Carbon nanotubes induce oxidative DNA damage in RAW 264.7 cells

The induction of DNA and chromosome damage following in vitro exposure to carbon nanotubes (CNT) was assessed on the murine macrophage cell line RAW 264.7 by means of the micronucleus (MN) and the comet assays. Exposures to two CNT preparations (single‐walled CNT (SWCNT > 90%) and multiwalled CNT (MWCNT > 90%) were performed in increasing mass concentrations (0.01–100 μg/ml). The frequency of micronuclei was significantly increased in cells treated with SWCNT (at doses above 0.1 μg/ml), whereas MWCNT had the same effect at higher concentrations (1 μg/ml) (P < 0.05). The results of the comet assay revealed that the effects of treatment with SWCNT were detectable at all concentrations tested (1–100 μg/ml); oxidized purines increased significantly, whereas pyrimidines showed a significant increase (P < 0.001) only at the highest concentration (100 μg/ml). In cells treated with MWCNT, an increase in DNA migration due to the oxidative damage to purines was observed at a concentration of 1 and 10 μg/ml, whereas pyrimidines showed a significant increase only at the highest mass concentration tested. However, both SWCNT and MWCNT induced a statistically significant cytotoxic effect at the highest concentrations tested (P < 0.001). These findings suggest that both the MN and comet assays can reliably detect small amount of damaged DNA at both chromosome and nuclear levels in RAW 264.7 cells. Moreover, the modified version of the comet assay allows the specific detection of the induction of oxidative damage to DNA, which may be the underlying mechanism involved in the CNT‐associated genotoxicity. Environ. Mol. Mutagen., 2010. © 2010 Wiley‐Liss, Inc.

[1]  V. Castranova,et al.  Vitamin E deficiency enhances pulmonary inflammatory response and oxidative stress induced by single-walled carbon nanotubes in C57BL/6 mice. , 2007, Toxicology and applied pharmacology.

[2]  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.

[3]  V. Castranova,et al.  Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron. , 2006, Toxicology letters.

[4]  W. Stark,et al.  The degree and kind of agglomeration affect carbon nanotube cytotoxicity. , 2007, Toxicology letters.

[5]  Peter Wick,et al.  Reviewing the environmental and human health knowledge base of carbon nanotubes. , 2007, Ciencia & saude coletiva.

[6]  R. Nemanich,et al.  Surfactant effects on carbon nanotube interactions with human keratinocytes. , 2005, Nanomedicine : nanotechnology, biology, and medicine.

[7]  Gunnar Brunborg,et al.  The comet assay: topical issues. , 2008, Mutagenesis.

[8]  F. Coppedè,et al.  Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients , 2005, Neurobiology of Aging.

[9]  A. Collins,et al.  Oxidative damage to DNA: do we have a reliable biomarker? , 1996, Environmental health perspectives.

[10]  L. Migliore,et al.  Oxidative damage and cytogenetic analysis in leukocytes of Parkinson’s disease patients , 2002, Neurology.

[11]  Ivana Fenoglio,et al.  Reactivity of carbon nanotubes: free radical generation or scavenging activity? , 2006, Free radical biology & medicine.

[12]  P. Baron,et al.  Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. , 2005, American journal of physiology. Lung cellular and molecular physiology.

[13]  Raffaella Corvi,et al.  ECVAM retrospective validation of in vitro micronucleus test (MNT) , 2008, Mutagenesis.

[14]  Vicki Stone,et al.  An in vitro study of the potential of carbon nanotubes and nanofibres to induce inflammatory mediators and frustrated phagocytosis , 2007 .

[15]  H. Byrne,et al.  In vitro toxicity evaluation of single walled carbon nanotubes on human A549 lung cells. , 2007, Toxicology in vitro : an international journal published in association with BIBRA.

[16]  R. Hillamo,et al.  Effects of solubility of urban air fine and coarse particles on cytotoxic and inflammatory responses in RAW 264.7 macrophage cell line. , 2008, Toxicology and applied pharmacology.

[17]  Mark D. Hoover,et al.  Raw Single-Wall Carbon Nanotubes Induce Oxidative Stress and Activate MAPKs, AP-1, NF-κB, and Akt in Normal and Malignant Human Mesothelial Cells , 2008, Environmental health perspectives.

[18]  M. Fenech.  The micronucleus assay determination of chromosomal level DNA damage. , 2008, Methods in molecular biology.

[19]  H. Krug,et al.  Oops they did it again! Carbon nanotubes hoax scientists in viability assays. , 2006, Nano letters.

[20]  A. Pietroiusti,et al.  Airway barrier dysfunction induced by exposure to carbon nanotubes in vitro: which role for fiber length? , 2009, Human & experimental toxicology.

[21]  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.

[22]  M. Fenech.  Cytokinesis-block micronucleus cytome assay , 2007, Nature Protocols.

[23]  V. Granato,et al.  Genotoxicity induced by fine urban air particulate matter in the macrophages cell line RAW 264.7. , 2006, Toxicology in vitro : an international journal published in association with BIBRA.

[24]  J. Nagy,et al.  Structural defects play a major role in the acute lung toxicity of multiwall carbon nanotubes: physicochemical aspects. , 2008, Chemical research in toxicology.

[25]  M. Roller,et al.  Carcinogenicity of inhaled nanoparticles , 2009, Inhalation toxicology.

[26]  Julie W. Fitzpatrick,et al.  Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy , 2005, Particle and Fibre Toxicology.

[27]  A. L. Le Faou,et al.  Macrophage Culture as a Suitable Paradigm for Evaluation of Synthetic Vitreous Fibers , 2008 .

[28]  M. Fenech,et al.  HUman MicroNucleus project: international database comparison for results with the cytokinesis‐block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei , 2001, Environmental and molecular mutagenesis.

[29]  Robert A Hoke,et al.  Development of a base set of toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk management. , 2007, Toxicology letters.

[30]  Stefano Bellucci,et al.  Non-functionalized multi-walled carbon nanotubes alter the paracellular permeability of human airway epithelial cells. , 2008, Toxicology letters.

[31]  M. Tornow,et al.  High-aspect-ratio nanogap electrodes for averaging molecular conductance measurements. , 2007, Small.

[32]  Gwi-Nam Bae,et al.  Monitoring Multiwalled Carbon Nanotube Exposure in Carbon Nanotube Research Facility , 2008 .

[33]  N. Monteiro-Riviere,et al.  Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. , 2009, Toxicology and applied pharmacology.

[34]  H. Krug,et al.  Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants. , 2007, Toxicology letters.

[35]  Laetitia Gonzalez,et al.  Genotoxicity of engineered nanomaterials: A critical review , 2008 .

[36]  H. Norppa,et al.  Induction of micronuclei by five pyrethroid insecticides in whole-blood and isolated human lymphocyte cultures. , 1995, Mutation research.

[37]  Minnamari Vippola,et al.  Genotoxicity of nanomaterials: DNA damage and micronuclei induced by carbon nanotubes and graphite nanofibres in human bronchial epithelial cells in vitro. , 2009, Toxicology letters.

[38]  H. Karlsson,et al.  Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes. , 2008, Chemical research in toxicology.