The AusD Study: a population-based study of the determinants of serum 25-hydroxyvitamin D concentration across a broad latitude range.

Observational studies suggest that people with a high serum 25-hydroxyvitamin D (25(OH)D) concentration may have reduced risk of chronic diseases such as osteoporosis, multiple sclerosis, type 1 diabetes, cardiovascular disease, and some cancers. The AusD Study (A Quantitative Assessment of Solar UV Exposure for Vitamin D Synthesis in Australian Adults) was conducted to clarify the relationships between ultraviolet (UV) radiation exposure, dietary intake of vitamin D, and serum 25(OH)D concentration among Australian adults residing in Townsville (19.3°S), Brisbane (27.5°S), Canberra (35.3°S), and Hobart (42.8°S). Participants aged 18-75 years were recruited from the Australian Electoral Roll between 2009 and 2010. Measurements were made of height, weight, waist:hip ratio, skin, hair, and eye color, blood pressure, and grip strength. Participants completed a questionnaire on sun exposure and vitamin D intake, together with 10 days of personal UV dosimetry and an associated sun-exposure and physical-activity diary that was temporally linked to a blood test for measurement of 25(OH)D concentration. Ambient solar UV radiation was also monitored at all study sites. We collected comprehensive, high-quality data from 1,002 participants (459 males, 543 females) assessed simultaneously across a range of latitudes and through all seasons. Here we describe the scientific and methodological issues considered in designing the AusD Study.

Prolonging hormone sensitivity in prostate cancer xenografts through dual inhibition of AR and mTOR.

BACKGROUND: To determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer (PCa), we investigated the relationship between the androgen receptor (AR) and mTOR pathways and the impact of inhibiting both pathways in androgen-dependent and castration-resistant PCa models. EXPERIMENTAL DESIGN: Androgen-dependent (LNCaP) and castration-resistant PCa (HP-LNCaP) cells were grown as tumours in SCID mice. Once tumours reached 500 mm(3), animals were grouped and injected subcutaneous with vehicle, our novel anti-androgen/androgen synthesis inhibitor, VN/124-1, bicalutamide, and everolimus. Tumour volumes were measured biweekly. The PSA and protein analyses were performed after completion of the treatment. RESULTS: The addition of everolimus to bicalutamide treatment of resistant tumours significantly reduced tumour growth rates and tumour volumes. Anti-androgen treatment also increased protein expression of multiple signal transduction pathways earlier than vehicle-treated control xenografts. VN/124-1 plus everolimus acted in concert to reduce tumour growth rates in our castration-resistant xenograft model. CONCLUSIONS: This study suggests that dual inhibition of AR and mTOR in castration-resistant xenograft models can restore sensitivity of tumours to anti-androgen therapy. Furthermore, after bicalutamide failure, dual inhibition with VN/124-1 and everolimus was the most effective treatment.

A versatile synthesis of 17-heteroaryl androstenes via palladium-mediated Suzuki cross-coupling with heteroaryl boronic acids.

Suzuki coupling of 17-iodoandrosta-5,16-dien-3beta-ol (1) and 17-iodoandrosta-4,16-dien-3-one (2) with nine heteroaryl boronic acids (mainly 2- or 3-furanyl, thienyl, benzofuranyl and benzothienyl boronic acid derivatives) were carried out under normal Suzuki condition (Pd(PPh(3))(4), 2M Na(2)CO(3) and MeOH), generally yielded C(17)-heteroaryl steroids in moderate (10-60%) yields, but furanyl-2- and 5-chlorothienyl-2-boronic acid did not give any coupling product.

Overview of recent development of aromatase inhibitors.

Since the first publication in 1973 concerning aromatase inhibitors, several effective compounds have been reported by a number of investigators. Our studies with 4-hydroxyandrostene-3,17-dione, 4-acetoxyandrostene-3,17-dione, and 1,-4,6-androstatrienedione indicate that these compounds cause rapid competitive inhibition of the enzyme. Aminoglutethimide binds competitively to cytochrome P-450 and inhibits a number of steroid hydroxylations but is more active as an aromatase inhibitor. 16 alpha-Bromoandrogens and 7 alpha-(4'-amino)phenylthioandrostenedione are also reported to be aromatase inhibitors. As yet, only some of these compounds have been evaluated in vivo, but all appear to be similarly effective in inhibiting aromatization in breast tumors in vitro. Recent interest has focused on enzyme inactivators or "suicide" inhibitors. Such compounds act as substrates for the enzyme but are converted by the normal catalytic mechanism of the enzyme to reactive intermediates. These then bind covalently to the active site of the enzyme causing loss of activity. Active site-directed inhibitors are usually quite specific and have long-lasting effects in vivo. A number of new compounds, as well as some of the above compounds, appear to be inactivators of aromatase and are potentially interesting as agents for hormone-dependent breast cancer therapy.

Effects of aromatase inhibitors, aminoglutethimide, and 4-hydroxyandrostenedione on cyclic rats and rats with 7,12-dimethylbenz(a)anthracene-induced mammary tumors.

4-Hydroxyandrostenedione (4-OHA) is a more potent and specific inhibitor of aromatase (estrogen synthetase) than aminoglutethimide (AG). The two inhibitors were compared in rats with 7,12-dimethylbenz(a)anthracene-induced, hormone-dependent tumors and in normal cyclic rats treated for 4 and 2 weeks, respectively. Ovarian estradiol levels and aromatase activities were not consistently reduced, and tumors regressed in only two of eight rats treated with AG. In animals treated with 4-OHA or 4-OHA:AG, the total tumor volume, estradiol levels, and aromatase activity decreased by greater than 70%. Ovarian weights and plasma luteinizing hormone (LH) levels were also reduced by 4-OHA but increased by AG. Uterine weights were not altered by AG treatment but were increased by 4-OHA. Similar but more consistent results were obtained with these treatments in normal, cyclic rats. In ovariectomized rats, AG had no effect, whereas 4-OHA decreased LH levels and increased uterine weights. The results suggest that, although AG reduces ovarian estrogen secretion by aromatase inhibition, this may lead to an increase in LH secretion. Increased LH may promote ovarian growth and aromatase synthesis, counteracting the inhibitory action of AG to some extent. 4-OHA which inactivates aromatase may also prevent new enzyme synthesis by directly inhibiting gonadotropins. This would result in more effective reduction in ovarian estrogen production by 4-OHA than AG during long-term treatment.

Effect of 4-hydroxyandrostenedione on murine Leydig tumor cell steroidogenesis.

The murine Leydig cell tumor (M5480A) possesses high levels of estrogen receptor and is known to produce estrogens. In these studies we examined the effects of the potent aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) on Leydig tumor cell steroidogenesis both in vitro and in vivo. The addition of 4-OHA to Leydig tumor cells in primary culture resulted in a dose- and a time-dependent decrease in media progesterone levels. The observed decrease was most likely due to impaired synthesis of progesterone, inasmuch as no alteration in progesterone metabolism was seen when progesterone levels were diminishing. However, 4-OHA inhibited progesterone conversion to testosterone following 1 h of incubation, but this effect disappeared coincident with 4-OHA metabolism. Analysis of pregnenolone production revealed a biphasic dose-dependent effect of 4-OHA. At low doses (0.01-0.1 microM), 4-OHA was found to decrease pregnenolone concentrations, while at higher doses (1-10 microM) pregnenolone levels were elevated. Therefore, the actions of 4-OHA on Leydig cell steroidogenesis in vitro appear to be multifocal. Other experiments were performed to evaluate the effects of 4-OHA on tumor-bearing male mice in vivo. In these studies, the predominant effects of 4-OHA were to act as an aromatase inhibitor and to inhibit progesterone production. Thus, while 4-OHA is a potent aromatase inhibitor, we have found that this compound may alter steroidogenesis in Leydig tumor cells at several sites prior to aromatization.

Antiandrogenic effects of novel androgen synthesis inhibitors on hormone-dependent prostate cancer.

We have found that in addition to being potent inhibitors of 17alpha-hydroxylase/C17,20-lyase and/or 5alpha-reductase, some of our novel androgen synthesis inhibitors also interact with the mutated androgen receptor (AR) expressed in LNCaP prostate cancer cells and the wild-type AR expressed in hormone-dependent prostatic carcinomas. The effects of these compounds on the proliferation of hormone-dependent human prostatic cancer cells were determined in vitro and in vivo. L-2 and L-10 are delta4-3-one-pregnane derivatives. L-35 and L-37 are delta5-3beta-ol-androstane derivatives, and L-36 and L-39 are delta4-3-one-androstane-derived compounds. L-2, L-10, and L-36 (L-36 at low concentrations) stimulated the growth of LNCaP cells, indicating that they were interacting agonistically with the mutated AR expressed in LNCaP cells. L-35, L-37, and L-39 acted as LNCaP AR antagonists. To determine whether the growth modulatory effects of our novel compounds were specific for the mutated LNCaP AR, competitive binding studies were performed with LNCaP cells and PC-3 cells stably transfected with the wild-type AR (designated PC-3AR). Regardless of AR receptor type, all of our novel compounds were effective at preventing binding of the synthetic androgen methyl-trienolone[17alpha-methyl-(3H)-R1881 to both the LNCaP AR and the wildtype AR. L-36, L-37, and L-39 (5.0 microM) prevented binding by >90%, whereas L-35 inhibited binding by 30%. To determine whether the compounds were acting as agonists or antagonists, LNCaP cells and PC-3AR cells were transfected with the pMAMneoLUC reporter gene. When luciferase activity was induced by dihydrotestosterone, all of the compounds were found to be potent inhibitors of transcriptional activity, and the pattern of inhibition was similar for both receptor types. However, L-2, L-10, and L-36 were determined to be AR agonists, and L-35, L-37, and L-39 were wild-type AR antagonists. When tested in vivo, L-39 was the only AR antagonist that proved to be effective at inhibiting the growth of LNCaP prostate tumor growth. L-39 slowed tumor growth rate in LNCaP tumors grown in male SCID mice to the same level as orchidectomy, significantly reduced tumor weights (P < 0.05), significantly lowered serum levels of prostate-specific antigen (P < 0.02), and significanty lowered serum levels of testosterone (P < 0.05). L-39 also proved to be effective when tested against the PC-82 prostate cancer xenograft that expresses wild-type AR. These results show that some of our compounds initially developed to be inhibitors of androgen synthesis also interact with the human AR and modulate the proliferation of hormone-dependent prostatic cancer cells. Therefore, compounds such as L-39, which have multifunctional activities, hold promise for the treatment of androgen-dependent prostate tumors.

Effect of treatment with aromatase inhibitor 4-hydroxandrostenedione on the nonhuman primate menstrual cycle.

4-Hydroxyandrostenedione (4-OHA) is a potent inhibitor of rat ovarian and human placental aromatase activity as assessed in vitro and has been shown to suppress ovarian estrogen secretion in vivo in rats. The compound inhibited extragonadal aromatization in male rhesus monkeys and reduced plasma levels of estradiol in postmenopausal women with advanced, metastatic breast cancer. Furthermore, 4-OHA caused disease remission in these patients. Prior to using this compound in premenopausal patients, the present study was carried out to determine whether 4-OHA affects the menstrual cycle by inhibiting ovarian estrogen production in nonhuman primate species. Four baboons (Papio anabis) exhibiting regular menstrual cycles were studied. Blood samples were collected daily throughout a control menstrual cycle and during treatment with daily s.c. injections of 4-OHA. Menstruation was not observed in three of four animals during treatment and did not resume until cessation of 4-OHA administration. The midcycle surge of estradiol which averaged 411 pg/ml in the control menstrual cycle was reduced during 4-OHA treatment by a mean of 49% of control. Thereafter, with continued 4-OHA treatment, serum concentrations of estradiol were significantly reduced below the basal control level [75.3 +/- 5.6 (SE) pg/ml] and were very low or undetectable. Perineal turgescence, an index of estrogenicity, was inhibited by 4-OHA administration. The preovulatory surge of serum luteinizing hormone, which averaged 114 ng/ml during the control cycle, appeared normal in three of four animals following 12 to 14 days of 4-OHA administration, but no luteinizing hormone surge occurred with continued 4-OHA administration. The results indicate that, although 4-OHA reduces ovarian estrogen formation during the first cycle, treatment for a second consecutive cycle appears to be necessary for maximal suppression.

Lack of evidence for aromatase in human prostatic tissues: effects of 4-hydroxyandrostenedione and other inhibitors on androgen metabolism.

The effects of 4-hydroxyandrostenedione (4-OHA) and other aromatase inhibitors, 10-propargylestr-4-ene-3,17-dione and imidazo[1,5-alpha]-3,4,5,6-tetrahydropyrin-6-yl-(4-benzonitrile), as well as 5 alpha-reductase inhibitors N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxyamide and 4-methyl-3-oxo-4-aza-androsta-5-ene-17-ol were investigated in prostatic tissue from six patients with benign prostatic hypertrophy and seven patients with prostatic cancer, and from normal men at autopsy. We attempted to measure aromatase activity in the tissue incubations by quantitating 3H2O released from androstenedione or testosterone labeled at the C-1 position. High performance liquid chromatography and thin layer chromatography were used to isolate steroid products. Although the amount of 3H2O released was at least twice that of the heat-inactivated tissue samples, no estrone or estradiol was detected on high performance liquid chromatography. The 3H2O release was significantly inhibited by 4-OHA and N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxyamide, but not by the other aromatase inhibitors. 4-OHA also inhibited 5 alpha-reductase in both benign prostatic hypertrophy and cancer tissue, although to a lesser extent than N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxyamide. The other aromatase inhibitors were without effect on 5 alpha-reductase. Our results indicate that 3H2O released from [1 beta-3H]androstenedione and [1,2,6,7-3H]androstenedione does not correlate with estrogen formation and may be the result of other metabolic reactions. Although it appears that the prostate lacks aromatase, 4-OHA may be of benefit in patients with benign prostatic hypertrophy or prostatic cancer by inhibiting this enzyme in peripheral tissue.

Effects of aromatase inhibitor 4-hydroxyandrostenedione and other compounds in the 7, 12-dimethylbenz(a)anthracene-induced breast carcinoma model.

Aromatase inhibitor, 4-hydroxyandrostene-3,17-dione (4-OHA), is a highly effective treatment in rats with 7,12-dimethylbenz(a) anthracene-induced hormone-dependent mammary tumors. Over 90% of tumors regress to less than one-half of their original size, and a high proportion regress completely. Treatment of rats with other inhibitors, 4-acetoxyandrostene-3,17-dione and 1,4,6-androstatriene-3,17-dione produce similar results. In comparison with other aromatase inhibitors, the compounds reduced ovarian estrogen secretion in the rat to the same extent as aminoglutethimide, whereas Teslac was without effect. The latter two compounds caused little and no regression of DMBA-induced mammary tumors, respectively. Our recent studies with 4-OHA, 4-acetoxyandrostene-3,17-dione, and 1,4,6-androstatriene-3,17-dione indicate that they interact with aromatase by a two-component mechanism, a rapid competitive inhibition, and a slower enzyme inactivation. Treatment of rats with 4-OHA also caused greater than 80% loss of ovarian aromatase activity in vivo and a reduction in ovarian estrogen secretion, which are maintained for at least 48 hr after injection. Although 4-OHA is cleared rapidly in vivo, the above results suggest that the compound has a sustained effect. Thus, when 7,12-dimethylbenz(a)anthracene-induced tumor-bearing rats were treated with 4-OHA injections on alternate weeks, tumor regression continued to occur during weeks without treatment. The overall regression was similar to that with continuous treatment. 4-OHA is also effective and similar to ovariectomy in rats with hormone-dependent metastatic mammary tumors produced by nitrosomethylurea. Our results indicate that mammary tumor regression induced by 4-OHA is mainly the result of the inhibition of aromatization, although other activities of the compound may also contribute.

Treatment of advanced postmenopausal breast cancer with an aromatase inhibitor, 4-hydroxyandrostenedione: phase II report.

4-Hydroxyandrostenedione (4-OHA), a potent new aromatase inhibitor, was given i.m. (500-1000 mg) to 58 patients with advanced postmenopausal breast cancer. Of 52 assessable patients 14 responded (27%), in 10 (19%) the disease stabilized, and in 28 (54%) the disease progressed. Sterile abscesses occurred at the injection site in 6 patients and painful lumps were found in a further 3 patients. Two patients developed allergic-type reactions and 4 developed lethargy, suspected to be treatment induced. Plasma estradiol levels were suppressed from a mean of 7.2 +/- 0.8 (SE) pg/ml before treatment to 2.6 +/- 0.2, 2.7 +/- 0.2, and 2.8 +/- 0.3 pg/ml after 1, 2, and greater than 4 months, respectively, of treatment and remained suppressed in patients whose disease relapsed. No significant fall in estrone levels was seen. Similarly, dehydroepiandrosterone sulfate, sex hormone binding globulin, and gonadotrophin levels were unaltered after 6 months of treatment. Plasma 4-OHA levels were measured in a radioimmunoassay for androstenedione after chromatographic separation of 4-OHA from androstenedione. Drug concentrations ranged from 0.7 to 23.2 (7.8 +/- 1.1) ng/ml after 2 months on treatment. 4-OHA is an effective drug in the management of postmenopausal patients with breast cancer and does not produce notable systemic side effects.

Use of the aromatase inhibitor 4-hydroxyandrostenedione in postmenopausal breast cancer: optimization of therapeutic dose and route.

4-Hydroxyandrostenedione (4-OHA) is a potent inhibitor of estrogen production by aromatase and causes suppression of plasma estradiol levels and disease regression in postmenopausal breast cancer patients. Groups of patients were given p.o. or parenteral 4-OHA, and plasma estradiol and 4-OHA levels were measured to enable the delineation of the minimal effective dose and optimal therapeutic regimen. A single injection of 500 mg i.m. suppressed estradiol levels to a mean 36.3 +/- 3.3% (SE) (n = 14) of base line after 4 to 7 days and maintained this suppression in six of seven patients for greater than 14 days. The half-life of 4-OHA was approximately 8 days, and when the level had fallen to less than 3 ng/ml, estradiol levels began to rise. Similar suppression was achieved by a single i.m. injection of 125 mg of 4-OHA and by 500 mg of 4-OHA p.o. daily after 1 wk, but escape from suppression was more rapid.

Growth inhibition of human prostate cells in vitro by novel inhibitors of androgen synthesis.

The long-standing strategy for the treatment of metastatic prostate cancer has been to reduce androgenic stimulation of tumor growth by removal of the testes, the primary site of testosterone synthesis. However, a low level of androgenic stimulation may continue, even after castration, by the conversion of adrenal androgens to 5alpha-dihydrotestosterone (DHT) in the prostate tumor cells. Two important enzymes of the androgen biosynthetic pathway are 17alpha-hydroxylase/C17,20-lyase, which regulates an early step in the synthesis of testosterone and other androgens in both the testes and adrenal glands, and 5alpha-reductase, which converts testosterone to the more potent androgen, DHT, in the prostate. We have identified new inhibitors of these enzymes that may be of use in achieving a more complete ablation of androgens in the treatment of metastatic prostate cancer. Three derivatives of androstene were shown to inhibit 17alpha-hydroxylase/C17,20-lyase with potencies 2-20-fold greater than that of ketoconazole, a previously established inhibitor of this enzyme. Derivatives of pregnane and pregnene displayed activities against 5alpha-reductase that were comparable to that of N-(1,1-dimethyl-ethyl)-3-oxo-4-aza-5alpha-androst-1-ene-17beta-car boxamide. All of the 5alpha-reductase inhibitors were able to at least partially inhibit the mitogenic effect of testosterone in either histocultures of human benign prostatic hypertrophic tissue or in cultures of the LNCaP human prostatic tumor cell line. For these compounds, it appears that this inhibition can be attributed to a reduction of DHT synthesis in these cultures, because no inhibitory effect was observed in DHT-treated cultures, and none of the compounds had a cytotoxic effect. Surprisingly, one of the inhibitors of 17alpha-hydroxylase/C17,20-lyase, 17beta-(4-imidazolyl)-5-pregnen-3beta-ol, was also able to inhibit the mitogenic effect of testosterone in both the histoculture and cell culture assays and had an effect against DHT as well. In transcriptional activation assays, it was found that this compound is an antagonist of both the wild-type androgen receptor and the mutant androgen receptor, which is present in LNCaP cells. In conclusion, the abilities of these compounds to inhibit androgen synthesis and, in some cases, to exert antiandrogen activity, did in fact translate to an inhibitory effect on the growth of human prostatic tissue in vitro, suggesting their potential utility in the treatment of prostatic cancer.

Aromatase, its inhibitors and their use in breast cancer treatment.

Aromatase, a cytochrome P450 enzyme, catalyses the rate-limiting step in the biosynthesis of estrogens. Many processes in male and female development and reproduction and especially in the growth of hormone-dependent cancers, are dependent on estrogens. Therefore, controlling estrogen production by inhibition of aromatase is a logical treatment strategy. Two classes of aromatase inhibitors, steroidal and non-steroidal compounds, are now coming into use. Among the steroid substrate analogs, 4-hydroxyandrostenedione has been shown to be effective in breast cancer patients with advanced disease and was recently approved for treatment in the United Kingdom. Several highly potent and selective non-steroidal inhibitors are now in clinical trials. The variety of compounds that act as aromatase inhibitors should provide breast cancer patients with a number of new treatment options.

Inhibition of peripheral aromatization by aromatase inhibitors, 4-hydroxy- and 4-acetoxy-androstene-3,17-dione.

To determine the effect of aromatase inhibitors on peripheral aromatization, male rhesus monkeys were infused with [7-3H]androstenedione and [4-14C]estrone before and during treatment with 4-hdyroxy-androstene-3,17-dione (50 mg/kg each; four monkeys) or 4-acetoxy-androstene-3,17-dione (700 mg each; two monkeys). In all six monkeys, no specific effects on the MCRs of androstenedione and estrone, the interconversion of androgens or estrogens, or androstenedione conversion to dihydrotestosterone were noted. In five of the six animals, aromatization rates were reduced by up to 97% of control values. The results demonstrate that these compounds inhibit peripheral aromatization and, together with our other results, suggest that aromatase inhibitors may be a useful treatment for estrogen-dependent cancer.

Aromatase inhibitors prevent granulosa cell differentiation: an obligatory role for estrogens in luteinizing hormone receptor expression.

To determine the role of newly synthesized estrogens in LH receptor expression, granulosa cells from diethylstilbestrol-implanted immature rats were cultured with FSH plus aromatase inhibitors. When present throughout the 48-h culture period, 4-hydroxy-4-androstene-3,17-dione (4-OHA; greater than or equal to 100 microM) and 1,4,6-androstatriene-3,17-dione (greater than or equal to 5 microM) inhibited FSH-induced LH receptor formation by 40% and 90%, respectively. Both aromatase inhibitors caused relatively greater inhibition of LH receptor formation when added from 20-48 h of culture, the period during which FSH-stimulated estrogen synthesis occurs (85% maximal inhibition with 4-OHA and 95% with 1,4,6-androstatriene-3,17-dione). Addition of estradiol, but not androstenedione, reversed the reduction of LH receptor formation by 4-OHA, indicating that the effects of the aromatase inhibitors were specifically related to their blockade of estradiol synthesis. The stimulation of estrogen production by FSH alone (8-fold) or with androstenedione (80-fold) during the 48-h culture period was prevented by 4-OHA. FSH-stimulated cAMP production was initially enhanced by 4-OHA from 0-20 h of culture, but was reduced from 20-48 h. Lower concentrations of 4-OHA (less than or equal to 50 microM) amplified FSH-stimulated cAMP production and LH receptor formation. However, these responses were blocked by the antiestrogen keoxifene or the antiandrogen flutamide, indicating that 4-OHA or a metabolite may have partial estrogenic or androgenic properties. The inhibitory effects of higher concentrations of 4-OHA on LH receptor expression were potentiated by keoxifene or flutamide. These results indicate that estrogen production and action are necessary for LH receptor expression in the granulosa cell.

Regulation of ovarian steroid biosynthesis by estrogen during proestrus in the rat.

Studies were performed to test the hypothesis that the rapid decline in estradiol (E2) levels on proestrus before ovulation was due to a reduction in androgen substrate for aromatase, and that this decline in androgen was regulated by an estrogen receptor-mediated mechanism. Aromatase activity, concentrations of E2, androstenedione (A), testosterone (T), and progesterone (P4) in follicular, corpora lutea, and ovarian homogenates as well as peripheral E2, A, P4, and LH were measured in cycling rats from 1400-2000 h on proestrus. These parameters were also recorded after the expected E2 surge in animals treated at 1900 h on diestrous day 2 with the antiestrogen keoxifene (20 mg/kg), with or without an ovulatory dose of PMSG at 1600 h on proestrus. In a second experiment, P-450-17 alpha-hydroxylase/C17,20-lyase (P-450(17 alpha] activity was measured in a group of control rats at 1500, 1700, and 1900 h. Aromatase activity remained unchanged, even though serum and ovarian E2 levels were reduced from peak values at 1500 h to basal values at 1800 h (P less than 0.01). Peripheral A as well as ovarian androgens (specifically follicular but not luteal) A and T were also reduced over this time period (P less than 0.01). Although total ovarian P4 remained unchanged, follicular levels rose from 1400-2000 h (P less than 0.01). These reductions in androgens and E2 levels coincided with a marked reduction in follicular P-450(17 alpha) activity. Treatment with keoxifene with or without PMSG prevented the fall in peripheral E2 and A and the increase in peripheral P4 seen in controls. Ovarian and follicular E2, A, and to a lesser extent T were also remained at values similar to those during the E2 surge. Follicular P4 was reduced by both treatments. Neither treatment had any effect on aromatase. These results indicate that the fall in peripheral and ovarian E2 levels before ovulation was due to a decline in aromatizable androgen, through an inhibition of follicular P-450(17 alpha) enzyme activity, which appears to be mediated by an estrogen receptor-regulated mechanism.

Differential responses of sex steroid target tissues of rats treated with 4-hydroxyandrostenedione.

4-Hydroxyandrostene-3,17-dione (4-OHA) inhibits ovarian aromatase activity and causes regression of carcinogen-induced hormone-dependent mammary tumors in rats. Although estrogen levels were reduced, LH levels did not increase nor did uterine weight decline in 4-OHA-treated animals. These findings are in contrast to those in animals deprived of estrogen by ovariectomy. The possible direct action of 4-OHA on gonadotropin secretion and uterine growth was, therefore, investigated in ovariectomized rats not treated with the carcinogen. Treatment with 4-OHA for 2 weeks prevented regression of the uterus and the increase in gonadotropin secretion in ovariectomized rats in a dose-dependent manner. The effect on gonadotropin secretion of 4-OHA at 50 mg/kg.day was similar to that of dihydrotestosterone at 0.5 mg/kg.day and could be completely antagonized by administration of the antiandrogen flutamide. The stimulation of uterine growth by 4-OHA was also blocked by flutamide, but not by the antiestrogen enclomiphene. The trophic action of 4-OHA at 50 mg/kg.day was equivalent to that of 1.8 mg/kg.day dihydrotestosterone. Furthermore, treatment with 4-OHA caused a reduction in uterine estrogen receptor and progesterone receptor levels. The reduction in uterine estrogen and progesterone receptor levels was also counteracted by the concomitant injection of flutamide, but not by enclomiphene. The results suggest that in the rat 4-OHA has multiple actions on sex steroid target tissues in addition to inhibition of aromatase. The effects appear to be related to the androgenic rather than estrogenic activity of the compound. Inhibition of gonadotropins may help maintain reduced ovarian estrogen secretion and contribute to the antitumor activity of this compound.

Somatomedin-C-mediated potentiation of follicle-stimulating hormone-induced aromatase activity of cultured rat granulosa cells.

We have recently observed that nanomolar concentrations of exogenously added somatomedin-C (Sm-C) are capable of synergizing with FSH in the induction of cultured rat granulosa cell progesterone biosynthesis and LH receptors without altering granulosa cell survival or replication. To further characterize the cytodifferentiative properties of Sm-C, we have undertaken to investigate whether the acquisition of granulosa cell aromatase activity is also subject to modulation by this intraovarian peptide. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were initially cultured for up to 3 days in an androstenedione-free medium, during which time aromatase activity was induced by FSH in the absence or presence of Sm-C (treatment interval). At the conclusion of this period, the cells were washed and reincubated for an additional 8-h test interval, during which time aromatase activity was estimated. Basal aromatase activity, as assessed by the conversion of unlabeled androstenedione (10(-7) M) to radioimmunoassayable estrogen, was negligible, remaining unaffected by treatment with highly purified Sm-C (50 ng/ml) alone. However, concurrent treatment with Sm-C (50 ng/ml) produced a 7.0-fold increase in the FSH (100 ng/ml; NIH FSH S14)-stimulated accumulation of estrogen. Similarly, Sm-C produced a 6.1-fold increase in FSH-induced aromatase activity, as assessed by the stereospecific generation of tritiated water from [1 beta-3H]androstenedione substrate. Sm-C-potentiated aromatase activity was dose and time dependent, with an apparent median effective dose of 5.0 +/- 1.9 (+/- SE) ng/ml and a minimal time requirement of 24 h or less, but was independent of the FSH dose employed. Although bovine insulin and multiplication-stimulating activity, like Sm-C, proved capable of augmenting aromatase activity (albeit at a substantially reduced potency), little or no effect was observed for either porcine or rat relaxin, a distantly related member of the insulin-like growth factor family. Examination of the apparent kinetic parameters of the aromatase enzyme revealed that the Sm-C-mediated potentiation of aromatase activity was due to enhancement of the apparent maximal reaction velocity, but not substrate affinity (Km = 2.8 X 10(-8) M). Our findings indicate that nanomolar concentrations of exogenously added Sm-C synergize with FSH in the enhancement of the maximal reaction velocity, but not Km, of granulosa cell aromatase in a dose- and time-dependent fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of an aromatase inhibitor, 4-hydroxy-4-androstene-3,17-dione, on estrogen-dependent processes in reproduction and breast cancer.

4-Hydroxy-f-androstene-3,17-dione (4-OH-A) when tested at various concentrations was found to inhibit markedly the conversion of 4-andorstene-3,17-dione to estrogens inhuman placental and rat ovarian microsomes. To obtain evidence that estrogen biosynthesis could also be reduced in vivo with 4-OH-A, rats were treated sc at a dose level of 50 mg/kg body weight. After 3 h the ovarian veins were cannulated and blood collected. Estradiol concentrations in the plasma were reduced by 80% compared to control values during the proestrous surge and on Day 4 of pregnancy. 4-OH-A was also found to be effective in controlling estrogen-dependent reproductive and neoplastic processes. In rats treated from Day 2-7 of pregnancy, implantation of fertilized ova was completely prevented in some rats, while in others either implantation was delayed or the development of implants was retarded. 4-OH-A treatment of rats having estrogen-dependent breast tumors induced by 7,12-dimethylbenz(a)anthracene caused 80% of the tumors to regress significantly in 4 weeks of treatment; 42% of these regressed completely.