Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles

Abstract Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs – as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines – demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

[1]  M. Dusinska,et al.  Genotoxicity testing of PLGA-PEO nanoparticles in TK6 cells by the comet assay and the cytokinesis-block micronucleus assay. , 2012, Mutation research.

[2]  H. M. Nielsen,et al.  In vitro placental model optimization for nanoparticle transport studies , 2012, International journal of nanomedicine.

[3]  Maria Dusinska,et al.  Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests , 2015, Nanotoxicology.

[4]  C. Serhan,et al.  Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin A4. , 1999, American journal of physiology. Cell physiology.

[5]  Lucienne Juillerat-Jeanneret,et al.  Evaluation of uptake and transport of ultrasmall superparamagnetic iron oxide nanoparticles by human brain-derived endothelial cells. , 2012, Nanomedicine.

[6]  Markus Schulz,et al.  Genotoxicity investigations on nanomaterials: methods, preparation and characterization of test material, potential artifacts and limitations--many questions, some answers. , 2009, Mutation research.

[7]  A. Collins,et al.  Detection of Oxidised Purines and UV-induced Photoproducts in DNA of Single Cells, by Inclusion of Lesion-specific Enzymes in the Comet Assay , 1996 .

[8]  M. Dusinska,et al.  Comet assay in human biomonitoring studies: Reliability, validation, and applications , 1997, Environmental and molecular mutagenesis.

[9]  Awadhesh N Jha,et al.  Hydroxyl radicals (*OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells. , 2008, Mutation research.

[10]  H. Karlsson,et al.  The comet assay in nanotoxicology research , 2010, Analytical and bioanalytical chemistry.

[11]  Sonja Boland,et al.  Toxicity evaluation of engineered nanoparticles for medical applications using pulmonary epithelial cells , 2015, Nanotoxicology.

[12]  E Ingham,et al.  Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness. , 2011, Nature nanotechnology.

[13]  G. Pojana,et al.  Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model , 2015, Nanotoxicology.

[14]  Lucienne Juillerat-Jeanneret,et al.  Induction of oxidative stress, lysosome activation and autophagy by nanoparticles in human brain-derived endothelial cells. , 2012, The Biochemical journal.

[15]  Maria Dusinska,et al.  Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles , 2014, Nanotoxicology.

[16]  Helinor Johnston,et al.  Development of in vitro systems for nanotoxicology: methodological considerations , 2009, Critical reviews in toxicology.

[17]  Maria Dusinska,et al.  Toxicological aspects for nanomaterial in humans. , 2013, Methods in molecular biology.

[18]  J. Castell,et al.  Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: a quantitative method for oxidative stress assessment of nanoparticle-treated cells. , 2013, Toxicology in vitro : an international journal published in association with BIBRA.

[19]  Helena Geys,et al.  Recommendations on the statistical analysis of the Comet assay , 2011, Pharmaceutical statistics.

[20]  D. Friend,et al.  HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS , 1969, The Journal of cell biology.

[21]  Maria Dusinska,et al.  Impact of agglomeration and different dispersions of titanium dioxide nanoparticles on the human related in vitro cytotoxicity and genotoxicity. , 2012, Journal of environmental monitoring : JEM.

[22]  Maria Dusinska,et al.  Iron oxide nanoparticle toxicity testing using high-throughput analysis and high-content imaging , 2015, Nanotoxicology.

[23]  H. Mcardle,et al.  Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo). , 2006, American journal of physiology. Endocrinology and metabolism.

[24]  G. Pojana,et al.  Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles , 2015, Nanotoxicology.

[25]  A. Jha,et al.  Titanium dioxide induced cell damage: a proposed role of the carboxyl radical. , 2009, Mutation research.

[26]  G. Jenkins,et al.  In vitro genotoxicity testing strategy for nanomaterials and the adaptation of current OECD guidelines , 2012, Mutation research.

[27]  H. Karlsson,et al.  DNA damage induced by micro- and nanoparticles--interaction with FPG influences the detection of DNA oxidation in the comet assay. , 2012, Mutagenesis.

[28]  M. Saunders,et al.  The toxicity, transport and uptake of nanoparticles in the in vitro BeWo b30 placental cell barrier model used within NanoTEST , 2015, Nanotoxicology.

[29]  Awadhesh N. Jha,et al.  Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on fish cells in vitro , 2008, Ecotoxicology.

[30]  Ken Donaldson,et al.  Possible genotoxic mechanisms of nanoparticles: Criteria for improved test strategies , 2010, Nanotoxicology.

[31]  Xavier Montet,et al.  Interaction of Functionalized Superparamagnetic Iron Oxide Nanoparticles with Brain Structures , 2006, Journal of Pharmacology and Experimental Therapeutics.

[32]  Damjana Drobne,et al.  Experimental evidence of false-positive Comet test results due to TiO2 particle – assay interactions , 2013, Nanotoxicology.

[33]  Maria Dusinska,et al.  Can Standard Genotoxicity Tests be Applied to Nanoparticles? , 2012, Journal of toxicology and environmental health. Part A.

[34]  A. Collins,et al.  The comet assay in human biomonitoring: gene-environment interactions. , 2008, Mutagenesis.

[35]  A. Collins,et al.  The comet assay for DNA damage and repair , 2004, Molecular biotechnology.

[36]  E. Teller,et al.  ADSORPTION OF GASES IN MULTIMOLECULAR LAYERS , 1938 .

[37]  Alexandra Kroll,et al.  Interference of engineered nanoparticles with in vitro toxicity assays , 2012, Archives of Toxicology.

[38]  Maria Dusinska,et al.  Biological impact assessment of nanomaterial used in nanomedicine. Introduction to the NanoTEST project , 2015, Nanotoxicology.

[39]  Stephen Mann,et al.  Nanoparticles can cause DNA damage across a cellular barrier. , 2009, Nature nanotechnology.