Differences between human and rat 5α-Reductase 1 inhibition by phenolic disinfectants: Comparative kinetics and...

5α-Reductase 1 (SRD5A1), a key enzyme in neurosteroid and androgen biosynthesis, is expressed in both human and rat brains as well as androgen-dependent tissues. Despite its physiological significance, the inhibitory effects of phenolic disinfectants on SRD5A1 activity remain unknown. This study aimed to evaluate the inhibitory potential of eight phenolic disinfectants on SRD5A1 (using human SF126 microsomes) and rat brain microsomes. Among the tested compounds, triclosan emerged as the most potent inhibitor of human SRD5A1 (IC₅₀ = 6.62 μM), followed by 2-phenylphenol (18.69 μM), dichloroxylenol (21.10 μM), amylmetacresol (21.43 μM), and chloroxylenol (29.04 μM). In contrast, rat SRD5A1 was inhibited exclusively by triclosan (IC₅₀ = 7.67 μM) and dichloroxylenol (40.64 μM), suggesting difference in inhibition patterns. Surface plasmon resonance analysis revealed that triclosan binds with high affinity (KD = 4.07 μM) to the NADPH-binding site of human SRD5A1, reinforcing its role as a mixed/noncompetitive inhibitor. 2-Phenylphenol had a moderate binding affinity (KD = 19.2 μM). Kinetic studies using enzyme kinetics inhibition (mixed inhibition model) confirmed mixed/noncompetitive inhibition across both species, indicating allosteric interference with enzymatic activity. Functional assays in SF126 glial cells demonstrated that triclosan, amylmetacresol and 2-phenylphenol at 1 and 10 μM significantly suppressed dihydrotestosterone production without inducing cytotoxicity, highlighting their endocrine-disrupting potential at physiologically relevant concentrations. Structural analyses identified Glu60 as a conserved catalytic residue in SRD5A1 but revealed notable differences in substrate-binding pockets between human and rat isoforms. Molecular docking simulations showed that triclosan forms a hydrogen bond with Glu60 in human SRD5A1, whereas its binding mode in rat SRD5A1 involves additional hydrophobic interactions, explaining the observed species-dependent inhibitory profiles. 3D-QSAR modeling further correlated inhibitor potency with hydrophobicity (logP), generating a validated pharmacophore model (R² = 0.81) for future inhibitor screening. These findings emphasize the critical need for species-specific assessments when evaluating the endocrine-disrupting effects of environmental chemicals, particularly phenolic disinfectants, to ensure accurate risk assessment and regulatory decision-making.