Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver

The metabolism of diazinon, an organo-phosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague-Dawley (SD) rats or SD rats treated with different P450-specific inducers (phenobarbital, dexamethasone, beta-napthoflavone, and pyrazole). Results obtained indicate an involvement of CYP2C11, CYP3A2, and CYP2B1/2, whereas CYP2E1 and CYP1A1 do not contribute to the pesticide oxidative metabolism. Indeed, diazinon was metabolized by microsomes from control rats; among the inducers, phenobarbital and dexamethasone only increased the production of either metabolites, although to different extents. The production of the two metabolites is self-limiting, due to P450 inactivation; therefore, the inhibition of CYP-specific monooxygenase activities after diazinon preincubation has been used to selectively identify the competent CYPs in diazinon metabolism. Results indicate that, after diazinon preincubation, CYP3A2-catalyzed reactions (2beta- and 6beta-testosterone hydroxylation) are very efficiently inhibited; CYP2C11- and CYP2B1/2-catalyzed reactions (2alpha- and 16beta-testosterone hydroxylation, respectively) are weakly inhibited, while CYP2E1-, CYP2A1/2-, and CYP1A1/2-related activities were unaffected. Results obtained by using chemical inhibitors or antibodies selectively active against specific CYPs provide a direct evidence for the involvement of CYP2C11, CYP3A2, and CYP2B1/2, indicating that each of them contributed about 40-50% of the diazinon metabolism, in hepatic microsomes from untreated, phenobarbital-, and dexamethasone-treated rats, respectively. The higher diazoxon/pyrimidinol ratio observed after phenobarbital-treatment together with the significantly more effective inhibition toward diazoxon production exerted by metyrapone in microsomes from phenobarbital-treated rats supports the conclusion that CYP2B1/2 catalyze preferentially the production of diazoxon.