Metabolism of Endosulfan-Alpha by Human Liver Microsomes and its Utility as a Simultaneous In Vitro Probe for CYP2B6 and CYP3A4
Abstract
Endosulfan-alpha was metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 muM, V(sub max) = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (rCYP) isoforms, we identified CYP2B6 (K(sub m) = 16.2 muM, V(sub max) = 11.4 nmol/nmol CYP/min) and CYP3A4 (K(sub m) = 14.4 muM, V(sub max) = 1.3 nmol/nmol CYP/min) as the primary enzymes catalyzing the metabolism of endosultan-alpha, albeit CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(sub int) = 0.70 muL/min/pmol CYP) than CYP3A4 (CL(sub int) = 0.09 muL/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 (r(exp 2) = 0.79) while a moderate correlation with testosterone 6-beta-hyroxylase activity of CYP3A4 (r(exp 2) = 0.54) was observed. Ticlopidine (5 muM), a potent CYP2B6 inhibitor, and ketoconazole (10 muM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha 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-alpha. 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-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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 30, 2006
- Accession Number
- ADA445178
Entities
People
- Andrew D. Wallace
- Ernest Hodgson
- Randy L. Rose
- Richard C. Casabar
Organizations
- North Carolina State University