The long-term goal of this proposal is to identify genes influencing murine aging through the use of quantitative trait loci (QTL) analysis. To do so, several molecular markers associated with oxidative stress will be measured across the lifespan in a cross-sectional study at 150, 450, and 750 days of age using C57Bl/6 DBA/2 (BXD) recombinant inbred (RI) strains and BXD F2 mice; quantitative differences in these markers among strains and individuals will then be correlated with genotyping data. In this resubmission, major revisions in the overall study design and valuable suggestions by the initial reviews have led to a broadening of the analytic approach for this subproject. We have expanded the panel of oxidative stress markers to include those which affect 3 major classes of macromolecules in 3 organs, including brain, and which have been shown to change significantly with age in mice. Specifically, oxidative damage to DNA in cardiac tissue will be assessed by quantitation of mitochondrial DNA deletions by PCR and measurement of 8 hydroxyl-2'-deoxyguanosine by HPLC. Protein levels of the antioxidant enzymes copper-zinc containing superoxide dismutase (CuZn-SOD) and manganese containing superoxide dismutase (Mn-SOD) will be measured in liver by Western blot. Lipid peroxidation will also be assessed in liver with the thiobarbituric acid assay. Protein oxidation in cerebellum will be quantified as protein carbonyl content. Quantitation of these markers of oxidative stress will then be correlated with polymorphic genetic markers to identify chromosomal regions (QTL) influencing these age- related phenomena.
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