total 283,000 Reproduced with permission from [76]; copyright 2006, World Health Organization.

Hemoglobin production in man is complicated. The type of hemoglobin produced in fetal life is altered after birth (Figure 18) as a result of the sequential suppression and activation of individual genes [75]. Clearly, there are a number of possible hemoglobin disorders that can result from this genetic framework and a breakdown of these disorders as monitored by the annual number of births is given in Table 7 [76]. 8 Iron: Effect of Deficiency and Overload 261

The thalassemias are characterized by a reduced rate of synthesis of one or more globulin chain(s); α°- and β°-thalassemias are associated with no production of α- or β-chains, respectively, α+- and β+-thalassemias are associated with a reduced production of α- or β-chains. These conditions all lead to an imbalanced globin chain production, which is associated with ineffective erythropoiesis and shortened red blood cell survival. Because thalassemias occur in populations in which hemoglobin variants are also common, some children inherit thalassemia from one parent and a hemoglobin variant from the other. These interactions produce disorders of varying severity. Thus, sickle cell/β-thalassemia may be as severe as sickle cell anemia and hemoglobin E/β-thalassemia can produce similar pathology to that of thalassemia major. Hemoglobin E is inefficiently synthesized and hence produces a mild form of β-thalassemia [75].

3.3.1.1 Thalassemia

Although the α-thalassemias are more common than the β-thalassemias, severe forms lead to intrauterine death and so do not pose a major burden on health care. It is the β-thalassemias and their combination with hemoglobin E that produce severe anemia. These diseases are particularly common throughout the Indian subcontinent and South East Asia. Over 180 different mutations have been identified amongst theβ globin chains of β-thalassemia patients. The bulk consists of single base changes which lead to different degrees of reduction in the synthesis of α-chains, thereby leading to the clinical diversity β-thalassemia. The reduction of α-chain production leads to an excess of α-chains, which are unstable and tend to precipitate, preventing the normal maturation and survival of erythrocytes [77]. β-Thalassemia major (β°) is associated with the total absence of β-subunits and consequently is fatal in the absence of regular blood transfusion, which in turn is associated with systemic iron overload. With untreated patients, death generally occurs in the second decade of life as a result of infection or heart disease [78]. Iron chelation therapy prevents the development of iron overload and as a result, the life style of thalassemia patients has dramatically improved over the past 50 years. Desferrioxamine (DFO, 7) a natural siderophore was introduced in the clinic by SephtonSmith in 1962 [79]. It scavenges and binds iron(III) extremely tightly, leading to the formation of a stable non-toxic iron complex (Figure 19) [80] which is excreted via the bile. Unfortunately DFO is not orally active and has to be administered parenterally over prolonged time periods, as it is rapidly cleared by the kidney [81]. Never-the-less it has been a remarkably successful pharmaceutical and has extended the lives of thousands of β-thalassemia major patients. There is a wide pathological diversity of β-thalassemia patients due to the large number of different mutations, for instance the inheritance of a severe mutation from one parent and a mild mutation from the second parent or the inheritance of β-thalassemia from one parent and an abnormal Hb from the other [82]. As a group, 262 Hider and Kong

Figure 19 Ferroxamine, the iron complex of desferrioxamine [80].

these patients are described as suffering from β-thalassemia intermedia. Management depends on the severity of the disease and may involve transfusion and chelation or in some cases, chelation therapy alone (vide infra) [82].

3.3.1.2 Sickle Cell Disease

Expression of sickle cell disease, like β-thalassemia is highly variable, ranging from mild phenotypes (mostly SC and Sβ+ genotypes) to severe disease (mostly SS and Sβ° genotypes). Transfusion therapy is a key component of patient management and is an effective treatment for chest syndrome, heart failure, and stroke. Monthly transfusions decrease the risk of recurrent stroke. Straight transfusion is used when the Hb level is less than 8 g dL–1 and exchange transfusion is recommended with normal Hb levels [83]. The target percentage of HbS in patients receiving regular transfusions is 30% [84]. Both straight and exchange transfusion lead to iron overload and although the endocrinopathology is less marked in sickle cell anemia patients when compared with β-thalassemia patients [85], iron overload should be treated by chelation (vide infra).

3.3.2 Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a diverse group of hematological disorders that lead to ineffective production of myeloid blood cells with a risk of transformation to leukemia. This bone marrow stem cell disorder is associated with ineffective erythropoiesis and leads to anemia. Diagnosis of MDS is made typically between 60 and 75 years, diagnoses are rare in children. Many MDS patients become dependent on blood transfusion and develop transfusional iron overload [86] which is associated with cardiac problems [87]. Although this iron overload can be treated with 8 Iron: Effect of Deficiency and Overload 263 desferrioxamine, the demanding continuous parenteral treatment leads to difficulties with compliance, particularly with this typically elderly group of patients [86]. Treatment with orally active iron chelators has good potential (vide infra).

3.4 Hereditary Disorders of Mitochondrial Iron Overload

Several iron loading disorders are characterized by mitochondrial accumulation in specific tissues, without systemic iron overload. They result from mutations in proteins involved in either heme or iron-sulfur cluster biosynthesis. These two metabolic pathways occur in the mitochondria and under normal conditions consume the majority of cellular iron.

3.4.1 Sideroblastic Anemia

Sideroblastic anemias are a group of disorders that are associated with the formation of a large number of ringed sideroblasts in the marrow. Sideroblasts are eryth- roblasts (nucleated red blood cells) which contain precipitates of non-heme iron aggregates deposited within the cristae of mitochondria [68]. X-linked sideroblastic anemia (XLSA) is the most common form of sideroblastic anemia which results from defects in 5-aminolevulinate synthase, a key enzyme in heme biosynthesis. Management of this disease frequently requires blood transfusion which leads to iron overload [68].

3.4.2 Friedreich’s Ataxia

Friedreich’s ataxia is an autosomal recessive neurodegenerative disorder linked to the functional inactivation of frataxin (FXN) [88,89]; a mitochondrial protein which has a critical role in the assembly of iron-sulfur clusters [90]. An expanded GAA triplet repeat is found in both alleles of the FXN gene. This triplet repeat leads to decreased RNA transcription and lower levels of frataxin. The degree to which transcription is suppressed is proportional to the length of the GAA repeat; with frataxin levels, 5% of normal, being associated with long GAA repeats and the association of higher frataxin levels (30% of normal) with shorter repeats [91]. Thus, patients with shorter GAA repeats generally have a less severe phenotype. Frataxin deficiency is associated with decreased ATP synthesis and elevated mitochondrial iron levels. The tissues most severely influenced are cardiac [92], gastrointestinal and brain [93]. Treatment with orally active iron chelators can be beneficial vide( infra). 264 Hider and Kong

3.4.3 Glutaredoxin-5 Deficiency

Glutaredoxin-5 (Grx 5) is a protein cofactor essential for the iron-sulfur cluster assembly pathway [94]. Its absence is causatively linked to microcytic anemia with a sideroblastic-like phenotype [95]. The disease requires regular blood transfusion which leads to systemic iron overload. The pathogenic mechanism involves inhibition of heme synthesis in erythroid precursor cells via accumulation of apo-IRP1 that represses 5-aminolevulinate synthase (ALAS) mRNA translation [96].

3.5 Animal Models of Iron Overload

Over the past two decades genetic analysis of patients with inherited iron homeostasis disorders and the analysis of mutant mice, rats, zebra fish, and fruit flies has greatly facilitated the present understanding of iron metabolism [97]. These animal models are excellent systems for investigating iron homeostasis and for the identification of new therapeutic agents. Although iron metabolism in zebra fish and the fruit fly is not understood in detail, iron metabolism in rodents is similar to that in man. Models have been developed which simulate iron deficiency anemia, sideroblastic anemia, various forms of defective iron transport, hemochromatosis, and Friedreich’s ataxia (Table 8) [21,98–100,104–120]. The first mutant to provide a useful insight into iron transport was the hypotrans- ferrinaemic mouse (hpx) [98,101], although the mk mouse was discovered in 1970 [102] and the Belgrade rat in 1969 [103]. One of the benefits from this work will be the accurate diagnosis of human iron deficiency and iron overload disorders; for instance, the clinical approach to hemochromatosis has been strongly influenced by diagnostic testing [68].

3.6 Genetic Screening for Thalassemia

The improved understanding of the pathophysiology of the thalassemias has led to improved symptomatic treatment by blood transfusion and chelation therapy. However, this therapy is expensive and complicated due to the increasing problem of obtaining blood that is free from HIV and hepatitis. Because thalassemia and sickle cell heterozygotes are relatively easy to identify, these diseases are suitable for investigation by prenatal diagnosis and termination of pregnancies of homozygote babies. In the 1970’s with the advent of fetal blood sampling, it became possible to undertake such diagnosis by monitoring hemoglobin chain synthesis [121] and later by DNA analysis [122,123]. Screening was soon established in several countries, for instance Sardinia and Cyprus, with remarkable success (Figure 20) [124]. There has now been success in reducing the number of babies born with thalassemia major in Greece, Italy and United Kingdom [121]. 8 Iron: Effect of Deficiency and Overload 265 _ Sideroblastic anemia Sideroblastic anemia riedreich’s ataxia F riedreich’s Porphyria Hemochromatosis Hemochromatosis (Type 4) Hemochromatosis (Type Hemochromatosis (Type 3) Hemochromatosis (Type Hemochromatosis (Type 2B) Hemochromatosis (Type Hemochromatosis (Type 2A) Hemochromatosis (Type Hemochromatosis (Type 1) Hemochromatosis (Type Atransferrinemia Human disease hematopoietic tissues in non- Anemia Microcytic anemia Microcytic Profound anemia, Embryonic death Progressive neurodegeneration and cardiac pathology neurodegeneration Progressive Increased mitochondrial iron anemia Embryonic lethal (day 12) microcytic Microcytic anemia and porphyria Microcytic Kupffer cells and low transferrin cells and low Kupffer Decreased hepcidin Increased body iron, decreased hepcidin Heterozygous mice have iron loading of Heterozygous mice have Increased body iron, decreased hepcidin Increased body iron, absence of hepcidin Increased body iron, decreased hepcidin Embryonic death with severe anemia Embryonic death with severe anemia Embryonic death with severe Severe iron deficiency anemia iron deficiency Severe anemia iron deficiency Severe anemia Iron deficiency Microcytic anemia Microcytic Impaired ferric reductase activity in transferrin endosome Impaired ferric reductase activity Increased body iron, decreased hepcidin Severe anemia, iron overload anemia, iron overload Severe Loss of function (mice) [ 114 ] mutant constructs [ 115 ] Zebra fish (frascati) [ 120 ] ) [ 118 ] Grxs zebra fish ( shiraz knockout (mice) [ 116 ] Mfrn 1 knockout knockout (mice) with human Fxn knockout knockout (mice) [ 113 ] Alas2 knockout Alas2 zebra fish ( sauternes ) [ 117 ] Fech F lat iron mouse Zebra fish ( weissherbst ) [ 21 ] Smad 4 (mice) [ 111 ] Slc40al [ 110 ] Tfr 2 (mice) [ 109 ] Hamp 1 (mice) [ 108 ] Hjv (mice) [ 106 , 107 ] knockout (mice) [ 99 ] Tfr knockout ) [ 120 ] Zebra fish ( chianti mk (mice) [ 100 ] Belgrade rat [ 100 ] ) [ 119 ] Zebra fish ( chardonnay sla (mice) [ 104 ] knockout (mice) [ 112 ] Steap 3 knockout Hfe (mice) [ 105 ] hpx (mice) [ 98 ] Mutation Mutations in iron metabolism genes. Glutaredoxin 5 Mitoferrin 1 F rataxin Mutations causing altered mitochondrial metabolism mitochondrial Mutations causing altered acid synthase 5-Aminolevulinic F errochelatase Smad 4 F erroportin Transferrin receptor 2 Transferrin Hamp 1 (hepcidin synthesis) Hemojuvelin Transferrin receptor 1 Transferrin Divalent metal transporter 1 Divalent Hephaestin Steap 3 (ferroxidase) Mutations causing hemochromatosis Hfe protein Mutations causing anemia Transferrin Protein Table 8 Table 266 Hider and Kong

Figure 20 number of births of new cases of β-thalassemia in Sardina since a screening and prenatal diagnosis program was developed in the mid-1970s [124]. Reproduced with permission from [124]; copyright 1998, Elsevier.

4 Iron-Selective Chelators with Therapeutic Potential

4.1 Design Features

Desferrioxamine-B (DFO) (7), the most widely used iron chelator in hematology over the past 40 years, has a major disadvantage of being orally inactive [125]. In order to identify an ideal iron chelator for clinical use, careful design consideration is essential; a range of specifications must be considered such as metal selectivity and affinity, kinetic stability of the complex, bioavailability, and toxicity. This field has recently been reviewed [126,127].

4.1.1 Metal Selectivity

Chelating agents can be designed for either the iron(II) or iron(III) oxidation state. High-spin iron(III) is a tripositive cation of radius 0.65 Å, and forms most stable bonds with charged oxygen atoms, such as those found in DFO (7). In contrast, the iron(II) cation, which has a lower charge density, prefers chelators containing nitrogen such as 1,10-phenanthroline. Ligands that prefer iron(II) retain an appreciable affinity for other biologically important bivalent metals such as copper(II) and zinc(II) ions. In contrast, iron(III)-selective ligands, typically oxyanions and notably hydroxamates and catecholates, are generally more selective for tribasic metal cations over dibasic cations and as most tribasic cations, for instance aluminium(III) and gallium(III), are not essential for life, iron(III) provides the best target for 8 Iron: Effect of Deficiency and Overload 267

‘iron chelator’ design under biological conditions. An additional advantage of high-affinity iron(III) chelators is that, under aerobic conditions, they will chelate iron(II) and rapidly autoxidize it to the corresponding iron(III) species [128].

4.1.2 Ligand Selection

Catechol moieties possess a high affinity for iron(III). This extremely strong interaction with tripositive metal cations results from the high electron density of both oxygen atoms. However, this high charge density is also associated with the high affinity for protons (pKa values of 12.1 and 8.4). Thus the binding of cations by catechol has marked pH sensitivity. The hydroxamate moiety possesses a lower affinity for iron than catechol, but the selectivity of hydroxamates, like catechols, favors tribasic cations over dibasic cations. Due to the lower value of the protonation constant (pKa ~9 ), competition with hydrogen ions at physiological pH is less pronounced than for that of catechol ligands. Hydroxypyridinones (Figure 21) combine the characteristics of both hydroxamate and catechol groups, forming 5-membered chelate rings in which the metal is coordinated by two vicinal oxygen atoms. The hydroxypyridinones are mono- protic acids at pH 7.0 and thus, like hydroxamates, form neutral tris-iron(III) complexes. 3-Hydroxypyridin-4-ones are highly selective for tribasic metal cations over dibasic cations as indicated by the low reduction potential of iron complexes (−620 mV versus NHE). 8-Hydroxyquinoline binds iron(II) more tightly than 3-hydroxypyridin-4-ones as indicated by its higher redox potential of the iron complex (−150 mV versus NHE). Never-the-less it is capable of scavenging iron under biological conditions, forming a 3:1 complex with iron(III) at pH 7.0. Although aminocarboxylates and hydroxycarboxylates bind iron(III) they are less selective, frequently possessing appreciable affinities for calcium(II) and magnesium(II), in addition to zinc(II) and copper(II).

4.1.3 Hexadentate, Tridentate, and Bidentate Iron(III) Complexes

The coordination requirements of high spin iron(III) are best satisfied by six donor atoms ligating in an octahedral fashion to the metal center, the affinity for the ligand generally decreasing in the sequence; hexadentate > tridentate > bidentate (Figure 22). The overall stability constant trends for bidentate and hexadentate ligands are typified by the bidentate ligand N,N-dimethyl-2,3-dihydroxybenzamide (DMB) and the hexadentate congener MECAM (Figure 21) where a differential of 6 log units in stability is observed (40.2 versus 46). Although MECAM binds iron(III) more tightly than its bidentate analogue DMB, other hexadentate catechols, for instance, enterobactin (Figure 21), bind iron(III) even more tightly. The smaller the conformational space of the free ligand, the higher the stability of the complex; as the difference between the flexibility of the ligand and its corresponding iron complex decreases, so does the entropy difference. 268 Hider and Kong

Bidentate Ligands

O OH

N O NMe2 H OH

N,N-Dimethyl-2,3- Acetohydroxamic acid dihydroxybenzamide (DMB)

O OH

N N OH 8-Hydroxyquinoline 3-Hydroxy-1,2-dimethylpyridin -4(1H)-one (Deferiprone)

Tridentate ligands HOOC

OH N N

N N N CH3 HO S COOH OH

Desferrithiocin Desferasirox (ICL670)

Hexadentate ligands

O OH OH NH OH OH O HN OH O H O N O O

O HN O O NH HO N O HO O HO H O HO HO OH MECAM Enterobactin

Figure 21 Iron(III) chelating agents. 8 Iron: Effect of Deficiency and Overload 269

O O O O O O N O O Fe Fe Fe O O N O O O O O O

Bidentate ligand Tridentate ligand Hexadentate ligand 3:1 complex 2:1 complex 1:1 complex

Figure 22 Schematic representation of chelate ring formation in metal-ligand complexes.

3+ Table 9 pFe values of Iron(III) chelator pFeIII selected iron(III) chelators. Bidentate Dimethyl-2,3-dihydroxybenzamide DMB 15 Acetohydroxamic acid 13 3-Hydroxypyridinone (Deferiprone) 20.5 8-Hydroxyquinoline 20.6 Tridentate Desferrithiocin 20.4 Desferasirox 22.5 Hexadentate Desferrioxamine-B (7) 26.6 MECAM 28 Enterobactin 31.5 III 3+ 3+ –6 pFe = −log[Fe ], when [Fe ]total = 10 M, [Ligand]total = 10–5 M at pH 7.4. The structures of the chelators are given in Figure 21.

Thus enterobactin (log stability constant = 48) can be considered to possess a degree of preorganisation in contrast to MECAM which does not [129]. Under biological conditions, a comparison standard which is generally more useful than the stability constant is the pFe3+ value [130]. pFe3+ is defined as the negative logarithm of the concentration of the free iron(III) in solution. Typically pFe3+ values are calculated for total [ligand] = 10–5 M, total [iron] = 10–6 M at pH 7.4. The comparison of ligands under these conditions is useful, as the pFe3+ value, unlike the stability constants log K or log β3, takes into account the effects of ligand protonation and denticity as well as differences in metal-ligand stoichiometries. Comparison of the pFe3+ values for hexadentate and bidentate ligands reveals that hexadentate ligands are far superior to their bidentate counterparts under typical in vivo conditions. The values for DMB, MECAM, and enterobactin being 15, 28, and 31.5, respectively (Table 9) [130]. 270 Hider and Kong

The formation of a complex will also be dependent on both free metal and free ligand concentration and as such will be sensitive to concentration changes. The degree of dissociation for a tris-bidentate ligand-metal complex is dependent on the cube of [ligand] whilst the hexadentate ligand-metal complex dissociation is only dependent on [ligand]. Hence the dilution sensitivity to complex dissociation for ligands follows the order hexadentate < tridentate < bidentate. It is for this reason that the majority of natural siderophores are hexadentate compounds and can therefore scavenge iron(III) efficiently at low metal concentrations [131]. In general, pFe3+ values follow the trend hexadentate > tridentate >bidentate as exemplified by the examples in Table 9.

4.1.4 Critical Features for Clinical Application: Molecular Size and Hydrophobicity

In order to achieve efficient oral absorption, the chelator should possess appreciable lipid solubility which may facilitate the molecule to penetrate the gastrointestinal tract (partition coefficient betweenn -octanol and water greater than 0.2) [132]. Molecular size is also a critical factor which influences the rate of drug absorption [133]. Indeed, it has been proposed by Lipinski et al. that the molecular weight should not exceed 500 in order to achieve efficient oral absorption [134]. This molecular-weight limit provides a considerable restriction on the choice of chelator and may effectively exclude hexadentate ligands from consideration; most siderophores, for instance DFO (7) and enterobactin (Figure 21) have molecular weights in the range 500–900. In contrast, bidentate and tridentate ligands, by virtue of their much lower molecular weights, tend to possess higher absorption efficiencies.

4.1.5 Toxicity of Chelators and Their Iron Complexes

The toxicity associated with iron chelators originates from a number of factors; including inhibition of metalloenzymes, lack of metal selectivity, redox cycling of iron complexes between iron(II) and iron(III), thereby generating free radicals, and the kinetic lability of the iron complex leading to iron redistribution. Enzyme inhibition: In general, iron chelators do not directly inhibit heme-containing enzymes due to the inaccessibility of porphyrin-bound iron to chelating agents. In contrast, many non-heme iron-containing enzymes such as the lipoxygenase and aromatic hydroxylase families and ribonucleotide reductase are susceptible to chelator- induced inhibition [135]. Lipoxygenases are generally inhibited by hydrophobic chelators, therefore, the introduction of hydrophilic characteristics into a chelator tend to minimize such inhibitory potential [136]. Stereochemistry can also limit chelator access to the metal binding center and the introduction of a rigid side chain close to the chelating center of the molecule can reduce inhibitory properties [137]. Thus, careful control of the bulk and shape of iron chelators leads to minimal inhibitory influence of many metalloenzymes. 8 Iron: Effect of Deficiency and Overload 271

Metal selectivity: An ideal iron chelator should be highly selective for iron(III) in order to minimize chelation of other biologically essential metal ions which could lead to deficiency with prolonged usage. Many ligands that possess a high affinity for iron(III) also have appreciable affinities for other metals such as zinc(II), this being especially so with carboxylate- and nitrogen-containing ligands. However, this factor is less of a problem with the bidentate catechol, hydroxamate, and hydroxypyridinone ligand groups, which possess a strong preference for tribasic over dibasic cations. In principle, competition with copper(II) could be expected to be a problem, however under most biological conditions this is not so, as copper is extremely tightly bound to chaperone molecules [138]. Iron-complex structure and redox activity: In order to prevent free radical production, iron should be coordinated in such a manner as to avoid direct access of oxygen and hydrogen peroxide, and to possess a redox potential which cannot be reduced under biological conditions. Most hexadentate ligands with oxygen containing ligands such as DFO are kinetically inert and reduce hydroxyl radical production to a minimum by failing to redox cycle. Chelators that are capable of binding both iron(II) and iron(III) at neutral pH values have potential to redox cycle. This is an undesirable property for iron scavenging molecules, as redox cycling can also lead to the production of reactive oxygen radicals (Figure 23). Significantly, the high selectivity of siderophores for iron(III) over iron(II) renders redox cycling under biological conditions unlikely. Thus the iron complexes of enterobactin and desferrioxamine are extremely low, namely −750 and −468 mV (versus NHE) [130]. In similar fashion, iron-deferiprone has a low redox potential (−620 mV versus NHE) [139]. Iron complexes with redox potentials above −200 mV (versus NHE) are likely to redox cycle under aerobic conditions. Kinetic lability of iron complexes: Hexadentate ligand iron complexes tend to be inert, the rate of dissociation of the complex being vanishingly small at neutral pH values. This renders such molecules ideal scavengers of iron. In contrast, bidentate and tridentate ligands are less kinetically stable and are able to dissociate at appreciable rates, thereby possibly facilitating iron redistribution. Such a property is

Figure 23 Redox cycling of an iron complex. 272 Hider and Kong undesirable for most therapeutic applications, where efficient iron excretion is required. In order to avoid appreciable redistribution of iron in mammalian body tissues, chelators possessing a high iron(III) affinity are required; generally a pFe value ≥20 appears to be sufficient to minimize the redistribution of iron.

4.2 Orally Active Iron Chelators in Current Use

Iron chelation therapy prevents the development of iron overload and as a consequence the life style of thalassemia major patients has been dramatically improved with the application of DFO (7) (Section 3.3.1.1). However, DFO is not an ideal therapeutic chelator due to its oral inactivity and rapid renal clearance (plasma half- life of 5–10 min) [140]. In order to achieve sufficient iron excretion, it has to be administered subcutaneously or intravenously for 8–12 h/day, 5–7 days/week [141]. Patient compliance with this regimen is frequently poor. Furthermore, NTBI (Section 3.1) is present in such patients whenever the plasma DFO level is low, rapidly reappearing on the cessation of DFO perfusion (Figure 24) [142]. As DFO is typically infused for 5 nights, this only provides protection for 40h per week; that is approximately 25% of the time. As transferrin is saturated in most of these patients, NTBI is present for 75% of the time and therefore has the possibility of gradually loading the heart and endocrine tissue with iron, even in well chelated patients. A large proportion of patients treated with DFO suffer from adverse cardiovascular events [143].

4.2.1 Tridentate Chelators

Unlike hexadentate and bidentate molecules, it is difficult to design tridentate ligands which only possess oxygen anion coordination sites [144], the central ligand typically being nitrogen (Figure 22).

Figure 24 The effect of DFO infusion at 50 mg/ kg/24h (intravenous) on NTBI is shown in a single patient with thalassemia major both on starting the DFO infusion and on stopping the infusion at 48 hours [142]. 8 Iron: Effect of Deficiency and Overload 273

Desferrithiocins: Desferrithiocin (DFT) (11) is a siderophore isolated from Streptomyces antibioticus. It forms a 2:1 complex with iron(III) at neutral pH using a phenolate oxygen, a carboxylate oxygen, and a nitrogen atom as ligands [145]. It possesses a high affinity for ferric iron (pFe3+ = 20.4). Long term studies of DFT in normal rodents and dogs at low doses have shown toxic side effects, such as reduced body weight and neurotoxicity [146]. However, a range of synthetic analogues of DFT have been prepared in an attempt to design molecules lacking renal and neurotoxicity [147] and two such molecules have been identified, namely deferitrin (12) and FBS0701 (13). Deferitrin (12), was found to be highly effective when given orally to rats and primates. Histopathological analysis indicated some nephrotoxicity but much less than that arising from DFT [148]. Phase I clinical trials demonstrated

OH HO OH

N N N CH3 CH3 S COOH S COOH

11 12 good oral absorption, however the compound was not progressed beyond Phase II clinical trials due to nephrotoxicity. FBS0701 (13) also binds iron(III) with high affinity and in contrast to deferitrin, demonstrated no observable toxicity at a predicted dose level range in preclinical studies [149]. The compound has entered clinical trials sponsored by Ferrokin Biosciences [150], where it has been shown to be well tolerated and to possess favorable pharmacokinetics [151]. FBS0701 is currently in Phase II clinical trials. Triazoles: Triazoles have been investigated as potential ligands by Novartis [152]. These molecules chelate iron(III) with two phenolate oxygens and one triazolyl nitrogen. The lead compound deferasirox (14) possesses a pFe3+ value of 22.5 and is extremely hydrophobic, with a log Poctanol/water value of 3.8. As a result, it can penetrate membranes easily and possesses good oral availability. Indeed, when orally administered to hypertransfused rats, deferasirox promotes the excretion of chelatable iron from hepatocellular iron stores four to five times more effectively than DFO [153].

HOOC O O O O OH N N N CH3 N HO S COOH OH 13 14 274 Hider and Kong

By virtue of a high proportion of both the free ligand and the 2:1 iron complex binding to albumin (greater than 98%), the ligand possesses low toxicity despite its strong lipophilic character. The extreme hydrophobicity of this chelator necessitates formulation in dispersion tablets, containing the disintegrants, SDS, povidone, and crospovidone. Thus, deferasirox is typically given once daily each morning as a dispersed solution in water, half an hour before breakfast. Deferasirox has been demonstrated to be efficient at removing liver iron from regularly transfused patients [154] but is apparently less effective at removing cardiac iron [155]. Deferasirox (14) forms a 2:1 iron complex which possesses a net charge of 3– and a molecular weight over 800. Should such a complex form intracellularly, it is possible that the iron will remain trapped within the cell. The redox potential of the 2:1 iron complex is −600 mV (versus NHE) confirming that deferasirox is highly selective for iron(III) and that it will not redox cycle under biological conditions. As with all therapeutic iron chelators there are side effects associated with deferasirox [156], kidney toxicity being particularly prevalent [157].

4.2.2 Bidentate Chelators

On the basis of selectivity and affinity, particularly considering pFe3+ values, 3-hydroxypyridin-4-one (Figure 21) is the optimal bidentate ligand for the chelation of iron(III) over the pH range of 6.0–10.0 and to date is the only bidentate class to have been subjected to extensive clinical study. Dialkylhydroxypyridinones: The 1,2-dimethyl derivative (deferiprone, Ll, CP20) (15) is marketed by Apotex Inc. Toronto, Canada, as FerriproxTm. Deferiprone was first reported as a potential orally active iron chelator in 1984 [158] and demonstrated to be active in man in 1987 [159]. It was licensed for use in India in 1994 and in Europe in 1999, receiving full marketing authorization in 2002. The FDA provided approval for its use in 2011. There are numerous reports indicating the comparative effectiveness of desferrioxamine and deferiprone [160]. A particularly important property of deferiprone is its ability to penetrate cells, coordinate iron, forming a neutral complex, which is also capable of permeating membranes. Thus, iron can be readily removed from iron-loaded cells including those of cardiac tissue (Figure 25) [161]. This ability extends to the clinical situation [162,163], where it has been demonstrated that deferiprone therapy is associated with significantly greater cardiac protection than DFO in patients with thalassemia major [143,164]. Unfortunately, the dose required to keep a previously well chelated patient in negative iron balance with deferiprone is relatively high, in the region of 75–100 mg kg–1 day–1. One of the major reasons for the limited efficacy of deferiprone in clinical use is that it undergoes extensive metabolism in the liver. The use of deferiprone has a range of associated side effects [165], the most important being a low incidence of reversible agranulocytosis [166]. 8 Iron: Effect of Deficiency and Overload 275

Figure 25 Schematic representation of the penetration of deferiprone [LH]0 through the plasma membrane. The bidentate ligand scavenges loosely bound intracellular iron, forming the 3:1 complex, which also carries zero net charge. Efflux as the iron complex leads to iron excretion.

O OH O O O OH OH OH OH N H OH N N CH N N CH 3 H3C 3 CH3 OH CH3 CH3 CH3 O

15 16 17 18

High iron affinity hydroxypyridinones: In order to further improve chelation efficacy, considerable effort has been applied to the design of novel hydroxypyridinones with enhanced pFe3+ values [167]. Novartis synthesized a range of bidentate hydroxypyridinone ligands, which possess an aromatic substituent at the 2-position. The lead compound (16) was found to be orally active and highly effective at removing iron from both the iron-loaded rat [168] and marmoset [169]. In similar fashion, Hider and coworkers have demonstrated that the introduction of either a l′-hydroxyalkyl group (17) [170] or an amido function (18) [171] at the 2-position of 3-hydroxypyridin-4-ones enhances the affinity for iron(III) over the pH range 5–8. These changes result in an increase in the corresponding pFe3+ values due to the reduced competition with hydrogen ions; thus the 2-amidopyridin-4-one (18) has a pFe value of 21.7 as compared with that of the analogous deferiprone (15) which possesses a pFe value of 20.5. In practical terms this means that at pH 7.4 (18) binds iron over ten times more tightly than deferiprone. These novel high pFe3+ HPOs show great promise in their ability to remove iron under in vivo conditions. Detailed dose-response studies suggest that chelators with high pFe3+ values scavenge iron more effectively at lower doses when compared 276 Hider and Kong with simple dialkyl substituted hydroxypyridinones and so in principle can be used at the lower dose of 20 mg kg–1. A number of related compounds are currently undergoing preclinical evaluation. Combined therapy with desferrioxamine and hydroxypyridinones: By virtue of its small size and ability to penetrate cells, deferiprone has the capability of efficiently scavenging excess iron. However due to its bidentate nature, the ability of deferiprone for iron(III) at neutral pH values is highly concentration-dependent and at relatively low concentrations (<5 μM) the iron deferiprone complex will donate iron to competing ligands [172]. If deferiprone is used together with a high affinity hexadentate chelator such as desferrioxamine, the deferiprone iron complex will readily donate iron to the kinetically more stable desferrioxamine [173]. Indeed, deferiprone enhances plasma NTBI removal in the presence of desferrioxamine [174]. Early clinical studies indicated that such combination therapy is effective at increasing iron excretion [175]. These observations led to more extensive clinical investigations using deferiprone and desferrioxamine in sequential fashion and resulted with beneficial effects in survival, iron removal and cardiac disease [176,177].

4.2.3 Use of Iron Chelators to Treat Diseases Other than Thalassemia

4.2.3.1 Sickle Cell Anemia

Transfusion has been introduced for the treatment of sickle cell anemia, due to its beneficial effect in the treatment of crises and in the reduction of the incidence of stroke [178] (Section 3.3.1.2). Again an effective orally active iron chelator permits regular transfusion of such patients without the worry of inducing an associated iron overloaded state. There have been a number of trials comparing the efficacy of different chelators and the outcome of these studies reviewed [179]. At the present time there is no clear preference for a particular chelator. In a multicenterd study of iron overload [180], three patient groups have been compared (transfused thalassemia patients, transfused sickle cell patients and non-transfused sickle cell patients). There were more endocrine problems in the transfusion-dependent thalassemia patients (56.3%) than in both transfused sickle cell patients (13.1%) and non- transfused sickle cell patients (7.8%) and it has been suggested that this difference may relate to the different NTBI levels and hence speciation (Section 3.1). Generally, NTBI levels in thalassemia patients are higher than those of sickle cell patients [181]. This finding could also explain the low incidence of cardiac disease in transfused sickle cell patients [180]. There was a relatively poor compliance observed with deferasirox in the sickle cell anemia group [182].

4.2.3.2 Myelodysplastic Syndromes

Many patients with myelodysplastic syndromes (MDS) become dependent on blood transfusions (Section 3.3.2) and so treatment by an orally active iron chelator is appropriate. The use of deferiprone is not ideal in view of the risk of agranulocytosis 8 Iron: Effect of Deficiency and Overload 277 which may be higher in this patient group [183]. In contrast, deferasirox has found useful application in removing excess iron from transfused MDS patients [184,185]. As cardiac problems are the most frequent serious complications associated with iron overload in MDS patients [86], procedures designed to maintain NTBI levels at a low level should be adopted. Daily administration of deferasirox is thus likely to provide a useful option.

4.2.3.3 Friedreich’s Ataxia

Friedreich’s ataxia involves the accumulation of iron in mitochondria; cardiac, gastrointestinal and brain tissues being most severely affected (Section 3.4.2). In principle, an iron chelator which can readily cross membranes, gain access to the mitochondrial matrix, scavenge inappropriately deposited iron and efflux from the mitochondrion as a stable iron complex, should find application in the treatment of this disease. Deferiprone is one such compound (Figure 25) as demonstrated by Cabantchik and coworkers [46,186]. Treatment of Friedreich’s ataxia patients with deferiprone for 6 months led to a decrease in iron levels that had specifically accumulated in dentate nuclei of the brain and also improved gait and control of the gastrointestinal tract [93]. This preliminary clinical study has now been extended to a multicenter investigation.

5 Neuropathology and Iron

Neurodegeneration is a complicated multifaceted process which leads to many chronic disease states. A broad classification can be achieved on the basis of neuropatho- logical changes: (i) Disorders characterized by the accumulation of abnormal proteins leading to a selective loss of neurons; examples include Alzheimer’s disease (Aβ amyloid plaques), Parkinson’s disease (Lewy bodies), Huntington disease (Huntingtin aggregates), and Pick’s disease (Pick bodies). (ii) Disorders resulting from dysfunction of motor neurons; examples include multiple sclerosis, amyotrophic lateral sclerosis, Friedreich’s ataxia, and progressive supranuclear palsy. Significantly, all these disease states have been associated with elevated levels of iron in the brain resulting from a loss of control of iron homeostasis in particular areas of the brain [187]. The control of neuronal cytosolic iron(II) levels is achieved by an interplay of fluxes centered on membrane transporters, mitochondria, lysosomes, and ferritin (Figure 8) together with neuromelanin which acts as an additional storage site for iron and other transition metals [188]. The concentration of iron in the cerebrospinal fluid, and hence the interstitial fluid, ranges between 0.2 and 1.1 μM, whereas transferrin levels are low, typically 0.25 μM [189]. Thus, CSF iron levels will frequently exceed the binding capacity of transferrin and in view of the relatively high levels of citrate, form complexes 9 and 10. 278 Hider and Kong

The levels of this non-transferrin bound iron will influence cytosolic levels of iron(II). Abnormal iron accumulation induces the oxidation of reduced glutathione in human neuronal cells [190] and this in turn, by virtue of the involvement of glutathione in iron sulfur cluster synthesis [8,191], can lead to a reduction in the respiratory Complex I activity (Complex I contains 8 Fe-S clusters). Reduction of Complex I activity can result in vicious cycles of increased oxidative stress, increased iron accumulation, and decreased GSH content [189]. Significantly, defects in mitochondrial electron transport have been reported for Alzheimer’s disease, Parkinson’s disease, Huntington disease, and Friedreich’s ataxia [192].

5.1 Alzheimer’s Disease

Alzheimer’s disease is the most common form of dementia, there being over 24 million sufferers at the present time and by 2040 it has been estimated that there will be 80 million worldwide. Central to the disease is the formation of amyloid plaques which predominately consist of Aβ amyloid peptide, a 42 membered peptide resulting from the breakdown of a membrane-bound protein (APP) (Figure 26) [193]. There is continual production of Aβ42 in normal individuals, but in Alzheimer’s disease patients, proteosome processing becomes less efficient and the peptide accumulates both in the membrane and as soluble oligomers. The latter aggregate to form amyloid plaques (Figure 26). The oligomers and the membrane bound Aβ42 molecules possess an iron binding site [194,195] which endows the peptide with

Figure 26 Amyloidogenic processing of amyloid precursor protein (APP). APP is cleaved to produce membrane bound Aβ42 peptide, which is in equilibrium with water soluble oligomers of Aβ42 peptide. The oligomers aggregate to form amyloid plaques. 8 Iron: Effect of Deficiency and Overload 279 pro-oxidant activity [196,197]. It has recently been proposed that APP possesses ferroxidase activity, which is inhibited in Alzheimer’s disease, thereby facilitating neuronal iron accumulation [198,199]. Brain iron accumulation has been demonstrated in Alzheimer’s disease patients by both MRI [200] and ICP-MS [201]. This sequence of events could lead to mitochondrial toxicity as outlined in Section 5.1. Clearly there is potential for iron chelation therapy, both in prophylactic and therapeutic treatments of Alzheimer’s disease (Section 5.5).

5.2 Parkinson’s Disease

Parkinson’s disease, like Alzheimer’s disease, tends to be more common in the elderly, about 4% of those over 80 years experience symptoms. Although stiffness and tremor are common symptoms, there are many features of the disease [202], for instance the nigrostriatal and caudate nucleus dopaminergic neurons are adversely effected leading to cell death and neuronal loss. Genetic factors, environmental factors and aging can all influence the onset of the disease [203]. Lewy bodies appear and there is multiple evidence of both damage induced by free radicals [204,205] and for an increased nigral iron content [206,207]. In particular, there are reduced glutathione levels in the nigral brain region and the mitochondrial Complex I is inhibited [205]. As with Alzheimer’s disease there is clear potential for iron chelation therapy (Section 5.5).

5.3 Pantothenate Kinase-2 Deficiency

Pantothenate kinase-2 (PANK 2) deficiency is a relatively uncommon autosomal recessive trait which is characterized by progressive Parkinson-like rigidity, together with mental and emotional retardation (previously termed Hallervorden-Spatz syndrome) [208]. Onset is in late childhood, death typically occurring within 10 years. The globus pallidus and substantia nigra possess elevated levels of iron, mostly located in microglia and macrophages giving rise to the ‘the eye of the tiger’ MRI brain image [209,210]. PANK 2 is the first enzyme involved in the biosynthesis of coenzyme A and is located in the mitochondria. Patients with PANK 2 deficiency suffer from a coenzyme A deficiency and a related elevation of cysteine [211] which has been suggested to increase iron levels by chelation [212].

5.4 Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the developed nations, in people aged 65 and older [213]. Oxidative stress and free radical damage have been implicated in the pathogenesis of AMD [214] 280 Hider and Kong and iron is a likely oxidant, as AMD-affected maculars possess a higher iron content than healthy age-matched maculars, as demonstrated by Perl’s stain [215]. Whereas iron accumulation is observed in AMD retinas, it is unclear whether iron is directly involved in AMD pathogenesis or is simply a byproduct of AMD pathology. However, there are several lines of evidence that link iron with AMD pathogenesis; for instance, the levels of bone morphogenetic protein 6 (BMP 6), a major regulator of systemic iron (Section 1.4.1), is increased in advanced AMD eyes [216] and hepcidin-knockout mice experience age-dependent increases in retinal iron followed by retinal degeneration [217]. It is significant that retinal iron accumulation resulting from Friedreich’s ataxia and PANK 2 disease is associated with retinal degeneration [218].

5.5 Potential of Iron Chelators for the Treatment of Neurodegeneration

A critical prerequisite for a chelator capable of treating various forms of neurodegeneration is to be orally active and to readily permeate the blood brain barrier or in the case of the eye, the blood-retinal barrier. Thus, the chelator should have a molecular weight <500, be sufficiently hydrophobic to partition in membranes and yet be sufficiently hydrophilic to achieve water solubility at pharmacological levels [219]. Of the three chelators currently used in the clinic, only deferiprone possesses these properties (Figure 25), which accounts for why it has been so extensively investigated for the treatment of neurodegenerative diseases [93,220–223]. A range of 8-hydroxyquinoline derivatives, including clioquinol (19) [224], VK-28 (20) [225], and an iron-binding peptide (21) [226], have also been investigated.

OH OH N N N Cl

S I N N OH OH NAPCSIPE

19 20 21

Deferiprone (15), VK-28 (20), and clioquinol (19) have each demonstrated neuroprotective action in various models of Alzheimer’s disease [227–229] and clioquinol has been investigated in a related clinical trial [230]. A similar situation exists with Parkinson’s disease, where a number of successful studies have been achieved in various animal models with both 8-hydroxyquinolines [225,231] and deferiprone [220]. Deferiprone is currently under clinical investigation for efficacy and safety in Parkinson’s disease [232,233]. Deferiprone has also been demonstrated to have a beneficial effect in the clinical treatment of age-dependent retinal degeneration [221,234] and PANK 4 disease [222,223]. 8 Iron: Effect of Deficiency and Overload 281

Various strategies have been investigated in an attempt to enhance the transfer of chelators across the BBB. In principle pendant glucose molecules can be attached to molecules in order to enhance their ability to permeate the BBB. Due to the critical requirement of the brain for glucose, the BBB is endowed with a high density of GLUT 1 hexose transporter protein. With this strategy in mind, glucose conjugates (22) and (23) have been synthesized [235,236], but to date have not been demonstrated to cross the BBB [237]. Another approach involves the attachment of chelators to nanoparticles [238]. Chelator hybrid molecules are also under current investigation in a range of neurodegenerative models, for instance, radical scavengers (24) [239] and monoamine oxidase inhibitors (25) [240]. This field has recently been reviewed [241].

O OH

N OH O O O O HO HO OH NH HO HO HO N O OH R

22 23

O OH

N N N

HO N OH

24 25

6 The Role of Iron Chelation in Cancer Therapy

The importance of iron in tumor cell growth has been discussed over the past 50 years [242], high levels of dietary iron having been linked to increased tumor development [243,244]. More recently an iron regulatory gene signature has been linked to the incidence of breast cancer [245], and both gastrointestinal and liver cancer have been specifically associated with dietary iron [246]. Cell proliferation is dependent on a plentiful supply of iron and iron chelators can interfere with the cell cycle, 282 Hider and Kong

causing G1/S arrest by removing iron from ribonucleotide reductase [247] and deoxyhypusine hydroxylase [248]. Many chelating agents have been investigated for their potential to selectively inhibit tumor cell growth, in particular desferrioxamine [249], spermidine catecholamides [250], and PIH analogues [247]. At the present time it has not been possible to achieve an acceptable selective toxicity against tumor cells in the clinic.

N N O O

HN HN N OH N OH N N N NH N S N OH

26 27 28

Pyridoxal isonicotinoyl hydrazine (PIH) derivatives (26) show potential in this regard and Richardson and his coworkers have thoroughly investigated this group of chelators. The β-napthol derivative (27) has been reported to be a particularly effective antiproliferative agent [251], as have the closely related thiosemicarbazones (28) [252]. The precise role of iron in the cell-cycle control remains unclear, but the posttranscriptional regulation of a cyclin-dependent kinase inhibitor may also feature in this network of iron-dependent reactions [253].

7 Iron and Infection

Iron is essential for the growth of almost all microorganisms and thus bacteria and fungi have evolved strategies to scavenge iron from the soil, fresh and marine water, and living organisms. One of the most common strategies is siderophore production. Siderophores are low molecular weight compounds (500–1500 dal- tons) which possess a high affinity and selectivity for iron. There are over 500 different siderophores 270 of which have been structurally characterized [131]; desferrioxamine (7), desferrithiocin (11), and enterobactin (Figure 21) are typical examples. Pathogenic bacteria and fungi have developed the means of survival in animal tissue. They may invade the gastrointestinal tract (Escherichia, Shigella, and Salmonella), the lung (Pseudomonas, Bordatella, Streptococcus, and Corynebacterium), skin (Staphylococcus) or the urinary tract (Escherichia and Pseudomonas). Such bacteria may colonize wounds (Vibrio and Staphylococcus) and be responsible for septicaemia (Yersinia and Bacillus). Some bacteria survive for long periods of time in intracellular organelles, for instance Mycobacterium. Because of this continual risk of bacterial and fungal invasion, animals have 8 Iron: Effect of Deficiency and Overload 283

developed a number of lines of defence based on immunological strategies, the complement system, the production of iron-siderophore binding proteins and the general “withdrawal” of iron [254]. There are two major types of iron-binding proteins present in most animals that provide protection against microbial invasion – extracellular protection is achieved by the transferrin family of proteins and intracellular protection is achieved by ferritin (Section 1.2.4). Under normal conditions transferrin is about 25–40% saturated, which means that any freely available iron in the serum will be immediately scavenged – thus preventing microbial growth. Most siderophores are unable to remove iron from transferrin, although some, for instance aerobactin, can compete [255,256]. Mammals also produce lactoferrin, which is similar to serum transferrin but possesses an even higher affinity for iron [257]. Lactoferrin is present in secretory fluids, such as sweat, tears and milk, thereby minimizing bacterial infection. Ferritin is present in the cytoplasm of cells and limits the intracellular iron level to approximately 1 μM. Siderophores are unable to mobilize iron from ferritin. In addition to these two classes of iron binding proteins, hepcidin is involved in controlling the release of iron from absorptive enterocytes, iron-storing hepatocytes, and macrophages (Section 1.4.1). Infection leads to inflammation and the release of interleukin-6 (IL-6) which stimulates hepcidin expression. In humans, IL-6 production results in low serum iron, making it difficult for invading pathogens to infect. In addition to these “iron withdrawal” tactics, mammals produce an iron- siderophore binding protein, siderochelin [258]. Siderocalin is a potent bacteriostatic agent against E. coli. As a result of infection, it is secreted by both macrophages and hepatocytes, enterobactin being scavenged from the extracellular space. Indeed, mice are highly susceptible to E. coli infections when the siderocalin gene is “knocked- out” [259]. Siderocalin binds a wide range of tris-catecholate siderophores.

7.1 Tuberculosis

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis infects one- third of the world population. Despite the development of new drugs, the incidence of TB continues to increase. Increased iron status enhances tuberculosis infection [260]. Siderophore molecules used by Mycobacterium for iron acquisition are potential therapeutic targets, as are synthetic iron chelators which are capable of outcompeting siderophores for iron.

7.2 Malaria

Many metabolic components of the erythrocytic malaria parasite are dependent on iron, including ribonucleotide reductase, and mitochondrial function. Several studies have indicated that the erythrocyte labile iron pool is the iron source for 284 Hider and Kong malaria parasites (Section 1.3) [246]; a finding somewhat confirmed by the observation that iron chelators suppress the growth of P. falciparum in human erythrocytes. A wide range of chelators have been demonstrated to inhibit the growth of erythrocytic malaria parasites under in vitro conditions [261,262], however with the exception of desferrioxamine, extension to animal studies has proved to be less impressive [262]. There have been a number of clinical investigations centered on the use of desferrioxamine, where it has displayed an appreciable antimalarial activity but failed to effect a cure [246].

8 Overview and Future Developments

The development of iron biochemistry has been dramatic over the past 50 years. A large range of iron-dependent enzymes have been characterized, the chemistry of electron transfer proteins, containing iron-sulfur clusters, is beginning to be understood and oxygen-binding iron centers are very well characterized. The highly controlled transport of iron throughout multicellular organisms and the mechanism of intracellular distribution is now understood, although there is one tissue where iron distribution is far from clear: the brain. Transferrin levels are much lower in CNS than in blood and there are a number of brain proteins which appear to be influenced by iron levels and yet currently have no known function, for instance amyloid precursor protein and α-synuclein. Clearly, this is an area that deserves intensive investigation, particularly as the progression of many forms of neurodegeneration appears to be enhanced by elevated iron levels. Over the same period iron chelators have emerged as an important therapeutic class. For many years the orally inactive desferrioxamine was the only iron chelator available for clinical use, but during the past twenty years two other chelators have been introduced, deferiprone and deferasirox. Both are orally active and this has rendered the treatment of iron overload to be more “patient friendly” thereby enabling clinicians to investigate the use of iron chelation for diseases other than systemic iron overload, for instance Parkinson’s disease and macular degeneration. There are more iron chelators under development and it is likely that in years to come there will be a selection of iron chelators available for clinical use that will cover ranges of both iron affinity and membrane penetrative ability. Studies are in progress to design lysosomotrophic and mitochondrotrophic chelators. Improved therapeutic control of a wide range of anemias has been accomplished over the past 50 years and in particular the development of a range of relatively nontoxic parential iron preparations has proved to be highly beneficial. Surprisingly, the most common oral supplement for the treatment of iron deficiency anemia is ferrous sulfate, which was first introduced to medicine in 1832. There is a very clear requirement for the introduction of a replacement oral therapy due to the various toxic side effects of ferrous sulfate. Hopefully one or more such replacements will be developed in the near future. 8 Iron: Effect of Deficiency and Overload 285

Abbreviations

ALAS 5-amino levulinate synthase AMD age-related macular degeneration APP amyloid precursor protein (Fig. 26) AscH– ascorbic acid ATP adenosine 5′-triphosphate BBB blood brain barrier BMP bone morphogenetic protein CD91 lipoprotein receptor CNS central nervous system CSF cerebrospinal fluid DCYTB duodenal cytochrome b DF desferrithiocin DFO desferrioxamine DMT1 divalent metal-iron transporter 1 EDTA ethylenediamine-N,N,N′,N′-tetraacetic acid FBXL5 f-box and leucine-rich repeat protein 5 FDA food and Drug Administration FPN1 ferroportin FXN frataxin GDF15 growth differentiation factor 15 GLUT 1 glucose transporter 1 Grx 5 glutaredoxin-5 HAMP hepcidin synthesis gene Hb hemoglobin (Fig. 16) HCP1 heme carrier protein 1 HEIDI 2,2′-(2-hydroxyethylazanediyl)diacetic acid HFE hemochromatosis protein HIV human immunodeficiency virus HPO hydroxypyridinone ICP-MS inductively coupled plasma mass spectrometry IL-6 interleukin 6 IRE iron responsive elements IRP iron responsive proteins JAK/STAT janus kinase/signal transducer and activator of transcription MDS myelodysplastic syndrome MECAM N,N′-(5-((2,3-dihydroxycyclohexa-1,3-dienecarboxamido)methyl)- 1,3-phenylene)bis(methylene)bis(2,3-dihydroxybenzamide) MHC major histocompatibility complex MRI magnetic resonance imaging MTP metal transporter protein NHE normal hydrogen electrode NTBI non-transferrin bound iron 286 Hider and Kong

PANK 2 pantothenate kinase-2 PIH pyridoxal isonicotinoyl hydrazine SDS sodium dodecyl sulfate SMAD 4 tumor suppressor gene SS sickle cell disease TB tuberculosis TfR1 transferrin receptor 1 TfR2 transferrin receptor 2 WHO World Health organization XLSA X-linked sideroblastic anemia

References

1. W. Schneider, Chimia 1988, 42, 9–20. 2. R. R. Crichton, Iron Metabolism, 3rd edn., Wiley, Chichester, 2009. 3. A. K. Powell, S. L. Heath, D. Gatteschi, L. Pardi, R. Sessoli, G. Spina, F. Del Giallo, F. Pieralli, J. Am. Chem. Soc. 1995, 117, 2491–2502. 4. W. R. Harris, in “Molecular and Cellular Iron Transport”, Ed D. M. Templeton, Marcel Dekker, Inc., New York, 2002, pp. 1–40. 5. B. Halliwell, J. M. C. Gutteridge, Arch. Biochem. Biophys. 1986, 246, 501–514. 6. A. Ozer, R. K. Bruick, Nature Chem. Biol. 2007, 3, 144–153. 7. C. Andreini, I. Bertini, G. Cavallaro, R. J. Najmanovich, J. M. Thornton, J. Mol. Biol. 2009, 388, 256–380 8. R. C. Hider, X. Kong, Dalton Trans. 2013, 42, 3220–3229. 9. K. Mizutani, M. Toyoda, B. Mikami, Biochim. Biophys. Acta 2012, 1820, 203–211. 10. M. K. Mayle, A. M. Le, D. T. Kamei, Biochim. Biophys. Acta 2012, 1820, 264–281. 11. W. R. Harris, Biochim. Biophys. Acta 2012, 1820, 348–361. 12. P. M. Harrison, P. Arosio, Biochim. Biophys. Acta 1996, 1275, 161–203. 13. G. A. Clegg, J. E. Fitton, P. M. Harrison, A. Treffry, Progr. Biophys. Mol. Biol.1981, 36, 53–86. 14. S. Roberts, A. Bomford, J. Biol. Chem. 1988, 241, 3630–3637. 15. R. C. Hider, X. Kong, BioMetals 2011, 24, 1179–1187. 16. H. Gonshin, B. Mackenzie, U. V. Berger, Y. Gunshin1, M. F. Romero, W. F. Boron, S. Nussberger, J. L. Gollan, M. A. Hediger, Nature 1997, 388, 482–488. 17. M. Shayeghi, G. O. Latunde-Dada, J. S. Oakhill, A. H. Laftah, K. Takeuchi, N. Halliday, Y. Khan, A. Warley, F. E. McCann, R. C. Hider, D. M. Frazer, G. J. Anderson, C. D. Vulpe, R. J. Simpson, A. T. McKie, Cell 2005, 122, 789–801. 18. P. N. Paradkar, K. B. Zumbrennen, B. H. Paw, D. M. Ward, J. Kaplan, Mol. Cell. Biol. 2009, 29, 1007–1016. 19. W. Chen, H. A. Dailey, B. H. Paw, Blood 2010, 116, 628–630. 20. A.T. McKie, P. Marciani, A. Rolfs, K. Brennan, K. Wehr, D. Barrow, S. Miret, A. Bomford, T. J. Peters, F. Farzaneh, M. A. Hediger, M. W. Hentze, R. J. Simpson, Mol. Cell 2000, 5, 299–309. 21. A. Donovan, A. Brownlie, Y. Zhou, J. Shepard, S. J. Pratt, J. Moynihan, B. H. Paw, Anna Drejer, B. Barut, A. Zapata, T. C. Law, C. Brugnara, S. E. Lux, G. S. Pinkus, J. L. Pinkus, P. D. Kingsley, J. Palis, M. D. Fleming, N. C. Andrews, L. I. Zon, Nature 2000, 403, 776–781. 22. S. Abboud, D. J. Haile, J. Biol. Chem. 2000, 275, 19906–19912. 23. M. U. Muckenthaler, R. Hill in Iron Physiology and Pathophysiology in Humans, Eds J. G. Anderson, G. D. McLaren, Humana Press, New York, 2012, pp. 27–50. 8 Iron: Effect of Deficiency and Overload 287

24. E. W. Mullner, B. Neupert, L. C. Kuhn, Cell 1989, 58, 373–382. 25. K. J. Addess, J. P. Basilion, R. D. Klausner, T. A. Rouault, A. Pardi, J. Mol. Biol. 1997, 274, 72–83. 26. D. J. Haile, M. W. Hentze, T. A. Rouault, J. B. Harford, R. D. Klausner, Mol. Cell. Biol. 1989, 9, 5055–5061. 27. T. Ganz, S.Vaulont, in Iron Physiology and Pathophysiology in Humans, Eds G. J. Anderson, G. D. McLaren, Humana Press, New York, 2011, pp. 173–190. 28. M. W. Hentze, M. U. Muckenthaler, B. Galy, C. Camaschella, Cell 2010, 142, 24–38. 29. J. J. Bullen, E. Griffiths, Iron and Infection, 2nd edn., Wiley Press, New York, 1999. 30. P. R. Dallman, M. A. Siimes, A. Stekel, Am. J. Clin. Nutr. 1980, 33, 86–118. 31. W. I. Leong, B. Lönnerdal in Iron Physiology and Pathophysiology in Humans, Eds G. J. Anderson, G. D. McLaren, Humana Press, New York, 2011, pp. 81–99. 32. World Health Organisation, Iron Deficiency Anaemia: Assessment, Prevention and Control. A Guide to Programme Managers, Geneva, WHO, 2001. 33. R. D. Baynes, T. H. Bothwell, Ann. Rev. Nutr. 1990, 10, 133–148. 34. F. E. Viteri, B. Torun, in Clinics in Haematology, Vol 3, Ed L. Garby, Saunders, London, 1974, pp 609–626. 35. G. W. Gardner, V. R. Edgerton, B. Senewiratne, R. J. Barnard, Y. Ohira, Am. J. Clin. Nutr. 1977, 30, 910–917. 36. The British Nutrition Foundation, Iron, Nutritional and Physiological Significance, Chapman and Hall, London, 1995, pp. 65–78. 37. J. D. Cook, M. B. Reddy, J. Burri, M. A. Juillerat, R. F. Hurrell, Am. J. Clin. Nutr. 1997, 65, 964–969. 38. P. Blaud, Rev. Med. Franç. Etranger, 1832, 45, 341–367. 39. I. H. Stockley, Stockley’s Drug Interactions, 6th edn, Pharmaceutical Press, London, 2002. 40. D. M. Reffitt, T. J. Burden, P. T. Seed, J. Wood, R. P. H. Thompson, J. Powell, Ann. Clin. Biochem. 2000, 37, 457– 466. 41. P. Jacobs, G. Johnson, L. Wood, J. Medicine 1984, 15, 367–377. 42. W. Schneider, W. Forth, Arzneim. Forsch. Drug Res. 1987, 37, 89–95. 43. C.N. Roy, in Iron Physiology and Pathophysiology in Humans, Eds G. J. Anderson, G. D. McLaren, Humana Press, New York, 2011, pp. 303–320. 44. C. N. Roy, D. A. Weinstein, N. C. Andrews, Pediatr. Res. 2005, 53, 507–512. 45. E. Nemeth, S. Rivera, V. Gabayan, C. Keller, S. Taudorf, B. K. Pedersen, T, Ganz, Blood 2003, 101, 2461–2463. 46. Y. S. Sohn, W. Brever, A. Munnich, Z. I. Cabantchik, Blood 2008, 111, 1690–1699. 47. R. W. Evans, X. Kong, R. C. Hider, Biochim. Biophys. Acta 2012, 1820, 282–290. 48. J. P. Kaltwasser, U. Kessler, R. Gottschalk, G. Stucki, B. Moller, J. Rheumatol. 2001, 28, 2430–2436. 49. L. T. Goodnough, B. Skikne, C. Brugnara, Blood 2000, 96, 823–833. 50. W. Y. Qunibi, Arzneimittelforschung 2010, 60, 399–412. 51. P. Geisser, S. Burckhurdt, Pharmaceutics 2011, 3, 12–33. 52. L. J. Noetzli, S. M. Carson, A. S. Nord, T.D. Coates, J. C. Wood, Blood 2009, 114, 4021–4026. 53. M. N. Silva, X. Kong, M. C. Parkin, R. Cammack, R. C Hider, Dalton Trans. 2009, 40, 8616–8625. 54. J.L. Pierre, I. Gautier-Luneau, BioMetals 2000, 13, 91–96. 55. R. W. Evans, R. Rafique, A. Zarea, C. Rapisarda, R. Cammack, P. J. Evans, J. B. Porter, R. C. Hider, J. Biol. Inorg. Chem. 2008, 13, 57–74. 56. M. N. Silva, R. C. Hider, Biochim. Biophys. Acta 2009, 1794, 1449–1458. 57. P. Brissot, M. Ropert, C. Le Lan, O. Loreal, Biochim. Biophys. Acta 2012, 1820, 403–410. 58. P. C. Adams, J. C. Barton, Lancet 2007, 370, 1855–1860. 59. N. C. Andrews, New Engl. J. Med. 1999, 341, 1986–1995. 60. A. Pietrangelo, New Engl. J. Med. 2004, 350, 2383–2397. 288 Hider and Kong

61. J. N. Feder, A. Gnirke, W. Thomas, Z. Tsuchihashi, D. A. Ruddy, A. Basava, F. Dormishian, R. Jr Domingo, M. C. Ellis, A. Fullan, L. M. Hinton, N. L. Jones, B. E. Kimmel, G. S. Kronmal, P. Lauer, V. K. Lee, D. B. Loeb, F. A. Mapa, E. McClelland, N. C. Meyer, G. A. Mintier, N. Moeller, T. Moore, E. Morikang, C. E. Prass, L. Quintana, S. M. Starnes, R. C. Schatzman, K. J. Brunke, D. T. Drayna, N. J. Risch, B. R. Bacon, R. K. Wolff, Nature 1996, 13, 399–408. 62. M. Simon, M. Bourel, R. Fauchet, B. Genetet, Gut 1976, 17, 332–334 63. J. N. Feder, Z. Tsuchihashi, A. Irrinki, V. K. Lee, F. A. Mapa, E. Marikang, C. E. Prass, S. M. Starnes, R. K. Wolff, S. Parkkilay, W. S. Sly, R. C. Schatzman, J. Biol. Chem. 1997, 272, 14025–14028. 64. K. J. Allen, L. C. Gurrin, C. C. Constantine, N. J. Osborne, M. B. Delatycki, A. J. Nicol, C. E. Mcharen, M. Bahlo, A. E. Nisselle, C. D. Vulpe, G. J. Anderson, M. C. Southey, G. G. Giles, D. R. English, J. L. Hopper, J. K. Olynk, L. W. Powell, D. M. Gertig, New Engl. J. Med. 2008, 358, 221–230. 65. P. L. Lee, E. Beutler, Annu. Rev. Pathol. Mech. Dis. 2009, 4, 489–515. 66. T. J. Sproule, E. C. Jazwinska, R. S. Britton, B. R. Bacon, R. E. Fleming, W. S. Sly, D. C. Roopeniom, Proc. Natl. Acad. Sci. USA 2001, 98, 5170–5174. 67. C. Camaschella, A. Roetto, M. Cicilano, P. Pasquero, S. Bosio, L. Gubetta, F. Di Vito, D. Girelli, A. Totaro, M. Carella, A. Grifa, P. Gasparini, Eur. J. Hum. Genet. 1997, 5, 371–375. 68. G. Sebastiani, K. Pantopoulos, Metallomics 2011, 3, 971–986. 69. L. P. Yang, S. J. Keam, G. M. Keating, Drugs 2007, 67, 2211–2230. 70. S. Rivella, in Iron Physiology and Pathophysiology in Humans, Eds G. J. Anderson, G. D. McLaren, Humana Press, New York, 2012, pp. 321–341. 71. D. J. Weatherall, Ann. New York Acad. Sci. 2010, 1202, 17–23. 72. J. B. S. Haldane, Hereditas 1949, 35, 267–273. 73. D. J. Weatherall, Brit. J. Haematol. 2008, 141, 276–286. 74. H. F. Bunn, Blood 2013, 121, 20–25. 75. D. J. Weatherall, J. B. Clegg, Thalassaemia Syndromes, 3rd edn, Blackwell, London, 1981. 76. B. Model, M. Darlison, Bull. World Health Organ. 2008, 86, 480–487. 77. D. J. Weatherall, Brit. J. Heamatol. 2000, 321, 1117–1120. 78. G. M. Brittenham, in Hematology: Basic Principles and Practice, Eds R. Hoffman, E. Benz, S. Shattil, B. Furie, H. Cohen, Churchill Livingstone, New York, 1991, pp. 327–342. 79. R. Sephton Smith, Brit. Med. J. 1962, 1577–1580. 80. S. Dhungana, P. S. White, A. L. Crumbliss, J. Biol. Inorg. Chem. 2001, 6, 810–818. 81. R. D. Propper, S. B. Schurin, D. G. Nathan, New Engl. J. Med. 1976, 294, 1421–1423. 82. A. T. Taher, K. M. Musallam, M. D. Cappellini, D. J. Weatherall, Brit. J. Haematol. 2010, 152, 512–523. 83. S. Claster, E. P. Vichinsky, Brit. Med. J. 2003, 327, 1151–1155. 84. M. J. Stuart, R. L. Nagel, Lancet 2004, 364, 1343–1360. 85. E. B. Fung, P. R. Harmatz, P. D. K. Lee, M. Milet, R. Bellevue, M. R. Jeng, K. A. Kalinyak, M. Hudes, S. Bhatia, E. P. Vinchinsky, Brit. J. Haematol. 2006, 135, 574–582. 86. N. Galtermann, E. A. Rachmilewitz, Ann. Hematol. 2011, 90, 1–10. 87. N. B. A. Roy, S. Myerson, A. H. Schuh, P. Bignell, R. Patel, J. S. Wainscoat, S. McGowan, E. Marchi, W. Atoyebi, T. Littlewood, J. Chacko, P. Vyas, S. B. Killick, Brit. J. Haematol. 2011, 154, 521–524. 88. v. Campuzano, L. Montermini, M.D. Molto, L. Pianese, M. Cossee, F. Cavalcanti, E. Monros, F. Rodius, F. Duclos, A. Monticelli, F. Zara, J. Cañizares, H. Koutnikova, S. Bidichandani, C. Gellera, A. Brice, P.Trouillas, G. De Michele, A. Filla, R. De Frutos, F. Palau, P. I. Patel, S. D. Donato, J. L. Mandel, S. Cocozza, M. Koenig, M. Pandolfo, Science 1996, 271, 1423–1427. 89. M. Babcock, D. deSilva, R. Oaks, S. Davis-Kaplan, S. Jiralerspong, L. Montermini, M. Pandolfo, J. Kaplan, Science 1997, 276, 1709–1712. 90. T. L. Stemmler, E. Lesuisse, D. Pain, A. Dancis, J. Biol. Chem. 2010, 285, 26737–26743. 8 Iron: Effect of Deficiency and Overload 289

91. V. Campuzano, L. Montermini, M. D. Moltò, L. Pianese, M. Cossée, F. Cavalcanti, Hum. Mol. Genet. 1997, 6, 1771–1780. 92. D. R. Lynch, S. R. Regner, K. A. Schadt, L. S. Friedman, K. Y. Lin, M. G. St John Sutton, Expert Rev. Cardiovasc. Ther. 2012, 10, 767–777. 93. N. Boddaert, K. H. Le Quan Sang, A. Rötig, A. Leroy-Willig, S. Gallet, F. Brunelle, D. Sidi, J. Thalabard, A. Munnich, Z. I. Cabantchik, Blood 2007, 100, 401–408. 94. M. T. Rodríguez-Manzaneque, J. Tamarit, G. Bellí, J. Ros, E. Herrero, Mol. Biol. Cell. 2002, 13, 1109–1121. 95. C. Camaschella, A. Campanella, L. D. Falco, L. Boschetto, R. Merlini, L. Silvestri, S. Levi, A. Iolascon, Blood 2007, 110, 1353–1358. 96. H. Ye, Jeong, M. C. Ghosh, G. Kovtunovych, L. Silvestri, D. Ortillo, N. Uchida, J. Tisdale, C. Camaschella, T. A. Rouault, J. Clin. Invest. 2010, 1120, 1749–1761. 97. N. C. Andrews, Nat. Rev. Genetics 2000, 1, 208–217. 98. C. C. Trenor, D. R. Campagna, V. M. Sellers, N. C. Andrews, M. D. Fleming, Blood 2000, 96, 1113–1118. 99. J. E. Levy, O. Jin, Y. Fujiwara, F. Kuo, N. C. Andrews, Nat. Genetics, 1999, 21, 396–399. 100. M. D. Fleming, M. A. Romano, M. A. Su, L. M. Garrick, M. D. Garrick, N. C. Andrews, Proc. Natl. Acad. Sci. USA 1998, 95, 1148–1153. 101. S. E. Bernstein, J. Lab. Clin. Med. 1987, 110, 690–705. 102. E. S. Russell, E. C. McFarland, E. L. Kent, Transpl. Proc. 1970, 2, 144–151. 103. D. Sladic-Simic, P. N. Martinovich, N. Zivkovic, D. Pavic, J. Martinovic, M. Kahn, H. M. Ranney, Ann. N. Y. Acad. Sci. 1969, 165, 93–99. 104. C. D. Vulpe, Y. M. Kuo, T. L. Murphy, L. Cowley, C. Askwith, N. Libina, J. Gitschier, G. J. Anderson, Nat. Genet. 1999, 21, 195–199. 105. X. Y. Zhou, S. Tomatsu, R. E. Fleming, S. Parkkila, A. Waheed, J. Jiang, Y. Fei, E. M. Brunt, D. A. Ruddy, C. E. Prass, R. C. Schatzman, R. O’Neill, R. S. Britton, B. R. Bacon, W. S. Sly, Proc. Natl. Acad. Sci. USA 1998, 95, 2492–2497. 106. V. Niederkofler, R. Salie, S. Arber, J. Clin. Invest. 2005, 115, 2180–2186. 107. F. W. Huang, J. L. Pinkus, G. S. Pinkus, M. D. Fleming, N. C. Andrews, J. Clin. Invest. 2005, 115, 2187–2191. 108. G. Nicolas, M. Bennoun, I. Devaux, C. Beaumont, B. Grandchamp, A. Kahn, Proc. Natl. Acad. Sci. USA 2001, 98, 8780–8785. 109. R. E. Fleming, J. R. Ahmann, M. C. Migas, A. Waheed, H. P. Koeffler, H. Kawabata, R. S. Britton, B. R. Bacon, and W. S. Sly, Proc. Natl. Acad. Sci. USA 2002, 99, 10653–10658. 110. I. E. Zohn, I. D. Domenico, A. Pollock, D. M. Ward, J. F. Goodman, X. Liang, A. J. Sanchez, L. Niswander, J. Kaplan, Blood 2007, 109, 4174–4180. 111. R. H. Wang, C. Li, X. Xu, Y. Zheng, C. Xiao, P. Zerfas, S. Cooperman, M. Eckhaus, T. Rouault , L. Mishra, C. X. Deng, Cell Metab. 2005, 2, 399–409. 112. R. S. Ohgami, D. R. Campagna, E. L. Greer, B. Antiochos, A. McDonald, J. Chen, J. J. Sharp, Y. Fujiwara, J. E Barker, M. D. Fleming, Nature Genet. 2005, 37, 1264–1269. 113. M. Yamamoto, O. Nakajima, Int. J. Hematol. 2000, 72, 157–164. 114. S. Lyoumi, M. Abitbol, V. Andrieu, D. Henin, E. Robert, C. Schmitt, L. Gouya, H. D. Verneuil, J. C. Deybach, X. Montagutelli, C. Beaumont, H. Puy, Blood 2007, 109, 811–818. 115. S. Al-Mahdawi, R. M. Pinto, D. Varshney, L. Lawrence, M. B. Lowrie, S. Hughes, Z. Webster, J. Blake, J. M. Cooper, R. King, M. A. Pook, Genomics 2006, 88, 580–590. 116. W. Chen, P. N. Paradkar, L. Li, E. L. Pierce, N. B. Langer, N. Takahashi-Makise, B. B. Hyde, O. S. Shirihai, D. M. Ward, J. Kaplan, B. H. Paw, Proc. Natl. Acad. Sci. USA 2009, 106, 16263–16268. 117. A. Brownlie, A. Donovan, S. J. Pratt, B. H. Paw, A. C. Oates, C. Brugnara, H. E. Witkowska, S. Sassa, L. I. Zon, Nature Genet. 1998, 20, 244–250. 118. R. A. Wingert, J. L. Galloway, B. Barut, H. Foott, P. Fraenkel, J. L. Axe, G. J Weber, K. Dooley, A. J. Davidson, B. Schmidt, B. H. Paw, G. C. Shaw, P. Kingsley, J. Palis, H. Schubert, O. Chen, J. Kaplan, L. I. Zon, Nature 2005, 436, 1035–1039. 290 Hider and Kong

119. A. Donovan, A. Brownlie, M. Dorschner, Y. Zhou, S. J. Pratt, B. H. Paw, C. B. Thisse, B. Thisse, L. I. Zon, Blood 2002, 100, 4655–4659. 120. G. C. Shaw, J. J. Cope, L. Li, K. Corson, C. Hersey, G. E. Ackermann, B. Gwynn, A. J. Lambert, R. A. Wingert, D. Traver, N. S. Trede, B. A. Barut, Y. Zhou, E. Minet, A. Donovan, A. Brownlie, R. Balzan, M. J. Weiss, L. L. Peters, J. Kaplan, L. I. Zon, B. H. Paw, Nature 2006, 440, 96–100. 121. M. Petrou, B. Modell, Prenatal Diagnosis 1995, 15, 1275–1295. 122. Y. W. Kan, M. S. Golbus, A. M. Dozy, N. Eng. J. Med. 1976, 295, 1165–1167. 123. Y. W. Kan, A. M. Dozy, Lancet(II) 1978, 910–913. 124. A. Cao, R. Galanello, M. C. Rosatelli, Clin. Haematol. 1993, 6, 263–286. 125. C. Hershko, A. M. Konijn, G. Link, Br. J. Haematol. 1998, 101, 399–406. 126. P. C. Sharpe, D. R. Richardson, D. S. Kalinowski, P. V. Bernhardt, Curr. Top. Med. Chem. 2011, 11, 591–607. 127. Y. Ma, T. Zhou, X. Kong, R. C. Hider, Curr. Med. Chem. 2012, 19, 2816–2927. 128. D. C. Harris, P. Aisen, Biochim. Biophys. Acta 1973, 329, 156–158. 129. S. J. Rodgers, C. W. Lee, C. Y. Ng, K. N. Raymond, Inorg. Chem. 1987, 26, 1622–1625. 130. K. N. Raymond, C. J. Carrano, Acc. Chem. Res. 1979, 12, 183–190. 131. R. C. Hider, X. Kong, Nat. Prod. Reports 2010, 27, 637–657. 132. G. S. Tilbrook, R. C. Hider, in Iron Transport and Storage in Microorganisms, Plants and Animals, Vol. 35 of Metal Ions in Biological Systems, Marcel Dekker, New York, 1998, 691–730. 133. U. Fagerholm, D. Nilsson, L. Knutson, H. Lennernas, Acta Physiol. Scand. 1999, 165, 315–324. 134. C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug Delivery Rev. 1997, 23, 3–25. 135. R. C. Hider, Toxicol. Lett. 1995, 82–3, 961–967. 136. R. D. Abeysinghe, P. J. Robert, C. E. Cooper, K. H. MacLean, R. C. Hider, J. B. Porter, J. Biol. Chem. 1996, 271, 7965–7972. 137. Z. D. Liu, R. S. Kayyali, R. C. Hider, J. B. Porter, A. E. Theobald, J. Med. Chem. 2002, 45, 631–639. 138. L. Banci, I. Bertini, S. Ciofi-Baffoni, T. Kozyreva, K. Zovo, P. Palumaa, Nature 2010, 465, 645–648. 139. M. Merkofer, R. Kissner, R.C. Hider, W. H. Koppenol, Helv. Chim. Acta 2004, 87, 3021–3034. 140. M. R. Summers, A. Jacobs, D. Tudway, P. Perera, C. Ricketts, Br. J. Haematol. 1979, 42, 547–555. 141. M. J. Pippard, S. T. Callender, D. J. Weatherall, Clin. Sci. Mol. Med. 1978, 54, 99–106. 142. J. B. Porter, R. D. Abeysinghe, S. Singh, R. C. Hider, Blood, 1996, 88, 705–713. 143. C. Borgna-Pignatti, M. D. Cappellini, P. De Stefano, G. C. Del Vecchio, G. L. Forni, M. R. Gamberini, R. Ghilardi, A. Piga, M. A. Romeo, H. Zhao, A. Cnaan, Blood, 2006, 107,3733–3737. 144. M. Y. Moridani, G. S. Tilbrook, H. H. Khodr, R. C. Hider, J. Pharm. Pharmacol. 2002, 54, 349–364. 145. F. N. Hahn, T. J. McMurry, A. Hugi, K. N. Raymond, J. Am. Chem. Soc. 1990, 112, 1854–1860. 146. L. C. Wolfe, Sem. Hematol. 1990, 27, 117–120. 147. R. J. Bergeron, J. Wiegand, W. R. Weimar, J. R. T. Vinson, J. Bussenius, G. W. Yao, J. S. McManis, J. Med. Chem. 1999, 42, 95–108. 148. R. J. Bergeron, J. Wiegand, J. S. McManis, B. H. McCosar, W. R. Weimar, G. M. Brittenham, R. E. Smith, J. Med. Chem. 1999, 42, 2432–2440. 149. J. C. Barton, Drugs 2007, 10, 480–490. 150. R. J. Bergeron, J. Wiegand, N. Bharti, J. S. McManis, S. Singh, BioMetals 2011, 24, 239–258. 151. H. Y. Rienhott, V. Viprakasit, L. Tay, P. Harmatz, E. Vichinsky, D. Chirnomas, J. L. Kwiatkowski, A. Tapper, W. Kramer, J. B. Porter, E. Neufeld, J. Haematol. 2011, 96, 521–525. 8 Iron: Effect of Deficiency and Overload 291

152. u. Heinz, K. Hegetschweiler, P. Acklin, B. Faller, R. Lattmann, H. P. Schnebli, Angew. Chem. Int. Ed. 1999, 38, 2568–2570. 153. C. Hershko, A. M. Konijn, H. P. Nick, W. Breuer, Z. I. Cabantchik, G. Link, Blood, 2001, 97, 1115–1122. 154. M. D. Cappellini, J. Porter, A. El-Beshlawy, C. K. Li, J. F. Seymour, M. Elalfy, N. Gattermann, S. Giraudier, J. W. Lee, L. L. Chan, K. H. Lin, C. Rose, A. Taber, S. W. Thein, V. Viprakasit, D. Habr, G. Domokos, B. Roubert, A. Kattamis, Haematologica, 2010, 95, 557–566. 155. J. C. Wood, B. P. Kang, A. Thompson, P. Giardina, P. Harmatz, T. Glynos, C. Paley, T. D. Coates, Blood 2010, 116, 537–543. 156. R. Galanello, A. Piga, D. Alberti, M. C. Rouan, H. Bigler, R. Sechaud, J. Clin. Pharmacol. 2003, 43, 565–572. 157. C. Rafat, F. Fakhouri, J. A. Ribeil, R. Delarue, M. LeQuintrec, Amer. J. Kidney Dis. 2009, 54, 931–934. 158. R. C. Hider, G. Kontoghiorghes, J. Silver, M. A. Stockham, G. B. Patent 1984, 2118176. 159. G. J. Kontoghiorghes, M. A. Aldouri, A. V. Hoffbrand, J. Barr, B. Wonke, T. Kourouclaris, L. Sheppard, Br. Med. J. 1987, 295, 1509–1512. 160. A. Maggio, G. D. Amico, A. Morabito, M. Capra, C. Ciaccio, P. Cianciulli, F. DiGregorio, G. Garozzo, R. Malizia, C. Magnano, A. Mangiagli, G. Quarta, M. Rizzo, D. G. D`Ascola, A. Rizzo, M. Midiri, Blood Cells, Mol. Dis. 2002, 28, 196–208. 161. H. Glickstein, R. BenEl, G. Link, W. Breuer, A. M. Konijn, C. Hershko, H. Nick, Z. I. Cabantchik, Blood 2006, 108, 3195 –3203. 162. D. J. Pennell, V. Berdoukas, M. Karagiorga, V. Ladis, A. Piga, A. Aessopos, E. D. Gotsis, M. A. Tanner, G. C. Smith, M. A. Westwood, B. Wonke, Blood, 2006, 107, 3738 –3744. 163. C. Hershko, M. D. Cappellini, R. Galanello, A. Piga, G. Tognoni, G. Masera, Br. J. Haematol. 2004, 125, 545–551. 164. A. Pepe, A. Meloni, M. Capra, P. Cianciulli, L. Prossomariti, C. Malaventura, M. C. Putti, A. Lippi, M. A. Romeo, M. G. Bisconte, A. Filosa, V. Caruso, A. Quarta, L. Pitrolo, M. Missere, M. Midiri, G. Rossi, V. Positano, M. Lombardi, A. Maggio, Haematologica 2011, 96, 41–47. 165. A. Cohen, R. Galanello, A. Piga, V. Sanctis, F. Tricta, Blood 2003, 102,1583–1587. 166. A. Ceci, P. Baiardi, M. Felisi, M. D. Cappellini, V. Carnelli, V. De Sanctis, R. Galanello, A. Maggio, G. Mosera, A. Piga, F. Schettini, I. Stefano, F. Tricta, Br. J. Haematol. 2002, 118, 330–336. 167. R.C. Hider, G. S. Tilbrook, Z. D. Liu, International Patent WO98/54138 (1998). 168. N. Lowther, P. Fox, B. Faller, H. Nick, Y. Jin, T. Sergejew, Y. Hirschberg, R. Oberle, H. Donnelly, Pharm. Res. 1999, 16, 434 –440. 169. T. Sergejew, P. Forgiarini, H. P. Schnebli, Br. J. Haematol. 2000, 110, 985–92. 170. Z. D. Liu, H. H. Khodr, D. Y. Liu, S. L. Lu, R. C. Hider, J. Med.Chem. 1999, 42, 4814–4823. 171. S. Piyamongkol, Y. M. Ma, X. L. Kong, Z. D. Liu, M. D. Aytemir, D. van der Helm, R. C. Hider, Chem. Eur. J. 2010, 16, 6374–6381. 172. L. D. Devanur, H. Neubert, R. C. Hider, J. Pharm. Sci. 2008, 97, 1454–1467. 173. L. D. Devanur, R. W. Evans, P. J. Evans, R. C. Hider, Biochem. J. 2008, 409, 439–441. 174. P. Evans, R. Kayyali, R. C. Hider, J. Eccleston, J. B. Porter, Transl. Res. 2010, 156, 55–61. 175. P. J. Giardina, R. W. Grady, Semin. Hematol. 2001, 38, 360–366. 176. M. E. Lai, R. W. Grady, S. Vacquer, A. Pepe, M. P. Carta, P. Bina, F. Saw, P. Cianciulli, A. Maggio, R. Galanello, P. Farci, Blood Cells Mol. Dis. 2010, 45, 136–143. 177. v. Berdoukas, G. Chouliaras, P. Moraitis, K. Zannikos, E. Berdoussi, V. Ladis, J. Cardiovasc. Magn. Reson. 2009, 28, 11–20. 178. W. Hagar, E. Vichinsky, Brit. J. Haematol. 2008, 141, 346–356. 179. G. Lucania, A. Vitrano, A. Filosa, A. Maggio, Brit. J. Haematol. 2011, 154, 545–555. 180. E. B. Fung, P. Harmatz, M. Milet, S. K. Ballas, L. DeCastro, W. Hagar, L. Hilliard, W. Owen, N. Olivieri, K. Smith-Whitley, D. Darbari, W. Wang, E. Vichinsky, Amer. J. Hematol. 2007, 82, 255–265. 181. W. C. Wang, N. Ahmed, M. Hanna, J. Pediat. 1986, 108, 552–557. 292 Hider and Kong

182. E. Vichinsky, F. Bernaudin, G. L. Forni, R. Gardner, K. Hassell, M. M. Heeney, B. Inusa, A. Kutlar, P. Lane, L. Mathias, J. Porter, C. Tebbi, F. Wilson, L. Griffel, W. Deng, V. Giannone, T. Coates, Br. J. Haematol. 2011, 154, 387–397. 183. M. Jaeger, C. Anul, D. Sohngen, U. Germing, W. Scheier, Drugs Today 1992, 28, 143–147. 184. G. Metzgeroth, D. Dinter, B. Schultheis, A. Dorn-Beineke, K. Lutz, O. Leismann, R. Hehlmann, J. Hastka, Ann. Hematol. 2009, 88, 301–310. 185. N. Galtermann, C. Finelli, M. D. Porta, P. Fenaux, A. Ganser, A. Guerci-Bresler, M. Schmid, K. Taylor, D. Vassilieff, D. Habr, G. Domokos, B. Roubert, C. Rose, Leuk. Res. 2010, 34, 1143–1150. 186. O. Kakhlon, H. Manning, W. Breuer, N. Melamed-Book, C. Lu, G. Cortopassi, A. Munnich, Z. I. Cabantchik, Blood, 2008, 112, 5219–5227. 187. A. Gaeta, R. C. Hider, Brit. J. Pharmacol. 2005, 146, 1041–1059. 188. K. L. Duble, M. Gerlach, V. Schunemann, A. X. Trautwein, L. Zecca, M. Gallorini, M. B. Youdim, P. Riederer and D. Ben-Shachar, Biochem. Pharmacol. 2003, 66, 489–494. 189. M. T. Núñez, P. Urrutia, N. Mena, P. Aguirre, V. Tapia, J. Salazar, BioMetals 2012, 25, 761–776. 190. M. T. Núñez, V. Gallardo, P. Muñoz, V. Tapia, A. Esparza, J. Salazar, H. Speisky, Free Radic. Biol. Med. 2004, 37, 953–960. 191. R. Lill, U. Mühlenhoff, Annu. Rev. Biochem. 2008, 77, 669–700. 192. A. H. Schapira, Lancet 2006, 368, 70–82. 193. K. Blennow, M. J. de Leon, H. Zetterberg, Lancet 2006, 368, 387–403. 194. R. C. Hider, Y. M. Ma, F. Molina-Holgado, A. Gaeta, S. Roy, Biochem. Soc. Trans. 2008, 36, 1304–1308. 195. F. Bousejra-ElGarah, C. Bijani, Y. Coppel, P. Faller and C. Hureau, Inorg. Chem. 2011, 50, 9024–9030. 196. M. A. Smith, P. L. R. Harris, L. M. Sayre, G. Perry, Proc. Natl. Acad. Sci. USA 1997, 94, 9866–9868. 197. A. Kontush, Free Radic. Biol. Med. 2001, 31, 1120–1131. 198. J. A. Duce, A. Tsatsanis, M. A. Cater, S. A. James, E. Robb, K. Wikhe, S. L. Leong, K. Perez, T. Johanssen, M. A. Greenough, H. Cho, D. Galatis, R. D. Moir, C. L. Masters, C. McLean, R. E. Tanzi, R. Cappai, K. J. Barnham, G. D. Ciccotosto, J. T. Rogers, A. I. Bush, Cell 2010, 142, 857–867. 199. P. Lei, S. Ayton, D. I. Finkelstein, L. Spoerri, G. D. Ciccotosto, D. K. Wright, B. X. W. Wong, P. A. Adlard, R. A. Cherny, L. Q. Lam, B. R. Roberts, I. Volitakis, G. F. Egan, C. A. McLean, R. Cappai, J. A. Duce, A. I. Bush, Nature Medicine 2012, 18, 291–296. 200. J. F. Schenck, E. A. Zimmerman, Z. Li, S. Adak, A. Saha, R. Tandon, K. M. Fish, C. Belden, R. W. Gillen, A. Barba, D. L. Henderson, W. Neil, T. O’Keefe, Top. Magn. Reson. Imaging 2006, 17, 41–50. 201. H. Akatsu, A. Hori, T. Yamamoto, M. Yoshida, M. Mimuro, Y. Hashizume, I. Tooyama, E. M. Yezdimer, BioMetals 2012, 25, 337–350. 202. C. E. Clarke, Brit. Med. J. 2007, 335, 441–445. 203. y. Agid, Lancet 1991, 337, 1321–1324. 204. P. Jenner, Acta Neurol. Scond. 1991, 84 Suppl. 136, 6–15. 205. P. Jenner, Lancet, 1994, 344, 796–798. 206. D. T. Dexter, F. R. Wells, F. Agid, Y. Agid, A. J. Lees, P. Jenner, C. D. Marsden, Lancet 1987, 330, 1219–1220. 207. E. Sofic, P. Riederer, H. Heinsen, H. Beckmann, G. P. Reynolds, G. Hebenstreit, M. B. Youdim, J. Neural. Transm. 1988, 74, 199–205. 208. J. Hallervorden, H. Spatz, Z. Ges. Neurol. Psychiat. 1922, 79, 254–302. 209. S. J. Hayflick, J. M. Penzien, W. Michl, U. M. Sharif, N. P. Rosman, P. G. Wheeler, Pediatric Neurology 2001, 25, 166–169. 210. A. Gregory, S. J. Hayflick, Folia Neuropathologica 2005, 43, 286–296. 211. n. Gordon, Eur. J. Pediatr. Neurol. 2002, 6, 243–247. 8 Iron: Effect of Deficiency and Overload 293

212. T. L. Perry, M. G. Norman, V. W. Yong, S. Whiting, J. C. Crichton, S. Hansen, S. J. Kish, Ann. Neurol. 1985, 482–489. 213. R. Klein, Q. Wang, B. E. K. Klein, S. E. Moss, S. M. Meuer, Invest. Ophthalmol. Vis. Sci. 1995, 36, 182–191. 214. M. A. Zarbin, Arch. Ophthalmol. 2004, 122, 598–614. 215. P. Haly, A. H. Milam, J. L. Dunaief, Arch. Ophthalmol. 2003, 121, 1099–1105. 216. M. Hadziahmetovic, Y. Song, N. Wolkow, J. Iacovelli, L. Kautz, M. P. Roth, J. L. Dunaief, Amer. J. Pathol. 2011, 179, 335–348. 217. M. Hadziahmetovic, Y. Song, P. Ponnuru, J. Iacovelli, A. Hunter, N. Haddad, J. Beard, J. R. Connor, S. Vaulont, J. L. Dunaief, Invest. Ophthalmol. Vis. Sci. 2011, 52, 109–118. 218. X. He, P. Hahn, J. Iacovelli, R. Wong, C. King, R. Bhisitkul, M. Massaro-Giordano, J. L. Dunaief, Prog. Retin. Eye Res. 2007, 26, 649–673. 219. T. Zhou, Y. Ma, X. Kong, R. C. Hider, Dalton. Trans. 2012, 41, 6371–6389. 220. D. T. Dexter, S. A. Statton, C. Whitmore, W. Freinbichler, P. Weinberger, K. F. Tipton, L. Della Corte, R. J. Ward, R. R. Crichton, J. Neural. Transm. 2011, 118, 223–231. 221. M. Hadziahmetovic, Y. Song, N. Wolkow, J. Iacovelli, S. Grieco, J. Lee, A. Lyubarsky, D. Pratico, J. Connelly, M. Spino, Z. L. Harris, J. L. Dunaief, Invest. Ophthalmol. Vis. Sci. 2011, 52, 959–968. 222. G. L. Forni, M. Balocco, L. Cremonesi, G. Abbruzzese, R. C. Parodi, R. Marchese, Movement Disorders 2008, 23, 904–907. 223. G. Abbruzzese, G. Cossu, M. Balocco, R. Marchese, D. Murgia, M. Melis, R. Galanello, S. Barella, G. Matta, U. Ruffinengo, U. Bonuccelli, G. L. Forni, Haematologica 2011, 96, 1708–1711. 224. A. I. Bush, Neurobiology of Aging 2002, 23, 1031–1038. 225. H. Zheng, L. M. Weiner, O. Bar-Am, S. Epsztejn, Z. I. Cabantchik, A. Warshawsky, M. B. H. Youdim, M. Fridkin, Bioorg. Med. Chem. 2005, 13, 773–783. 226. H. Zheng, D. Blat, M. Fridkin, J. Neural. Trans. 2006, 71, 163–172. 227. F. Molina-Holgado, A. Gaeta, P. T. Francis, R. J. Williams, R. C. Hider, J. Neurochem. 2008, 105, 2466–2476. 228. Y. Avramovich-Tirosh, T. Amit, O. Bar-Am, H. Zheng, M. Fridkin, M. B. Youdim, J. Neurochem. 2007, 100, 490–502. 229. R. A. Cherny, C. S. Atwood, M. E. Xilinas, D. N. Gray, W. D. Jones, C. A. McLean, K. J. Barnham, I. Volitakis, F. W. Fraser, Y. Kim, X. Huang, L. E. Goldstein, R. D. Moir, J. T. Lim, K. Beyreuther, H. Zheng, R. E. Tanzi, C. L. Masters, A. I. Bush, Neuron 2001, 30, 665–676. 230. C. W. Ritchie, A. I. Bush, A. Mackinnon, S. Macfarlane, M. Mastwyk, L. MacGregor, L. Kiers, R. Cherny, Q. X. Li, A. Tammer, D. Carrington, C. Mavros, I. Volitakis, M. Xilinas, D. Ames, S. Davis, K. Beyreuther, R. E. Tanzi, C. L. Masters, Arch. Neurol. 2005, 60, 1685–1691 231. D. Kaur, F. Yantiri, S. Rajagopalan, J. Kumar, J.Q. Mo, R. Boonplueang, V. Viswanath, R. Jacobs, L. Yang, M. F. Beal, D. DiMonte, I. Volitaskis, L. Ellerby, R. A. Cherny, A. I. Bush, J. K. Andersen, Neuron 2003, 37, 899–909. 232. University Hospital Lille Trial (FAIR-PARK1), http://clinicaltrials.gov/ct2/show/ NCT00943748?term=FAIR-PARK-I&rank=1 233. Imperial College London Trial (DeferipronPD), http://clinicaltrials.gov/ct2/show/NCT01539 837?term=deferiprone&rank=4 234. D. Song, Y. Song, M. Hadziahmetovic, Y. Zhong, J. L. Dunaief, Free Rad. Biol. Med. 2012, 53, 64–71. 235. T.P. Kruck, J.G. Cui, M.E. Percy, W.J. Lukiw, Cell Mol. Neurobiol. 2004, 24, 443–459. 236. H. Schugar, D. E. Green, M. L. Bowen, L. E. Scott, T. Storr, K. Bohmerle, F. Thomas, D. D. Allen, P. R. Lockman, M. Merkel, K. H. Thompson, C. Orvig, Angew. Chem. Int. Ed. 2007, 46, 1716–1718. 237. R. C. Hider, S. Roy, Y. M. Ma, X. L. Kong, J. Preston, Metallomics 2011, 3, 239–249. 294 Hider and Kong

238. G. Liu, P. Men, W. Kudo, G. Perry, M. A. Smith, Neurosci. Lett. 2009, 455, 187–190. 239. D. Bebbington, C. E. Dawson, S. Gaur, J. Spencer, Bioorg. Med. Chem. Lett. 2002, 12, 3297–3300. 240. H. Zheng, M. B. H. Youdim, M. Fridkin, J. Med. Chem. 2009, 52, 4095–4098. 241. L. R. Perez, K. J. Franz, Dalton Trans. 2010, 39, 2177–2187. 242. S. V. Torti, F. M. Torti, Cancer Res. 2011, 71, 1511–1514. 243. R. G. Stevens, B. I. Graubard, M. S. Micozzi, K. Neriishi, B. S. Blumberg, Int. J. Cancer 1994, 56, 364–369. 244. A. G. Smith, J. E. Francis, P. Carthew, Carcinogenesis 1990, 11, 437–444. 245. L. D. Miller, L. G. Coffman, J. W. Chou, M. A. Black, J. Bergh, R. D’Agostino, Jr., S. V. Torti, F. M. Torti, Cancer Res. 2011, 71, 6728–6737. 246. S. Nekhai, V. R. Gardeuk, in Iron Physiology and Pathophysiology in Humans, Eds G. J. Anderson, G. D. McLaren, Humana Press, New York, 2012, 477–495. 247. D. B. Lovejoy, D. R. Richardson, Curr. Med. Chem. 2003, 10, 1035–1049. 248. H.M. Hanauske-Abel, M.-H. Park, A.-R. Hanauske, A.M. Popowicz, M. Lalande, J.E. Folk, Biochim. Biophys. Acta, 1994, 1221, 115–124. 249. C. Hershko, Mol. Asp. Med. 1992, 13, 113–165. 250. R. J. Bergeron, Trends Biol. Sci. 1986, 11, 133–136. 251. J. Gao, D. R. Richardson, Blood 2001, 98, 842–850. 252. J. Yuan, D. B. Lovejoy, D. R. Richardson, Blood 2004, 104, 1450–1458. 253. D. Fu, D. R. Richardson, Blood 2007, 110, 752–761. 254. E. D. Weinberg, Biochim. Biophys. Acta 2009, 1790, 600–605. 255. N. H. Carbonetti, S. Boonchai, S. H. Parry, V. Vaisanenrhen, T. K. Korhonen, P. H. William, Infection and Immunity 1986, 51, 966–968. 256. S. Ford, R. A. Cooper, R. W. Evans, R. C. Hider, P. H. Williams, Eur. J. Biochem. 1988, 178, 477–481. 257. I. A. García-Montoya, T. S. Cendón, S. Arévalo-Gallegos, Q. Rascón-Cruz, Biochim. Biophys. Acta 2012, 1820, 226–236. 258. K. N. Raymond, E. A. Dertz, S. S. Kim, Proc. Natl. Acad. Sci. USA 2003, 100, 3584–3588. 259. M. A. Holmes, W. Paulsene, X. Jide, C. Ratledge, R. K. Strong, Structure 2005, 13, 29–41. 260. J. R. Boelaert, S. J. Vandecasteele, R. Appelberg, V. R. Gordeuk, J. Infect. Dis. 2007, 195, 1745–1753. 261. R. C. Hider, Z. Liu, J. Pharm. Pharmacol. 1997, 49, 59–64. 262. G. F. Mabeza, Pharmacol. Ther. 1999, 81, 53–75. Chapter 9 Cobalt: Its Role in Health and Disease

Kazuhiro Yamada

Contents ABSTRACT ...... 296 1 INTRODUCTION ...... 296

1.1 Cobalt and Vitamin B12 Deﬁ ciency ...... 296 2 COBALAMIN, VITAMIN B12 ...... 297 2.1 Biochemistry of Cobalamin ...... 297 2.2 Cobalamin Binding Proteins and Transporting System ...... 298 2.2.1 Overview of the Cobalamin Absorption and Delivering System ...... 298 2.2.2 Absorption of Cobalamin ...... 298 2.2.3 Intracellular Processing of Cobalamin ...... 300 2.3 Cobalamin-Dependent Enzymes in Mammals ...... 303 2.3.1 An Overview of the Cobalamin-Dependent Enzymes ...... 303 2.3.2 Methylmalonyl-Coenzyme A Mutase and Related Metabolism in Mammals ...... 303 2.3.3 Methionine Synthase and Related Metabolism in Mammals ...... 306

3 VITAMIN B12 DEFICIENCY AND DISEASE ...... 310 3.1 Vitamin B12 Deﬁ ciency...... 310 3.2 Methylmalonic Aciduria ...... 311 3.3 Hyperhomocysteinemia ...... 311 3.4 Megaloblastic Anemia ...... 312 3.5 Cobalamin Neuropathy ...... 312

3.6 Other Diseases Related to Vitamin B12 Deﬁ ciency ...... 312 3.7 Animal Models ...... 313 4 NON-CORRINOID COBALT ...... 314 4.1 Non-corrinoid Cobalt-Containing Proteins ...... 314 4.2 Overload of Cobalt ...... 315 5 IMPLICATIONS AND FUTURE DEVELOPMENT ...... 315 ABBREVIATIONS ...... 316 ACKNOWLEDGMENT ...... 317 REFERENCES ...... 317

K. Yamada (*) Department of Biochemistry , Uniformed Services University of the Health Sciences , 4301 Jones Bridge Road , Bethesda , MD 20814 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 295 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_9, © Springer Science+Business Media Dordrecht 2013 296 Yamada

Abstract The primarily function of cobalt in humans is based on its role in cobalamin

(Cbl, vitamin B12 ). Therefore, this chapter will focus on the physiological roles of Cbl and the importance of cobalt in human health. Cbl acts as the cofactor for two enzymes, i.e., methylmalonyl-CoA mutase and methionine synthase, in humans. Both enzymes are important for health. In addition, unlike other water-soluble vitamins, there is a unique absorption, delivery, and activation system for Cbl in mammals. Therefore, this chapter will also review the literature on the Cbl transporting system, which is crucial for Cbl function.

Keywords cobalamin • methionine synthase • methylmalonyl-CoA mutase

• vitamin B12

Please cite as: Met. Ions Life Sci. 13 (2013) 295–320

1 Introduction

1.1 Cobalt and Vitamin B12 Deﬁ ciency

Cobalt (Co) is an essential (mineral) micronutrient for humans. Historically, however, obvious nutritional Co defi ciency has not occurred in humans. This could imply that cobalt is not an essential factor for human health, per se. In contrast, Co defi ciency has been identifi ed in some ruminant mammals, including sheep, goat, and cattle. Ruminants raised in areas where cobalt is scarce show reduced food intake and growth retardation. This problem has been historically recognized by dairy farmers. Later, this phenomenon was realized to be vitamin B12 (B12 ) defi ciency rather than simple Co defi ciency, and that it was related to the synthesis of cobalamin (Cbl) by microorganisms in the stomach. Moreover, scientists working in veterinary nutrition had identifi ed an “animal protein factor”, that is, a nutritional growth factor that is exclusively present in animal foods, B12 being the strongest animal protein factor [ 1 ]. These observations indicate that the primary function of cobalt in mammals is in the form of Cbl. B12 is an essential micronutrient for humans, and Cbl is its functional unit. Therefore, this chapter focuses on the function of Cbl as the primary role for cobalt in humans. The Recommended Dietary Allowance of Cbl for the adult human is 2.4 μg per day, which is the lowest of all nutrients. Cbl and the related substances, corrinoids, can only be produced by certain microorganisms. Cbl found in food is originally from these bacteria, in which the complex molecule is synthesized using at least 25 genes in the Cob operon [ 2]. The B 12 content in food is very low. Its greatest abundance is in meat, fi sh, and milk products; it is generally absent from fruits and vegetables, but nori, an edible green and purple seaweed, is a non-dairy product that contains a signifi cant amount of B12 [ 3 ]. It is therefore an important potential source of B12 for strict vegetarians. However, the bioavailability of B12 in seaweed is still controversial because seaweed may contain pseudovitamin B12 , which is an inactive B 12 analog [ 3]. Humans require an authentic form of Cbl, and a human possesses an ingenious system for the precise selection and absorption of Cbl. 9 Cobalt: Its Role in Health and Disease 297

Cbl acts as the cofactor for two enzymes present in humans, i.e., methionine synthase and methylmalonyl-CoA mutase. One of the best-characterized human diseases caused by B12 deﬁ ciency is megaloblastic anemia, also known as pernicious anemia. It is thought that the inactivation of methionine synthase is responsible for this disease. Dysfunction of Cbl-dependent enzymes can be caused by inadequate intake of B12 . However, it can even occur in the presence of adequate amounts of B12 , due to inheritance of gene mutations of Cbl-dependent enzymes or failure of the Cbl delivery system. Thus, functioning of these proteins is also essential for proper Cbl function in humans.

2 Cobalamin, Vitamin B12

2.1 Biochemistry of Cobalamin

Cbl has been called nature’s most beautiful cofactor [ 4 ] and was identiﬁ ed as the anti-pernicious anemia factor from liver in 1948 [5 , 6]. Since then, many studies on the chemical properties of Cbl have been reported. The structure was solved by Hodgkin et al. using X-ray structural analysis in 1956 (Figure 1 ) [7 ]. As the structure shows, Cbl is a large and complex molecule. The characteristic tetrapyrrole ring is called the corrin ring, and compounds containing the corrin ring are called corrinoids. Unlike iron in the porphyrin ring of heme, whose tetrapyrrole ring is similar to the corrin ring, cobalt in Cbl is not interchangeable with other metals. Cobalt cannot be released from the ring unless the ring is broken. To investigate the chemical properties of cobalt bound to Cbl, cobaloxime can be used as a model compound, although it does not itself possess B12 activity. Cbl contains a nucleotide loop connected to the D ring of the corrin ring, and the dimethylbenzimidazole (DBI) base in the tail of the nucleotide loop is coordinated to the cobalt atom (Figure 1 ). The DBI coordinating side of the corrin ring is referred to as the lower axial position. Cobinamide and cobamide are corrinoids that lack the

R CONH2

B H2NOC CONH2 A N H2NOC N Figure 1 Structure of Co N N C D cobalamin. Vitamin B 12 H2NOC (cyanocobalamin, α α CONH2 Co - [ -(5,6-demethylbenzi- O midazolyl)]-Coβ-cyano- N cobamide) has the CN NH N group in the upper ligand. HO Methylcobalamin (R = CH -), cyanocobalamin, R= CN- 3 O O and adenosylcobalamin methylcobalamin, R= CH3- O ′ O P adenosylcobalamin, R= 5’-deoxyadenosyl- (R = 5 -deoxyadenosyl-) - are the cofactor forms. O HO 298 Yamada nucleotide loop and the DBI moiety (compared to Cbl), respectively. In the cob(III)- alamin state (which indicates a +3 oxidation state of cobalt), Co in Cbl is six-coordinate. It has an upper ligand, e.g., methyl, 5′-deoxyadenosyl, water, or cyano groups, for methylcobalamin (CH3 -Cbl), adenosylcobalamin (AdoCbl), aquacobalamin, or cyanocobalamin (CN-Cbl), respectively. CH3 -Cbl and AdoCbl are the cofactor forms for methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), respectively. CN-Cbl is a largely artifi cial form of Cbl, produced and purifi ed industrially, that can be converted to active cofactor forms in the body. In the strictest sense, B12 is CN-Cbl, as this is the commercially most available form. Cobalt in the corrin ring can be reduced to the +2 or +1 states, called cob(II)alamin and cob(I)alamin, respectively. Cob(II)alamin contains fi ve-coordinate cobalt without any upper ligand. Cob(I)- alamin is four-coordinate, so neither the DBI moiety nor an upper ligand are coordinated to Co. Cob(I)alamin is known as a hypernucleophilic species [ 8]. Other variants of cobamide can be found in nature. For example, the DBI moiety can be replaced by adeninyl, 2-methyladeninyl, 5-hydroxybenzimidazolyl, or methoxybenzimidazolyl groups in pseudoB12 , in factor A, factor III, or 5′-methoxybenzimidazole cobamide, respectively. For mammalian nutrition, Cbl is the most active form of B 12 ; other cobamides show very weak or no B12 function.

2.2 Cobalamin Binding Proteins and Transporting System

2.2.1 Overview of the Cobalamin Absorption and Delivering System

The famous discovery of B12 as the anti-pernicious anemia factor was conducted by Minot and Murphy [9 ]; they treated pernicious anemia patients with large amounts of animal liver. Liver contains quantities of B12 that are approximately 10 times greater than in other meats. The B12 content in calf liver, which is one of the richest

B12 -containing foods, is ~50 μg/100 g. In general, however, it is not necessary for normal adults to eat large amounts of liver. To take advantage of the precious nutrient Cbl, a specifi c transportation system is available immediately after intake of food. In all steps for Cbl transportation, there are specifi c proteins, whose function is essential for proper delivery and processing of Cbl. Hence, dysfunction of any of these proteins caused by gene mutations may result in functional B12 defi ciency. While the discovery of the Cbl absorption and transportation system is an old story, a new era has emerged as genes coding for the proteins responsible for intracellular processing of Cbl have recently been identifi ed.

2.2.2 Absorption of Cobalamin

The pioneer work on absorption of Cbl was done by Castle. In 1936, Castle and Ham reported that administration of a digested mixture of beef meat and gastric juice to pernicious anemia patients provided an effective cure [10 ]. The substances in 9 Cobalt: Its Role in Health and Disease 299

Figure 2 Crystal structures of Cbl binding proteins and Cbl binding forms. (a ) Intrinsic factor- Cbl complex (PDB 2PMV). ( b) Transcobalamin-Cbl complex (PDB 2BB5). (c ) CblC protein, the MMACHC gene product (PDB 3SC0). Proteins are shown in the ribbon models and Cbl is illustrated in the stick mode. Figures are generated by PyMol [127 ] .

beef meat and gastric juice were named the external and internal factors, respectively. Later, both factors were identifi ed: the external factor was shown to be Cbl and the intrinsic factor to be a glycoprotein secreted from the stomach. The glycoprotein was later called intrinsic factor (IF). There is another Cbl-binding protein, haptocorrin (also known as R-binder), present in the digestive tracts of humans. However, IF shows the highest affi nity for Cbl, and a lower affi nity for other corrinoids. Binding of IF is quite selective for the lower ligand of corrinoids, which contain the imidazo- lyl group as lower ligand [11 ]. The crystal structure of the IF-Cbl complex is shown in Figure 2a [12 ].

This complex may provide the initial selection of Cbl as B 12 because there are many other B12 analogues, such as the aforementioned pseudoB12 contained in seaweed. After release of Cbl from food protein or from the Cbl-protecting protein haptocorrin, Cbl is captured by the IF. In the ileum, the Cbl-IF complex is recognized by cubam, the receptor for the IF-Cbl complex consisting of a heterodimer of amnionless and cubilin [13 , 14 ]. Mutation of genes for cubam may cause the Gräsbeck-Imerslund syndrome [15 , 16], which is characterized by malabsorption of Cbl with normal IF production and function. If a patient has an autoantibody against IF due to autoimmune destruction of gastric parietal cells by an atrophic gastritis, 300 Yamada

Cbl binding to IF or interaction between the IF-Cbl complex and the IF receptor can be inhibited, potentially resulting in malabsorption of Cbl. Once formed, the complex of the IF-Cbl-IF receptor is absorbed by endocytosis. IF is degraded in the lysosome and Cbl passes through the cytosol of the ileal epithelial cell to the bloodstream. Cbl is released from ileal cells by MRP1, a multidrug resistance protein [17 ], although an alternative pathway has been proposed [18 ]. In blood, Cbl binds with either transcobalamin (TC) or to haptocorrin. The crystal structure of the complex of TC-Cbl is shown in Figure 2b [19 ]. The Cbl-TC complex is then captured by the TC receptor on target cells.

2.2.3 Intracellular Processing of Cobalamin

Cbl must undergo cellular transport and activation by proteins before it can bind to Cbl-dependent enzymes. Patients with hereditary B12 defi ciency due to defects in Cbl utilization have been identifi ed. To date, nine complementation groups for impaired cellular Cbl metabolism, CblA-CblG, CblJ, and mut , have been identifi ed. (The CblH complementation group could be a subclass of the CblD complementation group [20 ]). All genes corresponding to each group have been identifi ed in the last 10 years. With the exception of the MS and MCM proteins, complementary genes were identifi ed prior to isolation of the protein because the complementary proteins could not be detected in cell extracts, even when 57 Co-labeled Cbl was used [21 ,22 ]. The functions of most of these gene products are still under active investigation. Cellular Cbl processing is summarized in Figure 3 . The Cbl-TC complex is recognized by the TC receptor on the cell surface and absorbed into the cell by endocytosis. Cbl is released from TC in the lysosome, and then it is transferred to cytosol. From lysosome to cytosol, there are two genes, LMBD1 and ABCD4, which are responsible for the CblF and CblJ complementation groups, respectively. These genes encode membrane proteins. The CblF protein (the LMBD1 gene product) is identifi ed as a lysosomal membrane exporter of Cbl [23 ]. The protein shows signifi - cant homology to the lipocalin-1 interacting membrane receptor: a cell receptor for internalization of lipocalins. Lipocalins are small proteins carrying hydrophobic molecules in body fl uids. The CblJ protein (the ABCD4 gene product) is an ABC transporter, and it is suggested that ATPase activity of the protein is required for Cbl processing [24 ]. The CblC protein, the gene product from MMACHC (m ethyl m alonic a ciduria, cbl C type, and h omo c ystinuria) [25 ], may have a role in accepting Cbl from these membrane proteins [26 ], although there is no evidence for the direct interaction of two membrane proteins and the CblC protein. The crystal structure of the CblC protein has been solved [27 ] (Figure 2c ). Unlike IF and TC, the lower ligand of Cbl is dissociated upon binding to the CblC protein. On the one hand the conformation of Cbl in solution and upon binding to IF and TC is called the DBI-on form, and on the other that of Cbl in the CblC protein is referred to as the DBI-off form. The fi rst observation of the DBI-off form of Cbl was in the Cbl-binding domain of MS [28 ]. While both MCM and MS have the signature amino acid sequence motif, DxHxxG 9 Cobalt: Its Role in Health and Disease 301

CN TC•Cbl complex Co TC receptor DBI

cytosol me oso lys TC endocytosis degradation CblF CN 1 Co LMBD

DBI CblJ ABCD4

mitochondrion CN CblC MMACHC

Co CblG

DBI mtr MS DBI

Co Ado CblB Ado Co MMAB Co

DBI CblD DBI Ado MMADHC His

DBI Co

CblA inactivation CblE MMAA Co mtrr MSR DBI

Ado His

DBI Co Co

DBI Ado CH His 3 mut Co DBI MCM His

Figure 3 Cellular cobalamin metabolism. Nine complementation groups, CblA~G, CblJ , and mut , are shown in bold letters and the responsible genes in light letters (see text for details). for the DBI-off form of Cbl binding, the CblC protein does not contain the motif. CblC exhibits the catalytic function for the reductive de-cyanation of CN-Cbl in the presence of methionine synthase reductase (MSR), a dual fl avoprotein similar to the P450 reductase family (see Section 2.3.3), and NADPH [26 ]. MSR cannot be the 302 Yamada physiological partner protein for the reduction, so the reducing protein has yet to be identifi ed. Mutations in LMBD1, ABCD4, and MMACHC genes cause both methylmalonic aciduria and homocysteienemia because their impairment causes dysfunction of both MCM and MS. In contrast, the phenotype of patients in the CblD complementation group varies; it can cause methylmalonic aciduria (MMA) or homocysteienemia, or both. Hence, the function of the CblD protein must be down-stream of that of the CblC protein, and the CblD protein controls the fate of Cbl; to be transported into the mitochondrion or to remain in the cytosol. The gene, MMADHC ( m ethyl m alonic a ciduria, cbl D type, and h omo c ystinuria), corresponding to the CblD group, was cloned in 2008 [29 ]. The biochemical structure and function of CblD have not yet been reported. Because a reductive partner is required for the reduction of CN-Cbl bound to the CblC protein, the CblD protein might have reductase activity in addition to controlling the branching point of the Cbl delivery pathway (Figure 3 ). In mitochondria, Cbl has to be processed to become the active cofactor form, AdoCbl. MCM is the product of the mut gene, and the mut 0 and mut – groups are subtypes of patients that represent complete and partial defi ciency of MCM enzyme activities, respectively. Since MCM strictly requires AdoCbl for its activity, it was thought that CblA and CblB proteins should have both adenosyltransferase activity and Cbl reductase activity. The CblB protein, which is the product of the MMAB gene, shares homology with bacterial ATP-cob(I)alamin adenosyltransferase [30 ]. Crystal structures of human ATP-cob(I)alamin adenosyltransferase [31 ] and the bacterial homologue with Cbl [32 ] have been solved. The four-coordinate cob(II)alamin already mentioned facilitates the reduction of cob(II)alamin in catalysis because the enzyme needs to generate cob(I)alamin for the reaction. The purifi ed CblB protein can produce AdoCbl from cob(II)- alamin and ATP in the presence of MSR and NADPH [ 33 ]. MSR cannot be the physiological reductase because the CblE complementation group is independent from MCM function. The physiological reductase has yet to be identifi ed. The CblA protein, which is the product of the MMAA gene [34 ], was initially postulated to be the Cbl reductase. However, the enzyme property has recently been proposed as a molecular chaperone. The protein is similar to members of the GTPase family of metal insertion proteins. The protein function was proposed using bacterial MCM and MeaB, an orthologue of MMAA in bacteria, as a model (see the next section). In cytosol, Cbl fi nally binds to MS. MS is encoded in the mtr gene, which corresponds to the CblG complementation group [ 35 – 37]. For MS, CH 3-Cbl is the active cofactor, but the non-methyl form of Cbl, i.e., cob(II)alamin, can also bind and act as cofactor because MS can regenerate the active cofactor. For this reaction, an electron donor is required. The reducing equivalent is supplied from NADPH via MSR. The gene encoding MSR is identifi ed as mtrr , which corresponds to the CblE complementation group [38 ] (see the next section). 9 Cobalt: Its Role in Health and Disease 303

2.3 Cobalamin-Dependent Enzymes in Mammals

2.3.1 An Overview of the Cobalamin-Dependent Enzymes

There are two enzymes that use Cbl as a cofactor in humans, MCM and MS. AdoCbl is utilized by MCM and CH3 -Cbl is the active cofactor for MS. Binding of Cbl to both enzymes is as the DBI-off form [ 28 , 39 ], which is shown in Figure 4a and 4b . The biochemical properties of these Cbl-dependent enzymes have been reviewed recently in this series [40 ]. This section will therefore focus on the physiological aspects of these enzymes.

2.3.2 Methylmalonyl-Coenzyme A Mutase and Related Metabolism in Mammals

MCM catalyzes the rearrangement of the carbon backbone in the conversion of methylmalonyl-CoA to succinyl-CoA (Figure 5 ). MCM requires AdoCbl to catalyze this reaction. Although there are more AdoCbl-dependent enzymes known in the microbial world, MCM is the only AdoCbl-dependent enzyme in mammals. All AdoCbl-dependent enzymes catalyze difﬁ cult reactions that require the production of reactive radical species. The production of reactive radical species is accomplished using AdoCbl as the radical generator [41 ] (Figure 5 ). AdoCbl undergoes homolytic cleavage of the Co-C bond and yields a 5′-deoxyadenosyl radical and cob(II)alamin. The radical abstracts a hydrogen atom from the substrate to form deoxyadenosine. The radical on the substrate rearranges the carbon backbone, and then the radical on the product is returned to the cofactor. Because of the high reactivity of the radical intermediate, AdoCbl-dependent enzymes are sensitive to inactivation due to unexpected side reactions. Auxiliary reactivating proteins have been studied using AdoCbl- dependent diol dehydratase, a bacterial enzyme. The reactivation factors for diol dehydratase from Klebsiella oxytoca have been identiﬁ ed and characterized [42 – 46 ]. The overall reaction of the reactivation consists of the replacement of the inactivated cofactor binding to the inactive holoenzyme by the authentic cofactor, AdoCbl, in the presence of ATP. The molecular chaperone function of the reactivating factor can be found in other bacterial AdoCbl-dependent enzymes, such as glycerol dehydratase and ethanolamine ammonia-lyase, which bind AdoCbl in the DBI-on form. Banerjee and colleagues have reported that MeaB, which is a G-protein and shares homology to the human MMAA protein, acts as the molecular chaperone for MCM [47 – 49]. Three auxiliary functions of MeaB for MCM have been proposed: (i) “Editing”, MCM-MeaB-GTP prevents binding of cob(II)alamin to apoMCM using the binding energy of GTP by MeaB. (ii) “Gating”, MCM-MeaB-GTP

Figure 4 Protein structures of methylmalonyl-CoA mutase and methionine synthase. (a ) The cobalamin binding domain of human MCM (monomer) is shown in red. The substrate binding domain is illustrated in cyan with transparency. Cbl is shown in stick mode (PDB 2XIQ). ( b) The cobalamin binding domain of E. coli MetH (methionine synthase). The cap structure is shown in transparency (PDB 1BMT). ( c ) The overall structure of the MCM dimer. The color scheme is the same as in panel ( a). One of two subunits is shown transparent. ( d ) Structures of bacterial methionine synthase. The substrate homocysteine and folate binding domains of T. maritima MetH are shown in yellow and green, respectively (PDB 1Q8J). The Cbl binding domain (red) and the AdoMet binding domain (blue) of E. coli MetH are drawn in the reactivation conformation (PDB 1K7Y). -- Proteins and Cbl are illustrated in ribbon models and in the stick mode, respectively. Figures are generated by PyMol [127 ] . 9 Cobalt: Its Role in Health and Disease 305

S CoA O H -OOC H 2 H H methylmalonyl-CoA

SCoA O H -OOC 1 Ado H Ado II H II AdoCbl Cbl Cbl 3 MCM MCM MCM O SCoA

-OOC H H H

O H S CoA 4 -OOC H H H succinyl-CoA

Figure 5 Reaction mechanism of methylmalonyl-CoA mutase. (1 ) Unfavorable equilibrium of the homolytic cleavage of AdoCbl. (2 ) A hydrogen atom abstraction forms the substrate, methylmalonyl- CoA. (3 ) Rearrangement of the carbon skeleton and the radical migration. (4 ) A hydrogen atom abstraction by the product radical from 5′-deoxyadenosine. Ado• = 5′-deoxyadenosyl radical, AdoCbl = adenosylcobalamin, CblII = cob(II)alamin, MCM = methylmalonyl-CoA mutase.

introduces AdoCbl to apoMCM (cofactor loading) with GTP hydrolysis. (iii) “Rescue”, MCM-MeaB-GTP displaces the inactive cofactor, Cbl without the 5′-deoxyadenosyl group. Crystal structures of the MeaB protein from Methylobacterium extorquens AM1 [50 ] and the human MMAA protein [51 ] have been reported. The crystal structure of MCM has been solved using Propionibacterium shermanii MCM [39 ], and the structure of the human enzyme was recently solved (Figure 4c ) [51 ]. The overall structure of the catalytic subunits of both MCM is similar. However, there are important structural differences between the bacterial and human proteins; for example, the bacterial MCM forms a heterodimer, while the mammalian enzyme is a homodimer. Biochemical study of the structure of the human MCM and MMAA proteins reveals that the basic role of the molecular chaperone in bacterial systems is almost adaptable to the human system, although the detailed mechanism could be different [51 ]. Under physiological condition, the mammalian MCM protein exists mostly as apoprotein, which lacks the AdoCbl cofactor. Even when sufﬁ cient amounts of Cbl (25 μg CN-Cbl/kg diet) were added to the diet of rats, the ratio of holo- to total MCM is less than 5% [ 52]. The low ratio of holoMCM has been observed in sheep

[ 53] and fruit bat’s tissues [54 ]. During B12 defi ciency, MCM activity is lower, due to the lack of cofactor. Interestingly, the amount of MCM protein in the liver of the B12 -defi cient rats is increased [ 52]. While the physiological signifi cance of apoMCM is unknown, it stands in stark contrast to that of the apoMS protein, which is quite unstable. 306 Yamada

Valine, isoleucine, odd-chain fatty acids

- HCO3 O O O O O O - 123- O S CoA -O S CoA O SCoA SCoA O CH3 CH3 ATP ADP propionyl-CoA (S)-methyl- (R )-methyl- succinyl-CoA malonyl-CoA malonyl-CoA

4 HS CoA TCA cycle O O HO OH CH3 methylmalonic acid

Figure 6 Propionyl-CoA metabolism: (1 ) Biotin-dependent propionyl-CoA carboxylase. (2 ) Methylmalonyl-CoA racemase. ( 3) Methylmalonyl-CoA mutase. (4 ) ( S )-Methylmalonyl-CoA hydrolase.

MCM is involved in propionyl-CoA catabolism to succinyl-CoA, which includes branched-chain amino acid and odd-chain fatty acid metabolisms (Figure 6 ). − Propionyl-CoA is combined with HCO3 in the presence of ATP, forming (S )-methylmalonyl-CoA by the reaction catalyzed by propionyl-CoA carboxylase, a biotin-dependent enzyme. The product, (S )-methylmalonyl-CoA, is converted to (R )-methylmalonyl-CoA by methylmalonyl-CoA racemase. (R )-methylmalonyl- CoA is the only substrate capable of binding in the substrate-binding pocket to MCM. Dysfunction of MCM elevates the level of methylmalonic acid in blood and urine, which are known as methylmalonic anemia and MMA, respectively. Methylmalonic acidemia is organic acidemia, which is often diagnosed in the early neonatal period, and this symptom is one of the hallmark characteristics of B12 deﬁ ciency.

2.3.3 Methionine Synthase and Related Metabolism in Mammals

The reaction catalyzed by MS is shown in Figure 7 . Overall, the reaction consists of 5 the transfer of the N -methyl group of methyltetrahydrofolate (CH3 -H4 folate) to the thiol group of homocysteine. CH 3-Cbl acts as the intermediate for this reaction, which is divided into two-steps: (i) the methyl group of CH3 -Cbl is donated to homocysteine, forming methionine and cob(I)alamin; (ii) the hypernucleophilic cob(I)alamin accepts the methyl group from CH3 -H4 folate to regenerate CH3 -Cbl. This methyl transfer reaction proceeds by the ping-pong mechanism [ 55 ]. 9 Cobalt: Its Role in Health and Disease 307

H R O H N - N O O HN - O O COO- NH NH NN 2 H + O H S NH R= 3 H4 folate Homocysteine

CH H R O 3 N 1 2 CH -Cbl · MS N 3 COO- HN + CH3 S NH3 NH N N 2 H methionine

CH3-H4folate CblI · MS AdoHcy

e- e- + AdoMet CblII · MS

Figure 7 Reaction catalyzed by methionine synthase. The solid lines (reactions 1 and 2) show the catalytic cycle of the enzyme. The broken line indicates inactivation of the enzyme. The reductive methylation is shown with the dotted line. Three different methyltransfer reactions are catalyzed by methionine synthase: (1 ) Transmethylation from CH3 -Cbl to homocysteine. (2 ) Transmethylation from CH3 -H 4 folate to cob(I)alamin. (3 ) Reductive methylation of cob(II)alamin to regenerate the CH3 -Cbl cofactor. Methionine synthase is a multi-modular protein, which consists of four domains: The homocysteine, folate, cobalamin, and AdoMet binding domains in a single polypeptide from the N-terminus (Figure 4d ). The enzyme orchestrates the domain arrangements for each reaction.

Because of the reactivity of the intermediate, cob(I)alamin loses an electron once in approximately every 2000 turnovers [56 , 57 ]. Since the oxidized cofactor, cob(II)- alamin, is inactive, the enzyme needs to reactivate the cofactor. MS is reactivated by reductive methylation, in which one electron and a methyl donor are required to reconstitute CH3 -Cbl. The reactivating methyl donor is S -adenosylmethionine (AdoMet). MSR, a dual ﬂ avoprotein similar to the P450 reductase family, and NADPH are needed for the physiological electron donor for mammalian MS

[ 58 , 59 ]. Free CH3 -Cbl can successfully bind to apoMS, forming holoMS. However, aquacobalamin is much less effective to form the holoenzyme. When aquacobalamin is reduced to cob(II)alamin in the presence of MSR and NADPH, the cofactor can be efﬁ ciently loaded into apoMS [59 , 60 ]. MSR demonstrates the holoMS synthase function, which was proposed for bacterial MS holoenzyme formation [61 , 62 ]. Biochemical properties and structural features of methionine synthase have been studied using the MetH protein from Escherichia coli, a homologue of human MS. Although this is a bacterial protein, the amino acid sequence of E. coli MetH [ 63 ] shares very high homology (55% in identity) to that of human MS. Thus, E. coli 308 Yamada

MetH provides a model to understand the biochemical properties and catalytic function of human MS. E. coli MetH has four domains; the N-terminus MetH contains the homocysteine, folate, Cbl, and AdoMet binding domains [64 ]. Crystal structures of the Cbl and AdoMet domains from E. coli have been solved [ 28 , 55 , 65 ] as have the homocysteine and folate-binding domains from Thermotoga maritima MetH, which is similar to E. coli MetH and human MS [66 ] (Figure 4d ). The structure of the Cbl-binding domain from E. coli MetH was the fi rst solved for any Cbl-binding proteins [28 ]. Upon binding to MetH, Cbl undergoes a large conformational change; the DBI base is replaced by the His residue from the protein and coordinated to the cobalt atom. Even though the nucleotide loop would appear to have no function because Cobinamide, which lacks the nucleotide loop, cannot act as cofactor [57 ]. The ribose moiety of Cbl could act as a spacer[67 ], and the phosphodiester group is necessary for catalytic function [57 ]. Cobinamide methylphosphate, the Cbl analogue, which lacks ribose and the DBI moieties but has the phosphodiester group, shows catalytic function. The analogue is, however, easily released from the enzyme, whereas the Cbl-MS holoenzyme complex is quite stable. Protein structures of each domain of bacterial MS have been solved (Figure 4d ), however, the full-length protein structure has yet to be determined. The structural analysis of bacterial MS implies large domain rearrangements during the catalytic turnover of MS because the Cbl-binding domain has to interact with three independent binding domains for each substrate to accomplish the methyl transfer reaction. Cbl plays an important role not only for the methyl transfer reaction, but also for domain rearrangement during catalysis through the His ligand of the protein [ 68– 70 ]. Because of the high homology between E. coli MetH and human MS, the properties and reaction mechanisms are likely to be quite similar. For the reactivation of E. coli MetH, reduced fl avodoxin is required as the electron donor. MSR is homologous to the P450 reductase family of enzymes, which contains FMN and FAD as prosthetic groups. Although the FMN domain in MSR is homologous to fl avodoxin, reduced-fl avodoxin is unable to reactivate oxidized human MS [59 ], indicating that specifi c protein-protein interactions are important. MS is involved in folate and methionine metabolism, which is quite complex

(Figure 8 ). The dominant form of folic acid in blood is CH3 -H4 folate, which is absorbed into cells. MS is the only enzyme capable of metabolizing CH3 -H4 folate in mammalian cells. Thus, the reaction catalyzed by MS is the ﬁ rst step for folate metabolism. The product, tetrahydrofolate (H4 folate), is important as a carrier for a C1 unit, which is a functional group consisting of a single carbon atom, such as formyl or methylene groups. The C1 unit on 10-formyltetrahydrofolate is used in purine synthesis and the 5-methyl group of thymidine monophosphate (dTMP) is derived from methylenetetrahydrofolate (CH2 -H4 folate). Thus, the folate C1 unit is important for de novo synthesis of nucleic acids, precursors of DNA and RNA. H4 folate is also important for glycine, serine, and histidine metabolism. Thus, cellular storage of reduced folates, except CH3 -H4 folate, is often referred to as the “functional folate pool”. CH3 -H4 folate is produced by the reaction catalyzed by methylenetetrahydrofolate 9 Cobalt: Its Role in Health and Disease 309

transmethylation cysteine CH3-X X polyamine synthesis

cystathionine AdoHcy AdoMet 4 Ado 3 2 ATP serine homocysteine methionine

1 CH3-H4folate CH3-H4folate H4folate 6 serine glycine 5 CHO-H4folate 8

CH -H folate purine base 2 4 synthesis H2folate extracellular fluid (blood) intracellular fluid (cytosol) dUMP7 dTMP

Figure 8 Folate and methionine metabolism. (1 ) Methionine synthase. (2 ) Methionine adenosyltransferase. ( 3) Adenosylhomocysteine hydrolase. (4 ) Cystathionine-β-synthase. ( 5 ) Methylene- tetrahydrofolate reductase. ( 6) Serine hydroxymethyltransferase. (7 ) Thymidylate synthase. (8 ) Dihydrofolate reductase. reductase (MTHFR). This reaction is important to produce methionine using the C1 unit as the methyl source for the reaction of MS. Mammalian MTHFR activity is strongly inhibited by AdoMet [71 ]. While S -adenosylhomocysteine (AdoHcy) is not an activator for MTHFR, the enzyme’s activity is restored because AdoHcy competes with the AdoMet binding site on MTHFR. This negative feedback is important for the regulation of methionine production. Inactivation of MS can happened due to the lack of Cbl, dysfunction of MSR, or upon nitrous oxide (N 2O) exposure. N2 O, known as laughing gas, is used for anesthetic purposes. It is known that long-time exposure to N 2 O can cause megaloblastic anemia. N2 O exposure inactivates MS but not MCM [72 ]. The mechanism of inactivation was studied using E. coli MetH. The analysis revealed that inactivation is based on the irreversible chemical modiﬁ cation of the MS protein

[73 –75 ]. To fully recover MS activity in tissues caused by N2 O inactivation, it takes approximately 48 hr [76 ]. The long recovery time to restore MS activity is also observed in B12 - deﬁ cient rats. In B12 -deﬁ cient rat liver, MS activity is lowered and MS protein levels are decreased while MS mRNA levels are not changed [77 ]. Mammalian apoMS is quite unstable compared to holoMS [57 , 78 ].

Hence, administration of Cbl to B12 - deﬁ cient rats does not allow for a quick recovery due to lack of apoMS. To restore enzyme activity in both N2 O-exposed and B 12 -deﬁ cient rats, it takes a period of time necessary to synthesize new MS protein. This is different from MCM, which recovers activity quickly because the MCM apoenzyme is stable in mitochondria [52 ]. 310 Yamada

Dysfunction of MS inﬂ uences not only methionine production but also folate metabolism. The MS-deﬁ cient mouse (Mtr −/− mouse) is embryonic lethal even when various nutritional diets were fed, indicating the fundamental importance for this enzyme [ 79]. Methionine is an essential building block for proteins. Moreover, it is generated from AdoMet, which is the major methyl donor for the biological methyl transferase reaction and is a substrate for polyamine synthesis. For folate metabolism, MS introduces the reduced folate into the cellular “functional folate pool”.

When MS is inactivated, production of H4 folate and methionine is decreased. The reduced-methionine level lowers AdoMet synthesis. Since AdoMet is a strong inhibitor for MTHFR, MTHFR increases its activity, forming CH 3 -H4 folate. Additionally, accumulation of homocysteine leads to increased AdoHcy, which competes with the

AdoMet binding site of MTHFR. However, the product, CH 3 -H4 folate, can only be utilized by MS, which is being inactivated, resulting in the accumulation of CH 3 - H4 folate. This is called the “methyl(folate)-trap hypothesis” [ 80 ,81 ]. Reduced folate, such as H4 folate, is trapped as the CH 3 -H4 folate form, and thus the “functional folate pool” is depleted. While administration of folic acid can increase the cellular folate pool, it does not provide the fundamental solution. Methionine supplementation also masks

B12 -deﬁ cient symptoms, such as Cbl neuropathy [82 , 83] and testicular damage [78 ]. In this case, the “methyl-trap hypothesis” provides the following explanation: Dietary methionine provides a source of AdoMet, which inhibits MTHFR activity, thus

CH3 -H4 folate is not produced and folate can be retained in the “functional folate pool”. It seems that the methyl-trap hypothesis rationally explains the mechanism, although it is still controversial. There is a report that demonstrates that amounts of dNTPs, precursors of DNA, are normal [ 84] and the net amount of CH 3 -H4 folate is not increased during B12 deﬁ ciency [85 ].

3 Vitamin B12 Deﬁ ciency and Disease

3.1 Vitamin B12 Deﬁ ciency

We know how to diagnose B12 defi ciency and how to effectively treat it. Even though vitamin defi ciency and the corresponding disease can be treated effectively, we still do not understand which exact biochemical mechanism underlies most cases. Despite that administration of Cbl is effective to megaloblastic anemia and Cbl neuropathy, we still do not know what causes these diseases. That is so because Cbl is not the direct trigger for megaloblastic anemia and Cbl neuropathy, as well as for other symptoms of Cbl defi ciency, even though they are well-established symptoms of B 12 defi ciency. The secondary effects of B12 defi ciency, i.e., metabolic imbalances, could be responsible. Cbl-dependent enzymes, especially methionine synthase, are important to maintain a healthy metabolism. There are still many things remaining to identify the exact biochemical mechanisms. To that end, the typical B12 defi ciency symptoms will be discussed. 9 Cobalt: Its Role in Health and Disease 311

3.2 Methylmalonic Aciduria

Methylmalonic aciduria is a characteristic symptom caused by defects of MCM (Figure 6). Accumulated methylmalonyl-CoA is degraded to MMA and CoA by enzymatic hydrolysis. (S )-methylmalonyl-CoA hydrolase has been purifi ed from rat liver [ 86 ]. Shimomura et al. [ 87 ] have reported purifi cation and characterization of rat liver 3-hydroxyisobutyryl-CoA hydrolase, which is involved in valine metabolism. They observed that the enzyme showed the ability to hydrolyze methylmalonyl-CoA and that it showed properties similar to the previously purifi ed ( S )-methylmalonyl-CoA hydrolase. When MCM is inactivated, propionyl-carnitine in blood is increased. Accumulation of methylmalonyl-CoA can produce odd chain fatty acids. Although odd-chain fatty acids may cause some symptoms of B12 defi ciency, the exact biochemical mechanism has not been established. There are several reasons for dysfunction of MCM, such as inadequate intake of B12 , dysfunction of the Cbl absorption or cellular processing systems or due to MCM gene mutations. If methylmalonic aciduria were caused by low intake of B 12, or impaired AdoCbl synthesis, administration of AdoCbl would rescue the phenotype. Null mutations of MCM result in neonatal lethality, if not properly treated. Protein restriction treatment is most often chosen. Carnitine administration is effective, but the utility as a long-term treatment is unknown.

3.3 Hyperhomocysteinemia

During the last two decades, homocysteine metabolism has been actively studied by many researchers because hyperhomocysteinemia, the elevated level of homocysteine in blood, was reported to be an independent risk factor for cardiovascular disease. Epidemiological studies showed a relationship between hyperhomocysteinemia and many other diseases, such as schizophrenia [88 ], Alzheimer’s disease [89 ], and osteoporosis [90 ]. Homocysteine is a substrate for three enzymes, MS, betaine-homocysteine methyltransferase (BHMT), and cystathionine β-synthase (CBS) (Figure 8 ). Although BHMT is a liver-specifi c enzyme, MS is ubiquitously expressed in nearly all human tissues. CBS catalyzes the reaction forming cystathionine from homocysteine and serine. While homocysteine is not a direct substrate for MTHFR, the enzyme catalyzes the reduction of CH2 -H4 folate to produce CH3 -H4 folate, which is the other substrate for MS. Thus, dysfunction of MTHFR also causes hyperhomocysteinemia. Affi nity for homocysteine is highly variable; enzymes in the methionine recycling pathway, i.e., MS and BHMT, show high affi nity (low K m values) for homocysteine, while CBS in the catabolic pathway has low affi nity. Thus, dysfunction of CBS causes higher levels of homocysteine in blood than do MS and MTHFR. Folic acid fortifi cation in food has been conducted and has succeeded in lowering plasma homocysteine concentrations. However, recent epidemiological reports disprove 312 Yamada

“homocysteinemia as the risk factor for cardiovascular disease” [91 – 94]. Even so, elevated-levels of homocysteine certainly indicate the failure to regulate folate and methionine metabolism. It should be noted that lowering of homocysteine by folic acid fortiﬁ cation may not be the solution because it fails to solve the disruption of the methionine cycle due to B12 dependency.

3.4 Megaloblastic Anemia

Folate deﬁ ciency also causes megaloblastic anemia (formerly pernicious anemia). It is thought that the cause is impaired DNA synthesis due to decreased de novo dTMP synthesis, which results from a depleted “functional folate pool” due to dysfunction of MS, i.e., the “methyl-trap” (see Section 2.3.3 , Figure 8 ). “Functional folate” deﬁ ciency can explain the observed symptoms. Because CH 2 -H4 folate is essential for dTMP production as a methyl donor, dTMP production from the folate cycle would be reduced, which results in decreased DNA synthesis.

Administration of folate to a B12 -deﬁ cient patient with megaloblastic anemia could be effective because it increases the folate pool. In addition, methionine supplementation can alter the metabolic balance because it contributes to the production of AdoMet, which is an inhibitor for MTHFR. While the methyl-trap hypothesis is consistent with this mechanism, it is still controversial. Although B12 was initially identiﬁ ed as the anti-pernicious anemia factor, the fundamental biochemical basis of megaloblastic anemia is still unknown.

3.5 Cobalamin Neuropathy

Cbl neuropathy is an abnormality of the peripheral nervous system found in B 12 - deﬁ cient patients. N2 O-exposed patients show Cbl neuropathy, suggesting that dysfunction of MS is related to this disease. Although Cbl neuropathy is caused by impaired transmethylation [82 , 83], the cause is not yet fully understood. Interestingly, despite the fact that this symptom responds to Cbl administration, signiﬁ cant numbers of Cbl neuropathy patients show serum Cbl contents in the lower normal range. Thus, it is suggested that serum MMA and homocysteine levels are better indicators for biochemical diagnosis of Cbl neuropathy [ 95 – 97 ].

3.6 Other Diseases Related to Vitamin B12 Deﬁ ciency

A relationship between pernicious anemia and pregnancy has been suspected since the mid-1930’s. However, the results of early clinical trials using Cbl treatment were mixed, so this idea remains controversial. In 1962, Watson [98 ] and Sharp and 9 Cobalt: Its Role in Health and Disease 313

Witts [99 ] reported the relationship between serum B12 content and sperm maturation. The effect of B12 defi ciency on sperm maturation in humans can be reproduced in dietary B12 -defi cient rats [100 ] and in the N 2O-exposed rat model [101 ]. Both rat models show catastrophic testicular damage, including aplasia of sperm and spermatids. The testicular damage of the B12 -defi cient rat can be prevented by methionine supplementation to the B12 -defi cient diet, indicating that dysfunction of MS is responsible [78 ].

B12 defi ciency in the elderly remains a signifi cant concern. Clinical reports indicate that B12 defi ciency is related to dementia [102 , 103]. This could be due to malnutrition or dysfunction of the Cbl absorbing system caused by autoimmunity.

Even in patients who are not B12 -deﬁ cient, it is known that administration of B12 is an effective way to correct the circadian rhythm [104 , 105 ]. CH3 -Cbl is the most effective [106 ], so MS might be the responsible target. The biochemical basis of this observation has not yet been clearly demonstrated.

3.7 Animal Models

Whole animal models are useful for understanding the functions of nutrients. B12 - deﬁ cient animal models have been reported that allow us to understand the role of

Cbl in human health. It is especially diffi cult to establish a B12 -defi cient animal model using classical nutritional methods (i.e., by which animals are fed B 12 - defi cient diets) because Cbl shows biological activity in a very small amount. Despite this diffi culty, there are many potential animal models that allow investigation of dietary B12 defi ciency, including monkeys, pigs, rats, mice, fruit bats, etc.. Co-defi cient sheep are also used as B12 -defi cient animal models. Some animals, such as rats, show coprophagia, and therefore need very careful handling to prevent fecal recycling. Furthermore, the biological half-life of Cbl is quite long, and is estimated to be approximately one year [107 ]. These obstacles hamper investigation of Cbl function in normal animals. Since N2 O is known as specifi c inhibitor for MS, N2 O-exposed animals have been frequently used as MS-impaired animal models. Recently, an experimental autoimmune gastritis mouse model was developed as a megaloblastic anemia model [108 , 109 ], although a dietary B12 defi ciency- or an N2 O exposure-induced megaloblastic anemia animal model has not been reported. For Cbl neuropathy, it has been reported that monkeys

[110 , 111], fruit bats [ 82], and pigs [ 83] develop Cbl neuropathy when B12 -defi cient diets were fed or animals were exposed to N2 O. Genetic engineering and developmental biology have permitted the production of transgenic and targeted gene-disrupted mice by many laboratories. These should allow for investigations beyond that allowed by classical nutritional models. Gene- disrupted mice targeted for proteins in the Cbl transporting system have been reported only for TC receptor and megalin [112 ]. Targeting of cellular Cbl-delivering and -processing protein genes have not been reported, to date. As described above, gene disruption of Mtr (gene for MS) is embryonic lethal [79 ]. Dietary supplementation, 314 Yamada such as with methionine, betaine, and/or, folic acid, did not rescue the phenotype. Elmore et al. reported that intercross mating of a heterozygous Mtrr -deletional mouse model could not produce homozygous Mtrr -defi cient mice [113 ], indicating that function of both MS and MSR are absolutely necessary for development. However, a hypomorphic mouse model, i.e., a reduced-function MSR model, has been reported [113 ]. The Mtrr gene in this mouse model is interrupted by β-galactosidase/neomycin phosphotransferase gene, a “gene trap” which provides the marker for screening. Therefore, the Mtrr gt/gt homozygote mouse can produce the fusion protein of the FMN domain of MSR and β-galactosidase/neomycin phosphotransferase. While the intact FMN domain of the fusion protein has a function, the Mtrr gt/gt mouse shows disrupted methionine metabolism [113 ]. Although the hypomorphic mouse provides a model for reduced function of MS and MSR, phenotypes for B12 -defi cient symptoms, megaloblastic anemia, and neurological abnormality, are not yet characterized. A MCM knock-out mouse model has also been reported [114 ]. The knock-out mouse shows increased MMA in urine and neonaternal lethality, which resembles human mut0 patients (complete defi ciency of MCM enzyme activities). The MCM knock-out mouse was used to produce a humanized MCM-defi cient mouse model, in which the MCM gene from human mut0 patients (due to the missense mutation at Arg403) was introduced [115 ]. Because the mouse mimics human MCM defi ciency both at the phenotypic and genotypic levels, this model allows evaluation of possible treatments for MCM-defi cient human patients. Effect of depletion of enzymes involved in the propionyl-CoA pathway on methylmalonyl-CoA metabolism has also been reported using Caenorhabditis elegans and RNA interference techniques for gene knock-down [116 ]. While C. elegans is a non-vertebrate, the well-characterized genetic and genomic information could provide a model for further investigation.

4 Non-corrinoid Cobalt

4.1 Non-corrinoid Cobalt-Containing Proteins

Non-corrinoid cobalt-containing enzymes are found in bacteria [117 ]. For example, Co-dependent nitrile hydratase [ 118] is important for acrylamide production [119 ]. In this enzyme, the cobalt atom is placed in the active site with the unique post- translational modiﬁ cation on the cysteine residues [120 ]. Whereas homologous genes for nitrile hydratase are found in certain eukaryotes, such as Monosiga brevicollis [121 , 122 ], it is not present in the human genome. In mammals, function of non-corrinoid cobalt in enzymes are rarely reported. Methionine aminopeptidase has been puriﬁ ed from porcine liver and characterized [ 123]. Activity of the mammalian methionine aminopeptidase can be stimulated in the presence of Co 2+ ion. However, the enzyme is also active in the presence of other divalent ions such as Mg 2+, and therefore does not exclusively require Co 2+ for activity. 9 Cobalt: Its Role in Health and Disease 315

4.2 Overload of Cobalt

While non-corrinoid cobalt-speciﬁ c proteins are not found in humans, there are many reports using CoCl2 . For example, CoCl2 is often used as a simulative hypoxia- inducing reagent for cell culture [124 ]. Co2+ can substitute Fe2+ in the porphyrin ring of heme. This alters the heme protein conformation to the deoxygenated form by mimicking the lowered afﬁ nity for oxygen. Thus, it can cause hypoxia, resulting in activation of hypoxia response genes, such as the erythropoietin gene. Nickel also shows the same effect. Amounts necessary to see these effects in cell culture are massive (~100 μM). Toxicity of high doses of CoCl 2 has been recently reviewed [ 125 ]. Moreover, toxicity of nanocompounds containing cobalt are a novel concern [126 ].

5 Implications and Future Development

Roles of Cbl in health and disease were reviewed because the biological function of cobalt is predominantly as Cbl. Functions of Cbl in humans have been studied by many researchers since the isolation of Cbl. However, genes for intercellular transporting proteins were only recently discovered. These absorbing, transporting, and activating proteins are important for the function of Cbl because B12 is contained in very low amounts in food. Proper function of Cbl requires many auxiliary proteins. Currently, analyzing the functions of transporting and activating proteins involved with Cbl is under investigation in many laboratories. There might be even more intercellular Cbl-processing proteins that remained so far undiscovered, for example a mitochondrial transporter. These efforts should help to clarify the biochemical roles of Cbl-binding proteins in the near future. For these studies, understanding of protein-protein interactions is likely the key. Understanding the contributions of Cbl for human health remains elusive because the action of Cbl may not be the direct cause for B12 -deﬁ cient symptoms, with the exception of methylmalonic aciduria and hyperhomocysteinemia. Although it might be possible that completely new physiological functions of Cbl are discovered at this moment, the characterized function of Cbl is its cofactor role for the two enzymes discussed. Dysfunction of Cbl-dependent enzymes disrupts normal metabolism, and the impaired metabolism could be causal for disease. Especially, dysfunction of MS causes disruption of many cellular processes. Hyperhomocysteinemia could be the excellent biomarker for disturbed folate and methionine metabolism, which could indicate the patient’s health is at risk. However, it is difﬁ cult to establish links to certain diseases, as we have observed between hyperhomocysteinemia and cardiovascular disease. Folate and methionine metabolism is important for supplying substrates for other processes, such as methyl groups and precursors for DNA and RNA, and for providing necessary amounts. Moreover, many nutrients affect global metabolism and the requirements of metabolism could vary between individual persons. Such complexity 316 Yamada

precludes a defi nitive explanation for even well recognized characteristics of B12 defi ciency after many years of research. Using combinations of current experimental techniques and animal models, however, it should be possible to reveal the biochemical mechanisms underlying B12 defi ciency and disease, and to provide rational explanations for infl uences of gene mutations and nutrients intake for the diseases. Such knowledge would contribute to our overall wellness and quality of life.

Abbreviations

ABCD4 ATP-binding cassette transporter, D subfamily 4 AdoCbl adenosylcobalamin AdoHcy S -adenosylhomocysteine AdoMet S -adenosylmethionine ATP adenosine 5′-triphosphate

B12 vitamin B12 BHMT betaine-homocysteine methyltransferase Cbl cobalamin CBS cystathionine β-synthase

CH 2 -H4 folate methylenetetrahydrofolate

CH 3 Cbl methylcobalamin CH3 -H4 folate methyltetrahydrofolate CN-Cbl cyanocobalamin DBI dimethylbenzimidazole dNTP 2′-deoxynucleoside 5′-triphosphate dTMP thymidine 5′-monophosphate F A D ﬂ avin adenine dinucleotide FMN ﬂ avin mononucleotide GTP guanosine 5′-triphosphate

H4 folate tetrahydrofolate IF intrinsic factor LMBD1 LMBR1 (limb region 1 homolog) domain containing 1 MCM methylmalonyl-CoA mutase MMA methylmalonic acid MMAA methylmalonic aciduria, CblA type MMAB methylmalonic aciduria, CblB type MMACHC methylmalonic aciduria, CblC type, and homocysteinuria MMADHC methylmalonic aciduria, CblD type, and homocysteinuria MRP1 multidrug resistance protein MS methionine synthase MSR methionine synthase reductase MTHFR methylenetetrahydrofolate reductase

N2 O nitrous oxide NADPH nicotinamide adenine dinucleotide phosphate (reduced) TC transcobalamin 9 Cobalt: Its Role in Health and Disease 317

Acknowledgment The author acknowledges Dr. C. L. Elmore (US Food and Drug Administration) for reading and editing the English.

References

1. W. H. Ott, E. L. Rickes, T. R. Wood, J. Biol. Chem. 1948 , 174 , 1047. 2. J. R. Roth, J. G. Lawrence, T. A. Bobik, Annu. Rev. Microbiol. 1996 , 50 , 137–181. 3. F. Watanabe, Exp. Biol. Med. (Maywood) 2007 , 232 , 1266–1274. 4. J. Stubbe, Science 1994 , 266 , 1663–1664. 5. E. Rickes, N. Brink, F. Koniuszy, T. Wood, K. Folkers, Science 1948 , 107 , 396–397. 6. E. Smith, Nature 1948 , 161 , 638. 7. D. Hodgkin, J. Kamper, M. Mackay, J. Pickworth, K. Trueblood, J. White, Nature 1956 , 178 , 64–66. 8. K. P. Jensen, J. Phys. Chem. B 2005 , 109 , 10505–10512. 9. G. Minot, W. Murphy, J. Am. Med. Assoc. 1926 87 , 470–476. 10. W. B. Castle, T. H. Ham, J. Am. Med. Assoc. 1936 , 107 , 1456–1463. 11. E. Stupperich, E. Nexø, Eur. J. Biochem. 1991 , 199 , 299–303. 12. F. S. Mathews, M. M. Gordon, Z. Chen, K. R. Rajashankar, S. E. Ealick, D. H. Alpers, N. Sukumar, Proc. Natl. Acad. Sci. USA 2007 , 104 , 17311–17316. 13. S. K. Moestrup, R. Kozyraki, M. Kristiansen, J. H. Kaysen, H. H. Rasmussen, D. Brault, F. Pontillon, F. O. Goda, E. I. Christensen, T. G. Hammond, P. J. Verroust, J. Biol. Chem. 1998 , 273 , 5235–5242. 14. J. C. Fyfe, M. Madsen, P. Hojrup, E. I. Christensen, S. M. Tanner, A. de la Chapelle, Q. He, S. K. Moestrup, Blood 2004 , 103 , 1573–1579. 15. R. Grasbeck, R. Gordin, I. Kantero, B. Kuhlback, Acta Med. Scand. 1960 , 167 , 289–296. 16. O. Imerslund, Acta Paediatr. Suppl. 1960 , 49 (Suppl. 119) , 1–115. 17. R. Beedholm-Ebsen, K. van de Wetering, T. Hardlei, E. Nexø, P. Borst, S. K. Moestrup, Blood 2010 , 115 , 1632–1639. 18. N. P. Shah, C. M. Beech, A. C. Sturm, S. M. Tanner, Blood 2011 , 117 , 4397–4398. 19. J. Wuerges, G. Garau, S. Geremia, S. N. Fedosov, T. E. Petersen, L. Randaccio, Proc. Natl. Acad. Sci. USA 2006 , 103 , 4386–4391. 20. E. Moras, A. Hosack, D. Watkins, D. S. Rosenblatt, Mol. Genet. Metab. 2007 , 90 , 140–147. 21. J. F. Kolhouse, R. H. Allen, Proc. Natl. Acad. Sci. USA 1977 , 74 , 921–925. 22. I. S. Mellman, P. Youngdahl-Turner, H. F. Willard, L. E. Rosenberg, Proc. Natl. Acad. Sci. USA 1977 , 74 , 916–920. 23. F. Rutsch, S. Gailus, I. Miousse, T. Suormala, C. Sagnè, M. Toliat, G. Nürnberg, T. Wittkampf, I. Buers, A. Shariﬁ , M. Stucki, C. Becker, M. Baumgartner, H. Robenek, T. Marquardt, W. Höhne, B. Gasnier, D. Rosenblatt, B. Fowler, P. Nürnberg, Nat. Genet. 2009 , 41 , 234–239. 24. D. Coelho, J. C. Kim, I. R. Miousse, S. Fung, M. du Moulin, I. Buers, T. Suormala, P. Burda, M. Frapolli, M. Stucki, P. Nurnberg, H. Thiele, H. Robenek, W. Hohne, N. Longo, M. Pasquali, E. Mengel, D. Watkins, E. A. Shoubridge, J. Majewski, D. S. Rosenblatt, B. Fowler, F. Rutsch, M. R. Baumgartner, Nat. Genet. 2012 , 44 , 1152–1155. 25. J. P. Lerner-Ellis, J. C. Tirone, P. D. Pawelek, C. Dore, J. L. Atkinson, D. Watkins, C. F. Morel, T. M. Fujiwara, E. Moras, A. R. Hosack, G. V. Dunbar, H. Antonicka, V. Forgetta, C. M. Dobson, D. Leclerc, R. A. Gravel, E. A. Shoubridge, J. W. Coulton, P. Lepage, J. M. Rommens, K. Morgan, D. S. Rosenblatt, Nat. Genet. 2006 , 38 , 93–100. 26. J. Kim, C. Gherasim, R. Banerjee, Proc. Natl. Acad. Sci. USA 2008 , 105 , 14551–14554. 27. M. Koutmos, C. Gherasim, J. L. Smith, R. Banerjee, J. Biol. Chem. 2011 , 286 , 29780–29787. 28. C. Drennan, S. Huang, J. Drummond, R. Matthews, M. Lidwig, Science 1994, 266, 1669–1674. 29. D. Coelho, T. Suormala, M. Stucki, J. Lerner-Ellis, D. Rosenblatt, R. Newbold, M. Baumgartner, B. Fowler, N. Engl. J. Med. 2008 , 358 , 1454–1464. 30. C. M. Dobson, T. Wai, D. Leclerc, H. Kadir, M. Narang, J. P. Lerner-Ellis, T. J. Hudson, D. S. Rosenblatt, R. A. Gravel, Hum. Mol. Genet. 2002 , 11 , 3361–3369. 318 Yamada

31. H. L. Schubert, C. P. Hill, Biochemistry 2006 , 45 , 15188–15196. 32. M. St. Maurice, P. Mera, K. Park, T. C. Brunold, J. C. Escalante-Semerena, I. Rayment, Biochemistry 2008 , 47 , 5755–5766. 33. N. A. Leal, H. Olteanu, R. Banerjee, T. A. Bobik, J. Biol. Chem. 2004 , 279 , 47536–47542. 34. C. M. Dobson, T. Wai, D. Leclerc, A. Wilson, X. Wu, C. Dore, T. Hudson, D. S. Rosenblatt, R. A. Gravel, Proc. Natl. Acad. Sci. USA 2002 , 99 , 15554–15559. 35. D. Leclerc, E. Campeau, P. Goyette, C. E. Adjalla, B. Christensen, M. Ross, P. Eydoux, D. S. Rosenblatt, R. Rozen, R. A. Gravel, Hum. Mol. Genet. 1996 , 5 , 1867–1874. 36. L. H. Chen, M. L. Liu, H. Y. Hwang, L. S. Chen, J. Korenberg, B. Shane, J. Biol. Chem. 1997 , 272 , 3628–3634. 37. Y. N. Li, S. Gulati, P. J. Baker, L. C. Brody, R. Banerjee, W. D. Kruger, Hum. Mol. Genet. 1996 , 5 , 1851–1858. 38. D. Leclerc, A. Wilson, R. Dumas, C. Gafuik, D. Song, D. Watkins, H. H. Heng, J. M. Rommens, S. W. Scherer, D. S. Rosenblatt, R. A. Gravel, Proc. Natl. Acad. Sci. USA 1998 , 95 , 3059–3064. 39. F. Mancia, N. H. Keep, A. Nakagawa, P. F. Leadlay, S. McSweeney, B. Rasmussen, P. Bosecke, O. Diat, P. R. Evans, Structure 1996 , 4 , 339–350. 40. R. G. Matthews, Met. Ions Life Sci. 2009 , 6 , 53–114. 41. R. A. Kwiecien, I. V. Khavrutskii, D. G. Musaev, K. Morokuma, R. Banerjee, P. Paneth, J. Am. Chem. Soc. 2006 , 128 , 1287–1292. 42. K. Mori, T. Tobimatsu, T. Hara, T. Toraya, J. Biol. Chem. 1997 , 272 , 32034–32041. 43. T. Toraya, K. Mori, J. Biol. Chem. 1999 , 274 , 3372–3377. 44. K. Mori, T. Toraya, Biochemistry 1999 , 38 , 13170–13178. 45. N. Shibata, K. Mori, N. Hieda, Y. Higuchi, M. Yamanishi, T. Toraya, Structure 2005 , 13 , 1745–1754. 46. K. Mori, Y. Hosokawa, T. Yoshinaga, T. Toraya, FEBS J. 2010 , 277 , 4931–4943. 47. D. Padovani, R. Banerjee, Biochemistry 2006 , 45 , 9300–9306. 48. D. Padovani, T. Labunska, B. A. Palfey, D. P. Ballou, R. Banerjee, Nat. Chem. Biol. 2008 , 4 , 194–196. 49. D. Padovani, R. Banerjee, Proc. Natl. Acad. Sci. USA 2009 , 106 , 21567–21572. 50. P. Hubbard, D. Padovani, T. Labunska, S. Mahlstedt, R. Banerjee, C. Drennan, J. Biol. Chem. 2007 , 282 , 31308–31316. 51. D. Froese, G. Kochan, J. Muniz, X. Wu, C. Gileadi, E. Ugochukwu, E. Krysztoﬁ nska, R. Gravel, U. Oppermann, W. Yue, J. Biol. Chem. 2010 , 285, 38204–38213. 52. M. Nakao, S. Hironaka, N. Harada, T. Adachi, T. Bito, Y. Yabuta, F. Watanabe, T. Miura, R. Yamaji, H. Inui, Y. Nakano, Br. J. Nutr. 2009 , 101 , 492–498. 53. D. G. Kennedy, A. Cannavan, A. Molloy, F. O’Harte, S. M. Taylor, S. Kennedy, W. J. Blanchﬂ ower, Br. J. Nutr. 1990 , 64 , 721–732. 54. E. Vieira-Makings, N. Chetty, S. C. Reavis, J. Metz, Biochem. J. 1991 , 275 , 585–590. 55. V. Bandarian, R. G. Matthews, Biochemistry 2001 , 40 , 5056–5064. 56. J. T. Drummond, S. Huang, R. M. Blumenthal, R. G. Matthews, Biochemistry 1993 , 32 , 9290–9295. 57. K. Yamada, S. Yamada, T. Tobimatsu, T. Toraya, J. Biol. Chem. 1999 , 274 , 35571–35576. 58. H. Olteanu, R. Banerjee, J. Biol. Chem. 2001 , 276 , 35558–35563. 59. K. Yamada, R. Gravel, T. Toraya, R. Matthews, Proc. Natl. Acad. Sci. USA 2006 , 103 , 9476–9481. 60. K. Wolthers, N. Scrutton, FEBS J. 2009 , 276 , 1942–1951. 61. F. Rosales, S. J. Ritari, W. Sakami, Biochem. Biophys. Res. Commun. 1970 , 40 , 271–276. 62. N. Brot, H. Weissbach, J. Biol. Chem. 1966 , 241 , 2024–2028. 63. R. V. Banerjee, N. L. Johnston, J. K. Sobeski, P. Datta, R. G. Matthews, J. Biol. Chem. 1989 , 264 , 13888–13895. 64. C. W. Goulding, D. Postigo, R. G. Matthews, Biochemistry 1997 , 36 , 8082–8091. 65. M. M. Dixon, S. Huang, R. G. Matthews, M. Ludwig, Structure 1996 , 4 , 1263–1275. 66. J. Evans, D. Huddler, M. Hilgers, G. Romanchuk, R. Matthews, M. Ludwig, Proc. Natl. Acad. Sci. USA 2004 , 101 , 3729–3736. 9 Cobalt: Its Role in Health and Disease 319

67. A. Ishida, H. Kanefusa, H. Fujita, T. Toraya, Arch. Microbiol. 1994 , 161 , 293–299. 68. V. Bandarian, M. L. Ludwig, R. G. Matthews, Proc. Natl. Acad. Sci. USA 2003 , 100 , 8156–8163. 69. A. S. Fleischhacker, R. G. Matthews, Biochemistry 2007 , 46 , 12382–12392. 70. M. D. Liptak, A. S. Fleischhacker, R. G. Matthews, T. C. Brunold, Biochemistry 2007 , 46 , 8024–8035. 71. C. Kutzbach, E. Stokstad, Biochim. Biophys. Acta 1971 , 250 , 459–477. 72. R. Deacon, M. Lumb, J. Perry, I. Chanarin, B. Minty, M. J. Halsey, J. F. Nunn, Lancet 1978 , 2 (8098) , 1023–1024. 73. V. Frasca, B. Riazzi, R. Matthews, J. Biol. Chem. 1986 , 261 , 15823–15826. 74. J. T. Drummond, R. G. Matthews, Biochemistry 1994 , 33 , 3732–3741. 75. J. T. Drummond, R. G. Matthews, Biochemistry 1994 , 33 , 3742–3750. 76. J. B. Brodsky, J. M. Baden, M. Serra, Y. Kundomal, Anesthesiology 1984 , 61 , 66–68. 77. K. Yamada, T. Kawata, M. Wada, T. Isshiki, J. Onoda, T. Kawanishi, A. Kunou, T. Tadokoro, T. Tobimatsu, A. Maekawa, T. Toraya, J. Nutr. 2000 , 130 , 1894–1900. 78. K. Yamada, T. Kawata, M. Wada, K. Mori, H. Tamai, N. Tanaka, T. Tadokoro, T. Tobimatsu, T. Toraya, A. Maekawa, J. Nutr. Sci. Vitaminol. (Tokyo) 2007 , 53 , 95–101. 79. D. Swanson, M. Liu, P. Baker, L. Garrett, M. Stitzel, J. Wu, M. Harris, R. Banerjee, B. Shane, L. Brody, Mol. Cell. Biol. 2001 , 21 , 1058–1065. 80. J. Noronha, M. Silverman, “On Folic Acid, Vitamin B12, Methionine and Formiminoglutamic Acid Metabolism”, Proceedings of the Second European Symposium on Vitamin B12 and Intrinsic Factor , 1962, pp. 728–736. 81. V. Herbert, R. Zalusky, J. Clin. Invest. 1962 , 41 , 1263–1276. 82. J. van der Westhuyzen, J. Metz, Br. J. Nutr. 1983 , 50 , 325–330. 83. D. G. Weir, S. Keating, A. Molloy, J. McPartlin, S. Kennedy, J. Blanchﬂ ower, D. G. Kennedy, D. Rice, J. M. Scott, J. Neurochem. 1988 , 51 , 1949–1952. 84. N. Iwata, M. Omine, H. Yamauchi, T. Maekawa, Blood 1982 , 60 , 918–923. 85. G. E. Davidson, D. G. Weir, J. M. Scott, Biochim. Biophys. Acta 1975 , 392 , 207–215. 86. R. J. Kovachy, S. D. Copley, R. H. Allen, J. Biol. Chem. 1983 , 258 , 11415–11421. 87. Y. Shimomura, T. Murakami, N. Fujitsuka, N. Nakai, Y. Sato, S. Sugiyama, N. Shimomura, J. Irwin, J. W. Hawes, R. A. Harris, J. Biol. Chem. 1994 , 269 , 14248–14253. 88. B. R. Hutto, Compr. Psychiatry 1997 , 38 , 305–314. 89. R. Clarke, A. D. Smith, K. A. Jobst, H. Refsum, L. Sutton, P. M. Ueland, Arch. Neurol. 1998 , 55 , 1449–1455. 90. J. B. van Meurs, R. A. Dhonukshe-Rutten, S. M. Pluijm, M. van der Klift, R. de Jonge, J. Lindemans, L. C. de Groot, A. Hofman, J. C. Witteman, J. P. van Leeuwen, M. M. Breteler, P. Lips, H. A. Pols, A. G. Uitterlinden, N. Engl. J. Med. 2004 , 350 , 2033–2041. 91. J. F. Toole, M. R. Malinow, L. E. Chambless, J. D. Spence, L. C. Pettigrew, V. J. Howard, E. G. Sides, C. H. Wang, M. Stampfer, J. Am. Med. Assoc. 2004 , 291 , 565–575. 92. E. Lonn, S. Yusuf, M. J. Arnold, P. Sheridan, J. Pogue, M. Micks, M. J. McQueen, J. Probstﬁ eld, G. Fodor, C. Held, J. Genest, Jr., N. Engl. J. Med. 2006 , 354 , 1567–1577. 93. K. H. Bonaa, I. Njolstad, P. M. Ueland, H. Schirmer, A. Tverdal, T. Steigen, H. Wang, J. E. Nordrehaug, E. Arnesen, K. Rasmussen, N. Engl. J. Med. 2006 , 354 , 1578–1588. 94. R. Clarke, D. A. Bennett, S. Parish, P. Verhoef, M. Dotsch-Klerk, M. Lathrop, P. Xu, B. G. Nordestgaard, H. Holm, J. C. Hopewell, D. Saleheen, T. Tanaka, S. S. Anand, J. C. Chambers, M. E. Kleber, W. H. Ouwehand, Y. Yamada, C. Elbers, B. Peters, A. F. Stewart, M. M. Reilly, B. Thorand, S. Yusuf, J. C. Engert, T. L. Assimes, J. Kooner, J. Danesh, H. Watkins, N. J. Samani, R. Collins, R. Peto, PLoS Med. 2012 , 9 , e1001177. 95. I. C. Balfour, D. W. Lane, Lancet 1980 , 2 , 1246. 96. J. Lindenbaum, D. G. Savage, S. P. Stabler, R. H. Allen, Am. J. Hematol. 1990 , 34 , 99–107. 97. D. G. Savage, J. Lindenbaum, S. P. Stabler, R. H. Allen, Am. J. Med. 1994 , 96 , 239–246. 98. A. A. Watson, Lancet 1962 , 2 , 644. 99. A. Sharp, L. Witts, Lancet 1962 , 2 , 779. 320 Yamada

100. T. Kawata, T. Takada, F. Morimoto, N. Fujimoto, N. Tanaka, K. Yamada, M. Wada, T. Tadokoro, A. Maekawa, J. Nutr. Sci. Vitaminol. (Tokyo) 1992 , 38 , 305–316. 101. B. J. Kripke, A. D. Kelman, N. K. Shah, K. Balogh, A. H. Handler, Anesthesiology 1976 , 44 , 104–113. 102. J. Selhub, L. C. Bagley, J. Miller, I. H. Rosenberg, Am. J. Clin. Nutr. 2000 , 71 , 614S–620S. 103. P. Sipponen, F. Laxen, K. Huotari, M. Harkonen, Scand. J. Gastroenterol. 2003 , 38 , 1209–1216. 104. B. Kamgar-Parsi, T. A. Wehr, J. C. Gillin, Sleep 1983 , 6 , 257–264. 105. M. Okawa, K. Mishima, T. Nanami, T. Shimizu, S. Iijima, Y. Hishikawa, K. Takahashi, Sleep 1990 , 13 , 15–23. 106. G. Mayer, M. Kroger, K. Meier-Ewert, Neuropsychopharmacology 1996 , 15 , 456–464. 107. J. F. Adams, Nature 1963 , 198 , 200. 108. F. Alderuccio, J. W. Sentry, A. C. Marshall, M. Biondo, B. H. Toh, Clin. Immunol. 2002 , 102 , 48–58. 109. D. L. Greenwood, J. W. Sentry, Clin. Immunol. 2007 , 122 , 41–52. 110. D. P. Agamanolis, E. M. Chester, M. Victor, J. A. Kark, J. D. Hines, J. W. Harris, Neurology 1976 , 26 , 905–914. 111. J. M. Scott, J. J. Dinn, P. Wilson, D. G. Weir, Lancet 1981 , 2 , 334–337. 112. S. C. Lai, Y. Nakayama, J. M. Sequeira, B. J. Wlodarczyk, R. M. Cabrera, R. H. Finnell, T. Bottiglieri, E. V. Quadros, FASEB J. 2013 . 113. C. L. Elmore, X. Wu, D. Leclerc, E. D. Watson, T. Bottiglieri, N. I. Krupenko, S. A. Krupenko, J. C. Cross, R. Rozen, R. A. Gravel, R. G. Matthews, Mol. Genet. Metab. 2007 , 91 , 85–97. 114. H. Peters, M. Nefedov, J. Sarsero, J. Pitt, K. J. Fowler, S. Gazeas, S. G. Kahler, P. A. Ioannou, J. Biol. Chem. 2003 , 278 , 52909–52913. 115. N. E. Buck, H. Dashnow, J. J. Pitt, L. R. Wood, H. L. Peters, PLoS One 2012 , 7 , e44974. 116. R. J. Chandler, V. Aswani, M. S. Tsai, M. Falk, N. Wehrli, S. Stabler, R. Allen, M. Sedensky, H. H. Kazazian, C. P. Venditti, Mol. Genet. Metab. 2006 , 89 , 64–73. 117. M. Kobayashi, S. Shimizu, Eur. J. Biochem. 1999 , 261 , 1–9. 118. T. Nagasawa, K. Takeuchi, H. Yamada, Eur. J. Biochem. 1991 , 196 , 581–589. 119. H. Yamada, M. Kobayashi, Biosci. Biotechnol. Biochem. 1996 , 60 , 1391–1400. 120. A. Miyanaga, S. Fushinobu, K. Ito, T. Wakagi, Biochem. Biophys. Res. Commun. 2001 , 288 , 1169–1174. 121. K. U. Foerstner, T. Doerks, J. Muller, J. Raes, P. Bork, PLoS One 2008 , 3 , e3976. 122. A. O. Marron, M. Akam, G. Walker, PLoS One 2012 , 7 , e32867. 123. R. L. Kendall, R. A. Bradshaw, J. Biol. Chem. 1992 , 267 , 20667–20673. 124. M. A. Goldberg, S. P. Dunning, H. F. Bunn, Science 1988 , 242 , 1412–1415. 125. M. De Boeck, M. Kirsch-Volders, D. Lison, Mutat. Res. 2003 , 533 , 135–152. 126. S. Alariﬁ , D. Ali, A. O. Y, M. Ahamed, M. A. Siddiqui, A. A. Al-Khedhairy, Int. J. Nanomedicine 2013 , 8 , 17–23. 127. W. DeLano, DeLano Scientiﬁ c LLC, 2008 . Chapter 10 Nickel and Human Health

Barbara Zambelli and Stefano Ciurli

Contents ABSTRACT ...... 322 1 INTRODUCTION: THE DOUBLE FACE OF NICKEL IN BIOLOGICAL SYSTEMS ...... 322 2 NICKEL HAZARD FOR HUMAN HEALTH ...... 324 2.1 Nickel-Induced Carcinogenesis ...... 325 2.1.1 The Carcinogenic Potential of Nickel ...... 325 2.1.2 Molecular Mechanisms for Nickel-Induced Neoplastic Transformation ...... 326 2.2 The Different Faces of Nickel Allergy ...... 332 2.2.1 Nickel Effects on Immune Response ...... 332 2.2.2 The Impact of Nickel-Induced Dermatitis ...... 334 3 NICKEL-DEPENDENT INFECTIOUS DISEASES ...... 336 3.1 Nickel-Dependent Enzymes in Pathogenic Microorganisms ...... 336 3.1.1 Glyoxalase I ...... 336 3.1.2 Acireductone Dioxygenase ...... 337 3.1.3 Urease ...... 338 3.1.4 [NiFe]-Hydrogenase ...... 339 3.2 Molecular Regulation of Nickel Homeostasis in Pathogenic Microorganisms ...... 340 3.2.1 Nickel Membrane Transporters ...... 341 3.2.2 Nickel Molecular Chaperones and Metallo-Chaperones ...... 342 3.2.3 Nickel Sensors ...... 343 3.3 Nickel-Obligate Microorganisms with Severe Impact on Human Health...... 344 3.3.1 Helicobacter pylori as a Nickel-Dependent Pathogen: A Possible Correlation between Nickel Intake and Cancer Development ...... 344 3.3.2 Nickel Homeostasis and Intracellular Parasitism: Eukaryotic and Prokaryotic Pathogens ...... 346

B. Zambelli • S. Ciurli (*) Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology , University of Bologna , Bologna , Italy e-mail: [email protected]; [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 321 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_10, © Springer Science+Business Media Dordrecht 2013 322 Zambelli and Ciurli

4 NICKEL ESSENTIALITY IN ANIMALS AND HUMANS ...... 348 4.1 Effects of Nickel Depletion in Higher Organisms ...... 348 4.2 Inﬂ uence of Nickel Availability on Gut’s Microﬂ ora ...... 349 5 CONCLUSIONS AND OUTLOOK ...... 350 ABBREVIATIONS ...... 351 REFERENCES ...... 352

Abstract This review focuses on the impact of nickel on human health. In particular, the dual nature of nickel as an essential as well as toxic element in nature is described, and the main forms of nickel that can come in contact with living systems from natural sources and anthropogenic activities are discussed. Concomitantly, the main routes of nickel uptake and transport in humans are covered, and the potential dangers that nickel exposure can represent for health are described. In particular, the insurgence of nickel-derived allergies, nickel-induced carcinogenesis as well as infectious diseases caused by human pathogens that rely on nickel-based enzymes to colonize the host are reviewed at different levels, from their macroscopic aspects on human health to the molecular mechanisms underlying these points. Finally, the importance of nickel as a beneﬁ cial element for human health, especially being essential for microorganisms that colonize the human guts, is examined.

Keywords human health • nickel • nickel allergy • nickel carcinogenesis • nickel homeostasis

Please cite as: Met. Ions Life Sci. 13 (2013) 321–357

1 Introduction: The Double Face of Nickel in Biological Systems

Nickel is the 24th most abundant element regarding the natural abundance in the Earth’s crust [1 ]. This metal exists in nature either in insoluble particles, which are components of fumes and dusts, like nickel sulﬁ des (NiS, Ni3 S2 ), oxides (NiO), and silicates, or in water-soluble nickel compounds, such as nickel acetate, nickel chloride, and nickel sulfate. Natural sources of nickel include dusts from volcanic emissions and weathering of rocks and soils [2 ]. Soluble and insoluble nickel compounds are also found in soils and in waters [3 ]. In water, nickel ions are generally 2+ divalent, present as the greenish hexa-hydrated [Ni(H2 O)6 ] ion. The unique physical and chemical properties of nickel – low thermal and electrical conductivities, high resistance to corrosion and oxidation, excellent strength and toughness at elevated temperatures, and capability of being magnetized – make this metal and its compounds suitable materials for many applications widely found in modern industry [4 ]. Human activities, such as emission of nickel-containing fuel, industrial nickel production and utilization of nickel compounds, concur to the environmental release of nickel and to pollution by nickel and its products. 10 Nickel and Human Health 323

Human exposure to nickel occurs primarily via inhalation, ingestion, and dermal absorption [5 ]. Insoluble particulate nickel enters the vertebrate cells by phagocytosis, whereas nickel carbonyl is soluble in lipids and permeates the plasma membrane. Soluble nickel is transported into cells of vertebrate organisms by diffusion or through calcium channels and/or divalent cation transporters (DMT-1), involved in iron uptake [6 ]. Transport of nickel in blood plasma is mediated by binding to albumin and some small ligands, such as amino acids (e.g., histidine) and small peptides [ 7 ,8 ]. The Ni2+ -L-histidine complex is the major form of nickel transport across the cell membrane, and the Ni2+ -albumin complex is the form for systemic transport [9 ]. Exposure to nickel compounds yields a variety of adverse effects on humans. Nickel immune reaction, as a form of dermatitis, is one of the most common allergies in the modern world [ 10]. In addition, chronic nickel exposure can produce serious respiratory, cardiovascular, and kidney diseases. Some alterations in immune response in animal models have been observed as a result of nickel contact [11 ]. Nickel induces the production of reactive oxygen species (ROS), like the superox- −· ide radical (O2 ), hydrogen peroxide (H2 O2 ), and hypochlorous acid (HOCl) by several cells, such as in neutrophils and monocytes. This ultimately causes apoptosis in a number of cellular types, including human neutrophils and T-cells [12 , 13 ]. High exposure to nickel impairs the normal homeostasis of essential metal ions, decreasing the levels of calcium, magnesium, manganese, and zinc in different tissues [ 14] and possibly interfering with normal iron cofactor binding to specifi c proteins [15 –17 ]. Finally, the most serious concerns of nickel for human health is the nickel-induced teratogenicity and carcinogenesis, documented by the International Agency for Research on Cancer (IARC) in 1990 [18 ]. Despite its poisoning potential, nickel plays a fundamental role in living organisms, revealing its double faced nature of both as an essential and toxic element [19 ]. The importance of nickel for plants, bacteria, archaea, and unicellular eukaryotes is well documented. In these organisms, the choice of nickel as catalyst for important biological reactions is related to its fl exible coordination geometry, which makes this metal a very versatile element for many biological applications [1 ]. Nickel is a necessary component in the active site of several essential metalloenzymes in bacteria and lower eukaryotes. So far, eight microbial nickel-containing enzymes have been identifi ed, which include urease, hydrogenase, CO dehydrogenase, acetyl-CoA synthase, methyl-CoM reductase, Ni-superoxide dismutase, acireductone dioxygenase, and glyoxalase I, while a few other possible nickel-dependent enzymes are emerging [20 ]. The majority of known nickel-dependent enzymes have been structurally determined and nickel ions have been demonstrated to play an essential role in their enzymatic catalysis. In higher eukaryotes, the only known nickel-depending enzyme is plant urease. Some plant species that live in serpentine soils have evolved to hyperaccumulate nickel ions, creating complex systems of metal detoxifi cation and homeostasis that constitute appealing systems for phytoremediation of contaminated environments [21 ]. 324 Zambelli and Ciurli

So far, no nickel-containing enzyme or cofactor is known in higher animals. However, this metal has been included in the group of “possibly essential elements” for animals and humans as early as the 1970s [19 ]. Many experiments on animal models showed that nickel could be beneﬁ cial, if not essential, for optimal reproductive function, bone composition and strength, energy metabolism, and sensory function. The reasons of this essentiality remain obscure, even if some hypotheses have been suggested [5 , 22 ]. The double face of nickel, being both a poison and an essential micronutrient, implies that nickel-dependent organisms have developed tightly regulated systems for nickel handling, delivery it into the correct cellular location, and avoiding its dangerous potential. Bacteria that rely on nickel ions for environmental colonization and growth represent a good model to study nickel metabolism and homeostasis in nickel-dependent organisms. Due to the relative paucity of known nickel-dependent enzymes, the study of intracellular nickel homeostasis may represent a paradigm to study the general handling of dangerous and essential metal ions in vivo . In this chapter we review the present knowledge on the impact that nickel ions have on human health, focusing on the dangerous potential of nickel, leading to allergy, carcinogenesis, and infectious diseases, and the proofs of its essentiality, and on what is currently known on the molecular mechanisms underlying these points.

2 Nickel Hazard for Human Health

The most diffuse hazardous health effects caused by nickel exposure are nickel- induced carcinogenesis and allergy. They are both mediated by active changes in metabolic pathways that underlie infl ammation, stress response, oxidative stress, cell proliferation, and cell death [23 ]. As no protein specifi c for nickel homeostasis is known in mammals, one would not expect a specifi c nickel-mediated change of gene expression and metabolism. Indeed, many of the nickel effects on cells are triggered by non-specifi c interactions of nickel ions with macromolecules and general formation of reactive compounds that mediate cellular damage. Furthermore, the cellular response to nickel is related to signal transduction cascades such as second messengers, protein kinases, phosphatases or transcription factors, which are involved in general metal ion response. Notably, nickel exposure produces a rather specifi c pattern of gene expression. Nickel-driven alteration of transcription of genes involved in oxygen defi ciency has been extensively studied in vitro for its relevance to nickel carcinogenesis [23 ]. On the other hand, the activation of the infl ammatory response, with the induction of genes for chemokines and cytokines correlated with nickel-induced allergy and asthma, has been studied mostly in vivo [23 ]. Additionally, few proteins with nickel-binding motifs have been identifi ed and the effect of nickel binding to these proteins can be related to the specifi c cellular response to nickel exposure. A schematic representation of nickel uptake routes, intracellular distribution, and major effects on human cells metabolism, which will be discussed in detail in the following sections, is presented in Figure 1 . 10 Nickel and Human Health 325

Figure 1 Schematic representation of known nickel uptake routes, intracellular distribution, and its major effects on cellular metabolism in humans. ROS = reactive oxide species; HIF = hypoxia- inducible factor; HRE = hypoxia-responsive enhancer; GSH = reduced glutathione; NF-κB = nuclear factor κ-B; AP-1 = activating protein 1; TGF-β = transforming growth factor β; NF-AT = nuclear factor of activated T cells; DMT-1 = divalent cation transporter 1; NDRG-1 = N-myc downstream regulated gene 1, DAN = differential screening-selected gene aberrative in neuroblastoma.

2.1 Nickel-Induced Carcinogenesis

2.1.1 The Carcinogenic Potential of Nickel

The propensity of nickel workers to develop cancers in the respiratory tract was fi rstly reported in 1933 [24 ]. Subsequent epidemiological studies and carcinogenic assays in animals corroborated the carcinogenicity of nickel compounds, which is now generally accepted [18 ]. Epidemiological studies have reported an increased risk of lung and nasal cancers among nickel mining, smelting, and refi nery workers [ 25 ]. For many years, it was believed that only water-insoluble nickel components of fumes and dusts, like nickel sulfi des and oxides, were carcinogenic. Subsequent epidemiological studies indicated instead that aerosols of water-soluble nickel compounds, such as nickel acetate, nickel chloride and sulfate, are also carcinogenic in vivo , although with a lower potential [ 24]. Nickel present in endoprostheses, bone-fi xing plates and screws, and other implanted medical devices made of metal alloys, has been suspected, but not proven, to be the cause of sporadic local tumors. There is no epidemiological evidence on possible cancer risk from general environmental and dietary nickel exposures. 326 Zambelli and Ciurli

In animal models, nickel compounds induce tumors at virtually all sites of applications. Rats are more susceptible to nickel poisoning effects than mice, hamsters, or rabbits. The reasons for these differences may refl ect different abilities of the phagocytes to ingest and metabolize nickel-containing particles, the different mechanisms of nickel uptake, transport, distribution, and retention in the animal body, and the differences in the capacity of antioxidant systems among animals of different species and strains [23 ]. This suggests that genetic predispositions, including metabolic variations of different species and strains of animals, may also play an important role in nickel carcinogenesis. Similar genetic predispositions possibly also occur in human populations. The routes of administration that were shown to produce tumors include inhalation, intramuscular, intrarenal, intraperitoneal, intraocular, and subcutaneous applications [23 ]. The injection of crystalline Ni2 S 3 or NiS into experimental animals resulted in a high incidence of tumors in the application sites, while no tumor was induced in animals treated with soluble NiSO4 . In rats, intraocular and intramuscular injections generated the highest tumor incidences, yielding tumors in all animals, often readily metastasized to the lung and other organs [23 ]. Similarly, only water-insoluble nickel compounds were found to be carcinogenic in rats via the inhalation route, while soluble NiSO4 was not found to be carcinogenic [26 ]. Notably, intrarenal nickel injection, beside causing kidney tumors in rats, also caused erythrocytosis due to induction of erythropoietin, a part of the hypoxia- mimicking effect of nickel [23 ]. Insoluble nickel compounds are generally considered stronger carcinogens than soluble compounds. The difference of their carcinogenic activity is related to the faster clearance of soluble nickel ions from the tissues, which in turn retain insoluble nickel particles longer [5 ]. These data indicate that prolonged exposure to a nickel carcinogen is critical for tumor development. Nickel compounds also transform human cells in vitro [ 27]. Crystalline nickel particles, phagocytized by cultured cells, exist in intracellular vacuoles with internal acid pH that facilitates nickel solubilization, creating local high concentrations of soluble nickel inside the cell. Nickel-fi lled vacuoles easily migrate and reach the cellular nucleus, potentially exerting their effect on DNA and nuclear proteins. On the contrary, exposure of cells to water-soluble salts results in low nuclear and high cytosolic nickel contents [28 ]. Nickel compounds have been shown to produce a signifi cant synergistic effect, when co-administrated with other carcinogens, in enhancing cell transformation both in vitro and in vivo . On the other hand, essential metals such as manganese, magnesium, and zinc, co-administered with Ni3 S2 to animal models, reduced local tumor incidence in a dose-dependent manner. Magnesium was the strongest and zinc was the weakest inhibitor. Separate administration of the essential metals did not produce this effect [23 ].

2.1.2 Molecular Mechanisms for Nickel-Induced Neoplastic Transformation

The molecular basis of nickel carcinogenesis remains elusive, as data obtained by different groups are diffi cult to reconcile, and a general consensus on the mechanisms has still to be reached. To be defi ned carcinogenic, a compound should provoke two 10 Nickel and Human Health 327 effects fundamental for tumor genesis, that is, heritable changes in gene expression and cell proliferation. In most instances, carcinogenic compounds provide the fi rst condition by interacting with DNA and DNA-binding proteins, changing DNA structure and inducing mutations in its sequence, usually during replication. Often, these sequence changes alter the expression level or function of tumor suppressor genes or oncogenes. However, nickel does not behave like a typical mutagen, because it does not show high affi nity for DNA nor displays mutagenic potential in most assays on bacteria, fruit fl y, mammalian cells, and whole animals [29 – 33 ]. Therefore, alternative routes for nickel to change the gene expression levels and cellular phenotypes have been described. Nickel has been found to act at the DNA level, mostly through epigenetic mechanisms. Nickel promotion of tumors, on the other hand, occurs mostly at the protein level, and involves DNA-binding proteins such as transcription factors, metal-binding proteins, and proteins participating in important cellular pathways. The result is a change of the general cellular metabolism and a deregulation of cellular homeostasis inducing carcinogenic transformations in the cells.

2.1.2.1 Effects of Nickel on DNA Structure and Gene Expression

(i) Genotoxic effects. Nickel compounds have been reported to be mildly clastogenic, causing extensive DNA damage and chromosome aberrations, particularly in the heterochromatic region of the genome [5 ]. Nickel generates oxides and reactive species that produce DNA-protein cross-links and oxidative DNA damages [ 34 ]. This mechanism is typical of several transition metals that can generate reactive oxygen species in biological fl uids at physiological pH. However, this effect cannot fully explain the high carcinogenic potential of nickel: this metal is a weaker generator of redox-active species, but it is as good a carcinogen as chromium, which is very active in ROS production [35 ]. In addition, highly redox-active metals, such as copper, which also binds DNA more avidly than nickel, are only weakly or not carcinogenic [ 36 ]. The ability of carcinogenic metals to facilitate DNA damage through inhibition of DNA-repair enzymes or binding to histones can also explain its genotoxic activity [37 , 38 ]. (ii) Epigenetic effects. Further evidence from epidemiological, animal, and cellular studies shows a role of epigenetics in nickel carcinogenesis, in addition to genetic- based mechanisms. One major requirement for nickel carcinogenicity is the prolonged action on the target tissue, performed by compounds with limited solubility and long retention in biologic fl uids [24 ], which is typical of tumor promoters acting through epigenetic mechanisms, rather than tumor initiators, which are usually mutagenic. Indeed, nickel was found to synergistically increase the tumorigenic potential of several carcinogenic agents. The nickel-induced epigenetic changes include silencing of genes for DNA repair and tumor suppressors, mostly occurring through nickel-driven DNA methylation, which can modify the chromatin structure and eventually the genetic expression. In DNA of higher eukaryotes the methylation of CpG dinucleotides is an important modifi cation that leads to modulation of gene expression. In general, 328 Zambelli and Ciurli increased cytosine methylation represses transcription, and it is thus more abundant in the heterochromatin regions of chromosomes [39 ]. Exposure to nickel compounds enhances DNA methylation, leading to inactivation of gene expressions [40 ]. The nickel-induced silencing of a tumor suppressor gene, occurring through promoter hypermethylation, has been associated with cellular transformations in vivo [41 ]. Although the mechanisms by which nickel induces DNA hypermethylation are presently unknown, a possible model includes the ability of Ni2+ to substitute Mg2+ , normally bound to DNA, seeding chromatin condensation and triggering de novo DNA methylation by methylases that recognize newly generated heterochromatic and unmethylated DNA [ 40 ]. This property is related to the same charge and very similar ionic radius of Ni2+ (69 pm) and Mg2+ (71 pm). In addition to DNA methylation, nickel epigenetic effects are related to the ability of nickel to affect the global levels of histone modifi cations, implicating global deregulation of gene expression. Indeed, acetylation in the N-terminal tail of histone proteins is a well-known mechanism to regulate the chromatin transcriptional state, important to control the access of regulatory proteins to DNA. Conversely, histone methylation results in more pronounced chromatin and gene silencing. Nickel decreases the acetylation levels of histones H2A, H2B, H3 and H4, and increases H3K9 dimethylation, and the ubiquitination of H2A and H2B [42 ]. The higher methylation of the histones occurs through the nickel-induced inhibition of specifi c Fe2+ and α-ketoglutarate-dependent demethylases belonging to the family of Jmjc-domain containing histone demethylases (JHDM), such as JHMD1 and JMJD1A [ 43, 44]. The inhibited acetylation of histone H4 is thought to occur through the direct interaction of the metal ion with His18 of the histone itself, which would prevent its interaction with the specifi c acetyl-transferase enzyme [45 ]. Low levels of histone acetylation in nickel-exposed cells can also result from low levels of acetyl-CoA, a universal donor of acetyl groups, due to the nickel-dependent inhibition of pyruvate dehydrogenase kinase that converts pyruvate into acetyl-CoA [42 ]. The increase in H2A/H2B ubiquitination has been correlated to nickel up-regulation of the ubiquitin-conjugating enzyme H6 (UbcH6) E2 ligase or the inhibition of an unidentifi ed histone de-ubiqui- tinating activity [45 ]. Moreover, nickel induces the truncation, at the N-terminus of the histone H2A, and, both at N- and C-termini, of the histone H2B, possibly through the activation of specifi c nuclear proteolytic enzymes belonging to the calpain family [46 ]. The importance of deregulation of histone modifi cations by nickel has been demonstrated by observing that nickel- transformed cells, treated with the histone deacetylase inhibitor, namely trichostatin A, revert the phenotype to that of untrans- formed cells along with the gene expression profi le, suggesting that histone acetylation might be inhibited during nickel- induced transformation [47 ]. In addition to changing gene expression levels, epigenetic effects also promote cell proliferation: recent studies have demonstrated that the unlimited proliferative character of a nickel-transformed cell line could be reversed by induction of senescence. The senescence gene is located in the donor X chromosome, and its expression is regulated by DNA methylation. These data indicate that transformation by nickel may involve DNA methylation and silencing of a senescence gene in the X chromosome [48 ]. 10 Nickel and Human Health 329

2.1.2.2 Effects of Nickel on Proteins, Transcriptional Regulators, and Metabolic Pathways

(i) Disruption of calcium homeostasis. Nickel blocks calcium channels and disturbs intracellular calcium homeostasis. This results in the experimentally observed rapid proliferation of nickel-transformed cells in low-calcium media [49 ]. Since cytoplasmic calcium levels regulate expression of genes associated with cell growth, differentiation, and apoptosis, derangement of calcium regulation would impact the entire cellular metabolism [49 ]. In particular, nickel was shown to increase intracellular calcium levels: nickel likely uses calcium channels to enter the cells, and induces calcium release from intracellular stores, possibly through a cell surface receptor. Therefore, changes of calcium homeostasis invoked by nickel exposure may change the cellular expression induced by other signalling pathways, eventually leading to malignant transformation [23 ]. (ii) Oxidative damage. Soluble and insoluble nickel compounds can be redox-active at physiological pH, although to a lesser extent than iron and copper complexes, and they can generate reactive oxygen species. This is possible when the redox couple Ni 3+ /Ni2+ is formed, which usually only occurs when nickel is bound by certain natural ligands like peptides and proteins, especially those forming square planar nickel complexes. Reactions of such complexes with O 2 or H 2 O2 yield the hydroxyl radical · OH and other radicals. The oxidation of water-insoluble nickel sulfi des may involve both nickel and sulfur and lead to generation of not only nickel-, but also sulfur-derived ROS and other reactive intermediates (e.g. the sulfi te anion). This enables the sulfi des to produce a wider spectrum of oxidative damage than other nickel compounds and may be responsible for their high carcinogenic activity. In addition, nickel can deplete some important antioxidant ligands, such as ascorbate and reduced glutathione (GSH). Nickel is capable of depleting intracellular ascorbate through catalytic oxidation and hydrolysis of both ascorbic and dehydroascorbic acid, and inhibition of ascorbic acid transporters [23 ]. GSH depletion is likely the result of a cellular response to the ROS species generated by nickel. Coherently, resistance to nickel toxicity is usually associated to high levels of GSH [50 ]. Finally, the enzymatic components of cellular antioxidant defence, such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, are also affected by nickel exposition [23 ]. The nickel-induced oxidative stress can activate some transcriptional pathways through some oxidation-sensitive transcription factors. ROS created by nickel exposition result in lipid peroxidation, whose products can create adducts with DNA, thus altering gene expression. Protein oxidation, leading to protein fragmentations and cross-linking with other molecules (e.g., with DNA) and oxidative DNA and chromatin damage are also consequences of ROS generated by nickel. The presence of cross-links in chromatin may lead to morphologic aberrations of chromosomes.

In vitro, nickel was found to promote DNA cleavage by H2 O2 predominantly at the cytosine, thymine, and guanine bases [51 ]. ROS attack on DNA’s sugar moiety produces apurinic sites in DNA and mediates in vitro hydrolysis of 2′-deoxyguanosine. The depurination occurs concurrently with DNA strand scission and fragmentation. 330 Zambelli and Ciurli

Some compounds generated by oxidative stress, like 8-oxoguanine, may also misdirect DNA methylation and perturb orderly binding of transcription factors to DNA. (iii) Activation of hypoxia signalling . Nickel exposure produces a rather specifi c pattern of gene expression, which involves the same activation pathways of the response to hypoxia [52 ], and in particular the activation of the HIF-1 transcription factor. This protein exists as a HIF-1α/HIF-1β (ARNT) hetero-dimer, with the α subunit being the regulatory unit, formed in response to low oxygen tension in the cells. Under normal oxygen concentrations, HIF-1α is virtually undetectable in most cells [ 53]. In these conditions, the protein interacts with the tumor suppressor protein VHL, a part of the ubiquitin-ligase complex that induces the ubiquitination of HIF-1α and its rapid degradation. The structural basis for specifi c interaction of HIF-1α and VHL is provided by the introduction of the hydroxyl group at the C4 position of Pro402 and Pro564 [54 ], which facilitates hydrogen bonding with Ser111 and His115 in VHL. On the other hand, hydroxylation of Asp803 prevents complex formation between HIF-1α and the transcriptional co-activators CBP and P300, providing a second mechanism by which HIF-mediated transcription is regulated [55 ]. The hydroxylation reactions are carried out by specifi c hydroxylases that employ both Fe 2+ and ascorbate as cofactors to split dioxygen into two oxygen atoms, one of them converted into hydroxide. Ascorbate is a reducing agent needed to avoid iron oxidation, and to maintain the metal ion bound to the enzyme as Fe 2+ . Hypoxia signalling reduces the HIF-1α hydroxylation, therefore stabilizing HIF-1α and allowing it to join HIF-1α. The heterodimer translocates into the nucleus, where it binds the hypoxia-responsive enhancers (HREs) and recruits the co-activator acetyltransferase P300 [ 56 ]. Similarly to hypoxia, nickel was found to be a strong stabilizer of the HIF-1α protein and an activator of HIF-dependent transcription, inhibiting its enzymatic hydroxylation [56 ]. This likely occurs through a depletion of intracellular ascorbate that follows nickel-driven oxidation and/or uptake inhibition [ 57 ]. This results in the inactivation of the hydroxylases, followed by the induction of HIF-1 and activation expression of hypoxia-inducible genes [56 ]. The activation of the hypoxic signalling pathway and the switch of cellular metabolism to a state that mimics permanent hypoxia may be a part of nickel-induced carcinogenesis [42 ]. Indeed, hypoxia is a common state in tumors because transformed cells grow faster than the blood vessels providing them with oxygen. This state can activate genes that enable cells to overcome nutritive deprivation, to escape from the hostile metabolic microenvironment, and to stimulate angiogenesis. Additionally, cellular responses to hypoxic stress include inhibition of cell proliferation and, when cell damage is irreversible, apoptosis. Therefore, imitation of the state of hypoxia by nickel may provide the conditions for the selection of cells that have altered energy metabolism, changed growth control and/or have become resistant to apoptosis. A result of nickel-induced hypoxia response is the induction of numerous genes involved in glucose transport and glycolysis, coding for carbonic anhydrase IX, ceruloplasmin, erythropoietin, inducible nitric oxide synthase, vascular endothelial growth factor (VEGF), and many others [23 ]. 10 Nickel and Human Health 331

(iv) Additional alterations of other signalling pathways. Exposure of cells to nickel also induces a change of gene expression that yields cells with spectra of expressed genes similar to cancer cells. The proteins responsible for this effect are some transcription factors, involved in different metabolic pathways, such as oxidative stress defence (NF-κB, AP-1), infl ammatory response (NF-κB, TGF-β), apoptosis (p53), angiogenesis (ATF-1), calcium homeostasis (NF-AT), all activated by nickel [23 ]. Nickel also induces activation of the K-ras oncogene, and inhibits tumor suppressor genes, such as Rb, p16, FHIT, Zac-1, and Gas-1 [23 ]. (v) Interaction with proteins. The gene coding for the Cap43 protein (also called N-myc downstream-regulated gene 1 (NDRG1)) is induced by nickel through the hypoxia pathway [58 ]. This protein is usually expressed at low levels in normal tissues, but it is over-expressed in a variety of cancers, including lung, brain, melanoma, liver, prostate, breast and renal cancers, and has been often used as a marker of tumor progression [59 ]. The physiological function of this protein is not clear so far, but likely it acts as a tumor suppressor protein. The fact that this protein is ubiquitously expressed and highly conserved in all multicellular organisms, and that its expression is regulated by different central pathways that respond to several stress and growth signals, such as cellular proliferation, differentiation, growth arrest, neoplasia, tumor progression and metastasis, hypoxia, heavy metal response, favors a central role in cellular metabolism [60 ]. Impairing of calcium homeostasis, also triggered by nickel, induces Cap43/NDRG1 production. Interestingly, three repeats of the GTRSRSHTSE sequence are found in the C-terminal tail of Cap43/NDRG1, which are not present in the other family members, and have been postulated to serve as nickel binding motifs, and can bind one nickel ion each [61 , 62]. It is noteworthy that these repeats fall into a region that in all NDRG family members is predicted to be unfolded [63 ], therefore possibly involved in regulation through interaction with a partner or cofactor such as nickel. The fact that the 30-amino acid fragment of the C-terminal tail of the nickel-induced Cap43/NDRG1 is able to bind up to three nickel ions could shed new light onto the complex mechanism of nickel toxicity, in particular in relation to the physiological role exerted by the stress protein genes up-regulated by metal exposure, suggesting a possible role of Cap43/NDRG1 as a detoxifi cation agent and a feedback mode of nickel sensing. The crystal structure of a homologue, NDRG2, only expressed in adult skeletal muscle and brain, has been recently reported, and the protein was thought to serve as molecular interactor for regulating cell proliferation and cell differentiation [64 ]. Beside Cap43/NDRG1 protein and histones, nickel has been reported to form complexes with other proteins, possibly signifi cantly altering their conformations, interactions, functionality, and eventually cellular homeostasis, triggering the observed adverse effects. At physiological pH, the strength of Ni2+ interactions with proteins depends on the type of amino acid residues, their positions relative to each other, and their accessibility in the protein molecule. The greatest affi nity for Ni2+ is shown by histidine and cysteine residues in proteins. One of these proteins, named DAN, possessing a Ni2+ binding motif at the C-terminal region, shows a tumor suppressor activity. Interaction of DAN with Ni2+ can impair its activity triggering the 332 Zambelli and Ciurli tumorigenic phenotype [65 ]. Similarly, cullin-2, a component of the complex that serves HIF-1α ubiquitination, features three Ni 2+ and Co 2+ binding sites [ 66 ]. Metal binding prevents ubiquitination, therefore contributing to the typical hypoxia response. Nickel can also bind the iron regulatory protein 1 (IRP-1), a central regulator of iron homeostasis [67 ]. Replacement of one Fe2+ with Ni2+ in the 4Fe-4S cluster inactivates the protein enzymatic activity and may contribute to the nickel- induced hypoxic signalling. The ability of Ni2+ and Cu 2+ to bind neuromedin C, a neuropeptide, may represent the overlap between the metabolisms of these two metal ions, and may imply that Ni2+ is able to impair copper homeostasis in the brain [68 ].

2.2 The Different Faces of Nickel Allergy

2.2.1 Nickel Effects on Immune Response

The immune response triggered by nickel exposure is generally important, and responsible of allergic contact dermatitis (ACD), one of the most signifi cant consequence of occupational exposure to nickel. The fi rst event in nickel immunological response is a silent sensitization phase, which is initiated upon the fi rst contact with the antigen, leading to the generation of allergen-specifi c T-cells, and lymphocyte activation. Upon re-exposure to the allergen, an elicitation phase occurs, resulting in clinically apparent infl ammation. In this second phase, the T-cells exert cytotoxic functions and secrete infl ammatory mediators, such as chemokines and cytokines, to amplify the infl ammatory response and produce eczematous skin reaction [69 ]. As many ACDs, nickel allergy requires two events for being established both for sensitization and elicitation, that is (i) the activation of antigen-specifi c T-cells, and (ii) a non-specific proinflammatory microenvironment necessary for the development of a hypersensitivity response, also called innate immune signal [70 ]. In humans, nickel ions can trigger both these events, directly activating proinfl ammatory pathways [70 ].

2.2.1.1 How Nickel Activates Antigen-Speciﬁ c T-Cells

T-cells are the major effectors in Ni2+ hypersensitivity. These cells are usually activated when a peptide, recognized as non-self, is bound to a major histocompatibility complex (MHC) protein of the membrane of an antigen-presenting cell (APC), and interacts with a T-cell receptor (TCR) [71 ]. This is the signal that activates the lymphocytes and subsequently the immune cascade. Ni2+ ions, as many other immuno- logically active low molecular chemicals, are defi ned as haptens, that is antigens generally invisible to the immune system by themselves, becoming visible only when bound to proteins or peptides [ 72]. While, generally, hapten recognition by T-cells requires covalent hapten attachment to MHC-associated peptides, transition metal ions such as Ni2+ do not form stable covalent protein modifi cations, they 10 Nickel and Human Health 333 rather produce geometrically highly defi ned coordination complexes with reversible binding [73 ]. During the immune response, Ni2+ has been found to be reversibly bound to a particular MHC-II molecule, called HLA-DR52c, and an unknown MHC-bound peptide via a pH-sensitive interaction, consistent with metal ion coordination to amino acid side chains of histidine or acidic residues [ 74 ]. The structure of HLA- DR52c in complex with a self-peptide deriving from the Tu elongation factor (pTu), has been reported [75 ]. Although the HLA-DR52c/pTu complex is not able to bind Ni2+ , this structure is important to understand how Ni2+ can be presented in this complex. In particular, four residues (His81 , Asp55 , Gln70 , and Gln74 ) have been indicated as being potentially involved in metal coordination. However, these are not suffi cient to allow Ni2+ binding, coherently with the observation that a specifi c peptide is needed to form the Ni/HLA-DR52c/peptide complex [75 ]. How the metal ion infl uences TCR binding to the Ni/HLA-DR52c/peptide complex is still unknown. It has been proposed that the metal ion binds to the interface between MHC and TCR proteins, sterically infl uencing the interaction [76 ]. Based on current biochemical data, hypothetically the metal ion can bind the MHC and become the ligand of T-cells at least in four different ways [77 ]: (a) Ni2+ could bind the MHC only; (b) Ni 2+ can bind both the presented peptide and the MHC molecule; (c) Ni 2+ can interact with the presented peptide only; (d) Ni 2+ can induce a conformational change of the peptide and/or of the MHC, which are therefore recognized as neo- antigens. A further possibility is that Ni 2+ ions interfere with the processing of self-proteins in the APC and the exposure of cryptic self-peptides, which can be recognized as non- self by TCR [78 ]. Besides activating T-cells in a Ni/MHC/peptide ternary complex, Ni2+ appears to serve as a direct and peptide-independent linker between TCR and MHC, bearing certain similarities to superantigen-mediated activation [79 ]. Potential Ni2+ contacts both in the TCR and in the specifi c MHC molecule are responsible for Ni2+ reactivity independently from protein processing by antigen-presenting cells and peptide binding to MHC. This new type of Ni 2+ -induced TCR-MHC crosslinking might explain the high frequency of Ni-reactive T cells in the human population [79 ].

2.2.1.2 How Nickel Activates the Innate Immune Signal

Ni2+ is the only allergen for which a direct mechanism of innate immune activation has conclusively been demonstrated and unravelled at the molecular level. In vitro and in vivo experiments showed that treatment of human endothelial cells with Ni2+ triggers rapid expression of the surface molecules VCAM1, ICAM1, and E-selectin and monocyte-attracting chemokine MCP-1 [80 –82 ], required for leukocyte adhesion and infl ammation. On the other hand, expression of lymphocyte-attracting cytokines such as IP10, Mig, MDC, PARC, and TARC, occurs at later stages and correlates well with the infi ltration of T-cells into the dermis and epidermis [82 ]. Molecular analysis revealed that, in humans, Ni 2+ activates the IKK2/NF-κB and the MAPK/p38 signalling pathways, both regulating gene expression leading to 334 Zambelli and Ciurli infl ammation [83 , 84 ]. These activations occur through Ni2+ interaction with the membrane toll-like receptor 4 (TLR4), in combination with its co-receptor MD2, which induces receptor dimerization probably bridging two TLR4 monomers. Coherently, human cells expressing TLR4 and MD2 including macrophages, fi broblasts, and dendritic cells were able to induce a proinfl ammatory response upon Ni2+ treatment [ 85 ]. The non-conserved His456 and His458 residues in human TLR4 are critically required for Ni2+ -induced proinfl ammatory gene expression, possibly acting as specifi c ligands for the metal ion [85 ]. Notably, mouse TLR4, which lacks these histidine residues, is not able to produce the proinfl ammatory pathway in response to Ni 2+ exposition. Activation of innate immunity in mice rather needs co-stimulating adjuvants, such as the bacterial cell wall component lipopolysaccharide (LPS). Another pathway responsible for Ni2+ -induced ACD is the death receptor- mediated or extrinsic apoptosis pathway. It was reported that Ni 2+ transcriptionally represses expression of cFLIP, a cellular antagonist of the pro-apoptotic caspase-8, in both primary human keratinocytes and endothelial cells [86 ], which, as a result, are strongly sensitized to apoptosis. Coherently, there is evidence for an increased occurrence of death ligand-mediated keratinocyte apoptosis in the course of Ni 2+ - induced ACD in sensitized individuals [87 ]. Enhanced cell death of keratinocytes is predicted to impair the barrier function of the skin. Hence, Ni2+ -mediated cFLIP down-regulation might tip the balance towards increased apoptosis of certain skin cell populations, which successively may augment the severity of the ACD response during the elicitation phase by increasing the effi cient concentration of Ni2+ arriving in the epidermis [70 ]. In addition, nickel is able to induce apoptosis in a number of immune cells, including human neutrophils and T-cells, through the mitochondrial pathway that activates caspase-3, likely as a response to Ni 2+ -induced oxidative stress [12 ,13 ]. The production of ROS by Ni 2+ exposition also acts in concert with the mechanisms described above to produce and amplify the infl ammatory response. In particular, ROS act as signalling molecules and are recognized as important inducers of the proinfl ammatory response [69 ].

2.2.2 The Impact of Nickel-Induced Dermatitis

The immunological effects of nickel described above are responsible for allergic contact dermatitis (ACD), which is the most spread dermatitis over the world and is constantly increasing, reaching 20% of the human population. It was discovered for the fi rst time in 1930 [ 88]. It is caused by Ni 2+ ions solubilized from nickel- containing alloys by sweat and other body fl uids that serve as sensitizing allergen and come in contact with skin. Although the risk of occupational disability is an issue for a relatively large group of professionals, such as metal workers, cashiers, or hairdressers, the major problem associated with Ni2+ -induced contact hypersensitivity is the wide presence of this metal ion in modern industrial products, so that it is very common to come in contact with the allergen. Ni 2+ is released from coins, earrings, watches, belt buckles, bras, mobile phones, dental and orthopedic 10 Nickel and Human Health 335 implants, and cardiovascular stents [70 ]. In Europe, currently around 65 million individuals are sensitized to Ni2+ [ 89 ]. Legislative interventions limiting the amount of Ni2+ release from products intended for prolonged skin contact, such as the Danish nickel regulation in 1990 [90 ] and the EU directive 94/27/EG in 2001 (also known as “Nickel Directive”), result in decreasing sensitization rates, but prevalence of contact eczema to Ni2+ are still considerably high [ 91]. In its classic description, nickel ACD involves the hands and forearms mainly after occupational exposure. Sensitized individuals generally experience a predictable localized response following cutaneous exposure to nickel, including erythema, vesicle formation, scaling, and pruritus [ 92]. A nickel-free diet remains controversial in the treatment of ACD. In addition, inhaled nickel in ambient air might be a risk factor for nickel sensitization [ 93 ]. The most important way of preventing nickel allergy is the avoidance of exposure. Rubber gloves are ineffi cient for prevention of nickel contact, as metal ions penetrate through them. Polyurethane coating of metal items can be benefi cial in protection from nickel contact, as are barrier creams containing sodium EDTA [ 94 ]. Nickel is present in most dietary items, and food is considered to be a major source of exposure to nickel for the general population. Certain foods, such as green beans, broccoli, peas, canned vegetables and spaghetti, canned fruit, dried fruit, nuts, cocoa, and chocolate are routinely found to be high in nickel content. Nickel present in the diet of a nickel-sensitive person can provoke systemic reactions, called systemic contact dermatitis (SCD). SCD is manifested as generalized eczematous reactions, maculopapular rash and vasculitis-like skin, along with systemic symptoms such as headache, malaise, diarrhea, fever, and arthralgia [95 ]. The exact mechanisms of systemic contact dermatitis are still not known. The eczema fl are at sites of previous exposure suggests T cells resting at the area or homing the site upon systemic allergen exposure [96 ]. Circulating immune complexes together with non-specifi c cytokine release by the hapten stimulation can be responsible for the generalized reactions [95 , 96 ]. In patients with SCD, adherence to a low-nickel diet and avoidance of local exposure to metal objects result in the disappearance of skin symptoms [92 ]. Another therapeutic approach, studied for both ACD and SCD, is nickel hypo- sensitization, that is the procedure allowing sensitized patients affected by ACD to safely make contact with nickel containing objects, and those with SCD to freely eat nickel-containing foods. This aim is pursued through oral administration of cap- sules containing nickel to ACD and SCD patients. For ACD, the clinical studies, performed with oral supplementation of high nickel doses, are still preliminary, as they have been conducted with a limited number of subjects and for a limited period of treatment, but have given promising results [97 ]. In the case of SCD, the studies of oral hypo-sensitization with very low doses of nickel for a prolonged time led to an improvement of the symptoms and a complete remission in 50% of the patients. No side effects are observed during and after the treatment. The hypo-sensitization treatment exerts its clinical effects through a modulation of the allergic immune reaction specifi c for nickel [98 ]. 336 Zambelli and Ciurli

3 Nickel-Dependent Infectious Diseases

Nickel in the active site of several enzymes is responsible for the catalysis of important biological reactions, such as urea hydrolysis, hydrogen metabolism, methane formation, CO/CO2 inter-conversion, superoxide metabolism, and detoxiﬁ cation of methylglyoxal [99 ]. For many bacteria, archaea and unicellular eukaryotes these nickel-dependent processes render possible the colonization of environments that are inhospitable and hostile. These include parts of human and animal bodies, where these nickel-obligate pathogens grow and survive in virtue of nickel-catalyzed reactions. Consistently, some nickel-dependent enzymes are virulence factors for pathogenic organisms. As no nickel-dependent enzyme is known in vertebrates, catalyzed nickel-dependent processes and mechanisms of nickel delivery into speciﬁ c enzymes can be regarded and employed as possible selective targets to control the pathogenesis of nickel-obligate organisms.

3.1 Nickel-Dependent Enzymes in Pathogenic Microorganisms

The biological role of nickel is essentially defi ned by its use as a cofactor of enzymes found in all phyla of life (plants, fungi, eubacteria, and archaea). All known nickel enzymes involve the transformation and/or production of gases (ammonia, carbon monoxide, carbon dioxide, methane, dihydrogen, and dioxygen) involved in the geo-biological cycles of carbon, nitrogen, and oxygen. The nickel ion in the active sites of these enzymes exhibits a very large fl exibility in metal coordination and redox chemistry, spanning coordination numbers from 4 to 6 and oxidation states from +1 to +3, with potentials ranging from +900 to −600 mV [100 ]. In these enzymes, nickel often is inserted in a multinuclear metal cluster that also includes modifi ed amino acids and/or exogenous ligands [20 ]. The importance of nickel enzymes for human health is related to the fact that, among eight known nickel-dependent enzymes, four (glyoxalase I, acireductone dioxygenase, hydrogenase, and urease) are present in pathogenic microorganisms and are often essential for their growth and pathogenesis. For example, infections by urease-dependent organisms represent a widespread source of diseases, ranging from tuberculosis to urinary tract infections, hepatic coma, and kidney stones [101 ]. The glyoxalase enzyme of the protozoan parasites has been regarded as a potential chemotherapeutic target against eukaryotic pathogens, such as Trypanosoma or Leishmania [102 ].

3.1.1 Glyoxalase I

Glyoxalase (Glx) I and GlxII catalyze the conversion of methylglyoxal, a toxic species that forms covalent adducts with DNA, to lactate (Figure 2 ) [ 103 ]. For a long time, all GlxI enzymes were believed to be Zn2+ enzymes, like the human GlxI. 10 Nickel and Human Health 337

Figure 2 Reaction catalyzed by GlxI and GlxII, and schematic structure of the nickel-containing active site.

However, it was later shown that some GlxI enzymes are more active with Ni2+ in vitro . These include the GlxI from some bacteria, such as the pathogen Pseudomonas aeruginosa, and trypanosomatids, responsible for several diseases, such as Chagas’ disease and skin sores called leishmaniasis. It is not clear whether this metal ion is functional for GlxI in vivo [ 104 ]. A single Ni2+ ion in an octahedral coordination environment composed by 2 His, 2 Glu, and two water molecules, acts as a Lewis acid catalyst and remains in the 2+ oxidation state throughout the isomerization reaction (Figure 2 ). On the other hand, the inactive Zn 2+ enzyme is ﬁ ve-coordinated [103 ].

3.1.2 Acireductone Dioxygenase

Acireductone dioxygenase (ARD) catalyzes the incorporation of two oxygen atoms into the substrate acireductone, the penultimate intermediate of the methionine salvage pathway (Figure 3 ) [105 ,106 ]. The latter is a ubiquitous process that regulates methionine levels, thus playing an essential role in several biosynthetic processes. In Klebsiella ATCC 8724, this enzyme is a monomeric protein, showing a classic jellyroll structural motif. The active site, relatively well exposed to the solvent, contains Ni2+ in an octahedral high-spin coordination center, involving three His, one Asp, and two water molecules in equatorial positions (Figure 3 ). The Ni 2+ -bound enzyme catalyzes the so-called “off-pathway” reaction, leading to the formation of relatively large amounts of carbon monoxide. Peculiarly, this enzyme can also bind Fe2+ in the same coordination site. Fe 2+ binding stabilizes a different protein form that catalyzes the “on-pathway” reaction, leading to production of methionine. The Ni2+ - and Fe2+ -bound forms differ constitutionally only in the identity of the metal ion bound. Interestingly, this difference is sufﬁ cient to induce signiﬁ cant structural 338 Zambelli and Ciurli

Figure 3 Reaction catalyzed by acireductone dioxygenase, and schematic structure of the nickel- containing active site. changes resulting from differential packing of a compact hydrophobic core of the protein, accompanied by extensive changes in secondary structure and ordering of nearby structural features [107 ].

3.1.3 Urease

Urease is key to the global nitrogen cycle as it catalyzes the hydrolysis of urea, produced by vertebrates, to ammonia and carbamate, which then spontaneously decomposes to give a second molecule of ammonia and bicarbonate (Figure 4 ) [108 ]. The subsequent hydrolysis of the reaction products induces an overall pH increase that has negative implications both in human and animal health, as it is used by several pathogens, such as Helicobacter pylori , to colonize hostile acid habitats. In addition, this reaction represents a nitrogen source for several organisms that infect humans. Therefore, urease is a virulence factor for pathogens in the animal gut, urinary tract, and stomach. Microbial ureases are generally heteropolymeric proteins with a quaternary structure (αβγ)3 . In some bacteria, such as those of the genus Helicobacter , the trimer is of the type (αβ) 3 , with the β subunit corresponding to the fused β and γ subunits normally found in other bacteria, and presents a higher level of oligomerization that leads to an enzyme with a quaternary structure ((αβ)3 )4 . On the other hand, plant ureases are hexameric proteins α 6 , each α subunit being highly homologous to each (αβγ) assembly of microbial ureases [109 ]. Several structures of ureases are available. In all cases, the active site contains two Ni2+ ions bridged by the carboxylate group of a carbamylated lysine and by a hydroxide ion (Figure 4 ). Each Ni is also coordinated by two histidines and one water molecule, whereas Ni(2) is further bound to an aspartate. This results in a penta-coordinate Ni(1) and hexa-coordinate Ni(2). In the resting state of the enzyme from Bacillus pasteurii, the active site accommodates a fourth water molecule, completing a tetrahedral cluster of solvent molecules [110 ]. The access to the active site is regulated by a ﬂ exible helix-loop-helix segment, with the position of amino acids involved in the catalysis being critically affected by the ﬂ ap movement [111 ]. 10 Nickel and Human Health 339

Figure 4 Reaction catalyzed by urease, and schematic structure of the nickel- containing active site.

The structures of B. pasteurii urease (BPU), in the native hydrated form and complexed with several inhibitors of different chemical classes, suggest a structure- based reaction mechanism for urease, and highlight the importance of both metal ions in the reactivity of the enzyme [109 ]. The structures of BPU bound with borate [112 ], a substrate analogue, and with diamidophosphate [110 ], a transition state analogue, are particularly signiﬁ cant to understand the reaction mechanism [ 109 , 113 ].

3.1.4 [NiFe]-Hydrogenase

Hydrogenase enzymes catalyze the formation of hydrogen gas from protons and electrons and/or the reverse reaction, oxidizing H 2 as a source of reducing power, and coupling it to the reduction of various terminal electron acceptors (e.g., O 2 , − 2 − NO3 , SO4 , CO2 , and fumarate), in energy-conserving pathways (Figure 5 ). They can act as sensors for the availability of hydrogen gas [114 ]. The [NiFe]-hydrogenases are a class of hydrogenase enzymes that contain nickel and iron in the active site and are widely distributed in bacteria and archaea, including some pathogens [115 ]. A [NiFe]-hydrogenase is required for efﬁ cient colonization by H. pylori and Salmonella enterica serovar Typhimurium, a food poison, Campylobacter jejuni , a bacterium closely related to Helicobacter , as well as many 340 Zambelli and Ciurli

Figure 5 Reaction catalyzed by [NiFe]-hydrogenases, and schematic structure of the nickel-containing active site.

enteric bacteria (e.g., E. coli , Shigella , and Yersinia species) [20 ,116 ]. These infectious organisms use the hydrogen produced by other microorganisms in the body, a resource not used by the human host, as a supply of energy for their respiratory pathway. This permits the growth and maintains effi cient virulence during animal infections [116 ]. [NiFe]-hydrogenases are usually composed of two subunits. The smaller β subunit contains three closely spaced FeS clusters (two [4Fe-4S] and one [3Fe-4S]), linearly arranged, that transfer electrons to and from the active site. The active site is constituted by a hetero-nuclear bimetallic [NiFe] center, buried at the bottom of a hydrophobic channel in the larger α subunit. The nickel-containing hydrogenases can be classifi ed in two different categories, differing by ligation of the [NiFe] cluster found in the active site to either cysteine or seleno-cysteine. In the oxidized form of the enzyme, the Ni and Fe atoms are bridged by two cysteines and a third, non-protein ligand, possibly a sulfi de or an oxide (Figure 5 ). The Ni atom is bound to two additional cysteines, forming the equatorial plane of a distorted square pyramidal coordination geometry with the O/S bridging atom being the fi fth axial ligand. In [NiFeSe]-hydrogenase, selenium, in the form of selenocysteine, is a ligand to Ni. The Fe atom resides in a pseudo-octahedral coordination geometry, additionally bound to CO, and two CN– . In the structure of the reduced enzyme, the non-protein bridging ligand in the bimetallic center is absent (Figure 5 ), leading to the conclusion that activation of

[NiFe]-hydrogenases by H2 involves the removal of such a bridge, thereby causing the opening of binding sites on both Ni and Fe atoms. The subsequent steps of the catalytic mechanism have not been fully elucidated to date, but they likely include nickel, iron, or cysteine thiolates as potential substrate-binding sites and redox centers [ 115 ].

3.2 Molecular Regulation of Nickel Homeostasis in Pathogenic Microorganisms

The essentiality of nickel for nickel-dependent organisms, together with the environmental scarcity of this metal ion, led nickel-obligate pathogens to evolve a tight system for nickel homeostasis with mechanisms that correlate Ni2+ availability with 10 Nickel and Human Health 341

Figure 6 Schematic representation of bacterial Ni2+ homeostasis, intracellular transport, and utilization. ABC = ATP-binding cassette; NiCoT = nickel cobalt transporter; RcnA = resistance to cobalt and nickel A; CznA = cadmium-zinc-nickel resistance A; MCR = methyl coenzyme-M reductase; ADR = acireductone dioxygenase; GlxI = glyoxalase I. the expression of proteins involved in nickel transport and utilization. In particular, attaining suffi ciently high intracellular nickel concentrations to meet the demand of the nickel enzymes and, at the same time, protecting the cell by the poisoning potential of nickel excess, requires (i) an effi cient nickel uptake/effl ux system, ii) molecular chaperones and metallo-chaperones, and (iii) nickel sensors (Figure 6 ).

3.2.1 Nickel Membrane Transporters

Ni2+ ions should enter into the cytoplasm in order to be inserted into the active site of nickel-dependent enzymes. On the other hand, nickel excess must be extruded from the intracellular location, in order to prevent nickel-related danger at toxic metal concentrations. Therefore, the metal ions must be able to pass through the cytoplasmic membrane entering or exiting the cell according to the intracellular nickel abundance. Low levels of nickel can travel through the plasma membrane by way of multiple, possibly nonspecifi c, systems. However, in many of the microorganisms that require this metal ion, dedicated nickel uptake transporters have been 342 Zambelli and Ciurli identifi ed that belong mainly to two different classes, the NikABCDE import pumps and the nickel/cobalt permeases (NiCoT). The former, fi rstly characterized in E. coli, belongs to the family of ATP-binding cassette (ABC) transporters, and couples the translocation of a substrate to the hydrolysis of ATP. NikB and NikC are transmembrane proteins that form a nickel pore. NikD and NikE are proteins that bind to and hydrolyse ATP [117 ]. NikA is a periplasmic protein that binds one nickel per protein in the context of a nickelophore. The nature of this complex is controversial, but recent structural data have identifi ed two free histidines coordi- 2+ 2+ nating Ni in the metal binding site of NikA, suggesting that Ni -(L-His)2 is the nickelophore recognized by the periplasmic transporter [118 ]. This transport system has been found in several pathogenic organisms, such as Brucella suis , Vibrio parahemolyticus , Helicobacter hepaticus , Yersinia sp., and Staphylococcus aureus [119 ]. The NiCoT family is composed by monomeric single component permeases with eight transmembrane helices, found in many bacteria, such as the pathogen H. pylori , as well as several archaea and fungi. While some family members are specifi c for Ni 2+ , other permeases transport both Ni2+ and Co2+ , with the preference of one metal over the other [20 ]. In gram-negative bacteria, some nickel-specifi c importers have been found in the outer membrane. In particular, in H. pylori FecA3 and FrpB4 have been suggested to be involved in TonB-dependent transport of nickel [20 ]. The most common mechanism for metal resistance is metal effl ux, made by exporters that, in several organisms, have been predicted and/or demonstrated to pump nickel out of the cytoplasm and of the periplasm. Only in a few cases the transporters are specifi c for nickel [120 ]. In particular, nickel exporters have been identifi ed for H. pylori and E. coli . In H. pylori , CznABC is proposed to be a novel system that pumps Cd2+ , Zn2+ , and Ni2+ across both the inner and outer membranes [ 121]. In E. coli, RcnA is a membrane transporter with six trans-membrane segments, with only limited homology with other transporters families, involved in specifi c export of Ni2+ and Co2+ [122 ].

3.2.2 Nickel Molecular Chaperones and Metallo-Chaperones

Nickel activation pathways imply nickel delivery into the buried cavity of speciﬁ c enzymes, usually synthesized as apo-proteins undergoing activation through metal ion incorporation in a post-translational regulation mechanism of the enzymatic activity. Some of these pathways, usually involving a multistep, tightly regulated mechanism with several accessory proteins, have been extensively studied in bacteria and in some cases they even overlap (e.g., hydrogenase and urease pathways) [ 99]. The functional, biochemical and structural properties of these chaperones have been investigated in the case of urease (UreDEFG proteins), hydrogenase (HypABCDEF and SlyD), carbon monoxide dehydrogenase (CooCTJ), acetyl-CoA decarbonylase synthase (AcsF), and superoxide dismutase (SodX and CbiXhp) [99 ]. No accessory protein is known for acireductone dioxygenase and for glyoxalase so far. 10 Nickel and Human Health 343

Even though many aspects of these processes remain largely obscure, some general aspects of the roles performed by the accessory proteins are maintained throughout the investigated systems. In particular, (i) nickel storage, (ii) nickel delivery, and (iii) nucleotide triphosphate hydrolysis have been found in several nickel-driven enzyme activations. These three functions are carried out by specifi c protein domains, organized in a modular fashion, either in a single protein or in separated chaperones. The nickel storage role is normally carried out by a His-rich pattern: this motif may constitute a whole protein, such as in the case of Hpn or HspA, two nickel accumulators found in Helicobacter pylori [123 – 125], or may be located at the C- or at the N-terminus of a specifi c chaperone, as in the case of several UreE [126 ] and of UreG from Mycobacterium tuberculosis [ 127 ], Streptomyces coelicolor and Glycine max [ 128 ] for urease, HypB from Bradyrhizobium japonicum [129 ] and SlyD from Escherichia coli [ 130] for hydrogenase, and CooJ from Rhodospirillum rubrum [ 131 ] for CO dehydrogenase. On the other hand, the nickel delivery function is performed using nickel binding sites on specifi c metallo-chaperones, such as UreE for urease [109 , 132 – 134 ], HypA, and possibly HypB, for hydrogenase [20 ], and CooJ for carbon monoxide dehydrogenase [131 ]. Finally, hydrolysis of nucleotide triphosphates is required to complete the biosynthesis of the enzyme with a reaction catalyzed by homologous P-loop GTPases: HypB intervenes in the activation of both hydrogenase and urease [ 135 ], AcsF plays its function in the activation of acetyl-CoA decarbonylase synthase [136 ], CooC is involved in the assembly of carbon monoxide dehydrogenase [ 137 ], while UreG is the GTPase essential for urease assembly [109 ]. UreG from many organisms, including the pathogenic bacteria M. tuberculosis and H. pylori, has been reported to be an intrinsically disordered protein, existing in a fl exible behavior in vitro in the absence of other protein partners [127 , 138 – 141 ]. This observation reveals that, at least for urease, protein disorder plays a role in the protein-protein interaction network leading to enzyme activation, and represents a further post-translational mechanism to regulate the Ni2+ -dependent enzymatic activity. Furthermore, intrinsic disorder has been found also in some portions of other urease proteins, such as in the C-terminal sequence of UreE involved in the protein interaction with metal ions [134 , 142 ], which in turn regulates UreE interaction with UreG [142 ], and in the C-terminal sequence of UreF, which is disordered in the free form of the protein and becomes correctly folded upon UreF-UreD interaction [ 143 ].

3.2.3 Nickel Sensors

The crucial players of nickel homeostasis networks are specifi c nickel-responsive transcriptional regulators, which specifi cally bind promoters of genes involved in Ni2+ homeostasis, such as nickel importers or effl ux pumps, nickel storage proteins, nickel chaperones and nickel-dependent enzymes. These proteins couple specifi c metal ion binding with a change in their DNA binding affi nity and/or specifi city, 344 Zambelli and Ciurli thus translating the concentration of a certain metal ion into a change in transcriptional response. Several nickel sensors, belonging to different families of regulators, have been discovered and characterized, including NikR (NikR family), RcnR (RcnR/ CsoR family), NmtR (ArsR/SmtB family), KmtR (ArsR/SmtB family), SrnRQ (ArsR/SmtB family), and Nur (Fur family) [144 ]. These proteins exist as homo-dimers or homo-tetramers and usually act as transcriptional repressors and negatively control mRNA synthesis, preventing RNA polymerase from initiating the transcription at the promoter. Ni2+ ions bind in regulatory sites, acting (i) as co-repressors, increasing the affi nity of the protein repressor for the DNA operator sequences: this is the case of proteins that regulate genes for metal ion effl ux, storage, traffi cking, and tolerance (RcnR, NmtR, KmtR); (ii) as inducers, decreasing the affi nity of the repressor for the DNA operator sequences, eventually leading to transcriptional activation of genes for membrane uptake systems (NikR, SrnRQ, Nur). Only in the case of NikR from H. pylori the metal- responsive transcriptional regulator act as pleiotropic regulator, exerting a dual control, both as activator and repressor, on different promoters [144 ]. Nickel-protein interactions, selectively driven by the coordination chemistry and geometry of metal binding sites, usually octahedral or square planar, are propagated away from the specifi c metal binding site through changes in protein structure and/ or dynamics, along the protein backbone, resulting in a modifi cation of the DNA binding affi nity of the protein. For example, binding of metal ions to their specifi c coordination sites within the ArsR/SmtB family drives an allosteric change, with the stabilization of a protein conformer with low affi nity for DNA, and decreases the internal dynamics of the protein backbone, leading to the unavailability, in energetic terms, of the conformer that features high affi nity for DNA [145 ]. On the other hand, in case of Hp NikR, the presence of bound Ni2+ [ 146 , 147 ] does not induce, by itself, the stabilization of a specifi c conformer, and increases the protein dynamics unlocking inter-domain motions, supporting the view that the likely mechanism of interaction of the protein with its operator DNA sequence involves a selection of the correct conformation coupled with an induced fi t mechanism facilitated by the presence of bound Ni2+ [148 , 149 ]. These events produce a fi nely tuned metabolic response driven by Ni2+ ions, including the coordinated control of the entire machinery of metallo-enzyme synthesis and activation, as well as the systems of homeostasis that involve competitive Ni2+ uptake, intracellular accumulation, and extrusion.

3.3 Nickel-Obligate Microorganisms with Severe Impact on Human Health

3.3.1 Helicobacter pylori as a Nickel-Dependent Pathogen: A Possible Correlation between Nickel Intake and Cancer Development

Helicobacter pylori is a Gram-negative bacterium and the principal causative agent of many acute and chronic gastric pathologies, including peptic ulcer. It is a major factor of risk for the insurgence of gastric carcinomas and lymphomas [ 150 ]; 10 Nickel and Human Health 345 accordingly, the WHO classifi ed the bacterium as a class 1 carcinogen in 1994. After the colonization, untreated H. pylori infections persist for the entire life of the host because of the inability of the human immune response to effi ciently counter the bacterium [151 ]. H. pylori is widespread worldwide, and infects up to 50% and 80% of adults in industrialized and developing countries, respectively. Consistent with the hostility of the human stomach as a habitat for bacterial growth, H. pylori has set up a number of adaptive mechanisms, allowing the bacterium to promptly respond to environmental stresses, such as mild to strong acidity, fl uctuating nutrient availability and osmolarity, oxygen tension and host immune responses. These adaptive responses rely on transcriptional regulatory networks that control coordinated expression of tolerance and virulence genes in space and time [ 152 ]. Key virulence determinants for the processes of bacterial colonization include two essential nickel enzymes, that is, Ni 2+-dependent urease, which allows buffering of the acidic gastric environment, and a [NiFe]-hydrogenase, which allows consumption of energy-yielding hydrogen, freely available in the gastric niche. These enzymes were shown to be required for full colonization of mouse stomach. The importance of Ni2+ ions in H. pylori physiology is emphasized by the number and diversity of proteins involved in nickel homeostasis, which control the activity of nickel enzymes at the cellular level with three different processes: (i) regulation of transcription and expression of genes encoding metallo-enzymes and metal traffi cking proteins, performed by the pleiotropic Ni2+ sensor NikR; (ii) specifi c delivery of the metal ion cofactors to the protein active sites and metal-binding pockets, performed by the urease and hydrogenase chaperones; and (iii) intracellular metal ion accumulation and storage, performed by the outer membrane transporters FrpB4 and FecA3, the Ni2+ permease NixA, the Ni 2+ effl ux system CznABC, the cytoplasmic Ni2+ accumulators Hpn, Hpn-like, and HspA. Due to their importance for bacterial pathogenesis, these processes all represent possible targets for the development of alternative antibacterial strategies. Coherently, ureG -negative mutants (which can synthesize apo-urease but are unable to incorporate Ni2+ into the active site) are defi cient in colonizing the gastric mucosa of nude mice, thereby highlighting a link between Ni activation of urease and host colonization [153 ]. As urease is one of the most abundant enzymes in H. pylori, representing up to 10% of the bacterial protein content, it is likely that the demand for nickel is high in this microorganism. Similarly to other pathogens, metal ion starvation triggers the expression of toxins and metal ion scavengers in H. pylori , allowing the pathogen to compete with the host for these essential nutrients. Accordingly, infections by H. pylori have been epidemiologically linked to certain forms of anemia and impaired iron metabolism in hosts, proving that bacterial infection can signifi cantly impact on the balance of human metal ion homeostasis [154 ]. Nickel is naturally abundant in many types of food [5 ], therefore, it is plausible that H. pylori enters in contact with the necessary amount of Ni2+ to satisfy its metabolic demand in the stomach. The absence of known enzymes that require nickel as an essential cofactor in higher eukaryotes suggests that there is no competition for nickel between the host and the bacterium. However, the fl ux of nickel is probably not continuous and occurs in successive batch-like conditions rather than a constant stream of metals. In addition, nickel availability is also a function of pH, which fl uctuates widely within the gastric 346 Zambelli and Ciurli compartments. As a consequence, H. pylori must be able to accumulate Ni2+ when relatively high exogenous concentrations are available. This intracellular nickel reservoir, most likely bound to Ni2+ storage proteins, would in turn be available for urease or/and hydrogenase maturation when Ni2+ concentrations are limited. This was demonstrated in a recent study, aimed to correlate Ni 2+ levels introduced with diet, and colonization of H. pylori [155 ]. Even in Ni2+ -depleted diet, wild-type H. pylori is able to colonize the host stomach because it can store Ni2+ ions in the intracellular environment, relying on Hpn and Hpn-like Ni 2+ accumulators. If these proteins are mutated, the colonization levels of Ni-depleted animals are statistically lower than colonization levels of Ni-fed animals.

3.3.2 Nickel Homeostasis and Intracellular Parasitism: Eukaryotic and Prokaryotic Pathogens

Some nickel-dependent prokaryotic and eukaryotic microorganisms infect the human body as intracellular parasites. The most important eukaryotic pathogens are Trypanosomatids, such as Trypanosoma cruzi and Leishmania spp., which are protozoa causing Chagas’ disease and leishmaniasis, respectively. Their life cycle includes an intracellular stage in the mammalian host. These organisms produce a Ni2+ -dependent glyoxalase I, which is essential for pathogen survival as it serves to detoxify methylglyoxal, a toxic product of glycolysis and other metabolic pathways (see Section 3.1 ) [156 ]. The use of Ni2+ ions for glyoxalase activity in trypanosomatids is exceptional, as in other eukaryotes the enzyme cofactor is zinc. This refl ects distinctive substrate selectivity between the enzyme of the pathogen and human host, indicating that the enzyme could be a potential chemotherapeutic target against the protozoan parasite [157 ]. One of the most important intracellular infective bacteria worldwide is Mycobacterium tuberculosis, the etiological agent of tuberculosis. This bacterium establishes infection in adult humans primarily in the lungs by infecting macrophages, where it can remain asymptomatic or can become active manifesting the clinical symptoms. Currently, the WHO estimates that one-third of the world’s population is latently infected with tuberculosis, with 9.27 million new cases and 1.76 million deaths annually [158 ]. Upon infection, M. tuberculosis transfers into a bactericidal phagosome that is subsequently fused to a lysosome, creating a vacuole characterized by an acidic (pH 5.5), highly nitro-oxidative, nutrient-limiting, and possibly hypoxic microenvironment. In this hostile environment, M. tuberculosis relies on its exceptional metabolic fl exibility and ability to adapt, replicate and/or persist within changing and adverse microenvironments. Initially, it has been proposed that M. tuberculosis Ni2+ -dependent urease contributes to alkalize the mycobacterium- containing vacuole in macrophages, promoting a more favorable environment for the intracellular persistence of the bacilli. However, a recent study indicated that urease activity does not infl uence the general bacterial fi tness in vitro. In addition, the alkalizing effect of the M. tuberculosis urease activity has only been found in resting macrophages, while it is not present in the more acidic microenvironment of 10 Nickel and Human Health 347 the phagolysosomal compartment of activated macrophages, suggesting that the alkalizing effect provided by the mycobacterial urease activity is somehow modest [159 ]. On the other hand, the observation that M. tuberculosis urease expression and activity increase upon nitrogen deprivation [160 ], suggests that urea may be a potential source of nitrogen for M. tuberculosis. Consistently, this bacterium assimilates urea, as major nitrogen source, in an urease-dependent manner, this process generating ammonia, which is one of the key precursors for the biosynthesis of essential amino acids such as glutamate [159 ]. The apparent dispensability of urease activity in a mouse model suggests that other readily available nitrogen sources within the host cell could bypass the need for M. tuberculosis to metabolize urea, with a general functional redundancy, metabolic versatility, and compensatory mechanisms that characterize M. tuberculosis ability to adapt to virtually any microenvironment encountered in its host, in which carbon and nitrogen sources may vary qualitatively and quantitatively [159 ]. In addition, the ability of M. tuberculosis to infect extra-pulmonary sites may require the urease activity for bacillus persistence or replication at specifi c sites of infection within the host. In this case, the ability to utilize urea as a source of nitrogen could be critical at specifi c sites of infection where other sources of nitrogen are limited [159 ]. The mycobacterial urease activity may also have a role in evading the immune response of the host in response to the bacterial infection. Indeed, it is known that exogenous NH4 Cl blocks phagosome-lysosome fusion and promotes phagosome- endosome fusion in mouse mononuclear phagocytes [161 , 162]. This suggests that ammonia production by M. tuberculosis may partly slow down the phagolysosome formation, providing the bacterium with less hostile environmental conditions. In addition, M. bovis was demonstrated to attenuate the MHC-II molecule expression in infected macrophages, with consequent inhibition of CD4+ T-cell activation and general suppression of the host immune system [163 , 164 ]. The ability of the mycobacterial urease to alkalize the microenvironment has been exploited to develop a novel vaccine candidate, based on M. bovis urease, consisting of a urease-inactive M. bovis bacterium that expresses the Listeria mono- cytogenes listeriolysin [165 ]. In the absence of the mycobacterial urease activity, the mild acidic pH of the bacillus-containing vacuole provides an ideal pH environment for listeriolysin perforation of the vacuole membrane. The phagosome lysis promotes mycobacterial translocation into the macrophage cytoplasm, leading to cellular apoptosis and stronger immune responses that provide better protection upon M. tuberculosis reinfection. The importance of metal ion homeostasis for M. tuberculosis is underlined by the observation that its genome encodes an atypically large number (at least twelve) metal sensors of the ArsR-SmtB family [144 ], allowing to speculate that these proteins enable the pathogen to respond rapidly to metal fl uxes in the phagosome [166 ]. Notably, two of these metal-dependent transcription factors, named NmtR and KmtR, specifi cally respond to Ni2+ and Co2+ concentrations and regulate the expression of membrane proteins – an ATPase effl ux pump (NmtA) and a cation diffusion facilitator (CDF), respectively – that control metal ion effl ux from the cell [166 ]. 348 Zambelli and Ciurli

The reason of having two Ni2+ /Co2+ sensors for metal ion effl ux systems has been explained with the different affi nity observed for these two proteins and their cog- nate metal ions, which allows a fi ne modulation of transcriptional response [166 ]. In particular, KmtR appears to have higher affi nity for Ni 2+ and Co 2+, and it is therefore able to detect the basal level of the two metal ions, thus de-repressing the gene of CDF transport at low metal concentrations. Only a higher cytoplasmic metal ion threshold is able to bind NmtR, which presents a lower affi nity for Ni2+ and Co2+ as compared to KmtR, allowing the expression of the NmtA effl ux pump [166 ]. These data support the signifi cance of these metal ions for this pathogen as well as the bacterial ability to respond to metal fl uxes, depending on two levels of Ni2+ or Co 2+ sensing.

4 Nickel Essentiality in Animals and Humans

Nickel is classiﬁ ed as a “possibly essential element” for animals and humans since the 1970s [19 ]. Nickel deﬁ ciency in humans has never been reported, as, in general, human nickel intake greatly exceeds the requirements, which have been estimated between 5 and 50 μg per day [ 22 ].

4.1 Effects of Nickel Depletion in Higher Organisms

The essentiality of Ni2+ for higher eukaryotes, fi rstly proposed in 1920s, is still debated [5 ]. The importance of this metal for animals and humans has been tested evaluating the effect of nickel defi ciency in animals. Rats grown in the absence or in low-abundance of nickel exhibit very severe consequences, as depressed growth, low hemoglobin, red blood cell counts and activity of several liver and kidney enzymes, as well as the presence of urea, ATP, and glucose in serum, and also glucose, glycogen, and triglycerides in the liver [ 167 ]. The growth dependence on nickel was more signifi cant in the second depleted generation, which also showed anemia, manifested in decreased hemoglobin and hematocrit values [5 ]. Nickel deprivation during reproduction in rats increased perinatal mortality [168 ], while in breeding goats it signifi cantly decreased the success of fi rst insemination and conception rate and increased the number of breeding attempts to achieve pregnancy and the abortion rate [ 169 ]. This effect seems to be related to the ability of nickel to be involved in cyclic nucleotide- gated (CNG) channel functions [ 170]. CNG channels are located in a number of organs, including the central nervous, urogenital, and reproductive systems. The CNG channels also have an important role in kidney function and, thus, in sodium metabolism [170 ]. Additionally, nickel defi ciency impairs the absorption of iron from the intestine and the concentrations of several metals, such as iron, copper, and zinc, were also 10 Nickel and Human Health 349 decreased in the liver of nickel-depleted animals [ 171 , 172 ]. Nickel defi ciency also results in lowered specifi c activities of many enzymes involved in carbohydrate and amino acid metabolism. It was suggested that the essentiality of nickel for higher organisms is linked to its modulation of gene expression in eukaryotic cells [ 58 ]. Nickel has been proposed to be also involved in membrane stability or in lipid metabolism [173 ].

4.2 Inﬂ uence of Nickel Availability on Gut’s Microﬂ ora

The wide presence of Ni2+ -dependent enzymes in prokaryotes and unicellular eukaryotes raises the possibility that nickel, not required by the human and animal body itself, is rather needed for the normal development of the gut’s microfl ora, which impacts host metabolism in a variety of ways and is responsible for several metabolic and cardiovascular diseases [174 ]. The gut microbi- ota comprises the largest microbial community in the human body and represents one of the highest cellular densities in natural ecosystems, reaching 1011 to 10 12 cells/mL of luminal content, and includes species of all kingdoms of life, Eukaryotes, Bacteria , and Archaea [175 ]. The bacterial composition is made mostly by the Firmicutes and by the Bacteroidetes phyla and includes Proteobacteria and Actinobacteria [ 176 ]. This population comes in contact with all the nutrients that are ingested through diet, creating a symbiotic equilibrium with the host for the utilization of micronutrients, including metal ion cofactors. In particular, in the human intestinal tract, there are large populations of bacterial cells with the potential to bind and sequester metals that enter the body, such as the ones from the Lactobacillus group [ 177 ]. Therefore, it has been postulated that gut microorganisms may play a role in protecting the host from the toxic potential of metal ions ingested with diet, preventing their entry to the body from the intestine, and this could also be the case with Ni 2+ ions [ 177 ]. Accordingly, while Ni 2+ ions administered by other routes, such as inhalation or dermal exposure, show a carcinogenic potential, there is no epidemiological evidence on possible cancer risk from dietary nickel exposures, and this may be due to the protective effect of the gut microfl ora. Besides this function, it is plausible that Ni 2+-dependent enzymes play important roles also for the intestine community, and that Ni 2+ homeostasis in some of these microorganisms is important for developing a health-promoting gut microfl ora. Indeed, early work reported that several bacterial species found in the human and animal intestine show consistent urease activity [178 ]. Among them, some species of the genus Bifi dobacterium , such as B. bifi dum and B. longum, are noteworthy [179 ], because of the known probiotic effects of some types of Bifi dobacteria, that for decades have been added to food to promote healthy effects [ 180]. Other urease-producing species, coming from the genus Lactobacillus, such as L. fermentum , are interesting as they codify an acid urease which possesses an optimum of pH at 3–4 [178 ]. 350 Zambelli and Ciurli

Figure 7 Reaction catalysed by methyl coenzyme-M reductase, and schematic structure of the nickel-containing active site.

Methanogenic archaea compose an important component of the microorganisms living in human and animal guts, where they use dihydrogen as reductant to produce

CH4 [181 ]. In humans, Methanobrevibacter smithii is the main actor of the metha- nogen population. Methanogens have been proposed to play a key role in the pathogenesis of irritable bowel syndrome and chronic constipation [181 ]. The last step of methane formation is catalyzed by methyl coenzyme-M reductase [ 182]. The reaction involves methane formation concomitantly with the formation of a disulﬁ de bond between coenzyme M and coenzyme B (Figure 7 ). The active site of this enzyme contains coenzyme F430, a Ni tetrapyrrole hydrocorphin with the metal in the Ni(I) oxidation state in the active form. Structural data on the active site has been obtained for the inactive oxidized Ni(II) state (Figure 7 ). This correlates the presence of nickel in the diet with the healthy methanogenic activity in the guts.

5 Conclusions and Outlook

The wide use of nickel in many modern technologies and in objects of common use increases the amount of nickel release and accumulation into the environment and the possibility for humans to come in contact with this metal. Indeed, nickel-containing objects, such as coins, are the cause of severe occupational health problems, whose social costs have to be considered worldwide. The double nature of nickel, that is, acting both as a toxic and a beneﬁ cial element for human health, was proposed already in the early 1900s. The requirement of nickel as a cofactor in the active sites of enzymes has been recognized in 1975 and since then several studies have been conducted, which were aimed to describe 10 Nickel and Human Health 351 the molecular mechanisms of the effects of nickel on all forms of life, including higher organisms. However, the rationale of nickel carcinogenesis and allergy in humans, as well as the cascade of events involving metal ion-dependent gene regulation and protein- protein interactions leading to nickel homeostasis in eukaryotes, is yet largely unknown. A full understanding of the molecular aspects of the effects of nickel on human health represents therefore an important challenge and a future task for bioinorganic chemistry, with the potential to have a signiﬁ cant impact for the human population.

Abbreviations

ABC ATP- binding cassette ACD allergic contact dermatitis acetyl-CoA acetyl-coenzyme A APC antigen-presenting cell Ard acireductone dioxygenase ARNT aryl hydrocarbon receptor nuclear translocator BPU Bacillus pasteurii urease CDF cation diffusion facilitator CNG cyclic nucleotide-gated DMT-1 divalent cation transporter EDTA ethylenediamine-N,N,N′,N′-tetraacetate Glx glyoxalase GSH glutathione (reduced) GTP guanosine 5′-triphosphate HIF hypoxia-inducible factor HRE hypoxia-responsive enhancer IKK Iκ-B kinase IRP iron-regulatory protein LPS lipopolysaccharide MAPK mitogen-activated protein kinase MHC major histocompatibility complex NDRG N-myc downstream regulated gene 1 NF-κB nuclear factor κ-B NiCoT nickel/cobalt permease ROS reactive oxygen species SCD systemic contact dermatitis TCR T-cell receptor TLR toll-like receptor VEGF vascular endothelial growth factor VHL von Hippel-Lindau tumor suppressor 352 Zambelli and Ciurli

References

1. S. Ciurli, S. Mangani, in Handbook on Metalloproteins , Eds. I. Bertini, A. Sigel, H. Sigel, Marcel Dekker, New York, USA, 2001, pp. 669–708. 2. E. Merian, Toxicol. Envir. Chem. 1984 , 8 , 9–38. 3. R. G. Garrett, in Metal Ions in Biology and Medicine, Eds J. A. Centeno, P. Collery, G. Fernet, R. B. Finkelman, H. Gibb, J.-C. Etienne; John Libbey Eurotext, Paris, 2000, Vol. 6, pp. 508–510. 4. C. L. Coyle, E. I. Stiefel, in The Bioinorganic Chemistry of Nickel, Ed J. R. Lancaster, Jr., VCH, New York, 1988, pp. 1–28. 5. E. Denkhaus, K. Salnikow, Crit. Rev. Oncol. Hematol. 2002 , 42 , 35–56. 6. D. G. Barceloux, J. Toxicol. Clin. Toxicol. 1999 , 37 , 239–258. 7. M. Van Soestbergen, F. W. Sunderman, Jr., Clin. Chem. 1972 , 18 , 1478–1484. 8. N. Asato, M. Soestbergen, F. W. Sunderman, Jr., Clin. Chem. 1975 , 21 , 521–527. 9. J. D. Glennon, B. Sarkar, Biochem. J. 1982 , 203, 15–23. 10. F. Torres, M. das Gracas, M. Melo, A. Tosti, Clin. Cosmet. Investig. Dermatol. 2009 , 2 , 39–48. 11. R. B. Schiffer, F. W. Sunderman, Jr., R. B. Baggs, J. A. Moynihan, J. Neuroimmunol. 1991 , 34 , 229–239. 12. M. Freitas, P. Barcellos-de-Souza, C. Barja-Fidalgo, E. Fernandes, BioMetals 2013 , 26 , 13–21. 13. A. Au, J. Ha, M. Hernandez, A. Polotsky, D. S. Hungerford, C. G. Frondoza, J. Biomed. Mater. Res. A 2006 , 79 , 512–521. 14. M. Anke, A. Trüpschuch, G. Gunstheimer, in Trace Elements in Man and Animals 10, Eds A. M. Roussel, R. A. Anderson, A. E. Favrier, Kluwer Academic, 2002 , pp. 685–686. 15. S. G. Schafer, W. Forth, Ecotoxicol. Environ. Saf. 1983 , 7 , 87–95. 16. J. Tallkvist, A. M. Wing, H. Tjalve, Pharmacol. Toxicol. 1994 , 75 , 244–249. 17. M. Muller-Fassbender, B. Elsenhans, A. T. McKie, K. Schumann, Toxicology 2003 , 185 , 141–153. 18. International Agency for Research on Cancer, "Chromium, Nickel and Welding", Proceedings of the Monographs on the Evaluation of Carcenigenic Risks to Humans , Lyon, France, 1990. 19. F. H. Nielsen, D. A. Ollerich, Fed. Proc. 1974 , 33 , 1767–1772. 20. Y. Li, D. B. Zamble, Chem. Rev. 2009 , 109 , 4617–4643. 21. M. Yusuf, Q. Fariduddin, S. Hayat, A. Ahmad, Bull. Environ. Contam. Toxicol. 2011 , 86 , 1–17. 22. EMEA, European Medicines Agency, 2008; http://www.ema.europa.eu/ema . 23. K. S. Kasprzak, K. Salnikow, in Nickel and Its Surprising Impact in Nature, Vol. 2 of Metal Ions in Life Sciences, Eds A. Sigel, H. Sigel, R. K. O. Sigel, Wiley, Chichester, 2007, Vol. 2, pp. 581–618. 24. K. S. Kasprzak, F. W. Sunderman, Jr., K. Salnikow, Mutat. Res. 2003 , 533 , 67–97. 25. H. M. Shen, Q. F. Zhang, Environ. Health Perspect. 1994 , 102 (Suppl. 1), 275–282. 26. A. R. Oller, M. Costa, G. Oberdorster, Toxicol. Appl. Pharmacol. 1997 , 143, 152–166. 27. K. A. Biedermann, J. R. Landolph, Cancer Res. 1987 , 47 , 3815–3823. 28. M. Costa, T. L. Davidson, H. Chen, Q. Ke, P. Zhang, Y. Yan, C. Huang, T. Kluz, Mutat. Res. 2005 , 592 , 79–88. 29. N. W. Biggart, M. Costa, Mutat. Res. 1986 , 175 , 209–215. 30. J. B. Little, J. M. Frenial, J. Coppey, Teratog. Carcinog. Mutagen. 1988 , 8 , 287–292. 31. T. Miura, S. R. Patierno, T. Sakuramoto, J. R. Landolph, Environ. Mol. Mutagen. 1989 , 14 , 65–78. 32. F. Z. Arrouijal, H. F. Hildebrand, H. Vophi, D. Marzin, Mutagenesis 1990 , 5 , 583–589. 33. B. Kargacin, C. B. Klein, M. Costa, Mutat. Res. 1993 , 300 , 63–72. 34. M. Freitas, A. Gomes, G. Porto, E. Fernandes, J. Biol. Inorg. Chem. 2010 , 15 , 1275–1283. 35. T. J. O’Brien, S. Ceryak, S. R. Patierno, Mutat. Res. 2003 , 533 , 3–36. 36. B. Desoize, In Vivo 2003 , 17 , 529–539. 10 Nickel and Human Health 353

37. A. Hartwig, T. Schwerdtle, Toxicol. Lett. 2002 , 127 , 47–54. 38. W. Bal, R. Liang, J. Lukszo, S. H. Lee, M. Dizdaroglu, K. S. Kasprzak, Chem. Res. Toxicol. 2000 , 13 , 616–624. 39. L. Lande-Diner, J. Zhang, T. Hashimshony, A. Goren, I. Keshet, H. Cedar, Cold Spring Harb. Symp. Quant. Biol. 2004 , 69 , 131–138. 40. Y. W. Lee, C. B. Klein, B. Kargacin, K. Salnikow, J. Kitahara, K. Dowjat, A. Zhitkovich, N. T. Christie, M. Costa, Mol. Cell. Biol. 1995 , 15 , 2547–2557. 41. B. Govindarajan, R. Klafter, M. S. Miller, C. Mansur, M. Mizesko, X. Bai, K. LaMontagne, Jr., J. L. Arbiser, Mol. Med. 2002 , 8 , 1–8. 42. K. Salnikow, A. Zhitkovich, Chem. Res. Toxicol. 2008 , 21 , 28–44. 43. H. Chen, Q. Ke, T. Kluz, Y. Yan, M. Costa, Mol. Cell. Biol. 2006 , 26 , 3728–3737. 44. H. Chen, N. C. Giri, R. Zhang, K. Yamane, Y. Zhang, M. Maroney, M. Costa, J. Biol. Chem. 2010 , 285 , 7374–7383. 45. R. Martinez-Zamudio, H. C. Ha, Epigenetics 2011 , 6 , 820–882. 46. A. A. Karaczyn, F. Golebiowski, K. S. Kasprzak, Chem. Res. Toxicol. 2005 , 18 , 1934–1942. 47. Q. Zhang, K. Salnikow, T. Kluz, L. C. Chen, W. C. Su, M. Costa, Toxicol. Appl. Pharmacol. 2003 , 192 , 201–211. 48. C. B. Klein, K. Conway, X. W. Wang, R. K. Bhamra, X. H. Lin, M. D. Cohen, L. Annab, J. C. Barrett, M. Costa, Science 1991 , 251 , 796–799. 49. S. Orrenius, B. Zhivotovsky, P. Nicotera, Nat. Rev. Mol. Cell. Biol. 2003, 4, 552–565. 50. K. Salnikow, M. Gao, V. Voitkun, X. Huang, M. Costa, Cancer Res. 1994 , 54 , 6407–6412. 51. W. Bal, A. M. Protas, K. S. Kasprzak, in Metal Ions in Toxicology: Effects, Interactions, Interdependencies, Vol. 8 of Metal Ions in Life Sciences, Eds A. Sigel, H. Sigel, R. K. O. Sigel, Royal Society of Chemistry, Cambridge, 2011, pp. 319–373. 52. K. Salnikow, M. V. Blagosklonny, H. Ryan, R. Johnson, M. Costa, Cancer Res. 2000 , 60 , 38–41. 53. S. Salceda, J. Caro, J. Biol. Chem. 1997 , 272 , 22642–22647. 54. W. C. Hon, M. I. Wilson, K. Harlos, T. D. Claridge, C. J. Schoﬁ eld, C. W. Pugh, P. H. Maxwell, P. J. Ratcliffe, D. I. Stuart, E. Y. Jones, Nature 2002 , 417 , 975–978. 55. S. J. Freedman, Z. Y. Sun, F. Poy, A. L. Kung, D. M. Livingston, G. Wagner, M. J. Eck, Proc. Natl. Acad. Sci. USA 2002 , 99 , 5367–5372. 56. P. Maxwell, K. Salnikow, Cancer Biol. Ther. 2004 , 3 , 29–35. 57. K. Salnikow, S. P. Donald, R. K. Bruick, A. Zhitkovich, J. M. Phang, K. S. Kasprzak, J. Biol. Chem. 2004 , 279 , 40337–40344. 58. D. Zhou, K. Salnikow, M. Costa, Cancer Res. 1998 , 58 , 2182–2189. 59. H. Cangul, K. Salnikow, H. Yee, D. Zagzag, T. Commes, M. Costa, Cell. Biol. Toxicol. 2002 , 18 , 87–96. 60. T. P. Ellen, Q. Ke, P. Zhang, M. Costa, Carcinogenesis 2008, 29 , 2–8. 61. M. A. Zoroddu, M. Peana, T. Kowalik-Jankowska, H. Kozlowski, M. Costa, J. Inorg. Biochem. 2004 , 98 , 931–939. 62. M. A. Zoroddu, M. Peana, S. Medici, R. Anedda, Dalton Trans. 2009, 5523–5534. 63. J. G. Burchﬁ eld, A. J. Lennard, S. Narasimhan, W. E. Hughes, V. C. Wasinger, G. L. Corthals, T. Okuda, H. Kondoh, T. J. Biden, C. Schmitz-Peiffer, J. Biol. Chem. 2004 , 279 , 18623–18632. 64. J. Hwang, Y. Kim, H. B. Kang, L. Jaroszewski, A. M. Deacon, H. Lee, W. C. Choi, K. J. Kim, C. H. Kim, B. S. Kang, J. O. Lee, T. K. Oh, J. W. Kim, I. A. Wilson, M. H. Kim, J. Biol. Chem . 2011 , 286 , 12450–12460. 65. K. Kondo, T. Ozaki, Y. Nakamura, S. Sakiyama, Biochem. Biophys. Res. Commun. 1995 , 216 , 209–215. 66. K. Kanaya, A. L. Tsai, T. Kamitani, Biochem. Biophys. Res. Commun. 2002 , 290 , 294–299. 67. S. Oshiro, K. Nozawa, M. Hori, C. Zhang, Y. Hashimoto, S. Kitajima, K. Kawamura, Biochem. Biophys. Res. Commun. 2002 , 290 , 213–218. 68. C. Harford, B. Sarkar, Biochem. Biophys. Res. Commun. 1995 , 209 , 877–882. 69. S. F. Martin, P. R. Esser, F. C. Weber, T. Jakob, M. A. Freudenberg, M. Schmidt, M. Goebeler, Allergy 2011 , 66 , 1152–1163. 354 Zambelli and Ciurli

70. M. Schmidt, M. Goebeler, J. Mol. Med. (Berl.) 2011 , 89 , 961–970. 71. P. S. Friedmann, Toxicol. Lett. 2006 , 162 , 49–54. 72. K. Landsteiner, J. Jacobs, J. Exp. Med. 1935 , 61 , 643–656. 73. H. J. Thierse, K. Gamerdinger, C. Junkes, N. Guerreiro, H. U. Weltzien, Toxicology 2005 , 209 , 101–107. 74. L. Lu, J. Vollmer, C. Moulon, H. U. Weltzien, P. Marrack, J. Kappler, J. Exp. Med. 2003 , 197 , 567–574. 75. S. Dai, F. Crawford, P. Marrack, J. W. Kappler, Proc. Natl. Acad. Sci. USA 2008 , 105 , 11893–11897. 76. F. Sinigaglia, J. Invest. Dermatol. 1994 , 102 , 398–401. 77. Y. Wang, S. Dai, Immunol. Res. 2013 , 55 , 83–90. 78. P. Griem, C. von Vultee, K. Panthel, S. L. Best, P. J. Sadler, C. F. Shaw, 3rd, Eur. J. Immunol. 1998 , 28 , 1941–1947. 79. K. Gamerdinger, C. Moulon, D. R. Karp, J. Van Bergen, F. Koning, D. Wild, U. Pﬂ ugfelder, H. U. Weltzien, J. Exp. Med. 2003 , 197 , 1345–1353. 80. O. Wildner, T. Lipkow, J. Knop, Exp. Dermatol. 1992 , 1 , 191–198. 81. M. Goebeler, G. Meinardus-Hager, J. Roth, S. Goerdt, C. Sorg, J. Invest. Dermatol. 1993 , 100 , 759–765. 82. M. Goebeler, A. Trautmann, A. Voss, E. V. Brocker, A. Toksoy, R. Gillitzer, Am. J. Pathol. 2001 , 158 , 431–440. 83. M. Goebeler, J. Roth, E. B. Brocker, C. Sorg, K. Schulze-Osthoff, J. Immunol. 1995 , 155 , 2459–2467. 84. M. Goebeler, R. Gillitzer, K. Kilian, K. Utzel, E. B. Brocker, U. R. Rapp, S. Ludwig, Blood 2001 , 97 , 46–55. 85. M. Schmidt, B. Raghavan, V. Muller, T. Vogl, G. Fejer, S. Tchaptchet, S. Keck, C. Kalis, P. J. Nielsen, C. Galanos, J. Roth, A. Skerra, S. F. Martin, M. A. Freudenberg, M. Goebeler, Nat. Immunol. 2010 , 11 , 814–819. 86. M. Schmidt, M. Hupe, N. Endres, B. Raghavan, S. Kavuri, P. Geserick, M. Goebeler, M. Leverkus, J. Cell. Mol. Med. 2010 , 14 , 1760–1776. 87. A. Trautmann, M. Akdis, D. Kleemann, F. Altznauer, H. U. Simon, T. Graeve, M. Noll, E. B. Brocker, K. Blaser, C. A. Akdis, J. Clin. Invest. 2000 , 106 , 25–35. 88. S. Rothman, Dermatol. Wochenschr. 1930 , 2 , 98–99. 89. R. Spiewak, J. Pietowska, K. Curzytek, Expert Rev. Clin. Immunol. 2007, 3, 851–859. 90. T. Menne, K. Rasmussen, Contact Dermatitis 1990 , 23 , 57–58. 91. J. P. Thyssen, K. Ross-Hansen, T. Menne, J. D. Johansen, Contact Dermatitis 2010 , 63 , 102–106. 92. Y. Yoshihisa, T. Shimizu, Dermatol. Res. Pract. 2012 , 2012 , 749561. 93. E. Mann, U. Ranft, G. Eberwein, D. Gladtke, D. Sugiri, H. Behrendt, J. Ring, T. Schafer, J. Begerow, J. Wittsiepe, U. Kramer, M. Wilhelm, Contact Dermatitis 2010 , 62 , 355–362. 94. R. Darlenski, J. Kazandjieva, K. Pramatarov, Int. J. Dermatol. 2012 , 51 , 523–530. 95. J. P. Thyssen, T. Menne, Chem. Res. Toxicol. 2010 , 23 , 309–318. 96. R. I. Nijhawan, M. Molenda, M. J. Zirwas, S. E. Jacob, Dermatol. Clin. 2009 , 27 , 355–364, 97. D. Bonamonte, A. Cristaudo, F. Nasorri, T. Carbone, O. De Pita, G. Angelini, A. Cavani, Contact Dermatitis 2011 , 65 , 293–301. 98. M. Minelli, D. Schiavino, F. Musca, M. E. Bruno, P. Falagiani, G. Mistrello, G. Riva, M. Braga, M. C. Turi, V. Di Rienzo, C. Petrarca, C. Schiavone, M. Di Gioacchino, Int. J. Immunopathol. Pharmacol. 2010 , 23 , 193–201. 99. Nickel and Its Surprising Impact in Nature, Eds A. Sigel, H. Sigel, R. K. O. Sigel, John Wiley & Sons, Chichester, Met. Ions Life Sci. 2007 , 2 , pp. 1–702. 100. S. W. Ragsdale, J. Biol. Chem. 2009 , 284 , 18571–18575. 101. H. L. Mobley, M. D. Island, R. P. Hausinger, Microbiol. Rev. 1995 , 59, 451–480. 102. S. Wyllie, A. H. Fairlamb, Semin. Cell. Dev. Biol. 2011 , 22 , 271–277. 103. N. Sukdeo, E. Daub, J. F. Honek, in Nickel and Its Surprising Impact in Nature, Vol. 2 of Metal Ions in Life Sciences , Eds A. Sigel, H. Sigel, R. K. O Sigel, John Wiley & Sons, Chichester, 2007, pp. 445–472. 10 Nickel and Human Health 355

104. N. Sukdeo, J. F. Honek, Biochim. Biophys. Acta 2007 , 1774 , 756–763. 105. T. C. Pochapsky, T. Ju, M. Dang, R. Beaulieu, G. M. Pagani, B. OuYang, in Nickel and Its Surprising Impact in Nature , Vol. 2 of Metal Ions in Life Sciences , Eds A. Sigel, H. Sigel, R. K. O Sigel, John Wiley & Sons, Chichester, 2007, Vol. 2, pp. 473–500. 106. Q. Xu, R. Schwarzenbacher, S. S. Krishna, D. McMullan, S. Agarwalla, K. Quijano, P. Abdubek, E. Ambing, H. Axelrod, T. Biorac, J. M. Canaves, H.-J. Chiu, M.-A. Elsliger, C. Grittini, S. K. Grzechnik, M. DiDonato, J. Hale, E. Hampton, G. W. Han, J. Haugen, M. Hornsby, L. Jaroszewski, H. E. Klock, M. W. Knuth, E. Koesema, A. Kreusch, P. Kuhn, M. D. Miller, K. Moy, E. Nigoghossian, J. Paulsen, R. Reyes, C. Rife, G. Spraggon, R. C. Stevens, H. van den Bedem, J. Velasquez, A. White, G. Wolf, K. O. Hodgson, J. Wooley, A. M. Deacon, A. Godzik, S. A. Lesley, I. A. Wilson, Proteins: Structure, Function, and Bioinformatics 2006 , 64 , 808–813. 107. T. Ju, R. B. Goldsmith, S. C. Chai, M. J. Maroney, S. S. Pochapsky, T. C. Pochapsky, J. Mol. Biol. 2006 , 363 , 823–834. 108. S. Ciurli, in Nickel and Its Surprising Impact in Nature, Vol. 2 of Metal Ions in Life Sciences , Eds A. Sigel, H. Sigel, R. K. O Sigel, Wiley, Chichester, 2007, pp. 241–278. 109. B. Zambelli, F. Musiani, S. Benini, S. Ciurli, Acc. Chem. Res. 2011 , 44 , 520–530. 110. S. Benini, W. R. Rypniewski, K. S. Wilson, S. Miletti, S. Ciurli, S. Mangani, Structure Fold. Des. 1999 , 7 , 205–216. 111. F. Musiani, E. Arnoﬁ , R. Casadio, S. Ciurli, J. Biol. Inorg. Chem. 2001, 3, 300–314. 112. S. Benini, W. R. Rypniewski, K. S. Wilson, S. Mangani, S. Ciurli, J. Am. Chem. Soc. 2004 , 126 , 3714–3715. 113. S. Ciurli, S. Benini, W. R. Rypniewski, K. S. Wilson, S. Miletti, S. Mangani, Coord. Chem. Rev. 1999 , 190–192 , 331–355. 114. W. Lubitz, M. van Gastel, W. Gartner, Nickel and Its Surprising Impact in Nature , Vol. 2 of Metal Ions in Life Sciences , Eds A. Sigel, H. Sigel, R. K. O Sigel, John Wiley & Sons, Chichester, 2007, pp. 279–322. 115. P. M. Vignais, B. Billoud, Chem. Rev. 2007 , 107 , 4206–4272. 116. R. J. Maier, Biochem. Soc. Trans. 2005 , 33 , 83–85. 117. C. Navarro, L.-F. Wu, M.-A. Mandrand-Berthelot, Mol. Microbiol. 1993 , 9 , 1181–1191. 118. H. Lebrette, M. Iannello, J. C. Fontecilla-Camps, C. Cavazza, J. Inorg. Biochem. 2013 , 121 , 16–18. 119. L. Macomber, R. P. Hausinger, Metallomics 2011 , 3 , 1153–1162. 120. D. H. Nies, FEMS Microbiol. Rev. 2003 , 27 , 313–339. 121. F. N. Stahler, S. Odenbreit, R. Haas, J. Wilrich, A. H. Van Vliet, J. G. Kusters, M. Kist, S. Bereswill, Infect. Immun. 2006 , 74 , 3845–3852. 122. A. Rodrigue, G. Effantin, M. A. Mandrand-Berthelot, J. Bacteriol . 2005 , 187 , 2912–2916. 123. R. Ge, Y. Zhang, X. Sun, R. M. Watt, Q. Y. He, J. D. Huang, D. E. Wilcox, H. Sun, J. Am. Chem. Soc . 2006 , 128 , 11330–11331. 124. R. J. Maier, S. L. Benoit, S. Seshadri, BioMetals 2007 , 20 , 655–664. 125. Y. B. Zeng, D. M. Zhang, H. Li, H. Sun, J. Biol. Inorg. Chem. 2008 , 13 , 1121–1131. 126. F. Musiani, B. Zambelli, M. Stola, S. Ciurli, J. Inorg. Biochem. 2004 , 98 , 803–813. 127. B. Zambelli, F. Musiani, M. Savini, P. Tucker, S. Ciurli, Biochemistry 2007 , 46 , 3171–3182. 128. R. Real-Guerra, F. Staniscuaski, B. Zambelli, F. Musiani, S. Ciurli, C. R. Carlini, Plant Mol. Biol . 2012 , 78 , 461–475. 129. J. W. Olson, R. J. Maier, J. Bacteriol. 2000 , 182 , 1702–1705. 130. C. Wulﬁ ng, J. Lombardero, A. Pluckthun, J. Biol. Chem. 1994, 269 , 2895–2901. 131. R. K. Watt, P. W. Ludden, J. Biol. Chem. 1998 , 273 , 10019–10025. 132. H.-K. Song, S. B. Mulrooney, R. Huber, R. P. Hausinger, J. Biol. Chem. 2001 , 276 , 49359–49364. 133. H. Remaut, N. Safarov, S. Ciurli, J. Van Beeumen, J. Biol. Chem. 2001 , 276 , 49365–49370. 134. K. Banaszak, V. Martin-Diaconescu, M. Bellucci, B. Zambelli, W. Rypniewski, M. J. Maroney, S. Ciurli, Biochem. J. 2012 , 441 , 1017–1026. 135. N. Mehta, J. W. Olson, R. J. Maier, J. Bacteriol. 2003 , 185 , 726–734. 136. H. K. Loke, P. A. Lindahl, J. Inorg. Biochem. 2003 , 93 , 33–40. 356 Zambelli and Ciurli

137. R. L. Kerby, P. W. Ludden, G. P. Roberts, J. Bacteriol. 1997 , 179 , 2259–2266. 138. P. Neyroz, B. Zambelli, S. Ciurli, Biochemistry 2006 , 45 , 8918–8930. 139. B. Zambelli, M. Stola, F. Musiani, K. De Vriendt, B. Samyn, B. Devreese, J. Van Beeumen, P. Turano, A. Dikiy, D. A. Bryant, S. Ciurli, J. Biol. Chem. 2005 , 280 , 4684–4695. 140. B. Zambelli, P. Turano, F. Musiani, P. Neyroz, S. Ciurli, Proteins 2009 , 74 , 222–239. 141. B. Zambelli, N. Cremades, P. Neyroz, P. Turano, V. N. Uversky, S. Ciurli, Mol. Biosyst. 2012 , 8 , 220–228. 142. M. Bellucci, B. Zambelli, F. Musiani, P. Turano, S. Ciurli, Biochem. J. 2009 , 422 , 91–100. 143. Y. H. Fong, H. C. Wong, C. P. Chuck, Y. W. Chen, H. Sun, K. B. Wong, J. Biol. Chem. 2011 , 286 , 43241–43249. 144. B. Zambelli, F. Musiani, S. Ciurli, Met. Ions Life Sci. 2012 , 10 , 135–170. 145. Z. Ma, F. E. Jacobsen, D. P. Giedroc, Chem. Rev. 2009 , 109 , 4644–4681. 146. B. Zambelli, M. Bellucci, A. Danielli, V. Scarlato, S. Ciurli, Chem. Commun. 2007 , 3649–3651. 147. B. Zambelli, A. Danielli, S. Romagnoli, P. Neyroz, S. Ciurli, V. Scarlato, J. Mol. Biol. 2008 , 383 , 1129–1143. 148. F. Musiani, B. Bertosa, A. Magistrato, B. Zambelli, P. Turano, V. Losasso, C. Micheletti, S. Ciurli, P. Carloni, J. Chem. Theor. Comp. 2010 , 6 , 3503–3515. 149. S. Benini, M. Cianci, S. Ciurli, Dalton Trans. 2011 , 40 , 7831–7833. 150. J. G. Kusters, A. H. van Vliet, E. J. Kuipers, Clin. Microbiol. Rev. 2006 , 19 , 449–490. 151. H. de Reuse, S. Bereswill, FEMS Immunol. Med. Microbiol. 2007 , 50 , 165–176. 152. A. Danielli, G. Amore, V. Scarlato, PLoS Pathog. 2010 , 6 , e1000938. DOI: 10.371/journal. ppat.1000938. 153. K. A. Eaton, S. Krakowka, Infect. Immun. 1994 , 62 , 3604–3607. 154. K. Muhsen, D. Cohen, Helicobacter 2008 , 13 , 323–340. 155. S. L. Benoit, E. F. Miller, R. J. Maier, Infect. Immun. 2013 , 81 , 580–584. 156. M. Sousa Silva, A. E. Ferreira, R. Gomes, A. M. Tomas, A. Ponces Freire, C. Cordeiro, Int. J. Med. Microbiol. 2012, 302 , 225–229. 157. S. C. Chauhan, P. K. Padmanabhan, R. Madhubala, Curr. Drug Targets 2008 , 9 , 957–965. 158. WHO, "Global tuberculosis control report 2011", World Health Organization, Geneva, Switzerland, 2011. 159. W. Lin, V. Mathys, E. L. Ang, V. H. Koh, J. M. Martinez Gomez, M. L. Ang, S. Z. Zainul Rahim, M. P. Tan, K. Pethe, S. Alonso, Infect. Immun. 2012 , 80 , 2771–2779. 160. D. L. Clemens, B. Y. Lee, M. A. Horwitz, J. Bacteriol. 1995 , 177 , 5644–5652. 161. A. H. Gordon, P. D. Hart, M. R. Young, Nature 1980 , 286 , 79–80. 162. P. D. Hart, M. R. Young, J. Exp. Med. 1991 , 174 , 881–889. 163. K. Sendide, A. E. Deghmane, J. M. Reyrat, A. Talal, Z. Hmama, Infect. Immun. 2004 , 72 , 4200–4209. 164. T. Mukai, Y. Maeda, T. Tamura, Y. Miyamoto, M. Makino, FEMS Immunol. Med. Microbiol. 2008 , 53 , 96–106. 165. L. Grode, P. Seiler, S. Baumann, J. Hess, V. Brinkmann, A. Nasser Eddine, P. Mann, C. Goosmann, S. Bandermann, D. Smith, G. J. Bancroft, J. M. Reyrat, D. van Soolingen, B. Raupach, S. H. Kaufmann, J. Clin. Invest. 2005 , 115 , 2472–2479. 166. D. R. Campbell, K. E. Chapman, K. J. Waldron, S. Tottey, S. Kendall, G. Cavallaro, C. Andreini, J. Hinds, N. G. Stoker, N. J. Robinson, J. S. Cavet, J. Biol. Chem. 2007 , 282 , 32298–32310. 167. A. Schnegg, M. Kirchgessner, in In Trace Element Metabolism in Man and Animals-3 , Ed M. Kirchgessner, Proceedings of the 3rd Intern. Symposium, Technische Universität, Munchen, Germany, 1978, pp. 236–243. 168. F. H. Nielsen, D. R. Myron, S. H. Givand, T. J. Zimmerman, D. A. Ollerich, J. Nutr. 1975 , 105 , 1620–1630. 169. M. Szilagyi, M. Anke, I. Balogh, Acta Vet. Hung. 1991 , 39 , 231–238. 170. K. Yokoi, E. O. Uthus, F. H. Nielsen, Biol. Trace Elem. Res. 2003 , 93 , 141–154. 171. F. H. Nielsen, T. R. Shuler, T. G. McLeod, T. J. Zimmerman, J. Nutr. 1984, 114 , 1280–1288. 10 Nickel and Human Health 357

172. J. W. Spears, R. W. Harvey, L. J. Samsell, J. Nutr. 1986 , 116 , 1873–1882. 173. G. I. Stangl, M. Kirchgessner, J. Nutr. 1996 , 126 , 2466–2473. 174. V. Tremaroli, F. Backhed, Nature 2012 , 489 , 242–249. 175. C. Palmer, E. M. Bik, D. B. DiGiulio, D. A. Relman, P. O. Brown, PLoS Biol. 2007 , 5 , e177. 176. P. B. Eckburg, E. M. Bik, C. N. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S. R. Gill, K. E. Nelson, D. A. Relman, Science 2005 , 308 , 1635–1638. 177. M. Monachese, J. P. Burton, G. Reid, Appl. Environ. Microbiol. 2012 , 78 , 6397–6404. 178. K. Suzuki, Y. Benno, T. Mitsuoka, S. Takebe, K. Kobashi, J. Hase, Appl. Environ. Microbiol. 1979 , 37 , 379–382. 179. F. Turroni, E. Foroni, P. Pizzetti, V. Giubellini, A. Ribbera, P. Merusi, P. Cagnasso, B. Bizzarri, G. L. de’Angelis, F. Shanahan, D. van Sinderen, M. Ventura, Appl. Environ. Microbiol . 2009 , 75 , 1534–1545. 180. I. S. Arvanitoyannis, M. Van Houwelingen-Koukaliaroglou, Crit. Rev. Food Sci. Nutr. 2005 , 45 , 385–404. 181. M. Pimentel, R. P. Gunsalus, S. S. Rao, H. Zhang, Am. J. Gastroenter. Suppl. 2012 , 1 , 28–33. 182. B. Jaun, R. K. Thauer, in Nickel and Its Surprising Impact in Nature, Vol. 2 of Metal Ions in Life Sciences, Eds A. Sigel, H. Sigel, R. K. O Sigel, John Wiley & Sons, Ltd., Chichester, UK, 2007, pp. 323–356. Chapter 11 Copper: Effects of Deﬁ ciency and Overload

Ivo Scheiber , Ralf Dringen , and Julian F. B. Mercer

Contents ABSTRACT ...... 360 1 INTRODUCTION ...... 360 2 COPPER BIOCHEMISTRY AND HOMEOSTASIS ...... 361 2.1 Copper-Dependent Enzymes ...... 361 2.1.1 Cytochrome c Oxidase ...... 362 2.1.2 Copper/Zinc Superoxide Dismutase ...... 362 2.1.3 Ceruloplasmin ...... 362 2.1.4 Lysyl Oxidase ...... 363 2.1.5 Tyrosinase ...... 363 2.1.6 Dopamine-β-Monoxygenase and Peptidylglycine α-Amidating Monoxygenase ...... 364 2.2 Cellular Copper Homeostasis ...... 364 2.2.1 Copper Uptake...... 364 2.2.2 Copper Sequestration and Storage ...... 366 2.2.3 Intracellular Copper Traffi cking ...... 367 2.2.4 Copper Effl ux ...... 368 2.3 Systemic Copper Homeostasis ...... 369 3 COPPER DEFICIENCY DISORDERS ...... 371 3.1 Nutritional Copper Defi ciency ...... 371 3.1.1 Anemia ...... 371 3.1.2 Neuropathies ...... 372 3.1.3 Connective Tissues and Vascular System ...... 372 3.1.4 Immune System ...... 373 3.1.5 Copper and Development ...... 374

I. Scheiber Department of Parasitology, Faculty of Science , Charles University , Prague , Czech Republic R. Dringen Centre for Biomolecular Interactions Bremen, University of Bremen , Bremen , Germany J. F. B. Mercer (*) Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences , Deakin University , Burwood, Victoria 3125 , Australia e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 359 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_11, © Springer Science+Business Media Dordrecht 2013 360 Scheiber, Dringen, and Mercer

3.2 Genetic Copper Deﬁ ciencies ...... 374 3.2.1 Menkes Disease and Occipital Horn Syndrome ...... 374 3.2.2 Distal Hereditary Peripheral Neuropathy ...... 375 4 COPPER OVERLOAD DISORDERS ...... 375 4.1 Excessive Copper Intake ...... 375 4.2 Genetic Copper Overload ...... 375 4.2.1 Wilson Disease ...... 375 4.2.2 Copper-Associated Infantile Cirrhosis ...... 376 5 NEUROPATHOLOGY AND COPPER...... 376 5.1 Alzheimer Disease ...... 376 5.2 Parkinson Disease ...... 377 5.3 Huntington Disease ...... 378 5.4 Motor Neuron Disorders ...... 378 5.5 Prion Diseases ...... 379 6 OVERVIEW AND FUTURE DEVELOPMENTS ...... 380 ABBREVIATIONS ...... 380 ACKNOWLEDGMENT ...... 381 REFERENCES ...... 381

Abstract Copper is an essential trace metal that is required for the catalysis of several important cellular enzymes. However, since an excess of copper can also harm cells due to its potential to catalyze the generation of toxic reactive oxygen species, transport of copper and the cellular copper content are tightly regulated. This chapter summarizes the current knowledge on the importance of copper for cellular processes and on the mechanisms involved in cellular copper uptake, storage and export. In addition, we will give an overview on disturbances of copper homeostasis that are characterized by copper overload or copper deﬁ ciency or have been connected with neurodegenerative disorders.

Keywords ATP7A • ATP7B • copper chaperones • copper homeostasis • Ctr1 • glutathione • Menkes disease • metallothioneins • Wilson disease

Please cite as: Met. Ions Life Sci. 13 (2013) 359–387

1 Introduction

Copper became widely bioavailable about 2–3 billion years ago with the advent of an oxygen atmosphere that allowed for the conversion of Cu+ to the more soluble Cu2+ ion [ 1]. Because of the ready interconversion between these two oxidation states, copper has become an essential element for all organisms that have an oxidative metabolism. In humans, it represents the third most abundant essential transition metal [2 ]. As a cofactor of several enzymes and/or as structural component, copper is involved in many physiological pathways. Furthermore, copper is associated with important biological processes including angiogenesis, response to hypoxia and neuromodulation. 11 Copper: Effects of Deﬁ ciency and Overload 361

In recent years the importance of copper in human health has become increasingly recognized, and it has moved from a minor element, affected in a few rare conditions, to one that may be of vital importance in the pathology of several important neurological diseases. This recognition has largely come about as a consequence of the rapid advances in understanding of the molecular basis of copper homeostasis. This chapter will review some of the biological roles of copper and the diseases that are known, or plausibly proposed to involve defects in copper homeostatic mechanisms. Particular emphasis will be placed on the neurological disorders.

2 Copper Biochemistry and Homeostasis

2.1 Copper-Dependent Enzymes

Copper is an essential cofactor and/or a structural component in a number of important enzymes of plants and animals (Table 1 ). In general, these enzymes are involved in redox reactions [3 ]. The relatively high redox potential for the Cu2+ /Cu+ system is utilized by many enzymes for oxidation reactions, for example superoxide by superoxide dismutase and catechols by tyrosinase. Among others, copper-dependent enzymes participate in biological processes such as energy metabolism (e.g., cytochrome c oxidase), antioxidative defence (e.g., Zn,Cu-superoxide dismutase) and iron metabolism (e.g., ceruloplasmin) [2 ]. On the basis of their optical and electron paramagnetic resonance (EPR) features, copper-dependent enzymes are classiﬁ ed as type 1 (blue copper site), 2 or 3 copper enzymes [3 ]. Most copper enzymes contain only one type of copper center, but in some (e.g., ceruloplasmin, cytochrome c oxidase) more than one type can be found. Type 1 copper sites, also known as blue copper sites, exclusively function in single electron transfers [3 , 4 ]. Type 2 copper sites lack unique features in their UV/Vis and EPR spectra and catalytically activate enzyme substrates by direct interaction rather

Table 1 Mammalian copper-dependent enzymes. Enzyme Function Cytochrome c oxidase Oxidative phosphorylation Cu,Zn superoxide dismutase (SOD1) Superoxide detoxiﬁ cation, signaling Ceruloplasmin (Cp) Ferroxidase Lysyl oxidase (LOX) Crosslinking of collagen and elastin Tyrosinase Melanin synthesis Dopamine-β-monoxygenase (DβM) Norepinephrine synthesis Peptidylglycine α-amidating enzyme (PAM) Activation of peptide hormones Copper amine oxidase Deamination of amines Hephaestin Ferroxidase Coagulation factors V and VIII Blood clotting 362 Scheiber, Dringen, and Mercer than being involved in electron transfer [3 ]. In contrast to type 1 and 2 sites, type 3 copper sites are binuclear. These copper sites are constituted of two closely spaced coupled copper ions, each of them coordinated by three histidines, which can be reversibly bridged by dioxygen. The function of type 3 copper sites is the activation and transport of oxygen [3 ].

2.1.1 Cytochrome c Oxidase

Cytochrome c oxidase is a member of the super family of heme-copper containing oxidases. It is embedded in the mitochondrial inner membrane where it catalyzes the electron transfer from reduced cytochrome c to dioxygen in the fi nal step of mitochondrial oxidative phosphorylation [ 5]. Mammalian cytochrome c oxidase is a multimeric protein complex consisting of 13 subunits, encoded by both the mitochondrial and nuclear genome. Biogenesis of the functional holoprotein is a complicated process that requires several specifi c proteins, so-called assembly factors, including Cox17, Sco1, and Sco2 [6 , 7 ]. Cytochrome c oxidase defi - ciency is one of the most common causes of respiratory chain defects in humans. Pathological features range from metabolic acidosis, weakness and cardiomyopathy to neurodegeneration [6 ,7 ].

2.1.2 Copper/Zinc Superoxide Dismutase

The members of the ubiquitous family of superoxide dismutases (SODs) convert superoxide to dioxygen and hydrogen peroxide for further disposal by catalase and glutathione peroxidase. Superoxide is produced during the reduction of dioxygen that occurs in respiration and during autoxidation of catecholamines as well as its metabolites. Excess amounts of superoxide can lead to the formation of highly reactive oxygen species (ROS) that would damage cellular constituents [8 ]. Three distinct types of SOD are found in mammals: copper/zinc superoxide dismutase (Cu/Zn-SOD; SOD1), manganese superoxide dismutase (Mn-SOD, SOD2) and extracellular superoxide dismutase (EC-SOD; SOD3) [9 ]. SOD1 is a homodimeric protein located largely in the cytosol. SOD2 contains manganese as metal cofactor while both SOD1 and SOD3 contain catalytic copper and structural zinc ions in their active sites [10 ]. Mutations in SOD1 have been linked to the motor neuron disease, amyotrophic lateral sclerosis (ALS) [11 ].

2.1.3 Ceruloplasmin

Ceruloplasmin (Cp) belongs to the family of multicopper oxidases. Cp contains 6 copper atoms per molecule: three type 1 copper sites, a single type 2 copper ion and a binuclear type 3 copper site. Cp has a ferroxidase activity and has a critical role in iron homeostasis [12 ]. The majority of Cp is synthesized by hepatocytes and 11 Copper: Effects of Defi ciency and Overload 363 secreted into circulation [ 12]. In the human central nervous system and testes a glycosylphosphatidylinositol (GPI)-anchored form of Cp that is generated by alternative splicing has been identifi ed in astrocytes and Sertoli cells, respectively [13 ]. During biosynthesis copper insertion into apo-Cp takes place late in the secretory pathway [14 ]. In hepatocytes the copper transporting ATPase ATP7B and the Niemann-Pieck C1 protein are required for proper metallation of Cp [15 , 16 ]. Cp is an acute phase response protein whose synthesis and secretion can be strongly increased during pregnancy, infl ammation, infection, and in diseases such as diabetes, cancer as well as cardiovascular diseases [ 12 ]. Copper defi ciency does not affect the rates of biosynthesis and release of Cp by hepatocytes but results in an increase of unstable apo-Cp in the plasma leading to lowering of Cp protein and oxidase activity [17 ]. Aceruloplasminemia is an autosomal recessive disorder resulting from a loss of function mutation in the Cp gene [18 ]. Due to the importance of Cp in iron homeostasis, the lack of functional Cp in affected individuals is accompanied by excessive iron accumulation in most tissues leading to neurological symptoms such as retinal degeneration, mild dementia, dysarthria, dystonia as well as diabetes mellitus [18 ].

2.1.4 Lysyl Oxidase

Lysyl oxidase (LOX) has a crucial role in the formation, maturation, and stabilization of connective tissue by catalyzing the cross-linking of elastin and collagen [19 ]. Copper incorporation into LOX propeptide takes place in the trans -Golgi network (TGN) where it is delivered by the copper transporting ATPase, ATP7A [20 ]. Accordingly, LOX activity is low in patients suffering from Menkes disease (MD) (Section 3.2.1 ), which is caused by mutations in the ATP7A gene and patients have marked connective tissue dysfunctions [21 , 22]. Dietary copper status also affects LOX activity, but does not alter tissue levels of the LOX protein [19 ].

2.1.5 Tyrosinase

Tyrosinase is the key enzyme in the biogenesis of melanin pigments. In mammals, tyrosinase is mainly expressed in melanocytes and retinal pigment epithelium cells where it is localized to specialized organelles known as melanosomes [ 23]. One of the important functions of tyrosinase is the catalysis of the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the biosynthesis of melanins and dopamine, and its subsequent oxidation to DOPA quinone [ 24 ]. During trafﬁ cking from TGN to melanosomes, tyrosinase loses its copper and must be reloaded within melanosomes to sustain its activity. For both the TGN and melanosome compartments, copper loading into tyrosinase depends on the copper transporting ATPase ATP7A [ 25]. Consequently, mutations 364 Scheiber, Dringen, and Mercer in ATP7A such as found in patients with MD and in the mottled mouse mutants cause hypopigmentation, a feature found also in nutritionally copper deﬁ cient animals [21 ].

2.1.6 Dopamine-β-Monoxygenase and Peptidylglycine α-Amidating Monoxygenase

Dopamine-β-monoxygenase (DβM) and peptidylglycine α-amidating monoxygenase (PAM) belong to a small class of copper proteins found exclusively in animals [26 ]. DβM catalyzes the oxidative hydroxylation of dopamine to norepinephrine and thus plays an important role in the metabolism of these catecholamines [ 26 ]. PAM exclusively catalyzes the C-terminal α-amidation of various glycine-extended propeptides, a post-translational modifi cation essential for the bioactivity of diverse physiological regulators including peptide hormones, neurotransmitters, and growth factors [27 ]. Due to the physiological importance of PAM, lack of functional PAM in mice is embryonic lethal [28 ]. Proper metallation of DβM and PAM is essential for their activity. In MD patients plasma catechol levels are altered and levels of amidated peptides are low, refl ecting DβM and PAM defi ciency, respectively [29 , 30]. Thus, copper loading of both, DβM and PAM, is likely to depend on ATP7A. In support of this view, PAM activity is compromised in cells lacking functional ATP7A, although expression levels of PAM are normal [30 , 31 ].

2.2 Cellular Copper Homeostasis

Given the requirement for copper on one hand and the potential toxicity of copper on the other, cells have evolved mechanisms to regulate cellular copper concentrations. Many of the components involved in cellular copper homeostasis are well known at the molecular level. These include transporters that mediate the uptake and efﬂ ux of copper, biomolecules that sequester and store copper and specialized proteins called copper chaperones that guide copper to copper-dependent enzymes and to organelles (Figure 1 , Table 2 ).

2.2.1 Copper Uptake

Members of the copper transporter receptor (Ctr) family that were fi rst described for Saccharomyces cerevisiae [32 ], play a key role in the uptake of copper in eukaryotic cells. In humans, the two Ctr-members hCTR1 and hCTR2 have been identifi ed [ 33]. CTR1 is a 190 amino acid transmembrane protein and is considered as the major contributor to high-affi nity copper uptake in mammalian cells [34 ]. Monovalent copper is thought to be the copper species transported by CTR1 [35 ]. CTR2 has been localized to late endosomes and lysosomes and may play a role in copper recycling after degradation of copper enzymes [36 ]. 11 Copper: Effects of Defi ciency and Overload 365

Figure 1 Cellular copper homeostasis. Copper uptake is predominately mediated by the copper transporter receptor 1 (Ctr1). Since Ctr1 has been reported to transport Cu + , this substrate is provided by a cuprireductase and/or by chemical reduction with reducing agents such as ascorbate. In cells, copper is sequestered as GSH complex or stored in metallothioneins. The delivery of essential copper to copper-containing enzymes is mediated by speciﬁ c copper chaperones, such as by CCS to SOD1, by Cox17 to cytochrome c oxidase or by Atox1 to ATP7A. In many cell types ATP7A transports copper into the TGN for incorporation into LOX and other secreted copper- dependent enzymes. In hepatocytes, ATP7B supplies copper to ceruloplasmin. Elevated cellular copper concentrations induce a reversible translocation of ATP7A to the plasma membrane and ATP7B to subapical vesicles that allows direct export of copper.

Table 2 Proteins involved in mammalian copper homeostasis. Protein Function Copper transporter receptor 1 (Ctr1) Copper uptake Copper transporter receptor 2 (Ctr2) Copper uptake Divalent metal transporter 1 (DMT1) Copper uptake Copper chaperone for superoxide dismutase (CCS) Intracellular copper traffi cking ATOX1 Intracellular copper traffi cking Cox17 Intracellular copper traffi cking Glutathione (GSH) Intracellular copper traffi cking, storage, and detoxifi cation Metallothionein (MT) Storage and detoxifi cation ATP7A Copper export ATP7B Copper export

The extracellular N-terminus of hCTR1 contains two histidine-rich regions and two methionine motifs [33 ]. Mutation analysis revealed that deletion of the fi rst methionine motif and/or of the His-rich regions has almost no effect on copper transport activity of hCTR1 [ 37 , 38]. In contrast, mutation or deletion of methionine residues 366 Scheiber, Dringen, and Mercer in the second methionine motif had a strong inhibitory effect on hCTR1- mediated copper uptake [37 ]. Structural studies suggest that CTR1 is active as a trimer, forming a pore for the passage of copper across the lipid bilayer [39 ]. Mammalian Ctr1 is ubiquitously expressed; however, expression levels are tissue- specifi c, being highest in the liver, kidney, and intestine. In some tissues the expression level of Ctr1 depends on the copper status and is infl uenced by the physiological state, such as pregnancy and lactation [ 40 ]. Ctr1 plays an essential role in embryonic development as deletion of Ctr1 in mice is embryonic lethal, most likely due to an insuffi cient supply of the developing embryo with copper [41 ]. In cultured cells, Ctr1 is typically observed at the plasma membrane and in cytoplasmic vesicles and specifi c localization depends on the cell type [ 42]. In some cells, addition of copper stimulates the endocytosis of the protein which is likely to be a homeostatic control mechanism to prevent excessive copper uptake and potential copper toxicity [36 ]. There has been some controversy about Ctr1 localization in polarized cells such as the intestinal enterocytes, but in this cell type, the current evidence supports an apical location which is expected if the protein plays a role in copper uptake from the diet [43 ].

2.2.2 Copper Sequestration and Storage

The accumulation of copper in the cytosol induces a risk for copper-mediated oxidative damage and binding of copper to essential biomolecules. However, under physiological conditions the concentration of free copper within the cell has been calculated to be around 10–18 M which amounts to less than one free copper ion per cell [44 ]. Such low concentrations of free copper are maintained by binding of copper to metallothioneins (MTs) and ligands of low molecular mass such as glutathione (GSH). MTs and GSH also represent the major molecules involved in the intracellular sequestering and storing of excess copper. In addition, mitochondria have been suggested to contribute to the cellular copper buffering capacity [45 ]. The tripeptide GSH is the most abundant low-molecular-weight thiol in cells, being present in millimolar concentrations [46 ]. GSH is essential for the detoxifi cation of reactive oxygen species, maintains the cellular thiol reduction potential in a strongly reduced state and is involved in redox regulation and signalling [46 ]. In addition, GSH has been linked to the transport and the detoxifi cation of metal ions including copper [47 ]. Indeed, the majority of cytosolic copper is bound to GSH [48 ] and copper in the form of a Cu(I)-GSH complex is believed to be a major contributor to the copper exchangeable pool in the cytosol [49 ]. In addition, GSH can participate in cellular copper homeostasis by regulating the activities of copper transport proteins such as ATP7A and ATP7B via glutathionylation/deglutathionylation [50 ]. Metallothioneins constitute a heterogeneous family of low-molecular-weight, cysteine-rich proteins found in all eukaryotes [51 ]. In addition to a presumed role in zinc and copper homeostasis, MTs have been implicated in the detoxifi cation of non-essential metals such as cadmium, protection against ROS, maintenance of the intracellular redox balance, regulation of cell proliferation and apoptosis, as well as 11 Copper: Effects of Defi ciency and Overload 367 in neuroprotection [51 – 56 ]. Four mammalian MT isoforms exist that are denoted MT-1 to MT-4. The predominant isoforms MT-1 and MT-2 are ubiquitously expressed in almost all organs and tissues, being most abundant in liver and kidney [ 51 ]. The expression of MT-3 and MT-4 is confi ned mainly to the brain and stratifi ed epithelium, respectively [57 , 58]. MT-3 and MT-4 are constitutively expressed, while MT-1 and MT-2 are both basally expressed and inducible by various stressors, including heavy metals, oxidative stress and pro-infl ammatory cytokines [51 ]. The expression of MTs is induced by an excess of copper [59 ]. The excess hepatic copper found in patients with Wilson disease (WD), and in the toxic milk mouse model of this disease, is bound to MTs [ 60 , 61]. Since MTs are capable of binding excess cellular copper, an increase in the cellular MT content confers resistance against copper-induced toxicity [62 ]. Hence, the rise in MT levels refl ects an adaptation of cells to copper overload conditions.

2.2.3 Intracellular Copper Trafﬁ cking

The intracellular traffi cking of copper is mediated by a group of proteins termed copper chaperones. These specialized proteins shuttle copper to specifi c cellular targets thus avoiding toxicity to other cellular components [63 ]. Antioxidant protein 1 (Atx1) is a small cytoplasmic copper-binding protein originally identifi ed as a copper-dependent suppressor of oxygen toxicity in yeast strains lacking both SOD1 and SOD2 [ 64]. In yeast, Atx1 shuttles copper to the copper transporting P-type ATPase Ccc2 for subsequent transport into the secretory pathway and delivery to the ferroxidase Fet3p needed for high affi nity iron uptake [65 ]. The human homologue of Atx1, termed HAH1 or ATOX1, is a 68 amino acid protein that shares 47% amino acid identity Atx1 [64 ]. Atox1 has been demonstrated to bind and transfer Cu(I) to the N-terminal metal-binding domains (MBDs) of the copper transporting P-type ATPases, ATP7A and ATP7B [66 – 69]. Mutations in ATP7B that lead to an impairment of protein-protein interactions with ATOX1 have been identifi ed in patients suffering from WD [67 ]. This fi nding highlights the importance of specifi c protein-protein interactions in the transfer of copper from ATOX1 to the copper transporting P-type ATPases as well as the signifi cance of ATOX1 in cellular copper homeostasis. The copper chaperone for superoxide dismutase (CCS) is involved in the maturation of SOD1 by inserting copper into this enzyme [70 ]. Although SOD1 from most species can be activated independently of CCS, maximal SOD1 activity in the majority of organisms relies on the presence of CCS [71 ]. CCS is primarily localized to the cytosol and has a similar cellular distribution as its target protein [ 70 ]. The expression level of CCS depends on the cellular copper content [ 72 ]. Copper defi ciency has been demonstrated to result in an increase in CCS protein abundance due to a lowered rate of proteosomal degradation of CCS [73 ]. Biogenesis of cytochrome c oxidase requires the assembly of 13 subunits into a multimeric protein complex and the concomitant insertion of cofactors, including three copper ions, two heme a groups, one zinc ion and a magnesium ion [6 ]. 368 Scheiber, Dringen, and Mercer

Formation of the CuB and CuA site in the mitochondrial encoded subunits Cox1 and Cox2 takes place within the mitochondrial intermembrane space (IMS), and thus requires both the delivery of copper into this mitochondrial compartment as well as the insertion of copper into the two copper centers. A number of proteins have been identiﬁ ed so far to be involved in the insertion of copper ions into mammalian cytochrome c oxidase [63 ]. The initial event in the transfer of copper to Cox1 and Cox2 is the Cox17-mediated transfer of copper to the Sco proteins Sco1 and Sco2 and

Cox11. This is followed by the subsequent insertion of copper into the nascent CuA and CuB sites [63 ]. Cox17, initially identiﬁ ed in a yeast mutant displaying a respiratory defect, is essential for the metallation of eukaryotic cytochrome c oxidases [74 ]. Cox17 is a small cysteine-rich, hydrophilic protein localized in the IMS and in the cytosol of cells [74 , 75 ]. Although this dual localization suggests a role of Cox17 in the shuttling of copper from the cytosol into the IMS, the primary function of Cox17 is the transfer of copper to Sco1, Sco2, and Cox11 within the IMS [75 ]. Sco proteins are required for the formation of the CuA site of cytochrome c oxidase [76 ]. In humans, the two homologs hSco1 and hSco2 contribute to this process by transferring copper to the binuclear copper center and by acting as thiol-disulﬁ de oxidoreductases [77 ].

Consistent with their critical role in the formation of the binuclear CuA center, mutations in Sco1 and Sco2 cause severe cytochrome c oxidase deﬁ ciencies [6 ].

2.2.4 Copper Efﬂ ux

Cellular copper efﬂ ux in mammals relies on the function of two proteins, ATP7A and ATP7B. Mutations in ATP7A cause the human copper deﬁ ciency disorder MD (Section 3.2.1 ) [78 –80 ] and mutations in ATP7B cause the human toxicosis disorder

WD [ 81 , 82 ]. These proteins belong to the protein family of P1B -type ATPases, which have key functions in metal homeostasis in most organisms [83 ]. P 1B -type ATPases are a subgroup of P-type ATPases that use the energy of ATP hydrolysis to transport heavy metals across cellular membranes [83 ]. In addition to their critical function in the effl ux of excess cellular copper, ATP7A and ATP7B shuttle copper to the secretory pathway for incorporation into copper-dependent enzymes such as tyrosinase, PAM, DβM, LOX, and Cp (reviewed in [ 84 , 85]). The importance of these proteins in the maintenance of copper homeostasis is illustrated by the severe clinical phenotypes manifest by MD and WD (Sections 3.2.1 and 4.2.1 ) [86 , 87 ]. Human ATP7A and ATP7B are large multispanning membrane proteins that share 50–60% amino acid sequence homology [85 ]. Their overall structure consists of a cytosolic amino-terminus, eight transmembrane helices, an ATP-binding domain an actuator domain, and a cytosolic carboxyl-terminus [88 ]. The N-terminal tail of human ATP7A and ATP7B harbors six metal binding domains (MBDs), each capable of binding one Cu+ ion [ 89 ]. Only the MBDs closest to the membrane, MBD5 and MBD6, are important for effi cient copper transport [90 – 92] while MBD1-4 primarily function in the regulation of the catalytic activity in response to copper [ 88 ]. The eight transmembrane helices (TMs) of ATP7A and ATP7B are involved in the formation of the copper translocation channel [88 ]. Specifi c residues within TM6-TM8 contribute to the intramembrane copper coordination 11 Copper: Effects of Defi ciency and Overload 369 sites required for copper transmembrane transport. Mutation of the cysteines in a conserved CPC motif in TM6 and mutation of Met1393 in TM8 have been shown to result in an impaired catalytic activity of human ATP7B and murine ATP7A [93 ]. The ATP-binding domain of both ATP7A and ATP7B, located between TM6 and TM7, comprises a nucleotide-binding domain (N-domain) and a phosphorylation domain (P-domain) containing the DKTG, TGDN, and GDGxND signature motifs of P-type ATPases [88 ]. The invariant Asp residue in the DKTG sequence motif of the P-domain is crucial for the catalytic cycle of P-type ATPases [94 ]. In the case of ATP7A and ATP7B, it accepts the γ-phosphate from ATP upon binding of ATP to the N-domain and copper to the intramembrane copper sites [89 ]. The formation of this phosphorylated intermediate induces conformational changes that allow the copper ion to be released on the other side of the membrane. The catalytic cycle is closed by the hydrolysis of the aspartyl phosphate bond and the return of the enzyme to its initial state. The dephosphorylation step is facilitated by the actuator domain (A-domain) linked to TM4 and TM5. This domain harbors the TGE signature motif of the P-type ATPases that is strictly required for their phosphatase activity [ 89 ]. Consequently, mutations of the TGE motif in ATP7A and ATP7B result in hyper- phosphorylated and catalytic inactive proteins [92 , 94 ]. ATP7A continuously recycles between the TGN and the plasma membrane, whereas ATP7B traffi cs between the TGN and a cytosolic vesicular compartment [95 ,96 ]. When copper levels are normal, both ATP7A and ATP7B have steady state localization at the TGN, where they transport copper from the cytosol to the TGN lumen for incorporation into copper-dependent enzymes. A rise in cytosolic copper levels induces a shift in the steady state distribution from the TGN to the plasma membrane and/or to a distinct cytosolic vesicular compartment in close proximity to the plasma membrane (reviewed in [ 84 , 85]). Redistribution of ATP7A and ATP7B back to the TGN occurs when cellular copper levels return to normal [95 , 96 ]. The ability of ATP7A and ATP7B to effl ux copper is linked to their ability to undergo copper-induced redistribution [84 ]. Mutations that impair the copper-dependent traffi cking of these proteins have been associated with MD and WD [91 , 97 , 98 ].

2.3 Systemic Copper Homeostasis

The key features of systemic copper homeostasis are shown in Figure 2 . Overall balance of copper in the body is achieved by regulation of the rate of uptake of copper in the small intestine and effl ux of copper from the liver in the bile. Most dietary copper is absorbed in the small intestine [2 ] and current evidence suggests that the copper transporter Ctr1 is responsible for the apical uptake of copper. However, the role and cellular localization of Ctr1 in this process has been somewhat controversial. Some studies have reported that Ctr1 is basolateral in the enterocytes and the apical copper entry into intestinal cells was thought to be mediated by a copper transport system other than Ctr1 [99 ]. Nose et al. have shown that intestinal-specifi c knock out of Ctr1 in mice produces a severe copper defi ciency and death at about 3 weeks of age showing that Ctr1 is a major component of the dietary uptake of copper [100 ]. Intestinal epithelial cells generated from these mice hyperaccumulated copper, 370 Scheiber, Dringen, and Mercer

Figure 2 Systemic copper homeostasis. The overall status and distribution of copper is regulated primarily by the concerted action of CTR1 and ATP7A/B. Uptake of dietary copper occurs in the small intestine where copper is taken into the intestinal enterocytes by CTR1 and is efﬂ uxed into the circulation by ATP7A. Most of the newly absorbed copper is taken up by the liver, which excretes excess copper in the bile, mediated by ATP7B. Copper crosses the blood brain barrier via ATP7A, and this protein also plays a key part in supply of copper to fetus. ATP7B is responsible for supplying copper to the milk.

whereas all organs tested suffered from a severe copper defi cit [100 ]. Despite the up to 10 times higher copper levels in these cells compared to that of intestinal epithelial cells from control animals, activities of copper-dependent enzymes were strongly reduced and levels of the copper chaperone for superoxide dismutase dramatically increased. Thus, it appears that Ctr1 in the enterocytes is required for copper to be bioavailable. In a subsequent paper Nose et al. demonstrated that Ctr1 is located on the apical surface of enterocytes consistent with its role in uptake of dietary copper [ 43 ]. The amount of Ctr1 on the apical surface of the enterocytes was increased when a low copper diet was supplied to the animals, consistent with this being part of the systemic homeostatic regulation of copper uptake which has been demonstrated in humans by Turnlund et al. [101 ]. It is unclear why reports of the cellular localization of Ctr1 have been so contradictory, however, it seems probable that problems with the antibodies in use could be responsible [43 ]. The copper effl ux protein ATP7A is responsible for the transport of copper across the basolateral surface of the enterocyte, and its intracellular location is altered by changes in dietary copper. In mice, it has been shown that increasing dietary copper causes ATP7A to traffi c from the TGN to vesicles close to the basolateral surface of the enterocyte [102 ]. Presumably this process increases the copper delivery to the portal circulation, allowing the excess copper to be eliminated by biliary excretion, which is the major path of copper elimination from the body [ 2 ]. ATP7A has a role in the distribution of copper around the body and in the maintenance of safe copper levels in many cell types. It is expressed in most tissues, including intestine, skeletal muscle, placenta, brain, heart, and kidney, but its expression in liver is very low [78 , 80 , 103 ]. In contrast, ATP7B is abundantly expressed in the liver and at lower 11 Copper: Effects of Defi ciency and Overload 371 levels in kidney, placenta, brain, lung, and heart [ 81 , 82]. ATP7B is the transporter responsible for the regulation of biliary excretion of copper, and excess copper in the hepatocyte stimulates traffi cking of this protein from the TGN, where it supplies copper to ceruloplasmin, to vesicles close to the apical membrane of the hepatocyte that abuts the biliary caniliculus [95 ]. This copper- induced traffi cking of ATP7B is the principle homeostatic mechanism for removing excess copper from the body. The difference in expression patterns between ATP7A and ATP7B correlates well with the observed alterations in body copper homeostasis seen in MD and WD. Inactivation of ATP7A in MD results in systemic copper defi ciency due to diminished copper export from the intestine into the portal blood and defects in copper effl ux from other tissues such as the blood brain barrier and the kidney [104 , 105 ], while failure of biliary copper excretion by ATP7B in WD leads to copper overload in liver and other tissues [106 ].

3 Copper Deﬁ ciency Disorders

3.1 Nutritional Copper Deﬁ ciency

Copper defi ciency in humans can occur through multiple mechanisms [107 , 108 ]. It has been observed in premature and low-birth-weight infants who are born with low hepatic copper stores [109 ], in individuals receiving total parenteral nutrition without adequate copper supplementation [110 ], in malnourished infants [111 ], and in persons with malabsorption syndromes [112 ]. Low copper intakes in the diet can result in marginal copper defi ciency and a signifi cant proportion of the population consuming a typical Western diet is estimated to have inadequate copper intake [113 ]. Secondary copper defi ciency can occur as consequence of high dietary intake of zinc and this has occurred even in patients with WD [114 ] after pharmacological treatments with copper chelating agents such as D-penicillamine or tetrathiomolybdate and following gastrointestinal surgery [115 ]. The clinical symptoms of copper defi ciency in humans are numerous [107 , 116 ]. Early and common signs of acquired copper defi ciency are hematological manifestations such as anemia, leukopenia, neutropenia, and pancytopenia [117 ]. Bone abnormalities including osteoporosis, bone fractures, and bone malformation have often been observed in copper-defi cient low-birth-weight infants and young children [118 ]. Acquired copper defi ciency may manifest with neurological symptoms, the clinical presentation resembling that of myeloneuropathy observed in vitamin

B 12 deﬁ ciency [119 ].

3.1.1 Anemia

Although the link between copper defi ciency and anemia was fi rst proposed in the 19th century and followed up by a series of studies in the early 20th century, copper defi ciency remains an under-recognized cause of anemia in humans [120 ]. 372 Scheiber, Dringen, and Mercer

Severe copper defi ciency is relatively rare, but it is nevertheless a signifi cant cause of reversible refractory anemia and leukopenia, particularly neutropenia, and is often misdiagnosed as myelodysplastic syndrome (MDS). The link between copper and iron metabolism has been known for many years, but the detailed molecular basis of this link is being clarifi ed [ 121]. Nevertheless, the reason for anemia in copper defi ciency is complex and remains somewhat enigmatic [ 121, 122]. The common view is that copper defi ciency leads to reduced activity of the multicopper oxidases, ceruloplasmin and hephaestin, which are required to oxidize iron(II) to iron(III) for binding to transferrin. But as Prohaska elegantly discusses, this common hypothesis does not explain all the available evidence and other factors, perhaps in the bone marrow, are probably responsible [122 ]. Similarly, the mechanism of neutropenia remains unknown, however, it is possible that copper defi ciency results in the inhibition of differentiation and self-renewal of CD34(+) hematopoietic progenitor cells [ 123 ].

3.1.2 Neuropathies

The fi rst evidence of a link between copper and a neurological disease was provided by the discovery that a demyelinating disease in sheep known as swayback was due to copper defi ciency [124 ]. More recently a similar condition which resembles motor neuron disease, but resulting from chronic copper defi ciency, has been described in humans [119 ]. In a review of 55 patients, Jaiser and Winston identifi ed risk factors that include upper gastrointestinal surgery, zinc overload and malabsorption syndromes [119 ]. All of these factors result in reduced copper absorption. There have been a number of case reports of neurological complications following gastric bypass surgery for obesity [125 , 126]. The prevalence and incidence of copper defi ciency following gastric banding surgery for obesity was found to be about 9.6% and many of these patients showed hematological changes. These results suggest that frequent monitoring of the copper status of gastric banded patients is warranted [119 ]. The mechanistic basis of demyelination due to copper defi ciency is not understood, however, reduced cytochrome oxidase activity is a possible cause. Interestingly, older mice lacking axonal prion protein (PrPc , see Section 5.5 ) develop late-onset peripheral nerve demyelination [127 ] and since PrP c binds copper, this mechanism could be involved in the demyelination due to copper defi ciency. Interestingly, the neurologic defi cits caused by copper defi ciency are clinically indistinguishable from the neuropathology in vitamin B 12 defi ciency [123 ].

3.1.3 Connective Tissues and Vascular System

Clear evidence for the importance of copper for correct formation and function of connective tissues and the vascular system is seen in patients with MD and in a marked form in the allelic variant, occipital horn syndrome (OHS) [21 ]. MD and 11 Copper: Effects of Deﬁ ciency and Overload 373

OHS patients have defective cross-linking of collagen and elastin due to low activity of the copper-dependent lysyl oxidase [128 ]. OHS is a milder variant of MD, and features predominately defects in connective tissue; patients have skin and joint laxity and can die from aortic aneurysms. In addition, cardiovascular complications appear to be connected to copper defi ciency as MD patients are at higher risk of congenital heart defects [129 ]. Low activity of lysyl oxidase has been proposed to underlie the osteoporosis found in MD patients, as well as copper-defi cient animals [ 21 ]. However, a recent observation that SOD1-defi cient mice developed osteoporosis suggests another mechanism [130 ]. Nutritional copper defi ciency has been found to result in metabolic bone disease including osteoporosis in children of low birth weight, which can be mistaken for child abuse [131 ]. Both osteoporosis and cardiovascular defects have long been known to occur in copper-defi cient domestic animals. In the 1930s there were reports of sudden death in cattle (“falling disease”), attributed to cardiac failure [132 ]. Skeletal abnormalities have been widely reported in animals (reviewed in [ 133]), but evidence for copper defi ciency in human cases of osteoporosis remains unresolved [134 ,135 ]. A relative new contributor to the copper effects on both the vascular and connective tissue systems is extracellular SOD3. This enzyme receives its copper from ATP7A which is found to be highly expressed in the vascular system [ 136 ]. Hypertension induced by angiotensin II was more pronounced in a mouse mutant with a mutation in ATP7A and SOD3 is an important modulator of oxidative stress-dependent hypertension [137 ]. Another link of copper and cardiovascular disease is provided by the fi nding of high levels of ATP7A expression in macrophages from murine atherosclerotic lesions, and down-regulation of the copper transporter reduced the amount of low density lipoprotein oxidation [138 ].

3.1.4 Immune System

In 1981 Prohaska and Lukasewycz reported that copper-defi cient mice had an impaired humoral-mediated immune response [139 ]. Earlier data had demonstrated that copper-defi cient rats were more sensitive to Salmonella infection [140 ]. A number of studies in the 1980s showed that copper-defi cient farm animals had impaired immune responses. Boyne and Arthur demonstrated that neutrophils from copper-defi cient cattle had reduced ability to kill Candida albicans [ 141]. Babies with MD are susceptible to infection [ 142]. However, the exact role of copper in immune function is still unclear but more recent studies have begun to shed light on the mechanisms. Copper defi ciency was shown to reduce interleukin production in T-lymphocytes [143 ] and secretion of interleukin-1α is dependent upon copper [144 ]. Copper has been used as an antimicrobial agent throughout recorded history and its ability to kill microorganisms is presumably related to the generation of hydroxyl radicals via Fenton chemistry [145 ]. This same mechanism is most likely responsible for the killing of pathogens by macrophages. These cells were found to increase their copper uptake in response to interferon-γ stimulation and most signifi cantly ATP7A was induced to traffi c to the phagosomal compartment. It was proposed that 374 Scheiber, Dringen, and Mercer

ATP7A pumps copper into this compartment where it generates hydroxyl radicals, thus, killing the ingested microorganism, but further studies are needed to establish whether this mechanism applies to a wider range of microbial killing [146 ].

3.1.5 Copper and Development

Early evidence for the importance of copper in mammalian development was found in sheep grazing on copper-defi cient soils in Australia. Lambs from copper- defi cient mothers were born with a disease termed swayback, characterized by paralysis of the hind limbs, convulsions, and blindness [124 ]. This disorder is a defect of myelination, which has been found in copper-defi cient humans (see Section 3.1.2 ). Copper supplementation of the mothers prevented this disorder [124 ]. Copper defi ciency during embryonic and fetal development can result in numerous gross structural and biochemical abnormalities which can result from impaired free radical defense or connective tissue abnormalities [147 ]. Mutations in ATP7A result in fetal copper defi ciency since this copper transporter is required for movement of copper across the placenta [148 ]. Babies with MD have characteristic dysmorphic features and other abnormalities at birth due to copper defi ciency during gestation [21 ]. Mice with mutations in the Menkes gene orthologue display a range of developmental effects ranging from fetal death, neonatal death or longer term survival with connective tissue abnormalities depending on the degree of impairment of the activity of ATP7A [ 149]. The critical importance of copper in embryogenesis was demonstrated by the death of mouse embryos at midgestation of development following ablation of the gene for the copper transporter Ctr1 [41 ]. Copper is also critical during postnatal development. Adequate copper supplies in milk are essential, and mutations in the copper transporter ATP7B, which is involved in secretion of copper into milk [ 150 ], result in death of suckling mice [151 ]. Recent studies on the conditional knockout of ATP7A have further reaffi rmed the vital importance of this transporter and hence, copper for the developing mammal [152 , 153 ]. In humans, copper defi ciency due to inadequate nutrition is mostly manifest in babies, particularly premature infants. The lack of copper can result in anemia, neutropenia, osteoporosis, and neurological problems [107 ].

3.2 Genetic Copper Deﬁ ciencies

3.2.1 Menkes Disease and Occipital Horn Syndrome

As noted previously, severe copper defi ciency is a hallmark of the X-linked recessive diseases MD and occipital horn syndrome; both are caused by genetic defects in the copper transporting ATPase, ATP7A [22 ]. The clinical features of MD include severe progressive neurological degeneration, connective tissue abnormalities, muscular hypotonia, and hypopigmentation of skin and hair [ 21]. Many of the clinical symptoms of acquired copper defi ciency and MD can be attributed to a decrease in the activities of copper-dependent enzymes. Thus, hypopigmentation of skin and 11 Copper: Effects of Defi ciency and Overload 375 hair results from reduced tyrosinase activity and abnormalities of bone and connective tissue are principally due to lowered LOX activity [21 , 22 , 154 , 155 ]. While the exact mechanism of copper defi ciency myelopathy is not known [119 ], neurological degeneration in MD is suggested to be primarily caused by a decrease in the activity of neuronal cytochrome c oxidase [87 ]. Other pathogenic mechanisms probably contribute to the extensive neurodegeneration in MD, for example, copper is known to be neuroprotective against glutamate excitotoxicity [156 ]. OHS is primarily a connective disorder, due to specifi c effects of the specifi c mutations of ATP7A on LOX activity. OHS is mostly caused by splice site mutations that allow the production of a small amount of otherwise normal ATP7A. Possibly, when ATP7A is present in only small amounts, it is primarily located on the plasma membrane rather than the TGN, thus explaining the specifi c reduction in LOX which receives copper in the TGN [157 ].

3.2.2 Distal Hereditary Peripheral Neuropathy

Two novel mutations in ATP7A were recently found to cause a form of distal hereditary motor neuropathy [158 ]. Although affecting the same copper transporter that is mutated in MD, the clinical phenotype is remarkably distinct. The disease primarily causes gradual die-back of the long motor neurons in the legs. The reason for the specifi c death of the axons of the motor neurons is unclear, but possibly involves reduction in the protective effect of copper against glutamate excitotoxicity as noted for MD. The long axons may be specifi cally sensitive to the subtle traffi cking defects found for the mutant ATP7As [158 ].

4 Copper Overload Disorders

4.1 Excessive Copper Intake

Acute copper toxicity has been described for individuals that accidentally or with suicidal intention ingested high doses of copper [159 ]. For copper doses up to 1 gram, gastrointestinal symptoms predominate. With higher doses, nausea, vomiting, headache, diarrhea, hemolytic anemia, gastrointestinal hemorrhage, liver and kidney failure as well as death may occur [159 ].

4.2 Genetic Copper Overload

4.2.1 Wilson Disease

Chronic copper toxicity with predominant effects on the liver is a feature of the autosomal recessive disorder WD, as well as the probable genetic overload disorders, Indian childhood cirrhosis and idiopathic chronic toxicosis [86 , 160]. WD is caused 376 Scheiber, Dringen, and Mercer by mutations in the copper transporting ATPase ATP7B that is responsible for biliary excretion of copper. As a result copper accumulates in the liver to such high levels that hepatocytes die and release copper that accumulates in the central nervous system resulting in neurological abnormalities. Thus, WD can manifest as a liver or neurological condition [21 ]. The disease is fatal unless measures are taken to remove the excess copper from the body, either by copper chelation or blocking intestinal uptake of copper with zinc [86 , 160 ].

4.2.2 Copper-Associated Infantile Cirrhosis

Indian childhood cirrhosis and idiopathic chronic toxicosis are severe chronic liver diseases that are characterized by excessive hepatic copper accumulation in infancy (as opposed to WD in which the copper accumulation occurs later in life) [160 ]. The etiology of these rare and usually fatal diseases has been hypothesized to be a combination of an unknown genetic defect affecting the copper metabolism and high dietary copper intake [160 – 162 ].

5 Neuropathology and Copper

Impairment of copper homeostasis can lead to neurodegeneration, as exempliﬁ ed by both MD and WD [21 ] and as previously noted, severe nutritional copper deﬁ ciency can lead to motor neuropathies (Section 3.1.2 ). Alterations of copper homeostasis have also been associated with neurodegenerative diseases such as prion diseases, Alzheimer disease (AD), Parkinson disease (PD) or Huntington disease (HD) but the exact role of copper in these important neurological diseases remains unclear.

5.1 Alzheimer Disease

Alzheimer Disease is an irreversible and progressive disease that causes memory loss and psychiatric disturbance. Aside from age, other risk factors include genetic factors, gender and environmental factors [163 ]. The pathological hallmarks of AD are the extracellular senile plaques and the intracellular neurofi brillary tangles in brain [164 ]. The principal constituents of senile plaques are amyloid-β (Aβ) peptides of 40 and 42 residues, which are generated form the integral membrane amyloid precursor protein by the consecutive action of β- and γ-secretase [163 ]. There is strong evidence that AD involves disturbances of copper homeostasis in the brain. The senile plaques in AD brains are strongly enriched in copper and this accumulation of copper could deplete adjacent neurons [165 ]. Results on copper concentrations in the brain of AD patients have been somewhat contentious due to 11 Copper: Effects of Defi ciency and Overload 377 technical issues, a recent study has shown a reduction of about 50% in copper in the amygdala and hippocampus in AD brains [166 ]. Studies of compounds which affect copper distribution in the brain as potential therapeutics for AD have shown promising results [167 ]. Although copper is depleted in some brain regions in AD patients, the amyloid plaques have been shown to accumulate the metal [168 ], suggesting that the binding of copper to the plaques is actually depleting copper from other brain regions [169 ]. Copper has been shown to precipitate Aβ peptides in vitro and it has been suggested that copper triggers the formation of senile plaques [ 169 ]. In support of this view, Aβ deposition begins within the glutamatergic synapse [ 170 ], where both copper [171 , 172 ] and Aβ [ 173 ] are released during synaptic transmission. However, although accumulation of Aβ peptides in the form of senile plaques is the most prominent feature of AD, it is now widely accepted that soluble oligomeric Aβ species are the most toxic form of Aβ peptides [169 ]. Since Aβ can mediate the reduction of Cu2+ to Cu+ , copper may promote the toxicity of such Aβ oligomers through the formation of ROS [174 ]. While the enhancement of Aβ toxicity by copper in vitro suggests a detrimental role of copper in AD, the observed lower copper contents in the brain of AD patients [166 , 175 ] and mouse models for AD [ 176 , 177 ] as compared to controls rather argue for a copper defi cit contributing to the neurodegeneration in AD. Copper supplementation in a mouse AD model improved the survival of these animals [176 ]. Improved cognitive functions were also observed in another mouse model of AD following administration of Cu(gtsm) as copper source [178 ]. However, intake of copper had no effect on cognition in patients with mild AD in a phase 2 clinical trial [ 179]. Mechanistically, copper defi ciency may exacerbate disease progression by infl uencing amyloid precursor protein processing and Aβ metabolism [170 ]. In addition, copper defi ciency may also infl uence the activity of copper-dependent enzymes. In this regard low activities of cytochrome c oxidase [180 ] and SOD1 [181 ] have been reported for the AD brain. Therapeutic strategies aiming to restore the normal copper distribution in AD brain are currently under investigation [ 174 ] with promising results [182 , 183 ].

5.2 Parkinson Disease

Parkinson disease is characterized by a complex motor disorder known as Parkinsonism that manifests with resting tremor, bradykinesia, rigidity, and postural instability and is the second most common neurodegenerative disease in humans [184 ]. The pathological hallmarks of PD are the loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta and the presence of intracellular inclusions, called Lewy bodies consisting of the protein α-synuclein [ 184]. The majority of cases are idiopathic with less than 10% of PD having a strict familial etiology. The underlying mechanisms of idiopathic PD are not fully understood. Among others mitochondrial dysfunction, oxidative stress, and inﬂ ammation have been suggested in the pathogenesis of PD [184 ]. 378 Scheiber, Dringen, and Mercer

There is accumulating evidence for dyshomeostasis of copper in PD. Parkinsonism is frequently present in patients with neurological WD [185 ] and copper has been demonstrated to accelerate aggregation of α-synuclein [186 ]. While the total copper content in brains of PD patients does not differ signiﬁ cantly from healthy controls, copper levels were found to be substantially lower (45%) in substantia nigra of PD patients [187 , 188]. This reduction in the copper content of the substantia nigra in PD has been discussed to result in an impairment of copper-dependent pathways, thereby contributing to the pathogenesis of PD [189 ]. In support of this view, copper supplementation has been shown to prevent the increase in lipid peroxidation, striatal dopamine depletion, and the reduction in the activity of tyrosine hydroxylase in an animal model for PD [190 ], while copper chelation was reported not to be protective in PD animal models [ 191 ]. Recently, the hypoxia imaging agent, Cu(II)(atsm) has been found to be neuroprotective in several animal models of PD possibly by inhibiting the nitration of α-synuclein [192 ].

5.3 Huntington Disease

Huntington disease is a rare autosomal-dominant, progressive neurodegenerative disease that results in motor, cognitive, and psychiatric abnormalities [193 ]. The genetic defect underlying HD is an expansion of a CAG repeat in exon 1 of the huntingtin gene that is translated into an expanded polyglutamine domain at the N-terminus of the huntingtin protein [194 ]. The exact pathogenic mechanism in HD remains to be elucidated. Accumulation of copper in the HD brain has been hypothesized to promote disease progression by promoting the aggregation of the huntingtin protein [195 ]. In addition treatment with the copper chelator tetrathiomolybdate delayed the decline in motor function in mouse models for HD, further supporting a potential role of copper in disease progression [196 ].

5.4 Motor Neuron Disorders

Motor neuron disorders (MNDs) involve both the central and peripheral nervous systems and are relatively common diseases. The anterior horn of the spinal cord contains the cell bodies of the motor neurons whose axons can extend for considerable distances (up to 1 m) to distal limb muscles. The degeneration of these neurons leads to peripheral motor neuropathies. Inherited peripheral neuropathies can affect sensory and autonomic nerve cells as well as motor neurons [197 ]. The best known MND is amyotrophic lateral sclerosis (ALS). ALS is a progressive neurodegenerative disease preferentially but not exclusively affecting motor neurons in the spinal cord, brainstem, and brain [ 198 ]. About 10% of cases are genetic, and the most common genetic form is due to mutations 11 Copper: Effects of Defi ciency and Overload 379 in the copper/zinc SOD1. This fi nding and the demonstration of abnormalities of copper homeostasis in ALS were the fi rst indications that copper was playing a role in MNDs [199 ]. At least 10 genes of diverse function are known to cause hereditary peripheral neuropathies [200 ] but there is no obvious common pathological mechanism. The recent fi nding that the two specifi c mutations in ATP7A (T994I and P1386S) causing distal hereditary neuropathy, result in mislocalization and defective intracellular traffi cking of ATP7A in patient’s fi broblasts suggest that abnormal copper homeostasis may lead to the degeneration of the distal motor neurons [158 ]. Given the role of SOD1 in ALS and ATP7A in peripheral neuropathy, together with many other studies linking copper more broadly with other neurological diseases, further studies to clarify the role of copper homeostasis in neurological health and disease are warranted.

5.5 Prion Diseases

Refolding of the normal prion protein (PrPc ) into an abnormal conformation (PrPsc ) has been associated with transmissible neurodegenerative diseases, such as Creutzfeld-Jacob disease, Kuru and fatal familial insomnia in humans, bovine spongiform encephalopathy in cattle and scrapie in sheep, which are summarized as prion diseases or transmissible spongiform encephalopathies [201 ]. Most attention has been paid to the role of the abnormal prion protein in disease, and there is much less data relating to the normal physiological role of the cellular prion protein (PrP c ), but evidence is growing that its role involves copper in some capacity. The PrPc is ubiquitously expressed but most abundantly in neurons [202 ]. The N-terminal region of the protein contains four to fi ve octapeptide repeats, which have copper binding sites of various affi nities and binding of copper induces a conformation change in the protein [203 ]. Cells expressing PrPc are much more resistant to copper-treatment than PrPc - defi cient cells [204 ]. Copper has been shown to stimulate endocytosis of PrP c [ 205 ]. Based on this observation the PrP c has been suggested to serve as a receptor for cellular uptake or effl ux of copper [ 205]. The copper contents of synaptosomes of PrP-defi cient mice were found to be lower compared to that of wild-type mice, which has led to the proposal that the PrP c may play a role in regulating copper release at the synapse [206 ]. Evidence for a role of the prion protein in copper homeostasis remains controversial, however, recently it was found that PrPc increased when cells were copper defi cient and this resulted in an increase in copper uptake [207 ]. Relevant to the role of copper in neurological diseases, the prion protein has been found to regulate the activity of the N-methyl-D-aspartate (NMDA) receptor and indeed it has been proposed that copper modulation of the NMDA receptor possibly by PrPc could be a unifying theme in many neurological disorders [208 ]. Intriguingly, it has recently been found that mice with a mutation in ATP7A have a delayed onset of prion disease [209 ]. 380 Scheiber, Dringen, and Mercer

6 Overview and Future Developments

Copper has been known to be an essential element since the 1920s but despite many decades of research, the full biological roles of this element have yet to be clarifi ed. With the advent of molecular tools, the intricate nature of copper homeostasis has become clear. The isolation of the copper effl ux genes ATP7A and ATP7B in the early 1990s was a turning point for the fi eld and presaged the heady decades since, when so many of the molecular players that regulated copper status have been identifi ed. The interactions between many trace elements and copper are being identifi ed at a molecular level, particularly with iron and zinc. We have briefl y touched on some areas, where the importance of adequate dietary copper is underappreciated, such as cardiovascular diseases and immune system function. This is an area that promises to develop over the next few years, leading to wider appreciation among health professionals of the importance of adequate copper intake. Over the last 10 years the neurological importance of copper has begun to be appreciated. It is very exciting that abnormalities of copper homeostasis are apparently playing an important role in neurological disorders such as AD, PD, and motor neuron disorders. We anticipate that many of the hints of copper involvements in these diseases will become solid evidence in the next decade, and it is even possible that therapies based on modifi cation of copper metabolism will lead to cures of these devastating conditions.

Abbreviations

A β amyloid β AD Alzheimer disease ALS amyotrophic lateral sclerosis ATOX 1 human antioxidant protein 1 (also known as HAH1) ATP7A a copper transporting ATPase ATP7B a copper transporting ATPase atsm diacetyl bis ( N (4)methylthiosemicarbazonato) Atx1 antioxidant protein1 CCS copper chaperone for superoxide dismutase Cp ceruloplasmin Ctr1 copper transporter 1 DOPA 3,4-dihydroxyphenylalanine D βM dopamine-β-monoxygenase EPR electron paramagnetic resonance GPI glycosylphosphatidylinositol GSH glutathione gtsm glyoxal bis ( N (4)-methylthiosemicarbazonato) hCTR human copper transporter 11 Copper: Effects of Deﬁ ciency and Overload 381

HD Huntington disease IMS intermembrane space LOX lysyl oxidase MBD metal binding domain MD Menkes disease MDS myelodysplastic syndrome MNDs motor neuron disorders MT metallothionein NMDA N-methyl-D-aspartate OHS occipital horn syndrome PAM peptidylglycine α-amidating monoxygenase PD Parkinson disease PrPc normal prion protein PrP SC pathogenic prion protein ROS reactive oxygen species SOD superoxide dismutase SOD1 Cu/Zn superoxide dismutase SOD2 Mn superoxide dismutase SOD3 extracellular superoxide dismutase = EC-SOD TGN trans -Golgi network TM transmembrane helices WD Wilson disease

Acknowledgment J.M. is grateful for funding support from the National Health and Medical Research Council of Australia grant APP1003903.

References

1. R. R. Crichton, J. L. Pierre, BioMetals 2001 , 14 , 99–112. 2. M. C. Linder, The Biochemistry of Copper , Plenum Press, New York, 1991. 3. W. Kaim, J. Rall, Angew. Chem., Int. Ed. 1996 , 43–60. 4. D. J. Kosman, J. Biol. Inorg. Chem. 2010 , 15 , 15–28. 5. D. M. Popovic, I. V. Leontyev, D. G. Beech, A. A. Stuchebrukhov, Proteins 2010 , 78 , 2691–2698. 6. F. Diaz, Biochim. Biophys. Acta 2010 , 1802 , 100–110. 7. I. Hamza, J. D. Gitlin, J. Bioenerget. Biomembr. 2002 , 34 , 381–388. 8. B. Halliwell, Annu. Rev. Nutr. 1996 , 16 , 33–50. 9. J. J. Perry, D. S. Shin, E. D. Getzoff, J. A. Tainer, Biochim. Biophys. Acta 2010 , 1804 , 245–262. 10. L. Miao, D. K. St Clair, Free Rad. Biol. Med. 2009 , 47 , 344–356. 11. D. R. Rosen, T. Siddique, D. Patterson, D. A. Figlewicz, P. Sapp, A. Hentati, D. Donaldson, J. Goto, J. P. O'Regan, H. X. Deng, Z. Rahmani, A. Krizus, D. McKenna-Yasek, A. Cayabyab, S. M. Gaston, R. Berger, R. E. Tanzi, J. J. Halperin, B. Herzfeldt, R. van den Bergh, W.-Y. Hung, T. Bird, G. Deng, D. W. Mulder, C. Smyth, N. G. Laing, E. Soriano, M. A. Pricak-Vance, J. Haines, G. A. Rouleau, J. S. Gusella, H. R. Horvitz, R. H. Brown, Jr., Nature 1993, 362 , 59–62. 12. N. E. Hellman, J. D. Gitlin, Annu. Rev. Nutr. 2002 , 22 , 439–458. 13. L. W. Klomp, Z. S. Farhangrazi, L. L. Dugan, J. D. Gitlin, J. Clin. Invest. 1996 , 98 , 207–215. 382 Scheiber, Dringen, and Mercer

14. M. Sato, J. D. Gitlin, J. Biol. Chem. 1991 , 266 , 5128–5134. 15. K. Terada, N. Aiba, X.-L. Yang, M. Iida, M. Nakai, N. Miura, T. Sugiyama, FEBS Lett. 1999 , 448 , 53–56. 16. C. Yanagimoto, M. Harada, H. Kumemura, H. Koga, T. Kawaguchi, K. Terada, S. Hanada, E. Taniguchi, Y. Koizumi, S. Koyota, H. Ninomiya, T. Ueno, T. Sugiyama, M. Sata, Exper. Cell Res. 2009 , 315 , 119–126. 17. M. Broderius, E. Mostad, K. Wendroth, J. R. Prohaska, Comp. Biochem. Physiol. Toxicol. Pharmacol. 2010 , 151 , 473–479. 18. Z. L. Harris, Y. Takahashi, H. Miyajima, M. Serizawa, R. T. MacGillivray, J. D. Gitlin, Proc. Nat. Acad. Sci. USA 1995 , 92 , 2539–2543. 19. R. B. Rucker, T. Kosonen, M. S. Clegg, A. E. Mitchell, B. R. Rucker, J. Y. Uriu-Hare, C. L. Keen, Am. J. Clin. Nutr. 1998 , 67 , 996s–1002s. 20. T. Kosonen, J. Y. Uriu-Hare, M. S. Clegg, C. L. Keen, R. B. Rucker, Biochem. J. 1997 , 327 , 283–289. 21. D. M. Danks, in The Metabolic and Molecular Basis of Inherited Disease , Vol. 1, Eds C. R. Scriver, A. L. Beaudet, W. M. Sly, D. Valle, McGraw-Hill, New York, 1995, pp. 2211–2235. 22. S. G. Kaler, Nature Reviews. Neurology 2011 , 7 , 15–29. 23. M. J. Petris, D. Strausak, J. F. B. Mercer, Hum. Mol. Genet. 2000 , 9 , 2845–2851. 24. C. Olivares, F. Solano, Pigment Cell & Melanoma Research 2009 , 22 , 750–760. 25. S. R. Setty, D. Tenza, E. V. Sviderskaya, D. C. Bennett, G. Raposo, M. S. Marks, Nature 2008 , 454 , 1142–1146. 26. J. P. Klinman, J. Biol. Chem. 2006 , 281 , 3013–3016. 27. D. Bousquet-Moore, J. R. Prohaska, E. A. Nillni, T. Czyzyk, W. C. Wetsel, R. E. Mains, B. A. Eipper, Neurobiology of Disease 2010 , 37 , 130–140. 28. D. Bousquet-Moore, R. E. Mains, B. A. Eipper, J. Neurosci. Res. 2010 , 88 , 2535–2545. 29. S. G. Kaler, C. S. Holmes, D. S. Goldstein, J. Tang, S. C. Godwin, A. Donsante, C. J. Liew, S. Sato, N. Patronas, New England J. Med. 2008 , 358 , 605–614. 30. R. El Meskini, V. C. Culotta, R. E. Mains, B. A. Eipper, J. Biol. Chem. 2003 , 278 , 12278–12284. 31. M. J. Niciu, X. M. Ma, R. El Meskini, J. S. Pachter, R. E. Mains, B. A. Eipper, Neurobiology of Disease 2007 , 27 , 278–291. 32. A. Dancis, D. S. Yuan, D. Haile, C. Askwith, D. Eide, C. Moehle, J. Kaplan, R. D. Klausner, Cell 1994 , 76 , 393–402. 33. B. Zhou, J. Gitschier, Proc. Nat. Acad. Sci. USA 1997 , 94 , 7481–7486. 34. B. E. Kim, T. Nevitt, D. J. Thiele, Nature Chem. Biol. 2008 , 4 , 176–185. 35. J. Lee, M. M. Pena, Y. Nose, D. J. Thiele, J. Biol. Chem. 2002 , 277 , 4380–4387. 36. Y. Wang, V. Hodgkinson, S. Zhu, G. A. Weisman, M. J. Petris, Adv. Nutr. 2011 , 2 , 129–137. 37. J. F. Eisses, J. H. Kaplan, J. Biol. Chem. 2005 , 280 , 37159–37168. 38. S. Puig, J. Lee, M. Lau, D. J. Thiele, J. Biol. Chem. 2002 , 277 , 26021–26030. 39. S. G. Aller, V. M. Unger, Proc. Nat. Acad. Sci. USA 2006 , 103 , 3627–3632. 40. Y. M. Kuo, A. A. Gybina, J. W. Pyatskowit, J. Gitschier, J. R. Prohaska, J. Nutr. 2006 , 136 , 21–26. 41. J. Lee, J. R. Prohaska, D. J. Thiele, Proc. Natl. Acad. Sci. USA 2001 , 98 , 6842–6847. 42. A. E. Klomp, B. B. Tops, I. E. Van Denberg, R. Berger, L. W. Klomp, Biochem. J. 2002 , 364 , 497–505. 43. Y. Nose, L. K. Wood, B. E. Kim, J. R. Prohaska, R. S. Fry, J. W. Spears, D. J. Thiele, J. Biol. Chem. 2010 , 285 , 32385–32392. 44. T. D. Rae, P. J. Schmidt, R. A. Pufahl, V. C. Culotta, T. V. O'Halloran, Science 1999 , 284 , 805–808. 45. P. A. Cobine, L. D. Ojeda, K. M. Rigby, D. R. Winge, J. Biol. Chem. 2004 , 279 , 14447–14455. 46. J. Hirrlinger, R. Dringen, Brain Res. Rev. 2010 , 63 , 177–188. 47. K. Jomova, D. Vondrakova, M. Lawson, M. Valko, Mol. Cell. Biochem. 2010 , 345 , 91–104. 48. J. H. Freedman, M. R. Ciriolo, J. Peisach, J. Biol. Chem. 1989 , 264 , 5598–5605. 49. L. Banci, I. Bertini, S. Cioﬁ -Baffoni, T. Kozyreva, K. Zovo, P. Palumaa, Nature 2010 , 465 , 645–648. 11 Copper: Effects of Deﬁ ciency and Overload 383

50. W. C. Singleton, K. T. McInnes, M. A. Cater, W. R. Winnall, R. McKirdy, Y. Yu, P. E. Taylor, B. X. Ke, D. R. Richardson, J. F. Mercer, S. La Fontaine, J. Biol. Chem. 2010 , 285 , 27111–27121. 51. M. Vašak, G. Meloni, J. Biol. Inorg. Chem. 2011 , 16 , 1067–1078. 52. J. Hidalgo, M. Aschner, P. Zatta, M. Vašak, Brain Res. Bull. 2001 , 55 , 133–145. 53. W. Maret, J. Biol. Inorg. Chem. 2011 , 16 , 1079–1086. 54. R. A. Colvin, W. R. Holmes, C. P. Fontaine, W. Maret, Metallomics 2010 , 2 , 306–317. 55. N. Thirumoorthy, A. Shyam Sunder, K. Manisenthil Kumar, M. Senthil Kumar, G. Ganesh, M. Chatterjee, World J. Surg. Oncol. 2011 , 9 , 54. 56. M. G. Cherian, Y. J. Kang, Exp. Biol. Med. (Maywood) 2006 , 231 , 138–144. 57. B. A. Masters, C. J. Quaife, J. C. Erickson, E. J. Kelly, G. J. Froelick, B. P. Zambrowicz, R. L. Brinster, R. D. Palmiter, J. Neurosci. 1994 , 14 , 5844–5857. 58. C. J. Quaife, S. D. Findley, J. C. Erickson, G. J. Froelick, E. J. Kelly, B. P. Zambrowicz, R. D. Palmiter, Biochemistry 1994 , 33 , 7250–7259. 59. S. A. Wake, J. F. Mercer, Biochem. J. 1985 , 228 , 425–432. 60. J. F. Mercer, A. Grimes, H. Rauch, J. Nutr. 1992 , 122 , 1254–1259. 61. T. P. Mulder, A. R. Janssens, H. W. Verspaget, J. van Hattum, C. B. Lamers, J. Hepatol. 1992 , 16 , 346–350. 62. J. H. Freedman, J. Peisach, Biochim. Biophys. Acta 1989 , 992 , 145–154. 63. N. J. Robinson, D. R. Winge, Ann. Rev. Biochem. 2010 , 79 , 537–562. 64. S. J. Lin, V. C. Culotta, Proc. Nat. Acad. Sci. USA 1995 , 92 , 3784–3788. 65. S. J. Lin, R. A. Pufahl, A. Dancis, T. V. O'Halloran, V. C. Culotta, J. Biol. Chem. 1997 , 272 , 9215–9220. 66. L. Banci, I. Bertini, F. Cantini, A. C. Rosenzweig, L. A. Yatsunyk, Biochemistry 2008 , 47 , 7423–7429. 67. I. Hamza, M. Schaefer, L. W. Klomp, J. D. Gitlin, Proc. Nat. Acad. Sci. USA 1999 , 96 , 13363–13368. 68. K. Szigeti, J. R. Lupski, Eur. J. Hum. Genet. 2009 , 17 , 703–710. 69. D. Strausak, M. K. Howie, S. D. Firth, A. Schlicksupp, R. Pipkorn, G. Multhaup, J. F. Mercer, J. Biol. Chem. 2003 , 278 , 20821–20827. 70. V. C. Culotta, L. W. Klomp, J. Strain, R. L. Casareno, B. Krems, J. D. Gitlin, J. Biol. Chem. 1997 , 272 , 23469–23472. 71. J. M. Leitch, P. J. Yick, V. C. Culotta, J. Biol. Chem. 2009 , 284 , 24679–24683. 72. J. R. Prohaska, M. Broderius, B. Brokate, Arch. Biochem. Biophys. 2003 , 417 , 227–234. 73. A. L. Caruano-Yzermans, T. B. Bartnikas, J. D. Gitlin, J. Biol. Chem. 2006 , 281 , 13581–13587. 74. D. M. Glerum, A. Shtanko, A. Tzagoloff, J. Biol. Chem. 1996 , 271 , 14504–14509. 75. A. B. Maxﬁ eld, D. N. Heaton, D. R. Winge, J. Biol. Chem. 2004 , 279 , 5072–5080. 76. S. C. Leary, P. A. Cobine, B. A. Kaufman, G. H. Guercin, A. Mattman, J. Palaty, G. Lockitch, D. R. Winge, P. Rustin, R. Horvath, E. A. Shoubridge, Cell Metab. 2007 , 5 , 9–20. 77. S. C. Leary, F. Sasarman, T. Nishimura, E. A. Shoubridge, Hum. Mol. Genet. 2009 , 18 , 2230–2240. 78. J. Chelly, Z. Turmer, T. Tonnerson, A. Petterson, Y. Ishikawa-Brush, N. Tommerup, N. Horn, A. P. Monaco, Nat. Genet. 1993 , 3 , 14–19. 79. J. F. Mercer, J. Livingston, B. Hall, J. A. Paynter, C. Begy, S. Chandrasekharappa, P. Lockhart, A. Grimes, M. Bhave, D. Siemieniak, T. W. Glover, Nat. Genet. 1993 , 3 , 20–25. 80. C. Vulpe, B. Levinson, S. Whitney, S. Packman, J. Gitschier, Nat. Genet. 1993 , 3 , 7–13. 81. P. C. Bull, G. R. Thomas, J. M. Rommens, J. R. Forbes, D. C. Cox, Nat. Genet. 1993 , 5 , 327–337. 82. R. E. Tanzi, K. Petrukhin, I. Chernov, J. L. Pellequer, W. Wasco, B. Ross, D. M. Romano, E. Parano, L. Pavone, L. M. Brzustowicz, M. Devoto, J. Peppercorn, A. I. Bush, I. Sternlieb, M. Pirastu, J. F. Gusella, O. Evgrafov, G. K. Penchaszadeh, B. Honig, I. S. Edelman, M. B. Soares, I. H. Scheinberg, T. C. Gilliam, Nat. Genet. 1993 , 5 , 344–350. 384 Scheiber, Dringen, and Mercer

83. S. Lutsenko, N. L. Barnes, M. Y. Bartee, O. Y. Dmitriev, Physiol. Rev. 2007, 87 , 1011–1046. 84. S. La Fontaine, J. F. Mercer, Arch. Biochem. Biophys. 2007 , 463 , 149–167. 85. S. La Fontaine, M. L. Ackland, J. F. Mercer, Int. J. Biochem. Cell Biol. 2010 , 42 , 206–209. 86. D. Huster, Best Practice & Research. Clinical Gastroenterology 2010 , 24 , 531–539. 87. S. G. Kaler, Adv. Pediat. 1994 , 41 , 263–304. 88. A. N. Barry, U. Shinde, S. Lutsenko, J. Biol. Inorg. Chem. 2010 , 15 , 47–59. 89. S. Lutsenko, E. S. LeShane, U. Shinde, Arch. Biochem. Biophys. 2007 , 463 , 134–148. 90. D. Strausak, S. La Fontaine, J. Hill, S. D. Firth, P. J. Lockhart, J. F. Mercer, J. Biol. Chem. 1999 , 274 , 11170–11177. 91. J. R. Forbes, D. W. Cox, Hum. Mol. Genet. 2000 , 9 , 1927–1935. 92. M. A. Cater, J. Forbes, S. La Fontaine, D. Cox, J. F. Mercer, Biochem. J. 2004 , 380 , 805–813. 93. I. Voskoboinik, J. Mar, D. Strausak, J. Camakaris, J. Biol. Chem. 2001 , 276 , 28620–28627. 94. M. J. Petris, I. Voskoboinik, M. Cater, K. Smith, B. E. Kim, R. M. Llanos, D. Strausak, J. Camakaris, J. F. Mercer, J. Biol. Chem. 2002 , 277 , 46736–46742. 95. M. A. Cater, S. La Fontaine, K. Shield, Y. Deal, J. F. Mercer, Gastroenterology 2006 , 130 , 493–506. 96. M. J. Petris, J. F. Mercer, J. G. Culvenor, P. Lockhart, P. A. Gleeson, J. Camakaris, EMBO J. 1996 , 15 , 6084–6095. 97. B. E. Kim, K. Smith, M. J. Petris, J. Med. Genet. 2003 , 40 , 290–295. 98. L. Ambrosini, J. F. Mercer, Hum. Mol. Genet. 1999 , 8 , 1547–1555. 99. A. M. Zimnicka, E. B. Maryon, J. H. Kaplan, J. Biol. Chem. 2007 , 282 , 26471–26480. 100. Y. Nose, B. E. Kim, D. J. Thiele, Cell Metab. 2006 , 4 , 235–244. 101. J. R. Turnlund, W. R. Keyes, H. L. Anderson, L. L. Acord, Am. J. Clin. Nutr. 1989 , 49 , 870–878. 102. J. F. Monty, R. M. Llanos, J. F. Mercer, D. R. Kramer, J. Nutr. 2005 , 135 , 2762–2766. 103. J. A. Paynter, A. Grimes, P. Lockhart, J. F. B. Mercer, FEBS Lett. 1994 , 351 , 186–190. 104. H. Kodama, C. Fujisawa, W. Bhadhprasit, Brain Devel. 2011 , 33 , 243–251. 105. Z. Tumer, L. B. Moller, Eur. J. Hum. Genet. 2010 , 18 , 511–518. 106. R. F. Pfeiffer, Seminars in Neurology 2007 , 27 , 123–132. 107. D. M. Danks, Ann. Rev. Nutr. 1988 , 8 , 235–257. 108. D. L. de Romana, M. Olivares, R. Uauy, M. Araya, J. Trace Elem. Med. Biol. 2011 , 25 , 3–13. 109. P. A. Walravens, Clin. Chem. 1980 , 26 , 185–189. 110. M. Shike, Gastroenterology 2009 , 137 , S13–17. 111. A. Cordano, Am. J. Clin. Nutr. 1998 , 67 , 1012S–1016S. 112. S. Jameson, K. Hellsing, S. Magnusson, Sci. Tot. Envir. 1985 , 42 , 29–36. 113. L. M. Klevay, J. Trace Elem. Med. Biol. 2011 , 25 , 204–212. 114. J. Horvath, P. Beris, E. Giostra, P. Y. Martin, P. R. Burkhard, J. Neurol., Neurosurg., Psych. 2010 , 81 , 1410–1411. 115. K. B. O'Donnell, M. Simmons, Nutr. Clin. Pract. 2011 , 26 , 66–69. 116. R. Uauy, M. Olivares, M. Gonzalez, Am. J. Clin. Nutr. 1998 , 67 , 952s–959s. 117. T. R. Halfdanarson, N. Kumar, C. Y. Li, R. L. Phyliky, W. J. Hogan, Eur. J. Haematol. 2008 , 80 , 523–531. 118. A. M. Sutton, A. Harvie, F. Cockburn, J. Farquharson, R. W. Logan, Arch. Dis. Childhood 1985 , 60 , 644–651. 119. S. R. Jaiser, G. P. Winston, J. Neurol. 2010 , 257 , 869–881. 120. P. L. Fox, BioMetals 2003 , 16 , 9–40. 121. J. F. Collins, J. R. Prohaska, M. D. Knutson, Nutr. Rev. 2010 , 68 , 133–147. 122. J. R. Prohaska, Adv. Nutr. 2011 , 2 , 89–95. 123. J. Lazarchick, Curr. Opin. Hematol. 2012 , 19 , 58–60. 124. H. W. Bennetts, F. E. Chapman, Aust. Vet. J. 1937 , 13 , 138–149. 125. N. Kumar, J. E. Ahlskog, J. B. Gross, Jr., Clin. Gastroenterol. Hepatol. 2004 , 2 , 1074–1079. 126. J. C. Tan, D. L. Burns, H. R. Jones, J. Parent. Enter. Nutr. 2006 , 30 , 446–450. 127. J. Bremer, F. Baumann, C. Tiberi, C. Wessig, H. Fischer, P. Schwarz, A. D. Steele, K. V. Toyka, K. A. Nave, J. Weis, A. Aguzzi, Nature Neurosci. 2010 , 13 , 310–318. 11 Copper: Effects of Deﬁ ciency and Overload 385

128. H. Kuivaniemi, L. Peltonen, A. Palotie, I. Kaitila, K. I. Kivirikko, J. Clin. Invest. 1982 , 69 , 730–733. 129. J. D. Hicks, A. Donsante, T. M. Pierson, M. J. Gillespie, D. E. Chou, S. G. Kaler, Clin. Dysmorphol. 2012 , 21 , 59–63. 130. H. Nojiri, Y. Saita, D. Morikawa, K. Kobayashi, C. Tsuda, T. Miyazaki, M. Saito, K. Marumo, I. Yonezawa, K. Kaneko, T. Shirasawa, T. Shimizu, J. Bone Mineral Res. 2011 , 26 , 2682–2694. 131. M. L. Marquardt, S. L. Done, M. Sandrock, W. E. Berdon, K. W. Feldman, Pediatrics 2012 , 130 , e695–698. 132. L. M. Klevay, J. Nutr. 2000 , 130 , 489S–492S. 133. E. J. Underwood, in Trace Elements in Human and Animal Nutrition , Academic Press, New York, 1977, pp. 56–108. 134. J. Aaseth, G. Boivin, O. Andersen, J. Trace Elem. Med. Biol. 2012 , 26 , 149–152. 135. J. J. Strain, Med. Hypoth. 1988 , 27 , 333–338. 136. Z. Qin, S. Itoh, V. Jeney, M. Ushio-Fukai, T. Fukai, FASEB J. 2006 , 20 , 334–336. 137. Z. Qin, M. C. Gongora, K. Ozumi, S. Itoh, K. Akram, M. Ushio-Fukai, D. G. Harrison, T. Fukai, Hypertension 2008 , 52 , 945–951. 138. Z. Qin, E. S. Konaniah, B. Neltner, R. A. Nemenoff, D. Y. Hui, N. L. Weintraub, J. Lipid Res. 2010 , 51 , 1471–1477. 139. J. R. Prohaska, O. A. Lukasewycz, Science 1981 , 213 , 559–561. 140. P. M. Newberne, C. E. Hunt, V. R. Young, Brit. J. Exper. Pathol. 1968 , 49 , 448–457. 141. R. Boyne, J. R. Arthur, J. Comp. Pathol. 1981 , 91 , 271–276. 142. J. Kreuder, A. Otten, H. Fuder, Z. Tumer, T. Tonnesen, N. Horn, D. Dralle, Eur. J. Pediatr. 1993 , 152 , 828–832. 143. R. G. Hopkins, M. L. Failla, J. Nutr. 1997 , 127 , 257–262. 144. I. Prudovsky, A. Mandinova, R. Soldi, C. Bagala, I. Graziani, M. Landriscina, F. Tarantini, M. Duarte, S. Bellum, H. Doherty, T. Maciag, J. Cell Sci. 2003 , 116 , 4871–4881. 145. V. Hodgkinson, M. J. Petris, J. Biol. Chem. 2012 , 287 , 13549–13555. 146. C. White, J. Lee, T. Kambe, K. Fritsche, M. J. Petris, J. Biol. Chem. 2009 , 284 , 33949–33956. 147. C. L. Keen, J. Y. Uriu-Hare, S. N. Hawk, M. A. Jankowski, G. P. Daston, C. L. Kwik-Uribe, R. B. Rucker, Am. J. Clin. Nutr. 1998 , 67 , 1003S–1011S. 148. B. Hardman, U. Manuelpillai, E. M. Wallace, S. van de Waasenburg, M. Cater, J. F. Mercer, M. L. Ackland, Placenta 2004 , 25 , 512–517. 149. J. F. B. Mercer, Am. J. Clin. Nutr. 1998 , 5 , 1027S–1028S. 150. A. A. Michalczyk, J. Rieger, K. J. Allen, J. F. Mercer, M. L. Ackland, Biochem. J. 2000 , 352 Pt 2 , 565–571. 151. M. B. Theophilos, D. W. Cox, J. F. B. Mercer, Hum. Mol. Genet. 1996 , 5 , 1619–1624. 152. Y. Wang, S. Zhu, V. Hodgkinson, J. R. Prohaska, G. A. Weisman, J. D. Gitlin, M. J. Petris, Am. J. Physiol. Gastrointest. Liver Physiol. 2012 , 303 , G1236–1244. 153. Y. Wang, S. Zhu, G. A. Weisman, J. D. Gitlin, M. J. Petris, PloS one 2012 , 7 , e43039. 154. H. Kodama, C. Fujisawa, W. Bhadhprasit, Brain Dev. 2011 , 33 , 243–251. 155. S. G. Kaler, Nat. Rev. Neurol. 2011 , 7 , 15–29. 156. M. L. Schlief, J. D. Gitlin, Mol. Neurobiol. 2006 , 33 , 81–90. 157. J. F. Mercer, Trends Mol. Med. 2001 , 7 , 64–69. 158. M. L. Kennerson, G. A. Nicholson, S. G. Kaler, B. Kowalski, J. F. Mercer, J. Tang, R. M. Llanos, S. Chu, R. I. Takata, C. E. Speck-Martins, J. Baets, L. Almeida-Souza, D. Fischer, V. Timmerman, P. E. Taylor, S. S. Scherer, T. A. Ferguson, T. D. Bird, P. De Jonghe, S. M. Feely, M. E. Shy, J. Y. Garbern, Am. J. Hum. Genet. 2010 , 86 , 343–352. 159. N. Franchitto, P. Gandia-Mailly, B. Georges, A. Galinier, N. Telmon, J. L. Ducasse, D. Rouge, Resuscitation 2008 , 78 , 92–96. 160. I. H. Scheinberg, I. Sternlieb, Am. J. Clin. Nutr. 1996 , 63 , 842S–845S. 161. T. Muller, W. Muller, H. Feichtinger, Am. J. Clin. Nutr. 1998 , 67 , 1082S–1086S. 162. M. S. Tanner, Am. J. Clin. Nutr. 1998 , 67 , 1074S–1081S. 163. R. J. O'Brien, P. C. Wong, Ann. Rev. Neurosci. 2011 , 34 , 185–204. 386 Scheiber, Dringen, and Mercer

164. C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones, Lancet 2011 , 377 , 1019–1031. 165. M. A. Lovell, J. D. Robertson, W. J. Teesdale, J. L. Campbell, W. R. Markesbery, J. Neurol. Sci. 1998 , 158 , 47–52. 166. H. Akatsu, A. Hori, T. Yamamoto, M. Yoshida, M. Mimuro, Y. Hashizume, I. Tooyama, E. M. Yezdimer, BioMetals 2012 , 25 , 337–350. 167. A. I. Bush, Trends Neurosci. 2003 , 26 , 207–214. 168. L. M. Miller, Q. Wang, T. P. Telivala, R. J. Smith, A. Lanzirotti, J. Miklossy, J. Struct. Biol. 2006 , 155 , 30–37. 169. B. R. Roberts, T. M. Ryan, A. I. Bush, C. L. Masters, J. A. Duce, J. Neurochem. 2012 , 120 , 149–166. 170. M. A. Cater, K. T. McInnes, Q. X. Li, I. Volitakis, S. La Fontaine, J. Mercer, A. I. Bush, Biochem. J. 2008 , 412, 141–152. 171. A. Hopt, S. Korte, H. Fink, U. Panne, R. Niessner, R. Jahn, H. Kretzschmar, J. Herms, J. Neurosci. Methods 2003 , 128 , 159–172. 172. J. Kardos, I. Kovacs, F. Hajos, M. Kalman, M. Simonyi, Neurosci. Lett. 1989 , 103 , 139–144. 173. S. Lesne, C. Ali, C. Gabriel, N. Croci, E. T. MacKenzie, C. G. Glabe, M. Plotkine, C. Marchand-Verrecchia, D. Vivien, A. Buisson, J. Neurosci. 2005 , 25 , 9367–9377. 174. V. B. Kenche, K. J. Barnham, Brit. J. Pharmacol. 2011 , 163 , 211–219. 175. P. A. Adlard, A. I. Bush, J. Alzheimer's Dis. 2006 , 10 , 145–163. 176. T. A. Bayer, S. Schafer, A. Simons, A. Kemmling, T. Kamer, R. Tepest, A. Eckert, K. Schussel, O. Eikenberg, C. Sturchler-Pierrat, D. Abramowski, M. Staufenbiel, G. Multhaup, Proc. Natl. Acad. Sci. USA 2003 , 100 , 14187–14192. 177. C. J. Maynard, R. Cappai, I. Volitakis, R. A. Cherny, A. R. White, K. Beyreuther, C. L. Masters, A. I. Bush, Q. X. Li, J. Biol. Chem. 2002 , 277 , 44670–44676. 178. P. J. Crouch, L. W. Hung, P. A. Adlard, M. Cortes, V. Lal, G. Filiz, K. A. Perez, M. Nurjono, A. Caragounis, T. Du, K. Laughton, I. Volitakis, A. I. Bush, Q. X. Li, C. L. Masters, R. Cappai, R. A. Cherny, P. S. Donnelly, A. R. White, K. J. Barnham, Proc. Nat. Acad. Sci. USA 2009 , 106 , 381–386. 179. H. Kessler, T. A. Bayer, D. Bach, T. Schneider-Axmann, T. Supprian, W. Herrmann, M. Haber, G. Multhaup, P. Falkai, F. G. Pajonk, J. Neural Transmission 2008 , 115 , 1181–1187. 180. I. Maurer, S. Zierz, H. J. Moller, Neurobiology of Aging 2000 , 21 , 455–462. 181. D. L. Marcus, C. Thomas, C. Rodriguez, K. Simberkoff, J. S. Tsai, J. A. Strafaci, M. L. Freedman, Exper. Neurol. 1998 , 150 , 40–44. 182. V. B. Kenche, K. J. Barnham, Br. J. Pharmacol. 2011 , 163 , 211–219. 183. P. Zatta, D. Drago, S. Bolognin, S. L. Sensi, Trends Pharmacol. Sci. 2009 , 30 , 346–355. 184. B. Thomas, M. F. Beal, Human Mol. Genet. 2007 , 16 Spec. No. 2 , R183–194. 185. M. T. Lorincz, Ann. NY Acad. Sci. 2010 , 1184 , 173–187. 186. X. Wang, D. Moualla, J. A. Wright, D. R. Brown, J. Neurochem. 2010 , 113 , 704–714. 187. D. T. Dexter, F. R. Wells, A. J. Lees, F. Agid, Y. Agid, P. Jenner, C. D. Marsden, J. Neurochem. 1989 , 52 , 1830–1836. 188. D. A. Loefﬂ er, P. A. LeWitt, P. L. Juneau, A. A. Sima, H. U. Nguyen, A. J. DeMaggio, C. M. Brickman, G. J. Brewer, R. D. Dick, M. D. Troyer, L. Kanaley, Brain Res. 1996 , 738 , 265–274. 189. K. L. Double, Parkinsonism Relat. Disorders 2012 , 18 , S52–54. 190. M. Alcaraz–Zubeldia, M. C. Boll-Woehrlen, S. Montes-Lopez, F. Perez-Severiano, J. C. Martinez-Lazcano, A. Diaz-Ruiz, C. Rios, Rev. Invest. Clin. 2009 , 61 , 405–411. 191. M. B. Youdim, E. Grunblatt, S. Mandel, J. Neural Transmission 2007 , 114 , 205–209. 192. L. W. Hung, V. L. Villemagne, L. Cheng, N. A. Sherratt, S. Ayton, A. R. White, P. J. Crouch, S. Lim, S. L. Leong, S. Wilkins, J. George, B. R. Roberts, C. L. Pham, X. Liu, F. C. Chiu, D. M. Shackleford, A. K. Powell, C. L. Masters, A. I. Bush, G. O'Keefe, J. G. Culvenor, R. Cappai, R. A. Cherny, P. S. Donnelly, A. F. Hill, D. I. Finkelstein, K. J. Barnham, J. Exper. Med. 2012 , 209 , 837–854. 11 Copper: Effects of Deﬁ ciency and Overload 387

193. K. M. Shannon, Handbook of Clinical Neurology 2011 , 100 , 3–13. 194. K. N. McFarland, J. H. Cha, Handbook of Clinical Neurology 2011 , 100 , 25–81. 195. J. H. Fox, J. A. Kama, G. Lieberman, R. Chopra, K. Dorsey, V. Chopra, I. Volitakis, R. A. Cherny, A. I. Bush, S. Hersch, PloS one 2007 , 2 , e334. 196. S. J. Tallaksen-Greene, A. Janiszewska, K. Benton, G. Hou, R. Dick, G. J. Brewer, R. L. Albin, Neurosci. Lett. 2009 , 452 , 60–62. 197. A. E. Harding, P. K. Thomas, J. Med. Genet. 1980 , 17 , 329–336. 198. A. Bento-Abreu, P. Van Damme, L. Van Den Bosch, W. Robberecht, Eur. J. Neurosci. 2010 , 31 , 2247–2265. 199. E. Tokuda, E. Okawa, S. Ono, J. Neurochem. 2009 , 111 , 181–191. 200. N. D. Merner, P. A. Dion, G. A. Rouleau, Clin. Genet. 2011 , 79 , 23–34. 201. R. C. Holman, E. D. Belay, K. Y. Christensen, R. A. Maddox, A. M. Minino, A. M. Folkema, D. L. Haberling, T. A. Hammett, K. D. Kochanek, J. J. Sejvar, L. B. Schonberger, PloS one 2010 , 5 , e8521. 202. W. Hu, B. Kieseier, E. Frohman, T. N. Eagar, R. N. Rosenberg, H. P. Hartung, O. Stuve, J. Neurol. Sci. 2008 , 264 , 1–8. 203. C. S. Burns, E. Aronoff-Spencer, G. Legname, S. B. Prusiner, W. E. Antholine, G. J. Gerfen, J. Peisach, G. L. Millhauser, Biochemistry 2003 , 42 , 6794–6803. 204. C. L. Haigh, D. R. Brown, J. Neurochem. 2006 , 98 , 677–689. 205. L. R. Brown, D. A. Harris, J. Neurochem. 2003 , 87 , 353–363. 206. D. R. Brown, K. Qin, J. W. Herms, A. Madlung, J. Manson, R. Strome, P. E. Fraser, T. Kruck, A. von Bohlen, W. Schultz-Schaeffer, A. Giese, D. Westaway, H. Kretzschmar, Nature 1997 , 390 , 684–687. 207. E. Urso, D. Manno, A. Serra, A. Buccolieri, A. Rizzello, A. Danieli, R. Acierno, B. Salvato, M. Mafﬁ a, Cell. Mol. Neurobiol. 2012 , 32 , 989–1001. 208. P. K. Stys, H. You, G. W. Zamponi, J. Physiol. 2012 , 590 , 1357–1368. 209. O. M. Siggs, J. T. Cruite, X. Du, S. Rutschmann, E. Masliah, B. Beutler, M. B. Oldstone, Proc. Nat. Acad. Sci. USA 2012 , 109 , 13733–13738. Chapter 12 Zinc and Human Disease

Wolfgang Maret

Contents ABSTRACT ...... 390 1 INTRODUCTION ...... 390 2 ZINC BIOCHEMISTRY ...... 391 2.1 Zinc in Enzymes and Proteins ...... 392 2.1.1 Catalytic Zinc ...... 392 2.1.2 Structural Zinc ...... 392 2.1.3 Regulatory Zinc ...... 393 2.2 Zinc in Vesicles: Intracellular and Intercellular Signaling with Zinc(II) Ions ...... 393 2.3 Cellular Zinc Homeostasis ...... 394 2.4 Zinc and Oxidoreduction (Redox) ...... 395 2.5 Global Functions of Zinc ...... 396 3 ZINC IN ORGAN PATHOPHYSIOLOGY ...... 398 3.1 Liver and Gastrointestinal System ...... 398 3.2 Cardiovascular and Pulmonary System ...... 399 3.3 Immune System ...... 400 3.4 Central and Peripheral Nervous System ...... 401 3.5 Reproductive System ...... 402 3.6 Sensory Systems ...... 403 3.7 Other Systems ...... 403 4 ZINC IN DISEASE ...... 404 4.1 Genetic Disease ...... 404 4.2 Metabolic and Chronic Disease ...... 404 4.2.1 Diabetes ...... 404 4.2.2 Cancer ...... 406 4.2.3 Neurodegeneration ...... 406 4.3 Infectious Disease ...... 407 5 GENERAL CONCLUSIONS ...... 407

W. Maret (*) King’s College London, School of Medicine, Diabetes and Nutritional Sciences Division, Metal Metabolism Group, London, SE1 9NH, UK e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 389 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_12, © Springer Science+Business Media Dordrecht 2013 390 Maret

ABBREVIATIONS ...... 409 ACKNOWLEDGMENT ...... 409 REFERENCES ...... 409

Abstract The vast knowledge of the physiologic functions of zinc in at least 3000 proteins and the recent recognition of fundamental regulatory functions of zinc(II) ions released from cells or within cells links this nutritionally essential metal ion to numerous diseases. However, this knowledge so far has had remarkably limited impact on diagnosing, preventing, and treating human diseases. One major road- block is a lack of suitable biomarkers that would detect changes in cellular zinc metabolism and relate them to specifi c disease outcomes. It is not only the right amount of zinc in the diet that maintains health. At least as important is the proper functioning of the dozens of proteins that control cellular zinc homeostasis, regulate intracellular traffi c of zinc between the cytosol and vesicles/organelles, and determine the fl uctuations of signaling zinc(II) ions. Cellular zinc defi ciencies or overloads, a term referring to zinc concentrations exceeding the cellular zinc buffering capacity, compromise the redox balance. Zinc supplementation may not readily remedy zinc defi ciency if other factors limit the capability of a cell to control zinc. The role of zinc in human diseases requires a general understanding of the wide spectrum of functions of zinc, how zinc is controlled, how it interacts with the metabolism of other metal ions, in particular copper and iron, and how perturbation of specifi c zinc-dependent molecular processes causes disease and infl uences the progression of disease.

Keywords human diseases • zinc • zinc homeostasis • zinc metalloproteins • zinc signaling

Please cite as: Met. Ions Life Sci. 13 (2013) 389–414

1 Introduction

The biochemistry of zinc deserves much more attention than it generally receives in textbooks in the biomedical sciences and in some parts of the scientifi c literature. There are historic reasons for this lack of coverage. Unlike iron, which was easily detected and analyzed in blood and tissues due to the color of its complexes, zinc compounds are colorless. Therefore the fi eld of zinc biology developed much later and only with the advent of new methods to analyze zinc in biological material. Also, unlike iron, where a large amount is found in heme, zinc is not predominantly part of a single substance but instead serves as a cofactor of at least 3000 human proteins. This distribution among so many proteins dilutes zinc and requires sensitive methods for the speciation and characterization of proteins. Zinc is involved in a much wider variety of processes and molecular mechanisms than vitamins and other cofactors with more specifi c chemical functions. The notion of zinc being a 12 Zinc and Human Disease 391 trace metal also somewhat confuscates the issues at hand. While the total amount of zinc in a human (70 kg) is 2–3 g and about as much as that of iron, cellular zinc ion concentrations are rather high, almost as high as those of major metabolites such as ATP. A multi-authored text summarizes the knowledge accumulated since the fi eld was last reviewed two decades ago in terms of the biochemical basis of zinc physiology as it relates to the numerous functions of zinc in metalloenzymes and transcription factors [1 , 2 ]. What has changed in the time between these accounts is the increase of the number of zinc proteins by one order of magnitude, demonstrating a much more general role of zinc in protein structure and in protein-protein interactions, an understanding of the biological and chemical aspects of how cellular zinc is controlled (zinc homeostasis), and the discovery that zinc(II) ions function in cellular regulation and information transfer. All these advances demonstrate that the present knowledge has by far surpassed the already impressive zinc biochemistry, which in 1993 was deemed to be, based on functions of zinc, “too numerous to cite” [2 ]. The implications of zinc biology for human health are enormous as about half of the world’s population is believed to be at risk for zinc defi ciency [3 ]. The World Health Organization (WHO) has identifi ed zinc defi ciency as the fi fth most important risk factor for morbidity and mortality in developing countries (11th worldwide) [4 ]. The fi gure translates into 3.2% of all lost disability-adjusted life years (DALYs). These estimates are derived primarily from the incidence of infectious and parasitic diseases due to compromised immune functions in zinc defi ciency. Clearly, this measured outcome is far from inclusive. It does not take into account the functions of zinc in human memory acquisition and storage, behavior, growth retardation and development, delayed wound healing, the effects of environmental exposures to substances interfering with zinc metabolism, or the role of zinc in aging and in chronic diseases, such as cancer, diabetes, and neurodegeneration. Rather than trying to summarize all the functions of zinc in physiology, which would be an immense task well beyond the scope of this chapter, the subject matter is approached by discussing the general signifi cance of zinc in biochemistry for health and the specifi c involvement of zinc in pathophysiology and diseases at the molecular level. This approach will necessitate a certain degree of selectivity when referencing from the immense literature.

2 Zinc Biochemistry

Almost all our knowledge about the molecular roles of zinc is based on the interaction of zinc with proteins. Whether any interactions with other biomolecules are important is not known. Zinc has a role in enzymatic catalysis and in the structure and regulation of proteins. The coordination chemistry of zinc in proteins has been discussed in detail [5 ]. A major aspect of the cellular biology of zinc includes the storage of zinc(II) ions in cellular vesicles/organelles, in which relatively high concentrations can be reached and from which zinc(II) ions are released in a 392 Maret controlled way. In this regard, zinc resembles calcium and is quite different from iron, which uses redox chemistry of the central atom and a protein (ferritin) for storage and release.

2.1 Zinc in Enzymes and Proteins

Discoveries of zinc in numerous proteins demonstrated the key role of zinc for life. They occurred in the order of (i) zinc as a catalytic ion in enzymes, (ii) zinc in the structure of proteins, and (iii) zinc in the regulation of proteins [6 ]. By analyzing sequences of proteins from databases and detecting signatures for metal-binding sites with characteristically spaced amino acids providing the ligands, it became possible to estimate the number of zinc proteins in genomes, the so-called zinc proteome [7 ]. The estimate is about 3000 human zinc proteins [8 ].

2.1.1 Catalytic Zinc

The ﬁ eld of zinc metalloproteins began with the discovery of carbonic anhydrase as a zinc enzyme in 1939 [ 9]. Gradually, it became known that every enzyme class contains zinc enzymes. By far the largest number of zinc enzymes is in the class of hydrolases in the form of hundreds of zinc proteinases. Many proteinases are called metalloproteinases, e.g., matrix metalloproteinases (MMP), when in fact “metallo” stands for zinc only. For the most part, zinc enzymes seem to be absent in the major biochemical pathways of intermediary metabolism. This absence does not mean, however, that zinc has no roles in metabolism. It appears that rather than being a permanent constituent of metalloenzymes in these pathways, zinc has a role in controlling some of them.

2.1.2 Structural Zinc

Only a few structural zinc sites in enzymes were known before zinc fi nger proteins were discovered in 1986 [ 10]. The discovery revealed a new principle, namely the widespread use of zinc in proteins – which for the most part are not enzymes – to form domains for interacting with and recognizing DNA (transcription factors)/ RNA, other proteins, or lipids. Many additional “zinc fi nger motifs” were found, thus establishing zinc as an important metal ion in the tertiary structure of proteins and for the interaction of proteins, the interactome. Also, zinc participates in the interaction between peptide chains and determines the quaternary structure of some proteins. How many of these zinc-binding sites at the interface between subunits exist is presently unknown, leaving a fi nal count open with regard to the already remarkably high number of zinc-requiring proteins. 12 Zinc and Human Disease 393

2.1.3 Regulatory Zinc

A regulatory role of zinc in protein structure – as opposed to a role of zinc as a permanent cofactor in regulatory proteins – is not as ﬁ rmly deﬁ ned as the literature indicates. In contrast to catalytic and structural zinc in proteins, where sites are thought to be always fully occupied with zinc, regulation requires zinc association and dissociation. With the exception of metallothionein, variable zinc contents of proteins as a function of physiological changes have not been established unequivocally. Since there is now evidence for zinc(II) ions being signaling ions in the cell, this issue is receiving renewed scrutiny. Some zinc-binding sites satisfy criteria for serving in regulation, which occurs at remarkably low zinc(II) ion concentrations and with controlled release of zinc(II) ions. The proteins targeted and their coordination sites are the subject of recent investigations and include many proteins that were not known to be zinc proteins but bind zinc in their active sites very tightly and need to be activated by removal of the inhibitory zinc [11 , 12 ].

2.2 Zinc in Vesicles: Intracellular and Intercellular Signaling with Zinc(II) Ions

Mainly by employing histological staining techniques, it was recognized that many cells contain zinc(II) ions that apparently are not protein-bound [13 ]. These zinc(II) ions are found predominantly in intracellular compartments. From vesicular/organellar stores, tightly controlled processes release zinc(II) ions into the cytosol or into the extracellular space. Examples of zinc secretion from cells are the exocytosis of vesicles loaded with zinc in specialized neurons in the hippocampus, in pancreatic β-cells of the islets of Langerhans, and in epithelial cells of mammary glands [14 ]. Other cells secreting zinc(II) ions include somatotrophic cells in the pituitary gland, pancreatic acinar cells, Paneth cells in the crypts of Lieberkühn, cells of the tubulo- acinar glands of the prostate, epithelial cells of the epididymal ducts, and osteoblasts [13 ]. Zinc(II) ions are also released intracellularly from the endoplasmatic reticulum [15 ]. Together, these and additional ﬁ ndings led to the concept that zinc(II) ions are messengers in cellular control and in intra- and intercellular communication [16 , 17]. The targets of these regulatory zinc(II) ions seem to be primarily proteins, again linking zinc to the functions of proteins. The types of functions of zinc(II) ions secreted from cells are the subject of present investigations. They include the modulation of postsynaptic receptors (neurons), supplying the milk with zinc (mammary gland epithelial cells), and, more speculative, keeping secreted enzymes inhibited (pancreatic acinar cells and prostate epithelial cells), preventing proteins from forming amyloids (pancreatic β-cells) or simply being bactericidal. Within cells, the released zinc(II) ions affect many signaling pathways [18 ]. Altered signal transduction activity associated with changed phosphorylations of proteins and the very tight inhibition of several protein tyrosine phosphatases indicate a role 394 Maret of zinc in phosphorylation signaling and suggests even more abundant interactions of zinc with proteins than based on the estimate of 3000 human proteins [19 – 24 ]. The regulatory functions of zinc depend on how cellular zinc homeostasis is controlled and at which amplitudes and frequencies zinc(II) ion transients occur [25 ].

2.3 Cellular Zinc Homeostasis

Proper control of cellular zinc is critical for the balance between health and disease. How this control is achieved at the molecular level has been the subject of the latest phase of biological zinc research. Very tight control of cellular zinc is necessary to make the right amount of zinc available for protein structure and function, folding, and aggregation and to prevent zinc from interfering with the metabolism of other metal ions. The number of proteins involved in controlling cellular zinc and the fact that proteins control zinc subcellularly is remarkable and attests to the importance of this transition metal in biology. In humans, ten proteins of the ZnT family (SLC30A) export zinc from the cytosol, either out of the cell or into vesicles/organelles, fourteen proteins of the Zip family (SLC38A) import zinc into the cytsol from the extracellular space or from vesicles/organelles, and at least a dozen metallothioneins (MTs) buffer and translocate zinc [26 – 28]. Another major factor in the control of cellular zinc is the role of metal response element-binding transcription factor-1 (MTF-1) [29 ], which is a sensor of elevated zinc(II) ion concentrations and regulates zinc-dependent gene expression. In addition to this direct role of zinc in gene expression, there are multiple effects on signal transduction pathways. Accordingly, many investigations using transcriptomics demonstrated that variation of zinc concentrations affects a myriad of gene products. The zinc homeostatic proteins have dynamic coordination environments with specifi c mechanisms for handling transition metal ions [30 , 31]. MTs, for instance, have different binding constants for the seven zinc(II) ions and carry, release, and accept zinc ions dependent on cellular conditions [32 , 33 ]. In addition, they are redox-active zinc proteins. Zinc itself, on the contrary, is redox-inert. In MTs, the oxidation of the sulfur donors in the cysteine ligands of zinc causes zinc dissociation while the reduction of the oxidized cysteines generates zinc-binding capacity [ 34 ]. This property establishes redox cycles that link redox changes and the availability of cellular zinc [35 , 36 ]. The zinc homeostatic proteins are integrated into metabolism and exquisitely controlled by major signal transduction pathways. Thus, they do not work in isolation and are not only involved in housekeeping of zinc but control zinc(II) ion fl uxes for specifi c cellular functions. Aside from compiling the “parts list” of proteins involved in cellular zinc homeostasis, signifi cant advances have been made in understanding the concentrations at which cellular zinc is controlled. In contrast to other metal ions such as magnesium and calcium, most of the zinc is protein-bound with high affi nity. The consequence is that only picomolar concentrations are in the form of non-protein-bound “free” zinc(II) ions [37 ]. But this pool of zinc is not negligible and under- 12 Zinc and Human Disease 395 goes controlled fl uctuations [38 ]. Minute increases of cytosolic free zinc(II) ion concentrations have potent biological effects, which led to the concept of free zinc(II) ions being cellular signaling ions at much lower concentrations than signaling calcium(II) ions. In fact, the two ions complement each other in signaling and their different coordination chemistries allow signaling with metal ions over a wide range of concentrations [39 ]. Zinc buffering determines the amplitudes of the zinc(II) ion transients, and ultimately cellular zinc defi ciencies and overloads [37 ]. Owing to the fact that many proteins bind zinc, the overall cellular zinc buffering capacity is high but only the cellular zinc buffering capacity in the range of physiological zinc(II) ion concentrations is important. This buffering capacity is rather limited. Compromising it, e.g., by decreasing the concentrations of critical sulfhydryls involved in binding zinc, results in higher free zinc(II) ion concentrations with functional consequences. About a third of the cellular zinc buffering capacity relies on sulfhydryl donors (thiols) as zinc-binding ligands [21 ]. Some environmental agents and therapeutic drugs react with thiols and make fewer thiols available for zinc buffering. Such reactions change the zinc buffering capacity and increase the availability of free zinc(II) ions, which then bind to and change the functions of proteins that are not targeted under physiological conditions. This issue can hardly be over-emphasized because it demonstrates that factors other than zinc itself affect zinc- dependent functions. The concept of metal buffering in biology includes dynamic changes in buffering capacity. A unique feature in the cellular control of zinc and calcium is muffl ing, which refers to the transport of zinc(II) ions into vesicles/organelles and out of the cell. Muffl ing also contributes to buffering because the activity of transporters increases or decreases the cellular metal ion concentrations [ 28]. Thus, the capacities of the transport systems and the vesicular stores also contribute to zinc buffering. A central regulatory hormone of zinc metabolism akin to hepcidin in iron metabolism is not known. Knowledge about systemic control of zinc is lacking except for the acute phase response where adrenocorticotropic hormone (corticotropin, ACTH) decreases zinc in the blood [40 ].

2.4 Zinc and Oxidoreduction (Redox)

Zinc occurs as the redox-inert zinc(II) ion in biology. Because of this, the often quoted antioxidant properties of zinc can be indirect only, i.e., pro-antioxidant [41 ]. Zinc has this property only in a certain range of concentrations. Outside this range, it is a pro-oxidant, also by indirect mechanisms [41 ]. Cellular zinc deﬁ ciency and zinc overload cause oxidative stress. These opposing effects demonstrate a major function of zinc in redox metabolism and how important it is to control cellular zinc(II) ion concentrations in the correct range. The pro-antioxidant effects of zinc are due to (i) binding to and protecting free sulfhydryls against oxidation, (ii) competing with redox-active transition metal ions and suppressing the production of damaging free radicals, and (iii) inducing the synthesis of antioxidants, such as the expression of genes coding for antioxidant enzymes through MTF-1 and Nrf-2 396 Maret

(NF-E2-related factor) dependent gene transcription. Zinc defi ciency compromises these three functions and therefore constitutes a pro-oxidant condition that can trigger a cascading effect: oxidative stress releases zinc from zinc/thiolate coordination environments in proteins, such as metallothioneins and increases the oxidative stress through the actions of the released zinc if the buffering capacity is too weak. Pro-oxidant effects of zinc(II) ions at higher than normal concentrations have been linked to zinc inhibition of antioxidant enzymes, the mitochondrial respiratory chain with concomitant increased formation of reactive species, and other proteins, for example the cellular iron exporter ferriportin [42 ]. Biomarkers of oxidative stress or infl ammation decreased when normal healthy, middle aged or elderly humans were supplemented with zinc [ 43 , 44 ]. Suffi cient zinc may need to be present in cells before an oxidative insult occurs in order to support an antioxidant effect, e.g., liver protection against alcohol, cardiac protection (infarct size), vascular protection (postischemic injury), and protection of tissues against the oxidative stress of diabetic complications. In isolated cells and in mice, zinc defi ciency exacerbates endothelial cell dysfunction through proinfl ammatory pathways that involve NF-κB and peroxisome proliferator-activated receptor (PPAR) signaling, while prior zinc supplementation reduces fatty acid or tumor necrosis factor α-induced oxidative stress [45 , 46 ].

2.5 Global Functions of Zinc

Globally, the pro-oxidant effects express themselves as cytotoxic, proinfl ammatory, and pro-apoptotic functions while the pro-antioxidant effects translate into cytoprotective, anti-infl ammatory, and anti-apoptotic functions [41 ]. With molecular functions in so many proteins in metabolism and signal transduction, it is understandable that zinc is involved in proliferation, differentiation, and apoptosis of all cells with profound implications for healthy growth, renewal, and repair of cells. Zinc defi - ciency retards growth, compromises the immune and nervous systems, and affects virtually any other organ system. The most cited clinical manifestations in humans are skin lesions, depressed mental functions, impaired night vision, anorexia, hypogonadism, depressed wound healing, and changed immune functions. In animals, reduced growth, decreased food intake, alopecia, skin lesions (parakeratosis and hyperkeratinization), impaired skeletal development and abnormal gait and stance are observed. Zinc is involved in normal development through many specifi c zinc fi nger transcription factors, in genetics and epigenetics through zinc enzymes that control methylation of DNA and methylation and acetylation of histones, and in maintaining the integrity of DNA through its role in DNA repair enzymes. The involvement of zinc in the NF-κB pathway of infl ammation serves to illustrate the widespread role of zinc in many signaling pathways. In this pathway, no less than 24 zinc(II) ions are involved in various aspects of protein structure (Figure 1 ). Human IKKβ has a zinc-binding site in its kinase domain, thus increasing the number 12 Zinc and Human Disease 397

α TNFα TNFα TNF TNF Receptor

E1, E2 ITCH RNF11 Cell membrane TAX1BP1 RIP 1 TRADD TRADD A20 Cytoplasm TRAF2 TRAF2 K48 K48 RIP 1 RIP 1 K48 DeUbiquitinates RIP1 K63 Re-Ubiquitinates with K48 linked Lys

K63 Tab2 or 3 TAK1 Targets mRNA for degradation K63

Zn TNFα Zn Nup475/TTP/Tis11

/Nemo IKKα Degradation via the γ P /Nemo IKKα γ 26S Proteasome IKK IKKβ IKK IKKβ K63 K63 Activated IKK Complex IKK Complex

Ser NFkB starts transcription P Ser of genes for many pro- Ser P P Ser Ser inflammatory proteins IkB E1, E2, E3 P including TNFα, A20, IkB Ser Nup475/TTP/Tis11. P65 P50 P65 P50 NLS IkB mRNA P65 P50 NFkB K48 Phosphorylated NFkB Cytoplasm K48 K48

NLS Nucleus P65 P50

Figure 1 Zinc proteins involved in turning off the NF-κB pathway of infl ammation. A balance sheet of the number of zinc-containing proteins is given. The fi gures were kindly provided by Barbara Amann, Department of Chemistry, Goucher College, Towson, MD, USA. of zinc(II) ions in this pathway to 25 [ 47]. This complexity and the pleiotropic functions of zinc demonstrate the inherent diffi culties that investigators face in defi ning single modes of action for zinc or fi nding suitable biomarkers for either the cellular zinc status or specifi c zinc-dependent events. 398 Maret

One of the major issues in overcoming this diffi culty is the lack of knowledge about the hierarchy of cellular zinc distribution under zinc-limiting conditions. Do some proteins hold on to their zinc whereas others yield their zinc to support more crucial functions? Or are all zinc-dependent proteins affected to the same extent? Are vesicular pools of stored zinc(II) ions and signaling functions affected fi rst before the functions of zinc metalloproteins are compromised? Of course, such questions need to be asked for systemic zinc homeostasis as well: Which organs/ tissues yield their zinc fi rst and are therefore primarily affected by zinc defi ciency? Compartmental models have described re-distribution of zinc from the bone/skeleton to the liver, but answers lie in the affi nities of zinc for cellular proteins and the kinetics of cellular proteins that re-distribute zinc. With this knowledge, it is clear that the subject of zinc in organ pathophysiology and disease remains largely phenomenological as it has rarely been possible to relate pathology to single or specifi c zinc-dependent molecular events. It would be, however, a severe mistake to conclude that the zinc status is not a major determinant in the etiology and the progression of diseases. Unfortunately, such a conclusion seems to be the prevailing assessment of a large part of the medical community due to the absence of any suitable clinical marker of cellular zinc status.

3 Zinc in Organ Pathophysiology

The following short summaries are merely snapshots of examples where the fi eld has matured to indicate specifi c roles of zinc in organ pathophysiology and diseases. In most instances, pathways common to all cells are discussed in the literature of specifi c disciplines, but the knowledge applies across disciplines and will become the focus of zinc biology in the years to come. Challenges remain to tease apart the specifi c roles of zinc in zinc-dependent molecular pathways from the myriad of cellular functions of zinc. The involvement of zinc in the NF-κB pathway serves to illustrate this point (see Figure 1 ). The following summaries remain limited in scope as they focus on recently emerging general principles involving zinc(II) ion fl uxes rather than focusing on zinc metalloproteins or the many interactions of zinc with membrane ion channels and other membrane proteins. Some topics will be treated only cursorily in order to keep a focus on molecular events and to avoid too much phenomenology. Comprehensive reviews on most of the topics will be cited.

3.1 Liver and Gastrointestinal System

The liver is a central organ in zinc distribution. Therefore, liver disease can affect zinc-dependent functions of many other organs and can lead to zinc defi ciency [48 ]. In zinc defi ciency caused by factors other than liver disease, the liver is less pro- 12 Zinc and Human Disease 399 tected against various insults. Early observations indicated marked abnormalities of zinc metabolism in post-alcoholic (Laennec’s) cirrhosis with low serum and liver zinc and zincuria that correlated with the severity of the disease. A considerable improvement of liver function was noted when patients were supplemented with zinc and it was suggested that alcoholic liver disease is a conditioned zinc defi ciency [49 , 50]. These observations have been confi rmed repeatedly and demonstrate the therapeutic potential of zinc [51 ]. Alcohol consumption is also a risk factor for the severity of hepatitis C. The hepatitis C virus increases hepatic mitochondrial oxidative stress, thereby sensitizing hepatocytes to further oxidative insults from excessive alcohol consumption [ 52]. Nutritional and conditioned zinc defi ciencies, which are pro-oxidative conditions, are therefore cumulative risk factors for liver disease. Zinc is effective in treating alcoholic and viral liver disease. In liver regeneration, the zinc transporter Zip14, which also transports non-transferrin bound iron, is upregulated. The additional cellular infl ux of zinc affects the phosphorylation of the hepatocyte growth factor receptor c-Met via inhibiting its dephosphorylation by protein tyrosine phosphatase-1B (PTP-1B) [53 ]. The gastrointestinal tract is equally important in the systemic control of zinc because it is the main organ for zinc uptake, which increases under zinc defi ciency, as well as zinc excretion. Body zinc is a rather closed system where only a few milligrams (one thousandth of the total of 2–3 g) need to be replenished every day. Additional conservation of body zinc is afforded through re-uptake of the zinc secreted from the salivary glands, stomach, bile, and pancreas. Paneth cells located in the crypts of Lieberkühn in the small intestine secrete zinc(II) ions that are thought to be adjuvants to the microbicidal properties of secreted defensin peptides (cryptidins) [54 ].

3.2 Cardiovascular and Pulmonary System

The cellular role of zinc in protecting the vasculature and the extracellular role of zinc in hemostasis have implications for atherosclerosis and thrombosis. Zinc defi - ciency is associated with bleeding and clotting abnormalities through the involvement of zinc in platelet aggregation, coagulation, anticoagulation and fi brinolysis [55 ]. Beyond these roles with signifi cance for cardiovascular disease, zinc has been implicated in congestive heart failure, myocardial infarction, arrhythmias, and in diabetic cardiomyopathy through its involvement in diabetes [56 ]. Numerous investigations support a role of zinc in the pathways leading to heart disease. Some human zinc supplementation trials have shown positive effects on clinical parameters related to heart disease although the lack of suitable biomarkers of zinc status remains a serious issue in interpreting the fi ndings [57 ]. Experiments with rats demonstrated that suboptimal dietary intake of zinc promotes vascular infl ammation and atherogenesis by affecting lipoprotein levels and by enhancing proliferation of vascular smooth muscle cells [58 , 59]. A proteomics analysis of the rats has already provided signifi cant insights into the pathways involved [60 ]. 400 Maret

The lung epithelium and endothelium have been the subject of extensive investigations with regard to zinc [61 , 62]. As is the case in many other tissues, zinc chelation and zinc deﬁ ciency render the lung endothelium susceptible to injury, whereas zinc(II) ions released in the cell have a protective effect, though this effect is clearly a matter of the amount of zinc released because amounts that exceed the cellular zinc buffering capacity cause injury. The investigations have demonstrated that the zinc/thiolate clusters of metallothionein are oxidized in vivo and serve as a source of zinc(II) ions by converting redox signals into zinc signals [63 , 64]. Hormones and agents that stimulate the cellular production of nitric oxide (NO), hydrogen peroxide, or other reactive species release cellular zinc(II) ions. The pathway with oxidative mobilization of zinc from metallothionein and subsequent zinc inhibition of enzymes operates in many tissues [11 ]. Hormone → NO (reactive species) → Zn/S (metallothionein) → Zn 2+ → inhibition of enzymes The central role of metallothionein in this pathway draws attention to the many factors involved when it serves as a source of zinc released in cells. The differential gene expression of the about twelve human metallothioneins in tissues, their amounts, metal loads, and genetics, and their different reactivites towards thiol- reactive agents all determine the balance between cytoprotective and cytotoxic functions of zinc [65 , 66 ]. The roles of metallothioneins in diseases cover most of the areas where zinc is involved and are not discussed here as they have been the subject of a recent monograph [67 ].

3.3 Immune System

Zinc has extensive roles in both the adaptive (specifi c) and the innate (non-specifi c) immune response at multiple levels, including the development of immune cells and gene expression in these cells that either affects the cells themselves or other cells through secreted cytokines [ 68]. Investigations with rodents demonstrated that immune responses decline signifi cantly (>50%) in zinc defi ciency [69 ]. Human zinc defi ciency leads to atrophy of the thymus with apoptotic cell death of precursor lymphocytes as well as to defi cits in erythropoiesis resulting in anemia [70 ]. T- and B-cells are the basis of the adaptive immune system. Zinc defi ciency affects T-cell function more readily than B-cell function, but there are fewer B-cells formed because of the pro-apoptotic effect of zinc defi ciency in lymphopoiesis. In human zinc defi ciency, there is a shift in T-cell populations (Th1/Th2 balance) with the result of less interferon γ, interleukin-2, and TNFα being produced [71 ]. T-cell receptor-mediated T-cell activation also causes infl ux of extracellular zinc via the zinc transporter Zip6. The zinc inhibits subsynaptically the recruitment of the protein tyrosine phosphatase SHP-1 to the T-cell receptor [72 ]. Zinc is also needed to link the T-cell receptor CD4/CD8 and the tyrosine kinase Lck at protein interface sites between the two proteins and to induce the subsequent dimerization of this protein complex [73 , 74 ]. 12 Zinc and Human Disease 401

The innate immune response and infl ammation constitutes the fi rst line of defense of the host. It includes granulocytes, monocytes/macrophages, dendritic cells, and natural killer cells. Zinc is involved in the development, maturation, and function of all these cells. Zip6 is downregulated and cellular zinc decreases when toll-like receptor-4 of dendritic cells is stimulated [75 ]. Zip6 is needed to initiate zinc-dependent expression of major histocompatibility class II molecules. Zinc signaling in the immune system recently gained additional attention when a role of a protein kinase C-induced zinc(II) ion signal in the formation of neutrophil extracellular traps (NET) was discovered [76 ]. NETosis is a process, in which components such as DNA, chromatin, and proteins are released from cells to capture bacteria. At the molecular level, a re-distribution of zinc is important for the immune response. The acute phase response to injury or infection decreases plasma zinc and increases cellular zinc [40 ]. In the liver, the pro-infl ammatory cytokine interleukin- 6 induces the expression of Zip14 and metallothionein, resulting in zinc infl ux and binding of zinc [77 ]. Subsequent to restriction of zinc in the blood, induced cellular zinc(II) ion fl uxes are critical for functions of immune cells. FcεR1 receptor stimulation of mast cells increases cellular zinc(II) ions and so does stimulation of Jurkat T cells and monocytes [78 , 79 ]. The increase in mast cell zinc is important for allergic and autoimmune reactions (anaphylaxis, asthma, atopic dermatitis). Under conditions of zinc defi ciency, the cellular re-distribution of zinc cannot take place, compromising the function of immune cells and increasing morbidity and mortality, especially in the critically ill with sepsis. Another important observation is that the zinc transporter Zip8 is a transcriptional target of NF-κB [ 47 ]. Zinc transport through Zip8 suppresses proinfl ammatory conditions by zinc-dependent down-regulation of IκB (IKK) kinase activity. Zinc defi ciency, in contrast, results in increased infl ammation. Zip8 transports iron in addition to zinc [80 ]. Control of zinc homeostasis must be intact for proper immune functions. Zinc supplementation must be chosen carefully. Too much zinc causes copper defi ciency, leukopenia, inhibits immune functions, and counteracts the acute phase response, which removes zinc from the circulation so that an invading pathogen does not have access to the zinc it needs. Zinc defi ciency, on the other hand, increases bacterial invasion, in particular through an infl ammatory response and the damaging effects on mucosal functions in the gastrointestinal and respiratory tracts [ 81]. These effects of zinc on the immune system are important for autoimmune diseases and neoplastic growth, and the effi cacy of vaccinations in zinc-defi cient individuals. Since zinc affects B-cells indirectly through its effect on T-cells, T-cell function should be optimal prior to vaccination.

3.4 Central and Peripheral Nervous System

In addition to the functions of zinc in every nerve cell, specialized neurons in the cerebral cortex store zinc in synaptic vesicles. Upon neuronal stimulation, zinc(II) ions are released from these vesicles and have multiple effects on the postsynaptic 402 Maret neurons. Therefore, control of synaptic zinc homeostasis is critical [82 ]. ZnT3 and MT3 (growth inhibitory factor) participate in the loading of the synaptic vesicles with zinc. The role of zinc(II) ions in synaptic neurotransmission and in excitotoxicity has been investigated extensively [83 , 84]. In a provocative article entitled “Do we need zinc to think?” the role of synaptic zinc was discussed [85 ]. It is becoming clear that synaptic zinc is involved in cortical plasticity affecting learning and memory and thus critical to the function of the hippocampus [86 – 88 ]. Stroke leads to ischemic neuronal injury and neurodegeneration [89 ]. Release of vesicular zinc associated with a stroke either affects the receptors at the postsynaptic neuron, such as NMDA channels, acid-sensing channels or GABAA receptors, or enters the postsynaptic neuron through the AMPA receptor and other calcium channels and acts intracellularly. In the neuron, zinc is also released from proteins by oxidative/nitrosative stress and acidosis, both of which are consequences of ischemia. The zinc(II) ions then enter mitochondria and inhibit the respiratory chain and antioxidant enzymes in the matrix with the result of mitochondria churning out more reactive oxygen species. Reperfusion following ischemia may augment the injury. The fi ne balance between zinc being released for protective functions and zinc being neurotoxic and the timing of the events make it very diffi cult to intervene therapeutically with either chelating agents or with zinc supplementation. The protective functions of zinc are evident in preconditioning that lowers the damage of an ensuing ischemic insult. Zinc(II) ions released from vesicles through exocytosis and from cellular proteins by oxidative and nitrosative stress also contribute to neurotoxicity in traumatic brain injury and seizures [90 – 92 ]. For human brain health and for public health in general, it is important to realize that subclinical zinc defi ciency impairs brain function [93 ].

3.5 Reproductive System

The observation of hypogonadism in zinc defi ciency underscores the role of zinc in the reproductive system. The prostate, seminal fl uid, and sperm are all very rich in zinc. Testicular zinc is required for spermatogenesis. The high amount of zinc in the prostate has been linked to the inhibition of mitochondrial aconitase for the production of high concentrations of citrate, which together with secrected zinc(II) ions is important for the physiology of the seminal fl uid [94 ]. The high concentrations of zinc in prostate epithelial cells has been suggested to have antiproliferative and antitumor effects [94 ]. Signifi cant advances have been made recently in elucidating the role of zinc in oocytes. At the end of maturation, mouse oocytes accumulate zinc and arrest at metaphase II after the fi rst meiotic division. The fertilized oocytes then secrete zinc(II) ions with characteristic “zinc sparks” into the environment in order to lower intracellular 12 Zinc and Human Disease 403 zinc as a requirement for resuming the meiotic cell cycle [95 ]. Zinc is also involved in prophase I arrest through its effect on the MOS-MAPK pathway [96 ]. The authors comment on these remarkable fi ndings in the following way: “These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the fi rst and second meiotic arrest points.” Oocytes interact with cumulus cells and their cellular zinc content is intri- cately linked to the control of zinc homeostasis in cumulus cells [97 ]. These recent discoveries will impact signifi cantly our knowledge about fertility, reproductive health, and embryonic development.

3.6 Sensory Systems

Among the sensory systems, most work has focused on the eye, where the retina and the retinal pigment epithelium/choroid complex are particularly rich in zinc [98 , 99 ]. Drusen, deposits in the retina, accumulate metal ions, suggesting a pathology similar to that of perturbation of metal homeostasis in the deposits of Alzheimer’s disease (see below). Dietary supplementation with zinc has become a method of treatment for age-related macular degeneration. Loss of taste acuity is a clinical sign of zinc deﬁ ciency; hearing and smelling may also be affected. The olfactory bulb has very high zinc concentrations. Zinc is stored in vesicles of olfactory sensory neurons and can be released by electrical stimulation [100 ].

3.7 Other Systems

Other organ pathologies are also linked to zinc. There is a relative extensive literature on the role of zinc in skin diseases, wound healing, and bone health. One additional general subject is the role of zinc in growth. Stunting is a consequence of zinc defi ciency. Hypopituitarism was observed when human zinc defi - ciency was fi rst described [101 ]. Therefore, research has addressed the role of zinc in the growth hormone (somatostatin) – insulin-like growth factor-1 (IGF-1) axis. Neuroendocrine cells store secreted proteins in dense cores of secretory granules. Zinc has a role in the secretory pathway of growth hormone [102 ]. It binds to growth hormone in a stoichiometry close to 1:1 in the secretory granules of the pituitary glands and induces the dimerization of the hormone [ 103 , 104 ]. And anterior pituitary cells, which release growth hormone, secrete zinc(II) ions [ 13 ]. Growth hormone then stimulates the synthesis of IGF-1 in the liver. The function of IGF-1 in tissues (muscle, bone) is zinc-dependent in pathways that also involve Zip7, Zip13, and Zip14 [19 , 105 – 107 ]. 404 Maret

4 Zinc in Disease

4.1 Genetic Disease

In addition to the many changes in mRNA levels of zinc transporters observed in diseases, a number of mutations in zinc transporters are associated with human diseases (Table 1 ). A polymorphism of MT1A, an Asn27Thr substitution, changes the zinc-binding properties of the protein and is associated with type 2 diabetes and coronary heart disease [108 ]. Therefore, not only the availability of zinc in the diet but also the proteins that control cellular zinc homeostasis are important for health. Very little is known, however, about genetically determined differences in requirements for zinc or sensitivities towards an excess of zinc, nor have the genetics of the about 3000 human zinc proteins been much explored with regard to disease-causing mutations. In addition to the metabolic effects of mutations in zinc homeostatic proteins, the effect of such mutations on mental health are quite remarkable (Table 1 ). There are individuals with hyperzincemia [109 ]. Their high zinc in the blood is associated with calprotectinemia. Calprotectin is a protein of the S100 family that binds zinc. It is released from leuko- cytes in the acute phase response, and is involved in the innate immune response by binding manganese to deprive invading bacteria of this metal ion [ 110 ]. Hyperzincemic individuals present with the same symptoms as those with zinc deﬁ ciency.

4.2 Metabolic and Chronic Disease

4.2.1 Diabetes

A role of zinc in diabetes was considered for a long time but did not receive much attention because insights into molecular mechanisms were lacking, the many key functions of zinc in proteins and in the control of metabolism were not yet known,

Table 1 Polymorphisms/mutations in human zinc transporters causing, or being associated with, human diseases. ZnT2 Transient neonatal zinc defi ciency [111 ] ZnT8 Risk for diabetes type 2 [112 ] ZnT10 Broad phenotype with neurologic, hepatic, and hematologic disturbances, hypermanganesemia, obesity [113 , 114 ] Zip2 Carotid artery disease [115 ] Zip3 Bipolar disorder [116 ] Zip4 Acrodermatitis enteropathica [117 , 118 ] Zip8 Schizophrenia, obesity (dyslipidemia), coronary artery disease [119 , 120 ] Zip11 Major depressive disorder [121 ] Zip12 Schizophrenia [122 ] Zip13 Spondylocheiro dysplastic form of Ehlers–Danlos syndrome (SCD-EDS) [123 , 124 ] 12 Zinc and Human Disease 405 and cause versus effect could not be addressed [ 125 – 127]. Already in the 1930s, it was reported that the pancreas of cadavers from diabetics has only about half the amount of zinc compared to a healthy pancreas. Also, diabetics have signifi cant hyperzincuria. However, a zinc defi ciency is not readily established as reliable clinical markers of cellular zinc status are not available. Molecular studies in three areas have now signifi cantly strengthened the link between zinc and diabetes. Zinc enhances the insulin action in peripheral tissues; it has a role in insulin storage and secretion in pancreatic β-cells; and it has pro-antioxidant functions in protecting the endocrine pancreas and peripheral tissues. The following summaries are based on recent reviews that cite the original work [112 , 128 , 129 ].

4.2.1.1 Zinc in Pancreatic β-Cell Physiology

In the granules of human pancreatic β-cells, human insulin is stored as a crystalline hexamer with two bound zinc ions. The zinc(II) ions that are co-secreted with insulin may affect glucagon secretion from α-cells, channel proteins, and/or prevent amyloidogenesis of co-secreted proteins. For instance, zinc inhibits the fi brillation of monomeric insulin and the formation of the dimer of the human islet amyloid polypeptide (IAPP, amylin). The zinc transporter, ZnT8 provides zinc to the insulin-storing granules. A strong association between a polymorphism in the SLC30A8 gene (coding for ZnT8) and type 2 diabetes exists in different populations. ZnT8 with an Arg instead of a Trp at position 325 in its cytoplasmic domain increases the risk for diabetes by 53%. The risk allele is the most prevalent in populations, 55% in Asians, 75% in Europeans, and 95% in Africans. There is another variant with Gln at position 325. ZnT8 is also a signifi cant autoantigen in type 1 diabetes and the single nucleotide polymorphism modulates the ZnT8 autoantibody specifi cities, indicating that the amino acid substitution has probably a critical role. Mice with a β-cell specifi c knock-out of ZnT8 have decreased zinc in the granules, altered insulin secretion, and mild glucose intolerance. Other zinc transporters participate in zinc metabolism of β-cells and some of them are associated with diabetes type 2.

4.2.1.2 Zinc in the Physiology of Cells Targeted by Insulin

Already in the 1960s it was shown that zinc has an insulin-sparing effect and that zinc- deﬁ cient rats are less sensitive to insulin. Zinc stimulates lipogenesis in isolated adipose tissue and affects glucose uptake in target tissues. Remarkably, for the culture of some mammalian cells, zinc can replace insulin in serum-free media. These insulin- mimetic effects of zinc are intracellular. Zinc increases the phosphorylation of the insulin/IGF-1 receptor and hence protein phosphorylations downstream in insulin signal transduction. One molecular target of zinc is the protein tyrosine phosphatase PTP-1B, a major regulator of the phosphorylation state of the insulin receptor. Zinc inhibits this enzyme with an apparent zinc binding constant of 15 nM [19 , 130 ]. 406 Maret

4.2.1.3 Zinc and Oxidative and Carbonyl Stress

Diabetic hyperglycemia causes the glycation of proteins. The resulting advanced glycation end products (AGEs) increase reactive carbonyls, resulting in so-called carbonyl stress, which modiﬁ es, among other targets, sulfhydryl groups, lowers the zinc buffering/binding capacity, and mobilizes zinc from proteins [131 ]. Oxidative stress is a cause of insulin resistance [132 ]. The role of zinc in mediating oxidative stress thus establishes a causal link between zinc and insulin resistance.

4.2.2 Cancer

The roles of zinc in the immune system and thus immune surveillance and in maintaining genome stability are all relevant to cancer [133 ]. In addition, metastasis and angiogenesis require zinc. Matrix metalloproteinases are involved in metastasis. Cancer is often associated with low serum zinc, and with increased or decreased zinc in the malignant tissue. Zinc defi ciency is a major risk factor for esophageal and oral small cell carcinoma and is involved in the development of chemically induced esophageal cancer [134 ,135 ]. It causes a pro-infl ammatory environment through the expression of onco-micro RNAs [136 ]. The diminished cytoprotection in zinc defi ciency constitutes a risk factor for reactions of biomolecules with environmental carcinogens and therefore has general implications for carcinogenesis. Zinc chelation arrests cell growth. Zinc is required for cells to pass through the G1 and G2 restriction points of the cell cycle and for differentiation. In addition, mRNAs of specifi c cyclins decrease under zinc defi ciency [137 ]. Fluctuations of free cellular zinc(II) ions have been observed at the two restriction points [38 ]. Changes in zinc and in zinc homeostatic proteins (zinc transporters and metallothioneins) have also been observed in many cancers. Zinc transporters have a role in cancer signaling through the apparent activation of protein tyrosine kinases [ 138]. Very tight zinc inhibition of protein tyrosine phosphatases supports such activation [22 ]. Zip6 (LIV-1) is an estrogen-induced protein in breast cancer cells [139 ]. Zip6 is regulated by the transcription factor STAT3 and has a role in the EMT (epithelial to mesenchymal transition) in metastasis [140 ]. The increase of cellular zinc(II) ions caused by Zip6 induction inhibits downstream events, in particular the expression of E-cadherin, which is involved directly in cell detachment and hence metastasis [141 ].

4.2.3 Neurodegeneration

Among the neurodegenerative diseases, Alzheimer’s disease (AD) received the most attention with regard to zinc’s involvement in both the Aβ and tau protein pathologies. The reason is that zinc and other metals (Fe, Cu) were found in the amyloid plaques of AD at relatively high concentrations [142 ]. This fi nding led to the hypothesis that Aβ disrupts the control of neuronal zinc homeostasis by sequestering metals and that inappropriately localized zinc leads to a loss of zinc’s 12 Zinc and Human Disease 407 function in neurotransmission. Zinc induces the aggregation of Aβ into an insoluble form, the processing of amyloid precursor protein (APP), the metabolism of iron and copper, and interferes with microtubule-associated tau, the protein forming neurofi brillary tangles, the second hallmark of AD pathology [143 ]. Elevated levels of zinc inhibit the iron exporter ferroportin, possibly leading to iron overload and associated oxidative stress in neurons [42 ]. A quite remarkable observation is the shared phylogenetic origin of some Zip proteins and the prion proteins [144 ]. A role of the prion protein in uptake of zinc into neuronal cells indicates functional signifi cance for neurodegeneration [145 ].

4.3 Infectious Disease

With compromised immunity of the host in zinc defi ciency, infection with parasites is a major health concern, in particular for those at risk: children, pregnant women, and the elderly. Treating and preventing diarrhea in children under the age of five with zinc is robust and saves a high number of lives in the developing world. There is correlative evidence between zinc defi ciency and malaria, measles, HIV, tuberculosis, and respiratory tract infections such as pneumonia. However, evidence for effi cacy of the treatment and prevention of these diseases with zinc is less clear [146 ].

5 General Conclusions

The importance of proper control of cellular zinc is being recognized in several specialized disciplines of academic medicine. Zinc is involved in pathways common to all cells and in virtually all aspects of molecular and cellular biology. While changed levels of zinc in diseases were mostly considered consequences of the diseases, causation is now indicated strongly in many cases. Zinc biology, therefore, will have a major impact on the practice of medicine in many disciplines, including those where the roles of zinc are not considered at present. Translation of the knowledge into practice has been, and continues to be, hampered by the lack of suitable clinical markers, especially markers for milder forms of zinc defi ciency [147 ]. Biomarker discovery can be better pursued now because many additional and wider functions of zinc are known. In particular the knowledge about proteins involved in zinc homeostasis raise expectations that biomarkers refl ecting the zinc status akin to transferrin and ferritin in iron metabolism will be discovered. The majority of clinical cases of zinc defi ciency present themselves with overt signs. Mild zinc defi ciency, though known to affect the nervous and immune system, is generally not addressed in the clinical setting. Blood zinc represents only about 0.1% of total body zinc and corresponds to about the amount of zinc replenished every day, but the requirements are different 408 Maret

Table 2 Causes for zinc deﬁ ciency [149 ]. Nutritional General malnutrition but also wrong diets, including diets high in phytate; total parenteral nutrition Conditioned Diseases: Liver disease (cirrhosis, chronic active hepatitis, fatty liver disease) and intestinal disease (Crohn’s disease, colitis ulcerosa, celiac disease), pancreas disease (chronic pancreatitis), terminal kidney insufﬁ ciency, acute myocardial infarct, infections, tumors, diabetes mellitus, collagenoses Iatrogen: Treatment with penicillamine, contraceptives, corticoids, and certain antibiotics Genetic Sickle cell disease and inherited diseases of zinc metabolism

for children, pregnant and lactating women, the elderly, healthy and sick humans, and populations that are at risk for zinc defi ciency. As a systemic marker, blood zinc it is not a sensitive marker for the cellular zinc status, in particular since acute infections can lower serum zinc and increase cellular zinc, cells differ in their zinc requirements and kinetics, and major functions of zinc are within cells. Zinc therapy is already proven to be effective in diseases, such as intestinal diseases, acrodermatitis enteropathica, transient neonatal zinc defi ciency, and Wilson’s disease. Although many defi cits have been linked unequivocally to zinc defi ciency and zinc appears to be a panaceum for a large number of ailments, zinc supplementation is often ineffective in reversing defi cits [ 148]. This failure has perplexed some and is sometimes used to refute the hypothesis that zinc defi ciency is the cause of a condition. However, there are multiple reasons for the occasional failure of reversing symptoms with zinc supplementation. The control of zinc metabolism is remarkably complex and encompasses competition with and interactions among other metal ions; zinc supplementation depends on the dose as too much zinc may have the opposite effects; and last but not least, restoration of function may require additional factors because other micronutrient defi ciencies often accompany zinc defi ciency. Supplementing only zinc may not restore the control of cellular zinc homeostasis. Additional zinc may be a pro-oxidant and harmful under conditions of sustained oxidative stress when free zinc(II) ion concentrations are already higher than normal and the zinc buffering capacity is reduced. Under conditions such as oxidative stress, the binding capacity of cellular thiols is reduced and supplemental zinc may not be retained in the cells or bind to non-physiological targets with adverse side effects. Restoring control of cellular, not necessarily systemic, zinc homeostasis, restoring the cellular redox state, and addressing the factors that condition zinc defi - ciency (Table 2 ) seem to be a prerequisites to successful zinc therapy in the many conditions that may require zinc. With the discovery of the many proteins controlling cellular zinc homeostasis and the genetics of these proteins, the focus of attention shifted from the presence or absence of zinc to the functions of the proteins that determine the cellular alloca- tion of zinc to zinc proteins, redistribute zinc under changing conditions, and, importantly, regulate the functions of zinc in intra- and intercellular communication. Specifi c roles of zinc in cellular signaling pathways are being identifi ed and 12 Zinc and Human Disease 409 examined. Changed functions of zinc homeostatic proteins affect the redox state, infl ammation, genetic stability, and cellular signaling and metabolism through altered cellular zinc metabolism.

Abbreviations

ACTH adrenocorticotrophic hormone, corticotropin AD Alzheimer’s disease AGE advanced glycation end product AMPA α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid APP amyloid precursor protein DALY disability-adjusted life years EMT epithelial-to-mesenchymal transition GABAA γ -aminobutyric acid A HIV human immunodeﬁ ciency virus IAPP islet amyloid polypeptide, amylin IGF-1 insulin-like growth factor 1 IKKβ I κB kinase β MMP matrix metalloproteinase MOS-MAPK mos (a protein serine/threonine kinase)-mitogen-activated protein kinase MRE metal response element MT metallothionein MTF-1 metal response element (MRE)-binding transcription factor-1 NET neutrophil extracellular trap NF-κB nuclear factor κ light-chain-enhancer of activated B cells NMDA N-methyl D-aspartate PPAR peroxisome proliferator-activated acceptor PTB-1B protein tyrosine phosphatase-1B TNFα tumor necrosis factor α WHO World Health Organization Zip Zrt-, Irt-like proteins ZnT zinc transporter

Acknowledgment I thank Dr. Barbara Amann, Department of Chemistry, Goucher College, Towson, MD, for providing the ﬁ gures.

References

1 . Zinc in Human Health , Ed L. Rink, IOS Press, Amsterdam, 2011, pp. 1–577. 2. B. L. Vallee, K. H. Falchuk, Physiol. Rev. 1993 , 73 , 79–118. 3. K. H. Brown, S. E. Wuehler, Zinc and Human Health: The Results of Recent Trials and Implications for Program Interventions and Research , Ottawa: Micronutrient Initiative, 2000, pp.1–69. 410 Maret

4. World Health Report 2002 – Reducing Risks, Promoting Healthy Life. Geneva: WHO, 2002, pp. 1–250. 5. D. S. Auld, BioMetals 2001 , 14 , 271–313. 6. W. Maret, Adv. Nutr. 2013 , 4 , 82–91. 7. W. Maret, J. Anal. At. Spectrom. 2004 , 19 , 15–19. 8. C. Andreini, L. Banci, I. Bertini, A. Rosato, J. Proteome Res. 2006 , 5 , 196–201. 9. D. Keilin, T. Mann, Nature 1939 , 144 , 442–443. 10. J. Miller, A. D. McLachlan, A. Klug, EMBO J. 1985 , 4 , 1609–1614. 11. W. Maret, C. Jacob, B. L. Vallee, E. H. Fischer, Proc. Natl. Acad. Sci. USA 1999 , 96 , 1936–1940. 12. W. Maret, BioMetals 2013 , 26, 197–204. 13. G. Danscher, M. Stoltenberg, J. Cytochem. Histochem. 2005 , 53 , 141–153. 14. S. L. Kelleher, N. H. McCormick, V. Velasquez, V. Lopez, Adv. Nutr. 2011 , 2 , 101–111. 15. K. M. Taylor, S. Hiscox, R. I. Nicholson, C. Hogstrand, P. Kille, Sci. Signal. 2012 , 5 :ra11. 16. H. Haase, W. Maret, in Cellular and Molecular Biology of Metals, Eds R. Zalups, J. Koropatnick, Taylor and Francis, Boca Raton, 2010, pp. 179–210. 17. T. Fukada, S. Yamasaki, K. Nishida, M. Murakami, T. Hirano, J. Biol. Inorg. Chem. 2011 , 16 , 1123–1134. 18. D. Beyersmann, H. Haase, BioMetals 2001 , 14 , 331–341. 19. H. Haase, W. Maret, Exp. Cell Res. 2003 , 291 , 289–298. 20. H. Haase, W. Maret, J. Trace Elem. Med. Biol. 2005 , 19, 37–42. 21. A. Krezel, Q. Hao, W. Maret, Arch. Biochem. Biophys. 2007 , 463 ,188–200. 22. M. Wilson, C. Hogstrand, W. Maret, J. Biol. Chem. 2012 , 287 , 9322–9326. 23. W. Maret, Pure Appl. Chem. 2008 , 80 , 2679–2687. 24. W. Maret, Metallomics 2010 , 2 , 117–125. 25. W. Maret, BioMetals 2009 , 22 , 149–157. 26. L. A. Lichten, R. J. Cousins, Annu. Rev. Nutr. 2009 , 29 , 153–176. 27. T. Fukada, T. Kambe, Metallomics 2011 , 3 , 662–674. 28. R. A. Colvin, W. R. Holmes, C. P. Fontaine, W. Maret, Metallomics 2010 , 2 , 306–317. 29. V. Günther, U. Lindert, W. Schaffner, Biochim. Biophys. Acta 2012 , 1823 , 1416–1425. 30. W. Maret, Y. Li, Chem. Rev. 2009 , 109 , 4682–4707. 31. W. Maret, J. Inorg. Biochem. 2012, 111 , 110–116. 32. A. Krężel, W. Maret, J. Am. Chem. Soc. 2007 , 129 , 10911–10921. 33. W. Maret, J. Biol. Inorg. Chem. 2011 , 16 , 1079–1086. 34. W. Maret, B. L. Vallee, Proc. Natl. Acad. Sci. USA 1998 , 95 , 3478–3482. 35. W. Maret, Biochemistry 2004 , 43 , 3301–3309. 36. W. Maret, Antioxid. Redox Signal. 2006 , 8 , 1419–1441. 37. A. Krezel, W. Maret, J. Biol. Inorg. Chem. 2006 , 11 , 1049–1062. 38. Y. Li, W. Maret, Exp. Cell Res. 2009 , 315 , 2463–2470. 39. W. Maret, Proc. Natl. Acad. Sci. USA 2001 , 98 , 12325–12327. 40. K. H. Falchuk, New Engl. J. Med. 1977 , 269 , 1129–1134. 41. W. Maret, Exp. Gerontol. 2008 , 43 , 363–369. 42. J. A. Duce, A. Tsatsanis, M. A. Cater, S. A. James, E. Robb, K. Wikhe, S. L. Leong, K. Perez, T. Johanssen, M. A. Greenough, H.-H. Cho, D. Galatis, R. D. Moir, C. L. Masters, C. McLean, R. E. Tanzi, R. Cappai, K. J. Barnham, G. D. Ciccotosto, J. T. Rogers, A. I. Bush, Cell 2010 , 142 , 857–867. 43. A. S. Prasad, B. Bao, F. W. J. Beck, P. Kucuk, F. H. Sarkar, Free Radic. Biol. Med. 2004 , 37 , 1182–1190. 44. A. S. Prasad, F. W. J. Beck, B. Bao, J. T. Fitzgerald, D. C. Snell, J. D. Steinberg, L. J. Cardozo, Am. J. Clin. Nutr. 2007 , 85 , 837–844. 45. B. Hennig, P. Meerarani, M. Toborek, C. J. McClain, Am J. Coll. Nutr. 1999 , 18 , 152–158. 46. H. Shen, E. Oesterling, A. Stromberg, M. Toborek, R. MacDonald, B. Hennig, J. Am. Coll. Nutr. 2008 , 27 , 577–587. 12 Zinc and Human Disease 411

47. M.-J. Liu, S. Bao, M. Galvez-Peralta, C. J. Pyle, A. C. Rudawsky, R. E. Pavlovicz, D. W. Killilea, C. Li, D. W. Nebert, M. E. Wewers, D. L. Knoell, Cell Reports 2013 , 3 , 386–400. 48. K. Grüngreiff, D. Reinhold, in Zinc in Human Health , Ed L. Rink, IOS Press, Amsterdam, 2011, pp. 473–492. 49. B. L. Vallee, W. E. C. Wacker, A. F. Bartholomay, E. D. Robin, New Engl. J. Med. 1956 , 255 , 403–408. 50. B. L. Vallee, W. E. C. Wacker, A. F. Bartholomay, F. L. Hoch, New Engl. J. Med. 1956 , 257 , 1056–1065. 51. M. K. Mohammad, Z. Zhou, M. Cave, A. Barve, M. J. McClain, Nutr. Clin. Pract. 2012 , 27 , 8–20. 52. M. Korenaga, M. Okuda, K. Otani, T. Wang, Y. Li, S. A. Weinman, J. Clin. Gastroenterol. 2005 , 39 , S162–166. 53. T. B. Aydemir, H. S. Sitren, R. J. Cousins, Gastroenterology 2012 , 142 , 1536–1546. 54. L. J. Giblin, C. J. Chang, A. F. Bentley, C. Frederickson, S. J. Lippard, C. J. Frederickson, J. Histochem. Cytochem. 2006 , 54 , 311–316. 55. T. T. Vu, J. C. Fredenburgh, J. I. Weitz, Thrombosis Haemost. 2013 , 109 , 421–430. 56. P. J. Little, R. Bhattacharya, A. E. Moreyra, I. L. Korichneva, Nutrition 2010 , 26 , 1050–1057. 57. M. Foster, P. Petocz, S. Samman, Atherosclerosis 2010 , 210 , 344–352. 58. J. H. Beattie, M.-J. Gordon, S. J. Duthie, C. J. McNeil, G. W. Horgan, G. F. Nixon, J. Feldmann, I.-S. Kwun, Mol. Nutr. Food Res. 2012 , 56, 1097–1105. 59. E. H. Alcantara, M. Y. Shin, J. Feldmann, G. F. Nixon, J. H. Beattie, I. S. Kwun, Atherosclerosis 2013 , 228 , 46–52. 60. J. H. Beattie, M.-J. Gordon, G. J. Rucklidge, M. D. Reid, G. J. Duncan, G. W. Horgan, Y.-E. Cho, I.-S. Kwun, Proteomics 2008 , 8 , 2126–2135. 61. A. Q. Truong-Tran, J. Carter, R. Rufﬁ n, P. D. Zalewski, Immunol. Cell Biol. 2001 , 79 , 170–177. 62. K. Thambiayya, A. M. Kaynar, C. M. St.Croix, B. R. Pitt, Pulm. Circ. 2012 , 2 , 443–451. 63. W. Maret, J. Nutr. 2000 , 130 , 1455S–1458S. 64. W. Maret, J. Nutr. 2003 , 133 , 1460S–1462S. 65. Y. Chen, Y. Irie, W. M. Keung, W. Maret, Biochemistry 2002 , 41 , 8360–8367. 66. Y. Li, W. Maret, J. Anal. At. Spectrom. 2008 , 23 , 1055–1062. 67. Metallothioneins in Biochemistry and Pathology , Ed P. Zatta, World Scientiﬁ c Publishing Co., Singapore, 2008, pp. 1–320. 68. L. Rink, H. Haase, Trends Immunol. 2007 , 28 , 1–4. 69. P. J. Fraker, S. M. Haas, R. W. Luecke, J. Nutr. 1977 , 107 , 1889–1895. 70. P. J. Fraker, L. E. King, Annu. Rev. Nutr. 2004 , 24 , 277–298. 71. F. W. Beck, A. S. Prasad, J. Kaplan, J. T. Fitzgerald, G. J. Brewer, Am. J. Physiol. 1997 , 272 , E1002–1007. 72. M. Yu, W.-W. Lee, D. Tomare, S. Pryshchep, M. Czyzesnikiewicz-Guzik, D. L. Lamar, G. Li, K. Singh, L. Tian, C. M. Weyand, J. J. Goronzy, J. Exp. Med. 2011 , 208 , 775–785. 73. P. W. Kim, Z.-Y. J. Sun, S. C. Blacklow, G. Wagner, M. J. Eck, Science 2003 , 301 , 1725–1728. 74. J. Romir, H. Lilie, C. Egerer-Sieber, F. Bauer, H. Sticht, Y. A. Muller, J. Mol. Biol. 2007 , 365 , 1417–1428. 75. H. Kitamura, H. Morikawa, H. Kamon, M. Iguchi, S. Hojyo, T. Fukada, S. Yamashita, T. Kaisho, S. Akira, M. Murakami, T. Hirano, Nat. Immunol. 2006 , 7 , 971–977. 76. R. Hasan, L. Rink, H. Haase, Innate Immun. 2012, 19 , 253–264. 77. J. P. Liuzzi, L. A. Lichten, S. Rivera, R. K. Blanchard, T. B. Aydemir, M. D. Knutson, T. Ganz, R. J. Cousins, Proc. Natl. Acad. Sci. USA 2005 , 102 , 6843–6848. 78. S. Yamasaki, K. Sakata-Sogawa, A. Hasegawa, T. Suzuki, K. Kabu, E. Sato, S. Kurasaki, M. Yamashita, K. Tokunaga, K. Nishida, T. Hirano, J. Cell Biol. 2007 , 177 , 637–645. 79. H. Haase, J. L. Ober-Blöbaum, G. Engelhardt, S. Hebel, A. Heit, H. Heine, L. Rink, J. Immunol. 2008 , 181 , 6491–6502. 412 Maret

80. C.-Y. Wang, S. Jenkitkasemwong, S. Duarte, B. K. Sparkman, A. Shawki, B. Mackenzie, M. D. Knutson, J. Biol. Chem. 2012 , 287 , 34032–34043. 81. D. L. Knoell, B. Y. Besecker, in Zinc in Human Health , Ed L. Rink, IOS Press, Amsterdam, 2011, pp. 254–267. 82. A. Takeda, M. Nakamura, H. Fujii, H. Tamano, Metallomics 2013 , 5 , 417–423, DOI 10.1039. c3mt20269k 83. S. L. Sensi, P. Paoletti, A. I. Bush, I. Sekler, Nat. Rev. Neurosci. 2009 , 10 , 780–791. 84. C. J. Frederickson, J.-Y. Koh, A. I. Bush, Nat. Rev. Neurosci. 2005 , 6 , 449–462. 85. Y. V. Li, C. J. Hough, J. M. Sarvey, SciSTKE 2003 (182), pe19. 86. A. S. Nakashima, H.-P. Wu, R. Dyck, in Zinc in Human Health, Ed L. Rink, IOS Press, Amsterdam, 2011, pp. 389–402. 87. E. Pan, X. Zhang, Z. Huang, A. Krezel, M. Zhao, C. E. Tinberg, S. J. Lippard, J. O. McNamara, Neuron 2011 , 71 , 1116–1126. 88. K. Toth, Annu. Rev. Nutr. 2011 , 31 , 139–153. 89. C. W. Shuttleworth, J. H. Weiss, Trends Pharmacol. Sci. 2011 , 32 , 480–486. 90. C. J. Frederickson, W. Maret, M. P. Cuajungco, The Neuroscientist 2004 , 10 , 18–25. 91. Y. Li, B. E. Hawkins, D. S. Prough, D. S. DeWitt, W. Maret, Brain Res. 2010 , 1330 , 131–141. 92. D. R. Morris, C. W. Levenson, Hindawi Publishing Corporation, J. Toxicology 2012 , Article ID 785647. 93. H. Sandstead, J. Trace Elem. Biol. Med. 2012 , 26 , 70–73. 94. R. B. Franklin, L. C. Costello, Arch. Biochem. Biophys. 2007 , 463 , 211–217. 95. A. M. Kim, M. L. Bernhardt, B. Y. Kong, R. W. Ahn, S. Vogt, T. K. Woodruff, T. V. O’Halloran, ACS Chem. Biol. 2011 , 6 , 716–723. 96. B. Y. Kong, M. L. Bernhardt, A. M. Kim, T. V. O’Halloran, T. K. Woodruff, Biol. Reprod. 2012 , 87, 11 , 1–12. 97. R. S. Lisle, K. Anthony, M. A. Randall, F. J. Diaz, Reproduction 2013 , 145, 381–390. 98. M. Ugarte, N. N. Osborne, Prog. Neurobiol. 2001 , 64 , 219–249. 99. N. Barzegar-Befroei, S. Cahyadi, A. Gango, T. Peto, I. Lengyel, in Zinc in Human Health , Ed L. Rink, IOS Press, Amsterdam, 2011, pp 530–553. 100. L. J. Blakemore, E. Tomat, S. J. Lippard, P. Q. Trombley, Metallomics 2013 , 5 , 208–213. 101. H. H. Sandstead, Adv. Nutr. 2013 , 4 , 76–81. 102. V. Petkovic, M. C. Miletta, P.-E. Mullis, in Developmental Biology of GH Secretion, Growth and Treatment , Vol. 23 of Endocr. Dev. , Ed P.-E. Mullis, Karger, Basel, 2012, pp. 96–108. 103. P. C. Cunningham, M. G. Mulkerrin, J. A. Wells, Science 1991 , 253 , 545–548. 104. O. Thorlacius-Ussing, Neuroendocrinology 1987 , 45 , 233–242. 105. R. S. McDonald, J. Nutr. 2000 , 130 , 1500S–1508S. 106. K. M. Taylor, P. Kille, C. Hogstrand, Cell Cycle 2012 , 11 , 1–2. 107. T. Fukada, S. Hojyo, T. Furuichi, J. Bone Miner. Metab. 2013 , 31, 129–135. 108. R. Giacconi, A. R. Bonﬁ gli, R. Testa, C. Sirolla, C. Cipriano, M. Marra, E. Muti, M. Malavolta, L. Costarelli, F. Piacenza, S. Tesei, E. Mocchegiani, Mol. Genet. Metab. 2008, 94, 98–104. 109. B. Sampson, M. K. Fagerhol, C. Sunderkötter, B. E. Golden, P. Richmond, N. Klein, I. Z. Kovar, J. H. Beattie, B. Wolska-Kusnierz, Y. Saito, J. Roth, Lancet 2002 , 360 , 1742–1745. 110. S. M. Damo, T. E. Kehl-Fie, N. Sugitani, M. E. Holt, S. Rathi, W. J. Murphy, Y. Zhang, C. Betz, L. Hench, G. Fritz, E. P. Skaar, W. J. Chazin, Proc. Natl. Acad. Sci. USA 2013 , 110 , 3841–3846. 111. W. Chowanadisai, B. Lönnerdal, S. Kelleher, J. Biol. Chem. 2006 , 281 , 39699–39707. 112. F. Chimienti, J. Diabetes Invest. 2013 , 3 , 202–2011. 113. M. Quadri, A. Federico, T. Zhao, G. J. Breedveld, C. Battisti, C. Delnooz, L. A. Severijnen, L. Di Toro Mammarella, A. Mignarri, L. Monti, A. Sanna, P. Lu, F. Punzo, G. Cossu, R. Willemsen, F. Rasi, B. A. Oostra, B. P. van de Warrenburg, V. Bonifati, Am. J. Hum. Genet. 2012 , 90 , 467–477. 114. K. S. Burgdorf, A. P. Gjesing, N. Grarup, J. M. Justesen, C. H. Sandholt, D. R. Witte, T. Jorgensen, S. Madsbad, T. Hansen, O. Pedersen, Diabetologia 2012 , 55 , 105–113. 12 Zinc and Human Disease 413

115. R. Giacconi, E. Muti, M. Malavolta, M. Cardelli, S. Pierpaoli, C. Cipriano, L. Costarelli, S. Tesei, V. Saba, E. Mocchegiani, Rejuvenation Res. 2008 , 11 , 297–300. 116. H. M. Ollila, P. Soronen, K. Silander, O. M. Palo, T. Kieseppä, M. A. Kaunisto, J. Lönnqvist, L Peltonen, T. Partonen, T. Paunio, Mol. Psychiatry 2009 , 14 , 351–353. 117. K. Wang, B. Zhou, Y. M. Kuo, J. Zemansky, J. Gitschier, Am. J. Hum. Genet. 2002 , 71 , 66–73. 118. S. Kury, B. Dreno, S. Bezieau, S. Giraudet, M. Kharﬁ , R. Kamoun, J. Moisan, Nature Genet. 2002 , 31 , 239–240. 119. D. M. Waterworth, S. L. Ricketts, K. Song, L. Chen, J. H. Zhao, S. Ripatti, Y. S. Aulchenko, W. Zhang, X. Yuan, N. Lim, J. Luan, S. Ashford, E. Wheeler, E. H. Young, D. Hadley, J. R. Thompson, P. S. Braund, T. Johnson, M. Struchalin, I. Surakka, R. Luben, K. T. Khaw, S. A. Rodwell, R. J. Loos, S. M. Boekholdt, M. Inouye, P. Deloukas, P. Elliott, D. Schlessinger, S. Sanna, A. Scuteri, A. Jackson, K. L. Mohlke, J. Tuomilehto, R. Roberts, A. Stewart, Y. A. Kesäniemi, R. W. Mahley, S. M. Grundy; Wellcome Trust Case Control Consortium, W. McArdle, L. Cardon, G. Waeber, P. Vollenweider, J. C. Chambers, M. Boehnke, G. R. Abecasis, V. Salomaa, M. R. Järvelin, A. Ruokonen, I. Barroso, S. E. Epstein, H. H. Hakonarson, D. J. Rader, M. P. Reilly, J. C. Witteman, A. S. Hall, M. J. Samani, D. P. Strachan, P. Barter, C. M. van Duijn, J. S. Kooner, I. Peltonen, N. J. Wareham, R. McPherson, V. Mooser, M. S. Sandhu, Artherioscler. Thromb. Vasc. Biol. 2010 , 30 , 2264–2276. 120. N. Carrera, M. Arrojo, J. Sanjuán , R. Ramos-Ríos, E. Paz, J. J. Suárez-Rama, M. Páramo, S. Agra, J. Brenlla, S. Martínez, O. Rivero, D. A. Collier, A. Palotie, S. Cichon, M. M. Nöthen, M. Rietschel, D. Rujescu, H. Stefansson, S. Steinberg, E. Sigurdsson, D. St Clair, S. Tosato, T. Werge, K. Stefansson, J. C. González, J. Valero, A. Gutiérrez-Zotes, A. Labad, L. Martorell, E. Vilella, A. Carracedo, J. Costas, Biol. Psychiatry 2012 , 71 , 169–177. 121. P. Muglia, F. Tozzi, N. W. Galwey, C. Francks, R. Upmanyu, X. Q. Kong, A. Antoniades, E. Domenici, J. Perry, S. Rothen, C. L. Vandeleur, V. Mooser, G. Waeber, P. Vollenweider, M. Preisig, S. Lucae, B. Müller-Myhsok, F. Holsboer, L. T. Middleton, A. D. Roses, Mol. Psychiatry 2010 , 15 , 589–601. 122. M. Bly, Schizophr. Res. 2006 , 81 , 321–322. 123. T. Fukada, Y. Asada, K. Mishima, S. Shimoda, I. Saito, J. Oral Biosci. 2011 , 53 , 1–12. 124. J. Jeong, J. M. Walker, F. Wang, J. G. Park, A. E. Palmer, C. Giunta, M. Rohrbach, B. Steinmann, D. J. Eide, Proc. Natl. Acad. Sci. USA 2012 , 78 , E3530–3538. 125. A. B. Chausmer, J. Am. Coll. Nutr. 1998 , 17 , 109–115. 126. C. G. Taylor, Biometals 2005 , 18 , 305–312. 127. W. Maret, Biometals 2005 , 18 , 293–294. 128. G. A. Rutter, Islets 2010 , 2 , 1–2. 129. W. Maret, “Zinc and Diabetes”, Encyclopedia of Metalloproteins, Eds V. N. Uversky, R. H. Kretsinger, E. A. Permyakov, Springer Science + Business Media LLC, Dordrecht, 2013, 2371–2375. 130. A. Krezel, W. Maret, J. Biol. Inorg. Chem. 2008 , 13 , 401–409. 131. Q. Hao, W. Maret, FEBS J. 2006 , 273 , 4300–4310. 132. N. Houstis, E. D. Rosen, E. S. Lander, Nature 2006 , 440 , 944–948. 133. R. Sharif, P. Thomas, P. Zalewski, M. Fenech, Mutation Res. 2012 , 733 , 111–121. 134. P. M. Newberne, S. Broitman, T. F. Schrager, Pathobiology 1997 , 65 , 253–263. 135. L. Y. Y. Fong, L. Zhang , Y. Jiang, J. L. Farber, J. Natl. Cancer Inst. 2005 , 97 , 40–50. 136. H. Alder, C. Taccioli, H. Chen, Y. Jiang, K. J. Smalley, P. Fadda, H. G. Ozer, K. Huebner, J. L. Farber, C. M. Croce, L. Y. Y. Fong, Carcinogenesis 2012 , 33 , 1736–1744. 137. J. K. Chesters, L. Petrie, J. Nutr. Biochem. 1999 , 10 , 279–290. 138. C. H. Hogstrand, P. Kille, R. I. Nicholson, K. M. Taylor, Trends Mol. Med. 2009 , 15 , 101–111. 139. K. M. Taylor, S. Hiscox, R. I. Nicholson, Trends Endocrinol. Metabol. 2004 , 15 , 461–463. 140. S. Yamashita, C. Miyagi, T. Fukuda, N. Kagara, Y. S. Che, T. Hirano, Nature 2004 , 429 , 298–302. 414 Maret

141. K. Taylor, J. Gee, P. Kille, in Zinc in Human Health, Ed L. Rink, IOS Press, Amsterdam, 2011, pp. 283–304. 142. S. Ayton, P. Lei, A. I. Bush, Free Radic. Biol. Med. 2013 , 62, 76–89. 143. T. J. A. Craddock, J. A. Tuszynski, D. Chopra, N. Casey, L. E. Goldstein, S. A. Hameroff, R. E. Tanzi, PloSOne 2012 , 7(3)e33552. 144. S. Ehsani, H. Huo, A. Salehzadeh, C. L. Pocanschi, J. C. Watts, H. Wille, D. Westaway, E. Rogaeva, P. H. St. George-Hyslop, G. Schmitt-Ulms, Prog. Neurobiol. 2011 , 93 , 405–420. 145. N. T. Watt, D. R. Taylor, T. L. Kerrigan, H. H. Grifﬁ ths, J. V. Rushworth, I. J. Whitehouse, N. M. Hooper, Nat. Commun. 2012 , 3 :1134. 146. C. L. Fischer Walker, L. Lamberti, D. Roth, R. E. Black, in Zinc in Human Health , Ed L. Rink, IOS Press, Amsterdam, 2011, pp. 234–253. 147. N. M. Lowe, K. Fekete, T. Decsi, Am. J. Clin. Nutr. 2009 , 89 , 1S–12S. 148. W. Maret, H. H. Sandstead, J. Trace Elem. Med. Biol. 2006 , 20 , 3–18. 149. K. Grüngreiff, Zink und Leber , Dr. Falk Pharma GmbH, Freiburg, Germany, 2012, pp. 1–84. Chapter 13 Molybdenum in Human Health and Disease

Guenter Schwarz and Abdel A. Belaidi

Contents ABSTRACT ...... 416 1 INTRODUCTION ...... 417 1.1 Chemistry and Biology of Molybdenum ...... 417 1.2 Molybdenum Uptake ...... 417 1.3 Molybdenum Toxicity ...... 418 1.4 Molybdenum Enzymes...... 418 2 DEFICIENCIES IN MOLYBDENUM ENZYMES ...... 419 2.1 Xanthine Dehydrogenase and Oxidase ...... 420 2.1.1 Structure and Function ...... 420 2.1.2 Xanthinuria Type I ...... 420 2.1.3 Xanthinuria Type II ...... 421 2.1.4 Hyperuricemia ...... 421 2.2 Aldehyde Oxidase ...... 422 2.3 Sulfi te Oxidase and Sulfi te Toxicity ...... 423 2.3.1 Cysteine Catabolism ...... 423 2.3.2 Sulfi te Oxidase Localization, Structure, and Function ...... 425 2.3.3 Sulfi te Toxicity and Sulfi te Oxidase Defi ciency...... 425 2.4 Mitochondrial Amidoxime-Reducing Component ...... 426 3 MOLYBDENUM COFACTOR DEFICIENCIES ...... 426 3.1 Biochemistry of Molybdenum Cofactor Biosynthesis ...... 427 3.1.1 Pterin Synthesis ...... 427 3.1.2 Dithiolene Synthesis ...... 428 3.1.3 Molybdate Insertion ...... 429 3.1.4 Maturation of Moco ...... 430 3.2 Genetics of Human Molybdenum Cofactor Synthesis ...... 430 3.2.1 Structure and Organization of Moco Synthesis Genes ...... 430 3.2.2 Mutations in Molybdenum Cofactor-Defi cient Patients ...... 432 3.2.3 Biochemical Classifi cation of Molybdenum Cofactor Defi ciency ...... 434

G. Schwarz (*) • A. A. Belaidi Institute of Biochemistry, Department of Chemistry, Center for Molecular Medicine , University of Cologne , Zülpicher Strasse 47 , D-50674 Köln , Germany e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 415 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_13, © Springer Science+Business Media Dordrecht 2013 416 Schwarz and Belaidi

3.3 Pathophysiology of Molybdenum Cofactor Defi ciency ...... 434 3.3.1 Clinical Presentation of Patients with Molybdenum Cofactor Defi ciency ...... 434 3.3.2 Molecular Basis of Neurodegeneration ...... 435 3.4 Animal Models ...... 436 3.4.1 Mocs1-Defi cient Mice ...... 436 3.4.2 Gephyrin-Defi cient Mice...... 437 3.5 Treatment of Molybdenum Cofactor Defi ciency ...... 437 3.5.1 Treatment of Mocs1-Defi cient Mice with cPMP ...... 437 3.5.2 Treatment of MoCD Type A Patients with cPMP ...... 438 3.5.3 Treatment of MoCD Type B and C Patients ...... 439 3.5.4 Dietary Restriction and Treatment of Sulfi te Oxidase Defi ciency ...... 439 4 ASSOCIATION OF MOLYBDENUM WITH OTHER DISORDERS ...... 440 4.1 Copper Homeostasis Disorders ...... 440 4.2 Epilepsy and Neuropsychiatric Disorders ...... 441 4.3 Ethylmalonic Encephalopathy ...... 441 5 CONCLUDING REMARKS AND FUTURE DEVELOPMENTS ...... 442 ABBREVIATIONS AND DEFINITIONS ...... 443 ACKNOWLEDGMENTS ...... 444 REFERENCES ...... 444

Abstract Molybdenum is an essential trace element and crucial for the survival of animals. Four mammalian Mo-dependent enzymes are known, all of them harboring a pterin-based molybdenum cofactor (Moco) in their active site. In these enzymes, molybdenum catalyzes oxygen transfer reactions from or to substrates using water as oxygen donor or acceptor. Molybdenum shuttles between two oxidation states, MoIV and Mo VI . Following substrate reduction or oxidation, electrons are subsequently shuttled by either inter- or intra-molecular electron transfer chains involving prosthetic groups such as heme or iron-sulfur clusters. In all organisms studied so far, Moco is synthesized by a highly conserved multi-step biosynthetic pathway. A defi ciency in the biosynthesis of Moco results in a pleitropic loss of all four human Mo-enzyme activities and in most cases in early childhood death. In this review we fi rst introduce general aspects of molybdenum biochemistry before we focus on the functions and defi ciencies of two Mo-enzymes, xanthine dehydrogenase and sulfi te oxidase, caused either by defi ciency of the apo-protein or a pleiotropic loss of Moco due to a genetic defect in its biosynthesis. The underlying molecular basis of Moco defi ciency, possible treatment options and links to other diseases, such as neuropsychiatric disorders, will be discussed.

Keywords cyclic pyranopterin monophosphate • mitochondria • molybdenum cofactor • neurodegeneration • sulﬁ te oxidase • S-sulfocysteine • substitution therapy

Please cite as: Met. Ions Life Sci. 13 (2013) 415–450 13 Molybdenum in Human Health and Disease 417

1 Introduction

1.1 Chemistry and Biology of Molybdenum

Molybdenum is the only metal of the 2nd row (4d) of the periodic table with biological activity. It is found in nature in different oxidation states ranging from zero to six and forms complexes with organic and inorganic ligands with coordination numbers between four and eight. Molybdenum is the 25th most abundant element in seawater at an average concentration of 100 nM; whereas its concentration in continental water is much lower (5 nM). In living organisms, molybdenum is present at low concentrations. In humans, highest Mo levels are found in kidney, liver, small intestine, and adrenals [1 ]. In serum, the concentration is about 0.6 ng mL –1 [ 2], but depends VI 2– on dietary intake [ 3]. The oxyanion molybdate [Mo O4 ] is the only known form that cells can take up from the environment. In solution, molybdate can be chemically adsorbed onto positively charged iron, aluminium or manganese oxides [ 4 ]. Its availability increases as the pH increases, mainly due to decreased association with metal oxides. The discovery of molybdenum in enzymes such as nitrogenase, nitrate reductase (NR), and xanthine oxidase (XO) demonstrated the biological importance of molybdenum as catalyst in the active site of those enzymes. Up to now, more than 50 different Mo-dependent enzymes have been found in all kingdoms of life [5 ]. Most of them are of bacterial origin and, except for nitrogenase, they all bind molybdenum via a pterin-based prosthetic group forming the so-called molybdenum cofactor (Moco). In eukaryotes, Moco (Figure 1a ) is composed of a fully reduced pterin backbone with a C6-substituted four-carbon side chain forming a third pyran ring that hosts a terminal phosphate and the unique dithiolene group, which binds molybdenum [ 5 ].

1.2 Molybdenum Uptake

All organisms, that depend on molybdenum, take up the oxyanion molybdate as only source of molybdenum. In prokaryotes, molybdate enters the cell through the action of proteins belonging to the ATP-binding cassette (ABC) transporters family [ 6]. In contrast to bacteria, molybdate transport in eukaryotes is poorly understood and the fi rst eukaryotic molybdate transporters were identifi ed only recently [7 , 8]. Genome-wide sequence analysis revealed that orthologues of bacterial ABC-type molybdate transporters are absent in eukaryotes. Given the known interference between molybdate, sulfate, and phosphate transport in plant cells, molybdate transporters were identifi ed within the large and heterogeneous family of sulfate transporters [9 ]. The transporter MOT1 ( MO lybdate T ransporter, type 1 ) was simultaneously identifi ed in the alga Chlamydomonas 418 Schwarz and Belaidi reinhardtii and the plant Arabidopsis thaliana [7 , 8 ]. MOT1 exhibits both a high-specifi city and high-affi nity transport for molybdate. Physiological studies in Chlamydomonas suggested the presence of a second transporter, which was recently identifi ed as fi rst member of the MOT2 family of molybdate transporters. MOT2 belongs to the ubiquitous major facilitator superfamily (MFS) of transporters with orthologues in plants and animals. Saccharomyces expressing the human member of the MOT2 family (HsMOT2) shows molybdate uptake activity comparable to cells expressing MOT1 or MOT2 from Chlamydomonas

( K m = 546 nM) [ 10]. Therefore, HsMOT2 represents the ﬁ rst animal protein able to facilitate molybdate transport. Future studies are required to characterize the cellular localization and functional relevance of MOT2 proteins within the ssMo homeostasis in higher eukaryotes.

1.3 Molybdenum Toxicity

Severe molybdenum toxicity has only been reported in ruminants. Under conditions of high molybdate uptake, the reducing sulfi de-rich gastrointestinal track promotes the 2 − I formation of tetrathiomolybdate (MoS4 ), which in turn readily reacts with Cu or Cu II to form insoluble copper-thiolate-molybdenum complexes [11 ]. Therefore, high molybdenum intake causes a secondary copper defi ciency and is called molybdenosis or hypocuprosis [12 ]. Molybdenum toxicity in ruminants is characterized by severe diarrhea, anorexia, greying of hair, anemia, and leg stiffness accompanied with infertility or sterility. These symptoms are readily reversed by copper supplementation. The ability of tetrathiomolybdates to interact with copper has been used in the treatment of copper-dependent disorders such as Wilson’s disease, which in the absence of treatment results in hepatic and neurological dysfunctions due to intracellular copper accumulation [13 ]. Monogastric animals are less sensitive to molybdenum toxicity. In humans, cases of molybdenum toxicity are extremely rare and confi ned to geographic areas with high amounts of molybdenum in drinking water or soils [14 ]. In some regions of Armenia the population is exposed to high dietary molybdenum intake (10–15 mg/day as compared to 1–2 mg/day under normal conditions). In those individuals aching joints and symptoms resembling gout have been reported, probably due to increased XDH/XO activity causing elevated uric acid production, the major gout (see Section 2.1.4). The tolerable intake for molybdenum has been estimated to be 9 μg Mo kg–1 day–1 [ 15 ].

1.4 Molybdenum Enzymes

Five Mo-enzymes are found in eukaryotes that belong to two families (Figure 1 ). Nitrate reductase, sulﬁ te oxidase (SO), and the amidoxime-reducing component 13 Molybdenum in Human Health and Disease 419

Figure 1 Structure of Moco in the two families of Mo-enzymes and the domain organization of the four mammalian Mo-enzymes. ( a ) Chemical structure of Moco found in sulfi te oxidase (SO), with cysteine ligation, and Moco found in xanthine oxidase (XO), with a terminal sulfi do ligand. (b ) Domain structure of mammalian SO, mitochondrial amidoxime-reducing component (mARC), xanthine oxidoreductase (XOR), and aldehyde oxidase (AOX). Domains are depicted according to their relative size. Moco and dimerization domains with similar structures are identifi ed with the same grade of gray shade. Other domains binding prosthetic groups are shown as white boxes.

D: dimerization domain, b 5 : cytochrome b 5 domain, Fe-S cluster domain, FAD domain.

(mARC) are members of the SO family. In this family, Moco is covalently bound to the enzyme via an invariant cysteine residue forming the third S-ligand in the molybdenum coordination sphere. The other so-called XO family is formed by two very homologous members, xanthine dehydrogenase (XDH) or oxidase (XO), and aldehyde oxidase (AOX) [16 ]. Eukaryotic Mo-enzymes are involved in key processes in the global carbon, nitrogen, and sulfur cycles, such as nitrate reduction, sulﬁ te detoxiﬁ cation, and purine catabolism. Except nitrate reductase, which is only found in autotroph organisms such as plants, fungi, and algae, all other four Mo-enzymes are expressed in humans and will be discussed in the following sections (Figure 1 ).

2 Deﬁ ciencies in Molybdenum Enzymes

Defi ciencies in one or more Mo-enzymes have been described in humans with different pathophysiologies ranging from normal life to neonatal death. All defi - ciencies belong to the large family of inborn errors in metabolism; however, the numbers of known cases for the individual disorders are very low. Therefore, reliable projections on incidence and prevalence cannot be drawn for defi ciencies affecting 420 Schwarz and Belaidi

Mo-enzymes. In addition, considerable heterogeneity in symptoms and disease progression further contribute to misdiagnoses and limited understanding of the underlying pathophysiology. Only recently, novel therapeutic approaches have fostered systematic analyses of patient cohorts. In the following sections we will introduce the four mammalian Mo-enzymes, focus on the individual pathways they are involved in, and discuss the clinical and cellular consequences of their dysfunction.

2.1 Xanthine Dehydrogenase and Oxidase

2.1.1 Structure and Function

Eukaryotic XDH and XO (EC 1.17.1.4) are key enzymes in the degradation of purines, catalyzing the two terminal steps in purine catabolism, the oxidation of hypoxanthine to xanthine and xanthine to uric acid. The enzyme can function either as a dehydrogenase using NAD+ as electron acceptor or, upon reversible oxidation of two conserved cysteines, as an oxidase using dioxygen as terminal electron acceptor. In contrast, proteolytic cleavage of XDH converts the enzyme irreversibly into the XO form [17 , 18]. The protease, responsible for this cleavage remained unknown until today, but it is thought to be localized to the mitochondrial inner membrane space and might be released upon apoptotic permeabilization of mitochondria [19 ]. In the following we will use the term xanthine oxidoreductase (XOR) for both forms of the enzyme. XORs are homodimeric enzymes harboring three cofactor-binding domains (Fe-S clusters, FAD, and Moco) and an additional domain important for dimerization [ 20] (Figure 1b ). Hydroxylation of purine substrates causes two-electron reduction of Moco and the electrons are subsequently transferred singly via two [2Fe-2S] clusters to the FAD cofactor where either NAD+ or dioxygen is reduced in XDH or XO, respectively. The latter co-substrate produces superoxide anions, which renders XO an important target for cellular stress responses. For more details on structure-function relations in XOR see the latest review of Hille, Nishino, and Bittner [21 ].

2.1.2 Xanthinuria Type I

Defi ciency of XOR results in the accumulation of xanthine in urine (xanthinuria) [ 22] with hypoxanthine being also elevated. Hereditary type I xanthinuria lacks only XOR activity, while type II and molybdenum cofactor defi ciency (MoCD) lack two or more Mo enzyme activities, respectively. Type I xanthinuria patients carry mutations in the XDH gene [23 ]; up to date only a handful cases have been genetically classifi ed. The combined incidence for classical type I and type II xanthinuria has been estimated to 1/69,000 [24 ]. 13 Molybdenum in Human Health and Disease 421

The affected individuals by xanthinuria type I may develop urinary tract calculi, acute renal failure, or myositis due to tissue deposition of xanthine, while some subjects with homozygous xanthinuria remain asymptomatic [25 ]. The fact that xanthine accumulation is much higher than the one of hypoxanthine suggests an increased salvage of hypoxanthine, which was experimentally proven by feeding studies using radio-labeled purines [26 ]. Given the relatively broad substrate speci- ﬁ city, XOR can hydroxylate a number of exogenous substrates such as thiopurines, which are chemotherapeutics used for the treatment of acute lymphoblastic leukemia and autoimmune diseases [27 ]. Therefore polymorphisms in the XDH gene may increase the toxicity of drugs such as 6-mercaptopurine.

2.1.3 Xanthinuria Type II

Xanthinuria type II is characterized by the simultaneous loss of two Mo-enzyme activities, XOR and AOX [23 ]. The molecular basis of this dual loss of Mo-enzyme function is due to a mutation in the MCSU gene, encoding for a two-domain protein catalyzing the sulfuration of Moco in enzymes of the XO family [28 ] (for details see Section 3.1.4 ). Both types of hereditary xanthinuria are clinically similar, but patients with type I retain their ability to metabolize allopurinol ( via the activity of AOX, see Section 2.1.4 ), those with type II xanthinuria cannot. Both types result in plasma uric acid levels below 5 mmol/L and elevated plasma xanthine concentrations (>10 mmol/L). Less than half of the affected people develop symptoms, which are caused by deposition of xanthine in the urinary tract. This often results in haematuria or renal colic, and rarely, in acute renal failure or chronic complications related to urolithiasis [23 ]. In very few cases, muscle pains caused by xanthine deposition have been reported [ 29 ]. In one case with hereditary xanthinuria type II the association with mental delay, autism, cortical renal cysts, osteopenia, hair and teeth defects, and various behavioral symptoms was observed [30 ]. Although the underlying disease-causing mechanism remains unclear, xanthinuria joins the growing list of metabolic disorders, such as phenylketonuria, histidinaemia, dihydropyrimidine dehydrogenase deﬁ ciency, and 5’-nucleotidase superactivity, contributing to the development of complex neuropsychiatric disorders [31 ].

2.1.4 Hyperuricemia

Hyperuricemia is characterized by an accumulation of uric acid resulting in an increased urinary excretion. The major cause of the disease is seen in an imbalance between the rates of production and excretion of uric acid. Primary hyperuricemia is due to a decreased renal excretion of uric acid, while causes of secondary hyperuricemia are more complex and include increased XOR activity, increased purine release due to chronic infl ammation, increased purine synthesis and high dietary intake of purines. Longstanding hyperuricemia leads to gout, which is characterized by the deposition of urate crystals in the joints and periarticular structures. Hyperuricemia 422 Schwarz and Belaidi and gout have been associated with the development of cardiovascular disease. Epidemiological studies have shown that certain foods increase the risk of developing gout and hyperuricemia. Low-fat dairy products, purine-rich vegetables, whole grains, nuts and legumes, and less sugary fruits, coffee, and vitamin C supplements decrease the risk, whereas intake of red meat, fructose-containing beverages, and alcohol increase the risk of gout [32 ]. Since more than 50 years, gout is effectively treated with allopurinol, a suicide inhibitor of XOR, but recently, new drugs, developed by structure-based drug design, have entered the clinics [33 ]. Recent data indicate that XOR also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, infl ammatory diseases, as well as chronic heart failure [34 ]. Therefore, uric acid and other purine-related biomarkers gain increased interest in epidemiological studies of such diseases. Furthermore, XOR has also been implicated in nitrite-dependent synthesis of nitric oxide (NO) [ 35], which in turn would counteract hyperuricemia-related cardiovascular disorders. However, pharmacological studies do not support such fi ndings [36 ]. Future studies are needed to increase our understanding of the roles of foods, urate transporters and other molecular mechanisms on the risk of developing gout as well as hyperuricemia and their relation to cardiovascular disorders.

2.2 Aldehyde Oxidase

AOX enzymes (EC 1.2.3.1) originate from a duplication of the xdh gene in eukaryotes before the origin of multi-cellularity [37 ]. Therefore, both enzymes contain the same cofactor-binding domains (Fe-S clusters, FAD, and Moco) as well as a dimerization domain (Figure 1b ). The human genome harbors a single AOX gene together with two pseudogenes clustered on chromosome 2q. However, mouse and rat genomes express four aox genes giving rise to four iso-enzymes [38 ]. The crystal structure of AOX3 from mouse has been recently determined showing high structural similarity to XOR [39 ]. Similar to XOR, mammalian AOX produces superoxide and hydrogen peroxide as secondary products. However, the major difference between the two enzymes relies on substrate specifi city. In fact, AOX exhibits a broad substrate spectrum including heterocycles, aldehydes, purines, and pteridines [40 ]. AOX substrates are often involved in the metabolism of drugs and xenobiotics and the presence of high levels of human AOX in the liver renders this class of enzymes highly interesting in the fi eld of drug discovery [41 ]. Up to now, the physiological function and endogenous substrates of AOXs are unknown, although results from AOX-knockout mice revealed a signifi cant role in the synthesis and biodisposition of endogenous reti- noids in the Hardarian glands and skin [42 ]. In addition, AOX may participate in the oxidation of endogenous products involved in various metabolic pathways such as neurotransmitters (i.e., serotonin), the conversion of the hydroquinone-precursor gentisate aldehyde into gentisate, the catabolism of valine, leucine or isoleucine as well as vitamins (nicotinamide and pyridoxal) [ 40]. Besides acting as oxidase, a secondary function as reductase was also attributed to 13 Molybdenum in Human Health and Disease 423

AOX as it was found to catalyze the reduction of N-oxides, sulfoxides, nitro compounds, and heterocycles under certain conditions [43 – 45 ]. However, the reductase activity of AOX was only observed in vitro and its physiological relevance remains unclear. This ample substrate speciﬁ city and diverse functionality of either the oxidase or reductase rendered AOX very attractive in medical chemistry and toxicology. Thus, its function as a drug-metabolizing enzyme has been conﬁ rmed for different xenobiotics such as the antitumor agents, methotrexate [46 ], and 6-mercaptopurine [47 ], the antidepres- sant, citalopram [48 ], and other compounds of medical relevance [41 ].

2.3 Sulﬁ te Oxidase and Sulﬁ te Toxicity

2.3.1 Cysteine Catabolism

The intracellular pool of cysteine is relatively small as compared to the much larger pool of glutathione (GSH) [ 49 ]. Under oxidizing extracellular conditions, cysteine is oxidized to cystine. Thus, the plasma cysteine concentration is low (10–25 μM), compared with that of cystine (50–150 μM) [ 50]. Cysteine and cystine are transported by different membrane carriers, and cells have typically different transport affi nities for one or the other compound [51 ]. Hepatocytes have low or no capacity for import of cystine. However, GSH is abundant in the liver and reduces cystine extracellularly to cysteine, which is then imported into hepatocytes [50 ]. Cysteine undergoes catabolism via two pathways: an oxidative pathway involving cysteine dioxygenase (CDO; EC 1.13.11.20) and a non-oxidative pathway involving several enzymes (Figure 2). In mammals, the major route of cysteine catabolism follows the oxidation to cysteine sulfi nic acid (CSA) catalyzed by CDO [ 52 ]. This irreversible reaction involves the transfer of two oxygen atoms to the sulfhydryl group of cysteine. CSA can be either decarboxylated in the cytosol to form hypotaurine, which is further oxidized to taurine (Figure 2 ). Taurine is the most abundant non-proteinogenic amino acid in the body and is mainly produced in the liver [ 53 ]. Alternatively, CSA can be deaminated yielding the putative compound β-sulfi nylpyruvate, which spontaneously decomposes into pyruvate and sulfi te [ 52 ]. The latter undergoes oxidation to sulfate catalyzed by SO (see Section 3.3.2 ). The non-oxidative degradation pathway of cysteine involves the contribution of one of the three enzymes, cystathionine γ-lyase (CSE; EC 4.4.1.1), cystathionine β-synthase (CBS; EC 4.2.1.22), and 3-mercaptopyruvate sulfurtransferase (MSPT; EC 2.8.1.2) (Figure 2). Given that all three enzymes are physiologically active with other substrates, high K m values were found for cysteine, which do not correlate with the physiological concentration of cysteine [54 ]. However, all three enzymes have been shown to be involved in the cysteine-dependent production of hydrogen sulfi de [ 55 ], which was considered to be toxic as reported in several poisoning cases [ 56]. However, the fact that signifi cant levels of hydrogen sulfi de have been found in the brain of rats, humans, and cattle [ 57 – 59] suggested a functional role as neural messenger [60 ]. 424 Schwarz and Belaidi

Figure 2 Cysteine catabolism and altered metabolites in sulfi te oxidase defi ciency. Components of the transsulfuration pathway (methionine to cysteine; light orange box), cysteine oxidative (CDO-dependent; gray box), and non-oxidative catabolism (green box) are summarized with all involved enzymes and metabolites. Changes in MoCD are highlighted in blue with corresponding arrows indicating an increase or decrease in concentration in comparison to healthy controls. Enzyme abbreviations are: MAT, methionine-S-adenosyl transferase; MT, methyl transferase; SAHH; S-adenosylhomocysteine hydrolase; BHMT, betaine-homocysteine methyl transferase; MS, methionine synthase; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase (cystathionase) GCS, γ-glutamylcysteine synthetase; GS, glutathione synthetase CDO, cysteine dioxygenase; CSD, cysteinesulfi nate decarboxylase; AAT, aspartate aminotransferase; SO, sulfi te oxidase; MPST, 3-mercaptopyruvate sulfurtransferase; SQR, quinone oxidoreductase; SDO, sulfur dioxygenase; ST, sulfur transferase; KG, α-ketoglutarate.

Hydrogen sulfi de is further oxidized to thiosulfate in mitochondria by three sequential enzymatic reactions (Figure 2 ) [61 ]. First, the mitochondrial membrane fl avoprotein quinone oxidoreductase (SQR) converts sulfi de to a protein-bound persulfi de and transfers two electrons to the ubiquinone pool [ 62 ]. Next, the persulfi de is handed over to a sulfur dioxygenase, which converts the persulfi de molecule to sulfi te using molecular oxygen. Sulfi te, generated by sulfur dioxygenase may also 13 Molybdenum in Human Health and Disease 425 be converted to sulfate by the action of SO in mitochondria [ 62]. Finally, a sulfur transferase adds a second persulfi de molecule from SQR to sulfi te yielding the fi nal product thiosulfate [61 ]. The relative contribution of the non-oxidative pathway in cysteine catabolism is usually low and insensitive to cysteine dietary intake [63 ]. In contrast, CDO concentration and activity increases signifi cantly in response to a higher dietary sulfur- containing amino acid intake. Thus, it has been assumed that cysteine levels in the body are predominantly controlled via CDO [ 64 ]. However, non-oxidative cysteine catabolism is increased if CDO activity is lost as observed in CDO−/− mice, which in addition to increased plasma cysteine and sulfate levels, revealed high concentrations of hydrogen sulfi de [65 ].

2.3.2 Sulﬁ te Oxidase Localization, Structure, and Function

In vertebrates, SO catalyzes the two-electron oxidation of sulfi te to sulfate coupled to the reduction of two molecules of cytochrome c [66 ]. Sulfi te oxidation represents the terminal step in the oxidative degradation of cysteine. Vertebrate SO forms homodi- mers and each monomer harbors an N-terminal cytochrome b5 -type heme domain, a catalytic Moco domain, and a C-terminal dimerization domain [67 ]. In vertebrates, SO is localized in the mitochondrial intermembrane space where electrons derived from sulfi te oxidation are directly passed to the physiological electron acceptor cytochrome c [68 ]. Recently, the maturation of mammalian SO has been clarifi ed showing that it combines a conventional leader sequence-based translocation mechanism with the folding trap mechanism for which the presence of Moco is strictly required [69 ]. The catalytic mechanism of SO involves transfer of two electrons from sulfi te to Moco (MoVI → Mo IV) followed by two one-electron transfer steps via the heme domain to cytochrome c . Within the crystal structure of chicken SO a large distance of 30 Å has been found between the heme and Moco domain [67 ], which would not support the high electron transfer rate observed between the two domains by electrochemical methods [70 ]. Based on the inhibition of internal electron transfer by solution viscosity [ 71 ] and the use of deletions of the solvent- exposed tether connecting the heme and Moco domain, it was shown that during catalysis both domains undergo conformational changes in order to enable effi cient electron transfer [72 ]. SO is structurally very similar to nitrate reductase [73 ], which is only found in plants, fungi, and algae. Regardless of the high structural similarity, SO is unable to reduce nitrate to nitrite unless key residues are replaced by site-directed mutagenesis [74 ]. Interestingly, we recently found that SO is able to reduce nitrite to nitrate under anaerobic conditions [75 ], providing a fi rst and novel hint towards a second function of SO in nitrite-dependent NO synthesis [76 ].

2.3.3 Sulfi te Toxicity and Sulfi te Oxidase Defi ciency

Sulfi te toxicity is characterized by an accumulation of sulfi te, which can be detected in urine of affected patients using commercial sulfi te strip tests. Sulfi te accumulation and toxicity is observed either in isolated SO defi ciency (SOD) caused by mutations 426 Schwarz and Belaidi in the SUOX gene resulting in a loss of SO activity or in MoCD caused by mutations in any of the genes within the Moco biosynthetic pathway resulting in loss of activity of all Mo-enzymes [5 , 77]. SOD is less prevalent than MoCD and approximately 30 cases are reported up to now [78 ]. SUOX mutations affecting substrate binding, specifi city or catalytic activity were biochemically characterized [67 , 79 , 80 ]. SOD and MoCD are clinically nearly indistinguishable and patients of both groups are characterized by a severe neurodegenerative phenotype manifested in the early infancy by intractable seizures, hyper- and hypotonus, mental retardation, developmental delay, and lens dislocation and patients usually die in early childhood (more details below) [77 ]. Given the overlapping clinical phenotype between SOD and MoCD one can assume that SO is the most critical Mo-enzyme to ensure survival during the neonatal period of life.

2.4 Mitochondrial Amidoxime-Reducing Component

Recently, two isoforms of the mitochondrial amidoxime-reducing component have been identiﬁ ed in mammals, thus constituting a novel type of Mo-containing enzymes [81 ]. Mouse mARC1 localizes to the outer mitochondrial membrane [82 ]. It is targeted by a bipartite N-terminal signal peptide leading to its tail-anchored integration with cytosolic exposure of the protein. Besides a role of mARC2 in the regulation of NO synthesis [ 83], no primary physiological substrates are known for mARC proteins [84 ]. Human mARC proteins seem to be involved in pro-drug metabolism given their ability to reduce several N-hydroxylated substances commonly used as pro-drugs. All mARC proteins contain a C-terminal Moco-binding domain, which has been proposed to form a novel class of molybdo-enzymes in plants [85 ]. In contrast, studies in C. reinhardtii have demonstrated that a highly conserved cysteine residue present in all mARC proteins is essential for catalytic activity, which is a typical feature present in proteins of the SO family (Figure 1b ) [86 ]. So far, all characterized mARC proteins require the formation of complexes with cytochrome b 5 and with a NADH/cytochrome b 5 reductase for catalytic activity suggesting the formation of an intermolecular electron transfer chain [87 ].

3 Molybdenum Cofactor Deﬁ ciencies

MoCD is characterized by the simultaneous loss of all Mo-enzyme activities due to a mutational block in the biosynthesis of Moco. Human MoCD is a rare autosomal recessive disorder, which mostly affects neonates and is characterized by progressive brain injury leading to early childhood death [88 ]. In the following sections we ﬁ rst introduce the biochemistry of Moco and the genetics underlying MoCD before the pathophysiology of the disease, models and treatment options will be discussed. 13 Molybdenum in Human Health and Disease 427

3.1 Biochemistry of Molybdenum Cofactor Biosynthesis

The structure and function of Moco is universal in all eukaryotes (Figure 1a ) [5 ]. First genetic and later biochemical studies in fungi, bacteria, plants, and fi nally animals (including humans) demonstrated that also the biosynthesis of Moco is highly conserved in all kingdoms of life [89 ]. Eukaryotic Moco biosynthesis can be divided into three major steps based on the two fi rst identifi ed intermediates cyclic pyranopterin monophosphate (cPMP) [ 90 ], previously named Precursor Z [ 91] and the metal-binding pterin (MPT) [ 92]. Each of the steps involve the action of one or more proteins producing additional reaction intermediates (pyranopterin triphosphate [93 ], thio-pyranopterin phosphate [94 ], and adenylated MPT [95 ]) (Figure 3 ).

3.1.1 Pterin Synthesis

Similar to the biosynthesis of other pterins and fl avins, Moco synthesis starts with GTP [96 ]. Labeling studies in E. coli proposed a complex and unique rearrangement mechanism where the C8 atom of the purine base is removed as a formyl group and subsequently inserted between the 2’ and 3’ ribose carbon atoms resulting in the formation of the four-carbon pyran ring [97 ,98 ] present in both, cPMP and Moco. This reaction leads to the formation of the pterin backbone of the cofactor (Figure 3 ). cPMP is the most stable biosynthetic intermediate with a half-life of several hours, depending on the environment [ 99 ]. Its chemical structure has been resolved fi rst by high-resolution mass spectrometry and 1H NMR spectroscopy and confi rmed by 13 C NMR showing its pyranopterin nature with an unusual geminal diol [90 ,99 ]. The proteins MoaA and MoaC, catalyzing cPMP synthesis, have been best studied in bacteria. Human proteins are encoded by the MOCS1 gene and will be discussed later (Section 3.2.1 ). MoaA and homologous proteins are members of S-adenosylmethionine (SAM)-dependent radical enzymes containing one or two [4Fe-4S] clusters [100 , 101 ]. The N-terminal [4Fe-4S] cluster in MoaA promotes SAM cleavage to generate a 5’-deoxyadenosyl radical, which initiates the transformation of 5’-GTP (bound to the C-terminal [4Fe-4S] cluster) by abstracting the 3’ proton from the ribose resulting in the formation of pyranopterin triphosphate (Figure 3 ) [ 93]. This mechanism is believed to be conserved, given that plant and human orthologues are able to complement E. coli moa A mutants [102 , 103 ]. The second protein essential for cPMP synthesis is MoaC and it is involved in pyrophosphate release and formation of the geminol diol [ 96]. Plant proteins catalyzing cPMP synthesis have been found to localize to mitochondria [104 ], which is consistent with a recent fi nding for human MOCS1 proteins (see Section 3.2.1). The biogenesis of Fe-S clusters and high abundance of GTP within mitochondria might have been the driving force for the subcellular localization of these proteins in eukaryotes [105 ]. 428 Schwarz and Belaidi

Figure 3 Biosynthesis of the molybdenum cofactor. Major and transient intermediates of the three steps are shown. Co-substrates for each reaction step are depicted at the arrows.

3.1.2 Dithiolene Synthesis

In the second step of Moco biosynthesis two sulfur atoms are incorporated into cPMP to form the dithiolene function in MPT. The reaction is catalyzed by MPT synthase, a heterotetrameric complex built of two small and two large subunits that 13 Molybdenum in Human Health and Disease 429 stoichiometrically converts cPMP into MPT (Figure 3 ). The reaction mechanism of MPT synthesis involves a stepwise transfer of sulfur, which is accompanied by the formation of a mono-sulfurated intermediate (Figure 3 ) and hydrolysis of the cyclic phosphate (thio-pyranopterin phosphate) [94 ]. Each small subunit of MPT synthase carries a sulfur atom as thiocarboxylate [106 ], which is transferred by an ATP- dependent reaction involving an adenylyltransferase [107 ]. While in bacteria separate cysteine desulfurases can provide sulﬁ de for the subsequent sulfuration reaction following the hydrolysis of the protein-adenylate, in eukaryotes a rhodanese-like domain (MOCS3) binds sulfur as persulﬁ de at a conserved cysteine, which is believed to mediate the subsequent sulfuration of the small subunit [108 ].

3.1.3 Molybdate Insertion

Once MPT is formed, it is ready to coordinate molybdenum via the dithiolene function. However, prior to molybdenum coordination, MPT is activated by adenylylation. While in bacteria two different proteins (MogA and MoeA) catalyze MPT adenylylation [109 ] and molybdenum insertion [ 110], respectively, eukaryotic organisms mostly use two-domain proteins (Figure 3 ) in which both activities have been fused and in case of mammals, including humans, the resulting gephyrin represents a multi-functional protein [111 ]. Gephyrin’s G-domain binds MPT with high afﬁ nity and forms adenylylated MPT as demonstrated by functional and structural studies [95 , 112 ]. Furthermore, in the complex structure of the gephyrin- homologous Cnx1 G-domain with either MPT or MPT-AMP, a bound copper was found at the dithiolene [ 95 ]. A possible role of this copper can be seen either in protecting MPT from oxidation or in facilitating molybdenum insertion by providing a suitable leaving group. Following its synthesis by the G-domain, MPT-AMP is transferred to the gephyrin E-domain where MPT-AMP is subsequently hydrolyzed in a reaction that is molybdate- and Zn2+ - or Mg2+ -dependent (Figure 3 ) [113 ]. Adenylylated molybdate has been proposed as reaction intermediate in analogy to the adenylylated sulfate in sulfate assimilation [113 ]. Based on the fusion of two catalytic domains in gephyrin, intramolecular product- substrate-channeling has been proposed. Recently, we developed a fully deﬁ ned in vitro system for Moco biosynthesis allowing the direct comparison of the reaction rates of the individual gephyrin domains with holo-gephyrin [114 ]. In contrast to the isolated domains, holo-gephyrin exhibited a 300-fold increased

ATP-dependent Moco synthetic activity with a K m for molybdate being close to the intracellular concentration of molybdate as well as the K m of the respective molybdate transporter [114 ]. As side effect of the orientation of the fused domains in gephyrin, novel functions have evolved, one of which rendering gephyrin an instructive and essential protein in synaptogenesis [111 ]. Therefore, in the central nervous system, gephyrin functions in addition to Moco synthesis, as scaffolding protein at inhibitory synapses where it binds to glycine and γ-amino butyric acid type A receptors [115 ]. 430 Schwarz and Belaidi

3.1.4 Maturation of Moco

Following the release of Moco from the biosynthetic machinery, the cofactor either binds directly to enzymes of the SO family or it undergoes additional modifi cation, in case of enzymes of the XO family. In those cases, one of three oxo ligands of Moco is replaced by a terminal sulfi do ligand. In contrast, in enzymes of the SO family the third S-ligand is derived from the apo-enzyme by non-enzymatic ligation to a conserved cysteine residue. In all cases, the third sulfur ligand occupies one of the four corners of the square pyramidal coordination of molybdenum ligands (Figure 1 ). In humans, the sulfuration of Moco is catalyzed by a Moco sulfurase (MCSU) [ 116 ]. In vitro studies using the plant orthologue (ABA3) demonstrated its ability to sulfurate Moco in holo-enzymes of the XO family [117 ], while in vivo it remains unclear whether the transfer of sulfur to Moco takes place before or after cofactor insertion into apo-enzymes. Human MCSU and orthologues present two-domain proteins with an N-terminal cysteine desulfurase domain and a Mo co sulf urase C -terminal domain (MOSC) that binds Moco and is believed to mediate protein- protein interactions with apo-enzymes [118 , 119 ]. Following the desulfuration of L-cysteine, a protein-bound persulfi de is formed on a conserved cysteine and subsequently transferred to bound Moco [120 ].

3.2 Genetics of Human Molybdenum Cofactor Synthesis

Human genes and gene products involved in Moco synthesis are named according to the MOCS nomenclature (Mo Cofactor S ynthesis) [121 ] with the exception of GEPHYRIN, which has been identiﬁ ed earlier as neuronal scaffolding protein [ 122]. Due to its “bridging” function between inhibitory neuroreceptors and the subcellular cytoskeleton, the greek word for bridge “gephos” has been chosen and kept, regardless of the identiﬁ cation of GEPHYRIN’s primary function in Moco synthesis.

3.2.1 Structure and Organization of Moco Synthesis Genes

Shortly after the identifi cation of the fi rst human Moco synthesis gene MOCS1 [121 ], the other three genes in the pathway ( MOCS2 , MOCS3 , and GEPHYRIN ) have been identifi ed based on high homologies of their gene products to plant and bacterial Moco synthetic proteins [107 , 111 , 123]. Remarkably, MOCS1 as well as MOCS2 show a bicistronic gene structure suggesting a highly coordinated expression of the encoded gene products. In contrast to MOCS2 , the predicted expression of two open reading frames from the MOCS1 mRNA, MOCS1A and MOCS1B, encoding for MoaA- and MoaC-homologous proteins could not be verifi ed [103 ]. Instead, alternative splicing of the MOCS1A transcript was found to result in the 13 Molybdenum in Human Health and Disease 431

Figure 4 Types of molybdenum cofactor and Mo-enzyme defi ciencies. (a ) Three major steps of Moco synthesis, the involved genes and their translation products. Note, GEPH-G and GEPH-E represent the two functional domains of GEPHYRIN catalyzing two separate steps in Moco synthesis, respectively. MCSU catalyzes the sulfuration of Moco, which is essential for XOR and AOX activities. Patients with mARC and AOX defi ciencies have not been reported yet. (b ) MRI scans of a MoCD type A patient recorded at an age of 12 and 27 days showing the rapidly progressing brain damage resulting in brain atrophy and cystic changes in the cerebral cortex. expression of either MOCS1A alone (splice type I) or MOCS1AB fusion proteins (splice type II and III, Figures 3 and 4 ) [124 ]. The latter only harbors MOCS1B activity due to the truncation of functionally important C-terminal residues within the C-terminus of MOCS1A [103 ]. Besides alternative splicing of exon 9 (producing splice types II and III) [ 124], additional splice variants resulting from alternative splicing of exon 1 have been found [125 , 126 ]. Very recently, we could demonstrate that MOCS1 proteins are localized to mitochondria (similar to their plant orthologues, Roeper and Schwarz, unpublished results). Mitochondrial translocation of MOCS1A is dependent on exon 1a, while MOCS1AB fusion proteins are translocated also in the absence of exon 1a using another internal targeting signal encoded by exon 10, which drives MOCS1AB into the inner mitochondrial membrane. Human MPT synthase is encoded by the bicistronic MOCS2 gene. The two overlapping open reading frames (MOCS2A and MOCS2B) are translated by a ribosomal leaky scanning mechanism producing both subunits in an approximately equimolar ratio (Figure 4 ) [ 123 ]. Similar to MOCS1 , a fusion of both proteins would 432 Schwarz and Belaidi abolish the function of the fi rst translation product (small subunit of MPT synthase), due to the functional requirement of the conserved C-terminal double glycine motif in thiocarboxylation and subsequent sulfur transfer. MOCS3 encodes for the Moco sulfurase and has been identifi ed based on sequence homology to plant and E. coli proteins [107 ]. Both, MPT synthase (MOCS2A-MOCS2B) as well as the sulfurase (MOCS3) are localized in the cytosol. The GEPH gene encodes for the multi-domain cytosolic protein GEPHYRIN composed of an N-terminal G-domain (GEPH-G), central C-domain, and a C-terminal E-domain (GEPH-E, Figure 4 ), which is membrane-associated in the central nervous system due to its interaction with glycine and GABA type A receptors [115 ]. In addition to brain and spinal cord, high levels of GEPHYRIN expression have been identifi ed in liver and kidney [127 ]. The GEPH gene is highly mosaic with 27 exons distributed over 760 kb of genomic DNA on chromosome 14q32. At least six of these exons are subject to alternative splicing producing more than 10 different splice variants [128 ]. Functional diversity of GEPHYRIN is believed to rely on a tissue- specifi c expression of alternatively spliced transcripts [115 ]. For example, variants containing the C3 cassette are highly abundant in liver, the organ with highest Moco synthesis, while variants containing different forms of the C4 cassette were found in brain and spinal cord, where gephyrin is mainly functioning as receptor scaffold [128 , 129 ]. This fi nding is supported by recent studies showing that different gephyrin splice variants, which were expressed in Sf9 insect cells, bind to the glycine receptor with different affi nities [ 130 ].

3.2.2 Mutations in Molybdenum Cofactor-Deﬁ cient Patients

Following the identifi cation of MOCS1 and MOCS2 genes, mutations in patients with MoCD have been identifi ed [121 , 131 ] as well as prenatal diagnosis in carrier families has been successfully established [132 ]. Today more than 64 disease- causing mutations have been identifi ed in MOCS1 , MOCS2 and GEPHYRIN , representing over 100 reported cases worldwide [133 ]. In nearly all cases, disease-causing mutations result in a complete loss of enzyme function due to frame shifts, splice site and nonsense mutations, or missense mutation affecting highly conserved or invariant residues (Figure 5 ). The majority of MoCD patients carry mutations in the MOCS 1 gene affecting either MOCS1A function or the MOCS1B domain in splice type II and III translation products (Figure 4 ) [134 ], representing approximately two thirds of all MOCD patients identifi ed so far. Up to date at least 40 disease-causing MOCS 1 mutations have been found. In most cases homozygous mutations were found due to the high consanguinity of heterozygous parents, while compound heterozygous cases are the minority. Approximately one third of MOCD patients are affected in the second step of Moco synthesis due to mutations in the MOCS 2 gene. 23 disease-causing mutations have been found in both open reading frames affecting either the small (MOCS2A) or large subunit (MOCS2B) of MPT synthase. Interestingly, up to date not a single 13 Molybdenum in Human Health and Disease 433

Figure 5 Multiple sequence alignment of MOCS1A and MOCS1B translation products with plant Cnx2 and Cnx3 as well as E. coli MoaA and MoaC proteins, respectively. Identiﬁ ed mutations resulting in amino acid substitutions, frame shifts (fs) or deletions are shown above the aligned sequences. Note, missense mutations were only found at invariant positions.

MoCD patient has been identifi ed with a MOCS 3 mutation. Given the dual function of MOCS3 in Moco synthesis as well as tRNA thiolation [135 ], a combination of both might not be vital. Mutations in GEPH are very rare. Only two cases have been described so far. The fi rst patient was the last of three affected infants born to a Danish mother and father who were cousins. All three infants died in the neonatal period (day 12, 29, and 3, respectively), with typical symptoms of MoCD [136 ]. Genetic analysis revealed an early stop codon resulting in a total loss of GEPHYRIN expression. As a result, 434 Schwarz and Belaidi both functions of GEPHYRIN, Moco synthesis as well as glycine and GABA receptor clustering are impaired. In light of the neurological phenotype of both disorders (MoCD and impaired synaptic inhibition) [77 , 137 ], a worsening of each of the two isolated conditions can be assumed (see also next section). A second case with missense mutation in GEPH affects an invariant aspartate residue (Asp580) within the active site of the GEPHYRIN E-domain. This patient presented typical symptoms of MoCD, was two years old at the time of report [138 ] with severe axial hypotonia, peripheral hypertonia, and lack of head control and visual contact. Based on the clinical presentation as well as the underlying genetic defect, one can assume that in this patient Moco synthesis, namely the hydrolysis of MPT- AMP and molybdenum insertion is prohibited while GEPHYRIN’s function in receptor clustering is retained given that the binding site of both, glycine [ 139] and GABA receptors [140 ] are distinct from Moco synthesis [113 ]. The vast majority of MoCD patients present a very severe neurological phenotype. Only a handful cases have been reported with mild forms of the disease. To our knowledge, only one mild presentation was reported for MoCD type A defi ciency affecting the splicing of MOCS1 exon 9 probably affecting the mitochondrial maturation and/or targeting of MOCS1AB translation products, thus leading to a reduced but not completely absent cPMP synthetic activity. All other mild cases carry mutations either in MOCS2 , thus showing only partially impaired MPT synthesis [141 ] (G. Schwarz, unpublished results) or mutations in the SUOX gene [142 – 144 ].

3.2.3 Biochemical Classiﬁ cation of Molybdenum Cofactor Deﬁ ciency

MoCD can be grouped into three types according to the underlying genetic defect (Figure 4a ). Type A defi ciency affects two-thirds of all patients and is caused by mutations in the MOCS1 gene [133 ]. While type A patients lack the fi rst Moco intermediate cPMP, type B patients accumulate cPMP due to defects in the MOCS2 gene, which encodes the MPT-synthase [132 ]. Biochemical analysis of urine samples using HPLC and reverse phase chromatography can discriminate between type A and type B patients by quantifying the cPMP oxidation product compound Z, which accumulates in urine of type B patients and is completely absent in type A patients. The two GEPHYRIN-defi cient cases belong to type C MoCD defi ciency [136 , 138 ].

3.3 Pathophysiology of Molybdenum Cofactor Deﬁ ciency

3.3.1 Clinical Presentation of Patients with Molybdenum Cofactor Deﬁ ciency

Duran et al. described the fi rst case of a human patient with MoCD in 1978 [ 145 ]. The patient presented in his neonatal period initial feeding diffi culties, therapy- resistant seizures, high pitch crying followed by severe neurological abnormalities, 13 Molybdenum in Human Health and Disease 435 lens dislocation of the eyes, and major dysmorphic features of the head. At the time of identifi cation, the chemical nature of Moco was not known, neither its biosynthesis. Based on the identifi ed alterations in the biomarkers of two Mo-dependent enzymes, XO and SO, a defect in either molybdenum metabolism or transport has been proposed [145 ]. Since then more than 100 cases with MoCD have been reported [77 , 134 ], which nearly all share a predominant deterioration of the central nervous system as main disease feature mimicking hypoxic ischemic encephalopathy during the fi rst days of presentation [146 ]. In general, fi rst symptoms are observed within the fi rst days of life, which are initially presented by feeding diffi culties accompanied with intractable seizures with a predominant opisthotonus and an exaggerated startle reaction [138 ]. Disease progression is accompanied by psychomotor retardation due to progressive cerebral atrophy and ventricular dilatation, which are typical in brain MRI of patients (Figure 4b ). In addition, major radiological features of the disease include global cerebral edema, cystic encephalomalacia, cortical and white matter atrophy, focal or bilateral changes within the globi pallidi and subthalamic regions, dysgenesis of the corpus callosum, and ventriculomegaly [146 – 148 ]. Patients that survive the neonatal period show essentially no neuronal development, are unable in any coordinated movements, require tube feeding and show no signs of communication with their environment and usually die within their fi rst years of life [77 ].

3.3.2 Molecular Basis of Neurodegeneration

In humans, MoCD is clinically nearly indistinguishable from the less prevalent isolated SOD implicating sulfi te toxicity as a major underlying cause triggering neurotoxicity in MoCD patients. SO, which is localized in the mitochondrial intermembrane space oxidizes sulfi te to sulfate, thus no accumulation of sulfi te in the cytosol or extracellular compartments is seen under wild-type conditions [67 ]. In MoCD and SOD, sulfi te accumulates fi rst in mitochondria where it has been shown to increase reactive oxygen species [149 ]. Sulfi te also decreases ATP synthesis in mitochondria when respiring on glutamate (which is the case in the brain), while using other respiratory substrates such as malate, α-ketoglutarate, and succinate did not show any sulfi te vulnerability [149 ]. The mechanism underlying the inhibition of ATP synthesis has been related to glutamate dehydrogenase inhibition by sulfi te, which in brain, due to the fact that glutamate dehydrogenase operates in the direction of oxidative deamination, will lead to a decrease in the availability of α-ketoglutarate and other tricarboxylic acids resulting in an overall decrease in ATP synthesis [149 ]. Knowing that glutamate itself is neuroexcitotoxic and is the precursor of the inhibitory neurotransmitter GABA, inhibition of glutamate dehydrogenase may also affect the metabolism of those neurotransmitters, which would be one explanation for the accelerated injury in neuronal rather than non-neuronal tissue in MoCD. In the absence of SO activity sulfi te crosses the outer mitochondrial membrane and accumulates in cytosol and later in plasma. Sulfi te is a strong reductant and will therefore reduce disulfi de bridges, primarily in membrane and extracellular proteins, thus affecting their folding, stability, and activity. Probably fi rst, sulfi te 436 Schwarz and Belaidi reacts with cystine leading to the formation of the secondary metabolite S-sulfocysteine (SSC) [5 ]. SSC is very abundant in MoCD patients and its excretion in urine is detectable shortly after birth and increases with age [ 150 ], which supports the view that sulfi te is cleared during maternal gestation [151 ]. SSC is structurally similar to glutamate, is able to bind to NMDA receptors and therefore postulated as main agent responsible for seizure development and subsequent brain damage in MoCD [152 ]. In fact, early studies in rats demonstrated that subcutaneous administration of SSC induces the same type of brain damage that glutamate and other excitatory amino acids are known to cause [152 ]. In contrast to the neurotransmitter glutamate, whose release in the extracellular compartment is highly controlled by vesicular fusion and cellular re-uptake, SSC is continuously produced by sulfi te accumulation. In the absence of a specifi c transporter, SSC is assumed to persist in the extracellular compartment thus potentiating its excitotoxic effect leading to neuronal death. Besides SSC, taurine is also elevated in MoCD [ 153 ] and known to be neuroprotective by playing an important role in glutamate and GABA signaling [154 ], however, this positive effect seems to be erased by SSC toxicity. SSC formation goes hand in hand with cystine depletion [77 ]. Cystine is the major transport form of cysteine in plasma. In the brain, cystine is taken up into glial cells, where it is reduced and incorporated into glutathione, the major antioxidant in neuronal tissue and most abundant low-molecular-weight thiol in animal cells (0.5–10 mM) [50 ]. Most of the GSH (85–90%) is present in the cytosol, while relatively low GSH concentrations are found in plasma (2–20 μM) [155 , 156]. An increase in cysteine supply via oral or intravenous administration enhances GSH synthesis and prevents GSH defi ciency in humans [157 ]. Thus, cysteine is generally considered to be the limiting amino acid for GSH synthesis in humans, rats, and pigs [ 49 , 158 , 159] and its depletion will have a major impact on cell viability. Despite the dramatically reduced levels of plasma cystine in MoCD, no information is available regarding GSH concentrations in affected patients, which together with SSC, cystine, and glutamate levels in cerebrospinal fl uid may further contribute to the understanding of the mechanism of neurodegeneration in MoCD.

3.4 Animal Models

3.4.1 Mocs1-Deﬁ cient Mice

Due to the high prevalence for type A MoCD, a mocs1 -knockout mouse was generated by homologous recombination with a targeting vector [160 ]. Homozygous mocs1 −/− mice displayed a severe phenotype characterized by a retarded growth, abnormal behavior, lack of feeding and they died within the ﬁ rst 11 days of life with an average life span of 7.5 days [ 160]. As observed in humans, biochemical characterization of mocs1 −/− mice revealed markedly elevated urinary sulﬁ te and xanthine levels while uric acid was undetectable. Furthermore, neither MPT nor Moco could be detected in homozygous mice and as a result, Mo-enzyme activities were absent. 13 Molybdenum in Human Health and Disease 437

In contrast to humans, no brain atrophy or any other kind of histological damage could be observed, which could be due to a different developmental stage of mice in comparison to humans, presenting the last trimester of human brain development in comparison to neonatal mice. However, in terms of biomarkers and disease progression (survival), mocs1 −/− mice present a suitable animal model to study the molecular basis and treatment of human MoCD.

3.4.2 Gephyrin-Deﬁ cient Mice

Gephyrin-deﬁ cient mice present a severe phenotype resembling that of humans with hereditary MoCD and hyperekplexia, a failure of inhibitory neurotransmission [ 161 ]. This animal model was instrumental in demonstrating the dual role of gephyrin in Moco synthesis [111 ] and receptor clustering. Geph −/− neonates appeared externally normal, and failed to suckle. In response to mild tactile stimuli, they retained rigid with a hyperextended posture and exhibited apnea (difﬁ culty in breathing) by 12 hours after birth being consistent with impairment of inhibitory glycinergic inputs to motoneurons. Given the hyperekplectic presentation and a maximal survival of 1 day after birth renders the gephyrin deletion clearly more severe than MoCD as seen for mocs1 −/− mice. On the other hand, motor defects seen in geph−/− mice occurred earlier with more severe presentation than those observed in mutant mice that lack the glycine receptor α1 subunit or have reduced levels of the GlyR β subunit [ 162 ]. In light of both observations and the underlying pathomechanism of MoCD, we propose that the excitotoxic action of accumulating SSC due to gephyrin-dependent MoCD is worsened by the lack of appropriate synaptic inhibition, caused by the loss of gephyrin-dependent receptor immobilization in the central nervous system.

3.5 Treatment of Molybdenum Cofactor Deﬁ ciency

3.5.1 Treatment of Mocs1-Deﬁ cient Mice with cPMP

Following the establishment of an animal model (mocs1 −/− mice) for human MoCD type A, a cPMP fermentation and purifi cation procedure from E. coli has been developed [ 90 ]. To probe that mocs1−/− mice are still able to convert cPMP into MPT and to determine the required dose, purifi ed cPMP was titrated to crude liver extracts of mocs1−/− mice and in vitro Moco synthesis was quantifi ed as a function of cPMP added. Based on those results, an initial dose of 1 μg cPMP per animal was determined and injected in the liver of Mocs1-defi cient mice. cPMP-treated Mocs1- defi cient mice developed normally, gained weight and reached adulthood, were fertile and not distinguishable from their wild-type littermates [163 ]. Furthermore, improvement of the treated animals was directly correlated with cPMP injections as withdrawal of cPMP caused death within 10–14 days following the fi nal injection. 438 Schwarz and Belaidi

Consequently, a dose and treatment interval-dependent restoration of Moco synthesis as well as Mo-enzyme activity was observed. In conclusion, a ﬁ rst experimental and causal treatment approach using cPMP substitution has been established for type A MoCD [163 ].

3.5.2 Treatment of MoCD Type A Patients with cPMP

Based on the promising results of cPMP substitution therapy in Mocs1 -defi cient mice, cPMP fermentation and purifi cation has been upscaled allowing a fi rst human exposure following intensive discussion with the Regulatory Authorities [150 ]. Prior to treatment, patient’s urine and plasma levels revealed markedly elevated levels of SSC, thiosulfate, and xanthine, low uric acid, and elevated urine sulfi te and undetectable cPMP in urine, being indicative for MoCD type A, which has been confi rmed by genetic analysis [150 ]. The devastating character of the disease was manifested by a rapid and continued increase of urinary sulfi te, thiosulfate, and SSC levels during the fi rst 36 days of life before starting the treatment. Furthermore, magnetic resonance imaging showed diffuse cerebral edema with an elevated lactate peak in the magnetic resonance spectroscopy at day six and the infant had a markedly abnormal electro encephalogram at seven days of age. Treatment of the patient started on day 36 of life and sulfi te, SSC, xanthine, and uric acid were used as biomarkers to monitor treatment effi cacy. As starting dose, 80 μg cPMP per kg body weight were chosen based on previous studies in Mocs1 - defi cient mice [164 ] and applying the conversion factor from “mouse to men” of 12.3. Within days after treatment started, the patient showed a remarkable normalization of MoCD biomarkers with sulfi te disappearing after 3 days from urine and SSC dropping continuously during the fi rst week of treatment and reaching near normal levels following a stepwise dose adjustments to 240 μg/kg body weight. Xanthine and uric acid normalization was observed within two weeks of treatment. Clinically, the patient became more alert 48 hours after treatment started; convulsions and twitching disappeared within the fi rst two weeks and epileptic discharges were markedly reduced [150 ]. Today, the patient is more than fi ve years of age, shows a delayed neurological development with profound cerebral palsy due to the preexisting brain damage before treatment was initiated. The patient is alert, interacts with family members, can sit and stand with support and shows normal sleeping pattern (Veldman et al., unpublished results). Based on the fi rst index case, a treatment plan has been developed for cPMP therapy of MoCD patients and currently six MoCD patients receive cPMP treatment, all showing a similar biochemical and clinical improvement as the index case (Schwahn et al., unpublished results). Depending on the time of treatment initiation, patients can reach an almost normal neurodevelopmental outcome [165 ]. Very recently, the chemical synthesis of cPMP has been reported, providing a key milestone in the clinical development of cPMP therapy [166 ]. 13 Molybdenum in Human Health and Disease 439

3.5.3 Treatment of MoCD Type B and C Patients

Treatment of the most frequent MoCD type A defi ciency was fostered by the chemical nature of cPMP, which presents a fully reduced pterin with a relatively long half-life [99 ] as compared to other reduced and clinically relevant pterins such as tetrahydrobiopterin [167 ]. In contrast, MoCD type B patients are unable to synthesize MPT and therefore a substitution therapy with either MPT or mature Moco would be required. Neither MPT nor Moco have been stably isolated in a protein- free form yet and are therefore not available for substitution therapies [5 ]. MoCD type C has only been reported in two cases [ 134 , 136]. Given the overall increased severity of a gephyrin loss of function in both, an animal model [ 161 ] as well as a patient [ 136], we assume that most cases remain undiagnosed due to their early neonatal death. Biochemical studies with gephyrin-defi cient fi broblasts from either mouse (L929 cells) [168 ] or human [ 136 ] suggest that in the complete absence of gephyrin, molybdate excess results in the formation of Moco by chemical ligation of molybdenum to MPT. However, this reaction is only possible, when MPT is present in high quantities, while MPT-AMP, the reaction product of the G domain of gephyrin cannot be activated by molybdate. Therefore, the second gephyrin patient carrying a point mutation within the E-domain [ 138] would not have benefi ted from molybdate substitution therapy. In conclusion, we propose that gephyrin patients with missense mutation within the G-domain that do not impair the synaptic function of gephyrin, should be considered for molybdate supplementation.

3.5.4 Dietary Restriction and Treatment of Sulﬁ te Oxidase Deﬁ ciency

Knowing that sulfi te is the main toxic compound accumulating in MoCD and SOD, early attempts to reduce its formation by reducing the dietary intake of sulfur amino acids in patients have been conducted. Boles and colleagues reported a short-term rapid decrease in urinary sulfi te following methionine restriction and cysteine supplementation in a single MoCD patient [169 ]. However, this improvement could not be reproduced in a SOD patient [170 ]. In contrast, increase in urinary sulfi te was observed upon cysteine supplementation and treatment was stopped after four weeks [170 ]. Investigation of cysteine catabolism in mammals clearly demonstrated that increase in dietary cysteine levels are directly correlated with an increased activity of CDO as the fi rst and rate-limiting cysteine-catabolizing enzyme [171 ]. Following CDO reaction, either sulfi te or taurine are formed when cysteine is supplemented [171 ]. Thus, it is not surprising that on a long-term therapy, a continuous cysteine supplementation will lead to an increase in the formation of sulfi te and S-sulfocysteine. The short-term response to an increased cysteine supplementation reported in a SOD patient is probably due to the sulfi te-chelating capacity of cysteine, which initially leads to a rapid decrease in sulfi te concentration. In contrast, a low protein diet with reduced intake of both sulfur amino acids methionine and cysteine were 440 Schwarz and Belaidi effective in reducing sulfi te, thiosulfate and S-sulfocysteine in two patients with a mild form of SOD [ 143 ]. Furthermore, both patients grew normally without any sign for neurological deterioration and show evidence of progress in psychomotor development. Thus, a control of plasma cysteine and methionine levels should be considered in both MoCD and SOD. As reduction of methionine and cysteine also lead to sulfate and taurine decrease, a supplementation with those compounds may reduce possible side effects resulting from their defi ciencies [143 ]. A patient with SO defi ciency presenting a mild phenotype was recently reported, who showed a beneficial development after a restricted diet in sulfur amino acids, suggesting a residual activity of SO in that case [144 ].

4 Association of Molybdenum with Other Disorders

4.1 Copper Homeostasis Disorders

Copper has been found to bind to MPT and MPT-AMP [95 ]. It is known that molybdenum can act antagonistically to copper. The shortage of molybdate in Australian farmland triggered excessive fertilization, resulting in molybdate overload of the soil that caused pathologic symptoms of molybdenosis in animals, which in particular in ruminants triggered secondary copper defi ciency [172 ]. Later, these Mo-induced conditions of copper defi ciency revealed the pathology of two human Cu homeostasis disorders: Menkes (Cu defi ciency) and Wilson’s (Cu overload) diseases [173 ]. Consequently, potent Cu chelators such as tetrathiomolybdates were used to treat Wilson’s disease and a number of other disorders that are linked to Cu homeostasis, such as neurodegeneration, cancer, and infl ammation [174 ]. For example, tetrathiomolybdates are used to inhibit metastatic cancer progression, however, the molecular mechanism is poorly understood [175 ]. The underlying chemistry of the chelation of either copper or Mo to dithiolates most likely explains their antago- nistic function towards each other. The function of copper in Moco synthesis has not been clarifi ed yet. Therefore, future studies are needed to probe the impact of copper homeostasis on Moco synthesis and Mo-enzyme activities. Our own in vitro synthesis studies using protein- bound MPT-AMP showed an inhibition of Moco synthesis in the presence of 1 μM

CuCl2 , providing a link between Mo and copper metabolism [95 ]. Copper inhibition of Moco synthesis can be explained by inhibition of the Mg-dependent molybdate insertion reaction. This fi nding might suggest that Moco biosynthesis might be affected under conditions when cellular copper concentrations are increased, as seen in patients with Wilson’s disease [173 ], where copper accumulates in liver and brain, resulting in severe damage of both organs. In contrast, copper defi ciency might also impact Moco biosynthesis. Patients with impaired copper uptake (Menkes disease) are characterized by hypotonia, seizures, mental retardation, 13 Molybdenum in Human Health and Disease 441 developmental delay and other neurological features. It remains to be elucidated to which extend an underlying defi ciency in Moco synthesis might contribute to the disease pathology.

4.2 Epilepsy and Neuropsychiatric Disorders

Given the dual functions of gephyrin in Moco synthesis and receptor clustering, the spectrum of altered gephyrin function is very complex. While deletion or missense mutations in the GEPH gene result in a very severe MoCD phenotype with loss of synaptic inhibition, there are also subtle cases of altered GEPHYRIN expression resulting in various neuropsychiatric disorders. Mutations in the GEPH gene that do not affect its catalytic function in MoCD result in hyperekplexia, an impairment in inhibitory synaptic transmission [ 176 ]. The GEPH gene has also been associated with cancer development, given that a fusion with the mixed lineage leukemia gene results in leukemia by transforming hematopoietic progenitor cells [177 ]. We have found that stress-induced mis-splicing of GEPH transcript results in the exclusion of exons 4 to 8 producing truncated GEPHYRINS, which are able to curtail the function of wild-type GEPHYRIN by dominant negative interactions [178 ]. As a result, individuals expressing such GEPHYRIN variants develop temporal lobe epilepsy. In another study, rare genomic deletions of GEPH have been found in patients with autism, schizophrenia, and seizures [179 ]. Therefore, GEPHYRIN represents a novel contributor to the development of complex neuropsychiatric disorders. The investigation of GEPHYRIN’s Moco biosynthetic activity in such diseases might help to develop new biomarkers for the diagnosis and progression of neuropsychiatric disorders.

4.3 Ethylmalonic Encephalopathy

Ethylmalonic encephalopathy (EE) is similarly to MoCD an autosomal recessive inborn error of metabolism caused by defects in the ETHE 1 gene, which encodes a β-lactamase-like, iron-coordinating metalloprotein localized within the mitochondrial matrix [180 ]. As observed in MoCD, EE symptoms develop shortly after birth and include typical neurological features such as delayed development, encephalopathy, seizures, as well as microangipathy, hypotonia, and chronic diarrhea. ETHE 1 is a mitochondrial sulfur dioxygenase involved in hydrogen sulfi de metabolism and loss of its function leads to an accumulation of hydrogen sulfi de and inhibition of cytochrome c oxidase and short-chain fatty acid oxidation [180 ]. Besides increased hydrogen sulfi de levels, ETHE -knockout mice and human patients are characterized by a massive excretion of thiosulfate in urine, a biochemical feature, which is shared with MoCD. However, hydrogen sulfi de and thiosulfate increase was not accompanied by sulfi te increase in ETHE 1-knockout mice [62 ]. 442 Schwarz and Belaidi

5 Concluding Remarks and Future Developments

Molybdenum is crucial for the survival of all mammals. A defi ciency in one of the four Moco-dependent enzymes can either be asymptomatic in some cases (XDH defi ciency) or lethal in other cases (SOD). MoCD, however, is in nearly all cases a severe inborn error in metabolism and characterized by a rapidly progressing neurodegeneration. Although we begin to understand the underlying mechanism causing the catastrophic brain damage, future studies are needed to identify key players in metabolism that initiate neuronal cell death. Here, mitochondrial dysfunction is very likely to present a crucial entry point of signals contributing to cell death. Furthermore, the pathogenesis of multi-factorial neurodegenerative disorders such as Huntington’s disease might be dependent on an altered basic metabolism that involves Mo-dependent enzymes. In addition, cysteine homeostasis, which is dependent on a tight control of numerous S-containing intermediates might contribute to the pathogenesis of such disorders too. In this context, longevity has been show to be dependent on dietary restriction in methionine intake [181 ]. This in turn suggests that high sulfur turn over, probably via the oxidative catabolism of cysteine, is a negative predictor of life span, which is supported by the fi nding that longevity is increased when the trans-sulfuration pathway is increased. In conclusion, SO-dependent sulfi te detoxifi cation might be a key regulator of cell survival in health and disease. Besides purine catabolism, XO has been implicated in the generation of reactive oxygen species. Studies using animal models of mild hyperuricemia provided evidence for a pathogenic role of uric acid in the development of hypertension, vascular disease, and renal disease [ 182 ]. The physiological role of other Mo-enzymes such as AOX and mARC are still poorly understood and novel animal models are required to identify their primary function in metabolism [ 183]. SO is the most important Mo-enzyme in humans. Recently, we found nitrite reductase activity of SO under hypoxic conditions. In the future it will be important to learn more about SO’s role in nitrite-dependent NO signaling using novel animal-based approaches. Finally, Moco biosynthesis provided the evolutionary origin for numerous eukaryote- specifi c functions and pathways, such as radical SAM-dependent reactions, the ubiquitin-proteasome pathway including other thiolation reactions, and synaptic clustering of receptors. While the biochemistry of Moco biogenesis is well understood, the underlying cell biology, the organization and regulation of intermediate fl uxes and the assembly and turn over of different Mo-enzymes are still poorly understood. Not addressed at all is the catabolism of Moco, resulting in the excretion of a thiolated oxidized pterin, called urothione, whose identity has been uncovered more than 40 years ago [184 ]. In future studies, the catabolic machinery, including enzymes catalyzing the methylation and dephosphorylation of the cofactor need to be identifi ed. 13 Molybdenum in Human Health and Disease 443

Abbreviations and Deﬁ nitions

Proteins and genes written in capital letters refer to humans. AAT aspartate aminotransferase ABA3 plant orthologue of human Moco sulfurase ABC ATP-binding cassette AMP adenosine 5′-monophosphate AOX aldehyde oxidase ATP adenosine 5′-triphosphate BHMT betaine-homocysteine methyl transferase CBS cystathionine β-synthase CDO cysteine dioxygenase cPMP cyclic pyranopterin monophosphate CSA cysteine sulfi nic acid CSD cysteinesulfi nate decarboxylase CSE cystathionine γ-lyase (cystathionase) EE ethylmalonic encephalopathy F A D fl avin adenine dinucleotide GABA γ-amino butyric acid GCS γ-glutamylcysteine synthetase Glu glutamic acid Gly glycine GS glutathione synthetase GSH glutathione GTP guanosine 5′-triphosphate HPLC high performance liquid chromatography KG α-ketoglutarate mARC mitochondrial amidoxime reducing component MAT methionine-S-adenosyl transferase MCSU Moco sulfurase MFS major facilitator superfamily MoCD molybdenum cofactor defi ciency Moco molybdenum cofactor MOCS molybdenum cofactor synthesis MOSC Moco sulfurase C-terminal domain MOT1 molybdate transporter type 1 MPST 3-mercaptopyruvate sulfurtransferase MPT metal-binding pterin (or molybdopterin) MRI magnetic resonance imaging MS methionine synthase NAD+ nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide (reduced) NO nitric oxide NR nitrate reductase 444 Schwarz and Belaidi

PPi inorganic diphosphate (= pyrophosphate) SAHH S-adenosylhomocysteine hydrolase SAM S-adenosyl methionine SDO sulfur dioxygenase Ser serine SO sulfi te oxidase SOD sulfi te oxidase defi ciency SQR quinone oxidoreductase SSC S-sulfocysteine ST sulfur transferase XDH xanthine dehydrogenase XO xanthine oxidase XOR xanthine oxidoreductase

Acknowledgments The great work of all current and past postdocs, graduate, master, and bachelor students as well as technicians is gratefully acknowledged. This work would not have been possible without many collaborators that helped to build bridges of true interdisciplinarity. Research funding by the German Science Foundation (DGF), the Federal Ministry of Education and Research (BMBF), the FP7 EU funding (Marie Curie Program), the Fonds der Chemischen Industrie (FCI), and the Center for Molecular Medicine Cologne (CMMC) is gratefully acknowledged.

References

1. J. L. Burguera, M. Burguera, J. Trace Elem. Med. Biol. 2007 , 21 , 178–183. 2. J. Versieck, J. Hoste, F. Barbier, L. Vanballenberghe, J. Derudder, R. Cornelis, Clin. Chim. Acta 1978 , 87 , 135–140. 3. J. R. Turnlund, W. R. Keyes, J. Nutr. Biochem. 2004 , 15 , 90–95. 4. W. L. Lindsay, Chemical Equilibria in Solis , John Wiley & Sons, New York, 1979. 5. G. Schwarz, R. R. Mendel, M. W. Ribbe, Nature 2009 , 460 , 839–847. 6. A. M. Grunden, K. T. Shanmugam, Arch. Microbiol. 1997 , 168 , 345–354. 7. M. Tejada-Jimenez, A. Llamas, E. Sanz-Luque, A. Galvan, E. Fernandez, Proc. Natl. Acad. Sci. USA 2007 , 104 , 20126–20130. 8. H. Tomatsu, J. Takano, H. Takahashi, A. Watanabe-Takahashi, N. Shibagaki, T. Fujiwara, Proc. Natl. Acad. Sci. USA 2007 , 104 , 18807–18812. 9. M. J. Hawkesford, Physiol. Plant. 2003 , 117 , 155–163. 10. M. Tejada-Jimenez, A. Galvan, E. Fernandez, Proc. Natl. Acad. Sci. USA 2011 , 108 , 6420–6425. 11. G. N. George, I. J. Pickering, H. H. Harris, J. Gailer, D. Klein, J. Lichtmannegger, K. H. Summer, J. Am. Chem. Soc. 2003 , 125 , 1704–1705. 12. G. M. Ward, J. Anim. Sci. 1978 , 46 , 1078–1085. 13. G. J. Brewer, Expert Opin. Investig. Drugs 2009 , 18 , 89–97. 14. A. Vyskocil, C. Viau, J. Appl. Toxicol. 1999 , 19 , 185–192. 15. T. V. Fungwe, F. Buddingh, D. S. Demick, C. D. Lox, M. T. Yang, S. P. Yang, Nutr. Res. 1990 , 10 , 515–524. 16. R. Hille, Chem. Rev. 1996 , 96 , 2757–2816. 17. Y. Amaya, K. Yamazaki, M. Sato, K. Noda, T. Nishino, J. Biol. Chem. 1990 , 265 , 14170–14175. 18. T. Nishino, J. Biol. Chem. 1997 , 272 , 29859–29864. 13 Molybdenum in Human Health and Disease 445

19. M. Saksela, R. Lapatto, K. O. Raivio, FEBS Lett. 1999 , 443 , 117–120. 20. C. Enroth, B. T. Eger, K. Okamato, T. Nishino, T. Nishino, E. F. Pai, Proc. Natl. Acad. Sci. USA 2000 , 97 , 10723–10728. 21. R. Hille, T. Nishino, F. Bittner, Coord. Chem. Rev. 2011 , 255 , 1179–1205. 22. C. E. Dent, G. R. Philpot, Lancet 1954 , 1 , 182–185. 23. K. Ichida, Y. Amaya, N. Kamatani, T. Nishino, T. Hosoya, O. Sakai, J. Clin. Invest. 1997 , 99 , 2391–2397. 24. R. A. Harkness, G. M. McCreanor, D. Simpson, I. R. Macfadyen, J. Inher. Metab. Dis. 1986 , 9 , 407–408. 25. H. A. Simmonds, S. Reiter, T. Nishino, in The Metabolic and Molecular Bases of Inherited Disease , Eds C. R. Scriver, A. L. Beaudet, W. S. Sly, D. Valle, McGraw-Hill, New York, 1995, pp. 1781–1797. 26. F. A. Mateos, J. G. Puig, M. L. Jimenez, I. H. Fox, J. Clin. Invest. 1987 , 79 , 847–852. 27. M. Kudo, T. Sasaki, M. Ishikawa, N. Hirasawa, M. Hiratsuka, Drug Metab. Pharmacokin. 2010 , 25 , 361–366. 28. T. Watanabe, N. Ihara, T. Itoh, T. Fujita, Y. Sugimoto, J. Biol. Chem. 2000 , 275 , 21789–21792. 29. H. Peretz, M. S. Naamati, D. Levartovsky, A. Lagziel, E. Shani, I. Horn, H. Shalev, D. Landau, Mol. Genet. Metab. 2007 , 91 , 23–29. 30. R. Zannolli, V. Micheli, M. A. Mazzei, P. Sacco, P. Piomboni, E. Bruni, C. Miracco, M. M. de Santi, P. Terrosi Vagnoli, L. Volterrani, L. Pellegrini, W. Livi, B. Lucani, S. Gonnelli, A. B. Burlina, G. Jacomelli, F. Macucci, L. Pucci, M. Fimiani, J. A. Swift, M. Zappella, G. Morgese, J. Med. Gen. 2003 , 40 , e121. 31. T. Page, M. Coleman, Biochim. Biophys. Acta-Molecular Basis of Disease 2000 , 1500 , 291–296. 32. K. D. Torralba, E. De Jesus, S. Rachabattula, Int. J. Rheum. Dis. 2012 , 15 , 499–506. 33. P. Pacher, A. Nivorozhkin, C. Szabo, Pharmacol. Rev. 2006 , 58 , 87–114. 34. P. Pacher, A. Nivorozhkin, C. Szabó, Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol Rev. 2006, 58(1), 87–114. 35. H. T. Li, H. M. Cui, T. K. Kundu, W. Alzawahra, J. L. Zweier, J. Biol. Chem. 2008 , 283 , 17855–17863. 36. A. Dejam, C. J. Hunter, C. Tremonti, R. M. Pluta, Y. Y. Hon, G. Grimes, K. Partovi, M. M. Pelletier, E. H. Oldﬁ eld, R. O. Cannon, III, A. N. Schechter, M. T. Gladwin, Circulation 2007 , 116 , 1821–1831. 37. F. Rodriguez-Trelles, R. Tarrio, F. J. Ayala, Proc. Natl. Acad. Sci. USA 2003 , 100 , 13413–13417. 38. E. Garattini, M. Fratelli, M. Terao, Hum. Genomics 2009 , 4 , 119–130. 39. C. Coelho, M. Mahro, J. Trincao, A. T. Carvalho, M. J. Ramos, M. Terao, E. Garattini, S. Leimkuhler, M. J. Romao, J. Biol. Chem. 2012 , 287 , 40690–40702. 40. E. Garattini, R. Mendel, M. J. Romao, R. Wright, M. Terao, Biochem. J. 2003 , 372 , 15–32. 41. E. Garattini, M. Terao, Expert Opin. Drug Discov. 2013 , 8 , 641–654. 42. M. Terao, M. Kurosaki, M. M. Barzago, M. Fratelli, R. Bagnati, A. Bastone, C. Giudice, E. Scanziani, A. Mancuso, C. Tiveron, E. Garattini, Mol. Cell Biol. 2009 , 29 , 357–377. 43. S. Kitamura, K. Tatsumi, Biochem. Biophys. Res. Commun. 1984 , 120 , 602–606. 44. S. Kitamura, K. Tatsumi, Biochem. Biophys. Res. Commun. 1984 , 121 , 749–754. 45. R. A. Dick, D. B. Kanne, J. E. Casida, Chem. Res. Toxicol. 2006 , 19 , 38–43. 46. C. G. Jordan, M. R. Rashidi, H. Laljee, S. E. Clarke, J. E. Brown, C. Beedham, J. Pharm. Pharmacol. 1999 , 51 , 411–418. 47. M. R. Rashidi, C. Beedham, J. S. Smith, S. Davaran, Drug Metab. Pharmacokinet. 2007 , 22 , 299–306. 48. B. Rochat, M. Kosel, G. Boss, B. Testa, M. Gillet, P. Baumann, Biochem. Pharmacol. 1998 , 56 , 15–23. 49. T. K. Chung, M. A. Funk, D. H. Baker, J. Nutr. 1990 , 120 , 158–165. 446 Schwarz and Belaidi

50. G. Wu, Y. Z. Fang, S. Yang, J. R. Lupton, N. D. Turner, J. Nutr. 2004 , 134 , 489–492. 51. S. C. Lu, Curr. Top. Cell Regul. 2000 , 36 , 95–116. 52. M. H. Stipanuk, Annu. Rev. Nutr. 1986 , 6 , 179–209. 53. I. Ueki, M. H. Stipanuk, J. Nutr. 2007 , 137 , 1887–1894. 54. M. H. Stipanuk, K. M. King, Comp. Biochem. Physiol. B 1982 , 73 , 595–601. 55. P. Kamoun, Amino Acids 2004 , 26 , 243–254. 56. B. Nam, H. Kim, Y. Choi, H. Lee, E. S. Hong, J. K. Park, K. M. Lee, Y. Kim, Ind. Health 2004 , 42 , 83–87. 57. L. R. Goodwin, D. Francom, F. P. Dieken, J. D. Taylor, M. W. Warenycia, R. J. Reiffenstein, G. Dowling, J. Anal. Toxicol. 1989 , 13 , 105–109. 58. M. W. Warenycia, L. R. Goodwin, C. G. Benishin, R. J. Reiffenstein, D. M. Francom, J. D. Taylor, F. P. Dieken, Biochem. Pharmacol. 1989 , 38 , 973–981. 59. J. C. Savage, D. H. Gould, J. Chromatogr. 1990 , 526 , 540–545. 60. D. E. Baranano, C. D. Ferris, S. H. Snyder, Trends Neurosci. 2001 , 24 , 99–106. 61. T. M. Hildebrandt, M. K. Grieshaber, FEBS J. 2008 , 275 , 3352–3361. 62. O. Kabil, R. Banerjee, J. Biol. Chem. 2010 , 285 , 21903–21907. 63. P. J. Bagley, M. H. Stipanuk, J. Nutr. 1994 , 124 , 2410–2421. 64. M. H. Stipanuk, I. Ueki, J. Inherit. Metab. Dis. 2011 , 34 , 17–32. 65. I. Ueki, H. B. Roman, A. Valli, K. Fieselmann, J. Lam, R. Peters, L. L. Hirschberger, M. H. Stipanuk, Am. J. Physiol. Endocrinol. Metab. 2011 , 301, E668–E684. 66. C. Feng, G. Tollin, J. H. Enemark, Biochim. Biophys. Acta 2007 , 1774 , 527–539. 67. C. Kisker, H. Schindelin, A. Pacheco, W. A. Wehbi, R. M. Garrett, K. V. Rajagopalan, J. H. Enemark, D. C. Rees, Cell 1997 , 91 , 973–983. 68. H. J. Cohen, S. Betcher-Lange, D. L. Kessler, K. V. Rajagopalan, J. Biol. Chem. 1972 , 247 , 7759–7766. 69. J. M. Klein, G. Schwarz, J. Cell Sci. 2012 , 125 , 4876–4885. 70. A. Pacheco, J. T. Hazzard, G. Tollin, J. H. Enemark, J. Biol. Inorg. Chem. 1999 , 4 , 390–401. 71. C. Feng, R. V. Kedia, J. T. Hazzard, J. K. Hurley, G. Tollin, J. H. Enemark, Biochemistry 2002 , 41 , 5816–5821. 72. K. Johnson-Winters, G. Tollin, J. H. Enemark, Biochemistry 2010 , 49 , 7242–7254. 73. K. Fischer, G. G. Barbier, H.-J. Hecht, R. R. Mendel, W. H. Campbell, G. Schwarz, Plant Cell 2005 , 17 , 1167–1179. 74. J. A. Qiu, H. L. Wilson, K. V. Rajagopalan, Biochemistry 2012 , 51 , 1134–1147. 75. J. Wang, K. Fischer, X. J. Zhao, J. Tejero, E. E. Kelley, L. Wang, S. Shiva, S. Frizzell, Y. Z. Zhang, P. Basu, G. Schwarz, M. T. Gladwin, Free Rad. Biol. Med. 2010 , 49 , S122–S122. 76. A. Dejam, C. J. Hunter, A. N. Schechter, M. T. Gladwin, Blood Cells Molecules and Diseases 2004 , 32 , 423–429. 77. J. L. Johnson, M. Duran, in The Metabolic and Molecular Bases of Inherited Disease , Eds C. Scriver, A. Beaudet, W. Sly, D. Valle, McGraw-Hill, New York, 2001, pp. 3163–3177. 78. W. H. Tan, F. S. Eichler, S. Hoda, M. S. Lee, H. Baris, C. A. Hanley, P. E. Grant, K. S. Krishnamoorthy, V. E. Shih, Pediatrics 2005 , 116 , 757–766. 79. C. Feng, H. L. Wilson, J. K. Hurley, J. T. Hazzard, G. Tollin, K. V. Rajagopalan, J. H. Enemark, Biochemistry 2003 , 42 , 12235–12242. 80. H. L. Wilson, K. V. Rajagopalan, J. Biol. Chem. 2004 , 279 , 15105–15113. 81. A. Havemeyer, F. Bittner, S. Wollers, R. Mendel, T. Kunze, B. Clement, J. Biol. Chem. 2006 , 281 , 34796–34802. 82. J. M. Klein, J. D. Busch, C. Potting, M. J. Baker, T. Langer, G. Schwarz, J. Biol. Chem. 2012 , 287 , 42795–42803. 83. J. Kotthaus, B. Wahl, A. Havemeyer, D. Schade, D. Garbe-Schonberg, R. Mendel, F. Bittner, B. Clement, Biochem. J. 2011 , 433 , 383–391. 84. B. Plitzko, G. Ott, D. Reichmann, C. J. Henderson, C. R. Wolf, R. Mendel, F. Bittner, B. Clement, A. Havemeyer, J. Biol. Chem. 2013 , 288, 20228–20237. 13 Molybdenum in Human Health and Disease 447

85. B. Wahl, D. Reichmann, D. Niks, N. Krompholz, A. Havemeyer, B. Clement, T. Messerschmidt, M. Rothkegel, H. Biester, R. Hille, R. R. Mendel, F. Bittner, J. Biol. Chem. 2010 , 285 , 37847–37859. 86. A. Chamizo-Ampudia, A. Galvan, E. Fernandez, A. Llamas, Eukaryotic Cell 2011 , 10 , 1270–1282. 87. A. Havemeyer, J. Lang, B. Clement, Drug Metab. Rev. 2011 , 43 , 524–539. 88. G. Schwarz, Cell. Mol. Life Sci. 2005 , 62 , 2792–2810. 89. R. R. Mendel, G. Schwarz, Met. Ions Biol. Syst. 2002 , 39 , 317–368. 90. J. A. Santamaria-Araujo, B. Fischer, T. Otte, M. Nimtz, R. R. Mendel, V. Wray, G. Schwarz, J. Biol. Chem. 2004 , 279 , 15994–15999. 91. M. M. Wuebbens, K. V. Rajagopalan, J. Biol. Chem. 1993 , 268 , 13493–13498. 92. J. L. Johnson, B. E. Hainline, K. V. Rajagopalan, B. H. Arison, J. Biol. Chem. 1984 , 259 , 5414–5422. 93. A. P. Mehta, J. W. Hanes, S. H. Abdelwahed, D. G. Hilmey, P. Hanzelmann, T. P. Begley, Biochemistry 2013 , 52, 1134–1136. 94. M. M. Wuebbens, K. V. Rajagopalan, J. Biol. Chem. 2003 , 278 , 14523–14532. 95. J. Kuper, A. Llamas, H. J. Hecht, R. R. Mendel, G. Schwarz, Nature 2004 , 430 , 806–806. 96. P. Hanzelmann, H. Schindelin, Proc. Natl. Acad. Sci. USA 2006 , 103 , 6829–6834. 97. M. M. Wuebbens, K. V. Rajagopalan, J. Biol. Chem. 1995 , 270 , 1082–1087. 98. C. Rieder, W. Eisenreich, J. O’Brien, G. Richter, E. Götze, P. Boyle, S. Blanchard, A. Bacher, H. Simon, Eur. J. Biochem. 1998 , 255 , 24–36. 99. J. A. Santamaria-Araujo, V. Wray, G. Schwarz, J. Biol. Inorg. Chem. 2012 , 17 , 113–122. 100. H. J. Soﬁ a, G. Chen, B. G. Hetzler, J. F. Reyes-Spindola, N. E. Miller, Nucleic Acids Res. 2001 , 29 , 1097–1106. 101. P. Hänzelmann, H. Schindelin, Proc. Natl. Acad. Sci. USA 2004 , 101 , 12870–12875. 102. T. Hoff, K. M. Schnorr, C. Meyer, M. Caboche, J. Biol. Chem. 1995 , 270 , 6100–6107. 103. P. Hänzelmann, G. Schwarz, R. R. Mendel, J. Biol. Chem. 2002 , 277 , 18303–18312. 104. J. Teschner, N. Lachmann, J. Schulze, M. Geisler, K. Selbach, J. Santamaria-Araujo, J. Balk, R. R. Mendel, F. Bittner, Plant Cell 2010 , 22 , 468–480. 105. R. Lill, Nature 2009 , 460 , 831–838. 106. G. Gutzke, B. Fischer, R. R. Mendel, G. Schwarz, J. Biol. Chem. 2001 , 276 , 36268–36274. 107. A. Matthies, K. V. Rajagopalan, R. R. Mendel, S. Leimkuhler, Proc. Natl. Acad. Sci. USA 2004 , 101 , 5946–5951. 108. A. Matthies, M. Nimtz, S. Leimkuhler, Biochemistry 2005 , 44 , 7912–7920. 109. L. E. Bevers, P. L. Hagedoorn, J. A. Santamaria-Araujo, A. Magalon, W. R. Hagen, G. Schwarz, Biochemistry 2008 , 47 , 949–956. 110. J. Nichols, K. V. Rajagopalan, J. Biol. Chem. 2002 , 277 , 24995–25000. 111. B. Stallmeyer, G. Schwarz, J. Schulze, A. Nerlich, J. Reiss, J. Kirsch, R. R. Mendel, Proc. Natl. Acad. Sci. USA 1999 , 96 , 1333–1338. 112. A. Llamas, R. R. Mendel, G. Schwarz, J. Biol. Chem. 2004 , 279 , 55241–55246. 113. A. Llamas, T. Otte, G. Multhaup, R. R. Mendel, G. Schwarz, J. Biol. Chem. 2006 , 281 , 18343–18350. 114. A. A. Belaidi, G. Schwarz, Biochem. J. 2013 , 450 , 149–157. 115. J. M. Fritschy, R. J. Harvey, G. Schwarz, Trends Neurosci. 2008 , 31 , 257–264. 116. K. Ichida, T. Matsumura, R. Sakuma, T. Hosoya, T. Nishino, Biochem. Biophys. Res. Commun. 2001 , 282 , 1194–1200. 117. F. Bittner, M. Oreb, R. R. Mendel, J. Biol. Chem. 2001 , 276 , 40381–40384. 118. S. Wollers, T. Heidenreich, M. Zarepour, D. Zachmann, C. Kraft, Y. Zhao, R. R. Mendel, F. Bittner, J. Biol. Chem. 2008 , 283 , 9642–9650. 119. R. R. Mendel, G. Schwarz, Coord. Chem. Rev. 2011 , 255 , 1145–1158. 120. M. Lehrke, S. Rump, T. Heidenreich, J. Wissing, R. R. Mendel, F. Bittner, Biochem. J. 2012 , 441 , 823–832. 121. J. Reiss, N. Cohen, C. Dorche, H. Mandel, R. R. Mendel, B. Stallmeyer, M. T. Zabot, T. Dierks, Nat. Genet. 1998 , 20 , 51–53. 448 Schwarz and Belaidi

122. P. Prior, B. Schmitt, G. Grenningloh, I. Pribilla, G. Multhaup, K. Beyreuther, Y. Maulet, P. Werner, D. Langosch, J. Kirsch, H. Betz, Neuron 1992 , 8 , 1161–1170. 123. B. Stallmeyer, G. Drugeon, J. Reiss, A. L. Haenni, R. R. Mendel, Am. J. Hum. Genet. 1999 , 64 , 698–705. 124. T. A. Gray, R. D. Nicholls, RNA 2000 , 6 , 928–936. 125. S. Gross-Hardt, J. Reiss, Mol. Gen. Metab. 2002 , 76 , 340–343. 126. M. Arenas, L. D. Fairbanks, K. Vijayakumar, L. Carr, E. Escuredo, A. M. Marinaki, J. Inherit. Metab. Dis. 2009 , 32 , 560–569. 127. M. Ramming, S. Kins, N. Werner, A. Hermann, H. Betz, J. Kirsch, Proc. Natl. Acad. Sci. USA 2000 , 97 , 10266–10271. 128. M. I. Rees, K. Harvey, H. Ward, J. H. White, L. I. Evans, I. C. Duguid, C. C. Hsu, S. L. Coleman, J. Miller, K. Baer, H. J. Waldvogel, F. Gibbon, T. G. Smart, M. J. Owen, R. J. Harvey, R. G. Snell, J. Biol. Chem. 2003 , 278 , 24688–24696. 129. B. Smolinsky, S. A. Eichler, S. Buchmeier, J. C. Meier, G. Schwarz, J. Biol. Chem. 2008 , 283 , 17370–17379. 130. J. Herweg, G. Schwarz, J. Biol. Chem. 2012 , 287, 12645–12656. 131. J. Reiss, E. Christensen, G. Kurlemann, M.-T. Zabot, C. Dorche, Hum. Genet. 1998 , 103 , 639–644. 132. J. Reiss, C. Dorche, B. Stallmeyer, R. R. Mendel, N. Cohen, M. T. Zabot, Am. J. Hum. Genet. 1999 , 64 , 706–711. 133. J. Reiss, J. L. Johnson, Hum. Mutat. 2003 , 21 , 569–576. 134. J. Reiss, R. Hahnewald, Hum. Mutat. 2011 , 32 , 10–18. 135. M. M. Chowdhury, C. Dosche, H. G. Lohmannsroben, S. Leimkuhler, J. Biol. Chem. 2012 , 287 , 17297–17307. 136. J. Reiss, S. Gross-Hardt, E. Christensen, P. Schmidt, R. R. Mendel, G. Schwarz, Am. J. Hum. Genet. 2001 , 68 , 208–213. 137. C. Mulhardt, M. Fischer, P. Gass, D. Simon-Chazottes, J. L. Guenet, J. Kuhse, H. Betz, C. M. Becker, Neuron 1994 , 13 , 1003–1015. 138. J. Reiss, U. Lenz, C. Aquaviva-Bourdain, S. Joriot-Chekaf, K. Mention-Mulliez, M. Holder- Espinasse, Clin. Genet. 2011 , 80 , 598–599. 139. N. Schrader, E. Y. Kim, J. Winking, J. Paulukat, H. Schindelin, G. Schwarz, J. Biol. Chem. 2004 , 279 , 18733–18741. 140. H. M. Maric, J. Mukherjee, V. Tretter, S. J. Moss, H. Schindelin, J. Biol. Chem. 2011 , 286 , 42105–42114. 141. J. L. Johnson, K. E. Coyne, K. V. Rajagopalan, J. L. Van Hove, M. Mackay, J. Pitt, A. Boneh, Am. J. Med. Genet. 2001 , 104 , 169–173. 142. C. Barbot, E. Martins, L. Vilarinho, C. Dorche, M. L. Cardoso, Neuropediatrics 1995 , 26 , 322–324. 143. G. Touati, E. Rusthoven, E. Depondt, C. Dorche, M. Duran, B. Heron, D. Rabier, M. Russo, J. M. Saudubray, J. Inherit. Metab. Dis. 2000 , 23 , 45–53. 144. M. Del Rizzo, A. P. Burlina, J. O. Sass, F. Beermann, C. Zanco, C. Cazzorla, A. Bordugo, L. Giordano, R. Manara, A. B. Burlina, Mol. Genet. Metab. 2013 , 108 , 263–266. 145. M. Duran, F. A. Beemer, C. van de Heiden, J. Korteland, P. K. de Bree, M. Brink, S. K. Wadman, I. Lombeck, J. Inherit. Metab. Dis. 1978 , 1 , 175–178. 146. K. Vijayakumar, R. Gunny, S. Grunewald, L. Carr, K. W. Chong, C. DeVile, R. Robinson, N. McSweeney, P. Prabhakar, Pediatr. Neurol. 2011 , 45 , 246–252. 147. E. Bayram, Y. Topcu, P. Karakaya, U. Yis, H. Cakmakci, K. Ichida, S. H. Kurul, Eur. J. Paediatr. Neurol. 2013 , 17 , 1–6. 148. S. Arslanoglu, M. Yalaz, D. Goksen, M. Coker, S. Tutuncuoglu, M. Akisu, S. Darcan, N. Kultursay, M. Ciris, E. Demirtas, Brain Dev. 2001 , 23 , 815–818. 149. X. Zhang, A. S. Vincent, B. Halliwell, K. P. Wong, J. Biol. Chem. 2004 , 279 , 43035–43045. 150. A. Veldman, J. A. Santamaria-Araujo, S. Sollazzo, J. Pitt, R. Gianello, J. Yaplito-Lee, F. Wong, C. A. Ramsden, J. Reiss, I. Cook, J. Fairweather, G. Schwarz, Pediatrics 2010 , 125 , e1249–1254. 13 Molybdenum in Human Health and Disease 449

151. A. A. Belaidi, S. Arjune, J. A. Santamaria-Araujo, J. O. Sass, G. Schwarz, J. Inherit. Metab. Dis. Rep. 2012 , 5 , 35–43. 152. J. W. Olney, C. H. Misra, T. de Gubareff, J. Neuropathol. Exp. Neurol. 1975 , 34 , 167–177. 153. A. A. Belaidi, G. Schwarz, Adv. Exp. Med. Biol. 2013 , 776 , 13–19. 154. A. El Idrissi, E. Trenkner, J. Neurosci. 1999 , 19 , 9459–9468. 155. O. W. Grifﬁ th, Free Radic. Biol. Med. 1999 , 27 , 922–935. 156. D. P. Jones, Methods Enzymol. 2002 , 348 , 93–112. 157. D. M. Townsend, K. D. Tew, H. Tapiero, Biomed. Pharmacother. 2003 , 57 , 145–155. 158. F. Jahoor, A. Jackson, B. Gazzard, G. Philips, D. Sharpstone, M. E. Frazer, W. Heird, Am. J. Physiol. 1999 , 276 , E205–211. 159. J. Lyons, A. Rauh-Pfeiffer, Y. M. Yu, X. M. Lu, D. Zurakowski, R. G. Tompkins, A. M. Ajami, V. R. Young, L. Castillo, Proc. Natl. Acad. Sci. USA 2000 , 97 , 5071–5076. 160. H.-J. Lee, I. M. Adham, G. Schwarz, M. Kneussel, J.-O. Sass, W. Engel, J. Reiss, Hum. Mol. Gen. 2002 , 11 , 3309–3317. 161. G. Feng, H. Tintrup, J. Kirsch, M. C. Nichol, J. Kuhse, H. Betz, J. R. Sanes, Science 1998 , 282 , 1321–1324. 162. E. S. Simon, Movement Disorders 1997 , 12 , 221–228. 163. G. Schwarz, J. A. Santamaria-Araujo, S. Wolf, H. J. Lee, I. M. Adham, H. J. Grone, H. Schwegler, J. O. Sass, T. Otte, P. Hanzelmann, R. R. Mendel, W. Engel, J. Reiss, Hum. Mol. Gen. 2004 , 13 , 1249–1255. 164. S. Kugler, R. Hahnewald, M. Garrido, J. Reiss, Am. J. Hum. Genet . 2007 , 80 , 291–297. 165. M. M. Hitzert, A. F. Bos, K. A. Bergman, A. Veldman, G. Schwarz, J. A. Santamaria-Araujo, R. Heiner-Fokkema, D. A. Sival, R. J. Lunsing, S. Arjune, J. G. Kosterink, F. J. van Spronsen, Pediatrics 2012 , 130 , e1005–1010. 166. K. Clinch, D. K. Watt, R. A. Dixon, S. M. Baars, G. J. Gainsford, A. Tiwari, G. Schwarz, Y. Saotome, M. Storek, A. A. Belaidi, J. A. Santamaria-Araujo, J. Med. Chem. 2013 , 56, 1730–1738. 167. N. Blau, B. Thöny, R. G. H. Cotton, K. Hyland, in The Metabolic and Molecular Bases of Inherited Disease , Eds C. Scriver, A. L. Beaudet, W. S. Sly, D. Valle, McGraw-Hill, New York, 2001, pp. 1725–1776. 168. F. Falciani, M. Terao, S. Goldwurm, A. Ronchi, A. Gatti, C. Minoia, M. Li Calzi, M. Salmona, G. Cazzaniga, E. Garattini, Biochem. J. 1994 , 298 , 69–77. 169. R. G. Boles, L. R. Ment, M. S. Meyn, A. L. Horwich, L. E. Kratz, P. Rinaldo, Ann. Neurol. 1993 , 34 , 742–744. 170. J. O. Sass, A. Gunduz, C. Araujo Rodrigues Funayama, B. Korkmaz, K. G. Dantas Pinto, B. Tuysuz, L. Yanasse Dos Santos, E. Taskiran, M. de Fatima Turcato, C. W. Lam, J. Reiss, M. Walter, C. Yalcinkaya, J. S. Camelo Junior, Brain Dev. 2010 , 32 , 544–549 171. M. H. Stipanuk, I. Ueki, J. E. Dominy, Jr., C. R. Simmons, L. L. Hirschberger, Amino Acids 2009 , 37 , 55–63. 172. J. Mason, Toxicology 1986 , 42 , 99–109. 173. J. F. Mercer, Trends Mol. Med. 2001 , 7 , 64–69. 174. G. J. Brewer, Drug Discov. Today 2005 , 10 , 1103–1109. 175. H. M. Alvarez, Y. Xue, C. D. Robinson, M. A. Canalizo-Hernandez, R. G. Marvin, R. A. Kelly, A. Mondragon, J. E. Penner-Hahn, T. V. O’Halloran, Science 2010 , 327 , 331–334. 176. R. J. Harvey, M. Topf, K. Harvey, M. I. Rees, Trends in Genetics 2008 , 24 , 439–447. 177. M. Eguchi, M. Eguchi-Ishimae, M. Greaves, Blood 2004 , 103 , 3876–3882. 178. B. Forstera, A. A. Belaidi, R. Juttner, C. Bernert, M. Tsokos, T. N. Lehmann, P. Horn, C. Dehnicke, G. Schwarz, J. C. Meier, Brain 2010 , 133 , 3778–3794. 179. A. C. Lionel, A. K. Vaags, D. Sato, M. J. Gazzellone, E. B. Mitchell, H. Y. Chen, G. Costain, S. Walker, G. Egger, B. Thiruvahindrapuram, D. Merico, A. Prasad, E. Anagnostou, E. Fombonne, L. Zwaigenbaum, W. Roberts, P. Szatmari, B. A. Fernandez, L. Georgieva, L. M. Brzustowicz, K. Roetzer, W. Kaschnitz, J. B. Vincent, C. Windpassinger, C. R. Marshall, 450 Schwarz and Belaidi

R. R. Triﬁ letti, S. Kirmani, G. Kirov, E. Petek, J. C. Hodge, A. S. Bassett, S. W. Scherer, Hum. Mol. Genet. 2013 , 22 , 2055–2066. 180. V. Tiranti, C. Viscomi, T. Hildebrandt, I. Di Meo, R. Mineri, C. Tiveron, M. D. Levitt, A. Prelle, G. Fagiolari, M. Rimoldi, M. Zeviani, Nat. Med. 2009 , 15 , 200–205. 181. R. C. Grandison, M. D. W. Piper, L. Partridge, Nature 2009 , 462 , 1061–1121. 182. R. J. Johnson, D. H. Kang, D. Feig, S. Kivlighn, J. Kanellis, S. Watanabe, K. R. Tuttle, B. Rodriguez-Iturbe, J. Herrera-Acosta, M. Mazzali, Hypertension 2003 , 41 , 1183–1190. 183. H. Kabil, O. Kabil, R. Banerjee, L. G. Harshman, S. D. Pletcher, Proc. Natl. Acad. Sci. USA 2011 , 108 , 16831–16836. 184. M. Goto, A. Sakurai, K. Ota, H. Yamakami, J. Biochem. 1969 , 65 , 611–620. Chapter 14 Silicon: The Health Beneﬁ ts of a Metalloid

Keith R. Martin

Contents ABSTRACT ...... 452 1 INTRODUCTION ...... 452 2 SILICON BIOCHEMISTRY ...... 453 2.1 Silicon Distribution and Prevalence in Nature ...... 453 2.1.1 Dietary Sources ...... 454 2.1.2 Non-dietary Sources ...... 456 2.2 Silicon Chemical Speciation as Silicates ...... 456 2.3 Silicon Chemistry and Effects on Bioavailability ...... 456 3 SILICON AND ITS POTENTIAL HEALTH BENEFITS ...... 457 3.1 Bone Health and Skeletal Development ...... 458 3.2 Vascular Disease and Atherosclerosis ...... 459 3.3 Neurodegenerative Disease (Alzheimer’s Disease) ...... 460 3.4 Diabetes ...... 462 3.5 Wound Healing ...... 462 4 TOXICOLOGY OF SILICON AND SILICA ...... 463 4.1 Chemical Forms Contributing to Toxicity...... 463 4.2 Routes of Exposure and Safety ...... 464 4.2.1 Inhalation and Asbestosis ...... 464 4.2.2 Inhalation and Silicosis ...... 464 4.3 Mechanisms of Toxicity ...... 465 4.3.1 Oxidative Stress ...... 465 4.3.2 Inﬂ ammation ...... 466 5 POTENTIAL MEDICINAL USES OF SILICON AND SILICATES...... 467 6 SUMMARY AND FUTURE DIRECTIONS ...... 468 ABBREVIATIONS ...... 469 REFERENCES ...... 469

K. R. Martin (*) School of Nutrition and Health Promotion, Healthy Lifestyles Research Center , Arizona State University , 500 North 3rd Street , Phoenix , AZ 85004 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 451 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_14, © Springer Science+Business Media Dordrecht 2013 452 Martin

Abstract Silicon is the second most abundant element in nature behind oxygen. As a metalloid, silicon has been used in many industrial applications including use as an additive in the food and beverage industry. As a result, humans come into contact with silicon through both environmental exposures but also as a dietary component. Moreover, many forms of silicon, that is, Si bound to oxygen, are water- soluble, absorbable, and potentially bioavailable to humans presumably with biological activity. However, the specifi c biochemical or physiological functions of silicon, if any, are largely unknown although generally thought to exist. As a result, there is growing interest in the potential therapeutic effects of water-soluble silica on human health. For example, silicon has been suggested to exhibit roles in the structural integrity of nails, hair, and skin, overall collagen synthesis, bone mineralization, and bone health and reduced metal accumulation in Alzheimer’s disease, immune system health, and reduction of the risk for atherosclerosis. Although emerging research is promising, much additional, corroborative research is needed particularly regarding speciation of health-promoting forms of silicon and its relative bioavailability. Orthosilicic acid is the major form of bioavailable silicon whereas thin fi brous crystalline asbestos is a health hazard promoting asbestosis and signifi cant impairment of lung function and increased cancer risk. It has been proposed that relatively insoluble forms of silica can also release small but meaningful quantities of silicon into biological compartments. For example, colloidal silicic acid, silica gel, and zeolites, although relatively insoluble in water, can increase concentrations of water-soluble silica and are thought to rely on specifi c structural physicochemical characteristics. Collectively, the food supply contributes enough silicon in the forms aforementioned that could be absorbed and signifi cantly improve overall human health despite the negative perception of silica as a health hazard. This review discusses the possible biological potential of the metalloid silicon as bioavailable orthosilicic acid and the potential benefi cial effects on human health.

Keywords asbestos • dietary silica • medicine • orthosilicic acid • silicon • therapy

Please cite as: Met. Ions Life Sci. 13 (2013) 451–473

1 Introduction

Silicon is the second most prevalent element in the earth’s crust existing primarily as oxygen-containing silica and silicates and accounting for around 27% of elemental mass with oxygen comprising approximately 45% [1 – 3]. Silica is omnipotent being present in almost all of the earth’s minerals, rocks, sands, and clays and exists in myriad chemical forms expressed as quartz, emerald, feldspar, serpentine, mica, talc, clay, asbestos, and glass all of which have different uses [ 4 , 5 ]. Overall, quartz and aluminosilicates are the two most predominant silicates [6 ]. As an element, silicon has found widespread use in industrial applications often as a component of fabricated steel, a component of abrasives (silicon carbide), a building block of transistors (along with boron, gallium, arsenic, etc.), solar cells, rectifi ers, and other 14 Silicon: The Health Benefi ts of a Metalloid 453 electronic solid-state devices [7 ]. Industrial applications also include synthesis of glass when derived from sand-based silica, production of computer chips, and as a fi ller for paint and rubber ceramics, in lubricants, concrete and bricks, as well as being used for medical devices such as silicone implants [5 ]. Although silicon is used frequently for technical applications, its exposure to humans is fairly limited and largely in chemical forms that are not readily absorbed nor bioavailable. Silica is used widely in the food and beverage industry as a food additive, i.e., anti-caking agent in foods, clarifying agent in beverages, viscosity controlling agent, as an anti-foaming agent, dough modifi er, and as an excipient in drugs and vitamins [5 ]. Thus, silicon as silica is a dietary component although largely assumed to be inert when provided in forms typically used in the aforementioned applications. Nonetheless, humans are exposed to diet-derived forms of silica suggesting potential capacity for absorption and ultimate bioavailability, which raises the question of whether silicon as a molecular component can exert benefi cial, biological effects in humans. Currently, silicon is not recognized as a nutrient in humans although emerging research suggests benefi t from consumption of water-soluble forms. To that end, there is renewed growing interest in the potential benefi cial effects of silica on human health. Regarding inadvertent environmental exposure, previous research, in large part, has explored the toxic effects of inhaled crystalline silica and silica-derived asbestos. In fact, silicon has long been recognized as a pulmonary carcinogen with resultant silicosis or asbestosis developing upon prolonged and/or heavy exposure to airborne material [8 ]. Silicosis is a disease of the lungs caused by continued inhalation of the dust of minerals that contain silica and is characterized by progressive fi brosis and a chronic shortness of breath [9 ]. Asbestosis is similar in etiology and pathology but distinct as an exposure. While there are intrinsic dangers associated with inhalation of crystalline silica, there are multiple forms of silica in nature that are not toxic. Although non-toxic, the question remains as to the relative water- solubility of different compounds, relative amounts ingested, effi ciency of absorption and overall bioavailability. Low-molecular-weight silica can dissolve in water as silicic acid rendering it bioavailable and potentially a benefi cial component in humans. Collectively, the lack of understanding of the relative dependence of the physicochemical structure of silica and silicates on water-solubility for absorption has limited overall research interest in aqueous silica. As a result, a clearer understanding of the chemistry of silica, specifi cally of aqueous orthosilicic acid, is critical to fostering much needed research on potential health benefi ts.

2 Silicon Biochemistry

2.1 Silicon Distribution and Prevalence in Nature

Chemically, silica is an oxide of silicon and represented by silicon dioxide (SiO 2 ). Silicon itself is a tetravalent metalloid with chemical properties somewhere in between that of a metal and non-metal element. Its presence is second only to oxygen 454 Martin in its abundance on earth comprising almost a third of the earth’s crust. In its pure form, silicon typically does not exist in a natural elemental state due to its extreme propensity to undergo reactions with ambient oxygen and water. For example, silica,

SiO2 , and other oxides, are ubiquitously found in polymerized combinations with metals and embedded in geologic rock formations. Given its omnipotence, overall prevalence and reactivity with other elements, it clearly exists in myriad forms with differing physicochemical properties with some that are toxic and others that are seemingly critical for health. Silica is largely present in geographical formations and not readily released from these substrates except through natural, but signiﬁ cant, weathering of these structures. Overall, the forms and resultant molecular sizes of polymers and aggregates are dependent on pH and concentrations in aqueous matrices [10 ]. For example, at low concentrations (<2 mM) silicon exists in a monomeric acidic form (pK a 9.6) as orthosilicic acid, which imparts a fair degree of water solubility and certainly more than the higher-molecular-weight forms. As concentrations increase, polymerization will occur to form oligomers and eventually colloids, then aggregates and solid amorphous precipitates with a clear concentration dependence on solubility. As one might surmise, the increasing molecular weight and structural complexity restricts water solubility and, as a result, limits potential absorption by humans and animals.

2.1.1 Dietary Sources

Silica exists in the food chain with concentrations tending to be much higher in plant-based foods, i.e., phytolithic, than animal foods [11 ]. Beverages, however, are the major contributor to dietary silica, or silicon, and include water, coffee, and beer (due to barley, hops, etc.) where fl uid ingestion alone can account for ≥20% of intake [12 – 14]. Beer is the major source of bioavailable silicon for males with concentrations of 9–39 mg/L [14 – 16 ]. Silica is also prevalent in municipal water supplies but is particularly high in bottled spring and artesian waters depending on the respective geological source [17 ]. In fact, beverages alone contribute up to 55% of total dietary intake of silicon as water-soluble silica. Dietary grains and grain products including cereals, oats, barley, wheat fl our, pasta, pastries, and polished rice contribute 14% of ingested silicon and vegetables contribute 8% [18 ]. In the Western diet, major sources of silicon are cereals (30%) followed by fruits, beverages, and vegetables, which together make up 75% of total silicon intake [19 ]. Processing and refi nement of grains remove silicon during the processes but silica-derived food additives can replace the stripped silicon and increase the content although the relative absorptivity of added silicon is questionable. Overall, estimation of dietary intake from all sources is approximately 20–50 mg silicon/day for Western populations but up to ~200 mg/day for populations consuming a more plant-based diet such as populations from India and China [12 , 18 , 20 – 22 ]. The presence of large amounts of silica in geological formations contributes greatly to the silica content of water. For example, in the United Kingdom, silicon concentrations are ≤2.5 mg/L in north and west Britain but up to 14 mg/L in south 14 Silicon: The Health Benefi ts of a Metalloid 455 and east Britain [ 23– 25]. Silica is found in fresh water at concentrations of 1–100 mg/L depending on the geographical location, e.g., soil content. Typical municipal water supplies can provide 4–11 mg/L of aqueous silica as noted in a study of the large cities of France. Levels of around 18–20 mg/L occur in the water of large cities of the United States. Bottled waters also contain modest concentrations of silica ranging from 8 to 36 mg/L as noted for the French brands Badoit, Vichy Celestian, and Volvic [26 ]. Interestingly, bottled water from Malaysia contains 30–40 mg/L silica and from the Fiji Islands contains 85 mg/L silica, more than four times the levels found in fresh water and municipal supplies and over twice that of other bottled waters, presumably due to the leaching of water-soluble silica from volcanic rock. Collectively, aqueous sources provide a wide range of concentrations of water- soluble, bioavailable silica. There are other dietary sources of silicon including primarily food additives and dietary supplements. For foods, silicon may be added to processed, manufactured, and distributed foods as anticaking agents, thickeners, stabilizers, and clarifying agents, signifi cantly increasing the overall silicon concentration [27 ]. However, silicates are generally considered to be inert and, as a result, not absorbed to any great extent by humans. In particular, polymeric silicic acids and amorphous silicon dioxide are poorly absorbed. Dietary supplements are an alternative silicon source containing orthosilicic acid or other forms that are presumably modifi ed to a form that is water-soluble, absorbed, and bioavailable although this does not universally apply [28 , 29 ]. The estimated overall bioavailability of silicon from supplements ranges from <1 to >50%, a remarkably wide range, and depends on the formulation and concentration [6 ]. Silica is prevalent in the typical human diet at around 10–25 mg/day and generally considered safe, even if indigestible and non-absorbable. Although a biomarker of silicon status has yet to be developed, approximately 41% of ingested silicon is excreted in urine, which is signifi cantly correlated with dietary consumption of silicon [20 ,30 ]. The lack of clear understanding of the myriad of chemical forms of silica and signifi cant, widely communicated likelihood of increased risk of cancer has unduly overshadowed the study of the potential protective effects of silica on human health. It is the intent of this review to provide insight into the chemical properties of silica that may render it bioavailable and benefi cial to human health. Although there are dietary sources of silicon which are thought to exert benefi - cial effects in humans, there is no recommended dietary allowance (RDA) for silicon and, in fact many do not recognize silicon as a micronutrient essential for life, although 1–2 g is present in the human body [31 , 32 ]. However, if one considers the risk assessment of amorphous silicon dioxide as a common silicon source, although non-absorbed, the safe tolerable upper intake level (TUL), a component of the Dietary Reference Intakes (DRIs), is estimated to be 700 mg/day for adults, which is equivalent to 12 mg silicon/kg body weight/day for a 60 kg adult [33 ]. However, only minimal amounts of silicon become water-soluble and ultimately absorbed, thus the systemic plasma concentration does not increase signifi cantly. The mean dietary silicon intake reported for a Finnish population was 29 mg silicon/day and 456 Martin for a typical British diet 20–50 mg/day corresponding to 0.3–0.8 mg silicon/kg body weight/day [14 , 18 , 34 , 35]. The estimated dietary intake in the US is 24–33 mg silicon/day with males generally consuming more [20 ].

2.1.2 Non-dietary Sources

Given the relative prevalence and widespread use of silica, it seems reasonable that there are myriad, diverse sources of and exposures to non-dietary silica/silicon. These occur primarily from exposure to dust, pharmaceuticals, cosmetics, medical implants, and medical devices. Often the forms of silicon occur as silicates or “sili- cones,” synthetic organosilicon compounds that, for the most part, are sparse in the human diet and contribute little silicon overall. Moreover, the forms that do result in exposure are not readily absorbed or biologically useful. For example, some pharmaceuticals can increase exposure of silicon to >1 g/d but the molecular species are largely inert and not absorbed to any signiﬁ cant extent. Examples of silicates include talc, kaolin, and magnesium, calcium, and sodium salts. This seems to be the case with other non-dietary sources such as toiletries, e.g., toothpaste, lipstick, etc., and detergents, tissue implants, etc. [6 ].

2.2 Silicon Chemical Speciation as Silicates

Silicon is the second most abundant element on earth with properties that are a mixture of both metals and non-metals resulting in classiﬁ cation as an elemental metalloid. As stated previously, silicon is rarely found in its elemental form but rather complexed with oxygen and/or other elements forming silica and silicates. Silicon dioxide, SiO2 , is the oxide of silicon most commonly found in nature as sand or quartz. Generally, a silicate is any compound containing silicon and oxygen as an 2 − anion, SiO4 , with most in nature existing as oxides although the non-oxygen con- 2− taining hexaﬂ uorosilicate anion, [SiF6 ] , is also often included as a silicate. Chemically, silicate anions can form compounds with numerous, diverse cations, thus this chemical class of compounds is large with formation of aluminosilicates being the most prevalent in nature [36 ]. Aluminum is the third most prevalent element in the earth’s crust and exists in combination with >270 other minerals.

2.3 Silicon Chemistry and Effects on Bioavailability

Silica, SiO2 , is a silicic acid anhydride of monomeric orthosilicic acid (H 4 SiO4 ) which is water-soluble and stable in aqueous solutions when relatively dilute. Several other low-molecular-weight, but hydrated forms, of silicic acid exist in 14 Silicon: The Health Beneﬁ ts of a Metalloid 457

aqueous solutions and include metasilicic acid (H2 SiO3 ), lower-molecular-weight oligomers such as disilicic acid (H2 Si 2 O5 ) and trisilicic acid (H2 Si3 O7 ), as well as their hydrated forms pentahydro- and pyrosilicic acids [1 ]. Depending on the environmental conditions (temperature, pH, and presence of other ions), concentrations, and exposure time, formation of numerous potential polymerized silicic acids is possible through chemical condensation and cross-linking resulting in colloids and gels [37 ]. It is the lower molecular weight forms, especially the orthosilicic acid that is of the greatest research interest in exerting beneﬁ cial effects since this form is preferentially absorbed [38 ]. In fact, a small human study showed that ingestion of polymeric forms of silicic acid did not increase urinary levels suggesting little absorption, but 53% of ingested orthosilicic acid did increase urinary output indicating absorption [ 39 ]. Interestingly, most aqueous silica, i.e., seawater, freshwater, soil water, etc., occurs as orthosilicic acid (H4 SiO4 ) making it an important environmental exposure in the context of biological systems due both to its water-solubility and bioavailability [4 , 40 ]. As previously noted, orthosilicic acid is water-soluble at relatively low concentrations but polymerizes readily at higher concentrations in excess of 100–200 ppm to form colloids and gels, which are less bioavailable. However, more concentrated solutions of orthosilicic acid can be stabilized to avoid polymerization. In fact, choline- stabilized orthosilicic acid, a liquid formulation, has been developed and approved for human consumption. It is considered non-toxic at high doses with a lethal dose exceeding 5,000 mg/kg body weight in humans and 6,640 mg/kg body weight in animals [41 , 42 ]. For a 70 kg human, this translates to a safe level of consumption of 350 g. This stabilized form currently represents the most bioavailable source of supplemental silicon.

3 Silicon and Its Potential Health Beneﬁ ts

Silicon is the third most abundant trace element in the human body [ 20 , 43 ]. It is present at 1–10 ppm in hair, nails, the epidermis, and epicuticle of hair [44 – 46 ]. Considering the natural abundance, presence of bioavailable chemical forms, exposure to humans through diet, it seems more than plausible that there could, and likely is, potential benefi t to humans. Whether silicon is an essential micronutrient continues to be debated. It has, however, been reported in the peer-reviewed literature that silicon is actively involved, and perhaps integral, in bone mineralization and prevention of osteoporosis, collagen synthesis, and prevention of the aging of skin, overall condition of hair and nails, reduced risk of atherosclerosis and Alzheimer’s disease, as well as other biological effects [47 – 51 ]. Interestingly, serum levels are similar to other trace elements and appear to be dependent on life stage, age, and sex with levels of 11–31 μg/dL depending on population assessed and means of analysis [23 , 52 ]. A recent study by Jugdaohsingh et al. evaluated host factors potentially infl uencing the absorption and excretion of dietary silicon. Serum and urine samples were collected from 26 participants 458 Martin followed by a single ingestion of 17 mg orthosilicic acid. Analyses of samples over the subsequent 6 hours indicated that participant age, sex, and estrogen status did not infl uence absorption or excretion suggesting more research is needed to better understand the effects of host factors on disposition of dietary silica [53 ].

3.1 Bone Health and Skeletal Development

Osteoporosis is a leading cause of morbidity and mortality in the elderly and markedly affects overall quality of life, as well as life expectancy. As a result, there is considerable interest in elucidation and use of specifi c nutrients, non-nutritive dietary components, and/or bioactive compounds of natural origin singly or in combination as a means of mitigating or preventing disease, as well as for maintenance of bone health. Calcium and vitamin D have largely been the primary focus of nutritional prevention of osteoporosis, however, supplementation with other vitamins including B, C, and K has been an area of increased research as well as the use of silicon for maintenance of bone health [54 ,55 ]. Osteoporosis is defi ned as a progressive, debilitating skeletal disorder characterized by low bone mass and deterioration of the microarchitecture of bone [56 , 57 ]. Indeed, several key animal studies dating back four decades clearly showed that dietary silicon defi ciency caused abnormalities and dysfunction in connective tissues and bone function [58 –62 ]. Numerous human studies have supported a role for dietary silicon in bone health including reduction of the risk for osteoporosis. In a retrospective, clinical study by Eisinger and Clairet, dietary silicon administration induced signifi cant increases in bone mass and bone mineral density of the femur in human females [47 ]. Moukarzel et al. have also shown a direct relationship between silicon intake and bone mineral density [63 ]. In osteoporotic participants, supplementation with silicon increased trabecular bone volume and femoral bone mineral density [47 , 64 ]. Spector et al. showed in osteopenic and osteoporotic study participants an increase in bone formation markers, i.e., collagen synthesis, and signifi cant increases in femoral bone mineral density [65 ]. Maehira et al. [66 ] have shown in mice fed fi ve different calcium sources with differing silicon concentrations that soluble silicate and coral sand, with the highest silicon content, signifi - cantly improved bone biochemical and mechanical properties through induced gene expression encouraging correction of the imbalance between bone-forming osteoblastogenesis and suppression of bone-resorbing osteoclastogenesis [66 – 68 ]. Others have shown in human osteoblasts that orthosilicic acid-releasing zeolites could induce osteoblastogenesis, formation of extracellular matrix, induced synthesis of ostecalcin and activity of alkaline phosphatase both produced by osteoblasts and refl ecting biosynthetic activity of bone formation [ 69 – 71 ]. It has also been shown that silicon supplementation increased hip bone mineral density in men and pre-menopausal, but not post-menopausal, women although a subsequent study showed increased bone mineral density in the spine and femur of both pre- and post-menopausal women currently taking hormone replacement therapy [15 , 72 ]. 14 Silicon: The Health Benefi ts of a Metalloid 459

Compelling evidence demonstrates that silicon localizes to bone and that dietary silicon can strengthen bones and, as a result, reduce the risk of osteoporosis [73 ]. As mentioned before, stabilized preparations of silicic acid have been developed, e.g., choline-stabilized orthosilicic acid, permitting water-soluble preparations with higher concentrations and also markedly enhanced bioavailability. In a randomized controlled animal study, long-term treatment with choline-stabilized orthosilicic acid prevented partial femoral bone loss and exerted a positive, benefi cial effect on bone turnover and ultimately bone mineral density [74 ]. In this study, ovariecto- mized aged rodents were used suggesting a potential interrelationship between estrogen and bone health and silicon metabolism. A subsequent study by Macdonald et al. found that dietary silicon interacts with estrogen to benefi cially affect bone health [72 ]. Silicon has previously been shown to signifi cantly enhance the rate of bone mineralization and calcifi cation much like vitamin D, although functioning independently [75 ]. There are potentially confl icting reports since Jugdaohsingh et al. found that silicon supplementation in drinking water did not signifi cantly alter silicon concentrations in the bones of rodents suggesting an additional nutritional cofactor might be absent such as vitamin K in rodents fed a low silicon diet [76 ].

3.2 Vascular Disease and Atherosclerosis

It has been reported that there are higher incidences of sudden death, cerebrovascular diseases, arterial hypertension, and coronary heart disease in soft water areas of the United States suggesting, in part, that the absence of components presence in hard water, i.e., minerals, may be contributors. As a result, a major research effort has been devoted to identifying potential protective factors in hard water including calcium, magnesium, manganese, and silicon, as examples, all of which are considered potentially benefi cial [77 ]. Silicon is recognized by epidemiologic and biochemical studies as a protective trace element in atherosclerosis. Moreover, the observed decrease in silicon concentrations with increasing age has been suggested to contribute to chronic diseases such as atherosclerosis. The highest concentrations of silica in the human occur in connective and elastic tissues and especially the normal human aorta where it appears to function as a crosslinking agent that stabilizes collagen and presumably strengthens the vasculature [49 , 78]. Atherosclerosis signifi cantly decreases silicon levels in arterial walls. Moreover, silicon levels decrease just prior to plaque development, which may indicate that silicon defi ciencies cause inherent weaknesses in blood vessel walls. In a study by Trinca et al., the antiatheromatous effect of sodium silicate was tested in rabbits given a standard control diet, an atherogenic diet, and a sodium silicate- supplemented atherogenic diet. Levels of total lipids, cholesterol, triglycerides, free fatty acids, and phospholipids remained unchanged in sodium silicate supplemented rabbits fed an atherogenic diet [79 ]. In a subsequent study, silicon administered orally or intravenously in rabbits inhibited experimental atheromas normally induced by an 460 Martin atheromatous diet, decreasing the number of atheromatous plaques and lipid deposits. It was proposed that the preservation of elastic fi ber architecture, as well as of ground substance and the lack of free fatty acid accumulation in the aortic intima decreased plaque formation [80 ]. In a study by Maehira et al. using soluble silica and coral sand, as a natural silicon-containing material, the effect on hypertension, a contributing factor to atherosclerosis, was evaluated in spontaneously hypertensive rats. In rats fed 50 mg/kg dietary silicon for 8 weeks, systolic blood pressure was signifi cantly lowered by 18 mmHg. Provision of soluble dietary silica also suppressed the aortic gene expression of angiotensinogen and growth factors related to vascular remodeling. Silicon also stimulated the expression of peroxisome proliferator-activated receptor-γ, which has antiinfl ammatory and antihypertensive effects on vascular cells [81 ]. In a study by Oner et al., dietary silica modifi ed the characteristics of endothelial dilation in aortic rings from rats with modulation of endothelial relaxants and attenuation of smooth muscle cell responsiveness to nitric oxide [82 ]. Silicon has also been suggested to exert a protective role in atherosclerosis through its effects on blood vessel-associated glycosaminoglycans and collagen integrity and function via its crosslinking capacity [ 19]. Glycosaminoglycans are long unbranched (linear) polysaccharides consisting of repeating disaccharide units including hyaluronan, chondroitin, dermatan, heparan, and keratan. Silicon is also a constituent of the enzyme prolyl hydroxylase, which synthesizes collagen and glycosaminoglycans. Dietary silicon may facilitate the formation of glycosaminoglycans and collagen and/or serve a structural role as a component of glycosaminoglycans where it crosslinks, and strengthens, polysaccharide chains. Nakashima et al. have noted that the glycosaminoglycan content of the aorta was inversely correlated with the severity of atherosclerosis. Interestingly, they showed that the silicon content in fatty streaks and/or atheroma was signifi cantly higher than in normal human aortic intimal regions suggesting that the increase of silicon in the aortic intima is related to the occurrence and/progression of atherosclerosis [83 ].

3.3 Neurodegenerative Disease (Alzheimer’s Disease)

Metals that can cross the blood brain barrier and generate directly or indirectly oxidative stress can cause signiﬁ cant damage to the neuronal structure of the brain. Aluminum is abundant in the environment but is not a micronutrient. However, ingestion and/or exposures can cause deposition and accumulation in the body, e.g., brain, where it can cause considerable damage. Aluminum, a nonredox-active metal, is a well-known toxicant and its salts can accelerate oxidative damage of neurons. Oxidative stress is one of the critical features in the pathogenesis of Alzheimer’s disease and has been demonstrated in brain tissue from Alzheimer’s patients. Aluminum is a contributing factor to oxidative stress, as it generates reactive oxygen species (ROS) shown to cause oxidative damage to neurons through interaction with iron, a redox-active metal, and promotion of free radical-generating Fenton reactions, which can increase hallmark aggregation and accumulation of β-amyloid. 14 Silicon: The Health Beneﬁ ts of a Metalloid 461

Collectively, studies clearly indicate that aluminum promotes oxidative stress capable of damaging neuronal cell death [84 ]. The molecular pathogenesis of Alzheimer’s disease includes many risk factors including extracellular deposition of β-amyloid, accumulation of intracellular neurofi brillary tangles, oxidative neuronal damage and activation of infl ammatory cascades [85 ]. Although the subject of continuing scientifi c debate, aluminum has been detected in neurofi brillary tangles in the brains of both Alzheimer’s and Parkinson’s disease patients with dementia and is proposed to play crucial roles as a crosslinker in β-amyloid oligomerization [86 – 88 ]. Although the neurotoxicity of aluminum is well-documented, the association with neurodegenerative disorders is the subject of debate as is the potential benefi t of consuming silica [89 ]. Some epidemiological studies, but not all, suggest that silica could be protective against aluminum damage, because silica reduces oral absorption of aluminum and/or enhances its excretion [90 –92 ]. Studies have suggested that oligomeric but not monomeric, viz., orthosilicic acid, silica can prevent aluminum absorption through the gastrointestinal (GI) tract reinforcing the importance of chemical speciation [39 ]. Silicon readily complexes with aluminum and, in fact, aluminosilicates are the most prevalent silicates in nature. A silicate is any of numerous compounds containing silicon, oxygen, and one or more metals forming essentially a salt of silicic acid. Aluminum silicates are water-insoluble and although the processes involved in aluminum bioavailability are unclear regarding its transport into the central nervous system, numerous reports show that silicic acid can, in fact, reduce aluminum absorption and ultimately deposition and accumulation within the brain. In an epidemiological study, Rondeau et al. examined associations between exposure to aluminum or silica from drinking water and risk of cognitive decline, dementia, and Alzheimer’s disease among 1,925 elderly subjects followed for 15 years. The authors concluded that cognitive decline with time was greater in subjects with a higher daily intake or geographic exposure to aluminum from drinking water. An increase of 10 mg/day in silica intake was signifi - cantly associated with a reduced risk of dementia [93 ]. Thus, it appears that the relative concentration of both aluminum and silica in drinking water are important in determining benefi t or detriment regarding the risk and/or exacerbation of Alzheimer’s disease [94 ]. Interestingly, soft water contains less silica acid and more aluminum while the converse is true for hard water [ 25]. In a study by Exley et al. introduction of hard water rich in silica signifi cantly reduced overall aluminum levels in the body presumably through reduced absorption of aluminum as supported by reduced urinary concentrations [95 ]. A subsequent study showed that drinking up to 1 L of a silicon-rich mineral water daily for 12 weeks fostered urinary removal of aluminum in both control and Alzheimer patient groups without increasing urinary excretion of the micronutrients iron and copper [96 ]. Moreover, there were clinically relevant increases in cognitive performance in 20% of participants. Gonzalez-Munoz et al. have shown that beer consumption, a rich bioavailable source of silicic acid, can reduce cerebral oxidation caused by aluminum toxicity by, interestingly, modulating gene expression of pro-infl ammatory cytokines and antioxidative enzymes [51 ]. 462 Martin

3.4 Diabetes

Type 2 diabetes is a disorder of glycemia based largely on the development of insulin resistance. It has been noted that micronutrients can regulate metabolism and gene expression associated with glycemia thereby potentially inﬂ uencing the development and progression of diabetes [ 97]. In a report by Oschilewski et al., administration of silica to BB-rats, prone to spontaneous diabetic syndrome, completely prevented the development of diabetes [98 ]. Rats were treated with 100 mg silica/kg body weight via intraperitoneal and intravenous routes and observed for weight changes, glycosuria, and ketonuria. The authors showed nearly complete inhibition of the development of diabetes (1 of 31 in treated group versus 9 of 31 for control group) and attribute the protection of silica to reduced inﬁ ltration of pancreatic islets by macrophages. Kahn and Zinman showed in a previous study exploring bone health that dietary silicon suppressed bone marrow-derived peroxisome- proliferator receptor-γ, which regulates bone metabolism, but also regulates glucose metabolism where it is a ligand-activated transcription factor and a molecular target of a class of insulin-sensitizing drugs referred to as thiazolidinediones [99 ]. In the subsequent study, the antidiabetic effects of silicon were investigated in obese diabetic KKAy mice prone to hyperleptinemia, hyperinsulinemia, and hyperlipidemia (50 ppm silicon for 8 weeks). Interestingly, silicon and coral sand, a rich source of silicon, displayed antidiabetic effects through blood glucose reductions and increases in insulin responsiveness, as well as improvement in the responses to the adipokines leptin and adiponectin [ 100]. The authors report this as a novel function of anti-osteoporotic silicon and suggest use of silicon as a potential antidiabetic agent capable of reducing plasma glucose and reducing the risk of diabetic glomerulonephropathy. There is clearly a need for research into the potential novel therapeutic applications of silicon, as silica, for prevention and management of diabetes.

3.5 Wound Healing

Silica already fi nds widespread use in medical and surgical applications including tissue engineering for regeneration of tissues, e.g., wound repair and organs. This typically is in the form of collagen scaffolds, which are used as sponges, thin sheets or gels. Collagen, as a long fi brous structural protein, possesses the appropriate properties for tissue regeneration including optimal pore structure, permeability, hydrophilicity and stability in vivo. As a result, collagen scaffolds permit deposition and growth of cells, e.g., osteoblasts and fi broblasts, promoting normal tissue growth and restoration [101 ]. There are studies that suggest that dietary silicon can also exert benefi cial effects on wound repair. The successful healing of wounds requires local synthesis of signifi cant amounts of collagen with its high hydroxyproline content drawing upon amino acid precursors 14 Silicon: The Health Benefi ts of a Metalloid 463 such as proline and ornithine [102 ]. In animal studies, silica-defi cient diets result in poor formation of connective tissues including collagen and ultimate structural damage. Silica maintains the health of connective tissues due, in part, to its interaction with the formation of glycosaminoglycans where silicon is consistently found and presumed to have an active role. As a result, a defi ciency in silica could result in reduced skin elasticity and wound healing due to its role in collagen and glycosaminoglycan formation. Seaborn and Nielsen have reported that silicon deprivation decreases collagen formation in wounds and bone, and decreases ornithine transaminase enzyme activity in liver [103 ]. In a rodent study, silicon deprivation affected collagen formation at several different stages of bone development, the activities of collagen-forming enzymes, and consequent collagen deposition on other tissues. This has major implications suggesting that silicon is important in wound healing and supports that dietary silicon, as silicic acid, can exert therapeutic effects for this use.

4 Toxicology of Silicon and Silica

4.1 Chemical Forms Contributing to Toxicity

As previously discussed, elemental silicon exists primarily as an oxide largely in the form of silicon dioxide. Silica, SiO 2, is a silicic acid anhydride of monomeric orthosilicic acid (H 4 SiO4 ), which is water-soluble and stable in aqueous solutions when relatively dilute but can polymerize and complex with numerous minerals to form silicates with aluminum silicate being the most prevalent. Several other low- molecular- weight, but hydrated forms, of silicic acid exist in aqueous solutions and are non-toxic. Forms of silicon that are toxic include long fi brous crystalline forms such as asbestos. Asbestos is a group of crystalline 1:1 layer hydrated silicate fi bers that are classifi ed into six types based on different physicochemical features [104 ]. These include chrysotile [Mg 6 Si4 O10 (OH)8 ], the most common and economically important asbestos in the Northern Hemisphere, and the amphi- 3+ 2+ boles: crocidolite [Na2 (Fe )2 (Fe )3 Si8 O22 (OH)2 ], amosite [(Fe,Mg)7 Si 8 O22 (OH)2 ], anthophyllite [(Mg,Fe)7 Si8 O22 (OH)2 ], tremolite [Ca 2 Mg5 Si8 O22 (OH)2 ], and actinolite [Ca2 (Mg,Fe)5 Si8 O22 (OH)2 ]. Silica occurs in both non-crystalline and crystalline forms where crystalline silica is a basic component of soil, sand, granite, and many other minerals. Crystalline forms technically are physical states in which the silicon dioxide molecules are arranged in a repetitive pattern with unique spacing, lattice structure and angular relationship of the atoms. Crystalline silica forms, viz., polymorphs, include quartz, cristobalite, tridymite, keatite, coesite, stishovite, and moganite. Silicosis largely occurs due to inhalation of one of the forms of crystalline silica, most commonly quartz. All three forms may become respirable size particles when workers chip, cut, drill, or grind objects that contain crystalline silica. 464 Martin

4.2 Routes of Exposure and Safety

The most noted toxicity associated with silica and asbestos are silicosis and asbestosis, respectively. The key route of exposure leading to toxicity is respiratory with progressive, debilitating damage from lengthy and/or heavy inhalation of the dust of silica. In fact, the International Agency for Research on Cancer (IARC) classifi es silica as a “known human carcinogen” based on inhalation as a route of exposure. Regarding dietary exposure, there is no evidence of carcinogenesis when silica was fed to rodents for ~2 years (effectively the whole life span) supporting that the route of exposure is more critical than the chemical form. There are reports that magnesium trisilicate (6.5 mg elemental silicon) when used as an antacid in large amounts for years may be associated with the development of urolithiasis due to formation, in vivo , of silicon-containing stones although fewer than 30 cases have been reported in the last 80 years [105 ]. There are other reports of toxicity from oral ingestion of crystalline and amorphous silicates. For example, nephropathy can result from fi nely ground silicates and nephritis from long-term use of high dose, silica-containing medications as well as kidney damage and kidney stones [ 106]. There are some reports of increased risk of cancer (esophagus and skin) from silica-rich materials such as millet and seeds [ 14, 107 , 108]. It is proposed that the overall limitation in absorption of silicon, regardless of level of dietary intake, coupled with effi cient elimination signifi cantly limits the potential toxicity of silica. Circumventing this defense mechanism via peritoneal injections of silicon as shown in animals can easily exceed expected urinary output beyond that associated with presumed silicon adequacy [30 ,109 ].

4.2.1 Inhalation and Asbestosis

When asbestos fi bers are inhaled, most fi bers are expelled, but some can become lodged in the lungs and remain there throughout life increasing the risk of asbestosis. Asbestosis is a chronic infl ammatory and fi brotic disease affecting the parenchymal tissue of the lungs, referred to as interstitial fi brosis, caused by the inhalation and deposition of fi brous asbestos. Manifestation of the disease occurs typically after high intensity and/or long-term exposure to asbestos as a specifi c group of airborne crystalline silicate fi bers. Asbestos fi bers are invisible without magnifi cation because their size is approximately 3–20 μm wide but as small as 0.01 μm. For reference, human hair has a width of ~20–180 μm. Given the omnipotence of asbestosis in technical applications, it is considered an occupational lung disease.

4.2.2 Inhalation and Silicosis

Silicosis is also a form of irreversible occupational lung disease, technically a type of pneumoconiosis that is caused by inhalation of small particles of crystalline silica dust. Inhaling ﬁ nely divided crystalline silica dust even in small quantities 14 Silicon: The Health Beneﬁ ts of a Metalloid 465

(the Occupational Safety and Health Administration (OSHA) allows 0.1 mg/m3 ) over time can lead to silicosis, bronchitis, or cancer, as the dust becomes lodged in the lungs causing chronic irritation with reduced lung capacity. It is marked by infl ammation, pulmonary edema, scarring of the lungs, and formation of nodular lesions in the upper lobes of the lungs with resultant diffi culty in breathing. There are several different clinical and pathologic varieties of silicosis, including simple (nodular) silicosis, acute silicosis (silicoproteinosis), complicated pneumoconiosis (progressive massive fi brosis), and true diffuse interstitial fi brosis [110 ].

4.3 Mechanisms of Toxicity

The molecular mechanism of silica and asbestos-induced carcinogenesis is complex and unclear. Clearly, inhalation is the primary route of exposure leading to toxicity and depends on the shape and size of silica fi bers, duration of exposure, and relative dose, as well as lung clearance capacity and individual genetics [111 , 112 ]. Several mechanisms have been proposed including the adsorption, chromosome tangling, and oxidative stress hypotheses. The adsorption theory posits that the surface of asbestos has a high natural affi n- ity for proteins and other biomolecules and presumably disrupts cell function. The chromosome tangling hypothesis argues that asbestos can interact with chromosomes and “tangle” them during cellular division causing clastogenic damage. Probably the most compelling mechanism at this time is the oxidative stress theory, which purports that iron associated with asbestos fi bers, once internalized, can contribute to Fenton chemistry with generation of reactive, damaging free radicals and reactive oxygen species. Moreover, deposition of asbestos and silica particles in the lungs can initiate chronic infl ammation via involvement of phagocytic macrophages, which also produces copious ROS. Although discussed separately, oxidative stress and infl ammation are intimately linked and often occur concurrently, thus both occur concomitantly in lung disease.

4.3.1 Oxidative Stress

Cumulative supporting evidence suggests a role for ROS and reactive nitrogen species in the pathogenesis of asbestos- and silica-induced diseases [110 ,113 ]. Oxidative damage to the lungs can occur directly through highly reactive hydroxyl radical formation via the Fenton and Haber-Weiss reactions with fi ber surface iron, and indirectly through infl ammation [114 – 116]. This route involves the recruitment and activation of ROS-producing infl ammatory cells, such as macrophages. Other cell types also participate in the process including mesothelial cells and lung fi broblasts, which also produce ROS species in response to silica and/or asbestos. Numerous in vitro studies have shown the involvement of oxidative stress in damage caused by silica. For example, Liu et al. tested the effects of silica nanoparticles on 466 Martin endothelial cells by measuring ROS generation, apoptosis and necrosis, proinfl ammatory and prothrombic properties and the levels of the apoptotic signaling proteins and the transcription factors after exposure to silica nanoparticles (25–200 μg/mL) for 24h [117 ]. Silica nanoparticles markedly induced ROS production, mitochondrial depolarization and apoptosis in endothelial cells. Others have shown similar results with primary endothelial cells exposed to silica nanoparticles with activation and dysfunction of endothelial cells shown by release of von Willebrand factor and necrotic cell death [ 118]. In a study of mesothelial cells, exposure to crocidolite asbestos induced oxidative stress, caused DNA damage and induced apoptosis demonstrating that phagocytosis was important for asbestos-induced injury to mesothelial cells [119 ]. Several human studies have been conducted to determine if oxidative stress results from asbestos exposure using a relatively new biomarker of exposure. Measurement of exhaled breath condensate for markers of oxidative stress is one of the most promising methods available for determining pulmonary damage from environmental exposures [120 ]. An increase in the exhaled breath condensate concentrations of 8-isoprostane, an oxidative stress marker, has been observed in patients with idiopathic pulmonary fi brosis and in a limited study with asbestos- exposed subjects. Pelclova et al. measured 8-isoprostane, in 92 former asbestos workers with an average exposure of 24 years [114 ]. The results indicated higher levels of 8-isoprostane in exposed subjects compared to control subjects (69.5 versus 47.0 pg/mL) supporting asbestos-induced oxidative stress. In a study involving 83 patients (45 with asbestosis and hyalinosis and 37 with silicosis), concentrations of 8-isoprostane and hydroxynonenal, an oxidative degradation product, were measured in urine and exhaled breath condensate. The results indicated that most markers correlated positively and signifi cantly with lung function impairment [121 ]. These markers as well as others have been effectively developed to detect and confi rm oxidative stress in patients with asbestosis and silicosis [122 , 123 ].

4.3.2 Inﬂ ammation

There is growing evidence that amorphous silica can cause an infl ammatory response in the lung. These crystalline silicates are phagocytozed by macrophages that then release cytokines that attract and stimulate other immune cells including fi broblasts, which are responsible for the excessive production of collagen (fi brotic tissue) that is characteristic of silicosis [10 ]. In a study by McCarthy et al., exposure of human lung submucosal cells to SiO2 nanoparticles (10–500 nm) for up to 24 hours increased cyotoxicity and cell death, induced pro-infl ammatory gene expression and release of pro-infl ammatory IL-6 and IL-8, and upregulation of pro-apoptotic genes indicating oxidative stress-associated injury [124 ]. Bauer et al. also showed that silica nanoparticles caused dysfunction and cytoxicity through exocytosis of von Willebrand factor and necrotic cell death in primary human endothelial cells [118 ]. In the study by Liu et al., incubation of endothelial cells with 200 μg/mL silica caused increased cell death and the release of numerous, diverse proinfl ammatory mediators (TNF, IL-6, IL-8, and MCP-1) by remaining viable cells [117 ]. 14 Silicon: The Health Benefi ts of a Metalloid 467

Silica nanoparticles also activated pro-infl ammatory gene expression, e.g., NF-κB, and suppressed antiinfl ammatory gene expression, e.g., Bcl-2. The study collectively showed that silica nanoparticles damaged endothelial cells through oxidative stress via changes in gene expression associated with infl ammation. Others have shown the role of IFN-γ in the development of murine bronchus-associated lymphoid tissues induced by silica and activation of NF-κB in silica-induced IL-8 production by bronchial epitehelial cells [125 ]. Clearly, silicosis is characterized by mononuclear cell aggregation and lymphocytes are abundant in these lesions [ 126 ]. Ironically, short-term studies in rodents exposed to crystalline quartz suggested that silicon exposure stimulated the immune system and respiratory defense through activation of neutrophils, T lymphocytes, and NK cells with subsequent increased production of ROS [127 – 129 ]. This is thought to enhance the pulmonary clearance of microbes. Intriguingly, silica was shown, at least in rats, to activate and increase proliferation of CD8+ and CD4+ T cells suggesting potential therapeutic use in the future as an immunostimulant for pulmonary disorders. Recently a supplemental anionic alkali mineral complex containing sodium silicate (60% of mass) has been developed and is currently used as immunostimulant in animals including horses, pigs, etc. [ 130 ]. The mechanism of action is not known, however, it has been suggested that orthosilicic acid-generating sodium silicate is the bioactive agent responsible for the immunostimulation. Sodium metasilicate has also been shown to be immunostimulatory [131 ]. The seemingly dichotomous actions of silica represent a conundrum with excessive immunostimulation in silicosis and asbestosis clearly being detrimental but an apparent capacity of silica to also benefi cially boost the immune system with consequent ROS production.

5 Potential Medicinal Uses of Silicon and Silicates

The potential medicinal uses of silicon in the form of silica have only recently been recognized particularly with respect to bone health and prevention of neurodegenerative diseases. Data are preliminary yet supportive of potential roles in reducing the occurrence of type 2 diabetes and preserving and producing collagen, e.g., wound repair. Silicon is environmentally prevalent representing the second most abundant element yet the biological availability of silica is limited and distributed unevenly based largely on geographic location and source. As discussed previously, it is the orthosilicic acid that is water-soluble and bioavailable yet overall intake and absorption could be improved. Thus, orthosilicic acid will likely be a prominent therapeutic medicinal agent and, in fact, many potential therapeutic applications have already been presented. For example, silicon appears to play a signifi cant role in maintaining bone health through increased bone formation and increased bone mineral density and maintenance of connective tissues. Silicon, as dietary silica, also inhibits absorption of toxic aluminum, which may contribute to the development of Alzheimer’s disease. This occurs at a time when there is increased prevalence of osteoporosis and Alzheimer’s disease as populations worldwide become older. Other potential uses include enhancement of immune 468 Martin function, preservation and health of skin, hair, and nails, and use as potential antidiabetic and anticancer agents. The development of new formulations of orthosilicic acid or orthosilicic acid- releasing compounds is a promising means of delivering increased concentrations of bioavailable and safe silicon. Choline-stabilized orthosilicic acid is a newly developed, concentrated solution of orthosilicic acid in a choline and glycerol matrix and is promoted as biologically active and the most bioavailable form of silicon. Moreover, choline-stabilized orthosilicic acid has been approved for human consumption and is considered relatively non-toxic with a tolerable upper limit exceeding 5 g/kg body weight [ 28 , 41]. There are many other silicon supplements available including extracts of horsetail, which contains 12 mg silicon per tablet of which 85% is suggested to be bioavailable [28 , 29 , 65 , 74 ,132 ]. Overall, results of the NHANES III study indicate a median intake of silicon from supplements to be 2 mg/d, but with preparations such as the aforementioned could markedly increase. A particularly interesting area of research and development has been the emergence and/or use of orthosilicic acid-releasing compounds. Specifi cally, certain types of zeolites, a class of aluminosilicates with well-described ion (cation)- exchange properties have been shown to release orthosilicic acid [1 ]. Overall, 191 unique zeolites have been described with over 40 naturally occurring zeolites identifi ed. These are already widely employed in chemical and food industries, agriculture, and environmental technologies but could fi nd much greater use as medicinal and/or nutritional agents. In fact, the biomedical applications of zeolites include, in part, modulation of enzyme kinetics, use in hemodialysis, prevention of diabetes, increased bone formation, function as an antidiarrheal and antibacterial agent and as vaccine and tumor adjuvants [ 1]. The numerous biological activities of some types of zeolites documented so far is thought to be due, in large part, to the orthosilicic acid-releasing property.

6 Summary and Future Directions

In conclusion, silicon, as silica and silicates, represents a very large family of molecules with potential health benefi ts but also with potential toxic effects depending on the form, water-solubility, route of exposure, and amount consumed. For example, inhaled particulate fi brous crystalline silica can be toxic and depends heavily on route of exposure and chemical form. Silica can also dissolve in water to form non- toxic bioavailable silicic acids and specifi cally orthosilicic acid. This form of absorbable silica found in foods and water sources, is readily absorbed, reaches key tissue and organ target sites of action, and is effi ciently excreted. The lack of apparent toxicity of water-soluble forms that are consumed, as opposed to inhaled, and the ongoing debate regarding essentiality as a micronutrient have obscured the relative importance of chemical speciation and potential contributions of silica. Even though water-soluble to some degree, there are limitations to absorption dictated largely by chemical instability, e.g., propensity to polymerize, and maximum 14 Silicon: The Health Benefi ts of a Metalloid 469 allowable concentrations of water-soluble orthosilicic acids. However, there has been development of acid forms with markedly increased stability and, as a result, signifi cant increased concentrations and bioavailability of silicon. Choline chloride- stabilized orthosilicic acid is a pharmaceutical formulation that is particularly promising but other forms exist including sodium or potassium silicates, and orthosilicic acid-releasing forms such as zeolites. Further research on silicon is critically needed particularly focusing on the physiological roles of silicon and how this relates to human health, as well as the dependence on chemical speciation. Specifi cally, ample data exist to support a possible role of silicon in wound repair, atherosclerosis and hypertension, diabetes, several bone and connective tissue disorders, neurodegenerative diseases, e.g., Alzheimer’s and Parkinson‘s disease, and other conditions that occur particularly in the aging population. It is also important to further elucidate biochemical mechanisms of action of silicon-containing molecules, as silicic acids, and to extend testing more into whole body systems. Specifi cally, larger studies with humans are needed to explore the medicinal and nutritional potential of silicon.

Abbreviations

DRI dietary reference intake IARC International Agency for Research on Cancer IFN-γ interferon-γ IL interleukin MCP-1 monocyte chemoattractant protein-1 NF-κB nuclear factor B NHANES National Health and Nutrition Examination Survey NK cells natural killer cells NTF tumor necrosis factor OSA orthosilicic acid RDA recommended dietary allowance ROS reactive oxygen species TUL tolerable upper limit

References

1. L. Jurkic, I. Cepanec, S. Pavelic, K. Pavelic, Nutr. Metab. 2013, 10, 2, DOI: 10.1186/1743-7075-10-2. 2. C. Exley, J. Inorganic. Biochem. 1998 , 69 , 123–139. 3. S. Sjoberg, J. Non-Crystalline Solids 1996 , 196 , 51–57. 4. C. Perry, Prog. Mol. Subcell. Biol. 2009 , 47 , 295–313. 5. K. Martin, J. Nutr. Health Aging 2007 , 11 , 94–97. 6. R. Jugdaohsingh, J. Nutr. Health Aging 2007 , 11 , 99–110. 7. A. Elmore, Cosmetic Ingredient Review Expert Panel , Int. J. Toxicol. 2003 , 22 , 37–102. 470 Martin

8. R. Merget, T. Bauer, H. Kupper, S. Philippou, H. Bauer, R. Breitstadt, T. Bruening, Arch. Toxicol. 2002 , 75 , 625–634. 9. U. Saffi otti, L. Daniel, Y. Mao, X. Shi, A. Williams, M. Kaighn, Environ. Health Perspect. 1994 , 102 , 159–163. 10. R. K. Iler, The Chemistry of Silica: Solubility, Polymerization, Colloid and Surface Properties and Biochemistry of Silica , John Wiley & Sons, New York, 1979, pp. 866. 11. J. J. Powell, S. A. McNaughton, R. Jugdaohsingh, S. H. C. Anderson, J. Dear, F. Khot, L. Mowatt, K. L. Gleason, M. Sykes, R. P. H. Thompson, C. Bolton-Smith, M. J. Hodson, Br. J. Nutr. 2005 , 94 , 804–812. 12. S. McNaughton, C. Bolton-Smith, G. Mirshra, R. Jugdaohsingh, J. Powell, Br. J. Nutr. 2005 , 94 , 813–817. 13. L. Burns, M. Ashwell, J. Berry, C. Bolton-Smith, A. Cassidy, M. Dunnigan, K.T. Khaw, H. Macdonald, S. New, A. Prentice, J. Powell, J. Reeve, S. Robins, B. Teucher, Br. J. Nutr. 2003 , 89 , 835–840. 14. J. Bellia, J. Birchall, N. Roberts, Lancet 1994 , 343 , 235. 15. R. Jugdaohsingh, K.L. Tucker, N. Qiao, L. A. Cupples, D. P. Kiel, J. J. Powell, J. Bone Mineral Res. 2004 , 19 , 297–307. 16. S. Sripanyakorn, R. Jugdaohsingh, H. Elliott, C. Walker, P. Mehta, S. Shoukru, R. Thompson, J. Powell, Br. J. Nutr. 2004 , 91 , 403–409. 17. P. R. Barnett, M. Skougstad, K. Miller, J. Am. Water Works Assoc. 1969 , 61 , 61–68. 18. J. Pennington, Food Addit. Contam. 1991 , 8 , 97–118. 19. A. Mancinella, Clin. Ter. 1991 , 137 , 343–350. 20. R. Jugdaohsingh, S. H. Anderson, K. L. Tucker, H. Elliott, D. P. Kiel, R. P. Thompson, J. J. Powell, Am. J. Clin. Nutr. 2002 , 75 , 887–893. 21. F. Chen, P. Cole, L. Wen, Comm. Int. Nutr. 1994 , 124 , 196–201. 22. A. Anasuya, S. Bapurao, P. Paranjape, J. Trace Elements Med. Biol. 1996 , 10 , 149–155. 23. J. Dobbie, M. Smith, Scottish Med. J. 1982 , 27 , 10–16. 24. J. Birchall, Ciba Found Symp. 1992 , 169 , 50–61. 25. G. Taylor, A. Newens, J. Edwardson, D. Kay, D. Forster, J. Epidemiol. Comm. Health 1995 , 49 , 323–328. 26. S. Gillette-Guyonnet, S. Andrieu, F. Nourhashemi, V. de La Gueronniere, H. Grandjean, B. Vellas, Am. J. Clin. Nutr. 2005 , 81 , 897–902. 27. R. Villota, J. Hawkes, Crit. Rev. Food Sci. Nutr. 1986 , 23 , 289–321. 28. K. Van Dyck, R. Van Cauwenbergh, H. Robberecht, H. Deelstra, Fresenius J. Analytical Chem. 1999 , 363 , 541–544. 29. A. Barel, M. Calomme, A. Timchenko, K. De Paepe, N. Demeester, V. Rogiers, P. Clarys, D. Vanden Berghe, Arch. Dermatol. Res. 2005 , 297 , 147–153. 30. F. Nielsen, Gastroenterol. 2009 , 137 , S55–S60. 31. European Food Safety Authority, The EFSA Journal 2004 , 60 , 1–11. 32. Reports of the Scientifi c Committee for Food, 31st series, Nutrient and Energy Intakes for the European Community , Commission of the European Communities, Luxembourg, 1993 . 33. Food and Nutrition Board, Institute of Medicine, Arsenic, Boron, Nickel, Silicon and Vanadium, In: Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc, Washington National Academy Press 2000 , Chapter 13, 502–553. 34. P. Varo, P. Koivistoinen, Acta Agric. Scand. 1980 , 22 , 165–171. 35. H. Bowen, A. Peggs, J. Sci. Food Agric. 1984 , 35 , 1225–1229. 36. C. Willhite, G. Ball, C. McLellan, Crit. Rev. Toxicol. 2012 , 42 , 358–442. 37. C. Perry, T. Keeling-Tucker, J. Biol. Inorg. Chem. 2000 , 5 , 537–550. 38. J. Popplewell, S. King, J. Day, P. Ackrill, L. Fifi eld, R. Cresswell, M. di Tada, K. Liu, J. Inorg. Biochem. 1998 69 , 177–180. 39. R. Jugdaohsingh, D. M. Reffi tt, C. Oldham, J. P. Day, L. K. Fifi eld, R. P. Thompson, J. J. Powell, Am. J. Clin. Nutr. 2000 , 71 , 944–949. 14 Silicon: The Health Benefi ts of a Metalloid 471

40. P. Treguer, D. Nelson, A. Van Bennekom, D. DeMaster, A. Leynaert, B. Queguiner, Science 1995 , 268 , 375–379. 41. European Food Safety Authority, The EFSA Journal 2009 , 948 , 1–23. 42. G. Berlyne, A. Adler, N. Ferran, S. Bennett, J. Holt, Nephron 1986 , 43 , 5–9. 43. D.M. Refﬁ tt, R. Jugdaohsingh, R.P. Thompson, J.J. Powell, J. Inorg. Biochem. 1999 , 76 , 141–147. 44. B. L. Smith, Trace Elements in Man and Animals, Vol. 8, Eds M. Anke, D. Meissner, C. F. Mills, Kluwer, New York, 1993, 1091–1093. 45. J. Austin, Nobel Symp. 1997 , 255–268. 46. S. Fregert, J. Invest. Dermatol. 1958 , 31 , 95–96. 47. J. Eisinger, D. Clairet, Magnesium Res. 1993 , 6 , 247–249. 48. A. Lassus, J. Int. Med. Res. 1993 , 21 , 209–215. 49. K. Schwarz, Lancet 1977 , 1 , 454–457. 50. J. Candy, J. Edwardson, J. Klinowski, A. Oakley, E. Perry, R. Peny, Senile Dementia of the Alzheimer Type, Eds J. Traber, W. H. Gispen, Springer, Heidelberg, 1985 ,183–197. 51. M. Gonzalez-Munoz, I. Meseguer, M. Sanchez-Reus, A. Schultz, Food Chem. Toxicol. 2008 , 46 , 1111–1118. 52. E. Bisse, T. Epting, A. Bell, G. Lindinger, H. Lang, H. Wieland, Anal. Biochem. 2005 , 337 , 130–135. 53. R. Jugdaohsingh, S. Sripanyakorn, J. Powell, Br. J. Nutr. 2013 , 25 , 1–7. 54. M. Rondanelli, A. Opizzi, S. Perna, M. Faliva, Endocrinol. Nutr. 2013 , 60 , 197–210. 55. C. Price, J. Langford, F. Liporace, Open Orthop. J. 2012 , 6 , 143–149. 56. L. Russell, Rheum. Dis. Clin. North Am. 2010 , 36 , 665–680. 57. R. Marcus, J. Clin. Endocrinol. Metab. 1996 , 81 , 1–5. 58. K. Schwarz, D. Milne, Nature 1972 , 239 , 333–334. 59. E. Carlisle, Science 1972 , 178 , 619–621. 60. E. Carlisle, J. Nutr. 1976 , 106 , 478–484. 61. E. Carlisle, J. Nutr. 1980 , 110 , 352–359. 62. E. Carlisle, J. Nutr. 1980 , 110 , 1046–1056. 63. A. Moukarzel, M. Song, A. Buchman, M. Ament, J. Am. Coll. Nutr. 1992 , 11 , 584. 64. A. Schiano, F. Eisinger, P. Detolle, A. Laponche, B. Brisou, J. Eisinger, Rev. Rhum. Mal. Osteoastic. 1979 , 46 , 483–486. 65. T. Spector, M. Calomme, S. Anderson, R. Swaminathan, R. Jugdaohsingh, C. Van Hoorebeke, J. Powell, J. Bone Mineral Res. 2005 , 20 , S172. 66. F. Maehira, I. Miyagi, Y. Eguchi, Nutrition 2009 , 25 , 581–589. 67. P. Sambrook, C. Cooper, Lancet 1997 , 367 , 2010–2018. 68. S. Robins, S. New, Proc. Nutr. Soc. 1997 , 56 , 903–914. 69. M. Brady, P. Dobson, T. M. J. Kanis, J. Bone Mineral Res. 1991 , S139. 70. P. Keeting, M. Oursler, K. Wiegand, S. Bonde, T. Spelsberg, B. Riggs, J. Bone Mineral Res. 1992 , 7 , 1281–1289. 71. D. M. Refﬁ tt, N. Ogston, R. Jugdaohsingh, H. F. J. Cheung, B. A. J. Evans, R. P. H. Thompson, J. J. Powell, G. N. Hampson, Bone 2003 , 32 , 127. 72. H. Macdonald, A. Hardcastle, R. Jugdaohsingh, W. Fraser, D. Reid, J. Powell, Bone 2012 , 50 , 681–687. 73. W. Landis, D. Lee, J. Brenna, S. Chandra, G. Morrison, Calcif. Tissue Int. 1986 , 38 , 52–59. 74. M. Calomme, P. Geusens, N. Demeester, G. Behets, P. D’Haese, J. Sindambiwe, V. Van Hoof, D. Vanden Berghe, Calcif. Tissue Int. 2006 , 78 , 227–232. 75. E. Carlisle, Science 1970 , 167 , 279–280. 76. R. Jugdaohsingh, M. Calomme, K. Robinson, F. Nielsen, S. Anderson, P. D’Haese, P. Geusens, N. Loveridge, R. Thompson, J. Powell, Bone 2008 , 43 , 596–606. 77. S. Tubek, Biol. Trace Elem. Res. 2006 , 114 , 1–5. 78. K. Schwarz, B. Ricci, S. Punsar, M. Karvonen, Lancet 1977 , 1 , 538–539. 79. L. Trinca, O. Popescu, I. Palamaru, Rev. Med. Chir. Soc. Med. Natl. Iasi 1999 , 103 , 99–102. 472 Martin

80. J. Loeper, J. Goy-Loeper, L. Rozensztajn, M. Fragny, Atherosclerosis 1979 , 33 , 397–408. 81. F. Maehira, K. Motomura, N. Ishimine, I. Miyagi, Y. Eguchi, S. Teruya, Nutr. Res. 2011 , 31 , 147–156. 82. G. Oner, S. Cirrik, M. Bulbul, S. Yuksel, Endothelium 2006 , 13 , 17–23. 83. Y. Nakashima, A. Kuroiwa, M. Nakamura, Br. J. Exp. Pathol. 1985 , 66 , 123–127. 84. V. Bala Gupta, S. Anitha, M. Hegde, L. Zecca, R. Garruto, R. Ravid, S. Shankar, R. Stein, P. Shanmugavelu, K. Jagannatha Rao, Cell. Mol. Life Sci. 2005 , 62 , 143–158. 85. K. Chopra, S. Misra, A. Kuhad, Expert Opin. Ther. Targets 2011 , 15 , 535–555. 86. M. Kawahara, M. Kato-Negishi, Int. J. Alzheimers Dis. 2011 , 2011 , 1–17. 87. D. Perl, A. Brody, Science 1980 , 208 , 297–309. 88. D. Perl, Environ. Health Perspect. 1985 , 63 , 149–153. 89. V. Rondeau, Rev. Environ. Health 2002 , 17 , 107–121. 90. V. Frisardi, V. Solfrizzi, C. Capurso, P. Kehoe, B. Imbimbo, A. Santamato, F. Dellegrazie, D. Seripa, A.C. Pilotto, A, F. Panza, J. Alzheimer’s Disease 2010 , 20 , 17–30. 91. E. Carlisle, Microbiol. Aging 1986 , 7 , 545–546. 92. J. Edwardson, P. Moore, I. Ferrier, Lancet 1993 , 342 , 211–212. 93. V. Rondeau, H. Jacqmin-Gadda, D. Commenges, C. Helmer, J. Dartigues, Am. J. Epidemiol. 2009 , 169 , 489–496. 94. C. Seaborn, F. Nielsen, Biol. Trace Elem. Res. 1994 , 41 , 295–304. 95. C. Exley, M. Schneider, F. Doucet, Coord. Chem. Rev. 2002 , 228 , 127–1335. 96. S. Davenward, P. Bentham, J. Wright, P. Crome, D. Job, A. Polwart, C. Exley, J. Alzheimer’s Dis. 2013 , 33 , 423–430. 97. B. O’Connell, Diabetes Spectrum 2001 , 14 , 133–148. 98. U. Oschilewski, U. Kiesel, H. Kolb, Diabetes 1985 , 34 , 197–199. 99. S. Kahn, B. Zinman, Diabetes Care 2007 , 30 , 1672–1676. 100. F. Maehira, N. Ishimine, I. Miyagi, Y. Eguchi, K. Shimada, Nutrition 2010 , 27 , 488–495. 101. S. Oliveira, R. Ringshia, R. Legeros, E. Clark, L. Terracio, C. Teixeira, M. Yost, J. Biomed. Materials 2009 , 371–379. 102. J. Albina, J. Abate, B. Mastrofrancesco, J. Surg. Res. 1993 , 55 , 97–102. 103. C. Seaborn, F. Nielsen, Biol. Trace Elem. Res. 2002 , 89 , 251–261. 104. T. Sporn, Recent Results Cancer Res. 2011 , 189 , 1–11. 105. F. Haddad, A. Kouyoumdjian, Urol. Int. 1986 , 41 , 70–76. 106. J. Dobbie, M. Smith, Ciba Found. Symp. 1986 , 121 , 194–213. 107. I. Yu, L. Tse, T. Wong, C. Leung, C. Tam, A. Chan, Int. J. Cancer 2005 , 114 , 479–483. 108. R. Newman, Nutr. Cancer 1986 , 8 , 217–221. 109. F. Sauer, D. Laughland, D. Davidson, Can. J. Biochem. Physiol. 1959 , 37 , 183–191. 110. B. Mossman, A. Churg, Am. J. Respir. Crit. Care Med. 1998 , 157 , 1666–1680. 111. S. Toyokuni, Nagoya J. Med. Sci. 2009 , 71 , 1–10. 112. G. Liu, P. Cheresh, D. Kamp, Ann. Rev. Pathol. 2013 , 8 , 161–187. 113. A. Shukla, M. Gulumian, T. Hei, D. Kamp, Q. Rahman, B. Mossman, Free Rad. Biol. Med. 2003 , 34 , 1117–1129. 114. D. Pelclova, Z. Fenclova, P. Kacer, M. Kuzma, T. Navratil, J. Lebedova, Industrial Health 2008 , 46 , 484–489. 115. D. Kamp, S. Weitzman, Thorax 1999 , 54 , 638–652. 116. B. Fubini, E. Giamello, M. Volante, V. Bolis, Toxicol. Ind. Health 1990 , 6 , 571–598. 117. X. Liu, Y. Xue, T. Ding, J. Sun, Part. Fibre Toxicol. 2012 , 9 , 36. 118. A. Bauer, E. Strozyk, C. Gorzelanny, C. Westerhausen, A. Desch, M. Schneider, S. Schneider, Biomaterials 2011 , 32 , 8385–8393. 119. W. Liu, J. Ernst, V. Broaddus, Am. J. Respir. Cell. Mol. Biol. 2000 , 23 , 371–378. 120. M. Corradi, P. Gergelova, A. Mutti, Curr. Opin. Allergy Clin. Immunol. 2010 , 10 , 93–98. 121. D. Pelclova, Z. Fenclova, K. Sysclova, S. Vlckova, J. Lebedova, O. Pecha, J. Belachek, T. Navratil, M. Kuzma, P. Kacer, Industrial Health 2011 , 49 , 746–754. 14 Silicon: The Health Beneﬁ ts of a Metalloid 473

122. K. Syslova, P. Kacer, M. Kuzma, V. Najmanova, Z. Fenclova, S. Vickova, J. Lebedova, D. Pelclova, J. Chromatograph. Anal. Technol. Biomed. Life Sci. 2009 , 877 , 2477–2486. 123. K. Syslova, P. Kacer, M. Kuzma, A. Pankracova, Z. Fenclova, S. Vlckova, J. Lebedova, D. Pelclova, J. Breath Res. 2010 , 4, 1–8. 124. J. McCarthy, I. Inkielewicz-Stepniak, J. Corbalan, M. Radomski, Chem. Res. Toxicol. 2012 , 25 , 2227–2235. 125. M. Desaki, H. Takizawa, T. Kasama, K. Kobayashi, Y. Morita, K. Yamamoto, Cytokine 2000 , 12 , 1257–1260. 126. G. Davis, C. Holmes, L. Pfeiffer, D. Hemenway, J. Environ. Pathol. Toxicol. Oncol. 2001 , 20 , 53–65. 127. J. Antonini, J. Roberts, H. Yang, M. Barger, D. Ramsey, V. Castranova, Lung 2000 , 178 , 341–350. 128. J. Antonini, H. Yang, J. Ma, J. Roberts, M. Barger, L. Butterworth, Inhal. Toxicol. 2000 , 12 , 1017–1036. 129. R. Kumar, Am. J. Pathol. 1989 , 135 , 605–614. 130. H. Koo, S. Ryu, H. Ahn, W. Jung, Y. Park, N. Kwon, Clin. Vaccine Immunol. 2006 , 13 , 1255–1266. 131. G. Oner, S. Cirrik, O. Bakan, Kidney Blood Press. Res. 2005 , 28 , 203–210. 132. M. Calomme, P. Cos, P. D’Haese, R. Vingerhoets, L. Lamberts, M. De Broe, C. Van Hoorebeke, D. Vanden Berghe, in Metal Ions in Biology and Medicine, Eds P. Collery, P. Brätter, V. Negretti de Brätter, L. Khassanova, J. C. Etienne, John Libbey Eurotext, Paris, 1998 , Vol. III, 228–232. Chapter 15 Arsenic. Can This Toxic Metalloid Sustain Life?

Dean E. Wilcox

Contents ABSTRACT ...... 476 1 INTRODUCTION ...... 476 1.1 Overview ...... 476 1.2 Chemical Properties of Arsenic ...... 477 1.3 Environmental Properties of Arsenic ...... 479 1.4 Biological Properties of Arsenic ...... 480 2 TOXICITY ...... 481 2.1 Acute Toxicity ...... 481 2.2 Chronic Toxicity ...... 481 3 SUSTAINING ROLES ...... 484 3.1 Nutritional Need for Arsenic? ...... 484 3.2 Hormesis and Arsenic ...... 485 3.3 Surviving High Levels of Arsenic ...... 486 3.3.1 Microorganisms ...... 486 3.3.2 Humans ...... 490 4 BENEFICIAL USES ...... 490 4.1 Pesticides ...... 490 4.2 Pharmaceuticals...... 491 4.2.1 Antibiotics ...... 491 4.2.2 Chemotherapeutics ...... 492 5 SUMMARY ...... 492 ABBREVIATIONS ...... 494 ACKNOWLEDGMENTS ...... 494 REFERENCES ...... 494

D. E. Wilcox (*) Department of Chemistry , Dartmouth College , Hanover , NH 03755 , USA e-mail: [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 475 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_15, © Springer Science+Business Media Dordrecht 2013 476 Wilcox

Abstract It was recently reported that a bacterium, Halomonas species GFAJ-1, isolated from arsenic-rich Mono Lake and further selected for growth under conditions of high arsenate and low phosphate, is able to grow using arsenic instead of phosphorus. This claim, and subsequent studies to evaluate GFAJ-1, has brought new attention to the question of whether arsenic can play an essential or sustaining role for living organisms. If true, this would be in stark contrast to the well known toxicity of this element and its ability to cause a number of diseases, including cancer of the skin, lung, bladder, liver, and kidney. However, while deadly at high doses, arsenic oxide is also an approved and effective chemotherapeutic drug for the treatment of acute promyelocytic leukemia (APL). This review examines the evidence that arsenic may be a beneﬁ cial nutrient at trace levels below the background to which living organisms are normally exposed. It also examines whether arsenic can be used to sustain organisms growing under high arsenic conditions, speciﬁ cally the results from recent studies of arsenic biochemistry motivated by the report of GFAJ-1. Both of these topics are considered in the context of the toxicity of this element and its ability to cause cancer and other diseases, yet its Janus-faced ability to effectively treat APL.

Keywords acute promyelocytic leukemia • arsenic • beneﬁ cial nutrient • GFAJ-1 • toxicity

Please cite as: Met. Ions Life Sci. 13 (2013) 475–498

1 Introduction

1.1 Overview

The biochemical and physiological properties of arsenic (As) are invariably linked with the toxicity of this element. From earliest human use of arsenic as a constituent of bronze and decorative pigments, there has been a collateral risk of toxicity for all who worked with it. Even modern benefi cial uses of arsenic are predominantly associated with its lethality for organisms that affect crops, livestock, and human health. Recent efforts to understand the correlation between chronic exposure to arsenic, primarily through drinking water and food, and various diseases, including cancer and diabetes, reinforces the detrimental side of this element. So, what is a chapter on arsenic doing in a volume entitled, “Interrelations between Essential Metals Ions and Human Diseases”? In contrast to well-known essential trace elements, such as Cu, Mn, I, Mo, and Se, a number of elements that are commonly found in living organisms at ultra-trace levels, such as B, Si, V, Ni, and As, have not been recognized as essential for humans. It has been diffi cult to establish whether or not there is a requirement for arsenic at ultra-trace levels because of its prevalence in the environment from natural and anthropomorphic sources, its ubiquity in living organisms, and the onset of its toxic 15 Arsenic. Can This Toxic Metalloid Sustain Life? 477 effects. Because arsenic has some chemical properties that are similar to those of essential phosphorus, a relevant question is the interrelationship between these two elements for living organisms, including the possibility that arsenate may substitute for phosphate under certain conditions. Arsenic is certainly associated with human disease, both as a causative agent and as a therapeutic agent. Consumption of sub-lethal doses leads to a variety of symptoms that are given the broad medical designation arsenicosis, but a clear correlation has now been established between chronic exposure to arsenic and cancer of the skin, lung, bladder, liver, and kidney, type II diabetes, depressed cardiovascular function, and peripheral neuropathy. However, arsenic has been used to treat human diseases since early times, and As-based Salvarsan® , which Ehrlich developed for the treatment of syphilis, was one of the fi rst modern pharmaceuticals. Although As-based drugs have been largely replaced due to their toxicity, recently arsenic oxide (As2 O3 ), which was used in traditional Chinese medicine, has been approved for treatment of acute promyelocytic leukemia (APL). Although perhaps not a particularly good fi t for this volume, the aim of this contribution is to (i) summarize the evidence for benefi cial or sustaining roles for arsenic in living organisms, including its substitution for phosphorus, and (ii) summarize its Janus-faced role in both causing and treating human disease. Tying these together is the unifying theme of the toxicity of this element. After a brief introduction to the relevant chemical properties of arsenic, its availability to living organisms, and its uptake, metabolism and excretion, I begin with an overview of arsenic toxicity and its association with a number of diseases. This is followed by the limited evidence for a benefi cial role for arsenic, including the phenomenon known as hormesis. Microorganisms living in environments with high levels of arsenic have adapted to use its chemical properties for their energy-generating pathway, but it appears not, as reported recently [1 ], by substituting arsenate for phosphate in DNA and other biological molecules. I fi nish with the benefi cial roles of arsenic in modern society, and its use in treating human disease, including its remarkable therapeutic role in treating APL.

1.2 Chemical Properties of Arsenic

Two oxidation states of arsenic, As3+ (r = 0.58 Å) and As5+ (r = 0.46 Å), are found under environmental and biological conditions. Arsenic(III) is normally three- coordinate with pyramidal structures due to a stereochemical lone pair of electrons (Figure 1). Because of its size and polarizability, As 3+ has a relatively high stability with softer S- and Se-donating species. In aqueous solution it is found as the neutral tris-hydroxo arsenite, As(OH)3 whose lowest p K a is 9.2. Arsenic(V) is normally four-coordinate with tetrahedral structures. Its greater charge density leads to a higher stability with harder O-donating species and its prevalence in aqueous 3 − solution as arsenate, AsO4 in a pH-dependent protonation state (pK a ’s 2.3, 7.0, and 11.5). Comparison of arsenic and phosphorus is instructive, due to the similarity 3 − between arsenate and phosphate, PO 4 (p Ka ’s 2.1, 7.2, 12.7), which have a similar

Figure 1 Structural representations of environmentally, biologically and medically important arsenic molecules: 1. arsenite (arsenous acid) (monomethyl- and dimethylarsenite have one and two –CH3 ’s replacing –OH’s), 2. arsenate (arsenic acid) (monomethyl- and dimethylarsenate have one and two –CH 3’s replacing –OH’s), 3. arsenobetain (AsB), 4. arsenocholine (AsC), 5. arsenosugars, 6. arsenolipids, 7. roxarsone, 8. Salvarsan® (mixture of trimer and pentamer), 9. melarsoprol. 15 Arsenic. Can This Toxic Metalloid Sustain Life? 479 structure and size. However, the arsenate 2-electron reduction potential, ε°’ = +140 mV (pH 7.0, 25°C, versus NHE) is signiﬁ cantly higher than that of phosphate (ε°’ = –690 mV), resulting in the prevalence of both arsenite and arsenate under environmental and biological conditions. Since arsenic is larger and has longer (weaker) bonds than those of phosphorus, substitution reactions of arsenate species are faster than those of the corresponding phosphate species. Arsenic in both oxidation states forms stable bonds with carbon, most prevalent of which are those with one to three methyl groups (Figure 1 ). Thus, the environmental and biological chemistry of arsenic can be divided into that of inorganic (arsenite and arsenate) and various organic species, which lack and contain stable As-C bonds, respectively.

1.3 Environmental Properties of Arsenic

Although more than 20 years old, the review by Cullen and Reimer [ 2 ], recently augmented by their review of organoarsenic species in this series [3 ], provides a thorough overview of arsenic in the environment. Thus, only the prevalence, distribution, and availability of arsenic will be mentioned here. While not an insigniﬁ cant component of the earth’s crust at 1.8 ppm, arsenic is unevenly distributed. Although found in the sulﬁ de ores orpiment, As2 S3 (yellow arsenic) and realgar, AsS (red arsenic), arsenic is more commonly a constituent of the mineral arsenopyrite, FeAsS, and associated with various iron oxides. The release of arsenic from these minerals and geological formations is governed typically by redox processes. The concentration of arsenic in the ocean is 1–3 µg/L (ppb), but in typical freshwater and aquifers it has a wide range, 0.1–80 µg/L, depending on the local geology and geological processes. However, some conditions result in even higher local concentrations, such as 700 µg/L in certain aquifers in Taiwan and 15 mg/L (ppm) in Mono Lake in California due to evaporative concentration. The predominant arsenic species in natural waters depends on the redox potential, which is normally determined by the amount of dissolved oxygen, and the pH. Arsenate will predominate under aerobic conditions (e.g., surface water), while arsenite will predominate under anoxic conditions (e.g., subsurface aquifers). Although the relative amount of each species can be predicted from thermodynamic considerations, kinetic barriers and biological redox processes can alter the arsenate-arsenite ratio. Organoarsenic species can contribute to the total arsenic when biological activity (predominantly methylation) is prevalent, particularly in marine environments. Human activities can alter local and regional levels and the distribution of inorganic arsenic (e.g., mining and processing As-containing ores, burning As-rich coal) and organic arsenic (e.g., agricultural application of organoarsenic pesticides). Arsenic is injected into the atmosphere by volcanoes and combustion and by microbial activity that generates volatile arsine (AsH3 ) and methylated arsenic species. However, the atmo- spheric residence time of arsenic species is relatively short and the arsenic concentration is generally low (0.02 µg/m3 ), except in the vicinity of these sources. 480 Wilcox

1.4 Biological Properties of Arsenic

Arsenate and arsenite are taken up by microorganisms, plants (root cells), and animals (intestinal cells) with different mechanisms. Arsenate is actively imported by two pathways used for the essential and structurally similar phosphate, as shown in competition uptake studies [4 , 5 ]. The low affi nity phosphate inorganic transport (Pit) pathway uses energy from the trans-membrane proton gradient, while the high affi nity phosphate specifi c transport (Pst) pathway confers some selectivity for phosphate over arsenate with a periplasmic phosphate binding protein (PBP) and an ATP-hydrolyzing membrane transporter [6 ]. Neutral arsenite, however, diffuses through membrane-spanning channels created by aquaglyceroporin proteins, which allow the diffusion of water, glycerol, and other neutral species [7 ]. The properties of organoarsenic species are modulated by their organic substituent(s), and this affects their uptake by these or other pathways. Due to the prevalence of arsenic there is selective pressure for resistance to this toxic element, and microorganisms have genomic and plasmid-encoded mechanisms for exporting arsenic upon its inevitable import. The best studied of these involves proteins encoded by the ars operon, and include the minimal set of ArsR, a DNA-binding repressor protein that suppresses ars expression until arsenite binds and it is released from the operator DNA, ArsC, which is a cytoplasmic glutathione- dependent arsenate reductase, and ArsB , which is a membrane-spanning protein that exports arsenite using energy from the trans-membrane proton gradient [8 ]. Some ars operons also code for a membrane-associated ArsA, which couples ATP hydrolysis to arsenite export by ArsB for more effi cient removal of arsenic. Certain bacteria, as well as higher organisms from fungi to humans, are capable of methylating arsenic by a mechanism originally outlined by Challenger [ 9 ], based on oxidative addition from the activated methyl donor S-adenosyl methionine. Enzymes that catalyze this coupling reaction have been isolated [ 10 ] and this appears to be part of a detoxifi cation mechanism, as glutathione complexes of these methylated species are exported by multi-drug resistance protein (MRP) transporters [11 ]. Certain microorganisms are capable of demethylating organoarsenic species by a mechanism that may involve the reverse process of reductive elimination [ 12 ]. Some plants are capable of growing on soils that have high levels of arsenic, and these species often hyperaccumulate arsenic, which is transported to the leaves where it is sequestered in vacuoles [ 13]. Certain marine plants have pathways to synthesize various organoarsenic species, such as arsenocholine (AsC) and arsenobetaine (AsB) (Figure 1 ), which is found in seaweed and may not only be a product of arsenic detoxifi cation but also help to maintain osmotic balance. These and other organoarsenic species (e.g., arsenosugars, arsenolipids) (Figure 1) become widely distributed in marine food webs [14 ]. Normal human blood levels of arsenic are 0.3–2 µg/L but can be 1–2 orders of magnitude higher when elevated levels of arsenic are being consumed in drinking water or the diet. The overall half-life of arsenic in humans is about 10 hours, though elimination appears to be triphasic; while some may be retained for longer periods, there is no biological sink or pool where arsenic accumulates. The urine of individuals 15 Arsenic. Can This Toxic Metalloid Sustain Life? 481 consuming elevated levels of inorganic arsenic contains monomethyl (MMAs) and predominantly dimethyl (DMAs) arsenic species [15 ]. These are enzymatically formed primarily in liver cells by arsenic methyltransferases, As3MT [ 16 ]. Although readily absorbed from the diet, organoarsenic species are rapidly excreted in the urine, as are the intra-cellularly methylated species MMAs and DMAs after their active export by MRPs or diffusion out through aquaglyceroporins [17 ].

2 Toxicity

The toxicity of arsenate is due to its competition with isostructural phosphate, yet hydrolytic instability of arsenoester bonds results in unstable species, such as the ATP analog ADP-arsenate [18 , 19]. Arsenate lowers ATP levels by uncoupling its synthesis through a general mechanism known as arsenolysis. However, the toxicity of arsenite, which predominates under reducing intracellular conditions, is due to its afﬁ nity for functionally and structurally important thiols. This makes it an effective inhibitor of enzymes such as pyruvate dehydrogenase, which is required for the citric acid cycle [20 ]. Alternatively, redox generation of reactive oxygen species (ROS) has been suggested as a mechanism for this inhibition [ 21]. Methylation of arsenic modulates its properties and thus its toxicity that, depending on the organism (e.g., tissue culture, animal model) and the mode of exposure (e.g., oral, injection), can be higher or lower [22 ]. The toxicity of organoarsenic species is dictated to a large extent by the organic substituent(s). AsB and AsC, like various arsenosugars and arsenolipids, lack exchangeable valence sites and have low toxicity, particularly AsB, which is abundant in seafood. A Provisional Tolerable Daily Intake (PTDI) for inorganic arsenic has been set at 2.1 μg/kg/day [ 23], though this has recently been withdrawn [114 ].

2.1 Acute Toxicity

Ingestion of overtly toxic amounts of inorganic arsenic species (60–120 mg is the estimated lethal dose of As2 O3 ) leads to gastroenteritis, characterized by vomiting, abdominal pain, and bloody diarrhea, which lead to dehydration, shock, convulsions, coma, and death. Doses that overwhelm cellular and tissue mechanisms that remove arsenic also result in circulatory collapse and depression of the central nervous system.

2.2 Chronic Toxicity

Chronic arsenic exposure leads to symptoms given the general designation arsenicosis, which affects millions worldwide and arises primarily through contaminated drinking water [24 , 25]. Since arsenic occurs at elevated concentrations in many 482 Wilcox natural waters and is colorless and odorless, people were unaware when their water had high levels of arsenic and the consequences of chronic arsenic exposure were largely unknown. Water testing is now common and the consequences of exposure are well documented. Nevertheless millions remain exposed because of signifi cant socio-economic barriers to providing them with clean water. One of the fi rst correlations between human disease and arsenic exposure was the large-scale incidence of black foot disease (BFD) in southwestern Taiwan [26 ]. This condition, which is due to peripheral vascular disease, is manifest as a discol- oration and blackening of the extremities, especially the feet. Subsequently it was shown that exposure to elevated arsenic in drinking water from deep artesian wells of the BFD area of Taiwan correlates with increased incidence of cancer of the skin [27 ], diabetes [ 28 ] and cardiovascular disease [29 ,30 ]. Epidemiological studies from Taiwan formed the basis for the reduction of the US drinking water limit from 50 μg/L to 10 μg/L, which fi nally came into effect in 2006. Similar relationships between cancer and arsenic exposure through drinking water have been documented in Argentina [31 , 32], Chile [33 , 34], and, most recently, Bangladesh [35 ]. Epidemiological results from the Antofagasta region of Chile show that in utero and early life exposure to arsenic predisposes individuals to increased incidence of lung cancer and bronchiectasis in later life [ 36 ]. For the well-studied cases in Taiwan and Chile, exposure occurred in the early to mid 20th Century and drinking water has since been switched to lower arsenic sources. In contrast, many millions in Bangladesh and West Bengal are still exposed to high levels of arsenic from drinking water obtained from deep tube wells [ 37, 38 ]. The Bangladesh situation has been widely described as the worst mass poisoning in human history, and came about through the installation of an extensive series of tube wells in the 1970’s sponsored by UNICEF in an effort to reduce childhood disease and mortality from drinking microbially-contaminated surface water [ 39 ]. The mechanism(s) by which arsenic causes these diseases from chronic exposure is not yet well understood. With regard to cancer, while it is genotoxic, arsenic also affects cell proliferation, cell signaling, DNA structure (methylation), epigenetic regulation, DNA repair, and apoptosis [40 ]. With so many effects, some of which may originate from the non-specifi c generation of ROS [41 ], it is diffi cult to identify molecular targets for arsenic, and the mechanism by which arsenic causes cancer is still poorly understood [42 ]. One case where a specifi c arsenic target has been identifi ed is its effect on hormone-regulated pathways. Arsenite is known to disrupt steroid hormone function at the non-cytotoxic level of 1–5 μM, and in vitro and intracellular studies of the glucocorticoid receptor (GR) have traced arsenic effects to its DNA-binding domain (DBD) [43 ]. This ~90 residue domain has 10 cysteines that bind two Zn 2+ ions to stabilize a folded structure that is competent to bind to its target DNA, the glucocorticoid response element (GRE) (Figure 2 [ 44 ]). The hypothesis that arsenite displaces Zn2+ from the thiols and disrupts the active structure of the domain was tested and shown to be valid for MMAsIII , but not arsenite [45 ], consistent with the higher MMAsIII stabilities with small thiols [46 ]. Further, based on the measured stability constants and cellular conditions, it was 15 Arsenic. Can This Toxic Metalloid Sustain Life? 483

450 C G H S Y I K R A G D R E V I K 490 D L I N S T 480 CC C C N D V G P Zn R Zn L S A G A CC440 460 CC K 470 L R 500 510 VVFFKAR E GQQHNY Y R K CL A GMNLEAR

445 486 N-term 482 448 457 492 476

C-term

508 469 471

Figure 2 Amino acid sequence (top ) and the NMR-determined 2° and 3° structure (bottom ) of the rat GR-DBD (residues 440-510) with the two Zn2+ ions indicated by spheres. Reprinted with permission from [ 44 ]; copyright 1993 American Chemical Society.

estimated that approximately 0.5 μM MMAsIII would displace an essential Zn2+ and inhibit (IC50 ) GR binding to GRE. While arsenite may have other effects on the steroid hormone pathway [47 ], GR-DBD and homologous DBDs of other steroid hormone receptors appear to be a target for the monomethylated metabolite of arsenite [48 ]. Similarly, MMAsIII is able to compete with an essential Zn2+ for Cys thiols of the nucleotide excision repair protein Xeroderma pigmentosum group A [49 ], thereby identifying a potential target for arsenic in its known role as a co-carcinogen. With multiple potential mechanisms of action for arsenic-induced disease, health risks from exposure to arsenic are currently based on a “linear, no threshold” (LNT) dose-response model from epidemiology data for populations exposed to elevated levels in Taiwan and Chile [50 ]. However, some of these data have been re-analyzed 484 Wilcox and this model called into question [ 51 ]. Extrapolation to the low level of exposure that is widely encountered leads to uncertainty in risk for those populations with low background levels of arsenic in their drinking water and diet [52 ].

3 Sustaining Roles

3.1 Nutritional Need for Arsenic?

The currently recognized essential nutrients for humans, besides glucose and the essential amino acids and fatty acids, includes 13 vitamins (one of which is

Co-containing vitamin B12 , required in ultra-trace amounts), six elements (Ca, P, Mg, Na, K, Cl) required in macroscopic amounts, fi ve elements (Fe, Zn, Cu, Mn, F) required at trace levels (~1–10 mg/day), and four elements (I, Se, Mo, Cr) required at ultra-trace levels (<1 mg/day). In addition, there is some evidence that certain other elements found in humans at ultra-trace levels, including B, Si, Ni, V, and As, may play an essential biochemical role, but the evidence in each case is not compelling enough for a human nutritional requirement. Some of these, such as Ni, B, and Si, are required by plants and/or microorganisms. What is the evidence for any nutritional need for arsenic? Early studies with rats investigated arsenic defi ciency but these tended to be compromised by high background levels and elevated (toxic?) control (suffi ciency) levels. Careful studies of arsenic deprivation (<50 ng As/g ration) with goats, minipigs, chicken, rats, and hamsters have shown depressed growth and abnormal reproduction [53 , 54 ]. The latter includes low fertility, elevated perinatal mortality (spontaneous abortions), maternal death during lactation, and higher offspring mortality. Post-mortem analysis of arsenic-defi cient goats found compromised mitochondrial membranes in their myocardial tissue [53 ]. Extrapolation of results from these animal studies leads to 12–25 μg/day as an estimated human dietary requirement [53 ], which is well below the previous PTDI (150 μg/day for a 70 kg adult). Since market basket analysis shows that typical dietary arsenic is <10% of the previous PTDI [55 ] and typical arsenic intake in water and food is estimated to be 12–60 μg/day [56 ], diets not meeting such a need would be very rare. However, two caveats should be noted. First is a caution about extrapo- lating results from animal studies to humans. It is known that arsenic metabolism is different in common animal models: arsenic is uniquely sequestered in the erythrocytes of rats [ 57], and certain primates do not methylate ingested arsenic [ 58 ]. Second is the absence of evidence for an essential arsenic role in patients undergoing long- term total parenteral nutrition (TPN) therapy (intravenous delivery of nutrients). Such an individual provided the fi rst human evidence that chromium plays a role in regulating blood sugar levels [59 ]. Nevertheless, a need for trace amounts of arsenic might be missed in TPN therapy due to trace impurities of arsenic in the nutrients for these individuals [60 ]. Further, animal studies show that arsenic defi ciency 15 Arsenic. Can This Toxic Metalloid Sustain Life? 485 affects growth, development, and reproduction, and most TPN patients are adults where these effects could be missed. Studies by Nielsen, Uthus and coworkers at the Grand Forks Human Nutrition Research Center have sought the metabolic or biochemical origin of the effects of arsenic deprivation. Lower levels of taurine (H2 NCH2 CH2 SO3 H), S-adenosyl methionine and polyamines, and elevated S-adenosyl homocysteine are detected in rats fed a low arsenic (<50 ng/g) diet, suggesting that arsenic may play a role in the metabolism of methionine or modulation of methylation capacity [ 54 ]. Arsenic deprivation also affects enzymes involved in the biosynthesis of phosphatidylcholine [61 ] that, along with lower levels of taurine, could affect mitochondrial membranes, as found in the myocardial tissue of As-deprived goats [53 ]. Whether or not there is any benefi cial role for arsenic in human physiology and biochemistry is probably a moot point because it is ubiquitous and prevalent at the very low levels that might be required for roles in growth, development, and reproduction, as found in animals. No well-defi ned biochemical role has been found for arsenic that would explain these effects of its deprivation. Ironically, several of the adverse outcomes on reproduction found with As-deprived animals (spontaneous abortion, offspring mortality) are also found in human populations exposed to toxic levels of arsenic [40 ].

3.2 Hormesis and Arsenic

It has been noted that low levels of some toxic species have a stimulatory effect on certain biochemical processes and the growth of some organisms, prior to the onset of toxic effects at higher levels. Known as hormesis, and characterized by an upside down U response curve, this phenomenon has been found with arsenic in studies involving microorganisms, plants, invertebrates, and human cell cultures. Several early studies of the effect of arsenate, and in some cases arsenite, on the growth of crop plants (pea, wheat, potato, bean, oats) show enhanced growth at low arsenic concentrations, followed by depressed growth at higher concentrations [62 ]. More recently this has been found for salt marsh grass (Spartina alternifl ora ), where enhanced growth at low arsenate or arsenite (0.2 mg/L) is associated with higher levels of phosphorus in the roots [ 63]. Similar results were found with an As-accumulating plant, the Chinese brake fern (Pteris vittata L), but not its non- hyperaccumulating cousin (Pteris ensiformis L) [64 ]. Again, the As-stimulated growth was associated with higher phosphorus in the roots, as well as elevated arsenic transported to the fronds, as is common for hyperaccumulating plants. Finally, in a model of As-contaminated Taiwanese aquaculture ponds, the growth of algae (Microcystis aeruginosa ) showed stimulated growth at 0.1 μM arsenate but suppressed growth at higher concentrations [65 ]. The connection with phosphorus metabolism in some of these studies is noteworthy. While arsenic could be mobilizing growth-limiting phosphorus from soil, the salt marsh grass study was conducted with hydroponically grown plants. Thus, arsenic stimulation of growth may be due to increased phosphate uptake. It has 486 Wilcox been suggested that arsenate competition and uncoupling of phosphate-requiring energy-generating pathways (arsenolysis) is biochemically equivalent to a phosphate defi ciency and stimulates phosphate uptake [66 ]. A stimulated response at low arsenic levels has also been found in cell culture and animal studies. This has been reported for: DNA synthesis in human lymphocyte cell cultures [67 ]; growth of various cell types, where the As-enhanced growth correlated with lower levels of some cytogenetic effects [68 ]; base excision repair in human lung fi broblasts and keratinocytes, where low levels of arsenic (<1 μM) correlate with elevated levels of DNA polymerase-β [69 ]; and estrogen receptor-alpha expression and activity in breast cancer cells [70 ]. Arsenite stimulation at low doses (<1 μM) has also been reported for the intracellular activity of GR (Figure 3 ) and other steroid hormone receptors [48 ]. The freshwater amphipod (Hyalella azteca ), which accumulates arsenic and metals, shows a maximum stimulation of its growth under conditions where it has accumulated arsenic to a level of ~5 ppm [71 ]. Finally, a modest suppression of DNA damage in liver and lung tissue was found in rats exposed to low levels of arsenite [72 ]. Enhanced DNA synthesis and repair upon exposure to low levels of arsenic, which are then overwhelmed at higher levels, may explain some of these results. Thus, there could be a common basis for certain cases of arsenic hormesis, which may originate from a general response to toxic species, such as stimulated production of ROS [41 ]. However, it should be noted that hormesis is not a scientifi cally well-defi ned phenomenon based on known mechanisms and is controversial [73 ].

3.3 Surviving High Levels of Arsenic

3.3.1 Microorganisms

Certain ecological conditions, such as those found in or near volcanoes, highly saline lakes, and gold mines that are rich in arsenopyrite, contain high levels of arsenic, yet some microorganisms survive under these conditions. A major challenge for these organisms is dealing with the toxicity of arsenate imported by their essential phosphate pathways, the low affi nity Pit pathway and the high affi nity Pst pathway, though the latter provides some selectivity for phosphate over arsenate. Microorganisms use various strategies to survive under these conditions, including more effi cient arsenic export pathways, such as that encoded by the ars operon, and oxidizing arsenite in the immediate vicinity to the less toxic arsenate.

3.3.1.1 Redox

Certain microorganisms found under conditions of high arsenic levels are able to exploit its redox properties for their energy-generating pathways. All living organisms require the energy from hydrolysis of ATP, which is regenerated by ATP synthase using the trans-membrane proton gradient created by the electron transport 15 Arsenic. Can This Toxic Metalloid Sustain Life? 487

Figure 3 Effect of low a 2.00 concentrations of arsenite on Human WT human (a ) and rat (b ) GR 1.75 activation of transcription of a GRE- and luciferase- 1.50 containing reporter gene construct transfected into 1.25 EDR3 hepatoma cells. Reprinted with permission 1.00 from [43 ]; copyright 2004 American Chemical Society. 0.75

0.50

0.25

0.00 0.0 0.5 1.0 1.5 2.02.5 3.0 As (μM) b 1.75 Rat WT 1.50

1.25

1.00

0.75 Relative Activity Relative Activity 0.50

0.25

0.00 0.0 0.5 1.0 1.5 2.02.5 3.0 As (μM) pathway. This pathway starts with the oxidation of low potential electron donor species (i.e., reduced carbon molecules) and ends with the reduction of a high potential electron acceptor (i.e., oxygen). Since the arsenate reduction potential lies near the middle of the biologically useful range, depending on the availability of 3+ reductants (e.g., reduced carbon species) and oxidants (e.g., O 2), arsenite (As ) could serve as the electron donor or arsenate (As5+ ) could serve as the electron acceptor for the electron transport pathway. The ﬁ rst of these two roles was found for chemolithoautotrophs that can use arsenite oxidation as a source of electrons and CO 2 as a carbon source [ 74]. These were organisms found initially in cattle dipping solutions, where arsenic was used to kill ticks on livestock, and later in gold mine tailings. The second of these two roles was found initially for Eubacteria species [75 ], and later other bacteria and archaea, that 488 Wilcox use arsenate as a terminal electron acceptor to support dissimilatory respiratory [76 ]. However, the mechanism by which arsenate reduction is coupled to the generation of a trans-membrane potential gradient is not known. In each of these cases, abundant arsenic can be used to sustain the organism by its contribution to the essential energy-generating pathway.

3.3.1.2 Phosphate Substitute?

Since arsenate and phosphate have similar structural properties, it may be possible for the former to substitute for one or more of the essential roles that the latter plays in biology. Westheimer considered this notion over 25 years ago [77 ] and dismissed it for sound chemical reasons, specifi cally the rapid hydrolysis of arsenate ester bonds in aqueous solution. Thus, there was considerable surprise a few years ago when it was reported that a microorganism, Halomonas strain GFAJ-1, isolated from As-rich Mono Lake and selected for growth on high arsenic and low phosphorus, grew in the presence of arsenate and absence of phosphate [1 ]. Further, evidence was provided that arsenate was associated with the DNA of this organism, suggesting that arsenate replaced at least some of the phosphate. Several critiques of the experimental conditions, data analysis, and conclusions, as well as alternate explanations and contradictory chemical properties (e.g., reduction of arsenate to arsenite under intracellular conditions), and the authors’ response to these criticisms, accompanied publication of the original report [78 ]. Additional critiques that elaborated these and other points followed [79 – 82]. Three types of studies were motivated by the original report, those aimed to refute the original results, those aimed to provide alternate explanations, and those aimed to address the possibility that arsenate could substitute for phosphate. The fi rst type focused on the unique organism, GFAJ-1, which was made available to the scientifi c community. One of these publications showed that its DNA was hydrolytically stable, contrary to the expectation with arsenate diester linkages, and mass spectral data indicated the absence of arsenic in the DNA [83 ]. Another showed only small amounts of arsenic in various phosphorus metabolites and provided elemental analysis indicating the absence of arsenic in the GFAJ-1 DNA [84 ]. In addition, the genome of this organism was mapped and no unusual operons or genetic properties were found [85 ]. Two recent studies suggest alternate explanations for results in the original report. The fi rst provides evidence for arsenic destruction of bacterial ribosomes, which would liberate enough phosphorus for a small population of As-resistant organisms to grow slowly after a lag period [86 ], as reported for the growth of GFAJ-1. This toxic effect of As had not been reported previously, and may relate to arsenic effects on known metabolic factors (e.g., starvation) that lead to ribosome destruction [87 ]. The second addressed the ability of GFAJ-1 to grow, albeit slowly, under such high As:P ratios, and focused on the induction and activity of its periplasmic PBP’s, fi nding one from GFAJ-1 with a 10-fold higher phosphate-arsenate discrimination than PBP’s from other organisms [88 ]. In addition, this study reported the high-resolution phosphate- and arsenate-bound structures of the Pseudomonas 15 Arsenic. Can This Toxic Metalloid Sustain Life? 489

Figure 4 High resolution a O X-ray crystal structures of δ1 phosphate (top) and arsenate D62 (bottom) bound to the P. C ﬂ uorescens PBP, indicating γ their hydrogen bonding 122.0° interaction with Asp-62 2.51 Å of the protein. Reprinted Oδ2 with permission from [88 ]; O2 copyright 2012 Nature 179.1° Publishing Group. 108.7° P O1 O3 O4

b Oδ1 D62

Cγ

127° 162° Oδ2 O2 2.50 Å 95.4°

O1 O3 O4

fl uorescens PBP, which identifi ed a unique low-energy hydrogen bond that appears to provide the selectivity for phosphate (Figure 4). However, the molecular origin of the unusually high phosphate selectivity of the GFAJ-1 PBP has yet to be determined. While little support remains for the original claim that GFAJ-1 substitutes arsenate for phosphate in its DNA, this report motivated several efforts to examine this possibility, which may be relevant to alternate biochemistry of extra-terrestrial organisms. In particular, computational studies have examined the consequences of a P → As swap in DNA. While the structural differences are modest [ 89 – 91 ], the arsenate diester bonds are weaker [92 ] and their hydrolysis would be signifi cantly faster than that of native DNA [93 ], as predicted [77 ] based on data for small arsenate complexes [94 ]. DNA, with an estimated hydrolytic half-life of 30 million years [95 ], and the information encoded therein would not be sustainable for life forms that use arsenic instead of phosphorus in an aqueous environment. Thus, certain microorganisms can not only survive exposure to high levels of arsenic, but some take advantage of the unique reduction potential of this generally toxic element and can use abundant arsenite as an electron donor (chemolithoautotrophs) or arsenate as a dissimilatory electron acceptor under anaerobic conditions for their electron transport pathway. To survive exposure to this toxic element, organisms 490 Wilcox employ one or more mechanisms, including a higher phosphate selectivity in their phosphate import pathway, more effi cient or active arsenic export pathways, sequestration of arsenic in specialized organelles, and increased repair of damage to DNA and other biomolecules caused by arsenic.

3.3.2 Humans

Despite the well-known toxicity of arsenic, there are instances, mostly from the 18th and 19th Centuries, of arsenic being used therapeutically as a general tonic, in some cases at levels higher than believed to be fatal. The best example of these so- called ‘arsenic eaters’ is the inhabitants of Styria, now part of Austria. Their story was reported in the scientifi c literature at the time, formed the basis for a 1939 Ph.D. thesis, and has been summarized recently [96 ]. Certain individuals from regions in the Alps began eating arsenic in the belief that it increased the ability to breathe easily, improved the complexion, aided digestion, and protected against infectious disease. These individuals began by consuming small amounts of arsenic oxide (As2 O3 ) and gradually increased the amount consumed, such that they were eventually consuming doses that would otherwise be considered fatal. Stories of these arsenic eaters were greeted with skepticism at the time, and it was claimed that the consumed substances contained only minor amounts of arsenic or the arsenic solids were poorly adsorbed during digestion. However, a relatively compelling case has been made that there was likely some truth about the arsenic eaters of Styria. Indeed it has recently been suggested that arsenic exposure might lead to epigenetic changes that favor increased tolerance to altitude sickness [97 ]. Survival of supra-toxic doses of arsenic suggests a conditioned enhancement of detoxifi cation mechanisms, possibly overexpression of the arsenic methylation enzyme, As3MT, and/or MRP that exports arsenic-glutathione conjugates. A contemporary example of deliberate arsenic consumption is associated with the Chinese Dragon Boat Festival [98 ]. Part of the festivities involves consumption of wine with high levels of realgar (AsS) and face-painting with realgar-based paints. While this arsenic sulfi de is particularly insoluble and, therefore, presumably less bioavailable, elevated arsenic levels in the urine of children after face painting and adults after drinking the realgar wine are reported. These elevated urinary levels were reported to persist for quite some time, suggesting that at least some of the arsenic is bioavailable and absorbed dermally.

4 Beneﬁ cial Uses

4.1 Pesticides

Arsenic compounds have a long history of use as pesticides in the US and indeed worldwide. In the early 20th Century lead arsenate and calcium arsenate were commonly used in agriculture, most notably against insects in apple orchards [ 99 ]. Use of these pesticides decreased after the introduction of DDT but they were not 15 Arsenic. Can This Toxic Metalloid Sustain Life? 491 offi cially banned in the US until the 1990’s. The use of these arsenical pesticides has created a problem of legacy arsenic, and lead, in old orchard soils, which has human health implications when this land is used for urban development [100 ]. These contaminated soils are also potential point sources for wider distribution of arsenic in the environment [101 , 102]. Copper chrome arsenate (CCA) is a wood preservative compound that has been used extensively to suppress fungal rot in exterior lumber for residential and non-residential use. CCA is no longer registered for residential use in the US but can still be used to treat other lumber products. Various organoarsenicals have been developed and used for their benefi cial properties. MMAs and DMAs were used extensively as herbicides in cotton production and on golf courses. In the environment these organoarsenic species are less toxic than inorganic arsenic, but the possibility for their microbial demethylation into inorganic species [12 ] has prompted environmental concerns. Consequently, these two organoarsenic compounds were not re-registered in the US, effectively banning them from future use. Various phenyl-arsenic compounds, such as roxarsone, 3-nitro-4-hydroxyphenylarsonic acid (Figure 1 ), have been used as feed additives in poultry and swine production, as they control the parasitic protozoan disease coccidiosis in poultry and increase growth, presumably by lowering enteric microfl ora levels and thereby increasing nutrient absorption. There has been a concern about the potential for arsenic contamination of edible chicken meat. However, the US Food and Drug Administration (FDA) has a requirement for the withdrawal of arsenic growth promoters from chicken feed fi ve days before slaughter, and regulations for the maximum arsenic content in edible muscle and liver. Because poultry manure contains high levels of unabsorbed roxarsone and is mostly applied back to agricultural land as a soil amendment, there is concern about the fate of this organoarsenic species, including its microbial degradation to inorganic arsenic [ 103], and localized arsenic loading of soil. In 2011 roxarsone was voluntarily withdrawn from use as a feed additive in an agreement between the US FDA and the poultry industry. Hence, the widespread use of arsenic in agriculture has now largely been discontin- ued, at least in the US. While none of these widespread benefi cial uses of arsenic have a specifi c relationship to human disease, they do indicate the anthropomorphic distribution of arsenic that contributes to its background level and, depending on the extent and duration of exposure, its potential for toxic disease-causing impact.

4.2 Pharmaceuticals

4.2.1 Antibiotics

The toxicity of arsenic was the basis for its earlier use in formulations found to be effective against various microbial-based diseases, beginning with Ehrlich’s development of Salvarsan® , arsphenamine (Figure 1 ), to treat syphilis [104 ]. Although largely replaced by therapeutic agents that target speciﬁ c microbial pathways and lack arsenic’s known toxicity, the organoarsenical melarsoprol (Figure 1 ) is still used to treat trypanosome infections in Africa. 492 Wilcox

4.2.2 Chemotherapeutics

Arsenic compounds have been a component of traditional Chinese medicines, including those for the treatment of cancer, and Fowler’s solution (1% potassium arsenite) was in use by the mid-18th Century to treat a number of diseases and conditions, including leukemia. Largely replaced by other chemotherapeutic agents in the mid-20th Century, recently arsenic was found to be remarkably effective against one type of leukemia, acute promyelocytic leukemia (APL), leading to high rates of remission and survival [105 ]. Arsenic is known to affect a number of cellular processes, many of which are associated with elevated ROS levels, that result in apoptosis [ 106]. However, the effi cacy of As 2 O3 in treating APL at relatively low doses suggests there is a specifi c molecular target. This form of leukemia arises from a chromosomal translocation that results in a fusion protein that is generally formed between the promyelocytic leukemia protein (PML) and the retinoic acid receptor α (RARα). It was shown that arsenic decreases the level of this oncogenic PML-RARα fusion protein by inducing its degradation, which begins with the attachment of ubiquitin or the small ubiquitin-like protein (SUMO) [107 ]. This targeting for degradation is associated with the PML protein, which contains three cysteine-rich Zn-binding sequences, one of which binds two Zn2+ in a structure known as a RING domain. Both in vitro and in vivo results [108 ] suggest that As3+ binding to this domain affects the SUMOylation and/or ubiquitination of the protein, which labels it for degradation, consistent with an X-ray crystal structure showing the interaction between a RING domain and a ubiquitin conjugase [ 109] (Figure 5). The level of arsenic required for this therapy is not overtly toxic, in contrast to levels that are required to affect the progression of other cancers by alternate pathways, including those that induce apoptosis of cancer cells. Recently it has been shown that arsenic oxide is also effective against chronic myeloid leukemia (CML) and that a target protein containing a RING domain appears to be involved [ 111 ]. A chromosome translocation associated with this cancer also leads to an oncogenic fusion protein, but here the putative RING-containing target protein is a ligase that can ubiquitinate the CML fusion protein and whose activity is altered by arsenite. Thus, for APL, and possibly CML, there appears to be a specifi c molecular target for arsenic that is based on the thiophilic nature of arsenite and its competition with Zn2+ .

5 Summary

It has been claimed that arsenic has killed more human beings than any other substance [112 ]. Whether or not this is true, the hallmark of this element is its toxicity, which is still a problem. Efforts to provide clean water for large populations in Bangladesh and nearby Indian provinces have exposed millions to elevated levels of arsenic and its toxic effects, including higher risk for several types of cancer, diabetes, and cardiovascular and neurological problems. This has created one of the largest, if not the largest, public health incident of modern times. 15 Arsenic. Can This Toxic Metalloid Sustain Life? 493

Figure 5 X-ray crystal structure of the ternary complex of the RING-containing ligase c-Cbl, the ubiquitin-conjugating UbcH7, and a peptide from ZAP-70 tyrosine kinase [ 109], which serves as a model for the interaction between the RING domain of PML and the conjugase protein Ubc9 [110 ]. Reprinted with permission from [109 ]; copyright 2000 Elsevier.

This review has considered the opposite side of arsenic and reviewed the literature for evidence of any beneﬁ cial roles of this element. Animal studies indicate that very low levels of arsenic may promote normal growth and development, and its absence is detrimental to both the offspring and the mother during reproduction. However, the levels of arsenic that may be required are below the levels to which humans are normally exposed in their drinking water and diet, and below the level that has been directly associated with disease. In fact, a study of a Danish population suggests that there may be a reduced risk for non-melanoma skin cancer from low background levels of arsenic in drinking water [113 ]. Toxic effects of this element are clearly correlated with exposure levels above this background, and cause a variety of diseases by mechanisms that are still not well understood but in some cases can be correlated with the unique chemical properties of this element. Can arsenic sustain life? Under certain unique situations, microorganisms can use arsenite or arsenate for their essential energy-generating pathway. However, the report of a microorganism, GFAJ-1, that can grow using arsenic instead of phosphorus has not been supported by subsequent studies. Thus, there is still no evidence that life under terrestrial conditions, including those with very high levels of arsenic, can be sustained by substituting arsenate for phosphate. Nevertheless, for humans suffering from APL, and possibly CML, arsenic can send their disease into remission, thereby extending and sustaining their life. 494 Wilcox

Abbreviations

APL acute promyelocytic leukemia AsB arsenobetaine AsC arsenocholine BFD black foot disease CCA copper chrome arsenate CML chronic myeloid leukemia DBD DNA-binding domain DDT 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane DMAs dimethyl arsenic (As3+ or As5+ ) FDA Food and Drug Administration GR glucocorticoid receptor GRE glucocorticoid response element LNT linear, no threshold MMAs monomethyl arsenic (As3+ or As5+ ) MRP multi-drug resistance protein NHE normal hydrogen electrode PBP phosphate binding protein Pit phosphate inorganic transport PML promyelocytic leukemia protein Pst phosphate speciﬁ c transport PTDI provisional tolerable daily intake RARα retinoic acid receptor α RING really interesting new gene ROS reactive oxygen species SUMO small ubiquitin-like protein TPN total parenteral nutrition UNICEF United Nations Children’s Fund

Acknowledgments I thank Brian Jackson for his contributions to the sections on chronic toxicity ( 2.2 ), deliberate human exposure (3.3.2 ), and pesticides (4.1 ). I am grateful for previous support from the Dartmouth Superfund Research Program, which is supported by the NIH (P42 ES07373). This contribution is dedicated to the late Paul Saltman.

References

1. F. Wolfe-Simon, J. S. Blum, T. R. Kulp, G. W. Gordon, S. E. Hoeft, J. Pett-Ridge, J. F. Stolz, S. M. Webb, P. K. Weber, P. C. W. Davies, A. D. Anbar, R. S. Oremland, Science 2011 , 332 , 1163–1166. 2. W. R. Cullen, K. J. Reimer, Chem. Rev. 1989 , 89 , 713–764. 3. K. J. Reimer, I. Koch, W. R. Cullen, in Organometallics in Environment and Toxicology, Vol. 7 of Metal Ions in Life Sciences, Eds A. Sigel, H. Sigel, R. K. O. Sigel, Royal Society of Chemistry, Cambridge, 2010, pp. 165–229. 15 Arsenic. Can This Toxic Metalloid Sustain Life? 495

4. G. R. Willsky, M. H. Malamy, J. Bacteriol. 1980 , 144 , 356–365. 5. G. R. Willsky, M. H. Malamy, J. Bacteriol. 1980 , 144 , 366–374. 6. N. N. Rao, A. Torriani, Mol. Microbiol. 1990 , 4 , 1083–1090. 7. Z. J. Liu, J. Shen, J. M. Carbrey, R. Mukhopadhyay, P. Agre, B. P. Rosen, Proc. Natl. Acad. Sci. USA 2002 , 99 , 6053–6058. 8. H. Fu, X. Jiang, B. P. Rosen, in Biological Chemistry of Arsenic, Antimony and Bismuth , Ed H. Sun, Wiley, Hoboken, 2011, pp. 181–207. 9. F. Challenger, Chem. Rev. 1945 , 36 , 315–361. 10. S. Lin, Q. Shi, F. B. Nix, M. Stybo, M. A. Beck, K. M. Herbin-Davis, L. L. Hall, J. B. Simeonsson, D. J. Thomas, J. Biol. Chem. 2002 , 277 , 10795–10803. 11. E. M. Leslie, A. Haimeur, M. P. Waalkes, J. Biol. Chem. 2004 , 279 , 32700–32708. 12. T. Maki, N. Takeda, H. Hasegawa, K. Ueda, Appl. Organomet. Chem. 2006 , 20 , 538–544. 13. W. J. Fitz, W. W. Wenzel in Managing Arsenic in the Environment , Eds R. Naidu, E. Smith, G. Owens, P. Bhattacharya, P. Nadebaum, CSIRO, Collingwood, 2006, pp. 209–222. 14. K. A. Francesconi, D. Kuehnelt, in Environmental Chemistry of Arsenic , Ed W. T. Frankenberger, Jr., Dekker, New York, 2002, pp. 51–94. 15. H. V. Aposhian, E. S. Gurzau, X. C. Le, A. Gurzau, S. M. Healy, X. F. Lu, M. S. Ma, L. Yip, R. A. Zakharyan, R. M. Maiorino, R. C. Dart, M. G. Tircus, D. Gonzalez-Ramirez, D. L. Morgan, D. Avram, M. M. Aposhian, Chem. Res. Toxicol. 2000 , 13 , 693–697. 16. Z. Drobna, W. B. Xing, D. J. Thomas, M. Stybo, Chem. Res. Toxicol. 2006 , 19 , 894–898. 17. J. M. Carbrey, L. Song, Y. Zhao, M. Yoshinaga, A. Rojek, Y. Wang, Y. Liu, H. L. Lujan, S. E. DiCarlo, S. Nielsen, B. P. Rosen, P. Agre, R. Mukhopadhyay, Proc. Natl. Acad. Sci. USA 2009 , 106 , 15956–15960. 18. S. A. Moore, D. M. Moennich, M. J. Gresser, J. Biol. Chem. 1983 , 258 , 6266–6271. 19. Y. Xu, B. Ma, R. Nussinov, J. Phys. Chem. B 2012 , 116 , 4801–4811. 20. J. S. Pedrick, B. Jagadish, E. A. Marsh, H. V. Aposhian, Chem. Res. Toxicol. 2001, 14, 651–656. 21. T. Samikkannu, C.-H. Chen, L.-H. Yih, A. S. Wang, S.-Y. Lin, T.-C. Chen, K.-Y. Jan, Chem. Res. Toxicol. 2003 , 16 , 409–414. 22. M. F. Hughes, Toxicol. Lett. 2002 , 133 , 1–16. 23. World Health Organization, Evaluation of Certain Food Additives and Contaminants. Thirty- third Report of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1989, WHO Technical Report Series, No. 776. 24. D. K. Nordstrom, Science 2002 , 296 , 2143–2145. 25. A. H. Smith, P. A. Lopipero , M. N. Bates, C. M. Steinmaus, Science 2002, 296 , 2145–2146. 26. C. H. Tseng, C. K. Chong, C. J. Chen, T. Y. Tai. Atherosclerosis 1996 , 120 , 125–133. 27. H. Y. Chiou, S. T. Chiou, Y. H. Hsu, Y. L. Chou, C. H. Tseng, M. L. Wei, C. J. Chen, Am. J. Epidemiol. 2001 , 153 , 411–418. 28. C. H. Tseng, T. Y. Tai, C. K. Chong, C. P. Tseng, M. S. Lai, B. J. Lin, H. Y. Chiou, Y. M. Hsueh, K. H. Hsu, C. J. Chen, Environ. Health Perspect. 2000 , 108 , 847–851. 29. C. H. Tseng, C. K. Chong, C. P. Tseng, Y. M. Hsueh, H. Y. Chiou, C. C. Tseng, C. J. Chen, Toxicol. Lett. 2003 , 137 , 15–21. 30. Y. K. Huang, C. H. Tseng, Y. L. Huang, M. H. Yang, C. J. Chen, Y. M. Hsueh, Toxicol. Appl. Pharmacol. 2007 , 218 , 135–142. 31. M. Bates, O. Rey, M. L. Biggs, C. Hopenhayn, L. F. Moore, D. Kalman, C. Steinmaus, A. H. Smith, Epidemiology 2003 , 14 , S123. 32. C. Steinmaus, Y. Yuan, D. Kalman, O. A. Rey, C. F. Skibola, D. Dauphine, A. Basu, K. E. Porter, A. Hubbard, M. N. Bates, M. T. Smith, A. H. Smith, Toxicol. Appl. Pharmacol . 2010 , 247 , 138–145. 33. A. H. Smith, M. Goycolea, R. Haque, M. L. Biggs, Am. J. Epidemiol . 1998 , 147 , 660–669. 34. C. Ferreccio, C. Gonzalez, V. Milosavjlevic, G. Marshall, A. M. Sancha, A. H. Smith, Epidemiology 2000 , 11 , 673–679. 35. M. Argos, T. Kalva, P. J. Rathouz, Y. Chen, B. Pierce, F. Parvez, T. Islam, A. Ahmed, M. Rakibuz-Zaman, R. Hasan, G. Sarwar, V. Slavkovich, A. van Green, J. Graziano, H. Ahsan, Lancet 2010 , 376 , 252–258. 496 Wilcox

36. A. H. Smith, G. Marshall, J. Liaw, Y. Yuan, C. Ferreccio, C. Steinmaus, Environ. Health Perspect. 2012 , 120 , 1527–1531. 37. R. T. Nickson, J. M. McArthur, W. G. Burgess, K. M. Ahmed, P. Ravenscroft, M. Rahman, Nature 1998 , 395 , 338. 38. R. T. Nickson, J. M. McArthur, P. Ravenscroft, W. G. Burgess, K. M. Ahmed, Appl. Geochem. 2000 , 15 , 403–413. 39. A. H. Smith, E. O. Lingas, M. Rahman, Bull. World Health Org. 2000 , 78 , 1093–1103. 40. M. F. Naujokas, B. Anderson, H. Ahsan, H. V. Aposhian, J. H. Graziano, C. Thompson, W. A. Suk, Environ. Health Perspect. 2013 , 121 , 295–302. 41. L. M. Del Razo, B. Quintanilla-Vega, E. Brambila-Colombres, E. S. Calderon-Aranda, M. Manno, A. Albores, Toxicol. Appl. Pharmacol. 2001 , 177 , 132–148. 42. K. T. Kitchen, R. Conolly, Chem. Res. Toxicol. 2010 , 23 , 327–335. 43. J. E. Bodwell, L. A. Kingsley, J. W. Hamilton, Chem. Res. Toxicol. 2004 , 17 , 1064–1076. 44. H. Baumann, K. Paulsen, H. Kovacs, H. Berglund, A. P. H. Wright, J.-A. Gustafsson, T. Hard, Biochemistry 1993 , 32, 13463–13471. 45. A. M. Spuches, D. E. Wilcox, J. Am. Chem. Soc. 2008 , 130 , 8148–8149. 46. A. M. Spuches, H. G. Kruszyna, A. M. Rich, D. E. Wilcox, Inorg. Chem. 2005 , 44 , 2964–2972. 47. F. D. Barr, L. J. Krohmer, J. W. Hamilton, L. A. Sheldon, PLoS One 2009 , 4(8): e6766. doi: 10.1371/journal.pone.0006766 . 48. J. E. Bodwell, J. A. Goss, A. P. Nomikos, J. W. Hamilton, Chem. Res. Toxicol. 2006 , 19 , 1619–1629. 49. K. Piatek, T. Schwerdtle, A. Hartwig, W. Bal, Chem. Res. Toxicol. 2008 , 21 , 600–606. 50. National Research Council, Arsenic in Drinking Water 2001 Update , National Academy, Washington, 2001. 51. S. H. Lamm, A. Engel, C. A. Penn, R. Chen, M. Feinleib, Environ. Health Perspect. 2006 , 114 , 1077–1082. 52. E. T. Snow, P. Sykora, T. R. Durham, C. B. Klein, Toxicol. Appl. Pharmacol. 2005 , 207 , S557–S564. 53. M. Anke in Trace Elements in Human and Animal Nutrition, Vol 2, Ed W. Mertz, Academic, Orlando, 1986, pp. 347–372. 54. E. O. Uthus, Environ. Geochem. Health 1992 , 14 , 55–58. 55. S. S.-H. Tao, P. M. Bolger, Food Add. Contam. 1998 , 16 , 465–472. 56. F. H. Nielsen, J. Trace Elem. Exp. Med. 2000 , 13 , 113–129. 57. M. Lu, H. Wang, X.-F. Li, L. L. Arnold, S. M. Cohen, X. C. Le, Chem. Res. Toxicol. 2007 , 20 , 27–37. 58. M. Vahter, Sci. Prog. 1999 , 82 , 69–88. 59. K. N. Jeejeebhoy, R. C. Chu, E. B. Marliss, G. R. Greenberg, A. Bruce-Robertson, Am. J. Clin. Nutr. 1977 , 30 , 531–538. 60. M. M. Pluhator-Murton, R. N. Fedorak, R. J. Audette, B. J. Marriage, R. Y. Yatscoff, L. M. Gramlich, J. Parenteral Enteral Nutr. 1999 , 23 , 222–227. 61. W. E. Cornatzer, E. O. Uthus, J. A. Haning, F. H. Nielsen, Nutr. Reports Intl. 1983 , 27 , 821–829. 62. E. J. Calabrese, L. A. Baldwin, Crit. Rev. Toxicol. 2003 , 33 , 215–304. 63. A. A. Carbonell, M. A. Aarabi, R. D. DeLaune, R. P. Gambrell, W. H. Patrick, Jr., Sci. Total Environ. 1998 , 217 , 189–199. 64. N. Singh, L. Q. Ma, Environ. Poll. 2006 , 141 , 238–246. 65. Y. Gong, H.-N. Chou, C.-d. Tu, X. Liu, J. Liu, L. Song, J. Appl. Phycol. 2009 , 21 , 225–231. 66. M. S. Cox, P. E. Bell, J. Plant Nutr. 1996 , 19 , 1599–1610. 67. Z. Meng, N. Meng, Biol. Trace Elem. Res. 1994 , 42 , 201–208. 68. T.-C. Lee, M. Oshimura, J. C. Barrett, Cacrinogenesis 1985 , 6 , 1421–1426. 69. P. Sykora, E. T. Snow, Toxicol. Appl. Pharmacol. 2008 , 228 , 385–394. 70. A. Stoica, E. Pentecost, M. B. Martin, Endocrinology 2000 , 141 , 3595–3602. 71. W. P. Norwood, U. Borgmann, D. G. Dixon, Environ. Poll. 2007 , 147 , 262–272. 15 Arsenic. Can This Toxic Metalloid Sustain Life? 497

72. J. L. Brown, K. T. Kitchen, Cancer Lett. 1996 , 98 , 227–231. 73. P. Mushak, Environ. Health Perspect. 2007 , 115 , 500–506. 74. J. M. Santini, L. I. Sly, R. D. Schnagl, J. M. Macy, Appl. Environ. Microbiol. 2000 , 66 , 92–97. 75. D. Ahmann, A. L. Roberts, L. R. Krumholz, F. M. Morel, Nature 1994 , 371 , 750. 76. R. S. Oremland, J. F. Stolz, Science 2003 , 300 , 939–944. 77. F. H. Westheimer, Science 1987 , 235 , 1173–1178. 78. Science 2011 332 , 1149a-j. 79. M. I. Fekry, P. A. Tipton, K. S. Gates, ACS Chem. Biol. 2011 , 6 , 127–130. 80. D. S. Tawffi k, R. E. Viola, Biochemistry 2011 , 50 , 1128–1134. 81. S. Silver, L. T. Phung, FEMS Microbiol. Lett. 2011 , 315 , 79–80. 82. B. P. Rosen, A. A. Ajees, T. R. McDermott, Bioessays 2011 , 33 , 350–357. 83. M. L. Reaves, S. Sinha, J. D. Rabinowitz, L. Kruglyak, R. J. Redfi eld, Science 2012 , 337 , 470–473. 84. T. J. Erb, P. Kiefer, B. Hattendorf, D. Gunther, J. A. Vorholt, Science 2012 , 337 , 467–470. 85. L. T. Phung, S. Silver, W. L. Trimble, J. A. Gilbert, J. Bacteriol. 2012 , 194 , 1835–1836. 86. G. N. Basturea, T. K. Harris, M. P. Deutscher, J. Biol. Chem. 2012 , 287 , 28816–28819. 87. M. A. Zundel, G. N. Basturea, M. P. Deutscher, RNA 2009 , 15 , 977–983. 88. M. Elias, A. Wellner, K. Goldin-Azulay, E. Chabriere, J. A. Vorholt, T. J. Erb, D. S. Tawfi k, Nature 2012 , 491 , 134–137. 89. E. J. Denning, A. D. MacKerell, Jr, J. Am. Chem. Soc. 2011 , 133 , 5770–5772. 90. A. Mladek, J. Sponer, B. G. Sumpter, M. Fuentes-Cabrera, J. E. Sponer, J. Phys. Chem. Lett. 2011 , 2 , 389–392. 91. J. Gu, J. Wang, Y. Xie, J. Leszczynski, H. F. Schaefer, III, J. Comp. Chem. 2012 , 33 , 817–821. 92. M. Toulouze, J. Pilme, F. Pauzat, Y. Ellinger, Phys. Chem. Chem. Phys. 2012 , 14 , 10515–10522. 93. J. Sponer, A. Mladek, J. E. Sponer, D. Svozil, M. Zgarbova, P. Panas, P. Jurecka, M. Otyepka, Phys. Chem. Chem. Phys. 2012 , 14 , 15257–15277. 94. C. D. Baer, J. O. Edwards, P. H. Rieger, Inorg. Chem. 1981 , 20 , 905–907. 95. G. K. Schroeder, C. Lad, P. Wyman, N. H. Williams, R. Wolfenden, Proc. Natl. Acad. Sci. USA 2006 , 103 , 4052–4055. 96. G. Przygoda, J. Feldmann, W. R. Cullen, Appl. Organomet. Chem . 2001 , 15 , 457–462. 97. L. Tremolizzo, M. A. Riva, C. Ferrarese, Med. Hypothesis 2010 , 75 , 677. 98. Y.-N. Zhang, G.-X. Sun, Q. Huang, P. N. Williams, Y.-G. Zhu, Environ. Intl. 2011 , 37 , 889–892. 99. E. A. Murphy, M. Aucott, Sci. Total Environ. 1998 , 218 , 89–101. 100. L. L. Embrick, K. M. Porter, A. Pendergrass, D. J. Butcher, Microchem. J. 2005 , 81 , 117–121. 101. D. C. Elfving, K. R. Wilson, J. G. Ebel, K. L. Manzell, W. H. Gutenmann, D. J. Lisk, Chemosphere 1994 , 29 , 407–413. 102. C. E. Renshaw, B. C. Bostick, X. Feng, C. K. Wong, E. S. Winston, R. Karimi, C. L. Folt, C. Y. Chen, J. Environ. Qual. 2006 , 35 , 61–67. 103. B. P. Jackson, J. C. Seaman, P. M. Bertsch, Chemosphere 2006 , 65 , 2028–2034. 104. S. Gibaud, G. Jaouen, Top. Organomet. Chem. 2010 , 32 , 1–20. 105. Z. X. Shen, G. Q. Chen, J. H. Ni, X. S. Li, S. M. Xiong, Q. Y. Qui, J. Zhu, W. Tang, G. L. Sun, K. Q. Yang, Y. Chen, L. Zhou, Z. W. Fang, Y. T. Wang, J. Ma, P. Zhang, T. D. Zhang, S. J. Chen, Z. Chen, Z. Y. Wang, Blood 1997 , 89 , 3354–3360. 106. L. C. Platanias, J. Biol. Chem. 2009 , 284 , 18583–18587. 107. J. Zhu, M. H. M. Koken, F. Quignon, M. K. Chelbi-Alix, L. Degos, Z. Y. Wang, Z. Chen, H. de The, Proc. Natl. Acad. Sci. USA 1997 , 94 , 3978–3983. 108. X.-W. Zhang, X.-J. Yan, Z.-R. Shou, F.-F. Yang, Z.-Y. Wu, H.-B. Sun, W.-X. Liang, A.-X. Song, V. Lallemand-Breitengach, M. Jeanne, Q.-Y. Zhang, H.-Y. Yang, Q.-H. Huang, G.-B. Zhou, J.-H. Tong, Y. Zhang, J.-H. Wu, H.-Y. Hu, H. de The, S.-J. Chen, Z. Chen, Science 2010 , 328 , 240–243. 498 Wilcox

109. N. Zhang, P. Wang, P. D. Jeffrey, N. P. Pavletich, Cell 2000 , 102 , 533–539. 110. S.-J. Chen, G.-B. Zhou, X.-W. Zhang, J.-H. Mao, H. de The, Z. Chen, Blood 2011 , 117 , 6425–6437. 111. J.-H. Mao, X.-Y. Sun, J.-X. Liu, Q.-Y. Zhang, P. Liu, Q.-H. Huang, K. K. Li, Q. Chen, Z. Chen, S.-J. Chen, Proc. Natl. Acad. Sci. USA 2010 , 107 , 21683–21688. 112. J. O. Nriagu, in Environmental Chemistry of Arsenic , Ed W. T. Frankenberger, Jr., Dekker, New York, 2002, pp. 1–26. 113. R. Baastrup, M. Sorensen, T. Balstrom, K. Frederiksen, C. L. Larsen, A. Tjonneland, K. Overvad, O. Raaschou-Nielsen, Environ. Health Perspect. 2008 , 116 , 231–237. 114. World Health Organization, Summary and Conclusions of the 72nd Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 2010. Chapter 16 Selenium. Role of the Essential Metalloid in Health

Suguru Kurokawa and Marla J. Berry

Contents ABSTRACT...... 500 1 Introduction...... 501 1.1 History of Selenium...... 501 1.2 Identification of Selenium as an Essential Micronutrient...... 501 2 Selenium in Biomolecules...... 502 2.1 Selenocysteine, the 21st Amino Acid in Proteins...... 502 2.2 Selenocysteine tRNA and Biosynthesis of Selenocysteine...... 503 2.3 Selenoprotein mRNAs...... 506 2.4 Selenocysteine Incorporation into Proteins...... 507 3 Function of Selenoproteins...... 509 3.1 Human Selenoproteins...... 509 3.1.1 Glutathione Peroxidases (Gpx1, Gpx2, Gpx3, and Gpx6)...... 509 3.1.2 Thyroid Hormone Deiodinases (DI1, DI2, and DI3)...... 511 3.1.3 Thioredoxin Reductases (TR1, TR2, and TR3)...... 512 3.1.4 Methionine-R-Sulfoxide Reductase (MsrB1)...... 513 3.1.5 15kDa Selenoprotein (Sep15)...... 513 3.1.6 Selenophosphate Synthetase 2 (SPS2)...... 513 3.1.7 Selenoprotein P (Sepp1)...... 514 3.1.8 Selenoprotein W (SelW)...... 514 3.1.9 Selenoprotein V (SelV)...... 514 3.1.10 Selenoprotein T (SelT)...... 515 3.1.11 Selenoprotein M (SelM)...... 515 3.1.12 Selenoprotein H (SelH)...... 515

S. Kurokawa • M.J. Berry (*) Department of Cell & Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA e-mail: [email protected]; [email protected]

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 499 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8_16, © Springer Science+Business Media Dordrecht 2013 500 Kurokawa and Berry

3.1.13 Selenoprotein O and I (SelO and SelI)...... 515 3.1.14 Selenoprotein S (SelS)...... 515 3.1.15 Selenoprotein K (SelK)...... 516 3.1.16 Selenoprotein N (SelN)...... 516 4 Selenium and Disease...... 516 4.1 Overview of Selenium-Related Diseases...... 516 4.1.1 Selenium Deficiency...... 517 4.1.2 Selenium Toxicity (Selenosis)...... 517 4.2 Selenium in Brain Function...... 518 4.3 Selenium in Diabetics...... 518 4.4 Selenium in Reproduction...... 519 5 Health Benefits of Selenium in Humans...... 520 5.1 Molecular Forms of Selenium in Diet...... 520 5.2 Selenium Transport in Mammals...... 521 5.2.1 Tissue-Oriented Selenium Transport...... 521 5.2.2 Selenium Excretion...... 523 5.3 Human Dietary Standards for Selenium...... 524 6 General Conclusions...... 525 Abbreviations and Definitions...... 525 Acknowledgment...... 527 References...... 527

Abstract Selenium is an essential micronutrient in mammals, but is also recognized as toxic in excess. It is a non-metal with properties that are intermediate between the chalcogen elements sulfur and tellurium. Selenium exerts its biological functions through selenoproteins. Selenoproteins contain selenium in the form of the 21st amino acid, selenocysteine (Sec), which is an analog of cysteine with the sulfur- containing side chain replaced by a Se-containing side chain. Sec is encoded by the codon UGA, which is one of three termination codons for mRNA translation in non-selenoprotein genes. Recognition of the UGA codon as a Sec insertion site instead of stop requires a Sec insertion sequence (SECIS) element in selenoprotein mRNAs and a unique selenocysteyl-tRNA, both of which are recognized by specialized protein factors. Unlike the 20 standard amino acids, Sec is biosynthesized from serine on its tRNA. Twenty-five selenoproteins are encoded in the human genome. Most of the selenoprotein genes were discovered by bioinformatics approaches, searching for SECIS elements downstream of in-frame UGA codons. Sec has been described as having stronger nucleophilic and electrophilic properties than cysteine, and Sec is present in the catalytic site of all selenoenzymes. Most selenoproteins, whose functions are known, are involved in redox systems and signaling pathways. However, several selenoproteins are not well characterized in terms of their function. The selenium field has grown dramatically in the last few decades, and research on selenium biology is providing extensive new information regarding its importance for human health.

Keywords essential nutrient • selenium • selenocysteine • selenoprotein

Please cite as: Met. Ions Life Sci. 13 (2013) 499–534 16 Selenium. Role of the Essential Metalloid in Health 501

1 Introduction

1.1 History of Selenium

Selenium is a non-metal element, but sometimes considered as a metalloid, with the symbol Se and atomic number 34. It was first described in 1818 by the Swedish chemist Jöns Jacob Berzelius (1779–1848). He named this element selenium (Greek σελήνη selene meaning Moon) after the Greek moon goddess Selene (reviewed in [1]). Berzelius was investigating the cause of illness among workers at a sulfuric acid manufacturing plant when he found the element in the bottom sludge of a sulfuric acid preparation. He reported that selenium had similarities with the previously known element tellurium (named for the Earth). He also reported close similarities between selenium and sulfur. At that time, selenium was thought to be a toxic element.

1.2 Identification of Selenium as an Essential Micronutrient

Most of the early research on selenium was done with the goal of addressing selenium toxicity. In the 1930s, selenium was found to cause poisoning of livestock in areas with high selenium content in the soil. In the mid 20th century, selenium was recognized as a micronutrient and its biological function was studied with regard to its importance in human nutrition. In 1957, Klaus Schwarz, a German scientist working at the National Institutes of Health in Bethesda first reported on the health benefits of selenium. Schwarz had studied yeast as a protein source in Germany during World War II and continued the study in the United States, eventually discov- ering that feeding torula yeast instead of brewer’s yeast as a protein source to vitamin E-deficient rats led to necrotic liver formation. Schwarz and Foltz announced that the selenium contained in the fractionated brewer’s yeast was responsible for preventing liver necrosis [2]. Selenium deficiency was also recognized in studies in Oregon [3], which showed a myopathy known as ‘white muscle disease’ in calves and lambs to be associated with selenium-depleted soil. Selenium supplementation to livestock has subsequently had great economic impacts in several countries, including New Zealand and Finland. In 1979 in China, a congestive cardiac myopathy termed Keshan disease was the first reported human disease associated with selenium deficiency4 [ ]. Keshan County in northeastern China, for which the disease was named, is a predominantly rural region where the diet consisted almost entirely of food produced locally on selenium-deficient land. The disease was also reported inN ew Zealand and Finland where the level of selenium in the soil is low [5]. Further details are discussed in Section 4.1. In the 1970s selenium was found to be present in glutathione peroxidase as the amino acid selenocysteine (Sec) [6], and the focus on selenium studies shifted to the field of molecular biology. As a micronutrient, a recommended dietary allowance 502 Kurokawa and Berry

(RDA) for selenium was established in 1989 (70 μg/d for men and 55 μg/d for women) [7] and revised in 2000 (55 μg/d) [8]. In 1996, dietary recommendations from the World Health Organization (WHO) were issued (34 μg/d for men and 26 μg/d for women) [9].

2 Selenium in Biomolecules

2.1 Selenocysteine, the 21st Amino Acid in Proteins

Selenoproteins are defined as proteins containing the 21st amino acid, Sec 10[ ]. The discovery of selenoproteins occurred in 1973, when Hoekstra and coworkers at the University of Wisconsin identified the presence of selenium in glutathione peroxidase as the first animal selenoprotein 6[ ]. Research then focused on the catalytic role of the amino acid in the active site of selenoproteins. In 1976, Thressa Stadtman et al. identified glycine reductase as a selenoprotein [11] and in the 1980s, Böck and colleagues identified additional selenoproteins in bacteria 12[ ]. The application of bioinformatics and SECIS specific algorithms allowed for identification of selenoprotein genes from the expressed sequence tags (ESTs) database [13,14]. All of the 25 selenoprotein genes in humans were thus identified in 2003 15[ ]. Sec (Figure 1, left) contains a selenol group which is highly reactive at physiological pH. The reactivity of the thiol group (pKa 8.3) of cysteine is modulated by microenvironmental conditions, and when deprotonated, is nucleophilic and easily oxidized. Since the selenol group of Sec has a lower pKa (5.47), it is fully deprotonated. Due to the higher reduction potential of Sec, it is more efficient in participating in redox reactions, and is specifically used as a catalytic amino acid in most selenoproteins. The other selenium-containing amino acid is selenomethionine (Figure 1, right). Like methionine, selenomethionine is not synthesized de novo in humans, but is supplied from plants. Selenium is misincorporated at random in place of sulfur in methionine biosynthesis, followed by the ribosome failing to distinguish between

Figure 1 Selenium-containing amino acids in mammals. Selenocysteine is found in selenoproteins. At the physiologic pH, its selenol is mostly deprotonated. Sec can be synthesized by mammals. Selenomethionine is classified as non-polar amino acid. Like the essential amino acid methionine, selenomethionine is also not synthesized de novo in mammals. Selenomethionine appears to be distributed nonspecifically in the proteins in place of methionine residues. 16 Selenium. Role of the Essential Metalloid in Health 503 selenomethionine- and methionine-loaded tRNA during translation [16]. Since its selenium is covalently bound between two carbon atoms, selenomethionine is considerably less reactive than Sec.

2.2 Selenocysteine tRNA and Biosynthesis of Selenocysteine

Sec is a naturally occurring amino acid in eukaryotes, archaea, and eubacteria. Sec is cotranslationally incorporated into selenoproteins by Sec tRNA decoding of UGA, which is normally a termination codon [10,17,18]. In 1970, a seryl-tRNA was identified that specifically decoded the stop codon,U GA [19]. In assessing whether nonsense suppressor tRNAs occurred in mammalian cells, the minor seryl-tRNA was identified as a possible candidate. Extensive characterization of this tRNA subsequently revealed it to be Sec tRNA[Ser] (reviewed in [20]). Unlike the other 20 amino acids, the biosynthesis of Sec occurs on its transfer RNA (tRNA[Ser]Sec) [21]. The biosynthesis of Sec was first characterized in bacteria [22] by Böck and coworkers in the late 1980s to early 1990s [23,24], and subsequently in mammalian cells [21]. In the first step, tRNA[Ser]Sec is aminoacylated with serine, providing the carbon backbone for Sec, thus the tRNA has been designated as tRNA[Ser]Sec [21,25]. The pathway for mammalian Sec biosynthesis has been elucidated more recently, and is discussed below. Sec tRNA[Ser]Sec has been isolated and sequenced from bovine liver [19,26,27], rat liver [28], mouse liver, and HeLa cells [29]. tRNA[Ser]Sec is the longest tRNA in mammals with a length of 90 nucleotides [26,27,30], and contains several modified bases. It was the first mammalian tRNA shown to contain mcm5U34 and mcm5Um34 [28] (Figure 2). Full expression of selenoproteins requires modification of tRNA[Ser]Sec [31]. Interestingly, two major isoforms of tRNA[Ser]Sec differ by a single methyl group at the wobble position (Um34) and synthesize different subclasses of selenoproteins [20]. The non-Um34 isoform supports the synthesis of a subclass of selenoproteins, designated housekeeping, while the Um34 isoform supports the expression of another subclass, designated stress-related selenoproteins [32]. The modification of 6i A37 is also required for stress-related selenoproteins [33]. Um34 methylation of tRNA[Ser]Sec requires aminoacylated tRNA[Ser]Sec, most likely with Sec [34]. Recently, it was shown that N6-isopentenylation of base A37 is catalyzed by Trit1, a dimethylallyl:tRNA[Ser]Sec-transferase [35]. In the first step of Sec biosynthesis, seryl-tRNA synthetase (SerRS) attaches serine to Sec tRNA in the presence of ATP and Mg2+ as follows [36–38]:

[]SerSec []SerSec tRNA +serine+ATPs←→ eryl-tRNA+AMP+PPi In 1970, a kinase that phosphorylated what was presumed to be a minor species of seryl-tRNA to form O-phosphoseryl-tRNA was reported. Because it recognized the stop codon UGA, it was initially thought to be a suppressor tRNA [19,39]. This seryl-tRNAUGA was eventually identified as Sec tRNA[Ser]Sec, and the kinase originally found by Mäenpää and Bernfield was shown to be O-phosphoseryl-tRNA[Ser]Sec 504 Kurokawa and Berry

Figure 2 Cloverleaf model of bovine liver Sec tRNA[Ser]Sec and its modifications. Sec tRNA[Ser]Sec sequences in mammals are 90 nucleotides long. The tRNA contains base modifications at position 34 (methyl carboxymethyl-5’-uridine; mcm5U), 37 (isopentenyladenosine; i6A), 55 (pseudouridine; Ψ) and 58 (N1-methyladenosine; m1A). The two isoforms differ from each other by a single methyl group on the position 34 (mcm5U or mcm5Um).

kinase (PSTK) [40]. PSTK was identified using a comparative genomics approach that searched completely sequenced genomes of archaea for a kinase-like protein that was present in those organisms (Methanococcus jannaschii and Methanopyrus kandleri) that utilized the selenoprotein synthesizing machinery and was absent in those that did not. Two kinase-like genes were identified in the two archaea that synthesize selenoproteins. However, they were not found in the other twelve archaea which lack that ability. Further comparison was carried out in eukaryotic genomes that synthesize selenoproteins (nematodes, Drosophila, and mice) and those that do not (yeasts) for homologous sequences to the two candidate genes. A single candidate was detected and the putative pstk was cloned from mouse genomic DNA [40]. The protein’s biochemical properties showed it to be phosphoseryl-tRNA kinase (PSTK). The reaction is as follows:

[]SerSec []SerSec seryl-tRNA +ATP ←→ O-phosphoseryl-tRNA+ADP 16 Selenium. Role of the Essential Metalloid in Health 505

Figure 3 Biosynthesis of Sec and de novo synthesis of cysteine. Sec is synthesized on tRNA[Ser]Sec by generation of selenophosphate from selenide and ATP (upper portion of the figure for the final steps in Sec biosynthesis). The de novo synthesis of cysteine on Sec tRNA[Ser]Sec occurs when sulfide replaces selenide (lower portion of the figure for the final steps in cysteine biosynthesis).

The selenium donor is monoselenophosphate, which is formed from selenite and ATP by selenophosphate synthetase (SPS). SPS has two homologs, SPS1 and SPS2 [41,42]. The gene product of SPS2 is a selenoprotein [42]. Biochemical analysis demonstrated that mouse SPS2 generates selenophosphate in the presence of selenide and ATP [43]. The reaction is as follows:

selenide+ATP → selenophosphate+AMP +Pi Sec synthetase (SecS) then mediates the generation of selenocysteyl-tRNA[Ser]Sec and inorganic phosphate. Hatfield’s group identified SecS using a computational and comparative genomic strategy, similar to that used to identify pstk, in searching for a SecS gene in eukaryotes [44]. The sequence of the purported mammalian SecS matched a 48 kDa soluble liver antigen (SLA) [45], which was previously reported as an antigen in patients with autoimmune chronic hepatitis and co-precipitated with Sec-tRNA[Ser]Sec in extracts from such patients. SecS binds tightly with phosphoseryl-tRNA[Ser]Sec[46]. The reaction is as follows (Figure 3):

O-phosphoseryl-tRNA+[]SerSec selenophosphate → selenocysteyl-ttRNA[]SerSec + PPi

Recently, it was shown that cysteine can be synthesized on the tRNA[Ser]Sec by replacing selenide with sulfide in the Sec biosynthetic pathway 47[ ] (de novo cysteine biosynthesis is discussed in Section 3.1.6). 506 Kurokawa and Berry

2.3 Selenoprotein mRNAs

The specific, cotranslational incorporation of Sec into eukaryotic proteins requires the presence of a SECIS element within the selenoprotein mRNA. The SECIS element was identified through bioinformatic and functional studies of the selenoprotein genes encoding cytosolic glutathione peroxidase (Gpx1) and type 1 deiodinase (DI1) [48,49]. Deletion analyses of the respective 3’UTRs resulted in production of truncated selenoproteins where the Sec UGA codon was recognized as a stop codon. Analysis of the DI1 and Gpx1 cDNAs from multiple species revealed the consensus SECIS element to be a conserved stem-loop structure containing three short, highly conserved nucleotide sequences [48]. Typical SECIS elements are depicted in Figure 4 and are based on an experimental secondary structure model [50]. Alignment of selenoprotein 3’-UTRs revealed the consensus sequences of AUGA, AAA, and UGAU as the SECIS core [48]. The core (quartet non-Watson-Crick base pairs; two shared tandem G•A, A•G motif) is a conserved feature of SECIS elements which introduces a kink- turn like structure [50]. This structure is recognized by SECIS binding protein 2 (SBP2) and plays a key role for SECIS function [51]. For example, mutation of the SECIS core of selenoprotein N (AUGA to ACGA) causes myopathy [52]. The secondary structure of the SECIS element shows conserved AA residues in an apical loop or internal bulge [53]. This motif is essential and is recognized by the RNA binding protein nucleolin [54] (SECIS binding proteins are further discussed in Section 2.4). The sequences flanking the SECIS element need to be open and non-base paired. The stem length of the SECIS is between 9 to 15 Watson-Crick base pairs, a distance which approximates a single turn of an A-form double helix. Interestingly, human selenoprotein P (Sepp1), which has 10 UGA Sec codons, has two distinct SECIS elements, both necessary for synthesis of the full-length protein. The 5’ element has an extended apical loop (SECIS1, form 2) and the 3’ element has the basic stem-loop structure (SECIS2, form 1) [55]. These Sepp1-SECISs have distinct functions, SECIS1 being more efficient than SECIS2 56[ ]. SECIS elements have been utilized to develop software-based search algorithms in an attempt to identify potential selenoprotein cDNAs in the GenBank [13]. The algorithms search databases for the primary sequence and secondary structure features of SECIS elements, calculating the free energy of the secondary structures. This strategy was initially used to identify several selenoproteins (SelT and SelR [13]) (SelX, SelN, and SelZ [14]) and the SECIS elements activity was validated by incorporation of Sec into Gpx1 protein [57]. A more sophisticated approach was subsequently developed, searching for SECIS elements, conservation of open reading frames downstream of UGA codons between species (which identified SelH, SelI, SelK, SelO, SelS, and SelV), and conservation between Sec and Cys-containing orthologs (Gpx6) [15]. These novel proteins indeed contain selenium, which was verified by subsequent radioactive 75Se labeling analysis. 16 Selenium. Role of the Essential Metalloid in Health 507

Figure 4 Diagram of two classes of eukaryotic SECIS elements. Secondary structure models of Form 1 and 2 SECIS. The critical structural features are labeled. The eukaryotic SECIS element is located in 3’ UTR. N, any nucleotide; A/G and A/C indicate that A is the prevalent base.

2.4 Selenocysteine Incorporation into Proteins

SECIS element and RNA binding protein interaction is required for decoding of UGA codons as Sec. SECIS binding protein-2 was identified by Copeland et al. as a 94 kDa protein that specifically recognizes the UA GA core of SECIS elements [51,58]. SBP2 works as a factor for the recoding of an in-frame UGA as Sec, activity uncovered by an in vitro translation system in rabbit reticulocyte lysate. SBP2 contains an RNA binding domain found in several ribosomal proteins and eukaryotic translation termination release factor 1. Sec incorporation requires the eukaryotic selenocysteyl-tRNA-specific elongation factor (eEFSec). eEFSec was identified using searches of the murine and human EST databases for homology to a putative archaeal alternative translational elongation factor SELB sequence [59,60]. eEFSec reveals a high degree of conservation in the amino-terminal elongation factor domain and purified recombinant eEFSec protein specifically binds to selenocysteyl-tRNA but not seryl-tRNA [59]. This discrimination 508 Kurokawa and Berry between correctly charged selenocysteyl-tRNA and seryl-tRNA reveals the mechanism for preventing misincorporation of serine at UGA codons instead of Sec. eEFSec has been shown to interact with SBP2 in an RNA-dependent manner [59]. Electrophoretic mobility shift assays (EMSA) with in vitro transcribed 32P-labeled SECIS elements and recombinant purified proteins showed SBP2 and eEFSec bind the SECIS element independently. In contrast, co-immunoprecipitation studies of the mutant SECIS elements with eEFSec and SBP2 revealed a lack of ability to bind the SECIS element. This result suggests eEFSec has the ability to bind SECIS elements and SBP2 enhances its binding specificity. L30 is a component of the large ribosomal subunit that binds to a specific sequence in 28S rRNA. It is a ubiquitously expressed abundant protein in mammalian tissues, and it has been demonstrated to overlap with SBP2 in binding to SECIS elements [61]. In vitro binding studies demonstrated that L30 binds to the SECIS element and forms a kinked conformer to facilitate SBP2 binding since SBP2 only binds to the kinked SECIS form. Eukaryotic initiation factor 4A-III (eIF4a3) has been identified as a selenium status sensitive RNA-binding protein [62]. eIF4a3 is ubiquitously expressed and belongs to the DEAD box family of RNA-dependent ATPases [63]. Nucleolin, a protein that facilitates ribosome biogenesis in the nucleolus, has been demonstrated to exhibit SECIS element binding [64]. This protein alters mRNA stability or translation in the cytoplasm of specific selenoprotein mRNAs. It binds the upper part of the basal stem of SECIS elements but the consensus sequence for nucleolin binding in SECIS elements has not been identified [54]. siRNA knockdown of nucleolin demonstrated synthesis of essential selenoproteins was reduced but without effect on non-essential selenoprotein synthesis [54]. It is postulated that nucleolin may bind to the essential selenoproteins’ SECIS element and facilitate SECIS element-protein interactions with SBP2 or other factors in the Sec incorporation pathway (Figure 5). Supramolecular protein-protein and protein-RNA interactions are implicated in both the biosynthesis of selenocysteyl-tRNA and the incorporation of Sec [65]. Co-immunoprecipitation studies described the following interactions that have been characterized to date. Protein:RNA interactions SecS-selenocysteyl-tRNA Secp43- selenocysteyl-tRNA eEFsec- selenocysteyl-tRNA SBP2-SECIS mRNA and rRNA L30- SECIS mRNA and rRNA Protein:protein interactions SBP2-eEFSec SecS-SPS1 SecS-Secp43 As several selenoprotein biosynthesis factors have both nuclear localization signals and nuclear export signals, this suggests that these factors may shuttle between 16 Selenium. Role of the Essential Metalloid in Health 509

Figure 5 A model of Sec biosynthesis and incorporation. Aminoacylation of tRNA[Ser]Sec and its conversion to Sec-tRNA[Ser]Sec is depicted along the top. Recruitment of transcription factors (SBP2, eEFSec) are depicted top right. Shuttling of the complex of Sec-tRNA[Ser]Sec into the nucleus and the association with eEFSec, SBP2, and SECIS elements are depicted along the right bottom. Cytoplasmic export and translation are shown along the left bottom. the cytoplasm and the nucleus. SPS1, Secp43, and eEFSec were observed in both the cytoplasmic and nuclear fractions [65]. In summary, the co-immunoprecipitation studies suggest that SPS1, SecS, Secp43, and selenocysteyl-tRNA may form a complex in the cytoplasm that subsequently enters the nucleus. SecS may then leave the complex, replaced by eEFSec and SBP2 and subsequent shuttling to the cytoplasm.

3 Function of Selenoproteins

Twenty-five selenoproteins have been identified in human genome databases15 [ ]. Comparative genomic analyses of selenoproteins provide insights into the biological functions of selenium. Figure 6 summarizes the human selenoprotein families.

3.1 Human Selenoproteins

3.1.1 Glutathione Peroxidases (Gpx1, Gpx2, Gpx3, and Gpx6)

In the early 1970s, glutathione peroxidase was identified as the first true selenoprotein [6]. The Gpx family has 8 known homologous proteins (Gpx1–Gpx8) and in humans, Gpx1, Gpx2, Gpx3, Gpx4, and Gpx6 are Sec-containing. Gpxs break down hydroperoxides in a reduced glutathione (GSH)-dependent reaction. The selenolate 510 Kurokawa and Berry

Figure 6 Selenoproteins found in humans. SECIS type and Sec residues belong to the thioredoxin motif as shown (C; cysteine, x: any amino acid, U; Sec residue). On the right, relative size of selenoproteins are shown (relative to a 100 amino acid scale). The location of Sec within the protein sequence is shown by a black line.

(R-Se-) in Gpx is oxidized by hydroperoxide and forms seleninic acid (R-SeOH). The seleninic acid reacts with a GSH to form the GS-Se-R. A second GSH reduces the GS-Se-R intermediate back to R-SeH and releases GSSG and water as the by-product. The reaction is a ping-pong mechanism:

−− Gpx-Se +H22OG→ px-SeOH+OH − + Gpx-SeO+H+GSHG→ px-Se-SG+HO2 Gpx-SSe-SG+GSHGpx-Se +H+ +GSSG → Gpx kinetics are identical for Gpx1, Gpx3, and Gpx4. The oxidizing substrates for Gpx2 have not been identified. Gpx1 is expressed in all cells and is more abundant in liver and kidney. It is a homotetramer and localizes to cytosol and mitochondria, reacting with hydrogen peroxide and soluble low molecular mass hydroperoxides such as t-butyl hydroperoxide, cumene hydroperoxide, hydroperoxy fatty acids and 16 Selenium. Role of the Essential Metalloid in Health 511 hydroperoxy lysophosphatides. In tissues, which do not express GSH synthetase, Gpx1 can use γ-glutamylcysteine as a reductant for H2O2. Gpx1 knockout mice show increased susceptibility of liver and lung to ROS [66,67]. Gpx2 is expressed in the gastrointestinal epithelium [68]. It is a homotetramer and localizes to the cytosol. Gpx2 is upregulated in epithelium-derived tumors which include colon adenocarcinoma [69–72], Barrett’s esophagus [73], squamous cell carcinoma [74], and lung adenocarcinomas of smokers [75]. It is suggested to act as a barrier against absorption of food-born hydroperoxides. Gpx2-knockout mice display an increased rate of spontaneous apoptosis at crypt bases, an effect that is stimulated under restricted selenium supply [68,76]. Since selenium supplementation partially prevented the spontaneous apoptosis in crypt bases, Gpx1 might be compensating the Gpx2 depletion. Gpx1/Gpx2 double-knockout mice spontaneously develop colitis and intestinal cancer, a model now used to study spontaneous inflammatory bowel disease and predisposition to intestinal cancer [77]. Gpx3 is a tetramer, primarily synthesized in kidney proximal tubule cells and secreted into plasma [78,79]. Gpx3 produced by the kidney binds to renal proximal tubules, basement membranes of epithelial cells throughout the intestine, the epididymis, bronchi, and lung type II pneumocytes [80,81]. The epididymis synthesizes Gpx3 and releases it into its lumen [81]. Gpx3 knockout mice did not show an obvious phenotype. Since the apparent Km of Gpx3 for GSH (5.3 mM) [82] is significantly higher than plasma GSH concentrations (<0.5μ M) [83], Gpx3’s enzymatic function is unknown. Down-regulation of Gpx3 is observed in many types of cancer and hypermethylation of the Gpx3 promoter is detected in patients with Barrett’s esophagus cancer [84] and prostate cancer [85]. Gpx3 has been suggested to be a novel tumor suppressor. Gpx4 has 3 isoforms (cytosolic, sperm nuclear, and mitochondrial forms), which catalyze reduction of lipid peroxides and cholesterol ester hydroperoxides inside cellular membranes. Gpx4 is a monomer protein [86] that reacts with GSH and can use protein thiols instead of GSH when GSH is limiting. This has been shown for Gpx4 reactivity with sperm mitochondria-associated cysteine rich protein and also for Gpx4 against itself [87]. The mitochondrial form of Gpx4 has vital function in the sperm midpiece serving a structural role during spermatogenesis [87]. While total Gpx4 knockout is lethal [88,89], mitochondrial Gpx4-knockout mice developed normally [90]. Male mitochondrial Gpx4 mice are infertile since Gpx4 is necessary for sperm as an inactive structural protein. Mice in which the nuclear form of Gpx4 was knocked out were not only viable but also fully fertile [91]. Gpx6 is closely related to Gpx3, and is a homodimer selenoprotein only in humans [15].

3.1.2 Thyroid Hormone Deiodinases (DI1, DI2, and DI3)

The iodothyronine deiodinase family has three homologues (DI1, DI2, and DI3) in mammals. DIs catalyze reductive deiodination of thyroid hormones, regulating their activity. T3 enters the nucleus and binds to thyroid hormone receptors which 512 Kurokawa and Berry bind T3-responsive genes and regulate their transcription [92]. Thyroxin (T4) is predominantly produced in the thyroid gland but its affinity for the thyroid hormone receptors is about tenfold lower than T3 [93]. DI1 and DI2 are involved in activation of the thyroid hormone by outer ring deiodination of T4 to produce T3 [94]. DIs are homodimeric thioredoxin-like fold membrane-spanning proteins. DI1 and DI3 are found in the plasma membrane, while DI2 is present in endoplasmic reticulum (ER) [95–97]. DI1 is expressed at highest levels in liver and kidney, and produces most of the circulating T3 [98] while DI2 is most abundant in thyroid, heart, skeletal muscle, brown adipose tissue, and the central nervous system. DI2 on the ER may preferentially supply T3 to the nucleus. Interestingly, human DI2 has an active site Sec and a second Sec 7 amino acids from the C-terminus. The second Sec and the remaining seven C-terminal amino acids are not critical for DI2 enzyme activity and the function of the C-terminal region is unknown [99]. DI2 is expressed in the brown adipose tissue (BAT) and its activity increases in BAT during cold stress, resulting in increased intracellular T3 levels [100–102]. DI2-knockout mice exhibit a mild phenotype; they are unable to control normal body temperature following cold exposure and also show bone development defects [103,104]. Further analysis of DI2-knockout mice on a high fat diet showed a tendency of weight gain and development of insulin resistance [105]. On the other hand, DI3 inactivates T3 and T4 to biologically inactive T2 or reverse T3 (rT3) by preferentially removing the iodine from the inner ring of the molecule. DI3 is found in fetal tissue and placenta and is thought to function in protecting tissues from premature exposure to T3 [106].

3.1.3 Thioredoxin Reductases (TR1, TR2, and TR3)

The thioredoxin reductase (TR) family has three selenoprotein homologues (TR1, TR2, and TR3). TRs reduce oxidized thioredoxin (thioredoxin-S2) with NADPH as a cofactor. A C-terminal conserved motif (-GCUG) contains Sec, which is crucial for the enzyme activity. Reduced thioredoxin is reoxidized by disulfides in proteins generating thiols. thioredoxin-S+NADPH+Ht+ hioredoxin- SH + NADP+ catalyzed by TTR 2 → ()2 () thioredoxin- SH +protein-S thioredoxin-S+protein- SH ()2 22→ ()2

TRs also have broad substrate specificity, being able to use hydrogen peroxide, selenite, lipoic acid, ascorbate, and ubiquinone [107] as substrate. TR1 is localized in the cytosol. TR2, known as thioredoxin/glutathione reductase (TGR) [108], has the function of formation/isomerization of disulfide bonds during sperm maturation [109]. TR3 is a mitochondrial protein. Knockout of either TR1 or TR3 in mice is fatal [110,111]. Knockout of the TR1 gene results in early embryonic death at day 6.5 (E6.5). TR3-knockout mice develop exencephaly and die during midgestation (E10.5). Thus both genes are indispensable for mouse embryo development. 16 Selenium. Role of the Essential Metalloid in Health 513

3.1.4 Methionine-R-Sulfoxide Reductase (MsrB1)

Methionine, a sulfur containing amino acid, is highly susceptible to accumulated reactive oxygen species. Since sulfur of methionine is a prochiral atom, oxidized methionine generates two diastereomers, methionine-S-sulfoxide and methionine- R-sulfoxide. Methionine sulfoxide reductases (Msrs) reduce oxidized methionine residues in proteins and free methionine sulfoxides. Msrs have two stereospecific families, MsrA (reduces S-form of methionine sulfoxide) and MsrB (reduces R-form of methionine sulfoxide) [112]. Oxidized methionine may lead to conformational changes of proteins, e.g., ribosomal protein L12 and calmodulin are impaired by oxidation of methionines and restored by MsrA. On the other hand, methionine oxidation of calcium/calmodulin-dependent protein kinase II in the absence of calcium activates the protein. MsrB1 was identified through bioinformatics and initially designated as selenoprotein X but was subsequently renamed selenoprotein R [13,14]. MsrB1 efficiently acts on methionine sulfoxide in protein but it has very low activity on the free form of methionine sulfoxide. MsrB1 is a zinc-containing protein, primarily synthesized in liver and kidney, and localizes in the cytosol and nucleus. Its activity is the highest of the Msrs because of the presence of Sec in its active site. MsrB1 expression and activity is highly dependent on selenium status, and selenium deficiency decreases MsrB1 activity. Although MsrB1 affects redox regulation in liver and kidney, knockout of the gene in mice showed that MsrB1 is not an essential selenoprotein for development.

3.1.5 15kDa Selenoprotein (Sep15)

Sep15 has an ER signal peptide and localizes in the ER [113]. The C-terminal domain has a thioredoxin-like fold [114,115]. Sep15 has been suggested to take part in the process of rearrangement of disulfide bonds or reduction of incorrectly formed disulfide bonds in misfolded glycoproteins bound to UDP-glucose:glycoprotein glucosyltransferase [114,116,117]. There are several studies suggesting an association of Sep15 with cancer, but reports are contradictory as to whether it promotes or restricts cancer growth. Earlier studies had shown that Sep15 expression was reduced substantially in a malignant prostate cell line and in hepatocarcinoma [115]. An increase of Sep15 expression in colon cancer has been found [118], and targeted down-regulation of Sep15 inhibited growth of colon cancer cells [119]. Additionally, Sep15 knockout mice form significantly fewer carcinogen-induced aberrant crypt foci in colonic epithelia in vivo compared to controls [120]. Thus, the tumor- suppressor activity or oncogenic activity of Sep15 may be tissue-dependent.

3.1.6 Selenophosphate Synthetase 2 (SPS2)

SPS2 is a factor for selenoprotein biosynthesis, with an enzymatic activity to synthesize selenophosphate from ATP and selenite [44]. SPS2 could serve as an 514 Kurokawa and Berry autoregulator of selenoprotein synthesis because it is a selenoprotein [42]. Sodium sulfide is also a substrate for SPS2 and generates thiophosphate that would be used as an active sulfur donor in making Cys attached to tRNA[Ser]Sec. Mass spectrometry (MS) analysis of purified TR1 and TR3 from livers of mice that had been fed selenium-deficient (0 ppm selenium), or selenium-adequate (0.1 ppm selenium) diets showed Cys in place of Sec [47]. Cys was not detected in these selenoproteins on a selenium-enriched (2.0 ppm selenium) diet. Cysteine insertion instead of Sec occurs by sulfide competing with selenide in generating the active donor catalyzed by SPS2.

3.1.7 Selenoprotein P (Sepp1)

Sepp1 is synthesized primarily in the liver and secreted into the plasma. Sepp1 is the only selenoprotein with multiple Sec residues. Human Sepp1 has 10 Sec residues [55]. The N-terminal domain contains a Sec residue in a thioredoxin-like motif and the C-terminal domain contains nine Sec residues in human Sepp1. Four isoforms of Sepp1 have been identified; the shortest isoform terminates at the second UGA, and has been verified as encoding Sec by mass spectrometry of this isoform purified from rat plasma [121]. Sepp1 is a secreted, heparin-binding glycoprotein. Two histidine-rich domains separate the N-terminal and C-terminal regions. Sepp1 delivers selenium to organs where apolipoprotein E receptor 2 or megalin are expressed [122–124]. More details on selenium transport via Sepp1 receptor mechanisms are provided in Section 5.2.

3.1.8 Selenoprotein W (SelW)

SelW was identified in 1993 as a 6 kDa protein, the smallest selenoprotein in mammals. SelW is primarily expressed in muscle, where its absence was notable in muscle of Se-deficient sheep. The expression level of SelW in vertebrates is highly sensitive to dietary Se intake. SelW has a thioredoxin-like fold structure and a Sec- containing redox center located in an exposed loop [125]. SelW was first identified in Se-deficient sheep [126]. SelW contains a CXXU redox motif (where C is Cys, X is any amino acid, and U is Sec) that is conserved among various mammalian species. SelW may be involved in oxidative metabolic pathways and functions as an antioxidant protein [127,128]. SelW was the first selenoprotein to be linked to muscular disorders [125].

3.1.9 Selenoprotein V (SelV)

SelV is a homologue of SelW with additional N-terminal sequence and unknown function. SelV is primarily expressed in testis [15]. 16 Selenium. Role of the Essential Metalloid in Health 515

3.1.10 Selenoprotein T (SelT)

SelT is highly expressed in kidney, brain, heart, thymus, and testis [129]. SelT has a thioredoxin-like fold and belongs to a new redox protein family. SelT is likely an ER resident protein and the thioredoxin fold domain is exposed to the ER or cytosol. Recently, the role of SelT in regulation of calcium homeostasis and neuroendocrine secretion in response to a pituitary adenylate cyclase-activating polypeptide was demonstrated [130].

3.1.11 Selenoprotein M (SelM)

SelM is a homologue of Sep15 and has an ER signal [15]. SelM contains a thioredoxin fold motif and is abundantly expressed in the brain [116]. SelM knockdown experiments in cell culture revealed a role for SelM in calcium regulation [131]. Overexpression of SelM reduced peroxide-induced calcium influx in a neuronal cell line [131]. Knockdown of SelM increased cytosolic calcium concentrations and resulted in apoptotic cell death [131].

3.1.12 Selenoprotein H (SelH)

SelH has a thioredoxin-like fold motif with a small DNA-binding domain (AT-hook motif) and is localized in the nucleus [132]. SelH is highly responsive to selenium status and is upregulated under conditions of elevated copper in mouse liver [133]. SelH overexpression was shown to upregulate expression levels of genes involved in de novo GSH synthesis and phase II detoxification [134]. Chromatin immunoprecipitation experiments demonstrated SelH binds to sequences containing heat shock and/or stress response elements, suggesting SelH may function in a regulatory role in response to redox status. Overexpression of SelH demonstrates neuroprotection against UVB-induced cell death in neurons in culture and increases the levels of mitochondrial biogenesis regulators, mitochondrial cytochrome c content, mitochondria mass and enhanced respiration [135]. SelH may transduce oxidant signals by modulating gene expression.

3.1.13 Selenoprotein O and I (SelO and SelI)

The functions of SelO and SelI are unknown. SelI localizes in the ER.

3.1.14 Selenoprotein S (SelS)

SelS has an ER signal and is induced by ER stress [15,136,137]. SelS is a component of the ER-associated protein degradation (ERAD) system [138,139]. ERAD 516 Kurokawa and Berry protects cells from accumulation of misfolded proteins, transferring these proteins from the ER to the proteasome [140,141]. SelS interacts with Derlin-1, a ER membrane integral protein [141]. The specific function of SelS in the ERAD system is unknown. An association of SelS expression and type 2 diabetes has been reported [142]. Further details are discussed in Section 4.3.

3.1.15 Selenoprotein K (SelK)

SelK has been postulated to function in regulation of endoplasmic reticulum stress [143] and facilitation of calcium flux in immune cells 144[ ]. SelK mRNA is expressed in immune cells and lymphoid tissues, spleen, intestine, and testis. SelK predominantly localizes to the ER but has no ER localization signal [144]. Thus, the protein may be bound by a chaperone or other ER-localized protein for insertion into the ER. Studies with SelK-knockout mice suggest that this protein is not required for growth or development of mice [144]. SelK is cleaved by calpain and the cleaved form is highly abundant in unactivated macrophages [145]. SelK cleavage is inhibited by upregulation of the Toll-like receptor-induced calpain inhibitor, calpastatin [145].

3.1.16 Selenoprotein N (SelN)

SelN is ubiquitously expressed with highest expression in muscle [146] and is localized in the ER membrane [146]. It has a predicted redox active CUGS motif. Loss of SelN function is associated with congenital muscular dystrophy [147]. SelN has been reported to interact with ryanodine receptors, and may affect calcium flux [148]. Another study demonstrated SelN deficiency was associated with oxidative stress [149,150].

4 Selenium and Disease

4.1 Overview of Selenium-Related Diseases

Recently, selenium supplementation trials found that moderately higher selenium intake may influence redox status through selenoprotein synthesis to cause type 2 diabetes. Thus selenium homeostasis needs to be tightly regulated for healthy life. The range of selenium intake for optimal health in humans and animals is narrow, such that low selenium intake is associated with developmental defects and disease states and high selenium results in toxicity. We discuss below the conditions associated with both cases, specifically myopathies, selenosis, brain degeneration, type 2 diabetes, and male infertility. 16 Selenium. Role of the Essential Metalloid in Health 517

4.1.1 Selenium Deficiency

Three diseases have been reported to be associated with severe selenium deficiency, due to their occurrence in areas with selenium poor soils and their reversal upon selenium supplementation. It should be noted that selenium may be a cofactor in these diseases, with other factors contributing to their incidence or severity. (1) Keshan disease, was first described as a juvenile cardiomyopathy in the early 1930s in the Chinese medical literature [151]. Women and children were susceptible to the development of Keshan disease, which had a high mortality rate. Supplementation of individuals with sodium selenite tablets could prevent the development of Keshan disease [4]. Since the incidence of Keshan disease fluc- tuated seasonally and annually, viral infection was also considered as a possible cofactor [152]. Heart tissues from Keshan disease victims were subsequently shown to contain coxackie viruses. Further, studies in selenium-deficient mice demonstrated that coxsackie virus B4 infection induced severe heart pathology [153]. Selenium-adequate mice showed mild heart pathology when infected with the virus, which suggests that selenium deficiency in combination with coxsackie virus infection was required for the development of Keshan disease. (2) Kashin-Beck disease is a chronic, endemic osteochondropathy, accompanied by joint necrosis [154]. This syndrome affects individuals in specific regions of Tibet, northeastern to southwestern China, Siberia, and North Korea. While individuals with this disease show skeletal pathology, they are not reported to develop dysfunction of other organs or tissues. Kashin-Beck disease may require other factors since the disease is clustered within specific regions and/ or families. A polymorphism in the Gpx1 gene was reported as a potential genetic risk factor for Kashin-Beck disease [155]. (3) Myxedematous endemic cretinism, which is induced by thyroid atrophy, results in mental retardation [156,157]. Myxedematous endemic cretinism is highly prevalent in central Africa, where iodine and selenium-poor areas overlap with thiocyanate-rich areas.

4.1.2 Selenium Toxicity (Selenosis)

Blood selenium levels greater than 100 μg/dL can lead to selenosis. Symptoms of selenosis include hair loss, white blotchy nails, a garlic breath, gastrointestinal disorders, fatigue, irritability, and neurological damage [158]. Selenosis in humans is a rare event except in very high selenium areas. Extreme cases of selenosis can be fatal, due to cirrhosis of the liver [159]. Elemental selenium and metallic selenides have relatively low toxicities because of their low bioavailability. Selenates and selenites are very toxic. Organic selenium compounds which occur in metabolic processes, such as Sec, selenomethionine, and methylated selenium compounds are toxic in large doses. In the 10th edition of RDAs in 1989, it was pointed out that sensitive biochemical indices of selenium 518 Kurokawa and Berry overexposure were not available and no attempt was made to establish an upper limit of selenium intake [7]. In 2000, The Institute of Medicine of the National Academy of Science provided a DRI, which set the tolerable upper intake levels of selenium to 400 μg/d [8]. The Lowest Observed Adverse Effect Level is 910 μg/d [160], and the No Observed Adverse Effect Level is 200 μg/d.

4.2 Selenium in Brain Function

The supply of selenium to the brain is prioritized for normal development and brain function. There is a “hierarchy” of tissues with respect to selenium supply under low selenium status, whereby brain tends to maintain its selenium compared to other tissues [161,162]. Brain expresses almost all selenoproteins, especially within neurons [163]. Sepp1-knockout mice produce severe brain selenium deficiency when maintained on a selenium-deficient diet, with neurological impairments that lead to death within weeks [164,165]. Sepp1-knockout mice fed a 0.10 ppm selenium diet developed spasticity and abnormal movements in addition to poor performance on the rotarod test and pole climb. Sepp1-knockout mice fed 0.25 ppm selenium diet produced no neurological dysfunction. Recently, syndromes of congenital selenoprotein biosynthetic deficiency have been discovered. Progressive cerebellar-cerebral atrophy (PCCA) was identified in several non-consanguineous Jewish Sephardic families of Moroccan or Iraqi ances- try [166]. The syndrome was mapped to homozygous or compound heterozygous missense mutations of the SecS gene, with no enzymatic activity. PCCA involves mental retardation, progressive microcephaly, and spasticity [166]. PCCA represents the first clinical syndrome related to selenocysteine biosynthesis in humans. Clinically, cerebral and cerebellar atrophy involves gray and white matter [166].

4.3 Selenium in Diabetics

Body glucose homeostasis is regulated by functional insulin circulation and signaling. Type 2 diabetes is characterized by high blood glucose in the context of insulin resistance. The Nutrition Prevention Cancer (NPC) trial which is a double-blind, randomized, placebo-controlled clinical trial to test micronutrients for cancer prevention revealed a more than two-fold increase in type 2 diabetes incidence in the selenium-supplemented compared to the placebo group [167–169]. The Selenium and Vitamin E Cancer Prevention Trial (SELECT) revealed a similar trend [170], however non-significant in a 10-year follow-up, but still concerning enough to lead to the trial’s termination. A recent study reported that Sepp1 is associated with development of type 2 diabetes [171]. Sepp1 mRNA levels were increased in people with insulin resistance. Hepatic Sepp1 mRNA is upregulated by glucose and hepatic Sepp1 mRNA levels correlated with insulin resistance [171]. Furthermore, insulin suppressed 16 Selenium. Role of the Essential Metalloid in Health 519

Sepp1 protein expression in hepatocytes. It is postulated that Sepp1 induces insulin resistance in liver and muscle, resulting in hyperglycemia [171]. Targeted removal of the tRNA[Ser]Sec gene in hepatocytes revealed increases in plasma apolipoprotein E and cholesterol levels, up-regulation of cholesterol biosynthesis genes and down-regulation of cholesterol metabolism or transport genes in the hepatocytes [172]. These results suggest hepatic selenoproteins are responsible for ApoE and cholesterol metabolism. Recently, overproduction of the antioxidant selenoprotein, Gpx1 in mice resulted in a type 2 diabetes-like phenotype [173]. Lei’s group developed Gpx1-overproducing mice, which became obese at 6 months of age. Gpx1 catalyzes the reduction of hydrogen peroxide and organic hydroperoxides using GSH as the cofactor. Insulin production is regulated by pancreatic duodenal homeobox 1, forkhead box A2, and mitochondrial uncoupling protein 2, expression and function of which are affected by intracellular ROS [174,175]. Pancreatic β cells express relatively low amounts of Gpx1, and may be susceptible to oxidative stress [176]. ER stress caused by a disruption in ER homeostasis is associated with type 2 diabetes [177]. Up-regulation of SelS mRNA in liver, adipose tissue and skeletal muscle is associated with type 2 diabetes in Psammomys obesus [142]. Hepatic SelS mRNA expression and protein content are increased by deprivation of glucose in P. obesus [137] but not in non-diabetic P. obesus [142]. In addition, a high concentration of glucose reduces SelS mRNA expression in cultured hepatocytes [142]. A recent study demonstrated that SelS mRNA expression was increased by insulin stimulation in human subcutaneous adipocytes from type 2 diabetic patients but not in nondia- betic subjects [178]. Thus, SelS may be involved in development of type 2 diabetes. ER stress arrests translation in DI2 synthesis, which leads to a reduction in T3 production [179]. Chemical chaperones (tauroursodeoxycholic acid or 4-phenylbutyric acid) can resolve ER stress and restore glucose tolerance in a DI2-dependent manner [180]. DI2 is necessary for T3-induced adaptive thermogenesis [103] and DI2- knockout mice showed obesity and glucose intolerance when placed on thermoneu- trality and a high-fat diet [180]. The SNP of DI2 (A/G) at codon 92 has been identified [181] and this SNP leads to the Thr92Ala variant, which strongly associates with insulin resistance [182] and subsequently type 2 diabetes [181]. DI3-knockout mice were found to be glucose-intolerant and exhibited a reduction in pancreatic β-cell mass and insulin content [183]. The absence of DI3 in the β-cells exposes them to T3 and leads to impaired β-cell function [183] and insulin secretion.

4.4 Selenium in Reproduction

For at least five decades, selenium was recognized as an important factor for male fertility in rats, mice, and livestock. Selenium deficiency in human reproduction data is contradictory because of the limited number of patients analyzed. Thus, the role of selenium in human reproduction was largely inferred from studies in laboratory animals. Testis uptake and retain selenium even under conditions of substantial 520 Kurokawa and Berry selenium deficiency. Feeding a selenium-deficient diet for two generations generated abnormal spermatozoa in rats [184,185]. In severe selenium deficiency, male rats and mice become sterile as spermatogenesis is arrested, the seminiferous epithelium is degenerated and abnormal sperm morphology is observed. Interestingly, when 75Se was injected into rats, most of the selenium accumulated in the mid-piece of the spermatozoon that harbors the helix of mitochondria embedded in a keratin- like matrix [186]. Recently, TR2 was shown to be abundant in elongating spermatids at the site of the mitochondrial sheath formation [109]. In 1999 the Ursini and Flohé laboratories identified Gpx4 as the major component of the mitochondrial capsule [87]. Gpx4 activity is not detected with the specific substrate phosphatidylcholine hydroperoxide in spermatozoa but immunohistochemical staining of Gpx4 was observed. Gpx4 in the mitochondrial mid-piece of mature spermatozoa is a chemically inactive form, likely due to disulfide and selenyl sulfide bridges that form high molecular weight complexes with other capsular proteins [87]. The Gpx4 protein can be solubilized out of this complex by strong reduction and chaotropic agents and detected by MALDI-TOF mass spectrometry or Western blotting [87]. Prolonged preincubation with 0.1M DTT or mercaptoethanol leads to recovery of enzymatic activity. Unlike Gpx1-3, Gpx4 can accept electrons from protein thiols instead of GSH when GSH is limiting. As the spermatids mature, GSH and protein thiols decrease, resulting in Gpx4 polymerization. The inactive Gpx4 complex contains sperm mitochondrion-associated cysteine- rich protein fragments (SMCP), voltage-dependent anion channel and three types of keratins (type II keratin kb1, keratin k5, and acidic keratin complex I) [187]. SMCP with its 30% cysteine residues is the most likely candidate to react with Gpx4. SMCP-knockout mice showed infertility and asthenozoospermia in some mice, but marginal disturbance of spermatogenesis in others [188]. Reduced Gpx4 production in spermatozoa resulted in bent tails with slight angu- lations to hairpin structures and abnormal kinking at the mid-pieces [189]. Knockout of the mitochondrial Gpx4 isoform resulted in viability but male infertility, and the spermatozoa presented severe morphological abnormalities [90]. Knockout of the entire Gpx4 gene was embryonic lethal, whereas nuclear Gpx4-knockout mice are viable and fertile but exhibit transient nuclear instability and delay in chromatin condensation of spermatozoon [91]. Since cytosolic Gpx4 exists in the nucleus, lack of nuclear Gpx4 function may be compensated by cytosolic Gpx4 [91,190].

5 Health Benefits of Selenium in Humans

5.1 Molecular Forms of Selenium in Diet

Selenium exists in the environment in several inorganic and organic forms. Elemental selenium exists stably as selenite and selenate. Organic forms of selenium are found in biological matter, and include the methylated selenium compounds, selenoamino acids, and selenoproteins. However selenium is ubiquitous, and the amounts can vary widely [191,192]. 16 Selenium. Role of the Essential Metalloid in Health 521

Selenium is primarily supplied in diets from grains and animal products [193]. Plants have no true selenoproteins. Selenomethionine is produced in plants due to indiscriminate substitution of selenium for sulfur in methionine biosynthesis. Drinking water contains very low amounts of water-soluble inorganic forms of selenium (0.12 to 0.44 μg/L) [194,195] and this contribution to selenium as a dietary source is very minor. In the United States, the amount of selenium in water is regulated by the Environmental Protection Agency under the Safe Drinking Water Act. The federal standards allow up to 50 mg/L in drinking water [196]. Grains, wheat, and corn used for breads and other food products contain selenomethionine (~55%) as a bioavailable selenium source [197]. Sec and selenite/selenate are also detectable in substantial amounts in wheat (~20% respectively). The content of selenium in plants can vary widely, as much as 500-fold, depending on the soil selenium. In regions where soil selenium is low, such as Southwestern Oregon in the United States, Northeastern China, Finland, and New Zealand, sodium selenite is added to fertilizers as well as animal feed [198,199]. High-selenium regions, where soils exceed 600 mg/kg selenium, are found in parts of North and South America, China, and Russia [200,201]. In regions with high selenium in the soil, plants may accumulate up to 3,000 μg selenium per gram and may potentially be toxic [202]. Selenium absorption by plants depends on the pH of the soil, i.e., selenite in acidic soils and selenate in alkaline soils. Because selenate is a more water-soluble form of selenium, it is thought to be more available for plant uptake [203,204]. Fruit and vegetables contain only small amounts of selenium. Some vegetables can grow under selenium-rich soil and accumulate selenium (onions, leeks, garlic, and broccoli) [205]. These vegetables accumulate selenium up to 50-fold or higher. Vegetables grown in high-selenium soil contain mostly selenomethionine, methylselenocysteine, and γ-glutamyl-methylselenocysteine [206,207]. Fungi, such as mushrooms and yeast, can accumulate selenium and may contain more than 20 selenium-containing compounds (Sec, selenomethionine, methylselenocysteine, and Se-adenosylselenohomocysteine) [208]. Meats are good sources of selenium, but the selenium content in livestock is dependent on diet and the region in which the animals feed. Selenium supplementation of cattle, hogs, and chicken is a common practice [209]. Animal meat contains mostly selenomethionine (up to 60%) and Sec (up to 50%). The remaining selenium species are small selenium-containing molecules. These ratios can vary depending on what form of selenium is consumed. Selenite or selenate in food will be converted into Sec. Animals fed selenomethionine-containing food increase the content of selenomethionine and Sec in the meat [210].

5.2 Selenium Transport in Mammals

5.2.1 Tissue-Oriented Selenium Transport

Selenium is differentially distributed into different organs. It has been proposed there is a hierarchy of selenium requirements for selenium in tissues. Brain and testis tend to preserve selenium for their essential functions under selenium 522 Kurokawa and Berry

deficiency [161,211]. Dietary selenium is absorbed in intestine and transferred into liver. The body selenium content is regulated by hepatic production of methylated selenium compounds and its urinary excretion, not by intestinal absorption of selenium. Selenium is primarily transported in the plasma to the organs via Sepp1 [164,165]. Kidney expresses Sepp1 at 38% of the liver level. Skeletal muscle, heart, and testis followed with 10, 6, and 6%, respectively, in mouse [212]. Brain expresses Sepp1 at less than 2% of the liver level. Sepp1 is also expressed in many tissues at very low levels. The human plasma concentration of Sepp1 is approximately 5.6 mg/L. Since the liver exports selenium in the form of Sepp1, dietary selenium deficiency dramatically decreases liver selenium concentrations. As discussed above, Sepp1-knockout mice exhibited a severe phenotype, including neurological dysfunction and male infertility. Testis requires selenium for sperm maturation, and testis of Sepp1-knockout mice becomes severely selenium-deficient unless a high-selenium diet is fed. Testis selenium concentrations in Sepp1- knockout mice decrease to 8% of the wild-type value, whereas brain retains 56% of the wild-type value when mice were fed 0.25 ppm selenite diet for 4 weeks after weaning, a diet considered as selenium-adequate. Deletion of Sepp1 causes increased excretion of selenium in the urine [213]. Selective deletion of Sepp1 in hepatocytes showed liver selenium is maintained but whole-body selenium concentration decreased to 58% of the control value in mice fed 0.25 ppm selenium diet for 4 weeks beginning at weaning [212]. However. in other tissues selenium concentrations also decreased to varying degrees, but brain and testis retained selenium better than other tissues. Under conditions of selenium deficiency, selective deletion of Sepp1 in hepatocytes resulted in elevation of liver selenium concentration to 500% of the control value, accounting for 53% of whole-body selenium. These results demonstrate the central role of Sepp1 production by hepatocytes and the critical role of secretion of Sepp1 from liver in maintaining selenium homeostasis [212]. Sepp1 is observed in testis Sertoli cells in endocytosed vesicles. A lipoprotein receptor family member, apolipoprotein E receptor-2 (apoER2) was the first identified Sepp1 receptor in testis Sertoli cells [122]. ApoER2-knockout mice exhibit very low testis selenium concentrations. Thus, most of the selenium in testis is taken up in the form of Sepp1 by apoER2. ApoER2 is also highly expressed in the brain [214], where it was shown to play a crucial role in uptake of Sepp1 and preservation of selenium when dietary selenium is limiting [123]. Megalin, also a lipoprotein receptor family member, was identified as a Sepp1 receptor in kidney brush border of proximal convoluted tubule (PCT) cells [124]. Several isoforms of Sepp 1 have been identified in rat [121,215]. Shortened isoforms may result from termination of Sepp1 translation at UGAs in the open reading frame of Sepp1 mRNA. One of the shortest isoforms of mouse Sepp1Δ240–361 is small enough to pass through the renal glomerulus. Since megalin-knockout mice lose Sepp1 in urine, megalin prevents loss of selenoproteins in urine [216]. However, another plasma selenoprotein, Gpx3, which accounts for 21% of selenium in plasma [217], is not a selenium source of selenoproteins [218]. 16 Selenium. Role of the Essential Metalloid in Health 523

Figure 7 Selenium containing compounds in mammals. Methylselenocysteine is supplied by plants. Selenosugar is a major urinary selenium compound which is synthesized in liver. Dimethylselenide is found in breath.

When selenium intake is high, non-Sepp1 selenium forms including low molecular weight selenium compounds are taken up by kidney. Under selenium deficiency, low molecular weight selenium compounds are not sufficient to support tissue selenium requirements [212]. In summary, selenium is transported to tissues primarily via Sepp1 and small molecules. Plasma Sepp1 is an efficient form of selenium transporter. Gpx3 is another plasma selenoprotein but does not appear to transport selenium for specific uptake by cells. Small selenium compounds can transfer selenium but this requires much higher selenium intake and the pathway appears to be nonspecific. The functions of small molecule selenium compounds need further characterization.

5.2.2 Selenium Excretion

Selenium is excreted in urine, in feces, and by other routes, which include exhalation in breath and loss through hair and skin cells. Urine. Once selenium is absorbed by the body, it is excreted mostly into the urine. Urinary selenium excretion increased with increases in dietary selenium intake. Trimethylselenonium ion was identified as a prominent form of selenium in rat urine [219], and is the major excreted form when selenium is in excess [220]. Recently, Suzuki’s group identified the major selenium metabolite in urine as 1β-methylseleno-N-acetyl-D-galactosamine (selenosugar) within the required to low-toxic range [221]. This selenosugar is synthesized in liver [221] (Figure 7). Breath. Volatilization of selenium into breath is observed only at high selenium intakes [222]. The volatile compound dimethylselenide was identified as one of the methylated forms of selenium that account for most selenium excretion in urine and breath [223]. Feces. Fecal selenium excretion was regulated by dietary selenium intake at deficient to moderately high selenium intakes. Fecal selenium excretion plateaued at moderately high selenium intake [224]. Characterization of fecal selenium excretion has been relatively minimal. 524 Kurokawa and Berry

5.3 Human Dietary Standards for Selenium

In 1980, the National Research Council (NRC) established an estimated safe and adequate daily dietary intake for selenium in humans [225]. The recommendation for adults was set from 50 to 200 μg/d based on extrapolations from animal studies. In 1989, the Dietary Reference Intake (DRI) was established for selenium, with a RDA of 70 μg/d for men and 55 μg/d for women in accordance with the World Health Organization [7]. Selenoproteins are the major form of functional selenium, thus selenium nutritional requirements have been assessed through selenoprotein optimization [226]. Plasma contains two selenoproteins, Gpx3 and Sepp1. Plasma Gpx activity and Sepp1 concentration decrease to less than 5% of selenium-replete values in animals with severe selenium deficiency. Thus, plasma levels of these selenoproteins are used primarily as nutritional biomarkers of selenium. In 2001, Burk’s group carried out a study in a low-selenium area of China [227]. They concluded that full expression of glutathione peroxidase was achieved with 37 μg Se/d as selenomethionine and with 66 μg/d as selenite. However, full expression of selenoprotein P was not achieved at the highest doses of either form. There are several forms of selenium that exist in dietary food and supplements (e.g., high- selenium yeast and selenomethionine). Yeast contains selenium mostly as selenomethionine but has a significant amount of selenium in other forms. Burk’s group carried out a study supplementing moderate (approximately 200 μg/d) to high levels (approximately 600 μg/d) of selenium supplements in three forms (selenized yeast, selenomethionine, selenite) to selenium-replete individuals in the US [228]. Since selenomethionine is nonspecifically incorporated to proteins, high-yeast selenium supplement and selenomethionine raised the plasma selenium concentration in a dose-dependent manner but plasma selenoproteins did not respond to selenium supplementation in selenium-replete individuals. Selenite intake did not increase plasma selenium concentration and was excreted into urinary selenium compounds. In the study, total intakes of over 800 μg/d selenium for 16 weeks showed no signs of selenium toxicity. The authors concluded that the 800 μg/d can be used safely in studies of limited duration if the subjects are monitored closely for signs of selenium toxicity. In 2010, the Burk group further studied the effect of selenium supplementation in a selenium-deficient human population in China [229]. They studied healthy Chinese individuals who had a daily dietary selenium intake of 14 μg/d and showed that supplementation with 35 μg selenium/d for 40 weeks optimized the Sepp1 in the healthy selenium-deficient Chinese individuals. Gpx activities were optimized by a total intake of 35 μg selenium/d. The investigators concluded that adjustment for the difference in weight between the Chinese subjects (58 kg) and US residents (76 kg) and for variation among individuals would yield a selenium requirement for US adults of ≈75 μg/d. These studies indicate that once the selenium requirement has been met, selenoproteins are not increased and plasma Sepp1 concentration is the best marker of human selenium nutritional status. 16 Selenium. Role of the Essential Metalloid in Health 525

6 General Conclusions

It has been two centuries since the identification of selenium by Berzelius. Selenium is essential for life processes. Although it is a rare element, many organisms have evolved to maximize selenium’s properties. It is integrated into the biology of many life forms, to the extent of being critical for life. The selenium field has been dramatically expanding over the last few decades. However, functions of most of the selenoproteins and selenium containing molecules still remain unclear. Continued research of the biochemical properties of selenium will hopefully lead to new discoveries to improve human health.

Abbreviations and Definitions

Ψ55 tRNA pseudouridine position at 55 28S 28S ribosomal RNA ADP adenosine 5′-diphosphate AMP adenosine 5′-monophosphate apoER2 apolipoprotein E receptor-2 ATP adenosine 5′-triphosphate BAT brown adipose tissue cDNA complementary DNA Cys cysteine DI iodothyronine deiodinase DRI dietary reference intake DTT dithiothreitol EF elongation factor eEFSec eukaryotic selenocysteyl-tRNA-specific elongation factor eIF4a3 eukaryotic initiation factor 4A-III EMSA electrophoretic mobility shift assays ER endoplasmic reticulum ERAD ER-associated protein degradation EST expressed sequence tag γ-GCS γ-glutamylcysteine synthetase Gpx glutathione peroxidase GS glutathione synthetase GSH reduced form of glutathione or γ-glutamylcysteinylglycine GSSG oxidized form of glutathione, glutathione disulfide i6A isopentenyladenosine kDa kilo dalton L12 large ribosomal subunit protein L12 L30 large ribosomal subunit protein L30 m1A N1-methyladenosine MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight 526 Kurokawa and Berry mcm5U methylcarboxymethyl-5’-uridine mcm5Um methylcarboxymethyl-5’-uridine-2’-O-methylribose mRNA messenger ribonucleic acid MS mass spectrometry Msr methionine sulfoxide reductase mV millivolts NADP+ nicotinamide adenine dinucleotide phosphate NADPH nicotinamide adenine dinucleotide phosphate (reduced) NPC nutrition Prevention Cancer NRC national Research Council Nrf2 leucine zipper transcription factor NF-E2 factor 2 PCCA progressive cerebellar-cerebral atrophy PCT proximal convoluted tubule pKa acid dissociation constant PPi pyrophosphate (diphosphate) PSTK O-phosphoseryl-tRNA[Ser]Sec kinase RDA recommended dietary allowance ROS reactive oxygen species rRNA ribosomal ribonucleic acid rT3 reverse triiodothyronine SBP2 SECIS binding protein-2 Sec selenocysteine SECIS Sec insertion sequence Secp43 Sec tRNA[Ser]Sec associated 43 kDa protein SecS Sec synthetase Sel(X) selenoprotein X (X is any selenoprotein) SELB Sec-specific translation elongation factor SELECT Selenium and Vitamin E Cancer Prevention Trial selenosugar1 β-methylseleno-N-acetyl-D-galactosamine Sep15 15 kDa selenoprotein Sepp1 human selenoprotein P siRNA small interfering RNA SLA soluble liver antigen SMCP sperm mitochondrion-associated cysteine-rich protein SPS selenophosphate synthetase T3 3,3′,5-triiodo-L-thyronine or triiodothyronine T4 thyroxin TGR thioredoxin/glutathione reductase TR thioredoxin reductase Trit1 tRNA isopentenyltransferase, mitochondrial tRNA transfer ribonucleic acid UDP-glucose uridine diphosphate glucose Um34 single methyl group on the ribosyl moiety at position 34 UTR untranslated region UVB ultraviolet, 315-280 nm wave length WHO World Health Organization 16 Selenium. Role of the Essential Metalloid in Health 527

Acknowledgment This work was supported by National Institutes of Health grants R01DK047320 to MJB.

References

1. L. Schomburg, U. Schweizer, J. Köhrle, Cell Mol. Life Sci. 2004, 61, 1988–1995. 2. K. Schwarz, C. M. Foltz, J. Am. Chem. Soc. 1957, 79, 3292–3293. 3. O. H. Muth, J. E. Oldfield, L. F. Remmert, J. R. Schubert,Science 1958, 128, 1090. 4. Chin. Med. J. (Engl) 1979, 92, 471–476. 5. A. M. van Rij, C. D. Thomson, J. M. McKenzie, M. F. Robinson, Am. J. Clin. Nutr. 1979, 32, 2076–2085. 6. J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, W. G. Hoekstra, Science 1973, 179, 588–590. 7. National Research Council, Recommended Dietary Allowances:10th Edn, The National Academies Press, 1989. 8. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, The National Academies Press, 2000. 9. WHO, “Trace Elements in Human Nutrition and Health”, Geneva, 1996. 10. D. L. Hatfield, V. N. Gladyshev, Mol. Cell Biol. 2002, 22, 3565–3576. 11. J. E. Cone, R. M. Del Río, J. N. Davis, T. C. Stadtman, Proc. Natl. Acad. Sci. USA 1976, 73, 2659–2663. 12. F. Zinoni, A. Birkmann, T. C. Stadtman, A. Böck, Proc. Natl. Acad. Sci. USA 1986, 83, 4650–4654. 13. G. V. Kryukov, V. M. Kryukov, V. N. Gladyshev, J. Biol. Chem. 1999, 274, 33888–33897. 14. A. Lescure, D. Gautheret, P. Carbon, A. Krol, J. Biol. Chem. 1999, 274, 38147–38154. 15. G. V. Kryukov, S. Castellano, S. V. Novoselov, A. V. Lobanov, O. Zehtab, R. Guigó, V. N. Gladyshev, Science 2003, 300, 1439–1443. 16. R. F. Burk, K. E. Hill, A. K. Motley, Biofactors 2001, 14, 107–114. 17. M. Birringer, S. Pilawa, L. Flohé, Nat. Prod. Rep. 2002, 19, 693–718. 18. T. C. Stadtman, Annu. Rev. Biochem. 2002, 71, 1–16. 19. D. L. Hatfield, F. H. Portugal,Proc. Natl. Acad. Sci. USA 1970, 67, 1200–1206. 20. D. L. Hatfield, B. A. Carlson, X. M. Xu, H. Mix, V. N. Gladyshev, Prog. Nucleic Acid Res. Mol. Biol. 2006, 81, 97–142. 21. B. J. Lee, P. J. Worland, J. N. Davis, T. D. Stadtman, D. L. Hatfield, J. Biol. Chem. 1989, 264, 9724–9727. 22. W. Leinfelder, T. C. Stadtman, A. Böck, J. Biol. Chem. 1989, 264, 9720–9723. 23. A. Böck, K. Forchhammer, J. Heider, C. Baron, Trends Biochem. Sci. 1991, 16, 463–467. 24. K. Forchhammer, K. Boesmiller, A. Böck, Biochimie 1991, 73, 1481–1486. 25. R. A. Sunde, J. K. Evenson, J. Biol. Chem. 1987, 262, 933–937. 26. A. Diamond, B. Dudock, D. L. Hatfield,Cell 1981, 25, 497–506. 27. R. Amberg, C. Urban, B. Reuner, P. Scharff, S. C. Pomerantz, J. A. McCloskey, H. J. Gross, Nucleic Acids Res. 1993, 21, 5583–5588. 28. A. M. Diamond, I. S. Choi, P. F. Crain, T. Hashizume, S. C. Pomerantz, R. Cruz, C. J. Steer, K. E. Hill, R. F. Burk, J. A. McCloskey, J. Biol. Chem. 1993, 268, 14215–14223. 29. N. Kato, H. Hoshino, F. Harada, Biochem. Int. 1983, 7, 635–645. 30. D. L. Hatfield, A. Diamond, B. Dudock,Proc. Natl. Acad. Sci. USA 1982, 79, 6215–6219. 31. G. J. Warner, M. J. Berry, M. E. Moustafa, B. A. Carlson, D. L. Hatfield, J. R. Faust, J. Biol. Chem. 2000, 275, 28110–28119. 32. B. A. Carlson, M. E. Moustafa, A. Sengupta, U. Schweizer, R. Shrimali, M. Rao, N. Zhong, S. Wang, L. Feigenbaum, B. J. Lee, V. N. Gladyshev, D. L. Hatfield, J. Biol. Chem. 2007, 282, 32591–32602. 33. B. A. Carlson, X. M. Xu, V. N. Gladyshev, D. L. Hatfield, J. Biol. Chem. 2005, 280, 5542–5548. 528 Kurokawa and Berry

34. J. Y. Kim, B. A. Carlson, X. M. Xu, Y. Zeng, S. Chen, V. N. Gladyshev, B. J. Lee, D. L. Hatfield,Biochem. Biophys. Res. Commun. 2011, 409, 814–819. 35. N. Fradejas, B. A. Carlson, E. Rijntjes, N. P. Becker, R. Tobe, U. Schweizer, Biochem. J. 2013, 450, 427–432. 36. X. Q. Wu, H. J. Gross, Nucleic Acids Res. 1993, 21, 5589–5594. 37. T. Ohama, D. C. Yang, D. L. Hatfield,Arch. Biochem. Biophys. 1994, 315, 293–301. 38. R. Amberg, T. Mizutani, X. Q. Wu, H. J. Gross, J. Mol. Biol. 1996, 263, 8–19. 39. P. H. Mäenpää, M. R. Bernfield, Proc. Natl. Acad. Sci. USA 1970, 67, 688–695. 40. B. A. Carlson, X. M. Xu, G. V. Kryukov, M. Rao, M. J. Berry, V. N. Gladyshev, D. L. Hatfield, Proc. Natl. Acad. Sci. USA 2004, 101, 12848–12853. 41. S. C. Low, J. W. Harney, M. J. Berry, J. Biol. Chem. 1995, 270, 21659–21664. 42. M. J. Guimarães, D. Peterson, A. Vicari, B. G. Cocks, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, D. A. Ferrick, R. A. Kastelein, J. F. Bazan, A. Zlotnik, Proc. Natl. Acad. Sci. USA 1996, 93, 15086–15091. 43. X. M. Xu, B. A. Carlson, R. Irons, H. Mix, N. Zhong, V. N. Gladyshev, D. L. Hatfield, Biochem. J. 2007, 404, 115–120. 44. X. M. Xu, B. A. Carlson, H. Mix, Y. Zhang, K. Saira, R. S. Glass, M. J. Berry, V. N. Gladyshev, D. L. Hatfield,PLoS Biol. 2007, 5, e4. 45. C. Gelpi, E. J. Sontheimer, J. L. Rodriguez-Sanchez, Proc. Natl. Acad. Sci. USA 1992, 89, 9739–9743. 46. A. A. Turanov, X. M. Xu, B. A. Carlson, M. H. Yoo, V. N. Gladyshev, D. L. Hatfield,Adv. Nutr. 2011, 2, 122–128. 47. X. M. Xu, A. A. Turanov, B. A. Carlson, M. H. Yoo, R. A. Everley, R. Nandakumar, I. Sorokina, S. P. Gygi, V. N. Gladyshev, D. L. Hatfield, Proc. Natl. Acad. Sci. USA 2010, 107, 21430–21434. 48. M. J. Berry, L. Banu, Y. Y. Chen, S. J. Mandel, J. D. Kieffer, J. W. Harney, P. R. Larsen, Nature 1991, 353, 273–276. 49. M. J. Berry, L. Banu, P. R. Larsen, Nature 1991, 349, 438–440. 50. R. Walczak, E. Westhof, P. Carbon, A. Krol, RNA 1996, 2, 367–379. 51. P. R. Copeland, D. M. Driscoll, J. Biol. Chem. 1999, 274, 25447–25454. 52. V. Allamand, P. Richard, A. Lescure, C. Ledeuil, D. Desjardin, N. Petit, C. Gartioux, A. Ferreiro, A. Krol, N. Pellegrini, J. A. Urtizberea, P. Guicheney, EMBO Rep. 2006, 7, 450–454. 53. D. Fagegaltier, A. Lescure, R. Walczak, P. Carbon, A. Krol, Nucleic Acids Res. 2000, 28, 2679–2689. 54. A. C. Miniard, L. M. Middleton, M. E. Budiman, C. A. Gerber, D. M. Driscoll, Nucleic Acids Res. 2010, 38, 4807–4820. 55. K. E. Hill, R. S. Lloyd, R. F. Burk, Proc. Natl. Acad. Sci. USA 1993, 90, 537–541. 56. Z. Stoytcheva, R. M. Tujebajeva, J. W. Harney, M. J. Berry, Mol. Cell Biol. 2006, 26, 9177–9184. 57. W. Zhang, C. S. Ramanathan, R. G. Nadimpalli, A. A. Bhat, A. G. Cox, E. W. Taylor, Biol. Trace Elem. Res. 1999, 70, 97–116. 58. P. R. Copeland, J. E. Fletcher, B. A. Carlson, D. L. Hatfield, D. M. Driscoll,EMBO J. 2000, 19, 306–314. 59. R. M. Tujebajeva, P. R. Copeland, X. M. Xu, B. A. Carlson, J. W. Harney, D. M. Driscoll, D. L. Hatfield, M. J. Berry,EMBO Rep. 2000, 1, 158–163. 60. D. Fagegaltier, N. Hubert, K. Yamada, T. Mizutani, P. Carbon, A. Krol, EMBO J. 2000, 19, 4796–4805. 61. L. Chavatte, B. A. Brown, D. M. Driscoll, Nat. Struct. Mol. Biol. 2005, 12, 408–416. 62. M. E. Budiman, J. L. Bubenik, A. C. Miniard, L. M. Middleton, C. A. Gerber, A. Cash, D. M. Driscoll, Mol. Cell 2009, 35, 479–489. 63. Q. Li, H. Imataka, S. Morino, G. W. Rogers, N. J. Richter-Cook, W. C. Merrick, N. Sonenberg, Mol. Cell Biol. 1999, 19, 7336–7346. 16 Selenium. Role of the Essential Metalloid in Health 529

64. R. Wu, Q. Shen, P. E. Newburger, J. Cell. Biochem. 2000, 77, 507–516. 65. A. Small-Howard, N. Morozova, Z. Stoytcheva, E. P. Forry, J. B. Mansell, J. W. Harney, B. A. Carlson, X. M. Xu, D. L. Hatfield, M. J. Berry, Mol. Cell Biol. 2006, 26, 2337–2346. 66. W. H. Cheng, Y. S. Ho, B. A. Valentine, D. A. Ross, G. F. Combs, X. G. Lei, J. Nutr. 1998, 128, 1070–1076. 67. Y. Fu, W. H. Cheng, J. M. Porres, D. A. Ross, X. G. Lei, Free Radic. Biol. Med. 1999, 27, 605–611. 68. F. F. Chu, J. H. Doroshow, R. S. Esworthy, J. Biol. Chem. 1993, 268, 2571–2576. 69. H. Mörk, O. H. Al-Taie, K. Bähr, A. Zierer, C. Beck, M. Scheurlen, F. Jakob, J. Köhrle, Nutr. Cancer 2000, 37, 108–116. 70. S. Florian, K. Wingler, K. Schmehl, G. Jacobasch, O. J. Kreuzer, W. Meyerhof, R. Brigelius- Flohé, Free Radic. Res. 2001, 35, 655–663. 71. Y. M. Lin, Y. Furukawa, T. Tsunoda, C. T. Yue, K. C. Yang, Y. Nakamura, Oncogene 2002, 21, 4120–4128. 72. S. T. Chiu, F. J. Hsieh, S. W. Chen, C. L. Chen, H. F. Shu, H. Li, Cancer Epidemiol. Biomarkers Prev. 2005, 14, 437–443. 73. H. Mörk, M. Scheurlen, O. Al-Taie, A. Zierer, M. Kraus, K. Schöttker, F. Jakob, J. Köhrle, Int. J. Cancer 2003, 105, 300–304. 74. M. M. Serewko, C. Popa, A. L. Dahler, L. Smith, G. M. Strutton, W. Coman, A. J. Dicker, N. A. Saunders, Cancer Res. 2002, 62, 3759–3765. 75. M. Woenckhaus, L. Klein-Hitpass, U. Grepmeier, J. Merk, M. Pfeifer, P. Wild, M. Bettstetter, P. Wuensch, H. Blaszyk, A. Hartmann, F. Hofstaedter, W. Dietmaier, J. Pathol. 2006, 210, 192–204. 76. S. Florian, S. Krehl, M. Loewinger, A. Kipp, A. Banning, S. Esworthy, F. F. Chu, R. Brigelius- Flohé, Free Radic. Biol. Med. 2010, 49, 1694–1702. 77. M. A. Hahn, T. Hahn, D. H. Lee, R. S. Esworthy, B. W. Kim, A. D. Riggs, F. F. Chu, G. P. Pfeifer, Cancer Res. 2008, 68, 10280–10289. 78. N. Avissar, D. B. Ornt, Y. Yagil, S. Horowitz, R. H. Watkins, E. A. Kerl, K. Takahashi, I. S. Palmer, H. J. Cohen, Am. J. Physiol. 1994, 266, C367–375. 79. J. C. Whitin, S. Bhamre, D. M. Tham, H. J. Cohen, Am. J. Physiol. Renal Physiol. 2002, 283, F20–28. 80. G. E. Olson, J. C. Whitin, K. E. Hill, V. P. Winfrey, A. K. Motley, L. M. Austin, J. Deal, H. J. Cohen, R. F. Burk, Am. J. Physiol. Renal Physiol. 2010, 298, F1244–1253. 81. R. F. Burk, G. E. Olson, V. P. Winfrey, K. E. Hill, D. Yin, Am. J. Physiol. Gastrointest. Liver Physiol. 2011, 301, G32–38. 82. K. Takahashi, N. Avissar, J. Whitin, H. Cohen, Arch. Biochem. Biophys. 1987, 256, 677–686. 83. A. Wendel, P. Cikryt, FEBS Lett. 1980, 120, 209–211. 84. O. J. Lee, R. Schneider-Stock, P. A. McChesney, D. Kuester, A. Roessner, M. Vieth, C. A. Moskaluk, W. El-Rifai, Neoplasia 2005, 7, 854–861. 85. Y. P. Yu, G. Yu, G. Tseng, K. Cieply, J. Nelson, M. Defrances, R. Zarnegar, G. Michalopoulos, J. H. Luo, Cancer Res. 2007, 67, 8043–8050. 86. F. Ursini, M. Maiorino, M. Valente, L. Ferri, C. Gregolin, Biochim. Biophys. Acta 1982, 710, 197–211. 87. F. Ursini, S. Heim, M. Kiess, M. Maiorino, A. Roveri, J. Wissing, L. Flohé, Science 1999, 285, 1393–1396. 88. L. J. Yant, Q. Ran, L. Rao, H. Van Remmen, T. Shibatani, J. G. Belter, L. Motta, A. Richardson, T. A. Prolla, Free Radic. Biol. Med. 2003, 34, 496–502. 89. H. Imai, F. Hirao, T. Sakamoto, K. Sekine, Y. Mizukura, M. Saito, T. Kitamoto, M. Hayasaka, K. Hanaoka, Y. Nakagawa, Biochem. Biophys. Res. Commun. 2003, 305, 278–286. 90. M. Schneider, H. Förster, A. Boersma, A. Seiler, H. Wehnes, F. Sinowatz, C. Neumüller, M. J. Deutsch, A. Walch, M. Hrabé de Angelis, W. Wurst, F. Ursini, A. Roveri, M. Maleszewski, M. Maiorino, M. Conrad, FASEB J. 2009, 23, 3233–3242. 530 Kurokawa and Berry

91. M. Conrad, S. G. Moreno, F. Sinowatz, F. Ursini, S. Kölle, A. Roveri, M. Brielmeier, W. Wurst, M. Maiorino, G. W. Bornkamm, Mol. Cell Biol. 2005, 25, 7637–7644. 92. P. M. Yen, Physiol. Rev. 2001, 81, 1097–1142. 93. P. R. Larsen, T. E. Dick, B. P. Markovitz, M. M. Kaplan, T. G. Gard, J. Clin. Invest. 1979, 64, 117–128. 94. B. Gereben, A. M. Zavacki, S. Ribich, B. W. Kim, S. A. Huang, W. S. Simonides, A. Zeöld, A. C. Bianco, Endocr. Rev. 2008, 29, 898–938. 95. G. D. Sagar, B. Gereben, I. Callebaut, J. P. Mornon, A. Zeöld, W. S. da Silva, C. Luongo, M. Dentice, S. M. Tente, B. C. Freitas, J. W. Harney, A. M. Zavacki, A. C. Bianco, Mol. Cell Biol. 2007, 27, 4774–4783. 96. C. Curcio-Morelli, B. Gereben, A. M. Zavacki, B. W. Kim, S. Huang, J. W. Harney, P. R. Larsen, A. C. Bianco, Endocrinology 2003, 144, 937–946. 97. J. L. Leonard, T. J. Visser, D. M. Leonard, J. Biol. Chem. 2001, 276, 2600–2607. 98. A. C. Bianco, D. Salvatore, B. Gereben, M. J. Berry, P. R. Larsen, Endocr. Rev. 2002, 23, 38–89. 99. D. Salvatore, J. W. Harney, P. R. Larsen, Biochimie 1999, 81, 535–538. 100. J. E. Silva, P. R. Larsen, J. Clin. Invest. 1985, 76, 2296–2305. 101. A. C. Bianco, J. E. Silva, J. Clin. Invest. 1987, 79, 295–300. 102. A. C. Bianco, J. E. Silva, Am. J. Physiol. 1988, 255, E496–503. 103. L. A. de Jesus, S. D. Carvalho, M. O. Ribeiro, M. Schneider, S. W. Kim, J. W. Harney, P. R. Larsen, A. C. Bianco, J. Clin. Invest. 2001, 108, 1379–1385. 104. J. H. Bassett, A. Boyde, P. G. Howell, R. H. Bassett, T. M. Galliford, M. Archanco, H. Evans, M. A. Lawson, P. Croucher, D. L. St Germain, V. A. Galton, G. R. Williams, Proc. Natl. Acad. Sci. USA 2010, 107, 7604–7609. 105. A. Marsili, C. Aguayo-Mazzucato, T. Chen, A. Kumar, M. Chung, E. P. Lunsford, J. W. Harney, T. Van-Tran, E. Gianetti, W. Ramadan, C. Chou, S. Bonner-Weir, P. R. Larsen, J. E. Silva, A. M. Zavacki, PLoS One 2011, 6, e20832. 106. D. L. St Germain, V. A. Galton, Thyroid 1997, 7, 655–668. 107. E. S. Arnér, A. Holmgren, Eur. J. Biochem. 2000, 267, 6102–6109. 108. Q. A. Sun, L. Kirnarsky, S. Sherman, V. N. Gladyshev, Proc. Natl. Acad. Sci. USA 2001, 98, 3673–3678. 109. D. Su, S. V. Novoselov, Q. A. Sun, M. E. Moustafa, Y. Zhou, R. Oko, D. L. Hatfield, V. N. Gladyshev, J. Biol. Chem. 2005, 280, 26491–26498. 110. M. Conrad, C. Jakupoglu, S. G. Moreno, S. Lippl, A. Banjac, M. Schneider, H. Beck, A. K. Hatzopoulos, U. Just, F. Sinowatz, W. Schmahl, K. R. Chien, W. Wurst, G. W. Bornkamm, M. Brielmeier, Mol. Cell Biol. 2004, 24, 9414–9423. 111. C. Jakupoglu, G. K. Przemeck, M. Schneider, S. G. Moreno, N. Mayr, A. K. Hatzopoulos, M. H. de Angelis, W. Wurst, G. W. Bornkamm, M. Brielmeier, M. Conrad, Mol. Cell Biol. 2005, 25, 1980–1988. 112. H. Y. Kim, V. N. Gladyshev, Mol. Biol. Cell 2004, 15, 1055–1064. 113. V. N. Gladyshev, K. T. Jeang, J. C. Wootton, D. L. Hatfield, J. Biol. Chem. 1998, 273, 8910–8915. 114. V. M. Labunskyy, D. L. Hatfield, V.N . Gladyshev, IUBMB Life 2007, 59, 1–5. 115. E. Kumaraswamy, A. Malykh, K. V. Korotkov, S. Kozyavkin, Y. Hu, S. Y. Kwon, M. E. Moustafa, B. A. Carlson, M. J. Berry, B. J. Lee, D. L. Hatfield, A. M. Diamond, V. N. Gladyshev, J. Biol. Chem. 2000, 275, 35540–35547. 116. K. V. Korotkov, S. V. Novoselov, D. L. Hatfield, V. N. Gladyshev, Mol. Cell Biol. 2002, 22, 1402–1411. 117. K. V. Korotkov, E. Kumaraswamy, Y. Zhou, D. L. Hatfield, V.N . Gladyshev, J. Biol. Chem. 2001, 276, 15330–15336. 118. R. Irons, P. A. Tsuji, B. A. Carlson, P. Ouyang, M. H. Yoo, X. M. Xu, D. L. Hatfield, V. N. Gladyshev, C. D. Davis, Cancer Prev. Res. (Phila) 2010, 3, 630–639. 119. P. A. Tsuji, S. Naranjo-Suarez, B. A. Carlson, R. Tobe, M. H. Yoo, C. D. Davis, Nutrients 2011, 3, 805-817. 16 Selenium. Role of the Essential Metalloid in Health 531

120. P. A. Tsuji, B. A. Carlson, S. Naranjo-Suarez, M. H. Yoo, X. M. Xu, D. E. Fomenko, V. N. Gladyshev, D. L. Hatfield, C. D. Davis,PLoS One 2012, 7, e50574. 121. S. Ma, K. E. Hill, R. M. Caprioli, R. F. Burk, J. Biol. Chem. 2002, 277, 12749–12754. 122. G. E. Olson, V. P. Winfrey, S. K. Nagdas, K. E. Hill, R. F. Burk, J. Biol. Chem. 2007, 282, 12290–12297. 123. R. F. Burk, K. E. Hill, G. E. Olson, E. J. Weeber, A. K. Motley, V. P. Winfrey, L. M. Austin, J. Neurosci. 2007, 27, 6207–6211. 124. G. E. Olson, V. P. Winfrey, K. E. Hill, R. F. Burk, J. Biol. Chem. 2008, 283, 6854–6860. 125. A. Lescure, M. Rederstorff, A. Krol, P. Guicheney, V. Allamand, Biochim. Biophys. Acta 2009, 1790, 1569–1574. 126. P. D. Whanger, Biochim. Biophys. Acta 2009, 1790, 1448–1452. 127. Y. J. Kim, Y. G. Chai, J. C. Ryu, Biochem. Biophys. Res. Commun. 2005, 330, 1095–1102. 128. J. Loflin,N . Lopez, P. D. Whanger, C. Kioussi, J. Inorg. Biochem. 2006, 100, 1679–1684. 129. A. Dikiy, S. V. Novoselov, D. E. Fomenko, A. Sengupta, B. A. Carlson, R. L. Cerny, K. Ginalski, N. V. Grishin, D. L. Hatfield, V. N. Gladyshev, Biochemistry 2007, 46, 6871–6882. 130. Y. Tanguy, A. Falluel-Morel, S. Arthaud, L. Boukhzar, D. L. Manecka, A. Chagraoui, G. Prevost, S. Elias, I. Dorval-Coiffec, J. Lesage, D. Vieau, I. Lihrmann, B. Jégou, Y. Anouar, Endocrinology 2011, 152, 4322–4335. 131. M. A. Reeves, F. P. Bellinger, M. J. Berry, Antioxid. Redox Signal 2010, 12, 809–818. 132. S. V. Novoselov, G. V. Kryukov, X. M. Xu, B. A. Carlson, D. L. Hatfield, V.N . Gladyshev, J. Biol. Chem. 2007, 282, 11960–11968. 133. J. L. Burkhead, M. Ralle, P. Wilmarth, L. David, S. Lutsenko, J. Mol. Biol. 2011, 406, 44–58. 134. J. Panee, Z. R. Stoytcheva, W. Liu, M. J. Berry, J. Biol. Chem. 2007, 282, 23759–23765. 135. N. Mendelev, S. Witherspoon, P. A. Li, Exp. Neurol. 2009, 220, 328–334. 136. N. Fradejas, M. D. Pastor, S. Mora-Lee, P. Tranque, S. Calvo, J. Mol. Neurosci. 2008, 35, 259–265. 137. Y. Gao, H. C. Feng, K. Walder, K. Bolton, T. Sunderland, N. Bishara, M. Quick, L. Kantham, G. R. Collier, FEBS Lett. 2004, 563, 185–190. 138. Y. Ye, Y. Shibata, C. Yun, D. Ron, T. A. Rapoport, Nature 2004, 429, 841–847. 139. B. N. Lilley, H. L. Ploegh, Nature 2004, 429, 834–840. 140. B. Meusser, C. Hirsch, E. Jarosch, T. Sommer, Nat. Cell Biol. 2005, 7, 766–772. 141. B. N. Lilley, H. L. Ploegh, Proc. Natl. Acad. Sci. USA 2005, 102, 14296–14301. 142. K. Walder, L. Kantham, J. S. McMillan, J. Trevaskis, L. Kerr, A. De Silva, T. Sunderland, N. Godde, Y. Gao, N. Bishara, K. Windmill, J. Tenne-Brown, G. Augert, P. Z. Zimmet, G. R. Collier, Diabetes 2002, 51, 1859–1866. 143. S. Du, J. Zhou, Y. Jia, K. Huang, Arch. Biochem. Biophys. 2010, 502, 137–143. 144. S. Verma, F. W. Hoffmann, M. Kumar, Z. Huang, K. Roe, E. Nguyen-Wu, A. S. Hashimoto, P. R. Hoffmann, J. Immunol. 2011, 186, 2127–2137. 145. Z. Huang, F. W. Hoffmann, R. L. Norton, A. C. Hashimoto, P. R. Hoffmann, J. Biol. Chem. 2011, 286, 34830–34838. 146. n. Petit, A. Lescure, M. Rederstorff, A. Krol, B. Moghadaszadeh, U. M. Wewer, P. Guicheney, Hum. Mol. Genet. 2003, 12, 1045–1053. 147. P. Castets, A. T. Bertrand, M. Beuvin, A. Ferry, F. Le Grand, M. Castets, G. Chazot, M. Rederstorff, A. Krol, A. Lescure, N. B. Romero, P. Guicheney, V. Allamand, Hum. Mol. Genet. 2011, 20, 694–704. 148. M. J. Jurynec, R. Xia, J. J. Mackrill, D. Gunther, T. Crawford, K. M. Flanigan, J. J. Abramson, M. T. Howard, D. J. Grunwald, Proc. Natl. Acad. Sci. USA 2008, 105, 12485–12490. 149. S. Arbogast, M. Beuvin, B. Fraysse, H. Zhou, F. Muntoni, A. Ferreiro, Ann. Neurol. 2009, 65, 677–686. 150. S. Arbogast, A. Ferreiro, Antioxid. Redox Signal 2010, 12, 893–904. 151. G. S. Li, F. Wang, D. Kang, C. Li, Hum. Pathol. 1985, 16, 602–609. 152. M. A. Beck, O. A. Levander, J. Handy, J. Nutr. 2003, 133, 1463S–1467S. 153. O. A. Levander, M. A. Beck, Biol. Trace Elem. Res. 1997, 56, 5–21. 532 Kurokawa and Berry

154. R. Moreno-Reyes, F. Mathieu, M. Boelaert, F. Begaux, C. Suetens, M. T. Rivera, J. Nève, N. Perlmutter, J. Vanderpas, Am. J. Clin. Nutr. 2003, 78, 137–144. 155. Y. M. Xiong, X. Y. Mo, X. Z. Zou, R. X. Song, W. Y. Sun, W. Lu, Q. Chen, Y. X. Yu, W. J. Zang, Osteoarthritis Cartilage 2010, 18, 817–824. 156. J. E. Dumont, B. Corvilain, B. Contempre, Mol. Cell Endocrinol. 1994, 100, 163–166. 157. J. Golstein, B. Corvilain, F. Lamy, D. Paquer, J. E. Dumont, Acta Endocrinol. 1988, 118, 495–502. 158. S. B. Goldhaber, Regul. Toxicol. Pharmacol. 2003, 38, 232–242. 159. R. D. Lillie, M. I. Smith, Am. J. Pathol. 1940, 16, 223–228. 160. J. N. Hathcock, Am. J. Clin. Nutr. 1997, 66, 427–437. 161. D. Behne, H. Hilmert, S. Scheid, H. Gessner, W. Elger, Biochim. Biophys. Acta 1988, 966, 12–21. 162. D. Behne, T. Höfer-Bosse, J. Nutr. 1984, 114, 1289–1296. 163. Y. Zhang, Y. Zhou, U. Schweizer, N. E. Savaskan, D. Hua, J. Kipnis, D. L. Hatfield, V. N. Gladyshev, J. Biol. Chem. 2008, 283, 2427–2438. 164. K. E. Hill, J. Zhou, W. J. McMahan, A. K. Motley, J. F. Atkins, R. F. Gesteland, R. F. Burk, J. Biol. Chem. 2003, 278, 13640–13646. 165. L. Schomburg, U. Schweizer, B. Holtmann, L. Flohé, M. Sendtner, J. Köhrle, Biochem. J. 2003, 370, 397–402. 166. O. Agamy, B. Ben Zeev, D. Lev, B. Marcus, D. Fine, D. Su, G. Narkis, R. Ofir, C. Hoffmann, E. Leshinsky-Silver, H. Flusser, S. Sivan, D. Söll, T. Lerman-Sagie, O. S. Birk, Am. J. Hum. Genet. 2010, 87, 538–544. 167. L. C. Clark, G. F. Combs, B. W. Turnbull, E. H. Slate, D. K. Chalker, J. Chow, L. S. Davis, R. A. Glover, G. F. Graham, E. G. Gross, A. Krongrad, J. L. Lesher, H. K. Park, B. B. Sanders, C. L. Smith, J. R. Taylor, J. Am. Med. Ass. 1996, 276, 1957–1963. 168. S. Stranges, J. R. Marshall, M. Trevisan, R. Natarajan, R. P. Donahue, G. F. Combs, E. Farinaro, L. C. Clark, M. E. Reid, Am. J. Epidemiol. 2006, 163, 694–699. 169. S. Stranges, J. R. Marshall, R. Natarajan, R. P. Donahue, M. Trevisan, G. F. Combs, F. P. Cappuccio, A. Ceriello, M. E. Reid, Ann. Intern. Med. 2007, 147, 217–223. 170. S. M. Lippman, E. A. Klein, P. J. Goodman, M. S. Lucia, I. M. Thompson, L. G. Ford, H. L. Parnes, L. M. Minasian, J. M. Gaziano, J. A. Hartline, J. K. Parsons, J. D. Bearden, E. D. Crawford, G. E. Goodman, J. Claudio, E. Winquist, E. D. Cook, D. D. Karp, P. Walther, M. M. Lieber, A. R. Kristal, A. K. Darke, K. B. Arnold, P. A. Ganz, R. M. Santella, D. Albanes, P. R. Taylor, J. L. Probstfield, T. J. Jagpal, J. J. Crowley, F. L. Meyskens, L. H. Baker, C. A. Coltman, J. Am. Med. Ass. 2009, 301, 39–51. 171. H. Misu, T. Takamura, H. Takayama, H. Hayashi, N. Matsuzawa-Nagata, S. Kurita, K. Ishikura, H. Ando, Y. Takeshita, T. Ota, M. Sakurai, T. Yamashita, E. Mizukoshi, M. Honda, K. Miyamoto, T. Kubota, N. Kubota, T. Kadowaki, H. J. Kim, I. K. Lee, Y. Minokoshi, Y. Saito, K. Takahashi, Y. Yamada, N. Takakura, S. Kaneko, Cell Metab. 2010, 12, 483–495. 172. A. Sengupta, B. A. Carlson, V. J. Hoffmann, V. N. Gladyshev, D. L. Hatfield, Biochem. Biophys. Res. Commun. 2008, 365, 446–452. 173. X. D. Wang, M. Z. Vatamaniuk, S. K. Wang, C. A. Roneker, R. A. Simmons, X. G. Lei, Diabetologia 2008, 51, 1515–1524. 174. U. Ahlgren, J. Jonsson, L. Jonsson, K. Simu, H. Edlund, Genes Dev 1998, 12, 1763–1768. 175. L. K. Olson, A. Sharma, M. Peshavaria, C. V. Wright, H. C. Towle, R. P. Rodertson, R. Stein, Proc. Natl. Acad. Sci. USA 1995, 92, 9127–9131. 176. M. Tiedge, S. Lortz, J. Drinkgern, S. Lenzen, Diabetes 1997, 46, 1733–1742. 177. U. Ozcan, Q. Cao, E. Yilmaz, A. H. Lee, N. N. Iwakoshi, E. Ozdelen, G. Tuncman, C. Gorgun, L. H. Glimcher, G. S. Hotamisligil, Science 2004, 306, 457–461. 178. M. Olsson, B. Olsson, P. Jacobson, D. S. Thelle, J. Björkegren, A. Walley, P. Froguel, L. M. Carlsson, K. Sjöholm, Metabolism 2011, 60, 114–120. 16 Selenium. Role of the Essential Metalloid in Health 533

179. E. D. R. Arrojo, T. L. Fonseca, M. Castillo, M. Salathe, G. Simovic, P. Mohacsik, B. Gereben, A. C. Bianco, Mol. Endocrinol. 2011, 25, 2065–2075. 180. M. Castillo, J. A. Hall, M. Correa-Medina, C. Ueta, H. W. Kang, D. E. Cohen, A. C. Bianco, Diabetes 2011, 60, 1082–1089. 181. L. H. Canani, C. Capp, J. M. Dora, E. L. Meyer, M. S. Wagner, J. W. Harney, P. R. Larsen, J. L. Gross, A. C. Bianco, A. L. Maia, J. Clin. Endocrinol. Metab. 2005, 90, 3472–3478. 182. D. Mentuccia, L. Proietti-Pannunzi, K. Tanner, V. Bacci, T. I. Pollin, E. T. Poehlman, A. R. Shuldiner, F. S. Celi, Diabetes 2002, 51, 880–883. 183. M. C. Medina, J. Molina, Y. Gadea, A. Fachado, M. Murillo, G. Simovic, A. Pileggi, A. Hernandez, H. Edlund, A. C. Bianco, Endocrinology 2011, 152, 3717–3727. 184. S. H. Wu, J. E. Oldfield, P. D. Whanger, P. H. Weswig,Biol. Reprod. 1973, 8, 625–629. 185. A. S. Wu, J. E. Oldfield, L. R. Shull, P. R. Cheeke,Biol. Reprod. 1979, 20, 793–798. 186. D. G. Brown, R. F. Burk, J. Nutr. 1973, 103, 102–108. 187. M. Maiorino, A. Roveri, L. Benazzi, V. Bosello, P. Mauri, S. Toppo, S. C. Tosatto, F. Ursini, J. Biol. Chem. 2005, 280, 38395–38402. 188. K. Nayernia, I. M. Adham, E. Burkhardt-Göttges, J. Neesen, M. Rieche, S. Wolf, U. Sancken, K. Kleene, W. Engel, Mol. Cell Biol. 2002, 22, 3046–3052. 189. H. Imai, N. Hakkaku, R. Iwamoto, J. Suzuki, T. Suzuki, Y. Tajima, K. Konishi, S. Minami, S. Ichinose, K. Ishizaka, S. Shioda, S. Arata, M. Nishimura, S. Naito, Y. Nakagawa, J. Biol. Chem. 2009, 284, 32522–32532. 190. M. Maiorino, P. Mauri, A. Roveri, L. Benazzi, S. Toppo, V. Bosello, F. Ursini, FEBS Lett. 2005, 579, 667–670. 191. C. D. Reilly, Selenium in Food and Health, Springer, New York, 2006. 192. C. D. Reilly, Selenium in Food and Health, Blackie Academic & Professional, London, 1996. 193. J. W. Finley, Ann. Bot. 2005, 95, 1075–1096. 194. M. Deveau, J. Toxicol. Environ. Health A 2010, 73, 235–241. 195. G. A. Cutter, in Occurence and Distribution of Selenium, Ed M. Ihnat, CRC Press, Boca Raton, 1989. 196. Environmental Protection Agency USA, “National Primary and Secondary Drinking Water Regulations, Final Rule, Selenium, January, U.S. EPA”, Washington, 1991. 197. W. R. Wolf, R. J. Goldschmidt, Anal. Bioanal. Chem. 2007, 387, 2449–2452. 198. W. C. Wang, A. L. Mäkelä, V. Näntö, P. Mäkelä, H. Lagström, Eur. J. Clin. Nutr. 1998, 52, 529–535. 199. J. H. Watkinson, New Zeal. Vet. J. 1983, 31, 78–85. 200. T. C. Madison, Statistical Report on the Sickness and Mortality in the Army of the United States, January 1855 to January 1860, Washington, DC, 1860. 201. K. W. Franke, J. Nutr. 1934, 8, 597–608. 202. Agricultural Research Service, USA, Department of Agriculture, Selenium-Accumulating Plants, 2006, http://www.ars.usda.gov/Services/docs.htm?docid=9979 203. S. J. Fairweather-Tait, Y. Bao, M. R. Broadley, R. Collings, D. Ford, J. E. Hesketh, R. Hurst, Antioxid. Redox Signal. 2011, 14, 1337–1383. 204. M. Navarro-Alarcon, C. Cabrera-Vique, Sci. Total Environ. 2008, 400, 115–141. 205. J. S. Edmonds, M. Morita, Appl. Organomet. Chem. 2000, 14, 133–145. 206. M. Kotrebai, M. Birringer, J. F. Tyson, E. Block, P. C. Uden, Analyst 2000, 125, 71–78. 207. I. Arnault, J. Auger, J. Chromatogr. A 2006, 1112, 23–30. 208. R. Lobinski, J. S. Edmonds, K. T. Suzuki, P. C. Uden, Pure Appl. Chem. 2000, 72, 447–461. 209. V. I. Fisinin, T. T. Papazyan, P. F. Surai, Crit. Rev. Biotechnol. 2009, 29, 18–28. 210. C. D. Davis, J. W. Finley, Chemical versus Food Forms of Selenium in Cancer Prevention, in Functional Foods and Nutraceuticals in Cancer Prevention and Treatment, Iowa State University Press, Ames, 2003. 211. A. Nakayama, K. E. Hill, L. M. Austin, A. K. Motley, R. F. Burk, J. Nutr. 2007, 137, 690–693. 534 Kurokawa and Berry

212. K. E. Hill, S. Wu, A. K. Motley, T. D. Stevenson, V. P. Winfrey, M. R. Capecchi, J. F. Atkins, R. F. Burk, J. Biol. Chem. 2012, 287, 40414–40424. 213. R. F. Burk, K. E. Hill, A. K. Motley, L. M. Austin, B. K. Norsworthy, Biochim. Biophys. Acta 2006, 1760, 1789–1793. 214. D. H. Kim, H. Iijima, K. Goto, J. Sakai, H. Ishii, H. J. Kim, H. Suzuki, H. Kondo, S. Saeki, T. Yamamoto, J. Biol. Chem. 1996, 271, 8373–8380. 215. S. Himeno, H. S. Chittum, R. F. Burk, J. Biol. Chem. 1996, 271, 15769–15775. 216. J. Chiu-Ugalde, F. Theilig, T. Behrends, J. Drebes, C. Sieland, P. Subbarayal, J. Köhrle, A. Hammes, L. Schomburg, U. Schweizer, Biochem. J. 2010, 431, 103–111. 217. K. E. Hill, J. Zhou, L. M. Austin, A. K. Motley, A. J. Ham, G. E. Olson, J. F. Atkins, R. F. Gesteland, R. F. Burk, J. Biol. Chem. 2007, 282, 10972–10980. 218. S. Kurokawa, K. E. Hill, W. H. McDonald, R. F. Burk, J. Biol. Chem. 2012, 287, 28717–28726. 219. J. L. Byard, Arch. Biochem. Biophys. 1969, 130, 556–560. 220. K. T. Suzuki, K. Kurasaki, N. Okazaki, Y. Ogra, Toxicol. Appl. Pharmacol. 2005, 206, 1–8. 221. Y. Kobayashi, Y. Ogra, K. Ishiwata, H. Takayama, N. Aimi, K. T. Suzuki, Proc. Natl. Acad. Sci. USA 2002, 99, 15932–15936. 222. H. E. Ganther, O. A. Levander, C. A. Baumann, J. Nutr. 1966, 88, 55–60. 223. K. P. McConnell, O. W. Portman, J. Biol. Chem. 1952, 195, 277–282. 224. L. F. Pedrosa, A. K. Motley, T. D. Stevenson, K. E. Hill, R. F. Burk, Biol. Trace Elem. Res. 2012, 149, 377–381. 225. National Research Council, “Recommended Dietary Allowances / Committee on Dietary Allowances, 9th edn”, Washington, D.C., 1980. 226. J. G. Yang, K. E. Hill, R. F. Burk, J. Nutr. 1989, 119, 1010–1012. 227. Y. Xia, K. E. Hill, D. W. Byrne, J. Xu, R. F. Burk, Am. J. Clin. Nutr. 2005, 81, 829–834. 228. R. F. Burk, B. K. Norsworthy, K. E. Hill, A. K. Motley, D. W. Byrne, Cancer Epidemiol. Biomarkers Prev. 2006, 15, 804–810. 229. Y. Xia, K. E. Hill, P. Li, J. Xu, D. Zhou, A. K. Motley, L. Wang, D. W. Byrne, R. F. Burk, Am. J. Clin. Nutr. 2010, 92, 525–531. Index

A disturbance , 40 A β amyloid plaques, 277 imbalance , 43, 44

metabolism , 377 Acid dissociation constants (pK a ) , 146, 148, ABC. See ATP-binding cassette 151, 267, 454, 477, 502 superfamilies (ABC) Acidosis , 40–42, 44, 402 Absorption of Acid phosphatase , 86, 150 calcium , 84, 85, 127 Acid-sensing channel , 402 chromium , 176, 177 Acinetobacter baumannii , 11 cobalamin , 296, 298–300, 311, 313 Acireductone dioxygenase , 323, 336–338, iron , 242, 243, 247, 253–255, 257, 341, 342 258, 348 ACOG. See American College of Obstetrics potassium , 34 and Gynecology (ACOG) silicon , 464 Aconitase , 246, 402 sodium , 37 Acrodermatitis enteropathica , 11, Accumulation of 404, 408 calcium , 70, 89, 97, 119 Acrylamide production , 314 copper , 366, 376, 378 ACTH (adrenocorticotropic hormone) , 395 glycogen , 155 Actin , 108, 109, 112, 125 hydrogen sulﬁ de , 441 Actinobacteria , 349 iron , 208, 256, 277–280, 363 Actinolite , 463 magnesium , 51, 54, 55, 63–65 Action of manganese , 204, 208, 209, 213, 216 potassium on membranes , 32, 33 methyltetrahydrofolate , 310 sodium on membranes , 32, 33 sulﬁ te , 425, 435, 436 Active sites , 106, 150, 151, 159, 217, 314, uric acid , 421 323, 336–341, 345, 350, 362, 393, 416, xanthine , 420, 421 417, 434, 502, 512, 513 ACD. See Allergic contact dermatitis (ACD) Acute Aceruloplasminemia , 363 lymphoblastic leukemia , 421 Acetate , 14, 256, 322, 325 promyelocytic leukemia (APL) , 476, 477, Acetohydroxamate (and acid) , 234, 268, 269 492, 493 Acetylation of histones , 328, 396 renal failure , 38, 40, 41, 45, 421 Acetylcholine , 33 toxicity of arsenic , 481 AcetylCoA/CoA ratio , 107 AD. See Alzheimer’s disease (AD) Acetyl-CoA decarbonylase synthase , 342, 343 Addison disease , 44 Acid-base Adenosine balance , 31, 37 deoxy- , 303, 427

A. Sigel, H. Sigel, and R.K.O. Sigel (eds.), Interrelations between Essential 535 Metal Ions and Human Diseases, Metal Ions in Life Sciences 13, DOI 10.1007/978-94-007-7500-8, © Springer Science+Business Media Dordrecht 2013 536 Index

Adenosine 5′-diphosphate (ADP), 97, 107, Agency for Healthcare Research 111, 306, 481, 504 and Quality , 183 -arsenate , 481 Age-related macular degeneration , 279, Adenosine 5′-monophosphate (AMP) 280, 403 -activated protein kinase Aging , 64, 254, 255, 391, 457, 469 (AMPK), 115, 187, 189 Agranulocytosis , 274, 276 c- , 53–55, 68, 100, 428, 429, 431, 432, 434 Agricultural laborers , 251 Adenosine 5′-triphosphate (ATP) , 33, 51, Akt , 154–156, 159, 161, 186, 187, 189, 190 52, 54, 55, 65, 95, 104, 106–108, Albumin , 3, 10, 15, 18, 19, 21, 53, 73, 145, 111, 114, 124, 206, 209, 215, 217, 155, 203, 206, 207, 256, 274, 323 263, 302, 303, 306, 309, 341, 342, parv- , 52, 119 348, 368, 369, 391, 417, 429, 435, Alcohol , 58, 65, 257, 396, 399, 422 480, 481, 486, 503–505, 513 dehydrogenase , 65 ADP ratio , 107, 111 Alcoholism , 58, 65, 66 -cob(I)alamin adenosyltransferase , 302 Alcohol liver disease (ALD) , 65 hydrolysis , 108, 342, 368, 480, 486 Aldehyde oxidase (AOX) , 419, 421–423, production , 106, 108, 215 431, 442 -sensitive K + channels , 111 defi ciency , 431 synthase , 107, 486 -knockout mice , 422 synthesis , 108, 215, 263, 435 Aldosterone , 32, 33, 36, 37, 42–44, 59, 62, Adenosylcobalamin (Ado-Cbl) , 297, 298, 302, 67, 68 303, 305, 311 defi ciency , 44 S-Adenosyl-homocysteine , 309 Alga , 160, 161, 165, 417 hydrolase , 309, 424 Alkaline phosphatase (ALP) , 87, 88, 201, 458 S-Adenosyl-methionine (AdoMet/SAM) , 304, Alkalosis , 39, 41–44, 67 307–310, 312, 427 Allergen , 332–335 -dependent enzymes , 236 Allergic contact dermatitis (ACD) , Adenosyltransferase , 302, 309 332, 334, 335 Adenylate (or adenylyl) cyclase , 66, 515 Allopurinol , 421, 422 Adenylyltransferase , 429 ALS , 118, 121, 122, 212, 213, 362, 378, 379 Adequate intake , 173–175, 202 Aluminosilicates , 452, 456, 461, 468 ADH. See Antidiuretic hormone (ADH) Aluminum(III) , 456, 460, 461, 463, 467 Adhesives , 200 from drinking water , 461 Adipocytes , 64, 155, 180, 186–189, 519 oxides , 417 3T3-L1 , 187, 189 Alzheimer’s disease (AD) , 102, 118, 120–122, Adiponectin , 462 213, 277–280, 311, 376, 377, 380, 403, Adipose tissue , 145, 146, 405, 512, 519 406, 407, 452, 457, 460, 461, 467, 469 Ado-Cbl. See Adenosylcobalamin Amanita muscaria , 141–143 AdoMet. See S-Adenosyl-methionine Amavadin , 141–143 ADP. See Adenosine 5′-diphosphate (ADP) American College of Obstetrics and Adrenal cortex , 37 Gynecology (ACOG) , 71 β-Adrenergic stimulation , 33, 36 American Diabetes Association , 184 Adrenocorticotropic hormone (ACTH) , 395 American trypanosomiasis , 162 Adults , 3, 10, 17, 18, 20, 21, 30, 34, 41–43, 2-Amidopyridin-4-one, 275 101, 152, 173, 179, 181, 183, 191, Amiloride , 53 202, 204, 214, 247–249, 253, 260, Amino acid(s) (see also individual names) 296, 298, 331, 345, 346, 455, 484, hydroxylation , 237 485, 490, 524 metabolism , 349 Aerobactin , 283 mutations , 68 Aerosols , 143, 325 sequences , 300, 307, 368, 483 A f fi nity constants (see also Binding constants, γ-Aminobutyric acid (GABA) , 432, 434–436 Formation constants, and Stability Aminoglycoside , 58 constants), 233, 234, 245 5-Aminolevulinate (δ-aminolevulinate) Africa , 13, 253, 491, 517 synthase , 246, 263, 264 Index 537

Ammonia , 205, 303, 336, 338, 347 Anti-epidermal growth factor receptor Amoebae , 141, 162, 163 antibodies , 71–73 Amoebiasis , 140, 162–165 Antifungal Amoebocidal drug , 163 properties , 5 Amosite , 463 therapy , 9 AMP. See Adenosine 5′-monophosphate Antigen-specifi c T-cells , 332, 333 AMP-activated protein kinase (AMPK) , Antiinfl ammatory agents , 71, 217 115, 187, 189 Antimicrobial activity , 161 AMPA receptor , 402 Antioxidant(s) , 3, 4, 15–18, 213, 215, 218, Amphotericin , 58 326, 329, 436 Amylin , 405 enzymes , 16, 21, 201, 395, 396, 402 β-Amyloid or Amyloid-beta (Aβ) , 120, 213, supplementation , 17 277, 377, 460, 461 Antiparasitic properties of vanadium , 162 Amyloid precursor protein (APP) , 120, 278, Antiretroviral therapy , 16 279, 284, 376, 377, 407 Antituberculosis drugs , 162 Amyotrophic lateral sclerosis (ALS) , Antitumor activity , 147, 157 118, 121, 122, 212, 213, 277, Antiviral activity , 159, 161 362, 378, 379 APL. See Acute promyelocytic Anaphylaxis , 401 leukemia (APL) Anemia , 7, 8, 18, 64, 65, 231, 345, 348, 371, Apolipoprotein E (ApoE) , 514, 519, 522 372, 374, 400, 418 receptor-2 knockout mice , 522 of chronic disease , 8, 254, 255 Apoptosis , 65, 101, 113–116, 121, 123, 157, Anesthetic drugs , 124 158, 215, 218, 323, 329–331, 334, 347, Angiogenesis , 70, 72, 159, 330, 331, 360, 406 366, 396, 400, 466, 482, 492, 511, 515 Angiotensin II , 36, 37, 373 Apparent binding constants , 177 Animal(s) (see also individual names) zinc , 405 guts , 338, 350 Aquacobalamin , 298, 307 nutrition , 173 Aquaglyceroporins , 480, 481 studies , 6, 22, 284, 458, 459, 463, 484, Arabidopsis thaliana , 418 486, 493, 524 Archaea , 323, 336, 339, 342, 349, 350, 487, Animal models , 45, 65, 172, 212, 214, 280, 503, 504 313, 314, 316, 323, 324, 326, 436, 437, Argentina , 482 439, 442, 481, 484 Arginase , 20–22, 205 for Parkinson Disease , 378 Arginine vasopressin (AVP) , 39 mouse. See Mice and Mouse Aromatic amines , 233 of iron overload , 264 ArsA , 480 rat. See Rat Arsenate , 477–481, 485–490, 493 rodent , 193, 203, 250 diester bonds , 489 Annexins , 51, 87, 91, 92 lead , 490 Anorexia , 4, 42, 45, 396, 418 reductase , 480 Antacid , 56, 58, 250, 464 Arsenic , 452, 476–493 Anthophyllite , 463 -accumulating plants , 485 Antibacterial acid , 478 activity , 160 -based drugs , 477 agent , 468 chemical properties , 477–479 properties , 5 chronic exposure , 476, 477, 481, 482 Antibiotics , 12, 13, 58, 254, 273, 408, 491 defi ciency , 484 Anticoagulation , 399 dimethyl-(DMAs) , 481, 491 Antidiabetic eaters , 490 agents , 64, 462 excretion , 477 effect of silicon , 462 half-life in humans , 480 vanadium compounds , 154 in agriculture , 491 Antidiarrheal agent , 468 in drinking water , 482, 493 Antidiuretic hormone (ADH) , 36–38, 40 inorganic , 479, 481, 491 538 Index

Arsenic (cont. ) H + - , 99 in the environment , 479, 491 Menkes P-type , 18 levels in urine , 490 ATP7B , 363, 365–371, 374, 376, 380 metabolism , 484 ATP-binding cassette superfamilies (ABC) , methyltransferases (As3MT) , 481, 490 206, 209, 341, 417 organo- , 479, 491 ABC transporter , 300, 342, 417 stimulation of growth , 485 Atrophic gastritis , 299 toxicity , 477, 481 Australia , 374, 440 uptake , 477 Austria , 490 yellow , 479 Autoimmune Arsenic(III) , 477, 487, 492 diseases , 106, 255, 401, 421 Arsenic(V) , 477, 487 gastritis mouse model , 313 Arsenicosis , 477, 481 Autoimmunity , 313

Arsenic oxide (As2 O3 ) , 477, 490, 492 Autophagy , 98, 113–116 lethal dose , 481 Autosomal recessive disease , 11 Arsenite , 478–483, 485–489, 492, 493 Autoxidation of Arsenobetain (AsB) , 478, 480, 481 catecholamines , 362 Arsenocholine (AsC) , 478, 480, 481 iron(II) , 240 Arsenolipids , 478, 480 AV P . See Arginine vasopressin (AVP) Arsenopyrite , 479, 486 Azotobacter , 141 Arsenosugars , 478, 480 Arsenous acid , 478

Arsine (AsH3 ) , 479 B Arsphenamine , 491 Bacillus , 282 ArsR , 344, 347, 480 Bacillus pasteurii , 338 AsB. See Arsenobetain (AsB) Bacteria(l) , 3, 5, 6, 22, 141, 165, 205, 282, Asbestos , 452, 453, 463–467 296, 302, 314, 323, 324, 327, 336–340, -induced carcinogenesis , 465 342, 343, 346, 349, 401, 404, 417, 427, workers , 466 429, 480, 487, 502, 503 Asbestosis , 453, 464, 466, 467 gram-negative , 8, 161 AsC. See Arsenocholine (AsC) gram-positive , 161 Ascidia , 112 infection , 159–161, 283, 345, 347 Ascophyllum nodosum , 165 methionine synthase , 304, 307, 308 Ascorbate or ascorbic acid , 20, 147, 148, 176, nitrogen-fi xing , 141 191, 253, 329, 330, 365, 512 Bacterium bifi dum , 349 Asia , 13 Bangladesh , 164, 482, 492 As3MT. See Arsenic methyltransferases Barium(II) , 206, 208 (As3MT) Barrett’s esophagus cancer , 511 Aspergillus , 9 Bartter’s syndrome , 39, 41 Asthma , 144, 324, 401 BB-rats , 462 Astrocytes , 96, 122, 158, 201, 205, 210, 215, BCB. See Blood-cerebrospinal 216, 218, 363 fl uid barrier (BCB) Astrocytosis , 204, 212, 213, 215, 216 β-cells, 63, 111, 393, 400, 401, Ataxia , 116–118 405, 519

Atherosclerosis , 61, 62, 64, 399, 452, 457, B 12 -deﬁ cient rats , 305, 309, 313 459, 460, 469 Beef meat , 298, 299 Atopic dermatitis , 401 Beer , 38, 454, 461 ATP. See Adenosine 5′-triphosphate (ATP) Belgrade rat , 264, 265 ATP7A , 18, 363–371, 373–375, 379 Beta blockers , 37 ATP/ADP ratio , 107, 111 Beta-globulin , 203 ATPases , 82, 93, 109, 300, 347 Betaine , 314 Ca2+ /Mn2+ , 203 Beverages , 143, 422, 453, 454 efﬂ ux pump , 347 BFD. See Black foot disease (BFD) Index 539

Biﬁ dobacterium longum , 349 formation , 86, 87, 201, 458, 467, 468 Bile , 18, 20, 21, 202, 203, 369, 370, 399 health , 403, 452, 458, 459, 462, 467 excretion of copper , 371, 376 malformation , 371 Bilirubin , 3 marrow , 247, 262, 372, 462 Binding constants (see also Afﬁ nity constants, mass , 458 Formation constants, and Stability mineral density , 51, 458, 459, 467 constants), 177, 405 mineralization , 84, 452, 457, 459 Bioavailability (of) , 153, 253, 254, 266, 296, morphogenetic protein 6 , 280 452, 453, 455–457, 459, 461, 469, 517 Bordatella , 282

B 12 , 296 Borrelia burgdorferi , 5 selenium , 517 Bovine silica , 455 liver , 503, 504 Bioinformatics , 502, 506, 513 spongiform encephalopathy , 379 Biomarkers (for) , 10, 15, 22, 390, 422, 437, Bradycardia , 56 438, 466 Bradyrhizobium japonicum , 343 chromium , 178, 179 Brain , 21, 85, 101, 105, 120, 146, 158, 180, folate metabolism , 315 201–208, 210, 213, 214, 216, 217, 251, inﬂ ammation , 396 263, 277, 281, 284, 331, 332, 367, 370, methionine metabolism , 315 371, 376–378, 423, 432, 435, 436, 440, molybdenum-dependent enzymes , 435 460, 461, 515, 518, 521, 522 neuropsychiatric disorders , 441 cattle , 423 oxidative stress , 14 degeneration , 516 selenium , 524 human , 215, 402, 437 silicon , 455 injury , 402, 426 zinc , 396, 399, 407 rat , 423 Biomineralization , 86–88 Breast Biosynthesis of cancer , 281, 406, 486 phosphatidylcholine , 485 tumors , 156 selenocysteine , 503–505, 509 Brewer’s yeast , 174, 183, 186, 501 selenocysteyl-tRNA , 505, 507–509 Brody disease , 124, 125 the molybdenum cofactor , 427–430 Bromoperoxidase , 165 Biotin , 306 Bronchitis , 144, 465 Bipolar disorder , 404 Bronze , 476 Black foot disease (BFD) , 482 Brucella suis , 342 Blood Buffers , 31, 93, 394 clotting , 201, 361 HEPES , 191 donation , 249, 250 Bulimia , 42 fat , 173 Bumetanide , 39 manganese , 21, 202, 204, 217 Burns , 3, 4, 16–19, 22, 44 plasma , 15, 83, 84, 143, 145, 146, 323 patients , 13, 15 potassium , 41 pressure , 36, 37, 44, 45, 60, 61, 110, 173, 460 C -retinal barrier , 280 Cadmium , 5, 21, 207 selenium , 15, 517 cADPR. See Cyclic ADP-ribose (cADPR) sugar , 31, 184, 185, 484 Caenorhabditis elegans , 218, 314 transfusion , 258, 261–264, 276 CAKI-1 mice , 157 Blood-brain barrier (BBB) , 206, 207, 209, Calbindins , 84, 87 210, 281 Calcineurin (Cn) , 101, 104–106 Blood-cerebrospinal ﬂ uid barrier (BCB) , 207 Calcitonin , 84 Bone(s) , 50, 56, 83–87, 126, 146, 147, 324, Calcium(II), Ca 2+ , 33, 39, 45, 81–127, 325, 375, 463, 469 201, 206, 212, 392, 394, 395, 456, 458, composition , 324 459, 513, 515 540 Index

Calcium(II), Ca2+ ( cont.) Cancer (see also Carcinoma and Tumor) , 42, absorption , 84, 85, 127 69, 70, 72, 73, 102, 113, 114, 141, accumulation , 70, 89, 97, 119 144, 156–159, 163–165, 231, 254, and bioenergetics , 106–108 255, 331, 344–346, 349, 363, 391, arsenate , 490 440, 441, 452, 455, 464, 465, 468, as antagonist of Na+ , 88–93 476, 492, 493, 513, 518 as regulator , 100–116 Barrett’s esophagus , 511 as signaling agent , 88–93 bladder , 477 -ATPase , 36, 61, 98 breast , 281, 406, 486 -binding proteins , 96, 98 cervical , 157 carbonates , 83, 85 colon , 69, 70, 73, 157, 513 cytosolic , 88, 89, 95, 96, 100, 108, 111, gastric , 102 115, 119, 120, 125 gastrointestinal , 281 free , 61, 83, 87, 89, 93, 96, 111, 113 intestinal , 511 homeostasis , 86, 102, 113–115, 117, kidney , 157, 477 119–121, 124, 215 liver , 281, 477 hydroxyapatite , 85 lung , 144, 477, 482 in biological ﬂ uids , 84, 85 nasal , 325 in blood , 83, 84 ovarian , 72, 157 -induced Ca2+ release (CICR) , 95, 99, prostate , 511 109–112, 124 renal , 331 in plasma , 83, 84 respiratory tract , 325 Mn 2+ -ATPases , 203 skin , 477, 482 overload , 86, 88, 89, 96, 102, 113, 116, testicular , 157, 158 119, 122, 126 Candida , 9 oxalate , 83 albicans , 373 phosphate , 83, 85, 87, 88 Carbohydrate metabolism , 31, 107, 175, 201 picolinate , 83 Carbonates , 83, 85 regulation , 97, 116, 329, 515 Carbon dioxide , 336, 487 release , 33, 94–96, 98, 99, 104, 109–111, Carbonic anhydrase IX , 330 113, 120, 124, 125 Carbon monoxide , 140, 336, 337, 342, 343 secretion , 110–111 dehydrogenase , 342, 343 signaling , 94, 97, 98, 100, 116, Carboxylates , 233, 237, 271, 273, 338 118, 121 Carcinogenicity , 192, 325, 327, 345, 465 sulfate , 83 Carcinoma (see also Cancer and Tumor) transport , 96–98, 106, 208 colon adeno- , 511 uniporter , 93, 203, 215 esophageal , 406 uptake , 94, 99 gastric , 344 Calcium channel(s) , 59, 85, 87, 92, hepato- , 513 95–100, 109, 111, 117–120, 124, liver , 256 206, 209 lung adeno- , 511 L-type , 100, 109, 119 oral small cell , 406 Calmodulin , 52, 91, 105, 106, 513 squamous cell , 511 -dependent kinase (CaMK) , 100, 101, Cardiac 104, 105 arrest , 31, 41, 43, 56, 57 Calpains , 101–103, 119, 126 arrhythmias , 42, 59, 60 Calpastatin , 103, 516 diseases , 123, 124, 276 Calprotectin , 5, 404 hypertrophy , 159 Calprotectinemia , 404 iron , 274 Calretinin , 84, 119 muscles , 109, 123 cAMP. See Cyclic adenosine monophosphate myocytes , 63, 95, 98, 105, 109 (cAMP) surgery , 17 Campylobacter jejuni , 339 tissue , 274 Index 541

Cardiomyopathies , 102, 123, 124, 256, 257, 362 Sertoli , 363, 522 Cardio-protective effects of vanadium , 160 T- , 17, 101, 105, 106, 160, 163, 323, 325, Cardiovascular 332–335, 347, 400, 401, 467 diseases , 60, 161, 184, 185, 311, 312, 315, Cellular 349, 363, 373, 380, 399, 422, 482 calcium , 66, 94, 102, 115 effects , 159–162 homeostasis , 30, 114, 201, 327, 331 β-Carotene, 17 iron transport , 241–244 Carotid artery disease , 404 magnesium , 52, 54, 55, 58–65, 69, 73 Caspases , 113, 334 potassium , 63 Catalases , 3, 4, 15, 235, 329, 362 sodium , 60, 63 Cataract formation , 102 Central core disease , 124, 125 Catecholamine , 55, 58, 59, 210, 211, 214, Central nervous system (CNS) , 31, 203, 205, 362, 364 210, 244, 256, 284, 363, 376, 429, 432, Catechols , 267, 271, 361, 364 435, 437, 461, 481, 512 Cathepsin K , 86 Ceramics , 200, 453 Cattle , 296, 373, 379, 423, 487, 521 Ceramide , 66 Cbl. See Cobalamin (Cbl) Cereals , 10, 249, 252, 253, 454 CblC protein , 299–302 Cerebral CBS. See Cystathionine β-synthase (CBS) atrophy , 435 C2C12 skeletal muscle cells , 187 edema , 435, 438 cDNA , 69, 506 palsy , 71, 438 CDO. See Cysteine dioxygenase (CDO) Cerebrospinal ﬂ uid (CSF) , 203, 212, 277, 436 Celiac Ceruloplasmin (Cp) , 4, 18, 19, 203, 244, 330, disease , 250, 408 361–363, 365, 371, 372 sprue , 59 Cervical cancer , 157 Cell(s) , 18, 21, 30, 31, 34, 36, 42–44, 54, 55, Chagas’ disease , 162, 163, 165, 337, 346 61, 69, 82–85, 88, 89, 92, 93, 95, 97, Channels 99, 101, 106, 113, 114, 116, 117, acid-sensing , 402 119–121, 126, 176, 186–189, 192, 201, calcium. See Calcium channels 210, 213, 215, 216, 234, 235, 238, 240, ion , 95, 105, 208, 398 247, 248, 274, 283, 323, 324, 327, ionotropic glutamate receptor , 209 329–331, 366, 367, 379, 390, 393, 396, ligand-gated , 95 398, 406–408, 423, 462, 510, 516, 523 magnesium , 51 β , 63, 111, 393, 400, 401, 405, 519 membrane , 93–95, 112 C2C12 skeletal muscle , 187 N-methyl-D-aspartate , 402 cycle , 69, 281, 403, 406 nucleotide-gated , 348 cycle arrest , 158 ORAI1 , 97, 98 death , 37, 94, 101, 102, 113–116, 121, potassium , 54, 111 127, 214, 216, 279, 324, 442, 466, 515 ryanodine receptor , 95, 98, 99, 109 EDR3 hepatoma , 487 sodium , 32, 118, 119 endothelial , 61, 70, 206, 208, 247, 333, store-operated , 97, 98, 209 334, 396, 466, 467 transient receptor potential (TRP) , 51 erythroid , 241, 244 two-pore (TPC) , 94, 95, 99, 118 eukaryotic , 92, 349, 364 voltage-regulated , 97, 206, 208, 209, 520 lysis , 33, 37 Chaperones , 98, 114, 342, 343, 516

membrane , 3, 31–33, 51, 53, 55, 65, 92, C H3 -Cbl. See Methylcobalamin (CH3 -Cbl) 120, 146, 154, 190, 208, 209, 323 Chelating agents , 266, 268, 270, 282, natural killer (NK cells) , 10, 12, 15, 371, 402 401, 467 Chemokines , 324, 332 nerve , 31, 32, 378, 401 Chemolithoautotrophs , 487, 489 neuroendocrine , 96, 403 Chest syndrome , 262 proliferation , 69, 70, 158, 281, 324, 327, CHF. See Congestive heart failure (CHF) 328, 330, 331, 366, 482 Chicken , 42, 425, 484, 491, 521 542 Index

Childhood hepatitis B , 19 diarrhea , 12 infl ammation , 14, 421, 465 early death , 426 kidney disease (CKD) , 44, 45, 57 Children , 7, 12, 13, 16, 18–20, 40–42, 153, myeloid leukemia (CML) , 492, 493 202, 249, 258, 261, 262, 371, 373, 407, nickel exposure , 323 408, 490, 517 toxicity of arsenic , 481 Chile , 253, 482, 483 Chrysotile , 463 Chinese hamster ovary cells , 187 CICR. See Ca2+ -induced Ca2+ release (CICR) Chlamydomonas , 418 Cirrhosis , 18, 38, 39, 44, 204, 256, 375, 376, reinhardtii , 417, 418, 426 399, 408, 517 Chloride wasting , 67 Cisplatin , 43, 54, 58, 157, 158, 165 Chlorothiazide diuretics , 38 Citalopram , 423 Cholestasis , 18, 20, 21 Citrate , 53, 83, 148, 209, 232, 256, 277, 402 Cholesterol , 61, 180–182, 189, 190, 193, 201, Citric acid cycle , 106, 107, 481 459, 511, 519 CKD. See Chronic kidney disease (CKD) metabolism , 193, 519 Clastogenic , 191, 465 Choline transporters , 209 Claudin , 54, 68 Chondrocytes , 87 Clay , 452 Chromate , 191 Cleavage of Chromatin , 213, 215, 327–329, 401, DNA , 329 515, 520 the Co-C bond , 303 damage , 329 Clinical trials , 273, 377 Chromium, Cr , 19, 20, 171–193, 327, 484 phase I , 273 51 Cr , 176 phase II , 273, 377 absorption , 176, 177 Clioquinol , 280 biomarkers , 178, 179 Clusters , 86, 88, 95, 98, 109, 159, 243, 244, defi ciency , 173–177, 179, 192 264, 278, 336, 338, 400, 419, 420, 422 detoxifi cation , 189 [2Fe-2S] , 236, 420 excretion , 177, 178 [3Fe-4S] , 340 histidine , 186, 187 4Fe-4S , 236, 246, 332, 340, 427 in infectious diseases , 20 iron-sulfur , 240, 241, 263, 284, 416

picolinate ([Cr(pic)3 ]) , 173, 181, 183–187, CML. See Chronic myeloid leukemia (CML) 189–192 CN-Cbl. See Cyanocobalamin (CN-Cbl) supplementation , 179, 182–184 13 C NMR , 427 -vanadium-iron alloys , 164 CNS. See Central nervous system (CNS) Chromium(III) , 172–174, 176, 177, 179–181, CoA. See Coenzyme A (CoA) 183, 185, 186, 188, 190–193 Coagulation , 92, 399 in drinking water , 175 factors , 361 intake , 174, 175, 177, 178 Cobalamin (Cbl) , 296–310, 312, 313, 315 nicotinate , 183, 192 absorption , 296, 298–300, 311, 313 supplementation , 178, 179, 181–185, 189, adenosyl-(Ado-Cbl) , 297, 298, 302, 303, 192, 193 305, 311 Chromium(VI) , 188 biochemistry , 297, 298 Chromodulin , 188 biological half-life , 313 Chromosome binding proteins , 298–302, 308, 315 aberrations , 191, 192, 327, 329 57 Co-labeled , 300 translocation , 492 cyano-. See Cyanocobalamin (CN-Cbl) Chronic -dependent enzymes , 297, 300, arsenic exposure , 476, 477, 481, 482 303–310, 315 constipation , 350 in food , 296 diseases , 8, 254, 255, 277, 391, metabolism , 300, 301 404–407, 459 neuropathy , 310, 312, 313 heart failure , 422 reductase , 302 hemodialysis , 18 transport , 298, 313 Index 543

Cob(I)alamin , 298, 302, 306, 307 Contraceptives , 408 Cob(II)alamin , 298, 302, 303, 305, 307 Convulsions , 68, 146, 374 Cob(III)alamin , 298 Copper, Cu , 3–5, 10, 13, 15, 17–19, 22, Cobaloxime , 297 149, 205, 208, 214, 231, 266, 267, Cobalt, Co2+ , 5, 206, 242, 296–316, 332, 341, 271, 327, 329, 332, 348, 360–380, 342, 347, 348 401, 406, 407, 418, 429, 440, 441, 57 Co-labeled cobalamin , 300 461, 476, 484, 491, 515 defi ciency , 296, 297 accumulation , 366, 376, 378 essentiality , 296 amine oxidase , 361 overload , 315 biochemistry , 361–371 Cobamide , 297, 298 chaperones , 364, 365, 367, 370 Cobinamide , 297, 308 chelating agents , 371 Coccidiosis , 491 chelation , 376, 378 CO dehydrogenase , 323, 343 chrome arsenate (CCA) , 491 Coenzymes defi ciency , 4, 18, 363, 367–369, 371–377, A (CoA) , 107, 279, 311 401, 418, 440 B , 350 defi cient rats , 373 M , 341, 350 detoxifi cation , 366 Coffee , 252, 422, 454 effl ux , 364, 368–371, 379, 380 Colitis , 59, 408, 511 excretion , 371, 376 ulcerosa , 408 homeostasis , 18, 332, 361, 364–371, 376, Collagen , 86, 88, 361, 363, 373, 457–460, 379, 380, 440, 441 462, 463, 466, 467 -induced toxicity , 367 Colon in infectious diseases , 19 adenocarcinoma , 511 overload , 367, 371, 375, 376, 440 cancer , 69, 70, 73, 157, 513 sequestration , 366–367 Coma , 336, 481 storage , 18, 19, 366–367 Combustion of petroleum , 143 supplementation , 371, 374, 378 Committee on the Scientifi c traffi cking , 18, 365, 367–368 Evaluation of Dietary Reference uptake , 364–366, 370, 373, 379, 440 Intakes of the National Academies zinc ratio , 4, 19 of Science, 180 Copper(I), Cu + , 360, 361, 364, 365, 368, Common cold , 12, 13, 18 377, 418 Complex I , 119, 215, 278, 279, 520 -GSH , 366 Complexins , 110, 111 Copper(II), Cu 2+ , 332, 360, 361, 377 Computer chips , 453 Cu 2+ /Cu+ redox system , 361 Concrete , 453 Copper transporter receptor 1 (Ctr1) , 364–366, Conditional stability constant , 238 369, 370, 374 Conformational changes , 102, 103, 111, 163, Copper/zinc superoxide dismutase (SOD1, 164, 189, 308, 333, 369, 425 Cu/Zn-SOD), 3, 10, 18, 21, 149, 212, Congenital adrenal hyperplasia , 43 361, 362 Congestive Corallina inaequalis , 160, 161 cardiac myopathy , 501 Coral sand , 458, 460, 462 heart failure (CHF) , 38, 39, 43, 59–61, 72, 399 Corn , 521 Connective tissues , 201, 372–375, 458, 463, Coronary diseases , 18, 60, 404, 459 467, 469 Corrinoids , 296, 297, 299 dysfunction , 363 Corrin ring , 297, 298 Conn syndrome , 43 Corticotropin , 395 Contaminated Corynebacterium , 282 drinking water , 481 Cosmetics , 456 environments , 323 Countries. See individual names soils , 491 Coxsackie virus , 517 surface water , 482 Coxsackievirus B4 , 161 Continents. See individual names Cp. See Ceruloplasmin (Cp) 544 Index

cPMP. See Cyclic pyranopterin Cytokines , 3, 4, 12, 17, 65–67, 70, 71, 86, monophosphate (cPMP) 190, 255, 324, 332, 333, 335, 367, Creutzfeld-Jacob disease , 379 400, 401, 466 Critically ill patients , 3, 4, 13, 14, 16, 17 Cytoplasm , 53–55, 65, 86, 96, 100, 103, 113, Crocidolite , 463, 466 114, 122, 208, 244, 283, 341, 342, Crohn’s disease , 59, 250, 408 347, 508, 509 Crude oil , 142 Cytoslic Cryptidins , 399 calcium , 88, 89, 95, 96, 100, 108, 111, Cryptococcus , 9 115, 119, 120, 125 Crystalline silica , 453, 463, 464, 468 glutathione peroxidase (Gpx1) , 506 Crystal structures of AdoMet , 308 Cbl , 308 D Cbl binding proteins , 299 DAergic Ctr1. See Copper transporter receptor 1 (Ctr1) cell death , 214 Cunninghamella bertholletiae , 9 neurons , 210, 211, 214, 217, 218 Cushing syndrome , 43 DAG. See Diacylglycerol (DAG) Cyanide , 340 Daily Cyanocobalamin (CN-Cbl), 297, 298, 301, 302 intake of manganese , 202 Cyclic iron requirements , 249 adenosine monophosphate (cAMP) , 53–55, Dantrolene , 124, 125 68, 100 D A T . See Dopamine transporters (DAT) ADP-ribose (cADPR) , 97, 112 db/db mouse , 181 pyranopterin monophosphate (cPMP) , DBI. See Dimethylbenzimidazole (DBI) 427–429, 434, 437–439 D βM. See Dopamine-β-monooxygenase Cyclosporin , 58, 106 (DβM) Cynomologous macaques , 213 DDT. See Dichlorodiphenyltrichloroethane Cyprus , 264 (DDT) Cystathionine Deafness , 117, 123 β-synthase (CBS), 309, 311, 423, 424 Deferasirox , 9, 258, 273, 274, 276, 277, 284 γ-lyase (cystathionase) (CSE), 423, 424 Deferiprone , 9, 269, 274–277, 280, 284 Cysteine Deferitrin , 273 biosynthesis , 505 Deferoxamine , 9 catabolism , 423–425, 439 D e ﬁ ciency (of) dietary intake , 425 aldehyde oxidase , 431 dioxygenase (CDO) , 423–425, 439 aldosterone , 44 homo- , 311 arsenic , 484

methylseleno- , 521, 523 B12 , 296, 297, 300, 310–314, 371, 372 seleno-. See Selenocysteine chromium , 173, 175, 179 sulﬁ nic acid (CSA) , 423 cobalt , 296, 297 S-sulfo-(SSC) , 436–440 copper , 4, 18, 363, 367–369, 371–377, supplementation , 439 401, 418, 440 Cystic folate , 312 encephalomalacia , 435 frataxin , 263 ﬁ brosis , 11–12, 17, 18, 22 glutaredoxin , 264 Cytochrome(s) iron , 7, 18–21, 203, 248–255, 264, a , 235 265, 284 b , 235 magnesium , 56–58

b 5 , 419, 425, 426 manganese , 202, 205 deﬁ ciency , 362, 368 mARC , 431 P450 , 235 methylmalonyl-CoA mutase , 302, 314 Cytochrome c , 113, 215, 235, 362, 425, 515 molybdenum cofactor , 426, 431, 435, 437 oxidase , 235, 361, 362, 365, 367, oxygen , 324 368, 375, 377, 441 phosphate , 486 Index 545

potassium , 31, 35 Dichlorodiphenyltrichloroethane (DDT) , selenium , 15–17, 501, 513, 517–520, 32, 490 522–524 Dietary intake of sulﬁ te oxidase , 424–426, 439, 440 copper , 18, 363, 369, 370, 376, 380 thiamine , 42, 58 low protein , 439 xanthine oxidoreductase , 420 magnesium , 51, 52, 54, 57, 58, 62, 63, 66 zinc , 391, 395, 396, 398–408 manganese , 20, 22, 201–203 Dehydratases , 236 nickel , 325, 349 Dehydration , 39, 44, 481 potassium , 30 Dehydrogenases reference intake (DRI) , 455, 518, 524 alcohol , 65 selenium , 14, 514, 522–524 Delirium tremens , 43, 58, 65 silica , 454, 458, 460, 467 Dementia , 120, 278, 313, 363, 461 silicon , 454, 455, 457–460, 462, 463 Demethylating organoarsenic species , 480 sodium , 30 Demethylation of histones , 237 vanadium , 140, 143, 144, 146 Dendritic cells , 334, 401 zinc , 10, 371, 399 Dengue fever , 159, 161 Digitalis , 44, 58, 60 Dentin , 85–87 Dihydrofolate reductase , 309 Deoxyadenosine , 303 Dihydrogen , 336, 350 5 ′-Deoxyadenosyl radical, 427 DI2-knockout mice , 512, 519 Depression , 41, 42, 56, 58, 71, 92, 404, 481 DI3-knockout mice , 519 Dermal absorption of Dimethylarsenic (DMAs) , 481, 491 manganese , 201 Dimethylbenzimidazole (DBI) , 297, nickel , 349 298, 308 Dermatitis , 11, 201, 323, 334, 335, 404, 408 N,N-Dimethyl-2,3-dihydroxybenzamide atopic , 401 (DMB), 267–269 Desferrioxamine (DFO) , 261–263, 266, Dimethylselenide , 523 270–274, 276, 282, 284 Diol dehydratase , 303 -B , 233–235, 266, 269 Dioxygen. See Oxygen Desferrithiocin (DF) , 268, 269, 273, 282 Dioxygenases Detoxiﬁ cation (of) , 147, 189, 239, 323, 331, acireductone , 323, 336–338, 341, 342 365, 366, 419, 442, 480, 490, 515 cysteine , 423–425, 439 arsenic , 480 Diphosphate (pyrophosphate) (PPi), 87, cadmium , 366 88, 427 methylglyoxal , 336 Disease(s) (see also individual names) , 3–22, DF. See Desferrithiocin (DF) 29–45, 50–73, 83–127, 141, 147, 153, DFO. See Desferrioxamine (DFO) 159–165, 173, 180, 181, 184, 185, DI1. See Type 1 deiodinase (DI1) 200–219, 250, 253–255, 257, 258, Diabetes , 8, 18, 38, 40, 43, 62–64, 72, 172, 260–265, 276–280, 284, 296–315, 323, 173, 179, 186, 256, 391, 399, 404–406, 324, 336–349, 361–363, 367, 372–380, 462, 468, 469, 476, 482, 492 390–409, 417–442, 453, 457–461, 464, mellitus , 11, 39, 44, 152–156, 363, 408 465, 467, 469, 476, 477, 482, 483, type 1 , 11, 63, 152, 154, 181, 405 490–493, 501, 511, 516–520 type 2, 153, 165, 177, 182–185, 190, 193, 519 Addison , 44 Diabetic(s) , 8, 9, 11, 20, 37, 41, 44, 59, 63, 64, alcohol liver (ALD) , 65 152, 165, 178, 182–185, 193, 396, 399, Alzheimer’s disease (AD) , 102, 118, 405, 406, 462, 518, 519 120–122, 213, 277–280, 311, 376, 377, animals , 63, 165 380, 403, 406, 407, 452, 457, 460, 461, rats , 153, 177 467, 469 Diacylglycerol (DAG) , 104, 112 autoimmune , 106, 255, 401, 421 Dialysis patients , 7 autosomal recessive , 11 Diarrhea , 11, 34, 38–41, 58, 59, 163, 335, 375, black foot , 482 407, 418, 441, 481 Brody , 124, 125 childhood , 12 cardiovascular. See Cardiovascular diseases 546 Index

Disease(s) (cont. ) Diuresis , 36, 37, 40, 41, 43 cardiac , 123, 124, 276 Divalent carotid artery , 404 cation transporter , 208, 323, 325 celiac , 250, 408 metal transporter 1 (DMT1) , 5, 20, 203, central core , 124, 125 241, 242, 246, 250, 254, 265, 365 Chagas , 162, 163, 165, 337, 346 Dj-1-knockout mice , 217 chronic , 8, 44, 45, 57, 254–255, 277, 391, DMAs. See Dimethylarsenic (DMAs) 404–407, 459 DMB. See N,N-dimethyl-2,3- coronary , 18, 60, 404, 459 dihydroxybenzamide (DMB) Creutzfeld-Jacob , 379 DMD. See Duchenne muscular dystrophy Crohn’s , 59, 250, 408 (DMD) falling , 373 DMT1. See Divalent metal transporter 1 ferroportin , 257, 258 (DMT1) heart , 61, 173, 261, 399 DN. See Dopaminergic neurons (DN) Huntington’s (HD) , 118, 120, 121, 213, DNA , 3, 11, 100, 101, 106, 121, 141, 149, 376, 378 151, 158, 159, 163–165, 215, 264, 308, infectious , 1–23, 322, 324, 407, 409 310, 315, 326–330, 336, 343, 344, 392, inﬂ ammatory bowel , 254, 511 396, 401, 432, 477, 480, 482, 488, 489, Kashin-Beck , 517 504 Keshan , 501, 517 c , 69, 506 kidney , 44, 185, 323 cleavage , 329 liver , 10, 58, 65, 257, 376, 398, damage , 3, 146, 147, 327, 329, 466, 486, 490 399, 408 fragmentation , 215, 329 lyme , 5 methylation , 328, 330, 396, 482 metabolic , 349, 373, 404–407 polymerase , 151, 486 motor neuron , 362, 372 repair , 146, 327, 396, 482, 486 Mseleni , 202 strand scission , 329 muscle , 124–126, 501 synthesis , 51, 69, 151, 312, 486 neurodegenerative , 94, 118–122, 280, 376, Dogs , 273 379, 406, 467 Dopamine , 211, 214, 218, 251, 363, 364, 378 neurological , 39, 117, 201, 361, 372, - β-monooxygenase (DβM) , 361, 364 376, 379 transporters (DAT) , 209 Niemann-Pick , 118 Dopaminergic neurons (DN) , 119, 279, 377 pancreatic , 10 Down’s syndrome , 202 Parkinson’s (PD) , 118, 119, 121, 122, 206, DRI. See Dietary reference intake (DRI) 208, 211, 212, 214, 216–218, 277–280, Drinking water , 142–144, 175, 418, 459, 461, 284, 376–378, 380, 461 476, 480–482, 484, 493, 521 Perthest , 202 Drosophila , 191, 192, 504 Picks , 118, 277 Drugs (see also individual names) , 16, 36, 39, prion , 376, 379 44, 53, 54, 56, 58, 64, 106, 124, 125, pulmonary , 39 141, 158, 160, 162–165, 172, 217, 270, renal , 44, 45, 57, 67, 255, 442 283, 395, 421, 422, 453, 462, 477 selenium-related , 516–518 absorption , 270 sickle cell , 14, 22, 258, 262, 408 amoebocidal , 163 skeletal muscle , 124–126 anesthetic , 124 vascular , 459, 460, 482 antituberculosis , 162 Whipple’s , 59 arsenic-based , 477 Distribution of immunosuppressive , 106 magnesium , 50, 51 -induced disorders , 38 potassium , 34, 41 metabolism , 422 sodium , 34 toxicity , 421 vanadium , 141–147 Dry cell batteries , 200, 201 Disulﬁ de bonds , 350, 512, 513 dTMP. See Thymidine 5′-monophosphate Dithiolene synthesis , 428, 429 (dTMP) Index 547

Duchenne muscular dystrophy (DMD) , Endocytosis , 146, 176, 177, 186, 208, 300, 124–126 366, 379 Dust , 143, 322, 325, 453, 456, 464, 465 Endonucleases , 119 Dysarthria , 363 Endoplasmic reticulum (ER) , 54, 65, 93–99, Dysentery , 163 104, 106, 111, 113–117, 120, 121, 512, Dysfunction of 513, 515, 516, 519 cystathione β synthase , 311 Endothelial cells , 61, 70, 206, 208, 247, 333, methionine synthase , 302, 310, 312, 313 334, 396, 466, 467 methylenetetrahydrofolate reductase , 311 Energy metabolism , 31, 51, 108, 201, 324, Dystonia , 212, 217, 363 330, 361 Dystrophin , 125, 126 England , 164 Enstatite , 143 Entamoeba histolytica , 163 E Enterobactin , 267–271, 282, 283 Early childhood death , 426 Enterocyte membranes , 203 Earth’s crust , 83, 142, 143, 200, 231, 322, 452, Enteroviruses , 39 454, 456, 479 Environmental manganese exposure , 201 Eating disorders , 42 Environmental Protection Agency , 521 ECF. See Extracellular ﬂ uid (ECF) Enzymes (see also individual names) , 5, 10, EC-SOD. See Extracellular superoxide 18, 30, 44, 51, 54, 63, 70, 86, 96, 98, dismutase (EC-SOD, SOD3) 99, 101–104, 106–108, 119, 120, Eczematous skin reaction , 332 141, 150, 151, 160, 189, 191, 202, Edema , 31, 38, 39, 435, 438, 465 204, 205, 214, 217, 231, 236–238, EDTA. See Ethylenediamine-N,N,N′,N′- 240, 243, 244, 246, 247, 263, 279, tetraacetic acid (EDTA) 297, 300, 302–311, 314, 315, 323, EE. See Ethylmalonic encephalopathy (EE) 324, 327, 328, 330, 336–343, 345, eEFSec. See Eukaryotic selenocysteyl-tRNA- 346, 348–350, 360–365, 367–370, speciﬁ c elongation factor (eEFSec) 373, 374, 377, 392, 393, 400, 405, EF. See Elongation factor (EF) 417, 418–424, 427, 430, 432, 435, EF-hand proteins , 84, 90, 100, 105 439, 442, 460, 461, 463, 468, 480, EGF. See Epidermal growth factor (EGF) 481, 485, 490, 512 Ehlers-Danlos syndrome , 404 antioxidant , 16, 21, 201, 395, 396, 402 Ehrlich ascites tumor cells , 156 inhibition , 270 Elastin , 361, 363, 373 Epidermal growth factor (EGF) , 54, 69, 72, 73 Electrical potential , 31, 32, 42, 51 signaling , 69 Electrode coating , 200 Epigenetic effects in nickel Electrolyte(s) , 30–32, 56 carcinogenesis, 327 disorder , 38, 43, 44 Epilepsy , 202, 441 disturbance , 40, 42, 57 seizures , 69 Electron paramagnetic resonance (EPR) , Epinephrine , 36, 61 188, 361 EPR. See Electron paramagnetic Electron transport , 30, 235, 278, 486, 487, 489 resonance (EPR) chain , 106, 215 ER. See Endoplasmic reticulum (ER) Electrophoretic mobility shift assays Erythrocytes , 6, 15, 21, 60, 146, 250, 254, (EMSA) , 508 255, 261, 283, 284, 484 Elongation factor (EF) , 91, 92, 98, 100, Erythroid cells , 241, 244 102–106, 108, 109, 122, 507 Erythrophagocytosis , 244 Embryo development , 112, 512 Erythropoiesis , 247, 250, 255, 258, 261, Emerald , 452 262, 400 EMSA. See Electrophoretic mobility shift Erythropoietin assays (EMSA) gene , 315 Enamel , 42, 85, 86 therapy , 250 Encephalopathy , 179, 204, 379, 435 Escherichia coli , 6, 8, 283, 304, 307–309, 340, Endocrine tissue , 241, 255, 258, 272 342, 343, 427, 432, 433, 437 548 Index

Esomeprazole , 72 F Esophageal carcinoma , 406 FAD. See Flavin adenine dinucleotide (FAD) Essential elements , 141, 147, 172, 175–177, Falciparum , 13 179, 180, 189, 192, 201, 324, 348, Falling disease , 373 360, 380 Familial Essentiality of , 201–206, 324, 468 fatal insomnia , 379 chromium , 173 hemiplegic migraine (FHM) , 118 nickel , 340, 348–350 hypomagnesemia , 68 (micro)nutrients , 5, 201, 296, 324, 345, Fan worms , 143 455, 457, 484, 501, 502 Farm animals , 173, 185, 373 trace elements , 173, 174, 176, 476 Fatal familial insomnia , 379 vanadium , 165 Fatty acids , 3, 153, 311, 396, 441, 459, 460, Estimated safe and adequate daily dietary 484, 510 intake of metabolism , 189–190, 306 chromium , 173 Fatty liver disease , 408 selenium , 524 FDA. See Food and Drug Administration (FDA) Estrogens , 52, 190, 218, 458, 459, 486 Fecal ESTs. See Expressed sequence tags selenium excretion , 523 database (ESTs) zinc loss , 11 Ethanolamine ammonia-lyase , 303 Federal Trade Commission of the United Ethanol metabolism , 65 States, 173 Ethylenediamine-N,N,N′,N′-tetraacetic acid Feed additives , 491 (EDTA) , 234, 235, 253, 335 Feldspar , 452 Ethylmalonic encephalopathy (EE) , 441 Females , 21, 155, 156, 161, 177, 191, 192, Eubacteria , 336, 487, 503 249, 458 Eukaryotes , 314, 323, 327, 336, 345, 346, Fenton-like reaction, 146, 166 348, 349, 351, 366, 417, 418, 427, Fenton reaction , 233, 460 429, 503, 505 Feroxamine , 9 Eukaryotic Ferredoxins , 236 cells , 92, 349, 364 Ferriportin , 396 pathogens , 336, 346 Ferritin , 6, 9, 18, 146, 158, 202, 237–240, 246, SECIS elements , 507 247, 277, 283, 392, 407 selenocysteyl-tRNA-speciﬁ c elongation mRNA , 246 factor (eEFSec) , 507–509 Ferrochelatase , 244, 265 Europe , 163, 257, 274, 335 Ferroportin-1 (Fpn) , 20, 209, 210 European Food Safety Authority , 192 Ferroportin disease , 257, 258 Excitotoxicity , 102, 117, 118, 122, 205, 212, Ferrovanadin , 144 218, 375 Ferroxidase , 4, 19, 238, 239, 244, 265, 279, Excretion of 361, 362, 367 calcium , 127 FHM. See Familial hemiplegic migraine copper , 18, 371, 376 (FHM) silicon , 457 Fibrinolysis , 92, 399 vanadium , 141, 145 Fibrosis , 62, 256, 453, 465, 466 Exocytosis , 110–112, 115, 393, 402, 466 Fiji Island , 455 Experimental animals (see also Animal Finland , 501, 521 studies) , 326 Fireworks , 200 Exposure to nickel , 323, 324, 328, 335 Fish , 95, 112, 247, 296 Expressed sequence tags database (ESTs) , Flagellates , 141, 162, 163 502, 507 Flavin adenine dinucleotide (FAD) , 148, 308, Extracellular 419, 420, 422 ﬂ uid (ECF) , 31, 32, 34, 38–41, 43, 50, Flavin mononucleotide (FMN) , 308, 314 85, 87, 122 Fluorophores , 201 superoxide dismutase Fly agaric , 141–143 (EC-SOD, SOD3) , 362 FMN. See Flavin mononucleotide (FMN) Index 549

Folate Gastrointestinal defi ciency , 312 absorption of manganese , 21, 202 metabolism , 308, 310, 315 cancer , 281 Folic acid hemorrhage , 375 fortifi cation , 311, 312 surgery , 371, 372 in blood , 308 tract , 10, 11, 18, 34, 41, 144, 203, 250, Food , 143, 163, 174, 175, 180, 183, 250, 252, 270, 277, 282, 399, 401, 418, 461 253, 296, 298, 299, 311, 315, 335, 339, Gelatinase , 86 345, 349, 396, 422, 468, 476, 480, 484, Gene 501, 521, 524 expression , 100, 106, 113, 121, 155, additive , 453–455 324, 327–331, 333, 334, 345, 349, Food and Drug Administration (FDA) , 33, 72, 394, 395, 400, 458,460–462, 466, 184, 185, 192, 202, 274, 491 467, 515 Food and Nutrition Board of the Institute of mutations , 256, 297, 299, 311, 316 Medicine of the National Academy of transcription , 10, 99–101, 105, 115, 121, Sciences, 174 324, 395 Forkhead transcription factor , 101 Genetic Formation constants (see also Affi nity copper defi ciencies , 374, 375 constants, Binding constants, copper overload , 375, 376 and Stability constants) , 151, 152 engineering , 313 Forsterite , 143 hearing loss , 122, 123 Fowler’s solution , 492 Genomes , 101, 314, 327, 347, 362, 392, 406, Fpn. See Ferroportin-1 (Fpn) 422, 488, 504, 509 France , 165, 455 Genotoxic effects of nickel , 327 Frataxin (FXN) , 244, 263, 265 Gentamicin , 43, 54, 58 defi ciency , 263 Gentisate aldehyde , 422 Free radicals , 4, 14, 201, 214, 217, 244, 270, Gephyrin , 429–434, 441 271, 279, 374, 395, 465 -defi cient mice , 437 Friedreich’s ataxia , 263–265, 277, 278, 280 loss , 439 Frogs , 95, 112 GFAJ-1 , 488, 489, 493 Fruit Gitelman/Bartter’s syndrome , 59 bats , 305, 313 Gitelman’s syndrome , 67, 68 fl ies , 191, 264, 327 Global cycles Fuel additives , 200 carbon , 419 Fumes , 216, 217, 322, 325 nitrogen , 338, 419 Function of selenoproteins , 509–516 sulfur , 419 Fungi (or fungal) , 5, 6, 9, 150, 282, 336, Globulins 342, 419, 425, 427, 480, 521 β , 203 infections , 8, 9 β micro-, 257 Fura-2 , 201 macro- , 10, 206 Furosemide , 39 Glomerulonephropathy , 462 FXN. See Frataxin (FXN) Glucagon , 55, 405 Glucocorticoid receptor (GR) , 482, 483, 486, 487 G response element (GRE) , 482, 483, 487 GABA. See γ-Amino butyric acid (GABA) Glucose , 8, 39, 63, 153–156, 165, 177–184, Galactomannan , 164 281, 330, 348, 484 Gallium(III) , 266 homeostasis , 153, 518 Gambia , 7 intolerance , 11, 20, 174, 179, 405, 519 Gastric metabolism , 20, 31, 153, 174, 175, 179, cancer , 102 183, 186, 188, 205, 462 carcinomas , 344 tolerance , 173–176, 178, 181, 182, 519 lymphomas , 344 tolerance factor (GTF) , 20, 172, 174, Gastroenteritis , 481 186, 188 550 Index

Glucose (cont .) GPx. See Glutathione peroxidase (GPx) transporter (GLUT4) , 111, 154, 156, 187, GR. See Glucocorticoid receptor (GR) 189, 190 Gram-negative bacteria , 8, 161 uptake , 64, 111, 153–155, 186, 187, 189, 405 Gram-positive bacteria , 161 GLUT4. See Glucose transporter (GLUT4) Granulocytes , 14, 401 Glutamate , 96, 117, 118, 160, 188, 201, 205, Gräsbeck-Imerslund syndrome , 299 215, 216, 218, 347, 375, 435, 436 GRE. See Glucocorticoid response element -activated ionic channels , 208 (GRE) aspartate transporter (GLAST) , 216 Greece , 264 dehydrogenase , 435 Growth synthetase , 20 hormone , 403 (excito)toxicity , 117, 118, 122, 218, 375 retardation , 12, 296, 391 traffi cking , 205 GS. See Glutamine synthetase (GS) Glutamatergic signaling , 120, 205 GTF. See Glucose tolerance factor (GTF) Glutamine , 117, 201 GTP. See Guanosine 5′-triphosphate (GTP) synthetase (GS) , 201, 205, 424 GTPase , 302, 343 transporter , 216 Guanine Glutaredoxins , 241 8-oxo- , 330 defi ciency , 264 Guanosine 5′-triphosphate (GTP), Glutathione (GSH) , 3, 147, 148, 241, 242, 303, 427 278, 365, 366, 423, 436, 480 hydrolysis , 303 reduced , 278, 279, 325, 329, 509 Guatemala , 251 reductase (TGR) , 329, 512 Gut’s microfl ora , 349, 350 synthesis , 436, 515 synthetase , 511 S-transferase , 69 H Glutathione peroxidase (GPx) , 4, 15, 16, 329, Haber-Weiss-type reaction , 214, 465 362, 501, 502, 506, 509–511, 524 Haematuria , 421 Gpx1 , 506, 509–511, 519 Hallervorden-Spatz syndrome , 279 Gpx1-knockout mice , 511 Halomonas , 476, 488 Gpx4-knockout mice , 511, 520 Haloperoxidases , 141, 150, 151, 160 GlxI. See Glyoxalase I (GlxI) Hamsters , 326, 484 Glycated hemoglobin , 64, 180, 182–184 Haptocorrin , 299, 300 Glycemia , 63, 462 Haptoglobin , 244 Glycerol Harvard Stop Test , 250 dehydratase , 303 HCP-1 transporter , 252 diacyl-(DAG) , 104, 112 HD. See Huntington’s diseases (HD) Glycine max , 343 Headache , 31, 118, 335, 375 Glycine Heart (see also Cardiac and Cardiovascular) , metabolism , 308, 309 22, 30–32, 63, 66, 96, 108, 109, reductase , 502 123, 126, 145, 146, 239, 255, 256, Glycogen accumulation , 155 272, 370, 371, 512, 515, 522 Glycoprotein , 15, 203, 244, 299, 513, 514 damage , 42, 124 Glycosuria , 40, 59, 462 disease , 61, 173, 261, 399 Glycosylphosphatidylinositol (GPI) , 363 failure , 38, 43, 44, 124, 126, 163, Glycosyl transferase , 205 254, 262 Glyoxalase (Glx) , 336 muscle , 108 I (GlxI) , 323, 336, 337, 341, 346 transplant , 254 II (GlxII) , 336, 337 HEK 293 T cell model , 209 Goats , 296, 348, 484, 485 HeLa cells , 157, 163, 503 Gold mines , 486, 487 Helicobacter Golgi system , 89, 93 hepaticus , 342 Gout , 418, 421, 422 pylori , 250, 338, 339, 342–346 GPI. See Glycosylphosphatidylinositol (GPI) Hematopoietic stem cell transplantation G proteins , 96, 118, 303 (HSCT), 8, 9 Index 551

Hematuria , 45 cellular , 30, 114, 201, 327, 331 Heme , 6, 236, 240, 242, 252, 263, 264, 297, copper , 18, 332, 361, 364–371, 376, 379, 315, 367, 390, 416, 425 380, 440, 441 -containing proteins , 235, 236, 247, 315 glucose , 153, 518 oxidase , 244 iron , 5, 146, 244, 246, 247, 254, 264, 277, oxygenase , 242 332, 362, 363 Hemochromatosis magnesium , 54–56, 62–64, 73, 206 juvenile , 257, 258 manganese , 202, 203, 206, 210, 213 Hemodialysis , 4, 7, 18, 57, 468 molybdenum , 418 Hemoglobin (Hb) , 6, 235, 236, 244, 247, 248, nickel , 324, 340–349, 351 250, 252, 260, 261, 264, 348 potassium , 32–37, 41 disorders , 258, 260 sodium , 34, 36, 37 glycated , 64, 180, 182–184 zinc , 10, 366, 391, 394, 395, 398, 401–404, production , 260 406–408 Hemoglobinopathies , 258–263 Homocysteine metabolism , 311 Hemojuvelin , 257, 265 Homocysteinemia , 311–312, 315 Hemolysis , 57, 244 Homocystinuria , 300, 302 Hemopexin , 6, 244 Homolytic cleavage of AdoCbl , 305 Hepatic Hormesis , 477, 485, 486 cirrhosis , 38, 204, 217 Hormones , 39, 44, 50, 53, 55, 63, 68, 83, 84, dysfunction , 418 127, 180, 400, 403, 458 encephalopathy , 204, 211 adrenocorticotropic (ACTH) , 395 Hepatitis C , 16, 399 antidiuretic (ADH) , 36–38, 40 Hepatocarcinoma , 513 growth , 403 Hepcidin , 9, 247–248, 255–258, 265, 283, 395 parathyroid (PTH) , 53, 68, 72, 84 -knockout mice , 280 replacement therapy , 458 HEPES buffer , 191 steroid , 482, 483, 486 Hephaestin , 244, 265, 361, 372 thyroid , 511, 512 Herbicides , 491 HPLC. See High performance liquid Hereditary chromatography (HPLC)

B 12 defi ciency , 300 HPO. See Hydroxypyridinones (HPO) hemochromatosis , 9, 255–258 HRE. See Hypoxia-responsive enhancers (HRE) molybdenum cofactor defi ciency , 437 HSCT. See Hematopoietic stem cell peripheral neuropaty , 375, 379 transplantation (HSCT) Herpes simplex , 161 Htt. See Huntingtin (Htt) HFE hemochromatosis , 257 Human , 2, 3, 5, 6, 15, 18, 21, 22, 57, 62, 84, Hfe protein , 257, 265 123, 139, 141, 153, 174, 175, 179, 180, High performance liquid chromatography 185, 201, 202,204, 205, 246, 247, 283, (HPLC), 434 296, 297, 302, 303, 315, 360, 362, 364, Histidine , 141, 144, 145, 153, 186, 187, 234, 368, 370–372, 374, 377, 394, 396, 400 237, 238, 323, 331, 333, 334, 338, 362 457, 484, 490, 493, 502, 511, 516 metabolism , 308 blood , 177 Histone(s) blood levels of arsenic , 480 acetylation , 328, 396 brain , 215, 402, 437 methylation , 237, 328 central nervous system , 363 modifi cations , 328 dietary standards for selenium , 524 Histoplasma , 9 endothelial cells , 333, 466 HIV. See Human immunodefi ciency genome , 101, 314, 422, 500, 509 virus (HIV) glucocorticoid receptor , 486, 487 1 H NMR , 427 health , 30, 200, 218, 296, 313, 315,

H2 O2 . See Hydrogen peroxide (H2 O2 ) 321–351, 361, 391, 415–444, 452, HOCl. See Hypochlorous acid (HOCl) 453, 455, 469, 476, 494, 500, 525 Homeostasis , 34, 52, 240, 323, 324 immune response , 345 calcium , 86, 102, 113–115, 117, 119–121, kidney cancer cells , 157 124, 215 lymphocyte cells , 486 552 Index

Human (cont .) 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic mARC , 426 acid (HEPES), 191 MCM , 303–305, 314 Hydroxylation of amino acids , 237 methionine synthase , 307, 308 Hydroxyl radicals , 214, 233, 234, 271, 329, molybdenum cofactor deﬁ ciency , 373, 374, 465 426, 431, 435, 437 Hydroxypyridinones (HPO) , 267, 271, molybdenum cofactor synthesis , 430–434 275, 276 molybdenum levels , 417 8-Hydroxyquinoline , 267–269, 280 MPT synthase , 431 Hyperaccumulation of serum transferrin , 239 copper , 369 stomach , 345 nickel , 323 testes , 363 Hyperaldosteronism , 44, 58, 62 urine , 177 Hyperalgesia , 64 Human immunodeﬁ ciency virus Hypercalcemia , 39, 84 (HIV) , 4, 7, 8, 15, 16, 19, 140, 160, Hypercalciuria , 68 165, 254, 264, 407 Hypercholesterolemia , 155 HIV-1 , 15, 16, 159–161 Hyperekplexia , 437, 441 HIV-2 , 160, 161 Hyperglycemia , 38, 39 Human pharmacokinetics and pharmacology Hyperhomocysteinemia , 311–312 (of), 5, 10, 11, 14, 15, 18–21, 144–147 Hyperinsulinemia , 462 chromium , 19, 20 Hyperkalemia , 30, 31, 43–45, 56 copper , 18, 19 Hyperleptinemia , 155, 462 iron , 5 Hyperlipidemia , 39, 155, 179, 462 manganese , 20, 21 Hypermagnesemia , 55–57 selenium , 14, 15 Hypermagnesuria , 67 zinc , 10, 11 Hypernatremia , 30, 31, 40, 41, 44 Huntingtin (Htt) , 120, 121, 213 Hypernucleophilic , 298, 306 Huntington’s disease (HD) , 118, 120, 121, Hyperparathyroidism , 45, 57 213, 376, 378 Hypertension , 37, 40, 44, 58–62, 64, 72, 373, Hut/CCR5 cells , 160 442, 460, 469 Hyalella azteca , 486 Hypertrophy , 124 Hydrogen Hyperuricemia , 421, 422, 442 bonds , 158, 245, 330, 489 Hyperzincemia , 404 metabolism , 336 Hypoalbuminemia , 10, 40, 73 Hydrogenases , 323, 336, 339, 342, 343, 346 Hypoaldosteronism , 44 chaperones , 345 Hypocalcemia , 40, 56, 72, 73 [NiFe] , 339, 340, 345 Hypochlorous acid (HOCl) , 323

Hydrogen peroxide (H2 O2 ) , 4, 20, 152, Hypocuprosis , 418 158, 190, 191, 205, 214, 233, Hypogonadism , 256, 396, 402 235, 271, 323, 329, 362, 400, 422, Hypokalemia , 30, 39–45, 67, 72, 73 510–512, 519 Hypomagnesemia , 39, 42, 52, 55–73 Hydrogen sulﬁ de , 423–425, 441 Hypomorphic mouse model , 314 accumulation , 441 Hyponatremia , 30, 31, 38–41, 44 metabolism , 441 Hypophosphatemia , 42, 58 Hydrolases , 205, 306, 392 Hypopigmentation , 364 Hydrolysis of Hypopigmentation of hair , 374 ATP , 108, 342, 368, 480, 486 Hypopigmentation of skin , 374 cisplatin , 158 Hypotension , 38, 56 triphosphate , 343 Hypotonia , 56, 440, 441 urea , 336, 338 Hypotonicity , 38, 39 Hydrolytic cleavage , 149 Hypoxanthine , 420, 421 Hydroxide , 147, 232, 233, 338 Hypoxia , 315, 330, 331, 360 Hydroxyapatite , 50, 86–88, 146, 147 -responsive enhancers (HRE) , 325, 330 3-Hydroxy-1,2-dimethylpyridin-4(1H)-one , 268 Hypoxic ischemic encephalopathy , 435 Index 553

I iNOS. See Inducible nitric oxide synthase IARC. See International Agency for Research (iNOS)

on Cancer (IARC) Inositol 1,4,5-triphosphate (InsP3 ) , 93–98, ICP-MS. See Inductively coupled plasma mass 112–114, 121

spectrometry (ICP-MS) Inositol 1,4,5-triphosphate receptor (InsP3 R) , ICU. See Intensive care unit (ICU) 94, 95, 97–99, 110, 114, 115, 117, 120, Idiopathic 121, 124

chronic toxicosis , 375, 376 InsP3 . See Inositol 1,4,5-triphosphate (InsP3 ) Parkinson’s disease , 211 InsP3 R. See Inositol 1,4,5-triphosphate IF. See Intrinsic factor (IF) receptor (InsP3 R) IFN. See Interferon (IFN) Institute of Medicine of the National Academy Ig. See Immunoglobulins (Ig) of Science, 174, 518 IGF-1. See Insulin-like growth factor 1 Insulin , 20, 33, 43, 55, 63, 152, 153, 177, 178, (IGF-1) 181, 183, 519 IL-1. See Interleukin-1 (IL-1) -enhancing action , 141, 164 Imidazoles , 233, 237 -like growth factor 1 (IGF-1) , 403, 405 Immune receptor (IR) , 152–154, 156, 186, 189, cascade , 332 190, 405 function , 11, 15, 373, 391, 396, 401 sensitivity , 63, 172, 175, 176, 178, response , 12, 17, 65, 323, 332, 333, 347, 184–186, 189, 190, 193 400, 401 signaling , 186–189 system , 10, 17, 21, 71, 164, 332, 373, 374, storage , 405 380, 400, 401, 406, 407, 452, 467 Integrins , 69, 205 Immunoglobulins (Ig) , 10, 145 Intensive care unit (ICU) , 13–16, 19, 58 Immunosuppressive drugs , 106 sepsis , 16–17 Implanted medical devices , 325 Intercellular signaling with zinc(II) , India , 14, 164, 274, 454 393, 394 childhood cirrhosis , 375, 376 Interference between molybdate, sulfate, Indomethacin , 217 and phosphate transport, 417 Inducible nitric oxide synthase (iNOS) , 216 Interferon (IFN) , 17 Inductively coupled plasma mass spectrometry - α (IFN-α) , 4 (ICP-MS), 279 - γ (IFN-γ) , 373, 400, 467 Industry , 183, 322, 452, 453, 468 therapy , 19 workers , 201 Interleukin (IL) , 4, 373 Infants , 11, 71, 211, 248, 249, 371, 374, 433, 438 -1 (IL-1) , 11, 71, 373 Infections -2 (IL-2) , 10, 400 bacterial , 159–161, 283, 345, 347 International Agency for Research on Cancer Infectious diseases , 1–23, 322, 324, 407, 409 (IARC), 323, 464 Infertility , 418, 516, 520, 522 Interrelationship between arsenic and I n fl ammation , 6, 19, 62, 64–67, 70, 126, 190, phosphorus , 477 283, 333, 334, 363, 377, 396, 401, 409, Interstitial fi brosis , 464, 465 440, 465–467 Intestinal I n fl ammatory bowel disease , 254, 511 cancer , 511 I n fl uenza , 140, 159, 165 cells , 52, 53, 176, 369, 480 virus , 161 infl uenza , 34 Inhalation of Intestine , 34, 42, 53, 56, 58, 68, 72, 73, 84, 85, crystalline silica , 453 104, 109, 127, 144, 163, 348, 349, 366, manganese , 201 371, 511, 516, 522 nickel , 323, 326, 349 small , 10, 18, 19, 21, 51, 86, 176, 203, silica dust , 464 250, 369, 370, 399, 417 vanadium , 144 Intracellular Inner membranes , 94, 107, 108 calcium , 61, 64, 93–99, 112, 114, 116, Inorganic diphosphate (= pyrophosphate) 120, 206, 329 (PPi) , 87, 88, 427 copper traffi cking , 365, 367–368 554 Index

Intracellular (cont .) autxidation , 240 distribution of nickel , 324, 325 fumarate , 253, 254 processing of cobalamin , 298, 300–302 gluconate , 254 signaling with zinc(II) , 393–394 sulfate , 232, 253, 254, 284 traffi c of zinc , 390 Iron(III), Fe 3+ , 6, 144, 146, 179, 203, 206, 208, Intrinsic factor (IF), 299, 300 231–235, 237–239, 241, 242, 244, -cobalamin , 299 252–254, 256, 261,266, 267, 269–276, 372 receptor , 300 chelating agents , 268 Iodine , 512, 517 hydroxide , 254 Ion oxide , 255 channels , 95, 105, 398 polymaltose , 254 gradients , 33, 210 pyrophosphate , 253 Ionotropic glutamate receptor channels , 209 uptake , 206, 208 IR. See Insulin receptor (IR) Iron(IV) , 231, 237 IRE. See Iron responsive elements (IRE) Iron defi ciency anemia , 248–255, 264, 265, 284 Iron, Fe , 2–9, 15, 18–22, 83, 146, 176, 177, Iron-regulatory protein 1 (IRP-1) , 246, 332 179, 186, 200, 202–210, 229–286, 297, Iron responsive elements (IRE) , 245, 246 323, 329, 339, 340,348, 380, 390–392, Iron responsive protein (IRP) , 241, 245 401, 406, 407, 460, 461, 463, 465, 484 Iron-sulfur cluster(s) , 240, 241, 263, 284, 416 absorption , 242, 243, 247, 253–255, 257, biosynthesis , 263 258, 348 Iron-sulfur proteins , 236 accumulation , 208, 256, 277–280, 363 IRP. See Iron responsive protein (IRP) administration , 6 IRP-1. See Iron-regulatory protein 1 (IRP-1) chelators or chelation therapy , 230, 231, Irritable bowel syndrome , 350 258, 261, 263, 266, 267, 270–277, 279, Ischemic neuronal injury , 402 281, 282, 284 Isocitrate dehydrogenase , 106, 204 defi ciency , 7, 1821, 203, 248–255, 264, 8-Isoprostane , 466 265, 284 Italy , 264 -defi cient rats , 08 -dependent enzymes , 237, 238, 240, 284 detoxifi cation , 239 J effl ux , 244, 246, 248 JCR:LA-cp rats , 180, 185 excretion , 272, 275, 276 Juvenile hemochromatosis , 257, 258 export , 240, 244, 247 homeostasis , 5, 146, 244, 246, 247, 254, 264, 277, 332, 362, 363 K infection , 282–284 Kaolin , 456 in food , 252, 253 Karelianite , 143 in infectious diseases , 6–9 Kashin-Beck disease , 517 in the brain , 277 Kenya , 253 in the cerebrospinal fl uid , 277 Keratinocytes , 334, 486 metabolism , 244–246, 257, 264, 345, 361 Keshan disease , 501, 517 overload , 8, 9, 241, 255–257, 261–264, Ketoaciduria , 59 272, 276, 284, 407 α-Ketoglutarate , 106, 424, 435 oxides , 479 Ketonuria , 462 physiology , 246–248 Kidney , 10, 32, 34–37, 39, 40, 42, 50, 52, 58, production , 283 67, 72, 73, 84, 86, 127, 145, 146, 177, scavengers , 271 261, 366, 367, 370, 371, 417, 432, 476, supplementation , 7, 8, 231, 253–255, 257 477, 510–513, 515, 522, 523 supplementation therapy , 231 cancer , 157 toxicity , 270–272 cells , 241 transport , 240–246, 257, 264 disease , 44, 185, 323 Iron(II), Fe2+ , 5, 106, 158, 208, 231–235, failure , 56, 375 237–242, 244, 252–254, 266, 267, 270, stones , 336, 464 271, 278, 315, 328,330, 332, 337, 372 transplant , 59 Index 555

Kinases Lipid(s) , 146, 175, 177, 183, 201, 274, 323, AMP-activated protein (AMPK) , 366, 392, 459, 460, 481 115, 187, 189 biosynthesis , 98 calcium-dependent (CaMK) , 100, 101, metabolism , 172, 181, 186, 349 104, 105 peroxidation , 190, 214, 329, 378 mitogen-activated protein (MAPK) , peroxides , 511 66, 187, 190, 333 Lipocalins , 6, 300 myosin light chain (MLCK) , 104, 109 Lipoic acid , 512 pantothenate kinase-2 (PANK 2) , 279, 280 Lipopolysaccharides (LPS) , 14, 66, 334 phosphatidylinositol 3-kinase (PI3K, Akt) , Lipoprotein receptor , 244, 522 154, 156, 187 Lipoxygenase , 270 phospho- , 147 Listeria monoctyogenes , 347 phosphorylase (PhK) , 104 Listeriolysin , 347 O-phosphoseryl-tRNA[Ser]Sec (PSTK) , Lithium, Li + , 114 503, 504 batteries , 165 protein. See Protein kinase Liver , 9–11, 15, 18, 19, 21, 22, 31, 63, 104, pyruvate dehydrogenase , 328 145, 146, 158, 163, 164, 177, 202–204, tyrosine , 153, 186, 189, 400, 406, 493 210, 239, 252, 255–258, 297, 298, 305, Kissing bugs , 163 309, 311, 314, 331, 348, 349, 366, 367, KK-Ay mice , 155, 462 369–371, 375, 396, 401, 403, 417, 422, KK/HIJ mice , 187 423, 432, 437, 440, 453, 476, 477, 481, Klebsiella 486, 491, 501, 503–505, 510–515, 517, oxytoca , 303 518, 522, 523 pneumoniae , 8 cancer , 281 degeneration , 173 disease , 10, 58, 65, 257, 376, 398, 399, 408 L disorder , 174 Lactate , 144, 148, 336, 438 failure , 38 Lactating women , 408 iron , 274 Lactobacillus , 349 transplantation , 9, 10 fermentum , 349 Livestock , 476, 487, 501, 519, 521 Lactoferrin , 6, 283 Loop of Henle , 34–37, 39, 58 Laennec’s cirrhosis , 399 Low chromium rodent diets , 173–176 Lambs , 374, 501 Low density lipoprotein (LDL) , 373 Latin America , 163 LOX. See Lysyl oxidase (LOX)

Laughing gas (N2 O) , 309, 313 LPS. See Lipopolysaccharides (LPS) Laxative , 41, 42, 56 Lung LDL. See Low density lipoprotein (LDL) adenocarcinoma , 511 Lead arsenate , 490 cancer , 144, 482 Legumes , 252, 422 Lyases , 205 Leishmania , 163, 164, 336, 346 Lyme disease , 5 amazonensis, 164 Lymph nodes , 15 Leishmaniasis, 140, 162, 164, 165, 337, 346 Lymphocytes , 10, 14, 65, 332, 333, 400, 467, 486 Lens dislocation , 426, 435 T- , 19, 160, 373, 467 Leptin , 180, 181, 462 Lymphoma

Lethal dose of As2 O3 , 481 gastric , 344 Leukemia Lysyl oxidase (LOX) , 361, 363, 365, 368, 375 acute lymphoblastic , 421 cells , 164 promyelocytic (APL) , 476, 477, 492, 493 M Leukocyte antigen , 251 Macroglobulin , 10

Leukopenia , 372, 401 α2 - , 206 Levodopa , 217 Macrophages , 5, 8, 10, 65, 163, 190, 206, 242, Lewy bodies , 119, 212, 216, 277, 279, 377 244, 247, 248, 255, 279, 283, 334, 346, Lichen , 150, 154 347, 373, 401, 462, 465, 466, 516 556 Index

Macular degeneration , 279, 280, 284, 403 copper homeostasis , 365

MAC value for V2 O5 , 144 cytochrome c oxidase , 362, 368 Magnesium, Mg2+ , 43, 49–75, 83, 86, diet , 173, 174 88–90, 143, 205, 206, 212, 217, metallothioneins , 367 267, 323, 326, 328, 367, 394, molybdenum-dependent enzymes , 416 456, 459, 503 tissues , 238, 241, 508 absorption , 51–54, 58, 63, 72, 73 Mammary glands , 393 accumulation , 51, 54, 55, 63–65 Manganese, Mn , 20–22, 199–220, 323, 326, channels , 51 362, 404, 459 chloride , 53 54 Mn , 204, 206 citrate , 53 absorption , 20, 21, 202, 203, 208 defi ciency , 56–58 accumulation , 204, 208, 209, 213, 216 -defi cient animals , 63, 66 biochemistry , 204–206 dietary intake , 51, 52, 54, 57, 58, 62, blood , 21, 202, 204, 217 63, 66 -citrate , 206, 209 excretion , 58–60 daily intake , 202 free , 52–54, 61, 65 defi ciency , 202, 205 gluconate , 53 deposition in the brain , 203 homeostasis , 54–56, 62–64, 73, 206 dietary intake , 20, 22, 201–203 hydroxide , 53 effl ux , 209, 210 in disease , 55–73 essentiality , 201, 202 level in blood , 56 excretion , 21, 202, 203 loss , 59, 65 homeostasis , 202, 203, 206, 210, 213 oxalate , 53 in infectious diseases , 21, 22 oxide , 53 neurotoxicity , 214, 215, 218 storage , 56 oxides , 417

sulfate (MgSO4 ) , 53, 66, 70, 71 pharmacokinetics , 202–204 supplementation , 58, 60–62, 64–66, 72, 73 physiology , 204–206 transport , 53–55 toxicity , 206, 212, 216, 218 trisilicate , 464 trafﬁ cking , 206, 210 Magnetic resonance imaging (MRI) , 200, 204, transport , 20, 206–210 279, 431, 435, 438 uptake , 207–209 Magnetic susceptibility , 188 Manganese(II), Mn2+ , 5, 21, 98, 151, 200, 203, Malabsorption of 206–208, 214, 215, 217 cobalamin , 299, 300 detoxiﬁ cation , 203 zinc , 11 Manganese(III) , 200, 203, 208, 214, 215 Malaria , 7, 8, 13, 22, 231, 253, 258, 259, 283, Manganese(IV) , 200, 203 284, 407 Manganese(V) , 200 resistance , 258 Manganese(VI) , 200 Malate , 108, 435 Manganese(VII) , 200 Malaysia , 455 Manganese superoxide dismutase (Mn-SOD, MALDI-TOF mass spectrometry , 520 SOD2), 3, 4, 20, 22, 205, 212, 213, Males , 21, 34, 155, 175, 177, 192, 252, 253, 362, 367 454, 456, 511, 516, 519, 520, 522 Manganism , 204, 206, 210–212, 216–218 Malignancies , 9, 18, 254 Manganoproteins , 205 Malignant hyperthermia (MH) , 124, 125 Manitol diuresis , 40 Malnutrition , 40, 58, 164, 250, 313, 408 Manufacture of glass , 200 Mammal(ian) , 84, 112, 114, 172, 175, 192, MAPK. See Mitogen-activated protein kinases 247, 283, 296, 303–310, 314, 324, 363, (MAPK) 374, 423, 426, 429, 439, 442, 502–504, mARC. See Mitochondrial amidoxime- 511, 514, 521–523 reducing component (mARC) brain , 201 Marine cells , 50, 51, 53, 99, 186, 191, 240, 241, algae , 141, 150 243, 244, 308, 327, 364, 405, 503 food webs , 480 Index 557

Mass spectrometry (MS) , 188, 427, 514, 520 Metabolism (of) Matrix a β amyloid plaques, 377 -assisted laser desorption/ionization amino acids , 349 time-of-fl ight (MALDI-TOF) , 520 arsenic , 484 metalloproteinases (MMP) , 392, 406 carbohydrates , 31, 107, 175, 201 vesicles (MV) , 87 catecholamines , 364 MCM. See Methylmalonyl-CoA mutase cholesterol , 193, 519 (MCM) cobalt , 300, 301 MCU. See Mitochondrial Ca2+ uniporter drugs , 422 (MCU) energy , 31, 51, 108, 201, 324, 330, 361 MDS. See Myelodysplastic syndrome (MDS) ethanol , 65 MeaB , 302, 303, 305 fatty acids , 189, 190, 306 Measles , 407 folate , 308, 310, 315 Meat , 252, 257, 296, 298, 299, 422, 491, 521 glucose , 20, 31, 153, 174, 175, 179, 183, MECAM , 267–269 186, 188, 205, 462 Mechanisms (of) glycine , 308, 309 neurodegeneration , 436 histidine , 308 ping-pong , 306, 510 homocysteine , 311 reaction , 305 hydrogen sulfi de , 441 toxicity , 214–216, 465–467 iron , 244–246, 257, 264, 345, 361 Medicinal use of lipids , 172, 181, 186, 349 silica , 467 metal , 3, 368, 403 silicon , 467, 468 methionine , 315, 485 Megalin , 313, 514, 522 methylmalonyl-CoA , 314 Megaloblastic anemia model , 297, 309, 310, methylmalonyl-CoA mutase , 303–306 312–314 nickel , 324 Meiotic cell cycle , 403 phosphate , 141, 147 Melanin phosphorus , 485 neuro- , 277, 377 propyonyl-CoA , 306 synthesis , 361 proteins , 201 Melanocytes , 363 serine , 308 Melanoma , 331, 493 sodium , 348 Melarsoprol , 478, 491 superoxide , 336 Melastatin , 51 valine , 311 Melissa offi cinalis , 218 zinc , 11, 391, 395, 399, 405, 408, 409 Membrane Metal-binding pterin (MPT) , 215, 216, channels , 93–95, 112 427–429, 431, 432, 436, 437 potential , 31, 94, 96, 107, 110, 206, 215, -AMP , 429, 434, 439, 440 243, 488 synthase , 428, 429, 431, 432, 434 traffi cking , 92 synthesis , 429, 434 vesicles , 63, 92 Metallic selenides , 517 Menkes P-type ATPase , 18 Metallo-chaperones , 341–343 Menstruation , 247, 250 Metalloproteinase 9, 86 Mental retardation , 69, 426, 440, 517, 518 Metallothioneins (MTs) , 365–367, 394, 396, 6-Mercaptopurine , 421, 423 400, 406, 424 3-Mercaptopyruvate sulfurtransferase (MSPT) , MT-1 , 367 423, 424 MT-2 , 367 Mercurial diuretics , 38 MT-3 , 367 Mesothelial cells , 465, 466 MT-4 , 367 Metabolic Metal response element-binding transcription acidosis , 40, 41, 45, 362 factor-1 (MTF-1) , 394, 395 alkalosis , 39, 41, 42, 44, 67 Metastases , 70 bone disease , 373 Meteorites , 143 diseases , 349, 404–407 Metformin , 64 558 Index

MetH , 304, 307–309 Mexico , 164 Methane formation , 336, 350 MH. See Malignant hyperthermia (MH) Methanobrevibacter smithii , 350 Mica , 452 Methanococcus jannaschii , 504 Mice (see also Mouse) , 6, 9, 15, 21, 62, 68, 70, Methanogenic archaea , 350 117, 118, 122, 123, 153, 155–157, 163, Methanopyrus kandleri , 504 187, 192, 203, 208, 212, 213, 217, 218, Methionine 257, 264, 265, 280, 283, 313, 326, 334, adenosyl-transferase , 309 345, 364, 366, 369, 370, 372–374, 396, aminopeptidase , 314 405, 422, 425, 436–438, 441, 458, 504, biosynthesis , 502, 521 511–514, 516–520, 522 -loaded tRNA , 503 CAKI-1 , 157 metabolism , 308, 309, 312, 314, 315, 485 DI2-knockout , 512, 519 -R-sulfoxide reductase (MsrB1) , 513 DI3-knockout , 519 seleno- , 16, 502, 503, 517, Dj-1-knockout , 217 521, 524 Gpx1-knockout , 511 supplementation , 310, 312, 313 Gpx4-knockout , 511, 520 Methionine synthase (MS) , 297, 298, KK-Ay mice , 155, 462 300–304, 306–315, 424, 514 KK/HIJ mice , 187 -deﬁ cient mouse , 310 Mtrr-deﬁ cient , 314 metabolism , 309 mutant SOD1 , 122 reductase (MSR) , 301, 302, 307–309, 314 nude , 345 Methotrexate , 423 obese diabetic KKAy , 462 Methylation of histones , 237 parvalbumin null , 52

Methylcobalamin (CH 3 -Cbl) , 297, 298, 302, prion protein-deﬁ cient mice , 379 303, 306, 307, 313 SelK-knockout , 516 Methyl-cyclopentadienyl manganese Sep15 knockout , 513 tricarbonyl (MMT), 200 Sepp1-knockout , 518, 522

Methylenetetrahydrofolate (CH2 -H4 folate) , septic , 6 308, 309, 311, 312 SOD1-deﬁ cient , 373 reductase (MTHFR) , 309–312 transferrin knockout , 265 Methylmalonic acidemia , 306 Micelles , 83, 149, 190

Methylmalonic aciduria , 300, 302, 311, 315 Michaelis-Menten constant, K m , 107, 418, 423, CblA type, (MMAA) , 302, 303, 305 429, 511 CblB type, (MMAB) , 302 Microangipathy , 441 CblC type and homocysteinuria, Microcystis aeruginosa , 485 (MMACHC) , 299, 300, 302 Microcytic anemia , 264, 265 CblD type and homocysteinuria, Microcytosis , 250

(MMADHC), 302 β2 -Microglobulin , 257 Methylmalonic anemia , 306 Micronutrients , 3, 16, 17, 296, 324, (S)-Methylmalonyl-CoA hydrolase , 306, 311 349, 408, 455, 457, 460–462, 468, Methylmalonyl-CoA metabolism , 314 501, 502, 518 Methylmalonyl-CoA mutase (MCM) , 297, Microorganisms (see also individual names 298, 300–306, 309, 311, 314 and species), 3, 83, 248, 256, 282, 296, deﬁ ciency , 302, 314 336–346, 349, 350, 373, 374, 477, 480, knock-out mouse model , 314 484–490, 493 metabolism , 303–306 Migraine , 118 reaction mechanism , 305 Milk , 83, 187, 248, 283, 296, 367, 370, Methylobacterium extorquens , 305 374, 393 Methylselenocysteine , 521, 523 Minasragrite , 143

Methyltetrahydrofolate (CH3 -H4 folate) , Mineralization 306–311 bones , 51, 84, 452, 457–459, 467 accumulation , 310 Mineralized tissues , 85, 88 Metronidazole , 163 Miners , 201, 211 Metvan , 156, 158 Mining , 144, 325, 479 Index 559

Mitochondria(l) , 3, 20, 22, 54, 55, 65, 88, 89, Monkeys , 204, 213, 313 94, 97, 98, 101, 106–108, 115, Monoamine oxidase , 281 118–122, 125, 149, 203, 204, 211, 215, Monoclonal antibodies , 69, 70 217, 243, 263, 277, 279, 302, 309, 366, Monocytes , 65, 66, 71, 190, 323, 401 402, 420, 424, 425, 427, 431, 435, 510, Mono Lake , 479, 488 515, 520 Monomethylarsenic (MMAs) , 481, 491 amidoxime-reducing component (mARC) , MMAsIII , 482, 483 419, 426, 431, 442 Monosiga brevicollis , 314 Ca 2+ uniporter (MCU) , 97 Morocco , 253 damage , 119 MOT1. See Molybdate transporter type 1 dysfunction , 3, 212, 215, 216, 219, (MOT1) 377, 442 Motor neuron disease , 362, 372 iron transport , 243, 244 Mouse ( see also Mice) , 9, 88, 117, 155, 181, membranes , 96, 113, 204, 484, 485 187, 208, 213, 265, 310, 313, 314, 334, oxidative phosphorylation , 362 345, 347, 367, 373, 374, 377, 378, 402, Mitogen-activated protein kinases (MAPK) , 422, 436, 438, 439, 503–505, 512, 522 66, 187, 190, 333 adipocytes , 155 mk mouse , 264 db/db , 181 MMAs. See Monomethylarsenic (MMAs) genome , 504 MMP. See Matrix metalloproteinases (MMP) liver , 503, 515 Mn-SOD. See Manganese superoxide mARC , 426 dismutase (Mn-SOD, SOD2) methionine synthase-defi cient , 310 MoCD. See Molybdenum cofactor defi ciency mk , 264 (MoCD) Mocs1 –/– , 436, 437 Moco. See Molybdenum cofactor (Moco) Mocs1 -knockout , 436 Mocs1 -knockout mouse , 436 mottled , 364 Mocs1 –/– mice , 436, 437 Mouse models (for/of) , 9, 117, 213, 313, 314, MOCS1 protein , 427, 431 347, 367 Models of Parkinson’s disease , 208 Alzheimer’s disease , 377 Molecular oxygen , 233, 235, 424 diabetes , 181 Molybdate , 417, 418, 429, 439, 440 Huntington’s disease , 378 overload , 440 Wriggle Sagami , 117 supplementation , 439 YAC128Q , 213 Molybdate transporter , 417, 418, 429 MPT. See Metal-binding pterin (MPT) type 1 (MOT1) , 417, 418 MRI. See Magnetic resonance imaging (MRI) type 2 (MOT2) , 418 mRNA Molybdenosis , 418, 440 synthesis , 344 Molybdenum, Mb , 415–442 translation , 66, 264 enzymes , 418–426, 431, 442 MS. See Methionine synthase (MS) homeostasis , 418 Mseleni disease , 202 in drinking water , 418 MSR. See Methionine synthase in soils , 418 reductase (MSR) toxicity , 418 MTF-1. See Metal response element-binding uptake , 417–418 transcription factor-1 (MTF-1) Molybdenum(IV) , 425 MTHFR. See Methylenetetrahydrofolate Molybdenum(VI) , 425 reductase (MTHFR) Molybdenum cofactor (Moco), 417–420, 422, MTs. See Metallothioneins (MTs) 425–442 Mucin , 203 biosynthesis , 427–430 Mucopolysaccharide synthesis , 205 defi ciency (MoCD) , 420, 424, 426, Mucor , 9 431–442 Mucormycosis , 9 maturation , 430 Multicopper oxidases , 362, 372 structure , 419 Multi-drug resistance protein (MRP) , 300, sulfurase , 430, 432 480, 481, 490 560 Index

Multi-mineral dietary supplements , 53 Natural resistance-associated macrophage Multiple sclerosis , 277 protein (NRAMP), 5, 206, 208 Multi-vitamin dietary supplements , 53 Nausea , 13, 31, 41, 45, 56, 118, 375 Muscle Necrosis , 37, 123, 466, 501, 517 cells , 33, 37, 62, 65, 66, 70, 94, 109, 110, liver , 173, 501 124, 187, 399, 460 Neonates , 20, 21, 426 contractions , 30, 31, 108–110, 115, 123, 149 death , 374, 419, 439 disease , 124–126, 501 Nepal , 7, 40 ﬁ ber , 33, 125 Nephrocalcinosis , 68 stiffness , 125 Nephron , 51, 53, 54, 62, 67 weakness , 31, 56, 58, 125 Nephropathy , 42, 64, 464 Muscular Nerve cells , 31, 32, 378, 401 disorders , 514 Nervous system , 32, 58, 201, 396 dystrophy , 102, 124–126, 516 Neurasthenia , 165 hypotonia , 374 Neurodegeneration , 208, 214, 216, 230, 265, system , 30, 33 277, 280–281, 284, 362, 375–377, 391, Mushrooms , 141, 521 402, 406, 407, 435–436, 440, 442 Mutant SOD1 mice , 122 Neurodegenerative diseases , 94, 118–122, Mutation of 280, 376, 379, 406, 467 DMT1 gene , 250 Neuroendocrine cells , 96, 403 glutaredoxin-5 gene , 250 Neuroexcitotoxicity , 435 MV. See Matrix vesicles (MV) Neuroﬁ brillary tangles , 120, 376, 407, 461 Mycobacterial urease , 347 Neurological diseases , 39, 117, 201, 361, 372, Mycobacterium , 282, 283 376, 379 bovis , 347 Neuromedin C , 332 tuberculosis , 161, 162, 283, 343, 346, 347 Neuromelanin , 277, 377 Myelodysplastic syndrome (MDS) , 8, 258, Neuromodulation , 360 262–263, 276–277, 372 Neuronal Myeloma cells , 157 death , 212, 436 Myeloneuropathy , 371 diseases , 116–118 Myocardial infarction, 38, 43, 58–61, 159, 399 excitotoxicity , 102 Myoglobin , 235, 250, 252 iron accumulation , 279 Myosin , 51, 104, 108, 109, 123, 149 Neurons , 33, 58, 65, 94–96, 98, 101, 110, light chain kinase (MLCK) , 104, 109 117–122, 146, 205, 209–212, 216–218, Myositis , 421 277, 376, 379, 393, 401–403, 407, 460, Myxedemateous endemic cretinism , 517 515, 518 Neuropathy , 64, 179, 372, 376, 378, 379 Neuropsychiatric disorders , 421, 441 N Neurotoxicity , 205, 211–218, 273, 402 NAADP. See Nicotinic acid adenine of aluminum , 461 dinucleotide phosphate (NAADP) Neurotransmitters , 93, 96, 105, 118, 122, 201, NADH. See Nicotinamide adenine 206, 364, 422, 435, 436 dinucleotide, reduced (NADH) Neurotrophins , 205 Nails , 201, 452, 457, 468, 517 Neutropenia , 9, 18, 371, 372, 374 Nanoparticles , 281 Neutrophils , 10, 323, 334, 373, 467 Nasal cancer , 325 New Zealand , 501, 521 National Academy of Science , 4, 174, 202, N F - κB pathway , 396–398 518 Nickel, Ni , 315, 321–351 National Research Council (NRC) , 202, 524 acetate , 322, 325 National Research Council of the National allergy , 332–335 Academies of Science, 173 availability , 345, 349, 350 Natriuresis , 36, 37, 39, 43 carbonyl , 323 Natural killer cells (NK cells) , 10, 12, 15, carcinogenicity , 327 401, 467 chloride , 322, 325 Index 561

cobalt/permeases (NiCoT) , 342 signaling , 205, 442 -containing active site , 337–340, 350 synthase (NOS) , 21, 109, 159, 161, 216, -containing alloys , 334 218, 330, 425, 426 -containing fuel , 322 synthesis , 425, 426 -dependent infectious diseases , 336–348 Nitrile hydratase , 314 excess , 341 Nitrilotriacetate , 234 exposure , 323, 324, 328, 335 Nitrite , 422, 425, 442 genotoxic effects , 327 Nitrogen-ﬁ xing bacteria , 141 homeostasis , 324, 340, 343, 345–348, 351 3-Nitro-4-hydroxyphenylarsonic homeostasis in pathogenic acid , 491 microorganisms , 340–344 Nitrosative stress , 402

immune reaction , 323 Nitrous oxide (laughing gas) (N 2 O) , 309, 313 -induced carcinogenesis , 324–332 -exposed rat model , 313 -induced neoplastic transformation , 326–332 NK cells. See Natural killer cells (NK cells) -induced teratogenicity , 323 N-methyl-D-aspartate (NMDA) , 101, 105, membrane transporters , 341, 342 120, 121, 218, 379, 402, 436 metabolism , 324 channel , 402 mining , 325 NMR. See Nuclear magnetic resonance molecular chaperones , 342, 343 NO. See Nitric oxide (NO)

oxides (NiO) , 322 N2 O. See Nitrous oxide (laughing gas) (N 2 O) -protein interactions , 344 Non-corrinoid cobalt , 314–316 sensors , 331, 341, 343, 344 -containing proteins , 314 silicates , 322 Non-heme iron smelting , 325 absorption , 253 storage , 343 aggregates , 263 sulfate , 322 enzymes , 237 sulﬁ des , 322, 325, 329 Non-transferrin bound iron (NTBI) , 238, 241, toxicity , 329, 331 255, 256, 258, 272, 276–278, 399 transport , 341 Norepinephrine synthesis , 361 uptake , 323–326, 341 Nori , 296 Nickel(I) , 350 Normal hydrogen electrode (NHE) , 267, 271, Nickel(II), Ni2+ , 206, 208, 328, 331–335, 337, 274, 479 338, 340–348 North America , 163 accumulators , 345, 346 North Korea , 517 homeostasis , 341, 343, 349 NR. See Nitrate reductase (NR) Ni 2+ /Co2+ sensors , 348 NRAMP. See Natural resistance-associated Ni 3+ /Ni2+ redox couple , 329 macrophage protein (NRAMP) permease , 345 NRC. See National Research Council (NRC) uptake , 344 NTBI. See Non-transferrin bound iron (NTBI) utilization , 341 Nuclear factor B (NFκB) , 71, 217, 218, 325, Nicotinamide adenine dinucleotide (NAD) , 396, 401, 467 107, 148, 420 Nuclear magnetic resonance (NMR) , 188 Nicotinamide adenine dinucleotide, reduced 1 H , 427 (NADH) , 107, 108, 147, 214 Nuclear transcription factor NFκB , 4 Nicotinic acid adenine dinucleotide phosphate Nucleic acids. See DNA and RNA (NAADP) , 97, 99, 112 Nucleolin , 213, 506, 508 Niemann-Pick disease , 118 Nucleotide(s) (see also individual names) [NiFe]-hydrogenase , 339, 340, 345 -gated channel functions , 348 Nifurtimox , 164 triphosphate hydrolysis , 343 Nigeria , 40 Nutrients , 3–6, 111, 114, 116, 139, 172, 201, − Nitrate (NO3 ) , 339 202, 204, 296, 298, 313, 315, 316, 345, Nitrate reductase (NR) , 417, 419, 425 346, 349, 353, 458, 484, 491, 501, 502 Nitric oxide (NO) , 21, 22, 61, 216, 400, Nutrition , 3, 107, 173, 186, 253, 296, 298, 374 422, 460 copper deﬁ ciency , 371–374, 376 562 Index

Nutritional immunity , 4, 5 Oxidases Nutrition Prevention Cancer (NPC) trial , 518 aldehyde (AOX) , 419, 421–423, 431, 442 multicopper , 362, 372 Oxidative stress , 3, 19, 22, 43, 64, 119, 121, O 190, 205, 208, 211–219, 278, 279, Obesity , 62, 64, 153, 173, 181, 184, 372, 324, 329–331, 334, 367, 373, 377, 404, 519 395, 396, 399, 406–408, 460, 461, -related insulin resistance , 181 465–467, 516, 519 Occipital horn syndrome (OHS) , biomarker , 14 372, 374–375 Oxidovanadium(IV) , 142, 144, 146, 153, 158, Occupational exposure to 160, 165 manganese , 201 Oxidovanadium(V) , 141, 158 nickel , 335 Oxoglutarate dehydrogenase , 107 vanadium , 144 8-Oxoguanine , 330

Occupational Safety and Health Oxygen (O2 ) , 107, 146, 147, 235, 237, 329, Administration (OSHA), 465 330, 336, 339, 362, 420, 487 Odontoblasts , 86, 87 -containing free radicals , 244 O f ﬁ ce of Dietary Supplements deﬁ ciency , 324 of the National Institutes singlet , 161 of Health, 182 transfer , 423 OHS. See Occipital horn syndrome (OHS) Oligodendrocytes , 210 Oliguria , 44, 45 P Omeprazole , 58 Paints , 200, 453, 490 OMM. See Outer mitochondrial membrane Pancreatic (OMM) β-cells, 110, 111, 393, 405, 519 Oncogenes , 327, 331 disease , 10 Onco-micro RNA , 406 Pancreatitis , 13, 59, 408 Oocytes , 96, 112, 402, 403 Pancytopenia , 371 ORAI1 channels , 97, 98 Paneth cells , 393, 399 ORAI proteins , 98 Pantothenate kinase-2 (PANK 2) , 279, 280 Oral small cell carcinoma , 406 Para-aminosalicilic acid , 217 Organ dysfunction , 3 Paracellin-1 , 54, 68 Organoarsenicals , 491 Paracoccidioides , 9 pesticides , 479 Paralysis , 41, 43, 146, 374 Ornithine transaminase , 463 Paraquat , 119 Orpigment , 479 Parasites , 141, 162–165, 248, 283, 284, 336, Orthosilicic acid (OSA) , 453–459, 463, 346, 407 467–469 Parasitic infection , 15, 249 Osmotic gradient , 36 Parathyroid Osteoblastogenesis , 458 gland , 87 Osteoblasts , 84, 86, 87, 393, 458, 462 hormone (PTH) , 53, 68, 72, 84 Osteochondropathy , 517 Parenchymal cells , 255, 256 Osteoclastogenesis , 458 Parenteral nutrition , 4, 19, 21, 211 Osteoclasts , 84, 86–88 PARK9 , 20, 209, 218 Osteoporosis , 72, 202, 311, 371, 373, 374, Parkin , 119, 216–218 457–459, 467 Parkinsonian Outer membrane , 6, 98, 113, 342 disturbances , 201 transporters , 345 -like symptoms , 211, 217 Outer mitochondrial membrane (OMM) , Parkinsonism , 209, 211, 377, 378 97, 426, 435 Parkinson’s disease (PD) , 118, 119, 121, 122, Ovarian cancer , 72, 157 206, 208, 211, 212, 214, 216–218, Oxalate , 53, 83, 176, 234 277–280, 284, 376–378, 380, 461 Oxaloacetate , 205 PARK2 mutations , 216 Index 563

Parvalbumin, 52, 119 ester hydrolysis , 150 null mice , 52 metabolism , 141, 147 Pathogenic bacteria , 282, 343 Phosphatidylcholine Pathogens , 4, 6, 12, 22, 283, 336, 338–340, biosynthesis , 485 345, 373 Phosphatidylinositol 3-kinase (PI3K, Akt) , Pathology associated with 154, 156, 187 potassium , 41–45 Phosphatidylserine , 87 sodium , 38–41 Phosphocitrate (PC) , 88 Patronite , 143 Phosphodiester , 149, 308 PD. See Parkinson’s disease (PD) Phosphoester hydrolysis , 151 Penicillamine , 371, 408 Phosphoester linkage , 158 Penicillin , 43 Phosphokinases , 147 Pentamidine , 58 Phospholipase (PL) , 92, 112, 119 Peptic ulcer , 344 Phosphorus metabolism , 485 Peptidylglycine α-amidating enzyme (PAM) , Phosphorylase kinase (PhK) , 104 361, 364, 368 O-Phosphoseryl-tRNA [Ser]Sec kinase (PSTK) , Periplasmic phosphate binding protein (PBP) , 503, 504 480, 488, 489 Phosphotyrosyl phosphatase , 150 Pernicious anemia , 297, 298, 312 Photophobia , 118 Peroxidases Phytates , 10, 176, 252, 408 bromo-165 Phytoremediation , 323 Peroxide , 147, 159, 191 Pick’s Peroxynitrite , 21, 218 bodies , 277 Perthest disease , 202 disease , 118, 277 Pesticides , 119, 479, 490–491 Pigments , 363, 403, 476 PET. See Positron emission tomography (PET) Pigs , 313, 436, 467 Phagocytes , 8, 326, 347 liver , 314 Phagocytosis , 244, 323, 466 PI3K. See Phosphatidylinositol 3-kinase Pharmaceuticals , 44, 72, 153, 165, 261, 456, (PI3K, Akt) 469, 477, 491, 492 Ping-pong mechanism , 306, 510 Pharmacodynamics , 141, 144–147 Pioglitazone , 64 Pharmacokinetics , 5, 10–15, 18–20, 141, Pituitary cells , 403 144–147, 158, 204, 273 Placebo , 3, 9, 12–14, 16, 17, 182, 184, Pharmacological agents , 71–73 185, 518 Pharmacology , 192, 193 Placenta , 7, 370, 371, 374, 512 Phase I clinical trials , 273 Plant(s) , 10, 83, 143, 201, 205, 252, 323, 336, Phase II clinical trials , 273, 377 361, 417–419, 425–427, 430–433, 480, 1,10-Phenanthroline , 156, 159, 164, 234, 266 484, 485, 501, 502, 521, 523 Phenoxide , 233 urease , 323, 338 PhK. See Phosphorylase kinase (PhK) Plaques , 120, 377, 459, 460 Phlebotominae , 164 Plasma , 2, 10, 15, 21, 22, 39, 44, 87, 203, 206, Phlebotomy , 257, 258 209, 272, 311, 363, 435, 436, 511, 514, Phonophobia , 118 522, 524 Phosphatases , 88, 104–107, 140, 147, 151, calcium , 83, 84, 87 156, 158, 164, 186, 324, 369 cysteine/cystine , 423, 425, 436, 440 acid , 86, 150 insulin , 175 alkaline (ALP) , 87, 88, 201, 458 iron , 255 serine/threonine , 205 lipid , 181 Phosphate , 45, 85–89, 93, 108, 141, 143, manganese , 21, 202, 203 146–152, 165, 233, 256, 417, 427, 429, membrane , 63, 82, 85–87, 93–100, 102, 476, 477, 479–481, 485, 486, 488–490, 104, 105, 108–113, 115, 117, 122, 126, 493, 505 189, 209, 257, 275, 323, 341, 365, 366, deﬁ ciency , 486 369, 375, 512 di-. See Pyrophosphate potassium , 30 564 Index

Plasma (cont .) Pregnancy , 7, 8, 16, 18, 38, 202, 249, 250, selenium , 15, 16 264, 312, 348, 363, 366, 407 sodium , 30 Prenatal diagnosis , 264, 266, 432 sulfate , 425 Preterm labor , 66, 70, 71 uric acid , 421 Primates , 174, 204, 216, 273, 484 vanadium , 145 Prion diseases , 376, 379 zinc , 10–12 Prion protein (PrPc) , 372, 379, 407 Plasmodium falciparum , 284 -defi cient mice , 379 Platelets , 60 Production of sulfuric acid , 165 aggregation , 399 Progressive Platinum(II) , 158 cerebellar-cerebral atrophy (PCCA) , 518 PMCA pump , 93, 94, 109, 117, 122, 123 microcephaly , 518 Pneumoconiosis , 464, 465 supranuclear palsy , 277 Pneumocystis , 9 Proinfl ammatory cytokines , Pneumonia , 12, 13, 15, 17, 19, 144, 407 65, 70, 71, 255 Poisoning of Prokaryotic pathogens , 346–348 livestock , 501 Prolyl hydroxylase , 460 nickel , 323, 326, 341 Propionibacterium shermanii , 305 Pollution of nickel , 322 Propionyl Polycistin-2 , 94 -carnithine in blood , 311 Poly-glutamine neurological diseases , 117 -CoA carboxylase , 306 Polymerases -CoA metabolism , 306 DNA , 151, 486 Prostaglandin , 217 Polyphenols , 252 Prostate cancer , 511 Polyuria , 41, 45, 67 Prostatitis , 15 Porcine liver , 314 Proteases , 102, 117, 119, 121, 126, 420 Porphyria , 265 Protein(s) (see also individual names) , 205 Porphyrins , 6, 142, 160, 235, 297, 315 CblC, 299–302 Positron emission tomography (PET) , 200 EF-hand , 84, 90, 100, 105 Potassium, K + , 29–45, 50, 60, 63, G , 96, 118, 303 88, 210, 215 Hfe , 257, 265 absorption , 34 natural resistance-associated macrophage arsenite , 492 (NRAMP), 5, 206, 208 blood levels , 41 prion (PrPc) , 372, 379, 407 channels , 54, 111 -RNA interactions , 508 defi ciency , 31, 35 S100 , 52 -defi cient diet , 42 seleno-. See Selenoproteins excretion , 34, 35, 44 SIBLING family , 87 homeostasis , 32–37, 41 Protein data bank , 91 intake , 34, 35, 61 PDB 2BB5 , 299 membrane action , 32, 33 PDB 1BMT , 304 permanganate , 200 PDB 1K7Y , 304 secretion , 34 PDB 2PMV , 299 supplementation , 60 PDB 1Q8J , 304 Potentials PDB 3SC0 , 299 electrical , 31, 32, 42, 51 PDB 2XIQ , 304 membrane , 31, 94, 96, 107, 110, 206, 215, Protein dephosphorylation , 243, 488 103–106, 399 redox , 148, 233–235, 267, 271, 274, 361, Protein kinase(s) , 92, 158, 324 366, 479, 502 A (PKA) , 104, 105 Poultry industry , 491 B (PKB) , 154–156, 159 PPi. See Pyrophosphate (PPi) C (PKC) , 52, 65, 66, 92, 104 P450 reductase , 301, 307, 308 C signaling , 55, 65 Pre-eclampsia , 56 Protein phosphatase-1 , 205 Index 565

Protein phosphorylation , 103–106, 405 R Protein tyrosine phosphatase (PTPase) , Rabbits , 21, 326, 459 154, 158, 159, 393, 406 Radicals Proteinuria , 45 5 ′-deoxyadenosyl, 427 Proteobacteria , 349 free , 4, 14, 201, 214, 217, 244, 270, 271, Proteolysis , 101 279, 374, 395, 465 Proton hydroxyl , 146, 214, 233, 234, 271, 329, gradient , 99, 480, 486 373, 374, 465 pump inhibitors (PPI) , 58, 71–73 superoxide , 20, 214 Protonation constant (see also Acid tyrosyl , 237 dissociation constants), 267 Ramalina celastri , 164 Protoporphyrin IX , 252 Rapamycin , 116 Protozoa(n) , 7, 346 Rat(s) , 21, 42, 161, 173–178, 185, 192, 204, parasites , 141, 163, 336, 346 208, 217, 218, 273, 326, 348, 399, 436, Provisional Tolerable Daily Intake for 460, 467, 484–487, 519, 520, 522 inorganic arsenic (PTDI) , 481, 484 adipocytes , 155, 188 PrPc. See Prion protein (PrPc) BB , 462

Psammomys obesus , 519 B12 -defi cient rats , 305, 309, 313 Pseudomonas , 282 Belgrade , 264, 265 aeruginosa , 161, 337 brain , 423 fl uorescens , 488, 489 diabetic , 153, 177 PTDI. See Provisional Tolerable Daily Intake genome , 422 for inorganic arsenic (PTDI) GR , 483 Pterin synthesis , 427–428 JCR:LA-cp , 180, 185 Pteris liver , 309, 311, 503 ensiformis , 485 models , 146, 180, 181, 313 vittata , 485 vitamin E-defi cient , 501 PTH. See Parathyroid hormone (PTH) Wistar , 175 PTPase. See Protein tyrosine phosphatase RDA. See Recommended daily (PTPase) allowance (RDA) P-type ATPase , 367–369 Reactive transmembrane , 209 nitrogen species (RNS) , 3, 4, 465 Pulmonary oxygen species (ROS) , 3, 61, 66, 119, carcinogen , 453 126, 140, 146–148, 157, 158, 166, diseases , 39 190, 214–216, 323, 325, 327, 329, edema , 465 334, 362, 366, 377, 402, 435, 442, infections , 13 460, 465–497, 511, 513 malfunctions , 144 Realgar , 479, 490 Purine-related biomarkers, 422 Receptor-operated Ca2+ channels (ROCCs) , 96 Purkinje cells , 117 Recommended daily or dietary allowance Pyridoxal isonicotinoyl hydrazine (RDA) for , 455, 501, 502, 524 (PIH), 282 cobalamin , 296 Pyrophosphate (PPi) , 87, 88, 427 magnesium , 51 Pyruvate selenium , 501, 502 carboxylase , 20, 204, 205 Red arsenic , 479 decarboxylase , 205 Red blood cells , 9, 21, 181, 244, 261, 263, 348 dehydrogenase , 107, 481 transfusions , 9 dehydrogenase kinase , 328 Redox Pythium , 9 agents , 148 cycle , 235, 271, 274, 394 potentials , 148, 233–235, 267, 271, 274, Q 361, 366, 479, 502 Quartz , 452, 456, 463 Reference daily intake for Mn , 202 Quinines , 214 R e fi nery workers , 325 566 Index

Regeneration of tissues , 462 Ryanodine receptor (RyR) , 94, 109, 120, 121, Regulation of 124, 516 iron metabolism , 244–246 channels , 95, 98, 99, 109, 110 magnesium transport , 55 the calcium signal , 97–99 Renal S cancer , 331 Saccharomyces cerevisiae , 364 cells , 52 Safe Drinking Water Act , 521 disease , 44, 45, 57, 67, 255, 442 Salivary glands , 192, 399 dysfunction , 20 Salmonella , 39, 282, 373 failure , 7, 18, 38–41, 44, 45, 57, 421 enterica serovar Typhimurium , 339 Renin , 37, 42–44, 67 typhii , 8 -angiotensin-aldosterone system , 32, 44 Salt marsh grass , 485 Reperfusion injury , 161 Salvarsan ® , 478, 491 Reproductive SAM. See S-Adenosyl-methionine function , 324 Sandfl ies , 164 system , 348, 402–403 Sarcoplasmic reticulum , 93, 95 Respiratory Sardinia , 264 alkalosis , 43 SARS. See Severe acute respiratory syndrome chain , 94, 97, 107, 117, 119, 235, 362, (SARS) 396, 402 SCD. See Systemic contact dermatitis (SCD) tract , 144, 325, 401 Schizophrenia , 311, 404, 441 tract infections , 11, 12, 407 Sea Retina(l) , 210, 403 squirts , 141, 143 degeneration , 280, 363 urchins , 112 Retinopathy , 64, 153, 185 Seafood , 252, 481 Reverse phase chromatography , 434 Seawater , 142, 417, 457 Rheumatism , 165 Seaweed , 296, 299, 480 Rheumatoid arthritis , 254, 255 Sec. See Selenocysteine (Sec) Rhinitis , 144 Second messengers , 93, 95, 99, 112, 324 Rhizopus , 8, 9 Seizures , 31, 205, 402, 426, 435, 436, 440, 441 oryzae , 8, 9 SELECT. See The Selenium and Vitamin E Rhodospirillum rubrum , 343 Cancer Prevention Trial (SELECT) Ribonucleotide reductase , 237, 238, 270, Selenide , 505, 514 282, 283 dimethyl- , 523 RNA , 3, 51, 102, 149, 245, 246, 308, 314, Seleninic acid , 510 315, 392 Selenite , 505, 512, 513, 517, 520, 521, 524 -dependent ATPases , 508 Selenium, Se , 4, 13–19, 22, 58, 174, 340, 476, m- , 66, 263, 264, 344, 506–508 484, 499–526 onco-micro , 406 75 Se labeling , 506 protein interactions , 507 administration , 17 ribosomal (rRNA) , 508 absorption , 521 small interfering (siRNA) , 114, 508 bioavailability , 517 tRNA[Ser]Sec , 503–505, 509, 514, 519 biomarker , 524 ROCCs. See Receptor-operated Ca2+ channels blood , 15, 517 (ROCCs) defi ciency , 15–17, 501, 513, 517–520, Rodents , 95, 174, 185, 217, 264, 273, 400, 522–524 459, 463, 464, 467 -defi cient diet , 518, 520 models , 193, 203, 250 -defi cient sheep , 514 ROS. See Reactive oxygen species (ROS) -depleted soil , 501 Rotenone , 119 excretion , 523 Roxarsone , 478, 491 exhalation , 523 Ruminants , 296, 418, 440 in infectious diseases , 15–18 Russia , 521 lowest observed adverse effect level , 518 Index 567

-related diseases , 516–518 Serine , 104, 154, 308, 311, 503, 508 supplementation , 15–17, 501, 511, 516, hydroxymethyltransferase , 309 517, 521, 524 metabolism , 308 therapy , 17 Serine/threonine phosphatase , 205 toxicity , 501, 517, 518, 524 Serotonin , 251, 422 transporter , 523 Serpentine , 323, 452 transport in mammals , 521–523 SerRS. See Seryl-tRNA synthetase (SerRS) Selenocysteine (Sec) , 502–503, 506, 510, Sertoli cells , 363, 522 512–514, 517, 521 Serum biosynthesis , 503–505, 509 albumin , 15, 145

insertion sequence (SECIS) , 500, 502, B12 , 313 506–510 calcium , 201 methyl- , 521, 523 cholesterol , 201 synthetase (SecS) , 505, 509, 518 chromium , 20, 179 tRNA , 503–505 cobalamin , 312 Selenocysteyl copper , 4, 18 -tRNA , 507–509 creatinine , 44 -tRNA [Ser]Sec , 505 magnesium , 53, 56–60, 62, 64, 68, 71–73 Selenomethionine , 16, 502, 503, 517, 521, 524 phosphorus , 201 Selenophosphate synthetase (SPS) , 505 selenium , 14–16 Selenoprotein(s) , 15, 16, 502–516, 518, sodium , 31, 38, 40, 45 519–525 trace elements , 4 15kDa (Sep15) , 513, 515 transferrin , 238, 239, 283 biosynthesis , 508, 513 zinc , 4, 18 H (SelH) , 506, 515 Seryl-tRNA , 503, 507, 508 I (SelI) , 506 synthetase (SerRS) , 503 K (SelK) , 506, 516 Severe acute respiratory syndrome (SARS) , M (SelM) , 515 159, 161 mRNA , 506–508 Sheep , 296, 305, 313, 372, 374, N (SelN) , 506, 516 379, 514 O , 515 Shigella , 39, 40, 282, 340 P (Sepp1) , 15, 16, 506, 514, 518, 519, Shock , 44 522–524 SHR rats , 62 S (SelS) , 506, 515, 516, 519 SH-SY5Y cells , 216, 218 T (SelT) , 506, 515 Siberia , 517 V (SelV) , 506, 514 SIBLING family of proteins , 87 W (SelW) , 514 Sickle cell disease , 14, 22, 258, 262, 408 Selenosis , 516–518 Sideroblastic anemia , 263–265 Seminal ﬂ uid , 402 Siderocalin , 6, 283 Senile plaques , 213, 376, 377 Siderochelin , 283 Sensors Siderophores , 3, 6, 9, 261, 270, 271, 273, Ni 2+ /Co2+ , 348 282, 283 Sensory function , 324 Signaling , 55, 89, 91, 92, 98, 112, 152, 189, Sep15 knockout mice, 513 334, 361, 366, 398, 518 Sepp1. See Selenoprotein P (Sepp1) agents , 88–93 Sepp1-knockout mice, 518, 522 calcium(II) , 395 Sepsis , 6, 11, 13, 15, 401 pathways , 54, 66, 72, 115, 329–331, 393, Septic 396, 409 mice , 6 Signal transduction , 154, 324, 396 patients , 14 Silica shock , 16, 22, 66 bioavailability , 455 Sequence alignment of MOCS1A , 433 content of water , 454 SERCA pump , 93, 98, 109, 113, 114, crystalline , 453, 463, 464, 468 124, 125 -deﬁ cient diet , 463 568 Index

Silica ( cont.) intake , 30, 34, 36, 37, 40, 61 from drinking water , 461 membrane action , 32, 33 gel , 452 metabolism , 348 -induced carcinogenesis , 465 Na+ /Ca2+ exchangers , 86, 93, 94, 100, 109, nanoparticles , 465–467 115, 119 Silicates , 322, 452, 453, 455, 456, 461, 463, Na + /Mg2+ exchanger , 53–55, 62, 467, 468 63, 65 alumino- , 452, 456, 461, 468 physiology , 32–37 Silicic acid , 453, 455–457, 459, 461, 463, -potassium ATPase , 37, 43 468, 469 -potassium ATPase (Na+ ,K+ -ATPase) , Silicon(es) , 451–469 36, 37, 43, 216 absorption , 464 -potassium pump , 31, 32, 118, 165 biomarker , 455 secretion , 35–37 carbide , 452 selenite , 17, 517, 521

dioxide (SiO 2 ) , 453–456, 463 sulﬁ de , 514 distribution , 453–456 wasting , 67 implants , 453 Soft tissues , 50, 51 in urine , 455 Soil selenium , 521 Silicosis , 453, 463–467 Solar cells , 452 Silymarin , 218 Solid tumors , 70 Single-photon emission computed tomography Somatostatin , 403 (SPECT) , 200 South Africa , 253 Singlet oxygen , 161 South America , 521

SiO 2 . See Silicon dioxide (SiO2 ) South East Asia , 261 SIRS. See Systemic infl ammatory response Spartina alternifl ora , 485 syndrome (SIRS) Spasticity , 518 Site-directed mutagenesis , 425 Sperm , 112, 313, 402, 511, 520 Skeletal maturation , 313, 512, 522 development , 396, 458–459 Spermidine catecholamides , 282 muscle diseases , 124–126 Spinal cord , 116, 118, 121, 212, 251, muscles , 42, 50, 51, 63, 65, 95, 104, 108, 378, 432 109, 124, 179, 186, 187, 331, 370, 512, Spironolactone , 59, 68 519, 522 Spleen , 15, 146, 164, 516 Skin , 13, 31, 41, 85, 86, 144, 163, 282, 332, S100 protein , 52 334, 335, 337, 373, 374, 396, 403, 422, SPS. See Selenophosphate synthetase (SPS) 457, 463, 464, 468, 477, 482, 493, 523 Squamous cell carcinoma , 511 eczematous , 332 Stability constants (see also Affi nity constants, Small interfering RNA (siRNA) , 114, 508 Binding constants, and Formation Small intestine , 10, 18, 19, 21, 51, 86, 176, constants), 234, 238, 267, 269, 482 203, 250, 369, 370, 399, 417 conditional or apparent , 177, 238, 405 Smelters , 201 π-Stacking, 159 Smokers , 511 Staphylococcus , 282 Smooth muscles, 30, 62, 65, 66, 104, 108–110, aureus , 5, 6, 8, 13, 161, 342 399, 460 Starfi sh , 112 SO. See Sulfi te oxidase (SO) Starvation , 4, 6, 42, 345, 488 SOD1. See Superoxide dismutase 1 (SOD1) Steel , 174, 452 SOD2. See Superoxide dismutase 2 (SOD2) production , 200 SOD1-defi cient mice , 373 Stem cell Sodium, Na+ , 30–45, 53, 59–63, 67, 88, 95, disorder , 262 99, 119, 142 transplantation , 8 absorption , 37 Sterility , 191, 418 channel , 32, 118, 119 Steroid(s) , 218, 235 excretion , 30, 35–37, 39, 44 hormone , 482, 483, 486 homeostasis , 34, 36, 37 STIM protein , 97, 98, 120 Index 569

Stomach , 18, 42, 101, 140, 144, 148, with silicon , 458 174, 192, 250, 296, 299, 338, 345, Swayback , 372, 374 346, 399 Sweat , 35, 283, 334 Store-operated channels (SOCCs) , Sweden , 164 97, 98, 209 α-Syn , 119 calcium , 98, 120, 206 Synaptic vesicles , 105, 401, 402 plasma membrane , 93 Synaptotagmins , 91, 110, 111 Streptococcus , 282 α-Synuclein , 119, 212, 216, 284, 377, 378 pneumoniae , 8 Syphilis , 477, 491 Streptomyces , 150 Systemic antibioticus , 273 contact dermatitis (SCD) , 335 coelicolor , 343 infl ammatory response syndrome (SIRS) , Streptozotocin (STZ) , 155, 181 3, 4, 18 rats , 155 iron overload , 255–266, 284 Stroke , 65, 262, 276, 402 Strontium, Sr 2+ , 208 STZ. See Streptozotocin (STZ) T Substitution therapy , 438, 439 T4. See Thyroxine (T4) Succinate , 435 Tachycardia , 43, 58 dehydrogenase , 246 Taiwan , 479, 482, 483 Succinyl-CoA , 306 Talc , 452, 456 Sucrose , 6 Tamoxifen , 218 Sulfate, 45, 83, 146, 325, 326, 417, 425, 429, Tanning of leather , 200 435, 440 cTannins , 252 transporters , 417 Tanzania , 7 Sulfhydryl groups , 233, 406, 423 Tau protein , 120, 406, 407 Sulfi de , 141, 329, 340, 423–425, 429, 441, Taurine , 61, 423, 436, 439, 440, 485 479, 490, 505, 514, 520 TB. See Tuberculosis (TB) di- , 350, 512, 513 TC. See Transcobalamin (TC) hydrogen , 441 TCA cycle , 205, 246, 306 nickel , 322, 325, 326, 329 T-cells , 17, 101, 105, 106, 160, 163, Sulfi te 323, 325, 332–335, 347, accumulation , 425, 435, 436 400, 401, 467 toxicity , 423–426, 435 T1DM. See Type 1 diabetes (T1DM) Sulfi te oxidase (SO) , 418, 419, 423–426 T2DM. See Type 2 diabetes (T2DM) d e fi ciency , 424–426, 439–440 Tea , 153, 155, 252 S-Sulfocysteine (SSC) , 436–440 Teeth , 42, 83, 85–87, 421 Sulfuric acid manufacturing , 501 Tellurium , 501 Supercoiled plasmid DNA , 163 TEs. See Trace elements (TEs) Superoxide , 4, 146, 147, 158, 163, 205, 214, Testicular 215, 233, 361, 362, 420, 422 cancer , 157, 158 detoxifi cation , 361 damage , 310, 313 metabolism , 336 Testis , 105, 514–516, 519, 521, 522 radicals , 20, 214 Tetany , 41, 58, 68 Superoxide dismutases (SOD) , 15, 205, 215, Tetrahydrobiopterin , 439 329, 342, 361, 362, 367, 370 Tetrahydrofolate 1 (SOD1) , 121, 122, 212, 361, 362, 365, methyl- , 306–311 367, 377, 379 Tetrapyrrole ring , 297 2 (SOD2) , 3, 4, 20, 22, 205, 212, 213, Tetrathiomolybdate , 378, 418, 440 362, 367 Tf. See Transferrin (Tf) 3 (SOD3) , 362, 373 TfR. See Transferrin receptor (TfR) Supplementation TGR. See Thioredoxin/glutathione of vitamin B , 458 reductase (TGR) of vitamin K , 458 Thailand , 253 570 Index

Thalassemia , 8, 258, 260–262, 264–266, 272, excito- , 102, 117, 118, 122, 205, 212, 274, 276, 277 218, 375 α- , 259, 261 nanocompounds containing cobalt , 315 β- , 259–262, 266 Toxicology of major , 260, 261, 264, 272, 274 silica , 463–467 Thapsigargin , 114 silicon , 463–467 Therapeutic agents , 33, 172, 264, 477, 491 Toxins , 6, 12, 33, 208, 345 Thermotoga martima , 304, 308 TPC. See Two-pore channel (TPC) The Selenium and Vitamin E Cancer TPN. See Total parenteral nutrition (TPN) Prevention Trial (SELECT) , 518 TR. See Thioredoxin reductase (TR) Thiamine deﬁ ciency , 42, 58 Trace elements (TEs) , 3, 4, 15, 17, 18, 22, Thiazide diuretics , 38, 39, 43, 58, 60, 68 172–174, 176, 380, 457, 459, 476 Thiocyanate , 517 Traditional Chinese medicine , 477, 492 Thiol(s) , 3, 214, 216, 306, 366, 395, 408, 436, Transcobalamin (TC) , 299, 300 481–483, 502, 511, 512, 520 receptor , 300, 313, 332, 400 -disulﬁ de oxidoreductase , 368 Transcription factor(s) , 4, 100, 101, 105, 324, Thioredoxin , 510, 512, 515 327, 329–331, 347, 391, 392, 396, 406, glutathione reductase (TGR) , 512 462, 466, 509 reductase (TR) , 512 NFAT , 106 Thiosemicarbazones , 162, 282 SMAD4 , 248 Thiosulfate , 424, 425, 438, 440, 441 Transferases Threonine , 51, 104, 114, 205, 206 adenosyl- , 302, 309 Thrombosis , 399 adenylyl- , 429 Thymidine 5′-monophosphate (dTMP), arsenic methyl-(As3MT) , 481, 490 308, 312 -cob(I)alamin adenosyl- , 302 Thymidylate synthase , 309 glutathione S- , 69 Thymulin , 12 glycosyl , 205 Thymus , 400, 515 3-mercaptopyruvate sulfur-(MSPT) , Thyroid 423, 424 atrophy , 517 Transferrin (Tf) , 6, 9, 19, 21, 144–146, Thyroid hormone(s) , 511, 512 176–179, 186, 188, 202, 203, 208, 238, deiodinases , 511–512 241–242, 244, 247, 248, 250, 255, 256, receptor , 511, 512 265, 272, 277, 283, 284, 372, 407 Thyroxine (T4) , 254, 512 Transferrin receptor (TfR) , 20, 206, 208, 209, Tissue 238, 241, 242, 244, 246 adipose , 145, 146, 405, 512, 519 knockout mice , 265 distribution of vanadium , 141 Transfusion therapy , 262 magnesium , 52, 59, 63 Transgenic mice , 217 TNF. See Tumor necrosis factor (TNF) Transglutaminase , 21, 203 Tobramycin , 58 Transient receptor potential channels Tocolytic , 66, 70, 71 (TRP) , 51 Tolerable intake for molybdenum , 418 Trans-membrane proton gradient , 480, 486 Tolerable upper intake level (TUL) , 455 Transmissible spongiform of selenium , 518 encephalopathies, 379 TonB-dependent transport of nickel , 342 Transplants , 59, 106, 254 Torula yeast , 174, 501 Transport of Total parenteral nutrition (TPN) , 13, 18, calcium , 93, 94, 106, 208 179–180, 371, 408, 484, 485 chromium , 176, 179 Toxemia , 38 iron , 176, 241, 284 Toxicity of iron-loaded transferrin , 241–242 arsenate , 481, 486 manganese , 20, 98, 202, 206–210 arsenite , 481 nickel , 323, 341, 342 chelators , 270–272 oxygen , 250, 362 drugs , 421 zinc(II) , 401 Index 571

Trauma , 3, 4, 16, 18, 20, 22, 43, 250, 254 Type 2 diabetes (T2DM) , 43, 63, 64, 102, Traumatic brain injury , 402 152–154, 180–182, 184, 185, 404, 405, Treatment of 462, 467, 516, 518, 519 cancer , 156–159, 231, 492 Tyrosinase , 361, 363–364, 368, 375 childhood diarrhea , 12 Tyrosine kinase , 153, 186, 189, malaria , 13 400, 406, 493 manganism , 217 Tyrosine phosphatases , 158, 159, 161 MoCD type A , 438 phosphatase-1B (PTP-1B) , 150, 154, 156, MoCD type B , 439 186, 399, 405 molybdenum cofactor defi ciency , 437–440 Tyrosyl radical , 237 sulfi te oxidase defi ciency , 439–440 Tremolite , 463 Triatominae , 163 U Triazoles , 273 Ubiquitin , 492 Tricarboxylic acid cycle (TCA) , 205, 246 UNICEF. See United Nations Children’s Trichosporon , 9 Fund (UNICEF) Trichostatin A , 328 United Kingdom , 17, 264, 454 Triglycerides , 180–182, 348, 459 United Nations Children’s Fund Trimethylselenonium , 523 (UNICEF), 482 TR3-knockout mice, 512 United States , 34, 173, 184, 455, 459, tRNA[Ser]Sec , 503–505, 509, 514, 519 501, 521 Tropomyosin , 108, 109, 123 Urea Troponin , 52, 108, 109 cycle , 205 TRP. See Transient receptor potential channels hydrolysis , 336, 338 (TRP) Urease , 323, 336, 338–339, 342, 343, TRPM6 , 51, 52, 54, 55, 62, 63, 68, 69, 72 345–347, 349 TRPM7 , 51, 52, 55, 62, 63, 72, 206 chaperones , 345 Trypanasoma cruzi , 163, 164, 346 Uric acid , 3, 419–422, 436, 438, 442 Trypanosome infections , 491 accumulation , 421 Trypanosomiasis , 15, 162 Urinary American , 162 chromium loss , 20, 175, 177, 178 Tuberculosis (TB) , 4, 7, 16, 19, 162, 165, 254, excretion of zinc , 10 283, 336, 346, 407 potassium , 42 Tubular necrosis , 59 selenium , 523, 524 Tubulins , 149 sulfi te level , 436, 438 TUL. See Tolerable upper intake level (TUL) xanthine level , 436 Tumor(s) , 165, 325–327, 330, 331, 408, Urinary tract 468, 511 calculi , 421 breast , 156 infections , 19, 336 cells , 69, 70, 72, 141, 147, 156, 157, Urogenital system , 348 164, 281, 282 Urolithiasis , 421, 464 necrosis factor (TNF) , 4, 64, 190, Urothione , 442 396, 466 UV/Vis , 361 solid , 70 Tumor suppressor , 102, 327, 330, 511, 513 genes , 158, 327, 328, 331 V Tungstate(VI) , 160 Vaccine , 347, 468 Turkey , 40, 252 Valine metabolism , 311 Two-pore channel (TPC) , 94, 95, 99, 118 Vanadate(V) , 141–144, 146–161, 164, 165, Type 1 copper enzymes , 361 187, 191 Type 2 copper enzymes , 361 inorganic , 158 Type 3 copper enzymes , 361 -phosphate antagonism , Type 1 deiodinase (DI1) , 506, 511–512 141, 147–152 Type 1 diabetes (T1DM) , 63, 152, 181, 405 Vanadinite , 143, 164 572 Index

Vanadium (in) , V, 140–166 E , 4, 17, 518 antidiabetic compounds , 154 E-deﬁ cient rats , 501

antiparasitic properties , 162 Vitamin B12 , 297–310, 484 blood , 146 bioavailability , 296 cardio-protective effects , 160 deﬁ ciency , 296, 297, 300, 310–314, cycling , 142–147 371, 372 food , 143 deﬁ cient rats , 305, 309, 313 freshwater , 142 VOCCs. See Voltage-operated Ca2+ channels intake , 140, 143, 144, 146 (VOCCs) minerals , 143 Volcanic emissions , 143, 322 nitrogenase , 140, 141 Voltage-dependent anion channels (VDAC) , overload , 143, 144 97, 520 oxides , 143, 144, 146, 164 Voltage-gated K+ channels , 111 oxides in breathing air , 146 Voltage-operated Ca2+ channels (VOCCs) , poisoning , 146 96, 111 rocks , 142, 143 Voltage-regulated seawater , 142 Ca2+ channels , 206 sediments , 142, 143 channels , 208, 209

VOx , 143, 144, 164 Vomiting , 31, 38, 39, 41, 45, 56, 118, 375, 481 Vanadium(II) , 143 Vanadium(III) , 141, 143, 147 Vanadium(IV) , 141–148, 151–154, 157, 158, W 160, 162, 165, 166 Watson-Crick base pairs , 506 Vanadium(V) , 141–154, 156, 157, 161, 162 Welders , 201, 211, 212 Vanadocene , 156, 158, 159, 165 Welding fumes , 216, 217 Vanadyl sulfate , 153 Western blotting , 520 Vascular Wheat , 454, 485, 521 disease , 459, 460, 482 Whipple’s disease , 59 dysfunction , 62 White muscle disease , 501 endothelial growth factor (VEGF) , 330 WHO. See World Health Organization (WHO) smooth muscle cells , 62, 65, 66 Whole blood , 15, 21, 217 Vasopressin , 32, 53, 55, 59 Wistar rats , 175 VDAC. See Voltage-dependent anion channel Wood preservative , 491 (VDAC) World Health Organization (WHO) , 253, 260, VEGF. See Vascular endothelial growth factor 345, 346, 391, 502, 524 (VEGF) Wound healing , 13, 462–463 Verapamil , 125 Wriggle Sagami mouse model , 117 Vertebrates , 5, 6, 8, 85, 141, 206, 323, 336, 338, 425, 514 Vibrio X cholerae , 39 Xanthine parahemolyticus , 342 accumulation , 420, 421 vulniﬁ cus , 9 dehydrogenase (XDH) , 419–422 Vietnam , 253 oxidase (XO) , 191, 417–421, 430, Viomycin , 58 435, 442 Viral Xanthine oxidoreductase (XOR) , 419–422, infections , 15, 517 431 liver disease , 399 deﬁ ciency , 420 Viruses , 17, 141, 159–161, 165, 517 Xanthinuria Vitamin(s) , 3, 310, 390, 422, 453, 458, 484 type 1 , 420, 421 B6 , 58 type 2 , 421 C , 4, 17, 253, 422 XDH. See Xanthine dehydrogenase (XDH) D , 52, 58, 84–86, 459 Xenobiotics , 33, 36, 235, 422, 423 dietary supplements , 53 Xeroderma pigmentosum group A , 483 Index 573

XO. See Xanthine oxidase (XO) defi ciency , 391, 395, 396, 398–408 XOR. See Xanthine oxidoreductase (XOR) diet , 404 X-ray excretion , 10 absorbance , 188 fi nger proteins , 10, 392 X-ray crystal structure of fi nger transcription factors , 396 arsenate , 489 free , 394, 395, 408 cobalamin , 297 growth , 403 phosphate , 489 homeostasis , 10, 366, 391, 394, 395, 398, 401–404, 406–408 infectious diseases , 11–14 Y metabolism , 11, 391, 395, 399, 405, 408, 409 YAC128Q mouse model , 213 -metallo β-lactamases, 11 Yeast , 182, 218, 367, 368, 504, 521, 524 metalloproteins , 392, 398 Brewer’s , 174, 183, 186, 501 -metallothionein (Zn-MT) , 11 torula , 174, 501 overload , 372, 395 Yellow arsenic , 479 proteome , 392 Yersinia , 9, 282, 340, 342 regulatory , 393 enterocolitica , 8 release , 393 signaling , 393, 394, 396, 400, 401 sulfate , 12 Z supplementation , 11–14, 396, 399, 401, Zebra fi sh , 264, 265 402, 408 Zeolites , 458, 468, 469 therapy , 408 Zinc(II) (in), Zn2+ , 4, 5, 10–15, 17–19, 21, 22, transporters , 11, 208, 399–401, 404–406 88, 149, 153, 208, 213, 231, 250, 266, vesicles , 393, 394 267, 271, 323, 326, 336, 337, 342, 346, Zincuria , 399 348, 361, 362, 366, 367, 371, 376, 380, ZIP. See Zrt-,Irt-like proteins (ZIP) 390–409, 429, 482–484, 492 Zip family , 394 acetate , 14 Zn-MT. See Zn-metallothionein (Zn-MT) binding constant , 405 ZnT family , 394 biomarker , 396, 399, 407 Zrt-,Irt-like proteins (ZIP), 407 blood , 395, 401, 404 Zucker diabetic fatty rats , 175, 177, 180, 185