Mitochondria-derived reactive intermediate species mediate asbestos-induced genotoxicity and oxidative stress-responsive signaling pathways.

Abstract

The incidence of asbestos-induced human cancers is increasing worldwide, and considerable evidence suggests that reactive oxygen species (ROS) are important mediators of these diseases. Our previous studies suggested that mitochondria might be involved in the initiation of oxidative stress in asbestos-exposed mammalian cells.
We investigated whether mitochondria are a potential cytoplasmic target of asbestos using a mitochondrial DNA-depleted (ρ(0)) human small airway epithelial (SAE) cell model: ρ(0) SAE cells lack the capacity to produce mitochondrial ROS.
We examined nuclear DNA damage, micronuclei (MN), intracellular ROS production, and the expression of inflammation-related nuclear genes in both parental and ρ(0) SAE cells in response to asbestos treatment.
Asbestos induced a dose-dependent increase in nuclear DNA oxidative damage and MN in SAE cells. Furthermore, there was a significant increase in intracellular oxidant production and activation of genes involved in nuclear factor κB and proinflammatory signaling pathways in SAE cells. In contrast, the effects of asbestos were minimal in ρ(0) SAE cells.
Mitochondria are a major cytoplasmic target of asbestos. Asbestos may initiate mitochondria-associated ROS, which mediate asbestos-induced nuclear mutagenic events and inflammatory signaling pathways in exposed cells. These data provide new insights into the molecular mechanisms of asbestos-induced genotoxicity.

Full text

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volume 120 | number 6 | June 2012 • Environmental Health Perspectives
Research
Asbestos fibers are well-defined environmental
and occupational carcinogens in humans and
remain widely used in many developing coun-
tries (Nishikawa et al. 2008; Straif et al. 2009).
Currently, the worldwide incidence of asbes-
tos-induced cancer and other diseases is still on
the rise because of their long latency periods
(Nishikawa et al. 2008). Consequently, there is
great urgency and importance in studying the
molecular mechanisms underlying asbestos-
related diseases for a better understanding of
disease prevention and treatment strategy.
There is considerable evidence that
asbestos-initiated chronic oxidative stress
contributes to carcinogenesis and fibro-
sis by promoting oxidative DNA damage
and regulating redox signaling pathways
in exposed cells (Huang et al. 2011; Kamp
and Weitzman 1999; Shukla et al. 2003;
Toyokuni 2009; Xu et al. 1999, 2002).
Asbestos fibers may initiate reactive oxy-
gen species (ROS) production by multiple
mechanisms. The surface iron associated with
asbestos generates hydroxyl radicals either
through a redox reaction or by catalyzing a
Fenton-like reaction in exposed cells (Pezerat
et al. 1989; Weitzman and Graceffa 1984).
In addition, the uptake of asbestos fibers can
stimulate phagocytic cells to release extra-
cellular ROS through membrane-associated
NADPH (nicotinamide adenine dinucleotide
phosphate) oxidase (Kamp et al. 1992).
A possible important source of intra cellular
ROS generation in asbestos-exposed cells is
mitochondrial oxidative phosphorylation.
Mitochondria consume about 90% of
the oxygen used by cells. In normal physio-
logical conditions, mitochondrial oxidative
phosphorylation represents the major site of
endogenous oxidant generation. In patho-
physio logical conditions, certain chemical
xeno biotics are known to use the mitochondrial
respiratory chain to amplify reactive oxidant
production (Gutierrez 2000). Two studies
found that mitochondria-derived oxidative
stress was a mediator of asbestos-induced
apoptosis and cell toxicity in alveolar epithelial
cells (Panduri et al. 2003, 2004).
Thus far, it is not clear whether mito-
chondrial-originated reactive oxidants medi-
ate asbestos-induced nuclear DNA mutagenic
events. Our previous study demonstrated
that cytoplasmic components could initiate
asbestos-induced oxidative stress and promote
nuclear mutagenesis in AL (human–hamster
hybrid) cells (Xu et al. 2007). In the present
study, we investigated whether mitochondria
might be the potential cytoplasmic target of
asbestos fibers. We examined the effects of
chrysotile and crocidolite asbestos treatment
on intracellular ROS production in human
small airway epithelial (SAE) cells and found
that mitochondrial-originated reactive oxidants
were involved in mediating asbestos- induced
nuclear DNA damage and mutagenesis. We
also found that these oxidants altered the
expression of 178 inflammation- and immune-
related nuclear genes involved in signaling
pathways known to be activated in response to
oxidative stress in mammalian cells (Martindale
and Holbrook 2002).
Materials and Methods
Asbestos fibers, cell culture, and treatments. We
used UICC (Union Internationale Contre le
Cancer) standard reference samples of chryso-
tile and crocidolite asbestos in the present
study. Stock solutions (1 mg/mL) were pre-
pared as described previously (Xu et al. 2002).
The human telomerase reverse transcriptase–
immortalized human SAE cells were previ-
ously generated (Piao et al. 2005). Cells were
maintained in complete small airway growth
medium supplemented with growth factors
(Lonza Group Ltd., Basel, Switzerland) at
37°C in a humidified 5% CO2 atmosphere.
Typically, 70–80% confluent cell cultures were
treated with chrysotile or crocidolite at concen-
trations of 0.5, 1, 2, and/or 4 µg/cm2 for 12,
24, or 48 hr. Hydrogen peroxide (H2O2) was
added at final concentrations ranging from 100
to 500 µM for 30 min. In some experiments,
0.5% (vol/vol) dimethyl sulfoxide (DMSO;
Sigma, St. Louis, MO) was added before
and concurrently with asbestos treatment.
Methods for assessing the toxicity of asbestos
are described in Supplemental Material (http://
dx.doi.org/10.1289/ehp.1104287).
Address correspondence to T.K. Hei, Center for
Radiological Research, College of Physicians and
Surgeons, Columbia University, 630 West 168th St.,
VC-11-205, New York, NY 10032 USA. Telephone:
(212) 305-8462. Fax: (212) 305-3229. E-mail:
tkh1@columbia.edu
*Current address: Regeneron Pharmaceuticals,
Tarrytown, NY, USA.
Supplemental Material is available online (http://
dx.doi.org/10.1289/ehp.1104287).
We thank T. Templin and L. Smilenov for gener-
ously providing gene expression and analysis systems;
P. Grabham, S. Jockusch, J.Y.-C. Chen, E. Hernandez-
Rosa, S. Krishna, and W. Walker for expert technical
assistance; and Y. Wei for advice on statistical analysis.
This work was supported by National Institutes
of Health grants ES 05786, ES 12888, Superfund
grant P42 ES 10349, and National Institute of
Environmental Health Sciences Environmental
Center grant ES 09089.
The authors declare they have no actual or potential
competing financial interests.
Received 1 August 2011; accepted 7 March 2012.
Mitochondria-Derived Reactive Intermediate Species Mediate Asbestos-Induced
Genotoxicity and Oxidative Stress–Responsive Signaling Pathways
Sarah X.L. Huang,1 Michael A. Partridge,2* Shanaz A. Ghandhi,2 Mercy M. Davidson,3 Sally A. Amundson,2
and Tom K. Hei1,2,3
1Department of Environmental Health Sciences, Mailman School of Public Health, 2Center for Radiological Research, and 3Department of
Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
Background: The incidence of asbestos-induced human cancers is increasing worldwide, and
considerable evidence suggests that reactive oxygen species (ROS) are important mediators of these
diseases. Our previous studies suggested that mitochondria might be involved in the initiation of
oxidative stress in asbestos-exposed mammalian cells.
oBjective: We investigated whether mitochondria are a potential cytoplasmic target of asbestos
using a mitochondrial DNA–depleted (ρ0) human small airway epithelial (SAE) cell model: ρ0 SAE
cells lack the capacity to produce mitochondrial ROS.
Methods: We examined nuclear DNA damage, micronuclei (MN), intracellular ROS produc-
tion, and the expression of inflammation-related nuclear genes in both parental and ρ0 SAE cells in
response to asbestos treatment.
results: Asbestos induced a dose-dependent increase in nuclear DNA oxidative damage and MN
in SAE cells. Furthermore, there was a significant increase in intracellular oxidant production and
activation of genes involved in nuclear factor κB and proinflammatory signaling pathways in SAE
cells. In contrast, the effects of asbestos were minimal in ρ0 SAE cells.
conclusions: Mitochondria are a major cytoplasmic target of asbestos. Asbestos may initiate
mitochondria-associated ROS, which mediate asbestos-induced nuclear mutagenic events and
inflammatory signaling pathways in exposed cells. These data provide new insights into the molecu-
lar mechanisms of asbestos-induced genotoxicity.
key words: asbestos, DNA oxidative damage, genotoxicity, mitochondria, oxidative stress–
responsive signaling pathways, reactive oxygen species. Environ Health Perspect 120:840–847
(2012). http://dx.doi.org/10.1289/ehp.1104287 [Online 7 March 2012]

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841
Mitochondrial-DNA (mtDNA)–depleted
SAE cell generation. The mtDNA–depleted
(ρ0) SAE cell line was generated from the
parental human SAE cells by ethidium bro-
mide (EtBr) treatment, which is a standard
method for generating ρ0 SAE cell lines
from human cells (King and Attardi 1989).
Cells were treated with 50 ng/mL EtBr for
2 months in ρ0 medium: complete small
airway growth medium containing uridine
(50 µg/mL), sodium pyruvate (1 mM),
HEPES (20 mM), and glucose (4.5 g/L; all
chemicals from Sigma). After this treatment,
cells were maintained in ρ0 medium with-
out EtBr. The ρ0 SAE cells generate energy
through glycolysis using uridine and pyru-
vate supplements in the ρ0 media (King and
Attardi 1989). The ρ0 status was verified by
measuring mtDNA content and four mito-
chondrial functional markers: mitochondrial
membrane potential, oxygen consumption
rate, cytochrome c oxidase (COX) activity,
and intracellular superoxide content [see
Supplemental Material, pp. 3–4 (http://
dx.doi.org/10.1289/ehp.1104287)].
Immunofluorescence for 8-hydroxy-
deoxyguanosine (8-OHdG) detection. We deter-
mined oxidative DNA damage by measuring
8-OHdG levels using monoclonal antibody 1F7
(a gift from R. Santella, Columbia University).
After 48 hr of treatment, control and treated
cells were fixed and permeabilized following
the protocol described previously (Partridge
et al. 2007). Cells were incubated with 1F7
at 1:20 dilution for 1.5 hr at 37°C, followed
by incubation with Alexa Fluor 488 goat anti-
mouse IgG [Invitrogen (Life Technologies,
Grand Island, NY)] for 30 min and propidium
iodide (PI; BD Biosciences Pharmingen, San
Diego, CA) for 10 min. Samples were visual-
ized and images were captured on a confocal
microscope (Nikon Eclipse TE2000-U; Nikon
Corporation, Tokyo, Japan). The mean ± SD
green fluores cence intensity per cell was
obtained from approximately 200 cells per sam-
ple using Image-Pro Plus, version 6.0 (Media
Cybernetics Inc., Bethesda, MD).
Micronuclei (MN) induction. Chromosomal
damage of nuclear DNA was examined by
assaying the frequency of MN (Fenech 2000).
Control and asbestos-treated cells were incu-
bated with 0.5 ng/µL cytochal asin B for 48 hr
and then fixed and permeabilized. Cellular
nuclei were stained with PI and cytoplasm
was counterstained with Alexa Fluor 488 phal-
loidin (Invitrogen). Samples were visualized
and images were captured with a fluorescence
microscope [Olympus Bh-2 equipped with
Olympus MicroSuite FIVE software (Olympus
America, Center Valley, PA)]. About 500
binucleated cells per sample were examined,
and cells with MN were scored manually. The
percentage of cells containing MN was calcu-
lated for each sample.
5´,6´-Chloromethyl-2´,7´dichloro dihydro-
fluorescein diacetate (CM-H2DCFDA). The
intracellular oxidant level was determined by
CM-H2DCFDA (Invitrogen), a nonfluores-
cent probe that can be converted by oxidants
into a green fluorescent product, CM-DCF
(5´,6´-chloromethyl-2´,7´dichlorofluorescein).
Parental and ρ0 SAE cells were treated with
0.5 µg/cm2 chrysotile for 24 hr. H2O2-
exposed cells served as positive controls, and
DMSO was used as a potential radical scaven-
ger. Control and treated cells were labeled with
Cell Tracker Red (Invitrogen) followed by
15 µM CM-H2DCFDA treatment for 45 min.
Cells were immediately visualized and images
were captured on the Nikon confocal micro-
scope. All images were focused during brief
illumination with a laser beam and collected
with a single scan using a low laser power to
avoid photooxidation of CM-H2DCFDA,
which has been reported previously (Chen
et al. 2005). The green fluorescence intensity
was quantified by Image-Pro Plus and normal-
ized to the Cell Tracker staining to account
for the difference in cell thickness between
the parental and ρ0 SAE cells. The mean ± SD
fluorescence intensity per cell was determined
by measuring about 500 randomly selected
cells per sample per experiment.
5,5´,6,6´-Tetrachloro-1,1´,3,3´-tetra ethyl-
benzimi-dazolylcarbocyanine iodide (JC-1).
We used JC-1, a membrane potential–sen-
sitive fluorescent probe (Life Technologies),
to determine the mitochondrial membrane
potential. JC-1 accumulates as orange aggre-
gates in the mitochondria of cells with nor-
mal mitochondrial function and membrane
potential and as green monomers in the mito-
chondria of cells with impaired mitochondrial
function and membrane potential.
RNA purification and reverse- transcription
polymerase chain reaction (PCR). Exponentially
growing parental and ρ0 SAE cells were
treated with 1 µg/cm2 asbestos for 12, 24, or
48 hr. Total cellular RNA was purified with
the Ambion® mirVana™ miRNA Isolation
Kits (Life Technologies). RNA concentra-
tion was measured with a NanoDrop 3300
fluoro spectrometer (Thermo Fisher Scientific,
Waltham, MA), and RNA quality was checked
with an Agilent microfluidic RNA 6000 Nano
Chip kit on the 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA). cDNA was
generated by reverse transcription using High
Capacity cDNA Reverse Transcription Kits
[Applied Biosystems (Life Technologies)]. The
reaction was carried out in 60 µL 1× buffer con-
taining 10 ng/µL RNA template. The reverse-
transcription- PCR conditions were 25°C for
10 min followed by 37°C for 120 min.
Gene expression by TaqMan low- density
array. The mRNA expression level of 93 inflam-
mation-related and 93 immune- responsive
genes (178 in total, with eight overlapping
genes) was assayed with Applied Biosystems’
TaqMan Gene Signature human immune and
inflammation arrays on the 7900HT Fast Real-
Time PCR System controlled by SDS soft-
ware, version 2.3 [Applied Biosystems (Life
Technologies)]. For each sample, a mixture of
TaqMan Gene Expression Master Mix and
cDNA equivalent to 250 ng total RNA was
loaded onto the array and subjected to the
amplification of 93 immune- or inflammation-
related genes plus three endogenous controls.
Statistical analysis. Gene expression results
were analyzed using RealTime StatMiner, ver-
sion 4.1 (Integromics Inc., Granada, Spain).
The relative quantity of each gene in the
asbestos-treated group compared with the
corresponding gene in the control group was
calculated for all samples using 18S rRNA
(ribosomal RNA) and GAPDH (glyceraldehyde
3-phosphate dehydrogenase) as endogenous
controls. Gene clustering using single linkage
and Pearson correlation was performed to visu-
alize differences in gene expression. Network
generation was carried out using Ingenuity
Pathway Analysis (IPA) software, version 6.0
(Ingenuity Systems Inc., Redwood, CA). The
correlation between asbestos doses and MN
yields was examined using the Pearson cor-
relation test. All other results were analyzed
using one-way analysis of variance followed by
Dunnett’s multiple comparison test. p-Values
< 0.05 were considered statistically significant.
Results
Characterization of parental and ρ0 SAE cells
and cellular toxicity of asbestos. Real-time PCR
results showed that after 2 months of 50 ng/mL
EtBr treatment to deplete mtDNA, the ratio of
mtDNA to nuclear DNA in ρ0 SAE cells was
0.2 ± 0.08% of the ratio in parental SAE cells
[see Supplemental Material, Table 1 (http://
dx.doi.org/10.1289/ehp.1104287)]. Because
mtDNA-encoded proteins are important
components of mitochondrial respiration, we
further examined mitochondrial functional
markers in ρ0 SAE cells. The ρ0 status was con-
firmed by a loss of mitochondrial membrane
potential gradient (Figure 1A), significantly
decreased COX activity (Figure 1B), and oxy-
gen consumption rate (Figure 1C), as well as
a decrease in intracellular superoxide content
(Figure 1D). All the experiments in the present
study used ρ0 SAE cells within five passages to
guarantee a true ρ0 status (see Supplemental
Material, Table 1). For details regarding cell
and mitochondria morphology, growth curves,
and results of asbestos toxicity assays for both
cell lines, see Supplemental Material, Figure 1.
Asbestos fibers induced nuclear DNA oxi-
dative damage. Initially, we examined the role
of mitochondria in asbestos-induced oxida-
tive damage of the nuclear genome. To do
this, both parental and ρ0 SAE cells were
subjected to various doses of chrysotile and

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Huang et al.
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volume 120 | number 6 | June 2012 • Environmental Health Perspectives
crocidolite exposure for 48 hr. The forma-
tion of 8-OHdG, a DNA lesion known to
form upon oxidative damage, was measured
by immunofluorescence staining. Importantly,
the 8-OHdG staining colocalized with nucleus
staining by PI (data not shown). A higher base-
line 8-OHdG level was observed in untreated
ρ0 SAE cells (20.96 ± 7.72; Figure 2B,D) com-
pared with the parental SAE cells (7.45 ± 2.05;
Figure 2A,C). However, asbestos-induced
8-OHdG levels were much lower in ρ0 SAE
cells (Figure 2D) than in parental SAE cells
(Figure 2C; p < 0.01, Dunnett’s test). In addi-
tion, at a dose of 2 µg/cm2, chrysotile induced
a higher level of 8-OHdG than crocidolite
in both parental and ρ0 SAE cells (p < 0.05,
Dunnett’s test; Figure 2C,D).
Asbestos fibers induced chromosomal
breaks. Asbestos is a well-established chro-
mosomal mutagen that induces multilocus
deletions and MN (Jaurand 1997). We deter-
mined the asbestos-induced MN yield in
both parental and ρ0 SAE cells to confirm
the role of mitochondria in asbestos- mediated
chromosomal damage. MN were scored on
binucleated ρ0 or parental SAE cells after
48 hr of asbestos exposure (Fenech 2000).
Representative photo micrographs indicate
MN formation in 2 µg/cm2 chrysotile– treated
SAE cells (Figure 2F) but not untreated
controls (Figure 2E). The results show that
ρ0 SAE cells had significantly higher back-
ground MN (9.1 ± 2.34%) than did parental
SAE cells (4.9 ± 1.14%; p < 0.05, Dunnett’s
test). Asbestos treatment induced significant
increases in MN yield in parental SAE cells
(Figure 2G). The frequency of MN-positive
SAE cells increased in a dose-dependent man-
ner, from 6.7 ± 1.9% after 0.5 µg/cm2 to
17.6 ± 4.3% after 4 µg/cm2 chrysotile expo-
sure (r = 0.89, Pearson correlation), and from
1.7 ± 0.5% after 0.5 µg/cm2 to 5 ± 1.2% after
4 µg/cm2 crocidolite exposure (r = 0.9, Pearson
correlation). Although chrysotile induced a
slight dose-dependent (r = 0.97, Pearson cor-
relation) increase in MN yield among ρ0 SAE
cells (range, 1.2 ± 0.4% to 7.6 ± 0.2%), MN
yield was not affected by crocidolite treatment
at any dose examined (Figure 2G). These
results show that mitochondria-dysfunctional
ρ0 SAE cells were significantly less sensitive to
chrysotile- and crocidolite-induced MN for-
mation than were parental SAE cells (p < 0.01,
parental vs. ρ0 SAE cells for each of the doses
examined, Dunnett’s test). Chrysotile fibers
were generally more potent than crocidolite
in inducing MN in both cell lines (p < 0.01,
chrysotile vs. crocidolite for each of the doses
examined, Dunnett’s test).
Increase of intracellular oxidants in asbestos-
treated SAE cells. To verify the contribution
of mitochondria-derived ROS to asbestos-
induced nuclear oxidative damage and lesions,
we compared asbestos-induced intracellular
oxidant production in ρ0 versus parental SAE
cells. The overall ROS level was determined
using CM-H2DCFDA, a cellular membrane–
permeable non fluorescent probe that can be
irreversibly oxidized by intra cellular ROS into
a green fluorescent product, CM-DCF. In SAE
cells, 24-hr asbestos treatment tripled CM-DCF
fluorescence level over background (p < 0.05,
Dunnett’s test; Figure 3A,C). Pre treating and
co-treating cells with the radical scavenger
DMSO prevented induction of oxyradicals
by asbestos, as indicated by the decrease in
average fluorescence per cell (Figure 3A,C;
p < 0.05, Dunnett’s test). In contrast, although
the baseline ROS level was higher in ρ0 SAE
cells than in parental SAE cells, treatment
with asbestos induced little or no increase in
intracellular oxidants (Figure 3B,D). DMSO
treatment slightly reduced the fluorescence
level in asbestos- exposed ρ0 SAE cells with
Figure 1. Verification of ρ0 status. (A) ρ0 SAE cells showed a loss of mitochondrial membrane potential as indicated by JC-1 staining. JC-1 formed orange aggre-
gates in parental SAE cells but remained as green monomers in ρ0 SAE cells. (B) Compared with parental cells, ρ0 SAE cells showed a significant decrease in
COX activity [an enzyme complex (IV) encoded jointly by mtDNA and nuclear DNA] but showed no change in succinate dehydrogenase activity (an enzyme com-
plex encoded entirely by nuclear DNA). (C) ρ0 SAE cells have a significantly reduced oxygen consumption rate compared with the parental cells. (D) Intracellular
superoxide content is indicated by dihydro ethidium (DHE) fluorescence. ρ0 SAE cells (3) showed a decreased level compared with parental SAE cells (4). Each of
the experiments shown in A–D was repeated three times.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
20
15
10
5
0
550
450
350
250
150
Parental
ρ0
Parental
3. ρ0
4. parental
ρ0 SAE cellsParental SAE cells
Parental
ρ0
ρ0
Femtomole/cell/min
(% of control)
Oxygen consumption
Cytochrome c oxidaseSucccinate dehydrogenase
Intracellular superoxide content
1. & 2. Non-stained parental and ρ0 cells
Oxidized cytochrome c
(nmol/min/mg protein)
Oxidized succinate
(nmol/min/mg protein)
No. of cells
Fluorescent intensity (DHE)SAE cells
SAE cellsSAE cells
100
101
102
103
104

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Mitochondrial oxidants and genotoxicity of asbestos
Environmental Health Perspectives • volume 120 | number 6 | June 2012
843
no statistical significance (Figure 3B,D).
Notably, H2O2 induced an abundant increase
of intracellular ROS in both parental and ρ0
SAE cells (Figure 3). This clearly indicates that
H2O2 induced ROS via a non- mitochondrial–
related mechanism, in contrast with asbestos.
Gene expression and clustering analysis of
all genes examined. The above experiments
indicated that mitochondria-derived ROS
were a major factor mediating direct nuclear
damage in asbestos-treated SAE cells. Next, we
wanted to determine whether mitochondria-
derived ROS could also induce relevant nuclear
signaling pathways. We analyzed the mRNA
expression of 178 genes [see Supplemental
Material, Table 2 (http://dx.doi.org/10.1289/
ehp.1104287)] involved in immune and inflam-
mation pathways by TaqMan low-density
arrays. The expression of these genes was exam-
ined at 12, 24, and 48 hr in four experimental
groups: a) untreated SAE cells, b) 1-µg/cm2
asbestos-treated SAE cells, c) untreated ρ0 SAE
cells, and d) 1-µg/cm2 asbestos-treated ρ0 SAE
cells. Gene clustering analysis was used to com-
pare the global gene expression patterns among
different samples. It was performed on raw data
from three independent biological repetitions.
Figure 2. ρ0 SAE cells were significantly less sensitive than parental SAE cells to asbestos-induced 8-OHdG and MN formation. (A–D) Detection of 8-OHdG by
immuno fluorescence. (A,B) Representative images from untreated and treated (0.5 and 2 µg/cm2 for 48 hr; H2O2 treatment, 100 µM for 30 min) samples: parental
cells (A) and ρ0 SAE cells (B). (C,D) Results of chrysotile and crocidolite treatments plotted with a horizontal jitter; each dot represents a single cell quantified.
The mean ± SD fluorescence intensity per cell shown at the top was obtained from approximately 200 cells per sample. H2O2-treated group, 35.8 ± 6.52 (not plot-
ted here). (E,F) Untreated (E) and 2-µg/cm2 chrysotile–treated (F) SAE cells. Arrows indicate MN. (G) Asbestos-induced MN-positive cells, background subtracted
(mean ± SD). Pooled data were obtained from three independent experiments. Chrysotile (left) induced MN in both parental and ρ0 SAE cells in a dose-dependent
manner (r = 0.89 and 0.9, respectively, Pearson correlation). Crocidolite (right) induced a dose-dependent increase (r = 0.97, Pearson correlation) in the frequency
of MN-positive cells in parental cells but had no effect on ρ0 SAE cells.
*p < 0.05, and **p < 0.01, Dunnett’s test compared with untreated SAE cells. #p < 0.05, and # #p < 0.01, Dunnett’s test compared with untreated ρ0 SAE cells.
Untreated
H2O2 (100 µM)Crocidolite (0.5 µg/cm2) Crocidolite (2 µg/cm2)
Chrysotile (0.5 µg/cm2)Chrysotile (2 µg/cm2)
H2O2 (100 µM)Crocidolite (0.5 µg/cm2) Crocidolite (2 µg/cm2)
Chrysotile (0.5 µg/cm2)Chrysotile (2 µg/cm2)Untreated
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
Average fluorescnce/cell
(arbitrary unit)
Average fluorescnce/cell
(arbitrary unit)
7.5 ± 2.1
21 ± 7.721 ± 7.721.8 ± 6.825.9 ± 6.920.5 ± 5.6 35.1 ± 9.1##
7.5 ± 2.129.5 ± 5.6**
32.3 ± 6.8**
37.5 ± 8.5**
44.4 ± 7.9**
Control0.5
Chrysotile (µg/cm2)Crocidolite (µg/cm2)
Crocidolite (µg/cm2)
220.5Control
Control0.5
Chrysotile (µg/cm2)
220.5Control
Untreated
Asbestos treated
25
20
15
10
5
0
7
5
3
1
–1
0.51240.5124
Background subtracted
Parental SAE cells – background = 4.9% ± 1.14%
ρ0 SAE cells – background = 9.1% ± 2.34%
*
**
**
**
*
*
#
Chrysotile (µg/cm2)
Induced MN-positive cells/
binucleated cells (%)
Crocidolite (µg/cm2)
Parental SAE cells
ρ0 SAE cells

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The result shows that SAE cells clustered by
treatment, an indication that asbestos had a
substantial effect on gene expression (Figure 4A,
right). In contrast, asbestos treatment did not
affect gene expression in ρ0 SAE cells to the
same extent, and samples are poorly clustered
(Figure 4A, left).
Differentially expressed genes in asbestos-
treated groups versus controls. Genes differ-
entially expressed (significantly induced or
suppressed by asbestos, paired t-test, p < 0.05)
in asbestos-treated groups versus untreated
controls were identified for parental and ρ0
SAE cells, respectively. The relative quantity
for each of the differentially expressed genes
at 12, 24, and 48 hr is plotted in Figure 4B. A
detailed list of all of the differentially expressed
genes in SAE cells is provided in Supplemental
Material, Table 3 (http://dx.doi.org/10.1289/
ehp.1104287). Asbestos induced a time-
dependent increase in the expression of a
subset of immune- and inflammation-related
genes in SAE cells (Figure 4B, left). Although
12-hr treatment affected the expression of only
a few genes, with 24- and 48-hr treatments,
the expression of 26 and 32 genes, respec-
tively, was significantly altered (mostly up-
regulated; paired t-test, p < 0.05) by asbestos.
About half the genes differentially expressed at
24 hr were consistently up-regulated at 48 hr
in asbestos-treated SAE cells (Figure 4B, left;
see also Supplemental Material, Table 3).
In contrast, asbestos had little effect on gene
expression in ρ0 SAE cells (Figure 4B, right).
Six up-regulated genes (paired t-test, p < 0.05)
were identified at 48 hr, but the fold changes
were all < 2 except for MC2R (melanocortin 2
receptor; ratio, 5.66) and CCR4 (chemokine
receptor 4; ratio, 3.24; Figure 4B, right; see
also Supplemental Material, Table 4).
It should be noted that the baseline expres-
sion of some genes in ρ0 SAE cells differed
from that in parental SAE cells [Supplemental
Material, Table 5 (http://dx.doi.org/10.1289/
ehp.1104287)]. Because the background intra-
cellular oxidant level is increased in ρ0 SAE
cells (Figure 3B,D), it is necessary to examine
whether the baseline gene expression pattern
in untreated ρ0 SAE cells is similar to that
in asbestos-treated SAE cells where oxidative
stress is induced (Figure 3A,C). Importantly,
our data did not reveal such a similarity. This
suggests that the failure of asbestos to induce
any big change in gene expression in ρ0 SAE
cells was not due to the pre activation of these
genes during the process of ρ0 SAE cell genera-
tion, but was most likely a consequence of the
lack of mtDNA and mitochondrial function.
Network analysis. We observed significant
induction of 27 genes and down-regulation
Figure 3. Asbestos-induced intracellular ROS in parental but not ρ0 SAE cells. (A,B) Representative photomicrographs of parental (A) and ρ0 SAE cells (B):
Untreated, controls; asbestos treated, 0.5 µg/cm2 chrysotile for 24 hr; asbestos + DMSO, 0.5% DMSO for 24 hr, followed by 0.5 µg/cm2 chrysotile plus 0.5% DMSO for
24 hr; H2O2, 0.2 mM H2O2 for 30 min. CM-DCF fluorescence (green) indicates intracellular ROS level; red indicates Cell Tracker Red counterstaining. (C,D) Relative
quantification of CM-DCF fluorescence: parental (C) and ρ0 SAE cells (D). The green fluorescence was normalized to the Cell Tracker staining to account for the dif-
ference in cell thickness between the parental and ρ0 SAE cells. The mean ± SD fluorescence per cell was obtained from three independent experiments.
*p < 0.05; Dunnett’s test compared with untreated SAE cells; #p < 0.05, Dunnett’s test compared with asbestos-treated SAE cells.
Untreated
Parental SAE cells
ρ0 SAE cells
Asbestos treatedAsbestos + DMSO
Asbestos treatedAsbestos + DMSO
H2O2
UntreatedH2O2
35
30
25
20
15
10
5
0
35
30
25
20
15
10
5
0
3.43
23.72
10.25
*
#
4.02
Untreated
Untreated
AsbestosH2O2
Asbestos
+ DMSO
5.53
6.80
14.67
4.95
AsbestosH2O2
Asbestos
+ DMSO
Average fluorescence/cell
(arbitrary unit)
Average fluorescence/cell
(arbitrary unit)

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Mitochondrial oxidants and genotoxicity of asbestos
Environmental Health Perspectives • volume 120 | number 6 | June 2012
845
of 5 genes in SAE cells 48 hr after asbestos
treatment [Figure 4B; see also Supplemental
Material, Table 3 (http://dx.doi.org/10.1289/
ehp.1104287)]. Gene ontology–related path-
way analysis using IPA software showed that
these 32 genes were significantly associated
with induction of oxidative stress, xenobiotic
metabolic processes, pro apoptosis, and other
processes (Supplemental Material, Table 6).
Furthermore, a group of highly inter connected
networks of the 32 asbestos- responsive
genes in SAE cells was constructed by IPA
(Figure 5), based on direct interactions stored
in the Ingenuity Knowledge Base (Ingenuity
Systems Inc., Redwood City, CA) a collec-
tion of experimentally confirmed relationships
between molecules. The networks demonstrate
that the 32 genes were directly related to acti-
vation of transcription factors nuclear factor
κB (NF-κB) and activator protein-1 (AP-1)
signaling pathways, and to downstream sig-
naling including various interleukins (ILs),
tumor necrosis factor (TNF) and TNF recep-
tor (TNFR), colony stimulating factors 1–3
(CSFs), and other pro- inflammatory cytokines
or growth factors (Figure 5). These signaling
pathways are known to be oxidative stress–
responsive and have been consistently reported
to be activated by asbestos exposure (Manning
et al. 2002; Martindale and Holbrook 2002).
Network analysis of significant genes from the
24-hr group of SAE cells show similar results
(data not shown). By contrast, IPA revealed
that the 12 genes differentially expressed in
ρ0 SAE cells at 48 hr after asbestos treatment
(Figure 4; see also Supplemental Material,
Table 4) were associated with an inhibition of
mitogen- activated protein kinase, AP-1/JUN,
and TNF signaling cascades (Supplemental
Material, Figure 2).
Discussion
The biological effects of oxidative stress in
many disease states manifest as both direct
damage of macromolecules and modification
of redox signaling pathways in target cells
(Valko et al. 2007). In the present study, both
effects were detected in parental human SAE
cells, whereas minimal effects were observed
in mitochondria- dysfunctional ρ0 human
SAE cells. These results collectively provide
direct evidence that mitochondria may play
an essential role in regulating asbestos- induced
genotoxicity and cell signaling alterations.
These processes are most likely mediated
through mitochondria- originated ROS. To
our knowledge, this is the first report of the
direct involvement of mitochondria in asbes-
tos genotoxicity and pathogenicity in relevant
human target cells.
Consistent with our previous findings on
the role of oxyradicals and extranuclear targets
in fiber mutagenesis in AL cells (Xu et al. 1999,
2002, 2007), the present study found asbestos
fiber treatment of mitochondria- dysfunctional
ρ0 human SAE cells, which consume 10-fold
less oxygen than parental SAE cells, caused sig-
nificantly lower intracellular oxidant produc-
tion, nuclear oxidative damage (i.e., 8-OHdG),
and MN induction than observed in asbestos-
treated parental SAE cells. These data strongly
suggest that mitochondria- associated oxidants
are a main contributor to asbestos-induced
intracellular oxidative stress, and that reduc-
tions in asbestos-induced oxidative and chro-
mosomal damage in ρ0 SAE cells compared
with parental SAE cells were due to the lack of
mitochondria-derived ROS. The oxidation of
Figure 4. Asbestos treatment induced an increase in the expression of immune- and inflammation-related genes in parental but not ρ0 SAE cells. (A) Heatmap
depicting the expressions of all genes measured by low-density array. The gene expression levels for each sample were mean centered and hierarchically clus-
tered according to similarity in overall gene expression pattern. The plotted data represent results from three independent biological repetitions. The color scale
bar indicates the fold decrease (green) or increase (red) in gene expression compared with the mean expression level (black) of all genes examined. (B) A list
of genes that contributed to the clustering in A, that is, genes significantly induced or suppressed by asbestos (differentially expressed in response to 1-µg/cm2
asbestos treatment). Columns represent the relative quantity of asbestos-induced differentially expressed genes at 12, 24, and 48 hr for both parental (left) and
ρ0 (right) SAE cells. Genes consistently differentially expressed across time points appear in the same colors. White (parental cells) and black (ρ0 SAE cells) col-
umns indicate genes that were differentially expressed at one time point only. Data were obtained from three biological repetitions. A detailed list of all differen-
tially expressed genes is available in Supplemental Material, Table 3 (SAE cells) and Table 4 (ρ0 SAE cells) (http://dx.doi.org/10.1289/ehp.1104287).
100
10
1
0.1
100
10
1
0.1
100
10
1
0.1
100
10
1
0.1
100
10
1
0.1
100
10
1
0.1
Hierarchal clustering dendritic trees
–3 +27.5
T10
ρ0 SAE cells
Parental SAE cells
ρ0 SAE cellsParental SAE cells
T20
T1 T2C20C10
C2 C1T30
T3C30
C3
T = asbestos treated;
C = untreated control;
“0” indicates ρ0 cells;
“1, 2, 3” indicates each of the three experimental repeats
ANXA3
BAX
CACNB4
CCR7
CD34CD40
CSF1CSF2
ECE1
EDN1
HMOX1
HRH1HRH2
ICAM1
IL1A
IL6
NFKB2
PTAFR
PTGER2
PTGFR
PTGIR
PTGS2
TBXAS1
TGFB1
TNF
VEGF
ADRB2
AGTR1
CES1
CSF2
EDN1
HMOX1
HRH1
ICAM1
LTA
PTAFR
RPL3L
TBXAS1
TFRC
VEGF
CSF1CSF2
EDN1
FAS
IL10
IL1A
PLCE1
PTGER2
ADRB2
ANXA5
BAX
BCL2
CACNB4
CCL2
CCR4
CD4
CSF1 CSF2CSF3
CYSLTR1
FAS
FN1
HLA-DRA
HMOX1
HRH1
ICAM1
IL1A
IL6 IL8
LTA
NFKB2 PLCD1
PLCE1
PTGFR
SELE
TBXA2R
TFRC
TNF
TNFRSF1B
VEGF
ANXA3
B2M
CACNB4
CSF1
HMOX1
PLCB4PLCG1
PTGER2
PTGIS
TFRC
TNFRSF1B
ADRB2
CACNA2D1
CCR4
CD68
CSF3
EDN1
HMOX1
HRH1
ITGB2
MAPK1
MC2R
PLCE1
Relative quantityRelative quantity
Relative quantityRelative quantity
Relative quantityRelative quantity

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Huang et al.
846
volume 120 | number 6 | June 2012 • Environmental Health Perspectives
CM-H2DCFDA by reactive radical species, as
detected using confocal microscopy, provided
strong evidence that asbestos induced ROS and
reactive nitrogen species that could be inhibited
by DMSO (Xu et al. 2007).
Reported human asbestos exposure levels
vary depending on the sources and types of
exposure (Dodson et al. 1997; Roggli et al.
2004). Asbestos concentrations used in this
study ranged from 0.5 to 4 µg/cm2, well below
the reported dose (10 µg/cm2) that can cause
overload, i.e., a retained burden of asbestos
fibers resulting from an impairment of the
asbestos clearance capacity, for cultured cells
(Faux et al. 2003; International Life Sciences
Institute 2000). Although crocidolite has
been established by numerous studies to be
more carcinogenic than chrysotile in humans
(Berman and Crump 2008; Hodgson and
Darnton 2000), in the present study chrysotile
was found to be more DNA damaging than
crocidolite fibers. Our results show that chryso-
tile, but not crocidolite, can induce a signifi-
cant increase in 8-OHdG and MN formation
in ρ0 SAE cells at 2 and 4 µg/cm2, respectively.
It is possible that the chemical and physical
properties of the material used in our study
were modified during fiber processing and
may vary from other batches. Furthermore,
our findings suggest that in addition to ROS
production, chrysotile might induce nuclear
mutation through other mechanisms.
Notably, baseline levels of 8-OHdG, MN,
and intracellular oxidants in ρ0 SAE cells were
higher than corresponding background lev-
els in parental SAE cells. MN may have been
increased because of EtBr treatment (McCann
et al. 1975), although the mutagenic effect of
EtBr in mammalian systems remains unclear
(National Toxicology Program 2005). The
need for extended EtBr treatment is a poten-
tial drawback of the ρ0 SAE cell model;
nevertheless, growth kinetics in ρ0 SAE cells
are similar to those in wild-type cells. Until
the ideal mitochondria- dysfunctional cell
model is generated, ρ0 SAE cells represent the
next best alternative model available. EtBr is
a known selective inhibitor of mitochondrial
transcription and replication processes that has
no effect on nuclear DNA replication at the
concentration used in the study (Leibowitz
1971; Seidel-Rogol and Shadel 2002). EtBr
has effectively been used to reduce the copy
number of mtDNA in proliferating cells (King
and Attardi 1996). The effect of EtBr is revers-
ible, so that once EtBr is removed from the cul-
ture medium, cells are able to repopulate their
mtDNA (King and Attardi 1989). As such,
the present study used ρ0 SAE cells within
five passages to guarantee a true ρ0 status. The
oxidants in untreated ρ0 SAE cells may have
been derived from sources other than the mito-
chondrial respiration chain because the baseline
intracellular superoxide content in ρ0 SAE cells
was low compared with parental SAE cells.
This unidentified source of oxidants may also
have contributed to the substantially increased
background levels of 8-OHdG in ρ0 SAE cells.
It is possible that in ρ0 SAE cells the DNA
repair capacity is decreased or the antioxidant
enzymes are impaired because similar results
were observed in the nucleus of ρ0 HeLa cells
(Delsite et al. 2003). In the near future, we will
try to use genetic tools to block mitochondrial
ROS production and identify the other sources
of ROS production in ρ0 SAE cells.
Using gene arrays and pathway analyses,
we showed that mitochondria-associated oxi-
dants were able to mediate redox signaling in
response to asbestos exposure. Chrysotile asbes-
tos induced a significant increase in the expres-
sion of immune- and inflammation- related
genes in normal SAE cells but had mini mal
effect on gene expression in ρ0 SAE cells in
terms of both the number of genes affected
and the magnitude of up- or down- regulation
observed in affected genes. Asbestos-responsive
genes in parental SAE cells were significantly
associated with activation of cellular signal-
ing pathways, including NF-κB, AP-1, and
pro- inflammatory cytokines, chemokines, and
growth factors. These data provide additional
support for our hypothesis that mtDNA and
mitochondria-derived ROS are important in
mediating asbestos-induced nuclear DNA and
signaling alterations. Future gene knock-down
studies in parental SAE cells or gene over-
expression in ρ0 SAE cells may further illumi-
nate the role of these signaling pathways in the
asbestos-induced cellular response.
Conclusion
The link between asbestos exposure and the
development of lung cancer and mesothe-
lioma in humans is unequivocal, but the
underlying carcinogenic mechanism is not
Figure 5. Network of genes mediated by asbestos after 48 hr of treatment in SAE cells. The color scale indi-
cates the fold change in gene expression level for the 32 genes: green, down-regulation; red, up-regulation;
uncolored molecules, expression levels were not examined in the present study.
Extracellular space
Plasma membrane
Cytoplasm
Nucleus
Fold change: – 3+ 702
Transmembrane receptor
Group/complex
Kinase
Enzyme
Transcription regulator
Transporter
Growth factor
G-protein coupled receptor
Cytokine
Ligand-dependent nuclear receptor
Iron channel
Relationship
Others

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Mitochondrial oxidants and genotoxicity of asbestos
Environmental Health Perspectives • volume 120 | number 6 | June 2012
847
known. Using newly generated mtDNA-de-
ficient (ρ0) human SAE cells, we showed that
mitochondria are a major cytoplasmic target
of asbestos. Our earlier studies using asbestos
fiber–treated cytoplasts and fusion with non-
treated karyoplasts provided circumstantial
evidence of the role of mitochondria in fiber
genotoxicity (Xu et al. 2007). Results of the
present study expand on our earlier findings
and suggest that mitochondria-associated
oxidants mediate asbestos-associated nuclear
damage and initiate redox-responsive signal-
ing cascades in target cells. These data provide
new insights into the molecular mechanisms
of asbestos-induced genotoxic responses and
provide a base for better prevention and treat-
ment of fiber-mediated diseases.
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