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.
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