Effect of growth hormone and ectopic transplantation of pituitary gland on sex-specific forms of cytochrome P-450 and testosterone and drug oxidations in rat liver

The effects of growth hormone and ectopic transplantation of pituitary gland on the amounts of sex-specific cytochrome P-450, P-450-male and P-450-female, and the activities of testosterone and drug hydroxylases in male rat liver microsomes were studied. Hypophysectomy decreased the content of P-450-male, without changing the total cytochrome P-450 level. The continuous infusion of growth hormone into hypophysectomized rats and the transplantation of pituitary gland under the renal capsule caused a further decrease in P-450-male content and an expression of P-450-female. In contrast, the intermittent injection of growth hormone into hypophysectomized rats increased P-450-male content to the level seen in intact male rats. The activities of testosterone 2 alpha- and 16 alpha-, but not 6 beta-, 7 alpha-, or 15 alpha-hydroxylase, were changed in association with the level of P-450-male by these treatments. Anti-P-450-male immunoglobulin G inhibited testosterone 2 alpha- and 16 alpha-hydroxylations, but not 6 beta-, 7 alpha- or 15 alpha-hydroxylation. These results indicate that growth hormone regulates the expression of P-450-male responsible for testosterone 2 alpha- and 16 alpha-hydroxylations. The metabolism of 7-propoxycoumarin, benzo(a)pyrene and aminopyrine also changed with the content of P-450-male, although the correlation was less than that observed with testosterone 2 alpha- and 16 alpha-hydroxylation.     

Tissue-specific regulation of cytochrome P-450 dependent testosterone 15 alpha-hydroxylase

Testosterone 15 alpha-hydroxylase activity in kidney microsomes is higher in male mice than in female mice, while in the liver the activity is higher in females than in males. Cytochrome P-450 15 alpha, a specific form of cytochrome P-450 having testosterone 15 alpha-hydroxylase activity, accounts for virtually all of the testosterone 15 alpha-hydroxylase activity in female kidney microsomes, while other isozymes of testosterone 15 alpha-hydroxylase are present in male kidney microsomes. In female kidney, P-450 15 alpha expression is regulated by a single sex-dependent locus, called Rsh for "regulation of steroid hydroxylase." The higher level of P-450 15 alpha expression in male kidneys is dependent on androgens. Of all mice strains, 129/J seems to be the least dependent on androgens to maintain a high expression of P-450 15 alpha in male kidneys. Castration of male mice lowers kidney levels of P-450 15 alpha but in the liver, P-450 15 alpha levels rise after castration. This reciprocal regulation of P-450 15 alpha genes in liver and kidney was investigated by isolating cDNA clones encoding P-450 15 alpha from liver and kidney cDNA libraries. Two highly homologous cDNA clones encoding P-450 15 alpha designated type I and type II were identified, and levels of type I and type II mRNA in liver and kidney were determined by differential restriction mapping of double-stranded cDNA prepared from mRNA from these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Female-predominant expression of testosterone 16 alpha-hydroxylase ("I"-P-450(16)alpha) and its repression in strain 129/J

Using specific testosterone 16 alpha-hydroxylase activity as the basis for selection of fractions during purification, the cytochrome P-450 ("I"-P-450(16)alpha) has been isolated from livers of phenobarbital-treated 129/J female mice [K. Devore, N. Harada, and M. Negishi (1985) Biochemistry, 24, 5632-5637]. An antibody elicited in rabbits to "I"-P-450(16)alpha was used to determine the amount of hepatic microsomal 16 alpha-hydroxylase activity due to "I"-P-450(16)alpha in untreated females and males of the two mouse strains, 129/J and BALB/cJ. The activities inhibited were 0.03 and 0.3 nmol/min/mg protein in the 129/J and BALB/cJ females, respectively. No significant level of "I"-P-450(16)alpha-dependent activity was detected in the microsomes from males of either mouse strain. Immunoblotting of microsomal proteins with the antibody to "I"-P-450(16)alpha revealed approximately a 10-fold greater amount of a 54-kDa protein in the microsomes from BALB/cJ than from 129/J females (0.03 and 0.26 pmol/micrograms protein, respectively). A cDNA clone (R17) for phenobarbital-inducible rat cytochrome P-450 selected "I"-P-450(16)alpha mRNA of mice, indicating a high degree of homology between the mRNAs of mouse "I"-P-450(16)alpha and phenobarbital-inducible rat cytochrome P-450s. Northern and dot hybridization of total mouse liver poly(A)+ RNA with the R17 cDNA probe indicated that the specific content of the hybridizable mRNA was more than 10 times higher in BALB/cJ females than in males, and that the mRNA level in female 129/J mice was very similar to that of 129/J and BALB/cJ males. The repression of "I"-P-450(16)alpha in 129/J females was inherited as an autosomal recessive trait in 129/J and BALB/cJ pairs as indicated by the levels of mRNA in female F1 offspring and the "I"-P-450(16)alpha-dependent hydroxylase activity. Female and male mice of eight more inbred strains (AKR/J, DBA/2J, C57BL/6J, C3H/HeJ, NZB/J, A/J, CBA/CaJ, and P/J) were tested for levels of mRNA. The results showed that the levels of mRNA were always 5- to 10-fold greater in the females than in the corresponding males, although there was some variation in the mRNA content in the males from the different strains. 129/J females appear to be a genetic variant where the female-predominant expression of the mRNA is repressed.     

Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16 alpha-hydroxylase cytochrome P-450 apoprotein in the rat

The hormonal regulation of the sexually differentiated cytochrome P-450 isozyme which catalyzes 16 alpha-hydroxylation of testosterone and 4-androstene-3,17-dione in male rat liver (P-450(16) alpha) was investigated. Estradiol valerate injection of male rats caused a decrease in P-450(16) alpha levels to almost the female level, while methyltrienolone injection had the reverse effect in female animals. Hypophysectomy abolished the sex difference in P-450(16) alpha levels. Human growth hormone infusion into male rats, mimicking the female pattern of growth hormone secretion, caused a feminization of P-450(16) alpha levels. The same effect was also seen in hypophysectomized rats of both sexes. In contrast, a different administration schedule involving 12 h injections of human growth hormone, mimicking the male pattern of growth hormone secretion, caused a masculinization of P-450(16) alpha levels in hypophysectomized rats, at a daily dose which causes feminization when given by infusion. Thus, the level of expression of P-450(16) alpha in the liver is dependent on the temporal pattern of blood growth hormone levels. While infusion of rat growth hormone into male rats also feminized the P-450(16) alpha levels, infusion of ovine prolactin had no effect. Ontogenic studies showed that the developmental pattern of P-450(16) alpha expression in the liver coincided with the known pattern of development of the sexual differentiation of hepatic steroid 16 alpha-hydroxylase activity and of the diurnal pattern of growth hormone secretion.     

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.     

Genetic regulation of testosterone 15 alpha-hydroxylase (cytochrome P-450(15)alpha) in renal microsomes of female mice

P-450(15)alpha is a form of cytochrome P-450 purified from liver microsomes of female 129/J mice that is specific for oxidation of testosterone to its 15 alpha-hydroxylated product. Testosterone 15 alpha-hydroxylase activity that was inhibited by anti-P-450(15)alpha antibody was approximately 50 times higher in renal microsomes from 129/J than in BALB/cJ females. Western blots of renal microsomes using anti-P-450(15)alpha antibody showed the presence of immunoreactive protein with a molecular weight identical with that of hepatic P-450(15)alpha in 129/J but not in BALB/cJ female mice. To investigate the genetic basis for the strain differences in this activity, the distribution of P-450(15)alpha-dependent testosterone 15 alpha-hydroxylase activity in renal microsomes from individual females of 129/J and BALB/cJ, of F1 offspring of these strains, and of F1 back-crosses to the progenitor strains were determined. The results were consistent with a sex-related autosomal dominant regulation of the higher activity in 129/J females by a single locus, designated Rsh (regulation of steroid hydroxylase). The amounts of immunochemically cross-reactive P-450(15)alpha protein were linearly correlated with testosterone 15 alpha-hydroxylase activities in renal microsomes from Rsh heterozygotes and homozygotes. At least twice as much mRNA, which hybridized with the cDNA clone for hepatic P-450(15)alpha, was detected in 129/J and 129CF1/J compared to BALB/cJ female kidneys. The evidence suggests a pretranslational regulation of the P-450(15)alpha isozyme in the female mouse kidney by the Rsh locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Characterization and regulation of sex-specific mouse steroid hydroxylase genes

We characterized the genes of the male-specific mouse steroid 16 alpha-hydroxylase (C-P-45016 alpha) and the female-specific mouse steroid 15 alpha-hydroxylase (P-45015 alpha) within two distinct gene families. In spite of the high structural identities within each family, the expression of the hydroxylase genes is uniquely regulated. Moreover, the other family members encode the P-450s which are structurally very similar to the hydroxylases but are not able to catalyze steroid hydroxylase activities. For example, only a single amino acid substitution creates steroid 15 alpha-hydroxylase activity in another family-member P-450coh, which catalyzes coumarin 7-hydroxylase but little steroid hydroxylase activity. It appears, therefore, that the mouse P-450 gene families evolved through gene duplication and selective mutation to create new P-450s structurally as well as to establish novel regulatory elements for the gene expressions.     

Effects of cytochrome P450-inducing agents on the monooxygenation of testosterone in long-term cultures of hepatocytes from male and female rats

Hepatocytes from male or female rats were cultured for up to 2 weeks in a modified Waymouth medium supplemented with 0.1 or 1.0 microM dexamethasone, 10 nM insulin, and 0.1 nM glucagon with or without addition of phenobarbital, methylcholanthrene, or isoniazid. The activities of testosterone hydroxylases were measured in the intact cell monolayer and in the corresponding microsomal fraction. Aniline hydroxylase was measured in cell homogenates. In the presence of 0.1 microM dexamethasone the testosterone hydroxylase activities varied differently in hepatocytes from male and female rats during the culture period. The activities of 6 beta- and 15 alpha-hydroxylases increased in female and were unchanged in male hepatocytes, while 16 alpha-hydroxylase activity increased in female and decreased in male, and 2 alpha- and 7 alpha-hydroxylase activities were unchanged in both male and female hepatocytes during the culture period. Increasing the dexamethasone concentration to 1.0 microM caused an increase in 6 beta- and 15 alpha-hydroxylase activities in cultures of hepatocytes from both sexes, whereas an increase of 2 alpha- and a decrease of 7 alpha- and 17-hydroxylase activities were found only in cultures of hepatocytes from female rats. Addition of phenobarbital caused an increase in the activity of 7 alpha-hydroxylase in both male and female hepatocytes, while the effect on the other hydroxylases differed with the sex. In hepatocytes from male rats phenobarbital addition decreased the activities of 2 alpha- and 16 alpha-hydroxylases, while these were increased or stable after addition of phenobarbital to hepatocytes from female rats. The activity of aniline hydroxylase was increased at Day 1 and declined afterward. The results demonstrate that the activities of different steroid hydroxylases are inducible and can be directly measured in monolayers of hepatocytes from rats.     

Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16 alpha-hydroxylation in Saccharomyces cerevisiae: vitamin D3 25-hydroxylase and testosterone 16 alpha-hydroxylase are distinct forms of cytochrome P-450

Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.     

Adult male-specific and neonatally programmed rat hepatic P-450 forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone for expression

Rat hepatic cytochrome P-450 form RLM2 is a testosterone 15 alpha-hydroxylase reported to be male-specific on the basis of purification studies (Jansson, I., Mole, J., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 7084-7093). The sex dependence, developmental regulation, xenobiotic induction, and hormonal control of P-450 RLM2 expression were studied using P-450 form-specific immunochemical and catalytic assays. Polyclonal antibodies raised to rat hepatic P-450 3 (P-450 gene IIA1) were found to cross-react strongly with P-450 RLM2, but not with 10 other rat P-450 forms, suggesting that P-450 3 and P-450 RLM2 are highly conserved in primary structure. Western blotting of liver microsomes under conditions where P-450s 3 and RLM2 are resolved electrophoretically revealed that P-450 RLM2 is markedly induced at puberty in male rats, with no protein detected (less than or equal to 5% of adult male levels) in adult females or immature animals of either sex. A similar developmental dependence was observed for hepatic microsomal testosterone 15 alpha-hydroxylase activity, which was found to be catalyzed primarily by P-450 RLM2. P-450 RLM2 was resistant to induction by several xenobiotics and in the case of phenobarbital and beta-naphthoflavone, was suppressed by 50-60%. Studies on the steroid hormonal regulation of P-450 RLM2 revealed that its adult male-specific expression is imprinted (programmed) in response to neonatal testosterone exposure. Ovariectomy studies demonstrated that suppression by estrogen does not contribute significantly to the absence of P-450 RLM2 in adult female rats. Although the male-specific developmental induction of P-450 RLM2 in response to neonatal testosterone is strikingly similar to that of P-450 2c (testosterone 2 alpha/16 alpha-hydroxylase; gene IIC11), P-450 RLM2 expression is not dependent on the pulsatile pituitary growth hormone secretion required for P-450 2c synthesis. Rather, hypophysectomy of adult male rats increased P-450 RLM2 and its associated testosterone 15 alpha-hydroxylase activity by 50-100%.(ABSTRACT TRUNCATED AT 400 WORDS)     

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).     

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.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver

The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories.     

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.     

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.     

Reciprocal regulation of sex-dependent expression of testosterone 15 alpha-hydroxylase (P-450(15 alpha)) in liver and kidney of male mice by androgen. Evidence for a single gene

Testosterone 15 alpha-hydroxylase activities and its mRNA levels are higher in kidneys than in livers from male 129/J mice. Castration of 129/J male mice resulted in repression of P-450(15 alpha) in kidney, but increased it in liver. Two types of cDNA (p15 alpha-29 (Type I) and -15 (Type II)) encoding P-450(15 alpha) were previously cloned from 129/J female livers (Burkhart, B.A., Harada, N., and Negishi, M. (1985) J. Biol. Chem. 260, 15357-15361). With the use of p15 alpha-29 as a probe, Type I and II P-450(15 alpha) cDNAs were isolated from libraries of 129/J kidney poly(A)+ RNA. The nucleotide sequences of the cDNAs showed that Type I and II cDNAs from liver and kidney were identical and shared 98.3% similarity. The deduced amino acid sequence from a full-length Type I cDNA indicated that Type I P-450(15 alpha) consists of 494 amino acids with a molecular weight of 56,594. Nine amino acid substitutions were found in the Type II clone in 432 amino acids overlapping Type I. Type I cDNA clones accounted for approximately 90% P-450(15 alpha) clones isolated from a male kidney library, whereas approximately 90% of cDNA clones in a female kidney library were Type II. Liver cDNA libraries from males and females contained similar ratios of Type I and II. Effects of castration on Type I and II mRNAs were determined by Southern hybridization of a 32P-labeled ClaI-ClaI fragment from p15 alpha-29 to cDNAs synthesized from kidney and liver poly(A)+ RNAs prepared from sham-operated, castrated 129/J mice. The double-stranded cDNAs were digested with ClaI and PstI prior to gel electrophoresis to create the diagnostic restriction fragments specific for Type I or II. Castration resulted in decreased levels of Type I mRNA in male kidney. In male liver, only Type I mRNA rose significantly in response to castration. Testosterone administration returned the Type I mRNA to normal levels in castrated mice. It therefore appears that the high levels of P-450(15 alpha) in male kidney were due to androgen-dependent induction of Type I mRNA. Both Types I and II were repressed in male liver, which results in decreased levels of P-450(15 alpha). Androgen was responsible for the repression and expression of Type I in liver and kidney, but not Type II.