Specific gene mutations in L5178Y cells in culture.

The predominant test system uses a near-diploid L5178Y mouse lymphoma cell line and is based on the quantitation of forward mutations occurring at the heterozygous thymidine kinase (TK) locus (TK+/- leads to TK-/-). (Other markers, such as ouabain- or methothrexate-resistance and alanine independence, in other L5178Y mouse lymphoma cells were also examined, but our criteria for the acceptability of data or the paucity of data considerably reduced the value of these mutagenesis test systems to this study.) The biochemical basis for the L5178Y/TK+/- assay depends on the ability of TK-competent cells to phosphorylate 5-bromo-2'-deoxyuridine or trifluorothymidine. The phosphorylated product or its metabolites kill these cells, thus, medium containing 5-bromo-2'-deoxyuridine or trifluorothymidine is capable of selecting for cells that are lacking the TK enzyme (TK-/-). TK-/- cells eventually give rise to a bimodal distribution of colony sizes. The relative proportion of small and large colonies appears to be characteristic of the mutagen, its dose, and the length of the expression period. A total of 108 references were reviewed and 48 chemical agents were evaluated. Of these, in vivo carcinogenicity data existed for 44 and covered a wide variety of chemical classes (43 compounds) and a complex mixture. In this system, 39 agents were positive, 1 was negative, and 4 yielded inconclusive results. The 44 test substances evaluated were insufficient to single out agents or agent classes for which the assay was particularly well suited; however, with the exception of thymidine analogs, the system seems to be versatile. The correlation of the TK locus assay results with the carcinogenicity data revealed that 2 agents were definite false positives (sodium azide and methotrexate) and 1 agent was a definite false negative (1,1-dimethylhydrazine). Further evaluation suggested that 4-acetylaminofluorene and diphenylnitrosamine were questionable false positives, while benzo[e]pyrene was a questionable false negative. (The term questionable was used to imply uncertainties concerning the mutagenicity and/or carcinogenicity data). Thus, the assay is of value in the battery approach to mutagenicity/carcinogenicity screening.