Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of delta 4-steroid 5 alpha-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450

Neonatal gonadectomy studies and hormonal replacement regimens were employed to characterize the regulation of delta 4-steroid 5 alpha-reductase, microsomal flavin-containing monooxygenase, and several forms of rat hepatic microsomal cytochrome P-450, including three that are sexually differentiated. Rats of both sexes that had been gonadectomized at birth were either untreated or were administered testosterone propionate or estradiol benzoate neonatally (subcutaneous injection on days 1 and 3 of life), postpubertally (an implant of a hormone-packed capsule at 5 weeks of age), or both neonatally and postpubertally. At the age of 10 weeks, all rats were killed, and several liver microsomal enzymes were assayed using immunochemical and catalytic techniques. Expression in the 10-week-old male and female rats of two male-specific cytochrome P-450 forms, termed P-4502c/UT-A and P-4502a/PCN-E, and their associated respective 16 alpha- and 6 beta-steroid hydroxylase activities could either be imprinted (programmed) by androgen exposure during the early neonatal period or, alternatively, could be stimulated by continuous hormone treatment after the age of 5 weeks. By contrast, hepatic expression of two female-specific enzymes, P-4502d/UT-1 and delta 4-steroid 5 alpha-reductase, was only partially dependent on estradiol; birth-gonadectomized rats expressed as much as 30-50% of the enzyme levels present in untreated adult females. Expression of both female-specific enzymes was fully suppressed upon postpubertal exposure to testosterone. In another study, birth sham-operated female rats were administered testosterone using the same regimens described above for the birth-gonadectomized rats. Although neonatal testosterone treatment alone did not affect the expression in these females of the four sex-specific enzymes examined in this study, it did enhance significantly the masculinization effected by postpubertal androgen exposure. This resulted in expression of the male-specific enzymes P-4502c/UT-A and P-4502a/PCN-E in these females at levels comparable to those found in adult males, while simultaneously suppressing the two female-specific enzymes, P-4502d/UT-I and delta 4-steroid 5 alpha-reductase, by approximately 70-75% to levels characteristic of prepubertal rats of either sex. The levels of another microsomal enzyme, flavin-containing monooxygenase, were also measured and found to be regulated by testosterone, but the ontogenic profiles and the effects of gonadectomy and hormone replacement indicated clear differences in its regulation when compared to the other male-specific enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Hormonal regulation of rat renal cytochrome P450s by androgen and the pituitary.

The hormonal regulation of rat renal cytochrome P450s, P450 4A2 (K-5) and K-2, was investigated. The level of P450 4A2 in male rats was five times that in female rats and accounted for some 90% of total cytochrome P450, measured photometrically. Lauric acid omega- and (omega-1)-hydroxylation activities of renal microsomes of male rats were also higher than those of female rats. The sex differences in lauric acid hydroxylation activity seemed to arise from the differences in P450 4A2 concentrations, according to an immunochemical study. P450 K-2 was a female-dominant form in rat kidneys. The level of P450 K-2 in renal microsomes of male rats was one-tenth that of P450 4A2. Castration of male rats decreased the levels of P450 4A2 and treatment of castrated male rats with testosterone reversed the decrease. The castration of male rats decreased the lauric acid hydroxylation of the renal microsomes to the level of female rats. The administration of testosterone to castrated male rats reversed the decrease. Hypophysectomy of male rats decreased the level of P450 4A2 and the administration of growth hormone reversed the decrease when intermittent injections mimicking the male secretory pattern were given, although continuous administration mimicking the female secretory pattern did not. Castration of male rats did not affect the level of P450 K-2, but testosterone decreased its level. Hypophysectomy of male rats increased the level of P450 K-2 and growth hormone decreased its level in hypophysectomized rats. These results suggested that the expression of P450 4A2 was regulated by androgen or growth hormone and regulation of P450 4A2 was different from that of P450 K-2. To explore the regulation of renal cytochrome P450 further, testosterone was given to control (intact) or hypophysectomized adult female rats. P450 4A2 was induced in the kidneys of both control and hypophysectomized female rats to close to the level of male rats. Thus, P450 4A2 was directly regulated by testosterone as well as growth hormone, and the regulation of the male-dominant form in rat kidneys was different from that of the male-specific form in the rat liver, which is regulated mostly by growth hormone.     

Age- and sex-related expression of cytochromes p450f and P450g in rat liver

We have previously shown that rat hepatic cytochromes P450f, P450g, P450h, and P450i possess a high degree of immunochemical and, presumably, structural relatedness. Polyclonal antibodies directed against cytochromes P450f and P450g were made monospecific by immunoabsorption against the cross-reactive proteins. The specificity of the immunoabsorbed antibodies was established by using Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays (ELISA), and immunoblots. Since factors regulating the expression of cytochromes P450f and P450g are unknown, a competitive ELISA employing the monospecific antibodies was developed to quantitate each of these isozymes in hepatic microsomes from control and treated rats. The results obtained showed that expression of cytochrome P450f is developmentally regulated in both male and female rat liver. Cytochrome P450f levels rise from less than 1% in young animals to approximately 7 and 14% of total cytochrome P450 in adult male and female rats, respectively. Cytochrome P450g is sex-specific since it is expressed only in male rat liver where it also is developmentally regulated. Levels of cytochrome P450g rise from less than 1% in 3-week-old male rats to an average value of 17% of total cytochrome P450 in 6-week-old adult animals. However, there appear to be at least two subpopulations of adult male Long Evans rats, one of which expresses low levels (less than 1%) of cytochrome P450g and the other high levels (greater than or equal to 10%). This expression appears to be independent of serum testosterone levels. Treatment of immature and adult male rats with 20 xenobiotics that are known inducers of certain cytochrome P450 isozymes revealed that cytochromes P450f and P450g are relatively refractory to induction, although Kepone appears to be a weak inducer of cytochrome P450f.     

Perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters sex-dependent expression of hepatic CYP2C11

The cytochrome P450 (CYP) isoform CYP2C11 is specifically expressed in the liver of adult male rats, and 5alpha-reductase is specifically expressed in the liver of the adult female rats. The sexually dimorphic expressions of these hepatic enzymes are regulated by the sex-dependent profiles of the circulating growth hormone (GH). However, it is not well known whether hormonal imprinting or activation factors in the neonatal brain influence the sexually dimorphic expression patterns of hepatic enzymes. We therefore examined the effect of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sex-dependent expressions of hepatic enzymes. Pregnant rats were treated with TCDD at a dose of 0, 200, or 800 ng/kg on gestation day 15, exposing the pups to the chemical. Although the expression of CYP2C11 protein in the livers of male pups on postnatal day (PND) 49 was significantly higher than that of the controls, but the 5alpha-reductase activities in the livers of female pups were not altered by exposure to TCDD. Focusing on perinatal periods, testosterone and estrogen levels significantly increased in the brain of male pups on PND 2. The results suggest that the alteration of testosterone and estrogen levels affect hormonal imprinting in the neonatal brain of male pups, and thus induces a change in the level of male-specific hepatic CYP2C11. We conclude that perinatal exposure to TCDD at low doses may change the sexual differentiation of the neonatal brain in male rats.     

Identification of cytochrome P450a (P450IIA1) as the principal testosterone 7 alpha-hydroxylase in rat liver microsomes and its regulation by thyroid hormones

In the preceding paper, evidence was presented that rat liver microsomes contain two structurally related isozymes of cytochrome P450, namely cytochromes P450a and P450m, that can both catalyze the 7 alpha-hydroxylation of testosterone. The aim of the present study was to determine the extent to which these two P450 isozymes are responsible for the 7 alpha-hydroxylation of testosterone catalyzed by rat liver microsomes. Four monoclonal antibodies against cytochrome P450a, designated A2, A4, A5, and A7, were prepared in BALB/c mice. Monoclonal antibodies A2 (an IgM), A4 (an IgG2b), and A5 (an IgG1) were determined to be distinct immunoglobulins, whereas A7 could not be distinguished from A5. All of the antibodies were highly specific for cytochrome P450a; none cross-reacted with cytochrome P450m or with 10 other P450 isozymes purified from rat liver microsomes. Competition experiments between unlabeled and horseradish peroxidase-conjugated antibodies revealed that each of the monoclonal antibodies recognized the same epitope on cytochrome P450a. None of the monoclonal antibodies bound to denatured cytochrome P450a, suggesting that they each bound to a spatial epitope. A monospecific, polyclonal antibody against cytochrome P450a was also prepared, as described in the preceding paper. The levels of cytochrome P450a in liver microsomes were determined by single radial immunodiffusion, Western immunoblot (with polyclonal antibody), and enzyme-linked immunosorbent assay with monoclonal antibody. The levels of cytochrome P450a declined with age in male but not female rats, and were inducible up to 10-fold by treatment of rats with various xenobiotics. The levels of cytochrome P450a (but not cytochrome P450m) were also elevated (approximately 3-fold) by thyroidectomy of mature male rats. Near normal levels of cytochrome P450a were restored by treatment of athyroid rats with triiodothyronine, whereas treatment with thyroxine was less effective in this regard. These changes in the levels of cytochrome P450a were highly correlated (r = 0.995) with changes in testosterone 7 alpha-hydroxylase activity. None of the monoclonal antibodies inhibited the catalytic activity of cytochrome P450a when reconstituted with NADPH-cytochrome P450 reductase and lipid. In contrast, the polyclonal antibody not only inhibited the catalytic activity of purified cytochrome P450a, but also completely inhibited (greater than 96%) the 7 alpha-hydroxylation of testosterone catalyzed by liver microsomes from immature and mature rats of both sexes and by liver microsomes from male rats treated with a variety of cytochrome P450 inducers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of sex hormones on ethanol-induced gastric haemorrhagic erosions in rats.

The role of sex hormones in the pathogenesis of ethanol-induced gastric erosions was investigated following the recent observation that ethanol generates more severe gastric damage in male rats. Female and male Wistar rats aged 110 +/- 6 days were used. Intact female, ovariectomized female, intact male, orchidectomized male and cyproterone acetate-pretreated (this compound a testosterone antagonist) male rats were investigated. 1 ml of 75% ethanol was used to induce gastric lesions. The extent of the erosions was determined planimetrically 60 min after ethanol administration. The plasma testosterone and 17-beta-oestradiol levels were checked by radioimmunoassay (RIA) in gonadectomized rats. Ethanol generates more severe lesions in male rats. Orchidectomy and cyproterone acetate treatment each reduced the extent of ethanol-induced gastric erosions in male rats. Ovariectomy had no effect in this model. The plasma testosterone and 17-beta-oestradiol levels were significantly reduced after gonadectomy. It is concluded that endogenous testosterone plays an aggressive role in the pathogenesis of ethanol-induced gastric erosions in rats.     

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)     

Does neonatal gonadectomy affect the sexual differentiation of quail?

This experiment was designed to determine the contribution, if any, of posthatching gonadal hormones to sexual differentiation of behavior in Japanese quail (Coturnix coturnix japonica). Males and females were gonadectomized or sham-operated (controls) prior to age 7 days posthatching. At age 4-9 weeks controls were gonadectomized. All birds were then given 2 weeks of testosterone propionate injections and tested for sexual behavior with female partners. Neonatally gonadectomized females exhibited more male-typical copulatory behavior than control females, but this effect was not statistically significant. Neonatal gonadectomy had no effect on males, and neonatally gonadectomized males exhibited significantly more male-typical copulatory behavior than neonatally gonadectomized females. Although the process of sexual differentiation may extend to a minor degree into the posthatching period in females, nonetheless it is largely complete at hatching in this species.     

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.     

Hepatic P450 expression in hypothyroid rats: differential responsiveness of male-specific P450 forms 2a (IIIA2), 2c (IIC11), and RLM2 (IIA2) to thyroid hormone

Studies carried out in hypophysectomized adult rats have demonstrated that both thyroid hormone and GH can suppress hepatic expression of the steroid 6 beta-hydroxylase P450 2a (IIIA2). The present study further characterizes the influence of thyroid hormone on the expression of P450 2a and two other male-specific hepatic P450s, a steroid 2 alpha/16 alpha-hydroxylase, designated P450 2c (IIC11), and a steroid 15 alpha-hydroxylase, designated P450 RLM2 (IIA2). These studies were carried out in rats rendered hypothyroid by treatment with methimazole, which allows for the nonsurgical depletion of circulating T4, and in hypophysectomized rats. Hypothyroidism led to an increase in hepatic P450 2a (IIIA2) protein and mRNA in both male and female rats that was fully reversed by T4 replacement. In contrast, hypothyroidism decreased by 70-80% the expression of P450 2c (IIC11) activity and mRNA, but did not significantly alter the expression of P450 RLM2 (IIA2). The decrease in P450 2c (IIC11) was not reversed by T4 replacement, suggesting that it is a consequence of the loss of plasma GH pulses that occurs secondary to hypothyroidism. In agreement with these findings, T4 given to hypophysectomized rats partially suppressed the expression of P450 2a (IIIA2) mRNA, but not P450 2c (IIC11) or P450 RLM2 (IIA2) mRNA. A more complete suppression of P450 2a (IIIA2) mRNA as well as P450 2c (IIC11) mRNA was achieved when the hypophysectomized rats were treated with T3 at a supraphysiological, receptor-saturating dose. Although GH administered to intact male rats by continuous infusion fully suppressed all three male-specific P450 proteins and their mRNAs, the same treatment given to hypothyroid rats was only partially suppressive in the case of P450 2a (IIIA2) and P450 RLM2 (IIA2), unless combined with T4. In the case of P450 2c (IIC11), substantial suppression of the residual P450 present in hypothyroid rats was achieved by treatment with GH alone, despite persistent thyroid hormone deficiency. These studies demonstrate that while thyroid hormone is a negative regulator of P450 2a (IIIA2) expression and is required for the full suppression of that P450 and P450 RLM2 (IIA2) by the continuous plasma GH profiles associated with adult female rats, the suppression of P450 2c (IIC11) by continuous plasma GH is largely independent of the presence of thyroid hormone.     

Developmental regulation of hepatic testosterone hydroxylases: simultaneous activation and repression of constitutively expressed cytochromes P450 in senescent rats

The aging process is generally associated with marked decreases in the activities of numerous enzymes as well as lower levels of sex hormones such as testosterone. We therefore examined testosterone metabolism in liver microsomes from individual 3- and 24-month-old male rats. Although the old rats exhibited lower 16 alpha-, 6 beta-, and 2 alpha-hydroxylase activities than the young rats, the old rats had a higher 7 alpha-hydroxylase activity. Immunoquantitation of P450a, a known 7 alpha-hydroxylase, showed that the level of this protein was elevated in the old rats, and was correlated with 7 alpha-hydroxylase activity. The mRNA for P450a was measured with a cDNA probe and its level was fivefold higher in the old rats, whereas levels of mRNA coding for a 6 beta-hydroxylase P450 were markedly decreased. The increased expression of cytochrome P450a demonstrates that the observed common decrease in cytochrome P450-catalyzed activities with senescence is not a universal phenomenon. Thus, constitutive expression of specific cytochrome P450 genes is repressed or activated in senescent rats.     

Gender dependent effects of testosterone and 17β-estradiol on bone growth and modelling in young mice

This study examined the effects of estrogen (17β-estradiol) and testosterone on the growth of long bones in male and female mice, with and without gonadectomy. Weight and nose-to-tail length were determined at 3 weeks of age at time of gonadectomy, 7 days later at the onset of hormone therapy, and throughout the treatment period. Gonadectomized mice exhibited an initial weight gain during the pretreatment period but length was unaffected. Hormone treatment altered weight gain in surgical and intact animals in a gender- and hormone-dependent manner. Estradiol enhanced weight gain in intact mice, but inhibited weight gain in ovariectomized mice. Lower doses of estradiol increased weight gain in orchiectomized mice at early time points. Testosterone increased weight in intact females and males, but not in gonadectomized mice. Estradiol increased nose-to-tail length in intact females at early time points, but inhibited length in ovariectomized females at later times, and it decreased length in intact males. Testosterone increased length in normal females and normal males. Serum Ca was unaffected by ovariectomy, but orchiectomy resulted in decreased levels. Estradiol reduced serum Ca in gonadectomized animals; serum Ca was increased by estradiol treatment in intact females. Changes in tibial bone weight, ash weight and mineral composition, and relative sizes of epiphyseal and metaphyseal bone were gender-, gonadectomy- and hormone-specific. Bone weight was greater in ovariectomized mice. Ash weight per bone was comparable, but there was an increase in Ca and P content with ovariectomy. Estradiol increased bone weight, ash content, and bone Ca and P in ovariectomized and intact females. Orchiectomy alone did not alter bone weight, ash content, or Ca and P, but orchiectomized mice were sensitive to estradiol; all parameters were increased in the orchiectomized animals treated with estradiol. Analysis of the ash content and Ca and P per mg bone, rather than per bone, demonstrated estradiol and testosterone alter net bone formation, but not the amount of mineral per unit bone. Ovariectomy increased hypertrophic cartilage. While estradiol did not alter tibial area in ovariectomized mice, it caused an increase in intact females. The total amount of growth plate cartilage in ovariectomized animals was decreased by estradiol to levels typical of intact animals due to a greater decrease in the hypertrophic cartilage in the ovariectomized mice, as well as a greater increase in metaphyseal bone area. Testosterone had no effect on these parameters in the females. Orchiectomy decreased the amount of growth plate cartilage, but increased the hypertrophic zone. Estradiol increased growth plate cartilage in intact male mice, but decreased it in orchiectomized mice. This difference was also seen in the hypertrophic zone. Total growth plate cartilage and hypertrophic cartilage were increased by testosterone in intact males, whereas metaphyseal and epiphyseal bone area were decreased. The results show for the first time that there is a gender-specific response in both male and female mice to both estradiol and testosterone, whether or not the animals have been gonadectomized. For many parameters, orchiectomized mice behave like females in response to both sex steroids, indicating that the male gonad is needed for mouse bone to exhibit the male phenotypic response to estradiol and testosterone.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

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.     

Regulation of the expression of the sex-specific isoforms of cytochrome P-450 in rat liver

The hepatic metabolism of steroid hormones and of xenobiotics frequently depends on the expression of the sex-specific isoforms of cytochrome P-450 and on differences in sex hormones. Following biochemical, immunological and molecular biological investigations, it was shown that in adult rat liver there exist at least four male-specific and one female-specific isoforms of cytochrome P-450. The designation of these sex-specific genes is IIC11, IIIA2, IIC13 and IIA2 in males, and IIC12 in females. The irreversible programming of the expression of these isoforms of cytochrome P-450 in adulthood occurs during the perinatal period of life, and is named enzyme imprinting. One of the main factors that regulates the expression of the sex-specific isoforms of cytochrome P-450 is the level of androgens in the blood. Castration of adult rats decreased the level of the male isoforms of cytochrome P-450 and the activity of the monooxygenase enzyme system that remained higher than in intact females. The mechanism of enzyme imprinting can be explained as follows: neonatal androgens program the secretion of hypothalamic hormones, somatostatin and growth-hormone-releasing factor. These factors determine the type of growth hormone secretion in adult rats, and this controls the type of sex-specific isoforms of cytochrome P-450 expressed in adulthood. Metabolic regulation similar to that outlined above was shown to occur for several metabolism-dependent chemical carcinogens. Such a pathway may explain the different sensitivity displayed by male and female rats to treatment with these carcinogenic agents. One possible way of modulating the expression of some isoforms of cytochrome P-450 in adult rats is by treating neonates with specific xenobiotics that change the constitutive expression of neonatal androgens. It appears that this enzyme imprinting plays an important role in determining the individual sensitivity to the carcinogenic effects of chemicals.     

Age-dependent exression 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 2α-, 2β-, 15α-, 16α-, and 16β-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 7α-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 form) 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 6β-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 of male-specific cytochrome P450 2c and its mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl in rat liver is not causally related to changes in serum testosterone

Rat cytochrome P450 2c (P450 gene IIC11) is a constitutive, male-specific hepatic enzyme which is suppressed greater than 90% by treatment with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) [H. N. Yeowell et al. (1987) Mol. Pharmacol. 32, 340-347]. HCB also decreases serum testosterone levels in adult male rats (greater than 98% loss). The present study assesses whether the suppression of P450 2c by HCB is a direct result of its effects on serum testosterone levels. Further, the site along the hypothalamic-pituitary-testicular axis at which HCB acts to depress testosterone secretion was examined. Administration of the synthetic androgen methyltrienolone to HCB-treated rats failed to prevent the suppression of P450 2c mRNA and its associated microsomal steroid 16 alpha-hydroxylase activity under conditions where it effectively reversed the large decrease in P450 2c mRNA and steroid 16 alpha-hydroxylase activity produced by castration. Hepatic steroid 6 beta-hydroxylase activity, which is catalyzed primarily by P450 2a (P450 gene IIIA2), was also suppressed by HCB and was not protected by methyltrienolone. Administration of either human chorionic gonadotropin, an analog of pituitary-derived luteinizing hormone, or the hypothalamic luteinizing hormone releasing hormone elevated serum testosterone levels to a much smaller extent in HCB-treated rats than in control rats. These results indicate that the effects of HCB on serum testosterone levels reflect its effects on testicular function rather than the pituitary or hypothalamus. However, the present study demonstrates that the consequential reduction in serum testosterone levels in HCB-treated rats is not causally related to the reduction in hepatic P450 2c levels. Thus, HCB must also act on some other regulatory mechanism involved in the expression of this protein.