NITRIC OXIDE-INDUCED MODIFICATION OF DNA AND PROTEIN

我们的总体假设是，NO会在生物系统中产生活性物质，破坏DNA和蛋白质，并与谷胱甘肽等关键分子反应。反过来，这些反应将导致暴露的哺乳动物细胞的死亡和突变。在上一个赠款期间，我们在支持这一假设方面取得了重大进展。 我们未来研究的总体目标将是继续在更复杂的实验系统中测试这一假设，包括人类疾病的动物模型。长期目标将是通过使用最终可应用于人类临床和流行病学研究的生物标志物，在动物模型中建立NO与疾病之间的关系。这些生物标志物的另一个好处是它们最终将应用于动物和人类的化学预防研究。我们将遵循的策略将是分析NO衍生的反应物，亚硝酸酐和过氧亚硝酸盐与DNA的化学反应。脱氧核苷、寡核苷酸和靶基因中碱基和糖损伤的产物将被定性和定量地表征。一个主要的目标将是确定在动物模型中NO诱导的靶基因突变是否反映了这些位点的化学损伤。将应用化学光谱和分子生物学技术。与此同时，我们将测量一系列生物标志物，表征NO化学在动物模型中，包括NO生物合成，氧化应激，蛋白质硝化，DNA氧化和脱氨基的标志物。

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.