title: Genotoxicity of TiO2 nanoparticles 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 TiO2nanomaterials (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 TiO2materials 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.
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 171 77 Stockholm, Sweden and.
authors.forename: Sebastiano
authors.initials: S
authors.lastname: Di Bucchianico
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 171 77 Stockholm, Sweden and.
authors.forename: Francesca
authors.initials: F
authors.lastname: Cappellini
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 171 77 Stockholm, Sweden and.
authors.forename: Florane
authors.initials: F
authors.lastname: Le Bihanic
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 171 77 Stockholm, Sweden and.|Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, School of Chemical Science and Engineering, Teknikringen 42, 100 44 Stockholm, Sweden.
authors.forename: Yuning
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 171 77 Stockholm, Sweden and.
authors.forename: Kristian
authors.initials: K
authors.lastname: Dreij
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 171 77 Stockholm, Sweden and Hanna.L.Karlsson@ki.se.
authors.forename: Hanna L
authors.initials: HL
authors.lastname: Karlsson
mesh_terms: D001980:Bronchi / Q000187:drug effects; D002460:Cell Line; D020552:Comet Assay; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced*; D015162:Micronucleus Tests; D014025:Titanium / Q000494:pharmacology / Q000633:toxicity
chemical_list: C009495:titanium dioxide; D004247:DNA; D014025:Titanium