The in vitro synthesis of 7-hydroxy dehydroepiandrosterone by human mammary tissues

Normal and tumorous human mammary tissues were incubated in vitro with [7α-³H] dehydroepiandrosterone and [7α-³H] dehydroepiandrosterone sulphate. Tritium-labelled 7α-hydroxy dehydroepiandrosterone was identified as a principal metabolite from four of the five studies with normal tissue and all seven studies with tumorous tissue.     

The conversion of testosterone to 7α-hydroxy-testosterone by incubated rat testes

Incubations of testes of adult rats with testosterone yield rather important amounts of a very polar metabolite which is identified as 7α-hydroxytestosterone. The identification of the metabolite is based on chromatography, spectrophotometry, fluorimetry, counter current distribution and NMR spectrometry.     

High performance liquid chromatography of steroid metabolites in the pregnenolone and progesterone pathways

Rapid separation of gonadal steroids in the progesterone(Δ⁴) and pregnenolone pathway (Δ⁵) has been accomplished by the use of high performance liquid chromatography (HPLC). Two HPLC systems are utilized. The first requires the use of two separate radial compression columns (C-18 and C-8), with steroids being eluted with a methanol-water gradient. The second employs a stainless steel C-18 (reversed phase) column with a 12% octadecylsilane coating. The latter system separates seven of the eight steroids in the Δ⁴ and Δ⁵ pathways in thirty-five minutes. For the quantitation of steroids directly, integration of the peak areas, using 254 nm absorption for the Δ⁴ pathway steroids (5 ng minimum limit), and 210 nm absorption for the Δ ⁵ pathway steroids (25 ng minimum limit) is used. For the quantitation of radiolabeled metabolites resulting from incubation of gonadal tissue with radiolabeled steroid precursors, either one of two methods is used: (1) the eluent can be recovered from the HPLC using a fraction collector, and counted in liquid scintillation counter or (2) the entire eluent (or a portion of it) can be counted immediately by directing the flow through a radioactivity detector.     

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate.

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2 degrees C) for the synthetic androgen R1881 (17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17alpha,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 +/- 1.4 and 14.3 +/- 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 +/- 0.8 and 1.6 +/- 0.4 x 10(8) M-1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20-24 hr incubation at 15 degrees C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 +/- 3.3 fmoles per mg cytosol protein and 2.7 +/- 0.6 x 10(7) M-1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components.     

Steroid specificity of human placental 5-ene-3β-hydroxysteroid oxidoreductase

The properties of 5-ene-3β-hydroxysteroid oxidoreductase (3β-HSD) from human placental homogenates were studied invitro. The apparent Michaelis constants for 3β-HSD with the substrates pregnenolone (Δ⁵P) and dehydroepiandrosterone (DHA) were 170 nM and 40 nM respectively. The optimal pH for both these substrates was between 10 and 12. With NAD as the substrate, the Km for pregnenolone was 20 μM and for DHA, 17 μM. The activity of 3β-HSD was inhibited by various steroids. Competitive inhibitors (pregnenolone substrate) included: ethynylestradiol (inhibition constant Ki=7.3 nM), DHA (Ki=46 nM), estradiol-17β (Ki=46 nM), cholesterol (Ki=0.68 μM) and 16α-hydroxydehydroepiandrosterone (16αOHDHA) (Ki=2.2 μM). When the substrate was DHA, competitive inhibition occurred with the following steroids: ethynylestradiol (Ki=6.4 nM), estradiol-17β (Ki=69 nM), pregnenolone (Ki=91 μM), cholesterol (Ki=1.3 μM) and 16αOHDHA (Ki=1.9 μM). 4-Ene-3-ketosteroids such as androstenedione, progesterone (Δ⁴P), norethindrone and chlormadinone acetate acted as noncompetitive inhibitors towards both substrates.     

Studies on the mechanism of action of 1 alpha,24-dihydroxyvitamin D3. III. The specific binding of 1 alpha,24-dihydroxyvitamin D3 to the receptor of chick parathyroid gland

Three protein fractions of the cytosol of the chick parathyroid glands, which had the sedimentation constants of 2.5 S, 3.7 S and 5.5 S, were found to bind with 1 alpha,25-dihydroxyvitamin D3. Among these proteins, the 3.7 S protein was assumed to be the specific receptor protein. The 3.7 S receptor protein was also capable of binding to 1 alpha,24-dihydroxyvitamin D3 but not 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor protein was estimated to be 1.2 times greater than that of 1 alpha,25-dihydroxyvitamin D3, while 1 alpha,25-dihydroxyvitamin D3 bound to the receptor protein about 10 times stronger than 1 alpha,24(S)-dihydroxyvitamin D3. The dissociation constant for the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 2.7 x 10(-11) M, the dissociation constants were calculated to be 2.2 x 10(-11) M and 2.6 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3.     

Interactions of various 19-nor steroids with human placental microsomal cytochrome P-450 (P-450hpm).

Each of seven 19-nor steroids exhibited the capacity to facilitate the binding of carbon monoxide (CO) to human placental microsomal cytochrome P-450 although quantitative differences were shown to exist. In every case the facilitation was antagonized by androstenedione. 19-Norandrostenedione produced the most pronounced effect followed by 19-nortestosterone, nandrolone decanoate, norethandrolone, norgestrel, norethynodrel and norethindrone in that order. All steroids investigated produced typical type I binding spectra when added to placental microsomes. Scatched plots also indicated binding of each steroid to two sites--a high-affinity, low-capacity binding site and a low-affinity, high-capacity binding site. Correlations between affinity for either site and capacity to facilitate binding of CO to the cytochrome were not observed nor were there good correlations between maximal absorbance differences (approximately390-420 nm) producible and facilitation capacity. It was therefore concluded that no definitive relationships existed between facilitation capacity and qualitative or quantitative aspects of the steroid-binding spectra. The capacity to facilitate CO binding appeared to reside in the absence of a chemical group substituted at the 10 position on molecules of androgenic steroids since all investigated steroids possessing 10-methyl or other 10-substituted groups either had no effect on the CO-binding spectrum or caused a displacement of CO from ferrous heme. In contrast, all steroids studied that lacked a substitution at C-10 (19-nor steroids) produced a facilitating effect on heme-ligand binding.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Production of antisera against contraceptive steroids

A four step synthesis of 6-(O-carboxymethyl)oximinoethynylestradiol is reported. This compound, 6-(O-carboxymethyl)oximinomestranol, the 3-(O-carboxymethyl)oximes of norethindrone and norgestrel and the 3-hemisuccinate of ethynylestradiol were synthesized and conjugated with bovine serum albumin. Rabbits were immunized at 3 dose levels of haptene (20, 66 and 200 nmoles) and eight weeks later with a booster containing 66 nmoles of haptene. The antibody titer and association constant of responding rabbits was nearly independent of dose although most antibody production occurred after the booster injection. Antibodies to mestranol crossreacted more than 100% with ethynylestradiol and to a small extent with norethindrone and norgestrel.     

Specific antisera for the radioimmunoassay of estriol 3-sulfate

Antisera were raised in male guinea pigs against 6-oxoestriol 3-sulfate O-carboxymethyloxime-bovine serum albumin (BSA) conjugate. The antisera to this antigen exhibited high affinity (Ka=4.7 X 10(9)M-1) and excellent specificity for estriol 3-sulfate, showing slight cross-reactions (less than 0.43%) with other estrogen sulfates, and no cross-reactivities with free estrogens and other steroids (less than 0.01%) except cholesterol sulfate (0.22%). A standard curve using [6, 7-3H]-estriol 3-sulfate as the radioactive ligand showed high sensitivity in the range of 10-1000 pg estriol 3-sulfate.     

Sites of in vivo extraction and interconversion of estrone and estradiol in the dog

The interconversion and extraction of estrone and estradiol-17β across and within different tissues or areas have been studied in the dog by the constant infusion technique. The results were calculated using the ³H/¹⁴C ratios and radioactive concentrations of estrone and estradiol obtained from afferent and efferent blood and tissues at equilibrium. From these results it is concluded that: (1) there is no significant difference between metabolic clearance rates of estrone and estradiol, (2) blood transfer constants indicate a higher conversion of estradiol to estrone than of estrone to estradiol, (3) the transtissue interconversion favors the formation of estrone while the intratissue interconversion favors the formation of estradiol, (4) no interconversion of the two estrogens is observed in adipose tissue, (5) the extraction of estradiol entering a tissue was lower than the extraction of estradiol formed in these tissues, (6) calculation of the tissue metabolic clearance rates show that 63% and 61% of the total metabolism of estrone and estradiol, respectively, occurs in the splanchnic bed, and (7) the contribution of each tissue to the total interconversion of estrone and estradiol show that more than 90% of this interconversion occurs extrahepatically.     

Changes in plasma levels of estrone sulfate and estrone in the pregnant ewe around parturition

A method for the extraction, separation and measurement of estrone sulfate and estrone in a single plasma sample is described. The method has been applied to the determination of plasma levels of estrone sulfate and estrone in pregnant ewes over the period 60 hr before to 20 hr after parturition. The study revealed that the plasma levels of estrone sulfate and estrone began to increase about 40 hr before parturition, reached a peak at parturition and then declined rapidly to levels below the sensitivity of the method by 15 hr postpartum. The peak level of estrone sulfate recorded at parturition was 103 pmol (38 ng) per ml of plasma which was approximately 30 times greater than the corresponding peak level of estrone.     

Failure to detect delta 5-3 beta-hydroxysteroid oxidoreductase activity in the preimplantation rabbit embryo

Preimplantation rabbit embryos were incubated with pregnenolone and dehydroisoandrosterone under conditions which gave formazan precipitation by the histochemical technique. The metabolic fate of the labeled steroids was assessed simultaneously. There was no concomitant transformation of pregnenolone to progesterone and dehydroisoandrosterone was not transformed to androstenedione. It is concluded that the formazan precipitation is coupled with an "activity" other than delta 5-3 beta-hydroxysteroid oxidoreductase.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.