ras gene activation in rat tumors induced by benzidine congeners and derived dyes

Dimethoxybenzidine (DMO) and dimethylbenzidine (DM) are used to synthesize dyes such as C.I. Direct Blue 15 and C.I. Acid Red 114, respectively. These commercially used dyes are metabolically degraded to DMO or DM in the intestinal tract of rodents and subsequently DMO and DM are absorbed into the blood stream. Animals were exposed to DMO, DM, or the dyes in the drinking water. Tumors obtained from control and chemical-treated animals were examined for the presence of activated oncogenes by the NIH 3T3 DNA transfection assay. Activated oncogenes were detected in less than 3% (1/38) of the tumors from control animals whereas 68% (34/50) of the tumors from chemical-treated animals contained detectable oncogenes. Activated oncogenes were detected in both malignant (25/36) and benign (9/14) tumors from the chemically treated animals but only in one of 13 malignant tumors from the control animals. The presence of oncogenes in the chemically induced benign tumors suggests that oncogene activation was an early event in those tumors. Southern blot analysis of transfectant DNA showed that the transforming properties of the chemically induced rat tumor DNAs were due to the transfer of an activated H-ras (31/34) or N-ras (3/34) gene. One spontaneous rat tumor DNA was found to contain an activated H-ras gene. Oligonucleotide hybridization analysis indicated that the H-ras oncogenes from chemical-associated tumors contained mutations at codons 12, 13, or 61 whereas the spontaneously activated H-ras gene contained a point mutation at codon 61. These data suggest that activation of cellular ras genes by point mutation is an important step in the induction of tumors, at least in rats, by this class of benzidine-derived dyes. Moreover, in light of common histogenesis of the normal counterparts of many of the chemically induced neoplasms and histological evidence of varied tissue differentiation in some basal cell neoplasms, it is possible that most or all of the chemically induced neoplasms were derived from a common epidermal progenitor stem cell population.