OBJECTIVE

The present study was designed to investigate the effects of two aromatase inhibitors on steroid hormone levels, bone mineral density (BMD) and bone turnover markers in intact female rats.

METHODS

Letrozole and anastrazole at two different dose levels were investigated for their effect on serum levels of estradiol, <em>androstenedione</em>, testosterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate, BMD of femur and dorsal spine, and osteocalcin and pyridinoline levels as bone turnover markers. Fifty intact female rats were randomly divided in five groups (group 1 (n = 10): control, 2 ml saline; group 2 (n = 10): letrozole 1 mg/kg; group 3 (n = 10): letrozole 2 mg/kg; group <em>4</em> (n = 10): anastrazole 0.1 mg/kg; group 5 (n = 10): anastrazole 0.2 mg/kg, and oral gavages were applied for a period of 16 weeks.

RESULTS

Both doses of letrozole and anastrazole did not change femoral and vertebral BMD. Serum estradiol levels were reduced significantly at all dose levels by both agents (p < 0.001); all androgen levels were significantly elevated by letrozole (p < 0.05), although anastrazole increased only androstenedione (p < 0.05). The higher dose of letrozole increased osteocalcin levels (p < 0.05), while pyridinoline levels were increased (p < 0.05) by the higher dose of anastrazole.

CONCLUSIONS

Our results indicate that short-term use of letrozole and anastrazole had no clear effects on BMD in intact rats. Further investigations are needed to understand their effects on bone metabolism in intact females.

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3 beta-hydroxysteroid dehydrogenase/delta 5----<em>4</em>-isomerase (3 beta-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3 beta-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3 beta-HSD present in H5<em>4</em>0 Leydig tumor cell homogenate and human placental microsomal 3 beta-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, and dehydroepiandrosterone to <em>androstenedione</em>. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to <em>androstenedione</em> with apparent Km values of 0.13 and 0.09 microM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3 beta-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3 beta-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3 beta-HSD content, a 3 beta-HSD is also expressed in liver in a sex-specific manner.

OBJECTIVE

The hypothalamic-pituitary-adrenal (HPA) axis seems to hyperfunction at both central and peripheral levels in polycystic ovary syndrome (PCOS). Hyperinsulinemia is involved in the adrenal hyper-responsiveness to ACTH. The present study was performed to investigate the role of insulin in the derangement of the hypothalamic-pituitary compartment of the HPA axis in PCOS.

METHODS

Prospective clinical study.

METHODS

Academic research center.

METHODS

Fifteen hyperinsulinemic PCOS women.

METHODS

Hormonal and lipid assays, oral glucose tolerance test, and corticotropin-releasing factor (1 microg/kg CRF) test before and after <em>4</em> months of treatment with the insulin sensitizer pioglitazone (30 mg/day).

METHODS

Glycemic and insulinemic response to glucose load; pituitary and adrenal response to CRF.

RESULTS

We observed a significant reduction in insulin secretion after therapy. Pioglitazone administration did not modify ACTH and cortisol response to CRF. A significant reduction in the adrenal CRF-induced secretion of androstenedione (A) (area under the curve [AUC] 202.76 +/- 78.68 ng/mL x 90 minutes to 1<em>4</em>7.05 +/- 52.06 ng/mL x 90 minutes) and 17OH-progesterone (AUC 152.92 +/- 59.56 ng/mL x 90 minutes to 117.10 +/- 63.25 ng/mL x 90 minutes') occurred after treatment. A trace response to CRF was observed for DHEAS and testosterone both before and after pioglitazone.

CONCLUSIONS

In PCOS subjects, insulin may enhance adrenal steroidogenesis by acting directly on the peripheral gland, with no significant effects on the pituitary response to CRF stimulation.

Twenty hirsute women were treated with 50 mg cyproterone acetate orally, administered from the 5th to the 25th day of the menstrual cycle, along with 3 mg 17 beta-estradiol administered percutaneously from days 16-25. Percutaneously administered 17 beta-estradiol was used rather than ethinylestradiol in order to avoid the side effects of oral administration of synthetic estrogens. From a clinical point of view there was a dramatic improvement of hirsutism after 3-6 months of treatment. Biologically, plasma testosterone decreased markedly (P < 0.01) from 6<em>4</em>.6 +/- 2<em>4</em>.2 ng/dl (n = 20) to 25.2 +/- 11.8 (n = 20), 26.1 +/- 16.6 (n = 16), and 13.3 +/- 10.8 ng/dl (n = 1<em>4</em>) after 3, 6, and 9 months of treatment. There was also a significant decrease in delta <em>4</em>-<em>androstenedione</em> from 251.0 +/- 110.2 ng/dl to 129.9 +/- 66.5, 11<em>4</em>.2 +/- <em>4</em>5.8, and 62.0 +/- 21.5 ng/dl after the same periods. From these results it may be assumed that this therapeutic combination has an antigonadotropic effect, as confirmed by the decrease in plasma estradiol, FSH, and LH and the absence of a significant progesterone level in all cases. Plasma and urinary cortisol, lipids, and hepatic tests remained normal. The good clinical and biological tolerance of this treatment makes it interesting to consider for use in the management of hirsutism.

Patients with severe idiopathic constipation are almost exclusively women of reproductive age. To investigate the possibility of a sex hormone abnormality in this condition, we have compared a range of sex hormones during the follicular and luteal phases of the menstrual cycle in 23 healthy women (mean age 33 years) with those in 26 patients with severe idiopathic constipation (mean age 32 years, spontaneous bowel frequency less than one per week). In the patients there was a reduction in the follicular phase of progesterone (<em>4</em>.5 v <em>4</em> nmol/l, p = 0.006, median value, controls v patients), 17 hydroxyprogesterone (9.7 v 5.8 nmol/l, p = 0.01), cortisol (387 v 2<em>4</em>5 nmol/l, p = 0.008), testosterone (2.3 v 1.8 nmol/l, p less than 0.001), <em>androstenedione</em> (10.3 v 8.<em>4</em> nmol/l, p = 0.02), and dehydroepiandrosterone sulphate (5.1 v 3.0 mumol/l, p = 0.03). In the luteal phase there was a reduction of oestradiol (<em>4</em>83 v 350 pmol/l, p = 0.015), cortisol (322 v 2<em>4</em>2 nmol/l, p = 0.0<em>4</em>7), and testosterone (2.<em>4</em> v 1.7 nmol/l, p = 0.003). The concentrations of sex hormone binding globulin, prolactin, luteinising hormone, and follicle stimulating hormone were not significantly different in either phase of the cycle. Women with severe idiopathic constipation have a consistent reduction in steroid hormones.

Potentiation of the anthelmintic action of benzimidazole carbamates, such as fenbendazole [methyl 5(6)-(phenylthio)-1H-benzimidazol-2-ylcarbamate], has been noted during concurrent administration of benzimidazoles that possess no intrinsic anthelmintic activity. This study investigated the possibility that inhibition of P<em>4</em>50 enzymes by fenbendazole and its metabolites could play a role in the potentiation phenomenon. Fenbendazole underwent P<em>4</em>50-mediated oxidation in microsomes from untreated rat liver to the sulfoxide and (<em>4</em>'-hydroxyphenyl)thio metabolites [2.92 and 2.87 nmol/(mg of protein.h)]. Pretreatment of rats with phenobarbital or dexamethasone enhanced sulfoxidation by 1.9- and 2.9-fold, respectively. <em>4</em>'-Hydroxylation was increased slightly (by 28%) by phenobarbital and decreased slightly (by <em>4</em>1%) by dexamethasone. Induction also promoted further metabolism of the sulfoxide to fenbendazole sulfone. Immunoinhibition and chemical inhibition studies suggested that P<em>4</em>50 3A proteins and the flavin-containing monooxygenase are involved in sulfoxide and sulfone formation whereas <em>4</em>'-hydroxylation involved the P<em>4</em>50s 2C11, 2C6, and 2B1, depending on the type of induction. In untreated rat liver, the sulfoxide and (<em>4</em>'-hydroxyphenyl)thio metabolites of fenbendazole were relatively potent inhibitors of P<em>4</em>50-mediated <em>androstenedione</em> 16 alpha-, 16 beta-, and 6 beta-hydroxylation (IC50 values of <em>4</em>2, 36, and 7<em>4</em> microM, respectively); 7 alpha-hydroxylase activity was uninhibited. In contrast, fenbendazole and its sulfone metabolite were not inhibitors of these reactions. Mixed-function oxidase activities in phenobarbital-induced rat hepatic microsomes were refractory to inhibition by most compounds, but P<em>4</em>50 1A1 mediated activities in microsomes from beta-naphthoflavone-induced rat liver were quite susceptible to inhibition by fenbendazole sulfoxide. Studies with two analogous sulfoxides yielded similar findings.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the capability of hypothalamic tissue from young (3-<em>4</em> months) and old (22-2<em>4</em> months) male rats to aromatize androgens to estrogens. Aromatase activity was measured in homogenates of brain tissue by using a radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]<em>androstenedione</em> as an index of estrogen formation. Despite the <em>4</em>0% drop in circulating testosterone (T) levels associated with aging in males, we found that hypothalamic aromatase activity was unaffected by age. This finding suggested that chronic exposure to low levels of circulating T can maintain brain aromatase activity in aged male rats. In an experiment designed to examine the acute response of hypothalamic aromatase activity to induction by T, we found a significant positive correlation between circulating T levels and hypothalamic aromatase activity in both age groups. These results demonstrate that T remains an effective regulator of hypothalamic aromatase in old male rats.

In order to assess the androgenic potency of physiological plasma concentrations of the adrenal steroids dehydroepiandrosterone (DHEA) and <em>androstenedione</em> (delta <em>4</em>-dione) in the rat prostate, these two steroids were released from Silastic tubings of appropriate length and size in castrated male rats. Implants of DHEA led to plasma levels of DHEA and 5-androsten-3 beta,17 beta-diol covering the range of concentrations found in adult men while no significant change was observed in plasma levels of delta <em>4</em>-dione, testosterone and dihydrotestosterone (DHT). delta <em>4</em>-Dione implants, on the other hand, led to a parallel increase in plasma delta <em>4</em>-dione and testosterone levels at all doses used while plasma DHT only increased at supraphysiological doses of delta <em>4</em>-dione. At plasma concentrations comparable to those found in adult men; delta <em>4</em>-dione (0.8 ng/ml) and DHEA (3.<em>4</em> ng/ml) stimulated prostate weight 3.7- and 2.1-fold, respectively. In the same groups, prostatic DHT levels were elevated at <em>4</em>.<em>4</em>8 +/- 0.05 and 2.70 +/- 0.73 ng/g tissue, respectively. A close parallelism was observed between prostatic DHT levels and prostatic weight in all groups. The present data show that in the rat, a species having no significant secretion of adrenal androgens, plasma concentrations of DHEA and delta <em>4</em>-dione maintained within the range of those found in adult men are efficiently converted into DHT and act as potent androgenic stimuli in prostatic tissue. The castrated rat bearing Silastic implants releasing constant and predetermined amounts of adrenal steroids offers a good model to study the recently identified role of adrenal steroids in peripheral tissues.

Using radioimmunoassay method, we have estimated the levels of cortisol (C), pregnenolone (delta 5P), 17-hydroxypregnenolone (17-OH-delta 5P), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17-OH-P), <em>androstenedione</em> (A), testosterone (T) and dihydrotestosterone (DHT) in peripheral plasma samples collected at short-time (15 min) intervals from 10 fertile men, during two <em>4</em>-h periods (06.00 to 10.00 and 18.00 to 22.00). In addition, the levels of biologically active luteinizing hormone (LH) were measured by an in vitro bioassay method in 9 of the subjects. The levels of all steroids studied exhibited diurnal variation with higher levels during the morning and lower levels during the evening period. The cortisol and the delta 5-steroid levels also exhibited individual short-term episodic spikes during the 2 periods. No short-term variation was observed in the levels of 17-OH-P, A, T and DHT. Statistically significant correlations were found between the levels of C and those of the delta 5-steroids and A in most of the subjects. No correlation was found between the above steroid levels and those of 17-OH-P, T and DHT. Also the LH levels exhibited episodic spikes of 60 to 90 min duration, but no diurnal variation. When the LH levels were related to those of T found in the same samples or in samples withdrawn 15 to 810 min afterwards, a significant positive correlation was found on repeated occasions in 5 of the 9 subjects. No systematic negative correlation was found when the T levels were related to those of LH in the same sequential fashion. Whereas the positive correlations found between LH and T levels in some of the subjects might suggest that physiological changes in peripheral LH levels are instrumental in regulating T-secretion, the rather consistent lack of significant negative correlation between T and LH levels seems to favour the view that the release of LH is not modulated by peripheral testosterone levels alone.

To study the bioavailability of dehydroepiandrosterone (DHEA) administered by the oral and percutaneous routes, three groups of 12 postmenopausal women aged 60-70 years received two capsules of 50mg of DHEA orally before breakfast daily for 1<em>4</em> days or applied <em>4</em> g of a 10% DHEA cream or gel at the same time of the day on a 30 cm x 30 cm surface area on the thighs. Detailed serial blood sampling over 2<em>4</em>h was performed following 1st and 1<em>4</em>th DHEA administration for measurement of DHEA and nine of its metabolites by liquid chromatography tandem mass spectrometry (LC-MS/MS) or gas chromatography mass spectrometry (GC-MS). Serum levels of estrone (E1) and estradiol (E2) did not change following DHEA administration by any of the three formulations, while serum <em>androstenedione</em> (<em>4</em>-dione), testosterone, DHEA sulfate (DHEA-S), E(1)-S, androsterone glucuronide (ADT-G) and 3alpha-androstanediol-G (3alpha-diol-G), increased in all cases, the effect on these parameters being more important after oral than percutaneous administration due to the metabolism of DHEA into these metabolites in the gastrointestinal tract and liver. No qualitative differences in DHEA metabolism are observed between the oral and percutaneous routes of DHEA administration while the levels of all steroids remain on a plateau during the 2<em>4</em>h period during chronic percutaneous DHEA administration. The present data show that DHEA is transformed into active androgens and estrogens in peripheral intracrine tissues with no or minimal release of the active steroids E(1), E(2) or testosterone in the circulation. Moreover, DHEA is preferentially transformed into androgens rather than into estrogens. Most importantly, the present data show that changes in serum DHEA following oral or percutaneous DHEA administration are not a valid parameter of DHEA action since the increase in serum DHEA is at least 100% greater than the increase in the formation of active androgens and estrogens and thus much higher than the potential physiological effects.

The response of plasma testosterone (T), delta <em>4</em>-<em>androstenedione</em>, and cortisol (F) to the administration of synthetic Zn beta 1-2<em>4</em> ACTH (0.5 mg/m2 im every 12 h for 3 days) was ascertained in 20 infants, 35 prepubertal children, <em>4</em> early pubertal boys, and 15 adults. At all ages and in both sexes, a significant rise in delta <em>4</em>-<em>androstenedione</em> and F was observed (P less than 0.00001), whereas the response of T showed a sex difference: T levels increased in females (P less than 0.001) at all ages in response to ACTH, while they decreased (P less than 0.01) in males at periods of active testicular secretion (early infancy, puberty, and adulthood). In prepubertal boys, in the absence of significant Leydig cell activity, T levels increased after ACTH, as they did in girls. The post-ACTH values of T, expressed as percent-ages of the control levels, were significantly lower (P less than 0.01) in infants (3.17 +/- 16.7%) than in pubertal boys (59.5 +/- 1<em>4</em>.6%) or adult men (57.9 +/- 7.7%). F levels were significantly higher after ACTH stimulation at 1-<em>4</em> months of age (253 +/- 129 micrograms/dl) than at any later age studied (6<em>4</em> +/- 17 micrograms/dl). It would seem, therefore, that the suppressive effect of ACTh on testicular secretion might be glucocorticoid mediated and its magnitude might be related to circulating levels of F.

OBJECTIVE

To clarify, whether uterine endothelial proliferation could be regulated via an autocrine estrogen producing mechanism or direct actions of testosterone.

METHODS

In vitro study.

METHODS

Tertiary care facility.

METHODS

Human myometrial tissue obtained from <em>4</em>0 women undergoing hysterectomy without further intrauterine pathology.

METHODS

Cell culture, proliferation assay and CYP19 activity assay on human myometrial endothelial cells treated with testosterone, estradiol, letrozole, flutamide, PD98059, MG-132 alone or in combination.

METHODS

We analyzed whether aromatase is expressed in human myometrial microvascular endothelial cells (HMMECs) and whether it affects proliferation and converts androgens to estrogens. In addition, we aimed to define whether or not T could have a direct capability to affect HMMEC proliferation.

RESULTS

Using quantitative real-time PCR and Western analysis, primary passage four HMMECs were shown to express low levels of aromatase mRNA and protein, respectively. However, HMMECs were unable to convert radioactively labeled 3∗H-1β-<em>androstenedione</em> to estrogen. Pharmacologic doses of T (10(-6) and 10(-<em>4</em>) M) increased HMMEC proliferation, assessed through a bromodeoxyuridine ELISA. This effect of T on proliferation could not be blocked after pretreatment of cells with the aromatase inhibitor letrozole. In addition, HMMECs were found to express androgen receptors (ARs), and the AR antagonist flutamide abolished T-dependent proliferation. T was shown to increase AR protein levels, which was due to T-dependent receptor stabilization and not activation of gene transcription.

CONCLUSIONS

We conclude that myometrial endothelial proliferation is not regulated through myometrial endothelial estrogen production. However, pharmacologic doses of T increase myometrial endothelial proliferation through a receptor-dependent and -stabilizing mechanism.

Plasma testosterone, 5 alpha-dihydrotestosterone (DHT), delta <em>4</em>-<em>androstenedione</em>, dehydroepiandrosterone (DHA) and oestradiol-17 beta concentrations of crab-eating macaques after birth were analysed by RIA. The profiles of plasma testosterone and DHT exhibited four phases: (1) a neonatal phase (0 to 3-<em>4</em> months of age) with considerable synthetic testicular activity; (2) a phase of 'infancy' (generally up to 29 months of age) during which the values of both androgens were low; (3) a prepubertal phase (generally up to <em>4</em>3 months of age) when circulating values oscillated with wider individual variations, and (<em>4</em>) a pubertal phase when the concentrations increased in parallel and concomittantly with the onset of meiosis and the establishment of spermatogenesis. The testosterone values continued to increase, reaching adult values at about 5-6 years of age, whereas DHT levels tended to stabilize from <em>4</em>-5 years. Relatively high <em>androstenedione</em> values during the neonatal phase decreased progressively until puberty, then increased again slowly up to the adult stage when they plateaued at about neonatal levels. The DHA levels were high during the first months, decreased at about 1 year, remained stable during infancy and prepuberty and then declined again during puberty. At about 5 years, the values were 28% of those in neonates. There was no evidence of an adrenarche before the first signs of sexual maturity were observed. Oestradiol-17 beta concentrations were high at birth and until 3 months, then decreased and remained steady from 1 year of age until adulthood, except at the onset of puberty (27-30 months of age) when high values were again noted. Our results show that, during the neonatal period, the testis exhibited considerable secretory activity.

Previous studies of Delta <em>4</em>-androstene-3,17-dione (<em>4</em>-<em>androstenedione</em>) administration in men have not demonstrated sustained increments in testosterone levels, fat-free mass (FFM), and muscle strength, and failure to demonstrate <em>androstenedione</em>'s androgenic/anabolic effects has stifled efforts to regulate its sales. To determine whether <em>4</em>-<em>androstenedione</em> has androgenic/anabolic properties, we evaluated its association with androgen receptor (AR) and its effects on myogenesis in vitro. Additionally, we studied the effects of a high dose of <em>4</em>-<em>androstenedione</em> on testosterone levels, FFM, and muscle strength in hypogonadal men. We determined the dissociation constant (K(d)) for <em>4</em>-<em>androstenedione</em> using fluorescence anisotropy measurement of competitive displacement of fluorescent androgen from AR ligand-binding domain. AR nuclear translocation and myogenic activity of <em>androstenedione</em> were evaluated in mesenchymal, pluripotent C3H10T1/2 cells, in which androgens stimulate myogenesis through an AR pathway. We determined effects of a high dose of <em>androstenedione</em> (500 mg thrice daily) given for 12 wk on FFM, muscle strength, and hormone levels in nine healthy, hypogonadal men. <em>4</em>-<em>Androstenedione</em> competitively displaced fluorescent androgen from AR ligand-binding domain with a lower affinity than dihydrotestosterone (K(d), 6<em>4</em>8 +/- 21 and 10 +/- 0.<em>4</em> nm, respectively). In C3H10T1/2 cells, <em>4</em>-<em>androstenedione</em> caused nuclear translocation of AR and stimulated myogenesis, as indicated by a dose-dependent increase in myosin heavy chain II+ myotube area and up-regulation of MyoD protein. Stimulatory effects of <em>4</em>-<em>androstenedione</em> on myosin heavy chain II+ myotubes and myogenic determination factor expression were attenuated by bicalutamide, an AR antagonist. Administration of 1500 mg <em>4</em>-<em>androstenedione</em> daily to hypogonadal men significantly increased serum <em>androstenedione</em>, total and free testosterone, estradiol, and estrone levels and suppressed SHBG and high-density lipoprotein cholesterol levels. <em>4</em>-<em>androstenedione</em> administration was associated with significant gains in FFM (+1.7 +/- 0.5 kg; P = 0.012) and muscle strength in bench press (+<em>4</em>.3 +/- 3.1 kg; P = 0.006) and leg press exercises (+18.8 +/- 17.3 kg; P = 0.0<em>4</em>5). <em>4</em>-<em>androstenedione</em> is an androgen that binds AR, induces AR nuclear translocation, and promotes myogenesis in vitro, with substantially lower potency than dihydrotestosterone. <em>4</em>-<em>androstenedione</em> administration in high doses to hypogonadal men increases testosterone levels, FFM, and muscle strength, although at the dose tested, the anabolic effects in hypogonadal men are likely because of its conversion to testosterone.

Food availability is an important synchronizer of the pituitary-adrenal axis and daytime restriction of food access phase-shifts the diurnal periodicity of plasma corticosterone (B) concentration in rats. However, little is known about the synchronizers of circulating androgens in male rats. We studied intact and castrated male rats with free access to food (control groups, C) and with access to food only from 0900 to 1100 hr (food-restricted groups, FR) for 1<em>4</em> days. Blood samples were collected on the 15th day by decapitation at <em>4</em>-hr intervals. Plasma B concentration in C groups presented diurnal variation with higher values at 2000 than at 0800 hr. In the FR groups there was a 12-hr shift of peak B values. Apparently, castration had no effect on plasma B diurnal variation. In intact rats, plasma testosterone presented similar diurnal variation in both the C and the FR groups. In castrated rats, plasma testosterone was undetectable. Plasma <em>androstenedione</em> similarly varied over time in both C and FR intact rats. However, in castrated animals, the diurnal variation of plasma <em>androstenedione</em> was abolished. Our results indicate that the daily variation of plasma testosterone and <em>androstenedione</em> is dependent on testicular secretion and is not influenced by food availability in the male rat.

OBJECTIVE

Because the large increase in luteinizing hormone secretion induced by flutamide in the intact rat is not found in men, we have used castrated rats and mice supplemented with <em>androstenedione</em> (<em>4</em>-dione) instead of intact animals to measure the activity of the pure antiandrogens flutamide and Casodex.

METHODS

We first compared the effect of different schedules of administration of various doses of the two antiandrogens on prostate and seminal vesicle weights in the castrated rat and mice models.

RESULTS

For both flutamide and Casodex, no consistent difference was found between the effects of once daily and thrice daily oral dosing in the rat. It was observed, however, that flutamide, especially at the high and therapeutically more effective doses, is about three times more potent than Casodex under both schedules of dosing. When flutamide was administered subcutaneously three times a day, twice a day, once a day, or once every second day in rats and mice, no difference was observed in the degree of inhibition achieved on prostate and seminal vesicle weights.

CONCLUSIONS

The present data show that Casodex is about three times less potent than flutamide on the well-recognized parameters of androgen responsiveness in the rat, namely prostate and seminal vesicle weights. Another finding is that once daily dosing with flutamide exhibits an effectiveness comparable to thrice daily dosing; such data may have potential significance in facilitating compliance by administration of flutamide once daily instead of the current thrice daily schedule in men. Moreover, these data, if obtained in a reliable in vivo model, should be helpful in determining the choice of an appropriate dose of Casodex for the treatment of prostate cancer.

Goldfish (Carassius auratus) use reproductive hormones as endocrine signals to synchronize sexual behavior with gamete maturation and as exogenous signals (pheromones) to mediate spawning interactions between conspecifics. We examined the differential effects of two hormonal pheromones, prostaglandin F(2alpha) (PGF(2alpha)) and 17alpha,20beta-dihydroxy-<em>4</em>-pregnen-3-one (17,20beta-P) on neurogenesis, neurotransmission, and neuronal activities, and on plasma <em>androstenedione</em> (AD) levels. Exposure to waterborne PGF(2alpha) induced a multitude of changes in male goldfish brain. Histological examination indicated an increase in the number of dividing cells in male diencephalon (p < 0.05, Kruskal-Wallis test). Real-time quantitative PCR tests showed elevated levels of transcripts for the salmon gonadotropin-releasing hormone (GnRH) in the male telencephalon and cerebellum (p < 0.005, one-way ANOVA) and for ChAT (choline acetyltransferase) in the male vagal lobe and the brainstem underneath the vagal lobe (p < 0.05, one-way ANOVA). Therefore, PGF(2alpha) seemed to modulate male brain plasticity that coincided with behavioral changes during spawning season. Exposure to waterborne 17,20beta-P, however, increased circulatory levels of immunoreactive AD in males and the transcripts of androgen receptor and cGnRH-II (chicken-II GnRH) in the female cerebellum (p < 0.05, one-way ANOVA). PGF(2alpha) and 17,20beta-P thereby seemed to act through distinct pathways to elicit different responses in the neuroendocrine system. This is the first finding that links a specific pheromone molecule (PGF(2alpha)) to neurogenesis in a vertebrate animal.

The hemoprotein component of human placental aromatase (estrogen synthetase) has been purified to a high degree of homogeneity by a combination of affinity and adsorption chromatography on aminohexyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The monomeric form of the enzyme has an Mr of 55000 +/- 1000 as estimated by sodium dodecyl sulfate gel electrophoresis. Its absolute spectrum shows a high-spin Soret band at 39<em>4</em> nm while its reduced, CO-difference spectrum has a maximum at <em>4</em><em>4</em>7 +/- 1 nm. Full reconstitution of aromatase activity was obtained when it was recombined with a homogeneous preparation of the higher-Mr form of either human placental, or bovine hepatic NADPH-cytochrome P-<em>4</em>50 reductase. Critical factors for purification of the very unstable, membrane-bound hemoprotein with good retention of activity were, besides the chromatographic sequence, the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) during the solubilization, and the stabilizing effect of the aromatase substrate, <em>4</em>-androstene-3,17-dione, throughout the procedure. In the presence of NADPH, the reconstituted enzyme system smoothly aromatizes 19-oxo<em>androstenedione</em>, 19-hydroxy<em>androstenedione</em> and <em>androstenedione</em> in this order of reactivity. The same reconstituted system also aromatized testosterone, but it was inactive towards 19-nor<em>androstenedione</em>. Known cytochrome P-<em>4</em>50 inhibitors decreased its activity. We conclude: (a) the terminal oxidase of human placental aromatase is indeed a cytochrome P-<em>4</em>50-type monooxygenase; (b) the multistep aromatization reaction of C19 androstenes is catalyzed by a single enzyme; (c) aromatization of 19-norsteroids reported by other authors must be due to a different aromatase. Experimental data obtained with the reconstituted enzyme are fully compatible with the concept of a reaction mechanism for the aromatization sequence involving an all-trans, antiparallel elimination of the 19-methyl group, the 2 beta proton and the 1 alpha proton, rather than the 1 beta proton, as generally assumed.

The metabolism of various radiolabeled steroids by cultured human epidermal keratinocytes was studied in an attempt to identify the steroid-metabolizing enzymes present in these cells. Sulfatase activity was demonstrated in keratinocytes with either E1S or DS as substrates. The products of sulfatase action were E1 and DHEA, respectively. The specific activity of the enzyme was approximately 5- to 1<em>4</em>-fold greater with E1S as the substrate compared with DS, and the rates of hydrolysis were linear with incubation time up to 3 h. The metabolism of DHEA by the keratinocyte 17 beta-HSOR-catalyzed reaction resulted in the predominant formation of 5-androstene-3 beta,17 beta-diol. The rate of formation of 5-androstene-3 beta,17 beta-diol was linear with time of incubation up to 18 h, and the specific activity of 17 beta-HSOR, with DHEA as the substrate, was greater in keratinocytes maintained in culture for <em>4</em> weeks compared with keratinocytes kept in culture for 1 week. <em>Androstenedione</em> was a minor product of DHEA metabolism. The metabolism of DHT by epidermal keratinocytes resulted in the formation of 5 alpha-androstanedione, 5 alpha-androstane-3 alpha,17 beta-diol, 5 alpha-androstane-3 beta,17 beta-diol, androsterone, and isoandrosterone: the rates of formation of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol were linear with incubation time up to 2<em>4</em> h, and the specific activities of 3 alpha-HSOR and 3 beta-HSOR did not appear to change with keratinocyte time in culture up to 3 weeks. The metabolism of DOC by epidermal keratinocytes resulted in 5 alpha-dihydrodeoxycorticosterone production: the rate of formation of this metabolite was linear with incubation time up to <em>4</em> h. The metabolism of E1 by epidermal keratinocytes yielded E2, and that of E2 resulted in the formation of E1. The rate of E1 formation from E2, was approximately 10-fold greater than the rate of formation of E2 from E1; these rates were linear with incubation time up to <em>4</em> h. Epidermal keratinocytes maintained in culture did not metabolize <em>androstenedione</em> to either E1 or E2, and pregnenolone was not metabolized by these cells. This study serves to ascertain that epidermal keratinocytes express steroid 5 alpha-reductase, 17 beta-HSOR, 3 beta-HSOR, 3 alpha-HSOR, 3 beta-hydroxysteroid oxidoreductase-delta 5----<em>4</em>-isomerase, and sulfatase activities.

Aromatization of <em>androstenedione</em> to estrone in peripheral tissues is the major source of estrogen in postmenopausal women. The aromatase enzyme complex, which mediates the conversion of <em>androstenedione</em> to estrone, is present in several tissues, including adipose tissue and normal and malignant breast tissues. Aromatase activity is detectable in 50-60% of breast tumors, but the contribution that tumor aromatase makes to estrogen concentration in tumors and whether the estrogen formed is biologically important remains a controversial matter. Since concentrations of <em>androstenedione</em> are higher in tumors than in blood, and tumor aromatase activity in vivo may be enhanced by growth factors and by cytokines, the contribution of tumor aromatase to tumor estrogen levels may be higher than suggested by the original calculations. Measurements of tumor aromatase, tumor estrone concentrations, and DNA polymerase alpha activity (a marker of cellular proliferation), in samples obtained before and after treatment with the aromatase inhibitor <em>4</em>-hydroxy<em>androstenedione</em>, lend some support to a biological role for estrone formed locally.

Estrogens provide the major hormonal support for endocrine-dependent human mammary neoplasms. In postmenopausal women, the extraglandular aromatization of the adrenal prehormone, <em>androstenedione</em> to estrone is the major pathway for estrogen biosynthesis. Estrone can then be converted into estradiol or into an inactive conjugate, estrone sulfate. Recent data suggest that the estrogens may also be synthesized in situ by human breast tumors, either from <em>androstenedione</em> via aromatase, or from estrone sulfate via the enzyme, sulfatase. Our enzyme kinetic studies support the predominance of the sulfatase pathway for in situ estrogen biosynthesis. The ability of estrone sulfate to stimulate colony formation of the nitrosomethylurea-induced rat mammary tumor in the clonogenic assay, suggests that this in situ pathway has biologic relevance. Aromatase inhibitors can be used to suppress the levels of circulating estrone, estrone sulfate, and estradiol in postmenopausal women. Aminoglutethimide, the major inhibitor currently used clinically, acts in a competitive fashion and blocks cholesterol side chain cleavage and 11 beta-hydroxylase as well as aromatase. Clinical studies indicate that the combination of aminoglutethimide plus replacement glucocorticoid causes breast tumor regression with the same frequency and for the same duration as surgical ablative therapies such as adrenalectomy or hypophysectomy. Aminoglutethimide also induces a similar rate of tumor regression as achieved with the antiestrogen, tamoxifen. However, because tamoxifen is associated with fewer side effects, this antiestrogen is to be preferred over use of aminoglutethimide as first-line hormonal treatment for women with breast cancer. Several specific suicide inhibitors of aminoglutethimide such as <em>4</em>-hydroxy-<em>androstenedione</em> are being developed and have proven effective in early clinical trials with breast cancer patients. Further development of active aromatase inhibitors should allow precise control of estradiol levels in women with breast cancer. This ability to perform an 'estrogen clamp' may allow new strategies to be developed in which hormone depletion followed by repletion can produce a synchronization of tumor cell DNA synthesis. If achievable, such manipulations may allow potentiation of the effects of cytotoxic chemotherapy. This latter concept is currently being rigorously tested in basic and in clinical investigative studies.

The alpha ACTH-(1-2<em>4</em>) threshold dose and the response slope were determined for cortisol (F), delta <em>4</em>-<em>androstenedione</em> (A), and dehydroepiandrosterone (DHEA) in 10 normal and 16 obese eumenorrheic nonhirsute women matched for age. Each woman received 1 mg dexamethasone at 2300 h and again at 0700 h the next morning. At 0700 h, a continuous alpha ACTH-(1-2<em>4</em>) infusion was begun at an initial dose of 30 ng/1.5 m2 body surface area X hr. The ACTH infusion rate was doubled every hour for 5 consecutive h to a maximum dose of <em>4</em>80 ng/1.5 m2 X h. Blood samples were collected for steroid assays before the infusion and at the end of each hour. The ACTH threshold dose was defined as the dose that produced a steroid response significantly above the basal level. The ACTH threshold dose for serum F and DHEA stimulation was not different between the groups, but the threshold dose for A was significantly lower in the obese women. Basal and stimulated serum DHEA to F ratios were significantly higher in the obese women. In both groups, the mean F response slope was significantly higher than that for DHEA, which in turn, was significantly higher than that for A. The mean DHEA response slope was significantly greater in the obese women. The F and A response slopes were not different between the groups. We conclude that the relative responsivity of the steroids to ACTH was the same in both groups: F greater than DHEA greater than A; in the obese women, the ACTH threshold dose for F stimulation was lower (greater sensitivity) than for DHEA or A stimulation; and in the obese women, the ACTH threshold dose for A was significantly lower (increased sensitivity) and the slope of the DHEA response to ACTH was steeper (greater responsivity) than in normal women.

Leydig cell tumors are rare ovarian steroid cell neoplasms. More than 75% of patients show signs of virilization due to overproduction of testosterone. We report a case of an 81-year-old woman with progressive signs of virilization, and presenting vaginal bleeding. Clinical analyses revealed high levels of serum testosterone, delta <em>4</em>-<em>androstenedione</em> and estradiol, and also inappropriate low levels of gonadotrophins for a post-menopausal woman. Transvaginal ultrasound showed no evidence of ovarian tumor, but pelvic and abdominal computerized axial tomography imaging revealed a left ovarian solid nodule, and no evidence of alteration in the adrenal glands. Total hysterectomy and bilateral salpingoophorectomy were performed. Histopathology and immunohistochemistry confirmed the diagnosis of Leydig cell tumor. After surgery, androgen levels returned to normal, and there was regression of the signs of virilization.

Progesterone (P) is a strong mineralocorticoid receptor (MR) antagonist in vitro. The high P concentrations seen in normal pregnancy only moderately increase renin and aldosterone concentrations. In previous in vitro studies we hypothesized that this may be explained by intrarenal conversion of P to less potent metabolites. To investigate the in vivo anti-MR potency of P, we performed an infusion study in patients with adrenal insufficiency (n = 8). They omitted 9alpha-fluorocortisol for <em>4</em> d and hydrocortisone for 0.5 d before a continuous iv infusion of aldosterone for 8.5 h, with an additional iv P infusion commenced at <em>4</em> h. During aldosterone infusions the initially elevated urinary sodium to potassium ratio decreased significantly. Despite the 1000-fold excess of P over aldosterone, the urinary sodium to potassium ratio and urinary sodium excretion increased only slightly after 3 h of P infusion. We detected inhibition of renal 11beta-hydroxysteroid dehydrogenase type 2 by P, thus giving cortisol/prednisolone access to the MR. Urinary and plasma concentrations of 17alpha-hydroxyprogesterone, a major metabolite of renal P metabolism, and those of serum <em>androstenedione</em> and deoxycorticosterone, a mineralocorticoid itself, increased significantly during P infusion. This supports the hypothesis of an effective protection of the MR from P by efficient extraadrenal downstream conversion of P.