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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 3β-hydroxysteroid dehydrogenases (3β-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 3β-HSD2 and rat 3β-HSD1, with ferbam demonstrating the highest potency. The mode of action for these compounds was characterized, showing mixed inhibition for human 3β-HSD2 and mixed/noncompetitive inhibition for rat 3β-HSD1. Additionally, it was observed that dithiothreitol dose-dependently reversed the inhibitory effects of dithiocarbamates on both human and rat gonadal 3β-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 3β-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 3β-HSDs, suggesting that these compounds likely exert their inhibitory effects through binding to cysteine residues.

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.

Prenatal exposure to diethylhexyl phthalate (DEHP) has been linked with a decline in testosterone levels in adult male rats, but the underlying mechanism remains unclear. We investigated the potential epigenetic regulation, particularly focusing on N6-methyladenosine (m6A) modification, as a possible mechanism. Dams were gavaged with DEHP (0, 10, 100, and 750 mg/kg/day) from gestational day 14 to day 21. The male offspring were examined at the age of 56 days. Prenatal DEHP administration at 750 mg/kg/day caused a decline in testosterone concentrations, an elevation in follicle-stimulating hormone, a downregulated expression of CYP11A1 HSD3B2, without affecting Leydig cell numbers. Interestingly, Methyltransferase Like 4 (METTL4), an m6A methyltransferase, was downregulated, while there were no changes in METTL3 and METTL14. Moreover, CYP11A1 showed m6A reduction in response to prenatal DEHP exposure. Additionally, METTL4 expression increased postnatally, peaking in adulthood. Knockdown of METTL4 resulted in the downregulation of CYP11A1 and HSD3B2 and an increase in SCARB1 expression. Furthermore, the increase in autophagy protection in adult Leydig cells induced by prenatal DEHP exposure was not affected by 3-methyladenosine (3MA) treatment, indicating a potential protective role of autophagy in response to DEHP exposure. In conclusion, prenatal DEHP exposure reduces testosterone by downregulating CYP11A1 and HSD3B2 via m6A epigenetic regulation and induction of autophagy protection in adult Leydig cells as a response to DEHP exposure.

Per- and polyfluoroalkyl (PFAS) substances are enduring industrial materials. 17β-Hydroxysteroid dehydrogenase isoform 1 (17β-HSD1) is an estrogen metabolizing enzyme, which transforms estrone into estradiol in human placenta and rat ovary. Whether PFAS inhibit 17β-HSD1 and what the structure-activity relationship (SAR) remains unexplored. We screened 18 PFAS for inhibiting human and rat 17β-HSD1 in microsomes and studied their SAR and mode of action(MOA). Of the 11 perfluorocarboxylic acids (PFCAs), C8-C14 PFCAs at a concentration of 100 μM substantially inhibited human 17β-HSD1, with order of C11 (half-maximal inhibition concentration, IC50, 8.94 μM) > C10 (10.52 μM) > C12 (14.90 μM) > C13 (30.97 μM) > C9 (43.20 μM) > C14 (44.83 μM) > C8 (73.38 μM) > others. Of the 7 per- and poly-fluorosulfonic acids (PFSAs), the potency was C8S (IC50, 14.93 μM) > C7S (80.70 μM) > C6S (177.80 μM) > others. Of the PFCAs, C8-C14 PFCAs at 100 μM markedly reduced rat 17β-HSD1 activity, with order of C11 (IC50, 9.11 μM) > C12 (14.30 μM) > C10 (18.24 μM) > C13 (25.61 μM) > C9 (67.96 μM) > C8 (204.39 μM) > others. Of the PFSAs, the potency was C8S (IC50, 37.19 μM) > C7S (49.38 μM) > others. In contrast to PFOS (C6S), the partially fluorinated compound 6:2 FTS with an equivalent number of carbon atoms demonstrated no inhibition of human and rat 17β-HSD1 activity at a concentration of 100 μM. The inhibition of human and rat enzymes by PFAS followed a V-shaped trend from C4 to C14, with a nadir at C11. Moreover, human 17β-HSD1 was more sensitive than rat enzyme. PFAS inhibited human and rat 17β-HSD1 in a mixed mode. Docking analysis revealed that they bind to the NADPH and steroid binding site of both 17β-HSD1 enzymes. The 3D quantitative SAR (3D-QSAR) showed that hydrophobic region, hydrogen bond acceptor and donor are key factors in binding to 17β-HSD1 active sites. In conclusion, PFAS exhibit inhibitory effects on human and rat 17β-HSD1 depending on factors such as carbon chain length, degree of fluorination, and the presence of carboxylic acid or sulfonic acid groups, with a notable V-shaped shift observed at C11.

Soil salinization poses a threat to the sustainability of agricultural production and has become a global issue. Cotton is an important cash crop and plays an important role in economic development. Salt stress has been harming the yield and quality of many crops, including cotton, for many years. In recent years, soil salinization has been increasing. It is crucial to study the mechanism of cotton salt tolerance and explore diversified materials and methods to alleviate the salt stress of cotton for the development of the cotton industry. Nanoparticles (NPs) are an effective means to alleviate salt stress. In this study, zinc oxide NPs (ZnO NPs) were sprayed on cotton leaves with the aim of investigating the intrinsic mechanism of NPs to alleviate salt stress in cotton. The results show that the foliar spraying of ZnO NPs significantly alleviated the negative effects of salt stress on hydroponic cotton seedlings, including the improvement of above-ground and root dry and fresh weight, leaf area, seedling height, and stem diameter. In addition, ZnO NPs can significantly improve the salt-induced oxidative stress by reducing the levels of MDA, H2O2, and O2− and increasing the activities of major antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Furthermore, RNA-seq showed that the foliar spraying of ZnO NPs could induce the expressions of CNGC, NHX2, AHA3, HAK17, and other genes, and reduce the expression of SKOR, combined with the CBL-CIPK pathway, which alleviated the toxic effect of excessive Na+ and reduced the loss of excessive K+ so that the Na+/K+ ratio was stabilized. In summary, our results indicate that the foliar application of ZnO NPs can alleviate high salt stress in cotton by adjusting the Na+/K+ ratio and regulating antioxidative ability. This provides a new strategy for alleviating the salt stress of cotton and other crops, which is conducive to the development of agriculture.