The study of dynamic properties of metabolic and signaling networks is hindered by the lack of methods for imaging metabolic fluxes in individual intact cells. We describe a novel optical approach for measuring the changes of metabolic fluxes in cells, based on a two-substrate competition between a physiological substrate and a fluorogenic reporter substrate. We have constructed a model cell system for a two-step metabolic pathway involved in the metabolism of testosterone. Potent androgen testosterone is converted by steroid <em>5α</em>-reductase to <em>DHT</em> (<em>5α</em>-dihydrotestosterone), which is subsequently metabolized to 3α-diol (3α,17β-androstanediol) by the reductase AKR1C2 (aldo-ketoreductase 1C2), for which we have previously developed the fluorogenic reporter substrate Coumberone. Despite the medicinal importance of <em>5α</em>-reductase, there are presently no probes or methods for the continuous activity readout of this enzyme in cells. We show that the activity of <em>5α</em>-R1 (<em>5α</em>-reductase type 1) can be measured in COS-1 cells via the changes of <em>DHT</em> flux. Our system enables a measurement of <em>5α</em>-reductase activity in cells, via either fluorimetry or fluorescence microscopy, with a wide dynamic range of activities, and provides a continuous optical assay for evaluation of small molecule inhibitors for this important enzyme. Furthermore, this paper demonstrates a novel optical approach to measuring metabolic flux changes in living cells and expands the utility of fluorogenic enzyme reporter substrates: optical reporters can measure not only the activity of the target enzyme but also the activity of other enzymes upstream in the pathway, for which there are no probes available.

The steroid <em>5α</em>-reductase type II enzyme catalyzes the conversion of testosterone (T) to dihydrotestosterone (<em>DHT</em>), and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid <em>5α</em>-reductase type II gene (SRD5A2) was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA>> CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD) for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing <em>5α</em>-reductase type II enzyme deficiency.

Cytochrome P450 17A1 (P450c17) is the key enzyme required for the production of androgenic sex steroids by converting progestogens to androgens. <em>5α</em>-reductases are enzymes that convert testosterone (T) to dihydrotestosterone (<em>DHT</em>), which has a greater affinity for androgen receptors (AR) and stronger action than T. Our previous studies revealed that the scented glands of male muskrats expressed AR during the breeding and nonbreeding seasons. To further seek evidence of the activities of androgens in scented glands, the expression patterns of P450c17 and <em>5α</em>-reductase 2 were investigated in the scented glands of male muskrats during the breeding and nonbreeding seasons. The weight and size of scented glands in the breeding season were significantly higher than those of the nonbreeding season. Immunohistochemical data showed that P450c17 and <em>5α</em>-reductase 2 were presented in the glandular cells and epithelial cells of scented glands in both the seasons. The protein and mRNA expression of P450c17 and <em>5α</em>-reductase 2 were significantly higher in the scented gland during the breeding season than those during the nonbreeding season. In addition, the levels of <em>DHT</em> and T in the scented gland were remarkably higher during the breeding season. Taken together, these results suggested that the scented glands of male muskrats were capable of locally synthesizing T and <em>DHT</em>, and T and <em>DHT</em> might play an important role in the scented glandular function via an autocrine or paracrine manner.

BACKGROUND

Studies regarding genetic and clinical characteristics, gender preference, and gonadal malignancy rates for steroid 5-alpha-reductase type 2 deficiency (<em>5α</em>-RD2) are limited and they were conducted on small number of patients.

OBJECTIVE

To present genotype-phenotype correlation, gonadal malignancy risk, gender preference, and diagnostic sensitivity of serum testosterone/dihydrotestosterone (T/DHT) ratio in patients with <em>5α</em>-RD2.

METHODS

Patients with variations in the SRD5A2 gene were included in the study. Demographic characteristics, phenotype, gender assignment, hormonal tests, molecular genetic data, and presence of gonadal malignancy were evaluated.

RESULTS

A total of 85 patients were included in the study. Abnormality of the external genitalia was the most dominant phenotype (92.9%). Gender assignment was male in 58.8% and female in 29.4% of the patients, while it was uncertain for 11.8%. Fourteen patients underwent bilateral gonadectomy, and no gonadal malignancy was detected. The most frequent pathogenic variants were p.Ala65Pro (30.6%), p.Leu55Gln (16.5%), and p.Gly196Ser (15.3%). The p.Ala65Pro and p.Leu55Gln showed more undervirilization than the p.Gly196Ser. The diagnostic sensitivity of stimulated T/DHT ratio was higher than baseline serum T/DHT ratio, even in pubertal patients. The cut-off values yielding the best sensitivity for stimulated T/DHT ratio were ≥ 8.5 for minipuberty, ≥ 10 for prepuberty, and ≥ 17 for puberty.

CONCLUSIONS

There is no significant genotype-phenotype correlation in <em>5α</em>-RD2. Gonadal malignancy risk seems to be low. If genetic analysis is not available at the time of diagnosis, stimulated T/DHT ratio can be useful, especially if different cut-off values are utilized in accordance with the pubertal status.

Testosterone (T) is known to play an important masculinizing role in the developing brain of rat, including the regulation of <em>5α</em>-reductase (<em>5α</em>-R) isozymes. However, the effects of dihydrotesterone (<em>DHT</em>), a more potent androgen than T, have not been elucidated. In this study, <em>DHT</em> was administered from day 5 through day 20 of postnatal life (period of postnatal sexual differentiation of the central nervous system) at doses of: 12 mg/kg/d on days 5, 6, 7, 8, 19, and 20; 15 mg/kg/d on days 9, 10, 11, 12, 16, 17, and 18; and 18 mg/kg/d on days 13, 14, and 15. In adulthood, quantitative RT-PCR was used to measure mRNA levels of <em>5α</em>-R1 and <em>5α</em>-R2 isozymes in the prefrontal cortex (PFC) of male and female rats with varied androgenic status. Under our study conditions, neonatal <em>DHT</em> administration influenced on adult PFC <em>5α</em>-R isozymes levels and their regulation pattern by androgens, and this pattern was the inverse of that reported in adult neonatally T-treated rats.

Stanozolol (STZ) is a drug used to treat serious disorders like aplastic anemia and hereditary angioedema. It is also indicated as an adjunct therapy for the treatment of vascular disorders and growth failures. Encouraging results obtained using animal models demonstrated that STZ increases bone formation and mineralization, thus improving both density and biomechanical properties. Like natural androgens, such as TST and <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>), STZ binds androgen receptor (AR) to activate AR-mediated signalling. Despite its therapeutic effects, this synthetic anabolic-androgenic steroid (AAS), or <em>5α</em>-<em>DHT</em> derivative, due to its high lipophilicity, is poor soluble in water. Thus, to increase the water solubility and stability of STZ, as well as its bioavailability and efficacy, an innovative PEGylated STZ (STZ conjugated with (MeO-PEG-NH₂)_10kDa, (MeO-PEG-NH)_10kDa-STZ) was synthesized. As confirmed by chromatography (RP-HPLC) and spectrometry (ATR-FTIR, ¹H-NMR, elemental CHNS(O) analysis, MALDI-TOF/TOF) analyses, a very pure, stable and soluble compound was obtained. Acetylcholinesterase (AChE) competitive ELISA kit demonstrated that the resulting PEGylated STZ competes against biological TST, especially at lower concentrations. Cytotoxicity of increasing concentrations (1, 10, 25 or 50 µM) of STZ and/or (MeO-PEG-NH)_10kDa-STZ was also evaluated for up 80 h by performing the MTT assay on human osteosarcoma Saos-2 cells, which express AR and are responsive to STZ. PEGylation mitigated cytotoxicity of STZ, by increasing the cell viability values, especially at higher drug concentrations. Furthermore, these results suggest that (MeO-PEG-NH)_10kDa-STZ is a promising and reliable drug to be used in clinical conditions in which TST is required.

It has previously been demonstrated that the intratumoral generation of the potent androgen dihydrotestosterone (<em>DHT</em>), contributes critically to the progression of prostate cancer and its castration-resistant form, castration-resistant prostate cancer (CRPC). Circulating testosterone is converted into <em>DHT</em> by <em>5α</em>-reductase (SRD5A). Dutasteride is a dual inhibitor of type I and II SRD5A. The present study assessed the effectiveness of dutasteride in the treatment of CRPC. Between 2010 and 2013, CRPC was diagnosed in 41 patients at the Tokyo Metropolitan Tama Medical Center. Following diagnosis, the patients received 0.5 mg dutasteride daily. The patients' median age was 77.3 years (range, 63-90). Bone metastases were recognized in 12 patients. All the patients received dexamethasone. Twenty-four (59%) patients had previously undergone chemotherapy, while 11 (27%) received docetaxel, and 24 (59%) estramustine. The prostatic-specific antigen (PSA) level declined in 17 (41%) patients from the baseline value, following dutasteride treatment. The median value for the PSA decrease was 23% (range, 4.3-89.8%), and the median duration of the response was 4 months (range, 1-10). The PSA response rate (defined as >50% decline in PSA from the baseline value) was recognized in 7 (17%) patients. The median duration of the response was 3 months (range, 2-10). Dutasteride was efficacious against CRPC in certain patients and may be a promising option in CRPC treatment. A prospective randomized trial is necessary to verify the efficacy of dutasteride.

Neoadjuvant androgen deprivation therapy (NADT) is one strategy for the treatment of early-stage prostate cancer; however, the long-term outcomes of NADT with radical prostatectomy including biochemical failure-free survival are not promising. One proposed mechanism is incomplete androgen ablation. In this study, we aimed to evaluate the efficiency of serum hydroxy-androgen suppression in patients with localized high-risk prostate cancer under NADT (leuprolide acetate plus abiraterone acetate and prednisone) and interrogate the primary sources of circulating hydroxy-androgens using our recently described stable isotope dilution liquid chromatography mass spectrometric method. For the first time, three androgen diols including 5-androstene-3β,17β-diol (5-adiol), <em>5α</em>-androstane-3α,17β-diol (3α-adiol), <em>5α</em>-androstane-3β,17β-diol (3β-adiol), the glucuronide or sulfate conjugate of 5-adiol and 3α-adiol were measured and observed to be dramatically reduced after NADT. By comparing patients that took leuprolide acetate alone vs leuprolide acetate plus abiraterone acetate and prednisone, we were able to distinguish the primary sources of these androgens and their conjugates as being of either testicular or adrenal in origin. We find that testosterone, <em>5α</em>-dihydrotestosterone (<em>DHT</em>), 3α-adiol and 3β-adiol were predominately of testicular origin. By contrast, dehydroepiandrosterone (DHEA), epi-androsterone (epi-AST) and their conjugates, 5-adiol sulfate and glucuronide were predominately of adrenal origin. Our findings also show that NADT failed to completely suppress DHEA-sulfate levels and that two unappreciated sources of intratumoral androgens that were not suppressed by leuprolide acetate alone were 5-adiol-sulfate and epi-AST-sulfate of adrenal origin.

Epidemiological studies demonstrate an apparent sex-based difference in the prevalence of asthma, with a higher risk in boys than girls, which is reversed post-puberty, where women become more prone to asthma than men, suggesting a plausible beneficial role for male hormones, especially androgens as a regulator of pathophysiology in asthmatic lungs. Using a murine model of asthma developed with mixed allergen (MA) challenge, we report a significant change in airway hyperresponsiveness (AHR), as demonstrated by increased thickness of epithelial and airway smooth muscle layers and collagen deposition, as well as Th2/Th17 biased inflammation in the airways of non-gonadectomized (non-GDX) and gonadectomized (GDX) male mice. Here, compared to non-GDX mice, MA induced AHR and inflammatory changes were more prominent in GDX mice. Activation of androgen receptor (AR) using <em>5α</em>-Dihydrotestosterone (<em>5a</em>-<em>DHT</em>, AR agonist) resulted in decreased Th2/Th17 inflammation and remodeling associated changes, resulting in improved lung function compared to MA alone challenged mice, especially in GDX mice. These changes were not observed with Flutamide (Flut, AR antagonist). Overall, we show that AR exerts a significant and beneficial role in asthma by regulating AHR and inflammation.

<strong class="sub-title"> Keywords: </strong> <em>5α</em>-Dihydrotestosterone; Flutamide; Lung function; Sex differences; Sex-steroids.

The pyrethroid insecticide, cypermethrin has been demonstrated to be an environmental anti-androgen in the androgen receptor (AR) reporter gene assay. The amino- and carboxyl-terminal (N/C) interaction is required for transcription potential of the AR. In order to characterize the anti-androgen effects of cypermethrin involved in the N/C interaction of AR, the mammalian two-hybrid assay has been developed in the study. The fusion vectors pVP16-ARNTD, pM-ARLBD and the pG5CAT Reporter Vector were cotransfected into the CV-1 cells. The assay displayed appropriate response to the potent, classical AR agonist <em>5α</em>-dihydrotestosterone (<em>DHT</em>) and known AR antagonist nilutamide. The N/C interaction was induced by <em>DHT</em> from 10(-11)M to 10(-5)M in a dose-dependent manner. Nilutamide did not activate N/C interaction, while inhibited <em>DHT</em>-induced AR N/C interaction at the concentrations from 10(-7)M to 10(-5)M. Treatment of CV-1 cells with cypermethrin alone did not activate the reporter CAT. Cypermethrin significantly decreased the <em>DHT</em>-induced reporter CAT expression at the higher concentration of 10(-5)M. The mammalian two-hybrid assay provides a promising tool both for defining mechanism involved in AR N/C interaction of EDCs and for screening of chemicals with androgen agonistic and antagonistic activities. Cypermethrin exhibits inhibitory effects on the <em>DHT</em>-induced AR N/C interaction, while the potency is weaker than that of nilutamide.

Elevated expression of the efflux transporter, ATP-binding cassette subfamily G isoform 2 (ABCG2) on the plasma membrane of cancer cells contributes to the development of drug resistance and is a key characteristic of cancer stem cells. In this study, gene expression analysis identified that treatment of the MCF-7 and T-47D breast cancer cell lines with the androgen, <em>5α</em>-dihydrotestosterone (<em>DHT</em>), and the Hedgehog signaling inhibitor, cyclopamine downregulated ABCG2 mRNA levels. In MCF-7 cells, and in Hoechst 33342(lo) /CD44(hi) /CD24(lo) breast cancer stem-like cells isolated from MCF-7 cultures, ABCG2 was accumulated in cell-to-cell junction complexes and in large cytoplasmic aggresome-like vesicles. <em>DHT</em> treatments, which decreased cellular ABCG2 protein levels, led to diminished ABCG2 localization in both cell-to-cell junction complexes and in cytoplasmic vesicles. In contrast, cyclopamine, which did not alter ABCG2 protein levels, induced accumulation of ABCG2 in cytoplasmic vesicles, reducing its localization in cell-to-cell junction complexes. The reduced localization of ABCG2 at the plasma membrane of MCF-7 cells was associated with decreased efflux of the ABCG2 substrate, mitoxantrone, and increased sensitivity of cyclopamine-treated cultures to the cytotoxic effects of mitoxantrone. Together, these findings indicate that <em>DHT</em> and cyclopamine reduce ABCG2 activity in breast cancer cells by distinct mechanisms, providing evidence to advocate the adjunct use of analogous pharmaceutics to increase or prolong the efficacy of breast cancer treatments. J. Cell. Biochem. 117: 2249-2259, 2016. © 2016 Wiley Periodicals, Inc.

This work investigated the spectrum-effect relationships between high performance liquid chromatography (HPLC) fingerprints and the anti-benign prostatic hyperplasia activities of aqueous extracts from Saxifraga stolonifera. The fingerprints of S. stolonifera from various sources were established by HPLC and evaluated by similarity analysis (SA), hierarchical clustering analysis (HCA) and principal component analysis (PCA). Nine samples were obtained from these 24 batches of different origins, according to the results of SA, HCA and the common chromatographic peaks area. A testosterone-induced mouse model of benign prostatic hyperplasia (BPH) was used to establish the anti-benign prostatic hyperplasia activities of these nine S. stolonifera samples. The model was evaluated by analyzing prostatic index (PI), serum acid phosphatase (ACP) activity, concentrations of serum dihydrotestosterone (<em>DHT</em>), prostatic acid phosphatase (PACP) and type II <em>5α</em>-reductase (SRD5A2). The spectrum-effect relationships between HPLC fingerprints and anti-benign prostatic hyperplasia activities were investigated using Grey Correlation Analysis (GRA) and partial least squares regression (PLSR). The results showed that a close correlation existed between the fingerprints and anti-benign prostatic hyperplasia activities, and peak 14 (chlorogenic acid), peak 17 (quercetin 5-O-β-d-glucopyranoside) and peak 18 (quercetin 3-O-β-l-rhamno-pyranoside) in the HPLC fingerprints might be the main active components against anti-benign prostatic hyperplasia. This work provides a general model for the study of spectrum-effect relationships of S. stolonifera by combing HPLC fingerprints with a testosterone-induced mouse model of BPH, which can be employed to discover the principle components of anti-benign prostatic hyperplasia bioactivity.

It is well known that testosterone (T) under the influence of <em>5α</em>-reductase enzyme is converted to dihydrotestosterone (<em>DHT</em>), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a-i, 6 and 7) having potential inhibitory activity against the <em>5α</em>-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for <em>5α</em>-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4 g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4 g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f-h), could have anticancer properties.

Electrophysiological recordings were used to investigate the role of the local synthesis of 17β-estradiol (E2) and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) on synaptic long-term effects induced in the hippocampal CA1 region of male rat slices. Long-term depression (LTD) and long-term potentiation (LTP), induced by different stimulation patterns, were examined under the block of the <em>DHT</em> synthesis by finasteride (FIN), and the E2 synthesis by letrozole (LET). We used low frequency stimulation (LFS) for LTD, high frequency stimulation (HFS) for LTP, and intermediate patterns differing in duration or frequency. We found that FIN reverted the LFS-LTD into LTP and enhanced LTP induced by intermediate and HFSs. These effects were abolished by exogenous <em>DHT</em> at concentration higher than the basal one, suggesting a stimulus dependent increase in <em>DHT</em> availability. No effect on the synaptic responses was observed giving <em>DHT</em> alone. Moreover, we found that the inhibition of E2 synthesis influenced the HFS-LTP by reducing its amplitude, and the exogenous E2 either enhanced HFS-LTP or reverted the LFS-LTD into LTP. The equivalence of the E2 concentration for rescuing the full HFS-LTP under LET and reverting the LFS-LTD into LTP suggests an enhancement of the endogenous E2 availability that is specifically driven by the HFS. No effect of FIN or LET was observed on the responses to stimuli that did not induce either LTD or LTP. This study provides evidence that the E2 and <em>DHT</em> availability combined with specific stimulation patterns is determinant for the sign and amplitude of the long-term effects.

Medroxyprogesterone acetate (MPA) is a first generation progestin that has been in clinical use for various hormonal conditions in women since the 1960s. Although developed as a progesterone receptor (PR) agonist, MPA also has strong binding affinity for other steroid receptors. This promiscuity confounds the mechanistic action of MPA in target cells that express multiple steroid receptors. This study is the first to assess the relative contribution of progesterone, androgen and glucocorticoid receptors in mediating the transcriptional activity of MPA on endogenous targets in breast cancer cells that endogenously express all three receptors at comparable levels. Gene expression profiling in estrogen receptor positive (ER+) ZR-75-1 breast cancer cells demonstrated that although the MPA-regulated transcriptome strongly overlapped with that of Progesterone (PROG), <em>5α</em>-dihydrotestosterone (<em>DHT</em>) and Dexamethasone (DEX), it clustered most strongly with that of PROG, suggesting that MPA predominantly acts via the progesterone receptor (PR) rather than androgen receptor (AR) or glucocorticoid receptor (GR). Subsequent experiments manipulating levels of these receptors, either through specific culture conditions or with lentiviral shRNAs targeting individual receptors, also revealed a stronger contribution of PR compared to AR and GR on the expression of endogenous target genes that are either commonly regulated by all ligands or specifically regulated only by MPA. A predominant contribution of PR to MPA action in ER + T-47D breast cancer cells was also observed, although a stronger role for AR was evident in T-47D compared to that observed in ZR-75-1 cells. Network analysis of ligand-specific and commonly regulated genes demonstrated that MPA utilises different transcription factors and signalling pathways to inhibit proliferation compared with PROG. This study reaffirms the importance of PR in mediating MPA action in an endogenous breast cancer context where multiple steroid receptors are co-expressed and has potential implications for PR-targeting therapeutic strategies in ER + breast cancer.

Bisphenol A (BPA), one of the highest production volume chemicals, is a typical endocrine-disrupting chemical (EDC) that exhibits antiandrogenic activity. However, how BPA antagonizes androgen effects remains ambiguous. In this study, the in silico and in vitro assays were carried out to explore the molecular mechanism(s) of BPA on androgen receptor (AR) antagonism. In reporter gene assay, BPA caused a significant antagonistic effect on <em>5α</em>-dihydrotestosterone (<em>DHT</em>)-induced AR transcriptional activity at concentrations of 10^-9 M-10^-5 M. The results of molecular docking and molecular dynamics simulations indicated the availability of BPA binding to the ligand binding domain of AR. BPA treatment prevented the inhibition of receptor degradation caused by <em>DHT</em> binding to AR. BPA exposure also abolished <em>DHT</em>-dependent dissociation of AR from its co-chaperone, 90-kDa heat shock protein (Hsp90), and resulted in the blockage of <em>DHT</em>-induced AR nuclear translocation. This is the first report to show that BPA inhibited the <em>DHT</em>-induced stabilization of AR and the <em>DHT</em>-induced dissociation of AR-Hsp90 complex. This study provided new evidence for further understanding the precise mechanisms of antagonism of BPA on AR.

OBJECTIVE

To evaluate the effect of finasteride on serum androst-4-ene-3,17-dione (androstenedione) and its association with prostate cancer risk among subjects who participated in the Prostate Cancer Prevention Trial.

METHODS

We analyzed serum androstenedione levels in 317 prostate cancer cases and 353 controls, nested in the Prostate Cancer Prevention Trial, a randomized placebo-controlled trial that found finasteride decreased prostate cancer risk. Androstenedione is the second most important circulating androgen in men besides testosterone and also a substrate for <em>5α</em>-reductase enzyme.

RESULTS

We observed a 22% increase in androstenedione levels compared with the baseline values in subjects who were treated with finasteride for 3 years. This significant increase did not vary by case-control status. Adjusted odds ratio and 95% confidence interval for the third tertile of absolute change in androstenedione levels compared with the first tertile were 0.42 (95% confidence interval, 0.19-0.94) for low-grade (Gleason score <7) cases. Similar results were observed when analyzed using percent change. There were no significant associations between serum androstenedione levels and the risk of high-grade disease.

CONCLUSIONS

The results of this nested case-control study confirm that finasteride blocks the conversion of testosterone to dihydrotestosterone (DHT) and of androstenedione to <em>5α</em>-androstanedione-3,17-dione, which also leads to the reduction of DHT formation. This decrease in DHT may help reduce the risk of low-grade prostate cancer in men. Our data on a differential effect of androstenedione also suggest that some high-grade prostate cancers may not require androgen for progression.

Castration resistant prostate cancer (CRPC), the fatal form of prostate cancer, remains androgen dependent despite castrate levels of circulating testosterone (T) and <em>5α</em>-dihydrotestosterone (<em>DHT</em>). To investigate mechanisms by which the tumor can synthesize its own androgens and develop resistance to abiraterone acetate and enzalutamide, methods to measure a complete androgen profile are imperative. Here, we report the development and validation of a stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometric (SID-LC-ESI-MS/MS) method to quantify nine human hydroxy-androgens as picolinates, simultaneously with requisite specificity and sensitivity. In the established method, the fragmentation patterns of all nine hydroxy-androgen picolinates were identified, and [13C3]-<em>5α</em>-androstane-3α, 17β-diol and [13C3]-<em>5α</em>-androstane-3β, 17β-diol used as internal standards were synthesized enzymatically. Intra-day and inter-day precision and accuracy corresponds to the U.S. Food and Drug Administration Criteria for Bioanalytical Method Validation. The lower limit of quantitation (LLOQ) of nine hydroxy-androgens is 1.0pg to 2.5pg on column. Diols which have been infrequently measured: 5-androstene-3β, 17β-diol and <em>5α</em>-androstane-3α, 17β-diol can be determined in serum at values as low as 1.0pg on column. The method also permits the quantitation of conjugated hydroxy-androgens following enzymatic digestion. While direct detection of steroid conjugates by electrospray-ionization tandem mass spectrometry has advantages the detection of unconjugated and conjugated steroids would require separate methods for each set of analytes. Our method was applied to pooled serum from male and female donors to provide reference values for both unconjugated and conjugated hydroxy-androgens. This method will allow us to interrogate the involvement of the conversion of 5-androstene-3β, 17β-diol to T, the backdoor pathway involving the conversion of <em>5α</em>-androstane-3α, 17β-diol to <em>DHT</em> and the inactivation of <em>DHT</em> to <em>5α</em>-androstane-3β, 17β-diol in advanced prostate cancer.

Changes of androgen receptor (AR) and insulin-like growth factor-1 (IGF-1) were investigated in LNCaP cells treated with <em>5α</em>-dihydrotestosterone (<em>DHT</em>), estrone and flutamide. Real-time PCR, immunocytochemistry and western blotting were used to detect the expression of AR and IGF-1 in the presence or absence of various kinase inhibitors. Low concentrations of <em>DHT</em>, estrone and flutamide increased the expression of AR and IGF-1, especially estrone, with concentration and time dependence. With <em>DHT</em> and flutamide, there was a significant alteration in AR expression (p<0.001). The results indicated expression of AR and IGF-1 genes to be influenced by <em>DHT</em>, estrone and flutamide in LNCaP cells, regulated by multiple signal pathways.

Literature supports findings about a gender specific outcome following multiple trauma. Male sex hormones such as dihydrotestosterone (<em>DHT</em>) exert deleterious effects on the posttraumatic immune response whereas increased estradiol concentrations are correlated with improved outcome. Pretreatment with the <em>5α</em>-reductase inhibitor finasteride resulted in an improved outcome following trauma-hemorrhage (TH) in mice. The present study tested the hypothesis that finasteride exerts beneficial effects on the posttraumatic immune response also in a combined setting of TH and sepsis when administered during the resuscitation process.

METHODS

Male C57BL/6N-mice were subjected to TH (blood pressure, 35 mm Hg, 60 min) followed by finasteride application and fluid resuscitation. Thereafter, finasteride was administered every 12h. 24h after TH, sepsis was induced by cecal ligation and puncture (CLP) or sham operation was performed. Plasma cytokines (MIP-1α, MIP-1β, TNF-α, MCP-1, IL-6), productive capacity by alveolar macrophages (AM) and systemic estradiol levels were determined 4 h thereafter. The expression of pro-inflammatory mediators in lung tissue was evaluated by PCR. Pulmonary infiltration of PMN was determined by immunohistochemical staining.

RESULTS

Finasteride treatment resulted in a reduced posttraumatic cytokine secretion of AM as well as in a decreased concentration of MCP-1 and MIP-1β in lung tissue. Systemic estradiol levels were increased following finasteride treatment.

CONCLUSIONS

Finasteride mediates salutary effects on the pulmonary immune response using a therapeutical approach following TH-CLP in mice. Thus, finasteride might represent a relevant therapeutic substance following major trauma also in the clinical setting.

In adult songbirds, the telencephalic song nucleus HVC and its efferent target RA undergo pronounced seasonal changes in morphology. In breeding birds, there are increases in HVC volume and total neuron number, and RA neuronal soma area compared to nonbreeding birds. At the end of breeding, HVC neurons die through caspase-dependent apoptosis and thus, RA neuron size decreases. Changes in HVC and RA are driven by seasonal changes in circulating testosterone (T) levels. Infusing T, or its metabolites <em>5α</em>-dihydrotestosterone (<em>DHT</em>) and 17 β-estradiol (E2), intracerebrally into HVC (but not RA) protects HVC neurons from death, and RA neuron size, in nonbreeding birds. The phosphoinositide 3-kinase (PI3K)-Akt (a serine/threonine kinase)-mechanistic target of rapamycin (mTOR) signaling pathway is a point of convergence for neuroprotective effects of sex steroids and other trophic factors. We asked if mTOR activation is necessary for the protective effect of hormones in HVC and RA of adult male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii). We transferred sparrows from breeding to nonbreeding hormonal and photoperiod conditions to induce regression of HVC neurons by cell death and decrease of RA neuron size. We infused either <em>DHT</em> + E2, <em>DHT</em> + E2 plus the mTOR inhibitor rapamycin, or vehicle alone in HVC. Infusion of <em>DHT</em> + E2 protected both HVC and RA neurons. Coinfusion of rapamycin with <em>DHT</em> + E2, however, blocked the protective effect of hormones on HVC volume and neuron number, and RA neuron size. These results suggest that activation of mTOR is an essential downstream step in the neuroprotective cascade initiated by sex steroid hormones in the forebrain.

<AbstractText>What prevents the fall in anti-Müllerian hormone (AMH) levels in polycystic ovary syndrome (PCOS) and what are the consequences of this for follicle progression in these ovaries?</AbstractText><AbstractText>Exposure of granulosa cells (GCs) to high levels of androgens, equivalent to that found in PCOS, prevented the fall in AMH and was associated with dysregulated AMH-SMAD signalling leading to stalled follicle progression in PCOS.</AbstractText><AbstractText>In normal ovaries, AMH exerts an inhibitory role on antral follicle development and a fall in AMH levels is a prerequisite for ovulation. Levels of AMH are high in PCOS, contributing to the dysregulated follicle growth that is a common cause of anovulatory infertility in these women.</AbstractText><AbstractText>Human KGN-GC (the cell line that corresponds to immature GC from smaller antral follicles (AF)) were cultured with a range of doses of various androgens to determine the effects on AMH production. KGN-GC were also treated with PHTPP (an oestrogen receptor β (ERβ) antagonist) to examine the relationship between AMH expression and the ratio of ERα:ERβ. The differential dose-related effect of AMH on gene expression and SMAD signalling was investigated in human granulosa-luteal cells (hGLC) from women with normal ovaries, with polycystic ovarian morphology (PCOM) and with PCOS. KGN-GC were also cultured for a prolonged period with AMH at different doses to assess the effect on cell proliferation and viability.</AbstractText><AbstractText>AMH protein production by cells exposed to androgens was measured by ELISA. The effect of PHTPP on the mRNA expression levels of AMH, ERα and ERβ was assessed by real-time quantitative PCR (qPCR). The influence of AMH on the relative mRNA expression levels of aromatase, AMH and its receptor AMHRII, and the FSH and LH receptor (FSHR and LHR) in control, PCOM and PCOS hGLCs was quantified by qPCR. Western blotting was used to assess changes in levels of SMAD proteins (pSMAD-1/5/8; SMAD-4; SMAD-6 and SMAD-7) after exposure of hGLCs from healthy women and women with PCOS to AMH. The ApoTox-Glo Triplex assay was used to evaluate the effect of AMH on cell viability, cytotoxicity and apoptosis.</AbstractText><AbstractText>Testosterone reduced AMH protein secreted from KGN-GC at 10-9-10-7 M (P < 0.05; P < 0.005, multiple uncorrected comparisons Fishers least squares difference), but at equivalent hyperandrogenemic levels no change was seen in AMH levels. <em>5α</em>-<em>DHT</em> produced a significant dose-related increase in AMH protein secreted into the media (P = 0.022, ANOVA). Increasing the mRNA ratio of ERα:ERβ produced a corresponding increase in AMH mRNA expression (P = 0.015, two-way ANOVA). AMH increased mRNA levels of aromatase (P < 0.05, one-way ANOVA) and FSHR (P < 0.0001, one-way ANOVA) in hGLCs from women with PCOM, but not from normal cells or PCOS (normal n = 7, PCOM n = 5, PCOS n = 4). In contrast to hGLCs from ovulatory ovaries, in PCOS AMH reduced protein levels (cell content) of stimulatory pSMAD-1/5/8 and SMAD-4 but increased inhibitory SMAD-6 and -7 (P < 0.05, normal n = 6, PCOS n = 3). AMH at 20 and 50 ng/ml decreased KGN-GC cell proliferation but not viability after 8 days of treatment (P < 0.005, two-way ANOVA).</AbstractText><AbstractText>N/A.</AbstractText><AbstractText>Luteinised GC from women undergoing IVF have a relatively low expression of AMH/AMHRII but advantageously continue to display responses inherent to the ovarian morphology from which they are collected. To compensate, we also utilised the KGN cell line which has been characterised to be at a developmental stage close to that of immature GC. The lack of flutamide influence on testosterone effects is not in itself sufficient evidence to conclude that the effect on AMH is mediated via conversion to oestrogen, and the effect of aromatase inhibitors or oestrogen-specific inhibitors should be tested. The effect of flutamide was tested on testosterone but not <em>DHT</em>.</AbstractText><AbstractText>Normal folliculogenesis and ovulation are dependent on the timely reduction in AMH production from GC at the time of follicle selection. Our findings reveal for the first time that theca-derived androgens may play a role in this model but that this inhibitory action is lost at levels of androgens equivalent to those seen in PCOS. The AMH decline may either be a direct effect of androgens or an indirect one via conversion to oestradiol and acting through the upregulation of ERα, which is known to stimulate the AMH promoter. Interestingly, the ability of GCs to respond to this continually elevated AMH level appears to be reduced in cells from women with PCOS due to an adaptive alteration in the SMAD signalling pathway and lower expression of AMHRII, indicating a form of 'AMH resistance'.</AbstractText><AbstractText>This study was funded by the Thomas Addison Scholarship, St Georges Hospital Trust. The authors report no conflict of interest in this work and have nothing to disclose.</AbstractText><AbstractText>N/A.</AbstractText>

Anti-androgenic substances, mainly prostate <em>5α</em>-reductase inhibitors, used in the treatment of benign prostatic hyperplasia (BPH) have been associated with side effects in man and animals. To reduce these side effects as well as suppress BPH development, the management of the condition has come to include dietary interventions. This study investigated the effect of some cooking oils on testosterone-induced hyperplasia of the prostate in rats. Male Sprague-dawley rats were distributed into eighteen groups (n=6) as A-R. A negative control group was injected subcutaneously with soya oil; while prostatic hyperplasia was induced subcutaneously in groups B-R with 3mg/kg testosterone daily for 14days. Group B was the positive control (BPH group) while groups C-R were also administered orally 800mg/kg of coconut, castor, canola, cottonseed, pomegranate, blackseed, sheabutter, olive oil, codliver, sardine, palm, repeatedly heated palm (RHPO), vegetable, repeatedly-heated vegetable (RHVO), sesame, and groundnut oils respectively, daily, for 14 days. Blood sample was drawn via retro-orbital sinus for the estimation of serum testosterone(T) and dihydrotestosterone (<em>DHT</em>) level and rats were thereafter euthanized to obtain the prostates for T and <em>DHT</em> determination as well as tissue weights. Data are mean ± SEM, compared by ANOVA. The oils significantly reduced the increase in prostate weight (PW) to body weight (BW) ratio induced by testosterone. Apart from the fact that all the oils reduced the PW:BW ratio, the blackseed, sheabutter, sardine, vegetable and groundnut oils suppressed the <em>DHT</em> level in the serum, while pomegranate, olive, RHPO reduced <em>DHT</em> level in the prostate compared to the BPH rats. This study suggests that blackseed, sheabutter, sardine, vegetable, groundnut, pomegranate, olive, and RHPO oils could inhibit testosterone-induced hyperplasia of the prostate and therefore may be beneficial in the management of BPH.

We performed an exploratory study by analyzing the correlation of 46, XY disorders of sex development (46, XY DSD) with androgen receptor (AR) and steroid <em>5α</em>-reductase-2 (SRD5A2) gene mutations and a safety analysis of dihydrotestosterone (<em>DHT</em>) gel treatment for pediatric micropenis. We collected samples from 76 pediatric patients with 46, XY DSD and 50 healthy adult men with normal fertility as the control group. The pediatric patients were treated with <em>DHT</em> gel (0.1-0.3 mg/kg/day) for three to six months. The extended penis length, testicular volume, and multiple blood parameters were collected before treatment and one, three, and six months after treatment. Of the 76 cases with 46, XY DSD, 31.58% had hypospadias with micropenis and 6.58% had male pseudohermaphroditism. Through AR gene screening, it was found that 14 patients had AR point mutations and 22 patients had SRD5A2 mutations. After treatment with <em>DHT</em>, the penis length of the patients significantly improved after one, three, and six months of treatment, with longer treatment times resulting in greater improvement. Before treatment with <em>DHT</em>, the average serum <em>DHT</em> value of patients with 46, XY DSD was 24.29 pg/mL. After one, three, and six months of treatment, this value increased to 430.71, 328.9, and 323.6 pg/mL, respectively. We conclude that for pediatric patients who have male hermaphroditism or hypospadias with micropenis, AR and SRD5A2 gene mutation detection should be performed. Local application of <em>DHT</em> gel can promote penis growth effectively without systemic adverse reactions.