Neurotoxicological concerns: Azole fungicides as mixed/noncompetitive inhibitors of human and rat 5α-reductase 1

Azole fungicides are utilized in global agriculture to protect rice, vegetables, and fruits from fungal pathogens, yet their capacity to interfere with neurosteroid biosynthesis has raised significant toxicological concerns. In this study, we investigated the effects of eleven representative azole fungicides on the enzymatic activity of 5α-reductase 1 (SRD5A1) using both human and rat models. To assess inhibition, we employed HPLC-MS/MS to precisely measure testosterone to dihydrotestosterone conversion, complemented by SPR, intact cell assays, in silico docking, molecular dynamics, and 3D-QSAR modeling. Our screening revealed that three azoles inhibited the human isoform, whereas four inhibited the rat counterpart, with ipconazole demonstrating the strongest potency (IC₅₀ = 3.40 μM for human, 15.24 μM for rat). SPR confirmed its tight binding (KD = 2.12 μM) and NADPH disruption. Kinetic analyses classified these as mixed/noncompetitive inhibitors. In SF126 cells, 10 and 100 μM exposures markedly reduced DHT output. SAR correlated efficacy with LogP, and 3D-QSAR highlighted hydrophobic interactions as critical. Docking revealed that azoles occupy the NADPH-binding site via van der Waals forces, hydrogen bonds, hydrophobic contacts, and charge interactions. Collectively, our findings demonstrate that azole fungicides inhibit SRD5A1 across species, potentially causing neural disorders by depleting neurosteroids, underscoring the need for further neurotoxicological research.