Carbon nanotubes (CNTs) belong to a specific class of nanomaterials with unique properties. Because of their anticipated use in a wide range of industrial applications, their toxicity is of increasing concern. In order to determine whether specific physicochemical characteristics of CNTs are responsible for their toxicological effects, we investigated the cytotoxic and genotoxic effects of eight CNTs representative of each of the commonly encountered classes: single- SW-, double- DW-, and multiwalled (MW) CNTs, purified and raw. In addition, because most previous studies of CNT toxicity were conducted on immortalized cell lines, we decided to compare results obtained from V79 cells, an established cell line, with results from SHE (Syrian hamster embryo) cells, an easy-to-handle normal cell model. After 24 hours of treatment, MWCNTs were generally found to be more cytotoxic than SW- or DWCNTs. MWCNTs also provoked more genotoxic effects. No correlation could be found between CNT genotoxicity and metal impurities, length, surface area, or induction of cellular oxidative stress, but genotoxicity was seen to increase with CNT width. The toxicity observed for some CNTs leads us to suggest that they might also act by interfering with the cell cycle, but no significant differences were observed between normal and immortalized cells.
Carbon nanotubes (CNTs) belong to the nanomaterials family [
The biodurability and high length-to-width aspect ratio of CNTs have raised questions related to their toxicity and effects on human health. Their fibrous nature has led to particular concern surrounding the CNTs, and parallels have been made with asbestos fibres and their effects on humans [
During the last decade, many toxicological studies have been published on the potential health effects of CNTs, but the results have been sometimes conflicting. The discrepancy is mainly a result of differences in the type of CNT used (shape, diameter, and being single-walled or multiwalled), the concentrations used, or the dispersion methods employed. Moreover, few studies have analysed SW- and MWCNTs in the same experimental model [
To illustrate this complexity, CNTs have been shown to induce
The main objective of the present study was to determine the toxicological effects of CNTs according to their physicochemical characteristics. However, as the majority of previous studies were conducted on immortalized cell lines and as Syrian hamster embryo cells (SHE) are normal and easily implemented, we also compare the toxicological effects of CNTs on SHE cells and on immortalized Chinese hamster lung fibroblast V79 cells. This comparison will enable us to determine whether a normal cell model is more suitable than an immortalized cell line for evaluating the toxic effects of CNTs.
For this purpose, five commercially available CNTs (one SWCNT, two DWCNTs, and two MWCNTs), which can potentially be found in the workplace, were tested in V79 and SHE cells for their
Physicochemical characteristics of carbon nanotube samples.
Name | Type | Carbon purity (%)2 | Amorphous carbon1,2 | Nb. of walls1 | Ext. diameter (nm)1 | Ext. diameter (nm)2 | Length ( |
SSA (m2/g)3 | Chemical content (% of mass)4 |
---|---|---|---|---|---|---|---|---|---|
1100 | SW purified | >70 | Yes | 1-2 | 1.5–4 | 2 | >1 | 1128 | 3.15Si; 1.44Co; 0.14Mg |
2100 | DW purified | >90 | Yes | <5 | 3–7 | 3.5 | 1–10 | 626 | 2.69Mo; 1.79Fe; 0.16Si; 0.11Ca |
2150 | DW short purified | >90 | Yes | <5 | 3–7 | 3.5 | >1 | 611 | 2.48Mo; 1.40Fe; 0.10Si; 0.12Ca |
DWEF | DW purified (80% DW, 15% SW, and 5% TW) | ~90,5 | n.d. | 2 | 1.6–3.4 | 1–3 | 1–20 | 985 | 9.5Co2 |
3100 | MW purified | >95 | n.d. | 4-5 | 11-12 | 9.5 | 1.5 | 333 | 0.22Fe; 0.1Co |
3150 | MW short purified | >95 | n.d. | 4-5 | 15–19 | 9.5 | <1 | 308 | 0.21Fe |
SBb | MW raw | >88 | <2% | 4–10 | 15–68 | 15–50 | >0.8 | 151 | 7.22Al; 4.15Fe |
SBp | MW purified | >98 | n.d. | 4–10 | 9–77 | 15–50 | >0.8 | 168 | 0.86Fe |
2Manufacturer data.
3BET analysis.
4ICP-MS analysis (Ag, AL, As, B, Ba, Be, Bi, Ca, Cd, Ce, Co, Cr, Cu, Fe, K, La, Li, Mg, Mn, Mo, Ni, Pb, Sb, Se, Si, Sn, Sr, Ti, V, Zn, and U).
The single- and double-walled samples analysed in this study included a purified single-walled carbon nanotube (SWCNT 1100, Nanocyl, Belgium); a purified double-walled carbon nanotube (DWCNT 2100, Nanocyl, Belgium); a short, purified double-walled carbon nanotube (DWCNT 2150, Nanocyl, Belgium) derived from grinding DWCNT 2100; a purified double-walled carbon nanotube (DWEF), donated by E. Flahaut of CIRIMAT/UMR CNRS 5085, Toulouse, France [
Two multiwalled carbon nanotubes were also tested: a purified multiwalled carbon nanotube (MWCNT 3100, Nanocyl, Belgium); a short, purified multiwalled carbon nanotube (MWCNT 3150, Nanocyl, Belgium), derived from grinding of MWCNT 3100;
Two other MWCNT samples were provided by Dr. D. Begin (LMSPC-UMR 7515-Strasbourg), synthesized according to Gulino et al. [ a raw multiwalled carbon nanotube (MWCNT SBb); a purified multiwalled carbon nanotube (MWCNT SBp).
Several criteria guided our choice of CNT samples. First, five of the samples are commercially available (samples 1100, 2100, 2150, 3100, and 3150) and can therefore be encountered in the workplace. The other three samples (DWEF, SBb, and SBp) were synthesized in research laboratories. Second, each of the large CNT families is represented (single-, double-, and multiwalled CNTs). Third, both short and long CNTs were obtained in order to determine the biological effect of CNT length (2100 versus 2150; 3100 versus 3150). Finally, both raw and purified samples were chosen in order to determine the impact of the presence of chemical products other than carbon on cellular toxicity (2100 versus DWEF; SBb versus SBp).
The chemical contents of CNT samples were analysed by inductively coupled plasma mass spectrometry (ICP-MS) (Spectro Ciros CCD, Germany). Nanotube diameters and the number of walls present were measured by transmission electron microscopy (TEM) (Philips CM20, The Netherlands). Specific surface area was determined using the BET technique [
In order to obtain a homogeneous suspension (estimated visually), the samples were placed in complete medium at the highest concentration used in
DLS (dynamic light scattering) analysis was done to determine the agglomeration status of suspensions using a Zetasizer Nano ZS apparatus (Malvern, France), but as mentioned before by Tavares et al. [
The SHE and V79 cells were treated with CNTs at concentrations ranging from 0.27 to 2.1
V79 cells (lung fibroblast from Chinese hamster, ATCC, USA, reference CCL-93) were selected for this study as they are one of the cell models recommended in OCDE guideline number 487 for use in the
Syrian hamster embryo (SHE) cell cultures were used as they are normal diploid cells, nongenetically modified, metabolically competent, and p53 effective and there is no known difference with those constituting the organism where they come from. They have been demonstrated to be suitable for genotoxicity assays [
1 × 103 cells/mL (V79) or 1.5 × 103 cells/mL (SHE) were seeded in 48 wells on a 96-well plate for 24 h. The cell cultures were then treated for 24 h with culture medium (control) or with sample suspensions in final concentrations between 0.23 and 3.75
Data were expressed as % of control ± SEM for each treatment concentration and compared using an ANOVA-LSD test (Fisher’s least significant difference) (Statgraphics Centurion, Statpoint Technologies, USA).
Cell counting for the comet assay was performed with a Coulter Z1 (Beckman Coulter, France) (data not shown).
5 × 104 V79 or SHE cells were treated with 0.27 to 2.1
Potential interference between CNTs and DCF was tested by acellular assays, mixing H2DCF (obtained by NaOH treatment of H2DCF-DA) or DCF fluorescent probe (Sigma-Aldrich, France) and CNTs at different concentrations (from 1 to 250
The Fpg modified comet assay was used to evaluate oxidative DNA damage. The Fpg enzyme, a glycosylase, recognizes and specifically cuts modified bases such as 8-oxoguanine from DNA, producing apurinic sites that are converted into strand breaks by the associated AP-endonuclease activity. Therefore, DNA strand breaks detected by the Fpg modified comet assay provide a measure of oxidative DNA damage [
In brief, two duplicate comet slides were made for each treatment: one slide was treated with Fpg and the other with the Fpg buffer only. The two slides were subsequently treated in the same manner.
The SHE (2 × 105) or V79 (1 × 105) cells were treated for 24 hours either with CNTs at concentrations ranging from 0.23 to 3.75
Approximately 20,000 cells were mixed in 600
The slides were drained and incubated in the dark for 30 min at 37°C, either in enzyme buffer alone or in Fpg (5 U/mL) in enzyme buffer (40 mM HEPES, 0.1 M KCl, and 0.5 mM Na2EDTA; pH 8). The slides were immersed in cold alkaline solution (300 mM NaOH, 1 mM Na2EDTA; pH 13) for 20 min and electrophoresis was then performed in the same buffer at 0.7 V/cm for 40 min to allow the fragments of damaged DNA to migrate towards the anode. The slides were then washed with 0.4 M Tris-HCl for 15 min and stained with propidium iodide (2.5
Slides were examined at 200x magnification under a fluorescence microscope. Images of 100 randomly selected comets were acquired and analyzed for each sample (comet assay IV, Perceptive Instruments, UK) in order to evaluate the % tail DNA used as a measure of DNA damage. The presence of CNTs did not interfere with the reading at the concentrations tested. The experiment was repeated three times independently. Statistical analyses were performed on means using the ANOVA-LSD test (Statgraphics Centurion, Statpoint Technologies, USA). The
Approximately 2.5 × 104 V79 cells and 5 × 104 SHE cells were seeded in Labtek slides (Nunc A/S, Denmark) with 1 mL of culture medium. After 24 h, the cells were treated either with CNTs at concentrations ranging from 0.23 to 3.75
The single-walled 1100 CNT sample contained 3.15 wt. % silica and 1.44 wt. % cobalt. The double-walled 2100 and 2150 CNT samples contained 2.69 and 2.48 wt. % molybdenum and 1.79 and 1.4 wt. % iron, respectively. The double-walled DWEF CNT sample contained 9.5 wt. % cobalt. The multiwalled 3100, 3150, and SBp samples contained few impurities, but the MWCNT SBb contained 7.22 wt. % aluminium and 4.15 wt. % iron. The TEM analyses revealed that most of the metal catalysts were located inside the carbon nanotubes. Specific surface areas were higher for single- (1128 m2/g) and double-walled CNT (611 to 985 m2/g) than for the multiwalled CNT (between 150 and 330 m2/g). Due to the association of carbon nanotubes in bundles, it was not possible to accurately measure their lengths. The external diameters of the carbon nanotube samples ranked from small to large as follows: 1100 (1.5–4 nm) < DWEF (1.6–3.4 nm) < 2100–2150 (3–7 nm) < 3100–3150 (11–19 nm) < SBb-SBp (9–77 nm).
After dispersion in complete medium, optical microscopy observations showed that the MWCNTs were better dispersed than both the double-walled and single-walled CNT (1100), even though bundles were present in all samples.
The production of reactive oxygen species (ROS) is often associated with toxicological effects of particles or fibres. In order to address this issue, we performed ROS detection in cells after treatment with CNTs, using the cell-permeable DCFH-DA fluorogenic probe. As shown in Figures
Oxidative stress after 24 h of treatment with CNTs, expressed as fluorescence intensity (% of control ± SD) in V79 and SHE cells. Fluorescence intensity with single- (1100) or double-walled carbon nanotubes (2100–2150, DWEF) in (a) V79 cells and (b) SHE cells and with multiwalled carbon nanotubes (3100–3150, SBb and SBp) in (c) V79 cells and (d) SHE cells. C.: control (medium alone); TiO2: positive control. Two independent experiments with duplicate were realized for every point. Data were expressed as the mean fluorescence intensity of the two independent experiments ± SD. Sample concentrations are expressed as
Cell viability was assessed after 24 h treatment with the carbon nanotube samples (Figure in V79 cells: 1100–2100 (100–102% of control) < 2150 (77%) < DWEF (74%) < 3100-SBb (66%) < 3150 (64%) < SBp (59%); in SHE cells: 1100 (106%) < 2100 (87%) < DWEF (74%) < 3100–3150 (67%) < 2150 (63%) < SBp (50%) < SBb (47%).
Effect of carbon nanotubes on cell viability assessed by the WST assay. Results are expressed as the percentage of delta OD (OD 450 nm–OD 690 nm) in treated cells ± SD compared to control cells (100 %) after 24 h of treatment with CNT samples. Single- or double-walled carbon nanotubes in (a) V79 cells and (b) SHE cells. Multiwalled carbon nanotubes in (c) V79 cells and (d) SHE cells. Sample concentrations are expressed as
In conclusion, the MWCNTs were found to be more cytotoxic than both the SW- or DW-nanotubes.
Two types of assay were used to evaluate the genotoxicity of carbon nanotubes in V79 and SHE cells: the comet assay and the micronucleus assay.
Results obtained following 24-hour treatment with 1100, 2100, 2150, and DWEF samples are presented in Figure
DNA damage in cells after 24-hour treatment with SW- and DWCNTs, expressed as tail DNA (%) ± SEM. For each CNT, the small histogram represents the results obtained with the negative (medium) and positive (0.125 mM MMS) control, both with (filled histogram) and without (open histogram) the Fpg enzyme. The large histogram represents data obtained for different concentrations of CNT. Sample concentrations are expressed as
With the exception of sample 2150, treatment of the cells with the SW- or DWCNT samples induced no effect in either V79 or SHE cells, with or without Fpg treatment. Sample 2150 induced a significant increase in the number of DNA breaks at 1.87 and 3.75
Negative results were also obtained in V79 cells with MWCNTs (Figure
DNA damage in cells after 24-hour treatment with MWCNTs, expressed as tail DNA (%) ± SEM. For each CNT, the small histogram represents the results obtained with negative (medium) and positive (0.125 mM MMS) control, both with (filled histogram) and without (open histogram) the Fpg enzyme. The large histogram represents data obtained for different concentrations of CNT. Sample concentrations are expressed as
Induction of micronucleated cells after 24 h of treatment with CNTs in V79 and SHE cells.
Chemical | Concentration ( |
% of cells with MN | Mitotic index (%) | ||
---|---|---|---|---|---|
V79 cells | SHE cells | V79 cells | SHE cells | ||
Control | 0 | 1.8 | 5.1 | 6.1 | 1.5 |
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MMS | 0.25 mM | 18.5* | 14.1* | 3.5* | 2.6* |
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1100 | 0.23 | 2,2 | 4,9 | 4,8 | 1,8 |
0.47 | 2,2 | 5,3 | 5,1 | 1,9 | |
0.94 | 2,6* | 4,5 | 5,8 | 1,7 | |
1.87 | 2,7* | 3,7 | 7,5 | 2,0 | |
3.75 | 2,1 | 4,8 | 5,4 | 1,9 | |
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2100 | 0.23 | 2,6* | 5,3 | 4,4* | 1,7 |
0.47 | 2,3 | 5,6 | 5,1 | 1,9 | |
0.94 | 2,7* | 5,1 | 5,0 | 1,9 | |
1.87 | 1,9 | 5,3 | 4,3* | 1,4 | |
3.75 | 1,9 | 4,7 | 4,4* | 1,8 | |
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2150 | 0.23 | 2,6* | 5,7 | 6,4 | 1,9 |
0.47 | 2,2 | 5,9 | 4,4* | 1,9 | |
0.94 | 1,6 | 4,9 | 4,4 | 1,3 | |
1.87 | 2,0 | 4,7 | 5,0 | 0,8* | |
3.75 | 1,5 | 4,1 | 4,7* | 1,3 | |
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DWEF | 0.23 | 2,2 | 6,8* | 5,8 | 1,5 |
0.47 | 2,7* | 5,9 | 5,0 | 1,7 | |
0.94 | 2,5* | 5,6 | 6,1 | 1,2 | |
1.87 | 2,0 | 4,7 | 5,5 | 1,3 | |
3.75 | 1,8 | 4,7 | 6,1 | 1,1 | |
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3100 | 0.23 | 2,9* | 6,3 | 4,7* | 1,6 |
0.47 | 3,0* | 6,8* | 4,6* | 1,4 | |
0.94 | 2,4 | 6,2 | 2,9* | 2,0 | |
1.87 | 2,5* | 5,5 | 3,0* | 1,2 | |
3.75 | 1,5 | 5,4 | 3,1* | 1,2 | |
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3150 | 0.23 | 2,6* | 7,7* | 4,4 | 1,4 |
0.47 | 2,0 | 7,1* | 3,0* | 1,6 | |
0.94 | 2,9* | 6,4* | 3,2* | 1,3 | |
1.87 | 2,5* | 5,8 | 3,1* | 1,2 | |
3.75 | 3,2* | 5,9 | 3,6* | 1,2 | |
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SBb | 0.23 | 3,1* | 6,6* | 5,1 | 1,9 |
0.47 | 3,6* | 6,7* | 5,1 | 0,9* | |
0.94 | 4,6* | 6,5* | 4,3* | 0,8* | |
1.87 | 5,5* | 6,4* | 4,4* | 0,3* | |
3.75 | 5,6* | 4,4 | 3,5* | 0,2* | |
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SBp | 0.23 | 3,8* | 8,0* | 6,1 | 1,1 |
0.47 | 3,4* | 6,7* | 4,6* | 1,0 | |
0.94 | 3,8* | 6,9* | 4,8 | 0,8* | |
1.87 | 3,7* | 5,6 | 5,0 | 0,7* | |
3.75 | 2,7* | 3,4 | 2,8* | 0,2* |
Data presented were established with at least 3000 cells derived from three independent assays. *Statistically significant (
In V79 cells, sample 1100 induced a significant increase in micronucleated cells at concentrations of 0.94 and 1.87
The 3100 MWCNT induced a significant increase in the number of micronucleated cells at concentrations of 0.23, 0.47, and 1.87
The 3150 MWCNT exhibited similar genotoxic potential in that three (0.23, 0.94, and 1.87
The V79 mitotic index shows that all CNTs with the exception of the 1100 and DWEF samples induce a decrease in the number of cells in mitosis. This effect was more pronounced for the MWCNTs than for the single- or double-walled CNTs and correlates with cell viability if sample DWEF is excluded (Figure
The specific physicochemical properties of carbon nanotubes, associated with their high aspect ratios, have led many laboratories to initiate and conduct
We have shown that, in our experimental conditions, MWCNTs were more cytotoxic than their single- or double-walled equivalents in both cell types. SHE cells and V79 cells do not present any great differences in terms of sensitivity. Because the SB samples induced 50–55% cytotoxicity at concentrations of 3.75
Even though comparison with other studies is complex and risky because of differences between the materials and methods used, we note that our results differ from those obtained after 24-hour treatment in both macrophage NR8383 [
Cell number decrease, as measured by the WST viability test, and the decrease in cell mitosis for the majority of samples in both cell types, as measured by the mitotic index, suggest that CNTs can act on the cell cycle and block cell division, as was observed in C6 rat glioma cells with MWCNTs [
MWCNTs, on the whole, also had greater effects than SW- and DWCNTs in the genotoxicity assays. However, unlike in the cytotoxicity assays, some differences were observed in the responses of the two cell types. In the comet assay, none of the CNTs induced a significant increase in DNA damage in V79 cells, whereas SBb and SBp (MWCNTs) and 2150 (DWCNT) induced significant increases in the number of DNA breaks at the two highest concentrations in SHE cells. Treatment with the Fpg enzyme increased the level of DNA breakage in both cell types (see control assays with and without Fpg in Figures
An increase in the number of DNA breaks induced by the Fpg enzyme is often associated with the presence of oxidized bases. Oxidative stress and production of reactive oxygen species are described as cytotoxic and genotoxic effectors which can lead to the production of oxidized bases. This was demonstrated in different cellular types with the cell-permeable DCFH-DA fluorogenic probe after treatment with SWCNTs [
CNT samples were also shown in our study to be capable of inducing micronucleated cells in both cell types, and the effect was seen to be more pronounced with MWCNTs. The most genotoxic CNTs were the 3150 MWCNT, whose length is described as short by the supplier, the raw and the purified SB samples. The decrease in micronucleated cell frequency at the highest concentrations may be explained by a cell cycle arrest, as was also suggested by the decrease in the mitotic index (see the previous section). Our results from the micronucleus assay corroborate those obtained by others with both SW- and MWCNTs [
To summarize, our results show that some CNTs, and mainly the MWCNTs, can induce cytotoxicity and genotoxicity in SHE and V79 cells. Furthermore, because the CNTs induced more micronucleated cells than DNA damage and as CNT exposure provoked a cell cycle arrest as revealed by the evaluation of the mitotic index, we can hypothesize that CNTs may act on the apparatus spindle during cell division.
When looking at the
A number of comments can be made regarding the responses of the two cellular types, taking into account that CNTs are present in both cellular types as early as 3 h of treatment (electronic microscopy analysis, data not shown): (i) CNT cytotoxicity is at almost the same level in both cell types; (ii) more ROS were generated in V79 cells than in SHE cells exposed to CNTs; (iii) more micronucleated cells were observed after CNT treatment in V79 cells, but no DNA damage was revealed by the comet assay; the opposite of that was observed in SHE cells for SBb and SBp.
V79 cells are immortalized cells. As they can undergo an infinite number of cell divisions and even though no genotyping or metabolism data were available for this clone, the enzymatic content and gene expression profile for V79 cells are most probably modified at the level of cell cycle checkpoints and DNA repair pathways. These differences can explain both the higher level of ROS production compared to SHE cells and the higher background of DNA breaks observed in control V79 compared to normal SHE cells. However, the p53 protein, which mediates the cellular response to DNA damage and is involved in cell cycle regulation, apoptosis, and DNA repair [
To examine this further, as we suggested earlier, additional experiments should be conducted to investigate the cell cycle, spindle apparatus, and effectiveness of the DNA repair system. An analysis, at the mRNA and protein levels, of p53 and mdm2 (E3 ubiquitin ligase that inactivates p53 by binding directly) in SHE cells, could be also beneficial to better understanding of the response of these cells.
As mentioned before, our results show that, for a given CNT, the ROS generation can be different according to the cell type. In acellular assay, we have shown that all CNTS were able to induce DCF fluorescence in phosphate buffer up to 25 mg/mL (corresponding to 5.8
However, our results nevertheless suggest that no large differences exist between the V79 cell line and the SHE normal cells after CNT treatment. The two cellular types are thus complementary and a benefit can certainly be gained in using SHE cells as they are normal cells that are appropriate for the evaluation of nanomaterial cytotoxicity and genotoxicity.
Regarding the physicochemical properties and biological effects of CNTs, the most pronounced cytotoxic and genotoxic effects were obtained with the multiwalled SBb and SBp samples, and the least toxic CNTs in our experiments were the SW- and DWCNTs.
Our data also demonstrate that, in our experimental conditions, there is no relationship between the toxicological effects of CNTs and their metal contaminants. Indeed, SBb, which contains 7.22% aluminium and 4.15% iron, presented near-identical toxicological effects to SBp, which contains only 0.86% iron. Concerning surface area, our results suggest that increased toxicity is not correlated with a higher specific surface area. However, it is important to note that the BET method uses a gas to determine the surface area, and therefore the value obtained does not reflect the real surface area in contact with a liquid or biomolecules. Furthermore, the agglomeration status of the suspension used, which we were unable to determine in this study, could directly influence the biological response.
The biological impact of CNT length is also unclear from our experiments. Even though the “shorter” 2150 sample was found to be more cytotoxic and induced more ROS than the “longer” 2100 sample, the two samples exhibited near-identical genotoxic effects. The “long” 3100 and “short” 3150 samples also presented no differences.
In our study, the only physical parameters that we were able to partially link to toxic effects were the number of walls and the outer diameters of the CNTs. Certainly, the thickest CNT samples (SBb and SBp) produced the most toxic effects (in terms of both cytotoxicity and genotoxicity). The importance of CNT diameter as a parameter to be considered in toxicology assessment has previously been suggested in the work of Fenoglio et al. [
In conclusion, this
Because of their different physicochemical properties, CNTs have different toxicological profiles. This suggests that it is not possible to draw any general conclusions regarding the toxicity of these nanomaterials.
The authors report no conflict of interests. The paper was reviewed and approved by the Scientific Direction of INRS (National Research and Safety Institute) prior to submission.
The authors are grateful to Mrs. C. Eypert-Blaison, INRS, for the SEM analyses, Mr. D. Rousset, INRS, for ICP-MS analyses, and Mrs. V. Fierro, Institut Jean Lamour, Nancy Université, for the BET analyses. The authors are also grateful to Dr. Emmanuel Flahaut (CIRIMAT/LCMIE, UMR CNRS 5085, Toulouse, France) for providing the DWEF sample and to Dr. D. Begin and Dr. C. Pham-Huu (LMSPC-UMR 7515, Strasbourg, France) for providing the SBb and SBp samples.