i THE ROLE OF DIETARY EXPOSURE TO HETEROCYCLIC AROMATIC AMINES AND GENETIC ...

350 Pages·2014·5.91 MB·English

THE ROLE OF DIETARY EXPOSURE TO HETEROCYCLIC AROMATIC AMINES
AND GENETIC SUSCEPTIBILITY IN COLORECTAL ADENOMA ETIOLOGY

By

Vikki Ho

A thesis submitted to the Department of Public Health Sciences
in conformity with the requirements for the
Degree of Doctor of Philosophy

Queen’s University
Kingston, Ontario, Canada
April, 2014

Copyright © Vikki Ho, 2014

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Abstract
Background: Meat consumption is associated with an elevated risk of colorectal cancer (CRC);
exposure to heterocyclic aromatic amines (HAAs), carcinogens produced when meat is cooked at
high temperatures, is one hypothesized explanation for this relationship. HAAs form adducts
with DNA; left unrepaired, DNA adducts can induce mutations which may initiate and/or
promote the development of colorectal adenomas, precursors to the vast majority of CRCs.
Along this continuum, genetic differences in the ability to biotransform or metabolize HAAs and
repair DNA is postulated to modify the HAA-CRC relationship.

Methods: This thesis examined the HAA-CRC relationship in two studies (Phase 1 and 2). In a
cross-sectional study of 99 healthy volunteers, Phase 1 investigated the relationship between
dietary exposure to HAAs and the levels of bulky DNA adducts in blood leukocytes. In Phase 2,
a cross-sectional study examined the relationships between dietary exposures to: a) HAAs and;
b) meat mutagenicity, and the prevalence of colorectal adenomas among 342 patients undergoing
a screening colonoscopy. Both Phase 1 and 2 examined potential gene-diet interactions between
dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair.

Results: In Phase 1, an interaction was observed for dietary HAAs and NAT1 polymorphisms
where a positive association between HAA intakes and bulky DNA adduct levels was found
among those with the NAT1 slow acetylator genotype, hypothesized to confer a lower ability to
biotransform HAAs. In Phase 2, polymorphisms in genes involved in the biotransformation of
HAAs (CYP1B1 rs10012 and rs1056827) and DNA repair (XPC rs2228001) were found to
determine colorectal adenoma risk. As well, gene-diet interactions were observed for dietary
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HAAs/meat mutagenicity exposures and polymorphisms in CYP1B1 and XPD (rs13181 and
rs1799793). Overall, a higher risk of colorectal adenoma was observed with higher HAA and/or
meat mutagenicity exposures among those with polymorphisms which confer a greater activity to
biotransform HAAs and/or a lower ability to repair DNA.

Conclusion: This research supports the contribution of dietary HAAs and genetic susceptibility
to the risk of developing colorectal adenomas and highlighted bulky DNA adduct formation as a
potential biologic pathway through which HAAs may influence cancer risk.

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Acknowledgements
People always say that acknowledgements and thank you’s should be short and sweet. It
is hard to stick to such a notion since throughout this journey I was supported, encouraged and
inspired by so many people – and as I write I feel how words fall short on expressing gratitude
on this scale of enormity.
I first and foremost must acknowledge my supervisors Will King and Tom Massey. Will,
it is hard to sort out how much of who I am as a researcher was inspired by you. You have a
humble approach to mentoring that made me chase after every idea and inspiration, and your
support has been felt every step of the way. Your love of science is infectious and I thank you
for always challenging me to see the “big” picture and insisting that the path less taken is always
worth it in the end. It is hard to find one’s calling in life – especially one so fitting and
challenging, and I not only had one great supervisor to guide me, but two. Tom, you are ever-
wise and knowing; you set the best example of balance. Your students’ respect of you (including
mine) is so apparent and you deserve every bit of it.
The Department of Public Health Sciences at Queen’s houses the best faculty in the
world and has been a second home to me. Harriet Richardson started it all by writing my letter
of recommendation for Grad Studies at Queen’s – thank you Harriet, for always going above and
beyond in supporting me through my graduate journey. To my cherished professors, Kristan
Aronson, Duncan Hunter, Patti Groome and Will Pickett: a big thank you for shaping my mind
and always forcing me to take a leap.
I have met some of the greatest people and dearest friends here at my time at Queen’s.
Janet Ashbury: you are forever my Epidemiology mom; Sarah Peacock: my partner in crime and
my rock; and Celine Simmers: my bestest friend who always believe in me more than I deserve.
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Alyson Mahar: you kept me smiling and in love with science; Marianne Gee and Anne Grundy: I
could never have asked for better study buddies; and all the rest of the gang, you kept me sane
and relentless.
For my PhD, I was fortunate to be able to work on (what I hope) is the beginning of my
life’s work. This work was made possible by funding support from Cancer Care Ontario and the
Canadian Cancer Society. As well, I must acknowledge the valued participants of both my
projects without which this research would not have been possible.
Finally in this “short” acknowledgement, to my parents whose only requirement for me
was to find happiness in life – thank you for the impossible task that keeps evolving day by day
but of which I never lose sight of. To my sisters, as a middle child it is easy to thank the older
one for the wisdom and the younger one for keeping me cool. And, I must acknowledge my
husband Fred: you know all my faults and darkest doubts but still believe in me every day. You
let me chance my dreams and paths that don’t always work out so well but in the end you were
always there and for that you get half a PhD.

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Table of Contents
Abstract ...................................................................................................................................... ii
Acknowledgements .................................................................................................................... iv
Table of Contents ....................................................................................................................... vi
List of Tables ............................................................................................................................. xv
List of Figures ............................................................................................................................ xix
List of Abbreviations ................................................................................................................. xxi
CHAPTER 1: General Introduction ......................................................................................... 1
1.1 Summary of Research ....................................................................................................... 1
1.2 Rationale ........................................................................................................................... 4
1.3 Objectives and Hypotheses ............................................................................................... 5
i. Phase 1: Dietary HAA Intake and Bulky DNA Adducts ............................... 6
ii. Phase 2: Dietary HAA Intake and the Risk of Colorectal Adenoma ............. 7
1.4 Context of Research .......................................................................................................... 7
1.5 Thesis Outline ................................................................................................................... 8
1.6 References ......................................................................................................................... 9
CHAPTER 2: Literature Review – Heterocyclic Aromatic Amine-Colorectal Cancer
Relationship ................................................................................................................................ 10
2.1 Colorectal Adenoma – A Surrogate Endpoint for Colorectal Cancer ............................... 10
2.2 Meat Consumption and Colorectal Cancer Risk ............................................................... 10
2.3 Exposure Assessment Framework for Heterocyclic Aromatic Amines ............................ 11
2.4 Colorectal Cancer .............................................................................................................. 13
2.4.1 Descriptive Epidemiology ..................................................................................... 13
2.4.2 Risk Factors for Colorectal Cancer ....................................................................... 13
2.4.3 The Colorectal Adenoma-Carcinoma Sequence ................................................... 14
2.5 Heterocyclic Aromatic Amines ......................................................................................... 15
2.5.1 Meat Consumption and Colorectal Cancer ........................................................... 15
2.5.2 Heterocyclic Aromatic Amines – A Potential Causal Factor Underlying the
Meat-Colorectal Cancer Relationship ................................................................... 16
2.5.3 Assessment of Heterocyclic Aromatic Amine Content and Meat Mutagenicity in
Epidemiologic Research ........................................................................................ 17
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2.5.4 Heterocyclic Aromatic, Meat Mutagenicity and Colorectal Cancer –
Epidemiologic Evidence ....................................................................................... 19
2.5.5 Heterocyclic Aromatic and Colorectal Cancer – Toxicologic Evidence .............. 24
2.5.6 Biotransformation of Heterocyclic Aromatic Amines .......................................... 24
2.5.7 Heterocyclic Aromatic Amine-DNA Adducts – Relevance for
Colorectal Cancer .................................................................................................. 27
i. Top-down versus Bottom-up Hypothesis ...................................................... 28
2.6 Heterocyclic Aromatic Amines and Colorectal Adenoma Risk – A Molecular
Epidemiologic Approach .................................................................................................. 31
2.6.1 DNA Adducts – Measurement Methods ............................................................... 31
i. Mass Spectrometry Versus 32P-Postlabelling ................................................. 32
2.6.2 DNA Adducts –Validity and Reliability ............................................................... 33
2.6.3 DNA Adducts – Selection of Biologic Matrix ...................................................... 34
2.6.4 DNA Adducts – Within-subject Variability and Timeframe of Exposure
Representation ....................................................................................................... 35
i. Short-term Variability of DNA Adducts ........................................................ 35
ii. Long-term Variability of DNA Adducts Introduced by Cell Turnover ......... 35
iii. Long-term Stability of Dietary Habits ........................................................... 36
2.6.5 Determinants of Bulky DNA Adducts Measured by 32P-Postlabelling –
Epidemiologic Evidence ....................................................................................... 37
2.6.6 Meat Consumption and DNA Adduct – Epidemiologic Evidence ....................... 37
2.6.7 DNA Adduct and Colorectal Adenoma Risk –Epidemiologic Evidence .............. 38
2.7 Conclusions and Perspectives ........................................................................................... 39
2.8 References ......................................................................................................................... 41
CHAPTER 3: Literature Review – Effect Modification in the Heterocyclic Aromatic Amine
– Colorectal Cancer Relationship .............................................................................................. 49
3.1 The Investigation of Gene-Environment Interactions in the HAA-CRC Relationship .... 49
3.2 Epidemiologic Principles Underlying Gene Selection Strategies .................................... 50
3.3 Phase 1: Dietary HAA Intake and Bulky DNA Adducts .................................................. 53
3.3.1 Phase 1: Gene and Polymorphism Selection Strategy .......................................... 54
i. Polymorphism in Genes Involved in Biotransformation of HAAs ................. 55
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ii. Polymorphism in Genes Involved in the Repair of DNA Adducts ................. 55
3.3.2 Phase 1: Polymorphisms in Genes Involved in Biotransformation of HAAs ....... 58
i. Bioactivation via CYP 450 ............................................................................. 58
ii. Bioactivation by N-acetyltransferases ............................................................ 59
iii. Bioactivation by Sulfotransferase .................................................................. 59
iv. Detoxification by Glutathione S-Transferase ................................................. 59
v. Detoxification by UDP-glucuronsyltransferase ............................................. 60
3.4 Phase 1: Repair of DNA adducts by Nucleotide Excision Repair and Base Excision
Repair ............................................................................................................................... 60
3.4.1 Phase 1: Polymorphisms in Genes Involved in Repair of DNA Adducts ............ 61
i. Xeroderma pigmentosum group A.................................................................. 61
ii. Xeroderma pigmentosum group D.................................................................. 62
iii. X-ray repair cross-complementing group 1 .................................................... 62
3.5 Summary of Polymorphisms of Interest to Phase 1 .......................................................... 62
3.6 Phase 2: Dietary HAA Intake and Risk of Colorectal Adenomas .................................... 63
3.6.1. DNA Adducts, Mutations and Cancer ................................................................... 64
3.6.2 DNA Damage Assays ............................................................................................ 64
i. Micronucleus Formation ................................................................................ 64
ii. DNA Strand Breaks ........................................................................................ 65
iii. Sister Chromatid Exchanges .......................................................................... 65
3.6.3 Phase 2: DNA Repair Polymorphism Selection Strategy ..................................... 65
3.7 Overview of DNA Damage Response .............................................................................. 70
3.7.1 DNA Repair Mechanisms ..................................................................................... 70
i. Base-Excision Repair ..................................................................................... 70
ii. Nucleotide-Excision Repair ........................................................................... 71
iii. Homologous Recombination .......................................................................... 71
3.7.2 Cell Cycle Checkpoints ......................................................................................... 71
3.7.3 Phase 2: Polymorphisms in Genes Involved in the Repair of Damage Induced by
HAAs ..................................................................................................................... 72
3.7.4 Phase 2: Polymorphisms in Genes Involved in Base Excision Repair ................. 72
i. X-ray repair cross-complementing group 1 ................................................... 72
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ii. ADP-ribosyltransferase .................................................................................. 73
3.7.5. Phase 2: Polymorphisms in Genes Involved in Nucleotide Excision Repair ........ 73
i. Xeroderma pigmentosum group D ................................................................. 73
ii. Xeroderma pigmentosum group C ................................................................. 74
3.7.6. Phase 2: Polymorphism in Genes Involved in Homologous Recombination ....... 74
i. X-ray repair cross-complementing group 3 (XRCC3) ................................... 74
3.8 Summary of Polymorphisms of Interest to Phase 2 .......................................................... 74
3.9 Conclusions and Perspectives ........................................................................................... 75
3.10 References ........................................................................................................................ 77
Chapter 4: Phase 1 Study Designs and Methods...................................................................... 83
4.1 Phase 1: Dietary HAA Intake and Bulky DNA Adducts ................................................. 83
4.1.1 Study Subjects ....................................................................................................... 84
4.1.2 Recruitment ........................................................................................................... 84
4.1.3 Data Collection ...................................................................................................... 85
i. Meat Consumption Module ............................................................................ 85
ii. Questionnaire ................................................................................................. 85
iii. Blood Sample ................................................................................................. 85
4.1.4 Exposure Assessment – Dietary Heterocyclic Aromatic Amines ......................... 86
i. Development of Meat Consumption Module ................................................. 86
ii. Derivation of Dietary Heterocyclic Aromatic Amines Exposures ................. 87
4.1.5 Outcome Assessment – Bulky DNA Adduct in Blood Leukocytes ...................... 87
i. 32P-Postlabelling ............................................................................................ 88
ii. Assessment of Reliability/Quality Control .................................................... 88
4.1.6 Genetic Assessment – Genes Involved in the Biotransformation of HAAs and the
Repair of DNA Adducts ........................................................................................ 89
4.1.7 Covariate Assessment ........................................................................................... 89
i. Characterization of Personal Characteristics.................................................. 90
ii. Characterization of Lifestyle and Dietary Factors ......................................... 91
iii. Characterization of Genetic Factors ............................................................... 92
4.1.8 Data Validity ......................................................................................................... 94
i. Genetic Data ................................................................................................... 94
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4.1.9 Data Management ................................................................................................. 94
4.1.10 Data Analysis ........................................................................................................ 95
i. Outcome Representation ................................................................................ 95
ii. Exposure Representation ................................................................................ 95
iii. Main Analysis ................................................................................................ 96
iv. Assessment of Confounding .......................................................................... 98
v. Effect Modification ........................................................................................ 98
4.1.11 Sample Size and Power ......................................................................................... 98
4.1.12 Additional Analysis ............................................................................................ 100
i. Sex-Specific Analysis ................................................................................. 100
4.2 Ethical Considerations ................................................................................................... 100
4.3 References ...................................................................................................................... 102
CHAPTER 5: Phase 1 Results ............................................................................................... 105
5.1 Phase 1: Dietary HAA Intake and Bulky DNA Adducts ............................................... 105
5.1.1 Phase 1 Objectives ............................................................................................. 105
5.2 Descriptive Statistics ...................................................................................................... 105
5.2.1 Exposure Distribution - Dietary Heterocyclic Aromatic Amine Intake ............. 105
5.2.2 Outcome Distribution - Bulky DNA Adduct Levels in Blood Leukocytes ....... 108
5.2.3 Covariates ........................................................................................................... 111
5.2.4 Covariate Representation ................................................................................... 113
5.2.5 Genetic Factors ................................................................................................... 113
5.3 Covariate Associations with Bulky DNA Adduct Levels .............................................. 115
5.4 Covariate Associations with Dietary Heterocyclic Aromatic Amine Intake ................. 117
5.5 Main Analysis ................................................................................................................ 121
5.6 Multivariable Analysis ................................................................................................... 123
5.7 Genetic Analysis ............................................................................................................ 127
5.7.1 Genetic Determinants of Bulky DNA Adduct Concentration in
Blood Leukocytes ............................................................................................... 127
5.7.2 Gene-Diet Interaction Analysis .......................................................................... 129
5.8 Additional Analysis ....................................................................................................... 133
5.8.1 Sex-Specific Analysis ........................................................................................ 133
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