To precise the location of PS-NH2 nanobeads within and/or around Calu-3 cells, we also performed confocal fluorescence microscopy analysis. We confirmed that PS-NH2 nanobeads were mainly located around Calu-3 cell is lets at 1 and 2h, faintly detected at 4 h, and again detected at 24h. Similar experiments were performed on THP-1 macrophages and showed that PS-NH2 nanobeads were effectively internalized and mainly located in the cytoplasm rather than in the nuclei, corroborating data from the literature. We then investigated the genotoxic effects of these nanobeads GW7647 together with reduced GSH dosage, as genotoxicity and oxidative stress could be related. The GSH dosage method was chosen rather than the ROS approach due to strong interferences observed between PS nanobeads fluorescence and CM-H2DCFDA or Mitosox probes. Indeed, the excitation and emission wave lengths of PS nanobeads and ROS GSK0660 probes are very close and prevents to accurate study measurements. In order to study genotoxicity we analyzed ��-H2Axfoci, which is a very sensitive method to detect DNA double strand breaks and recently described as a powerful method to predict in vivo genotoxicity. As seen in Table2, NPs functionalization strongly impacted genotoxicity, however with variation depending on the dose of PS nanobeads. PS-NF nanobeads did not significantly induce genotoxicity for concentrations up to 8.1 ��g/cm2 in both cell lines. PS-COOH nanobeads were not genotoxic for Calu-3 cells but genotoxic for THP-1 macrophages. These data should be interpreted together with PS nanobeads uptake since PS-COOH were detected into macrophages and faintly in Calu-3 cells. However, we found that PS-NH2 nanobeads induced DNA double strand breaks while being either mainly around or within the cells. In addition, it was published that NPs were able to generate oxidative stress, which can lead to ROS generation and GSH depletion. NH2 functionalization was shown to lead to the highest GSH depletion both on Calu-3 cells and THP-1 macrophages.Interestingly, these results are in complete agreement with ��-H2Ax-foci results, since the highest number of DNA double strand breaks was observed after PS-NH2 nanobeads exposure.