Nine female rhesus monkeys (four in the follicular and five in the luteal phases of their cycles) had catheters implanted and were infused iv with [7-3H]<em>androstenedione</em> (A) and [<em>4</em>-1<em>4</em>C]estrone (E1) for <em>4</em> h. Blood samples were drawn at intervals from the hepatic, renal, jugular, uterine, and brachial veins and the femoral artery. The samples were analyzed for radioactivity as A and E1. The mean +/- SE MCRs for A and E1 were 280 +/- <em>4</em>0 and 270 +/- 30 liters/day, respectively. The mean extractions across the liver measured in six of the monkeys were 0.83 +/- 0.03 for A and 0.71 +/- 0.06 for E1. The percentage of A entering each tissue, which was measured as E1 leaving the tissue (pA,E1AV), was 0.20 +/- 0.10 for splanchnic, 0.21 +/- 0.11 for renal, 0.<em>4</em>6 +/- 0.21 for jugular, 2.36 +/- 1.27 for arm, and 0.35 +/- 0.10 for uterine veins. Because of the sampling technique, the value for the uterus may be a reflection of ovarian blood admixture with uterine blood. There were no apparent differences in tissue aromatization between values in the follicular and luteal phases of the cycle. The overall mean value for the percentage of A infused and measured as E1 in arterial blood (pA,E1BB) was 1.01 +/- 0.38%. Using previously reported tissue blood flow, we calculate that the contributions to the overall aromatization rate of tissues drained by the brachial, renal, jugular, hepatic, and uterine veins are 23%, 5%, 5%, <em>4</em>%, and 0.2%, respectively. Thus, the splanchnic tissue is a minor site for extraglandular aromatization of androgens in the rhesus monkey. An important site appears to be the arm, which reflects aromatization in adipose tissue, muscle, skin, and supporting structures.

Oral DHEA administration to patients with hypoadrenalism, in addition to glucocorticoid and mineralcorticoid replacement, may improve both well-being and hormonal/metabolic parameters. Twenty patients (13 men, 7 women, 26-76 yr, 11 with Addison's disease, 9 with central hypoadrenalism) were recruited in a placebo-controlled, randomized study. Hormone levels, carbohydrate and lipid parameters, bone metabolism, body composition and psychological parameters were evaluated at baseline and after treatment with DHEA 50 mg/day or placebo for <em>4</em> months. After <em>4</em> months of DHEA administration, serum DHEAS levels raised both in men (from 0.71+/-0.18 to 8.28+/-1.66 micropmol/l, p<0.005) and in women (from 0.25+/-0.07 to 5.65+/-1.93 micromol/l, p<0.05). Only in hypoadrenal women an increase in testosterone (T; from 0.<em>4</em>+/-0.1 to 1.<em>4</em>5+/-0.26 nmol/l, p<0.05) and <em>androstenedione</em> (A; from 0.86+/-0.3<em>4</em> to 2.05+/-0.29 nmol/l, p<0.05) levels was observed. In men no significant modifications in T and 17-hydroxyprogesterone (17-OHP) levels were found, whereas serum SHBG significantly decreased. As far as the metabolic parameters are concerned, only in patients with Addison's disease a significant decrease in total cholesterol and in low-density lipoproteins after <em>4</em> months of DHEA administration was found. No changes in glucose metabolism and insulin sensitivity were observed. In basal conditions, mean serum osteocalcin (OC) was normal and significantly decreased after DHEA treatment. A significant reduction in body fat mass percentage (BF%) after DHEA administration was observed. As far as well-being is concerned, DHEA replacement did not cause any relevant variation of subjective health scales and sexuality in both sexes. Our study confirms that DHEA may be beneficial for female patients with hypoadrenalism, mainly in restoring androgen levels. Concerning the health status, more sensitive and specific instruments to measure the effects of DHEA treatment could be necessary.

Tumor necrosis factor-alpha (TNF) induces clustering of theca-interstitial cells (TIC) isolated from immature, hypophysectomized rats, while inhibiting luteinizing hormone (LH)-stimulated <em>androstenedione</em> in vitro. Stimulators of PKC, 1-oleoyl-2-acetyl-sn-glycerol (OAG, 50 and 100 microM) and phorbol-12-myristate-13-acetate (PMA, 50 nM), caused TIC clustering by 6 days in vitro. Clustering induced by these compounds resembled that induced by TNF. The protein kinase inhibitor, staurosporine at 1 and 10 nM, impaired TNF-induced TIC clustering for 6 days, as did the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H-7); conversely, the protein kinase inhibitor, chelerythrine chloride (0.1, 1.0 or 10 microM), did not attenuate TNF-directed clustering. The protein kinase inhibitors did not reverse the suppression of LH-stimulated <em>androstenedione</em> by TNF. Inhibitors of the EGF receptor PTK, A23 (10, 50, or 100 microM) and A<em>4</em>6 (0.1, 1.0, 10, or 50 microM), impaired TNF-induced TIC clustering, while TNF suppression of LH-directed <em>androstenedione</em> was unaffected. EGF-induced TIC clustering was also impaired by A<em>4</em>6, while A23 was less effective. Both A23 and A<em>4</em>6 blocked EGF attenuation of LH-directed <em>androstenedione</em> after <em>4</em> days. When challenged with TNF (1 ng/ml) or PMA (50 nM), PKC activity increased in TIC. A23 (50 microM) and A<em>4</em>6 (10 microM) each alone blocked the TNF-associated increase in PKC activity; however, PKC activity attributable to PMA was unaffected by A<em>4</em>6. Together, these results suggest that TNF-induced TIC clustering involves activation of PTK which directs subsequent increases in PKC activity; however, mechanisms by which TNF inhibits LH-stimulated steroidogenesis remains elusive.

To investigate some biochemical changes during bovine follicle development, ovaries were obtained from cyclic heifers (7 to 11 heifers/d on each day of the 21-d estrous cycle; N = 152). Follicular fluid from the two largest follicles from both ovaries and a pool from small follicles (N = 30/cow) were collected from each animal and analyzed for ionic, enzymatic and endocrine changes in relation to day of the estrous cycle, follicle size, rank and atretic or growing status. Follicular fluid alkaline phosphatase activity and ascorbate concentrations were highest in all follicular sizes during the earlier portion of the estrous cycle (d 1 to 12; P less than .05), then decreased to the lowest levels (d 13 to 21). As follicular size (diameter) increased lactate dehydrogenase (LDH), acid and alkaline phosphatase activity was reduced in follicular fluid (P less than .05). Alkaline phosphatase and LDH activity tended to be increased in atretic follicles (P less than .10), and was correlated with increased progesterone and androgen concentrations of follicular fluid (r = .<em>4</em>, P less than .05). Both albumin and total protein concentrations decreased as follicular diameter increased (P less than .05). Sodium concentrations in follicular fluid were greater in growing-antral than atretic follicles, and increased with follicular enlargement (P less than .05). Follicular potassium concentrations increased as the estrous cycle progressed (P less than .05), and tended to be elevated in atretic follicles (nonsignificant). Both Ca and Mg concentrations increased with follicular enlargement (P less than .05). Dehydroepiandrosterone and testosterone were the predominant androgens in follicular fluid (<em>androstenedione</em>, the lowest concentration); their concentration decreased with follicle development (P less than .05), but were quite variable. Estradiol was increased in growing follicles (P less than .01). Estrone and estradiol concentrations increased as ovulation approached, particularly in small follicles (less than or equal to <em>4</em> mm diameter). Changes of biochemical components found in follicular fluid that relate to the growth and atresia process may provide a more sensitive and accurate method to classify follicle status, and thus aid in understanding the complexity of events associated with maturation of the bovine follicle and oocyte.

The objective of this study was to evaluate the levels of several pituitary and gonadal hormones in pancreatic adenocarcinoma. We examined circulating levels of LH, FSH, Testosterone, Oestradiol, Progesterone and delta <em>4</em>-<em>Androstenedione</em> in 36 patients with pancreatic adenocarcinoma, 12 patients with chronic pancreatitis and 87 age matched controls. According to our findings males with pancreatic cancer were found to have higher levels of FSH (p < 0.01). LH and oestradiol (p < 0.001) and lower levels of progesterone (p < 0.01) and testosterone (p < 0.05) than the controls. Female patients with pancreatic cancer were found to have higher levels of oestradiol (p < 0.001) and lower levels of LH, FSH and progesterone (p < 0.001), compared with group of healthy volunteers. Our data provide evidence of a generalised dysfunction of the hypothalamic-hypophysial-gonadal axis in pancreatic cancer patients.

Evidence that transforming growth factor-beta (TGF beta) is produced by thecal-interstitial cells (TIC) has suggested the hypothesis that TGF beta may be an autocrine regulator of TIC function. The purpose of these studies is to begin to test this hypothesis. In the present experiments we tested the effects of TGF beta on steroid production by TIC isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When TIC (10(<em>4</em>) viable cells/well) were cultured in serum-free medium (0.2 ml in 96-well plates), low amounts of androsterone (less than <em>4</em> ng/ml) were produced at 2, <em>4</em>, and 6 days. TGF beta (0.01-100 ng/ml) did not change basal androsterone production. Treatment with LH (50 ng/ml) stimulated a 100-fold increase in androsterone at 2 days and 60-fold increases at <em>4</em> and 6 days. Concomitant treatment with TGF beta (10 ng/ml) caused a 65% inhibition (ED50 = 2.3 +/- 0.7 ng/ml) of androsterone production at each time period. Analysis of key steroid metabolites demonstrated that androsterone and <em>androstenedione</em> were inhibited equally, while progesterone was significantly increased (ED50 = 1.2 +/- 0.2 ng/ml). Time-course studies revealed that TGF beta alone did not alter progesterone production at 2 days, but markedly increased progesterone (10-fold) above control levels at <em>4</em> and 6 days. Dose-response experiments showed that TGF beta did not alter the sensitivity of the TIC to LH stimulation, indicating that LH activation of the intracellular signaling pathway was not blocked by TGF beta. Treatment with insulin-like growth factor-I (IGF-I) together with LH caused a synergistic increase in androsterone production. The synergistic stimulation of LH action by IGF-I could be blocked by TGF beta. Interestingly, TIC were more sensitive to TGF beta in the presence of IGF-I (ED50 = 0.18 +/- 0.0<em>4</em> ng/ml). In contrast, TGF beta enhanced progesterone production only at the highest dose of TGF beta (10 ng/ml). To further elucidate the mechanism of TGF beta action, the effects of TGF beta on the TIC content of 17 alpha-hydroxylase/C17-20 lyase (P<em>4</em>50(17)alpha) and cholesterol side-chain cleavage (P<em>4</em>50scc) were analyzed by immunoblotting. TGF beta alone or in combination with LH stimulated an increase in P<em>4</em>50scc content, but did not alter P<em>4</em>50(17 alpha content. These results lead us to conclude that 1) the TIC are targets for TGF beta; 2) IGF-I increases the sensitivity of TIC to TGF beta action; and 3) TGF beta acts directly on TIC to stimulate progesterone while inhibiting androgen production.

Progesterone, <em>4</em>-<em>androstenedione</em>, testosterone, dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol), estrone, and estradiol levels were determined by radioimmunoassay in the different lobules of the testis of Salamandra salamandra throughout the year according to the seasonal cycle. 3 beta-diol levels were not detectable. High levels of steroids were found in the grandular tissue (enlarged pericystic cells after spermiation) and large variations were showed for progesterone, <em>4</em>-<em>androstenedione</em>, testosterone, 3 alpha-diol, and estrone. In the mature lobule (formed by cysts with mature spermatozoa), only testosterone showed seasonal variations and in the immature lobule (with early stages of meiosis), 3 alpha-diol showed fluctuations. The major estrogen found in the testis of Salamandra was estrone; estradiol stayed at a low level throughout the cycle. The steroids fluctuation seems to be related to the histological evolution of the testis throughout the cycle. The present data were the first on steroid seasonal variations in the testis of an urodele.

Evodiamine, a bioactive component isolated from the Chinese medicine Wu-chu-yu, exhibits vasodilative and antianoxic action. Although evodiamine indeed has many biological effects, its effects on the endocrine system are not clear. The present study explored the effects of evodiamine on testosterone secretion in vitro. Rat collagenase-dispersed testicular interstitial cells (TICs) were incubated with evodiamine (0 to 10(-<em>4</em>) mol/L) in the presence or absence of human chorionic gonadotropin (hCG), forskolin, 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), or steroidogenic precursors (including 25-hydroxycholesterol, pregnenolone, progesterone, 17alpha-hydroxyprogesterone, and <em>androstenedione</em>) at 3<em>4</em> degrees C for 1 hour. The testosterone concentration in the media samples was measured by radioimmunoassay. Evodiamine 10(-<em>4</em>) mol/L was effective to reduce both basal and hCG-stimulated testosterone secretion in rat TICs after 1, 2, or <em>4</em> hours of incubation. The stimulatory effect of forskolin on testosterone release in TICs was prevented by administration of evodiamine. Evodiamine 10(-<em>4</em>) mol/L also decreased 8-Br-cAMP- and <em>androstenedione</em>-stimulated testosterone secretion. These results suggest that evodiamine reduces testosterone secretion in rat TICs via a mechanism involving reduced activity of cAMP-related pathways and 17beta-hydroxysteroid dehydrogenase (17beta-HSD).

Virilization may occur during pregnancy as the result of an ovarian Krukenberg tumor. mechanism of the androgen overproduction in this exceptional condition is still poorly understood. A new case is reported in which only in the postpartum clinical, endocrine, and endoscopic studies led to the diagnosis of an ovarian Krukenberg tumor secondary to a gastric carcinoma. In the mother, basal hormonal studies were done 1 and <em>4</em> weeks after delivery, then after gastric and ovarian surgery. Three months after delivery, ovarian steroid response to hCG (priming dose, 5000 IU; then 1500 IU every other day for 12 days) and a study of progesterone (P) metabolism at a steady state after a constant infusion of [3H]P and cold P (92 micrograms/min leads to blood production rate (BPR) of 152 mg/day designed to reproduce the BPR of P usually seen in pregnancy) were successively performed. Hormones were measured by specific RIAs after chromatographical purification. Basal hormonal levels were normal in the child. In the mother, on the 5th day postpartum, mean hormone levels (in nanograms per dl) were: testosterone (T), <em>4</em>181; <em>androstenedione</em> (delta <em>4</em>), 8876; 17 alpha-hydroxyprogesterone (17-OHP), 97<em>4</em>6; P 1075; estrone (E1), 195; and estradiol (E2), 151. One month later, levels were normal for the follicular phase; T, <em>4</em>0; delta <em>4</em>, 1<em>4</em>6; P, 52; E2, 9; and E2, <em>4</em>.5. At both times, dehydroepiandrosterone was normal (703-750). Hormone levels increased progressively during hCG stimulation but their time course was different between hormones. At the end of the test, T. 1<em>4</em><em>4</em>; delta <em>4</em>, 7<em>4</em>6;' 17-OHP, 789; P, 723; E1, 37; and E2, 20. The MCR of P was decreased, 1<em>4</em>50 liters/day (normal, 2020). Conversion ratios between products and precursor during constant infusion were normal. From these data, obtained in four different conditions (postpartum period, hCG stimulation, progesterone infusion, and after oophorectomy), the following can be concluded: adrenal production of dehydroepiandrosterone was normal; the ovarian overproduction of androgens likely resulted from the excessive reductive metabolism of both placental and ovarian P along the delta <em>4</em> steroid biosynthetic pathway by an hypertrophic stromal compartment; and HCG stimulation seems to be the necessary stimulus for this condition. The enhancement by T on its own peripheral production is also discussed.

The aim of this paper was to investigate the relationship between sex hormones and fat distribution in premenopausal obese women. Serum concentrations of sex hormones, glucose tolerance and fat distribution were determined in a population of non-diabetic obese women, in the outpatient clinic of University Hospital, Bari, Italy. The subjects were <em>4</em>0 consecutive premenopausal obese women (BMI>> 25). The amounts of visceral, abdominal subcutaneous, and femoral subcutaneous fat, and the visceral to abdominal subcutaneous fat ratio were measured by ultrasound techniques. Serum concentrations of total testosterone (T), free testosterone (FT), dehydroepiandrosterone sulphate (DHEAS), delta <em>4</em>-<em>androstenedione</em> (A), 17-beta-estradiol (E2), sex hormone binding globulin (SHBG), and the FT to DHEAS molar ratio were measured during the follicular phase. Plasma glucose and insulin concentrations were evaluated during an oral glucose tolerance test. Of all sex hormones, the FT/DHEAS molar ratio was the parameter that most closely related to the amount of visceral fat (r: 0.5<em>4</em><em>4</em>, P < 0.001), and this positive association was maintained (P < 0.01) after adjustment for age, BMI and insulin levels (fitted model: R2 adjusted: 0.50<em>4</em>; F ratio: 1<em>4</em>.73; P-value: < 0.0001). DHEAS was inversely correlated with the amount of visceral fat (r: -0.32<em>4</em>, P < 0.05). T was inversely correlated with the amounts of both abdominal subcutaneous (r: -0.<em>4</em>09, P < 0.01) and visceral fat (r: -0.32<em>4</em>, P < 0.05). The FT to DHEAS molar ratio is the androgenic parameter that most closely relates to the accumulation of visceral fat in premenopausal obese women.

OBJECTIVE

The aim of this investigation is to study androgen metabolism in gingival fibroblasts in response to phenytoin, oestradiol and the antioestrogen tamoxifen, in order to establish the possible role of hormones in the aetiopathogenesis of phenytoin-induced gingival overgrowth.

METHODS

Six cell lines of human gingival fibroblasts were established in monolayer culture in Eagle's minimum essential medium. Duplicate incubations were performed independently with radiolabelled testosterone and <em>4</em>-<em>androstenedione</em>, respectively (1<em>4</em>C-T/1<em>4</em>C-<em>4</em>-A), with optimal concentrations of phenytoin, oestradiol and tamoxifen alone and in combination. At the end of a 2<em>4</em>-h incubation period, the medium was solvent extracted for steroid metabolites, which were separated by thin layer chromatography and quantified using a radioisotope scanner.

RESULTS

The substrates were metabolised mainly to the diols, 5alpha-dihydrotestosterone (DHT) and <em>4</em>-<em>androstenedione</em> or testosterone, with the two substrates used. The trends were that phenytoin and oestradiol significantly elevated the yields of the androgens DHT, diols and <em>4</em>-A/testosterone from both substrates while tamoxifen inhibited the stimulatory effects of oestradiol and phenytoin alone and in combination (n=6; p<0.01, one-way anova).

CONCLUSIONS

Specific hormone-mediated activity in response to phenytoin could contribute to the pathogenesis of gingival overgrowth, which can be decreased by the anti oestrogen tamoxifen.

Physiological and many pathological changes in plasma sex-hormone-binding globulin (SHBG) levels have been attributed to the opposing effects of androgens which lower, and oestrogens which elevate, levels. We examined four clinical situations in which changes in SHBG levels may not be explained by sex steroid alterations. (1) Dexamethasone caused an increase in SHBG levels in hyperandrogenaemic hirsute women whether or not androgens were suppressed. (2) In male patients with untreated isolated gonadotrophin deficiency there was a highly significant correlation between SHBG levels and age, but there was no relationship between the levels of SHBG and those of plasma testosterone, <em>androstenedione</em> or DHEAS. (3) Two <em>4</em>6-XY siblings, phenotypic female subjects with complete androgen insensitivity, demonstrated a marked decline in SHBG levels between the ages of 9-13 and 12-16 years. (<em>4</em>) SHBG was suppressed in obese oligomenorrhoeic women while plasma concentrations of testosterone, <em>androstenedione</em> and oestradiol were normal and that of oestrone was elevated; however, the testosterone:SHBG ratio, an index of free testosterone, was elevated. These observations indicate that the decline in SHBG levels which normally occurs in men during the second decade of life is independent of androgen activity and is under the influence of as yet unidentified factors. Glucocorticoids in small doses under the influence of as yet unidentified factors. Glucocorticoids in small doses increase SHBG levels independently of sex steroid alterations while elevated free testosterone concentration may contribute to suppression of SHBG in obesity.

Serum androstanediol glucuronide (3 alpha-diol G), a metabolite of the active androgens dihydrotestosterone and androstanediol, was elevated in 28 consecutive women with idiopathic hirsutism (IH). The mean 3 alpha-diol G level in the women with IH was <em>4</em>87 +/- 192 (+/- SD) ng/dL compared to 119 +/- 37 ng/dL in normal women (n = 50), and only 1 patient had a value overlapping with the normal range. Since 3 alpha-diol G appears to be formed entirely in target organs and has a long serum half-life, we studied its clinical usefulness by following women with IH during treatment. In 15 of 17 women with IH treated for 1-<em>4</em> yr with glucocorticoids, contraceptives, or spironolactone, serum 3 alpha-diol G levels changed concordantly with clinical responses, in contrast to the poor concordance of serum testosterone (5 of 17), free testosterone (7 of 17), and <em>androstenedione</em> (7 of 17). Specifically, in IH patients treated with spironolactone, serum testosterone, free testosterone, and <em>androstenedione</em> levels changed little, yet clinical improvement frequently occurred, and this improvement was reflected by concomitantly lowered 3 alpha-diol G levels. Further, in <em>4</em> IH patients, discontinuation of effective therapy resulted in prompt increases in serum 3 alpha-diol G as harbingers of worsening hair growth. We, thus, conclude that serum 3 alpha-diol G measurements are clinically useful in evaluating hirsute women and correlate with the clinical responses to therapy.

Many studies indicate that obesity is associated with postmenopausal breast cancer and cancer of the endometrium. The mechanisms by which obesity contributes to cancer risk is not known, although increases in serum estrone from delta <em>4</em>-<em>androstenedione</em> by the adipose tissue have been implicated in postmenopausal women. Blood estrogens increase with the degree of obesity and aging. In animal experiments the confounding of high fat, low carbohydrate, and high calorie diets needs to be defined. The effects of diet on estrogen metabolism; the relationship of fatty acids from animal, vegetable, and marine sources to tumor formation; and the mechanisms by which energy intake influences cancer risk need to be precisely defined. Any estimate of the contribution of heredity to the burden of human cancer is impossible until we have a better understanding of genetic and environmental interactions.

The peripheral aromatization ([rho]BM) of <em>androstenedione</em> (A) and testosterone (T) was measured before and after administration of the aromatase inhibitor 10-(2 propynyl)estr-<em>4</em>-ene-3,17-dione (MDL-18,962) to five mature female baboons, Papio annubis. The measurements were made by infusing [3H]<em>androstenedione</em>/[1<em>4</em>C]estrone or [3H]testosterone/[1<em>4</em>C]estradiol for 3.5 h and collecting blood samples during the infusions and all urine for 96 h from the start of the infusion. Blood samples were analyzed for radioactivity as infused and product steroids, and the data were used to calculate MCRs. An aliquot of the pooled urine was analyzed for the glucuronides of estrone and estradiol and used to calculate the [rho]BM. MDL-18,962 was administered as a pulse in polyethylene glycol-<em>4</em>00 (1-5 ml) either iv or via gastric tube 30 min before administration of the radiolabeled steroids. Control studies were done with and without polyethylene glycol-<em>4</em>00 administration. When MDL-18,962 was given iv at <em>4</em> mg/kg, the aromatization of A was decreased 91.8 +/- 0.9% from the control value of 1.23 +/- 0.13% to 0.11 +/- 0.01%. At the same dose, aromatization of T was decreased 82.0 +/- 7.1%, from a control value of 0.20 +/- 0.03% to 0.037 +/- 0.018%. When MDL-18,962 was given iv at doses of 0.<em>4</em>, 0.1, 0.0<em>4</em>, and 0.01 mg/kg, the values for aromatization of A were 0.16 +/- 0.03%, 0.18 +/- 0.06%, 0.37 +/- 11%, and 0.65 +/- 0.09%, respectively. The administration of MDL-18,962 via gastric tube at <em>4</em> mg/kg as a pulse decreased the aromatization of A from 1.35 +/- 0.06% to 0.<em>4</em>3 +/- 0.12%, an inhibition of 67.2 +/- 10.7%. When administered via gastric tube daily for 5 days at <em>4</em> mg/kg, the aromatization of A fell from 1.35 +/- 0.06% to 0.063 +/- 0.003%, an inhibition of 8<em>4</em>.<em>4</em> +/- 0.5%. The MCRs of A and estrone were not altered by any dose of MDL-18,962 regardless of the mode of administration, but there was an increase in the MCRs of T and estradiol at the only dose (<em>4</em> mg/kg, iv) at which these steroids were infused. The interconversions between the androgens and between the estrogens were not altered by the administration of MDL-18,962 at <em>4</em> mg/kg, iv. The enzyme-activated inhibitor MDL-18,962 is an effective inhibitor of [rho BM] in female baboons and could prove to be a useful therapeutic agent in treating estrogen-dependent breast cancer.

In postmenopausal women with breast cancer, aromatase, which is the enzyme converting <em>androstenedione</em> to estrone and testosterone to estradiol, is the rate-limiting step in estrogen biosynthesis. The currently available aromatase inhibitor, aminoglutethimide, effectively blocks estrogen production and products tumor regression in patients previously treated with tamoxifen. This drug, however, produces frequent side effects and blocks steroidogenic steps other than the aromatase enzyme. Thus, newer aromatase inhibitors with greater potency and specificity are under intense study. More than 20 such compounds have recently been developed. In several clinical trials, <em>4</em>-hydroxy<em>androstenedione</em>, given parenterally, has been highly active and specific for aromatase inhibition in patients with breast cancer. In two large recent studies, one-third of heavily pretreated woman experienced objective tumor regression with this therapy. CGS 169<em>4</em>9A, a newer agent, is also in Phase III clinical trials. This compound is an imidazole derivative with nearly 1000-fold greater potency than aminoglutethimide. An initial Phase I study compared the potency of 0.6-16 mg daily in 12 postmenopausal women and found maximal suppression of urinary and plasma estrogens with 2 mg daily. The degree of inhibition was similar to that induced by aminoglutethimide or by surgical adrenalectomy. No CNS, hematologic or biochemical toxicity was observed. A larger Phase II study in 5<em>4</em> patients confirmed this high degree of potency of CGS since a plateau effect was observed at the 1.8, 2 and <em>4</em> mg daily doses. The endocrine effects were not absolutely specific as a blunting of ACTH-stimulated but not basal aldosterone levels were observed. This and other emerging aromatase inhibitors offer promise as pharmacologic methods to inhibit estrogen production specifically and without side effects.

OBJECTIVE

To study redox responses of cultured osteoblasts, mediated by bacterial lipopolysaccharide (LPS), glucose (G), glucose-oxidised low density lipoprotein (GLDL) and minocycline (M) using radiolabelled steroid markers of redox status and wound healing. The clinical relevance of this concept in periodontitis patients with cardiometabolic risk markers is addressed.

METHODS

A well differentiated osteoblastic cell-line was cultured in Eagle's MEM in confluent monolayer, in 2<em>4</em> well multiwell plates. Radiolabelled testosterone was used as the steroid substrate. Experiments were set up with controls in the absence of agents, optimal concentrations (previously determined) of G, GLDL, LPS, M, GLDL+LPS and the latter combined with M (n = 8). At the end of a 2<em>4</em>h incubation period, the reaction was terminated and the medium analysed for yields of the steroid metabolite 5α-dihydrotestosterone (DHT), the redox marker relevant to wound healing, the weaker androgen <em>4</em>-<em>androstenedione</em> (<em>4</em>-A) and the diols. Analysis entailed thin layer chromatography and radioisotope scanning.

RESULTS

The yields of DHT showed 1.<em>4</em>-fold and 2.3-fold decreases in response to GLDL and LPS respectively and a 1.3-fold reduction in response to the combination, when compared with controls in the absence of agents. Minocycline stimulated the yield of DHT by 1.<em>4</em>-fold, and when combined with GLDL+LPS, the decreased yield was overcome and raised to 2-fold above the combination in response to the addition of minocycline (n = 8; p < 0.001), when compared with controls. The trends in the yields of <em>4</em>-A and diols were inversely related to each other with increases and decreases over controls respectively, in keeping with enzymic pathways.

CONCLUSIONS

Decreased yields of the oxidative stress marker DHT in response to LPS, G and GLDL were overcome in the presence of minocycline, which demonstrates its potential role as an adjunctive therapeutic agent in an environment of oxidative stress. These applications could be extrapolated to periodontal disease and co-existing cardiometabolic risk markers, in the context of its antiinflammatory and antioxidant actions relevant to healing. In this paper, recent patents relevant to adjunctive therapeutic management of periodontal disease co-existing with cardiometabolic risk markers are addressed. There have been significant advances in therapeutic interventions for overcoming oxidative stress-inducing mechanisms that are common to these disease entities.

We measured follicular fluid hormone levels in <em>4</em>8 normally cycling infertile women who underwent follicle puncture and oocyte retrieval during diagnostic laparoscopy at time-bracketed intervals after an endogenous LH surge. Follicular fluid LH, FSH, PRL, estrone (E1), estradiol (E2), progesterone (P), <em>androstenedione</em> (A), and testosterone (T) concentrations and P/E2 and A/E2 ratios were determined. Oocytes were classified as germinal vesicle (gv), metaphase I (mI), metaphase II (mII), or degenerating (dg). Follicular fluid (ff) hormone levels then were correlated with the stage of oocyte maturation. There were no differences in ff E1 or E2 levels at any stage of oocyte maturation, except that the mean ff E2 concentration was significantly (P less than 0.05) lower in ff containing dg oocytes [2,<em>4</em>7<em>4</em> +/- 1,<em>4</em>35 (+/- SE) nmol/L] than in those containing the other oocyte stages. The mean P levels were significantly (P less than 0.0001) higher in ff containing mI (<em>4</em>8,781 +/- 10,2<em>4</em>0 nmol/L) and mII (<em>4</em>1,801 +/- 11,098 nmol/L) oocytes than in ff containing gv oocytes (1371 +/- 696 nmol/L). The mean A level was highest (P less than 0.01) in dg-associated ff. Similarly, T was highest (P less than 0.05) in ff containing dg (52 +/- 1<em>4</em> nmol/L) oocytes than in ff containing mI (10.7 +/- 10.1 nmol/L) or mII (10.1 +/- <em>4</em> nmol/L) oocytes, and it was also elevated (P less than 0.05) in gv ff (72 +/- 33 nmol/L) compared to mII ff. The above differences also were reflected in the P/E2 ratio, which was significantly higher (P less than 0.05) in mI and mII ff, as well as in the A/E2 ratio, which was higher (P less than 0.05) in ff containing mI and mII oocytes compared to ff containing gv or dg oocytes. These data define the evolving changes in the microenvironment of the follicular fluid of preovulatory follicles of normally cycling women. They also provide reference points for analysis of ff obtained from women during stimulated cycles intended for in vitro fertilization.

Time-mated baboons (n = 8) were bled throughout the luteal phase of the cycle of conception, and an equal number of nonpregnant animals were studied as controls. A significant increase in plasma testosterone and <em>androstenedione</em> was seen in the cycle of conception prior to expected menses, whereas the levels in nonpregnant baboons were unchanged throughout the luteal phase. Plasma testosterone and <em>androstenedione</em> continued their rise in three pregnant baboons sampled between days 16 and 33 of gestation. In an additional three baboons bled at <em>4</em>-day intervals from day 35 through day 75, there was a further increase to about day <em>4</em>0, but by day 50 the androgen levels had declined to nonpregnant luteal phase levels and remained constant. Treatment with human chorionic gonadotropin during the nonpregnant luteal phase caused increases in both testosterone and <em>androstenedione</em>. Removal of the ovary bearing the corpus luteum at day 20 of gestation resulted in abortion and a sharp drop in plasma progesterone and estradiol. One baboon had a dramatic decline of the elevated plasma androgen following oophorectomy, while another that did not have elevated androgen levels showed only a trivial decline. Pregnancy continued in two baboons in which the corpus luteum-bearing ovary was removed at day 25 or day 30. There was only a slight drop in plasma progesterone postoperatively and a rapid return to normal levels. A similar decline, with a more gradual recovery, was noted in plasma estradiol and androgen levels. In one animal the androgen levels were increased by about day <em>4</em>0 and subsequently declined by day 50, just as did the androgens of unoperated pregnant baboons. Estrogen administration in early pregnancy causes a suppression of the normal increases in plasma estradiol and androgen levels.

Potency and selectivity of aromatase inhibition are parameters which ultimately influence the therapeutic efficacy of aromatase inhibitors. This report describes an in vitro model which allows an assessment of the selectivity with which aromatase inhibitors inhibit estrogen biosynthesis. Estrogen production was stimulated by incubating adult female hamster ovarian tissue with ovine LH. The production rates of estrogens (E), testosterone (T) and progesterone (P) were determined using radioimmunoassays to measure the amount of these steroids released into the incubation medium over a <em>4</em>-hour incubation period. The selectivity of aromatase inhibition was assessed by determining the IC50S with which each inhibitor inhibited the production of E (end product), T (immediate precursor of E) and P (early precursor of E). Selectivity was studied for each of the <em>4</em> aromatase inhibitors, CGS 169<em>4</em>9A (a new non-steroidal compound), <em>4</em>-OH-<em>androstenedione</em>, aminoglutethimide and testolactone. CGS 169<em>4</em>9A was the most potent of the four, followed by <em>4</em>-OH-<em>androstenedione</em>, aminoglutethimide and testolactone. As far as selectivity was concerned, both CGS 169<em>4</em>9A and <em>4</em>-OH-<em>androstenedione</em> selectively inhibited aromatase judging from the IC50s for E and P production (CGS 169<em>4</em>9A: IC50 for E & P = 0.03 & 160 microM, resp.; <em>4</em>-OH-<em>androstenedione</em>: IC50 for E & P = 0.88 & greater than or equal to 330 microM, resp.). Aminoglutethimide was the least selective inhibitor of aromatase (IC50 for E & P = 13 & 60 microM, resp.). For testolactone, the least potent of the four (IC50 for E = 130 microM), no conclusive data were obtained concerning the selectivity of aromatase inhibition. Thus a simple, effective and reproducible method is described for assessing the selectivity with which aromatase inhibitors inhibit aromatase.

Sex hormone binding globulin (SHBG) capacity was reduced in 9 of 31 patients with polycystic ovarian (PCO) disease and the mean level in PCO patients was significantly less (p less than 0.001) than normal. Serum testosterone levels were elevated in 21 of 32 PCO patients and the mean level was significantly elevated (p less than 0.001). Serum <em>androstenedione</em> values were raised in 17 of 31 patients and the mean value was also significantly raised (p less than 0.001). Serum dehydroepiandrosterone sulphate (DHAS) concentrations were elevated in only 2 of 1<em>4</em> patients. Urinary 17-oxo and 17-oxogenic steroids were normal in all patients studied. Basal follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were normal but LH release following injection of luteinizing hormone-releasing hormone (LH-RH) was enhanced. A highly significant negative correlation (r=--0.<em>4</em><em>4</em>9; p less than 0.01) was found between the logarithm of testosterone and the logarithm of LH levels. Serum prolactin concentrations were elevated in <em>4</em> of 21 PCO patients. Thyroid-stimulating hormone (TSH) values were normal. Eighteen of 20 patients ovulated following treatment with clomiphene and nine became pregnant. Five of 12 of patients treated with oestrogen/progesterone preparations noticed an improvement in their hirsutism. It is suggested that the normal cyclical release of LH is inhibited in PCO disease by a negative feedback by androgens to the hypothalamus or the pituitary, and that wedge resection should be reserved for patients in whom other forms of treatment have failed.

The present report deals with the concentrations of C-21, C-19 and C-18 steroids as well as steroid glucuronides, namely androstane-3 alpha,17 beta-diol glucuronide, androsterone glucuronide, estradiol glucuronide, and estrone glucuronide (E1-G) in breast cyst fluid (BCF). The concentration of the gross breast cystic disease protein 15 (GCDFP-15) was also measured and its value was correlated with concentrations of unconjugated steroids as well as steroid conjugates. The present data indicate that for a large number of unconjugated steroids (namely pregnenolone, progesterone, dehydroepiandrosterone (DHEA), androsterone, androstane-3 alpha,17 beta-diol, estrone and estradiol) there is an important accumulation in BCF. Our data permit us to demonstrate a statistical relationship between the concentrations of DHEA and its metabolites (namely, <em>androstenedione</em> [<em>4</em>-ene-Dione]), thus suggesting an activity of the enzyme 3 beta-hydroxysteroid dehydrogenase-<em>4</em>-ene-5-ene-isomerase in the breast tissue. Further examination of the relationship between the concentrations of <em>4</em>-ene-Dione and its metabolites, namely testosterone and 5 alpha-reduced steroid metabolites, as well as their glucuronide derivatives, strongly suggests that the metabolism of androgens in the breast tissue occurs mainly via 5 alpha-reductase and glucuronyl transferase activities. The concentration of GCDFP-15 in BCF found in the present investigation was 27<em>4</em>5 +/- 23<em>4</em> micrograms/ml and we have shown that a negative relationship exists in the BCF between E1-G and GCDFP-15 levels.

OBJECTIVE

This study sought to examine corticosteroidogenic enzyme activities in normo- and hyperandrogenic polycystic ovary syndrome (PCOS) patients.

METHODS

This cohort study included 81 patients with biochemical hyperandrogenism and <em>4</em>1 patients with normal androgen levels. Enzyme activities were assessed according to the serum steroid product/precursor ratios at baseline and after adrenal stimulation.

RESULTS

At baseline, in the delta <em>4</em> (Δ<em>4</em>) pathway, hyperandrogenic patients showed greater 17-hydroxylase and 17,20 lyase activities in converting progesterone (P<em>4</em>) into 17-hydroxyprogesterone (17-OHP<em>4</em>) and 17-hydroxypregnenolone (17-OHPE) into <em>androstenedione</em> (A) (p = 0.0005 and p = 0.0<em>4</em>7, respectively) compared to normoandrogenic patients. In the delta 5 (Δ5) pathway, the 17-hydroxylase and 17,20 lyase enzymes showed similar activities in both groups. Hyperandrogenic patients presented lower 21-hydroxylase, lower 11β-hydroxylase (p = 0.0001), and statistically significant increases in 3β-hydroxysteroid dehydrogenase II (3β-HSDII) activities (p < 0.0001). Following tetracosactrin stimulation, only the 17,20 lyase activity remained up-regulated in the Δ<em>4</em> pathway (p < 0.0001).

CONCLUSIONS

Hyperandrogenic patients had higher 17,20 lyase activity, both at baseline and after adrenal stimulation. Greater conversion of dehydroepiandrosterone (DHEA) into A with normal conversion of 17-OHPE to 17-OHP<em>4</em> in hyperandrogenic PCOS patients indicated different levels of 3β-HSDII activity in adrenal cells, and hyperandrogenic patients had lower 11β-hydroxylase and 21-hydroxylase activities.

The plasma concentrations of testosterone (T), dehydroepiandrosterone (D), <em>androstenedione</em> (A), pregnenolone (delta 5P), progesterone (P), 17-hydroxypregnenolone (17-delta 5P), 17-hydroxyprogesterone (17-P), 11-deoxycortisol (S), and cortisol (F) were measured before, and 30 and 60 minutes after, a bolus intravenous injection of 25 units of adrenocorticotropic hormone (ACTH) in nine normal women and in fifteen patients with a variety of manifestations of androgen excess. Patients with androgen excess demonstrated significantly higher mean baseline levels of T, D, A, delta 5P, 17-delta 5P, and 17-P. After a bolus intravenous injection of 25 units of ACTH, higher-than-normal increments were noted for the following steroids: delta 5P (one patient), 17-delta 5P (one patient), D (two patients), P (one patients), 17-P (two patients), and S (two patients). Following ACTH injections, the ratios of increments in plasma steroid pairs were computed to estimate the efficiency of several adrenal enzymes, and evidence suggesting partial deficiency of 3 beta-hydroxysteroid dehydrogenase delta <em>4</em>-5 isomerase (five patients) and 11 beta-hydroxylase (five patients) was found. In addition, 6 of the 15 patients with androgen excess exhibited an abnormally high increment in D relative to the increment in F. The data show that apparent abnormalities in adrenal steroid biosynthesis are a frequent occurrence in patients with hyperandrogenism.