Investigation on the mechanism of non-photocatalytically TiO₂-induced reactive oxygen species and its significance on cell cycle and morphology

Titanium dioxide (TiO₂) nanoparticles are widely used in daily human life, and were reported to elicit biological effects such as oxidative stress either generating reactive oxygen species (ROS) or causing cell necrosis without generating ROS, whose underlying molecular mechanisms are not yet known. In this study, the role of dissolved oxygen in TiO₂ catalytic activity in dark environment, and long-term cytotoxic effects of TiO₂ exposure were investigated. To determine the effect of dissolved oxygen, the anatase-TiO₂ nanoparticle suspension was prepared both in deoxygenated and regular MilliQ water, and a ~ 9-fold higher ROS in regular MilliQ samples was observed compared to deoxygenated samples while in the dark, which suggested dissolved oxygen as the driving agent behind the TiO₂ catalytic reaction. On the other hand, the differential cell viability and endogenous ROS activity was demonstrated through a sensitive macrophage-based assay, on a dose- and time-dependent manner. Both the cell number and endogenous ROS activity increased with increase in time till 48 h, followed by a reduction at 72 h exposure period. Long-term exposures to these nanoparticles even at low concentrations were found detrimental to cells, where late apoptosis until 48 h and necrosis at 72 h leading to cell death were noted. Late apoptotic events and cell membrane cytoskeletal actin rearrangement observed were hypothesized to be induced by particle-mediated cellular ROS. This in addition to radical generation ability of TiO₂ in the dark will help further in better understanding of the toxicity mechanism in cells beyond ROS generation. Copyright © 2016 John Wiley & Sons, Ltd.