EVALUATION OF THE IN-VIVO GENOTOXIC POTENTIAL OF 3 CARCINOGENIC AROMATIC-AMINES USING THE BIG-BLUE(TM) TRANSGENIC MOUSE MUTATION ASSAY

Three genotoxic mouse carcinogens, 4-chloro-o-phenylenediamine (4-C-o- PDA), 2-nitro-p-phenylenediamine (2-N-p-PDA), and 2,4-diaminotoluene ( 2,4-DAT), were tested in the Big Blue(TM) transgenic mouse mutation as say. Each experiment consisted of a vehicle control group with ten Big Blue(TM) C57BL/6 mice, five of either sex, and an equally sized group treated with a high dose of the test chemical. In addition, four anim als were treated with the vehicle and six animals with the test compou nd For the measurement of bromodeoxyuridine (BrdU) incorporation to de termine cellular proliferation. Prior to the mutagenicity experiments, the maximally tolerated dose of each compound was determined using no ntransgenic C57BL/6 mice. Based on these results the doses used in the main study were 200 mg/kg/day for 4-C-o-PDA, 150 mg/kg/day for 2-N-p- PDA, and 80 mg/kg/day For 2,4-DAT. Animals were treated for 10 days ov er a 2 week period and were killed 10 days after the last treatment. i n an additional experiment with 2,4-DAT, animals were killed 28 days a fter treatment. Since all three chemicals are liver carcinogens in the mouse, the DNA of the liver was analyzed using the standard procedure s for the Big Blue(TM) assay. Hepatocyte proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen ( PCNA) and, in some studies, by measuring BrdU incorporation. 4-C-o-PDA and 2-N-p-PDA did not induce an increase in PCNA expression when meas ured 10 days after the last treatment. There was no increase in BrdU i ncorporation immediately after treatment with 4-C-o-PDA or with 2,4-DA T. However, 10 days after the last treatment with 2,4-DAT, a strong mi togenic effect was found with both techniques, i.e., in the PCNA and B rdU assays. 4-C-o-PDA, a liver carcinogen in both genders of mice, ind uced a small, statistically significant increase of the mutant frequen cies in females. No increase was found in males. 2-N-p-PDA, which has been reported to induce liver tumors only in females, was Found positi ve in males and was clearly negative in females. 2,4-DAT, a liver carc inogen in female mice, was positive in females and negative in males w hen the animals were killed 10 days after the last treatment. After an expression time of 28 days, 2,4-DAT induced a statistically significa nt increase in both sexes. The effect in females was marginally strong er than after 10 days' expression time and almost identical to the eff ect observed in males under these test conditions. In conclusion, the experiments showed that the Big Blue(TM) assay detects the genotoxicit y of the three carcinogenic monocyclic aromatic amines tested. However , it seems that the sex specificity of the carcinogenic effects of the se compounds is not reflected by the mutagenicity data in Big Blue(TM) mice. (C) 1996 Wiley-Liss, Inc.