The pathogenesis of hepatic injury in cholestatic liver disease is poorly understood. The major objective of this proposal is to test the hypothesis that oxidative (free radical) damage to the liver, and specifically hepatic mitochondria, plays a major role in the pathogenesis of these diseases. It is postulated that hydrophobic, or toxic, bile acids that accumulate in the cholestatic liver are responsible to a large part for hepatocyte injury in cholestasis. To determine if toxic bile acids stimulate generation of free radicals and subsequent liver injury, lipid peroxidation as a marker of free radical injury (analyzed by four different methods) will be assessed in rat liver microsomes and mitochondria isolated after intravenous infusion of toxic bile acids and after bile duct-ligation; the effect of in vitro incubation of bile acids upon superoxide generation by isolated hepatocytes, Kupffer cells, and hepatic endothelial cells will be assessed; and the effect of free radical scavengers and inhibitors on bile acid toxicity to isolated rat hepatocytes at physiologic oxygen tensions will be examined. To determine the biochemical source of free radicals generated in the cholestatic liver, three potential pathways will be studied in intact liver and isolated hepatocytes: xanthine oxidase generation of superoxide; changes in cytosolic low molecular weight, catalytically- active iron or copper that may increase generation of the hydroxyl radical; and increased leak of superoxide from hepatic mitochondria. To determine the role of endogenous antioxidant defenses in protecting the hepatocyte from bile acid toxicity, primary cultured rat hepatocytes will be incubated with a series of inhibitors of antioxidant enzymes followed by bile acids; changes in cell toxicity and lipid peroxidation compared to control cells will be assessed and related to the hydrophobicity of the bile acids. To determine if the toxicity of bile acids to hepatic mitochondria is related to alterations in the oxidative metabolism of mitochondria, isolated mitochondria will be incubated with bile acids and the effect on mitochondrial respiration, the oxidoreductase activity of the four electron transport protein complexes, and mitochondrial glutathione status will be evaluated and compared to the degree of lipid peroxidation generated in the mitochondria. The effect of in vivo cholestasis on mitochondrial function will be assessed by measuring changes in mitochondrial respiration, ATP generation, activity of the electron transport protein complexes, and levels of cytochromes a + a3, b, c1 and c in mitochondria isolated from bile acid-infused rats. The role of hepatic metallothionein as an endogenous antioxidant that protects the liver during cholestasis and bile acid toxicity will be examined. Metallothionein mRNA and protein levels will be measured in liver from bile acid-infused and bile duct-ligated rats to determine if acute cholestasis leads to the induction of this protein. Metallothionein will then be induced in rats by zinc chloride or cadmium chloride injections, and the effect of increased hepatic metallothionein levels on in vitro bile acid toxicity to isolated hepatocytes and on in vivo liver injury after bile acid-infusion or bile duct ligation will be assessed. If the results of these studies show that free radicals are involved in the pathogenesis of cholestatic liver injury, new therapies for these disorders might be developed based on this new knowledge.
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