The purpose of this research is to explore possible mechanisms of age-associated intensification of oxidative damage to the liver. Previous results from the investigator demonstrated that vulnerability of isolated rat hepatocytes to diquat, a toxicant that acts through oxidative mechanisms, was enhanced as a function of aging. The increase in toxicity could not be explained on the basis of age- related changes in classical antioxidant defense systems, thus other possibilities were indicated. The proposed research will test the hypothesis that increased availability of iron for stimulation of lipid and protein oxidation and/or a deficiency of glutathione (GSH)-dependent protective factors make(s) the aged liver more susceptible to damage from oxidative chemicals. Three age groups of the Fisher 344 rat, representing young adulthood, middle age, and old age, and both sexes will be studied. The importance of iron availability (Aim 1) will be examined using ferritin isolated from livers of male and female rats at 5, 15, and 28 months of age. The purified ferritin preparations will be tested for their ability to release iron when subjected to a diquat redox cycling system and their ability to enhance diquat-induced lipid and protein oxidation in vitro.
In Aim 2, the effects of age and gender on the activities of GSH- dependent protective factors will be determined. An ADP- iron/ascorbate microsomal lipid peroxidation system will be used for measuring inhibition of lipid peroxidation and protein carbonyl formation by liver cytosolic and microsomal fractions from aging rats of both sexes. It has been suggested that the factors responsible for GSH-dependent protection against lipid peroxidation are the thiol-disulfide exchange enzymes, thioltransferase and protein disulfide isomerase; therefore, the influence of age and gender on the activities of these enzymes also will be determined (Aim 3).
In Aim 4, thioltransferase and protein disulfide isomerase will be purified from rat liver and added to the ADP-Fe/ascorbate lipid peroxidation system to see whether either or both of these enzymes confers GSH-dependent protection and is able to restore the protection that is lost as a consequence of aging. These studies should provide knowledge about the effects of aging on the vulnerability of the liver to oxidative stress.
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