The need for long-term treatment in the mouse lymphoma assay

The L5178Y tk +/- mouse lymphoma assay (MLA) has been widely used as a genotoxicity test for the detection of mutagens and clastogens. The standard MLA, as well as other mammalian cell gene mutation assays, usually employs a short treatment period (3-6 h). Our previous report, however, suggested that such short treatments may be insufficient for detecting some clastogens and spindle poisons. For the present study, we introduced and evaluated a longer treatment (24 h) in the MLA. We examined 15 chemicals which were evaluated as negative or inconclusive in the short-term study. Cells were exposed to the chemical for 24 h without S9 mix, cultured for 2 days and then thymidine kinase-deficient mutants were selected in 96-well microtiter plates under trifluorothymidine. Eleven chemicals yielded positive responses in the 24 h treatment MLA. They included nucleoside analogs (2'-deoxycoformycin and dideoxycytidine), a base analog (1,3-dimethylxanthine) and spindle poisons (colchicine and vinblastine sulfate), all of which do not directly affect DNA, but bring about mutations and chromosome alterations through nucleoside metabolism and chromosome segregation. Because the mutagenicities of these non-DNA targeting chemicals appear to be cell cycle dependent, treatment extending over more than one cell cycle may be required for their effect. Combining results from the present and previous studies, 31 of 34 (91%) chromosome aberration-positive chemicals exhibited positive responses in the MLA, suggesting that the sensitivity of the MLA with 24 h treatment periods approaches that of the chromosome aberration test.