1C, left). Livers from ethanol-fed rats showed dilated ER, a large number of electron-dense mitochondria, abundant micro- and macrovesicular steatosis, and disrupted cellular membranes

Selleckchem BIBW2992 (Fig. 1C, right). All these parameters were good indicators of mitochondrial and ER impaired function, which play a role in the development of ALD. To identify proteins participating in ethanol hepatotoxicity and their link with signaling pathways involved in ALD, particularly NO· production, next we used a combination of proteomics along with a systems biology approach. To this end, first we used the mass spectrometry-based isotope-coded affinity tag (ICAT) proteomics technique to identify differentially expressed proteins in hepatocytes from ethanol-fed rats (HEthanol) compared with hepatocytes from control rats (HControl). Second, to dissect the differentially regulated proteins in the context of protein interaction networks we used the Institute for Systems Biology Trans-Proteomics Pipeline (Seattle) and the Gaggle 14 computer platform. Lastly, we narrowed down our search by focusing on the subproteome of mitochondrial and/or cytosolic proteins of potential significance for the development of ALD. The ICAT labeling methodology

and the proteomics analysis identified multiple differentially expressed proteins selleck products in HControl versus HEthanol with probability scores >0.5 (<5% error rate). Among them, there were several well-known alcohol-regulated proteins such as cytochrome P450 2E1 (CYP2E1) and NOS2, which were validated by western blot analysis, whereas other proteins, such as DYNAMIN and HSP70, decreased by ethanol (Fig. 1D). The acquired dataset was further analyzed on the Gene Ontology Categories, Kyoto Encyclopedia of Genes MCE and Genomes Pathways, and Protein Interaction Networks using the Gaggle platform. 14 The systems-based quantitative

proteomics analysis led us to focus on the urea and the L-citrulline/NO· cycles as likely impaired under ethanol consumption because a potential link with NO· production could be established. ASS, a novel ethanol-specific induced protein, was identified in the proteomics analysis (Fig. 1E). Hence, we explicitly selected it as a protein of interest in the follow-up analysis because it could play a role in ALD by regulating de novo biosynthesis of L-arginine from L-citrulline for high-output NO· generation by NOS2. Next, ASS expression was validated by western blot analysis in hepatocytes. We found a 4.2-fold increase in HEthanol versus HControl (Fig. 2A). To better understand the potential physiological role of the induction of ASS, other enzymes from the urea cycle and/or the L-citrulline/NO· cycle were studied. Western blot analysis showed that ethanol induced ASL (2.2-fold), ARG1 (2.3-fold), NOS2 (2.