Chain-length matters: How benzylalkyldimethylammonium quaternary disinfectants disrupt gonadal steroidogenesis via 3β-hydroxysteroid dehydrogenase inhibition

Benzylalkyldimethylammonium (BAC) quaternary disinfectants are extensively used as antimicrobial agents. However, the specific BACs responsible for inhibition and their structure-activity relationship (SAR) for human 3β-hydroxysteroid dehydrogenase 2 (3β-HSD2) and its homolog rat 3β-HSD1, the critical enzyme in gonadal steroidogenesis, remain poorly understood. This study evaluated nine BACs for their inhibitory effects on human 3β-HSD2 and rat 3β-HSD1, determining potency (IC 50 ), inhibition kinetics, cellular impact, and binding interactions via molecular docking and 3D-QSAR. Inhibitory potency (IC 50 ) revealed C18 (11.84 μM) > C16 (15.51 μM) > C14 (25.25 μM) > C12 (30.63 μM) > C10 (56.86 μM) > C1-C8 BACs (no inhibition at 100 μM) against human 3β-HSD2 and C18 (8.23 μM) > C16 (10.74 μM) > C14 (12.21 μM) > C12 (14.38 μM) > C10 (26.62 μM) > C1-C8 BACs (no inhibition at 100 μM) against rat 3β-HSD1. Both enzymes exhibited mixed-type inhibition. Cellular suppression showed that C14, C16, and C18 BACs inhibited progesterone synthesis in human KGN granulosa tumor cells at ≥ 5 μM. Docking and SAR revealed binding at the NAD+ /steroid site via hydrogen bonding, hydrophobic, and van der Waals interactions. Lipophilicity (LogP), steric bulk, Fsp3, and flexibility correlated positively with potency, indicating that longer alkyl chains (≤C18) enhance inhibition. 3D-QSAR confirmed the importance of hydrophobic regions in binding affinity due to long alkyl chain. In conclusion, BACs inhibit gonadal 3β-HSD activity in a chain-length-dependent manner (C10-C18), with lipophilicity as a key determinant. These findings suggest that BAC disinfectants may act as potential endocrine disruptors by interfering with steroidogenesis. • Alkyl chain length of BACs dictates their inhibition against gonadal 3β-HSD. • BACs bind human and rat 3β-HSD NAD + binding sites as mixed inhibitors. • Inhibitory potency against 3β-HSD increased with increasing chain length of C10-C18 BACs.