11β-hydroxysteroid Dehydrogenase Research Articles

Ecdysteroids from the Korean Endemic Species Ajuga spectabilis with Activities against Glucocorticoid Receptors and 11β-Hydroxysteroid Dehydrogenase Type 1.

Two new ecdysteroids, spectasterone A (1) and spectasterone B (2), together with four known ecdysteroids, breviflorasterone (3), ajugalactone (4), 20-hydroxyecdysone (5), and polypodine B (6) were isolated from the Korean endemic plant Ajuga spectabilis using feature-based molecular networking analysis. The chemical structures of 1 and 2 were determined based on the interpretation of NMR and mass spectrometric data. Their absolute configurations were established using 3JH,H coupling constants, NOESY interactions, Mosher's method, and ECD and DP4+ calculations. To identify their biological target, a machine learning-based prediction system was applied, and the results indicated that ecdysteroids may have 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)-related activity, which was further supported by molecular docking results of ecdysteroids with 11β-HSD1. Following this result, all the isolated ecdysteroids were tested for their ability to affect the expression of 11β-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptors (GRs) in HaCaT cells irradiated with UVB. Compounds 2-5 exhibited inhibition of 11β-HSD1 expression and increases in GR activity.

Psoralen prevents the inactivation of estradiol and treats osteoporosis via covalently targeting HSD17B2.

Psoralea corylifolia L. seeds (P. corylifolia), popularly known as Buguzhi in traditional Chinese medicine, are often used to treat osteoporosis in China. Psoralen (Pso) is the key anti-osteoporosis constituent in P. corylifolia, however, its targets and mechanism of action are still unclear. The purpose of this study was to explore the interaction between Pso and 17-β hydroxysteroid dehydrogenase type 2 (HSD17B2), an estrogen synthesis-related protein that inhibits the inactivation of estradiol (E2) to treat osteoporosis. Tissue distribution of Pso was analyzed by in-gel imaging after oral administration of an alkynyl-modified Pso probe (aPso) in mice. The target of Pso in the liver was identified and analyzed using chemical proteomics. Co-localization and cellular thermal shift assays (CETSA) were used to verify the key action targets. To detect the key pharmacophore of Pso, the interaction of Pso and its structural analogs with HSD17B2 was investigated by CETSA, HSD17B2 activity assay, and in-gel imaging determination. Target competitive test, virtual docking, mutated HSD17B2 activity, and CETSA assay were used to identify the binding site of Pso with HSD17B2. A mouse model of osteoporosis was established by ovariectomies, and the efficacy of Pso in vivo was confirmed by micro-CT, H&E staining, HSD17B2 activity, and bone-related biochemical assays. Pso regulated estrogen metabolism by targeting HSD17B2 in the liver, with the α, β-unsaturated ester in Pso being the key pharmacophore. Pso significantly suppressed HSD17B2 activity by irreversibly binding to Lys236 of HSD17B2 and preventing NAD+ from entering the binding pocket. In vivo studies in ovariectomized mice revealed that Pso could inhibit HSD17B2 activity, prevent the inactivation of E2, increase levels of endogenous estrogen, improve bone metabolism-related indices, and play a role in anti-osteoporosis. Pso covalently binds to Lys236 of HSD17B2 in hepatocytes to prevent the inactivation of E2, thereby aiding in the treatment of osteoporosis.

Potent Dual Inhibitors of Steroid Sulfatase and 17β-Hydroxysteroid Dehydrogenase Type 1 with a Suitable Pharmacokinetic Profile for In Vivo Proof-of-Principle Studies in an Endometriosis Mouse Model.

Treating estrogen-dependent diseases like endometriosis with drugs suppressing local estrogen activation may be superior to existing endocrine therapies. Steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) are key enzymes of local estrogen activation. We describe the rational design, synthesis, and biological profilation of furan-based compounds as a novel class of dual STS/17β-HSD1 inhibitors (DSHIs). In T47D cells, compound 5 showed irreversible inhibition of STS and potent, reversible inhibition of 17β-HSD1. It was selective over 17β-HSD2 and displayed high metabolic stabilities in human and mouse liver S9 fractions. No effect on cell viability was detected up to 31 μM (HEK293) and 23 μM (HepG2), respectively, and there was no activation of the aryl hydrocarbon receptor (AhR) up to 3.16 μM. Single daily application to mice revealed steady-state plasma levels high enough to make this compound eligible for an in vivo proof-of-principle study in a mouse endometriosis model.

Bisphenol S stimulates Leydig cell proliferation but inhibits differentiation in pubertal male rats through multiple mechanisms.

Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 μM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 μM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17β-hydroxysteroid dehydrogenase 3 activity at 10-100 μM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17β-hydroxysteroid dehydrogenase 3 activity.

α-Linolenic acid-regulated testosterone biosynthesis via activation of the JNK-SF-1 signaling pathway in primary rooster Leydig cells

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. Methods: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 μmol/L) or pretreated with a p38 inhibitor (50 μmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 μmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 μmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). Results: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 μmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression significantly increased (P < 0.05) in the 40 μmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 μmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 μmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 μmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3β-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3β-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3β-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). Conclusion: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3β-HSD, and P450c17 expression in primary rooster Leydig cells.

Development of submicromolar 17β-HSD10 inhibitors and their in vitro and in vivo evaluation

17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a multifunctional mitochondrial enzyme and putative drug target for the treatment of various pathologies including Alzheimer's disease or some types of hormone-dependent cancer. In this study, a series of new benzothiazolylurea-based inhibitors were developed based on the structure-activity relationship (SAR) study of previously published compounds and predictions of their physico-chemical properties. This led to the identification of several submicromolar inhibitors (IC50 ∼0.3 μM), the most potent compounds within the benzothiazolylurea class known to date. The positive interaction with 17β-HSD10 was further confirmed by differential scanning fluorimetry and the best molecules were found to be cell penetrable. In addition, the best compounds weren't found to have additional effects for mitochondrial off-targets and cytotoxic or neurotoxic effects. The two most potent inhibitors 9 and 11 were selected for in vivo pharmacokinetic study after intravenous and peroral administration. Although the pharmacokinetic results were not fully conclusive, it seemed that compound 9 was bioavailable after peroral administration and could penetrate into the brain (brain-plasma ratio 0.56).

Zinc protects against lead-induced testicular damage via modulation of steroidogenic and xanthine oxidase/uric acid/caspase 3-mediated apoptotic signaling in male Wistar rats

Aim This study evaluated the effect of lead, with or without zinc co-administration, on steroidogenic and xanthine oxidase (XO)/uric acid (UA)/caspase 3-mediated apoptotic signaling in the testis. Materials and methods Forty male Wistar rats were divided into four groups at random; vehicle-treated control, zinc-treated, lead-treated, and lead + zinc-treated groups. Results Lead exposure significantly lowered overall weight gain, testicular, epididymal, seminal vesicle, and prostate weights. Also, lead decreased sperm count, viability and motility but increased the fraction of sperm with aberrant morphology. In addition, lead caused a marked rise in the level of UA and XO activity but a decrease in nuclear factor erythroid 2-related factor 2 (Nrf2), reduced glutathione (GSH) as well as total antioxidant capacity (TAC) levels, and superoxide dismutase (SOD) and catalase activities. Furthermore, lead increased the testicular levels of nuclear factor kappa B (NFkB), interleukin-1beta (IL-1β), and tumour necrotic factor-alpha (TNF-α), which were associated with an increase in testicular caspase 3 activity and DNA fragmentation as well as a decline in circulating gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). These were associated with lead-induced degenerative changes in testicular tissues evidenced by shrunken seminiferous tubules, degeneration and sloughing of germ cells. Co-administration of zinc prevented lead-induced testicular injury by ameliorating oxidative stress, apoptosis, and inflammation through downregulation of XO/UA/caspase 3 pathway and upregulation of testicular 3β-HSD/17β-HSD. Conclusion This study demonstrated that zinc protected against lead-induced testicular toxicity via the downregulation of XO/UA/caspase 3 signaling.

Contribution of local regeneration of glucocorticoids to tissue steroid pools.

11β-Hydroxysteroid dehydrogenase 1 (11βHSD1) is a drug target to attenuate adverse effects of chronic glucocorticoid excess. It catalyses intracellular regeneration of active glucocorticoids in tissues including brain, liver and adipose tissue (coupled to hexose-6-phosphate dehydrogenase, H6PDH). 11βHSD1 activity in individual tissues is thought to contribute significantly to glucocorticoid levels at those sites, but its local contribution vs glucocorticoid delivery via the circulation is unknown. Here, we hypothesised that hepatic 11βHSD1 would contribute significantly to the circulating pool. This was studied in mice with Cre-mediated disruption of Hsd11b1 in liver (Alac-Cre) vs adipose tissue (aP2-Cre) or whole-body disruption of H6pdh. Regeneration of [9,12,12-2H3]-cortisol (d3F) from [9,12,12-2H3]-cortisone (d3E), measuring 11βHSD1 reductase activity was assessed at steady state following infusion of [9,11,12,12-2H4]-cortisol (d4F) in male mice. Concentrations of steroids in plasma and amounts in liver, adipose tissue and brain were measured using mass spectrometry interfaced with matrix-assisted laser desorption ionisation or liquid chromatography. Amounts of d3F were higher in liver, compared with brain and adipose tissue. Rates of appearance of d3F were ~6-fold slower in H6pdh-/- mice, showing the importance for whole-body 11βHSD1 reductase activity. Disruption of liver 11βHSD1 reduced the amounts of d3F in liver (by ~36%), without changes elsewhere. In contrast disruption of 11βHSD1 in adipose tissue reduced rates of appearance of circulating d3F (by ~67%) and also reduced regenerated of d3F in liver and brain (both by ~30%). Thus, the contribution of hepatic 11βHSD1 to circulating glucocorticoid levels and amounts in other tissues is less than that of adipose tissue.

Bisphenol A Analogues Inhibit Human and Rat 11β-Hydroxysteroid Dehydrogenase 1 Depending on Its Lipophilicity.

Bisphenol A (BPA) analogues substituted on the benzene ring are widely used in a variety of industrial and consumer materials. However, their effects on the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) remain unclear. The inhibitory effects of 6 BPA analogues on the inhibition of human and rat 11β-HSD1 were investigated. The potencies of inhibition on human 11β-HSD1 were bisphenol H (IC50, 0.75 µM) > bisphenol G (IC50, 5.06 µM) > diallyl bisphenol A (IC50, 13.36 µM) > dimethyl bisphenol A (IC50, 30.18 µM) > bisphenol A dimethyl ether (IC50, 33.08 µM) > tetramethyl bisphenol A (>100 µM). The inhibitory strength of these chemicals on rat 11β-HSD1 was much weaker than that on the human enzyme, ranging from 74.22 to 205.7 µM. All BPA analogues are mixed/competitive inhibitors of both human and rat enzymes. Molecular docking studies predict that bisphenol H and bisphenol G both bind to the active site of human 11β-HSD1, forming a hydrogen bond with catalytic residue Ser170. The bivariate correlation of IC50 values with LogP (lipophilicity), molecular weight, heavy atoms, and molecular volume revealed a significant inverse regression and the correlation of IC50 values with ΔG (low binding energy) revealed a positive regression. In conclusion, the lipophilicity, molecular weight, heavy atoms, molecular volume, and binding affinity of a BPA analogue determine the inhibitory strength of human and rat 11β-HSD isoforms.

Tissue Glucocorticoid Metabolism in Adrenal Insufficiency: A Prospective Study of Dual-release Hydrocortisone Therapy.

Patients with adrenal insufficiency (AI) require life-long glucocorticoid (GC) replacement therapy. Within tissues, cortisol (F) availability is under the control of the isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD). We hypothesize that corticosteroid metabolism is altered in patients with AI because of the nonphysiological pattern of current immediate release hydrocortisone (IR-HC) replacement therapy. The use of a once-daily dual-release hydrocortisone (DR-HC) preparation, (Plenadren®), offers a more physiological cortisol profile and may alter corticosteroid metabolism in vivo. Prospective crossover study assessing the impact of 12 weeks of DR-HC on systemic GC metabolism (urinary steroid metabolome profiling), cortisol activation in the liver (cortisone acetate challenge test), and subcutaneous adipose tissue (microdialysis, biopsy for gene expression analysis) in 51 patients with AI (primary and secondary) in comparison to IR-HC treatment and age- and BMI-matched controls. Patients with AI receiving IR-HC had a higher median 24-hour urinary excretion of cortisol compared with healthy controls (72.1 µg/24 hours [IQR 43.6-124.2] vs 51.9 µg/24 hours [35.5-72.3], P = .02), with lower global activity of 11β-HSD2 and higher 5-alpha reductase activity. Following the switch from IR-HC to DR-HC therapy, there was a significant reduction in urinary cortisol and total GC metabolite excretion, which was most significant in the evening. There was an increase in 11β-HSD2 activity. Hepatic 11β-HSD1 activity was not significantly altered after switching to DR-HC, but there was a significant reduction in the expression and activity of 11β-HSD1 in subcutaneous adipose tissue. Using comprehensive in vivo techniques, we have demonstrated abnormalities in corticosteroid metabolism in patients with primary and secondary AI receiving IR-HC. This dysregulation of pre-receptor glucocorticoid metabolism results in enhanced glucocorticoid activation in adipose tissue, which was ameliorated by treatment with DR-HC.

367-OR: E-cigarette Vaping Exposure Increases Adipose 11ß-HSD1 Expression and Disturbs Glucose Homeostasis in Mice

Electronic cigarette (E-cigs) use may increase the prediabetic risk, but the underlying mechanisms remain underexplored. Pre-receptor activation of glucocorticoids (GCs) via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in adipose tissues has been identified as an important mediator of insulin resistance and type 2 diabetes. However, little is known about the effects of E-cig vaping on 11β-HSD1 and glucose homeostasis. To address this issue, we conducted animal model studies with mice exposed to aerosolized PBS, nicotine-free or nicotine-containing E-cigs, with concurrent exposure to either vehicle or the GC receptor (GR) antagonist RU486. We observed that exposure of E-cig vaping nicotine for 4 weeks increased 11β-HSD1 expression and induced lipase HSL and ATGL abundance within the adipose tissues with induction of hypercortisolemia in response to elevated plasma nicotine levels in mice compared with those of aerosolized E-cig vaping vehicle or PBS controls. Induction of adipose 11β-HSD1 was correlated with elevated the expression of hepatic gluconeogenic enzymes PEPCK and G6Pase and corresponded to the elevated fasting glucose levels. Moreover, E-cig vaping nicotine also increased glucose intolerance, but decreased the glucose-lowing effects of insulin and elevated plasma FFA levels in mice compared with PBS controls. However, E-cig vapor without nicotine did not affect these metabolic parameters. Furthermore, RU486 treatment attenuated E-cig vaping nicotine-induced adipose 11β-HSD1 and lipase expression. In addition, RU486 also attenuated the E-cig-mediated activation of hepatic PEPCK and G6Pase and decreased plasma FFA levels, but did not change the elevated plasma corticosterone levels in mice on E-cig vaping nicotine. These data indicate that E-cig vapor exposure harmfully affects glucose homeostasis and these effects may arise, in part, from aerosol nicotine-induced adipose 11β-HSD1 and GR expression. Disclosure J.Wang: None. T.Friedman: None. M.Jiang: None. Y.Liu: None. J.Liu: None. Y.Wang: None. S.C.J.Kim: None. E.Espinal: None. R.Yang: None. H.Zhong: None. X.M.Shao: None. K.Lutfy: None. Funding Tobacco-Related Disease Research Program (T31IR1603)

Recycling glucocorticoids: therapeutic implications of the 11β-HSD1 enzyme system.

Endogenous glucocorticoids and commonly used oral glucocorticoids have the property of existing in an inactive and active form in vivo. The inactive form can be converted back to the active form, or 'recycled' in cells and tissues that express the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. This recycling provides an important contribution to the action of glucocorticoids. This review examines the literature relating to the importance of 11β-HSD1 activity during glucocorticoid treatment, with an emphasis on studies examining bone and joint disease and the ability of glucocorticoids to suppress inflammatory damage in models of arthritis. Animal models with global or selective deletion of 11β-HSD1 have determined the extent to which this recycling is important in normal physiology and during treatment with oral glucocorticoids. These studies demonstrate that 11β-HSD1-mediated recycling of inactive glucocorticoids has a substantial action and indeed is responsible for the majority of the effects of orally administered glucocorticoids on a range of tissues. Importantly, the anti-inflammatory actions of glucocorticoids appear largely through this mechanism such that mice that lack 11β-HSD1 are resistant to the anti-inflammatory actions of glucocorticoids. The recognition that to a large extent the circulating inactive counterpart of these glucocorticoids is more important to anti-inflammatory effects than the active glucocorticoid presents novel opportunities to more selectively target glucocorticoids to tissues or to reduce the likely side effects.

Early life corticosteroid overexposure: Epigenetic and fetal origins of adult diseases.

The relationship between events occurring during intrauterine development and later-life predisposition to long-term disease, has been described. The fetus responds to excess intrauterine exposure to high levels of corticosteroids, modifying their physiological development and stopping their growth. Fetal exposure to elevated levels of either endogenous (alterations in fetal hypothalamic-pituitary-adrenal axis) or synthetic corticosteroids, is one model of early-life adversity; to developing adult disease. At the molecular level, there are transcriptional changes in metabolic and growth pathways. Epigenetic mechanisms participate in transgenerational inheritance, not genomic. Exposures that change 11β-hydroxysteroid dehydrogenase type 2 enzyme methylation status in the placenta can result in transcriptional repression of the gene, causing the fetus to be exposed to higher levels of cortisol. More precise diagnosis and management of antenatal corticosteroids for preterm birth, would potentially decrease the risk of long-term adverse outcomes. More studies are needed to understand the potential roles of factors to alter fetal corticosteroid exposure. Long-term infant follow-up is required to determine whether methylation changes in placenta may represent useful biomarkers of later disease risk. This review, summarize recent advances in the programming of fetal effects of corticosteroid exposure, the role of corticosteroids in epigenetic gene regulation of placental 11β-hydroxysteroid dehydrogenase type 2 enzyme expression and transgenerational effects.

3βHSD activity saturates at physiological substrate concentrations in intact cells.

Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) or to Δ5 -androstenediol by 17βHSD. To better understand this process, we studied the kinetics of these reactions in cells. Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. The two reactions displayed very different saturation profiles, with only the 3βHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3βHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17βHSD-catalyzed conversion to Δ5 -androstenediol. Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3βHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

IN-SILICO SCREENING OF PHYTOCHEMICAL COMPOUNDS IN CAESALPINIA BONDUCELLA L. SEEDS AGAINST THE GENE TARGETS OF OVARIAN STEROIDOGENESIS PATHWAY

Polycystic ovary syndrome (PCOS) is the most common gynaecological disorder among reproductive-age women. Impaired metabolism of androgens and estrogens is one of the leading causes of PCOS. In India, medicinal herbs are being explored for their anti-androgenic and anti-estrogenic properties. In this study, we have screened the seed extracts of the herbal plant, Caesalpinia bonducella for potent inhibitors of estrogen and testosterone biosynthesis and assimilation. Methanol extract of C. bonducella seed kernels were subjected to gas chromatography - mass spectrometry (GC-MS) to identify the phytochemical constituents. Out of forty-three phytochemical compounds identified from the extract, eight compounds were selected based on Lipinski's rule of five for molecular docking. The selected phytochemical compounds were docked against specific targets of ovarian steroidogenesis pathway; human aromatase (CYP19A1), human 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1), human androgen receptor and estrogen receptor α. Further, the nature of these compounds was validated using density functional theory (DFT) calculations and ADME/T studies. As per the molecular docking output, compounds 33, 35, 38, 40, and 43 exhibited higher binding affinities against the four selected targets. Phytochemical compounds were optimized using Gaussian 16 with the B3LYP function and the 6-31G(d, p) basis set and were correlated with docking results. ADME/T helps in identifying the potential drug candidates from a pool of drugs. Five phytochemical compounds, 33, 35, 38, 40, and 43 were found to have the ability to bind and inhibit appropriate targets in the ovarian steroidogenesis pathway. Hence, these compounds can be further characterized in vitro and in vivo for alleviating PCOS.

Subacute exposure to Roundup® changes steroidogenesis and gene expression of the glutathione-glutaredoxin system in rat ovaries: Implications for ovarian toxicity of this glyphosate-based herbicide

Studies have indicated that glyphosate induces endocrine disruption and may adversely affect the male reproductive system. However, evidence of its effects on ovarian function is poorly understood so far, making further studies necessary on the mechanisms of the glyphosate toxicity in the female reproductive system. The aim of this work was to evaluate the effect of a subacute exposure (28 days) to the glyphosate-based formulation Roundup® (1.05, 10.5 and 105 μg/kg b.w. of glyphosate) on steroidogenesis, oxidative stress, systems involved in cell redox control and histopathological parameters in rat ovaries. Hence we quantify plasma estradiol and progesterone by chemiluminescence; non-protein thiol levels, TBARS, superoxide dismutase and catalase activity by spectrophotometry; gene expression of steroidogenic enzymes and redox systems by real-time PCR; and ovarian follicles by optical microscopy. Our results demonstrated that oral exposure increased progesterone levels and the mRNA expression of 3β-hydroxysteroid dehydrogenase. Histopathological analysis revealed a decrease in the number of primary follicles and an increase in the number of corpus luteum in rats exposed to Roundup®. An imbalance of the oxidative status was also evidenced by decreasing the catalase activity at all groups exposed to the herbicide. Increased lipid peroxidation and gene expression of glutarredoxin and decreased of glutathione reductase were also observed. Our results indicate that Roundup® causes endocrine disruption of hormones related to female fertility and reproduction and changes the oxidative status by altering antioxidant activity, inducing lipid peroxidation, as well as changing the gene expression of the glutathione-glutarredoxin system in rat ovaries.

Interplay of Cortisol, Testosterone, and Abdominal Fat Mass in Normal-weight Women With Polycystic Ovary Syndrome.

Ovarian and adrenal steroidogenesis underlie endocrine-metabolic dysfunction in polycystic ovary syndrome (PCOS). Adipocytes express aldo-keto reductase 1C3 and type 1 11β-hydroxysteroid dehydrogenase, which modulate peripheral androgen and cortisol production. To compare serum adrenal steroids, including 11-oxygenated androgens (11-oxyandrogens), cortisol, and cortisone between normal-weight women with PCOS and body mass index- and age-matched ovulatory women with normal-androgenic profiles (controls), and assess whether adrenal steroids associate with abdominal adipose deposition. Prospective, cross-sectional, cohort study. Academic medical center. Twenty normal-weight women with PCOS and 20 body mass index-/age-matched controls. Blood sampling, IV glucose tolerance testing, and total-body dual-energy x-ray absorptiometry. Clinical characteristics, hormonal concentrations, and body fat distribution. Women with PCOS had higher serum total/free testosterone (T) and androstenedione (A4) levels and a greater android/gynoid fat mass than controls (androgens P < .001; android/gynoid fat mass ratio, P = .026). Serum total/free T and A4 levels correlated positively with android/gynoid fat mass ratio in all women combined (P < .025, all values). Serum 11ß-hydroxyA4, 11-ketoA4, 11ß-hydroxyT, 11-ketoT, cortisol, and cortisone levels were comparable between female types and unrelated to body fat distribution. Serum 11-oxyandrogens correlated negatively with % total body fat, but lost significance adjusting for cortisol. Serum cortisol levels, however, correlated inversely with android fat mass (P = .021), with a trend toward reduced serum cortisol to cortisone ratio in women with PCOS vs controls (P = .075), suggesting diminished 11β-hydroxysteroid dehydrogenase activity. Reduced cortisol may protect against preferential abdominal fat mass in normal-weight PCOS women with normal serum 11-oxyandrogens.

Hyperglycemia-activated 11β-hydroxysteroid dehydrogenase type 1 increases endoplasmic reticulum stress and skin barrier dysfunction

The diabetes mellitus (DM) skin shows skin barrier dysfunction and skin lipid abnormality, similar to conditions induced by systemic or local glucocorticoid excess and aged skin. Inactive glucocorticoid (GC) is converted into active glucocorticoid by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Hyperglycemia in DM and excessive GC are known to increase endoplasmic reticulum (ER) stress. We hypothesized that hyperglycemia affects systemic GC homeostasis and that the action of skin 11β-HSD1 and GC contributes to increased ER stress and barrier defects in DM. We compared 11β-HSD1, active GC, and ER stress between hyperglycemic and normoglycemic conditions in normal human keratinocytes and db/db mice. 11β-HSD1 and cortisol increased with time in keratinocyte culture under hyperglycemic conditions. 11β-HSD1 siRNA-transfected cells did not induce cortisol elevation in hyperglycemic condition. The production of 11β-HSD1 and cortisol was suppressed in cell culture treated with an ER stress-inhibitor. The 14-week-old db/db mice showed higher stratum corneum (SC) corticosterone, and skin 11β-HSD1 levels than 8-week-old db/db mice. Topical 11β-HSD1 inhibitor application in db/db mice decreased SC corticosterone levels and improved skin barrier function. Hyperglycemia in DM may affect systemic GC homeostasis, activate skin 11β-HSD1, and induce local GC excess, which increases ER stress and adversely affects skin barrier function.

Mechanisms of biphasic surges of testosterone in response to human chorionic gonadotrophin (hCG) in stallions

Human chorionic gonadotrophin (hCG) is included in stimulation tests for diagnosis of cryptorchidism and endocrine function of testes in stallions. Although the exact mechanisms are unknown, circulating testosterone (T) concentration shows a clear biphasic increase in response to a single administration of hCG (Tsunoda. et. al. Animal Reproduction Science. 2010; 121S: S165-S167). In the first experiment, concentrations of circulating T and estradiol-17β (E) in response to intravenous administration of 10,000IU hCG were measured. Concentrations of circulating testosterone were also measured in adult Thoroughbred stallions (n=4). In the second experiment, the same experiment was done using adult male golden hamsters (n=45) to figure out mechanisms of biphasic response of T. Adult male golden hamsters were used as a model of stallions, because golden hamsters are typical long day seasonal breeders same as stallions. In the first experiment, blood samples were taken hourly for 6 hours, and daily for 7 days after administration of hCG. Circulating T concentrations in stallions showed a clear biphasic increase within 6 hours and between 2 and 4 days after administration of hCG. Circulating T concentrations at the second peak were significantly higher than those at the first peak. Circulating E concentrations after hCG administration was similar to that of T at the first peak, though the second increase was not detected. Circulating hCG concentrations sharplyincreased just after administration, then followed by an abrupt decline, and low levels were maintained until day 7. It indicated that concentrations of hCG decreased completely when the second peak of T concentrations appeared in circulation. In the second experiment, 25 male golden hamsters were administered 100IU of hCG, and 20 hamsters were administered saline. Blood samples and testes were taken from 5 golden hamsters 5 hours, 24 hours, 3, 5, and 7 days after administration, respectively. Testicular tissues were investigated by real-time PCR for cytochrome P450scc (P450scc), 3β-hydroxy-steroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and receptors of luteinizing hormone (LHR). Testes were also histologically evaluated by hematoxylin-eosin staining. The results of changes in circulating T and hCG concentrations in male golden hamsters were similar to those in stallions. Expression of P450cc, 3β-HSD, 17β-HSD and number of LHR in testes 3 days after administrations of hCG were significantly higher as compared with those at 0 hour. The number of Leydig cells in the histological section 3 days after administration of hCG were also higher compared to 0 hour. The present study strongly suggests that the increase in numbers of LHR and Leydig cells, together with upregulation of steroid enzymes in testes causes the second increase of T plasma concentration after hCG in stallions.

Relationship between androgen biosynthesis linked to 3βHSD1 and resistance to radiotherapy: A germline biomarker for combined androgen blockade with radiation in high-risk prostate cancer.

e17084 Background: Around 50% of men with advanced prostate cancer (PCa) have at least one germline copy of the adrenal-permissive (1245C) HSD3B1 allele. This allele leads to higher levels of the steroid biosynthesis enzyme, 3β-hydroxysteroid dehydrogenase (3βHSD1). Inheriting this allele is linked to worse outcomes in men with advanced PCa. To determine if (1245C) HSD3B1 could be causing early resistance to combined androgen deprivation and radiotherapy in localized PCa, we studied its role in modulating radioresistance. Methods: PCa cell lines were used to investigate the reciprocal effects of 3βHSD1 knockdown and overexpression in intrinsically high and low 3βHSD1 lines, respectively. PCa xenografts were used to confirm the results in vivo. The connection between androgen receptor (AR) expression and elevated DNA Damage Response (DDR) gene expression was validated using transcriptomic data from 680 radical prostatectomy specimens The ability of enzalutamide, a non-steroidal anti-androgen, to restore radiosensitivity in 1245C expressing lines was interrogated in vitro and in vivo. Results: 1245C HSD3B1 expressing PCa lines displayed increased clonogenic survival after ionizing radiation. Effects were dependent on the 3βHSD1 substrate, DHEA, confirming intratumoral androgen metabolism was required for radioresistance. Resistant lines showed enhanced DNA double-strand break (DSB) repair and heightened DDR gene expression. PCa xenografts with 1245C HSD3B1 were similarly more resistant to radiation using murine models that faithfully mimic human adrenal physiology. A correlation between AR expression and increased DDR gene expression was confirmed in 680 patient samples. Enzalutamide pretreatment resulted in a decrease in DSB repair ability and re-sensitized 1245C HSD3B1 PCa cells to radiation. Conclusions: 1245C HSD3B1 fuels prostate cancer treatment resistance by elevating regional androgen biosynthesis from adrenal steroid precursors, leading to DDR gene overexpression and enhanced DNA DSB repair kinetics. This work supports clinical validation of biomarker informed selective intensification strategies, e.g., combined androgen blockade, for high-risk men with the 1245C HSD3B1 allele receiving curative intent radiotherapy.