Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats

The functional importance of sex steroid hormones (testosterone and estrogens), derived from extragonadal tissues, has recently gained significant appreciation. Circulating dehydroepiandrosterone (DHEA) is peripherally taken up and converted to testosterone by 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, and testosterone in turn is irreversibly converted to estrogens by aromatase cytochrome P-450 (P450arom). Although sex steroid hormones have been implicated in skeletal muscle regulation and adaptation, it is unclear whether skeletal muscles have a local steroidogenic enzymatic machinery capable of metabolizing circulating DHEA. Thus, here, we investigate whether the three key steroidogenic enzymes (3beta-HSD, 17beta-HSD, and P450arom) are present in the skeletal muscle and are capable of generating sex steroid hormones. Consistent with our hypothesis, the present study demonstrates mRNA and protein expression of these enzymes in the skeletal muscle cells of rats both in vivo and in culture (in vitro). Importantly, we also show an intracellular formation of testosterone and estradiol from DHEA or testosterone in cultured muscle cells in a dose-dependent manner. These findings are novel and important in that they provide the first evidence showing that skeletal muscles are capable of locally synthesizing sex steroid hormones from circulating DHEA or testosterone.     

Testosterone and DHEA activate the glucose metabolism-related signaling pathway in skeletal muscle

Circulating dehydroepiandrosterone (DHEA) is converted to testosterone or estrogen in the target tissues. Recently, we demonstrated that skeletal muscles are capable of locally synthesizing circulating DHEA to testosterone and estrogen. Furthermore, testosterone is converted to 5alpha-dihydrotestosterone (DHT) by 5alpha-reductase and exerts biophysiological actions through binding to androgen receptors. However, it remains unclear whether skeletal muscle can synthesize DHT from testosterone and/or DHEA and whether these hormones affect glucose metabolism-related signaling pathway in skeletal muscles. We hypothesized that locally synthesized DHT from testosterone and/or DHEA activates glucose transporter-4 (GLUT-4)-regulating pathway in skeletal muscles. The aim of the present study was to clarify whether DHT is synthesized from testosterone and/or DHEA in cultured skeletal muscle cells and whether these hormones affect the GLUT-4-related signaling pathway in skeletal muscles. In the present study, the expression of 5alpha-reductase mRNA was detected in rat cultured skeletal muscle cells, and the addition of testosterone or DHEA increased intramuscular DHT concentrations. Addition of testosterone or DHEA increased GLUT-4 protein expression and its translocation. Furthermore, Akt and protein kinase C-zeta/lambda (PKC-zeta/lambda) phosphorylations, which are critical in GLUT-4-regulated signaling pathways, were enhanced by testosterone or DHEA addition. Testosterone- and DHEA-induced increases in both GLUT-4 expression and Akt and PKC-zeta/lambda phosphorylations were blocked by a DHT inhibitor. Finally, the activities of phosphofructokinase and hexokinase, main glycolytic enzymes, were enhanced by testosterone or DHEA addition. These findings suggest that skeletal muscle is capable of synthesizing DHT from testosterone, and that DHT activates the glucose metabolism-related signaling pathway in skeletal muscle cells.     

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.     

Neurosteroid biosynthesis in the quail brain: a review

The brain traditionally has been considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have shown that the brain itself also has the capability of forming steroids de novo, the so-called "neurosteroids". De novo neurosteroidogenesis in the brain from cholesterol is a conserved property of vertebrates. Our studies using the quail, as an excellent animal model, have demonstrated that the avian brain possesses cytochrome P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/c17,20-lyase (P450(17alpha,lyase)), 17beta-HSD, etc., and produces pregnenolone, progesterone, 3beta, 5beta-tetrahydroprogesterone, androstenedione, testosterone and estradiol from cholesterol. However, the biosynthetic pathway of neurosteroids in the avian brain from cholesterol may be still incomplete, because we recently found that the quail brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid. This paper summarize the advances made in our understanding of biosynthesis of neurosteroids in the avian brain.     

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.     

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism

Introduction

Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.

Methods

We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.

Results

Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻⁷ M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.

Conclusions

We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Intracrinology and the skin

The skin, the largest organ in the human body, is composed of a series of androgen-sensitive components that all express the steroidogenic enzymes required to transform dehydroepiandrosterone (DHEA) into dihydrotestosterone (DHT). In fact, in post-menopausal women, all sex steroids made in the skin are from adrenal steroid precursors, especially DHEA. Secretion of this precursor steroid by the adrenals decreases progressively from the age of 30 years to less than 50% of its maximal value at the age of 60 years. DHEA applied topically or by the oral route stimulates sebaceous gland activity, the changes observed being completely blocked in the rat by a pure antiandrogen while a pure antiestrogen has no significant effect, thus indicating a predominant or almost exclusive androgenic effect. In human skin, the enzyme that transforms DHEA into androstenedione is type 1 3beta-hydroxysteroid dehydrogenase (type 1 3beta-HSD) as revealed by RNase protection and immunocytochemistry. The conversion of androstenedione into testosterone is then catalyzed in the human skin by type 5 17beta-HSD. All the epidermal cells and cells of the sebaceous glands are labelled by type 5 17beta-HSD. This enzyme is also present at a high level in the hair follicles. Type 1 is the 5alpha-reductase isoform responsible in human skin for the conversion of testosterone into DHT. In the vagina, on the other hand, DHEA exerts mainly an estrogenic effect, this effect having been demonstrated in the rat as well as in post-menopausal women. On the other hand, in experimental animals as well as in post-menopausal women, DHEA, at physiological doses, does not affect the endometrial epithelium, thus indicating the absence of DHEA-converting enzymes in this tissue, and avoiding the need for progestins when DHEA is used as hormone replacement therapy.     

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 alpha-hydroxylase cytochrome P-450 in bovine ovarian follicles

Follicles were collected from cows and processed for electron microscopy and for immunofluorescent staining at the light microscope level. Key regulatory steroidogenic enzymes cholesterol side-chain-cleavage cytochrome P-450 (P-450scc) and 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) were immunolocalized using specific IgG fractions raised against these enzymes. In larger follicles in which the theca interna had differentiated, positive staining for cytochromes P-450scc and P-450(17) alpha was observed in the cells of the theca interna. Electron microscopic examination showed that these cells were rich in endoplasmic reticulum, mainly rough, and had moderate numbers of mitochondria with tubular and lamellar cristae. Positive staining was also present in the theca of follicles undergoing atresia. Positive staining for cytochrome P-450(17) alpha was not observed in the membrana granulosa but cytochrome P-450scc was present in the membrana granulosa in some follicles, particularly in the larger antral follicles. By contrast, positive staining for both enzymes was not observed in stroma, surface epithelium or in small preantral follicles in which the theca interna had not differentiated. These results indicate good agreement between the type(s) of steroidogenic enzyme(s) present in tissues and the type(s) of steroid hormone(s) produced. It is concluded that regulation of steroid hormone production involves, at least in part, regulation of the levels of steroidogenic enzymes.     

Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells

Testosterone biosynthesis in Leydig cells is dependent on two cytochrome P450 enzymes, cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha]. The expression of these two enzymes is differentially regulated by LH acting via its second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), and by specific steroid hormones. P450scc is constitutively expressed in normal mouse Leydig cells and in MA-10 tumor Leydig cells. Chronic cAMP stimulation increases the steady state levels of P450scc mRNA and de novo P450scc protein synthesis. In contrast, cAMP is obligatory for de novo synthesis of P450(17 alpha) in normal mouse Leydig cells; P450(17 alpha) synthesis ceases in the absence of luteinizing hormone or cAMP. MA-10 tumor Leydig cells do not express P450(17 alpha) even after treatment with cAMP. The amount of P450(17 alpha) in Leydig cells is negatively regulated by testosterone acting by two distinct mechanisms. At low concentrations, testosterone acts via the androgen receptor to repress cAMP-induced synthesis of P450(17 alpha), whereas at high concentrations this steroid increases the rate of degradation of the enzyme by an oxygen-mediated mechanism. Both constitutive and cAMP-induced synthesis of P450scc protein and steady state levels of mRNA are modulated by glucocorticoids. In normal mouse Leydig cells, glucocorticoids repress P450scc synthesis and steady state levels of P450scc mRNA, whereas glucocorticoids stimulate P450scc synthesis and levels of P450scc mRNA in the tumor Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of testicular cytochrome P-450-dependent steroid biosynthesis by cis-platinum. Reversal by human chorionic gonadotropin

The treatment of rats with cis-platinum for 7 days caused a profound, and seemingly selective, decrease (70-80%) in the microsomal cytochrome P-450 levels in the testis. This decrease was accompanied by marked reductions (70-80%) in steroid 17 alpha-hydroxylase activity and in plasma testosterone concentration. The treatment of rats with human chorionic gonadotropin partially restored the cytochrome P-450 concentration and 17 alpha-hydroxylase activity and permitted the plasma testosterone level to approach control values. The effect of cis-platinum on the testicular cytochrome P-450 appeared unrelated to deficiencies in heme metabolic processes, in so far that neither was the activity of delta-aminolevulinate synthetase decreased, nor was that of heme oxygenase increased. These enzymes are rate-limiting in heme biosynthesis and degradation pathways, respectively. Also, the activities of uroporphyrinogen I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the concentration of total porphyrins in the testis remained unchanged. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation did not reveal a diminished level of apocytochrome; however, in this preparation, heme could not be detected in molecular weight regions corresponding to cytochrome P-450. The microsomal cytochrome b5 and the mitochondrial heme concentrations were not decreased in cis-platinum-treated rats. It is suggested that the mechanism of depletive action of cis-platinum on microsomal cytochrome P-450 involves an impairment of the effective assembly of heme and apoprotein moieties. It is further suggested that the anterior pituitary hormones control the factor(s) involved in this assembly, a process which is interrupted by cis-platinum.     

Relation of the pituitary gland to gonadal hormone effects on the adrenocortical 11beta-hydroxylase system in rats.

Studies were conducted to further examine the mechanisms responsible for gonadal hormone effects on the rat adrenocortical 11beta-hydroxylase system. Despite higher concentrations of cytochrome P-450 and larger 11-deoxycorticosterone (DOC)-induced difference spectra in adrenal mitochondria from females than males, no sex difference in 11beta-hydroxylase activity was observed. The pregnenolone-induced difference spectrum, indicative of cholesterol binding to cytochrome P-450, also was similar in males and females. Testosterone administration to castrated males lowered both 11beta-hydroxylase activity and mitochondrial cytochrome P-450 content. Estradiol produced the opposite effects in castrated females. However, when given to ACTH-replaced hypophysectomized rats, neither testosterone nor estradiol affected cytochrome P-450 levels or the rate of 11beta-hydroxylation. These observations, taken with the known effects of estradiol and testosterone on ACTH secretion in rats and the effects of ACTH on 11beta-hydroxylation, indicate that gonadal hormone effects on the 11beta-hydroxylase system are mediated by ACTH.     

Steroid interactions with cytochrome P-450 from testis microsomes

The cytochrome P-450 of gonadal microsomes is an integral component of the steroid converting enzymes, 17 alpha-hydroxylase and 17,20-lyase. Interaction of the steroid substrates with this cytochrome results in a shift in the Soret band as measured by difference spectroscopy. In these studies it is shown that in contrast to placental microsomal cytochrome P-450 which binds C19 steroids, testis microsomal cytochrome P-450 primarily binds C21 steroids. However, addition of a 17 alpha- methyl, 17 beta-acetate or a 17 beta-benzoate group to testosterone permits interaction. The addition of hydroxyl or methyl groups to other positions does not affect binding. The presence of multiple oxygen functions on C21 steroids, as in cortisol and corticosterone, precludes interaction. At least one oxygen function seems necessary for binding as 5 alpha- and 5 beta-pregnane do not bind whereas 20-deoxypregnenolone (5-pregnen-3 beta-ol) does bind. These findings indicate that factors in addition to hydrophobic interactions dictate the binding of steroid substrates to testis microsomal cytochrome P-450.     

Steroid metabolism and profile of steroidogenic gene expression in Episkin: high similarity with human epidermis

The skin is a well-recognized site of steroid formation and metabolism. Episkin is a cultured human epidermis. In this report, we investigate whether Episkin possesses a steroidogenic machinery able to metabolize adrenal steroid precursors into active steroids. Episkin was incubated with [14C]-dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) and their metabolites were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS). The results show that the major product of DHEA metabolism in Episkin is DHEA sulfate (DHEAS) (88% of the metabolites) while the other metabolites are 7alpha-OH-DHEA (8.2%), 4-dione (1.3%), 5-androstenediol (1.3%), dihydrotestosterone (DHT) (1.4%) and androsterone (ADT) (2.3%). When 4-dione is used as substrate, much higher levels of C19-steroids are produced with ADT representing 77% of the metabolites. These data indicate that 5alpha-reductase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 3alpha-hydroxysteroid dehdyrogenase (3alpha-HSD) activities are present at moderate levels in Episkin, while 3beta-HSD activity is low and represents a rate-limiting step in the conversion of DHEA into C19-steroids. Using realtime PCR, we have measured the level of mRNAs encoding the steroidogenic enzymes in Episkin. A good agreement is found between the mRNAs expression in Episkin and the metabolic profile. High expression levels of steroid sulfotransferase SULT2B1B and type 3 3alpha-HSD (AKR1C2) correspond to the high levels of DHEA sulfate (DHEAS) and ADT formed from DHEA and 4-dione, respectively. 3beta-HSD is almost undetectable while the other enzymes such as type 1 5alpha-reductase, types 2, 4, 5, 7, 8, and 10 17beta-HSD and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) (AKR1C1) are highly expressed. Except for UGT-glucuronosyl transferase, similar mRNA expression profiles between Episkin and human epidermis are observed.     

Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein (StAR)

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.     

Feminization of rat hepatic P-450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid-metabolizing enzymes

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-450 activity was studied in adult male rat liver using P-450 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-450 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-450 forms designated P-450 2c (P-450 gene IIC11), P-450 2a (gene IIIA2), and P-450 RLM2 were decreased by 70-90% after 7-14 days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-450 3 (gene IIA1) and P-450j (gene IIE1) were elevated approximately 2-4-fold. The female-specific microsomal enzyme androstenedione 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-450 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-450 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-450 enzyme expression in adult male rats.