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Selective association of germ-free (GF) rats with dehydroepiandrosterone sulfate (DHEAS) desulfating bacteria allowed us to assess the exact impact of intestinal bacterial desulfation on the excretion and enterohepatic circulation of orally administered DHEAS. Germ-free rats selectively associated with the DHEAS-desulfating strain Peptococcus niger H4 (H4 rats) excreted 50% of the total label recovered within 17 h vs 21 h in GF rats and 13 h 23 min in conventional (CV) rats. Germ-free rats excreted 30% of the total label recovered via their urine. However, association of GF rats with the desulfating microorganism increased urinary excretion to 46%, comparable to the 45.5% found in CV rats. Fractionation of fecal label yielded 70% sulfoconjugated DHEAS and 2% unconjugated dehydroepiandrosterone in GF rats vs 5 and 77% in CV rats, and 55 and 14% in H4 rats, respectively. Our results demonstrate that the intestinal bacterial desulfation of DHEAS stimulated the enterohepatic circulation of DHEAS. This in turn increased the urinary excretion of label resulting in an accelerated elimination of labeled DHEAS from the body.

It has been reported that human-oocyte complexes (COC) retrieved at a stimulated cycle manifest an asynchrony between oocyte meiotic maturation and cumulus mucification. However, when mature COC were subdivided into subtypes marked by the culture morphology of their cumulus cells following 3 days' culture, successful fertilization and cleavage were approximately 1.5-fold lower in mature COC yielding cumulus cells aggregated into clumps (type A and B COC) than in mature COC yielding homogeneously spread cells (type C-D COC). To determine whether the existing relationship between cumulus culture morphology and oocyte functionality in the various COC types (A-D) could be extended to another follicular compartment--the granulosa-lutein (G-L) cells--basal steroid secretion by the corresponding G-L cells was evaluated within 5 days of culture. Over the first 3 days of culture, secretion of progesterone was 3-fold lower and secretion of testosterone (T) was 2.5-fold higher in cultures of G-L cells from follicles yielding type A COC than in type C-D COC. During days 4 and 5 of culture, G-L cells were incubated with or without 10(-7) M 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), dehydroepiandrosterone (DHA), 4-androstene-3,17-dione (androstenedione), or T. The pattern of progesterone level noted over the first 3 days of culture was not altered in the presence of pregnanolone. DHA, androstenedione, or T. Addition of pregnenolone, DHA, androstenedione, or T increased T level 2.5-, 5.6-, 7.3-, and 17.7-fold, respectively, in cultures of G-L cells from follicles yielding type A COC, but did not significantly alter T level in cultures of G-L cells from follicles yielding type C-D COC. In cultures of G-L cells from follicles yielding type A COC, addition of androgens unsaturated at position 4 preferentially increased oestradiol-17 beta (E2) level, whereas in cultures of G-L cells of type C-D COC, DHA and androstenedione preferentially increased E2 level. Taken together, the asynchrony between oocyte and cumulus activity could be diminished when the activity of various follicular cell compartments is evaluated according to cumulus culture morphology rather than cumulus expansion and mucification. The present study suggests that follicles yielding mature COC represent a non-homogenous population in which G-L cells from follicles yielding type A-B COC manifest a less luteinized state than those from follicles yielding type C-D COC.

Steroid sulfatase activity was measured in 89 human samples, using dehydroepiandrosterone sulfate (DHEAS) as substrate. The lowest activity was that of follicular fluid which was significantly lower than that of other tissues tested (each P less than 0.01). The steroid sulfatase activity of ovarian tissue taken collectively (corpus luteum, stroma, and follicles) was higher than that of other tissues taken collectively (abdominal skin, uterus, and fallopian tube) (P less than 0.001), and the steroid sulfatase activity of either the follicle (P less than 0.01) or the stroma (P less than 0.05) was significantly greater than that of the corpus luteum. The geometric mean steroid sulfatase activity of the placenta was significantly higher than other tissues tested (each P less than 0.01) and was 22-fold higher than that of the follicle, the tissue with the next highest activity. These data indicate that the human ovary (particularly the stroma and follicle) is capable of utilizing DHEAS, an adrenal product, as a substrate for production of other androgens such as dehydroepiandrosterone (DHEA), androstenedione, and testosterone.

Several lines of evidence in animal and human studies, including measurements of elevated adrenal androgens in salt wasting syndromes suggest that adrenal androgen levels may in part be regulated by angiotensin II. This possibility was tested directly by measurement of cortisol and dehydroepiandrosterone (DHA) in canine adrenal cell suspensions after addition of angiotensin II and ACTH, separately and together. Minimal cortisol and no DHA stimulation was obtained using 4 x 10(-10) to 4 x 10(-5) M concentrations of angiotensin II alone. However, increased cortisol and DHA secretion were observed beginning at 4 x 10(-10) M concentrations of angiotensin II when it was combined with 10(-13) MACTH. These are physiological concentrations of both stimuli and may explain the increased adrenal androgens seen in some pathological situations characterized by elevated PRA.

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.

Trilostane is known as an inhibitor of 3 beta-hydroxysteroid dehydrogenase. Conflicting data published on this drug led us to look for the effects of 0.02 to 0.5 mM of trilostane on the in vitro steroid synthesis in sheep adrenals and human adrenals (Cushing's or Conn's syndrome) in the presence of an NADPH-generating system. The synthesis of 4-androstenedione, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione were studied either from dehydroepiandrosterone or 4-androstenedione or 11 beta-hydroxyandrostenedione. The synthesis of 11-deoxycortisol, cortisol, cortisone, 4-androstenedione, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione were studied either from 17-hydroxyprogesterone or 11-deoxycortisol or cortisol. This study showed that trilostane inhibited 3 beta-hydroxysteroid dehydrogenase whereas it had no effect on 21-, 11- and 17-hydroxylase. Trilostane was responsible for an increased 11 beta-hydroxysteroid dehydrogenase activity in vitro, resulting in low yields of cortisol and 11 beta-hydroxyandrostenedione, and high yields of cortisone and 11-ketoandrostenedione. This unexpected effect of trilostane allowed us to show that erroneous conclusions (in this case: pseudo inhibition of 11 beta-hydroxylase) can be drawn if all the metabolic pathways from a determined precursor are not exhaustively documented when studying the effects of drugs on steroid synthesis in vitro. The decrease of cortisol synthesis by trilostane may thus be related to the effects of the drug on both 3 beta-hydroxysteroid-dehydrogenase (inhibitory effect) and 11 beta-hydroxysteroid-dehydrogenase (stimulatory effect). This latter effect was found to be species-dependent.

After incubating a dehydroepiandrosterone solution with an enzymatic preparation from Helix pomatia, the steroid recovery was markedly decreased. The transforming activity resulted specific for some 3 beta-hydroxysteroids and it appeared to lower after storing the preparation at 4 degrees C for some days. The presence of 4-androsten-3,17-dione within the incubation products suggests that the enzymatic preparation contains at least 3 beta-hydroxysteroid: NAD oxydoreductase and 3-oxosteroid-5-4-ene isomerase. The use of large amounts of freshly dissolved Helix pomatia juice solution may lead to unreliable results in steroid analyses.

The long-term effects of ACTH on steroidogenesis in bovine adrenocortical cells maintained in primary culture have been investigated. Cells in monolayer culture were incubated in the presence or absence of ACTH for up to 72 h, and the steroid content of the incubation medium was assayed at 12 h intervals. During the first 12 h, adrenocortical cells incubated in the presence of ACTH (10(-9) M and 10(-6) M) produced substantially more cortisol and corticosterone than did cells incubated in the absence of ACTH. The production of steroidogenic intermediates such as pregnenolone, progesterone, and 17 alpha-hydroxypregnenolone, as well as 17 alpha-hydroxyprogesterone, 11-deoxycortisol, and 11-deoxycorticosterone also was increased by short-term (12 h) treatment with ACTH. Thereafter, corticosteroid production by cells incubated in the continued presence of ACTH decreased in a time and concentration dependent fashion. The maximal rate of cortisol production by cells incubated in the presence of ACTH (10(-9) M and 10(-6) M) for 72 h was only one third that of cells incubated in the presence of ACTH for 12 h. More dramatically, by 36 h, corticosterone secretion by cells incubated in the presence of ACTH (10(-6) M) declined to less than 20% of that of nontreated cells, and the production of 11-deoxycorticosterone was no longer detectable. ACTH also induced refractoriness in the production of other C21-steroids (pregnenolone, progesterone, 17 alpha-hydroxypregnenolone, 17 alpha-hydroxyprogesterone, and 11-deoxycortisol) as well as of C19-steroids (dehydroepiandrosterone, androstenedione, and 11 beta-hydroxyandrostenedione). The ACTH-induced refractoriness in the production of C21-steroids lacking a 17 alpha-hydroxyl group occurred earlier than that of 17-hydroxylated C21-steroids. Despite the decline in total corticosteroid production, the long term effect of ACTH was to enhance the relative secretion of 17 alpha-hydroxylated steroids and C19-steroids. Adrenocortical cells incubated for 72 h in the presence of ACTH continued to secrete cortisol, 17 alpha-hydroxyprogesterone, 11-deoxycortisol, and 11 beta-hydroxyandrostenedione in increased amounts. In fact, 11-deoxycortisol became a major secretory product of the ACTH-refractory adrenocortical cell. These results are indicative that ACTH acts in diverse manners on the bovine adrenocortical cell to affect corticosteroid secretion. The initial stimulation of corticosteroid production appears to be reflective of an increase in overall substrate (cholesterol) utilization and probably is mediated, in part, by an increase in cholesterol side chain cleavage activity. The secretion of 17 alpha-hydroxysteroids and C19-steroids is enhanced further by an action of ACTH to increase 17 alpha-hydroxylase activity and possibly also 17,20-lyase activity. The ACTH-induced refractoriness in corticosteroid production, on the other hand, appears to result primarily from a decline in precursor (cholesterol) utilization.

After the rat preputial gland was treated with collagenase and trypsin, five bands of cells were isolated by centrifugation in Ficoll gradients. Homogenates of the heavier cells (Bands IV and V) which contained less lipids, were more active than the homogenates of the lighter cells (Bands I, II and III) in transforming [1,2-3H]-dehydroepiandrosterone ([1,2-3H]-DHA) into [3H]-androstenedione and [3H]-testosterone and the latter into [3H]-dihydrotestosterone (DHT). In the presence of NAD, NADH and NADPH-generating system, [1,2-3H]-DHA was transformed into [3H]-DHT in 50-60% yield by homogenates of cells in Bands IV and V. DHT levels in the preputial gland were measured by radioimmunoassay. The levels in female rats reduced by 77% from 3.14 +/- 0.27 to 0.72 +/- 0.10 pg/mg tissue after adrenalectomy, and by 45% to 1.71 +/- 0.10 pg/mg tissue after ovariectomy. In male rats, the level reduced by 15% from 4.58 +/- 0.55 to 3.88 +/- 0.62 pg/mg tissue after adrenalectomy and by 40% to 2.74 +/- 0.21 pg/mg tissue after orchidectomy. These results demonstrated the transformation of DHA into DHT in the preputial gland of the rat, and that the adrenal is an important source of precursor steroid (DHA) for DHT formation in the preputial gland.

The inhibition of estrogen 2-hydroxylase by androgens was demonstrated in screening assays and has been further investigated under initial velocity conditions. The ability of testosterone, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone to block the conversion of estradiol to 2-hydroxyestradiol by male rat liver microsomal preparations was determined using two radiotracer methods--the conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol and the release of 3H2O from [2-3H]estradiol. The apparent Ki's for the androgens ranged from 12.0 to 14.0 microM, with the apparent Km for the substrate estradiol in these assays of 2.08 microM. Multiple inhibition studies with the androgens and 2-bromoestradiol, an effective estrogen inhibitor, in male rat liver microsomes resulted in Dixon plots consisting of a series of nonparallel, intersecting lines. Thus, the androgens and 2-bromoestradiol are non-exclusive inhibitors, i.e. the binding of one compound to the enzyme does not interfere with the binding of the other. These interactions of androgens suggest that the steroid hormonal environment be considered in the examination of the physiological role(s) of the estrogen 2-hydroxylase and the catechol estrogen products.

The A-549 cell line was initiated from an explant of human lung carcinoma tissue. The biochemical characteristics of these cells are similar to those of normal alveolar type II epithelial cells. To gain some insight into the steroid-metabolizing capabilities of A-549 cells, the metabolism of tritium-labeled dehydroisoandrosterone and androstenedione by these cells was studied. The metabolism of dehydroisoandrosterone led to the exclusive formation of 5-androstene-3 beta,17 beta-diol. The major product of androstenedione metabolism was testosterone; and, 5 alpha-reduced steroids also were formed, viz. 5 alpha-androstane-3,17-dione, androsterone, isoandrosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol. Estrogens, viz., estrone and estradiol-17 beta, were not products of androstenedione metabolism by A-549 cells. The rates of metabolite formation from either dehydroisoandrosterone or androstenedione were linear as a function of incubation time up to 3 h, and with cell number up to 1 X 10(6) cells/ml. The apparent Km of 17 beta-hydroxysteroid oxidoreductase for dehydroisoandrosterone was 11 microM, and that for androstenedione was 13 microM. The predominant formation of 5-androstene-3 beta,17 beta-diol from dehydroisoandrosterone, and testosterone from androstenedione is a likely indication that the principal C19-steroid-metabolizing enzyme in A-549 cells is 17 beta-hydroxysteroid oxidoreductase; the other steroid-metabolizing enzymes expressed in these cells are 5 alpha-reductase, 3 beta-hydroxysteroid oxidoreductase and 3 alpha-hydroxysteroid oxidoreductase. The findings of this study demonstrate that A-549 cells express steroid-metabolizing enzymatic activities that are qualitatively similar to those found in other human pneumonocytes and human lung tissue, except for 3 beta-hydroxysteroid oxidoreductase-5----4-isomerase activity, which is not expressed in these cells with dehydroisoandrosterone as the substrate.

Although androstanediol (AD) and androstanediolglucuronide (ADG) are generally considered to be parameters of peripheral androgen action, their plasma levels do not always vary in parallel, suggesting that they may have different precursors. Few hard data being available concerning ADG precursors in women, we studied in postmenopausal women with absent or suppressed adrenal function, the blood conversion rates of testosterone (T), dihydrotestosterone (DHT), androstenedione (A) and dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) to AD and ADG respectively, as well as the conversion of AD to ADG. Moreover conversions of these precursors to testosteroneglucuronide (TG) and dihydrotestosteroneglucuronide (DHTG), respectively, were also studied. Our data show that, whereas plasma A and DHT are the major precursors of AD, plasma DHEAS and A are the major precursors of plasma ADG, accounting for 50 and 15%, respectively, of plasma ADG, A being the major precursor of plasma TG and DHTG, respectively. When the conversion rates, obtained in this study, were applied to the plasma concentration of precursors found in normal young and postmenopausal women, respectively, the calculated concentration of product steroids accounted for almost the totality of the actual plasma levels of ADG, TG and DHTG respectively. The difference in relative importance of their precursors, explains that plasma concentrations of AD and ADG do not always vary in parallel; moreover, the importance of DHEAS as precursor of ADG explains the suppression by dexamethasone and the increase after adrenocortical stimulation of plasma ADG levels.

Steroid alcohol sulfotransferase (SAS) has been isolated from the cytosol of a human breast carcinoma cell line, MCF-7. This enzyme from Sephadex G-200 chromatography displayed a mol. wt of 118 KDa. The conditions for optimal enzymic activity of SAS were determined to be 20 min incubations at 45 degrees C in 0.2 M Tris buffer (pH 7.5) containing 0.06 M Mg2+. Chromatofocusing chromatography also yielded a single peak of SAS with a pI of 5.8. Results from the incubations of a series of androstane analogues revealed that SAS required a 3 beta-hydroxyl on a steroid with the trans bridge between the A and B rings. Neither the 3 beta-allylic hydroxyl group nor the A-ring phenolic 3-hydroxyl accepted the sulfate group from 3'-phosphoadenosine-5'-phosphosulfate. D-ring beta-hydroxyl groups were tolerated by the enzyme, however, alpha-hydroxyl groups on the D-ring appeared to interfere with the reaction. Sulfurylation of steroids by SAS was related inversely to the sum of the displacements of the 3-hydroxyl plus that of the 17-hydroxyl groups relative to the plane of symmetry of the dehydroepiandrosterone nucleus. This enzyme was also capable of sulfurylating short chain aliphatic alcohols, although at greatly reduced rates. 3 beta-Chloro-5-androstene-17-one and 2-nitroestradiol. 17 beta proved to be the best inhibitors of SAS.

Three multivariate statistical techniques, which have been found very useful in the analysis of hormone data, are demonstrated. The value of these techniques as a means of compressing the information contained in a complex array of experimental units and variates into a few "dimensions" is emphasised.

In order to quantitate the extent of intracellular metabolic conversions of C19 steroids in human endometrium, specimens of proliferative and secretory tissue were superfused at a constant rate with several pairs of labeled compounds at low concentrations. About 16% of dehydroepiandrosterone sulfate interacting with endometrial cells was converted to dehydroepiandrosterone and about 3% of this compound was converted to androstenedione. Androstenedione was reversibly reduced to testosterone and the extent of this conversion was shown to be several-fold higher in secretory than in proliferative tissue. About 1% of testosterone entering the cells was reduced to 5 alpha-dihydrotestosterone. These results demonstrate that the conversion of the main circulating C19 steroids in women, i.e. dehydroepiandrosterone sulfate and androstenedione, to 5 alpha-dihydrotestosterone, the compound considered to be the true intracellular androgen, is very small. In contrast, formation of testosterone from androstenedione is extensive and increases during the luteal phase under the influence of progesterone, a hormone known to stimulate the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrium.

The in vitro testicular steroidogenesis of male rats, androgenized on the third postnatal day by a single injection of 1 mg testosterone propionate, was investigated when the animals were 100 days old. The neonatal androgenization resulted in a 25% lower testes weight, significantly increased plasma levels of FSH (P less than 0.01) and LH (P less than 0.02), and normal levels of testosterone. Although the testes were hypotrophic, the incubation of the testes pairs yielded the same amounts of testosterone, 7 alpha-hydroxytestosterone and 5 alpha-androstane-(3 alpha + 3 beta), 17 beta-diol as in the control animals. However, the steroidogenic response to an acute hCG stimulation was reduced. From incubations of testes homogenates with various labelled steroid precursors it could be inferred that the activity of the 17 alpha-hydroxylase, the 3 beta-hydroxysteroid dehydrogenase-isomerase and the 17 beta-hydroxysteroid dehydrogenase, expressed per unit of incubated protein, was significantly increased in the testes of the androgenized rats. These data indicate that the basal steroidogenesis in neonatally androgenized male rats is maintained by an increased synthesis per unit of tissue, possibly under influence of an increased gonadotrophic stimulus, but that the maximum steroidogenic capacity is reduced.

Previous studies have shown that the C19 adrenal steroid 5-androstene-3 beta, 17 beta-diol (5-ene-diol), a metabolite of dehydroepiandrosterone (DHEA), can stimulate typical estrogenic responses in target tissues. Since estrogens are known to cause a specific stimulatory effect on LHRH-induced LH release in rat anterior pituitary cells in culture, we have taken advantage of the precision of this system to study the effect of 5-ene-diol or DHEA on this precise estrogen-sensitive parameter. Pretreatment for 48 h with 17 beta-estradiol (E2), 5-ene-diol or DHEA induces a 2.4-, 2.7- and 2.6-fold stimulation of LH release induced by 0.3 nM LHRH, the effect being exerted at respective 50% maximally effective concentrations (ED50 values) of 0.015, 45 and 115 nM. Following a 48-h preincubation with 10 nM E2, 1 microM 5-ene-diol or 1 microM DHEA, the maximal LH and FSH responses to LHRH are increased by approx 50% above control. On the other hand, the sensitivities of the LH and FSH responses to LHRH as assessed by ED50 values of LHRH action are increased by 3.3- to 7.5-fold. As further proof of the estrogenic nature of the effect of 5-ene-diol and DHEA, the effects of E2, 5-ene-diol and DHEA are inhibited competitively by simultaneous incubation with the antiestrogen LY156758 (keoxifene). The 2-fold stimulation of LHRH-induced LH release caused by DHEA-S, at concentrations within the range found in the plasma of women, is also completely blocked by 120 nM LY156758. In direct binding studies, 5-ene-diol and DHEA or DHEA-S have approx 85- and greater than 10,000 lower affinities than E2, respectively, for the estrogen receptor in rat anterior pituitary homogenate and human breast carcinoma cytosol. The present data clearly show that 5-ene-diol, DHEA and DHEA-S can exert full estrogenic activity in rat gonadotrophs, thus supporting the potential estrogenic role of these C19 adrenal steroids in estrogen-dependent processes, especially breast cancer.

In order to elucidate the role of G6PD inhibition by DHEA on erythrocyte redox metabolism, we measured: (1) G6PD activity in erythrocytes collected at different times after injection of synthetic ACTH in 13 normal subjects; (2) G6PD activity and GSH levels in erythrocytes incubated in the presence of different DHEA concentrations; (3) DHEA distribution and metabolic clearance in red cells, together with G6PD activity; (4) GSH regeneration after hydroperoxide oxidative stress in red cells preincubated in the presence of DHEA. In vivo DHEA increase elicits a clear-cut inhibition of red cell G6PD activity. Decreasing DHEA goes in step with the recovery of enzyme activity. In vitro G6PD inhibition by DHEA reaches its maximum within 10-15 min (20-25% inhibition at 10(-7) M DHEA concentration) and the recovery time is dose-dependent. More than 2/3 of DHEA is concentrated in the red cell after 5 min incubation. GSH levels change in step with G6PD activity. After oxidative stress by BHP, DHEA-treated cells fail to restore normal GSH concentrations. These results show that DHEA inhibits human erythrocyte G6PD activity at concentrations usually observed after ACTH plasma increase and increases red cell sensitivity to oxidant agents. Moreover, it is possible that the high DHEA concentrations present in target tissues may interfere with metabolic pathways in which NADPH is the cofactor.

The purified multifunctional enzyme, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3 beta-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.

An experimental model for in vivo screening of aromatase inhibitors was developed which overcomes the interference of compounds centrally active via the pituitary-gonad axis. Mature female surgically or chemically hypophysectomised (hypx) rats were treated with the oestrogen precursor dehydroepiandrosterone sulphate (DHEAS), immediately followed by administration of the test compound. During the treatment period vaginal smears were prepared daily. In the hypx rats DHEAS was metabolised to oestrogens, which induced vaginal cornification. By determining oestradiol levels it was shown that the aromatase inhibitors tested antagonised oestrogen synthesis and, as a result, cornification was counteracted. 4-Hydroxyandrostenedione and SH 489 showed equipotent aromatase inhibition, whereas 19-mercapto-androstenedione (ORG 30365) was at least twice as potent as the former compounds. By using various oestrogen precursors the inhibition of the enzyme aromatase was demonstrated. For in vivo screening of compounds on their aromatase inhibiting activity the assay in hypx rats appeared to be very suitable and selective but, because anti-oestrogens also antagonise vaginal cornification, anti-oestrogenic activity has to be excluded.

Radioimmunoassay methods for measuring dehydroepiandrosterone (DHA) and its sulphate (DHAS) in human plasma and tissues were developed and validated. Plasma levels were measured in men, pre- and postmenopausal women, and patients with endometrial or breast cancer. In the patients, tissue concentrations were also measured. Plasma DHA levels fluctuated synchronously with cortisol, but DHAS levels were less labile, and reached maximum levels during the day and were lowest at night. No obvious pattern was seen in relation to the menstrual cycle. Both DHA and DHAS levels fell with age, but DHA levels reached a plateau at about 60 years. No significant effect of weight on plasma levels was found. Plasma levels in the cancer patients were not significantly different from age-matched controls either when single samples were compared, or when mean 24 h levels were used. Endometrial tissue levels of DHA fell with age, unlike DHAS. In breast tumour tissue, DHA, but not DHAS concentrations were higher than in normal breast tissue. A good correlation between plasma and tissue DHAS levels was found, and DHA levels were also correlated in plasma and tissue. The correlations between plasma and tissue were not observed in tumour tissue, which may be due to altered tissue metabolism.

Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.

This investigation was designed to evaluate the possibility that the therapeutic benefits of dehydroepiandrosterone (DHA)-feeding in mice is mediated by (1) a metabolite of DHA formed in liver or (2) by way of the obligatory hepatic metabolism of DHA fed in large amounts. We found that the pattern of metabolism of DHA is strikingly different when DHA in low (tracer) quantities is incubated with mouse liver compared with that found when DHA in high concentrations is incubated with this tissue. In the former case, the principal metabolites are sulfoconjugates and other polar compounds, e.g. hydroxylated products. In the case of DHA metabolism when the substrate is present in high concentrations (1-100 microM), the principal metabolite formed is delta 5-androstenediol. And in this case, there is the formation of very nonpolar metabolites, which we have identified as the acetates of DHA and delta 5-androstenediol. We find further that the acetates are formed by a transacetylation mechanism in which performed acetylated compounds, e.g. pregnenolone acetate and ethyl acetate, can serve directly as co-substrates; but (at least in short-term incubations), Na+-acetate and acetyl CoA do not serve as co-substrates. We suggest that the therapeutic benefits of DHA-feeding in mice may be, in part, the result of alterations in hepatic intermediary metabolism that is obliged by the metabolism of DHA when this otherwise inert agent is fed in large amounts. Thus, DHA-feeding may serve to cause changes similar to those that are beneficial or therapeutic with dietary manipulations including caloric restriction.

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.

Sulphotransferase activity has been assayed in porcine liver and testis cytosol using either 3'-phosphoadenosine-5'-phospho [35S]sulphate (PAPS) or unlabelled PAPS as sulphate donors. In porcine liver the sulphotransferase for DHA was linear for up to 10 min, the optimum pH was 7.7 and optimum temperature, 37 degrees C. The apparent Km value was found to be 91 mumol/l and the activity was inhibited non-competitively by 5 alpha-androst-16-en-3 beta-yl sulphate, with all concentrations used (0.02-25 mumol/l) inhibiting the enzyme to the same extent. Time courses for sulphoconjugation of pregnenolone and 5 alpha-androst-16-en-3 beta-ol were linear for up to at least 10 min or up to only 5 min, respectively. The optimum pH values and temperatures were pH 8.0 and 37 degrees C in each case. The porcine testicular sulphotransferase activity for DHA as substrate was linear with time up to 10 min, the apparent Km for the reaction was 2 mumol/l and apparent Vmax 10 nmol/l/mg/min. 5 alpha-Androst-16-en-3 beta-yl sulphate (11.3-45.2 mumol/l) failed to inhibit the enzyme activity. The time-course for the reaction, when pregnenolone was used as substrate, was also linear up to 10 min at the optimum pH 8.0 but, in contrast to the reaction when DHA was the substrate, had an apparent Km of 20 mumol/l and was inhibited by pregnenolone sulphate, 5 alpha-androst-16-en-3 beta-yl sulphate, DHA and 5 alpha-androst-16-en-3 beta-ol, but not by DHA sulphate. 5 alpha-Androst-16-en-3 beta-yl sulphate inhibited the reaction non-competitively and to the same extent at concentrations over the range 11.3-45.2 mumol/l. These data suggest that DHA and pregnenolone may not be sulphoconjugated by the same sulphotransferase. With 5 alpha-androst-16-en-3 beta-ol as substrate, the time-course for its sulphate formation was linear up to 15 min, and this reaction could explain the quantities of 5 alpha-androst-16-en-3 beta-yl sulphate that are found endogenously in porcine testis. Our results further suggest that these quantities could well inhibit the sulphation of pregnenolone in porcine testis in vivo, and the possibility of control of sulphoconjugation in this tissue is discussed. Having regard to the smaller quantities of 5 alpha-androst-16-en-3 beta-yl sulphate present in porcine liver, our results suggest that the sulphation of DHA there may not be so much affected.

The effects of a single im injection of 10,000 IU of hCG on testicular steroid secretions were studied in 15 normal men and 54 patients with varicocele. Plasma levels of the following steroids were determined by radioimmunoassay: T, DHT, A, DHA, 5Adiol, E1, E2, 5P, 170H5P, 170HP, E1S, E2S, TE1, TE2, DHAS, 5AdiolS and hCG as well as FSH and LH in baseline samples. In normal men, peripheral venous blood was collected at 0, 1, 2, 4, 6, 8, 24, 36, 48, 72 and 96 h after hCG. Three types of steroid response were observed. The first, biphasic with an early peak within 2 h and a delayed one after 24 h, was shown by T, its unconjugated precursors and DHT. The second with an early rise plateauing for 6 h and followed by a 36 h peak characterized the estrogen pattern. Finally DHAS did not demonstrate a significant increase but 5AdiolS fluctuated at higher levels than the baseline ones. A significant correlation was only observed between hCG maximum value and E2 and TE1 maximum relative responses. In the patient group, peripheral blood was collected at 0, 1, 1.5, 2, 4, 6, 8, 10, 24 and 48 h after hCG. Baseline levels of all steroids were similar in the two groups. After hCG the same delayed pattern was observed as in the controls. The early relative response was low for T while A, DHT, E2 and E1S levels decreased. Conversely 170HP was higher until 24 h. Spermatic vein blood was collected before and at 1.5, 24 and 48 h after hCG. Steroid baseline levels in the varicocele side were similar to the other. After hCG, despite very large variations, general steroid patterns were comparable to those observed in peripheral vein blood. The important fall at 24 h of T/170HP and T/170H5P concomitant with that of T/E2 might be related to an inhibition of C17-20 lyase activity. The testicular secretion of all the measured steroids, including E1S, could be demonstrated except for DHAS. From our findings, a biogenetic defect evidenced by the fast response to hCG administration might exist in varicocele patients. In view of these results, the protocol of the hCG test should be improved using more frequent blood sampling and determining, besides T, its precursors and estrogen conjugates.

The enzyme 17 beta-hydroxysteroid dehydrogenase (17OHSD) was studied in human endometrium and adrenal cortex with respect to the metabolism of 5-androstene-3 beta,17 beta-diol (androstenediol) and dehydroepiandrosterone (DHA). The aim was to provide further information concerning the origin and biological significance of these androgens in endometrium, particularly the increased concentrations of the secretory phase and to compare the characteristics of the enzyme in the two tissues. In both endometrium and adrenal cortex the metabolism of androstenediol to DHA was linear with time and increasing enzyme concentration. The preferred cofactor was NAD and the apparent Km values were 3.4 +/- 0.2 (SD) microM (n = 3) for endometrium and 30.5 +/- 6.1 microM (n = 3) for adrenal cortex. In endometrium DHA was not metabolised to androstenediol in the presence of either NADH or NADPH whereas in the adrenal cortex both cofactors were utilised. However, the concentration of NADH required to achieve maximum enzyme activity was 10-fold higher (1 mM) than for NADPH (0.1 mM) and maximum activity with NADH was only 30% of that using NADPH. The apparent Km was 125 microM DHA (n = 2). The study indicates that androstenediol in endometrium does not arise from DHA metabolism but that its presence could be due to a binding protein particularly during the secretory phase. Our findings also suggest that the enzyme of endometrium differs from that of the adrenal cortex and that the kinetic properties may be related to the physiological requirements of the two tissues.

To elucidate the main metabolic pathways which lead to the foeto-placental biosynthesis of estetrol (I), we investigated the 15 alpha- and 16 alpha-hydroxylations of potential precursors of this estrogen in the human fetal liver. We determined the 15 alpha- and 16 alpha-hydroxylation capacity of the fetal liver for each precursor by GC-MS. The results suggest that estetrol is derived only from estradiol sulfate (II) and DHEA sulfate (III). 15 alpha-Hydroxy-androstenedione (IV) can no longer be regarded as a good precursor of estetrol. The phenolic pathway appears to be a more likely route than the neutral pathway, even when derived from DHEA sulfate.

Subcellular fractions of purified pig Leydig cells from 7 different animals have been investigated with respect to their abilities to catalyze the sulfation of several steroids and the hydrolysis of the sulfated forms of these same steroids. Considerable estrone sulfate sulfohydrolase of pH optimum 7.5 and high apparent Km was found to be concentrated in the 105,000 g pellet but no evidence was obtained, in any subcellular fraction, for the presence of any activity toward the 3-sulfate of pregnenolone, dehydroepiandrosterone (DHA) or delta 5-androstene-3 beta,17 beta-diol (androstenediol). Cytosolic sulfotransferase activity toward estrone, pregnenolone, DHA and androstenediol was present in each animal. The activity toward these 4 substrates was eluted from a gel filtration column as a single peak of apparent molecular weight 43 KDa. Upon chromatofocusing, a sharp estrogen sulfotransferase peak of apparent pI 6.1 and pH optimum 9.5, was clearly separated from the neutral steroid sulfotransferase which eluted over a more acidic pH range in a manner suggestive of the presence of several isozymes. This latter, which exhibited a wide pH optimum range between 6 and 8.5, was most active toward androstenediol, and least active toward pregnenolone. The estrogen sulfotransferase exhibited Michaelis-Menten kinetics (apparent Km = 4 microM). The neutral steroid sulfotransferase activity increased in velocity with increasing androstenediol or DHA concentration up to 1 microM beyond which considerable substrate inhibition occurred. It appears from these data that neutral steroid sulfates synthesized in the pig Leydig cell are not subject to enzymic desulfation in the same cells.