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.

Significance: Embryonic development is one of the most amazing miracles in nature. The proteins and signaling events driving this highly complex process are far from being elucidated completely. For a long time, an important role of protein reduction and oxidation during development has been assumed. Here, we demonstrate the essential role of such a regulation during cardiovascular development: The modification of a single cysteine in the protein sirtuin 1 by the vertebrate-specific oxidoreductase glutaredoxin 2 is required for vessel formation and guidance. Our data indicate that this redox-signaling pathway based on glutaredoxin-dependent reversible S -glutathionylation may be also important for diseases of the cardiovascular system and pathological situations connected to angiogenesis, e.g., malignancies.