Genotoxicity of multi-walled carbon nanotubes at
occupationally relevant doses
Katelyn J Siegrist1
Steven H Reynolds1
Michael L Kashon1
David T Lowry1
Ann F Hubbs1
Jeffrey L Salisbury3
Dale W Porter1
Stanley A Benkovic1
Michael J Keane1
John T Mastovich5
Kristin L Bunker5
Lorenzo G Cena1
Mark C Sparrow5
Jacqueline L Sturgeon5
Cerasela Zoica Dinu2*
* Corresponding author
Linda M Sargent1*
* Corresponding author
1 National Institute for Occupational Safety and Health, Morgantown, WV 26505,
2 Department of Chemical Engineering, Benjamin M. Statler College of
Engineering and Mineral Resources, West Virginia University, Morgantown, WV
3 Department of Biochemistry, Mayo Clinic, 2001st Street NW, Rochester, MN
4 Department of Occupational & Environmental Health Sciences, West Virginia
University, Morgantown, WV 26506, USA
5 RJ Lee Group Inc, 300 Hockenberg Drive, Monroeville, PA 15146, USA
Carbon nanotubes are commercially-important products of nanotechnology; however, their
low density and small size makes carbon nanotube respiratory exposures likely during their
production or processing. We have previously shown mitotic spindle aberrations in cultured
primary and immortalized human airway epithelial cells exposed to single-walled carbon
nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes
(MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of
exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose
responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid
chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 µg/cm2 MWCNT.
Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional
reconstructions of 0.1 µm optical sections showed carbon nanotubes integrated with
microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a
greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated
compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar
phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp
contrast to the multi-polar spindle and G2 block in the cell cycle previously observed
following exposure to SWCNT. One month following exposure to MWCNT there was a
dramatic increase in both size and number of colonies compared to diluent control cultures,
indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate
significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure
Carbon nanotubes (CNT) are used in many consumer and industrial products including
electronic devices, protective clothing, sports equipment and medical devices as well as
vehicles for drug delivery [1-3]. Due to the wide variety of applications, the nanotechnology
industry is predicted to grow to one trillion dollars by 2015 . The low density and small
size of carbon nanotubes make respiratory exposure likely during production and processing.
Indeed, recent investigations have shown that carbon nanotubes can be aerosolized under
workplace conditions [5-8]. Although carbon nanotubes have a large variety of applications,
their potential health effects have not been fully investigated.
The low density, fiber-like geometry and durability of carbon nanotubes are characteristics
shared with asbestos [9,10]. Single-walled and multi-walled carbon nanotubes have been
shown to enter cells and induce DNA damage, sister chromatid exchange, chromosome
damage and micronuclei in vitro in human keratinocytes, human breast cancer cell lines,
human lung cancer epithelial cells and immortalized mouse fibroblasts (Balb/3 T3 cells) [11-
15]. Micronuclear formation can result from either a high level of chromosome damage or
mitotic spindle disruption. Research by Di Giorgio et al., 2011 demonstrated significant
chromosome breakage by analysis of chromosome spreads as well as DNA damage by the
comet assay in a mouse macrophage cell line 24–48 hours after exposure to MWCNT (10–25
nm) and SWCNT (0.7-1.2 nm) material . The carbon nanotube-exposed cells also had
high levels of intracellular reactive oxygen species suggesting that carbon nanotubes can
cause chromosome damage through reactive oxygen species . Increased DNA damage
due to oxygen radicals was also observed in imprinting control region mice (ICR) mice in
vivo following intratracheal installation of 0.05 or 0.2 mg MWCNT/mouse . Carbon
nanotubes bind to DNA at G-C rich regions in the chromosomes including telomeric DNA
[17,18]. The interaction with the DNA results in a conformational change. DNA intercalation
and telomeric binding can induce chromosome breakage suggesting that interaction of the
nanotubes with the DNA may also be a source of chromosome damage. Recent investigations
have shown that acid-washed single-walled carbon nanotubes of 1–4 nm in diameter and one
micron in length induce centrosome fragmentation, multipolar mitotic spindles and errors in
chromosome number in cultured immortalized and primary lung epithelial cells .
Furthermore, exposure of cancer cell lines to MWCNT of 5–10 nm diameter and one micron
in length also results in multipolar mitotic spindles .
Mitotic spindle disruption and aneuploidy are a concern because these effects have been
observed with the carcinogenic fiber, asbestos. In vitro investigations have demonstrated that
chrysotile asbestos exposure causes multipolar mitotic spindles and a G2/M block similar to
SWCNT and vanadium pentoxide exposure [19,21-24]. Asbestos exposure disrupts the
mitotic spindle and causes aneuploidy through amplification of the centrosome [21,22]. By
contrast, the mitotic disruption and aneuploidy resulting from vanadium pentoxide and
SWCNT is associated with fragmented centrosomes [19,23]. Furthermore, in vitro
examinations of asbestos and vanadium pentoxide potency have demonstrated that the
disruption of the mitotic spindle and aneuploidy in cultured cells is strongly correlated with in
vivo carcinogenesis [25-28]. Together these investigations indicate the importance of
genotoxicity in carcinogenesis as well as validating the significance of culture models to
To simulate aerosol exposures in the workplace, rodents have been exposed to high aspect
ratio particles by inhalation, pharyngeal aspiration or intratracheal installation. In a manner
similar to asbestos, rodent pulmonary exposure to biopersistant carbon nanotubes has been
shown to result in lung inflammation, epithelial cell proliferation, cellular atypia and
mutations in the K-ras gene [29-32]. The lung is the principal site of carbon nanotube
deposition and toxicity following aspiration or inhalation [31,33]. In vivo investigations have
demonstrated that carbon nanotube exposure can cause macrophages without nuclei as well
as dividing macrophages connected by nanotubes [30,31]. Exposure of rats to the MWCNT
by pharyngeal aspiration has been shown to result in micronuclei formation in Type II
epithelial cells further indicating the potential for genetic damage . Inflammation, cellular
proliferation, cellular atypia, mitotic spindle disruption, centrosome fragmentation and errors
in chromosome number are linked with the development of cancer [34-40]. Chronic
exposures to asbestos particles which induce strong inflammatory, proliferative and
genotoxic responses in the lung are associated with an increased incidence of lung cancer in
rodents [41,42]. Although the lung is the key target organ for particle toxicity, high aspect
ratio carbon nanotubes have been shown to translocate to the subpleural space indicating that
the mesothelial cells are also a potential target [43,44].
The overall objective of our study was to examine the role of CNT diameter in the nanotube-
induced genetic damage using carbon nanotubes prepared with the same acid washing
procedure and one micron length used in our previous studies to evaluate the potential
genotoxicity of the narrower SWCNT [24,45]. Because vanadium pentoxide has been
demonstrated to induce aneuploidy and mitotic spindle disruption through fragmentation of
the centrosome, we selected vanadium as the positive control for genotoxicity. Immortalized
and primary lung epithelial cells were examined for the potential of MWCNTs to cause
aneuploidy, mitotic spindle disruption, centrosome fragmentation, and cell cycle distribution
following exposure of primary and immortalized human epithelial cells to occupationally
relevant doses of 10–20 nm diameter MWCNT. Primary cells were used in the assays since
the normal karyotype made it possible to determine changes in chromosome number after
exposure. The concentrations chosen for the current investigation were selected to be relevant
to previous in vivo exposure doses of MWCNT of 10–40 µg/mouse (0.5 µg, 1 µg, and 2 µg/kg
respectively) reported by Porter et al. . In brief, the mouse lung burdens per alveolar
epithelial surface area of 500 cm2/mouse lung  correspond to in vitro concentrations of
0.02–0.08 µg/cm2. The minimal in vitro dose of 0.02 µg/cm2 MWCNT would require 4
weeks of exposure at the Occupational Safety and Health Administration (OSHA)
permissible exposure limit for particles with an aerodynamic diameter of 5 microns or less of
5 mg/m3 [47,48]. NIOSH has recently reduced the REL from 7 µg/m3 to 1 µg/m3  .
Although exposure to concentrations of carbon nanotubes equivalent to the current NIOSH
REL of 1 µg/m3 would require 34 years to yield a equivalent exposure of the 0.024 µg/cm2,
levels of MWCNT between 0.7 and 331 µg/m3 have been measured in workplace air [6,7,50-
Characterization of carbon nanotubes
Raman spectroscopy was used to characterize the structure of pristine and acid-washed
MWCNTs and to determine the degree of MWCNTs functionalization after acid treatment.
Figure 1A shows the Raman spectra of pristine and acid-washed MWCNT. There are 4 bands
identified in both pristine and acid-washed MWCNTs samples, i.e. D band around 1350 cm-1
that reflects the level of disorder in the sample, the G band around 1585 cm-1 indicative of the
high degree order and well-structured samples, the G’ band around 2690 cm-1 representing
the binary disordered band and lastly the peak around 2930 cm-1 indicative of the oxidation
level of the sample being characterized. As shown, the D band was wider and had a higher
frequency for the acid-washed sample when compared to the pristine MWCNTs. The shift in
the D band indicates that the acid treatment minimally altered the chemical structure of
MWCNTs by disrupting the structured walls and introducing additional functional groups
(carboxylic acid groups) . For the acid-washed MWCNTs there was also a shift of G’
band towards higher frequency; this may be due to the removal of metal catalysts, increase in
the number of functional groups having electron accepting ability and decrease in the
amorphous carbon. The ratio of intensity of D to G peaks indicate the degree of
functionalization [54-56] and was 0.59 for pristine and 0.81 for 1 hr. acid-washed MWCNTs.
This also confirms that the acid treatment increased the number of functional groups (i.e. free
carboxylic acid groups) on the walls of the MWCNTs samples. Energy dispersive X-ray
spectroscopy (EDX) confirmed the increase in the oxygen content due to the acid treatment
and thus the increase in the MWCNTs degree of functionalization with free carboxylic acid
groups as shown in Additional file 1. Further, the acid washing also reduced the catalyst
content in the sample (Fe, 0.81). The content of the iron, cobalt and nickel were further
analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). Specifically, the
MWCNT by ICP-MS contained 0.03% Fe ±0 .001, 0% cobalt, and 0% Nickel .
Figure 1 Raman characterization, electron microscopy analysis and length distribution
of MWCNTs. Figure 1A. The figure is a histogram of the Raman spectra of pristine (black)
and one hour acid-washed carbon nanotubes (red). Four independent bands have been
identified for both samples, i.e., D band around 1350 cm-1, G band at 1585 cm-1, G’ band
around 2690 cm-1, and an additional band around 2930 cm-1. Shifts in these bands are
noticed for samples that have been treated with acid for 1 h. Figure 1B. Histograms of length
distribution of pristine (a) and 1 h acid-washed MWCNTs (b) as identified by tapping mode
Atomic force microscopy (AFM). At least 30 nanotubes have been analyzed for each one of
the samples. Figure 1C, D, E, F: Figure 1C shows a representative bright-field image and
Figure 1D shows the corresponding dark-field image of the MWCNT sample. The images
demonstrated that the MWCNTs have a diameter of 10–20 nm and a typical multi-walled
tubular morphology. Figure 1D shows representative dark-field STEM (DF-STEM) image of
the native MWCNT sample that was acquired. The analysis demonstrated low amounts of the
iron catalyst. Figure 1E shows a representative bright-field image and Figure 1F shows the
corresponding dark-field image of the MWCNT sample. The dark-field image provides
atomic number contrast information. The bright 10 nm particle at the end of the MWCNT in
Figure 1F is a catalyst particle. Energy dispersive X-ray spectroscopy (EDS) showed that the
catalyst particle was iron-rich. Further analysis of the MWCNT sample identified low
amounts of the iron catalyst.
The length distribution of pristine and 1 h acid-washed MWCNT respectively is shown in
Figure 1B (at least 30 individual MWCNTs were measured for each sample). AFM analysis
showed that pristine MWCNT samples had an average length of 5499 ± 3009 nm while 1 h
acid-washed MWCNTs had an average length of 825 ±585 nm respectively indicating that
acid treatment led to shortening of the nanotubes. The pristine and acid washed MWCNT had
a diameter of 15 ±5 nm. Moreover, acid washing also increased nanotube solubility in
DMEM + FBS by two-fold compared to pristine MWCNT  as a result of the addition of
the free carboxylic acid groups .
Mitotic spindle disruption
Two human epithelial cell populations were examined to determine whether MWCNT
induced genetic damage. Immortalized respiratory epithelial cells (BEAS-2B) were used to
determine the effects of MWCNT on the mitotic spindle. Primary respiratory epithelial cells
(SAEC) were included in the analysis to determine whether MWCNT induced errors in
chromosome number. Treatment with acid-washed MWCNT induced a dose dependent
mitotic spindle disruption (Figure 2A). The disrupted mitotic spindles were predominantly
monopolar (Figure 2B). Figure 2C shows a 20X photomicrograph of the cultured cells with
three monopolar mitotic spindles in one 40X field. Only 5-10% of the disrupted mitotic
spindles were multipolar (Figure 2D).
Figure 2 Mitotic disruption following treatment with MWCNTs. 2A: the bar graph
demonstrates the mitotic disruption 24 hours following exposure to MWCNT. Mitotic spindle
abnormalities are expressed as a percent of total mitotic figures. The abnormalities are
separated into monopolar and multipolar mitotic spindles. The multipolar spindles include
tripolar and quadrapolar mitotic spindles. * indicates significantly different from the
unexposed control cells at p < .01; ± standard deviation. Figure 2B: The bar graph
demonstrates the distribution of the mitotic spindle abnormalities in BEAS-2B cells
following exposure to MWCNT. The white bars indicate the percent of mitotic cells with one
mitotic spindle pole. The solid bars indicate the percent of total mitotic cells that had a
multipolar mitotic spindle apparatus. The grey bars indicate the percent of mitotic cells with
either a multipolar mitotic spindle or a monopolar mitotic spindle to show the percent of cells
with any disruption of the mitotic spindle apparatus. *indicates significance at p <0.01.; ±
standard deviation. Figure 2C: The photomicrograph of a culture exposed to 0.24 µg/cm2
MWCNT using a 40X objective. The yellow arrows indicate monopolar mitotic spindles.
This figure demonstrates the typical monopolar phenotype of the cultures following exposure
to MWCNT. Figure 2D: The bar graph demonstrates the percent of SAEC with an aneuploid
chromosome number after a 24 hour exposure to MWCNT or the positive control V205. The
solid bars indicate the level of apoptosis in the exposed and control BEAS-2B. The hatched
bars indicate the level of apoptosis in the exposed SAEC. MWCNT exposure induced a
dramatic elevation of chromosome loss and gain at all doses of exposure at levels equal to the
positive control V205. .* indicates significantly different from the unexposed control cells at p
Primary SAEC cells from a normal donor were used to investigate the effects of MWCNT on
the chromosome number. The normal karyotype of the primary cells made it possible to
evaluate the treatment related changes in chromosome number. FISH analysis for either
chromosome 1 or 4 demonstrated a 2.25 ± 1.0% aneuploidy in the untreated SAEC cells
(Table 1). The frequency of the cells with abnormal chromosome number is within the range
reported in adult human cells in culture [59,60]. By contrast, the MWCNT-treated SAEC
cells had a level of aneuploidy that was comparable to the vanadium pentoxide-treated
positive control cells (Figure 2D; Table 1). Abnormal chromosome number was significantly
elevated following MWCNT treatment as follows: 62 ± 7.0%, 24 µg/cm2; 59.0 ± 6.0%, 2.4
µg/cm2; 49 ± 6.0%, 0.24 µg/cm2 and 42 ± 10%, 0.024 µg/cm2 compared with control
incidence of 2.25 ± 1.0%. Treatment with 0.31 µg/cm2 V205 resulted in 67 ± 6.0% aneuploid
cells. The chromosome alterations in the MWCNT treated cells were predominantly gains of
either chromosome 1 or 4 (Table 1). The chromosome losses accounted for 24%, 24 µg/cm2;
13%, 2.4 µg/cm2; 8%, 0.24 µg/cm2 and 12%, 0.024 µg/cm2. Chromosomal gains accounted
for over 70% of the aneuploidy (Table 1). There was also a dose-dependent increase in the
number of cells with gains of both chromosomes 1 and 4 indicating an increase in polyploid
cells. The number of alterations of chromosome 1 was not statistically different than the
alterations of chromosome 4, therefore; there was not a bias for a change of either
Table 1 Percent of chromosome errors in SAEC cells following treatment with MWCNT
Diluent 2.25 ± 1.0 1.0 ± 1.0% 1.0 ± 1.0%
0.024 42 ±10* 2.0 ± 1.26 15.0 ± 2.0*
0.24 49 ±6.0 1.7 ± 0.7* 23.7 ± 5.0*
2.4 59.0 ± 6.0* 3.4 ± 0.8* 26.0 ± 3.0*
24 62 ±7.0* 7 ± 3.0%* 49.3 ± 4.0%*
0.31 69.0 ±7.0* 23.0 ± 5.0* 35.0 ± 9.0*
* Statistically significant at p < .05.
The distribution of the aneuploidy that was contributed by chromosome 1 and by
chromosome 4 is detailed in the table as “Total% aneuploid cells”. The percent of cells with a
gain in chromosome 1 and/or of chromosome 4 are indicated in the table under “Gain” of
each chromosome. Cells with both chromosomes gained are indicated by “Gain of both
chromosomes”. Cells with a loss of chromosome 1 and/or chromosome 4 are indicated in the
table under “Loss” of each chromosome. *: p <0.05 of the treated cells compared to diluent
control exposed cultures; ± standard deviation.
Total% % Loss of % Gain of % Loss of
1.25 ± 1.0%
3.0 ± 1.26*
2.0 ± 10*
4.3 ± 1.2*
% Gain of
16.4 ± 2.0*
25 ± 4.0*
25 ± 10*
53.3 ± 5%*
% Gain of both
12.0 ± 3.0*
18 ± 6.0*
23 ± 5.0*
44 ± 5.0%*
25.0 ± 11* 34.0 ± 7* 19.0 ± 6*
Interaction of carbon nanotubes with mitotic spindle apparatus
The MWCNTs were 10–20 nanometers in width. Nanotubes of 10 nanometers or greater can
be observed using differential interference contrast imaging. MWCNTs were observed in the
cytoplasm and the nucleus (Figure 3A). The MWCNTs also had a strong association with the
centrosomes as shown in Figure 3B. The high frequency of monopolar mitotic spindles
allowed confirmation of the monopolar phenotype by transmission electron microscopy
(TEM) as shown in Figure 3C. The 3D reconstructed image demonstrates strong physical
associations between the carbon nanotubes, the microtubules and DNA and the centrosomes
(4A- B). The 3D reconstruction further demonstrated that MWCNTs not only associated with
the centrosome but inside the centrosomal structure (Figure 4C).
Figure 3 MWCNT-treated cell with one spindle pole. The photographs in Figure 3A-C
show a monopolar mitotic spindle with one pole rather than the two poles which would be
expected in a normal cell. The details of the detection protocol for the mitotic spindle
components and the photography using the Zeiss Confocal are in the methods section. The
tubulin in 3A was stained red using Spectrum red and indirect immunofluorescence. The
DNA was detected by DAPI and was blue. The nanotubes were imaged using differential
interference contrast and are black. In Figure 3B, the nanotubes can be seen in the nucleus, in
association with microtubules, the DNA and the centrosome. Serial optical sections at 0.1
micron intervals using confocal microscopy confirmed the location of the nanotubes in the
nuclear DNA and the tubulin including the microtubules of the mitotic spindle. Figure 3C is a
high resolution TEM of a monopolar mitosis. The image was photographed at 11000X
Figure 4 A, B and C: Three-dimensional reconstruction of a MWCNT-treated mitotic
cell. Figure 4A: This 3-dimension reconstruction was created from serial optical laser
scanning confocal microscopy sections using immunofluorescence to identify centrosomes
and microtubules while differential interference contrast was used to visualize aggregated
MWCNT as previously described . Briefly, nanotubes of 10 nanometers or greater could
be visualized by their interference with transmitted light using DIC imaging. Because the
nanotubes block the light, the nanotubes produce a black image. The reconstructed image
shows aggregated nanotubes which appear as irregular tangled black structures located inside
the cell in association with the centrosomes (green), the microtubules (red) and the DNA
(blue). In this cell, the one spindle pole, the doughnut shaped DNA arrangement and the
disruption of microtubule attachments to clustered centrosome fragments into a monopolar
spindle apparatus suggest major perturbations in cell division. The yellow arrows indicate
nanotubes in association with mitotic spindle and the DNA. Figure 4B: The yellow arrows
indicate the nanotubes (black) in association with the centrosomes (green) and the
microtubules (red). Figure 4C: The yellow arrows indicate nanotubes (black) inside the
centrosome structure (green).
Viability and clonal growth
Exposure to MWCNT did not reduce viability 24 hours after treatment in either the primary
SAEC or the immortalized BEAS-2B cells (Figure 5A). Vanadium pentoxide treatment
resulted in reduced viability in both SAEC and the BEAS-2B cells. Seventy-two hours
following exposure, the viability of the SAEC cells was significantly reduced in cells exposed
to 0.024, 0.24, 2.4 or 24 µg/cm2 MWCNT (Figure 5 B). Three weeks following exposure, the
BEAS-2B cells had a small increase in colony formation at 0.024 µg/cm2 (Figure 5C). One
month following exposure, the SAEC cells had a reduced number of colonies at the highest
dose; however, exposure to 0.024, 0.24 and 2.4 µg/cm2 resulted in a dramatic increase in
colony formation (Figure 5C).
Figure 5 A, B and C: Clonal growth and viability of BEAS-2B and SAEC cells. Figure
5A: The bar graph represents viability of BEAS-2B and SAEC cells 24 hours following
exposure to MWCNT or V205. The white bar indicates viability of BEAS-2B cells. The black
bar indicates viability of SAEC cells. The viability was not reduced in either the BEAS-2B or
the SAEC cells. Figure 5B: The bar graph represents the viability of BEAS-2B and SAEC
cells 72 hours following exposure to MWCNT. The white bar indicates the viability of
BEAS-2B cells and the black bar indicates viability of SAEC cells. MWCNT exposure
resulted in reduced viability in the SAEC and the BEAS-2B at 0.024, 0.24, 2.4 and 24 µg/cm2
compared to control cells. The exposure to V205 resulted in reduced viability in SAEC treated
cells at all doses. * indicates statistical significance of the treated cells compared to control
cells at p <0.05. Figure 5C: The bar graph demonstrates the clonal growth in BEAS-2B cells
3 weeks following MWCNT exposure and SAEC cells 4 weeks following exposure. The
black bars indicate the mean number of colonies of BEAS-2B cells and the white bars
indicate mean number of colonies in SAEC cells. *indicates significance at p <0.05 of treated
cells compared to control cultures.
The impact of MWCNT-treatment on the cell cycle was evaluated by Click-iT EdU Flow
Cytometry assay. Treatment with 24ug/cm2 MWCNT induced a statistically significant
increase in the percent of cells in S phase from 32.11% (PBS-treated) to 40.1% (Table 2).
When the cells in G2 phase of the cell cycle were compared, exposure to the positive control,
arsenic, resulted in 32.1% of the cells in G2 compared to 18.30% of the cells in the PBS
control group thus indicating an arsenic-induced block in G2 (Table 2, p < .05).
Table 2 Distribution of the cell cycle in BEAS-2B cells 24 hours after treatment
24 hour PBS 43.25 ± 5.6
24 hour As 35.6 ± 6.9
24 hour MWCNT 39.8 ± 4.0
The table demonstrates the mean of percent of cells in G1, S and G2 phase of the cell division
24 hours following treatment with media, 5 µM arsenic or to 24 µg/cm2 MWCNT. The data is
based on replicates of 6 that were repeated in 9 independent experiments.
*: p <0.05 of the treated cells compared to diluent control exposed cultures.
32.11 ± 6.5
26.38 ± 7.9
40.1 ± 5.6*
18.30 ± 5.3
32.10 ± 6.7*
15.90 ± 3.3
Since their discovery in 1991  carbon nanotubes have been used for a variety of
applications including fiber optics , conductive plastics, molecular electronics as well as
biological and biomedical applications . Although the durability and fiber-like structure
of carbon nanotubes have raised concerns that carbon nanotubes may have effects similar to
asbestos, the health effects have not been fully investigated [64,65]. Our data reported here
are the first to show induction of monopolar mitotic spindles, aneuploidy, and a G1/S block
in the cell cycle as well as a dramatic increase in colony formation following exposure to 10–
20 nm diameter MWCNT. Exposure to 0.024 µg MWCNT/cm2 resulted in errors in
chromosome number and mitotic spindle aberrations in greater than 40% of the cells
examined. The dramatic increase in MWCNT-induced colony formation and aneuploidy
observed in the primary SAEC cells was significantly higher than was previously observed in
SWCNT-treated cells. The proliferation of cells with a high degree of genetic damage could
result in the expansion of a population of genetically-altered cells. Cell proliferation is
important in the second stage of pulmonary carcinogenesis, tumor promotion, while genetic
instability is observed during the progression of preneoplastic cells to frank neoplasia [40,66].
During the progression of neoplastic disease, centrosome disruption is observed. The degree
of centrosome disruption and aneuploidy is important because it is correlated with tumor
The level of centrosome fragmentation, mitotic spindle damage and aneuploidy following
MWCNT exposure was similar to the effects of the known carcinogen and positive control,
vanadium pentoxide. MWCNTs were found in association with the DNA, the microtubules,
the centrosomes as well as inside the centrosome structure. A previous investigation has
shown that MWCNT are incorporated into the microtubules during polymerization thus
forming a microtubule/nanotube hybrid . The mitotic disruption that was observed
following exposure to MWCNT may be due to a number of factors including incorporation of
the nanotubes into the centrosome and microtubules of the mitotic spindle resulting in failed
cytokinesis, failed centrosome duplication or inhibited centrosome separation. If two spindle
poles are not formed during cell division, the chromosomes are not divided equally and
chromosome errors occur.
Exposures that induce monopolar mitotic spindles produce daughter cells that fail to undergo
cytokinesis and have double the number of chromosomes (polyploid) [71-73]. Although the
data from the current investigation demonstrated that the aneuploidy was predominantly due
to a gain of chromosomal material or polyploidy, the chromosomes were also lost in a
significant number of cells suggesting that the genetic damage was due to more than a failure
of cytokinesis. Asakura et al.  observed polyploid cells in cancer cell lines following
exposure to 0.25 to 50 µg MWCNT of 80 nm diameter . Although detailed analysis of
chromosome loss and gain was not possible in a cancer cell line, the study demonstrated a
significant number of polyploid cells which they attributed to a failure of cytokinesis. Carbon
nanotubes have been observed in the bridge separating dividing cells . Three dimensional
reconstruction of MWCNT-exposed cells in the current study and of previously published
SWCNT-exposed mitotic figures have shown carbon nanotubes integrated with the
microtubules, the DNA and within the centrosome structure [19,24]. The disruption of cell
division that has been observed following carbon nanotube exposure may be due to the
incorporation of the carbon nanotubes into the microtubules that make up the division
In this study, we observed fragmented centrosomes clustered into a single pole. These results
are in sharp contrast to the multipolar mitotic spindles that have been observed with narrower
Centrosomes are duplicated in early G1/S of the cell cycle. The separation of the mother and
daughter centrosomes by proteolytic enzymes is necessary for the exit from S phase and the
formation of a bipolar mitotic spindle . Incorporation of the stiff MWCNT into the
centrosome may have resulted in a more rigid centrosomal structure which fractured during
mitosis. In addition, the integration of the nanotubes into the centrosome structure could have
prevented the proteolysis of the linker connecting duplicated mother and daughter centrioles
in G1/S thereby preventing the centrosome separation necessary for the formation of a
bipolar spindle . Furthermore the excess of cells in the S phase and significantly lower
number of cells in the G2 phase in the MWCNT-treated compared to the control cells in the
current investigation indicate a G1/S block and a failure to progress to G2. Interaction of the
nanotubes into the microtubules would potentially impact many cellular process including
cellular transport of organelles (lysosomes, mitochondria, Golgi apparatus and endoplasmic
reticulum), RNA and protein transport as well as phagocytosis and cell movement .
Kinesin and dynein motors move the organelles, chromosomes, proteins and RNA. Defects in
the microtubule surface have been reported to result in detaching of the motors from the
microtubule and interruption of cell signaling [77-80]. Aberrant cell signaling is a concern
because it is important in the progression of carcinogenesis [81-83].
Although both SWCNT and MWCNT had a strong association with the microtubules that
make up the mitotic spindle and induced aberrant mitotic spindles, the data suggests that the
type of damage may be determined by the diameter of the carbon nanotubes. SWCNT of 1–2
nm in diameter , MWCNT of 5–10 nm  and the NanoLabs 10–20 nm MWCNT form
hybrids with microtubules . Both the SWCNT and the 10–20 nm MWCNT are
incorporated into the centrosome structure. The stiffness of the nanotubes is determined by
their diameter . Although, carbon nanotubes have similar mechanical properties to the
microtubules, the stiffness of the carbon nanotubes is a thousand-fold greater than that of the
microtubules . The incorporation of the more rigid MWCNT into the microtubules that
make up the mitotic spindle fibers and the centrosome may reduce the elasticity of the mitotic
spindle apparatus to a greater degree than the SWCNT. The elasticity of the mitotic apparatus
is a critical factor in the separation of the centrosomes to organize two spindle poles as well
as in the separation of the chromosomes during cell division.
Evidence from rodent exposure studies has demonstrated that high aspect ratio nanoparticles
have carcinogenic properties [9,64,86,87]. Inhalation exposure is the route that most closely
resembles occupational exposure. The lung is the principal target organ for carbon nanotube
exposure . The long thin carbon nanotubes induce inflammation, cell proliferation of type
II epithelial cells and cellular atypia [30,31,33]. Recent investigations have shown that
inhaled MWCNT migrate to the subpleural wall [44,88]. The fiber-like structure, evidence of
carbon nanotube-induced inflammation, proliferation and cellular atypia in the lung as well as
migration to the subpleural space, inflammation, macrophage injury and evidence of
genotoxic damage have raised concerns that the material has carcinogenic properties similar
to asbestos [44,64,89]. The lung as well as the parietal pleura is the sites of asbestos-induced
carcinogenesis [64,90-93]. Injection of high doses of 100 nm diameter MWCNT into the
abdominal cavity of p53 +/− mice has been shown to induce mesothelioma on the surface of
the diaphragm . In a more recent investigation of p53 +/− mouse exposure, Takagi et al.
demonstrated a dose response of mesothelioma development after peritoneal injection of 3–
300 micrograms of Mitsui-7 MWCNT . Nagi et al. investigated the role of nanotube
diameter in the development of mesothelioma in a rat model . Greater inflammation and
mesothelioma development were observed with the 50 nm diameter Mitsui-7 MWCNT of 10
microns or less in length compared to nanotubes of 145 nm diameter and similar length .
The mouse studies were criticized due to the route of exposure and the sensitivity of the
genetically modified p53 knock-out mouse strain; however, the induction of mesothelioma
was significant. The demonstration of mesothelioma at high exposures combined with our
findings revealing disruption of the integrity of the division apparatus further suggest a
carcinogenic potential for MWCNT. A manuscript in press by Sargent et al. has demonstrated
that inhaled Mitsui-7 MWCNT material promoted the formation of lung adenocarcinomas in
B6C3F1 hybrid mice following 3-methylcholanthrene (MCA) initiation . While the data
did not indicate tumor initiation by MWCNT, the exposure resulted in lung adenocarcinoma
and adenoma in 90.5% of the mice exposed to MCA followed by inhaled MWCNT. The
mouse lung tumors were large and 15% of the tumors were metastatic indicating tumor
progression with some forms of MWCNT. Furthermore, the strong MWCNT-induced tumor
promotion was observed in a hybrid mouse that is intermediate in sensitivity to lung cancer
[98,99]. The exposure dose of the tumor promotion study of 32 µg/mouse is only 2.6 fold
higher than the dose of the current in vitro investigation that shows significant chromosomal
and mitotic spindle effects at the lowest administered dose of 0.024 µg/cm2 . Although
lung cancer or mesothelioma have not been observed in humans exposed to MWCTs,
centrosome disruption, aneuploidy and mitotic spindle aberrations as well as recent data
indicating mesothelioma as well as lung tumor promotion and progression are a concern and
indicate that caution should be used to prevent respiratory exposure to workers during the
production or use of commercial products.
Materials and methods
Multi-walled carbon nanotubes acid washing
Multi-walled carbon nanotubes produced by chemical vapor deposition (Nanolab Inc.
PD15L5-20) were acid-washed to remove iron catalyst. The MWCNT were suspended in a
mixture of 3:1 v/v sulfuric acid (H2SO4) (Fisher Scientific, Pittsburgh, PA): nitric acid
(HNO3) (69.5%, Fisher Scientific, Pittsburgh, PA) for 1 hour in a water bath sonicator
(Branson 2510, Fisher, Pittsburgh, PA) over ice. The mixture was subsequently diluted in
deionized water (2 L) and filtered through a 0.2 µm polycarbonate membrane filter
(Millipore, USA); the filtration step was repeated 6 times to remove catalysts or impurities.
All cell exposure experiments were performed with one hour acid-washed MWCNT
Characterization of MWCNT
Atomic force microscopy (AFM) was used to investigate the length of both pristine and acid-
washed MWCNT. Commercial Si tips (Asylum Research, AC240TS, USA) were used at
their original resonance frequency, varying from 50 to 90 kHz. Pristine or acid-washed
nanotubes (10 µg/ml) were deposited on mica surfaces (9.5 mm diameter, 0.15-0.21
thickness, Electron Microscopy Sciences, USA) and dried overnight under vacuum. Scans of
10 µm x 10 µm were acquired using tapping mode in air. At least 30 individual MWCNTs
were analyzed to determine their length.
Raman spectroscopy was used to characterize the structure of both pristine and acid-washed
MWCNTs. Raman analyses were performed at room temperature using a Renishaw InVia
Raman Spectrometer (CL532-100, 100 mW, USA). The excitation source used an argon ion
(Ar+) laser operating at 514.5 nm. MWCNT (pristine or acid-washed, 1 mg) were mounted on
a clean glass slide (Fisher, Pittsburgh, PA) and a 20x microscope objective was used to focus
the laser beam to a spot size of < 0.01 mm2 and to collect the scattered light. Low energy
laser of < 0.5 mV and an exposure time of 10 sec were used to prevent unexpected heating
effects of the MWCNT samples being analyzed. Detailed scans ranging from 100 to 3200 cm-
1 were acquired.
The elemental analysis of the pristine and acid-washed carbon nanotubes was examined by
energy dispersive X-ray spectroscopy (EDX). Both pristine and acid-washed MWCNT (1
mg/ml in water) were vacuum-dried on silica wafers. The experiments were performed using
a Hitachi S-4700 Field Emission Scanning Electron Microscope (USA) and backscattered
(BSE) electron detection in a single unit and operating at 20 kV.
ICP-MS) was performed to further analyze the chemical composition of the nanotubes as
described previously. Carbon nanotubes were suspended in pure H2O (18.2 MΩ–cm) at a
concentration of 1.0 mg/ml. One ml of each vortexed suspension was added to a 100 ml
polytetrafluoroethylene digestion tube (CEM, Matthews, NC) along with 9.0 ml of ultrapure
HNO3 and 1.0 ml of ultrapure H2O2 (Fisher Optima, Fisher Scientific, Pittsburgh, PA). Three
replicate samples for each nanotube type were digested in the Microwave-Assisted Reaction
System (CEM, Matthews, NC) by ramping up to 200°C for 15 min., holding at 200°C for 30
minutes, then cooling to 22°C, adapting a procedure as previously described . There was
no visible carbonaceous material remaining in any of the samples after digestion. After
suspension (1 mg/ml), the metal content of the nanotubes was analyzed by ICP-MS using the
Perkin-Elmer Nexion 300D , using 54Fe, 60Ni, and 59Co isotopes. Standards were
certified multi-element standards in 1% HNO3.
The dispersity of pristine MWCNTs and acid-washed MWCNTs in Phosphate buffered
Saline (PBS, Fisher, Pittsburgh, PA) was determined by centrifuging the corresponding
suspensions (initial concentration 5 mg/mL for both pristine and acid-washed MWCNTs) at
3000 rpm for 5 min. Subsequently, 0.8 mL of the supernatant mixture was filtered through a
0.2 µm filter membrane. After complete drying under vacuum, the amount of pristine
MWCNTs or acid-washed MWCNTs on the filter membrane was measured and the dispersity
was calculated based on the starting volumes. The obtained values do not reflect the
Two human respiratory epithelial cell populations were used to examine the potential genetic
damage to MWCNT exposure. Immortalized human bronchial epithelial cells (BEAS-2B,
ATCC, Manassas, VA) cultures of passage 4–6 were used to examine the mitotic spindle
integrity. The high mitotic rate of the BEAS-2B cells allows examination of sufficient
number of mitotic spindles following treatment. BEAS-2B cells grown in serum enriched
media double every 18–20 hours and have normal mitotic spindle morphology. The high
mitotic index of the BEAS-2B cells made it possible to analyze a sufficient number of mitotic
spindles during the 24 hour exposure. Primary small airway respiratory epithelial cells
(SAEC; Lonza, Walkersville, MD) from a normal human donor were used to determine the
response of a normal cell population. In addition, the normal karyotype of the primary cells
was essential for the examination of aneuploidy. The SAEC cells double every 20–24 hours
which allowed analysis of a potential change in chromosome number and centrosome
morphology of cells that have divided during the 24 hour exposure. The low mitotic index of
the SAEC cells (0.5%) prevented the analysis of mitotic spindle integrity in this cell
population. The BEAS-2B and SAEC cells were therefore analyzed 24 hours after exposure
to allow a sufficient number of cells that have gone through division.
BEAS-2B cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media
supplemented with 10% serum (Invitrogen, Grand Island, NY). The SAEC cultures were
cultured following manufacturer’s directions and using Cabrex media (Lonza, Walkersville,
MD). The cell cultures were examined by electron microscopy and cytokeratin 8 and 18
staining to verify the epithelial phenotype of the cells as described previously .
The immortalized BEAS-2B and the primary SAEC were exposed in parallel culture dishes
to MWCNT or to the positive control, vanadium pentoxide (Sigma St. Louis, MO). Three
independent experiments were performed for each exposure for SAEC and BEAS-2B
respectively. MWCNT and vanadium control were suspended in media and sonicated over
ice for 5 minutes and 30 minutes respectively. The cells were seeded in dishes and exposed 0,
0.024, 0.24, 2.4 and 24 µg/cm2 MWCNT or to 0.031 µg/cm2 vanadium pentoxide when the
cells were 70% confluent. The one milliliter culture was treated with 0.024, .24, 2.4 and 24
µg/ml respectively. Twenty-four hours after exposure all cells were prepared for analysis of
apoptosis and necrosis, integrity of the mitotic spindle, as well as the centrosome and
chromosome number as described below.
Viability and apoptosis
Triplicate cultures were prepared in 96 well plates (Becton Dickinson Franklin Lakes, NJ) for
the analysis of viability using the Alamar Blue bioassay (Invitrogen, Carlsbad, CA),
following manufactures directions as described previously . Eight wells were performed
for each treatment and dose. Three independent experiments were performed for the analysis
of cellular toxicity by Alamar Blue. Parallel cultures were also prepared in duplicate in one
milliliter chamber slides (Nunc Rochester, NY) for the analysis of apoptosis using the
TUNEL assay following the manufacturer’s directions (Roche, Inc., Indianapolis, IN) with
some modifications outlined previously . A minimum of 100 cells were analyzed for each
sample; experiments were repeated three times for a total of 300 cells for each treatment and
dose, respectively for the analysis of apoptosis by the TUNEL assay. An additional positive
control, 1.68 Molar DNase (Sigma St. Louis, MO) was used for the analysis of apoptosis.
Twenty-four hours after dosing, cells in the chamber slides were fixed in 4%
paraformaldehyde in phosphate buffer (Sigma St. Louis, MO) and stained with DAPI
(Millipore Billerica, MA). The resulting stained samples were fluorescently analyzed using a
Zeiss Axiophot fluorescent microscope (Carl Zeiss Microimaging Inc. Thornwood, NY).
Mitotic spindle analysis
BEAS-2B was cultured in 1 milliliter chamber slides as described previously. Dual chambers
were prepared for each treatment and each cell type. Three independent experiments were
prepared for each cell type and treatment . A minimum of 100 cells of good centrosome
and mitotic spindle morphology were analyzed for each sample; experiments were repeated
three times for a total of 300 cells for each treatment and dose, respectively. The centrosome
integrity as well as the dispersion of carbon nanotubes in the cell cultures was evaluated The
spindle integrity of the BEAS-2B cells was examined using dual-label immunofluorescence
for tubulin and centrin to detect the mitotic spindle and the centrosomes, respectively.
Primary rabbit anti-beta tubulin (Abcam, La Jolla, CA, USA) and mouse anti-centrin
antibodies (a generous gift from Dr. Jeff Salisbury), and secondary Rhodamine Red goat anti-
rabbit IgG and Alexa 488 goat anti-mouse IgG antibodies (Invitrogen, Carlsbad, CA) were
used. The mitotic spindle and centrosome morphology were analyzed in the BEAS-2B cells
using a laser scanning confocal microscope (LSM 510, Carl Zeiss MicroImaging Inc.,
Thornwood, NY) as previously described . Briefly, a monopolar or multipolar mitotic
spindle was counted as disrupted. The location of MWCNT was determined by differential
interference contrast. Because the nanotubes block the light, the nanotubes produce a black
image. To determine the association of the MWCNT with the microtubules of the mitotic
spindle and the centrosome, serial optical slices was obtained to create a z-stack and permit
three-dimensional reconstruction using LightWave software  by TEM following
methods outlined previously . Briefly, cells were fixed in 2% glutaraldehyde in sodium
phosphate buffer, pH 7.2, for 2 h, postfixed in osmium tetroxide, dehydrated through an
ethanol series, and embedded in Spurr’s resin (Sigma, St Louis, MO). Silver-gold sections
were stained in 2% aqueous uranyl acetate and Reynolds’ lead citrate, observed using a JEOL
1200 EX electron microscope and recorded digitally.
Chromosome number by fluorescence in situ hybridization (FISH)
Due to the necessity of a normal diploid karyotype for the analysis of chromosome number,
the SAEC cells were prepared for analysis of the chromosome number. Fluorescence in situ
hybridization (FISH) for human chromosomes 1 and 4 was used to determine the
chromosome number (Abbott Molecular, Des Plaines, IL) according to the guidelines of the
American College of Medical Genetics . Three independent experiments for a total of
300 cells were evaluated for each treatment and dose. A minimum of 100 interphase cells of
good FISH morphology were analyzed to determine the number of chromosome 1 and 4. The
SAEC cells were photographed using a Zeiss Axiophot microscope and Genetix Cytovision
software. Cells with three copies or greater than 4 copies of chromosome 1 or 4 were
recorded as a gain for that chromosome. Cells with less than two copies of chromosome 1 or
4 were recorded as a loss of that chromosome. The loss and gain of both chromosomes were
added to obtain the errors in chromosome number (aneuploidy).
Triplicate cultures of SAEC cells were grown in T25 flasks. When the cells were 70%
confluent they were treated with MWCNT. After 24 hours, the cells were trypsinized,
counted and plated at 500 cells/well in 6-well plates for analysis of colony formation. One
month following exposure, the cells were washed with PBS, stained with 10% crystal violet
solution in neutral buffered formalin (Sigma, Saint Louis, MO) and colonies counted.
Cell cycle analysis for DNA content
BEAS-2B cells were grown in six parallel T25 flasks. A total of 9 independent experiments
were performed for the analysis of cell cycle. Twenty-four hours after exposure to 24 µg/cm2
MWCNT or to the positive control, 5 µM arsenic (Sigma, St Louis MO), the cells were
washed twice with PBS and removed from the dishes with 0.25% trypsin prior to detection of
the cell cycle. The cells were stained according to (Invitrogen) manufacturer’s instructions.
EdU (5-ethynyl-2′-deosyuridine) is a nucleoside analog of thymidine and is incorporated into
DNA during active DNA synthesis. Detection is based on a click reaction- a copper catalyzed
covalent reaction between an azide and an alkyne. Twenty-four hours after exposure to
MWCNT, the cells were washed twice with PBS and incubated with EdU for 2 hours to
detect cells in S-phase. Following incubation, the cells were removed from the plate using
0.25% trypsin. After fixation and Click-iT Saponin permeabilization, CuSO4 was added to
the cells to detect the EdU signal. The total amount of DNA was analyzed following
incubation with 7AAD (7-aminoactinomycin D) using a LSR II flow cytometer (BD
Biosciences Immunocytometry Systems, San Jose, CA). Data were analyzed and plotted
using FlowJo v7.2.5 software.
All analyses were performed using SAS/STAT (Version 9.3) for Windows. Chi-square
analysis was used to determine statistical significance for the scoring of the mitotic spindle
abnormalities and the number of cells with abnormal chromosome number. The number of
viable and apoptotic cells were analyzed using analysis of variance (ANOVA). The mean of
duplicate samples were used for the analysis. For cell cycle analysis, a mixed model ANOVA
was used to compare the proportion of cells in G1, S and G2/M phase across treatment
groups. Experimental block was utilized as a random factor. All differences were considered
statistically significant at p < 0.05.
The authors declare that they have no competing interests.
KJS contributed to the study design, writing of the manuscript, conducted experiments and
analyzed FISH signals. LMS conceived of and designed the study, analyzed the experimental
results and drafted the manuscript. SHR and MLK contributed to the experimental design,
acquisition of funding and writing of the manuscript. MLK also analyzed the data for
statistical significance. DTL contributed to the study design, conducted the experiments as
well as contributed to the analysis of the data. CD acid washed the MWCNT and performed
analysis of the material. AFH contributed to the study design and writing of the manuscript
and acquisition of funding. JLS was involved in acquisition of funding and writing of the
manuscript. DWP contributed to the study design and calculations of the dose for exposure.
CZD contributed to the preparation and characterization of the MWCNT and drafting the
description of the manuscript. MK performed ICP-MS and drafting of the manuscript. JM,
KB, MS and JS contributed to writing of the manuscript and materials characterization. LC
assisted with MWCNT characterization and photography, writing of the manuscript and
preparation of the figures. All authors read and approved the final manuscript.
The authors would like to thank Mike Gipple, Scientific Arts, LLC, Morgantown, WV for his
help with three dimensional reconstructions, Kimberly Clough-Thomas for her help with the
images and Adrienne McGraw, WVU for her help with scanning electron microscopy
imaging. This work was supported by NIOH NORA 9927Z8V and NSF EPS-1003907.
Research findings and conclusions are those of the authors and do not necessarily represent
the views of the National Institute for Occupational Safety and Health.
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Additional_file_1 as DOC
Additional file 1 Metal composition of Pristine and Acid-washed MWCNT. Table: The table
demonstrates the metal composition of the pristine and 1h acid-washed MWCNTs as
measured by energy dispersive X-ray spectroscopy (EDX).
Additional files provided with this submission: Download full-text
Additional file 1: 5478824610164458_add1.doc, 32K