Genotoxicity of TiO₂nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry

Abstract

The widespread production and use of nanoparticles calls for faster and more reliable methods to assess their safety. The main aim of this study was to investigate the genotoxicity of three reference TiO2 nanomaterials (NM) within the frame of the FP7-NANoREG project, with a particular focus on testing the applicability of mini-gel comet assay and micronucleus (MN) scoring by flow cytometry. BEAS-2B cells cultured under serum-free conditions were exposed to NM100 (anatase, 50–150nm), NM101 (anatase, 5–8nm) and NM103 (rutile, 20–28nm) for 3, 24 or 48h mainly at concentrations 1–30 μg/ml. In the mini-gel comet assay (eight gels per slide), we included analysis of (i) DNA strand breaks, (ii) oxidised bases (Fpg-sensitive sites) and (iii) light-induced DNA damage due to photocatalytic activity. Furthermore, MN assays were used and we compared the results of more high-throughput MN scoring with flow cytometry to that of cytokinesis-block MN cytome assay scored manually using a microscope. Various methods were used to assess cytotoxic effects and the results showed in general no or low effects at the doses tested. A weak genotoxic effect of the tested TiO2 materials was observed with an induction of oxidised bases for all three materials of which NM100 was the most potent. When the comet slides were briefly exposed to lab light, a clear induction of DNA strand breaks was observed for the anatase materials, but not for the rutile. This highlights the risk of false positives when testing photocatalytically active materials if light is not properly avoided. A slight increase in MN formation for NM103 was observed in the different MN assays at the lower doses tested (1 and 5 μg/ml). We conclude that mini-gel comet assay and MN scoring using flow cytometry successfully can be used to efficiently study cytotoxic and genotoxic properties of nanoparticles.

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells

“…For micronucleus analysis, 2% phytohemagglutinin (Murex, Dartford, Kent, UK) was added to stimulate lymphocyte proliferation. To reduce the TiO 2 photocatalytic activity (Di Bucchianico et al, ) that can create extracellular ROS, we kept particle suspensions and PBMC cultures after treatment, in dark conditions.…”

“…To reduce the TiO 2 photocatalytic activity (Di Bucchianico et al, 2017) that can create extracellular ROS, we kept particle suspensions and PBMC cultures after treatment, in dark conditions.…”

Genotoxic and mutagenic properties of Ni and NiO nanoparticles investigated by comet assay, γ‐H2AX staining, Hprt mutation assay and ToxTracker reporter cell lines

“…The cells were washed with PBS (Gibco) and harvested with trypsin (Gibco) and then processed for the comet assay. The mini‐gel comet assay as previously described [Di Bucchianico et al, ] was used and cells exposed to hydrogen peroxide (0.05 M) for 5 min on ice were used as a positive control. In short, following exposure, cells were embedded in 1.1% agarose mini‐gels on pre‐coated (0.5% agarose) microscope slides.…”

Effect of the Compositions on the Biocompatibility of New Alumina–Zirconia–Titania Dental Ceramic Composites

“…Their results, based on DNA damage, mutagenicity, and cancerogenetic potential in mammalian cells, confirmed that ZTA material could be considered suitable for biomedical applications. The addition of titania could induce non-significant genotoxic effects [50][51][52]. However, as observed by Lu et al [53], who studied the effects of titania on the induction of sister chromatid exchanges and micronuclei in Chinese hamster ovary-K1 cells, the titania could have a potentially genotoxic effect.…”