Ann. Occup. Hyg., Vol. 54, No. 5, pp. 532–544, 2010
Ó The Author 2010. Published by Oxford University Press
on behalf of the British Occupational Hygiene Society
doi:10.1093/annhyg/meq012
In Vitro Cytotoxicity and Transforming Potential
of Industrial Carbon Dust (Fibers and Particles)
in Syrian Hamster Embryo (SHE) Cells
C. DARNE1*, F. TERZETTI1, C. COULAIS1, J. FOURNIER2, Y. GUICHARD1,
L. GATÉ1 and S. BINET1
1
De´partement Polluants et Sante´, Institut National de Recherche et de Se´curite´ (INRS),
54519 Vandoeuvre Cedex, France; 2Laboratoire de re´activite´ de surface, UMR-CNRS 7609,
Universite´ Pierre et Marie Curie, 75252 Paris Cedex 05, France
Received 12 May 2009; in final form 11 February 2010; published online 10 March 2010
Carbon fibers have many applications, mainly in high-tech industries such as the aviation
industry. Eleven carbon samples (fibers and particles) coming from an aeronautic group were
tested for their cytotoxicity and carcinogenic potential using in vitro short-term assays in
Syrian hamster embryo cells. These samples were taken during each important step of the
process, i.e. from the initial heating of polyacrylonitrile fibers to pure carbon fibers. They were
compared to an asbestos fiber, an amorphous silica, and two commercial graphite powders.
Their physical–chemical characteristics and their capacity to release reactive oxygen species
(ROS) were determined. This study showed that none of the carbon samples was able to
generate ROS as measured by Electron Paramagnetic Resonance analysis, and in our biological assays, they demonstrated no morphological transformation potential and low cytotoxicity
compared to positive control (chrysotile asbestos).
Keywords: carbon fibers; carbon particles; cytotoxicity; peroxidation; reactive oxygen species; SHE cell transformation
obtain pure carbon fibers. These intermediates can
be released into the occupational environment as fibers
or particles. Exposure may also occur during fabrication of composite structures and when repairing damaged structures. All these carbon dusts, which can
have different structures and chemical compositions,
may have different biological effects.
Surprisingly, despite the current widespread use of
carbon fibers, toxicological studies have mainly focused on nanomaterials and carbon nanotubes, and
only a few studies have evaluated carbon fiber and
particle toxicity. In vitro short-term studies, e.g. cytotoxicity, genotoxicity, and cell transformation studies
related to assessing reactive oxygen species (ROS)
generation, have contributed to an understanding of
the action mechanisms of fibers and particles.
In the case of carbon fibers, the toxicological studies published present conflicting results (for a review
see Thomson, 1989). Risks associated with pitch-
INTRODUCTION
Carbon fibers are mostly used in advanced composite
materials to improve strength, stiffness, durability,
electrical conductivity, or heat resistance, mainly in
high-tech industries. Carbon fibers can be synthesized
from polyacrylonitrile (PAN) or from petroleum pitch.
PAN-based fibers are the most commonly used precursors. The fabrication process of a carbon piece requires
several heating steps. Initially, precursors (PAN and
pitch) are oxidatively stabilized and dehydrogenated
at a moderate temperature (200–300°C). The fibers
are then carbonized at a temperature of 750–1300°C
in a non-oxidizing atmosphere, and production may
involve a secondary heating phase at .1400°C,
known as graphitization, in an inert atmosphere to
*Author to whom correspondence should be addressed.
Tel: þ33-3-83-50-20-00; fax: þ33-3-83-50-20-96;
e-mail: [email protected]
532
In vitro SHE transformation assay
based carbon fibers have been suspected since positive effects were obtained with in vitro genotoxicity
tests and the dermal painting test in mice, but there
was no evidence that PAN-based carbon fibers could
have adverse health effects.
In this study, different PAN-based carbon dusts
were sampled during a production process of pure
carbon pieces. All the samples came from the same
industrial group except the two samples of carbon
particles used as another source of carbon. We examined the cytotoxicity and carcinogenic potential of
these samples in relation to their ability to generate
ROS in a liquid system. The cytotoxicity and transforming potential, as well as the oxidizing activity,
were compared to asbestos chrysotile, the positive
control for our experiments.
MATERIAL AND METHODS
General experimental design
The effects of fibers/particles on cell growth and
viability depend on cellular system, chemical reactivity of particles, treatment concentrations, etc.
and can be expressed by mitogenic, cytostatic,
and/or cytotoxic responses (Fubini, 1998; Fubini
et al., 1999; Fubini and Otero-Aréan, 1999; Elias
et al., 2000). The assays used in this study to
measure alterations in Syrian Hamster Embryo
(SHE) cell functions and the cytotoxic potential
of samples were:
(i) ‘Cell proliferation assay’: this assay provides
information on the ability of proliferating culture cells to divide up to a stationary state.
Treatments inducing inhibition of cell proliferation were determined for each sample.
(ii) ‘Cloning efficiency (CE)’: this assay measures
the ability of isolated cells to give rise to
colonies of daughter cells at the end of several
days. The cell death, as the final consequence
of irreversible cell damage induced by the
samples, was determined by the decrease in
cell colony number versus that of control cells.
These data were obtained from the SHE cell
transformation assay, which has the advantage
of assessing both cytotoxicity and morphological transformation within the same cell culture
and after the same incubation time.
The transforming potency of the carbon samples
was assessed using: ‘SHE cell transformation assay’:
among the current in vitro genotoxicity assays, cell
transformation is the most relevant for assessing
533
the in vivo carcinogenesis process, involving many
of the same genetic and epigenetic mechanisms.
The SHE cell system, the most extensively used
transformation assay, is applied both to understand
the mechanisms of carcinogenesis and to detect carcinogens (Yamasaki, 1996). We used the SHE cell
transformation assay in combination with the other
endpoints because cellular proliferation and viability
are involved in the expression of induced transformation. This approach also distinguishes between
a cytotoxic, but not transforming, sample and a transforming and/or not cytotoxic one.
To avoid misinterpretation of the results, all assays
were performed on the same cell type, SHE cells, using pre-selected batches (i.e. cells giving expected
results with a positive and a negative control). The
treatment concentrations of the carbon samples used
in each assay were set after preliminary experiments
and are indicated below.
Samples
Samples E1 to E11 were collected during the
fabrication process of carbon pieces in Safran aeronautic plants (Messier-Bugatti and Snecma Propulsion Solide). E1 to E5 corresponded to the first
steps of the process, i.e. after heating ,1000°C. E6
to E9 corresponded to the intermediate steps, i.e.
heating .1000°C. E10 corresponded to dust that
was sampled during machining of a final carbon
piece, and E11 to dust coming from a test bench.
In the context of the fabrication process, atmospheric sampling seemed to be difficult to achieve
and, further, was not able to provide enough material. Sampling was then made directly on material
generated by each step of the process. As collected,
samples sizes were not compatible with biological
assays and could not be considered as ‘respirable
size’. To get samples that could be closer to those potentially found in the various services operations and
thereby represent respirable size, samples E1 to E9
were crushed in a corundum jar with corundum balls.
E10 and E11 were used as they were after
collection.
Alpha corundum Al2O3, Graphite G28286-3, and
Graphite G49659-6 were from Sigma Aldrich,
France.
Chrysotile (Laboratory code: C19) was obtained
from Union Internationale Contre le Cancer (Johannesburg, South Africa). This sample was used
as a positive control in our assays.
An amorphous biogenic diatomaceous earth (DE)
was used as a positive control for cytotoxicity and
a negative control for the transformation assay.
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C. Darne et al.
Physicochemical characterization of the samples
Granulometry. Particles and fibers size distribution was measured using an electron scanning microscopy (JEOL 840A, Japan). Analysis was made
at least on 100 fibers or particles.
Chemical impurities. Impurities were analyzed
first by inductively coupled plasma spectrometry
(Spectro Ciros CCD, Germany) and after grinding
by thermogravimetric analysis (Setaram TGA24S,
France) to evaluate corundum aluminum contamination (samples E1 to E9).
Electron paramagnetic resonance detection of
ROS. The release of ROS from aqueous buffered
suspensions of the particles in the presence of hydrogen peroxide (H2O2) as the oxygenated target was
monitored by Electron Paramagnetic Resonance
(EPR) spectroscopy, using a spin trapping agent:
5,59-dimethyl-1-pyrroline-N-oxide (DMPO). In absence of formate anions, the hydroxyl radical
(HO ) is detected in the form of a radical adduct
[DMPO,HO] . The use of formate anions revealed
all the very electrophilic radicals and non-radical
species that react with HCO2 to form the carboxylate radical anion (CO2). DMPO reacts with CO2 to form [DMPO,CO2] .
The EPR tests were conducted in accordance with
the experimental procedure detailed previously
d
d
d
d
Table 1. Physicochemical analysis of samples
Sample
Chemical impurities
(% mass)a
ROS
generationb
DE (amorphous silica)
1.4 Al, 1.3 Fe,
2.2 Ca, 0.3 Ti,
0.2 Mg
Chrysotile—C19
1.9 Fe
þþ
Graphite (49659-6)
—
Graphite (28286-3)
0.2 Fe
Al2O3 (corundum)
—
/þ
E1
1.1 Al2O3
/þ
E2
3.3 Al2O3
E3
E4
11 Al2O3
3 Al2O3
E5
47.9 Al2O3
E6
2.8 Al2O3
E7
12 Al2O3
E8
3.4 Al2O3
E9
5.7 Al2O3
E10
0.02 Al
/þ
E11
0.3 Fe, 0.2 Ca
a
Inductively coupled plasma and/or TGA analysis.
EPR analysis and MDA detection; , negative; /þ,
weakly positive; þþ, positive.
b
(Zalma et al., 1987). Forty-five milligram of dust
was added to a solution containing 0.5 M sodium phosphate buffer (pH 7.4) from a mixture of sodium dihydrogen phosphate and sodium hydrogen phosphate
(99% pure, Sigma, St. Quentin Fallavier, France),
0.15%/v H2O2 solution (VWR-Prolabo, Fontenay
sous bois, France), and 50 mM DMPO (97% pure, Aldrich, France). For the tests in the presence of formate
anions, 1 M sodium formate (99% pure, Merck, Fontenay sous bois, France) was added to this mixture.
The suspension was stirred in a reactor protected from
light exposure at 37°C. Aliquots were withdrawn at
different incubation times (5 and 30 min without
formate; 30 and 60 min with formate) and were then
filtered (porosity 0.22 lm). Blanks containing no solid
material were done in the same experimental conditions. For each assay, result reproducibility was
checked (at least twice for each assay).
The presence of [DMPO,HO] and [DMPO,CO2]
radicals was detected by EPR spectroscopy (ESP300E, Brucker, Rheinstetten, Germany). The conditions to obtain the signal of these radical adducts were
as follows: central field, 3390 G; scan range, 100 G;
microwave power, 10 mW; frequency, 9.65 GHz (X
band); and time constant, 82 ms.
Production of these radicals is based on the measurement of EPR signal intensity (in arbitrary units,
a.u.) and are given as the mean of the adduct signal
after the two incubation periods, which the blank
was subtracted from.
Linolenic acid peroxidation. Linolenic acid, polyunsaturated fatty acid, has been used as a model
molecule to study peroxidation in the presence of
solid particles in an aqueous buffered medium containing H2O2. The peroxidation mechanism is very
complex and implies the presence of ROS species
which is revealed by the detection of some degradation products from this reaction [for example
malonaldehyde (MDA), total monoaldehydes, etc.]
(Halliwell and Gutteridge, 1986; Chouchane et al.,
2000). MDA, known for its toxicity, is considered
to be one of the main indices of peroxidation
(Fournier et al., 1995).
The procedure for the detection of MDA was carried out from solids suspensions in aqueous and aerated medium, according to the same experimental
protocol previously detailed (Chouchane et al., 2000).
The peroxidation test was performed as follows: in
a reactor kept at constant temperature (37°C) and
protected from external light, the sample (45 mg)
was added to an aqueous phosphate-buffered
(0.25 mM, pH 7.4) medium containing 0.15% (v)
H2O2 solution and 1 mM purified linolenic acid or
9,12,15-octadécatrienoic acid (99% pure, Sigma,
d
d
In vitro SHE transformation assay
France). The suspension was stirred for 18 h. After
this incubation period, 0.2%/w of butylated hydroxytoluene (99.5% pure, Sigma, France) in ethanol was
added to the suspension to stop the oxidation reaction, then a solution containing 15% (w/v) of
trichloroacetic acid (99.5% pure, Sigma, France)
and 0.375% (w/v) of 2-thiobarbituric acid (TBA;
98% pure, Sigma, France) in hydrochloric acid
(0.25 M). The mixture was heated to 100°C then
cooled in an ice bath. The chromogen formed,
TBA–MDA–TBA (pink color) (Marcuse and
Johansson, 1973), was extracted by 1-butanol
(99.5% pure, Merck, France) after centrifugation
(8000 r.p.m.). The organic phase was then analyzed
by UV/vis spectroscopy (V-550, Jasco, France).
The absorbance was measured at 534 nm for TBA–
MDA–TBA against 1-butanol as the reference.
Blanks were measured in the absence of solid material under the same experimental conditions. For
each assay, the reproducibility of the results was
checked (at least twice for each incubation time).
The results, from which the blank has been subtracted, are presented in the form of absorbance
per milliliter of butanol phase as a function of the
samples.
The samples were not submitted to any preliminary sterilization.
Cell cultures
SHE cell cultures were established from individual 13-day gestation fetuses (inbred colony, INRS,
Vandoeuvre les Nancy, France). The culture medium
was Dulbecco’s modified Eagle’s medium (Invitrogen, Cergy Pontoise, France), pH 7 supplemented
with 20% pre-selected fetal calf serum (Dutscher,
Brumath, France) and 2 mM L-glutamine (Invitrogen, France) with antibiotics (50 units ml1 Penicillin, 50 lg ml1 Streptomycin; Invitrogen, France).
The cells were incubated at 37°C, 10% CO2. Cryopreserved primary cultures were selected for cell
growth, CE, and spontaneous and induced morphological transformation. Primary and secondary cultures from a batch yielding results consistent with
historical range were used in the study.
Cell proliferation assay. Cells (30 000 cells ml1)
were cultured for 24 h at 37°C, 10% CO2 in culture
medium. The cell cultures were then treated with
culture medium (control) or carbon suspensions in final concentrations ranging between 0.045 and 28.58
lg cm2 for up to 3 days. On the day of treatment
and at 72 h post treatment, the cells were removed
by trypsination and counted (Coulter Z1, Beckman
Coulter, Villepinte, France). Cell viability was determined by the trypan blue exclusion method. At least
535
four concentrations of carbon samples were analyzed and triplicate cell cultures were used for each
treatment concentration and time. The concentration
that reduced cell proliferation to 50% of control, inhibitory concentration 50 (IC50), was calculated
from cell proliferation curves obtained from four to
six individual experiments. The mean number of
cells – SEM for each treatment concentration and
time was calculated and compared to that of the control using the Student’s t-test.
CE and transformation assay. The assay was performed as described previously (Elias et al., 1989;
Elias et al., 2000). X-irradiated SHE feeder cells
were seeded at 3 104 cells ml1 in 60 mm dishes.
After 24 h of incubation (37°C, 10% CO2), 600 SHE
target cells per dish were seeded onto the feeder
cells. The cells were incubated for 24 h at 37°C,
10% CO2 and then exposed to at least four sample
concentrations ranging from 4.75 to 28.5 lg cm2.
The control cells received culture medium alone.
After 7 days of incubation at 37°C, 10% CO2, the
dishes were washed (Hanks’ phosphate-buffered saline, Invitrogen) and the colonies were fixed (absolute
methanol) and stained (10% Giemsa). The colonies
were counted and examined for morphological transformation in a stereomicroscope (Wild, Germany).
Ten dishes of cell cultures were used per treatment
concentration and control. The following were scored
for each treatment concentration and control of an individual assay: (i) total colony number; (ii) CE 5 (total
colony number/total target cell number seeded) 100;
(iii) relative CE 5 (CE of treated cells/CE of the control) 100; (iv) cytotoxicity 5 100 relative CE; (v)
number of morphologically transformed colonies; and
(vi) transformation frequency (TF) 5 (the number of
transformed colonies/total number of colonies) 100.
The treatment concentration that induced a 50% reduction in relative CE, the lethal concentration 50 (LC
50), was calculated from the relative CE curve. The
total number of colonies analyzed was between
400 and 10 000 per treatment concentration. The
mean CE – SEM of the control cultures was 23.98
– 2.0% (n 5 16). Only five spontaneous transformed
colonies were recorded in 16 experiments. For each
treatment concentration, the data reported the pooled
results of three to eight individual assays. The TF was
compared to control using the Khi2 test.
RESULTS
Physicochemical characterization of the samples
All the characteristics of the samples used in this
study are presented in Table 1 and Table 2.
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C. Darne et al.
Table 2. Size of fibers and particles in the samples
Sample
DE (amorphous silica)
Particle
GMD (lm)
1.65
GSD
Fibers
(% in number)
(L/D 3)
GMD
(lm)
GSD
Fiber
geometric
mean
length (lm)
—
GSD
1.38
—
—
—
Chrysotile—C19
—
—
96.20
0.13
1.78
Graphite (49659-6)
10.46
1.70
—
—
—
—
Graphite (28286-3)
9.43
1.68
—
—
—
—
—
Al2O3 (corundum)
0.30
2.43
—
—
—
—
—
E1
E2
1.53
3.93
2.43
1.86
4.3
25
1.48
1.94
4.5
2.03
4.86
10.56
4.44
2.29
E3
2.91
1.88
10.2
1.45
1.74
7.69
1.70
E4
3.79
2.13
8.4
2.21
3.14
8.27
3.12
E5
2.19
2.53
5.8
2.96
3.68
17.25
5.44
E6
2.40
1.76
4.6
1.01
1.85
3.53
1.81
E7
1.08
2.88
4
0.40
2.13
1.44
2.08
E8
1.16
2.34
17.6
0.66
2.11
2.41
2.16
E9
E10a
2.47
40.0
2.06
3.90
8.5
26.1
1.03
9.36b
2.12
2.22
4.00
78.58
2.30
2.34
E11
5.19
1.86
5.8
3.82
2.11
18.59
4.09
1.00
—
2.16
—
a
‘Particles’ and ‘fibers’ found in this sample are fibers bundle fragments and fiber bundles.
Fiber GMD for fiber bundles. The GMD of fibers constituting the bundles is 7.37 lm with a GSD at 1.15.
b
The asbestos sample mainly contained small fibers
[geometric mean diameter (GMD) 0.13 lm and geometric mean length 1 lm]. The amorphous silica
particles (DE) had a GMD of 1.65 lm [geometric
standard deviation (GSD) of 1.38]. The GMD of
the two commercial graphites (G49659-6 and
G28286-3) used as other sources of carbon particles
were 10.46 lm (GSD 1.70) and 9.43 lm (GSD 1.68),
respectively. The corundum aluminum oxide was 0.3
lm in GMD (GSD 2.43).
The majority of carbon samples (E1 to E11) contains fibers. Their percentage in number in the samples
vary from 4 to 26.1%, if we consider that a fiber is
defined as having a L (length)/D (diameter) ratio 3.
For E1 to E9, the GMD of particles ranged between 1.08 lm (E7) to 3.93 lm (E2); the GMD of
fibers ranged from 0.4 lm (E7) to 2.96 lm (E5)
(GSD 2.13 and 3.68, respectively) with a geometric
mean length ranged from 1.44 lm (GSD 2.08) for E7
to 17.25 lm (GSD 5.44) for E5.
Grinding samples E1 to E9, with corundum, has
conducted to their contamination with aluminum oxide (corundum form). The percentage of aluminum
oxide ranged from 1.1 to 47.9 of sample mass, as
shown in Table 1.
E10, sampled during machining of a final carbon
piece, was heterogenous, mainly constituted of fibers
bundles and part of fibers bundle. There was
a bimodal distribution for the particle fraction (71%
of particles between 8 and 36 lm and 29% .50 lm
and up to 500 lm). The overall particle GMD was
39.99 lm (with a GSD at 3.9). The GMD of fiber bundles was 9.36 lm (GSD 2.22) with a GMD for fibers
constituting bundles at 7.37 lm and a GSD at 1.15.
E11 was homogenous with a mean diameter of 5.19
lm (GSD 1.86) for particles and a fibers GMD of 3.82
lm (GSD 2.11) and a geometric mean length of 18.59
(GSD 4.09). Sample E11 contained 0.3% of iron.
Release of ROS species from fibers/particles surface
Fig. 1 reveals that carbon samples G49659-6,
G28286-3, E2 to E9, and E11 are completely
inactive, contrary to chrysotile. For samples E1 and
E10, a weak production of electrophilic ROS can be
noted but remains negligible compared to chrysotile.
Results from the linolenic acid peroxidation, in the
presence of hydrogen peroxide in aqueous buffered
medium, after 18 h of sample incubation, are illustrated in Fig. 2.
As shown, all the carbon samples are inactive in
the MDA production. The quantity of MDA formed
in the presence of alumina is very low compared to
the activity of chrysotile.
From all these data (Figs 1 and 2), carbon and alumina samples can be considered inactive in the production of ROS (Table 1).
In vitro SHE transformation assay
Cell proliferation
Controls. As shown in Fig. 3, chrysotile and amorphous silica DE induced a concentration-dependent
decrease in cell proliferation. The cytotoxicity of
chrysotile was higher than that of DE with an IC50
537
(inhibition concentration giving a 50% decrease in
cells) of 1.63 lg cm2 versus 6.8 lg cm2 after
72 h of treatment.
Effect of aluminum oxide on cell proliferation. Because samples E1 to E9 contained aluminum oxide
Fig. 1. Formation of the radicals, HO and CO2, from materials in the presence of H2O2 (0.15%/v) in phosphate-buffered
medium (0.5 M). Each bar represents the average error from two independent measurements.
Fig. 2. Formation of MDA from materials in the presence of H2O2 (0.15%/v) in phosphate-buffered medium (0.5 M) after 18 h of
incubation. Each bar represents the average error from two independent measurements.
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C. Darne et al.
Fig. 3. Cell proliferation: controls. Percentage of SHE cells (–SEM) after 72 h of treatment with silica DE, chrysotile C19, Al2O3,
Graphite G49659-6, and G28286-3. Sample concentrations are expressed as microgram per square centimeter of cell culture
surface. Asterisk indicates statistically significant (P , 0.05) decrease in cell number compared to control.
(corundum form), we looked at its possible interference with other particles on cell proliferation.
Alone, as shown in Fig. 3, corundum aluminum
oxide induces a decrease in cell proliferation after
exposure for 72 h (69% of control) at the highest
concentration tested, but there is no clear concentration-dependent growth inhibition.
Aluminum oxide did not change the effect of chrysotile on cell proliferation, as shown in Fig. 4, even at
the highest concentration. Therefore, it can be assumed
that corundum aluminum oxide did not modify the effect of the other particles in the cell proliferation assay.
Carbon particle samples We used two commercial samples of graphite (named Graphite 28286
and Graphite 49659) as another source of carbon.
As shown in Fig. 3, the smallest graphite
(G28286-3) was the most cytotoxic, with a decrease
of 44% of cell growth at 72 h. (versus 13.5%), but the
IC50 was not reached even at the highest dose.
For carbon samples E1 to E11 (Figs 5 and 6), none
of the concentration tested allowed the IC50 to be
reached, even the highest (28.58 lg cm2). On this
basis, the inhibitory potency of the samples at
28.58 lg cm2 ranked in the following order: E2
(49% inhibition) . E9 (42%) . E7 (39%) . E11
(38.5%) . E8 (33%) . E6 (31%) . E4 (27%) .
E3 (21%) . E1, E5 (16%) . E10.
No relationship could be established between cell
proliferation and fiber percentage or with fiber/particle size. Likewise, there was no obvious relationship
with ROS generation (E1 and E10, which were
weakly positive in the ROS generation assay, showed
a low cell proliferation inhibition potential).
CE and TF of SHE cells (Table 3 and 4)
Controls. The positive control, chrysotile, induced a concentration-dependent increase in cell lethality from 0.95 lg cm2. The first transformed
colonies were observed at 0.38 lg cm2 and the cell
TF increased with the increasing concentration.
As previously shown (Elias et al., 2006), the amorphous silica DE was not able to induce significant
cell transformation.
Effect of aluminum oxide on cell transformation.
Corundum aluminum oxide was used at concentrations
of 4.75, 9.5, 19, and 28.5 lg cm2. As shown in Table 3,
aluminum oxide induced no cell lethality and cell
transformation, even at the highest concentration.
We also examined the possibility of interference
between corundum aluminum oxide and the other
particles in relation to their cell transformation potential. To this end, chrysotile (0.95 and 1.9 lg
cm2) was mixed with increasing concentrations
(0.095, 0.238, 0.476 lg cm2 and 0.19, 0.476, 0.95
lg cm2, respectively) of corundum aluminum oxide (equivalent of 1, 25, or 50% of chrysotile mass).
Despite the presence of 1, 25, or 50% aluminum
oxide, there was no modification of the relative CE
or the percentage of transformed cells. As shown
for cell proliferation, there was no influence of corundum aluminum oxide on cell transformation.
Therefore, it can be assumed that corundum aluminum oxide did not modify the effect of the other particles in the cell transformation assay.
Effect of carbon samples on cell transformation
(Tables 3 and 4). The cytotoxicity of samples after
7 days of treatment was fairly well correlated with
In vitro SHE transformation assay
539
Fig. 4. Cell proliferation: Effect of Al2O3 on chrysotile C19. Percentage of mean number of treated cells with respect to the mean
number of control cells (100%) after 72 h of treatment with chrysotile C19 and Al2O3. Sample concentrations are expressed as
microgram per square centimeter of cell culture surface. Asterisk indicates statistically significant (P , 0.05) decrease in cell
number compared to control.
Fig. 5. Cell proliferation: Carbon samples. Percentage of mean number of treated cells with respect to the mean number of control
cells (100%) after 72 h of treatment with carbon particles E1, E2, E3, E4, E5, and E6. Sample concentrations are expressed as
microgram per square centimeter of cell culture surface. Asterisk indicates statistically significant (P , 0.05) decrease in cell
number compared to control.
the inhibition of cell proliferation after 3 days. On
the basis of a concentration of 28.5 lg cm2, the cytotoxicity of the samples ranked in the following order: E11 (60.81% of cell lethality) . E9 (48.5%) .
E8 (40.8%) . E7 (34.7%) . E6 (32.9%) . E2
(9.6%) . E3, 5, 10, 4, 1 (no lethality).
None of the tested samples led to a significant cell
transformation, except for four transformed colonies
found with commercial graphite G28296 in only one
of the three experiments.
DISCUSSION
Carbon fiber composite materials are used in a
variety of industrial applications, and machining
processes often aerosolize these materials in workplaces. Worker exposure can occur not only during
the manufacturing of composite structures involving
carbon fibers and during the repair of such structures
but also at the different stages of the carbon fiber process and more precisely during machinery servicing
540
C. Darne et al.
Fig. 6. Cell proliferation. Percentage of mean number of treated cells with respect to the mean number of control cells (100%) after
72 h of treatment with carbon particles E7, E8, E9, E10, and E11. Sample concentrations are expressed as microgram per square
centimeter of cell culture surface. Asterisk indicates statistically significant (P , 0.05) decrease in cell number compared to control.
and oven cleaning. Only three epidemiological studies have been conducted on carbon fibers. In these
studies, analyzing the results was difficult: exposed
worker number was low or the exposures were complex with several chemicals involved. Despite the fact
that the exposure was not very well characterized, the
only health effects observed was some dermal irritation (Jones et al., 1982; Mathias, 1987; Eedy, 1996).
The data available for PAN-based carbon fibers/
particles, coming from experimental studies, have
shown no significant adverse effects. But these in vivo
studies remain few and far between; the main body of
work was done with often non-respirable carbon fibers and not always well characterized (for review:
Thomson, 1989; Warheit et al., 2001).
In order to evaluate the toxicity of carbon dust
possibly released into the work atmosphere during
the manufacturing process, 11 samples of carbon
fibers/particles were taken at each step of the process. The first nine samples were ground. The
remaining two samples were used as collected. We
investigated the cytotoxicity and carcinogenic potential of these different samples of carbon fibers/
particles using in vitro short-term assays.
In vivo, fiber or particle toxicity can often be
related to their physical–chemical properties, including diameter, length, morphology, chemical composition, and ROS production capacity. The role of
ROS in the inflammatory and fibrogenic activity of
particles is well established (Fubini, 1997; Jaurand,
1997; Fubini and Hubbard, 2003). The role of ROS
in the induction of cell transformation in vitro by
quartz, cristobalite, and diatomite earth samples
(Elias et al., 2000; Fubini et al., 2001; Elias et al.,
2002) has also been shown in some of our previous
studies. A linear correlation was found between the
amount of OH released by particles and TF in
SHE cells (Fubini et al., 2001). Here, none of the
carbon samples led to an inhibitory effect of 50%
of proliferation, even at the highest concentration
tested. The results did not permit to establish a relationship between particle size, percentage of fiber or
ROS production capacity, and the biological effects.
Compared to asbestos chrysotile or amorphous silica
DE, carbon samples showed no significant effect on
cell proliferation or on high cytotoxicity (only one
led to a loss of 50% of cell colonies as evaluated
by the cell transformation assay (E11, LC50:
23.56 lg cm2)). Chrysotile was the only sample
that induced transformed colonies with a dose–response relationship, and it was the only sample with
a ROS generation capacity. All the carbon samples
were negative. Based on the DE data, and to a lesser
extent to carbon samples data, the cytotoxic and
transforming potencies of the samples examined
seem to be unrelated, suggesting different mechanisms triggered by different surface properties in
the two effects elicited. This was also the case for
other compounds, as previously reported (Elias
et al., 2000; Elias et al., 2006). Cytotoxicity might
not be, or not only, related to the generation of free
radicals but to other physicochemical features such
In vitro SHE transformation assay
541
Table 3. Morphological transformation into SHE cells following 7 days treatment with references
Sample
Dose
(lg cm2)
Morphological
TF (%)
Number of
transformed
colonies
Total
number of
colonies
Relative
plating
efficiency (%)
Culture medium
C19
0.19
0.043
0.066
5
2
11 512
3028
100
105
C19
0.38
0.128*
4
3120
108
C19
0.95
0.396*
8
2018
C19
1.9
0.646*
43
6652
46*
C19
3.8
0.826*
9
1089
38*
40*
C19 0.95 lg cm2
Al2O3 0.095
0.276*
4
1447
100a
2
Al2O3 0.238
0.452*
7
1548
107a
2
C19 0.95 lg cm
C19 1.9 lg cm2
Al2O3 0.476
Al2O3 0.19
0.429*
0.836*
6
7
1400
837
97a
84b
C19 1.9 lg cm2
Al2O3 0.476
0.569*
5
878
88b
2
Al2O3 0.952
0.719*
7
974
97b
C19 0.95 lg cm
C19 1.9 lg cm
Al2O3
4.75
0.000
0
743
Al2O3
9.5
0.000
0
709
99
0.000
0
748
104
Al2O3
19
Al2O3
28.5
DE
DE
4.75
9.5
103
0.000
0
738
103
0.000
0.000
0
0
856
926
119
129
DE
19
0.393*
2
509
71
DE
28.5
0.000
0
366
51*
G49659-6
3.8
0.000
0
1469
102
G49659-6
7.6
0.039
1
2540
118
G49659-6
15.2
0.043
1
2302
110
G49659-6
30.4
0.044
1
2290
106
G28286-3
G28286-3
3.8
7.6
0.000
0.170*
0
4
1445
2352
100
109
G28286-3
15.2
0.000
0
2294
106
G28286-3
30.4
0.000
0
1752
81
a
2
Percentage calculated in comparison with C19 0.95 lg cm .
Percentage calculated in comparison with C19 1.9 lg cm2.
*Statistically significant (P , 0.05) compared to control (culture medium).
b
as micromorphology, surface charges, and hydrophilicity (Fubini, 1998; Fubini et al., 1999).
Two other ‘carbon’ samples were collected on the
floor in a workshop where secondhand pieces are reconditioned. These samples were not representative
of the carbon samples due to their complex composition [carbon ,55%; lots of metal (3–15% iron,
0.3% Zn, 0.2% Pb, etc.), and probably organic compounds] and were not included in this article. Nevertheless, it is interesting to point out that these
samples were found to be able to induce transformed
colonies with no cell proliferation inhibition. When
analyzed by EPR, these samples were also able to
induce ROS production (probably because of the
metallic compounds), suggesting a relationship be-
tween cell transformation potency and the capacity
to generate ROS, as it has been shown for chrysotile
and silica (present data; Elias et al., 2006). The results obtained with these samples, compared to those
coming from pure carbon particles (reference graphites, E10, E11), and also to a lesser extent with the
others samples, led to our considering that that the
induction of transformed colonies did not come from
carbon fibers/particles.
In summary, in our conditions, the carbon fiber/
particle samples tested showed no carcinogenic potential, as evaluated by the SHE cell transformation
assay and had low cytotoxicity and little effect on
cell proliferation compared to chrysotile asbestos
fibers. Our results were close to some others
542
C. Darne et al.
Table 4. Morphological transformation into SHE cells following 7 days treatment with carbon samples
Sample
Culture medium
E1
E1
Dose
(lg/cm2)
4.75
9.5
Morphological
TF (%)
Number of
transformed
colonies
Total
number of
colonies
Relative
plating
efficiency (%)
0.043
0.000
5
0
11512
2635
100
122
0.038
1
2606
121
E1
19
0.042
1
2367
110
E1
28.5
0.044
1
2260
105
E2
4.75
0.041
1
2447
113
E2
9.5
0.000
0
2354
109
E2
19
0.000
0
2225
103
E2
E3
28.5
4.75
0.000
0.040
0
1
1951
2531
90
117
E3
0.040
1
2478
115
E3
19
9.5
0.043
1
2342
109
E3
28.5
0.046
1
2175
101
E4
4.75
0.041
1
2463
114
E4
9.5
0.000
0
2471
114
E4
19
0.000
0
2505
116
E4
E5
28.5
4.75
0.000
0.000
0
0
2255
2550
104
118
E5
0.040
1
2508
116
E5
19
9.5
0.042
1
2383
110
E5
28.5
0.046
1
2195
102
E6
4.75
0.048
1
2075
96
E6
9.5
0.000
0
1950
90
E6
19
0.054
1
1868
87
E6
E7
28.5
4.75
0.000
0.051
0
1
1449
1965
67*
91
E7
0.051
1
1976
92
E7
19
9.5
0.000
0
1752
81
E7
28.5
0.000
0
1410
65*
E8
4.75
0.000
0
2089
97
E8
9.5
0.000
0
1848
86
E8
19
0.000
0
1705
79*
E8
E9
28.5
4.75
0.000
0.000
0
0
1277
2010
59*
93
E9
0.000
0
1823
84
E9
19
0.000
0
1539
71*
E9
28.5
E10
E10
9.5
0.000
0
1111
4.75
0.000
0
2331
108
51*
9.5
100
0.047
1
2150
E10
19
0.047
1
2141
99
E10
E11
28.5
4.75
0.046
0.000
1
0
2197
2051
102
95
9.5
E11
0.000
0
1769
82
E11
19
0.000
0
1295
60*
E11
28.5
0.000
0
846
39*
*Statistically significant (P , 0.05) compared to control (culture medium).
In vitro SHE transformation assay
(Thomson, 1989) reporting that PAN-based carbon
fibers were negative in the chromatid exchange assay
(sister chromatin exchange assay), the unscheduled
DNA synthesis assay (UDS), and also in the Ames
test. It should also be noted that the results are different with pitch-based carbon fibers (SCE positive and
UDS positive). The results differ also from smaller
materials such as carbon blacks or carbon nanotubes.
Indeed, IARC (1996, 2006) has classified carbon
blacks in the Group 2B. Their evaluation was based
on results of animals’ studies, with ultrafine materials or nanomaterials, showing an increase in lung tumor after treatment, results coming from in vitro
studies being much debated since Ames, thimidine
kinase, and SCE assays were negative while hypoxanthine-phosphoribosyl transferase and micronucleus assays were positive for ultrafine carbon
black. In vitro evaluation of carbon nanotubes gives
also different results (compared to what we obtained
with our carbon dust): they are mostly cytotoxic and
they could conduct to alteration of the DNA (positive
for micronucleus and comet assays) (Muller et al.,
2008; Lindberg et al., 2009). In vivo, they have
shown to be able to produce an inflammatory response associated with granuloma and fibrosis after
intratracheal instillation (Muller et al., 2005). But it
should be mentioned that overall conclusion is not
that simple, the carbon nanotubes family is quite
large and results are sometime conflicting (Shvedova
et al., 2009). Another carbon materials are fullerenes
and they seem to be non-cytotoxic and they don’t induce mutations (Gharbi et al., 2005; Jia et al., 2005;
Mori et al., 2006).
Our samples of fibers and particles of carbon were
generated by one type of process; it is therefore necessary to consolidate these results by studying other
samples, on the one hand coming from other firms
using the same manufacturing technique and on the
other hand generated by other methods.
FUNDING
Safran (C03094 and C05194a01).
Acknowledgements—The authors are grateful to Safran group,
and more precisely to Messier-Bugatti and Snecma Propulsion
Solide for provided samples. The authors are grateful to
Mrs C. Eypert-Blaison, INRS, for the SEM analysis, to
Mr B. Morin, Laboratoire de Réactivité de Surface, for
EPR technical assistance and to Mrs M. Roussel, INRS, for
manuscript preparation.
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