CYP2A6 and CYP2B6 Genetic Variation, and Tobacco Use. Behaviours and Biomarkers in Alaska Natives

Transcription

1 CYP2A6 and CYP2B6 Genetic Variation, and Tobacco Use Behaviours and Biomarkers in Alaska Natives By Matthew Binnington A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Pharmacology and Toxicology University of Toronto Copyright by Matthew Binnington, 2011.

2 CYP2A6 and CYP2B6 Genetic Variation, and Tobacco Use Behaviours and Biomarkers in Alaska Natives Matthew Binnington 2011 Master of Science Department of Pharmacology and Toxicology, University of Toronto ABSTRACT The impact of CYP2A6 and CYP2B6 genetic variation on nicotine metabolism, tobacco use behaviours, and nicotine biomarkers was investigated in a group of Alaska Natives (n = 400). CYP2A6 and CYP2B6 allele frequencies were unique and associations of CYP2A6 genotype and CYP2A6 activity (plasma and urine trans 3!- hydroxycotinine/cotinine (3HC/COT) ratios) were robust. Notably, this population possessed a more rapid rate of CYP2A6 activity (higher plasma 3HC/COT) when compared to CYP2A6 wild-type individuals in other ethnic groups (ANOVA P < 0.001). Also demonstrated was a significant difference in urine total nicotine equivalents by CYP2A6 activity median split (t-test P < 0.01), the first evidence of nicotine titration by CYP2A6 activity within a light smoking population. Overall, this population possessed a distinctive pattern of CYP2A6 and CYP2B6 variant frequencies and a faster rate of nicotine metabolism, which may in part explain higher levels of tobacco use prevalence and tobacco-related disease risk. ii

3 ACKNOWLEDGEMENTS I would like to take this opportunity to especially thank a few of the numerous individuals who assisted, encouraged, and challenged me through the course of graduate study and completion of my Masters degree. Primarily I would like to thank my supervisor Dr. Rachel Tyndale for her excellent guidance, patience, and support in this endeavor. I benefitted greatly from her personal time and input, but even more so from her natural ambition, dedication, and professionalism. Her example as a lifelong scholar and scientist is one I can only hope to emulate. Thank you. I am also extremely grateful for the time, care, and teaching expertise of both Ewa Hoffman and Qian Zhou. Their support was invaluable in helping me complete my project, especially during the most challenging times when experiments just seemed to refuse to go as planned! That they were able to do this while continually providing a positive and encouraging atmosphere within the lab is a testament to their patience and professionalism. I am not the first, and certainly will not be the last, to owe a great deal to their excellent assistance. I must thank all lab members, past and present, for their friendship and advice; it was a great benefit to join a group with such intelligent, humble, and caring people as yourselves! Further, I owe a special thanks to both Andy Zhu and Catherine Wassenaar for their insight and example; they are both superb graduate students and certainly scientists in their own right. Finally, I am deeply indebted to my family and friends for their continued support, especially my wife Catherine, parents Mark and Lee Anne, and siblings Luke and Brittany Anne. I cannot begin to express my gratitude for all your love, care, and encouragement; my sincerest thanks. iii

4 TABLE OF CONTENTS Abstract Acknowledgments Table of Contents List of Tables List of Figures List of Abbreviations 1. Introduction 1.1. Alaska Native Tobacco Use 1.2. Light Smoking 1.3. Smokeless Tobacco 1.4. Health Disparities of Alaska Native Tobacco Users 1.5. Nicotine: The Main Addictive Substance in Tobacco Nicotine Titration Nicotine Reinforcement Measures of Nicotine Dependence 1.6. Nicotine Pharmacokinetics Absorption and Distribution Metabolism and Elimination 1.7. CYP2A The Role of CYP2A6 in Nicotine Metabolism The Influence of CYP2A6 Genetic Variation on Nicotine Metabolism The CYP2A Gene Subfamily Specific CYP2A6 Gene Variants and their Functional Impact CYP2A6 Genotype Grouping Strategy Non-Genetic Variables that Impact CYP2A6 Function 1.8. CYP2B The Role of CYP2B6 in Nicotine Metabolism The Influence of CYP2B6 Genetic Variation on Nicotine Metabolism 1.9. The Role of Other Enzymes in Nicotine Metabolism CYP2A6 Impact on Tobacco-Related Behaviours and Biomarkers Tobacco Use and Dependence ii iii iv vii ix xi iv

5 Biomarkers of Tobacco Use Cigarettes Per Day/Smokeless Tobacco Per Day Plasma Cotinine Urine Total Nicotine Equivalents Cancer Risk Statement of Problem Rationale, Objectives, and Hypotheses 2. Methods 2.1. Study Overview 2.2. Study Design Subject Recruitment and Screening Assessments Eligibility Screening and Structured Interview Specimen Collection and Analysis 2.3. CYP2A6 Genotyping DNA Extraction Genotyping Overview Assays, Primer Sets, and Reaction Conditions Gel Electrophoresis and Visualization CYP2A6 Genotype Grouping 2.4. Statistical Analysis 3. Results 3.1. CYP2A6 and CYP2B6 Allele and Genotype Group Frequencies 3.2. Association Between CYP2A6 Genotype and Plasma CYP2A6 Activity 3.3. Variables Independent of CYP2A6 Genotype that Impact Plasma 3HC/COT 3.4. CYP2A6 and CYP2B6 Gene Interaction and Haplotyping 3.5. Participant Demographic Characteristics by CYP2A6 Genotype Grouping 3.6. Association Between CYP2A6 Genotype and Urinary CYP2A6 Activity v

6 3.7. Variables Independent of CYP2A6 Genotype that Impact Urinary 3HC/COT 3.8. Modeling of the Plasma and Urinary 3HC/COT Ratios 3.9. Comparisons of Yupik Plasma 3HC/COT Ratios to Studied Ethnicities Tobacco Use Variables Participant Baseline Characteristics Impact of CYP2A6 Genotype Impact of Plasma 3HC/COT Comparison of Nicotine Dose Biomarkers Impact of CYP2A6 Genotype and Plasma 3HC/COT on NNAL Level Modeling NNAL Level 4. Discussion 4.1. CYP2A6 and CYP2B6 Genetic Variation Among Yupik 4.2. CYP2A6 Genotype Association with Plasma 3HC/COT 4.3. CYP2A6 Genotype Association with Urine 3HC/COT 4.4. CYP2B6 Genotype Association with 3HC/COT 4.5. Ethnic Differences in Plasma 3HC/COT 4.6. The Impact of CYP2A6 Genetic Variation and CYP2A6 Enzyme Activity on Yupik Tobacco Use Behaviors 4.7. Carcinogen Exposure Association with CYP2B6, and not CYP2A6 Genotype 4.8. Limitations of Study 5. General Conclusions 6. References vi

7 LIST OF TABLES Table 1.1 The functional consequences of relevant CYP2A6 gene variants, and their allele frequencies in various populations Table 2.1 Listing of primers for each CYP2A6 genotyping assay Table 2.2 CYP2A6 genotyping assay reaction conditions for all variants studied Table 2.3 CYP2A6 genotyping assay PCR conditions for all variant assays performed Table 2.4 CYP2A6 genotyping gel visualization parameters (loading dye and agarose percentage) for all variant assays performed Table 3.1 Observed CYP2A6 and CYP2B6 allele frequencies of all Alaska Native (n = 400), Yupik (n = 361), and all Yupik with 4 Alaska Native grandparent participants Table 3.2 Observed CYP2A6 and CYP2B6 allele frequencies of all Alaska Native (n = 400), Yupik (n = 361), and all Yupik with 4 Alaska Native grandparent participants Table 3.3 Frequency of CYP2A6 genotypes among Yupik (n = 361), and the Yupik tobacco user subset (n = 265) Table 3.4 Frequency of CYP2A6 genotypes and their associated plasma 3HC/COT ratios among Yupik tobacco users (n = 265) Table 3.5 Factors that influence the plasma 3HC/COT ratio in CYP2A6*1/*1 Yupik tobacco users (n = 147) Table 3.6 Baseline characteristics for Yupik tobacco users (n = 265) by CYP2A6 genotype grouping Table 3.7 Frequency of CYP2A6 genotypes and their associated urine 3HC/COT ratios among Yupik tobacco users (n = 265) Table 3.8 Factors that influence the urine 3HC/COT ratio in CYP2A6*1/*1 Yupik tobacco users (n = 147) Table 3.9 Factors that influence the plasma and urine 3HC/COT ratios in Yupik tobacco users (n = 246) Tale 3.10 Baseline characteristics for Yupik tobacco users (n = 265) and Yupik smokers (n = 143) vii

8 Table 3.11 Baseline characteristics by product use group for Yupik tobacco users (n = 265) Table 3.12 Measures of tobacco use by CYP2A6 genotype grouping among Yupik tobacco users (n = 265) Table 3.13 Measures of tobacco use and dependence by CYP2A6 genotype grouping among Yupik smokers (n = 143) Table 3.14 Baseline characteristics for all Yupik tobacco users (n = 265) and Yupik smokers (n = 143) by plasma 3HC/COT median split Table 3.15 Comparisons of urine NNAL level and urine NNAL/TNE ratio by CYP2A6 genotype and plasma 3HC/COT ratio in Yupik tobacco users (n = 265) and Yupik smokers (n = 143), and by distinct Yupik tobacco use groups Table 3.16 Factors that influence urine NNAL levels in Yupik tobacco users (n = 246) differentiated by a) CYP2A6 genotype grouping or b) 3HC/COT median split viii

9 LIST OF FIGURES Figure 1.1 Major pathways of human nicotine metabolism Figure 1.2 The CYP2ABFGST gene cluster on chromosome 19q13.2 Figure 1.3 A schematic illustrating the formation of each CYP2A6*4 variant subtype, and the reciprocal CYP2A6*1X2 products Figure 3.1 Yupik possess a unique pattern of CYP2A6 and CYP2B6 allele frequencies when compared to other ethnicities Figure 3.2 Association of CYP2A6 genotype with the plasma 3HC/COT ratio among Yupik tobacco users (n = 265) Figure 3.3 Association of CYP2A6 genotype groupings with the plasma 3HC/COT ratio among all Yupik tobacco users (n = 265) Figure 3.4 Association of CYP2B6 genotype groupings with the plasma 3HC/COT ratio among Yupik tobacco users (n = 265) Figure 3.5 Association of CYP2B6 genotype with the plasma 3HC/COT ratio results from interaction with the CYP2A6 gene Figure 3.6 Interaction of the CYP2A6 and CYP2B6 genes in Yupik (n = 361) Figure 3.7 Correlation of plasma 3HC/COT and urine 3HC/COT ratios in Yupik tobacco users (n = 265) Figure 3.8 Association of CYP2A6 genotype with the urinary 3HC/COT ratio among Yupik tobacco users (n = 265) Figure 3.9 Association of CYP2A6 genotype groupings with the urine 3HC/COT ratio among Yupik tobacco users (n = 265) Figure 3.10 Association of CYP2B6 genotype groupings with the urine 3HC/COT ratio among Yupik tobacco users (n = 265) Figure 3.11 Yupik possess an elevated rate of CYP2A6 activity compared to characterized ethnicities Figure 3.12 Differences in mean plasma nicotine metabolite levels between Yupik and characterized ethnicities ix

10 Figure 3.13 No difference in daily product consumption was found by CYP2A6 genotype grouping among Yupik smokers (n = 143), commercial chew users (n = 73), and iqmik users (n = 20) Figure 3.14 No difference in daily product consumption was found by plasma 3HC/COT median split among Yupik smokers (n = 143), commercial chew users (n = 73), and iqmik users (n = 20) Figure 3.15 Association of urine TNE with plasma 3HC/COT median split among Yupik tobacco users (n = 265) and Yupik smokers (n = 143) Figure 3.16 Correlation of plasma COT with urine TNE among Yupik smokers (n = 143) Figure 3.17 Correlation of CPD with a) plasma COT and b) urine TNE among Yupik smokers (n = 143) Figure 3.18 Correlation of CPD with a) plasma COT/CPD and b) urine TNE/CPD among Yupik smokers (n = 143) Figure 3.19 Association of a) urinary NNAL and b) urinary NNAL/TNE with tobacco product type among Yupik tobacco users (n = 265) Figure 3.20 Association of CYP2B6 genotype with urinary NNAL level among Yupik tobacco users (n = 265) and Yupik smokers (n = 143) Figure 4.1 The utility of plasma cotinine as a biomarker of nicotine exposure is reduced in Yupik smokers (n = 143) among those with decreased CYP2A6 activity x

11 LIST OF ABBREVIATIONS 3HC COT 3HC/COT BMI bp CPD CO CYP DSM-IV FTND FMO1 ICD-10 IM kb nachr NM NNAL NNK NNN PAH PCR RM SM SNP ST TNE TSNA UGT trans 3-Hydroxycotinine Cotinine trans 3-Hydroxycotinine/Cotinine Body Mass Index base pairs Cigarettes Per Day Carbon Monoxide Cytochrome P450 Diagnostic and Statistical Manual of the American Psychiatric Association Version IV Fagerström Test of Nicotine Dependence Flavin-containing Monooxygenase International Classification of Diseases 10 Intermediate Metabolizer kilo bases Nicotinic Acetylcholine Receptor Normal Metabolizer 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone N-nitrosonornicotine Polycyclic Aromatic Hydrocarbon Polymerase Chain Reaction Reduced Metabolizer Slow Metabolizer Single Nucleotide Polymorphism Smokeless Tobacco Total Nicotine Equivalents Tobacco-Specific Nitrosamine Uridinediphosphate-glucuronosyltransferase xi

12 1. INTRODUCTION 1.1 Alaska Native Tobacco Use American Indians and Alaska Natives currently report the highest rates of cigarette smoking and smokeless tobacco use of any ethnic minority group in the United States (Beauvais et al. 2007; Carmona et al. 2004; USDHHS 1998; USDHHS 2007a). In comparisons of Alaska Natives to non-native residents of Alaska, a consistently greater prevalence of tobacco use is observed in Alaska Natives, with rates being nearly twice as high (Peterson et al. 2004; Redwood et al. 2010; Smith et al. 2010; USDHHS 2007b). The majority of participants in the current study population self-identified as members of the Yupik tribe, and much work has been performed recently on tobacco use within this Alaska Native sub-population. Yupik are found mainly in the Yukon-Kuskokwim delta, an area of approximately square miles in the southwest region of Alaska (Wolsko et al. 2009). The Yupik people total approximately (USCB 2000) and constitute the main inhabitants in the area (85%) (Sepez et al. 2005), the majority of whom live in geographically isolated villages of between residents (Wolsko et al. 2009). Notably, reports indicate that the Yupik are the most linguistically and culturally intact Alaska Native group throughout the entirety of Alaska (Sepez et al. 2005). Figures compiled by the Alaska Department of Health and Social Services demonstrate that 43.5% of Yupik regularly smoke cigarettes and 14.2% regularly use smokeless tobacco (Peterson et al. 2004). A recent study by Wolsko et al. (2009) of Yupik within eight Yukon-Kuskokwim delta villages observed that over a quarter of participants smoked cigarettes (28.8%), over half used some form of smokeless 1

13 tobacco (69.9% - combined chew and snuff tobacco users), and 19.2% reported using more than one tobacco type. Interestingly, smokeless tobacco use was more prevalent among women than men in Yupik (Wolsko et al. 2009; Smith et al. 2010). This contrasts with findings in both the total U.S. population and among non-native Alaskans, where current smokeless tobacco use is far more prevalent in males (7% and 9%) than in females (0.3% and 0.1%) (USDHHS 2006; Wells 2004). Also noteworthy is that the average figure for daily cigarettes in Yupik falls below the general U.S. population 9.9 cigarettes per day (CPD) and 8.5 for Yupik men and women, compared to 14.2 and 12.1, respectively (Smith et al. 2010; USDHHS 2003). The traditional cutoff to differentiate light smokers from typical levels of consumption is 10 CPD (Ho et al. 2009b), making the Yupik a light smoking population. 1.2 Light Smoking The prevalence of cigarette use among Yupik is notable as smoking is the single greatest preventable cause of death in the world and is responsible for over five million deaths annually (WHO 2008). It has been directly linked with several types of morbidities, including: tobacco-related cancers, cardiovascular, respiratory, dental, and eye diseases, and reductions in reproductive health and immunity (CDCP 2004). Recent North American estimates of smoking prevalence suggest a growing proportion of the smoking population is characterized by a non-daily, or light smoking pattern (defined as 10 or fewer CPD) similar to that seen in Yupik (Kandel and Chen 2000; Trinidad et al. 2009). Despite a decreasing overall prevalence of smoking (Benowitz 2010; NCI 2010), the proportion of light smokers has increased. The percentage of current adult smokers in the United States reporting light use has risen 2

14 from 16% in 1998 to 22% in 2009 (CDCP 2010; Wortley et al. 2003). Accompanying the increase in non-daily smoking has been a decrease in CPD number in daily smokers. In the U.S. the average CPD number smoked by adults has decreased significantly; in 1980 mean CPD in U.S. males and females was 25.1 and 21.5, and average consumption has subsequently dropped to 16.7 and 13.2 CPD respectively in 2000 (Duval et al. 2008). Additionally, wide variation is seen in the level of cigarette consumption between different ethnic groups in the United States. Among Caucasian current smokers the percentage of users consuming less than 10 CPD is approximately 40%, while a greater proportion of light use is found in African- American (67%), Asian-American (72%), and Hispanic-American (76%) current smokers (Trinidad et al. 2009). Compared to never-smokers, light smokers maintain an increased risk of mortality, particularly from cardiovascular disease and cancer. Smokers consuming one to four CPD in Norway were found at 3 times greater risk of death from ischemic heart disease, 5 times greater risk from lung cancer, and 1.5 times greater risk from any cause when compared to never smokers (Bjartveit and Tverdal 2005). The risk for myocardial infarction is also 2.1 times greater in light smokers (specifically shown in a group consuming 3-9 CPD) (Prescott et al. 2002). Further, light smoking puts individuals at higher risk of gastrointestinal cancers, respiratory illnesses, compromised reproductive health, and cataracts (Schane et al. 2010). Not surprisingly, light smoking behavior challenges current theories on tobacco dependence, as smoking at regular intervals throughout the day is thought to be critical for the avoidance of withdrawal symptoms. The few studies analyzing tobacco 3

15 dependence among Caucasian light smokers have found that they do not exhibit the features characteristic of tobacco dependence seen in moderate to heavy Caucasian smokers (Shiffman 1989; Shiffman et al. 1995). Among light smokers, these include reduced withdrawal symptoms during smoking abstinence, fewer cravings and smoking urges between cigarettes, a longer time until the first cigarette after waking, and lesser likelihood of smoking even when unwell or where it is prohibited (Shiffman 1989; Shiffman and Paty 2006). In a population of African-Americans, maintenance of light smoking was increased in males, lower education brackets, divorced individuals, and current alcohol and marijuana users (Mwenifumbo et al. 2008c). Continued light smoking was influenced by many of the same factors in a recently studied population of Alaska Natives, as significant associations of current smoking were also seen in males, lower education brackets, unmarried individuals, and current users of alcohol (Smith et al. 2010). An increased rate of smoking was also observed in those with an income below $25 000/year, and younger individuals (Smith et al. 2010). Due to the continued persistence in smoking prevalence and its deleterious impact on human health, an abundance of work has been performed on the etiology of this behavior. The factors leading to habitual cigarette use are complex and result from genetic predisposition as well as environmental influences such as peers and culture. Family, adoption, and twin studies suggest genetic factors play a major role in smoking (Swan et al. 1996; Swan et al. 1997). It is estimated that 60-70% of the variability in smoking persistence and nicotine dependence, 45-86% of the variability in cigarettes smoked, and 26-48% of the variability in nicotine withdrawal symptoms are due to heredity (Broms et al. 2006; Carmelli et al. 1992; Koopmans et al. 1999). 4

16 1.3 Smokeless Tobacco In addition to cigarettes Yupik exhibit a high prevalence of smokeless tobacco (ST) use, including various forms of chew and snuff, and a unique form of ST called iqmik. Like smoking, the use of smokeless tobacco is addictive (USDHHS 1986) and similarly, withdrawal symptoms among smokeless tobacco users resemble those observed in smokers (Hatsukami et al. 1987). The aforementioned chew and snuff are the two major types of smokeless tobacco sold in North America and differ from each other mainly in cut and consumption method. For chew tobacco individuals typically place an approximately 8 g piece of crudely processed tobacco in the mouth and lightly chew to manipulate the product and saturate it with saliva (Benowitz et al. 1988), while snuff is a more finely cut, sticky mixture of tobacco which individuals use by placing a small pinch (of 1-3 g) between the cheek and gum to gently suck for consumption (Hatsukami et al. 1988). Iqmik is a unique homemade smokeless tobacco product used by Alaska Natives, made from a mixture of fire-cured tobacco leaves with the ash generated from burning the woody fungus Phellinus igniarius (or punk fungus) (Hurt et al. 2005; Renner et al. 2005). Punk fungus can be harvested from the bark of local birch trees, or purchased in stores as either the fungus or ash (Blanchette et al. 2002). Iqmik is most commonly prepared by women, who combine tobacco leaves with the punk fungus ash and pre-chew it in their mouth to form individual chews (Renner et al. 2005). Alternatively, the tobacco is cut into small pieces and mixed with punk ash, followed by stirring in a bowl of water or blender (Hurt et al. 2005). Individual rates for the prevalence of iqmik use are unknown, but use rates of all types of smokeless 5

17 tobacco are high among Yupik women with estimates in excess of 50% (Renner et al. 2005). Yupik women also perceive fewer health risks with iqmik use compared to smoking and commercial ST (Renner et al. 2004), and as a result this product is consistently used throughout pregnancy (Hurt et al. 2005). The prevalence of all ST use in the United States remains important, as 7.0% of men and 0.3% of women are regular consumers (USDHHS 2006). Specifically, current ST use is more commonly observed in young non-hispanic whites, those with a high school diploma or lower level of education, in Southern U.S. states, and within rural areas (Tomar 2003). Though the health risks associated with smokeless tobacco are fewer than with smoking, due to the lack of exposure to combustion products (Boffetta et al. 2008), several morbidities are linked to ST use. In excess of 30 documented human carcinogens are found in smokeless tobacco (IARC 2008). Particularly, nitrosamines in ST are at the highest non-occupational exposure levels known; contained within every gram of common North American ST are 1 5 mg of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) (IARC 2008; Stepanov et al. 2006). As a result, ST users have a significantly greater risk of both oral and lung cancer than non-users; in one study comparing current male smokers who switched to ST use versus male smokers who quit smoking entirely, the relative oral cancer risk was 2.6 for the switchers (95% CI = ) and relative lung cancer risk was 1.5 (95% CI = ) (Henley et al. 2007). ST is also associated with a higher occurrence of oral soft tissue lesions (regarded as cancer precursors) and gingival recession (IARC 2008). 6

18 Despite the health concerns associated with ST, much consideration in recent years has been given to implementing ST use as a harm reduction method for cigarette smokers who are unable to quit using tobacco. The proposed use of ST as a harm reduction approach is based mainly on successes in Sweden, where significant reductions in the rate of tobacco-related morbidity and mortality have been attributed to replacement of cigarette smoking with low-nitrosamine oral snuff (in contrast to the high nitrosamine content of North American ST) among male tobacco users (Levy et al. 2006; Ramstrom 2000). Swedish cohort studies suggest most successful smoking cessation attempts by men occurred with co-use of ST (Lindstrom and Isacsson 2002a; Lindstrom and Isacsson 2002b; Rodu et al. 2003; Tillgren et al. 1996). In addition to proof-of-principle studies among Swedish smokers, the theoretical basis for ST as a harm reduction method among smokers is three-fold. First, fewer deleterious health effects are associated with ST use as compared to smoking. It has been estimated that North American ST use poses approximately 2% of the total mortality risk of smoking based on the aforementioned lack of exposure to tobacco combustion products and the difference in administration route for the tobacco-specific toxins (Rodu and Cole 1994; Rodu and Cole 1999). Second, the potential for abuse liability with ST compared to cigarettes may be lower due to differences in the route of administration resulting in a slower rate of nicotine absorption. The speed of a drug!s delivery has been attributed to its abuse liability (Balster and Schuster 1973; Henningfield et al. 1985), and thus it is notable that all tested brands of ST in the U.S. have a slower rate of nicotine absorption than cigarettes (Hatsukami et al. 2004). Third, use of ST in place of cigarettes may assist in cessation attempts, based on the 7

19 lower number of behavioral cues accompanying consumption. Although similar types of behavioral cues are seen in ST and cigarette users (Hatsukami and Severson 1999), fewer are experienced daily by average treatment-seeking ST users than smokers as they typically take 10 dips per day compared to cigarettes on average (Hatsukami et al. 2004). Although a biological, experimental, and statistical rationale exists for increased use of ST as a harm-reduction method, further study of commonly used types of ST has been proposed (Hatsukami et al. 2004) prior to the widespread implementation of ST-assisted cessation programs. 1.4 Health Disparities of Alaska Native Tobacco Users Consistent with a high prevalence of tobacco use, significant levels of tobaccorelated death and disease are observed among Alaska Natives, although rates of disease appear to be disproportionate to the population!s mean level of tobacco consumption. Cancer is the leading cause of death in Alaska Natives, with a rate 1.3 times greater than the national rate of the United States (Day and Lanier 2003). In fact, from 1994 to 1998 a reduction in the rate of cancer-related mortality occurred among most American ethnic groups, while the cancer-related death rate for Alaska Natives grew (Ehrsam et al. 2001). Lung cancer is responsible for the greatest number of Alaska Native cancer deaths (Lanier et al. 2008), and represents 20% of all cancers diagnosed (Friborg and Melbye 2008). Compared with the worldwide incidence rate of lung cancer, the prevalence in Alaska Natives is within the highest range, with rates among Alaska Native women the highest reported (Parkin et al. 2002). The rate of Alaska Native oral cancer is also higher than rates seen among non-native residents of Alaska, and has been linked to the elevated frequency of regular smokeless 8

20 tobacco use (Lanier et al. 2006). The high prevalence of both tobacco use and obesity among Alaska Natives has also been associated with the high rate of chronic diseases among tribal communities compared to other US ethnic minorities (Day and Lanier 2003; USDHHS 2008), including cerebrovascular diseases and chronic liver disease (USDHHS 2008). 1.5 Nicotine: The Main Addictive Substance in Tobacco Nicotine Titration Tobacco contains thousands of chemicals which range from toxins and carcinogens such as polycyclic aromatic hydrocarbons and aromatic amines (Moritsugu 2007) to known psychoactive constituents including nicotine and related alkaloids (such as nornicotine), acetaldehyde, and several inhibitors of monoamine oxidase (Dome et al. 2009). Of these, nicotine is the main addictive compound in tobacco and is responsible for its pharmacological effect, dependence, and maintenance of use (Henningfield et al. 1985); a conclusion which is supported by a number of lines of evidence. First, tobacco consumption is titrated by users to ensure an optimal level of nicotine in both the plasma and brain (McMorrow and Foxx 1983). Second, when the nicotine content of cigarettes is decreased a compensatory change in smoking behavior is seen in smokers to obtain greater nicotine, including increased cigarette consumption, greater puff volume, and higher puff frequency (Djordjevic et al. 1995a; Kassel et al. 2007; Scherer 1999). A similar change in smoking behavior is also observed when renal nicotine clearance is increased through acidification of urine (Benowitz and Jacob 1985). Third, nicotine is preferred over saline in studies of IV self-administration among current smokers (Harvey et al. 2004; Sofuoglu et al. 2008). 9

21 1.5.2 Nicotine Reinforcement The psychoactive characteristics of nicotine are due to its reinforcing effects on the endogenous reward system within the brain, and this effect explains in part how tobacco dependence is established and maintained (Corrigall and Coen 1989; Le Foll and Goldberg 2006; Le Foll et al. 2007). Nicotine readily crosses the blood-brain barrier, activating!4"2 nicotinic acetylcholine receptors (nachrs) on dopamine neurons in the ventral tegmental area (Lockman et al. 2005; Picciotto et al. 1998; Tapper et al. 2004; Wonnacott 1997), and triggering increased dopamine release in the nucleus accumbens, which stimulates the mesolimbic reward system of the brain (Laviolette and van der Kooy 2003; Rossi et al. 2005; Stein et al. 1998). Chronic nicotine exposure results in!4"2 nachr upregulation within the brain, a response theorized to be due to long-term desensitization and inactivation of these same nachrs by nicotine (Fenster et al. 1999; Quick and Lester 2002). Due to the longterm inactivation of neural!4"2 nachrs, upregulation of these receptors results in increasing tolerance to the rewarding effects of nicotine, as the inactive proportion of!4"2 nachrs grows (Dani and Heinemann 1996). As well, increased withdrawal symptoms are observed and include craving, anxiety, difficulty concentrating, impatience, and restlessness (Hughes et al. 1991) Measures of Nicotine Dependence Diagnosis of nicotine dependence is typically determined by means of standardized psychiatric measures, such as the American Psychiatric Association!s Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) or the tenth revision of the International Classification of Diseases (ICD-10) by the World Health 10

22 Organization. However, administering DSM-IV and ICD-10 interviews in a research setting often proves challenging because they are lengthy and require trained staff for implementation, also making them expensive (Etter 2008; Kawakami et al. 1999). Self-rating questionnaires for subjects based on DSM-IV and ICD-10 criteria for nicotine dependence have been developed that are more frequently provided due to their brevity and ease-of-use. The six-item Fagerström Test for Nicotine Dependence (FTND) is one such example. Itself a revision of the Fagerström Tolerance Questionnaire (FTQ), the FTND is the most widely used research and clinical test of nicotine dependence as it appears to best capture the critical aspects of physical dependence on nicotine (Stavem et al. 2008). Examples of questions include: How soon after you wake up do you smoke your first cigarette?, How many cigarettes per day do you smoke?, and Do you find it difficult to refrain from smoking in places where it is forbidden? (Stavern et al. 2008). 1.6 Nicotine Pharmacokinetics Absorption and Distribution A cigarette contains between 10 and 14 mg of nicotine while the nicotine dose available on average in chewing tobacco is 16.8 mg/g of chew and in daily snuff is 10.5 mg/g of chew (Benowitz et al. 1990). The average systemic nicotine dose resulting from comparable products differs as well: mg per cigarette, 0.6 mg/g from chewing tobacco consumed over 30 minutes, and 1.4 mg/g from daily snuff placed in the mouth for 30 minutes (Benowitz et al. 1988). Nicotine is rapidly absorbed from the small airway and alveoli of the lung during smoking, while oral absorption of smokeless tobacco occurs more slowly; peak plasma nicotine concentrations are 11

23 observed within 10 minutes of smoking a cigarette while the corresponding t max in smokeless tobacco users is 30 minutes (Benowitz et al. 1988; Hukkanen et al. 2005). The rapidity with which brain nicotine concentrations rise in smokers allows them to titrate nicotine dose with greater ease than users of other tobacco products, and contributes to smoking causing the highest levels of reinforcement of any tobacco type (Benowitz 1990; Henningfield and Keenan 1993). Nicotine is a weak base, with a pk a of approximately 8.0, and its absorption across biological membranes is highly dependent on ph (Hukkanen et al. 2005). The ph of most cigarette smoke ranges from resulting in some buccal absorption as the level of nicotine in its unionized form varies (Pankow et al. 2003). Upon reaching the small airways and alveoli of the lung nicotine is easily absorbed due to the huge surface of these tissues and dissolution of nicotine in the ph 7.4 pulmonary fluid (Hukkanen et al. 2005). Considerable oral absorption is observed with smokeless tobacco due to its higher ph (Henningfield et al. 1995), and substantial gastrointestinal absorption is also seen because some saliva containing product constituents is swallowed (Benowitz et al. 1989). Smokeless tobacco products range widely in ph and great variation is seen in nicotine bioavailability due to differences in the unionized proportion of nicotine, a property key to its oral absorption. A study of six distinct smokeless tobacco products, with ph , found a 17-fold difference in the amount of free nicotine in solution, ranging from 0.53 mg/g to 9.03 mg/g (Henningfield et al. 1995). Additional work has determined that the percentage of unionized nicotine in 17 smokeless tobacco products ranges from % (Djordjevic et al. 1995b). Compared to other smokeless tobacco products iqmik has an exceptionally high ph, 12

24 as the basic punk fungus ash raises the measured level to Thus, almost 100% of nicotine in iqmik is found in the unionized form (Renner et al. 2005). As a result, enhanced oral nicotine absorption is observed in iqmik users; serum nicotine concentrations of Alaska Native iqmik users exceed those who use other forms of tobacco (Hurt et al. 2005). Plasma levels of nicotine decay quickly following use due to extensive distribution throughout the body (distribution half-life of minutes), with particular affinity for the liver, kidney, brain, lung, and spleen and lowest for adipose tissue (Urakawa et al. 1994). Steady state plasma nicotine concentrations sampled from regular daily smokers in the afternoon typically fall between ng/ml (Benowitz et al. 1990), and similar nicotine levels are seen in smokeless tobacco users who use at regular intervals throughout the day (Benowitz et al. 1989). The plasma elimination half-life of nicotine is approximately 1-2 hours (Benowitz et al. 1982; Jarvis 2004), and the terminal half-life based on urinary metabolites reaches approximately 11 hours due to slow nicotine release from high-affinity body tissues (Jacob et al. 1999) Metabolism and Elimination Nicotine undergoes rapid and extensive metabolism by the liver. Nicotine is a high extraction drug, and if given orally (as in smokeless tobacco use) a significant first-pass effect is observed with bioavailability reduced to 20 to 45% of total dose (Benowitz et al. 1991). In humans, approximately 80% of nicotine is metabolically inactivated to cotinine (COT via C-oxidation) with the hepatic enzyme cytochrome P450 2A6 (CYP2A6) mediating 90% of this conversion (Benowitz and Jacob 1994; Messina et al. 1997; Nakajima et al. 1996b). The major pathway of cotinine 13

25 metabolism, oxidation to trans-3!-hydroxycotinine (3HC), is mediated exclusively by CYP2A6 (Dempsey et al. 2004; Nakajima et al. 1996a). Notably, a high degree of variation is observed in nicotine metabolism among individuals and between ethnicities (Benowitz and Jacob 1994; Benowitz et al. 1995; Benowitz et al. 1997; Cholerton et al. 1994; Nakajima and Yokoi 2005). Interindividual differences in pharmacokinetic parameters, such as hepatic blood flow and urine ph, are known to influence nicotine clearance rates (Benowitz et al. 1997; Cholerton et al. 1994; Hukkanen et al. 2005), and sex-related variation in nicotine clearance is also seen (Benowitz et al. 2006a). In vitro estrogen is known to induce CYP2A6 transcription (Higashi et al. 2007), consistent with the finding that females possess significantly faster in vivo nicotine metabolism than males (Benowitz et al. 2006a; Ho et al. 2009b). 1.7 CYP2A The Role of CYP2A6 in Nicotine Metabolism The conversion of nicotine to cotinine to 3HC is quantitatively the most important pathway in human nicotine metabolism. The sum of nicotine, cotinine, 3HC and their glucuronide conjugates in the urine of tobacco users accounts for 73 96% of the excreted dose (Benowitz et al. 1994; Byrd et al. 1992) (Figure 1.1). Cotinine has a much longer plasma half-life than nicotine (14 20 hours) and its concentration remains relatively stable throughout the day in regular smokers (Benowitz and Jacob 1994; Dempsey et al. 2004; Zevin et al. 1997). Plasma cotinine concentration can 14

26 15 Figure 1.1 Major pathways of human nicotine metabolism. Metabolites are encircled and accompanied by the percentages of total nicotine dose excreted in 24 h urine on average. Shaded circles indicate metabolites that compose the urine total nicotine equivalents measure. This figure is adapted and modified from Hukkanen et al. (2005). 15

27 therefore be used as a rudimentary marker of nicotine intake in moderate to heavy smokers (Benowitz and Jacob 1994). The plasma half-life of directly administered 3HC is 5-6 hours, but since its formation from nicotine is dependent on cotinine metabolism it maintains a stable plasma concentration as seen with cotinine (Benowitz and Jacob 2001). Further, the ratio of 3HC to cotinine in the plasma of smokers remains constant throughout daily smoking (Lea et al. 2006). As the formation of 3HC from cotinine is catalyzed entirely by CYP2A6, the plasma 3HC/COT ratio is an effective biomarker for CYP2A6 activity and correlates highly with both cotinine clearance and cotinine half-life among smokers at steady state (Dempsey et al. 2004). The plasma 3HC/COT ratio derived from ad libitum smoking also strongly correlates with the same ratio following oral nicotine administration, and both ratios correlate highly with oral nicotine clearance (Dempsey et al. 2004), indicating hepatic first pass metabolism does not confound measurement. Thus, plasma 3HC/COT may be used as a relevant biomarker of nicotine metabolism by CYP2A6 in regular smokers with nicotine intake at steady state (Dempsey et al. 2004), where a low plasma ratio of 3HC to COT reflects reduced CYP2A6 activity and a high plasma ratio of 3HC to COT reflects enhanced CYP2A6 activity. In addition to measurement in plasma, the 3HC/COT ratio can also be quantified in urine and saliva samples. To truly represent CYP2A6 activity with urine nicotine metabolites the denominator of the ratio must include only free urine cotinine, as opposed to total cotinine level. Total cotinine includes both freely excreted cotinine and the proportion that is deconjugated from glucuronides. Since cotinine-glucuronide is not further metabolized by CYP2A6 to 3HC before excretion the inclusion of this 16

28 metabolite in the ratio would not reflect CYP2A6 activity (Swan et al. 2009). The urinary total 3HC to free COT ratio correlates highly with the same ratio derived from plasma (R = 70) (Swan et al. 2009), as well as with measures of tobacco dependence in groups of adult Caucasian and adolescent mixed-ethnicity smokers (Benowitz et al. 2003; Kandel et al. 2007). Notably, total population variation in the urinary 3HC/COT ratio is much greater than in plasma (Benowitz et al. 2003; Kandel et al. 2007; Swan et al. 2003). In a genetic heritability study of smoking twins, Swan et al. (2009) determined that though variation in plasma and urinary 3HC/COT was affected by the same genetic influences, the urinary ratio exhibited more variability due to a greater impact of non-shared environmental factors. Examples of environmental parameters that affect the urinary 3HC/COT ratio include renal COT and 3HC clearance, which in turn vary due to cotinine reabsorption or urine flow rates (Benowitz et al. 1983; Swan et al. 2009). Some environmental influence on the urinary 3HC/COT ratio can be mitigated by controlling for urine flow rate through measuring renal creatinine excretion (Benowitz et al. 2009a). Creatinine correction has been demonstrated to improve correlation of the plasma and urinary 3HC/COT ratios (Benowitz et al. 2009a). Extensive variation has been observed in interindividual CYP2A6 expression level and activity (Shimada et al. 1996), in vitro cotinine formation (30-fold) (Messina et al. 1997), and the conversion of cotinine to 3HC (Messina et al. 1997; Nakajima et al. 1996a). At the population level, considerable variation in nicotine metabolism and CYP2A6 activity is seen between ethnicities. When compared to Caucasians, Japanese possess reduced CYP2A6 expression and activity (Shimada et al. 1996), African-American populations exhibit lower cotinine clearance (Perez-Stable et al. 17

29 1998), and Chinese-Americans have decreased clearance of both nicotine and cotinine (Benowitz et al. 2002b). This disparity in CYP2A6 activity is at least partially due to differential frequencies of CYP2A6 genetic polymorphisms in these populations (Mwenifumbo and Tyndale 2007; Nakajima et al. 2001; Nakajima et al. 2000) The Influence of CYP2A6 Genetic Variation on Nicotine Metabolism The CYP2A Gene Subfamily The CYP2 gene family (Figure 1.2) is the largest and most complex of the cytochromes P450; it consists of multiple subfamilies, which cumulatively form one of the most important CYP families for xenobiotic metabolism (Hoffman et al. 2001). In humans, the CYP2 family genes are found clustered on chromosome 19, where the CYP2A, CYP2B, and CYP2F subfamilies map to a 350 kb region within 19q12 19q14 (Hoffman et al. 1995). Four genes compose the human CYP2A subfamily: CYP2A6 and CYP2A13 express functional enzymes, while CYP2A7 and CYP2A18 are pseudogenes (Hoffman et al. 2001). The shared exonic sequence identities of CYP2A6 with CYP2A7 and CYP2A13 are 97 and 85%, respectively (Hoffman et al. 2001). CYP2A6 is the primary metabolizing enzyme of nicotine, cotinine, and coumarin, and also contributes to the biotransformation of the antineoplastic tegafur, anticoagulant (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM ), anesthetic methoxyflurane, and tobacco-specific nitrosamines such as 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Daigo et al. 2002; Ikeda et al. 2000; Kharasch et al. 1995; Nunoya et al. 1996; Nunoya et al. 1999b; Pelkonen et al. 2000; Yamazaki et al. 1992). 18

30 19 Figure 1.2 The CYP2ABFGST gene cluster on chromosome 19q13.2. Each arrow represents one gene with the arrow orientation representing the 5! to 3! direction of transcription of the labeled gene; genes within the sub-family share the same shading pattern. Several genes in the CYP2 family are pseudogenes, including: CYP2T2P, CYP2F1P, CYP2A7, CYP2G1P, CYP2B7P, and CYP2G2P. CYP2A18 is also a pseudogene that is divided into two portions (C and N) by an insertion of the CYP2B subfamily. This figure is adapted and modified from Hoffman et al. (2001), and is not drawn to scale. 19

31 CYP2A13 mrna is expressed at considerable levels in respiratory tissues where a high activity toward tobacco-related nitrosamines has been documented (Su et al. 2000). However, hepatic CYP2A13 expression is significantly lower than that of CYP2A6 (Zhang et al. 2007) and, based on quantification of nicotine metabolites in vivo in individuals whose CYP2A6 gene has been deleted, CYP2A13 does not impact systemic nicotine and cotinine levels (Yamanaka et al. 2004). The CYP2A7 pseudogene expresses full-length hepatic mrna transcripts (Ding et al. 1995), but the corresponding enzyme is inactive due to an inability to incorporate the critical cytochrome P450 constituent heme (Yamano et al. 1990). The human CYP2A18 pseudogene is separated by the insertion of the CYP2B gene subfamily, a 112 kb region containing one functional gene (CYP2B6) and one pseudogene (CYP2B7P). The CYP2B6 gene spans approximately 27 kb and is made up of nine exons (Hoffman et al. 2001) Specific CYP2A6 Gene Variants and Their Functional Impact The CYP2A6 gene possesses numerous allelic variants and single nucleotide polymorphisms (SNPs), as categorized on the CYP nomenclature website Several well-documented variants with an established impact on CYP2A6 enzyme function are detailed below, however CYP2A6 gene variants with low frequency (CYP2A6*3, *5, *6, *11, *13, *15), minimal or unclear impact on CYP2A6 enzyme function (CYP2A6*14), low frequency and impact (CYP2A6*16, *18, *19, *21), or an uncharacterized influence on function (CYP2A6*22, *29, *30, *31, *32, *33, *34, *36, *37) are not discussed (Al Koudsi et al. 2006; Fukami et al. 2005; Mwenifumbo et al. 2008a; Nakajima et al. 2006; Schoedel et al. 2004; 20

32 Yoshida et al. 2002). In addition, CYP2A6 polymorphism frequencies vary across ethnicities (Malaiyandi et al. 2005; Nakajima et al. 2006; Schoedel et al. 2004) and variants found in African-Americans but at very low frequencies (CYP2A6*23, *24, *25, *26, *27, *28) are not described. The functional consequences of relevant CYP2A6 variants (CYP2A6*1X2, *1B, *2, *4, *7, *8, *9, *10, *12, *17, *35) and their respective allele frequencies in different ethnic populations are described below and summarized in Table 1.1. CYP2A6*1B is characterized by a 58 base pair (bp) CYP2A7 gene conversion occurring within the 3! untranslated region (UTR) of CYP2A6 (Yamano et al. 1990). This variant is highly prevalent in a number of world populations; its allele frequency in Caucasians ranges from %, in African-Americans from %, in Japanese from %, in Koreans from 57.0%, and in Chinese from % (Mwenifumbo and Tyndale 2007). Elevated luciferase reporter gene activity and mrna stabilization has been reported with CYP2A6*1B in vitro (Wang et al. 2006). As a result, CYP2A6*1B has been associated with greater levels of CYP2A6 mrna and protein expression, as well as higher enzyme activity, in liver microsomes (Wang et al. 2006). However, inconsistencies exist in the literature regarding the in vivo impact of CYP2A6*1B. Some studies have found an association of this variant with increased CYP2A6 activity, including nicotine clearance (Mwenifumbo et al. 2008b), the ratio of plasma cotinine to nicotine (Yoshida et al. 2002), and the plasma 3HC/COT ratio (Ho et al. 2009b). In contrast, a relationship between CYP2A6*1B and increased CYP2A6 activity has not been replicated in all work (Al Koudsi et al. 2009b; Mwenifumbo et al. 2008a; Nakajima et al. 2006; Yoshida et al. 2003). 21

33 22 CYP2A6 Allele Nucleotide Change Protein Change Increased or Unchanged Function *1X2A Gene duplication! expression *1X2B Gene duplication! expression 58 bp *1B CYP2A7 conversion! expression in 3!-UTR *8 6600G>T (exon 9) R485L Reduced Function -48T>G " expression *9 (5!-flanking (" mrna region) expression) CYP2A6/ *12 CYP2A7 hybrid Loss-of-Function *2 *4 1799T>A (exon 3) Gene deletion 10 amino acid substitution - " expression L160H No heme incorporation Allele Frequencies (%) Functional Consequence Japanese Korean Chinese! in vivo nicotine metabolism! in vivo nicotine metabolism! or unchanged in vivo nicotine metabolism (study dependent) Unchanged in vivo nicotine metabolism " in vitro coumarin activity; " in vivo nicotine metabolism " in vitro coumarin activity; " in vivo nicotine metabolism African American Caucasian Inactive protein No translation Inactive

34 23 CYP2A6 Allele *7 *10 *17 *35 Nucleotide Change 6558T>C (exon 9) 6558T>C 6600G>T (exon 9) 5065G>A (exon 7) 6458A>T (exon 9) Protein Change I471T I471T R485L V365M N438Y Allele Frequencies. (%) Functional Consequence Japanese Korean Chinese " in vitro nicotine activity; " in vivo nicotine metabolism " in vivo nicotine metabolism " in vitro nicotine activity; " in vivo nicotine metabolism " in vitro nicotine activity; " in vivo nicotine metabolism African American Caucasian Table 1.1 The functional consequences of relevant CYP2A6 gene variants, and their allele frequencies in various world populations. This table was adapted and modified from Mwenifumbo and Tyndale (2007). References: (Yamano et al. 1990; Hadidi et al. 1997; Oscarson et al. 1998; Nunoya et al. 1999; Oscarson et al. 1999a; Oscarson et al. 1999b; Ariyoshi et al. 2000; Rao et al. 2000; Ariyoshi et al. 2001; Pitarque et al. 2001; Ariyoshi et al. 2002; Benowitz et al. 2002; Oscarson et al. 2002; Xu et al. 2002; Yoshida et al. 2002; Kiyotani et al. 2003; Yoshida et al. 2003; Ariyoshi et al. 2004; Fukami et al. 2004; Pitarque et al. 2004; Schoedel et al. 2004; Fukami et al. 2005; Haberl et al. 2005; Mwenifumbo et al. 2005; Benowitz et al. 2006b; Malaiyandi et al. 2006; Nakajima et al. 2006; Audrain-McGovern et al. 2007; Fukami et al. 2007; Ho et al. 2008; Mwenifumbo et al. 2008; Al Koudsi et al. 2009). 23

35 Seventeen different CYP2A6*1B variants sharing the 3!-UTR CYP2A7 conversion have been identified in the literature (CYP2A6*1B1-17), but are distinguished by the presence of additional SNPs found in CYP2A6 5!-flanking, 3!- flanking, and intronic regions (Haberl et al. 2005; Mwenifumbo et al. 2008a; Mwenifumbo et al. 2008b; Nakajima et al. 2006). In assessments of in vivo activity, all individuals carrying the CYP2A6*1B 3!-UTR CYP2A7 conversion are typically grouped together (Al Koudsi et al. 2009b; Ho et al. 2009b; Mwenifumbo et al. 2008a; Mwenifumbo et al. 2008b). However, the functional impacts of all 17 CYP2A6*1B variants have not yet been characterized, and the frequencies of different subtypes of CYP2A6*1B alleles likely vary between ethnicities (Nakajima et al. 2006). These variables may clarify in part the conflicting results observed regarding the in vivo functional impact of the CYP2A6*1B variant. An alternative explanation may be underestimation of CYP2A6*1B variant subtype frequencies due to SNPs in haplotype with the 3!-UTR CYP2A7 conversion occurring within CYP2A6 primer annealing sites, thus confounding genotyping results (Mwenifumbo et al. 2008b). The CYP2A6*2 allele is due to a 1799T>A SNP (L160H) in which the resulting CYP2A6 enzyme is incapable of incorporating heme, rendering it inactive in vivo (Hadidi et al. 1997; Oscarson et al. 1998). CYP2A6*2 is a low prevalence allele in Caucasians ( % allele frequency) and African-Americans ( %), and is at an extremely low prevalence in Asian populations (Ho et al. 2009b; Lerman et al. 2010; Malaiyandi et al. 2006b; Mwenifumbo et al. 2008a; Rao et al. 2000; Schoedel et al. 2004). 24

36 Seven distinct CYP2A6*4 alleles have been identified to date (CYP2A6*4A/C, B, D, E, F, G, H) (Figure 1.3) and all result in functional deletion of the CYP2A6 gene due to a non-homologous crossover with the CYP2A7 pseudogene (Nunoya et al. 1999a; Oscarson et al. 1999b). In all seven variants of this hybrid allele CYP2A7 sequence is found toward the 5! end of the gene while CYP2A6 sequence is located 3!. CYP2A6*4A/C results from a crossover in the 3!-flanking region of CYP2A6 and is composed of CYP2A7 exons 1 9 and the 3!-UTR of CYP2A6 (Nunoya et al. 1999a; Nunoya et al. 1999b; Oscarson et al. 1999b). The CYP2A6*4B variant results from a CYP2A7 crossover location occurring downstream of both genes and lacks all CYP2A6 exons and 3!-flanking sequence (Ariyoshi et al. 2004; Ariyoshi et al. 2002). CYP2A6*4D results from a CYP2A7 crossover in either intron 8 or exon 9 of CYP2A6, and retains exon 9 and the 3!-UTR of CYP2A6 while exons 1-8 are of CYP2A7 origin (Oscarson et al. 1999a). Following identification of extensive sequence variation in specific individuals in both coding and non-coding sections of CYP2A6 alleles, CYP2A6*4E and *4F were characterized. These variants resemble CYP2A6*4A/C due to a non-homologous crossover with CYP2A7 found in the 3!-flanking region of CYP2A6, differing only in the precise location of the CYP2A7 crossover junction (Mwenifumbo et al. 2008a). Finally, the CYP2A6*4G and *4H alleles were recently identified in a smoking population of Black African descent and bear similarity to CYP2A6*4B in their crossover location downstream of both the CYP2A6 and CYP2A7 genes, although at novel locations (Mwenifumbo et al. 2010). The frequency of the CYP2A6*4 variants is high in Asian populations, ranging from % in Japanese, Koreans, and Chinese, but is much less prevalent in Caucasians and 25

37 26 Figure 1.3 A schematic illustrating the formation of each CYP2A6*4 variant subtype, and the reciprocal CYP2A6*1X2 duplication products. The top of the diagram displays a magnified version of the CYP2A6 and CYP2A7 genes and their relative position to one another, with the numbers identifying each exon. Below is a representation of the different homologous recombinations that occur with the approximate crossover region for each CYP2A6*4 variant denoted by an X. The crossover junctions for CYP2A6*4A/C, *4E, *4F, *4G, *4H are located in the 3!-UTR of CYP2A6, the crossover junction for CYP2A6*4B is located in the downstream region of CYP2A6, the crossover junction for CYP2A6*4D is located in intron 8/exon 9. This figure is modified and adapted from Hoffman et al. (2001) and Mwenifumbo et al. (2010). 26

38 African-Americans ( % and % allele frequencies, respectively)(ho et al. 2009b; Malaiyandi et al. 2006b; Mwenifumbo et al. 2008a; Mwenifumbo et al. 2010; Rao et al. 2000; Schoedel et al. 2004). The reciprocal products of the CYP2A6*4 deletion are the CYP2A6 duplication alleles CYP2A6*1X2A and *1X2B (Fukami et al. 2007; Rao et al. 2000; Yoshida et al. 2002). The same non-homologous crossover mechanism that results in the CYP2A6*4 gene deletion also produces an alternative allele that possesses two distinct copies of the CYP2A6 gene (and one copy of CYP2A7) (Figure 1.3). CYP2A6*1X2A and *1X2B result in greater in vivo nicotine metabolism and have been collectively found at low frequencies in Caucasians (0 0.7%), African-Americans (1.7%), and Asian populations (0 1.5%) (Fukami et al. 2007; Lerman et al. 2010; Mwenifumbo and Tyndale 2007; Rao et al. 2000; Yoshida et al. 2002). CYP2A6*7 is characterized by a T>C SNP at position 6558 (I471T) that severely decreases in vitro metabolism of nicotine, and in vivo reduces nicotine metabolism (Ariyoshi et al. 2001; Xu et al. 2002; Yoshida et al. 2002). CYP2A6*8 contains a non-synonymous SNP within 50 bp of CYP2A6*7 6600G>T (R485L) but does not have a detectable effect on nicotine biotransformation in vivo (Ariyoshi et al. 2001; Xu et al. 2002). However, CYP2A6*7 and *8 can occur in haplotype on the same chromosome to form CYP2A6*10, an allele that has been determined to be essentially inactive for nicotine metabolism in vivo (Mwenifumbo et al. 2005; Xu et al. 2002; Yoshida et al. 2002). Notably, CYP2A6*7 and *10 occur as unique Asian variants, and are found at a considerable frequency in Japanese (12.5% and 3.2%, respectively), Koreans (9.4% and 4.1%), and Chinese ( % and %) but 27

39 are extremely rare in African-Americans and Caucasians (Mwenifumbo et al. 2005). The CYP2A6*8 variant is essentially absent in all ethnicities (Mwenifumbo et al. 2005). The CYP2A6*9 allele is caused by a -48T>G SNP in the TATA box of the CYP2A6 5!-flanking region (Pitarque et al. 2001). In vitro results for CYP2A6*9 impact demonstrate lower CYP2A6 mrna and protein expression due to decreased transcription activity in human livers (Kiyotani et al. 2003). Functional consequences of CYP2A6*9 include reduced in vitro activity toward coumarin (Haberl et al. 2005; Kiyotani et al. 2003; Yoshida et al. 2003), as well as lower nicotine metabolism in vivo (Benowitz et al. 2006b; Yoshida et al. 2003). A high CYP2A6*9 prevalence is observed in Caucasians ( %), African-Americans ( %), and especially Asian groups ( %) (Ho et al. 2009b; Lerman et al. 2010; Malaiyandi et al. 2006a; Mwenifumbo et al. 2008a; Pitarque et al. 2001; Schoedel et al. 2004; Yoshida et al. 2003). CYP2A6*12 is a hybrid allele of CYP2A6 and CYP2A7, in which gene exons 1 2 are of CYP2A7 origin and exons 3 9 come from CYP2A6 (Oscarson et al. 2002). The unequal crossover event in intron 2 of CYP2A6 leading to this variant results in a series of 10 amino acid substitutions (Oscarson et al. 2002). Lower in vitro coumarin metabolism and reduced biotransformation of both coumarin and nicotine in vivo are seen with CYP2A6*12 (Benowitz et al. 2006b; Oscarson et al. 2002). Appreciable allele frequencies of CYP2A6*12 are seen in Caucasians ( %), while this variant is nearly absent in African-Americans (0 0.4%) and Asians (0 0.8%) (Ho et al. 2009b; Malaiyandi et al. 2006b; Mwenifumbo et al. 2008a; Oscarson et al. 2002; Schoedel et al. 2004). 28

40 CYP2A6 alleles *17 and *35 are loss of function variants with a substantial prevalence in African-American populations and are nearly undetectable in other ethnicities (Al Koudsi et al. 2009a; Fukami et al. 2004). The CYP2A6*17 allele is characterized by a G>A SNP at position 5065 (V365M) resulting in decreased in vitro CYP2A6 activity toward coumarin and nicotine and also reduced nicotine metabolism in vivo (Fukami et al. 2004; Ho et al. 2008). The frequency of this variant is % in African-Americans but is essentially absent in Caucasian, Japanese, and Korean populations (Fukami et al. 2004; Ho et al. 2009b; Mwenifumbo et al. 2008a). CYP2A6*35 contains a non-synonymous 6458A>T SNP (N438Y) leading to reduced nicotine metabolism observed in vitro and in vivo (Al Koudsi et al. 2009a). The African- American allele frequency of CYP2A6*35 is %, and it is found at negligible levels in Asians ( %) and not yet in Caucasians (Al Koudsi et al. 2009a; Ho et al. 2009b) CYP2A6 Genotype Grouping Strategy Individuals can be grouped according to their total CYP2A6 genotype (Benowitz et al. 2006b), similar to categorization performed for other cytochromes P450 such as CYP2D6 (Kirchheiner et al. 2011). Three CYP2A6 genotype groupings are used, slow, intermediate, and normal metabolizers (SMs, IMs, NMs), that differ significantly by CYP2A6 activity measured by fractional clearance of nicotine to cotinine or plasma 3HC/COT ratio phenotypes (Benowitz et al. 2006b; Dempsey et al. 2004; Ho et al. 2009b). The normal metabolizer group consists of individuals without the presence of any detected reduced or loss-of-function alleles (i.e. a wild-type CYP2A6*1/*1 genotype), and whose hypothesized CYP2A6 activity is at the normal population level. 29

41 Normal metabolizers possess greater CYP2A6 activity and a higher 3HC/COT ratio compared to intermediate and slow metabolizers, a relationship observed consistently in several studied populations and ethnicities (Benowitz et al. 2006b; Ho et al. 2009b; Lerman et al. 2010; Malaiyandi et al. 2006b). The intermediate metabolizer group refers to individuals possessing one copy of a known CYP2A6 reduced function allele, such as CYP2A6*9 or *12, whose hypothesized CYP2A6 activity would be approximately 75% of wild-type levels (Benowitz et al. 2006b; Ho et al. 2009b; Oscarson et al. 2002). The slow metabolizer group contains individuals hypothesized to have! 50% of wild-type CYP2A6 activity due to the presence of one or more copies of known CYP2A6 loss-of-function alleles (CYP2A6*2, *4, *7, *9, *10, *17, *35), two copies of known reduced function CYP2A6 alleles, or a combination of reduced and loss-of-function alleles (Benowitz et al. 2006b; Ho et al. 2009b; Lerman et al. 2010; Malaiyandi et al. 2006b). Intermediate and slow metabolizer groups may also be collapsed into the composite reduced metabolizer (RM) group, containing individuals with any decreased function CYP2A6 variant (Lerman et al. 2010) Non-Genetic Variables that Impact CYP2A6 Function Environmental factors also contribute substantially to interindividual differences in CYP2A6 function. CYP2A6 activity is inducible, and can be increased through exposure to a range of pharmaceutical inducers, including oral contraceptives (Benowitz et al. 2006a), rifampin (Rae et al. 2001), dexamethasone (Onica et al. 2008), and phenobarbital (Itoh et al. 2006). CYP2A6 can likewise be inhibited in vivo by compounds such as methoxsalen and tryptamine (Kharasch et al. 2000; Zhang et al. 2001). Lower nicotine clearance rates are also seen in smokers as compared to 30

42 non-smokers (Benowitz and Jacob 1993), and several compounds within tobacco smoke have been assessed as potential causal agents. In vivo nicotine clearance in smokers was unaffected by co-administration of intravenous cotinine (Zevin et al. 1997), or exposure to inhaled carbon monoxide levels similar to those seen in smokers (Benowitz and Jacob 2000). However, CYP2A6 can be effectively inhibited in vitro by the minor tobacco alkaloid "-nicotyrine (Denton et al. 2004) and CYP2A6 down-regulation in vivo was observed following 21 days of nicotine administration to monkeys due to reduced CYP2A6 mrna expression and liver protein (Schoedel et al. 2003). Though nicotine reduced CYP2A6 protein levels in monkeys, when transdermal nicotine was given to healthy, abstinent smokers no effect was observed on nicotine or cotinine clearance (Hukkanen et al. 2010). The compound and mechanism responsible for reduced nicotine clearance rate in smokers thus remains unidentified. 1.8 CYP2B The Role of CYP2B6 in Nicotine Metabolism CYP2A6 is the main enzyme responsible for nicotine C-oxidation to cotinine, performing 80-90% of this reaction, but a minor contribution (10-20%) is also made by additional enzymes (Messina et al. 1997; Dicke et al. 2005). cdna expressed CYP2B6 is capable of nicotine C-oxidation, although with a 10-fold lower affinity (10- fold higher Km) than cdna expressed CYP2A6 (Dicke et al. 2005; Yamazaki et al. 1999). Addition of anti-cyp2b6 antibody to liver microsomal activity studies resulted in immunochemical inhibition of cotinine formation ranging from 0 to 35% (Dicke et al. 2005), similar to the impact noted by a specific CYP2B6 chemical inhibitor (mean 20%) and anti-cyp2b6 antibody (0 25%) published in an earlier study (Messina et 31

43 al. 1997). However, a more recent in vitro study of the CYP2B6 contribution to nicotine C-oxidation indicated a minor role (Al Koudsi and Tyndale 2010). Specifically CYP2B6 expression significantly correlated with CYP2A6 protein and nicotine C-oxidation in human livers, but correlation of CYP2B6 with cotinine formation was abrogated when controlling for CYP2A6 expression (Al Koudsi and Tyndale 2010). Thus, CYP2B6 likely has a minor role with respect to in vivo nicotine metabolism. Still, low plasma cotinine has been detected among smokers that lack CYP2A6 enzyme expression and therefore when CYP2A6 activity is decreased considerably CYP2B6 may make a significant contribution to cotinine formation (Yamanaka et al. 2004). One explanation for the lack of consistency in reports of CYP2B6 contribution to nicotine metabolism may be co-regulation of CYP2A6 and CYP2B6 expression. Correlation of CYP2A6 and CYP2B6 protein levels has been noted (Al Koudsi and Tyndale 2010; Forrester et al. 1992), and a similar relationship has been demonstrated in mrna expression (Miles et al. 1989). Thus, the perceived impact of CYP2B6 on nicotine pharmacokinetics may be due to an association of CYP2B6 expression with the underlying contribution by CYP2A6. The variation in nicotine metabolism seen by CYP2B6 may be an artificial relationship predicated on similar CYP2B6 and CYP2A6 levels in human livers The Influence of CYP2B6 Genetic Variation on Nicotine Metabolism Like CYP2A6, the CYP2B6 gene contains numerous single nucleotide polymorphisms similarly categorized on the CYP nomenclature website As CYP2B6 contributes to the metabolism of greater than fifty compounds, the impact of genetic variation is of considerable import. 32

44 Substrates include the procarcinogen aflatoxin B1 (Aoyama et al. 1990), anticancer drug cyclophosphamide (Roy et al. 1999), drug of abuse methylenedioxymethamphetamine (MDMA ecstasy ) (Kreth et al. 2000), antiretroviral efavirenz (Ward et al. 2003), and antidepressant and smoking cessation drug bupropion (Faucette et al. 2000). Three major CYP2B6 variant alleles have been studied with respect to their influence on human nicotine metabolism rate. The SNP 785A>G (K262R) occurs alone as CYP2B6*4 or in multiple haplotypes to form distinct CYP2B6 alleles (CYP2B6*7, *13, *16, *19, *20, *26). However, the 785A>G SNP is highly prevalent in linkage disequilibrium with the 516G>T (Q172H) SNP (CYP2B6*9 when occurring alone) combining as CYP2B6*6, and resulting in a low frequency of 785A>G (CYP2B6*4) on its own (Klein et al. 2005; Zanger et al. 2007). In human livers CYP2B6*4 has been associated with higher CYP2B6 protein expression (Lang et al. 2001) and a reduced efavirenz area-under-the-concentration-time-curve (AUC) (Rotger et al. 2007). In vivo, this allele associated with elevated bupropion clearance (Kirchheiner et al. 2003), Taken together the in vivo evidence suggests CYP2B6*4 may serve as a gain of function allele. Notably, the CYP2B6*4 allele has been associated with an elevated plasma 3HC/COT ratio in moderate to heavy smokers (Johnstone et al. 2006). This finding was unexpected, as the conversion of cotinine to 3HC in humans is mediated exclusively by CYP2A6 (Dempsey et al. 2004; Nakajima et al. 1996a). Linkage disequilibrium was assessed between CYP2A6 and CYP2B6 in this study, but significant associations were not found for the variant impacting plasma 3HC/COT within the study, CYP2B6*4 (Johnstone et al. 2006). 33

45 The CYP2B6*6 combination haplotype allele is associated with lower CYP2B6 protein expression, lower efavirenz hydroxylation, and reduced bupropion hydroxylation in human livers (Desta et al. 2007; Hesse et al. 2004). Reduced CYP2B6 expression likely results from an aberrant mrna splicing mechanism removing exons 4-6 during transcription (Hofmann et al. 2008). Conflicting reports exist as to the influence of CYP2B6*6 on nicotine metabolism. A study by Ring et al. (2007) has reported a faster nicotine and cotinine clearance rate in CYP2B6*6 individuals, with greater strength of association in those bearing CYP2A6 reduced function alleles. Yet, Lee et al. (2007) observed no significant difference in nicotine plasma level by CYP2B6 genotype among transdermal nicotine users, even following population stratification by CYP2A6 genotype. Further, no difference in nicotine C- oxidation by CYP2B6*6 genotype was observed in human liver analyses, even when samples were stratified by CYP2A6 genotype groups (Al Koudsi and Tyndale 2010). Together these findings suggest CYP2B6 does not appreciably contribute to in vivo nicotine metabolism even when the activity of CYP2A6 is significantly decreased. 1.9 The Role of Other Enzymes in Nicotine Metabolism Beyond cotinine, additional nicotine metabolites produced by other xenobioticmetabolizing enzymes are found in the urine of tobacco users. For example nicotine is also N-demethylated to form nornicotine, and N-oxidized to nicotine-1!-n-oxide via hepatic flavin-containing monooxygenase 1 (FMO1) (Hinrichs et al. 2011). Cotinine is also converted to multiple minor metabolites through CYP2A6-mediated (norcotinine), and CYP2A6-independent paths (cotinine-1!-n-oxide, and 5!-hydroxycotinine) (Benowitz et al. 1994; Murphy et al. 1999). 34

46 As mentioned previously, a considerable proportion of plasma nicotine, cotinine, and 3HC undergoes glucuronidation prior to elimination in humans. These phase II metabolic reactions are catalyzed by the family of UDPglucuronosyltransferase (UGT) enzymes and create the more polar products nicotine- N-glucuronide, cotinine-n-glucuronide, and 3HC-O-glucuronide. In the urine of smokers approximately 5, 9, and 17% of total nicotine dose is recovered as these three glucuronide conjugates (Benowitz et al. 1994; Byrd et al. 1992). The glucuronidation activities of nicotine and cotinine are highly correlated in both in vitro and in vivo studies, while the rate of 3HC conjugation does not associate with either nicotine or cotinine glucuronidation (Benowitz et al. 1994; Ghosheh and Hawes 2002a; Ghosheh and Hawes 2002b; Kuehl and Murphy 2003a; Kuehl and Murphy 2003b; Nakajima et al. 2002). Thus, the same UGT enzymes are responsible for catalyzing glucuronidation of nicotine and cotinine whereas 3HC is glucuronidated by distinct UGT isoforms. Traditionally, the glucuronidation of nicotine and cotinine has been attributed to UGT1A4 with UGT1A9 serving a more minor role (Kuehl and Murphy 2003a; Nakajima et al. 2002), while the enzymes thought to be responsible for 3HC O-glucuronidation are UGT2B7 and UGT2B17 (Chen et al. 2010; Yamanaka et al. 2005). However, updated analytical methods have identified UGT2B10 as the main enzyme responsible for nicotine and cotinine N-glucuronidation (Kaivosaari et al. 2007). In vitro, human livers homozygous for the reduced function gene variant UGT2B10*2 have a lower nicotine and cotinine glucuronidation rate (Chen et al. 2007), and corresponding results are seen in vivo, as smokers heterozygous for the UGT2B10*2 allele exhibit 35

47 reduced nicotine and cotinine conjugation (Berg et al. 2010). The proportions of total nicotine dose accounted for by nicotine, cotinine, and 3HC glucuronide conjugates are 3-5%, 12-17%, and 7-9%, respectively (Benowitz et al. 1994) CYP2A6 Impact on Tobacco-Related Behaviors and Biomarkers Tobacco Use and Dependence The pattern of tobacco craving, withdrawal, and repeated use is related to nicotine brain and blood levels (Benowitz and Jacob 1985; Djordjevic et al. 1995a; Jarvik et al. 2000; Kassel et al. 2007; McMorrow and Foxx 1983; Scherer 1999). As CYP2A6 is the primary mediator of nicotine plasma clearance genetic variation in this enzyme, affecting the rate of nicotine metabolism, may result in an altered risk for nicotine dependence and may impact frequency of tobacco use. It has been hypothesized that individuals who exhibit low CYP2A6 activity would smoke with reduced frequency, as nicotine plasma levels would remain above the threshold of withdrawal for a greater length of time compared to those with a normal CYP2A6 metabolism rate. In fact, several studies have reported an association between reduced CYP2A6 activity and lower daily cigarette consumption among moderate to heavy Caucasian and Asian smokers (Derby et al. 2008; Johnstone et al. 2006; Minematsu et al. 2006; Rao et al. 2000; Schoedel et al. 2004), while this type of analysis has not yet been performed in smokeless tobacco users. However, not all studies have detected a relationship between CYP2A6 and cigarette consumption (Ando et al. 2003). Notably one study of tobacco cessation within a population of African-American light smokers, whose mean CPD at baseline closely approximated that observed in Alaska Natives, did not observe an association between CYP2A6 36

48 genotype grouping and cigarette consumption (Ho et al. 2009). Further, a significantly higher plasma nicotine level was observed in SMs within this light smoking population, suggesting titration of nicotine intake was not occurring to compensate for differential metabolism rates (Ho et al. 2009). In addition to CPD, lower levels of the tobacco use biomarker exhaled carbon monoxide (CO) were seen in SMs among moderate to heavy Caucasian smokers (Rao et al. 2000), while no difference was observed between CYP2A6 genotype groupings in exhaled CO among African-American light smokers (Ho et al. 2009). The extent of tobacco dependence by CYP2A6 genotype has also been documented previously in smoking cohorts. Among Caucasians a significantly greater proportion of individuals with a CYP2A6 SM genotype was found in the non-smoking population compared to the group of DSM-IV dependent moderate to heavy smokers (Schoedel et al. 2004). In Japanese moderate to heavy smokers, individuals predicted to have less then 50% of wild-type CYP2A6 activity exhibited significantly lower FTND scores than the high activity genotype group (Kubota et al. 2006). Significantly lower FTND scores were also recently reported by CYP2A6 RMs when compared to NMs in a population of Caucasian moderate to heavy smokers (Wassenaar et al. 2011). Unlike moderate to heavy smokers, in a population of African-American light smokers CYP2A6 genotype grouping had no impact nicotine dependence scores as assessed by both FTND and the Cigarette Dependence Scale (Ho et al. 2009b). 37

49 Biomarkers of Tobacco Use Cigarettes Per Day/Smokeless Tobacco Per Day Individual rates of cigarettes smoked per day, or smokeless tobacco consumed, are often used as a proxy measure for nicotine exposure, level of tobacco dependence, and disease risk. However, the self-report of cigarette or smokeless tobacco consumption as representative of nicotine exposure is confounded by several limitations: smoking topography varies widely between individuals (Scherer 1999), as does nicotine content and bioavailability between types of smokeless tobacco (Djordjevic et al. 1995b; Henningfield et al. 1995). An additional factor is the nonlinear relationship between objective biomarkers such as plasma cotinine and self report measures such as cigarettes per day, whereby a plateau effect is observed at higher consumption levels (greater than CPD) (Joseph et al. 2005). This is likely the result of heavy smokers that are consuming in excess of 20 CPD reducing the intensity with which they smoke each cigarette compared to lighter smokers (Joseph et al. 2005; Malaiyandi et al. 2006a). When comparing self-reported CPD and plasma cotinine among smokers, the highest variability in plasma cotinine level per cigarette is seen in lighter smokers (Malaiyandi et al. 2006; Ho et al. 2009). This suggests there may be great variation in nicotine intake in lighter smoking, in which individuals reporting the same CPD consumption may actually vary widely in nicotine intake. It would seem that CPD (and likely daily smokeless tobacco use as well) provides a particularly poor representation of exposure level at the low and high ends of the consumption level spectrum. 38

50 Plasma Cotinine Following nicotine exposure cotinine is detectable in several biological fluids. Its long plasma half-life (14 20 hours) and specificity to nicotine exposure (Benowitz et al. 2002a; Benowitz and Jacob 1994) make cotinine a useful biomarker of use, especially given levels are still detectable 3 4 days following any previous exposure (Benowitz et al. 2002a; Hukkanen et al. 2005). A traditional plasma cotinine cutoff value for distinguishing tobacco users from non-users is 14 ng/ml (Benowitz et al. 2002a; Jarvis et al. 1987) as individuals can be exposed to low levels of nicotine through their diet (Davis et al. 1991; Domino et al. 1993) or via second hand smoke (Benowitz et al. 2009b). Importantly, this threshold was highly successful in differentiating smokers from non-smokers even among African-American light smokers who on average consumed 7.2 CPD; only 3.1% of smoking subjects possessed plasma cotinine concentrations below the 14 ng/ml level (Ho et al. 2009a). However, this plasma cotinine level cutoff has been shown to classify a much greater proportion of individuals as smokers than the US national average, and in some cases directly opposes an individual!s self-report of active smoking (Benowitz et al. 2009b). Several previous studies in Caucasian moderate to heavy smokers have observed strong correlations between plasma cotinine and CPD measures (correlation coefficients ranging from 0.3 to 0.8) (Domino and Ni 2002; Mustonen et al. 2005; Perez-Stable et al. 1995; Scherer 2006), illustrating the strength of association between these two nicotine dose biomarkers during high tobacco consumption. The association of plasma cotinine with CPD in a population of African-American light smokers (7.2 mean CPD) was also significant, although a weak correlation was seen 39

51 (R = 0.39) (Ho et al. 2009a). Only approximately 17% of the variation in CPD level was explained by plasma cotinine in regression analysis of this population (Ho et al. 2009a), and together these results suggest that at lighter levels of cigarette consumption additional factors complicate the association between plasma cotinine and CPD. These may include irregular or intermittent smoking at lower consumption levels, differences in smoking topography between individuals, or inconsistencies in reporting of daily cigarette use. Like self-report of daily tobacco use, the use of plasma cotinine as a biomarker for tobacco consumption does have additional limitations. For example, plasma cotinine is negatively correlated with BMI (Ahijevych et al. 2002; Ho et al. 2009a; Perez-Stable et al. 1995), and concentrations can be highly influenced by rates of CYP2A6 enzyme activity (Benowitz et al. 2006b; Ho et al. 2009a). When administered an intravenous infusion of nicotine, individuals (smokers and non-smokers) with reduced or loss-of-function CYP2A6 variants exhibited approximately 35% lower cotinine clearance rates than wild-type participants (Benowitz et al. 2006b). In a study of African-American light smokers, subjects with slow CYP2A6 activity had 20-30% higher plasma cotinine concentrations while reporting no difference in CPD from those with normal CYP2A6 activity (Ho et al. 2009b) Urine Total Nicotine Equivalents Urine measurement of nicotine metabolites is preferable to plasma cotinine in quantifying nicotine dose as 1) urine collection is less invasive than blood sampling, 2) urine metabolite concentrations (ng/ml) are four- to five-fold higher than those in plasma, and unlike plasma cotinine, 3) the total sum urine nicotine metabolites is 40

52 unaffected by variability in CYP2A6 metabolism rate (Benowitz et al. 2009a; Benowitz et al. 2010; Benowitz et al. 2006b). The major metabolites of nicotine found in the urine include nicotine (8 10% of total nicotine dose), nicotine glucuronide (3 5%), cotinine (10 15%), cotinine glucuronide (12 17%), 3HC (33 40%), and 3HC glucuronide (7 9 %), (Hukkanen et al. 2005), and the sum of these six metabolites correlates very highly with nicotine dose (R = 0.96) (Benowitz et al. 2010). By also including nicotine N-oxide (4 7% of total nicotine dose), cotinine N-oxide (2.5%), and nornicotine ( %)(Hukkanen et al. 2005), 98% of the total nicotine dose from ad libitum smoking is accounted for in 24 hour urine (Benowitz et al. 1994). Urinary total nicotine equivalents (TNE) strongly correlate with self-reported CPD (Joseph et al. 2005; Scherer et al. 2007) and plasma cotinine (Benowitz et al. 2009a). Notably, correlation of urinary TNE with nicotine dose is improved by creatinine correction for urine flow rate (Benowitz et al. 2010), and is stronger than the correlation seen between plasma cotinine and nicotine dose (Benowitz et al. 2010) Cancer Risk A number of procarcinogenic tobacco-specific nitrosamines (TSNAs) are present in tobacco products and require metabolic activation by CYPs to form their carcinogenic reactive intermediates (Guo et al. 1992; Hecht 1998; Hecht et al. 1980). Multiple studies have demonstrated that TSNAs NNN and NNK can induce cancers in a variety of species (Hecht 1998). In rodents NNN can induce tumors of the esophageal and nasal tracts, and NNK has been shown to selectively and potently cause pulmonary adenocarcinomas (Furukawa et al. 1994; Hecht 1998; Padma et al. 1989). In vitro, cdna-expressed CYP2A6 and CYP2A13 have been shown to catalyze 41

53 NNN 5!-hydroxylation with similar efficiency (Wong et al. 2005). CYP2A6 and CYP2B6 were found to possess the highest affinity for NNK activation (Dicke et al. 2005; Patten et al. 1996), yet cdna-expressed CYP2A13 possesses a rate of catalytic efficiency 30- to 50-fold higher than CYP2A6 toward NNK, and the presence of CYP2A13 at an appreciable concentration within the pulmonary system indicates a potentially significant role for local NNK bioactivation and targeted carcinogenesis (He et al. 2004; Su et al. 2000; Zhang et al. 2007). In vitro analyses with lung microsomes showed the rate of NNK activation correlates significantly with CYP2A13 expression, and not CYP2A6, within samples expressing high levels of CYP2A13 (Zhang et al. 2007). Despite strong correlative analysis, CYP2A13 was only detected in 12% of analyzed samples whereas CYP2A6 was detected in 90% (Zhang et al. 2007). This would suggest that individuals with an elevated CYP2A13 expression level may possess increased risk for development of tobacco-related lung cancers, but that CYP2A6 may contribute more to this morbidity in the majority of individuals. Due to the role of CYP2A6 in procarcinogenic TSNA activation, CYP2A6 slow metabolizers may be at decreased risk for tobacco-related cancer as a result of decreased carcinogen generation, as well as reduced cigarette consumption and shorter lifetime smoking duration (Schoedel et al. 2004). At present, 13 studies have analyzed the association of differential lung cancer risk with CYP2A6 gene variation. As hypothesized, CYP2A6 variants resulting in reduced activity or loss-of-function are associated with protection against lung cancer development (Rodriguez-Antona et al. 2010; Rossini et al. 2008). Through meta-analysis, deletion of enzyme activity via the CYP2A6*4 allele significantly reduced risk for tobacco-related cancers in Asian 42

54 populations when compared to the CYP2A6 wild-type allele (OR = 0.25, 95% CI = ) (Rodriguez-Antona et al. 2010). The results examining CYP2A6 genetic protection by decreased activity against tobacco-related esophageal, nasopharyngeal, neuroblastomal, oral, colorectal, and stomach cancers remain inconclusive (Rossini et al. 2008), and other studies have found an increased or similar pulmonary cancer risk among CYP2A6 SMs (London et al. 1999; Loriot et al. 2001; Tan et al. 2001; Wang et al. 2003). However, these earlier studies were performed in Caucasian smokers and may have been underpowered due to a low frequency of CYP2A6 RMs (London et al. 1999; Loriot et al. 2001), or they examined Asian smokers for the CYP2A6*4 gene deletion only, and therefore may have classified some individuals with ungenotyped reduced function variants as NMs (Tan et al. 2001; Wang et al. 2003). In a more recent case-control study of Caucasian moderate to heavy smokers with a considerable CYP2A6 RM proportion, CYP2A6 RMs consuming <20 CPD possessed significantly lower lung cancer risk, even when controlling for variation in the duration of lifetime smoking (Wassenaar et al. 2011) Statement of Problem The goal of this study was to characterize CYP2A6 and CYP2B6 genetic variation in a population of Alaska Natives and determine its effect on nicotine metabolism, as the tobacco use prevalence and tobacco-related disease rates of this group are among the highest of any US minority. The primary aims of this study were: 1. To determine the extent of genetic variation in the CYP2A6 and CYP2B6 genes among Alaska Natives and conduct comparisons to previously characterized comparator ethnicities. 43

55 2. To estimate the rate of nicotine metabolism in Alaska Natives and determine its association with CYP2A6 and CYP2B6 genotypes. 3. To determine the influence of genetic variation in nicotine metabolism on biomarkers of nicotine exposure, tobacco use behaviors, and level of carcinogen exposure in Alaska Natives Rationale, Objectives and Hypotheses Primary Aim 1 To determine the level of genetic variation in the CYP2A6 and CYP2B6 genes among Alaska Natives and conduct comparisons to previously characterized comparator ethnicities. Rationale Alaska Natives are known to exhibit the highest prevalence of tobacco use of any ethnic minority within the United States (Carmona et al. 2004; USDHHS 2007), and suffer from disproportionately higher rates of tobacco-related disease (Lanier et al. 2008). Genetic variation in the CYP2A6 gene has been previously shown in other ethnicities to significantly alter rates of nicotine metabolism (Ho et al. 2009b; Nakajima et al. 2006), translating to differences in tobacco use behaviors (Liu et al. 2011; Malaiyandi et al. 2006b; Schoedel et al. 2004). Additionally, genetic variation in the CYP2B6 gene has been associated with differences in nicotine metabolism (Johnstone et al. 2006; Ring et al. 2007). However, there is an absence of published literature describing genetic variation in these relevant nicotine metabolism genes in Alaska Natives, and this variability may influence the rates of tobacco use and tobacco-related disease observed in this population. Additionally, CYP2A6 and CYP2B6 represent two highly polymorphic genes previously unstudied in Alaska 44

56 Natives and will provide further data for comparisons of genetic variation to previously characterized world populations. Objective To quantify levels of genetic variation in the CYP2A6 and CYP2B6 genes among Alaska Natives and directly compare them to levels published in comparator ethnicities, including: Japanese, Koreans, Chinese, African-Americans, and Caucasians. Allele frequencies of CYP2A6 and CYP2A6 gene variants will be used to make these comparisons. Hypotheses 1) Alaska Native CYP2A6 and CYP2B6 variant allele frequencies will resemble those measured in Asian populations, exhibiting no difference from those in Chinese. 2) When compared to Caucasians a higher proportion of Alaska Natives will possess reduced function CYP2A6 variants and a lower proportion will possess reduced function CYP2B6 variants. Reasoning for Hypotheses 1 & 2: Since the theorized ancestors of Alaska Natives have a central Asian origin (Crawford 1998; Schurr 2004), it is expected that their CYP2A6 and CYP2B6 variant allele frequencies will be the same as a modern ethnicity residing in this region (Chinese). Alaska Natives are also expected to exhibit higher total frequencies of genotyped CYP2A6 reduced function variants and lower frequencies of CYP2B6 variants than Caucasians. This is based on anticipated similarity to Asians, who possess the same differences in CYP2A6 and CYP2B6 allele frequencies when compared to Caucasians (Mwenifumbo and Tyndale 2007; Nakajima et al. 2006; Zanger et al. 2007). 45

57 Primary Aim 2 To estimate the rate of nicotine metabolism in Alaska Natives and determine its association with CYP2A6 and CYP2B6 genotypes. Rationale The plasma ratio of 3HC/COT has been shown to associate strongly with CYP2A6 genotype, and characterized CYP2A6 variant alleles are associated with consistent impacts on enzyme activity in multiple studied populations of varying ethnic composition (Benowitz et al. 2006b; Ho et al. 2009b; Lerman et al. 2010; Malaiyandi et al. 2006b; Mwenifumbo et al. 2008a; Schoedel et al. 2004). Further, when participants are stratified by established CYP2A6 genotype groupings (NMs, IMs, SMs) a significant difference in plasma and urinary 3HC/COT was seen (BenowItz et al. 2006b; Ho et al. 2009b; Lerman et al. 2010; Mwenifumbo et al. 2008a). An association of nicotine metabolism with CYP2A6 genotype has not been previously investigated in Alaska Natives, but if found CYP2A6 genetic variation may assist in explaining the unique tobacco use prevalence and cancer risk in this population. As plasma 3HC/COT is known to vary by ethnicity (Benowitz et al. 2002b), the mean Alaska Native ratio will be compared to previously published data from other ethnic groups. The evidence supporting a role for CYP2B6 in nicotine metabolism is less consistent than that for CYP2A6, but in specific studies CYPB26 genotype was associated with nicotine and cotinine plasma clearance (Ring et al. 2007) as well as plasma 3HC/COT (Johnstone et al. 2006). However, CYP2B6 genotype has also been found to have a negligible effect on in vitro nicotine metabolism (Al Koudsi and Tyndale 2010) and nicotine plasma level in a clinical trial of nicotine replacement therapy users (Lee et al. 2007). Investigation of CYP2B6 genetic variation in a novel 46

58 population of Alaska Natives may yield more information regarding any influence of this gene on in vivo nicotine metabolism, and whether a CYP2B6 effect is due to a potential co-regulation with CYP2A6. Objective To determine the relationship between CYP2A6 and CYP2B6 genotype and nicotine metabolism in Alaska Natives by assessing the associations of plasma and urinary 3HC/COT ratios with known CYP2A6 and CYP2B6 functional variants and CYP2A6 genotype groupings. Mean plasma and urinary 3HC/COT ratios will be compared by CYP2A6 and CYP2B6 genotypes within Alaska Natives, and then between Alaska Natives and previously characterized ethnic groups (Caucasians and African-Americans). Hypotheses 3) The Alaska Native plasma and urinary 3HC/COT ratios will be significantly associated with CYP2A6, and not CYP2B6, genotypes. 4) Alaska Natives will possess a lower plasma 3HC/COT ratio than Caucasians, based on a greater frequency of reduced function CYP2A6 alleles. Reasoning for Hypotheses 3 & 4: A far greater amount of in vitro and in vivo evidence demonstrates a critical role for CYP2A6 in nicotine metabolism (Benowitz and Jacob 1994; Dempsey et al. 2004; Messina et al. 1997; Nakajima et al. 1996a; Nakajima et al. 1996b). Similar to previously studied ethnicities, it is expected that CYP2A6 genotype will associate strongly with plasma and urinary 3HC/COT in Alaska Natives. As it has been demonstrated that when controlling for CYP2A6 genotype, CYP2B6 genotype has little impact on in vitro and in vivo nicotine 47

59 metabolism (Al Koudsi and Tyndale 2010; Lee et al. 2007), a similar effect is anticipated in Alaska Natives. It is possible the CYP2B6 genotype effect on plasma nicotine clearance, cotinine clearance, and 3HC/COT found previously (Johnstone et al. 2006; Ring et al. 2007) is due to linkage disequilibrium between the genes due to their close proximity (Hoffman et al. 2001). Evidence supports linkage relationships between these genes (Haberl et al. 2005), though when analyzed specifically in relation to a CYP2B6 genotype effect on nicotine metabolism, causal linkage disequilibrium between CYP2A6 and CYP2B6 variants was not detected (Johnstone et al. 2006). CYP2A6 genetic variation in Alaska Natives is expected to resemble levels seen in Chinese, a population possessing a greater proportion of CYP2A6 reduced metabolizers than Caucasians given their higher frequencies of CYP2A6 variants (Nakajima et al. 2006; Mwenifumbo and Tyndale 2007). Given this hypothesis, the mean plasma 3HC/COT ratio quantified in Alaska Natives is expected to fall below that of Caucasians, since a greater percentage of Alaska Natives are theorized to exhibit decreased CYP2A6 activity. Primary Aim 3 To determine the influence of variation in nicotine metabolism on tobacco use behaviors, biomarkers of nicotine exposure, and the level of carcinogen exposure in Alaska Natives. Rationale Differences in self-reported CPD have been observed between CYP2A6 genotype groups with significantly different mean plasma 3HC/COT ratios in populations of moderate to heavy smoking Caucasian and Asian subjects (Liu et al. 48

60 2011; Malaiyandi et al. 2006b; Schoedel et al. 2004; Wassenaar et al. 2011). In moderate to heavy smokers CYP2A6 genotype has also been shown to influence nicotine dependence (Kubota et al. 2006; Schoedel et al. 2004). However, light smokers exhibited no difference in cigarette consumption or nicotine dependence by CYP2A6 genotype and plasma 3HC/COT (Ho et al. 2009b). Biomarkers of nicotine exposure (in addition to CPD), such as plasma cotinine and exhaled carbon monoxide (CO) are also differentially affected by CYP2A6 genotype and plasma 3HC/COT depending on the level of cigarette consumption (Malaiyandi et al. 2006a; Ho et al. 2009a). An assessment of smoking biomarker utility in African-American light smokers by Ho et al. (2009a) observed no difference in exhaled CO consistent with the non-significant difference in cigarette consumption by self-reported CPD. However, despite no apparent nicotine intake difference by CPD or CO, significantly higher levels of plasma COT were found in those with reduced nicotine metabolism by CYP2A6 genotype grouping and plasma 3HC/COT. CYP2A6 can also metabolize the carcinogenic tobacco-specific nitrosamine NNK, resulting in its activation to a mutagenic intermediate (Dicke et al. 2005; Patten et al. 1996). Perhaps related to a reduction in NNK activation, individuals possessing the reduced function allele CYP2A6*9 and loss-of-function allele CYP2A6*4 are at significantly lower risk for lung cancer in case-control studies (Ariyoshi et al. 2002; Fujieda et al. 2004; Wassenaar et al. 2011). This reduction in risk can be attributed partially to differences in cigarette consumption by CYP2A6 genotype (Wassenaar et al. 2011), but may also reflect differential carcinogen activation in individuals with reduced CYP2A6 activity. 49

61 Objective To determine whether variation in nicotine metabolism, assessed by CYP2A6 genotype or plasma 3HC/COT ratio, affects tobacco use behaviors, biomarkers of nicotine exposure, and carcinogen exposure. Mean daily product consumption, nicotine dependence, plasma cotinine, urine total nicotine equivalents, and urine NNK metabolite concentrations will be compared between CYP2A6 genotype groupings and by plasma 3HC/COT median split. Hypotheses 5) No significant differences in daily product consumption (whether cigarette, commercial chew, or iqmik), and nicotine dependence will be found by CYP2A6 genotype grouping or plasma 3HC/COT median split in Alaska Natives. 6) Plasma cotinine and urine total nicotine equivalents will be elevated when comparing those with slower nicotine metabolism to those with a faster rate, using either CYP2A6 genotype or plasma 3HC/COT in Alaska Natives. Reasoning for Hypotheses 5 & 6: Alaska Natives represent a light smoking population (fewer than 10 CPD) (Smith et al. 2010; USDHHS 2003) and measures of tobacco use behaviors and nicotine biomarkers are expected to resemble a previously studied African-American light smoking population (Ho et al. 2009a; Ho et al. 2009b). As outlined above, no association of CYP2A6 genotype or plasma 3HC/COT was seen with CPD or nicotine dependence in this light-smoking group and the same effect is anticipated in Alaska Natives. Increased plasma COT was also observed in African-American light smokers when comparing CYP2A6 SMs to NMs and those with highest plasma 3HC/COT to those with the lowest (Ho et al. 50

62 2009a). A similar association of nicotine metabolism rate with plasma COT is expected in Alaska Natives. Like plasma COT, urine TNE is a measure of nicotine dose in tobacco users (Benowitz et al. 2010). It is anticipated the association of this nicotine dose biomarker with CYP2A6 genotype and plasma 3HC/COT will resemble the increased plasma cotinine level of individuals with reduced CYP2A6 activity in African-American light smokers (Ho et al. 2009a. 7) NNK activation will be reduced in Alaska Native subjects with a lower rate of nicotine metabolism compared to those with a higher rate when assessed by CYP2A6 genotype and plasma 3HC/COT. Reasoning for Hypothesis 7: CYP2A6 is capable of activating the tobacco carcinogen NNK to a genotoxic intermediate product. However, carbonyl reductases also compete for NNK to convert it to the metabolite 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) for glucuronidation and excretion (Ter-Minassian et al. 2011). NNAL is a highly specific urinary biomarker of NNK, and can be measured as an estimate of carcinogen exposure (Goniewicz et al. 2009; Ter-Minassian et al. 2011). As Alaska Natives are light smokers, the population is expected to show no difference in tobacco consumption (Ho et al. 2009a), and as a result NNK intake, by CYP2A6 genotype and plasma 3HC/COT. It is therefore hypothesized that Alaska Native individuals with reduced CYP2A6 activity will exhibit greater conversion of NNK to NNAL due to a lower proportion of NNK being activated by CYP2A6 (Ter-Minassian et al. 2011). In other words, a greater proportion of NNK dose in subjects with higher CYP2A6 activity will be bioactivated, leaving a lower amount for conversion to NNAL. 51

63 2. METHODS 2.1 Study Overview The current study was part of a cross-sectional review of Alaska Native tobacco users at the Bristol Bay Area Health Corporation (BBAHC) led by Caroline Renner (M.P.H.) and Dr. Anne Lanier. The study was supported by NIDA/NCI NARCH III (Indian Health Service Grant) U26IHS30001/01, and was approved by the Health Sciences Research Ethics Board of the University of Toronto - # Caroline Renner and Dr. Anne Lanier conducted participant recruitment, gathering of subject data, and sample collection and preparation. Blood samples were sent to the lab of Dr. Rachel Tyndale for DNA extraction and genotyping of CYP2A6 and CYP2B6. Plasma NIC metabolites and urinary carcinogen levels were measured by Dr. Neal Benowitz at the University of California San Francisco. Urine NIC metabolites were quantified at the Center for Disease Control and Prevention by Dr. Cliff Watson. 2.2 Study Design Subject Recruitment and Screening Subjects were recruited via advertisements at the BBAHC Kanakanak Hospital in Dillingham, Alaska and also local village visits in the Bristol Bay area; enrolment took place from Jan June 31, Male and female current users of tobacco (cigarettes, commercial chew tobacco, iqmik, mixed products), former users, and never users aged were eligible for the study. Exclusion criteria included female subjects who were pregnant, planning pregnancy, or lactating; individuals currently using medications that affect NIC metabolism, (ex. rifampin, dexamethasone, coumarin, benzodiazepines); current involvement in a tobacco cessation program or 52

64 use of nicotine replacement therapy; use of marijuana in the seven days previous to the study day; alcohol consumption on the study day; and use of inhalants, cocaine, heroin, methamphetamine, amphetamines, or other illicit drugs in the month prior to the study day. Non-users must not have used any tobacco for the previous 12 months. At the closing of recruitment 400 Alaska Native individuals were enrolled in the study with the following sample baseline characteristics: gender 220 female, 180 male; tribal ethnicity 19 Aleut, 11 Athabascan, 1 Cupik, 4 Inupiaq, 1 Tlingit, 361 Yupik, 3 unknown; tobacco product use 164 smokers, 77 commercial chew users, 20 iqmik users, 29 mixed product users, 82 former tobacco users, 28 non-users; number of Alaska Native grandparents 15 with 2/4, 50 with 3/4, 335 with 4/ Assessments Eligibility Screening and Structured Interview Interested participants were required to complete a screening questionnaire to determine eligibility for the study and then provide informed consent to enroll. Selfreported demographic measures (age, sex, BMI, ethnicity of parents and grandparents, marital status, level of education), health status, tobacco use history (age of initiation, duration of tobacco use, number and length of previous quit attempts), an estimate of second-hand smoke exposure, dietary practices, and beliefs and attitudes toward tobacco products were recorded via a structured interview conducted by a trained research assistant. Current users of tobacco reported their frequency of use, portion size (for smokeless tobacco), and time of last cigarette or chew. Cigarette smokers were also administered the six-item FTND questionnaire to assess their level of NIC dependence (Heatherton et al. 1991). Iqmik users were 53

65 asked to provide further information on their procedure for making the product, including proportional amounts of tobacco and ash, any side effects of use, and whether the iqmik is premasticated prior to consumption Specimen Collection and Analysis Urine and blood samples for all participants (n = 400) were collected by Kanakanak Hospital Laboratory staff working with a trained research assistant. All samples were aliquoted by BBAHC staff and stored at -80 C prior to shipment. Participants using commercial chew tobacco or iqmik also provided a tobacco product sample of similar size to chews typically consumed for analysis. CYP2B6 genotyping was performed by Andy Zhu in the laboratory of Dr. Rachel Tyndale for purposes of measuring its impact as a covariate of nicotine and nitrosamine metabolism. Dr. Neal Benowitz at the University of California San Francisco measured concentrations of COT and 3HC in plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Dempsey et al. 2004). Dr. Benowitz also employed LC-MS/MS to quantify the concentrations of established tobacco carcinogens, including NNAL, polycyclic aromatic hydrocarbons, benzene, acrolein, and butadiene, in the urine samples of participants (Jacob et al. 2008; Jacob et al. 2007). Urinary NIC, COT, and 3HC concentrations, in addition to minor NIC metabolites nornicotine, nicotine-n-oxide, and cotinine-n-oxide were measured by Dr. Cliff Watson at the Center for Disease Control and Prevention (Xia et al. 2001). Measurements were obtained by high performance liquid chromatography (HPLC), including incubation with "-glucuronidase to cleave the respective proportion of NIC, COT, and 3HC conjugated to glucuronic acid. The combination of free plus 54

66 glucuronidated metabolite measure equaled its total concentration; total and free NIC, COT, and 3HC were measured directly and glucuronidated metabolite concentrations were determined by subtracting the free amount from total amount. The 9-item measure of urinary TNE constituted the sum of free nicotine, free cotinine, free 3HC, glucuronidated nicotine, glucuronidated cotinine, glucuronidated 3HC, nornicotine, nicotine-n-oxide, and cotinine-n-oxide. 2.3 CYP2A6 Genotyping DNA Extraction Blood samples (400) were delivered to the lab of Dr. Tyndale and stored at - 20 C. Genomic DNA was extracted from plasma samples through use of the G1N70 GenElute miniprep kit purchased from Sigma-Aldrich (St. Louis, MO). Included reagents were elution solution (containing 10 mm TRIS-HCl, 0.5 mm EDTA, ph 9.0), column preparation solution, lysis solution, wash solution, and proteinase K (20 mg/ml). 20 µl proteinase K and 200 µl lysis solution were added to 200 µl of each whole blood sample and the mixtures incubated at 70 C for 10 minutes. Following, 200 µl of 95% ethanol was added to the mixtures and the lysates were then applied to GenElute binding columns included in the kit. Following three sequential purification steps of centrifugation (8000 rpm 1 minute, 8000 rpm 1 minute, rpm 3 minutes) and placement in new 2 ml collection tubes, lysate columns were placed in a final 2 ml collection vial and 50 µl of elution solution (warmed to 70 C) was added to lysates. After a final centrifugation performed at 8000 rpm for one minute, eluted DNA was diluted to 50 ng/ml and stored at - 25 C. 55

67 2.3.2 Genotyping Overview Following isolation of participant DNA, genotyping was successfully completed for all 400 subjects. Assays were utilized to detect CYP2A6 variants through two-step allele-specific polymerase chain reaction (PCR). The initial step involved amplifying the CYP2A6 region containing the variant of interest and was gene-specific. The second step used the template resulting from the first amplification to determine if the specific variant was present. Two parallel allele-specific reactions were performed, one reaction testing for the wild-type allele and the other for the variant. For each assay water was used as a negative control and positive controls were selected from samples in previous data sets whose CYP2A6 genotype were known: a homozygote wild-type individual, an individual heterozygous for the genotype, and a homozygote variant. Allele-specific results from the second PCR amplification were then electrophoresed through ethidium bromide-stained agarose gel prior to visualization under UV light Assays, Primer Sets, and Reaction Conditions All DNA samples were analyzed for CYP2A6*1X2A, *1X2B, *1B, *2, *4, *7, *8, *9, *10, *12, *17, and *35. These CYP2A6 variants have differing prevalences among previously studied ethnic groups and many have been used to characterize CYP2A6 among specific ethnicities (Ho et al. 2009b; Mwenifumbo et al. 2005; Schoedel et al. 2004): Caucasians by CYP2A6*2, *4, *12, African-Americans by CYP2A6*17, *35, and Asian populations by CYP2A6*7, *8, *10. Additionally, the functional impact of each of the listed alleles has been established in prior work. CYP2A6 primers were 56

68 ordered from ACGT Corporation in Toronto, ON and the specific sets used for each genotyping assay are listed in Table 2.1. All PCR reaction reagents were purchased from Fermentas (Burlington, ON) including Taq polymerase enzyme, 10X Taq PCR KCl buffer (containing 100 mm TRIS-HCl (ph 8.8 at 25 C), 500 mm KCl, and 0.8% Nonidet P40), 10X Taq (NH 4 ) 2 S0 4 buffer, and 25 mm MgCl 2. For the CYP2A6*1X2B assay, requiring first step amplification of the entire CYP2A6 gene, a different kit purchased from Fermentas was used; the long PCR kit contained Long Taq polymerase enzyme mix, 10X long PCR buffer with 15 mm MgCl 2, and DMSO. Fermentas also supplied the dntp set (25 mm of each nucleotide) and 1-kb Gene Ruler DNA ladder. The first and second amplification reaction conditions for each CYP2A6 genetic variant are listed in Table 2.2, and PCR conditions in Table 2.3. In all PCR primary amplifications 50 ng of genomic DNA was used, and 1.0 µl of the first amplification then served as a template for the second amplification. A PTC-200 Peltier Thermal Cycler (BioRad, Toronto, ON) was used to perform PCR amplifications. The listed steps for all PCR reactions were: 1) initial denaturation; 2) denaturation; 3) annealing; 4) extension; 5) an allele-specific number of cycled repeats of steps 2-4 (denaturation, annealing, extension); and 6) final extension (first amplification only) Gel Electrophoresis and Visualization Following its second amplification, the resulting total PCR product volume (25 µl) was mixed with either 2.5 µl of 0.25% Bromophenol Blue or 0.25% Xylene Cyanol FF loading dye (both containing 30% glycerol) and 20 µl of the mixture was loaded onto an allele-specific percentage agarose gel (Table 2.4). Selection of loading dye 57

69 CYP2A6 Allele & PCR Amp. Primer Name Primer Sequence (5! 3!) Gene Loc. *1X2A-1 *1X2A-2 *1X2B-1 *1X2B-2 *1B-1 *1B-2 *2-1 *2-2 *4-1 *4-2 *7-1 *7-2 *8-1 *8-2 *9-1 *9-2 2Aex7F GGC CAA GAT GCC CTA CAT G Exon 7 2A7R11 GTG CAG AGG TTT TTG TGT GAC TG 3!-UTR 2A7In7F1 CCC CAT TAG AAG CTT TCT ACT CA Intron 7 2A6In7F1 ACC CAC ATT AGA AGC TTT CTA GA Intron 7 2A7R12 TTC GTC TTC CAA AGT AGC TGT GC 3!-UTR 2A6F3 TAG ACA GAT TCT TAA AAA GCA CCT 3!-UTR 2A6/7R CTG GAT TCT TGG GCA TTC AAC CCA 3!-UTR 2A6F0 TGA GTA CAA AAC TTC TAG AAG ATA AT 3!-UTR 2A6Rdup AAT TCC TGG ATT GAC AAG AG 3!-UTR 2A7Rdup AAT TCC TGG ATT GAC GAG AC 3!-UTR 2A6In6F1 ATT TCC TGC TCT GAG ACC Intron 6 2A6R6 TAA TTG GGT TGT TTT CTA TTG AGT 3!-UTR 2A6*1Bwt ACT GGG GGC AGG ATG GC 3!-UTR 2A6*1Bmut AAT GGG GGG AAG ATG CG 3!-UTR 2A6R0 AGG TCA TCT AGA TTT TCT CCT ACA 3!-UTR 2A61F GCT GAA CAC AGA GCA GAT GTA CA Exon 1 2A61R GGA GGT TGA CGT GAA CTG GAA GA Exon 4 2A62wtF CTC ATC GAC GCC CT Exon 3 2A62v1F CTC ATC GAC GCC CA Exon 3 E3R-1 AAC GCA CGC GGG TTC CTC GT Intron 3 2Aex7F GGC CAA GAT GCC CTA CAT G Exon 7 2A6R11 CAT CAA GCC CTG CCG TAT 3!-UTR 2A6In7F1 ACC CAC ATT AGA AGC TTT CTA GA Intron 7 2A6In7F1 ACC CAC ATT AGA AGC TTT CTA GA Intron 7 2A6R12 ATT GTC TTT CAA AGT AGC TGT GT 3!-UTR 2A6In6F1 ATT TCC TGC TCT GAG ACC Intron 6 2A6R6 TAA TTG GGT TGT TTT CTA TTG AGT 3!-UTR 2A6*7FWT-M TCC CAG TCA CCT AAG GAA AT Exon 9 2A6*7FV-M TCC CAG TCA CCT AAG GAA AC Exon 9 2A6R0 AGG TCA TCT AGA TTT TCT CCT ACA 3!-UTR 2A6In6F1 ATT TCC TGC TCT GAG ACC Intron 6 2A6R6 TAA TTG GGT TGT TTT CTA TTG AGT 3!-UTR 2A6*8WTF GCT TTG CCA CGA TCC CAC G Exon 9 2A6*8VF GCT TTG CCA CGA TCC CAC T Exon 9 2A6R0 AGG TCA TCT AGA TTT TCT CCT ACA 3!-UTR 2A65Pr1F ACC TAG ACT TAA TCT TCC CGT ATA C 5!-UTR 2A6In1R CCC AAG ATC CTG TCT TTC TGA T 5!-UTR 2A6-460F ATC CTC CAC AAC AGA AGA CCC CTA A 5!-UTR 2A6-17RA ACG GCT GGG GTG GTT TGC CTT TA 5!-UTR 2A6-17RC ACG GCT GGG GTG GTT TGC CTT TC 5!-UTR 58

70 CYP2A6 Allele & PCR Amp. Primer Name Primer Sequence (5! 3!) Gene Loc. *10-1 *10-2 *12-1 *12-2 *17-1 *17-2 *35-1 *35-2 2A6In6F1 ATT TCC TGC TCT GAG ACC Intron 6 2A6R6 TAA TTG GGT TGT TTT CTA TTG AGT 3!-UTR 2A6*7FWT-M TCC CAG TCA CCT AAG GAA AT Exon 9 2A6*7FV-M TCC CAG TCA CCT AAG GAA AC Exon 9 2A6*8Rwt-L GGA AGC TCA TGG TGT AGT TTC Exon 9 2A6*8Rv-L GGA AGC TCA TGG TGT AGT TTA Exon 9 2AF GCA CCC CTC CTG AGG TAC CAC 5!-UTR 2A6ex3R1 GTC CCC TGC TCA CCG CCA Exon 3 2A61F-L TGG CTG TGT CCC AAG CTA GGC A 5!-UTR 2A71F-L TGG CTG TGT CCC AAG CTA GGT G 5!-UTR 2A6ex3R2 CGC TCC CCG TTG CTG AAT A Exon 3 2A6In6F1 ATT TCC TGC TCT GAG ACC Intron 6 2A6R6 TAA TTG GGT TGT TTT CTA TTG AGT 3!-UTR 2A6*17Fwt-M GAG ATC CAA AGA TTT GGA GCC G Exon 7 2A6*17Fv-M GAG ATC CAA AGA TTT GGA GCC A Exon 7 2A6In7AS CTG AGA TTT CTG TCC CTA T Intron 7 2A6In6F1 ATT TCC TGC TCT GAG ACC Intron 6 2A6R6 TAA TTG GGT TGT TTT CTA TTG AGT 3!-UTR 2A6in8ex9 Intron 8/ TCC TCA GGA AAG CGG A F6458W Exon 9 2A6in8ex9 Intron 8/ TCC TCA GGA AAG CGG T F6458V Exon 9 2A6R0 AGG TCA TCT AGA TTT TCT CCT ACA 3!-UTR Table 2.1 Listing of primers for each CYP2A6 genotyping assay. All assays include a gene-specific initial amplification step (-1) and variant-specific second amplification (-2). The nucleotide sequence and CYP2A6 location for binding of each primer are listed. 59

71 CYP2A6 Allele *1X2A *1B *2 *4 *7 *8 *9 *10 *12 *17 *35 CYP2A6 Allele *1X2B PCR Amp. PCR Buffer (µl) dntps 25mM (µl) Reaction Conditions MgCl 2 (µl) Primer (µl) Taq (units) DNA (NH 4 ) 2 SO ng (NH 4 ) 2 SO µl KCl ng KCl µl KCl ng KCl µl (NH 4 ) 2 SO ng (NH 4 ) 2 SO µl KCl ng KCl µl KCl ng (NH 4 ) 2 SO µl KCl ng (NH 4 ) 2 SO µl KCl ng (NH 4 ) 2 SO µl KCl ng (NH 4 ) 2 SO µl KCl ng (NH 4 ) 2 SO µl KCl ng KCl µl Reaction Conditions PCR dntps PCR Buffer DMSO Primer Taq Amp. 10mM with MgCl 2 (µl) (µl) (units) (µl) DNA ng µl Table 2.2 CYP2A6 genotyping assay reaction conditions for all variants studied. 60

72 61 Initial Denaturation Denaturation Annealing Extension Final Extension CYP2A6 PCR Allele Amp Temp. Time Temp. Time Temp. Time Temp. Time Cycles Temp. Time ( C) (min.) ( C) (min.) ( C) (min.) ( C) (min.) ( C) (min.) *1X2A : : : : : : : : :30 28 *1X2B a : : : : : : : : : : : : : : : :00 *1B : : : : : : : : :00 18 * : : : : : : : : :45 22 * : : : : : : : : :00 23 * : : : : : : : : :00 30 * : : : : : : : : :00 20 * : : : : : : : : : * : : : : : : : : :30 30 * : : : : : : : : :00 20 * : : : : : : : : :00 23 * : : : : : : : : :00 20 Table 2.3 CYP2A6 genotyping assay PCR conditions for all variant assays performed. a The CYP2A6*1X2B assay involves two sequential cycled stages of denaturation, annealing, and extension. 61

73 CYP2A6 Allele Second Amplification Product Size (base pairs) Loading Dye Gel Agarose Composition (%) *1X2A 3151 Bromophenol Blue 1.2 *1X2B 7353 Bromophenol Blue 1.0 *1B 1060 Bromophenol Blue 1.2 *2 97 Xylene Cyanol FF 2.0 * Bromophenol Blue 1.2 * Bromophenol Blue 1.2 * Bromophenol Blue 1.2 *9 408 Xylene Cyanol FF 1.2 *10 82 Xylene Cyanol FF 2.0 * Bromophenol Blue 1.2 * Xylene Cyanol FF 1.2 * Bromophenol Blue 1.2 Table 2.4 CYP2A6 genotyping gel visualization parameters (loading dye and agarose percentage) for all variant assays performed. 62

74 and agarose gel percentage was dependent on second PCR amplification product size. Agarose was supplied by ONBIO Inc. (Richmond Hill, ON) and 10X TAE buffer plus ethidium bromide (10mg/mL solution) from Sigma Aldrich (St. Louis, MO). Gels were prepared with a total volume of 350 ml 1X TAE buffer (0.4M TRIS base, 0.02 M acetic acid, M EDTA) and stained with 20 µl ethidium bromide (210 µg). Upon gel loading, samples were electrophoresed at 96V for 60 minutes and visualized with the AlphaDigiDoc real-time imaging system by Alpha Innotech Fisher Scientific (Ottawa, ON). The presence or absence of a band in the gel lanes for each sample indicated whether an individual possessed the tested CYP2A6 variant. Once all CYP2A6 assays were complete a total genotype was determined for each individual CYP2A6 Genotype Grouping All subjects were categorized by the CYP2A6 genotype grouping strategy used previously (Benowitz et al. 2005; Ho et al. 2009; Lerman et al. 2010; Liu et al. 2011; Mwenifumbo et al. 2008a). Individuals with one copy of the reduced activity CYP2A6*9, or *12 alleles were grouped as intermediate metabolizers (IMs). Those with two copies of CYP2A6*9 and *12 or one or more copies of the loss-of-function CYP2A6*2, *4, *7, *10, *17, *35 alleles were grouped as slow metabolizers (SMs). All participants who did not possess one of the aforementioned variants, including those having CYP2A6*1X2A, *1X2B, *1B, or *8, were considered normal metabolizers (NMs). Reduced metabolizers (RMs) consisted of individuals classified as IMs or SMs combined together for the purpose of comparison to the NM group. 63

75 2.4 Statistical Analysis Assessments of Hardy-Weinberg equilibrium for CYP2A6 and CYP2B6 genotype frequencies and comparisons of allele frequencies previously reported for alternative ethnicities were analyzed by chi-square test. Baseline demographic characteristics were compared between CYP2A6 genotype groupings, 3HC/COT median split strata, or tobacco product use groups by chi-square test if categorical and unpaired t-test or one-way ANOVA with Bonferonni!s correction for multiple comparisons (when P < 0.05) if continuous. Plasma COT, 3HC, urine TNE, urine NNAL, urine TNE/NNAL, and the plasma and urine 3HC/COT ratios were not normally distributed and therefore log-transformed prior to statistical analysis. The plasma and urine 3HC/COT ratios for each CYP2A6 genotype were compared to the CYP2A6 wild-type reference group by unpaired t-test or one-way ANOVA with Bonferonni!s correction for multiple comparisons (when P < 0.05). Comparisons of plasma and urine 3HC/COT ratios by CYP2A6 genotype grouping, by CYP2B6 genotype, and of Yupik to previously studied ethnicities were made by unpaired t-test or one-way ANOVA with Bonferroni!s correction for multiple comparisons (when P < 0.05). Haplotype analysis of CYP2A6 and CYP2B6 was conducted using the Haploview! program, obtained from the Broad Institute (Cambridge, MA) (Barrett et al. 2005). Linear regression modeling of factors influencing the plasma and urine 3HC/COT ratios and NNAL level in tobacco users was performed using SPSS! on logtransformed values (IBM Somers, NY). The impact of CYP2A6 genotype grouping, CYP2B6 genotype, and plasma 3HC/COT ratio on smoking variables was determined by unpaired t-test. Comparisons of smoking variables and metabolite measures 64

76 between product groups were made by one-way ANOVA with Bonferroni!s correction for multiple comparisons (if P < 0.05). Correlations of plasma and urine 3HC/COT ratio, and between nicotine use biomarkers were determined by the Pearson!s product-moment correlation coefficient of log-transformed values. All statistical tests were performed using GraphPad Prism" version 5.0 or SPSS" version

77 3. RESULTS 3.1 CYP2A6 and CYP2B6 Allele and Genotype Group Frequencies During this study 400 Alaska Native blood samples (361 Yupik, 19 Aleut, 11 Athabascan, 4 Inupiaq, 1 Cupik, 1 Tlingit, 3 unknown ethnicity) were received, and extracted DNA was successfully genotyped for CYP2A6*1X2A, *1X2B, *1B, *2, *4H, *7, *8, *9, *10, *12, *17, *35 and CYP2B6*4, *6, *9. Allele frequencies were calculated for the total study population (n = 400) and separately among Yupik (n = 361) and Yupik with four Alaska Native grandparents (n = 309). These allele frequencies are listed in Table 3.1 and Table 3.2, and include comparisons to frequencies previously published for other ethnic groups. Further genotypic and phenotypic analyses are restricted to the Yupik subgroup as 1) the low frequency of individuals in other distinct tribal groups limited any characterizations of the Alaska Native population as a whole and 2) allele frequencies are very similar between the total Alaska Native population and Yupik. Also, comparisons were performed with both the total Yupik population and the subset of Yupik individuals with four Alaska Native grandparents to determine if admixture with local non-alaska Native populations contributed to differences in allele frequency. Chi-square tests were used to contrast observed Yupik frequencies for selected alleles with those previously reported for distinct ethnicities, including Japanese, Korean, Chinese, African-American, and Caucasian (Figure 3.1). As few distinct differences in allele frequencies, and their significance level versus comparator groups, were noted between the total Yupik and the subset with four Alaska Native grandparents further analysis is based on all Yupik (n = 361). Significant deviation from Hardy-Weinberg equilibrium was not seen for any Yupik genotype (#2 P > 0.20). 66

78 67 Allele CYP2A6 CYP2A6*1X2A CYP2A6*1X2B CYP2A6*1B CYP2A6*2 Present Study Comparator Ethnic Groups Alaska Natives Japanese Korean Chinese Observed Observed 2 Observed 2 Observed 2 Total!!! Alleles a Frequency Frequency Frequency Frequency % (n) (%) b P Value (%) b P Value (%) b P Value CYP2A6*4 c CYP2A6*7 CYP2A6*8 CYP2A6* (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 63.5 (508) 65.4 (472) 66.5 (411) 0.4 (3) 0.1 (1) 0.2 (1) 14.5 (116) 14.5 (105) 15.4 (95) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 8.5 (68) 8.4 (61) 9.2 (51) < < < < < < < < < < < < < < < < < < < < < < < < < 0.01 <

79 68 Allele CYP2A6*10 CYP2A6*12 CYP2A6*17 CYP2A6*35 CYP2B6 CYP2B6*4 CYP2B6*6 CYP2B6*9 Present Study Comparator Ethnic Groups Alaska Natives Japanese Korean Chinese Observed Observed 2 Observed 2 Observed 2!!! Frequency Frequency Frequency Frequency % (n) (%) b P Value (%) b P Value (%) b P Value (20) (20) (18) Total Alleles a (3) 0.1 (1) 0.2 (1) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 49.1 (393) 51.7 (373) 53.7 (332) 0.0 (0) 0.0 (0) 0.0 (0) < < < < < < < < < < < < < < < < < < < < <

80 Table 3.1 Observed CYP2A6 and CYP2B6 allele frequencies of all Alaska Native (n = 400), all Yupik (n = 361), and Yupik with 4 Alaska Native grandparent (n = 309) participants compared to Asian populations. Allele frequencies from the current study are reported and compared to previous studies by! 2 test. a CYP2A6 and CYP2B6 allele frequencies were calculated among the total Alaska Native population (n = 400; 800 total alleles), Yupik (n = 361; 722 total alleles), and Yupik with four Alaska Native grandparents (n = 309; 618 total alleles). b The allele frequencies reported in comparator ethnicities were sampled from a number of studies based on the CYP2A6 and CYP2B6 variants genotyped. The total alleles tested were study-dependent and the ranges for each ethnicity follow: Japanese (n = 50 92; total alleles); Korean (n = ; total alleles); Chinese (n = ; total alleles). c The current study utilized the CYP2A6*4H assay, which detected the CYP2A6*4A, *4D, *4E, *4F, *4G and*4h variants. The previous studies in Japanese, Koreans, and Chinese used the CYP2A6*4A&D assay, which detects *4A and *4D only. References: (Al Koudsi et al. 2009a; Fukami et al. 2004; Mwenifumbo et al. 2005; Mwenifumbo and Tyndale 2007; Mwenifumbo et al. 2008b; Minematsu et al. 2006; Nakajima et al. 2006; Oscarson et al. 1999a; Oscarson et al. 1999b; Oscarson et al. 2002; Pitarque et al. 2004; Zanger et al. 2007) 69 69

81 70 Allele CYP2A6 CYP2A6*1X2A CYP2A6*1X2B CYP2A6*1B CYP2A6*2 Present Study Comparator Ethnic Groups Alaska Natives African-American Caucasian Observed Observed 2 Observed 2 Total!! Alleles a Frequency Frequency Frequency % (n) (%) b P Value (%) b P Value CYP2A6*4 c CYP2A6*7 CYP2A6*8 CYP2A6* (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 63.5 (508) 65.4 (472) 66.5 (411) 0.4 (3) 0.1 (1) 0.2 (1) 14.5 (116) 14.5 (105) 15.4 (95) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 8.5 (68) 8.4 (61) 9.2 (51) < < < < < < < < < < < < < 0.01 < 0.01 < 0.01 < < <

82 71 Allele CYP2A6*10 CYP2A6*12 CYP2A6*17 CYP2A6*35 CYP2B6 CYP2B6*4 CYP2B6*6 CYP2B6*9 Present Study Comparator Ethnic Groups Alaska Natives African-American Caucasian Observed Observed 2 Observed! Frequency Frequency Frequency n (%) (%) b P Value Total Alleles a (20) 2.8 (20) 2.9 (18) 0.4 (3) 0.1 (1) 0.2 (1) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 49.1 (393) 51.7 (373) 53.7 (332) 0.0 (0) 0.0 (0) 0.0 (0) < 0.01 < 0.01 < < < < < < < 0.001! (%) b P Value < < < < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < < < < < < <

83 Table 3.2 Observed CYP2A6 and CYP2B6 allele frequencies of all Alaska Native (n = 400), Yupik (n = 361), and Yupik with 4 Alaska Native grandparent (n = 309) participants compared to Caucasian and African-American populations. Allele frequencies from the current study are reported and compared to previous studies by! 2 test. a CYP2A6 and CYP2B6 allele frequencies were calculated among the total Alaska Native population (n = 400; 800 total alleles), Yupik (n = 361; 722 total alleles), and Yupik with four Alaska Native grandparents (n = 309; 618 total alleles). b The allele frequencies reported in comparator ethnicities were sampled from a number of studies (see references 1-12) based on the CYP2A6 and CYP2B6 variants genotyped. The total alleles tested were study-dependent and the ranges for each ethnicity follow: African-American (n = ; total alleles); Caucasian (n = ; total alleles). c The current study utilized the CYP2A6*4H assay, which detected the CYP2A6*4A, *4D, *4E, *4F, *4G and*4h variants. The previous studies in Caucasians used the CYP2A6*4A&D assay, which detects *4A and *4D only. 72 References: (Al Koudsi et al. 2009a; Ho et al. 2009b; Malaiyandi et al. 2006b; Mwenifumbo and Tyndale 2007; Mwenifumbo et al. 2008a; Mwenifumbo et al. 2008b; Mwenifumbo et al. 2010; Nakajima et al. 2006; Oscarson et al. 1999a; Oscarson et al. 1999b; Oscarson et al. 2002; Pitarque et al. 2004; Schoedel et al. 2004; Zanger et al. 2007) 72

84 30 CYP2A6*4 Allele Frequency (% + 95% CI) *** *** *** Yupik Japanese Korean Chinese AA Caucasian 30 CYP2A6*7 Allele Frequency (% + 95% CI) *** *** *** 0 Yupik Japanese Korean Chinese AA Caucasian 73

85 30 CYP2A6*9 Allele Frequency (% + 95% CI) *** *** ** 0 60 Yupik Japanese Korean Chinese AA Caucasian CYP2B6*6 Allele Frequency (% + 95% CI) *** *** *** *** *** 0 Yupik Japanese Korean Chinese AA Caucasian Figure 3.1 Yupik possess a unique pattern of CYP2A6 and CYP2B6 allele frequencies when compared to other ethnicities. Yupik individuals; n = 361. Alleles depicted were selected based on prevalence in Yupik subjects. *P < 0.05, **P < 0.01, ***P < when compared to Yupik in individual! 2 tests. 74

86 Observed allele frequencies demonstrate the unique CYP2A6 and CYP2B6 genetic profile of the Yupik, as great variability was seen in comparisons to published ethnic data. Particularly, the pattern of Yupik frequencies was not the same as that seen in Chinese, refuting the a priori hypothesis. For example, the Yupik frequency of the CYP2A6*4 allele was similar to levels found in the Asian groups (Japanese, Korean, Chinese) and exceeds values reported for African-Americans and Caucasians. However, when comparing the CYP2A6*9 allele the Yupik frequency resembles African-Americans and Caucasians but is significantly lower than the prevalence reported for Asians. Additionally, the complete absence of the CYP2A6*7 allele alone in Yupik while in Asian groups this variant is high frequency is notable, especially as CYP2A6*10 levels (the combination CYP2A6*7 and *8 haplotype) are not distinct between the Yupik and Asian populations. Further, the significantly higher frequencies of the CYP2A6*1B and CYP2B6*6 variants in Yupik versus all ethnic comparator groups provide further evidence of their unique genetic position. Frequencies of individual CYP2A6 genotypes for all Yupik (n = 361) and among the subset of Yupik individuals who used tobacco (n = 265) are shown in Table 3.3. The Yupik tobacco user subgroup was included, as this subgroup consists of all individuals possessing valid CYP2A6 activity phenotype measures necessary to assess the impact of CYP2A6 genotype on plasma and urinary 3HC/COT ratios in this population. As little difference in the proportion of genotype frequencies is observed, it is assumed that further analysis within the Yupik tobacco user subgroup accurately represents the Yupik population as a whole, if all individuals were exposed to appreciable levels of nicotine. 75

87 76 Yupik Yupik Tobacco Users CYP2A6! 2 Genotype Allele Observed Observed % % P Value Frequency (n) Frequency (n) Wild-type Reference Group *1/* *2 *1/* *4 *1/* *4/* *9 *1/* *9/* *10 *1/* *12 *1/* Compound *4/* Heterozygotes *9/* Total Table 3.3 Frequency of CYP2A6 genotypes among Yupik (n = 361), and the Yupik tobacco user subset (n = 265). Genotype frequencies are reported and compared by! 2 test. 76

88 3.2 Association Between CYP2A6 Genotype and Plasma CYP2A6 Activity A distribution scatterplot of plasma 3HC/COT ratios for each CYP2A6 genotype in Yupik tobacco users (n = 265) is depicted in Figure 3.2 and summarized in Table 3.4. Comparisons of mean plasma 3HC/COT to the reference CYP2A6*1/*1 group were performed by one-way ANOVA or student!s t-test, if heterozygotes and homozygotes were found or if only heterozygotes were found respectively, for each CYP2A6 genotype. The CYP2A6*1B variant has been shown previously to associate with enhanced CYP2A6 enzyme activity in European American and African American populations (Mwenifumbo et al. 2007; Ho et al. 2009). While CYP2A6*1B/*1B individuals trended toward having a higher plasma 3HC/COT ratio than CYP2A6*1/*1 and CYP2A6*1/*1B subjects, this difference was not significant (ANOVA P = 0.24). The mean plasma 3HC/COT ratio was significantly lower for individuals with the CYP2A6*4 (ANOVA P < 0.001), CYP2A6*9 (ANOVA P < 0.001), CYP2A6*10 (t-test P < 0.001), and those possessing two distinct variant alleles (compound heterozygotes t-test P < 0.001). As seen previously (Ho et al. 2009; Lerman et al. 2010), the CYP2A6*9 allele resulted in reduced enzyme activity (72% of wild-type activity for heterozygotes and 20% for variant homozygotes) and the *4 deletion allele led to loss of enzyme function (51% of wild-type activity for heterozygotes and 2% for variant homozygotes). Thus the impact of these CYP2A6 variants in Yupik and their suggested genotypes are consistent with previous characterization of their effect on in vivo enzyme function. As only two participant tobacco users possessed the CYP2A6*2 and *12 alleles respectively, testing for difference of these variants from the CYP2A6*1/*1 group was not possible. 77

89 HC/COT (mean) *1/*1 *1/*2 *1/*4 *4/*4 *1/*9 *9/*9 *1/*10 *1/*12 *4/*9 CYP2A6 Genotype Figure 3.2 Association of CYP2A6 genotype with the plasma 3HC/COT ratio among Yupik tobacco users (n = 265). CYP2A6 genotypes and their associated plasma ratio; each dot represents the pre-log transformed value for one individual and the line represents the mean 3HC/COT ratio of each genotype group. The CYP2A6*1/*1 group (n = 147) includes individuals possessing the CYP2A6*1B allele. Mean 3HC/COT ratios (± SD) for each variant genotype group, with comparisons by ANOVA to the CYP2A6*1/*1 wild-type individuals, are listed in Table

90 CYP2A6 Allele *1B Genotype Observed frequency (n) Mean Plasma 3HC/COT SD % of CYP2A6*1/*1 Plasma 3HC/COT *1/* *1/*1B P Wild-type Reference Group *1B/*1B *1/* *2 *1/* *4 *9 *1/* *4/* *1/* *9/* <0.001 <0.001 *10 *1/* <0.001 *12 *1/* Compound Heterozygotes *4/* <0.001 Table 3.4 Frequency of CYP2A6 genotypes and their associated plasma 3HC/COT ratios among Yupik tobacco users (n = 265). The baseline mean plasma 3HC/COT ratios for each genotype group are listed along with the percentage of the CYP2A6 wild-type reference group. Prior to statistical analysis values were log-transformed as they were not normally distributed. Comparisons were made using the CYP2A6*1/*1 individuals as a reference group, including those with CYP2A6*1B alleles as they did not differ significantly, and this is the usual reference group composition used in the literature (Ho et al. 2009). 79

91 As the CYP2A6 genotypes in Yupik tobacco users had a similar impact on plasma 3HC/COT to published data in multiple ethnic groups (African-Americans - Ho et al. 2009b, Caucasians - Lerman et al. 2010), the same validated CYP2A6 metabolizer grouping strategy was used. The mean plasma 3HC/COT ratios differed for each CYP2A6 metabolizer group within all Yupik tobacco users (n = 265) (Figure 3.3). Compared to NMs, mean plasma 3HC/COT ratios were significantly lower in IMs (P < 0.01), SMs (P < 0.001), and RMs (the combined IM + SM population - P < 0.001). A significant difference was also observed when comparing IMs and SMs (P < 0.001). 3.3 Variables Independent of CYP2A6 Genotype that Impact Plasma 3HC/COT To discern any impact of factors independent of CYP2A6 genotype within Yupik tobacco users, analysis of the plasma 3HC/COT ratio was conducted in CYP2A6*1/*1 tobacco users only. Among these individuals the 3HC/COT ratio was higher in female participants, individuals with increasing age, and those with decreasing BMI (Table 3.5). These impacts on plasma 3HC/COT are consistent with previous findings (Ho et al. 2009). No difference in 3HC/COT ratio was found by CYP2B6 genotype. Based on our univariate analysis, CYP2B6 genotype did not impact the 3HC/COT ratio in CYP2A6*1/*1 individuals (n = 147), however when participants possessing CYP2A6 variants were also included a significant association with CYP2B6 genotype was seen (Figure 3.4). One-way ANOVA with Bonferroni!s multiple comparison test on the mean plasma 3HC/COT ratios of the CYP2B6*6 genotype groups revealed a significant effect (P < 0.001). However, the effect was lost when CYP2A6 genotype was controlled for (P = 0.99), suggesting the association of CYP2B6 with plasma 3HC/COT was dependent on CYP2A6 genotype (Figure 3.5). 80

92 0.8 *** ** *** 3HC/COT (mean + 95% CI) % *** 73% 100% 51% 0.0 SM IM NM RM (n = 87) (n = 31) (n = 147) (n = 118) CYP2A6 Genotype Figure 3.3 Association of CYP2A6 genotype groupings with the plasma 3HC/COT ratio among all Yupik tobacco users (n = 265). The plasma 3HC/COT ratio significantly associated with current CYP2A6 genotype grouping strategy. Mean plasma 3HC/COT values for each CYP2A6 group are (mean ± 95% CI): NM = 0.61 ± 0.05, IM = 0.44 ± 0.06, SM = 0.26 ± 0.03, and RM (IM + SM) = 0.31 ± Prior to statistical analysis via one-way ANOVA with Bonferroni!s correction for multiple comparisons values were log-transformed as they were not normally distributed. The number of individuals is listed on the x-axis. **P < 0.01, ***P <

93 Gender n Mean 3HC/COT SD P value Males Females Age < * * r 0.31 < BMI a Low (< 27) High (" 27) r CYP2B6 Genotype CYP2B6*1/* CYP2B6*1/* CYP2B6*6/* Table 3.5 Factors that influence the plasma 3HC/COT ratio in CYP2A6*1/*1 Yupik tobacco users (n = 147). * Significant difference (P < 0.05) from the age group. r = Spearman!s correlation coefficient between age and 3HC/COT. a Body mass index (BMI) was categorized by median split at 27.0; group sizes following split are unequal due to whole-number reporting of BMI. BMI was not available for 10 individuals. r = Spearman!s correlation coefficient between BMI and 3HC/COT. 82

94 0.8 * *** 3HC/COT (mean + 95% CI) % 124% * 100% 0.0 *6/*6 *1/*6 *1/*1 (n = 68) (n = 138) (n = 59) CYP2B6 Genotype Figure 3.4 Association of CYP2B6 genotype groupings with the plasma 3HC/COT ratio among Yupik tobacco users (n = 265). Mean 3HC/COT values for each CYP2B6 genotype group are (mean ± 95% CI): CYP2B6*1/*1 = 0.38 ± 0.06, CYP2B6*1/*6 = 0.46 ± 0.05, and CYP2B6*6/*6 = 0.60 ± Prior to statistical analysis values were log-transformed as they were not normally distributed. Significance was maintained when controlling for gender, age, and BMI (ANOVA P < 0.001) but the impact of CYP2B6 genotype was negated after inclusion of CYP2A6 genotype as a co-factor (P = 0.99). The number of individuals is listed on the x-axis. *P < 0.05, **P < 0.01, ***P <

95 Figure 3.5 Association of CYP2B6 genotype with the plasma 3HC/COT ratio results from interaction with the CYP2A6 gene. The majority of variability in the 3HC/COT ratio results from CYP2A6, and not CYP2B6, genotype. Mean plasma 3HC/COT ratios were stratified by CYP2A6 and CYP2B6 genotype together for comparison. A greater effect on mean plasma 3HC/COT is observed when comparing column size by CYP2A6 metabolizer grouping along the z-axis than CYP2B6 genotype along the x-axis. Note no subjects possessed the combination of CYP2A6 IM and CYP2B6*6/*6 genotypes. 84

96 3.4 CYP2A6 and CYP2B6 Gene Interaction and Haplotyping To better characterize a possible interaction between CYP2A6 and CYP2B6 genotypes, linkage disequilibrium was assessed through haplotyping analysis, shown in Figure 3.6. CYP2A6 and CYP2B6 are located within the CYP2 gene cluster, on the anti-sense and sense strands respectively, and are separated by a distance of less than 350 kb (Hoffman et al. 2001); the close proximity of these genes suggests the possibility of historical recombination. The CYP2B6*6 allele was significantly associated with CYP2A6*1B providing evidence of an interaction between CYP2B6 and CYP2A6 genotypes in this population. Thus given the high percentage of CYP2A6*1B among Yupik (65.4% allele frequency - Table 3.1, 3.2) it is likely that linkage disequilibrium between these variants is responsible for the high plasma 3HC/COT ratio observed in CYP2B6*6 individuals. In addition, CYP2A6*7 and *8 (together CYP2A6*10) and CYP2B6*4 and *9 (together CYP2B6*6), were found to be in full linkage disequilibrium as these single variants were not found alone in any subject. Associations with CYP2B6*6 were also observed for CYP2A6*4 and *10. Linkage disequilibrium between CYP2A6*4 and CYP2B6*6 did not result in the association of elevated plasma 3HC/COT ratio with CYP2B6*6, as CYP2A6*4 leads to loss of CYP2A6 activity (seen in impact of CYP2A6*4 on plasma 3HC/COT Figure 3.2, Table 3.4). Further, the low number of Yupik individuals possessing the CYP2A6*10 variant (2.8% allele frequency Table 3.1, 3.2) indicated it was also not the variant causing an interaction with CYP2B6. 85

97 a) b) 86 Figure 3.6 Interaction of the CYP2A6 and CYP2B6 genes in Yupik (n = 361). Haplotype analysis of genotyped variants within CYP2A6 and CYP2B6 displayed by a) D! score and b) R2 value. D! and R2 values are provided for each pair of SNPs analyzed, ranging from 0 (no linkage disequilibrium) to 100 (full linkage disequilibrium) for both measures. Haplotype map cells are also shaded based on the strength of corresponding disequilibrium relationships. Note a D! score of 100 indicates that only two or three haplotypes were observed for a pair of SNPs, and a score <100 signifies the occurrence of four haplotypes, indicating historical recombination has occurred. R2 describes the statistical correlation of SNPs at two loci and a score of 100 indicates just two haplotypes are observed (Wall and Pritchard 2003). Tag SNPs were utilized for CYP2A6*1B, CYP2A6*4, and CYP2A6*12. 86