Integration of the rat liver micronucleus assay into a 28-day treatment protocol: testing the genotoxicity of four small-molecule nitrosamines with different carcinogenic potencies and tumor target specificities

Several potent carcinogenic nitrosamines, including N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA), induce micronuclei in the micronucleated hepatocyte (MNHEP) assay but not in the micronucleated reticulocyte (MNRET) assay. However, the MNHEP assay is not as frequently used as the MNRET assay for evaluating in vivo genotoxicity. The present study evaluated MN formation in the liver of Big Blue transgenic rats exposed to four small-molecule nitrosamines, NDMA, N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisoporpylamine (NEIPA), and N-nitrosomethylphenylamine (NMPA), using a repeat-dose protocol typically used for in vivo mutagenicity studies. NDMA is a potent liver carcinogen, while NDIPA and NEIPA are relatively weak liver carcinogens, and NMPA primarily produces esophageal tumors. Seven-week-old rats were treated with the nitrosamines for 28 consecutive days; liver was harvested three days after the last dose and used for conducting the flow-cytometry-based MNHEP assay. All four nitrosamines induced dose-dependent increases in %MNHEP and the magnitude of the responses correlated with their carcinogenicity in rat liver. These results indicate that the flow-cytometry-based MNHEP assay can be successfully integrated into 28-day repeat-dose studies, and that the MNHEP assay may be useful for evaluating the genotoxicity of nitrosamines that have different carcinogenic potencies and different tumor target specificities.