Metabolism of Endosulfan-Alpha by Human Liver Microsomes and its Utility as a Simultaneous In Vitro Probe for CYP2B6 and CYP3A4
NORTH CAROLINA STATE UNIV AT RALEIGH
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Endosulfan-alpha was metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes Km 9.8 muM, Vsub max 178.5 pmolmgmin. With the use of recombinant cytochrome P450 rCYP isoforms, we identified CYP2B6 Ksub m 16.2 muM, Vsub max 11.4 nmolnmol CYPmin and CYP3A4 Ksub m 14.4 muM, Vsub max 1.3 nmolnmol CYPmin as the primary enzymes catalyzing the metabolism of endosultan-alpha, albeit CYP2B6 had an 8-fold higher intrinsic clearance rate CLsub int 0.70 muLminpmol CYP than CYP3A4 CLsub int 0.09 muLminpmol 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 rexp 2 0.79 while a moderate correlation with testosterone 6-beta-hyroxylase activity of CYP3A4 rexp 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.
- Anatomy and Physiology