Aging-independent and size-dependent genotoxic response induced by titanium dioxide nanoparticles in mammalian cells

Titanium dioxide nanoparticles (TiO₂ NPs) are subjected to various transformation processes (chemical, physical and biological processes) in the environment, potentially affecting their bioavailability and toxic properties. However, the size variation of TiO₂ NPs during aging process and subsequent effects in mammalian cells are largely unknown. The aim of this study was to illustrate the adverse effects of TiO₂ NPs in different sizes (5, 15 and <100 nm) during aging process on human-hamster hybrid (A_L) cells. There was an aging-time dependent enhancement of average hydrodynamic size in TiO₂ NPs stock suspensions. The cytotoxicity of fresh TiO₂ NPs increased in a size-dependent manner; in contrast, their genotoxicity decreased with the increasing sizes of NPs. No significant toxicity difference was observed in cells exposed to either fresh or 60 day-aged TiO₂ NPs. Both Fresh and aged TiO₂ NPs efficiently induced mitochondrial dysfunction and activated Caspase-3/7 in a size-dependent manner. Using mitochondrial-DNA deficient (ρ⁰) A_L cells, we further discovered that mitochondrial dysfunction made significant contribution to the size-dependent toxicity induced by TiO₂ NPs during the aging process. Taken together, our data indicated that TiO₂ NPs could significantly induced the cytotoxicity and genotoxicity in an aging time-independent and size-dependent manner, which were triggered by mitochondrial dysfunction. Our study suggested the necessity to include size as an additional parameter for the cautious monitoring of TiO₂ NPs disposal before entering the environment.