Cytotoxicity of peroxynitrite and nitric oxide induced oxidative stress on 3T3 fibroblast cell line

Official URL: http://risc01.sabanciuniv.edu/record=b1082169 (Table of Contents)

In the present study, the oxidative and cytotoxic effects of two biologically important molecules, NO and ONOŌ have been studied in 3T3 Fibroblast cell line. NO is a key molecule in many physiological pathways, but also its reactivity gives it the potential to cause considerable damage to cells and tissues. NȮ reacts rapidly with superoxide anion (O².̄) to form ONOŌ which is a powerful oxidant. Cell morphology and viability studies showed that both NO. and ONOŌ caused significant loss of viability in 3T3 fibroblast cell line. Intracellular ROS production has been monitored by a fluorescent probe, DCFH-DA. When cells were treated with of NO, a significant increase in fluorescence intensity has been observed when compared to the control cells and this was supported with flourometric analysis results. Similar results have been observed when cells were incubated with ONOŌ at the same concentration range. To further studies, cells were stained by a fluorescent probe Hoechst 33342 (HO) to identify apoptotic cells; both NO and ONOŌ induced apoptosis. In molecular studies, cells were incubated with the oxidant molecules, NȮ and ONOŌ for different time periods and at all relevant doses. In all conditions, DNA remained intact; indicating that cytotoxic effect of NO and ONOŌ were merely due to a mechanism other than apoptosis. This phenomena requires further mechanistic studies. A potential antioxidant molecule, Catechin has been studied as a preventive molecule against cytotoxicity. In chemical model system Catechin was left to react with ONOŌ and FT-IR and NMR analysis of the end product gave us preliminary information about its structure. In cellular studies, the effect of Catechin on NO. and ONOŌ induced cytotoxicity was investigated. The optimum determined conditions for preventive effect of Catechin were 5 mM of Catechin for 50 mM of NO. and 200 mM of ONOŌ. These results were discussed in the light of ROS induced cytotoxicity in cellular signaling mechanisms.