3β-hydroxysteroid Dehydrogenase Activity Research Articles

Boldenone and Testosterone Production from Phytosterol via One-Pot Cascade Biotransformations

Testosterone (TS) and its 1(2)-dehydrogenated derivative boldenone (BD) are widely used in medicine, veterinary science and as precursors in organic synthesis of many therapeutic steroids. Green production of these compounds is possible from androstenedione (AD) enzymatically, or from phytosterol (PS) using fermentation stages. In this study, the ascomycete Curvularia sp. VKM F-3040 was shown to convert androstadienedione (ADD, 4 and 10 g/L) to yield 97% and 78% (mol/mol) of BD, respectively. Based on its high 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, a novel cascade biotransformation of PS was developed for production of TS and BD. At the first stage, the strains of Mycolicibacterium neoaurum VKM Ac-1815D or M. neoaurum VKM Ac-1816D converted PS (5 or 10 g/L) into AD or ADD (each in a concentration of 2.5 or 5 g/L), respectively. At the second stage, mycelium of the fungus under the revealed optimal conditions reduced AD or ADD with more than 90% efficiency to form TS or BD, respectively. Based on transcriptome analysis, six candidate genes that might encode 17β-HSDs in the Curvularia sp. genome were revealed. Along with 17β-HSDs, the fungus possessed inducible P450cur 7-monooxygenase, which led to the accumulation of 7α-hydroxytestosterone (7α-OH-TS) as a major product from AD (up to 83% within 24 h after mycelium addition at the second stage of cascade biotransformation). The presence of protein synthesis inhibitor cycloheximide (CHX) prevented 7α/β-hydroxylation due to inhibition of de novo synthesis of the enzyme in the fungal cells. The results demonstrate the high biotechnological potential of the Curvularia sp. strain and open up prospects for the synthesis of valuable 17β-reduced and 7-hydroxylated steroids by cascade biotransformations.

African walnut (Plukenetia conophora) oil promotes glucose uptake while improving energy metabolism and steroidogenesis and maintaining surface architecture in rat testes.

African walnut (Plukenetia conophora) oil (AWO) has been reported for its nutritional and medicinal properties and has been employed for the management of metabolic diseases including hyperglycemia-mediated ailments. In the present study, AWO was investigated for its ability to stimulate glucose uptake and its effect on energy metabolism, steroidogenesis, and tissue morphology in isolated testes of Wistar rats. Isolated testes were incubated with AWO (30-240 μg/mL) in the presence of 11.1 mMol glucose at 37°C for 2 h. Control consisted of testes incubated with glucose only, while normal control consisted of testes not incubated with AWO and/or glucose. The standard antidiabetic drug was metformin. Incubation with AWO led to significant increase in glucose uptake, hexokinase, glyoxalase 1, glutathione reductase, glutathione peroxidase, 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase activities, GLUT4, glutathione, and ATP levels while concomitantly suppressing glucose-6-phosphatase, fructose-1,6-biphosphatase, glycogen phosphorylase, aldose reductase, polyol dehydrogenase, E-NTPDase, and ATPase activities. Furthermore, incubation with AWO led to improved testicular morphology while elevating testicular levels of magnesium, sulfur, potassium, calcium, and iron. Fatty acid profiling with GC-MS revealed linoleic acid and linolenic acid as the predominant essential fatty acids in AWO. Molecular docking analysis revealed potent molecular interactions of linoleic acid and linolenic acid with GLUT4. These results suggest the ability of AWO to improve testicular glucose metabolism and steroidogenesis and can be explored in the management of male infertility.

Structural determinants of parabens in inhibiting human and rat gonadal 3β-hydroxysteroid dehydrogenase

This study delved into the impacts of 10 parabens on the activity of human and rat gonadal 3β-hydroxysteroid dehydrogenase (3β-HSD) within human KGN cell and rat testicular microsomes, as well as on the secretion of progesterone in KGN cells and the inhibitory potency was compared between human and rats. Intriguingly, the outcomes revealed that ethyl, propyl, butyl, hexyl, heptyl, nonyl, phenyl, and benzyl parabens displayed varying IC50 values for human 3β-HSD2, from 4.15 to 139.96 μM, demonstrating characteristics of mixed inhibitors. Notably, within KGN cells, all examined parabens, excluding nonyl and phenyl parabens, significantly inhibited progesterone secretion at 5–50 μM. In the case of rats, the IC50 values for these parabens on gonadal 3β-HSD1 fluctuated between 7.15 and 110.76 μM, likewise functioning as mixed inhibitors. Through docking analysis, it was proposed that most parabens effectively bind to NAD+ and/or steroid binding site. Moreover, bivariate correlation analysis unveiled an inverse correlation between IC50 values and structural characteristics such as LogP, molecular weight, heavy atom number, and carbon number within the alcohol moiety of parabens. 3D-QSAR elucidated pivotal regions, comprising hydrogen bond donor, hydrogen bond acceptor, and hydrophobic region, with the most potent inhibitor nonyl paraben engaging with all regions, while the weakest inhibitor ethyl paraben interacted with the regions except for the hydrophobic region. In conclusion, this investigation underscored the inhibitory effects imparted by several parabens on both human and rat gonadal 3β-HSD activity, with their inhibitory potency being modulated by aspects of hydrophobicity and carbon chain length.

Evaluation of 3β-hydroxysteroid dehydrogenase activity using progesterone and androgen receptors-mediated transactivation.

3β-Hydroxysteroid dehydrogenases (3β-HSDs) catalyze the oxidative conversion of delta (5)-ene-3-beta-hydroxy steroids and ketosteroids. Human 3β-HSD type 2 (HSD3B2) is predominantly expressed in gonadal and adrenal steroidogenic cells for producing all classes of active steroid hormones. Mutations in HSD3B2 gene cause a rare form of congenital adrenal hyperplasia with varying degree of salt wasting and incomplete masculinization, resulting from reduced production of corticoids and androgens. Therefore, evaluation of the HSD3B2 enzymatic activity in both pathways for each steroid hormone production is important for accurately understanding and diagnosing this disorder. Using progesterone receptor (PR)- and androgen receptor (AR)-mediated transactivation, we adapted a method that easily evaluates enzymatic activity of HSD3B2 by quantifying the conversion from substrates [pregnenolone (P5) and dehydroepiandrosterone (DHEA)] to (progesterone and androstenedione). HEK293 cells were transduced to express human HSD3B2, and incubated medium containing P5 or DHEA. Depending on the incubation time with HSD3B2-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the PR/AR expression vector and progesterone-/androgen-responsive reporter. Culture media from human and other mammalian HSD3B1-expressing cells also increased the luciferase activities. HEK293 cells expressing various missense mutations in the HSD3B2 gene revealed the potential of this system to evaluate the relationship between the enzymatic activities of mutant proteins and patient phenotype.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Histochemical analysis of 3β-hydroxysteroid dehydrogenase activity in some organs not classified as producing steroid hormones

Histochemical analysis of 3β-hydroxysteroid dehydrogenase activity in some organs not classified as producing steroid hormones

Chalcones from plants cause toxicity by inhibiting human and rat 11β-hydroxysteroid dehydrogenase 2: 3D-quantitative structure-activity relationship (3D-QSAR) and in silico docking analysis

Chalcones from licorice and its related plants have many pharmacological effects. However, the effects of chalcones on the activity of human and rat 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), and associated side effects remain unclear. The inhibition of 11 chalcones on human and rat 11β-HSD2 were evaluated in microsomes and a 3D-quantitative structure-activity relationship (3D-QSAR) was analyzed. Screening revealed that bavachalcone, echinatin, isobavachalcone, isobavachromene, isoliquiritigenin, licochalcone A, and licochalcone B significantly inhibited human 11β-HSD2 with IC50 values ranging from 15.62 (licochalcone A) to 38.33 (echinatin) μM. Screening showed that the above chemicals and 4-hydroxychalcone significantly inhibited rat 11β-HSD2 with IC50 values ranging from 6.82 (isobavachalcone) to 72.26 (4-hydroxychalcone) μM. These chalcones acted as noncompetitive/mixed inhibitors for both enzymes. Comparative analysis revealed that inhibition of 11β-HSD2 depended on the species. Most chemicals bind to the NAD+ binding site or both the NAD+ and substrate binding sites. Bivariate correlation analysis showed that lipophilicity and molecular weight determine inhibitory strength. Through our 3D-QSAR models, we identified that the hydrophobic region, hydrophobic aliphatic groups, and hydrogen bond acceptors are pivotal factors in inhibiting 11β-HSD2. In conclusion, many chalcones inhibit human and rat 11β-HSD2, possibly causing side effects and there is structure-dependent and species-dependent inhibition on 11β-HSD2.

Comparative effects of chemical and green zinc oxide nanoparticles in caprine testis: ultrastructural and steroidogenic enzyme analysis

ABSTRACT Recent advancements in nanotechnology has opened up enormous possibilities in diverse sectors such as industries, agriculture, environmental remediation, electronics, medicine and varied industries. Among metal oxide nanoparticles zinc oxide nanoparticles has gained considerable attention due to their fascinating physiochemical properties. Rapid growth in the use of zinc oxide nanoparticles (ZnONPs) in daily household products, food and feed additives, biological products, medicine, as antimicrobial agents, electronics and agriculture, creates serious toxic potential risks of these engineered nanoparticles on living organisms. The aim of present study was to assess the effects of synthesized chemical ZnONPs and green ZnONPs on testicular tissue of Capra hircus (goat) in vitro. The reproductive stress was analyzed by ultrastructural damage, change in frequency of apoptotic cells and alteration in steroidogenic enzyme activity. The transmission electron micrographs of testicular cells after treatment with chemical and green ZnONPs at three doses (10 µg/ml, 20 µg/ml and 30 µg/ml) for exposure duration 4 h and 8 h illustrated that chemical nanoparticles induced more alterations, identified as ruptured nuclear membrane, condensation and margination of chromatin material in somatic cells and germ cells in the seminiferous tubules, presence of apoptotic bodies in nucleus of spermatocytes and spermatids, reduction in number of cell organelles, vacuolization and hyalinization of cytoplasm. Maximum damage was observed after treatment of testicular tissues with 30 µg/ml of chemical ZnONPs for 8 h exposure duration. However, the green ZnONPs were found to be less toxic as evidenced by few apoptotic characteristics in testicular cells. The results of fluorescence assay by acridine orange staining showed significant increase in the percentage of apoptotic cells in chemical treated groups as compared to green and control groups. Decreased enzyme activity of 3β-Hydroxysteroid dehydrogenase and 17β-Hydroxysteroid dehydrogenase was assayed in chemical ZnONPs than green ZnONPs treated groups. Our results confirm that chemical ZnONPs are significantly more toxic in comparison to green ZnONPs and adversely affects the male fertility.

Changes of androgen and corticosterone metabolites excretion and conversion in cystic fibrosis.

Cystic fibrosis (CF) is a life-threatening inherited disease related to a mutation in the CFTR gene, that leads to serious health complications such as chronic pulmonary infections, pancreatic insufficiency, dysfunction of the sweat glands and reproductive system. For the first time, we have described the profile of corticosterone and androgen metabolites in urine, as well as the activity of enzymes involved in steroid genesis and metabolism in people with CF, using gas chromatography/mass spectrometry. A significant reduction in the excretion of most of the measured metabolites in CF was found. These differences were observed in the group of progestagen metabolites, as well as among metabolites of corticosterone and androgens. We revealed higher activities of 17β-hydroxysteroid dehydrogenase and 17,20-lyase in the Δ4 pathway compared with controls, what can promote the androgen synthesis through the backdoor androgen pathway. We have also found the increased conversion activity of 11-oxyganated steroids by 5a-reductase in backdoor pathway. Levels of the most potent and vital androgens (testosterone and dihydrotestosterone) are comparable in both groups. However, the excretion of dehydroepiandrosterone was lower in CF. Decreased cholesterol lipoprotein levels may contribute to limited intracellular cholesterol supply and reduced adrenal steroidogenesis in CF individuals. Changes in the activity of some steroidogenesis enzymes may suggest the presence of some peripheral adaptive mechanisms in CF to maintain androgen balance in the body despite the limited sufficiency of secretion by the adrenal cortex.

Changes in cortisol and cortisone in hair of pigs reared under heat stress conditions.

Heat stress accounts for millions of dollars in losses for swine producers worldwide. The aim of the present study was to determine and evaluate cortisol and cortisone in hair as indicators of thermal stress in growing pigs reared under high environmental temperatures. The study was carried out in two independent batches of commercial crosses of Lean Duroc and Pietrain in trials 1 and 2, respectively, during the growing period (from 40 to 100 kg; 81 days in trial 1 and 77 days in trial 2) in the same commercial farm in Spain during the summers of 2020 and 2021. In both cases, four rooms were used. In Trial 1, Room 1 had cooling and 11 pigs per pen; Room 2 had no cooling and 13 pigs per pen; Room 3 had no cooling and 11 pigs per pen, and Room 4 had cooling and 13 pigs per pen. In Trial 2, Rooms 2 and 3 had cooling and rooms 1 and 4 had no cooling, and all of them had 13 pigs per pen. Mean THI value was higher (p < 0.0001) in rooms without cooling systems (75.0 trial 1; 74.9 trial 2) than with them (71.3 trial 1; 71.7 trial 2). A total of four pens per room (16 in total) was selected for analysis of hair corticoids and all pigs inside were sampled at the end of the study. Fifty percent of the pigs were males (castrated and intact in trial 1 and 2, respectively) and 50% females. In total, 44, 52, 44, and 52 pigs, respectively, were sampled in four rooms from the first trial and 52 for each of four rooms in Trial 2. Cortisol concentrations in hair did not show any significant change in relation to cooling-non-cooling in any trial. However, hair cortisone concentration was 172.3 pg./mg and 105.8 pg./mg less (p < 0.001) in pigs housed with cooling systems compared to those without them in Trial 1 and 2, respectively. In addition, the cortisone/cortisol ratio, which is an estimator of the activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2, was also greater in rooms without cooling than in rooms with cooling in both trials (p < 0.0001 and p = 0.0105 for Trials 1 and 2, respectively). In relation to the sex effect, the results showed greater levels in females than in castrated males both in cortisone and the cortisol/cortisone ratio while cortisol hair levels were greater in intact males than in females. Therefore, the use of cortisone and the estimation of 11β-HSD type 2 activity in hair is recommended to evaluate the chronic stress produced by high environmental conditions in pigs instead of using hair cortisol concentrations alone.

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.

The analysis of pesticides and fungicides in the inhibition of human and rat placental 3β-hydroxysteroid dehydrogenase activity: Mode of inhibition and mechanism

3β-Hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase 1 (3β-HSD1) plays a critical role in the biosynthesis of progesterone from pregnenolone in the human placenta to maintain normal pregnancy. Whether they inhibit placental 3β-HSD1 and mode of inhibition remains unclear. In this study, we screened 21 pesticides and fungicides in five classes to inhibit human 3β-HSD1 and compared them to rat homolog 3β-HSD4. 3β-HSD activity was measured by catalyzing pregnenolone to progesterone in the presence of NAD+. Of the 21 chemicals, azoles (difenoconazole), thiocarbamates (thiram and ferbam) and organochlorine (hexachlorophene) significantly inhibited human 3β-HSD1 with half maximal inhibitory concentration (IC50) values of 2.77, 0.24, 0.68, and 17.96 μM, respectively. We also found that difenoconazole, ferbam and hexachlorophene are mixed/competitive inhibitors of 3β-HSD1 while thiram is a mixed/noncompetitive inhibitor. Docking analysis showed that difenoconazole and hexachlorophene bound steroid-binding site. Difenoconazole and hexachlorophene except thiram and ferbam also significantly inhibited rat 3β-HSD4 activity with IC50 of 1.12 and 2.28 µM, respectively. Thiram and ferbam significantly inhibited human 3β-HSD1 possibly by interfering with cysteine residues, while they had no effects on rat 3β-HSD4. In conclusion, some pesticides potently inhibit placental 3β-HSD, leading to the reduction of progesterone formation.

Protective effects and mechanisms of the Erzhi formula on glucocorticoid induced primary cortical neuron injury.

High concentrations of glucocorticoids (GC) can cross the blood-brain barrier into the brain parenchyma, triggering a stress state that can lead to a range of physiological changes. This study investigated whether Erzhi formula has neuroprotective effects against glucocorticoid damage by establishing a dexamethasone-induced primary cortical neuron injury model in vitro. The results showed that Erzhi formula could reduce dexamethasone-induced apoptosis in primary cultured cortical neurons and improve synaptic damage. Further, network pharmacological analysis revealed that Erzhi formula may exert antidepressant effects by multi-component, multi-target, and multi-pathway characteristics, in which Salidroside, Biochanin-A and other ingredients are key components, HSD11B1, NR3C1, and other proteins are key targets, and steroid metabolism may be a key process in its action. Moreover, our study found that the neuroprotective effect of Erzhi formula might be related to the 11β-HSD1-GC/glucocorticoid receptor (GR) signaling pathway. The Erzhi formula could significantly inhibit the activity of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in vitro using homogeneous time-resolved fluorescence. In addition to providing evidence for the pharmacological effects of the Erzhi formula, the present study lays down the foundation for subsequent experiments.

Pre-puberty cannabichromene exposure modulates reproductive function via alteration of spermatogenesis, steroidogenesis, and eNOS pathway metabolites.

Cannabis and cannabinoids affect almost every system of the body and exert systemic effects such as alterations in memory and cognitive functions, neurotransmission impediment, as well as obstruction of endocrine and reproductive system functions. Reproduction is a complicated phenomenon that integrates biological, psychological and behavioural aspects, hence susceptible to intracellular and extracellular modulations by numerous chemicals and toxicants like cannabis. The effects of early-life exposure to cannabis on reproductive function biomarkers and genes were investigated in male and female Wistar rats in this study. An initial computational analysis (molecular docking and induced fit docking) of some cannabinoids with reproductive enzymes; androgen and follicle stimulating hormone receptors was conducted. Overall, cannabichromene (CBC) had the best IFD scores and binding free energies for the two proteins studied and it interacted with notable amino acids within their active sites. Subsequently, forty (40) Wistar rats, 20 male and 20 female (24-28 days old, weighing 20-28±2g) were divided into two groups each and orally administered CBC for 21 days. Penile tissues, testes and ovaries, were collected for biochemical analysis (hormonal assays, enzyme activities, and metabolite concentrations), gene expressions, and histological evaluations. Activities of arginase and phosphodiesterase-5 in the penile tissue were significantly increased, while nitric oxide and calcium levels were significantly (p<0.05) decreased in the CBC-exposed groups relative to the control group. Semen analysis showed significantly more abnormalities and decreased concentration of spermatozoa in the CBC-exposed group compared to the control. Activities of 17β-hydroxysteroid dehydrogenase and cholesterol level were decreased in both testes and ovaries of CBC-exposed groups. Furthermore, levels of testosterone, progesterone, luteinizing, and follicle-stimulating hormones were reduced in the serum of CBC rats. Moreover, relative expressions of androgen receptor and follicle-stimulating hormone receptor genes were significantly downregulated in the CBC-exposed groups. Histological evaluations revealed lesions, tubular necrosis, and cellular congestions in both the testes and ovaries. This study suggests that pre-puberty exposure to cannabis modulates reproductive functions via cannabichromene inhibition of steroidogenesis, stimulation of erectile dysfunction (modulation of intermediates and enzymes of the endothelial nitric oxide synthase (eNOS) pathway in the penile tissue), and downregulation of the expressions of genes associated with reproduction.

Creation and Characterization of Mycolicibacterium Smegmatis mc2155 with Deletions in Genes Encoding Sterol Oxidation Enzymes

The fast-growing saprotrophic strain Mycolicibacterium smegmatis mc2155 is capable of utilizing plant and animal sterols and can be used for creation of genetically engineered strains producing biologically active steroids. Oxidation of the 3β-hydroxyl group and Δ5(6)→Δ4(5) double bond isomerization followed by formation of stenones from sterols are considered as the initial stage of steroid catabolism in some actinobacteria. The study of the mechanism of steroid nucleus 3β-hydroxyl group oxidation is relevant for the creation of a method of the microbiological production of valuable 3β-hydroxy-5-en-steroids. A mutant strain of M. smegmatis with deletions in three genes (MSMEG_1604, MSMEG_5228 and MSMEG_5233) encoding known enzymes exhibiting 3β-hydroxysteroid dehydrogenase activity was constructed by homologous recombination coupled with double selection. The resulting mutant retained macromorphological properties and the ability to convert cholesterol. 3-Keto-4-en-steroids were found among the sterol catabolism intermediates. Experimentally obtained data indicate the presence of a previously undetected intracellular enzyme that performs the function of 3β-hydroxysteroid dehydrogenase/Δ5(6)→Δ4(5) isomerase.

Spontaneous Remission of Primary Aldosteronism with Mineralocorticoid Receptor Antagonist Therapy: A Review.

In this review, we describe previous basic and clinical studies on autonomous aldosterone production. Over the past decades, mineralocorticoid receptor antagonists (MRAs) have been found to concentration-dependently inhibit steroidogenesis in different degrees. However, many studies have proven the suppressive effects of MRAs on the activities of hormone synthase. The probable factors of cytochrome P-450 reduction, both in microsomes and mitochondria, have also been considered: (1) one of the spironolactone metabolite forms had destructive function, except canrenone, (2) 7α-thio-spironolactone was an obligatory intermediate in the spironolactone-induced CYP450 decrease, and (3) the contributing steroids should have 7α-methylthio or 7α-methylsulfone groups. In previous clinical research, spironolactone-body-containing cells showed a type II pattern of enzyme activity (i.e., enhanced 3β-hydroxysteroid dehydrogenase, glucose-6-phosphate, and NADP-isocitrate dehydrogenase activities and weaken succinate dehydrogenase activity), and the subcapsular micronodules composed of spironolactone-body-containing cells also exhibited a type II pattern and excess aldosterone secretion, indicating that the subcapsular micronodules might be the root of aldosterone-producing adenoma. Moreover, combined with the potential impeditive function to aldosterone secretion, a few cases of spontaneous remission of primary aldosteronism, with normal ranges of blood pressure, plasma potassium, plasma renin activity, and aldosterone renin ratio, have been reported after long-term treatment with MRAs.

Bioproduction of testosterone from phytosterol by Mycolicibacterium neoaurum strains: “one-pot”, two modes

The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first “oxidative” stage, phytosterol (5–10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17β-hydroxysteroid dehydrogenase activity in the strains (“reductive” mode). The conditions favorable for “oxidative” and “reductive” stages have been revealed to increase the final testosterone yield. Glucose supplement and microaerophilic conditions during the “reductive” mode ensured increased testosterone production by mycolicibacteria cells. Both strains effectively produced testosterone from phytosterol, but highest ever reported testosterone yield was achieved using M. neoaurum VKM Ac-1815D: 4.59 g/l testosterone was reached from 10 g/l phytosterol thus corresponding to the molar yield of over 66%. The results contribute to the knowledge on phytosterol bioconversion by mycolicibacteria, and are of significance for one-pot testosterone bioproduction from phytosterol bypassing the intermediate isolation of the 17-ketoandrostanes.Graphical

Effects of phenolic compounds on 3β-hydroxysteroid dehydrogenase activity in human and rat placenta: Screening, mode of action, and docking analysis

Human 3β-hydroxysteroid dehydrogenase type I (HSD3B1) and rat type IV (HSD3B4) in placentas catalyze the conversion of pregnenolone to progesterone, which plays a key role in maintaining pregnancy. Many phenolic compounds potentially inhibit HSD3B in placentas as endocrine disruptors. In this study, the effects of 16 phenolic compounds on the activity of human HSD3B1 and rat HSD3B4 were determined and the structure-activity relationship was compared. HSD3B1 in human placental microsomes and HSD3B4 in rat placental microsomes were used to measure their activities and pregnenolone and NAD+ were used as substrates. Of the 16 phenolic compounds, 4-nonylphenol, pentabromophenol, and 2-bromophenol resulted in residual human HSD3B1 activity lower than 50 % and 4-nonylphenol and pentabromophenol resulted in residual rat HSD3B4 activity lower than 50 %. 4-Nonylphenol, pentabromophenol, and 2-bromophenol were mixed inhibitors of human HSD3B1, with Ki values of 2.31, 3.58 and 4.86 µM, respectively, while 4-nonylphenol and pentabromophenol were noncompetitive inhibitors of rat HSD3B4 with Ki values of 20.86 and 141.8 µM. Molecular docking showed that 4-nonylphenol, pentabromophenol, and 2-bromophenol docked to the active sites of human HSD3B1 and rat HSD3B4, and the shift of residue S125 in human HSD3B1 to T125 in rat HSD3B4 could explain the species-dependent difference in their inhibitory potency and mode of action. This study demonstrates that 4-nonylphenol, pentabromophenol, and 2-bromophenol are mixed inhibitors of human placental HSD3B1, while 4-nonylphenol and pentabromophenol are noncompetitive inhibitors of rat HSD3B4, possibly blocking the placental steroidogenesis.

Structure-activity relationship analysis of perfluoroalkyl carbonic acids on human and rat placental 3β-hydroxysteroid dehydrogenase activity

Placenta contains 3β-hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase (HSD3B), which catalyzes pregnenolone to progesterone for maintaining pregnancy. Perfluoroalkyl carbonic acids (PFC) are subclass of perfluoroalkyl substances containing 4–14 carbons (C4-C14) in the carbon backbone and are potential endocrine disruptors. Whether PFC inhibit HSD3B and structure-activity relationship (SAR) remains unclear. Herein, we screened 11 PFC for inhibiting human type I HSD3B (HSD3B1) and rat type IV HSD3B (HSD3B4) activities and determined SAR and mode of inhibition. HSD3B was measured by converting pregnenolone to progesterone assisted by NAD+ in placental microsomes. Of the 11 PFC, C9-C14 significantly inhibited human HSD3B1 activity at 100 μM. Half-maximal inhibitory concentration (IC50) values of C9-C14 compounds were 363.56 ± 12.14, 12.78 ± 0.69, 6.54 ± 0.65, 20.88 ± 0.41, 118.35 ± 0.16, and 149.26 ± 21.67 μM, respectively. We determined Ki values and mode of inhibition of three most potent PFC (C10-C12), and found that they were mixed inhibitors against pregnenolone, with Ki values of 5.57 ± 4.37, 2.04 ± 2.26, and 9.93 ± 7.71, respectively. Docking analysis showed that they bound steroid-binding site. Effects of PFC on rat placental HSD3B4 were performed. Of the 11 PFC, C10-C12 significantly inhibited rat HSD3B4 activity at 100 μM. IC50 values of C10-C12 compounds were 45.85 ± 1.49, 36.08 ± 1.50, and 88.74 ± 1.99 µM, respectively. Ki values and inhibition modes of the three most potent PFC (C10-C12) were studied. It was found that they were mixed inhibitors against pregnenolone, with Ki values of 48.16 ± 20.44, 36.28 ± 53.07, and 91.79 ± 21.75 μM, respectively. Docking analysis showed that they bound steroid-binding site of rat HSD3B4. In conclusion, PFC showed significant SAR differences. The potency of inhibiting HSD3B activity increased from C9 to C11, and then declined. Human HSD3B1 was more sensitive to the inhibition of rat HSD3B4.

Gas chromatography-mass spectrometry based steroid metabolomics in women with different phenotypes of polycystic ovarian syndrome and normal body weight

Objective: to study the steroid metabolomics in women with normal body weight and various PCOS phenotypes by gas chromatography-mass spectrometry (GC-MS). Materials and methods: forty-eight(48)women with PCOS aged 25±0,3 yearswith a BMI less than 25 kg/m2 were examined. The control group (CG) consisted of twenty-five (25) healthy women aged 26±0,6 years with a BMI of 23 (21-24) kg/m2. Immunoassays were used to determine the levels of hormones in serum. Urinary steroid profiles (USP) were studied by GC-MS method. Statistical data processing was performed using the software system STATISTICA for WINDOWS (ver. 10). Results: the article provides an analysis of the metabolism of androgens, glucocorticoids and progestogens in women with different phenotypes of polycystic ovary syndromeaccording to gas chromatography-mass spectrometry. Summary: the urinary excretion of androstenedione metabolites was increased in PCOS patients with androgen excess and anovulation (A and B phenotypes), dehydroepiandrosterone metabolites - in PCOS patients with androgen excess (A, B and C phenotypes). PCOS women with phenotype C showed raised urinary excretion of 11-oxo-pregnanetriol, pregnanetriol and 17-hydroxypregnanolone, a decrease in the ratios of the sum of tetrahydro derivatives of cortisol and cortisone to these progestogens, as well as determination of tetrahydro-21-deoxycorticol and nonclassical 5-ene-pregnenes according to GC-MS data. In fact, it indicated to deficiency of the 21-hydroxylase enzyme in these patients. It was found PCOS patients with androgen excess (A, B and C phenotypes) had the signs of insufficient 3β-hydroxysteroid dehydrogenase activity. PCOS women with phenotype A were revealed deficiency of 11β-hydroxysteroid dehydrogenase (type 1).