In vitro autoradiographic methods have been developed for selective measurement of occupied and unoccupied estrogen receptors (ERs) in brain tissue sections. Addition of protamine sulfate traps unoccupied ERs in the tissue sections, allowing them to be detected after a short period of incubation with labeled estrogen. Occupied ERs are assessed, after washing in buffer without protamine to eliminate unoccupied receptor, by incubating the sections for 2 h at 37 C to exchange isotopically labeled steroid for the endogenous unlabeled ligand. Total ER binding capacity is estimated by summing the values for occupied and unoccupied ER. In all brain regions of normal females, ER occupation is low at estrus, reflecting the very low levels of circulating estradiol present at this stage of the estrous cycle, rising to approximately 50% of binding capacity at proestrus. By contrast, in intact males ER occupation varies considerably between brain regions, from a high of 55% of binding capacity in the bed nucleus of the stria terminalis to a low of 21% in the hypothalamic arcuate nucleus. Gonadectomy or treatment of intact males with the aromatase inhibitor <em>4</em>-hydroxy <em>androstenedione</em> greatly reduces or eliminates ER occupation, depending on the brain region. In both sexes, changes in levels of endogenous gonadal steroids have little effect on total (occupied plus unoccupied) ER concentrations, with the exception of the hypothalamic ventromedial nucleus of the female, in which total ER concentration declines at estrus. These results are consistent with the hypothesis that local aromatization may be the primary determinant of regional ER occupation in the brain of the male rat, in contrast to the female, in which high levels of ER occupation are found only during the preovulatory estrogen surge. Although physiological changes in circulating estradiol and aromatizable androgen concentrations induce large changes in ER occupation, they have little effect on total ER content in most regions of the brain, suggesting that previous reports of changes in ER messenger RNA levels under different conditions of gonadal steroid exposure may not be directly reflected in steady state levels of the cognate receptor site.

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-<em>4</em>50 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.<em>4</em>-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.<em>4</em>5% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-<em>4</em>50 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-<em>4</em>50 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and <em>4</em> of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-<em>4</em>50 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-<em>4</em>50 reductase and at least one form of cytochrome P<em>4</em>50 (P-<em>4</em>50IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-<em>4</em>50 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-<em>4</em>50 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of <em>androstenedione</em> and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Plasma 17 alpha-hydroxyprogesterone (17-OHP), <em>androstenedione</em> and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/L (r=0.999). Recoveries (S.D.) of 17-OHP, <em>androstenedione</em> and testosterone in plasma were 100% (5), 102% (2) and 92% (<em>4</em>), respectively. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y=1.9<em>4</em>2 x+0.255 nmol/L (r=0.695; n=97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for testosterone was y=0.963 x+0.035 nmol/L (r=0.955; n=107). Preliminary reference intervals for children were determined as a function of age and sex. The sensitivity and specificity of the LC-MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.

Platelet-derived growth factor (PDGF) is a potent mitogenic factor for ovarian thecal cells cultured in vitro. PDGF binds to and induces homo- or heterodimerization of PDGF receptor-a or -beta (PDGF-Ralpha or PDGF-Rbeta). Despite this, little information is available about which PDGF receptors are expressed in the ovary, what signaling cascades are activated by PDGF, and the effects of PDGF on thecal cell steroidogenesis. The present study demonstrates the expression of immunoreactive PDGF-Rbeta, but not PDGF-Ralpha, in the thecal and stromal compartments of intact porcine ovaries as well as in cultured porcine thecal cells. Treatment of porcine thecal cells in vitro with PDGF resulted in rapid and sustained tyrosine phosphorylation of PDGF-Rbeta, activation of Src tyrosine kinase and phosphatidylinositol-3-kinase (PI3-kinase), and serine <em>4</em>73 phosphorylation of Akt/protein kinase B. In addition, PDGF stimulated an increase in GTP-Ras (activated Ras) and extracellular signal-regulated kinase (ERK) phosphorylation. Both forms of PDGF, AB and BB, stimulated thecal cell growth approximately 3- to <em>4</em>-fold over controls and inhibited LH-stimulated progesterone and <em>androstenedione</em> secretion. Blockade of PI3-kinase activation with wortmannin had no effect on PDGF-stimulated thecal cell growth or PDGF inhibition ofLH-stimulated steroid secretion, indicating that PI3-kinase activation is not necessary for PDGF-stimulated thecal cell growth or inhibition of LH-stimulated steroidogenesis. Conversely, blockade of the MEK-ERK pathway with PD98059 completely blocked PDGF-stimulated cell growth, indicating that activation of the MEK-ERK pathway is required for PDGF-stimulated thecal cell growth. Additionally, the MEK inhibitor PD98059 restored LH-stimulated steroid secretion, demonstrating that activation of the MEK-ERK pathway can lead to inhibition of LH-stimulated steroid secretion. The present study demonstrates that PDGF acts on ovarian thecal cells via activation of the PDGF beta-receptor and stimulates thecal cell growth via activation of a Rasmitogen-activated protein kinase-dependent, PI3-kinase-independent pathway. The strong expression of PDGF-Rbeta and the potent effects of PDGF on thecal cell growth and steroidogenesis suggest an important role for PDGF in thecal cell recruitment and growth during follicular development in vivo.

Twenty-one postmenopausal women with advanced breast cancer were treated with monthly 3.6-mg sc injections of the LHRH agonist goserelin [D-Ser-(But)6, Azgly10-LHRH] to determine whether the resultant endocrine changes could provide an explanation for the clinical responses that occur during therapy with this agent. After <em>4</em> weeks, serum gonadotropin levels were less than 10% of pretreatment levels, whereas serum PRL levels did not change. A significant decrease in serum testosterone occurred in 19 of 20 patients; this fall was associated with a 22% fall in serum estradiol levels. Serum <em>androstenedione</em> levels also decreased, but serum estrone and dehydroepiandrosterone sulfate (DHAS) levels did not. The lack of fall in serum DHAS levels indicates that the changes in androgen levels were a result of reduced ovarian secretion, and the reduced estradiol levels were a consequence of reduced precursor (i.e. testosterone) availability. The continued dependence of ovarian androgen secretion on gonadotropin stimulation after the menopause may explain the responses of some patients to LHRH agonists and some other therapeutic agents of unknown or uncertain modes of action.

The purpose of the study was to measure the concentrations of estradiol, its primary precursors, and factors with which it interacts in the breast, and determine their sources of variation. Nipple aspirate fluid (NAF) was collected from premenopausal women during the mid-luteal phase of the menstrual cycle. The fluid was diluted and unconjugated steroids were extracted. Estradiol was further purified by a solvent partition into aqueous NaOH. Androgens were measured in the non-phenolic fraction. Water-soluble, conjugated steroids and proteins were measured in the aqueous residue. All analytes were measured by immunoassays. Permutation methods were used to determine the correlations over multiple periods of time. The average concentration of estradiol in NAF was <em>4</em>35 pmol/L after purification but was many times higher when assayed without purification. Estrone and dehydroepiandrosterone (DHEA) sulfates were present in 3.7 and 75 micromol/L concentrations, respectively, while unconjugated <em>androstenedione</em> and DHEA were present in nanomole per liter concentrations. Lack of the steroid sulfates in NAF in 19% of subjects had no effect on NAF estradiol levels but was associated with a 77% lower concentration of unconjugated DHEA. Progesterone was present in concentrations that were 3- to <em>4</em>-fold higher than normal serum concentrations (mean: 291 nmol/L). Cathepsin D, epidermal growth factor, and interleukin 6 had average values of 3.<em>4</em> microg/mL, <em>4</em>2<em>4</em> ng/mL, and 1.7 ng/mL, respectively. Correlations between breasts were between 0.57 and 0.8<em>4</em> for the several analytes; correlations over time ranged from 0.6<em>4</em> and 0.93 with estrone sulfate highest in both categories. The lower correlation between breasts than within breasts indicates that local factors play an important role in determining the levels of many of these analytes in the breast. The high stability of the concentrations of several analytes over time indicates that fluctuations in environmental factors have little immediate effect on levels in the breast, and portends their utility as surrogate breast cancer risk markers.

BACKGROUND

Androstenedione, a precursor to testosterone, is marketed to increase blood testosterone concentrations as a natural alternative to anabolic steroid use. However, whether androstenedione actually increases blood testosterone levels or produces anabolic androgenic effects is not known.

OBJECTIVE

To determine if short- and long-term oral androstenedione supplementation in men increases serum testosterone levels and skeletal muscle fiber size and strength and to examine its effect on blood lipids and markers of liver function.

METHODS

Eight-week randomized controlled trial conducted between February and June 1998.

METHODS

Thirty healthy, normotestosterogenic men (aged 19-29 years) not taking any nutritional supplements or androgenic-anabolic steroids or engaged in resistance training.

METHODS

Twenty subjects performed 8 weeks of whole-body resistance training. During weeks 1, 2, 4, 5, 7, and 8, the men were randomized to either androstenedione, 300 mg/d (n = 10), or placebo (n = 10). The effect of a single 100-mg androstenedione dose on serum testosterone and estrogen concentrations was determined in 10 men.

METHODS

Changes in serum testosterone and estrogen concentrations, muscle strength, muscle fiber cross-sectional area, body composition, blood lipids, and liver transaminase activities based on assessments before and after short- and long-term androstenedione administration.

RESULTS

Serum free and total testosterone concentrations were not affected by short- or long-term androstenedione administration. Serum estradiol concentration (mean [SEM]) was higher (P<.05) in the androstenedione group after 2 (310 [20] pmol/L), 5 (300 [30] pmol/L), and 8 (280 [20] pmol/L) weeks compared with presupplementation values (220 [20] pmol/L). The serum estrone concentration was significantly higher (P<.05) after 2 (153 [12] pmol/L) and 5 (142 [15] pmol/L) weeks of androstenedione supplementation compared with baseline (106 [11] pmol/L). Knee extension strength increased significantly (P<.05) and similarly in the placebo (770 [55] N vs 1095 [52] N) and androstenedione (717 [46] N vs 1024 [57] N) groups. The increase of the mean cross-sectional area of type 2 muscle fibers was also similar in androstenedione (4703 [471] vs 5307 [604] mm2; P<.05) and placebo (5271 [485] vs 5728 [451] mm2; P<.05) groups. The significant (P<.05) increases in lean body mass and decreases in fat mass were also not different in the androstenedione and placebo groups. In the androstenedione group, the serum high-density lipoprotein cholesterol concentration was reduced after 2 weeks (1.09 [0.08] mmol/L [42 (3) mg/dL] vs 0.96 [0.08] mmol/L [37 (3) mg/dL]; P<.05) and remained low after 5 and 8 weeks of training and supplementation.

CONCLUSIONS

Androstenedione supplementation does not increase serum testosterone concentrations or enhance skeletal muscle adaptations to resistance training in normotestosterogenic young men and may result in adverse health consequences.

BACKGROUND

Heavy acute alcohol drinking decreases blood testosterone in men due to an effect on the testicular level. An acute increase in blood testosterone levels after a low alcohol dose has, however, recently been reported in women. The objective of this investigation was to study the effect of a low alcohol dose on testosterone in men and further elucidate the mechanism behind the effect by using <em>4</em>-methylpyrazole, an inhibitor of alcohol metabolism.

METHODS

A double-blind placebo-controlled interventional crossover trial in random order (n = 13).

RESULTS

After intake of alcohol (0.5 g/kg, 10% w/v), an acute increase in plasma testosterone (from 13.5 +/- 1.2 nmol/liter to 16.0 +/- 1.6 nmol/liter, mean +/- SEM; p < 0.05), a decrease in androstenedione (from 5.1 +/- 0.<em>4</em> nmol/liter to <em>4</em>.0 +/- 0.3 nmol/liter; p < 0.05), and an increase in the testosterone:androstenedione ratio (from 2.8 +/- 0.3 to <em>4</em>.2 +/- 0.<em>4</em>; p < 0.01) were observed. The effects were not observed during pretreatment with <em>4</em>-methylpyrazole (10-15 mg/kg orally), which inhibited the ethanol elimination rate by 37 +/- 3%.

CONCLUSIONS

Alcohol intake affects the androgen balance in men through an effect mediated by the alcohol-induced change in the redox state in the liver.

The effect of treatment with the aromatase inhibitor, <em>4</em>-hydroxy<em>androstenedione</em> (<em>4</em>-OHA) on the peripheral conversion of <em>androstenedione</em> to estrone has been examined in eight postmenopausal women with advanced breast cancer. Before treatment conversion of <em>androstenedione</em> to estrone ([p]AEIBB) ranged from 0.81 to 3.7% and was almost completely inhibited after treatment with <em>4</em>-OHA (two doses of 500 mg i.m. with an interval of 12 days between doses). Transfer constants were also measured by the urinary method ([p]AEIBU) for some subjects and decreased from 2.3 +/- 0.52% to 0.2<em>4</em> +/- 0.11% after treatment, a mean reduction of 90%. Mean plasma concentration of estradiol (37.<em>4</em> +/- 16.6 pmol/liter) and estrone (99.0 +/- 32.2 pmol/liter) decreased significantly (P less than 0.01) to 15.7 +/- <em>4</em>.6 pmol/liter and 52.<em>4</em> +/- 8.9 pmol/liter, respectively, after treatment. Aromatase and DNA polymerase alpha (a marker of cell proliferation) activities were measured in seven samples of breast tumor tissue obtained before and after treatment. For three samples there was a marked (67 +/- 17%) decrease in tumor aromatase activity after treatment, for two, little change occurred, while tumor aromatase activity in the other two samples appeared to be resistant to the effect of <em>4</em>-OHA. The correlation between tumor aromatase and DNA polymerase alpha activities (r = 0.<em>4</em>5) failed to reach a significant level.

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 2<em>4</em> h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-<em>4</em> (1-<em>4</em> layers granulosa cells; no theca) were unaffected for up to <em>4</em>8 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 2<em>4</em> h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 2<em>4</em>-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 2<em>4</em> h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, <em>androstenedione</em> and oestradiol production. Follicles (Stages 1-<em>4</em>) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.

Greater offspring predation favors evolution of faster development among species. We hypothesized that greater offspring predation exerts selection on mothers to increase levels of anabolic androgens in egg yolks to achieve faster development. Here, we tested whether (1) concentrations of yolk androgens in passerine species were associated with offspring predation and (2) embryo and nestling development rates were associated with yolk androgen concentrations. We examined three androgens that increase in potency along the synthesis pathway: <em>androstenedione</em> (A(<em>4</em>)) to testosterone (T) to 5 alpha -dihydrotestosterone (5 alpha -DHT). Concentrations of none of these steroids were related to clutch size; only A(<em>4</em>) was allometrically related to egg volume. Species that experience greater predation showed higher yolk concentrations of T and 5 alpha -DHT. Higher concentrations of T and particularly 5 alpha -DHT were strongly correlated with faster development during the embryo period and less so during the nestling period. Development rates were most strongly correlated with 5 alpha -DHT, suggesting that potency increases along the androgen synthesis pathway and that effects are mediated by the androgen receptor pathway. These results are consistent with the hypothesis that selection for faster development by time-dependent offspring mortality may be achieved epigenetically by varying embryo exposure to maternal anabolic steroids.

In 3 groups of human newborns, 5 sex hormones were assayed from samples of umbilical-cord blood, and concentrations were analyzed by the sex and birth order of the infants. The 5 hormones assayed were testosterone, <em>androstenedione</em>, estrone, estradiol, and progesterone. Concentrations of testosterone were significantly greater in males than females. The other <em>4</em> hormones did not differ significantly by sex. In both sexes, firstborns had significantly more progesterone and estrogens, with progesterone showing the largest birth-order effects. Among male infants, firstborns had higher concentrations of testosterone. The higher concentrations of progesterone in firstborns of both sexes, and of testosterone in firstborn boys, were found not to be due to length of labor, birth weight, or maternal age. However, they were a function of temporal spacing of childbirths. Later borns who were closely spaced in relation to their next-older siblings had lower concentrations of hormones. The effect of temporal separation was greater on male than female infants for each of the 5 hormones studied. The results are discussed in terms of the possible effects of hormone "depletion" on the psychological development of closely spaced later borns.

This report describes aggregate time trend effects of advancing gestational age on circulating maternal concentrations of 17beta-estradiol (E2), estriol (E3), dehydroepiandrosterone (D), dehydroepiandrosterone sulfate (D-S), delta 5-androstenediol (delta 5 diol), delta <em>4</em>-<em>androstenedione</em> (delta <em>4</em> A), testosterone (T), and dihydrotestosterone (DHT) in a sequential series of 155 blood samples obtained from 19 normal pregnant women ranging from 26-<em>4</em>0 weeks gestational age. Only E2, E3, and D-S show aggregate time trend effects. Log (E2) plots as a linear positive sloping curve from 26-<em>4</em>0 weeks. Log (E3) plots as a positive sloping curve that is significantly steeper than log (E2) (P less than 0.05). Log (D-S) plots into a negative sloping curve which mirrors the pattern for log (E2) but cannot be statistically associated with log (E2) except for the opposite sign of their slopes, which are both significantly different from a zero slope (P less than 0.05). delta <em>4</em> A, T, DHT, delta 5 diol, and D show no aggregate time trends; however wide, comoving undulations for delta <em>4</em> A, T, DHT, and delta 5 diol between 26-28 and 38-<em>4</em>0 weeks are confirmed in time by comparison of log mean plots and in magnitude by regressing the C19 steroids on one another. D shows virtually no association with the other C19 steroids. All C19 steroids, except for T, circulate at nonpregnant concentrations, implying that there is little placental secretion of these steroids into the maternal circulation.

Aromatase, the key enzyme in estrogen biosynthesis, converts <em>androstenedione</em> to estrone and testosterone to estradiol. The enzyme is expressed in various tissues such as ovary, placenta, bone, brain, skin, and adipose tissue. Aromatase enzyme is encoded by a single gene CYP 19A1 and its expression is controlled by tissue-specific promoters. Aromatase mRNA is primarily transcribed from promoter I.<em>4</em> in normal breast tissue and physiological levels of aromatase are found in breast adipose stromal fibroblasts. Under the conditions of breast cancer, as a result of the activation of a distinct set of aromatase promoters (I.3, II, and I.7) aromatase expression is enhanced leading to local overproduction of estrogen that promotes breast cancer. Aromatase is considered as a potential target for endocrine treatment of breast cancer but due to nonspecific reduction of aromatase activity in other tissues, aromatase inhibitors (AIs) are associated with undesirable side effects such as bone loss, and abnormal lipid metabolism. Inhibition of aromatase expression by inactivating breast tumor-specific aromatase promoters can selectively block estrogen production at the tumor site. Although several synthetic chemical compounds and nuclear receptor ligands are known to inhibit the activity of the tumor-specific aromatase promoters, further development of more specific and efficacious drugs without adverse effects is still warranted. Plants are rich in chemopreventive agents that have a great potential to be used in chemotherapy for hormone dependent breast cancer which could serve as a source for natural AIs. In this brief review, we summarize the studies on phytochemicals such as biochanin A, genistein, quercetin, isoliquiritigenin, resveratrol, and grape seed extracts related to their effect on the activation of breast cancer-associated aromatase promoters and discuss their aromatase inhibitory potential to be used as safer chemotherapeutic agents for specific hormone-dependent breast cancer.

Wastewater impoundments at concentrated animal feeding operations (CAFOs) represent a potential source of veterinary pharmaceuticals and steroid hormone contamination to shallow groundwater. This study investigates the occurrence of seventeen veterinary pharmaceuticals and thirteen steroid hormones and hormone metabolites in lagoons and adjacent groundwater at operating swine and beef cattle facilities. These sites were chosen because subsurface geology and previous monitoring of nitrate, ammonia and chloride levels in shallow ground water strongly indicated direct infiltration, and as such represent worst cases for ground water contamination by waste water. Pharmaceutical compounds detected in samples obtained from cattle facilities include sulfamerazine; sulfamethazine; erythromycin; monensin; tiamulin; and sulfathiazole. Lincomycin; ractopamine; sulfamethazine; sulfathiazole; erythromycin; tiamulin and sulfadimethoxine were detected in wastewater samples obtained from swine facilities. Steroid hormones were detected less frequently than veterinary pharmaceuticals in this study. Estrone, testosterone, <em>4</em>-<em>androstenedione</em>, and androsterone were detected in wastewater impoundments at concentrations ranging from 30 to 3600ng/L, while only estrone and testosterone were detected in groundwater samples at concentrations up to 390ng/L. The co-occurrence of veterinary pharmaceutical and steroid hormone contamination in groundwater at these locations and the correlation between pharmaceutical occurrence in lagoon wastewater and hydraulically downgradient groundwater indicates that groundwater underlying some livestock wastewater impoundments is susceptible to contamination by veterinary pharmaceuticals and steroid hormones originating in wastewater lagoons.

There is evidence suggesting that local intracrine formation of sex steroids from inactive precursors, dehydroepiandrosterone (DHEA), its sulfate (DHEA-S) and <em>4</em>-<em>androstenedione</em> (<em>4</em>-DIONE) plays an important role in the regulation of growth and function of peripheral target tissues. Moreover, human solid tumors are often infiltrated by stromal/immune cells secreting a wide spectra of cytokines. These cytokines might in turn regulate the activity of both immune and neoplastic cells. Our data demonstrate that the potent regulatory effects of interleukin-<em>4</em> (IL-<em>4</em>) and IL-6 on both estrogenic and androgenic 17beta-HSD/KSR activities in breast cancer cells depend on the cell-specific gene expression of various types of 17beta-HSD/KSR enzymes. However, in both estrogen-receptor (ER)-positive (ZR-75-1, T-<em>4</em>7D) and ER-negative (MDA-MB-231, BT-20) human breast cancer cells, exposure to IL-<em>4</em> and IL-13 caused a rapid and potent induction of 3beta-HSD type 1 gene expression. Such an induction was also observed in normal human mammary and prostate epithelial cells in primary culture as well as in human HaCaT immortalized keratinocytes, ME-180 cervix cancer cells, and HT-29 colon cancer cells. The DNA-binding activity of Stat6, a member of the Signal Transducers and Activators of Transcription gene family, was activated after a 30 min exposure to IL-<em>4</em> in all the cell types where IL-<em>4</em> induced 3beta-HSD expression, but not in those that failed to respond to IL-<em>4</em>. Our data therefore suggest that IL-<em>4</em> and IL-13 may play a role in the biosynthesis of active sex steroids from the inactive adrenal steroid DHEA, not only in breast cells but also in various cell types derived from peripheral target tissues.

Menstrual dysfunction is common among athletes with very low body mass, such as long distance runners and dancers, and is usually associated with hypothalamic dysfunction. The purpose of this study was to investigate the menstrual status of swimmers, in whom exercise is nonweight bearing and thinness is, thus, not essential. Questionnaires recording the menstrual history of 69 young competitive swimmers (aged 16.<em>4</em> +/- 0.5) were compared to those of 279 age-matched controls. Age of menarche (M) was significantly (P < 0.005) delayed among swimmers (13.8 +/- 0.2 yr) compared to controls (13.0 +/- 0.1 yr). Eighty-two percent of swimmers had menstrual irregularities after M compared to <em>4</em>0% of control, with longer duration of these irregularities (16 vs. <em>4</em> months; P < 0.005). A subset of 2<em>4</em> swimmers was studied further for body composition, pubertal stage, and reproductive hormone levels. Estradiol levels were normal in all post-M swimmers (273 +/- 20 pmol/L) and higher than average in pre-M (383 +/- <em>4</em><em>4</em> pmol/L). FSH levels were normal in all subjects (10.7 +/- 1.6 IU/L), LH was mildly elevated (17.1 +/- 1.2 IU/L), and the LH/FSH ratio was 1.7. Levels of dehydroepi<em>androstenedione</em> sulfate and <em>androstenedione</em>, but not testosterone, were higher than average in all groups of swimmers. The results of this study indicate that female competitive swimmers are vulnerable to delayed puberty and menstrual irregularities, but the associated hormonal profile is very different from the hypothalamic amenorrhea described in dancers and runners. We, therefore, suggest a different mechanism for reproductive dysfunction in swimmers that is associated not with hypoestrogenism, but, rather, with mild hyperandrogenism. A distinction among the various types of athletic amenorrhea should be made based on hormonal profiles with attention to their weight and somatotype.

Amniotic fluid (AF) levels of all steroids leading from pregnenolone (delta 5Preg) to androgens and estrogens of both the delta 5 and the delta <em>4</em> pathways and those of cortisol and cortisone have been determined in 63 normal pregnancies (12-19 weeks gestation). The 12 unconjugated steroids [delta 5Preg, 17 alpha-hydroxypregnenolone, progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone (DHA), delta <em>4</em>-<em>androstenedione</em>, delta 5-androstene-3 beta,17 beta-diol (delta 5Adiol), testosterone (T), estrone, 17 beta-estradiol (E2), cortisol, and cortisone] were measured by specific RIAs after appropriate purification by Celite or LH-20 column chromatography, while the sulfates of DHA and delta 5Preg were assayed directly on diluted samples. There were distinct sex differences; T and delta <em>4</em>-<em>androstenedione</em> levels were higher (P < 0.001) in males than in females, while AF levels of delta 5Preg, 17 alpha-hydroxypregnenolone, 17-hydroxyprogesterone, DHA, and delta 5Adiol were higher (P < 0.05) in females than in males. AF levels of E2 were significantly higher in females only between 15 and 19 weeks gestation. There was no difference between sexes in AF levels of progesterone, estrone, cortisone, and cortisol. AF levels of T, delta <em>4</em>-<em>androstenedione</em>, and E2 decreased with age in males, and AF levels of DHA increased in females during the period examined. These observed sex differences suggest that T and delta <em>4</em>-<em>androstenedione</em> may reflect fetal testicular activity, while E2 and 17-hydroxyprogesterone might reflect fetal ovarian activity. Determination of AF levels of T would appear to be a valuable screening test for antenatal diagnosis of sex (predictive error, less than or equal to 15%), but not in the presence of steroidogenic enzyme defects. Elevated levels of 17-hydroxyprogesterone were found in the AF of two fetuses with either a 17-20 desmolase defect or 21-hydroxylase deficiency; AF levels of androgens were low in the former and high in the latter.

The fact that certain ultraviolet (UV) filters used in cosmetics display estrogenic activity prompted us to study potential actions on androgen receptors (AR) in the human breast carcinoma cell line MDA-kb2, which expresses functional endogenous AR and glucocorticoid receptors (GR) and is stably transfected with a luciferase reporter plasmid. Dihydrotestosterone (DHT), methyltrienolone (R1881), methyltestosterone, danazol, and <em>androstenedione</em> increased luciferase activity, with EC50 values between 0.11 nM (R1881), 0.1<em>4</em> nM (DHT), and 73.5 nM (<em>androstenedione</em>). DHT-induced luciferase gene expression was inhibited by nonsteroidal antiandrogens, hydroxyflutamide, flutamide, bicalutamide, and vinclozolin. In contrast, the steroidal AR agonist/antagonist cyproterone actetate showed agonistic activity in the absence and presence of DHT, which was not blocked by hydroxyflutamide and thus seems not to be mediated by AR. GR-mediated activation of luciferase by dexamethasone was 100 times less potent than DHT and was not antagonized by hydroxyflutamide. The cell line was used for screening of UV filters, benzophenone-3 (Bp-3), benzophenone-<em>4</em>, 3-benzylidene camphor, <em>4</em>-methylbenzylidene camphor, butyl-methoxy-dibenzoylmethane, homosalate (HMS), octyl-dimethyl-PABA, and octyl-methoxycinnamate. Two of these, Bp-3 and HMS, antagonized DHT-induced AR activation below cytotoxic concentrations, with IC50 of 5.57 10-6 M (HMS) and <em>4</em>.98 10-6 M (Bp-3). None of the eight UV filters displayed agonistic activity when tested alone, but high concentrations of Bp-3 induced an increase of luciferase activity in the presence of dexamethasone, which was not blocked by hydroxyflutamide or the estrogen antagonist, ICI 182,780. These data indicate that the UV filters Bp-3 and HMS possess antiandrogenic activity in vitro in addition to estrogenic activity.

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P<em>4</em>50 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P<em>4</em>50 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-<em>4</em>-ene steroids (progesterone and <em>androstenedione</em>); and P<em>4</em>50(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and <em>androstenedione</em>). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P<em>4</em>50(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P<em>4</em>50(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P<em>4</em>50(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P<em>4</em>50(scc) and 3 beta-HSD was restricted to mitochondria, while P<em>4</em>50(c17) immunoreactivity was exclusively observed in ER. In the ovary, P<em>4</em>50(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P<em>4</em>50(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P<em>4</em>50(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P<em>4</em>50(scc), 3 beta-HSD and P<em>4</em>50(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

Many fish use steroid hormones as pheromones to initiate behavioral and physiological changes during spawning. To assess the occurrence of steroid hormones with pheromonal properties in the aquatic environment and to evaluate the possibility that municipal wastewater discharges contain compounds that could affect fish reproduction by interfering with pheromones, several estrogens, androgens, and progestins were quantified by gas chromatography/tandem mass spectroscopy in effluent samples from 12 municipal wastewater treatment plants. Samples also were analyzed from an engineered treatment wetland, three groundwater wells, and one reservoir. Estrogens (17beta-estradiol and estrone) were detected in wastewater effluent at maximum concentrations of <em>4</em> and 12 ng/L, respectively. Androgens (testosterone and <em>androstenedione</em>) were detected at concentrations as high as 6.1 and <em>4</em>.5 ng/L, respectively, whereas the synthetic progestin medroxyprogesterone was detected at concentrations up to 15 ng/L. Data from an effluent-receiving engineered treatment wetland and shallow groundwater wells suggested that these compounds were not rapidly attenuated. The measured concentrations of steroids often exceeded olfactory detection thresholds at which fish detect these steroids, and in several cases, the steroid concentrations were comparable to levels at which pheromonal responses have been observed in fish.

Activities of several steroid metabolizing enzymes (steroid sulfate-sulfatase, 17 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase, and 3 alpha beta-hydroxysteroid dehydrogenase) as well as total tissue content and subcellular distribution (nuclear-extranuclear) of several androgen precursors, active androgens, and androgen deactivation products (DHEA sulfate, DHEA, 5-androstenediol, <em>4</em>-<em>androstenedione</em>, testosterone, DHT, and 3 alpha-androstanediol) were quantified in primary tumors and lymph node metastases of human prostatic cancer obtained from patients without previous endocrine manipulation. Primary tumors were compared to benign parts of the same prostates, and the metastases were compared to their primary tumors. All enzymes and steroids found in benign prostatic tissues could also be detected in the malignant tissues. Even the capacity to accumulate active androgens in the nuclei was found to be unchanged in nearly all of the samples. Lower activities of hormone-dependent enzymes were observed in the cancers, suggesting a less efficient utilization of hormonal stimuli. Most striking changes found in the malignant tissues were a subtotal loss of 5 alpha-reductase activity and a metabolic shift to testosterone, which was more pronounced in samples from metastatic disease as compared to samples from non-metastatic disease. In conclusion, primary tumors and metastases of prostatic cancers not treated by endocrine manipulations retain their androgen receptor system and possess the same capacity to metabolize adrenal androgen precursors along the pathway to DHT as benign prostatic tissue. Consequently, they should be able to use at least <em>androstenedione</em> for production of active androgens directly in the target tissue.

OBJECTIVE

To evaluate the effects of D-Chiro-Inositol in women affected by polycystic ovary syndrome (PCOS).

METHODS

We enrolled <em>4</em>8 patients, with homogeneous bio-physical characteristics, affected by PCOS and menstrual irregularities. These patients underwent treatment with 1 gr of D-Chiro-Inositol/die plus <em>4</em>00 mcg of Folic Acid/die orally for 6 months. We analyzed pre-treatment and post-treatment BMI, Systolic and Diastolic blood pressure, Ferriman-Gallwey score, Cremoncini score, serum LH, LH/FSH ratio, total and free testosterone, DHEA-S, Δ-<em>4</em>-<em>androstenedione</em>, SHBG, prolactin, glucose/IRI ratio, HOMA index, and resumption of regular menstrual cycles.

RESULTS

We evidenced a statistically significant reduction of systolic blood pressure, Ferriman-Gallwey score, LH, LH/FSH ratio, total Testosterone, free Testosterone, ∆-<em>4</em>-Androstenedione, Prolactin, and HOMA Index; in the same patients, we noticed a statistically significant increase of SHBG and Glycemia/IRI ratio. Moreover, we observed statistically significant (62.5%; p < 0.05) post-treatment menstrual cycle regularization.

CONCLUSIONS

D-Chiro-Inositol is effective in improving ovarian function and metabolism of patients affected by PCOS.

At approximately 8.5 mm in diameter, the future dominant follicle is "selected" for continued growth in cattle. In the present study, cows were treated with a gonadotropin-releasing hormone receptor antagonist, acyline, just before follicle selection (near 7.8 mm) to investigate the role of LH in changing mRNA concentrations during selection of a dominant follicle. The ovaries containing the expected dominant follicle (EDF; first largest follicle) and expected largest subordinate follicle (ESF) were removed after 12 or 2<em>4</em> h of treatment. Real-time PCR was used to determine mRNA concentrations. ELISA was used to measure testosterone and 17beta-estradiol (E(2)) and radioimmunoassay to measure <em>androstenedione</em> (A(<em>4</em>)) in follicular fluid. Concentrations of E(2) were greater in EDF than in ESF of untreated cows near the time of follicle selection (12 h) or at 12 h after selection (2<em>4</em> h). Testosterone, E(2), and A(<em>4</em>) were all dramatically decreased by acyline treatment at both times. In theca cells, acyline treatment reduced CYP17A1 (P<em>4</em>50 17alpha) in EDF and STAR (steroidogenic acute regulatory protein) in both EDF and ESF but did not alter CYP11A1 (P<em>4</em>50scc). In granulosa cells (GCs), LHCGR (luteinizing hormone [LH] receptor) was much greater in EDF than in ESF at both time of selection (739% greater) and 12 h after selection (2837% greater) and was decreased by acyline in EDF (87% decrease). The mRNA for CYP19A1 (cytochrome P<em>4</em>50 aromatase) and PAPPA (pregnancy-associated plasma protein-A) tended to be greater in EDF than in ESF at follicle selection, and both mRNAs were much greater at 12 h after selection, with acyline significantly decreasing PAPPA mRNA after 2<em>4</em> h of treatment. The mRNA for FSHR (follicle-stimulating hormone receptor) was not different in EDF versus ESF and was not altered by acyline. Thus, induction of LHCGR mRNA in GCs is an early event during the follicle selection process, and surprisingly, expression of LHCGR mRNA is dependent on circulating LH. Production of follicular A(<em>4</em>), testosterone, and E(2) are also acutely related to LH but due to changes in expression of STAR and CYP17A1 in TC.