The classical in vitro two-stage transformation method was modified for high transformation frequency, and the promotional effect of cadmium was evaluated. In this study, we reconfirmed the usefulness of the replating method and the optimal duration time between the initiator and promoter treatments for the optimal transformation of the Balb/3T3 cells. The results also showed that subsequent exposure to CdCl2 for 2 weeks after initial exposure to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) markedly enhanced the transformation frequency. At the concentration of 360 ng/ml, the transformation frequency was increased by 35-fold that of the cultures treated only with MNNG, and was higher than that of the positive control group treated with 100 ng/ml 12-O-tetradecanoyl phorbol-13-acetate (TPA) after MNNG treatment. This transformation frequency was higher than that reported previously. Therefore, this in vitro two-stage transformation method can be used efficiently for the screening of genotoxic and non-genotoxic carcinogens and the study of multistage carcinogenesis. These results also indicate that cadmium has a strong potency as a promoter, and the promotional effect of cadmium is higher than that of TPA.
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