The overall objective of this proposal is to advance our understanding of the pathogenesis of alcoholic liver disease, a major cause of death in Western countries. We have identified an ethanol-induced defect in the uptake of glutathione (GSH) by hepatic mitochondria which impairs the defense against oxidative stress. This occurs early and predominantly in perivenous hepatocytes. To elucidate the mechanism and importance of this defect, the following aims are proposed; (1) Cloning and molecular characterization of the mitochondrial GSH transporter of rat liver (RmGshT). We have functionally expressed the hepatic mitochondrial transport of GSH using Xenopus laevis oocytes microinjected with total rat liver mRNA. We have identified the size-class canalicular GSH carriers. Following similar strategy as we used recently in the cloning of the two plasma membrane GSH carriers, we plan to isolate the cDNA encoding the mitochondrial GSH carrier to determine the nucleotide sequence, deduced amino acid sequence, hydropathy analysis and homologies to other cloned sequences. We will perform in vitro translation studies, organ distribution by Northern blot analyses, and generate polyclonal antibodies using peptides or recombinant protein. Also, large scale production of recombinant transporter will be established. (2) Mechanism of the defective transport of GSH in hepatic mitochondria caused by chronic ethanol feeding. We will determine the abundance of RmGsh T transcript (Northern blot), and, if abnormal, quantify nuclear run-off transcription, and asses polypeptide abundance (Western blots) in cellular and isolated mitochondrial extracts of ethanol-fed rats and controls using the Lieber- DiCarli Model. We will assess the insertion of newly synthesized RmGshT in the inner membrane using isolated hepatic mitochondria from ethanol and pair fed rats. We will also examine effects of mitochondrial lipid in reconstitution experiments with the recombinant transporter to determine the effect of changes of physical-chemical properties of mitochondrial lipids induced by ethanol in contributing to the defect in GSH transport. (3) Role of the defective mitochondrial GSH transport in the pathogenesis of experimental alcohol liver disease. The work in this aim will be undertaken using the Tsukamoto-French intragastric feeding model. We will determine the temporal relationship between mitochondrial and cellular functions and the defect of the mitochondrial GSH carrier. We will examine manifestations of mitochondrial oxidative stress on mitochondrial functions, mitochondrial electron carriers, activation of nuclear transcription factor (NF-kB) and gene regulation, as well as the effect of depletion of mitochondrial GSH in hepatocytes on the susceptibility to cytotoxic effects of cytokines such as TNF-alpha and the relationship between prevention of liver disease by antibiotics, saturated fat, or phosphatidylcholine and the defect in mitochondrial GSH. Finally, the ability of S-adenosylmethionine (SAM) to prevent both the GSH defect and liver-disease will be assessed.
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