Identification of Cytochrome P450 Isozymes Involved in Enantioselective Metabolism of Fipronil in Fish Liver: In Vitro Metabolic Kinetics and Molecular Modeling

Abstract

Fipronil has been frequently detected in waterways worldwide at concentrations that threaten aquatic organisms, yet the metabolic behavior of fipronil enantiomers in aquatic organisms is largely unknown, which is of significance in enantioselective toxicity evaluation. We quantitatively identified the specific cytochrome P450 (CYP) isozymes involved in metabolizing fipronil enantiomers in tilapia by combining in vitro metabolic kinetic assays and molecular docking. Inhibition studies suggested that CYP1A enzyme was the main isoform catalyzing metabolism of fipronil and that CYP3A contributed in a limited way to the metabolism in fish liver S9. Both the dissipation rate constant and the maximum metabolic velocity of R-(−)-fipronil were greater than those of S-(+)-fipronil in tilapia liver S9, suggesting that tilapia selectively metabolized R-(−)-fipronil. The CYP1A1 isozyme exhibited the highest binding capacity to R-(−)-fipronil and S-(+)-fipronil (binding energy -9.39 and -9.17 kcal/mol, respectively), followed by CYP1A2 (-7.30 and -6.94 kcal/mol, respectively) and CYP3A4 (-7.16 and -6.91 kcal/mol, respectively). The results of in vitro metabolic assays and molecular docking were consistent, that is, CYP1A, specifically CYP1A1, exhibited a higher metabolic capacity to fipronil than CYP3A, and fish liver S9 selectively metabolized R-(−)-fipronil. The present study provides insight into the enantioselective metabolic behavior and toxicological implications of the in vitro metabolic kinetics of fipronil in fish.