Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs

The steroid androstenedione has been shown to be a valuable tool for the study of the selective inactivation of cytochrome P-450 isozymes in intact rat liver microsomes. The validity of this approach was investigated using microsomes, purified cytochrome P-450 isozymes, antibodies to particular cytochromes P-450, and the known mechanism-based inactivator chloramphenicol. Enzyme inactivation and antibody inhibition studies show that microsomes from both phenobarbital- and non-phenobarbital-treated rats are needed to accurately monitor the inactivation of the major phenobarbital-inducible cytochrome P-450 isozyme (PB-B) and of the major constitutive androstenedione 16 alpha-hydroxylase (UT-A). Similar experiments indicate that, although isozyme P-450g does catalyze the 6 beta-hydroxylation of androstenedione in a reconstituted system, this cytochrome appears to make only a minimal contribution to microsomal 6 beta-hydroxylase activity, which reflects instead the activity of pregnenolone-16 alpha-carbonitrile-induced isozymes. With these parameters investigated, initial enzyme inactivation studies showed that the antibiotic chloramphenicol caused different rates of NADPH-dependent enzyme inactivation among the four androstenedione hydroxylases monitored (16 beta greater than 6 beta greater than 16 alpha greater than 7 alpha). Based on these data, 12 chloramphenicol analogs were examined, and the results with these compounds show that their selectivity as cytochrome P-450 inactivators is a function of at least three structural features: 1) the number of halogen atoms, 2) the presence of a para-nitro group on the phenyl ring, and 3) substitutions on the ethyl side chain. For example, the compound N-(2-phenethyl)dichloroacetamide was shown to reversibly inhibit but not inactivate the cytochrome(s) P-450 responsible for androstenedione 6 beta-hydroxylase activity, whereas N-(2-p-nitrophenethyl) and N-(1,2-diphenethyl)dichloroacetamide rapidly inactivated the 6 beta-hydroxylase. The ability to monitor the activity of multiple isozymes with a single substrate should allow the development of a systematic approach to the design of selective inactivators of rat liver cytochromes P-450.     

Immunochemical identity of the 2,3,7,8-tetrachlorodibenzo-p-dioxin- and beta-naphthoflavone-induced cytochrome P-450 arachidonic acid epoxygenases in chick embryo liver: distinction from the omega-hydroxylase and the phenobarbital-induced epoxygenase.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) cause marked induction of cytochrome P-450 (P-450)-mediated arachidonic acid metabolism in chick embryo liver. We show here that the P-450 arachidonic acid epoxygenases induced by TCDD and beta NF are immunochemically indistinguishable from each other and unrelated to the arachidonic acid epoxygenase induced by PB. On Western blots, IgG from an antiserum against beta NFAA, a 55-kDa P-450 arachidonic acid epoxygenase purified from beta NF-treated chick embryo liver, immunoreacted selectively and to the same extent with a 55-kDa band in liver microsomes from chick embryos treated with TCDD or beta NF. It failed to react with proteins from untreated, solvent-treated, or PB-treated embryos on immunoblots or to immunoinhibit PB-induced arachidonic acid metabolism. Anti-beta NFAA IgG immunoinhibited all arachidonic acid metabolism by reconstituted beta NFAA and formation of arachidonic epoxides (EETs) and monohydroxylated derivatives (HETEs) by microsomes from TCDD- and beta NF-treated livers; it did not inhibit omega-hydroxylation. In contrast, IgG from an antiserum against the major PB-induced chicken P-450s, 2H1 and 2H2, immunoreacted with two major PB-induced P-450s, of 48 and 49 kDa, on Western blots. It also immunoinhibited formation of EETs and HETEs by PB-treated microsomes entirely and omega-hydroxylation by 50%. It failed to react with TCDD- or beta NF-induced P-450s on Western blots or to immunoinhibit TCDD- or beta NF-induced arachidonic acid metabolism. Because other P-450s with which anti-beta NFAA and anti-PB IgG cross-reacted were inactive in arachidonic acid epoxygenation, the findings are consistent with beta NFAA being principally responsible for the epoxygenation induced by TCDD and beta NF and 2H1 and/or 2H2 being responsible for epoxygenation induced by PB. Further, the P-450 arachidonate omega-hydroxylase and the epoxygenase in livers of TCDD- or beta NF-treated embryos are immunochemically unrelated, whereas those in livers of PB-treated embryos may be partly related.     

Role of isozymes of cytochrome P-450 in the metabolism of N,N-dimethyl-4-aminoazobenzene in the rat

The metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) was investigated in vitro by use of hepatic 10,000g supernatant fraction, microsomes, and purified cytochromes P-450 prepared from rats. Position-selective metabolism was studied in response to induction by 3-methylcholanthrene (MC), phenobarbital (PB), beta-naphthoflavone (BNF), and pregnenolone-16 alpha-carbonitrile (PCN) as well as inhibition by SKF 525-A, metyrapone, alpha-naphthoflavone, and piperonyl butoxide. The principal phase I pathways are demethylation of the tertiary (DAB) and secondary (MAB) amines and ring hydroxylation. When metabolism was measured with 10,000g supernatant fractions, each pathway responded differently and often independently to the inducers and inhibitors, suggesting that they are catalyzed preferentially by different isozymes of cytochrome P-450. Microsomes from PB-treated animals demethylated and hydroxylated DAB at the same rate as did control microsomes, based on cytochrome P-450 content, whereas microsomes from BNF- or MC-treated animals demethylated more rapidly and hydroxylated more slowly. Microsomes from PB-treated animals demethylated the secondary amine, MAB, more rapidly than the tertiary amine, DAB. Purified cytochrome P-448 from MC-treated animals catalyzed DAB demethylation very readily but hydroxylation very poorly. The turnover number was 10 times that seen in microsomes from MC-treated animals. Only one of the four cytochrome P-450 fractions isolated from PB-treated animals had significant activity with DAB and the turnover number of one of these (fraction B) was approximately that seen in microsomes. This study supports the concept of selectivity of various isozymes of cytochrome P-450 for the different steps in phase I metabolism of DAB. Furthermore, it is apparent that the association of certain inhibitors with specific isozymes of cytochrome P-450 is a generalization that requires qualification in terms of the substrates(s) involved.     

Cytochrome P-450-dependent nicotine oxidation by liver microsomes of guinea pigs. Immunochemical evidence with antibody against phenobarbital-inducible cytochrome P-450

When guinea pigs were treated with phenobarbital (PB), the specific activity of liver microsomal nicotine oxidase increased by 42%. PB-inducible cytochrome P-450 (PB-P-450) was purified to homogeneity from liver microsomes of PB-treated guinea pigs. Purified PB-P-450 catalyzed nicotine oxidation when reconstituted with NADPH-P-450 reductase and phospholipid system. Antibody prepared against the purified PB-P-450 formed single precipitation lines with both purified PB-P-450 and microsomal components in livers of PB-treated guinea pigs, and both precipitation lines fused. The antibody against PB-P-450 strongly inhibited nicotine oxidation in the reconstituted system. The antibody also inhibited liver microsomal nicotine oxidase activities in PB-treated and untreated guinea pigs by about 30% and less than 5% respectively. About 45% of total P-450 in liver microsomes of PB-treated guinea pigs was precipitated by the antibody. These results show that PB-P-450 participates in liver microsomal nicotine oxidation in PB-treated guinea pigs but not in untreated control animals.     

Relation between hepatic microsomal metabolism of N-nitrosamines and cytochrome P-450 species

Effects of SKF 525A (0.1 mM), metyrapone (0.1 mM), alpha-naphthoflavone (ANF) (0.5 mM) and pyrazole (1.0 mM) on N-nitrosodimethylamine (NDMA), N-nitrosomethylbutylamine (NMBuA) and N-nitrosomethylbenzylamine (NMBeA) metabolism by hepatic microsomes from rats pretreated with inducers were investigated. NDMA demethylation was weakly increased by phenobarbital (PB) treatment. The demethylation was inhibited by SKF 525A and enhanced by metyrapone in non-treated and PB-treated microsomes, and weakly inhibited by ANF in 3-methylcholanthrene(MC)-treated microsomes. NMBuA demethylation was increased by PB treatment and inhibited by SKF 525A in all microsomes. Metyrapone inhibited the demethylation in PB-treated microsomes. NMBuA debutylation was increased by PB and MC treatments, and inhibited by metyrapone in all microsomes. The strongest inhibition by metyrapone was observed in PB-treated microsomes. The debutylation was inhibited by SKF 525A in non-treated and PB-treated microsomes and by ANF in MC-treated microsomes. NMBeA demethylation was decreased by MC treatment and weakly inhibited by SKF 525A in all microsomes. The effects of the inducers and inhibitors on NMBeA debenzylation were almost the same as those on NMBuA debutylation except that the increasing effect of MC was small. Pyrazole was a relatively selective inhibitor of NDMA demethylation. These results suggest the following: NDMA demethylation is catalyzed by PB-induced cytochrome P-450 species (P450-PB) and MC-induced cytochrome P-450 species (P448-MC). But their specific activity is low and the other cytochrome P-450 species demethylate NDMA. NMBuA demethylation is catalyzed by P450-PB. But the specific activity is not high and the other cytochrome P-450 species also demethylate NMBuA. NMBuA debutylation is catalyzed by P450-PB and P448-MC. Almost all of NMBeA demethylation is catalyzed by cytochrome P-450 species other than P450-PB and P448-MC. NMBeA debenzylation is catalyzed by P450-PB and P448-MC, but the specific activity of P448-MC is not high.     

Studies of the mechanisms of metabolism of diethyl p-nitrophenyl phosphorothionate (parathion) by rabbit liver microsomes

Based on the protein content of microsomes, the administration of 3-methylcholanthrene (3-MC) and phenobarbital (PB) to adult rabbits leads to an increased rate of metabolism of parathion (diethyl 4-nitrophenyl phosphorothionate) by rough-surfaced and whole microsomes but not by smooth-surfaced microsomes. Although prior administration of both PB and 3-MC increased the cytochrome P-450 content of the microsomes, when the rate of metabolism of parathion was calculated on the basis of the concentration of cytochrome P-450 in these microsomes, there is no difference in the rate of metabolism of parathion by rough-surfaced and smooth-surfaced microsomes from the untreated, 3-MC-treated and PB-treated animals. However, based on the cytochrome P-450 concentration, the rate of metabolism of parathion by whole microsomes from 3-MC and PB-treated animals is less than the rate with whole microsomes from untreated animals. Further studies have shown there is no correlation between the concentration of high spin or low spin cytochrome P-450 in any of the microsomal fractions or subfractions and the rate of metabolism of parathion to paraoxon or diethyl phosphorothionate.     

Selective inactivation by chlorofluoroacetamides of the major phenobarbital-inducible form(s) of rat liver cytochrome P-450

Five N-monosubstituted chlorofluoroacetamides have been tested as potential specific irreversible inhibitors of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P450IIB1). In vitro, N-(2-phenethyl)chlorofluoroacetamide was ineffective in causing a time-dependent loss of P450IIB1-mediated androstenedione 16 beta-hydroxylase activity in liver microsomes from phenobarbital-treated rats. However, addition of a nitro or bromo substitutent at the para position of the phenyl group or addition of a second phenyl group at the 1- or 2-position on the phenethyl side chain yielded compounds that caused a selective time-dependent decrease in androstenedione 16 beta-hydroxylase activity relative to four other P-450 form-specific androstenedione or progesterone hydroxylase activities monitored. The two compounds that were the most effective in inactivating P450IIB1 in vitro, N-(2-p-bromophenethyl) and N-(2-p-nitrophenethyl)chlorofluoroacetamide were also administered ip to phenobarbital-treated rats, and inhibition of cytochromes P-450 was assessed by in vitro assays of steroid and R- and S-warfarin hydroxylation in subsequently prepared hepatic microsomes. Both compounds selectively inhibited P450IIB1, and at a dose (200 mg/kg) of N-(2-p-nitrophenethyl)chlorofluoroacetamide that reduced androstenedione 16 beta-hydroxylase activity to approximately one-third of the control level, only two other activities, both attributable to P450IIB1, were decreased. In contrast, steroid and warfarin hydroxylase activities indicative of at least five other cytochromes P-450 were unaffected by the compound. These results indicate the feasibility of an empirical approach to the design of specific cytochrome P-450 inactivators.     

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of NADPH-cytochrome P-450 reductase

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.     

Interspecies homology of cytochrome P-450: inhibition by anti-P-450-male antibodies of testosterone hydroxylases in liver microsomes from various animal species including man

P-450-male is one of the male-specific forms of cytochrome P-450 in liver microsomes of adult rats and functions as testosterone 2 alpha- and 16 alpha-hydroxylases. The purpose of this study was to examine whether forms of cytochrome P-450 cross-reactive with anti-P-450-male antibodies are present in liver microsomes of other animal species, such as male and female mice, rabbits, guinea pigs, hamsters, dogs and humans. The antibodies cross-reacted with a protein(s) in the liver microsomes of all animal species to show one or more bands on nitrocellulose paper by Western blot peroxidase-anti-peroxidase staining analysis. Qualitative as well as quantitative species differences were noted in the testosterone hydroxylases. The antibodies inhibited 2 alpha-hydroxylase in male rats, 16 alpha-hydroxylase in male rats and male and female dogs, 7 alpha-hydroxylase in female rats and male and female mice and hamsters, and 15 alpha-hydroxylase in female mice and hamsters. No clear inhibition of 6 beta-hydroxylase, which was present in all animal species, was observed. These results indicate that forms of cytochrome P-450 that are immunochemically related with P-450-male catalyze the hydroxylation of testosterone at varying positions depending on the animal species, with the exception of 6 beta-hydroxylation, which may be catalyzed by a distinct form of cytochrome P-450.     

Nature of N-nitrosodimethylamine demethylase

The nature of enzymes involved in demethylation of N-nitrosodimethylamine (NDMA) was investigated in hepatic microsomes of rats. Compared to the other cytochrome P-450-dependent enzymes. NDMA demethylase had anomalous properties as reported in the literature. However, kinetic analysis suggested a qualitative change in NDMA demethylase induced by phenobarbital (PB) and 3-methylcholanthrene (MC) pretreatment. The inhibition of demethylase by -naphthoflavone in MC-treated microsomes also suggested that cytochrome P-450 species induced by MC are active in demethylating NDMA. The enhancement of NDMA demethylase activity by metyrapone in PB-treated microsomes was greater than in non-treated ones, and was not observed in MC-treated ones. The result is almost the same as in acetanilide hydroxylation, depending on cytochrome P-450. Pyrazole, tranylcypromine, and aminoacetonitrile, which are selective inhibitors of NDMA demethylation, interacted with cytochrome P-450 species to produce type-II spectra, and typical type-II compounds (aniline, imidazole, and nicotinamide) were inhibitors of the NDMA demethylation. Tranylcypromine irreversibly inhibited microsomal monoamine oxidase [EC 1.4.3.4], but not NDMA demethylase. Semicarbazide (a copper- and pyridoxal-containing amine oxidase [EC 1.4.3.6] inhibitor) had no effect on demethylation. From these results it is concluded that NDMA demethylation depends only on cytochrome P-450-dependent monooxygenases.     

Rat lung and liver cytochrome P-450 isozymes involved in the hydroxylation of m-xylene

The primary metabolism of m-xylene in rat lung and liver microsomes was investigated. The ratio of side chain to aromatic hydroxylation was found to be approximately 1:1 in lung microsomes from untreated rats and in a reconstituted system containing the major cytochrome P-450 isozyme induced in rat liver by phenobarbital, cytochrome P-450-PB-B2, as compared to 8:1 in liver microsomes. Antibody inhibition studies showed the major importance of cytochrome P-450-PB-B2 for the formation of both primary m-xylene metabolites (3-methylbenzylalcohol and 2,4-dimethylphenol) in lung microsomes. Antibodies to the major cytochrome P-450 isozyme induced in rat liver by beta-naphthoflavone, P-450-BNF-B2, did not inhibit m-xylene metabolism in either liver or lung microsomes from beta-naphthoflavone treated rats although this isozyme efficiently catalyzed m-xylene hydroxylation in a reconstituted system. m-Xylene metabolism by purified P-450-BNF-B2 appeared to cause rapid inactivation of the enzyme.     

Effect of congestive heart failure on the intrinsic metabolic capacity of the liver in the dog.

The intrinsic metabolic capacity of the liver at end-stage heart failure in the pacing overdrive dog model of congestive heart failure was evaluated ex vivo. Congestive heart failure was induced in seven adult mongrel dogs (20-30 kg) by cardiac electrical pacing at a frequency of 240 stimuli/min until the development of overt heart failure; seven other dogs served as controls. The animals were then anesthetized and the right ventricular papillary muscles and samples from the left lateral hepatic lobes were collected. The degree of myocardial dysfunction as well as the total amount and the activities of cytochromes P-450 were evaluated. Tension, maximum rate of tension rise, and Vmax were significantly lower (40-60%) in the paced than in the control dogs, indicating a marked myocardial dysfunction. Moreover, significant decreases in total cytochrome P-450 (0.31 +/- 0.04 vs. 0.53 +/- 0.03 nmol/mg of microsomal protein, p less than 0.01) and in the intensity of four different electrophoretic protein bands (molecular masses of 46, 48, 50, and 59 kDa) occurred in the dogs with congestive heart failure. The decrease in total cytochrome P-450 was accompanied by a significant reduction in aminopyrine N-demethylase activity (1.74 +/- 0.25 vs. 2.91 +/- 0.40 nmol/min/mg of microsomal protein, p less than 0.05). Immunoblot analysis using antibodies to two different dog liver phenobarbital-inducible cytochromes P-450 demonstrated that PBD-1 (a P-450IIIA) was not affected by congestive heart failure, whereas PBD-2 (a P-450IIB) was markedly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel form of cytochrome P-450 in beagle dogs. P-450-D3 is a low spin form of cytochrome P-450 but with catalytic and structural properties similar to P-450d

A form of cytochrome P-450, P-450-D3, cross reactive with antibodies to rat P-450d was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated female Beagle dogs to an electrophoretic homogeneity. Judging from the result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of P-450-D3 was estimated to be 54,000. The oxidized form of P-450-D3 showed a peak at 416 nm indicating that the cytochrome is mostly in a low spin state. The carbon monoxide bound reduced form of P-450-D3 showed a peak at 448 nm. In a reconstituted system, P-450-D3 catalyzed drug oxidations including benzphetamine and aminopyrine N-demethylations, 7-ethoxycoumarin and p-propoxyaniline O-dealkylations, and aniline and benzo(a)pyrene hydroxylations. The rate of aniline hydroxylation catalyzed by P-450-D3 was similar to that catalyzed by P-450c which is a low spin form of cytochrome P-450 purified from liver microsomes of PCB-treated rats, whereas the catalytic activities of P-450-D3 for 7-ethoxycoumarin O-deethylation and benzo(a)pyrene hydroxylation were considerably lower than those of P-450c. The amino terminal portion of P-450-D3 was found to be highly similar to those of P-450d, human P3-450 and P3-450 when four amino acid deletions were tentatively inserted between fifth and sixth amino acids from the N-terminal, but not that of P-450c which is a low spin form of cytochrome P-448 purified from rat liver microsomes. These results indicate that Beagle dogs possess a low spin form of cytochrome P-450 with spectral properties similar to P-450c but with catalytic and structural properties similar to P-450d.     

Oxidation of butylated hydroxytoluene to toxic metabolites. Factors influencing hydroxylation and quinone methide formation by hepatic and pulmonary microsomes.

The effects of inducing agents and inhibitors on the cytochrome P-450-catalyzed oxidations of butylated hydroxytoluene (BHT) to form three metabolites were investigated with liver and lung microsomes from rats and mice. These compounds, the quinone methide (QM) formed by two-electron oxidation of BHT, the hydroxy-tert-butyl analog of BHT (BHT-OH) resulting from aliphatic hydroxylation, and the hydroxy-quinone methide (QM-OH) derived from BHT-OH, have been implicated previously as intermediates or products involved in BHT bioactivation and toxicity. Although there was little or no increase in BHT metabolism in pulmonary microsomes from either species following phenobarbital (PB) administration, 6- to 37-fold enhancements occurred in the transformation of BHT to QM, and the conversion of BHT-OH to QM-OH with hepatic microsomes from both species. The first step in QM-OH formation, hydroxylation of BHT to BHT-OH, is a minor pathway with hepatic microsomes from treated or untreated rats, thereby explaining the lack of QM-OH formation from BHT by that species. The two-step oxidation of BHT to QM-OH, however, is a relatively important metabolic pathway with hepatic microsomes from PB-treated mice, due to an unusually large (111-fold) PB-induced increase in the tert-butyl hydroxylation step. These results demonstrate that pulmonary microsomes from mice, but not rats, have relatively high constitutive P-450 activity for the formation of QM-OH from BHT, supporting the proposal that this metabolite is involved in BHT-induced pneumotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro formation of an inhibitory complex between an isosafrole metabolite and rat hepatic cytochrome P-450 PB-B

A series of in vitro studies was performed, in rat liver microsomes, in which metabolite intermediate (MI) complexation of cytochrome P-450 (P-450) by the methylenedioxyphenyl compound isosafrole was related to P-450 isozyme-specific inhibition of drug oxidation. The C19-steroid androst-4-ene-3,17-dione was selected for initial study because the stereoselective hydroxylation of this substrate is specific for certain P-450s. In control microsomes only the 6 beta- and 16 beta-hydroxylations of the steroid (catalyzed, respectively, by the P-450s PCN-E and PB-B) were inhibited by isosafrole (I50 = 100 and 110 microM). In contrast, the 7 alpha- and 16 alpha-hydroxylases (P-450 UT-F- and UT-A-mediated, respectively) were refractory to inhibition. After phenobarbital (PB) induction, steroid 6 beta- and 16 beta-hydroxylase activities were again inhibited (I50 = 170 and 190 microM) but, in addition, the 16 alpha-hydroxylase pathway was also inhibited (I50 = 200 microM). Spectral studies revealed that MI complexation of P-450 in untreated microsomes was minimal but was enhanced markedly after PB induction (up to 50% of the total P-450 content complexed). Thus, it is apparent that a PB-inducible P-450 is involved in MI complex formation under these conditions. Indeed the I50 of isosafrole toward steroid 16 beta-hydroxylase activity was decreased if the inhibitor was preincubated with NADPH-fortified PB-induced microsomes prior to substrate addition; the preincubation step did not enhance the inhibition of any other steroid hydroxylase pathway by isosafrole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies to phenobarbital-induced rat liver cytochrome P-450

Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified phenobarbital-induced rat liver cytochrome P-450 (PB-P-450). Hybridomas were selected in HAT medium, and the monoclonal antibodies (MAbs) produced were screened for binding to the PB-P-450 by radioimmunoassay, for immunoprecipitation of the PB-P-450, and for inhibition of PB-P-450-catalyzed enzyme activity. In two experiments, MAbs of the IgM and IgG1 were produced that bound and, in certain cases, precipitated PB-P-450. None of these MAbs, however, inhibited the PB-P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity. In two other experiments, MAbs to PB-P-450 were produced that bound, precipitated and, in several cases, strongly or completely inhibited the AHH and 7-ethoxycoumarin deethylase (ECD) activities of PB-P-450. These MAbs showed no activity toward the purified 3-methylcholanthrene-induced cytochrome P-450 (MC-P-450), β-naphthoflavone-induced cytochrome P-450 (BNF-P-450) or pregnenolone 16-α-carbonitrile-induced cytochrome P-450 (PCN-P-450) in respect to RIA determined binding, immunoprecipitation, or inhibition of AHH activity. One of the monoclonal antibodies, MAb 2-66-3, inhibited the AHH activity of liver microsomes from PB-treated rats by 43% but did not inhibit the AHH activity of liver microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 also inhibited ECD in microsomes from PB-treated rats by 22%. The MAb 2-66-3 showed high cross-reactivity for binding, immunoprecipitation and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver (PB-P-450_LM2). Two other MAbs, 4-7-1 and 4-29-5, completely inhibited the AHH of the purified PB-P-450. MAbs to different cytochromes P-450 will be of extraordinary usefulness for a variety of studies including phenotyping of individuals, species, and tissues and for the genetic analysis of P-450s as well as for the direct assay, purification, and structure determination of various cytochromes P-450.     

Differential metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate by rat and chicken hepatic microsomes

The in vitro hepatic metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated in the hen (a species that is sensitive to EPN delayed neurotoxicity) and the rat (an insensitive species). EPN, which produced a Type I binding spectrum on incubation with cytochrome P-450, was converted by liver microsomes from both species to its oxygen analog, O-ethyl O-4-nitrophenyl phenylphosphonate (EPNO), and to p-nitrophenol (PNP). The formation of EPNO and PNP was dependent on the presence of NADPH in the reaction mixture and could be inhibited by either SKF-525A or by anaerobic conditions. The rates of EPNO and PNP formation by rat liver microsomes were, however, 3- and 20-fold higher, respectively, than the rates of formation by chicken liver microsomes. There was also a 4-fold difference in the cytochrome P-450 contents of the liver microsomes. The EPNO-hydrolyzing activity of rat liver microsomes was much greater than that of chicken liver microsomes. EPNO metabolism, in contrast to EPN metabolism, did not require NAPDH nor was it inhibited by SKF-525A or by anaerobic conditions. Prior exposure of rats to phenobarbital (PB) or Arochlor 1254 resulted in an increase in hepatic microsomal EPN metabolism and cytochrome P-450 content. On the other hand, 3-methylcholanthrene (3-MC) treatment elevated microsomal cytochrome P-450 but did not increase EPNO or PNP formation. Pretreatment with EPN did not alter either microsomal EPN metabolism or cytochrome P-450 levels. In chickens, prior exposure to PB, 3-MC or 100 mg/kg EPN increased EPNO and PNP formation by liver microsomes as well as cytochrome P-450 levels; prior exposure of chickens to 15 mg/kg EPN did not alter these variables. The λ_max Soret bands of the reduced hepatic cytochrome P-450 complexes from these animals differed as follows (rat then chicken): untreated, 450 vs 452 nm; PB-treated, 450 vs 451 nm; and 3-MC-treated, 448 vs 449 nm. None of the above treatments had an effect on EPNO metabolism by liver microsomes.     

Selective inactivation of rat liver cytochromes P-450 by 21-chlorinated steroids

The inactivation by 21-chlorinated steroids of rat liver cytochromes P-450 involved in the hydroxylation of progesterone and androstenedione has been investigated. Preincubation of intact liver microsomes from phenobarbital-treated rats with 21-chloropregnenolone, 21,21-dichloropregnenolone, or 21,21-dichloroprogesterone in the presence of NADPH caused a time-dependent decrease in progesterone 21-hydroxylase and in progesterone or androstenedione 6 beta-hydroxylase activity but had negligible or only minor effects on five other steroid hydroxylases. The compounds differed, however, with regard to the relative rate constants for inactivation of the 21- and 6 beta-hydroxylases. For example, 21,21-dichloroprogesterone and 21,21-dichloropregnenolone inactivated the progesterone 6 beta-hydroxylase at similar rates, but the dichloroprogesterone was a more effective inactivator of the 21-hydroxylase. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few isozymes of cytochrome P-450 may be a rational means of designing isozyme-selective inhibitors but that target and nontarget enzymes may not totally retain the regioselectivity they exhibit towards the underivatized substrate.     

Evidence for the stability and cytochrome P450 specificity of the phenobarbital-induced reductive halothane-cytochrome P450 complex formed in rat hepatic microsomes

The hypothesis that the reduced spectral halothane-cytochrome P450 complex formed in rat hepatic microsomes is a stable cytochrome P450 specific species was examined. Comparisons of the cytochrome P450 inducers, phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN) and beta-naphthoflavone (beta-NF) showed that PB was the most effective inducer of the halothane-cytochrome P450 complex and the cytochrome P450 which liberates the halothane metabolites, 2-chloro-1,1-difluoroethene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE). However, the ratio of CDE produced to quantity of complex was found to be reduced 70-77% in these microsomes. A large portion of total microsomal cytochrome P450 was destroyed upon halothane reduction (up to 39%), yet the complexed cytochrome P450, particularly in microsomes from PB-treated animals, was resistant to the irreversible inactivation mechanisms of halothane reduction. The effects of reductive halothane metabolism on subsequent warfarin metabolism showed that 7-hydroxywarfarin formation from either (R)- or (S)-warfarin in microsomes from PCN-treated, PB-treated or untreated rats was highly susceptible to irreversible inhibition. In microsomes from PB-treated, but not PCN or untreated rats, the formation of one warfarin metabolite, 4'-hydroxywarfarin from (R)-warfarin, could be shown to be increased when complex was eliminated by photodissociation. These results suggest that PB-B is preferentially bound as complex and resistant to inactivation because of complex stability, and that halothane reduction readily destroys the cytochrome P450 form, PB-C.     

Inhibition of mono-oxygenase activities by 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrase, in rat liver microsomes.

Addition of 1,1,1-trichloropropene 2,3-oxide (TCPO), an inhibitor of microsomal epoxide hydrase, to rat liver microsomes caused a type I spectral change, and its magnitude was increased by pretreatment of animals with phenobarbital (PB) but not with 3-methylcholanthrene and polychlorinated biphenyls. TCPO inhibited aminopyrine N-demethylation competitively and prevented covalent binding of 2,4,2',4'-tetrachlorobiphenyl to macromolecules catalyzed by liver microsomes, although it stimulated benzolalpyrene hydroxylation significantly. It is suggested that TCPO interacts with cytochrome P-450, especially a species of the cytochrome which is inducible by PB administration, and thus inhibits mono-oxygenase activities of liver microsomes.