Experimental evidence of false-positive Comet test results due to TiO2 particle--assay interactions

We have studied the genotoxicity of TiO2 particles with a Comet assay on a unicellular organism, Tetrahymena thermophila. Exposure to bulk- or nano-TiO2 of free cells, cells embedded in gel or nuclei embedded in gel, all resulted in a positive Comet assay result but this outcome could not be confirmed by cytotoxicity measures such as lipid peroxidation, elevated reactive oxygen species or cell membrane composition. Published reports state that in the absence of cytotoxicity, nano- and bulk-TiO2 genotoxicity do not occur directly, and a possible explanation of our Comet assay results is that they are false positives resulting from post festum exposure interactions between particles and DNA. We suggest that before Comet assay is used for nanoparticle genotoxicity testing, evidence for the possibility of post festum exposure interactions should be considered. The acellular Comet test described in this report can be used for this purpose.