Overestimation of nanoparticles-induced DNA damage determined by the comet assay

Abstract

The increasing use of engineered nanoparticles (NPs) in a wide range of commercial products raises concern about the possible risks that NPs pose to human health. Many aspects of the interaction between living cells and NPs are still unclear, and a reliable assessment of NP genotoxicity would be important. One of the most common tests used for genotoxicity is the comet assay, a sensitive method measuring DNA damage in individual cells. The assay was originally developed for soluble molecules, but it is also used in the assessment of genotoxicity of NPs. However, concerns have been raised recently about the reliability of this test in the case of NPs, but no conclusive results have been presented. Using nuclei isolated from human epithelial cells incubated with NPs, we obtained clear evidence of overestimation of NP genotoxicity by the comet assay in the case of CeO₂, TiO_2, SiO₂, and polystyrene NPs. Removal of the NPs in the cytoplasm was effective in eliminating this genotoxicity overestimation (ex post damage) and determining the actual damage produced by the NPs during incubation with the cells (ex ante damage). This method could improve significantly the determination of NP genotoxicity in eukaryotic cells.

• Genotoxicity
• nanotoxicology
• nuclei isolation
• single-nucleus gel electrophoresis assay

Acknowledgements

The authors thank Dr. Enrico Solcia and Dr. Monica Savio (University of Pavia, Department of Molecular Medicine) for the ultrastructural cell analysis and for useful insights on comet assay, respectively.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by Fondazione Cariplo (Grants no. 2011-2095).

Supplementary material available online

Supplementary Figures S1–S5