Mechanisms of unconjugated bilirubin toxicity: The contribution of oxidative stress and apoptosis

The experiments in this thesis investigate the molecular mechanisms of toxicity of unconjugated bilirubin (UCB). Elevated concentrations of UCB are responsible for neonatal jaundice and can eventually lead to kernicterus and even death. Apoptotic cell death has previously been demonstrated in Hepa-1c1c7 cells exposed to muM concentrations of UCB. In this thesis, the apoptotic signaling pathway initiated during UCB-mediated cell death was determined to be via the intrinsic mitochondrial pathway. Supporting this conclusion, depolarization of the mitochondrial membrane potential and activation of caspase-9 then caspase-3 were observed by 3 h post-treatment. A significant oxidative stress response was also observed following treatment with muM concentration of UCB as demonstrated by increased generation of reactive oxygen species and depletion of intracellular GSH.;The oxidative stress response was further characterized using novel, redox sensitive green fluorescent proteins to detect changes in intracellular redox state and 2D redox gel electrophoresis to detect changes in the disulphide proteome. A pro-oxidant concentration of UCB (50 muM) was found to cause early, reversible oxidation of the intracellular environment that was followed by a second phase of enhanced oxidation as well as changes to the disulphide proteome. Conversely, an antioxidant concentration of UCB (70 nM) was demonstrated to cause reduction of the intracellular environment.;Global changes in gene regulation were analyzed following treatment of Hepa-1c1c7 cells with both pro- and antioxidant concentrations of UCB. 50 muM UCB was found to upregulate a number of genes involved in the cellular response to ER stress and to downregulate a number of genes involved in the adaptive response to oxidative stress. Changes in ER stress genes were validated at the protein level indicating that UCB is able to induce ER stress in Hepa-1c1c7 cells. Decreased procaspase-12 content was also observed implicating a role for ER stress in UCB mediated apoptosis.;The research findings presented in this thesis represent novel contributions to the understanding of the mechanism of toxicity of UCB while also identifying novel signaling pathways and molecular components which may one day prove to be effective therapeutic targets for the treatment and prediction of UCB-mediated toxicity.;Keywords. apoptosis, aryl hydrocarbon receptor, caspase, disulphide proteome, endoplasmic reticulum stress, glutathione, Hepa-1c1c7, mouse, oxidative stress, reactive oxygen species, unconjugated bilirubin.