The use of multiple sampling times in in vitro chromosome assays

Asynchronously growing V79 hamster cells were treated with hypotonic solutions of different osmolalities, with ethyl methanesulfonate (EMS) or a combination of these 2 agents. Four different fixation times (8, 10, 14, 18 h) were tested. Hypotonic treatment alone induced chromosomal aberrations (CA), leading to very high frequencies at low osmolalities (50 mOsm/kg H2O). EMS also induced CA, but a comparison of EMS and hypotonicity revealed that EMS induced fewer aberrations at the maximum doses tested. A combined treatment produced contradictory results, depending on the sampling time: 8 and 14 h led to a clear elevation of EMS-induced CA by hypotonicity, 10- and 18-h sampling times led to a decrease in the aberration frequency in EMS and hypotonically treated cells. The observation that the choice of sampling time significantly influences the incidence of chromosomal aberrations is of particular interest for in vitro assays. In this study the 4 different sampling times are not enough to allow meaningful conclusions about additive or synergistic effects of the mutagens tested. These contradictory results are caused, on the one hand, by the very steep dose-response curve after hypotonic treatment and on the other hand by the varying degree of cell-cycle delay after combination treatment. This makes a valid comparison of the CA frequencies impossible because no 2 sampling times contain the same mixture of cells.