The pathogenesis of liver injury in human cholestatic diseases is poorly understood. Current therapies for these disorders are inadequate, thus progression to end-stage liver disease is common. Developing novel treatment approaches will require an expanded understanding of the fundamental mechanisms causing hepatocyte necrosis and apoptosis in this condition. The overall objective of this proposal is to test the hypothesis that oxidative stress and hepatic mitochondrial dysfunction are key elements involved in the pathogenesis of liver injury in cholestasis. In this grant renewal, we will expand our previous investigations to now examine mechanisms involved in hepatocellular apoptosis and necrosis induced by toxic bile acids that accumulate in the liver during cholestasis. We will test the following interrelated hypotheses, using freshly isolated rat and human hepatocytes, hepatic mitochondria, an intravenous bile acid toxicity model developed in our laboratory, the bile duct-ligated model of cholestasis and transgenic mice. To determine if generation of reactive oxygen species (ROS) are a proximate event in bile acid-induced hepatocyte necrosis and apoptosis, the time-sequence of related intracellular events will be carefully assessed during these two forms of cell death in isolated hepatocytes and, by using various inhibitors of critical processes involved, the role of ROS generation will be evaluated. Digitized confocal fluorescent microscopy will also be used to better define the role of mitochondria in these processes. To determine the role played by the hepatic mitochondrion in bile acid-induced liver injury, we will investigate the role of the mitochondrial permeability transition, cytochrome c release, ROS generation, activation of caspases, translocation of Bcl-2 family proteins, ATP concentrations and mitochondrial iron in bile acid-induced necrosis and apoptosis. To determine the role played by nitric oxide (NO) in this injury, generation of NO and its metabolites and their effect on cellular steps of apoptosis and necrosis will be studied in isolated hepatocytes and mitochondria undergoing bile acid-induced injury. To explore oxidative mechanisms to explain the increased susceptibility of the neonatal liver to cholestatic injury, we will investigate developmental differences of mitochondrial processes involved in necrosis and apoptosis in hepatic mitochondria isolated from the developing rat. The results from these investigations will improve our fundamental understanding of the mechanisms of hepatocellular necrosis and apoptosis in cholestasis, and may form the basis for the development and application of new therapies for these disorders.
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