New methodological developments for testing the in vitro genotoxicity of nanomaterials: Comparison of 2D and 3D...

Nanomaterials (NMs) are defined as materials with at least one external dimension below 100 nm. Their small size confers them interesting unique physico-chemical properties, hence NMs are increasingly used in a diversity of applications. However, the specific properties of NMs could also make them more harmful than their bulk counterparts. Therefore, there is a crucial need to deliver efficient NM hazard assessment in order to sustain the responsible development of nanotechnology. This study analysed the genotoxic potential of several NMs: one titanium dioxide (TiO₂) and two zinc oxide NMs (ZnO) that were tested up to 100 μg/mL on 2D and 3D hepatic HepaRG models. Genotoxicity analysis was performed comparing the alkaline comet assay in classical and high throughput formats. Moreover, oxidative DNA lesions were investigated with the Fpg-modified comet assay. Results showed that TiO₂ NMs were not cytotoxic and not genotoxic in either cell model, although a small increase in the % tail DNA was observed in 3D HepaRG cells at 100 μg/mL in the classical format. The two ZnO NMs (ZnO S. NMs a commercial suspension and NM110 provided by the European Union Joint Research Centre) induced a concentration-dependent increase in cytotoxicity that was more pronounced in the 2D (>20% cytotoxicity was observed for ZnO S. at concentrations greater than 25 μg/mL, and for NM 110 at 50 μg/mL) than in the 3D model (more than 20% cytotoxicity for ZnO S. NMs at 50 μg/mL). While ZnO S. NMs induced DNA damage associated with cytotoxicity (at 25 and 50 μg/mL in 2D and 50 μg/mL in 3D), NM110 showed a clear genotoxic effect at non-cytotoxic concentrations (25 μg/mL in 2D and at 25 and 50 μg/mL in 3D). No major differences could be observed in the comet assay in the presence or absence of the Fpg enzyme. High throughput analysis using CometChip® mostly confirmed the results obtained with the classical format, and even enhanced the detection of genotoxicity in the 3D model. In conclusion, this study demonstrated that new approach methodologies (NAMs), 3D models and the high throughput format for the comet assay, were more efficient in the detection of genotoxic effects, and are therefore promising approaches to improve hazard assessment of NMs.