Our overall hypothesis is that NO will generate reactive species in biological systems that damage DNA and proteins and react with critical molecules such as glutathione. in turn these reactions will contribute to death and mutations in exposed mammalian cells. in the last grant period we have made major progress in support of that hypothesis. The overall objective of our future research will be to continue to test this hypothesis in more complex experimental systems including animal models of human disease. The long-term goal will be to establish the relationship between NO and disease in an animal model through the use of biomarkers that can eventually be applied to human clinical and epidemiological studies. An additional benefit of these biomarkers will be their eventual application to studies on chemoprevention in animals and humans. The strategy we will follow will be to analyze the chemical reactions of the NO-derived reactants, nitrous anhydride, and peroxynitrite, with DNA. Products of base and sugar damage will be characterized qualitatively and quantitatively in deoxynucleosides, oligonucleotides, and target genes. A major goal will be to determine whether NO-induced mutations in target genes in animal models reflect the chemical damage at those sites. Both chemical spectroscopic and molecular biological techniques will be applied. At the same time we will measure a series of biomarkers that characterize NO chemistry in animal models, including markers of NO biosynthesis, oxidative stress, protein nitration, and DNA oxidation and deamination.
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