Linda B. von Weymarn

CYP2A13: Variable Expression and Role in Human Lung Microsomal Metabolic Activation of the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

CYP2A13 is the most efficient cytochrome P450 enzyme in the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen. The aims of this study were to determine the levels of CYP2A13 protein in human lung microsomes and to ascertain whether CYP2A13 plays any role in lung microsomal NNK metabolic activation. The expression of CYP2A6 and CYP2A13 was examined using a high-resolution immunoblotting method, following immunopurification with an anti-CYP2A5 antibody. We found that, of 116 human lung microsomal samples analyzed, ∼90% had detectable CYP2A6, whereas only 12% had detectable CYP2A13 with a detection limit of ∼2 fmol of CYP2A/mg protein. For the majority of microsomal samples analyzed, the level of CYP2A13 was found to be lower than the level of CYP2A6; overall, the highest level of CYP2A13 found (∼20 fmol/mg protein) was ∼10-fold lower than the highest level of CYP2A6 detected. Quantitative RNA-polymerase chain reaction analysis confirmed that the highly variable expression of the CYP2A proteins was consistent with variations in the levels of the corresponding CYP2A mRNAs in the same tissue samples. It is noteworthy that the level of CYP2A13, but not CYP2A6, was correlated with lung microsomal NNK metabolic activation activity. Furthermore, the addition of 8-methoxypsoralen, a CYP2A inhibitor, led to greater inhibition of NNK metabolic activation in microsomes containing relatively high levels of CYP2A13 than in samples containing no detectable CYP2A13. Taken together, these data indicate that human lung microsomal CYP2A13 is active in NNK metabolic activation. Therefore, individuals having relatively high levels of CYP2A13 expression will likely have an increased risk of developing smoking-related lung cancer.

The contribution of common CYP2A6 alleles to variation in nicotine metabolism among European-Americans.

Objective To study the association between cytochrome P450 2A6 (CYP2A6) genotype and metabolism of nicotine to cotinine, identify functional polymorphisms, and develop a predictive genetic model of nicotine metabolism. Methods 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, (i) single time point measures of D2-cotinine/(D2-cotinine+D2-nicotine) after oral administration were used as a metric of CYP2A6 activity; (ii) the impact of CYP2A6 haplotype was treated as acting multiplicatively; (iii) 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 (iv) a minimum number of predictive polymorphisms were justified to be included in the model based on statistical evidence of differences between haplotypes. Results The final model includes seven polymorphisms and fits the phenotype, 30-min after 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 the 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′-untranslated region conversion has negligible impact on metabolism. After controlling for CYP2A6 genotype, modest associations were found between increased metabolism and both female sex (P=4.8×10−4) and current smoking (P=0.02). Conclusion Among European–Americans, seven polymorphisms in the CYP2A6 gene explain the majority of variability in the metabolism of nicotine to cotinine after oral administration. Parameters determined from this in-vivo experiment can be used to predict nicotine metabolism based on CYP2A6 genotype.

Eukaryotic Initiation Factor 4E Binding Protein Family of Proteins: Sentinels at a Translational Control Checkpoint in Lung Tumor Defense

The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here, we show that the primary negative regulators of these oncogenic initiation factors--the 4E-BP protein family--operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK), 4ebp1(-/-)/4ebp2(-/-) mice showed increased sensitivity to tumorigenesis compared with their wild-type counterparts. The 4E-BP-deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape, with translational activation of genes governing angiogenesis, growth, and proliferation, and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense.

UGT2B10 Genotype Influences Nicotine Glucuronidation, Oxidation, and Consumption

Background: 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-glucuronsyltransferase, UGT2B10, on nicotine metabolism and consumption. Methods: 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. Results: Individuals heterozygous for the Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than did wild-type, resulting in a 60% lower ratio of cotinine glucuronide to cotinine, a 50% lower ratio of nicotine glucuronide to nicotine, and increased cotinine and trans-3′-hydroxycotinine. Nicotine equivalents, a robust biomarker of nicotine intake, were lower among Asp67Tyr heterozygotes compared with individuals without this allele: 58.2 (95% confidence interval, 48.9-68.2) versus 69.2 nmol/mL (95% confidence interval, 64.3-74.5). Conclusions: 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. Impact: UGT2B10 genotype influences nicotine metabolism and should be taken into account when characterizing the role of nicotine metabolism on smoking. Cancer Epidemiol Biomarkers Prev; 19(6); 1423–31. ©2010 AACR.

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol

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 was an inactivator 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.

Chronic Nicotine Consumption Does Not Influence 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone-Induced Lung Tumorigenesis

Nicotine replacement therapy is often used to maintain smoking cessation. However, concerns exist about the safety of long-term nicotine replacement therapy 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 mL per 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 vs. 0.53 ± 0.1 tumors per mouse). Lung tumor multiplicity in NNK-treated mice was 18.4 ± 4.5 and was not different 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 that nicotine stimulates cancer cell growth. Cancer Prev Res; 4(11); 1752–60. ©2011 AACR.

Effects upon in-vivo nicotine metabolism reveal functional variation in FMO3 associated with cigarette consumption

Background 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-oxidation 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. Methods 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. Results FMO3 haplotype, based on all common coding variants in Europeans, significantly predicts nicotine metabolism and accounts for ∼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. 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 in nicotine-dependent European Americans (n=1025, P=0.04), and significantly interact (P=0.016) with CYP2A6 genotype to predict cigarettes per day. Conclusion These findings suggest that common polymorphisms in FMO3 influence nicotine clearance and that these genetic variants in turn influence cigarette consumption.

CYP2A6 and CYP2A13-catalyzed metabolism of the nicotine Δ5'(1')iminium ion

Nicotine, the major addictive agent in tobacco, is metabolized primarily by CYP2A6-catalyzed oxidation. The product of this reaction, 5′-hydroxynicotine, is in equilibrium with the nicotine Δ5′(1′)iminium ion and is further metabolized to cotinine. We reported previously that both CYP2A6 and the closely related extrahepatic enzyme CYP2A13 were inactivated during nicotine metabolism; however, inactivation occurred after metabolism was complete. This led to the hypothesis that oxidation of a nicotine metabolite, possibly the nicotine Δ5′(1′)iminium ion, was responsible for generating the inactivating species. In the studies presented here, we confirm that the nicotine Δ5′(1′)iminium ion is an inactivator of both CYP2A6 and CYP2A13, and inactivation depends on time, concentration, and the presence of NADPH. Inactivation was not reversible and was accompanied by a parallel loss in spectrally active protein, as measured by reduced CO spectra. These data are consistent with the characterization of the nicotine Δ5′(1′)iminium ion as a mechanism-based inactivator of both CYP2A13 and CYP2A6. We also confirm that both CYP2A6 and CYP2A13 catalyze the metabolism of the nicotine Δ5′(1′)iminium ion to cotinine and provide evidence that both enzymes catalyze the sequential metabolism of the nicotine Δ5′(1′)iminium ion. That is, a fraction of the cotinine formed may not be released from the enzyme before further oxidation to 3′-hydroxycotinine.

The contribution of common UGT2B10 and CYP2A6 alleles to variation in nicotine glucuronidation among European Americans.

Background To develop a predictive genetic model of nicotine metabolism. UDP-glucuronosyltransferase-2B10 (UGT2B10) is the primary catalyst of nicotine glucuronidation. Materials and methods 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. Results The variant, rs61750900T (D67Y) (minor allele frequency=10%), is confirmed to abolish nicotine glucuronidation activity. Another variant, rs112561475G (N397D) (minor allele frequency=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. Conclusion 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 undeuterated (D0) nicotine glucuronidation in individuals smoking ad libitum. We find no evidence for further common variation markedly influencing hepatic UGT2B10 expression in European Americans.

Quantitation of the Minor Tobacco Alkaloids Nornicotine, Anatabine, and Anabasine in Smokers’ Urine by High Throughput Liquid Chromatography–Mass Spectrometry

Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids; among these are nornicotine, anatabine, and anabasine. We developed and applied a 96 well plate-based capillary LC-tandem mass spectrometry method for the analysis of nornicotine, anatabine, and anabasine in urine. The method was validated with regard to accuracy and precision. Anabasine was quantifiable to low levels with a limit of quantitation (LOQ) of 0.2 ng/mL even when nicotine, which is isobaric, is present at concentrations >2500-fold higher than anabasine. This attribute of the method is important since anatabine and anabasine in urine have been proposed as biomarkers of tobacco use for individuals using nicotine replacement therapies. In the present study, we analyzed the three minor tobacco alkaloids in urine from 827 smokers with a wide range of tobacco exposures. Nornicotine (LOQ 0.6 ng/mL) was detected in all samples, and anatabine (LOQ, 0.15 ng/mL) and anabasine were detected in 97.7% of the samples. The median urinary concentrations of nornicotine, anatabine, and anabasine were 98.9, 4.02, and 5.53 ng/mL. Total nicotine equivalents (TNE) were well correlated with anatabine (r(2) = 0.714) and anabasine (r(2) = 0.760). TNE was most highly correlated with nornicotine, which is also a metabolite of nicotine. Urine samples from a subset of subjects (n = 110) were analyzed for the presence of glucuronide conjugates by quantifying any increase in anatabine and anabasine concentrations after β-glucuronidase treatment. The median ratio of the glucuronidated to free anatabine was 0.74 (range, 0.1 to 10.9), and the median ratio of glucuronidated to free anabasine was 0.3 (range, 0.1 to 2.9). To our knowledge, this is the largest population of smokers for whom the urinary concentrations of these three tobacco alkaloids has been reported.