Search

Background: Hirsutism is the excessive growth of terminal hair in a male pattern in a female. In most hirsute women, hirsutism is caused by increased androgens. However, not all women with hirsutism actually show elevated levels of circulating androgens with standard laboratory tests, in which case we speak of idiopathic hirsutism (IH)., Objectives: The aim of this paper is to investigate whether there are biochemical markers that can be used to unravel the cause in IH., Methods: An electronic search through the PubMed database was conducted to find studies describing potential biomarkers for IH., Results: The majority of included studies claimed an increased 5α-reductase (5α-RD) activity in women with IH by means of increased DHT metabolite levels. Studies investigating abnormalities of the androgen receptor (AR) and serum levels of indirect markers showed no significant differences., Conclusions: Our literature search showed that polymorphisms of the AR as well as indirect markers seem to be nonspecific, but that the dihydrotestosterone-reduced metabolite 5α-androstane-3α,17β-diol glucuronide is markedly enhanced in women with IH, suggesting an increased 5α-RD activity in these women. Further studies need to be performed to determine the clinical usefulness of 3α-diol G as a biomarker for IH., (Copyright © 2022. Published by Elsevier B.V.)

5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17β-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4- 14 C]-3α-diol from [4- 14 C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC 50  = 1.2-14.1 μM). The most potent inhibitor 25 (IC 50  = 1.2 μM) possesses a 4-(4-CF 3 -3-CH 3 O-benzyl)piperazinyl methyl side chain at C3β and 17β-OH/17α-CCH functionalities at C17 of a 5α-androstane core., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of the present study was to identify microRNAs (miRNAs) that regulate the proliferation and differentiation of Leydig cells (LCs) of rat. Three small RNA libraries derived from progenitor LCs (PLCs), immature LCs (ILCs) and adult LCs (ALCs) were analyzed by microarrays. In total, 68 differentially expressed miRNAs (DEMs) were identified. Based on the trend of DEM expression from PLCs to ALCs, primary LCs were transfected with miRNA mimics or inhibitors. Five miRNAs (miR-30a-5p, miR-3585-5p, miR-212-3p, miR-369-5p and miR-434-3p) promoted PLC proliferation, and 3 miRNAs (miR-17-5p, miR-532-3p and miR-329-3p) activated caspase-3, which triggered LC apoptosis. For steroidogenesis, 18 miRNAs could elevate or inhibit androsterone release at the PLC stage. Eleven and 9 miRNAs inhibited the production of 5α-androstane-3α,17β-diol in ILCs and testosterone in ALCs, respectively. miR-17-5p, miR-29a-3p and miR-299a-5p decreased androgen production by LCs at all developmental stages. Furthermore, the miR-299a-5p-mediated decrease in androgen production by the LC lineage was primarily achieved by downregulating the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). These findings provide insights into the regulatory roles of miRNAs during the postnatal development of LCs and suggest potential strategies for the treatment of steroid-related disorders., (Copyright © 2019 Elsevier B.V. All rights reserved.)

DHRS7 (SDR34C1) has been associated with potential tumor suppressor effects in prostate cancer; however, its function remains largely unknown. Recent experiments using purified recombinant human DHRS7 suggested several potential substrates, including the steroids cortisone and Δ4-androstene-3,17-dione (androstenedione). However, the substrate and cofactor concentrations used in these experiments were very high and the physiological relevance of these observations needed to be further investigated. In the present study, recombinant human DHRS7 was expressed in intact HEK-293 cells in order to investigate whether glucocorticoids and androgens serve as substrates at sub-micromolar concentrations and at physiological cofactor concentrations. Furthermore, the membrane topology of DHRS7 was revisited using redox-sensitive green-fluorescent protein fusions in living cells. The results revealed that (1) cortisone is a substrate of DHRS7; however, it is not reduced to cortisol but to 20β-dihydrocortisone, (2) androstenedione is not a relevant substrate of DHRS7, (3) DHRS7 catalyzes the oxoreduction of 5α-dihydrotestosterone (5αDHT) to 3α-androstanediol (3αAdiol), with a suppressive effect on androgen receptor (AR) transcriptional activity, and (4) DHRS7 is anchored in the endoplasmic reticulum membrane with a cytoplasmic orientation. Together, the results show that DHRS7 is a cytoplasmic oriented enzyme exhibiting 3α/20β-hydroxysteroid dehydrogenase activity, with a possible role in the modulation of AR function. Further research needs to address the physiological relevance of DHRS7 in the inactivation of 5αDHT and AR regulation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Intratumoural dihydrotestosterone (DHT) synthesis could be an explanation for castration resistance in prostate cancer (PC). By using liquid chromatography-mass spectrometry, we evaluated the intratumoral DHT synthesis from 5α-androstane-3β,17β-diol (3β-diol), which is inactive androgen metabolized from DHT. 3β-diol had biochemical potential to be converted to DHT via three metabolic pathways and could stimulate PC cell growth. Especially, 3β-diol was not only converted back to upstream androgens such as dehydroepiandrosterone (DHEA) or Δ5-androstenediol but also converted directly to DHT which is the main pathway from 3β-diol to DHT. Abiraterone had a significant influence on the metabolism of DHEA, epiandrosterone and 3β-diol, by the inhibition of the intratumoural 3β-hydroxysteroid dehydrogenase (3β-HSD) activities which is one of key catalysts in androgen metabolic pathway. The direct-conversion of 3β-diol to DHT was catalysed by 3β-HSD and abiraterone could inhibit this activity of 3β-HSD. These results suggest that PC had a mechanism of intratumoural androgen metabolism to return inactive androgen to active androgen and intratumoural DHT synthesis from 3β-diol is important as one of the mechanisms of castration resistance in PC. Additionally, the inhibition of intratumoural 3β-HSD activity could be a new approach to castration-resistant prostate cancer treatment.

Recently, we created a unique gain-of-function mouse model with Sertoli cell-specific transgenic androgen receptor expression (TgSCAR) showing that SCAR activity controls the synchronized postnatal development of somatic Sertoli and Leydig cells and meiotic-postmeiotic germ cells. Moderate TgSCAR (TgSCAR(m)) expression reduced testis size but had no effect on male fertility. Here, we reveal that higher TgSCAR expression (TgSCAR(H)) causes male infertility. Higher SCAR activity, shown by upregulated AR-dependent transcripts (Rhox5, Spinw1), resulted in smaller adult TgSCAR(H) testes (50% of normal) despite normal or elevated circulating and intratesticular testosterone levels. Unlike fertile TgSCAR(m) males, testes of adult TgSCAR(H) males exhibited focal regions of interstitial hypertrophy featuring immature adult Leydig cells and higher intratesticular dihydrotestosterone and 5α-androstane 3α,17β-diol levels that are normally associated with pubertal development. Mature TgSCAR(H) testes also exhibited markedly reduced Sertoli cell numbers (70%), although meiotic and postmeiotic germ cell/Sertoli cell ratios were twofold higher than normal, suggesting that elevated TgSCAR activity supports excessive spermatogenic development. Concurrent with the higher germ cell load of TgSCAR(H) Sertoli cells were increased levels of apoptotic germ cells in TgSCAR(H) relative to TgSCAR(m) testes. In addition, TgSCAR(H) testes displayed unique morphological degeneration that featured accumulated cellular and spermatozoa clusters in dilated channels of rete testes, consistent with reduced epididymal sperm numbers. Our findings reveal for the first time that excessive Sertoli cell AR activity in mature testes can reach a level that disturbs Sertoli/germ cell homeostasis, impacts focal Leydig cell function, reduces sperm output, and disrupts male fertility., (Copyright © 2016 the American Physiological Society.)

Aim: Observationally, low serum testosterone among men is associated with cardiovascular diseases and its risk factors, but it is unclear whether raising endogenous androgens would be protective. To clarify the role of androgens, the association of two different androgen biomarkers (serum testosterone and androstanediol glucuronide) with cardiovascular disease risk factors and mortality was examined in a nationally representative sample of US men., Research Design and Methods: Multivariable linear and proportion hazards regression were used to examine the adjusted associations of serum testosterone and androstanediol glucuronide with cardiovascular disease risk factors and death from major cardiovascular diseases in 1460 men from NHANES III phase 1 (1988-1991) followed-up through 2006., Results: Serum testosterone and androstanediol glucuronide were weakly correlated (0·13). Serum testosterone was associated with healthier values of most cardiovascular disease risk factors but not with death from ischaemic heart disease or stroke, adjusted for age, education, race/ethnicity, smoking and alcohol use. Similarly adjusted, androstanediol glucuronide was associated with unhealthier values of some cardiovascular risk factors and death from ischaemic heart disease (hazard ratio 1·16, 95% confidence interval 1·003-1·33 per standard deviation)., Conclusions: Androgen biomarkers had inconsistent associations with cardiovascular disease risk factors and ischaemic heart disease. Androstanediol glucuronide, rather than serum testosterone, had associations with cardiovascular disease risk factors more similar to those seen in randomized controlled trials of testosterone therapy, with corresponding implications for raising androgens., (© 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Rationale: Neurosteroids are steroids synthesized within the brain with rapid effects on neuronal excitability. Allopregnanolone, allotetrahydrodeoxycorticosterone, and androstanediol are three widely explored prototype endogenous neurosteroids. They have very different targets and functions compared to conventional steroid hormones. Neuronal γ-aminobutyric acid (GABA) type A (GABA(A)) receptors are one of the prime molecular targets of neurosteroids., Objective: This review provides a critical appraisal of recent advances in the pharmacology of endogenous neurosteroids that interact with GABA(A) receptors in the brain. Neurosteroids possess distinct, characteristic effects on the membrane potential and current conductance of the neuron, mainly via potentiation of GABA(A) receptors at low concentrations and direct activation of receptor chloride channel at higher concentrations. The GABA(A) receptor mediates two types of inhibition, now characterized as synaptic (phasic) and extrasynaptic (tonic) inhibition. Synaptic release of GABA results in the activation of low-affinity γ2-containing synaptic receptors, while high-affinity δ-containing extrasynaptic receptors are persistently activated by the ambient GABA present in the extracellular fluid. Neurosteroids are potent positive allosteric modulators of synaptic and extrasynaptic GABA(A) receptors and therefore enhance both phasic and tonic inhibition. Tonic inhibition is specifically more sensitive to neurosteroids. The resulting tonic conductance generates a form of shunting inhibition that controls neuronal network excitability, seizure susceptibility, and behavior., Conclusion: The growing understanding of the mechanisms of neurosteroid regulation of the structure and function of the synaptic and extrasynaptic GABA(A) receptors provides many opportunities to create improved therapies for sleep, anxiety, stress, epilepsy, and other neuropsychiatric conditions.

Androst-5-ene-3β,17β-diol (ADIOL) and 5α-androstane-3β,17β-diol (3β-DIOL), metabolites of dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), respectively, are known to possess estrogenic properties. To better understand their hormonal action and roles in the proliferation of breast cancer (BC) cells, we studied their binding to sex-hormone receptors in estrogen receptor (ER)-positive (ZR-75-1 and T-47D) and ER-negative (MDA-MB-231) human BC cells. The results demonstrated that estradiol (E2), ADIOL and 3β-DIOL stimulated the proliferation of ZR-75-1 and T-47D cells, but had no effect on ER-negative cells. In the presence of estradiol, ADIOL and 3β-DIOL inhibited the estrogen-stimulated BC cell growth. This inhibition was counteracted by anti-androgens, which were unable to affect the ADIOL and 3β-DIOL stimulatory effects in E2-free medium. On the other hand, in the presence of tamoxifen, ADIOL and 3β-DIOL showed an additional anti-proliferative activity on hormone-sensitive BC cells compared with tamoxifen treatment alone. These results are similar to previous reports obtained using MCF-7 cells, which confirmed that ADIOL and 3β-DIOL stimulated estrogen-dependent BC cell growth via ERs, but inhibited growth via androgen receptors (ARs). Several steroids bind to both ER and AR in a different preference and degree, i.e. E2>estrone (E1)>ADIOL>3β-DIOL>testosterone (T)>DHT for ER and DHT>T>3β-DIOL>ADIOL>E1>E2 for AR. The relative binding affinities of ADIOL, 3β-DIOL, and E2 corresponded well to their respective potential in stimulating cell proliferation of ZR-75-1 and T-47D cells in our results. The intrinsic relationship between cell proliferation effects and binding affinities for receptors of several steroids was revealed here by a combined binding and cell study. This article is part of a Special Issue entitled 'Synthesis and biological testing of steroid derivatives as inhibitors'., (Copyright © 2013. Published by Elsevier Ltd.)

Aromatase inhibitors (AIs) have been reported to exert their antiproliferative effects in postmenopausal women with hormone receptor-positive breast cancer not only by reducing estrogen production but also by unmasking the inhibitory effects of androgens such as testosterone (TS) and dihydrotestosterone (DHT). However, the role of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-Androstane-3β,17β-diol (3β-diol) generated from DHT by 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) shows androgenic and substantial estrogenic activities, representing a potential mechanism of AI resistance. Estrogen response element (ERE)-green fluorescent protein (GFP)-transfected MCF-7 breast cancer cells (E10 cells) were cultured for 3 months under steroid-depleted, TS-supplemented conditions. Among the surviving cells, two stable variants showing androgen metabolite-dependent ER activity were selected by monitoring GFP expression. We investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms in these variant cell lines. The variant cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes after stimulation with androgens or 3β-diol. Further analysis suggested that increased expression of HSD3B1 and reduced expression of androgen receptor (AR) promoted adaptation to androgen-abundant conditions, as indicated by the increased conversion of DHT into 3β-diol by HSD3B1 and AR signal reduction. Furthermore, in parental E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to androgen-abundant conditions. Coculture with stromal cells to mimic local estrogen production from androgens reduced cell sensitivity to AIs compared with parental E10 cells. These results suggest that increased expression of HSD3B1 and reduced expression of AR might reduce the sensitivity to AIs as demonstrated by enhanced androgen metabolite-induced ER activation and growth mechanisms. Androgen metabolite-dependent growth of breast cancer cells may therefore play a role in AI-resistance.

A deproteinized natural cancellous bone mineral (B) was studied in a cell culture model for its anabolic potential using two radiolabelled steroid substrates, 14C-testosterone (14C-T) and 14C-4-androstenedione (14C-4-A) independently; in the presence or absence of the anti-androgen finasteride (F) and minocycline (M). Culture medium was assayed for the biologically active metabolite 5 alpha-dihydrotestosterone (DHT) a marker of regenerative potential and wound healing. Confluent monolayer cultures of human periosteal fibroblasts were incubated in Eagle's minimum essential medium with each of the substrates 14C-T and 14C-4-A. Incubations were performed with previously established optimal concentrations of B5 (milligrams/ml), M25 (μg/ml) and F5 (μg/ml) alone and in combination (n=6) for 24h. The eluent was solvent extracted with ethyl acetate (2 ml x 2) and subjected to TLC in a benzene/acetone solvent system (4:1 v/v) for separation of metabolites; they were quantified using a radioisotope scanner. The yield of DHT was increased over controls in response to B and M with both substrates 14C-T and 14C-4-A by 1.7, 1.8-fold and 1.7, 1.6-fold respectively (n=6; p<0.001; one way ANOVA). Combined incubations of B and M resulted in similar yields. F inhibited DHT yields with both radiolabelled substrates by 2-3-fold (n=6; p<0.001) which was overcome by a combined incubation of F+B to values similar to those of controls (p<0.01). Documented pro-anabolic effects of minocycline were applicable as a standard for confirmation of responses to B. Significant increases in yields of DHT in response to B and M with both substrates indicate their anabolic potential in periosteal fibroblasts with implications for wound healing., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Testosterone has been shown to suppress the acute stress-induced activation of the hypothalamic-pituitary-adrenal axis; however, the mechanisms underlying this response remain unclear. The hypothalamic-pituitary-adrenal axis is regulated by a neuroendocrine subpopulation of medial parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN). These neurons are devoid of androgen receptors (ARs). Therefore, a possibility is that the PVN target neurons respond to a metabolite in the testosterone catabolic pathway via an AR-independent mechanism. The dihydrotestosterone metabolite, 5α-androstane-3β,17β-diol (3β-diol), binds and activates estrogen receptor-β (ER-β), the predominant ER in the PVN. In the PVN, ER-β is coexpressed with oxytocin (OT). Therefore, we tested the hypothesis that 3β-diol regulates OT expression through ER-β activation. Treatment of ovariectomized rats with estradiol benzoate or 3β-diol for 4 days increased OT mRNA selectively in the midcaudal, but not rostral PVN compared with vehicle-treated controls. 3β-Diol treatment also increased OT mRNA in the hypothalamic N38 cell line in vitro. The functional interactions between 3β-diol and ER-β with the human OT promoter were examined using an OT promoter-luciferase reporter construct (OT-luc). In a dose-dependent manner, 3β-diol treatment increased OT-luc activity when cells were cotransfected with ER-β, but not ER-α. The 3β-diol-induced OT-luc activity was reduced by deletion of the promoter region containing the composite hormone response element (cHRE). Point mutations of the cHRE also prevented OT-luc activation by 3β-diol. These results indicate that 3β-diol induces OT promoter activity via ER-β-cHRE interactions.

A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR.

Intratumoral synthesis of dihydrotestosterone (DHT) from precursors cannot completely explain the castration resistance of prostate cancer. We showed that DHT was intratumorally synthesized from the inactive androgen metabolites 5α-androstane-3α/β,17β-diol (3α/β-diol) in prostate cancer cells via different pathways in a concentration-dependent manner. Additionally, long-term culture in androgen-deprived media increased transcriptomic expression of 17β-hydroxysteroid dehydrogenase type 6 (HSD17B6), a key enzyme of oxidative 3α-HSD that catalyzes the conversion of 3α-diol to DHT in prostate cancer cells. Correspondingly, the score for HSD17B6 in tissues of 42 prostate cancer patients undergoing androgen deprivation therapy (ADT) was about 2-fold higher than that in tissues of 100 untreated individuals. In men receiving ADT, patients showing biochemical progression had a higher HSD17B6 score than those without progression. These results suggested that 3α/β-diol also represent potential precursors of DHT, and the back conversion of DHT from androgen derivatives can be a promising target for combination hormone therapy.

P-glycoprotein (Pgp), a multiple drug resistance transporter expressed by vascular endothelial cells, is a key component of the blood-brain barrier and has been shown to increase after inflammation. The nonaromatizable androgen, dihydrotestosterone (DHT), decreases inflammatory markers in vascular smooth muscle cells, independent of androgen receptor (AR) stimulation. The principal metabolite of DHT, 5α-androstane-3β,17β-diol (3β-diol), activates estrogen receptor (ER)β and similarly decreases inflammatory markers in vascular cells. Therefore, we tested the hypothesis that either DHT or 3β-diol decrease cytokine-induced proinflammatory mediators, vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2), to regulate Pgp expression in male primary human brain microvascular endothelial cells (HBMECs). Using RT-qPCR, the mRNAs for AR, ERα, and ERβ and steroid metabolizing enzymes necessary for DHT conversion to 3β-diol were detected in male HBMECs demonstrating that the enzymes and receptors for production of and responsiveness to 3β-diol are present. Western analysis showed that 3β-diol reduced COX-2 and Pgp expression; the effect on Pgp was inhibited by the ER antagonist, ICI-182,780. IL-1β-caused an increase in COX-2 and VCAM-1 that was reduced by either DHT or 3β-diol. 3β-diol also decreased cytokine-induced Pgp expression. ICI-182,780 blocked the effect of 3β-diol on COX-2 and VCAM-1, but not Pgp expression. Therefore, in cytokine-stimulated male HBMECs, the effect of 3β-diol on proinflammatory mediator expression is ER dependent, whereas its effect on Pgp expression is ER independent. These studies suggest a novel role of 3β-diol in regulating blood-brain barrier function and support the concept that 3β-diol can be protective against proinflammatory mediator stimulation.

The endocrine component of the stress response is regulated by glucocorticoids and sex steroids. Testosterone down-regulates hypothalamic-pituitary-adrenal (HPA) axis activity; however, the mechanisms by which it does so are poorly understood. A candidate testosterone target is the oxytocin gene (Oxt), given that it too inhibits HPA activity. Within the paraventricular nucleus of the hypothalamus, oxytocinergic neurons involved in regulating the stress response do not express androgen receptors but do express estrogen receptor-β (ERβ), which binds the dihydrotestosterone metabolite 3β,17β-diol (3β-diol). Testosterone regulation of the HPA axis thus appears to involve the conversion to the ERβ-selective ligand 5α-androstane, 3β-diol. To study mechanisms by which 3β-diol could regulate Oxt expression, we used a hypothalamic neuronal cell line derived from embryonic mice that expresses Oxt constitutively and compared 3β-diol with estradiol (E2) effects. E2 and 3β-diol elicited a phasic response in Oxt mRNA levels. In the presence of either ligand, Oxt mRNA levels were increased for at least 60 min and returned to baseline by 2 h. ERβ occupancy preceded an increase in Oxt mRNA levels in the presence of 3β-diol but not E2. In tandem with ERβ occupancy, 3β-diol increased occupancy of the Oxt promoter by cAMP response element-binding protein and steroid receptor coactivator-1 at 30 min. At the same time, 3β-diol led to the increased acetylation of histone H4 but not H3. Taken together, the data suggest that in the presence of 3β-diol, ERβ associates with cAMP response element-binding protein and steroid receptor coactivator-1 to form a functional complex that drives Oxt gene expression.

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report, we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17β-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17β-HSD6 in prostate-derived cell lines and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor-bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis., (©2011 AACR.)

Aim: The purpose of this study was to provide a quantitative assessment of female rat sexual behaviors after acute exposure to the A-ring reduced testosterone metabolite, androstanediol (3α-Diol), through the nucleus accumbens (NA) shell., Main Outcome Measures: Quantitative analyses of female rat sexual behaviors and assessment of protein levels for the enzyme glutamic acid decarboxylase isoform 67 (GAD67) and gephyrin, a protein that participates in the clustering of GABA-A receptors in postsynaptic cells, were accomplished., Methods: Female rats were ovariectomized and primed with estrogen and progesterone to induce sexual behaviors. Females received a 3α-Diol infusion via guided cannula that aimed to the NA shell five minutes prior to a sexual encounter with a stud male. The following parameters were videotaped and measured in a frame by frame analysis: lordosis quotient (LQ), Lordosis rating (LR), frequency and duration of proceptive behaviors (hopping/darting and ear wiggling). Levels of GAD67 and gephyrin were obtained by Western blot analysis two or twenty-four hours after the sexual encounter., Results: Acute exposure to 3α-Diol in the NA shell enhanced LR, ear wiggling, and hopping/darting but not LQ. Some of these behavioral effects were counteracted by co-infusion of 3α-Diol plus the GABAA-receptor antagonist GABAzine. A transient reduction of GAD67 levels in the NA shell was detected., Conclusions: The testosterone metabolite 3α-Diol enhances sexual proceptivity, but not receptivity, when infused into the NA shell directly. The GABAergic system may participate in the androgen-mediated enhancement of female rat sexual motivation., (© 2010 International Society for Sexual Medicine.)

Four isomers of 5α-androstan-3,17-diol have been transformed by the filamentous fungus Aspergillus tamarii, an organism which has the ability to convert progesterone to testololactone in high yield through an endogenous four step enzymatic pathway. The only diol handled within the lactonization pathway was 5α-androstan-3α,17β-diol which, uniquely underwent oxidation of the 17β-alcohol to the 17-ketone prior to its Baeyer-Villiger oxidation and the subsequent production of 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one. This demonstrated highly specific stereochemical requirements of the 17β-hydroxysteroid dehydrogenase for oxidation of this specific steroidal diol to occur. In contrast, the other three diols were transformed within the hydroxylation pathway resulting in functionalization at C-11β. Only 5α-androstan-3β,17α-diol could bind to the hydroxylase in multiple binding modes undergoing monohydroxylation in 6β and 7β positions. Evidence from this study has indicated that hydroxylation of saturated steroidal lactones may occur following binding of ring-D in its open form in which an α-alcohol is generated with close spatial parity to the C-17α hydroxyl position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR and DEPT analysis and further characterized using infra-red, elemental analysis and accurate mass measurement., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

17beta-Hydroxysteroid dehydrogenase type 7 (17beta-HSD7) catalyzes the reduction of estrone (E(1)) into estradiol (E(2)) and of dihydrotestosterone (DHT) into 5alpha-androstane-3beta,17beta-diol (3beta-diol), therefore modulating the level of mitogenic estrogens and androgens in humans. By classical and parallel chemistry, we generated several 4-methyl-4-aza-5alpha-androstane derivatives differing in their C-17 substituent: 17beta-formamide, 17beta-benzamide, and 17beta-tertiary amine. Best candidates in each category had demonstrated good inhibitory potency toward the conversion of E(1) into E(2) (IC(50) = 189-451 nM) and also toward the conversion of DHT into 3beta-diol (69-91% at 3 microM). Inhibition assays with 17beta-HSD1, 17beta-HSD5, 5alpha-reductase (5alpha-R) 1 and 5alpha-R2 revealed that 17beta-HSD7 inhibitors with a 4-methyl-4-aza nucleus were also able to inhibit 5alpha-Rs but not the other enzymes tested. Two 4-aza-5alpha-androstane inhibitors were, however, selective and still showed good inhibition of 17beta-HSD7. First selective and efficient inhibitors of 17beta-HSD7 are now available for additional mechanistic and therapeutic studies.

The current study presents data indicating that 5alpha-androstane-3alpha,17beta-diol (3alpha-Adiol) undergoes a previously unknown metabolism into hydroxymetabolites, catalyzed by CYP7B1. 3alpha-Adiol is an androgenic steroid which serves as a source for the potent androgen dihydrotestosterone and also can modulate gamma-amino butyric acid A (GABA(A)) receptor function in the brain. The steroid hydroxylase CYP7B1 is known to metabolize cholesterol derivatives, sex hormone precursors and certain estrogens, but has previously not been thought to act on androgens or 3alpha-hydroxylated steroids. 3alpha-Adiol was found to undergo NADPH-dependent metabolism into 6- and 7-hydroxymetabolites in incubations with porcine microsomes and human kidney-derived HEK293 cells, which are high in CYP7B1 content. This metabolism was suppressed by addition of steroids known to be metabolized by CYP7B1. In addition, 3alpha-Adiol significantly suppressed CYP7B1-mediated catalytic reactions, in a way as would be expected for substrates that compete for the same enzyme. Recombinant expression of human CYP7B1 in HEK293 cells significantly increased the rate of 3alpha-Adiol hydroxylation. Furthermore, the observed hydroxylase activity towards 3alpha-Adiol was very low or undetectable in livers of Cyp7b1(-/-) knockout mice. The present results indicate that CYP7B1-mediated catalysis may play a role for control of the cellular levels of androgens, not only of estrogens. These findings suggest a previously unknown mechanism for metabolic elimination of 3alpha-Adiol which may impact intracellular levels of dihydrotestosterone and GABA(A)-modulating steroids.

It is well known that fluoxetine (Fluox), a selective serotonin reuptake inhibitor, increases the brain content of allopregnanolone (AP), a potent neuroactive steroid that positively modulates the action of gamma-aminobutyric acid (GABA) at the GABA type A receptors, but the influence of Fluox on the brain and serum levels of a neuroactive androgen, 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), is poorly understood. In this study, we examined the Fluox-evoked changes in the 3alpha,5alpha-Adiol levels and compared the level changes of 3alpha,5alpha-Adiol with those of AP. The brain and serum 3alpha,5alpha-Adiol and AP levels were determined using previously developed LC-MS/MS. The ratio of the brain 3alpha,5alpha-Adiol to the serum 3alpha,5alpha-Adiol concentrations (B/S value) was significantly elevated in the rats intraperitoneally administered Fluox (10 mg/kg). Although the magnitude of the Fluox-evoked level change in 3alpha,5alpha-Adiol was much lower than that in AP, this study demonstrated that the 3alpha,5alpha-Adiol content is also influenced by Fluox. The most probable cause for the increase in the B/S value by the Fluox treatment is the activation of the 3alpha-hydroxysteroid dehydrogenase enzyme followed by the promotion of the de novo biosynthesis of 3alpha,5alpha-Adiol in the brain.

Aldo-keto reductase (AKR) 1C2 is a human, cytosolic enzyme that has an important role in the deactivation of the potent androgen dihydrotestosterone (DHT). AKR1C2 can regulate the extent and duration of activation of the androgen receptor by catalyzing the reduction of DHT to the less potent receptor ligand 3alpha-diol. In this study, we functionally characterize in vitro the effect of 11 naturally occurring nonsynonymous single nucleotide polymorphisms on the ability of AKR1C2 to reduce DHT to 3alpha-diol. The wild-type and variant enzymes were expressed using a transfected insect cell system, and their kinetic activities were measured using both a specific fluorogenic probe and DHT as substrates. This functional characterization demonstrates that several variant AKR1C2 proteins have significantly reduced or altered reductase activities as shown by their measured kinetic parameters. Data from our two separate in vitro studies revealed significant reductions in V(max) for two variants (F46Y and L172Q) and significantly lower apparent K(m) values for three variants (L172Q, K185E, and R258C) compared with the wild type. These results provide evidence that several naturally occurring nonsynonymous single nucleotide polymorphisms in AKR1C2 result in reduced enzyme activities. These variant AKR1C2 alleles may represent one factor involved in the variable degradation of DHT in vivo.

The aromatase inhibitors (AIs) are used to treat estrogen receptor-positive (ER+) breast tumors in post-menopausal women, and function by blocking the conversion of adrenal androgens to estrogens by the enzyme CYP19 aromatase. Breast cancer patients receiving AI therapy have circulating estrogen levels below the level of detection; however, androgen concentrations remain unchanged. We were interested in studying the effects of androgens on breast cancer cell proliferation under profound estrogen-deprived conditions. Using in vitro models of estrogen-dependent breast cancer cell growth we show that the androgens testosterone and 5alpha-dihydrotestosterone induce the growth of MCF-7, T47D and BT-474 cells in the absence of estrogen. Furthermore, we demonstrate that under profound estrogen-deprived conditions these breast cancer cells up-regulate steroidogenic enzymes that can metabolize androgens to estrogens. Lastly, we found that the downstream metabolite of 5alpha-dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), is estrogenic in breast cancer cells, and induces growth and ER-signaling via activation of ERalpha. In conclusion, our results show that breast cancer cells deprived of estrogen up-regulate steroidogenic enzymes and metabolize androgens to estrogen-like steroids. The generation of estrogen-like steroids represents a potential mechanism of resistance to aromatase inhibitors.

Cytochrome P4507B1 7alpha-hydroxylates dehydroepiandrosterone (DHEA), epiandrosterone (EpiA) and 5alpha-androstane-3beta,17beta-diol (Adiol). 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) interconverts 7alpha- and 7beta-forms. Whether the interconversion proceeds through oxido-reductive steps or epimerase activity was investigated. Experiments using [(3)H]-labelled 7beta-hydroxy-DHEA, 7beta-hydroxy-EpiA and 7beta-hydroxy-Adiol showed the (3)H-label to accumulate in the 7-oxo-DHEA trap but not in 7-oxo-EpiA or 7-oxo-Adiol traps. Computed models of 7-oxygenated steroids docked in the active site of 11beta-HSD1 either in a flipped or turned form relative to cortisone and cortisol. 7-Oxo-steroid reduction in 7alpha- or 7beta-hydroxylated derivatives resulted from either turned or flipped forms. 11beta-HSD1 incubation in H(2)(18)O medium with each 7-hydroxysteroid did not incorporate (18)O in 7-hydroxylated derivatives of EpiA and Adiol independently of the cofactor used. Thus oxido-reductive steps apply for the interconversion of 7alpha- and 7beta-hydroxy-DHEA through 7-oxo-DHEA. Epimerization may proceed on the 7-hydroxylated derivatives of EpiA and Adiol through a mechanism involving the cofactor and Ser(170). The physiopathological importance of this epimerization process is related to 7beta-hydroxy-EpiA production and its effects in triggering the resolution of inflammation.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is a basic response of animals to environmental perturbations that threaten homeostasis. These responses are regulated by neurones in the paraventricular nucleus of the hypothalamus (PVN) that synthesise and secrete corticotrophin-releasing hormone (CRH). Other PVN neuropeptides, such as arginine vasopressin and oxytocin, can also modulate activity of CRH neurones in the PVN and enhance CRH secretagogue activity of the anterior pituitary gland. In rodents, sex differences in HPA reactivity are well established; females exhibit a more robust activation of the HPA axis after stress than do males. These sex differences primarily result from opposing actions of sex steroids, testosterone and oestrogen, on HPA function. Ostreogen enhances stress activated adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) secretion, whereas testosterone decreases the gain of the HPA axis and inhibits ACTH and CORT responses to stress. Data show that androgens can act directly on PVN neurones in the male rat through a novel pathway involving oestrogen receptor (ER)beta, whereas oestrogen acts predominantly through ERalpha. Thus, we examined the hypothesis that, in males, testosterone suppresses HPA function via an androgen metabolite that binds ERbeta. Clues to the neurobiological mechanisms underlying such a novel action can be gleaned from studies showing extensive colocalisation of ERbeta in oxytocin-containing cells of the PVN. Hence, in this review, we address the possibility that testosterone inhibits HPA reactivity by metabolising to 5alpha-androstane-3beta,17beta-diol, a compound that binds ERbeta and regulates oxytocin containing neurones of the PVN. These findings suggest a re-evaluation of studies examining pathways for androgen receptor signalling.

Testicular 5alpha-reduced androgens, largely 5alpha-androstane-3alpha,17beta-diol (androstanediol), are responsible for virilisation of pouch young in one marsupial (the tammar wallaby), but are not formed until later in development in another marsupial (the brushtail possum) and in rodents. Because the mechanism of virilisation of the urogenital tract in the grey short-tailed opossum Monodelphis domestica has never been defined, androgen formation and metabolism were investigated in this species. Testis fragments from grey short-tailed opossums of a wide range of ages were incubated with [3H]-progesterone and the metabolites were separated by high-performance liquid chromatography (HPLC). The only 19-carbon metabolites identified in the youngest ages (5-26 days) and the major metabolites in adult testes were testosterone and androstenedione. At 30, 42 and 49 days of age, dihydrotestosterone and small amounts of androstanediol were present. Time-sequence studies indicated that dihydrotestosterone and androstanediol were formed from the 5alpha-reduction (and 3-keto reduction) of testosterone. In a second series of experiments, tissue fragments of a variety of urogenital tract tissues were incubated with [3H]-testosterone and the metabolites separated by HPLC. During the interval in which male urogenital tract differentiation takes place in this species (between Days 15 and 28), the major metabolite identified was dihydrotestosterone. We conclude that the timing of 5alpha-reductase expression in the testes of the grey short-tailed possum resembles that of rodents and the brushtail possum rather than that of the tammar wallaby and that dihydrotestosterone is probably the intracellular androgen responsible for virilisation of the urogenital tract in this species.

Di-2-ethylhexyl (DEHP) phthalate, one of the phthalates most widely distributed in our general environment, causes reproductive toxicity that is attributable to the action of its primary metabolite, mono(2-ethylhexyl) phthalate (MEHP). Here, we have investigated the effects of MEHP on steroidogenesis by primary cultures of immature and adult rat Leydig cells. In both cases MEHP (250muM) was found to inhibit stimulation of androgen production evoked by human chorionic gonadotropin (hCG). This was associated with decreased expression of steroidogenic acute regulatory (StAR) protein and reduced transport of cholesterol into mitochondria but no detectable adverse effect on steroidogenic enzymes. Moreover, upon exposure to MEHP alone, 5alpha-reductase activity was decreased in immature, but not in adult Leydig cells. All together, our findings demonstrate that MEHP exerts suppressive effects on hCG-activated steroidogenesis in primary cultures of immature and adult rat Leydig cells and suppresses 5alpha-reductase activity in immature and not of adult rat cells. This may partly explain the anti-androgenic effects of DEHP in vivo and indicate a higher susceptibility in younger subjects.

Objective: Testosterone is a commonly abused androgen in sports and in the gym culture of the society. Its abuse is conventionally disclosed by urinary assay of the testosterone/epitestosterone (T/E) glucuronide ratio, which should not exceed 4. A noteworthy number of athletes, however, have higher natural ratios than 4, most likely because of decreased excretion of epitestosterone glucuronide. Falsely positive doping test results are of great concern for the legal rights of the sportsman. Our objective was to study the genetic aspects of epitestosterone formation, and to elucidate the impact of genetic variation in androgen-metabolizing enzymes., Methods: Urine from different study populations was analysed for androgen glucuronides by gas chromatography-mass spectrometry. All men were genotyped for the uridine diphospho-glucuronosyltransferase (UGT) 2B17 deletion polymorphism and single nucleotide polymorphisms in the cytochrome P-450c17alpha (CYP17), UGT2B15 and UGT2B7 genes. Expression of UGT2B15 mRNA in human liver samples was analysed using real-time PCR., Results: A T>C (A1>A2) promoter polymorphism in the CYP17 gene was associated with the urinary glucuronide levels of epitestosterone and its putative precursor androstene-3beta, 17alpha-diol, resulting in 64% higher T/E ratios in A1/A1 homozygotes. Individuals devoid of UGT2B17 had significantly higher UGT2B15 mRNA levels in liver than individuals carrying two functional UGT2B17 alleles., Conclusion: The CYP17 promoter polymorphism may partly explain high natural (>4) T/E ratios. Our data indicate that 5-androstene-3beta, 17alpha-diol is an important precursor of epitestosterone and that CYP17 is involved in its production. In addition, we found that lack of the UGT2B17 enzyme may be compensated for by increase in UGT2B15 transcription.

Healthy postmenopausal women aged 60-65 years (n=150) were randomized to receive twice daily application on the skin of 3g of a 0.3% dehydroepiandrosterone (DHEA) or placebo emulsion for 12 months. Serum DHEA and eleven of its metabolites were measured at screening and on day 1, as well as at 1, 3, 6, 9 and 12 months to study long-term metabolism. While serum DHEA and androst-5-ene-3beta, 17beta-diol (5-diol) increased by 203% and 178%, respectively, on average, during the 12-month period, the sum of concentrations of the metabolites of androgens, namely androsterone glucuronide (ADT-G), androstane-3alpha,17beta-diol-3G and -17G increased by only 71% while usually non statistically significant changes of 30%, 17% and 20% were observed for estrone (E(1)), estradiol (E(2)) and E(1) sulfate (E(1)-S), respectively. Despite the return of serum DHEA to normal premenopausal values with the present DHEA treatment regimen, the 65% decrease in the androgen pool found in this group of postmenopausal women is in fact corrected by only 24%, thus remaining 41% below the values found in normal premenopausal women. In fact, the changes in serum DHEA observed after percutaneous DHEA administration are a 186% overestimate of the true changes in androgen formation while the overestimate of estrogen production is even much higher. On the other hand, the pharmacokinetics of the steroids are stable over the 12-month period with no significant induction or decrease of activity of the enzymatic systems transforming DHEA predominantly into androgens.

CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metabolism involving dehydroepiandrosterone or 3beta-Adiol may play an important role for estrogen receptor beta-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor beta activation. In the present study, we investigated CYP7B1-mediated conversions of dehydroepiandrosterone and 3beta-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3beta-Adiol (a CYP7B1 substrate) and 7alpha-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor beta activation. The data obtained indicated that 3beta-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor beta activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehydroepiandrosterone and 3beta-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the K(cat)/K(m) values were in the same order of magnitude. A high dehydroepiandrosterone/3beta-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3beta-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3beta-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-mediated metabolism of dehydroepiandrosterone or 3beta-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence estrogen receptor beta signaling.

Steroid hormones play a key role in the pathophysiology of several brain disorders. Testosterone modulates neuronal excitability, but the underlying mechanisms are obscure. There is emerging evidence that testosterone-derived "androgenic neurosteroids", 3alpha-androstanediol and 17beta-estradiol, mediate the testosterone effects on neural excitability and seizure susceptibility. Testosterone undergoes metabolism to neurosteroids via two distinct pathways. Aromatization of the A-ring converts testosterone into 17beta-estradiol. Reduction of testosterone by 5alpha-reductase generates 5alpha-dihydrotestosterone, which is then converted to 3alpha-androstanediol, a powerful GABA(A) receptor-modulating neurosteroid with anticonvulsant properties. Although the 3alpha-androstanediol is an emerging neurosteroid in the brain, there is no specific and sensitive assay for determination of 3alpha-androstanediol in biological samples. This article describes the development and validation of mass spectrometric assay of 3alpha-androstanediol, and the molecular mechanisms underlying the testosterone modulation of seizure susceptibility. A liquid chromatography-tandem mass spectrometry assay to measure 3alpha-androstanediol is validated with excellent linearity, specificity, sensitivity, and reproducibility. Testosterone modulation of seizure susceptibility is demonstrated to occur through its conversion to neurosteroids with "anticonvulsant" and "proconvulsant" actions and hence the net effect of testosterone on neural excitability and seizure activity depends on the levels of distinct testosterone metabolites. The proconvulsant effect of testosterone is associated with increases in plasma 17beta-estradiol concentrations. The 5alpha-reduced metabolites of testosterone, 5alpha-dihydrotestosterone and 3alpha-androstanediol, had powerful anticonvulsant activity. Overall, the testosterone-derived neurosteroids 3alpha-androstanediol and 17beta-estradiol could contribute to the net cellular actions of testosterone in the brain. Because 3alpha-androstanediol is a potent positive allosteric modulator of GABA(A) receptors, it could serve as an endogenous neuromodulator of neuronal excitability in men. The 3alpha-androstanediol assay is an important tool in this area because of the growing interest in the potential to use adjuvant aromatase inhibitor therapy to improve treatment of epilepsy.

Prostate is one of the major targets for dihydrotestosterone (DHT), however this gland is also recognized as a nonclassical target for estrogen as it expresses both types of estrogen receptors (ER), especially ERbeta. Nevertheless, the concentrations of aromatase and estradiol in the prostate are low, indicating that estradiol may not be the only estrogenic molecule to play a role in the prostate. It is known that DHT can be metabolized to 5alpha-androstane-3beta,17beta-diol (3beta-diol), a hormone that binds to ERbeta but not to AR. The concentration of 3beta-diol in prostate is much higher than that of estradiol. Based on the high concentration of 3beta-diol and since this metabolite is a physiological ERbeta ligand, we hypothesized that 3beta-diol would be involved in the regulation of ERbeta expression. To test this hypothesis, adult male rats were submitted to castration followed by estradiol, DHT or 3beta-diol replacement. ERbeta and AR protein levels in the prostate were investigated by immunohistochemistry and Western blotting assays. The results showed that after castration, the structure of the prostate was dramatically changed and ERbeta and AR protein levels were decreased. Estradiol had just minor effects on the parameters analyzed. DHT-induced partial recovery of ERbeta while it was the most effective inductor of AR expression. Replacement with 3beta-diol-induced the highest levels of ERbeta, but was comparatively less effective in recovering the AR expression and the gland structure. These results offer evidence that one functional role of 3beta-diol in the prostate may be autoregulation of its natural receptor, ERbeta.

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder associated with reduced very long-chain fatty acid beta-oxidation, mainly affecting the nervous system, the adrenal cortex and the testes. The clinical manifestations of hypogonadism, alopecia and the impairment of the enzyme 5alpha-reductase, which converts testosterone into dihydrotestosterone, clearly point to an involvement of androgens in this pathology. The disease is characterized by mutations in the ABCD1 gene, which codes for the peroxisomal ABC half-transporter ALDP, and by a broad range of clinical manifestations. The altered function of ALDP can be compensated by the overexpression of proteins belonging to the same family of ABC half-transporters. A promising therapeutic approach is represented by the activation of these proteins by specific agonists. In this study we evaluated the effect of the testosterone metabolite dihydrotestosterone (DHT) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) on the expression of the ABC half-transporters encoded by the ABCD2 and ABCD3 genes, in fibroblasts drawn from controls and from two affected brothers. The two patients presented the same mutation in exon 9 but had different clinical manifestations, one patient being asymptomatic and the second one severely affected. When the cells were stimulated with testosterone metabolites, only the severely affected patient showed a significant increase in ABCD2 mRNA levels, while the ABCD3 expression remained unchanged in both patients.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of the rat brain 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol) has been developed and validated. The brain extract was purified using solid-phase extraction cartridges, derivatized with isonicotinoyl azide, and subjected to LC-MS/MS. The method was accurate and reproducible, and the limit of quantitation was 0.1 ng/g tissue when a 100-mg tissue sample was used. The change in the brain 3alpha,5alpha-Adiol level by immobilization stress was also analyzed using the developed method.

Objective: The study objective was to assess the role of 3alpha-androstanediol glucuronide (3alpha-diolG) as the marker of peripheral androgen action in the young women with hirsutism diagnosed as polycystic ovary syndrome (PCOS), in patients with idiopathic hirsutism (IH) and in normal non-hirsute women., Study Design: Fifty-nine young women with mean age 21.90+/-3.52 years suffered from hirsutism were included in the study. Among these 59 hirsute women 31 women with mean age 21.60+/-3.56 years were diagnosed as PCOS and 28 women with mean age 22.20+/-3.59 years were classified as idiopathic hirsutism patients. Twenty-seven normal women, age-matched (mean age 22.60+/-2.90 years), without signs of hirsutism and with normal menstrual cycle served as control for this study. Serum was collected from women with hirsutism (due to PCOS or idiopathic hirsutism) and from non-hirsute women. Serum levels of 3alpha-androstanediol glucuronide (3alpha-diolG), main androgens such as: testosterone, free testosterone, dehydroepiandrosterone (DHEAS) and also others hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and sex hormone-binding globuline (SHBG) were measured using radioimmunoassay (RIA) method in the study and control group. Hirutism was assessed using modified Ferriman-Gallwey method. Serum 3alpha-diolG levels in PCOS women were significantly higher than in controls., Results: There were no significant differences between serum 3alpha-diolG levels in PCOS group and IH group. Similarly, there were significant differences between serum 3alpha-diolG levels in IH group and control subjects., Conclusion: We conclude that 3alpha-diolG is not useful as the marker for peripheral androgen metabolism and for differentiation between idiopathic hirsutism and PCO-related hirsutism.

We examined 5alpha-dihydrotestosterone (5alpha-DHT) inactivation and the expression of several steroid-converting enzymes with a focus on aldoketoreductases 1C (AKR1C), especially AKR1C2, in abdominal adipose tissue in men. AKR1C2 is mainly involved in the conversion of the potent androgen 5alpha-DHT to its inactive forms 5alpha-androstane-3alpha/beta,17beta-diol (3alpha/beta-diol). Subcutaneous (s.c.) and omental (Om) adipose tissue biopsies were obtained from 21 morbidly obese men undergoing biliopancreatic derivation surgery and 11 lean to obese men undergoing general abdominal surgery. AKR1C2 mRNA and 5alpha-DHT inactivation were detected in both s.c. and Om adipose tissue. After incubation of preadipocytes with 5alpha-DHT, both 3alpha-diol and 3beta-diol were produced through 3alpha/beta-ketosteroid reductase (3alpha/beta-HSD) activity. In preadipocyte cultures, 3alpha-reductase activity was significantly predominant over 3beta-reductase activity in cells from both the s.c. and Om compartments. Expression levels of AKR1C1, AKR1C3 and of the androgen receptor were significantly higher in s.c. versus Om adipose tissue while mRNA levels of 17beta-HSD-2 (hydroxysteroid dehydrogenase type 2) and 3(alpha-->beta)-hydroxysteroid epimerase were significantly higher in Om fat. 3Alpha/beta-HSD activity was mainly detected in the cytosolic fraction, suggesting that AKR1C may be responsible for this reaction. Experiments with isoform-specific AKR1C inhibitors in preadipocytes showed that AKR1C2 inhibition significantly decreased 3alpha-HSD and 3beta-HSD activities (3alpha-HSD: 30 +/- 24% of control for s.c. and 32 +/- 9% of control for Om, 3beta-HSD: 44 +/- 12% of control for s.c.). When cells were incubated with both AKR1C2 and AKR1C3 inhibitors, no significant additional inhibition was observed. 5Alpha-DHT inactivation was significantly higher in mature adipocytes compared with preadipocyte cultures in s.c. adipose tissue, as expressed per microgram total protein (755 +/- 830 versus 245 +/- 151 fmol 3alpha/beta-diol per microg protein over 24 h, P < 0.05 n = 10 cultures). 5Alpha-DHT inactivation measured in tissue homogenates was significantly higher in the s.c. depot compared with Om fat (117 +/- 39 versus 79 +/- 38 fmol 3alpha/beta-diol per microg prot over 24 h, P < 0.0001). On the other hand, Om 3alpha/beta-HSD activity was significantly higher in obese men (body mass index (BMI) >or= 30 kg/m2) compared with lean and overweight men (84 +/- 37 versus 52 +/- 30 fmol 3alpha/beta-diol per microg protein over 24 h, P < 0.03). No difference was found in s.c. 3alpha/beta-HSD activity between these groups. Positive correlations were found between s.c. 5alpha-DHT inactivation rate and circulating levels of the androgen metabolites androsterone-glucuronide (r = 0.41, P < 0.02) and 3alpha-diol-glucuronide (r = 0.38, P < 0.03) and with the adrenal precursor androstenedione (r = 0.42, P < 0.02). In conclusion, androgen inactivation was detected in abdominal adipose tissue in men, with higher 3alpha/beta-HSD activity in the s.c. versus Om depot. Higher Om 5alpha-DHT inactivation rates were found in obese compared with lean men. Further studies are required to elucidate whether local androgen inactivation in abdominal adipose tissue is involved in the modulation of adipocyte metabolism and regional fat distribution in men.

Androgens as well as monohydroxy-fatty acids are implicated in the pathogenesis of prostate cancer. Like a huge variety of endo- and xenobiotics, they are eliminated as glucuronide conjugates formed by uridine diphosphate-glucuronosyltransferase (UGT) enzymes. In the present study, we observe that treatment of the prostate cancer cells LNCaP with natural and synthetic androgens, IL-1alpha, or epidermal growth factor (EGF) differently modulates the glucuronidation of androgen and bioactive lipid metabolites. Indeed, glucuronidation of 5alpha-androstane-3alpha,17beta-diol and 13-hydroxyoctadecadienoic acid was drastically reduced, whereas 12-hydroxyeicosatetraenoic acid conjugation by UGT was increased after androgen treatment. These effects reflected the reduction of UGT2B10, -B15, and -B17 enzyme expression, and the activation of the UGT2B11 gene. In human prostate epithelial cells, only UGT2B11 and -B15 mRNAs are detected and are regulated by androgens in a similar manner as in LNCaP cells. In LNCaP cells, IL-1alpha and EGF also regulate UGT2B expression in an isoform-specific manner; IL-1alpha induced UGT2B10 and reduced UGT2B17, while having no effects on UGT2B11 mRNA levels. EGF treatment resulted in a decreased UGT2B17 expression, whereas UGT2B10 and -B11 mRNA remained at their basal levels. Overall, these results demonstrate that in the human prostate, androgens do not only affect their own inactivation but also influence the levels of monohydroxy-fatty acids by regulating the expression of UGT2B enzymes in an isoform-specific manner. These differential effects of androgens, IL-1alpha, and EGF on lipid metabolism likely constitute an additional mechanism by which these endogenous factors promote prostate cancer development.

Background: Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite., Methods: To test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay., Results: Data show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay., Conclusion: This is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid.

The liquid chromatography-mass spectrometry (LC-MS) methods were developed and validated for the determination of testosterone (T) in the brain and serum of rats and of 5alpha-androstane-3alpha,17beta-diol (ADIOL), a metabolite of T, in the brain of rats. After derivatization of T with 2-hydrazino-1-methylpyridine and of ADIOL with p-nitrobenzoyl chloride, the detection sensitivities of T and ADIOL using LC-MS were increased 70- and 400-times superior to those of intact T and intact ADIOL, respectively. Those LC-MS methods are specific and reliable for the analysis of trace amounts of T and ADIOL in small amounts of samples. The animal studies using the developed methods showed that the brain and serum levels of T and the brain levels of ADIOL were not changed by stress or ethanol administration but the concentration ratio of the brain T to serum T in the stressed rats was higher than that in untreated rats. The low levels of endogenous AIDOL in brain of stressed and unrestrained rats found in this study demonstrated that the contribution to anesthetic and anxiolytic effects of ADIOL via gamma-aminobutyric acid type A receptors may be negligible.

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.

3alpha-Hydroxysteroid dehydrogenase catalyzes the transformation of 3-ketosteroids into 3alpha-hydroxysteroids, thus playing an important role in androgen and progesterone metabolism. So far, mouse cDNA and gene encoding 3alpha-HSD has not been reported. In this report, we describe the isolation of a mouse 3alpha-HSD cDNA and the characterization of its substrate specificity and tissue distribution. Sequence analysis indicates that m3alpha-HSD shares 87% amino acid identity with rat 3alpha-HSD. Cells stably transfected with this enzyme catalyze the transformation of dihydrotestosterone (DHT), 5alpha-androstanedione (5alpha-dione) and dihydroprogesterone (DHP) into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), androsterone (ADT) and 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone), respectively. Quantification of mRNA expression levels of this enzyme was determined in male and female mouse sex-specific tissues using quantitative Realtime PCR. We show that this enzyme is mainly expressed in female-specific tissues while being almost absent from male-specific tissues. In the liver, the same expression level is seen in both male and female, while there is 6-fold higher expression level in female pituitary than in male. These results strongly suggest that m3alpha-HSD could play an important role in the female mouse physiology similar to that of type 1 5alpha-reductase with which it works in tandem. This role could be related to the inactivation of excess of androgen and progesterone that are more severely regulated than in man.

Although testosterone (T) may have effects to enhance analgesia and reduce anxiety, its effects and mechanisms are not well understood. We hypothesized that if T's anti-anxiety and analgesic effects are due in part to actions of its 5alpha-reduced metabolite (dihydrotestosterone-DHT) and/or its 3alpha-hydroxysteroid dehydrogenase reduced metabolite (3alpha-androstanediol-3alpha-diol), in the hippocampus, then androgen regimens that increase levels of these metabolites in the hippocampus should produce anti-anxiety behavior, and analgesic effects, in gonadectomized (GDX) male rats. In Experiment 1, GDX rats were administered T, DHT, 3alpha-diol (1 mg/kg, SC), or vehicle. In Experiment 2, GDX rats had T, DHT, 3alpha-diol-containing inserts, or empty control inserts applied to the dorsal hippocampus immediately prior to behavioral testing. Androgen-administered rats (SC or intrahippocampal) showed significantly more exploratory behavior in the open field and elevated plus maze, less freezing in response to shock, and longer tailflick and pawlick latencies. These findings suggest that T's anti-anxiety effects may be due in part to actions of its 5alpha-reduced metabolites in the dorsal hippocampus.

The testicular androgen 5alpha;-androstane-3alpha,17beta-diol (androstanediol) mediates virilisation in pouch young of a marsupial, the tammar wallaby, and is the principal androgen formed in immature rodent testes. To chart the pattern of androstanediol formation in another marsupial species, the testes or fragments of testes from brushtail possums (Trichosurus vulpecula) that spanned the age range from early pouch young to mature adults were incubated with (3)H-progesterone and the products were identified by high-performance liquid chromatography. The only 19-carbon steroids identified in pouch young and adult testes were the Delta(4)-3-keto-steroids testosterone and androstenedione. However, androstanediol and another 5alpha-reduced androgen (androsterone) were synthesised by testes from Day 87-200 males and these appeared to be formed from the 5alpha-reduction and 3-keto reduction of testosterone and androstenedione. In the prostate and glans penis of the immature male, (3)H-androstanediol was converted to dihydrotestosterone. We conclude that the timing of androstanediol formation in the possum testis resembles the process in rodents rather than in the tammar wallaby and that any androstanediol in the circulation probably acts in target tissues via conversion to dihydrotestosterone.

3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the rate-determining step in this physiologically important reaction, rat liver 3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. Using single and multiple turnover experiments to monitor the NADPH-dependent reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 at pL 6.0, suggest that the slow chemical transformation is significantly rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, single and multiple turnover experiments showed a k(lim) and burst kinetics consistent with product release as being rate-limiting overall. When NAD(+) was substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was identical to k(cat). Thus with the physiologically relevant substrates 5alpha-DHT plus NADPH and 3alpha-diol plus NAD(+), the slowest event is chemistry. R276 forms a salt-linkage with the phosphate of 2'-AMP, and when it is mutated, tight binding of NAD(P)H is no longer observed [Ratnam, K., et al. (1999) Biochemistry 38, 7856-7864]. The R276M mutant also eliminated the burst phase kinetics observed for the NADP(+)-dependent oxidation of 3alpha-diol. The data with the R276M mutant confirms that the release of the NADPH product is the slow event; and in its absence, chemistry becomes rate-limiting. W227 is a critical hydrophobic residue at the steroid binding site, and when it is mutated to alanine, k(cat)/K(m) for oxidation is significantly depressed. Burst phase kinetics for the NADP(+)-dependent turnover of 3alpha-diol by W227A was also abolished. In the W227A mutant, the slow release of NADPH is no longer observed since the chemical transformation is now even slower. Thus, residues in the cofactor and steroid-binding site can alter the rate-determining step in the NADP(+)-dependent oxidation of 3alpha-diol to make chemistry rate-limiting overall.

Androgens may improve cognitive performance; however, these effects and mechanisms of androgens are not well understood. Whether testosterone's (T) effects on cognitive performance are mediated by its 5alpha-reduced, non-aromatizable metabolite dihydrotestosterone (DHT) and/or its 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) reduced metabolite 3alpha-androstanediol (3alpha-diol), was investigated. In Experiment 1, male rats that were gonadally intact, or gonadectomized (GDX) and DHT-replaced with a silastic capsule, had better performance in the inhibitory avoidance task, and higher plasma DHT and 3alpha-diol levels, compared to GDX rats. In Experiments 2-4, intra-hippocampal indomethacin, a 3alpha-HSD inhibitor, to intact or DHT-replaced, but not GDX, rats decreased performance in the inhibitory avoidance task and reduced hippocampal 3alpha-diol levels compared to that observed in rats with control implants. Thus, the 5alpha-reduced androgen DHT has cognitive-enhancing effects, independent of E(2), which are attenuated by a 3alpha-HSD inhibitor, indomethacin. These results suggest that 5alpha-reduced androgens may have actions in the hippocampus to improve cognitive performance.

5alpha-Androstane-3alpha,17beta-diol (androstanediol) is the predominant androgen in immature mouse testes, and studies were designed to investigate its pathway of synthesis, the steroid 5alpha-reductase isoenzyme involved in its formation, and whether testicular androstanediol is formed in embryonic mouse testes at the time of male phenotypic development. In 24-26-day-old immature testes, androstanediol is formed by two pathways; the predominant one involves testosterone --> dihydrotestosterone --> androstanediol, and a second utilizes the pathway progesterone --> 5alpha-dihydroprogesterone --> 5alpha-pregnane-3alpha-ol-20-one --> 5alpha-pregnane-3alpha,17alpha-diol-20-one --> androsterone --> androstanediol. Formation of androstanediol was normal in testes from mice deficient in steroid 5alpha-reductase 2 but absent in testes from mice deficient in steroid 5alpha-reductase 1, indicating that isoenzyme 2 is not expressed in day 24-26 testes. The fact that androstenedione and testosterone were the only androgens identified after incubation of day 16 and 17 embryonic testes with [3H]progesterone implies that androstanediol formation in the testis plays no role in male phenotypic differentiation in the mouse.

Virilization of the urogenital tract is under the control of testicular androgens in all mammals. In tammar young, prostate differentiation begins between d 20 and d 40 under the control of the testicular androgen 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol), but uncertainties exist about the control of penile development. We performed longitudinal studies up to d 150 of pouch life to define normal penile development and the effects of androgen administration and castration. In control animals the male phallus was longer than the female phallus by d 48. Closure of the urethra in males begins around d 60 and continues to at least d 150. Administration of supraphysiological doses of testosterone to females caused penile development equivalent to that of the male and also induced partial closure of the urethral groove by d 150. Castration of male pouch young at d 25 prevented penile development, whereas the penis in males castrated at d 40, 80, or 120 had partial closure of the urethral groove. Administration of 5alpha-adiol to females from d 20-40 also caused partial closure of the urethral groove and some growth of the phallus at d 150, whereas 5alpha-adiol treatment from d 40-80 or 80-120 caused some penile growth but had little effect on urethral development. These findings, together with the fact that we found no sex differences in plasma levels of testosterone, dihydrotestosterone, 5alpha-adiol, dehydroepiandrosterone, or androstenedione from d 51-227, clearly indicate that the action of 5alpha-adiol between d 20 and 40 imprints later differentiation of the male penis.

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.