The overall hypothesis is to be tested in this program is that DNA damage, mutation, and cytotoxicity and cellular proliferation will arise as a result of nitrosative deamination, NO radical reactions, and oxygen radical damage when target cells are exposed to generator cells that over produce NO. Depending upon the dose rate, total dose, types of cells, and other circumstances, NO may drive cells into apoptosis through multiple pathways, or inhibit apoptosis and enhance mutation through damage to bases and formation of strand breaks, and crosslinks. This hypothesis will be tested in vitro and in vivo in cell culture and in animal (mouse) models. In Dr. Deen's project mathematical models will be developed to predict the concentrations of NO and related compounds in aqueous solutions, cell cultures, and tissues. This information on the rates of NO synthesis and diffusion will be combined with other kinetic and morphometric data to estimate the in vivo concentrations of NO and its products. Dr. Tannenbaum's Project will develop biomarkers for DNA and protein damage derived from the reactive species N2O3, NO2, ONOO, and HOC1. The biomarkers will be applied to mouse models to define the relative roles of macrophages and neutrophils in the inflammatory process. Dr. Wogan's Project will characterize mechanisms of mutagenicity, apoptosis and homologous recombination resulting from DNA damage induced by NO, with particular emphasis on how specific types of damage may contribute to tumor initiation and development. Dr. Erdman's Project will characterize the role of nitric oxide (NO) and NO-derived species in mouse models of inflammatory bowel disease (IBD) and cancer. These studies will use Rag-2 KO mice and T cell receptor (TCR) alpha-beta KO mice. Histopathologic lesions and the pattern of macrophage and neutrophil infiltration will be correlated with biomarkers for nitration, oxidation, and halogenation of DNA and proteins in Rag-2 KO mice. To further characterize the role of NO and NO-derived species in IBD, we will compare pharmacologic inhibition of iNOS with genetic inactivation of the enzyme by generating iNOS-deficient TCR alpha-beta KO mice. In vitro analysis of somatic mutations arising in vivo in the presence and in the absence of NO and NO-derived species will also be performed. Core 2 is the center of development and application of new animal models. Together the individual projects and cores will create a useful paradigm for dealing with pathophysiological situations characterized by overproduction of NO. This is important for many health problems, including cancer. Just as our work in the past led to useful and widely used assays for nitrate, nitrite, and N-nitroso compounds, the Current Program is expected to lead to the development of useful biomarkers for studying the molecular epidemiology of inflammatory diseases.
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