Improvement of carcinogen identification in BALB/3T3 cell transformation by application of a 2-stage method

The present studies intend to heighten the sensitivity of BALB/3T3 cells to chemical carcinogens in a transformation assay, by including exposure of carcinogen-treated cells to a tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA). In the assay, cells were first treated with a known or suspected carcinogen for 72 h, cultured in normal medium for 3 days, exposed to media with and without TPA for 2 weeks, and cultured in normal medium for an additional 3 weeks. Benzo[a]pyrene, a potent carcinogen with a polycyclic aromatic hydrocarbon structure, caused transformation in the presence and absence of TPA. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), a carcinogen with direct-acting alkylating ability, did not induce significant transformation without TPA, while treatment with MNNG followed by TPA produced numerous transformed foci, classifying MNNG as an initiating agent of transformation under the condition presented in this report. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), sodium nitrite and butylated hydroxyanisole (BHA), which are carcinogenic and/or mutagenic, produced transformed foci in significant numbers of treated dishes in the presence but not in the absence of TPA. Butylated hydroxytoluene (BHT) and sodium saccharin, which are considered to be a modifier and a promoter of carcinogenesis, did not cause significant transformation with or without TPA treatment. These studies suggest that this 2-stage transformation system is capable of detecting a wider range of chemical carcinogens as initiating agents than the standard assay. Studies on the transformation assay schedule revealed that the proportion of dishes with foci, the number of foci per dish and sizes of foci all increased in the normal medium after the termination of TPA treatment. Therefore, transformed cells appear to proliferate independently of TPA after those cells are released by TPA from postconfluence inhibition of cell division.