Metabolic activation of pentachlorophenol: TCBQ as a potent inhibitor of gonadal steroidogenesis via 3β-hydroxysteroid dehydrogenase NAD+ binding-site thiolalkylation

Pentachlorophenol (PCP) is a pesticide and persistent endocrine disruptor. This study examined how PCP and its metabolite tetrachloro-1,4-benzoquinone (TCBQ) inhibit human (h3β-HSD2) and rat (r3β-HSD1) gonadal steroidogenic enzymes. In vitro assays using human KGN cells and rat testicular microsomes showed PCP moderately inhibited h3β-HSD2 (IC₅₀: 22.55 μM, mixed inhibitor), while TCBQ was more potent (IC₅₀: 6.42 μM). PCP (25-50 μM) significantly reduced progesterone in KGN cells, whereas TCBQ suppressed it at ≥ 1 μM. In rats, PCP weakly inhibited r3β-HSD1 (IC₅₀: 32.03 μM), but TCBQ was far stronger (IC₅₀: 41 nM, mixed/noncompetitive). Molecular docking revealed both compounds bind to the enzyme's steroid/NAD⁺ site, with TCBQ covalently attaching to Cys123 via Michael addition. Dithiothreitol (DTT) partially reversed TCBQ's inhibition but not PCP's, suggesting differing mechanisms. Findings indicate PCP's metabolic activation to TCBQ drives its stronger endocrine-disrupting effects, highlighting TCBQ's role as a potent 3β-HSD inhibitor in both species.