title: Development of a non-high pressure liquid chromatography assay to determine [14C]chlorzoxazone 6-hydroxylase (CYP2E1) activity in human liver microsomes.
abstract: The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, Center for Environmental and Occupational Health, University of Kansas Medical Center, Kansas City 66160-7417, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Draper
authors.forename: A
authors.initials: A
authors.lastname: Madan
authors.forename: J
authors.initials: J
authors.lastname: Latham
authors.forename: A
authors.initials: A
authors.lastname: Parkinson
mesh_terms: D000818:Animals; D002753:Chlorzoxazone / Q000032:analysis / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D019392:Cytochrome P-450 CYP2E1 / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D019392:Cytochrome P-450 CYP2E1; D002753:Chlorzoxazone