Proteomic identification of potential susceptibility factors in drug-induced liver disease.
ABSTRACT Drug-induced liver disease (DILD) causes significant morbidity and mortality and impairs new drug development. Currently, no known criteria can predict whether a drug will cause DILD or what risk factors make an individual susceptible. Although it has been shown in mouse studies that the disruption of key regulatory factors, such as cyclooxygenase-2 (COX-2), interleukin (IL)-6, and IL-10, increased susceptibility to DILD caused by acetaminophen (APAP), no single factor seems to be absolute. As an approach to better understand the multifactorial basis of DILD, we compared the hepatic proteome of mice that are resistant (SJL) and susceptible (C57Bl/6) to APAP-induced liver disease (AILD), using solution-based isotope-coded affinity tag (ICAT) liquid chromatography mass spectrometry. Several novel factors were identified that were more highly expressed in the livers of SJL mice, including those involved in stress response, cell proliferation and tissue regeneration, and protein modification, implicating these proteins as potential hepatoprotective factors. There was also a selective loss of several mitochondrial proteins from the livers of the susceptible C57Bl/6 mice, suggesting that the loss of functional mitochondria may indeed play a role in AILD. These findings indicate that comparative hepatic proteomic analyses of susceptible and resistant mouse strains may provide a global approach for identifying potential risk factors and mechanistic pathways responsible for DILD.
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ABSTRACT: The mechanism by which acetaminophen (APAP) causes liver damage evokes many aspects drug metabolism, oxidative chemistry, and genetic-predisposition. In this study, we leverage the relative resistance of female C57BL/6 mice to APAP-induced liver damage (AILD) compared to male C57BL/6 mice in order to identify the cause(s) of sensitivity. Furthermore, we use mice that are either heterozygous (HZ) or null (KO) for glutamate cysteine ligase modifier subunit (Gclm), in order to titrate the toxicity relative to wild-type (WT) mice. Gclm is important for efficient de novo synthesis of glutathione (GSH). APAP (300 mg/kg, ip) or saline was administered and mice were collected at 0, 0.5, 1, 2, 6, 12, and 24 h. Male mice showed marked elevation in serum alanine aminotransferase by 6 h. In contrast, female WT and HZ mice showed minimal toxicity at all time points. Female KO mice, however, showed AILD comparable to male mice. Genotype-matched male and female mice showed comparable APAP-protein adducts, with Gclm KO mice sustaining significantly greater adducts. ATP was depleted in mice showing toxicity, suggesting impaired mitochondria function. Indeed, peroxiredoxin-6, a GSH-dependent peroxiredoxin, was preferentially adducted by APAP in mitochondria of male mice but rarely adducted in female mice. These results support parallel mechanisms of toxicity where APAP adduction of peroxiredoxin-6 and sustained GSH depletion results in the collapse of mitochondria function and hepatocyte death. We conclude that adduction of peroxiredoxin-6 sensitizes male C57BL/6 mice to toxicity by acetaminophen.Redox biology. 01/2014; 2:377-87.
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ABSTRACT: Tolbutamide is used as a first line oral antihyperglycemic drug for type 2 diabetes. One side effect of this drug, hepatotoxicity, is well recognized; however, the precise mechanisms underlying tolbutamide-induced hepatotoxicity remain unclear. In this respect, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxicity induced by this drug. In this study, we aimed to identify molecular pathways based on proteins responding to cellular toxicity in tolbutamide-treated primary hepatocytes, using nano UPLC-MS/MS analysis. Rat primary hepatocytes were treated with an IC(20) concentration for 24 h to study the hepatotoxic effects of tolbutamide. For high-throughput label-free quantitation, tryptic-digested peptides of proteins from cell lysates were analyzed using LC-MS/MS and quantitated using the IDEAL-Q software, in which several parameters, such as assisted sequence, elution time, and mass-to-charge ratio were included. We quantified a total of 330 distinct proteins from the tolbutamide-treated hepatocytes and identified 55 upregulated and 82 downregulated proteins with expression changes. Among these differentially expressed proteins, we focused mainly on the 18 upregulated proteins belonging to xenobiotic cytochrome P450 (CYP), drug metabolism/detoxification, oxidative stress/antioxidant response, and cell damage pathway. CYP2D1, CYP2C11, UDP-glucuronosyltransferase 2B (UGT2B), superoxide dismutase 2 (SOD2), 60 kDa heat shock protein (HSPD1), heat shock protein 90 (HSP90), and catalase (CAT) were confirmed by Western blot analysis. In addition, various xenobiotic CYP proteins upregulated in the tolbutamide-treated group, CYP2D1, CYP2C13, and CYP2C11 were confirmed by reverse transcriptase-PCR analysis. Our results offer important new insights into the molecular mechanisms of tolbutamide-induced hepatotoxicity.Electrophoresis 07/2012; 33(18):2806-17. · 3.26 Impact Factor
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ABSTRACT: 1. Drug induced organ injury is multifaceted, encompassing a spectrum of cell types and numerous networks reflecting cell-cell and cell-matrix interactions. Characterization of drug induced side effects and human response can be addressed in organ slice models. 2. The application of human tissue to various organ slice models including liver, intestine, kidney, liver-blood co-cultures and thyroid enhances our ability to focus on the clinical relevance of side effects identified in animal studies for human, and to evaluate potential biomarkers of the side effects. Dose-response relationships can help discern drug concentrations which alter organ function or affect morphology, to identify drug concentrationswhich could pose a risk for humans. 3. Insight into pathways of organ injury, by incorporating gene and protein expression profiling, with functional measurements and morphology, aid to define species differences and sensitivity. 4. Human organ slice studies are valuable for bridging the extrapolation of animal derived data and for identifying mechanisms relevant for humans, thereby expanding the scope of translational research for drug safety assessment.Xenobiotica 10/2012; · 1.98 Impact Factor