) DNA adduction resulting from covalent interaction with electrophiles and oxygen free radicals is recognized to play a critical role in degenerative diseases, including cancer. Endogenously formed and exogenous genotoxic agents can damage DNA and are a constant threat to genome integrity. If not repaired before the onset of DNA replication, or repaired erroneously, the DNA lesions can lead to mutations, and if such alterations occur in growth-controlling genes, these mutations can lead to cell transformations and ultimately malignancy. Therefore, progressive accumulation of both premutational DNA lesions and genetic mutations is central to understanding the process of tumorigenesis. A number of observations suggest causal and quantitative relationship between DNA adduct formation, mutagenicity, and carcinogenicity. Our working hypothesis is that significant interindividual differences occur in the accumulation of DNA adducts presumably due to differences in uptake, activation and detoxification of carcinogens, and DNA repair efficiency, among others. And these differences could explain the wide range of susceptibility seen in the induction of certain forms of human cancers such as in cigarette smoking and lung cancer. Sensitive methods are therefore required for detecting and identifying trace level DNA adducts produced in the human scenario. The major objectives of this research proposal are to 1) develop a sensitive technology based on 32p-postlabeling assay for detecting structurally diverse, premutational DNA lesions: hydroxylated bases, delaminated products, methylated bases, abasic sites, lipid peroxidation-induced cyclic adducts, polyaromatics adducts, DNA-DNA and DNA-protein cross-links, glucose-6-P-DNA complex, and indigenous products; 2) further advance and apply liquid chromatography/mass spectrometry (LC/MS) and capillary electrophoresis (CE)/MS techniques for adducts detected by 32P-postlabeling; and 3) apply these techniques in a pilot study to human lung tissues and WBC as a surrogate. Significant efforts will be applied to supplanting TLC techniques with HPLC in attempts to make the postlabeling technology more routine and less labor intensive. The technologies can then be used in future to associate accumulation of premutational DNA lesions with that of genetic mutations in specific chromosomes or chromosomal regions carrying cancer- and other disease-related genes as human genome project advances further. A marriage of 32P-postlabeling and LC(CE)/MS technologies will make a major impact in understanding the role of oxidative and other DNA lesions with risks associated with degenerative diseases.
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