Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.     

The effect of testosterone propionate, 5 alpha-dihydrotestosterone and 5 alpha-androstan-3 beta, 17 beta-diol on acid phosphatase activity in the prostate of castrated rats

The acid phosphatase is studied in the prostate gland of rats. It is shown that 10 days after gonadectomy the activity of acid phosphatase lowered considerably. Administration of testosterone propionate or 5 alpha-dihydrotestosterone to castrated animals restored the enzyme activity whereas 5 alpha-androstan-3 beta,17-diol was not effective. Administration of testosterone propionate with one of its metabolites increased the activity of acid phosphatase in the prostate gland, however to a less extent than with the use of the hormone itself. The lowest activity is detected with the simultaneous application of three androgens.     

Adult sheep blood metabolizes dihydrotestosterone to 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol

The metabolism of 5 alpha-dihydrotestosterone by adult sheep blood was investigated. Erythrocytes contain 3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities. The mean rate of reduction of 5 alpha-dihydrotestosterone by erythrocytes established in 15-min incubations was 0.66 +/- 0.36 (s.d.) mumol ml-1 erythrocytes h-1 and at equilibrium after a 60-min incubation, 90.6 +/- 5.1% of the substrate was reduced. The reduction of 5 alpha-dihydrotestosterone was shown to be dependent upon extracellular glucose and the intracellular cofactor NADPH. The proportion of the two reduction products was determined at equilibrium after separation by paper partition, chromatography and favoured 5 alpha-androstane-3 alpha, 17 beta-diol (96.0%) to 5 alpha-androstane-3 beta, 17 beta-diol (4.0%). The identities and proportions of the two products were confirmed by recrystallization procedures. The fact that erythrocytes can significantly metabolize the androgen 5 alpha-dihydrotestosterone is evidence for the recognition of blood as a major component of steroid endocrine homeostasis in sheep.     

Metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one and 5 alpha-androstan-3 alpha,17 beta-diol in the rat.

Significant metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one was recorded in several tissues and organs from rats and humans. This bioconversion was further investigated in rat testis homogenates. 5 alpha-Androstane-3 beta,17 beta-diol was readily metabolized to 17 beta-hydroxy-5 alpha-androstan-3-one with NAD and/or NADP added as cofactors. When a NADPH generating system was included in the incubation, 5 alpha-androstane-3 beta,17 beta-diol was metabolized to 5 alpha-androstan-3 alpha,17 beta-diol. Only small amounts of 17 beta-hydroxy-5 alpha-androstan-3-one accumulated under the latter condition.     

5alpha-Androstane-3beta,17beta-diol (3beta-diol), an estrogenic metabolite of 5alpha-dihydrotestosterone, is a potent modulator of estrogen receptor ERbeta expression in the ventral prostrate of adult rats

Prostate is one of the major targets for dihydrotestosterone (DHT), however this gland is also recognized as a nonclassical target for estrogen as it expresses both types of estrogen receptors (ER), especially ERbeta. Nevertheless, the concentrations of aromatase and estradiol in the prostate are low, indicating that estradiol may not be the only estrogenic molecule to play a role in the prostate. It is known that DHT can be metabolized to 5alpha-androstane-3beta,17beta-diol (3beta-diol), a hormone that binds to ERbeta but not to AR. The concentration of 3beta-diol in prostate is much higher than that of estradiol. Based on the high concentration of 3beta-diol and since this metabolite is a physiological ERbeta ligand, we hypothesized that 3beta-diol would be involved in the regulation of ERbeta expression. To test this hypothesis, adult male rats were submitted to castration followed by estradiol, DHT or 3beta-diol replacement. ERbeta and AR protein levels in the prostate were investigated by immunohistochemistry and Western blotting assays. The results showed that after castration, the structure of the prostate was dramatically changed and ERbeta and AR protein levels were decreased. Estradiol had just minor effects on the parameters analyzed. DHT-induced partial recovery of ERbeta while it was the most effective inductor of AR expression. Replacement with 3beta-diol-induced the highest levels of ERbeta, but was comparatively less effective in recovering the AR expression and the gland structure. These results offer evidence that one functional role of 3beta-diol in the prostate may be autoregulation of its natural receptor, ERbeta.     

Suppression of 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activity in rat pituitary by cobalt protoporphyrin: implications for testosterone homeostasis.

Cobalt protoporphyrin (CoPP) administered subcutaneously to adult male rats caused a marked reduction in the conversion of 5 alpha-androstane-3 beta-17 beta-diol (3 beta-adiol) to its main triol derivative (6 alpha-atriol) by homogenates of the pituitary but not of the prostate or brain (ventromedial hypothalamus and cortex). No effect in the brain was observed when this heme analogue was infused intracerebroventricularly. 3 beta-adiol hydroxylase, the enzyme responsible for the reaction and whose main function is thought to be the elimination of dihydrotestosterone and its metabolites from target tissues, was also inhibited by CoPP and SKF-525A added in vitro. The reaction was microsomal and dependent on NADPH. It is proposed that the lack of reciprocal elevation of luteinizing hormone in the face of the low testosterone levels observed following treatment with CoPP may be due, in part, to increased levels of androstanediols. These metabolites accumulate because of increased production from testosterone and decreased conversion to their triol derivatives in the pituitary.     

Pituitary metabolism of 5alpha-androstane-3beta-17beta-diol: intense and rapid conversion into 5alpha-androstane-3beta,6alpha,17beta-triol and 5alpha-androstane-3beta,7alpha, 17beta-triol.

In the male rat pituitary, 5alpha-androstane-3beta, 17beta-diol (3beta-diol) is extensively metabolized into polar steroids. They were identified as 5alpha-androstane-3beta, 6alpha-17beta-triol (6alpha-triol) and 5alpha-androstane-3beta, 7alpha, 17beta-triol (7alpha-triol). 6-alpha-Triol represents 53% and 7alpha-Triol 28% of the total 3beta-diol metabolites. The remaining percentage is related to 6beta and 7beta isomers. The biological role of triols is still unknown.     

Synthesis of deuterium-labeled 5 alpha-androstane-3 alpha, 17 beta-diol and its 17 beta-glucuronide.

Using unlabeled androsterone as starting material, 5 alpha-[16,16-2H2]androstan-3 alpha-ol-17-one was synthesized by exchange using deuterated potassium methoxide. This labeled androsterone product was reduced by sodium borodeuteride, which gave predominantly trideuterated 5 alpha-androstane-3 alpha, 17 beta-diol. The labeled androstanediol was conjugated with glucuronide by using the Koenig-Knorr reaction with methyl-1-bromo-1-deoxy-2,3,4-tri-O-acetyl-alpha-D-glucopyranosuronate . The dominant product was identified by thermospray high-performance liquid chromatography/mass spectrometry (MS) and electrospray MS as 5 alpha-[16,16,17-2H3]androstane-3 alpha, 17 beta-diol, 17 beta-glucuronide.     

Metabolism of testosterone to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol in alveolar macrophages from rat lung--II. Effects of intratracheal instillation of 3.4 mumol K2Cr2O7

Activity of the steroid 5 alpha-reductase in pulmonary alveolar macrophages from adult male rats has been investigated in vitro. Intratracheal instillation of 3.4 mumol K2Cr2O7 lowered the enzyme activity within 6 h, and the reduction was significant on the subsequent 2, 4 and 7 days. The activity of this enzyme was significantly decreased only 6 and 24 h after instillation when measured in the 800 g supernatant fraction of whole lung. Instillation of 3.4 mumol K2Cr2O7 increased serum levels of corticosterone. Serum levels of triiodothyronine and thyroxine decreased except for a transient increase 3 h after the K2Cr2O7 instillation. Subcutaneous administration of 200 micrograms dexamethasone/100 g b.wt, 200 micrograms/100 g b.wt of testosterone, 17 beta-hydroxy-5 alpha-androstane-3-one (5 alpha-DHT), dehydroepiandrosterone or corticosterone had no effect on the 5 alpha-reductase activity of the pulmonary alveolar macrophages within 12 h. The combined treatment with dexamethasone s.c. and intratracheal instillation of 3.4 mumol K2Cr2O7 reduced the steroid 5 alpha-reductase activity in the pulmonary alveolar macrophages to about 25% of controls. Measurement of the steroid 5 alpha-reductase activity in pulmonary alveolar macrophages as an index of lung damage when exposed to toxic material is discussed.     

An alternate pathway for androgen regulation of brain function: activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5alpha-androstane, 3beta,17beta-diol (3beta-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3beta-Diol is an important modulator of the stress response mediated by the hypothalmo-pituitary-adrenal axis. Furthermore, the actions of 3beta-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.     

Chemical synthesis and biological activities of 16alpha-derivatives of 5alpha-androstane-3alpha,17beta-diol as antiandrogens

In our efforts to develop compounds with therapeutic potential as antiandrogens, we synthesized a series of 5alpha-androstane-3alpha,17beta-diol derivatives with a fixed side-chain length of 3-methylenes at C-16alpha, but bearing a diversity of functional groups at the end. Among these, the chloride induced the best antiproliferative activity on androgen-sensitive Shionogi cells. Substituting the OH at C-3 by a methoxy group showed the importance of the OH. Moreover, its transformation into a ketone increased the androgen receptor (AR) binding but decreased the antiproliferative activity and induced a proliferative effect on Shionogi cells. These results confirm the importance of keeping a 5alpha-androstane-3alpha,17beta-diol nucleus instead of a dihydrotestosterone nucleus. Variable side-chain lengths of 2-, 3-, 4-, and 6-methylenes at C-16alpha were investigated and the optimal length was found to be 3-methylenes. Although exhibiting a weak AR binding affinity, 16alpha-(3'-chloropropyl)-5alpha-androstane-3alpha,17beta-diol (15) provided an antiproliferative activity on Shionogi cells similar to that of pure non-steroidal antiandrogen hydroxy-flutamide (77% and 67%, respectively, at 0.1 microM). The new steroidal compound, 15, thus constitutes a good starting point for development of future antiandrogens with a therapeutic potential against prostate cancer.     

Binding of estradiol and 5 alpha-androstane-3 beta-17 beta-diol by the male rat enriched gonadotrope cells

Dispersed pituitary cells from 42-day old male rats were separated using centrifugal elutriation. Based on LH and PRL cellular contents, fractionated cells were pooled into two fractions: "Lactotrope++ population" and "gonadotrope++ population". Estradiol and 5 alpha-androstane-3 beta, 17 beta-diol binding was measured in these fractions. Results revealed that: (1) The steroid receptors are not destroyed by cell dispersion and elutriation. (2) The estradiol receptor content is higher in gonadotrope++ cells than in lactotrope++ cells. (3) The number of binding sites for the two steroids changes in the different fractions: whereas it is exactly similar in "lactotrope++ population", it is much higher for estradiol than for 5 alpha-androstane-3 beta, 17 beta-diol in "gonadotrope++ population". These results suggest two different species--or conformations--of receptor binding sites for estradiol in the male rat pituitary; the first one could link both steroids, the second one would be specific for estradiol.     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: effects of antibodies and chemical inhibitors of cytochrome P450 enzymes.

The purpose of the present study was to test the hypothesis that rat prostate microsomes contain a single cytochrome P450 enzyme responsible for the conversion of 5 alpha-androstane-3 beta,17 beta-diol to a series of trihydroxylated products. The three major metabolites formed by in vitro incubation of 5 alpha-[3H]androstane-3 beta,17 beta-diol with rat prostate microsomes were apparently 5 alpha-androstane-3 beta,6 alpha,17 beta-triol, 5 alpha-androstane-3 beta,7 alpha,17 beta-triol, and 5 alpha-androstane-3 beta,7 beta,17 beta-triol, which were resolved and quantified by reverse-phase HPLC with a flow through radioactivity detector. The ratio of the three metabolites remained constant as a function of incubation time, microsomal protein concentration, ionic strength, and substrate concentration. The ratio of the three metabolites was dependent on pH, apparently because the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol shifted from the 6 alpha- to the 7 alpha-position with increasing pH (6.8-8.0). The V(max) values were 380, 160, and 60 pmol/mg microsomal protein/min for the rate of 6 alpha-, 7 alpha-, and 7 beta-hydroxylation, respectively. Similar Km values (0.5-0.7 microM) were measured for enzymatic formation of all three metabolites, which suggests that formation of all three metabolites was catalyzed by a single, high-affinity enzyme. Testosterone, 5 alpha-dihydrotestosterone, and 5 alpha-androstane-3 alpha,17 beta-diol did not appreciably inhibit the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol, suggesting that this enzyme exhibits a high degree of substrate specificity. Formation of all three metabolites was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase (85%) and by a 9:1 mixture of carbon monoxide and oxygen (60%). Several chemical inhibitors of cytochrome P450 enzymes, especially the antimycotic drug clotrimazole, also inhibited the formation of all three metabolites. Polyclonal antibodies that recognize liver cytochrome P450 1A, 2A, 2B, 2C, and 3A enzymes did not inhibit 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activity. Overall, these results are consistent with the hypothesis that the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes is catalyzed by a single, high-affinity P450 enzyme. This cytochrome P450 enzyme appears to be structurally distinct from those in the 1A, 2A, 2B, 2C, and 3A gene families.     

Periovulatory changes in serum concentration and ovarian content of 5 alpha-androstane-3 alpha, 17 beta-diol in the adult rat

The high amounts of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) present in immature female rats decline towards first ovulation, but on the day of first proestrus a peak is seen. This raises the possibility that during adulthood similar proestrous peaks may occur. Therefore, serum concentrations and ovarian content of 3 alpha-diol were estimated every two hours between 0900 and 2100 h in adult cyclic rats on the day of proestrus. In the same rats, serum concentrations of estradiol (E2), progesterone (P) and luteinizing hormone (LH) were measured, as were ovarian contents of E2 and P. A significant elevation in ovarian 3 alpha-diol was found between 0900 and 1700 h proestrus, whereas serum concentrations of 3 alpha-diol were elevated from 1300 to 2100 h. The high morning values of ovarian 3 alpha-diol correlated with those for ovarian E2 (p less than 0.005); the elevated serum concentrations of 3 alpha-diol during the afternoon correlated with serum P (p less than 0.005) and with serum LH concentrations (p less than 0.005). Serum and ovarian values were positively correlated for P and E2, but not for 3 alpha-diol. The rise in serum 3 alpha-diol could be prevented by blocking the LH surge with sodium pentobarbitone (Nembutal; 35 mg/kg b.w.) administered at 1300 h. In Nembutal-treated rats, the concentration of 3 alpha-diol at 1700 h (886 pg/ml) was significantly lower than in saline-treated control rats (1135 pg/ml; p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)