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Title: Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos.

Authors: D'Agostino, Jaime; Zhang, Haoming; Kenaan, Cesar; Hollenberg, Paul F

Published In Chem Res Toxicol, (2015 Jul 20)

Abstract: Chlorpyrifos (CPS) is a commonly used pesticide which is metabolized by P450s into the toxic metabolite chlorpyrifos-oxon (CPO). Metabolism also results in the release of sulfur, which has been suggested to be involved in mechanism-based inactivation (MBI) of P450s. CYP2B6 was previously determined to have the greatest catalytic efficiency for CPO formation in vitro. Therefore, we characterized the MBI of CYP2B6 by CPS. CPS inactivated CYP2B6 in a time- and concentration-dependent manner with a kinact of 1.97 min(-1), a KI of 0.47 μM, and a partition ratio of 17.7. We further evaluated the ability of other organophosphate pesticides including chorpyrifos-methyl, diazinon, parathion-methyl, and azinophos-methyl to inactivate CYP2B6. These organophosphate pesticides were also potent MBIs of CYP2B6 characterized by similar kinact and KI values. The inactivation of CYP2B6 by CPS was accompanied by the loss of P450 detectable in the CO reduced spectrum and loss of detectable heme. High molecular weight aggregates were observed when inactivated CYP2B6 was run on SDS-PAGE gels indicating protein aggregation. Interestingly, we found that the rat homologue of CYP2B6, CYP2B1, was not inactivated by CPS despite forming CPO to a similar extent. On the basis of the locations of the Cys residues in the two proteins which could react with released sulfur during the metabolism of CPS, we investigated whether the C475 in CYP2B6, which is not conserved in CYP2B1, was the critical residue for inactivation by mutating it to a Ser. CYP2B6 C475S was inactivated to a similar extent as wild type CYP2B6 indicating that C475 is not likely the key difference between CYP2B1 and CYP2B6 with respect to inactivation. These results indicate that CPS and other organophosphate pesticides are potent MBIs of CYP2B6 which may have implications for the toxicity of these pesticides as well as the potential for pesticide-drug interactions.

PubMed ID: 26075493 Exiting the NIEHS site

MeSH Terms: Animals; Chlorpyrifos/chemistry; Chlorpyrifos/metabolism*; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2B1/chemistry; Cytochrome P-450 CYP2B1/metabolism; Cytochrome P-450 CYP2B6/chemistry; Cytochrome P-450 CYP2B6/genetics; Cytochrome P-450 CYP2B6/metabolism*; Electrophoresis, Polyacrylamide Gel; Heme/metabolism; Humans; Insecticides/chemistry; Insecticides/metabolism*; Kinetics; Mutagenesis, Site-Directed; Protein Binding; Rats; Spectrometry, Mass, Electrospray Ionization

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