Evaluation of DNA damage in murine fibroblasts treated with cigarette smoke condensate

CSC is a complex chemical mixture containing about 4800 compounds, many of them have cytotoxic and mutagenic activities on mammalian cells. Most of these compounds are able to interact with DNA at different levels. Cells may respond to DNA damage by following different pathways, such as the DNA repair processes and the cell cycle and DNA damage checkpoint activation. To the aim to evaluate the biological effects of CSC on cells, alkaline comet assay and flow cytofluorimetry were used to examine DNA damage/repair and cell cycle progression. All experiments were performed by using CSC from standard cigarettes in the range of doses 30-180g/ml and Swiss 3T3 murine fibroblasts. Results obtained by comet assay showed that CSC induces DNA strand breaks, significantly higher after 90 min of treatment than 3hrs. This difference, particularly evident at 100 and 150g/ml, it is probably due to a fast repair that can be explained by the oxidative component of the DNA damage CSC-induced. To clarify these results, further investigations on the evaluation of the oxidative damage are in progress by applying two different methods. one is the detection of the oxidised bases on the DNA by using the modified protocol of Comet assay with FPG and endo III, the second is the analysis of the intracellular levels of ROS, measured as the ability of treated cells to oxidise a fluorogenic dye, is going to be carried out. Previous results of long-term survival showed that cells lose their ability to form colonies in dose-dependent manner, after 24hrs of CSC treatment and 168 hrs of culture. However, the cytofluorimetric analysis showed that a fraction of cells, blocked in G2/M immediately after 24hrs of treatment, are gradually granted to continue the cell cycle, after incubation for further 6hrs in medium CSC-free. Further investigations on the cell cycle alteration are on going.