The objective of the studies proposed in this competing continuation application remains the elucidation of the biochemical mechanisms whereby partially reduced and thereby activated oxygen species lethally injure cells in general and hepatocytes in particular. The progress made in the analysis of the cell injury produced by partially reduced oxygen species in the last 4 years and detailed in the Progress Report allows the formulation of specific concerns with respect to the killing of liver cells by an acute oxidative stress. Thus, it is proposed to pursue the following specific aims: l) the intracellular localization and regulation of the requisite cellular pool of ferric iron; 2) the control of lipid peroxidation by the major endogenous antioxidants, reduced glutathione and vitamins E and C; 3) the structural and functional consequences of membrane damage by lipid peroxidation; and 4) the nature and functional consequences of non-peroxidative mitochondrial injury. Pursuit of the first aim will entail purification of the protein(s) to which the cellular pool of non-ferritin ferric iron is bound. The purified protein will be used to isolate the encoding gene and for the reparation of a polyclonal antibody. The gene will be used to study the conditions that regulate the expression of the iron binding protein under varying conditions of iron metabolism and sensitivity to oxidative stress. The antibody will be used to determine the intracellular localization of the iron pool.
The second aim will address the disposition of vitamin C in cultured hepatocytes under conditions of oxidative stress, and the nature of the interactions between vitamins C and E and the GSH redox cycle.
The third aim will pursue the presence the lipid domains of differing molecular order in peroxidized hepatocyte membranes by using electron spin resonance spectroscopy and digital imaging fluorescence microscopy.
The final aim i s concerned with the role of the mitochondrial permeability transition in lethal oxidative cell injury of liver cells.
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