No cytotoxicity or genotoxicity of graphene and graphene oxide in murine lung epithelial FE1 cells in vitro

Graphene and graphene oxide receive much attention these years, because they add attractive properties to a wide range of applications and products. Several studies have shown toxicological effects of other carbon‐based nanomaterials such as carbon black nanoparticles and carbon nanotubes in vitro and in vivo. Here, we report in‐depth physicochemical characterization of three commercial graphene materials, one graphene oxide (GO) and two reduced graphene oxides (rGO) and assess cytotoxicity and genotoxicity in the murine lung epithelial cell line FE1. The studied GO and rGO mainly consisted of 2–3 graphene layers with lateral sizes of 1–2 µm. GO had almost equimolar content of C, O, and H while the two rGO materials had lower contents of oxygen with C/O and C/H ratios of 8 and 12.8, respectively. All materials had low levels of endotoxin and low levels of inorganic impurities, which were mainly sulphur, manganese, and silicon. GO generated more ROS than the two rGO materials, but none of the graphene materials influenced cytotoxicity in terms of cell viability and cell proliferation after 24 hr. Furthermore, no genotoxicity was observed using the alkaline comet assay following 3 or 24 hr of exposure. We demonstrate that chemically pure, few‐layered GO and rGO with comparable lateral size (> 1 µm) do not induce significant cytotoxicity or genotoxicity in FE1 cells at relatively high doses (5–200 µg/ml). Environ. Mol. Mutagen. 57:469–482, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

[1]  Meng Li,et al.  Deciphering the underlying mechanisms of oxidation-state dependent cytotoxicity of graphene oxide on mammalian cells. , 2015, Toxicology letters.

[2]  A. Cornet,et al.  The Importance of Interbands on the Interpretation of the Raman Spectrum of Graphene Oxide , 2015 .

[3]  Robert Langer,et al.  In vivo compatibility of graphene oxide with differing oxidation states. , 2015, ACS nano.

[4]  Andrew Williams,et al.  MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. , 2015, Toxicology and applied pharmacology.

[5]  Per Axel Clausen,et al.  Characterization of genotoxic response to 15 multiwalled carbon nanotubes with variable physicochemical properties including surface functionalizations in the FE1‐Muta(TM) mouse lung epithelial cell line , 2015, Environmental and molecular mutagenesis.

[6]  Hui‐Ming Cheng,et al.  The global growth of graphene. , 2014, Nature nanotechnology.

[7]  Martin Pumera,et al.  Synthetic routes contaminate graphene materials with a whole spectrum of unanticipated metallic elements , 2014, Proceedings of the National Academy of Sciences.

[8]  Bengt Fadeel,et al.  Classification framework for graphene-based materials. , 2014, Angewandte Chemie.

[9]  M. Riediker,et al.  Detecting the oxidative reactivity of nanoparticles: a new protocol for reducing artifacts , 2014, Journal of Nanoparticle Research.

[10]  Ying Tang,et al.  Distribution of Graphene Oxide and TiO2-Graphene Oxide Composite in A549 Cells , 2014, Biological Trace Element Research.

[11]  M. Pumera,et al.  The CVD graphene transfer procedure introduces metallic impurities which alter the graphene electrochemical properties. , 2014, Nanoscale.

[12]  Ning Zhang,et al.  Graphene oxide can induce in vitro and in vivo mutagenesis , 2013, Scientific Reports.

[13]  Robert H. Hurt,et al.  All in the graphene family - A recommended nomenclature for two-dimensional carbon materials , 2013 .

[14]  Dongmei Wu,et al.  Transcriptomic Analysis Reveals Novel Mechanistic Insight into Murine Biological Responses to Multi-Walled Carbon Nanotubes in Lungs and Cultured Lung Epithelial Cells , 2013, PloS one.

[15]  U. Vogel,et al.  Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay , 2013, Mutagenesis.

[16]  W. Duan,et al.  Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells , 2013, Journal of applied toxicology : JAT.

[17]  J. M. Navas,et al.  Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2 , 2013, Particle and Fibre Toxicology.

[18]  N. Jacobsen,et al.  Black tattoo inks induce reactive oxygen species production correlating with aggregation of pigment nanoparticles and product brand but not with the polycyclic aromatic hydrocarbon content , 2013, Experimental dermatology.

[19]  M. Pumera,et al.  The toxicity of graphene oxides: dependence on the oxidative methods used. , 2013, Chemistry.

[20]  Kostas Kostarelos,et al.  Purified Graphene Oxide Dispersions Lack In Vitro Cytotoxicity and In Vivo Pathogenicity , 2013, Advanced healthcare materials.

[21]  S. Khondaker,et al.  Oxygenated Functional Group Density on Graphene Oxide: Its Effect on Cell Toxicity , 2013 .

[22]  Omid Akhavan,et al.  Size-dependent genotoxicity of graphene nanoplatelets in human stem cells. , 2012, Biomaterials.

[23]  M. Pumera,et al.  Graphite oxides: effects of permanganate and chlorate oxidants on the oxygen composition. , 2012, Chemistry.

[24]  P. Møller,et al.  Carbon black nanoparticles and vascular dysfunction in cultured endothelial cells and artery segments. , 2012, Toxicology letters.

[25]  S. Bellucci,et al.  Comparative cyto-genotoxicity assessment of functionalized and pristine multiwalled carbon nanotubes on human lung epithelial cells. , 2012, Toxicology in vitro : an international journal published in association with BIBRA.

[26]  A. T. Saber,et al.  Inflammatory and genotoxic effects of nanoparticles designed for inclusion in paints and lacquers , 2012, Nanotoxicology.

[27]  Wei Wei,et al.  The role of the lateral dimension of graphene oxide in the regulation of cellular responses. , 2012, Biomaterials.

[28]  Ken Donaldson,et al.  Graphene-based nanoplatelets: a new risk to the respiratory system as a consequence of their unusual aerodynamic properties. , 2012, ACS nano.

[29]  J. K. Nøjgaard,et al.  Oxidative stress, genotoxicity, and vascular cell adhesion molecule expression in cells exposed to particulate matter from combustion of conventional diesel and methyl ester biodiesel blends. , 2011, Environmental science & technology.

[30]  Nicklas Raun Jacobsen,et al.  Mutation spectrum in FE1‐MUTATMMouse lung epithelial cells exposed to nanoparticulate carbon black , 2011, Environmental and molecular mutagenesis.

[31]  C. Fan,et al.  Protein corona-mediated mitigation of cytotoxicity of graphene oxide. , 2011, ACS nano.

[32]  Yanli Chang,et al.  In vitro toxicity evaluation of graphene oxide on A549 cells. , 2011, Toxicology letters.

[33]  M. M. Lucchese,et al.  Evolution of the Raman spectra from single-, few-, and many-layer graphene with increasing disorder , 2010 .

[34]  Gaetano Granozzi,et al.  Evolution of Electrical, Chemical, and Structural Properties of Transparent and Conducting Chemically Derived Graphene Thin Films , 2009 .

[35]  A. Reina,et al.  Large area, few-layer graphene films on arbitrary substrates by chemical vapor deposition. , 2009, Nano letters.

[36]  S. Banerjee,et al.  Large-Area Synthesis of High-Quality and Uniform Graphene Films on Copper Foils , 2009, Science.

[37]  Y. Kim,et al.  In vivo immunological toxicity in mice of carbon nanotubes with impurities , 2009 .

[38]  R. Ruoff,et al.  Chemical methods for the production of graphenes. , 2009, Nature nanotechnology.

[39]  Antonio Marcomini,et al.  Genotoxicity, cytotoxicity, and reactive oxygen species induced by single‐walled carbon nanotubes and C60 fullerenes in the FE1‐Muta™Mouse lung epithelial cells , 2008, Environmental and molecular mutagenesis.

[40]  U. Vogel,et al.  Diesel exhaust particles are mutagenic in FE1-MutaMouse lung epithelial cells. , 2008, Mutation research.

[41]  Nicklas Raun Jacobsen,et al.  Increased mutant frequency by carbon black, but not quartz, in the lacZ and cII transgenes of muta™mouse lung epithelial cells , 2007, Environmental and molecular mutagenesis.

[42]  L. Forró,et al.  Cellular toxicity of carbon-based nanomaterials. , 2006, Nano letters.

[43]  S. Kyrtopoulos,et al.  Mutagenesis by man-made mineral fibres in the lung of rats. , 2006, Mutation research.

[44]  B. Halliwell,et al.  Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? , 2004, British journal of pharmacology.

[45]  K. Donaldson,et al.  Interactions between ultrafine particles and transition metals in vivo and in vitro. , 2002, Toxicology and applied pharmacology.

[46]  L. Napolitano.  Materials , 1984, Science.

[47]  L. Du,et al.  In vitro toxicity evaluation of graphene oxide on human RPMI 8226 cells. , 2014, Bio-medical materials and engineering.

[48]  N. Chatterjee,et al.  A systems toxicology approach to the surface functionality control of graphene-cell interactions. , 2014, Biomaterials.

[49]  Carbon black, titanium dioxide, and talc. , 2010, IARC monographs on the evaluation of carcinogenic risks to humans.

[50]  P. White,et al.  Development and characterization of a stable epithelial cell line from Muta™Mouse lung , 2003, Environmental and molecular mutagenesis.

[51]  J. Higginson,et al.  International Agency for Research on Cancer. , 1968, WHO chronicle.