A PROTEOMIC STUDY OF OXIDATIVE STRESS IN ALCOHOLIC LIVER DISEASE

Alcoholic steatosis (AS) is the initial pathology associated with early stage alcoholic liver disease and is characterized by the accumulation of fat in the liver. AS is considered clinically benign as it is reversible, as compared with alcoholic steatohepatitis (ASH) which is the next stage of alcoholic liver disease (ALD), and mostly irreversible. Proteomics were used to investigate the molecular basis of AS to determine biomarkers representative of AS. Liver tissue proteins at different stages of steatosis from a rodent model of AS were separated by two dimensional electrophoresis (2DE), followed by MALDI mass spectrometry (MS) identification of significantly expressed proteins. Expression levels of several proteins related to alcohol induced oxidative stress, such as peroxiredoxin 6 (PRDX6) and aldehyde dehydrogenase 2 (ALDH2) were reduced by 2 to 3-fold in ethanol fed rats, and suggested an increase in oxidative stress. Several proteins involved in fatty acid and amino acid metabolism were found at increased expression levels, suggesting higher energy demand upon chronic exposure to ethanol. In order to delineate between the effects of fat accumulation and oxidative stress, an in vitro hepatocyte cell culture model of steatosis was developed. HepG2 cells loaded with oleic acid surprisingly demonstrated lower cytotoxicity upon oxidative challenge (based on lactate dehydrogenase activity) and inflammation (based on TNF-? induced activation of the pro-inflammatory transcription factor NF-?B). We also examined the effect of oleic acid loading in HepG2 cells on protein carbonylation, which is an important irreversible protein modification during oxidative stress that leads to protein dysfunction and disease. Fat-loaded hepatocytes exposed to oxidative stress with tert-butyl hydroperoxide (TBHP) contained 17% less carbonylated proteins than the non-fat loaded control. Mass spectrometric analysis of carbonylated proteins indicated that known classical markers of protein carbonylation (e.g., cytoskeletal proteins, chaperones) are not carbonylated in oleic acid loaded HepG2 cells, and suggests that the protective effect of fat loading is through interference with protein carbonylation. While counterintuitive to the general concept that AS increases oxidative stress, our fat loading results suggests that low levels of fat may activate antioxidant pathways and ameliorate the effect of subsequent oxidative or inflammatory challenge.