Received for publication March 31, 2006.
Revised July 15, 2006.
Accepted for publication July 18, 2006.

METABOLISM OF ENDOSULFAN- BY HUMAN LIVER MICROSOMES AND ITS UTILITY AS A SIMULTANEOUS IN VITRO PROBE FOR CYP2B6 AND CYP3A4

Abstract

Endosulfan- 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 (rCYP)isoforms, we identified CYP2B6 (K_m = 16.2 µM, V_max = 11.4 nmol/nmol CYP/min) and CYP3A4 (K_m = 14.4 µM, V_max = 1.3 nmol/nmol CYP/min) as the primary enzymes catalyzing the metabolism of endosulfan-, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL_int = 0.70 µL/min/pmol CYP) than CYP3A4 (CL_int = 0.09 µL/min/pmol CYP). Using 16 individual human liver microsomes (HLM), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N demethylase activity of CYP2B6 (r² = 0.79) while a moderate correlation with testosterone 6--hyroxylase 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-metabolism in HLMs. Using six HLM samples, the percent total normalized rate (% TNR) was calculated to estimate the contribution of each CYP in the total metabolism of endosulfan-. In five of the six HLMs used, the percent inhibition (% I) with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan- 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.

Key words: CYP2B, CYP3A, cytochrome P450, enzyme inhibitors, human CYP enzymes, insecticides, liver microsomes