Oxidative injury has been implicated in the pathophysiology of liver injury during xenobiotic metabolism, ischemia/reperfusion injury, and neutrophil activation. Oxidative injury occurs by a variety of mechanisms and has been studied with several diverse models involving different agents and biological preparations. My previous work shows that fructose, an effective glycolytic substrate in liver, protects against variety of oxidant chemicals. This suggests that mitochondrial injury is involved in oxidative stress. Acidotic extracellular pH also provides substantial protection against oxidant chemicals. These findings form the basis for this application. My overall goal is to understand the cellular mechanisms underlying toxic injury during oxidative stress. Specifically, I will evaluate the role of the mitochondrial permeability transition in toxic injury induced tert-butylhydroperoxide (t-BuOOH), menadione and extracellular ATP. In cultured rat hepatocytes, I will evaluate the following hypotheses: 1) These toxicants cause a mitochondrial permeability transition by promoting opening of permeability transition pores or megachannels in the inner mitochondrial membrane. Different agents may promote channel opening by different mechanisms, such as increases of intramitochondrial Ca2+ or pH, oxidation of mitochondrial glutathione (GSH) or pyridine nucleotides, or production of reactive oxygen species. 2) Megachannel opening leads to mitochondrial depolarization and uncoupling of oxidative phosphorylation. The resulting bioenergetic deficit may serve as a common final pathway leading to cell death for different oxidant chemicals. Glycolytic substrates like fructose rescue hepatocytes from lethal injury by providing an alternate source of cellular ATP. 4) Acidotic pH, which is protective against lethal cell injury caused by a wide variety of oxidant chemicals, inhibits the pore opening and prevents onset of the mitochondrial permeability transition. Accordingly, l will characterize cellular injury to cultured rat hepatocytes from t-BuOOH, menadione and extracellular ATP in terms of dose-response, nutritional status and pH-dependence. Utilizing laser scanning confocal microscopy, l will measure changes of cytosolic and mitochondrial ions (Ca2+, H+, Na+, Mg2+), cytosolic and mitochondrial GSH and NAD(P)H, cytosolic and mitochondrial reactive oxygen species, and delta-psi in response to the onsets of permeability transition and cell death. The project will provide fundamental new information concerning mechanisms underlying cell death resulting from oxidative injury.
