Metabolism of endosulfan-α by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4

Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes ( K-m = 9.8 mu M, V-max = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 ( P450) isoforms, we identified CYP2B6 ( K-m = 16.2 mu M, V-max = 11.4 nmol/nmol P450/min) and CYP3A4 ( K-m = 14.4 mu M, V-max = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate ( CLint = 0.70 mu l/min/pmol P450) than CYP3A4 ( CLint = 0.09 mu l/min/pmol P450). Using 16 individual human liver microsomes ( HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 ( r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 ( r(2) = 0.54) was observed. Ticlopidine ( 5 mu M), a potent CYP2B6 inhibitor, and ketoconazole ( 10 mu M), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.