Effect of dietary fat on chronic ethanol-induced oxidative stress in hepatocytes

Background: Although oxidative stress and deficits in hepatic energy metabo lism have been implicated as important factors in the initiation of alcohol ic liver disease, their relative contribution to ethanol-induced cell death is not known. The purpose of this study was to examine the effects of chro nic ethanol administration on hepatocyte reactive oxygen species (ROS) gene ration,energy state, and viability, as well as the effect of dietary fat on these parameters. Methods: Male Sprague-Dawley rats were fed liquid diets that provided 36% t otal calories as ethanol with fat as either 12% (low fat) or 35% (high fat) of total calories. Pair-fed controls received liquid diets in which maltos e-dextrin was substituted for ethanol calories. The fluorescent probe 2',7' -dichlorofluorescin diacetate was used to detect ROS, lactate dehydrogenase leakage was used to assess viability, and ATP levels were used as a measur e of the energy state. The effect of chronic ethanol feeding on these param eters was determined by incubating hepatocytes under a 5% oxygen-containing atmosphere or an atmosphere less than or equal to 1% oxygen for 60 min. Results: In general, chronic ethanol feeding stimulated ROS production and decreased ATP concentrations, which were associated with decreased viabilit y in hepatocytes isolated from rats fed either high- or low-fat, ethanol-co ntaining diets, compared to the corresponding controls. Incubation under an atmosphere less than or equal to 1% oxygen and/or ethanol (10 mM) augmente d these effects in both high- and low-fat control and ethanol-fed hepatocyt es. The addition of antimycin to the incubations increased ROS production, decreased ATP concentrations, and accelerated loss of hepatocyte viability. Viability loss under all conditions used in this study was correlated with decreases in cellular ATP. Conclusions: Comparisons of incubations performed under the two oxygenation conditions revealed that viability loss was inversely associated with ROS production, which indicates that ATP loss and not ROS production was a bett er predictor of loss in cell integrity. This study also demonstrates that t he level of dietary fat has only minor effects on generation of ROS and the cellular energy state. In contrast, ethanol consumption had significant ef fects on generation of ROS, energy state, and hepatocyte viability.