Species-specific inhibition of human and rat gonadal 3β-hydroxysteroid dehydrogenases by emerging organophosphate esters: A multiscale approach combining in vitro assays and computational modeling

Organophosphate esters (OPEs) are widely used as flame retardants and plasticizers and are considered emerging environmental pollutants. However, whether they affect human and rat gonadal 3β-hydroxysteroid dehydrogenase (3β-HSD) isoforms, crucial enzymes in steroidogenesis, remains unclear. A total of 22 trialkyl OPEs were comprehensively screened for their inhibitory effects on human and rat gonadal 3β-HSDs, and structure-activity relationships (SAR), mode of action, and in silico docking were explored. C4-C6 alkyl OPEs inhibited human KGN cell 3β-HSD2 activity with the inhibitory strength order of trihexyl (IC 50 : 20.92 µM) > triamyl (52.58 µM) > tributyl (90.16 µM), with a V-shaped turn observed for C7-C10 trialkyl OPEs, and the same pattern was seen for the inhibition on rat testicular 3β-HSD1 enzyme. Most trialkyl OPEs functioned as mixed inhibitors and reduced progesterone secretion in human KGN cells. Rat 3β-HSD1 was found to be more susceptible than human 3β-HSD2. Docking studies showed that OPEs bound to the NAD + and steroid binding sites. SAR and 3D-quantitative SAR indicated that hydrophobicity and carbon chain length were critical for the inhibition activity and the V-shaped pattern while docking analysis emphasized the importance of the optimal size of trialkyl OPEs for binding affinity to human 3β-HSD2 active sites. In conclusion, trialkyl OPEs can inhibit human and rat gonadal 3β-HSDs, depending on factors such as carbon chain length and lipophilicity, with a distinct V-shaped shift at C6. These findings suggest that OPEs may have endocrine-disrupting properties and pose risks to reproductive health.