Flow Cytometric Evaluation of Nanoparticles Using Side-Scattered Light and Reactive Oxygen Species-Mediated Fluorescence-Correlation with Genotoxicity

We recently clarified that the side-scatter(ed) light (SSC) of flow cytometry (FCM) could be used as a guide to measure the uptake potential of nanoparticles [Suzuki et al. Environ. Sci. Technol. 2007, 41, 3018-3024]. In this paper, the method was improved so as to be able to determine simultaneously the uptake potential of nanoparticles and the production of reactive oxygen species (ROS), and correlations with genotoxicity were evaluated. In the FCM analysis, SSC and fluorescence of 6-carboxy-2,7'-diclorodihydro-fluorescein diacetate, di(acetoxy ester) based on ROS production were concurrently detected after treatments with ZnO, CuO, Fe3O4, TiO2, and Ag nanoparticles. The ZnO and CuO nanoparticles caused high ROS production, which was more significant in the cells with higher SSC intensity. The increase of SSC intensity was more significant for TiO2 than ZnO and CuO, whereas ROS production was higher for ZnO and CuO than TiO2, suggesting that the extent of ROS production based on the uptake of nanoparticles differed with each kind of nanoparticle. ROS production was correlated with generation of the phosphorylated histone H2AX (gamma-H2AX), a marker of DNA damage, and an antioxidant, n-acetylcysteine, could partially suppress the gamma-H2AX. This method makes it possible to predict not only uptake potential but also genotoxicity.