Screening PFAS-Mediated Inhibition of Human Cytochrome P450 Enzyme Activity Reveals Nanomolar Potency against CYP2C9

Abstract Cytochrome P450 (CYP) enzymes catalyze the biotransformation of environmental toxicants, drugs, and endogenous substrates. Per- and polyfluoroalkyl substances (PFAS) accumulate in human liver tissue, yet the capacity of PFAS to inhibit hepatic CYP activity at human-relevant concentrations remains poorly characterized. This study tested the hypothesis that structurally diverse PFAS reversibly inhibit seven major human CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5) in pooled human liver microsomes (HLM) and examined their relevance to concentrations measured in human livers. Thirteen structurally diverse PFAS were evaluated (ηpfc = 4–11). Perfluorooctanesulfonic acid (PFOS) and perfluorooctane sulfonamides inhibited CYP2C9 at nanomolar concentrations, with 50% inhibitory concentrations (IC50) of 29–230 nM that exceeded the potency of sulfaphenazole, the reference CYP2C9 probe inhibitor (IC50 = 380 nM), and also exceeded several clinically used CYP2C9 inhibitors. A subset of long-chain PFAS inhibited CYP2C19, CYP2C8, and CYP2B6 at low-micromolar concentrations, while the tested PFAS inhibited CYP1A2, CYP2D6, and CYP3A4/5 generally weakly (IC50 typically ≥ 10 μM). Substrate-dependent experiments supported competitive CYP2C9 inhibition by PFOS and FOSA (nominal Ki = 16 and 57 nM) and mixed inhibition by EtFOSA (13 nM). Exploratory quantitative in vitro–in vivo extrapolation using unbound hepatic PFAS concentrations estimated from concentrations measured in 211 U.S. donors showed that hepatic PFAS burdens remained well below inhibitory thresholds for most individuals. However, 19% of individuals had hepatic PFOS within 10-fold of the CYP2C9 10% inhibitory concentration, and the equimolar 13-PFAS mixture inhibited CYP2C9 within 1.7-fold of the concentration-addition prediction, indicating that cumulative exposure can further narrow these margins. Because PFAS exposures are chronic and cumulative, sustained low-level CYP2C inhibition by PFAS may disrupt endogenous lipid signaling and contribute to interindividual variability in drug metabolism, particularly in subpopulations with elevated hepatic PFAS burdens or reduced baseline CYP2C9 function from genetic polymorphisms or liver disease.