Abiraterone Acetate for Cushing Syndrome: Study in a Canine Primary Adrenocortical Cell Culture Model.

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer but could also be used to treat patients with Cushing syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM to 10 µM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50, 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In summary, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model.

TSPYL Family Regulates CYP17A1 and CYP3A4 Expression: Potential Mechanism Contributing to Abiraterone Response in Metastatic Castration-Resistant Prostate Cancer.

The testis-specific Y-encoded-like protein (TSPYL) gene family includes TSPYL1 to TSPYL6. We previously reported that TSPYL5 regulates cytochrome P450 (CYP) 19A1 expression. Here we show that TSPYLs, especially TSPYL 1, 2, and 4, can regulate the expression of many CYP genes, including CYP17A1, a key enzyme in androgen biosynthesis, and CYP3A4, an enzyme that catalyzes the metabolism of abiraterone, a CYP17 inhibitor. Furthermore, a common TSPYL1 single nucleotide polymorphism (SNP), rs3828743 (G/A) (Pro62Ser), abolishes TSPYL1's ability to suppress CYP3A4 expression, resulting in reduced abiraterone concentrations and increased cell proliferation. Data from a prospective clinical trial of 87 metastatic castration-resistant prostate cancer patients treated with abiraterone acetate/prednisone showed that the variant SNP genotype (A) was significantly associated with worse response and progression-free survival. In summary, TSPYL genes are novel CYP gene transcription regulators, and genetic alteration within these genes significantly influences response to drug therapy through transcriptional regulation of CYP450 genes.

Phase I and II therapies targeting the androgen receptor for the treatment of castration resistant prostate cancer.

INTRODUCTION: Prostate cancer is the most common cancer in elderly males. Regardless of the initial hormonal treatment in metastatic disease, a significant proportion of patients develop castration resistant prostate cancer (CRPC). A better understanding of the molecular mechanisms behind castration resistance has led to the approval of oral medications such as abiraterone acetate and enzalutamide. Relevant research is accelerated with numerous agents being tested for the management of CRPC. AREAS COVERED: The authors present Phase I and II studies targeting the androgen receptor for the treatment of CRPC. Three groups of agents are identified according to the mechanism of action. These include the CYP-17 modulators (Orteronel, Galeterone, VT-464 and CFG-920), novel antiandrogens (Apatorsen, ARN-509, ODM-201, EZN-4176, AZD-3514) and bipolar androgen therapy. EXPERT OPINION: Further understanding of the mechanisms leading to castration resistance in prostate cancer can reveal potential targets for the development of novel anti-cancer agents. Except for the development of novel antiandrogens and CYP-17 modulators, bipolar androgen therapy is an interesting therapeutic approach. The combinations of the novel agents tested in Phase I and II studies with established agents is another field of interest. The real challenge is to distinguish a novel anti-cancer agent with acceptable tolerability and the best outcome.

Steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation.

OBJECTIVE: To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation. METHODS: Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal) for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip(®) Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip(®) Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip) software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry. RESULTS: We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group. CONCLUSION: Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis.

Effects of monocrotophos pesticide on steroidogenesis and transcription of steroidogenic enzymes in rainbow trout RTG-2 cells involving the protein kinase A signal pathway.

Monocrotophos (MCP) pesticide, listed as a UNEP Prior Informed Consent chemical, has been proved to exert toxic effects on the reproductive system of teleost fishes by changing the balance of sex steroid hormones. To investigate the effects of MCP on steroidogenesis in vitro, the rainbow trout (Oncorhynchus mykiss) gonadal cell line RTG-2 was exposed to different MCP concentrations for 48 h. The levels of 17 ß-estradiol (E(2)) and testosterone in the medium were measured by radioimmunoassay and the expression of steroidogenic acute regulatory protein and cytochrome P450 enzymes CYP11A1, CYP17, and CYP19A was detected by quantitative real-time PCR. The results showed that 1.0 and 10.0 µg/L MCP pesticide induced E(2) levels and promoted steroidogenic enzyme expression. The possible mechanisms of MCP steroidogenic activity were investigated using inhibitors of protein kinase A (PKA) and protein kinase C. The PKA inhibitor H-89 abrogated the 10.0 µg/L MCP-induced transcriptional up-regulation of steroidogenic enzymes, suggesting an involvement of PKA-dependent mechanism in the disruption of steroidogenesis by the MCP pesticide in rainbow trout RTG-2 cells.

Targeting the androgen receptor.

Androgen receptor (AR)-mediated signaling is critical to the growth and survival of prostate cancer. Although medical castration and antiandrogen therapy can decrease AR activity and lower PSA, castration resistance eventually develops. Recent work exploring the molecular structure and evolution of AR in response to hormonal therapies has revealed novel mechanisms of progression of castration-resistant prostate cancer and yielded new targets for drug development. This review focuses on understanding the mechanisms of persistent AR signaling in the castrate environment, and highlights new therapies either currently available or in clinical trials, including androgen synthesis inhibitors and novel direct AR inhibitors.

Management of docetaxel failures in metastatic castrate-resistant prostate cancer.

The treatment of metastatic castration-resistant prostate cancer has evolved since the approval of docetaxel-based therapy. Since docetaxel approval, three new agents have gained approval for this indication: sipuleucel-T, cabazitaxel, and abiraterone. Recent Phase III trials have also demonstrated survival benefits for MDV-3100 and radium-223 though regulatory approval ispending. Practicing physicians face the challenge of determining the optimal sequencing of these new agents. This dilemma is particularly relevant to the post-docetaxel setting, in which the indication for several of these agents overlaps. This article details the efficacy and safety of these agents to provide a framework for their clinical use.

Chemopreventive actions by enterolactone and 13 VIOXX-related lactone derivatives in H295R human adrenocortical carcinoma cells.

Cytochrome P450c17 (CYP17) has been linked to various hormone-related diseases, including breast cancer, thus being a potential target for cancer chemoprevention. We studied the naturally occurring phytochemical enterolactone (ENL) and 13 VIOXX-related lactone derivatives (CRI-1 to CRI-13) for their effects on CYP17 activity and expression and on cell cycle status in the human H295R adrenocorticocarcinoma cell line. Of the tested compounds, only CRI-3, -7, -10 and -12 showed to be inhibitors of CYP17 activity in H295R cells. This inhibition was not due to decreased mRNA expression, but was apparently caused by post-translational modification of the CYP17 enzyme. The MAPK kinase (MEK) inhibitor PD98059 induced CYP17 activity by 24%, while co-incubation of the CRI-s with PD98059, reduced CYP17 activity even further than the reduction caused by the CRI-s alone. In addition, CRI-3, -7, -10 and -12 arrested the cell cycle in the G(2)/M phase. The structure-activity similarities of the CRI-s with known micro-tubule binding agents strongly suggest that cell cycle arrest is a result of interaction with tubulin. We conclude that the proposed cancer chemopreventive actions of ENL are not mediated through interaction with CYP17 or cell cycle status. Of the VIOXX-related lactone derivatives, CRI-7 could prove useful in the prevention of hormone-dependent cancers, such as breast cancer, since in vitro it shows low cytotoxicity, it is a potent inhibitor of CYP17 activity and strong inducer of cell cycle arrest.

Species-specific regulation of PXR/CAR/ER-target genes in the mouse and rat liver elicited by o, p'-DDT.

BACKGROUND: Dichlorodiphenyltrichloroethane (DDT) is a persistent estrogenic organochlorine pesticide that is a rodent hepatic tumor promoter, with inconclusive carcinogenicity in humans. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated activity, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in tumor promotion in the rat liver. To further characterize species-specific hepatic responses, gene expression analysis, with complementary histopathology and tissue level analyses were investigated in immature, ovariectomized C57BL/6 mice treated with 300 mg/kg o, p'-DDT, and compared to Sprague-Dawley rat data. RESULTS: Rats and mice exhibited negligible histopathology with rapid o, p'-DDT metabolism. Gene expression profiles were also similar, exhibiting PXR/CAR regulation with the characteristic induction of Cyp2b10 and Cyp3a11. However, PXR-specific target genes such as Apoa4 or Insig2 exhibited more pronounced induction compared to CAR-specific genes in the mouse. In addition, mouse Car mRNA levels decreased, possibly contributing to the preferential activation of mouse PXR. ER-regulated genes Cyp17a1 and Cyp7b1 were also induced, suggesting o, p'-DDT also elicits ER-mediated gene expression in the mouse, while ER-mediated effects were negligible in the rat, possibly due to the inhibitory effects of CAR on ER activities. In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor. Furthermore, elevated blood DHEA-S levels at 12 h after treatment in the mouse may also contribute to the endocrine-related effects of o, p'-DDT. CONCLUSION: Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

Targeting CYP17: established and novel approaches in prostate cancer.

There is a growing body of evidence that although medical or surgical castration blocks the generation of gonadal testosterone in prostate cancer, androgens originating from other sources may continue to drive androgen receptor (AR) signaling. Recent studies have demonstrated high intratumoral levels of androgens and continued AR signaling in castration-resistant prostate cancer (CRPC), suggesting that androgens may also be synthesized de novo. Inhibiting the systemic biosynthesis of androgens in CRPC by targeting CYP17 may thus represent a rational therapeutic approach since this enzyme catalyses two key steroid reactions involving 17alpha-hydroxylase and C(17,20)-lyase in the androgen biosynthesis pathway. This review will discuss the rationale for and implications of targeting CYP17 in CRPC and focus on established and novel CYP17 inhibitors, including ketoconazole, abiraterone acetate, and VN/124-1, which are agents currently at different stages of development.

Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds.

The H295R cell bioassay was used to evaluate the potential endocrine disrupting effects of 18 of the most commonly used pharmaceuticals in the United States. Exposures for 48 h with single pharmaceuticals and binary mixtures were conducted; the expression of five steroidogenic genes, 3betaHSD2, CYP11beta1, CYP11beta2, CYP17 and CYP19, was quantified by Q-RT-PCR. Production of the steroid hormones estradiol (E2), testosterone (T) and progesterone (P) was also evaluated. Antibiotics were shown to modulate gene expression and hormone production. Amoxicillin up-regulated the expression of CYP11beta2 and CYP19 by more than 2-fold and induced estradiol production up to almost 3-fold. Erythromycin significantly increased CYP11beta2 expression and the production of P and E2 by 3.5- and 2.4-fold, respectively, while production of T was significantly decreased. The beta-blocker salbutamol caused the greatest induction of CYP17, more than 13-fold, and significantly decreased E2 production. The binary mixture of cyproterone and salbutamol significantly down-regulated expression of CYP19, while a mixture of ethynylestradiol and trenbolone, increased E2 production 3.7-fold. Estradiol production was significantly affected by changes in concentrations of trenbolone, cyproterone, and ethynylestradiol. Exposures with individual pharmaceuticals showed the possible secondary effects that drugs may exert on steroid production. Results from binary mixture exposures suggested the possible type of interactions that may occur between drugs and the joint effects product of such interactions. Dose-response results indicated that although two chemicals may share a common mechanism of action the concentration effects observed may be significantly different.

Fetal pituitary gonadotropin as an initial target of dioxin in its impairment of cholesterol transportation and steroidogenesis in rats.

Reproductive and developmental disorders are the most sensitive toxic effects caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is thought to produce many, if not all, of these toxic effects by impairing steroidogenesis and/or steroid action during the prenatal or early postnatal stages. However, the mechanism of the antisex steroid effect of TCDD is not well understood. This study revealed that steroidogenic acute-regulatory protein (StAR), a key transporter of cholesterol for steroidogenesis, in the testes of fetal rats are down-regulated by maternal exposure to TCDD. It was also shown that many mRNAs of steroidogenetic enzymes, including cytochromes P450 11A1, 17, and 11B1 and 3beta-hydroxysteroid dehydrogenase, are reduced in fetuses of TCDD-treated dams in a testis-specific manner. The same was also observed for the expression of estrogen-alpha receptors and androgen receptors. Whereas StAR expression was not affected by TCDD in cultured fetal testis, the fetal serum content of LH, a pituitary regulator of StAR, was significantly reduced by TCDD. In agreement with this, pituitary expression of LHbeta subunit mRNA in fetuses was reduced by maternal exposure to TCDD, whereas the alpha-subunit remained unchanged. The reduction in LHbeta is suggested to occur by a mechanism different from the reduction in the GnRH level. Direct supply of exogenous gonadotropin to TCDD-exposed fetuses completely abolished the reduction of StAR expression. Taken together, these results demonstrate that TCDD impairs steroidogenesis in the fetus by targeting pituitary gonadotropins.

Differential steroidogenic gene expression in the fetal adrenal gland versus the testis and rapid and dynamic response of the fetal testis to di(n-butyl) phthalate.

The phthalate ester di(n-butyl) phthalate (DBP) causes feminization of male rats upon in utero exposure by repressing expression of genes required for testicular steroidogenesis. Previous work in our laboratory has shown that repression of gene expression and steroidogenesis in the fetal testis is apparent within a few hours of DBP exposure. The purpose of this study was to determine the precise timing of DBP-associated gene expression changes in the fetal testis using transcriptional profiling and to determine whether DBP exerts similar effects on steroidogenesis in the fetal adrenal. A DBP time-course experiment showed that testicular steroidogenesis was decreased within 1 h of DBP exposure and that this decrease preceded the repressed transcription of Star (steroidogenic acute regulatory protein); Scarb1 (scavenger receptor class B, member 1; also know as Sr-b1); Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1; also known as P450SCC); and Cyp17a1 (cytochrome P450 family 17, subfamily a, polypeptide 1; also known as Cyp17). Gene expression profiling demonstrated rapid (within 1 to 3 h) and transient induction of immediate early genes in the fetal testis after administration of DBP to the pregnant dam. There was a statistically insignificant decrease in corticosterone production by the fetal adrenal after in utero exposure to DBP from Gestation Day 12 to Gestation Day 19. The extent of steroidogenesis diminution was much less in the adrenal than in the testis (approximately 45% decrease in the adrenal versus 87% decrease in the testis) and expression of genes required for steroidogenesis in the adrenal was unaffected by DBP. Together, these studies demonstrate that DBP initiates a rapid and dynamic change in gene expression in the fetal testis that likely plays a role in the reduction in steroidogenesis that is unique to the fetal testis relative to the steroidogenically active fetal adrenal.

Effects of anticonvulsants on human p450c17 (17alpha-hydroxylase/17,20 lyase) and 3beta-hydroxysteroid dehydrogenase type 2.

PURPOSE: Women with epilepsy apparently have a higher incidence of polycystic ovary syndrome (PCOS) than do women without epilepsy. Whether the underlying disease or the antiepileptic drug (AED) treatment is responsible for this increased risk is unknown, although clinical reports implicate valproic acid (VPA) as a potential cause. The steroidogenic enzymes 3beta HSDII (3beta-hydroxysteroid dehydrogenase) and P450c17 (17alpha-hydroxylase/17,20 lyase) are essential for C19 steroid biosynthesis, which is enhanced during adrenarche and in PCOS. METHODS: To determine whether the AEDs VPA, carbamazepine (CBZ), topiramate (TPM), or lamotrigine (LYG) directly affect the activities of human 3beta HSDII and P450c17, we added them to yeast expressing human P450c17 or 3beta HSDII and assayed enzymatic activities in the microsomal fraction. RESULTS: Concentrations of VPA < or = 10 mM had no effect on activities of P450c17; however, VPA inhibited 3beta HSDII activity starting at 0.3 mM (reference serum unbound concentration, 0.035-0.1 mM) with an IC50 of 10.1 mM. CBZ, TPM, and LTG did not influence 3beta HSDII or P450c17 activities at typical reference serum unbound concentrations, but did inhibit 3beta HSDII and P450c17 at concentrations >10-fold higher. CONCLUSIONS: None of the tested AEDs influenced 3beta HSDII or P450c17 activities at concentrations normally used in AED therapy. However, VPA started to inhibit 3beta HSDII activity at concentrations 3 times above the typical reference serum unbound concentration. Because inhibition of 3beta HSDII activity will shift steroidogenesis toward C19 steroid production when P450c17 activities are unchanged, very high doses of VPA may promote C19 steroid biosynthesis, thus resembling PCOS. CBZ, TPM, and LTG influenced 3beta HSDII and P450c17 only at toxic concentrations.

Steroidogenic activities in MA-10 Leydig cells are differentially altered by cAMP and Müllerian inhibiting substance.

In addition to causing Müllerian duct regression in fetal males, Müllerian inhibiting substance (MIS) inhibits the expression of the bifunctional cytochrome P450, C17 hydroxylase/C(17-20) lyase (Cyp17), the enzyme that catalyzes the committed step in sex steroid synthesis. To investigate the paracrine effects of MIS on steroidogenic activity, we have performed assays with microsomes from mouse MA-10 Leydig cells. With microsomes from untreated MA-10 cells, progesterone was largely metabolized by 5alpha-reductase and subsequently converted by 3-keto steroid reductases to allopregnanolone and epiallopregnanolone. Addition of cAMP to the cells shifted microsomal steroid production to the Cyp17 product androstenedione and its 5alpha,3beta-reduced form, epiandrosterone. Microsomes from MIS-treated cells were less active with the progesterone substrate than those of untreated cells but co-treatment of the cells with both MIS and cAMP mitigated the cAMP-induced shift of the microsomes to androstenedione production. Quantitative analyses of steroid production by Cyp17 showed that cAMP decreased the amount of 17-hydroxyprogesterone produced relative to the androstenedione, suggesting that cAMP signaling lowers the efficiency of the Cyp17 hydroxylase activity or else increases the efficiency of its lyase activity. Addition of MIS to the cAMP-treated cells partially reversed this effect, as well. These results indicate that cAMP induces MA-10 cells to switch from producing 5alpha-reduced progesterone metabolites to producing androstenedione and its metabolites by increasing Cyp17 expression and its relative lyase activity, both of which are inhibited by MIS.

Marked inhibition of testosterone biosynthesis by the hepatotoxin nodularin due to apoptosis of Leydig cells.

We previously observed that the serum testosterone level was greatly reduced in the course of diethylnitrosamine-nodularin-induced hepatocarcinogenesis in Fischer 344 male rats (Lim et al., Gastroenterological Carcinogenesis, 1999). As an extension of this observation, this study was undertaken to investigate the molecular mechanism of downregulation of testosterone and its effect on target organs in Fischer 344 male rats treated with the hepatotoxin nodularin. After treating the rats with nodularin, a marked reduction of the testosterone level was noted in both serum and testis, with an accompanying accumulation of cholesterol in serum. Reduction of serum testosterone was not due to increased degradation of testosterone in the liver but to impaired biosynthesis in the testes, reduced activities of the cholesterol side chain cleavage enzyme and 17alpha-hydroxylase, and decreased expression of the steroidogenic acute regulatory protein gene, all of which constitute rate-limiting steps for testosterone biosynthesis in the testes. Intraperitoneal injection of nodularin into rats induced cuboidal changes of glandular epithelium in ventral prostates and apoptotic changes of spermatogonium, for example, nuclear chromatin condensation, shrinkage, and detachment from Sertoli cells, which included many lysosomal granules. Leydig cells also showed evidence of chromatin condensation and significant induction of peroxisome proliferation. In conclusion, the potential causes of impaired testosterone biosynthesis might have been apoptosis of Leydig cells induced by direct toxicity of the hepatotoxin on testes or hypothalamopituitary dysfunction.

Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.

Lack of an ovarian function influence on the increased adrenal androgen secretion present in women with functional ovarian hyperandrogenism.

OBJECTIVE: To evaluate whether ovarian function might have an influence on the adrenal hyperandrogenism present in patients with functional ovarian hyperandrogenism. DESIGN: Controlled clinical study. SETTING: Tertiary institutional hospital. PATIENT(S): Twenty-nine hirsute women with functional ovarian hyperandrogenism and 12 normal controls. INTERVENTION(S): The ACTH and GnRH tests were performed before and during triptorelin-induced ovarian suppression in patients. The normal women served as controls for the ACTH test. MAIN OUTCOME MEASURE(S): Basal and ACTH-stimulated steroid values. RESULT(S): All patients presented elevated T and free androgen index, which normalized after triptorelin. Patients with functional ovarian hyperandrogenism and adrenal hyperandrogenism, defined by elevated basal DHEAS (n = 10), presented enhanced delta 4-17, 20-lyase activity, which persisted during ovarian suppression. delta 4-17,20-lyase activity was normal in the functional ovarian hyperandrogenism patients without adrenal hyperandrogenism (n = 19). No correlation was observed between the any of the indexes of the adrenal enzymatic activities evaluated and plasma E2 or T. CONCLUSION(S): Increased adrenal delta 4-17,20-lyase activity is present in functional ovarian hyperandrogenism women with adrenal hyperandrogenism. No influence of the excess ovarian androgens or estrogens was found on any of the adrenal enzymatic pathways explored.

Direct inhibitions of the activities of steroidogenic cytochrome P-450 mono-oxygenase systems by anticonvulsants.

The effects of anticonvulsants on the activities of cytochromes P-450(17alpha,lyase) (CYP17), P-450arom (CYP19), P-450C21 (CYP21), P-450SCC (CYP11A1), and P-450(11beta) (CYP11B1) mono-oxygenase systems were studied using rat testicular microsomes, human placental microsomes, bovine adrenocortical microsomes, bovine adrenocortical mitochondria and purified cytochrome P-450(11beta). Phenytoin, clonazepam and carbamazepine inhibited the steroidogenesis catalysed by these cytochrome P-450 mono-oxygenase systems and the Ki values for each anticonvulsant were determined. Neither hydantoin nor sodium valproate inhibited the activities of steroidogenic cytochromes P-450. When the activities of cytochromes P-450arom and P-450C21 were measured in the presence of anticonvulsants, the Ki values (0.15 mM) for phenytoin were close to the plasma concentration of phenytoin under therapeutic conditions. Phenytoin, clonazepam and carbamazepine directly inhibited the monooxygenase activities of cytochromes P-450, because they did not affect the activities of NADPH-cytochrome P-450 reductase, NADPH-adrenoferredoxin reductase and adrenoferredoxin.

Splenic macrophages can modify steroidogenesis of Leydig cells.

A large amount of LH/hCG treatment given to male rats is known to suppress the enzyme activity of cytochrome P450c17 in Leydig cells for 48 h. A high dose LH/hCG injection is also known to allow immunocytes, such as macrophages, to migrate into the testicular interstitial compartment. It has not been known, however, whether these cells play a role in that suppression. In this study, we examined if splenic macrophages have any effects on testosterone secretion from Leydig cells by culturing rat testicular interstitial cells (TIC). Splenic macrophages co-cultured with TIC significantly suppressed testosterone secretion. Macrophages co-cultured reduced both progesterone to testosterone conversion and the amount of cytochrome P450c17 mRNA. The conditioned medium (SMCM), prepared by culturing macrophages for 12 h, significantly reduced either testosterone secretion from TIC or progesterone to testosterone conversion by TIC. These results indicate that splenic macrophages suppress testosterone secretion from Leydig cells by suppressing the cytochrome P450c17 enzyme in vitro, and that this effect is mediated at least in part by some soluble factors secreted from macrophages. Splenic macrophages migrating into the testis after LH/hCG stimulation could play a role in suppressing cytochrome P450c17 in Leydig cells.