Our goal for the proposed grant period is to ascertain whether oxygen free radical-induced damage accumulates in DNA during aging, and whether capacity to repair this damage diminishes with age. During the previous grant period, we identified lesions in DNA, i.e., characteristic chemical modifications and mutations, that provide specific markers for genomic alterations caused by oxygen free radicals. We have three specific aims. Having established that CC->TT tandem substitutions are produced in bacteria exposed to oxygen free radicals, we will utilize a new PCR-based assay to quantitate the frequency of CC->TT mutations in mammalian cells and tissues. Having documented that 5-hydroxy 2'-deoxycytidine is produced by oxygen free radicals and is highly mutagenic in bacteria, we will measure the content, repair and mutagenicity of this adduct in mammalian cells. Having introduced a flow cytometric assay to measure DNA damage during the cell cycle, we will utilize this assay to quantitate oxygen- mediated DNA damage and repair in cells and tissues. We will obtain tissue from mice (ages 6-8, 16-18 and over 24 months), as well as kidney and lymphocytes from human subjects of various ages. We will also utilize lymphoblast cell lines from patients with Fanconi's anemia which are uniquely sensitive to oxygen damage. These model systems are established in the laboratories that constitute this program project grant. We will also obtain tissue from transgenic mice that overexpress human catalase alone or both human SOD-1 and catalase, and from avian tissue as it becomes available from project 1. We will utilize the three assays mentioned in the specific aims to examine these model systems. Our observations will allow us to assess the accumulation of oxidative damage to DNA as a function of age and oxidative state in three species.
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