Variation in Smoking and Nicotine Metabolism Among American Indians: Novel Influences on In Vivo and In Vitro Nicotine Metabolism Phenotypes by Julie-Anne Tanner A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Pharmacology and Toxicology University of Toronto © Copyright by Julie-Anne Tanner 2017 Variation in Smoking and Nicotine Metabolism Among American Indians: Novel Influences on In Vivo and In Vitro Nicotine Metabolism Phenotypes By Julie-Anne Tanner Doctor of Philosophy Department of Pharmacology and Toxicology University of Toronto 2017 Abstract Tobacco use and associated disease risk vary widely among different populations and ethnic/racial groups. While American Indian and Alaska Native populations collectively have the highest prevalence of smoking, recent studies have highlighted disparities in smoking and disease risk across different American Indian/Alaska Native populations. The Northern Plains American Indian tribal populations of South Dakota have a much higher smoking prevalence, smoke more cigarettes per day, and have higher lung cancer risk than the Southwest American Indian tribe of Arizona. Underlying reasons behind the differences in smoking and lung cancer risk between these tribes remain unknown. We sought to biochemically characterize the level of tobacco exposure among smokers and non-smokers in the Northern Plains and Southwest tribes, using cotinine as a biomarker. We demonstrated that despite both tribes being relatively light smoking populations, secondhand tobacco smoke exposure is high among non-smokers, highlighting a potential source of increased risk for tobacco-related disease. As variation in the rate of nicotine metabolism (i.e. CYP2A6 activity) has been associated with differences in smoking behaviours and lung cancer risk in other ethnic/racial groups, we then evaluated variation in the CYP2A6 gene and the rate of nicotine metabolism in the Northern Plains and ii Southwest tribes. We identified distinct patterns of CYP2A6 genetic variation across the two tribes, and an overall faster rate of nicotine metabolism in the Northern Plains compared to the Southwest tribe. We then used a human liver bank to explore additional sources (both genetic and non-genetic) of variation in CYP2A6 activity. We identified several independent and shared sources of variation that could translate to variable hepatic clearance of nicotine, and may impact smoking behaviour. Lastly, we searched for CYP2A6 gain of function genetic variants in order to identify sources of faster CYP2A6 activity. Through investigation of novel uncharacterized variation at the CYP2A6 locus using next-generation sequencing, we identified seven novel high frequency CYP2A6 genetic variants and a five-SNP diplotype, which were associated with faster CYP2A6 activity in vitro and in vivo. Overall, our findings provide insight into the patterns of tobacco exposure, CYP2A6 genetic variation, and nicotine metabolism rate in American Indian tribal communities, and expand on our knowledge of genetic and non-genetic sources of variation in nicotine metabolism rate. These findings could improve our understanding of lung cancer risk in these American Indian populations, and help to inform future tobacco cessation efforts. iii ACKNOWLEDGEMENTS To the many people who have supported me throughout my PhD – thank you! There are a few people in particular to whom I would like to extend special thanks. First, I would like to thank my supervisor, Dr. Rachel Tyndale, for her guidance and invaluable expertise. Under your mentorship, I have been able to grow as a scientist, writer, presenter, and independent thinker. I am grateful to have been pushed outside of my comfort zone, as this has shown me that there are no limits to my capabilities. I truly appreciate the opportunity to learn from you and collaborate with you, and I thank you for your encouragement along the way. I would also like to acknowledge the collaborators with whom I have been fortunate enough to work. I am thankful to Dr. Jeffrey Henderson, Dr. Kenneth Thummel, and Dr. Steven Scherer for their insight and advice, stemming from their extensive experience and accomplishments in the fields of public health, pharmacology, and genomics. I also thank my PhD supervisory committee members, Dr. Denis Grant and Dr. Christian Hendershot, for the helpful ideas and feedback that they have provided throughout this entire process. They have both challenged and encouraged me to expand on the interpretations of my data. I was lucky to have them as committee members. I would also like to extend my thanks to the entire Tyndale lab. Qian Zhou, Ewa Hoffman, and Dr. Zhao Bin were a key part of my learning to conduct quality experimental work within the lab. All members of the Tyndale lab have been a great support system throughout my PhD, but I have to give a special thank you to Dr. Meghan Chenoweth and (soon-to-be) Dr. Doug McMillan for ensuring that there was never a dull moment and for allowing me to sit between you and distract you for five whole years. In addition to the support received from the Tyndale lab and research community, my friends and family have been a constant source of support and love. Gill and Hélène, you have been along for the ride since long before I embarked on becoming a scientist, and I could not have completed this journey without your friendship and belly laughs. iv To my soon-to-be family, the Martelli’s, thank you for welcoming me in to your family and for always showing interest in my work, even if it was a tad confusing. I have always appreciated your support and look forward to boring you with more scientific tidbits in the future. Of course, I want to thank my parents for making all of this possible. Saying “thank you” just doesn’t seem sufficient. You have made so many sacrifices for Lindsay and me, and have always put our happiness and education first. Mom, you have picked me up when I have stumbled and cheered me on when I needed that extra boost. Dad, you have shown me the meaning of hard work and integrity. Thank you both for the huge role you played in getting me here. Thank you to my sister, Lindsay, for being my partner in crime since birth and for always looking out for me. I also want to thank Gram for encouraging me to learn more about the world around me. Lastly, I must say thank you to my better half, someone who inspires me to be my best self every day. Mike, you have showered me in love and support for nearly nine years. You are one of the most hard working and passionate people that I know, and saying “yes” to you was one of the smartest (and easiest) decisions I have ever had to make. We have created an incredible life together with our little Bear, and I can’t wait take on this next chapter with you. v Table of Contents ACKNOWLEDGEMENTS............................................................................... iv TABLE OF CONTENTS................................................................................... vi LIST OF PUBLICATIONS............................................................................... xi LIST OF TABLES............................................................................................ xiii LIST OF FIGURES…....................................................................................... xv LIST OF ABBREVIATIONS.......................................................................... xix LIST OF APPENDICES.................................................................................. xxi STATEMENT OF RESEARCH PROBLEM............................................... xxii MAIN RESEARCH OBJECTIVES............................................................... xxv 1 GENERAL INTRODUCTION........................................................................ 1 1.1 Epidemiology of Tobacco Use........................................................................................ 1 1.1.1 Prevalence………….……………….…………………………..…………...…… 1 1.1.2 Health Consequences……………….…………………………..……………...… 1 1.1.2.1 Lung Cancer……..……..……………………………………………...… 3 1.1.2.2 Chronic Obstructive Pulmonary Disease………………………………… 5 1.1.2.3 Cardiovascular Disease………………………………………………...… 6 1.1.2.4 Other Health Effects……………………………………………....…...… 6 1.1.3 Vulnerable Populations…………………………………………..………………. 8 1.1.3.1 Low Income and Socioeconomic Status………………..………………... 8 1.1.3.2 Mental Illness and Substance Use Disorders.…………..………………... 9 1.1.3.3 Racial Minorities…………………………………………..…………… 11 1.2 Interethnic Tobacco Use and Associated Disease…..……………..………………... 13 1.2.1 Trends in Whites……………………………….……………..…………….…... 13 1.2.1.1 Prevalence and Characteristics of Tobacco Use………..………………. 13 1.2.1.2 Tobacco-Related Disease Risk………………..………………………... 16 1.2.2 Trends in African Americans………………..……………………………….…. 16 1.2.2.1 Prevalence and Characteristics of Tobacco Use………………..………. 16 1.2.2.2 Tobacco-Related Disease Risk………………..………………………... 18 1.2.3 Trends in Asian Americans …………………………...…….……………..…… 18 vi 1.2.3.1 Prevalence and Characteristics of Tobacco Use………………..………. 18 1.2.3.2 Tobacco-Related Disease Risk………………..………………………... 19 1.2.4 Trends in American Indians/Alaska Natives.………………..………..………... 20 1.2.4.1 Prevalence and Characteristics of Tobacco Use………………..………. 20 1.2.4.3 Ceremonial Tobacco Use………………..…………………..………….. 21 1.2.4.4 Tobacco-Related Disease Risk………………..………………………... 22 1.2.4.5 Education and Research Toward Health (EARTH) Study……………... 23 1.3 Pharmacology of Smoking…..……………………………………..……………….... 24