An in vivo pilot study characterizing the new CYP2A6*7, *8, and *10 alleles.

We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced.     

Influence of the genetic polymorphism in the 5'-noncoding region of the CYP1A2 gene on CYP1A2 phenotype and urinary mutagenicity in smokers

The functional significance of genetic polymorphisms on tobacco smoke-induced CYP1A2 activity was examined. The influence of three polymorphisms of the cytochrome P450 1A2 gene (CYP1A2) (-3860 G-->A (allele *1C), -2467 T-->delT (allele *1D), -163C-->A (allele *1F)), located in the 5'-noncoding promoter region of the gene, on CYP1A2 activity (measured as caffeine metabolic ratio, CMR), was studied in Caucasian current smokers (n=95). Tobacco smoke intake was calculated from the number of cigarettes/day. Also, studied was the influence of these CYP1A2 genotypes on smoking-associated urinary mutagenicity, detected in Salmonella typhimurium strain YG1024 with S9 mix, considering the urinary excretion of nicotine plus its metabolites as an internal indicator of tobacco smoke exposure. Smokers with at least one of the variant alleles CYP1A2 -3860A and -2467 delT showed a significantly increased CYP1A2 CMR (-3860 G/A versus G/G, p<0.05; -2467 delT/delT versus T/delT and T/T, p<0.01). Multiple regression analysis showed that the increase in CYP1A2 CMR (ln values) was again significantly related to the presence of CYP1A2 variants -2467delT and also to variant -163A (p<0.05), but moderately to -3860A (p=0.084). No influence of the number of cigarettes smoked per day by each subject was found. Heavy smokers (n=48, with urinary nicotine plus its metabolites>or=0.69 mg/mmol creatinine) with variant allele -2467delT or -163A had significantly increased urinary mutagenicity (p<0.01 and <0.05). CYP1A2 genetic polymorphisms are shown to influence the CYP1A2 phenotype in smokers, -2467 T-->delT having the main effect. This information is of interest for future studies assessing the possible role of tobacco smoke-inducible CYP1A2 genotypes as individual susceptibility factors in exposure to carcinogens.     

Expression of cytochrome P450 2A6 in Escherichia coli: purification, spectral and catalytic characterization, and preparation of polyclonal antibodies.

Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines. In this paper expression of CYP2A6 in Escherichia coli is reported. In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis. The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture. Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein. Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked. The N-terminal formylmethionine block was removed by mild acid treatment. Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K(m) = 1.48 +/- 0.37 microM, V(max) = 3.36 +/- 0.18 nmol product/min/nmol CYP). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies. Availability of this expression system and specific antibodies should facilitate characterization of the role of CYP2A6 in the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of this enzyme.     

Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism.

The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C-oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re-evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3' flanking region of 30-40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.     

Identification of a single nucleotide polymorphism in the TATA box of the CYP2A6 gene: impairment of its promoter activity

Human cytochrome P450 2A6 (CYP2A6) constitutes the major nicotine oxidase, and large interindividual differences are seen in the levels of this enzyme, to a great extent caused by the distribution of several different polymorphic gene variants mainly located in the open reading frame (ORF). In the present study, we report a common polymorphism located in the 5' flanking region of CYP2A6 affecting its expression. DHPLC analysis and complete sequence of the open reading frame of the gene from a Turkish individual revealed a -48T > G substitution disrupting the TATA box. Using dynamic allele-specific hybridization (DASH), genotyping of this novel variant (named CYP2A6*9) was carried out in 116 Swedish, 132 Turkish, and 102 Chinese subjects, and the allele frequencies were found to be 5.2, 7.2, and 15.7%, respectively. The significance of the polymorphism was investigated by the construction of luciferase reporter plasmids containing 135 or 500 bp of the 5'-upstream region of the gene transfected into human hepatoma B16A2 cells. The constructs carrying the -48T > G mutation were only expressed at about 50% of the wild-type alleles. It is concluded that the CYP2A6*9 allele might be one of the most common CYP2A6 variants in Caucasians that alters the levels of enzyme expression.     

Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation

Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val(117)--> Ala and Arg(372)--> His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala(117) --> Val and His(372) --> Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.     

3'-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression

Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in human and participates in the metabolism of drugs and precarcinogens. The CYP2A6 gene is highly polymorphic and more than 22 different alleles have been described. We here focused on the polymorphism in the 3'-UTR region, in particular the common CYP2A6*1B allele, carrying an unequal crossover element from the pseudogene CYP2A7. Analysis of CYP2A6 expression in a human liver bank (n=46) revealed that the protein level and catalytic activity using coumarin as a substrate were all higher, following a linear gene-dose relationship, in livers carrying one or two copies of CYP2A6*1B, as compared to other CYP2A6 allelic variants. Different variants of the CYP2A6 3'-UTR were cloned into a modified pGL3 plasmid downstream of the luciferase reporter gene. The plasmids, having the proximal promoter of CYP2A6 gene, were transfected into HeLa cells or injected into the tail veins of male CD1 mice. In both systems, the 3'-UTR CYP2A6*1B constructs caused higher reporter gene activity and the CYP2A7 3'-UTR construct lower activity, compared to the CYP2A6*1 3'-UTR constructs. Two SNPs differentiating the 3'-UTR between CYP2A7 and CYP2A6*1B were found to be of importance for the expression in both systems. Analysis of reporter enzyme degradation in HeLa cells showed that luciferase-3'-UTR-CYP2A6*1A had a half-life of approximately 4.9h as compared to 6.3h for luciferase-3'-UTR-CYP2A6*1B. In conclusion, we identified polymorphic motifs in the CYP2A6 3'-UTR of importance for CYP2A6 mRNA stabilization and enzyme expression. Such polymorphism has been described to influence the in vivo rate of nicotine elimination and possibly the cigarette consumption and risk of smoking induced lung cancer.     

'Smoking genes': a genetic association study

Some controversy exists on the specific genetic variants that are associated with nicotine dependence and smoking-related phenotypes. The purpose of this study was to analyse the association of smoking status and smoking-related phenotypes (included nicotine dependence) with 17 candidate genetic variants: CYP2A6*1×2, CYP2A6*2 (1799T>A) [rs1801272], CYP2A6*9 (-48T>G) [rs28399433], CYP2A6*12, CYP2A13*2 (3375C>T) [rs8192789], CYP2A13*3 (7520C>G), CYP2A13*4 (579G>A), CYP2A13*7 (578C>T) [rs72552266], CYP2B6*4 (785A>G), CYP2B6*9 (516G>T), CHRNA3 546C>T [rs578776], CHRNA5 1192G>A [rs16969968], CNR1 3764C>G [rs6928499], DRD2-ANKK1 2137G>A (Taq1A) [rs1800497], 5HTT LPR, HTR2A -1438A>G [rs6311] and OPRM1 118A>G [rs1799971]. We studied the genotypes of the aforementioned polymorphisms in a cohort of Spanish smokers (cases, N = 126) and ethnically matched never smokers (controls, N = 80). The results showed significant between-group differences for CYP2A6*2 and CYP2A6*12 (both P<0.001). Compared with carriers of variant alleles, the odds ratio (OR) for being a non-smoker in individuals with the wild-type genotype of CYP2A6*12 and DRD2-ANKK1 2137G>A (Taq1A) polymorphisms was 3.60 (95%CI: 1.75, 7.44) and 2.63 (95%CI: 1.41, 4.89) respectively. Compared with the wild-type genotype, the OR for being a non-smoker in carriers of the minor CYP2A6*2 allele was 1.80 (95%CI: 1.24, 2.65). We found a significant genotype effect (all P≤0.017) for the following smoking-related phenotypes: (i) cigarettes smoked per day and CYP2A13*3; (ii) pack years smoked and CYP2A6*2, CYP2A6*1×2, CYP2A13*7, CYP2B6*4 and DRD2-ANKK1 2137G>A (Taq1A); (iii) nicotine dependence (assessed with the Fagestrom test) and CYP2A6*9. Overall, our results suggest that genetic variants potentially involved in nicotine metabolization (mainly, CYP2A6 polymorphisms) are those showing the strongest association with smoking-related phenotypes, as opposed to genetic variants influencing the brain effects of nicotine, e.g., through nicotinic acetylcholine (CHRNA5), serotoninergic (HTR2A), opioid (OPRM1) or cannabinoid receptors (CNR1).     

Comparison of "high throughput" micromethods for determination of cytochrome P450 activities with classical methods using HPLC for product identification

Enzyme activities of the CYP enzymes (CYP3A4, CYP2C9 and CYP2A6) were determined using classical substrates (testosterone, diclofenac and coumarin, respectively) as well as with luminogenic or fluorogenic substrates in micromethod arrangement. The luciferin-based luminogenic substrates for CYP3A4 and CYP2C9 as well as coumarin in micromethod for assay of CYP2A6 activity gave results well comparable with the classical methods with determination of reaction products by the HPLC.     

Heterologous expression of CYP102A5 variant from Bacillus cereus CYPPB-1: Validation of model for predicting drug metabolism of human P450 probe substrates

CYP102A5 variant (ADL27534) from isolated Bacillus cereus CYPPB-1 was heterologously expressed in Escherichia coli Top 10 cells. Comparative sequence analysis of purified CYP102A5 variant with respect to reported CYP102A5 (AAP10153) from Bacillus cereus ATCC 14579 revealed amino acid sequence changes at positions P245S and M318I of heme domain. The binding affinities of 15 selected human P450 probe substrates towards isolated CYP102A5 were analyzed in silico using a homology model together with molecular docking techniques to predict the human drug metabolism. In vitro analysis suggested that the purified CYP102A5 metabolizes typical substrates of human CYP2C9, CYP2D6, CYP2E1, and CYP3A4, such as coumarin, propranolol, aniline, chlorzoxazone, p-nitrophenol, and nifedipine. The calculated K _M values for propranolol, chloroxazone, coumarin, aniline, and 4-nitrophenol were calculated to be 0.962?±?0.041, 1.254?±?0.057, 2.859?±?0.083, 2.732?±?0.106, and 2.528?±?0.11 mM, respectively. Importantly, taking a ChemScore cutoff value of ?31 kJ/mol, substrate binding at active site and in vitro activity as the distinguishing lines between “substrates” and “nonsubstrates” revealed one false-positive and one false-negative results out of the 15 compounds examined. This is the first report on validation of CYP102A family homology model for in silico prediction of human drug metabolism.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

The significance of the homozygous CYP2A6 deletion on nicotine metabolism: a new genotyping method of CYP2A6 using a single PCR-RFLP.

A convenient and specific CYP2A6 genotyping method was developed in this study. This method consisting of a single PCR-RFLP is capable of resolving the genotype into either CYP2A6*1 (wild type), CYP2A6*2, or CYP2A6*3. Among 252 Japanese persons genotyped, 241 were genotyped as the wild type, 1 as an unknown variant, and none as either CYP2A6*2 or CYP2A6*3. A homozygous deletion was found in the 10 remaining subjects. To clarify the metabolic significance of this deletion in the whole human body, urinary cotinine, the principal metabolite of nicotine, was analyzed subsequent to smoking. Cumulated urinary cotinine excretion in the homozygously CYP2A6-deleted individuals was about one-seventh compared to the control group (wild type). This study provides a firm experimental basis for correlating genotypic characterization of CYP2A6 with phenotypic expression of nicotine metabolism.     

Cytochrome P4502C9 genotype in Southeast Anatolia and possible relation with some serum tumour markers and cytokines

Substrates for CYP2C9 include fluoxetine, phenytoin, warfarin, losartam and numerous nonsteroidal anti-inflammatory drugs. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino-acid residues 144 Arg/Cys and 359 Ile/Leu of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, the frequency of this allele is, however, rather low. Consistently with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. The polymorphic enzyme CYP2C9 takes part in the metabolism of alkylating agents and polycyclic aromatic hydrocarbons like benzo(a)pyrene, a carcinogen present in tobacco smoke. Although the impact of impaired enzyme activity in metabolism of carcinogens and procarcinogens has not been fully defined, an association of CYP2C9 variant alleles to DNA adduct levels in lung tissues as well as to lung cancer risk have been reported. In this study 64 healthy subjects (44M/22F) were analysed for CYP2C9 genotype with PCR-RFLP and for serum carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA 19-9, CA 15-3, ferritin, IL-6, IL-8 concentrations by chemiluminescence or electrochemiluminescence methods. CYP2C9*1 was found to be the most prevalent allele and CYP2C9*1/CYP2C9*1 was the most frequent genotype represented in 64% of the population in southeastern Anatolia (Gaziantep). Although slight differences in serum tumour marker and cytokine concentrations were observed for CYP2C9 genotypes the differences were statistically insignificant (P > 0.05). This could be due to the complexity of the role of CYP2C9 in benzo(a)pyrene metabolism as well as from other contributing factors like interindividual variability of diverse enzymes participating in the same metabolic pathway, unequal expression of the variant alleles and differences in exposure to carcinogens. However, determination of CYP2C9 phenotypes in a larger group of subjects might clarify these slight differences.     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis

Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k(cat) and Km) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (k(cat)/Km) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher Km values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations.     

Rational design of novel CYP2A6 inhibitors

Inhibition of CYP2A6-mediated nicotine metabolism can reduce cigarette smoking. We sought potent and selective CYP2A6 inhibitors to be used as leads for drugs useful in smoking reduction therapy, by evaluating CYP2A6 inhibitory effect of novel formyl, alkyl amine or carbonitrile substituted aromatic core structures. The most potent CYP2A6 inhibitors were thienopyridine-2-carbaldehyde, benzothienophene-3-ylmethanamine, benzofuran-5-carbaldehyde and indole-5-carbaldehyde, with IC₅₀ values below 0.5 μM for coumarin 7-hydroxylation. Nicotine oxidation was effectively inhibited in vitro by two alkyl amine compounds and benzofuran-5-carbonitrile. Some of these molecules could serve as potential lead molecules when designing CYP2A6 inhibitory drugs for smoking reduction therapy.     

A Genetic Polymorphism in Coumarin 7-Hydroxylation: Sequence of the Human CYP2A Gnes and Identification of Variant CYP2A6 Alleles

A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes—CYP2A6, CYP2A7, and CYP2A13—plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6ν2. Sequence differences in the CYP2A6ν2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6ν2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6ν2 allele, and a variant—designated CYP2A6ν1—that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6ν1 and CYP2A6ν2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.     

Interethnic and intraethnic variability of CYP2C8 and CYP2C9 polymorphisms in healthy individuals

Cytochrome P450 (CYP) superfamily members CYP2C8 and CYP2C9 are polymorphically expressed enzymes that are involved in the metabolic inactivation of several drugs, including, among others, antiepileptics, NSAIDs, oral hypoglycemics, and anticoagulants. Many of these drugs have a narrow therapeutic index, and growing evidence indicates a prominent role of CYP2C8 and CYP2C9 polymorphisms in the therapeutic efficacy and in the development of adverse effects among patients treated with drugs that are CYP2C8 or CYP2C9 substrates. In this review, we summarize present knowledge on human variability in the frequency of variant CYP2C8 and CYP2C9 alleles. Besides an expected interethnic variability in allele frequencies, a large intraethnic variability exists. Among Asian subjects, for example, statistically significant differences (p < 0.0001) in CYP2C9*3 allele frequencies between Chinese and Japanese individuals have been reported. In addition, individuals from East Asia present different allele frequencies for CYP2C9*2 and CYP2C9*3 compared with South Asian subjects (p < 0.0001). Among Caucasian Europeans, statistically significant differences for the frequency of CYP2C8*3, CYP2C9*2, and CYP2C9*3 exist (p < 0.0001). This indicates that Asian individuals or Caucasian European individuals cannot be considered as homogeneous groups regarding CYP2C8 or CYP2C9 allele frequencies. Caucasian American subjects also show a large variability in allele frequencies, which is likely to be related to ethnic ancestry. A higher frequency of variant CYP2C8 and CYP2C9 alleles is expected among Caucasian Americans with South European ancestry than in individuals with North European ancestry. The findings summarized in this review suggest that among individuals with Asian or European ancestry, intraethnic differences in the risk of developing adverse effects with drugs that are CYP2C8 or CYP2C9 substrates are to be expected. In addition, the observed intraethnic variability reinforces the need for proper selection of control subjects and points against the use of surrogate control groups for studies involving association of CYP2C8 or CYP2C9 alleles with adverse drug reactions or spontaneous diseases.     

Identification of a null allele of cytochrome P450 3A7: CYP3A7 polymorphism in a Korean population

Cytochrome P450 3A7 (CYP3A7) is expressed in the human fetal liver and plays a role in the metabolism of hormones, drugs, and toxic compounds. Genetic variants of CYP3A7 are associated with serum estrone level, bone density, and hepatic CYP3A activity in adults. We analyzed the genetic variations of CYP3A7 in a Korean population. From direct sequencing of all exons and flanking regions of the CYP3A7 gene in 48 Koreans, we found five genetic variants, including three novel variants. One variant, a thymidine insertion in exon 2 (4011insT), causes premature termination of CYP3A7 translation, which may result in a null phenotype. The novel variant was assigned to the CYP3A7*3 allele by the CYP allele nomenclature committee. For further screen of this novel variant in other ethnic populations, we used pyrosequencing to analyze an additional 185 Koreans, 100 African Americans, 100 Caucasians, and 159 Vietnamese for the presence of this variant. The variant was not found in any other individuals, except for one Korean subject. The frequencies of two known functional alleles, CYP3A7*2 and CYP3A7*1C, were 26 and 0%, respectively, in Koreans. The frequencies of the functional CYP3A7 polymorphisms in Koreans were significantly different from those in Caucasians and African Americans. This is the first report of a null-type allele of the CYP3A7 gene. It also provides population-level genetic data on CYP3A7 in Koreans to reveal the wide ethnic variation in CYP3A7 polymorphism.     

Functional analysis of CYP2D6.31 variant: homology modeling suggests possible disruption of redox partner interaction by Arg440His substitution

Cytochrome P450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous CYP2D6 alleles have been characterized at a functional level, although the consequences for expression and/or catalytic activity of a substantial number of rare variants remain to be investigated. One such allele, CYP2D6*31, is characterized by mutations encoding three amino acid substitutions: Arg296Cys, Arg440His and Ser486Thr. The identification of this allele in an individual with an apparent in vivo poor metabolizer phenotype prompted us to analyze the functional consequence of these substitutions on enzyme activity using yeast as a heterologous expression system. We demonstrated that the Arg440His substitution, alone or in combination with Arg296Cys and/or Ser486Thr, altered the respective kinetic parameters [Km (microM) and kcat (min(-1))] of debrisoquine 4-hydroxylation (wild-type, 25; 0.92; variants, 43-68; 0.05-0.11) and dextromethorphan O-demethylation (wild-type, 1; 4.72; variants, 12-23; 0.64-1.43), such that their specificity constants (kcat/Km) were decreased by more than 95% compared to those observed with the wild-type enzyme. The rates of oxidation of rac-metoprolol at single substrate concentrations of 40 and 400 microM were also markedly decreased by approximately 90% with each CYP2D6 variant containing the Arg440His substitution. These in vitro data confirm that the CYP2D6*31 allele encodes an enzyme with a severely impaired but residual catalytic activity and, furthermore, that the Arg440His exchange alone is the inactivating mutation. A homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 locates Arg440 on the proximal surface of the protein. Docking the structure of the FMN domain of human cytochrome P450 reductase to the CYP2D6 model suggests that Arg440 is a key member of a cluster of basic amino acid residues important for reductase binding.