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

Abstract

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 APAPinduced 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. Acetaminophen (APAP)3 overdose causes acute liver and kidney failure (1, 2). Because of its clinical importance, APAPinduced acute toxicity has become an indispensable model for studying drug-induced liver and kidney injury. Over the past 40 years, numerous efforts have been undertaken to understand the molecular mechanism of this toxicological event. Results from those studies indicated that the toxicity is initiated by P450-mediated reactions that convert APAP to the reactive electrophile, N-acetyl-p-benzoquinone imine (NAPQI), causing glutathione depletion and covalent binding (3). Subsequent damage to mitochondria, cell membranes, and nuclei, as well as the disruption of cell death-and survival-related signaling pathways, lead to massive necrosis and apoptosis (4). Besides the P450-catalyzed oxidation reactions, detoxicating reactions including sulfation, glucuronidation, and glutathione conjugation also significantly contribute to the biotransformation of APAP and NAPQI. The balance between these activation and detoxication routes can largely determine the consequences of APAP treatment. Severa...

CD36 Protein Influences Myocardial Ca2+ Homeostasis and Phospholipid Metabolism

“…8C). PCA (58), an unbiased data processing method that evaluates the contributions of individual species to profiles, separation was used to identify the changes that define the phospholipid profile of the CD36 Ϫ/Ϫ myocardium in the fed and fasted states. The first two PCA components separated mice into four distinct groups corresponding to genotype (x axis) and dietary state (y axis) (Fig.…”

TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Δ9-tetrahydrocannabiorcol

“…TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid ∆ The metabolism of APAP is complex and the effective dose is high and dependent on the species and route of administration [19][20][21] . Previous studies on APAP metabolites have largely focused on their toxic actions.…”

“…These early experiments and the subsequent findings that TRPA1 is activated by electrophilic compounds 4,5 led us to speculate that p-AP and APAP indirectly activate TRPA1 on primary sensory neurons, after their conversion to electrophilic compounds such as p-benzoquinone (p-BQ) and N-acetyl-p-benzoquinoneimine (NAPQI). The formation of these electrophilic compounds in vivo is catalysed by several enzymes, including cytochrome P450 (CYP450) monoxygenases, peroxidases and COX, many of which are present in the central nervous system [19][20][21][24][25][26][27][28][29] . NAPQI is believed to mediate the well-known hepato-and nephro-toxic effects of APAP.…”

Therapeutic paracetamol treatment in older persons induces dietary and metabolic modifications related to sulfur amino acids

“…Eleven metabolites acquired in positive and negative modes were detected only at d14 (Table 5), 9 of them were surely related to paracetamol metabolism: paracetamol, paracetamol sulfate, paracetamol glucuronide, cysteine paracetamol, N-acetylcysteine paracetamol, cysteine paracetamol glucuronide, 3-methoxyparacetamol glucuronide, 3-methoxy-paracetamol sulfate and the benzothiazine compound; the latter originated from deacetylated paracetamol (Chen et al 2008). The unknown metabolite with RT of 6.6 and m/z 449.045 was likely related to paracetamol detoxification, since its fragmentation spectrum showed a paracetamol Ta b l e 4 P a r a c e t a m o l metabolites, sulfur and nitrogen in urines collected over d14…”

“…Metabolites contributing to the discrimination between baseline and d14 samples were identified on the basis of their exact masses which were compared to those registered in Kyoto Encyclopedia of Genes and Genomes database (http://www.genome.jp/kegg/) or in the Human Metabolome Database (www.hmdb.ca) (Wishart et al 2007) and to those of expected metabolites of paracetamol as described in the literature (Johnson and Plumb 2005;Chen et al 2008). Identification was confirmed using appropriate standards when available, isotopic patterns and mass fragmentation analyses.…”