The long-term goal of this project is to investigate the role of oxidative stress in chronic alcohol-induced liver injury. Studies related to this goal will be implemented to establish the central role of the mitochondrion in the development of alcoholic liver disease. The working hypothesis of this application is that chronic ethanolrelated alterations in mitochondrial structure and function influence the production of reactive oxygen species and the activity of antioxidant defense mechanisms in mitochondria, as well as in other subcellular regions within the hepatocyte. Specifically, these investigations will be performed to determine whether chronic ethanol consumption (1) increases reactive oxygen species production primarily within the mitochondrion, (2) decreases the activity of glutathione peroxidase-1 by irreversible oxidation of the active site selenocysteine residue or depressing liver selenium concentrations, and (3) alters the activity of phospholipid hydroperoxide glutathione peroxidase. To address these Specific Aims, the effect of chronic ethanol consumption on the liver will be studied. For these studies, rats will be pair-fed ethanol and control liquid diets. Confocal microscopy will be used to determine whether chronic ethanol-related increases in reactive oxygen species are primarily localized to the mitochondrion in isolated rat hepatocytes. Reactive oxygen species production will be monitored using 2'Tdichlorofluorescin diacetate. Mitochondria will be localized using MitoTracker Red CM-H2XRos. The rate and magnitude of ROS production will be compared between ethanol- and control-fed hepatocytes. Ethanolinduced alterations in the oxidation state of the active site selenocysteine residue of glutathione peroxidase-1 will be determined by quantifying the incorporation of '4C-iodoacetate into the active site of the reduced (undenatured) mitochondrial and cytosolic forms of the enzyme. Liver selenium concentrations will be determined by inductively coupled plasma atomic emission spectrometry. Ethanol- related alterations in the activity and protein level of mitochondrial and cytosolic phospholipid hydroperoxide glutathione peroxidase will be determined after chronic ethanol exposure. These studies are designed to identify (1) the site(s) of chronic ethanol-induced reactive oxygen species production within the hepatocyte and (2) the mechanisms responsible for depressed antioxidant function following ethanol exposure. This investigation should thus provide information on ethanol-elicited alterations that contribute to oxidative damage in the liver.
