MUTATIONAL SPECTRA INDUCED BY NITRIC OXIDE, PEROXYNITRITE AND REACTIVE OXIDANTS

该项目的目标是表征由NO诱导的DNA损伤的潜在致突变后果，特别关注特定类型的损伤如何促进肿瘤的发生和发展。巨噬细胞和中性粒细胞过量产生NO和活性氧的能力已得到证实，这些活性氧诱导致死性和诱变性DNA损伤的能力也已得到证实。NO诱导细胞损伤的化学过程虽然复杂，但可能只经过两个基本过程，即与氧反应生成亚硝化物质和与超氧化物反应生成过氧亚硝酸盐（ONOO-）。该项目旨在通过表征化学定义系统中NO和ONOO-以及活化的吞噬细胞产生的DNA损伤的致突变后果来验证这一假设。我们的第一个具体目标将是确定二氧化碳和羟基自由基损伤对ONOO-诱导的pSP189 supF基因在人细胞中复制的致突变性和突变谱的影响；最近，其他人已经确定，浓度为mM的CO2在某种程度上降低过氧亚硝酸盐的寿命，从而可能极大地改变其DNA破坏特性。其次，我们将在体外与小鼠RAW264.7巨噬细胞共培养的细胞中表征靶基因的突变，这些细胞被激活产生NO和活性氧。我们的第三条研究线将探索与转基因SJL小鼠NO过量产生相关的细菌转基因的诱变，这是我们为研究体内一氧化氮毒理学而开发的系统。在我们的最后一个具体目标中，我们建议鉴定导致NO处理细胞中观察到的突变的DNA损伤。将合成含有由NO形成的修饰碱基的寡核苷酸。这些寡核苷酸将被插入到M13衍生物的基因组中，并在大肠杆菌中复制。最后，基因变化的类型和数量（如果有的话）将被描述。此外，将定义特定病变致突变的遗传要求。这些数据将对上述实验中观察到的突变的前体可能涉及的特定病变进行排序。

The goal of this project is to characterize the potential mutagenic consequences of DNA damage induced by NO, with particular attention to how specific types of damage may contribute to tumor initiation and development. The ability of macrophages and neutrophils to overproduce NO and reactive oxygen species is well established, as is the ability of these reactive species to induce lethal and mutagenic DNA damage. The chemistry of cellular damage induced by NO, although complex, may result from only two fundamental processes, reaction with oxygen to form nitrosating species and reaction with superoxide to form peroxynitrite (ONOO-). This project is designed to test this hypothesis through characterization of the mutagenic consequences of DNA damage produced by NO and ONOO- in chemically defined systems, as well as by activated phagocytes. Our first specific aim will be to determine the effects of CO2 and hydroxyl radical damage on mutagenicity and mutational spectrum induced by ONOO- in the supF gene of pSP189 replicated in human cells; it recently has been established by others that CO2 at mM concentration decreases the lifetime of peroxynitrite in a manner that may greatly alter its DNA damaging properties. Second, we shall characterize mutations in target genes within cells co-cultivated in vitro with mouse RAW264.7 macrophages activated to produce NO and reactive oxygen species. Our third line of investigation will probe mutagenesis in bacterial transgenes associated with NO overproduction in genetically altered SJL mice, a system we have developed for the study of nitric oxide toxicology in vivo. In our last specific aim, we propose to identify the DNA lesion(s) giving rise to the mutations observed in NO treated cells. Oligonucleotides will be synthesized containing modified bases formed by NO. These oligonucleotides will be inserted into the genome of an M13 derivative and replicated within E. coli. Finally, the type and amount of genetic change induced, if any, will be characterized. in addition, the genetic requirements for mutagenesis by specific lesions will be defined. These data will rank the possible involvement of specific lesions as the precursors to the mutations observed in the experiments described above.