Mutagenesis Advance Access originally published online on April 7, 2005
Mutagenesis 2005 20(3):165-171; doi:10.1093/mutage/gei023
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DNA damage induced by a quinoxaline 1,4-di-N-oxide derivative (hypoxic selective agent) in Caco-2 cells evaluated by the comet assay
Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain, 1Department of Biochemistry and Molecular Biology, University of Barcelona, Faculty of Chemistry, C/Martí Franqués 1, 08028 Barcelona, Spain and 2Department of Toxicology and Applied Hygiene, University Bordeaux 2, V. Ségalen, 146 Rue Léo Saignat, 33076 Bordeaux CEDEX, France
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 comet 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 HCl acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HCl itself may be acting.
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