Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

To investigate the potential interaction of the various pathways of androgen hydroxylation, we have conducted studies to identify the profile of products formed during the time course of metabolism of androst-4-ene-3,17-dione (AD). Incubates containing AD, NADPH, and liver microsomes (from rats pretreated with phenobarbital) were sampled at times between 0 and 20 min and the metabolites resolved by reverse-phase (C18) high-performance liquid chromatography. By this method, the pattern of formation and of utilization of eight major primary and secondary metabolites of AD was determined. We report here the formation of two previously unidentified major metabolites of AD: 6 beta,16 alpha-dihydroxyandrost-4-ene-3,17-dione and 6 beta,16 beta-dihydroxyandrost-4-ene-3,17-dione. We propose that liver microsomal cytochromes P-450 can sequentially hydroxylate a single molecule of AD at multiple sites. These hydroxylase activities are presumably a result of multiple cytochrome P-450 isozymes acting on AD resulting in a transient time course for the appearance of some monohydroxylated metabolites. In addition, a unidirectional conversion of the metabolite 16 alpha-hydroxyandrost-4-ene-3,17-dione to 16 beta-hydroxyandrost-4-ene-3,17-dione is described. Evidence is provided to support the role of cytochrome P-450 in catalyzing this reaction.     

Metabolism of valproic acid by hepatic microsomal cytochrome P-450

Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid ω-1 and ω hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the β-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes ω-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxyvalproic acid, should not be used to assess in vivo metabolism of valproate via the β-oxidation pathway.     

Comparative characteristics of isolated forms of cytochrome P-450 induced by phenobarbital and perfluorodecalin

Two forms of cytochrome P-450 were isolated from liver microsomes of perfluorodecalin-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from phenobarbital-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromatographic behaviour on 1.8-diaminooctyl-Sepharose 4B and DEAE-Sephacel columns, molecular mass determined by SDS polyacrylamide gel electrophoresis, spectral properties, immunoreactivity, peptide mapping, catalytic activity. These findings suggest that in rat liver microsomes perfluorodecalin and phenobarbital which differ in their chemical structure induce identical forms of cytochrome P-450.     

3,5-Diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine inactivates rat liver cytochrome P-450c, but not its orthologue, rabbit lung form 6

Various rat liver cytochrome P-450 (P-450) isozymes are targets for mechanism-based inactivation by 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (4-ethyl DDC). Unlike rat liver, which contains multiple P-450 isozymes, rabbit lung contains only three major isozymes referred to as forms 2, 5, and 6. We have examined the ability of 4-ethyl DDC to destroy P-450 heme in hepatic and pulmonary microsomes from untreated and beta-naphthoflavone (beta NF)-treated rabbits. This compound destroyed 31% of the P-450 in either hepatic microsomal preparation, but was ineffective at lowering P-450 and heme levels in pulmonary microsomes when examined at a range of concentrations (0.45-5.0 mM). These data suggest that rabbit pulmonary P-450 forms 2, 5, and 6 are not targets for destruction by 4-ethyl DDC, despite the ability of this compound to inactivate rat liver P-450c, the orthologue of rabbit lung form 6.     

Participation of two structurally related enzymes in rat hepatic microsomal androstenedione 7 alpha-hydroxylation.

Rat hepatic cytochrome P-450 form 3 (testosterone 7 alpha-hydroxylase; P-450 gene IIA1) and P-450 form RLM2 (testosterone 15 alpha-hydroxylase; P-450 gene IIA2) are 88% identical in primary structure, yet they hydroxylate testosterone with distinct and apparently unrelated regioselectivities. In this study, androstenedione and progesterone were used to assess the regioselectivity and stereospecificity of these two P-450 enzymes towards other steroid substrates. Although P-450 RLM2 exhibited low 7 alpha-hydroxylase activity with testosterone or progesterone as substrate (turnover number less than or equal to 1-2 nmol of metabolite/min per nmol of P-450), it did catalyse androstenedione 7 alpha-hydroxylation at a high rate (21 min-1) which exceeded that of P-450 3 (7 min-1). However, whereas P-450 3 exhibited a high specificity for hydroxylation of these steroids at the 7 alpha position (95-97% of total activity), P-450 RLM2 actively metabolized these compounds at four or more major sites including the nearby C-15 position, which dominated in the case of testosterone and progesterone. The observation that androstenedione is actively 7 alpha-hydroxylated by purified P-450 RLM2 suggested that this P-450 enzyme might make significant contributions to microsomal androstenedione 7 alpha-hydroxylation, an activity that was previously reported to be associated with immunoreactive P-450 3. Antibody inhibition experiments were therefore carried out in liver microsomes using polyclonal anti-(P-450 3) antibodies which cross-react with P-450 RLM2, and using a monoclonal antibody that is reactive with and inhibitory towards P-450 3 but not P-450 RLM2. P-450 3 was thus shown to catalyse only around 35% of the total androstenedione 7 alpha-hydroxylase activity in uninduced adult male rat liver microsomes, with the balance attributed to P-450 RLM2. The P-450-3-dependent 7 alpha-hydroxylase activity was increased to approximately 65% of the total in phenobarbital-induced adult male microsomes, and to greater than 90% of the total in untreated adult female rat liver microsomes. These observations are consistent with the inducibility of P-450 3 by phenobarbital and with the absence of P-450 RLM2 from adult female rat liver respectively. These findings establish that P-450 RLM2 and P-450 3 can both contribute significantly to microsomal androstenedione 7 alpha-hydroxylation, thus demonstrating that the 7 alpha-hydroxylation of this androgen does not serve as a specific catalytic monitor for microsomal P-450 3.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.     

Purification and characterization of liver microsomal cytochromes p-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or beta-naphthoflavone

Eight different forms of cytochrome P-450 (P-450) were purified to electrophoretic homogeneity by a common procedure from liver microsomes of rats treated with phenobarbital or beta-naphthoflavone. Antibodies were prepared to seven of these forms in rabbits. The eight P-450s were distinguished by spectral properties of the ferric, ferrous, and ferrous carbonyl forms, apparent monomeric molecular weights, peptide mapping, immunological reactivity as discerned by double-diffusion immunoprecipitin analysis and crossed immunoelectrophoresis, and catalytic activities toward the substrates acetanilide, aminopyrine, aniline, benzo[a]-pyrene, d-benzphetamine, N,N-dimethylnitrosamine, 7-ethoxycoumarin, 7-ethoxyresorufin, ethylmorphine, p-nitroanisole, testosterone, and (R)- and (S)-warfarin. Crossed sodium dodecyl sulfate-polyacrylamide gel immunoelectrophoresis was used to estimate the levels of each of the eight forms of P-450 present in the liver microsomes of untreated rats and rats treated with phenobarbital, 5,6-benzoflavone, pregnenolone-16 alpha-carbonitrile, isosafrole, or the polychlorinated biphenyl mixture Aroclor 1254. In each situation, the sum of the levels of these eight P-450s was at least as high as the spectrally determined P-450 content. The results clearly demonstrate that individual forms of P-450 can be induced by different compounds and that a single compound can lower the level of one form of P-450 while inducing one or more other forms of P-450. Catalytic activities toward each of the substrates observed with microsomal preparations are compared to rates predicted on the basis of the content of each of the eight P-450s. These studies provide a basis for further studies on the regulation of individual P-450s, the physical properties of the different P-450s, and the metabolic consequences of changes in the forms of P-450 in rat liver models.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Multiple forms of cytochrome P-450 from microsomes of rat liver, kidney, and lung resolved by high-performance liquid chromatography

The amount of cytochrome P-450 in microsomes of rat kidney and lungs was 23 and 7%, respectively, of the amount in microsomes of the liver. HPLC profiles of solubilized microsomes showed that there were five or more forms of cytochrome P-450 in microsomes of kidneys and also of lungs of untreated rats. While 3-methylcholanthrene induced a new form of cytochrome P-450 in kidney and lung microsomes, phenobarbital caused no major change in the HPLC profiles of these microsomes.     

Rat hepatic cytochrome P-450 isoenzyme 2c. Identification as a male-specific, developmentally induced steroid 16 alpha-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme

Rat hepatic cytochrome P-450 isoenzyme 2c, purified to homogeneity from uninduced, adult rat liver (Waxman, D.J., Ko, A., and Walsh, C. (1983) J. Biol. Chem. 258, 11937-11947), was shown to exhibit a unique NH2-terminal amino acid sequence as well as distinctive peptide maps and immunochemical properties when compared to seven other purified rat liver P-450 isoenzymes. P-450 2c was an efficient monooxygenase catalyst with several xenobiotic substrates; P-450 2c also catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone, androst-4-ene-3,17-dione and progesterone (total turnover = 7-9 min-1 P-450(-1) at 25 microM steroid substrate) with the ratio of 2 alpha to 16 alpha hydroxylation varying from less than or equal to 0.02 to 1.6 depending on the steroid's C-17 substituent. Six different microsomal steroid hydroxylase activities characteristic of purified P-450 2c and sensitive to specific inhibition by anti-P-450 2c antibody were induced at puberty in male but not female rat liver. Microsomal steroid hydroxylations catalyzed by other P-450 isoenzymes exhibited age and sex dependencies distinct from those of the P-450 2c-mediated activities. Immunochemical analyses confirmed that this sex dependence and developmental induction reflected alterations in P-450 2c polypeptide levels. Attempts to chromatographically detect P-450 2c in either immature male or adult female microsomes were unsuccessful and led to purification of P-450 2d (female), a catalytically distinct and female-specific form. Peptide mapping and immunochemical analyses suggested significant structural homologies between the two sex-specific isoenzymes, P-450 2c and P-450 2d (female). A significant suppression of P-450 2c levels (up to 70-80%) was observed upon administration of several classical P-450 inducers. These studies establish that P-450 2c corresponds to the male-specific and developmentally-induced steroid 16 alpha-hydroxylase of rat liver and suggest that the expression of P-450 2c versus P-450 2d (female) may provide a biochemical basis for the sex differences characteristic of rat liver xenobiotic metabolism.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats

Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats.     

Purification and characterization of two forms of cytochrome P-450 with omega-hydroxylase activities toward prostaglandin A and fatty acids from rabbit liver microsomes

Two forms of cytochrome P-450 (P-450), designated as P-450LPGA omega 1 and P-450LPGA omega 2, have been purified to specific contents of 17.9 and 11.1 nmol P-450/mg protein, respectively, from liver microsomes of rabbits treated with di(2-ethylhexyl)phthalate (DEHP), a peroxisomal proliferator. The purified P-450LPGA omega 1 and P-450LPGA omega 2 were found to have apparent molecular weights of 52,500 and 53,000, respectively. They showed absorption maxima at 451 and 450 nm in the carbon monoxide-difference spectra for their reduced forms, respectively. The two P-450s both efficiently catalyzed the omega-hydroxylation of prostaglandins A1 (PGA1) and A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate. In a reconstituted system, various metal ions such as Na+ and Mg2+ stimulated these reactions. The P-450s exhibited no detectable activity toward several xenobiotics tested. The two P-450s showed different peptide map patterns following limited proteolysis with Staphylococcus aureus V8 protease or papain. The NH2-terminal amino acid sequences (ALNPTRLPGSLSGLLQVAGL and ALSLTRLPGSFSGFLQAxGLLGLLL) of P-450LPGA omega 1 and P-450LPGA omega 2 were identical at 18/20 and 19/24 positions with that of the lung prostaglandin omega-hydroxylase from pregnant rabbits, respectively. An antibody against P-450LPGA omega 2 recognized a 52,000-53,000 molecular weight protein(s) in rabbit liver microsomes. The intensity of the immunoblot was significantly increased in liver microsomes from rabbits treated with DEHP, but not with phenobarbital or 3-methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Inhibition of CCl4 metabolism by oxygen varies between isoenzymes of cytochrome P-450

Oxygen inhibition of CCl4 metabolism by different isoenzymes of cytochrome P-450 was assessed by studying liver microsomes isolated from control rats and rats treated with phenobarbital or isoniazid. Rates of CCl4 metabolism were similar for all microsomes under a nitrogen atmosphere. An air atmosphere inhibited metabolism by microsomes from control rats to 12% of the value under nitrogen and metabolism by microsomes from rats treated with phenobarbital to 5%. It inhibited metabolism by microsomes from rats treated with isoniazid only to 32%. Rats treated with phenobarbital, which increases hepatic cytochrome P-450 content, or isoniazid, which does not increase hepatic cytochrome P-450 content, both metabolized more CCl4 than control rats as indicated by exhalation of greater quantities of CCl4 metabolites and by an increase in CCl4 toxicity. These results indicate that some isoenzymes of cytochrome P-450 are more effective than others in metabolizing CCl4 when oxygen is present.     

Cytochrome P-450C-M/F, a new constitutive form of microsomal cytochrome P-450 in male and female rat liver with estrogen 2- and 16 alpha-hydroxylase activity

A new cytochrome P-450 isozyme, P-450C-M/F, has been purified from untreated rat liver microsomes. The purified preparation was electrophoretically homogeneous and contained 12-15 nmol of P450/mg of protein and had a minimum molecular weight of 48,500. The NH2-terminal amino acid sequence of P-450C-M/F was different from that of other P-450's. Immunoblot analysis of microsomes demonstrated that P-450C-M/F was present in the liver of untreated male as well as female rats. Treatment of rats with phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone did not induce P-450C-M/F. Cytochrome P-450C-M/F exhibited little activities of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation or hydroxylation of arylhydrocarbon, testosterone, androstenedione, and progesterone. In contrast, it was highly active in N-demethylation of ethylmorphine and benzphetamine and in 2- and 16 alpha-hydroxylation of estrogens, particularly that of estradiol. These studies establish that cytochrome P-450C-M/F is constitutively present in both male and female rats and suggest that it may be involved in the oxidative metabolism of estradiol, particularly in the formation of estriol, the uterotropic metabolite of estradiol.     

Interaction of 9-hydroxyellipticine with two forms of partially purified rabbit lung cytochrome P-450. Inhibition of lung microsomal benzo[a]pyrene hydroxylase.

9-Hydroxyellipticine (9-OHE), a potent inhibitor of rat liver monooxygenase activities, binds to the various forms of partially purified lung cytochromes P-450 from untreated and 3-methylcholanthrene (3-MC)-treated rabbits. The spectral data (lambda max: 428 nm (ox.), 447 nm (red.), Ks: 10 microM and 5 muM for cytochrome I and cytochrome II from 3-MC-treated rabbits respectively) resemble those obtained with cytochrome P-450 purified from liver of Aroclor 1254-pretreated rats (lambda max: 428 nm (ox.), 445 nm (red.), Ks: 8 microM). 9-OHE has been shown to inhibit the benzo[a]pyrene hydroxylase activity of rat and rabbit lung microsomes. The inhibitory effect was higher towards the 3-MC-induced lung microsomes than with the control microsomes. However, the lung microsomes, as well as the liver microsomes of rabbits were less sensitive to inhibition by 9-OHE than the corresponding microsomes from rats. These results suggest that rabbit and rat cytochromes P-450 have subtle structural differences.     

Constitutive testosterone 6 beta-hydroxylase in rat liver

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.