Analysis of oxidative damage in cells in different phases of the cell cycle

In vivo and in vitro cell populations exhibit heterogeneous responses to ma ny genotoxic agents which can be partially explained by the relative positi on of the cells in the mitotic cycle. In this study, the possibility to sim ultaneously detect oxidative DNA damage and cell cycle position has been in vestigated in C3H10T1/2 cell line. Exponential and plateau phase cells were treated with hydrogen peroxide and DNA damage and repair were evaluated by the alkaline comet assay and the micronucleus test. Our results show a dos e-related increase of DNA damage after H2O2 treatment in both culture condi tions. The percentage of cells in the various phases of cell cycle determined by c omet assay was compared with the data obtained by DNA content flow cytometr ic analysis, and a good agreement between the results of the two techniques was found. Untreated exponentially growing cells in G1 phase shaved a lowe r tail moment than S and G2/M cells. The same cell cycle dependence was evi denced in cells treated with low doses of H2O2, while, at higher doses, all cells showed a similar high level of damage. DNA repair was assessed analyzing cells by comet assay 2 h after treatment, The results showed that DNA repair was almost complete for all H2O2 doses tested. These results confirm the sensitivity of the Comet Assay in assessing DNA d amage and repair, and support its usefulness in evaluating cell cycle-relat ed differential sensitivity to genotoxic agents.