Metabolism of dehydroisoandrosterone and androstenedione by human A-549 alveolar type II epithelial-like cells

The A-549 cell line was initiated from an explant of human lung carcinoma tissue. The biochemical characteristics of these cells are similar to those of normal alveolar type II epithelial cells. To gain some insight into the steroid-metabolizing capabilities of A-549 cells, the metabolism of tritium-labeled dehydroisoandrosterone and androstenedione by these cells was studied. The metabolism of dehydroisoandrosterone led to the exclusive formation of 5-androstene-3 beta,17 beta-diol. The major product of androstenedione metabolism was testosterone; and, 5 alpha-reduced steroids also were formed, viz. 5 alpha-androstane-3,17-dione, androsterone, isoandrosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol. Estrogens, viz., estrone and estradiol-17 beta, were not products of androstenedione metabolism by A-549 cells. The rates of metabolite formation from either dehydroisoandrosterone or androstenedione were linear as a function of incubation time up to 3 h, and with cell number up to 1 X 10(6) cells/ml. The apparent Km of 17 beta-hydroxysteroid oxidoreductase for dehydroisoandrosterone was 11 microM, and that for androstenedione was 13 microM. The predominant formation of 5-androstene-3 beta,17 beta-diol from dehydroisoandrosterone, and testosterone from androstenedione is a likely indication that the principal C19-steroid-metabolizing enzyme in A-549 cells is 17 beta-hydroxysteroid oxidoreductase; the other steroid-metabolizing enzymes expressed in these cells are 5 alpha-reductase, 3 beta-hydroxysteroid oxidoreductase and 3 alpha-hydroxysteroid oxidoreductase. The findings of this study demonstrate that A-549 cells express steroid-metabolizing enzymatic activities that are qualitatively similar to those found in other human pneumonocytes and human lung tissue, except for 3 beta-hydroxysteroid oxidoreductase-5----4-isomerase activity, which is not expressed in these cells with dehydroisoandrosterone as the substrate.     

Steroid 5 alpha-reductase activity in endothelial cells from human umbilical cord vessels

The metabolism of radiolabeled progesterone and androstenedione was evaluated in endothelial cells from human umbilical cord vein and arteries maintained in culture. The predominant metabolite of progesterone was 5 alpha-pregnane-3,20-dione and that of androstenedione was 5 alpha-androstane-3,17-dione. Thus, the major pathway of progesterone and androstenedione metabolism within these cells is via steroid 5 alpha-reductase. The rate of formation of 5 alpha-pregnane-3,20-dione from progesterone by venous endothelial cells was linear with incubation time up to 4 h and with cell number up to 1.6 X 10(6) cells/ml. The apparent Km of 5 alpha-reductase for progesterone was 0.4 microM; and, the Vmax was 55 pmol 5 alpha-pregnane-3,20-dione formed/mg protein X h. The rate of 5 alpha-androstane-3,17-dione formation from androstenedione also was linear with incubation time up to 4 h. In addition to 5 alpha-androstane-3,17-dione, the metabolism of androstenedione by either venous or arterial cells resulted in the formation of various minor metabolites, including testosterone and 5 alpha-reduced steroids, viz. 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, and 5 alpha-androstane-3 beta, 17 beta-diol. Estrogens (i.e. estradiol-17 beta and estrone) were not detected as products of androstenedione metabolism. The formation of these metabolites are indicative that the steroid-metabolizing enzymes present in endothelial cells are: 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase, 3 alpha-hydroxysteroid oxidoreductase, and 3 beta-hydroxysteroid oxidoreductase.     

Androgen metabolism in somatic and germinal tissues of the sea star Asterias vulgaris.

1. Cell-free homogenates of male and female pyloric caeca, body wall, testis and ovary were incubated with radiolabeled 3H-androstenedione. 2. Pyloric caeca had highest rates of androstenedione conversion. The predominant metabolites in the pyloric caeca were testosterone, 5 alpha-androstane-3 beta, 17 beta-diol and 5 beta-androstane-3 beta, 17 beta-diol. 3. In body wall, testicular and ovarian homogenates, androstenedione was converted primarily to testosterone and also to 5 alpha-androstanedione and epiandrosterone. 4. Qualitative and quantitative differences in androgen metabolism in somatic and germinal tissues may be related to tissue-specific regulation of cellular metabolism.     

Androstenedione metabolism in human lung fibroblasts

Human lung fibroblasts in culture metabolized [3H]androstenedione to a number of different compounds, including testosterone, 5 alpha-androstanedione, androsterone, 5 alpha-dihydrotestosterone, isoandrosterone, and 5 alpha-androstane-3 alpha,-17 beta-diol. The major products were 5 alpha-androstanedione and testosterone. Estrone, estradiol-17 beta and 5 beta-reduced steroids were not formed. The production rates of testosterone and 5 alpha-androstanedione from [3H]androstenedione by lung fibroblasts were studied both as a function of incubation time and substrate concentration. The rates of formation of testosterone and 5 alpha-androstanedione remained linear with time up to 4 h. The apparent Km of human lung fibroblast 5 alpha-reductase was 1 microM, and that of 17 beta-hydroxysteroid oxidoreductase was 11 microM. The findings of this study suggest that mesenchyma may contribute to the metabolism of androstenedione in human lung tissue.     

Guinea pig liver 3-hydroxyhexobarbital dehydrogenase. Purification and properties.

3-Hydroxyhexobarbital dehydrogenase, which catalyzes the reversible oxidation of 3-hydroxyhexobarbital to 3-oxohexobarbital, has been purified 470-fold from the soluble fraction of guinea pig liver with a yield of 47%. The specific activity of the purified enzyme is 9.4 units/mg of protein. Results of polyacrylamide gel disc electrophoresis and isoelectric focusing indicated that the purified enzyme preparation is a single and homogeneous protein. NADP+ served as preferred co-factor, but NAD+ is also utilized in the presence of phosphate ion. The guinea pig liver enzyme possessed a relatively narrow substrate specificity in comparison with the rabbit liver enzyme. It is very distinctive that guinea pig liver 3-hydroxyhexobarbital dehydrogenase catalyzes the dehydrogenation of 17beta-hydroxysteroids such as testosterone, 4-androstene-3beta,17beta-diol, 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol, 5alpha-androstan-17beta-ol-3-one, and 5beta-androstane-3alpha,17beta-diol.     

Influence of 5 alpha-reduced androgens on oestradiol secretion by granulosa cells of pregnant mare serum gonadotrophin treated immature rats

The effect of aromatizable androgens (testosterone and androstenedione) and naturally occurring 5 alpha-androstane, 3 alpha 17 beta-diol and 5 alpha-androstane, -3 beta, 17 beta-diol on oestradiol secretion by granulosa cells isolated from preovulatory follicles of PMSG-primed immature rats was investigated. The amount of oestradiol secreted by granulosa cells in the absence of exogenous aromatizable androgen in a 4h incubation was negligible. However, the addition of testosterone or androstenedione resulted in concentration dependent increases in oestradiol secretion. The 5 alpha-reduced androgens inhibit oestradiol and oFSH-stimulated oestradiol secretion by the granulosa cells in the presence of exogenous testosterone. The least potent of the androgens tested in causing this effect being the 5 alpha-androstane-3 alpha, 17 beta-diol. This result suggests that the naturally occurring 5 alpha-reduced androgens have a direct effect on androgen-aromatizing enzymes. The effect of these androgens may have an important connotation with respect to the control of the onset of puberty and regulation of ovarian oestradiol secretion within the microenvironment of an ovarian follicle.     

Age dependent changes in androgen metabolism in the rat prostate

Oxidation and reduction of androstenedione, testosterone, dihydrotestosterone (DHT), 5 alpha-androstan-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol (3 alpha- and 3 beta-A'diol) were measured in homogenates from the ventral prostate (VP), dorsal prostate (DP), lateral prostate (LP), the coagulating gland (CG) and seminal vesicles (SV) in intact rats of different ages from young mature (3-6 months) to senescent rats (20-30 months). Some very old intact rats (30-32 months) were treated with testosterone in order to rule out the effect of this hormone on androgen metabolism. The enzymatic activities for young mature rats were significantly altered by increasing age, both with regard to differences between the various organs as well as differences in cofactor requirement. With increasing age, the specific activity of most enzymes gradually decreased. With testosterone as substrate, 5 alpha-reductase activity was significantly reduced in the old rats in all tissues studied and was undetectable in the oldest animals in the VP and the SV. On the other hand, 5 alpha-reductase could not be recorded in any tissue in any tissue in old rats when androstenedione was the substrate. 3 alpha-Hydroxysteroid oxidoreductase (3 alpha-HSOR) in the VP was the only enzyme which did not decrease in activity by increasing age. In the other lobes this enzyme activity decreased similar to 3 beta-hydroxysteroid oxidoreductase (3 beta-HSOR) and the 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) activity. Administration of testosterone to old rats increased the specific activity of most of the enzymes studied.     

In vitro metabolism of 4-androstene-3,17-dione and testosterone by rat submaxillary gland.

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of male and female rats. The metabolism was predominately reductive. In 15 and 180 min incubations submaxillary tissue converted 4-androstene-3,17-dione chiefly to androsterone. Less testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, and 4-androstene-3 alpha, 17 beta-diol were also identified. Testosterone was converted to the same products plus 4-androstene-3,17-dione. 5 alpha-Androstane-3 alpha, 17 beta-diol was the major testosterone metabolite. Qualitatively the metabolism by male and female submaxillary gland was similar.     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5 alpha-reductase inhibitors.

5 alpha-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5 alpha-reductase. The inactivation of 5 alpha-dihydrotestosterone involves reversible reduction to 5 alpha-androstane-3 beta,17 beta-diol by 3 beta-hydroxysteroid oxidoreductase followed by 6 alpha-, 7 alpha-, or 7 beta-hydroxylation. 5 alpha-Androstane-3 beta,17 beta-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity (Km approximately 0.5 microM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5 alpha-reductase (4-azasteroid analogs; 10 microM) or inhibitors of 3 beta-hydroxysteroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 microM) had no appreciable effect on the 6 alpha-, 7 alpha-, or 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (10 microM) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1-10 microM) all markedly inhibited 5 alpha-androstane-3 beta,17 beta-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1-10 microM) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole greater than clotrimazole greater than ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a Ki of 0.03 microM. The imidazole-type antimycotic drugs inhibited all three pathways of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.     

In vitro metabolism of testosterone by cultured Sertoli cells and the effect of FSH.

Cultures of Sertoli cells isolated from testes of 18-and 36-day-old Long Evans rats were used to investigate their capacity to metabolize testosterone and the effect of FSH on such metabolism. Three different approaches were used: 1) investigation of the metabolism of radiolabeled testosterone under saturating substrate conditions; 2) study of the metabolism of radiolabeled testosterone utilizing trace amounts of high specific activity substrates; 3) the utilization of radioimmunoassay for measurement of estradiol-17 beta. The following steroids were isolated and identified by recrystallization to constant specific acitvity from the control and FSH-treated cultures; testosterone (unconverted substrate), androstenedione, dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha, 17 beta-diol. Radioimmunoassay data suggests that the Sertoli cells produce an estradiol-17 beta-like compound from unlabeled testosterone and that this production is stimulated by FSH. However, the radioactive metabolite from all our studies that behaved chromatographically like estradiol--17 beta failed to crystallize to constant specific activity, while in each experiment, authentic radiolabeled estradiol-17 beta added as recovery tracer did. The data demonstrate that : 1) cultures of Sertoli cells from immature rats have 5 alpha-reductase, 3 alpha- and 17 beta-hydroxysteroid oxidoreductase activities; 2) these enzymes may be affected by FSH; 3) based on radiolabeled metabolic techniques, Sertoli cells were unable to biotransform testosterone to estradiol-17 beta even in the presence of FSH.     

Quantitative assessment of endogenous testicular and adrenal sex steroids and of steroid metabolizing enzymes in untreated human prostatic cancerous tissue

Total tissue content and subcellular distribution of DHEA sulfate, DHEA, androst-5-ene-3 beta,17 beta-diol, androst-4-ene-3,17-dione, testosterone, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol as well as the activities of steroid sulfate-sulfatase, 17 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase, 3 alpha/beta-hydroxysteroid dehydrogenase, and creatine kinase were quantified in 12 untreated primary tumors of prostatic cancer. Samples were obtained by radical prostatectomy and serial sections, and were alternately used for either biochemical or morphological evaluation. The results were compared with values determined in benign parts of the same prostates. Qualitatively, all enzymes and steroids found in the benign tissues could also be demonstrated in the cancers. Steroid patterns showed individual quantitative variation but no general differences between the carcinomas and the benign tissues. Enzymes showed a tendency to lower activities in the cancers, particularly when expressed per DNA. Substantial diminutions of creatine kinase and 5 alpha-reductase activity, the latter being often accompanied by an increased testosterone/DHT ratio, were the most striking differences seen in most of the cases between malignant and nonmalignant tissues. Some interesting individual parallels of morphological and biochemical aspects were seen, but there was no obvious general parallelism between the histological picture and endocrinological characteristics.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Administration of pregnenolone and dehydroepiandrosterone to guinea pigs and rats causes the accumulation of fatty acid esters of pregnenolone and dehydroepiandrosterone in plasma lipoproteins.

Steroids were administered continuously to guinea pigs and rats using subcutaneously applied silastic tubing implants, and the effects on circulating steroid and steroid conjugate levels were monitored. Using implants filled with pregnenolone, we observed that pregnenolone had a marked effect on increasing the levels of its fatty acid-esterified derivative, while dehydroepiandrosterone-releasing implants produced a rise in circulating nonconjugated dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, androstenedione, testosterone, and lipoidal derivatives of both dehydroepiandrosterone and androst-5-ene-3 beta,17 beta-diol. Implants filled with androstenedione produced a 20-fold increase in plasma androstenedione levels relative to untreated controls and a corresponding five-fold increase over control testosterone levels. No fatty acid-esterified derivative of testosterone could be detected within the plasma. Lipoproteins were isolated from both rats and guinea pigs treated with implants filled with pregnenolone or dehydroepiandrosterone. The steroid and steroid fatty acid esters present in each fraction were analyzed, revealing that approximately 75% of all the fatty acid esters of pregnenolone recovered in the lipoproteins was localized within the high-density lipoprotein (HDL) fraction of both guinea pig and rat plasma. Similarly, lipoidal dehydroepiandrosterone was found associated predominantly with the low-density lipoprotein and HDL fractions in the guinea pig, while in the rat this steroid conjugate was exclusively within the HDL fraction. High-density lipoprotein-incorporated tritiated pregnenolone fatty acid esters and dehydroepiandrosterone fatty acid esters were injected into castrated male guinea pigs to study the fate of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol directly increases dihydrotestosterone conversion primarily to 5 alpha-androstan-3 beta, 17 beta-diol in rat Leydig cells

Our studies demonstrate that direct stimulation of dihydrotestosterone metabolism by ethanol (2.2 - 65 mM) in rat Leydig cells primarily involves an increase in 5 alpha-androstan-3 beta, 17 beta-diol. Although the enzyme catalyzing this conversion, 5 alpha-androstane-3 beta-hydroxysteroid dehydrogenase, is localized in the microsomal fraction of Leydig cells, ethanol does not increase 5 alpha-androstan-3 beta, 17 beta-diol formation in isolated microsomes, presumably because of the removal of soluble alcohol dehydrogenase activity, which we propose mediates this action. Because 5 alpha-androstan-3 beta, 17 beta-diol is generally considered a weak or inactive androgen, this effect may function to decrease dihydrotestosterone secretion by Leydig cells and/or to reduce the availability of this androgen in responsive tissues.     

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.     

Reductive metabolism of 5 alpha-dihydrotestosterone by rat ventral and dorsolateral prostate: kinetic parameters of the enzymes

In male sex accessory organs the active androgen 5 alpha-dihydrotestosterone (DHT) is metabolized to 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) by the reductase activities of 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR; EC 1.1.1.50) and 3 beta-hydroxysteroid oxidoreductase (3 beta-HSOR; EC 1.1.1.51). After separation of radiosubstrate and products by HPLC, these enzymes activities in subcellular preparations of rat ventral and dorsolateral prostate were determined from the conversion of [3H]DHT to the radiometabolites 3 alpha-diol and 3 beta-diol and 3 beta-triols (5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol plus 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol). Whereas both enzymes were found in the dorsolateral prostate, 3 beta-HSOR reductase activity was near the limit of detection in ventral prostate. Unlike the equal distribution of 3 alpha-HSOR reductase between the microsomal and cytosol fractions of the ventral prostate, both 3 alpha- and 3 beta-HSOR reductase activities of the dorsolateral prostate are mainly confined to its cytosol fraction. Km and Vmax of the 3 alpha- and 3 beta-HSOR reductases in dorsolateral prostate cytosol were 1.8 microM, 24.6 pmol.mg-1 min-1 and 25.4 microM, 45.7 pmol.mg-1 min-1, respectively. We surmise from these and earlier studies that 3 beta-HSOR reductase is the rate-limiting prostatic enzyme in the catabolic disposition of intracellular DHT.     

Effect of neonatal estrogenization on testosterone metabolism in the prostate and in the epididymis of the rat

The present experiments were performed in order to analyze whether the administration of estrogens (single injection of 500 micrograms of estradiol benzoate s.c.) to neonatal male rats might modify the weight of the ventral prostate and the epididymis as well as the metabolism of testosterone in these two organs. The metabolism of testosterone was evaluated in vitro using 14C-radiolabelled testosterone as the substrate. The metabolites dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), androstenedione, 5 alpha-androstane-3,17-dione (5-A-dione) and 3 alpha-hydroxy-5 alpha-androstane-17-one (androsterone) were quantified. After neonatal estrogen administration animals were killed on days 22 and 90 of age. The following changes were observed: (1) the body weight, the weight of the testes and of the ventral prostate were lower than in controls on both day 22 and 90; (2) the weight of the epididymides was higher than in controls on day 22 and lower on day 90; (3) in the ventral prostate the in vitro formation of DHT was lower and that of the diols was higher than in control tissue on day 22 of age; (4) the in vitro formation of alpha-reduced metabolites of the 17-keto series (5 alpha-A-dione + androsterone) was higher in ventral prostate of treated animals than in that of controls on day 22; (5) in treated animals, no formation of DHT in the caput epididymis was observed at day 22. On the contrary, at the same age the formation of androstenedione was higher than in controls; on day 90 of age the formation of DHT, androstenedione and the 5 alpha-reduced metabolites of the 17-keto series was identical in caput epididymis of the treated animals and of the controls, while the formation of the diols was higher in the treated than in the controls. The data indicate that neonatal estrogenization may induce important changes in testosterone metabolism in the prostates and in the epididymides of the rat.     

Multiple steroid metabolic pathways in ZR-75-1 human breast cancer cells

In order to characterize the main enzymatic systems involved in androgen and estrogen formation as well as metabolism in ZR-75-1 human breast cancer cells, incubation of intact cells was performed for 12 or 24 h at 37 degrees C with tritiated estradiol (E2), estrone (E1), androst-5-ene-3 beta, 17 beta-diol (5-ene-diol), dehydroepiandrosterone (DHEA), testosterone (T), androstenedione (4-ene-dione), dihydrotestosterone (DHT) or androsterone (ADT). The extra- and intracellular steroids were extracted, separated into free steroids, sulfates and non-polar derivatives (FAE) and identified by HPLC coupled to a Berthold radioactivity monitor. Following incubation with E2, 5-ene-diol or T, E1, DHEA and 4-ene-dione were the main products, respectively, thus indicating high levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). When 4-ene-dione was used, on the other hand, a high level of transformation into 5 alpha-androstane-3,17-dione (A-dione), Epi-ADT and ADT was found, thus indicating the presence of high levels of 5 alpha-reductase as well as 3 alpha- and 3 beta-hydroxysteroid dehydrogenase. Moreover, some T was formed, due to oxidation by 17 beta-HSD. No estrogen was detected with the androgen precursors T or 4-ene-dione, thus indicating the absence of significant aromatase activity. Moreover, significant amounts of sulfates and non-polar derivatives were found with all the above-mentioned substrates. The present study shows that ZR-75-1 human breast cancer cells possess most of the enzymatic systems involved in androgen and estrogen formation and metabolism, thus offering an excellent model for studies of the control of sex steroid formation and action in breast cancer tissue.     

Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.     

Influence of neonatal androgenization on the testicular steroidogenesis in the adult rat

The in vitro testicular steroidogenesis of male rats, androgenized on the third postnatal day by a single injection of 1 mg testosterone propionate, was investigated when the animals were 100 days old. The neonatal androgenization resulted in a 25% lower testes weight, significantly increased plasma levels of FSH (P less than 0.01) and LH (P less than 0.02), and normal levels of testosterone. Although the testes were hypotrophic, the incubation of the testes pairs yielded the same amounts of testosterone, 7 alpha-hydroxytestosterone and 5 alpha-androstane-(3 alpha + 3 beta), 17 beta-diol as in the control animals. However, the steroidogenic response to an acute hCG stimulation was reduced. From incubations of testes homogenates with various labelled steroid precursors it could be inferred that the activity of the 17 alpha-hydroxylase, the 3 beta-hydroxysteroid dehydrogenase-isomerase and the 17 beta-hydroxysteroid dehydrogenase, expressed per unit of incubated protein, was significantly increased in the testes of the androgenized rats. These data indicate that the basal steroidogenesis in neonatally androgenized male rats is maintained by an increased synthesis per unit of tissue, possibly under influence of an increased gonadotrophic stimulus, but that the maximum steroidogenic capacity is reduced.