Oxidant damage to DNA is involved in molecular processes of cancer and aging. Fixation of DNA damage mediated by nitric oxide (NO) may lead to alteration o the cellular phenotype.
The specific aims are: 1. To characterize, quantitate and compare NO-mediated DNA damage in isolated DNA, isolated mitochondria, and intact cells. 2. To measure DNA repair in mitochondrial and nuclear DNA of cultured cells. 3. To determine whether inflammatory cell DNA damage and repair correlates with in vitro NO experiments.
In Specific aim 1, DNA damage will be characterized and quantitated. The first approach is DNA digestion to nucleosides, separation by HPLC, with UV and electrochemical detection. The second approach will use bacterial reversion assays, where specific DNA damage results in antibiotic resistance, allowing selection o mutants (revertants). These approaches will identify relevant DNA damage for repair studies (Specific Aim 2).
In Specific aim 2, NO mediated DNA damage and repair in cultured cells will be characterized by gene specific repair. In this method, DNA lesion- specific enzymes introduce DNA strand breaks which are characterized by DNA mobility in alkaline gels (shorter strands migrate further) and following Southern blotting nuclear as well as mitochondrial DNA repair is analyzed. Repair in mitochondrial DNA will be compared to repair in isolated mitochondria supplemented with different mitochondrial electron transport substrates. The effect of NO on cellular proliferation and/or cytotoxicity will be studied. Thus, these studies will develop endonuclease assays for NO mediated DNA damage and characterize the cellular response to NO mediated damage.
In Specific aim 3, established methods will be used to study whether cellular DNA damage and repair correlates inflammatory oxidants. In these studies, DNA repair as well as cytotoxic responses such as apoptosis will be measured in cultured cells exposed to activated mouse peritoneal phagocytes to establish if a correlation exists between inflammation, NO generation and cellular responses to NO.
| Djuric, Z; Potter, D W; Taffe, B G et al. (2001) Comparison of iron-catalyzed DNA and lipid oxidation. J Biochem Mol Toxicol 15:114-9 |
