Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells.

Abstract

The aim of this study was to evaluate adverse effects of multiwalled carbon nanotubes (MWCNT), produced for industrial purposes, on the human epithelial cell line A549. MWCNT were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in two other media: ethanol (EtOH) and phosphate-buffered saline (PBS). Effects of MWCNT were also compared to those of two asbestos fibers (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells but also in mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. MWCNT formed agglomerates on top of both cell lines (surface area 15-35 microm(2)) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 microg/ml MWCNT induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNT cellular internalization nor oxidative stress was observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects. In conclusion, this study demonstrates that MWCNT produced for industrial purposes exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.

Full text

Page 1

PLEASE SCROLL DOWN FOR ARTICLE
This article was downloaded by: [University of Lausanne]
On: 27 November 2008
Access details: Access Details: [subscription number 786945812]
Publisher Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Journal of Toxicology and Environmental Health, Part AJournal of Toxicology and Environmental Health, Part A
Publication details, including instructions for authors and subscription information:
http://www.informaworld.com/smpp/title~content=t713667303
Adverse Effects of Industrial Multiwalled Carbon Nanotubes on HumanAdverse Effects of Industrial Multiwalled Carbon Nanotubes on Human
Pulmonary CellsPulmonary Cells
Lyes Tabet a; Cyrill Bussy a; Nadia Amara a; Ari Setyan b; Alain Grodet c; Michel J. Rossi d; Jean-Claude
Pairon e; Jorge Boczkowski af; Sophie Lanone a
a INSERM, Unité 700 and Université Paris 7, Faculté de Médecine, Paris, France b Institut universitaire
romand de Santé au Travail (Institute for Work and Health), Université de Lausanne et Université de Genève,
Lausanne, Switzerland c INSERM, Unité 773, Université Paris 7, Faculté de Médecine, Paris, France d EPFL
(Ecole Polytechnique Fédérale de Lausanne), LPAS (Laboratoire de Pollution Atmosphérique et Sol),
Lausanne, Switzerland e INSERM, Unité 841, Université Paris 12, Faculté de Médecine, and CHI Creteil,
Service pneumologie et pathologie professionnelle, Créteil, France f Assistance Publique-Hôpitaux de Paris,
Hôpital Bichat, Paris, France
Online Publication Date: 01 January 2009
To cite this ArticleTo cite this Article Tabet, Lyes, Bussy, Cyrill, Amara, Nadia, Setyan, Ari, Grodet, Alain, Rossi, Michel J., Pairon, Jean-Claude,
Boczkowski, Jorge and Lanone, Sophie(2009)'Adverse Effects of Industrial Multiwalled Carbon Nanotubes on Human Pulmonary
Cells',Journal of Toxicology and Environmental Health, Part A,72:2,60 — 73
To link to this Article: DOI: To link to this Article: DOI: 10.1080/15287390802476991
URL: URL: http://dx.doi.org/10.1080/15287390802476991
Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf
This article may be used for research, teaching and private study purposes. Any substantial or
systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or
distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses
should be independently verified with primary sources. The publisher shall not be liable for any loss,
actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly
or indirectly in connection with or arising out of the use of this material.

Page 2

60
Journal of Toxicology and Environmental Health, Part A, 72: 60–73, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 1528-7394 print / 1087-2620 online
DOI: 10.1080/15287390802476991
UTEH
Adverse Effects of Industrial Multiwalled Carbon Nanotubes
on Human Pulmonary Cells
Toxic Effects of Industrial Carbon Nanotubes
Lyes Tabet1, Cyrill Bussy1, Nadia Amara1, Ari Setyan2, Alain Grodet3,
Michel J. Rossi4, Jean-Claude Pairon5, Jorge Boczkowski1,6, and Sophie Lanone1
1INSERM, Unité 700, Paris, France, and Université Paris 7, Faculté de Médecine, Paris, France,
2Institut universitaire romand de Santé au Travail (Institute for Work and Health), Université de
Lausanne et Université de Genève, Lausanne, Switzerland, 3INSERM, Unité 773, Paris, France and
Université Paris 7, Faculté de Médecine, Paris, France, 4EPFL (Ecole Polytechnique Fédérale de
Lausanne), LPAS (Laboratoire de Pollution Atmosphérique et Sol), Lausanne, Switzerland, 5INSERM,
Unité 841, Créteil, France and Université Paris 12, Faculté de Médecine, and CHI Creteil, Service
pneumologie et pathologie professionnelle, Créteil, France, and 6Assistance Publique-Hôpitaux de
Paris, Hôpital Bichat, Paris, France
The aim of this study was to evaluate adverse effects of multi-
walled carbon nanotubes (MWCNT), produced for industrial
purposes, on the human epithelial cell line A549. MWCNT were
dispersed in dipalmitoyl lecithin (DPL), a component of pulmo-
nary surfactant, and the effects of dispersion in DPL were
compared to those in two other media: ethanol (EtOH) and phos-
phate-buffered saline (PBS). Effects of MWCNT were also com-
pared to those of two asbestos fibers (chrysotile and crocidolite)
and carbon black (CB) nanoparticles, not only in A549 cells but
also in mesothelial cells (MeT5A human cell line), used as an
asbestos-sensitive cell type. MWCNT formed agglomerates on top
of both cell lines (surface area 15–35 mm2) that were significantly
larger and more numerous in PBS than in EtOH and DPL. What-
ever the dispersion media, incubation with 100 mg/ml MWCNT
induced a similar decrease in metabolic activity without changing cell
membrane permeability or apoptosis. Neither MWCNT cellular
internalization nor oxidative stress was observed. In contrast,
asbestos fibers penetrated into the cells, decreased metabolic
activity but not cell membrane permeability, and increased apop-
tosis, without decreasing cell number. CB was internalized with-
out any adverse effects. In conclusion, this study demonstrates
that MWCNT produced for industrial purposes exert adverse
effects without being internalized by human epithelial and
mesothelial pulmonary cell lines.
Carbon nanotubes (CNT) are cylinders of one or several
(up to 20) graphite layers (single- or multiwall carbon nano-
tubes, respectively: SWCNT and MWCNT). Their diameter
is in the order of the nanometer, and they can measure up to
several micrometers in length. Because of their unique elec-
trical properties, unusual strength, and particular effective-
ness in heat conduction, CNT are particularly promising
nanomaterials for industrial use in medical as well as non-
medical applications (see http://www.nanotechproject.org/44/
for inventory). However, the same novel properties that make
CNT interesting raise concerns about their potential adverse
effects on biological systems, which may lead to health
issues.
Pulmonary effects of CNT have been evaluated in a num-
ber of in vivo and in vitro studies. Mice and rats exposed by
the respiratory route showed acute and chronic pulmonary
inflammation with and without fibrosis (Lam et al., 2004;
Li et al., 2007; Muller et al., 2005; Shvedova et al., 2005;
Warheit et al., 2004). Extrapulmonary effects of respiratory
administered CNT were also recently reported, with the
presence of aortic mitochondrial DNA damage after a single
intrapharyngeal installation of mice to SWCNTs (Li et al.,
Received 16 May 2008; accepted 10 September 2008.
Lyes Tabet is a recipient of a joint grant from ADEME (Agence de
l’Environnement et de la Maitrîse de l’Énergie) and ARKEMA. Part
of this work was supported by the French ANR through
RESPINTTOX project (SEST program) and by the Région Ile-de-France
in the frame-work of C’nano-IdF, NANOTUBTOX project. C’Nano-IdF
is the nanoscience competence center of Paris Region, supported by
CNRS, CEA, MESR, and Région Ile-de-France. Nadia Amara is
supported by Chancellerie des Universités de Paris (legs Poix) and
Jorge Boczkowski by INSERM and Assistance Publique-Hôpitaux
de Paris (Contrat d’Interface). The authors thank Marie-Annick
Billon-Galland for her help and expertise in electronic microscopy,
Marie-Claude Jaurand for providing the Met5A cells, and Hélène
Desquerroux (ADEME) for her helpful comments. Ari Setyan and
Michel J. Rossi acknowledge partial support from the French
NANOTOX (ANR) program.
Address correspondence to Jorge Boczkowski, Inserm U700,
Faculté de Médecine, Paris 7, site X. Bichat, BP416 75870, Paris
Cedex 18, France. E-mail: jorge.boczkowski@inserm.fr
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 3

TOXIC EFFECTS OF INDUSTRIAL CARBON NANOTUBES
61
2007). Interestingly, a study by Shvedova et al. (2005)
found the number of alveolar type II cells increased in
response to SWCNT, which highlights the importance of
this cell type in the pulmonary response to CNT exposure.
In vitro studies demonstrated that CNT induced cytotoxicity
and/or inflammatory responses in different cell types
(Bottini et al., 2006; Cui et al., 2005; Ding et al., 2005;
Jia et al., 2005; Kagan et al., 2006; Kisin et al., 2007;
Manna et al., 2005; Monteiro-Riviere et al., 2005a, 2005b;
Sayes et al., 2006; Tian et al., 2006). However, among these
studies, very few examined the effects of CNT on alveolar
type II cells.
Accumulating evidence shows that adverse effects of
CNT, as well as those of other nanomaterials, are related to
their physicochemical properties (Smart et al., 2006). There-
fore, effects observed with one CNT may not necessarily be
extrapolated to another CNT, even if both are single- or mul-
tiwalled. In this context, and from a public health perspective,
it is critical to analyze potential adverse effects of CNT
produced in large amounts for industrial applications. Indeed,
although providing valuable information, several of the
published studies investigated the effects of CNT produced in
limited amounts in research laboratories. Other studies inves-
tigated the effects of CNT produced by small industrial
companies, but further modified in research laboratories. All
of these CNT might differ physically and chemically from the
ones produced in large amounts for different industrial
applications.
Therefore, the aim of the present study was to evaluate
adverse effects of MWCNT produced for industrial purposes.
These CNT were produced by chemical vapor deposition
(CVD) in a French facility (ARKEMA France). Toxicological
effects (cell viability, apoptosis, and oxidative stress) as well as
cellular internalization of CNT were analyzed in the human
lung epithelial cell line A549, as representative of human alve-
olar type II cells (Foster et al., 1998).
To simulate human respiratory exposure, MWCNT were
dispersed in dipalmitoyl lecithin (DPL), a component of pul-
monary surfactant (Lu et al., 1994). Since the dispersion status
affects biological effects of nanomaterials (Monteiro-Riviere
et al., 2005b), the effects of dispersion in DPL were compared
with those of dispersion in two other media: phosphate-buffered
saline (PBS) or ethanol (EtOH).
Concern exists about whether fiber-shaped nanoscale
particles formed from carbon and other materials behave
like asbestos, a toxic and carcinogenic fiber (Mohr et al.,
2005; Takagi et al., 2008; Poland et al., 2008). Therefore, in
the present study, effects of MWCNT were compared to
those of two asbestos fibers, chrysotile and crocidolite, as
well to those of carbon black (CB) nanoparticles. This com-
parison was performed not only using A549 cells, but also
in mesothelial cells (human MeT5A cell line), which are
sensitive to asbestos fibers (Mohr et al., 2005; Nymark
et al., 2007).
METHODS
Experimental Design
When intending to evaluate the potential adverse effects of
nanomaterials, it is of prime importance to obtain a thorough
knowledge of the nanomaterials used. Therefore, a thorough
physicochemical characterization of MWCNT (in powder, in
solution, and after contact with cells) was performed, using
electronic microscopy techniques and chemical analytic tools.
In order to evaluate the potential adverse effects of nanomateri-
als, a comprehensive approach was used, aimed to evaluate both
cytotoxic effects and the underlying mechanisms (apoptosis,
proliferation, oxidative stress, internalization).
Particles
MWCNT (Graphistrength C100, ARKEMA, France) were
produced by CVD on a supported catalyst in a fluidized bed.
Graphistrength C100 is produced as material composed of
MWCNT entangled around the supported catalyst. These
spherical heaps of MWCNT are about few hundred microme-
ters in diameter and form a free-flowing powder.
The effects of MWCNT were compared to those of two
types of asbestos fibers—chrysotile (20 nm diameter) and cro-
cidolite (80 nm diameter), from UICC (Union Internationale
Contre le Cancer) (Kido et al., 2008; Dopp et al., 1997)—and
to nanosized carbon black (CB, FR101, primary particles of
95 nm diameter; Degussa/Evonik, Germany; Pigmentrusse/
pigment blacks, Technische Daten Europa/Technical Data
Europe, Degussa AG, Advanced Fillers & Pigments, 2006).
CNT Physicochemical Characterization
MWCNT dimensions were measured by transmission elec-
tron microscopy (TEM). Chemical composition and carbon
content were determined by inductively coupled plasma
spectroscopy (ICP-MS), electron spectroscopy for chemical
analysis (ESCA), and scanning electron microscopy (SEM)
and TEM analysis (Figure 1A). Specific surface area was mea-
sured using the Brunauer–Emmett–Teller (BET) method using
adsorption isotherms of nitrogen at 77K. The surface composi-
tion in terms of functional groups was examined by surface
titration using six probe gases undergoing heterogeneous reac-
tions in a Knudsen flow reactor (Demirdjian & Rossi, 2005).
Particle Suspensions
MWCNT were suspended at 10 mg/ml in aqueous DPL,
PBS, or EtOH. Asbestos fibers were suspended in culture
medium, and CB particles in PBS. Particle solutions were
vortexed for 1 min and sonicated (RLI 275 sonication bath,
LIREC, France) for 30 min under cooling conditions, with
30-s interruption every 10 min for vortex at maximum speed.
Immediately after the end of sonication, particle solutions
were vortexed for 1 min at maximum speed and diluted in
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 4

62
L. TABET ET AL.
culture medium. The particle size distribution functions
(PSD) of the different particle suspensions were analyzed
using a Malvern Mastersizer S and were represented by the
volume distribution functions in the range 50 nm to 880 μm.
For these experiments, particles were also suspended in pure
H2O as a control. The results were analyzed using the spheri-
cal particle approximation with refractive indices of 1.5295 and
1.3300 for CNT and H2O, respectively. The measurement of the
PSD in the submicrometer range in terms of a number rather
than volume distribution was attempted using a SMPS system
(TSI, Inc.) in which the particle suspension was atomized after
suitable sonication. However, the solvent blanks resulted in sev-
eral 104 counts in the 20 to 200 nm diameter range, presumably
owing to solutes giving rise to aerosol particles after evaporation
of H2O, so that this effort was abandoned.
The formation of agglomerates in suspension was character-
ized by TEM analysis. An aliquot of solutions was deposited
on 47-mm-diameter polycarbonate filters (0.2 μm pores) and
further analyzed by TEM with a JEOL 1200 EX II microscope
(Figure 1B). In addition, agglomerates formation after cell
incubation with particles was measured by optical microscopy
(see later description).
Cell Culture and Stimulation
Human alveolar epithelial cells (A549 cell line, ATCC,
France) and mesothelial cells (MeT5A cell line, ATCC,
France) were cultured as previously described (Amara et al.,
2007; Nymark et al., 2007), seeded in 96-well plates at 60,000
cells/ml, and grown to confluence (48 to 72 h later). Cells were
exposed for 6, 24, 48, or 72 h to serum-free medium, 0.1 to
100 μg/ml (0.02–20 μg/cm2) of MWCNT, or 100 μg/ml
(20 μg/cm2) asbestos fibers or CB nanoparticles. The final con-
centration of DPL, PBS, or EtOH in the culture medium was
1% for each condition of stimulation, a concentration that did
not elicit any cell toxicity (data not shown). As MeT5A cells
were used only to verify the main results obtained with A549
cells; these cells were exposed only to DPL-suspended
MWCNT at different concentrations (0.1 to 100 μg/ml).
Assessment of Cell Morphology and Agglomerates
Formation After Cell Incubation With Particles
Cell morphology was assessed by optical microscopy in
cells stained with Harris hematoxylin–phloxin. During the
observations, it was noted that MWCNT appeared as agglom-
erates attached to cells. These agglomerates were further quan-
tified in A549 and MeT5A cells exposed for 48 h to 100 μg/ml
MWCNT suspended in different media. For each condition of
stimulation, 10 fields (magnification 10×) were selected from
the top to the bottom across the vertical diameter of the culture
well. The following parameters were analyzed by use of a
video microscope coupled to the AnalySIS 3.0 software (Soft
Imaging System GmbH, Germany): (1) proportion of the field
surface occupied by cells, (2) number of agglomerates present
in each examined field, and (3) surface area of each agglomer-
ate. An agglomerate was defined as a black individualized
area, of more than 0.20 μm2. Analysis was performed blinded
by two independent observers (LT and NA). The coefficient of
variation for measurement of the 3 parameters was <5%.
Assessment of Cell Viability
Two methods were used to evaluate changes in cell viabil-
ity: MTT, and neutral red assays. These tests were performed
FIG. 1.
Transmission electronic microscopy (TEM) images of MWCNT powder (A) or in suspension (B) in the different media (1, DPL; 2, EtOH; 3, PBS).
A
B
1 µm
1 µm
1 µm1 µm
1
2
3
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 5

TOXIC EFFECTS OF INDUSTRIAL CARBON NANOTUBES
63
as previously described (Davoren et al., 2007; Monteiro-
Riviere et al., 2005a, 2005b; Worle-Knirsch et al., 2006).
Results of cell viability are expressed as the means of at least
three independent experiments, each of six replicates, given as
the ratio of the mean for each condition to the mean of the con-
trol condition (cells cultured in media containing the respective
suspension agent). Since nanomaterials might interfere with
cytotoxicity tests (Monteiro-Riviere & Inman, 2006; Worle-
Knirsch et al., 2006), the assays were performed with and with-
out 100 μg/ml MWCNT during incubation with the dye and
measured absorbance. No interference of MWCNT with MTT
and neutral red assays was observed (data not shown).
Assessment of Cell Number
Cell number was assessed by quantifying DNA content with
the fluorescent dye bisbenzimide H33258 (Hoechst 33258,
Sigma, France), as described previously (Bussy et al., 2008).
The dye was added to cells at the end of the stimulation period
(24, 48, or 72 h) and fluorescence was measured at 460 nm
(excitation: 360 nm). Interference of MWCNT with the fluo-
rescent dye was evaluated by the incubation of a known con-
centration of DNA in the presence or absence of 100 μg/ml of
MWCNT. No interference was observed (data not shown).
Assessment of Apoptosis
DAPI Staining
Apoptosis was examined by 4′,6-diamidino-2-phenylindole
dihydrochloride (DAPI) staining (Sigma-Aldrich, France) cou-
pled to fluorescence microscopy analysis in the same experi-
mental setting used for assessment of cell number. Cells were
seeded onto sonic seals and were exposed for 24, 48, or 72 h to
100 μg/ml of particles or suspension medium alone. At the end
of stimulation, the cells were fixed with 4% paraformaldehyde
in PBS for 25 min at room temperature. DAPI solution (1 μg/ml)
was added for 5 min at 37°C. Observations involved use of a
fluorescence video microscope (Leica DMIRB) (excitation 358
nm; emission 461 nm). For each condition of stimulation, 10
fields (magnification 63×) were selected from top to bottom
across the vertical diameter of the culture well. The percentage
of apoptotic cells was calculated as follows: Percent apoptotic
cells=(total number of cells with apoptotic nuclei/total number
of counted cells)×100. Analysis was performed in a blind way
by two independent observers (LT and NA). The coefficient of
variation for measurement of the 2 parameters was <5%.
DNA Laddering
Genomic DNA (250 ng) of cells exposed for 72 h to DPL at
1% or 100 μg/ml MWCNT, crocidolite, or etoposide as a positive
control was extracted and purified using DNAeasy Blood &
Tissue QIAGEN kit (Qiagen, France) as per the manufacturer’s
instructions. Finally, degradation of DNA was visualized with
SYBR green on a 1.5% agarose gel.
Assessment of Cell Proliferation
The effects of nanomaterials on cell proliferation were
determined by the bromodeoxyuridine (BrdU) cell prolifera-
tion enzyme-linked immunosorbent assay (ELISA; Roche
Applied Science, Germany). The assay is based on the immun-
odetection of BrdU incorporated into the genomic DNA in
place of thymidine of proliferating cells. Cells were cultured in
96-well plates. Upon confluence, they were exposed, in serum-
free medium, for 24, 48, or 72 h to particles from 0.1 to 100
μg/ml or to suspension medium alone. BrdU was added to the
medium 20 h before the end of stimulation. At the end of incu-
bation, the labeling solution was removed, and 200 μl of
FixDenat solution was added and incubated for 30 min at room
temperature. After removing FixDenat, 100 μl anti–BrdU-POD
solution was added and incubated for 90 min at room tempera-
ture. After washing 3 times with 200 μl/well of washing buffer,
100 μl tetramethylbenzidine (TMB) substrate solution was
added and incubated for 5 to 30 min at room temperature until
color development was sufficient for photometric detection.
The reaction was stopped with the addition of 25 μl/well
H2SO4 (1 M). The absorbance of the samples was measured in
a microplate reader at 450–690 nm within 5 min after adding
H2SO4.
Assessment of Internalization of Nanomaterials
Cells exposed for 48 h to different nanomaterials at 100 μg/ml
were analyzed by transmission electronic microscopy (TEM).
Cells were adherent on their plastic surface and fixed in situ or
were trypsinized and fixed in suspension in a mix of 2%
paraformaldehyde/0.5% glutaraldehyde, postfixed in 1% osmic
acid, and embedded in Epon. Fine (1 μm thick) and ultrafine
(60 nm thick) slices were cut, stained with uranyl acetate and
lead salt, and observed under Jeol 1010 TEM (60 keV). As
observations were similar after both fixation processes (in situ or
after trypsinization), only in situ-fixed cells are shown.
Markers of Oxidative Stress
Oxidative stress was evaluated by analyzing mRNA expres-
sion of the anti- and pro-oxidant systems HO-1, SOD2, GPx,
and NOX4, respectively (Amara et al., 2007; Ryter & Choi,
2005; Sumimoto et al., 2005) by quantitative real-time reverse-
transcription polymerase chain reaction (RT-PCR) by use of
the PCR ABI 7700 apparatus (Applied Biosystems), after
exposure of cells for 6 or 24 h up to 100 μg/ml nanoparticles.
The following sets of primers were used: HO-1, 5′-TTCT-
TCACCTTCCCCAACATTG-3′ and 5′-CAGCTCCTGCAACTC-
CTCAAA-3′; SOD2, 5′-GAACGAGCATCCTGTCTTCG-3′ and
5′-CCAAATGATGAGCTTGGGATC-3′; GPX-2, 5′-GCCCTG-
GAACCTCACATCAAC-3′ and 5′-CGGCTCAGGTTGT-
TCACGTAG-3′; and NOX4, 5′-CTCAGCGGAATCAATCAGCTGTG-3′
and 5′-AGAGGAACA CGACAATCAGCCTTAG-3′. Ubiquitin
was used as housekeeping gene: 5′-CACTTGGTCCTGCGCTTGA-3′
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 6

64
L. TABET ET AL.
and 5′-TTTTTTGGGAATGCAACAACTTT-3′. Expression of
mRNA was normalized to that of ubiquitin. In previous experi-
ments it was verified that ubiquitin mRNA expression did not
change in the different experimental conditions (data not
shown).
Statistical Analysis
Experiments were performed at least in triplicate. Values are
given as mean ± SEM of values obtained for each experiment.
The data were analyzed by nonparametric tests (GraphPad Prism
software). A value of p < .05 was considered significant.
RESULTS
Physicochemical Characteristics of CNT
MWCNT present an average diameter of 12 nm (size distri-
bution: 0–15 nm, 85%; 15–30 nm, 13%; and >30 nm, 2%) and
a length of 0.1 up to 13 μm as observed by TEM (Figure 1A).
No free amorphous carbon was detectable by SEM and TEM,
and ESCA revealed the presence of graphitic carbon, with only
0.8 at% of oxygen. Metallic impurities, measured by ICP-MS,
were aluminum 2.4% and iron 2%. BET analysis under
nitrogen gave a specific surface area value of 219.2 m2/g.
Surface chemical analysis of MWCNT was based on titra-
tion of surface functional groups using six probe gases
[N(CH3)3, NH2OH, HCl, CF3COOH, O3, NO2] that specifi-
cally interacted with the MWCNT sample located in the
sample compartment of a Knudsen flow reactor. Table 1 indi-
cates the number of functional groups located on the surface of
MWCNT that are “interrogated” by the probe gases per
milligram and per square centimeter of MWCNT or in terms of
a formal monolayer of probe gas adsorbed on MWCNT. As an
example, the base N(CH3)3 interacts with acidic surface
groups. Four main conclusions may be drawn from the results
displayed in Table 1: (1) There are very few acidic sites inter-
acting with N(CH3)3, significantly less than on diesel soot
(A. Setyan, personal communication); (2) the degree of partial
oxidation of MWCNT, that is, the amount of carbonyl groups
interacting with NH2OH, is comparable to that of commer-
cially available amorphous carbons such as CB, but smaller
than for diesel soot or secondary organic aerosol (SOA)
(Setyan, personal communication), which indicates a moderate
degree of surface oxidation; (3) there is a high degree of unsat-
uration present on the surface as measured by O3 uptake, which
may be due either to the presence of olefinic double bonds or
polycyclic aromatic hydrocarbon moieties with double-bond
localization, such as acenaphthylene, pentalene, or azulene;
and (4) the surface of MWCNT is multifunctional, indicating
apparent coexistence of acidic and basic sites as well as
partially oxidized and reduced (unsaturated) sites on the sur-
face of MWCNT without apparent internal acid–base or redox
reactions.
Particle Agglomerate Formation in Suspension
and Over Exposed Cells
The results of the PSD measurement of the suspensions
using light scattering may be summarized as follows: None of
the suspensions (pure double-distilled H2O, 1% DPL, 1%
EtOH, 1% PBS) showed the presence of primary particles of
CNT, undoubtedly owing to lack of sensitivity of the instru-
ment. However, after sonication, the suspensions in pure H2O
and 1% EtOH showed the presence of submicrometer particles
with a volume fraction of 13.6 and 2.5% integrated around the
maximum at 60 ± 5 nm, which corresponds to the presence of
109 and 2.5×1010 submicrometer particles of 60 nm to 1 parti-
cle at 100 and 650 μm, respectively. The sensitivity of the used
instrument is apparently not sufficient to detect the volume
fraction of the primary CNT particles in the suspensions of
DPL and PBS, even after sonication. Interestingly enough, the
presence of the additives seems to affect primarily the size of
the agglomerates, whose size changes upon sonication except
in suspensions containing 1% DPL. The latter is characterized
by a significant volume fraction of heavy particles at 22.5 and
480 μm, frequently detectable even by the naked eye. Based on
the sonication experiments a large number of primary CNT
particles was expected even in this case.
TABLE 1
Characterization of Surface Functional Groups Present on MWCNT using a Knudsen Flow Reactor
Gas
[gas-phase
probe molecules]
N(CH3)3
[acidic sites]
HCI
[basic sites]
CF3COOH
[basic sites]
NH2OH
[carbonyl
functions]
O3
[oxidizable sites]
NO2
[oxidizable sites]
no/mga
no/cm2b
MLc
3.0 ⋅1015
1.4 ⋅1012
0.28%
8.1 ⋅1016
3.7 ⋅1013
7.4%
2.4 ⋅1016
1.1 ⋅1013
2.2%
2.4 ⋅1017
1.1 ⋅1014
22%
3.9 ⋅1018
1.8 ⋅1015
360%
1.2 ⋅1016
5.3 ⋅1012
1.1%
Results are expressed as indicated in a, b and c and using a BET surface of 219.2 m2/g.
aNumber of probe molecules taken up per mg of deposited nanoparticles.
bNumber of probe molecules taken up per square cm of MWCNT (number/cm2).
cNumber or fraction of formal monolayer (ML) of reacted probe gas using an average of 5 × 1014 molecules cm−2 as one formal ML.
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 7

TOXIC EFFECTS OF INDUSTRIAL CARBON NANOTUBES
65
Transmission Electron Microscopy (TEM)
As shown in Figure 1B, MWCNT form agglomerates that
either preexisted in solution, irrespective of the dispersion media,
or that were generated during solvent evaporation in the course of
the preparation of the TEM sample. Moreover, individual
MWCNT less than 100 nm in dimension were also observed in all
three media. This diameter from TEM imaging is commensurate
with the MWCNT monomer, that is, primary particle diameter
from light scattering experiments (see earlier description).
Optical Microscopy
Optical microscopy examination of cells exposed to 100 μg/ml
MWCNT, asbestos, or CB nanoparticles for 48 h revealed no
change in the morphology of exposed cells. Control and
exposed A549 cells showed typical cuboidal shape, indicative
of type II alveolar cell morphology (Figure 2A) (Nardone &
Andrews, 1979), and MeT5A cells showed typical features of
mesothelial cells (Figure 2B) (Mutsaers, 2004). MWCNT
formed agglomerates (black area on the field), which seem to
FIG. 2.
CB (2), chrysotile (3), crocidolite fibers (4), or MWCNT suspended in DPL, EtOH, or PBS (5, 6, and 7 respectively). Magnification: ×20.(B) MeT5A cells
exposed for 48 h to medium alone (1), 100 μg/ml CB (2), crocidolite (3), or MWCNT suspended in DPL (4). Magnification: ×20.
Representative optical microscopy images of cells exposed to the different particles. (A) A459 cells exposed for 48 h to medium alone (1), 100 μg/ml
1
3
57
4
2
6
A
B
1
234
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 8

66
L. TABET ET AL.
be on the top of the cells, because they strictly colocalized with
the cells despite thorough washing before the fixation/staining
procedure. Exposure of A549 cells to three different suspen-
sions of MWCNT led to formation of agglomerates (range 15
to 30 μm2) that were significantly larger in PBS than in DPL
and EtOH (Table 2). The results from inspection of optical
microscopy images displayed in Table 2 are in rough qualita-
tive agreement with the PSD obtained in MWCNT suspensions
despite the disparate nature of the experiments. Light scattering
resulted in apparent diameters of 22.5, 21, and 100 μm for
DPL-, EtOH-, and PBS-containing suspensions of MWCNT,
respectively. In this comparison, the largest particles recorded
in the course of light-scattering experiments were omitted owing
to their rareness in terms of number concentration compared to
the large number of smaller particles. The mean number of
agglomerates was significantly lower when MWCNT were dis-
persed in EtOH than in the two other media (Table 2). The
fraction of the field surface occupied by cells did not vary with
the different experimental conditions (data not shown). The
size of agglomerates formed by MWCNT suspended in DPL
was similar over A549 and MeT5A cells. However, the num-
ber of agglomerates appeared less important in MeT5A cells.
Cell Viability
Cell viability was analyzed with the MTT and neutral red
assays, which determine mitochondrial metabolism and plasma
membrane permeability, respectively. For the MTT assay,
MWCNT effects on A549 cell viability were similar for the
three dispersion media (Figure 3A). Compared with respective
controls (cells cultured in media containing the respective sus-
pension agent without particles), MTT values decreased signif-
icantly with 10 μg/ml to 100 μg/ml incubation, reaching 60%
of control values for 100 μg/ml incubation at 48 h postexpo-
sure (for 10 and 100 μg/ml). Similar results were obtained after
exposure of MeT5A cells to DPL-suspended MWCNT (Figure 3B
and data not shown; for a better reading, only results obtained
after 48 h of incubation of MeT5A cells with the different
nanoparticles are shown subsequently). In both cell types,
incubation with 100 μg/ml chrysotile or crocidolite elicited
similar decreases in MTT, which were not different from that
induced by 100 μg/ml MWCNT after 24 h. No change in MTT
was observed when cells were incubated with CB nanoparti-
cles at 100 μg/ml, except for significant decrease after incuba-
tion of MeT5A cells with CB for 48 or 72 h (Figure 3).
In contrast, results of the neutral red assay showed no sig-
nificant alteration of A549 cell viability after incubation with
MWCNT in the three media, whatever the time point consid-
ered (data not shown). Similar results were observed with
MeT5A cells (data not shown). Moreover, neither asbestos
fibers nor CB nanoparticles induced significant diminution of
viability as assessed by neutral red assay, whatever the cell
type studied (data not shown). Interestingly, incubation of both
cell types with asbestos fibers induced an increase in neutral
red incorporation, as well as incubation of MeT5A cells with
CB nanoparticles for 6 or 24 h (data not shown). Collectively,
these results suggest that MWCNT and asbestos fibers alter
mitochondrial metabolism, with no apparent effect on cell
membrane permeability.
Cell Number, Apoptosis, and Proliferation
Whether the impaired mitochondrial metabolism might
result in decreased cell number after incubation with MWCNT
was next evaluated. This was first assessed by quantifying
DNA content. Incubation with 100 μg/ml MWCNT resulted in
a significant decrease in A549 cell number after 24, 48, and
72 h regardless of dispersion media, with a reduction of 15 to
20% of control cell number (Figure 4A). No such effect was
observed with MeT5A cells (Figure 4B and data not shown).
Moreover, neither asbestos fibers nor CB induced a significant
decrease in total DNA content, except after 24 h of treatment
of MeT5A cells with CB (Figure 4 and data not shown). Fur-
thermore, a significant increase in DNA content was observed
after incubation of A549 cells with crocidolite for 48 h.
To investigate the mechanism(s) involved in the reduced
A549 cell number after MWCNT incubation, apoptosis was
examined. DAPI staining showed a small proportion of con-
trol cells with apoptotic nuclei (1 to 2% of total cells) at 48
and 72 h, and this fraction did not change significantly in the
cells incubated with MWCNT (Figure 5 and data not shown).
In contrast, a significant increase in the proportion of apop-
totic cells was observed after 72 h incubation with crocidolite
(approximately 5% of total cells). Similar results were
obtained with MeT5A cells (Figure 5 and data not shown).
DNA laddering experiments confirmed those results in both
cell types, except for an absence of modification induced by
exposure to asbestos fibers (Figure 6 and data not shown).
Finally, no significant modification of proliferation was
observed, whatever the nanomaterials, type point, and cell
type (data not shown).
TABLE 2
Quantification of Agglomerates Formed after 48 hr Incubation
of A549 or MeT5A Cells with 100 mg/ml of MWCNT
Suspended in DPL, EtOH, or PBS
MWCNT
agglomerate area
(mm2)/field
MWCNT
agglomerate
number/field
A549 (DPL)
(EtOH)
(PBS)
Met5A (DPL)
16,64 ± 1,50
20,11 ± 2,01
31,08 ± 2,20*
14,29 ± 0,93
154,50 ± 7,23
99,60 ± 5,15#
155,40 ± 10,11
42,80 ± 2,09
Results are means ± SEM of the values obtained for 10 fields.
Asterisk indicates significant at p < .05 vs. DPL and EtOH; #signifi-
cant at p < .05 vs. DPL and PBS.
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 9

TOXIC EFFECTS OF INDUSTRIAL CARBON NANOTUBES
67
Cellular Internalization of Nanomaterials
Internalization of the different materials in A549 cells was
analyzed by TEM, which showed morphological features such
as lamellar body structure, microvilli, and tonofilament charac-
teristics of alveolar epithelial type II cells in control cell culture
(Figure 7). These morphological characteristics were similar in
cells incubated with 100 μg/ml MWCNT for 24 h. No nuclear
change revealing apoptosis was observed. No evidence of
MWCNT internalization, whatever the dispersion media used,
was found. In contrast, CB particles and chrysotile and crocidolite
FIG. 3.
μg/ml CB, chrysotile, crocidolite, or 0.1 to 100 μg/ml MWCNT in 1% DPL, EtOH, or PBS. (B) Viability, expressed as percent of control cell values, of MeT5A
cells after 48 h of exposure to 100 μg/ml CB, chrysotile, crocidolite, or 0.1 to 100 μg/ml MWCNT in 1% DPL. Asterisk indicates significant at p < .05 vs. control
condition.
Cell viability assessed by MTT assay. (A) Viability, expressed as percent of control cell values, of A549 cells after 6, 24, 48, or 72 h of exposure to 100
1% DP L
1% EtOH
1% PBS
48H
0
20
40
60
80
100
120
140
relative viability (% of control)
CB
chrysotile
crocidolite
100 g/ml 0.11 10
MWCNT
*
*
*
6H
0
20
40
60
80
100
120
140
relative viability (% of control)
CB
chrysotile
crocidolite
100 g/ml 0. 11 10
MWCN T
*
72H
0
20
40
60
80
100
120
140
relative viability (% of control)
CB
chrysotile
crocidolite
100 g/ml0.1110
MWCNT
*
24H
0
20
40
60
80
100
120
140
relative viability (% of control)
CB
chrysotile
crocidolite
100 g/ml 0. 11 10
MWCN T
*
*
A
48H
0
20
40
60
80
100
120
chrysotile
crocidolite
100 g/ml0.1110
MWCNT
CB
relative viability (% of control)
*
*
*
B
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 10

68
L. TABET ET AL.
fibers were observed inside cells incubated with these particles.
Similar results were obtained after exposure of MeT5A cells to
the different nanomaterials (data not shown).
Oxidative Stress
Since MWCNT were not internalized in cells, whether the
observed decrease in viability could be related to oxidative
stress was examined by analyzing mRNA expression of the
different genes implied in an oxidative response: HO-1, SOD2,
GPx, and NOX4 (Li & Shah, 2002; Taille et al., 2004). No sig-
nificant change in expression of HO-1 mRNA was observed in
cells incubated with MWCNT in the different media for 6 or 24 h
or with asbestos fibers (data not shown). The same results were
obtained for SOD2 and GPx genes (data not shown). Finally,
no change was observed in the mRNA of NOX4 (data not
shown), a pro-oxidant system involved in amplification of the
oxidative response in cells exposed to diesel exhaust particles
(Amara et al., 2007).
DISCUSSION
The MWCNT examined in the present study were produced
by CVD as spherical sets of agglomerates of primary MWCNT
particles in the range of several tens to several hundreds of
micrometers of diameter. In the present study, the aim was to
be as close as possible to an in vivo respiratory exposure and
therefore the CNT were dispersed in DPL, a component of pul-
monary surfactant. The issue of the biological relevance of dis-
persion media in the evaluation of the toxicological effects of
manufactured nanomaterials is critical (Fu & Sun, 2003;
Monteiro-Riviere et al., 2005b; Lanone & Boczkowski, 2006),
FIG. 4.
crocidolite, or 100 μg/ml MWCNT in 1% DPL, EtOH, or PBS. (B) DNA content, expressed as a percentage of control cell values, of MeT5A cells after 48 h of
exposure to 100 μg/ml CB, crocidolite, or 100 μg/ml MWCNT in 1% DPL. Asterisk indicates significant at p < .05 vs. control condition.
DNA content. (A) DNA content, expressed as percent of control cell values, of A549 cells after 24, 48, or 72 h of exposure to 100 μg/ml CB,
48H
0
20
40
60
80
100
120
140
*
crocidolite
CB
MWCNT
72H
24H
0
20
40
60
80
100
120
140
*
DNA content
(% of control)
DNA content
(% of control)
DNA content
(% of control)
crocidolite
CB MWCNT
1% DPL
1% EtOH
1% PBS
20
40
60
80
100
120
140
*
*
crocidolite
CB MWCNT
0
A
DNA content
(% of control)
48H
0
20
40
60
80
100
120
140
crocidolite
MWCNT
CB
B
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 11

TOXIC EFFECTS OF INDUSTRIAL CARBON NANOTUBES
69
and DPL has been extensively used in studies investigating the
respiratory effects of air pollution particles (Bachoual et al.,
2007; Baulig et al., 2003). Using this method of dispersion it
was observed that, in addition to the presence of primary
MWCNT particles, MWCNT agglomerates of micrometer size
were present in solution based on TEM image analysis, light
scattering of the suspension, and optical microscopy of
exposed cells. The relative contribution of the primary particles
to the agglomerates is not known when the particles come out
of production. Indeed, it is well documented that individual,
raw CNT form agglomerates in solution (Bianco et al., 2006).
Dispersing the MWCNT in the two other examined media
(PBS ad EtOH) resulted also in similar formation of agglomer-
ates accompanied by the presence of primary MWCNT parti-
cles. Although agglomerates in DPL were smaller than in PBS,
they were still in the micrometer range. In DPL two types of
aggregates corresponding to a bimodal distribution of 22.5 and
480 μm peak diameter in terms of volume fraction, were
observed, whereas in PBS a single broad particle distribution
from 10 to 400 μm was recorded that may have coalesced from
FIG. 5.
cells to 100 μg/ml nanomaterials for 72 h. (C and D) Representative microscopic images of MeT5A cells (C) and quantification of DAPI-positive cells (D), after
exposure of cells to 100 μg/ml nanomaterials for 72 h. Magnification ×60. Asterisk indicates significant at p < .05 vs. control.
Apoptosis assessment. (A and B) Representative microscopic images of A549 cells (A) and quantification of DAPI-positive cells (B), after exposure of
1% DPL
1% EtOH
1% PBS
A
crocidolite
CB MWCNT Controls
*
0
1
2
3
4
5
6
7
% of nuclear apoptosis
B
C
CB MWCNT Controls
0
1
2
3
4
5
6
7
8
9
crocidolite
*
% of nuclear apoptosis
D
MWCNT (DPL) crocidolite 0%FCS
MWCNT (DPL)
Crocidolite 0%FCS
FIG. 6.
1), 1% DPL (lane 2), 100 μg/ml crocidolite (lane 3), MWCNT in 1% DPL (lane 4), or etoposide used as positive control (lane 5).
DNA laddering. (A and B) Representative image for DNA laddering experiments in A549 (A) and MeT5A (B) cells exposed for 72 h to 0% FCS (lane
A B
1234512345
DNA
Fragmentation
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 12

70
L. TABET ET AL.
the bimodal PSD distribution function. It needs to be stressed,
however, that, irrespective of the dispersing media, not all the
MWCNT present in the solution formed agglomerates, since
individual CNT were observed by TEM analysis of the suspen-
sions. Light scattering reveals a significant relative number
concentration of primary MWCNT particles on the order of 108
to 2.5×1010 particles to 1, thereby reaching volume fractions
up to 13.6% at a modal diameter of 60 ± 5 nm in pure H2O
after sonication. Sonication experiments in 1% EtOH dispers-
ant reveal the partly reversible nature of aggregation and
breakup of the MWCNT aggregates into primary particles, but
also the re-agglomeration with time once sonication has been
halted.
Irrespective of the dispersing media, MWCNT induced a
concentration- and time-dependent decrease in mitochondrial
metabolism, as revealed by a significant decrease in MTT
reduction, but no effect on membrane permeability, as
revealed by neutral red assay. Gao et al. (2001) showed that
silica particles, when mixed with dipalmitoyl phosphatidyl-
choline (DPPC), lose their cytotoxic potential on NR8383 rat
alveolar macrophages. However, in the same study, Gao et al.
(2001) also fail to demonstrate such protective effect of
surfactant coating for kaolin particles. The absence of effect
of dispersion in DPL, as compared to other media, on
MWCNT-induced cytotoxicity is in agreement with data pub-
lished by Monteiro-Riviere and collaborators (2005b) and
Wick and collaborators (2007) showing that cytotoxicity of
MWCNT in human epidermal keratinocytes and mesothelial
cells was independent of their dispersion in different concen-
trations of surfactants. However, Monteiro-Riviere et al.
(2005b) showed that HEK cells exposed to CNT dispersed in
surfactant produced less IL-8 than cells exposed to CNT
alone. Therefore, if dispersion in various media, and in sur-
factant in particular, is an important point to study, biological
effects cannot be considered as the results of one single end-
point. An effect of MWCNT on mitochondrial metabolism
without any alteration in cell membrane integrity agrees with
results published by different groups examining separately
these parameters in A549 (Davoren et al., 2007) and other
cell types (Muller et al., 2005; Wick et al., 2007). This may
be explained by the different intracellular targets/mechanisms
of the two assays. The metabolic effect of MWCNT on A549
cells further resulted in a decreased cell number after 48 to 72 h
of incubation, as shown by decreased cellular DNA content,
FIG. 7.
CB (2), chrysotile (3), crocidolite (4), or MWCNT in DPL (5), EtOH (6), or PBS (7).
Representative transmission electronic microscopy (TEM) images of A459 cells exposed for 48 h to 100 μg/ml of culture medium alone (1), 100 μg/ml
5 µm
1
5 µm5µm
5 µm
5 µm5 µm
5 µm
4
3
2
5
6
7
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 13

TOXIC EFFECTS OF INDUSTRIAL CARBON NANOTUBES
71
without any apoptosis or necrosis. The decrease in cell
metabolism and number occurred essentially after 24 h of cell
incubation with MWCNT and was stable thereafter, suggest-
ing an initial, nonprogressive insult. Despite a similar degree
of metabolic effect of MWCNT on MeT5A cells, no change
in total cell number was observed as well as any sign of apop-
tosis. Since in both cells types, no compensatory modification
of proliferation was observed, the exact mechanisms implied
in these events still need to be elucidated. A possibility may
be a modification of cell architecture, as described by Kaiser
et al. (2008), that might induce subsequent modification in
cell physiology, specific to each cell type, or, as proposed by
Fung et al. (1997), differing repair capacities. However, these
possibilities need further study.
MWCNT internalization in exposed cells was not seen. It
is important to note, however, that while large agglomerates
may not get internalized, small individual tubes could be
(such as those identified by light scattering experiments
reported earlier in this study), although they may not be visi-
ble at light microscopy level or even with TEM. Few studies
investigated the relation between agglomerate formation and
CNT internalization. Monteiro-Riviere and coworkers
(2005a) showed that the cytotoxic effect of MWCNT
agglomerates to human epidermal keratinocytes resulted from
CNT internalization. However, Davoren and coworkers
(2007) found no SWCNT internalization in A549 cells,
using a different solubilization media than utilized here.
Wörle-Knirsch and colleagues (2006) showed SWCNT
present in A549 cells, but prior to cell exposure the CNT
underwent an acidic treatment that may have modified sur-
face reactivity (Fu & Sun, 2003) and subsequent CNT inter-
nalization. Indeed, CNT uptake by cells is currently being
discussed and might depend on the state of functionalization
and morphology of the material (Bianco et al., 2005). Collec-
tively, our results and those of Davoren and coworkers (2007)
suggest that CNT, either SW or MW, are not always internal-
ized in A549 cells, despite their differing degrees of agglom-
eration. Some studies showed that, when comparing different
aggregated nanomaterials, one cannot conclude on the rela-
tive cytotoxicity induced by those materials from the agglom-
erate size range (Soto et al., 2007). Finally, another important
issue is that internalization is not an endpoint for particles to
have a biological effect. Indeed, Ovrevik et al. (2006) showed
that silica particles induce a cascade of events prior to any
internalization of the particles in A549 cells. Therefore, the
relationship between agglomeration and internalization may
not be the only parameter relevant to understanding biologi-
cal effects of MWCNT.
The effect of our MWCNT on cell metabolism may rely on
mechanisms other than cell internalization. In this context,
occurrence of oxidative stress, postulated as a central mecha-
nism in the cellular toxic effects of nanoparticles was
explored (Nel et al., 2006), but expression neither of HO-1, a
redox-sensitive antioxidant system, nor of GPx or SOD in
MWCNT-exposed A549 or MeT5A cells was modified. Fur-
thermore, the expression of the pro-oxidant system NOX4,
induced by diesel exhaust particles in A549 cells (Amara et
al., 2007), was unchanged in these cells. Such results may be
associated with the cancerous nature of A549 cells, as cancer
cell lines are known to be refractory to oxidant stress. How-
ever, along with exposure to particles, cells were exposed to
hydrogen peroxide as a positive control for oxidative stress
generation, and an increase in oxidative stress markers was
found (data not shown). Therefore, other nonoxidant mecha-
nisms may also be important to the effects of MWCNT and
asbestos. Activation of cell-surface receptors, involving a
redox-independent signaling cascade, may be an alternative
explanation. Interestingly, Ovrevik and coworkers (2006)
recently showed that silica upregulates interleukin (IL)-8
release from A549 cells through interactions with membrane
components prior to particle internalization. However, such a
mechanism might not occur in our study since, as opposed to
silica, which is acidic, our industrially produced MWCNT
contain few acidic groups at their surface. The oxidable
groups present at their surface may potentially act as free rad-
ical scavengers, as shown by Fenoglio and collaborators
(2006). However, investigation of such mechanisms requires
further study.
As stated previously, data are scarce in the current literature
comparing the effects of CNT with those of asbestos fibers.
Wick and coworkers (2007) showed that agglomerates of CNT
induced a cytotoxic effect similar to that of crocidolite fibers in
the human mesothelioma cell line MSTO-211H, but the ultra-
structural basis and molecular basis underlying these effects
were not established. A recent pilot study, by Poland and
coworkers (2008), also showed similar effects of carbon nano-
tubes and asbestos fibers in mice in which the mesothelial lin-
ing of the body cavity was exposed, as a surrogate for the
mesothelial lining of the chest cavity. However, the exact
molecular pathways were not addressed. In the present study,
although both MWCNT and asbestos fibers induced similar
alterations in viability of A549 and MeT5A cells, (1) in con-
trast to MWCNT, asbestos fibers did not diminish cell number
but augmented apoptosis, and (2) MWCNT were not internal-
ized in cells whereas asbestos fibers were clearly internalized.
The nature of the effects induced by MWCNT and those
induced by asbestos fibers were different, although the present
study cannot provide definitive answers on that issue. In con-
clusion, this study shows that MWCNT produced by CVD for
industrial purposes as spherical sets of several hundreds of
micrometers exert adverse biological effects without being
internalized by human epithelial and mesothelial pulmonary
cells.
REFERENCES
Amara, N., Bachoual, R., Desmard, M., Golda, S., Guichard, C., Lanone, S.,
Aubier, M., Ogier-Denis, E., and Boczkowski, J. 2007. Diesel exhaust par-
ticles induce matrix metalloprotease-1 in human lung epithelial cells via a
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 14

72
L. TABET ET AL.
NADP(H) oxidase/Nox4 redox-dependent mechanism. Am. J. Physiol.
Lung Cell Mol. Physiol. 293:L170–L181.
Bachoual, R., Boczkowski, J., Goven, D., Amara, N., Tabet, L., On, D.,
Lecon-Malas, V., Aubier, M., and Lanone, S. 2007. Biological effects of par-
ticles from the Paris subway system. Chem. Res. Toxicol. 20:1426–1433.
Baulig, A., Sourdeval, M., Meyer, M., Marano, F., and Baeza-Squiban, A.
2003. Biological effects of atmospheric particles on human bronchial epi-
thelial cells. Comparison with diesel exhaust particles. Toxicol. In Vitro
17:567–573.
Bianco, A., Kostarelos, K., and Prato, M. 2005. Applications of carbon nano-
tubes in drug delivery. Curr. Opin. Chem. Biol. 9:674–679.
Bottini, M., Bruckner, S., Nika, K., Bottini, N., Bellucci, S., Magrini, A.,
Bergamaschi, A., and Mustelin, T. 2006. Multi-walled carbon nanotubes
induce T lymphocyte apoptosis. Toxicol. Lett. 160:121–126.
Bussy, C., Cambedouzou, J., Lanone, S., Leccia, E., Heresanu, V., Pinault, M.,
Mayne-L’hermite, M., Brun, N., Mory, C., Cotte, M., Doucet, J.,
Boczkowski, J., and Launois, P. 2008. Carbon nanotubes in macrophages:
Imaging and chemical analysis by X-ray fluorescence microscopy. Nano
Lett., 8:2659–2663.
Cui, D., Tian, F., Ozkan, C., Wang, M., and Gao, H. 2005. Effect of single wall
carbon nanotubes on human HEK293 cells. Toxicol. Lett. 155:73–85.
Davoren, M., Herzog, E., Casey, A., Cottineau, B., Chambers, G., Byrne, H. J.,
and Lyng, F. M. 2007. In vitro toxicity evaluation of single walled carbon
nanotubes on human A549 lung cells. Toxico.l In Vitro 21:438–448.
Demirdjian, B., and Rossi, M. J. 2005. The surface properties of SOA from
limonene and toluene using specific molecular probes: A new experimental
technique. Atmos. Chem. Phys. Discuss. 5:607–654.
Ding, L., Stilwell, J., Zhang, T., Elboudwarej, O., Jiang, H., Selegue, J. P.,
Cooke, P. A., Gray, J. W., and Chen, F. F. 2005. Molecular characteriza-
tion of the cytotoxic mechanism of multiwall carbon nanotubes and nano-
onions on human skin fibroblast. Nano Lett. 5:2448–2464.
Dopp, E., Schuler, M., Schiffmann, D., and Eastmond, D. A. 1997. Induction
of micronuclei, hyperdiploidy and chromosomal breakage affecting the
centric/pericentric regions of chromosomes 1 and 9 in human amniotic
fluid cells after treatment with asbestos and ceramic fibers. Mutat. Res.
377:77–87.
Fenoglio, I., Tomatis, M., Lison, D., Muller, J., Fonseca, A., Nagy, J. B., and
Fubini, B. 2006. Reactivity of carbon nanotubes: Free radical generation or
scavenging activity? Free Radical Biol. Med. 40:1227–1233.
Foster, K. A., Oster, C. G., Mayer, M. M., Avery, M. L., and Audus, K. L.
1998. Characterization of the A549 cell line as a type II pulmonary epithe-
lial cell model for drug metabolism. Exp. Cell Res. 243:359–366.
Fu, K., and Sun, Y. P. 2003. Dispersion and solubilization of carbon nano-
tubes. J Nanosci. Nanotechnol. 3:351–364.
Fung, H., Kow, Y. W., Van Houten, B., and Mossman, B. T. 1997. Patterns of
8-hydroxydeoxyguanosine formation in DNA and indications of oxidative
stress in rat and human pleural mesothelial cells after exposure to crocidol-
ite asbestos. Carcinogenesis 18:825–832.
Gao, N., Keane, M. J., Ong, T., Ye, J., Miller, W. E., and Wallace, W. E. 2001.
Effects of phospholipid surfactant on apoptosis induction by respirable
quartz and kaolin in NR8383 rat pulmonary macrophages. Toxicol. Appl.
Pharmacol. 175:217–225.
Jia, G., Wang, H., Yan, L., Wang, X., Pei, R., Yan, T., Zhao, Y., and
Guo, X. 2005. Cytotoxicity of carbon nanomaterials: Single-wall nano-
tube, multi-wall nanotube, and fullerene. Environ. Sci. Technol.
39:1378–1383.
Kagan, V. E., Tyurina, Y. Y., Tyurin, V. A., Konduru, N. V., Potapovich, A. I.,
Osipov, A. N., Kisin, E. R., Schwegler-Berry, D., Mercer, R., Castranova,
V., and Shvedova, A. A. 2006. Direct and indirect effects of single walled
carbon nanotubes on RAW 264.7 macrophages: Role of iron. Toxicol. Lett.
165:88–100.
Kaiser, J. P., Wick, P., Manser, P., Spohn, P., and Bruinink, A. 2008. Single
walled carbon nanotubes (SWCNT) affect cell physiology and cell archi-
tecture. J. Mater. Sci. Mater. Med. 19:1523–1527.
Kido, T., Morimoto, Y., Asonuma, E., Yatera, K., Ogami, A., Oyabu, T.,
Tanaka, I., and Kido, M. 2008. Chrysotile asbestos causes AEC apopto-
sis via the caspase activation in vitro and in vivo. Inhal. Toxicol.
20:339–347.
Kisin, E. R., Murray, A. R., Keane, M. J., Shi, X. C., Schwegler-Berry, D.,
Gorelik, O., Arepalli, S., Castranova, V.,, Wallace, W. E., Kagan, V. E.,
and Shvedova, A. A. 2007. Single-walled carbon nanotubes: Geno- and
cytotoxic effects in lung fibroblast V79 cells. J. Toxicol. Environ. Health A
70:2071–2079.
Lam, C., James, J. H., McCluskey, R., and Hunter, R. 2004. Pulmonary toxic-
ity of single-wall carbon nanotubes in mice 7 and 90 days after intratra-
cheal instillation. Toxicol. Sci. 77:126–134.
Lanone, S., and Boczkowski, J. 2006. Biomedical applications and potential
health risks of nanomaterials: Molecular mechanisms. Curr. Mol. Med.
6:651–663.
Li, J. G., Li, W. X., Xu, J. Y., Cai, X. Q., Liu, R. L., Li, Y. J., Zhao, Q. F., and
Li, Q. N. 2007. Comparative study of pathological lesions induced by mul-
tiwalled carbon nanotubes in lungs of mice by intratracheal instillation and
inhalation. Environ. Toxicol. 22:415–421.
Li, J. M., and Shah, A. M. 2002. Intracellular localization and preassembly of
the NADPH oxidase complex in cultured endothelial cells. J. Biol. Chem.
277:19952–19960.
Lu, J., Keane, M. J., Ong, T., and Wallace, W. E. 1994. In vitro genotoxicity
studies of chrysotile asbestos fibers dispersed in simulated pulmonary sur-
factant. Mutat. Res. 320:253–259.
Manna, S. K., Sarkar, S., Barr, J., Wise, K., Barrera, E. V., Jejelowo, O., Rice-
Ficht, A. C., and Ramesh, G. T. 2005. Single-walled carbon nanotube
induces oxidative stress and activates nuclear transcription factor-kappaB
in human keratinocytes. Nano Lett. 5:1676–1684.
Mohr, S., Keith, G., and Rihn, B. 2005. Asbestos and malignant pleural
mesothelioma: Molecular, cellular and physiopathological aspects. Bull.
Cancer 92:959–976.
Monteiro-Riviere, N. A., Inman, A. O., Wang, Y. Y., and Nemanich, R. J.
2005b. Surfactant effects on carbon nanotube interactions with human
keratinocytes. Nanomedicine 1:293–299.
Monteiro-Riviere, N., and Inman, A. 2006. Challenges for assessing carbon
nanomaterials toxicity to the skin. Carbon 44:1070–1078.
Monteiro-Riviere, N., Nemanich, R., Inman, A., Wang, Y., and Riviere, J.
2005a. Multi-walled carbon nanotube interactions with human epidermal
keratinocytes. Toxicol. Lett. 155:377–384.
Muller, J., Huaux, F., Moreau, N., Misson, P., Heilier, J. F., Delos, M., Arras, M.,
Fonseca, A., Nagy, J. B., and Lison, D. 2005. Respiratory toxicity of multi-
wall carbon nanotubes. Toxicol. Appl. Pharmacol. 207:221–231.
Mutsaers, S. E. 2004. The mesothelial cell. Int. J. Biochem. Cell. Biol. 36:9–16.
Nardone, L., and Andrews, S. 1979. Cell line A549 as a model of the type II
pneumocyte. Phospholipid biosynthesis from native and organometallic
precursors. Biochim. Biophys. Acta 573:276–295.
Nel, A., Xia, T., Madler, L., and Li, N. 2006. Toxic potential of materials at the
nanolevel. Science 311:622–627.
Nymark, P., Lindholm, P. M., Korpela, M. V., Lahti, L., Ruosaari, S., Kaski, S.,
Hollmen, J., Anttila, S., Kinnula, V. L., and Knuutila, S. 2007. Gene
expression profiles in asbestos-exposed epithelial and mesothelial lung cell
lines. BMC Genom. 8:62.
Poland, C. A., Duffin, R., Kinloch, I., Maynard, A., Wallace, W. A., Seaton, A.,
Stone, V., Brown, S., Macnee, W., and Donaldson, K. 2008. Carbon nano-
tubes introduced into the abdominal cavity of mice show asbestos-like
pathogenicity in a pilot study. Nat. Nanotechnol. 3:423–428.
Ovrevik, J., Refsnes, M., Namork, E., Becher, R., Sandnes, D., Schwarze, P. E.,
and Lag, M. 2006. Mechanisms of silica-induced IL-8 release from A549
cells: Initial kinase-activation does not require EGFR activation or particle
uptake. Toxicology 227:105–116.
Ryter, S. W., and Choi, A. M. 2005. Heme oxygenase-1: Redox regulation of a
stress protein in lung and cell culture models. Antioxid. Redox Signal.
7:80–91.
Sayes, C. M., Liang, F., Hudson, J. L., Mendez, J., Guo, W., Beach, J. M.,
Moore, V. C., Doyle, C. D., West, J. L., Billups, W. E., Ausman, K. D.,
and Colvin, V. L. 2006. Functionalization density dependence of sin-
gle-walled carbon nanotubes cytotoxicity in vitro. Toxicol. Lett. 161:
135–142.
Shvedova, A. A., Kisin, E. R., Mercer, R., Murray, A. R., Johnson, V. J.,
Potapovich, A. I., Tyurina, Y. Y., Gorelik, O., Arepalli, S., Schwegler-
Berry, D., Hubbs, A. F., Antonini, J., Evans, D. E., Ku, B. K., Ramsey, D.,
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008

Page 15

TOXIC EFFECTS OF INDUSTRIAL CARBON NANOTUBES
73
Maynard, A., Kagan, V. E., Castranova, V., and Baron, P. 2005. Unusual
inflammatory and fibrogenic pulmonary responses to single-walled
carbon nanotubes in mice. Am. J. Physiol. Lung Cell Mol. Physiol.
289:L698–L708.
Smart, S., Cassady, A., Lu, G., and Martin, D. 2006. The biocompatibility of
carbon nanotubes. Carbon 44:1034–1047.
Soto, K., Garza, K. M., and Murr, L. E. 2007. Cytotoxic effects of aggregated
nanomaterials. Acta Biomater. 3:351–358.
Sumimoto, H., Miyano, K., and Takeya, R. 2005. Molecular composition and
regulation of the Nox family NAD(P)H oxidases. Biochem. Biophys. Res.
Commun. 338:677–686.
Taille, C., El-Benna, J., Lanone, S., Dang, M. C., Ogier-Denis, E., Aubier, M.,
and Boczkowski, J. 2004. Induction of heme oxygenase-1 inhibits
NAD(P)H oxidase activity by down-regulating cytochrome b558
expression via the reduction of heme availability. J. Biol. Chem. 279:
28681–28688.
Takagi, A., Hirose, A., Nishimura, T., Fukumori, N., Ogata, A., Ohashi, N.,
Kitajima, S., and Kanno, J. 2008. Induction of mesothelioma in p53+/-
mouse by intraperitoneal application of multi-wall carbon nanotube. J.
Toxicol. Sci. 33:105–116.
Tian, F., Cui, D., Schwarz, H., Estrada, G. G., and Kobayashi, H. 2006. Cyto-
toxicity of single-wall carbon nanotubes on human fibroblasts. Toxicol. In
Vitro 20:1202–1212.
Warheit, D. B., Laurence, B. R., Reed, K. L., Roach, D. H., Reynolds, G. A.,
and Webb, T. R. 2004. Comparative pulmonary toxicity assessment of sin-
gle-wall carbon nanotubes in rats. Toxicol. Sci. 77:117–125.
Wick, P., Manser, P., Limbach, L. K., Dettlaff-Weglikowska, U., Krumeich, F.,
Roth, S., Stark, W. J., and Bruinink, A. 2007. The degree and kind of agglom-
eration affect carbon nanotube cytotoxicity. Toxicol. Lett. 168:121–131.
Worle-Knirsch, J. M., Pulskamp, K., and Krug, H. F. 2006. Oops they did it
again! Carbon nanotubes hoax scientists in viability assays. Nano Lett.
6:1261–1268.
Downloaded By: [University of Lausanne] At: 07:28 27 November 2008