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Aromatase (CYP19A1), a pivotal enzyme in the biosynthesis of estradiol from testosterone, is predominantly expressed in reproductive tissues including placentas. This study investigated the effects of paraben acid and nine parabens on the activity of human and rat CYP19A1 using microsomes derived from human and rat placentas and on estradiol secretion in human choriocarcinoma BeWo cells. The results showed that propyl, butyl, hexyl, heptyl, and nonyl parabens significantly inhibited human CYP19A1 activity, with IC50 values of 66.37, 61.08, 55.65, 48.26, and 27.24 μM, respectively. In BeWo cells, these parabens notably diminished estradiol secretion at concentrations of 100 μM. Similarly, rat CYP19A1 was inhibited by these parabens, with IC50 values of 98.07, 70.10, 41.30, 27.93, and 6.33 μM for propyl, butyl, hexyl, heptyl, and nonyl parabens, respectively. Kinetic analysis identified these compounds as mixed inhibitors. Bivariate correlation analysis revealed a negative correlation between the partition coefficient value, molecular weight, the number of carbon atoms in the alcohol moiety, as well as heavy atom number and IC50 values. Three-dimensional quantitative structure-activity relationship analysis highlighted the critical role of hydrophobic regions in determining inhibitory potency. Docking studies suggested that parabens interact with the heme-iron binding site of both human and rat CYP19A1. This study elucidates the inhibitory effects of various parabens on CYP19A1 and their binding mechanisms, thereby providing a deeper understanding of their potential impact on estrogen biosynthesis.

Bisphenol H (BPH) has emerged as a potential alternative to bisphenol A (BPA), which has been curtailed for use due to concerns over its reproductive and endocrine toxicity. This study investigates whether BPH exerts antiandrogenic effects by impairing Leydig cell function, a critical component in testosterone production. We administered orally BPH to adult male rats at doses of 0, 1, 10, and 100 mg/kg/day for 7 days. Notably, BPH treatment resulted in a dose-dependent reduction in testicular testosterone levels, with significant decreases observed at ≥ 1 mg/kg/day. Additionally, BPH affected the expression of key genes involved in steroidogenesis and cholesterol metabolism, including Nr5a1, Nr3c4, Lhcgr, Scarb1, and Star, at higher doses (10 and/or 100 mg/kg/day). The study also revealed alterations in antioxidant gene expression (Sod2 and Cat) and modulation of m6A-related genes (Ythdf1–3 and Foxo3) and their proteins. Through MeRIP-qPCR analysis, we identified increased m6A modifications in Scarb1 and Star genes following BPH exposure. In vitro experiments with primary Leydig cells confirmed that BPH enhanced oxidative stress and diminished testosterone production, which were partially mitigated by antioxidant vitamin E supplementation and Ythdf3 knockdown. Meanwhile, simultaneous administration of BPH and vitamin E to primary Leydig cells partially counteracted BPH-induced alterations in the Ythdf3 expression. Our findings underscore a novel mechanism by which BPH disrupts Leydig cell function through the oxidative stress-m6A modification-autophagy pathway, raising concerns about its potential reproductive toxicity.

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.