Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.     

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.     

Characterization of testosterone 16 alpha-hydroxylase (I-P-450(16) alpha) induced by phenobarbital in mice

Cytochrome P-450 (I-P-450(16) alpha), which is associated with phenobarbital-induced testosterone 16 alpha-hydroxylation activity, was purified from livers of phenobarbital-treated female 129/J mice on the basis of the specific hydroxylation activity in fractions eluted from columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified I-P-450(16) alpha fraction was 12.4 nmol/mg of protein, and it had an apparent molecular weight of 54K. The specific activity of reconstituted testosterone 16 alpha-hydroxylation activity with the purified I-P-450(16) alpha fraction was 6-8 nmol min-1 (nmol of cytochrome P-450)-1. Rabbit antibody raised against the purified I-P-450(16) alpha fraction inhibited nearly 100% of the 16 alpha-hydroxylation activity in liver microsomes of phenobarbital-treated female 129/J mice but did not affect hepatic microsomal 16 alpha-hydroxylation activity of untreated male and female 129/J mice at all. In hepatic microsomes of phenobarbital-treated male 129/J mice, 70% of the 16 alpha-hydroxylation activity, at most, was catalyzed by I-P-450(16) alpha, and the residual 30% of the activity was catalyzed by C-P-450(16) alpha. The increase of I-P-450(16) alpha by phenobarbital was due to de novo synthesis of I-P-450(16) alpha, and this induction was not sexually regulated in 129/J mice. Anti-C-P-450(16) alpha [Harada, N., & Negishi, M. (1984) J. Biol. Chem. 259, 12285-12290] did not inhibit the 16 alpha-hydroxylation catalyzed by I-P-450(16) alpha; thus, I-P-450(16) alpha and C-P-450(16) alpha are immunochemically distinct isozymes of testosterone 16 alpha-hydroxylase.     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

Testosterone metabolism by microsomal cytochrome P-450 in liver of rats treated with some inducers

1. The stereoselective hydroxylation of testosterone by microsomal cytochrome P-450 and the changes in level of components participated in the microsomal electron transport system were observed in the microsomes induced unique P-450 isozymes. 2. Flavone- and hesperetin-inducible P-450 catalyzed the hydroxylation of testosterone more effectively than other chemicals-inducible ones. 3. The P-450 in all the microsomal preparations tested most rapidly oxidized testosterone to 6 beta-monohydroxy form. 4. Particularly, MC- and BNF-inducible P-450 showed high stereoselectivity on C6-position of testosterone, and PB-, flavone- and hesperetin-inducible one showed that on C2-position of this compound, respectively. 5. This specificity of two flavonoid-inducible P-450 for the formation of 2 alpha- and 2 beta-epimer of monohydroxytestosterone was opposite to each other. 6. The content of P-450 and the activity of NADPH-cytochrome P-450 reductase were high in PB-, MC- and BNF-microsomes, whereas NADH-cytochrome b5 reductase activity was high in two flavonoid-microsomes and the content of cytochrome b5 was not changed except the PB-treated rats. 7. It is suggested that the increasing activities of testosterone hydroxylases in flavonoid-microsomes seems to be closely related to NADH-cytochrome b5 reductase.     

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.     

Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms

The "major" phenobarbital (PB)-induced cytochrome P-450 species present in livers of male Sprague-Dawley rats was resolved into two catalytically active heme-protein fractions on diethylaminoethyl cellulose. The two species, P-450 PB-4 (Mr = 49,000) and P-450 PB-5 (Mr = 51,000), were purified to homogeneity, and their chromatographic, spectral, catalytic, and structural properties were compared. P-450 BP-5 eluted earlier on hydroxylapatite and exhibited a more significant cholate-induced Type I spectral shift than P-450 BP-4. Very similar substrate specificity profiles were evident when the two isozymes were reconstituted with lipid, cytochrome P-450 reductase, and cytochrome b5 for oxidative metabolism of several xenobiotics, although P-450 PB-4 exhibited a higher specific catalytic activity (greater than or equal to 5-fold) with all substrates tested. Marked differences were also observed in the sensitivities of both isozymes to several P-450 inhibitors. In addition, P-450 PB-4 was greater than or equal to 10-fold more susceptible than P-450 PB-5 to suicide inactivation by two allyl-containing compounds, allylisopropylacetamide and secobarbital, providing a possible explanation of the previously observed partial inactivation by such compounds of phenobarbital-induced P-450 activity in liver microsomes. One-dimensional peptide maps of the two isoenzymes were highly similar. Antibody raised against purified Long Evans rat liver P-450b (Thomas, P. E., Korzeniowski, D., Ryan, D., and Levin, W. (1979) Arch. Biochem. Biophys. 192, 524-532) cross-reacted with P-450 PB-4 and P-450 PB-5. NH2-terminal sequence analysis demonstrated that the first 31 residues of both PB-4 and PB-5 were identical. These sequences indicated that a highly hydrophobic terminal segment, observed previously for other P-450s as well, is followed by a cluster of basic residues, suggesting that the NH2-terminal portion of these P-450s might be involved in membrane anchoring. Although it is unclear whether P-450 PB-4 and P-450 PB-5 are separate gene products or are related by post-translational modifications, this present demonstration of closely related isozymic forms suggests the possible added complexity of microheterogeneity for this family of microsomal monooxygenases.     

Functional characterization of two cytochrome P-450s within the mouse, male-specific steroid 16 alpha-hydroxylase gene family: expression in mammalian cells and chimeric proteins

Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.     

25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.     

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

Iodosylbenzene derivatives as oxygen donors in cytochrome P-450 catalyzed steroid hydroxylations.

The mechanism of cytochrome P-450 catalyzed steroid hydroxylations in rat liver microsomes has been investigated by employing derivatives of iodosylbenzene as oxygen donors. The model steroid substrate androstenedione which was hydroxylated in positions 7 alpha, 6 beta, and 16 alpha was used in reactions supported by NADPH, iodosylbenzene, and iodosylbenzene derivatives. Evidence for cytochrome P-450 involvement in iodosylbenzene-sustained androstenedione hydroxylation included inhibition by substrates and modifiers of cytochrome P-450. The most efficient oxygen donors were (diacetoxyiodo)-2-nitrobenzene greater than (diacetoxyiodo)-2-chlorobenzene greater than 2-nitroiodosylbenzene greater than (dinitratoiodo)-2-nitrobenzene greater than (diacetoxyiodo)benzene greater than (diacetoxyiodo)-2-methoxybenzene greater than 4-(diacetoxyiodo)toluene greater than iodosylbenzene. The capacity of the oxidation agents to serve as oxygen donors in cytochrome P-450 dependent steroid hydroxylation is probably dependent upon several factors such as the tendency of iodosyl compounds to associate, which decreases coordination with the heme iron, the presence of bulky substituents in the 2 position (decreases association), and the presence of electron-withdrawing substituents (tends to decrease coordination with the heme iron). The rates of 7 alpha, 6 beta, and i6 alpha hydroxylation of androstenedione catalyzed by (diacetoxyiodo)-2-nitrobenzene were 108-, 130-, and 167-fold higher, respectively, than the rates of the NADPH-supported reactions. These results strongly suggest that the rate-limiting step in NADPH-sustained cytochrome P-450 catalyzed reactions is the rate of reduction of cytochrome P-450.     

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.     

Mouse liver testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha). Purification, regioselectivity, stereospecificity, and immunochemical characterization

Microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) was purified from the livers of male 129/J mice based on enzyme activity in the eluates from columns of DEAE Bio-Gel A, hydroxylapatite, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified P-450(16) alpha fraction was 9.5 nmol/mg of protein. The specific testosterone 16 alpha-hydroxylation activity of the purified P-450(16) alpha fraction was 80 nmol/min/nmol of cytochrome P-450 or 764 nmol/min/mg of protein, and these values were about 40- and 400-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(16) alpha showed extremely high regioselectivity and stereospecificity for testosterone hydroxylation; more than 90% of the testosterone metabolites formed by the purified P-450(16) alpha fraction was 16 alpha-hydroxytestosterone. The purified anti-P-450(16) alpha antibody exhibited absolute specificity for inhibition of testosterone 16 alpha-hydroxytestosterone was inhibited by the anti-P-450(16) alpha. Anti-P-450(16) alpha inhibited the 16 alpha-hydroxylation activity of intact microsomes prepared from livers of male or female 129/J mice more than 90%, indicating that P-450(16) alpha is the major cytochrome P-450 isozyme catalyzing 16 alpha-hydroxylation activity of testosterone in these microsomal preparations. The purified P-450(16) alpha fraction also possessed high benzphetamine N-demethylation activity relative to the rates found with other xenobiotic substrates tested in this report.     

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes

Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes.     

Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase

We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).     

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)     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

A sex-specific form of cytochrome P-450 catalyzing propoxycoumarin O-depropylation and its identity with testosterone 6 beta-hydroxylase in untreated rat livers: reconstitution of the activity with microsomal lipids

Characteristics of a typical male-dominant reaction, dealkylation of n-propoxycoumarin, in rat livers were studied in relation to microsomal testosterone 6 beta-hydroxylase. The depropylation was more than 10-fold higher in the liver of male than female adult rats, but the sex-related difference was eliminated by neonatal castration. Hypophysectomy of adult male rats, which decreased the rates of male-specific P-450-male-dependent reactions, increased the depropylation of propoxycoumarin, while the rate was decreased by either intermittent injection or continuous infusion of human growth hormone to hypophysectomized rats. With regard to age-related difference, microsomal depropylation was detectable at neonate and reached a maximal level at 14 to 20 d of age, but was abruptly diminished only in female rats at puberty. These changes are in good agreement with those of testosterone 6 beta-hydroxylation and the content of a male-specific P-450(6)beta-1/PB-1. In reconstituted systems using extracted microsomal lipids, P-450(6)beta-1/PB-1 and P-450-male catalyzed the depropylation of propoxycoumarin. However, the microsomal depropylation was inhibited by antibodies which recognize P-450(6)beta-1/PB-1, but not P-450-male. These results indicate that microsomal depropylation of propoxycoumarin is catalyzed mainly by a male-specific P-450(6)beta-1/PB-1 in livers of untreated rats.