Changes in the isoenzyme pattern of cytochrome P<em>4</em>50 during culture were investigated in primary cultures of rat hepatocytes, measuring specific enzyme activities in microsomes prepared from cultured cells as well as in intact monolayers. Assays of 7-ethoxyresorufin O-deethylation (EROD), 7-pentoxyresorufin O-depentylation (PROD), aniline <em>4</em>-hydroxylation (AH) and the specific regioselective hydroxylation of testosterone were used as representatives of the activities of seven isoenzymes of cytochrome P<em>4</em>50. The isoenzyme profile expressed as catalytic activities was qualitatively and quantitatively similar in microsomes obtained from freshly isolated hepatocytes in comparison with microsomes obtained from whole livers of untreated rats. There was a relatively high activity in EROD, AH and the oxidation of testosterone at the 7 alpha, 2 alpha, 6 beta, 16 alpha and 17 sites (<em>androstenedione</em>). During culture, these microsomal enzyme activities declined at a similar rate to ca. 50% of the activities of microsomes prepared from freshly isolated hepatocytes after 2<em>4</em> hr and to 15% after 96 hr. The overall decline of cytochrome P<em>4</em>50-dependent activities during culture was not accompanied with gross changes in catalytic profile. Determining the same drug-metabolizing activities directly in intact hepatocyte monolayers revealed a much higher metabolic rate for all measured P<em>4</em>50-dependent activities. The profile of the catalytic activities was essentially the same as measured in microsomes prepared from cultured hepatocytes. The relatively low activity towards the 7 alpha site of testosterone measured in intact hepatocytes, however, remained constant during culture. Determination of enzyme activities directly in intact hepatocytes is a convenient way of studying changes in monooxygenase activities of different P<em>4</em>50 isoenzymes in vitro.

OBJECTIVE

In hyperandrogenic women, hyperinsulinaemia amplifies 17 α-hydroxycorticosteroid intermediate response to ACTH, without alterations in serum cortisol or androgen response to stimulation. The aim of the study is to assess whether acute hyperinsulinaemia determines absolute changes in either basal or ACTH-stimulated adrenal steroidogenesis in these subjects.

METHODS

Twelve young hyperandrogenic women were submitted in two separate days to an 8 h hyperinsulinaemic (80 mU/m² × min) euglycaemic clamp, and to an 8 h saline infusion. In the second half of both the protocols, a <em>4</em> h ACTH infusion (62.5 μg/h) was carried out. Serum cortisol, progesterone, 17 α-hydroxyprogesterone (17-OHP), 17 α-hydroxypregnenolone (17-OHPREG), DHEA and <em>androstenedione</em> were measured at basal level and during the protocols. Absolute adrenal hormone secretion was quantified by measuring C19 and C21 steroid metabolites in urine collected after the first <em>4</em> h of insulin or saline infusion, and subsequently after <em>4</em> h of concurrent ACTH infusion.

RESULTS

During insulin infusion, ACTH-stimulated 17-OHPREG and 17-OHP were significantly higher than during saline infusion. No significant differences in cortisol and androgens response to ACTH were found between the protocols. Nevertheless, urinary excretion of ACTH-stimulated C19 and C21 steroid metabolites was significantly higher during hyperinsulinaemia than at basal insulin levels (both P < 0.005). Changes in steroid metabolites molar ratios suggested stimulation by insulin of 5 α-reductase activity.

CONCLUSIONS

These in vivo data support the hypothesis that insulin acutely enhances ACTH effects on both the androgen and glucocorticoid pathways.

BACKGROUND

Currently available 5α-reductase inhibitors are not completely effective for treatment of benign prostate enlargement, prevention of prostate cancer (CaP), or treatment of advanced castration-recurrent (CR) CaP. We tested the hypothesis that a novel 5α-reductase, 5α-reductase-3, contributes to residual androgen metabolism, especially in CR-CaP.

METHODS

A new protein with potential 5α-reducing activity was expressed in CHO-K1 cellsandTOP10 E. coli for characterization. Protein lysates and total mRNA were isolated from preclinical and clinical tissues. Androgen metabolism was assessed using androgen precursors and thin layer chromatography or liquid chromatography tandem mass spectrometry.

RESULTS

The relative mRNA expression for the three 5α-reductase enzymes in clinical samples of CR-CaP was 5α-reductase-3 ≫ 5α-reductase-1> 5α-reductase-2. Recombinant 5α-reductase-3 protein incubations converted testosterone, <em>4</em>-androstene-3,17-dione (<em>androstenedione</em>) and <em>4</em>-pregnene-3,20-dione (progesterone) to dihydrotestosterone, 5α-androstan-3,17-dione, and 5α-pregnan-3,20-dione, respectively. 5α-Reduced androgen metabolites were measurable in lysates from androgen-stimulated (AS) CWR22 and CR-CWR22 tumors and clinical specimens of AS-CaP and CR-CaP pre-incubated with dutasteride (a bi-specific inhibitor of 5α-reductase-1 and 2).

CONCLUSIONS

Human prostate tissues contain a third 5α-reductase that was inhibited poorly by dutasteride at high androgen substrate concentration in vitro, and it may promote DHT formation in vivo, through alternative androgen metabolism pathways when testosterone levels are low.

Immature 27-day-old female Sprague-Dawley rats were administered daily subcutaneous injections of dehydroepiandrosterone (DHEA, 5 mg/100 g BW) to induce the formation of ovarian follicular cysts. Groups of rats were killed on days 0, 10, 15, 20, 25, and 30. Ovaries from each group of rats were processed for light and electron microscopy and for follicular or cystic fluid hormone analysis. Normal antral follicle fluid, PMSG-treated preovulatory follicular fluid, and cystic fluids were analyzed for progesterone (P), estrone (E1), estradiol (E2), testosterone (T), delta <em>4</em>-<em>androstenedione</em> (delta <em>4</em>-A), 5 alpha-dihydrotestosterone (DHT), luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL). DHEA induced anovulation, acyclicity, and the formation of follicular cysts. In certain antral follicles, there was a dramatic increase in the quantities of smooth endoplasmic reticulum (SER) in the granulosa cells and many mitochondria had tubular cristae. Further depletion of granulosa cell number was associated with intense blebbing of the cytoplasm into the follicle antrum. Formation of the ovarian follicular cyst was completed when the entire cyst was lined by a single layer of transformed granulosa cells in contact via adhering, gap, and tight junctions. These cells had little cytoplasm, mitochondria with lamellar cristae, vast basal and apical bands of microfilaments, and an extensive array of smooth-surfaced endocytotic invaginations on the basal plasma membrane. These endocytotic pits may subsequently form smooth-surfaced vesicles and thereby serve as one mechanism for moving fluid from the ovarian interstitium into the cyst. Theca interna cells were rarely observed in the peripheral regions of the cyst. Abundant smooth muscle cells were located beneath the basement membrane of the epithelial cells comprising the cyst wall. These acquired morphological and physiological features may ensure persistence of the ovarian cyst and thus potentiate a chronic pathological condition. In this study it was also shown that progesterone, estrone, and estradiol as well as androgen concentration increased in the follicle after PMSG treatment. With DHEA treatment, the follicular cystic fluid concentrations of these steroids progressively increased to extremely high levels concurrent with the development of the follicular cysts.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Androgens play important endocrine roles in development and physiology. Here, we characterize activities of two "Andro" prohormones, <em>androstenedione</em> (A-dione) and <em>4</em>-androsten-3beta,17beta-diol (A-diol) in MDA-MB-<em>4</em>53 (MDA) and LNCaP cells. A-dione and A-diol, like cyproterone acetate, were partial agonists of transfected mouse mammary tumor virus (MMTV) and endogenous prostate-specific antigen (PSA) promoters. Different from bicalutamide but similar to CPA, both are inducers of LNCaP cell proliferation with only mild suppression of 5alpha-dihydrotestosterone (DHT)-enhanced cell growth. Like bicalutamide and cyproterone acetate, A-dione and A-diol significantly antagonized DHT/R1881-induced PSA expression by up to 30% in LNCaP cells. Meanwhile, in MDA cells, EC(50)s for the MMTV promoter were between 10 and 100nM. Co-factor studies showed GRIP1 as most active for endogenous androgen receptor (AR), increasing MMTV transcription by up to five-fold, without substantially altering EC(50)s of DHT, A-dione or A-diol. Consistent with their transcriptional activities, A-dione and A-diol bound full-length endogenous AR from MDA or LNCaP cells with affinities of 30-70nM, although binding to expressed ligand-binding domain (LBD) was >20-fold weaker. In contrast, DHT, R1881, and bicalutamide bound similarly to LBD or aporeceptor. Together, these data suggest that A-dione and A-diol are ligands for AR with partial agonist/antagonist activities in cell-based transcription assays. Binding affinities for both are most accurately assessed by AR aporeceptor complex. In addition to being testosterone precursors in vivo, either may impart its own transcriptional regulation of AR.

The laboratory reliability and validity of sex hormone measurements were examined at multiple levels, including lower levels characteristic of children and postmenopausal women. Serum was drawn from four adult male and four adult female healthy volunteers. From each individual's serum pool, a medium- and a low-dilution pool were created. Biochemical analyses for total and non-sex hormone-binding globulin (SHBG)-bound estradiol, estrone, estrone sulfate, progesterone, and SHBG were performed on female samples. Male samples were analyzed for total and non-SHBG-bound testosterone, dihydrotestosterone, <em>androstenedione</em>, and dehydroepiandrosterone sulfate. Two aliquots from each pool were assayed twice in each of two labs. All assays except SHBG in one lab used RIA procedures. Reliability was assessed by variance components analyses and estimated coefficients of variation (CVs). Validity was assessed by comparing observed measurements versus expected values based on known dilution ratios. For the testosterone and dihydrotestosterone assays, CVs were usually less than 10%. For estradiol and progesterone, CVs were usually less than 15%. Assays with larger estimated CVs included <em>androstenedione</em>, dehydroepiandrosterone sulfate, estrone, and estrone sulfate. Absolute levels differed markedly between labs for most assays. Observed measurements generally agreed with values expected from the dilution ratios. A notable exception was the estrone assay at the lowest dilution level, where observed measurements were 2-<em>4</em> times those expected. A similar but less pronounced overestimation bias for the low levels of estradiol was also suggested. This intra- and interlaboratory variability and apparent low dilution overestimation should be accounted for in studies relating hormones to cancer risk, especially those involving children and postmenopausal women.

A deficiency of 21-hydroxylase activity is one of the most commonly inherited genetic disorders in man, with heterozygosity for CYP21 mutations affecting approximately 1 in 60 of the non-Jewish Caucasian population. We have hypothesized that heterozygosity for CYP21 mutations in women increases their risk of developing clinically evident hyperandrogenism, and that this risk is related to the severity of the mutation of CYP21 and/or the 17-hydroxyprogesterone (17-OHP) response to ACTH stimulation. To test these hypotheses, we studied 38 obligate carriers for 21-hydroxylase deficiency (i.e. mothers of children with congenital adrenal hyperplasia or nonclassic congenital adreanl hyperplasia), comparing them to 27 weight-, parity-, and age-matched controls. Premenopausal carriers, not receiving hormonal treatment (n = 27), had higher mean total and free testosterone [T; 2.02 +/- 0.55 vs. 1.56 +/- 0.65 nmol/L (P < 0.007) and 0.018 +/- 0.007 vs. 0.012 +/- 0.006 nmol/L (P < 0.007), respectively] and lower mean sex hormone-binding globulin (21<em>4</em> +/- 62 vs. 277 +/- 129 nmol/L; P < 0.03) levels compared to controls. There was no difference in the mean basal levels of dehydroepiandrosterone sulfate, <em>androstenedione</em> (A<em>4</em>), or 17-OHP between carriers and controls. As expected, carriers exhibited higher stimulated and net increment 17-OHP levels than controls [21.1 +/- 27.1 vs. 6.2 +/- 3.1 nmol/L (P < 0.01) and 19.0 +/- 26.5 vs. <em>4</em>.<em>4</em> +/- 2.8 nmol/L (P < 0.009), respectively]. However, no difference was observed in the response of A<em>4</em> to ACTH-(1-2<em>4</em>) stimulation. Of the 27 carriers studied biochemically, 2 (7.<em>4</em>%) had a stimulated 17-OHP value between 30.3-60.6 nmol/L, and 1 (3.7%) had a 17-OHP level above 60.6 nmol/L, suggestive of nonclassic adrenal hyperplasia. Of all carriers studied genetically (n = 36), 50.0% (18 of 36) had 1, 33% (12 of 36) had 2, and 16.7% (6 of 36) had 3 or more mutations. In 27.8% (10 of 36) of carriers, the mutations were contiguous, consistent with a large gene conversion. All 38 carriers were examined for historical and physical features of hyperandrogenism. Hirsutism was defined as a Ferriman-Gallwey score of 6 or more, menstrual/ovulatory dysfunction as a history of menstrual cycles of more than 35-day, and hyperandrogenemia as total or free T, A<em>4</em>, and/or dehydroepiandrosterone sulfate levels above the upper 95th percentile of control values. Further, defining functional androgen excess (FAE) as the presence of at least 2 of the 3 hyperandrogenic features, <em>4</em> of 38 (10.5%) of carriers appeared to be affected (95% confidence interval, 2.9-2<em>4</em>.8%). Assuming an expected prevalence rate of FAE in the general population of 5-20%, the frequency of FAE among our carriers was not significantly higher than expected. In conclusion, heterozygosity for CYP21 mutations does not appear to increase the risk of clinically evident hyperandrogenism, although carrying the defect was associated with higher mean and free T levels. Finally, due to the low frequency of androgen excess in our heterozygote population, we were unable to correlate the severity of the CYP21 mutation and/or the 17-OHP response to ACTH stimulation with the presence of the phenotype.

The androgen-secreting Leydig cells produce cGMP, but the pathways responsible for generation and actions of this intracellular messenger have been incompletely characterized in these cells. Here, we show the presence of mRNA transcripts for the membrane-bound and soluble guanylyl cyclases (sGC), the cGMP-specific phosphodiesterase 5, and the cGMP-dependent protein kinase I (PKG I) and PKG II in purified rat Leydig cells from adult animals. Stimulation of both guanylyl cyclases and inhibition of phosphodiesterase 5 in vitro were accompanied by elevations in cGMP and androgen production, whereas inhibition of sGC and PKG led to a decrease in steroidogenesis. The stimulatory action of cGMP on steroidogenesis was preserved in cells with inhibited cAMP-dependent protein kinases. Experiments with exogenously added substrates revealed the dependence of cGMP-induced progesterone and androgen synthesis on cholesterol but not on 22-OH cholesterol, pregnenolone, progesterone, and Delta(<em>4</em>)-<em>androstenedione</em>. Treatment with nitric oxide donor increased phosphorylation of the steroidogenic acute regulatory protein (StAR). In contrast, inhibition of sGC and PKG, but not protein kinase A, significantly reduced StAR phosphorylation. These results suggest that cGMP contributes to the control of basal steroidogenesis in Leydig cells through the PKG-dependent modification of the StAR protein.

BACKGROUND

Adults with congenital adrenal hyperplasia (CAH) are treated with a wide variety of glucocorticoid treatment regimens.

UNASSIGNED

To test whether drug dose and timing of glucocorticoid treatment regimen impacts on health outcomes. This was a cross-sectional study of 196 adult CAH patients in whom treatment and health outcomes were measured. Glucocorticoid dose was converted to prednisolone dose equivalent (PreDEq) using three published formulae. Associations between the type of glucocorticoid regimen and PreDEq with specific health outcome variables were tested using partial correlation and principal components analysis (PCA).

RESULTS

Patients on dexamethasone had lower androgens and ACTH but greater insulin resistance compared with those receiving hydrocortisone or prednisolone. Dexamethasone dose and once daily administration were associated with insulin resistance. Partial correlation analysis adjusted for age and sex showed PreDEq weakly correlated (r < 0·2) with blood pressure and <em>androstenedione</em>. Mutation severity was associated with increased PreDEq (F(3,1<em>4</em>1) = <em>4</em>·<em>4</em>, P < 0·01). In PCA, 3 PCs were identified that explained 62% of the total variance (r(2) ) in observed variables. Regression analysis (age and sex adjusted) confirmed that PC2, reflecting disease control (<em>androstenedione</em>, 17-hydroxypregesterone and testosterone), and PC3, reflecting blood pressure and mutations (systolic and diastolic blood pressure and mutation severity), related directly to PreDEq (r(2) = 23%, P < 0·001).

CONCLUSIONS

In adults with congenital adrenal hyperplasia, dexamethasone use was associated with lower androgens but greater insulin resistance, and increasing glucocorticoid dose associated with increased blood pressure, poor disease control and mutation severity.

The steroidogenic potential of various physiological compartments within the ovary of the hen were examined using in vitro systems. Three-hour incubations of individual whole small follicles (less than 1 mm-1 cm) or 100,000 collagenase-dispersed theca cells of the five largest ovarian follicles (F1-F5) were conducted in 1 ml of Medium 199 at 37 degrees C in the presence and absence of luteinizing hormone (LH) (0.39, 0.78, 1.56, 3.13 and 6.25 ng), progesterone (5 ng), and dehydroepiandrosterone (DHEA, 5 ng). Steroid output was measured by radioimmunoassay of incubation media. Progesterone was not produced by small follicles although they are a major source of DHEA and estradiol and a significant source of <em>androstenedione</em>. Output of DHEA, <em>androstenedione</em> and estradiol was highly stimulated by LH. The substrate for <em>androstenedione</em> and estradiol in small follicles is probably DHEA. Output of DHEA and <em>androstenedione</em> in theca cells of F2-F5 was stimulated by LH in a dose-related manner. A dose-response relationship between estradiol output and the concentration of LH in media was not apparent in theca cells from F2-F5. Steroidogenesis in theca tissue of large follicles occurs predominantly via the delta <em>4</em> pathway. The ability of these theca cells to metabolize progesterone to <em>androstenedione</em> is lost between 36 and 12 h before ovulation. Their ability to metabolize DHEA to <em>androstenedione</em> is still present 12 h before ovulation. Aromatase activity is significantly reduced between 36 and 12 h before ovulation. These data indicate that both large and small follicles can be stimulated by LH. The small follicles are the major source of estrogen. As the large yolky follicles mature, steroidogenesis shifts from the delta 5 to the delta <em>4</em> pathway. By 12 h before ovulation, the F1 follicle has lost the ability to convert progesterone to <em>androstenedione</em>. The inability of the largest ovarian follicle to convert progesterone to <em>androstenedione</em> contributes at least in part to the preovulatory increase in the plasma concentration of progesterone that generates the preovulatory LH surge by positive feedback.

Endogenous androgens (<em>androstenedione</em>, testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol), and some of their C21 precursors (pregnenolone, progesterone and 17-hydroxyprogesterone) were measured in rat testes between Day 18.5 of pregnancy and Day 6<em>4</em> postpartum, and correlated with numerical densities of Leydig cells. The latter parameter showed an early maximum on Day 19.5 of the fetal period, a nadir on Day 15 postpartum, and a gradual increase thereafter. The two dominating androgens, testosterone and 5 alpha-androstane-3 alpha,17 beta-diol, had similar levels until 15 days of age, but the 5 alpha-diol predominated thereafter. The total steroid content per Leydig cell was highest on Day 18.5 of gestation (77 ng/10(6) cells). A decline started already in utero, and reached a nadir of 5 ng/10(6) cells on Day 29. Thereafter, a slight increase occurred with advancing age. It is concluded that: The fetal testis has highest Leydig cell and endogenous steroid concentrations. A nadir in these parameters is seen 2-<em>4</em> wk postpartum. The Leydig cell concentration increases around puberty on Days <em>4</em>0-60, but only a slight concomitant increase occurs in steroids. A sharp decline in steroid content per Leydig cell occurs during the last fetal days, but the postnatal decline of testicular steroids is due to Leydig cell loss. The new Leydig cell generation after 15 days has a persistently low steroid concentration through puberty.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Resveratrol is a naturally occurring polyphenol with purported inhibitory effects on prostate growth and cancer development. A number of studies have demonstrated that resveratrol reduces prostate growth in animal models and reduces prostate cell growth in vitro. Based on these pre-clinical findings, interest in resveratrol is increasing in relation to the management of benign prostate hyperplasia (BPH) and prostate cancer. So far, no human trials have evaluated the effects of resveratrol on circulating androgens, prostate size, or biochemical markers of prostate size.

METHODS

In a randomized placebo controlled clinical study using two doses of resveratrol (150 mg or 1,000 mg resveratrol daily) for <em>4</em> months, we evaluated the effects on prostate size, prostate specific antigen (PSA) and sex steroid hormones in 66 middle-aged men suffering from the metabolic syndrome(MetS).

RESULTS

At baseline, prostate size and PSA were positively correlated (R = 0.3<em>4</em>, P < 0.007) as was prostate size and age (R = 0.37, P < 0.003). Prostate size did not correlate with testosterone, free testosterone, dihydrotestosterone (DHT), or any other androgen precursor at baseline. The highest dose of resveratrol lowered the serum level of androstenedione 2<em>4</em>% (P = 0.052), dehydroepiandrosterone (DHEA) <em>4</em>1% (P < 0.01), and dehydroepiandrosterone-sulphate (DHEAS) 50% (p<0.001), compared to the control group. However, prostate size and levels of PSA, testosterone, free testosterone and DHT remained unchanged.

CONCLUSIONS

In this population of middle-aged men suffering from MetS, high dose resveratrol (1,000 mg daily) administration for <em>4</em> months significantly lowered serum levels of the androgen precursors androstenedione, DHEA and DHEAS, whereas prostate size and circulating levels of PSA, testosterone, free testosterone, and dihydrotestosterone were unaffected. The present study suggests that resveratrol does not affect prostate volume in healthy middle-aged men as measured by PSA levels and CT acquired prostate volumes. Consequently, we find no support for the use of resveratrol in the treatment of benign prostate hyperplasia.

Female ring-tailed lemurs (Lemur catta) are Malagasy primates that are size monomorphic with males, socially dominate males, and exhibit a long, pendulous clitoris, channeled by the urethra. These masculine traits evoke certain attributes of female spotted hyenas (Crocuta crocuta) and draw attention to the potential role of androgens in lemur sexual differentiation. Here, hormonal correlates of prenatal development were assessed to explore the possibility that maternal androgens may shape the masculine morphological and behavioral features of developing female lemurs. Maternal serum 17α-hydroxyprogesterone, dehydroepiandrosterone sulphate (DHEA-S), ∆⁴ <em>androstenedione</em> (androst-<em>4</em>-ene-3,17,dione), testosterone, and 17β-estradiol were charted throughout the 19 pregnancies of 11 ring-tailed lemurs. As in spotted hyenas, lemur pregnancies were associated with an immediate increase in androgen concentrations (implicating early maternal derivation), followed by continued increases across stages of gestation. Pregnancies that produced singleton males, twin males, or mixed-sex twins were marked by greater androgen and estrogen concentrations than were pregnancies that produced singleton or twin females, especially in the third trimester, implicating the fetal testes in late-term steroid profiles. Concentrations of DHEA-S were mostly below detectable limits, suggesting a minor role for the adrenals in androgen biosynthesis. Androgen concentrations of pregnant lemurs bearing female fetuses, although less than those of pregnant hyenas, exceeded preconception and postpartum values and peaked in the third trimester. Although a maternal (and, on occasion, fraternal) source of androgen may exist for fetal lemurs, further research is required to confirm that these steroids would reach the developing female and contribute to her masculinization.

Androgens, in concert with lactogenic hormones, contribute to the maintenance of function of the corpus luteum (CL) in pregnant rats. Whereas some of the androgenic actions in the CL are clearly mediated by intracrine conversion to estrogen, pure androgenic effects are also implicated in the regulation of this transient endocrine gland. In this report, we have established, to our knowledge for the first time, the expression of androgen receptor (AR) mRNA and protein throughout gestation in the rat CL. We have found that the AR remains expressed in the CL of gestation on Day <em>4</em> postpartum and becomes expressed in the newly formed CL after postpartum ovulation. An AR immunoreactive protein was identified in the CL of pregnancy as well as in prostate and epididymis, which were used as positive controls. The luteal AR protein had mainly nuclear localization, yet some diffuse cytoplasmic staining was also observed. Moreover, we have established that <em>androstenedione</em>, the main circulating androgen in pregnant rats, significantly reduces the decline in luteal weight observed during postpartum structural regression. This effect was correlated with a decrease in the number of cells undergoing apoptosis and with enhanced levels of circulating progesterone. In addition, in vivo administration of <em>androstenedione</em> delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that the interference with apoptosis in vitro elicited by <em>androstenedione</em> is accompanied by an increased capacity of the CL to secrete progesterone. In summary, the results of this study have established that the rat CL expresses AR throughout pregnancy and after parturition, and they have defined a potential role for <em>androstenedione</em> in opposing postpartum luteal regression through inhibition of apoptosis and stimulation of progesterone production.

Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC-MS or GC-MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E2), estrone (E1), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3β, 17β-diol (5-diol), dehydroepiandrosterone (DHEA) and <em>androstenedione</em> (<em>4</em>-dione). The system used is a UPLC-MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid-liquid extraction and a simple selective derivatization for only E1 and E2. This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1pg/mL, <em>4</em>pg/mL, 50pg/mL, 10pg/mL, 100pg/mL, 500pg/mL and 100pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as <em>4</em>pg/mL with S/N≥5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1-8<em>4</em>.7% for E2, 83.6-9<em>4</em>.5% for E1, 88.2-90.3% for Testo, 82.0-90.6% for DHT, 8<em>4</em>.9-92.0% for 5-diol, 88.1-93.8% for DHEA and 86.2-90.3% for <em>4</em>-dione, respectively. A good linearity is obtained with R>0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1-8.9% for the low limits of quantification of all seven compounds with 0.7-6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC-MS/MS and LC-MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use.

Androgens have important effects on bone in vivo, possibly by direct activation of the androgen receptors in osteoblasts. To test this hypothesis, calcium homeostasis, bone mass, and bone turnover were evaluated in mature (<em>4</em>-month-old) androgen-resistant (testicular feminized, TFM) male rats. Data were compared with data from both female and male littermates of the same age and strain. Compared to normal males, TFM had similar serum testosterone, twofold higher estradiol and estrone, and sixfold higher <em>androstenedione</em> concentrations. Compared to normal females, TFM rats showed lower estradiol but also elevated concentrations of <em>androstenedione</em> and estrone. Despite similar free 1,25-(OH)2D3 concentrations, both TFM and male rats maintained higher serum calcium and phosphate concentrations than their female littermates. Serum IGF-I concentrations in TFM rats were decreased compared to male rats (-12%) or female rats (-27%). Serum osteocalcin concentrations, however, were twofold higher in TFM rats than in females but not significantly different from males. Femoral length, diameter, and cortical thickness were intermediate between those of males and females. The cancellous bone density of the femur and cancellous bone volume of the proximal metaphysis of the tibia, however, were not significantly different between groups. The ash weight of the tibia was also not significantly different, and the ash weight of the four distal lumbar vertebrae ranged between male and female values. Bone mechanical properties as measured by torsional strength and energy absorption of the femur were lower in TFM than in females but not different from males.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen plays a major role in bone mineral homeostasis, maintaining a balance between bone formation and bone resorption not only in women but also in men. Extraglandular aromatization of circulating androgen is the major source of estrogen in post-menopausal women and men. In order to assess the capacity of bone cells as a local source of estrogen, osteoblast-like cells (OLCs) were obtained from human fetal bone in mid-trimester by the explant method and by mechanical disaggregation. The integrity of OLCs was confirmed by their ability to produce alkaline phosphatase and osteocalcin in response to vitamin D3 and also by their ability to deposit mineral. Aromatase activity was assessed by the formation of estrone from [1,2,6,7-3H]<em>androstenedione</em> and by the release of tritium from [1beta-3H]<em>androstenedione</em> into [3H]water. Formation of estrone was confirmed by thin layer chromatography (TLC) in OLCs stimulated with dexamethasone (DEX) + oncostatin M. The aromatase activity was 10 x higher in non-passaged OLCs than in passaged cells in the presence or absence of the stimulants (DEX + IL-1beta). The apparent Km and Vmax estimated by the release of [3H]water was 5.8+/-0.6 nM and 10.8+/-1.<em>4</em> pmol/mg per 6 h in the presence of DEX + IL-1beta. The effects of several stimulants on aromatase activity in OLCs were examined: serum, IL-1beta, TNFalpha and type I cytokines stimulated activity in the presence of DEX, while PMA and PMA + dibutyryl cAMP did not. To confirm the expression of aromatase in OLCs, cells prepared from periosteal membranes were also examined: These cells in culture possessed aromatase activity corresponding to OLCs prepared from bone specimens. Moreover, the fresh periosteum expressed aromatase at higher levels than that of metaphyseal specimens. The aromatase gene employs several different promoters (I.1, 1.2, I.3, I.<em>4</em>, I.5, I.6, 2a, 1f and PII) and the usage of these promoters is known to be controlled in a tissue-specific fashion. Accordingly, promoter usage in OLCs and fetal long bone (tibia) tissue was examined using the 5' rapid amplification of cDNA ends (RACE) technique. The major promoter used was I.<em>4</em>, not only in stimulated and non-stimulated OLCs, but also in fetal tibia. Some minor transcripts were also found: 1f (brain-specific promoter), PII and I.6 in OLCs stimulated by DEX + IL-1beta, and PII and I.3 in OLCs stimulated by DEX + serum. Fetal tibia also expressed I.3 (15%) and I.6 (10%). Thus, regulation and promoter usage in OLCs was quite different from other tissues known as estrogen sources including adipose tissue, ovary and placenta. These results suggest that bone is an extraglandular source of local estrogen which plays an important role in bone mineral metabolism through autocrine and paracrine actions.

In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, <em>androstenedione</em>. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to <em>androstenedione</em> or any other product was detectable. Similarly, <em>4</em>-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-<em>4</em>50. Message levels for cholesterol side-chain cleavage P-<em>4</em>50 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-<em>4</em>50 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.

The effect of the adrenal steroids androst-5-ene-3 beta,17 beta-diol (delta 5-diol) and <em>androstenedione</em> (delta <em>4</em>-dione) was studied on the growth of mammary carcinoma induced in the rat by dimethylbenz[a]anthracene (DMBA). The plasma levels of the two steroids were maintained at values within the range of those found in the circulation of post-menopausal women by constant release from osmotic pumps in ovariectomized animals. delta 5-diol and delta <em>4</em>-dione, at the daily release rate of 500 micrograms, led to plasma levels of 1.26 +/- 0.19 and 1.72 +/- 0.75 ng/ml, respectively. At these physiologically relevant plasma concentrations, both delta 5-diol and delta <em>4</em>-dione caused a marked stimulation of tumor growth while having minimal or no effect on uterine weight or on plasma prolactin and LH levels. Concomitant treatment with the aromatase inhibitor aminoglutethimide completely blocked the stimulatory effect of delta <em>4</em>-dione released from silastic implants on tumor growth, while simultaneous administration of the antiandrogen flutamide had no significant effect. On the other hand, when aminoglutethimide was administered with delta 5-diol, the stimulatory effect of the adrenal steroid on tumor growth was not affected. Such data indicate that, under the present experimental conditions, transformation of delta <em>4</em>-dione into androgens plays a minor role, the predominant effect of the adrenal steroid being stimulation of tumor growth through conversion into estrogens, while delta 5-diol exerts a direct estrogenic effect independent from aromatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To evaluate the association between serum bisphenol-A (BPA) concentration and sex hormone levels in men.

METHODS

Cross-sectional study.

METHODS

Not applicable.

METHODS

A total of 290 men with or without BPA exposure in the workplace.

METHODS

None.

METHODS

Serum sex hormone levels.

RESULTS

After adjustment for potential confounders using linear regression, increasing serum BPA concentration was statistically significantly associated with [1] decreased <em>androstenedione</em> levels, [2] decreased free testosterone levels, [3] decreased free androgen index, and [<em>4</em>] increased sex hormone-binding globulin levels. Comparison of hormone levels between workers exposed and unexposed to BPA showed similar associations.

CONCLUSIONS

Exposure to a high BPA level may impact sex hormone levels in men.

Estrogens are involved in numerous physiologic processes and have crucial roles in particular disease states, such as mammary carcinomas. Estradiol, the most potent endogenous estrogen, is biosynthesized from androgens by the cytochrome P-<em>4</em>50 enzyme complex called aromatase. Aromatase is found in breast tissue, and the importance of intratumoral aromatase and local estrogen production is being unraveled. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in hormone-dependent breast cancer. Effective aromatase inhibitors have been developed as therapeutic agents for controlling estrogen-dependent breast cancer. Investigations into the development of aromatase inhibitors began in the 1970s and have expanded greatly in the past three decades. Competitive aromatase inhibitors are molecules that compete with the substrate <em>androstenedione</em> for noncovalent binding to the active site of the enzyme to decrease the amount of product formed. Steroidal inhibitors that have been developed to date build on the basic <em>androstenedione</em> nucleus and incorporate chemical substituents at varying positions on the steroid. The structure-activity relationships for steroidal inhibitors have become more refined in the past decade, and only some modifications can be made to the steroid and still keep its affinity for aromatase. Nonsteroidal aromatase inhibitors can be divided into three classes: aminoglutethimide-like molecules, imidazole/triazole derivatives, and flavonoid analogs. Mechanism-based aromatase inhibitors are inhibitors that mimic the substrate, are converted by the enzyme to a reactive intermediate, and result in the inactivation of aromatase. Aromatase inhibitors, both steroidal and nonsteroidal, have shown clinical efficacy for the treatment of breast cancer. The initial nonselective nature of nonsteroidal inhibitors such as aminoglutethimide has been greatly reduced in the later generations of inhibitors, anastrozole and letrozole. Mechanism-based steroidal inhibitors such as <em>4</em>-hydroxy<em>androstenedione</em> and exemestane produce prolonged aromatase inhibition in patients. The potent and selective third-generation aromatase inhibitors anastrozole, letrozole, and exemestane are approved for clinical use as second-line endocrine therapy in postmenopausal patients failing antiestrogen therapy alone or multiple hormonal therapies.

The plasma profiles of 8 hormones were followed over the course of prepuberty and puberty in 30 adolescent males who developed gynecomastia and 2<em>4</em> who did not. Throughout puberty, ratios of delta <em>4</em>-<em>androstenedione</em> to estrone (E1) and estradiol (E2) were significantly lower in the gynecomastia group than in the control group. Similarly, ratios of dehydroepiandrosterone-sulfate to E1 and E2 were significantly lower in the gynecomastia group. In contrast, ratios of plasma testosterone to E1 and E2 as well as plasma progesterone and PRL concentrations, were similar in both groups. Because of the adrenal origin of dehydroepiandrosterone and its sulfate, and of peripheral conversion of adrenal androgens to E1 and to E2, it appears that either decreased adrenal production of androgens and/or increased conversion of dehydroepiandrosterone-sulfate and delta <em>4</em>-<em>androstenedione</em> to estrogens cause transient gynecomastia in adolescent boys.

To evaluate the long-term effects of treatment with RU<em>4</em>86 and to test its efficacy in endometriosis, a 3-month trial was conducted to evaluate the effects of daily administration (100 mg/day or approximately 2 mg/kg/day) on the functional activity of the reproductive axis, as well as implants, in patients with symptomatic pelvic endometriosis. All women became amenorrhoeic and acyclic. However, ovarian suppression was incomplete. In 2<em>4</em> h sampling studies, mean luteinizing hormone (LH) and LH pulse amplitude were increased without a change in LH pulse frequency. Additionally, an antiglucocorticoid effect was demonstrated. Treatment resulted in improvement in pelvic pain in all subjects without significant changes in the extent of disease as evaluated by laparoscopy. We also attempted to reduce the growth of uterine fibroids by using 50 mg/day of RU<em>4</em>86 for 3 months in 10 patients. Myoma size decreased approximately 22% at <em>4</em> weeks, 39% at 8 weeks and <em>4</em>9% at 12 weeks. Serum concentrations of LH, <em>androstenedione</em> and testosterone increased in the first 3 weeks of treatment and then returned to baseline. In conclusion, daily administration of RU<em>4</em>86 resulted in ovarian inhibition and menstrual acyclicity and in an improvement in the pain associated with pelvic endometriosis and decreased the size of uterine fibroids. This ovarian inhibition was achieved without oestrogen deprivation and may provide a novel long-term approach to the treatment of ovarian steroid-dependent disease processes.

A regimen or aminoglutethimide in combination with replacement glucocorticoid has been used to suppress adrenal steroidogenesis in postmenopausal women with metastatic breast carcinoma. During acute and chronic treatment with aminoglutethimide, the levels of the delta <em>4</em>-steroids [progesterone (P), 17 alpha-hydroxyprogesterone (17-delta <em>4</em>-P), and <em>androstenedione</em> (delta <em>4</em>-A)] and the delta 5-steroids [dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S), and 17 alpha-hydroxypregnenolone (17-delta 5-P)] were determine. In the total group of women, the plasma levels of P and delta <em>4</em>-A increased 2- to 3-fold (P less than 0.05) while 17-delta <em>4</em>-P rose 10-fold (P less than 0.01) from basal concentrations of 0.65 +/- 0.07 to 6.<em>4</em>8 +/- 1.<em>4</em>6 ng/ml during the initial 2 weeks of therapy with aminoglutethimide (AG) and dexamethasone. These three steroids then fell to basal levels during chronic treatment (P and 17-delta <em>4</em>-P) or were suppressed (delta <em>4</em>-A; P less than 0.001). In contrast, the levels of delta 5-steroids (17-delta 5-P, DHEA, and DHEA-S) were reduced 3- to 5-fold during the initial 2 weeks of therapy and remained suppressed throughout. The relative levels of certain delta 5- and delta <em>4</em>-steroids pairs were then examined. The ratio of 17-delta 5-P to 17-delta <em>4</em>-P decreased from baseline values of 2.15 +/- 0.35 to 0.38 +/- 0.21 ng/ml (P less .02) with the initiation of therapy and remained low thereafter. A similar pattern for the ratios between DHEA and delta <em>4</em>-A, and DHEA-S and delta <em>4</em>-A was observed. This may indicate that the regimen of AG treatment utilized may facilitate the activity of the 3 beta-ol-dehydrogenase, delta 5- to delta <em>4</em>-isomerase, and accelerate the conversion of delta 5- to delta <em>4</em>-steroids. The patterns of suppression of the plasma delta <em>4</em>- and delta 5-steroids in oophorectomized and spontaneously postmenopausal patients with intact ovaries were analyzed separately. The plasma levels of progesterone were higher during the first 2 weeks of therapy in surgically castrate women than in spontaneously postmenopausal women (0.72 +/- 0.25 vs. 0.<em>4</em>7 +/- 0.20 ng/ml). A similar pattern was observed for 17-delta <em>4</em>-P, DHEA, and DHEA-S indicating that the adrenals might contribute to this increase. In contrast, during chronic treatment the levels of all steroids were lower in surgically castrate women than in those with intact ovaries. This suggested residual ovarian steroid during AG administration.