Genotoxicity of nanomaterials: DNA damage and micronuclei induced by carbon nanotubes and graphite nanofibres in human bronchial epithelial cells in vitro
Introduction
Because of the growing industrial use of nanomaterials, there is an urgent need for information on their potential health effects. Due to the distinct physico-chemical properties of many nanomaterials, their possible toxicity may differ from that of the bulk material of similar chemical nature. As the lungs are the primary route of entry for inhaled nanoparticles, lung toxicity is of particular concern. It would appear especially important to identify nanomaterials that could act as lung carcinogens. Some fibrous nanomaterials with a high aspect ratio, such as carbon nanotubes (CNTs), might be able to induce lung cancer and mesothelioma in a similar manner as asbestos. CNTs seem to be very stable in biological systems and form aggregates in the micrometer size range (Lam et al., 2006), thus resembling asbestos. Because of their unique electrical, chemical, and thermal properties, CNTs and other carbon nanomaterials have wide applications in different industrial fields and are among the most important nanomaterials presently manufactured. Recently, low-iron (0.35%) multi-walled CNTs (MWCNTs) were observed to induce mesothelioma in p53+/− mice upon a single intraperitoneal injection, in a similar way as crocidolite asbestos (Takagi et al., 2008).
The carcinogenic effect of biopersistent fibres such as asbestos has been suggested to be linked with the local generation of reactive oxygen and nitrogen species and inflammatory reactions (Takagi et al., 2008); genotoxic effects associated with these phenomena or occurring independently probably also play a role. Studies conducted on rats and mice have indicated that MWCNTs and single-walled CNTs (SWCNTs) can induce oxidative stress, inflammation, fibrosis, and granulomas in the lungs (Lam et al., 2006, Lam et al., 2004, Li et al., 2007a, Li et al., 2007b, Muller et al., 2005, Shvedova et al., 2005, Warheit et al., 2004). Inflammatory responses have not, however, been seen in all in vivo studies with CNTs. In mice, the lung fibrosis induced by SWCNTs was associated with intercellular structures composed of nanotubes bridging lung macrophages (Mangum et al., 2006). Mice exposed to MWCNTs showed systemic immune function alterations (Mitchell et al., 2007).
Several in vitro studies have demonstrated the toxic potential of SWCNTs, as concerns oxidative stress in human epidermal HaCaT keratinocytes (Shvedova et al., 2003) and rat lung epithelial cells (Sharma et al., 2007), cytotoxicity in guinea pig alveolar macrophages (Jia et al., 2005), and G1 arrest and apoptosis in HEK293 human embryo kidney cells (Cui et al., 2005) at relatively low doses. Also MWCNTs were cytotoxic to guinea pig alveolar macrophages (Jia et al., 2005), induced apoptosis in T lymphocytes (Bottini et al., 2006), and altered protein expression in human epidermal keratinocytes (Witzmann and Monteiro-Riviere, 2006). However, studies indicating a low in vitro cytotoxicity of CNTs have also been published. SWCNTs showed only low toxicity against human monocyte-derived macrophages (Fiorito et al., 2006) and human A549 lung cells (Davoren et al., 2007) with no intracellular localization of SWCNTs seen in the latter cells. No acute toxic effects or inflammatory mediators were produced in rat NR8383 macrophages or A549 cells after treatment with commercial SWCNTs or MWCNTs, although the rat macrophages took up the materials (Pulskamp et al., 2007). In both NR8383 and A549 cells, there was a dose- and time-dependent production of intracellular oxygen species and decrease in mitochondrial membrane potential after treatment with the commercial SWCNTs and MWCNTs, which disappeared when the materials were acid-purified to remove metal catalysts (such as Co). Thus, the diverging findings might partly be explained by the catalyst metals in the CNTs used.
Fibre carcinogenesis is probably a complex process also involving a long-term genotoxic stress. In principle, fibrous carbon nanomaterial could be genotoxic via direct interaction with DNA or with the mitotic apparatus or indirectly via oxidative stress and inflammatory responses. Specific cationic functionalized CNTs are able to condense DNA. Nanotube surface area and charge density are considered to be critical in determining electrostatic complex formation with DNA (Singh et al., 2005). Positively charged polyelectrolyte coating of nanotubes functions as a counterpart for negatively charged DNA, so that CNTs can be wrapped with DNA to produce DNA sensors (He and Bayachou, 2005). SWCNTs complexes with double- and single-stranded DNA and peptide nucleic acid (Rajendra et al., 2004, Rajendra and Rodger, 2005, Zheng et al., 2003).
At present, only a few studies on the genotoxicity of fibrous carbon nanomaterials have been published. Mice exposed by intrapharyngeal instillation to SWCNTs (10 and 40 μg/mouse) developed aortic mtDNA damage accompanied by changes in aortic mitochondrial glutathione and protein carbonyl levels at 7, 28, and 60 days after the exposure. In ApoE(−/−) mice fed an atherogenic diet, accelerated plaque formation in the aortas was accompanied by increased mtDNA damage but not inflammation (Li et al., 2007a, Li et al., 2007b). A single intra-tracheal administration of MWCNTs increased the frequency of micronucleated type II pneumocytes in rat lungs in vivo in connection with a marked lung inflammatory response (Muller et al., 2008). Also in vitro, there was a significant increase of micronuclei in rat lung epithelial RLE cells and human MCF-7 epithelial cells treated with MWCNTs, with an induction of both centromere-positive and -negative micronuclei as judged by fluorescence in situ hybridization in MCF-7 cells (Muller et al., 2008). MWCNTs accumulated in cultured mouse embryonic stem (ES) cells and induced apoptosis, p53 activation, increased expression of DNA repair proteins, and a twofold increase in adenine phosphoribosyltransferase mutations (Zhu et al., 2007). SWCNTs produced no mutations in Salmonella typhimurium strains YG1024 or YG1029, but induced DNA damage (as measured by comet assay) in Chinese hamster V79 lung fibroblasts (Kisin et al., 2007); a non-significant increase in micronuclei was also seen. Xenopus laevis larvae grown in the presence of double-walled CNTs did not show an induction of micronuclei in blood erythrocytes, although gill toxicity and uptake of the test material to the organisms could be demonstrated (Mouchet et al., 2008).
The aim of the present study was to examine in vitro the potential genotoxicity of two commercially available fibrous carbon nanomaterials, CNTs (a mixture of SWCNTs and other CNTs) and graphite nanofibres (GNFs), in human bronchial epithelial cells (BEAS 2B). We applied the approach earlier proposed (Speit, 2002) for mutagenicity testing of fibres, utilizing the comet assay to assess DNA damage and the micronucleus assay to assess chromosomal damage.
Section snippets
Nanomaterials and dispersions
The carbon nanomaterials examined in the present study were commercially available carbon nanotubes (product no. 636797; according to product sheets: >50% single-walled, ∼40% other nanotubes; 1.1 nm × 0.5–100 μm) and graphite nanofibres (product no. 636398; according to product sheets: 95%; ∼4% catalyst metals; outer diameter 80–200 nm, inner diameter 30–50 nm, length 5–20 μm) purchased from Sigma–Aldrich (Steinheim, Germany).
The size and morphology of the nanomaterials were characterized by using a
Cell viability
Treatment of the BEAS 2B cells with CNTs (Fig. 2A) and GNFs (Fig. 2B) decreased cell viability in a dose-dependent manner after all the exposure times used (24, 48, and 72 h) when the cells were allowed to recover for 48 h post treatment. In general, the number of viable cells decreased with the incubation time in all series, including treatments with CNTs and GNFs as well as the control cultures (Fig. 2A and B). Both carbon nanomaterials showed similar toxicity, with the number of viable cells
Discussion
Both of the carbon nanomaterials examined were found to be genotoxic in human bronchial epithelial BEAS 2B cells, as measured by the alkaline comet assay and the micronucleus assay. Dose-dependent increases in DNA damage were seen in the comet assay with both materials, and the effect was more marked at the longer treatment times. Also in the micronucleus assay, the longer treatment times resulted in a clearer effect than the 24-h treatment, but no obvious dose-dependent effects were seen at
Conflict of interest
None.
Acknowledgements
This paper was presented in ECETOC Symposium on Nano(geno)toxicology at the 37th Annual Meeting of the European Environmental Mutagen Society, 9–13 September 2007. We thank the organisers for support. The paper was partly supported by Commission of the European Communities Contract No. NMP4-CT-2006-032777, “NMP4-CT-2006-032777” (NANOSH), Finnish Work Environment Fund, and the Academy of Finland. The views and opinions expressed in this paper do not necessarily reflect those of the European
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