Formation and turnover of long-chain fatty acid esters of 5-androstene-3 beta, 17 beta -diol in estrogen receptor positive and negative human mammary cancer cell lines in culture

Microsomal preparations derived from bovine placenta cotyledons, previously investigated as a convenient source of fatty acyl coenzyme A: estradiol-17 beta-acyl transferase, have been shown to acylate other steroids bearing 3 beta- or 17 beta-hydroxyl groups. In the presence of 0.1 mM oleoyl CoA, the apparent Km values for dehydroepiandrosterone, testosterone, and 5-androstene-3 beta,17 beta-diol (delta 5-DIOL) were 45, 67, and 20 microM, respectively. Acylation of delta 5-DIOL occurred at either the 3 beta- or 17 beta-positions to give monoesters. Testosterone, estradiol-17 beta, and delta 5-DIOL acted as competitive inhibitors for the acylation of the 3 beta-hydroxyl group of dehydroepiandrosterone (Ki values 71, 75, and 41 microM, respectively). Such data indicate that a single enzyme of wide substrate specificity may be involved in these acylation reactions. When estrogen receptor (ER) positive and negative human mammary cancer cell lines were incubated with 10 nM [3H]delta 5-DIOL, intracellular accumulation of delta 5-DIOL long-chain fatty acid esters occurred; rates being higher (p less than 0.001) in ER negative cells (MDA-MB-231 and MDA-MB-330) compared to MCF-7 cells (ER positive), and higher (P less than 0.005) in MDA-MB-231 cells compared to ZR-75-1 cells (ER positive). After exposure to 10 nM [3H]delta 5-DIOL for 16 h, the total labeled steroid fatty acid fraction was composed predominantly of delta 5-DIOL-3 beta- and 17 beta-monoesters (approximately 85%), the remainder containing approximately equal amounts of delta 5-DIOL-diesters and dehydroepiandrosterone-3 beta-esters. Subsequent transfer to medium lacking delta 5-DIOL was accompanied by a breakdown of the labeled esters, which was more rapid in the ER positive cell lines. During this period, intracellular free delta 5-DIOL levels rapidly declined in MDA-MB-330 cells but were maintained in MCF-7 cells, presumably by binding to ER. This behavior parallels that of estradiol-17 beta previously observed in these cell lines and further emphasizes the potential importance of the adrenal-derived estrogen delta 5-DIOL in consideration of a hormone-based etiology of human breast cancer.     

Serum 5-androstene-3 beta,17 beta-diol sulphate and 5 alpha-androstane-3 alpha,17 beta-diol sulphate in hirsute females with polycystic ovarian disease

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), unconjugated androstene-dione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI), 17 alpha-hydroxyprogesterone (17OHP), luteinising hormone (LH) and follicle stimulating hormone (FSH) were measured by specific radioimmunoassay in 28 hirsute women with polycystic ovarian disease (PCO) and in normal women (n = 73). Mean levels of steroids measured were significantly elevated, and SHBG significantly depressed, in the women with PCO with values (mean +/- SE) for 5-ADIOL-S (516 +/- 51 vs 267 +/- 10 nmol/l), 3 alpha-DIOL-S (130 +/- 9 vs 52 +/- 2 nmol/l), DHEA-S (7.3 +/- 0.5 vs 4.4 +/- 0.2 mumol/l), AD (11.3 +/- 1.1 vs 3.4 +/- 0.2 nmol/l), T (3.3 +/- 0.2 vs 1.5 +/- 0.1 nmol/l) and 17OHP (5.1 +/- 0.8 vs 2.8 +/- 0.2 nmol/l). SHBG levels were 31 +/- 2.9 vs 65 +/- 2.5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by (SHBG nmol/l)] was 12.5 +/- 1.4 vs 2.4 +/- 0.1. The mean LH to FSH ratio was also elevated at 2.8 +/- 0.3. These studies suggest that the measurement of 5-ADIOL-S and DHEA-S may indicate adrenal gland involvement in PCO while 3 alpha-DIOL-S appears to be a reflection of peripheral androgen metabolism. A comprehensive biochemical profile of PCO should thus include the analysis of these sulphoconjugates as well as unconjugated steroids.     

Radioimmunoassay of 5-androstene-3 beta,17 beta-diol in plasma and in breast cyst fluid

A simple and reliable radioimmunoassay for the determination of 5-androstene-3 beta, 17 beta-diol in peripheral plasma and in breast cyst fluid, after a chromatography on Celite microcolumn has been described and evaluated. The antiserum used was raised in rabbits injected with dehydroepiandrosterone-15 alpha-(O-carboxymethyl)-bovine serum albumin. In men below 40 years of age the levels ranged from 0.85 to 2.80 ng/ml (mean +/- SEM: 1.52 +/- 0.11; n = 24) and from 0.50 to 2.20 ng/ml (mean +/- SEM: 0.93 +/- 0.09; n = 20) in men aged between 41 and 62 years. The mean level was significantly different (P less than 0.001) between the 2 groups. A significant correlation (r = -0.56; P less than 0.01) was demonstrated between age and all male levels. In females the mean plasma level was in the follicular phase: 0.81 +/- 0.07 ng/ml (range: 0.40-1.50; n = 17; age: 19-41 years) and in the luteal phase: 0.83 +/- 0.05 ng/ml (range: 0.40-1.30; n = 29; age: 18-43 years). No cyclical change and no correlation with age could be evidenced. A significant difference (P less than 0.001) was shown between females and the young male group. In breast cyst fluid the levels ranged from 0.05 to 13.70 ng/ml (mean +/- SEM: 2.36 +/- 0.86; n = 20) whereas the sulfate concentrations ranged from 75 to 7500 ng/ml (mean +/- SEM: 1891 +/- 565; n = 15), thus demonstrating very wide inter-individual variations.     

Selective turnover of the essential fatty acid ester components of estradiol-17 beta lipoidal derivatives formed by human mammary cancer cells in culture

The properties of fatty acyl coenzyme A: estradiol-17 beta acyl transferase in microsomes derived from pooled human mammary cancer tissue have been examined. A pH optimum of 5.5 was found and addition of long-chained fatty acyl CoAs increased estradiol-17 beta (E2) 17-monoacyl ester synthesis; the apparent Km for E2 being 8 microM when oleoyl CoA, linolenoyl CoA or palmitoyl CoA were employed. Testosterone, dehydroepiandrosterone, and 5-androsterone-3 beta, 17 beta-diol acted as competitive inhibitors with Ki values of 36, 36 and 46 microM, respectively. The composition of E2 fatty acyl esters (E2-L) formed by incubation of [3H]E2 with human mammary cancer tissue and human mammary cancer cell lines has been determined by HPLC. Although the composition of E2-L in estrogen receptor negative cell lines (MDA-MB-231 and MDA-MB-330) was generally similar to that found for MCF-7 cells (estrogen receptor positive) and pooled human mammary cancer tissue, the former cell lines contained a 3-fold higher relative concentration of E2-17 beta stearate. MCF-7 cells were exposed to 30 nM [3H]E2 and the composition of the isolated [3H]E2-L fraction studied at various time intervals. At 0.5 h, the intracellular concentration of E2-L was 1.8 +/- 0.4 (SEM) pmol/mg DNA which increased to values of 3.6 +/- 0.6 and 4.3 +/- 0.5 at 4 h and 16 h, respectively. In the subsequent 3 h following transfer to medium lacking [3H]E2, the concentration of E2-L declined to 3.7 +/- 0.3 pmol/mg DNA. The subfraction of E2-L composed of E2-17 beta arachidonate, linolenate and docosahexaenoate, was seen to decline in relative abundance after 0.5 h and to reach significantly lower relative levels at 16 h, and again in the 3 h period following estrogen withdrawal. The data suggests that these components, derived from essential fatty acids, are more metabolically active. This may then provide a new lead to link these novel estrogen derivatives with the established relationship between unsaturated fatty acids and an increased mammary cancer incidence.     

Serum levels of 5-androstene-3 beta,17 beta-diol sulphate, 5 alpha-androstane-3 alpha, 17beta-diol sulphate and glucuronide, in late onset 21-hydroxylase deficiency

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), as well as 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (3 alpha-DIOL-G) and unconjugated androstenedione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI) and 17 alpha-hydroxyprogester-one (17OHP) were measured by specific radioimmunoassays (RIA) in 14 women with late-onset 21-hydroxylase deficiency (LOCAH), and in normal women (n = 73). The diagnosis of LOCAH was made on the finding of a (17OHP) response level greater than 30 nmol/l following ACTH stimulation, and/or an elevation of urinary metabolites of 17OHP. Mean values for serum concentrations of all steroids measured and the free androgen index (100 X T nmol/l divided by SHBG nmol/l) were significantly elevated, and SHBG levels depressed in patients with LOCAH. These studies show that in LOCAH, in addition to the unconjugated steroids AD and T, the sulphoconjugated steroids DHEA-S, 5-ADIOL-S and 3 alpha-DIOL-S are increased, as is the glucuronide conjugate 3 alpha-DIOL-G and the index of bioavailable testosterone (FAI), and that mean SHBG levels are depressed. These data suggest that as well as AD, 5-ADIOL-S and DHEA-S may act as pro-hormones for more potent steroids (T and 5 alpha-dihydrotestosterone) in peripheral tissues, while 3 alpha-DIOL-S and 3 alpha-DIOL-G may both reflect peripheral androgen metabolism in patients with LOCAH.     

Determination of 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol in human plasma by radioimmunoassay

5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) were measured in human peripheral plasma by radioimmunoassay using celite microcolumn purification. The antisera used for the assay were obtained by immunization of rabbits with 3 alpha,17 beta-dihydroxy-5 alpha-androstane-6-(O-carboxymethyl) oxime: BSA for 3 alpha-diol and 3 beta,17 beta-dihydroxy-5 alpha-androstane-15 alpha-carboxymethyl: BSA for 3 beta-diol. The concentrations (pg/ml +/- SD) of the two diols in normal male and female plasma are respectively: 216 +/- 51 and 49 +/- 32 for 3 alpha-diol, 239 +/- 76 and 82 +/- 45 for 3 beta-diol. Comparison of these results with published ones shows that 3 beta diol concentrations were significantly lower. The high specificity of the assay is due to chromatography on celite microcolumns, allowing elimination of 5-androstene-3 beta,17 beta-diol from the plasma sample.     

Radioimmunoassays for androsterone, 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol

Androsterone (3alpha-hydroxy-5alpha-androstan-17-one), 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol were conjugated at C-16 through sulfur to bovine and human serum albumin. Rabbits injected with these conjugates produced antibodies suitable for radioimmunoassays of these hormone metabolites. Samples were purified on Sephadex LH-20 columns. Levels of these steroids were measured in a rat blood serum pool and in ovarian tissue extract pools.     

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

The direct effect of ethanol on dihydrotestosterone (DHT) conversion to 5 alpha-androstan-3 beta,17 beta-diol (3 beta-diol) and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) by adult rat Leydig cells was examined. Concentrations of ethanol comparable to blood levels of alcoholic men (2.2 - 65 mM) increased DHT conversion to 3 beta - and 3 alpha-diol, in direct relation to the dose of ethanol added; a 2-fold or greater stimulation was observed. Because this effect was blocked by 4-methylpyrazole or a saturating NADH concentration, these results suggest that this action is mediated by Leydig cell alcohol dehydrogenase activity. These results may have significant impact in the testis and/or other DHT sensitive tissues because ethanol may decrease the availability of the proposed active androgen.     

Induction of thymidine kinase synthesis by 5-androstene-3 beta,17 beta-diol in the uterus of the immature rat

In uteri from immature female rats, thymidine kinase activity was largely increased by administration of 5-androstene-3 beta, 17 beta-diol. Kinetic studies showed that the enzyme activity reached its maximum level 30 h after hormone administration. That increase in thymidine kinase activity was dose-dependent and could be related to the synthesis of new molecules of enzyme. Moreover, it was exclusively observed in target-organs for estrogens. It was concluded that 5-androstene-3 beta, 17 beta-diol which results from the metabolism of dehydroepiandrosterone had estrogen-like properties with regard to the induction of thymidine kinase.     

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.     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

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.     

Age-related changes in conversion of 5 alpha-androstan-17 beta-ol-3-one to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol by rat testicular cells in vitro

Conversion of labelled 5 alpha-androstane-17 beta-ol-3-one (DHT) by isolated testicular cells from rats of different ages was examined under saturating substrate conditions in vitro (5--10 micrograms DHT/ml in a 24 h incubation). Two detectable metabolites of DHT were produced by testicular cells in vitro. 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol). Production of these diols during a 24 h period was linear, and the amounts formed were directly related to the cell number. The amount of 3 alpha- and 3 beta-diols formed by testicular cells of rats of different ages increased from Day 10 to Day 25, then declined. Testicular cells from rats 10 to 20 days of age converted DHT mainly to 3 alpha-diol, but thereafter 3 beta-diol was the predominant testicular metabolite of DHT.     

Ethanol directly stimulates dihydrotestosterone conversion to 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstan-3 beta, 17 beta-diol in rat liver

The present results demonstrate for the first time in rat liver, that low ethanol concentrations (2.2 and 22 mM) directly stimulate dihydrotestosterone conversion to 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstan-3 beta, 17 beta-diol. Because this effect was blocked by 4-methylpyrazole, an alcohol dehydrogenase inhibitor, or by the addition of a saturating NADH concentration, this action probably is mediated by hepatic alcohol dehydrogenase activity through elevation of the NADH/NAD+ ratio. It remains to be determined whether this effect of ethanol actually reduces circulating and/or target tissue dihydrotestosterone levels; nevertheless, it is tempting to speculate that this action, in part, is responsible for the reported adverse effects of alcohol on male reproductive functions.