DNA & Protein Reactions of NO, ONOO, Reactive Species

该计划的假设是巨噬细胞(一氧化氮、超氧化物)和中性粒细胞(HOC1、NO2‘)在炎症部位产生的化学物质与附近细胞中的DNA、脂类、碳水化合物和蛋白质反应产生大量毒素，导致细胞死亡和与恶性转化相关的突变。项目2的目标是开发这种损伤的生物标记物，并使用它们来确定发病机制。在上一次资助期间，我们开发了DNA脱氨基、氧化和硝化以及蛋白质硝化的分析方法。我们现在建议将这些方法应用于培养细胞。 (与项目3)以及炎症和癌症的动物模型。生物标记物将连接在这笔赠款的其他子项目中开发的化学模型，并使这些模型的测试和改进成为可能。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。具体目标如下： 目的1.确定分离的DNA、细胞核和培养中的细胞(核心1)产生的DNA和蛋白质损伤的谱。我们建议开发一系列DNA和蛋白质损伤的分析方法，然后将它们用于测试与NO‘衍生物种(N2O，NO2’，过氧亚硝酸盐)和HOC1反应有关的假说。选择用于研究的生物标志物涵盖了预计在炎症部位发生的损害的范围：碱基脱氨和G-G交联键；碱基氧化和硝化；脱氧核糖(M1G)和脂类(EpsilonA)氧化产生的DNA加合物(EpsilonA)；硝基酪氨酸；卤化碱基；以及碱性部位和链断裂。这些标记物将被用来确定DNA、核和暴露于受控NO‘和ONOO通量的细胞以及与激活的巨噬细胞共同培养的细胞中炎症化学的相对优势(项目3)。我们还将分析具体的 硝基酪氨酸的蛋白质，以确定硝化剂在细胞中的空间分布。 目的2.定义小鼠炎症模型组织中出现的损伤的谱系。这些生物标记物将应用于项目4和核心2中开发的小鼠炎症模型。我们将首先确定哪些生物标记物对SJL小鼠(脾和肝脏)和RAG-2小鼠(结肠和肝脏)的靶器官有用。接下来，这些生物标记物将用于确定巨噬细胞和中性粒细胞在炎症过程中的相对作用。最后，我们将量化DNA损伤和组织中的硝基酪氨酸，作为与所有其他项目协调努力的一部分，以确定炎症细胞的数量和空间分布，并开发产生活性氮和卤素的预测模型。

The hypothesis driving this Program is that chemical species generated by macrophages (nitric oxide, superoxide) and neutrophils (HOC1, NO2') at sites of inflammation react with DNA, lipids, carbohydrates and proteins in nearby cells to generate a host of toxins that lead to cell death and mutations associated with malignant transformation, The objective of Project 2 is to develop biomarkers of this damage and use them to define the mechanisms of pathogenesis. In the last grant period, we developed methods for analysis of DNA deamination, oxidation, and nitration, and protein nitration. We now propose to apply these methods to cultured cells (with Project 3) and animal models of inflammation and cancer. The biomarkers will link the chemical models developed in other subprojects in this grant and enable the testing and refinement of those models. An additional benefit of the biomarkers will be their eventual application to studies on chemoprevention in animals and humans. Specific Aims are as follows: Aim 1. Define the spectrum of DNA and protein lesions produced in isolated DNA, nuclei, and cells in culture (with Core 1). We propose to develop analytical methods for a series of DNA and protein lesions and then apply them to test hypotheses related to the reactions of NO'-derived species (N2O3, NO2', peroxynitrite) and HOC1. The biomarkers chosen for study cover the range of lesions expected to arise at sites of inflammation: base deamination and the G-G cross-link; base oxidation and nitration; DNA adducts derived from oxidation of deoxyribose (M1G) and lipids (epsilonA); nitro-tyrosine; halogenated bases; and abasic sites and strand breaks. These markers will be used to define the relative predominance of inflammation chemistries in DNA, nuclei and cells exposed to controlled fluxes of NO' and ONOO (Project 1), and to cells co-cultured with activated macrophages (Project 3). We will also analyze specific proteins for nitro-tyrosine to define the spatial distribution of nitrating agents in cells. Aim 2. Define the spectrum of lesions arising in tissues from mouse models of inflammation. The biomarkers will be applied to the mouse models of inflammation developed in Project 4 and Core 2. We will first determine which of the biomarkers are useful for target organs in the SJL mouse (spleen and liver) and the Rag-2 mouse (colon and liver). The biomarkers will next be used to define the relative roles of macrophages and neutrophils in the inflammatory process. Finally, we will quantify DNA lesions and nitro-tyrosine in tissues as part of a coordinated effort with all other projects to define the number and spatial distribution of inflammatory cells and develop predictive models for production of reactive nitrogen and halogen species.