DNA damage induced by a quinoxaline 1,4-di-Af-oxide derivative (hypoxic selective agent) in Caco-2 cells evaluated by the comet assay

Abstract

The DNA damage induced by 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2- quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride (Q-85 HCl) in Caco-2 cells under hypoxic and well-oxygenated conditions has been studied by using the comet assay. This compound has shown a good in vitro profile of high selective toxicity in hypoxia, but its mechanism of action is unknown. The DNA damage has been evaluated by performing the comet assay after a 2-h treatment with Q-85 HCl (0.1, 0.2, 0.4 μm in hypoxia; 20, 40 μm in well-oxygenated conditions). The number of cells in apoptosis has also been assessed by flow cytometry analysis of Annexin V-FITC staining. The capability of the cells to repair the DNA damage and the proliferation rate was evaluated at different times after the treatment (24-168 h). Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed (comet score: 132 ± 13, 343 ± 30 and 399 ± 1; control comeit score: 42 ± 14). Under well-oxygenated conditions, the number of nuclei with comet increased significantly with respect to the control (comet score: 273 ± 14 and 312 ± 9; control comet score: 27 ± 4). Cells in apoptosis were not detected by the comet assay nor by flow cytometry. The recovery from DNA damage was time- and concentration-dependent in hypoxia (cells treated with the highest concentration still showed DNA damage after 72 h) and rather time-dependent in well-oxygenated conditions (DNA was completely repaired after 24 h). In conclusion, Q-85 HC1 acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HC1 itself may be acting.