Nitric oxide and peroxynitrite-induced cellular damage

Since its initial discovery in vivo, nitric oxide (NO') has become one of the most highly studied and important biological molecules. Nitric oxide is a key participant in many physiological pathways in the body; however its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity. Although excess production of NO' can lead to damage and toxicity, the precise mechanisms underlying NO'-induced cellular toxicity are still very unclear. One of the objectives of this study was to analyze in detail some of the molecular targets of NO' within a cell. NO'-induced toxicity was investigated in two different cell lines, Chinese Hamster Ovary (CHO-AA8) and Human Lymphoblastoid (TK6) over a range of NO' doses (0-9 mM). Examples of damage parameters measured in this study include inhibition of DNA synthesis, damage to mitochondria, loss of cell membrane integrity, apoptosis, changes in cell cycle distribution and the occurrence of DNA strand breaks. Results indicate that NO'-induced toxicity is a complex process involving multiple pathways eventually leading to apoptotic cell death. The earliest observable effect of NO' exposure in both cell types is inhibition of DNA synthesis which appears to be the result of DNA damage ultimately leading to cell cycle arrest. Furthermore, results consistently demonstrate that TK6 cells are much more susceptible to NO'-induced toxicity than CHO-AA8 cells. In addition to investigating the toxic effects of NO', research was undertaken to better understand peroxynitrite (ONOO-) DNA damage chemistry at the level of deoxynucleosides, oligonucleotides, and plasmid DNA. Peroxynitrite, a powerful oxidant with hydroxyl radicallike reactivity, is formed in biological systems from the rapid reaction between nitric oxide and superoxide. Once formed, within or in close proximity to a target cell, ONOOcan react with multiple cellular components including lipids, proteins and DNA. Results indicate that exposure of DNA to ONOOcan lead to multiple DNA damage products including base modifications, abasic sites, and single-strand breaks. A more detailed analysis revealed that two pathways, direct sugar damage and cleavage of abasic sites, are most likely responsible for the observed strand breaks. Surprisingly, experiments performed with individual deoxynucleosides indicate that only deoxyguanosine shows significant reaction with ONOO (30%) while the remaining deoxynucleosides exhibit minimal reactivity (2-3%). Furthermore, oxidation and nitration products of deoxyguanosine, primarily 8-oxodG and 8-nitrodG, are even more reactive towards ONOOthan deoxyguanosine itself. Experiments with ONOO--treated oligonucleotides suggest that 8-oxoG may be the most important target of ONOOin DNA. The main enzyme responsible for the repair of the 8-oxoG/ONOOadducts, at least at low ONOOconcentrations, appears to be FaPy Glycosylase (Fpg). Also, no spontaneous depurination of the 8-oxoG/ONOOadducts was detected. Although both nitric oxide and peroxynitrite participate in key physiological processes, several aspects of their reactivity and interactions with biological molecules are not well understood. The results presented in this thesis provide new information on the potential impact of nitric oxide and peroxynitrite on living cells. Furthermore, results from this study should contribute towards a better understanding of the roles played by nitric oxide and peroxynitrite in advancing the onset of mutagenesis and cell death in target cells and the involvement of these processes in cancer and inflammatory diseases. Thesis Supervisor: Steven R. Tannenbaum Title: Professor of Chemistry and Toxicology