The major objective of this research proposal is to investigate in cultured cells (fibroblasts, pulmonary endothelial cells, type II lung epithelial cells, H2O2-resistant cell lines and O2-resistant cell lines) the involvement of diffusible cytotoxic by-products of cellular peroxidation reactions in mechanisms of oxygen toxicity. The diffusible cytotoxins of interest include toxic lipid aldehydes (4-hydroxyalkenals particularly 4-hydroxy-2-nonenal), toxic lipid hydroperoxides and H2O2. These substances were selected for study on the basis of l) direct evidence that peroxidation is accompanied by the formation of these toxins, 2) direct evidence that by-products of peroxidation are formed during exposure of cells to hyperoxia, and 3) indirect evidence that increased metabolic detoxification of these cytotoxins may represent an important mechanism for the protection of cells from O2-induced injury. The initial focus of this work will be to determine if differential cell susceptibility to oxygen toxicity is due to differential sensitivity to, metabolism of, and/or exposure to peroxidation derived cytotoxins (i.e., 4-hydroxy-2-nonenal). The degree of O2-induced injury or protection from injury will be determined using biochemical, cell physiological, and cell survival papameters. Differential cell sensitivity to O2-induced injury as a function of exposure time will be correlated with the production of cytotoxins [determined by gas chromatography/mass spectrometry (GC/MS)], the metabolism of cytotoxins (determined by GC/MS and HPLC), and the susceptibility to cytotoxins. The effects of clinically significant modifiers of oxygen induced injury (various polyunsaturated, monosaturated, and saturated fatty acids; lipid soluble antioxidants; and various inflammatory mediators including endotoxin, tumor necrosis factor, and interleukin- 1) will be examined with respect to their ability to alter the production of, susceptibility to, and/or metabolism of diffusible cytotoxins. The long term goal is to identify clinically significant nutritional and cellular modifications which will afford protection from lung injury in prematurely born infants exposed to hyperoxia based on a mechanistic understanding of the involvement of peroxidation derived diffusible cytotoxins in O2-induced injury.
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