PT  - JOURNAL ARTICLE
AU  - John P. Harrelson
AU  - Brendan D. Stamper
AU  - John D. Chapman
AU  - David R. Goodlett
AU  - Sidney D. Nelson
TI  - Covalent Modification and Time-Dependent Inhibition of Human CYP2E1 by the <em>meta</em>-Isomer of Acetaminophen
AID  - 10.1124/dmd.112.045492
DP  - 2012 Aug 01
TA  - Drug Metabolism and Disposition
PG  - 1460--1465
VI  - 40
IP  - 8
4099  - http://dmd.aspetjournals.org/content/40/8/1460.short
4100  - http://dmd.aspetjournals.org/content/40/8/1460.full
SO  - Drug Metab Dispos2012 Aug 01; 40
AB  - The hypothesis that N-acetyl-m-aminophenol (AMAP), the meta isomer of acetaminophen, will covalently bind to and inhibit human CYP2E1 in a time- and NADPH-dependent manner was investigated. Liquid chromatography/electrospray ionization-mass spectrometry analysis indicated that AMAP metabolites (i.e., AMAP*) selectively and covalently modified CYP2E1 apoprotein in a ratio of 1.4:1 (AMAP*/CYP2E1) in a reconstituted system. The deconvoluted spectra of CYP2E1 apoprotein from incubations containing NADPH and AMAP displayed mass shifts of 167.2 ± 7.1 and 334.4 ± 6.5 Da, suggesting the addition of one and two hydroxylated AMAP metabolites to CYP2E1, respectively. Mass shifts in cytochrome P450 reductase, cytochrome b5, and heme from these samples were not observed. CYP2E1 inhibition by AMAP increased with time in the presence of NADPH; a reversible inhibition component was also observed. The results support a bioactivation process that involves formation of a hydroquinone metabolite that undergoes further oxidation to a quinone, which reacts with CYP2E1 nucleophilic residues. The data are consistent with evidence from previous studies that identified hydroxylated AMAP glutathione conjugates collected from mice and indicate that cysteine residues are the most likely sites for adduct formation. This study reports the first direct evidence of AMAP-derived hydroquinone metabolites bound to human CYP2E1.