Development of a repeated-dose liver micronucleus assay using adult rats (II): Further investigation of 1,2-dimethylhydrazine and 2,6-diaminotoluene

Abstract

Detecting genotoxicity in the liver is considered an effective approach for predicting hepatocarcinogenicity, as many genotoxic chemicals in vivo may act as hepatocarcinogens in rodents. Here, a genotoxic rodent hepatocarcinogen, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), and a genotoxic (Ames positive) noncarcinogen, 2,6-diaminotolunene (2,6-DAT), were administered orally to rats for up to 28 days, and liver samples were then examined in a repeated-dose liver micronucleus (MN) assay, and additionally tested in the bone marrow (BM) MN assay concurrently. We recently established a simple method to isolate hepatocytes without in situ liver perfusion procedures, and applied this method in the liver MN assay. As a result, 1,2-DMH increased the proportion of micronucleated hepatocytes in both a dose- and duration-dependent manner at relatively low-dose levels that are routinely used in repeated-dose toxicity studies. In contrast to 1,2-DMH, 2,6-DAT did not have a detectable effect. In addition to these two chemicals, two genotoxic rodent hepatocarcinogens, diethylnitrosamine and 2,4-diaminotoluene, which gave positive responses in the liver MN assay in our previous investigation [Narumi et al., Mutat. Res. 747 (2012) 234-239], were subjected to the BM MN assay and histopathological evaluation. All four test chemicals gave negative responses in the BM MN assay. Furthermore, the three hepatocarcinogens displayed hepatotoxicity, including hepatocellular hypertrophy and anisokaryosis, but no abnormal findings were observed in the liver of rats treated with 2,6-DAT. Taken together, the present results indicate that the liver MN assay is effective for predicting hepatocarcinogenicity and may be integrated into repeated-dose toxicity studies without disturbing routine examinations, such as histopathology. Furthermore, with repeat-dose treatment protocols, our findings indicate that the liver MN assay is superior to the BM MN assay for detecting genotoxic or carcinogenic chemicals in rats.