Intracellular Generation of Superoxide by TiO₂ Nanoparticles Decreases Histone Deacetylase 9 (HDAC9), an Epigenetic Modifier.

Titanium dioxide (TiO₂) nanoparticles are used on a massive scale in commercial and industrial products. Of specific concern is how the inhalation of these nanoparticles in a manufacturing setting may affect human health. We examine the cellular response to TiO₂ nanoparticles using a combination of cell-free spectroscopic assays, fluorescence microscopy, Western blotting, and TiO₂ nanoparticle surface modifications. These experiments show that TiO₂ nanoparticles generate superoxide, both in solution and in cells, and this intracellular superoxide decreases expression of histone deacetylase 9 (HDAC9), an epigenetic modifier. We use protein coronas formed from superoxide dismutase (SOD) and catalase, enzymes that scavenge reactive oxygen species (ROS), to probe the relationship between TiO₂ nanoparticles, ROS, and the subsequent cellular response. These protein coronas provide nanoparticle-localized scavengers that demonstrate that the nanoparticles are the source of the intracellular superoxide. Importantly, the use of a SOD corona or surface passivated TiO₂ nanoparticles prevents the decrease of HDAC9. These experiments elucidate the underlying mechanism of TiO₂ nanoparticle-mediated cellular responses including oxidative stress and changes in gene expression. They also provide the first demonstration of a protein corona as a tool for probing cellular responses to nanoparticles. Overall, this research shows that low, nontoxic concentrations of TiO₂ nanoparticles alter an enzyme responsible for epigenetic modifications, which points to concerns regarding long-term exposures in manufacturing settings.