BIOMARKERS FOR CYP2E1 EXPRESSION AND ACTIVITY: CYP2E1 is a key enzyme in the metabolic activation of many low molecular weight toxicants and also an important contributor to oxidative stress. A noninvasive method to monitor CYP2E1 activity in vivo would be of great value for studying the role of CYP2E1 in chemical-induced toxicities and stress-related diseases. In this study, UPLC-ESI-QTOFMS was used to identify a metabolite biomarker of CYP2E1 through comparing the urine metabolomes of wild-type, Cyp2e1-null, and CYP2E1-humanized mice. Metabolomic analysis with multivariate models of urine metabolites revealed a clear separation of Cyp2e1-null mice from wild-type and CYP2E1-humanized mice in the multivariate models of urine metabolomes. Subsequently, 2-piperidone was identified as a urinary metabolite that inversely correlated to the CYP2E1 activity in the three mouse lines. Backcrossing of wild-type and Cyp2e1-null mice, together with targeted analysis of 2-piperidone in mouse serum, confirmed the genotype dependency of 2-piperidone. The accumulation of 2-piperidone in the Cyp2e1-null mice was mainly caused by the changes in the biosynthesis and degradation of 2-piperidone because compared with the wild-type mice, the conversion of cadaverine to 2-piperidone was higher, whereas the metabolism of 2-piperidone to 6-hydroxy-2-piperidone was lower in the Cyp2e1-null mice. Overall, untargeted metabolomic analysis identified a correlation between 2-piperidone concentrations in urine and the expression and activity of CYP2E1, thus providing a noninvasive metabolite biomarker that can be potentially used in to monitor CYP2E1 activity. METABOLISM OF ISONIAZID BY CYP2E1 LEADS TO CHOLESTASIS: Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition-induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives;these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria beta-oxidation. BIOMARKERS METABOLOMICS METHODS FOR TUMOR CELLS: An LC/MS-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the Panc-1 cancer cell line. Different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) with UPLC-QTOF-MS were also evaluated. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen to harvest Panc-1 cells which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was chosen as the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further global metabolomics studies on cancer cell lines. FOR SQUAMOUS CELL CARCINOMA GROWTH IN XENOGRAPHS: UPLC-ESI-QTOFMS-based metabolomics was used to explore metabolic signatures of tumor growth in mice. Urine samples were collected from control mice and mice injected with squamous cell carcinoma (SCCVII) tumor cells as a function of time. When tumors reached 2 cm, all mice were killed and blood and liver samples collected. The urine metabolites hexanoylglycine, nicotinamide 1-oxide, and 11beta, 20alpha-dihydroxy-3-oxopregn-4-en-21-oic acid, were elevated in tumor-bearing mice as was asymmetric dimethylarginine, a biomarker for oxidative stress. Interestingly, SCCVII tumor growth resulted in hepatomegaly, reduced albumin/globulin ratios, and elevated serum triglycerides suggesting liver dysfunction. Alterations in liver metabolites between SCCVII tumor-bearing and control mice confirmed the presence of liver injury. Hepatic mRNA analysis indicated that inflammatory cytokines, tumor necrosis factor alpha and transforming growth factor beta were enhanced in SCCVII tumor-bearing mice and cytochromes P450 expression were decreased in tumor-bearing mice. Further, genes involved in fatty acid oxidation were decreased, suggesting impaired fatty acid oxidation in SCCVII tumor-bearing mice. Additionally, activated phospholipid metabolism and disrupted tricarboxylic acid cycle were observed in SCCVII tumor-bearing mice. These data suggest that tumor growth imposes a global inflammatory response that results in liver dysfunction and underscores the use of metabolomics to temporally examine these changes and potentially use metabolite changes to monitor tumor treatment response. ROLE OF THIOREDOXIN AND ANTIOXIDANTS IN DRUG-INDUCED HEPATOTOXICITY: The importance of glutathione in mitigating the deleterious effects of electrophile generating drugs such as acetaminophen is well established. However, the role of other antioxidant systems, such as that provided by thioredoxin, has not been studied. Selenoprotein thioredoxin reductase 1 (Txnrd1) is important for attenuating activation of the apoptosis signaling-regulating kinase 1 and the c-Jun N-terminal kinase pathway caused by high doses of acetaminophen. Therefore, the role of Txnrd1 in acetaminophen -induced hepatotoxicity was investigated. Liver-specific Txnrd1 knockout mice were generated and treated with a toxic dose of acetaminophen. At 1 h post- acetaminophen treatment, total and mitochondrial glutathione levels in control and liver-specific Txnrd1 knockout mice were depleted. However, at 6 h post- acetaminophen treatment, liver-specific Txnrd1 knockout mice were resistant to acetaminophen toxicity. A compensatory up-regulation of many of the nuclear factor erythroid 2-related factor 2 target genes and proteins in liver-specific Txnrd1 knockout mice with and without acetaminophen treatment was found. Yet, c-Jun N-terminal kinase was phosphorylated to a similar extent in acetaminophen-treated control mice. These results suggest that Txnrd1 knockout mice mice are primed for xenobiotic detoxication primarily through nuclear factor erythroid 2-related factor 2 activation.
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