In Vitro Phototoxicity and Hazard Identification of Nano-scale Titanium Dioxide

Titanium dioxide nanoparticles (nano-TiO2) catalyze reactions under UV radiation and are hypothesized to cause phototoxicity. A human-derived line of retinal pigment epithelial cells (ARPE-19) was treated with six samples of nano-TiO2 and exposed to UVA radiation. The TiO2 nanoparticles were independently characterized to have mean primary particle sizes and crystal structures of 22 nm anatase/rutile, 25 nm anatase, 31 nm anatase/rutile, 59 nm anatase/rutile, 142 nm anatase, and 214 nm rutile. Particles were suspended in cell culture media, sonicated, and assessed for stability and aggregation by dynamic light scattering. Cells were treated with 0, 0.3, 1, 3, 10, 30, or 100 μg/ml nano-TiO2 in media for 24 hrs and then exposed to UVA (2 hrs, 7.53 J/cm2) or kept in the dark. Viability was assessed 24 hrs after the end of UVA exposure by microscopy with a live/dead assay (calcein-AM/propidium iodide). Exposure to higher concentrations of nano-TiO2 with UVA lowered cell viability. The 25 nm anatase and 31 nm anatase/rutile were the most phototoxic (LC50 with UVA < 5 μg/ml), while the 142 nm anatase and 214 nm rutile were the least phototoxic. An acellular assay ranked TiO2 nanoparticles for their UVA photocatalytic reactivities. The particles were found to be capable of generating thiobarbituric acid reactive substances (TBARS) under UVA. Flow cytometry showed that nano-TiO2 combined with UVA decreased cell viability and increased the generation of reactive oxygen species (ROS, measured by Mitosox). LC50 values under UVA were correlated with TBARS reactivity, particle size, and surface area.

► Nano-TiO2 enters cells within 24 hours ► Nano-TiO2 causes dose-dependent cytotoxicity greatly enhanced by UVA radiation ► Treatment with nano-TiO2 and UVA produces reactive oxygen species ► Phototoxicity is correlated with particle size, surface area, and TBARS reactivity