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.     

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.     

Neonatal testosterone imprinting of hepatic microsomal drug metabolism and a male-specific form of cytochrome P-450 in the rat

The effects of neonatal castration and treatment with testosterone on sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, were studied. Neonatal castration of male rats resulted in a change in the population of forms of cytochrome P-450. Castration 1 day after birth abolished the synthesis of P-450-male and stimulated the synthesis of P-450-female. The decrease in the amount of P-450-male as well as the activities of drug metabolizing enzymes was partially reversed by administration of testosterone after castration.     

Suppression of levels of phenobarbital-inducible rat liver cytochrome P-450 by pituitary hormone

The effect of pituitary factor on the constitutive and inducible levels of hepatic phenobarbital (PB)-inducible major cytochrome P-450, P-450b and P-450e, in male and female rat livers was studied by immunoblot analyses. Although only trace amounts (approximately 4 pmol/mg protein) of P-450b and P-450e were detected in untreated adult rats, hypophysectomy increased the contents of P-450b and P-450e 58- and 14-fold, respectively, in male rats and 118- and 30-fold, respectively, in female rats. The increases were also observed in treatment with dexamethasone, which suppressed the pituitary function. Treatment with PB increased more effectively the hepatic contents of P-450b and P-450e, but their contents were still 4-fold higher in the male than the female. Treatment of hypophysectomized female rats with PB increased the contents of P-450b and P-450e 4-fold higher than the contents in PB-treated nonhypophysectomized female rats. Consequently, the sex-related difference in their contents was reduced less than 1.4-fold in the hypophysectomized rats treated with PB. Similar results were also obtained from the quantitation of microsomal O-pentylresorufin O-depentylation and testosterone 16 beta-hydroxylation. Either intermittent injection or continuous infusion of human growth hormone, but not of ovine prolactin, into hypophysectomized male and female rats decreased the contents of both cytochromes. These results indicate that growth hormone acts as a repressive factor for the constitutive and inducible levels of P-450b and P-450e in a manner different from the regulation of P-450-male and P-450-female.     

Sex difference of cytochrome P-450 in the rat: Purification,characterization, and quantitation of constitutive forms of cytochrome P-450 from liver microsomes of male and female rats

One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti-P-450-male and anti-P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female 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.     

Studies on in vitro antipyrine metabolism by 13C,15N double labeled method

The capacities of forms of cytochrome P-450 to oxidize antipyrine were compared. An isotope dilution gas chromatography/mass spectrometry/selected ion monitoring assay was developed to quantify the three main metabolites, norantipyrine, 3-hydroxymethylantipyrine and 4-hydroxyantipyrine. 13C,15N-Double labeled antipyrine was used as a substrate and the metabolites were analyzed as their trimethylsilyl derivatives. Among forms of cytochrome P-450 examined, a male-specific form of P-450, namely P-450-male, showed higher activity to form all the three metabolites. The other forms were responsible only for the formation of norantipyrine and 4-hydroxyantipyrine. The activities of liver microsomes from untreated male and female rats and rats treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyl were expressed dependent on the activities of forms of cytochrome P-450 examined.     

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.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Age-related changes in the iron spin state of testosterone-binding rat liver microsomal cytochromes P-450

The aging process is accompanied by decreased drug metabolism as well as lower levels of sex hormones such as testosterone. We examined the age-dependence of liver microsomal cytochromes P-450 from young (3 months) and old (24 months) male rats by absorption and ESR spectroscopy. Spectral perturbations by testosterone were used to identify testosterone-specific P-450 forms. Absorption difference spectra indicated that testosterone induced a greater conversion of P-450 to the high spin form in young rats than in old rats. ESR signals corresponding to total low spin P-450 were of higher intensity in the young rats and were increased by testosterone. Testosterone also interconverted one low spin P-450 species to another. These results demonstrate age-related differences in the types and amounts of testosterone-specific P-450's in rats.     

Expression of cytochrome P-450 isozymes in the liver of hypophysectomized rats. Evidence for different regulation mechanisms concerning P450IIB and P450IIIA subfamilies

1. Monooxygenase activities have been examined in rat liver to determine the effects of castration and hypophysectomy on cytochrome P-450 species. In adult males, hypophysectomy caused a decrease of total P-450 concentration, aniline hydroxylase, benzopyrene hydroxylase, benzphetamine demethylase, testosterone hydroxylase and imipramine hydroxylase and demethylase activities. The treatment of hypophysectomized animals with human growth hormone or testosterone did not restore the full activity. 2. When probed with antibodies, microsomes from hypophysectomized males and females exhibited an intense reaction with a polyclonal anti-(phenobarbital-induced P-450) which was not observed with a monoclonal antibody of anti-(phenobarbital-induced P-450). 3. These microsomal preparations also reacted with an antibody raised against a developmentally regulated P-450. No sex difference could be detected with this antibody. Furthermore, administration of human growth hormone to hypophysectomized males prevented this immunoreaction. 4. Total RNA has been prepared from the same liver; when probed with cDNAs, no changes occurred in the content in P-450 b/e, PB 24 (a constitutive member of the phenobarbital subfamily) and phenobarbital-inducible mRNA for UDP-glucuronosyltransferase. 5. In contrast, P-450 mRNA induced by pregnenolone 16 alpha-carbonitrile was modulated by hormonal manipulations: lower in females and castrated males than in intact males, increased in both sexes after hypophysectomy. Treatment of hypophysectomized males with human growth hormone abolished this rise in pregnenolone-16 alpha-carbonitrile-induced P-450 mRNA accumulation. Data collected in this study support the assumption that hypophysectomy acts differently on the regulation of various P-450 isozymes and that this regulation clearly does not involve the phenobarbital subfamily of P-450s.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

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)     

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)     

Age-dependent expression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Suppression in the expression of a male-specific cytochrome P450, P450-male: difference in the effect of chemical inducers on P450-male mRNA and protein in rat livers

Hypophysectomy of male adult rats caused a 70% decrease in the hepatic level of mRNA hybridized to two specific oligonucleotide probes for the sequence of coding and 3'-noncoding regions of P450(M-1) (H. Yoshioka et al., (1987) J. Biol. Chem. 262, 1706-1711), which corresponds to P450-male. Treatment of hypophysectomized male and female rats with subcutaneous injection of human growth hormone twice a day for 7 days increased the mRNA to a level similar to that of normal male rats. In contrast, the mRNA was decreased by treatment with continuous infusion. These results correlated well with those on the amounts of P450-male protein, indicating that growth hormone regulates the hepatic level of P450-male protein mainly by acting at the pretranslational step. Treatment of adult male rats with phenobarbital (PB), dexamethasone (Dex), or 3-methylcholanthrene (MC) decreased the content of P450-male protein by 68, 36, and 46%, respectively. The content of P450-male protein was also decreased to 65% in Dex-treated hypophysectomized male rats, but was not changed by treatment of hypophysectomized male rats with PB or MC, suggesting that PB and MC decrease P450-male protein through a pituitary growth hormone-mediated process. However, the level of mRNA hybridizable to the P450-male oligonucleotide probe was not decreased, but rather it increased in PB- or Dex-treated hypophysectomized male rats. A similar inconsistent change in protein and mRNA was also observed in PB-treated normal rats. These results indicate that PB and Dex have an additional effect of increasing the hepatic level of the specific mRNA of P450-male/(M-1) or a closely related form. Noncoordinate changes in the level of P450-male protein and mRNA also suggest that the hepatic level of P450-male protein is regulated by plural mechanisms: pretranslational and translational regulation in which pituitary growth hormone and/or other endocrine factors are involved.     

Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.     

Role of growth hormone in modulating the constitutive and phenobarbital-induced levels of two P-450(6)beta (testosterone 6 beta-hydroxylase) mRNAs in rat livers

The role of growth hormone in the expression of two forms of hepatic cytochrome P-450(P-450), P-450(6)beta-1(6 beta-3), and P-450(6)beta-4, was investigated using RNA blots. The level of P-450(6)beta-1(6 beta-3) mRNA was twenty times higher than that of P-450(6) beta-4 mRNAs in untreated male rat livers. The levels of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were increased two fold and three fold, respectively, by hypophysectomy of adult male rats. By intermittent injection of human growth hormone (hGH) into hypophysectomized male rats, both mRNAs were decreased to the level of normal rats, and almost disappeared after continuous infusion of hGH. In female rats, these two mRNAs were not detected, but were increased remarkably by hypophysectomy. The increases in these mRNAs were almost abolished after continuous infusion of hGH in hypophysectomized female rats. The effect of hGH on PB-mediated induction of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs was also examined. The PB-mediated increases in P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were higher in hypophysectomized male rats (2.5-fold and 10.9-fold, respectively) than in normal male rats (1.5-fold and 5.2-fold, respectively). Thus, the levels of P-450(6)beta-1(6-beta-3) and P-450(6)beta-4 mRNAs were 4.1-fold and 7.3-fold, respectively, higher in PB-induced hypophysectomized rats than in normal male rats. Concerning the postnatal developmental profiles, P-450(6)beta-1(6 beta-3) mRNA was detectable at neonate and reached a maximal level at around 17 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of multiple forms of cytochrome P-450 in the occurrence of sex-related differences of drug metabolism in the rat

Multiple forms of cytochrome P-450 in liver microsomes of untreated male and female rats could be divided into several fractions by the use of ω-amino-n-octyl Seph. 4B and DE-52 columns. Male cytochrome P-450 fractions (I-b - I-e) differed from female fractions (I-b - I-e) with respect to absorption peaks in carbon monoxide difference spectra and 7-prop-oxycoumarin O-depropylation activities. Although male and female I-a fractions showed quite similar protein bands on SDS-polyacrylamide gel electrophoresis, some protein bands in other male fractions (I-b - I-e) were absent in corresponding female fractions. Immunochemical examinations using immunoglobulin G raised to cytochrome P-450 purified from untreated male rats also showed that liver microsomes from male and female rats contain different forms of cytochrome P-450. Based on these results, we propose that sex-related differences of drug metabolizing activities in liver microsomes are caused by multiple forms of cytochrome P-450.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)