Integration of in vivo genotoxicity and short-term carcinogenicity assays using F344 gpt delta transgenic rats: in vivo mutagenicity of 2,4-diaminotoluene and 2,6-diaminotoluene structural isomers

Abstract

An important trend in current toxicology is the replacement, reduction, and refinement of the use of experimental animals (the 3R principle). We propose a model in which in vivo genotoxicity and short-term carcinogenicity assays are integrated with F344 gpt delta transgenic rats. Using this model, the genotoxicity of chemicals can be identified in target organs using a shuttle vector lambda EG10 that carries reporter genes for mutations; short-term carcinogenicity is determined by the formation of glutathione S-transferase placenta form (GST-P) foci in the liver. To begin validating this system, we examined the genotoxicity and hepatotoxicity of structural isomers of 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT). Although both compounds are genotoxic in the Ames/Salmonella assay, only 2,4-DAT induces tumors in rat livers. Male F344 gpt delta rats were fed diet containing 2,4-DAT at doses of 125, 250, or 500 ppm for 13 weeks or 2,6-DAT at a dose of 500 ppm for the same period. The mutation frequencies of base substitutions, mainly at G:C base pairs, were significantly increased in the livers of 2,4-DAT-treated rats at all three doses. In contrast, virtually no induction of genotoxicity was identified in the kidneys of 2,4-DAT-treated rats or in the livers of 2,6-DAT-treated rats. GST-P-positive foci were detected in the livers of rats treated with 2,4-DAT at a dose of 500 ppm but not in those treated with 2,6-DAT. Integrated genotoxicity and short-term carcinogenicity assays may be useful for early identifying genotoxic and nongenotoxic carcinogens in a reduced number of experimental animals.

FIG. 1.

Histological comparison of rat livers treated with 0 ppm 2,4-DAT (A), 125 ppm 2,4-DAT (B), 250 ppm 2,4-DAT (C), 500 ppm 2,4-DAT (D), 500 ppm 2,6-DAT (E), and DEN (F). Hepatotoxicity was observed in rats administered 2,4-DAT and DEN. Bar = 100 μm.

FIG. 2.

Mutagenic activity of 2,4-DAT (A) and 2,6-DAT (B) in Salmonella typhimurium strains TA98 (circle) and YG1024 (rhombus). Filled circle and rhombus assayed with S9 mix; open circle and rhombus assayed without S9 mix.

FIG. 3.

MFs of gpt genes. Values represent mean SD (n = 5). Significant differences were observed in 2,4-DAT–treated livers compared to livers from rats fed negative control basal diet. *p < 0.05.