Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents

Abstract

Titanium dioxide nanoparticles (TiO₂-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1–50 μg/mL TiO₂-NPs, 86% anatase/14% rutile, 24 ± 6 nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO₂-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO₂-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO₂-NPs.

• Genotoxicity
• mechanistic toxicology
• nanoparticles
• TiO₂
• toxicity

Acknowledgement

The authors would like to thank Veronique Collin-Faure for flow cytometry analyses.

Declaration of interest

The authors report no conflicts of interest. The authors are responsible for the content and writing of this article.

This work was funded by CEA through the Nanoscience and Toxicology research programs, and via the European Commission’s 7th Framework Program project NanoMILE (Contract No. NMP4-LA-2013-310451). It is a contribution to the Labex Serenade (No. ANR-11-LABX-0064) funded by the “Investissements d’Avenir” French Government program of the French National Research Agency (ANR) through the A*MIDEX project (No. ANR-11-IDEX-0001-02). This research received support from the QualityNano Project (http://www.qualitynano.eu) which is financed by the European Community Research Infrastructures under the FP7 Capacities Program (Grant No. INFRA-2010-262163), and its partner University of Birmingham.

Supplementary material available online

Supplementary Tables S1–S6 and Figures S1–S3