title: Metabolism of endosulfan-alpha by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4.
abstract: Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/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 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha 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-alpha. 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-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.
authors.affiliation: Department of Environmental and Molecular Toxicology, Box 7633, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Richard C T
authors.initials: RC
authors.lastname: Casabar
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Wallace
authors.forename: Ernest
authors.initials: E
authors.lastname: Hodgson
authors.forename: Randy L
authors.initials: RL
authors.lastname: Rose
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001711:Biotransformation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004726:Endosulfan / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015394:Molecular Structure; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D013988:Ticlopidine / Q000494:pharmacology
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007306:Insecticides; D013431:Sulfates; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating; D004726:Endosulfan; D013988:Ticlopidine; D007654:Ketoconazole