Herbicides as Isoform-Selective Inhibitors of Human 3β-HSD1 and Rat 3β-HSD4: Structure–Activity Relationships and Implications for Progesterone Biosynthesis and Recurrent Pregnancy Loss

Environmental exposure to herbicides has raised significant concerns. However, whether herbicides affect placental 3β-hydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase (3β-HSD) remains unclear. This study investigated the inhibitory effects of 14 herbicides, especially dinitroanilines, on human (h3β-HSD1) and rat (r3β-HSD4), key enzymes in progesterone biosynthesis. In vitro assays showed isopropalin and oryzalin potently inhibited h3β-HSD1 (IC 50 39.56 and 52.99 μM) via mixed-type inhibition. Inplacental JAr cells, oryzalin and profluraline suppressed progesterone synthesis at 100 μM, while isopropalin had no effect. Species-dependent inhibition was observed; oryzalin was most potent against r3β-HSD4 (IC 50 33.52 μM), and cyanazine also inhibited the rat isoform. Molecular docking indicated their binding to the NAD + -binding pocket. SAR and pharmacophore modeling identified molecular volume and flexibility as key for h3β-HSD1 potency. Network toxicology linked herbicide exposure to recurrent pregnancy loss via steroidogenic genes. These findings provide mechanistic evidence that specific herbicides disrupt progesterone production and may contribute to adverse reproductive outcomes.