Asbestos surface provides a niche for oxidative modification.
ABSTRACT Asbestos is a potent carcinogen associated with increased risks of malignant mesothelioma and lung cancer in humans. Although the mechanism of carcinogenesis remains elusive, the physicochemical characteristics of asbestos play a role in the progression of asbestos-induced diseases. Among these characteristics, a high capacity to adsorb and accommodate biomolecules on its abundant surface area has been linked to cellular and genetic toxicity. Several previous studies identified asbestos-interacting proteins. Here, with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry, we systematically identified proteins from various lysates that adsorbed to the surface of commercially used asbestos and classified them into the following groups: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. The surfaces of crocidolite and amosite, two iron-rich types of asbestos, caused more protein scissions and oxidative modifications than that of chrysotile by in situ-generated 4-hydroxy-2-nonenal. In contrast, we confirmed the intense hemolytic activity of chrysotile and found that hemoglobin attached to chrysotile, but not silica, can work as a catalyst to induce oxidative DNA damage. This process generates 8-hydroxy-2'-deoxyguanosine and thus corroborates the involvement of iron in the carcinogenicity of chrysotile. This evidence demonstrates that all three types of asbestos adsorb DNA and specific proteins, providing a niche for oxidative modification via catalytic iron. Therefore, considering the affinity of asbestos for histones/DNA and the internalization of asbestos into mesothelial cells, our results suggest a novel hypothetical mechanism causing genetic alterations during asbestos-induced carcinogenesis.
- SourceAvailable from: Shinya Toyokuni[Show abstract] [Hide abstract]
ABSTRACT: Asbestos was used worldwide in huge quantities in the past century. However, because of the unexpected carcinogenicity to mesothelial cells with an extremely long incubation period, many countries face this long-lasting social problem. Mesothelioma is often diagnosed in an advanced stage, for which no effective therapeutic protocols are yet established. We previously reported based on animal experiments that the major pathology in asbestos-induced mesothelial carcinogenesis is local iron overload. Here we undertook to find an effective strategy to prevent, delay or lower the malignant potential of mesothelioma during asbestos-induced carcinogenesis. We used intraperitoneal injections of crocidolite to rats. We performed a 16-week study to seek the maximal tolerated intervention for iron reduction via oral deferasirox administration or intensive phlebotomy. Splenic iron deposition was significantly decreased with either method, and we found that Perls' iron staining in spleen is a good indicator for iron reduction. We injected a total of 10 mg crocidolite at the age of 6 weeks, and the preventive measures were via repeated oral administration of 25-50 mg/kg/day deferasirox or weekly to bimonthly phlebotomy of 4-10 ml/kg/day. The animals were observed till 110 weeks. Deferasirox administration significantly increased the fraction of less malignant epithelioid subtype. Although we found a slightly prolonged survival in deferasirox-treated female rats, larger sample size and refinement of the current protocol are necessary to deduce the cancer-preventive effects of deferasirox. Still, our results suggest deferasirox serves as a potential preventive strategy in people already exposed to asbestos via iron reduction.Cancer Prevention Research 09/2013; · 4.89 Impact Factor
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ABSTRACT: Malignant mesothelioma is strongly associated with asbestos exposure. Among asbestos fibers, crocidolite is considered the most and chrysotile the least oncogenic. Chrysotile accounts for more than 90% of the asbestos used worldwide, but its capacity to induce malignant mesothelioma is still debated. We found that chrysotile and crocidolite exposures have similar effects on human mesothelial cells. Morphological and molecular alterations suggestive of epithelial-mesenchymal transition, such as E-cadherin down-regulation and β-catenin phosphorylation followed by nuclear translocation, were induced by both chrysotile and crocidolite. Gene expression profiling revealed high-mobility group box-1 protein (HMGB1) as a key regulator of the transcriptional alterations induced by both types of asbestos. Crocidolite and chrysotile induced differential expression of 438 out of 28,869 genes interrogated by oligonucleotide microarrays. Out of these 438 genes, 57 were associated with inflammatory and immune response and cancer, and 14 were HMGB1 targeted genes. Crocidolite-induced gene alterations were sustained, whereas chrysotile-induced gene alterations returned to background levels within 5 weeks. Similarly, HMGB1 release in vivo progressively increased for 10 or more weeks after crocidolite exposure, but returned to background levels within 8 weeks after chrysotile exposure. Continuous administration of chrysotile was required for sustained high serum levels of HMGB1. These data support the hypothesis that differences in biopersistence influence the biological activities of these two asbestos fibers.American Journal Of Pathology 11/2013; 183(5):1654-66. · 4.60 Impact Factor
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ABSTRACT: Few people expected that asbestos, a fibrous mineral, would be carcinogenic to humans. In fact, asbestos is a definite carcinogen in humans, causing a rare but aggressive cancer called malignant mesothelioma (MM). Mesothelial cells line the three somatic cavities and thus do not face the outer surface, but reduce the friction among numerous moving organs. MM has several characteristics: extremely long incubation period of 30-40 years after asbestos exposure, difficulty in clinical diagnosis at an early stage, and poor prognosis even under the current multimodal therapies. In Japan, 'Kubota shock' attracted considerable social attention in 2005 for asbestos-induced mesothelioma and, thereafter, the government enacted a law to provide the people suffering from MM a financial allowance. Several lines of recent evidence suggest that the major pathology associated with asbestos-induced MM is local iron overload, associated with asbestos exposure. Preclinical studies to prevent MM after asbestos exposure with iron reduction are in progress. In addition, novel target genes in mesothelial carcinogenesis have been discovered with recently recognized mesothelioma-prone families. Development of an effective preventive strategy is eagerly anticipated because of the long incubation period for MM.Redox report: communications in free radical research 11/2013; · 1.51 Impact Factor
Asbestos surface provides a niche for oxidative
Hirotaka Nagai,1,2Toshikazu Ishihara,1Wen-Hua Lee,2Hiroki Ohara,1Yasumasa Okazaki,1Katsuya Okawa3and
1Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya;2Department of Pathology and Biology of
Diseases, Kyoto University Graduate School of Medicine, Kyoto;3Biomolecular Characterization Unit, Horizontal Medical Research Organization, Kyoto
University Graduate School of Medicine, Kyoto, Japan
(Received May 13, 2011 ⁄ Revised August 22, 2011 ⁄ Accepted August 27, 2011 ⁄ Accepted manuscript online September 2, 2011 ⁄ Article first published online September 27, 2011)
Asbestos is a potent carcinogen associated with increased risks of
malignant mesothelioma and lung cancer in humans. Although the
mechanism of carcinogenesis remains elusive, the physicochemical
characteristics of asbestos play a role in the progression of asbes-
tos-induced diseases. Among these characteristics, a high capacity
to adsorb and accommodate biomolecules on its abundant surface
area has been linked to cellular and genetic toxicity. Several previ-
ous studies identified asbestos-interacting proteins. Here, with the
use of matrix-assisted laser desorption ionization-time of flight
mass spectrometry, we systematically identified proteins from var-
ious lysates that adsorbed to the surface of commercially used
asbestos and classified them into the following groups: chroma-
tin⁄⁄nucleotide⁄⁄RNA-binding proteins, ribosomal proteins, cytopro-
tective proteins, cytoskeleton-associated proteins, histones and
hemoglobin. The surfaces of crocidolite and amosite, two iron-rich
types of asbestos, caused more protein scissions and oxidative
modifications than that of chrysotile by in situ-generated 4-
hydroxy-2-nonenal. In contrast, we confirmed the intense hemo-
lytic activity of chrysotile and found that hemoglobin attached to
chrysotile, but not silica, can work as a catalyst to induce oxidative
DNA damage. This process generates 8-hydroxy-2¢-deoxyguano-
sine and thus corroborates the involvement of iron in the carcino-
genicity of chrysotile. This evidence demonstrates that all three
types of asbestos adsorb DNA and specific proteins, providing a
niche for oxidative modification via catalytic iron. Therefore, con-
sidering the affinity of asbestos for histones⁄⁄DNA and the inter-
nalization of asbestos into mesothelial cells, our results suggest
a novel hypothetical mechanism causing genetic alterations
during asbestos-induced carcinogenesis. (Cancer Sci 2011; 102:
ity, heat resistance and low cost. However, it has become clear
that respiratory exposure to asbestos fibers, especially crocido-
lite and amosite, which have high biopersistence and contain
abundant iron, is associated with high risks of developing malig-
nant mesothelioma and lung cancer.(1–3)Many countries antici-
pate increased numbers of mesothelioma patients in the coming
decades because there is an extremely long incubation period
(30–40 years) for this fatal disease following asbestos expo-
The molecular mechanism of asbestos-induced carcinogenesis
remains elusive,(5)but both mesothelial cell injury and persistent
macrophage activation are thought to be essential, if not suffi-
cient, for mesotheliomagenesis.(6)These two events interact
in vivo, leading to genetic mutations, chromosomal aberrations
and aneuploidy in mesothelial cells. At least four major hypothe-
sesrelated tothe underlyingmechanisms have been proposed.(6,7)
First, the free radical theory postulates that DNA is injured by
reactive oxygen species generated through a foreign body
sbestos is a natural fibrous mineral that was heavily used
in industry during the past century because of its durabil-
reaction or catalytic action of the asbestos surface.(8–12)Asbes-
tos fibers of a large size, especially those that are quite long
(>15–20 lm), interrupt macrophage phagocytosis and prohibit
them from clearing fibers.(13)In this situation, activated macro-
phages release cytokines and oxidants, thereby inducing chronic
inflammation.(14)Even in the absence of activated phagocytes,
asbestos can produce free radicals via the Fenton reaction
because some types of amphibole asbestos, for example, crocid-
olite and amosite, include iron as an integral component of their
chemical structure and other types of asbestos contain iron as a
surface impurity.(8,15)Iron is the most abundant heavy metal in
the human body, but excess iron can work as a catalyst for the
generation of free radicals, leading to carcinogenesis.(16,17)In
experiments involving mammalian cells, asbestos fibers pro-
duced 8-hydroxy-2¢-deoxyguanosine (8-OHdG),(18–21)which is
a common oxidative modification of DNA involved in mutagen-
esis, carcinogenesis and aging.(22–24)Asbestos fibers also induce
clastogenic events in a free radical-dependent manner.(25)
Second, the mitotic disturbance theory proposes that asbestos
fibers physically interact with chromosomes directly and⁄or via
mitotic spindles, thereby inducing chromosomal aberrations.(26–31)
This is indeed a specific event caused by fibrous particles and
might be involved in the early induction of chromosomal aberra-
tions observed in Syrian hamster cells exposed to asbestos.(31)
Third, the molecule adsorption theory suggests that adsorption
of various molecules on the surface of asbestos fibers causes the
accumulation of intrinsic or extrinsic carcinogenesis-associated
molecules and interferes with intracellular signaling pathways.(7)
Certain carcinogenic molecules, such as benzo(a)pyrene in ciga-
rette smoke, are known to have high affinity for asbestos and
have a cooperative mutagenic effect.(32–34)Various endogenous
molecules, such as vitronectin and tubulin, are also likely to
interact with asbestos, playing important roles in fiber internali-
zation(35,36)and mitotic disturbances,(37)respectively. The depo-
sition of molecules on asbestos has also attracted attention as a
mechanism underlying the formation of asbestos bodies, a pro-
cess that was extensively investigated in previous studies.(38–49)
These studies suggested the involvement of acid mucopolysac-
the development of asbestos bodies. Among these components,
the pathobiological contribution of iron should be carefully con-
sidered. Iron exists in and near asbestos bodies in various forms:
hemosiderin,(52)ferritin(39,51)and colloidal iron.(43)Governa
et al.(42)discussed the reactivity of iron inside asbestos bodies
and concluded that it can catalyze free radical formation in
reductive conditions in vitro. Considering that hemosiderin is
capable of inducing hydroxyl radicals under physiological
conditions,(53,54)iron-rich asbestos bodies might play a role in
oxidative tissue damage.
4To whom correspondence should be addressed.
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ª ª 2011 Japanese Cancer Association
Finally, the chronic inflammation theory suggests that persis-
tently activated macrophages contribute to the initiation as well
as the progression of carcinogenesis.(55,56)Particularly in meso-
theliomagenesis, chronic inflammation is associated with asbes-
tos-induced genotoxicity in mesothelial cells. Yang et al.(57)
showed that tumor necrosis factor-a (TNF-a), a cytokine persis-
tently released from macrophages during inflammation, inhibits
asbestos-induced mesothelial cell death and might increase the
likelihood of transformation.
All of the aforementioned theories are clearly associated with
each other. However, currently there is little information avail-
able on asbestos-interacting proteins. Therefore, in the present
study we used matrix-assisted laser desorption ionization-time
of flight mass spectrometry (MALDI-TOF⁄MS) to systemati-
cally identify proteins that adsorb to the surface of different
asbestos fibers, investigated the modifications of adsorptive mol-
ecules and evaluated their possible involvement in mesothelial
Materials and Methods
Full materials and methods are provided in Data S1.
Materials. Three types of asbestos (chrysotile, crocidolite and
amosite) were acquired from Union for International Cancer
Control (UICC; Geneva, Switzerland) and suspended in saline.
MeT5A cells were obtained from the American Type Culture
Collection (Manassas, VA, USA). Silica (powder, 0.014 lm)
was purchased from Sigma Aldrich (St. Louis, MO, USA) and a
protease inhibitor cocktail (complete mini) was purchased from
Roche diagnostics (Basel, Switzerland).
Collection and analysis of asbestos-interacting proteins. We
modified a previously reported method(37)to collect proteins
adsorbed to the surface of asbestos fibers. Briefly, lysates(58)of
various rat tissue including lung, liver, kidney, brain and tunica
vaginalis or MeT5A human mesothelial cells (200–400 lg)
were mixed with each fiber (250 lg), and the total volume was
adjusted to 1 mL with radio-immunoprecipitation assay (RIPA)
buffer. After >3 h of incubation at 4 or 37?C, the mixture was
centrifuged (20 000g) at 4?C for 5 min. The supernatant was
discarded and the pellet was washed three times with RIPA buf-
fer. After the final centrifugation, we carefully discarded the
supernatant, directly added SDS-PAGE sample buffer, and
heated the sample at 95?C for 10 min. The samples were centri-
fuged (20 000g) at 4?C for 2 min, and the supernatant was ana-
lyzed using SDS-PAGE. We used a silver staining kit to stain
the SDS-PAGE gels but avoided using glutaraldehyde to mini-
mize unnecessary protein modifications.
Adsorption of specific proteins by asbestos. To collect pro-
teins adsorbed by asbestos fibers, we used an assay system simi-
lar to immunoprecipitation that we modified from a previously
described method.(37)Both silica and asbestos adsorbed a variety
of specific proteins (Fig. 1a). The total amount of proteins
bound to fibers was highest for chrysotile, followed by crocido-
lite, amosite and silica. We determined that approximately 1 lg
of protein had adsorbed onto 250 lg asbestos by comparison
with the original amount of rat lung lysate (2 lg, Fig. 1a). Cro-
cidolite and amosite fibers yielded similar protein profiles on
SDS-PAGE gels, whereas that of chrysotile was distinct,
although all three were different from the original profiles of the
untreated lysates. Much less protein was adsorbed on silica than
asbestos when a protease inhibitor cocktail, which protects pro-
teins from enzymatic degradation, was used for sample prepara-
tion (data not shown). Thus, proteins are adsorbed on the
surfaces of silica and asbestos via different mechanisms. To test
the specificity of the asbestos–protein interaction, we pre-incu-
bated the fibers with actin or albumin prior to the incubation
with tissue lysate. This treatment yielded essentially the same
protein profiles, indicating that neither actin nor albumin could
inhibit protein adsorption on silica or asbestos (data not shown)
and that the protein adsorption was specific. To further confirm
the specificity of asbestos–protein interaction, we incubated
chrysotile with a various amount of rat lung lysate (10, 100 and
400 lg) and found that there is a difference between proteins in
its affinity to asbestos (Fig. 1h). Therefore, there is a specific
preference of proteins to asbestos, even though the specificity
between proteins and asbestos is much weaker than that of
protein–protein interaction because there are many kinds of
proteins adsorptive to asbestos simultaneously.
Identification of asbestos-interacting proteins with MALDI-
TOF⁄⁄MS. To identify asbestos-interacting proteins, we performed
in-gel digestion and subjected the samples to MALDI-
TOF⁄MS.(60)More than 100 proteins were found to interact with
asbestos (Fig. 1a–g; Table 1; Table S1). We classified these
proteins into the following eight categories on the basis of their
cellular localization and function: chromatin⁄nucleotide⁄RNA-
binding proteins; ribosomal proteins; cytoprotective proteins;
cytoskeleton-associated proteins; histones; and hemoglobin.
Proteins adsorbed to asbestos are modified by 4-hydroxy-2-
nonenal (HNE). We studied the oxidative modifications of pro-
teins adsorbed to asbestos because asbestos can catalyze the
Fenton reaction.(61)We analyzed the presence of HNE(62)modi-
fications of the proteins using western blotting.(63,64)
Hydroxy-2-nonenal is a major lipid peroxidation end-product
associated with a variety of signaling pathways. We found that
all three types of asbestos induced HNE modification of
adsorbed proteins (Fig. 2a). In particular, crocidolite and amo-
site, which contain high amounts of iron (approximately 30%),
induced higher amounts of HNE modification than chrysotile.
Furthermore, actin incubated with amosite was degraded and
modified by HNE (Fig. 2b). Interestingly, this phenomenon of
actin degradation was only observed when we used amosite, not
chrysotile or crocidolite (data not shown), indicating that asbes-
tos–protein interactions can differ even between crocidolite and
amosite. Thus, asbestos not only adsorbed proteins on its surface
but also modified proteins via the HNE modification and
Among the various asbestos-interacting proteins, we focused
on histone H3 and hemoglobin to evaluate differences in adsorp-
tive activity and oxidative modification. We therefore incubated
asbestos with both histone H3 and hemoglobin and analyzed
various parameters (Fig. 2c). Hemoglobin showed a higher
binding specificity for chrysotile than histone H3, but the two
proteins had similar specificity values for crocidolite. Further-
more, histone H3 was modified by HNE, but hemoglobin was
not (Fig. 2c). Hemoglobin specifically bound to asbestos, but
not to silica. Finally, hemoglobin dimers were observed after
incubation with crocidolite or amosite (Fig. 2d).
Hemoglobin adsorption to asbestos results in high catalytic
activity.(59)Asbestos is known to cause hemolysis.(65)We con-
firmed this property using UICC asbestos and silica (Fig. 3a,b).
Silica exhibited the most potent hemolytic activity, followed by
chrysotile. Crocidolite and amosite were also hemolytic but with
a much lower activity (approximately 200-fold less). Silica,
chrysotile and crocidolite were then evaluated for catalytic
activity related to free radical generation in the presence of
bleomycin sulfate and DNA. Both asbestos fibers showed signif-
icantly higher catalytic activity(59)after incubation with hemo-
globin, whereas silica did not (Fig. 3c), consistent with the
result shown in Figure 2(d). These results suggest that asbestos
induces hemolysis, collects iron-containing proteins, namely
hemoglobin, on its surface and catalyzes free radical generation,
whereas silica causes hemolysis but lacks subsequent catalytic
Nagai et al. Cancer Sci|
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ª ª 2011 Japanese Cancer Association
surface. In addition to proteins, we also studied DNA adsorp-
tion to the asbestos surface. We quantified the amount of DNA
adsorbed on asbestos fibers (250 or 500 lg). Chrysotile most
effectively adsorbed DNA, followed by silica, crocidolite and
amosite (Fig. 4a). We then found that the DNA adsorbed by cro-
cidolite was oxidized to generate 8-OHdG(66)after incubation at
37?C for 3 h (Fig. 4b). Furthermore, we found that adding
hemoglobin to the reaction with chrysotile enhanced the oxida-
tion of DNA in the presence of hydrogen peroxide (Fig. 4c).
Collectively, every commercial type of asbestos, regardless of
its iron content, can utilize iron in various forms to induce
oxidative DNA damage.
lation. Finally, we investigated how asbestos fibers interact
with red blood cells in vivo. We instilled chrysotile or crocido-
adsorbsDNA and generates8-OHdG onits
lite suspension through the airways of mice or rats and 3 h later
their lungs were then collected and histopathological specimens
were prepared. We found that both types of asbestos fibers were
surrounded by inflammatory cells and red blood cells (Fig. 5,
top panels; hematoxylin and eosin-stained sections). Immuno-
histochemistry using an antibody against hemoglobin revealed
that chrysotile was directly interacting with red blood cells and
that hemoglobin was colocalized with crocidolite, suggesting
that the surface of asbestos fibers can accommodate erythrocytes
as well as hemoglobin in vivo.
We identified asbestos-interacting proteins using a method simi-
lar to immunoprecipitation. This method allowed us to measure
the selective adsorption of proteins on the asbestos surface using
inorganic material and silver staining was performed after SDS-PAGE. Asbestos exhibited higher adsorption than silica (Sil), and chrysotile (Chr)
was the most adsorptive. (a–h) The panels show the original gels subjected to matrix-assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF⁄MS) analysis. The amount of rat lung lysate (Lys) loaded in panel (a) was 2 lg. Numbers correspond to the identified
proteins listed in Table S1. The gels shown in each panel are different in the concentration of polyacrylamide and the combination of inorganic
material and cell⁄tissue lysate. The origin of the rat lysate (e.g. lung, kidney, liver, etc.) used for the incubation is shown at the top and the
species of the inorganic material is shown next to the top. (h) Chrysotile was incubated with 10, 100 or 400 lg of rat lung lysate and the
proteins adsorbed onto chrysotile were analyzed by the coupling of SDS-PAGE and silver staining. The square in a continuous line shows an
increase in the amount of asbestos-binding proteins as the total amount of proteins incubated increases. In contrast, the square in a dotted line
shows the opposite. Cro, crocidolite; Amo, amosite; Tunica v., tunica vaginalis; MeT5A-Memb., membrane fraction of MeT5A cells.
Adsorption of specific proteins by asbestos fibers. Lysates from rat tissues or MeT5A mesothelial cells were incubated with each
ª ª 2011 Japanese Cancer Association
cell or tissue lysates. All of the proteins identified in the present
study are listed in Table S1. We classified 99 out of 128
asbestos-interacting proteins into the following eight categories:
nine chromatin-binding proteins; 10 nucleotide-binding proteins;
14 RNA-binding proteins; 24 ribosomal proteins; nine cytopro-
tective proteins; 26 cytoskeleton-associated proteins; four
histones; and three hemoglobin subunits (Table S1). These
results indicate that not only DNA (Fig. 4a) but also many types
of DNA-interacting proteins have an affinity for asbestos.
Indeed, physical interactions between the mitotic spindle and
asbestos might cause mitotic disturbances and chromosomal
aberrations.(7,37)Moreover, our identification of RNA-binding
and ribosomal proteins as asbestos-interacting proteins indicates
that asbestos might interfere with chromosomal replication, tran-
scription and translation.
The different affinities of each asbestos type for DNA can be
partially explained by their surface charges. The surface of chrys-
otile is positively charged, whereas crocidolite and amosite are
negatively charged.(3)Thus, chrysotile provides more suitable
surface area for a negatively charged biomolecule such as DNA.
Among the cytoskeleton-associated proteins, tubulin, actin,
vimentin and cytoskeleton-associated protein 4 were previously
reported to interact with asbestos.(7,37)In addition, we identified
many other cytoskeleton-associated proteins, including a actinin
1 and 4, filamin-A, keratin 8 and 18, myosin 9, 10 and 11, septin
2 and 7, spectrin, ezrin, radixin and moesin. Adsorption of these
proteins onto the surface of asbestos might affect cytoskeletal
We identified several cytoprotective proteins that metabolize
reactive oxygen species (ROS), including manganese superoxide
dismutase, glutathione peroxidase 1, and peroxiredoxin 1 and
2,(67)suggesting that asbestos might disturb the redox state of
cells not only by generating ROS but also by adsorbing proteins
that metabolize and reduce ROS.
Hemoglobin was identified as an asbestos-interacting protein.
This interaction was specific to asbestos at body temperature
(37?C) in vitro and did not occur on silica. Hemoglobin is a
major oxygen-transporting protein and is released from red
blood cells during hemolysis. Adsorption of hemoglobin on the
asbestos surface has been mentioned previously,(65)but the pres-
ent study is the first to demonstrate that this event augments
asbestos-induced free radical generation (Figs 3c,4c). We also
investigated the direct interaction of red blood cells⁄hemoglobin
and asbestos fibers by immunohistochemistry (Fig. 5), although
to what extent this direct interaction contributes to oxidative
damage in surrounding tissue still remains elusive.
In addition to enhancement of oxidative damage, this direct
interaction might be the first step in the formation of asbestos
bodies. Governa et al.(50)suggested that bilirubin was present in
the innermost layer of the asbestos body. Because bilirubin is a
metabolite of heme, our results support this idea. Thus, we pro-
pose a model in which asbestos utilizes heme iron from erythro-
cytes to enhance free radical generation, thereby inducing DNA
damage in vivo, at least temporarily. Hemoglobin metabolism is
mediated by heme oxygenase-1, which is upregulated when ani-
mals or cells are treated with asbestos.(68,69)Therefore, this
Table 1. A summarized list of asbestos-binding proteins
ATP-dependent DNA helicase 2 subunit 2
Coiled-coil domain-containing protein 124
DNA replication licensing factor MCM6
DNA replication licensing factor MCM7
DNA-(apurinic or apyrimidinic site) lyase
Flap endonuclease 1
Interleukin enhancer-binding factor 2
Transcription initiation factor IIE subunit b
Cleavage and polyadenylation specificity factor subunit 5
Eukaryotic translation initiation factor 2 subunit 1
FUS glycine-rich protein
Heterogeneous nuclear ribonucleoprotein A0
Heterogeneous nuclear ribonucleoprotein A1
Heterogeneous nuclear ribonucleoprotein U
KH domain-containing RNA-binding signal
transduction-associated protein 1
Probable ATP-dependent RNA helicase DDX5
Putative pre-mRNA-splicing factor-ATP-dependent RNA
RNA-binding protein EWS
rRNA 2¢-O-methyltransferase fibrillarin
Splicing factor, proline and glutamine rich
THO complex subunit 4
ATP synthase subunit a
ATP synthase subunit O
Developmentally regulated GTP-binding protein 1
Elongation factor 1- a 1
Elongation factor 1- a 2
Elongation factor Tu
Glutamate dehydrogenase 1
78 kDa glucose-regulated protein
DnaJ homolog subfamily B member 13
Glutathione peroxidase 1
Heat shock 70 kDa protein 1⁄2
Heat shock cognate 71 kDa protein
Superoxide dismutase (Mn)
Cytoskeleton-associated protein 4
Keratin type I cytoskeletal 18
Keratin type II cytoskeletal 8
Myosin binding protein C
Myosin light polypeptide 6
Predicted: similar to Myosin 11
Predicted: similar to septin-11
Predicted: similar to tubulin
Tubulin b-5 chain
Histone H2A type 3
Histone H2B type 1
Hemoglobin subunit a 1⁄2
Hemoglobin subunit b 1
Hemoglobin subunit b 2
39S ribosomal protein L28
39S ribosomal protein L40
39S ribosomal protein L48
40S ribosomal protein S2
40S ribosomal protein S3a
40S ribosomal protein S4, X isoform
40S ribosomal protein S7
40S ribosomal protein S9
40S ribosomal protein S16
60S ribosomal protein L7a
60S ribosomal protein L8
60S ribosomal protein L9
60S ribosomal protein L10
60S ribosomal protein L13
60S ribosomal protein L17
60S ribosomal protein L18
60S ribosomal protein L18a
60S ribosomal protein L22
60S ribosomal protein L23a
60S ribosomal protein L31
Refer to Table S1 for details.
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enzyme might play a role in the conversion of hemoglobin into
bilirubin on asbestos.
We also provided evidence suggesting that asbestos can mod-
ify proteins with HNE. In particular, incubation with asbestos
promoted the accumulation of HNE modifications on both his-
tone H3 and actin, leading to subsequent degradation of the lat-
ter. We believe that membrane lipids in the lysate and
contaminant lipids in commercial proteins are the source of
HNE in this setting. Here, we propose that asbestos not only
adsorbs proteins but also provides a niche for oxidative reac-
tions. This situation is also true in the case of DNA. DNA is
adsorbed on the surface of asbestos (Fig. 4a) and oxidatively
modified to 8-OHdG (Fig. 4b,c). This reaction might occur in
the nuclei of dividing cells, leading to prominent genomic alter-
In conclusion, we have identified and classified a variety of
asbestos-interacting proteins. Among these proteins, we believe
that hemoglobin and chromatin constituents such as DNA and
histones are especially important; hemoglobin is an iron source
and histones lie in close proximity to genomic DNA. DNA also
showed a high affinity for asbestos, especially chrysotile. Taken
together, we propose that asbestos provides a niche for
Amosite + actin
incubation time (min)
5 30 180
Lys Sil Chr Cro Amo
asbestos. (a) After incubation of lung lysates with asbestos at 37?C,
notable increases in the 4-hydroxy-2-nonenal (HNE) modification were
observed in the samples incubated with crocidolite (Cro), amosite
(Amo) and chrysotile (Chr). IB, immunoblot. (b) After incubation with
amosite at room temperature for the indicated periods, actin was
degraded and modified by HNE in a time-dependent manner. (c)
Competition of histone H3 and hemoglobin for the asbestos surface.
When chrysotile (Chr) was used, hemoglobin had a higher affinity
than histone H3. Histone H3 was modified by HNE. When crocidolite
(Cro) was used, histone H3 and hemoglobin showed similar affinities,
although only histone H3 was modified by HNE. (d) All types of
asbestos fibers adsorbed hemoglobin, whereas silica showed no
4-Hydroxy-2-nonenal modification of proteins adsorbed on
3060 90120 150180
37°C Time (min)
4°C Time (min)
Amount of catalytic iron (ng)
catalytic iron resulting from adsorption of hemoglobin on the
asbestos surface. (a,b) Silica and chrysotile exhibited prominent
hemolysis, whereas crocidolite and amosite showed lower hemolytic
activity. Hemolysis was time-dependent and eventually reached a
plateau. Incubations were performed at 4 or 37?C as indicated. (c)
hemoglobin adsorption. Silica did not show catalytic activity even
after incubation with hemoglobin. The amount of catalytic iron was
calculated based on a standard curve using Fe(NO3)3. **P < 0.01
relative to the negative control (deionized water instead of asbestos
or iron). †P < 0.05 between the indicated groups. NS, no significant
Hemolytic activity of each type of asbestos and increases in
ª ª 2011 Japanese Cancer Association
oxidative reactions by specifically adsorbing various important
proteins and DNA and subsequently generating local iron over-
load (Fig. 6). Among the three types of asbestos, chrysotile
induced mesothelioma most rapidly when injected intraperitone-
ally to rats (Li Jiang, Hirotaka Nagai and Shinya Toyokuni, un-
published data, 2011). With these results, further studies are
necessary to re-evaluate the risk posed by chrysotile exposure in
the development of lung cancer and mesothelioma.
We thank Nobuaki Misawa (Toyokuni laboratory) and Yi Zhong (Gradu-
ate School of Medicine, Kyoto University) for technical assistance and
Seishiro Hirano (National Institute for Environmental Studies), Yasushi
Shinohara (National Institute of Occupational Health and Safety) and
Norihiko Kohyama (National Science Laboratory, Faculty of Economics,
Toyo University) for the kind gift of asbestos. We acknowledge the
Bright field8-OHdGBright field 8-OHdG
Chrysotile + H2O2 + DNA
4°C 9 h
37°C 9 h
37°C 3 h
4°C 3 h
Crocidolite + DNA
Sil 250 µg Sil 500 µg
Chr 250 µg
Chr 500 µg
Cro 250 µg
Cro 500 µg
Amo 250 µg
Amo 500 µg
adsorbed DNA (µg)
hydroxy-2¢-deoxyguanosine (8-OHdG) on asbestos fibers. (a) Asbestos
and silica adsorbed DNA after incubation at 37?C. Chrysotile was the
most potent DNA adsorbent, followed by silica, crocidolite and
amosite. *P < 0.05. **P < 0.01. (b) Crocidolite was incubated with
genomic DNA at 4 or 37?C for 3 h. Fluorescent immunohistochemistry
was performed after incubation to show the generation of 8-OHdG
on the asbestos surface (arrows). (c) Formation of 8-OHdG on the
surface of hemoglobin-treated chrysotile after incubation with DNA in
the presence of H2O2(arrows). Scale bar, 50 lm.
Simultaneous adsorption of DNA and generation of 8-
3 h after instillation
asbestos fibers in vivo. Three hours after instillation of asbestos fiber
suspension to the airways of mice or rats, asbestos fibers were
surrounded by inflammatory cells as well as red blood cells (in
hematoxylin and eosin [HE]-stained sections). Immunohistochemistry
to detecthemoglobin revealed
interacted with erythrocytes and that hemoglobin was colocalized
with crocidolite with serial sections. Arrowheads indicate asbestos
fibers. Hoechst, hoechst 33342; scale bar, 50 lm.
Directinteractionof redblood cells⁄hemoglobinwith
Nagai et al. Cancer Sci|
| vol. 102| no. 12|
ª ª 2011 Japanese Cancer Association
support of the Division for Medical Research Engineering, Nagoya
University Graduate School of Medicine, for fluorescent microscopy.
This study was supported by a MEXT grant (Special Coordination Funds
for Promoting Science and Technology), a research grant from the
Princess Takamatsu Cancer Research Fund (10-24213), a grant-in-aid
for cancer research from the Ministry of Health, Labour and Welfare of
Japan, a grant-in-aid from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, a grant from the Takeda Science
Foundation and a grant-in-aid from the Japan Society for the Promotion
of Science Fellows (H. N.).
The authors have no conflict of interest.
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Additional Supporting Information may be found in the online version of this article:
Table S1. A list of asbestos-binding proteins.
Data S1. Materials and Methods.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries
(other than missing material) should be directed to the corresponding author for the article.
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