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Reactive Nitrogen and Oxygen Species Derived from Inflammatory Cells

Research in the Wogan laboratory addresses two major topics,both dealing with cancer causation, with the long-range objective of contributing to the scientific foundation for preventive strategies. The first focus is on how chronic inflammation acts as a major risk factor for cancers of the stomach, colon, liver, urinary bladder and possibly prostate and other cancers as well. Cancers arise out of chronically inflamed tissues, in which infiltrating macrophages and neutrophils expose normal tissue cells to a myriad of highly reactive chemical species that cause DNA damage, protein modification, mutation and cell death through apoptosis and necrosis. Our work concerns DNA damage, mutations and apoptosis induced by nitric oxide and its reactive derivatives: NO2, N2O3, ONOO, carbonate radical and hydroxyl radical. These agents can either drive cells into programmed cell death (apoptosis) through multiple pathways, or inhibit apoptosis and enhance mutation through damage to DNA bases, strand breaks and cross links. Responses are highly dependent on cell type, exposure dose and dose-rate, and underlying mechanisms are poorly understood. We are systematically characterizing mutagenesis, mutation spectra and chemical damage to DNA in well-defined genetic targets exposed under controlled conditions to nitric oxide and derivatives. Mutation specta are compared with chemical damage induced in the target DNA, determined through collaborations with the Dedon and Tannenbaum groups, with the objective of identifying DNA modifications responsible for specific mutational events. In collaboration with the Essigmann group, this information forms the basis for synthesis of oligonucleotides containing appropriate modified DNA bases, in which mutations created during replication of the modified genomes in suitable host cells are unambiguously identified. Mutagenesis is also studied in target cells co-cultured with macrophages activated to produce nitric oxide over long periods of time, as surrogates for tissue cells exposed in vivo to prolonged inflammation.

Fluorescence Labeling of Nucleoside Adducts for Molecular Epidemiology

The second research focus is molecular markers of exposure to environmental carcinogens. Accurate assessment of cancer risks from carcinogen exposure requires quantitative data on individual human exposure to specific carcinogens. Detection and analysis of specific carcinogen-damaged DNA bases (DNA adducts) resulting from endogenous or exogenous exposures to carcinogens is essential not only for quantifying biologically effective doses, but also for establishing relationships between exposure and cancer risk.

To meet this need, we have developed and validated a sensitive and specific procedure based on fluorescence labeling of DNA adducts combined with HPLC-laser-induced fluorescence detection. The fluorescent dye BODIPY FL has been used to label deoxynucleoside adducts of the aromatic amine 4-aminobiphenyl, a bladder carcinogen widely disseminated in the environment, and PhIP, a heterocyclic amine formed during cooking of high-protein foods. The labeling reaction is carried out on adducts at picomolar to nanomolar concentrations, and the fluorescent product is detected and quantified by HPLC/laser-induced fluorescence. This analytical method is being used for analysis of aminobiphyenyl-deoxyguanosine levels in DNA isolated from urinary bladder in a case-control study of bladder cancer. The method will also be used in evaluating the significance of oxidative DNA damage products associated with chronic inflammation as well as other chemical carcinogens (heterocyclic amines) in the etiology of other major forms of cancer, including stomach and colon. Data from these studies will be important in developing preventive strategies to minimize the impact of these carcinogens as risk factors.


Copyright © 2003 Gerald N. Wogan, Ph.D. All Rights Reserved.
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