Insights into the inhibitory mechanisms of alkyl phosphate esters on 3β-hydroxysteroid dehydrogenases: Implications for placental reproductive toxicity.

Alkyl phosphate esters (APEs) are mass-produced substances characterized by a wide range of physicochemical properties and applications. Their endocrine-disrupting effects via inhibition of placental 3β-hydroxysteroid dehydrogenase (3β-HSD) remain unclear. The objective of this study was to examine the inhibitory effects of 22 APEs on human 3β-HSD1 (h3β-HSD1) its rat homolog r3β-HSD4, critical enzymes in progesterone synthesis, using placental microsomes and computational analysis. The results demonstrated that h3β-HSD1 was more sensitive to inhibition by 100 μM APEs, exhibiting a V-shaped potency trend. Among the tested APEs, triheptyl phosphate ester showed the strongest inhibition of h3β-HSD1 (IC50 = 3.33 μM), followed by trihexyl (IC50 = 5.53 μM), triamyl (IC50 = 22.67 μM), triethylhexyl (IC50 = 22.85 μM), and tributyl phosphate esters (IC50 = 102.53 μM). In contrast, r3β-HSD4 exhibited lower sensitivity, with significant inhibition observed for only three APEs: diethyl, triamyl, and trihexyl phosphate esters, with IC50 values of 38.94, 109.76, and 51.49 μM, respectively. All tested APEs functioned as mixed-type inhibitors, and they suppressed progesterone production in human JAr cells at 10 and/or 100 μM (noncytotoxic concentrations). Lipophilicity and molecular size were identified as key factors influencing inhibitory potency against h3β-HSD1, as supported by 3D-QSAR modeling and molecular docking. These findings suggest that the mode of inhibition involves interactions at the NAD+/steroid interface, including hydrogen bonds and hydrophobic forces. Network toxicology analysis highlighted the association of h3β-HSD1 with endometrial cancer and preeclampsia. This research provides mechanistic insights into the potential reproductive risks of APEs and emphasizes the need for further studies on their environmental and health impacts.