agar and Leukemia-L5178

Topics: Agar; Aneuploidy; Animals; Chromosome Aberrations; Cloning, Molecular; Leukemia L5178; Lymphoma; Mice; Mutagenicity Tests; Mutagens; Polymorphism, Restriction Fragment Length; Time Factors

The L5178Y tk +/- mouse lymphoma assay (MLA) has been validated as a sensitive and specific test system for the detection of mutagens/clastogens. There are currently two methodologies for performing the MLA: the original soft agar procedure and the newer microtitre procedure. While both procedures are considered acceptable, a limited amount of comparative information exists for the two methods. In this report the two methods were compared with regard to: (1) spontaneous and induced mutant frequencies; (2) cloning efficiencies; and (3) colony size distributions for mutants. In addition, small and large mutant colonies from microtitre wells were rechallenged for trifluorothymidine (TFT) resistance. In a majority of the cases, cloning efficiency values were higher for the microtitre as were the spontaneous and induced mutation frequency (MF) values. Nevertheless, when responses were compared according to mutation index (fold increase over background MF) the results from the two systems were often similar. More spontaneous small colonies were observed in the microtitre assay. While colony size distribution for induced mutant colonies was compound specific, generally, more small colonies were counted in microtitre. All mutant clones that were rechallenged with TFT demonstrated resistance. Aside from the differences mentioned above, both the microtitre and the soft agar procedures appear equally capable of identifying mutagenic agents.

Topics: Agar; Animals; Cell Division; Clone Cells; Culture Techniques; Drug Resistance, Neoplasm; Leukemia L5178; Mice; Mutagenicity Tests; Mutagens; Reproducibility of Results; Sensitivity and Specificity; Thymidine Kinase; Trifluridine; Tumor Cells, Cultured

Microtitration methods for assaying cell survival and mutation frequency to ouabain resistance, 6-thioguanine resistance and 1-beta-D-arabinofuranosyl cytosine resistance in L5178Y mouse lymphoma cells were compared to the standard agar cloning technique. The two methods gave essentially similar results for untreated cells, and after treatment with ethyl methanesulphonate and 4-nitroquinoline 1-oxide. Potential advantages of the microtitration method as a routine assay system are discussed.

Topics: Agar; Animals; Cell Survival; Clone Cells; Culture Media; Culture Techniques; Cytarabine; Leukemia L5178; Leukemia, Experimental; Mice; Mutation; Thioguanine