BACKGROUND

Biotransformation is an effective technique for the synthesis of libraries of bioactive compounds. Current study on microbial transformation of dihydrotestosterone (DHT) (1) was carried out to produce various functionalized metabolites.

RESULTS

Microbial transformation of DHT (1) by using two fungal cultures resulted in potent butyrylcholinesterase (BChE) inhibitors. Biotransformation with Macrophomina phaseolina led to the formation of two known products, 5α-androstan-3β,17β-diol (2), and 5β-androstan-3α,17β-diol (3), while biotransformation with Gibberella fujikuroi yielded six known metabolites, 11α,17β-dihydroxyandrost-4-en-3-one (4), androst-1,4-dien-3,17-dione (5), 11α-hydroxyandrost-4-en-3,17-dione (6), 11α-hydroxyandrost-1,4-dien-3,17-dione (7), 12β-hydroxyandrost-1,4-dien-3,17-dione (8), and 16α-hydroxyandrost-1,4-dien-3,17-dione (9). Metabolites 2 and 3 were found to be inactive, while metabolite 4 only weakly inhibited the enzyme. Metabolites 5-7 were identified as significant inhibitors of BChE. Furthermore, predicted results from docking simulation studies were in complete agreement with experimental data. Theoretical results were found to be helpful in explaining the possible mode of action of these newly discovered potent BChE inhibitors. Compounds 8 and 9 were not evaluated for enzyme inhibition activity both in vitro and in silico, due to lack of sufficient quantities.

CONCLUSIONS

Biotransformation of DHT (1) with two fungal cultures produced eight known metabolites. Metabolites 5-7 effectively inhibited the BChE activity. Cholinesterase inhibition is among the key strategies in the management of Alzheimer's disease (AD). The experimental findings were further validated by in silico inhibition studies and possible modes of action were deduced.

A cross-talk between androgen/androgen receptor signaling and the AP-1 transcription factor has been proposed. In this study, we asked whether activation of AP-1 modifies androgen-responsive gene transcription, and whether androgens effect AP-1-regulated gene transcription. We show that activation of AP-1 via expression of a constitutively active mutant of mitogen-activated/extracellular signal responsive kinase kinase (MEK) kinase-1 did not increase the activity of the androgen-responsive probasin promoter. Likewise, expression of a constitutively active mutant of the transcription factor c-Jun, which is a major constitutent of AP-1, did not increase the activity of the probasin promoter. In contrast, <em>5α</em>-dihydrotestosterone (<em>DHT</em>) activated both the probasin promoter and the AP-1-regulated collagenase promoter in LNCaP prostate cancer cells. The AP-1 binding site within the collagenase promoter was identified as <em>DHT</em>-responsive element. In line with this, <em>DHT</em> increased the activities of the c-Jun promoter and the tumor necrosis factor alpha promoter, which both contain AP-1 binding sites. The signal transduction pathway coupling <em>DHT</em> stimulation with AP-1 activation required c-Jun, MAP kinases and androgen receptors, but was independent of transient receptor potential melastatin-8 (TRPM8) channels, proposed to function as ionotropic testosterone receptors. Expression of the GTPase activating protein RGS2 attenuated <em>DHT</em>-induced activation of AP-1, indicating that the <em>DHT</em>-induced signaling cascade involves G proteins.

Cistanche salsa has been used in traditional medicine for the treatment of kidney deficiency, neurasthenia, sexual dysfunction diseases, and benign prostatic hyperplasia (BPH). The aim of this study was to investigate the mechanism by which C. salsa extract (CSE) elicits an anti-proliferative effect on the prostate tissue of BPH-induced rats. The effects of CSE on BPH were evaluated in terms of prostate weight, production of serum dihydrotestosterone (<em>DHT</em>), and the mRNA expression of <em>5α</em>-reductase type 1 and type 2 in the prostate tissue of BPH-induced rats. In addition, hematoxylin and eosin (H&E) staining was performed for histological examination of prostate gland morphology, and protein expression levels in prostate tissue were investigated by western blot analysis. CSE treatment decreased prostate weight, serum <em>DHT</em> concentration, and the mRNA expression of <em>5α</em>-reductase type 1 and type 2 in prostate tissue of BPH-induced rats. In addition, CSE treatment suppressed cell proliferation by regulating the expression levels of inflammatory-related proteins (inducible nitric oxide synthase and cyclooxygenase 2) and apoptosis-associated proteins (caspase-3 and Bcl-2 family proteins). CSE may be a potential therapeutic candidate for BPH owing to its ability to regulate the expression of inflammatory and apoptosis-related proteins.

Human aldo-keto reductase family 1 member C3 (AKR1C3) was initially identified as an enzyme in reducing <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) to <em>5α</em>-androstane-3α, 17β-diol (3α-diol) and oxidizing 3α-diol to androsterone. It was subsequently demonstrated to possess ketosteroid reductase activity in metabolizing other steroids including estrogen and progesterone, 11-ketoprostaglandin reductase activity in metabolizing prostaglandins, and dihydrodiol dehydrogenase x (DDx) activity in metabolizing xenobiotics. AKR1C3 was demonstrated in sex hormone-dependent tissues including testis, breast, endometrium, and prostate; in sex hormone-independent tissues including kidney and urothelium. Our previous study described the expression of AKR1C3 in squamous cell carcinoma and adenocarcinoma but not in small cell carcinoma. In this report, we studied the expression of AKR1C3 in normal tissue, adenocarcinomas (43 cases) and neuroendocrine (NE) tumors (40 cases) arising from the aerodigestive tract and pancreas. We demonstrated wide expression of AKR1C3 in superficially located mucosal cells, but not in NE cells. AKR1C3-positive immunoreactivity was detected in 38 cases (88.4%) of adenocarcinoma, but only in 7 cases (17.5%) of NE tumors in all cases. All NE tumors arising from the pancreas and appendix and most tumors from the colon and lung were negative. The highest ratio of positive AKR1C3 in NE tumors was found in tumors arising from the small intestine (50%). These results raise the question of AKR1C3's role in the biology of normal mucosal epithelia and tumors. In addition, AKR1C3 may be a useful adjunct marker for the exclusion of the NE phenotype in diagnostic pathology.

BACKGROUND

Many traditional Chinese medicines (TCM) have been used for hundreds of years for hair blackening and hair nourishing, and now many of them are commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth.

OBJECTIVE

The present study was performed to screen <em>5α</em>-reductase (<em>5α</em>R) inhibitors from traditional Chinese medicines, evaluate its hair growth promoting activity in vivo, and further investigate its effects on androgen metabolism and the expression of <em>5α</em>R II in hair follicles.

METHODS

Nine TCM which were dried, ground and extracted by maceration with 75% ethanol or distilled water were used for screening <em>5α</em>R inhibitors, and enzymes were extracted from the rat epididymis. The leaves of Platycladus orientalis (L.) Franco was used to evaluate the in vivo anti-androgenic activity. Skin color was observed daily and the hair re-growth was assessed by assigning the hair growth score. The longitudinal sections of hair follicles were used for observing follicle morphology, classifying of distinct stages of hair follicle morphogenesis and calculate the average score. The transverse sections were used for determination of hair follicle counts. Testosterone (T), Dihydrotestosterone (DHT) and Estradiol (E2) levels in serum and skin tissue were detected by ELISA kits. The immunofluorescence assay was used to detect the influence of CP-ext on <em>5α</em>R expression in dorsal skin.

RESULTS

We found the extract of Ganoderma lucidum (GL-ext), Polygonum multiflori (PM-ext), Cacumen platycladi (CP-ext) and Cynomorium songaricum (CS-ext) showed stronger <em>5α</em>R inhibitory activity. CP-ext (5mg and 2mg/mouse/day) could significantly shorten the time of the dorsal skin darkening and got longhaired (P<0.01), and showed high hair re-growth promoting activity. Furthermore the histological data of hair follicles in each group showed that CP-ext could promote the growth of hair follicle and slowed down hair follicles enter the telogen. What's more CP-ext significantly reduced DHT levels and down-regulated the expression of <em>5α</em>RⅡin skin (P<0.01).

CONCLUSIONS

GL-ext, PM-ext, CP-ext and CS-ext showed strong <em>5α</em>R inhibitory activity. CP-ext possesses high hair growth promoting activity in the in vivo androgen-sensitive mouse model via inhibiting the <em>5α</em>R activity, decreasing the DHT levels and in turn suppressing the expression of <em>5α</em>R. Our study may contribute to the development of a new generation of herbal supplements with clearer material basis of pharmacodynamic for treating androgenic alopecia (AGA).

Diabetes develops predominantly in males in experimental models, and extensive evidence suggests that 17β-estradiol (E2) modulates progression of diabetes in humans. We previously developed a severely diabetic transgenic (Tg) mouse model by β-cell-specific overexpression of inducible cAMP early repressor (ICER) and found that male ICER-Tg mice exhibit sustained severe hyperglycemia, but female ICER-Tg mice gradually became normoglycemic with aging. This implies that differences in circulating androgen and E2 levels might influence skeletal muscle glucose uptake and glycemic status. Here we examined whether a decrease of androgen or E2 excess can improve muscle glucose uptake in hyperglycemic male ICER-Tg mice and, conversely, whether a decrease of E2 or androgen excess can elevate blood glucose levels and impair muscle glucose uptake in normoglycemic female ICER-Tg mice. We treated hyperglycemic male ICER-Tg mice with orchiectomy (ORX) or ORX+E2 pellet implantation and normoglycemic female ICER-Tg mice with ovariectomy (OVX) or OVX+<em>5α</em>-<em>DHT</em> pellet implantation to alter the androgen to E2 ratio. ORX+E2 treatment of male ICER-Tg mice caused a rapid drop in blood glucose via both a dramatic increase of β-cells and significantly improved muscle glucose uptake due to the induction of glucose transporter type 4 (GLUT4) expression and translocation of GLUT4 to the cell membrane. In contrast, OVX+<em>5α</em>-<em>DHT</em>-treated female ICER-Tg mice showed an elevation of blood glucose without any decrease of β-cells; instead, they showed decreased muscle glucose uptake due to decreased activation of serine/threonine-specific protein kinase AKT and GLUT4 expression. These findings suggest that androgen (<em>5α</em>-<em>DHT</em>) promotes insulin resistance in females, whereas E2 improves insulin sensitivity in severely diabetic male mice.

Testosterone (T) affects β cell function in men and women. T is a pro-hormone that undergoes intracrine conversion in target tissues to the potent androgen dihydrotestosterone (<em>DHT</em>) via the enzyme <em>5α</em>-reductase (<em>5α</em>-R), or to the active estrogen 17β-estradiol (E2) via the aromatase enzyme. Using male and female human pancreas sections, we show that the <em>5α</em>-R type1 isoform (SRD5A1) and aromatase are expressed in male and female β cells. We show that cultured male and female human islets exposed to T produce <em>DHT</em> and downstream metabolites. In these islets, exposure to the <em>5α</em>-R inhibitors finasteride and dutasteride inhibited T conversion into <em>DHT</em>. We did not detect T conversion into E2 from female islets. However, we detected T conversion into E2 in islets from one out of four male donors. In this donor, exposure to the aromatase inhibitor anastrozole inhibited E2 production. Notably, in cultured male and female islets, T enhanced glucose-stimulated insulin secretion (GSIS). In these islets, exposure to <em>5α</em>-R inhibitors or the aromatase inhibitor both inhibited T enhancement of GSIS. In conclusion, male and female human islets convert T into <em>DHT</em> and E2 via the intracrine activities of SRD5A1 and aromatase. This process is necessary for T enhancement of GSIS.

Androgens are key mediators of prostate development and function, a role that extends to the development of prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In prostate, <em>DHT</em> is the major androgen and reduction and glucuronidation are the major metabolic pathways for <em>DHT</em> elimination. A streamlined method for quantitation of dihydrotestosterone (<em>DHT</em>), <em>5α</em>-androstan-3α,17β-diol (3α-diol), and 3α-diol glucuronide (diol-gluc) was established and validated for use with archived prostate tissue specimens to facilitate examination of the roles of the underlying metabolism. This involved a sequential 70/30 hexane/ethyl acetate (hex/EtOAc) extraction of steroids, followed by an ethyl acetate extraction for diol-gluc. Derivatization of the hex/EtOAc fraction with2-fluoro-1-methylpyridinium p-toluene-4-sulfonate (FMP) was used to enhance sensitivity for hydroxyl steroids and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized for analysis of both fractions. The method was validated with calibration standards followed by recovery assessment from spiked samples of BPH and normal prostate. Lower limits of quantitation (LLOQ) were 50 pg/g, 20 pg/g and 100 pg/g for <em>DHT</em>, 3α-diol and diol-gluc, respectively for extracts from 50mg equivalents of tissue. Prepared samples were stable for up to three weeks at 4 °C and 37 °C. The method provides excellent sensitivity and selectivity for determination of tissue levels of <em>DHT</em>, 3α-diol, and diol-gluc. Furthermore, this protocol can easily be extended to other hydroxyl steroids, is relatively straightforward to perform and is an effective tool for assessing steroid levels in archived clinical prostate samples.

Evidence shows neurosteroids play a key role in regulating epileptogenesis. Neurosteroids such as testosterone modulate seizure susceptibility through its transformation to metabolites which show proconvulsant and anticonvulsant effects, respectively. Reduction of testosterone by aromatase generates proconvulsant 17-β estradiol. Alternatively, testosterone is metabolized into <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) by <em>5α</em>-reductase, which is then reduced by 3α-hydroxysteroid oxidoreductase enzyme (3α-HSOR) to form anticonvulsant metabolite 3α-androstanediol (3α-Diol), a potent GABAA receptor modulating neurosteroid. The present study evaluated whether inhibition of aromatase inhibitor letrozole protects against seizures and neuronal degeneration induced by kainic acid (KA) (10 mg/kg, i.p.) in Swiss albino mice. Letrozole (1 mg/kg, i.p.) administered one hour prior to KA significantly increased the onset time of seizures and reduced the% incidence of seizures. Pretreatment with finasteride, a selective inhibitor of <em>5α</em>-reductase and indomethacin, a selective inhibitor of 3α-hydroxysteroid oxidoreductase enzyme (3α-HSOR), reversed the protective effects of letrozole in KA-induced seizures in mice. Microscopic examination using cresyl violet staining revealed that letrozole did not modify KA-induced neurotoxicity in the CA1, CA3 and DG region of the hippocampus. Letrozole treatment resulted in the reduced levels of 17-β estradiol and elevated the levels of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) and 3α-Diol in the hippocampus. Finasteride and indomethacin attenuated letrozole-induced elevations of <em>5α</em>-<em>DHT</em> and 3α-Diol. Our results indicate the potential anticonvulsant effects of letrozole against KA-induced seizures in mice that might be mediated by inhibiting aromatization of testosterone to 17β-estradiol, a proconvulsant hormone and by redirecting the synthesis to anticonvulsant metabolites, <em>5α</em>-<em>DHT</em> and 3α-Diol. Acute aromatase inhibition, thus, might be used as an adjuvant in the treatment of status epilepticus and can be pursued further.

BACKGROUND

Detecting prostate cancer before spreading or predicting a favorable therapy are challenging issues for impacting patient's survival. Presently, 2-[(18) F]-fluoro-2-deoxy-D-glucose ((18) F-FDG) and/or (18) F-fluorocholine ((18) F-FCH) are the generally used PET-tracers in oncology yet do not emphasize the T877A androgen receptor (AR) mutation being exclusively present in cancerous tissue and escaping androgen deprivation treatment.

METHODS

We designed and synthesized fluorinated <em>5α</em>-dihydrotestosterone (<em>DHT</em>) derivatives to target T877A-AR. We performed binding assays to select suitable candidates using COS-7 cells transfected with wild-type or T877A AR (WT-AR, T877A-AR) expressing plasmids and investigated cellular uptake of candidate (18) F-RB390. Stability, biodistribution analyses and PET-Imaging were assessed by injecting (18) F-RB390 (10MBq), with and without co-injection of an excess of unlabeled <em>DHT</em> in C4-2 and PC-3 tumor bearing male SCID mice (n = 12).

RESULTS

RB390 presented a higher relative binding affinity (RBA) (28.1%, IC50 = 32 nM) for T877A-AR than for WT-AR (1.7%, IC50 = 357 nM) related to DHT (RBA = 100%). A small fraction of (18) F-RB390 was metabolized when incubated with murine liver homogenate or human blood for 3 hr. The metabolite of RB390, 3-hydroxysteroid RB448, presented similar binding characteristics as RB390. (18) F-RB390 but not (18) F-FDG or (18) F-FCH accumulated 2.5× more in COS-7 cells transfected with pSG5AR-T877A than with control plasmid. Accumulation was reduced with an excess of DHT. PET/CT imaging and biodistribution studies revealed a significantly higher uptake of (18) F-RB390 in T877A mutation positive xenografts compared to PC-3 control tumors. This effect was blunted with DHT.

CONCLUSIONS

Given the differential binding capacity and the favorable radioactivity pattern, (18) F-RB390 represents the portrayal of the first imaging ligand with predictive potential for mutant T877A-AR in prostate cancer for guiding therapy. Prostate 75:348-359, 2015. © 2014 Wiley Periodicals, Inc.

The hypothesis that sexual ornaments are honest signals of quality because their expression is dependent on hormones with immune-depressive effects has received ambiguous support. The hypothesis might be correct for those signals that are carotenoid-dependent because the required carotenoid deposition in the signal, stimulated by testosterone, might lower the carotenoid-dependent immune defence of the organism. Two pathways underlying this androgen-dependent honest signaling have been suggested. Firstly, androgens that are needed for ornament expression may suppress immune defence, a cost that only high-quality animals can afford. Alternatively, immune activation may downregulate the production of androgens in low-quality individuals. Which of these alternatives is correct, and to what extent these effects are mediated by the different metabolites of androgens, remain open questions. To provide answers to these questions, we manipulated the levels of testosterone (T), <em>5α</em>-dihydrotestosterone (<em>DHT</em>), and 17-β-estradiol (E2) in diamond doves Geopelia cuneata, a species in which both sexes exhibit a carotenoid-dependent, androgen-regulated red-orange periorbital ring of bare skin. On the first day of the experiment (day 0), we inserted steroid-releasing implants into groups of birds and on day 14, we subjected half of the birds to an immunological challenge by immunizing them with sheep red blood cells (SRBC). In females, but not in males, androgen but not estradiol treatments reduced antibody production to SRBC. In addition, the immunological challenge reduced redness and size of the trait as well as androgens levels in both sexes and in all treatments. This indicates that an immunological challenge can lower circulating T at the cost of the trait expression. These findings are in accordance with both pathways postulated in the immunocompetence-handicap hypothesis, but do not entirely support the idea that the immunosuppressive effect of androgens yields honest signaling since both T and <em>DHT</em> were not immunosuppressive in males, for which sexual signaling is supposed to be especially important.

We previously identified the selective androgen receptor (AR) modulator S42, which does not stimulate prostate growth but has a beneficial effect on lipid metabolism. In the prostate cancer (PC) cell line LNCaP, S42 did not induce AR transactivation but antagonized <em>5α</em>-dihydrotestosterone (<em>DHT</em>)‒induced AR activation. Next, we investigated whether S42 suppresses the growth of PC cell lines. Basal growth of LNCaP cells was significantly suppressed by treatment with S42 compared with vehicle, as determined by cell counting and 5-bromo-2'-deoxyuridine assays. The suppressive effect of S42 on cell growth was evident in the AR-positive PC cells LNCaP and 22Rv1 and was slightly observed even in the AR-negative PC-3 cells. However, S42 did not induce apoptosis as determined by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. S42 had an even greater suppressive effect on <em>DHT</em>-dependent LNCaP cell proliferation than on basal proliferation (P < 0.05). <em>DHT</em> treatment increased the expression of phosphorylated extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK), a major signaling molecule for PC proliferation, and this was significantly inhibited by S42. <em>DHT</em> also significantly upregulated AR, insulinlike growth factor-1 receptor (IGF-1R), and insulin receptor (IR)-β protein levels, which were similarly reduced by S42 treatment. Importantly, S42 administration to mice attenuated the growth of LNCaP tumors and reduced tumor expression of the prostate-specific antigen, P504S, Ki67, and phosphorylated ERK-MAPK. These data suggest that S42 attenuates LNCaP tumor growth not by inducing apoptosis but by inhibiting the expression of proliferation-related receptors, including IGF-1R, IR, and AR, and by suppressing ERK-MAPK activation. S42 may thus be a feasible candidate for PC treatment.

<AbstractText><em>5α</em>-<em>DHT</em> can decrease the cell viability of the hair follicle stem cells (HFSCs) with CD34-positive and CD200-rich in bald scalp area of androgenic alopecia (AGA) patients and the apoptosis of HFSCs may be involved in the pathogenesis of AGA. The expression of Vascular endothelial growth factor (VEGF) turns to be weakened or disappeared in hair follicles of AGA patients.</AbstractText><AbstractText>To investigate whether VEGF is involved in the apoptosis of HFSCs induced by <em>5α</em>-<em>DHT</em> in the patients of AGA.</AbstractText><AbstractText>By <em>5α</em>-<em>DHT</em>, apoptosis of CD200-rich and CD34-positive HFSCs was induced and apoptotic rates up to 24 hours were assessed using flow cytometry. The expression grades of Bcl-2, Akt, caspase-3 and Bax were observed through Western blot analysis.</AbstractText><AbstractText>Vascular endothelial growth factor could cut <em>5α</em>-<em>DHT</em> induced apoptosis down substantially in a concentration-dependent manner. The <em>5α</em>-<em>DHT</em> induced decline in the rise of Bcl-2/Bax proportion and the increase in caspase-3 degrees were mostly reversed by using VEGF and the VEGF's anti-apoptotic actions were impeded through preventing the activation of phosphoinositide 3-kinase (PI3K)/Akt.</AbstractText><AbstractText>Vascular endothelial growth factor can protect CD200-rich and CD34-positive HFSCs from androgen induced apoptosis by means of the PI3K/Akt pathway.</AbstractText>

Androgen is an essential factor involved in masculinization of external genitalia. Failure of the exposure to <em>5α</em>-dihydrotestosterone (<em>DHT</em>) causes a hypoplastic penile size and urethral abnormality. The main pathology of hypospadias is defective urethral closure on the ventral side of the penis. Hormone-dependent genes are suggested as the causative factors. However, the detailed mechanisms of <em>DHT</em> functions on urethral tube formation remain unknown. Androgen is both a positive and negative regulator of cell proliferation. The roles of locally converted <em>DHT</em> in cell proliferation at the periurethral mesenchyme have not been elucidated. We revealed the expression pattern of <em>5α</em>-reductase type 2 mRNA (Srd5a2) and local <em>DHT</em> distribution by direct measurement in this study. We also analyzed periurethral mesenchymal cell proliferation status using systematic three-dimensional (3D) reconstruction analyses. A prominent Srd5a2 expression and localized <em>DHT</em> distribution on the ventral side of the genital tubercle were detected. Cell proliferation was reduced in this mesenchymal region during urethral formation. The current results suggest the presence of the possible negative regulation of cell proliferation by <em>DHT</em>. Moreover, cell proliferation related to urethral tube formation was revealed to be <em>DHT</em> dose dependent. These data are expected to contribute to the understanding of the mode of regulation of cell proliferation related to urethral tube formation by <em>DHT</em>. These findings may also offer insight into the understanding of human hypospadias and related hormone-dependent factors.

OBJECTIVE

Benign prostatic hyperplasia (BPH) is a common disease found in elderly men and <em>5α</em>-reductase (<em>5α</em>-R) inhibitors are a commonly used treatment option. <em>5α</em>-reduced steroids are compounds that play a role in several functions across different organs and systems. In the adult brain, <em>5α</em>-R accounts for neuroactive steroid production. Whether neuropsychological impairment could be due to dutasteride treatment, a <em>5α</em>-R inhibitor affecting the production of dihydrotestosterone (<em>DHT</em>), is still unknown. The aim of our study was to investigate neuropsychological features in men receiving dutasteride.

METHODS

The Mini Mental State Examination (MMSE), the Clock Drawing Test (CDT), the Frontal Assessment Battery (FAB), the Hamilton Anxiety Rating Scale (HAM-A), the Beck Depression Inventory second edition (BDI-II) and the Short Form-12 (SF-12) questionnaire were administered in order to explore both cognitive impairment and psychological features.

RESULTS

In a sample of BPH patients (n = 40; mean age 71.4 ± 7.4 years), men receiving dutasteride showed no significant differences during the neuropsychological assessment in comparison with an age-matched control group, consisting of BPH men not receiving dutasteride (p < 0.05). No significant associations were recorded between treatment duration and any of the administered tests.

CONCLUSIONS

This is the first study investigating the neuropsychological features in dutasteride users. Our preliminary data are consistent with the safety of dutasteride under a mental profile.

A systems-level mathematical model is presented that describes the effects of inhibiting the enzyme <em>5α</em>-reductase (5aR) on the ventral prostate of the adult male rat under chronic administration of the 5aR inhibitor, finasteride. 5aR is essential for androgen regulation in males, both in normal conditions and disease states. The hormone kinetics and downstream effects on reproductive organs associated with perturbing androgen regulation are complex and not necessarily intuitive. Inhibition of 5aR decreases the metabolism of testosterone (T) to the potent androgen <em>5α</em>-dihydrotestosterone (<em>DHT</em>). This results in decreased cell proliferation, fluid production and 5aR expression as well as increased apoptosis in the ventral prostate. These regulatory changes collectively result in decreased prostate size and function, which can be beneficial to men suffering from benign prostatic hyperplasia (BPH) and could play a role in prostate cancer. There are two distinct isoforms of 5aR in male humans and rats, and thus developing a 5aR inhibitor is a challenging pursuit. Several inhibitors are on the market for treatment of BPH, including finasteride and dutasteride. In this effort, comparisons of simulated vs. experimental T and <em>DHT</em> levels and prostate size are depicted, demonstrating the model accurately described an approximate 77% decrease in prostate size and nearly complete depletion of prostatic <em>DHT</em> following 21 days of daily finasteride dosing in rats. This implies T alone is not capable of maintaining a normal prostate size. Further model analysis suggests the possibility of alternative dosing strategies resulting in similar or greater effects on prostate size, due to complex kinetics between T, <em>DHT</em> and gene occupancy. With appropriate scaling and parameterization for humans, this model provides a multiscale modeling platform for drug discovery teams to test and generate hypotheses about drugging strategies for indications like BPH and prostate cancer, such as compound binding properties, dosing regimens, and target validation.

Androgens interact with catecholamines in the central nervous system (CNS) to regulate many physiological processes including blood pressure (BP). To test the hypothesis that testosterone (T) and <em>5a</em>-dihydrotestosterone (<em>DHT</em>) modulate CNS catecholamines and BP through androgen receptor (AR)-dependent and independent mechanisms, we used the testicular feminized male (Tfm) rat. Females that carry the AR mutation (Tfm mutation) on the X chromosome were bred with spontaneously hypertensive rat (SHR) males. The normal AR male and Tfm offspring were divided into groups: control, castrated, castrated, and T or (<em>DHT</em>) replacement. In both AR normal and Tfm males, BP was reduced by castration, but T restored BP in both groups. In the amygdale, castration decreased dopamine (DA) in both strains and both T and <em>DHT</em> restored it. In the bed nucleus of the stria terminalis castration increased DA which was further increased by <em>DHT</em> and reduced to normal by T in both strains. In the frontal cortex, castration reduced DA content in both strains but only T restored it to normal in SHR but not in Tfm. Brain norepinephrine (NE) content showed a significant strain effect for the preoptic area (POA), but no treatment effect. Although castration did not change NE in the amygdala or POA in either strain, both T and <em>DHT</em> increased NE in the Tfm castrates. Blood pressure was influenced by T manipulation and correlated most significantly with DA content in the amygdala, frontal cortex, and stria terminalis. These data demonstrate an action of androgen on brain catecholamines and BP, which is independent of the classic androgen receptor.

Androgens not only play an important role in the development and function of the prostate but they are also intimately involved in the development and progression of prostate cancer (PCa). Within the prostate, testosterone is converted to the more potent androgen dihydrotestosterone (<em>DHT</em>) via the action of <em>5α</em>-reductase enzymes. <em>DHT</em> is the primary prostatic androgen and promotes the growth and survival of normal, hyperplastic and malignant prostate tissues. Throughout the different stages of PCa [prostatic intraepithelial neoplasia (PIN), localised, recurrent, and metastatic] there is an increase in expression of <em>5α</em>-reductase enzymes, particularly in localised high-grade carcinoma. Specifically inhibiting <em>5α</em>-reductase may reduce the production of <em>DHT</em> in the prostate while maintaining other endogenous hormone levels. Clinical studies have shown significant PCa risk reduction by blocking this pathway with <em>5α</em>-reductase inhibitors (5ARIs). However, this comes at a risk, albeit low, with sexual side effects, gynaecomastia and cardiac failure. In addition, one study has shown a slight, but significant, risk of high-grade PCa. The currently available evidence does not support the routine use of <em>5α</em>-reductase inhibitors to prevent PCa in the general population. It could, however, be considered as an individual option for high-risk or concerned patients with appropriate education from the prescribing provider.

In addition to androgenic properties mediated via androgen receptors, dihydrotestosterone (<em>DHT</em>) also regulates estrogenic functions via an alternate pathway. These estrogenic functions of <em>DHT</em> are mediated by its metabolite <em>5α</em>-androstane-3β, 17β-diol (3β-diol) binding to estrogen receptor β (ERβ). CYP7B1 enzyme converts 3β-diol to inactive 6α- or 7α-triols and plays an important role as a regulator of estrogenic functions mediated by 3β-diol. Using a mutant mouse carrying a null mutation for the CYP7B1 gene (CYP7B1KO), we examined the contribution of CYP7B1 on physiology and behavior. Male, gonadectomized (GDX) CYP7B1KO and their wild type (WT) littermates were assessed for their behavioral phenotype, anxiety-related behavioral measures, and hypothalamic pituitary adrenal axis reactivity. No significant effects of genotype were evident in anxiety-like behaviors in open field (OFA), light-dark (L/D) exploration, and elevated plus maze (EPM). T significantly reduced open arm time on the EPM while not affecting L/D exploratory and OFA behaviors in CYP7B1KO and WT littermates. T also attenuated the corticosterone response to EPM in both genotypes. In GDX animals, T was able to reinstate male-specific reproductive behaviors (latencies and number of mounts, intromission, and ejaculations) in the WT but not in the CYP7B1KO mice. The male reproductive behavior defect in CYP7B1KO seems to be due to their inability to distinguish olfactory cues from a behavioral estrus female. CYP7B1KO mice also showed a reduction in androgen receptor mRNA expression in the olfactory bulb. Our findings suggest a novel role for the CYP7B1 enzyme in the regulation of male reproductive behaviors.

Neuroactive steroids such as progesterone, testosterone, and their derivatives have been widely studied for their neuroprotective roles in the nervous system. Autologous nerve transplantation is considered as the gold standard repair technique when primary suture is impossible; nevertheless, this method is far from ideal. In this study, we aimed to explore the impact of dihydrotestosterone (<em>DHT</em>), a <em>5α</em>-reduced derivative of testosterone, on the recovery of peripheral nerve injury treated with autologous nerve transplantation. Sprague-Dawley rats were subjected to a 10-mm right side sciatic nerve reversed autologous nerve transplantation and randomly divided into groups that received <em>DHT</em> or <em>DHT</em> + flutamide (an androgen receptor blocker) daily for 8 weeks after operation. Our results demonstrated that <em>DHT</em> could speed up the rate of axonal regeneration and increase the expression of myelin protein zero (P0) in autograft reversal sciatic nerves. Thus, our study provided new insights into improving the prognosis of patients with long gap peripheral nerve defects.

BACKGROUND

Hypospadias is one of the most common forms of congenital malformation of the male external genitalia worldwide. The ratio in the Iranian population is one in 250 live male births. The conversion of testosterone to dihydrotestosterone (<em>DHT</em>) in the presence of steroid <em>5α</em>-reductase 2, which is encoded by SRD5A2 gene, plays an important role in the normal development of the male reproductive system.

METHODS

We examined whether SRD5A2 gene mutations (V89L and A49T polymorphisms) are associated with the risk of hypospadias in the Iranian population. We performed exons sequencing for SRD5A2 gene in 109 hypospadias patients.

RESULTS

We identified two new mutations in the subgroups of affected cases: including a substitution of the nucleotide T>> A in the codon 73 [c.219T>> A (p.Leu73_Ser74insHisPro)] and an insertion of an extra A nucleotide in the codon 77 [c.229insA* (p.Gly77*)]. Additionally, we performed PCR-RFLP for the two identified polymorphisms and revealed that V89L [OR = 5.8, 95% CI (3.8-8.8), p value < 0.001] and A49T [OR = 10.16, 95% CI (3.94-26.25), p value < 0.001] are significantly associated with hypospadias occurrence in patients. Our haplotype analysis further indicated that the Leu-Ala haplotype increases risk of hypospadias; conversely, the Val-Ala haplotype decreases the risk of hypospadias in the studied patients.

CONCLUSIONS

This study demonstrates that polymorphisms in the SRD5A2 gene could be considered as a risk factor for hypospadias disease emergence.

OBJECTIVE

To investigate the effects of an antibiotic brefeldin A (BFA) on androgen-regulated cellular events in androgen-responsive prostate cancer cells, focusing on PSA (prostate-specific antigen) status, cell growth, and bioactivity of androgen receptor (AR).

METHODS

Androgen-responsive human prostate cancer LNCaP cells were employed and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) was used as an androgenic mediator to induce androgen-modulated cellular events. Effects of BFA on synthesis and secretion of PSA, cell growth, and AR activity were assessed using Tandem PSA assay, trypan blue exclusion method, and AR binding assay, respectively.

RESULTS

BFA (30 ng/ml) dramatically (90%) blocked secretion of PSA and also reduced cell growth by >75% under non-androgen-regulated condition. Under androgen-stimulated condition using DHT (1 nM), both the cellular and secreted PSA levels as well as cell growth was significantly elevated or stimulated by DHT (compared with controls); however, BFA was capable of completely inhibiting such DHT-stimulated cellular events. In addition, AR binding assay revealed that AR activity has been drastically (~90%) diminished by BFA, likely resulting in interruption of DHT-mediated events.

CONCLUSIONS

BFA is capable of attenuating androgenic regulation in LNCaP cells such as androgen-stimulated PSA synthesis/secretion and cell growth. This BFA-blocked androgen action appears to be primarily attributed to severe inactivation of AR with BFA because AR is a crucial factor for relaying androgenic messages (to DNA). Therefore, BFA could be considered a promising agent for a more effective treatment of hormone-dependent prostate cancer.

BACKGROUND

Exogenous androgen supplement is an optional treatment for micropenis; however, its use in childhood is controversial due to potential side effects.

METHODS

Twenty-three children (mean age: 4.07±3.4 years) with micropenis of unknown causes harboring the 46,XY karyotype were recruited in an open prospective study. Androgen receptor (AR), steroid <em>5α</em>-reductase-2 (SRD5A2), and SRY genes were sequenced; 2.5% dihydrotestosterone (<em>DHT</em>) transdermal gel (0.1-0.3 mg/kg/day) was applied and titrated within the normal <em>DHT</em> serum reference ranges. Stretched penile length (SPL) was measured before therapy, and after 1, 3 and 6 months of <em>DHT</em> gel treatment, respectively.

RESULTS

Two patients were found with AR gene mutations and five patients with SRD5A2 gene mutations. Average stretched penile lengths (SPLs) were 1.68±0.6 cm at baseline and 2.2±0.66 cm, 2.6±0.59 cm and 2.9±0.55 cm (mean ± 1 SD) after 1, 3 and 6 months of treatment, respectively. Fourteen cases (61%) reached standard penile length ranges >>-2.5 SD) and medication was discontinued; six cases (26%) were satisfied with the improved penile lengths despite failing to reach the aged matched standards. Three infants (13%) discontinued the medication after 3 months due to anxiety about the potential side effects. No significant side effects were found except the elevated DHT serum levels after therapy.

CONCLUSIONS

Short term and local application of DHT at low doses in patients with micropenis could accelerate penile growth effectively without evident side effects; however, precautions still need be taken due to the paucity of long term study and the lack of ideal DHT dosage.

Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen <em>5α</em>-<em>DHT</em> (<em>5α</em>-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with <em>5α</em>-<em>DHT</em> and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP(+)·A-dione (<em>5α</em>-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with <em>5α</em>-<em>DHT</em> in the presence of NADP(+) Although <em>5α</em>-<em>DHT</em> was introduced during the crystallization, oxidoreduction of <em>5α</em>-<em>DHT</em> occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for <em>5α</em>-<em>DHT</em> reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP(+)·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the <em>5α</em>-<em>DHT</em> concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for <em>5α</em>-<em>DHT</em> reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth.