APOPTOSIS AND 1-METHYL-2-NITROIMIDAZOLE TOXICITY IN CHO CELLS

The time course and characteristics of the selective hypoxic cytotoxic ity of the 2-nitroimidazole model compound 1-methyl-2-nitroimidazole ( INO2) were analysed during prolonged time periods (up to 5 days post t reatment). When central populations were seeded at the same cell densi ty as drug-treated cells, they entered confluency at day 3 and underwe nt apoptosis at day 5, which appeared to be mediated by an autocrine m echanism. in subsequent studies of drug-treated cells, the seeding den sity of treated cells was adjusted to avoid this cell confluency effec t. Treatment with a low INO2 concentration (2.5 mM) resulted in apopto tic DNA fragmentation (ladders), which was observed 4-5 days after an acute 6-h hypoxic drug exposure. In contrast, at a high INO2 concentra tion (40 mM) for 2 h, which was equitoxic to the low concentration, no characteristic DNA ladders were observed. Fluorescence microscopy rev ealed apoptotic bodies and pyknotic nuclei 5 days following hypoxic 2. 5 mM INO2 exposure, whereas 40 mM INO2 hypoxic treatment produced cell ular ghosts devoid of DNA 5 days after exposure, consistent with the D NA ladder results. However, characteristic apoptotic morphology was pr eviously observed immediately after the acute hypoxic exposure of 40 m M INO2. Cell cycle analysis and DNA fragmentation as measured by the T dT assay suggested that dose-dependent differences in the apoptotic re sponse occur post exposure after an equitoxic acute hypoxic exposure t o either the low or the high INO2 concentration. This dose-dependent d ifferential in response may be attributed to the degree of initial DNA damage as measured by the comet assay.