Nicotine metabolism and urinary elimination in mouse: in vitro and in vivo

This study aimed at elucidating the in vivo metabolism of nicotine both with and without inhibitors of nicotine metabolism. Second, the role of mouse CYP2A5 in nicotine oxidation in vitro was studied as such information is needed to assess whether the mouse is a suitable model for studying chemical inhibitors of the human CYP2A6. The oxidation of nicotine to cotinine was measured and the ability of various inhibitors to modify this reaction was determined. Nicotine and various inhibitors were co-administered to CD2F1 mice, and nicotine and urinary levels of nicotine and four metabolites were determined. In mouse liver microsomes anti-CYP2A5 antibody and known chemical inhibitors of the CYP2A5 enzyme blocked cotinine formation by 85–100%, depending on the pre-treatment of the mice. The amount of trans-3-hydroxycotine was five times higher than cotinine N-oxide, and ten times higher than nicotine N-1-oxide and cotinine. Methoxsalen, an irreversible inhibitor of CYP2A5, significantly reduced the metabolic elimination of nicotine in vivo, but the reversible inhibitors had no effect. It is concluded that the metabolism of nicotine in mouse is very similar to that in man and, therefore, that the mouse is a suitable model for testing novel chemical inhibitors of human CYP2A6.     

Characterization and comparison of nicotine and cotinine metabolism in vitro and in vivo in DBA/2 and C57BL/6 mice

DBA/2 and C57BL/6 are two commonly used mouse strains that differ in response to nicotine. Previous studies have shown that the nicotine-metabolizing enzyme CYP2A5 differs in coumarin metabolism between these two strains, suggesting differences in nicotine metabolism. Nicotine was metabolized to cotinine in vitro by two enzymatic sites. The high-affinity sites exhibited similar parameters (Km, 10.7 +/- 4.8 versus 11.4 +/- 3.6 microM; Vmax, 0.58 +/- 0.18 versus 0.50 +/- 0.07 nmol/min/mg for DBA/2 and C57BL/6, respectively). In vivo, the elimination half-lives of nicotine (1 mg/kg, s.c.) were also similar between DBA/2 and C57BL/6 mice (8.6 +/- 0.4 versus 9.2 +/- 1.6 min, respectively); however, cotinine levels were much higher in DBA/2 mice. The production and identity of the putative cotinine metabolite 3'-hydroxycotinine in mice was confirmed by liquid chromatography/mass spectrometry/mass spectrometry. The in vivo half-life of cotinine (1 mg/kg, s.c.) was significantly longer in the DBA/2 mice compared with the C57BL/6 mice (50.2 +/- 4.7 versus 37.5 +/- 9.6 min, respectively, p < 0.05). The in vitro metabolism of cotinine to 3'-hydroxycotinine was also less efficient in DBA/2 than C57BL/6 mice (Km, 51.0 +/- 15.6 versus 9.5 +/- 2.1 microM, p < 0.05; Vmax, 0.10 +/- 0.01 versus 0.04 +/- 0.01 nmol/min/mg, p < 0.05, respectively). Inhibitory antibody studies demonstrated that the metabolism of both nicotine and cotinine was mediated by CYP2A5. Genetic differences in Cyp2a5 potentially contributed to similar nicotine but different cotinine metabolism, which may confound the interpretation of nicotine pharmacological studies and studies using cotinine as a biomarker.     

Metabolism of nicotine in rat lung microvascular endothelial cells

The aim of this study was to examine whether cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas-blood barrier, have the ability to metabolize nicotine. Nicotine was biotransformed to cotinine and nicotine N'-oxide by cytochrome 450 (CYP) and flavin-containing monooxyganase (FMO), respectively, in rat LMECs. The intrinsic clearance (Vmax1/Km1) for the cotinine formation was about 20 times as high as that for the trans-nicotine N'-oxide formation in the low-Km phase, indicating that oxidation by CYP was much higher than that by FMO. On the other hand, as shown in Eadie-Hofstee plots, the formation of cis-nicotine N'-oxide was monophasic, whereas the plot for the trans-nicotine N'-oxide formation was clearly biphasic. These results suggest that nicotine N'-oxide was stereoselectively metabolized to cis and trans forms. However, in the high-Km phase there was no significant difference in N'-oxidation between the cis and trans forms. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to cotinine of nicotine in rat LMECs using the respective enzyme inhibitors (tranylcypromine and troleandomycine). On the other hand, methimazole (5 microM) caused 73 and 45% decreases in the formation of N'-oxides of cis- and trans- enantiomers, respectively, demonstrating the presence of FMO in rat LMECs. These results suggest that rat LMEC enzymes can convert substrates of exogenous origin such as nicotine for detoxication, indicating LMECs are an important barrier for metabolic products, besides hepatic cells.     

Metabolism of nicotine and cotinine by human cytochrome P450 2A13.

Nicotine, a major constituent of tobacco, plays a critical role in smoking addiction. In humans, nicotine is primarily metabolized to cotinine, which is further metabolized to trans-3'-hydroxycotinine. Recently, we have demonstrated that heterologously expressed human CYP2A13 is highly active in the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived carcinogen. In the present study, CYP2A13-catalyzed NNK metabolism was found to be inhibited competitively by nicotine and N'-nitrosonornicotine (NNN), suggesting that both nicotine and NNN are also substrates of CYP2A13. We have further demonstrated that human CYP2A13 is indeed an efficient enzyme in catalyzing C-oxidation of nicotine to form cotinine, with the apparent K(m) and V(max) values of 20.2 microM and 8.7 pmol/min/pmol, respectively. CYP2A13 also catalyzes the 3'-hydroxylation of cotinine to form trans-3'-hydroxycotinine, with the apparent K(m) and V(max) values of 45.2 microM and 0.7 pmol/min/pmol, respectively. The importance of CYP2A13-catalyzed nicotine and cotinine metabolism in vivo remains to be determined.     

Strain‐specific altered nicotine metabolism in 3,3′‐diindolylmethane (DIM) exposed mice

Two indole compounds, indole‐3‐carbinol (I3C) and its acid condensation product, 3,3′‐diindolymethane (DIM), have been shown to suppress the expression of flavin‐containing monooxygenases (FMO) and to induce some hepatic cytochrome P450s (CYPs) in rats. In liver microsomes prepared from rats fed I3C or DIM, FMO‐mediated nicotine N‐oxygenation was decreased, whereas CYP‐mediated nicotine metabolism to nicotine iminium and subsequently to cotinine was unchanged. Therefore, it was hypothesized that in mice DIM would also suppress nicotine N‐oxygenation without affecting CYP‐mediated nicotine metabolism. Liver microsomes were produced from male and female C57BL/6 J and CD1 mice fed 2500 parts per million (ppm) DIM for 14 days. In liver microsomes from DIM‐fed mice, FMO‐mediated nicotine N‐oxygenation did not differ from the controls, but CYP‐mediated nicotine metabolism was significantly increased, with results varying by sex and strain. To confirm the effects of DIM in vivo, control and DIM‐fed CD1 male mice were injected subcutaneously with nicotine, and the plasma concentrations of nicotine, cotinine and nicotine‐N‐oxide were measured over 30 minutes. The DIM‐fed mice showed greater cotinine concentrations compared with the controls 10 minutes following injection. It is concluded that the effects of DIM on nicotine metabolism in vitro and in vivo differ between mice and rats and between mouse strains, and that DIM is an effective inducer of CYP‐mediated nicotine metabolism in commonly studied mouse strains.     

Gene-gene interactions between CYP2B6 and CYP2A6 in nicotine metabolism

OBJECTIVES: CYP2A6 is the major enzyme involved in nicotine metabolism, yet large interindividual variations in the rate of nicotine metabolism exist within groups of individuals having the same CYP2A6 genotype. We investigated the influence of genetic variation in another potential nicotine-metabolizing enzyme, CYP2B6, and its interaction with CYP2A6, on the metabolism of nicotine. METHODS: Two hundred and twelve healthy Caucasian adult twin volunteers underwent an intravenous infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, for characterization of pharmacokinetic and metabolism phenotypes. Five CYP2B6 genetic polymorphisms causing amino acid substitutions (R22C, Q172 H, S259R, K262R, and R487C) were analyzed. RESULTS: We observed that the CYP2B6*6 haplotype (defined as having both Q172 H and K262R variants) was associated with faster nicotine and cotinine clearance, and that such associations were more prominent among individuals having decreased-activity CYP2A6 genotypes. Statistically significant interactions between CYP2B6 and CYP2A6 genotypes were observed (P<0.003 for nicotine clearance and P<0.002 for cotinine clearance). CONCLUSIONS: Our results indicate that CYP2B6 genetic variation is associated with the metabolism of nicotine and cotinine among individuals with decreased CYP2A6 activity. Further investigation of the roles of CYP2B6 and the interaction between CYP2B6 and CYP2A6 genotypes in mediating nicotine dependence and tobacco-related diseases is merited.     

Nicotine metabolism and CYP2A6 allele frequencies in Koreans.

CYP2A6 is a major catalyst of nicotine metabolism to cotinine. Previously, we demonstrated that the interindividual difference in nicotine metabolism is related to a genetic polymorphism of the CYP2A6 gene in Japanese. To clarify the ethnic differences in nicotine metabolism and frequencies of CYP2A6 alleles, we studied nicotine metabolism and the CYP2A6 genotype in 209 Koreans. The cotinine/nicotine ratio of the plasma concentration 2 h after chewing one piece of nicotine gum was calculated as an index of nicotine metabolism. The genotypes of CYP2A6 gene (CYP2A6*1A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4 and CYP2A6*5) were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific (AS)-PCR. There were ethnic differences in the allele frequencies of CYP2A6*1A, CYP2A6*1B, CYP2A6*4 and CYP2A6*5 between Koreans (45.7%, 42.8%, 11.0% and 0.5%, respectively) and Japanese (42.4%, 37.5%, 20.1% and 0%, respectively, our previous data). Similar to the Japanese, no CYP2A6*2 and CYP2A6*3 alleles were found in Koreans. The homozygotes of the CYP2A6*4 allele (four subjects) were completely deficient in cotinine formation, being consistent with the data among Japanese. The heterozygotes of CYP2A6*4 tended to possess a lower metabolic ratio (CYP2A6*1A/CYP2A6*4, 4.79 +/- 3.17; CYP2A6*1B/CYP2A6*4, 7.43 +/- 4.97) than that in subjects without the allele (CYP2A6*1A/CYP2A6*1A, 7.42 +/- 6.56; CYP2A6*1A/CYP2A6*1B, 9.85 +/- 16.12; CYP2A6*1B/CYP2A6*1B, 11.33 +/- 9.33). The subjects who possess the CYP2A6*1B allele appeared to show higher capabilities of cotinine formation. It was confirmed that the interindividual difference in nicotine metabolism was closely related to the genetic polymorphism of CYP2A6. The probit plot of the metabolic ratios in Koreans (8.73 +/- 11.88) was shifted to a higher ratio than that in the Japanese (3.78 +/- 3.09). In each genotype group, the Korean subjects revealed significantly higher metabolic ratios than the Japanese subjects. The ethnic difference in cotinine formation might be due to environmental and/or diet factors as well as genetic factors.     

Genetic variation in CYP2A6-mediated nicotine metabolism alters smoking behavior.

Approximately 50% of the initiation of tobacco dependence is genetically influenced, whereas maintenance of dependent smoking behavior and amount smoked have approximately 70% genetic contribution (1-5). Determining the variation in nicotine's inactivation is important because of nicotine's role in producing tobacco dependence and regulating smoking patterns (6-11). The genetically polymorphic CYP2A6 enzyme is responsible for the majority of the metabolic inactivation of nicotine to cotinine (12-14). Both in vitro and in vivo studies have demonstrated considerable interindividual variation in CYP2A6 activity (15-17). CYP2A6 is genetically polymorphic, individuals carrying inactive CYP2A6 alleles have decreased nicotine metabolism, are less likely to become smokers and if they do, they smoke fewer cigarettes per day (13,18,19). The decrease in smoking behavior was confirmed by measuring carbon monoxide (CO, a measure of smoke inhalation) levels, plasma and urine nicotine and cotinine levels, and cigarette counts (13,18,19). A duplication variant in the CYP2A6 gene locus has been identified which increases nicotine inactivation and increases smoking (19). CYP2A6 can also activate tobacco smoke procarcinogens (e.g. NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone); current studies are investigating the role of CYP2A6 in risk for lung cancer. Based on these epidemiologic data it was postulated that inhibition of CYP2A6 activity might be useful in a therapeutic context. Kinetic studies in humans indicated that selective CYP2A6 inhibitors decrease the metabolic removal of nicotine. It was also shown that inhibiting CYP2A6 in vivo (phenocopying, or mimicking the genetic defect) in smokers results in decreased smoking, making nicotine orally bioavailable, and the rerouting of procarcinogens to detoxifying pathways (20-22).     

Interindividual variability in nicotine metabolism: C-oxidation and glucuronidation

Nicotine has roles in the addiction to smoking, replacement therapy for smoking cessation, as a potential medication for several diseases such as Parkinson's disease, Alzheimer's disease, and ulcerative colitis. The absorbed nicotine is rapidly and extensively metabolized and eliminated to urine. A major pathway of nicotine metabolism is C-oxidation to cotinine, which is catalyzed by CYP2A6 in human livers. Cotinine is subsequently metabolized to trans-3'-hydroxycotinine by CYP2A6. Nicotine and cotinine are glucuronidated to N-glucuronides mainly by UGT1A4 and partly by UGT1A9. Trans-3'-hydroxycotinine is glucuronidated to O-glucuronide mainly by UGT2B7 and partly by UGT1A9. Approximately 90% of the total nicotine uptake is eliminated as these metabolites and nicotine itself. The nicotine metabolism is an important determinant of the clearance of nicotine. Recently, advances in the understanding of the interindividual variability in nicotine metabolism have been made. There are substantial data suggesting that the large interindividual differences in cotinine formation are associated with genetic polymorphisms of the CYP2A6 gene. Interethnic differences have also been observed in the cotinine formation and the allele frequencies of the CYP2A6 alleles. Since the genetic polymorphisms of the CYP2A6 gene have a major impact on nicotine clearance, its relationships with smoking behavior or the risk of lung cancer have been suggested. The metabolic pathways of the glucuronidation of nicotine, cotinine, and trans-3'-hydroxycotinine in humans would be one of the causal factors for the interindividual differences in nicotine metabolism. This review mainly summarizes recent results from our studies.     

Interindividual differences in nicotine metabolism and genetic polymorphisms of human CYP2A6

Nicotine is widely consumed throughout the world, and exerts a number of physiological effects. After nicotine is absorbed through the lungs by cigarette smoking, it undergoes extensive metabolism in humans. Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. CYP2A6 can metabolize some pharmaceutical agents such as halothane, valproic acid, and fadrozole, and activate tobacco-specific nitrosamines. There are large interindividual differences in nicotine metabolism, and it has been found that the interindividual differences are attributed to the genetic polymorphisms of CYP2A6 gene. This review describes the techniques for determination of in vivo nicotine metabolism, characteristics of each human CYP2A6 alleles, and ethnic differences. The relationship between CYP2A6 genetic polymorphism and potency of nicotine metabolism, smoking behavior, and cancer risk are extensively reviewed. Finally, the usefulness of nicotine metabolism for phenotyping of CYP2A6 in individuals and implication of the significance of CYP2A6 genetic polymorphism in a clinical perspective are discussed.     

Nicotine self-administration in mice is associated with rates of nicotine inactivation by CYP2A5

Rationale Cyp2a5, the mouse homologue of human CYP2A6, encodes for the enzyme responsible for the primary metabolism of nicotine. Variation in human CYP2A6 activity can alter the amount smoked such as number of cigarettes smoked per day and smoking intensity. Different mouse strains self-administer different amounts of oral nicotine and quantitative trait loci analyses in mice suggested that Cyp2a5 may be involved in differential nicotine consumption behaviors. Objectives The goal of this study was to examine whether in vivo nicotine consumption levels were associated with CYP2A5 protein levels and in vitro nicotine metabolism in mice. Methods F2 mice propagated from high (C57Bl/6) and low (St/bJ) nicotine consuming mice were analyzed for CYP2A5 hepatic protein levels and in vitro nicotine metabolizing activity. Results We found that F2 male high-nicotine (n=8; 25.1±1.2 μg nicotine/day) consumers had more CYP2A5 protein, compared to low (n=11; 3.8±1.4 μg nicotine/day) consumers (10.2±1.0 vs 6.5±1.3 CYP2A5 units). High consumers also metabolized nicotine faster than the low consumers (6 μM: 0.18±0.04 vs 0.14±0.07; 30 μM: 0.36± 0.06 vs 0.26±0.13; 60 μM: 0.49±0.05 vs 0.32±0.17 nmol/min/mg). In contrast, female high- (25.1±2.1 μg nicotine/day) and low-nicotine (4.7±1.4 μg nicotine/day) consumers did not show pronounced differences in nicotine metabolism or CYP2A4/5 protein levels; this is consistent with other studies of sex differences in response to nicotine. Conclusions These data suggested that among male F2 mice, increased nicotine self-administration is associated with increased rates of nicotine metabolism, most likely, as a result of greater CYP2A5 protein levels. This study was supported by CAMH, CIHR 14173 & 53248, CIHR–Special Training Program in Tobacco Use in Special Populations and Ontario Graduate Scholarship (ECKS) and a Canada Research Chair in Pharmacogenetics (RFT).     

A LC-MS/MS Method for Concurrent Determination of Nicotine Metabolites and Role of CYP2A6 in Nicotine Metabolism in U937 Macrophages: Implications in Oxidative Stress in HIV + Smokers

Nicotine, the major constituent of tobacco, is predominantly metabolized by liver CYP2A6 into cotinine and many other compounds, including nicotine-derived nitrosamine ketone (NNK), which is known to cause oxidative stress. We have recently shown that CYP2A6 is highly expressed in U937 monocyte-derived macrophages. In this study we investigated the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages. To study nicotine metabolism, we developed a highly sensitive LC-MS/MS method for simultaneous quantitative determination of nicotine, cotinine, and NNK. The LC-MS/MS analysis was carried out by multiple reaction monitoring mass transitions with m/z of 163.2/130.1, 177.4/98.3, and 208.4/122.1 for nicotine, cotinine, and NNK, respectively. The calibration curves were linear within 3.3–1028.1 ng/ml for nicotine and 0.3–652.6 ng/ml for cotinine and NNK. This novel method was then applied to quantify nicotine metabolites, cotinine and NNK, in nicotine-treated U937 macrophages. Cotinine and NNK initially formed at 30 min, followed by a peak at 2–3 h. The role of CYP2A6 in nicotine metabolism in U937 macrophages was further confirmed by using CYP2A6-selective inhibitor, tryptamine, which significantly decreased cotinine (70%) and completely inhibited NNK formations. Finally, we showed that nicotine-treated macrophages increase the formation of oxidant at 30–60 min, which is consistent with the initial formation of cotinine and NNK. In conclusion, we have developed a new LCMS/MS method for concurrent determination of nicotine metabolites and analyzed the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages, which may have implications in viral replication among HIV + smokers.     

Molecular orbital calculations and nicotine metabolism: a rationale for experimentally observed metabolite ratios

The results of molecular orbital calculations and molecular modelling studies on nicotine are reported. It is shown that the product ratio of nicotine metabolism can be directly related to HOMO electron densities on the relevant hydrogen atoms associated with oxidation sites in S-nicotine. In addition, molecular modelling of nicotine within the putative active site of CYP2A6, the enzyme most closely associated with nicotine metabolism, indicates that the substrate is orientated for oxidation at the 5'-position via a combination of hydrogen bonding and pi-pi stacking interactions. Alternative routes of metabolism may require rotation of the pyrrolidine ring system and could, therefore, involve a degree of re-orientation of the nicotine molecule which is energetically less favourable than the modelled interaction indicating formation of cotinine via 5'-oxidation.     

Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes.

In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5'-oxidation. The nicotine Delta(5'(1'))-iminium ion product of this reaction is further metabolized to cotinine by aldehyde oxidase. Previous investigators have reported kinetic parameters for cotinine formation using human liver cytosol as a source of aldehyde oxidase. Using [5-(3)H]nicotine and radioflow high-performance liquid chromatography analysis, we determined kinetic parameters for nicotine 5'-oxidation by P450 2A6 and the closely related human extrahepatic P450 2A13 as well as the rodent P450s 2A3, 2A4, and 2A5. The formation of both cotinine and nicotine Delta(5'(1'))-iminium ion was monitored. The K(m) and V(max) values for P450 2A6 were 144 +/- 15 muM and 1.30 +/- 0.05 pmol/min/pmol, respectively. Previously reported K(m) values for cotinine formation by P450 2A6 in the presence of cytosol were much lower, ranging from 11 to 45 muM. P450 2A13 was a somewhat better catalyst of nicotine Delta(5'(1'))-iminium formation, with 2-fold lower K(m) and 2-fold higher V(max) values than P450 2A6. The rat P450 2A3 and the mouse P450 2A5, which are 85 and 84% identical to P450 2A6, were much more efficient catalysts of nicotine 5'-oxidation. P450 2A4 was not an efficient catalyst of nicotine metabolism. Whereas 5'-oxidation was the major pathway of nicotine metabolism for all five P450 2A enzymes, these enzymes also catalyzed methyl oxidation. Nornicotine, the product of this reaction was detected as 5 to 15% of the total nicotine metabolites. Nornicotine is the amine precursor to the esophageal carcinogen N'-nitrosonornicotine. Therefore, methyl oxidation of nicotine by P450 2A6 or P450 2A13 followed by nitrosation of nornicotine are possible endogenous pathways of N'-nitrosonornicotine formation.     

Effects of whole deletion of CYP2A6 on nicotine metabolism in humans

We investigated the effects of CYP2A6 genotypes on nicotine metabolism, focused from nicotine to cotinine and its additional 3'-hydroxylating resulted in trans-3'-hydroxycotinine formation. In the subjects genotyped by PCR-RFLP method, one cigarette smoking experiment was performed and urine samples were collected for 24 h. In all subjects who smoked, we detected nicotine, cotinine and trans-3'-hydroxycotinine in urine by GC-MS analysis. In whole deletion of CYP2A6, urinary excretion amounts of cotinine and trans-3'-hydroxycotinine were significantly smaller than those in the wild-type of CYP2A6*1. A lack of CYP2A6 reduces the formation of cotinine and trans-3'-hydroxycotinine, but not entirely reduces the trans-3'-hydroxycotinine formation. Unknown cotinine 3'-hydroxylating activity except CYP2A6 are suspected in humans.     

In vitro metabolism of styrene to styrene oxide in liver and lung of Cyp2E1 knockout mice

Styrene is a widely used chemical. In mice it is both hepatotoxic and pneumotoxic, and this toxicity is thought to be associated with its metabolism to styrene oxide. In vitro studies by several investigators suggest that this bioactivation in mice is primarily due to CYP2E1 and CYP2F2. However, in vivo studies demonstrate that CYP2E1 knockout mice can metabolize styrene to a similar extent as the wild-type mice. The current studies compared the in vitro metabolism of styrene by hepatic and pulmonary microsomes from CYP2E1 knockout and wild-type mice. There was no difference in the hepatic microsomal metabolism of styrene to styrene oxide between the two strains. The metabolism of styrene was lower in the lungs of the knockout mice than in the wild-type. Chemical inhibitors were used to ascertain the contributions made by various cytochromes P-450: imipramine for CYP2C, alpha -methylbenzylaminobenzotriazole for CYP2B, alpha -naphthoflavone for CYP1A, 5-phenyl-1-pentyne for CYP2F2, and diethyldithiocar-bamate for CYP2E1. The data indicate that CYP2E1 and CYP2F2 may be important in wild-type mice, but they do not clearly indicate what cytochromes P-450 are responsible for the metab-olism in the knockout mice. Inhibition of styrene metabolism in the knockout mice by diethyl-dithiocarbamate indicates this inhibitor is not completely selective for CYP2E1. These in vitro data support the in vivo finding of styrene metabolism in CYP2E1 knockout mice and indicate that other enzymes are contributing to styrene metabolism in these mice.     

Characterization of the novel CYP2A6*21 allele using in vivo nicotine kinetics

Objective The impact of CYP2A6*21 (K476R) on in vivo nicotine metabolism and disposition was investigated.Methods A two-step allele-specific PCR assay was developed to detect the 6573A>G single nucleotide polymorphism (SNP) in CYP2A6*21. Nicotine metabolism phenotypes from a previously described intravenous labeled nicotine and cotinine infusion study [1] was used to assess the impact of CYP2A6*21. Genomic DNA samples from 222 (111 monozygotic and dizygotic twin pairs) Caucasian subjects were genotyped for CYP2A6 alleles (CYP2A6*1X2, -*1B, -*2, -*4, -*7, -*9, -*10, -*12, and -*21). The pharmacokinetic parameters were compared between individuals with no detected CYP2A6 variants (CYP2A6*1/*1, n=163) and individuals heterozygous for the CYP2A6*21 allele (CYP2A6*1/*21, n=9).Results The frequency of the CYP2A6*21 allele was found to be 2.3% in Caucasians (n=5/222 alleles, evaluated in one twin from each twin pair). In vivo pharmacokinetic parameters, such as nicotine clearance (1.32±0.37 vs. 1.18±0.20 L/min), fractional clearance of nicotine to cotinine (1.02±0.36 vs. 0.99±0.23 L/min), nicotine half-life (111±37 vs. 116±29 min), and the trans-3′-hydroxycotinine to cotinine ratio (1.92±1.0 vs. 1.55±0.58) indicated no substantial differences in nicotine metabolism between those without the variant (CYP2A6*1/*1, n=163) and those with the variant (CYP2A6*1/*21, n=9), respectively.Conclusions CYP2A6*21 does not have a detectable impact on nicotine metabolism in vivo. Our data suggest that CYP2A6*21 may not be important for future studies of nicotine metabolism and the resulting impacts on smoking behaviors.Nael Al Koudsi and Jill C. Mwenifumbo contributed equally to this work.     

Nicotine metabolism,human drug metabolism polymorphisms,and smoking behaviour

Large interindividual differences occur in human nicotine disposition, and it has been proposed that genetic polymorphisms in nicotine metabolism may be a major determinant of an individual's smoking behaviour. Hepatic cytochrome P4502A6 (CYP2A6) catalyses the major route of nicotine metabolism: C-oxidation to cotinine, followed by hydroxylation to trans-3'-hydroxycotinine. Nicotine and cotinine both undergo N-oxidation and pyridine N-glucuronidation. Nicotine N-1-oxide formation is catalysed by hepatic flavin-containing monooxygenase form 3 (FMO3), but the enzyme(s) required for cotinine N-1'-oxide formation has not been identified. trans-3'-Hydroxycotinine is conjugated by O-glucuronidation. The uridine diphosphate-glucuronosyltransferase (UGT) enzyme(s) required for N- and O-glucuronidation have not been identified. CYP2A6 is highly polymorphic resulting in functional differences in nicotine C-oxidation both in vitro and in vivo; however, population studies fail to consistently and conclusively demonstrate any associations between variant CYP2A6 alleles encoding for either reduced or enhanced enzyme activity with self-reported smoking behaviour. The functional consequences of FMO3 and UGT polymorphisms on nicotine disposition have not been investigated, but are unlikely to significantly affect smoking behaviour. Therefore, current evidence does not support the hypothesis that genetic polymorphisms associated with nicotine metabolism are a major determinant of an individual's smoking behaviour and exposure to tobacco smoke.