MUTATIONAL SPECTRA INDUCED BY NITRIC OXIDE, PEROXYNITRITE AND REACTIVE OXIDANTS

该项目的目标是表征NO诱导的DNA损伤的潜在致突变后果，特别注意特定类型的损伤如何可能有助于肿瘤的发生和发展。巨噬细胞和嗜中性粒细胞过度产生NO和活性氧的能力已得到充分证实，这些活性物质诱导致死性和致突变性DNA损伤的能力也是如此。由NO诱导的细胞损伤的化学过程虽然复杂，但可能仅由两个基本过程引起，即与氧反应形成亚硝化物质和与超氧化物反应形成过氧亚硝酸盐（ONOO-）。该项目旨在通过表征NO和ONOO-在化学定义的系统中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CO2和羟基自由基损伤的致突变性和突变谱诱导的ONOO-在supF基因的pSP 189在人类细胞中复制的影响;它最近已经建立了由其他人，在mM浓度的CO2降低过氧亚硝酸盐的寿命的方式，可能会大大改变其DNA损伤特性。 第二，我们将表征与小鼠RAW264.7巨噬细胞体外共培养的细胞内靶基因的突变，所述巨噬细胞被激活以产生NO和活性氧。我们的第三条调查线将探测诱变细菌转基因与NO过量生产的基因改变的SJL小鼠，我们已经开发了一个系统，在体内的一氧化氮毒理学研究。在我们的最后一个具体目标中，我们提出鉴定引起在NO处理的细胞中观察到的突变的DNA损伤。将合成含有NO形成的修饰碱基的寡核苷酸。这些寡核苷酸将插入M13衍生物的基因组中，并在E.杆菌最后，将描述诱导的遗传变化的类型和数量（如果有的话）。此外，还将确定特定病变致突变的遗传要求。这些数据将对特定病变作为上述实验中观察到的突变前体的可能参与进行排序。

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.