Abstract Cytochrome P450 2A6 (CYP2A6) is involved in the 7-hydroxylation of coumarin, C-oxidation of nicotine, and the metabolism of tobacco specific nitrosamines. Initially in 1995 Fernandez-Salguero et al. reported a genotyping method for three alleles: CYP2A6*1 (wild-type), CYP2A6*2 (variant 1), and CYP2A6*3 (variant 2). Later studies presented in this thesis indicated that the original genotyping method produces erroneous results for the CYP2A6*3 allele due to unspecific PCR conditions and previously unknown CYP2A6*1B allele. Furthermore, the CYP2A6*2 allele genotyping caused erroneous genotypes (CYP2A6*2/*2 was misclassified as CYP2A6*1/*2). In this work, new PCR based genotyping methods were developed for CYP2A6*2 and for several new alleles (CYP2A6*1B, CYP2A6*4A/*4D and CYP2A6*5). In population-based studies, the deletion alleles (pooled as CYP2A6*4) turned out to be more prevalent among Asians (15.1%) than Caucasians (0.5%). The frequencies of the other inactive alleles varied within 0–3% in both populations. Asians totally lacked the CYP2A6*2 allele, whereas Caucasians lacked the CYP2A6*5 allele. The frequencies of two wild-type alleles, CYP2A6*1A and CYP2A6*1B alleles were 66.5% and 30.0% in Caucasians, and 43.2% and 40.6% in Asians, respectively. Correlation studies between the phenotype, as tested by the administration of coumarin, and the genotype demonstrated that individuals with the CYP2A6*2/*2 genotype were totally defective, while CYP2A6*1/*2 subjects exhibited intermediate and CYP2A6*1/*1 subjects full capablility of producing 7-hydroxycoumarin. Upon phenotyping with nicotine, individuals with the CYP2A6*1/*2 or CYP2A6*1/*4 genotype were shown to have a lower enzyme activity (one fourth of the normal activity), compared to those with the CYP2A6*1/*1 genotype. Defective CYP2A6 activity has been hypothesised to reduce the risk of environmentally (especially tobacco smoke) induced diseases either by decreasing production of genotoxic metabolites or by preventing addiction to tobacco smoking. However, in our case-control studies on Spanish patients with liver cirrhosis (n = 83) and liver cancer (n = 90) and their controls (n = 237) no significant association between the CYP2A6 genotypes and disease proneness was found. The odds ratio (OR) for developing liver cancer was was 1.4 (95% confidence interval [CI] 0.5–3.7) for genotypes containing at least one CYP2A6*2 allele. For liver cancer the respective OR was 1.3 (95% CI 0.4–4.5). Similarly, no statistically association between CYP2A6 alleles and the risk of lung cancer was observed in our Finnish study population cinsisting of 177 cases and 1089 controls; the OR for combined CYP2A6 variant allele containing genotypes (CYP2A6*1/*2 and CYP2A6*1/*4) was 1.19 (95% CI 0.56–2.45). Our studies therefore do not indicate any major modifying role for the CYP2A6 genotypes in individual susceptibility to environmentally induced diseases.

>Magister Scientiae - MSc / Cancer is defined as the abnormal growth of genetically mutated or perturbant cells. Nicotine is a known cancer promoter and an apoptotic suppressor. This alkaloid acts on the nicotinic acetylcholine receptors which affects the ubiquitin-proteasome protein degradation pathway and ultimately hinders apoptosis. The endoplasmic reticulum (ER) is an interconnecting organelle which synthesises proteins and its quality control processes ensures the proper protein folding, post-translational modifications and conformation of secretory and trans-membrane proteins. Studies demonstrated that the antibiotic, Tunicamycin (Tm) and the sesquiterpene lactone, Thapsigargin (Tg) causes ER stress and consequently cellular arrest. Tm interferes with N-glycosylation of newly synthesised proteins triggering the unfolded protein response, while Tg inhibits intracellular Ca2+ ATPases resulting in increased cytosolic Ca2+. Studies showed that these compounds have potential pro-apoptotic effects. The combinatorial effects of nicotine, Tm and Tg may produce antagonistic or synergistic effects and provide a therapeutic tool against breast cancer. The aim of the study was to determine the apoptotic effects of nicotine, Tm, and Tg on human breast carcinoma (MCF-7) at various time intervals and further to elucidate whether selected ratios of their combinations resulted in synergistic or antagonistic effects.

Magister Artium - MA / Hookah pipe use is widely viewed as a safe alternative to cigarette smoking rather than a potential health-risk. In fact, for young people hookah pipe use may represent an initial stage of later addiction and the transition to cigarette smoking. Furthermore, studies conducted abroad, suggest that the use of the hookah pipe firstly started as a cultural phenomenon, and secondly, as with cigarette smoking, the hookah pipe has become a social phenomenon. Despite these challenges, studies provide sufficient evidence that hookah pipe use is a potential health risk. The primary aim of the study was to compare male and female university students’ knowledge, risk perceptions and behaviours concerning hookah pipe smoking. A quantitative methodological approach, with a cross-sectional design, was used to conduct the research study. A final self-selected sample of 389 participants voluntarily participated in this study. The final sample included 64% females and 36% males with a mean age of 22.2 years; with the mean age for first-time hookah pipe smoking was 15.7 years. The instrument used was a self-administered questionnaire constructed from The College Health Behavior Survey (2010-2011) which was developed at the University of Missouri-Columbia. Descriptive quantitative results were conducted using Statistical Package for Social Sciences (SPSS version 20) and presented. The results suggest 70% of hookah pipe users daily smoke the hookah pipe with more than 20% smoking on campus. This was similar for males and females. Users perceived the hookah pipe to be less harmful and less addictive than cigarette smoking. Furthermore, smoking the hookah pipe is considered socially acceptable and is also smoked in the family home. Implications for policy are stated.

Introduction: An active area of tobacco regulatory science research focuses on examining the effects of varying the nicotine content of cigarettes as part of a potential national policy to lower their nicotine content levels to reduce addiction potential. The present study examines differences in the behavioral effects of reduced nicotine content cigarettes related to their menthol status. Menthol is the only cigarette flavoring that is still legally permissible according to Food and Drug administration regulations. Methods: Participants were 26 current adult smokers from three populations especially vulnerable to tobacco use and addiction (economically disadvantaged women, opioid-dependent individuals, individuals with affective disorders) dichotomized as menthol (n=11) or non-menthol (n=15) smokers. Participants completed 14 experimental sessions following acute smoking abstinence (CO<50% baseline level). Across sessions, participants smoked four Spectrum research cigarettes (22nd Century Group, Clarence, NY) with varying nicotine content levels (0.4mg/g, 2.4 mg/g, 5.2 mg/g, 15.8 mg/g) or their usual brand cigarette. Research cigarettes were mentholated or non-mentholated corresponding to participants usual brand. Upon completion of smoking, participants completed tasks measuring reinforcing efficacy, subjective effects, topography, and withdrawal and craving measures. Repeated Measures Analysis of Variance was used for all analyses (p<.05). Results: Main effects of menthol status, as well as interactions of nicotine dose and menthol were noted across subscales of subjective effects and direct assessments of reinforcing efficacy. Usual brand mentholated cigarettes produced a profile of equal or greater relative reinforcing effects than usual brand non-mentholated cigarettes, while mentholated research cigarettes produced a profile of effects that fell below (i.e., lower relative reinforcing effects compared to usual brand or non-mentholated cigarettes) those of non-mentholated research cigarettes. Conclusions: Mentholated research cigarettes produce a lower profile of reinforcing and subjective effects, without discernible differences in smoking topography. The potential impact of mentholation on reinforcing efficacy and subjective effects should be considered when using Spectrum research cigarettes.

Maternal smoking during pregnancy has often been associated with numerous adverse outcomes for the offspring. However, its long-term effects are not well established. Given the high prevalence of maternal smoking during pregnancy, an understanding of these effects is essential. Therefore, the aim of the present dissertation was to shed light on the long-term neurophysiological effects of prenatal nicotine exposure on three different executive functioning processes by assessing participants in young adulthood, using functional magnetic resonance imaging (fMRI). Participants imaged were members of the Ottawa Prenatal Prospective Study, a longitudinal study that collected a unique body of information on participants from infancy to young adulthood, which allowed for the measurement of an unprecedented number of potentially confounding drug exposure variables. The dissertation consists of three separate original manuscripts. In manuscript 1, participants completed a response inhibition task, in manuscript 2 participants completed a verbal working memory task and in manuscript 3 participants completed a visuospatial working memory task. Taken together, results from all three manuscripts showed that prenatal nicotine exposure leads to altered neural functioning during executive functioning processing that continues into young adulthood. These significant results highlight the need for education about the repercussions of women smoking during pregnancy.

Cigarette smoking during pregnancy contributes to the development of neurological health problems in offspring. As a result, public health organizations are recommending NRT to pregnant women to wean them off tobacco. If nicotine itself is injurious to the developing brain, then nicotine substitution may not eliminate the deleterious health outcomes of maternal smoking. In studies of cognitive decline, estradiol elicits a neuroprotective effect through ER activation. However, the underlying mechanism remains unclear. Evidence suggests that estrogen-mediated neuroprotection is activated through glial cell interaction, mitigating inflammation and protecting neurons critical for learning and memory. If NRT antagonizes these cellular targets, it may put individuals at risk for future cognitive impairments. Randomly assigned nulliparous female Wistar rats were injected subcutaneously with 1 mg/kg/day of nicotine bitartrate or saline for 2 weeks before mating until weaning (PND 21). Pups (saline n=6 and nicotine, n=6) were sacrificed at 26 weeks of age and the hippocampal formation was processed for Nissl and immunohistochemical staining for GFAP and ERα. Gestational exposure to nicotine only produced a significant increase in the expression of ERα in the DG of the hippocampus. While additional research is needed, these findings suggest that NRT might indeed interfere with proper brain development, making offspring increasingly susceptible to long-term adverse health effects. Le tabac fumé pendant la grossesse affecte de manière importante le développement neurologique de la progéniture, y compris à long terme. C’est pourquoi, les autorités de la santé publique recommandent les substituts nicotiniques comme soutien au sevrage tabagique chez les femmes enceintes. Si le danger se situe dans la nicotine de la cigarette, alors les produits de substitution nicotiniques risquent également d’interférer avec le développement cérébral. De nombreuses données expérimentales convergent pour attribuer un rôle protecteur à l’oestradiol sur le fonctionnement cognitif. Par contre, le mécanisme sous-jacent est inconnu. Il se peut que l’oestradiol arrive à neutraliser la réaction inflammatoire provoquée par les cellules gliales, amoindrissant la détérioration des neurones impliqués au niveau de la mémoire. Ainsi, une perturbation de ce mécanisme par la nicotine pourrait engendrer une détérioration progressive des fonctions cognitives. Des rats femelles Wistars nullipares assignées de façon alléatoire à un groupe ont reçu soit une injection sous-cutanée de 1mg/kg/jour de nicotine bitartrate ou de saline, 2 semaines avant l’accouplement jusqu'au sevrage au jour 21 postnatal. A 26 semaines, les ratons furent sacrifiés (saline n=6 et nicotine, n=6) et une analyse du Nissl et immunohistochimique de GFAP et ERα furent réalisées sur les formations hippocampiques. L’exposition prénatale à la nicotine a seulement augmenté significativement l’expression de ERα dans le GD de l’hippocampe. Alors que des études plus approfondies sont nécessaires, ces résultats suggèrent que les substituts nicotiniques affectent le développement neurologique périnatal, ce qui risque d’entrainer des répercussions a long terme sur la santé.

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Background A growing body of research suggests the therapeutic use of cannabis may affect the use of other substances, including reducing the use of alcohol, tobacco and prescription drugs such as opioid analgesics. However, most of the evidence stems from small, cross-sectional surveys or population-level studies, both of which have significant limitations, including the inability to conclusively determine causality for behavioural changes. Furthermore, very little detail has been gathered on the factors that potentially impact substitution, including patient characteristics and patterns of cannabis use (e.g., X, Y, Z). Additionally, despite consistent calls by physicians, academics, patients and policy-makers around the globe citing the need for high quality studies to identify the risks and benefits of cannabis in both medical and non-medical applications, there are many pre-existing and ongoing challenges to conducting such research. These include shifting regulatory policies that may be impacting access to cannabis for both medical and non-medical use, and that could ultimately be affecting patient retention in prospective medical cannabis studies. In the interest of learning more about how the use of cannabis effects the use of alcohol, tobacco and other substances, and to better understand factors that may be impacting retention in prospective cannabis research, I designed and conducted two studies: 1. The Canadian Cannabis Patient Survey 2019 (CCPS 2019) was a national cross-sectional survey of 2102 Canadian medical cannabis patients that examined demographics, patient patterns of cannabis use, and self-reported changes in the use of alcohol, tobacco, prescription drugs and illicit substances following medical cannabis initiation. 2. The Tilray Observational Patient Survey (TOPS) was a prospective, multi-site, observational study examining the impact of medical cannabis products on quality of life and the use of prescription drugs of 1145 patients over a 6 month period, which provided an opportunity to conduct a survival analysis and other analyses to assess variables potentially impacting retention in longitudinal cannabis studies. Methods This dissertation includes three analyses of the data resulting from these studies in the form of one published and two submitted manuscripts. The first paper provides an overview of research to date examining the impact of cannabis and cannabinoids on alcohol use, followed by an analysis of the 973 CCPS 2019 participants who either previously or currently use alcohol. The questionnaire gathered a detailed inventory of alcohol use prior and post medical cannabis initiation using two separate but related measures: drinking days per month, and standard drinks per week. The analyses used descriptive statistics as well as univariate and multivariate regression analyses to explore patient characteristics and other variables potentially associated with changes in alcohol use post medical cannabis, including assessing the impact of “intent” to use medical cannabis to reduce alcohol use, as well as participation in other substance use treatment modalities. Findings suggest that medical cannabis initiation is associated with significant reductions in alcohol use, and that younger age (<55 years of age), specific intent to use medical cannabis to reduce alcohol use, and greater patterns of alcohol use prior to medical cannabis initiation were associated with greater odds of reducing alcohol. The second paper follows a similar methodology and format as the first paper, but with a focus on tobacco/nicotine (T/N) use. In this case, 650 survey participants reported past or current T/N use, and the analysis focused on assessing patient characteristics and other variables associated with changes in T/N uses per day, with the primary outcome of interest being no use in the 30 days prior to the survey, which was considered to be complete cessation of T/N use. The findings suggest that odds of T/N cessation were greater amongst those who were age 55 or older or that reported >25 T/N uses per day prior to initiating medical cannabis use, and that specific intent to use medical cannabis in T/N reduction/cessation efforts resulted in significantly greater odds of reducing T/N use, while involvement with traditional T/N cessation treatments (pharmacological or psychobehavioral) was negatively associated with T/N cessation. The third paper addresses the challenge of retaining patients in prospective observational medical cannabis studies at a time when there are major policy changes disrupting the legal supply while also increasing access options for adults who use cannabis. The Tilray Observational Patient Study (TOPS) was one of the largest national prospective medical cannabis studies ever conducted, taking place at 21 medical clinics in five provinces. The study was designed to assess the impact of medical cannabis on quality of life and prescription drug use over a six month period. However, initial data analysis on 1145 patients enrolled at/before Oct 15, 2018 that had completed at least one post baseline visit highlighted a significant rate of patients that were lost to follow up (LTFU). This high drop out rate, coupled with a compensation scheme that provided credits to help cover the cost of medical cannabis led to concerns of potential retention bias limited the conclusions that could be drawn from this data. However, the study and resulting data provided a unique opportunity to examine baseline patient characteristics that may have been protective of LTFU, so a survival analysis was conducted on this cohort. Additionally, since the study took place during the official launch of the legalization of adult non-medical use of cannabis in Canada on Oct. 17th, 2018, the potential impact of this significant increase in legal access options on the odds of study retention was the subject of additional analyses. The survival analysis found that baseline use of antidepressants or antiseizure medications, citing no preference for either THC or CBD, and inhalation as a primary method of use were associated with increased probability of survival/retention in the study at six months. Additionally, while the legalization of non-medical adult cannabis use in October 2018 resulted in more than three times the odds of participants being LTFU at six months, being under 55 years old, having a preference for THC, or citing inhalation as a primary method of use was partially protective of LTFU following legalization. Discussion The studies in this dissertation presented an opportunity to gather subjective and objective data on naturalistic patterns of medical cannabis use from large, heterogeneous cohorts of patients, and to explore associated impacts on the use of alcohol, tobacco and other substances. The results of these studies provide a more comprehensive understanding of the public health risks and benefits associated with the medical use of cannabis, and could subsequently inform policy decisions affecting access to cannabis vis-à-vis other drugs, private and public payer considerations related to cost-coverage for medical cannabis, and potentially lead to the development of novel alcohol and tobacco cessation strategies. Additionally, the survival analysis conducted on TOPS participants highlights some of the challenges of conducting medical cannabis research at a time when patients have a multitude of cannabis access options, including legal adult dispensaries and a still robust illicit market. Future longitudinal medical cannabis studies should consider the potential impact of policy changes effecting cannabis access on study retention/survival, and may want to focus on patient populations with characteristics associated with lower odds of LTFU. / Graduate / 2022-03-09