Elsevier

Toxicology

Volume 313, Issue 1, 8 November 2013, Pages 24-37
Toxicology

Genotoxicity of short single-wall and multi-wall carbon nanotubes in human bronchial epithelial and mesothelial cells in vitro

https://doi.org/10.1016/j.tox.2012.12.008Get rights and content

Abstract

Although some types of carbon nanotubes (CNTs) have been described to induce mesothelioma in rodents and genotoxic effects in various cell systems, there are few previous studies on the genotoxicity of CNTs in mesothelial cells. Here, we examined in vitro DNA damage induction by short multi-wall CNTs (MWCNTs; 10–30 nm × 1–2 μm) and single-wall CNTs (SWCNTs; >50% SWCNTs, ∼40% other CNTs; <2 nm × 1–5 μm) in human mesothelial (MeT-5A) cells and bronchial epithelial (BEAS 2B) cells, using the single cell gel electrophoresis (comet) assay and the immunoslot blot assay for the detection of malondialdehyde (M1dG) DNA adducts. In BEAS 2B cells, we also studied the induction of micronuclei (MN) by the CNTs using the cytokinesis-block method. The cells were exposed to the CNTs (5–200 μg/cm2, corresponding to 19–760 μg/ml) for 24 and 48 h in the comet assay and for 48 and 72 h in the MN and M1dG assays. Transmission electron microscopy (TEM) showed more MWCNT fibres and SWCNT clusters in BEAS 2B than MeT-5A cells, but no significant differences were seen in intracellular dose expressed as area of SWCNT clusters between TEM sections of the cell lines. In MeT-5A cells, both CNTs caused a dose-dependent induction of DNA damage (% DNA in comet tail) in the 48-h treatment and SWCNTs additionally in the 24-h treatment, with a statistically significant increase at 40 μg/cm2 of SWCNTs and (after 48 h) 80 μg/cm2 of both CNTs. SWCNTs also elevated the level of M1dG DNA adducts at 1, 5, 10 and 40 μg/cm2 after the 48-h treatment, but both CNTs decreased M1dG adduct level at several doses after the 72-h treatment. In BEAS 2B cells, SWCNTs induced a statistically significant increase in DNA damage at 80 and 120 μg/cm2 after the 24-h treatment and in M1dG adduct level at 5 μg/cm2 after 48 h and 10 and 40 μg/cm2 after 72 h; MWCNTs did not affect the level of DNA damage but produced a decrease in M1dG adducts in the 72-h treatment. The CNTs did not affect the level of MN. In conclusion, MWCNTs and SWCNTs induced DNA damage in MeT-5A cells but showed a lower (SWCNTs) or no (MWCNTs) effect in BEAS 2B cells, suggesting that MeT-5A cells were more sensitive to the DNA-damaging effect of CNTs than BEAS 2B cells, despite the fact that more CNT fibres or clusters were seen in BEAS 2B than MeT-5A cells. M1dG DNA adducts were induced by SWCNTs but decreased after a 3-day exposure to MWCNTs and (in MeT-5A cells) SWCNTs, indicating that CNTs may lead to alterations in oxidative effects within the cells. Neither of the CNTs was able to produce chromosomal damage (MN).

Introduction

Carbon nanomaterials showing unique physicochemical, electrical and mechanical properties are increasingly utilized in nanotechnology and in various consumer products. Their widespread industrial use has raised concern about their potential health effects. Carbon nanotubes (CNTs) with a high aspect ratio (length:thickness > 3:1) consisting of long (>5–20 μm) and rigid fibres are of particular concern, since they might be able to induce lung cancer and mesothelioma in a manner similar to asbestos (Linton et al., 2012, Takagi et al., 2012). The carcinogenic capacity of long multi-wall CNTs (MWCNTs) (MITSUI MWCNT-7; diameter 70–170 nm, length 1–20 μm) was demonstrated in p53+/− mice and Fisher 344 rats, where a single intraperitoneal injection (intrascrotal in rats) induced mesothelioma more effectively than crocidolite asbestos (Takagi et al., 2008, Sakamoto et al., 2009); a recent study showed that the mesothelioma induction in p53+/− mice was dose dependent (Takagi et al., 2012). The same type of needle-like MWCNTs was also able to activate the NLRP3 (NLR family, pyrin domain containing 3) inflammasome in human primary macrophages, in a similar manner as asbestos (Palomäki et al., 2011). Another study, exposing the mesothelial lining of the body cavity of C57BL/6 mice to MWCNTs, showed that needle-like MWCNTs, containing a substantial proportion of fibres longer than 20 μm produced asbestos-like, length-dependent, pathogenic alterations (Poland et al., 2008). However, no carcinogenic response was detected in rats after a single intraperitoneal injection of shorter and thinner MWCNTs (11.3 nm × 0.7 μm) (Muller et al., 2009). The authors suggested that the lack of response could be due to insufficient sustainability of the inflammatory reaction in the peritoneal cavity or to the fact that the MWCNTs used did not contain a sufficient number of long nanotubes (Muller et al., 2009).

Exposure of rats or mice to MWCNTs and single-wall CNTs (SWCNTs) by inhalation or intra-tracheal instillation caused inflammation, fibrosis, granuloma formation, and immunosuppression (Lam et al., 2004, Li et al., 2007a, Mitchell et al., 2007, Muller et al., 2005, Nygaard et al., 2009, Shvedova et al., 2005). However, studies indicating a low toxicity of CNTs have also been published (Davoren et al., 2007, Fiorito et al., 2006, Muller et al., 2009, Pulskamp et al., 2007). Several in vitro studies have demonstrated that both MWCNTs and SWCNTs can induce oxidative stress, inflammatory cytokines, cytotoxic effects, apoptosis, and altered protein expression in various cell types (Barillet et al., 2010, Bottini et al., 2006, Cui et al., 2005, Di Giorgio et al., 2011, Jia et al., 2005, Sharma et al., 2007, Shvedova et al., 2003, Witzmann and Monteiro-Riviere, 2006). It was recently suggested that relatively thin MWCNTs (diameter ∼50 nm) with high crystallinity enter mesothelial cells by piercing their membrane, are cytotoxic in vitro, and induce inflammation and mesothelioma in vivo, while thicker (diameter ∼150 nm) and thinner tangled (diameter ∼2–20 nm) MWCNTs are less effective (Nagai et al., 2011, Nagai and Toyokuni, 2012).

In assessing the carcinogenic hazard of CNTs, the evaluation of genotoxic potential is of crucial importance. The mechanisms by which CNTs could exert their possible genotoxic effects in biological systems are presently not well understood, but two main modes of action have been proposed: effect on the level of reactive oxygen species (ROS) and mechanical interference with cellular components. Increased levels of ROS could be generated by the particles themselves, upon particle-cell contact, or due to particle-elicited inflammation (Donaldson et al., 2010). On the other hand, MWCNTs with point defects in the carbon framework have been indicated to scavenge ROS levels, and point defects have been associated with genotoxicity and acute toxicity (Fenoglio et al., 2008, Haniu et al., 2011, Muller et al., 2008). The small size of CNTs may allow them to mechanically interact or interfere with cellular components of similar dimensions (Gonzalez et al., 2008). CNTs may traverse cell membranes and subsequently enter nuclei through several different mechanisms (Singh et al., 2009).

Peroxidation of polyunsaturated fatty acids of biological membranes can result in the generation of a number of potentially genotoxic compounds, notably malondialdehyde (MDA) which may also be formed as a by-product in arachidonic acid metabolism in the biosynthesis of prostaglandins (Singh et al., 2001). Induction of lipid peroxidation, including MDA formation, by various types of SWCNTs and MWCNTs was described in a number of different biological systems in vivo (Kato et al., 2012, Ravichandran et al., 2011, Reddy et al., 2011, Tyurina et al., 2011) and in vitro (e.g. Kagan et al., 2006, Pichardo et al., 2012, Reddy et al., 2011, Wang et al., 2011). MDA binds to DNA, but MDA adducts in DNA have not previously been examined in studies of CNTs or other nanomaterials. A major product formed in this reaction is cyclic N1-N2 malondialdehyde-2′-deoxyguanosine (M1dG), a highly fluorescent cyclic adduct which can be determined by an immunoslot blot method (Singh et al., 2001).

In vivo, intrapharyngeal instillation of SWCNTs (mean diameter 1–4 nm) to mice (10 and 40 μg/mouse) lead to aortic mtDNA damage 7, 28, and 60 days after the exposure; in apolipoprotein E knockout (ApoE−/−) mice fed an atherogenic diet, plaque formation in the aortas and increased mtDNA damage but no inflammation was observed (Li et al., 2007b). In C57BL/6 mice, inhalation of SWCNTs (diameter 0.8–1.2 nm; 5 mg/m3, 5 h/day, for 4 days) was more effective than SWCNT aspiration (5–20 μg/mouse) in causing K-ras mutations, an inflammatory response, and oxidative stress (Shvedova et al., 2008). Intratracheal instillation of SWCNTs (0.9–1.7 nm × ≤1 μm; 54 μg) produced inflammation and increased DNA damage in cells of bronchoalveolar lavage from ApoE−/− mice (Jacobsen et al., 2009). A single intra-tracheal administration of MWCNTs (11.3 nm × 0.7 μm; 0.5 or 2 mg) in rats induced a dose-dependent increase of micronuclei (MN) in type II pneumocytes (Muller et al., 2008). Intratracheal instillation of MITSUI MWCNT-7 (50 and 200 μg) in ICR mice induced DNA damage, DNA adducts related to oxidative stress (8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxodG) and lipid peroxidation (heptanone etheno-deoxyribonucleosides), and gene mutations in lung cells (Kato et al., 2012). In Sprague-Dawley rats, inhalation of newly-generated MWCNTs for 5 days (6 h/day) resulted in an increase in lung cell DNA damage that was still detectable one month postexposure (Kim et al., 2012). A single oral administration (0.064 or 0.64 mg/kg body weight) of SWCNTs (0.9–1.7 nm ×  < 1 μm) increased the level of 8-oxodG DNA adducts in the liver and lungs but not in colon mucosa of Fisher-344 rats (Folkmann et al., 2009). A 5-day intraperitoneal administration of carboxyl-functionalized (11.5 nm × 12 μm) and nonfunctionalized MWCNTs (15–30 nm × 15–20 μm) induced chromosomal aberrations, MN, and DNA damage in Swiss-Webster mice (Patlolla et al., 2010).

However, orally administered MWCNTs (diameter 69 and 70 nm; 10–20 mg/kg) or SWCNTs (3 nm ×  1.2 μm) did not induce MN in polychromatic erythrocytes of ICR mice (Ema et al., 2012a, Kim et al., 2011, Naya et al., 2011). In Fisher-344 rats, oral exposure to SWCNTs or MWCNTs did not increase urinary mutagenicity assessed by the Ames test (Szendi and Varga, 2008). Xenopus laevis larvae grown in the presence of double-walled CNTs showed no induction of MN in blood erythrocytes (Mouchet et al., 2008).

Several in vitro studies have shown the genotoxic potential of CNTs. SWCNTs increased DNA damage, as measured by the comet assay, in Chinese hamster V79 lung fibroblasts (Kisin et al., 2007) and by the level of 8-oxoguanine DNA glycosylase sensitive sites/oxidized purines in FE1-MutaTM mouse lung epithelial cells (0.9–1.7 nm ×  < 1 μm) (Jacobsen et al., 2008). Both SWCNTs (1.4 nm × 2–5 μm) and MWCNTs (81 nm × 8 μm) induced DNA damage and activation of the transcription factors H2AX (phosphorylated histone H2AX) and PARP (poly ADP ribose polymerase) in normal and malignant mesothelial cells (Pacurari et al., 2008a, Pacurari et al., 2008b) but did not produce 8-oxodG adducts in DNA of human mesothelial MeT-5A cells (Ogasawara et al., 2012). MWCNTs (dimensions not specified) were observed to accumulate in cultured mouse embryonic stem (ES) cells and induce apoptosis, p53 activation, increased expression of DNA repair proteins, and a twofold increase in gene mutations (Zhu et al., 2007). MWCNTs (20–40 nm × 0.5–200 μm) induced DNA damage (Cavallo et al., 2011, Karlsson et al., 2008) and MN (Kato et al., 2012) in A549 cells, and DNA damage was also produced in primary mouse embryo fibroblasts by moderately cytotoxic CNTs (type not specified) (Yang et al., 2008). Functionalized and nonfunctionalized MWCNTs (11.5 nm × 12 μm and 15–30 nm × 15–20 μm, respectively) induced DNA damage in cultures of human lymphocytes (Patlolla et al., 2010). In human epithelial MCF-7 cells and rat epithelial RLE cells, MWCNTs (11.3 nm × 0.7 μm) were observed to increase both centromere-positive and -negative MN, which suggested induction of both clastogenic and aneugenic events (Muller et al., 2008). In mouse RAW 264.7 cells, SWCNTs (1.1 nm × 0.5–100 μm) and MWCNTs (150 nm × 5–9 μm) induced MN, oxidative DNA damage, ROS release, chromosomal aberrations, and apoptosis (Di Giorgio et al., 2011, Migliore et al., 2010). SWCNTs (1.58 nm × 0.76 μm) were found to cause DNA damage, MN and oxidative stress in human gingival fibroblasts (Cicchetti et al., 2011).

MWCNTs or SWCNTs were not mutagenic to bacteria (Di Sotto et al., 2009, Ema et al., 2012a, Kim et al., 2011, Kisin et al., 2007, Naya et al., 2011) and did not affect the level of MN or sister chromatid exchanges in cultured human lymphocytes (Szendi and Varga, 2008) or structural chromosomal aberrations in Chinese hamster cell lines – although MWCNTs increased numerical chromosomal alterations (Asakura et al., 2010, Ema et al., 2012a, Kim et al., 2011, Naya et al., 2011, Wirnitzer et al., 2009). MWCNTs did not induce gene mutations in Chinese hamster lung CHL/IU cells (Asakura et al., 2010).

We have previously demonstrated that long SWCNTs (>50% single-walled, ∼40% other nanotubes; length 0.5–100 μm) and graphite nanofibres (95%; length 5–20 μm) induce DNA damage (comet assay) and MN (cytokinesis-block assay) in human bronchial epithelial BEAS 2B cells (Lindberg et al., 2009). In the present study, we applied the same assays to examine commercially available short (1–5 μm) SWCNTs and MWCNTs. These CNTs were earlier shown to induce chromosomal aberrations in human lymphocytes in vitro (Catalán et al., 2012) but no immunotoxic effects in mouse antigen presenting cells (Palomäki et al., 2010). In addition to BEAS 2B cells, we studied here DNA damage in human mesothelial MeT-5A cells which have earlier been used for this purpose only once (Ogasawara et al., 2012), although they provide an in vitro model for effects of CNTs on the mesothelium. We also assessed whether the short CNTs are able to induce MDA adducts associated with lipid peroxidation and determined the uptake of the materials by transmission electron microscopy (TEM). Results from the present study suggest that MeT-5A cells are more sensitive to the DNA-damaging effect of CNTs than BEAS 2B cells, although especially MWCNTs are taken up more efficiently by BEAS 2B cells.

Section snippets

Carbon nanotubes and dispersions

The CNTs examined in the present study were commercially available purified SWCNTs (product no. 900-1351; according to vendor: >90% CNT, >50% single-walled, ∼40% other nanotubes, <5% amorphous carbon, <2% ash; primary fibre dimensions < 2 nm × 1–5 μm) and purified MWCNTs (product no. 900-1260; >95% CNTs, <0.2% ash; primary fibre dimensions 10–30 nm × 1–2 μm) purchased from SES Research (Houston, USA). The materials were produced by the chemical vapour deposition (CVD) method. The characteristics of these

Cytotoxicity

The cytotoxic response of BEAS 2B and MeT-5A cells treated with SWCNTs and MWCNTs, as assessed by the Trypan blue exclusion assay, is shown in Fig. 1. In BEAS 2B cells, SWCNTs were clearly more cytotoxic than MWCNTs. A >50% decrease in living BEAS 2B cells treated with SWCNTs compared with the control was seen already at 40 μg/cm2 after the 48-h treatment and starting from 60 μg/cm2 after the 72-h treatment – with these longer treatment times only about 10% of the cells were alive at 100 μg/cm2

Discussion

Our results showed that SWCNTs were more cytotoxic than MWCNTs in BEAS 2B cells, as measured by the reduction of cell number. In MeT-5A cells, the difference between the materials was less clear, although SWCNTs were slightly more cytotoxic than MWCNTs. The underlying mechanisms behind the toxicity are presently unclear but the significantly larger surface area of SWCNTs (436 m2/g) than MWCNTs (60 m2/g) could have contributed to the higher cytotoxicity of the SWCNTs. Our findings are in agreement

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

The research leading to these results has received funding from the European Commission under grant agreement NMP4-CT-2006-032777 (NANOSH) and from the Academy of Finland under the FinNano programme (NANOHEALTH).

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