In vitro mechanistic study of human CYP2B6-catalyzed bioactivation and GSTs-mediated detoxification of chlorfenapyr.

Chlorfenapyr (CFP) is a halogenated pyrrole insecticide that requires metabolic activation to exert toxicity and has been associated with severe poisoning in humans. However, the metabolic pathways underlying CFP bioactivation and detoxification in humans remain poorly defined. In this study, we systematically investigated the metabolic fate of CFP and its toxic metabolite tralopyril (TLP) using in vitro human drug-metabolizing enzyme systems. Screening of ten human cytochrome P450 (CYP) isoforms at 5 and 50 µM CFP indicated that CYP2B6 is the major contributor to CFP bioactivation, which was further supported by selective inhibition and correlation analyses. CFP metabolism by CYP2B6 followed Michaelis-Menten kinetics, with showing a Km of 1.80 ± 0.57 μM, a Vmax of 0.24 ± 0.02 pmol/min/pmol P450, and an CLint of 139.78 ± 33.70 nL/min/pmol P450. In addition, incubations using 50 or 100 µM TLP and varying concentrations of glutathione (0.1 to 1 mM GSH) showed the formation of a novel GSH conjugate of TLP (TLP-GSH), of which level was markedly enhanced by human glutathione S-transferases (GSTs). Among six recombinant human GST isoforms tested, GSTA1, GSTA2, GSTM1, and GSTP1 showed significant catalytic activity in TLP-GSH formation compared with non-enzymatic reactions. Moreover, extracellular flux analysis revealed that TLP dose-dependently inhibited basal and maximal oxygen consumption rates, an effect exacerbated by GST inhibition, indicating a protective role of GSTs in TLP-induced mitochondrial toxicity. Together, these findings delineated metabolic enzymes-mediated bioactivation and detoxification pathways governing CFP toxicity in humans and provided a new perspective on clinical management strategies for CFP poisoning.