The aim of this project is to investigate the ability of chronic ethanol ingestion to stimulate hepatic production of chemically reactive aldehydic products having the potential to function as intermediates in alcohol- induced liver damage. The proposed studies are designed to identify correlations of alcohol-induced changes in lobular histology and immunocytochemistry with biochemical and molecular alterations at the level of zonal-specific hepatocytes and isolated mitochondria. Specifically, experiments are described to measure concentrations of """"""""free"""""""" malondialdehyde, 4-hydroxynonenal and hexanal in livers of rats following chronic ethanol feeding. Lipid- and protein-complexed carbonyls will also be measured to quantitate aldehydic functions hound to hepatocellular constituents. The mechanisms of production of these aldehydic products will be examined in isolated periportal and perivenous hepatocytes and isolated mitochondria obtained from rats chronically ingesting ethanol. The ability of ethanol and acetaldehyde metabolism to stimulate lipid peroxidation will be evaluated in zonal specific hepatocytes and isolated mitochondria. Steady-state concentrations of malondialdehyde, 4-hydroxynonenal and hexanal will be quantitated as indices of lipid peroxidation in hepatocytes and mitochondrial for determination of correlations between the magnitude of lipid peroxidation, alterations in glutathione concentrations and disruption of ethanol and acetaldehyde oxidation. The ability of aldehydic lipids to interfere with mitochondrial acetaldehyde oxidation will be evaluated in order to test the hypothesis that these biogenic aldehydes inhibit aldehyde dehydrogenase mediated oxidation of acetaldehyde. A final series of experiments are proposed to evaluate the hypothesis that specific proteins in perivenous or periportal hepatocytes are targets for adduct formation with reactive aldehydic products of lipid peroxidation. This will be determined by using antibodies against aldehydelysine and cysteine adducts for immunocytochemical identification of lobular, cellular and subcellular proteins predisposed for formation of aldehyde adducts. Collectively, the design and statistical analyses employed in the proposed experiments will allow identification of significant changes in lobular histochemistry and immunocytochemical parameters causally related to biochemical and molecular changes at the hepatocellular level. The long-term goal of this proposed research is to establish the role of aldehydic products of lipid peroxidation and their adducts in alcohol-induced liver injury and determine the utility of their measurement as an early indicator of hepatocellular liver damage.
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