The aromatic amines 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) are structural isomers that have been extensively studied for their mutagenic and carcinogenic characteristics. Both compounds are equally mutagenic in the Ames/Salmonella assay in the presence of S9. However, the differences in the results of chronic rodent carcinogen bioassays using these two compounds are significant, in that 2,4-DAT is a potent hepatocarcinogen, whereas 2,6-DAT does not produce an increased incidence of tumors in rats or mice at similar doses. The Big Blue transgenic B6C3F1 mouse carries multiple copies of bacteriophage lambda, each with a lacI mutational target gene, integrated into mouse chromosome 4. Our studies were designed to determine whether the Big Blue system could be used to detect differences in the in vivo mutagenic activity between the carcinogen/non-carcinogen pair 2,4- and 2,6-DAT and to determine whether the in vivo mutagenesis assay results correspond to the rodent carcinogen bioassay results. Male B6C3F1 transgenic mice were exposed to 2,4- or 2,6-DAT at 0 or 1000 p.p.m. in the diet for 30 and 90 days. Mice serving as positive controls were administered five daily i.p. injections of 6 mg/kg dimethylnitrosamine (DMN) in saline and were sacrificed 15 days following the last injection. Mutant frequencies at lacI were determined by recovering the genomically integrated lambda phage using an in vitro packaging reaction followed by infection of an appropriate Escherichia coli host. Complete non-sectored blue mutant plaques were scored against a background of clear non-mutant plaques. Mutant frequencies were nearly identical for all three groups at 30 days, while at 90 days the mutant frequency for the hepatocarcinogen 2,4-DAT (12.1 +/- 1.4 x 10(-5)) was significantly higher (P < 0.01) as compared with both age-matched (spontaneous) controls (5.7 +/- 2.9 x 10(-5)) and the 2,6-DAT-exposed group (5.7 +/- 2.4 x 10(-5)). Mutations at lacI arising ex vivo during replication in E. coli are observed in this system as sectored blue plaques. The sectored plaque frequency in this study was constant across all groups at approximately 9.0 x 10(-5). Results from this study demonstrate that the Big Blue transgenic mutation assay: (i) can distinguish differences in vivo between the mutagenic responses of a carcinogen and a non-carcinogen which elicited comparable mutagenic activity in S.typhimurium; (ii) is sensitive to mutagens through subchronic dietary exposure; and (iii) yields a differential response depending upon the length of time mice are exposed to a mutagen.

The glutathione S-transferases (GSTs) are a family of isoenzymes involved in the detoxication of a variety of electrophilic xenobiotics. The present investigation demonstrates that GST activity and the concentration of cytosolic GSTs in cerebellar cortex of Gunn rats were increased in hyperbilirubinaemic animals compared with non-jaundiced controls. Age-dependent and region-specific increases in GST isoenzymes were seen in three regions of the cerebellar cortex of jaundiced Gunn rats, whereas GST concentrations were not altered in the brainstem, thalamus/hypothalamus, cortex or liver. Cytosolic GST activity was increased 1.3-fold in the flocculus and lateral hemispheres of 20-day-old and 1.7-fold in the flocculus, lateral hemispheres and vermis of 60-day-old jaundiced (jj; homozygous) Gunn rats compared with non-jaundiced (Jj; heterozygous) Gunn rats. H.p.l.c. was used to determine the GST subunit protein concentrations in cytosolic fractions isolated from liver and brain regions of jaundiced and non-jaundiced animals. In all regions of the cerebellum from 20-day-old animals, the levels of Alpha-class GST subunits 2 (Yc1; 3.0-fold) and 8 (Yk; 2.0-fold) were increased in jaundiced rats. In 60-day-old animals, the concentrations of Alpha-class GST subunits 2 (Yc1; 5.0-fold) and 8 (Yk; 3.0-fold), Mu-class subunit 11 (Yo; 2.5-fold) and Pi-class subunit 7 (Yp; 2.0-fold) were increased in all regions of cerebellar cortex of jaundiced animals. In cerebellum of 10-, 20- and 60-day-old non-jaundiced and jaundiced Gunn rats, the flocculus had the highest concentration of Mu-class GST subunit 4 (Yb2) and vermis the lowest; hyperbilirubinaemia increased the concentration of subunit 4 (Yb2; 3- to 5-fold) in the flocculus and lateral hemispheres, but not the vermis, of 20- and 60-day-old rats. Intraperitoneal injection of sulphadimethoxine, a long-acting sulphonamide which displaces bilirubin from its albumin-binding sites and increases the bilirubin levels in tissues, further increased the already elevated concentrations of GST subunits in the lateral regions of cerebellar cortex of hyperbilirubinaemic rats. For example, the concentration of subunit 4 (Yb2) was increased 2.2-fold (compared with non-jaundiced controls) in Gunn rats injected with saline and 7.4-fold in rats injected with 100 mg of sulphadimethoxine/kg body weight. In contrast, GSTs in the vermis of jaundiced animals were not affected by sulphadimethoxine injection. Sulphadimethoxine had no effect on GST concentrations in lateral regions and vermis of heterozygous (Jj) Gunn rats.(ABSTRACT TRUNCATED AT 400 WORDS)

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The aromatic amines 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) are structural isomers that have been extensively studied for their mutagenic and carcinogenic characteristics. Both compounds are rapidly absorbed after oral administration and are equally mutagenic in the Ames test; however, 2,4-DAT is a potent hepatocarcinogen, whereas 2,6-DAT does not produce an increased incidence of tumors in rats or mice at similar doses. The Big Blue transgenic B6C3F1 mouse carries multiple copies of the lacl mutational target gene. Our studies were designed to determine whether the Big Blue system could be used to detect differences in the vivo mutagenic activity between the carcinogen-noncarcinogen pair 2,4-DAT and 2,6-DAT and to determine whether the in vivo mutagenesis assay results correspond to the rodent carcinogen bioassay results. Male B6C3F1 transgenic mice were exposed to 2,4-DAT or 2,6-DAT at 0 or 1,000 ppm in the diet for 30 and 90 days or to dimethylnitrosamine as a positive control. Mutant frequencies were nearly identical for all three groups at 30 days, while at 90 days the mutant frequency for the hepatocarcinogen 2,4-DAT (12.1 +/- 1.4 x 10(-5)) was significantly higher (p < 0.01) as compared to both age-matched (spontaneous) controls (5.7 +/- 2.9 x 10(-5)) and the 2,6-DAT-exposed group (5.7 +/- 2.4 x 10(-5)). Results from this study demonstrate that the Big Blue transgenic mutation assay can distinguish differences in vivo between the mutagenic responses of hepatic carcinogens ad a noncarcinogen; is sensitive to mutagens through subchronic dietary exposure; and yields a differential response depending upon the length of time mice are exposed to a mutagen.

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