Long-term objectives are (i) to advance knowledge of lipid peroxidation in animal tissues, with a major focus on the interaction of toxic initiators and antioxygenic protectors as determined by differential biochemical damage to lipids, proteins, and nucleic acids; and (ii) to evaluate antioxidant systems in living animals. Lipid peroxidation is involved in many disease processes and chemical toxicities. Protection is provided by biological antioxidants, and some requiring further evaluation will be studied.
Specific aims are: 1. To utilize in vitro systems to measure lipid peroxidation in animal tissues and cells. a. To compare the susceptibility of various animals organs to lipid peroxidation using tissue slices, organ homogenates,a nd hepatocytes as in vitro model test systems, with emphasis being placed on the tissue slice system. b. To determine the relative capacities of various toxic compounds as initiators of lipid peroxidation, and to determine the additive or synergistic effects of mixtures of toxic compounds as initiators of lipid peroxidation. c. To determine the relative protective capabilities of antioxidants and multiple antioxygenic systems against potent peroxidation initiators. 2. For application to a wide range of potential lipid peroxidation initiators, develop in vitro tissue assays that may become substitutes for or supplements to whole-animal assays currently used to determine some chemical toxicities. These assays will be used to evaluate single and multiple antioxygenic systems in tissues. 3. In tissue slices and hepatocytes, quantitate lipid peroxidation molecular damage to lipids, proteins and enzymes, nucleic acids, and other biomolecules. For screening initiators (halogenated hydrocarbons and peroxides) and antioxidants, thiobarbituric acid-reactive substances; release of ethane and pentane; aldehydes; decrease in polyunsaturated fatty acids; formation of conjugated dienes; release of free fatty acids; incorporation of radiolabeled amino acids into slices; oxygen consumption; efflux of glutathione; changes in mitochondrial enzymes; isolation of DNA for determination of template activity, electrophoresis of transcribed mRNA, DNA crosslinks, and tryptophan bound to DNA; oxidation of flavins; changes in heme pigments; electrophoretic changes in specific proteins that may be polymerized; oxidation of methionine; and release of hydrogen sulfide. 4. To determine in vivo protection against lipid peroxidation by biological compounds for which antioxygenic capacity has not been adequately determined, including beta-carotene, uric acid, coenzyme Q10, and (+)-catechin. 5. To quantitate additive effects or synergism among antioxidants, including beta-carotene, uric acid, coenzyme Q10, (+)-catechin, vitamin E, and selenium. To measure protection by dietary or injected antioxidants, high level lipid peroxidation will be produced in rats by injection of iron dextran or methyl ethyl ketone peroxide. Noninvasive measurements of expired ethane and pentane will be made, and hydrocarbon gas data will be correlated with data on erythrocyte hemolysis, peroxidizability of tissues, thiobarbituric acid-reactive substances in urine, and lipid-soluble fluorophores in spleen.
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