Androgenic effects of aqueous and methanolic extracts of Ficus asperifolia in male Wistar rats.

BACKGROUND: Androgen deficiency is a clinical syndrome resulting from the inability of the testes to produce physiological levels of testosterone due to a disturbance occurring at one or more levels of the hypothalamic-pituitary-testicular axis. The present study was undertaken to evaluate the androgenic properties of aqueous and methanolic extracts of Ficus asperifolia on normal and castrated immature rats. METHODS: Normal rats were treated either per os with aqueous or methanolic extract of Ficus asperifolia (100 mg/kg or 500 mg/kg b.w.), distilled water (10 ml/kg b.w.), 5% Tween 80 (10 ml/kg b.w.) or subcutaneously with testosterone propionate (0.5 mg/kg b.w.). Castrated rats were treated with plant extracts (100 mg/kg b.w. or 500 mg/kg b.w.) alone or with the co-administration of plant extracts and testosterone propionate (s.c., 0.5 mg/kg b.w.) or bicalutamide (2 mg/kg b.w. per os). Animals were treated once a day during four weeks. Body weight growth and relative sexual organ weights were recorded at the end of each treatment. Some biomedical parameters were measured in the plasma (proteins, cholesterol), testes (cholesterol) and epididymis (proteins). RESULTS: In normal rats, Ficus asperifolia significantly (p < 0.05) increased the relative weights of the testes and all sexual-dependent organs whereas total testicular cholesterol concentration was significantly (p < 0.05) decreased. In castrated groups, treatment with Ficus asperifolia was followed by an increase in the sexual organ weights, epididymal protein and prostatic acid phosphatase concentrations. The co-administration of testosterone and plant extracts significantly (p < 0.05) increased the weight of accessory sexual organs and epididymal protein contents. In the presence of bicalutamide (an anti-androgen), the sexual stimulating activity of Ficus asperifolia was diminished with remarkable effects on vas deferens weight (p < 0.05), plasma (p < 0.01) and epididymal (p < 0.05) protein contents. CONCLUSION: Ficus asperifolia possesses androgen-like activity through possible stimulation of cytoplasmic and/or nuclear receptors by the bioactive compounds found in its extracts.

Bringing GC-MS profiling of steroids into clinical applications.

Abnormalities of steroid biosynthesis and excretion are responsible for the development and prevention of endocrine disorders, such as metabolic syndromes, cancers, and neurodegenerative diseases. Due to their biochemical roles in endocrine system, qualitative and quantitative analysis of steroid hormones in various biological specimens is needed to elucidate their altered expression. Mass spectrometry (MS)-based steroid profiling can reveal the states of metabolites in biological systems and provide comprehensive insights by allowing comparisons between metabolites present in cells, tissues, or organisms. In addition, the activities of many enzymes related to steroid metabolism often lead to hormonal imbalances that have serious consequences, and which are responsible for the progress of hormone-dependent diseases. In contrast to immunoaffinity-based enzyme assays, MS-based methods are more reproducible in quantification. In particular, high-resolution gas chromatographic (GC) separation of steroids with similar chemical structures can be achieved to provide rapid and reproducible results with excellent purification. GC-MS profiling therefore has been widely used for steroid analysis, and offers the basis for techniques that can be applied to large-scale clinical studies. Recent advances in analytical technologies combined with inter-disciplinary strategies, such as physiology and bioinformatics, will help in understanding the biochemical roles of steroid hormones. Therefore, comprehensive analytical protocols in steroid analysis for different research purposes may contribute to the elucidation of complex metabolic processes relevant to steroid function in many endocrine disorders, and in the identification of diagnostic biomarkers.

Analysis of androgenic steroids in environmental waters by large-volume injection liquid chromatography tandem mass spectrometry.

A new method was developed for the analysis of natural and synthetic androgenic steroids and their selected metabolites in aquatic environmental matrixes using direct large-volume injection (LVI) high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). Method accuracy ranged from 87.6 to 108% for analytes with well-matched internal standards. Precision, quantified by relative standard deviation (RSD), was less than 12%. Detection limits for the method ranged from 1.2 to 360 ng/L. The method was demonstrated on a series of 1 h composite wastewater influent samples collected over a day with the purpose of assessing temporal profiles of androgen loads in wastewater. Testosterone, androstenedione, boldenone, and nandrolone were detected in the sample series at concentrations up to 290 ng/L and loads up to 535 mg/h. Boldenone, a synthetic androgen, had a temporal profile that was strongly correlated to testosterone, a natural human androgen, suggesting its source may be endogenous. An analysis of the sample particulate fraction revealed detectable amounts of sorbed testosterone and androstenedione. Androstenedione sorbed to the particulate fraction accounted for an estimated 5 to 7% of the total androstenedione mass.

Fate and levels of steroid oestrogens and androgens in waste stabilisation ponds: quantification by liquid chromatography-tandem mass spectrometry.

The capacity for removing wastewater-borne endocrine disrupting chemicals (EDCs) was investigated for two wastewater treatment plants (WWTPs) incorporating waste stabilisation ponds (WSPs) as the principal treatment technology. Samples were analysed for a number of steroidal oestrogens and androgens using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Removal efficiency for steroid androgens was high for both WWTPs (93-100%) but WSP treatment was observed to be less effective for removing steroid oestrogens, particularly oestriol.

Determination of escin content in androgenic embryos and hairy root culture of Aesculus hippocastanum.

Escin, a group of chemically related triterpenic glycosides, is widely used in commercial preparations for the treatment of venous insufficiency. Since the zygotic embryo cotyledons accumulate the highest amount of escin, it is currently extracted from the seeds of horse chestnut, Aesculus hippocastanum L. (Hippocastanaceae), on a large scale. As this material is available during only short period of the year, we studied the possibility of using plant tissue culture to obtain escin. For this purpose, the content of escin in androgenic embryos and hairy root cultures of horse chestnut was studied. Escin content was found to be dependent on the stage of androgenic embryo development and the type of phytoregulator supplemented to the nutritive medium. In the absence of phytoregulators, androgenic embryos at the globular stage of development contained approximately four times less escin than those at the cotyledonary stage. Inclusion of various phytoregulators in the nutritive media stimulated escin production. Among them, 2,4-dichlorophenoxyacetic acid (2,4-D) showed the most pronounced effect, with escin content almost reaching that found in zygotic embryos (6.77% versus 6.96%). Two hairy root clones produced substantial amounts of escin (3.57% and 4.09%), less than zygotic embryos, but higher than cotyledonary embryos on phytoregulator-free medium.

Anabolic and androgenic activities of Bulbine natalensis stem in male Wistar rats.

Aqueous extract of Bulbine natalensis Baker (Asphodelaceae) stem at 25, 50 and 100 mg/kg body weight was investigated for anabolic and androgenic effects in male Wistar rats. Sixty male rats were grouped into four (A-D) consisting of 15 each. Group A (control) was orally treated with 0.5 mL of distilled water for 14 days while groups B, C and D were treated like the control except they received 0.5 mL containing 25, 50, and 100 mg/kg body weight of the extract respectively. All the doses of the extract increased (P <0.05) the testicular-body weight ratio as well as alkaline phosphatase activity, glycogen, sialic acid, protein, and cholesterol content of the testes except the single administration of 100 mg/kg body weight which compared well (P>0.05) with the controls for glycogen and cholesterol. The testicular and serum testosterone concentration were increased except in the 100 mg/kg body weight where the effect on the tissue and serum hormone did not manifest until after the first and seven daily doses respectively. Testicular acid phosphatase activity, serum follicle stimulating and luteinizing hormone concentrations also increased at all the doses except in the 100 mg/kg body weight where the effect on the enzyme and the hormone did not manifest until after seven days. The increases were most pronounced in the 50 mg/kg body weight extract treated animals. The results indicate anabolic and androgenic activities of Bulbine natalensis stem in male rat testes with the 50 mg/kg body weight of the extract exhibiting the highest anabolizing and androgenic activities. These activities further support the folkloric use of the plant most especially at 50 mg/kg body weight in the management of male sexual dysfunction in South Africa.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators.

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newly developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.

Evaluation of a photocatalytic reactor membrane pilot system for the removal of pharmaceuticals and endocrine disrupting compounds from water.

A photocatalytic reactor membrane pilot system, employing UV/TiO(2) photocatalysis, was evaluated for its ability to remove thirty-two pharmaceuticals, endocrine disrupting compounds, and estrogenic activity from water. Concentrations of all compounds decreased following treatment, and removal followed pseudo-first-order kinetics as a function of the amount of treatment. Twenty-nine of the targeted compounds in addition to total estrogenic activity were greater than 70% removed while only three compounds were less than 50% removed following the highest level of treatment (4.24 kW h/m(3)). No estrogenically active transformation products were formed during treatment. Additionally, the unit was operated in photolytic mode (UV only) and photolytic plus H(2)O(2) mode (UV/H(2)O(2)) to determine the relative amount of energy required. Based on the electrical energy per order (EEO), the unit achieved the greatest efficiency when operated in photolytic plus H(2)O(2) mode for the conditions tested.

Assessment of trace organic chemical removal by a membrane bioreactor using gas chromatography/mass spectrometry and a yeast screen bioassay.

A membrane bioreactor (MBR) was assessed for the removal of estrogens, androgens, and a selection of pharmaceuticals and personal care products. The biomass and aqueous components of the MBR were investigated to determine whether removal was by biodegradation or by adsorption to the biomass. Removal was monitored using chemical analysis by gas chromatography/mass spectrometry (GC-MS) as well as biological analysis using estrogenic and androgenic yeast assays. Results showed that the MBR was effective in removing the compounds of concern from raw influent with removal rates between 78 and 99%. Removal efficiencies were comparable or better than those reported for conventional activated sludge systems, which was attributed to the relatively high sludge retention time of the MBR. The biomass component showed significant concentrations of salicylic acid, triclosan, and 4-tert-octylphenol. Estrogenic and androgenic activity was also measured in the biomass. Estrone was identified as the main compound responsible for the estrogenic activity. It was concluded that the main removal pathway was biodegradation, but sorption to biomass may also be important, particularly for triclosan and 4-tert-octylphenol.

Cynomorium songaricum Rupr demonstrates phytoestrogenic or phytoandrogenic like activities that attenuates benign prostatic hyperplasia via regulating steroid 5-α-reductase.

ETHNOPHARMACOLOGICAL RELEVANCE: Cynomorium songaricum Rupr. (CS) belongs to the genus of parasitic perennial flowering plants, mostly used in Chinese traditional medicine for benign prostatic hyperplasia (BPH) treatment. BPH is a chronic disease in men that both androgen and estrogen play a crucial role in promoting its development via their receptors. Previously we have showed that compounds from CS have the phytoestrogenic and/or phytoandrogenic activities that may have the potential suppressive effects on BPH, while the mechanism remains unclear. AIM OF THE STUDY: In this study, we aim to investigate the effect of CS and its derived compounds: luteolin (LUT), gallic acid (GA), protocatechuic acid (PA) and protocatechualdehyde (Pra) on inhibition of rat BPH and proliferation of BPH-1 cell line respectively, and further uncover whether it is related with the phytoestrogenic and / or phytoandrogenic activities. MATERIALS AND METHODS: Estradiol/testosterone (1:100) was subcutaneous injected to induce BPH in a castrated rat model, and CS was orally administrated for 45 days. Then the weights of the body and prostate were recorded, the pathogenesis changes of prostate were analyzed by Hematoxylin and eosin (H&E) and immunohistochemical (IHC). The levels of 17ß-estradiol (E2), testosterone, and dihydrotestosterone (DHT) from rats' serum were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, human benign prostatic epithelial cell BPH-1 was cultured and treated with or without different CS compounds and DHT or E2. MTT and CCK-8 assays were performed to detect the regulatory effects on cell proliferation. The expressions of PCNA, AR, ERα, ERß, and steroid 5-α-reductases (SRD5A1 and SRD5A2) were further analyzed by western blotting upon treatment. RESULTS: Treatment with CS significantly inhibited rat prostate enlargement, improved the pathological feature and reduced the thickness of smooth muscle layer. The up-regulated AR and ERα expressions and down-regulated ERß in BPH rat prostate were significantly blocked after CS administration. Moreover, the enhanced values of E2/testosterone and the level of DHT in serum were also strongly inhibited in CS group compared with those in BPH groups. In cellular level, LUT, GA, PA, or Pra significantly inhibited DHT- or E2- induced BPH-1 cell proliferation and PCNA expressions. Consistently with the data in vivo, compounds from CS interfered the DHT or E2-regulated AR, ERα and ERß expressions in BPH-1 cells as well. Importantly, the dramatic increased SRD5A1 and SRD5A2 expressions were observed in BPH rat prostates and DHT or E2-stimulated BPH-1 cells. However, treatment with CS in rat or with compounds isolated from CS in BPH-1 cells significantly blocked the induction of SRD5A1 and SRD5A2. CONCLUSIONS: CS suppressed BPH development through interfering with prostatic AR, ERα/ß, and SRD5A1/2 expressions, which provided evidence of CS for BPH treatment.

In vitro bioanalytical evaluation of removal efficiency for bioactive chemicals in Swedish wastewater treatment plants.

Chemical contamination of wastewater is a problem of great environmental concern, as it poses a hazard to both the ecosystem and to human health. In this study, we have performed a bioanalytical evaluation of the presence and removal efficiency for bioactive chemicals in wastewater treatment plants (WWTPs), using in vitro assays for toxicity endpoints of high relevance for human health. Water samples were collected at the inlet and outlet of five Swedish WWTPs, all adopting a treatment technology including pretreatment, primary treatment (sedimenation), seconday treatment (biological processes), post-sedimentation, and sludge handling. The water samples were analyzed for cytotoxicity, estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity, oxidative stress response (Nrf2) and the ability to activate NFĸB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. We observed clear androgenic and estrogenic activities in all inlet samples. Androgenic and estrogenic activities were also observed in all outlet samples, but the activities were lower than the respective inlet sample. AhR activity was observed in all samples, with higher activities in the inlet samples compared to the outlet samples. The removal efficiency was found to be high for androgenic (>99% for two plants and 50-60% for two plants) and estrogenic (>90% for most plants) compounds, while the removal efficiency for AhR-inducing compounds was 50-60% for most plants and 16% for one plant.

Isolation and quantification by high-performance liquid chromatography-ion-trap mass spectrometry of androgen sulfoconjugates in human urine.

Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography-ion-trap mass spectrometry (LC-MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C18 column (150 mm x 4.6 mm I.D., 5 microm) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89-107%), repeatability (3.4-22%) and intermediate precision (5.8-22%) over the range of 2-200 ng/mL (corresponding to 1.5-147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.

Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities.

Recently we constructed yeast cells that either express the human estrogen receptor alpha or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3'-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.

Bendigoles A approximately C, new steroids from Gordonia australis Acta 2299.

Bendigoles A approximately C are the first secondary metabolites to be isolated from a member of the actinomycete genus Gordonia. They were detected in a culture filtrate extract of Gordonia australis Acta 2299 by HPLC-diode array analysis and characterized as new steroids by mass spectrometry and NMR experiments. Bendigole C show binding affinity to the human progesterone and A approximately C to androgen receptor but are inactive at mineralocorticoid and estrogen receptors. In in vitro transactivation studies bendigoles A and C showed moderate and weak androgenic activities.

Novel phytoandrogens and lipidic augmenters from Eucommia ulmoides.

BACKGROUND: Plants containing compounds such as the isoflavonoids, with female hormone-like effects that bind to human estrogen receptors, are known. But none has been previously shown to have corresponding male hormone-like effects that interact with the human androgen receptor. Here, we report that the tree bark (cortex) of the Gutta-Percha tree Eucommia ulmoides possesses bimodal phytoandrogenic and hormone potentiating effects by lipidic components. METHODS: The extracts of E. ulmoides were tested using in-vitro reporter gene bioassays and in-vivo animal studies. Key compounds responsible for the steroidogenic effects were isolated and identified using solid phase extraction (SPE), high performance liquid chromatography (HPLC), thin layer chromatography (TLC), gas chromatography-mass spectroscopy (GC-MS), electron spray ionisation-mass spectroscopy (ESI-MS) and nuclear magnetic resonance (NMR). RESULTS: The following bioactivities of E. ulmoides were found: (1) a phenomenal tripartite synergism exists between the sex steroid receptors (androgen and estrogen receptors), their cognate steroidal ligands and lipidic augmenters isolated from E. ulmoides, (2) phytoandrogenic activity of E. ulmoides was mediated by plant triterpenoids binding cognately to the androgen receptor (AR) ligand binding domain. CONCLUSION: In addition to well-known phytoestrogens, the existence of phytoandrogens is reported in this study. Furthermore, a form of tripartite synergism between sex steroid receptors, sex hormones and plant-derived lipids is described for the first time. This could have contrasting clinical applications for hypogonadal- and hyperlipidaemic-related disorders.

Remediation efficiency of three treatments on water polluted with endocrine disruptors: Assessment by means of in vitro techniques.

Chemical substances with potential to disrupt endocrine systems have been detected in aquatic environments worldwide, making necessary the investigation about water treatments able to inhibit such potential. The present work aimed to assess the efficiency for removing endocrine disruptors (with estrogenic and androgenic activity) of three simple and inexpensive substrates that could be potentially used in sectors or regions with limited resources: powdered activated carbon (PAC), powdered natural zeolite (ZEO) (both at a concentration of 500 mg L-1) and natural aquatic humic substances (AHS) (at 30 mg L-1). MilliQ-water and mature water from fish facilities (aquarium water, AW), were artificially spiked with 17ß-estradiol (E2), 17α-ethinylestradiol and dihydrotestosterone. Moreover, effluent samples from waste water treatment plants (WWTP) were also submitted to the remediation treatments. Estrogenic and androgenic activities were assessed with two cell lines permanently transfected with luciferase as reporter gene under the control of hormone receptors: AR-EcoScreen containing the human androgen receptor and HER-LUC transfected with the sea bass estrogen receptor. PAC was efficiently removing the estrogenic and androgenic compounds added to milliQ and AW. However, androgenic activity detected in WWTP effluents was only reduced after treatment with ZEO. The higher surface area of PAC could have facilitated the removal of spiked hormones in clean waters. However, it is possible that the substances responsible of the hormonal activity in WWTP have adsorbed to micro and nanoparticles present in suspension that would have been retained with higher efficiency by ZEO that show pores of several microns in size.

Identification of neuroactive steroids and their precursors and metabolites in adult male rat brain.

Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3alpha,5alpha-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3alpha,5alpha-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5alpha-dihydroprogesterone. Furthermore, the presence of 3beta-, 11beta-, 17alpha-, and 20alpha-hydroxylated metabolites of 3alpha,5alpha-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20alpha-reduced metabolites could also be found for pregnenolone, progesterone, and 5alpha-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3alpha,5alpha-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.

Effects of ethanol on steroid profiles in the rat testis.

The effects of ethanol on the concentrations of steroids in testis was studied in adult rats. Testosterone, seven of its potential precursors, three of its metabolites, and estradiol were analyzed by gas chromatography-mass spectrometry of samples from testes removed 2 h after intraperitoneal administration of ethanol, 1.2 g/kg body weight. The same analyses were made on samples from control rats. Ethanol gave a marked increase of all 3 beta-hydroxy-delta 5 steroids analyzed: pregnenolone (60%), 17-hydroxypregnenolone (480%), dehydroepiandrosterone (430%) and 5-androstene-3 beta, 17 beta-diol (60%). This resulted in highly significant increases of the 3 beta-hydroxy-delta 5/3-oxo-delta 4 steroid ratios for all steroid couples analyzed. An analogous increase of the ratio between 5 alpha-androstane-3 beta, 17 beta-diol and dihydrotestosterone was also observed, whereas the ratio between androstenediol and dehydroepiandrosterone was decreased by ethanol. The concentration of estradiol was not affected. The results indicate that moderate doses of ethanol inhibit the conversion of 3 beta-hydroxy-delta 5 to 3-oxo-delta 4 steroids. This may be one mechanism by which ethanol decreases the production of testosterone.

Isotopic fractionation of endogenous anabolic androgenic steroids and its relationship to doping control in sports.

The use of gas chromatography (GC)-combustion (C)-isotope ratio mass spectrometry (IRMS) demonstrates that a single oral administration of dehydroepiandrosterone (DHEA, 100 mg) to a male subject significantly lowers the 13C content of etiocholanolone (Et) and androsterone (A) in the subject's urine. The difference in carbon isotope ratio (d13C per thousand) values between Et and A increases from 1.6 per thousand at the time of administration to 5.1 per thousand at 26 h post-administration, indicating preferential metabolism of administered DHEA to form Et in relation to A. Multiple oral administrations of DHEA to a male subject reveals lower d13C values during the excretion period of Et (-31.7 per thousand to -34.6 per thousand) and A (-31.4 per thousand to -33.0 per thousand) to that of the d13C value of the administered DHEA (-31.3 per thousand). Reference distributions of d13C Et and d13C A constructed from normal athlete populations within Australia and New Zealand show a small natural discrimination against 13C in the formation of Et relative to A (mean=0.3 per thousand, n=167, p=0.007). Amplified differences between d13C Et and d13C A, and in vivo 13C depletion measured by GC-C-IRMS are shown to be potentially useful for doping control.

Spironolactone does not affect in vitro androgen secretion by human granulosa-luteal cells.

Although spironolactone has been documented to decrease peripheral serum androgen levels in women treated for androgen excess of ovarian origin, the site of action does not appear to be the granulosa cell. In vitro cell culture studies using human granulosa-luteal cells isolated from women undergoing ovarian stimulation for IVF have shown no affect on T, delta 4A, or DHT secretion in the presence of clinically therapeutic levels of spironolactone (0-10(-6) M). In addition, neither P nor E2 production was affected by increasing concentrations of spironolactone.