SPECIFIC AIMS: In 1987 we reported the discovery of a selectively depleted pool of GSH in hepatic mitochondria in alcohol fed rats. Over the past decade we have made significant progress in elucidating the mechanism and significance of this defect in the transport of GSH from cytosol into mitochondria. This defect appears to be mainly due to an alteration of the physical-chemical properties of the inner mitochondrial membrane and renders hepatocytes extremely susceptible to the cytotoxic effects of tumor necrosis factor (TNF)-induced oxidative stress in mitochondria. 1. To clone and characterize the mitochondrial GSH transporter. Using the Xenopus oocyte system we will screen and subdivide liver cDNA libraries for expression of ATP-dependent GSH uptake in mitochondria. 2. To define the mechanism of the defective transport of GSH in mitochondria due to chronic ethanol exposure. We will examine HepG2 cells expressing ADH or CYP2E1 to see if exposure to ethanol induces the mitochondrial GSH transport defect and the relationship between GSH compartmentation and ROS production and NF-kappaB activation. We will assess the relationship between membrane permeability in isolated mitochondria and hepatocytes or KepG2 cells and GSH compartmentation. We will study the role of phospholipids in mitochondrial GSH transport by reconstitution experiments in proteoliposomes. Finally, we will examine the effect of chronic ethanol feeding on the newly described S-adenosyl-L-methionine (SM) transport system in the inner membrane and on the compartmentation of SAM and S- adenosylhomocysteine (SAH). 3.To determine the role of the defective mitochondrial GSH transport in the pathogenesis of experimental alcoholic liver disease. We will assess the role of NF-kappaB and expression of stress proteins and nitric oxide synthase in the susceptibility of hepatocytes from chronic ethanol-fed rats To the cytotoxicity of TNF. We will examine the role of caspase-3 and sphingomyelinases in the TNF signaling of cytotoxicity in alcohol-induced susceptible rat hepatocytes. We will determine if increased fluidity of the inner mitochondrial membrane of intact hepatocytes will reverse the defect in GSH compartmentation and the enhanced susceptibility to TNF. Finally, we will related the presence or absence of the mitochondrial GSH transport defect with protection against liver injury in the intragastric feeding model by saturated MCT, SAM, phosphatidylcholine, betaine and anti-TNF serum.
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