Human Liver Microsomal Cytochrome P450 Research Articles

Human liver microsomal cytochrome P450 and B5 act as reductases in the reductive metabolism of 3′-azido-3′-deoxythymidine to 3′-amino-3′deoxythymidine.

Human liver microsomal cytochrome P450 and B5 act as reductases in the reductive metabolism of 3′-azido-3′-deoxythymidine to 3′-amino-3′deoxythymidine.

21] Measurement of human liver microsomal cytochrome P450 2D6 activity using [O-methyl-14C]dextromethorphan as substrate

Publisher Summary This chapter describes a simple, rapid, and sensitive assay procedure for measuring CYP2D6-dependent monooxygenase activity in human liver microsomes employing [O-methyl- 14 C]dextromethorphan as substrate. The assay involves the radiometric measurement of [ 14 C]formaldehyde after a single-step extraction procedure and does not require the use of high-performance liquid chromatography (HPLC), an internal standard, or metabolite standards. The method circumvents problems with interfering compounds and may prove useful for the bulk screening of potential CYP2D6 cosubstrates and/or inhibitors. The CYP2D6 gene is inherited as an autosomal recessive trait and separates the population into extensive metabolizer and poor metabolizer (PM) phenotypes. PM subjects lack functional CYP2D6 protein and also have impaired metabolism of more than 30 drugs. In addition, if two or more drugs are metabolized by CYP2D6 in extensive metabolizer (EM) subjects, then the likelihood of a potentially hazardous drug-drug interaction is increased. The chapter discusses the human liver microsomal CYP2D6, but the method may also prove useful for measuring CYP2D activity in nonhuman species such as rat and monkey.

Effects of erythromycin and roxithromycin on oxidation of testosterone and nifedipine catalyzed by CYP3A4 in human liver microsomes.

Roxithromycin and erythromycin were incubated with rat and human liver microsomal or reconstituted cytochrome P450 (P450 or CYP) monooxygenase systems in the presence of an NADPH-generating system, and the effects of these chemicals on testosterone 6 beta-hydroxylation and nifedipine oxidation activities were compared with those of typical CYP3A4 inhibitors including ketoconazole, troleandomycin, and gestodene. Roxithromycin and erythromycin were found to be relatively weak inhibitors of testosterone 6 beta-hydroxylation and nifedipine oxidation activities by rat and human liver microsomes or by reconstituted systems containing CYP3A4/5. Formation of an inhibitory P450-metabolite complex was determined spectrally by incubating troleandomycin with human liver microsomes; the extents of the complex formation were lesser in liver microsomes of humans than those of rats treated with dexamethasone. Erythromycin and roxithromycin were also activated slightly by rat liver microsomes to form P450.Fe(II)-metabolite complex, although these chemicals caused very little or undetectable levels, respectively, of spectral changes by human liver microsomes even when a human sample which contained relatively high levels of CYP3A4 was used. These results suggested that roxithromycin and erythromycin were relatively less potent to inhibit CYP3A4-catalytic activities in human liver microsomes, because of their low capabilities to form P450.Fe(II)-metabolite complex.

Suicidal inactivation of haemoproteins by reductive metabolites of halomethanes: a structure-activity relationship study

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at μM concentrations with 1 mM carbon tetrachloride (CCl 4), trichlorobromomethane (CCl 3Br), chloroform (CHCl 3) or methylene chloride (CH 2Cl 2) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemoprotein systems, for CCl 3Br, CCl 4 and CHCl 3, but not CH 2Cl 2. For Hb, the loss was greater with CCl 3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl 4 (haem assay, 31%) or CHCl 3 (haem assay, 13%). On the other hand, with MHA, CCl 4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl 3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl 3. Finally, with microsomes, the inactivation was larger with CCl 4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl 3Br (CO binding, 33%; haem assay, 10%) or CHCl 3 (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent haemderived products during incubation of CCl 3Br with Hb or microsomes, and of CCl 3 with Hb. A correlation between potential for free radical formation ( CCl 3 Br > CCl 4 > CHCl 3 > CH 2 Cl 2) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.

Activation of trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene to genotoxic metabolites by rat and human cytochromes P450.

In order to address the hypothesis that 6-aminochrysene (6-AC) is converted to genotoxic products by cytochrome P450 enzymes via two activation pathways (N-hydroxylation and epoxidation), the activation of 6-AC and trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene (6-AC-diol) to genotoxic metabolites was examined in rat and human liver microsomal cytochrome P450 enzymes using Salmonella typhimurium TA1535/pSK1002 and TA1535/pSK1002/pNM12 (NM2009) as tester strains. The latter bacteria, an O-acetyltransferase-overexpressing strain, was highly sensitive to metabolites derived from activation of 6-AC, but not those from 6-AC-diol, using liver microsomes from phenobarbital-treated rats or a reconstituted monooxygenase system containing P4502B1 or -2B2, thus suggesting the roles of P450 and acetyltransferase systems in the activation process. 6-AC-diol, on the other hand, was activated very efficiently by liver microsomes prepared from beta-naphthoflavone-treated rats or a reconstituted system containing P4501A1 or -1A2; the activation reaction is considered to proceed through diol-epoxide formation. The contribution of rat P4501A enzymes towards activation of 6-AC-diol was confirmed by the inhibitory effects on the activation process of alpha-naphthoflavone, a specific inhibitor of P4501A-related activities, and antibodies raised against purified P4501A1 and -1A2. In humans, P4501A2 was found to be the major enzyme involved in the activation of 6-AC-diol to genotoxic metabolites while the parent compound 6-AC was activated mainly by P4503A4. Experiments using recombinant P450 proteins expressed in human lymphoblastoid cell lines showed that human P4501A1 could also activate 6-AC-diol to reactive metabolites at almost the same rate measured with P4501A2. In addition, P4502B6 was found to efficiently catalyze the activation of 6-AC to genotoxic metabolites, and P4503A4 was active in the activation of 6-AC-diol as well as 6-AC. Addition of purified rat epoxide hydrolase to the incubation mixture containing purified rat P4501A1 or microsomes expressing human P4501A1 caused inhibition of activation of 6-AC-diol. These results suggest the existence of different enzymatic activation pathways for 6-AC and 6-AC-diol. The former carcinogen may be N-hydroxylated principally by P4502B enzymes in rats and P4503A4 and -2B6 in humans and activation to its ultimate metabolites may proceed through esterification of the N-hydroxy metabolites by an N-acetyltransferase. The 6-AC-diol is metabolized to its ultimate diolepoxide product by P4501A enzymes in rat and human liver microsomes. P4503A4 (humans) and P4503A2 (rats) may also contribute to some extent in the activation of 6-AC-diol, albeit at lower rates than those of P4501A enzymes.

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.

Single-dose disulfiram inhibition of chlorzoxazone metabolism: a clinical probe for P450 2E1.

Disulfiram and its reduced metabolite diethyldithiocarbamate have been identified previously as selective mechanism-based inhibitors of human liver microsomal cytochrome P450 2E1 in vitro. In animals, a single oral dose of disulfiram has been shown to produce a rapid and selective inactivation of hepatic P450 2E1 content and catalytic activity in vivo. This investigation explored the efficacy of single dose disulfiram as an inhibitor of human P450 2E1 activity in vivo. Clinical P450 2E1 activity was assessed by the 6-hydroxylation of chlorzoxazone, a metabolic pathway catalyzed selectively by P450 2E1. Six healthy volunteers received 750 mg oral chlorzoxazone on two occasions in a crossover design, 10 hours after 500 mg oral disulfiram, or after no pretreatment (control subjects). Disulfiram pretreatment markedly decreased chlorzoxazone elimination clearance to 15% of control values (from 3.28 +/- 1.40 to 0.49 +/- 0.07 ml/kg/min, p < 0.005), prolonged the elimination half-life (from 0.92 +/- 0.32 to 5.1 +/- 0.9 hours, p < 0.001), and caused a twofold increase in peak plasma chlorzoxazone concentrations (20.6 +/- 9.9 versus 38.7 +/- 10.3 micrograms/ml, p < 0.001). Disulfiram also profoundly decreased the formation clearance of 6-hydroxychlorzoxazone, from 2.30 +/- 0.93 to 0.17 +/- 0.05 ml/kg/min (p < 0.005). These findings show that a single dose of disulfiram significantly diminishes the activity of human P450 2E1 in vivo. The efficacy of single-dose disulfiram as an inhibitor of human P450 2E1 suggests that this modality for manipulating clinical P450 2E1 activity may provide a useful probe for delineating P450 2E1 participation in human drug biotransformation or for the treatment of poisoning by P450 2E1-activated toxins.

Oxidation of dihydropyridine calcium channel blockers and analogs by human liver cytochrome P-450 IIIA4

A series of 21 different 4-substituted 2,6-dimethyl-3-(alkoxycarbonyl)-1,4-dihydropyridines was considered with regard to oxidation to pyridine derivatives by human liver microsomal cytochrome P-450 (P-450). Antibodies raised against P-450 IIIA4 inhibited the microsomal oxidation of nifedipine and felodipine to the same extent, as did cimetidine and the mechanism-based inactivator gestodene. Gestodene was approximately 10(3) times more effective an inhibitor than cimetidine, on a molar basis. When rates of oxidation of the 1,4-dihydropyridines were compared to each other in different human liver microsomal preparations, all were highly correlated with each other with the exceptions of a derivative devoid of a substituent at the 4-position and an N1-CH3 derivative. A P-450 IIIA4 cDNA clone was expressed in yeast and the partially purified protein was used in reconstituted systems containing NADPH-cytochrome P-450 reductase and cytochrome b5. This system catalyzed the oxidation of all of the 1,4-dihydropyridines except the two for which poor correlation was seen in the liver microsomes. Principal component analysis supported the view that most of these reactions were catalyzed by the same enzyme in the yeast P-450 IIIA4 preparation and in the different human liver microsomal preparations, or by a closely related enzyme showing nearly identical properties of catalytic specificity and regulation. The results indicate that the enzyme P-450 IIIA4 is probably the major human catalyst involved in the formal dehydrogenation of most but not all 1,4-dihydropyridine drugs.

Mechanism-based inactivation of human liver microsomal cytochrome P-450 IIIA4 by gestodene

A series of 17 alpha-acetylenic steroids was examined with regard to ability to inactivate human liver microsomal cytochrome P-450 (P-450) IIA4, an enzyme involved in the oxidation of a number of drugs, carcinogens, and steroids, including estrogens and progestogens. Of the eight compounds tested, gestodene was found to be particularly active as a mechanism-based inactivator of P-450 IIIA4. Inhibition of both microsomal nifedipine oxidation and 17 alpha-ethynylestradiol (EE) 2-hydroxylation was dependent upon NADPH and gestodene concentration. Rates of inactivation were pseudo first order-values of kinactivation = 0.4 min-1 and Ki = 46 microM and a partition ratio of 9 were calculated. The kinactivation is approximately 50-fold greater than estimated for EE and is one of the highest reported for P-450 mechanism-based inactivators. Spectrally detectable P-450 was also destroyed in microsomes, but several experiments indicate that little covalent binding to amino acid residues of P-450 IIIA4 occurs. Microsomal inactivation of P-450 could be blocked by the presence of other P-450 IIIA4 substrates, and several activities catalyzed by other P-450s were not inhibited under conditions in which greater than 90% of P-450 IIIA4 was inactivated. Consideration of structure/activity relationships among the 17 alpha-acetylenic steroids examined indicates that the delta 15 double bond is critical but is not in itself sufficient for the inactivation process, which is postulated to result from attack of P-450 on the substituted acetylenic carbon and lead to porphyrin N-alkylation. The effectiveness of this mechanism-based inactivator may account for reports of increased estrogen and steroid levels in some women using gestodene in oral contraceptives.

Metabolic deactivation of furylfuramide by cytothrome P450 in human and liver microsomes

Metabolic deactivation of furylfuramide by human and rat liver microsomal cytochrome P450 enzymes has been investigated in a system measuring induction of umu gene expression response in Salmonella typhimurium TA1535/pSK1002. Both human and rat liver microsomes catalyzed the metabolism of furylfuramide to inactive form(s) that are incapable of inducing umu gene expression in the tester strain. The reaction required an NADPH-generating system and molecular oxygen and was inhibited by carbon monoxide, suggesting that a cytochrome P450-linked mono-oxygenase system is prerequisite for the deactivation reaction. With liver microsomes from variously pretreated rats, 3-methylcholanthrene was found to be a powerful inducer for the furylfuramide-metabolizing activity, and antibodies raised against rat P450IA1(BNF-B, c) and P450IA2(ISF-G, d) inhibited the microsomal activity. Human liver microsomal furylfuramide-metabolizing activity was also inhibited significantly by anti-P450IA2 IgG but weakly by anti-P450IA1 IgG. In liver microsomes prepared from seven different human samples, the activities of deactivation of furylfuramide were found to correlate with the amounts of immunoreactive protein related to rat P450IA2 and with the monooxygenase activities of metabolic activation of 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ) and of ethoxyresorufin O-deethylation. These results suggest that P450IA1 and P450IA2 in rats, and P450PA (IA2, the phenacetin O-deethylase and ortholog of rat P450IA2) in humans are the major enzymes involved in the deactivation of furylfuramide in liver microsomes. The metabolic studies involving HPLC analysis of products followed by spectrophotometric examination have also suggested that furylfuramide can be degraded very rapidly through the aerobic metabolism by liver microsomes.

Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase.

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).

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.

Metabolic characterization of human liver microsomal cytochromes P-450 involved in the oxidation of debrisoquine, bufuralol and the carcinogen 2-acetylaminofluorene

Over the last two decades, considerable attention has been focused on the relationship between carcinogen metabolism and cancer risk in humans. In particular, the association between carcinogenesis and the enzyme system responsible for the metabolism of most lypophilic chemicals, the cytochrome P-450 monooxygenases, has been addressed (1-4). This enzyme system is composed of numerous cytochrome P-450 isoenzymes that differ with respect to both substrate specificities and in their responses to enzyme inducers and inhibitors (1-6). In the case of aromatic amines and amides the first and obligatory step in their metabolic activation involves N-hydroxylation, and for the model hepatocarcinogen, 2-acetylaminofluorene (AAF), we have shown that a distinct isoenzyme of cytochrome P-450 in rabbit liver is involved in this process (7). This high degree of regio-selectivity of cytochrome P-450 isoenzymes in the oxidation of model carcinogens such as AAF (7) and benzo(a)pyrene (3) has provided the basis for the notion that the relative composition of cytochrome P-450 isoenzymes may be a major determinant in individual susceptibility to cancer as well as in target organ selection upon exposure to chemical carcinogens. In man, numerous studies have documented large inter-individual differences both

Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism.

The metabolism of the dihydropyridine calcium antagonist and vasodilator nifedipine has been reported to exhibit polymorphism among individual humans (Kleinbloesem, C. H., van Brummelen, P., Faber, H., Danhof, M., Vermeulen, N. P. E., and Breimer, D.D. (1984) Biochem. Pharmacol. 33, 3721-3724). Nifedipine oxidation has been shown to be catalyzed by cytochrome P-450 (P-450) enzymes. Reconstitution, immunoinhibition, and induction studies with rat liver indicated that the forms designated P-450UT-A and P-450PCN-E are the major contributors to microsomal nifedipine oxidation. The P-450 which oxidizes nifedipine (P-450NF) was purified to electrophoretic homogeneity from several human liver samples. Antibodies raised to P-450NF were highly specific as judged by immunoblotting analysis and inhibited greater than 90% of the nifedipine oxidase activity in human liver microsomes. A monoclonal antibody raised to the human P-450 preparation reacted with both human P-450NF and rat P-450PCN-E. Immunoblotting analysis of 39 human liver microsomal samples using anti-P-450NF antibodies revealed the same 52,000-dalton polypeptide, corresponding to P-450NF, with only one of the microsomal samples showing an additional immunoreactive protein. The level of nifedipine oxidase activity was highly correlated with the amount of P-450NF thus detected using either polyclonal (r = 0.78) or monoclonal (r = 0.65) antibodies, suggesting that the amount of the P-450NF polypeptide may be a major factor in influencing the level of catalytic activity in humans as well as rats. Cytochrome b5 enhanced the catalytic activity of reconstituted P-450NF, and anti-cytochrome b5 inhibited nifedipine oxidase activity in human liver microsomes. P-450NF also appears to be a major contributor to human liver microsomal aldrin epoxidation, d-benzphetamine N-demethylation, 17 beta-estradiol 2- and 4-hydroxylation, and testosterone 6 beta-hydroxylation, the major pathway for oxidation of this androgen in human liver microsomes.

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction.

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.

Monoclonal antibodies against human liver cytochrome P-450

Monoclonal hydribomas which produce antibodies against human liver microsomal cytochrome P-450 were developed. Three similar hybridomas produced antibodies which recognized an epitope specific to a family of human P-450 isozymes (P-450 5). This epitope was also present on cytochrome P-450 PCN-E (pregnenolone-16α-carbonitrile induced) from rat liver microsomes, but this isozyme differed from the human P-450 5 by its molecular weight. These antibodies enabled us to quantify cytochrome P-450 5 in human liver microsomes and to demonstrate an important quantitative polymorphism in the human liver monooxygenase system.

Purification of a new cytochrome P-450 from human liver microsomes

Using a classical methodlogy of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulos) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450 9. It has been proven to be different from all preceedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450 9, does not recognize rat liver microsomes; thus this cytochrome P-450 9 is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quatitative polymorphism. In reconstituted system, cytochrome P-450 9 is able to hydroxylate all substrates tested but is not specific on any; its exatc role in xenobiotic metabolism in man remains to be elucidated.

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of ( S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [ Methyl- 14C]mephenytoin was synthesized by alkylation of S-nirvanol with 14CH 3I and used as a substrate. After incubation of [ methyl- 14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The K m and V max rates obtained with a human liver microsomal preparation were 0.1 m m and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basis system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.

Cholesterol 7 alpha-hydroxylase from human liver: partial purification and reconstruction into defined phospholipid-cholesterol vesicles.

Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme for bile acid synthesis, was shown to be copurified with human liver microsomal cytochrome P-450. When these cytochrome P-450 species were reconstituted in phospholipid-cholesterol vesicles together with NADPH-cytochrome P-450 reductase, high cholesterol 7 alpha-hydroxylase activity was obtained in the presence of NADPH. The activity represented a twofold enrichment relative to cytochrome P-450 and 43-fold enrichment relative to total microsomal protein. Availability of such a preparation will allow further characterization of the enzyme and will also allow studies of its mechanisms of regulation.

Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg −1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit M rs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo( a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450. These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion.