Miniaturized flow cytometry-based CHO-K1 micronucleus assay discriminates aneugenic and clastogenic modes of action

A well recognized advantage of the in vitro micronucleus assay is its ability to detect both aneugens and clastogens. This laboratory has previously described a flow cytometric approach for scoring in vitro micronuclei (MN)(Avlasevich et al. [2006]: Environ Mol Mutagen 47: 56–66). More recently, based on work with Chinese hamster cells, evidence has accumulated that the multiparametric data acquired by the flow cytometric process is capable of discriminating between aneugenic and clastogenic modes of action (MOA). That is, in the case of CHO-K1 cells, clastogens are observed to induce MN with minimal effects on the incidence of hypodiploid nuclei or the median size of MN (i.e., fluorescence intensity), whereas aneugens are observed to affect all three parameters. To systematically test whether these ‘‘signatures’’ are indeed reliable indicators of genotoxic MOA, CHO-K1 cells were treated with eight prototypical clastogens, eight an eugens, and 15 nongenotoxicants. Exposure was continuous (18–24 hrs) with harvest occurring immediately thereafter. Treatment and all subsequent processing and analysis steps occurred in the same 96-well plate, making this an efficient, miniaturized assay. The resulting flow cytometric MN data correlated well with expected in vitro cytogenetics: sensitivity 5 16/16, specificity 5 14/15. In addition, MOA signatures were identified that classified each of the 16 genotoxicants correctly as clastogenic or aneugenic. Taken together, these data indicate that flow cytometry represents an analytical platform that is capable of rapidly and objectively acquiring MN counts while simultaneously providing information on genotoxic MOA.