Cortexolone 17α-propionate (clascoterone) is a novel androgen antagonist that is currently being analyzed in a large phase 2 clinical trial for the topical treatment of androgenetic alopecia (AGA). While the pathogenesis of AGA is still debated, the consensus is that AGA is an androgen-dependent hair disorder with strong genetic links, and that the testosterone metabolite, dihydrotestosterone (<em>DHT</em>), plays a causal role in its development. <em>DHT</em> binds to the androgen receptor (AR) in scalp dermal papilla cells (DPC) to induce AR-mediated transcription of genes that contribute to AGA in genetically predisposed individuals.Several studies have established that clascoterone is a potent antiandrogen that is well tolerated and has selective topical activity. The study described herein elucidates a potential mechanism of clascoterone in AGA. Clascoterone was found to inhibit AR-regulated transcription in a reporter cell line with similar efficacy to the <em>5α</em>-reductase inhibitor, finasteride. More importantly, when compared with another direct AR antagonist, enzalutamide, clascoterone was significantly better at inhibiting IL-6 synthesis from <em>DHT</em>-stimulated primary cultures of human scalp DPC. Therefore, clascoterone may be an excellent candidate to be the first topical antiandrogen for treating AGA.J Drugs Dermatol. 2019;18(2):197-201.

SEPTIN12 (SEPT12) is a testis-enriched gene that is downregulated in the testis of infertile men with severe spermatogenic defects. While SEPT12 is involved in spermatogenic failure and sperm motility disorder, SEPT12 transcriptional regulation is still unknown. Here we report the promoter region of SEPT12 as a 245 bp segment upstream of the transcription start site. One androgen receptor (AR) and two estrogen receptor α (ERα) binding sites in this region were initially identified by bioinformatics prediction and confirmed by chromatin immunoprecipitation assay. Truncated ERα or AR binding sites decreased the promoter activity, which indicated that the ERα and AR are essential for the SEPT12 promoter. On the other hand, the promoter activity was enhanced by the treatment with 17β-estradiol (E2) and <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>). Thus, one androgen and two estrogen hormone responsive elements located in the promoter of SEPT12 gene can regulate SEPT12 expression. Two single nucleotide polymorphisms (SNPs), rs759992 T > C and rs3827527 C > T, were observed in the SEPT12 gene promoter region and were able to decrease the promoter activity. In conclusion, the current work identified the promoter of the human SEPT12 gene and provided key evidence about its transcriptional regulation via E2 and <em>5α</em>-<em>DHT</em>. Since SEPT12 has an important role in spermatogenesis, SEPT12 expression analysis can be developed as a potential tool for the assessment of environmental or food pollution by hormones or for the evaluation of the risk of endocrine-disrupting chemicals (EDCs) in general.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is the gold-standard approach for androgen analysis in biological fluids, superseding immunoassays in selectivity, particularly at low concentrations. While LC-MS/MS is established for analysis of testosterone and androstenedione, <em>5α</em>-dihydrotestosterone (<em>DHT</em>) presents greater analytical challenges. <em>DHT</em> circulates at low nanomolar concentrations in men and lower in women, ionizing inefficiently and suffering from isobaric interference from other androgens. Even using current LC-MS/MS technology, large plasma volumes (>0.5 mL) are required for detection, undesirable clinically and unsuitable for animals. This study investigated derivatization approaches using hydrazine-based reagents to enhance ionization efficiency and sensitivity of analysis of <em>DHT</em> by LC-MS/MS. Derivatization of <em>DHT</em> using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A method was validated using an UHPLC interfaced by electrospray with a triple quadruple mass spectrometer , analyzing human plasma (male and post-menopausal women) following solid-phase extraction. HMP derivatives were selected for validation affording greater sensitivity than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (<em>DHT</em>-HMP m/z 396→108; testosterone-HMP m/z 394→108; androstenedione-HMP m/z 392→108). Chromatographic separation of androgen derivatives was optimized, carefully separating isobaric interferents and acceptable outputs for precision and trueness achieved following injection of 0.4 pg on column (approximately 34 pmol/L). HMP derivatives of all androgens tested could be detected in low plasma volumes: male (100 µL) and post-menopausal female (200 µL), and derivatives were stable over 30 days at -20°C. In conclusion, HMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of <em>DHT</em>, testosterone and androstenedione in low plasma volumes, offering advantages in sensitivity over current methodologies.

<strong class="sub-title"> Keywords: </strong> <em>5α</em>-Dihydrotestosterone; Androstenedione; Derivatization; Liquid chromatography mass spectrometry; Testosterone.

Dehydroepiandrosterone (DHEA), testosterone (TES) and its 5-reduced metabolites induce a nongenomic vasorelaxation in several vascular beds of mammals; similarly these hormones produce systemic hypotensive and antihypertensive responses in normotensive and hypertensive male rats. Thus, it was hypothesized that the antihypertensive response of androgens, whose levels are elevated during gestation, protect against gestational hypertension. An animal model of preeclampsia was induced in female Wistar rats using DOCA-salt-treated pregnant (PT) and normal pregnant (NP) rats. In vivo experiments in conscious rats revealed that bolus intravenous injections of DHEA, TES, <em>5α</em>- or 5β-dihydrotestosterone (-<em>DHT</em>) log -1.0 to 2.0μmolk-1min-1, produced substantial transient reductions in arterial blood pressure (BP), without significant changes in heart rate (HR). Mean arterial blood pressure (MAP) was reduced significantly in both groups. PT rats were more sensitive to the antihypertensive responses of androgens than NP. DHEA and 5β-<em>DHT</em> were the most potent to reduce MAP: 66±07 and 69±2.0mmHg in PT but only 33±0.5 and 35±1.2mmHg in NP rats, respectively. In isolated aortas of PT and NP, the concentration-response curves to each androgen (0.1-100μM) indicated that KCl-induced pre-contraction is more sensitive to all androgens than phenylephrine (Phe) pre-contractions. Notably, 5β-<em>DHT</em> is the greatest vasorelaxant with KCl-induced contraction than with Phe contraction of both groups, suggesting a preferential blockade on L-VOCCs. TES exhibited minor vasorelaxing effect of aortas pre-contracted with KCl, compared to its precursor DHEA and its 5-reduced metabolites. These data show that these androgens exert acute vasorelaxing effects in vitro and remarkably, reduce the BP in vivo in PT and NP at term pregnancy. Moreover, a deficit in feto-placental androgen production during pregnancy may trigger the development of preeclampsia or gestational hypertension.

Hyper androgen state frequently can be diagnosed in bulimic women. Eating disorder not otherwise specified (EDNOS) recognized as a less severe form of bulimia nervosa (BN). The objective of the study was to determine whether androgen levels and androgen origin differs in bulimic women compared to control subjects. Forty-six women with bulimia nervosa (BN), 31 with eating disorder not otherwise specified, purging type (EDNOS P) and 56 matched healthy controls were studied with respect to serum testosterone (T), 5alpha-dihydrotestosterone (<em>DHT</em>), sex hormone-binding globulin (SHBG), deyhydroepiahndrosterone sulfate (DHEAS) and luteinizing hormone (LH) and to ovarian morphology. Despite all groups had almost identical androgen and SHBG levels; there were differences in the origin of circulating T and <em>DHT</em>. Correlation analysis suggest major differences in the formation of circulating testosterone (T) and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) with BN being more like the control subjects with peripheral formation from 4-androsterne-3,17-dione (A-4), dehydroepiandrosterone sulfate (DHEAS) and also from T. While in EDNOS group a possible direct ovarian T secretion and a DHEAS modulating action of androgens on pituitary gonadotropin secretion is present. The origin of circulating T and <em>DHT</em> differs between bulimics. Our findings do probably not reflect direct actions of circulating <em>DHT</em> on pituitary LH secretion in the women with EDNOS, but rather the effect of A-4, T via conversion to <em>DHT</em> in the central nervous system, indicating psych/endocrine differences between the two groups of bulimic women.

Tetrabromoethylcyclohexane (TBECH), an additive brominated flame retardant, has been shown to have an androgenic activity in vitro. In the present study, we aimed to investigate the effects of TBECH on gonadal differentiation and development in the frog Pelophylax nigromaculatus, an amphibian species sensitive to androgenic chemicals, and to assess the androgenic activity of TBECH in vivo. P. nigromaculatus tadpoles were exposed to TBECH (1, 10, 100nM) from Gosner stage 24 to complete metamorphosis, and to <em>5α</em>-dihydrotestosterone (<em>DHT</em>) as a positive control. We found that 1nM <em>DHT</em> resulted in 100% males, while the sex ratio in the solvent control group was close to 1:1. In all the TBECH treatment groups, sexually ambiguous gonads based on gross morphology and intersexualities with testicular and ovarian histological structures were found, but no abnormality occurred in the solvent control. In the 1, 10, 100nM TBECH treatment groups, the female percentages were 52%, 31%, 17%, with 36%, 56%, 66% for males and 12%, 13%, 17% for abnormal sexes, respectively. X2-test revealed significant differences in sex ratios between the three TBECH groups and the solvent control group, and the sex ratios in the two higher concentration groups were male-biased. These observations show that TBECH has a masculinizing effect on gonadal differentiation and development in P. nigromaculatus, suggesting an androgenic activity of TBECH in vivo. To our knowledge, this is the first study demonstrating that TBECH could induce gonadal masculinization in an animal, which raises new concerns for reproductive risk of TBECH exposure.

9-cis-Retinoic acid (9cRA), which binds to both retinoic acid receptors and retinoic X receptors, inhibits prostate cancer induction in rats and reduces growth of prostate cancer cells. However, the nature of this growth inhibition and the interactive influence of androgens are not well defined and are the subject of this report. LNCaP and PC-3 cells were cultured and treated with a range of 9cRA concentrations for 3-6 days in the absence or presence of <em>5α</em>-dehydrotestosterone. 9cRA inhibited cell proliferation in a dose-dependent manner, plateauing at 10 mol/l. Treatment of cells with 10 mol/l 9cRA inhibited <em>5α</em>-dihydroxytestosterone (<em>DHT</em>)-stimulated proliferation, the effect of which was maximal at 10 mol/l <em>DHT</em>. Treatment of <em>DHT</em> (10 mol/l)-exposed cells with 9cRA caused a dose-dependent increase in prostate-specific antigen in the medium after 6 days, but not 3 days. 9cRA caused a dose-dependent increase in apoptotic cells stained with H33258 after 3 days, but not 6 days; however, on using flow cytometry, apoptosis was apparent at both 3 and 6 days. Flow cytometry also revealed interference of G0/G1 to S phase transition by 9cRA. Inhibition by 9cRA of anchorage-independent growth of PC-3 cells was also found; LNCaP cells did not grow colonies in soft agar. 9cRA inhibited growth and induced differentiation of human LNCaP prostate cancer cells in vitro and inhibited anchorage-independent growth of PC-3 cells. Because 9cRA and 13-cis-retinoic acid, which is retinoic acid receptor-selective, prevent prostate carcinogenesis in rats, and 13-cis-retinoic acid also inhibits growth of human prostate cancer cells, the RAR is a potential molecular target for prostate cancer prevention and therapy.

<strong class="sub-title"> Background: </strong> The formation of the male urethra depends to enzyme-mediated testosterone (T) conversion into <em>5α</em>-dihydrotestosterone (<em>DHT</em>). Two metabolic pathways could be operating in the fetal testis to synthesize androgens: 1) the "classic" route (T→<em>DHT</em>) mediated by SRD5A2 and 2) a "backdoor" pathway in which <em>DHT</em> is synthesized by aldo-keto reductase family 1, member C2 (AKR1C2), AKR1C3, and AKR1C4 enzymes without formation of a T intermediate.

Objective: We studied four genes of the "backdoor" pathway in karyotypic males with hypospadias to ascertain whether gene defects in AKRs impair urethral DHT formation that result in hypospadias.

Design and patients: The coding regions of the AKR1C2-4 and HSD17B6 genes were analyzed by PCR-SSCP and sequencing in a cohort of 25 Mexican patients (0.3-9 year-old-children) with 46,XY-hypospadias. Chi-squared tests was performed to evaluate the distribution of genotypes, alleles, and the Hardy-Weinberg (H-W) equilibrium. The effect of the genetic variants was investigated by in silico studies.

Results: Screening studies revealed distinct genotypic patterns at different exons of AKR1C2-4 whereas HSD17B6 presented a wild-type sequence. The DNA analyses detected two synonymous variants (c.327C>T, c.666T>C/unreported) in AKR1C2. The AKR1C3 had two variants (c.15C>G, c.230A>G), two unreported variants (c.538T>C, c.596G>A), and one silent variant (c.312G>A). Two variants (c.434C>G, c.931C>G) were identified in AKR1C4. All variants were in H-W equilibrium without structural changes.

<strong class="sub-title"> Discussion: </strong> Hypospadias have been associated with defects that alter androgen biosynthesis in the human fetal testis, specifically <em>5α</em>-<em>DHT</em>. We selected four candidate genes involved in the "backdoor" pathway for the formation of <em>5α</em>-<em>DHT</em>. Molecular assays of the AKR1C2, AKR1C3, and AKR1C4 genes revealed a total of nine genetic single nucleotide variants. Several variants in the AKR1C genes have been associated with a variety of human pathologies. However, our studies suggest that active steroid biosynthesis via AKR1C might not be involved in hypospadias. Additionally, genetic research suggests a low involvement in the "backdoor" <em>5α</em>-<em>DHT</em> pathway during human sexual development, specifically, the differentiation of male external genitalia.

Conclusion: These results indicate that substitutions in AKR1C2-4 are polymorphisms and all genetic variants lacks deleterious significant association with hypospadias. The data suggest that inactivating mutations in the AKR1C2-4 and HSD17B6 genes are an infrequent cause of hypospadias, which might weaken the contribution of the "backdoor" pathway to embryonic urethral masculinization.

Keywords: 46,XY Disorders of sex development; Backdoor pathway; DHT; Hypospadias; Single-nucleotide polymorphism; Steroidogenesis.

Androgenetic alopecia, a major cause for baldness, is caused by the deposition of dihydrotestosterone (<em>DHT</em>) at the androgen receptors present in the pilosebaceous unit (PSU). Finasteride (FIN) is a potent <em>5α</em>-reductase inhibitor capable of preventing the conversion of testosterone to <em>DHT</em>. But, its oral administration in males causes infertility. An attempt was made to prepare ethosomes of FIN with a size range 100-300 nm to enhance its delivery to the PSU. Finasteride loaded ethosomes (FES) were prepared using an ultra-probe sonicator and characterized for its size, morphology, surface charge and entrapment efficiency. The ability of FES to permeate across rat skin and frontal scalp skin of human cadaver was also evaluated. The spherical shaped ethosomes of different batches were in the size range of 107.8 ± 2.50 to 220.4 ± 6.92 nm and showed good permeation across rat skin and frontal scalp skin of human cadaver when compared to the unencapsulated FIN. The results portrayed the ability of FES to permeate across the stratum corneum to reach the PSU of the hair follicle. Although additional use of permeation enhancer increases the permeation of FIN across the skin, its addition may not be a favourable option for the deposition of ethosomes in the PSU.

Inactivating mutations of the <em>5α</em>-steroid reductase type-2 (SRD5A2) gene result in a broad spectrum of masculinization defects, ranging from a male phenotype with hypospadias to a female phenotype with Wolffian structures. Molecular studies of the SRD5A2 revealed a new heterozygous gene variant within the coding region that results in phenotypic expression. A c.92C>T transition changing serine to phenylalanine at codon 31 of exon 1 (p.Ser31Phe) was identified in a patient with 46,XY disorder of sexual development who displayed glandular hypospadias with micropenis and bilateral cryptorchidism. The restoration of the p.Ser31Phe mutation by site-directed mutagenesis and transient expression assays using cultured HEK-293 cells showed that this novel substitution does not abolish but does deregulate the catalytic efficiency of the enzyme. Thus, the maximum velocity (V max) value was higher for the mutant enzyme (22.5 ± 6.9 nmol <em>DHT</em> mg protein(-1) h(-1)) than for the wild-type enzyme (9.8 ± 2.0 nmol <em>DHT</em> mg protein(-1) h(-1)). Increased in vitro activity of the p.Ser31Phe mutant suggested an activating effect. This case provides evidence that heterozygous missense mutations in SRD5A2 may induce the abnormal development of male external genitalia.

The androgens testosterone (T) and dihydrotestosterone (<em>DHT</em>), besides playing an important role in prostate development and growth, are also responsible for the development and progression of benign prostate hyperplasia (BPH) and prostate cancer. Therefore, the actions of these hormones can be antagonized by preventing the irreversible conversion of T into <em>DHT</em> by inhibiting <em>5α</em>-reductase (<em>5α</em>-R). This has been a useful therapeutic approach for the referred diseases and can be achieved by using <em>5α</em>-reductase inhibitors (RIs). Steroidal RIs, finasteride and dutasteride, are used in clinic for BPH treatment and were also proposed for chemoprevention of prostate cancer. Nevertheless, due to the increase in bone and muscle loss, impotency and occurrence of high-grade prostate tumours, it is important to seek for other potent and specific molecules with lower side effects. In the present work, we designed and synthesized steroids with the 3-keto-Δ(4) moiety in the A-ring, as in the <em>5α</em>-R substrate T, and with carboxamide, carboxyester or carboxylic acid functions at the C-17β position. The inhibitory <em>5α</em>-R activity, in human prostate microsomes, as well as the anti-proliferative effects of the most potent compounds, in a human androgen-responsive prostate cancer cell line (LNCaP cells), were investigated. Our results showed that steroids 3, 4 and 5 are good RIs, which suggest that C-17β lipophylic amides favour <em>5α</em>-R inhibition. Moreover, these steroids induce a decrease in cell viability of stimulated LNCaP cells, in a <em>5α</em>-R dependent-manner, similarly to finasteride.

Steroid <em>5α</em>-reductase type 2 deficiency (<em>5α</em>-RD2) is a rare autosomal recessive inherited disorder caused by mutations in the SRD5A2 gene. Its clinical features and pathogenesis in Chinese patients are poorly understood. This study aimed to characterize the clinical features and genetically analyze the SRD5A2 gene in three Chinese <em>5α</em>-RD2 patients. The patients were characterized by ambiguous genitalia and spontaneous virilization without breast development at puberty. Elevated post-human chorionic gonadotropin stimulation T/<em>DHT</em> ratios were useful indicators of <em>5α</em>-RD2 (with ratios of 20.4, 20.1, and 26.6 in the three patients, respectively). Two compound heterozygous mutations in the SRD5A2 gene were identified: p.G203S/p.R246Q in patients 1 and 2 and p.G203S/c.655delT in patient 3. The father and the mother of patients 1 and\xa02 were carriers of p.R246Q and p.G203S, respectively. p.G203S appears to be common in Chinese <em>5α</em>-RD2 patients. Early genetic analysis should be performed in suspected patients to improve prognosis.

We aimed to survey the monogenic causes of disorders of sex development (DSD) and thereby its prevalence in India. This study revealed mutations resulting in androgen insensitivity syndrome, <em>5α</em>-reductase type 2 deficiency, and gonadal dysgenesis were commonly reported. Intriguingly, AR deficits were the most prevalent (32 mutations) and of 11/26 missense mutations were in exons 4-8 (encoding ligand binding domain). The unique features of SRD5A2 defects were p.R246Q (most prevalent) and p.G196S could be mutational hotspots, dual gene defects (p.A596T in AR and p.G196S in SRD5A2) in a patient with hypospadias and novel 8 nucleotide deletion (exon 1) found in a patient with perineal hypospadias. Deficits in SRY, WT1, DHH, NR5A1, and DMRT1 caused 46,XY gonadal dysgenesis. Notably, mutations in AR, SRD5A2, MAMLD1, WT1, and MAP3K1 have led to hypospadias and only one CYP19A1 mutation caused aromatase deficiency was reported to date. Data mining from various databases has not only reinforced the role of well-established genes (e.g., SRY, WT1, DHH, NR5A1, DMRT1, AR, SRD5A2, MAMLD1) involved in DSD but also provided us 12 more potential candidate genes (ACVR1, AMHR2, CTNNB1, CYP11A1, CYP19A1, FGFR2, FGF9, PRKACA, PRKACG, SMAD9, TERT, ZFPM2), which benefit from a close association with the well-established genes involved in DSD and might be useful to screen owing to their direct gene-phenotype relationship or through direct functional interaction. As more genes have been revealed in relation to DSD, we believe ultimately it holds a better scenario for therapeutic regimen. Despite the advances in translational medicine, hospitals are yet to adopt genetic testing and counseling facilities in India that shall have potential impact on clinical diagnosis. Abbreviations: <em>5α</em>-RD2: <em>5α</em>-Reductase type 2; AIS: androgen insensitivity syndrome; AMH: antimullerian hormone; AMHR: antimullerian hormone receptor; AR: androgen receptor gene; CAH: congenital adrenal hyperplasia; CAIS: complete AIS; CAH: congenital adrenal hyperplasia; CHH: congenital hypogonadotropic hypogonadism; CXORF6: chromosome X open reading frame 6 gene; CYP19A1: cytochrome P450 family 19 subfamily A member 1 gene; <em>DHT</em>: dihydrotestosterone; DMRT1: double sex and mab-3 related transcription factor 1 gene; DSD: disorders of sexual development; GD: gonadal dysgenesis; HGMD: human gene mutation database; IH: isolated hypospadias; MAMLD1: mastermind like domain containing 1 gene; MIS: mullerian inhibiting substance; NTD: N-terminal domain; OT DSD: ovotesticular DSD; PAIS: partial AIS; SOX9: SRY-related HMG-box 9 gene; SRY: sex-determining region Y gene; STAR: steroidogenic acute regulatory protein gene; SRD5A2: steroid 5 alpha-reductase 2 gene; T DSD: testicular DSD; T: testosterone; WNT4: Wnt family member 4 gene; WT1: Wilms tumor 1 gene; Δ₄: androstenedione.

<em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) is the most potent natural androgen. <em>5α</em>-<em>DHT</em> elicits a multitude of physiological actions, in a host of tissues, including prostate, seminal vesicles, hair follicles, skin, kidney, and lacrimal and meibomian glands. However, the physiological role of <em>5α</em>-<em>DHT</em> in human physiology, remains questionable and, at best, poorly appreciated. Recent emerging literature supports a role for <em>5α</em>-<em>DHT</em> in the physiological function of liver, pancreatic b-cell function and survival, ocular function and prevention of dry eye disease and kidney physiological function. Thus, inhibition of <em>5α</em>-reductases with finasteride or dutasteride to reduce <em>5α</em>-<em>DHT</em> biosynthesis in the course of treatment of benign prostatic hyperplasia (BPH) or male pattern hair loss, known as androgenetic alopecia (AGA) my induces a novel form of tissue specific androgen deficiency and contributes to a host of pathophysiological conditions, that are yet to be fully recognized. Here, we advance the concept that blockade of <em>5α</em>-reductases by finasteride or dutasteride in a mechanism-based, irreversible, inhabitation of <em>5α</em>-<em>DHT</em> biosynthesis results in a novel state of androgen deficiency, independent of circulating testosterone levels. Finasteride and dutasteride are frequently prescribed for long-term treatment of lower urinary tract symptoms in men with BPH and in men with AGA. This treatment may result in development of non-alcoholic fatty liver diseases (NAFLD), insulin resistance (IR), type 2 diabetes (T2DM), dry eye disease, potential kidney dysfunction, among other metabolic dysfunctions. We suggest that long-term use of finasteride and dutasteride may be associated with health risks including NAFLD, IR, T2DM, dry eye disease and potential kidney disease.

OBJECTIVE

To measure T and DHT contents in normal and diseased human prostate tissues.

METHODS

Serum and prostatic T and DHT levels were measured in patients with normal, benign prostatic hyperplasia and prostate cancer.

RESULTS

A decline was observed in serum T level, but no change in DHT concentration with aging. There were no significant differences in both blood T and DHT levels between the patients with BPH or PCA and normal controls. Serum T level remained constant. There were excessive accumulation of DHT in BPH, and cancerous prostate tissues were responsible for the pathogenesis of BPH and PCA. Finasteride treatment did not produce a reduction in prostatic DHT content.

CONCLUSIONS

More than one form of 5a-reductases is responsible for the high level of DHT in the gland.

The impact of the brominated flame-retardant mixture, DE-71, on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined. A partial lifecycle study exposing S. tropicalis to varying concentrations of DE-71 (0.0, 0.65, 1.3, 2.5, and 5.0 μg/L [nominal]) was conducted from early gastrula-stage embryo to 150 d post-metamorphosis (dpm). Exposure of S. tropicalis to DE-71 induced liver necrosis and induced abnormal ovary development characterized by previtellogenic oocyte necrosis and arrested development of vitellogenic oocytes in females in a concentration-dependent manner. Decreased mean plasma <em>DHT</em> and T, gonad T, and increased mean plasma E2 levels were found in 150 dpm DE-71-treated male S. tropicalis compared to controls. Plasma E2 levels in females were not significantly altered compared to control S. tropicalis, although lower plasma and gonad T were detected. Mean gonadal CYP 19 aromatase activity in both male and female S. tropicalis exposed to DE-71 was not appreciably affected. Decreased mean male <em>5α</em>-reductase and CYP17 activities in both male and females were observed compared to control frogs. Overall, these studies suggested that PBDE exposure induced liver necrosis and abnormal ovary development; and reduced circulating and gonadal androgens resulting in a phenotypic skew in sex ratio toward the female sex in S. tropicalis.

In the fight against androgen-sensitive prostate cancer, the enzyme 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) is an attractive therapeutic target considering its key role in the formation of androgenic steroids. In this study, we attempted to assess the in vivo efficacy of the compound RM-532-105, an androsterone derivative developed as an inhibitor of 17β-HSD3, in the prostate cancer model of androgen-sensitive LAPC-4 cells xenografted in nude mice. RM-532-105 did not inhibit the tumor growth induced by 4-androstene-3,17-dione (4-dione); rather, the levels of the androgens testosterone (T) and dihydrotestosterone (<em>DHT</em>) increased within the tumors. In plasma, however, <em>DHT</em> levels increased but T levels did not. In troubleshooting experiments, the non-androgenic potential of RM-532-105 was confirmed by two different assays (LAPC-4 proliferation and androgen receptor transcriptional activity assays). The enzyme <em>5α</em>-reductase was also revealed to be the predominant enzyme metabolizing 4-dione in LAPC-4 cells, yielding <em>5α</em>-androstane-3,17-dione and not T. Other 17β-HSDs than 17β-HSD3 seem responsible in the androgen synthesis. From experiments with LAPC-4 cells, we fortuitously came across the interesting finding that 17β-HSD3 inhibitor RM-532-105 is concentrated inside tumors.

The androgens testosterone (T) and dihydrotestosterone (<em>DHT</em>) play a key role in the function and integrity of prostate tissue, but are also implicated in prostate cancer and benign prostatic hyperplasia (BPH). The reduction of androgen levels can be achieved by the inhibition of 3-oxo-<em>5α</em>-steroid-4-dehydrogenase (<em>5α</em>-reductase), which is responsible for the irreversible conversion of T into its more active metabolite <em>DHT</em>. In fact, the use of <em>5α</em>-reductase inhibitors (RIs), like finasteride, can be a valuable strategy for the treatment of BPH and in chemoprevention of prostate tumors. In this work a new method based on a dispersive liquid-liquid microextraction (DLLME) procedure, followed by gas chromatography-mass spectrometry (GC-MS), to evaluate the <em>5α</em>-reductase activity, by measuring the conversion percentage of T into <em>DHT</em> was optimized and validated. Enzymatic assays were carried out in human prostate microsomes, using T as substrate. T and <em>DHT</em> were extracted by the developed DLLME technique and quantified, after silylation, by GC-MS. Variables affecting the extraction efficiency and derivatization of T and <em>DHT</em> were evaluated. The optimized method showed good linearity (with correlation coefficients over 0.9994 for T and 0.9995 for <em>DHT</em>), good recoveries (higher than 80%), and good intra- and inter-day precision (below 13%, 3 levels, n=6). The detection limits for T and <em>DHT</em> were 0.5 nM and the limits of quantification were 5 nM. The new GC-MS method is a good alternative to the already described methods, to evaluate <em>5α</em>-reductase activity, since it avoids the use of radioactive compounds and corresponds to a fast and sensitive methodology with a good extraction efficiency, accuracy and high recovery. As this method allows the evaluation of <em>5α</em>-reductase activity, also permits the study of inhibitory efficacy of new molecules as potential RIs.

It is known that the growth of prostate metastatic bone tumor depends on androgens, and tumor formation can start from migratory malignant cells produced in that organ. These cells exhibit grater type 1 <em>5α</em>-reductase (<em>5α</em>-R1) activity than type 2 <em>5α</em>-reductase. Noteworthy, both isozymes convert testosterone (T) to the more active androgen dihydrotestosterone (<em>DHT</em>) in the target tissues. Thus, in order to potentially improve the prognosis of this disease, in this work, seven derivatives of 17-(1H-benzimidazol-1-yl)-16-formillandrosta-5,16-dien-3β-yl benzoate (4a-f) and 17-(1H-benzimidazol-1-yl)-3-hydroxy-16-formylandrost-5,16-diene (4) were synthesized, characterized and identified as inhibitors of type 1 <em>5α</em>-reductase (<em>5α</em>R1). These derivatives having the advantage of improved plasma half-life. The inhibitory activity of the compounds towards <em>5α</em>-R1 isoenzyme was determined by conversion of T into <em>DHT</em> in the presence or absence of compounds 4, 4a-f. Further, in vivo experiments were also carried out, treating gonadectomized hamsters with T and/or 4, 4a-f and evaluating their effect on the diameter of hamster flank organs and on the weight of the prostatic and seminal vesicles. Results indicated that compounds 4, 4b, 4c, served as in vitro inhibitors of the enzyme <em>5α</em>-R1 and pharmacological experiments showed that 4 and derivatives 4a-f decreased the diameter of the flank glands, the weight of the prostate and seminal vesicles of treated hamsters without any appreciable toxicity during observation. Noteworthy the fact that compound 4 is the product, in all cases, of the hydrolysis of the series of esters 4a-f, thus they can serve as precursors (prodrugs) of the active form 4.

Corpus luteum (CL) regression is a complex physiological process. Previous studies have shown that dihydrotestosterone (<em>DHT</em>) may be involved in regulating CL regression, but the mechanism is still unclear. In this study, we evaluated the localization of the two isoforms of <em>DHT</em> synthetase <em>5α</em>-reductase (<em>5α</em>-red1 and <em>5α</em>-red2) and androgen receptor (AR) in sheep CL, and investigated <em>5α</em>-red1, <em>5α</em>-red2, AR, and <em>DHT</em> levels at different luteal stages of CL (early, middle, and late phase) by immunohistochemistry, quantitative real-time polymerase chain reaction, and western blot analysis. Moreover, we cultured luteal cells from middle phase CL and treated them with different concentrations of <em>DHT</em> (10^-10 -10 ^-6 M) and the AR antagonist flutamide (10 ^-5 M), to evaluate whether <em>DHT</em> is involved in the regulation of progesterone (P4) secretion and progesterone nuclear receptor (PGR) expression and whether these effects are regulated by the AR pathway. We also investigated the effects of <em>DHT</em> and flutamide on prostaglandin F2α (PGF2α) secretion and apoptotic gene and protein expression. Our results showed that <em>5α</em>-red1, <em>5α</em>-red2, and AR were expressed in the CL, and their expression and <em>DHT</em> levels were changed during the luteal phase. <em>DHT</em> was involved in mediating P4 and PGF2α secretion and PGR and apoptotic gene and protein expression. The effects of <em>DHT</em> on CL were at least partially regulated by the AR pathway. This study reveals the mechanism of action of <em>DHT</em> on sheep CL regression and lays the foundation for further exploration of androgen regulation of CL function.

While the neuroprotective benefits of estrogen and progesterone in critical illness are well established, the data regarding the effects of androgens are conflicting. Surgical repair of congenital heart disease is associated with significant morbidity and mortality, but there are scant data regarding the postoperative metabolism of sex steroids in this setting. The objective of this prospective observational study was to compare the postoperative sex steroid patterns in pediatric patients undergoing major cardiac surgery (MCS) versus those undergoing less intensive non-cardiac surgery. Urinary excretion rates of estrogen, progesterone, and androgen metabolites (μg/mmol creatinine/m(2) body surface area) were determined in 24-h urine samples before and after surgery using gas chromatography-mass spectrometry in 29 children undergoing scheduled MCS and in 17 control children undergoing conventional non-cardiac surgery. Eight of the MCS patients had Down's syndrome. There were no significant differences in age, weight, or sex between the groups. Seven patients from the MCS group showed multi-organ dysfunction after surgery. Before surgery, the median concentrations of 17β-estradiol, pregnanediol, <em>5α</em>-dihydrotestosterone (<em>DHT</em>), and dehydroepiandrosterone (DHEA) were (control/MCS) 0.1/0.1 (NS), 12.4/11.3 (NS), 4.7/4.4 (NS), and 2.9/1.1 (p=0.02). Postoperatively, the median delta 17β-estradiol, delta pregnanediol, delta <em>DHT</em>, and delta DHEA were (control/MCS) 0.2/6.4 (p=0.0002), -3.2/23.4 (p=0.013), -0.6/3.7 (p=0.0004), and 0.5/4.2 (p=0.004). Postoperative changes did not differ according to sex. We conclude that MCS, but not less intensive non-cardiac surgery, induced a distinct postoperative increase in sex steroid levels. These findings suggest that sex steroids have a role in postoperative metabolism following MCS in prepubertal children.

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([(3)H] 4-dione) to [(3)H]dihydrotestosterone ([(3)H]<em>DHT</em>) in prostate nuclear membrane fractions, where the <em>5α</em>-reductase activity is present. The enzyme <em>5α</em>-reductase catalyzes the <em>5α</em>-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or <em>5α</em>-dione to dihydrotestosterone (<em>DHT</em>). Moreover, we also investigated the role of unlabeled <em>5α</em>-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [(3)H] 4-dione to [(3)H]<em>DHT</em>, homogenates of human prostate were incubated with [(3)H] 4-dione, NADPH and increasing concentrations of non-labeled <em>5α</em>-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of <em>DHT</em> was separated and the radioactivity determined. The results showed that the presence of [(3)H] 4-dione plus unlabelled <em>5α</em>-dione produced similar levels of <em>DHT</em> as compared to [(3)H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [(3)H] 4-dione to [(3)H]<em>DHT</em>, showing IC(50) values of 2 and 1.6 nM, respectively.

The zebra finch is a common model organism in neuroscience, endocrinology, and ethology. Zebra finches are generally considered opportunistic breeders, but the extent of their opportunism depends on the predictability of their habitat. This plasticity in the timing of breeding raises the question of how domestication, a process that increases environmental predictability, has affected their reproductive physiology. Here, we compared circulating steroid levels in various "strains" of zebra finches. In Study 1, using radioimmunoassay, we examined circulating testosterone levels in several strains of zebra finches (males and females). Subjects were wild or captive (Captive Wild-Caught, Wild-Derived, or Domesticated). In Study 2, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined circulating sex steroid profiles in wild and domesticated zebra finches (males and females). In Study 1, circulating testosterone levels in males differed across strains. In Study 2, six steroids were detectable in plasma from wild zebra finches (pregnenolone, progesterone, dehydroepiandrosterone (DHEA), testosterone, androsterone, and <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>)). Only pregnenolone and progesterone levels changed across reproductive states in wild finches. Compared to wild zebra finches, domesticated zebra finches had elevated levels of circulating pregnenolone, progesterone, DHEA, testosterone, androstenedione, and androsterone. These data suggest that domestication has profoundly altered the endocrinology of this common model organism. These results have implications for interpreting studies of domesticated zebra finches, as well as studies of other domesticated species.

The <em>5α</em>-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on <em>5α</em>-reduced progesterone, <em>5α</em>-dihydroprogesterone (DHP). Epididymis expresses <em>5α</em>-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for <em>5α</em>-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (<em>DHT</em>), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine <em>5α</em>-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for <em>5α</em>-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of <em>5α</em>-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and <em>DHT</em>, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine <em>5α</em>-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies.