OXIDATIVE DAMAGE INDUCED BY NO IN BACTERIAL AND TRANSGENIC MAMMALIAN CELLS

North Carolina Central University, Durham NC

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Abstract

The broad long-term objective of this research proposal is to investigate nitric oxide (NO)-induced oxidative DNA damage in bacterial and transgenic mammalian cells. The proposed research plan will involve (a) genotoxicity evaluations followed by molecular analysis of the NO- induced mutations to generate mutation spectra, the PI will focus on analyzing NO-induced mutations in bacterial (genes and alleles) and transgenic mammalian (transgene) systems, using the polymerase chain reaction (PCR) and DNA dideoxy sequencing; and (b) oxidative DNA damage analysis using high performance liquid chromatography and electrochemical detection (HPLC/EC) to determine NO-induced damage at the base level. The HPLC/EC analysis will proceed using the advanced tool of multichannel EC to generate a profile for NO-induced DNA damage. The significance of this research lies in the comprehensive approach it puts forth to determine the genotoxic risk associated with endogenously produced NO. This project also evaluates NO-toxicity by underscoring variations, similarities , as well as synergism in oxidative DNA damage that may exist among NO and other reactive oxygen species (ROS), namely hydrogen peroxide (H2O2). It is important to note that both NO and H2O2 are products of macrophage activation, thus effect cytostasis and cytoxicity in the inflammatory response, but in preliminary studies to date NO and H2O2 have been shown to have different genotoxic characteristics. Thus the central hypothesis to be tested is that for NO to perform differently then H2O2 as an oxidative mutagen, the NO oxidative assault must (a) target different DNA bases, and/or (b) cause base alterations that are influenced by different cellular repair mechanisms. The research plan is designed to achieve three Specific Aims: (1) To identify the intracellular factors, such as targe loci and repair in bacterial and transgenic mammalian cells that influence NO- induced mutagenesis, (2) To determine, using HPLC/EC, the nature of NO- induced damage on purified DNA Bases: and (3) To generate HPLC/EC profiles for NO-induced damage to cellular DNA. The project will utilize E. coli and Salmonella typhimunum strains; and the trangenic mammalian cell lines AS52 and G12, the Chinese hamster cells that allow for analysis of mutations generated under mammalian intracellular conditions against a simpler bacterial target (E. coli guanine phosphoribosyl transferase [gpt] transgene)