Human and rat gonadal 3ß-hydroxysteroid dehydrogenases are suppressed by dithiocarbamate fungicides via interacting with cysteine residues.

Dithiocarbamates have been widely used in various industrial applications, such as insecticides (ferbam) or drug (disulfiram). This study explored the inhibitory effects of dithiocarbamates on human and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD) and investigated the structure-activity relationship and mechanistic insights. The inhibitory activity of six dithiocarbamates and thiourea on the conversion of pregnenolone to progesterone was evaluated using human KGN cell and rat testicular microsomes, with subsequent progesterone measurement using HPLC-MS/MS. The study found that among the tested compounds disulfiram, ferbam, and thiram exhibited significant inhibitory activity against human 3ß-HSD2 and rat 3ß-HSD1, with ferbam demonstrating the highest potency. The mode of action for these compounds was characterized, showing mixed inhibition for human 3ß-HSD2 and mixed/noncompetitive inhibition for rat 3ß-HSD1. Additionally, it was observed that dithiothreitol dose-dependently reversed the inhibitory effects of dithiocarbamates on both human and rat gonadal 3ß-HSD enzymes. The study also delved into the penetration of these dithiocarbamates through the human KGN cell membrane and their impact on progesterone production, highlighting their potency in inhibiting human 3ß-HSD2. Furthermore, bivariate correlation analysis revealed a positive correlation of LogP (lipophilicity) with IC50 values for both enzymes. Docking analysis indicated that dithiocarbamates bind to NAD+ and steroid-binding sites, with some interactions with cysteine residues. In conclusion, this study provides valuable insights into the structure-activity relationship and mechanistic aspects of dithiocarbamates as inhibitors of human and rat gonadal 3ß-HSDs, suggesting that these compounds likely exert their inhibitory effects through binding to cysteine residues.

The impact of tetrachlorobisphenol A exposure during puberty: Altered Leydig cell development and induced endoplasmic reticulum stress in male mice.

Tetrachlorobisphenol A (TCBPA), a halogenated flame retardant and endocrine disruptor, has been detected in human urine and serum. While previous research has shown its impact on the reproductive system, investigations into its mechanisms during puberty remain limited. This study aims to explore the effects of TCBPA on Leydig cells in adolescent mice and potential underlying mechanisms. Male C57 mice of age 28 days were gavaged with 50, 100, and 200 mg/kg/day for 28 days. TCBPA did not alter body weight and testis weight but lowered testosterone levels at 100 and 200 mg/kg and reduced sperm count in the epididymis at 200 mg/kg. TCBPA lowered Leydig cell number at 200 mg/kg while it downregulated key Leydig cell gene (Lhcgr, Scarb1, Cyp11a1, Cyp17a1, Hsd3b6, Hsd17b3 and Insl3) as low as 50 mg/kg. Further study indicated that TCBPA induced reactive oxygen species and caused endoplasmic reticulum stress. In vitro study in TM3 mouse Leydig cells showed that TCBPA indeed induced reactive oxygen species and caused endoplasmic reticulum stress at 75 µM and inhibited testosterone production at this concentration and addition of antioxidant tocopherol can reverse it. These discoveries provide new insights and references for a deeper understanding of the toxic mechanisms of TCBPA on Leydig cells during puberty.

Halogenated bisphenol A derivatives potently inhibit human and rat 11ß-hydroxysteroid dehydrogenase 1: Structure-activity relationship and molecular docking.

Chlorinated bisphenol A (BPA) derivatives are formed during chlorination process of drinking water, whereas bisphenol S (BPS) and brominated BPA and BPS (TBBPA and TBBPS) were synthesized for many industrial uses such as fire retardants. However, the effect of halogenated BPA and BPS derivatives on glucocorticoid metabolizing enzyme 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) remains unclear. The inhibitory effects of 6 BPA derivatives in the inhibition of human and rat 11ß-HSD1 were investigated. The potencies for inhibition on human 11ß-HSD1 were TBBPA (IC50, 3.87 µM) = monochloro BPA (MCBPA, 4.08 µM) = trichloro BPA (TrCBPA, 4.41 µM) > tetrachloro BPA (TCBPA, 9.75 µM) > TBBPS (>100 µM) = BPS (>100 µM), and those for rat 11ß-HSD1 were TrCBPA (IC50, 2.76 µM) = MCBPA (3.75 µM) > TBBPA (39.58 µM) > TCBPA = TBBPS = BPS. All these BPA derivatives are mixed/competitive inhibitors of both human and rat enzymes. Molecular docking studies predict that MCBPA, TrCBPA, TCBPA, and TBBPA all bind to the active site of human 11ß-HSD1, forming hydrogen bonds with catalytic residue Ser170 except TCBPA. Regression of the lowest binding energy with IC50 values revealed a significant inverse linear regression. In conclusion, halogenated BPA derivatives are mostly potent inhibitors of human and rat 11ß-HSD1, and there is structure-dependent inhibition.

Benzene ring bisphenol A substitutes potently inhibit human, rat, and mouse gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Bisphenol A (BPA) is a chemical used in the production of certain plastics and resins. Recent research has found that BPA can inhibit the activity of 3ß-hydroxysteroid dehydrogenase/Δ5,4-isomerases (3ß-HSDs). Whether benzene ring BPA substitutes can inhibit human, rat, and mouse gonadal 3ß-HSDs, the structure-activity relationship and the underlying mechanism remain unclear. In this study, we compared 6 benzene ring BPA substitutes to BPA in the inhibition of human, rat, and mouse gonadal 3ß-HSDs and conducted structure-activity relationship and in silico docking analysis. The inhibitory activity (IC50) of human 3ß-HSD2 in KGN cells ranged from about 0.02 µM for bisphenol H to 8.75 µM for BPA, that of rat 3ß-HSD1 in testicular microsomes ranged from 0.099 µM for bisphenol H to 31.32 µM for BPA, and that of mouse 3ß-HSD6 ranged from 0.021 µM for BPH to ineffectiveness for 100 µM BPA. These compounds acted as mixed inhibitors with LogP inversely correlated with IC50 and ΔG positively correlated with IC50 value. Docking analysis showed that these compounds bind to the steroid active site of the 3ß-HSD enzymes. In conclusion, some benzene ring BPA substitutes potently inhibit gonadal 3ß-HSD in various species, and lipophilicity and binding affinity determine their inhibitory strength.

Citrinin inhibits the function of Leydig cells in male rats in prepuberty.

Citrinin, a mycotoxin existing in fruits, has nephrotoxicity, hepatotoxicity and embryotoxicity. The effects of citrinin on Leydig cell development in prepuberty remains unclear. Male Sprague-Dawley rats were gavaged with 0, 1, 2.5, and 5 mg/kg citrinin from postnatal days 21-28. Citrinin at 5 mg/kg significantly decreased serum testosterone levels, while increasing serum LH and FSH levels. Citrinin at 1-5 mg/kg markedly downregulated Hsd17b3 and HSD17B3 expression, while upregulating Srd5a1 (SRD5A1) and Akr1c14 (AKR1C14) expression at 2.5 and/or 5 mg/kg. Citrinin at 5 mg/kg also significantly increased PCNA-labeling index in Leydig cells. Citrinin at 5 mg/kg significantly raised testicular MDA amount, whiling at 2.5 and 5 mg/kg downregulating SOD1 and SOD2 expression. Citrinin at 5 mg/kg markedly decreased the ratio of Bcl2 to Bax, in consistent with the increased apoptosis in Leydig cells judged by TUNEL assay. Enzymatic assay revealed that citrinin inhibited rat testicular HSD3B1 activity at 100 µM and HSD17B3 activity at 10-100 µM. Citrinin at 50 µM and higher also induced reactive oxygen species (ROS) and apoptosis of R2C cell line. In conclusion, citrinin inhibits Leydig cell development at multiple levels via different mechanisms and oxidative stress partially plays a role.

Bisphenol A Analogues Inhibit Human and Rat 11ß-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 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) remain unclear. The inhibitory effects of 6 BPA analogues on the inhibition of human and rat 11ß-HSD1 were investigated. The potencies of inhibition on human 11ß-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 11ß-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 11ß-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 11ß-HSD isoforms.

UV-filter benzophenones suppress human, pig, rat, and mouse 11ß-hydroxysteroid dehydrogenase 1: Structure-activity relationship and in silico docking analysis.

Benzophenone chemicals (BPs) have been developed to prevent the adverse effects of UV radiation and they are widely contaminated. 11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) catalyze the conversion of inactive glucocorticoid to active glucocorticoid, playing critical role in many physiological function. However, the direct effect of BPs on human, pig, rat, and mouse 11ß-HSD1 remains unclear. In this study, we screened the inhibitory strength of 12 BPs on 4 species, and performed the structure-activity relationship (SAR) and in silico docking analysis. The inhibitory potency of BPs was: for human 11ß-HSD1, BP6 (IC50 = 18.76 µM) > BP8 (40.84 µM) > BP (88.89 µM) > other BPs; for pig 11ß-HSD1, BP8 (45.57 µM) > BP6 (59.44 µM) > BP2 (65.12 µM) > BP (135.56 µM) > other BPs; for rat 11ß-HSD1, BP7 (67.17 µM) > BP (68.83 µM) > BP8 (133.04 µM) > other BPs; and for mouse 11ß-HSD1, BP8 (41.41 µM) > BP (50.61 µM) > other BPs. These BP chemicals were mixed/competitive inhibitors of these 11ß-HSD1 enzymes. The 2,2'-dihydroxy substitutions in two benzene rings play a key role in enhancing the effectiveness of inhibiting 11ß-HSD1, possibly via increasing hydrogen bond interactions. Docking analysis shows that these BPs bind to NADPH/glucocorticoid binding sites and forms hydrogen bonds with catalytic residues Ser and/or Tyr. In conclusion, this study demonstrates that BP chemicals can inhibit 11ß-HSD1 from 4 species, and there are subtle species-dependent difference in the inhibitory strength and structural variations of BPs.

Triclosan inhibits testosterone biosynthesis in adult rats via inducing m6A methylation-mediated autophagy.

Triclosan is a potent antibacterial compound widely used in everyday products. Whether triclosan affects Leydig cell function in adult male rats remains unknown. In this study, 0, 50, 100, or 200 mg/kg/day triclosan was gavaged to Sprague-Dawley male rats from 56 to 63 days postpartum. Triclosan significantly reduced serum testosterone levels at ≥ 50 mg/kg/day via downregulating the expression of Leydig cell gene Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3 and regulatory transcription factor Nr3c2 at 100-200 mg/kg. Further analysis showed that triclosan markedly increased autophagy as shown by increasing LC3II and BECN1 and decreasing SQSTM1. The mRNA m6A modification analysis revealed that triclosan significantly downregulated Fto expression at 200 mg/kg while upregulating Ythdf1 expression at 100 and 200 mg/kg, leading to methylation of Becn1 mRNA as shown by MeRIP assay. Triclosan significantly inhibited testosterone output in rat R2C Leydig cells at ≥ 5 µM via downregulating Fto and upregulating Ythdf1. SiRNA Ythdf1 knockdown can reverse triclosan-mediated mitophagy in R2C cells, thereby reversing the reduction of testosterone output. In summary, triclosan caused Becn1 m6A methylation by downregulating Fto and upregulating Ythdf1, which accelerated Becn1 translation, thus leading to the occurrence of autophagy and the decrease of testosterone biosynthesis.

Demethoxylation of curcumin enhances its inhibition on human and rat 17ß-hydroxysteroid dehydrogenase 3: QSAR structure-activity relationship and in silico docking analysis.

Curcuminoids have many pharmacological effects. They or their metabolites may have side effects by suppressing 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3). Herein, we investigated the inhibition of curcuminoids and their metabolites on human and rat 17ß-HSD3 and analyzed their structure-activity relationship (SAR) and performed in silico docking. Curcuminoids and their metabolites ranked in terms of IC50 values against human 17ß-HSD3 were bisdemethoxycurcumin (0.61 µM) > curcumin (8.63 µM) > demethoxycurcumin (9.59 µM) > tetrahydrocurcumin (22.04 µM) > cyclocurcumin (29.14 µM), and those against rat 17ß-HSD3 were bisdemethoxycurcumin (3.94 µM) > demethoxycurcumin (4.98 µM) > curcumin (9.62 µM) > tetrahydrocurcumin (45.82 µM) > cyclocurcumin (143.5 µM). The aforementioned chemicals were mixed inhibitors for both enzymes. Molecular docking analysis revealed that they bind to the domain between the androstenedione and NADPH active sites of 17ß-HSD3. Bivariate correlation analysis showed a positive correlation between LogP and pKa of curcumin derivatives with their IC50 values. Additionally, a 3D-QSAR analysis revealed that a pharmacophore model consisting of three hydrogen bond acceptor regions and one hydrogen bond donor region provided a better fit for bisdemethoxycurcumin compared to curcumin. In conclusion, curcuminoids and their metabolites possess the ability to inhibit androgen biosynthesis by directly targeting human and rat 17ß-HSD3. The inhibitory strength of these compounds is influenced by their lipophilicity and ionization characteristics.

Azole fungicides inhibit human and rat gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Azole fungicides are widely used in the agricultural industry to control fungal infections in crops. However, recent studies have shown that some azole fungicides inhibit the activity of 3ß-hydroxysteroid dehydrogenases (3ß-HSDs) in the gonads. Out of the 16 azole fungicides tested, 8 were found to inhibit human KGN cell 3ß-HSD2 with IC50 values of less than 100 µM. The strongest inhibitor was difenoconazole, with an IC50 value of 1.88 µM. In contrast, only 3 of the azole fungicides inhibited rat testicular 3ß-HSD1, which was less sensitive to inhibition. Azole fungicides potently inhibited progesterone secretion by KGN cells under basal and forskolin stimulated conditions at ≥ 5 µM. The inhibitory strength of azole fungicides was determined by their lipophilicity (LogP), molecular weight, pKa, and binding energy. A pharmacophore analysis revealed that the hydrogen bond acceptor-lipid group was a critical feature required for inhibition. Overall, these findings suggest that the use of azole fungicides have unintended consequences on reproductive health due to their inhibition of gonadal 3ß-HSDs. Key words: Azole fungicides; steroid hormones; 3ß-hydroxysteroid dehydrogenase; docking analysis; lipophilicity.