Comparative In Vitro Biotransformation of Triphenyl Phosphate in Rats, Humans, and Trout: Michaelis–Menten Kinetics and Elucidation of Metabolic Pathways via Nontarget Analysis

Abstract

Interspecies differences in triphenyl phosphate (TPhP) biotransformation are critical for extrapolating toxicological outcomes but remain poorly characterized. This study compared TPhP biotransformation (0.1–10 μmol·L–1) in rat, human, and trout liver S9 fractions to determine kinetic parameters and metabolic pathways. Human S9 exhibited the highest intrinsic clearance, driven by strong substrate affinity, whereas rat S9 showed lower clearance despite higher catalytic capacity. In contrast, rainbow trout displayed markedly limited biotransformation, characterized by only 39.11% depletion at 2.5 μmol·L–1 and apparent zero-order kinetics with very low reaction velocity, indicating a pronounced metabolic bottleneck. Nontarget analysis revealed both conserved and species-specific biotransformation pathways. Hydrolysis, monohydroxylation, and glucuronidation of monohydroxylated metabolites were conserved across all species. Aromatic dihydroxylation and subsequent glucuronidation were restricted to mammalian models, consistent with higher cytochrome P450 activity measured in these species. Notably, a dihydrodiol metabolite was detected in all species, indicating a conserved bioactivation pathway likely involving arene oxide intermediates. Detoxification of this intermediate was species-dependent, with glutathione conjugation occurring in rat- and trout-S9 incubations while being absent in human-S9. Collectively, these results demonstrate species-specific metabolic biotransformation of TPhP and highlight the importance of integrating toxicokinetics and metabolomics to improve the risk assessment of emerging contaminants.