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.

Identification of inhibitors of the nicotine metabolising CYP2A6 enzyme--an in silico approach.

The compulsive nature of tobacco use is attributable to nicotine addiction. Nicotine is eliminated by metabolism through the cytochrome P450 2A6 (CYP2A6) enzyme in liver. Inhibition of CYP2A6 by chemical compounds may represent a potential supplement to anti-smoking therapy. The purpose of this study was to rationally design potent inhibitors of CYP2A6. 3D-QSAR models were constructed to find out which structural characteristics are important for inhibition potency. Specifically located hydrophobic and hydrogen donor features were found to affect inhibition potency. These features were used in virtual screening of over 60,000 compounds in the Maybridge chemical database. A total of 22 candidate molecules were selected and tested for inhibition potency. Four of these were potent and selective CYP2A6 inhibitors with IC(50) values lower than 1 muM. They represent novel structures of CYP2A6 inhibitors, especially N1-(4-fluorophenyl)cyclopropane-1-carboxamide. This compound can be used as a lead in the design of CYP2A6 inhibitor drugs to combat nicotine addiction.

New potent and selective cytochrome P450 2B6 (CYP2B6) inhibitors based on three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis.

BACKGROUND AND PURPOSE: The cytochrome P450 2B6 (CYP2B6) enzyme metabolises a number of clinically important drugs. Drug-drug interactions resulting from inhibition or induction of CYP2B6 activity may cause serious adverse effects. The aims of this study were to construct a three-dimensional structure-activity relationship (3D-QSAR) model of the CYP2B6 protein and to identify novel potent and selective inhibitors of CYP2B6 for in vitro research purposes. EXPERIMENTAL APPROACH: The inhibition potencies (IC(50) values) of structurally diverse chemicals were determined with recombinant human CYP2B6 enzyme. Two successive models were constructed using Comparative Molecular Field Analysis (CoMFA). KEY RESULTS: Three compounds proved to be very potent and selective competitive inhibitors of CYP2B6 in vitro (IC(50)<1 microM): 4-(4-chlorobenzyl)pyridine (CBP), 4-(4-nitrobenzyl)pyridine (NBP), and 4-benzylpyridine (BP). A complete inhibition of CYP2B6 activity was achieved with 0.1 microM CBP, whereas other CYP-related activities were not affected. Forty-one compounds were selected for further testing and construction of the final CoMFA model. The created CoMFA model was of high quality and predicted accurately the inhibition potency of a test set (n=7) of structurally diverse compounds. CONCLUSIONS AND IMPLICATIONS: Two CoMFA models were created which revealed the key molecular characteristics of inhibitors of the CYP2B6 enzyme. The final model accurately predicted the inhibitory potencies of several structurally unrelated compounds. CBP, BP and NBP were identified as novel potent and selective inhibitors of CYP2B6 and CBP especially is a suitable inhibitor for in vitro screening studies.

More potent inhibition of human CYP2A6 than mouse CYP2A5 enzyme activities by derivatives of phenylethylamine and benzaldehyde.

1. A rapid 96-well plate assay method was developed and validated to measure liver microsomal coumarin 7-hydroxylation in vitro. 2. The method was used to test inhibition of human and mouse CYP2A enzymes by three phenylethylamine derivatives 2-(p-tolyl)-ethylamine, amphetamine, 2-phenylethylamine and benzaldehyde, and two of its derivatives, 4-methylbenzaldehyde and 4-methoxybenzaldehyde. 3. The benzaldehyde derivatives were more potent inhibitors of CYP2A5 than the phenylethylamines. The K(ic) value of 4-methylbenzaldehyde was 3.4 micro M and for 4-methoxybenzaldehyde it was 0.86 micro M for CYP2A5. 4. Amphetamine is a weak inhibitor of CYP2A6, whereas benzaldehyde is a suicide inhibitor with K(inact) = 0.16 min(-1) and K(I) = 18 micro M. The K(ic) values of 2-phenylethylamine, 2-(p-tolyl)-ethylamine, 4-methylbenzaldehyde and 4-methoxybenzaldehyde were 1.13, 0.23, 0.36 and 0.73 micro M for CYP2A6, respectively. 5. Novel potent inhibitors were found for CYP2A6 and, except for 4-methoxybenzaldehyde, all the compounds inhibited CYP2A5 and CYP2A6 enzymes differentially. These data add to the refinement of CYP2A enzyme active sites and provide chemical leads for developing novel chemical inhibitors of the CYP2A6 enzyme.

Cytochrome P450 3A expression in the human fetal liver: evidence that CYP3A5 is expressed in only a limited number of fetal livers.

CYP3A is the major cytochrome P450 subfamily constitutively expressed in the human liver. CYP3A4 is the predominant hepatic P450 form in adults and it is expressed at high but very variable levels among individuals. The fetal liver contains mainly CYP3A7, while the presence of the other CYP3A enzymes in fetal liver has remained controversial. In this study, the relative levels of CYP3A4, CYP3A5 and CYP3A7 expression were determined in a panel of 9-11 fetal livers with a similar gestation age (9-12 weeks) and compared to adult livers. CYP3A7 was found to be the major CYP3A form in all the fetal liver samples. The abundance of CYP3A7 varied more at the mRNA (77-fold variation) than at the protein level (4.8-fold variation). CYP3A5 mRNA was also detected in all of the fetal liver samples, but the average level was 700-fold lower than that of CYP3A7. CYP3A5 protein was detected by immunoblot analysis in only 1 fetal liver out of the 9 investigated, the level of expression being moderately high in this sample. CYP3A4 mRNA was detected in only a subset of the fetal liver samples and its level was the lowest of the CYP3A forms. This is the first study to demonstrate the polymorphic expression of CYP3A5 and the variability of CYP3A7 expression in fetal liver and suggests that significant interindividual differences in the metabolism of xenobiotics may already exist at the prenatal stage. These differences may contribute to individual pharmacological and/or toxicological responses in the fetus.

Polymorphisms of CYP2A6 and its practical consequences.

CYP2A6 is an hepatic enzyme predominantly with some expression in specialized extrahepatic cell types. The CYP2A6 enzyme has a somewhat restricted active site, accepting only a few xenobiotics as substrates. Interest in CYP2A6 has risen considerably after nicotine and some tobacco specific nitrosamines were established as high-affinity substrates for this enzyme. Recently, the organization and structures of the CYP2A gene cluster and several polymorphic alleles of the CYP2A6 gene have been characterized. Two alleles with a point mutation and at least three different types of gene deletion, all leading to deficient gene function, have been found. The frequencies of these alleles vary considerably among different ethnic populations, the deletion alleles being most common in Orientals (up to 20%). The frequency of point mutations are low in all populations studied thus far (< 3%). Several case-control studies have addressed the relationship between CYP2A6 status and smoking habits as well as the role of CYP2A6 polymorphism in lung cancer risk. Studies in Japanese suggest that CYP2A6 poor metabolizer genotypes result in altered nicotine kinetics and may lower cigarette smoking elicited lung cancer risk, whereas similar studies in Caucasian populations have not revealed any clear associations between variant CYP2A6 genotypes and smoking behaviour or lung cancer predisposition.

Distribution of cytochrome P4501A (CYP1A) in the tissues of Baltic ringed and grey seals.

Information about the expression of CYP1A in wildlife species is essential for understanding the impact of organochlorine exposure on the health status of an exposed population. Therefore, we aimed at characterising a putative CYP1A enzyme expression in both hepatic and extrahepatic tissues of ringed and grey seals from the Baltic Sea and from less polluted waters. The cellular localisation of CYP1A was identified using a monoclonal antibody against scup P4501A1 (MAb 1-12-3). Immunohistochemical staining showed the highest level of CYP1A expression in liver hepatocytes, and the second highest level in the endothelial cells of capillaries and larger blood vessels in the liver and other organs. The most frequent and strongest staining was found in Baltic ringed seals. Although CYP1A-positive staining was observed in only a few tissues in the other seal populations, it was more intense in Baltic grey seals than in Canadian grey seals. The CYP1A enzyme activity, expressed as ethoxyresorufin O-deethylation (EROD), followed a similar tissue distribution and geographical pattern as the immunohistochemistry with clearly elevated EROD activities in most tissues of both Baltic seal populations. Immunochemical characterisation by immunoblotting confirmed the presence and elevation pattern of a putative CYP1A protein in ringed and grey seals, supporting our findings using other methods. The evenly distributed elevation of CYP1A expression among most of the tissues examined indicates that Baltic seals are exposed to CYP1A inducing agents affecting the whole body. This may result in an increased or decreased toxic potential of foreign substances, which may ultimately determine the biological effects of the contaminants.

Characterization of xenobiotic-metabolizing cytochrome P450 (CYP) forms in ringed and grey seals from the Baltic Sea and reference sites.

Earlier studies have shown that members of the cytochrome P4501 (CYP1) enzyme family are constitutively expressed, and are elevated in the livers of ringed seals (Phoca hispida) and grey seals (Halichoerus grypus) living in the heavily polluted Baltic Sea. In this study, we compared the expression profiles of several additional CYP enzymes in the liver and extrahepatic tissues of Baltic ringed and grey seals with the corresponding CYP expression in seals from relatively unpolluted waters. We used marker enzyme activity levels, diagnostic inhibitors and immunoblot analysis to assess members of the CYP2A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A sub-families. Coumarin 7-hydroxylation (COH), a marker of CYP2A activity, was high in the liver and the lungs of all the studied seal populations. The presence of a putative CYP2A form in these seals was further supported by the strong inhibition of COH activity by a chemical inhibitor and by an anti-CYP2A5 antibody. However, antibodies to human and rodent CYP2B, CYP2C and CYP2E forms did not recognize any proteins in these seal species. Dextromethorphan O-demethylation (marker for CYP2D activity) and chlorzoxazone 6-hydroxylation (marker for CYP2E activity) were measurable in the livers of all the seals we studied. Both activities were elevated in the Baltic seal populations, showed a strong positive correlation with CYP1A activity and were at least partly inhibited by a typical CYP1A inhibitor, alpha-naphthoflavone. Further studies are needed to determine the presence and characteristics of CYP2D and CYP2E enzymes in ringed and grey seals. Testosterone 6beta-hydroxylation, a CYP3A marker, showed a relatively high level of activity in the livers of both seal species and was potently inhibited by ketoconazole, a CYP3A-selective inhibitor. The putative CYP3A activity showed an opposing geographical trend to that of CYP2D and CYP2E, since it was elevated in the control area. CYP3A protein levels, revealed by immunoblotting, showed a positive correlation with testosterone 6beta-hydroxylation. We conclude tentatively that CYP2A- and CYP3A-like enzymes are expressed in ringed and grey seals, but that CYP2B- and CYP2C-like ones are not. Further information on the individual contaminant profile is needed before any conclusions can be drawn on a possible connection between the varying CYP expressions and the contaminant load.

cAMP mediated upregulation of CYP2A5 in mouse hepatocytes.

CYP2A5 is induced by a large number of chemicals including some cAMP modifiers. In a primary hepatocyte model, stimulation of the cAMP signal transduction pathway by glucagon and isoproterenol, acting via specific G-protein coupled plasma membrane receptors, produced up to 17-fold increases in the marker activity of CYP2A5, coumarin 7-hydroxylase. In contrast, glucagon and isoproterenol caused no significant effects on two other major CYP forms, CYP2B10 and CYP1A1/2. Phenobarbital (PB) elicited a 3-fold increase in CYP2A5 expression (catalytic activity and mRNA), while the cAMP and protein kinase A (PKA) stimulators dibutyryl-cAMP, forskolin and Sp-cAMPs caused up to 18-fold increases in the amount of CYP2A5 mRNA. Coadministration of PB and cAMP/PKA stimulating agents produced an additive inducing effect. The expression of CYP2A5, but not CYP2B10 or CYP1A1/2, in DBA/2 mice displayed a marked circadian rhythm, the level of expression being highest in the evening. These results suggest that among xenobiotic metabolizing CYP enzymes, CYP2A5 is uniquely upregulated by cAMP, possibly having the physiological function of priming the olfactory and digestive systems for nocturnal feeding.

Expression of xenobiotic-metabolizing enzymes in human pulmonary tissue: possible role in susceptibility for ILD.

The lung is a major target for all inhaled toxicants. Many inhaled chemicals are not hazardous as such, but are biotransformed to reactive intermediates. Therefore, the pathogenesis of interstitial and other lung diseases is intimately linked to exposure to environmental and other chemicals, which may be causative or modifying factors in the cellular pathways and mechanisms mediating oxidative stress and cell protection in the pulmonary tissue. Several different xenobiotic-metabolizing cytochrome P450 (CYP) and phase II enzymes (i.e. conjugation enzymes including several transferases) are present in the human lung and lung-derived cell lines, possibly contributing to in situ activation and inactivation of chemical toxicants. This paper describes the expression and localization of individual CYP-forms in the lung. Interindividual differences in the expression of these enzymes may contribute to the risk of developing interstitial and other lung diseases initiated by agents that require metabolic activation.

The expression of cytochrome P450 enzymes in human breast tumours and normal breast tissue.

Environmental chemicals are one of the risk factors in breast cancer genesis. Cytochrome P450 (CYP) enzymes play a major role in the activation of these chemicals. Using highly specific and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. the expression profile of all major xenobiotic metabolizing CYP forms was screened in breast tumour and surrounding tumour free (control) breast tissue in a series of 20 sample pairs obtained from females with infiltrating ductal carcinoma. The levels of CYPIAI mRNA were very low in both tumour and normal tissue. CYP1B1, CYP2B6, CYP2C, CYP2D6, CYP2E1, CYP4B1, and CYP11A1 expressions were positive in both tumours and control tissue. CYP2A6, CYP2A7, CYP2A13, CYP2F1, CYP3A4, CYP3A5. and CYP3A7 mRNAs were expressed neither in tumours nor in control tissue. These results show that several CYPs. responsible for the activation of a quite large number of procarcinogens and genotoxic estrogen metabolites. are expressed in breast tissue with a lack of qualitative differences in CYP expression at mRNA level between breast tumours and surrounding normal breast.

CYP2A5/CYP2A6 expression in mouse and human hepatocytes treated with various in vivo inducers.

Induction of coumarin 7-hydroxylation, catalyzed by CYP2A5 in mice and CYP2A6 in humans by various known in vivo murine inducers and modifiers, was compared in human and mouse hepatocytes in culture. Phenobarbital and rifampicin were efficient inducers (up to 10-fold induction) after 48-h treatment in murine cultured hepatocytes, whereas the enzyme activity in human hepatocytes was much more refractory to induction. However, a prolongation of incubation time to 72 h in human hepatocytes led to a modest restoration of inducibility by phenobarbital. Of the three porphyrinogenic inducers studied, griseofulvin induced the murine enzyme efficiently, but not the human enzyme, whereas aminotriazole and thioacetamide had no effect on either species. Pyrazole produced substantial induction in both human and murine hepatocytes, whereas cobalt chloride, which is also an in vivo inducer of the mouse enzyme, had no effect. Clofibric acid, an in vivo depressor of coumarin 7-hydroxylase, also depressed hepatocyte activities. In both murine and human hepatocytes, changes in CYP2A5/6 mRNA levels correlated roughly with enzyme changes, except in the case of cobalt chloride, which increased mRNA levels despite a lack of effect on enzyme activity. In general, human and mouse hepatocytes gave a similar response to CYP2A inducers. However, some differences were found, which means that, although CYP2A isozymes are probably regulated in a similar manner in both species, it is necessary to be cautious before extrapolating to human the results found in mouse models.

Expression and inducibility of members in the cytochrome P4501 (CYP1) family in ringed and grey seals from polluted and less polluted waters.

The expression and inducibility of cytochrome P4501A (CYP1A) were investigated in hepatic microsomes of ringed seals (Phoca hispida) and grey seals (Halichoerus grypus) from polluted (Baltic Sea) and less polluted (Svalbard and Sable Island) areas. Liver CYP1A protein levels and activities were assessed by immunoblot analysis and determining catalytic activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD). The amount of CYP1A apoprotein and its catalytic activity were increased up to 5-fold in Baltic ringed and grey seal populations in comparison with ringed seals from Svalbard and grey seals from Sable Island. EROD and PROD activities correlated in all seal groups, indicating catalysis by the same CYP form(s). Enzyme kinetic studies suggested that PROD activity is catalysed by CYP1A enzymes in both ringed and grey seals. In immunoblot analysis, a protein was revealed in liver with an antibody against human CYP1B1, indicating that a CYP1B1 like protein could be present in ringed and grey seals. In conclusion, these data strengthen the concept that CYP1A is markedly induced in seals living in polluted waters and that both EROD and PROD activities are mediated by CYP1A forms in the seal liver. In addition, this study provides the first evidence for the presence of a CYP1B like protein in seal liver.

Expression of CYP1A1, CYP1B1 and CYP3A, and polycyclic aromatic hydrocarbon-DNA adduct formation in bronchoalveolar macrophages of smokers and non-smokers.

Variability in the expression of enzymes metabolizing carcinogens derived from cigarette smoke may contribute to individual susceptibility to pulmonary carcinogenesis. This study was designed to determine the effects of smoking and 3 major cytochrome P450 (CYP) enzymes, i.e., CYP1A1, CYP1B1 and CYP3A, which metabolize polycyclic aromatic hydrocarbons (PAH) on PAH-DNA adduct formation in the bronchoalveolar macrophages (BAM) of 31 smokers and 16 non-smokers. CYP protein levels were determined by immunoblotting and PAH-DNA adduct levels by the nuclease P1 enhanced (32)P-postlabeling method. The expression of specific CYP forms was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) from 10 additional samples. CYP3A protein, CYP3A5 by RT-PCR, was detected in the majority of samples from smokers and non-smokers. The levels of CYP3A appeared to be lower in active smokers than in ex-smokers (p = 0.10) or never smokers (p = 0.02). CYP1A1 was not detectable by either immunoblotting or RT-PCR. The expression of CYP1B1 was low or undetectable in most samples. The PAH-DNA adduct levels were higher (mean 1.57/10(8) nucleotides) in samples from smokers compared with non-smokers (mean 0.42/10(8) nucleotides, p < 0.001) and the number of adducts correlated with the number of cigarettes smoked daily (regression analysis, p < 0. 001). Higher levels of adducts were detected in samples from smokers with a high level of CYP3A compared with those with a low level (regression analysis, p = 0.002). As CYP3A5 is abundant in both lung epithelial cells and BAM, its association with adduct formation suggests that this CYP form may be important in the activation of cigarette smoke procarcinogens.

CYP2A6: a human coumarin 7-hydroxylase.

Coumarin 7-hydroxylation is catalysed by a high-affinity CYP2A6 enzyme in human liver microsomes. CYP2A6 is the only enzyme catalysing this reaction and consequently the formation of 7-hydroxycoumarin can be used as 'an in vitro and in vivo probe' for CYP2A6. CYP2A6 is a major contributor to the oxidative metabolism of nicotine and cotinine, and it also contributes, to a larger or smaller extent, to the metabolism of a few pharmaceuticals (e.g. fadrozole), nitrosamines, other carcinogens (e.g. aflatoxin B1) and a number of coumarin-type alkaloids. CYP2A6 may be inducible by antiepileptic drugs and it is decreased in alcohol-induced severe liver cirrhosis. Several mutated or deleted CYP2A6 alleles have been characterized. Although CYP2A6 represent up to 15% of human microsomes P450 proteins, it is still one of the less well characterised cytochrome P450 enzymes.

Induction and regulation of xenobiotic-metabolizing cytochrome P450s in the human A549 lung adenocarcinoma cell line.

Several cytochrome P450 (CYP) enzymes are expressed in the human lung, where they participate in metabolic inactivation and activation of numerous exogenous and endogenous compounds. In this study, the expression pattern of all known xenobiotic-metabolizing CYP genes was characterized in the human alveolar type II cell-derived A549 adenocarcinoma cell line using qualitative reverse transcriptase/polymerase chain reaction (RT-PCR). In addition, the mechanisms of induction by chemicals of members in the CYP1 and CYP3A subfamilies were assessed by quantitative RT-PCR. The expression of messenger RNAs (mRNAs) of CYPs 1A1, 1B1, 2B6, 2C, 2E1, 3A5, and 3A7 was detected in the A549 cells. The amounts of mRNAs of CYPs 1A2, 2A6, 2A7, 2A13, 2F1, 3A4, and 4B1 were below the limit of detection. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1 and CYP1B1 mRNAs 56-fold and 2.5-fold, respectively. CYP3A5 was induced 8-fold by dexamethasone and 11-fold by phenobarbital. CYP3A4 was not induced by any of the typical CYP3A4 inducers used. The tyrosine kinase inhibitor genistein and the protein kinase C inhibitor staurosporine blocked TCDD-elicited induction of CYP1A1, but they did not affect CYP1B1 induction. Protein phosphatase inhibitors okadaic acid and calyculin A enhanced TCDD-induction of CYP1B1 slightly, but had negligible effects on CYP1A1 induction. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in human pulmonary epithelial cells and give the first indication of the induction of CYP3A5 by glucocorticoids in human lung cells. These results establish that having retained several characteristics of human lung epithelial cell CYP expression, the A549 lung cell line is a valuable model for mechanistic studies on induction of the pulmonary CYP system.

Antepartum glucocorticoid therapy suppresses human placental xenobiotic and steroid metabolizing enzymes.

We investigated the effects of maternal gestational corticosteroid therapy on placental xenobiotic and steroid metabolizing enzymes at term in 20 glucocorticoid/betamethasone treated (with various doses) and control (n=10) women. A single dose of betamethasone (12 mg i.m. twice at a 24-h interval) was given to 15 mothers at risk of preterm delivery to prevent respiratory syndrome in their premature newborns. Five mothers were treated more than once. The gestation time in mothers receiving the glucocorticoid therapy varied from 22-38 gestational weeks. Compared with controls, a significant decrease in placental aromatase activity (53.6+/-18.0 pmol/mg/min versus 119+/-30 pmol/mg/min, P=0.0007) and placental CYP19 mRNA content (by 50 per cent ) was observed in mothers treated with glucocorticoids. Also the formation of androstenedione (13.2+/-8.1 pmol/mg/min, steroids versus 30.03+/-5.2 pmol/mg/min, controls, P< 0.001), using testosterone as the substrate, and 7-ethoxycoumarin O-deethylase (P< 0.05) and 7-ethoxyresorufin O-deethylase (P< 0.09) were slightly decreased in the glucocorticoid treated compared to control patients' values. The changes were not dependent on the number of treatments or the time between treatment and delivery. Our results demonstrate that even a single dose of glucocorticoid given to expectant mothers is associated with diminished placental steroid hormone and xenobiotic metabolizing enzymes at term. Further studies are needed to assess whether these changes affect the well-being of the fetus and its later development.

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity.

OBJECTIVE: To determine the existence of mutant and variant CgammaP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression. METHODS: A bacterial artificial chromosome that contains the complete CgammaP3A4 gene was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation. RESULTS: To investigate the existence of mutant and variant CgammaP3A4 alleles in humans, all 13 exons and the 5'-flanking region of the human CgammaP3A4 gene in three racial groups were sequenced and four alleles were identified. An A-->G point mutation in the 5'-flanking region of the human CgammaP3A4 gene, designated CgammaP3A4*1B, was found in the three different racial groups. The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The CgammaP3A4*1B allele was not found in Chinese subjects. A second variant allele, designated CgammaP3A4*2, having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the CYP3A4*2 P450 had a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6beta-hydroxylation. Another rare allele, designated CgammaP3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450. CONCLUSIONS: These are the first examples of potential function polymorphisms resulting from missense mutations in the CgammaP3A4 gene. The CgammaP3A4*2 allele was found to encode a P450 with substrate-dependent altered kinetics compared with the wild-type P450.

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.