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J. Biol. Chem., Vol. 283, Issue 8, 4543-4559, February 22, 2008

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Identification of Novel Toxicity-associated Metabolites by Metabolomics and Mass Isotopomer Analysis of Acetaminophen Metabolism in Wild-type and Cyp2e1-null Mice^*

Chi Chen, Kristopher W. Krausz, Jeffrey R. Idle¹, and Frank J. Gonzalez²

From the Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the Institute of Pharmacology, 1st Faculty of Medicine, Charles University, 12800 Prague, Czech Republic

CYP2E1 is recognized as the most important enzyme for initiation of acetaminophen (APAP)-induced toxicity. In this study, the resistance of Cyp2e1-null mice to APAP treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known APAP metabolites in urine and serum revealed that the contribution of CYP2E1 to APAP metabolism decreased with increasing APAP doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of APAP overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis (PCA) model that delineated a relationship between urinary metabolomes and the responses to APAP treatment. Urinary ions high in wild-type mice treated with 400 mg/kg APAP were elucidated as 3-methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys-APAP transamination reaction in kidney), 3,3'-biacetaminophen (VIII, an APAP dimer), and a benzothiazine compound (IX, originated from deacetylated APAP), through mass isotopomer analysis, accurate mass measurement, tandem mass spectrometry fragmentation, in vitro reactions, and chemical treatments. Dose-, time-, and genotype-dependent appearance of these minor APAP metabolites implied their association with the APAP-induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated APAP metabolism might significantly contribute to APAP-induced toxicity. The combination of genetically modified animal models, mass isotopomer analysis, and metabolomics provides a powerful and efficient technical platform to characterize APAP-induced toxicity through identifying novel biomarkers and unraveling novel mechanisms.

Received for publication, July 31, 2007 , and in revised form, December 18, 2007.

^* This work was supported in part by the NCI Intramural Research Program of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S4.

¹ Supported by the U. S. Smokeless Tobacco Company for collaborative research.

² To whom correspondence should be addressed: Laboratory of Metabolism, NCI, NIH, 37 Convent Dr., Bethesda, MD 20892. Tel.: 301-496-9067; Fax: 301-496-8419; E-mail: fjgonz{at}helix.nih.gov.

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