title: Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines.
abstract: BACKGROUND
  The rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz).
  
  
  METHODS
  The metal oxide- and silver nanoparticles were characterized in terms of agglomeration and ion release in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay, γ-H2AX and RAD51 foci formation).
  
  
  RESULTS
  We show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles.
  
  
  CONCLUSIONS
  We conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress.
authors.affiliation: Nanosafety & Nanomedicine Laboratory, Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. Hanna.L.Karlsson@ki.se.
authors.forename: Hanna L
authors.initials: HL
authors.lastname: Karlsson
authors.forename: Anda R
authors.initials: AR
authors.lastname: Gliga
authors.forename: Fabienne M G R
authors.initials: FM
authors.lastname: Calléja
authors.forename: Cátia S A G
authors.initials: CS
authors.lastname: Gonçalves
authors.forename: Inger Odnevall
authors.initials: IO
authors.lastname: Wallinder
authors.forename: Harry
authors.initials: H
authors.lastname: Vrieling
authors.forename: Bengt
authors.initials: B
authors.lastname: Fadeel
authors.forename: Giel
authors.initials: G
authors.lastname: Hendriks
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005742:Gasoline / Q000633:toxicity; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057166:High-Throughput Screening Assays; D053768:Metal Nanoparticles / Q000633:toxicity*; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods*; D037742:Nanotubes, Carbon / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D010087:Oxides / Q000378:metabolism / Q000633:toxicity*; D010316:Particle Size; D011791:Quartz / Q000633:toxicity; D017382:Reactive Oxygen Species / Q000378:metabolism; D018570:Risk Assessment; D012834:Silver / Q000378:metabolism / Q000633:toxicity*; D012995:Solubility
chemical_list: D015415:Biomarkers; D005742:Gasoline; D037742:Nanotubes, Carbon; D010087:Oxides; D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins; D011791:Quartz; D012834:Silver