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1.  Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol 
Chemico-Biological Interactions  2012;197(2-3):87-92.
Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (β-nicotyrine) and (−)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (−)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2 μM and 110 μM, respectively). β-nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 μM). Neither menthol nor β-nicotyrine were inactivators of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106 μM, kinact was 0.61 min−1). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that β-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.
PMCID: PMC3362486  PMID: 22486895
Cytochrome P450; Inhibition; Inactivation; CYP2A6; CYP2A13; menthol; menthofuran; β-nicotyrine
2.  Cotinine and trans 3'-Hydroxycotinine in Dried Blood Spots as Biomarkers of Tobacco Exposure and Nicotine Metabolism 
Tobacco use is the major preventable cause of premature death in the United States. Secondhand smoke (SHS) exposure also contributes to a number of premature deaths as well as other negative health outcomes. An accurate assessment of tobacco smoke exposure is critical to understanding these disease processes. The plasma concentration of cotinine, the primary metabolite of nicotine, is widely accepted as a quantitative measure of tobacco and SHS exposure. However, it is not always feasible to collect plasma. Dried blood spots (DBS), which are collected routinely from newborns and often from young children for lead screening, provide an alternative sampling method. We have developed a quantitative high throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the analysis of cotinine in DBS. The limit of quantitation was 0.3 ng/g (~ 0.2 ng/ml plasma). Cotinine levels in DBS from 83 smokers and 99 nonsmokers exposed to SHS were determined. Plasma cotinine concentrations in these subjects ranged from <0.02 to 443 ng/ml. Cotinine was detected in DBS from 157 subjects, and the correlation between cotinine in plasma and DBS was excellent, 0.992 (p<0.001). We also determined the ratio of trans-3'-hydroxycotinine to cotinine, a measure of nicotine metabolism, in DBS from smokers. This ratio in DBS was well correlated with the ratio in plasma, 0.94 (p<0.001). In a small study we confirmed the feasibility of using extant DBS collected for lead screening to assess SHS exposure in children.
PMCID: PMC4048618  PMID: 23443235
Second hand smoke exposure; lead; smoking
3.  Effects upon in vivo nicotine metabolism reveal functional variation in FMO3 associated with cigarette consumption 
Pharmacogenetics and genomics  2013;23(2):62-68.
Flavin-containing monooxygenases (FMO) catalyze the metabolism of nucleophilic heteroatom containing drugs and xenobiotics including nicotine. Rare mutations in FMO3 are responsible for defective N-oxygenation of dietary trimethylamine leading to trimethylaminuria, and common genetic variation in FMO3 has been linked to interindividual variability in metabolic function that may be substrate specific.
A genetic model of CYP2A6 function is used as a covariate to reveal functional polymorphism in FMO3 that indirectly influences the ratio of deuterated nicotine metabolized to cotinine following oral administration. The association is tested between FMO3 haplotype and cigarette consumption in a set of nicotine dependent smokers.
FMO3 haplotype, based on all common coding variants in Europeans, significantly predicts nicotine metabolism and accounts for approximately 2% of variance in the apparent percent of nicotine metabolized to cotinine. The metabolic ratio is not associated with FMO2 haplotype or an FMO1 expression quantitative trait locus (eQTL). Cross validation demonstrates calculated FMO3 haplotype parameters to be robust and significantly improve the predictive nicotine metabolism model over CYP2A6 genotype alone. Functional classes of FMO3 haplotypes, as determined by their influence on nicotine metabolism to cotinine, are also significantly associated with cigarettes per day (CPD) in nicotine dependent European Americans (n=1,025, p=0.04), and significantly interact (p=0.016) with CYP2A6 genotype to predict CPD.
These findings suggest that common polymorphisms in FMO3 influence nicotine clearance, and that these genetic variants in turn influence cigarette consumption.
PMCID: PMC4583063  PMID: 23211429
4.  Chronic Nicotine Consumption does not Influence 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Induced Lung Tumorigenesis 
Nicotine replacement therapy (NRT) is often used to maintain smoking cessation. However, concerns exist about the safety of long term NRT use in ex-smokers and its concurrent use in smokers. In this study, we determined the effect of nicotine administration on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors in A/J mice. Female mice were administered a single dose of NNK (10 μmol) and 0.44 μmol/ml nicotine in the drinking water. Nicotine was administered 2 weeks prior to NNK, 44 weeks after NNK, throughout the experiment, or without NNK treatment. The average weekly consumption of nicotine-containing water was 15 ± 3 mls/mouse, resulting in an estimated daily nicotine dose of 0.9 μmol (0.15 mg) per mouse. Nicotine administration alone for 46 weeks did not increase lung tumor multiplicity (0.32 ± 0.1 tumor/mouse versus 0.53 ± 0.1 tumors/mouse). Lung tumor multiplicity in NNK-treated mice was 18.4 ± 4.5 and was not different than for mice consuming nicotine before or after NNK administration, 21.9 ± 5.3 and 20.0 ± 5.4 tumors per mouse, respectively. Lung tumor multiplicity in animals consuming nicotine both before and after NNK administration was 20.4 ± 5.4. Tumor size and progression of adenomas to carcinomas was also not affected by nicotine consumption. In addition, nicotine consumption had no effect on the level of O6-methylguanine in the lung of NNK-treated mice. These negative findings in a commonly used model of human lung carcinogenesis should lead us to question the interpretation of the many in vitro studies that find nicotine stimulates cancer cell growth.
PMCID: PMC3357110  PMID: 22027684
Nicotine; NNK; A/J mouse; O6-methylguanine
5.  Reduced nicotine content cigarettes: effects on toxicant exposure, dependence and cessation 
Addiction (Abingdon, England)  2010;105(2):343-355.
To examine the effects of reduced nicotine cigarettes on smoking behavior, toxicant exposure, dependence and abstinence.
Randomized, parallel arm, semi-blinded study.
University of Minnesota Tobacco Use Research Center.
Six weeks of: (i) 0.05 mg nicotine yield cigarettes; (ii) 0.3 mg nicotine yield cigarettes; or (iii) 4 mg nicotine lozenge; 6 weeks of follow-up.
Compensatory smoking behavior, biomarkers of exposure, tobacco dependence, tobacco withdrawal and abstinence rate.
Unlike the 0.3 mg cigarettes, 0.05 mg cigarettes were not associated with compensatory smoking behaviors. Furthermore, the 0.05 mg cigarettes and nicotine lozenge were associated with reduced carcinogen exposure, nicotine dependence and product withdrawal scores. The 0.05 mg cigarette was associated with greater relief of withdrawal from usual brand cigarettes than the nicotine lozenge. The 0.05 mg cigarette led to a significantly higher rate of cessation than the 0.3 mg cigarette and a similar rate as nicotine lozenge.
The 0.05 mg nicotine yield cigarettes may be a tobacco product that can facilitate cessation; however, future research is clearly needed to support these preliminary findings.
PMCID: PMC4565618  PMID: 20078491
Biomarkers of exposure; compensatory smoking; nicotine dependence; reduced nicotine cigarettes; tobacco cessation; tobacco withdrawal
6.  Exposure to a Tobacco-Specific Lung Carcinogen in Adolescent vs. Adult Smokers 
Previous studies with adult smokers have shown an association between number of cigarettes smoked per day (CPD) and levels of biomarkers of exposure to the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). This study compared carcinogen and nicotine exposure in adolescent and adult smokers across categories of CPD.
Baseline smoking history and biomarker data were merged from six studies to make two samples: one of adolescent smokers and one of adult smokers. Metabolites of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides (NNAL-Glucs) and total cotinine were quantified in urine.
CPD was stratified into categories of 5–10, 11–15, and 16–20 CPD. Adolescents tended to have lower mean levels of NNAL plus NNAL-Glucs (total NNAL) compared to adults, although differences were not significant overall. Adolescent mean levels of NNAL/CPD were significantly lower than adult levels only in the 11–15 CPD category (p = 0.045). However, a significant positive relationship was observed for total NNAL/CPD by age. No significant differences between adolescents and adults were found in mean levels of total cotinine or cotinine/CPD. A sub-sample of urines from adolescents and adults were analyzed for NNAL-Glucs and NNAL. Adolescents and adults did not significantly differ in the ratio of NNAL-Glucs to NNAL.
Adolescent uptake of NNK and nicotine tends to be lower, although not statistically different from adults. The lack of significant differences may be due to the wide variation in exposure in adolescents. Some adolescent smokers are exposed to lung carcinogens at levels similar to those of adults.
PMCID: PMC4547466  PMID: 19064548
Adolescent; Biomarkers; Toxicant Exposure; Tobacco; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL); Cotinine
7.  UGT2B10 genotype influences nicotine glucuronidation, oxidation and consumption 
Tobacco exposure is routinely assessed by quantifying nicotine metabolites in plasma or urine. On average, 80% of nicotine undergoes C-oxidation to cotinine. However, interindividual variation in nicotine glucuronidation is substantial and glucuronidation accounts for from 0 to 40% of total nicotine metabolism. We report here the effect of a polymorphism in a UDP-glucuronsyl transferase, UGT2B10, on nicotine metabolism and consumption.
Nicotine, cotinine, their N-glucuronide conjugates, and total trans-3'-hydroxycotinine were quantified in the urine (n=327) and plasma (n =115) of smokers. Urinary nicotine N-oxide was quantified in 105 smokers. Nicotine equivalents, the sum of nicotine and all major metabolites, were calculated for each smoker. The relationship of the UGT2B10 Asp67Tyr allele to nicotine equivalents, N-glucuronidation, and C-oxidation was determined.
Individuals heterozygous for the Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than wild-type, resulting in a 60% lower ratio of cotinine glucuronide:cotinine, a 50% lower ratio of nicotine glucuronide:nicotine and increased cotinine and trans-3'-hydroxycotinine. Nicotine equivalents, a robust biomarker of nicotine intake, were lower among Asp67Tyr heterozygotes compared to individuals without this allele; 58.2 nmol/ml (95% CI, 48.9 – 68.2) versus 69.2 nmol/ml (95% CI, 64.3 – 74.5).
Individuals heterozygous for UGT2B10 Asp67Tyr consume less nicotine than do wild type smokers. This striking observation suggests that variations in nicotine N-glucuronidation, as reported for nicotine C-oxidation, may influence smoking behavior.
UGT2B10 genotype influences nicotine metabolism and should be taken into account when characterizing the role of nicotine metabolism on smoking.
PMCID: PMC2882998  PMID: 20501767
Nicotine; glucuronide; UGT2B10; cotinine; smoking; glucuronidation
8.  Prenatal Tobacco Exposure and Cotinine in Newborn Dried Blood Spots 
Pediatrics  2014;133(6):e1632-e1638.
Tobacco smoking by pregnant women is a major public health hazard with both short- and long-term effects on offspring. This study describes the presence and level of the nicotine metabolite cotinine in newborn dried blood spots (DBS) and compares it with the reported maternal smoking recorded on state birth registries. We hypothesize that cotinine in DBS may be a useful measure of newborn in utero tobacco exposure.
An observational, cross-sectional study of 1414 DBS obtained from California, Michigan, New York, and Washington newborn screening programs was carried out. Cotinine levels in DBS were quantified by liquid chromatography tandem mass spectrometry analysis and compared with maternal smoking as reported in vital statistics data.
Cotinine ≥0.3 ng/g was detected in 35% of newborn DBS, including DBS of 29% of newborns whose mothers reportedly did not smoke cigarettes during pregnancy, some of whom were presumably exposed to environmental tobacco smoke. Twelve percent of the newborn DBS had cotinine levels that were ≥9.0 ng/g (equivalent to 6 ng/mL plasma, a level that indicates active smoking of the mother), although 41% of the mothers of these infants reportedly did not smoke.
These data confirm that reported smoking during pregnancy is an imperfect measure of prenatal tobacco smoke exposure. Cotinine assessment in newborns may improve surveillance of tobacco use during pregnancy.
PMCID: PMC4035592  PMID: 24819573
cotinine; dried blood spot testing; newborn
9.  Elevated Levels of Mercapturic Acids of Acrolein and Crotonaldehyde in the Urine of Chinese Women in Singapore Who Regularly Cook at Home 
PLoS ONE  2015;10(3):e0120023.
Lung cancer is unusually common among non-smoking women in Southeastern Asia but the causes of this frequently fatal disease are not well understood. Several epidemiology studies indicate that inhalation of fumes from high temperature Chinese style cooking with a wok may be a cause. Only one previous study investigated uptake of potential toxicants and carcinogens by women who cook with a wok. We enrolled three-hundred twenty-eight non-smoking women from Singapore for this study. Each provided a spot urine sample and answered a questionnaire concerning their cooking habits and other factors. The urine samples were analyzed by liquid chromatography-tandem mass spectrometry for mercapturic acid metabolites of acrolein (3-hydroxypropylmercapturic acid), crotonaldehyde (3-hydroxy-1-methylpropylmercapturic acid), and benzene (S-phenylmercapturic acid), accepted biomarkers of uptake of these toxic and carcinogenic compounds. We observed statistically significant effects of wok cooking frequency on levels of 3-hydroxypropylmercapturic acid and 3-hydroxy-1-methylpropylmercapturic acid, but not S-phenylmercapturic acid. Women who cooked greater than 7 times per week had a geometric mean of 2600 (95% CI, 2189-3090) pmol/mg creatinine 3-hydroxypropylmercapturic acid compared to 1901 (95% CI, 1510-2395) pmol/mg creatinine when cooking less than once per week (P for trend 0.018). The corresponding values for 3-hydroxy-1-methylpropylmercapturic acid were 1167 (95% CI, 1022-1332) and 894 (95% CI, 749-1067) pmol/mg creatinine (P for trend 0.008). We conclude that frequent wok cooking leads to elevated exposure to the toxicants acrolein and crotonaldehyde, but not benzene. Kitchens should be properly ventilated to decrease exposure to potentially toxic and carcinogenic fumes produced during Chinese style wok cooking.
PMCID: PMC4373935  PMID: 25807518
10.  Combined Analysis of the Tobacco Metabolites Cotinine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in Human Urine 
Analytical chemistry  2015;87(3):1514-1517.
Two of the most widely measured compounds in the urine of people who use tobacco products are cotinine, a major metabolite of the addictive constituent nicotine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of the powerful lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Thousands of analyses have been reported in the literature, carried out exclusively – to the best of our knowledge – by separate methods. In the study reported here, we have developed a sensitive, accurate, and precise liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring method for the combined analysis of total cotinine (the sum of cotinine and its glucuronide) and total NNAL (the sum of NNAL and its glucuronide). The new method quantifies naturally occurring [13C]cotinine to minimize problems associated with the vast differences in concentration of total cotinine and total NNAL in urine. This method should greatly facilitate future determinations of these important compounds.
PMCID: PMC4315695  PMID: 25544129
11.  The Contribution of Common UGT2B10 and CYP2A6 Alleles to Variation in Nicotine Glucuronidation among European Americans 
Pharmacogenetics and genomics  2013;23(12):706-716.
UDP-glucuronosytransferase-2B10 (UGT2B10) is the primary catalyst of nicotine glucuronidation. To develop a predictive genetic model of nicotine metabolism, the conversion of deuterated (D2)-nicotine to D2-nicotine-glucuronide, D2-cotinine, D2-cotinine-glucuronide, and D2-trans-3'-hydroxycotinine were quantified in 188 European Americans, and the contribution of UGT2B10 genotype to variability in first-pass nicotine glucuronidation assessed, following a procedure previously applied to nicotine C-oxidation. The proportion of total nicotine converted to nicotine-glucuronide (D2-nicotine-glucuronide/ (D2-nicotine +D2-nicotine-glucuronide +D2-cotinine +D2-cotinine-glucuronide +D2-trans-3'-hydroxycotinine)) was the primary phenotype. The variant, rs61750900T (D67Y) (minor allele frequency (MAF) = 10%), is confirmed to abolish nicotine glucuronidation activity. Another variant, rs112561475G (N397D) (MAF = 2%), is significantly associated with enhanced glucuronidation. rs112561475G is the ancestral allele of a well-conserved amino acid, indicating that the majority of human UGT2B10 alleles are derived hypomorphic alleles. CYP2A6 and UGT2B10 genotype explain 53% of the variance in oral nicotine glucuronidation in this sample. CYP2A6 and UGT2B10 genetic variants are also significantly associated with un-deuterated (D0) nicotine glucuronidation in subjects smoking ad libitum. We find no evidence for further common variation markedly influencing hepatic UGT2B10 expression in European Americans.
PMCID: PMC3919513  PMID: 24192532
UGT2B10; nicotine; cotinine; metabolism; glucuronidation; CYP2A6
12.  Developing the science base for reducing tobacco harm 
The University of Minnesota Transdisciplinary Tobacco Use Research Center has been examining the multiple dimensions and the scientific evidence required to determine the feasibility of tobacco harm reduction as a means to reduce tobacco-related mortality and morbidity. Because of the complexity associated with exploring this area, an interdisciplinary approach is necessary. The research components that have been of particular focus at our center include (a) developing and validating biomarkers of tobacco-related exposure and toxicity, (b) developing animal models and designing studies with humans to assess a variety of smoking reduction approaches and potential reduced exposure products, and (c) determining individual differences in response to these interventions and products. A description of the ongoing activities and challenges in these areas is provided, along with projected directions for the future.
PMCID: PMC4222243  PMID: 18067031
13.  Smoking reduction fails to improve clinical and biological markers of cardiac disease: A randomized controlled trial 
Cigarette reduction has been proposed as a treatment goal for smokers who are not interested in stopping completely. This randomized controlled trial was designed to determine the effect of a smoking reduction intervention on smoking behavior, symptoms of heart disease, and biomarkers of tobacco exposure. It included 152 patients with heart disease who did not intend to stop smoking in the next 30 days. Participants were randomly assigned to smoking reduction (SR) or usual care (UC). SR subjects received counseling and nicotine replacement therapy to encourage ≥50% reduction in cigarettes per day (CPD). They were followed at 1, 3, 6, 12 and 18 months to assess smoking, heart disease symptoms, quality of life and nicotine, cotinine, carbon monoxide (CO), white blood cell (WBC) count, fibrinogen, hs-C-reactive protein (hs-CRP), F2-isoprostane, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), and 1-hydroxypyrene (1-HOP). At 6 months SR participants reduced by 10.9 CPD, compared with 7.4 CPD in UC (difference NS). At 18 months, 9/78 SR vs. 9/74 UC participants quit smoking. There were no significant differences between treatment groups in angina, quality of life or adverse events, nicotine, cotinine, CO, WBC count, fibrinogen, hs-CRP, F2-isoprostane, total NNAL or 1-HOP levels at any time point. To determine if smoking reduction, regardless of treatment condition, was associated with improved outcomes, we compared all subjects at 6 months to baseline (mean reduction in CPD from 27.4 to 18.1, p<.01). There were no significant changes in outcome variables except CO, which decreased by 5.5 ppm (p<.01). There were also no significant improvements considering only subjects who reduced by ≥50%, or those who had no history of reduction prior to enrollment in the study. The SR intervention did not significantly reduce CPD or toxin exposure, or improve smoking cessation or clinical outcomes compared to UC. These results emphasize the importance of abstinence for smokers with heart disease to minimize health risks from tobacco.
PMCID: PMC4110889  PMID: 18324566
14.  Dose–Response Effects of Spectrum Research Cigarettes 
Nicotine & Tobacco Research  2012;15(6):1113-1121.
Experimental cigarettes are needed to conduct studies examining the effects of varying doses of nicotine content on smoking behavior. The National Institute on Drug Abuse contracted with Research Triangle Institute to make such cigarettes available to researchers. The goal of this study was to determine whether cigarettes that vary in nicotine content produce an expected dose–response effect.
Two studies were conducted. The first study recruited subjects from 3 sites and consisted of a single, within-subject laboratory session. Subjects first smoked 4 puffs on their usual-brand cigarette and then in double-blind, random-order, smoked 4 puffs on each experimental cigarette that contained either low nicotine (LN, 0.4mg/g), intermediate nicotine (IN, 5.7–5.8mg/g), or high nicotine (HN, 11.4–12.8mg/g). Each puffing bout was separated by a 30-min interval. Subjects completed questionnaires and were assessed for vital signs after each cigarette. The second study involved 1 site and used a between-subject design in which subjects were assigned to 1 of the 3 experimental cigarettes for 1 week. Subjective responses and biomarkers of exposure were assessed.
In the first study, significant dose–response effects were observed, particularly between the LN and HN cigarettes. The second study showed decreases in cigarette smoking and exposure biomarkers predominantly in the LN group, with no changes in the HN cigarette group.
These results are similar to those observed in prior literature, confirming that these experimental cigarettes can be used safely and with the expected pharmacological effects.
PMCID: PMC3646652  PMID: 23178320
15.  Reduced Nicotine Content Cigarettes and Nicotine Patch 
Reduced nicotine content (RNC) cigarettes have led to smoking fewer cigarettes, withdrawal relief, and facilitation of cessation. The aim of this study is to examine the effects RNC cigarettes with and without nicotine patch and patch alone on smoking behavior, toxicant exposure, withdrawal discomfort and as an exploratory analysis, on long-term abstinence.
This study involved a randomized, parallel arm design and six weeks of: 1) 0.05-0.09 mg nicotine yield cigarettes (N=79); 2) 21 mg nicotine patch (N=80) or 3) 0.05-0.09 nicotine yield cigarettes with 21 mg nicotine patch (N=76); all groups received six weeks of additional behavioral treatment with follow-ups up to six months.
Combination approach led to lower rates of smoking assigned cigarettes and hence lower CO levels than RNC cigarettes alone. Additionally, the combination approach was associated with less withdrawal severity when switching from usual brand to assigned product, and less smoking of usual brand cigarettes during treatment, but not after treatment compared to the other approaches.
Combining very low nicotine content cigarettes with nicotine patch may improve the acute effects resulting from switching to either of these products alone.
These findings may have implications for smoking cessation treatment or a policy measure to reduce nicotine content in cigarettes.
PMCID: PMC3681886  PMID: 23603206
reduced nicotine; cigarettes-nicotine; patch-tobacco; addiction-cigarette; consumption-biomarkers
16.  A compensatory effect upon splicing results in normal function of the CYP2A6*14 allele 
Pharmacogenetics and genomics  2013;23(3):107-116.
A synonymous variant in the first exon of CYP2A6, rs1137115 (51G > A), defines the common reference allele CYP2A6*1A, and is associated with lower mRNA expression and slower in-vivo nicotine metabolism. Another common allele, CYP2A6*14, differs from CYP2A6*1A by a single variant, rs28399435 (86G > A, S29N). However, CYP2A6*14 shows in-vivo activity comparable with that of full-function alleles, and significantly higher than CYP2A6*1A. rs1137115A is predicted to create an exonic splicing suppressor site overlapping an exonic splicing enhancer (ESE) site in the first exon of CYP2A6, whereas rs28399435A is predicted to strengthen another adjacent ESE, potentially compensating for rs1137115A. Using an allelic expression assay to assess cDNAs produced from rs1137115 heterozygous liver biopsy samples, lower expression of the CYP2A6*1A allele is confirmed while CYP2A6*14 expression is found to be indistinguishable from that of rs1137115G alleles. Quantitative PCR assays to determine the relative abundance of spliced and unspliced or partially spliced CYP2A6 mRNAs in liver biopsy samples show that *1A/*1A homozygotes have a significantly lower ratio, due to both a reduction in spliced forms and an increase in unspliced or partially spliced CYP2A6. These results show the importance of common genetic variants that effect exonic splicing suppressor and ESEs to explain human variation regarding clinically-relevant phenotypes.
PMCID: PMC3919506  PMID: 23292114
CYP2A6; exonic splice enhancer; exonic splice suppressor; nicotine metabolism; splicing
17.  Tobacco Smoke Biomarkers and Cancer Risk Among Male Smokers in the Shanghai Cohort Study 
Cancer letters  2012;334(1):34-38.
Metabolites of tobacco smoke constituents can be quantified in urine and other body fluids providing a realistic measure of carcinogen and toxicant dose in a smoker. Many previous studies have demonstrated that these metabolites – referred to as biomarkers in this paper – are related to tobacco smoke exposure. The studies reviewed here were designed to answer another question: are these substances also biomarkers of cancer risk? Using a prospective study design comparing biomarker levels in cancer cases and controls, all of whom were smokers, the results demonstrate that several of these biomarkers – total cotinine, total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), r-1-,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), and total N’-nitrosonornicotine (NNN) - are biomarkers of cancer risk. Therefore, these biomarkers have the potential to become part of a cancer risk prediction algorithm for smokers.
PMCID: PMC3648613  PMID: 22824243
tobacco smoke; biomarkers; cotinine; NNAL; PheT; NNN
18.  Exposure to Nicotine and a Tobacco-Specific Carcinogen Increase with Duration of Use of Smokeless Tobacco 
Tobacco control  2008;17(2):128-131.
Smokeless tobacco is an efficient delivery vehicle for nicotine and can contain significant amounts of carcinogens. However, few studies have examined factors that might moderate levels of nicotine or carcinogen exposure.
To determine the effect of duration of smokeless tobacco use on the uptake of nicotine and a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
Questionnaires on use of smokeless tobacco were administered, and urine samples from 212 smokeless tobacco users were analyzed for biomarkers of uptake of nicotine and NNK. The biomarkers were cotinine and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL).
Male smokeless tobacco users were recruited for studies designed to investigate methods of reducing smokeless tobacco use. The questionnaire and biomarker data were obtained at baseline, prior to reduction.
Levels of cotinine (p < 0.0001) and total NNAL (p = 0.0003) were significantly correlated with duration (in years) of use of smokeless tobacco products. Median cotinine and total NNAL were 2.4 and 2.1 times higher, respectively, in the 21 + year than in the 0-5 year category of use.
Smokeless tobacco users adjust their intensity of use with experience in order to increase their nicotine dose, resulting in a corresponding increase in exposure to NNK, a powerful carcinogen. These results indicate the importance of educating smokeless tobacco users about the effects of prolonged use of these products.
PMCID: PMC3889131  PMID: 18375734
19.  CYP2B6 Non-Coding Variation Associated with Smoking Cessation Is Also Associated with Differences in Allelic Expression, Splicing, and Nicotine Metabolism Independent of Common Amino-Acid Changes 
PLoS ONE  2013;8(11):e79700.
The Cytochrome P450 2B6 (CYP2B6) enzyme makes a small contribution to hepatic nicotine metabolism relative to CYP2A6, but CYP2B6 is the primary enzyme responsible for metabolism of the smoking cessation drug bupropion. Using CYP2A6 genotype as a covariate, we find that a non-coding polymorphism in CYP2B6 previously associated with smoking cessation (rs8109525) is also significantly associated with nicotine metabolism. The association is independent of the well-studied non-synonymous variants rs3211371, rs3745274, and rs2279343 (CYP2B6*5 and *6). Expression studies demonstrate that rs8109525 is also associated with differences in CYP2B6 mRNA expression in liver biopsy samples. Splicing assays demonstrate that specific splice forms of CYP2B6 are associated with haplotypes defined by variants including rs3745274 and rs8109525. These results indicate differences in mRNA expression and splicing as potential molecular mechanisms by which non-coding variation in CYP2B6 may affect enzymatic activity leading to differences in metabolism and smoking cessation.
PMCID: PMC3829832  PMID: 24260284
20.  Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6 
Human Molecular Genetics  2012;21(13):3050-3062.
This study demonstrates a novel approach to test associations between highly heterogeneous genetic loci and complex phenotypes. Previous investigations of the relationship between Cytochrome P450 2A6 (CYP2A6) genotype and smoking phenotypes made comparisons by dividing subjects into broad categories based on assumptions that simplify the range of function of different CYP2A6 alleles, their numerous possible diplotype combinations and non-additive allele effects. A predictive model that translates CYP2A6 diplotype into a single continuous variable was previously derived from an in vivo metabolism experiment in 189 European Americans. Here, we apply this model to assess associations between genotype, inferred nicotine metabolism and smoking behaviors in larger samples without direct nicotine metabolism measurements. CYP2A6 genotype is not associated with nicotine dependence, as defined by the Fagerström Test of Nicotine Dependence, demonstrating that cigarettes smoked per day (CPD) and nicotine dependence have distinct genetic correlates. The predicted metric is significantly associated with CPD among African Americans and European American dependent smokers. Individual slow metabolizing genotypes are associated with lower CPD, but the predicted metric is the best predictor of CPD. Furthermore, optimizing the predictive model by including additional CYP2A6 alleles improves the fit of the model in an independent data set and provides a novel method of predicting the functional impact of alleles without direct metabolism measurements. Lastly, comprehensive genotyping and in vivo metabolism data are used to demonstrate that genome-wide significant associations between CPD and single nucleotide polymorphisms are the result of synthetic associations.
PMCID: PMC3373237  PMID: 22451501
21.  Urinary Levels of Cigarette Smoke Constituent Metabolites Are Prospectively Associated with Lung Cancer Development in Smokers 
Cancer Research  2011;71(21):6749-6757.
Polycyclic aromatic hydrocarbons (PAH) are believed to be among the principal causative agents for lung cancer in smokers, but no epidemiologic studies have evaluated the relationship of PAH uptake and metabolism to lung cancer. In this study, we quantified prediagnostic urinary levels of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydro-phenanthrene (PheT), a validated biomarker of PAH uptake and metabolism, as well as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), and cotinine and its glucuronides (total cotinine), validated biomarkers of uptake of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and nicotine, respectively, in relation to lung cancer risk among current smokers in a nested case–control study within a cohort of 18,244 Chinese men in Shanghai, China. Urinary levels of PheT, total NNAL, and total cotinine were significantly higher in cases than controls (N = 476 matched pairs). ORs (95% confidence intervals) for lung cancer in the second, third, fourth, and fifth quintiles of PheT were 1.70 (1.00–2.88), 1.07 (0.62–1.84), 1.48 (0.86–2.53), and 2.34 (1.33–4.11), respectively, relative to the lowest quartile (Ptrend = 0.023) after adjustment for self-reported smoking intensity and duration and urinary total NNAL and total cotinine. This study also confirmed that urinary total NNAL and total cotinine are independently related to lung cancer risk.
PMCID: PMC3392910  PMID: 22028322
22.  The Contribution of Common CYP2A6 Alleles to Variation in Nicotine Metabolism Among European Americans 
Pharmacogenetics and genomics  2011;21(7):403-416.
Cytochrome P450 2A6 (CYP2A6) is the primary catalyst of nicotine metabolism. To develop a predictive genetic model of nicotine metabolism, the conversion of deuterated (D2)-nicotine to D2-cotinine was quantified in 189 European Americans and the contribution of CYP2A6 genotype to variability in first-pass nicotine metabolism was assessed. Specifically, 1) single time-point measures of D2-cotinine/(D2-cotinine + D2-nicotine) following oral administration were used as a metric of CYP2A6 activity; 2) the impact of CYP2A6 haplotype was treated as acting multiplicatively; 3) parameter estimates were calculated for all haplotypes in the subject pool, defined by a set of polymorphisms previously reported to affect function, including gene copy number; and 4) a minimum number of predictive polymorphisms are justified to be included in the model based on statistical evidence of differences between haplotypes. The final model includes seven polymorphisms and fits the phenotype, 30 minutes following D2-nicotine oral administration, with R2=0.719. The predictive power of the model is robust: parameter estimates calculated in men (n=89) predict the phenotype in women (n=100) with R2=0.758 and vice versa with R2=0.617; estimates calculated in current smokers (n=102) predict phenotype in former smokers (n=86) with R2=0.690 and vice versa with R2=0.703. Comparisons of haplotypes also demonstrate that CYP2A6*12 is a loss of function allele indistinguishable from CYP2A6*4 and CYP2A6*2 and that the CYP2A6*1B 5′ UTR conversion has negligible impact on metabolism. After controlling for CYP2A6 genotype modest associations were found between increased metabolism and both female gender (p= 4.8×10−4) and current smoking (p=0.02).
PMCID: PMC3116045  PMID: 21597399
CYP2A6; nicotine metabolism; cotinine
23.  Common polymorphisms in FMO1 are associated with nicotine dependence 
Pharmacogenetics and genomics  2011;21(7):397-402.
Cigarette smoking and other forms of tobacco use are the leading cause of preventable mortality in the world. A better understanding of the etiology of nicotine addiction may help increase the success rate of cessation and decrease the massive morbidity and mortality associated with smoking.
In order to identify genetic polymorphisms that contribute to nicotine dependence, our group undertook a genetic association study including three enzyme families that potentially influence nicotine metabolism: cytochrome P450 enzymes (CYP P450s), flavin monooxygenases (FMOs) and UDP-glucuronosyl transferases (UGTs).
Several polymorphisms in FMO1 showed association in a discovery sample and were tested in an independent replication sample. One polymorphism, rs10912765, showed association that remained significant after Bonferroni correction (nominal p=0.0067, corrected p=0.0134). Several additional polymorphisms in linkage disequilibrium with this SNP also showed association. Subsequent in vitro experiments characterized FMO1 as a more efficient catalyst of nicotine N-oxidation than FMO3. In adult humans, FMO1 is primarily expressed in the kidney and is likely to be a major contributor to the renal metabolism and clearance of therapeutic drugs. FMO1 is also expressed in the brain and could contribute to the nicotine concentration in this tissue.
These findings suggest that polymorphisms in FMO1 are significant risk factors in the development of nicotine dependence and that the mechanism may involve variation in nicotine pharmacology.
PMCID: PMC3248052  PMID: 21540762
FMO1; nicotine dependence; nicotine metabolism
24.  Metabolites of a Tobacco-Specific Lung Carcinogen in Children Exposed to Secondhand or Thirdhand Tobacco Smoke in Their Homes 
People exposed to secondhand tobacco smoke (SHS) inhale the lung carcinogen NNK which is metabolized to NNAL and its glucuronides. These urinary metabolites, termed total NNAL, can be quantified. A related compound, iso-NNAL, has been proposed as a biomarker for exposure to smoke constituent residues on surfaces (thirdhand tobacco smoke). There is limited information in the literature on levels of total NNAL in children exposed to SHS.
We recruited 79 parent child dyads from homes where the enrolled parent was a cigarette smoker, and visited their homes. Parents were asked questions, home ambient air quality was evaluated, and children provided urine samples. Urine was analyzed for total NNAL, total cotinine, total nicotine, and iso-NNAL.
Ninety percent of the children had detectable total NNAL in urine; total nicotine and total cotinine were also detected in most samples. There were significant positive relationships between biomarker levels and exposure of children in the home. Levels were highest in homes with no smoking restrictions. African-American children had significantly higher levels than other children. iso-NNAL was not detected in any urine sample.
There was nearly universal exposure of children to the lung carcinogen NNK, due mainly to exposure to SHS from adult smokers in their homes.
Homes with adult smokers should adopt restrictions to protect their children from exposure to a potent lung carcinogen.
PMCID: PMC3111852  PMID: 21467230
NNAL; secondhand tobacco smoke; thirdhand tobacco smoke; children’s exposure
25.  Effect of Oral Snus and Medicinal Nicotine in Smokers on Toxicant Exposure and Withdrawal Symptoms: A Feasibility Study 
Smokeless, spitless tobacco products are being introduced and marketed as cigarette substitutes. Data are needed regarding how smokers interested in cessation would use these products, the levels of resultant toxicant exposure and the feasibility of using these products as aids for tobacco cessation.
Smokers were randomized to receive Camel Snus (n=51), Taboka (n=52) or medicinal nicotine (n=27) and required to quit smoking for 4 weeks. Measures of toxicant exposure and symptoms of craving and withdrawal were assessed prior to and during product use.
Concentrations of exhaled carbon monoxide, urinary cotinine, urinary total NNAL and urinary total NNN were significantly (p values <0.05) lower at the end of treatment in each group except for total NNN in those receiving Camel Snus (p=0.066). A significant group × time effect was observed for total NNAL concentrations (p=0.002) with the decrease greatest in the medicinal nicotine group and smallest decrease in the Camel Snus group. No significant differences between groups were found in craving and withdrawal symptoms.
Enrolling smokers into a cessation study utilizing newer smokeless tobacco products is feasible. Camel Snus and Taboka use was not found to be superior to medicinal nicotine in reducing withdrawal symptoms but decreases in NNAL were smaller in users of Camel Snus.
This study demonstrates the feasibility of conducting a smoking cessation study utilizing these newer tobacco products. An appropriately powered study is needed assessing smoking cessation rates using these newer products compared with established, safer products such as medicinal nicotine.
PMCID: PMC3063713  PMID: 21068204
Smokeless tobacco; medicinal nicotine; biomarkers; craving; withdrawal

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