Quantitation of Ten Urinary Nicotine Metabolites, Including 4-Hydroxy-4-(3-pyridyl) Butanoic Acid, a Product of Nicotine 2'-Oxidation, and CYP2A6 Activity in Japanese Americans, Native Hawaiians, and Whites.

Smoking intensity varies across smokers and is influenced by individual variability in the metabolism of nicotine, the major addictive agent in tobacco. Therefore, lung cancer risk, which varies by racial ethnic group, is influenced by the primary catalyst of nicotine metabolism, cytochrome P450 2A6 (CYP2A6). In smokers, CYP2A6 catalyzes nicotine 5'-oxidation. In vitro, CYP2A6 also catalyzes, to a much lower extent, 2'-oxidation, which leads to the formation of 4-hydroxy-4-(3-pyridyl) butanoic acid (hydroxy acid). The urinary concentration of hydroxy acid has been quantified in only a few small studies of White smokers. To quantitatively assess the importance of nicotine 2'-oxidation in smokers, an LC-MS/MS-based method was developed for the analysis of nicotine and ten metabolites in urine. The concentrations of nicotine and these metabolites were measured in 303 smokers (99 Whites, 99 Native Hawaiians, and 105 Japanese Americans), and the relative metabolism of nicotine by four pathways was determined. Metabolism by these pathways was also compared across quartiles of CYP2A6 activity (measured as the plasma ratio of 3-hydroxycotinine to cotinine). As reported previously and consistent with their average CYP2A6 activity, nicotine 5'-oxidation was highest in Whites and lowest in Japanese Americans. Nicotine N-glucuronidation and N-oxidation increased with decreasing CYP2A6 activity. However, the relative urinary concentration of hydroxy acid (mean, 2.3%; 95% CI, 2.2-2.4%) did not vary by ethnic group or by CYP2A6 activity. In summary, CYP2A6 is not an important catalyst of nicotine 2'-oxidation in smokers, nor does nicotine 2'-oxidation compensate for decreased CYP2A6 activity.

Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells.

Occupational and tobacco exposure to aromatic amines (AAs) including 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are associated with bladder cancer (BC) risk. Several epidemiological studies have also reported a possible role for structurally related heterocyclic aromatic amines (HAAs) formed in tobacco smoke or cooked meats with BC risk. We had screened for DNA adducts of 4-ABP, 2-NA, and several prominent HAAs formed in tobacco smoke or grilled meats including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) in the bladder DNA of BC patients, using liquid chromatography/mass spectrometry. We detected DNA adducts of 4-ABP, but not adducts of the other carcinogens. In this study, we have examined the capacity of RT4 cells, an epithelial human bladder cell line, to bioactivate AAs and HAAs to DNA damaging agents, which may contribute to BC. 4-ABP and AαC formed DNA adducts, but DNA adducts of 2-NA, PhIP, and MeIQx were not detected. 4-ABP DNA adducts were formed at tenfold higher levels than AαC adducts. Pretreatment of RT4 cells with α-naphthoflavone (1-10 µM), a specific cytochrome P450 1 (CYP1) inhibitor, decreased AαC adduct formation by 50% but did not affect the level of 4-ABP adducts. However, cell pretreatment with 8-methoxypsoralen (0.1-1 µM), a potent inhibitor of CYP2A, resulted in a 90% decrease of 4-ABP DNA adducts levels. These data signify that CYP2A and CYP1A isoforms expressed in the target urothelium bioactivate 4-ABP and AαC, respectively, and may be a critical feature of aromatic amine-induced urinary bladder carcinogenesis. The bioactivation of other tobacco and environmental AAs by bladder CYPs and their ensuing bladder DNA damage warrants further study.

In Vivo Stable-Isotope Labeling and Mass-Spectrometry-Based Metabolic Profiling of a Potent Tobacco-Specific Carcinogen in Rats.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen that exerts its carcinogenic effects upon metabolic activation. The identification and quantitation of NNK metabolites could identify potential biomarkers of bioactivation and detoxification of this potent carcinogen and may be used to predict lung cancer susceptibility among smokers. Here, we used in vivo isotope-labeling and high-resolution-mass-spectrometry-based methods for the comprehensive profiling of all known and unknown NNK metabolites. The sample-enrichment, LC-MS, and data-analysis workflow, including a custom script for automated d0- d4- m/ z-pair-peak detection, enabled unbiased identification of numerous NNK metabolites. The structures of the metabolites were confirmed using targeted LC-MS2 with retention-time ( tR) and MS2-fragmentation comparisons to those of standards when possible. Eleven known metabolites and unchanged NNK were identified simultaneously. More importantly, our workflow revealed novel NNK metabolites, including 1,3-Diol (13), α-OH-methyl-NNAL-Gluc (14), nitro-NK- N-oxide (15), nitro-NAL- N-oxide (16), γ-OH NNAL (17), and three N-acetylcysteine (NAC) metabolites (18a-c). We measured the differences in the relative distributions of a panel of nitroso-containing NNK-specific metabolites in rats before and after phenobarbital (PB) treatment, and this served as a demonstration of a general strategy for the detection of metabolic differences in animal and cell systems. Lastly, we generated a d4-labeled NNK-metabolite mixture to be used as internal standards ( d4-rat urine) for the relative quantitation of NNK metabolites in humans, and this new strategy will be used to assess carcinogen exposure and ultimately to evaluate lung-cancer risk and susceptibility in smokers.

Influence of UGT2B10 Genotype on Urinary Excretion of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol- N-glucuronide by African American Smokers.

At similar smoking levels, African American's lung cancer risk is as much as twice that of whites. We hypothesized that racial/ethnic differences in UDP-glucuronosyltransferase (UGT)-catalyzed glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a detoxication pathway for the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) may contribute to this variable risk. UGT2B10 catalyzes NNAL- N-glucuronidation, and a UGT2B10 splice variant is common among African Americans. Smokers from two independent studies were genotyped for this variant (rs116294140) and an Asp67Tyr variant (rs61750900), and urinary NNAL and NNAL-glucuronide concentrations were quantified. In the first, no significant differences in NNAL- N-glucuronidation between African Americans ( n = 257) and whites ( n = 354) or between homozygous carriers of UGT2B10 variants (genetic score 2) and noncarriers (score 0) were detected. However, total NNAL glucuronidation by score 2 compared to score 0 smokers was lower (68.9 vs 71.2%, p < 0.0001). For NNAL- N-glucuronide to be more precisely quantified in a second study, a sensitive high-resolution LC-MS/MS-based method, which separated NNAL, NNAL- O-glucuronide, and NNAL- N-glucuronide prior to analysis, was developed. In this study, the excretion of total NNAL (free plus glucuronides) by African American ( n = 52) and white ( n = 54) smokers was not different; however, total NNAL glucuronidation by African Americans (64.0%) was slightly less than by whites (68.3%, p = 0.05). The mean NNAL- N-glucuronidation by African Americans was much lower than for whites (14 vs 24.9%, p < 0.00001), but the NNAL- O-glucuronidation was greater (50.0 vs 43.3%, p = 0.013). UGT2B10 genotype influenced NNAL- N-glucuronidation; the geometric mean percentage N-glucuronidation was 22.5% for smokers with genetic score 0 ( n = 57) and 11.2% for score 2 ( n = 11). In summary, the high prevalence of a UGT2B10 splice variant among African Americans results in lower NNAL- N-glucuronidation but only a small decrease in total NNAL glucuronidation. Therefore, despite the significant contribution of UGT2B10 to NNAL- N-glucuronidation, the UGT2B10 genotype does not play a large role in NNAL detoxication. Any decrease in N-glucuronidation was accompanied by a parallel increase in O-glucuronidation.

Dietary Dihydromethysticin Increases Glucuronidation of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol in A/J Mice, Potentially Enhancing Its Detoxification.

Effective chemopreventive agents are needed against lung cancer, the leading cause of cancer death. Results from our previous work showed that dietary dihydromethysticin (DHM) effectively blocked initiation of lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice, and it preferentially reduced 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-derived DNA adducts in lung. This study explored the mechanism(s) responsible for DHM's differential effects on NNK/NNAL-derived DNA damage by quantifying their metabolites in A/J mice. The results showed that dietary DHM had no effect on NNK or NNAL abundance in vivo, indicating that DHM does not affect NNAL formation from NNK. DHM had a minimal effect on cytochrome P450 2A5 (CYP2A5, which catalyzes NNK and NNAL bioactivation in A/J mouse lung), suggesting that it does not inhibit NNAL bioactivation. Dietary DHM significantly increased O-glucuronidated NNAL (NNAL-O-gluc) in A/J mice. Lung and liver microsomes from dietary DHM-treated mice showed enhanced activities for NNAL O-glucuronidation. These results overall support the notion that dietary DHM treatment increases NNAL detoxification, potentially accounting for its chemopreventive efficacy against NNK-induced lung tumorigenesis in A/J mice. The ratio of urinary NNAL-O-gluc and free NNAL may serve as a biomarker to facilitate the clinical evaluation of DHM-based lung cancer chemopreventive agents.

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 ß-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.

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.

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.

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 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 R=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 R=0.758 and vice versa with R=0.617; estimates calculated in current smokers (n=102) predict the phenotype in former-smokers (n=86) with R=0.690 and vice versa with R=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) 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.

Analysis of [3',3'-d(2)]-nicotine and [3',3'-d(2)]-cotinine by capillary liquid chromatography-electrospray tandem mass spectrometry.

A selective and sensitive LC/MS/MS assay was developed for the quantification of d(2)-nicotine and d(2)-cotinine in plasma of current and past smokers administered d(2)-nicotine. After solid phase extraction and liquid-liquid extraction, HPLC separation was achieved on a capillary hydrophilic interaction chromatography phase column. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated for d(2)-nicotine (0.03-6.0 ng/ml plasma) and d(2)-cotinine (0.15-25 ng/ml plasma). The lower limits of quantitation were 0.15 ng/ml and 0.25 ng/ml for d(2)-nicotine and d(2)-cotinine, respectively. The coefficient of variation was 3.7% for d(2)-nicotine and 2.5% for d(2)-cotinine. The method was applied to two ongoing studies of d(2)-nicotine metabolism in prior and current smokers. Preliminary analysis of a subset of subjects from these studies detected a significantly lower rate of nicotine conversion to cotinine by past smokers compared to current smokers.

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, ß-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 (ß-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). ß-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 µM). Neither menthol nor ß-nicotyrine was an inactivator of CYP2A13. Whereas, ß-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 ß-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 ß-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 O(6)-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.

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.

Effects of eleven isothiocyanates on P450 2A6- and 2A13-catalyzed coumarin 7-hydroxylation.

Many isothiocyanates (ITCs), both naturally occurring and synthetic, are potent and selective inhibitors of carcinogenesis in animal models and are now viewed as a class of promising chemopreventive agents. We have investigated the ability of 11 ITCs to inhibit and/or inactivate P450 2A6- and 2A13-mediated coumarin 7-hydroxylation. Two of these 11 ITCs, phenylpropyl isothiocyanate (PPITC) and phenylhexyl isothiocyanate (PHITC), were potent inhibitors of P450 2A13. The K I values for the inhibition of P450 2A13-mediated coumarin 7-hydroxylation by PPITC and PHITC were approximately 0.14 and 1.1 microM, respectively. P450 2A6 was also inhibited by these two ITCs; however, the K I values indicated they were approximately 10-20-fold less potent for P450 2A6 than for P450 2A13. Most of the ITCs tested, including PPITC and PHITC, showed some degree of inactivation of both P450s; however, only one compound, tert-butyl isothiocyanate (tBITC), showed significant inactivation of P450 2A13 at a concentration of 10 microM. None of the ITCs caused significant inactivation of P450 2A6 at this concentration. tBITC inactivated P450 2A13 with an apparent K I of 4.3 microM and a k inact of 0.94 min (-1). Inactivation of P450 2A6 by tBITC was observed only at high concentrations and long incubation times. The observed differences in inhibition and/or inactivation of P450 2A6 and 2A13 by a few of the isothiocyanates suggest that these compounds may be useful for structure-function studies.

Inactivation of CYP2A6 and CYP2A13 during nicotine metabolism.

Nicotine is the major addictive agent in tobacco. The primary catalyst of nicotine metabolism in humans is CYP2A6. However, the closely related enzyme CYP2A13 is a somewhat better catalyst. CYP2A13 is an extrahepatic enzyme that is an excellent catalyst of the metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK). Here we report that both CYP2A6 and CYP2A13 were inactivated during nicotine metabolism. Inactivation of both enzymes was dependent on NADPH and increased with time and concentration. Alternate substrates for CYP2A6 and CYP2A13 protected these enzymes from inactivation. Inactivation of CYP2A13 was irreversible upon extensive dialysis and seems to be mechanism-based. The K(I) of CYP2A13 inactivation by nicotine was 17 microM, the rate of inactivation, k(inact), was 0.1 min(-1), and the t(1/2) was 7 min. However, the loss in enzyme activity occurred after nicotine metabolism was complete, suggesting that a secondary or possible tertiary metabolite of nicotine may be responsible. [5-(3)H]Nicotine metabolism by CYP2A13 was monitored by radioflow high-pressure liquid chromatography during the course of enzyme inactivation; the major product was the Delta(1'(5'))iminium ion. However, cotinine was a significant metabolite even at short reaction times. The metabolism of the nicotine Delta(1'(5'))iminium ion to cotinine did not require the addition of aldehyde oxidase. CYP2A13 catalyzed this reaction as well as further metabolism of cotinine to 5'-hydroxycotinine, trans-3'-hydroxycotinine, and N-(hydroxymethyl)-norcotinine as enzyme inactivation occurred. Studies are on-going to identify the metabolite responsible for nicotine-mediated inactivation of CYP2A13.

Effects of benzyl and phenethyl isothiocyanate on P450s 2A6 and 2A13: potential for chemoprevention in smokers.

Isothiocyanates have been shown to be potent inhibitors of carcinogenesis in animals exposed to a number of chemical carcinogens including the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study the effects of benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), two naturally occuring isothiocyanates, on P450 2A6 and 2A13 were investigated. P450s 2A6 and 2A13 are thought to be the primary human P450 enzymes responsible for the in vivo metabolism of nicotine and NNK, respectively. In vitro, BITC and PEITC efficiently inhibited P450 2A6- and 2A13-mediated coumarin 7-hydroxylation. The inhibition of P450 2A6 and 2A13 by BITC was non-competitive with KI's of 4.1 and 1.3 microM, respectively. PEITC was a more potent inhibitor of both enzymes than BITC, with a KI of 0.37 microM for P450 2A6 and 0.03 microM for P450 2A13. P450 2A6-mediated metabolism of nicotine and P450 2A13-mediated alpha-hydroxylation of NNK were also inhibited significantly by these two isothiocyanates. Both BITC and PEITC were able to inactivate P450 2A6 and 2A13 in an NADPH-dependent manner potentially through the formation of adducts to the apoprotein. The potent inhibition of P450 2A6- and 2A13-mediated metabolisms together with the ability of BITC and PEITC to inactivate the enzymes suggests the possibility that these isothiocyanates could be developed as chemopreventive agents to protect smokers who are unwilling or unable to quit smoking against lung cancer.

Identification of N-(hydroxymethyl) norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism.

Cotinine formation is the major pathway of nicotine metabolism in smokers, and the primary pathway of cotinine metabolism is trans-3'-hydroxylation. trans-3'-Hydroxycotinine and its glucuronide conjugate account for up to 50% of the nicotine metabolites excreted by smokers. Minor metabolites of cotinine excreted by smokers include norcotinine and cotinine N-oxide, each of which account for <5% of the nicotine dose. It has been reported that P450 2A6 is the catalyst of cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown human metabolite of cotinine. N-(Hydroxymethyl)norcotinine was chemically synthesized, and its stability under the conditions of the enzyme reactions was confirmed. The products of P450 2A6-catalyzed [5-3H]cotinine metabolism were quantified by radioflow HPLC. The identification of N-(hydroxymethyl)norcotinine as the major metabolite was based on HPLC analysis on three unique systems and coelution with N-(hydroxymethyl)norcotinine standard. 5'-Hydroxycotinine and trans-3'-hydroxycotinine were minor products of P450 2A6-catalyzed cotinine metabolism, accounting for 14 and 8% of the total cotinine metabolites, respectively. N-(Hydroxymethyl)norcotinine was a product of cotinine metabolism by the extrahepatic P450, 2A13, but it was a minor one. The major product of P450 2A13-catalyzed cotinine metabolism was 5'-hydroxycotinine, which was formed at twice the rate of trans-3'-hydroxycotinine. The identification of all cotinine metabolites formed by both enzymes was confirmed by LC/MS/MS analysis. Kinetic parameters for cotinine metabolism were determined for P450 2A6 and P450 2A13. This work has confirmed that the major metabolite of cotinine in smokers, trans-3'-hydroxycotinine, is only a minor metabolite of P450 2A6-catalyzed cotinine metabolism.

Effects of 8-methoxypsoralen on cytochrome P450 2A13.

Cytochrome P450 2A13 efficiently catalyzes the bioactivation of several tobacco-specific nitrosamines in vitro. This efficient bioactivation together with the selective expression of P450 2A13 in the human lung suggests that this P450 may play an important role in the initiation of lung cancer in smokers. Therefore, the identification of potent and selective inhibitors/inactivators of P450 2A13 could potentially help to lower the risk of lung cancer in smokers. In this study, we investigated the ability of 8-methoxypsoralen (8-MOP), a known inhibitor of P450 2A6, to inhibit and inactivate the activities of heterologously expressed P450 2A13 in reconstituted systems. We found that 8-MOP is a potent inhibitor of P450 2A13-mediated metabolism of several compounds, including testosterone, which had not been known to be a P450 2A13 substrate. The K(I) for the non-competitive inhibition of P450 2A13-mediated coumarin 7-hydroxylation by 8-MOP was 0.11 microM. The inhibition of P450 2A13 was accompanied by inactivation of the enzyme. Therefore, the observed decrease in activity is most likely due to the inactivation of the enzyme together with competitive or non-competitive inhibition of P450 2A13 by 8-MOP. The inactivation did not result in a loss of native heme, or a significant change in the reduced-CO spectrum of the P450, and did not generate any detectable heme adducts. Instead, the inactivation of P450 2A13 by8-MOP occurred through the formation of an adduct to the apoprotein. LC/MS analysis of the adducted protein indicated an increase in the mass of 232 Da compared with the unadducted protein. This mass shift correlates with the addition of one molecule of 8-MOP plus one atom of oxygen atom to the P450 apoprotein.

Identification of amino acid residues involved in the inactivation of cytochrome P450 2B1 by two acetylenic compounds: the role of three residues in nonsubstrate recognition Sites.

The homologous rat cytochrome P450s 2B1 and 2B2 differ by 13 amino acids. A chimeric construct of P450 2B1/2B2 was used in conjunction with several site-directed mutants to identify key residues involved in the inactivation of P450 2B1 by two acetylenic compounds, 17alpha-ethynylestradiol (17EE) and tert-butyl 1-methyl-2-propynyl ether (tBMP). 17EE is a mechanism-based inactivator of P450 2B1 but not of P450 2B2. We show here that tBMP is also a mechanism-based inactivator of P450 2B1 and not P450 2B2. Minimal loss in 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) activity was observed when P450 2B1 G478A was incubated with either inactivator, suggesting that this residue plays a role in the inactivation. However, P450 2B2 A478G behaved like wild-type P450 2B2, indicating that this residue alone is not sufficient for inactivation. A chimeric construct of P450 2B1/2B2 that is essentially P450 2B1 with five residues of P450 2B2 (including residue 478), was not inactivated by either tBMP or 17EE, suggesting that these five residues are important for inactivation. Sequential mutagenesis of the chimeric construct to quadruple (S407T-N417D-A419T-G478A) and triple (S407T-N417D-A419T) mutants of P450 2B1 did not result in inactivation by either inactivator. However, the triple mutant with mutations only in non-substrate recognition site (SRS) regions still exhibits wild-type P450 2B1 7-EFC O-deethylation activity with a K(m) value of 25 microM and V(max) of 8 nmol/min/nmol P450. These results demonstrate that substitution of three non-SRS residues in P450 2B1 leads to protection against inactivation of 2B enzymes by these two acetylenic compounds.