Application of the DNA adductome approach to assess the DNA-damaging capability of in vitro micronucleus test-positive compounds

Abstract

The in vitro micronucleus (MN) test is widely used for screening genotoxic compounds, but it often produces false-positive results. To consider the significance of positive results, it is important to know whether DNA adducts are formed in the cells treated with the test compound. Recently, Matsuda et al. developed the DNA adductome approach to detect DNA adducts comprehensively ([4] Kanaly, et al., Antioxid. Redox Signal., 2006, 8, 993-1001). We applied this method to assess the DNA-damaging capability of in vitro MN test-positive compounds. CHL/IU cells were treated with compounds from three categories: (1) carcinogens causing DNA alkylation, ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; (2) carcinogens producing DNA bulky adducts, 2-amino-6-phenyl-1-methylimidazo[4,5-b]pyrene, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and 4-nitroquinoline-1-oxide, and (3) non-carcinogens, caffeine, maltol, and sodium chloride, with or without metabolic activation. With the conditions in which all test compounds gave positive results in the MN tests, DNA was extracted from the cells and hydrolyzed to deoxyribonucleosides, which were subsequently subjected to LC/ESI-MS/MS analysis. All carcinogens (categories 1 and 2) produced various DNA adduct peaks, and some of the m/z peak values corresponded to known adducts. No non-carcinogens produced DNA adducts, indicating that these compounds produced MN through different mechanisms from the adduct formation. These results indicate that the adductome approach is useful to demonstrate DNA damage formation of MN test-positive compounds and to understand their mechanisms of action.