Abstract

Endosulfan-α is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (K_m = 9.8 μM, V_max = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (K_m = 16.2 μM, V_max = 11.4 nmol/nmol P450/min) and CYP3A4 (K_m = 14.4 μM, V_max = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-α, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL_int = 0.70 μl/min/pmol P450) than CYP3A4 (CL_int = 0.09 μl/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 (r² = 0.79), whereas a moderate correlation with testosterone 6 β-hydroxylase activity of CYP3A4 (r² = 0.54) was observed. Ticlopidine (5 μM), a potent CYP2B6 inhibitor, and ketoconazole (10 μM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-α 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-α. 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-α 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.