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.     

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.     

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.     

Endometrial tissue and plasma concentrations of 5-androstene-3 beta, 17 beta-diol during the menstrual cycle in normal premenopausal and perimenopausal women

A radioimmunoassay for 5-androstene-3 beta, 17 beta-diol (ADIOL) in human endometrium and plasma is described. The recognised criteria of reliability have been fulfilled. Plasma and endometrial tissue concentrations of ADIOL were determined in samples obtained from normal premenopausal and perimenopausal women (average ages 37 and 48 years respectively) at different phases of the menstrual cycle. In perimenopausal women plasma concentrations of ADIOL did not vary throughout the cycle (proliferative phase: 411 +/- 95 (SEM) pg/ml; secretory phase: 462 +/- 28.5 (SEM) pg/ml). For the premenopausal group the pattern was similar (proliferative phase: 568.4 +/- 56.9 (SEM) pg/ml; secretory phase: 663.1 +/- 64.7 (SEM) pg/ml) although a significant difference (P less than 0.05) was noted between late proliferative and late secretory phase levels in these women. A different pattern was observed for endometrial tissue concentrations of ADIOL. In both groups of women a significant (3-4-fold) increase occurred during the secretory phase. There was no apparent relationship between plasma and tissue concentrations of ADIOL either during the proliferative or the secretory phase. There was, however, an age associated decrease for both tissue and plasma ADIOL. Theories are proposed to account for the increase in ADIOL concentration during the luteal phase.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

The proliferative effects of 5-androstene-3 beta,17 beta-diol and 5 alpha-dihydrotestosterone on cell cycle analysis and cell proliferation in MCF7, T47D and MDAMB231 breast cancer cell lines

Epidemiological studies suggest that precursor steroids are implicated in the aetiology of breast cancer. However, our understanding of the role of precursor steroids in breast cancer is complicated by fact that there are many precursor steroids, which are metabolically inter-related and have divergent proliferative activities on the growth of breast cancer cell lines. In this study the proliferative affects of 5 alpha-dihydrotestosterone and 5-androstene-3 beta,17 beta-diol, which may be considered true metabolites acting at a tissue level, on MCF7, T47D and MDAMB231 breast cancer cell lines have been examined by a flow cytometric technique. DNA cell cycle analysis demonstrates that 5-androstene-3 beta,17 beta-diol stimulates the proliferation of hormone-dependent cell lines at physiological levels by an oestrogen receptor mediated mechanism whereas 5 alpha-dihydrotestosterone does not affect the proliferation of MCF7 and T47D cell lines at physiological levels over short (48 h) incubations. Both 5 alpha-dihydrotestosterone and 5-androstene-3 beta,17 beta-diol stimulate proliferation of hormone-dependent cell lines at pharmacological levels via and interaction with the oestrogen receptor. In long (6-9 days) incubations both 5 alpha-dihydrotestosterone and 5-androstene-3 beta,17 beta-diol inhibit the 17 beta-oestradiol induced proliferation of MCF7 and T47D cell lines, however, 5 alpha-dihydrotestosterone inhibits while 5-androstene-3 beta,17 beta-diol stimulates basal proliferation. These cell line studies suggest a model for the role of precursor steroids in pre- and postmenopausal breast cancer.     

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.     

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.     

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.     

Formation of glucuronides of estradiol-17 beta by human mammary cancer cells

The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.     

Hydroxylation of [3H]5 alpha-androstane-3 beta,17 beta-diol by whole tissue, epithelial cells and fibroblasts from the same hyperplastic human prostate

Hydroxylations of 3 beta-hydroxy 5 alpha-dihydro C19-steroids are terminal reactions by which male accessory sex organs dispose of intracellular androgens. Cellular androgen egress is of particular interest in benign prostatic hyperplasia (BPH) where the elevated nuclear 5 alpha-dihydrotestosterone-receptor content may be implicated in the etiology of the disease. We here report substitution of hydroxyl groups at C-6 alpha, C-7 beta and predominantly at C-7 alpha of [3H]5 alpha-androstane-3 beta,17 beta-diol on incubation of 3 and 8.5 nM substrate concentrations with minced and explanted human BPH tissue. Fibroblasts isolated from the same prostatectomy specimen hydroxylated 3 nM radiosubstrate mainly at C-6 alpha, with extensive metabolism to 17-oxosteroids. Epithelial cells from the same tissue source substituted to the same extent at the three positions. Competing 3 beta-hydroxysteroid dehydrogenase exceeded hydroxylase activity only in epithelial-cell cultures. Our findings support previous evidence that prostatic epithelial and stromal cells make different contributions to androgen disposition by the 3 beta-hydroxysteroid pathway.