Improvement of carcinogen detection in the BALB/3T3 cell transformation assay by using a rich basal medium supplemented with low concentration of serum and some growth factors

To improve the detection sensitivity and reproducibility of the transformation assay using BALB/3T3 cells, we scrutinized a new assay method in which the cells were replated in a medium containing a low concentration of serum after carcinogen treatment. Dulbecco's modified Eagle's medium plus Ham F12 (DME.F12) supplemented with a mixture of insulin, transferrin, ethanolamine and sodium selenite (ITES) and a low concentration of fetal calf serum (FCS) caused transformation at a high frequency with a high reproducibility. The transformation frequency in the culture treated with N-methyl-N'-nitro-N-nitrosoguanidine was the highest in DME.F12 medium containing ITES and 2% FCS, and it decreased at either a lower or higher FCS concentration. Moreover, the transformation frequency was not markedly influenced by the difference in lot of FCS, probably due to reduction in FCS concentration. According to the present method, the transformation frequency was 2-times higher and transformed foci appeared much earlier than by the method using the original medium. Next, we examined some gene-and non-genotoxic carcinogens, and some genotoxic non-carcinogens to confirm the availability of this method for predicting potential carcinogens. This method could precisely identify not only genotoxic carcinogens but also non-genotoxic carcinogens and genotoxic non-carcinogens. In conclusion, these findings suggest that this method is useful for detection of chemical carcinogens because it provides high sensitivity, high reproducibility and accurate predictivity, without the requirement of 12-O-tetradecanoylphorbol 13-acetate as a promoter, making it harmless to examiner. (C) 1997 Elsevier Science B.V.