Abstract
Endosulfan-α is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 μM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 μM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 μM, Vmax = 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 (CLint = 0.70 μl/min/pmol P450) than CYP3A4 (CLint = 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.
Footnotes
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This work was supported by National Institute for Occupational Safety and Health Grant OH 07551-ECU. R.C. was a recipient of the Air Force Institute of Technology scholarship. Results were presented at the 13th annual meeting of ISSX in Maui, HI, Oct 23–27, 2005 (Drug Metab Rev37: 244).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.010199.
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ABBREVIATIONS: P450, cytochrome P450; rP450, recombinant P450; HLM, human liver microsome; % TNR, percentage total normalized rate; % I, percentage inhibition; ACN, acetonitrile; FMO, flavin-containing monooxygenase; rFMO, recombinant FMO; NR, normalized rate; pHLM, pooled human liver microsome; M-M, Michaelis-Menten; CLint, intrinsic clearance.
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↵1 This article is dedicated in memory of Dr. Randy Rose, who died in a tragic car accident.
- Received March 31, 2006.
- Accepted July 18, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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