Toxicology Letters 168 (2007) 5874

Carbon nanotubes show no sign of acute toxicity but induce

intracellular reactive oxygen species in
dependence on contaminants
Karin Pulskamp, Silvia Diabate , Harald F. Krug
Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, Environmental Molecular Toxicology,
Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
Received 7 July 2006; received in revised form 30 October 2006; accepted 2 November 2006
Available online 15 November 2006

Abstract
Today nanosciences are experiencing massive investment worldwide although research on toxicological aspects of these nanosized particles has just begun and to date, no clear guidelines exist to quantify the effects. In the present study, we focus on carbon
nanotubes (CNTs), which represent one of the most widely investigated carbon nanoparticles. The present data indicate that CNTs
are able to cross the cell membrane of rat macrophages (NR8383) and, therefore, might have an influence on cell physiology and
function. NR8383 and human A549 lung cells were incubated with commercial single-walled (NT-1) and multi-walled (NT-2, NT-3)
CNTs, carbon black and quartz as reference particles as well as an acid-treated single-walled CNT preparation (SWCNT a.t.) with
reduced metal catalyst content. We did not observe any acute toxicity on cell viability (WST-1, PI-staining) upon incubation with all
CNT products. None of the CNTs induced the inflammatory mediators NO, TNF- and IL-8. A rising tendency of TNF- release
from LPS-primed cells due to CNT treatment could be observed. We detected however, a dose- and time-dependent increase of
intracellular reactive oxygen species and a decrease of the mitochondrial membrane potential with the commercial CNTs in both
cell types after particle treatment whereas incubation with the purified CNTs (SWCNT a.t.) had no effect. This leads us to the
conclusion that metal traces associated with the commercial nanotubes are responsible for the biological effects.
2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Carbon nanotubes; Alveolar epithelial cells; Alveolar macrophages; Oxidative stress; TNF-; IL-8

1. Introduction
The knowledge on the adverse health effects induced
by the inhalation of very small particles was obtained
from several studies on ambient ultrafine particles (UFP)
unintendedly released into the atmosphere. Epidemiological surveys showed that increased levels of UFP

Corresponding author. Tel.: +49 7247 822692;

fax: +49 7247 823557.
E-mail address: silvia.diabate@itg.fzk.de (S. Diabate).

(less than 0.1 m in aerodynamic diameter) in the ambient atmosphere are associated with increased respiratory
and cardiovascular mortality and morbidity as well as
worsening of asthma. These effects were observed in particular in susceptible persons, in very young and older
people and those with a compromised respiratory or cardiovascular system (Ibald-Mulli et al., 2002; Samet et al.,
2000; Wichmann et al., 2000). The composition of ambient UFP includes organic and elemental carbon, metals,
chlorides, nitrates and sulphates. These tiny particles
pose the highest risk because of their ability to reach
every part of the lung and their interaction with cellular

0378-4274/$ see front matter 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2006.11.001

K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

functions, e.g. inflammation (Diabate et al., 2002) and

inhibition of clearance mechanisms (Saxena et al., 2003).
Induction of intracellular oxidative stress seems to
be a key event of the biological effects of many particle
types. Once inside the cell, particles may induce intracellular oxidative stress by disturbing the balance between
oxidant and anti-oxidant processes, e.g. the glutathione
system. The oxidative stress induced by exposure to
particles may also stimulate an increase of the cytosolic calcium concentration (Brown et al., 2004) or may
cause the translocation of transcription factors (e.g. NFB) to the nucleus, which regulate pro-inflammatory
genes, such as TNF- and iNOS (Castranova, 2004).
Exceeding oxidative stress may also modify proteins,
lipids and nucleic acids, which further stimulates the
anti-oxidant defence system or even leads to cell death.
Many diseases have been linked to oxidative stress and
increased formation of reactive oxygen/nitrogen species
(ROS/RNS). Among these are several lung, cardiovascular and autoimmune diseases as well as aging (Simm
and Bromme, 2005).
In this study, we focus on a rather new form of carbon
nanomaterial, called carbon nanotubes (CNTs). Carbon
science and technology have enlarged its scope following the discovery of fullerenes (carbon nanocages, C60 )
(Kroto, 2001) and the identification of CNTs (rolled
graphene sheets) and more recently nanofibres (graphene
wires).
By modification of the production method for carbon fibres, long cylindrical CNTs are formed (Iijima
et al., 1992). Single-walled CNTs (SWCNTs) have a

59

diameter of 12 nm and a length of up to 100 m.

Multi-walled CNTs (MWCNTs) consist of several layers of carbon cylinders, which increases the diameter
to 1030 nm. This new material has high potentials
for new commercial products because it exhibits very
interesting properties such as great tensile strength,
high conductivity or unique electronic features used in
aerospace, automotive and computer industry. It is predicted that tons of CNTs will be produced worldwide
every year (Ball, 2001). It is, therefore, highly desirable
and necessary to know more about a possible toxicity of
CNTs.
While the toxic effects of carbon black were studied
very intensely in the past, only few studies with toxicological background exist on C60 fullerenes (Adelmann et
al., 1994; Baierl and Seidel, 1996; Huczko et al., 1999)
and CNTs (Bottini et al., 2006; Huczko et al., 2001; Jia et
al., 2005; Manna et al., 2005; Muller et al., 2005; Murr,
2005; Shvedova et al., 2005; Worle-Knirsch et al., 2006).
In the case of CNTs, it is questioned if there are analogous mechanisms to those of other fibrous particles such
as asbestos (Shukla et al., 2003) and synthetic vitreous
fibers (SVFs), which penetrate into the lung and may
persist in the tissue.
In the production process of CNTs transition-metal
catalysts, predominantly Fe and Ni, are used. Manufacturing of CNTs leads to bundles, forming clumps and
aggregates (Fig. 1B). If they are inhaled or get into contact with skin during handling, the potential hazard will
strongly depend on the metal content and on the size of
the agglomerates.

Fig. 1. Transmission electron micrographs of CNTs: (A) SWCNT (a.t., 12 nm) (magnification: 20,000) and (B) MWCNT (NT-2, 1020 nm)
(magnification: 3000).

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K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

To date only a few studies report on toxic effects

of CNTs either in vivo (Lam et al., 2004; Shvedova
et al., 2005; Warheit et al., 2004) or in vitro (Cui et
al., 2005; Jia et al., 2005; Shvedova et al., 2003, 2005;
Worle-Knirsch et al., 2006) and the results are often
divergent. The bio-persistence, large aspect-ratio and
fibrogenic character of CNTs are important features
that may cause adverse health effects. However, the
publications available on this topic are often controversial. While some of them demonstrate no toxicity at all
(Huczko and Lange, 2001; Pantarotto et al., 2004b), others show various adverse effects within in vitro systems
(Bottini et al., 2006; Manna et al., 2005). Therefore,
evaluation and characterization of their toxic potential is
necessary.
The aim of this study was to investigate the in vitro
responses of lung macrophages and epithelial cells after
exposure to three types of commercially available CNTs,
as well as carbon black and quartz as reference particles.
A purified CNT product with reduced metal catalyst content was included to study the contribution of impurities
such as metal traces and amorphous carbon. We focused
on their effects on cell viability and production of ROS as
well as on the release of typical pro-inflammatory mediators such as TNF-, IL-8 and the expression of enzymes
like the inducible nitric oxide synthase (iNOS). The rat
alveolar macrophage cell line NR8383 and the human
lung epithelial cell line A549 were chosen as cellular
models for possible target cells.
2. Materials and methods
2.1. Cell culture
The rat alveolar macrophage cell line (NR8383) was
obtained from American Type Culture Collection (Rockville,

MD). NR8383 cells were grown in F-12K medium (Kaighns

Modification, Gibco) supplemented with 15% heat inactivated
fetal calf serum (FCS) 100 g/ml penicillin and 100 U/ml
streptomycin. The human alveolar epithelial cell line A549
(ATCC, CCL-185) (Giard et al., 1973; Lieber et al., 1976) was
obtained from American Type Culture Collection (Rockville,
MD). A549 cells were grown in Dulbeccos modified Eagles
medium (DMEM, Invitrogen, Karlsruhe) supplemented with
10% FCS, 2 mM l-glutamine, 100 g/ml penicillin and
100 U/ml streptomycin. The cells were grown in a humidified
incubator at 37 C (95% room air, 5% CO2 ).
2.2. Particles
Three different, commercially available CNTs obtained
from Nanostructured & Amorphous Materials Inc., Los
Alamos, USA (stock numbers 1280NMG, 1205YJ, 1231YJ)
were examined in this study. The nanotubes were produced
using chemical vapor deposition (Vajtai et al., 2004) and still
contain some metal catalyst impurities (see Table 1). In order
to analyze the influence of metal catalyst impurities, some
experiments were conducted using purified SWCNT a.t., which
were synthesized by F. Hennrich (Institute for Nanotechnology, Research Center Karlsruhe, Germany) with the earlier
described laser vaporisation method using a carbon composite rod containing a Ni/Co catalyst (Hennrich et al., 2003;
Lebedkin et al., 2002). This nanotube preparation was acidtreated to reduce the catalyst impurity and stored in a 1%
SDS (w/w) solution. Before use in the experiment, the acidtreated (a.t.) SWCNTs were acetone-precipitated (21,000 g),
resuspended in bidistilled water, sedimented again by centrifugation and finally suspended in growth media at the given
concentrations. Carbon black (CB14; primary particle size
14 nm, Printex90, Degussa) and crystalline quartz particles
(DQ12; particle size <5 m, kindly provided by Prof. Dr.
N.H. Seemayer, Dusseldorf, Germany) were used as welldefined reference particles with known biological properties.
All particle suspensions were prepared freshly before the
experiments in the respective complete culture medium and

Table 1
Concentration of selected elements in CNTs (wt.%)
Material

SWCNT a.t.a

NT-1b 1280NMG

C
O
N
Co
Cu
Ni
Fe
Mo
Amorphous carbon
Specific surface area (m2 /g)

65.7
25.3
1.5
1.3

84.95
3.08
0.12
2.8
0.03
<0.005
0.009
4.2

a
b
c

1.2

NT-1c 1280NMG

NT-2c 1205YJ

NT-3c 1231YJ

99.8

97.37

0.6
1.86
0.55
0.1
<5
400

40600

40600

Determined by IR and atomic emission spectroscopy by F. Hennrich (Institute for Nanotechnology, Research Center Karlsruhe).
Determined by ICP-OES by the Institute of Material Research, Research Center Karlsruhe.
Values according to the analysis certificate of the supplying company.

K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

sonified (6 30 s, 70 W, Branson Sonfier 250) to break up

agglomerates.
2.3. MTT assay
For determining the effect of particles on cell viability, different assays were used. For the MTT and WST assay 2.5 104
A549 or 1 105 NR8383 cells were seeded into each well
of a 96-well plate (Nunc, Wiesbaden) and grown overnight.
Cells were treated in triplicate with the particle suspensions in
concentrations of 5 g/ml, 10 g/ml, 50 g/ml and 100 g/ml
in complete culture medium for 2496 h. The culture supernatants were removed and frozen at 20 C for later analysis
of cytokines (see below). Then, 200 l MTT (3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution at
0.5 mg/ml in HBSS (Hanks balanced salt solution; Invitrogen, Karlsruhe) was added into each well and incubated
for 2 h at 37 C and 5% CO2 . Mitochondrial dehydrogenases
of viable cells reduce the yellowish water-soluble MTT to
water-insoluble formazan crystals, which were solublized with
isopropanol/HCl. The extracts without particles were transferred into a clean 96-well plate to prevent an interaction of the
light absorption with the particles and measured at 550 nm in
a microtiter plate reader (Molecular Devices, Ismaning, Germany). The results are given as relative values to the untreated
control in percent.
2.4. WST assay
Alternatively, the tetrazolium salt 2-(4-iodophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, better
known as WST-1 (Roche, Mannheim), was used to detect the
loss of viability. The reduced tetrazolium salt is water-soluble
in this assay and, therefore, no isopropanol/HCl extraction
is necessary. After treatment of the cells with particles as
described above, the supernatant medium was replaced by
WST-1 diluted 1:20 (v/v) with HBSS and incubated for 2 h.
The coloured supernatants without particles were transferred
into a clean 96-well plate and measured at 450 nm. The results
are given relative to the untreated control.

61

mitochondrial proton gradient. Measuring the loss of m

using TMRE was carried out as previously described (Kamp
et al., 1993; Oberle et al., 2005). Briefly, detached cells were
stained with TMRE (Molecular Probes, Leiden, The Netherlands) at 500 nM in HBSS for 30 min at 37 C. The TMRE
fluorescence intensity was measured with a LSR flow cytometer (BD Heidelberg) using an excitation wavelength of 488 nm
and an emission wavelength of 520 nm and data were analyzed
with CellQuestPro software (BD Heidelberg).
2.6. PI/annexin V staining
Cells were analyzed for annexin V binding and propidium
iodide (PI) incorporation to distinguish between apoptotic and
necrotic cells. 1 106 NR8383 cells were seeded into 3.5 cm
petri dishes (Nunc, Wiesbaden) and grown overnight. Cells
were treated with CNTs as described for the TMRE assay.
Exposure to 50 M etoposid was used as a positive control
for apoptosis. Harvested cells were washed in ice cold PBS,
taken up in 100 l calcium containing binding-buffer [10 mM
HEPES (pH 7.4), 140 mM NaCl, 5 mM CaCl2 )] and stained for
15 min with 5 l Annexin V-FITC (BD, Heidelberg) and PI at
1 g/ml. The stained cells were analyzed via FACS-analysis
of 10,000 cells at an excitation wavelength of 488 nm and
emission wavelengths of 530 nm for FITC fluorescence and
610 nm for PI fluorescence. The percentages of viable (PI,
Annexin), apoptotic (PI, Annexin+) and necrotic cells (PI+,
Annexin+) were evaluated with the CellQuestPro software
(BD Heidelberg). Since double labelling was performed, compensation was set using macrophages stained with PI or the
FITC-conjugated annexin V alone.
2.7. Determination of TNF- and IL-8
The culture supernatants obtained from the MTT and WST
assays were analyzed for the release of pro-inflammatory
cytokines. TNF- was determined in the supernatants of the
macrophages and IL-8 in the supernatants of the epithelial
cells by specific sandwich ELISAs (OptEIA, PharMingen,
Heidelberg), respectively, according to the manufacturers
instructions.

2.5. TMRE staining

2.8. Determination of nitrite
The effects of particles on the mitochondrial membrane
potential (MMP, m ) were determined utilizing a fluorometric assay with the dye tetramethylrhodamine ethyl ester
(TMRE) (Sigma, Taufkirchen, Germany). TMRE exclusively
stains the mitochondria and is not retained in cells upon collapse of the m , which is an early indication of an apoptotic
process. 1 106 NR8383 cells were seeded into 3.5 cm petri
dishes (Nunc, Wiesbaden) and grown overnight. Cells were
treated with CNTs for 2496 h. As a positive control 20 M carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)
(Sigma, Taufkirchen, Germany) was added to a separate group
of cells for 30 min to induce maximal loss of m by
uncoupling the oxidative phosphorylation and eliminating the

NO production of the NR8383 macrophages was detected

by analyzing nitrite in the culture supernatants by the Griess
reaction (Green et al., 1982). 1 105 cells were seeded into
each well of a 96-well plate (Nunc, Wiesbaden, Germany)
and grown overnight. Cells were treated in triplicate with the
particle suspensions at 5, 10, 50 and 100 g/ml in complete
culture medium in the absence or presence of lipopolysaccharide (LPS from Pseudomonas aeruginosa, Sigma, Taufkirchen,
Germany) at 0.1 g/ml for 24 h. Griess reagent, the mixture
of 0.1% N-(1-naphthyl)-ethylene-diamine and 1% sulfanilamide in 2% phosphoric acid, gave red-violet diazo dye with
nitrite, and was detected photometrically in the visible range at

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K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

540 nm. Nitrite concentrations were calculated from a sodium

nitrite standard curve using a linear curve fit (OD 590 nm,
SOFTmax PRO software, Molecular Devices).

and with particle suspension containing the same amount of

NAC.
2.11. Transmission electron microscopy (TEM)

2.9. Western blot analysis

For Western blot analysis, 1 106 NR8383 cells were
seeded into 3.5 cm petri dishes (Nunc, Wiesbaden) and grown
overnight. Afterwards, cells were treated with CNTs for 24 h
in the absence or presence of LPS at 0.1 g/ml. Cells were then
harvested with accutase, washed with cold PBS and whole cell
extracts were prepared by incubation in lysis buffer (150 mM
NaCl, 50 mM Tris pH 8, 5 mM EDTA, 1% (v/v) NP-40, 1 mM
PMSF) for 30 min on ice. The lysates were centrifuged at
12,000 g for 5 min at 4 C and supernatants were stored at
80 C. The protein contents of the lysates were measured
with the Bradford assay and 20 g of protein were loaded
on a 10% sodium dodecyl sulphate (SDS)-polyacrylamide
gel. After electrophoresis, the gel was transferred onto a
PVDF membrane (Schleicher & Schull, Dassel) and electroblotted. Immunodetection was carried out using antibodies
to iNOS (H-174) at 1/1500 dilution and to proliferation cell
nuclear antigen (PCNA, PC-10) at 1/2000 dilution (both from
Santa Cruz, Heidelberg, Germany) and corresponding HRPconjugated secondary anti-rabbit (Amersham, Freiburg) and
anti-mouse (DAKO, Hamburg) antibodies, respectively. Blots
were developed with ECL reagents (Amersham) according to
manufacturers instructions.

The particles alone and particle-treated cells were visualized by TEM. 2 106 NR8383 cells were seeded on
Transwell polycarbonate membranes with 0.4 m pores
(Corning, Wiesbaden, Germany) and exposed to particle suspensions of 100 g/ml. After 24 h, the membrane was cut out
and fixed in 2.5% (w/v) glutaraldehyde (Carl Roth, Karlsruhe)
for 1 h. The membrane was washed and subsequently postfixed in 1% (w/v) osmium tetroxide. Afterwards, the cells
were dehydrated in a graded series of ethanol (50, 70, 95,
and 100%). Next, membranes were infiltrated and embedded
with LR-White Embedding Media (Polysciences, Eppelheim,
Germany). A UC6 ultramicrotome (Leica, Bensheim) was
used for cutting the embedded cells into ultrathin sections of
70100 nm. They were picked on 75-mesh formvar coated copper grids, contrasted with 0.5% (w/v) uranyl acetate in 0.05 M
maleate and photographed with a Zeiss EM 109T transmission
electron microscope (Zeiss, Oberkochen, Germany).
2.12. Statistical analysis
Values are reported as mean standard error of the mean
(S.E.M.). Statistical analysis was performed in case when
experiments were carried out at least in triplicate using Students t-test.

2.10. DCF assay

3. Results
The formation of intracellular ROS was measured via monitoring the increasing fluorescence of 2 7 -dichlorofluorescein
(DCF). The cell-permeant 2 7 -dichlorodihydrofluorescein
(H2 DCF-DA, Molecular Probes, Leiden, The Netherlands)
enters the cell where intracellular esterases cleave off the
diacetate group. The resulting H2 DCF is retained in the cytoplasm and oxidized to DCF by ROS. The original method
described by Wan et al. (1993) was modified in that the particles were washed away before the incubation with H2 DCF-DA
because black particles strongly reduced the fluorescence signal. 5 104 A549 cells or 2 105 NR8383 cells were seeded
into each well of a 96-well plate and allowed to attach over
night. Subsequently, the cells were washed once with HBSS
and treated with 5, 10, 50 and 100 g/ml (3.1, 6.3, 31.3 and
62.5 g/cm2 ) of the described particles in HBSS. After the
incubation for different time periods from 10 min to 24 h,
cells were washed with HBSS, loaded with 50 M H2 DCFDA for 40 min and washed again with HBSS. After a further
incubation in HBSS for 1 h the fluorescence intensities were
measured at 488 nm excitation and 530 nm emission in the fluorescence reader (MWG-Biotech AG, Ebersberg, Germany) and
processed with the Lambda KC4 software (Version 2.7, MWGBiotech AG, Ebersberg, Germany). To study the effect of the
anti-oxidant N-acetyl-l-cysteine (NAC, Sigma, Taufkirchen,
Germany) cells were pretreated with NAC at 10 mM for 30 min

3.1. Characterization of the carbonaceous material

The physical and chemical properties of the three different purchased CNT samples and the acid-treated CNT
preparation are given in Table 1. According to the supplier, the material 1280NMG (NT-1) contains >90 vol.%
CNTs of which >50 vol.% are SWCNTs with an average
outside diameter of 12 nm. The material 1205YJ (NT-2)
consists of 95% of MWCNTs with an outside diameter of 1020 nm and 1231YJ (NT-3) are also MWCNTs
of 95% purity, however, with an outside diameter of
3050 nm. The analysis certificate of the supplier states
that NT-2 does not contain any metal impurities, but
in NT-1 and NT-3 traces of metal catalyst used for the
generation of the material were detected (Table 1). To
verify the declared values, the elemental composition
of NT-1 was analyzed by inductively coupled plasmaoptical emission spectroscopy (ICP-OES). As a result
of the analysis, additionally trace elements such as Cu
(0.03%) and Fe (0.009%) and a higher content of Co
(2.8%) and Mo (4.2%) were detected compared to the
declared values of 0.6% for Co and 0.1% for Mo. The

K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

purified SWCNT a.t. material is the same used in a previous study (Worle-Knirsch et al., 2006) where it was
compared to the non-purified material. The metal content (mainly Co and Ni) derived from the analysis by
atomic emission spectroscopy with standard dissolution
methods was 2.5 wt.% whereas the non-purified material contained approximately 8 wt.%. The carbon black
(CB14) preparation used for the experiments consists
of amorphous, colloidal carbon particles of 14 nm mean
diameter and has a specific surface area of 300 m2 /g.
This material is used in a number of studies as a model
for insoluble UFP.

63

CNTs were visualized by transmission electron

microscopy as shown in Fig. 1. The material consists of
large agglomerates forming bundles or ropes, however,
single nanotubes are clearly visible. The agglomerates
are tightly bound together even when the particle suspensions in water or medium were sonified prior to
use.
3.2. Uptake of CNTs
Light and electron microscopy demonstrated that
CNT agglomerates were taken up by rat alveolar

Fig. 2. Uptake of CNTs by NR8383 alveolar macrophages: (A) light microscopy image (magnification: 800) and (B) TEM image (magnification:
3000) of NR8383 cells incubated with 100 g/ml SWCNTs (NT-1) for 24 h.

Fig. 3. Effect of particles on the viability of NR8383 cells detected by the MTT assay. Cells were treated with 5 g/ml, 10 g/ml, 50 g/ml and
100 g/ml of CNTs (NT-1, NT-2, NT-3 and SWCNT a.t.), carbon black (CB14) and quartz (Q) for 24 h. The viability was measured with the MTT
assay and results are given in percent related to untreated controls. Results are the mean S.E.M. (vertical bars) of four experiments each carried
out in triplicate [* p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to untreated controls (dashed line)].

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K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

macrophages NR8383 (Fig. 2). The electron micrograph clearly shows large bundles of CNTs (NT-1)
inside the cell located within the cytoplasm (arrow) as
well as attached to the cell surface (arrow heads). Single
nanotubes could only rarely be observed inside the
cells due to their small size and carbons poor electron
density. They disappear in the background of other cell
components.
3.3. Acute toxicity of CNTs
The measurement of the activity of the mitochondrial dehydrogenases by the MTT assay after particle
exposure of NR8383 cells for 24 h implicated an obvious dose-dependent decrease of cell viability (Fig. 3).
NT-1 and NT-2 samples seemed to be more effective
than the NT-3 product reaching almost 6080% reduction after incubation with 100 g/ml CNTs. Exposure to
carbon black resulted in a decrease of viability similar
to NT-1 and NT-2, however, quartz was nearly ineffective. Similar effects on the viability were observed
with NR8383 cells in the presence of 0.1 g/ml LPS,
which was used to prime the cells for mimicking particle effects in the infected lung and with A549 alveolar
epithelial cells which exhibit a comparable reduction in
viability after treatment with the same particles (data not
shown).
The WST-assay also detects the viability of cells measuring the activity of the mitochondrial dehydrogenases
by formation of a formazan product, which is watersoluble in contrast to the formazan product generated

in the MTT assay. Surprisingly, this test did not agree

with the MTT assay (Fig. 4) concerning the effects of
the CNTs. The WST-assay did not indicate a loss of viability due to exposure to all kinds of CNTs; only a minor
reduction was detected at the highest dose of NT-1.
Only the results obtained for carbon black agreed
with those of the MTT assay. Additionally, when lactate
dehydrogenase (LDH) was determined in the medium
supernatant of NR8383 and A549 cells, only CB14 and
quartz caused a release of LDH in a dose-dependent manner (data not shown). This indicates agreement with the
results of the WST-assay.
Because of the divergent results of the MTT and the
WST tests, the effects of the particles on the cell membrane integrity were determined additionally with PI. PI
can only diffuse into the cell upon a loss of the cell membrane integrity and then it interacts with the DNA. Cells
were additionally stained with FITC-labelled annexin V
to detect cells in an early stage of apoptosis. NR8383
cells were incubated with 5 g/ml, 10 g/ml, 50 g/ml
and 100 g/ml particles for 24 h, 48 h and 72 h and
the PI-stained necrotic cells were differentiated from
annexin-stained and -unstained vital cells by FACS analysis. No significant decrease of cell membrane integrity
was detected for the tested nanotubes at different concentrations (Fig. 5) and at all time points. Furthermore,
the percentage of annexin-positive/PI-negative cells was
not changed compared to the untreated control, which
gave no hint of apoptotic processes (data not shown).
This again confirms the results of the WST-assay that
the tested CNTs did not induce acute toxicity.

Fig. 4. Effect of particles on the viability of NR8383 cells detected by the WST assay. Cells were treated with 5 g/ml, 10 g/ml, 50 g/ml and
100 g/ml of CNTs (NT-1, NT-2, NT-3 and SWCNT a.t.), carbon black (CB14) and quartz (Q) for 24 h. The viability was measured with the WST
assay and results are given in percent related to untreated controls. Results are the mean S.E.M. (vertical bars) of four experiments each carried
out in triplicate [* p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to untreated controls (dashed line)].

K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

Fig. 5. Effects of CNTs on the cell membrane integrity measured by PIstaining in NR8383 cells. Cells were treated with 5 g/ml, 10 g/ml,
50 g/ml and 100 g/ml of CNTs (NT-1, NT-2 and NT-3) for 24 h. The
viability was measured by PI-staining and results are given in percent
of viable cells (PI-negative) related to untreated controls. Results are
the mean S.E.M. (vertical bars) of four experiments each carried out
in triplicate.

65

demonstrates that all carbon materials enhance the intracellular ROS in NR8383 cells after 24 h incubation up
to four-fold at 50 g/ml of NT-1. This response starts
immediately after short time incubation of 10 min or
1 h (data not shown) in a dose-dependent manner; however, the specifically purified CNTs (SWCNT a.t.) with
reduced metal catalyst content were ineffective. Only
a minor DCF response was observed after exposure to
quartz particles. Similar doseresponse behaviour was
observed in A549 cells treated with the same particles
at the same concentrations (data not shown). The ROS
induction could be inhibited partly by co-incubating the
cells with 10 mM of the anti-oxidant N-acetyl-l-cysteine
(NAC) as shown in Fig. 7 with A549 cells which have
been treated with the described particles. In these experiments a 1 h incubation with 50 g/ml NT-1 results in
an eight-fold increase of ROS in comparison to control
cells. This effect could also be inhibited to some extent
by NAC.
3.5. Mitochondrial membrane potential

3.4. Formation of reactive oxygen species

The ability of CNTs to induce the formation of
intracellular ROS in NR8383 macrophages and A549
epithelial cells was assessed using DCF fluorescence
as a reporter of intracellular oxidant production. Fig. 6

TMRE is a quantitative marker for the maintenance of

the MMP (m ). Upon collapse of the m TMRE is
no longer retained in the mitochondria. This test does not
require a physiological action of the cell like the MTT
and the WST assays which use the enzymatic reduction

Fig. 6. Formation of ROS in NR8383 alveolar macrophages after particle incubation for 24 h. Cells were treated with 5 g/ml, 10 g/ml, 50 g/ml
and 100 g/ml of CNTs (NT-1, NT-2, NT-3 and SWCNT a.t.), carbon black (CB14) and quartz (Q) for 24 h. ROS were detected by fluorescence
measurement of the reporter DCF and results are given in fold of untreated controls. Results are the mean S.E.M. (vertical bars) of four experiments
each carried out in triplicate [* p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to untreated controls].

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K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

Fig. 7. Effect of the anti-oxidant N-acetyl-cysteine (NAC) on the formation of ROS in A549 lung epithelial cells by particle incubation for 1 h.
Cells were pretreated with or without 10 mM NAC for 30 min and then incubated with 50 g/ml of CNTs (NT-1, NT-2, NT-3 and SWCNT a.t.) and
carbon black (CB14) for 1 h. ROS were detected by fluorescence measurement of the reporter DCF and results are given in fold of untreated controls.
Results are the mean S.E.M. (vertical bars) of four experiments each carried out in triplicate [* p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to
untreated controls].

of a substrate. Treatment of cells with the different CNT

preparations induced a significant and dose-dependent
loss in m and, therefore, a loss of the functionality
of the mitochondria (Fig. 8) after 24 h as measured by
FACS analysis. Cells treated for 24 h with the SWCNT
product NT-1 at 100 g/ml showed the highest loss of
m of roughly 60% compared to control cells.

Fig. 8. Effects of different carbon nanotube preparations on the MMP

in NR8383 cells. Cells were treated with 5 g/ml, 10 g/ml, 50 g/ml
and 100 g/ml of CNTs (NT-1, NT-2 and NT-3) for 24 h. The MMP of
cells was measured by TMRE-staining and results are given in percent
of cells with high TMRE fluorescence (relative fluorescence intensity
2001000 units) related to untreated controls [* p < 0.05, ** p < 0.01,
*** p < 0.001 in comparison to untreated controls].

3.6. Nitrite formation

The supernatants of NR8383 cells which were treated
with particles for 24 h in the absence or presence of
0.1 g/ml LPS were used to measure the nitrite concentration as an indicator for the induction of the iNOS.
None of the tested particles alone induced a change of
the basal nitrite concentration of 1 M. LPS at 0.1 g/ml
stimulated NR8383 macrophages to release NO, which
converted to nitrite and accumulated to a concentration
of 37 2.14 M after 24 h. The LPS-induced nitrite concentration was reduced by 80% when the cells were
co-incubated with NT-2 or CB14 at 100 g/ml, but only
moderately after co-incubation with NT-1, NT-3 and
quartz (Fig. 9). The low nitrite concentration of cells
exposed to 100 g/ml CB14 may be attributed to the
reduction of viability measured with the WST assay (see
Fig. 2). However, the viability of NT-2-treated cells at
100 g/ml was not reduced. Here another mechanism
seems to evoke this effect than simply the loss of viability, which might be responsible for the reduction of
nitrite production in CB14-treated cells.
A possible explanation for the reduced ability of
LPS-induced NO formation after incubation with NT-2
nanotubes could be an interaction with the expression of the iNOS protein. Therefore, iNOS protein was
detected by immunoblotting of the lysates of NR8383
cells, which were stimulated with LPS and additionally
with CNTs at different concentrations. Fig. 10 shows

K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

67

Fig. 9. Effect of particles on LPS-induced nitrite production in alveolar macrophages NR8383. Cells were treated with 5 g/ml, 10 g/ml, 50 g/ml
and 100 g/ml of CNTs (NT-1, NT-2 and NT-3), carbon black (CB14) and quartz (Q) in the presence of 0.1 g/ml LPS for 24 h and the culture
supernatant was analyzed for the nitrite concentration. Results are the mean S.E.M. (vertical bars) of four experiments each carried out in triplicate
[* p < 0.05, ** p < 0.01 in comparison to untreated controls].

that iNOS was not present in unstimulated cells, but

was strongly induced by LPS treatment at 0.1 g/ml.
The LPS-induced iNOS protein level however, was not
reduced by simultaneous exposure to NT-2 nanotubes.
Similar results of iNOS protein level were obtained with
NT-1 and NT-3 (data not shown).
3.7. Inammatory response (TNF-, IL-8)
Pulmonary epithelial cells and macrophages are able
to secrete inflammatory mediators like cytokines upon
stimulation and interact by these mediators. CNTs were,
therefore, studied for their potential to interact with the
release of the typical pro-inflammatory cytokines TNF-

and IL-8. The particles under study were not able to

induce a release of TNF- from NR8383 macrophages.
The bacterial endotoxin LPS is well known to induce
TNF- in macrophages representing an important part
of the innate immune response. Therefore, we studied a possible influence of these particles on this
LPS-dependent response. The basal level of TNF-
in the medium supernatant of unstimulated NR8383
macrophages was 1.3 ng/ml. LPS at 0.1 g/ml induced
a strong increase of TNF- up to 37.4 8.5 ng/ml after
24 h (Fig. 11). Additional exposure to the CNTs NT-1,
NT-2, and NT-3 as well as to CB14 and quartz resulted in
an increase of TNF-, which was not statistically significant compared to the only LPS-treated cells. However,

Fig. 10. Effect of CNTs NT-2 on the LPS-induced expression of the iNOS in alveolar macrophages NR8383. Cells were treated with 5 g/ml,
10 g/ml, 50 g/ml, 100 g/ml and 200 g/ml of NT-2 CNTs in the presence of 0.1 g/ml LPS for 24 h. The cell lysates were analyzed for the
expression of iNOS protein by Western blotting. The detection of PCNA was used as loading control.

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K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

Fig. 11. Effect of particles on the LPS-induced TNF- release from alveolar macrophages NR8383. Cells were treated with 5 g/ml, 10 g/ml,
50 g/ml and 100 g/ml of CNTs (NT-1, NT-2 and NT-3), carbon black (CB14) and quartz (Q) in the presence of 0.1 g/ml LPS for 24 h and the
culture supernatant was analyzed for the TNF- concentration. Results are the mean S.E.M. (vertical bars) of four experiments each carried out
in triplicate.

a general tendency towards an increase of up to 60 ng/ml

TNF- could be observed.
The potential of the particles to induce the release of
the pro-inflammatory chemokine IL-8 in human alveolar
epithelial cells A549 was also investigated. The basal

IL-8 concentration in the medium supernatant, which

was 0.2 0.05 ng/ml, was only moderately enhanced by
carbon black and quartz treatment but not significantly
by the CNTs (Fig. 12). In the lung, macrophages are in
direct contact with pulmonary epithelial cells, which are

Fig. 12. Effect of particles on the IL-8 release from alveolar epithelial cells A549. Cells were treated with 5 g/ml, 10 g/ml, 50 g/ml and 100 g/ml
of CNTs (NT-1, NT-2 and NT-3), carbon black (CB14) and quartz (Q) for 24 h and the culture supernatant was analyzed for the IL-8 concentration.
Results are the mean S.E.M. (vertical bars) of five experiments each carried out in triplicate [* p < 0.05, ** p < 0.01 in comparison to untreated
controls].

K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

also involved in the inflammatory response by producing

IL-8 and other cytokines upon stimulation with TNF. We, therefore, studied if the particles influence the
TNF--induced generation of IL-8. We could not detect
a significant effect of the particles on the TNF--induced
IL-8 concentration, which was 17.9 4.7 ng/ml (data not
shown).
4. Discussion
CNTs, which possess unique physical and chemical
properties are promising nanomaterials in the electronic and computer industry, e.g. for flat panel displays,
bridges made out of nanotubes and even elevators into
space have been postulated by the NASA. The ideas
for their potential use never seem to cease. Another rising field for nanoparticles is in the cosmetic (Gelis et
al., 2003) and pharmaceutical industry. Because of their
small size and large surface area, they are promising
tools for new drug delivery devices and applications in
biotechnology and biomedicine (Pantarotto et al., 2004b;
Singh et al., 2005). But again as seen with other new
products, it is essential to look at both sides of the story in
order not to underestimate the harming potential of these
tiny particles. Nano-sized materials, which exhibit new
features not found in the bulk material might, therefore,
possess the potential to induce toxicological and environmental effects, which are not observed with larger
particles.
Recently, it has been published that CNTs exhibit
toxicity in vivo and in vitro (Barlow et al., 2005; Cui
et al., 2005; Jia et al., 2005; Lam et al., 2004; Manna
et al., 2005; Monteiro-Riviere et al., 2005; Shvedova et
al., 2005; Warheit et al., 2004). It is well described that
particles below the size of 2.5 m can reach the alveoli
of the lung, which allows them to interact with alveolar
epithelial cells and macrophages (Diabate et al., 2002;
Fujii et al., 2002). Once in the lung, particles can induce
the generation of ROS, which may lead to inflammation
or cytotoxicity (Brown et al., 2004). Particles may even
enter the blood system and gain access to other organs
of the body (Kreuter et al., 2002; Kreyling et al., 2002;
Oberdorster et al., 2004; Oberdorster et al., 2005).
In the case of CNTs, it is questioned if there are analogous mechanisms to those of other fibrous particles such
as asbestos and SVFs, which penetrate into the lung and
may persist in the tissue. Large epidemiology studies
of SVF manufacturing workers provided very little evidence of harmful effects in humans (Hesterberg and Hart,
2001). However, it is widely assumed that all biopersistent fibers may be harmful if inhaled in large enough
doses. Long and insoluble fibers such as asbestos are

69

difficult to clear by phagocytic cells. The macrophages

die after a long process of trying to engulf the fibers
and release inflammatory cytokines into the lung. This
may trigger the complex cellular response mechanisms
that cause cancer (Godleski, 2004). The potential exposure to CNTs has been demonstrated by Maynard et
al. who measured the aerosol mass and number concentration in three laboratories where SWCNTs were
generated by different processes and manually handled
(Maynard et al., 2004). They observed concentrations
of 0.753 g/m3 of nanotubes in the atmosphere and
considerable masses on gloves during handling.
While the toxic effects of carbon black were studied
very intensely in the past, only few studies with toxicological background exist on C60 fullerenes and CNTs.
Baierl et al. (1996) studied the effects of fullerenes in
primary bovine alveolar macrophages. The fullerenes
reduced the viability of the cells and induced increased
levels of TNF-, IL-6 and IL-8. But these effects are
significantly lower when compared to those of quartz.
Among the few in vitro studies on cytotoxic effects with
CNTs are data from Shvedova et al., which report a loss
on cell viability, induction of oxidative stress as well
as morphological changes upon SWCNT treatment in
human keratinocytes (Shvedova et al., 2003). Two independent studies with rats (Warheit et al., 2004) and mice
(Lam et al., 2004) report on the appearance of granuloma,
interstitial inflammation and obstruction of the airways
after instillation of high doses of aggregated CNTs. Just
recently another study with mice demonstrated unusual
pulmonary effects with progressive fibrosis and granuloma formation upon pharyngeal aspiration of SWCNTs
(Shvedova et al., 2005). The bio-persistence of SWCNTs and the induction of granulomatosis is an important
evidence for adverse health effects.
In the present study, we evaluate the toxicological
potential of CNTs on pulmonary epithelial cells as well
as macrophages by studying their effects on cell viability,
induction of oxidative stress and release of inflammatory mediators. Crystalline silica (quartz) and carbon
black were used to compare the results obtained with
CNTs with these well-characterized reference particles
(Lambert et al., 2003; Oberdorster, 1996; Srivastava et
al., 2002). Carbonaceous particles are difficult to detect
in cells and tissues by electron microscopy because of
their low contrast and small diameters. Nevertheless,
the TEM images shown in this paper demonstrate that
CNTs are able to enter the cell and can be found as bundles in the cytoplasm or organelles of macrophages and
epithelial cells and, therefore, might interact with the
cell physiology and cellular mechanisms. CNT uptake
(energy-dependent and -independent mechanisms) is

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K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

currently being discussed and might be dependent on the

state of functionalization and morphology of the material. Bianco et al. (2005) reported that functionalized
CNTs are able to cross the cell membrane in a passive
and endocytosis-independent way whereas Kam and Dai
(2005) propose an energy-dependent endocytosis mechanism.
The widely applied MTT assay, which was one of
the assays used to determine cell viability upon CNT
treatment in NR8383 alveolar macrophages and A549
epithelial cells, indicated a dose-dependent decrease of
the mitochondrial enzyme activity after 24 h similar to
results seen before in other published studies (Cui et al.,
2005; Jia et al., 2005; Murr, 2005). The WST-1 assay,
however, which is based on the same principle as the
MTT assay but results in a soluble reaction product,
showed no acute cytotoxicity of the nanotube preparations studied. There also seems to be an increase of
viability, which rather suggests an increase of proliferation upon particle stimulation. It appears that CNTs
attach to the insoluble MTT formazan product and
thereby disturb the test. This has been demonstrated
recently in our group with different cell lines (WorleKnirsch et al., 2006). Working with macrophages as a
test system in which oxidative burst might be expected
on exposure to particles, the results of the MTT and
WST-1 assay which both employ oxidative mechanisms
to generate the coloured product, should be reconsidered. Therefore, we checked the reliance of the viability
assays additionally by measuring the influence of CNTs
on the cell membrane integrity using PI and FACS analysis. This assay confirmed the results of the WST-1 test
in that almost no decrease in viability was observed after
24 h exposure of NR8383 cells to CNTs at a concentration of 100 g/ml. Even after long-term incubation to
CNTs with 100 g/ml for up to 72 h the cell viability
was still 7080%, which was comparable to the results
of the WST-1 assay. This is in accordance with other
published results that demonstrate no cytotoxic effect of
intact CNTs on cell membrane integrity by assessing the
LDH levels of the supernatants of macrophage cultures
(Muller et al., 2005).
When particles enter the cell, they may induce oxidative stress by disturbing the balance between oxidant and
anti-oxidant processes, e.g. the glutathione system. The
oxidative stress may also stimulate an increase of the
cytosolic calcium concentration (Brown et al., 2004).
Exceeding oxidative stress may also modify proteins,
lipids and nucleic acids, which further stimulates the
anti-oxidant defence system or even leads to cell death.
Oberdorster studied the effects of C60 fullerenes in fishes
(juvenile largemouth bass) as a model for the impact of

nanoparticles produced in bulk with the potential to be

released into the environment (Oberdorster, 2004). She
demonstrated that a 48 h exposure to fullerenes significantly increased the lipid peroxidation in the brain and
depleted the glutathione content in the gill.
The production of ROS upon particle treatment has
been described for many different forms of fine, ultrafine and nano-sized particles (Hiura et al., 1999; Martin
et al., 1997). On the other hand, we found a formation of
ROS in nanotube-treated cells only when raw material
was used. This effect was concentration-dependent and
comparable to the induction of carbon black (CB14).
Nevertheless, this effect was totally diminished when
nanotubes were purified and the transition metals were
extracted before treatment of the cells. The results for
intracellular ROS induction upon quartz treatment did
show little effect despite what is known from historical literature. Nevertheless, it has been discussed that
structural characteristics of individual specimen can contribute to a certain variability of quartz hazard (Fubini
and Hubbard, 2003) and also the surface changes by
aging of the particles may influence ROS induction to
some extent (Ding et al., 2001).
Since it has been shown that there is no acute effect
of CNTs on cell viability it needs to be determined if
the observed particle-induced increase of intracellular
ROS affect cell organelles such as the mitochondria.
A very important parameter of the mitochondria that
is connected to a variety of cellular mechanisms and
whose disturbance has severe consequences to the cell
is the MMP (m ). Our experiments result in a dosedependent decrease of this potential in NR8383 cells
indicating a loss in functionality of the mitochondria
after CNT-treatment. The SWCNT product (NT-1) had
the strongest effect, which confirms results described for
another SWCNT preparation before (Jia et al., 2005)
and even exhibited a time-dependent influence. It has
been proposed that UFP in ambient particulate matter
can enter the mitochondria and, therefore, are able to
induce oxidative stress (Li et al., 2003). This group discusses the possibility that ROS may lead to a destruction
of the mitochondrial membrane and that particles then
enter the cells or that the small size is the key to make an
entry into mitochondria possible which then causes the
release of ROS.
We recently reported that the purified SWCNTs also
used in this study as well as the SWCNTs with a high
metal content are not able to induce any effect on the
MMP of A549 cells (Worle-Knirsch et al., 2006). This
altogether indicates that CNT preparations vary in their
potential to induce the formation of ROS and to perturb
mitochondrial function in dependence of the nature and

K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

the concentration of the metal impurities and the used

cell line.
Metals such as Fe, Ni and Cu have been shown to
induce the formation of ROS through a Fenton-like reaction and to induce intracellular oxidative stress (Diabate
et al., 2002; Voelkel et al., 2003) either in a direct or
indirect way via extracellular pathways (Kagan et al.,
2006). Interactions between UFP and transition metals
have been shown to enhance the formation of ROS in
a cell-free assay using DCF in a potentiative manner as
well as inflammation after instillation into the lungs of
rats (Wilson et al., 2002). Indeed, our own observations
revealed that CNTs and carbon black are able to generate
ROS in a cell-free system with DCF in dependence of
the amount and type of impurities, which may also contribute to enhanced oxidative stress (data not shown). In
the production process of CNTs transition-metal catalysts, predominantly Fe, Co and Ni, are used. They are
removed from the raw product, however, part of the metal
is encased in the tubes and cannot be removed completely. If CNTs are inhaled or get contact to skin during
handling, the potential hazard will strongly depend on
the metal content, the size of the agglomerates (Dick et
al., 2003) and particle surface (Donaldson et al., 2003).
The biological effects of SWCNTs prior catalyst removal
(containing 30% Fe by mass) were studied in human keratinocyte cells (Shvedova et al., 2003). They observed
decreases in cell viability and glutathione (GSH) levels in a dose-dependent manner, which was dramatically
reversed by the metal chelator desferrioxamin. This indicates a significant role of Fe as cause of the biological
effects of the SWCNTs. They further confirmed oxidative stress in SWCNT-treated cells by the formation of
free radical species, increased lipid peroxidation and
decrease of the anti-oxidant reserve.
The particle-induced oxidative stress may lead to
changes of the activation status of proteins by conformational changes. Many of these proteins are receptors,
which transmit external signals into the cell and a conformational change of the receptor may activate an onset
of cellular responses. The activation of redox-sensitive
transcription factors, such as NF-B, AP-1 or Nrf2,
leads to their translocation into the nucleus and binding to the promotor regions of the genes regulated by
these transcription factors. In the case of NF-B these
genes include TNF-, IL-6, IL-8, ICAM-1, iNOS and
others, which are highly pro-inflammatory (Castranova,
2004).
In this study, the release of nitrite and TNF- from
macrophages and the production of IL-8 in epithelial cells were used as indicators for an inflammatory
response. Our results indicate that CNTs did not signifi-

71

cantly affect TNF- release of NR8383 cells when they

were additionally stimulated with LPS. A general tendency towards an increase of TNF- in LPS-stimulated
macrophages could be observed however these results
are not statistically significant. LPS was used to mimic a
pre-existing lung disease in vitro or the naturally attached
LPS on particles (Becker et al., 2002). Our results are in
accordance with those obtained with ultrafine and fine
particles (Brown et al., 2004; Diabate et al., 2002; van
Eeden et al., 2001) suggesting that pre-existing inflammation can potentially aggravate the toxicity of CNTs.
An increase of the TNF- release may lead to a systemic
effect on the cardiosvascular system and stimulation of
the bone marrow, which then could result in chronic
diseases (Mukae et al., 2000).
Similar to the results on the TNF- production there
was no nitrite production measurable upon CNT incubation without LPS co-stimulation in NR8383 cells. LPS
is a known inducer of iNOS mRNA and the subsequent
iNOS protein synthesis in alveolar macrophages (Chen
et al., 2003). LPS-stimulated cells treated with CNTs
and reference particles did hardly induce any changes
on nitrite concentrations compared to control. Since the
protein expression level of the LPS-induced iNOS did
not change upon particle treatment, any reduction on
nitrite release (NT-2, CB14) could be due to a change in
iNOS enzyme activity, which has to be elucidated. NO
is a signalling molecule with several different properties
in physiological and pathological conditions. Depending on cell type, concentration and time of release NO
can have either harmful or protective effects (Wolkow,
1998).
To date, only a few studies have investigated possible toxicological mechanisms of nanoparticles even
though there is no shortage of conceptions how to
use their potential to make things better, smaller and
more efficient, e.g. for drug delivery systems (Kreuter
et al., 2002). In this study, we demonstrate that CNTs
possess no acute toxicity in pulmonary epithelial and
macrophage cell lines. Like Shvedova et al. we hardly
observe any changes of TNF- production and NO
release in LPS-stimulated macrophages treated with
CNTs (Shvedova et al., 2005). Their potential to induce
oxidative stress is likely due to their content of impurities (Kagan et al., 2006). These findings are short-term
effects and the influence of chemically unmodified or
functionalized CNTs on DNA modifications upon entering the cell (Pantarotto et al., 2004a) still needs to be
investigated. For occupational safety purposes and environmental exposure studies, long-term treatment in vitro
as well as inhalation studies are inevitable. Thus, it seems
that pure CNTs without metal catalyst and amorphous

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K. Pulskamp et al. / Toxicology Letters 168 (2007) 5874

carbon impurities offer possibilities for promising new

biomedical applications.
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