Coumarin 7-hydroxylation Activity Research Articles

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes

The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes.CYP2A6 was highly active in oxidizing flavanone to form flavone, 2′-hydroxy-, 4′-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5.Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2′-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities.Molecular docking analysis showed that the C2′-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2′ and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively.These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities.

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

Inhibitory Potency of 8-Methoxypsoralen on Cytochrome P450 2A6 (CYP2A6) Allelic Variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22: Differential Susceptibility Due to Different Sequence Locations of the Mutations

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6*16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6*16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.

Corrigendum

British Journal of PharmacologyVolume 165, Issue 8 p. 2808-2808 Free Access Corrigendum This article corrects the following: Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo Yune-Fang Ueng, Chien-Chih Chen, Yu-Ting Chung, Tsung-Yun Liu, Yu-Ping Chang, Wei-Sheng Lo, Norie Murayama, Hiroshi Yamazaki, Pavel Souček, Gar-Yang Chau, Chin-Wen Chi, Ruei-Ming Chen, Ding-Tzai Li, Volume 163Issue 6British Journal of Pharmacology pages: 1250-1262 First Published online: June 27, 2011 First published: 23 March 2012 https://doi.org/10.1111/j.1476-5381.2012.01915.xCitations: 2AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Ueng et al. (2011) there were errors in the y-axes of Figure 1D–F. The corrected panels appear below. Figure 1 As a result of these errors the values of the inactivation rates (Kapp) at different chalepensin concentrations, kinact (maximal inactivation rate constant of chalepensin) and KI (chalepensin concentration required for half-maximal inactivation) were incorrect. Further errors appeared on pages 1250, 1254 and 1256 and are corrected by the following: In the Key Results section of the abstract on page 1250 the sentence beginning ‘This time-dependent inactivation . . .’ should read ‘This time-dependent inactivation (kinact 0.298 min−1; KI 4.8 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation.’ In the Data and kinetic analyses section of the Methods on page 1254 ‘Lineweaver-Burke plots’ should be ‘Lineweaver-Burk plots’. In the same section the sentence beginning ‘The kinact . . .’ should read ‘The kinact (maximal inactivation rate constant of chalepensin) and KI (chalepensin concentration required for half-maximal inactivation) values were estimated from nonlinear regression from the equation Kapp= (kinactI)/(I+KI) with the initial values calculated from the double reciprocal plot of inactivation rate versus chalepensin concentration (Silverman, 1995).’ In the The metabolism-dependence and kinetic analysis of the CYP2A6 inhibition by chalepensin section of the Results on page 1254 the paragraph beginning ‘Pre-incubation with NADPH and chalepensin . . .’ should read ‘Pre-incubation with NADPH and chalepensin induced a time-dependent inactivation of coumarin 7-hydroxylation activity with kinact of 0.298 ± 0.044 min−1 and apparent KI of 4.8 ± 1.8 µM (Figures 1D,E). Linear regression analysis of the double reciprocal plots for inactivation rate and chalepensin concentration generated the kinact and apparent KI values for CYP2A6 of 0.233 min−1 and 2.6 µM respectively (Figure 1F).’ In the Discussion and conclusions on page 1256 the sentence beginning ‘In the presence of NADPH . . .’ should read ‘In the presence of NADPH, the efficiency of enzyme inactivation by chalepensin, calculated as the ratio of kinact to KI was 62.1 min−1 mM−1.’ The errors in the figure and text do not change the conclusion of the study. Reference Ueng Y-F, Chen C-C, Chung Y-T, Liu T-Y, Chang Y-P, Lo, W-S et al. (2011). Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo. Br J Pharmacol 163: 1250– 1262. Citing Literature Volume165, Issue8Special Issue: Themed Section: Cannabinoids in Biology and Medicine, Part II. Guest Editors: Itai Bab and Steve AlexanderApril 2012Pages 2808-2808 ReferencesRelatedInformation

Spectral Modification and Catalytic Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2A6, and 2A13 by Four Chemopreventive Organoselenium Compounds

Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (K(s)) for the interaction of P450 1B1 with these chemicals were 3.6-5.7 μM; the values were lower than those with seen with P450 1A1 (19-30 μM) or 1A2 (6.3-13 μM). The K(s) values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 μM; the values were very low compared to those for the interaction of P450 2A6 with m-XSC (5.7 μM) and BSC (2.0 μM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC(50) values <1.0 μM, except for the inhibition of P450 1A2 by BSC (1.3 μM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC(50) values of 0.22-1.4 μM for P450 2A13 and 2.4-6.2 μM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes.

Mechanism‐based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC(50) value decreased by 98%). This time-dependent inactivation (k(inact) 0.044 min(-1) ; K(I) 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. 'Poor metabolizers' carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice.

Cytochrome P450-depedent Drug Oxidation Activity of Liver Microsomes from Microminipigs, A Possible New Animal Model for Humans in Non-clinical Studies

Small minipigs (Bland name, Micromini Pig; registered as a novel variety of pig in the Japanese Ministry of Agriculture, Forestry and Fisheries) were developed with the aim of non-clinical pharmacological/toxicological use. They were principally mated with<10 kg body weight at 7 months-old resulting in good handling. Cytochrome P450 (P450)-and flavin-containing monooxygenases (FMO)-dependent drug oxidation activity of liver microsomes prepared from male Microminipigs (8 months-old) was compared with that for pooled dogs, monkeys, and humans. High P450 2D-dependent bufuralol 1'-hydroxylation and FMO-dependent benzydamine N-oxygenation activity was observed in liver microsomes from Microminipigs. Typical P450 1A, 2B, 2C, 2E, and 3A-dependent drug oxidation activity was also seen in Microminipigs. However, occasional differences might give undetected low P450 2A-dependent coumarin 7-hydroxylation in Microminipigs at 8-months-old, in contrast to liver microsomes from one 10-days-old Microminipis and commercially available pooled minipigs which had low but detectable coumarin 7-hydroxylation activity. The present results suggest that there is some overlap in Microminipig and human P450 substrate specificity. These findings should provide important information for greater understanding of drug metabolism in Microminipigs, as an experimental animal model for non-clinical use.

Involvement of CYP2A6 in the Formation of a Novel Metabolite, 3-Hydroxypilocarpine, from Pilocarpine in Human Liver Microsomes

Pilocarpine is a cholinergic agonist that is metabolized to pilocarpic acid by serum esterase. In this study, we discovered a novel metabolite in human urine after the oral administration of pilocarpine hydrochloride, and we investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by liquid chromatography-tandem mass spectrometry and NMR analyses and by comparing to the authentic metabolite. To clarify the human cytochrome P450 (P450) responsible for the metabolite formation, in vitro experiments using P450 isoform-selective inhibitors, cDNA-expressed human P450s (Supersomes; CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4), and liver microsomes from different donors were conducted. The formation of 3-hydroxypilocarpine in human liver microsomes was strongly inhibited (>90%) by 200 microM coumarin. Other selective inhibitors of CYP1A2 (furafylline and alpha-naphthoflavone), CYP2C9 (sulfaphenazole), CYP2C19 [(S)-mephenytoin], CYP2E1 (4-methylpyrazole), CYP2D6 (quinidine), and CYP3A4 (troleandomycin) had a weak inhibitory effect (<20%) on the formation. The highest formation activity was expressed by recombinant CYP2A6. The K(m) value for recombinant CYP2A6 was 3.1 microM, and this value is comparable with that of human liver microsomes (1.5 microM). The pilocarpine 3-hydroxylation activity was correlated with coumarin 7-hydroxylation activity in 16 human liver microsomes (r = 0.98). These data indicated that CYP2A6 is the main enzyme responsible for the 3-hydroxylation of pilocarpine. In conclusion, we identified a novel metabolite of pilocarpine, 3-hydroxypilocarpine, and we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.

Characterization of Humanized Liver from Chimeric Mice Using Coumarin as a Human CYP2A6 and Mouse CYP2A5 Probe

Coumarin 7-hydroxylation (COH), which is catalyzed almost solely by human CYP2A6 and mouse CYP2A5, shows large differences in activity (humans>>mice) and inhibitor specificity between mice and humans. To differentiate human and mouse liver functions of chimeric mice (CM1, CM2 and CM3) prepared with hepatocytes from 3 donors, the microsomal COH activities were measured with and without benzaldehyde and undecanoic gamma-lactone as a specific inhibitor of human CYP2A6 and mice CYP2A5, respectively. The replacement % to human hepatocytes designated as replacement index (RI) was calculated from human specific cytokeratin 8/18 expression in the liver section. The COH activities correlated well with RIs in CM2 (R(2)=0.98) and CM3 (R(2)=0.94), except CM1 whose genotype of donor is CYP2A6*4/*4. However, the COH activities expressed as % of donor activities were not always coincident with RIs, and the inhibition pattern of CM2 and CM3 was human-type after RI exceeded approximately 50%. Subsequently, our attempts to use % of COH activities or inhibition patterns as an accurate functional replacement index were unsuccessful. Since the detection of human CYP2A6 protein in the liver and the steep increase of human albumin (hAlb) levels in the blood were begun from almost RI=50% similarly to the changes of inhibition pattern, RI=50% is the turning point for chimeric mice to have humanized liver function.

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.

Regulation of theCyp2a5Gene Involves an Aryl Hydrocarbon Receptor-Dependent Pathway

We have investigated the role of the aryl hydrocarbon receptor (AHR) in the regulation of the Cyp2a5 gene. The C57BL/6 and DBA/2 mouse strains with a genetically determined difference in AHR function were used to study the CYP2A5 induction by typical AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. The CYP2A5 mRNA up-regulation in these mouse strains showed a difference in response, typical for AHR-regulated genes, both by TCDD in cultured primary hepatocytes and by 3-methylcholanthrene in vivo. In primary hepatocytes, TCDD caused a 3-fold elevation of the CYP2A5 protein level and a similar induction of the CYP2A5-catalyzed coumarin 7-hydroxylation activity. In reporter gene assays, the Cyp2a5 promoter region -3033 to +10 mediated a 2- to 5-fold induction of luciferase activity by TCDD treatment in primary hepatocytes and in Hepa-1 hepatoma cells with an intact AHR/AHR nuclear translocator (ARNT) complex. In Hepa-1 variant cell lines with deficiencies in the AHR/ARNT complex, the absence of ARNT abolished the induction. A putative AHR response element (XRE) was identified in the Cyp2a5 promoter at the position -2514 to -2492 and found to interact with the AHR/ARNT heterodimer. Transfection experiments combined with mutation of the XRE site indicated that the site partly mediates the TCDD induction of Cyp2a5. An additional AHR-dependent mechanism also regulates the proximal promoter of the Cyp2a5 gene. In conclusion, our studies showed that AHR ligands up-regulate Cyp2a5 transcriptionally by an AHR/ARNT-dependent mechanism and established Cyp2a5 as a novel AHR-regulated gene.

P.6.032 Pattern of drug use among opioid addicts with comorbid attention deficit hyperactivity disorder and conduct disorder

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, α-naphthoflavone. Further studies are needed to determine the presence and characteristics of CYP2D and CYP2E enzymes in ringed and grey seals. Testosterone 6β-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 6β-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.

Identification of Val 117 and Arg 372 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 ( V max/ K m) 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.

Activities of Cytochrome P450 Enzymes in Liver and Kidney Microsomes from Systemic Carnitine Deficiency Mice with a Gene Mutation of Carnitine/Organic Cation Transporter

Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2 degrees strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6beta-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.

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, α-naphthoflavone. Further studies are needed to determine the presence and characteristics of CYP2D and CYP2E enzymes in ringed and grey seals. Testosterone 6β-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 6β-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.

Metabolism of 3-methylindole by porcine liver microsomes: responsible cytochrome P450 enzymes.

The role of different cytochrome P450 enzymes on the metabolism of 3-methylindole (3MI) was investigated using selective chemical inhibitors. Eight chemical inhibitors of P450 enzymes were screened for their inhibitory specificity towards 3MI metabolism in porcine microsomes: alpha-naphthoflavone (CYP1A1/2), 8-methoxypsoralen (CYP2A6), menthofuran (CYP2A6), diethyldithiocarbamate (CYP2A6), 4-methylpyrazole (CYP2E1), sulphaphenazole (CYP2C9), quinidine (CYP2D6), and troleandomycin (CYP3A4). The production of 3MI metabolites was only affected by the presence of inhibitors of CYP2A6 and CYP2E1 in the microsomal incubations. In a second experiment, a set of porcine microsomes (n = 30) was analyzed for CYP2A6 content by protein immunoblot analysis and for their coumarin 7-hydroxylation activity (CYP2A6 activity). Both CYP2A6 content and enzymatic activity were found to be highly and negatively correlated with 3MI fat content. The results of the present study indicate that the CYP2A6 porcine ortholog plays an important role in the metabolism of 3MI and that measurement of CYP2A6 levels and/or activity could be a useful marker for 3MI-induced boar taint.

CYP2A6 genetic polymorphisms and liver microsomal coumarin and nicotine oxidation activities in Japanese and Caucasians.

Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.

Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes: the role of the HBx protein

Hepatitis B virus (HBV) and aflatoxins are major risk factors for hepatocellular carcinoma (HCC) exhibiting a synergistic interaction in the development of this disease. The molecular mechanisms of this interaction remain to be elucidated but an altered carcinogen metabolism in the presence of hepatitis-induced liver injury is one hypothesis. The availability of biomarkers of aflatoxin exposure and metabolism permits this hypothesis to be examined in human populations whilst animal models, such as HBV transgenic mice permit parallel studies in an experimental setting. The hepatitis B virus X protein (HBx) is suspected to play a role in the hepatocarcinogenic process by virtue of its capacity to transactivate oncogenes and several other cellular genes via cis-acting elements. In previous studies in HBV transgenic mice expressing the HB surface antigen and X genes we observed a marked induction of specific cytochrome P450s (CYP) (Kirby et al., 1994a). In the current study we investigated the status of CYP, glutathione S-transferases (GST) and antioxidant enzymes in mice carrying only the X gene under the control of the α-1 antitrypsin regulatory elements (ATX mice). Livers of ATX mice showed no major pathological alterations compared to age-matched non-transgenic control mice. Immunohistochemical staining for CYP1A, 2A5 and GST expression and determination of related enzymatic activities (7-ethoxyresorufin O-deethylation, 7-methoxyresorufin O-deethylation, coumarin 7-hydroxylation and GST activities) revealed no differences between control and ATX mice. In addition, no differences in antioxidant enzymes were observed. Overall, these results support the conclusion that HBx expression alone is insufficient to induce transactivation of CYP and GST genes or to alter the antioxidant system and that the induction in other HBV models is a result of inflammatory injury in the liver, a feature absent in ATX mice. These data are compared to biomarker studies of enzyme activities in aflatoxin-exposed human populations with and without HBV infection.

Spectrofluorometric analysis of CYP2A6-catalyzed coumarin 7-hydroxylation.

A spectrofluorometric method is described for the determination of CYP2A6-catalyzed coumarin 7-hydroxylation. Following acidification of the reaction mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and assayed using an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This assay is applicable to enzymatic studies of cDNA-expressed CYP2A6 and can be used to monitor coumarin 7-hydroxylation activity in microsomes prepared from liver and other tissues and in isolated hepatocytes and cultured cells that express this cytochrome P450 activity.

Ethnic-related differences in coumarin 7-hydroxylation activities catalyzed by cytochrome P4502A6 in liver microsomes of Japanese and Caucasian populations

1. Interethnic differences in cytochrome P4502A6 (CYP2A6) levels and coumarin 7-hydroxylation activities were determined in liver microsomes of 30 Japanese and 30 Caucasians. 2. Although CYP2A6 levels and coumarin 7-hydroxylation activities varied very significantly in the 60 human samples examined, both CYP2A6 levels and coumarin 7hydroxylation activities were found to be higher in Caucasian than Japanese population. 3. Interestingly, eight of the 30 Japanese examined showed very low or undetectable levels of coumarin 7-hydroxylation activities as well as of CYP2A6 in liver microsomes. All of the Caucasians, however, had significant CYP2A6 levels and variable 7-hydroxylation activities. 4. Kinetic analvsis of coumarin 7-hydroxylation activities in liver microsomes of various human samples suggested that although there were 260-fold differences in Vmax's in 10 human samples examined, the Km's were very similar (2.1 + or - 107 mu M); a value consistent with that obtained (1.2 mu M) with purified CYP2A6 in reconstituted system. 5. The results suggest that CYP2A6 is actually involved in the 7-hydroxylation of coumarin in human liver microsomes, and that interethnic differences in coumarin 7-hydroxylation activities in Japanese and Caucasian population may be ascribed to the differences in expression of CYP2A6 protein.