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1.  Known and Novel Sources of Variability in the Nicotine Metabolite Ratio in a Large Sample of Treatment-Seeking Smokers 
The ratio of 3′hydroxycotinine to cotinine, or nicotine metabolite ratio (NMR), is strongly associated with CYP2A6 genotype, CYP2A6-mediated nicotine and cotinine metabolism, and nicotine clearance. Higher NMR (faster nicotine clearance) is associated retrospectively with heavier smoking and lower cessation rates.
NMR as a predictive biomarker of cessation outcomes is being investigated (NCT01314001). In addition to strong CYP2A6-genetic influences on NMR, demographic and hormonal factors alter NMR. Here we analyzed, for the first time together, these sources of variation on NMR in smokers screened for this clinical trial (N=1672).
Participants (mean age=45.9) were 65.1% Caucasian, 34.9% African American, and 54.8% male. Mean NMR (SD) was higher in Caucasians vs. African Americans (0.41(0.20) vs. 0.33(0.21); P<0.001), and in females vs. males (0.41(0.22) vs. 0.37(0.20); P<0.001). Among females, birth control pill use (N=17) and hormone replacement therapy (N=14) were associated with 19.5% (P=0.09) and 29.3% (P=0.06) higher mean NMR, respectively, albeit non-significantly. BMI was negatively associated with NMR (Rho=−0.14; P<0.001), while alcohol use (Rho=0.11; P<0.001) and cigarette consumption (Rho=0.12; P<0.001) were positively associated with NMR. NMR was 16% percent lower in mentholated cigarette users (P<0.001). When analyzed together in a linear regression model, these predictors (each ≤2%) accounted for <8% of total NMR variation.
While these factors significantly affected NMR, they contributed little (together <8%; each ≤2%) to total NMR variation.
Thus when using NMR, for example to prospectively guide smoking cessation therapy, these sources of variation are unlikely to cause NMR misclassification.
PMCID: PMC4154993  PMID: 25012994
Smoking; nicotine; metabolism; demographics; CYP2A6
2.  Association of CHRNA5-A3-B4 SNP rs2036527 with smoking cessation therapy response in African American smokers 
Associations between CHRNA5-A3-B4 variants and smoking behaviors exist, however the association with smoking abstinence is less understood, particularly among African Americans. In 1295 African Americans enrolled in two clinical trials, we investigated the association between CHRNA5-A3-B4 and smoking abstinence. Rs2056527[A] was associated with lower abstinence with active pharmacotherapy (during-treatment: OR=0.42&P<0.001; end of treatment (EOT): OR=0.55&P=0.004), or with nicotine gum alone (during-treatment: OR=0.31&P<0.001; EOT: OR=0.51&P=0.02), but not significantly with bupropion, although similar directions and magnitudes were observed (during-treatment: OR=0.54&P=0.05; EOT: OR=0.59&P=0.08). Additionally, rs588765[T] was associated with abstinence with gum during treatment (OR=2.31&P<0.01). Rs16969968 occurred at a low frequency and was not consistently associated with abstinence. CHRNA5-A3-B4 variants were not associated with tobacco consumption and adjustments for smoking behaviors did not alter the associations with smoking abstinence. Together, our data suggest that in African Americans CHRNA5-A3-B4 variants are not associated with baseline smoking, but can influence smoking abstinence during active pharmacotherapy.
PMCID: PMC4111775  PMID: 24733007
African Americans; CHRNA5-A3-B4; Smoking cessation; Nicotine; Bupropion; Tobacco Consumption
3.  Variation in P450 oxidoreductase (POR) A503V and flavin containing monooxygenase (FMO)-3 E158K is associated with minor alterations in nicotine metabolism but does not alter cigarette consumption 
Pharmacogenetics and genomics  2014;24(3):172-176.
Nicotine metabolism rates differ widely, even after controlling for genetic variation in the major nicotine metabolizing enzyme, CYP2A6. Genetic variants in an additional nicotine metabolizing enzyme, flavin containing monooxygenase (FMO)-3, and an obligate microsomal CYP-supportive enzyme, cytochrome P450 oxidoreductase (POR), were investigated. We examined the impact of FMO3 E158K and POR A503V, before and after stratifying by CYP2A6 metabolism group. In 130 non-smokers of African descent who received 4 mg oral nicotine, FMO3 158K trended towards slower nicotine metabolism in reduced CYP2A6 metabolizers (P=0.07) only, whereas POR 503V was associated with faster CYP2A6 activity (nicotine metabolite ratio) in normal (P=0.03), but not reduced, CYP2A6 metabolizers. Neither FMO3 158K nor POR 503V significantly altered the nicotine metabolic ratio (N=659), cigarette consumption (N=667), or urine total nicotine equivalents (N=418) in smokers of African descent. Thus, FMO3 E158K and POR A503V are minor sources of nicotine metabolism variation, insufficient to appreciably alter smoking.
PMCID: PMC3985268  PMID: 24448396
Tobacco; smoking; nicotine; metabolism; African Americans; genetic association studies
4.  CHRNA5-A3-B4 genetic variants alter nicotine intake and interact with tobacco use to influence body weight in Alaska-Native tobacco users 
Addiction (Abingdon, England)  2013;108(10):1818-1828.
Background and aims
Gene variants in CHRNA5-A3-B4, which encode for the α5, α3 and β4 nicotinic receptor subunits, are associated with altered smoking behaviors in European-Americans. Little is known about CHRNA5-A3-B4 and its association with smoking behaviors and weight in Alaska-Native people, which is a population with high prevalence but low levels of tobacco consumption, extensive smokeless tobacco use, and high rates of obesity. We investigated CHRNA5-A3-B4 haplotype structure and its association with nicotine intake and obesity in Alaska-Native people.
Design, Setting, Participants
A cross sectional study of 400 Alaska-Native individuals including 290 tobacco users.
CHRNA5-A3-B4 genotype, body weight, and tobacco consumption biomarkers such as plasma cotinine and urinary total nicotine equivalents (TNE).
Alaska-Native people have a distinct CHRNA5-A3-B4 haplotype structure compared with European/African-Americans. In 290 Alaska-Native tobacco users, the ‘G’ allele of rs578776, which tagged a 30kb haplotype in CHRNA5-A3-B4, was prevalent (16%) and significantly associated with nicotine intake (20% higher plasma cotinine, P<0.001, 16% higher TNE, P=0.076), while rs16969968 was not associated with nicotine intake. Rs578776 acted in combination with CYP2A6, the main nicotine-metabolizing enzyme, to increase nicotine intake by 1.8 fold compared with the low risk group (P<0.001). Furthermore rs2869950, a single nucleotide polymorphism 5′ to CHRNB4, was significantly associated with increased body mass index (P<0.01) in the tobacco users even after controlling for differences in nicotine intake (P<0.01).
Genetic variants in CHRNA5-A3-B4 alter nicotine intake and body mass index in a population of Alaska-Native people, who have a distinct haplotype structure, smoking behaviors and prevalence of obesity.
PMCID: PMC3775934  PMID: 23692359
Alaska-Native People; Smoking; CHRNA5-A3-B4; Obesity
5.  First demonstration that brain CYP2D-mediated opiate metabolic activation alters analgesia in vivo 
Biochemical pharmacology  2013;85(12):1848-1855.
The response to centrally-acting drugs is highly variable between individuals and does not always correlate with plasma drug levels. Drug-metabolizing CYP enzymes in the brain may contribute to this variability by affecting local drug and metabolite concentrations. CYP2D metabolizes codeine to the active morphine metabolite. We investigate the effect of inhibiting brain, and not liver, CYP2D activity on codeine-induced analgesia. Rats received intracerebroventricular injections of CYP2D inhibitors (20 μg propranolol or 40 μg propafenone) or vehicle controls. Compared to vehicle-pretreated rats, inhibitor-pretreated rats had: a) lower analgesia in the tail-flick test (p<0.05) and lower areas under the analgesia-time curve (p<0.02) within the first hour after 30 mg/kg subcutaneous codeine, b) lower morphine concentrations and morphine to codeine ratios in the brain (p<0.02 and p<0.05, respectively), but not in plasma (p>0.6 and p>0.7, respectively), tested at 30 min after 30 mg/kg subcutaneous codeine, and c) lower morphine formation from codeine ex vivo by brain membranes (p<0.04), but not by liver microsomes (p>0.9). Analgesia trended toward a correlation with brain morphine concentrations (p=0.07) and correlated with brain morphine to codeine ratios (p<0.005), but not with plasma morphine concentrations (p>0.8) or plasma morphine to codeine ratios (p>0.8). Our findings suggest that brain CYP2D affects brain morphine levels after peripheral codeine administration, and may thereby alter codeine's therapeutic efficacy, side-effect profile and abuse liability. Brain CYPs are highly variable due to genetics, environmental factors and age, and may therefore contribute to interindividual variation in the response to centrally-acting drugs.
PMCID: PMC3687523  PMID: 23623752
cytochrome P450; codeine; analgesia; drug metabolism; pharmacokinetics; neuropharmacology
6.  The ability of plasma cotinine to predict nicotine and carcinogen exposure is altered by differences in CYP2A6: the influence of genetics, race and sex 
Cotinine, a nicotine metabolite, is a biomarker of tobacco, nicotine and carcinogen exposure. However a given cotinine level may not represent the same tobacco exposure; for example, African Americans have higher cotinine levels than Caucasians after controlling for exposure.
Cotinine levels are determined by the amount of cotinine formation and the rate of cotinine removal which are both mediated by the enzyme CYP2A6. Since CYP2A6 activity differs by sex (estrogen induces CYP2A6) and genotype, their effect on cotinine formation and removal were measured in non-smoking Caucasians (Study 1, n=181) infused with labeled nicotine and cotinine. The findings were then extended to ad libitum smokers (Study 2, n=163).
Study 1: Reduced CYP2A6 activity altered cotinine formation less than cotinine removal resulting in ratios of formation to removal of 1.31 and 1.12 in CYP2A6 reduced and normal metabolizers (P=0.01), or 1.39 and 1.12 in males and females (P=0.001), suggesting an overestimation of tobacco exposure in slower metabolizers. Study 2: Cotinine again overestimated tobacco and carcinogen exposure by ≥25% in CYP2A6 reduced metabolizers (≈2 fold between some genotypes) and in males.
In people with slower, relative to faster, CYP2A6 activity cotinine accumulates resulting in substantial differences in cotinine levels for a given tobacco exposure.
Cotinine levels may be misleading when comparing those with differing CYP2A6 genotypes within a race, between races with differing frequencies of CYP2A6 gene variants (i.e. African Americans have higher frequencies of reduced function variants contributing to their higher cotinine levels) or between the sexes.
PMCID: PMC3617060  PMID: 23371292
Tobacco; Cotinine; CYP2A6; Polycyclic aromatic hydrocarbons; NNAL
7.  CYP2A6 slow nicotine metabolism is associated with increased quitting by adolescent smokers 
Pharmacogenetics and genomics  2013;23(4):232-235.
Variation in the CYP2A6 gene, that decreases the rate of nicotine metabolic-inactivation, is associated with higher adult smoking cessation rates during clinical trials. We hypothesized that slow metabolism is associated with increased quitting during adolescence. Caucasian adolescent smokers (N=308, aged 12 to 17, 36.3% male) from a cohort study were genotyped for CYP2A6 resulting in 7.8% slow, 14.0% intermediate and 78.2% normal metabolizers. Overall, 144 smokers quit smoking, as indicated by being abstinent for ≥12 months. In logistic regression analyses, the odds ratio for quitting was 2.25 (95% confidence interval 1.05, 4.80; P=0.037) for slow metabolizers relative to normal metabolizers. A linear trend toward increased quitting with decreasing CYP2A6 activity was also observed (odds ratio = 1.44, 95% confidence interval 1.02, 2.01; P=0.034). Thus, CYP2A6 slow metabolism is associated with increased adolescent smoking cessation, indicating that even early in the smoking history genetic variation is influencing smoking cessation.
PMCID: PMC3744214  PMID: 23462429
adolescent; longitudinal studies; epidemiology; smoking cessation; genetic association studies
8.  CYP2B6 and bupropion’s smoking cessation pharmacology: the role of hydroxybupropion 
Bupropion is indicated to promote smoking cessation. Animal studies suggest that bupropion’s major metabolite hydroxybupropion can mediate bupropion’s pharmacologic activity. We measured plasma bupropion and metabolite levels in a double-blind, placebo controlled, randomized smoking cessation trial. Among the treatment adherent individuals, higher hydroxybupropion concentrations (per µg/mL) resulted in better smoking cessation outcomes (Week 3, 7 and 26 OR=2.82, 2.96 and 2.37, P=0.005–0.040), this was not observed with bupropion levels (OR=1.00–1.03, P=0.59–0.90). Genetic variation in CYP2B6, the enzyme that metabolizes bupropion to hydroxybupropion, was identified as a significant source of variability in hydroxybupropion formation. Our data indicate that hydroxybupropion contributes to the pharmacologic effects of bupropion for smoking cessation, and that variability in response to bupropion treatment is related to variability in CYP2B6-mediated hydroxybupropion formation. These findings suggest dosing bupropion to achieve a hydroxybupropion level of 0.7 µg/ml or increasing bupropion dose for CYP2B6 slow metabolizers, could improve bupropion’s cessation outcomes.
PMCID: PMC3729209  PMID: 23149928
9.  Variation in Trans-3′-Hydroxycotinine Glucuronidation Does Not Alter the Nicotine Metabolite Ratio or Nicotine Intake 
PLoS ONE  2013;8(8):e70938.
CYP2A6 metabolizes nicotine to its primary metabolite cotinine and also mediates the metabolism of cotinine to trans-3′-hydroxycotinine (3HC). The ratio of 3HC to cotinine (the “nicotine metabolite ratio”, NMR) is an in vivo marker for the rate of CYP2A6 mediated nicotine metabolism, and total nicotine clearance, and has been associated with differences in numerous smoking behaviors. The clearance of 3HC, which affects the NMR, occurs via renal excretion and metabolism by UGT2B17, and possibly UGT2B10, to 3HC-glucuronide. We investigated whether slower 3HC glucuronidation alters NMR, altering its ability to predict CYP2A6 activity and reducing its clinical utility.
Plasma NMR, three urinary NMRs, three urinary 3HC glucuronidation phenotypes and total nicotine equivalents were examined in 540 African American smokers. The UGT2B17 gene deletion and UGT2B10*2 were genotyped.
The UGT2B17 gene deletion, but not UGT2B10*2 genotype, was associated with slower 3HC glucuronidation (indicated by three 3HC-glucuronidation phenotypes), indicating its role in this glucuronidation pathway. However, neither lower rates of 3HC glucuronidation, nor the presence of a UGT2B17 and UGT2B10 reduced function allele, altered plasma or urinary NMRs or levels of smoking.
Variation in 3HC glucuronidation activity, including these caused by UGT2B17 gene deletions, did not significantly alter NMR and is therefore unlikely to affect the clinical utility of NMR in smoking behavior and cessation studies. This study demonstrates that NMR is not altered by differences in the rate of 3HC glucuronidation, providing further support that NMR is a reliable indicator of CYP2A6 mediated nicotine metabolism.
PMCID: PMC3732272  PMID: 23936477
10.  Association of nicotine metabolite ratio and CYP2A6 genotype with smoking cessation treatment in African-American light smokers 
CYP2A6 is the main nicotine metabolizing enzyme in humans. We investigated the relationships between CYP2A6 genotype, baseline plasma 3HC/COT (a phenotypic marker of CYP2A6 activity), and smoking behaviors in African-American light smokers. Cigarette consumption, age of initiation, and dependence scores did not differ between 3HC/COT quartiles or CYP2A6 genotype groups. Slow metabolizers (both genetic and phenotypic) had significantly higher plasma nicotine levels suggesting cigarette consumption was not reduced to adjust for slower rates of nicotine metabolism. Individuals in the slowest 3HC/COT quartile had higher quit rates with both placebo and nicotine gum treatments (OR 1.85, 95% CI 1.08-3.16, p = 0.03). Similarly, the slowest CYP2A6 genotype group had higher quit rates, although this did not reach significance (OR 1.61, 95% CI 0.95-2.72, p = 0.08). 3HC/COT ratio, and possibly CYP2A6 genotype, may be useful in the future for personalizing the choice of smoking cessation treatment for African-American light smokers.
PMCID: PMC3698861  PMID: 19279561
Cytochrome P450 2A6; CYP2A6; nicotine; cotinine; trans-3′-hydroxycotinine; African-Americans; smoking; light smokers
11.  CYP2A6 and CYP2B6 genetic variation and its association with nicotine metabolism in South Western Alaska Native people 
Pharmacogenetics and Genomics  2012;22(6):429-440.
Alaska Native people (AN) have a high prevalence of tobacco use and associated morbidity and mortality when compared to the general U.S. population. Variation in the CYP2A6 and CYP2B6 genes, encoding enzymes responsible for nicotine metabolic inactivation and procarcinogen activation, has not been characterized in AN and may contribute to the increased risk.
AN people (n = 400) residing in the Bristol Bay region of South Western Alaska were recruited for a cross-sectional study on tobacco use. They were genotyped for CYP2A6*1X2A, *1X2B, *1B, *2, *4, *7, *8, *9, *10, *12, *17, *35 and CYP2B6*4, *6, *9 and provided plasma and urine samples for measurement of nicotine and metabolites.
CYP2A6 and CYP2B6 variant frequencies among the AN Yupik people (n=361) were significantly different from other ethnicities. Nicotine metabolism (as measured by the plasma and urinary ratio of metabolites trans-3’hydroxycotinine to cotinine [(3HC/COT)] was significantly associated with CYP2A6 (P< 0.001) but not CYP2B6 genotype (P = 0.95) when controlling for known covariates. Of note, plasma 3HC/COT ratios were high in the entire Yupik people, and among the Yupik CYP2A6 wild-type participants they were substantially higher than previously characterized racial/ethnic groups (P < 0.001 vs. Caucasians and African Americans).
Yupik AN people have a unique CYP2A6 genetic profile which associated strongly with in vivo nicotine metabolism. More rapid CYP2A6-mediated nicotine and nitrosamine metabolism in the Yupik people may modulate tobacco-related disease risk.
PMCID: PMC3349071  PMID: 22569203
CYP2A6; CYP2B6; nicotine; tobacco; smoking; genetic variation; Alaska Native people
12.  Relationship Between CYP2A6 and CHRNA5-CHRNA3-CHRNB4 Variation and Smoking Behaviors and Lung Cancer Risk 
Genetic variations in the CYP2A6 nicotine metabolic gene and the CHRNA5-CHRNA3-CHRNB4 (CHRNA5-A3-B4) nicotinic gene cluster have been independently associated with lung cancer. With genotype data from ever-smokers of European ancestry (417 lung cancer patients and 443 control subjects), we investigated the relative and combined associations of polymorphisms in these two genes with smoking behavior and lung cancer risk. Kruskal–Wallis tests were used to compare smoking variables among the different genotype groups, and odds ratios (ORs) for cancer risk were estimated using logistic regression analysis. All statistical tests were two-sided. Cigarette consumption (P < .001) and nicotine dependence (P = .036) were the highest in the combined CYP2A6 normal metabolizers and CHRNA5-A3-B4 AA (tag single-nucleotide polymorphism rs1051730 G>A) risk group. The combined risk group also exhibited the greatest lung cancer risk (OR = 2.03; 95% confidence interval [CI] = 1.21 to 3.40), which was even higher among those who smoked 20 or fewer cigarettes per day (OR = 3.03; 95% CI = 1.38 to 6.66). Variation in CYP2A6 and CHRNA5-A3-B4 was independently and additively associated with increased cigarette consumption, nicotine dependence, and lung cancer risk. CYP2A6 and CHRNA5-A3-B4 appear to be more strongly associated with smoking behaviors and lung cancer risk, respectively.
PMCID: PMC3168937  PMID: 21747048
13.  Utility and relationships of biomarkers of smoking in African-American light smokers 
While expired carbon monoxide (CO) and plasma cotinine (COT) have been validated as biomarkers of self-reported cigarettes per day (CPD) in heavy smoking Caucasians, their utility in light smokers is unknown. Further, variability in CYP2A6, the enzyme that mediates formation of COT from nicotine (NIC) and its metabolism to trans-3′-hydroxycotinine (3HC), may limit the usefulness of COT. We assessed whether CO and COT are correlated with CPD in African-American light smokers (≤10CPD, n=700), a population with known reduced CYP2A6 activity and slow COT metabolism. We also examined whether gender, age, BMI, smoking mentholated cigarettes or rate of CYP2A6 activity, by genotype and phenotype measures (3HC/COT), influence these relationships. At baseline, many participants (42%) exhaled CO ≤10ppm, the traditional cutoff for smoking, while few (3.1%) had COT below the cutoff of ≤14ng/ml; thus COT appears to be a better biomarker of smoking status in this population. CPD was weakly correlated with CO and COT (r = 0.32–0.39, p<0.001), and those reporting fewer CPD had higher CO/cigarette and COT/cigarette, although the correlations coefficients between these variables were also weak (r = −0.33 and −0.08, p < 0.05). The correlation between CPD and CO was not greatly increased when analyzed by CYP2A6 activity, smoking mentholated cigarettes or age, although it appeared stronger in females (r = 0.38 vs.0.21, p<0.05) and obese individuals (r = 0.38 vs.0.24, p<0.05). Together, these results suggest that CO and COT are weakly associated with self-reported cigarette consumption in African-American light smokers, and that these relationships are not substantially improved when variables previously reported to influence these biomarkers are considered.
PMCID: PMC2791893  PMID: 19959692
biomarkers; smoking; cotinine; carbon monoxide; African-Americans
14.  Pharmacogenetics: A Tool for Identifying Genetic Factors in Drug Dependence and Response to Treatment 
Pharmacogenetics research looks at variations in the human genome and ways in which genetic factors might influence how individuals respond to drugs. The authors review basic principles of pharmacogenetics and cite findings from several gene-phenotype studies to illustrate possible associations between genetic variants, drug-related behaviors, and risk for drug dependence. Some gene variants affect responses to one drug; others, to various drugs. Pharmacogenetics can inform medication development and personalized treatment strategies; challenges lie along the pathway to its general use in clinical practice.
PMCID: PMC3120126  PMID: 22002450
15.  A novel CYP2A6 allele (CYP2A6*35) resulting in an amino acid substitution (Asn438Tyr) is associated with lower CYP2A6 activity in vivo 
The pharmacogenomics journal  2009;9(4):274-282.
Cytochrome P450 2A6 (CYP2A6) is the primary human enzyme involved in nicotine metabolism. Our objective was to characterize two nonsynonymous single nucleotide polymorphisms (SNPs) in CYP2A6*24, 594G>C (Val110Leu) and 6458A>T (Asn438Tyr). We determined their haplotype, allele frequencies, effect on CYP2A6 activity in vivo, as well as their stability and ability to metabolize nicotine in vitro. CYP2A6*35 (6458A>T) occurred at a frequency of 2.5–2.9% among individuals of black African descent, 0.5–0.8% among Asians, and was not found in Caucasians. In addition, we identified two novel alleles, CYP2A6*36 (6458A>T and 6558T>C [Ile471Thr]) and CYP2A6*37 (6458A>T, 6558T>C, and 6600G>T [Arg485Leu]). In vivo, CYP2A6*35 was associated with lower CYP2A6 activity as measured by the 3HC/COT ratio. In vitro, CYP2A6.35 had decreased nicotine C-oxidation activity and thermal stability. In conclusion, we identified three novel CYP2A6 alleles (CYP2A6*35, *36, and *37); the higher allele frequency variant CYP2A6*35 was associated with lower CYP2A6 activity.
PMCID: PMC2922203  PMID: 19365400 CAMSID: cams1468
Nicotine; Cotinine; CYP2A6; Smoking; Pharmacogenetics
16.  Identification of novel CYP2A6*1B variants; the CYP2A6*1B allele is associated with faster in vivo nicotine metabolism 
Cytochrome P450 2A6 (CYP2A6) is the human enzyme responsible for the majority of nicotine’s metabolism. CYP2A6 genetic variants contribute to the inter-individual and inter-ethnic variation in nicotine metabolism. We examined the association between the CYP2A6*1B variant and nicotine’s in vivo metabolism.
Intravenous infusions of deuterium-labeled nicotine were administered to 292 volunteers, 163 of whom were White and did not have common CYP2A6 variants, other than CYP2A6*1B.
We discovered three novel CYP2A6*1B variants in the 3′-flanking region of the gene that can confound genotyping assays. We found significant differences between CYP2A6*1A/*1A, CYP2A6*1A/*1B and CYP2A6*1B/*1B groups in total nicotine clearance (17.2±5.2, 19.0±6.4 and 20.4±5.9, P < 0.02), nonrenal nicotine clearance (16.4±5.0, 18.5±6.2 and 19.8±5.7, P < 0.01) and the plasma 3HC/COT ratio (0.26±0.1, 0.26±0.1 and 0.34±0.1, P < 0.001). There were also differences in total nicotine (29.4±12.9, 25.8±0.12.9 and 22.4±12.4, P < 0.01), cotinine (29.2±8.1, 32.2±9.1 and 33.0±6.6, P < 0.01) and trans-3′-hydroxcotinine (32.4±9.1, 34.2±12.3 and 41.3±11.3, P < 0.001) excreted in the urine.
We report evidence that CYP2A6*1B genotype is associated with faster nicotine clearance in vivo, which will be important to future CYP2A6 genotype association studies.
PMCID: PMC2921956  PMID: 17522595 CAMSID: cams1335
17.  Utility and relationships of biomarkers of smoking in African-American light smokers 
While expired carbon monoxide (CO) and plasma cotinine (COT) have been validated as biomarkers of self-reported cigarettes per day (CPD) in heavy smoking Caucasians, their utility in light smokers is unknown. Further, variability in CYP2A6, the enzyme that mediates formation of COT from nicotine (NIC) and its metabolism to trans-3′-hydroxycotinine (3HC), may limit the usefulness of COT. We assessed whether CO and COT are correlated with CPD in African-American light smokers (≤10CPD, n=700), a population with known reduced CYP2A6 activity and slow COT metabolism. We also examined whether gender, age, BMI, smoking mentholated cigarettes or rate of CYP2A6 activity, by genotype and phenotype measures (3HC/COT), influence these relationships. At baseline, many participants (42%) exhaled CO ≤10ppm, the traditional cutoff for smoking, while few (3.1%) had COT below the cutoff of ≤14ng/ml; thus COT appears to be a better biomarker of smoking status in this population. CPD was weakly correlated with CO and COT (r = 0.32–0.39, p<0.001), and those reporting fewer CPD had higher CO/cigarette and COT/cigarette, although the correlations coefficients between these variables were also weak (r = −0.33 and −0.08, p < 0.05). The correlation between CPD and CO was not greatly increased when analyzed by CYP2A6 activity, smoking mentholated cigarettes or age, although it appeared stronger in females (r = 0.38 vs.0.21, p<0.05) and obese individuals (r = 0.38 vs.0.24, p<0.05). Together, these results suggest that CO and COT are weakly associated with self-reported cigarette consumption in African-American light smokers, and that these relationships are not substantially improved when variables previously reported to influence these biomarkers are considered.
PMCID: PMC2791893  PMID: 19959692 CAMSID: cams1333
biomarkers; smoking; cotinine; carbon monoxide; African-Americans
18.  A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent 
Pharmacogenetics and genomics  2008;18(1):67-75.
CYP2A6 is the main enzyme involved in nicotine metabolism in humans. We have identified a novel allele, CYP2A6*23 (2161C>T, R203C), in individuals of Black-African descent and investigated its impact on enzyme activity and association with smoking status.
Wildtype and variant enzymes containing amino acid changes R203C (CYP2A6*23), R203S (CYP2A6*16) and V365M (CYP2A6*17) were expressed in Escherichia coli. The effect of CYP2A6*23 in vivo was examined in individuals of Black-African descent given 4 mg oral nicotine.
CYP2A6*23 occurred at an allele frequency of 2.0% in individuals of Black-African descent (N = 560 alleles, 95% confidence interval 0.8%–3.1%) and was not detected in Caucasians (N = 334 alleles), Chinese (N = 288 alleles) or Japanese (N = 104 alleles). In vitro, CYP2A6.23 had greatly reduced activity towards nicotine C-oxidation similar to CYP2A6.17, as well as reduced coumarin 7-hydroxylation. Conversely, CYP2A6.16 did not differ in activity compared to the wildtype enzyme. The trans-3′-hydroxycotinine to cotinine ratio, a phenotypic measure of CYP2A6 activity in vivo, was lower in CYP2A6*1/*23 and CYP2A6*23/*23 individuals(mean adjusted ratio of 0.60, n = 5) compared to CYP2A6*1/*1 individuals (mean adjusted ratio of 1.21, n = 150) (p < 0.04). CYP2A6*23 trended towards a higher allele frequency in nonsmokers (3.1%, N = 9/286 alleles) compared to smokers (0.7%, N = 2/274 alleles) (p = 0.06).
These results suggest the novel CYP2A6*23 allele impairs enzyme function in vitro and in vivo and trends toward an association with lower risk of smoking.
PMCID: PMC2910083  PMID: 18216723 CAMSID: cams1332
CYP2A6; genetic polymorphisms; Black-Africans; nicotine; smoking
19.  Socioeconomic and drug use determinants of smoking status in a Canadian urban adult population of black African descent 
Our aim was to examine the influence that socioeconomics and drug use had on current smokers (n=137) and nonsmokers (n=143) from an urban adult population of black African descent. Participants were a median of 33 years (range 20–59). Smokers consumed a median of 8 cigarettes per day (range 0–35). Interestingly, 86% smoked <15 cigarettes per day and only 8% smoked menthol cigarettes. Socioeconomic and drug use variables that were significantly associated with smoking status in univariate analyses were included in a multiple logistic regression model that controlled for gender and age. Compared to nonsmokers, smokers were less likely to be university educated (adjusted odds ratio (AOR)=0.08 [95% CI: 0.02–0.43]), more likely to be divorced/separated/widowed (AOR=10.3 [2.8–37.6]), more likely to be current alcohol users (AOR=3.5 [1.8–7.0]) and more likely to be current marijuana users (AOR=7.1 [3.5–14.2]). Unexpectedly, household income and employment status were not associated with smoking status. Among current alcohol users, smokers consumed three times the number of drinks per month compared to nonsmokers (median 12 vs. 4, respectively; P<0.001). Among current marijuana users, smokers consumed over five times the number of joints per month compared to nonsmokers (median 22 vs. 4, respectively; P<0.001). Overall, lower education, divorce, alcohol and marijuana use were significantly associated with increased likelihood of smoking among this population.
PMCID: PMC2896966  PMID: 18686179 CAMSID: cams1336
black African descent; smoking; socioeconomics; drug use
20.  Drug Metabolizing Enzyme and Transporter Gene Variation, Nicotine Metabolism, Prospective Abstinence, and Cigarette Consumption 
PLoS ONE  2015;10(7):e0126113.
The Nicotine Metabolite Ratio (NMR, ratio of trans-3’-hydroxycotinine and cotinine), has previously been associated with CYP2A6 activity, response to smoking cessation treatments, and cigarette consumption. We searched for drug metabolizing enzyme and transporter (DMET) gene variation associated with the NMR and prospective abstinence in 2,946 participants of laboratory studies of nicotine metabolism and of clinical trials of smoking cessation therapies. Stage I was a meta-analysis of the association of 507 common single nucleotide polymorphisms (SNPs) at 173 DMET genes with the NMR in 449 participants of two laboratory studies. Nominally significant associations were identified in ten genes after adjustment for intragenic SNPs; CYP2A6 and two CYP2A6 SNPs attained experiment-wide significance adjusted for correlated SNPs (CYP2A6 PACT=4.1E-7, rs4803381 PACT=4.5E-5, rs1137115, PACT=1.2E-3). Stage II was mega-regression analyses of 10 DMET SNPs with pretreatment NMR and prospective abstinence in up to 2,497 participants from eight trials. rs4803381 and rs1137115 SNPs were associated with pretreatment NMR at genome-wide significance. In post-hoc analyses of CYP2A6 SNPs, we observed nominally significant association with: abstinence in one pharmacotherapy arm; cigarette consumption among all trial participants; and lung cancer in four case:control studies. CYP2A6 minor alleles were associated with reduced NMR, CPD, and lung cancer risk. We confirmed the major role that CYP2A6 plays in nicotine metabolism, and made novel findings with respect to genome-wide significance and associations with CPD, abstinence and lung cancer risk. Additional multivariate analyses with patient variables and genetic modeling will improve prediction of nicotine metabolism, disease risk and smoking cessation treatment prognosis.
PMCID: PMC4488893  PMID: 26132489
21.  The Relationship between the Nicotine Metabolite Ratio and Three Self-Report Measures of Nicotine Dependence Across Sex and Race 
Psychopharmacology  2014;231(12):2515-2523.
Variability in the rate of nicotine metabolism, measured by the nicotine metabolite ratio (NMR), is associated with smoking behavior. However, data linking the NMR with nicotine dependence measured by the Fagerström Test for Nicotine Dependence (FTND) are mixed. Few past studies have examined alternative measures of nicotine dependence and how this relationship may vary by sex and race.
Using data from smokers undergoing eligibility evaluation for a smoking cessation clinical trial (n=833), this study examined variability in the relationship between NMR and nicotine dependence across sex and race and using three measures of nicotine dependence: FTND, time-to-first-cigarette (TTFC), and the Heaviness of Smoking Index (HSI).
Controlling for sex and race, nicotine metabolism was associated with nicotine dependence only when using the HSI (p < .05). Male normal metabolizers of nicotine were more likely to have high nicotine dependence based on the FTND and HSI (p < .05), but NMR was not related to measures of nicotine dependence in women. For African Americans, the NMR was associated with nicotine dependence only for the TTFC (p < .05) but NMR was not associated with nicotine dependence among Caucasians. Post-hoc analyses indicated that the NMR was associated with cigarettes per day, overall and among men and Caucasians (p < .05).
While there was some variation in the relationship between nicotine metabolism and nicotine dependence across measures and sex and race, the results indicate that this relationship may be more attributable to the association between NMR and cigarettes per day.
PMCID: PMC4040302  PMID: 24402139
Nicotine metabolite ratio; nicotine dependence; race; sex; Fagerström; time-to-first cigarette; heaviness of smoking index; cigarettes per day
22.  Lack of Associations of CHRNA5-A3-B4 Genetic Variants with Smoking Cessation Treatment Outcomes in Caucasian Smokers despite Associations with Baseline Smoking 
PLoS ONE  2015;10(5):e0128109.
CHRNA5-A3-B4 variants, rs16969968, rs588765 and rs578776, are consistently associated with tobacco consumption among smokers, but the association with smoking cessation is less consistent. Among the studies that reported significant associations with cessation, the effects were observed in smokers treated with placebo treatment in some studies and conversely in those receiving active pharmacological therapy (bupropion and nicotine replacement therapies) in others. Thus, it remains unclear whether CHRNA5-A3-B4 is a useful marker for optimizing smoking cessation. Using data from 654 Caucasian smokers treated with placebo, nicotine patch or varenicline, we investigated whether CHRNA5-A3-B4 variants were associated with smoking cessation outcomes, and whether there were significant genotype-by-treatment or haplotype-by-treatment interactions. We observed no significant associations between CHRNA5-A3-B4 variants and smoking cessation, despite replicating previous associations with baseline tobacco consumption. At end of treatment the effect size on smoking cessation in the placebo, patch and varenicline groups for rs16969968 [GG vs. GA+AA] was OR = 0.66 (P = 0.23), OR = 1.01 (P = 0.99), and OR = 1.30 (P = 0.36) respectively, of rs588765 [CC vs. CT+TT] was OR = 0.96 (P = 0.90), OR = 0.84 (P = 0.58), and OR = 0.74 (P = 0.29) respectively, and for rs578776 [GG vs. GA+AA] on smoking cessation was OR = 1.02 (P = 0.95), OR = 0.75 (P = 0.35), and OR = 1.20 (P = 0.51) respectively. Furthermore, we observed no associations with cessation using the CHRNA5-A3-B4 haplotype (constructed using rs16969968 and rs588765), nor did we observe any significant genotype-by-treatment interactions, with or without adjusting for the rate of nicotine metabolism (all P>0.05). We also observed no significant genetic associations with 6 month or 12 month smoking abstinence. In conclusion, we found no association between CHRNA5-A3-B4 variants and smoking cessation rates in this clinical trial; however, as expected, significant associations with baseline tobacco consumption were replicated. Our data suggest that CHRNA5-A3-B4 gene variants do not exhibit a robust association with smoking cessation and are unlikely to be useful for clinically optimizing smoking cessation pharmacotherapy for Caucasian smokers.
PMCID: PMC4444267  PMID: 26010901
23.  Nicotine dependence as a moderator of genetic influences on smoking cessation treatment outcome☆ 
Drug and alcohol dependence  2014;138:109-117.
Genetic influences on smoking cessation treatment outcome may be affected by pretreatment patient characteristics. Nicotine dependence is arguably the most salient clinical factor in smoking cessation.
In this secondary analysis of clinical trial data (N = 793), we examined nicotine dependence severity as a moderator of the effects of 1198 single nucleotide polymorphisms (SNPs) in 53 biologically-relevant gene regions on smoking cessation outcomes. P-values were adjusted to account for multiple correlated SNPs within a gene region; corrected system-wide significance was 5 × 10−4.
SNP × nicotine dependence interactions reached region-wide significance for several SNPs in the Dopamine Beta Hydroxylase (DBH) locus (0.0005 < Adjusted-P < 0.05), including rs1541333, which reached system-wide significance for predicting end of treatment (EOT) abstinence (Adjusted-P = 0.0004). A haplotype including 6 DBH SNPs predicted abstinence at EOT (OR = 1.7, P = 0.001) and 6-month follow-up (OR = 1.6, P = 0.008) in those with high nicotine dependence (n = 526) but not in those with low dependence (n = 227). The DBH signal observed here may be distinct from a previously reported genome-wide significant signal for former smoking status and from the principal haplotype associated with plasma dopamine beta-hydroxylase activity. A haplotype within the Chromosome 15 Nicotinic Acetylcholine Receptor gene region predicted abstinence at EOT in those with high (OR = 2.0, P = 0.0004) but not low (P = 0.6) dependence in post hoc analyses.
Considering pre-treatment nicotine dependence level may optimize the prediction of genetic influences on cessation outcomes. If replicated, results like these may inform prognosticative genomic screening panels designed to identify smokers at high risk of relapse when coupled with severe nicotine dependence.
PMCID: PMC4095777  PMID: 24667010
Genetics; Smoking cessation; Nicotine dependence; Dopamine
24.  Pharmacokinetic and Pharmacodynamics Studies of Nicotine After Oral Administration in Mice: Effects of Methoxsalen, a CYP2A5/6 Inhibitor 
Nicotine & Tobacco Research  2013;16(1):18-25.
The use of novel oral nicotine delivery devices and compositions for human consumption and for animal research studies has been increasing in the last several years.
Studies were undertaken to examine whether the systemic administration of methoxsalen, an inhibitor of human CYP2A6 and mouse CYP2A5, would modulate nicotine pharmacokinetics and pharmacological effects (antinociception in the tail-flick, and hot-plate tests, and hypothermia) in male ICR mouse after acute oral nicotine administration.
Administration of intra peritoneal (ip) methoxsalen significantly increased nicotine’s Cmax, prolonged the plasma half-life (fourfold decrease) of nicotine, and increased its area under the curve (AUC) compared with ip vehicle treatment. Methoxsalen pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (15mg/kg, po) for periods up to 6- and 24-hr postnicotine administration, respectively. Additionally, methoxsalen potentiated nicotine-induced antinociception and hypothermia as evidenced by leftward shifts in nicotine’s dose–response curve. Furthermore, this prolongation of nicotine’s effects after methoxsalen was associated with a parallel prolongation of nicotine plasma levels in mice. These data strongly suggest that variation in the rates of nicotine metabolic inactivation substantially alter pharmacological effects of nicotine given orally.
We have shown that the pharmacological effects of inhibiting nicotine’s metabolism after oral administration in mice are profound. Our results suggest that inhibiting nicotine metabolism can be used to dramatically enhance nicotine’s bioavailability and its resulting pharmacology, which further supports this inhibitory approach for clinical development of an oral nicotine replacement therapy.
PMCID: PMC3864487  PMID: 23884323
25.  Factors Associated with Discontinuation of Bupropion and Counseling among African American Light Smokers in a Randomized Clinical Trial 
African Americans are at risk for inadequate adherence to smoking cessation treatment yet little is known about what leads to treatment discontinuation.
Examine the factors associated with discontinuation of treatment in African American light smokers (≤10 cigarettes per day).
Bupropion plasma levels and counseling attendance were measured among 540 African American light smokers in a placebo-controlled randomized trial of bupropion.
By Week 3, 28.0% of subjects in the bupropion arm had discontinued bupropion and only moderate associations were found between plasma levels and self-reported bupropion use (rs=0.38). By Week 16, 36.9% of all subjects had discontinued counseling. Males had greater odds of discontinuing medication (OR=2.02, 95% CI, 1.10–3.71, p=0.02) and older adults had lower odds of discontinuing counseling (OR=0.96, 95% CI, 0.94–0.97, p<0.0001).
Bupropion and smoking cessation counseling are underutilized even when provided within the context of a randomized trial. Future research is needed to examine strategies for improving treatment utilization among African American smokers.
PMCID: PMC3815499  PMID: 23733379
Smoking cessation; discontinuation of treatment; African Americans

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