Titanium dioxide particles phosphorylate histone H2AX independent of ROS production

► TiO 2 particles generated γ-H2AX, which was more remarkable with smaller particles. ► The generation of γ-H2AX was independent of cell cycle phases. ► ROS formation by TiO 2 particles was not involved in the generation of γ-H2AX. ► The surface condition of TiO 2 particles would be an important factor in the generation of γ-H2AX. With the rising use of nano-sized particles in nanotechnology, harmful effects of TiO 2 particles which have been recognized as a safe material, are of increasing concern. In this study, we examined the genotoxicity of two different sized TiO 2 particles in the lung adenocarcinoma epithelial cell line A549 based on the phosphorylation of histone H2AX (γ-H2AX), recently regarded as a sensitive marker for DNA damage. TiO 2 particles generated γ-H2AX, which was more remarkable with the smaller particles. Flow cytometric analysis showed that the generation was independent of cell cycle phases and cells which incorporated larger amounts of TiO 2 particles had more significant γ-H2AX. Although there are some reports that the incorporation of nanomaterials into cells generates reactive oxygen species (ROS), the level of ROS was low even if large amounts of TiO 2 particles were taken-up. In addition, the generation of γ-H2AX by TiO 2 particles was more significant than that after treatment with hydrogen peroxide. On the other hand, the generation of γ-H2AX was attenuated by coating the surface of TiO 2 particles with bovine serum albumin. These results suggested that smaller TiO 2 particles were easy to incorporate into cells and generated cell cycle phase-independent γ-H2AX, which was dependent on the condition of the TiO 2 surface but not on the formation of ROS.