J. Clin. Biochem. Nutr. | May 2012May 2012 | vol. 50 | no. 3 | 211–216doi: 10.3164/jcbn.11-70
JCBN Journal of Clinical Biochemistry and Nutrition0912-00091880-5086 the Society for Free Radical Research Japan Kyoto, Japanjcbn11-7010.3164/jcbn.11-70Original Article
Metal nanoparticle-induced micronuclei
and oxidative DNA damage in mice
Ming-Fen Song, Yun-Shan Li, Hiroshi Kasai and Kazuaki Kawai*
Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health,
1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
*To whom correspondence should be addressed.
(Received 17 May, 2011; Accepted 18 July, 2011)
Copyright © 200? JCBN200? This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unre-stricted use, distribution, and reproduction in any medium, pro-vided the original work is properly cited.
Several mechanisms regarding the adverse health effects of nano-
materials have been proposed. Among them, oxidative stress is
considered to be one of the most important. Many in vitro studies
have shown that nanoparticles generate reactive oxygen species,
deplete endogenous antioxidants, alter mitochondrial function and
produce oxidative damage in DNA. 8-Hydroxy-2'-deoxyguanosine
is a major type of oxidative DNA damage, and is often analyzed
as a marker of oxidative stress in human and animal studies. In
this study, we focused on the in vivo toxicity of metal oxide and
silver nanoparticles. In particular, we analyzed the induction of
micronucleated reticulocyte formation and oxidative stress in
mice treated with nanoparticles (CuO, Fe3O4, Fe2O3, TiO2, Ag). For
the micronucleus assay, peripheral blood was collected from the
tail at 0, 24, 48 and 72 h after an i.p. injection of nanoparticles.
Following the administration of nanoparticles by i.p. injection to
mice, the urinary 8-hydroxy-2'-deoxyguanosine levels were
analyzed by the HPLC-ECD method, to monitor the oxidative stress.
The levels of 8-hydroxy-2'-deoxyguanosine in liver DNA were also
measured. The results showed increases in the reticulocyte micro-
nuclei formation in all nanoparticle-treated groups and in the
urinary 8-hydroxy-2'-deoxyguanosine levels. The 8-hydroxy-2'-
deoxyguanosine levels in the liver DNA of the CuO-treated group
increased in a dose-dependent manner. In conclusion, the metal
nanoparticles caused genotoxicity, and oxidative stress may be
responsible for the toxicity of these metal nanoparticles.
Key Words:nanoparticles, metal oxide, silver, oxidative stress,
greatly impacted industrial technology. With the increasing
utilization of nanoparticles, people have a greater opportunity to
be exposed to nanoparticles through the occupational environment
and consumer products in daily life. At the same time, there has
been increasing concern about the adverse effects of nanoparticles
on human health. In this study, we especially focused on the
induction of reticulocyte micronuclei and oxidative DNA damage
in vivo caused by metal oxides (CuO, Fe2O3, Fe3O4, TiO2) and
CuO nanoparticles have many commercial applications, as
components in catalysts,(1) pigments, and antimicrobial textiles.(2)
However, there are only a few reports describing the toxicity of
CuO nanoparticles in bacteria(3,4) or human lung epithelial cells.(5)
Recently, it was reported that CuO nanoparticles were highly
toxic, as compared to other metal oxide nanoparticles in vitro.(6,7)
However, the amount of in vivo data is still insufficient.
Iron oxide nanoparticles have attracted much attention, not only
due to their superparamagnetic properties but also because they
hold great potential in many biomedical applications, such as drug
delivery, magnetic resonance imaging (MRI) contrast enhance-
ment,(8–10) and the targeted destruction of tumor tissue through
Significant advances in nanotechnology in recent years have
hyperthermia.(11) However iron oxide caused a significant increase
in DNA damage in A549 cells.(7)
Nanosized TiO2 is one of the most widely used nanomaterials.
TiO2 is a poorly soluble, biologically inert particulate, which is
broadly used as a white pigment, ultraviolet light blocker, or
catalyst in a number of products, such as paints, plastics, paper,
cosmetics, medicines, foods, and welding rods.(12,13) At the same
time, The International Agency for Research on Cancer classified
TiO2 as possibly carcinogenic to humans (class 2B), based on
sufficient evidence in experimental animals. The genotoxicity of
TiO2 was also reported in several previous studies.(14–16) In general,
nanosized TiO2 particles produce reactive oxygen species,
damage DNA, and induce oxidative DNA damage in vitro.(17,18)
On the other hand, negative genotoxicity results have also been
reported.(19,20) Therefore, additional information on the genotoxicity
of nanosized TiO2 is needed.
Silver nanoparticles are used most commonly in numerous
consumer products, including textiles, cosmetics, and health care
products, due to their strong antimicrobial activity. However,
despite their widespread use, there is a serious lack of information
concerning the toxicity of silver nanoparticles to humans.(21,22)
One of the most discussed mechanisms behind the health effects
induced by metal oxide and silver nanoparticles is their ability to
cause oxidative stress.(23–27) This mechanism is believed to be
important in the toxicity of manufactured nanoparticles. Further-
more, since oxidative stress is directly linked to DNA damage,
mutations, and cancer,(28–30) nanoparticles may affect on cancer
development. Although some in vitro studies have focused on
investigating and comparing metal oxide and silver nanoparticles,
regarding DNA damage and markers for oxidative stress, the
amount of in vivo research is still insufficient. We were parti-
cularly interested in the in vivo toxicities of these types of
Nanoparticles are generally considered to be problematic in a
toxicity assessment for several reasons.(23,24–27) The small size and
the relatively large surface area have been suggested to result in
increased toxicity, as compared to micrometer-sized particles.
However, it is not clear whether the increased toxicity is a
common feature of all kinds of nanoparticles with different
chemical compositions. In particular, metal nanoparticles easily
form micrometer sized aggregates. In this study, however,
commercially available nanoparticles were tested as-is, because
the toxicities of nanoparticles should be assessed using their
commercially available forms. With regards to human exposure,
it is therefore likely that under most circumstances, the nano-
materials will exist in the form of aggregates, rather than indi-
vidual units. The aggregated form of the material may be more
representative of workplace or consumer exposure scenarios.
The aim of this study was to investigate and compare different
nanoparticles, regarding their ability to cause micronucleus
formation and oxidative DNA damage in vivo. The study focused
on different metal oxides (CuO, TiO2, Fe2O3, Fe3O4) and Ag
Materials and Methods
from Kanto Chemical, Co. Inc. (Tokyo, Japan). The particle size
of CuO was 27.2–95.3 nm, with a surface area of 10–35 m2/g. The
size of the TiO2 nanopowder was 19.7–101.0 nm, with a surface
area of 15–77 m2/g. Fe2O3 and Fe3O4 nanoparticles were obtained
from JEF Chemical (Chiba, Japan). The sizes of the Fe2O3
nanopowder particles were 60–100 nm, with a surface area of
15.0 m2/g. The particle size of the Fe3O4 nanopowder was 80 nm,
with a surface area of 38 m2/g. The Ag nanoparticle powder was
purchased from Sigma-Aldrich, Co. (St. Louis, MO), and the
particle size was <100 nm, with a surface area of 5.0 m2/g.
Animals and treatment.
Female ICR mice (6 weeks old)
were purchased from SLC (Shizuoka, Japan), and were used for
the experiments at 7 weeks of age. They were given commercial
food (Dyets no. 110952; Dyets Inc., Bethlehem, PA) and water ad
libitum throughout the acclimatization and experimental periods.
Each type of nanoparticle was suspended in saline with 0.05%
Tween 80 and injected intraperitoneally.
Acridine orange (AO)-coated slides were
prepared as described previously.(31) Briefly, 10 μl of a 1 mg/ml
AO aqueous solution was spread homogeneously on a warmed
glass slide. A 5 μl portion of peripheral blood, collected by cutting
the ventral tail, was placed without any anticoagulant on the center
of an AO-coated glass slide and covered immediately with a
24 × 40 mm coverslip. AO supravitally stained reticulocytes were
examined by fluorescence microscopy, with a blue excitation and
a yellow barrier filter. The frequencies of micronucleated peri-
pheral reticulocytes (MNRETs) were recorded, based on the
observation of 1,000 reticulocytes per mouse.
Measurement of serum Cu level.
from the inferior vena cava, and were centrifuged for 10 min at
3,000 g to separate the serum. Serum samples were stored at
−80°C until the analyses. Serum samples were analyzed using
atomic absorption spectrometry with a graphite furnace instru-
ment, Z-8200, equipped with an SSC-300 autosampler (Hitachi,
Tokyo, Japan). The copper-specific hollow cathode lamp was used
at 324.8 nm, with a slit width of 1.3 nm and Zeeman background
correction. The atomization temperature was set to 2,400°C.
Samples were diluted 1:100 to 2,000 with 0.05 M nitric acid. A
commercial standard solution of copper was used for calibration.
Collection of urine.
For urine collection, the mice were
housed individually in glass metabolic cages (Sugiyama-Gen
Iriki Co., Tokyo, Japan), and 24-h urine outputs were collected
and stored at −20°C until analyzed. Time-based urine samples
were also collected, before and 24, 48, 72 h after the injection of
Isolation of nuclear DNA.
Mice were killed under deep
ether anesthesia at appropriate times after nanoparticle injection,
and the livers and bone marrows were promptly removed and
stored at −80°C until analyzed. The nuclear DNA of the mouse
tissues was isolated by the sodium iodide method, using a DNA
Extraction WB Kit (Wako Pure Chem. Ind., Ltd., Osaka, Japan).
To avoid oxidative DNA artifacts, 1 mM desferal (deferoxamine
mesylate, Sigma Chemical Co., MO) was added to the lysis
solution for the tissue homogenization and DNA extraction. A
50 mg portion of liver was homogenized with a teflon-glass
homogenizer, in 1 ml of ice-cold lysis solution. In the case of the
bone marrow, the total amount from a mouse was used, without
homogenization. Subsequent DNA isolation was performed
according to the manufacturer’s instructions.
CuO and TiO2 nanoparticles were purchased
Blood samples were drawn
Analysis of 8-OH-dG in DNA.
digested with 8 units of nuclease P1 (Yamasa Corp., Choshi,
Japan), in 100 μl of a solution containing 1 mM EDTA and
20 mM sodium acetate (pH 4.5). It was then treated with 2 units
of alkaline phosphatase (Roche Diagnosis GmbH, Mannheim,
Germany) in 250 mM Tris-HCl buffer (pH 8.0), to obtain a
deoxynucleoside mixture. The solution was filtered with a
0.45 μm filter (3CR, Japan PALL, Tokyo, Japan). The filtrate was
stored at −80°C until analysis. The filtrate was injected into an
HPLC column (Capcell Pak C18 MG, 3 μm, 4.6 × 250 (series-
connected 100 + 150) mm, Shiseido Fine Chemicals, Tokyo,
Japan) equipped with UV (UV-8020, Tosoh Co., Tokyo, Japan)
and ECD (ECD-300, Eicom Co., Kyoto, Japan, applied voltage:
550 mV) detectors. The mobile phase was 10 mM NaH2PO4,
containing 8% methanol and 0.13 mM Na2EDTA, delivered at a
flow rate of 0.7 ml/min. The column temperature was 32°C. The
amount of 8-OH-dG in the DNA was determined by comparison
to the authentic standards. The 8-OH-dG value in the DNA was
calculated as the number of 8-OH-dG per 106 deoxyguanosine (dG).
Analysis of 8-OH-dG in urine.
was determined using the previously described detection appa-
ratus,(32,33) with three pumps, the sampling injector, two valves,
the HPLC-1 column, the UV detector, the HPLC-2 column, and
the EC detector. The HPLC-1 column was set in a column oven
at 65°C, and the HPLC-2 column was set in a column oven at
50°C. Frozen urine samples were defrosted and mixed completely,
to form homogeneous suspensions. For the 8-OH-dG analysis, a
50 μl aliquot of each sample was mixed with the same volume
of a dilution solution, containing the ribonucleoside marker 8-
hydroxyguanosine (120 mg/ml) and 4% acetonitrile, in a solution
of 130 mM NaOAc (pH 4.5) and 0.6 mM H2SO4. The diluted urine
samples were centrifuged at 13,000 rpm for 5 min. A 70 μl aliquot
of each supernatant was transferred to a vial for analysis in the
apparatus. A 20 μl aliquot of the diluted urine sample was injected
into HPLC-1 (MCI GEL CA08F, 7 mm, 1.5 × 150 mm, solvent A,
50 ml/min) from the sampling injector, and the chromatogram
was recorded by a UV detector (245 nm). In this method, the 8-
OH-dG fraction was collected, depending upon the relative elution
position from the peak of the added marker, 8-hydroxyguanosine
(8-OH-Guo), and was automatically injected into the HPLC-2
column (Shiseido, Capcell Pak C18, 5 mm, 4.6 × 250 mm, solvent
B, 1 ml/min). This column was coupled with an ECD [Coulochem
III EC detector with a guard cell (5020) and an analytical cell
(5011) (applied voltage: guard cell, 400 mV; E1, 280 mV; E2,
350 mV)]. The solvents used were: solvent A, 2% acetonitrile in
0.3 mM sulfuric acid; solvent B, 10 mM sodium phosphate buffer
(pH 6.0), 5% methanol, plus an antiseptic, Reagent MB (100 ml/l).
The isolated DNA was
The urinary 8-OH-dG level
In vivo micronucleus test.
assay were determined after the i.p. administration of metal
nanoparticles at various doses (0, 1, 3 mg/mouse) to ICR female
mice. The time courses of the induction of micronucleated
reticulocytes (MNRETs) by the intraperitoneal administration of
nanosized CuO, Fe2O3 and Fe3O4 are shown in Fig. 1. These metal
nanoparticles induced significant increases in MNRETs. The peak
induction of MNRETs appeared 48 h after the administration. The
frequencies of MNRETs increased dose-dependently at 48 h after
administration of CuO, Fe2O3 and Fe3O4 (Fig. 2). Significant
increases of MNRETs were also obtained at 48 h after treatment
with nanosized TiO2 and Ag, in addition to CuO, Fe3O4 and Fe2O3,
as compared to the corresponding negative controls (Fig. 3).
Serum copper content.
CuO was injected i.p. into mice
once at a dose of 3 mg/mouse, as described in the Materials
and Methods. The copper concentrations in the serum varied
with time after the nanoparticle injection, as shown in Fig. 4. The
Copper concentration in the serum increased immediately after
The results of the micronucleus
J. Clin. Biochem. Nutr. | May 2012 May 2012 | vol. 50 | no. 3 | 213
M.-F. Song et al.
injection, and reached its maximum at 1 h. Thereafter, it gradually
decreased to nearly the control level in 24 h.
The urinary level of 8-OH-dG, corrected
by creatinine, was significantly increased by the CuO administra-
tion to mice at each time point of the urine analysis (Fig. 5).
However, the increases in the urinary levels of 8-OH-dG from the
other nanoparticle treatments were not significant. The analysis of
the 24 h urine collection with the metabolic cage revealed that the
8-OH-dG level was increased about 1.6-fold by the administration
of 3 mg of CuO (Fig. 6). In addition, the other nanoparticles
showed positive increases in the of 8-OH-dG levels.
DNA 8-OH-dG in bone marrow and liver.
levels in the bone marrow immediately increased after the
injection of CuO, and continued to increase up to 24 h after
administration (Fig. 7). The data in Fig. 8 show that the levels of
8-OH-dG in the DNA from the liver increased for 24 h after the
administration of CuO. The 8-OH-dG levels in the liver DNA of
the mice treated with 3 mg CuO were 5-fold higher than those in
i.p. administration of 3 mg metal oxide nanoparticles. Significant
differences exist between the control and the CuO or Fe2O3 treated
group. Each value represents the average of 4–6 mice and a 95%
confidence interval. In total, 3,000 reticulocytes from each mouse were
Time courses of micronucleated reticulocyte induction after
48 h after i.p. administration of metal oxide nanoparticles. Significant
differences exist between the 0 mg and 3 mg doses in the nanoparticle
treated groups. Each value represents the average of 4–6 mice and
95% confidence interval. In total, 3,000 reticulocytes from each mouse
Dose-response of micronucleated reticulocyte, induction at
after i.p. administration of 3 mg metal oxide nanoparticles. Each value
represents the average of 4–6 mice ± SE. *indicates significantly higher
levels as compared to the control, corresponding to p<0.05. In total,
3,000 reticulocytes for each mouse were analyzed.
Frequencies of micronucleated reticulocyte induction at 48 h
time point after i.p. administration of 3 mg metal oxide nanoparticles.
The data show the ratio of 8-OH-dG levels at each time point to that
at zero time, because each mouse had a different 8-OH-dG level
before administration. Each value represents the average of 3 mice ± SE.
*indicates significantly higher levels as compared to the control,
corresponding to p<0.05.
Urinary 8-OH-dG levels corrected by creatinine (ng/mg) at each
3 mg CuO. Each value represents the average of 3 mice ± SE.
Serum copper concentration after the i.p. administration of
the non-treated control mouse liver DNA (0.78 ± 0.13 8-OH-dG/
106dG). The other nanoparticles did not cause an increase in the
liver 8-OH-dG level at 24 h after administration. The 8-OH-dG
levels in the liver DNA increased for 72 h after the administration
of 1 and 3 mg doses of CuO (Fig. 9).
Manufactured nanoparticles and their applications are an
expanding field of technology, and as the use of these materials
increases, it is becoming more and more important to investigate
their possible adverse effects on human health as well as the
environment. This study described the genotoxic activities and
the induction of oxidative stress by metal oxide and silver
nanoparticles in vivo.
Although several studies have reported the toxicities of metal
oxide and silver nanoparticles in cultured cells, very little in vivo
study information is available.(22–24)
The CuO nanoparticles were much more genotoxic, as
compared to the other metal oxide nanoparticles, in the in vivo
micronuclei test.(6) These genotoxic tendencies of metal oxide
nanoparticles are similar to those recently reported by Karlsson
et al.,(6) based on an in vitro Comet assay. In our results, the
maximum rate of MNRETs occurred at 48 h, and thereafter
declined to the control level by 72 h after CuO or iron oxide
nanoparticle exposure. These micronuclei test results for the metal
oxide nanoparticles support the proposal that MNRETs originate
from lesions induced only during a short time after the administra-
tion of nanoparticles. Actually, the serum copper level increased
immediately after the i.p. injection of CuO particles, and peaked
at 1 h, followed by a gradual decrease with time. The copper
level in the serum was reduced to the control levels at 24 h after
administration. The increased copper concentrations in the blood
indicated that the i.p. injection of CuO nanoparticles allowed them
to enter the blood circulation and become distributed in the bone
marrow. The time-courses of micronuclei induction and the serum
metal levels seem to be intimately related to each other.
From a different standpoint, the proper timing of the peripheral
blood sampling is important to evaluate the genotoxity of metal
oxide nanoparticles by an in vivo micronuclei assay. Furthermore,
a dose-related increase in micronuclei induction was observed in
the peripheral blood of mice treated with CuO, Fe2O3 and Fe3O4
The actual reason why the CuO nanoparticles show the highest
toxicity is still unclear, although several possibilities have been
discussed, as follows. The CuO may have shown the highest
i.p. administration of 3 mg metal oxide nanoparticles. Each value
represents the average of 3 mice ± SE. *indicates significantly higher
levels as compared to the control, corresponding to p<0.05.
Urinary 8-OH-dG levels in 24 h urine samples, collected after
of 3 mg CuO nanoparticles. Each value represents the average of 3
mice ± SE. *indicates significantly higher levels as compared to the
control, corresponding to p<0.05.
8-OH-dG levels in bone marrow DNA after i.p. administration
3 mg metal oxide nanoparticles. Each value represents the average of 3
mice ± SE. *indicates significantly higher levels as compared to the
control, corresponding to p<0.05.
8-OH-dG levels in liver DNA 24 h after i.p. administration of
istration of 1 or 3 mg CuO nanoparticles. Each value represents the
average of 3 mice ± SE.
Time courses of 8-OH-dG levels in liver DNA after i.p. admin-
J. Clin. Biochem. Nutr. | May 2012May 2012 | vol. 50 | no. 3 | 215
M.-F. Song et al.
toxicity because copper, like iron, is a transition metal, and can
cause oxidative stress via the Fenton reaction. The Cu-ions
released in the cell may be an additional source of the toxicity, and
some studies have shown a strong relationship between cyto-
toxicity and nanoparticle solubility in vitro.(6,34) Another possi-
bility is that the CuO nanoparticles physically interacted with the
mitochondrial membranes. This structural damage would lead to
the loss of mitochondrial membrane potential, the opening of the
permeability transition pores, and ROS production.(35) Considering
these phenomena together, a key mechanism underlying the high
toxicity of CuO seems to be the ability to cause oxidative stress.
Oxidative stress is a redox imbalance within cells, and usually
results from increased intracellular reactive oxygen species (ROS)
and decreased antioxidants.(25) ROS are highly reactive molecules
that can disturb the homeostasis of the intracellular physiological
state by reacting with cellular macromolecules including DNA,
proteins and lipids. ROS-induced DNA damage is typified by
single- and double-stranded DNA breaks, base modifications
(e.g. the formation of 8-OH-dG adducts) and DNA cross-links,
all of which, if un-repaired, have the potential to initiate and
promote carcinogenesis.(36) 8-OH-dG(37) is one of the most well-
studied oxidative stress markers in investigations of the role of
oxidative stress in carcinogenesis. It is a useful marker to measure
the oxidative damage in DNA, and the urinary 8-OH-dG level is
also a common oxidative stress marker in living organisms.
Although many studies using comet assays suggested that metal
nanoparticles produce 8-OH-dG in DNA,(25–27) very few have
directly measured, reliable 8-OH-dG data.(38) Therefore, we
measured the 8-OH-dG levels in liver and bone marrow DNA and
urine after the administration of metal oxide and silver nano-
particles, by the HPLC-ECD method.(33)
The urinary 8-OH-dG levels, corrected by creatinine (8-OH-
dG/Cre), increased during 24–72 h after the CuO treatment
(Fig. 5). The high level of liver 8-OH-dG persisted even at 72 h
after the CuO administration (Fig. 9). Those data are in good
agreement with other data. The 8-OH-dG levels in bone marrow
immediately increased after the injection of CuO and continued to
increase up to 24 h after administration (Fig. 7). During the first
24 h after administration, not only CuO but also all of the other
metal compounds showed higher levels of urinary 8-OH-dG than
the control (Fig. 6). The maximum point of urinary 8-OH-dG
excretion was probably within 24 h after the injection of most of
the metal nanoparticles, with the exception of CuO. The amount of
8-OH-dG excreted into urine during the 24 h after CuO treatment
was rather low, in spite of its high toxicity. The degeneration of
the kidney tubule epithelium has been reported as one of the
toxic effects of copper.(39) As a result, the reduction of urine output
(data not shown) by CuO may be related to the low 8-OH-dG ex-
cretion. The results described above revealed that the i.p. injected
nanoparticles are quickly circulated through the blood system, and
then are rapidly sequestered. This is reasonable, since the micro-
nuclei induction peaked at 48 h after administration. Some metals,
such as Cu, may accumulate in the liver and affect it for a long
period of time. This may also be related to the continuously high
levels of urinary 8-OH-dG (Fig. 5). However, the effects on bone
marrow micronuclei formation are apparently short-lived.
The transition metals ions (such as copper, iron and titanium)
released from certain nanoparticles have the potential to cause the
conversion of cellular oxygen metabolic products, such as H2O2
and superoxide anions, to hydroxyl radicals (•OH), which are one
of the primary DNA damaging species. Fe(II) can also cause the
production of H2O2 from molecular O2, which can diffuse through
cellular and nuclear membranes to react with Fe bound to DNA,
resulting in the generation of •OH.
In conclusion, our study has shown that different nanoparticles
have the common feature of inducing chromosomal damage and
oxidative DNA damage in vivo. Among them, the CuO nano-
particles were the most potent, and exposure to these particles,
occupationally or via consumer products, may pose a health risk.
Iron oxide particles (Fe2O3, Fe3O4) also showed relatively high
toxicity. However, TiO2 and Ag particles showed low toxicity.
Taken together, our results suggest that metal oxide nanoparticles
cause genotoxic effects in vivo. Nevertheless, further studies are
needed to clarify the molecular mechanisms involved in the
genotoxicity of metal oxide nanoparticles, for safer and proper
This work was supported by Grants-in-Aid from the Ministry of
Health, Labor and Welfare of Japan, and by UOEH Grant for
Advanced Research (2009–2011).
•OH hydroxyl radicals
ROSreactive oxygen species
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