Metabolic Activation of Pesticide Isoprocarb Mediated by CYP3A4 and the Possible Correlation with Its Cytotoxicity

Abstract

Isoprocarb (IPC), one of the most important carbamate pesticides, is used to control pests, such as rice planthoppers in crops. Studies have found that IPC induced hepatotoxicity in poultry chicken. However, the mechanisms of IPC-induced hepatotoxicity are unclear. The objectives of this study were to characterize reactive metabolites of IPC in vitro and in vivo, to identify cytochrome P450 enzymes for metabolic activation, and to define a possible correlation between the metabolic activation and cytotoxicity of IPC. In GSH- or NAC-supplemented microsomal incubations, one GSH conjugate (M6) and two NAC conjugates (M7 and M8) were detected after exposure to IPC. The corresponding GSH conjugate and NAC conjugates were found in the liver homogenates and urine of mice after IPC administration. IPC was found to be metabolized to a quinone intermediate reactive to GSH in vitro and in vivo. IPC was found to induce marked cytotoxicity in cultured mouse primary hepatocytes. Ketoconazole, a selective CYP3A4/5 enzyme inhibitor, attenuated the susceptibility of hepatocytes to IPC cytotoxicity.

Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats

“…Boron tribromide (BBr 3 ) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). 3,3-Dimethyldioxirane (DMDO) was prepared in our laboratory as described early. , Rat (Sprague–Dawley, male) liver microsomes (RLMs) were prepared in our laboratory, according to a previous publication . All reagents and solvents used were of analytical grade or mass spectrometric grade.…”

“…The animals were administered ( i.g. ) GRM dissolved in CMC-Na at a dose of 100 mg/kg according to our early publiaction (there is no information about human exposure dosage available), and 0–6 h bile samples were collected. Rats of the other group ( n = 3) were given GRM at the same dose and then placed in metabolism cages.…”

“…Rat primary hepatocytes were seeded in 96-well plates at a density of 2 × 10 5 cells/well and precultured for 8 h in an incubator (37 °C and 5% CO 2 ). To investigate the mechanism of cytotoxicity induced by GRM or M1, we determined the final concentrations of above compounds as described in our previous study . Hepatocytes were mixed with GRM or M1 at final concentrations of 0, 25, 50, 100, 200, or 400 μM and cultured for 24 h. In a parallel experiment, hepatocytes were pretreated with KTC (10 μM) or a vehicle for 1 h. The resulting cells were further exposed to GRM or M1 as above at the same concentrations.…”

“…To investigate the mechanism of cytotoxicity induced by GRM or M1, we determined the final concentrations of above compounds as described in our previous study. 29 Hepatocytes were mixed with GRM or M1 at final concentrations of 0, 25, 50, 100, 200, or 400 μM and cultured for 24 h. In a parallel experiment, hepatocytes were pretreated with KTC (10 μM) or a vehicle for 1 h. The resulting cells were further exposed to GRM or M1 as above at the same concentrations. In another experiment, primary hepatocytes were pretreated with L-buthionine sulfoximine (BSO) at 1,000 μM or vehicle for 12 h. The resulting cells were further exposed to GRM.…”