Biomarkers of exposure to nitrosation products of amino acids and peptides.
Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester
by
Kathryn Lisa Harrison BSc. MSc. CMHT University of Leicester
July 1998 UMI Number: U536777
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Biomarkers of exposure to nitrosation products of amino acids and peptides. Kathryn L. Harrison.
The endogenous nitrosation of dietary amino acids and peptides may be a major source of genotoxic damage in the stomach and lower GI tract. Amongst the many N-alkyl-N- nitrosocompounds and related compounds, there are a number which share the common features of being derived from glycine and are carboxymethylating agents.
Novel immunoaffinity-RP-HPLC-fluorescence methods were developed allowing the concomitant detection of 0 6-carboxymethy 1-2 ’ -deoxyguanosine (0 6-CMdG) and 0 6-methyl- 2’-deoxyguanosine (0 6-MedG) with limits of detection of 0.128pmol 0 6-CMdG/mol dG and 0.064(j.mol 0 6-MedG/mol dG in lmg of DNA. N-(N’-acetyl-L-prolyl)-N-nitrosoglycine (APNG), azaserine and potassium diazoacetate (KDA) all reacted with calf thymus DNA in vitro to give the expected 0 6-CMdG as well as lesser amounts of 0 6-MedG. The ratios of O6- MedGto 0 6-CMdG were 1:9.73 (APNG), 1:16.12 (KDA) and 1:38.45 (azaserine). However, these nitrosated glycine derivatives showed large differences in their capacity to alkylate DNA with 0 6-alkylation formation ratios of 1:12.1:137.9 for 5mM azaserine, APNG and KDA respectively.
As 0 6-CMdG is not repaired by 0 6-alkylguanine alkyl transferases and is likely to be persistent in mammalian tissues, a sensitive immunoslotblot assay (0.32{imol 0 6-CMdG/mol dG) which required only lpg DNA for 0 6-CMdG was developed. 0 6-CMdG was detected in the stomach DNA of experimental animals treated with KDA or APNG by gastric intubation. Interestingly, control animals and non-target tissues of treated animals (liver, intestine) also had detectable levels of 0 6-CMdG.
Examination of the DNA from human gastric biopsies from 30 individuals involved in a H. pylori study, revealed detectable amounts of 0 6-CMdG in 27 samples ranging from 0.60- 19.79pmol/mol dG. No statistically significant difference was found between individuals with or without H. pylori infection on the basis of 0 6-CMdG levels, although the highest levels observed were all in infected individuals. 0 6-CMdG was also detected in human white blood cell DNA from the H. pylori and a diet study with levels ranging from 0.66-15.42jj.mol/mol dG.
1 Contents
ABSTRACT 1
CONTENTS 2
INDEX OF TABLES AND FIGURES. 9
ACKNOWLEDGMENTS 15
ABBREVIATIONS 16
CHAPTER 1. INTRODUCTION 19
1. Introduction. 20
1.1. Chemical carcinogenesis. 21 1.1.1. Historical perspective. 21 1.1.2. Chemical carcinogens: structure and activity. 22 1.1.3.Carcinogenesis is a multistage process. 23 1.1.3.1. Initiation and promotion. 24 1.1.3.2. Progression 25 1.1.4. Critical DNA targets during carcinogenesis. 26 1.1.4.1. Proto-oncogenes. 26 1.1.4.2. Tumour-suppressor genes. 27 1.1.4.3. Multiple genetic events occur during multistage carcinogenesis. 28
1.2. Alkylating Agents. 29 1.2.1. Biological consequences of alkylating exposure. 29 1.2.1.1. DNA Modification. 29 1.2.1.2. DNA Repair of Alkylated Bases 32 1.2.1.3. Non-random DNA alkylation and repair. 33 1.2.2. Human exposure to alkylating agents. 34 1.2.3. Use of Carcinogen-DNA Adducts as Biomarkers for Cancer. 35
2 Contents
1.3. Gastric Cancer. 38 1.3.1. Risk factors for Gastric Cancer. 3 8 1.3.2. Endogenous nitrosation. 39 1.3.2.1. Acid-catalysed endogenous nitrosation and its possible role in gastric cancer. 40 1.3.2.2. Bacterially mediated endogenous nitrosation and its possible role in gastric cancer. 41 1.3.3. The Correa model of gastric carcinogenesis. 42 1.3.3.1. Conditions with achlorhydria. 43 1.3.3.2. Helicobacter pylori and the importance of inflammation. 44 1.3.4. Measurements of N-nitroso compounds in gastric juice. 45 1.3.5. Precursors for endogenous nitrosation in the diet. 46 1.3.5.1. Amino acids and peptide nitrosation. 47
1.4. Biological activity of nitrosated peptides and compounds of related structure. 49 1.4.1. DNA alkylation adducts from nitrosated peptides and compounds of related structure. 50 1.4.2. Nitrosated glycine derivatives cause methylation as well as carboxymethylation of DNA. 52 1.4.3. The 0 6-carboxymethyldeoxyguanosine DNA adduct. 52 1.4.4. The 0 6-methyldeoxyguanosine DNA adduct. 53 1.4.5. Techniques available for the detection of DNA alkylation. 55
1.5. Aims of project. 57
CHAPTER 2. METHOD DEVELOPMENT 59
2.1. Introduction. 60
2.2. Synthesis of tritiated purine deoxynucleoside adducts. 61
2.3. 0 6-CMdG rabbit antiserum. 63
2.4. 0*-CMdG immunoaffinity column characterisation. 64
2.5. 0 6-MedG immunoaffinity column characterisation. 66
3 Contents
2.6. Preparation of N2-amino-S6-(carboxymethyl)-mercaptopurine for use as an Internal Standard. 68
2.7. HPLC optimisation for 0 6-alkylguanine adducts. 72
2.8. Digestion of DNA to nucleosides. 75
2.9. Sample preparation prior to HPLC analysis. 79 2.9.1. 0 6-CMdG immunoaffinity eluate 79 2.9.2. 0 6-MedG immunoaffinity eluate 80
2.10. Method validation 80
2.11. Discussion. 87
CHAPTER 3. IN VITRO STUDIES. 90
3.1. Introduction. 91
3.2. Concomitant formation of 0 6-CMdG and 0 6-MedG by N-carboxymethyl-N- nitrosocompounds and diazoacetic acid derivatives. 92 3.2.1. Analysis of 0 6-CMG and 0 6-MeG in DNA. 92 3.2.2. 0 6-alkylation by nitrosated glycine derivatives. 93
3.3. Mechanistic studies on 0 6-alkylation. 97
3.4. Discussion. 99
CHAPTER 4. IN VIVO ANIMAL STUDIES. 104
4.1. Introduction. 105
4.2. Development of the immunoslot blot assay for 0 6-CMdG. 107
4.3. In vivo APNG study. 110
4.4. In vivo KDA study. 112 4.4.1. Non-target tissue levels of 0 6-CMdG 4 hours after dosing with KDA. 112
4 Contents
4.4.2. Target tissue levels of 0 6-CMdG 4 hours and 24 hours after dosing with KDA. 114 .
4.5. Comparison of the immunoslot blot with the immunoafflnity-HPLC- fluorescence assay. 116
4.6. Discussion. 118
CHAPTER 5. HUMAN STUDIES. 123
5.1. Introduction. 124
5.2. H. pylori study. 125 5.2.1. 0 6-CMdG levels in gastric mucosa DNA. 125 5.2.2. 0 6-CMdG levels in white blood cell DNA. 129
5.3. Diet Study. 130
5.4. Discussion. 134
CHAPTER 6. DISCUSSION. 139
6.1 Discussion. 140
CHAPTER 7. MATERIALS AND METHODS 145
7.1. Materials and Methods for Chapter 2. 146 7.1.1. Apparatus. 146 7.1.2. Synthesis of tritiated purine deoxynucleoside derivatives. 146 7.1.2.1. Chemicals. 146 7.1.2.2. HPLC Systems. 146 7.1.2.3. Enzymatic Coupling. 147 7.1.2.3.1.Trial reaction: Chapeau & Mamett 147 7.1.2.3.2.Trial reaction: Stadler et al (1994) 147 7.1.2.3.3. [3H]-deoxyguanosine derivative synthesis. 148 7.1.2.4. Purification of [3H]-deoxynucleoside derivatives. 149 7.1.2.5. Quantitation of [3H]-deoxynucleoside derivatives. 150
5 Contents
7.1.3. Characterisation of rabbit 0 6-CMdG anti-serum. 152 7.1.4. Preparation and characterisation of immunoaffinity columns for 0 6-CMdG. 154 7.1.4.1. Materials and chemicals. 154 7.1.4.2. Preparation of columns 154 7.1.4.3. Characterisation of immunoaffinity columns for 0 6-CMdG. 155 7.1.4.4. Capacity determination for 0 6-CMdG immunoaffinity columns. 155 7.1.5. Preparation and characterisation of immunoaffinity columns for 0 6-MedG. 155 7.1.5.1. Materials and chemicals. 155 7.1.5.2. Preparation of columns. 156 7.1.5.3. Characterisation of immunoaffinity columns for 0 6-MedG. 156 7.1.5.4. Capacity determination for 0 6-MedG immunoaffinity columns. 156 7.1.6. Immunoaffinity work for S6-CMdG. 157 7.1.6.1. Binding and elution of [3H]-S6-CMdG using 0 6-CMdG immunoaffinity columns. 157 7.1.6.2. Determination of 0 6-CMdG column capacity for Oe-CMdG using [3H]- S6-CMdG as the marker compound. 157 7.1.6.3. Determination of binding of [3H]- S6-CMdG to the 0 6-CMdG columns in the presence of DNA digests. 157 7.1.7. HPLC optimisation for 0 6-alkylguanine adducts. 158 7.1.7.1. 0 6-CMG 158 7.1.7.2. 0 6-MeG 158 7.1.8. DNA digestion to nucleosides. 159 7.1.8.1. Chemicals. 159 7.1.8.2. Enzymatic digestion of DNA. 159 7.1.8.3. Acid hydrolysis of DNA. 160 7.1.8.4. HPLC analysis of DNA hydrolysates. 160 7.1.9. Eluate sample preparation prior to HPLC-analysis. 161 7.1.9.1. 0 6-CMdG eluate. 161 7.1.9.2. 0 6-MedG eluate. 161 7.1.10. Method Validation. 162 7.1.10.1. Isolation of 0 6-CMdG or 0 6-MedG from DNA digests by immunoaffinity purification. 162
6 Contents
7.1.10.2. Overall immunoaffinity purification protocol for 0 6-CMdG and 0 6-MedG from DNA. 162
7.2. Materials and Methods for Chapter 3. 163 7.2.1. Safety Warning. - 163 7.2.2. Chemicals and Apparatus. 163 7.2.3. Treatment of DNA with carboxymethylating agents. 163 7.2.4. Determination of 0 6-CMG and 0 6-MeG in DNA. 164 7.2.5. KDA pH study. 1 6.4
7.3. Materials and methods for Chapter 4. 165 7.3.1. The immunoslot blot assay. 165 7.3.1.1. Chemicals and apparatus. 165 7.3.1.2. Alkylated DNA standard 165 7.3.1.3. Immunoslot blot assay. 165 7.3.1.4. Densitometry of X-ray films. 166 7.3.2. In vivo APNG study. 167 7.3.3. In vivo KDA study. 167 7.3.4. DNA extraction from rat tissues. 168 7.3.4.1. Chemicals and Apparatus. 168 7.3.4.2. Phenol-chloroform extraction. 168 7.3.4. DNA quantitation for the immunoslot blot assay. 169 7.3.4.1. Chemicals and apparatus. 169 1 3 .4 2 . Enzymatic digestion of DNA to deoxynucleoside- 3 ’-monophosphates. 169 7.3.4.2.I. RP-HPLC-UV of 3’monophoshate deoxynucleosides. 171
7.4. Materials and Methods for Chapter 5. 171 7.4.1. Helicobacter pylori study. 171 7.4.1.1. Study design. 171 7.4.1.2. Samples collected. 172 7.4.1.3. Preparation of DNA Samples for the determination of DNA adducts. 172 7.4.2. Diet Study. 173 7.4.2.1. Study Protocol. 173 7.4.2.2. Diets. 173
7 Contents
7.4.2.3. Diet study sample. 174 7.4.3. Extraction and quantitation of DNA from whole blood. 174 7.4.4. Extraction and quantitation of DNA from gastric biopsies. 176
CHAPTER 8. REFERENCES. 177
8.0. REFERENCES. 178
CHAPTER 9. PUBLICATIONS AND PRESENTATIONS. 196
9. Publications. 197 9.1. Poster presentations. 197
9.2. Publications. 197
8 Tables and Fieures
Index of tables and figures.
Table 1.0. General classification of carcinogenic agents. 20
Table 1.1.2. Chemicals with carcinogenic activity in man. 23
Table 1.1.3. Characteristics and mechanisms of stages of carcinogenesis, and classification of carcinogens in relation to their action on stages of carcinogenesis. 24
Figure 1.1.3.2. Carcinogenesis is a multistage process. 26
Figure 1.1.4.3. Multiple genetic alterations occur in colorectal tumourigenesis. 28
Figure 1.2.1.1. Sites of alkylation in cellular DNA. 30
Table 1.2.1.1. Alkylation products in DNA generated by MNU, ENU, MMS and MNNG expressed as percent of the total alkylation. 31
Figure 1.2.2. Exogenous sources of exposure to NOC. 34
Figure 1.2.3. The biomonitoring of exposure to genotoxic compounds. 37
Table 1.3.1. Risk factors in Gastric Cancer. 39
Table 1.3.2.2. Sites in the body where bacterial N-nitrosation can occur. 41
Figure 1.3.3.The Correa model of gastric carcinogenesis. 43
Figure 1.3.5.1. The formation of N-nitroso and diazopeptides. 48
Figure 1.4.1. Formation of carboxymethylating agents from a range of nitrosated glycine derivatives. 51
Figure 1.4.4. Mechanisms of cell killing and mutation due to 0 6-MedG. 53
Table 1.4.4. 0 6-MedG detection in human tissues. 54
Table 1.4.5. Assays available for the quantitation of 0 6-MedG. 56
Figure 2.1. Principle of affinity chromatography. 60
9 Tables and Figures Figure 2.2. The synthesis of tritium labeled deoxynucleoside derivatives from the corresponding purine base. 62
Table 2.2. Enzymatic synthesis of [3H]-deoxynucleosides. 63
Figure 2.4.1. Elution of [3H]-06-CMdG from Oe-CMdG immunoaffinity columns. 65
Table 2.4.1. Elution conditions for 0 6-CMdG immunoaffinity columns. 65
Figure 2.4.2. Determination of 0 6-CMdG immunoaffinity column capacity. 66
Figure 2.5.1. Elution of 0 6-MedG immunoaffinity columns. 67
Figure 2.5.2. Determination of column capacity of immunoaffinity columns for 0 6-MedG. 68
Figure 2.6.1. Elution profile of 0 6-CMdG immunoaffinity columns with [3H]-S6-CMdG. 69
Figure 2.6.2. Determination of column capacity of 0 6-CMdG immunoaffinity columns using [3H]-S6-CMdG. 70
Figure 2.6.3.[3H]-S6-CMdG binding to 0 6-CMdG immunoaffinity columns in the presence of DNA digests. 70
Figure 2.6.4. HPLC chromatograms showing the hydrolysis products of [3H]-S6-CMdG and a standard of S6-CMG. 71
Table 2.7.1. HPLC optimisation for the 0 6-CMG adduct. 73
Table 2.7.2. HPLC optimisation for the 0 6-MeG adduct. 74
Figure 2.7.1. HPLC chromatogram for standard 0 6-CMG. 74
Figure 2.7.2. HPLC chromatogram for standard 0 6-MeG. 75
Figure 2.8.1. HPLC chromatogram of lpg digested DNA. 76
Figure 2.8.2. Calibration curve for 2’dG. 77
10 ______Tables and Figures Figure 2.8.3. HPLC chromatogram of acid hydrolysed DNA. 78
Figure 2.8.4. Calibration curve for guanine. 78
Figure 2.9.1. Time course for the hydrolysis of 0 6-CMdG to 0 6-CMG. 79
Figure 2.9.2. Time course for the hydrolysis of 0 6-MedG to 0 6-MeG. 80
Figure 2.10.1. Binding of [3H]-06-CMdG to IAC in the presence of digested DNA. 81
Figure 2.10.2. Binding of [3H]-06-MedG to IAC in the presence of digested DNA. 81
Figure 2.10.3. Overall scheme for immunoaffinity purification of 0 6-CMdG and 0 6-MedG from DNA digests. 83
Figure 2.10.4. Calibration curves for 0 6-CMG. 84
Figure 2.10.5. Calibration curves for 0 6-MeG. 84
Figure 2.10.6. Typical RP-HPLC chromatogram for 0 6-CMG isolated from DNA. 85
Figure 2.10.7. Typical RP-HPLC chromatogram for 0 6-MeG isolated from DNA. 86
Figure 3.2.1.1. RP-HPLC chromatogram of DNA treated with KDA and the determination of 0 6-CMG. 92
Figure 3.2.1.2. RP-HPLC chromatogram of DNA treated with KDA and the determination of 0 6-MeG. 93
Table 3.2.2.1. 0 6-Guanine alkylation by various nitrosated glycine derivatives at pH 7.4. 94
Figure 3.2.2.1. 0 6-Guanine alkylation of CT DNA by KDA. 95
Figure 3.2.2.2. 0 6-Guanine alkylationof CT DNA by Azaserine. 95
Figure 3.2.2.3. 0 6-Guanine alkylation of CT DNA by APNG. 96
Table 3.2.2.2. Ratios of 0 6-MeG to 0 6-CMG formation from a range of nitrosated glycine derivatives. 97
Figure 3.3.1. The formation of 0 6-MeG by KDA at varying pH. 98 ______Tables and Figures Figure 3.3.2. The formation of 0 6-CMG by KDA at varying pH. 98
Figure 3.3.3. The ratios of formation of 0 6-MeG /0 6-CMG by KDA at varying pH. 99
Figure 3.4.1. HPLC chromatograms of 0 6-CMG in calf thymus DNA treated with KDA (5'mM). * 100
Figure 3.4.2. Structures of APNG, Azaserine, KDA, mesyloxyacetic acid and the carboxymethyldiazonium ion. 100
Figure 3.4.3. Mechanisms of formation of 0 6-CMdG and 0 6-MedG in DNA from the carboxymethyldiazonium ion. 102
Figure 4.1. Schematic representation of the immunoslot blot assay. 106
Figure 4.2.1. Typical calibration curves for the determination of 0 6-CMdG in DNA. 108
Figure 4.2.2. Immunoslot blot filter. 109
Figure 4.3.1. Oe-CMdG levels in gastric DNA for APNG treated rats. I l l
Figure 4.3.2. 0 6-CMdG levels in intestine DNA for APNG treated rats. I l l
Figure 4.3.3. Oe-CMdG levels in liver DNA for APNG treated rats. 112
Figure 4.4.1. 0 6-CMdG levels in rat intestine and liver DNA after 4hr following KDA treatment. 113
Figure 4.4.2.1. Dose-dependent formation of 0 6-CMdG in rat gastric DNA following treatment with KDA. 114
Figure 4.4.2.2. Levels of 0 6-CMdG in gastric DNA for individual rats dosed with a range of concentrations of KDA after 4hr. 115
Figure 4.4.2.3. Levels of 0 6-CMdG in gastric DNA for individual rats dosed with a range of concentrations of KDA after 24hr. 115
Figure 4.5.1. Comparison of 0 6-CMdG levels determined by the immunoslot blot and by immunoaffinity-HPLC. 117
12 ______Tables and Figures Figure 4.5.2. HPLC chromatogram of the rat stomach DNA following immunoaffinity purification. 117
Figure 5.2.1.1. Immunoslot blot filter. 126
Figure 5.2.1.2. Calibration curve for 0 6-CMdG in DNA for the immunoslot blot shown in Figure 5.2.1.1. 127
Figure 5.2.1.3. Levels of 0 6-CMdG in Human gastric mucosa DNA. 127
Figure 5.2.1.4. Levels of 0 6-CMdG in gastric mucosa DNA according to Helicobacter pylori status. 128
Figure 5.2.2.1. 0 6-CMdG levels in the WBC and gastric mucosa DNA of individuals. 129
Figure 5.2.2.2. Correlation of levels of 0 6-CMdG in WBC DNA compared to that in gastric mucosa DNA. 130
Table 5.3. Diet regimes for the subjects. 130
Figure 5.3.1. Typical immunoslot blot filter for samples from the diet study and a range of standards. 131
Figure 5.3.2. Levels of 0 6-CMdG and N-nitroso compounds for CHX103 during different dietary regimes. 132
Figure 5.3.3.. Levels of 0 6-CMdG and N-nitroso compounds for CHX104 during different dietary regimes. 133
Figure 7.1.2.3.3. RP-HPLC -UV profiles showing the formation of [3H]-Oe-CMdG from 0 6-CMG in a TPase/PNPase catalysed reaction over time. 148
Figure 7.1.2.4.1. Isolation of [3H]-S6-CMdG. 149
Figure 7.1.2.4.2. HPLC chromatogram for the purified [3H]-S6-CMdG. 150
Figure 7.1.2.5.1. Calibration curve for 0 6-CMdG using RP-HPLC-fluorescence. 151
Figure 7.1.2.5.2. Calibration curve for 0 6-MedG using RP-HPLC-fluorescence. 151
13 Tables and Figures Figure 7.1.2.5.3. Calibration curve for S6-CMG. 152
Figure 7.3.4.2.1. Calibration curve for deoxyguanosine-3’-monophoshate. 170
Figure 7.3.4.2.2. HPLC-UV chromatogram of a rat stomach DNA digested to deoxynucleoside-3 ’ -monophosphates. “ 170
Table 7.4.2.2. Percentages of major food groups in the diets studied. 174
Figure 7.4.3. HPLC chromatogram of enzymatically digested blood DNA. 176
14 Acknowledgments
Acknowledgments
I would like to thank Dr D.E.G. Shuker for his expert supervision, guidance and assistance throughout the course of my PhD. I have also received a good deal of assistance and encouragement from the ‘MAFF’ group, and would like to thank in particular Drs C. Leuratti and R. Singh along with J. Crawley. I would also like to thank all of the members of the Biomarkers and Molecular Interactions section, both past and present for their friendship and support during my three years at the CMHT. Finally I am very grateful to ‘Stretch’ for his endless patience and encouragement during the writing up of this thesis, and for the use of his PC. I would like to dedicate this thesis to my partner and my mother whose continued support was much appreciated.
15 Abbreviations
Abbreviations
A - adenine APNG - N-(N'-acetyl-L-prolyl)-N-nitrosoglycine ATase - 0 6-alkyl guanine alkyl transferase
BDS- base deactivated silica BSA - bovine serum albumin
C - cytosine °C - degrees centigrade CAG - chronic atrophic gastritis CM agent - carboxymethylating agent cm - centimetre CRA - competitive repair assay CT - calf thymus
2’dA - 2’ deoxy adenosine 2’dC - 2’deoxycytidine ddH20 - double distilled water 2’dG - 2’deoxyguanosine DNA - deoxyribonucleic acid DMSO - dimethyl sulphoxide dpm - disintegrations per minute 2’dT - thymidine
K Coli- Escherichia coli EDTA - ethylenediamine tetra acetic acid ELISA - enzyme linked immunosorbent assay ENU - N’-ethyl- N-nitrosourea fmol - femtomoles g-gram G - guanine GBq - giga becquerel GI tract - gastro intestinal tract
[3H] - tritiated HC1 - hydrochloric acid HFBA - heptafluorobutyric acid HPLC - high performance liquid chromatography H. pylori - Helicobacter pylori hr - hour
16 Abbreviations
IAC - immunoaffinity column IgG - immunoglobulin G KDA - potassium diazoacetate L - litre
MBq - mega becquerel MeOH - methanol jig - microgram mg - milligram min - minute mL - millilitre jiL - microlitre mm - millimetre mM - millimolar JlM - micromolar mmol - millimoles jimol - micromoles MMS - methylmethane sulfonate MNNG - N-methyl-N’ -nitro-N-nitrosourea MNTJ - N-nitroso-N-methylurea
NBP - 4-(para-nitrobenzyl) pyridine NC - nitrocellulose NDMA - N-nitrosodimethylamine ng - nanogram nmol - nanomoles NOC - N-nitroso compounds NOGC - N-nitrosoglycocholic acid NOTC - N-nitrosotaurocholic acid NPRO - N-nitrosoproline
0 6-alkyIG - 0 6-alkylguanine 0 4-alkylT - 0 4-alkylthymine Ofi-CMdG - 0 6-carboxymethyl-2’deoxyguanosine 0 6-CMG - 0 6-carboxymethyl-guanine 0 6-CMG-0V - 0 6-carboxymethyl-guanine ovalbumin conjugate ODS - octadecyl silane 0 6-MedG - 0 6-methyl-25 deoxyguanosine Ofi-MeG - 0 6-methylguanine 0 4-MeT - 0 4-methylthymine OV - ovalbumin
PBS - Phosphate buffered saline PAH - polycyclic aromatic hydrocarbons pmol - picomoles PNPase - purine nucleoside phosphorylase PT - phosphate buffered saline-tween-20 (0.1%)
17 Abbreviations
RIA - radioimmunoassay RNA - ribonucleic acid RP-HPLC - reverse phase high performance liquid chromatography rpm - revolutions per minute S6-CMdG - N2-amino-S6-carboxymethyl-mercapto-deoxyguanosine S6-CMG - N2-amino-S6-carboxymethyl-mercaptopurine SDS - sodium dodecyl sulphate sec - second
T - thymine TEA - triethylammonium acetate TFA - trifluoroacetic acid TPase - thymidine phosphorylase TNOC - total N-nitrosocompounds
UV - ultraviolet
WBC - white blood cells yrs - years
18 Chapter 1. Introduction
19 Chapter 1. Introduction
1. In tro d u ctio n .
Cancer is a disease that has been known since ancient times (Pitot, 1986), and it is estimated that up to 30% of all individuals will present clinically with one of a wide variety of cancers at some time in their lives (World Cancer Research Fund, 1997). Cancer can be regarded as a disease of cells and is characterised by an excess of cells beyond that needed for normal function of the body organ affected, which results in a neoplasm (Pitot & Dragan, 1991). Cells within a neoplasm have undergone a series of fundamental, heritable and irreversible changes in their physiology and structure which are probably caused by an alteration to their genomic DNA. Alteration in the coding sequence of the bases on the DNA backbone can lead to abnormal proteins, with altered or disabled function, which may be inherited by the daughter cells during cell division, providing the cell with a growth advantage and thus allowing clonal expansion to occur. A variety of agents actively induce carcinogenesis, and these have been classified into four distinct categories; chemical, physical, biological and genetic (Table 1.0). This Chapter will consist of a review on DNA damage by chemical carcinogens, in particular the alkylating agents including the N-nitroso compounds and their possible role in the aetiology of certain cancers.
Class of Carcinogenic Agent Examples
Chemical Polycyclic aromatic hydrocarbons, aromatic amines and halides, diet, hormones, polymer surfaces and alkylating agents.
Physical Ionising (X and yray, particle radiation) and ultraviolet radiation.
Biological Papilloma, Epstein, retroviruses.
Genetic Transgenesis by enhancer-promoter-oncogene constructs; selective breeding.
Table 1.0. General classification of carcinogenic agents (Pitot & Dragan, 1991).
20 ______Chapter 1. Introduction 1.1. Chemical carcinogenesis.
“Given the ubiquitous nature of both natural and man-made carcinogenic substances in our environment, it is doubtful whether the genome of any adult man or woman has sustained no damage over the course of that individual’s lifetime.” (Spom, 1991).
1.1.1. Historical perspective.
Over the course of the last fifty years, interest in chemical carcinogenesis has increased dramatically and attracted attention from a variety of scientific disciplines such as organic chemistry, biochemistry, molecular biology and epidemiology. The aim of such research is to further elucidate how exposure to, and the mechanisms by which, a chemical agent may participate in the causation or development of neoplastic disease. It is ultimately hoped that by furthering our understanding of chemical carcinogenesis, cancer mortality and incidence will be reduced by improvements in treatment and prevention/control of exposure respectively.
Some of the first evidence that human cancer can be caused by chemical agents comes from epidemiological associations that first began over two centuries ago, when the physician John Hill observed that a high incidence of nasal cancers occurred as a consequence of using tobacco snuff (as reviewed by Miller, 1970). Over a decade later, in 1775, the surgeon Sir Percival Pott described the occurrence of cancer in the scrotum of young male patients who had previously been employed as chimney sweeps in their childhood and suggested that soot exposure was the causative agent (as reviewed by Miller, 1978). A century later, similar observations were made by Butlin (1892). Various other observations of increased cancer incidences in certain occupational groups (urinary bladder cancer correlated with aniline dye industry) were made shortly afterwards. As a consequence of these epidemiological observations that particular chemical exposures resulted in increased incidence of specific human cancers, researchers began to try to induce tumours in experimental animals. By 1915, Yamagiwa and Ichikawa had successfully induced carcinomas on the ears of rabbits following the topical application of coal tar, thereby clearly demonstrating that exposure to chemicals can result in neoplastic disease (as reviewed by Miller, 1978). The carcinogenic activity of a synthetic compound was first demonstrated in 1930 by Kennaway and Hieger for dibenz[a,h]anthracene. Shortly afterwards the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, was isolated from coal tar which led to extensive studies on the chemical
21 ______Chapter1. Introduction features that were required for the carcinogencity of PAHs. In the late 1940s, the aromatic amines (2-acetylaminofluorene), inorganic chemicals (zinc, beryllium silicate and beryllium oxide), nitrogen mustards and the wide range of alkylating agents were identified as having carcinogenic activity (as reveiwed by Miller, 1978).
Perhaps the most profound epidemiological association between exposure to chemicals and human cancer was made by Doll and Hill (1950), who suggested that tobacco smoking was the major cause of lung cancer. It has since been estimated that 30% of all cancers in the USA are associated with tobacco use and this clearly signifies the importance of chemical carcinogen involvement in human cancer (Wynder & Gori, 1977, Doll & Peto, 1981).
1.1.2. Chemical carcinogens: structure and activity.
Over the last fifty years, the list of compounds with carcinogenic properties has grown steadily longer (Miller, 1978). To date, however, clear associations between exposure to specific compounds and induction of human carcinogenesis have been rare due to the fact that humans are exposed to complex mixtures of chemicals, both naturally occurring and man- made, in the environmental milieu (Autrup, 1991). Table 1.1.2. contains examples of chemicals which have been classed as human carcinogens, and illustrates the diversity of these compounds as they range from organic compounds to metals and minerals. On the basis of experimental tumour induction studies a great many more compounds are considered to be potential human carcinogens (Cartwright, 1984). The majority of these compounds have been classified according to their chemical structures and/or modes of action. These include the polycyclic aromatic hydrocarbons (PAH’s), aromatic amines, N-nitroso compounds (NOC), azodyes, alkylating agents and a number of inorganic compounds. Many of these compounds (PAH’s, aromatic amines, alkylating agents and some NOC) share a common mechanism by which they exert their carcinogenic effect (Miller, 1978). Studies by different groups of researchers lead to the generalisation that these chemical carcinogens needed to be metabolised (“activated”) to the ultimate carcinogens, a highly reactive electrophilic species, by a category of enzymes, the cellular cytochrome P450 associated mixed-fimction oxidases (Cooper eta l, 1995). The known exceptions are the direct-acting alkylating or acylating agents. The electrophilic intermediate generated binds covalently to nucleophilic sites in cellular macromolecules, such as DNA, RNA and proteins (Dipple et al, 1984, Gamer et al ,
22 Chapter 1. Introduction 1984, Lawley, 1984). DNA is believed to be an important target for chemical carcinogens and the covalently bound products were referred to as DNA adducts (Brookes & Lawley, 1960 and 1961). Many animals studies have suggested that DNA adducts play a key role in the initiation of carcinogenesis (Section 1.1.3.1.). The detection and quantitation of DNA adducts are deemed to be important as they are thought to be relevant‘to the mechanism of chemical carcinogenesis as well as providing valuable information for the evaluation of human exposure to chemical carcinogens in molecular epidemiology studies (Perera, 1996).
Chemical Source of exposure Target organ(s)
aflatoxins environmental, occupational liver 4-aminobiphenyl occupational bladder arsenic compounds environmental, occupational, skin, lung, liver medicinal asbestos occupational lung, gastrointestinal tract benzene occupational haemopoietic system benzidine occupational bladder bis(chloromethyl)ether occupational lung chloramphenicol medicinal bladder chromium occupational lung, larynx cyclophosphamide medicinal haemopoietic system diethylstilboestrol medicinal uterus, vagina melphalan medicinal bladder mustard gas occupational lung, nasal cavity 2-naphthylamine occupational bladder nickel occupational lung, nasal cavity oxymetholone medicinal liver vinyl chloride occupational liver
Table 1.1.2. Chemicals with carcinogenic activity in man (adapted from Wigley & Balmain, 1991).
1.1.3.Carcinogenesis is a multistage process.
Carcinogenesis is thought to occur via multiple stages (Table 1.1.3.) and may occupy the life span of an individual (Pitot & Dragan, 1991; Weinstein, 1981) involving complex interactions between both exogenous (environmental) and endogenous (genetic, hormonal,
23 ______Chapter 1. Introduction etc.) factors. Many model systems have been used to study the process of tumour development in animals and in the 1940s two distinct stages termed initiation and promotion had been identified studying epidermal carcinogenesis in the mouse (Miller & Miller, 1981). Foulds (1954) further clarified the process of carcinogenesis, with a mouse mammary adenocarcinoma model, by modifying the concept of the promotion stage to include all events after initiation of the neoplastic process.
Stage of Characteristics and Classification of carcinogenesis mechanisms carcinogen
Initiation Irreversible, additive, no Incomplete carcinogen, capable threshold, requires fixation, of initiating cells only, (e.g. (preventable), simple mutations alkylating agent). in involving cellular genome, point mutations in protooncogenes. Promotion Reversible, threshold, maximal Capable of causing the reversible response, inhibition of expansion of initiated cell clones, apoptosis by promoting agent. (e.g. UVA radiation). Progression Irreversible, somatic Capable of converting an aneuploidy, progressive initiated cell or a cell in the stage karyotopic instability, complex of promotion to a potentially genetic alterations, irreversible malignant cell, (e.g. hepatitis B changes in gene expression, virus). selection of neoplastic cells for optimal growth.
Table 1.1.3. Characteristics and mechanisms of stages of carcinogenesis, and classification of carcinogens in relation to their action on stages of carcinogenesis (adapted from Pitot & Dragan, 1991).
1.1.3.1. Initiation and promotion.
Agents that initiate carcinogenesis do so by damaging/modifying cellular DNA. The modification of the DNA may cause a genetic change, mutation, which confers on the initiated cell an altered responsiveness to its microenvironment such that it possesses a proliferative advantage when compared to surrounding unaltered cells (Cohen & Ellwein, 1991, Harris, 1991). The irreversibility of the initiating stage is consistent with the mutational mechanism by which chemical carcinogens exert their actioni.e. DNA modification followed by DNA synthesis and cell division resulting in the generation of a heritable mutation (Ying
24 ______Chapter 1. Introduction et al, 1982). Thus, the efficiency of initiation is related to the cellular DNA repair processes, persistence of DNA damage and cellular replication rates (Swenberg et al , 1985). Inhibition of metabolism of chemicals to their ultimate carcinogen forms can occur resulting in the blocking of the stage of initiation (Wattenberg, 1978). The absence of threshold limits for initiating agents is evident from the studies on mutations that result from these agents, from the activation of proto-oncogenes to cellular oncogenes and from the deactivation of tumour suppressor genes (Pitot & Dragan, 1991).
Although the mutational mechanism for chemical carcinogenesis has been established for a range of compounds, evidence exists that certain substances e.g. saccharin (Cohen et al, 1987), may mediate their carcinogenic effect by epigenetic mechanisms (not involving DNA modification and mutation). These promoting agents increase the chance of full malignancy, as they increase the proliferation rate of normal cells, or cells that already contain carcinogen- induced or spontaneously derived mutations (Miller & Miller, 1981). Promotion has been demonstrated to be reversible in nature under certain conditions. This is evident in several model systems, such as mouse epidermal carcinogenesis where papillomas are shown to regress when administration of promoting agents is stopped, but then reappear when the promoting agent is re-administered (Reddy et al, 1987). However, after a period of time a certain proportion of the papillomas appear to behave in a promoter independent fashion. Apoptosis (programmed cell death) is thought to play a part in the regression of cells after withdrawal of a promoting agent, which has been shown to inhibit apoptosis in pre-neoplastic lesions (Schulte-Hermann et al, 1995).
I.I.3.2. Progression
After a certain period, some tumours do not require promotion in order to escape from normal cellular and tissue control mechanisms. This phenomenon is believed to be due to the accumulation of further genetic changes, which are directly related to the increased growth rate, invasiveness, metastatic capability and biological changes in the malignant cell (Vogelstein et al, 1988, Harris, 1991). Thus the development of irreversible, aneuploid malignant neoplasms and karyotypic instability distinguish the stage of progression. Therefore, carcinogenesis should be regarded as a multistage process (Figure 1.1.3.2.) where the initial interaction of the carcinogen with the target cell results in an altered genotype
25 ______Chapter 1. Introduction (initiation) which, after a certain period under appropriate stimuli (promotion), undergoes further genetic changes (progression) prior to the manifestation of clinical disease. The loss or altered expression of a number of cellular genes appears to be central to the initiation, promotion and progression scheme for carcinogenesis and are discussed in more detail in the next section.
MTLAIION PROMOTION
Carcinogen
Normal cell Initiated cell Fre-neoplastic lesion Malignant tumour
Figure I.I.3.2. Carcinogenesis is a multistage process (adapted from Harris, 1991). Multiple genetic and epigenetic changes are believed to occur before a carcinogenic cell develops into a clinical cancer.
1.1.4. Critical DNA targets during carcinogenesis.
Alterations in the genome of cells which influence the function and expression of proto- oncogenes and tumour suppressor genes are considered to be the main effectors of carcinogenesis (W einberg, 1991).
1.1.4.1. Proto-oncogenes.
“Proto-oncogenes are normal cellular genes that, when inappropriately activated as oncogenes, cause dysregulation of growth and differentiation pathways and enhance the probability of neoplastic transformation.” (Harris, 1991).
Specific genes with aberrant function have been detected in a variety of human tumours, and show functional diversity. However, mutated alleles of proto-oncogenes are believed to act in a dominant manner in order to produce a phenotypic effect (Bishop, 1991). The ras oncogene
2 6 Chapter 1. Introduction gene family has been extensively studied and these genes appear to be mutated in a range of human tumours (Bishop, 1991). ras proteins are structurally and functionally similar to G- proteins and the wild type ras protein may be involved in the transduction of growth factor signals. Mutated ras genes detected in human tumours predominantly contain point mutations in codohs 12, 13 or 61 (Bos, 1989). These mutations have also been detected after chemical carcinogen treatment in vivo and in vitro, and furthermore the particular base changes involved often reflect the mutational spectrum of the chemical compound under investigation (Balmain & Brown, 1988). In the mouse skin model it has been demonstrated that the retroviral transfection of mutated H -ras into epidermal cells followed by phorbol ester promotion resulted in carcinomas (Brown et al , 1986). This observation is consistent with the multistage model of tumour development and indicates that ras mutations may act as an initiating event in the carcinogenic process.
1.1.4.2. Tumour-suppressor genes.
Tumour-suppressor genes have anti-oncogenic properties acting as physiological barriers against clonal expansion and hinder growth and metastasis of cells driven to uncontrolled proliferation by oncogenes (Harris, 1995). However the loss of both functional alleles of a tumour-suppressor gene can result in neoplastic development (Harris, 1991). These genes are therefore vulnerable sites for DNA damage, and loss of tumour-suppressor function can occur via damage to the genome through mutation or chromosomal rearrangement. One of the most frequently mutated tumour-suppressor gene loci in human cancer is the p53 gene which is mutated in about half of human cancers (Hollstein et al, 1991). It is believed that the wild- type p53 protein is a nuclear phosphoprotein involved in transcriptional control of genes implicated in the regulation of the cell cycle (Vogelstein & Kinzler, 1992).
The range of p53 mutations induced in human cancer can help identify particular carcinogens responsible for the genetic lesions associated with a cancer. The frequency and type of p53 mutations can act as a molecular dosimeter of carcinogen exposure (Harris, 1995). For example, ultraviolet light is correlated with transition mutations at dipyrimidine sites; dietary aflatoxin Bj exposure is correlated with GC —> TA transversions that lead to serine substitution at residue 249 of p53 in hepatocellular carcinoma and exposure to tobacco smoke is correlated with GC —» TA transversions in lung carcinomas. These characteristics of p53
2 7 Chapter 1. Introduction mutations can then be combined to provide information about the molecular epidemiology of human cancer risk (Harris, 1995).
1.1.4.3, Multiple genetic events occur during multistage carcinogenesis.
The current understanding of the carcinogenic process centres on the hypothesis that during tumour initiation, promotion and progression, multiple genetic alterations, involving oncogenes and tumour suppressor-genes, occur prior to the manifestation of clinical disease. The latter stages of this process, that is from the initiated cell through to the carcinoma, have been characterised and delineated in some detail for human colorectal cancers by Vogelstein and co-workers and can be seen in Figure 1.1.4.3. (Vogelstein et al, 1988, Fearon & Vogelstein, 1990). It can be seen that tumour progression proceeds through a series of mutational events which may have ramifications in the understanding of chemical carcinogens in this disease process. Traditionally, chemical carcinogens were considered to be primarily involved in the initial mutational event during carcinogenesis. However, as human exposure to such agents is likely to be chronic (environmental), it is conceivable that exposure to chemical carcinogens “post-initiation” may play an important role in the generation of subsequent mutational events (Harris, 1991).
Chromosome 5q 12p 17p Alteration mutation or loss mutation loss Gene FAP K-ras p53
DNA other genetic hjpomethylation alterations
Figure I.I.4.3. Multiple genetic alterations occur in colorectal tumourigenesis. ( adapted from Fearon & Vogelstein, 1990)
28 ______Chapter 1. Introduction 1.2. Alkylating Agents.
There are many different classes of chemicals which are known to be carcinogenic and are efficient at causing point mutations, deletions, frameshift and chromosomal abnormalities (Harris,, 1991). The alkylating agents form a large diverse group of chemicals including the N-nitrosocompounds (NOC), alkyl alkanesulphonates, alkyl sulphates, dialkylhydrazines and alkyltriazines (Lawley, 1984). Irrespective of their chemical structure, alkylating agents are believed to exert their biological effects by the transfer of an alkyl group to nucleophilic sites on cellular macromolecules (Saffhill et al , 1985). NOCs represent the most extensively studied alkylating agents and can be subdivided into two groups; direct-acting compounds which spontaneously decompose (N-nitrosamides e.g. MNNG) or indirect-acting compounds which require metabolic activation (N-nitrosamines e.g. NDMA) to yield the reactive species (Shuker & Bartsch, 1994a, Beranek, 1990). As a consequence of the requirement for metabolism, the N-nitrosoamines exert their biological effects only in those tissues of experimental animals which contain the enzymes necessary for activation, whilst the N- nitrosamides tend to show less tissue specificity and exert their biological effects at the site of administration.
1.2.1. Biological consequences of alkylating exposure.
1.2.1.1. DNA Modification.
Alkylating agents modify DNA at a variety of nucleophilic sites (exocyclic nitrogen and oxygen atoms, and ring nitrogen atoms of the purine and pyrimidine bases) and these are illustrated in Figure 1.2.1.1. (Lawley and Brookes, 1963). The phosphate intemucleotide linkages also undergo alkylation, but are not represented in this figure. The most frequently modified sites are N7 and O6 of guanine and the N3 position of adenine. However, the relative proportions of alkylation at different sites are dependent on the particular alkylating agent responsible for the modification (Table 1.2.1.1.). This difference is particularly apparent when comparing NOC (e.g. MNU) and alkyl alkanesulphonates (e.g. MMS), although both classes of compounds preferentially modify the N7 position of guanine, there is a considerable variation in the reactivity at the oxygen atoms, with proportionally less O6- MeG being formed following MMS exposure than MNU. This can be explained by the mechanism of alkylation on DNA by simple alkylating agents which have been reviewed by Swenson (1983) and Beranek (1990) involving unimolecular (SN1) and bimolecular (SN2)
2 9 Chapter 1. Introduction substitution reactions and Swain-Scott factors. The former reactions follow first order kinetics and the rate of action is dependent on the formation of the reactive intermediate, which subsequently binds covalently to the nucleophilic sites in DNA. SN2 reactions, however, are dependent on steric accessibility and involve the formation of a nucleophile-electrophile transition complex which forms the alkylated product after the release of the leaving group. On this basis, MNU and MMS have been designated SN1 and SN2 compounds respectively. Saffhillet a l (1985) proposed an alternative mechanism involving the hard and soft acids {i.e. electrophilic species) and bases {i.e. nucleophilic species) theory of Pearson (1969). Hard acids have a preference for reaction with hard bases, while soft acids prefer soft bases. Alkylating species have been classified as intermediate in terms of their reactivity, as have the ring N and exocyclic oxygen atoms of purine and pyrimidine bases in DNA. Therefore the nitrogen and oxygen atoms will be preferentially alkylated in DNA. But oxygen atoms are harder bases than nitrogen atoms, and since electrophilic species formed by the N-nitroso compounds are harder acids than alkanesulphonates, N-nitroso compounds as opposed to the alkanesulphonates will preferentially bind to oxygen atoms.
I DNA Thymine - Adenine
DNA / N
N \ DNA
Cytosine - Guanine
Figure 1.2.1.1, Sites of alkylation in cellular DNA (indicated by arrows).
3 0 Chapter 1. Introduction
Percentage of Total Alkylation by:
Alkylated Bases MNU ENU MMS MNNG
Nl-alkyladenine 1.0 0.3 1.5 1.0 N3-alkyladenine 9.0 2.8-5.0 10.0 12 N7-alkyladenine 2 0.4 - 1.5 nd
N3 -alkylguanine 0.8 0.6 0.6 nd N7 -alkylguanine 66 11.0-14.0 84 67 0 6-alkylguanine 7.5 7.6-10.0 0.3 7.0
N3-alkylcytosine 0.5 0.2 0.5 2.0 C^-alkylcytosine nd 2.9-4.0 nd nd
N3 -alkylthymine 0.2 nd 0.1 nd CP-alkylthymine 0.1 7.0-7.8 nd nd 0 4-alkylthymine <0.1 1.0-4.3 nd nd
MNU, N’ -methyl-N’ -nitrosourea, ENU, ethylnitrosourea, MMS, methyl methanesulfonate; MNNG, N-methyl- N’-nitro-N’-mtrosoguanidine , nd, not detected.
Table I.2.I.I. Alkylation products in DNA generated by MNU, ENU, MMS and MNNG expressed as percent of the total alkylation (from Saffhill et al , 1985 and Beranek, 1990).
The pattern of DNA alkylation and the stabilities of the individual alkylation products are assumed to be essential factors in determining the carcinogenic and mutagenic effects of alkylating agents (Singer & Essigman, 1991). The alkylation of oxygens in DNA, notably O6- alkylguanine (0 6-alkylG) and 0 4-alkylthymine (04-alkylT) which lead to miscoding during DNA and RNA synthesis, are believed to be primarily responsible for the mutagenic and hence the carcinogenic effect of alkylating agents (Saffhill et al, 1985, Lawley, 1990,). O6- alkylG directs the misincorporation of a thymine residue causing a GC—»AT transition, and
0 4-alkylT may mispair with guanine resulting in an AT—>GC transition (Pegg, 1990). Although 0 4-alkylT is formed at lower levels than 0 6-alkylG, the contribution of this adduct to the mutagenic effects of alkylating agents may be significant due to its persistence (Pegg, 1990). Similarly ethylating agents are considered to be more mutagenic than their methyl counterparts due to the persistence of ethylated adducts compared to the corresponding methylated adducts (Den Engelse et al, 1986; Jansen et al, 1994). 31 Chapter 1. Introduction The predominant adduct formed following alkylating agents, N7-alkylguanine, is generally believed to be biologically innocuous as it does not appear to be directly pro-mutagenic or cytotoxic (Saffhill et al, 1985). However, the formation of apurinic sites following alkylation at N7 or N3 positions of guanine and adenine, may occur through the action of DNA glycosylase repair enzymes or as a consequence of the labilisation of the glycosidic bond and its subsequent spontaneous hydrolysis (Saffhill et al, 1985). Such apurinic sites are capable of acting as promutagenic lesions as they result in the misincorporation of adenine during DNA synthesis which leads to GC—»TA and AT—»TA transversion mutations in the case of N7- methylguanine and N3-methyladenine respectively (Loeb, 1985). Similarly, alkyl phosphotriesters are not considered to be directly pro-mutagenic. However, these lesions are thought to interfere with DNA-handling enzymes that regulate gene expression (Takeda et al, 1983).
1.2.1.2. DNA Repair o f Alkylated Bases
It is not only the formation of DNA damage but the persistence of these adducts which contributes to the carcinogenic effects of alkylating agents, implicating DNA repair processes as being an important factor in alkylating agent mutagenicity and carcinogenicity (Herron & Shank, 1981, Zaidi et al, 1993a and 1993b). Maintenance of the integrity of the genome is fundamental for survival and every type of organism so far tested has been found to possess efficient DNA repair mechanisms to ensure that particular alkylated oxygen and nitrogen atoms do not accumulate in the genome (Samson, 1992). Two main repair processes for alkyl adducts exist; i) direct reversal by removal of only a modified group and ii) base excision repair (Singer & Hang, 1997). DNA adducts occurring via other processes such as, acylation and free radical attack (bulky adducts or pyrimidine dimers by action of UV radiation), undergo nucleotide excision repair. Patients with Xeroderma pigmentosum are hypersensitive to UV-induced DNA damage due to a reduced capacity to repair pyrimidine dimers and tend to develop skin tumours (McGee et al, 1992).
0 6-alkylG adducts in DNA are repaired by the protein 0 6-alkylguanine-alkyltransferase (ATase), which transfers the alkyl group to a cysteine residue at the active site of the repair protein leaving behind an intact base. ATase is inactivated by this process and cannot be regenerated (Lindahl et al, 1988, Pegg, 1990). The first ATase genes to be isolated and
3 2 ______Chapter 1. Introduction characterised were the inducible ada and non-inducible ogt genes in E. coli (Margison et al, 1985, Margison & O’Connor, 1990). These genes code for proteins which repair 0 6-alkylG and 0 4-alkylT, but the ADA protein has a further active site which repairs methylphosphotriesters (Pegg, 1990). Mammalian ATases have a single active site, the principle substrate for which is 0 6-methylguanine (0 6-MeG), although repair of O4- methylthymine (0 4-MeT) has been shown to occur at a lower efficiency (Zak et al, 1994).
Some alkylpurines are repaired by DNA glycosylases which catalyse the hydrolytic cleavage of the N-glycosylic bond linking the damaged base to deoxyribose in DNA, producing an apurinic (AP) site. The undamaged DNA sequence is then restored by the consecutive action of AP endonuclease, exonuclease, DNA polymerase and DNA ligase enzymes (Friedberg, 1985). Human and mouse DNA glycosylase enzymes have been isolated and characterised and shown to repair N3-alkyladenine, N7-alkylguanine and N3-methylguanine (Mattes et al, 1996; Engelward et al, 1993).
1.2,1.3. Non-random DNA alkylation and repair.
The distribution of alkylation damage and repair is non-uniform showing heterogeneity at the level of the organ, cell and DNA sequence (Pegg, 1990). Organ and cell specificity can result from the requirement for some alkylating agents to undergo metabolism and this may also be influenced by the level of ATase in different cell types and tissues (Pegg, 1990). However, differential alkyl adduct formation and repair is also evident at a sub-cellular level, with the more accessible regions of the genome, e.g. transcribed chromatin, being damaged and repaired with greater efficiency than less accessible regions (Bohr et al, 1987). Alkylation damage has also been demonstrated to be sequence specific which can lead to carcinogens exhibiting a unique mutational spectrum, i.e. each carcinogen has its own mutational ‘hot spots’ and ‘cold spots’ throughout the nucleotide sequence. Thus, by determining the sequence distribution of DNA adducts in certain genes, it may be possible to correlate them with mutational ‘hot spots’ in specific cancers, allowing a causal link of a carcinogenic agent to a particular cancer to be made (Dennisenko et al, 1996).
33 ______Chapter 1. Introduction 1.2.2. Human exposure to alkylating agents.
The source and extent of human exposure to alkylating agents, particularly NOC, has become an active area of research due to their potent carcinogenicity in experimental systems (Bartsch & Spiegelhalder, 1996). Total human exposure to NOC involves exogenous and endogenous components, the latter of which will be discussed in more detail in Section 1.3 in relation to gastric cancer. The major sources of exogenous exposure to NOC are depicted below in Figure 1.2.2.
Figure 1.2.2. Exogenous sources of exposure to NOC (from top right, cigarette smoke, pharmaceuticals, industry, smoked fish, cured meats and beer).
The most widespread exogenous human exposure to high levels of volatile and non-volatile NOC is considered to be through the use of tobacco products (Challis, 1996). Various NOC have been identified, including the tobacco specific N-nitrosamine, 4-(N-methylnitrosamino)- l-(3-pyridyl)-l-butanone (NNK), a potent carcinogen in animals inducing lung and oral tumours, sites commonly linked to tobacco related cancers in humans (Hecht & Hoffman, 1989, Spiegelhalder & Bartsch, 1996). Estimates on the exposure level of NOC for smoking related products range from 17-200pg/person/day. Occupational exposure to high concentrations of N-nitrosoamines has been identified in several industries including rubber production, metal working, chemical production and leather tanning (Bartsch & Spiegelhalder, 1996). The level of exposure to NOC varies considerably, but regulatory
34 Chapter 1. Introduction measures have considerably decreased exposure levels in Western countries. However, in developing countries, where working conditions are poorly monitored/controlled, exposure of workers to carcinogenic NOC may still be high.
NOC have been detected in several human foods and beverages including nitrite-cured meat, smoked or salt-dried fish and beer with variable levels up to 2.3jig/person/day of volatile
NOC and 10-100p.g/person/day of non-volatile NOC for individuals in the UK (Hotchkiss, 19S9, Challis, 1996). An increased risk of cancer has been identified in certain populations which can be associated with the presence of NOC in various foodstuffs, e.g. a high incidence of naso-pharyngeal carcinoma in areas of China where a high intake of salted fish is consumed (Zou et al , 1994). The formation of NOC in food products primarily occurs during manufacture and there has been a reduction in the N-nitrosoamine levels by improved processing procedures (Challis, 1996). Many drugs carry secondary or tertiary amines which can undergo nitrosation to yield NOC such as the now banned analgesic drug, aminopyrine, which was shown to be nitrosated to yield the powerful carcinogen dimethylnitrosamine (Challis, 1996). Further sources of exposure to volatile NOC have been detected in pharmaceuticals, cosmetics and toiletries. However, the contribution that these sources make to cancer risk is not thought to be significant (Tricker et al , 1989).
1.2.3. Use of Carcinogen-DNA Adducts as Biomarkers for Cancer.
In human biomonitoring there are two types of measurements that can be made to determine the extent of human exposure to carcinogens. Firstly, the measurement of levels of chemicals, and their metabolites or derivatives in body fluids and tissue, and then secondly, the measurement of biological responses, such as mutations, sister chromatid exchanges and chromosome aberrations (Wogan & Gorelick, 1985). DNA adducts are thought to represent early events which may lead to mutation and/or malignant tumours, thus the measurement of DNA adducts would be a good indicator of exposure to carcinogenic agents (Shields & Harris, 1991). However, the presence of a DNA adduct does not necessarily indicate that the person is going to develop cancer but may be an indication that the individual is at risk.
The response of individuals to carcinogen exposure is variable and host factors, particularly genetic factors, can determine susceptibility (Shields & Harris, 1991). Genetic factors which
35 Chapter 1. Introduction may increase an individuals risk include polymorphisms in genes that encode for carcinogen metabolising or detoxifying enzymes as many chemicals require metabolic activation to exert their carcinogenic effects. The issue of measuring DNA adducts is further complicated by variation in DNA repair capacity and cell turnover rates, as the removal of adducts from DNA by chemical or enzymatic processes can occur at different rates, even within the same cell. If cells are highly efficient at DNA repair, adducts, although formed may go undetected and if cell replication is rapid, then only short-term exposures can be measured in the tissue. Hence, in the consideration of the production of adducts, dosimetry is important and two different terms have been defined: the internal dose (amount of genotoxic compound absorbed into the organism) and the biologically active dose (amount of chemical needed to induce a biological response, i.e. adduct). The amount of internal dose can be related to the biological dose for risk estimation, but to account for differences in genetic susceptibility, and differences in absorption, metabolism and excretion, the biological dose is more relevant. Dosimetry data in experimental systems and human pilot studies indicate that the degree of binding of a carcinogen to the macromolecular target is, in most cases directly proportional to the administered dose (especially at low doses) and that there is no threshold of exposure below which adducts are not formed (Perera, 1988, Reed et al, 1986, Souliotis et al, 1990). However the rate and accuracy of repair of adducts prior to cell replication as well as the specific type of adduct formed (including location in the tissue, cell type and location in the genome) are all important in determining the carcinogenic effect of DNA adducts.
Furthermore, the measurement of DNA adducts is all relative depending on what body tissue or fluid is examined. DNA adducts measured in situ, within the target cell, would give the most direct evidence of genotoxic exposure, whereas adducts measured in excreted body fluids represent total recent exposure for the individual. Even the measurement of DNA adducts within an organ/tissue may vary upon the exact location from which the sample is taken. In human lung samples the levels of N7-methyl- and N7-ethyldeoxyguanosine 3’- monophosphate are not distributed with any specific pattern, hence for most individuals a random lung sample would not be representative of other parts of the lungs (Blomeke et al, 1996). Thus care in tissue sampling protocols and care about the conclusions made from adduct levels detected in specific samples must be taken as tissues may be misclassified.
3 6 Chapter 1. Introduction DNA adduct measurements can be useful in studies of human carcinogenesis and in cancer prevention by providing direct evidence, not only that humans have been exposed to a specific chemical, but that potentially precarcinogenic lesions have been formed. Hence, for molecular epidemiology studies, DNA-adduct biomarkers allow for the possibility of the identification of early biological responses and preclinical rather than clinical events, providing an opportunity for intervention prior to the manifestation of disease (Perera, 1987). Thus considerable time can be saved and a full understanding of the complex link between DNA adduct and the ensuing cancer need not be necessary, providing an insight into the underlying aetiology is known.
EXPOSURE
Internal D ose
Biologically Active Dose
Individual S usceptibility
Biological Response
Changed Structure or Function
CANCER
Figure 1.2.3. The biomonitoring of exposure to genotoxic compounds. (Decaprio, 1997; Farmer ef al., 1996; Henderson, 1995)
3 7 Chapter 1. Introduction 1.3. Gastric Cancer.
In 1980, stomach cancer was estimated to be the single most common form of cancer in the world, accounting for 10.5% of all cancers. It is now the second most common, after lung cancer, and it is estimated that there were over 1 million new cases diagnosed worldwide in 1996 with incidence rates being 50% lower in women than in men. The incidence rate of stomach cancer has been declining in recent decades in developed countries, but it still remains one of the most serious health burdens throughout the world (Correa & Chen, 1994, Tomatis eta l, 1990, World Cancer Research Fund, 1997).
The majority of patients with gastric cancer have no identifiable genetic predisposition, although approximately 10% appear to have an increased genetic risk (Martin & Quirke, 1994). Mutations of the p53 gene are found in 30-57% of all gastric carcinomas and the most common genetic changes are GC—> AT base transitions (Seruca et al , 1992, Renault et al , 1993). World statistics on the incidence rates show a distinct geographical variation, with the highest rates being found in Japan, Central and South America, and eastern Asia, whereas Australia, Canada and the USA have the lowest rates (Parkin et al , 1992). This extreme variation in incidence rates around the world has led to the conclusion that environmental and/or lifestyle factors have a major role in the aetiology of gastric cancer (Doll & Peto, 1981). There are also marked variations by socioeconomic status, with a difference of almost three-fold increase in the mortality rates from the highest to the lowest categories of social status in England and Wales (Howson et al , 1986). Migrant population studies have also shown that the risk for stomach cancer decreases in populations that move from high-risk to lower-risk countries and there is evidence that changes in consumption of dietary components are linked to this effect (Kolonel et al, 1980).
1.3.1. Risk factors for Gastric Cancer.
The underlying aetiology of gastric cancer has not been unambiguously elucidated, however numerous epidemiological studies have highlighted the chief factors affecting stomach cancer risk (Table 1.3.1.). The evidence for dietary risk and protective factors has recently been reviewed by the World Cancer Research Fund, (1997) and Palli, (1994).The major findings of these reports are that diets high in vegetables and fruits protect against stomach cancer. This may in part be due to their vitamin C content and refrigeration that facilitates the year-round 38 ______Chapter 1. Introduction consumption of vegetables thus reducing the need for salt as a preservative. High salt consumption increases the risk of stomach cancer and of the non-dietary factors, Helicobacter pylori infection appears to be the most important determinant in the development of stomach cancer (Forman et aly 1991), whereas cigarette smoking is associated with a small excess risk (Noruma et al, 1990).
Factors associated with decreased Risk Factors associated with increased Risk
Vegetables and fruit Helicobacter pylori Refrigeration Salt Vitamin C Cigarette smoking
Table 1.3.1. Risk factors in Gastric Cancer.
1.3.2. Endogenous nitrosation.
The endogenous nitrosation and subsequent in situ formation of N-nitroso compounds (NOC) was proposed as a possible hypothesis for human gastric carcinogenesis by numerous authors in the 1970s (Correa et al, 1975, Mirvish, 1975). The demonstration of the endogenous nitrosation of proline in human subjects, strongly implied that endogenous formation of NOC could indeed occur (Ohshima & Bartsch, 1981). As a large number of NOC (300), including N-nitrosoamines, are known to be potent carcinogens in experimental animals and as amines are readily available in the diet their N-nitrosation has been the subject of much research (review, Magee, 1989). NOC are known to be toxic in human beings, causing both acute and subacute changes that closely resemble those found in experimental animals, and there is a strong probability that NOC are also human carcinogens and involved in gastric carcinogenesis (Magee, 1996).
N-nitrosation of primary and secondary amines readily occurs at acidic pH (<5) in the presence of nitrite ions. However, nitrosation can also occur at neutral and alkaline pH by reaction of the nitrogenous substrate with gaseous nitrogen oxides which may be derived from nitric oxide and oxygen (Leaf et al, 1989). Hence, endogenous N-nitrosation occurs under varying conditions of nitrite availability via two major routes, namely the acid-
3 9 ______Chapter 1. Introduction catalysed reaction and the bacterially mediated reaction. Another source of endogenous nitrosation is by NO formation during the inflammation process (Mirvish, 1995).
1.3.2.1. ’Acid-catalysed, endogenous nitrosation and its possible role in gastric cancer.
The rate of chemical nitrosation is pH-dependent, with the nitrosation of amines usually having an optimum around 3, while that of amides exhibits a continuous increase with decreasing pH (Mirvish, 1975). Therefore in practice, due to the low pH optimum of these reactions, the acid stomach is the only site in the body where acid catalysed N-nitrosation can occur (Hill, 1996). The major factors influencing the rate of NOC formation in the acid stomach are the pH, incubation time, nitrite concentration, nitrosatable nitrogen concentration and the presence of catalysts and inhibitors.
The normal acidic stomach is essentially sterile with a resting pH of 1.5-2.0, but this can rise transiently to higher values following the ingestion of food (Milton-Thompson, 1982). Dietary nitrite levels are small, hence intragastric nitrite is largely of salivary origin as salivary nitrate is reduced by bacterial action to nitrite and swallowed (Bartholomew & Hill, 1984). The incubation time available for nitrosation is dependent on the availability of intragastric nitrite. However, nitrite has a short half-life of less than 10 minutes in the stomach due to its high reactivity and physiological absorption (Kyrtopoulos, 1989). Catalysts of acid-catalysed N-nitrosation include thiocyanate (present in larger amounts in the saliva of smokers) and other anions such as chloride (Fan & Tannenbaum, 1973). Nitrosation inhibitors include compounds which scavenge the nitrosating agent such as the antioxidant vitamins ascorbic acid and tocopherol or polyphenolics such as tannins (Mirvish, 1996).
The formation of carcinogenic NOC in an acidic intragastric environment in high-risk populations begins early in life and these compounds possibly induce a series of changes in the gastric mucosa leading eventually to gastric cancer (Mirvish, 1975). As 25% of ingested nitrate is actively secreted into saliva and 5% of all nitrate entering the mouth is reduced to nitrite by oral bacteria and mostly swallowed, it has been calculated that this route of nitrite formation contributes about 3.5mg nitrite/person/day, which corresponds to over 80% of the gastric nitrite load of a person with normal gastric acidity (Kyrtopoulos, 1989). The rate of acid catalysed nitrosation is proportional to [nitrite]2 (Bartholomew & Hill, 1984), and it has
4 0 ______Chapter 1.Introduction been shown in some human studies that increased nitrate consumption in the diet increases acid-catalysed endogenous nitrosation resulting in increased formation of NOC (Moller et al, 1989, Tricker & Preussmann, 1987) .
I.3.2.2. Bacterially mediated endogenous nitrosation and its possible role in gastric cancer.
Bacterially-mediated nitrosation has an optimal pH of 6-8 and can occur anywhere in the body where bacteria, nitrate or nitrite and nitrosatable nitrogen co-exist (Table 1.3.2.2.). Bacterial overgrowth in the stomach is determined primarily by gastric pH, as once the pH rises above 4 bacterial colonisation occurs (Kyrptopoulos et al, 1985). Gastric nitrite comes from the diet and the saliva as in acid-catalysed nitrosation (see Section 1.3.2.), however the main source is from locally acting bacterial nitrate reductase which contributes over 90% of the total gastric nitrite load of a hypochlorhydric person (Kyrptopoulos, 1989). Hence it is not surprising that nitrite concentrations increase with increasing pH in gastric juice as not only is nitrite formed in situ but it is less likely to decompose (Xu & Reed, 1993).
Site Conditions for bacterial colonisation and nitrosation. Mouth Normal salivary or buccal flora, no significant nitrosation due to short incubation time
Stomach Hypochloihydria as a result of natural atrophy, Helicobacter pylori infection, gastric surgery, significant nitrosation and formation of NOC has been shown to occur
Urinary bladder Bladder infection, either simple, or secondary to bilharzia or paraplegia, significant nitrosation and formation of NOC has been shown to occur Vagina Vaginitis, including Trichomonas vaginalis infection, no nitrosation has been shown to occur in vivo
Small bowel Presence of ‘blind loops’, small bowel overgrowth as a result of stasis or uremia, no nitrosation has been shown to occur in vivo
Large bowel Normal colonic flora, NOC have been detected in faeces presumably derived from nitrate secreted into the colon
Table I.3.2.2. Sites in the body where bacterial N-nitrosation can occur (data from a review by Hill, 1996b) .
41 ______Chapter 1. Introduction 1.3.3. The Correa model of gastric carcinogenesis.
In 1975 Correa et a l proposed an hypothesis for human gastric carcinogenesis, a model that has been updated several times (1983, 1988 and 1992) to take into consideration various aetiological factors. In brief, exposure to gastric irritants such as salt, damages mucosal cells and enhances the generation or release of endogenous NO in the mucosa. This in turn leads to the initial stages of superficial gastritis which can progress to chronic atrophic gastritis (CAG) (Takeuchi et al, 1994). Bacterial infection with Helicobacter pylori results in chronic inflammation and superficial gastritis in virtually all infected individuals (Kuipers et al, 1995a). Helicobacter pylori infection does not resolve spontaneously and the development of CAG is prevalent (Kuipers et al, 1995b). Gastric cancer incidence in humans correlates positively with the prevalence of CAG, which has emerged as the key precursor event in human gastric carcinogenesis (Sirurala et al, 1974, Correa et al, 1990b).
CAG begins as a multifocal lesion which rapidly spreads to cover much of the gastric mucosa and is characterised by cellular atrophy, the loss of gastric glands and of their acid-secreting parietal cells and their gradual replacement by intestinal-type glands. This results in an appreciable increase in the pH of the bulk of the gastric juice and facilitates bacterial growth. CAG in high-risk populations is frequently followed by a series of cell transformations, usually interpreted as successive mutations. The first and most prevalent transformations result in intestinal metaplasia. While in most patients, intestinal metaplasia is well- differentiated, the cellular prototype in a few individuals becomes progressively displasic and may finally become neoplastic. These apparently progressive stages have been documented in several populations and form the backbone of the aetiological model outlined in Figure 1.3.3. (Correa, 1983).
The basis of this model is that conditions that lead to increased gastric pH, achlorhydria, allow the colonisation of the stomach by bacteria, as in normal gastric juice (pH < 3) bacteria entering the stomach with saliva are rapidly killed (excluding H. pylori). The microflora of the achlorhydric stomach can include up to 30 species of aerobic and anaerobic bacteria (Kyrtopoulos et al, 1985). While the frequency of occurrence of certain species depends on the cause of achlorhydria, the total bacterial counts depend only on high pH. The presence of bacteria capable of reducing nitrate to nitrite has been shown and some bacteria are also capable of catalysing amine nitrosation (Calmels et al, 1987). As a consequence, high 42 Chapter 1. Introduction concentrations of nitrite build up in the hypochlorhydric stomach which is postulated to give rise in turn to increased endogenous nitrosation of available precursors which results in in situ formation of NOC. The latter act on the cells of the gastric mucosa to bring about mutations leading to intestinal metaplasia and other changes which ultimately result in neoplasia.
Gastric lumen Tissue
salt
Figure 1.3.3.The Correa model of gastric carcinogenesis, (adapted from Correa, 1992)
1.3.3.1. Conditions with achlorhydria.
There are many disease states where loss of gastric acidity is observed. Such conditions include pernicious anemia, gastric cancers as well as gastric operations (Billroth I and Billroth II partial gastrectomies and truncal and proximal-gastral vagotomies) aimed at reducing gastric acid secretion. Many epidemiological studies are in agreement that individuals with a partial gastrectomy or pernicious anemia are at an increased risk of developing gastric as well as other forms of cancer 15-30 years later (Caygill et al , 1987). During the period 1976-1986
43 ______Chapter 1. Introduction more than 20 reports all showed that an increase in gastric juice pH was accompanied by a more profuse gastric flora (in particular the nitrate-reducing species) and an increased nitrite concentration in gastric juice (Hill, 1990). In only a few studies were N-nitroso compounds assayed in these diseases and the results were highly variable due to the difficulties in methodology (See Section 1.3.4.).
13,3.2. Helicobacter pylori and the importance of inflammation.
H. pylori infection causes chronic inflammation in the human stomach. As an established infection is rarely cleared spontaneously, the gastritis persists indefinitely and thus gives rise to cellular atrophy. By this means, H. pylori can be said to initiate the sequence of events leading to gastric cancer in the Correa model.
H. pylori infection is prevalent worldwide with estimates of 30% -50% of the population in developed countries thought to have been infected by the age of 50yrs (Forman et al, 1991, Sitas, 1991). Epidemiological studies have shown that there is a geographical association between areas of the world with high rates of gastric cancer and the prevalence of H. pylori infection (Forman et al, 1990, Eurogast, 1994). The high prevalence of H. pylori infection at an early age in population groups at high risk of gastric cancer has also been reported to be an important factor. Studies where blood was taken from healthy individuals, stored and H. pylori status assessed in those who later developed gastric cancer showed that infection was significantly more common in gastric cancer patients (Forman et al, 1991).
There are several other consequences of H. pylori infection apart from the causation of gastritis that are relevant to cancer. Some of these include the suppression of ascorbic acid excretion into the stomach (Sobala etal, 1991), which is normally a protective factor against the formation of NOC (Mirvish, 1994). Infection by H. pylori causes chronic inflammation which results in the presence of lymphocytes, neutrophils and macrophages in the affected mucosal tissue. The presence of macrophages and neutrophils can lead to NO synthesis when activated (Leaf et al, 1989) as well as the stimulation of free radical oxygen (such as superoxide and H202) production by macrophages (Davies & Rampten, 1994). The NO produced reacts with oxygen to form N203 and N204 which in turn react with water to generate additional nitrate and nitrite. N203 directly deaminates amino groups of DNA bases
4 4 ______Chapter 1. Introduction and reacts with 5-methylcytosine to form thymine which is not repaired due to this being a normal DNA base. Since 5-methylcytosine usually occurs immediately upstream from guanine, this may explain why CG —» TA transitions at CpG sites in the p53 gene are associated with H. pylori infection (Palli et al, 1997). The ability of activated macrophages to catalyse nitrosation reactions with amines has been described.'Thus higher levels of NOC may be expected due to inflammation (Liu & Hotchkiss, 1995). Active oxygen species that are produced during inflammation may also cause mutations by oxidising DNA guanine to form various adducts such as 8-hydroxyguanosine (Lunec et al, 1994). H. pylori has also been shown to catalyse nitrosation reactions under in vitro conditions (Ziebarth et al, 1997) and increased proliferation of epithelial cells also contributes to the cancer risk associated with H. pylori infection (Cahill et al, 1994).
There is, therefore, a good deal of consistent evidence to support H. pylori infection as an important risk factor for gastric cancer and its designation as a IARC Class 1 carcinogen for gastric cancer underlines this strong association.
1.3.4. Measurements of N-nitroso compounds in gastric juice.
Since Ohshima and Bartsch (1981) developed their N-nitrosoproline (NPRO) test for endogenous nitrosation, experiments have been carried out in many laboratories measuring urinary excretion of NPRO as an indicator of endogenous nitrosation. Interpretation of the data is not simple and it is thought that this test system is only reliable for predicting acid- catalysed nitrosation. However, all humans tested so far have detectable levels of NPRO in their urine (Tannenbaum, 1987).
Total NOC in gastric juice as a group have been measured using a method developed by Walters et al (1983), which has had modifications made to it by two subsequent research groups, Bavin et a l (1982) and Pignatelli et al, (1987), the latter being applicable to various biological and food matrixes. All of these methods suffer from the requirement to add either sulphamic acid or hydrazine to prevent in vitro nitrite formation which will denitrosate a significant proportion of the NOC, especially at higher pH. This has meant that due to different analysis procedures, study design, storage of samples and specificity of procedure, no consensus of the relationship between pH, total bacteria counts and nitrite levels with the
45 ______Chapter 1. Introduction NOC formation has been achieved (recent review of analytical procedures can be found in Pignatelli & Walters, 1996).
As the reliability of all available methods for measuring both stable and unstable NOC as a group in biological fluids had been questioned, a new method'for measuring total N-nitroso compounds (TNOC) in fresh gastric juice was developed (Xu & Reed, 1993). Overall, the studies employing this technique demonstrate a linear relationship between pH and nitrite, the existence of two TNOC peaks, one in the acidic range of pH 1.13-2.99 (mean value: 1.45jimol/L) and the other at pH 6.0-8.42 (mean value: 3.57|imol/L). These results confirm that both acid-catalysed and bacterially-catalysed N-nitrosation occur in the human stomach and that higher levels of TNOC are formed in the hypochlorhydric stomach.
Although these studies are good indicators of endogenous nitrosation occurring, they may not be indicative of the reactive species present in the gastric lumen that react with the gastric mucosa. In addition, many primary amines react with nitrosating agents to give biologically relevant diazo- and diazonium-compounds which would not release nitrite and thus escape detection by this methodology.
1.3.5. Precursors for endogenous nitrosation in the diet
The human diet contains a variety of nitrosatable precursors, which differ markedly with respect to their daily intake, rate of nitrosation and carcinogenicity of the nitroso derivative. These include amongst others alkylamines, amino acids, amines, urea, bile acids, guanidines and primary, secondary and tertiary amines (Shephard & Lutz, 1987). The nitrosation rates of individual precursor groups has been reviewed by Hill (1990a). The risk from endogenous exposure of any NOC formed from a precursor is hard to assess because of the wide range of NOC formed, and the largest proportion of the total exposure is from non-volatile NOC which are not easily analysed individually. Nitrite will be the limiting substrate for endogenous N-nitrosation as the levels of amino substrates are likely to be in large excess. For example, the level of peptide amino groups in gastric juice is estimated to be approximately 0.2M and the concentration of other amino substrates to be <10~4M (Challis et al, 1982). It is believed that mutations in the p53 tumour-suppressor gene at non-CpG sites
4 6 ______Chapter 1. Introduction are related to alkylating compounds from the diet and their endogenous nitosation (nitrite, protein, and fat, particularly from animal sources)(Palli et al , 1997).
1.3.5.1. -Amino acids and peptide nitrosation.
The nitrosation of amino acids, peptides and proteins has attracted interest due to their high gastric concentrations. Free amino acids in human gastric juice are derived from the blood plasma amino acid pool, secreted from the cells of the gastric mucosa or are liberated by the gastric proteolysis of proteins and peptides (Komorowska et al, 1981). Free amino acid levels in human gastric juice were thought to be in the order of 0.37mM. However, recently levels up to 8mM have been reported to be present in gastric juice (Wang et a/,1995). Glycine (12%), histidine (15.61%), phenylalanine, leucine and glutamic acid together represent more than 50% of the total free amino acid concentration in human gastric juice (Komorowska et al, 1981). Certain food stuffs that are associated with the diet in areas of high gastric cancer risk (China) have high levels of free amino acids (13-14mg/g in fish sauce, soy sauce and salted fish) and may therefore be involved in the aetiology of gastric cancer in these areas. Interestingly, it has been shown by Chen et al, (1995) that salted fish extracts under acidic nitrosation conditions result in the methylating mutagen 2-chloro-4-methylthiobutanic acid being formed from methionine and sodium chloride.
As has already been stated peptides represent the most abundant source of nitrosatable substrates in human gastric juice. In common with other amino compounds, peptides react with nitrosating agents under a range of conditions and several products arise depending on the amino acid constituents of the peptide. Two general reactions are found however, irrespective of these constituents (Figure 1.3.5.1.). The terminal primary amino group can undergo nitrosation to generate a diazopeptide or the peptide-N-atom can be nitrosated to give an N-nitrosopeptide (Challis, 1989).
4 7 Chapter 1. Introduction
R H
COOH COOH
NO-X diazopeptide
R NO
N-nitrosopeptide
Figure 1.3.5.1. The formation of N-nitroso and diazopeptides.
The nitrosation of glycylglycine which contains both primary amine and amide nitrogens, is dominated by reaction at the primary amine under simulated gastric conditions. When the primary amine is blocked by acetylation, the overall rate of nitrosation is much slower with peptide nitrosation being 20 times slower than amine diazotisation in the normal gastric range of pH (1-4) and a nitrite concentration of 10'4M. However, under acidic conditions the diazopeptide is unstable due to the rapid protonation of the diazoalkane to give a highly reactive diazonium ion which rapidly decomposes. N-nitrosopeptides have been shown to be more stable than diazopeptides at both acidic and neutral pH, but on decomposition generate either a diazoacetic acid or a diazopeptide (from longer chain N-nitrosopeptides). Both diazoacetic acids and diazopeptides are alkylating agents (Challis, 1989). Diazopeptides form rapidly at neutral pH from gaseous nitrogen oxides and are relatively stable under these conditions. The yields produced will depend on peptide concentration, N 02 level, pH and temperature.
4 8 Chapter 1. Introduction 1.4. Biological activity of nitrosated peptides and compounds of related stru c tu re .
The amide bonds of bile acid conjugates and peptides are susceptible to nitrosation to give the corresponding nitrosamide. However, due to their novelty, very little is known about human exposure to these compounds but studies have been carried out on their biological activity in in vitro cellular systems and animal models. The bile acid conjugates, glycocholic acid and taurocholic acid (both present in the gastrointestinal tract as part of normal enterohepatic circulation) can be nitrosated under simulated gastric conditions to form N-nitrosoglycocholic acid (NOGC) and N-nitrosotaurocholic acid (NOTC) (Shuker et al , 1981). NOGC and NOTC are equally potent mutagens to bacterial cells. However, NOGC was a more potent mutagen in mammalian cells (diploid human lymphoblasts) and the cytotoxicity of these two compounds was found to be comparable (Puju et al, 1982). NOGC and NOTC have been shown to induce hepatocellular and stomach tumours in CDF Fischer rats following gastric intubation (Busby eta l, 1985). Furthermore, alkaline phosphatase-positive foci, a putative early mucosal alteration which may precede intestinal metaplasia and ultimately neoplasia, were found in approximately 35% of the glandular stomachs of compound-treated rats. A series of N-nitrosopeptides including N-(N' -acetyl-L-prolyl)-N-nitrosoglycine (APNG) and N-(N-acetylva!yl)-N- nitrosoglycine were prepared and found to be direct-acting base-pair substituting mutagens in vitro to both bacteria and mammalian cells (Challis, 1989). APNG has also been shown to be carcinogenic in mice following oral or intraperitoneal administration, producing a range of tumours including pulmonary adenoma, squamous-cell carcinoma of the stomach and renal adenoma. (Anderson & Blowers, 1994). Since N- nitrosopeptides decompose to either diazotic or diazopeptides in vivo (Section 1.3.5.1.), their biological properties may well be due to these decomposition products. Although the exact mechanism of genotoxicity has still to be clarified for most N-nitrosopeptides evidence suggests that it is likely to be through DNA alkylation. The known DNA alkylation products of some of these compounds will be covered in Section 1.5.1.
Rather more is known about the biological properties of diazopeptides, particularly the N- (diazoacetyl)-glycine compounds. These compounds are directly acting mutagens and exhibit cytotoxic properties (reviewed by Challis, 1989). at least two are known to be carcinogenic (N-(diazoacetyl)-glycine amide or the analogous hydrazide), primarily to lung and
4 9 Chapter 1. Introduction haematopoietic system, following intraperitoneal injection in Swiss mice (Brambilla et al, 1972). It seems probable that cytotoxic and mutagenic properties of diazopeptides relate to their ability to alkylate DNA. However, no diazopeptide has yet been administered by intubation where it is likely that the acidic milieu of the gastric juice would convert the diazopeptide into locally-acting alkylating agents.
1.4.1. DNA alkylation adducts from nitrosated peptides and compounds of related structure.
Shephardet al (1987) performed in vitro assays to detect the alkylating activity of various amino acids and dipeptides in the presence of nitrite at pH 2.5 using the 4-(para-nitrobenzyl) pyridine (NBP) colourimetric test linked to an Ames test for the detection of mutagenic activity. The a-amino acid and their derivatives and dipeptide nitrosation products were shown to alkylate NBP which is in accordance with the results observed by Brambilla et al (1979) who found that N-(diazoacetyl)-glycine derivatives also alkylate NBP. The nitrosated a-amino acids and dipeptides also gave positive results on Ames testing indicating that the adduct formed had mutagenic potential. In order to identify the alkylating groups Wang et al (1995) carried out a series of experiments. These indicated that diazoacetic acids (N=N=CR* COOH) were the alkylating species involved. Hence for peptides with a C-terminal in glycine this would result in the carboxymethylation of nucleophiles. Azaserine (O-diazoacetyl-L- serine), a diazoacetic glycine derivative is a direct-acting bacterial mutagen and a pancreatic carcinogen in rats. Azaserine carboxymethylates DNA in vivo producing [14C]-N- 7(carboxymethyl)guanine which was detected in DNA extracted from pancreatic acinar cells treated with [14C]-azaserine (Zurlo eta l, 1982).
The possible mechanisms of genotoxicity of N-nitrosopeptides needs to be clarified. However, APNG (a glycine derivative) gave rise to a number of carboxymethylated products including 0 6-carboxymethyl and N-7-(carboxymethyl)guanine on reaction with calf thymus DNA in vitro (Fairhurst (thesis), 1994). Similarly, the nitrosated bile acid N- nitrosoglycocholic acid (NOGC), which is also a glycine derivative, reacts in vitro with DNA to give a number of carboxymethylated products including 0 6-carboxymethylguanine, N-7- (carboxymethyl) guanine and N-3-(carboxymethyl)adenine (Shuker et al, 1987).
50 Chapter 1. Introduction Furthermore, the administration of [14C]-NOGC to rats resulted in a dose dependent excretion of [14C]-N-7-(carboxymethyl)guanine (Shuker et al , 1987).
Hence, N-alkyl-N-nitrosocompounds that share the common feature of being derived from the simplest amino acid, glycine, are all, in principle, carboxymethylating agents (Challis, 1989). The proposed mechanism for the formation of a common intermediate, the carboxymethyl diazonium ion, from both diazoacetic acid and N-nitrosoglycine derivative is shown in Figure 1.4.1. It can be seen that NOGC, azaserine and APNG would undergo decomposition to produce the reactive species, whereas potassium diazoacetate only needs to be protonated, and hence would be expected to be a carboxymethylating agent (CM) by analogy with these known CM agents. Thus, a diverse range of N-nitrosated glycine derivatives may give rise to the same DNA adducts, 0 6-CMdG and related adducts (N-7- (carboxymethyl)guanine and N-3 -(carboxymethyl)adenine), which are all potential markers of exposure to this diet-related nitrosation pathway (Shuker at al , 1995).
Diazoacetic acid derivatives N-nitrosoglycine derivatives
N* * NO ° N /? 'H . Potassium diazoacetate H O Ac O APNG
N >pH 7
h2n oh Azaserine NOGC H+ and ester hydrolysis >pH 7 N E N ^ - ^ O *
carboxymethyldiazonium ion
Figure 1.4.1. Formation of carboxymethylating agents from a range of nitrosated glycine derivatives.
51 Chapter 1. Introduction 1.4.2. Nitrosated glycine derivatives cause methylation as well as carboxymethylation of DNA.
NOGC was shown to react with DNA in vitro to give a number of adducts. These were O6- carboxymethylguanine, N-7-(carboxymethyl)guanine and N-3-(carboxymethyl)adenine, as well as N-7-methylguanine and 0 6-methylguanine (06-MeG), the latter at approximately 65% of the 0 6-CMG level (Shuker & Margison, 1997). Azaserine treated rats were shown to have 0 6-MedG and Oe-CMdG lesions in their pancreatic DNA via immunohistochemical staining (personal correspondence with Dr. G. P. Margison). Thus, it would appear that not only is the carboxymethyldiazonium ion derived from these type of compounds (Section 1.4.1), but that a methyldiazonium ion may also be involved (Figure 1.4.2.). The extent to which different nitrosated glycine derivatives methylate or carboxymethylate DNA has yet to be established.
NO
L .COOH 'T' C H 2 o
| DN o c h 3 o c h 2co o h N ^n^N
06-MedG 06-CMdG
Figure 1.4.2. The formation of 0 6-CMdG and 0 6-MedG in DNA.
1.4.3. The 0 6-carboxymethyldeoxyguauosine DNA adduct.
0 6-CMdG has attracted interest because of its apparent lack of repair by bacterial and mammalian 0 6-alkylguanine alkyl transferases that repair a variety of 0 6-alkyl adducts to varying degrees (Shuker and Margison, 1997). ATase is known to repair presumably the O6- MedG produced by azaserine (Sedgwick, 1997). However, if cells lack nucleotide excision repair then an increased mutation rate by azaserine is seen (Kubitschek & Sepanski, 1982). 0 6-CMdG is refractory to DNA repair by ATase it has been suggested that this may be the mutagenic lesion repaired by nucleotide excision repair. Apart from the observation that O6-
52 Chapter 1. Introduction CMdG is not repaired by 0 6-alkylguanine-DNA-alkyl transferase, other possible biological characteristics of this lesion have yet to be established as all carboxymethylating agents so far tested also methylate DNA, although from the characteristics of other 0 6-alkyl-guanines it would not be unreasonable to anticipate that it would be promutagenic and possibly a cytotoxic lesion. Thus 0 6-CMdG offers the possibility of a group-specific and possibly persistent DNA adduct derived from nitrosated glycine derivatives.
1.4.4. The 0 6-methyldeoxyguanosine DNA adduct.
0 6-MedG is a well established promutagenic lesion, has the potential to activate oncogenes, and can also be responsible for other biological effects such as chromosome damage and cell killing (Cooper et al, 1995) (Figure 1.4.4 ). The epithelium of the gastrointestinal tract is particularly low in ATase (Kyrtopoulos et al, 1990) and since the methylated repair protein is inactivated and is only slowly resynthesised, episodic exposure to methylating agents could overwhelm this repair pathway which could result in dividing epithelial cells acquiring mutations.
‘Normal” base pairing (low frequency) Methylating agent 0 6-Me G = C CH
Chain termination \ 0 6-Me G = C Replication 0 6-MeG (unknown frequency)
A T ase Replication Q6-Me G = T
Transition mutation
Futile” repair Oe-Me G = (M M R ) loop
Engage apoptosis
Figure 1.4.4. Mechanisms of cell killing and mutation due to 0 6-MedG.
53 Chapter 1. Introduction Several reports are available for the presence of 0 6-MedG in human DNA from various tissues (Table 1.4.4.). Numerous projects have investigated target organ DNA alkylation in populations where an increased risk for cancer in certain tissues has been identified. For example, gastric cancer risk is high in Lin Xian county, where various food constituents have been found to contain several N-nitrosoamines and a high proportion of stomach mucosa DNA samples were found to contain 0 6-MedG in relatively large amounts compared to similar tissues obtained from European controls.
Exposure Population Tissue Method Proportion 0 #-MedG Reference alkylated* rangeb
(umol/mol dGor dA) Lin Xian (China) Oesophagus and RIA 18/26 0.016-0.26 Umbenhauer et stomach 9/11 0.024-0.14 al, (1985). Singapore (S.E. Oesophagus and RIA 27/53 0.016-0.08 Saffhill et al, Asia) stomach (1988).
Shanghai (China) Stomach RIA 15/53 0.053—0.718 Cooper et al, (1991). Nile deha (Egypt) bladder RIA 44/46 0.012-0.49 Badawi et al, (1992). General European (France Oesophagus, RIA 5/12 0.032-0.064 Umbenhauer et and Germany) stomach and colon al, (1985). survey Manchester (UK) Stomach and colon RIA 27/53 0.011->0.3 Hall et al, /controls" (1991). Manchester (UK) Bladder RIA 4/12 0.034-0.225 Badawi et al, (1992). Athens (Greece) Stomach CRA 1/20 0.083 Kyrtopoulos et al, (1990). Japan Liver HPLC/32 13/15 0.11-0.67 Kang et al, leukocytes PPL 15/15 0.007-0.046 (1995)
17 populations leukocytes CRA 21/413 0.085-0.42 Eurogast (1994)
Shanghi (China) Stomach IAC/32? 7/7 0.021-0.041 Povey & PL Cooper(1995) Manchester (UK) Stomach 5/5 0.028-0.07
USA Peripheral lung HPLC/32 s 5/11 0.1-5.2 Wilson et al, P-PL ns 3/6 <0.1-2.4 (1989). non-smokers USA Placenta ELISA s 2/10 C0.5-1.6 Foiles et al, ns 3/10 <0.1-1.6 (1988). (ns) France Oral mucosa RIA s 4/20 2.2-7.0 Wild et al, ns 0/20 - (1989).
Medical Dacarbazine Leucocytes CRA 10/11 0.72 Souliotis et al, (Greece) (1991). Procarbazine Leucocytes CRA 7/7 up to 0.45 Souliotis et al, (Greece) (1990).
* number of samples containing 0 6-MedG/total number of samples. b Amount of 0 6-MedG detected in positive samples. c Tissues taken from individuals at increased risk for developing cancer in specific tissues due to enviromental factors. d Tissues taken from individuals not known to be at an increased risk for developing cancer. RIA, radioimmunoassay, HPLC, high performance liquid chromatography, PL, postlabelling, ELISA, enzyme linked immunosorbent assay, IAC, immunoaffinity column, CRA, competitive repair assay.
Table 1.4.4. 0 6-MedG detection in human tissues. 54 Chapter 1. Introduction Similarly, the occurrence of 0 6-MedG in gastric tissues collected in Shanghai (an area of China with a high incidence of stomach cancer) has been investigated and a tendency for a higher frequency of alkylation in the samples obtained from patients suffering from gastritis was observed (Cooper et al, 1991). In studies concerning gastric cancer risk factors, elevated levels of 0 6-MedG in DNA extracted from gastric epithelial tissue of subjects with atrophic gastritis were observed (Hall et al, 1991). The Eurogast Study (1994) found detectable levels of 0 6-MedG in DNA from peripheral lymphocytes from subjects with low levels of serum pepsinogen A, a marker of severe chronic atrophic gastritis compared to undetectable levels in subjects with higher levels of serum pepsinogen A. It has also been observed that DNA treated in vitro with gastric juice contains 0 6-MedG (Kyrtopoulos, 1987). This evidence suggests that 0 6-MedG formation by methylating species in the stomach may be involved in the aetiology of gastric cancer.
The detection of 0 6-MedG in the DNA of control samples from a variety of sources has also been detected in almost all studies undertaken. This suggests that individuals presumed to be at no specific risk for a certain cancer/environmental exposure are also exposed to some extent to alkylating agents either endogenously or via their environment. The possibility that this exposure is due to the presence of N-nitrosoamines in tobacco smoke has stimulated investigations to compare the extent of DNA alkylation in target and non-target tissues from smokers and non-smokers. Interestingly the detectable levels of 0 6-MedG in lung or placenta showed no difference between the smokers and non-smokers (Wilson et al, 1989, Foiles et al, 1988), but Wild et a l (1989) found that buccal cells from smokers were more likely to contain 0 6-MedG than those from non-smokers.
1.4.5. Techniques available for the detection of DNA alkylation.
The desire to monitor levels of 0 6-MedG and other DNA adducts in tissues and cells of model systems and ultimately human tissues has led to the development of a number of methods for the detection of low levels of 0 6-MedG. Amongst the variety of techniques available (Table 1.4.5.) the detection of 0 6-MedG in human tissues has been accomplished primarily with immunoassays using highly specific mono- and polyclonal antibodies or via 32P-postlabelling (Wild, 1990,). Most of these techniques employ an adduct purification step
55 Chapter 1. Introduction (HPLC, TLC and immunoaffinity) to remove the large excess of unmodified bases (million fold) and thus increase the sensitivity and specificity of the assay.
The most widely used antibody-based assay involves the HPLC purification of 0 6-MedG prior to radioimmunoassay (RIA). Although this assay is sensitive with a detection limit of - 25fmol/mg DNA and has been used in human population studies (Umbenhauer et al, 1985, Saffhillet al, 1988, Hall et al, 1991, Badawi et al, 1992), it requires a large amount of DNA for analysis (l-10mg) thus limiting the applications for which it can be used. Greater sensitivity can be achieved using smaller amounts of DNA (50-100pg) by the 32P- postlabelling assay. However, this type of assay requires multiple chromatographic separations and therefore is extremely time consuming (Haque et al, 1994, Povey & Cooper, 1995).The competition repair assay which involves the direct quantitation of 0 6-MedG without the need for prior enrichment offers a sensitive and rapid assay (Kyrtopoulos et al, 1990). As this assay requires the incubation of DNA with ATase and radiolabelled oligonucleotides containing 0 6-MedG, the requirement for ATase (normally purified from cloned human genes and expressed in cells) is essential.
Method Detection limit (finol) Quantity DNA used (mg) Reference
RIA" 65 0.005b Wild et al, 1983
ELISA (hydrolysed 120 0.100 Foiles et al, 1985. DNA)*
RIA-HPLC 105 1.0-10.0 Umbenhauer et al, 1985.
ELISA-HPLC 720 1.0-10.0 Foiles etal, 1985.
Immunoslot blot 2.8 0.003b Ludeke & Kleihues, 1988.
Competitive repair assay 0.5 0.010 Kyrtopoulos et al, 1990. 32P Postlabelling-HPLC 1 0.1 Haque et al, 1994.
32P Postlabelling-IAC 0.18 0.05 Povey & Cooper, 1995
Electrochemical-HPLC 50 0.025 de Groot et al, 1994.
Fluorescence-HPLC 500 1.0 Belinsky et al, 1987. a Competitive immunoassay analysing directly DNA hydrolysates. b Quantity of DNA required for a single analysis. Normally the analysis will be performed in duplicate or triplicate on separate occasions thus requiring 4-6 times this quantity of DNA.
Table 1.4.5. Assays available for the quantitation of 0 6-MedG (adapted from Wild, 1990)
56 Chapter 1. Introduction 1.5. Aims of project
Diet/food is a very complex mixture which contains a large number of constituents which can be nitrosated in the gastro-intestinal tract to potentially carcinogenic nitrogenous compounds (Shephard & Lutz, 1989). This situation is also complicated by the fact that there is exposure to endogenous preformed NOC in certain foodstuffs, and via tobacco-specific nitrosamines (Preston-Martin & Correa, 1989). Confirmation has been obtained for human exposure to a total of 58 different N-nitrosamines and of these, 44 have been evaluated in long-term animal bioassays showing 30 to be carcinogenic (Tricker, 1995). Mechanistic information on the metabolism and biological activity of NOC is also extensive. However, the problem of the possible contribution of exposure to NOC to the pathogenesis of gastric cancer still remains largely unsolved. One main problem which hampers epidemiological investigations relates to the difficulty of accurately estimating human exposure to specific NOC or types of NOC, especially those derived by endogenous processes. Hence the utilisation of molecular biomarkers may present a solution to this problem. It is important, however, to be aware of the fact that over 200 different DNA binding agents (including N-nitrosamines) have been identified, but the total number of individual adducts formed by these compounds is small, since many compounds form the same adduct(s) (Tricker, 1995). For example N- nitrosodimethylamine forms a variety of covalent methyl adducts with DNA. However, 15 other N-nitrosamines to which man is exposed also methylate DNA. Therefore, the detection of a single adduct can not with certainty be related to exposure to a single NOC but may be indicative of a class of compounds. For this reason the identification and determination of specific biomarkers to particular classes of compounds or nitrosation pathways rather than to one compound are likely to be more useful for the measurement of food-related DNA- damage.
To this end the DNA adduct, 0 6-CMdG, may prove to be a useful biomarker for exposure to nitrosated glycine derivatives which themselves may be indicative for the endogenous nitrosation of proteins or amino acid in the gastric lumen.
The first aim of this study is to develop and apply methods which are capable of detecting and quantitating 0 6-carboxymethyl-2’deoxyguanosine and 0 6-methyl-2 ’ deoxyguanosine DNA adducts in in vitro mechanistic studies to establish the concomitant carboxymethylating
57 Chapter 1. Introduction and methylating properties of a range of nitrosated glycine derivatives. This will involve the development and validation of immunoaffmity purification protocols for both adducts followed by HPLC-fluorescence detection systems.
In order to ensure that these reagents cause the formation of 0 6-CMdG in vivo, potassium diazoacetate and APNG, the carcinogenic nitrosated peptide, will be administered by gastric intubation to rats and the determination of the adduct levels will be undertaken. This will be carried out using an immunoslot blot procedure, which will be developed and validated using a polyclonal anti-06-CMdG rabbit antibody. Published protocols for this technique indicate that only small quantities of DNA are required and the sensitivity is relatively high, making this type of assay ideal for use in human epidemiology studies where DNA sample size is limited.
As the 0 6-CMdG adduct may be the major and most persistent 0 6-guanine DNA base lesion produced by nitrosated glycine derivatives, it is potentially an ideal marker of the biologically effective dose arising from endogenous exposure to this class of compounds. As 0 6-CMdG has not as yet been detected in human DNA samples, this will be undertaken using the immunoslot blot assay. Hence two distinct studies involving human subjects were undertaken. A study to detect and quantitate the level of 0 6-CMdG in the DNA from the gastric mucosa of individuals who may be at an increased risk of gastric cancer due to H. pylori infection was carried out. Furthermore, white blood cell DNA was available from human subjects who participated in a diet study designed to establish if the increased faecal NOC levels observed on a high red meat diet (Bingham et al , 1997) can be attenuated by the dietary addition of vegetables and/or tea. Thus the effect of dietary alteration on the levels of 0 6-CMdG in white blood cell DNA was examined.
58 Chapter 2 . Method Development
59 Chapter 2. Method development
2.1. Introduction.
The quantitation of low levels of 0 6-alkylguanine adducts can be hindered by the presence of the large excess of normal nucleosides. It is therefore necessary to incorporate an enrichment procedure prior to detection of the adducts. Immunoaffinity chromatography is a method of fractionation which can provide dramatic purification of a substance of interest. The technique is extremely powerful and purification factors of 2000-20 000 fold are often possible. In fact it can be possible to achieve purification to homogeneity in a single step (Goding, 1986).
The principle of affinity chromatography is shown in Figure 2.1. A successful separation requires that a biospecific ligand (the antibody) is available and that it can be covalently attached to a matrix, rendering it insoluble. The soluble molecule (antigen) specifically interacts with the now insoluble antibody. This will temporarily render the antigen insoluble and allows it to be separated from soluble contaminants.
i. . . i
Immobilise the antibody on matrix I 3 IMPURITIES
Application of sample substance
Disruption of antibody-antigen bond
Figure 2.1. Principle of affinity chromatography. Where A is the antibody and B is the soluble molecule of interest (antigen). 60 Chapter 2. Method development Antibodies have been used in a range of methods to detect DNA adducts arising from a wide variety of agents (Wild, 1990). The immunoafflnity purification of modified bases from hydrolysed DNA has been achieved prior to quantitation by a variety of detection methods such as 32P-postlabelling (Povey & Cooper, 1995), gas chromatography-mass spectrometry (Shuker & Bartsch, 1994) or HPLC with electrochemical detection (Bianchini et al, 1993).
As this methodology is simple, quick and allows high purification of minor components of a mix of compounds, it was considered to be ideal for the purification of the DNA adducts O6- CMdG and 0 6-MedG from DNA digests. Antibodies to the two adducts were available (O6- CMdG polyclonal rabbit antiserum from previous work (N. Fairhurst) and monoclonal O6- MedG (D. Cooper)) and would need to be immobilised on a suitable matrix while retaining their specific binding affinity for the adduct of interest. Likewise a method for selectively dissociating the antibody/antigen complex formed would be explored, after washing away unbound material. In order to investigate these aspects of immunoafflnity purification radiolabelled antigens were synthesised as they are extremely useful.
As nucleosides and bases can be chromatographically quantitated by RP-HPLC (Lim, 1986) and the particular adducts of interest, 0 6-CMdG and 0 6-MedG, are naturally fluorescent (Hemminki, 1980), RP-HPLC -fluorescence was the method of choice for quantitation after immunoaffinity purification. Thus method development to gain maximum sensitivity to quantitate 0 6-CMdG and 0 6-MedG from the same DNA sample was undertaken.
2.2. Synthesis of tritiated purine deoxynucleoside adducts.
Marker compounds to be used for immunoaffinity column characterisation were prepared by the enzymatic coupling of purine analogues to deoxyribose as described by Chapeau & Mamett (1991) and Stadler et al (1994). Purine nucleoside phosphorylase (PNPase) catalyses the displacement of phosphate from deoxyribose-1-phosphate of purines and purine analogues which can be generated in situ by the phosphorolysis of thymidine, a reaction catalysed by thymidine phosphorylase (TPase) (Figure 2.2.). As [5'-3H] thymidine is commercially available it can be used as the pentosyl donor leading to the formation of a radiolabelled purine deoxynucleoside adduct.
61 Chapter 2. Method development
THYMIDINE
HPO where X corresponds to : TPase X-O-CHjCO^H for 0*-CMdG X = O-CHj for 0*-MedG X = S-CHjCO;H for S*-CMdG
OPO
deoxyribose-1 -phosphate
base
PNPase HPO?'
Tritium labeled deoxyguanosine
Figure 2.2. The synthesis of tritium labeled deoxynucleoside derivatives from the corresponding purine base. Where T is thymidine and X denotes one of the various adducts indicated in the figure.
Initial trial reactions (not incorporating [^-thym idine) for 0 6-carboxymethylguanine (O6- CMG) using a scaled, down version (1 : 20) of the Chapeau & Mamett (1991) protocol indicated that the reaction proceeded very slowly. However the synthesis method by Stadler et al (1994) led to a 21% coupling yield of 0 6-CMG in a 48hr period as determined by RP- HPLC, indicating that this base was a viable substrate for the PNPase/TPase reaction (Table
62 ______Chapter 2. Method development 2.2.). Unfortunately the Stadler et al (1994) method could not be employed for the synthesis of tritiated purine deoxynucleosides due to the large excess of thymidine required.
Subsequent optimisation of the reaction conditions led to the conversion of 0 6-methylguanine (0 6-MeG) to its 2’ -deoxynucleoside with a 74% yield after 24hr as monitored by RP-HPLC- UV detection. However, for 0 6-CMG and N2-amino-S6-(carboxymethyl)-mercaptopurine (S6- CMG), the relative conversion to the corresponding nucleosides reached 7.35% and 8.4% even after prolonged incubation times of up to 456hr as measured by RP-HPLC (Table 2.2.). Nevertheless, sufficient amounts of all the required radiolabelled nucleosides were purified by RP-HPLC for later procedures.
Base Coupling yield Duration of reaction (%) (hr) 0 6-MeG 74* 24
o 6-c m g 7.4“ 456 00 s6-c m g 336
0 6-CMGc 21k 48 a Yields reported as isolated. b Yields are estimated from HPLC analysis of reaction mixture. 0 Reaction conditions according to Stadler et al (1994)
Table 2.2. Enzymatic synthesis of [3H]-deoxynucleosides.
2.3. 0 6-CMdG rabbit antiserum.
In order to ensure that the 0 6-CMdG antiserum was still viable after long term storage (6yrs) a competitive ELISA procedure was undertaken. Optimal conditions for the competitive ELISA were found using a chequerboard procedure. At a level of coating antigen (06-CMG- ovalbumin) of 5ng/well and an antiserum dilution of 1: 2x106, a final absorbance at 450nm of approximately 0.7-0.9 was achieved. Under conditions of a competitive ELISA, 0 6-CMdG was tested over a wide range (10-106fmol/well) and the 50% inhibition was found to be 3pmol. Under the same conditions 2’deoxyguanosine was tested for cross-reactivity and a
63 ______Chapter 2. Method development substrate concentration for 50% inhibition of >104pmol/well was found. Therefore, it was apparent that the antiserum is selective for the molecule and of the same high quality as originally determined after its synthesis by N. Fairhurst (Harrison et al, 1997). Accordingly, the antiserum could be used to prepare immunoaffinity columns to selectively isolate O6- CMdG from enzymatically hydrolysed DNA.
2.4. 0 6-CMdG immunoafflnity column characterisation.
A crude ammonium sulfate precipitated total IgG fraction from 0 6-CMdG rabbit antiserum was covalently bound to Protein A-Sepharose using a bifunctional cross-linking agent, dimethylpimelimidate, which resulted in a stable immunoaffinity gel. Protein A-Sepharose was chosen as the antibodies bind on their Fc portions leaving the antigen-combining site in the correct orientation for the binding of the 0 6-CMdG, hence a highly active immunoadsorbent gel is obtained (Schneider et al, 1982). As was expected from the ELISA results (Section 2.3.) the resultant lmL columns retained [3H]-06-CMdG (Figure 2.4.1.)
Various approaches to eluting bound antigen were explored. The majority were ineffective giving poor, if any recovery, due to the binding of 0 6-CMdG being so strong (Table 2.4.1.). Nonetheless, using fairly drastic conditions (1M TFA) did result in complete elution with the majority of the bound antigen eluting in the initial 2mL of TFA. Under these conditions extensive hydrolysis of the glycosidic bond in 0 6-CMdG occurred to yield the base 0 6-CMG. However, since 0 6-alkylguanines are slightly more fluorescent than the corresponding nucleosides (Gaffney & Jones, 1982), this phenomenon was turned into an advantage to improve the ultimate sensitivity of the overall immunoaffinity-HPLC-fluorescence assay.
6 4 Chapter 2. Method development
1000
900
800
700
300
200 100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 fraction (ml)
Figure 2.4.1. Elution of [3H]-06-CMdG from Ofi-CMdG immunoaffinity columns. [3H]- 0 6-CMdG (17.2ng, 950d.p.m.) was in PBS/azide (2mL), and the columns washed with PBS/azide (lOmL) and 1M TFA (5mL). The column eluate was collected as lmL aliquots which were added to scintillation fluid and counted directly.
Eluate [3H]-Ofi-CMdG eluted (%) 1M TFA 100 0.1MTFA 98 0.05M TFA 76 0.1M acetic acid 0 1M acetic acid 0 1M formic acid 0 50% aqueous DMSO 0 50% aqueous MeOH 0 70% aqueous MeOH 30 80% aqueous MeOH 39 80% MeOH aqueous 0.1M TFA 48 80% MeOH aqueous 1M TFA 50 70% isopropanol 0 80% isopropanol 8
Table 2.4.1. Elution conditions for 0 6>CMdG immunoaffinity columns.
65 ______Chapter 2. Method development The capacity of the 0 6-CMdG immunoaffinity columns was determined using a simple saturation assay. Separate solutions each containing [3H]-06-CMdG (17.2ng, 950dpm) and increasing amounts of unlabelled 0 6-CMdG (O-lOOOng) in PBS/azide were applied to columns. The standard elution protocol was used, and the radioactivity eluting with 1M TFA was measured (Figure 2.4.2.). The capacity of the columns was 200-250ng (lnmol). The immunoaffinity columns can be recycled many times even with these strong elution conditions and successive use involving loading with [3H]-06-CMdG, 0 6-CMdG and DNA digests did not reduce the capacity for approximately 150 runs.
100
90
80 •8 70 J 60 2 0 50 1 40 VOa o 30 a S 2 0
0 200 400 600 800 1000 0 6-CMdG added (ng)
Figure 2.4.2. Determination of 0 6-CMdG immunoaffinity column capacity. [3H]-06- CMdG (17.2ng, 950dpm) was eluted through the columns in the presence of increasing amounts of unlabelled 0 6-CMdG (O-lOOOng). The 1M TFA eluate was collected and the radioactivity remaining on the column determined by scintillation counting.
2.5. 0 6-MedG immunoaffinity column characterisation.
Immunoaffinity columns were prepared by covalently linking an 0 6-MedG monoclonal antibody (supplied by Dr. D. Cooper) to either Protein A-Sepharose or Protein D-Sepharose (Friesen et a/,1991). Both gel types retained [3H]-06-MedG (Figure 2.5.1.) and had similar capacities and characteristics of 500-600ng ,(2nmol) (Figure 2.5.2.). Elution of 0 6-MedG was 66 ______Chapter 2. Method development achieved using methanol, and an 80% methanol solution was chosen as complete elution occurred in the initial 2mL compared to larger volumes which were required with reduced methanol concentrations (Figure 2.5.1.).
900
800
700 T3 600 400 O *5 300 200 100 0 0 5 10 15 2 0 fraction number (ml) Figure 2.5.1. Elution of 0 6-MedG immunoaffinity columns. [3H]-06-MedG (13.45ng, 900dpm) was in PBS/azide (2mL), and the column was washed with PBS/azide (3mL), water (lOmL), and eluted with 60% methanol (red squares), 70% methanol (closed circles) or 80% methanol (blue squares). The 0 6-MedG immunoaffinity columns could also be eluted using trifluoroacetic acid. However, this method was not chosen as upon drying down of the fraction, acid hydrolysis led to substantially decreased yields of the base adduct obtained as demethylation of the adduct occurred. Thus, combining the immunoaffinity gels for the 0 6-CMdG and 0 6-MedG was not appropriate. Although the columns could be recycled numerous times, this gel showed a sudden decrease in capacity after approximately 50 runs. 67 Chapter 2. Method development 100 80 70 U i o 50 ' 9 30 DC ^ 20 0 500 1000 1500 2000 2500 3000 0 6-MedG (ng) Figure 2.5.2. Determination of column capacity of immunoafflnity columns for O6- MedG. [3H]-06-MedG (13.45ng, 900dpm) was eluted through the columns in the presence of increasing amounts of unlabelled 0 6-MedG, 0-3000ng for Protein D- Sepharose (blue triangles) and 0-1500ng for Protein A-Sepharose (closed circles). 2.6. Preparation of N2-amino-S6-(carboxymethyI)-mercaptopurine for use as an Internal Standard. An internal standard method involves the ‘spiking’ prior to any pre-treatment of sample solutions with a constant amount of a compound called the internal standard. Ideally an internal standard should have the following characteristics : be recovered to a similar extent to the analyte of interest, be reliably resolved from the analyte peak and any other peaks and have a similar detector response. Due to the nature of immunoaffinity purification, finding an internal standard can be a difficult task, as the antibody has to recognise the proposed internal standard yet the internal 68 Chapter 2. Method development standard has to be different enough to be easily identified compared to the compound of interest. For the 0 6-CMdG immunoaffinity columns it is already known from work done by N. Fairhurst (Harrison et cd, 1997) that the 0 6-CMG antiserum shows little cross-reactivity with a range of substrates. Hence the internal standard should ideally be a compound that is not naturally present in DNA, unlikely to be formed by the action of carcinogens, be fluorescent for quantitation by HPLC and retained by the 0 6-CMdG immunoaffinity column. S6-CMdG was therefore an ideal candidate as an internal standard, as its structure is very similar to 0 6-CMdG and the base S6-CMG is fluorescent. In order to ascertain if S6-CMdG was retained by the 0 6-CMdG immunoaffinity column [3H]- S6-CMdG was synthesised and loaded onto the immunoaffinity columns in PBS. The standard elution protocol for 0 6-CMdG was followed and [3H]-S6-CMdG was retained by the columns (Figure 2.6.1.). A binding capacity study was undertaken by loading 0 6-CMdG in increasing amounts until breakthrough was seen, the results can be seen in Figure 2.6.2.. The column had a capacity of lOOng 0 6-CMdG. This is approximately half the capacity from when O6- CMdG alone is loaded, indicating that the antibody has a higher affinity for 0 6-CMdG than S6-CMdG. S6-CMdG would be retained by the immunoaffinity columns providing that the columns were not loaded with more than 50% of their capacity for 0 6-CMdG. 1400 a 1200 d, ■d 1000 800 Io 0 600 1u 400 NO cn 200 £ 0 0 5 10 15 20 Fraction (ml) Figure 2.6.1. Elution profile of 0 6-CMdG immunoaffinity columns with [3H]-S6-CMdG. [3H]-S6-CMdG (29.5ng, lOOOdpm) was in PBS/azide (2mL), and the columns washed with PBS/azide (lOmL) and 1M TFA (5mL). The column eluate was collected as lmL aliquots which were added to scintillation fluid and counted directly. 6 9 Chapter 2. Method development 0 100 200 300 400 500 0 6-CMdG added (ng) Figure 2.6.2. Determination of column capacity of 0 6-CMdG immunoafflnity columns using [3H]-S6-CMdG. [3H]-S6-CMdG (29.5ng, lOOOdpm) was eluted through the columns in the presence of increasing amounts of unlabelled 0 6-CMdG (0-500ng). In order to ensure that S6-CMdG bound in the presence of excess normal nucleosides, DNA digests (0.5 - 5mg) with [3H]-S6-CMdG were loaded onto the immunoaffinity columns. Up to 2mg of normal nucleoside did not appear to affect the binding of [3H]-S6-CMdG however with larger amounts of DNA some loss of binding was observed (Figure 2.6.3.). 100 90 80 70 60 50 ^ 40 J 30 C/3i 0 1 2 3 4 5 DNA (mg) Figure 2.6.3. [3H]-S6-CMdG binding to 0 6-CMdG immunoaffinity columns in the presence of DNA digests. [3H]-S6-CMdG (29.5ng, lOOOdpm) was eluted through the columns in the presence of increasing amounts digested DNA (0-5mg). 7 0 Chapter 2. Method development Further studies to ascertain if S6-CMdG could be incorporated as an internal standard were not carried out, as it was discovered that on hydrolysis in the elution conditions used (1M TFA (5ml) for the immunoaffinity columns for 0 6-CMdG) S6-CMdG did not hydrolyze cleanly to S6-CMG but gave several peaks detectable by HPLC-fluorescence (Figure 2.6.4). It seemed unlikely that the [3H]-S6-CMdG was not pure as a clean symmetrical peak was obtained on RP-HPLC analysis (Figure 7.1.2.4.2.). § in 0 5 10 15 20 Time (mins) Figure 2.6.4. HPLC chromatograms showing the hydrolysis products of [3H]-S6-CMdG and a standard of S6-CMG. RP-HPLC was performed as stated in the materials and methods chapter 7.1.2.2., system 1. Chromatogram A: hydrolysis products of [3H]-S6- CMdG, chromatogram B: standard S6-CMG. 71 ______Chapter 2. Method development 2.7. HPLC optimisation for 0 6-alkylguanine adducts. In order to gain the maximum sensitivity for the detection of 0 6-CMG and 0 6-MeG adducts following immunoaffinity purification it is essential that the liquid chromatography conditions are optimal for each adduct. This realistically requires the maximum peak height obtainable and hence rapid elution, with separation free from any interfering peaks. Initial work was using the standard RP-HPLC ODS (25cm x 4.5mm) 5pm column which incorporated a 0.1M TEA buffer system with a methanol gradient that had already been used for the isolation of the tritiated adducts in Section 2.2.. Although this gave a limit of quantitation of 2pmol for 0 6-CMG, this was five fold higher for 0 6-MeG and significant tailing of this peak was observed. To improve this sensitivity and chromatography it was decided to employ a RP-HPLC ODS (25cm x 2mm) HPLC column. Narrowbore columns can improve chromatography by allowing the eluate to be concentrated in a smaller volume of HPLC buffer. Thus on detection, the compound of interest should be more concentrated in the flow cell of the fluorescent detector and a narrower, sharper peak with increased height obtained. The flow rate is reduced for narrowbore columns and due to this factor an isocratic elution system was more appropriate as the pump mechanism/mixing chamber of the Waters HPLC system did not give a constant flow at low flow rates if gradient elution was tried. As can be seen from table 2.7.1. and table 2.7.2, this did increase the sensitivity of detection for both DNA adducts of interest. Stationary phases with 3pm diameter silica columns have higher efficiencies and with these columns separations almost equivalent to those obtainable with gradient elution can be achieved isocratically (Lim, 1986). The use of isocratic elution also means the system is simple and able to operate at higher sensitivity as less baseline noise will be produced. As there is no re-equilibration necessary the system has a higher throughput. Thus isocratic elution is ideal when high sensitivity is required for relatively simple separations. A shorter narrow bore column (10cm x 2mm) was then utilised to overcome some of the tailing that was still been observed by the 0 6-MeG peak. It was hoped that due to the shorter length of the HPLC column less retention of the adduct would occur and hence the tailing would be reduced. In order to overcome tailing of peaks the pH of the buffer can be adjusted, though for 0 6-MeG reducing the pH had not had any discernible effect down to pH 5. 7 2 ______Chapter 2. Method development However, the short narrowbore column did reduce peak tailing which in turn led to 100% increase in sensitivity for 0 6-MeG. As the 0 6-CMG peak was not showing any tailing, this short column did not improve the detection of this adduct. Although the limit of quantitation for both adducts was now lpmol using the short narrowbore column, the fluorescence of compounds caq alter with pH. Shuker and Margison (1997) employed a 0.1% HFBA system with methanol elution which is at a low pH. Subsequently the same system was examined on the current HPLC column. This resulted in a substantial 20 fold increase in the limit of quantitation of 0 6-MeG as this compounds natural fluorescence maximum occurs at a low pH (Belinsky et al, 1987). Similarly an increase of 10 fold in 0 6-CMdG detection was observed. This resulted in two HPLC-fluorescence protocols that had limits of quantitation of 0.075pmol and 0.05pmol for 0 6-CMG and 0 6-MeG respectively. Typical chromatograms using these conditions can be seen in Figure 2.7.1. and 2.7.2. HPLC column flow rate Buffer Limit of Retention system Quantitation time RP-ODS C l8 (25cm x lmL/min 0.1M TEA pH7 2pmol 7min 4.6mm) 5pm column methanol gradient RP-BDS C18(25cm x 0.2mL/min 0.1M TEApH7 lpmol 12min 2mm) 3 pm Shandon 10% methanol column RP-BDS C18 (10cm x 0.2mL/min 0.1M TEA pH7 lpmol llm in 2mm) 3 pm Hypersil 10% methanol column RP-BDS C18 (10cm x 0.2mL/min 0.1%aqueous 0.075pmol 9.1min 2mm) 3pm Hypersil HFBA, 10% column MeOH pH2 Table 2.7.1. HPLC optimisation for the 0 6-CMG adduct. 73 mV 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 — 140.00 160.00 — 160.00 — 180.00 RP-BDS C l8 (10cm x x (10cm l8 C RP-BDS 2mm) 3 2mm) Hypersil pm 3 2mm) Hypersil pm x (10cm C18 RP-BDS al ...HL piiainfrte06-MeG adduct. 0 the for optimization HPLC 2.7.2. Table column Figure 2.7.1. HPLC chromatogram for standard 0 6-CMG. RP-HPLC-fluorescence using using RP-HPLC-fluorescence 6-CMG. 0 standard for chromatogram HPLC 2.7.1. Figure column column 3 Shandon 2mm) pm C18(25cmx RP-BDS RP-ODS C18 (25cm x x (25cm C18 RP-ODS .m)5m column 5pm 4.6mm) HPLC column HPLC 0.00 — — a BDS C l8 (10cm x 2mm) 3pm column with 0.1%HFBA and 10% methanol, excitation excitation 10%methanol, and 0.1%HFBA with column 3pm 2mm) x (10cm l8 C a BDS at 286nm, emission at 378nm. at emission 286nm, at 00 .0 5 lwrt Buffer rate flow 0.2mL/min 0.2mL/min 0.2mL/min lmL/min Minutes FA 15% HFBA, MeOHpffi gradient 0.1%aqueous 0.1%aqueous 0.1MTEApH7 0.1MTEApH7 0.1M TEA pH7 TEA 0.1M methanol pH7 TEA 0.1M system 15%methanol 15%methanol 10.00 Limit of of Limit Quantitation 2pmol 0.05pmol lOpmol lpmol Chapter 2. Method development Method 2. Chapter 15.00 time Retention 9.7min 15min 15min 14min Chapter 2. Method development 3 5 0 .0 0 — 3 0 0 .0 0 — 2 5 0 .0 0 — > 200.00 — 1 5 0 .0 0 — 100.00 — 5 0 .0 0 0.00 0.00 5 .0 0 10.00 1 5 .0 0 M i n u t e s Figure 2.7.2. HPLC chromatogram for standard 0 6-MeG. RP-HPLC-fluorescence using a BDS C l8 (10cm x 2mm) 3pm column with 0.1%HFBA and 15% methanol, excitation at 286nm, emission at 378nm. 2.8. Digestion of DNA to nucleosides. In order for the immunoaffinity purification of 0 6-CMdG and 0 6-MedG the DNA needs to be digested to deoxynucleosides. Digestion of DNA to deoxynucleosides was performed as outlined by Beranek et a l (1980), which involves Nuclease and acid and alkaline phosphatases. Beranek et a l (1980) reported that DNA which underwent a neutral thermal hydrolysis, leading to apurinic DNA followed by enzyme hydrolysis released O6- alkyldeoxyguanosine. However, in this method no means of specifically isolating a particular adduct was employed prior to HPLC separation. Hence, the need to generate apurinic DNA prior to DNA digestion and immunoaffinity purification was not considered a necessary step. To this end control calf thymus DNA was enzymatically digested and analysed by RP-HPLC- UV (employing the system detailed in Section 7.1.8.4). As can be seen in Figure 2.8.1., the four normal deoxynucleosides were released cleanly, suggesting that the reaction had gone to 75 mV 0.00 20.00 00 .0 0 4 00 .0 0 6 0 .0 0 8 100.00 120.00 00— 0 .0 0 4 1 a 81% digestion, thus indicating the reaction had not run to completion. to run not had reaction the indicating thus digestion, 81% a quantitated by reference to a 2’dG standard curve (Figure 2.8.2.), and was found to equate equate to to found and was 2.8.2.), (Figure curve standard a 2’dG to reference by quantitated iue281 PCcrmtga flgdgse N.R-PCwso D l8 C a BDS on was RP-HPLC DNA. digested lpg of chromatogram HPLC 2.8.1. Figure completion. To ensure that this was indeed the case, the amount of 2’dG released was released 2’dG of amount the case, the indeed was this that ensure To completion. eeto a i U 20m.Odro lto, ek:2d,pa ’G ek3 : 3peak : 2’dG, 2 peak 2’dC, peakl: elution, of Order (260nm). UV via was Detection 2’dT and peak 4: 2’dA. 4: peak and 2’dT 0.00 (10cm x 2mm) 3pm Hypersil column using 0.1M TEA pH 5 with 5with pH TEA 0.1M using column Hypersil 3pm 2mm) x (10cm — — 00 .0 5 Minutes 76 10.00 Chapter 2. Method development Method 2. Chapter 00 .0 5 1 4% methanol. Chapter 2. Method development 4.5 3.5 s o & 2.5 i 2 t 15 0.5 0 200 400 600 800 1000 2'dG (pmol) Figure 2.8.2. Calibration curve for 2’dG. HPLC conditions are the same as for Figure 2.8.1. r2 = 0.9994, n = 3 ± SD. Hence, the reaction conditions were altered such that the incubation time was prolonged from 8hr to 13hr, and the enzyme concentration increased two fold with the prolonged incubation period. Both of these alterations still resulted in the same liberation of 2’dG as seen in the original digest. In order to ascertain if this was due to incomplete digestion or simply a reflection that the DNA solution (made-up according to weight of calf thymus DNA) was not pure and therefore did not contain the expected amount of DNA, the same solution was acid hydrolysed to its constituent bases. Upon RP-HPLC-UV (260nm) of the acid hydrolysed DNA (Figure 2.8.3.) and with reference to a guanine standard curve (Figure 2.8.4.) a similar yield of 83% of the expected level of guanine was observed. This result confirmed that the enzymatic digestion conditions were appropriate. A 100% yield was probably not achieved due to salt impurities in the calf thymus DNA. To overcome these discrepancies when estimating the amount of DNA in subsequent procedures, 2’dG was routinely analysed for each DNA digest by taking a small aliquot equivalent to lfig of the DNA digest for RP- HPLC-UV analysis. 7 7 Chapter 2. Method development 100.00 — 8 0 .0 0 £ ” 6 0 .0 0 4 0 .0 0 20.00 0.00 0.00 5 .0 0 10.00 Figure 2.8.3. HPLC chromatogram of acid hydrolysed DNA. RP-HPLC was on a BDS C18 (10cm x 2mm) 3pm Hypersil column using 0.1M TEA pH 5 with 4% methanol. Detection was via UV (260nm). Order of elution of peaks, guanine followed by adenine. 14 .. 12 .. 3 1 0 .. 2 .. 0 100 200 300 400 500 600 700 800 900 1000 guanine (pmol) Figure 2.8.4. Calibration curve for guanine. HPLC conditions are the same as for Figure 2.8.1. r2 = 0.9918 78 Chapter 2. Method development 2.9. Sample preparation prior to HPLC analysis. 2.9.1. 0 6-CMdG immunoaffinity eluate In Section 2.4. it was discovered that the elution conditions of the 0 6-CMdG immunoaffinity columns, 1M TFA, led to hydrolysis of the deoxynucleoside to the corresponding base. For the method to be quantitative this hydrolysis needed to be driven to completion by heating the eluate prior to HPLC analysis. To investigate what conditions would be appropriate, standard Oe-CMdG was added to 1M TFA and aliquots containing lOpmol were removed over a time course while the solution was heated at 50°C and immediately frozen in liquid nitrogen, to avoid further hydrolysis, and then freeze-dried. HPLC-fluorescence of the samples and the subsequent quantitation from an 0 6-CMG standard curve revealed that after 60min a 92% recovery of Oe-CMG was obtained. There were no other discernible peaks, most notably O6- CMdG and if the reaction was left for a further hour no further increase in yield was seen (Figure 2.9.1.). If this was decarboxylation no 0 6-MeG would be seen. However, the methyl adduct is labile and will undergo demethylation in strong acid conditions. Thus, hydrolysis for 60min at 50°C and immediate freezing in liquid nitrogen to avoid further hydolysis and freeze-drying prior to HPLC-fluorescence analysis were the optimum conditions found. 100 80 2 70 £ 60 O 50 ^3 40 S 30 VOV 20 O 10 0 20 40 60 80 100 120 time (min) Figure 2.9.1. Time course for the hydrolysis of 0 6-CMdG to 0 6-CMG. Single determination at each time point for the hydrolysis of 0 6-CMdG in 1M TFA at 50°C. Quantitation was via RP-HPLC fluorescence (detailed in section 7.1.7.1., system 4). 7 9 ______Chapter 2. Method development 2.9.2. 0 6-MedG immunoaffinity eluate As the eluate was in 4mL of 80% aqueous methanol this would be dried down in a speedvac and hydolysed to the base. Beranek et a l (1980) indicated that mild acid hydrolysis should liberate the base without causing demethylation of the adduct. A time course to investigate the appropriate conditions for hydrolysis to 0 6-MeG using RP-HPLC fluorescence (0.1% HFBA, 15% methanol) was undertaken and the results can be seen in Figure 2.9.2.. A quantitative hydrolysis occurred after 30min at 50°C in 0.1M HC1 when the sample had been re-dried and re-dissolved for injection in a 0.1% HFBA solution. The volumes of 0.1 M HC1 was kept to the minimum to speed the drying process and to reduce the possibility of further hydrolysis and possible demethylation. 100 90 80 s 30 20 0 5 152 0 30 Time (mins) Figure 2.9.2. Time course for the hydrolysis of 0 6-MedG to 0 6-MeG. Single determination at each time point for the hydrolysis of 0 6-MedG in 0.1M HC1 (IO jjL) at 50°C for up to 30min. Quantitation was via RP-HPLC-fluorescence ( detailed in section 7.1.7.2. system 4). 2.10. Method validation Having developed a possible purification protocol for both the adducts of interest, it was then necessary to determine that the conditions allowed for the specific isolation of both adducts from a DNA digest and that no other component would interfere with the chromatography. This proposed analytical method must then be validated. Initially the examination of the 80 ______:______Chapter 2. Method development ability of the 0 6-CMdG and 0 6-MedG immunoaffinity columns to isolate the relevant adduct from DNA digests and the determination of how much DNA could be loaded onto an immunoaffinity column before recovery was affected was undertaken. Samples of DNA (0.5- 3mg) with either [3H]-06-CMdG or [3H]-06-MedG were digested and passed through the immunoaffinity columns. The eluate fractions were collected and the radioactivity retained on the immmunoaffity column determined. No decrease in binding was seen up to this level of DNA loading (Figure 2.10.1. and 2.10.2.). 0 0.5 1 1.5 2 2.5 3 DNA (mg) Figure 2.10.1. Binding of [3H]-06-CMdG to IAC in the presence of digested DNA. Triplicate determination ± sd of [3H]-06-CMdG (17.2ng, 950dpm) bound to IAC in the presence of increasing amounts of digested DNA (0-3mg). 100 V? 90 80 4) 70 60 O 50 XI Figure 2.10.2. Binding of [3H]-06-MedG in the presence of digested DNA. Triplicate determination ± sd of [3H]-06-MedG (13.45ng, 900dpm) bound to IAC in the presence of increasing amounts of digested DNA (0-3mg). ______Chapter 2. Method development In order to ensure that when DNA digests were passed through the immunoaffinity columns and analysed by HPLC and that no interfering peaks were generated, control DNA digests were passed through the immunoaffinity columns, prepared as samples and injected onto the HPLC system. No peaks were seen in the chromatographic area for 0 6-CMG or 0 6-MeG. To demonstrate that there was a reliable baseline resolution for both adducts, DNA samples (2.5mg) containing either a) 0 6-CMdG (0-50pmol), b) 0 6-MedG (0-50pmol) or c) both O6- CMdG and 0 6-MedG (0-50pmol) were digested, passed through the first immunoaffinity column (0 6-CMdG) which eluted directly on to the second immunoaffinity column (O6- MedG) and washed with 3mL PBS/azide. The columns were then separated for independent washing and elution (Figure 2.10.3.). The eluate fractions were collected and prepared for HPLC analysis, where injection volumes containing the equivalent of lmg DNA were analysed in duplicate. For DNA solutions a) and c), a linear response was determined for 0 6-CMG between 0- 20pmol with a zero intercept. On comparison to a standard 0 6-CMG calibration curve the recovery was determined to be 59% with a limit of quantitation of 0. lpmol (Figure 2.10.4.). Similarly DNA solutions b) and c) gave a linear response for 0 6-MeG between 0-15pmol, with a zero intercept, a recovery of 79% and a limit of quantitation of 0.05pmol (Figure 2.10.5.) For the blank immunoaffinity runs in solutions a) and b) no additional peaks from background DNA were observed indicating that each immunoaffinity column did specifically recover one DNA adduct from the digested DNA. Typical RP-HPLC-fluorescence chromatograms from spiked DNA which was digested, immunoaffinity purified and analysed by HPLC can be seen in Figures 2.10.6 and 2.10.7. 82 Chapter 2. Method development j m DNA containing O’-GYHG and if- w MedG adducts ^s w%jr J ^ Enzymatic digestion ^ ^ ^ u t i o n 0M3VMG affinity coiuim O-M edG Elution affinity col uni Figure 2.10.3. Overall scheme for immunoaffinity purification of 0 6-CMdG and O6- MedG from DNA digests. 83 Chapter 2. Method development 100 T 90 .. 80 .. 70 .. 60 -- 50 .. 30 .. 20 .. 10 .. 0 6-CMG (pmol) Figure 2.10.4. Calibration curves for 0 6-CMG. Os-CMG purified from DNA digests (red), linear regression (r2) of 0.9997, standard 0 6-CMG (blue), r2 = 0.9977, n = 3 ± SD. 160 T 140 120 3 to % x100 80 s i 1 * 20 6 8 10 0 6-MeG (pmol) Figure 2.10.5. Calibration curves for 0 6-MeG. 0 6-MeG purified from DNA digests (red squares), linear regression (r2) of 0.999, standard 0 6-MeG (blue circles), r2 = 0.999, n = 3 ± SD. A reproducibility study where DNA digests (2.5mg) containing 25pmol of both 0 6-CMdG and 0 6-MedG demonstrated that this method was reproducible in that a lOpmol standard of 84 0.00 mV 20.00 40.00 60.00 80.00 0.00 MedG gave 7.96 ± 0.24pmol 0 6-MeG (n = 6), 2.97% coefficient of variance. of coefficient 2.97% 6), 6-MeG = (n 0 0.24pmol ± 7.96 gave MedG Chapter 2.Method development Figure 2.10.6. Typical RP-HPLC chromatogram for 0 6-CMG isolated from DNA. RP- DNA. from isolated 6-CMG 0 for chromatogram RP-HPLC Typical 2.10.6. Figure 0 6-CMdG resulted in 5.92 ± 0.17 0 6-CMG (n = 6), 2.87% coefficient of variance and O6- and variance of coefficient 2.87% 6-CMG6), = (n 0 0.17 ± 5.92 in 6-CMdG resulted 0 with 10% methanol. Detection was via fluorescence (excitation 286nm, emission emission 286nm, (excitation fluorescence via was Detection 10%methanol. with HPLC was on a BDS Cl 8 (10cm x 2mm) 3pm Hypersil column using 0.1% HFBA pH 2 pH HFBA 0.1% using column Hypersil 3pm 2mm) x (10cm 8 Cl a BDS on was HPLC corresponds to 5pmol recovered from lmg of DNA. of lmg from recovered 5pmol to corresponds and 6-CMGindicated is peak 0 The 4. system 7.1.7.1. section in detailed as 378nm) 00 .0 5 Minutes 10.00 85 00 .0 5 1 mV 20.00 0.00 40.00 100.00 120.00 60.00 80.00 140.00 — 140.00 0.00 — Figure 2.10.7. Typical RP-HPLC chromatogram for 0 6-MeG isolated from DNA. RP- DNA. from 6-MeG isolated 0 for chromatogram RP-HPLC Typical 2.10.7. Figure HPLC was on a BDS C l8 (10cm x 2mm) 3jLim 2mm) x 2(10cm pH HFBA 0.1% l8 using column C Hypersil a BDS on was HPLC corresponds to 3 pmol recovered from lmg of DNA. of lmg from 3 recovered to corresponds pmol emission 286nm, (excitation fluorescence via was Detection methanol. 15% with 378nm) as detailed in section 7.1.7.2. system 4. The 0 6-MeG peak is indicated and 6-MeG indicated is peak 0 The 4. system 7.1.7.2. section in detailed as 378nm) 10.00 Minutes 86 ChapterMethod 2. development 20.00 Chapter 2. Method development 2.11. Discussion. The enzyme catalysed synthesis of tritiated deoxynucleosides was undertaken to generate marker compounds for the characterisation of immunoaffinity columns. This approach is advantageous as it does not require tedious protection and deprotection measures or complex purification and chromatographic procedures. The synthesis of three tritiated deoxynucleoside derivatives was achieved, 0 6-CMdG, S6-CMdG and 0 6-MedG. However, only the latter gave a high yield (74%). Although sufficient quantities of all of these deoxynucleoside derivatives were purified for use as marker compounds in immunoaffinity procedures, the low yields of [3H]-06-CMdG and [3H]-S6-CMdG could be a problem if this synthesis was required to be used as a routine method. High yields for purine substituted in the 6-position with small alkyl groups have been reported by Chapeau and Mamett (1991) which does suggest that this type of compound should be a good substitute for purine nucleoside phosphorylase (PNPase). However, the catalytic efficiency might be a reflection not only of the ability of analogues to serve as substrates but also of their ability to stabilise the catalysts. It has also been proposed that enhanced stabilisation of the PNPase could occur with the enzyme, phosphate and purine base forming a “dead-end” complex (Krenitsky et al, 1981). Another factor that is known to affect yields is the phosphate concentration. Phosphate stabilises PNPase and TPase, but at high concentrations has an undesirable effect on the yield of the reaction. However, if the concentration is too low the time required to reach equilibrium would be significantly increased. Immunopurification procedures offer a rapid, selective method for the isolation of purified material for further analysis. Immunoaffinity columns were prepared on a batch basis for either 0 6-CMdG and 0 6-MedG and found to efficiently bind the appropriate deoxynucleoside derivative in the presence of excess normal deoxynucleosides. The capacities of the O6- CMdG columns (-lnmol) and 0 6-MedG columns (-2nmol) were found to be comparable with those prepared using antibodies against other DNA adducts (Friesen et al, 1991, Prevost et al, 1990 and Durand & Shuker, 1994). It is interesting to note that in these studies we have generated immunoaffinity columns using both monoclonal and polyclonal antibodies which both show good selectivity and capacity but with the monoclonal antibodies having the 87 ______Chapter 2. Method development advantage of continuous availability. 0 6-CMdG antibody has an apparent high affinity which was illustrated by the rather drastic conditions (1M TFA) required to elute 0 6-CMdG. This may be due to the fact that the polar (charged) carboxymethyl group is very strongly bound in the antigen-binding site of the antibody molecule as a result of a combination of van der Waals and electrostatic interactions. The behavior of polyclonal immunoaffinity columns is also dominated by the highest affinity subset of antibodies because this subset must be disrupted before all the antigen can be released. The elution curve for the 0 6-CMdG (polyclonal) columns has a sharp leading edge and a tailing edge, indicating that there are some higher affinity interactions. The situation with monoclonal antibodies is completely different. The homogenous nature of the interactions means that once appropriate elution conditions are found, the peak should have minimal tailing. This was found to be the case for the 0 6-MedG columns and the elution conditions were milder (80% aqueous methanol). The severe elution conditions for 0 6-CMdG from immunoaffinity columns gave rise to partial hydrolysis to Os-CMG. However, since the free base is slightly more fluorescent (10%) than the 2’deoxynucleoside (Gaffney & Jones, 1982), this proved advantageous for the sensitivity of the assay : the hydrolysis was driven to completion prior to evaporation and HPLC analysis. As the elution conditions were so severe compared to that for the 0 6-MedG columns it would be inappropriate to combine the two affinity gels as demethylation of the more labile 0 6-MedG adduct would be likely to occur. However, by allowing the eluate from one column to drip directly onto the second type of column followed by separate elution, the concomitant determination of both 0 6-CMdG and 0 6-MedG from a sample was still possible. In order to gain maximum sensitivity the 0 6-MedG eluate was also mildly acid hydrolysed to the more fluorescent base prior to HPLC analysis. The incorporation of an internal standard for the 0 6-CMdG determination in DNA was attempted with N2-amino-S6-(carboxymethyl)-mercapto -2’-deoxyguanosine (S6-CMdG). This would have allowed a reliable estimate of recovery of 0 6-CMdG to be made for every sample, rather than comparing calibration curves. Although S6-CMdG was retained by the 0 6-CMdG immunoaffinity columns (albeit with less affinity than for 0 6-CMdG), on hydrolysis, S6-CMdG forms various products which would interfere with the HPLC- fluorescent analysis of 0 6-CMG and quantitation of the internal standard itself would be impossible. 88 ______Chapter 2. Method development The method was validated using DNA samples spiked with 0 6-CMdG and 0 6-MedG, which were digested, immunopurified and analysed by HPLC-fluorescence. Good linear calibration curves were generated and the recovery of the adducts was estimated to be 59 and 79% for 0 6-CMdG and Ofi-MedG respectively. The limit of quantitation of the immunoafflnity-HPLC fluorescence assay was O.lpmol 0 6-CMG/injection and 0.05pmol Os-MeG/injection. If lmg of DNA hydrolysate was used per injection, the limit of detection of this assay corresponded to 0.128pmol Os-CMdG/mol dG and 0.064|imol 0 6-MedG/mol dG. This level of sensitivity is suitable for mechanistic experimental studies on the concomitant formation of both adducts by proposed carboxymethylating and methylating agents, as amounts of DNA for analysis are fairly large (>0.1mg). This method offers a reliable, reproducible, relatively sensitive and rapid procedure for the concomitant determination of two adducts from a DNA sample. Other assays that are available for the determination of 0 6-MedG have been summarised in Section 1. 4.5.. From these only a few would have been applicable for the concomitant determinant of 0 6-CMdG and 0 6-MedG. Two of these assays would have required the routine use of highly radioactive constituents, a radioimmunoassay (Umbenhauer et al, 1985) or 32P-postlabelling (Povey & Cooper, 1995) and would still require prepurification prior to final analysis. 32P-postlabelling requires the deoxyribonucleoside 3’-monophosphates for labeling, hence the synthesis of adducted standards would have needed to be undertaken and may have proved arduous for the carboxymethyl adduct. Ideally, for a specific and sensitive32P-postlabelling assay an immunoaffinity purification step is incorporated prior to postlabelling (Povey & Cooper, 1995). However, as has been stated already hydrolysis of 0 6-CMdG occurs during its elution and thus this approach could not have been taken. ELISA procedures would have been unlikely to show any improvement in sensitivity than the method developed here (Foiles et al, 1988) and HPLC-electrochemical detection (Groot et al, 1994) has a similar order of detection limit but requires another HPLC purification step which would be time consuming. 89 Chapter 3. In vitro studies. 90 Chapter 3. In vitro studies. 3.1. Introduction. Alkylation of DNA is considered to be a key step in the induction of cancer by different chemicals (Miller, 1978). For many compounds including N-alkyl-N-nitrosocompounds, alkylation at O6 of 2’-deoxyguanosine (dG) appears to be the major mutagenic lesion, although 0 4-alkylthymidines may also be mutagenic (Saffhill et al , 1985). In both prokaryotic and eukaryotic cells, efficient specific repair mechanisms exist for the removal of 0 6-MedG residues as well as some higher homologues, albeit more slowly (Karran & Lindahl, 1985). Among the many N-alkyl-N-nitroso compounds that are known to be carcinogenic, there are a number which are derived from the simplest amino acid, glycine and share a common feature of being carboxymethylating agents. N-nitrosoglycocholic acid (NOGC) is a carcinogenic and mutagenic derivative of the naturally occurring bile acid conjugate, glycocholic acid (Shuker et al, 1981, Song et al, 1982, Busby et al, 1985). NOGC has been shown to be a carboxymethylating agent in vitro giving rise to a range of carboxymethyl DNA adducts, including 0 6-CMdG (Shuker et al, 1987, Shuker & Margison, 1997). N- nitrosopeptides which are C-terminal in glycine, such as N-(N’ -acetyl-L-prolyl)-N- nitrosoglycine (APNG) are mutagenic and carcinogenic (Challis, 1989, Anderson & Blowers, 1994) and would be expected to be carboxymethylating agents by analogy with NOGC. Azaserine (O-diazoacetyl-L-serine), a pancreatic carcinogen (Longnecker et al, 1981) is also known to carboxymethylate DNA in vivo (Zurlo et al, 1982). In studies on the formation of DNA adducts by NOGC it has been observed that in an analogous manner to many N-nitroso compounds, NOGC gives rise to alkylation mainly at purine nitrogen atoms (Shuker & Margison, 1997). Although the formation of the carboxymethylated bases (0 6-CM and N7-carboxymethylguanine) in DNA was anticipated from the decomposition mechanism of NOGC the concomitant methylation (0 6-methyl and N7-methylguanine) of DNA observed by Shuker & Margison (1997) suggested that this type of carboxymethylating agent could also decompose to generate a methylating species. It was therefore of interest to see if DNA methylation was a common property of a range of carboxymethylating agents, and to gain an insight into the possible mechanism underlying the formation of the proposed carboxymethyldiazonium ion and methyldiazonium ion. 91 ______Chapter 3. In vitro studies. 3.2. Concomitant formation of 0 6-CMdG and 0 6-MedG by N- carboxymethyl-N-nitrosocompounds and diazoacetic acid derivatives. 3.2.1. Analysis of 0 6-CMG and Ofi-MeG in DNA. The immunoaffinity-HPLC-fluorescence protocol detailed in Section 7.2.4. was used for the analysis of 0 6-CMG and 0 6-MeG in DNA which had been treated with various carboxymethylating agents. Enzymatic hydrolysates of treated DNA were applied first of all to one immunoaffinity column and the eluate from that column passed directly onto the second immunoaffinity column. The two columns were then washed and eluted separately and the 0 6-CMG- and 0 6-MeG- containing extracts analyzed using different HPLC conditions. Typical RP-HPLC-fluorescence chromatograms for both adducts are shown in Figures 3.2.1.1. and 3.2.1.2. and were free from interfering peaks. Standard calibration curves for the quantitation of 0 6-CMG and 0 6-MeG were prepared as in Section 7.1.10.2. For the quantitation of normal nucleosides lpL aliquots of the enzymatic hydrolysate were separated on RP-HPLC and 2’-dG quantitated. 1 0 0 .0 0 — 80.00 60.00 % 40.00 20.00 0.00 0.00 5 .0 0 10.00 15.00 Minutes Figure 3.2.I.I. RP-HPLC chromatogram of DNA treated with KDA and the determination of 0 6-CMG. HPLC conditions as in Section 7.1.7.1., system 4. The upper trace is of an aliquot of DNA (25pg) treated with 5mM KDA, the lower trace of an equal quantity of untreated DNA. 92 Chapter 3. In vitro studies. 25.00 — 20.00 — 15.00 — £ 10.00 — 5.00 0.00 -5 .0 0 — -10.0Q- 0.00 5 .0 0 10.00 15.00 Minutes Figure 3.2.I.2. RP-HPLC chromatogram of DNA treated with KDA and the determination of 0 6-MeG. HPLC conditions as in Section 7.1.7.2, system 4. The lower trace is of an aliquot of DNA (50fig) treated with 5mM KDA, the upper trace of an equal quantity of untreated DNA. 3.2.2. 0 6-alkyIation by nitrosated glycine derivatives. Calf thymus DNA was treated with a range of concentrations of potassium diazoacetate (KDA) (0-5mM), APNG (0-5mM) and azaserine (0-10mM) at pH 7.4. Each dose level was incubated with DNA in triplicate and duplicate HPLC injections from each triplicate were analysed (Table 3.2.2.). In all cases dose dependent increases in the levels of 0 6-CMG and 0 6-MeG were seen Figures 3.2.2.1.-3.2.2.3. 93 Chapter 3. In vitro studies. Sample 0 6MeG 0 6CMG pmol/mol2'dG |imol/mol2’dG mean ± sd mean ± sd nd nd Control nd nd DNA nd nd KDA 0.5mM 3.07 57.13 3.84 3.31 ± 0.45 63.37 62.11 ± 4.49 3.03 65.86 ImM 12.48 204.08 11.25 12.04 ± 0.68 264.89 220.99 ± 38.34 12.38 194.01 2.5mM 25.26 479.21 26.25 25.84 ± 0.52 448.76 496.0 ± 58.02 26.02 562.04 5mM 69.16 948.21 54.16 60.74 ± 7.66 960.79 979.61 ± 44.01 58.90 1030.01 Azaserine ImM nd 1.44 nd 1.42 1.43 ± 0.01 nd 1.44 2.5mM nd 2.17 nd 3.47 2.59 ± 0.76 nd 2.12 5mM 0.18 7.68 0.26 0.19 ± 0.06 6.98 7.32 ± 0.35 0.13 7.31 lOmM 0.34 10.86 0.41 0.34 ± 0.07 15.23 12.69 ± 2.26 0.27 11.99 APNG 0.5mM 0.94 5.93 1.01 0.89 ± 0.13 5.76 5.96 ± 0.21 0.75 6.18 ImM 2.41 14.24 2.53 2.44 ± 0.08 15.08 14.77 ± 0.46 2.39 14.99 2.5mM 3.43 42.78 3.38 3.93 ± 0.91 37.96 39.93 ± 2.53 4.98 39.04 5mM 8.61 73.60 8.75 8.47 ± 0.37 81.52 82.44 ± 9.33 8.05 92.22 nd - < 0.07jj.mol/mol2’dG Table 3.2.2.I. 0 6-Guanine alkylation by various nitrosated glycine derivatives in calf thymus DNA at pH 7.4. 94 Chapter 3. In vitro studies. 1200 1000 - J g 800 ^4 CN 13 ’o o> g 600 s ° 3 400 200 - 1 2 3 4 5 KDA concentration (mM) Figure 3.2.2.I. 0 6-Guanine alkylation by KDA in calf thymus DNA at pH 7.4. The solid blue line represents 0 6-CMG formation, r2 = 0.997 and the dotted green line 0 6-MeG, r2 = 0.9929, n = 3 ± SD. 14 .. 4 -- 0 2 4 6 8 10 Azaserine concentration (mM) Figure 3.2.2.2. 0 6-Guanine alkylation by Azaserine in calf thymus DNA at pH 7.4. The solid blue line represents 0 6-CMG formation, r2 = 0.9887 and the dotted green line O6- MeG, r2 = 0.8984, n = 3 ± SD. 95 Chapter 3. In vitro studies. 100 I O 20 0 1 2 3 4 5 APNG concentration (mM) Figure 3.2.2.3. 0 6-Guanine alkylation by APNG in calf thymus DNA at pH 7.4. The solid blue line represents 0 6-CMG formation, r2 = 0.9992 and the dotted green line 0 6-MeG, i2 = 0.9875, n = 3 ± SD. Interestingly the relative amount of 0 6-alkylation (06-CMG plus 0 6-MeG) formed from these nitrosated glycine derivatives show that there are large differences in their capacity to alkylate DNA. The formation ratios observed for 0 6-alkylation are 1 : 12.1 : 137.9 for 5mM azaserine, APNG and KDA respectively. The relative capacity for each compound to generate 0 6-alkylation will depend on the ease with which it decomposes or rearranges to the ultimate reactive species. As KDA merely needs to be protonated to generate the carboxymethyldiazonium ion it is not surprising that it produces significantly more 0 6-alkylation than azaserine or APNG. Carboxymethylation by azaserine in vivo is thought to occur by the enzymatic a,p-elimination of diazoacetic acid (Zurlo et al, 1982). In this current in vitro study the liberation of diazoacetic acid, which is highly reactive, is solely dependent on spontaneous chemical decomposition of the original compound which explains why low levels of 0 6-CMG and 0 6-MeG were observed. Similarly for APNG, this compound needs to undergo either decomposition or rearrangement before 96 Chapter 3. In vitro studies. the ultimate reactive species, carboxymethyl diazonium ion or methyl diazonium ion (diazomethane), are produced. The relative proportions of methyl to carboxymethyl adducts formed also varied for each compound studied (Table 3.2.2.2.). This was of interest as although the 0 6-CMG is formed, presumably by the reaction of the carboxymethyl diazonium ion with DNA, the exact route via which methylation also occurs needed to be investigated. Compound 0 6-M eG /06-CMG ratio KDA 16.12 Azaserine 38.54 APNG 9.73 Table 3.2.2.2. Ratios of 0 6-MeG to 0 6-CMG formation in calf thymus DNA from a range of nitrosated glycine derivatives. 3.3. Mechanistic studies on O6~alkylation. In order to investigate the mechanism by which concomitant methylation and carboxymethylation occur, DNA was treated with KDA at different pH values and the amount of 0 6-CMG and 0 6-MeG produced determined in triplicate. As can be seen in Figure 3.3.1., 0 6-MeG levels increased linearly with increasing pH. However, the level of 0 6-CMG formation showed no alteration with differing pH values (Figure 3.3.2.). This therefore resulted in the ratio of 0 6-MeG / 0 6-CMG increasing with increasing pH (Figure 3.3.3.). Calf thymus DNA was also treated with mesyloxyacetic acid, a compound that is expected to carboxymethylate DNA, but is structurally different to the other carboxymethylating agents (KDA, azaserine and APNG) as it does not contain the diazo function (and hence diazonium ion). Mesyloxyacetic acid (5mM) produced 0.63 ± O.Olpmol 0 6-MeG/mol 2’dG and 11.45 ± 0.87jjjnol 0 6-CMG / mol 2’dG. This corresponds to a 0 6-MeG / Oe-CMG ratio of 18.15. 97 Chapter 3. In vitro studies. 80 T ’T3O 70 " 6 6.5 7 7.5 8 8.5 9 9.5 10 pH Figure 3.3.1. The formation of 0 6-MeG in calf thymus DNA by KDA at varying pH. r2 = 0.9843. n = 3 ± SD. 1200 T S' 1000 3 S 800 .. 3 £ 600 .. '•—'r t o 400 -- S u1 200 .. VO o o -I------1------1------1------1------1------1------1------1 6 6.5 7 7.5 8 8.5 9 9.5 10 PH Figure 3.3.2. The formation of 0 6-CMG in calf thymus DNA by KDA at varying pH. tz =0.0819, n = 3 ±SD. 98 Chapter 3. In vitro studies. 0.08 0.075 0.07 0.07 O s 006 . 0.05 o 0.04 9 0.03 V 0.02 0 pH 6.5 pH 7.5 pH 8.5 pH 9.5 pH Figure 3.3.3. The ratios of formation of 0 6-MeG /0 6-CMG in calf thymus DNA by KDA at varying pH. 3.4. Discussion. The utility of the combination of immunoaffmity purification of Oe-CMdG and 0 6-MedG with HPLC fluorescence is illustrated by the analysis of 0 6-CMG and 0 6-MeG in calf thymus DNA treated with APNG, a model nitrosopeptide which is a potent mutagen and carcinogen (Anderson & Blowers, 1994), Azaserine, a potent pancreatic carcinogen (Zurlo et al, 1982), KDA and mesyloxyacetic acid. KDA is particularly interesting because it is a simple nitrosated derivative of glycine, one of the most common amino acids. The advantage of using immunoaffmity purification prior to HPLC fluorescence, compared to no purification, is shown in Figure 3.4.1. Fluorescence due to the normal DNA bases interfered substantially with the quantitation of 0 6-CMG and 0 6-MeG. The results presented in this chapter show that concomitant methylation and carboxymethylation at O6 of guanine in DNA appears to be a general property of N- carboxymethyl-N-nitrosocompounds (APNG and NOGC) as well as diazoacetic acid derivatives (KDA and azaserine). All of these compounds would be expected to decompose to give rise to a common alkylating intermediate, the carboxymethyl diazonium ion. The structures of all these compounds can be seen in Figure 3.4.2. 99 mV - 20.00 0.00 00 — 40.00 100.00 00 — 60.00 — 80.00 120.00 140.00 — 140.00 —| 0 .0 0 6 1 180.00 — 180.00 20.00 Figure 3.4.2. Structures of APNG, Azaserine, KDA, mesyloxyacetic acid and the the and acid mesyloxyacetic KDA, Azaserine, APNG, of Structures 3.4.2. Figure Figure 3.4.1. HPLC chromatograms of 0 6-CMG in calf thymus DNA treated with KDA KDA with treated DNA thymus calf 6-CMG in 0 of chromatograms HPLC 3.4.1. Figure — 0.00 — — carboxymethyldiazonium ion. carboxymethyldiazonium (5mM). DNA was either acid hydrolyzed (10% HFBA, 100°C, 30min) and injected injected and 30min) 100°C, (10% HFBA, hydrolyzed acid either was DNA (5mM). line). In both cases 25jng of DNA was injected onto the HPLC column. HPLC the injected onto was DNA 25jng cases of both In line). directly (solid line) or enzymatically hydrolyzed and immunoaffmity purified (broken (broken purified immunoaffmity and hydrolyzed enzymatically or line) (solid directly c O Ac M e s y l o x y a c e t i c a c i d APING 00 .0 5 OH c n r b o x y m c t l i y l d i n z o i i i i i u i i o n Minutes 10.00 Azaserine 100 o Po t a ssium < l i ; i / . o : i c c t : i l c N II 5. 0 .0 15 O K+ Chapter 3. In vitro In studies. Chapter 3. In vitro studies. The detection of 0 6-MeG in DNA after treatment with agents which generate the carboxymethyldiazonium ion could be explained by a decarboxylation mechanism (Figure 3.4.3.). Some evidence for this comes from the measurement of the 0 6-MeG / 0 6-CMG ratios after the reaction of KDA with DNA at different pH value. It was observed that the ratios of 0 6-MeG /0 6-CMG increase with increasing pH, this was due to an overall increase in the amount of methylation indicating that the generation of the methyldiazonium ion was favored at alkaline pH which would be expected in a decarboxylation mechanism. In contrast, the rate of protonation of diazoacetate to give the carboxymethyldiazonium ion would be relatively constant over this pH range (Kreevoy & Konasewich, 1970) giving rise to the observed constant level of 0 6-CMdG. At a higher pH, KDA is more stable and this may result in a prolonged half-life of the carboxymethyldiazonium ion, thus this may possibly allow for its subsequent decarboxylation and the generation of the methyldiazonium ion. However, the fact that the different compounds studied yield different amounts of methyl and carboxymethyl adducts on reaction with DNA, may indicate several intermediates that are not in rapid equilibrium, and that these arise to varying degrees from the various precursors exist (Figure 3.4.3.). The partition of these intermediates between carboxymethylation and methylation reactions to different extents may therefore explain the different alkylation patterns observed. Interestingly this decarboxylation pathway is not limited to diazoacetic acid derivatives since mesyloxyacetic acid also gave rise to 0 6-CMdG and 0 6-MedG. Mesyloxyacetic acid was studied as an example of a SN2-type alkylating agent which may be expected to result in the formation of low levels of 0 6-CMdG. However, 5mM mesyloxyacetic acid produced less O6- alkylation than KDA, but a similar amount to that formed by ImM APNG, which is consistent with its potent mutagenicity (Osterman-Golkar et al, 1970) and suggests that the electrophilic carbon in mesyloxyacetic acid has a similar reactivity to the carboxymethyldiazonium ion. 101 Chapter 3. In vitro studies -senne •s N N HO I NO O" AS fic DNA DNA Q j y y ° I I ™ ° HO APNG -senne DNA c^sop-o^coo- CHjSop-c^ -CO- MAA Figure 3.4.3. Possible mechanisms of formation of 0 6-CMdG and 0 6-MedG in DNA from carboxymethylating agents . AS-azaserine, MAA-mesyloxyacetic acid. The finding that nitrosated glycine derivatives give rise to DNA methylation, in particular the promutagenic lesion 0 6-MedG, may be of major significance in our understanding of the aetiology of cancers of the gastrointestinal (GI) tract (Margison & O’Connor, 1990). A number of N-nitroso compounds are known to induce cancer of the stomach after oral administration to rodents and dogs, including N-methyl-N’-nitro-nitrosoguanidine (MNNG) and N-nitroso-N-methylurea (MNU) (Sugimura & Kawachi, 1973, Preussmann & Stewart, 1984). These are agents which either spontaneously generate methylating agents or do so after metabolic activation and result in the formation of DNA methyl adducts in the target organs of treated animals (Preussmann & Stewart, 1984). In combination with this experimental data there are a number of reports that 0 6-MedG levels, in either the gastric mucosa or in blood leukocyte DNA, are raised in human subjects at elevated risk of gastric cancer (Eurogast, 1994, Povey & Cooper, 1995 and Kyrtopoulos e ta l , 1990), which suggests that methylating 102 ______Chapter 3. In vitro studies. agents may be involved in the aetiology of this cancer. Furthermore, studies have indicated that intragastric nitrosation (Xu & Reed, 1993) and intraintestinal nitrosation (Bingham et al , 1996) of dietary precursors may be a significant source of alkylating agents. Although the ingestion of some preformed N-nitroscompounds does occur depending on an individual’s diet (Startin, 1996, Hotchkiss, 1989), the endogenous nitrosation of dietary amino acids and peptides is a more likely reaction due to their high gastric concentrations (Shepard & Lutz, 1989, Challis, 1989). As glycine is one of the most abundant amino acids in nature it would seem likely that its nitrosation products would constitute a major source of alkylating agents. Thus, the observation that nitrosated glycine derivatives, either N- nitrosopeptides or diazoacetic acid derivatives, decompose to yield DNA-methylating agents lends support to these hypotheses. Of additional interest is the fact that the major O6 guanine adduct generated by these nitrosated glycine derivatives, 0 6-CMG, is not repaired by 0 6-alkylguanine alkyl transferase (Margison & Shuker, 1997), and thus may accumulate in the DNA of GI tract tissues. 103 Chapter 4. In vivo animal studies. 104 ______Chapter 4. In vivo animal studies. A 4.1. Introduction. We have recently shown that KDA a nitrosated derivative of glycine, the simplest and most abundant a - amino acid, not only forms the expected carboxymethylated DNA adducts but also results in DNA methylation (Shuker & Margison, 1997, Harrison et al, 1997). Similarly the known carcinogenic and mutagenic N-nitrosopeptide APNG (Blowers & Anderson, 1988, Anderson & Blowers 1994), also shows concomitant carboxymethylation and methylation in vitro albeit to a lesser extent than KDA (Chapter 3). Both routes of adduct formation are particularly interesting because 0 6-CMdG is resistant to repair by 0 6-alkylguanine alkyltransferase (Shuker & Margison, 1997) and is likely to be persistent and potentially mutagenic, and the concomitant formation of 0 6-MedG, a known promutagenic lesion in DNA, has been proposed as the most likely initiating event in carcinogenesis by methylating agents (Pegg, 1984). The object of the work presented in this chapter was therefore to determine the extent and persistence of 0 6-CMdG formation in the gastrointestinal tract and non-target tissue of experimental animals with nitrosated glycine derivatives by gastric intubation. In order to investigate the presence of 0 6-CMdG in samples obtained from in vivo studies it was deemed necessary to develop a sensitive assay system that would require only small amounts of DNA. This method could then be validated by the experimental animal studies and would then have the potential for molecular dosimetry or epidemiology studies in human DNA samples where the amount of DNA available would be less than 50jj.g. To this end, the immunoslot blot technique described by Nehls et al , 1984 appeared to be particularly useful. This assay requires very small amounts of DNA and can be applied for the detection and quantitation of any heat-stable DNA adduct providing an appropriate antibody is available (Nehlset al, 1984, Ludeke & Kleihues, 1988). A schematic representation of the immunoslot blot assay can be seen in Figure 4.1. Modified DNA is first heat-denatured to yield single stranded DNA which can then be immobilised on nitrocellulose filters. The immobilised DNA is then firstly reacted with an antibody specifically directed against a particular modified DNA component, such as an alkyl- deoxynucleoside, and thereafter with a second antibody directed against the first one. The 105 Chapter 4. In vivo animal studies. second antibody is labeled with either 125I or linked to an enzyme complex, for example horseradish peroxidase, which is capable of eliciting a colour reaction with a suitable substrate which enables its detection. Adducted ds DNA ^ s u heatif Adducted ss DNA Light generating naaa system Primary antibody Secondary antibody X-ray film Nitrocellulose filter Figure 4.1. Schematic representation of the immunoslot blot assay. 106 Chapter 4. In vivo animal studies 4.2. Development of the immunoslot blot assay for 0 6-CMdG. An immunoslot blot assay to determine 0 6-CMdG in single stranded DNA was developed from the protocol outlined by Nehls et al (1984). An aliquot of DNA that had been carboxymethylated with KDA was quantitated for 0 6-CMdG by the immunoaffinity-HPLC fluorescence procedure detailed in Chapter 2 and the remaining DNA was used as standards for the immunoslot blot. Initial work was done using a Minifold 1 microfiltration apparatus to allow the immobilisation of single stranded DNA on the nitrocellulose filter. However, this apparatus led to irregular bands of DNA due to the nature of the equipment but the use of the Minifold II microfiltration apparatus eliminated this problem. Constant amounts of DNA (1 fig/slot) but containing different amounts of 0 6-CMdG (0-20fmol) were used to optimise the conditions for this assay. Various primary antibody (0 6-CMdG rabbit polyclonal) and secondary antibody (goat -anti-rabbit) IgG horseradish peroxidase conjugated) concentrations were tried. The washing, antibody incubation and non-specific blocking steps were optimised. The optimum conditions are detailed in Section 7.4.1. Visualisation of the specifically bound second antibody was achieved by the incubation of the nitrocellulose filter with chemiluminescence reagents (Amersham ECL or Pierce Ultrasignal), which allow the enzymatic activity associated with the conjugated antibody to generate a luminescent signal which can be detected by exposure of the filter to X-ray film. Typical immunoslot blot calibration curves for 0 6-CMdG in heat-denatured DNA are shown in Figure 4.2.1. using the two different chemiluminescence reagents. The volume intensity of the bands obtained by densitometric evaluation of the slots are plotted against the 0 6-CMdG content in each respective slot. Both chemiluminescence reagents gave a similar limit of quantitation, taken as three times the value for background binding observed to unmodified DNA, of ^ 0.25fmol 0 6-CMdG/ p.g DNA. This corresponds to 0.32jimol 0 6-CMdG/mol dG. This response was linear over a wide range of 0 6-CMdG concentrations, from 0.2fmol towards 20fmol. The Ultrasignal chemiluminescence reagent was found to yield a superior intensity of signal range before saturation occurred for the same range of 0 6-CMdG concentrations in DNA (Figure 4.2.1.), which allows for a more accurate determination of unknown samples and was thus the reagent of choice. Since the intensity of the chemiluminescence reaction showed day-to-day variations, we found it mandatory to include a complete set of standards, 107 Chapter 4. In vivo animal studies run in triplicate on each blot. A typical immunoslot blot filter which has been scanned by the densitometer can be seen in Figure 4.2.2. 5 0 0 0 4 5 0 0 4 0 0 0 3 5 0 0 a33 3 3 0 0 0 S3 2 5 0 0 5 0 0 0 0 10 2 0 3 0 4 0 50 0 6-CMdG (^mol/mol dG) Figure 4.2.1. Typical calibration curves for the determination of 0 6-CMdG in DNA. The volume intensities for slots (average of three densitometric readings) are plotted against the respective amounts of 0 6-CMdG. The red line indicates the use of Ultrasignal i2= 0.9983 , blue line indicates the use of Amersham ECL 12= 0.9912. n = 3 ± SD 108 Chapter 4. In vivo animal studies. 0.64 1.28 2.56 Standards 0 6-CMdG 6.4 Jimol/mol dG 12.81 19.20 25.6] ___ rat 13 rat 14 rat 15 Rat Stomach rat 16 DNA samples rat 17 rat 18 rat 19 rat 20 Figure 4.2.2. Immunoslot blot filter. Constant amounts of DNA (1 jig/s lot) containing either a range of concentrations of 0 6-CMdG in triplicates (as indicated in the figure alongside each triplicate) or in samples of DNA from rat stomach. Prior to immunoslot blot analysis, DNA was quantitated and the purity assessed accurately using a combination of UV spectrometric analysis and digestion of the DNA to the 3 ’mononucleosides and their quantitation by HPLC-UV for both standards and samples. The accurate quantitation of the DNA was essential as varying levels of DNA would have led to false positive or negative results due to the fact that background levels of non-specific 109 Chapter 4. In vivo animal studies. binding would alter. We found that UV spectrometric analysis was misleading as, an impure sample containing both protein and RNA can result in a absorbance ratio of 260/280nm between 1.7-1.9, which is normally considered to be pure DNA. This would then lead to an inaccurate quantitation of the amount of DNA present in a sample. Hence, a digestion protocol which allowed for any RNA present to be visualised "by the subsequent HPLC-UV analysis of the sample was incorporated, and the re-purification of samples contaminated with RNA could then be undertaken. 4.3. I n v i v o APNG study. The formation (4hr) and persistence (24hr) of 0 6-CMdG in the DNA of various rat organs following a single dose by gastric intubation of APNG (4mg) compared to background levels in control animals were determined by immunoslot blot and can be seen in Figures 4.3.1.- 4.3.3. for the stomach, intestine and liver respectively. In the potential target tissue of the stomach, a significant dose effect was seen between the levels of 0 6-CMdG formed in the gastric DNA of control and dosed (4hr and 24hr) animals (p < 0 .005, one way analysis of variance). After receiving APNG (4hr) the level of 0 6-CMdG was 13.42 ± 1.08jimol/mol dG which is a 6 fold increase from the background level of 2.20 ± 0.441pmol/mol dG observed in the control gastric DNA. The loss of 0 6-CMdG from gastric DNA can be seen in the 24hr measurements when the level of 0 6-CMdG expressed as a fraction of the initial (4hr) Oe-CMdG concentration was 62.8% which is still significantly higher than the control level. Although levels of Oe-CMdG were detectable in the intestine DNA for all 6 rats, they showed no significant dose or time effect. However, after 24hr the level of 0 6-CMdG does appear to show a slight rise of 1.6 fold, from the observed background level in intestine DNA but this was not statistically significant. In the liver DNA, 0 6-CMdG was detectable in 4 of the 6 animals and showed no relationship to APNG treatment. Interestingly the levels of 0 6-CMdG detected in control animals showed no significant difference between tissue types, but they did vary from being undetectable up to 2.94|imol/mol dG. 110 Chapter 4. In vivo animal studies. 16 T 14 - 1 12 - I o B 10 - o % 8 - O 6 - I 4 - u1 2 - o 0 control 4hr 24hr Time after APNG dose Figure 4.3.1. 0 6-CMdG levels in gastric DNA for APNG treated rats. The persistence of 0 6-CMdG in the gastric DNA of rats dosed with 4mg of APNG after 4hr and 24hr compared to control animals. Each bar represents one individual animal with the levels determined in triplicate ± SD. 0 2 10 10 3 1 0 T3 1 control 4hr 24hr Time after APNG dose Figure 4.3.2. 0 6-CMdG levels in intestine DNA for APNG treated rats. The persistence of 0 6-CMdG in the intestine DNA of rats dosed with 4mg of APNG after 4hr and 24hr compared to control animals. Each bar represents one individual animal with the levels determined in triplicate ± SD. I ll Chapter 4. In vivo animal studies. 12 T o T3 10 - 1 8 -- i s A 6 o 4 -- IT3 □ 2 -- NO1 o ND ND 0 -- □ control 4hr 24hr Time after APNG dose Figure 4.3.3. 0 6-CMdG levels in liver DNA for APNG treated rats. The persistence of O6- CMdG in the liver DNA of rats dosed with 4mg of APNG after 4hr and 24hr compared to control animals. Each bar represents one individual animal with the levels determined in triplicate ± SD. ND = not detectable. 4.4. In vivo KDA study. In a similar, but larger scale study to investigate if 0 6-CMdG formation showed a clear dose- response, female Fisher 344 rats were dosed by gastric intubation with KDA (0.5, 2.5 and 5mg) or with solvent control (PBS). Four hours after dosing, half of the rats were sacrificed and the liver, intestine and stomach frozen for DNA extraction. The remaining animals were culled 24hrs after dosing for the determination of 0 6-CMdG persistence. All tissues to be examined were double phenol chloroform extracted and the levels of 0 6-CMdG determined by the immunoslot blot method. 4.4.1. Non-target tissue levels of 0 6-CMdG 4 hours after dosing with KDA. The dose-dependent formation of 0 6-CMdG was studied in the intestine and liver after the initial 4hr period. For the intestine and liver DNA no dose response was found to occur after KDA treatment, although variations in the amounts of adducts present were apparent (Figure 4.4.1.). Levels of 0 6-CMdG varied in liver DNA from being undetectable to 9.09pmol/mol dG and in the intestine DNA also from being undetectable to 6.04fimol/mol dG. These variations however were not statistically significant (one way analysis of variance) 112 Chapter 4. In vivo animal studies in comparison to the control group or between treatment groups even if rat 5 was removed as it had unusually high levels of 0 6-CMdG in both of these non-target tissue DNAs. 10 - 9 - O T3 8 - O | 7 - 6 - J o 1 5m g KDA a 5 - vj± 1 2.5m g KDA o 4 - □ 0.5m g KDA T 3 i control s 3 - □ 2 - 1 - 0 - animal 9 -- a 5m g KDA 0 2.5m g KDA □ 0.5m g KDA m control VO 0 0 o Figure 4.4.1. 0 6-CMdG levels in rat intestine and liver DNA after 4hr following KDA treatment. The bars indicate 0 6-CMdG levels in the intestine (A) and in the liver (B) determined in triplicate ± SD. Rats 1-3 were control animals, rats 4-6 received 0.5mg KDA/per animal, rats 7-9 received 2.5mg KDA/per animal and rats 10-12 received 5mg KDA/per animal. 113 Chapter 4. In vivo animal studies. 4.4.2. Target tissue levels of 0 6-CMdG 4 hours and 24 hours after dosing with KDA. The dose-dependent formation of 0 6-CMdG was investigated in the target tissue of the stomach after both the initial 4hr period and at the end of the study (24hr). The linear regression lines obtained (Figure 4.4.2.1.) had different intercepts with the 4hr data being close to 1, indicating that this adducted base is formed strictly proportionally to dose over the entire dose range investigated (p < 0.005, one way analysis of variance). This dose- dependency effect is still apparent to some extent at 24hr after exposure to KDA. However, the linearity had decreased (r2 = 0.7621). In the animals allowed to survive for 24hr post administration of the single dose of KDA, the amounts of 0 6-CMdG in gastric DNA were generally higher than those seen after 4hr, but not statistically significant (p = 0.111, one way analysis of variance), when dose groups were averaged, indicating that this adduct may be persistent. However, if the individual animal results are examined it can be seen that there is a greater variation between the level of adducts in the gastric DNA between animals in a dose group at 24hr compared to the dose groups at 4hr (Figures 4.4.2.2.- 3.). These data therefore suggest that after a 24hr period the level of adducts within the DNA may be dependent on other factors and not merely the initial exposure to the carboxymethylating agent KDA. 0 0.5 1 1.5 2 2.5 3 3.54 4.5 5 KDA dose (mg/animal) Figure 4.4.2.1. Dose-dependent formation of 0 6-CMdG in rat gastric DNA following treatment with KDA. Rats were culled 4hr ( blue dashed line) r2 = 0.9436 and 24hr ( red line) linear regression r2 = 0.7621, after a single dose of KDA ranging from 0.5- 5mg/animal. Data represents mean ± SD of dose group of 3 animals. 114 Chapter 4. In vivo animal studies □ 5m g KDA 0 2.5mg KDA E3 0.5m g KDA n control 4 - VO ooo ts animal Figure 4.4.2.2. Levels of 0 6-CMdG in gastric DNA for individual rats dosed with a range of concentrations of KDA after 4hr. The levels of 0 6-CMdG in the gastric DNA of control animals (rats 1-3), 0.5mg KD A/animal (rats 4-6), 2.5mg KD A/animal (rats 7- 9) and 5mg KD A/animal (rats 10-12) determined in triplicate ± SD. @ 5m g KDA 0 2.5mg KDA □ 0.5m g KDA m control animal Figure 4.4.2.3. Levels of 0 6-CMdG in gastric DNA for individual rats dosed with a range of concentrations of KDA after 24hr. The levels of 0 6-CMdG in the gastric DNA of control animals (rats 13-16), 0.5mg KDA/animal (rats 17-19), 2.5mg KD A/animal (rats 20-22) and 5mg KDA/animal (rats 23-25) determined in triplicate ± SD. 115 Chapter 4. In vivo animal studies. In a sifhilar manner to the APNG rat in vivo study, background levels of 0 6-CMdG were seen in the tissues of control animals which had not been dosed with a carboxymethylating agent. These were comparable to those seen previously with the control tissues in the APNG study, and in fact on analysis no significant difference between control levels in the DNA of tissues from both in vivo studies was observed. A reproducibility study using rat gastric DNA samples (n = 10) where levels of the adduct ranged from 2.39-16.18|imol/mol dG showed that samples run on 3 separate blots on various days had a 10.92 ± 3.83 %CV. Samples run in triplicate on the same blot had a slightly lower %CV of 10.30 ±7.91. 4.5. Comparison of the immunoslot blot with the immunoaffinity-HPLC- fluorescence assay. The results obtained with this immunoslot blot assay were compared with those obtained using the combined immunoaffinity-HPLC-fluorescence assay (detailed in Chapter 2) to analyse DNA isolated from rat stomachs in the in vivo KDA study. In brief, rat DNA samples were digested enzymatically to nucleosides using Nuclease Pl5 acid phosphatase and alkaline phosphatase. The 0 6-CMdG was then isolated by the immunoaffmity columns and quantified by HPLC-fluorescence. The same rat DNA samples were run on an immunoslot blot which included gastric DNA from control animals, low dose (0.5mg KDA) animals 4hr and 24hr after dosing, medium dose (2.5mg KDA) 4hr after dosing and high dose (5mg KDA) after 4hr and 24hr following dosing. 0 6-CMdG levels determined by immunoaffinity-HPLC-fluorescence ranged from 3.86 to 18.41 and those by the immunoslot blot assay from 2.13 to 16.18jj.mol/mol dG. The results shown in Figure 4.5.1. indicate there is a good correlation of 0.907 (Pearson Correlation) between the two assays over this range of values and confirms that the immunoslot blot assay measures 0 6-CMdG accurately in rat DNA samples. The usefulness of the immunoslot blot assay for small amounts of DNA was also exemplified by this study as although sufficient amounts of DNA were available to determine 0 6-CMdG by immunoaffinity-HPLC (< 200jxg) this gave results close to the limit of quantitation (Figure 4.5.2.) and would not allow for 116 Chapter 4. In vivo animal studies. numerous analyses of a sample. However, the same quantity of DNA would allow for approximately 200 determinations of the 0 6-CMdG level by immunoslot blot. 8 .. 6 .. 4 2 -. &6-CMdG Determination by immunoslot blot1 f^rno 1/mol 20 dG) Figure 4.5.1. Comparison of 0 6-CMdG levels determined by the immunoslot blot and by immunoaffinity-HPLC. Samples of DNA were from the stomachs of rats (both control or dosed with KDA) R2 = 0.8817, Pearson Correlation 0.907. 20.00 — 15.00 — 5.00 0.00 -5.00 0.00 5.00 10.00 15.00 Minutes Figure 4.5.2. HPLC chromatogram of the rat stomach DNA following immunoaffinity purification. The amount of DNA injected onto the HPLC corresponds to lOOjig and the 0 6-CMG to 0.3pmol. HPLC conditions as detailed in Section 7.1.7.1., system 4. Chapter 4. In vivo animal studies. A 4.6. Discussion. One of the main purposes of the work described in this Chapter was to develop a rapid and highly sensitive assay which could reproducibly determine 0 6-CMdG levels in the amounts of DNA that could be obtained at biopsy (e.g. < 50p.g DNA) or from in vivo animal studies (< 500p.g). Hence, an immunoslot blot assay for the detection of 0 6-CMdG was developed and found to have a similar sensitivity to that achieved by Nehls et al (1984) for O6- ethyldeoxyguanosine and 0 4-ethylthymidine and by Ludeke & Kleihues (1988) for 0 6-(2- hydroxyethyl)-2 ’ -deoxyguanosine. Although Nehls et al (1984) had employed a murine monoclonal antibody we found that the use of a rabbit antisera was not disadvantageous in concurrence with the results obtained by Ludeke & Kleihues (1988). In preliminary experiments we used an Amersham ECL western blotting kit for the generation of a chemiluminescence signal from the horseradish peroxidase-conjugated second antibody. However, we found that the signal produced by the Supersignal reagent purchased from Pierce yielded a superior signal range. In its present form, the immunoslot blot assay enables the quantitation of 0 6-CMdG > 0.32pmol/ mol dG, which corresponds to > 0.25fniol 0 6-CMdG /l|ng DNA. Although this method is therefore slightly less sensitive than the immunoaffmity-HPLC-fluorescence method detailed in Chapter 2 for Os-CMdG (0.128pmol/ mol dG), improvements to the sensitivity could possibly be made by using up to 3 fig of DNA/per slot as indicated by the work of Nehls et a l (1984) providing a low signal to noise ratio could be maintained by varying the antibody concentrations. However, for use in biomonitoring of human individuals where DNA quantities are low, the current assay should prove extremely advantageous. In order to validate the immunoslot blot assay under in vivo conditions, DNA samples from rat stomachs (either control animals or those treated with KDA) were analysed by both the immunoaffinity-HPLC-fluorescence and immunoslot blot protocols and a good correlation between the two assays was found, which confirms that the immunoslot blot assay measures 0 6-CMdG accurately in rat DNA. The reproducibility of the assay was investigated using rat DNA samples and was found to have a %CV of 10.92 ± 3.83. 118 ______Chapter 4. In vivo animal studies. A As the nitrosated glycine derivatives KDA and APNG are known to carboxymethylate DNA in vitro giving rise to 0 6-CMdG, it was of interest to determine if this phenomenon could be detected in vivo after gastric intubation of rats with these agents. KDA and APNG both decompose to yield the highly reactive carboxymethyldiazonium ion. Therefore, following dosing by gastric intubation, one would expect the target tissue to be the stomach, and there to be little if any systemic caiboxymethyladon at other tissues such as the liver. However, depending on the gastric emptying rate and stability of the test compound some carboxymethylation may be expected in the small intestine. Following a single dose via gastric intubation of either 4mg APNG/animal or 0.5-5mg KDA/animal, the initial (4hr) levels of 0 6-CMdG were determined in the stomach, liver and intestine DNA. Only stomach DNA showed a statistically significant increase in 0 6-CMdG formation for both treatments compared to background levels of 0 6-CMdG in control animals. The levels of 0 6-CMdG at the highest doses of carboxymethylating agents werel2.13 ± 1.57}imoI/mol dG for KDA (5mg) and 13.41 ± 1.08jj.mol/mol dG for APNG (4mg). Interestingly, in vitro KDA produced significantly more 0 6-alkylation than APNG by a factor of 11 : 1 for 0 6-CMdG, whereas in this in vivo study it would appear that APNG is the more potent carboxymethylating agent when the actual moles of each reagent administered to the rats are taken into account. Thus at the highest dose of KDA (5mg) which corresponded to 40.32pmols compared to APNG (4mg) which corresponded to 16.46pmols, then the APNG in a molar ratio produced approximately 2.7 fold more 0 6-CMdG. The apparent disparity of these compounds’ carboxymethylating abilities in vivo compared to in vitro could be due to a variety of factors, but the most important one is likely to be their relative stability and hence the release of the carboxymethyldiazonium ion and its subsequent interaction with other molecules. The decomposition and hence rate of release of carboxymethyldiazonium ion will be dependent on the pH of the gastric lumen in vivo. Rat stomachs spontaneously secrete acid at a rate of 1.3pEq/ 5min under normal conditions (Takeuchi et al, 1994) and the luminal pH values of 3.4-3.8 or 1.5 have been reported in fasted rats (Takeuchi et al, 1994, Uchida et al, 1991). However, due to the dilution effect after fluid intubation and as the animals were allowed to ea tad libitum, the pH of the gastric contents would be expected to be slightly higher due to the buffering capacity of the food and 119 ______Chapter 4. In vivo animal studies. the saline (Ryden et al, 1990, Kyrtopoulos, 1989). KDA decomposes rapidly at low pH releasing the carboxymethyldiazonium ion which would then interact directly with the gastric contents, the mucus layer and the gastric mucosa. APNG decomposes more readily at pH values greater than 7 and hence it is likely to be more stable in the gastric lumen. However, at the gastric mucosal surface, pH values rise suddenly from approximately 1.5 to 7 due to a pH gradient across the mucus layer (Uchida et al, 1991). Mucosal pH values vary according to the area of the stomach studied from 5.17 in the forestomach to 6.89 in the fundic area. The nitrosated peptide, APNG, is therefore more likely to decompose and hence generate the carboxymethyl diazonium ion when within the epithelial cells of the gastric mucosa, which means that this reactive species is liberated in closer proximity to the cellular DNA and explains why this reagent is more potent in vivo than KDA. 0 6-CMdG was detected at similar levels in both the liver and intestine DNA of rats treated with either KDA and APNG. However, these levels were not significantly elevated from levels observed in the same tissues of control animals. The stomach DNA of control animals also had detectable levels of the Oe-CMdG adduct. The endogenous formation of N-nitroso compounds (NOC), such as N-nitrosopeptides, is likely to occur in the stomach providing that suitable conditions of acidity, nitrate/nitrite and peptide availability are met and this could account for the background levels of 0 6-carboxymethylation (Challis, 1989). The levels of 0 6-CMdG in the liver could be due to either the N-nitroso compound formation in the stomach or in the large intestine where intestinal bacteria can reduce nitrate to nitrite for NOC formation (Hill, 1996) and their systemic transfer from the digestive tract via the hepatic portal vein (Farris & Griffith, 1949). Alternatively free amino acids are available in plasma (Armstrong & Stave, 1973) and providing a source for their nitrosation is available then endogenous nitrosation could occur which may account for the observed 0 6-CMdG in the liver. For the KDA in vivo study a clear dose response for the 0 6-carboxymethylation after the initial 4hr could be seen in the stomach DNA. This was not however as pronounced as might have been expected from the in vitro rate of carboxymethylation seen in Chapter 3. This may be in part due to the stability of the KDA and its possible reaction with the gastric contents as discussed earlier, but may also be a reflection of the fact that the cells likely to contain the highest levels of DNA adducts, the mucosa cells bordering the luminal surface (Kobori et al, 120 ______Chapter 4. In vivo animal studies. 1988), will have been effectively diluted out with DNA from cells situated throughout the stomach tissue examined. It has also been observed that the pattern of alkylation after oral administration of A-methyl-A7 -nitro-A-nitrosoguanidine differs around the various regions of the rat stomach (Zaidi et al, 1993(a), Zaidi et al, 1993(b)) which could also contribute to observing a median level of adduct formation throughout the stomach, when specific regions could show larger dose responses. The APNG rat study indicated that 0 6-CMdG in rat stomach DNA may undergo repair. This was in contrast to the results observed following dosing with KDA, where no decrease after a 24hr period from the initial levels (4hr) occurred. In fact as can be seen in Figure 4.4.2.1. the level of 0 6-CMdG present in the stomach DNA slightly increased at 24hr compared to the level at 4hr, although individual variation between rats within each dose group was increased indicating that factors other than the original dose, such as the level of DNA repair or cell turnover rate may be involved in the level of adducts detected 24hr after the dosing (Swenberg et al, 1979). It is possible that the maximum 0 6-CMdG levels had not been reached at 4hr, although gastric emptying rates for the rat stomach have been estimated to be between 2-4hr in non-fasted animals (Zaidi e ta l, 1993(b), O’Neill eta l, 1987) allowing for the homogenous distribution of the agent throughout the stomach and its subsequent absorption (Pegg & Perry, 1981). However, if the absorbed KDA did not react immediately with the cellular DNA then a later increase in 0 6-CMdG levels could be expected. The rate of repair of and hence the persistence of 0 6-CMdG in rat stomach DNA are therefore difficult to estimate from these two studies, as they give contrasting results. It is known for other repair enzymes such as 0 6-alkylguanine-DNA-alkytransferase (ATase), which removes the methyl group from the Opposition of guanine that the various tissues of the gastrointestinal tract shows great variation in the cellular levels and inducibility (Zaidi et al, 1993 (b)) and this is dependent on the animal strain studied as up to ten fold differences can be observed. 0 6-MedG is removed from the colon with a half life of approximately 34hr and from the ileum 12hr. The rate of removal from the colon is thought to be accounted for prominent scheduled DNA synthesis and cell loss due to exfoliation (Swenberg et al, 1979, Herron & Shank, 1981). As the APNG study indicated that some removal/repair of the adduct had occurred by 24hr, this may indicate a repair process for this adduct. However, if the KDA study is correct and no repair has occurred by 24hr then the 0 6-CMdG could be extremely 121 Chapter 4. In vivo animal studies. persistent and its removal be dependent on nucleotide excision repair, base excision repair (glycosylases) or spontaneous chemical depurination. 122 Chapter 5. Human Studies. 123 Chapter 5. Human studies 5.1. Introduction. Cancers o f the gastrointestinal tract account for a substantial part o f the burden o f cancer worldwide (World Cancer Research Fund, 1997). Although diet is considered to play a major role in both causation and modulation of risk of these cancers there is very little known about the underlying mechanisms involved. Correa and collaborators (Correa et a/,1975, Correa, 1988, Correa, 1992) proposed that intragastric nitrosation o f dietary precursors provided a source of mutagenic and carcinogenic agents. Amongst the many nitrosatable precursors in the human diet, protein, peptides and amino acids represent the most abundant source o f nitrosatable substrates in gastric juice (Challis et al, 1982, Shepard & Lutz, 1989). Persistent infection with Helicobacter pylori , a known risk factor for gastric cancer (Correa et al , 1990) has been implicated in providing a source of nitrosating agents and has recently been shown to catalyse nitrosation (Correa et al, 1992, Ziebarth et al, 1997). There is some evidence in humans that gastric pathology which is associated with increased risk of gastric cancer, such as elevated pH and bacterial overgrowth, does result in nitrosation (Xu & Reed, 1993). Dietary protein which escapes digestion and absorption in the small intestine is utilised by the gut microflora as a source of energy, nitrogen and carbon (Cumming & Macfarlane, 1991). A number of metabolites that arise from the bacterial utilisation of protein may have a causative role in colorectal cancer. These include ammonia, a potential cancer promoter, and amines which can be nitrosated to N-nitroso compounds (NOC) (Cumming et al, 1979, Macfarlane et al, 1995). Meat is rich in protein and Bingham et al, (1996) have shown that increasing meat intake is associated with raised levels of total NOCs in the human intestine. The risk factors associated with colorectal cancer, including inheritance of mutations in specific genes such as the APC genes and hereditary non-polyposis colorectal cancer-related DNA mismatch-repair gene, ulcerative colitis and infection with Schistosoma sinensis (World Cancer Research Fund, 1997), have been considered to be quite different from those of the stomach although protective factors such as diets high in vegetables appear to be equally effective at both sites (Potter et al, 1994, World Cancer Research Fund, 1997). Fruits and 124 Chapter 5. Human Studies. vegetables contain many biologically active compounds that may be responsible for an anticarcinogenic effect including vitamin C which inhibits in vivo nitrosation (review, Mirvish, 1994). We have shown that administration of KDA and APNG, two nitrosated glycine derivatives, results in the formation of 0 6-CMdG in the gastric DNA of rats. As endogenous nitrosation > occurs in the human gastric lumen (Xu & Reed, 1993), then it is reasonable to expect the nitrosation of glycine and its derivatives to occur and result in the carboxymethylation of the gastric mucosa DNA. Human gastric mucosa biopsies and blood DNA samples were available from control subjects and individuals with H. pylori from a collaborative study with Dr Simon Everett (Leeds) and Prof. A.T.R. Axon, as were blood samples from a diet study organised by Dr. S. Bingham (Cambridge). The object of the work in this present Chapter was therefore to determine the extent of 0 6-CMdG present in the human DNA samples available and to examine if the disease or nutritional status of the individuals showed any correlation with the formation of this adduct. 5.2. H . p y lo r istu d y . 5.2.1.0 6-CMdG levels in gastric mucosa DNA. The immunoslot blot protocol detailed in Section 7.3.1.3. was used to determine the levels of 0 6-CMdG in 30 gastric biopsies obtained from patients attending the Centre for Digestive Diseases in Leeds, who had been included in the Helicobacter Pylori Study detailed in Section 7.4.2. A typical immunoslot blot filter showing human gastric mucosa DNA which has been scanned by the densitometer can be seen in Figure 5.2.1.1. and the corresponding standard curve for this filter is shown in Figure 5.2.1.2. 125 Chapter 5. Human Studies. 0 0.64 1.28 2.56 S ta n d a rd s 0 6-CMdG 6.4 Umol/mol dG 10.24 12.81 19.20 25.61 HB 118 HB 35 HB 112 Human gastric HB 117 DNA samples HB 123 HB 99 HB 85 HB 10 Figure 5.2.1.1. Immunoslot blot filter. Showing a series of standards and human gastric mucosa DNA all run in triplicate. 126 Chapter 5. Human Studies. 3000 T 2500 -- 2000 -- 1500 -- o 1000 500 5 10 15 20 25 30 0 6-CMdG (^mol/mol dG) Figure 5.2.I.2. Calibration curve for 0 6-CMdG in DNA for the immunoslot blot shown in Figure 5.2.1.1. All standards were run in triplicate ± SD. r2 = 0.981 Initial results on a set of 30 human gastric biopsy samples have shown only three samples with levels of 0 6-CMdG below the limit of quantitation (< 0.32pmol/mol dG (0.25fmol/pg DNA)), whilst the remaining samples had levels of 0.60 - 25.09p.mol/mol dG (Figure 5.2.1.3.). These samples were all run in triplicate and had an average %CV of 12.19 ± 10.9. -oS ' 25 o £ 20 - o 1 15 0 1 10 1 u 'OI O 5 - o in oo cm CM co^r CO CO OO O) oo CO X X X I X X xxxxxxxxxxx Gastric Biopsy Figure 5.2.I.3. Levels of 0 6-CMdG in Human gastric mucosa DNA. Each sample was run in triplicate ± SD, nd = below the limit of quantitation (< 0.32pmol/mol dG). Chapter 5. Human Studies. As these samples were run as part of a much larger study, various other factors such as mucosal and gastric juice vitamin C concentrations and the H. pylori status of the individuals are also to be determined. It was therefore of interest to see if the variation observed between the individual’s levels of 0 6-CMdG in the gastric mucosa DNA could be correlated to other factors. However, as this H. pylori study is being performed blind, the only other data for these initial results at the moment was the histology data. A summary of the analysis according to histology, normal gastric mucosa against H. pylori associated gastritis, can be seen in Figure 5.2.1.4. The medians of the two groups are similar, which are represented by the horizontal lines on the graph, with the normal group having a median value of 6.95 and theH. pylori positive group 7.05 0 6-CMdG fmol/pg DNA and are not statistically different. However, there does appear to be a trend towards those individuals with the highest level of adducts all being found in the H. pylori positive group. 17.5- 15,0- 2.5- 0.0 normal H.pylori positive Figure 5.2.I.4. Levels of 0 6-CMdG in gastric mucosa DNA according to Helicobacter pylori status. The medians of the two groups are represented by the horizontal lines on the graph, lfmol Q6-CMdG /fig DNA corresponds to 1.28pmol/mol dG of 0 6-CMdG. 128 Chapter 5. Human Studies. 5.2.2. 0 6-CMdG levels in white blood cell DNA. Blood samples were also available for 14 of the patients whose level of gastric biopsy O6- CMdG had been determined. Hence, DNA extraction and the subsequent measurement, by the immunoslot blot, of the 0 6-CMdG levels were also carried out for these available blood samples. All of these samples had detectable levels of 0 6-CMdG which showed a variation from 1.75 to 10.41p.mol/mol dG compared to the corresponding gastric DNA from the same individuals which had a variation from 0.60 to 19.79pmol/mol dG in those samples that had a detectable level (three samples were below the limit of quantitation) (Figure 5.2.2.1.). The white blood cell (WBC) DNA levels do not show as large a range of variation and showed no significant correlation (using the Pearson correlation test) with the gastric mucosa DNA O6- CMdG level for individual patients (Figure 5.2.2.2.). 25 20 O 'o Blood i Biopsy B 15 O B 2 10 I T s o I vo' O s nd nd : H85 H92 HS>3 H98 H99 H100 H112 H115 H117 H118 H120 H122 H123 Individual Figure 5.2.2.1. 0 6-CMdG levels in the WBC and gastric mucosa DNA of individuals. Samples were all determined in triplicate ± SD, nd = below the limit of quantitation (< 0.32pmol/mol dG of Oe-CMdG). 129 Chapter 5. Human Studies. —o 20 T S 0 16 .. 1 -oO s 12 u ^ o O ’O 8 C/3o O3 s 4 -- cdC /2 00 ♦ ♦ 10 12 WBC DNA (Q6-CMdG ^mol/mol dG) Figure 5.2.2.2. Correlation of levels of 0 6-CMdG in WBC DNA compared to that in gastric mucosa DNA. r2 = 0.0541. 5.3. Diet Study. WBC DNA was available from 2 male volunteers, CHX103 and CHX104, who underwent the complete diet study (detailed in Section 7.4.2.1. and summarised in Table 5.3.) at the Dunn Clinical Nutrition Centre (Cambridge). Both individuals had passed a health check prior to the start of the study which included liver function tests on their blood (albumin, calcium, phosphate, bilirubin, alkaline phosphatase and gamma glutamyl transferase) and all results were within the normal ranges. The subjects were aged 31 and 32 years old and were smokers. Diet Free subjects normal diet prior to study Meat 420g meat/day Meat + Vegetables 420g meat + 400g veg/day Meat + Tea 420g meat + 3g tea/day Meat + Vegetables + Tea 420g meat + 400g veg +3g tea /day Table 5.3. Diet regimes for the subjects. Each regime was for a period of 15 days. 130 ______Chapter 5. Human Studies. DNA from blood samples taken on days 13 and 15 of each dietary period were analysed for 0 6-CMdG by the immunoslot blot assay and a typical filter for these samples including a range of standards can be seen in Figure 5.3.1. 0.64 1.28 2.56 Standards 0 6-CMdG 3.84 pmol/mol dG 6.4 10.24 12.81 19.20_ 103 MT 103 MVT 103 MVT Human blood 104 MVT DNA samples 104 MT 103 MV 104 MT 104 M Figure 5.3.1. Typical immunoslot blot filter for samples from the diet study and a range of standards. 131 Chapter 5. Human Studies For both subjects, all the DNA samples showed detectable levels of 0 6-CMdG which varied from 5.79-15.42pmol/mol dG for CHX103 and 0.66-6.78pmol/mol dG for CHX104 (Figures 5.3.2 and 5.3.3.). However, when pooled 0 6-CMdG levels determined for the 2 volunteers showed no significant difference between dietary regimes and no correlation with faecal N- nitroso compounds (Pearson correlation, one way analysis of variance, [NOC], which were analysed by R. Hughes) when expressed as ng/g faeces or jig/day. Interestingly, the overall levels of Oe-CMdG adducts (9.45 ± 2.91pmol/mol dG) and NOC levels (318 ± 188ng/g faeces or 267 ± 128(j.g/day) for CHX103 were higher than those determined for CHX104 (2.80 ± 2.04|nmol 0 6-CMdG/mol dG and NOC levels of 92 ± 69ng/g faeces or 59 ± 39|ig/day). 14 —r- _ 800 12 -- 600 -a 10 O .. 500 ~ao ^-=• 8 - 2 u 400 mM-h O tJO £ 300 ^ =L 2 0 0 CJ 2 -- - 100 Free Meat Meat + Meat + Meat + vegetables tea vegetables + tea Figure 5.3.2. Levels of 0 6-CMdG and N-nitroso compounds for CHX103 during different dietary regimes. NOC (ng/g faeces) black line, NOC (fig/day) blue line. O6- CMdG levels were determined in triplicate ± SD. The individual measurements of both 0 6-CMdG and NOC (day 13 of the meat and tea diet NOC result was unavailable) for subject CHX103 can be seen above in Figure 5.3.2. The O6- CMdG and NOC (ng/g faeces) levels had a Pearson correlation of 0.781 or 0.572 if the NOC levels are expressed as pg/day. No statistically significant difference between any of the different dietary protocols was observed (one way analysis of variance). However, it can be 132 ______Chapter 5. Human Studies. seen that the meat diet did increase the level of 0 6-CMdG detected in the blood DNA from the free diet, and the supplementation of this diet with either vegetables or tea raised the level of adducts further, whereas, inclusion of both tea and vegetables had no apparent effect on the average level of adducts from that observed with meat alone. Interestingly the levels of O6- CMdG between the two points determined for each diet regime do appear to fluctuate more when the meat diet is supplemented when compared to meat alone for both individuals studied. For the subject CHX104 (Figure 5.3.3.) there was no correlation between the levels of O6- CMdG and NOC and unfortunately no sample from the free choice diet period was available. Variations in the levels of 0 6-CMdG on the diet supplemented with vegetables were apparent. A significant decrease from the level of 0 6-CMdG in the meat diet was observed for both the diet supplemented with tea (p < 0.05, one way analysis of variance) and the diet supplemented with tea and vegetables (p <0.01, one way analysis of variance). t 250 12 - *0 10 - g % 150 "o 8 CD s 1 4 - OI - 50 o VO o o 2 - £ Meat Meat + Meat + Meat + vegetables tea vegetables + tea Figure 5.3.3.. Levels of 0 6-CMdG and N-nitroso compounds for CHX104 during different dietary regimes. NOC (ng/g faeces) black line, NOC (pg/day) blue line. O6- CMdG levels were determined in triplicate ± SD 133 ______Chapter 5. Human Studies. 5.4. Discussion. The present results show that the immunoslot blot assay using the polyclonal antibody against 0 6-CMdG is sensitive enough to detect and quantitate the presence of 0 6-CMdG in human tissues. The extent of formation of adducts in DNA is believed to constitute a measure of the biologically significant exposure of a tissue to a carcinogen and may reflect the carcinogenic risk posed to the tissue by the particular exposure if factors such as repair and cell replication are taken into account. Examination of the DNA from gastric biopsies obtained from 30 individuals, revealed detectable amounts of the 0 6-CMdG adduct in all but three samples, ranging from 0.60- 19.79pmol/ mol dG. This level is considerably higher than that reported for 0 6-MedG in human gastric tissues, where levels ranging from ca 0.016-0.16(j.mol/mol dG have been reported (Umbenhauer et al, 1985, Hall eta l, 1991, Povey & Cooper, 1995, Kyrtopoulos et al, 1990, Haque et al, 1994). This higher level of 0 6-CMdG formation would be expected if this adduct accumulates to some extent due to a less rapid repair mechanism than that for O6- MedG (Shuker & Margison, 1997) or if compounds such as the nitrosated glycine derivatives are involved, as these compounds are known to produce more carboxymethylation than methylation at least in vitro (Chapter 3). 0 6-CMdG levels were also determined in white blood cell (WBC) DNA from two studies (H. pylori and diet) which showed a range of 0.665 to 15.42p.mol 0 6-CMdG /mol dG. 0 6-MedG has also been quantitated in WBC DNA and lymphocyte DNA with values of 0.006- 0.364pmol/mol dG being reported (Eurogast, 1994, Kang et al, 1995), which are generally lower than the levels reported for gastric tissue (0.0122-0.621 pmol/mol dG) (Cooper et al, 1991, Kyrtopoulos et al, 1990, Povey & Cooper, 1995, Hall et al, 1991). However, the adduct level range for 0 6-CMdG is very similar in both the gastric biopsy and WBC DNA, unlike the levels reported for 0 6-MedG. This could be due to cellular differences in repair capacity and the amount of exposure to the carboxymethylating agent, as it is possible that the WBC level is a composite measure of various cell types with differing lower repair capacities. In order to determine a tissue/WBC dose relationship further animal studies would need to be undertaken. 134 ______Chapter 5. Human Studies There is increasing epidemiological evidence that persistent infection with H. pylori is a risk factor for the development of gastric adenocarcinoma (Forman et al, 1990, Eurogast, 1994, Parsonnet et al, 1991), and molecular and pathological studies support its biological plausibility (Correa, 1992). From the results for the H. pylori gastric biopsy trial, it was seen that whilst the medians obtained for the patient groups based on histology (normal gastric mucosa, Helicobacter associated gastritis) are similar, two observations can be made. The first is that there seems to be a trend towards high levels of adduct in the Helicobacter associated gastritis patients and the second is that, whether or not this becomes statistically significant with more patients, the small number of patients with the highest level of adduct are found in the Helicobacter positive group. Since only a small number of patients with H. pylori gastritis go on to develop gastric cancer (Blaser et al, 1995, Forman et al, 1990), it is possible to hypothesize that it could be these patients with the highest level of adducts that are at increased risk. Several factors are thought to be important in the clinical outcome of H. pylori infection. These include the strain of H. pylori (in particular virulence factors such as vacuolating cytotoxin and cytotoxin-associated protein), the host’s immunological response to infection and enviromental factors such as diet. (Rudi et al, 1997, Kuipers et al, 1995, Mohammadi et al, 1996, Yokota et al, 1997, McColl & El-Omar, 1996). As the H. pylori negative subjects also had detectable levels of 0 6-CMdG, it is likely that the dietary habits of the individuals are likely to be a confounding factor in this study. However, plasma vitamin C levels are also being measured and should be available at completion of the trial. 0 6-CMdG levels in WBC DNA samples were analysed from some of the individuals whose gastric mucosa levels had been determined. However, no correlation between the adduct levels in these sources of DNA was seen. This result indicates that WBC DNA may not be a good surrogate marker for gastric mucosa DNA. However, further analyses need to be carried out to confirm this finding. There are several reasons why WBC DNA might not correlate with the levels in gastric mucosa DNA. Endogenous nitrosation may be occurring at sites other than the stomach, such as the colon, where nitrate can be reduced to nitrite by the colonic flora (Thompson, 1984, Calmels et al, 1985) or the gastric biopsy sample may not be representative of other areas within the stomach. Other studies in which total white blood cell DNA has been used have also reported no significant correlation with target tissue DNA 135 ______Chapter 5. Human Studies. adducts and blood DNA adducts (van Schooten et al, 1990). In this case, it was suggested that as short-lived granulocytes, which are the predominant nucleated cell type within whole blood (approximately 70%) (Souliotis et al, 1990) may not have persisted long enough (8- 12hr) to accumulate detectable levels of DNA adducts, whereas measurement of lymphocyte or total mononuclear cell DNA fractions (which account for approximately 30% of the leukocytes) which are longer lived cells than blood granulocytes appear to achieve good correlations with tissue adduct levels (Wiencke et al, 1995). Hence, the choice of which WBC type to measure will depend on the extent of exposure to the carcinogen and hence the level of DNA adduct, the repair and accumulation of the adduct within the WBC type and the sensitivity of the analysis method. Thus for 0 6-CMdG determination it may be more appropriate to measure one specific class of WBC which will be a better surrogate marker for gastric mucosa DNA. WBC DNA samples were analysed for their 0 6-CMdG level as part of a study being undertaken by Dr. S. Bingham (MRC, Cambridge) to investigate dietary supplementation with vegetables and black tea on the levels of faecal NOC levels in individuals on a high red meat diet. The 0 6-CMdG levels showed variations in the levels detected. As all other factors were kept constant, apart from the dietary supplementation, this could indicate that 0 6-CMdG variations are solely due to fluctuations in the endogenous formation of NOCs that carboxymethylate DNA. However, both subjects were smokers and it is known that tobacco smoke contains many preformed N-nitroso compounds (Spiegelhalder & Bartsch, 1996) and is a rich source of nitrogen oxides which can increase endogenous nitrosation (Bartsch et al, 1989, Carmella et al, 1997). It has also been suggested that there may be biological interactions between smoking and dietary protective factors such as vitamin C, in that smokers may require higher levels of vitamin C to achieve serum levels and presumably tissue levels comparable with non-smokers (Gonzalez & Agualo, 1994). As only two individuals were studied, general trends and conclusions about the possible protective roles of vegetables and tea in dietary supplementation can not be made. However, from these results some interesting observations have been seen. Firstly, in the individual CHX103 the level of faecal NOCs showed a good correlation with the levels of 0 6-CMdG over the dietary protocol, which indicate that 0 6-CMdG levels are a good marker for endogenous nitrosation. This individual had high levels of NOCs (personal correspondence 136 Chapter 5. Human Studies. with R. Hughes) and also had the higher levels of 0 6-CMdG that did not decrease upon any of the dietary supplementations, which suggests that this individual’s endogenous NOC formation capacity is high and may require higher levels of antioxidants to produce an effect. For the individual CHX104, lower levels of both NOCs and 0 6-CMdG were observed, which showed no correlation. Faecal NOC levels can show great variations and are not indicative of the exact nature of the individual NOC formed, hence at lower levels of faecal NOC formation the relationship with 0 6-CMdG levels in WBC DNA would appear to be not as clear cut as at higher faecal NOC levels. For this individual, supplementation of the basic meat diet with tea, and vegetables and tea produced a significant reduction in 0 6-CMdG levels. Numerous epidemiological studies have reported a decrease in risk for gastric or colon cancer with consumption of diets high in vegetables (recent review World Cancer Research Fund,1997), which may at least in part be due to the vitamin C content of these food stuffs which can inhibit nitrosation and also reduce free radicals to non-radical species (Mirvish, 1996). Black tea and vegetables also contain polyphenols which are phytoprotectants and have also been implicated in a reduced cancer risk (World Cancer Research Fund, 1997). Thus, the results for CHX104 would indicate that tea or the combination of tea and vegetables does reduce the exposure of WBC DNA to carboxymethylating agents. Interestingly, the initial level of 0 6-CMdG in the subject at day 13 of the meat and vegetable diet also showed a decrease from the meat diet. However, on day 15 the 0 6-CMdG level had increased to a higher level than observed with meat alone. Some vegetables contain high levels of nitrate and it is possible that this may be a contributory factor in endogenous nitrosation levels. Similarly, the 0 6-CMdG levels in the supplemented diets (not meat alone) appear to fluctuate more than on the basic meat diet for both subjects. This could be due to the dietary protocol. Although the same type and amount of vegetables were eaten each day, they were prepared differently on a three day rotational system and the tea was taken intermittently throughout the day. These dietary studies therefore indicate that diet may play a role in the level of 0 6-CMdG detected in WBC DNA, but undoubtedly this relationship is likely to be complicated and further studies are needed to elucidate the role of dietary constituents. This finding will also 137 Chapter 5. Human Studies have a significant impact on the use of 0 6-CMdG levels in both the gastric biopsies and WBC DNA used in the H. pylori study as the diet is likely to be a confounding factor. 138 Chapter 6. Discussion. 139 Chapter 6. Discussion 6.1 Discussion. In order to investigate the in vitro formation of 0 6-CMdG and 0 6-MedG in calf thymus DNA by a variety of nitrosated glycine derivatives, novel immunoaffinity-RP-HPLC fluorescence methods were developed. The absolute limits of detection of the HPLC-fluorescent assay were O.lpmol 0 6-CMG/injection and 0.05pmol 0 6-MeG/injection. If lmg of DNA hydrolysate was used per injection, the limit of detection of this assay corresponded to 0.128pmol 0 6-CMdG/mol dG and 0.064pmol 0 6-MedG/mol dG. For the 0 6-MedG adduct this immunoaffinity-HPLC-fJuorescence method has comparable or has increased sensitivity to other HPLC based assays (Belinsky et al, 1987, de Groot et al, 1994, Foiles et al, 1985). While this level of sensitivity for 0 6-CMdG is suitable for experimental studies where amounts of DNA for analysis are fairly large (>0. lmg) or adduct levels are high, it is not sufficient for human or animal studies where only small biopsies or blood volumes with possibly lower adduct levels and lower yields of DNA are available. The lack of cross reactivity of the 0 6-CMdG antiserum with normal bases (N. Fairhurst, (1990), Harrison et al, 1997) suggested that O6-0MdG could be detected in intact DNA. Accordingly, an immunoslot blot assay was developed, as described by Nehls et a l (1984), where an almost equivalent sensitivity of > 0.32pmol 0 6-CMdG/mol dG was obtained using only lpg DNA. The results presented in Chapter 3 showed that the concomitant methylation and carboxymethylation at the O6 position of guanine in in vitro calf thymus DNA appears to be a general property of N-carboxymethyl-N-nitroso compounds, APNG and NOGC (Shuker & Margison, 1997), as well as diazoacetic acid derivatives, KDA and azaserine. However, the ratios of O6 methylation/carboxymethylation and the capacity for their formation by each agent studied varied considerably, which probably reflects the ease of decomposition/ rearrangement to generate the ultimate reactive species, the carboxymethyl diazonium ion and the methyl diazonium ion. In order to investigate if these nitrosated glycine derivatives result in the carboxymethylation of DNA in vivo, two animal experiments were undertaken where either KDA or APNG were administered by gastric intubation to rats. 0 6-CMdG was detected in the potential target tissue, the stomach DNA, after both treatments with the KDA resulting in a clear dose response after 4hr. This finding illustrates that these nitrosated glycine 140 ______Chapter 6. Discussion derivatives are capable of carboxymethylating DNA under in vivo conditions in the stomach, the proposed site of their in situ formation. However, as yet, no studies to determine the in vivo ratio of O6 carboxymethylation to methylation have been undertaken as the immunoaffinity RP-HPLC-fluorescence assay would not be sensitive enough to detect the levels of 0 6-MedG adducts observed at the doses administered in these studies. It is possible that the caiboxymethylation/methylation ratio as well as the capacity to alkylate the cellular DNA may well differ from the results seen in the in vitro mechanistic studies for these agents. In fact, some indication of the possible differences between in vitro and in vivo results could be seen from the relative amounts of O6- CMdG formation by KDA and APNG in the experimental systems. KDA produced significantly more Oe-CMdG, with a molar ratio of 11 : 1 than APNG under in vitro conditions, whereas upon in vivo administration to rats APNG produced slightly more O6- CMdG in the stomach DNA than KDA. In order to gain a clearer insight into how these nitrosated glycine derivatives gain access to cellular DNA it would be advantageous to use cell culture systems. This would provide a rapid means of gaining information not only on the carboxymethylation to methylation potential within cells of these agents but the associated cellular toxicity could be assessed as dosing levels could possibly be increased and larger quantities of DNA could be recovered. This may be of particular interest in the case of azaserine, the potent pancreatic carcinogen, as studies with acinar cells indicate that the release of diazoacetic acid requires enzymatic a,[S elimination which is catalyzed by pyridoxal (Zurlo et al, 1982). Thus, azaserine is more likely to generate reactive species in cellular systems and therefore result in higher levels of adducts than those seen in the in vitro calf thymus DNA study. Although a clear increase in the levels of 0 6-CMdG from background levels in stomach DNA was seen in the in vivo KDA and APNG animal studies after 4hr, the persistence/repair of this adduct was far from clear cut at 24hr. The APNG animal study showed a decrease in O6- CMdG levels with 37.2% lower than those detected after 4hr, whereas the KDA animal study indicated that a slight increase in 0 6-CMdG levels was seen at 24hr from the 4hr levels. However, the interindividual variation in the dose groups for the KDA study at 24hr was much greater than that at 4hr. To ascertain why these studies showed different results, a variety of options are available. Firstly, more animal studies over a longer time scale may be 141 Chapter 6. Discussion appropriate with animals being sacrificed up to 8 days post dosing (Zaidi & O’Connor, 1995). A higher dose of the carboxymethylating agent may also result in clearer results. However, higher doses would not be as biologically relevant for extrapolation to human exposures. Secondly, immunohistochemical staining can be a powerful technique as it enables the identification and localisation of adducts at the level of individual cells within tissues. This can identify target cell populations and determine if there is any heterogeneity in adduct formation and if the immunohistochemical analysis is carried out some while after exposure to a carcinogen then repair-deficient cells can also be identified. Such heterogeneity in repair and formation has been noted for the 0 6-MedG adduct in rat stomach mucosae after dosing with MNNG (Zaidi & O’Connor, 1995, Zaidi et al, 1993 (b)). The 0 6-CMdG antiserum is of high enough affinity for immunohistochemical applications and has been validated on azaserine treated rat pancreatic tissue (personal correspondence with J. Bailey, CRC Paterson Institute Manchester). As sections from all of the rat tissues assayed for their 0 6-CMdG content in the APNG and KDA studies were fixed and sectioned, the immunohistochemical analysis of these sections may reveal why inconsistencies between the two studies occurred. Potentially these sections could also be stained for various other factors that are related to carcinogenic risk, such as proliferation markers Ki-67 (Kang et al, 1997) or repair enzymes like ATase and human apurinic endonuclease 1 (Zaidi et al, 1993 (a), Kakolyris et al, 1997). This may prove invaluable if Os-CMdG and its relationship to cancer risk is to be investigated, as even well characterised DNA lesions, such as the presence of 0 6-MedG in DNA, do not always correlate with cancer risk unless other factors are taken into account (Zaidi et al, 1992). Similarly, the non-target tissue sections could be analysed immunohistochemically to ascertain if certain cell populations within the liver and intestine show levels of 0 6-CMdG above that of surrounding cells. Persistent infection with H. pylori is a risk factor for the development of gastric adenocarcinoma which may be due to increased endogenous nitrosation (Correa, 1992). To this end 0 6-CMdG levels were determined in 15 WBC and 30 gastric biopsy DNAs from individuals participating in an H. pylori study. 0 6-CMdG was detected in 27 of the gastric biopsies with values ranging from 0.60-19.79|imol/mol dG and in all 15 of the WBC DNA with levels ranging from 1.75-10.4lpmol/mol dG. To my knowledge this is the first report to 142 ______Chapter 6. Discussion show the presence of this adduct in human tissues. There was no statistical difference in O6- CMdG levels from gastric biopsy DNA between individuals on the basis of H. pylori infection which may be due to the limited number of cases analysed in the present study. However, those individuals that had the highest levels of 0 6-CMdG were all H. pylori positive. No correlation was found between gastric and WBC'DNA 0 6-CMdG levels which suggests that WBC DNA would not be a good surrogate marker for the gastric mucosae. This may however be due to the relatively small number of cases where both types of sample DNA were available. Further investigations into the relationship between gastric DNA and DNA derived from blood could clarify this situation as animal experiments where some of the variable factors associated with this human study (different histologies, diet, smoking status) could be controlled. WBC DNA was also analysed for 0 6-CMdG to investigate the effect of dietary supplementation of high risk diets (high red meat) by vegetables and black tea (Bingham et al , 1996). Samples from the two individuals studied showed detectable levels of 0 6-CMdG. However, due to the small scale of this pilot study only the general trend that the levels did vary can be concluded. This finding is extremely interesting in itself, as this indicates that some lifestyle/diet factor is the source of the carboxymethylating agent and hence there is potential to limit this exposure. The levels of 0 6-CMdG detected in the human gastric biopsy samples were of a similar range to that observed for the rat stomach DNA following treatment with KDA (up to 5mg) or APNG (4mg).The level of peptide amino groups in gastric juice is estimated to be approximately 0.2M and recently levels up to 8mM for free amino acids have been reported with glycine being one of the most abundant (Challis et al, 1982, Wang et al, 1995). Hence the potential to form nitrosated glycine derivatives is high in the human stomach, and as the exposure will be chronic it is perhaps not surprising that similar levels between rat and human gastric 0 6-CMdG levels were observed. However, very litde is known about the repair and potential persistence of the lesion in cellular DNA, apart from the fact that it is not repaired by 0 6-alkylguanine alkyl transferase (Margison & Shuker, 1997). 143 Chapter 6. Discussion Now that methods are available for the reliable quantitation of 0 6-CMdG in DNA (Chapter 2) it should be possible to investigate the repair and hence the persistence of this adduct using either cell free extracts or cells that are deficient in a variety of repair enzymes. Sedgwick (1997) found that cells lacking nucleotide excision repair had an increased mutation rate following treatment by azaserine and suggested that this could be due to the 0 6-CMdG adduct. However, azaserine will produce a range of methyl and carboxymethyl adducts so this repair process may not be responsible for the repair of 0 6-CMdG. It would also be extremely useful to determine if the 0 6-CMdG adduct is promutagenic. This may require the synthesis of an oligonucleotide containing the adduct and its subsequent insertion into cells where following their replication, the isolation, amplification and sequencing of the DNA would allow any mutation to be detected and characterised. It is difficult to estimate the potential risk the presence of 0 6-CMdG indicates in human DNA due to the current lack of knowledge concerning the biological significance of this lesion. However, to date each of these carboxymethylating agents all of which are nitrosated glycine derivatives also methylate DNA which results in the formation of the known promutagenic lesion 0 6-MedG (Chapter 3). 0 6-MedG has been reported in human DNA from various sources including the gastrointestinal tract and lymphocyte DNA (Eurogast, 1994, Povey & Cooper, 1995 and Kyrtopoulos et a l , 1990). This suggests that methylating agents may be involved in the aetiology of these cancers. However, as nitrosated glycine derivatives cause both methylation and carboxymethylation then this type of compound which is likely to be formed by the endogenous nitosation of dietary amino acids and peptides is a possible candidate for the observed presence of 0 6-MedG (Harrison et al, 1997, Shuker and Margison, 1997). 144 Chapter 7. Materials and methods 145 Chapter 7. Materials and methods 7.1. M aterials and M ethods for Chapter 2. 7.1.1. Apparatus. Analytical and preparative HPLC instrumentation comprised of a Waters 600E pump with a Rheodyne 7125 injector system and either a Shimadzu SPO-GA UV spectrophotometric detector, or a Waters 470 scanning fluorescence detector connected to a Waters 745B data module or a Waters Millennium data system. Fractions were collected using a Pharmacia LKB-Helifrac fraction collector. Radioactivity, disintegrations per minute (dpm) were determined by liquid scintillation counting on a Wallac 1410 liquid scintillation counter using Hydrofluor. Samples were concentrated or dried down using either a Lyoprep 3000 freeze drier or a Savant speedvac concentrator. The plate reader for ELISA was a Labsystems Multiskan Plus (type 314). 7.1.2. Synthesis of tritiated purine deoxynudeoside derivatives. 7. 1.2.1. Chemicals. Thymidine, thymidine phosphorylase (TPase E.C.2.4.2.4), purine nucleoside phosphorylase (PNPase E.C.2.4.2.1), N2-amino- S6-(caiboxymethy l)-mercaptopurine (S6-CMG) and O6- MedG were purchased from Sigma Chemical Co. [5'-3H] thymidine with a specific activity of 481 GBq/mmol was obtained from Amersham International pic. 0 6-CMG, 0 6-CMdG and O6- MeG were available from previous work (Harrison et al , 1997). Buffer salts and solvents were purchased from Fisons and were of an analytical grade where possible. 7. 1.2.2. HPLC Systems. System 1 comprised of a Reverse-phase (R-P) HPLC using a Techsphere 5pm ODS reverse- phase column (250 x 4.6 mm) with a prefilter at a flow rate of 1 .OmL/min. A step gradient elution system was employed; 0-10 min 0.1M triethylammonium acetate (TEA) (pH 7) with 10% methanol, 10-20 min 0.1M TEA (pH 7) with 15% methanol, 20-30min 0.1M TEA (pH 7) with 20% methanol, going back to initial conditions over a further lOmin. UV-absorbance was monitored at a wavelength of 278nm. Injection loop volume was lOOpL. 146 ______;______Chapter 7. Materials and methods System 2 employed R-P HPLC incorporating a Techsphere 5pm ODS (250 x 4.6 mm) column with a prefilter and an isocratic flow at lmL/min with 10% methanol and 0.1M TEA buffer (pH 7). Peaks were monitored by fluorescence with excitation wavelength at 286nm and emission wavelength at 378nm. Injection loop volume was lOOpL. System 3 was for analytical HPLC and was carried out using a Phase Sep S5 BDS2 (250 x 2.0 mm) narrowbore column with a prefilter, with an isocratic flow rate of 0.2mL/min of 0.1M TEA with 15% methanol. Peaks were detected by fluorescence; excitation wavelength 286nm, emission wavelength 378nm. Injection loop volume was lOpL. 7.1.2.3. Enzymatic Coupling. 7.1.2.3.1. Trial reaction: Chapeau & Marnett This reaction was carried out essentially as described by Chapeau & Marnett (1991) but scaled down to a 1: 20, hence the following modifications: Os-CMG (1.07pmol, 50pL) from a stock solution (dissolved in a saturated sodium bicarbonate solution) and thymidine (3.3pmol,33pL) were dissolved in lmL of 20mM potassium phosphate buffer and the pH adjusted to 7.3 with a saturated potassium hydroxide solution. TPase (0.11 units, 22pL), PNPase (1.65 units, lOpL) dissolved in water were added. The solution was shaken at 38°C for up to 2 weeks, over which time the progress of each reaction was monitored by RP-HPLC (employing System 1 (Section 7.1.2.2.)) by taking aliquots of 50pL. The general order of elution from the reverse-phase column was thymine, starting base 0 6-CMG, thymidine and the adducted deoxynudeoside, 0 6-CMdG. 7.1.2.3.2. Trial reaction: Stadler et al (1994) Stadler e t a l (1994) also used a method developed from the Chapeau & Marnett (1990) procedure. However, there were some important differences between the two methods. Primarily the starting base was less concentrated, there was a 37 fold excess of thymidine, more of the enzymes were used and the pH was at 8. Hence a 1:6.2 scaled down version of this alternative method was undertaken. 0 6-CMG (0.107pmol, 5pL) and thymidine 147 Chapter 7. Materials and methods (3.99pmol) were added to 0.798mL of 50mM dipotassium hydrogen phosphate buffer pH8. TPase (4.28 units, lOpL) and PNPase (7.26 units, 20(j.L) were added. The solution was shaken at 38°C for up to 48hr, over which time the progress of each reaction was monitored by RP-HPLC (employing System 1 (Section 7.1.2.2.)) by taking aliquots of 20pL. 7.1.23.3. fH]-deoxygu.anosine derivative synthesis. This reaction was carried out essentially as described by Chapeau & Marnett (1991) with the following modifications: the purine of interest (0.214pmol) and thymidine (0.66fimol) were dissolved in 300pL of 20mM potassium phosphate buffer and the pH adjusted to 7.3 with a saturated potassium hydroxide solution. TPase (0.22 units), PNPase (6.6 units), sodium azide (to a final concentration of 0.05%) and 66pL [3H]-thymidine (2.442 MBq) were added. The solutions were shaken at 38°C for varying times (up to 3 weeks), over which time the progress of each reaction was monitored by RP-HPLC (employing System 1 (Section 7.1.2.2.)) using duplicate reaction mixtures which had the [3H]-thymidine replaced by the same volume of water. The general order of elution from the reverse-phase column was thymine, starting base, thymidine and the adducted deoxynudeoside (Figure 7.1.2.3.3.). I. Thymine Z.0*-CM0 3. Thymidine Id 13 20 23 30 Time (mint) Figure 7.1.2.3.3. RP-HPLC -UV profiles showing the formation of [3H]-06-CMdG from 0 6-CMG in a TPase/PNPase catalysed reaction over time. HPLC conditions are as described under Section 7.1.2.2. system 1. 148 Chapter 7. Materials and methods 7. 1.2.4. Purification o f f H]-deoxynucleoside derivatives. Each reaction was judged to have gone to completion by either no starting base being detectable, or there being no difference in the relative peak sizes of the starting base compared to the resulting deoxynucleoside observed over a period of 48hrs. Subsequently, 2|iL of the ‘hot’ reaction was made up to 20pL by the addition of 0.1M TEA and 5pL subsequently injected onto the HPLC (Section 7.1.2.2.,system 1) collecting 1ml fractions every minute directly into mini-scintillation vials. Hydrofluor (3mL) was added to each vial, and the radioactivity in each fraction determined. In each case the peak by HPLC-UV corresponding to, [3II]-06-CMdG, [3H]-06-MedG (retention time determined using standards) or [3H]-S6-CMdG (the only other discernible peak apart from thymine, S6-CMG and thymidine) showed a detectable elevated dpm compared to the base line on scintillation counting (Figure 7.1.2.4.1). Time i u». 0 9 19 19 29 29 HPLC fractions (mi) Figure 7.I.2.4.I. Isolation of [3H]-S6-CMdG. The main graph shows the dpm in each fraction (ml/min) collected from a HPLC analysis (Section 7.1.2.2.,system 1) of the reaction mixture. The insert is the HPLC trace corresponding to this analysis and shows four peaks, where peak 1 is thymine, peak 2 is S6-CMG, peak 3 is thymidine and peak 4 corresponds in retention time to the unidentified peak on scintillation counting. On the scintillation graph peak 1 is unincorporated [3H]-ribose, peak 2 [3H]-thymidine and peak 3 the product [3H]-S6-CMdG. 149 ______Chapter 7. Materials and methods For each reaction mixture : Aliquots of the reaction mixture (3x100jiL) were then loaded onto the HPLC ( Section 7.1.2.2.,system 1) and the peak corresponding to this increased radioactivity and known standard were collected, combined and concentrated by freeze drying. The fraction was then re-dissolved in lOOpL 0.1M TEA (pH7) and re-injected into the HPLC system (which had undergone extensive washing with methanol until any subsidiary background radioactivity was removed). Fractions were collected every 30sec for 30min, and 5pl of each (in 3ml liquid scintillation fluid) were then counted on a Wallac 1410 liquid scintillation counter. Each fraction corresponding in retention time to either 0 6-CMdG, 0 6-MedG or S6-CMdG showed an elevated dpm. Re-chromatography of this fraction in each case showed no other peaks were present except for the required deoxynucleoside derivative. The re-chromatogram for purified [3H]-S6-CMdG can be seen in Figure 7.1.2.4.2. C c3 od> G O Time Figure 7.1.2.4.2. HPLC chromatogram for the purified [3H]-S6-CMdG. HPLC conditions as in section 7.1.2.2., system 1. 7. 1.2.5. Quantitation o f fHJ-deoxynucleoside derivatives. [3H]-06-CMdG and [3H]-06-MedG were quantitated against calibration curves for standards of 0 6-CMdG and 0 6-MedG by the injection of IOjo L of each purified fraction onto RP-HPLC employing either system 2 (for 0 6-CMdG) or system 3 (for 0 6-MedG) detailed in Section 150 Chapter 7. Materials and methods 7.1.2.2.. Calibration curves were constructed by the injection of 10|aL of standard solutions of each compound over a range of 0-70pmols or O-lOOpmols and are shown in Figures 7.1.2.5.1. and 7.1.2.5.2. respectively. 9 8 7 6 5 4 1 0 0 10 20 30 40 50 60 7 0 0 6-CMdG (pmol) Figure 7.1.2.5.1. Calibration curve for 0 6-CMdG using RP-HPLC-fluorescence. HPLC conditions are detailed in section 7.1.2.2., system 2. r2 = 0.999. n = 3 ± SD 7 6 5 4 3 2 a . 1 0 0 20 40 6 0 80 100 Q6-MedG (pmol) Figure 7.1.2.5.2. Calibration curve for 0 6-MedG using RP-HPLC-fluorescence. HPLC conditions are detailed in section 7.1.2.2., system 3. r2 = 0.998 n = 3 ± SD. Quantitation of [3H]-S6-CMdG was attempted via hydrolysis in 0.1M TFA (lhr, 60°C) back to the original purine base as no standard S6-CMdG was available. Unfortunately, on hydrolysis several peaks were detectable using HPLC (Section 7.1.2.2.,system 3) (Figure 151 ______Chapter 7. Materials and methods 2.6.4.),and only an estimate of the yield could be achieved from a S6-CMG calibration curve (Figure 7.1.2.5.3.) 3 0 0 0 0 0 0 §> 2 5 0 0 0 0 0 2000000 Ou . S 1 5 0 0 0 0 0 a3 1000000 5 0 0 0 0 0 0 20 40 6 0 80 100 S6-CMG (pmol) Figure 7.I.2.5.3. Calibration curve for S6-CMG. HPLC-fluorescence conditions are detailed in Section 7.1.2.2., system 3. n = 3 ± SD. 7.1.3. Characterisation of rabbit 0 6-CMdG anti-serum. In order to study the occurrence of 0 6-CMdG in DNA the synthesis and characterisation of 0 6-CMdG and a method for its immunochemical detection has been undertaken by N. Fairhurst (Harrison et al, 1997). In brief, 0 6-CMG was conjugated to bound serum albumin (BSA) and ovalbumin (OV) which resulted in the formation of an antigenic molecule. Rabbits that were immunised with Oe-CMG-BSA produced an antiserum of high titre that was selective for 0 6-CMdG and showed little cross-reactivity with other modified and unmodified nucleosides. Hence prior to the preparation of 0 6-CMdG immunoaffinity columns these existing antibodies were characterised by ELISA procedures to ensure that they were of the same high quality as previously reported (Fairhurst, 1990). Optimal conditions for ELISA were determined using a chequerboard procedure in which coating-antigen levels of lng - 10p.g per well and antiserum dilutions of 1 in 10 to 1 in 106 were tested. A reasonable absorbance was found using 5ng 0 6-CMG-OV per well and a 152 ------Chapter 7. Materials and methods dilution of 1 in 2 x 105. Under conditions of a competitive ELISA, 0 6-CMdG and 2'- deoxyguanosine were tested over a wide range (10 - 106fmol/well). The ELISA protocol was as follows Polystyrene microtitre plates with 96 wells (Dynatech M129B) were filled with a solution of coating antigen (40jiL PBS containing 5ng 0 6-CMG-0V) and dried overnight at 37 °C. These plates were then stored at room temperature and protected from dust and light until use. The plates were then washed with PBS 0.005% Tween (6 times) and dried by tapping onto absorbent paper towels. Standard solutions of 0 6-CMdG or 2/-deoxyguanosine were prepared in PBS so that their concentrations varied between lOfmol and 106 fmol/25pL. An aliquot of 0 6-CMdG in PBS (25fiL) was pipetted onto the plate in rows (8 wells) for each of the standard solutions. Usually two rows on the ELISA plate (first and tenth rows) were used for controls and to each of these rows PBS (25|xL per well) was added. A reference row (eleventh row) containing PBS (50fiL per well) was also used. Polyclonal rabbit antiserum (25pL of a 1:2 x 105 dilution in PBS of the neat antiserum) was added to each well in the plate except those in the reference row. The plate was then incubated for 90 min at room temperature, after which time the supernatant liquid was decanted from the ELISA plate by inversion and a quick flick of the wrist, and the plate then washed in PBS/Tween six times. Horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (Sigma, 50pL of a 1:103 dilution in PBS) was added to every well on the plate. The plate was then re-incubated at room temperature for 90 min. After this time had elapsed, the supernatant was discarded and the plate washed with PBS/Tween six times in a new bath and once with distilled water. Enzyme substrate in aqueous citrate buffer pH 5.3 (50pL) from a solution (lOmL) containing 3',3',5',5'-tetramethylbenzidene ( prepared by the addition of lmg in lOOpL dimethylsulphoxide) and H20 2 (2p.L of a 30% w/w solution) was added to each well of the plate, and the plate incubated for 15min at room temperature to allow colour development to occur. 1M HC1 (50pL) was added to each well of the plate, the contents of which were then measured by an automatic plate reader and the data processed using the Genesis software package (Labsystems). Generally the control wells of the plate gave an optical density of approximately 0.7 after subtraction of the reference (blank) wells. 153 ------Chapter 7. Materials and methods 7.1.4. Preparation and characterisation of immunoaiTinity columns for 0 6-CMdG. 7.1.4.1. Materials and chemicals. Immunoaffmity column kits (polystyrene columns and polyethylene frits) and dimethylpimelimidate were purchased from Pierce. PBS was made up using tablets from Oxoid. Buffer salts, solvents and water were from Sigma and were all of an HPLC analytical grade. 7.1.4.2. Preparation o f columns The procedure for making gel sufficient for 5 x lmL columns was as follows: A crude IgG fraction was prepared according to the method of Friesen et al (1991). Briefly 0 6-CMdG rabbit antiserum (5mL) was placed in a small beaker (25mL) to which cold saturated ammonium sulphate was added to a final concentration of 40% (i.e. 3.33mL) and the solution stirred for 5 minutes. The solution was then transferred to a plastic centrifuge tube and centrifuged at 3000g for 15 minutes, after which the supernatant was discarded and the precipitate washed twice with cold 50% saturated ammonium sulphate. The pellet was then resuspended in PBS (5mL) and dialysed overnight against PBS (3L). The dialysate was centrifuged at 3000g to remove suspended matter and supernatant used directly in the next step. Protein A - Sepharose CL 4B (5mL) was washed well with Tris buffer (0.1 M, pH 7.4) and suspended in the same buffer to a total volume of lOmL. The IgG fraction (5mL) was added and the mixture stirred end over end for 30 minutes at room temperature. The gel was washed several times with Tris buffer to remove unbound IgG. The gel was then washed twice with 20mL of a triethanolamine solution (0.2 M, pH 8.2) and resuspended in dimethylpimelimidate solution (Pierce, 40mL, 20mM freshly made up in triethanolamine buffer), placed in a polypropylene screw-cap centrifuge tube and made up to lOOmL with the same solution. This solution was then stirred end over end for a further 45 minutes at room temperature. The gel was recovered by low-speed centrifugation and treated with aqueous buffered ethanolamine (lOOmL, 20mM in triethanolamine buffer) for 5 minutes to block unreacted cross-linking agents. The gel was then recovered and washed several times with PBS-azide (0.02%). The gel was poured into polystyrene minicolumns in lmL aliquots and 154 ______Chapter 7. Materials and methods maintained in place by use of hydrophobic plastic frits (both below and on top of the gel). Prior to use, the columns were washed extensively with PBS azide (0.02%) and stored at 4°C. 7.1.4.3. Characterisation o f immunoaffinity columns for Cf^CMdG. [3H]-06-CMdG (lOpJL, 17.2ng, 950dpm) in 2ml PBS/azide 0.02% was applied to a immunoaffinity column, followed by a further 3ml PBS/azide 0.02%. The column was then washed with lOmL of ultra pure water. One millilitre fractions of the column eluate were collected throughout directly into scintillation vials, and 3 ml Hydrofluor added prior to scintillation counting. Various elution conditions were tried, these included acetic acid (1M, 5mL), 50-80% aqueous methanol (5mL), 50% aqueous DMSO (5mL), formic acid (1M, 5mL), trifluoroacetic acid ((TFA) 1M, 5mL), TFA (0.1M, 5mL) and 70-80% isopropanol. Quantitative elution with no carry over of the [3H]-06-CMdG was obtained with 1M TFA (5mL). Regeneration of the columns was obtained by washing with PBS/azide 0.02% (15ml). 7.1.4.4. Capacity determination for Cf-CMdG immunoaffinity columns. The determination of the capacity for 0 6-CMdG was achieved using a simple saturation assay. PBS/azide 0.02% (2mL) containing [3H]-Oe-CMdG (17.2ng, 950dpm) and 0 6-CMdG (O-lOOOng) were applied to the immunoaffinity columns. The columns were then washed with PBS/azide 0.02% (3mL) and water (lOmL), elution with 1M TFA (5mL) was then carried out. The eluate was collected directly into scintillation vials. Liquid scintillation fluid (Hydrofluor, 3mL) was added to each vial, and the radioactivity determined by scintillation counting. 7.1.5. Preparation and characterisation of immunoaffinity columns for 0 6-MedG. 7.1.5.1. Materials and chemicals. Immunoaffinity column kits (polystyrene columns and frits) and dimethylpimelimidate were purchased from Pierce. PBS was made up using tablets from Oxoid. Buffer salts, solvents and water were from Sigma and were all of an HPLC analytical grade. HPLC-methanol was for fluorescence applications. 155 ------Chapter 7. Materials and methods 7.1.5.2. Preparation o f columns. 0 6-MedG monoclonal antibody (provided by D. Cooper) which had been freeze-dried was reconstituted in lmL of water to give a titre of approximately 1/10, 000 and a protein concentration of 1.7mg/mL. This solution was then placed in a small beaker (25mL) to which cold saturated ammonium sulphate was added to a final concentration of 40%, stirred for 5 minutes and transferred to a plastic centrifuge tube and centrifuged at 3000g for 15 minutes, after which, the supernatant was discarded and the precipitate washed twice with cold 50% saturated ammonium sulphate. The pellet was then resuspended in PBS (5mL) and dialysed overnight against PBS (3L). The dialysate was centrifuged at 3000g to remove suspended matter and supernatant used directly in the next step to make immunoaffinity columns. The same process as detailed in Section 7.1.4.2. was followed except that only 2mL of Protein A - Sepharose CL 4B or Protein D - Sepharose CL 4B were used and the protocol was scaled down accordingly. Once the affinity Protein A-Sepharose gel had been decanted it was diluted down with 3mL of Sepharose CL 4B, to enable 5 x lmL column to be made. 7.1.5.3. Characterisation o f immunoaffinity columns for Cf-MedG. Cooper et a l (1992) had already used this monoclonal antibody for 0 6-MedG to prepare immunoaffinity columns using a different approach and found elution could be achieved using a methanol solution. [3H]-06-MedG (10pL, 13.45ng, 900dpm) in 2ml PBS/azide 0.02% was applied to an immunoaffinity column, followed by a further 3ml PBS/azide 0.02%. The column was then washed with lOmL of ultra pure water. One millilitre fractions of the column eluate were collected throughout directly into scintillation vials, and 3ml Hydrofluor added prior to scintillation counting. Various aqueous methanol elution (60-80%) conditions were explored and quantitative elution of the [3H]-06-MedG was obtained with all of these methanol solutions. However, the 80% methanol resulted in the sharpest elution peak in the smallest volume and the columns could still be regenerated with 15mL PBS/azide 0.02%. 7.1.5.4. Capacity determination for Cf-MedG immunoaffinity columns. The determination of the capacity for 0 6-MedG was achieved using a simple saturation assay as in Section 7.1.4.4. [3H]-06-MedG (13.45ng, 900dpm) and 0 6-MedG (0-1500ng) were applied to the immunoaffinity columns which were then washed with PBS/azide 0.02% 156 ------Chapter 7. Materials and methods (3mL) and water (lOmL). Elution with 80% methanol (4mL) was then carried out. The eluate was collected directly into scintillation vials. Liquid scintillation fluid (Hydrofluor, 3mL) was added to each vial, and the radioactivity determined by scintillation counting. 7.1.6. Immunoaffinity work for S6-CMdG. 7.1.6.1. Binding and elution o f fHJ-S6-CMdG using Cf-CMdG immunoaffinity columns. [3H]-S6-CMdG (IOjlxL, 29.5ng, lOOOdpm) in 2ml PBS/azide 0.02% was applied to a immunoaffinity column, followed by a further 3ml PBS/azide 0.02%. The column was then washed with lOmL of ultra pure water. The standard 0 6-CMdG elution protocol was used, 5mL of 1M TFA and lmL fractions of the column eluate were collected throughout directly into scintillation vials, and had 3ml Hydrofluor added prior to scintillation counting. 7.1.6.2. Determination o f Of-CMdG column capacity for 0 6-CMdG using fH J- Sf-CMdG as the marker compound. [3H]-S6-CMdG (lOpL, 29.5ng, lOOOdpm) in 2ml PBS/azide 0.02% with Oe-CMdG (0-500ng) were applied to the immunoaffinity columns which were then washed with PBS/azide 0.02% (3mL) and water (lOmL). Elution with 1M TFA (5mL) was then carried out. The eluate was collected directly into scintillation vials. Liquid scintillation fluid (Hydrofluor, 3mL) was added to each vial, and the radioactivity determined by scintillation counting. 7.1.6.3. Determination o f binding o f fH ]- S6-CMdG to the Cf -CMdG columns in the presence o f DNA digests. Calf-thymus DNA (0.5-5mg) was digested as detailed in Section 7.1.8.2. and to each digest solution [3H]-S6-CMdG (10|iL, 29.5ng, lOOOdpm), 2mL PBS/azide 0.02% were added. These were then applied to 0 6-CMdG immunoaffinity columns and the standard washing and elution protocol carried out. Radioactivity eluting with the 1M TFA was determined by the collection of lmL fractions directly into scintillation vials to which 3mL of Hydrofluor was added. 157 ------Chapter 7. Materials and methods 7.1.7. HPLC Optimisation for 0 6-alkyIguanine adducts. 7.1.7.1. Of-CMG System 1. Reverse-phase (RP) HPLC was carried out using a Techsphere 5pm ODS reverse- phase column (25cm x 4.6 mm) with a prefilter at a flow rate of l.OmL/min. A step gradient elution system was employed; 0-10 min 0.1M triethylammonium acetate (TEA) (pH 7) with 10% methanol, 10-20min 0.1M TEA (pH 7) with 15% methanol, going back to initial conditions over a further lOmin. Peaks were monitored by fluorescence with excitation wavelength at 286nm and emission wavelength at 373nm. Injection loop volume was lOOpL. System 2. RP-HPLC was performed using a Shandon 3pm ODS (25cm x 2mm) column with a prefilter and an isocratic flow at 0.2mL/min with 10% methanol and 0.1M TEA buffer (pH 7). Peaks were monitored by fluorescence with excitation wavelength at 286nm and emission wavelength at 378nm. Injection loop volume was lOpL. System 3. Analytical RP-HPLC was carried out using a RP-BDS C18 Hypersil (10 cm x 2.0 mm) narrowbore column with a prefilter, with an isocratic flow rate of 0.2mL/min of 0.1M TEA with 10% methanol. Peaks were detected by fluorescence; excitation wavelength 286nm, emission wavelength 378nm. Injection loop volume was lOpL. System 4. Analytical RP-HPLC was carried out using a RP-BDS C18 Hypersil (10 cm x 2.0 mm) narrowbore column with a prefilter, with an isocratic flow rate of 0.2ml/min of 0.1% Heptafluorobutyric acid (HFBA) with 10% methanol. Peaks were detected by fluorescence; excitation wavelength 286nm, emission wavelength 378nm. Injection loop volume was lOpL. 7.1.7.2. Q*-MeG System 1. Reverse-phase (RP) HPLC was carried out using a Techsphere 5pm ODS reverse- phase column (25cm x 4.6 mm) with a prefilter at a flow rate of l.OmL/min. A step gradient elution system was employed; 0-10min 0.1M triethylammonium acetate (TEA) (pH 7) with 10% methanol, 10-20min 0.1M TEA (pH 7) with 15% methanol and 20-30min 0.1M TFA 158 Chapter 7. Materials and methods (pH 7) with 20% methanol going back to initial conditions over a further lmin. Peaks were monitored by fluorescence with excitation wavelength at 286nm and emission wavelength at 378nm. Injection loop volume was lOOpL. System 2. RP-HPLC was performed using a Shandon 3pm ODS (25cm x 2mm) column with a prefilter and an isocratic flow at 0.2mL/min with 15% methanol and 0.1M TEA buffer (pH 7). Peaks were monitored by fluorescence with excitation wavelength at 286nm and emission wavelength at 378nm. Injection loop volume was lOpL. System 3. Analytical RP-HPLC was carried out using a RP-BDS C18 Hypersil (10 cm x 2.0 mm) narrowbore column with a prefilter, with an isocratic flow rate of 0.2mL/min of 0.1M TEA with 15% methanol. Peaks were detected by fluorescence; excitation wavelength 286nm, emission wavelength 378nm. Injection loop volume was lOpL. System 4. Analytical RP-HPLC was carried out using a RP-BDS C l8 Hypersil (10 cm x 2.0 mm) narrowbore column with a prefilter, with an isocratic flow rate of 0.2mL/min of 0.1% Heptafluorobutyric acid (HFBA) with 15% methanol. Peaks were detected by fluorescence; excitation wavelength 286nm, emission wavelength 378nm. Injection loop volume was lOpL 7.1.8. DNA digestion to nucleosides. 7.1.8.1. Chemicals. Calf thymus DNA, Nuclease Pi (E.C.3.1.30.1.) stored at -20°C in lmMZnCl2, alkaline phosphatase type (111) (E. coli) (E.C.3.1.3.1.) and acid phosphatase type 1 from wheat germ (E.C.3.1.3.2.) stored at -20°C at a concentration of 125mg/mL in water were obtained from Sigma. All other chemicals were reagent or HPLC grade as required. 7.1.8.2. Enzymatic digestion o f DNA. Calf thymus DNA was dissolved in ddH20 (pH 7.4) to give a stock solution of DNA at a concentration of 5mg/mL. Enzyme hydrolysis was performed following the method outlined 159 ------Chapter 7. Materials and methods by Beranek et a l (1980). DNA samples were hydrolysed in 50mM BisTris /ImM MgCl2 (pH 6.5) at 50°C for 8h, using Nuclease P} (24 units), bacterial alkaline phosphatase (2.4 units) and wheat germ acid phosphatase (0.3 units) per mg DNA (to a final concentration of lmg DNA/mL). The reaction was stopped by heating at 100°C for 5min and was then centrifuged to remove the denatured enzyme proteins. HPLC analysis of standard nucleosides, as well as a sample of digested DNA, was performed on the RP-HPLC system detailed below in Section 7.1.8.4. Standard nucleosides are freely soluble in water and stock solutions were diluted down to generate a calibration curve for 2’dG in the range of 0-1000pmol. As Ijug of DNA contains 3.10559 x 10‘9 moles of normal nucleosides, lpg of DNA will contain 7.763 x 1010 mol 2’dG (776.3pmol). These indicated that after 8hr in the digestion conditions the DNA had indeed been hydrolysed to nucleosides. However, different reaction conditions were also employed, such th a t: a) incubation at 50°C for 13hr and b) all enzyme concentrations were doubled with incubation at 50°C for 13hr. 7. 1.8.3. Acid hydrolysis o f DNA. Calf thymus DNA (IO jjL, 50pg) from the stock prepared in Section 7.1.8.3. was acid hydrolysed by the addition of 0.24mL 0.1M HC1 and heating at 100°C for 30min. This solution was then neutralised prior to injection onto HPLC by the addition of 0.1M NaOH (0.25mL). An injection volume of lOpL should therefore contain lpg of acid hydrolysed DNA. A calibration curve to estimate the amount of guanine liberated was constructed from a stock solution of guanine (0.54mg initially dissolved in 0.1M HC1 (lOOpL) and then made up to lmL with water) which was serially diluted to give a range of 0-1000pmol. 7.1.8.4. HPLC analysis o f DNA hydrolysates. The HPLC conditions consisted of a reverse-phase (RP)-HPLC using a Hypersil 3pm BDS reverse-phase column (10cm x 2mm) with a prefilter at an isocratic flow rate of 0.2mL/min of 0.1M triethylammonium acetate (TEA) (pH 5) with 4% methanol. UV-absorbance was monitored at a wavelength of 260nm. Injection loop volume was lOpL. 160 Chapter 7. Materials and methods 7.1.9. Eluate sample preparation prior to HPLC-analysis. 7.1.9.1. (/-CM dG eluate. In order to ensure complete hydrolysis of the 0 6-CMdG in the immunoaffinity eluate prior to its quantitation, a study to simulate the immunoaffinity conditions was undertaken. To 5mL of 1M TFA, 0 6-CMdG (lOOpmol, lOpL) was added and vortexed. This solution was then heated at 50°C for 120min. Aliquots containing lOpmol of 0 6-CMdG (0.5mL) were removed at the following time points 0, 10, 20, 30, 45, 60 and 120min, pipetted into a 15mL tube and frozen in liquid nitrogen. Prior to the removal of an aliquot the solution was quickly vortexed. The samples were then freeze-dried, re-dissolved in lmL of water and brought to dryness on the speed-vac. Each sample was then, just prior to RP-HPLC analysis (Section 7.1.7.1.,system 4), dissolved in 20pL of 0.1% HFBA. Conditions of 50°C for 60min were found to be the most appropriate and were used as routine for all further immunoaffinity eluate sample preparations prior to analysis by HPLC. 7.1.9.2. (/-M edG eluate. A time course to investigate the conditions for the hydrolysis of 0 6-MedG to 0 6-MeG was performed. Standards of 0 6-MedG (20pmol,5pL) diluted with water from a stock solution of 0 6-MedG (0 6-MedG (Img) dissolved in lOOpL of 0.1M HC1 which was then diluted with PBS to give a solution which contained lmg/mL) were pipetted into 15mL tubes and taken to dryness using a speedvac.To each tube, 0.1M HC1 (lOpL) was added and the tube heated at 50°C for one of the following time durations, 0, 5, 15, 20 or 30mins, after which each tube was removed from the heating block and re-dried. Just prior to injection onto RP-HPLC (Section 7.1.7.2., system 4) the sample was redissolved in 20pL of 0.1% HFBA. Injection volumes of lOpL were analysed which would correspond to lOpmol 0 6-MeG when hydrolysis was complete. 161 Chapter 7. Materials and methods 7.1.10. Method Validation. 7.1.10.1. Isolation o f O6-CMdG or (f-M edGfrom DNA digests by immunoaffinity purification. Calf thymus DNA (0.5, 1, 1.5, 2, 2.5, 3mg) spiked with eitherIO jiL of [3H]-06-CMdG (17.2ng, 950dpm) or [3H]-06-MedG (13.45ng, 900dpm) were digested as described in Section 7.1.8.2. To each digest, PBS/azide 0.02% (2mL) was added, and the sample briefly vortexed before being loaded onto the appropriate immunoaffinity column. Blanks containing no DNA but the same amount of [3H]-06-CMdG (17.2ng, 950dpm) or [3H]-06-MedG (13.45ng, 900dpm) in PBS/azide 0.02% (2mL) were also loaded onto immunoaffinity columns. The standard washing protocols of a further 3mL of PBS/azide 0.02% and lOmL of water were carried out and elution of the immunoaffinity columns was achieved as detailed in Sections 7.1.4.3. and 7.1.5.3. with lmL fractions of the column eluate being collected throughout directly into scintillation vials. Each vial had 3mL of Hydrofluor added prior to scintillation counting. 7.1.10.2. Overall immunoaffinity purification protocol for 0 5-CMdG and 0 6-MedG from DNA. Calf thymus DNA (2.5mg) spiked with a) Os-CMdG (0-50pmol), b) 0 6-MedG (0-50pmol) or c) 0 6-CMdG (0-50pmol) and 0 6-MedG (0-50pmol) were digested as described in Section 7.1.8.2. To each digest, PBS/azide 0.02% (2mL) was added, and the sample briefly vortexed before being loaded onto the 0 6-CMdG immunoaffinity column which eluted directly onto a 0 6-MedG immunoaffinity column washed with a further 3mL of PBS/azide 0.02%. The columns were then separated for an independent wash of water (lOmL) and elution with either 1M TFA (0 6-CMdG) or 80% methanol (0 6-MedG). Eluates were collected in 15mL tubes. Each eluate was then prepared for RP-HPLC analysis as detailed in Sections 7.9.1.1. and 7.9.1.2. and redissolved in 0.1% HFBA (25pL). This allowed for duplicate injections of lOpL from each sample. 162 Chapter 7. Materials and methods 7.2. M aterials and Methods for Chapter 3. 7.2.1. Safety Warning. Reagents which generate carboxymethyl diazonium ions are alkylating agents and should be treated with extreme caution. Unused solutions of N-carboxymethyl-N-nitrosocompounds should be decomposed in 0.1M sodium hydroxide solutions overnight in a fume cupboard. Unused solutions of diazoacetic acid derivatives should be decomposed by treatment with 1M aqueous acetic acid overnight in a fume cupboard. 7.2.2. Chemicals and Apparatus. Azaserine, calf thymus DNA, 0 6-MedG and 2’-deoxynucleosides were purchased from Sigma (Dorset, UK). APNG was synthesised according to the published procedure (Challis & Latif, 1990) as was mesylooxyacetic acid (Havbrandt & Wachtmeister, 1968) by R. Jukes. Potassium diazoacetate (KDA) was synthesised by R. Jukes by the alkaline hydrolysis of ethyl diazoacetate according to Kreevoy (1970) and suitable dilutions of the resulting solution were used without further purification for reactions with DNA. Attempts to isolate the salt resulted in decomposition and polymerisation (Kreevoy, 1970). All solvents and buffers were of the highest analytical grade and were purchased from Sigma or Fisons. Apparatus was the same as detailed in Section 7.1. 7.2.3. Treatment of DNA with carboxymethylating agents. Calf thymus DNA (2.5mg) from a stock solution (5mg/ml in PBS, pH 7.4) was treated with either APNG or KDA to give 0.5,1, 2.5 and 5mM solutions or azaserine (1, 2.5, 5, and lOmM solutions) and left gently stirring at 37°C in the dark overnight. Each reaction was done in triplicate for each compound and treatment concentration. After treatment, DNA was precipitated from the reaction medium with sodium acetate (0.1 volume, 2.5M) and cold ethanol (2 volumes), centrifuged gently (3000g, 5mins) and the DNA washed several times with ethanol. The DNA pellet was recovered and evaporated down to dryness (speedvac) and resuspended in water to the original volume. 163 ______Chapter 7. Materials and methods 7.2.4. Determination of 0 6-CMG and 0 6-MeG in DNA. Enzyme hydrolysis was performed following the method outlined by Beranek et al (1980) detailed in Section 7.1.8.3. In order to quantitate 0 6-CMG by a molar ratio to the normal 2’deoxyguanosine in a sample, lOpL of the DNA hydrolysate was withheld from the immunoaffinity purification protocol for separate analysis as detailed in Section 7.1.8.4. For the determination of 0 6-CMG and 0 6-MeG in DNA the overall analytical protocol outlined in Section 7.1.10.2. was followed for both samples of DNA treated with carboxymethylating agents and DNA spiked with standards of 0 6-CMdG and 0 6-MedG for the generation of calibration curves. The method is summarised in Figure 2.10.3. The volume of 0.1% HFBA used to dissolve the sample for analysis by RP-HPLC was varied according to the amount of 0 6-CMG or 0 6-MeG present in the sample to keep the level of adduct in the middle of the calibration lines where possible. 7.2.5. KDA pH study. Calf thymus DNA (2.5mg) from a stock solution (5mg/mL in H20) was pH adjusted with either 2M sodium hydroxide or 2M aqueous phosphoric acid to give DNA solutions with pHs of 6.5, 7.5, 8.5 and 9.5. DNA aliquots (3 x 0.5mg) from each pH group, were then treated with KDA to give a final concentration of 5mM (3.14p.L of a 80mM stock) and left gently stirring at 37°C in the dark overnight. After treatment, DNA was precipitated from the reaction medium with sodium acetate (0.1 volume, 2.5M) and cold ethanol (2 volumes), centrifuged gently (3000g, 5mins) and the DNA washed several times with ethanol. The DNA pellet was recovered and evaporated down to dryness (speedvac) and resuspended in water to the original volume. The determination of the 0 6-CMG and 0 6-MeG was then undertaken as detailed previously in Section 7.2.4. 164 ______Chapter 7.Materials and methods 7.3. M aterials and methods for Chapter 4. 7.3.1. The immunoslot blot assay. 7.3.1.1. Chemicals and apparatus. Calf thymus DNA and Tween-20 were purchased from Sigma Chemical Co. Goat anti-rabbit IgG horseradish peroxidase conjugate and Supersignal ULTRA Chemiluminescent substrate were both obtained from Pierce. KDA and anti-rabbit 0 6-CMdG serum were available from previous work and Marvel (99% fat free) was from a local supermarket. The Minifold II microfiltration apparatus and nitrocellulose filters (0.1pm PH79) were from Schleicher & Schuell and the ECL (Western blotting detection reagent) and film (Hyperfilm-ECL) were purchased from Amersham Life Sciences. A Branson sonifier, Gallenkamp vacuum oven, Perkin Elmer UV/VIS spectrometer (type Lambda 2), Molecular Dynamics Computing Densitometer, X-OGRAPH Compact X2 film developer and Stuart Scientific 3D rocking platform were used. 7.3.1.2. Alkylated DNA standard Carboxymethylated standard DNA was prepared by reacting calf thymus DNA, 5mg (dissolved in PBS, pH 7.4, 5mg/mL) with KDA to a final concentration of 5mM. The carboxymethylation reaction was allowed to proceed at 37°C, overnight in the dark. The DNA was precipitated with sodium acetate and ethanol and washed three times with absolute ethanol. The concentration of 0 6-CMdG was determined by the immunoaffinity-HPLC fluorescence protocol detailed earlier (Section 7.2.4.) using an aliquot of DNA (0.5mg). A range of standards was achieved by diluting the treated DNA with control calf thymus DNA which was also quantitated spectrometrically and by HPLC-UV (Section 7.3.4.2.). 7.3.1.3. Immunoslot blot assay. Immunoslot blots were carried out essentially as described by Nehls et al, (1984) Briefly, DNA samples (3.5pg in 140pL of lOmM K2HP04, pH7) containing various amounts of carboxymethylated and / or unmodified DNA were sonicated for 10 seconds and then heat- denatured for lOmin in a boiling water bath, quickly chilled on ice and mixed with an equal 165 Chapter 7. Materials and methods volume of 2M ammonium acetate. Single-stranded DNA was then immobilised on nitrocellulose (NC) filters ( 0.1 p.m PH79) using a Minifold 11 microfiltration apparatus. NC filters were presoaked in 1M ammonium acetate. After application of 80fiL containing ljig of DNA, the slots were rinsed with 1M ammonium acetate (200pL). The filters were then removed from the support and baked at 80°C for lh, following which they were blocked for lhr with PBS-Tween-20 (0.1%) (PT)-5% marvel and washed twice for 5min with PT. The NC filters were then incubated overnight at 4°C with the anti-06-CMdG rabbit serum diluted 1:5000 in 20mL of PT containing 0.5% non-fat milk powder (Marvel). After the incubation with the primary antibody the NC filters were washed with PT in a small tray, for lmin and then twice for 5min using 30mL per time. The bound antibodies were then reacted with goat anti-rabbit IgG horseradish peroxidase conjugate , diluted 1:40,000 in 16 mL PT containing 0.5% non-fat milk powder. The incubation was carried out for lh at room temperature. The NC strips were then washed with 30mL PT (1 x 15 min, 2 x 5min) afterwards not letting the filter dehydrate. Enzymatic activity was visualised by bathing the NC filter in chemiluminiscence reagents prepared just prior to use by combining 5mL of reagent 1 with 5mL reagent 2 and incubating for the recommended time, either lmin (ECL) or 5min (Supersignal ULTRA). Initially the reagent used was ECL (Amersham), however a better response was found with the Supersignal ULTRA obtained from Pierce. Filters were then blotted and wrapped in clingfilm and exposed for varying times to a film (Hyperfilm- ECL; Amersham). These were subsequently developed using a X-OGRAPH Compact X2 film developer. 7.3.1.4. Densitometry o f X-ray films. Densitometric evaluation of x-ray films was performed using a Molecular Dynamics Densitometer with Image Quant software. Detected bands were then subsequently quantitated using the Spot Finder program. Standard curves were constructed by plotting light intensities against the known amount of 0 6-CMdG present in a band. 166 ______Chapter 7. Materials and methods 7.3.2. In vivo APNG study. Six female Fisher 344 rats, each weighing approximately 120g were obtained from the suppliers and left to acclimatise for 2 weeks prior to dosing. APNG was given in phosphate buffered saline by gastric intubation at a dose of 4mg per rat (in 0.5mL) to 4 rats. Two rats served as controls and received only phosphate buffered saline (0.5mL) by gastric intubation. Four hours after administration of the APNG, 2 dosed and 1 control animal were culled by C 02, and the liver, stomach and intestine tissues were excised, immediately frozen in liquid nitrogen and stored at -80°C. After 24hr the remaining animals were culled and the same organs removed and frozen for DNA extraction at a later date. 7.3.3. In vivo KDA study. Twenty five female Fisher 344 rats, each weighing approximately 150g were obtained from the suppliers and left to acclimatise for 2 weeks prior to dosing. Potassium diazoacetate was given in phosphate buffered saline by gastric intubation at doses of 0.5, 2.5, or 5mg per rat (in 0.5mL) to groups of 6 rats, where each group represented a single dose level. One further group of 7 rats served as controls and received only phosphate buffered saline by gastric intubation. The dosing was staggered by 30min between each dose level. The solutions of KDA were prepared immediately prior to administration and kept away from light to minimise decomposition. Exactly four hours after administration of the KDA half of each dose group and 3 control animals were anaesthetised and sacrificed. Subsequently, liver, stomach and intestine tissues were excised, slices of which were fixed in 70% ethanol for immunohistochemistry, and the remaining tissue immediately frozen in liquid nitrogen and stored at -80°C. Twenty four hours after dosing the remaining animals underwent the same protocol. Immunohistochemical samples were kept in 70% ethanol overnight with one change before being cassetted for slide preparation. 167 Chapter 7. Materials and methods 7.3.4. DNA extraction from rat tissues. 7. 3.4.1. Chemicals and Apparatus. Saturated phenol solution, phenol: chloroform: isoamyl alcohol (25:24:1), chloroform : isoamyl alcohol :(20:1), RNase A and Proteinase K were all obtained from Sigma Chemical Co. HPLC grade water was purchased from Fisons. A mechanical homogeniser was used and samples were centrifuged on either a Beckman GPR centrifuge or Hettich Zentrifugen EBA 12 desktop centrifuge depending on the tube size. 7.3.4.2. Phenol-chloroform extraction. Tissue samples were removed from the freezer and an appropriate amount weighed out, approximately 300mg of stomach and intestine or 200mg liver. The sample was then washed in lmL of ice cold Ten-9 buffer (50mM Tris base (50mL of a 1M Tris base stock solution) mixed with lOOmM EDTA in 800mL of water, adjusted to pH 8 with NaOH and made up to 1L) in a petri dish over ice, cut into small pieces and resuspended in 3mL of the same buffer. After thoroughly homogenising the tissue mechanically, the homogenate was transferred to a 15mL centrifuge tube and 250jig RNase A in lOpL distilled water added. This was mixed by inversion and the solution incubated at room temperature for lOmin, after which 250pL of a 10% SDS solution was added and incubated for a further lOmins at room temperature. The solution was then treated with Proteinase K (2.63mg) overnight at 37°C in a rocking incubator. The mixture was subsequently extracted with 5mL of a saturated phenol solution (rotated for lhr at room temperature) and centrifuged at 5000g for 15min at 4°C, following which the top layer was transferred to a clean tube to which 5mL of phenol: chloroform: isoamyl alcohol (25:24:1) was added. The mixture was again rotated and centrifuged and the top layer transferred to a clean tube followed by the addition of 5mL chloroform: isoamyl alcohol (20:1). This was rotated at room temperature for 30min and centrifuged at 5000g for 5min at 4°C. The top layer containing the DNA was transferred to a clean tube and precipitated by the addition of 3M sodium acetate (500pL) and isopropanol (4.4mL). The DNA was pelleted by centrifugation at 5000g for 15min at 4°C and the supernatant discarded. The pellet was 168 ------—— Chapter 7.Materials and methods transferred to an eppendorf tube in lOOpL of 70% ethanol, and re-centrifuged at 2000g for 6min, the supernatant removed and the pellet washed twice with absolute alcohol and allowed to air dry. The DNA was then dissolved in 400pL of Ten-9 buffer overnight, transferred to a 15mL tube and made up to 3mL with the same buffer and the above protocol repeated from the RNase A step, with the resultant DNA being dissolved in 500pL of HPLC grade water. 7.3.4. DNA quantitation for the immunoslot blot assay. 7.3.4.1. Chemicals and apparatus. Micrococcal nuclease was obtained from Sigma Chemical Co and calf spleen phosphodiesterase from Boehringer Mannheim. DNA quantitation required the use of a Perkin Elmer UV/VIS spectrometer (type Lambda 2) or HPLC instrumentation which comprised of a Waters 600E pump with a Rheodyne 7125 injector system and a Shimadzu SPO-GA UV spectrophotometric detector, connected to a Waters Millennium data system. 7.3.4.2. Enzymatic digestion o f DNA to deoxynucleoside- 3’-monophosphates. To determine the concentration and purity of DNA, an initial spectrophotometer scan was used and the ratio of A260nm/A280nm measured. For pure DNA this ratio should be between 1.7- 1.9. HPLC-UV analysis of the DNA digested to the deoxynucleoside-3’-monophosphates was also performed for a more accurate determination of concentration. This method of DNA digestion was chosen as the human DNA samples available (Chapter 5) were also to be used by a colleague using a 32P-postlabelling method for the detection of a different DNA adduct. From the spectrophotometric scan an approximate concentration of the DNA in a sample was determined. This enabled a volume equal to lOpg to be taken. This was then digested to the deoxynucleoside-3’-monophosphates by the method set up in the laboratory by Dr. C. Leuratti adapted from the published protocol by Jones et al (1991). To the DNA solution, 3jxL of SSCC buffer (lOOmM sodium succinate, 50mM calcium chloride, pH 6) was added, followed by 4pL of water, micrococcal nuclease (0.4 units/pL) and calf spleen phosphodiesterase (6 milliunits). The sample was then briefly vortexed, spun down and incubated at 37°C overnight, after which time the sample was lyophilised, redissolved in 169 Chapter 7. Materials and methods water (IOO jllL) and analysed by HPLC with the system described in Section 7.3.4.2. In order to quantitate the amount of DNA present a standard curve for deoxyguanosine-3’- monophoshate was generated (Figure 7.3.4.2.1.) and a typical chromatogram for the digestion of a rat stomach DNA can be seen in Figure 7.3.4.2.2. 20 .. 15 - I 10 .. 5 -- 0 200 400 600 800 1000 dGMP (pmol) Figure 7.3.4.2.I. Calibration curve for deoxyguanosine-3’-monophoshate. Points represent the mean of three injections ± SD. r2 = 0.9966 40.00 — 30.00 — I 2 0 .0 0 — 1 0 .0 0 — 0.00 0.00 5.00 10.00 Minutes Figure 7.3.4.2.2. HPLC-UV chromatogram of a rat stomach DNA digested to deoxynucleoside-3’-monophosphates. Peak 1; deoxycytidine-3’-monophoshate, peak 2; deoxyguanosine-3’-monophoshate, peak 3; thymidine-3’-monophoshate, peak 4; deoxyadenosine-3 ’-monophoshate. 170 ______Chapter 7. Materials and methods 7.3.4.2.1. RP-HPLC-UVof 3’monophoshate deoxynucleosides. The HPLC conditions consisted of a reverse-phase (RP)-HPLC using a Hypersil 3pm BDS reverse-phase column (10cm x 2mm) with a prefilter at a isocratic flow rate of 0.2mL/min of 0.1M triethylammonium acetate (TEA) (pH 5) with 4% methanol. UV-absorbance was monitored at a wavelength of 260nm. Injection loop volume was lOpL. 7.4. M aterials and Methods for Chapter 5. 7.4.1. Helicobacter pylori study. The overall purpose of this study was to compare the safety and efficacy of GR12231IX co prescribed with clarithromycin plus metronidazole or placebo on the long-term remission of symptoms and histological gastritis over 52 weeks in patients positive for Helicobacter pylori with non-ulcer dyspepsia. In conjunction with this, the measurement of DNA adducts in the gastric mucosa and white blood cells to assess the medium and long term effects of eradicating H. pylori was to be undertaken. This work was done in collaboration with Professors D. Forman and A.T.R. Axon and Drs P.Quirke, C. Schorah and S. Everett at The Centre for Digestive Diseases, Gastroenterology Unit, The General Infirmary, Leeds, to compare long-term symptom relief and resolution of gastritis following treatment with ranitidine bismuth citrate, clarithromycin plus metronidazole. 7.4.1.1. Study design. This was a multicentre, randomised, double-blind, placebo-controlled, parallel group study. The study involved patients diagnosed with non-ulcer dyspepsia and infected with Helicobacter pylori detected by a positive CLOtest™ on an antral or corpus biopsy, and subsequently confirmed by a 13C-urea breath test with H. /^/on-associated gastritis. The study included 320 patients (160 in each arm of the study) who were randomly allocated to receive either; 171 Chapter 7. Materials and methods 1) GR12231IX (ranitidine bismuth citrate) (400mg) bd co-prescribed with Clarithromycin (500mg) bd and Metronidazole (400mg) bd for 7 days or 2) Placebo for 7 days Patients then entered a 52 week follow-up phase. Ranitidine was provided as a rescue medication during the follow-up phase, one course (30 days of ranitidine (150mg) bd) was provided at the end of treatment and at 13, 26 and 39 weeks of follow-up. Patients attended for follow-up assessment (abdominal symptoms and H. pylori status by 13C-urea breath test) at 4, 13, 26, 39 and 52 weeks after the end of treatment or at any time if severe symptoms recurred. Endoscopy and histological gastritis was assessed pre-study and at 26 and 52 weeks after the end of the treatment, or at any time if severe symptoms recurred. 7.4.1.2. Samples collected. At the time of endoscopy on entry to the study, at 6 months and at 12 months, the following was performed. Seven antral biopsies were taken from within 5cm of the pylorus on the greater curvature and a further three from the corpus. One biopsy from each site was analysed by the CLOtest and a further 2 biopsies from each site were used for histological assessment, and two for the measurement of mucosal vitamin C. The remaining two antral biopsies were frozen and sent to Leicester for the measurement of DNA adducts. Five millilitres of gastric juice was aspirated through a fine bore catheter passed through the endoscope. Ten millilitres of blood were also taken for the measurement of white blood cells DNA adducts and plasma vitamin C. 7.4.1.3. Preparation o f DNA Samples for the determination o f DNA adducts. Extraction of DNA from the whole blood was carried out as detailed in Section 7.4.3. by J. Crawley and R. Singh and the subsequent quantitation of the DNA was also performed as detailed in Section 7.4.3. by K. Harrison. Gastric biopsies were pooled and the DNA extracted using the standard protocol detailed in the Qiagen Genomic DNA Handbook (pg 22-23) using a 20/G Qiagen Genomic-tip by either J. Crawley or R. Singh and quantitated using the methodology detailed in Section 7.4.3. by R. Singh. 172 ______Chanter 7. Materials and methods 7.4.2. Diet Study. This work was done in collaboration with S. Bingham and R. Hughes at the MRC Dunn Clinical Nutrition Centre (Cambridge). 7.4.2.1. Study Protocol Twelve healthy male volunteers were recruited and studied over four fifteen day dietary periods whilst living in the metabolic suite at the Dunn Clinical Nutrition Centre (Cambridge), where all food was provided and specimens could be collected. One volunteer dropped out following the first diet and was not replaced. Subjects were randomly assigned to each diet via a cross-over design. 7.4.2.2. Diets. Four dietary regimes were studied: 1. 420g meat 2. 420g meat with 400g of vegetables 3 .420g meat with 3g black tea 4. 420g meat with 400g vegetables and 3g black tea. The subjects are fed 420g of meat per day; lOOg at lunch (cold roast beef for sandwiches) + 320g for dinner as lasagna, sweet and sour pork or beefsteak on a three day rotation diet. For the meat and vegetable study, the meat diet is supplemented with 400g of frozen vegetables given as 133.3g petite pois, 133.3g brussel sprouts and 133.3g broccoli daily. This is given as a salad at lunch and a soup and side dish with the evening meal. Therefore, 1 day the subject will have pea salad, brussel sprout soup and broccoli with their evening meal, and this will be rotated the following day. During the meat and tea diet, the meat diet is supplemented with 3g of tea taken as 6 cups containing 500mg each (preweighed instant tea was given in sachets) and taken intermittently throughout the day. The meat, vegetable and tea diet involved the supplementing of the basic meat diet with both the vegetables and tea as stated above. The diets were all prepared in advance of the study and frozen until required, therefore the food used for the study was all from the same batch. Ultra pure deionised water was used for cooking and drinking throughout the study to control nitrate intake. 173 ______Chapter 7. Materials and methods The diets were kept isocaloric by using a caloric drink (Hycal) to make up the calories and the diets were also adjusted to meet the energy requirements of the volunteer. Fat intake was kept constant and the other major food groups varied as little as possible (Table 1A.2.2.) The body weight of the volunteers was recorded each day and should remain constant, if it did not increments were added to the diet to modify the body weight appropriately. Diet Protein Fat Carbohydrate NSP (% energy) (% energy) (% energy) (g/day) Meat 28 28 44 13 Meat + 31 28 41 27 Vegetables NSP - non-starch polysaccharides Table 7.4.2.2. Percentages of major food groups in the diets studied. 7.4.2.3. Diet study sample. Fasting blood samples were taken on days 13 and 15 of each dietary period and the DNA extracted at source by R. Hughes using the same protocol detailed in Section 7.4.3. The DNA samples were then sent to the Leicester MRC laboratory for quantitation as detailed in Section 7.4.3. and subsequently used for the determination of 0 6-CMdG in the DNA by the immunoslot blot assay (Section 7.3.1.3.). Determination of N-nitroso compounds (NOC) was performed by R. Hughes using the method of Pignatelli et al (1987). 7.4.3. Extraction and quantitation of DNA from whole blood. Frozen blood samples were defrosted and the DNA extracted according to the standard protocol detailed in the Qiagen Genomic DNA Handbook by J. Crawley, R. Singh, C. Leuratti and K. Harrison. In brief, lmL of ice-cold Cl Buffer (320mM, 5mM MgCl2, lOmM Tris/HCl, 1% Triton X-100, pH 7.5) and 3mL of ice-cold dH2Q were added to the whole 174 ______Chapter 7. Materials and methods blood (approximately lmL) and mixed by inverting the 15mL tube several times until the suspension became translucent. The sample was then incubated on ice for lOmin, after which the lysed blood was centrifuged at 1300g for 15min at 4°C and the supernatant discarded. The pelleted nuclei were then resuspended by vortexing in ice-cold Buffer Cl (lmL) and dH20 (3mL) and once again centrifuged as stated previously. If the pellet was not white after this stage, the wash step was repeated. The pellet was then re-suspended by vortexing in 5mL of Buffer G2 (800mM GuHCl, 30mM EDTA, 30mM Tris/HCl, 5% Tween-20, 0.5% Triton X- 100, pH 8) and 95jliL of Proteinase K (20mg/mL) was added and the sample incubated at 50°C for 60min. The sample was then vortexed for lOsec and applied to an equilibrated 100/G Qiagen Genomic-tip (equilibrated with 4mL of a buffer containing 750mM NaCl, 50mM MOPS, 15% ethanol, pH 7) and allowed to enter the resin by gravity flow. The Qiagen Genomic-tip was subsequently washed with 2 x 7.5mL of a wash buffer (1M NaCl, 50mM MOPS, 15% ethanol, pH 7) and then eluted into a 15mL tube with 5mL of elution buffer (1.25M NaCl, 50mM Tris/HCl, 15% ethanol, pH 8.5) which was wanned to 50°C. To precipitate the DNA, isopropanol (3.5mL) was added and the tube inverted for 10-20 times. The DNA was then pelleted (5000g, 4°C, 15min) and the supernatant discarded. The DNA was then transferred to an eppendorf tube in 0.5mL of ethanol, re-centrifuged and washed twice with ethanol (lmL). The pellet of DNA was then dissolved in ultra pure water (lOOpL) on a shaker overnight. The DNA was quantitated by taking an aliquot of the sample (5pL) diluted in water (45pL). This was then analysed spectrophotometrically and the A260nm and A2g0nm determined. This gave a rough guide to the purity and yield of DNA. Enzymatic digestion and RP-HPLC-UV analysis of the sample (5pL diluted in 5pL of water) according to the protocol detailed in Section 7.3.4.2. was also carried out and a typical chromatogram of purified white blood cell DNA can be seen in Figure 7.4.3. 175 mV 10.00 0.00 20.00 — 50.00 30.00 — 30.00 40.00 blood DNA samples (Section 7.4.3.). (Section samples DNA blood whole the asfor manner same the in blot immunoslot the to subjected being sample the to 70.00 — 70.00 60.00 — 60.00 handbook) using 20/G Qiagen Genomic-tips. Quantitation of the DNA was determined prior prior determined was DNA the of Quantitation Genomic-tips. Qiagen 20/G using handbook) Genomic Qiagen the of 22 (pg Isolation DNA Tissue for protocol Qiagen the using 7.4.4. Extraction and quantitation of DNA from gastric biopsies. gastric from DNA of quantitation and Extraction 7.4.4. Gastric pinch biopsies were pooled for an individual and the DNA extracted by J. Crawley J.Crawley by extracted DNA the and individual an for pooled were biopsies pinch Gastric Figure 7.4.3. HPLC chromatogram of enzymatically digested blood DNA. Peak 1; Peak DNA. blood digested enzymatically of chromatogram HPLC 7.4.3. Figure 0.00 thymidine-3’-monophoshate, peak 4; deoxyadenosine-3’-monophoshate. Digestion was was Digestion deoxyadenosine-3’-monophoshate. 4; peak thymidine-3’-monophoshate, deoxycytidine-3 ’-monophoshate, peak 2; deoxyguanosine-3 deoxyguanosine-3 ’ 3;2; peak peak -monophoshate, ’-monophoshate, deoxycytidine-3 carried out as detailed in Section 7.3.4.2. HPLC conditions as detailed in section 7.3.4.2. section in detailed as conditions HPLC 7.3.4.2. Section in detailed as out carried — 5.00 s e t u n i M 176 10.00 ChapterMaterials 7. and methods 15.00 Chapter 8. References. 177 Chapter 8. References 8.0. References. Anderson, D. and Blowers, S.D. (1994): Limited cancer bioassay to test a potential food chemical.Lancet. 344. 343-344. Armstrong M.D. and Stave, U. (1973): A study of plasma free amino acid levels. Metabolism. 22. 571-578. Autrup, H. (1991): Human exposure to genotoxic carcinogens: methods and their limitations. J. Cancer Res. Clin. Oncol. 117. 6-12. Balmain, A. and Brown, K. (1988): Oncogene activation in chemical carcinogenesis. Adv. Cancer Res. 51. 147-182. Badawi, A.F., Mostafa, M.H., Aboul-Azm, T., Haboubi, N.Y., O’Connor, P.J. and Cooper, D.P. (1992): Promutagenic methylation damage in bladder DNA from patients with bladder cancer associated with schistosomiasis and from normal individuals. Carcinogenesis. 13. 877- 881. Bartholomew, B. and Hill, M.J. (1984): The pharmacology of dietary nitrate and the origin of urinary nitrate. Food Chem. Toxicol. 22. 789-795. Bartsch, H., Ohshima, H., Pignatelli, B. and Calmels, S. (1989): Human exposure to endogenous N-nitroso compounds : quantitative estimates in subjects at high risk for cancer of the oral cavity, oesophagus, stomach and urinary bladder. Cancer Surv. 8. 335-362. Bartsch, H. and Spiegelhalder, B. (1996): Environmental exposure to N-nitroso compounds (NNOC) and precursors: an overview. Eur. J. Cancer Prevention. 5s. 11-18. Bavin, P.M.G., Darkin, D.W. and Viney, N.J. (1982): Total nitroso compounds in gastric juice. In: H. Bartsch, I.K. O’Neill, M. Castegnaro and M. Okada. (Eds.), N-nitroso compounds: Occurrence and biological effects. IARC Scientific Publications. 41. 337-344. Belinsky, S.A., White, C.M., Deveraux, T.R., Swenberg, J.A. and Anderson, M.W. (1987) : Cell selective alkylation of DNA in rat lung following low dose exposure to the tobacco specific carcinogen 4-(N-methyl-N-nitrosoamine)-l-(3-pyridyl)-l-butanone. Cancer Res. 47. 1143-1148. Beranek, D.T. (1990): Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutat. Res. 231. 11-30. Beranek, D.T., Weis, C.C. and Swenson, D.H. (1980): A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis. 1. 595-606. 178 Chapter 8. References Blaser, M.J., Perez-Perez, G.I., Kleanthous, H., Cover, T.L., Peek, R.M., Chyou, P H., Stemmermann, G.H. and Nomura, A. (1995) : Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res. 55. 2111-2115. Blowers, S.D. and Anderson, D. (1988): The in vivo genotoxic properties of the nitrosopeptide N-(N-acetyl-L-prolyl)-N-nitosoglycine. Food Chem. Toxicol. 26. 785-790. Bianchini, F., Montesano, R., Shuker, D.E.G, Cuzick, J., and Wild, C.P. (1993): Quantification of 7-methyldeoxyguanosines using immunoaffinity purification and HPLC with electrochemical detection. Carcinogenesis. 14. 1677-1682. Bingham, S.A., Pignatelli, B., Pollock, J.R.A., Ellul, A., Malaveille, C., Gross, G., Runswjck, S., Cummings, J.H., and O’Neill, I.K. (1996): Does increased endogenous formation of N- nitroso compounds in the human colon explain the association between red meat and colon cancer? Carcinogenesis. 17. 515-523. Bishop, J.M. (1991): Molecular themes in oncogenesis. Cell. 64. 235-248. Blomeke, B, Greenblatt, M J., Doan, V.V., Bowman, E.D., Murphy, S.E., Chen, C.C., Kato, S. and Shields, P.G. (1996): Distribution of 7-alkyl-2’-deoxyguanosine adduct levels in human lung. Carcinogenesis. 17. 741-748. Bohr, V.A., Phillips, D.H. and Hanawalt, P.C. (1987): Heterogenous DNA damage and repair in the mammalian genome. Cancer Res. 47. 6426-6436. Bos, J.L. (1989): Ras oncogenes in human cancer: a review. Cancer Res. 49. 4682-4689. Brambilla, G., Cavanna, M., Carlo, P., Finollo, R , Sciaba, L., Parodi, S. and Bolognesi, C. (1979): DNA damage and repair induced by diazoacetyl derivatives of amino acids with different mechanism of cytotoxicity. Correlations with mutagenicity and carcinogenicity. J. Cancer Res. Clin. Oncol. 94. 7-20. Bronstein, S.M., Cochrane, J.E., Hunter, F., Rhamakrishnan, T., Fontham, E. and Shi, T.Y. (1993): Quantification of 7-methyldeoxyguanosine using immunoaffinity purification and HPLC with electrochemical detection. Carcinogenesis. 14. 1677-1682. Brookes, P. and Lawley, P.D. (1960): The reaction of mustard gas with nucleic acid in vitro and in vivo. Biochem. J. 77. 478-484. Brookes, P. and Lawley, P.D. (1961): The reaction of mono- and di-functional alkylating agents with nucleic acids. Biochem. J. 80. 496-503. Brown, K., Quintanilla, M., Ramsden, M , Kerr, I.B., Young, S. and Balmain, A. (1986): v- ras genes from Harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis. Cell. 46. 447-456. 179 —------—-— —— ------Chapter 8. References Busby, W.F., Shuker, D.E.G., Chamley, G., Newbeme, P.M., Tannenbaum, S.R. and Wogan G.N. (1985): Carcinogenicity in rats of the nitrosated bile acid conjugates N- nitrosoglycocholic acid and N-nitrosotaurocholic acid. Cancer Res. 45. 1367-1371. Butlin, H.T. (1892): Cancer of the scrotum in chimney-sweeps and others. II. Why foreign sweeps do not suffer from scrotal cancer. Brit. Med. J. 2. 1-6. Cahill, R. J., Sant, S., Beattie, S., Hamilton, H. and O’Morain, C. (1994): Helicobacter pylori and increased epithelial cell proliferation: a risk factor for cancer. Eur. J. Gastroenterol. Hepatol. 6. 1123-1127. Calmels, S., Ohshima, H., Vincent, P., Gounot, A. and Bartsch, H. (1985): Screening of micro-organisms for nitrosation catalysis at pH 7. Carcinogenesis. 6. 911-915. Calmels, S., Ohshima, H., Rosenkranz, H., McCoy, E. and Bartsch, H. (1987): Biochemical studies on the catalysis of nitrosation by bacteria. Carcinogenesis. 8. 1085-1088. Carmella, S.G., Borukhova, A., Desai, D. and Hecht, S.S. (1997): Evidence for endogenous formation of tobacco-specific nitrosamines in rats treated with tobacco alkaloids and sodium nitrite. Carcinogenesis. 18. 587-592. Cartwright, R.A. (1984): Cancer epidemiology. Searle, C.E. (Ed). Chemical Carcinogens, vol. I. ACS symposium series No 182, American Chemical Society, USA. ppl-39. Caygill, C.P.J., Leach, S.A., Kiikham, J.S., Northfield, T.C., Hall, C.N. and Hill M.J. (1987): Gastric hypoacidity as a risk factor for gastric and other cancers. In: H. Bartsch, I.K. O’Neill and R. Schulte-Hermann (Eds.), The relevance o f N-nitroso compounds to human cancer. Exposures and mechanisms. IARC Scientific Publications. 84. 524-526. Challis, B.C., Lomas, S.J., Rzepa, H.S., Bavin, M.G., Darkin, D.W., Viney, N.J. and Moore, P. J. (1982): A kinetic model for the formation of gastric N-nitroso compounds. In: P.N. Magee (Ed.), Nitrosamines and human cancer. Banbury report 12. pp243-253. Challis, B.C. (1989): Chemistry and biology of nitrosated peptides. Cancer Surv. 8. 363-384. Challis, B.C. and Latif, F. (1990): Synthesis and characterisation of some new diazopeptides. J. Chem. Soc. Perkin. Trans. 1. 1005-1009. Challis, B.C. (1996): Environmental exposures to N-nitroso compounds and precursors: general review of methods and current status. Eur. J. Cancer Prevention. 5s. 19-26. Chan, W., Weisburger, J.H., Fiala, E.S., Carmella, S.G., Chen, D., Spratt, T.E. and Hecht, S.S. (1995): Unexpected mutagen in fish. Nature. 374. 599. Chapeau, M.C. and Mamett, L. J. (1991): Enzymatic synthesis of purine deoxynucleoside adducts. Chem. Res. Toxicol. 4. 636-638. Cohen, S.M. and Ellwein, L.B. (1991): Genetic errors, cell proliferation and carcinogenesis. Cancer Res. 51. 6493-6505.’ 180 Chapter 8. References Cohen, S.M., Ellwein, L.B. and Johansson, S.L. (1987): Bladder tumour promotion. In : Butterworth, B.E. and Slaga, T.J. (Eds) Non-genotoxic mechanisms in carcinogenesis, Banbury Report No 25, Cold Spring Harbor Laboratory, USA. pp55-67. Cooper, D.P., Yao, G-F., Qu, Y-H. and O’Connor, P.J. (1991): DNA methylation in individuals at high risk for stomach cancer. Brit. J. Cancer. 63. 65-68. Cooper, D.P., Griffith, K.A, and Povey, A.C. (1992): Immunoaffinity purification combined with 32P-postlabelling for the detection of 0 6-methyiguanine in DNA from human tissue. Carcinogenesis. 13. 469-475. Cooper, D.P., O’Connor, P.J., Povey, A.C. and Rafferty, J.A. (1995): Cell and molecular mechanisms in chemical carcinogenesis. In: Peckham M., Pinedo H and Veranesi U.,(Eds.) Oxford Textbook o f Oncology, 1,, Oxford University Press, Oxford. ppl35-147. Correa, P., Haenszel, W., Cuello, C., Tannenbaum, S. and Archer, M. (1975): A model for gastric cancer epidemiology. Lancet, ii. 58-60. Correa, P. (1983): The gastric precancerous process. Cancer Surv. 2. 437-450. Correa, P. (1988): A human model of gastric carcinogenesis. Cancer Res. 48. 3554-3560. Correa, P., Fox, J., Fontham, E., Ruiz, B., Lin, Y., Zavala, D., Taylor, N., Mackinley, D., de Lima., Portilla, H. and Zarama, G. (1990): Helicobacter pylori and gastric carcinoma : serum antibody prevalence in populations with contrasting cancer risks. Cancer. 66. 2569-2574. Correa, P., Haenszel, W., Cuello, C., Zavala, D., Fontham, E. and Zarama, G. (1990b). Gastric precancerous process in a high risk population cohort follow up. Cancer Res. 50. 4737-4740. Correa, P. (1992): Human gastric carcinogenesis: a multistep and multifactorial process. Cancer Res. 52. 6735-6740. Correa, P. and Chen, V.W. (1994): Trends in cancer incidence and mortality : Gastric cancer. Cancer Surv. 19/20. 55-74. Cummings, J.H., Hill, M.J., Bone, E.S., Branch, W.J. and Jenkins, D.J.A. (1979): The effect of meat protein and dietary fibre on colonic function and metabolism II Bacterial metabolism in faeces and urine. Am. J. Clin. Nutrition. 32. 2094-2101. Cummings, J.H. and Macflarlane, G.T. (1991): The control and consequences of bacterial fermentation in the human colon. J. Appl Bacteriol. 70. 443-459. Davies, G.R. and Rampton, D.S. (1994): Helicobacter pylori, free radicals and gastroduodenal disease. Eur. J. Gastroenterol. Hepatol. 6.1-9. Decaprio, A.P. (1997): Biomarkers: coming of age for environmental health and risk assessment. Environ. Sci. Tech. 31. 1837-1849. 181 ------_— ____ Chapter 8. References Den Engelse, L., Menkveld, G.J., De Brij ,R.-J. and Tates, A.D. (1986): Formation and stability of alkylated pyrimidines and purines (including imidazole ring-opened 7- alkylguanine) and alkylphosphotriesters in liver DNA of adult rats treated with ethylnitrosourea or dimethylnitrosamine. Carcinogenesis. 7. 393-403. Dennisenko, M.F., Pao, A., Tang, M.-S. and Pfeifer, G.P. (1996): Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science. 21 A.430-432. Dipple, A., Moschel, R.C. and Bigger, C.A.H. (1984): Polynuclear aromatic carcinogens. In: Searle, C.E. (Ed) Chemical carcinogens, vol I. ACS symposium series No 182. American Chemical Society, USA. 41-163. Doll, R and Hill, A.B. (1950): Smoking and carcinoma of the lung: preliminary report. Brit. Med. J. 2. 739-748. Doll, R and Peto, R. (1981): The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J. Natl. Cancer Inst. 66. 1191-1308. Durand, M.J. and Shuker, D.E.G. (1994): Preparation of compound-specific and group- specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification. Carcinogenesis. 15. 957-961. Engelward, B.P., Boosalis, M.S., Chen, B.J., Deng, Z., Siciliano, M.J. and Samson, L.D. (1993): Cloning and characterisation of a mouse 3-methyladenine/7-methylguanine/3- methylguanine DNA glycosylase cDNA whose gene maps to chromosome 11. Carcinogenesis. 14. 175-181. The Eurogast Study Group (1994): 0 6-methylguanine in blood leukocyte DNA: an association with the geographic prevalence of gastric cancer and with low levels of serum pepsinogen A, a marker of severe chronic atrophic gastritis. Carcinogenesis. 15. 1815-1820. Fan, T-Y., and Tannerbaum, S.R. (1973): Factors influencing the rate of formation of nitrosomorpholine from morpholine and nitrite : accelerated by thiocynanate and other anions. J. Agric. Food Chem. 21. 237-240. Farris, E. J. and Griffith, J.Q. (1949): The rat in laboratory investigation. Hafner press. Farmer, P.B., Sepai, O., Lawrence, R., Autrup, H., Nielsen, P.S., Vestergard, A.B., Waters, R., Leuratti, C., Jones, N.J., Stone, J., Baan, R.A., van Delft, J.H.M., Steenwinkel, M.J.S.T., Kyrtopoulos, S.A., Souliotis ,V.L., Theodorakopoulos, N., Bacalis, N.C., Natarajan, A.T., Tates, A.D., Haugen, A., Andreassen, A , 0vrebo, S., Shuker, D.E.G., Amaning, K.S., Schouft, A., Ellul, A., Gamer, R.C., Dingley, K.H., Abbondandolo, A., Merio, F., Cole, J., Aldrich, K., Beare, D., Capulas, E., Rowley, G., Waugh, A.P.W., Povey, A.C., Haque, K., Kirsch-Volders, M., Van Hummelen, P. and Castelain, P. (1996): Biomonitoring human exposure to environmental carcinogenic chemicals. Mutagenesis. 11. 363-381. Fearon, E.R. and Vogelstein B. (1990): A genetic model for colorectal tumorigenesis. Cell. 61. 759-767. 182 — ______Chapter 8.References Foiles, P.G., Miglietta, L.M., Shobha, A.A., Everson, R.B, and Hecht, S.S. (1988): Detection of 0 6-methyldeoxyguanosine in human placental DNA. Cancer Res. 48. 4184-4188. Forman, D., Sitas, F., Newell, D.G., Stacey, A.R., Boreham, J., Peto, R., Campbell, T.C., Li, J. and Chen, J. (1990): Geographic association of Helicobacter pylori antibody prevalence and gastric cancer mortality in rural China. Int. J. Cancer. 46. 608-611. Forman, D., Newell, D.G., Fullerton, F., Yamell, J.W.G., Stacey, A.R., Wald, N. and Sitas, F. (1991): Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation. Brit. Med. J. 302. 1302-1305. Foulds, L. (1954): The experimental study of tumour progression: a review. Cancer Res. 14. 327-339 Friedberg E.F. (1985): DNA Repair, Freeman, San Francisco, USA. Friesen, M.D., Garren, L., Prevost, V. and Shuker, D.E.G. (1991): Isolation of urinary 3- methyladenine using immunoaffinity columns prior to determination by low-resolution gas chromatography-mass spectrometry. Chem. Res. Toxicol. 4. 102-106. Gaffney, B.L., and Jones, R.A. (1982): Synthesis of 0 6-alkylated deoxyguanosine nucleosides. Tetrahedron Lett. 23. 2253-2256. Gamer, R.C., Martin, C.N. and Clayson, O.B. (1984): Carcinogenic aromatic amines and related compounds. In :Searle, C.E. (Ed) Chemical carcinogens, vol I. ACS symposium series No 182. American Chemical Society, USA. 175-275. Goding, J.W., (1986): Monoclonal antibodies. Principles and practice. Second edition. Academic press. Harcourt Brace Jovanovich, Publishers. pp219-240. de Groot, A.J.L., Jansen, J.G., van Valkenburg, C.F.M. and van Zeeland, A.A. (1994): Molecular dosimetry of 7-alkyl- and 0 6-alkylguanine in DNA by electrochemical detection. Mutat. Res. 307. 61-66 Gonzalez, C.A. and Agudo, A. (1994): Tobacco smoking and alcohol consumption. Eur. J. Gastroenterol. Hepatol. 6. 1083-1088. Hall, C.N., Badawi, A.F., O’Conner, P.J., Saffhill, R. (1991): The detection of alkylation damage in the DNA of human gastrointestinal tissues. Brit. J. Cancer. 64. 59-63. Haque, K., Cooper, D.P. and Povey, A.C. (1994): Optimisation of 32P-postlabelling assays for the quantitation of 0 6-methyl and N7-methyldeoxyguanosine-3’-monophosphates in human DNA. Carcinogenesis.IS. 2485-2490. Harris, C.C. (1991): Chemical and physical carcinogenesis: advances and perspectives for the 1990s. Cancer Res. 51. 5023S-5044S. Harris, C.C. (1995): 1995 Deichmann lecture - p53 tumour suppressor gene: at the cross roads of molecular carcinogenesis, molecular epidemiology and cancer risk assessment. Tax. Lett. 82/83. 1-7. 183 - — ------Chapter 8. References Harrison, K.L., Fairhurst, N., Challis, B.C., and Shuker, D.E.G. (1997): Synthesis, characterisation, and immunochemical detection of 0 6-(carboxymethyl)-2,-deoxyguanosine : A DNA adduct formed by nitrosated glycine derivatives. Chem. Res. Toxicol. 10. 652-659. Havbrandt, O. and Wachmeister, C.A. (1968): Synthesis of mesyloxyacetic acid and some derivatives. Acta. Chem. Scand. 22. 2043-2044. Hecht, S. S. and Hoffinan, D. (1989): The relevance of tobacco-specific nitrosamines to human cancer. Cancer Surv. 8. 273-294. Hemminki, K. (1980): Fluorescence properties of alkylated guanine derivatives. Acta Chem. Scand. B 34, 603-605. Henderson, RJF. (1995): Strategies for use of biological markers of exposure. Tox. Lett. 82/83. 379-383. Herron, D.C. and Shank, R.C. (1981): In vivo kinetics of 0 6-methylguanine and 7- methylguanine formation and persistence in DNA of rats treated with symmetrical dimethylhydrazine. Cancer Res. 41. 3967-3972. Hill, M.J. (1990): Mechanisms of endogenous N-nitrosation. In : S. Leach (Ed.) Nitrosamines toxicology and microbiology. Series in food science and technology. Ellis Horwood Chichester, pp69-87. Hill, M.J. (1996a): N-nitrosation at non-gastric sites. Eur. J. Cancer Prevention. 5. Suppl.l. 71-73. Hill, M.J. (1996b): Factors controlling endogenous N-nitrosation. Eur. J. Cancer Prevention. 5. Suppl.l. 71-75. Hollstein, M., Sidransky, D., Vogelstein, B. and Harris, C.C. (1991): p53 mutations in human cancers. Science. 253. 49-53. Hotchkiss, J.H. (1989): Preformed N-nitroso compounds in foods and beverages. Cancer Surv. 8. 295-321. Howson, G.R., Hiyama, T. and Wynder, E.L. (1986): The decline in gastric cancer : epidemiology of an unplanned triumph. Epidemiol Rev. 8. 1-27. Jansen, J.G., De Groot, A.J.L., Van Teijlingen, C.M.M., Lohman, P.H.M., Mohn, G.R. and Van Zeeland, A. A. (1994): Formation and persistence of DNA adducts in pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N- methyl-N-nitrosourea or N-ethyl-N-nitrosourea. Mutat. Res. 307. 95-105. Jones, N.J., Ellard, S., Waters, R and Parry, E.M. (1991): 32P-PostlabeIIing analysis and micronuclei induction in primary Chinese hamster lung cells exposed to tobacco particulate matter. Carcinogenesis. 12.1507-1514. 184 ------——-______Chapter 8. References Kakolyris, S., Kaklanmanis, L., Engels, K., Turly, T., Hickson, I.D., Gaiter, K.C. and Harris, A.L. (1997) : HAP1 expression in a colorectal adenoma-carcinoma sequence Cancer Res. 57. 1794-1797. Kang, H., Konishi, C., Kuroki, T. and Huh, N. (1995): Detection of 0 6-methylguanine, O4- methylthymine and 0 4-ethylthymine in human liver and peripheral blood leukocyte DNA. Carcinogenesis. 16. 1277-1280. Kang, M., Mitomi, H., Sada, M., Tokumchsu, Y., Takahashi, Y., Igarashi, M., Katsumata, T. and Okayasu, I. (1997) : Ki-67, p53 and Bcl-2 expression of serrated adenomas of the colon. Am. J. Surg. Path. 21. 417-423. Karran, P. and Lindahl, T. (1985): Cellular defense mechanisms against alkylating agents. Cancer Surv. 4. 585-599. Kobori, O., Schmerold, I., Ludeke, B., Ohgaki, H. and Kleihues, P. (1988): DNA methylation in rat stomach and duodenum following chronic exposure to N-methyl-N’ -nitro-N- nitrosoguanidine and the effect of dietary taurocholate. Carcinogenesis. 9. 2271-2274. Kolonel, L.N., Hinds, M.W. and Hankin, J.H. (1980): Cancer patterns among migrant and native-born Japanese in Hawaii in relation to smoking, drinking and dietary habits. In: H. V. Gelboin, B., MacMahon, T. Matsushima, T. Sugimura, S. Takayama and H. Takebe (Eds.) Genetic and environmental factors in experimental and human cancer. pp327-340. Komorowska, M., Szafran, H., Popiela, T. and Szafran, Z. (1981): Free amino acids of human gastric juice. Acta Physiol. Pol. 32.559-567. Kreevoy, M.M. and Konasewich, D.E. (1970): The mechanism of hydrolysis of diazoacetate ion. J. Phys. Chem. 74. 4464-4472. Krenitsky, T.A., Koszalka, G.W. and Tuttle, J.V. (1981): Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases. Biochemistry. 20. 3615-3621. Kubitschek, H.E. and Sepanski, R. J. (1982) : Azaserine: survival and mutation in Escherichia coli. Mutat. Res. 94. 31-38. Kuipers, E. J., Perez-Perez, G.I., Meuwissen, S.G.M. and Blaser, M.J. (1995a): Helicobacter pylori and atrophic gastritis : Importance of the cagA status. J. Natl. Cancer Inst. 87.1777- 1780. Kuipers, E.J., Thijs, J.C. and Festen, H.P. (1995b): The prevalence of Helicobacter pylori in peptic ulcer disease. Aliment. Pharmacol. Ther. 9. 59-69. Kyrtopoulos, S.A., Daskalakis, G. and Outram, J.R. (1985): Studies in gastric carcinogenesis. HI. The kinetics of nitrosation of gastric juice components in vitro and their implications for the in vivo formation of N-nitroso compounds in normal and in hypochlorhydric populations. Carcinogenesis. 6. 1141-1145. 185 Chapter 8. References Kyrtopoulos, S. A. (1987): Investigations of the DNA-damaging activity of human gastric juice. In: H. Bartsch, I.K. O’Neill and R. Schulte-Hermann (Eds.), The relevance o f N-nitroso compounds to human cancer. Exposures and mechanisms. IARC Scientific Publications. 84. pp507-510. Kyrtopoulos, S. A. (1989): N-nitroso compound formation in human gastric juice. Cancer Surv. 8. 423-442. Kyrtopoulos, S.A., Ampatzi, P., Davaris, P., Hartiopoulous, N., Golematis, B. (1990): Studies in gastric carcinogenesis. IV. 0 6-Methylguanine and its repair in normal and atrophic biopsy specimens of human gastric mucosa. Correlation of 0 6-alkylguanine-DNA alkyltransferase activities in gastric mucosa and circulating lymphocytes. Carcinogenesis. 11. 431-436. Lawley, P.D. (1990): N-nitroso compounds. In: Cooper, C.S. and Grover, P.L. (Eds). Chemical carcinogenesis and mutagenesis, Handbook o f experimental pharmacology. Springer-Verlag, Berlin, vol. 94. 409-469. Lawley, P.D. (1984): Carcinogenesis by alkylating agents. Searle, C.E. (Ed) Chemical carcinogens, vol I. ACS symposium series No 182. American Chemical Society, USA. pp325-484. Lawley P.D. and Brookes P. (1963): Further studies on the alkylation of nucleic acids and their constituent nucleotides. Biochem. J. 89. 127-138. Leaf, C.D., Wishnok, J.S. and Tannenbaum, S.R. (1989): Mechanisms of endogenous nitrosation. Cancer Surv. 8. 323-334. Liu, R.H. and Hotchkiss, J.H. (1995): Potential genotoxicity of chronically elevated nitric oxide: a review. Mutat. Res. 339. 73-89. Lim, C.K. (1986): HPLC o f small molecules. Series editors Rickwood, D. and Hames, B.D. ERL Press limited. Lindahl, T., Sedgwick, B., Sekiguchi, M. and Nakabeppu, Y. (1988): Regulation and expression of the adaptive response to alkylating agents. Annu. Rev. Biochem. 57. 133-157. Loeb, L.A. (1985): Apurinic sites as mutagenic intermediates. Cell. 40. 483-484. Longnecker, D.S., Roebuck, B.D., Yager, J.D., Lilja, H.S. and Siegmund, B. (1981): Pancreatic carcinoma in azaserine-treated rats. Cancer. 47. 1562-1572. Ludeke, B.I. and Kleihues, P. (1988): Formation and persistence of 0 6-(2-hydroxyethyl)-2'- deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2- hydroxyethyl)urea. An immuno-slot-blot study. Carcinogenesis. 9. 147-151. Lunec, J., Herbert, K., Blount, S., Griffiths, H R. and Emery, P. (1994): 8- Hydroxydeoxyguanosine. A marker of oxidative DNA damage in systemic lupus erythematosus. F E B SLetts. 348. 131-138. 186 ______Chapter 8.References Macfarlane, G.T., Gibson, G.R., Drasar, B.S. and Cummings, J.H. (1995): Metabolic significance of the gut microflora. In: Whitehead, R (Ed.) Gastrointestinal and oesophageal pathology. Churchill Livingson, London. pp249-273. Magee, P.N. (1989): The experimental basis for the role of nitrosocompounds in human cancer. Cancer Surv. 8. 207. Magee, P.N. (1996): Nitrosoamines and human cancer : introduction and overview. Eur. J. Cancer Prevention. 5. Suppl.l. 7-10. Margison, G.P, Cooper, D.P. and Brennand, J. (1985): Cloning of the E C o li O6- methylguanine and methylphosphotriester methyltransferase gene using a functional DNA repair assay. Nucleic Acids Res. 13. 1939-1952. Margison, G.P. and O’Connor, P.J. (1990): Biological consequences of reactions with DNA: role of specific lesions. In : P.L. Grover and C.S. Cooper (Eds.), Chemical carcinogenesis andMutagenesis. Handbook o f experimental pharmacology. 94/1. pp547-571. Martin, I.G. and Quirke P. (1994): Genetic predisposition and abnormalities in gastric cancer. Eur. J. Gastroenterol. Hepatol 6. 1111-1116. Mattes, W.B., Lee, C.-S., Laval, J. and O’Connor, T.R. (1996): Excision of DNA adducts of nitrogen mustards by bacterial and mammalian 3-methyladenine-DNA glycosylases. Carcinogenesis. 17. 643-648. McColl, K.E.L. and El-Omar, E. (1996): Helicobacter pylori and disturbance of gastric function associated with duodenal ulcer disease and gastric cancer. Scand J. Gastroenterol. 31. Suppl. 215. 32-37. McGee, J. O’D., Isaacson, P.G. and Wright, N.A. (1992): Oxford Textbook of Pathology ., led., Oxford University Press, Oxford, pp571-678. Miller, J.A. (1970): Carcinogenesis by chemicals: an overview - G.H.A. Clowes memorial lecture. Cancer Res. 30. 559-576. Miller, E.C.(1978): Some current perspectives on chemical carcinogenesis in humans and experimental animals: Presidential address. Cancer Res. 38. 1479-1496. Miller, E. and Miller, J.A. (1981): Mechanisms of chemical carcinogenesis. Cancer. 47. 1055-1064. Milton-Thompson, G.J., Lightfoot, N. and Ahmet, Z. (1982): Intragastric acidity, bacteria, nitrite and N-nitroso compounds before, during and after cimetidine treatment. Lancet, i. 1091-1095. Mirvish, S.S. (1975): Formation of N-nitroso compounds: chemistry, kinetics and in vivo occurrence. Toxicol. Appl. Pharmacol. 31. 325-351. 187 Chapter 8. References Mirvish, S. S. (1994): Experimental evidence for inhibition of N-nitroso compound formation as a factor in the negative correlation between vitamin C consumption and the incidence of certain cancers. Cancer Res. Suppl. 54. 1948-1951. Mirvish, S. S. (1995): Role of N-nitroso compounds and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Letts. 93. 17-48. Mirvish, S. S. (1996): Inhibition by vitamins C and E of in vivo nitrosation and vitamin C occurrence in the stomach. Eur. J. Cancer Prevention. 5. Suppl.l. 131-136. Mohammadi, M., Redline, R , Nedrud, J. and Czinn, S. (1996): Role of the host in pathogenesis of Helicobacter-associated gastritis : H. fe lis infection of inbred and congenic mouse strains. Infection and Immunity. 64. 238-245. Moller, H., Landt, J., Pedersen, E., Jensen, P., Autrup, H. and Jensen, O.L. (1989): Endogenous nitrosation in relation to nitrate exposure from drinking water and diet in a Danish rural population. Cancer Res. 49. 3117-3121. Nehls, P., Adamkiewicz, J. and Rajewsky, M.F. (1984): Jmmuno-slot-blot: A highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA. J. Cancer Res. Clin. Oncol. 108. 23-39. O’Neill, I.K., Castegnaro, M., Brouet, I. And Povey, A.C. (1987): Magnetic semi-permeable polyethyleneimine microcapsules for monitoring of N-nitrosation in the gastrointestinal tract. Carcinogenesis. 8. 1469-1474. Nomura, A., Grove, J.S., Stemmermann, G.N. and Severson, RK. (1990): A prospective study of stomach cancer and its relation to diet, cigarettes, and alcohol consumption. Cancer Res. 50. 627-631. Oshima, H. and Bartsch, H. (1981): Quantitative estimation of endogenous nitrosation in humans by monitoring N-nitrosoproline excreted in the urine. Cancer Res. 41. 3658-3662. Ostermann-Golkar, S., Ehrenberg, L. and Wachtmeister, C.A. (1970) : Reaction kinetics and biological action in barley of mono-functional methanesulfonic esters. Radiation Bot. 10. 303-327. Palli, D. (1994): Gastric cancer : Dietary factors. Eur. J. Gastroenterol. Hepatol. 6. 1076- 1082. Palli, D., Caporaso, N.E., Shiao, Y-H., Saiva, C., Amorosi, A., Masala, G., Rice, J.M. and Fraumeni, J.F. (1997): Diet, Helicobacter pylori , and p53 mutations in gastric cancer: A molecular epidemiology study in Italy. Cancer Epidemiology, Biomarkers & Prevention. 6. 1065-1069. Parkin, D.M., Muir, C.S., Whelan, S.L., Gao, Y.T., Ferley, J. and Powell, J. (Eds) (1992): Cancer incidence in five continents. IARC. 120, International Agency for Research on Cancer, Lyon. 188 Chapter 8. References Parsonnet, J., Friedman, G.D., Vandersteen, D.P., Chang, Y., Vogelman, J.H., Orentriech, N., and Sibley, R.K. (1991): Helicobacter pylori infection and the risk of gastric carcinoma N. Engl J. Med. 325. 1127-1131. Pegg, A.E. (1990): DNA repair and carcinogenesis by alkylating agents. In: Cooper, C.S. and Grover, P.L. (Eds). Chemical carcinogenesis and mutagenesis, Handbook o f experimental pharmacology. Springer-Verlag, Berlin, vol. 94. pp409-469. - Pegg, A.E. (1984): Methylation of the O6 position of guanine in DNA is the most likely initiating event in carcinogenesis by methylating agents. Cancer Investigation. 2. 223-231. Pegg, A.E. and Perry, W. (1981): Alkylation of nucleic acids and metabolism of small doses of dimethylnitrosamine in the rat. Cancer Res. 41. 3128-3132. Perera, F.P. (1987): Molecular cancer epidemiology: a new tool in cancer prevention. J. Natl. Cancer Inst. 78. 887-898. Perera, F.P. (1988): The significance of DNA and protein adducts in human biomonitoring studies. Mutat. Res. 205. 255-269. Perera, F. (1996): Molecular epidemiology: Insights into cancer susceptibility, risk assessment and prevention. J. Natl. Cancer Inst. 88. 496-509. Pignatelli, B., Richard, I., Bourgarde, M.-C. and Bartsch, H. (1987): Improved group determination of total N-nitroso compounds (NOC) in human gastric juice by chemical denitrosation and thermal energy analysis. Analyst. 112. 945-949. Pignatelli, B. and Walters C.L. (1996): Analytical techniques for total N-nitroso compounds. Eur. J. Cancer Prevention. 5. Suppl.l. 51-58. Pitot, H.C. (1986) : Fundamentals o f oncology. 3rd ed, Dekker, New York, USA. Pitot, H.C. and Dragan, Y.P. (1991): Facts and theories concerning the mechanism of carcinogenesis. The FASEB J. 5. 2280-2286. Potter, J.D., Slattery, M.L., Bostick, R.M. and Gapstur, S.M. (1994): Colon cancer, a review of the epidemiology. Epidemiol. Rev. 15. 1143S-1152S. Povey, A.C. and Cooper, D.P. (1995): The development, validation and application of a 32P- postlabelling assay to quantify 0 6-methylguanine in human DNA. Carcinogenesis. 16. 1665- 1669. Preston-Martin, S. and Correa, P. (1989): Epidemiological evidence for the role of nitroso compounds in human cancer. Cancer Surv. 8. 459-473. Prevost, V., Shuker, D.E.G., Friesen, M.D., Bartsch, H., Pastorelli, R., Stillwell, W.G., Trudel, L.J., and Tannenbaum, S.R. (1990): The determination of urinary 3-methyladenine by immunoaffinity chromatography-monoclonal antibody based ELISA: use in human biomonitoring studies. Carcinogenesis 11. 1747-1751. 189 ------—-______Chapter 8. References Preussmann, R., and Stewart B.W. (1984): N-nitroso carcinogens. In : Searle, C.E. (Ed), Chemical carcinogens. ACS Symposium Series Nol82, American Chemical Society, Washington DC. pp643-828. Reddy, A.L., Caldwell, M. and Fialkow, P.J. (1987): Studies of skin tumorigenesis in PGK mosaic mice: many promoter-independent papillomas and carcinomas do not develop from pre-existing promoter-dependent papillomas. Int. J. Cancer. 39. 261-265. Reed, E., Ozols, R.F., Fasy, T., Yuspa, S.H. and Poirier, M.C. (1986): Biomonitoring of cisplatin-DNA adducts in cancer patients receiving cisplatin chemotherapy. In: C. Ramel, B. Lambert, and J. Magnusson (Eds.) Genetic Toxicology o fEnvironmental Chemicals, Part B: Genetic Effects and Applied Mutagenesis. pp247-252. Renault, T.B., van den Broek, M., Fodde, R., Wijnen J., Pellegata, N.S., Amadori, D., Khan, P.M. and Ranzani, G.N. (1993): Base transitions are the most frequent genetic changes at p53 in gastric cancer. Cancer Res. 53. 2614-2617. Rudi, J., Kolb, C., Maiwald, M., Zuna, I., Herbay, A., Galle, P.R. and Stremmel, W. (1997): Serum antibodies against Helicobacter pylori proteins vacA and cagA are associated with increased risk for gastric adenocarcinoma. Digestive Diseases and Sciences. 42. 1652-1659. Ryden, E.B., Licht, W.R., Cabot, B. and Fox, J.G. (1990): Gastric nitrite processing in the surgically altered maximal and minimal bile reflux ferret model. Carcinogenesis. 11. 405- 409. Saffhill, R., Margison, G.P., and O’Connor, P.J. (1985): Cellular defense mechanisms of carcinogenesis induced by alkylating agents. Biochim. Biophys. Acta. 823. 111-145. Saffhill, R., Badawi, A.F. and Hall, C.N. (1988) : Detection of 0 6-methylguanine in human DNA. In : Bartsch, H., Hemminki, K. and O’Neil I.K (Eds.) Methods for detecting DNA damaging agents in humans. IARC Scientific Publication. 89. pp301-305. Samson, L.D. (1992): The repair of DNA alkylation damage by methyltransferases and glycosylases. In: Essays Biochem. 27. Biochemical Society, 69-78. Schneider, C., Newman, R.A., Sutherland, D.R., Asser, U., and Greaves, M.F. (1982): A one- step purification of membrane proteins using a high-efficiency immunomatrix. J. Biol. Chem. 257. 10766-10769. Schulte-Hermann, R , Bursch, W., Grasl-Kraupp, B., Torok, L., Ellinger, A. and Miillauer, L. (1995): Role of active cell death (apoptosis) in multi-stage carcinogenesis. Tox. Lett. 82/83. 143-148. Sedgwick, B. (1997) : Nitrosated peptides and polyamines as endogenous mutagens in O6- alkylguanine-DNA alkyltransferase deficient cells. Carcinogenesis. 18. 1561-1567. Seruca, R , Holm, D.L., Nesland, J.M., Fangan, B.M., Castedo, S., Sobrinho-Simoes, M. and Borresen, A.-L (1992): p53 mutations in gastric carcinomas. Br. J. Cancer. 65. 708-771. 190 Chapter 8. References Shephard, S.E., Schlatter, C. and Lutz, W.K. (1987): Model risk analysis of nitrosatable compounds in the diet as precursors of potential endogenous carcinogens. In: H. Bartsch, I.K. O’Neill and R. Schulte-Hermann (Eds.), The relevance o fN-nitroso compounds to human cancer. Exposures and mechanisms. IARC Scientific Publications. 84. 328-332. Shephard, S.E. and Lutz, W.K. (1989): Nitrosation of dietary precursors. Cancer Surv. 8. 401-421. Shields, P.G. and Harris, C.C. (1991): Molecular epidemiology and the genetics of environmental cancer. J. Am. Med Assoc. 266. 681-687. Shuker D.E.G. and Bartsch H. (1994a): DNA adducts of nitrosoamines. In: Hemminki, K., Dipple, A., Shuker, D.E.G. Kadlubar, F.F., Segerback, D. and Bartsch, H. (Eds.) DNA adducts: Identification and biological significance. IARC Scientific Publications No. 125. pp73-89. Shuker, D.E.G., and Bartsch, H. (1994b): Detection of human exposure to carcinogens by measurement of alkyl-DNA adducts using immunoaffinity clean-up in combination with gas chromatography-mass spectrometry and other detection methods. Mutat. Res. 313, 263-268. Shuker, D.E.G., Tannenbaum, S.R, and Wishnok, J.S. (1981): N-nitroso bile acid conjugates. I. Synthesis, chemical reactivity and mutagenic activity. J. Org. Chem. 46, 2092-2096. Shuker, D.E.G., Howell, J. and Street, B.W. (1987) The formation and fate of nucleic acid and protein adducts derived from N-nitroso bile acid conjugates. In: H. Bartsch, I.K. O’Neill and R. Schulte-Hermann (Eds.), The relevance o fN-nitroso compounds to human cancer. Exposures and mechanisms. IARC Scientific Publications. 84. 187-190. Shuker, D.E.G., Leuratti, C., Harrison, K.L., Conduah Birt, J. and Farmer, P.B. (1995) : Non invasive or minimally invasive biomarkers of exposure to genotoxic agents derived from food. In: Crews, H.M. & Hanley, A.B (Eds.) Biomarkers in Food Chemistry Risk Assessment. The Royal Society of Chemistry. pp73-79. Shuker, D.E.G., and Margison, G.P. (1997): Nitrosated glycine derivatives as a potential source of 0 6-methyguanine in DNA. Cancer Res. 57, 366-369. Singer, B. and Essigman, J.M. (1991): Site-specific mutagenesis: retrospective and prospective. Carcinogenesis. 12. 949-955. Singer, B. and Hang, B. (1997): What structural features determine repair enzyme specificity and mechanism in chemically modified DNA? Chem. Res. Toxicol. 10. 713-731. Sirulal, M. Lehtola, J. and fliamaki, T. (1974): Atrophic gastritis and its sequelae. Scand J. Gastroenterol. 9. 441-446. Sitas, F., Forman, D., Newell, D.G., Yamell, J.W.G., Burr, M.L., Elwood, P.C. and Pedley, S. (1991): Helicobacter pylori infection rates in relation to age and social class in a population of Welsh men. Gut. 32. 25-28. 191 ______Chapter 8. References Sobala, G.M., Pignatelli, B., Schorah, C.J., Bartsch, H., Sanderson, M., Dixon, M.F., Shires, S., King, R.F.G. and Axon, A.T.R. (1991): Levels of nitrite, nitrate, N-nitroso compounds, ascorbic acid and total bile acids in gastric juice of patients with and without precancerous conditions of the stomach. Carcinogenesis. 12. 193-198. Song, P., Shuker, D.E.G., Bishop, W.W., Falchuk, K.R., Tannenbaum, S.R. and Thilly, W.G. (1982): Mutagenicity of N-Nitroso bile acid conjugates in S. typhimurium and diploid human lymphoblasts. Cancer Res. 42. 3056-3058. Souliotis, V.L., Kaila, S., Boussiotis, V.A., Pangalis, G.A. and Kyrtopoulos, S.A. (1990): Accumulation of 0 6-methylguanine in human blood leukocyte DNA during exposure to procarbazine and its relationships with dose and repair. Cancer Res. 50. 2759-2764. Souliotis, V.L., Boussiotis, V.A., Pangalis, G.A. and Kyrtopoulos, S.A. (1991): In vivo formation and repair of 0 6-methylguanine in human leukocyte DNA after intravenous exposure to dacarbazine. Carcinogenesis. 12. 285-288. Spiegelhalder, B. and Bartsch, H. (1996): Tobacco-specific nitrosamines. Eur. J. Cancer Prevention. 5. Suppl.l. 33-38. Spom, M.B. (1991): Carcinogenesis and cancer: different perspectives on the same disease. Cancer Res. 51. 6215-6218. Stadler, R.H., Staempfli, A.A., Fay, L.B., Turesky, R.J. and Welti, D.H. (1994): Synthesis of multiply-labeled [15N3,13C1]-8-oxo-substituted purine bases and their corresponding 2'- deoxynucleosides. Chem. Res. Toxicol. 7. 784-791. Startin, J.R. (1996): N-nitroso compounds in foods and drinks. Eur. J. Cancer Prevention. 5. 39. Sugimura, T., and Kawachi T. (1973): Experimental stomach cancer. In : Busch, H. (ed), Methods in Cancer Research, 7, Academic Press, New York and London. 245-308. Swenberg, J.A., Cooper, H.K., Bucheler, J. and Kleihues, P. (1979): 1,2-Dimethylhydrazine- induced methylation of DNA bases in various rat organs and the effect of pretreatment with disulfiram. Cancer Res. 39. 465-467. Swenberg, J.A. Richardson, F.C., Boucheron, J.A. and Dyroff M.C. (1985): Relationship between DNA adduct formation and carcinogenesis. Enviro. Health. Perspect. 62. 177-183. Swenson D.H. (1983): Significance of electrophilic reactivity and especially DNA alkylation in carcinogenesis and mutagenesis. In: Hayes A.W., Schnell R.C. and Miya T.S., (Eds.), Developments in the Science and Practice o f Toxicology, Elsevier, Amsterdam, pp247-254. Takeda, Y., Ohlendorf, D.H., Anderson, W.F. and Mathews, B.W. (1983): DNA-binding proteins. Science. 221. 1020-1026. Takeuchi, K., Ohuchi, T. and Okabe, S. (1994): Endogenous nitric oxide in gastric alkaline response in the rat stomach after damage. Gastroenterol. 106. 367-374. 192 Chapter 8. References Tannenbaum, S.R. (1987): Endogenous formation of N-nitroso compounds: a current perspective. In: H. Bartsch, I.K. O’Neill and R Schulte-Hermann (Eds.), The relevance ofN - nitroso compounds to human cancer. Exposures and mechanisms. IARC Scientific Publications. 84. pp292-296. Thompson, M. (1984): Aetiological factors in gastrointestinal cacinogenesis. Scand J. Gastroenterol. Suppl.19 (104). 77-89. Tricker, A.R. and Preussmann, R. (1987): Influence of cysteine and nitrate on the endogenous formation of N-nitrosoamino acids. Cancer Letts. 34. 39-47. Tricker, A .R (1995): Tissue adducts of N-nitrosamines. Speakers abstract in: Proceedings o f the 13th AnnualE.C.P. Symposium. 29. Uchida, M., Misaki, N. and Kawano, O. (1991): A new method for measurement of blood flow, pH, and transmucosal potential difference in rat gastroduodenal mucosa by endoscopy. Digestive Diseases and Sciences. 36. 1537-1542. Umbenhauer, D., Wild, C.P., Montesano, R , Safhill, R , Boyle, J.M., Huh, N., Kirstein, U., Thomale, J., Rajewsky, M.F., and Lu, S.H. (1985): 0 6-methyldeoxyguanosines in oesophageal DNA among individuals at high risk of oesophageal cancer. IntJ. Cancer. 36. 661-665. Van Schooten, F.J., Hillebrand, M.J., van Leewen, F.E., Lutgerink, J.T., van Zandwijk, N., Jansen, H.M. and Kriek, E. (1990): Polycyclic aromatic hydrocarbon-DNA adducts in lung tissue from lung cancer patients. Carcinogenesis. 11. 1677-1681. Vogelstein, B. Fearson, E.R., Hamiliton, S.R, Kern, S.E., Preisinger, A.C., Leppert, M., Nakamura, Y., White, R , Smits, A.M. and Bos, J.L. (1988): Genetic alterations during colorectal-tumor development. N. Engl. J. Med. 319. 525-532. Vogelstein, B. and Kinzler, K.W. (1992): p53 function and dysfunction. Cell. 70. 523-526. Walters, C.L., Hart, R J. and Smith, P.L.R (1983): Analysis of total N-nitroso compounds as a group by denitrosation to NO with detection using a chemiluminescence analyzer. In: R. Preussmann, I.K. O’Neill, G. Eisenbrand, B. Spiegelhalder and H. Bartsch (Eds.), Environmental carcinogens, selected methods o f analysis for N-nitroso compounds. IARC Scientific Publications. 45. pp295-308. Wang, X., Chen, S.C., Perini, F., Gold, B.I. and Mirvish, S.S. (1995): Carboxyalkylating agents produced by nitrosation of a-amino acids. Poster abstract In: Proceedings o f the 13th AnnualE.C.P. Symposium. 35. Wattenberg, L.W. (1978): Inhibition of chemical carcinogenesis. J. Natl. Cancer Inst. 60. 11- 18. Weinberg, R.A. (1991): Tumour suppressor genes. Science. 254. 1138-1146. 193 Chapter 8. References Weinstein, I.B. (1981): Current concepts and controversies in chemical carcinogenesis. J. Supramol. Struct. Cell. Biochem. 17. 99-120. Wiencke, J.K., Kelsey, K.T., Varkonyi, A., Semey, K., Wain, J.C., Mark, E. and Christiani, D.C. (1995): Correlation of DNA adducts in blood mononuclear cells with tobacco carcinogen-induced damage in human lung. Cancer Res. 55. 4910-4914. Wigley, C. and Balmain, A. (1991): Chemical carcinogenesis and cancer. Franks, L.M. and Teich, N.M. (Eds). Introduction to the cellular and molecular biology o f cancer, Oxford University Press, UK. 148-174. Wild, C.P., Smart, G., Saffhill, R. and Boyle, J.M. (1983): Radioimmunoassay of O6- methyldeoxyguanosine in DNA of cells alkylated in vitro and in vivo. Carcinogenesis. 4. 1605-1609 Wild, C.P., Stich, H.F. and Montesano, R. (1989) : Presence of alkylated DNA in oral mucosa cells from cigarette smokers. Proc. Am. Ass. Cancer. Res. 30. 318-323. Wild, C.P. (1990): Antibodies to DNA alkylation adducts as analytical tools in chemical carcinogenesis. Mutat. Res. 233. 219-233. Wilson, V.L, Weston, A., Manchester, D.K., Trivers, G.E., Roberts, D.W., Kadlubar, F.F., Wild, C.P., Montesano, R., Willey, J.C., Mann, D.L. and Harris, C.C. (1989) : Alkyl and aryl carcinogen adducts detected in human peripheral lung. Carcinogenesis. 10. 2149-2153. Wogan, G.N. and Gorelick, N.J. (1985): Chemical and biochemical dosimetry of exposure to genotoxic chemicals. Environ. Health Perspec. 62. 5-18. World Cancer Research Fund (1997): Food, nutrition and the prevention o f cancer: a global perspective. Published by the American Institute for Cancer Research. Wynder, E.L. and Gori, G.B. (1977): Contribution of the environment to cancer incidence: an epidemiological exercise. J. Natl. Cancer Inst. 58. 825-832. Xu, G.P. and Reed, P.I. (1993): N-nitroso compounds in fresh gastric juice and their relation to intragastric pH and nitrite employing an improved analytical method. Carcinogenesis. 14. 2547-2551. Ying, T.S., Enomoto, K., Sarma, D.S. and Farber, E. (1982): Effects of delays in the cell cycle on the induction of preneoplastic and neoplastic lesions in rat liver by 1,2- dimethylhydrazine. Cancer Res. 42. 876-880. Yokota, S., Amano, K., Hayashi, S. and Fujii, N. (1997): Low antigenicity of the polysaccharide region of Helicobacter pylori lipopolysaccharide derived from tumours of patients with gastric cancer. Infection and Immunity. 65. 3509-3512. Zaidi, H.H. and O'Connor, P.J. (1995) : Identification in rat stomach mucosae of a cell population characterised by a deficiency for the repair of 0 6-methyldeoxyguanosine from DNA. Carcinogenesis. 16. 461-469. 194 Chapter 8. References Zaidi, N.H., Potten, C.S., Margison, G.P., Cooper, D P. and O’Connor, P.J. (1993a): Tissue and cell specific methylation, repair and synthesis of DNA in the upper gastrointestinal tract of Wistar rats treated with single doses of N-methyl-N”-nitro-N-nitrosoguanidine. Carcinogenesis. 14. 1981-1990. Zaidi, N.H., Potten, C.S., Margison, G.P., Cooper, D P. and O’Connor, P.J. (1993b): Tissue and cell specific methylation, repair and synthesis of DNA in the upper gastrointestinal tract of Wistar rats treated with N-methyl-N”-nitro-N-nitrosoguanidine via the drinking water. Carcinogenesis. 14. 1991-2001. Zak, P., KleibI, K. and Laval, F. (1994): Repair of 0 6-methylguanine and 0 4-methylthymine by the human and rat 0 6-methylguanine-DNA methyltransferases. J. Biol Chem. 269. 730- 733. Ziebarth, D., Spiegelhalder, B. and Bartsch, H. (1997): N-nitrosation of medicinal drugs catalyzed by bacteria from human saliva and gastro-intestinal tract, including Helicobacter pylori. Carcinogenesis. 18. 383-389. Zurlo, J., Curphey, T.J., Hiley, R. and Longnecker, D.S. (1982): Identification of 7- carboxymethylguanine in DNA from pancreatic acinar cells exposed to azaserine. Cancer Res. 42. 1286-1288. 195 Chapter 9. Publications and Presentations. 196 Chapter 9. Publications and Presentations. 9. Publications. 9.1. Poster presentations. The following four posters were presented at the various meetings indicated, and they consisted of work resulting from this PhD: 1) ‘Immunochemical detection of 0 6-carboxymethyl-25-deoxyguanosme.’ Harrison K.L. & Shuker, D.E.G. United Kingdom Environmental Mutagen Society (UKEMS) 1995, Leicester (UK). 2) ‘Formation of 0 6-carboxymethyl-2’-deoxyguanosine in DNA by nitrosated glycine derivatives and related compounds.’ Harrison K.L. & Shuker, D.E.G. 13th Annual European Cancer Prevention Symposium: N-Nitroso compounds in human cancer, current status and future trends, 1995, London (UK). 3) ‘Formation of 0 6-carboxymethyl- and 0 6-methyl-2’-deoxyguanosine in DNA by nitrosated glycine derivatives and related compounds.’ Harrison K.L., Cooper D.P. & Shuker, D.E.G. British Toxicology Society annual meeting, 1996, York (UK). 4) ‘Formation of 0 6-carboxymetbyl- and 0 6-methyl-2’-deoxyguanosine in DNA in vitro and in vivo by nitrosated glycine derivatives.’ Harrison K.L., Cooper D.P., Margison, G.P. & Shuker, D.E.G. American Association of Cancer Research (AACR) annual meeting, 1997, San Diego, (USA). 9.2. Publications. To date results generated from this PhD have contributed in two publications copies of which are attached overleaf. 197 Chapter 9. Publications and Presentations. Shuker, D.E.G., Leuratti, C., Harrison, K.L., Conduah Birt, J. and Farmer, P.B. (1995) : Non invasive or minimally invasive biomarkers of exposure to genotoxic agents derived from food. In Biomarkers in Food Chemistry Risk Assessment (eds. Crews, H.M. & Hanley, A.B.). The Royal Society of Chemistry, pp 73-79. Non-invasive or Minimally Invasive Biomarkers of Exposure to Genotoxic Agents Derived from Food D. E. G. Shuker, C. Leuratti, K. Harrison, J. Conduah Birt, and P. B. Farmer MRC TOXICOLOGY UNIT. HODGKIN BUILDING, UNIVERSITY OF LEICESTER. PO BOX 138, LANCASTER ROAD. LEICESTER LEI 9HN, UK 1 INTRODUCTION The incidence of some of the major cancers (of gastrointestinal tract [such as esophagus, stomach and colon/rectum] and breast) varies widely over the world1. The sometimes extreme extent of the variation (for example, of esophageal cancer within China where there is an 20-fold difference between the highest and lowest rates) has led to the conclusion that environmental and/or lifestyle factors have a major role in their etiology2. Striking evidence for this conclusion has come from studies of migrant populations, such as Japanese living in Hawaii or on the West Coast of the United States, where their cancer rates, at sites such as stomach, begin to reflect those of the local population within one generation3. There is good evidence that changes in consumption of specific dietary components are linked to these effects4. The underlying etiology of the major cancers noted above has not been unambiguously elucidated but many epidemiological studies have highlighted various dietary risk, and protective factors which account for some of the variability. High consumption of fat and red meat are risk factors for colorectal cancers and a lack o f fresh fruits and vegetables is associated with increasing risk of stomach cancer. Many of the known human carcinogens are DNA-damaging agents and there is increasing evidence that the formation o f covalent DNA adducts is a necessary, but probably not sufficent, factor in the mechanisms of carcinogenesis of these agents5. Given that the human diet is an extremely complex mixture of chemicals it is extremely unlikely that the risk o f cancer at any site will be associated with single food constituents. However, this can sometimes happen in the case of contamination of food with particularly potent carcinogens, such as the naturally occuring aflatoxins, which are responsible for otherwise rare cancers. It may be the effect of diet on the levels of endogenous DNA damage such as depurination, oxidation and deamination which accounts for the major cancer risks associated with diet6. Conversely, the modulation of these same endogenous processes by dietary factors may account for the protection against cancer provided by some foods such as fresh fruits and vegetables7. The unique aspects of dealing with food-related exposures to genotoxic agents has been succinctly stated by Saracci8, “Unlike xenobiotic agents which, if found to be carcinogenic, can at least in principle be dispensed with, food is indispensable and many individual nutrients cannot be dispensed with either. Furthermore, in contrast with a toxic xenobiotic agent for which 198 Chapter 9. Publications and Presentations. 74 liionm rkvrs in k'otnl C/icniic the'lowest possible exposure is best for health, an optimal range of intake exists for most nutrients, and this needs to be determined as accurately as possible in humans so that the levels above and below which harmful effects occur can be identified"'. We have recently begun a programme of work aimed at developing a number of biomarkers o f exposure to food-related DNA-damaging agents. The overall objectives of the programme are to develop biomarkers of DNA damage derived from macroscopic components o f diet (fat and protein) as well as certain individual food components consumed at a relatively high level, such as alcohol. The approaches that are being employed are based on the use of accessible bodily fluids (blood and urine) and the analysis o f covalently-modified macromolecules such as haemoglobin and excreted DNA adducts. These approaches have been reviewed in a number of articles9,10. The analytical techniques which have been most widely used in our laboratory have also been reviewed and include mass spectrometry11 and immunoaffinity purification in combination with various analytical methods12. 2 BIOMARKERS OF EXPOSURE TO NITROSATION PRODUCTS OF AMINO ACIDS AND PEPTIDES Amongst the many N-alkyl-N-nitroso compounds that are known to be mutagenic and carcinogenic, there are a number which share a common feature of being derived from the simplest amino acid glycine and are all, in principle, carboxymethylating agents13. N- nitrosoglycocholic acid (NOGC) is a carcinogenic and mutagenic derivative of the naturally occuring bile acid conjugate, glycocholic acid14,15,16. Incubation of NOGC with calf thymus DNA in vitro gave rise to N-7-carboxymethylguanine (7-CMG), N-3- carboxymethyladenine (3-CMA) and 0 -carboxymethylguanine6 .^17 (0 -CMG)‘ . Furthermore, administration of [14C]-NOGC to rats gave rise to dose dependent excretion o f 7-CMG in urine17. N-nitrosopeptides which are C-terminal in glycine, such as N-(N’- acetyl-L-prolyl)-N-nitrosoglycine (APNG), are mutagenic18 and carcinogenic19 and would be expected to be carboxymethylating agents by analogy with NOGC. Similarly, N- nitroso-N-carboxymethylurea is a gastrointestinal carcinogen20,21. Azaserine, a pancreatic carcinogen, is also known to carboxymethylate DNA in vivo and [14C]-7-CMG was detected in DNA extracted from pancreatic acinar cells treated with (14C]-azaserine22. Recently, 0 6-carboxymethyl-2’-deoxyguanosine (0 6-CMdG) has attracted our particular interest because of its apparent lack of repair by bacterial and mammalian O6- alkylguanine alkyl transferases (Shaker and Margison, unpublished results) and therefore offered the possibility of a group-specific and possibly persistent DNA adduct derived from nitrosated glycine derivatives. A specific antiserum to 0 6-CMdG was prepared and used to make reusable immunoaffinity columns which selectively retained the adduct from DNA hydrolysates. The binding of 0 6-CMdG was so strong that conditions used to elute the adduct (0.1 M trifluoroacetic acid) resulted in partial hydrolysis (becoming quantitative on heating) o f the glycosidic bond to give 0 6-CMG which is more fluorescent than the deoxynucleoside. DNA treated with APNG (5 mmol) afforded 0 6-CMdG at levels of 35 pmol/mg DNA. Similar results were obtained with potassium diazoacetate (Harrison et a i, manuscript in preparation). The antiserum was also found to be capable of detecting 0 6-CMdG in situ in tissue sections from azaserine-treated animals by immunohistochemical staining (Harrison et al, unpublished results). 199 rhfrpter 9 Publications and Presentations. Noti-iiivitsivc (Udmarkcrs o f Espt>sitiT to Gcnoio.xic Agents 75 In summary, a diverse range of N-nitrosoglycine or-nitrosated glycine derivatives all give rise to the same DNA adduct, 0 6-CMdG, via formation o f a common carboxymethylating intermediate (Scheme 1). 0 6-CMdG and related adducts (7-CMG and 3-CMA) are all potential markers of exposure to dieGrelated nitrosation pathways Diazoncetic acid derivatives N-nitrosoglycine derivatives N'* N Potassium diazoaeetate h 0 “ k + Ac APNG CH, OH HO HO''' OH NOGC N = N O carboxymethyldiazonium ion DNA | DNA— " O carboxymethylated DNA Scheme 1. Formation of carboxymethylating age?its from a range of nitrosated glycine derivatives 3 MALONDIALDEHYDE-DNA ADDUCTS AS BIOMARKERS OF LIPID PEROXIDATION Malondialdehyde (MDA) is the most abundant carbonyl compound and the major mutagenic and carcinogenic product generated by lipid peroxidation. The major DNA adduct formed at neutral pH is the highly fluorescent pyrimidopurinone product from 2’- deoxyguanosine (M,dG, Scheme 2)23,24 Recent evidence suggests that M,dG is present in human liver DNA at levels of 5-11 adducts per 107 bases25. The analytical method involved the isolation o f the base M,G from DNA hydrolysates followed by conversion to a pentafluorobenzyl derivative which was quantified by NICI GC-MS26. The limit of sensitivity o f the assay was approximately 2 adducts per 108 base pairs with 300 ^ig of DNA. Previous work had suggested that both rats and humans excreted M,G base in urine26,27 but this could not be satisfactorily reproduced in another laboratory28. 200 Chapter 9. Publications and Presentations. 76 Hmnuirkcrs in /■ <> M fdGM? has been detected in DNA using 32P-postlabelling but the currently available methods have some drawbacks. The procedure developed by Vaca et aJ:) does not, in our hands, satisfactorily separate M^GMP from the normal base dGMP. The recently published procedure of Wang et aZ30 would appear to'more suitable but still lacks the power to unambiguously quantify M,dG. We have improved the synthesis of M,dGMP for use as an authentic standard and developed conditions for purification of the adduct byHPLC with fluorescence detection prior to postlabelling. M,dGMP is phosphorylated by incubation with [y-32P]-ATP and T4 polynucleotide kinase and the resulting 3’,5’-bisphosphate separated by two- dimensional TLC. Based on our previous experience12 we are also attempting to prepare antibodies to M(dG in order to make immunoaffinity columns which will allow selective purification of the adduct. H O NH HO HO OH OH dG MjdG Scheme 2. Reaction of malondialdehyde with deoxygnanosine 4 STABLE ACETALDEHYDE-PROTEIN ADDUCTS AS BIOMARKERS OF ALCOHOL EXPOSURE Acetaldehyde is the highly reactive intermediate produced in the metabolism of ethanol to acetate mainly by the action of alcohol dehydrogenase and microsomal cytochrome P450IIE1. The toxic effects of excessive alcohol consumption including liver disease are thought to be due to acetaldehyde, which binds to hepatic as well as other proteins. A number of in vitro studies have shown that acetaldehyde forms stable and unstable adducts with haemoglobin and proteins, such as albumin, tubulin and collagen31. In the case of haemoglobin, several types of adducts have been identified as due to acetaldehyde modification. Various approaches such as hplc, nmr, ms and immunological assays have been used for detecting acetaldehyde adducts. Modified haemoglobin has been detected in red blood cells and blood from alcohol consuming individuals31,32’33. NMR and MS techniques have been utilized to confirm the formation and modification of haemoglobin and peptides34,35,36. In immunological studies, antibodies have been produced that recognize acetaldehyde modified proteins but the structures of the epitopes have not been 201 Chapter 9. Publications and Presentations. Non-inva.sivc liinmarkers of Exposure to Genotoxic Agents 7 ' characterised. However, at present there is no reliable quantitative method available foi measuring acetaldehyde adducts. The aim of this study was to locate a site on the haemoglobin chain where a reproducible adduct is formed, identify the structure and develop a suitable quantitative method for analysis. Preliminary studies were carried out using two model peptides, a dipeptide (valine- leucine), and a polypeptide (21 chain amino acid). Incubation of the two peptides with acetaldehyde yielded stable adducts which we were able to identify by mass spectrometry. Possible unstable adducts may have been produced as intermediates but these were not detected in these assays. From the tandem MS/MS analysis it appeared that the stable adducts were formed between the aldehyde and the amino group of the N-terminal valine o f each o f the peptides. San George and Hoberman34 found that on incubation of haemoglobin with acetaldehyde an imidazolidinone type adduct was produced at the N- terminal end o f haemoglobin and we have confirmed these results with the model peptides and shown by NMR that a diastereoisomeric mixture of adducts is formed (Scheme 3). Current work is aimed at developing a quantitative method for the analysis of the stable acetaldehyde adduct on the N-terminal valine of haemoglobin. 5 DISCUSSION The work described in this short review is at an early stage of development but the preliminary results suggest that informative biomarkers of human exposure to genotoxic compounds, particularly alkylating agents derived from major'constituents in diet, can be developed. The work of Ames and his collaborators6 on DNA oxidation suggests that this endogenous source of genotoxic damage can be strongly influenced by diet, particularly total caloric intake. However, the focus of our work is on alkylation damage from endogenous sources which occurs at levels not dissimilar to those observed for oxidative DNA damage. Thus, macroscopic components of the diet such as fat and protein can be converted by endogenous processes such as lipid peroxidation and peptide- and amino acid nitrosation to reactive alkylating agents (malondialdehyde and alkyldiazonium ions, respectively). Recent results suggest that the characteristic DNA adducts formed by these pathways can be detected with sufficient sensitivity in blood and urine samples to enable them to be used in studies on human subjects. Acknowledgements This work was supported by MAFF Contract No. 1A025 and the Medical Research Council. We gratefully acknowledge the help of Dr. Gavain Sweetman for help with mass spectral analyses and Ms. Rebekah Jukes for assistance with NMR spectra. 202 Chapter 9. Publications and Presentations. Dipeptide Acetalclelivde R" x „ . H,N A Nucleophiiic addition results V in tetrahedral intermediate HN HO CH. A -H,0 Schiff base Nucleophiiic attack of the CH. amide nitrogen of the peptide bond on the imine nitrogen o HN H HN CH. CH3 Monoethylated derivative 2-methylimidazolin-4-one Scheme 3. Reactions of acetaldehyde with the terminal amino 'group o f peptides (adaptedfrom San George and Hoberman4) References 1. IARC Scientific Publication No. 100 (1990), IARC, Lyon, France. 2. IARC Scientific Publication No. 120 (1992), IARC, Lyon, France. 3. D. M. Parkin, in IARC Publication No. 123 (1993), IARC, Lyon, France, Chap.l. 4. L. N. Kolonel, A. M. Y. Nomura, T. Hirohata, J. H. Hankin and M. W. Hinds, Am. J. Clin. Nutr., 1981, 34, 2478. 5. Consensus report in “Mechanisms of Carcinogenesis in Risk Identification” (H. Vainio, P. Magee, D. McGregor and A. J. McMichael, eds), IARC Scientific Publication No. 116, IARC, Lyon, France (1992) pp.9-54. 6. B. N. Ames, L. S. Gold and W. C. Willett, Proc. Natl. Acad Sci. USA 1995, 92, 5258. 203 Chapter 9. Publications and Presentations. ivii/i-///vu .M ir />/<'///(//a(V.v i)/ /•..v/ju.vm/v if> (.ic iio io m c A g e n t s I n 7. K. A. Steinmetz and J. D. Potter, Cancer Causes and Control, 1991, 2, 325. 8. R. Saracci,./. Intern. Med., 1993, 233, 41. 9. D. E. G. Shuker and P. B. Farmer, Chent. Res.Toxicol.. 1992, 5, 450. 10. P. B. Farmer, Clin. Chem., 1994, 40, 1438. 11. P. B. Farmer and G. M. A. Svveetman,./. Mass. Spectrom.. 1995, in press. 12. D. E. G. Shuker and H. Bartsch, Mutat. Res.. 1994, 313, 263. 13. B. C. Challis, Cancer Surveys, 1989,8,363. 14. D. E. G. Shuker, S. R. Tannenbaum and J. S. Wishnok,./. Org. Chent., 1981, 46, 2092. 15. S. Puju, D. E. G. Shuker, W. W. Bishop, K. R. Falchuk, S. R. Tannenbaum and W. G. Thilly, Cancer Res., 1982, 42, 2601. 16. W. F. Busby, Jr., D. E. G. Shuker, G. Charnley, P. M. Newberne, S. R. Tannenbaum and G. N. Wogan, Cancer Res., 1985, 45, 1367. 17. D. E. G. Shuker, J. R. Howell and B. W. Street, in ‘‘Relevance of N-Nitroso Compounds to Human Cancer: Exposures and Mechanisms” (H. Bartsch, I. K. O’Neill and R. Schulte-Hermann, eds), IARC Scientific Publication No. 84, IARC, Lyon, France, pp. 187-190. 18. D. Anderson, B. J. Phillips, B. C. Challis, A. R. Hopkins, J. R. Milligan and R. C. Massey. Food Chem. Toxicol., 1986, 24, 289. 19. D. Anderson and S. D. Blowers, Lancet, 1994, 344, 343. 20. O. Bulay, S. S. Mirvish, H. Garcia, A. F. Pelfrene and B. Gold, J. Natl. Cancer Inst., 1979, 62, 1523. 21. A. Maekaiva, T. Ogiu, C. Matsouka, H. Onedura, K. Furuta, H. Tanagawa and S. Odashima, J. Cancer Res. Clin. Oncol., 1983, 106, 12. 22. J. Zurlo, T. J. Curphey, R. Hiley and D. S. Longnecker, Cancer Res., 1982, 42, 1286. 23. H. Seto, T. Takesue and T. Ikemura, Bull. Soc. Chem. Jpn., 1985, 58, 3431. 24. L. J. Marnett, A. K. Basu, S. M. O’Hara, P. E. Weller, A. F. M. Maqsudur Rahman and J. P. Oliver, J. Amer. Chem. Soc., 1985, 108, 1348. 25. A. K. Chaudhary, M. Nokubo, G. R. Reddy, S. N. Yeola, J. D. Morrow, I. A. Blair and L. J. Marnett, Science (Washington), 1994, 265, 1580. 26. M. Hadley and H. H. Draper, Lipids, 1990, 25, 82. 27. S. Agarwal and H. H. Draper, Free Rad Biol. M ed, 1992, 13, 695. 28. H. K. Jajoo, P. C. Burcham, Y. Goda, I. A. Blair and L. J. Marnett, Chem. Res. Toxicol., 1992, 5, 870. 29. C. E. Vaca, P. Vodicka and K. Hemminki, Carcinogenesis, 1992, 13, 593. 30. M.-Y. Yang and J. G. Liehr, Arch. Biochem. Biophys., 1995, 316, 38. 31. M. D. Gross, S. M. Gapstur,J. D. Belcher, G. Scanlan and J. D. Potter, Alcohol. Clin. Exp. Res., 1992, 16, 1093. 32. V. J. Stevens, W. J. Fantl, C. B. Newman, R. V. Sims, A. Cerami and C. M. Peterson, J. Clin. Invest., 1981, 67, 361. 33. L. Itala, K. Seppa, U. Turpeinen, and P. Sillanaukee, Anal. Biochem., 1995, 224, 323. 34. R. San George, and H. D. Hoberman, J. Biol. Chem., 1986. 261, 6811. 35. K. P. Braun, R. B. Cody, D. R. Jones,and C. M. Peterson, J. Biol. Chem., 1995. 270,11263. 36. R. C. Lin, J. B. Smith, D. B. Radtke, and L. Lumeng, Alcohol. Clin. Exp. Res, 1995, 19, 314. 204 Synthesis, Characterization, and Immunochemical Detection of D6-(Carboxymethyl)-2'-deoxypuanosine: A DNA Adduct Formed by Nitrosated Glycine Derivatives Kathryn L. Harrison, Neil Fairhurst, Brian C. Challis, and David E. G. Shuker MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, U.K., and Department of Chemistry, The Open University, Walton Hall, Milton Keynes MK9 6AA, U.K. Chemical Research in Toxjcojogy Reprinted from Volume 10, Number 6, Pages 652-659 652 Chem. Res. Toxicol. 1997, 10, 652—659 Synthesis, Characterization, and Immunochemical Detection of 0 6-(Carboxymethyl)-2'-deoxyguanosine: A DNA Adduct Formed by Nitrosated Glycine Derivatives Kathryn L. Harrison,^ Neil Fairhurst,* Brian C. Challis,^ and David E. G. Shuker*’* MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester LEI 9HN, U.K., and Department of Chemistry, The Open University, Walton Hall, Milton Keynes MK9 6AA, U.K Received December 12, 1996® 0 6-(Carboxymethyl)-2'-deoxyguanosine (Oe-CMdG) is formed in DNA by nitrosated glycine derivatives and appears to be nonrepairable by Oe-alkylguamne transferases. Oe-CMdG has been synthesized by an unambiguous route involving the introduction of a methyl glycolate moiety at C6 of a 3',5'-bis-0-(methoxyacetyl)dGuo derivative by displacement of a quinuclid- inium ion. Methanolysis of the methoxyacetyl groups and calcium hydroxide-mediated hydrolysis of the methyl ester afforded the calcium salt of Oe-CMdG in good yield. A similar route was used to synthesize 0 6-(carboxymethyl)guanosine (06-CMGuo), which was used to prepare protein conjugates for immunization. Rabbit antisera were prepared, and a quantita tive competitive ELISA was developed which showed 50% inhibition at 2 pmol of 0 6-CMdG/ well. 0 6-CMGuo was 30 times less cross-reactive (50% inhibition at 60 pmol/well), and normal nucleosides and carboxymethylated purines did not cross-react to any significant extent. The antiserum was also used to prepare reusable immunoaffinity columns which retained Oe-CMdG. The binding of 0 6-CMdG was so strong that conditions used to elute the adduct (1 M trifluoroacetic acid) resulted in partial hydrolysis (becoming quantitative on heating) of the glycosidic bond to give Oe-CMguanine which was detected by HPLC with fluorescence detection. DNA treated with azaserine (5 mmol), IV-dV'-acetyl-L-prolyD-JV-nitrosoglycine (5 mmol), and potassium diazoacetate (5 mmol) afforded Oe-CMdG at levels of 7.3, 393.9, and 496 //mol of Oe-CMdG/mol of dG. The antiserum also recognized Oe-CMdG in intact DNA. Introduction to N -l-(carboxymethyl)guanine (7-CMG), JV-3-(carboxym- ethyl)adenine (3-CMA), and 0 6-(carboxymethyl)guanine Alkylation of DNA is considered to be a key step in (Oe-CMG) (7). Furthermore, administration of [14C]- the induction of cancer by many different chemicals (I). NOGC to rats gave rise to dose dependent excretion of For many compounds including N-alkyl-N-nitroso com 7-CMG in urine (7). A-Nitroso peptides which are pounds, alkylation at O6 of 2'-deoxyguanosine (dGuo) C-terminal in glycine, such as N-(N'-acetyl-L-pro\yl)-N- appears to be the major mutagenic lesion, although O4- nitrosoglycine (APNG), are mutagenic (8) and carcino alkylthymidines may also be mutagenic (2). In both genic (9) and would be expected to be carboxymethylating prokaryotic and eukaryotic cells, efficient specific repair agents by analogy with NOGC. Similarly, N-nitroso-JV- mechanisms exist for the removal of Os-methyldGuo (carboxymethyl)urea is a gastrointestinal carcinogen (10, residues as well as some higher homologues, albeit more 11). Azaserine (AS), a pancreatic carcinogen, is also slowly (3). known to carboxymethylate DNA in vivo. [14C]-7-CMG Among the many A-alkyl-iV-nitroso compounds that was detected in DNA extracted from acinar cells treated are known to be carcinogenic, there are a number which in vitro with [14C]azaserine (12). share a common feature of being carboxymethylating Recently, 0 6-(carboxymethyl)-2'-deoxyguanosine (O6- agents. N-Nitrosoglycocholic acid (NOGC1 ) is a carci nogenic and mutagenic derivative of the naturally oc CMdG) has attracted our interest because of its apparent curring bile acid conjugate glycocholic acid (4—6). Incu lack of repair by bacterial and mammalian Oe-alkylgua- bation of NOGC with calf thymus DNA in vitro gave rise nine alkyltransferases (13). In an analogous manner to many N -nitroso compounds, NOGC gives rise to alkyla tion mainly at purine nitrogen atoms, and the level of * Author to whom correspondence should be addressed. Oe-CMdG is about 10% of that at N -7 of guanine (13). In t University of Leicester. * The Open University. order to study the occurrence of Oe-CMdG in DNA, the ® Abstract published in Advance ACS Abstracts, May 1, 1997. synthesis and characterization of the previously unknown 1 Abbreviations: NOGC, iV-nitrosoglycocholic acid; 7-CMG, 7-(car- boxymethyl)guanine; 3-CMA, 3-(carboxymethyl)adenine; Oe-CMG, O6- Oe-CMdG were required for its use as an authentic (carboxymethyl)guanine; 0 6-CMGuo, Oe-(carboxymethyl)guanosine; compound. In addition, antibodies to 0 6-CMdG were APNG, N-(N'-acetyl-L-prolyl)-N-nitrosoglycine; AS, azaserine; O6- required for detection and analysis of this adduct in CMdG, 0 6-(carboxymethyl)guanine-2'-deoxyguanosine; BSA, bovine serum albumin; OV, ovalbumin; PBS, phosphate-buffered saline; TFA, human tissues. Previous experience (14,15) has shown trifluoroacetic acid; TEA, triethylammonium acetate; DBU, 1,8- that antibodies are extremely useful not only in quanti diazabicyclo[5.4.0]undecane; ELISA, enzyme-linked immunosorbant tation of adducts but also in the isolation, purification, assay; IgG, immunoglobulin G; TPase, thymidine phosphorylase; PNPase, purine nucleoside phosphorylase; KDA, potassium diazoac and concentration of the adducts using immunoaffinity etate. techniques. In the case of Oe-alkyldG adducts, antigens S0893-228x(96)00203-2 CCC; $14.00 © 1997 American Chemical Society Immunochemical Detection of Oe-CMdG Chem. Res. Toxicol., Vol. 10, No. 6, 1997 653 can be prepared from the corresponding guanosine 0 6-(Carboxymethyl)-2'-deoxyguano8ine, Methyl Ester analogue (16), and this approach is described along with (le). lb (0.3 g, 0.5 mmol) was dissolved in dry THF (4 mL), the use of the antibodies to immunopurify C^-CMdG prior quinuclidine (0.32 g, 2.5 mmol) was added, and the solution to detection by HPLC fluorescence. stirred at room temperature under dry nitrogen. Progress of the reaction was monitored by loss of lb and formation of a blue Materials and Methods fluorescent spot at the baseline (believed to be the quinuclid- inium ion intermediate lc), using silica gel TLC developed with W arning: APNG and AS have been shown to be carcinogenic EtOAc/CILOH (85:15, v/v). The formation of lc was usually in experimental animals and should be treated with extreme complete in ca. 3 h at room temperature. Dry methyl glycolate caution. Unused solutions of APNG were decomposed by (0.44 g, 5 mmol) in THF (3 mL) and l,8-diazabicyclo[5.4.0]- overnight treatment with 0.1 M NaOH in a fume cupboard. undecane (DBU; 0.24 g, 1.5 mmol) in THF (3 mL) were added Unused solutions of AS and potassium diazoacetate wereto the mixture, and the solution stirred at room temperature decomposed by overnight treatment with 10% aqueous aceticfor 3—4 h. After deprotection of Id using methanolic EtaN and acid. purification by column chromatography on silica gel (30 g) using General. Melting points were measured on a Gallenkamp CH3OH/EtOAc (15:85, v/v) as eluant, le was further purified hot- 8 tage apparatus and are uncorrected. Infrared spectra were by semipreparative LC on a Phase Sep S10 ODS2 C-18 silica recorded on Perkm-Elxner 298 and 1420 grating spectropho gel column, using CH 3CN/H20 (13:87, v/v) isocratic eluant tometers. 1H-NMR spectra were recorded on either a Jeol system. After removal of the eluant by lyophilization, le was FX90Q spectrometer or a Jeol FX400Q in the solvents indicated. obtained as a white crystalline solid: yield 0 .1 1 g ( 6 8 %); mp Mass spectra were recorded by using VG-7070 and VG-20-250 8 3 -8 4 °C; A*** (pH = 7) 248,281; vM (KBr) 3300 (N -H , O -H instruments. Microanalyses were provided by Medac Ltd., str), 2800 (C -H str),1752 (C =0 str), 1650 (C=N str), 1580 (C=C Brunei University, London. 2'-Deoxyguanosine and guanosine str), 1250 (C -O str), 1065 cm"1 (C -0 str); NMR (DMSO-d6) <5 were supplied by Cruachem Ltd. (Aired, Glasgow, U.K.). Methyl 2.30 (2H, m, 2'-H), 3.55 (2H, m, 5'-CH2), 3.61 (3H, s, OCH3), glycolate (Kodak) was purified by distillation. Quinuclidine 3.80 (1H, m, 4'-H), 4.35 (1H, m, 3'-H), 4.81 (2H, s, CH 2C 02CH3), (Aldrich) was purified by sublimation. Azaserine, bovine serum 5.0 (1H, t, 5'-OH), 5.30 (1H, d, 3'-OH), 6.22 (1H, t, l'-H), 6.45 albumin (BSA), ovalbumin (OV), horseradish peroxidase-linked (2H, brs, NH2), 8.10 (1H, s, 8 -H); MS m/z (FAB positive ion) goat anti-rabbit immunoglobulin G (IgG), thymidine, thymidine 340 (MH+) 10, 224 (MH+ - 116) 20, 152 (MH+ - alkyl - 116) phosphorylase (Tpase, E.C. 2.4.2.4), purine nucleoside phospho 32. rylase (PNPase, E.C. 2.4.2.1), calf thymus DNA, nuclease PI O6-(Carboxymethyl)-2'-deoxyguanosine (If), le (0.042 (E.C. 3.1.30.1), alkaline phosphatase type III (Escherichia coli, g, 0.12 mmol) was dissolved in aqueous 4 mM Ca(OH )2 (30 mL), E.C. 3.1.3.1), and acid phosphatase type I (E.C. 3.1.3.2) were and the solution stirred at room temperature. Loss of starting purchased from Sigma Chemical Co. [ 3H]Thymidine was pur material was monitored by TLC on silica gel using EtOAc/CH 3- chased from Amersham Life Sciences. Buffer salts and solvents OH (85:15, v/v) as eluant. After 18 h, lyophilization of the were purchased from Fisons and were of an analytical grade. reaction solution gave If as a white fluffy solid. The solid was 3',5'-Bis-0- (Methoxyacetyl) -2'-deoxyguanosine (la). 2'- dissolved in absolute ethanol, and the insoluble residue was Deoxyguanosine (1.0 g, 3.60 mmol) was suspended in anhydrous removed. The solvent was removed under reduced pressure, N^V-dimethylformamide (DMF) (16 mL) containing pyridine (4 to give I f as a white solid: yield 0.040 g (98%); mp >250 °C; (17) mLi), methoxyacetic anhydride (2.45 g, 15 mmol) was added, Amax (pH = 7) 248, 282; (KBr) 3300 (N -H , O -H br str), and the mixture was heated at 40 °C for 2 h. The solution 2860 (C-H str), 1650 (C=N str), 1620 (C=0 str), 1580 (C=C became homogeneous and was allowed to cool to room temper str), 1250 (C—O str), 1065 cm-1 (C—O str); NMR (DMSO-cfe) <5 ature. Methanol (0.5 mL) was then added, and the solution 2.30 (2H, m, 2'-H), 3.55 (2H, m, 5'-H), 3.80 (1H, m, 4'-H), 4.35 stirred for a further 30 min at room temperature. The m ixture (1H, m, 3'-H), 4.64 (2H, s, CH2), 5.0 (1H, t, 5'-OH), 5.30 (1H, d, was concentrated under reduced pressure, and the residue was 3'-OR), 6.22 (1H, t, l'-H), 6.45 (2H, brs, NH2), 8.09 (1H, s, 8 -H); filtered and then washed with cold ethanol. Recrystallization MS m /z (FAB negative ion) 324 (M~) 8,151 (M_ — alkyl — 116) from ethanol gave l a as white crystals: yield 1.18 g (81%); mp 20. Anal. Calcd for C^-MNioOiaCa^HaO: C, 39.78; H, 4.42; 176-177 °C; Vmax (Nujol) 3450 (N -H str), 3350 (N -H str), 1750 N, 19.34; 0 , 30.94; Ca, 5.52. Found: C, 39.60; H, 4.44; N, 19.30. (C=0 ester str), 1700 (C=0 amide str), 1230 cm -1 (C—O str); NMR (DMSO-de) <5 2.50 (1H, m, 2 '-H), 2.93 (1H, m, 2 '-H), 3.30 0 6-[(2,4,6-Trimethylphenyl)sulfonyl]-2',3',5'-tri-0- acetylguanosine (2b). 2a (18) (1.0 g, 2.4 mmol), 2-mesityl- (6 H, s, 2 x CHa), 3.92 (4H, 2s, 2 x CH2), 4.10 (2H, s, 5'-CH2), 4.30 (1H, m, 4'-H), 5.38 (1H, m, 3'-H), 6.17 (1H, t, l'-H), 6.63 sulfonyl chloride (1.05 g, 4.8 mmol), and 4-(dimethylamino)- (2H, brs, NH2), 7.95 (1H, s, 8 -H), 10.70 (1H, brs, NH); MS m /z pyridine (130 mg, 1.06 mmol) were suspended in CH3CN (12 (FAB positive ion) 412 (MH+) 20, 152 (MH+ - 260) 100. mL). Triethylamine (1.10 g, 9.6 mmol) was added dropwise, and 0 6-[(2,4,6-Trimethylphenyl)sulfonyl]-3,,5/-bis-0-(meth- the suspension stirred at room temperature. After 2 h, the oxyacetyl)-2/-deoxyguanosine (lb). 3',5'-Bis-0-(methoxy- residue was evaporated to dryness under reduced pressure, and acetyl)-2/-deoxyguanosine (1.0 g, 2.4 mmol), 2-mesitylsulfonyl the residue was redissolved in CHCI 3/CH3OH (90:10, v/v) (2 mL) chloride (1.2 g, 5.5 mmol), and 4-(dimethylamino)pyridine (15 and separated on silica gel (60 g) using CHCI 3 (70 mL) and then mg, 1.2 mmol) were suspended in CH 3CN (10 mL). Triethy- CHCla/CH3OH (97:3, v/v) as eluants, to give a brown solid which lam in e (1.15 g, 11.4 mmol) was added drop wise, and the was dissolved in the minimum volume of CH 2C12 and precipi suspension stirred at room temperature. After 2 h, this solution tated with petroleum ether (40—60 °C) to give 2b as yellow was concentrated under reduced pressure; the residue was crystals: yield 1.31 g (91%); mp 140—141 °C [lit. (17) mp 141— 142 °C]; (Nujol) 3450 (N-H str), 3350 (N-H str), 1730 redissolved in CHCI 3/CH3OH (90:10, v/v, 10 mL) and then (C=0 ester str), 1350 (S02—O str), 1230 (C—O str), 1160 cm-1 separated on silica gel (60 g) using CHCI 3 (70 mL) and then (S 02- 0 str); NMR (CDClg) <5 2.03 (3H, s, CH 3CO), 2.07 (3H, s, CHCI3/CH3OH (97:3, v/v) as eluants, to give the product lb as a brown glass. This glass was dissolved in the minimum volume CH3CO), 2.08 (3H, s, CH 3CO), 2.30 (3H, s, p-CH3), 2.70 ( 6 H, s, o-CHg), 4.35 (2H, brs, 5'-CH2), 5.60 (2H, m, 2'-H, 3'-H), 5.85 (1H, of CH 2CI2 and precipitated with petroleum ether (40—60 °C) to give lb as pale yellow crystals: yield 1.35 g (95%); mp 140— m, 2'-H), 6.00 (1H, brd), 6.50 (2H, brs, NH2), 6.9 (2H, s, arom), 152 °C; Vmax (Nujol) 3450 (N -H str), 3350 (N -H str), 1750 (C=G 7.8 (1H, s, m, 8 -H); MS m /z (FAB positive ion) 592 (MH+) 18, 334 (MH+ - 258) 50. ester str), 1350 (SOa-o str), 1230 (C -0 str), 1160 cm ' 1 (S 0 2- str); NMR (CDCI3) <5 2.31 (3H, s,p-CH3), 2.50 (1H, m, 2 /-H), 2.75 0®-(Carboxymethyl)guano8ine, Methyl Ester (2e). 2b (6 H, s, 2 x 0 -CH3), 2.93 (1H, m, 2 '-H), 3.35 (6 H, 2s, 2 x CH3), (0.3 g, 0.5 mmol) was dissolved in dry THF (4 mL), quinuclidine 3.92 (4H, 2s, 2 x CH2), 4.10 (2H, s, 5'-CH2), 4.40 (1H, m, 4'-H), (0.32 g, 2.5 mmol) was added, and the solution was stirred at 4.95 (2H, brs, NH2), 5.50 (1H, m, 3'-H), 6.30 (1H, t, l'-H), 6.95 room temperature under dry N2. Progress of the reaction was (2H, brs, arom), 7.90 (1H, s, 8 -H); MS m/z (El) 593 (M+) 12, monitored by loss of 2b and formation of a blue fluorescent spot 333 (M+ - 260) 30. at the baseline (presumably the quinuclidium salt 2c), using 654 Chem. Res. Toxicol., Vol. 10, No. 6, 1997 Harrison et al. silica gel TLC developed with EtOAc/CHaOH (85:15, v/v). The Purification of [3H]-Oe-CMdG: Aliquots of the reaction formation of the quinuclidine ion intermediate 2 c was usually mixture (3 x 100 fiL) were loaded onto the HPLC system (run complete in ca. 3—4 h at room temperature. Dry methyl conditions as above), and the peak corresponding to Oe-CMdG glycolate (0.44 g, 5 mmol) in THF (5 mL) and DBU (0.24 g, 1.5 was collected and concentrated by freeze-drying. These fractions mmol) in THF (3 mL) were added to the reaction mixture, and were then combined, concentrated to dryness, redissolved in 100 the solution stirred at room temperature until the blue fluo fiL of 0.1 M TEA (pH = 7), and reinjected onto the HPLC system. rescent spot disappeared. After 4 h, the solution was cooled to Fractions were collected every 30 s for 30 min, and 5 fiL of each room temperature, the solvent removed under reduced pressure, (in 3 mL of liquid scintillation fluid) was then counted on a and the resultant residue dissolved in methanol (5 mL), treated Wallac 1410 liquid scintillation counter. The fraction corre with 0.4 M methanolic triethylamine (9 mL), and then stirred sponding in retention time to 0 6-CMdG showed an elevated level at room temperature for 12 min. Evaporation of the solvent of radioactivity. Rechromatography of this fraction showed that under reduced pressure gave a residual oil, which was separated no other peaks were present except for Oe-CMdG, and on on silica gel (40 g) using EtOAc/CHaOH (85:15, v/v) as eluant. quantitation against a calibration curve, an isolated yield of The appropriate product-containing fractions identified by TLC 7.44% of [ 3H]-06-CMdG was achieved. on silica gel were combined, dried using Na 2SC>4, filtered, and Preparation of C^-CMGuo Conjugated with Ovalbumin evaporated under reduced pressure to give 2 e as a white solid. (OV) or Bovine Serum Albumin (BSA). 2f(Ca 2+ salt, lOmg) 2e was further purified by semipreparative HPLC on a Phase in H2O (0.5 mL) was treated with N aI0 4 (21.4 mg, 100 fiL) and Sep C-18 silica gel column using CH 3OH/H2O (12:88, v/v) as stirred at room temperature for 15 min. Ethylene glycol (5 fiL) eluant at 20 mL/min at room temperature. After removal of was added to stop the reaction, and this solution was added to the solvent by lyophilization, 2 e was isolated as a white OV or BSA (10 mg) in H 2O (1 mL) adjusted to pH = 9.5 with crystalline solid: yield 0.11 g (62%); mp 123—124 °C; (pH 0.2 M Na 2C0 3 and kept at room temperature for 4 min. The = 7) 248, 281; vma* (KBr) 3300 (N-H , O-H str), 2860 (C-H Schiff base was stabilized by addition of NaCNBIL (0.5 mL of str), 1752 (C=0 str), 1580 (C=C str), 1250 (C -0 str), 1065 cm "1 30 mg/mL freshly made in H 2O) and left at 4 °C for 3 h, with 1 (C -0 str); NMR (DMSO-dg) <5 3.58 (2H, m, 5'-H), 3.61 (3H, s, drop of octanol to prevent foaming. The resultant mixture was OCH3), 3.88 ( 1H, m, 4'-H), 4.10 (1 H, m, 3'-H), 4.46 (1H, m, 2 '- dialyzed overnight against PBS and then eluted through a H), 4.87 (2H, s, CH 2CO2CH3), 5.14 (2H, m, 5'-OH, 2'-OH), 5.40 Sephadex G-50 column using 0.15 M NaCl. The first eluting (1H, d, 3'-OH), 5.80 ( 1H, d, l'-H), 6.52 (2H, brs, NH2), 8.11 (1H, UV-absorbing fractions were collected and concentrated in a s, 8 -H); MS mix (FAB positive ion), 356 (MH+) 12, 224 (MH+ - Micro-ProDiCon apparatus (Spectrum Microgon, Laguna Hills, 132) 30. CA) overnight and the samples lyophilized. The hapten—carrier 0 6-(Carboxymethyl)guano8ine (2f). 2e (0.042 g, 0.11 ratio was determined by measuring the contribution of the mmol) was dissolved in 4 mM aqueous Ca(OH )2 solution (30 nucleoside to the absorbance at 280 nm. The ratios were 3 for mL), and the solution stirred at room temperature. Loss of 0 6-CMGuo—BSA and 1 for 0 6-CMGuo-OV. starting material was monitored by TLC on silica gel using Immunization of Rabbits and Preparation of Rabbit EtOAc/CHsOH (85:15, v/v) as eluant. After 18 h, lyophilization A ntisera. C^-CMGuo—BSA conjugate (2.5 mg) dissolved in of the reaction solution gave 2f as a white fluffy solid. The solid PBS (1.25 mL) was emulsified with Freunds complete adjuvant was dissolved in ethanol, and the insoluble residue was re (1.25 mL); 1 mL of the emulsion was injected into multiple sites moved. The solvent was removed under reduced pressure to on the shaved backs of two rabbits (New Zealand White, female). give 2f as a white solid: yield 0.04 g (97%); mp >250 °C; After 3 weeks, the rabbits were given another injection of O6- (pH = 7) 248 (10 020), 282 nm, (9880); Vmax (KBr) 3300 (O-H, CMGuo—BSA adduct (1 mg in PBS/Freunds incomplete adju N -H str), 2880 (C-H str),1650 (C=N str), 1620 (C=0 str), 1580 vant, 1 mL/rabbit) in 500 juL aliquots into the hind quarters. (C=C str), 1250 (C-O str), 1065 cm ' 1 (C -0 str); NMR (400 Two months later, the rabbits were given a further booster MHz, DMSO-de) <5 3.58 ( 2 H, m, 5'-CH2), 3.88 ( 1H, m, 4'-H), 4.10 injection of 0 6-CMGuo—BSA adduct (0.5 mg in PBS/Freunds (1H, m, 3'-H), 4.46 (1H, m, 2'-H), 4.62 (2H, s, CH2), 5.14 (2H, incomplete adjuvant, 1 mL/rabbit) in 500 fiL aliquots into the m, 5'-OH, 2-OH), 5.40 (1H, m, 3'-OH), 5.82 (1H, d, l'-H), 6.52 hind quarter. Two months later, the rabbits were given a (2H, brs, NH2), 8.09 (1H, s, 8 -H); MS m /z (FAB negative ion) further booster injection of C^-CMGuo—BSA adduct [1 mg in 340 (M“) 7, 151 (M“ - alkyl - 116) 2 0 . Anal. Calcd for PBS/Freunds incomplete adjuvant (1:1, v/v, 2 mL)], 1 mL/rabbit, C24H28NioOi4Ca*H20 : C, 39.02; H, 4.07; N, 18.97; 0 , 32.50; Ca, in 500 fiL aliquots into the hind quarter. Two weeks later the 5.42. Found: C, 38.90; H, 4.35; N, 18.90. rabbits were bled from the lateral ear vein, and the blood was Preparation of [ 3H ]-0 6-CMdG. Enzymatic Coupling; stored at 4 °C overnight. The sera were then stored at 37 °C This reaction was carried out essentially as described by for 1 h and centrifuged at lOOOg for 10 min. The supernatants Chapeau and Marnett (19) with the following modifications. O6- were decanted off and stored in 1 mL aliquots at —30 °C. CMG (0.214 /tmol; prepared by hydrolysis of Oe-CMdG in 10% ELISA Procedure. Optimal conditions for ELISA were HFBA followed by isolation using HPLC) and thymidine (0.66 determined using a checkerboard procedure in which coating jamol) were dissolved in 300 fiL of 20 mM potassium phosphate antigen levels of 1 ng —1 0 ^g/well and antiserum dilutions of 1 buffer, and the pH was adjusted to 7.3. Tpase (0.22 unit), in 10 to 1 in 10 6 were tested. A reasonable absorbance was PNPase ( 6 .6 units), sodium azide (final concentration 0.05%), found using 5 ng of 0 6-CMGuo-OV/well and a dilution of 1 in and 6 6 fiL of a solution containing [ 3H]thymidine (2.44 MBq) 200 000. Both antisera displayed similar properties in a were added. The mixture was incubated at 38 °C for 2 weeks, competitive ELISA for 0 6-CMdGuo. The ELISA protocol was over which time the progress of the reaction was monitored by as follows: RP-HPLC (employing the system outlined below) using a Polystyrene microtiter plates (96-well; Dynatech M129B) duplicate reaction mixture which had [ 3H]thymidine replaced were filled with a solution of coating antigen (40 fiL of PBS by the same volume of water. containing 5 ng of 0 6-CMGuo—OV) and dried overnight at 37 HPLC Conditions: Analytical and preparative HPLC were °C. Plates were stored at room temperature, protected from performed on a Waters HPLC system [600E system controller dust and in the dark. Plates were washed with PBS/0.005% and a UV spectrophotometric detector (Shimadzu SPD 6 A) at a Tween (6 x) and then dried by tapping onto absorbant paper wavelength of 278 nm] equipped with a Techspere 5 mm ODS towels. Standard solutions of 0 6-CMdG or other inhibitor were reverse-phase column (250 x 4.6 mm) at a flow rate of 1 mlV prepared in PBS so that the concentration varied between 0.1 min. A step gradient elution system was employed as follows: and 10 7 finol/25 fiL. An aliquot of 0«-CMdG in PBS (25 fiL) 0-10 min 10% MeOH in 0.1 M TEA (pH = 7), 10-20 min 15% was pipetted onto the plate in rows (eight wells) for each of the MeOH in 0.1 M TEA (pH = 7), 2 0 - 3 0 min 20% MeOH in 0.1 M standard solutions. Usually two rows on the ELISA plate were TEA (pH = 7), and going back to initial conditions over a further used for controls, and to each of these rows was added PBS (25 10 min. The order of elution was thymine, 0 6-CMG, thymidine, /iL/well). A reference row containing PBS (50 ,uL/well) was also and 0 6-CMdG. used. Polyclonal rabbit antiserum (CMG2; 25 fiL of a 1:200 000 Immunochemical Detection of Oe-CMdG Chem. Res. Toxicol., Vol. 10, No. 6, 1997 655 dilution in PBS of the neat antiserum) was added to each well to dryness (freeze-dried) and the residue redissolved in water in the plate except those in the reference row. The plate was (1 mL), which was in turn evaporated to dryness (Speedvac) then incubated at room temperature for 90 m in- The superna and redissolved in 0.1% HFBA (25 fiL) for HPLC analysis. The tant liquid was then decanted from the ELISA plate, and the above procedure was also applied to untreated DNA Analytical plate was washed in PBS/Tween ( 6 x). Horseradish peroxidase- HPLC was performed on a 100 x 2 mm Hypersil BDS C18 (3 linked goat anti-rabbit immunoglobulin G (Sigma; 50 fiL of a ftm) column for both 0®-CMG and dG quantitation. C^-CMG 1:1000 dilution in PBS) was added to every well on the plate. was quantitated by fluorescence detection (excitation at 286 nm, The plate was reincubated at room temperature for 90 m in emission at 378 nm) using isocratic conditions (0.1% HFBA/2.7 After 9 0 min, the supernatant was discarded and the plate mM EDTAmethanol, 90:10) at 0.2 mL/min. dG was determined washed with PBS/Tween ( 6 x) and once with distilled water. in aliquots of DNA hydrolysate (10 fiL) prior to immunoaffinity Aqueous citrate buffer, pH = 5 .3 (50 fiL), from a solution (10 treatment by detection at 260 nm using isocratic conditions ( 0.1 mL) containing 3/,3/,5/,5/-tetrametby1henzidine (prepared by M TEA, pH = 4.5:methanol, 96:4) at 0.2 m L/m in. addition of 1 mg in 100 fiL of dimethyl sulfoxide) and H 2O2 (2 fiL of 30%, w/w, solution), was added to each well of the plate, R e su lts and the plate was incubated for 15 m in at room temperature to allow color development to occur. HC1 (1 M 5 0 fiL) was added Synthesis of 0®-CMdG. The synthesis of Oe-CMdG to each well, and the optical density at 45 0 nm of each well of (If) is summarized in Scheme 1. The synthesis route was the plate was measured by an automatic plate reader. Gener based on the approach developed by Gaffney and Jones ally, the control wells of the plate gave cm optical density of ca. (24), in which an 0 6-arylsulfonate is displaced by a 0.7 after subtraction of the reference (blank) wells. trialkylamine, with the resulting trialkylammonium Preparation and Characterization of Immunoaffinity intermediate reacting with an alcohol to give the corre Columns for 0*-CMdG. Tmmnnoflffinity columns were pre sponding 0 6-alkylnucleoside. For our purposes, it was pared by covalently linking the ammonium sulfate-precipitated IgG fraction of sera to protein A-Sepharose CL4B and using the not necessary to protect the exocyclic A^-amino group, resultant gel to prepare small (1 mL) columns as described by especially since removal of the A^-acyl group by concen Friesen et al. (20). [3H]-Oe-CMdG (17.2 ng, 950 dpm) in PBS/ trated ammonia can result in substitution of the Os-alkyl 0.02% azide (2 mL) was applied to a column followed by a by an amino group (24). It was decided to protect the further 3 mL of PBS/0.02% azide. The column was then washed 3',5'-hydroxyl groups of dGuo with more readily hydro with water (10 mL). One milliliter fractions of the colum n lyzable methoxyacetyl as opposed to acetyl. However, as eluate were collected, and the radioactivity was measured by described below, acetyl protection is advantageous for the liquid scintillation counting using various elution conditions: more labile esters of 2'-hydroxyl groups in ribonucleo- 1 M acetic add (5 mL), 50% aqueous MeOH (5 mL), 50% aqueous sides. DMSO (5 mL), 1 M formic add (5 mL), 1 M trifluroacetic add (TFA) (5 mL), and 0.1 M TFA (5 mL). Quantitative elution of Conversion of 1 to lb proceeded cleanly in ca. 80% the [3H ]-06-CMdG was obtained by washing the colum n with overall yield. Attempts to introduce the methyl glycolate 1M TFA (5 mL) with most of the radioactivity eluting in the moiety via trimethylammonium or IV-methylpyrrolidin- first 2 mL. ium ion intermediates were unsuccessful and resulted D eterm in ation o f th e C olum n C apacity for C^-CMdG: in the formation of substantial amounts of 6-(dimethy- PBS/0.02% azide (2 mL) containing [ 3H]-Oe-CMdG (17.2 ng, 950 lamino)- and 6-pyrrolidinodG products, respectively (re dpm) and 0®-CMdG (0—1000 ng) was applied to immunoaffinity sults not shown). In contrast, the use of quinuclidine as columns. The colu m n s were washed with PBS/0.02% azide (3 tertiary amine resulted in a 65% yield of Id. The mL) and water (10 mL), elution with TFA (1 M, 5 mL) was then methoxyacetyl protecting groups were readily removed carried out, and the eluate was collected directly into scintilla by treatment with triethylamine/methanol at room tem tion vials. Liquid scintillation fluid (3 mL) was added to each perature to give le. However, at this stage, an HPLC vial and the radioactivity determined by scintillation counting. cleanup was carried out since a persistent contamination The results are expressed as the percentage of [ 3H]-06-CMdG retained on the column. with mesitylenesulfonic acid was detected in the mass spectrum of le despite recrystallization. Finally, If was Treatment of DNA with Various Nitrosated Glycine Derivatives. Calf thymus DNA was dissolved in PBS (pH = prepared as the dihydrated calcium salt by calcium 7) (5 mg/mL), and APNG (21), AS, or potassium diazoacetate hydroxide-mediated hydrolysis of le. (22) was added to give a 5 mM solution which was left gently Synthesis of (^-CMGuo for Use in Antiserum stirring (37 °C) in the dark overnight. After treatment, DNA Preparation. Oe-CMGuo (2f) was required in order to was precipitated from the reaction medium with sodium acetate prepare antibodies to If. Initially, the same procedure (0.1 volume, 2.5 M) and cold ethanol (2 volumes) and centrifuged for protection of ribose hydroxyls using methoxyacetic gently (300Gg for 5 min) and the DNA washed with ethanol. anhydride was used, but deprotection of the 2'-hydroxyl The DNA pellet was recovered, evaporated to dryness, and occurred in subsequent steps resulting in a mixture of resuspended in water to the original volume. products (data not shown). It was, therefore, decided to D eterm in a tio n o f Oe-CMdG in DNA: Enzyme hydrolysis use the more stable acetyl protection. Thus, guanosine was performed following the method described by Beranek et was acetylated to give 2a (18), and this was cleanly al. (23). DNA samples were hydrolyzed in 50 mM bisTris/1 mM converted to 2b in 90% yield. Introduction of the O6- MgCl 2 (pH = 6.5) at 50 °C for 8 h, using nuclease PI (24 units), bacterial alkaline phosphatase (4.8 units), and wheat germ add carboxymethyl moiety proceeded smoothly and was fol phosphatase (0.3 unit) per 1 mg of DNA (final concentration 1 lowed by deprotection of 2d without isolation and an mg of DNA/mL). The reaction was stopped by heating at 100 HPLC purification step to give 2e in 62% yield. Calcium °C for 5 m in, and the mixture was then centrifuged to remove hydroxide hydrolysis of 2e afforded 2f in 97% yield as the denatured enzyme protein. DNA hydrolysate (2.5 mg) in 2 the monohydrated calcium salt. m L of PBS/0.02% azide was loaded onto an 0 6-CMdG immu Preparation of an 0®-CMdGuo Antiserum and noaffinity column and then washed with PBS (3 mL) and water Development of an ELISA ProtocoL If was conju (10 mL). Elution was achieved with 1 M TFA (5 mL) which gated to BSA using the standard method of Muller and was collected and then heated for 1 h at 50 °C to quantitatively convert 06-CMdG to the more fluorescent base 06-CMG. The Rajewsky (16), which results in the formation of an immunoaffinity column was then washed with PBS/0.02% antigenic molecule with a structure resembling O6- azid e (15 m L) for further use. The TFA fraction was evaporated CMdGuo. Despite the low level of modification, both 656 Chem. Res. Toxicol., Vol. 10, No. 6, 1997 Harrison et al. Scheme 1. Synthetic Routes for the Preparation of 0®-CMGuo (2f) and 0®-CMdG (lf)a h 3c CH, C23 HO HO CH3 NHj NH. NH. OR” OR" OR” OR“ OH OR" °R" 1c,2c 1a, 2a 1,2 1b,2b NH. NH. nh2 OR” 0R" OH OH id ,2d 1e,2e 1 R=H, R-H, R"=methoxyacetyl 2 R=OH, R-acetoxy, R"=acetyl “ Conditions: (i) 1, methoxyacetic anhydride, DMF, 2, acetic anhydride, DMF; (ii) mesitylenesulfonyl chloride, triethylamine, 4 -(dimethylamino)pyridine, rt; (iii) quinuclidine, THF, rt; (iv) methyl glycolate, DBU, THF, 65 °C; (v) triethylamine, MeOH, rt; (vi) Ca(OH) 2- 100 100 r 75 75 2 c o 8 50 25 10-1 10° 101 102 103 104 105 1 08 1 07 0.0 1.0 10.0 Inhibitor (pmol/well) Inhibitor (fmol/well) Figure 2. Standard curve for 0 6-CMdGuo in a competitive Figure 1. Inhibition curve for Oe-CMdGuo in competitive ELISA ELISA using the conditions described in the Materials and Methods section. Table 1. Cross-Reactivity of Rabbit Anti serum CMG2 in a rabbits that were immunized produced antisera of similar Competitive ELISA titer. Optimal conditions for competitive ELISA were substrate conctn for found using a checkerboard procedure. At a level of 50% inhibition (pmol/well) coating antigen (0 6-CMGuo—OV) of 5 ng/well and an substrate antiserum dilution of 1 in 200 000, a final absorbance at 0 6-(carboxymethyl)- 2'-deoxyguanosine 2 450 nm of ca. 0.7—0.9 was obtained. Under conditions Oe-(carboxymethyl)guanosine 60 0 6-methyl-2 '-deoxyguanosine > 1 04 of a competitive ELISA, 0 6-CMdGuo was tested over a Oe-ethyl-2 / -deoxyguanosine > 1 0 * wide range (0.1—107 fmol/well), and the 50% inhibition 2 '-deoxyguanosine >104 was found to be 2 and 3 pmol for antisera CMG2 and 7-(carboxymethyLguanine >104 CMG1, respectively. Figure 1 shows the inhibition curve 3-(carboxymethyl)adenine >104 for CMG2. Under the same conditions, a number of 2 '-deoxyadenosine >104 2 '-deoxycytidine >104 purines and nucleosides were tested for cross-reactivity thymidine >104 using CMG2, and the results are summarized in Table 1. A standard curve for Oe-CMdGuo was constructed rabbit antiserum to Protein A-Sepharose (19). 0®-CMdG between 0.2 and 10 pmol/well and is linear over this was retained by the columns (Figure 3) with a capacity range (Figure 2). of ca. 1 nmol (Figure 4). The binding was so strong that Immunoaffinity Purification of 0®-CMdG and Its fairly drastic conditions (1 M TFA) were required for Detection in DNA. Immunoaffinity columns were elution. Nonetheless, the columns can be recycled many prepared by covalently linking a total IgG fraction from times using this elution solvent without apparent dete- Immunochemical Detection of Oe-CMdG Chem. Res. Toxicol., Vol. 10, No. 6, 1997 657 1000 6 5 | 800 ■o ■o 4 33 600 £ 10 3 CO£ o 400 JC 10 CO o a> 2 ■o a $ 200 1 0 r 0 5 10 15 20 10 15 20 O -CMdG (pmol/mg DNA) Fraction No (mL) Figure 5. Calibration line for the determination of added Figure 3. Elution of 0 6-CMdG from CMG2 immunoaffinity standards of Oe-CMdG in DNA following enzymatic hydrolysis, columns. [3H]-Oe-CMdG (17.2 ng, 950 dpm) was in PBS/azide immunoaffinity cleanup, and HPLC fluorescence. The details (2 mL), and the column was washed with PBS/azide (3 mL), are given in the Materials and Methods section. water (10 mL), and 1 M TFA (5 mL). The column eluate was collected as 1 mL aliquots which were added to scintillation fluid Table 2. Yield of 0*-CMdG in Calf Thymus DNA Treated and counted directly. with Various Nitrosated Glycine Derivatives compound0 Oe-CMdG (umol/mol o f dG)6 100 AS 7.3 ± 0.35 APNG 39.9 ± 2.5 KDA 496 ± 58 80 TJa> ° At a concentration of 5 mmol. 6 Mean ± SD of three separate c reactions. £ 60 0 TJ s 40 «o9 9 i 20 S3 200 400 600 800 1000 a Os-CMdG added (ng) o 4>a Figure 4. Determination of the column capacity of immunoaf V finity columns for 0®-CMdG. [3H]-Ofi-CMdG (17.2 ng, 950 dpm) uV was eluted through the column in the presence of increasing o amounts of unlabeled 0®-CMdG (0—1000 ng). s rioration in column performance. Under these conditions 0®-CMdG was extensively hydrolyzed to the correspond ing base Oe-CMG. However, since C^-alkylguanines are slightly more fluorescent then the corresponding deoxy- nudeosides (24), this phenomenon was turned into an advantage by heating the column eluate to drive the hydrolysis to completion prior to HPLC analysis. Stan 0 5 10 15 dard amounts of Oe-CMdG were added to samples of Time (min) DNA (0—20 pmol/mg) which were then hydrolyzed and analyzed using immunoaffinity purification with HPLC Figure 6. HPLC fluorescence traces of 0®-CMG in APNG- treated (solid line) and control (broken line) DNA. In each case fluorescence as described above. A linear response was the sample injected corresponded to 0.25 mg of DNA. The observed (Figure 5). conditions are described in the Materials and Methods section. Calf thymus DNA (5 mg/mL) which had been incubated with APNG (5 mmol), AS (5 mmol), or KDA (5 mmol) Discussion was analyzed using the above method and found to contain varying am ou n ts of 0®-CMdG (Table 2). The The synthesis of Oe-CMdG (If) has been achieved by HPLC fluorescence chromatograms were free of interfer unambiguous insertion of a glycolate moiety into the ing peaks, and a representative trace for APNG-treated 6-position of a suitably protected dG molecule (Scheme DNA is shown in Figure 6. 1). If was used as an authentic standard to confirm the 658 Chem. Res. Toxicol., Vol. 10, No. 6, 1997 Harrison et al. Chart 1. Structures of iV-(iV'-Acetyl-L-prolyl)-iV-nitrosoglycine (APNG), Azaserine (AS), and Potassium Diazoacetate (KDA) d CvJJL s X - X d I 0 NH, A ua APNG AS .ou 03 CU XX O K v d o Xfxa* KDA o J3 presence of the nonrepairable carboxymethylated adduct E in DNA treated with A-nitrosoglycocholic acid (13). An antiserum was raised against If by using the corresponding guanosine analogue bound to a carrier protein, and a sensitive competitive ELISA procedure was developed. Good recognition of Oe-CMdG was ob 0 10 20 tained compared to normal nucleosides and carboxym ethylated DNA bases. Interestingly, 0 6-CMGuo was 30 Tim e (m in) times less efficient as an inhibitor than Oe-CMdGuo, Figure 7. HPLC chromatograms of Oe-CMdG in calf thymus illustrating the selectivity of the antiserum. 0 6-Alkylated DNA treated with KDA (5 mmol). DNA was either add deoxyguanosine and unmodified nucleosides were at least hydrolyzed (10% HFBA, 100 °C, 30 min) and injected directly (solid line) or enzymatically hydrolyzed and immunoaffinity 5000 times less efficient as inhibitors, as were the purified as described in the Materials and Methods section carboxymethylated purine bases. It is apparent that the (broken line). In both cases the amount of hydrolysate injected antiserum is very selective for the Oe-CMdG molecule. onto the column was equivalent to 25 f i g of DNA Attempts to increase the sensitivity of the ELISA by conducting part of the assay at 4 °C only resulted in a modest (2.5-fold) improvement (data not shown), in 0 0 M (U 2 contrast to the much larger effects noted for alkylpurine ELISAs (26). This may be due to the fact that the polar (charged) carboxymethyl group is very strongly bound in SJ6 17.2 35.4 the antigen-binding site of the antibody molecule as a result of a combination of van der Waals and electrostatic i 1 1 I interactions (see below). Immunoaffinity columns were prepared and found to efficiently bind Oe-CMdG. The capacity of the columns Figure 8. Immunoslotblot assay of calf thymus DNA contain (~1 nmol) was found to be comparable with those ing increasing amounts of 0®-CMdG. KDA-treated calf thymus DNA was mixed with unmodified calf thymus DNA, and a prepared using antibodies against other DNA adducts constant amount of DNA (1 fig) was added to each well in (19,27—29). It is interesting to note that in these studies duplicate. The values indicated are the levels of 0 6-CMdG we have immunoaffinity columns which have good se determined by immunoaffinity HPLC fluorescence. lectivity and capacity using both monoclonal antibodies and polyclonal antisera but that monoclonal antibodies which decompose or rearrange to give carboxymethylat have the advantage of continuous availability. The ing agents resulting in the formation of (XCMdG but apparent high affinity of the antibody was illustrated by which also give rise to DNA methylation (13). the rather drastic conditions (1 M TFA) required to elute The advantage of using immunoaffinity purification Oe-CMdG giving rise to partial hydrolysis to Oe-CMGuo. prior to HPLC fluorescence, compared to no prepurifica However, since the free base is slightly more fluorescent tion, is shown in Figure 7. Fluorescence due to the than the 2'-deoxynucleoside (25), this proved advanta normal DNA bases interfered substantially with the geous for the sensitivity of the assay: the hydrolysis was quantitation of C^-CMdG. The absolute limit of detection driven to completion by heating the eluate prior to of the HPLC fluorescence assay was 0.1 pmol/injection. evaporation and HPLC analysis. The utility of the If 1 mg of DNA hydrolysate was used per injection, the combination of immunoaffinity purification of Oe-CMdG limit of detection of the combined immunoaffinity—HPLC with HPLC fluorescence is illustrated by analysis of O6- fluorescence assay corresponded to 1 0®-CMdG adduct/ CMdG in calf thymus DNA treated with APNG, a model 107 normal bases. While this level of sensitivity is nitroso peptide which is a potent mutagen and carcinogen suitable for experimental studies where amounts of DNA (9), AS, a potent pancreatic carcinogen (12), and KDA for analysis are fairly large (>0.1 mg), it is not sufficient (22) (Chart 1). KDA is particularly interesting because for human studies where only small biopsies or blood it is a stable nitrosated derivative of glycine, one of the volumes, which yield ca. 10 jug of DNA, are available. The most common dietaxy amino acids. APNG, AS, and KDA lack of cross-reactivity of antiserum CMG2 with normal are members of a family of nitrosated glycine derivatives DNA bases (Table 1) suggested that 0®-CMdG could be Immunochemical Detection of Oe-CMdG Chem. Res. Toxicol., Vol. 10, No. 6, 1997 659 detected in intact DNA. Accordingly, an immunoslotblot (13) Shuker, D. E. G., and Margison, G. P. (1997) Nitrosated glycine assay was developed where an equivalent sensitivity is derivatives as a potential source of 0 6-methylguanine in DNA. Cancer Res. 57, 366—369. obtained on small amounts (typically 1 fig) of DNA (14) Wild, C. P. (1990) Antibodies to DNA alkylation adducts as (Figure 8). The immunoslotblot assay was carried out analytical tools in chemical carcinogenesis. Mutat. Res. 2 3 3 ,219— as described by Nehls et al. (30), and full details will be 233. published elsewhere. (15) Shuker, D. E. G., and Bartsch, H. (1994) Detection of human exposure to carcinogens by measurement of alkyl-DNA adducts using immunoaffinity clean-up in combination with gas chroma- Acknowledgment. This work has been supported, tography-mass spectrometry and other methods of detection. in part, by the U.K. Ministry of Agriculture, Fisheries Mutat. Res. 313, 2 6 3 -2 6 8 . and Food (Contract No. 1A025) and the Cancer Research (16) Muller, R , and Rqjewsky, M. (1981) Immunological quantification Campaign. Neil Fairhurst gratefully acknowledges the by high affinity atibodies of 0 6-ethyldeoxyguanosine in DNA award of a European Science Foundation Toxicology exposed to ethylnitrosourea. Cancer Res. 41, 887—896. Programme Fellowship which enabled him to work on (17) Reese, C. B., Stewart, J. C. M., van Boom, J. H., de Leeum, H. P. M., Nagel, J., and de Rooy, J. F. M. (1975) The synthesis of the development of immunoassays at the IARC in Lyon, oligoribonucleotides. Part XI Preparation of ribonucleoside 2'- France. Some of the results in this paper are taken from acetyl-3'-esters by selective deacylation. J. Chem. Soc., Perkin the Ph.D. Thesis (University of London, 1990) of Neil Trans. 1 934-942. Fairhurst. We thank Dr. J. A. Challis for obtaining the (18) Bridson, P. K., Markiewicz, W. T., and Reese, C. B. (1977) mass spectra. Acylation of 2,,3',5'-tri-0-acetylguanosine. Chem. Commun. 791— 792. (19) Chapeau, M.-C., and Marnett, L. J. (1991) Enzymatic synthesis References of purine deoxynucleoside adducts. Chem. Res. Toxicol. 4, 636— 638. (1) Miller, E. C. (1978) Some current perspectives on chemical carcinogenesis in humans and experimental animals: address. (20) Friesen, M. D., Garren, L., Prevost, V., and Shuker, D. E. G. (1991) Cancer Res. 88,1479—1496. Isolation of urinary 3-methyladenine using immunoaffinity col (2) Saffhill, R., Margison, G. P., and O’Connor, P. J. (1985) Mecha umns prior to determination by low-resolution gas chromatogra nisms of carcinogenesis induced by alkylating agents. Biochim. phy-mass spectrometry. Chem. Res. Toxicol. 4, 102—106. Biophys. Acta 8 2 3 , 111—145. (21) Challis, B. C., M illigan, J. R., and Mitchell, R. C. (1990) Synthesis (3) Karran, P., and Lindahl, T. (1985) Cellular defense mechanisms of N-nitrosopeptides. J. Chem. Soc., Perkin Trans.3103—3107. 1 against alkylating agents. Cancer Surveys 4, 585—599. (22) Muller, E. (1908) tJber pseudo diazoessigsaure. (On pseudo (4) Shuker, D. E. G., Tannenbaum, S. R., and Wishnok, J. S. (1981) diazoacetic acid.) Chem. Ber. 41, 3116—3139. N-Nitrosobile add conjugates. I. Synthesis, chemical reactivity (23) Beranek, D. T., Weis, C. C., and Swenson, D. H. (1980) A and mutagenic activity. J. Org. Chem. 46, 2092—2096. comprehensive quantitative analysis of methylated and ethylated (5) Song P., Shuker, D. E. G., Bishop, W. W., Falchuk, K. R., DNA using high pressure liquid chromatography. Carcinogenesis Tannenbaum, S. R., and Thilly, W. G. (1982) Mutagenidty of 1, 595-606. N-nitrosobile add conjugates, N-nitrosoglycocholic add and N- (24) Gaffney, B. L., and Jones, R. A. (1982) Synthesis of Oe-alkylated nitrosotaurocholic add. Cancer Res. 42, 1367—1371. deoxyguanosine nucleosides. Tetrahedron Lett. 23, 2253—2256. (6) Busby, W. F., Jr., Shuker, D. E. G., Charnley, G., Newbeme, P. (25) Pauly, G. T., Powers, M., Pei, G. K , and Moschel, R. C. (1988) M., Tannenbaum, S. R., kand Wogan, G. N. (1985) Carcinogenicity Synthesis and properties of H- m s DNA sequences containing O6- of the nitrosated bile add conjugates, N-nitrosoglycocholic add substituted 2'-deoxyguanosine residues at the first, second or both and N-nitrosotaurocholic add. Cancer Res. 45, 1367—1371. positions of codon 12. Chem. Res. Toxicol. 1, 391—397. (7) Shuker, D. E. G., Howell, J. R., and Street, B. W. (1987) Formation (26) Hemminki, K (1980) Identification of guanine-adducts of car and fate of nudeic add and protein adducts from N-nitroso bile cinogens by their fluorescence. Carcinogenesis 1, 311—316. add conjugates. In Relevance of N-nitroso Compounds for Human Cancer: Exposures and Mechanisms, IARC Scientific Publications (27) Prevost, V., Shuker, D. E. G., Bartsch, H., Pastorelli, R., Stillwell, No. 84, (Bartsch, H., O’Neill, I. K , and Schiilte-Hermann, R., Eds.) W. G., Trudel, L. J., and Tannenbaum, S. R. (1990) The deter pp 187—190, International Agency for Research on Cancer, Lyon, mination of urinary 3-methyladenine by immunoaffinity chro France. matography-monoclonal antibody based ELISA: use in human (8) Challis, B. C. (1989) Chemistry and biology of nitrosated peptides. biomonitoring studies. Carcinogenesis 11, 1747—1751. Cancer Surveys 8, 363—384. (28) Prevost, V., Shuker, D. E. G., Friesen, M. D., Eberle, G., Rajewsky, (9) Anderson, D., and Blowers, S. D. (1994) Limited cancer bioassay M. F., and Bartsch, H. (1993) Immunoaffinity purification and to test a potential food chemical. Lancet 344, 343—344. gas chromatography-mass spectrometric quantification of 3-alky- (10) Bulay, O., Mirvish, S. S., Garcia, H., Pelfrene, A. F., and Gold, B. ladenines in urine: metabolism studies and basal excretion levels (1979) Carciogenicity test of six nitrosamides and a nitrosocy- in man. Carcinogenesis 14, 199—204. anamide administered orally to rats. J. Natl. Cancer Inst. 62, (29) Durand, M.-J., and Shuker, D. E. G. (1994) Preparation of 1523-1528. compound-specific and group-specific antibodies to 7-methylgua- (11) Maekaiva, A., Ogiu, T., Matsuoka, C., Onodera, H., Furuta, K., nine and related 7-alkylguanines and their use in immunoaffinity Tangawa, H., and Odashima, S. (1983) Induction of tumours in purification. Carcinogenesis 15, 957—961. the small intestine and mammary gland of female Donryu rats (30) Nehls, P., Adamkiewicz, J., and Rajewsky, M. F. (1984) Immuno by continuous oral administration of N-carboxymethyl-N-ni- slotblot: a highly sensitive immunoassay for the quantification trosourea. J. Cancer Res. Clin. Oncol. 106, 12—16. of carcinogen-modified nucleosides in DNA. J. Cancer Res. Clin. (12) Zurlo, J., Curphey, T. J., Hiley, R., and Longnecker, D. S. (1982) Oncol. 108, 2 3 -2 9 . Identification of 7-carboxymethylguanine in DNA from pancreatic acinar cells exposed to azaserine. Cancer Res. 4 2 ,1286—1288. T X 960203U