The synthesis of reduced metabolites of aldosterone by subcellular fractions of rat kidney: effects of antimineralocorticoids

Subcellular fractionation of male rat kidney revealed that the nuclear and plasma membrane fractions isolated from the 1,000 g pellet retained a significant proportion of the aldosterone ring-A reducing activity. Improved HPLC solvent systems separated all six possible ring-A reduced metabolites of aldosterone and revealed that 80-90% of the reduced metabolites synthesized by purified nuclei and plasma membranes were 5 alpha-reduced compounds consisting of 5 alpha-DHA and 3 alpha,5 alpha-THA in ratios of 1:2 (nuclei) and 1:1 (membranes). The 105,000 g cytosol also synthesized significant quantities of reduced, hydroxylated, and conjugated metabolites of aldosterone. In contrast, the majority of the reduced metabolites of aldosterone synthesized by kidney cytosol were 5 beta-products, consisting principally of 5 beta-DHA and smaller quantities of 3 alpha,5 beta-THA and 3 beta,5 beta-THA. The synthesis of reduced aldosterone metabolites in the cytosol, nuclear, and plasma membrane fraction was inhibited by both 5 and 50 microM concentrations of the antimineralocorticoids, progesterone, K+-canrenoate, and corticosterone. Progesterone was the strongest inhibitor of the synthesis of 5 alpha-DHA and 3 alpha,5 alpha-THA in both nuclei and plasma membranes. The overall order of inhibition of the synthesis of ring-A reduced metabolites in the kidney subcellular fractions was progesterone greater than K+-canrenoate greater than corticosterone; both progesterone and K+-canrenoate inhibited 5 alpha-reduction more than 5 beta-reduction.     

17 beta-hydroxysteroid and 20 alpha-hydroxysteroid dehydrogenase activities of human placental microsomes: kinetic evidence for two enzymes differing in substrate specificity

During storage at 4 degrees C, the 17 beta-hydroxysteroid dehydrogenase activity of human placental microsomes with estradiol-17 beta was more stable than that with testosterone. In order to evaluate the basis for this difference, kinetics with C18-, C19-, and C21- steroids as substrates and/or inhibitors was studied in conjunction with an analysis of the effects of detergents. Both 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities were detected. At pH 9.0, apparent Michaelis constants were 0.8, 1.3, and 2.3 microM for estradiol-17 beta, testosterone, and 20 alpha-dihydroprogesterone, respectively, 17 beta-HSD activity with testosterone was inhibited by estradiol-17 beta, 5 alpha-dihydrotestosterone, 5 beta-dihydrotestosterone, 20 alpha-dihydroprogesterone, and progesterone. In each case 90 to 100% inhibition was observed at 50 to 200 microM steroid. Activity with 20 alpha-dihydroprogesterone was similarly sensitive to inhibition by C19-steroids. By contrast, 25 to 45% of the activity with estradiol-17 beta was not inhibited by high concentrations of C19- or C21-steroids and differed from the 17 beta-HSD activity with testosterone and the major fraction of that with estradiol-17 beta by being insensitive to solubilization by detergent. These results are consistent with an association of two dehydrogenase activities with human placental microsomes. One recognizes C18-, C19-, and C21-steroids as substrates with comparable affinities. The second appears to be highly specific for estradiol-17 beta. The former activity may account for most if not all of the oxidation-reduction at C-17 of C19-steroids and at C-20 of C21-compounds at physiological concentrations by term placental tissue.     

Synthesis of aldosterone by a reconstituted system of cytochrome P-45011 beta from bovine adrenocortical mitochondria

When corticosterone was incubated with cytochrome P-45011 beta purified from bovine adrenocortical mitochondria in the presence of adrenodoxin, NADPH-adrenodoxin reductase and an NADPH generating system, aldosterone as well as 18-hydroxycorticosterone were formed with turnover numbers of 0.23 and 1.1 nmol/min/nmol P-450, respectively. Phospholipids extracted from adrenocortical mitochondria remarkably enhanced the activity of aldosterone formation by the cytochrome P-45011 beta-reconstituted system. The apparent Km and turnover number were estimated to be 6.9 microM and 2.0 nmol/min/nmol P-450 for aldosterone formation in the presence of the lipidic extract. When 18-hydroxycorticosterone was tested as a substrate, cytochrome P-45011 beta showed catalytic activity for aldosterone synthesis with an apparent Km and turnover number of 325 microM and 5.3 nmol/min/nmol P-450, respectively. Carbon monoxide and metyrapone inhibited the production of aldosterone from corticosterone and that from 18-hydroxycorticosterone. These results suggest that conversion of corticosterone and of 18-hydroxycorticosterone to aldosterone occurs through P-45011 beta-catalyzed reaction.     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.     

Enzyme induction in Streptomyces hydrogenans. V. Characterization of testosterone-17 beta-dehydrogenase and its induction by steroids]

Testosterone 17beta-dehydrogenase can be enriched from Streptomyces hydrogenans. The enzyme dehydrogenizes testosterone with Km=13muM and estradiol-17beta with Km=21muM to the corresponding 17-ketoderivatives. NAD forms NADH with Km=125muM. The enzyme is strongly inhibited by androstandione and 17alpha-methyltestosterone. The Ki for 17alpha-methyltestosterone is 18muM. The enzyme activity increases with increasing pH up to alkali-mediated denaturation at about pH 10. The optimum temperature is at 45 degrees C. If Streptomyces hydrogenans is cultivated in the absence of steroids, the specific activity of testosterone 17beta-dehydrogenase in the cytosol of the microorganisms amounts to 10 mU/mg protein, and increases up to 10-fold if the cells are cultivated in the presence of certain steroids. Testosterone, alpha-dihydrotestosterone, beta-dihydrotestosterone, estradiol-17beta, and 17alpha-methyltestosterone are very effective inducers. Thus, for the first time, the ability of estradiol-17beta to induce an enzyme synthesis in a microorganism is shown. The steroid-dependent induction is inhibited by testosterone acetate and rifamycin SV. Cyproterone, however, does not decrease the testosterone-dependent enzyme induction of testosterone 17beta-dehydrogenase.     

Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle-stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action

Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha-dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity.     

Kinetic analysis of human placental, ovarian, and adrenal 3 beta-hydroxysteroid dehydrogenase inhibition by epostane in vitro

The effect of epostane [(2 alpha,4 alpha,5 alpha,17 beta)-4,5-epoxy-17-hydroxy-4,17-dimethyl-3-oxo- androstane-2-carbonitrile] on the conversion of pregnenolone to progesterone and of dehydroepiandrosterone (DHA) to androstenedione was studied in human term placental microsomes and in comparison with human ovarian and adrenal microsomes. Using pregnenolone as substrate, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the three tissues had a similar Km (3-6 microM) but Vmax ranged from 1.3 nmol/mg protein per min in ovary to 10 nmol/mg protein per min in placenta. Epostane inhibited 3 beta-HSD activity in all three tissues with the characteristics of a pure competitive inhibitor: mean Ki values were 1.7 microM for placenta, 0.5 microM for adrenal and 0.1 microM for ovary. Moreover, in placental microsomes epostane inhibited the conversion of DHA to androstenedione with a Ki of 0.6 microM. The mechanism of action of epostane explains its effectiveness in blocking progesterone synthesis during the luteal phase and in pregnancy in women, and its strong anti-steroidogenic effect in other endocrine tissues in vitro.     

Estrogen-2/4-hydroxylase activities in rat brain and liver microsomes exhibit different substrate preferences and sensitivities to inhibition

NADPH-dependent estrogen-2/4-hydroxylase activities in rat brain and liver microsomes were compared with respect to the utilization of different estrogens as substrates and the inhibitory effects of alpha-naphthoflavone, metyrapone and steroids. Of 6 different estrogens used as substrates, only 17 beta- and 17 alpha-estradiol were transformed relatively effectively by brain microsomes. In contrast liver microsomes utilized these two estrogens as well as ethynyl estradiol, estrone and diethylstilbestrol effectively. Estriol was a poor substrate for estrogen-2/4-hydroxylase activity in both tissues. With 40 microM 17 beta-estradiol as substrate the estrogen-2/4-hydroxylase activities in brain and liver were inhibited by alpha-naphthoflavone, metyrapone, progesterone, 17 alpha-hydroxyprogesterone and testosterone. The brain enzyme activity appeared to be more sensitive than the liver enzyme to inhibition by alpha-naphthoflavone and metyrapone. Testosterone propionate (50-100 microM) stimulated the brain enzyme activity significantly. Progesterone and 17 alpha-hydroxyprogesterone were the most effective steroidal inhibitors of brain estrogen-2/4-hydroxylase activity. In the liver the inhibitory potencies of 3 different steroids varied, depending on the estrogen used as substrate. With 17 beta-estradiol, for example, progesterone was the most potent steroidal inhibitor, while corticosterone was the most potent inhibitor when diethylstilbestrol was used as substrate. These findings indicate that rat liver microsomes can utilize a wider range of different estrogens for catecholestrogen formation than brain microsomes and suggest that the profiles of catecholestrogen-forming P-450 isozymes in the two organs differ.     

Identification of radioactivity in cell nuclei from brain, pituitary gland and genital tract of male rhesus monkeys after the administration of [3H]testosterone

Enzymes are present in the primate brain that convert testosterone into 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone), estradiol-17 beta and 4-androstene-3,17-dione. To identify the metabolites of testosterone that accumulate in cell nuclei obtained from different regions of the brain, 9 adult castrated male rhesus monkeys were injected with 5 mCi [3H]testosterone as an intravenous bolus. After 1 h, brains were rapidly removed and the left halves were used for autoradiography while the right halves were dissected to provide 14 samples. Radioactive metabolites in cell nuclei were identified by high-performance liquid chromatography (HPLC) and by repeated recrystallization. In autoradiograms of brain, most of the labeled neurons were in the hypothalamus, preoptic area and amygdala. These three regions also had the highest levels of radioactivity. The major form of this radioactivity was [3H]estradiol-17 beta (Type I tissues) and the major radioactive androgen present was [3H]testosterone. In all other brain regions and pituitary gland, the major form of radioactivity was unchanged [3H]testosterone (Type II tissues). In genital tract structures, [3H]dihydrotestosterone predominated (Type III tissues). These results suggested that, in contrast to its actions on genital tract structures, testosterone acts on neuronal nuclei mainly in unmetabolized form or after conversion to estradiol-17 beta.     

In vitro and in vivo studies demonstrating potent and selective estrogen inhibition with the nonsteroidal aromatase inhibitor CGS 16949A

CGS 16949A inhibited the conversion of [4-14C]androstenedione (A) to [4-14C]estrone by human placental microsomes in a competitive manner (Ki = 1.6 nM). Aminoglutethimide, also a competitive inhibitor, had a Ki = 0.7 microM in this assay system. The Km for the aromatization of A was 0.11 microM. Using ovarian microsomes from immature rats primed with pregnant mare's serum gonadotrophin and using [4-14C]testosterone conversion to [4-14C]estradiol as a measure of aromatase activity, the Km was 42 nM. At a substrate concentration 3-fold the Km, CGS 16949A was 180 times more potent as an inhibitor than aminoglutethimide, exhibiting half-maximal inhibition at 1.7 nM as compared to 0.3 microM. In vivo CGS 16949A lowered ovarian estrogen synthesis by gonadotropin-primed, androstenedione treated, immature rats by 90% at a dose of 260 micrograms/kg (PO). A dose of 100 mg/kg of aminoglutethimide was needed to produce this same effect. CGS 16949A at a dose of 4 mg/kg (PO) induced uterine atrophy (aromatase inhibition) without inducing adrenal hypertrophy - indicating a lack of inhibition of corticosterone secretion, while aminoglutethimide at 40 mg/kg (PO) induced adrenal hypertrophy without inducing uterine atrophy. CGS 16949A was neither androgenic nor estrogenic in rats using standard bioassays. The data suggest that CGS 16949A may serve as a potent and selective agent for modulating estrogen-dependent functions.     

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.     

Steroid metabolism in human teratocarcinoma cell line PA 1

Human ovarian teratocarcinoma cells of line PA 1, (Zeuthen et al., 1979[1]) used as model for early embryonic cells, were analyzed for their in vitro capacity to convert steroids. The cells were incubated for 20 h with radioactive pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone or estradiol-17 beta, or with non-radioactive progesterone, 6 alpha- or 6 beta-hydroxyprogesterone, 3 beta-hydroxy-5 alpha-pregnan-20-one, dehydroepiandrosterone or estradiol-17 beta. The metabolites were analyzed by thin layer chromatography or studied by gas chromatography-mass spectrometry. The results indicate that PA 1 cells are able to metabolize, although to a restricted amount, a variety of steroids, most markedly progesterone. The metabolites were almost exclusively found in the medium. The main metabolite of progesterone was 3 beta, 6 alpha-dihydroxy-5 alpha-pregnan-20-one. Minor formation of progesterone from pregnenolone could be detected. Human chorionic gonadotropin did not have any effect on pregnenolone metabolism. No formation of estradiol-17 beta or estrone from dehydroepiandrosterone, androstenedione or testosterone could be detected. However, estradiol-17 beta was shown to be converted mainly to estrone. These findings indicate that undifferentiated PA 1 teratocarcinoma cells like certain mouse teratocarcinoma cells, seem not to be steroidogenic but are capable of metabolizing naturally occurring steroid hormones and their precursors.     

Production of testosterone from progesterone by rat testicular microsomes without release of the intermediates 17 alpha-hydroxyprogesterone and androstenedione

It has been shown that during the in vitro conversion of progesterone to androstenedione, 17 alpha-hydroxyprogesterone is not an obligatory intermediate which equilibrates with freely diffusible steroids in the incubation medium. Recently a cytochrome P-450 was purified that catalyzed, in addition to hydroxylase/lyase activities, reduction of androstenedione to testosterone. In order to determine whether progesterone could be transformed to testosterone without both intermediates (17 alpha-hydroxyprogesterone and androstenedione) being equilibrated with steroids in the medium, several double-label double-substrate experiments were performed. When rat microsomes were incubated with an equimolar mixture of [14C]progesterone and 17 alpha-hydroxy[3H]progesterone, androstenedione was isolated with a 11-fold higher 14C/3H ratio than 17 alpha-hydroxyprogesterone, indicating that androstenedione could not be produced from free, diffusible 17 alpha-hydroxyprogesterone. Incubation of an equimolar mixture of 17 alpha-hydroxy[3H]progesterone and [14C]androstenedione with testicular microsomes resulted in the incorporation of 3-4-fold more 17 alpha-hydroxyprogesterone into testosterone than of androstenedione, although the latter is the immediate precursor of testosterone. In an experiment in which equimolar concentrations of [3H]progesterone and [14C]androstenedione were incubated with testicular microsomes, the large pool of progesterone inhibited competitively lyase activity, but still the label of progesterone was incorporated into testosterone to the same extent as that of androstenedione. These results indicate that testosterone can be produced by immature rat testicular microsomes from added progesterone on an organized unit without the intermediates equilibrating with the incubation medium.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Intestinal and hepatic microsomal metabolism of testosterone and progesterone by a 3 alpha-hydroxysteroid dehydrogenase to the 3 alpha-hydroxy derivatives in the channel catfish,Ictalurus punctatus

Intestinal or hepatic microsomes from channel catfish converted [4-14C]-testosterone to three major metabolites: 6 beta-hydroxytestosterone, androstenedione and a third metabolite. Formation of the unknown metabolite required NADPH as cofactor. When incubated with 200 microM testosterone, the rate of formation of the unknown metabolite was 265+/-158 pmol/(min mg) protein (mean+/-S.D.) in microsomes from the proximal intestine, 515+/-93 pmol/(min mg) protein in distal intestine and 226+/-42 pmol/(min mg) protein in hepatic microsomes. Comparison of the chromatographic and spectral properties of the unknown metabolite with those of authentic testosterone derivatives showed that this metabolite corresponded to 4-androstene-3 alpha,17 beta-diol. No 3 alpha-reduced metabolite was formed in incubations of testosterone with catfish intestinal cytosol. Testosterone was reduced to 5 alpha-dihydrotestosterone primarily in the cytosolic fraction and not in microsomes. Incubation of progesterone with intestinal microsomes resulted in the formation of a metabolite with properties similar to that of the 3 alpha-reduced testosterone, and this metabolite was identified by co-chromatography with authentic standard as 3 alpha-reduced progesterone. Thus, 3 alpha-hydroxysteroid dehydrogenase is an important pathway in intestinal microsomes of the channel catfish.     

Kinetic analysis of 3 beta-hydroxysteroid dehydrogenase activity in microsomes from complete hydatidiform mole

Microsomes isolated from complete hydatidiform moles (CHM) were able to convert [3H]pregnenolone to [3H]progesterone which indicates the presence of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) activity. The kinetic parameters found (Km = 0.63 microM and Vmax = 1-3.05 nmol/min/mg of protein) were like those observed in microsomes from normal early placenta (NEP) of similar gestational age (herein) and term placenta suggesting that the enzymes from the three sources are kinetically similar. Testosterone, progesterone and estradiol in a dose range of 0.05-5 mumol/l inhibited differently the in vitro conversion of [3H]pregnenolone to [3H]progesterone in a dose-dependent manner. The steroid concentrations necessary to inhibit the conversion of pregnenolone to progesterone by 50% (ID50) in CHM were 0.1 microM for testosterone, 0.6 microM for progesterone and 3 microM for estradiol, whereas in NEP they were 2.5, 1 and 5 microM respectively. The Ki values calculated from these ID50 in CHM together with the reported levels of endogenous steroids indicate that the accumulation of testosterone and progesterone inside the molar vesicle could physiologically regulate the rate of further conversion of pregnenolone to progesterone. The present findings could provide an explanation for the low level of progesterone in patients with CHM in the second trimester of pregnancy which in turn may directly or indirectly affect the spontaneous expulsion of this aberrant tissue.     

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.     

Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.     

Progesterone Inhibits Folic Acid Transport in Human Trophoblasts

The aim of this work was to test the putative involvement of members of the ABC superfamily of transporters on folic acid (FA) cellular homeostasis in the human placenta. [(3)H]FA uptake and efflux in BeWo cells were unaffected or hardly affected by multidrug resistance 1 (MDR1) inhibition (with verapamil), multidrug resistance protein (MRP) inhibition (with probenecid) or breast cancer resistance protein (BCRP) inhibition (with fumitremorgin C). However, [(3)H]FA uptake and efflux were inhibited by progesterone (200 microM). An inhibitory effect of progesterone upon [(3)H]FA uptake and efflux was also observed in human cytotrophoblasts. Moreover, verapamil and ss-estradiol also reduced [(3)H]FA efflux in these cells. Inhibition of [(3)H]FA uptake in BeWo cells by progesterone seemed to be very specific since other tested steroids (beta-estradiol, corticosterone, testosterone, aldosterone, estrone and pregnanediol) were devoid of effect. However, efflux was also inhibited by beta-estradiol and corticosterone and stimulated by estrone. Moreover, the effect of progesterone upon the uptake of [(3)H]FA by BeWo cells was concentration-dependent (IC(50 )= 65 [range 9-448] microM) and seems to involve competitive inhibition. Also, progesterone (1-400 microM) did not affect either [(3)H]FA uptake or efflux at an external acidic pH. Finally, inhibition of [(3)H]FA uptake by progesterone was unaffected by either 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), a known inhibitor of the reduced folate carrier (RFC), or an anti-RFC antibody. These results suggest that progesterone inhibits RFC. In conclusion, our results show that progesterone, a sterol produced by the placenta, inhibits both FA uptake and efflux in BeWo cells and primary cultured human trophoblasts.