Abstract

Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo(e)dioxathiepin-3-oxide) is a broad-spectrum chlorinated cyclodiene insecticide. This study was performed to elucidate the stereoselective metabolism of endosulfan in human liver microsomes and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of endosulfan. Human liver microsomal incubation of endosulfan in the presence of NADPH resulted in the formation of the toxic metabolite, endosulfan sulfate. The intrinsic clearances (CL_int) of endosulfan sulfate from β-endosulfan were 3.5-fold higher than those from α-endosulfan, suggesting that β-endosulfan would be cleared more rapidly than α-endosulfan. Correlation analysis between the known P450 enzyme activities and the rate of the formation of endosulfan sulfate in the 14 human liver microsomes showed that α-endosulfan metabolism is significantly correlated with CYP2B6-mediated bupropion hydroxylation and CYP3A-mediated midazolam hydroxylation, and that β-endosulfan metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in human liver microsomes and the incubation study of cDNA-expressed enzymes also demonstrated that the stereoselective endosulfan sulfate formation from α-endosulfan is mediated by CYP2B6, CYP3A4, and CYP3A5, and that from β-endosulfan is mediated by CYP3A4 and CYP3A5. The total CL_int values of endosulfan sulfate formation catalyzed by CYP3A4 and CYP3A5 were consistently higher for β-endosulfan than for the α-form (CL_int of 0.67 versus 10.46 μl/min/pmol P450, respectively). CYP2B6 stereoselectively metabolizes α-endosulfan, but not β-endosulfan. These findings suggest that the CYP2B6 and CYP3A enzymes are major enzymes contributing to the stereoselective disposition of endosulfan.