BALB/c 3T3細胞を用いる形質転換試験の改良
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The in vitro cell transformation assay utilizing BALB/c 3T3 cells is regarded as a useful method for screening potential carcinogens. In spite of the expectation, however, its use for screening carcinogens has not been fully exploited. It is evident that some difficulties still exist in employing it as a routine procedure, and for this reason we considered it important to attempt further improvement of the method. In the present study, we examined basal culture conditions of the transformation assay using DME/F12 medium plus insulin-transferrin-ethanolamine-sodium selenite(ITES)and FCS, and optimal duration and timing of 12-O-tetradecanoylphorbol-13-acetate(TPA)addition to achieve transformation. The results showed that the use of T medium(modified DME/F-12)or DME/F12 supplemented with 1% ITES plus 2% FCS resulted in a high transformation frequency, but also well supported the growth of transformed foci. Transformation frequency was highest when TPA was started from 1 week after N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)treatment. Optimal duration of TPA-exposure on MNNG-initiated cells was at least 11 days. In addition, we tested several typical carcinogens and promoters in order to confirm the applicability of the new protocol to the two-stage transformation assay. MNNG and benzo[a]pyrene but not non-carcinogenic benzo[e]pyrene induced transformation foci. With the addition of a metabolic activation system, dimethylnitrosamine was a potent inducer of transformation. Okadaic acid and other tumor promoters enhanced transformation of MNNG-initiated cells. A collaborative study of the transformation assay was performed by a subgroup of the Non-Mutagenic Carcinogen Study Group, which is a suborganization of the Environmental Mutagen Society of Japan. The results showed that the performance characteristics of this transformation assay were very similar between laboratories.
- 日本環境変異原学会の論文
- 1998-07-21
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