Inhibition of rat liver microsomal cytochrome P-450 steroid hydroxylase reactions by imidazole antimycotic agents

The imidazole antimycotic agents ketoconazole, miconazole and clotrimazole were tested for their abilities to inhibit the reactions involved in the oxidative metabolism of androst-4-ene-3,17-dione by rat liver microsomal cytochromes P-450. All three compounds were found to function as potent inhibitors of steroid hydroxylase reactions, producing 50% inhibition of 6 beta-, 16 beta-, and 16 alpha-hydroxylase activities at concentrations between 10(-7) and 10(-5) M. The antimycotic agents, when added to liver microsomes, bound to cytochrome P-450 with high affinity to produce a "type II" spectral complex. These agents showed differential inhibition of the various steroid hydroxylases and were found not to affect the activities of the liver microsomal steroid 5 alpha-reductase or the androst-4-ene-3,17-dione 17-oxidoreductase. The results presented demonstrate an interaction of these imidazole antimycotic agents with the various cytochromes P-450 of liver microsomes, resulting in selective inhibition of monooxygenase activity.     

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)     

Inhibition of hepatic drug metabolism by phenothiazine tranquilizers: quantitative structure-activity relationships and selective inhibition of cytochrome P-450 isoform-specific activities

Twelve phenothiazine tranquilizers were investigated for the capacity to inhibit rat hepatic microsomal cytochrome P-450 (P-450) isoform-specific drug oxidation in vitro. All congeners were substituted in the 2-(carbocyclic) and 10- (thiazinyl nitrogen of the thiazine ring) positions. Cytochrome P-450 PB-B-mediated 7-pentylresorufin O-depentylase and P-450 BNF-B-mediated 7-ethylresorufin O-deethylase activities were effectively inhibited by most of the compounds. Structure-activity correlations revealed the apparent importance of the lipophilicity of the 2-substituent, and the negative effect of flexibility in the 10-position substituent, on anti-P-450 PB-B potency. On the other hand, inhibition of P-450 BNF-B activity was promoted by bulkiness and branching within the 10-substituent and the shape/bulk of the 2-group. From this analysis it is likely that the active site of P-450 PB-B is relatively small with at least one lipophilic region that may be involved in substrate and inhibitor binding. The active site of P-450 BNF-B is relatively large, and steric properties, rather than lipophilic character, appear to determine inhibition by phenothiazines. Derivatives with piperidinyl and piperazinyl ring systems in the 10-position were relatively active inhibitors of P-450 PCN-E (or an immunochemically related form of P-450) that catalyzes androst-4-ene-3,17-dione 6 beta-hydroxylation, and P-450 UT-A-mediated 16 alpha-hydroxylase activity. In contrast, steroid 7 alpha-hydroxylation (P-450 UT-F) and N-nitrosodimethylamine N-demethylation (P-450j) were refractory to inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioactivation and detoxication of the pyrrolizidine alkaloid senecionine by cytochrome P-450 enzymes in rat liver

Rats display a marked sex difference in the oxidation of the pyrrolizidine alkaloid senecionine, especially with respect to N-oxidation. This sex difference was largely eliminated following treatment with dexamethasone. These observations suggested the potential involvement of the male-specific cytochrome P-450 UT-A and the P-450 PCN-E in the metabolism of this pyrrolizidine alkaloid. Reconstituted rat P-450 UT-A exhibited a high rate of N-oxidation (15 nmol min-1 nmol P-450-1) which is almost 3-fold higher than the turnover number observed with male rat liver microsomes. In contrast, rat P-450 UT-A displayed a much lower activity toward necine pyrrole [+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, DHP) formation (1.0 nmol min-1 nmol P-450-1). The N-oxygenation and pyrrole formation activities displayed by rat cytochromes P-450 PB-B and P-450 BNF-B toward senecionine were low, with rates less than 1 nmol min-1 nmol P-450-1. Rabbit antibody to rat P-450 UT-A inhibited the senecionine-N-oxidation activity of untreated male rat liver microsomes by 60%, with lesser inhibition of DHP production. Rabbit antibody to human P-450NF (the human homologue to rat P-450 PCN-E) was a potent inhibitor of DHP production by untreated male rat liver microsomes. With microsomes from dexamethasone-pretreated rats, anti-P-450NF inhibited DHP and N-oxide production in parallel. We conclude that the large sex difference in senecionine N-oxidation probably is the result of the specificity of P-450 isozymes UT-A and PCN-E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substituted pyridines: nonsteroidal inhibitors of human placental aromatase cytochrome P-450.

The binding to human placental aromatase cytochrome P-450 and resulting extent of inhibition was examined for pyridine, pyridines substituted at the 2-, 3-, or 4-positions with phenyl or benzoyl groups, and the nonpyridinic structural analogs biphenyl and benzophenone. Spectral binding studies with partially purified aromatase indicated that pyridine, 3- and 4-phenylpyridines, and 3- and 4-benzoylpyridines interact at the active site via ligation of the pyridinic nitrogen to heme iron, as judged by the type II difference spectra. The apparent dissociation constants for these compounds are 2.1, 0.11, 0.09, 2.6, and 0.27 mM, respectively. Biphenyl, benzophenone, and 2-benzoylpyridine show exclusively a hydrophobic interaction in the presence of the substrate, androst-4-ene-3,17-dione, to give reverse type I difference spectra, with apparent spectral dissociation constants of 0.14, 0.42, and 1.8 mM, respectively. 2-Phenylpyridine shows a unique spectrum, previously not observed with cytochrome P-450, that has components of both type I and type II spectra. As measured by tritium ion release from [1 beta-3H]androst-4-ene-3,17-dione, competitive inhibition of human microsomal aromatase activity was observed with pyridine, 2-, 3-, and 4-phenylpyridines, biphenyl, and benzophenone, with Ki values of 320, 62, 1.48, 0.36, 750, and 130 microM, respectively. In contrast, 2-, 3-, and 4-benzoylpyridines exhibited complex kinetic behavior from which inhibition constants could not be obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Similarities and differences in the regulation of hepatic cytochrome P-450 enzymes by diabetes and fasting in male rats

The effects of streptozotocin-induced diabetes and fasting on hepatic cytochrome P-450 enzymes in sexually mature male rats were studied by immunochemical techniques and enzyme assays. The level of cytochrome P-450ac (an acetone/ethanol inducible form), 65 pmol/mg microsomal protein in control rats, increased 4- to 5-fold in diabetic rats and 3- and 5-fold in fasting rats. In contrast, P-450 UT-A (a male specific form) decreased drastically from 295 pmol/mg in the control group to about 10% of this value in diabetic rats and to 50% in fasting rats. P-450 PCN-E (a 16 alpha-cyanopregnenolone/dexamethasone inducible form), on the other hand, decreased from 151 pmol/mg to 38% in diabetic rats and increased 2-fold in fasting rats. These changes were also reflected in catalytic activities using N-nitrosodimethylamine, benzphetamine, and erythromycin as substrates. Slight changes in cytochromes P-450 UT-F, P-450 UT-I and P-450 PB-C were also observed under these conditions, but the biological significance is not known. These results suggest that different mechanisms exist for the regulation of the expression of cytochrome P-450 enzymes in diabetic and fasting rats.     

Effect of the suicide substrate 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine on the metabolism of xenobiotics and on cytochrome P-450 apoproteins

Treatment of rats with the cytochrome P-450 suicide substrate, 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), produced a 95% inhibition of the in vivo demethylation of either aminopyrine or morphine within 2 hr. One-carbon metabolism of formaldehyde or formate to carbon dioxide was not altered. DDEP also produced a time-dependent decrease in total hepatic microsomal cytochrome P-450 but had no effect on either NADPH-cytochrome c reductase or p-nitrophenol glucuronyl-transferase activities up to 24 hr after administration. A rapid decrease in rat liver microsomal aniline hydroxylation and ethoxyresorufin deethylation was observed in vitro following DDEP administration. Although in vitro testosterone metabolism to 16 alpha-, 16 beta-, and 2 alpha-hydroxy metabolites was depressed profoundly by DDEP in microsomes from untreated and 3-methylcholanthrene-treated animals, 7 alpha-hydroxylation of testosterone was much less affected. Immunochemical quantification of various microsomal cytochrome P-450 protein moieties showed that cytochromes P-450 beta NF-B, P-450UT-A, P-450PCN-E, and P-450PB-C were decreased in hepatic microsomes from DDEP-treated rats. However, the protein moiety of cytochrome P-450UT-H was not diminished and the immunoreactive protein for cytochromes P-450UT-F, P-450PB-B, and P-450ISF-G was only slightly decreased. These results show that DDEP treatment leads to marked decreases in holoprotein and apoproteins of many but not all hepatic microsomal cytochrome P-450 isozymes.     

In vitro inhibition of hepatic drug oxidation by thioridazine. Kinetic analysis of the inhibition of cytochrome P-450 isoform-specific reactions

The phenothiazine tranquilizer thioridazine has been associated with drug interactions in man. This study investigated the capacity of the drug to inhibit hepatic drug oxidations mediated by cytochromes P-450 (P-450) in microsomes in vitro. Thioridazine was a potent linear mixed-type inhibitor of P-450b-dependent 7-pentoxyresorufin O-depentylase activity in phenobarbital-induced rat liver. The kinetic analysis revealed the enzyme-substrate dissociation constant (Ks) to be 1.6 microM whereas the dissociation constant of the enzyme-inhibitor complex (Ki) was 0.11 microM. In contrast, 7-ethoxyresorufin O-deethylase activity (mediated by P-450c) in beta-naphthoflavone-induced rat hepatic microsomes was inhibited to a lesser extent (Ki = 2.4 microM) in relation to the Ks value (0.5 microM). Spectral studies indicated that the efficiency of thioridazine binding in phenobarbital-induced microsomes was about 25-fold greater than in microsomes from beta-naphthoflavone-induced rat liver. This finding is consistent with the relative capacity of thioridazine to inhibit oxidase activities catalyzed by P-450b and P-450c. Mixed-function oxidase activities catalysed by other P-450s were also inhibited by thioridazine, although to a lesser extent than those catalysed by forms b and c. Thus, the 6 beta- and 16 beta-hydroxylations of androst-4-ene-3,17-dione in hepatic microsomes from untreated rats were inhibited to a similar extent (I50S = 52 and 43 microM, respectively). The 7 alpha- and 16 alpha-hydroxylase pathways were approximately only half as susceptible to inhibition by thioridazine. These findings demonstrate the capacity of thioridazine to inhibit a range of P-450-dependent drug oxidations, with those catalysed by forms b and c most susceptible. The present study strongly suggests that drug interactions elicited by thioridazine are most likely a consequence of inhibitory interactions with P-450 enzymes.     

Monoclonal antibodies against human liver cytochrome P-450

Monoclonal hybridomas 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-16 alpha-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.     

In vitro inhibition of hepatic steroid hydroxylation by tamoxifen, a series of tamoxifen analogues and related compounds

The in vitro inhibition of the cytochrome P-450 (P-450) isozyme specific positional hydroxylation of androst-4-ene-3,17-dione (androstenedione) by the alkylamino containing compounds trans- and cis-tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, SKF 525-A and the non-alkylamino containing compounds tamoxifen metabolite E, and tamoxifen analogue U-23469 was assessed in pooled hepatic microsomes isolated from untreated male rats. P-450 IIA 1-mediated androstenedione 7 alpha-hydroxylation appeared refractory to inhibition, with the lowest I50s being approximately 200 microM (cis- and and trans-tamoxifen, 4-hydroxytamoxifen). (According to the recently recommended nomenclature for cytochromes P-450 (Nebert DW and Gonzalez FJ, Ann Rev Biochem 56: 945-993, 1987), rat hepatic cytochromes P-450 UT-A, PB-B, PCN-E and UT-F are encoded by genes IIC 11, IIB 1, IIIA 1/2 and IIA 1, respectively. I50s toward the P-450 IIC 11-, IIB 1-, and IIIA 1/2-catalysed reactions, androstenedione 16 alpha-, 16 beta- and 6 beta-hydroxylations, respectively, were generally in the range 70-190 microM. However, metabolite E exhibited a rather specific and potent capacity to inhibit androstenedione 16 alpha-hydroxylase activity (I50 = 18 microM). Since a number of alkylamine compounds have been shown to sequester microsomal P-450 as an inactive metabolite intermediate (MI), the tamoxifen analogues were investigated for their in vitro MI complexation capacity. However, spectral binding studies revealed that the incubation of these compounds with NADPH-fortified microsomal fractions did not result in MI complex formation. In binding experiments conducted with oxidised microsomal fractions it was apparent that most of the tamoxifen analogues are type I ligands of quite high affinity for ferric P-450 (Ks range 10-60 microM). It seems unlikely that MI formation is involved in the observed inhibition of androstenedione hydroxylation by tamoxifen and congeners. Instead, and in contrast to the situation observed with SKF 525-A, it would appear that the inhibitory capacity of the tamoxifen analogues is more closely related to type I binding capacity with ferric P-450. A finding of particular interest is that metabolite E, in which the alkylamino side-chain is absent, elicited a type I interaction of high capacity. The maximal absorbance change of the type I interaction of this compound with microsomal P-450 was about three-fold greater than the other compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of long-term choline deficiency on hepatic microsomal cytochrome P-450-mediated steroid and xenobiotic hydroxylases in the female rat

Total cytochrome P-450 levels decreased to about 80% of control in hepatic microsomes from female rats maintained for 30 weeks on a choline-deficient diet. Livers from these rats were fibrotic and had extensive fatty infiltration but, unlike livers of male rats on the same regimen, were not cirrhotic. Steroid hydroxylase activities were assessed in microsomes of female rats that received the choline-deficient diet and it was noted that the activity of the cytochrome P-450 UT-F-mediated steroid 7 alpha-hydroxylase was decreased to about 50% of the activity present in choline-supplemented control rat microsomes. Similar decreases were observed for microsomal androstenedione 6 beta-hydroxylase and aniline 4-hydroxylase activities. In female rat hepatic microsomes these two activities are probably mediated by the isozyme cytochrome P-450 ISF-G. In contrast to these findings, the activities of four other xenobiotic metabolising enzymes, as well as rates of microsomal steroid 16 alpha- and 16 beta-hydroxylation, were unchanged from control. Thus, in hepatic microsomes from choline-deficient female rats, it appears likely that levels of the non-sexually differentiated cytochromes P-450 UT-F and ISF-G are decreased. Unlike the situation in male rats, long term choline deficiency does not appear to influence levels of sexually-differentiated P-450 enzymes in the female rat.     

Drug metabolism in cirrhosis. Selective changes in cytochrome P-450 isozymes in the choline-deficient rat model

The effect of a choline-deficient diet on microsomal cytochrome P-450 and mixed-function oxidase (MFO) activity was investigated in relation to the development of nutritional cirrhosis. In rats that received the choline-deficient diet for 28 weeks cirrhosis was evident macroscopically and histologically; control rats that received an identical diet supplemented with choline had normal livers. Microsomal cytochrome P-450 and cytochrome b5 were reduced in cirrhotic liver to 50% of control levels. Three MFO activities (ethylmorphine N-demethylase, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase) were also reduced to 40-70% of control levels. However, the turnover number for the O-deethylation of 7-ethoxycoumarin was not reduced in cirrhotic liver. This finding suggested that certain drug oxidations may be selectively depressed in nutritional cirrhosis. To examine the possibility that selective changes in MFO activity may reflect the suppression of certain cytochrome P-450 isozymes, partially purified fractions of the cytochrome were prepared after solubilisation and hydrophobic affinity chromatography (on n-octylamino-Sepharose 4B) of cirrhotic and control liver microsomes. Analysis of these fractions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and laser densitometry indicated that a protein band of apparent minimum molecular weight 50.5 kD was primarily affected in cirrhotic rat liver microsomes. Levels of two other bands (apparent minimum molecular weight 48 and 52.5 kD) appeared essentially unaltered. Additional electrophoretic studies, conducted under non-reduced conditions, indicated the haemoprotein nature of protein bands in the 48-55 kD region. These data strongly suggest that cirrhosis produced in rats by a choline-deficient diet is associated with selective decreases in oxidative drug metabolism and individual cytochrome P-450 isozymes.     

Identification of the hepatic cytochrome P-450 isozymes induced and decreased by picloram

Microsomes from male rats treated with picloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16 alpha-hydroxylase activity when incubated with (4-14C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P-450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450 in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to picloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbital-inducible forms of cytochrome P-450. Third, hepatic microsomes from picloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98 revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from picloram-treated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.     

Preparation and characterization of monoclonal antibodies to pregnenolone 16-alpha-carbonitrile inducible rat liver cytochrome P-450

Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified rat hepatic pregnenolone 16-alpha-carbonitrile (PCN) induced cytochrome P-450 2a/PCN-E. The monoclonal antibodies (MAbs) thus obtained were screened for binding to the purified P-450 2a/PCN-E by radioimmunoassay. Eleven independent hybrid clones produced MAbs, each of which was of a single mouse immunoglobulin subclass of the IgG1, IgG2a or IgG2b type. Each of the MAbs produced by the eleven individual hybrid clones bound strongly to P-450 2a/PCN-E as assessed by radioimmunoassay and immunoprecipitation of P-450 2a/PCN-E in Ouchterlony double-immunodiffusion plates. Of the eleven MAbs, three also bound strongly to the phenobarbital-inducible rat liver cytochrome P-450 PB-4. Thus, two classes of MAbs were obtained, one class specific for P-450 2a/PCN-E and a second class that bound to both PCN- and phenobarbital-inducible P-450 forms. The reactivities of one MAb from each class toward eight highly purified rat hepatic cytochromes P-450 were examined using solid phase enzyme-linked immunosorbent analyses. The MAb designated C2 was found to be specific for P-450 2a/PCN-E and did not cross-react with seven other P-450 forms. This MAb was shown to be an effective probe for monitoring, by Western blotting, the induction of microsomal P-450 2a/PCN-E by PCN and phenobarbital. The MAb designated C1 reacted both with P-450 2a/PCN-E and with the two major phenobarbital-inducible P-450 forms, PB-4 and PB-5. None of the MAbs was inhibitory towards P-450 2a/PCN-E-dependent aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, ethoxycoumarin O-deethylase or ethymorphine N-demethylase activity, indicating that the epitopes recognized by these MAbs are not directly associated with catalytic activity. The strong reactivities of three of the MAbs with both P-450 2a/PCN-E and P-450s PB-4 and PB-5 indicate that these two structurally quite different cytochrome P-450 families share at least one common epitope. These new MAbs are additions to our library of MAbs to different cytochromes P-450 and should help further our understanding of the relationship of cytochrome P-450 phenotype and multiplicity to inter-individual differences in drug and carcinogen metabolism and sensitivity.     

Changes in the concentration of seven forms of cytochrome P-450 in primary cultures of adult rat hepatocytes

We prepared primary monolayer cultures of adult rat hepatocytes and measured the losses of cytochromes P-450 with the use of specific antibodies directed against purified forms of hepatic cytochrome P-450 which predominate in untreated rats (P-450UT-A, P-450UT-F) or in rats treated with phenobarbital (P-450PB-B/D, P-450PB-C, P-450PB/PCN-E) or with 3-methylcholanthrene (P-450 beta NF-B, P-450 beta NF/ISF-G). In hepatocytes prepared from an untreated rat and incubated in control medium, total cytochrome P-450, measured spectrally as CO-binding hemoprotein, declined 68% during the first 72 hr in culture. However, the sum of the immunoreactive cytochromes P-450 declined only 24%, indicating that loss of heme rather than of protein accounts for much of the well-known loss of cytochromes P-450 in hepatocyte cultures. In cultures prepared from untreated rats or from rats treated with phenobarbital or with 3-methylcholanthrene, individual forms of cytochrome P-450 declined at markedly differing rates. Incubation of cultures in three different media previously reported to maintain levels of total cytochrome P-450 failed to prevent the decline in total cytochrome P-450 during the first 24 to 72 hr in culture. However, in cultures incubated in medium containing metyrapone, the level of holocytochrome P-450 was maintained at the initial value during the first 72 hr, apparently by preventing the net loss of cytochrome P-450 heme and by increasing the concentrations of immunoreactive P-450PB/PCN-E and P-450 beta NF-B. Medium containing nicotinamide increased the proportion of P-450 beta NF-B relative to the other forms of cytochrome P-450, whereas cysteine-free medium increased P-450UT-F. We conclude that loss of cytochrome P-450 in cultured hepatocytes involves loss of its heme moiety coupled with changes in the concentrations of the individual forms. Recognition of these changes as influenced by specific components of the culture medium is important when using primary hepatocyte cultures for study of xenobiotic metabolism and toxicity in the liver.     

Role of the steroid 17 alpha-hydroxylase in spironolactone-mediated destruction of adrenal cytochrome P-450

Previous investigations have established that spironolactone (SL) is converted to a reactive metabolite by adrenal microsomal enzymes, resulting in the degradation of cytochrome P-450 (P-450). Deacetylation of SL to 7 alpha-thiospironolactone (7 alpha-thio-SL) is the first step in the activation pathway, but further NADPH-dependent metabolism of 7 alpha-thio-SL is required for P-450 destruction. Studies were done to evaluate the role of the steroid 17 alpha-hydroxylase in the activation of 7 alpha-thio-SL by adrenal microsomes. Incubation of guinea pig adrenal microsomes with 7 alpha-thio-SL in the presence of NADPH effected greater than 50% declines in P-450 content and in 17 alpha-hydroxylase activity but no change in the rate of 21-hydroxylation. Preincubation of the microsomes with antisera to the 17 alpha-hydroxylase P-450 isozyme (P-450(17 alpha,lyase)) decreased 17 alpha-hydroxylase but not 21-hydroxylase activity and prevented the degradation of P-450 by 7 alpha-thio-SL. Control IgG had no effect on 17 alpha-hydroxylase activity or on the 7 alpha-thio-SL-mediated destruction of P-450. When added to a purified P-450(17 alpha,lyase) preparation, 7 alpha-thio-SL and the endogenous substrate progesterone caused typical type I spectral changes, but SL did not. Incubation of a purified and reconstituted 17 alpha-hydroxylase system, consisting of P-450(17 alpha,lyase), NADPH-P-450 reductase, cytochrome b5, and dilauroylphosphatidylcholine, with 7 alpha-thio-SL plus NADPH effected the complete degradation of the P-450(17 alpha,lyase). Neither progesterone nor SL caused P-450 destruction with the reconstituted enzyme preparation. The results provide direct evidence for the activation of 7 alpha-thio-SL by the 17 alpha-hydroxylase and support the hypothesis that a mechanism-based inhibition of the enzyme occurs. The data also provide additional evidence that 7 alpha-thio-SL is an obligatory intermediate in the degradation of P-450 by SL.     

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.     

Effects of some common inducers on the hepatic microsomal metabolism of androstenedione in rainbow trout with special reference to cytochrome P-450-dependent enzymes

The effects of 16α-cyanopregnenolone, 3-methylcholanthrene (3-MC), Clophen A 50 (Cl 50) and phenobarbital on the total amount of cytochrome P-450 and the metabolism of 4-androstene-3,17-dione in liver microsomes from rainbow trout were studied. Only 3-MC and CI 50 caused marked changes in the cytochrome P-450 levels and the cytochrome P-450-dependent steroid hydroxylase activities. A varying response to 3-MC and Cl 50 was seen in fish of different ages and sex. Different responses of 6β-hydroxylase towards α-naphtoflavone, SKF 525-A and metyrapone in vitro were seen in 3-MC- and Cl 50-treated fish when compared to control fish. It is suggested that endocrine factors may be involved in the regulation of cytochrome P-450-mediated metabolism in fish and the presence of multiple forms of cytochrome P-450 in trout liver is indicated.     

Polymorphism of human cytochrome P-450

The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS)