The primary objective of the proposed research is to investigate alkylation damage and repair in several postmitotic tissues of the C57BL/6Nia inbred mouse strain as a function of age. An elucidation of DNA damage that persists and possibly accumulates during aging will provide a better understanding of the role normal internal processes play in the etiology of degenerative diseases and the pathogenesis of cancer. Among environmental factors which have been implicated as carcinogens and teratogens are N-nitroso compounds. N-nitroso compounds are common in our external and internal environments. It is known that some of these compounds are potent alkylating agents which are capable of damaging cellular DNA. However, the types of modified nucleotides, the extent of DNA damage and the repair of alkyl adducts as a consequence of normal aging have not been determined. This study proposes to investigate these areas. Alkylation damage will be studied in untreated mice obtained from the NIA colony where environmental conditions are stringently regulated, and therefore are relatively constant, throughout their lifespan. The proposed experiments will characterize modified nucleotides that we have recently described in senescent mouse myocardium. The effects of age on DNA repair of methylated nucleotides and the persistence of specific adducts will also be assessed. The objectives of the coming year will be to characterize several methylated nucleotides and to estimate the abundance of these nucleotides as a function of age in DNA of postmitotic cells of an inbred mouse model for aging studies. To achieve this objective, the types of lesions introduced into brain, liver and heart DNA by administration of N-methyl-N-nitrosourea (MNU) will be compared to lesions found in senescent mouse tissues. To identify specific adducts, these studies will employ a 32P-end labeling technique in which methylated nucleotides are resolved by tow- dimensional, thin-layer chromatography. Isolated adducts will then be characterized by high performance liquid chromatography. Studies with radiolabeled MNU will be initiated to follow DNA repair and persistence of methylated nucleotides in young and old animals.
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