Alcoholic liver disease is associated with an increase in oxidative stress and inflammatory mediators such as TNFalpha. Recently, work by this group has clearly demonstrated an essential role of oxidant production and TNFalpha signaling in the liver pathogenesis due to chronic ethanol consumption in a intragastric ethanol-feeding model in mice. Two primary approaches have been employed thus far to elucidate the mechanisms of alcohol-induced liver injury: viral vector-mediated gene transfer and mutant (i.e., knockout and transgenic) mouse technology. The work proposed here will continue to rely on the strengths of these approaches and build on our recent finding to test the unifying hypothesis that Kupffer cell-derived oxidants initiate a cascade of inflammatory responses including NFkappaB activation and TNFalpha production which subsequently induces mitochondrial dysfunction and oxidative stress in hepatocytes leading to tissue damage. First, isolated liver perfusion and superoxide production by cytochrome c reduction from transgenic and knock out mice following 4 weeks of intragastric ethanol feeding will be used to identify sources or oxidants (i.e., NADPH oxidase, CYP2E1, myeloperoxidase, iNOS, etc) and critical pathways responsible of hepatic oxidative stress due to alcohol (Aim 1). Next, in order to specifically target cellular and subcellular sources of oxidants, cell-specific gene delivery vectors will be used. Using recombinant adeno-associated viral (rAAV) serotypes and cell-specific promoter elements for Kupffercells or hepatocytes, optimized gene transfer to both Kupffer cells and hepatocytes in vivo will be achieved (Aim 2). Finally, using rAAV serotypes and cell-specific promoter elements, the over expression of cytosolic Cu/Zn-SOD, mitochondrial Mn-SOD and dominant-negative IkappaBalpha in each cell type will be evaluated in the mouse enteral ethanol feeding model to test the hypothesis that Kupffer cell-derived oxidants are initially responsible for activating an inflammatory cascade leading to mitochondrial oxidative stress and dysfunction and ultimately tissue damage (Aim 3). The experiments outlined in this proposal will not only provide important mechanistic data related to oxidant production and transcription factor activation involved in acute ethanol-induced liver injury, provide novel experimental tools that can be used to address numerous questions related to basic biology and disease of liver and potentially provide an effective therapeutic approach to treatment of alcoholic liver disease.
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