A single intratracheal instillation of single-walled carbon nanotubes ...

Arch Toxicol (2011) 85:1121–1131
DOI 10.1007/s00204-011-0655-8
ORGAN TOXICITY AND MECHANISMS
A single intratracheal instillation of single-walled carbon
nanotubes induced early lung fibrosis and subchronic tissue
damage in mice
Eun-Jung Park • Jinkyu Roh • Soo-Nam Kim •
Min-sung Kang • Young-Ah Han • Younghun Kim •
Jin Tae Hong • Kyunghee Choi
Received: 13 October 2010 / Accepted: 13 January 2011 / Published online: 7 April 2011
Ó Springer-Verlag 2011
Abstract Large amounts of nanomaterials may reach
both the natural and occupational environments. This represents
a potential health hazard. People have forecasted
that CNTs may lead to the toxicity such as mesothelioma
and fibrosis like asbestos. To identify dominant immune
responses induced by SWCNTs, we investigated the composition
of bronchioalveolar lavage (BAL) cells, the
secretion of cytokine and collagen, histopathology, protein
expression, and cell phenotypes over time after a single
administration of single-walled carbon nanotubes
(SWCNT). In our results, the number of total cells and
macrophages remained at the up-regulated level until Day
28, neutrophils rapidly increased at Day 1, and lymphocytes
increased from Day 7. In the BAL fluid, proinflammatory
cytokines rapidly increased at Day 1 and
remained at an up-regulated level throughout the
Electronic supplementary material The online version of this
article (doi:10.1007/s00204-011-0655-8) contains supplementary
material, which is available to authorized users.
E.-J. Park (&) K. Choi
Environmental Health Risk Research Department,
National Institute of Environmental Research, Kyungseo-dong,
Seo-gu, Incheon 404-708, Korea
e-mail: pej303@korea.kr
J. Roh Y. Kim
Department of Chemical Engineering, Kwangwoon University,
447-1, Wolgye-dong, Nowon-gu, Seoul 139-701, Korea
S.-N. Kim M. Kang Y.-A. Han
Inhalation Toxicology Center, KIT Jeongeup Campus,
1051, Shinjeong-dong, Jeongeup, Korea
J. T. Hong
College of Pharmacy and Medical Research Center,
Chungbuk National University, 410, Seongbong-ro,
Heungdeok-gu, Cheongju, Chungbuk 361-763, Korea
experimental period. IL-12 and IL-10 rapidly increased at
Day 1 after administration and remained at a similar level
until Day 28. IFN-c and IL-4 reached the maximum at Day
1, and IL-5, TGF-b, and collagen reached the maximum at
Day 7. IL-13 and IL-17 increased in a time-dependent
manner. The distribution of B cells and cytotoxic T cells
markedly increased at Days 7 and 14, and fibrotic lesions
were histopathologically observed at Days 7 and 14. The
expressions of caspase-3, p53, COL1A1, COX-2, iNOS,
MMP-9, and MMP-2 were also markedly increased at Days
7 and 14. In addition, the expression of mesothelin, iNOS,
MMP-9, and p53 was up-regulated until Day 28. Based on
these findings, we suggest that a single intratracheal
instillation of SWCNTs may induce early lung fibrosis and
subchronic tissue damage.
Keywords SWCNT Lung fibrosis Mesothelin
Cytokine Inflammation
Introduction
The use of manufactured nanomaterials is rapidly
increasing due to nanotechnology advances in different
industries and consumer products. Many feel that these
nanotechnologies have resulted in colossal benefits in
improving the quality of life. However, the large amounts
of nanomaterials may consequently reach the natural, as
well as occupational, environment and thus represent a
potential health hazard (Shvedova and Kagan 2010; Lam
et al. 2006; Maynard et al. 2004; Warheit et al. 2004). Song
et al. reported symptoms, including non-specific pulmonary
inflammation, pulmonary fibrosis, and foreign-body granulomas
of the pleura, in workers exposed to nanoparticles
for 5–13 months (Song et al. 2009).
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1122 Arch Toxicol (2011) 85:1121–1131
Some nanomaterials of importance are carbon nanotubes
(CNTs). CNTs have a unique length-to-diameter ratio,
which is significantly larger than that of any other material.
CNTs have been categorized as single-walled carbon
nanotubes (SWCNTs) and multi-walled carbon nanotubes
(MWCNTs), according to the number of layers, and both
types of CNTs are listed as high-priority groups in the
Organization for Economic Co-operation and Development
(OECD) Steering Group for Test Guidelines.
Many researchers have forecasted that CNTs may lead
to mesothelioma and fibrosis, similar to asbestos, due to
their unique morphology (Pacurari et al. 2008; Takagi et al.
2008; Sakamoto et al. 2009, Donaldson et al. 2010).
Mesothelioma is a cancer that develops from the protective
lining that covers many of the body’s internal organs, the
mesothelium, and it is most commonly generated in the
pleura, peritoneum, heart, pericardium, and tunica vaginalis
(Chang and Pastan 1996). Fibrosis is generated in
various organs, such as the lung, liver, heart, and intestine,
as a kind of autoimmune disease, and it forms excess
fibrous connective tissue in an organ or tissue as a reparative
or reactive process.
The outbreak of environment-related disease is caused
by the destruction of immune balance by the xenobiotics
inflowing into the body. In previous reports, some
researchers have suggested that SWCNT induces oxidative
stress and fibrotic inflammatory responses (Folkmann et al.
2009; Pacurari et al. 2008; Shvedova et al. 2008a, b; Chou
et al. 2008; Sharma et al. 2007; Manna et al. 2005). For
example, nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase-deficient C57BL/6 mice showed an
increase in the accumulation of neutrophils and a decrease
of fibrosis in the lung (Shvedova et al. 2008a, b), and the
pharyngeal aspiration of SWCNT elicited a robust but
acute inflammation with an early onset of progressive
fibrosis and granulomas in C57BL/6 mice (Shvedova et al.
2005). Furthermore, Chou et al. studied innate and adaptive
immune responses related to chronic pulmonary inflammation
and granuloma formation caused by SWCNT (Chou
et al. 2008). They suggested that the uptake of SWCNT
(500 lg) into the macrophages activates various transcription
factors, such as NF-kB and AP-1, and this leads to
oxidative stress, the release of pro-inflammatory cytokines,
the recruitment of leukocytes, the induction of protective
and anti-apoptotic gene expression, and the activation of T
cells.
The treatment of SWCNT together with OVA strongly
increased serum levels of OVA-specific IgE, the number of
eosinophils in bronchial alveolar lavage fluid (BALF), and
the secretion of Th2-associated cytokines in the mediastinal
lymph node (Nygaard et al. 2009). However, there is
still not enough data on the immunotoxicity of SWCNTs.
In this study, we investigated the change of cytokines and
cells (NK, NKT, B, and T) to identify whether SWCNTs
induce Th1 or Th2 response. Furthermore, we explored
proteins related to lung tissue damage.
Materials and methods
Animals
Male, 6-week-old (40–42 days old, 26 ± 1 g), ICR mice
were purchased from the Orient Bio INC. (Seongnam,
Gyunggi-do, Korea) and were acclimated the animal to
room conditions prior to the initiation of the study. The
environmental conditions were a temperature of 23 ± 1°C,
relative humidity of 55 ± 5%, and a 12-h light/dark cycle.
All the animals used in this study were cared for in
accordance with the principles outlined in the ‘‘Guide for
the Care and Use of Laboratory Animals’’ issued by the
Animal Care and Use Committee of the National Veterinary
Research and Quarantine Service (NVRQS). To
observe time-dependent change, 16 mice were treated per
time point.
Preparation of test material
SWCNTs (approximately 10% weight metal contents,
1.2 nm in diameter, 2–10 lm in length; ASP-100F, Hanhwa
Nanotech, Korea) were dispersed into DI water by
sonication. Because SWCNTs are very hydrophobic and
immiscible to water, 40 mg sodium dodecyl sulfate (SDS,
Sigma–Aldrich) was added to 200 ml of the SWCNTtreated
water as a stabilizer. Then, the solution was treated
with sonification (ULH-700S, ULSSO HI-Tech, Korea) for
30 min, 4 kHz (20% PWR), and was then diluted with 29
PBS. As shown in Fig. 1, SWCNT was relatively well
dispersed in the PBS buffer (A, JEM1010, JEOL, Japan),
showed the peak of graphite band in Raman spectroscopy
(B, T64000, HORIABA Jobin–Yvon, France), and the
average length was 0.76 lm (C, ELS-8000, Otuska Electronics,
Japan) (Yang et al. 2005; Yu et al. 2010; Wei et al.
2005).
Intratracheal instillation and sample preparation
Intratracheal instillation and sample preparation was conducted
by a special technician from the Korea Institute of
Toxicology, one of the GLP institutes in Korea. SWCNTs
were delivered using a 24-gauge catheter at a 100 lg/kg
dose by intratracheal administration under light tiletamine
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Arch Toxicol (2011) 85:1121–1131 1123
Fig. 1 Physicochemical properties of SWCNTs suspended in PBS a TEM image, b Raman spectroscopy, and c Size distribution
anesthesia. The animals were killed at Days 1, 7, 14, and 28
after treatment. The control group (4 mice per time point)
was treated with a vehicle control solution that was manufactured
by the same method.
At the selected time intervals after administration,
about 1.2 ml of blood was collected per mouse from the
saphenous vein. The samples harvested from the 16 mice
per time point were pooled to four test samples for
analysis of the BAL cell count, cell phenotype, cytokines,
and collagen (n = 4). Whole blood was centrifuged at
3,000 rpm for 10 min to make serum, and 400–450 ll of
serum was obtained from each mouse. BAL fluid was
obtained by cannulating the trachea and lavaging the
lungs with 1 ml of cold sterile (Ca2 plus Mg2)-free
PBS (0.15 M, pH 7.2). Approximately 500–600 ll of
BAL fluid was harvested per mouse, and this was centrifuged
at 3,000 rpm for 10 min.
BAL fluid analysis
Total cells in the BAL fluid were quantified by hemocytometric
counting. Cell differentials were performed on
cytocentrifuged preparations fixed in methanol and stained
with Diff-Quick (Thermo Shandon, PA, USA). Distributions
of the alveolar macrophages, neutrophils, and lymphocytes
were assessed by their characteristic cell shapes.
Measurement of cytokines
The concentrations of each cytokine in the supernatant of
the BAL fluid and serum were determined using commercially
available ELISA kits (eBioscience, San Diego,
CA, USA). First, each well of the microplates was coated
with 100 ll of capture antibody and incubated overnight at
4°C. After washing and blocking with assay diluent and
BAL fluid, serum or standard antibody was added to the
individual wells. The plates were then maintained at room
temperature for 2 h. Next, the plates were washed, and
biotin-conjugated detecting antibody was added to each
well. Then, the plates were incubated at room temperature
for 1 h. After incubation, the plates were washed again and
further incubated with avidin–HRP for 30 min before
detection using TMB solution. Finally, the reactions were
stopped by adding 1 M H 3 PO 4 , and the absorbance at
450 nm was measured with an ELISA reader (Molecular
Devices, Sunnyvale, CA, USA). The amount of cytokine
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1124 Arch Toxicol (2011) 85:1121–1131
was calculated from the linear portion of the generated
standard curve (Park et al. 2009a, b).
Immunophenotyping
All monoclonal antibodies were purchased from eBioscience
(San Diego, CA, USA). T cells (CD3, 1:50), B cells
(CD19, 1:50), NK cells (DX5, 1:100), CD4 T cells
(CD4 , 1:160), and CD8 T cells (CD8 , 1:50) were
identified using directly conjugated anti-mouse antibodies.
First, the blood samples were blocked with Fc-block (eBioscience,
San Diego, CA, USA) to reduce non-specific antibody
binding. The cells were then incubated in the dark with
10 ll of the appropriate fluorochrome-conjugated antibody
for 20 min at 4°C. The cells were then washed with fluorescence-activated
cell sorter (FACS) buffer. The blood was
lysed for 5 min with FACS lysis buffer (BD Bioscience,
Franklin Lakes, NJ, USA) at room temperature and then rewashed
with FACS buffer. Finally, each sample was fixed
with 1% paraformaldehyde until further analysis.
Flow cytometry analysis was performed using the
FACSCalibur system (BD Biosciences, Franklin Lakes, NJ,
USA). Control samples were matched for each fluorochrome.
Data were analyzed using CellQuest software
(Becton–Dickinson, Franklin Lakes, NJ, USA) (Silva et al.
2005; Vendrame et al. 2006).
Measurement of collagen
The collagen concentration was measured using a mouse
hydroxyproline ELISA kit (USCNLIFE SCIENCE and
TECHNOLOGY CO., LTD., Wuhan, China). First, 100 ll
of standard, blank, or sample was added per well and the
plates were incubated for 2 h at 37°C. Then, the liquid was
removed from each well and detection reagent A working
solution was added to each well. The plates were then
incubated at 37°C for 1 h. After washing, detection reagent
B working solution was added to each well and incubated
at 37°C for 1 h. After re-washing, substrate solution was
added to each well and incubated at 37°C for 15 min
protecting from light. Finally, the reaction was terminated
by the addition of stop solution, and the absorbance was
measured at 450 nm using a microplate reader (Molecular
Devices, Sunnyvale, CA, USA). The amounts of secreted
collagen were calculated from the linear portion of the
prepared standard curve.
Protein expression in tissue
Lung tissue was homogenized with a protein extraction
solution (PRO-PREP TM , Cat. No. 17081, iNtRON
biotechnology, Kyunggi, Korea), and lysates were centrifuged
at 13,000 rpm for 10 min. The protein concentration
was measured by the Bradford method (Bio-Rad
Protein Assay, Bio-Rad Laboratories Inc., Hercules, CA),
and equal amounts of proteins (40 lg) were separated on
a SDS/1%-polyacrylamide gel and then transferred to a
nitrocellulose membrane (Hybond ECL, Amersham
Pharmacia Biotech Inc., Piscataway, NJ, USA). Blots
were blocked for 2 h at room temperature with 5% (w/v)
non-fat dried milk in Tris-buffered saline (10 mM Tris,
pH 8.0, and 150 mM NaCl) solution containing 0.05%
Tween-20. The membranes were immunoblotted with
primary specific antibodies: rabbit polyclonal for rabbit
polyclonal of caspase-3 and ADP ribose polymerase
(PARP) (1:1,000 dilution; Cell Signaling Technology, Inc.
Beverly, MA, USA), COX-2 (1:500 dilution; Cayman
Chemical, MI, USA), and MMP-2 (1:500 dilution; Santa
Cruz Biotechnology Inc. CA, USA); rabbit monoclonal
for b-actin (1:2,000 dilution; Cell Signaling Technology,
Inc. Beverly, MA, USA); mouse monoclonal for mesothelin,
p53, and MMP-9 (1:1,000 dilution; Santa Cruz
Biotechnology Inc. CA, USA), and iNOS (1:1,000 dilution;
BD Biosciences, CA, USA); and goat polyclonal for
COL1A1 (1:1,000 dilution; Santa Cruz Biotechnology
Inc. CA, USA). The blots were then incubated with the
corresponding conjugated anti-mouse, anti-rabbit, or antigoat
immunoglobulin G-horseradish peroxidase (1:2,000
dilution; Santa Cruz Biotechnology Inc.). Immunoreactive
proteins were detected with the ECL western blotting
detection system.
Histopathology
Histopathological analysis was performed at the Korea
Institute of Toxicology (Yuseong-gu, Daejeon, Korea) on
tissue harvested from six mice per group. The lung, brain,
and thymus from mice in the control group and the treated
group were fixed with 10% neutral buffered formalin and
processed using routine histological techniques. After
paraffin embedding, 3 lm sections were cut and stained
with hematoxylin and eosin (H&E) for histopathological
evaluation.
Statistical analysis
The results obtained from the chemically treated groups
were compared to those of the control group. The values
were compared using Dunnett’s t test after one-way
ANOVA, and levels of significance were represented
compared to the control group.
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Arch Toxicol (2011) 85:1121–1131 1125
Results
Cell distribution in BAL fluid
The number of total cells recovered in the BAL fluid of
mice treated with SWCNTs did not significantly increase at
Day 1 after administration but did significantly increase by
Day 7 (Fig. 2a) compared with the control. When the
composition of cells in the BAL fluid was analyzed, the
percentage composition of neutrophils, which is characteristic
of the initial stage of inflammation, showed maximum
(8.41 ± 0.01%) increase at Day 1. The distribution
ratio of macrophages increased again by Day 7. The distribution
ratio of lymphocytes increased to 0.7 ± 0.0%,
1.8 ± 0.0%, 3.4 ± 0.0, and 2.1 ± 0.0% at Days 1, 7, 14,
and 28, respectively, compared to 0.4 ± 0.0% of the control
(Fig. 2b).
Cytokines in BAL
To identify inflammatory responses to the administration
of SWCNT, we measured the concentrations of proinflammatory
cytokines (IL-1, TNF-a, and IL-6), Th0
cytokine (IL-2), Th1-type cytokines (IL-12 and IFN-c),
and Th2-type cytokines (IL-4, IL-5, IL-10, IL-13, and
IL-17) in the BAL fluid over time (Table 1). Proinflammatory
cytokines, such as IL-1b, TNF-a, and IL-6,
rapidly increased at Day 1 after instillation and were upregulated
during the experimental period. IL-2 reached
the maximum at Day 7. IL-12 and IL-10 rapidly increased
at Day 1 and remained at a similar level until Day 28.
IFN-c and IL-4 reached the maximum at Day 1, and IL-5
reached the maximum at Day 7. IL-13 and IL-17
increased in a time-dependent manner.
Cytokines in the blood
As shown in Table 2, the level of IL-6 reached the maximum
at Day 7, and that of IL-12 reached the maximum at
Day 14 after treatment. IL-17 was up-regulated during the
experimental period. However, neither IL-1, TNF-a, IL-2,
IFN-c, IL-4, IL-5, IL-10, nor IL-13 was detected at any of
the designed time points.
Secretion of TGF-b and collagen
In the BAL fluid, the concentration of TGF-b was
103.3 ± 6.6 pg/ml, 146.2 ± 9.2 pg/ml, 106.9 ± 1.1 pg/ml,
and 117.6 ± 2.0 pg/ml at Days 1, 7, 14, and 28,
respectively, after treatment. The concentration for the
control was 7.2 ± 0.1 pg/ml (Fig. 3). In blood, the
concentration of TGF-b reached the maximum at Day 7
(45.1 ± 9.5 pg/ml).
Fig. 2 Changes of cell distribution in BAL fluid after treatment
SWCNTs (n = 4). Mice were treated with 100 lg/kg of SWCNTs
and then killed on the designated Day (1, 7, 14, or 28). Total cells in
the BAL fluid were quantified by hemocytometric counting a, and the
distributions of alveolar macrophages, neutrophils, and lymphocytes
were identified by their characteristic cell shapes b. Level of control
group is mean ± SD of the values measured at each time point.
*P \ 0.05; **P \ 0.01
The concentration of collagen following administration
of 100 lg/kg of SWCNT was also measured. As shown in
Fig. 4, the concentration of collagen in the BAL fluid and
in blood reached the maximum of 16.5 ± 0.4 ng/ml and
928.7 ± 150.5 ng/ml at Days 14 and 7, respectively. The
concentration of collagen in the control group was
2.81 ± 0.00 ng/ml and 659.59 ± 79.47 ng/ml in BAL and
blood, respectively.
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1126 Arch Toxicol (2011) 85:1121–1131
Table 1 Changes in cytokine levels in BAL fluid after a single treatment of SWCNTs (n = 4)
Control Day 1 Day 7 Day 14 Day 28
IL-1 b 19.9 ± 12.7 64.1 ± 9.5* 73.4 ± 19.3* 82.9 ± 29.4* 89.0 ± 41.0
TNF-a 16.4 ± 13.5 43.6 ± 12.8* 44.3 ± 4.4* 41.6 ± 8.8* 41.6 ± 6.8*
IL-6 22.0 ± 5.8 115.6 ± 16.8** 137.2 ± 11.9** 151.3 ± 45.2** 129.2 ± 24.7**
IL-2 5.1 ± 5.9 66.9 ± 23.0** 151.5 ± 27.0** 77.8 ± 3.5** 81.5 ± 22.9**
IL-12 14.1 ± 2.9 49.1 ± 14.7* 45.7 ± 6.0** 45.5 ± 8.1* 52.6 ± 12.3*
IFN-c 7.0 ± 2.3 32.2 ± 6.1** 16.9 ± 3.6* 26.1 ± 7.5* 23.5 ± 3.6*
IL-4 20.2 ± 1.9 59.8 ± 7.2* 47.6 ± 1.9** 50.7 ± 6.9* 48.6 ± 4.3*
IL-5 15.4 ± 1.4 35.8 ± 8.4* 45.8 ± 5.8** 36.4 ± 8.0* 41.5 ± 10.7*
IL-10 64.6 ± 2.7 100.0 ± 17.0* 97.0 ± 13.8* 93.7 ± 11.1* 120.1 ± 45.9
IL-13 13.1 ± 3.8 44.3 ± 5.3* 43.1 ± 4.1* 50.83 ± 4.4** 56.9 ± 4.6**
IL-17 6.1 ± 3.9 36.2 ± 7.4* 38.4 ± 8.0* 45.2 ± 23.5 48.2 ± 30.1
BAL fluid was harvested and pooled (400 ll per mouse) at Days 1, 7, 14, and 28 after treatment
Cytokine concentrations were determined using ELISA kits. Level of control group was calculated as mean ± SD of the values obtained from
4 mice per time point. * P \ 0.05; ** P \ 0.01
Table 2 Changes in cytokine levels in the blood after a single treatment of SWCNTs (n = 4)
Control Day 1 Day 7 Day 14 Day 28
IL-1 b ND ND ND ND 1.2 ± 0.0
TNF-a ND ND ND ND ND
IL-6 ND 4.5 ± 0.1** 26.6 ± 2.6** 12.6 ± 0.8** ND
IL-2 ND ND ND ND ND
IL-12 38.1 ± 2.3 58.5 ± 3.8* 46.5 ± 0.9* 76.1 ± 22.5 52.4 ± 16.9
IFN-c ND ND ND ND ND
IL-4 ND ND ND ND ND
IL-5 ND ND ND ND 4.15 ± 0.56
IL-10 ND ND ND ND ND
IL-13 1.5 ± 0.0 2.7 ± 0.0 3.3 ± 0.0 1.9 ± 0.0 2.4 ± 0.0
IL-17 52.1 ± 1.5 85.3 ± 31.6 62.3 ± 8.3 98.5 ± 1.9* 62.4 ± 12.5
Serum was harvested and pooled (400 ll per mouse) at Days 1, 7, 14, and 28 after treatment
Cytokine concentrations were determined using ELISA kits. Level of control group was calculated as mean ± SD of the values obtained from
4 mice per time point. * P \ 0.05; ** P \ 0.01
Lymphocyte phenotype
A single instillation of 100 lg/kg SWCNTs significantly
increased the proportion of T cells in blood at Day 1.
However, the proportion of B cells in blood lymphocytes
had rapidly increased by 85.80 ± 5.20%, 70.33 ± 2.68%,
and 62.80 ± 5.81% at Days 7, 14, and 28, respectively,
compared to 59.03 ± 0.95% of the control (Table 3;
Fig. 5a, b). The ratio of CD4/CD8 T cells also exhibited
a significant change compared to the control group
(Table 3; Fig. 5c, d). The ratio of CD4/CD8 T cells in
the blood lymphocytes of the SWCNT-treated group was
4.05 ± 0.11, 3.31 ± 0.09, 3.75 ± 0.03, and 4.98 ± 0.19 at
Days 1, 7, 14, and 28, respectively, while that of the control
group was 4.44 ± 0.20.
Histopathology of lung tissue
We observed histopathological changes in the lung, brain,
and thymus. Figure 6 and Table 4 show the histopathological
changes in the lung tissue following treatment with
SWCNTs. SWCNTs induced fibrotic pathology by Day 7,
but the severity of the symptoms decreased with time. At
Day 1, phagocytosis by macrophages was observed, and
cell infiltration and fibrosis were observed in one of the
samples tested. By Day 7, the number of individuals
showing cell infiltration and fibrosis had increased. No
pathological symptom, including phagocytosis, cell infiltration,
or fibrosis, was observed at Day 28. No pathological
changes were observed in the brain and thymus (data
not shown).
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Arch Toxicol (2011) 85:1121–1131 1127
Protein expression by SWCNT
As shown in Fig. 7, the expression of the apoptosis-related
proteins p53 and caspase-3 and the inflammatory proteins
COX-2 and iNOS showed maximum induction at Day 7
and Day 14, respectively. The expression of the tissue
damage-related proteins COL1A1, MMP-2, MMP-9, and
mesothelin also markedly increased until Day 28 after
treatment.
Discussion
Fig. 3 Levels of TGF-b in BAL fluid and in blood after a single
instillation of SWCNTs (n = 4). BAL fluid and serum were harvested
and pooled at Days 1, 7, 14, and 28 after treatment with 100 lg/kg of
SWCNTs. The TGF-b concentration in each sample was determined
using commercially available kits. Level of control group is
mean ± SD of the values measured at each time point. *P \ 0.05;
**P \ 0.01
Fig. 4 Levels of collagen in BAL fluid and in blood after a single
treatment with SWCNTs (n = 4). BAL fluid and serum were
harvested and pooled at Days 1, 7, 14, and 28 after treatment with
100 lg/kg of SWCNTs. The collagen concentration in each sample
was determined using commercially available kits. Level of control
group is mean ± SD of the values measured at each time point.
*P \ 0.05
Human beings are constantly exposed to environmental
pollutants since birth. The development of new technology
and materials has improved the quality of life for humans,
but it also entails the possibility of exposure to new
materials. One of the recent hot issues is nanoparticles,
including CNTs. Extensive toxicological studies with in
vitro and in vivo models are required for the safe application
of nanoparticles. In our study, 2 of the 64 mice died
at Day 3 instilled with SWCNTs, one death due to pigmented
histiocytes and the other due to a hemorrhage in the
lung.
Although the raw materials are same, nanoparticles can
have different physicochemical properties according to the
manufacturing process. The toxicity of nanoparticles
depends on various parameters, such as the purity of the
samples, structure, size distribution, surface area, surface
chemistry, surface charge, and agglomeration state (Kagan
et al. 2006; Murray et al. 2009; Herzog et al. 2009). In one
study, exposure of JB6P cells (murine epidermal cells)
to unpurified SWCNT (30% iron) resulted in the production
of ESR detectable hydroxyl radicals and caused a
significant dose-dependent activation of the transcription
factor, AP-1. However, no significant changes in AP-1
activation were detected when partially purified SWCNT
(0.23% iron) was introduced to the cells (Murray et al.
2009). In another study, SWCNT dispersed in dipalmitoylphosphatidylcholine,
the main component of lung lining
fluid, suppressed the production of inflammatory
mediators, such as IL-8, IL-6, TNF-a, and MCP-1, in
human lung epithelial cells (Herzog et al. 2009). In this
study, we used SWCNT dispersed by ultra-sonication and
used SDS as a surfactant.
Nanoparticles entering the body trigger the innate
immune system. This is usually resolved by macrophages
phagocyting the nanoparticles. Some researchers have
suggested that SWCNTs impair the phagocytic function of
macrophages in both a cytotoxic and a non-cytotoxic state
(Witasp et al. 2009; Nygaard et al. 2009; Shvedova et al.
2008a, b). For example, SWCNTs with low metal impurity
content did not exert direct cytotoxic effects on human
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1128 Arch Toxicol (2011) 85:1121–1131
Table 3 Lymphocyte phenotypes in blood after treatment with SWCNTs (n = 4)
Control Day 1 Day 7 Day 14 Day 28
NK cell 0.13 ± 0.06 0.63 ± 0.15 0.50 ± 0.10 0.63 ± 0.19 0.37 ± 006
NKT cell 0.03 ± 0.06 0.13 ± 0.06 0.10 ± 0.00 0.18 ± 0.05 0.27 ± 0.06
B cell 59.03 ± 0.95 51.63 ± 5.58 85.80 ± 5.20* 70.33 ± 2.68* 62.80 ± 5.81
T cell 40.77 ± 0.95 47.63 ± 5.71 13.60 ± 5.16** 28.88 ± 2.81* 36.57 ± 5.80
CD4/CD8 4.44 ± 0.20 4.05 ± 0.11 3.31 ± 0.09* 3.75 ± 0.03* 4.98 ± 0.19
Level of control group is mean ± SD of the values measured at each time point. * P \ 0.05; ** P \ 0.01
Fig. 5 Lymphocyte phenotypes
in blood after treatment with
SWCNTs. The experiment was
repeated 4 times using samples
pooled by n = 4 and
representative data are shown.
Control samples were matched
for each fluorochrome:
a lymphocyte of control,
b lymphocyte at Day 7,
c T subtype of control, and
d T subtype at Day 7. a and
b consist of Q1-NK, Q2-NKT,
Q3-B, and Q4-T cells
monocyte-derived macrophages (HMDM). However,
SWCNT suppressed the chemotaxis of primary human
monocytes. Moreover, macrophage engulfment of apoptotic
target cells was significantly impaired following the preincubation
of HMDM with SWCNT at non-cytotoxic
concentrations (Witasp et al. 2009).
Other researchers reported the inflow of immune cells
into target sites by the phagocytes of SWCNTs (Shvedova
et al. 2005; Inoue et al. 2008). The pharyngeal aspiration of
SWCNTs (about 500 lg/kg) induced an early neutrophil
accumulation in BAL fluid at Day 1, followed by lymphocytes
at Day 3 and macrophages at Day 7 (Shvedova
et al. 2005). The concentration of MIP-1a and MCP-1 in
BAL fluid significantly increased by about 32.2-fold and
10.7-fold, respectively, at 24 h after instillation with
4 mg/kg SWCNT compared to the vehicle control. The
number of total cells and neutrophils in BAL fluid also
significantly increased by about 1.7-fold and 22.4-fold,
respectively, compared to the vehicle control (Inoue et al.
2008). In the present study, the number of total cells in the
BAL fluid increased 1.7-fold, 3.9-fold, 4.8-fold, and 3.2-fold
at Days 1, 7, 14, and 28, respectively, after instillation with
100 lg/kg SWCNTs in relation to the control group. The
inflow of neutrophils was markedly increased at Day 1, and
the proportion of lymphocytes to the number of total cells
increased by 5.1-fold, 9.5-fold, and 5.7-fold at Days 7, 14,
and 28 after treatment, respectively, relative to the control.
Pro-inflammatory cytokines, such as IL-1b, TNF-a, and
IL-6, play an important role in the initiation of the immune
system when it comes to removing xenobiotics. In prior
reports, the secretion of TNF-a and IL-1b was elevated by
16-fold and tenfold, respectively, at Day 1 by the pharyngeal
aspiration of SWCNT (about 500 lg/kg) (Shvedova
et al. 2005). The concentration of IL-1b in BAL fluid
123

Arch Toxicol (2011) 85:1121–1131 1129
Fig. 6 Histopathological changes in lung tissue after a single intratracheal treatment of SWCNTs. Lung sections were stained with hematoxylin
and eosin stains (9200): a solvent control, b Day 1, c Day 7, and d Day 28. Tissue of control group was randomly chosen from each time point
Table 4 Histopathological changes in lung tissue after a single intratracheal treatment of SWCNTs (n = 6)
Control Day 1 Day 7 Day 14 Day 28
F I F I F I F I F I
Pigmented macrophage 4/6 ± or 4/6 ± or 5/6 ± or 2/6 ±
Inflammatory cell 1/6 ± 3/6 ± or 3/6 ± or
Fibrosis, interstitial 1/6 ± 3/6 ± 1/6 ±
Microfibrillar material 1/6 ±
Epithelial degeneration, bronchiole 1/6 ± 1/6 ±
Tissue of control group was randomly chosen from each time point. F Frequency of the events, I Intensity of the events, ± minimal; mild
significantly increased to about 6.8-fold at 24 h after
treatment with 4 mg/kg of SWCNT compared to the
vehicle control, but its level in the blood stream did not
increase (Inoue et al. 2008). In this study, the concentration
of IL-1b, TNF-a, and IL-6 rapidly increased by 3.2-fold,
2.7-fold, and 5.2-fold relative to the control at Day 1 after
instillation and remained at similar levels during the
experimental period.
In addition, macrophages secrete IL-2 to stimulate naïve
T cells to trigger the adaptive immune system. Following
this, activated T cells are differentiated into Th1 cells and
Th2 cells. These differentiated cells secrete IFN-c and
IL-10 to stimulate themselves or to inhibit the activation of
another, respectively. Th1 and Th2 cells have very different
functions. Th2 cells are the most effective activators of
B cells, especially in primary responses, whereas Th1 cells
123

1130 Arch Toxicol (2011) 85:1121–1131
Con. 1 7 14 28
β- Actin
COX-2
iNOS
Caspase-3
p53
MMP-9
MMP-2
COL1A1
Mesothelin
Fig. 7 Changes in protein expression following treatment with
SWCNTs. Results were confirmed by several separate experiments,
and representative images are shown. Tissue of control group was
pooled from all mice in the control group
are crucial for activating macrophages. However, under
many circumstances in vivo, there is a mixed Th1 and Th2
response, and the overall effect is determined by the
responses that are dominant. In this study, Th0-type cytokine
(IL-2), Th1-type cytokines (IL-12, IFN-c), and Th2-
type cytokines (IL-4, IL-5, IL-10, IL-13) were markedly
increased at Day 1 in the BAL fluid and steadily remained
at the up-regulated level during the experimental period.
We hypothesize that the sustained increase of pro-inflammatory
and Th1-type cytokine may be the effect of the
sustained inflow of macrophages into damaged sites.
Inoue et al. suggested that any histopathological opinion
on the attack of fibrotic lesions was not observed after the
administration of 4 mg/kg of SWCNTs. However, Shvedova
et al. (2005) suggested that the fibrotic response
proceeded with time, as the alveolar wall was significantly
thicker at 60 days than it was at 28 days postexposure to
roughly 2 mg/kg of SWCNTs. Moreover, the deposition of
collagen and elastin was observed in granulomatous
regions, as well as in the areas distant from granulomas. In
our study, the fibrotic lesions histopathologically peaked on
Day 7, and the severity of the symptoms decreased with
time. In addition, cell composition in lymphocytes shifted
from T cell dominant at Days 1 and 28 to B cell dominant
at Days 7 and 14. The population of B cells in lymphocytes
increased to 1.45-fold and 1.19-fold at Day 7 and Day 14,
respectively, compared to that of the control group. The
ratio of cytotoxic T cells relative to helper T cells also
significantly increased until Day 14 after treatment. Cytotoxic
T cells are capable of inducing death in somatic
tumor cells, infected cells, damaged cells, and dysfunctional
cells through the release of cytotoxins, such as perforins,
granzymes, and granulysin. The theory that early
outbreak of fibrosis is caused by exposure to SWCNTs is
also supported by the expression of inflammatory response-
(iNOS and COX-2), apoptosis- (p53 and caspase-3), and
matrix damage- (MMP-9, MMP-2, and COL1A1) related
protein, which peaked at Day 7 and Day 14.
Some researchers also have suggested that the retention
of serve is the initiator of mesothelial injury and inflammation
that over time leads to pleural pathology, including
mesothelioma (Pacurari et al. 2008; Takagi et al. 2008;
Sakamoto et al. 2009; Donaldson et al. 2010). TGF-b
exhibits three major activities: inhibition of cell proliferation,
immunosuppression, and enhancement of the formation
of the extracellular matrix (Lawrence 1996). Collagen
is the main protein of connective tissue and is related to the
repair of injured matrices (Kershenobich Stalnikowitz and
Weissbrod 2003; Karsenty and Park 1995; Cutroneo 2007).
It is known that the expression of mesothelin in normal
tissue is limited to mesothelial cells, but is over-expressed
in several human tumors, including mesothelioma and
ovarian, lung, and pancreatic adenocarcinomas (Hassan
and Ho 2008). In our study, the expression of mesothelin
increased in a time-dependent manner. Furthermore, at Day
28, although the fibrotic lesions were almost completely
resolved, the secretion of TGF-b and collagen and the
expression of p53 related to the repair of damaged DNA
were still being up-regulated.
Based on these findings, we suggest that a single intratracheal
instillation of SWCNTs may induce early lung
fibrosis and subchronic tissue damage. Thus, sufficient
verification of the toxicity of SWCNT is necessary before
the expansion of its market size to avoid unforeseen
consequences.
Acknowledgments This work was supported by Ministry of Environment
as the Eco-technopia 21 project and National Institute of
Environmental Research.
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