Androgen metabolism in male and female breast tissue

Incubation studies have been carried out using normal breast tissue and breast tissue from patients with gynecomastia, mammary dysplasia and breast carcinoma to determine the pattern of androstenedione metabolism. All tissues formed estrone (E₁) and testosterone (T) in all incubations. Estradiol (E₂) was isolated in incubations of tissue from 1 to 6 patients with mammary dysplasia, 5 of 6 patients with gynecomastia and in all incubations with normal and carcinoma tissue. Estrone formation was lowest in mammary dysplasia and gynecomastia, and higher in apparently normal breast tissue. The greatest E₁ formation was found in incubations with breast carcinoma tissue, although there was considerable variation within this tissue group. Estradiol formation was low in all tissues, with the highest conversion rates in carcinoma tissue. Testosterone formation in carcinoma tissue was greater than in mammary dysplasia or gynecomastia, but similar to apparently normal tissue. These results indicate that breast tissue from different pathological states varies in its capacity to aromatize androstenedione (A) to estrogenic products and to convert it to other androgens. They have also shown that the pattern of metabolism is distinctive for the nature of the pathological abnormality.     

A radioimmunoassay for the measurement of 5-androstene-3β,17β-diol in plasma

A reliable radioimmunoassay for the determination of 5-androstene-3β, 17β-diol in plasma is described. Antisera were obtained by immunization of rabbits with 3β,17β-dihydroxy-5-androsten-16-one coupled to bovine serum albumin in position 16. The antiserum was characterized by titer, affinity, and specificity. Only dehydroepi-androsterone (24.3 %) and pregnenolone (2.7 %) showed a small crossreactivity. The assay method consisted of extraction with ether, thin-layer chromatography and endpoint determination.The reliability of the method was studied. The interassay variability was 7.5 % at a concentration of 1.22 μg/l. The limit of detection was 0.068 μg/l. Specificity was achieved by Chromatographic separation of the crossreacting steroids. Mass recovery experiments with 250 and 500 pg were performed, in which 99.0 ± 4.6 % of the smaller and 97.6 ± 11.3 % of the greater mass were recovered. In 45 healthy adult males plasma concentrations between 0.44 and 1.80 μg/l were found. The median was 1.06 μg/l. Stimulation of the Leydig cells with human chorionic gonadotropin (HCG) increased plasma concentrations by 93 % (average in 12 males). Therapeutic castration in 8 men caused an average decrease of 55.4 % in plasma values.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

Developmental pattern of Δ5 3β-hydroxysteroid dehydrogenase activity in isolated rat Leydig cells

A direct method for determination of Δ⁵ 3β-hydroxysteroid dehydrogenase (3β-HSD) activity was employed in isolated Leydig cells (LC) derived from rats on fetal day 19 (F₁₉) and postnatal (N) days 1,12,24, 34 and 45 and adults. The activity of 3β-HSD in the adult LC was 1.15 ± 0.02 (μmole/μg DNA/hr, mean ± SEM, n = 73). Activities in the other groups, expressed as a percentage of the respective adult control, were: F₁₉-38%; N₁-39%; N₁₂-8%; N₂₄-89%; N₃₄-166%; and N₄₅-118%. A good correlation was found between histochemical staining for 3β-HSD and the quantitive method employed. Using (³H)-DHA as a substrate, LC isolated from F₁₉, n₁ and N₁₂ produced testosterone in appreciable amounts (41%, 55% and 20% of the toal products respectively) whereas at advanced stages of development (N₂₄ to adulthood) the major product was androstenedione (93 ± 1%). These findings may be explained by the observed decrease in 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, due to an insufficient supply of NADPH, in the older vs. earlier stages of development. This study indicates the presence of steroidogenic enzymatic activity in LC throughout development in the rat. It also provides a relatively simple in vitro model for studies of testicular regulation during development.     

Developmental pattern of testosterone production by the rat testis

We studied the steroidogenic activity of isolated Leydig ceils derived from rats on fetal day 19 (F₁₉) and postnatal (N) days 1, 12, 24, 34, 45 and adults. Leydig cells, isolated at all ages by the collagenase method, increased in number throughout development with a doubling time of 8 days. Testicular content and serum concentrations of testosterone showed parallel changes during development. Moderate values were found at the early stages (F₁₉ and N₁), with a nadir on day 12, followed by a progressive increment to reach maximal values in adulthood. A reduction in steroidogenic activity of the testis during neonatal life was confirmed by urlar|_vitro studies with isolated Leydig cells. Maximal activity was found in group F₉; testosterone production diminished after birth to reach a minimum in group N₃₄ and rose thereafter to adulthood. Leydig cells were responsive to hCG stimulation at all ages in the following order: N₁>N₃₄>N₁₂>F₁₉>N₂₄>N₄₅>adult. The present study demonstrates the existence of an active and hCG-responsive population of Leydig cells in the rat testis from fetal life to adulthood.     

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.     

Synthesis of new steroid haptens for radioimmunoassay. part I. 15β-Carboxyethylmercaptotestosterone-bovine serum albumin conjugate. measurement of testosterone in male plasma without chromatography

A radioimmunoassay (RIA) procedure has been developed for measurement of testosterone in male plasma after ether:chloroform (4:1) extraction of the plasma sample without resorting to chromatography. The highly specific anti-testosterone serum was generated from both rabbits and sheep immunized with 15β-carboxyethylmercapto-testosterone-BSA conjugate. The synthesis of 15β-carboxyethylmercaptotestosterone and the preparation of its BSA conjugate are described. The high affinity (K_a = 2.38 × 10⁹ liters/mole) antiserum binds 50% of 50 picograms of tritiated testosterone at working dilutions of 1:100,000 to 1:200,000. Both 5α and 5β-dihydrotestosterone compounds exhibited less than 2% cross-reaction. The only other steroids that showed minor cross-reaction were 11β-hydroxytestosterone (3.8%), progesterone (2.1%), corticosterone (1.6%), and deoxycorticosterone (7.7%).     

Effects of 4-hydroxy-4-androstene-3,17-dione and 10-propargylestr-4-ene-3,17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues

The effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 10-propargylestr-4-ene-3,17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 μM, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations.Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E₁) formation by 80% at a concentration of 0.085 μM, with maximum effect at 0.34 μM. 90% inhibition of estradiol (E₂) formation was observed at inhibitor concentrations of 0.17 μM or greater. T formation was slightly affected at 0.67 μM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 μM.Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 μM was sufficient to inhibit aromatization while T inhibition required 11 μM.4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.     

Steroid delta 4-5 beta-reductase in human mammary tumors.

Steroid delta 4-5 alpha- and delta 4-5 beta-reductase activity was determined in 16 human mammary tumors and 8 DMBA-induced rat mammary tumors using a spectrophotometric assay. Steroid delta 4-5 alpha-reductase was present in all tumors investigated while delta 4-5 beta-reductase was detected in only 6 estrogen receptor negative human breast tumors and absent in all estrogen receptor positive human breast tumors as well as in all rat mammary tumors. Further support for the presence of delta 4-5 beta-reductase was established by using a dual-labelling technique consisting of incubating tumor slices with [14C] testosterone and adding [3H] etiocholanolone, [3H] testosterone and [3H]-5 alpha-dihydrotestosterone at the end of the reaction. Following extraction and chromic acid oxidation, 4-androstenedione, 5 beta-androstanedione and 5 alpha-androstanedione were isolated and purified, and the constancy of the 14C/3H ratio was used as proof of 5 alpha-reductase and 5 beta-reductase. These results were shown to be consistent with the data obtained using the spectrophotometric assay.     

The effect of ACTH administration on plasma testosterone, dihydrotestosterone and serum LH concentrations in normal men

Plasma cortisol (F), testosterone (T), dihydrotestosterone (DHT) and serum luteinizing hormone (LH) concentrations were measured in 8 normal young men at 8 AM on two control days. Exogenous adrenocorticotrophin (ACTH) 20 U.S.P. units per m² of body surface area every 12 or 6 hours was administered intramuscularly for 4 days. Twenty-four hours after starting ACTH administration, the plasma T and DHT concentrations were significantly lower than those of the control days on a paired t test. No significant change in serum LH concentration could be demonstrated. Similar results were observed after 48, 72 and 96 hours of ACTH stimulation.     

A radioimmunoassay method for urinary catechol estrogens

A radioimmunoassay method for urinary catechol estrogens is described. The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 μg per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758.20 μg per 24 hours.     

Variations in the concentrations of androstenedione in peripheral plasma of women the day and during the menstrual cycle

The target tissues (e.g., hypothalamus, pituitary, uterus and vagina) of mature female ovariectomized rats show selective uptake of radioactivity in one hour after the injection of 6,7, ³H-estradiol-17β in a dose of 0.1 μg per 100 g body weight. Injection of 100 μg norethindrone or norgestrel per 100 g body weight 15 min before or 15 min after the administration of tritiated estradiol reduced the radioactivity in most target tissues, and also in the non-target tissues to a lesser extent. The uptake of radioactivity in the pituitary and uterus is reduced more by norethindrone than by norgestrel treatment when these Steroids were injected 15 min after estradiol-17β injection. It appears that there exists a competitive inhibition of estradiol-17β by these contraceptive Steroids in the rat. It is speculated that such competition with estradiol-17β may be an inherent property of the 17-substituted 19-nortestosterone group of Steroids.     

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.     

Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone.

Methods für the determination of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone (DOC) in rats are described. The free corticosteroids were measured in urine samples of 0.1–0.5 (2.0) ml by radioimmunoassay after purification by column chromatography. The validity of the methods is demonstrated by the data of the free urinary corticoids under basal conditions and after adrenal suppression and various forms of adrenal stimulation. The basal excretion of free corticosterone, free aldosterone and free DOC was 123.71 ± 15.31 (x? ± SD), 3.87 ± 1.29 and 10.61 ± 2.24 ng/day, respectively, exhibiting a decrease to 26.20 ± 5.21, 1.05 ± 0.47 and 1.35 ± 1.20 ng/day after adrenal suppression by dexamethasone. Irrespective of the mode of adrenal stimulation i.e., synthetic ACTH and systemic (cold, hunger) or neurotrophic (ether, reserpine) stress stimuli free corticosterone increased to about 450 ng/day, while free aldosterone excretion decreased during hunger and cold and was strongly enhanced after the application of reserpine. Furthermore, determination of urinary free DOC, which increased by a factor of 4, may be applied in the metyrapone test. There was a good correlation between the excretion of free corticosterone and that of free aldosterone and free DOC under basal conditions and after ACTH application, demonstrating that ACTH is responsible for the secretion of all the 3 corticoids measured. It is concluded, that the measurement of the urinary excretion of corticosterone, aldosterone and DOC is a valuable parameter of adrenal function in rats. Furthermore, in small laboratory animals like rats steroid measurements in urine are often more advantageous than Measurements in plasma.     

Aromatization of androgens by human breast cancer.

The metabolism of dehydroepiandrosterone and testosterone by human mammary tumor was investigated. Estrogen synthesis from dehydroepiandrosterone was observed in 9 of 10 estrogen-receptor-negative tumors and only in 2 of 8 receptor-positive tumors (p less than 0.025). Conversion of testosterone to estrogens was observed in 7 of 8 receptor-negative and 2 of 7 receptor-positive tumors. Tumors which are capable of transforming dehydroepiandrosterone to estrogens were also able to aromatize testosterone suggesting that the presence of the aromatase enzyme is inherent to certain tumor cells. No estrogen formation was detected by the mitochondrial-microsomal fraction of normal breast cells while fractions from both fat cell and tumor cell showed estrogen synthesis. Estrogen formation by tumor cell fraction ranged from 5 to 190 times that observed for fat cells. The physiological significance of these results in the neoplastic tissue and its relationship to hormone dependence are discussed.     

Synthesis of new steroid haptens for radioimmunoassay. Part II. 15beta-Carboxyethylmercaptodehydroepiandrosterone bovine serum albumin conjugate. Specific antiserum for solid-phase radioimmunoassay of dehydroepiandrosterone.

Antiserum for radioimmunoassay (RIA) of dehydroepiandrosterone (DHA) was raised in rabbits with the conjugate obtained by coupling 15beta-carboxyethylmercapto-dehydroepiandrosterone with bovine serum albumin. The antiserum proved to have high affinity and specificity for DHA and showed only minor cross-reaction to androst-5-ene-3beta, 17beta-diol (3.5%). The Rivanol-treated antiserum was covalently coupled to Enzacryl Polyacetal to evaluate its utility in solid-phase RIA.     

Influence of age on the formation of 5alpha-androstanediol and 7alpha-hydroxy-testosterone by incubated rat testes.

Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.     

Conjugation of androgens and estrogens by human breast tumors in vitro

The metabolism of ³H-androstenedione (Δ⁴ -A) and ³H-estriol (E³) was studied in 12 human breast tumors. Part of each tumor was analyzed for estrogen receptor content. Aliquots of tumor homogenates were incubated for 2 hr separately with ³H-δ⁴-A and ³H-E₃ in the presence of appropriate cofactors. No distinct differences emerged in the profiles of the unconjugated metabolites of ³H-δ⁴-A, the major compounds in the approximate order of descendence being androsterone, androstanedione, testosterone, 5α-androstane-3α,17β-diol, epiandrosterone, and dihydrotestosterone. One tumor homogenate from an infiltrating lobular carcinoma converted ³H-Δ⁴-A to glucosiduronate metabolites (11%), of which androsterone, 6.4%; testosterone, 1.6%; and androstanediol, 0.6% predominated. The homogenate of this tumor and two other tumors converted ³H-E₃ to ³H-E₃-3S. Conversions of E₃ to E₃-3S In the other tumor homogenates were less than 0.6%. No correlation between receptor content and the capability of the tumor to conjugate Δ⁴-A or E₃ evolved. However, correlations between steroid hormone metabolism and tumor histopathology may exist.     

Characterization of an androgen binding protein (ABP) in rat testis and epididymis

An androgen binding protein (ABP) was demonstrated in the 105,000 g supernatant of rat testis homogenate after charcoal extraction of endogenous steroids. Testis ABP proved to be identical to an ABP previously described in rat epididymis. It contained saturable high-affinity sites which exhibited binding specificity for dihydrotestosterone (6) and testosterone when measured by polyacrylamide gel electrophoresis or by competitive binding using charcoal adsorption. Binding to ABP was not affected by ribonuclease or neuraminidase but was decreased by the disulfide reducing agent, dithiothreitol and the sulfhydryl reagent, N-ethylmaleimide. Binding was abolished by treatment with proteolytic enzyme. The mean molecular radius of ABP was 2.92 nm as determined by the retardation of electrophoretic mobility in polyacrylamide gels of decreasing pore size. Assuming a partial specific volume of 0.66–0.74 the molecular weight was 86,000–91,000 for a spherical molecule. ABP binding was stable after treating at 45° C for 20 min. but was destroyed at 60° C. Binding was maximal between pH 7.5 and 9.0 and decreased at pH below 7.0.     

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730–740 day old) rats is decreased 50% when compared to intact, young mature (150–170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 μg testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.