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We previously reported that ethanol elicits an increased protein oxidation in the liver of rats receiving chronic ethanol by continuous intragastric infusion (Tsukamoto-French method). This accumulation of oxidized proteins could result from a decrease in the cytosolic proteolysis, related specifically to alkaline protease and its major components, the proteasomes. Because several studies suggest that intracellular proteolysis depends on the severity of oxidative stress, we investigated the cytosolic proteolytic activity under two chronic ethanol treatment paradigms associated with varying degrees of oxidative stress. For 4 weeks, male rats received chronic ethanol by continuous intragastric infusion or by oral administration (10% ethanol ad libitum as sole drinking fluid). A significant decrease was evident for alkaline protease activity as well as for sodium dodecyl sulfate (SDS)-activated latent 20S proteasome (chymotrypsine-like [ChT-L] and peptidylglutamyl peptide hydrolase [PGPH] activities) in the liver of rats receiving ethanol by continuous intragastric infusion. Free radical production and related processes appeared to be contributing events in proteolysis inhibition, because phenethyl isothiocyanate (PIC), an inhibitor of cytochrome P4502E1 (CYP2E1), reduced the inhibition of the ethanol-related ChT-L activity. Moreover, the lipid peroxidation level was inversely correlated with ChT-L activity. In contrast, no such changes were observed in ChT-L and PGPH activities or in cellular free radical targets following the oral ad libitum consumption of 10% ethanol. It appears, thus, that only the alcohol treatment paradigm associated with an overt oxidative stress produced a significant inhibition of the proteasome activity. The mechanisms of proteasome inhibition could involve the formation of an endogenous inhibitor such as protein aggregates or aldehyde-derivative peptides. Whatever the mechanism, the inhibition of cytosolic proteolysis and the subsequent accumulation of damaged proteins may be involved in the oxidatively challenged alcoholic livers and play a pathogenic role in experimental alcoholic liver disease.

Liver lipid peroxidation, nonheme iron, antioxidants, and protein oxidation were investigated in experimental alcohol-induced liver disease in the rat. Wistar male rats were intragastrically and continuously infused for 4 weeks with a high-fat diet plus an ethanol or an isocaloric amount of dextrose, maintaining a high blood alcohol level (200-300 mg%). This model induced fatty liver, spotty necrosis, and focal inflammation. This pathology was associated with an enhanced lipid peroxidation and a decrease in the major antioxidant factors. Hepatic alpha-tocopherol and glutathione concentrations were significantly decreased in ethanol-fed rats. Glutathione peroxidase (GPx) was also decreased, whereas glutathione S-transferase (GST) was unaffected. The nonheme iron level was significantly decreased. Protein oxidation was assessed through three parameters: protein thiols, protein carbonyl groups, and the activity of glutamine synthetase (GS), a centrilobular enzyme particularly susceptible to free-radical-mediated damage. Ethanol-fed rats had decreased protein thiol concentrations and reduced GS activity, together with increased protein carbonyls. A significant correlation between GS activity and the pathological score was observed. This study confirms the ethanol-related increase in lipid peroxidation and shows that ethanol impairs the hepatic antioxidant potential. Furthermore, evidence of oxidative protein damage is given, including decreased activity of a key enzyme of ammonia metabolism. These protein disturbances may contribute to the pathogenesis of the observed liver damage.