Forms Of Human Cytochrome P450 Research Articles

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways.In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays.Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion.The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Probiotic strain Lactobacillus casei does not markedly alter rat liver microsomal cytochromes P450

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1,1A2, 2A1, 2A2,2B1,2B2,2C6,2C7, 2C11,2C12,2C13,2E1,3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2,2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

Stereoselective inhibition of cytochrome P450 forms by lansoprazole and omeprazole in vitro

The stereoselectivity of the inhibitory interaction potential of lansoprazole and omeprazole isomers on six human cytochrome P450 forms was evaluated using human liver microsomes. Lansoprazole enantiomers showed stereoselective inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, CYP2C19-catalysed S-mephenytoin 4′-hydroxylation, CYP2D6-catalysed dextromethorphan O-demethylation, CYP2E1-catalysed chlorzoxazone 6-hydroxylation and CYP3A4-catalysed midazolam 1-hydroxylation, whereas omeprazole only inhibited CYP2C19 stereoselectively. Of the P450 forms tested, CYP2C19-catalysed S-mephenytoin 4′-hydroxylation was extensively inhibited by both the lansoprazole and omeprazole enantiomers in a competitive and stereoselective manner; the S-enantiomers of both drugs inhibited the hydroxylation more than the R-enantiomers. The estimated Ki values determined for CYP2C19-catalysed S-mephenytoin 4′-hydroxylation were 0.6, 6.1, 3.4 and 5.7 µM for S-lansoprazole, R-lansoprazole, S-omeprazole and R-omeprazole, respectively. The results indicate that although both lansoprazole and omeprazole are strong inhibitors of CYP2C19, the inhibition of CYP2C19 by lansoprazole is highly stereoselective, whereas the inhibition by omeprazole is less stereoselective. In addition, S-lansoprazole, the most potent CYP2C19 inhibitor, is not a good CYP2C19-selective inhibitor owing to its inhibition of other P450 forms.

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B 1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[ a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.

A commentary on the impacts of metals and metalloids in the environment upon the metabolism of drugs and chemicals

The salient feature of metals is that unlike organic compounds they do not degrade in the environment and barely move from one environmental matrix to another. Human interventions take these compounds from their stable and non-bioavailable geological matrix into situations of biological accessibility. Studies in the 1970s and the 1980s of metal bioavailability and impacts of metals and metalloids were driven by the process of abatement of lead in the environment. Humans have clear and identifiable sources of exposure from fuels, food and leaded water pipes to lead. Interventions started at that time have dramatically lowered human lead exposure. Attention has now shifted to other metals, in particular, cadmium, which has seen increasing use. It is generally accepted that food crops grown on cadmium containing soils or soils naturally rich in this metal are the major source of exposure to humans other than exposure from smoking of cigarettes. This mini-review gives a summary and commentary on early studies on effects of lead on haem metabolism that provide us the clue to why investigations of the impacts of other toxic heavy metals and metalloids such as cadmium and arsenic on different human cytochrome P450 forms have become of great interest at the current time.

Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds, purpurin and alizarin

Recently we have shown that anthraquinone food pigments such as purpurin and alizarin suppress the genotoxic activities of several mutagens including heterocyclic amines and polycyclic aromatic hydrocarbons in the Drosophila DNA repair test and in the Ames test. To investigate the mechanism of this inhibition, we have now examined the effects of these anthraquinone pigments on enzymes that metabolize xenobiotics. The activities of eight human recombinant cytochrome P450 (CYP) isozymes were measured in the presence of purpurin, alizarin or carminic acid. Purpurin and alizarin strongly inhibited the activities of CYP1A1, CYP1A2 and CYP1B1, and weakly suppressed those of CYP2A6 and CYP2E1 in a dose-dependent manner, but did not inhibit those of CYP2C19, CYP3A4 and CYP3A5. Carminic acid did not affect the activities of any CYPs tested. CYP1B1 was the most strongly affected CYP molecule by purpurin and alizarin among CYPs examined in this study. From kinetic analysis, it was shown that the inhibition by purpurin on CYP1B1 was both competitive and non-competitive, and that by alizarin was competitive. The values of slopes obtained from Lineweaver–Burk plots are proportional to the square of purpurin concentration. This observation suggests that two molecules of purpurin are interacting with one molecule of CYP1B1. The K m value of CYP1B1 was 11 μM, and the K i value of purpurin and alizarin against CYP1B1 was 0.7 μM 2 and 0.5 μM, respectively. We also examined the effects of these pigments on the mutagenicities of MeIQx and B[ a]P in the Ames test, using Salmonella typhimurium TA1538 co-expressing each form of human CYP and NADPH-cytochrome P450 reductase (OR). The mutagenicity of MeIQx in TA1538 1A2/OR or 1B1/OR was suppressed by purpurin and alizarin but not by carminic acid. Purpurin also reduced the mutagenicity of B[ a]P in TA1538 1A1/OR or 1B1/OR. These results suggest that the antigenotoxic activities of purpurin and alizarin can be explained by inhibition of CYP activities responsible for activating the mutagens.

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by human cytochrome P450-dependent monooxygenase systems.

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by monooxygenase systems dependent on 12 forms of human cytochrome P450 (CYP) was examined with the recombinant yeast microsomes containing each of the human CYP. The metabolites of PCDDs were analyzed by HPLC and GC-MS. Remarkable metabolism by the multiple CYP forms was observed toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, and hydroxylation with elimination of a chloride substituent. Although major CYPs in human liver such as CYP2C8, CYP2C9, and CYP3A4 showed no significant metabolism toward the PCDDs, CYP1A1 and CYP1A2 showed high catalytic activity toward DD and mono-, di-, and trichloroDDs. The kinetic parameters K(m)(app) and V(max) of the CYP1A1-dependent 8-hydroxylation activity of 2,3,7-trichloro-DD (2,3,7-triCDD) were estimated to be 0.30 microM and 51 (mol/min/mol of P450), respectively, suggesting that 2,3,7-triCDD was a good substrate for CYP1A1. However, none of the CYPs showed any detectable activity [<0.01 mol/min/mol of P450)] toward 2,3,7,8-tetraCDD. Substrate-induced absorption spectrum and inhibition studies indicated that CYP1A1 could bind 2,3,7,8-tetraCDD with considerably high affinity. It was strongly suggested that the long half-life (7.1 years) of 2,3,7,8-tetraCDD in humans was due to an extremely low activity of CYPs toward 2,3,7,8-tetraCDD in addition to its chemical stability.

Role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines: application of genetically engineered Salmonella typhimurium YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase

The role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines was examined by Ames test using genetically engineered Salmonella typhimurium ( S. typhimurium)YG7108 cells expressing each form of human CYP together with human NADPH-cytochrome P450 reductase (OR). The relationship between the structure of N-alkylnitrosamines and CYP form(s) involved in the activation was evaluated. Eleven strains of S. typhimurium YG7108 cells expressing each form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) were employed. Eight N-alkylnitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylethylamine (NMEA), N-nitrosomethylpropylamine (NMPA), N-nitrosomethylbutylamine (NMBA) and N-nitrosoethylbutylamine (NEBA) were examined. Minimal concentration (MC) value of a promutagen was defined as the concentration of a chemical giving a positive result. Mutagen-producing capacity of CYP, as indicated by induced revertants/nmol promutagen/pmol CYP, for an N-alkylnitrosamine was determined for all forms of CYP. These N-alkylnitrosamines were mainly activated by CYP2E1, CYP2A6 and CYP1A1. N-alkylnitrosamines with relatively short alkyl chains such as NDMA and NMEA were primarily activated by CYP2E1 as judged by mutagen-producing capacity. With the increase of the number of the carbon atoms of the alkyl chains, the contribution of CYP2A6 increased. CYP2A6 played major roles in the activation of NDEA, NDPA, NMPA, NMBA and NEBA. Interestingly, CYP1A1 became a molecular form of CYP playing a major role in the metabolic activation of NDBA.

Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase.

A series of Salmonella typhimurium (S. typhimurium) YG7108 strains, each coexpressing a form of human cytochrome P450 (CYP) (CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase (OR), was established. The parental S. typhimurium YG7108, derived from TA1535, lacks two O(6)-methylguanine-DNA methyltransferase genes, ada and ogt, and is highly sensitive to the mutagenicity of alkylating agents. The expression levels of CYP holo-protein in the genetically engineered S. typhimurium YG7108 cells, determined by carbon monoxide (CO) difference spectra, ranged from 62 nmol/L culture for CYP2C19 to 169 nmol/L culture for CYP3A4. The expression level of the OR varied, depending on the form of CYP coexpressed, and ranged from 214 to 1029 units/L culture. Each form of CYP expressed in the S. typhimurium YG7108 cells catalyzed the oxidation of a representative substrate at an efficient rate. The rates appeared comparable to the reported activities of CYP expressed in human liver microsomes or CYP in other heterologous systems, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. These S. typhimurium strains may be useful not only for predicting the metabolic activation of promutagens catalyzed by human CYP but also for identifying the CYP form involved.

Human liver cytochrome P450 enzymes involved in the 7-hydroxylation of R- and S-warfarin enantiomers

Human liver microsomes had about 8-fold higher 7-hydroxylation activities for S-warfarin than for R-warfarin. Activities of racemic warfarin 7-hydroxylation by liver microsomes of 35 human samples correlated more closely with those of S-warfarin 7-hydroxylation ( r = 0.95) than with those of R-warfarin 7-hydroxylation ( r = 0.69). The correlation coefficient between R-warfarin 7-hydroxylation and 7-ethoxyresorufin O-deethylation activities was 0.73 in these human samples, suggesting that R- and S-warfarin enantiomers are catalyzed by different forms of human cytochrome P450 (P450 or CYP) enzymes. Anti-CYP2C9 antibodies inhibited completely the 7-hydroxylation of S-warfarin, but not R-warfarin, catalyzed by human liver microsomes, while anti-CYPlA2 inhibited R-warfarin 7-hydroxylation by about 70%. Interestingly, the racemic warfarin 7-hydroxylation activities (turnover numbers of 1.6 ± 1.0 pmol/min/mg protein in 35 human samples) were found to be low compared with the S-warfarin 7-hydroxylation activities (4.1 ± 2.5 pmol/min/mg protein), indicating that R-warfarin may have affected the CYP2C9-dependent S-warfarin 7-hydroxylation activities when racemic warfarin was used as a substrate. Several P450 inhibitors, as well as R-warfarin, were examined for their abilities to inhibit S-warfarin 7-hydroxylation; we found that R-warfarin was a non-competitive inhibitor with a K i value of about 150 μM, whereas both tolbutamide and sulfaphenazole were competitive inhibitors with K i values of about 100 and 0.5 μM, respectively, for S-warfarin 7-hydroxylation activities. These results suggest that R- and S-warfarin enantiomers are catalyzed principally by CYP1A2 and CYP2C9, respectively, in human liver microsomes, and that the pharmacokinetic properties of S-warfarin may be altered by R-warfarin in vivo when racemic warfarin is administered clinically to humans.

Trimethadione metabolism, a useful indicator for assessing hepatic drug-oxidizing capacity

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1,1A2, 2A1, 2A2,2B1,2B2,2C6,2C7, 2C11,2C12,2C13,2E1,3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2,2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

Formation of genotoxicity by chlorine treatment of amino acids

The metabolic activation of procarcinogens and promutagens by human liver microsomal cytochrome P-450 has been investigated by means of a newly developed method measuring the induction of umu gene in Salmonella typhimurium TA1535/pSK1002 [T. Shimada and S. Nakamura, Biochem. Pharmac.36, 1979 (1987)]. The chemicals examined were aflatoxin B1 (AFB1), eight carcinogenic heterocyclic aromatic amines isolated from protein and amino acid pyrolysates, and 2-aminoanthracene. Liver microsomes from six patients catalyzed the metabolic activation of these chemicals; 2-amino-3,5-dimethylimidazo[4,5-|] quinoline (MeIQ) and AFB1 were most actively bioactivated, followed by 2-amino-3-methylimidazo [4,5-|]quinoline (IQ), 2-aminoanthracene (2-AA) and 2-amino-3,8-dimethyl-imidazo [4,5-|]quinoxaline. At least two forms of human cytochrome P-450 may be involved in the activation of these procarcinogens. This suggestion was supported by the following lines of evidence: (a) addition of non-ionic detergent Emulgen 913 to the incubation mixture caused a more profound inhibition of microsome-catalyzed activation of AFB1 than of MeIQ, IQ and 2AA, (b) 7,8-benzoflavone stimulated the activation of AFB1 by about 2.5-fold, whereas it inhibited significantly the reactions with MeIQ, IQ and 2AA, and (c) polyclonal antibodies against a 3-methylcholanthrene-inducible form of rat cytochrome P-450 (P-450d) caused a marked inhibition of the metabolic activation of MeIQ, IQ and 2-AA by human liver microsomes though they did not show any effects on the microsomal activation of AFB1. Data are also presented showing that none of the reactions catalyzed by human liver microsomes were inhibited by antibodies to a phenobarbital-inducible form of rat cytochrome P-450 (P-450b). These results suggest that the human cytochrome P-450 isozyme that is immunochemically similar and, thus, homologous to rat P-450d plays a major role in the metabolic activation of several procarcinogens examined, and that the activation of AFB1 is catalyzed by another and, possibly, not phenobarbital-inducible form(s) of human cytochrome P-450.

Immunochemical characterization and toxcological significance of P-450HFLb purified from human fetal livers

Immunochemical properties of P-450HFLb purified from human fetal livers were invested. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB 1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).

Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150–900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30–50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6β-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6β-hydroxylation as their major activity but, additionally, formed several minor products that include 2β-hydroxy and 15β-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.

Formation of cytotoxic metabolites from phenytoin, imipramine, desipramine, amitriptyline and mianserin by mouse and human hepatic microsomes

The effects of enzyme induction on the generation of cytotoxic metabolites from phenytoin, mianserin, imipramine, desipramine and amitriptyline by mouse liver microsomes has been investigated and then compared with the bioactivation mediated by human hepatic microsomes. Cytotoxicity was assessed by co-incubation of drug and microsomes with human mononuclear leucocytes which served as target cells. Enzyme induction was assessed by measurement of hepatic cytochrome P-450 content, and determination of alkoxycoumarin O-dealkylase activity. None of the compounds investigated were metabolized to cytotoxic metabolites in the presence of control mouse microsomes. However, significant bioactivation could be observed for each drug when incubated with microsomes prepared from mice pretreated with either phenobarbitone (60 mg/kg) or β-naphthoflavone (75 mg/kg). The rank order for metabolism-dependent cytotoxircity with phenobarbitone-induced mouse microsomes (expressed as % cell death) was phenytoin (14.6%) > desipramine (10.5%) > imipramine (7.5%) > mianserin (3.4%) > amitriptyline (3.1%). Expression of cytotoxidty with phenytoin required pre-exposure of the target cells to trichloropropane oxide, an epoxide hydrolase inhibitor. Only mianserin and desipramine were activated to cytotoxic metabolites by human liver microsomes. Analysis of stable metabolites revealed that mianserin underwent extensive (>80%) metabolism by both control and induced mouse microsomes and that the principal metabolites, 8-hydroxymianserin, desmethylmianserin and mianserin N-oxide, were the same as those produced by human liver microsomes. These data suggest that mianserin is activated to a cytotoxic metabolite selectively by a constitutive form of human cytochrome P-450, whereas phenytoin, amitriptyline and imipramine are selectively activated by forms of mouse cytochrome P-450 which are induced by either phenobarbitone or β-naphthoflavone.

Estradiol Metabolism by Complementary Deoxyribonucleic Acid-Expressed Human Cytochrome P450s

Twelve forms of human cytochrome P450 were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. The cDNA-expressed enzymes were tested for their ability to oxidize estradiol. Incubation of [14C]estradiol with cell lysates containing P450 IA2 resulted in the production of 2-hydroxy and 4-hydroxy metabolites with substrate turnovers of 2.74 and 0.27 min-1, respectively. P450s IIIA3 and IIIA4 yielded the same metabolites at about one third the rate of P450 IA2. Low levels of estradiol hydroxylation were also catalyzed by P450s IIC9, IIIA5, and IVB1. Six other P450 forms yielded no detectable metabolism. The roles of P450s IA2, IIA3, and IIIA4 were further established by immunoinhibition using antirat P450 antibodies. Antibody that specifically binds to P450 IIIA3 and IIIA4 inhibited 60-70% of estradiol hydroxylation, and antibody against P450 IA2 inhibited 20-40% of the estradiol hydroxylase activity in microsomes from two human liver specimens, suggesting that these enzymes constitute the major forms catalyzing estradiol oxidation in human liver. Immunoinhibition results also suggest that 2-hydroxy- and/or 4-hydroxycatechol estrogens are further metabolized to other yet uncharacterized metabolites by P450s IIIA3 and IIIA4.

The metabolism of 1-nitropyrene by human cytochromes P450

The metabolism of [3H]1-nitropyrene by specific forms of human cytochrome P450 was investigated in vitro using Vaccinia virus expression of P450 cDNAs in HepG2 cells. Cell lysates were injected individually with recombinant Vaccinia virus containing human P450 cDNA (P450 IA2, IIA3, IIB7, IIC8, IIC9, IID6, IIE1, IIF1, IIIA3, IIIA4, IIIA5 AND IVB1). Only IIIA3 and IIIA4 demonstrated significant activity in the C-oxidation of 1-nitropyrene. The principal metabolite from both P450 forms was 1-nitropyren-3-ol, produced in at least 4-fold greater abundance than the mixture of 1-nitropyren-6-ol and 1-nitropyren-8-ol, or the K-region dihydrodiols. This is in contrast to the metabolism in many species where 6-ol and 8-ol formation predominate over 3-ol formation. In fact, in rats and rabbits, P450 forms quite distinct from the IIIA P450s catalyze the majority of the metabolism of this pollutant. This is the first demonstration of the role of individual human P450 forms in the metabolism of a representative chemical in this important class of environmental pollutants. The importance of these observations in the overall carcinogenic risk of humans to these chemicals remains to be established. These studies furthermore establish a marked species difference in the metabolism of nitrated polycyclic aromatic hydrocarbons.

Characterization of a cDNA encoding a new member of the glucocorticoid-responsive cytochromes P450 in human liver

Adult human liver contains a form of cytochrome P450, termed HLp, that resembles the glucocorticoid-inducible cytochrome P450p in rat liver in its structure, function, and regulation and catalyzes the oxidation of such clinically important substrates as cyclosporin, nifedipine, erythromycin, and midazolam. Recent evidence, however, suggests that HLp may represent two or more closely related forms of cytochromes P450, one of which is termed P450nf. To search for additional members of the Class III human subfamily of HLp related genes, we screened a human liver cDNA library cloned in phage vector λgt11 with oligonucleotides and with a cDNA fragment related to HLp. We isolated a full-length cDNA (1709 nucleotides) encoding a new form of human cytochrome P450 termed HLp2. Analysis of HLp2 cDNA predicted a protein of 502 amino acids, weighing 57,294 Da 83% similar to HLp. HLp2 appears to represent a distinct gene as judged by partial sequence analysis of a cloned human gene and by hybridizations of Southern blots, under conditions of varying stringency, with a 3′-portion of HLp cDNA and with an oligonucleotide specific for HLp2. Northern blot analysis revealed that HLp/ P450nf was present in all samples of liver mRNA from adult patients not treated with inducers of HLp, whereas HLp2 mRNA was undetectable in more than two-thirds. Human fetal liver RNA contained mRNA species 2.1 and 1.9 kb which hybridized with an HLp2 oligonucleotide. We conclude that HLp2 represents a third member of the Class III glucocorticoid-responsive gene family that is expressed in both fetal and adult human liver and may account for polymorphism in metabolism of clinically important drugs.

Purification of cytochrome P-450 from polychlorinated biphenyl-treated crab-eating monkeys: high homology to a form of human cytochrome P-450

Cytochrome P-450, designated as P-450-MK2, was purified to an electrophoretic homogeneity from polychlorinated biphenyl (PCB)-treated female crab-eating monkeys. P-450-MK2 catalyzed nifedipine and nilvadipine oxidations, at a rate comparable to human P-450-HM1. The N-terminal amino acid sequence of P-450-MK2 was highly homologous to those of P-450-HM1 and NF 25. The antibodies to P-450-HM1 recognized P-450-MK2 and effectively inhibited the activity of testosterone 6 beta-hydroxylase in monkey liver microsomes. These results suggest that a form of cytochrome P-450 corresponding to human P-450-HM1 or P-450NF which belongs to the P450 III gene family is also present in liver microsomes of crab-eating monkeys.

Metabolic activation of environmental carcinogens and mutagens by human liver microsomes: Role of cytochrome P-450 homologous to a 3-methylcholanthrene-inducible isozyme in rat liver

The metabolic activation of procarcinogens and promutagens by human liver microsomal cytochrome P-450 has been investigated by means of a newly developed method measuring the induction of umu gene in Salmonella typhimurium TA1535/pSK1002 [T. Shimada and S. Nakamura, Biochem. Pharmac. 36, 1979 (1987)]. The chemicals examined were aflatoxin B 1 (AFB 1), eight carcinogenic heterocyclic aromatic amines isolated from protein and amino acid pyrolysates, and 2-aminoanthracene. Liver microsomes from six patients catalyzed the metabolic activation of these chemicals; 2-amino-3,5-dimethylimidazo[4,5-|] quinoline (MeIQ) and AFB 1 were most actively bioactivated, followed by 2-amino-3-methylimidazo [4,5-|]quinoline (IQ), 2-aminoanthracene (2-AA) and 2-amino-3,8-dimethyl-imidazo [4,5-|]quinoxaline. At least two forms of human cytochrome P-450 may be involved in the activation of these procarcinogens. This suggestion was supported by the following lines of evidence: (a) addition of non-ionic detergent Emulgen 913 to the incubation mixture caused a more profound inhibition of microsome-catalyzed activation of AFB 1 than of MeIQ, IQ and 2AA, (b) 7,8-benzoflavone stimulated the activation of AFB 1 by about 2.5-fold, whereas it inhibited significantly the reactions with MeIQ, IQ and 2AA, and (c) polyclonal antibodies against a 3-methylcholanthrene-inducible form of rat cytochrome P-450 (P-450d) caused a marked inhibition of the metabolic activation of MeIQ, IQ and 2-AA by human liver microsomes though they did not show any effects on the microsomal activation of AFB 1. Data are also presented showing that none of the reactions catalyzed by human liver microsomes were inhibited by antibodies to a phenobarbital-inducible form of rat cytochrome P-450 (P-450b). These results suggest that the human cytochrome P-450 isozyme that is immunochemically similar and, thus, homologous to rat P-450d plays a major role in the metabolic activation of several procarcinogens examined, and that the activation of AFB 1 is catalyzed by another and, possibly, not phenobarbital-inducible form(s) of human cytochrome P-450.