Combined effects of 60 Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells.
ABSTRACT Epidemiological studies have suggested that extremely low-frequency magnetic fields (ELF-MF) are associated with an increased incidence of cancer. Studies using in vitro systems have reported mixed results for the effects of ELF-MF alone, and the World Health Organization (WHO) Research Agenda published in 2007 suggested that high priority research should include an evaluation of the co-carcinogenic effects of ELF-MF exposure using in vitro models. Here, the carcinogenic potential of ELF-MF exposure alone and in combination with various stress factors was investigated in NIH3T3 mouse fibroblasts using an in vitro cellular transformation assay. NIH3T3 cells were exposed to a 60 Hz ELF-MF (1 mT) alone or in combination with ionizing radiation (IR), hydrogen peroxide (H₂O₂), or c-Myc overexpression, and the resulting number of anchorage-independent colonies was counted. A 4 h exposure of NIH3T3 cells to ELF-MF alone produced no cell transformation. Moreover, ELF exposure did not influence the transformation activity of IR, H₂O₂, or activated c-Myc in our in vitro assay system, suggesting that 1 mT ELF-MF did not affect any additive or synergistic transformation activities in combination with stress factors such as IR, H₂O₂, or activated c-Myc in NIH3T3 cells.
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ABSTRACT: The principal objective of this study was to assess the DNA damage in a normal cell line system after exposure to 60 Hz of extremely low frequency magnetic field (ELF-MF) and particularly in combination with various external factors, via comet assays. NIH3T3 mouse fibroblast cells, WI-38 human lung fibroblast cells, L132 human lung epithelial cells, and MCF10A human mammary gland epithelial cells were exposed for 4 or 16 h to a 60-Hz, 1 mT uniform magnetic field in the presence or absence of ionizing radiation (IR, 1 Gy), H2O2 (50 μM), or c-Myc oncogenic activation. The results obtained showed no significant differences between the cells exposed to ELF-MF alone and the unexposed cells. Moreover, no synergistic or additive effects were observed after 4 or 16 h of pre-exposure to 1 mT ELF-MF or simultaneous exposure to ELF-MF combined with IR, H2O2, or c-Myc activation.Biophysik 12/2013; · 1.70 Impact Factor
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ABSTRACT: Abstract Purpose: Genotoxic effects have been considered the gold standard to determine if an environmental factor is a carcinogen, but the currently available data for extremely low frequency time-varying magnetic fields (ELF-MFs) remain controversial. As an environmental stimulus, the effect of ELF-MF on cellular DNA may be subtle. Therefore, a more sensitive method and systematic research strategy are warranted to evaluate genotoxicity. Materials and methods: We investigated the effect of ELF-MFs in combination with ionizing radiation (IR) or H2O2 on the DNA damage response of expression of phosphorylated H2AX (γ-H2AX) and production of γ-H2AX foci in non-tumorigenic human cell systems consisting of human lung fibroblast WI38 cells and human lung epithelial L132 cells. Results: Exposure to a 60-Hz, 2 mT ELF-MFs for 6 h produced increased γ-H2AX expression, as well as γ-H2AX foci production, a common DNA double-strand break (DSB) marker. However, exposure to a 1 mT ELF-MFs did not have the same effect. Moreover, 2 mT ELF-MFs exposure potentiated the expression of γ-H2AX and γ-H2AX foci production when combined with IR, but not when combined with H2O2. Conclusions: ELF-MFs could affect the DNA damage response and, in combination with different stimuli, provide different effects on γ-H2AX.International Journal of Radiation Biology 01/2014; · 1.84 Impact Factor
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ABSTRACT: In the past century, there have been many attempts to treat cancer with low levels of electric and magnetic fields. We have developed noninvasive biofeedback examination devices and techniques and discovered that patients with the same tumor type exhibit biofeedback responses to the same, precise frequencies. Intrabuccal administration of 27.12 MHz radiofrequency (RF) electromagnetic fields (EMF), which are amplitude-modulated at tumor-specific frequencies, results in long-term objective responses in patients with cancer and is not associated with any significant adverse effects. Intrabuccal administration allows for therapeutic delivery of very low and safe levels of EMF throughout the body as exemplified by responses observed in the femur, liver, adrenal glands, and lungs. In vitro studies have demonstrated that tumor-specific frequencies identified in patients with various forms of cancer are capable of blocking the growth of tumor cells in a tissue- and tumor-specific fashion. Current experimental evidence suggests that tumor-specific modulation frequencies regulate the expression of genes involved in migration and invasion and disrupt the mitotic spindle. This novel targeted treatment approach is emerging as an appealing therapeutic option for patients with advanced cancer given its excellent tolerability. Dissection of the molecular mechanisms accounting for the anti-cancer effects of tumor-specific modulation frequencies is likely to lead to the discovery of novel pathways in cancer.Ai zheng = Aizheng = Chinese journal of cancer 11/2013; 32(11):573-581.
Epidemiological studies have suggested that extremely low-frequency magnetic fields (ELF-MF)
are associated with an increased incidence of cancer. Studies using in vitro systems have reported
mixed results for the effects of ELF-MF alone, and the World Health Organization (WHO) Re-
search Agenda published in 2007 suggested that high priority research should include an evalua-
tion of the co-carcinogenic effects of ELF-MF exposure using in vitro models. Here, the
carcinogenic potential of ELF-MF exposure alone and in combination with various stress factors
was investigated in NIH3T3 mouse fibroblasts using an in vitro cellular transformation assay.
NIH3T3 cells were exposed to a 60 Hz ELF-MF (1 mT) alone or in combination with ionizing
radiation (IR), hydrogen peroxide (H2O2), or c-Myc overexpression, and the resulting number of
anchorage-independent colonies was counted. A 4 h exposure of NIH3T3 cells to ELF-MF alone
produced no cell transformation. Moreover, ELF exposure did not influence the transformation
activity of IR, H2O2, or activated c-Myc in our in vitro assay system, suggesting that 1 mT ELF-
MF did not affect any additive or synergistic transformation activities in combination with stress
factors such as IR, H2O2, or activated c-Myc in NIH3T3 cells. Bioelectromagnetics 33:207–214,
? 2011 Wiley Periodicals, Inc.
Key words: extremely low frequency; combination with stress factors; cellular transformation
During the last two decades, concerns have ris-
en regarding a possible association between exposure
to extremely low-frequency magnetic fields (ELF-
MF) and the increased incidence of cancers such as
childhood acute leukemia, cancers of the nervous
system, and lymphomas. Epidemiological studies
have investigated the possible relationship between
to ELF-MF and the rate of cancers among those
exposed. In parallel with epidemiological studies,
mechanistic studies using in vitro and in vivo
model systems have been conducted to determine
whether a link exists between ELF-MF exposure
and mutagenesis, and to investigate possible mecha-
nisms of increased cancer risk. After conducting a
Grant sponsor: Power Generation and Electricity Delivery of the
Korea Institute of Energy Technology, Evaluation and Planning
(KETEP), funded by the Korean Ministry of Knowledge
Economy (Grantnumber: 2009101030003E);
Research Project of the Nuclear Research and Development
Program of the Korea Science and Engineering Foundation
(KOSEF), funded by the Korean government (MEST).
*Correspondence to: Yun-Sil Lee, College of Pharmacy and
Division of Life and Pharmaceutical Sciences, Ewha Womans
University, 11-1 Daehyun-Dong Seodaemun-Gu, Seoul 120-750,
Korea. E-mail: email@example.com
Received for review 23 November 2010; Accepted 26 July 2011
Published online 6 September 2011 in Wiley Online Library
comprehensive literature review that included epide-
miological reports, animal carcinogenicity data, and
the outcomes of in vitro studies [Testa et al., 2004;
Fatigoni et al., 2005; Ivancsits et al., 2005; Moretti
et al., 2005; Erdal et al., 2007], the International
Agency for Research on Cancer (IARC) has pro-
posed that ELF-MF be labeled as category 2B, a pos-
sible human carcinogen [IARC, 2002].
No carcinogenic effects of ELF-MF alone have
been reported in animal studies to date. Similarly, gen-
otoxicity studies have generally shown no effects of
ELF-MF alone, although extremely strong fields
(>50 mT) had effects. However, ELF-MF have been
shown to enhance the effects of known carcinogenic or
mutagenic chemicals or physical agents in animal and
in vitro studies [Kumlin et al., 1998; Harakawa et al.,
2005; Yokus et al., 2008; Kim et al., 2009, 2010;
Focke et al., 2010]. Furthermore, a systematic review
has suggested that the majority of such studies were
positive, supporting the conclusion that ELF-MF inter-
act with chemical and physical agents [Juutilainen
et al., 2000, 2006; Crumpton and Collins, 2004]. Stud-
ies using in vitro systems have reported mixed results
for the effects of ELF-MF, and the World Health
Organization (WHO) Research Agenda published in
2007 suggested that high priority research should in-
clude an evaluation of the co-carcinogenic effects of
ELF-MF exposure using in vitro models.
Anchorage independence determined as the
acquisition of colony-forming ability in a semisolid
medium provides a useful system for studying
neoplastic transformation. It is well known that
this characteristic is closely correlated with cellular
tumorigenicity [Suzuki et al., 1983]. Anchorage
dependence can be described in terms of primary
fibroblasts and other fibroblastic cell lines (e.g.,
BALB/c3T3, NIH-3T3, etc.) that must attach to a
solid surface before they can divide. They fail to
grow when suspended in a viscous fluid or gel (e.g.,
agar or agarose); however, when these cell lines are
transformed they are able to grow in a viscous fluid
or gel and become anchorage independent. The pro-
cess by which these phenotypic changes occur is
assumed to be closely related to the process of
in vivo carcinogenesis [Benigni and Bossa, 2011].
Because both anchorage-dependent and -independent
colonies are detected in traditional cell survival
assays, it is not difficult to differentiate between nor-
mal and tumorigenic cells. Therefore, these systems
are believed to be reasonably good predictors of
in vivo carcinogenic activity.
In this study, we evaluated the combined effects
of exposure to an ELF-MF and various stress factors
including ionizing radiation (IR), hydrogen peroxide,
and oncogenic c-Myc activation on cellular transfor-
mation in non-tumorigenic NIH3T3 cells, and found
that ELF-MF exposure with 1 mT for 4 h did not
increase any cellular transforming activity when
combined with these stress factors.
MATERIALS AND METHODS
ELF Magnetic Field Exposure System
The equipment for ELF-MF generation, shown
in Figure 1a,b, was designed and constructed by the
Korea Electrotechnology Research Institute (KERI,
Changwon, Korea). Magnetic field monitoring was
conducted by observing the current injected into the
exposure system because a magnetic field is propor-
tional to the injected current. The field generator con-
sisted of four square coils and one cage with three
testing floors (top, middle, and bottom floors). The
voltage fluctuation rate and harmonic rate of power
quality using a power amp was <1%.
Figure 1c shows the calculated magnetic field
distribution in the middle floor of the exposure sys-
tem. The magnetic field at the center of the middle
floor was fixed at 1 mT, and the fields at various
points were measured to determine the uniformity of
the ELF-MF exposure system (Table 1). The spatial
variation of the magnetic field was <3%, strongly
demonstrating that the field generator was applicable
for an in vitro study within a small area. Using a
water-jet cooling system, the temperature in the incu-
bator at 1 mT was maintained at 37 ? 0.3 8C during
the experimental period, as shown in Figure 1d. A
magnetic field shielding system using ferrite material,
which is commonly used for the shielding of low fre-
quency magnetic fields, was adopted to block strong
magnetic fields outside the ELF-MF exposure sys-
tem. The magnetic field exposure system generates
60 Hz magnetic fields up to 1 mT in the inside of
the system. This strong magnetic field should be
blocked by shielding materials, such as ferrite mate-
rials, to prevent it from leaking out of the exposure
system. Ferrite materials also block the external
magnetic fields from penetrating into the exposure
system. The shielding is above 90% at the power
Compared with the magnitude of the generated
magnetic fields by the exposure system, the magni-
tude of the AC background fields, which is usually
less than about 1 mG, is negligible and DC back-
ground fields are equal to the Earth’s magnetic field.
In Korea, the Earth’s magnetic field is about
350 mG. The entire ELF-MF exposure system, in-
cluding the incubator, is shown in Figure 1b.
Cell Culture and Transfection
NIH3T3 mouse fibroblast cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco–Invitrogen, Paisley, Scotland, UK) supple-
mented with 10% calf serum (Gibco–Invitrogen) at
37 8C in an incubator with a humidified atmosphere
of 95% air/5% CO2. Cells were transfected using
4 mg of c-Myc-V5 or pcDNA-V5 empty vector,
according to the manufacturer’s recommended proto-
col. Full-length c-Myc was cloned into the pcDNA3-
V5 plasmid, after which internal deletion mutants
were constructedin c-Myc-containing
vector (Invitrogen Life Technology, Carlsbad, CA)
in thec-Myc coding sequence
primers. This was followed by restriction and ligation
Dose Determinations for IR and H2O2Exposure
To determine the appropriate H2O2concentra-
tions and IR doses for examining the effects on cellu-
lar transformation, we cultured non-tumorigenic
NIH3T3 cells (2 ? 106/100 mm dish), incubated the
cells overnight, and then treated the cells with vari-
ous concentrations of H2O2for 4 h or various single
or fractionated doses of IR. We performed soft agar
assay using 5 ? 103cells per 60 mm dish after treat-
ment with IR or H2O2. The number of anchorage-
TABLE 1. Uniformity of ELF-MF Exposure System
Location 1 mT referenceUniformity
Fig.1. Schematic ofthe 60 Hz ELFmagnetic field exposure system for invitro studies. a:System
structure of the magnetic field generator. b: Sixty hertz magnetic field exposure system included
a power supply, magnetic field generating coils anda ferrite shielding systemin a CO2incubator.
c: ELF magnetic field distribution for the middle floor of the exposure system. d: Maintenance of
independent colonies was counted to determine cell
Exposure to ELF Magnetic Field, Irradiation,
Cells in 60 mm Petri dishes were placed in the
exposure chamber, allowed to equilibrate for 1 h, and
then exposed to a 60 Hz ELF-MF (0, 0.01, 0.5, or
1 mT) for 4 h. During the exposure time, the temper-
ature in the chamber was maintained at 37 ? 0.2 8C
by circulating water, and the temperature of the
culture medium was monitored every 10 min. For the
positive control, cells were exposed to various doses
of gamma radiation (0, 0.5, 1, 2, and 4 Gy as a single
or fractionated dose) from a137Cs gamma-ray source
(Elan 3000, Atomic Energy of Canada, Mississauga,
Canada) at a dose rate of 3.81 Gy/min. For H2O2
exposure, cells in 60 mm Petri dishes were treated
with H2O2(0, 50, 100, 150, and 200 mM) for 4 h.
For the combination experiments, there were three
conditions: cells exposed to an ELF-MF (1 mT) for
4 h, cells exposed to an ELF-MF (1 mT) for 4 h
immediately after irradiation with gamma rays
(2 Gy), and cells exposed to an ELF-MF (1 mT) for
4 h in the presence of H2O2(100 mM). Sham expo-
sure was sequentially performed just after the ELF-
MF exposure experiments using the same exposure
equipment without ELF-MF exposure.
Equal amounts of protein (40 mg) were dis-
solved in lysis buffer, the samples were boiled for
5 min, and the proteins were separated by electro-
phoresis in a 12% SDS–PAGE (sodium dodecyl
sulfate–polyacrylamide gel electrophoresis) gel. The
separated protein bands were transferred to a nitro-
cellulose membrane. After blocking with 5% skim
milk in PBS, the membrane was incubated with anti-
c-Myc antibody (1:1000 dilution; Santa Cruz Bio-
technology, Santa Cruz, CA) for 18 h at 4 8C,
washed, and then incubated with horseradish peroxi-
dase-conjugated anti-mouse secondary antibody. Im-
munoreactive bands were visualized using enhanced
chemiluminescence (Amersham International, Buck-
Soft Agar Transformation Assays
Cells (5 ? 103) in 2 ml of 0.9% low-melting
agarose were seeded over a layer of 1.8% agar in
DMEM with 10% calf serum and incubated for
21 days. Fresh DMEM without phenol red supple-
mented with 10% calf serum was added to the sur-
face of the agarose every 2–3 days. After 21 days,
the colonies were stained by adding 500 ml of PBS
containing 0.4% trypan blue to the surface of the
agarose and incubated for 2 h at 37 8C and 5% CO2.
The colonies consisting of >50 cells were counted
using a Fluorchem SP counter (Alpha Innotech, San
Leandro, CA). Each assay was performed in tripli-
cate and in more than three independent experiments.
The results are presented as mean ? SD.
Statistical analyses (one-way analysis of vari-
ance) were performed using SPSS software (SPSS
version 15.0, Chicago, IL). Statistical significance
was accepted for P values ?0.05.
Dose Dependency of Cellular Transformation by
IR and H2O2
For the examination of transforming activity,
we used NIH3T3 mouse fibroblast cells because
activity even in the weaker carcinogens [Isfort and
LeBoeuf, 1996; Mascolo et al., 2010]. Compared
with sham-exposed control cells, the cells exposed to
2 GyIRalone exhibited
Fig. 2. a: Anchorage-independent growth of NIH3T3 cellsin soft
agar after 4 h treatment of indicated concentrations of H2O2.
b:Exposuretoionizingradiation (IR) with a single or fractionated
dose.?Significantly different from untreated control cells at
P < 0.05(mean ? SD).
14.8 ? 0.9 colony-forming units (CFU) for control
and 2 Gy, respectively),
4 Gy IR slightly increased the colony number
(18.7 ? 4.6 CFU). There was no significant differ-
ence in the number of anchorage-independent colo-
nies between sham-exposed control cells and cells
exposed to 1 Gy IR. Within this group, transforma-
tion activity was higher with two exposures of 1 Gy
each than with a single exposure of 2 Gy. Cells ex-
posed to a fractionated dose totaling 4 Gy IR also
slightly increased colony number over cells exposed
to 4 Gy IR (21.4 ? 5.1, 2 Gy ? 2 vs. 18.7 ? 4.6,
4 Gy CFU). For a total dose of 4 Gy IR, transforma-
tion activity was similar between a single dose and
fractionated doses. However, for a total dose of 2 Gy,
transformation activity was higher with two expo-
sures of 1 Gy each than with a single 2 Gy exposure
(Fig. 2a). In the case of H2O2, transformation activity
was highest at 100 mM H2O2. Higher concentrations
of H2O2(150 and 200 mM) inhibited transformation
activity compared with that at 100 mM, suggesting
colonies(0.6 ? 1.1
cytotoxic effects (Fig. 2b). For subsequent experi-
ments we used 2 Gy IR or 100 mM H2O2.
ELF-MF Did Not Affect the Cellular Transformation
Activity Induced by IR or H2O2
When cells were exposed to ELF-MF alone,
the number of anchorage-independent colonies at ex-
posure intensities of 0.01, 0.5, and 1 mT were similar
to unexposed controls. All groups of CFU presented
few colonies (average 0.6 per plate) or none (data
not shown). To determine whether ELF-MF in com-
bination with IR or H2O2could affect cellular trans-
formation activity, NIH3T3 cells were treated with
2 Gy of fractionated IR (two doses of 1 Gy) or
100 mM H2O2in combination with exposure to an
ELF-MF (1 mT) for 4 h, and a cell transformation
assay was performed. As shown in Figure 3, the
IR or H2O2 did affect the number of anchorage-
independent colonies in NIH3T3 cells with and
without ELF-MF exposure (IR vs. IR þ ELF-MF:
P ¼ 0.287, 95% confidence interval (CI) ?2.66 to
0.857; H2O2vs. H2O2þ ELF-MF: P ¼ 0.709, 95%
CI ?2.95 to 1.79).
Fig. 3. a,b:Number of colonies with 4 h exposure of1 mT ELF with or without two doses of1 Gy
IR.c,d: Treatmentwith100 mMH2O2.b,d: TypicalmorphologyofcoloniesafterIRexposureorH2O2
treatmentisalsoshown(whitearrows).Scalebar ¼ 50 mm.?Significantlydifferentfromuntreated
controlcellsat P < 0.05 (mean ? SD).CON, unexposedcontrolcells; ELF,ELF-exposedcells; IR,
ionizingradiation-exposedcells;H2O2,H2O2-exposedcells;ELF þ IR,combinedexposureofELF
andIR;ELF þ H2O2,combinedexposureofELFandH2O2.
ELF-MF Did Not Affect Cell Transformation by
c-Myc Oncogene Activation
NIH3T3 cells overexpressing c-Myc (Fig. 4a)
produced a significantly increased number of colo-
nies produced by empty vector transfected control
cells (Fig. 4b). ELF-MF exposure had no effect on
the c-Myc-mediated transformation activity (c-Myc
vs. c-Myc with IR: P ¼ 0.870, 95% CI ?3.030 to
2.830). Moreover, IR exposure (two doses of 1 Gy)
significantly increased the transformation activity of
both c-Myc transfected and empty vector transfected
cells but this activity was not further affected by ex-
posure to ELF-MF (Fig. 4c; IR with c-Myc vs.
IR þ ELF with c-Myc: P ¼ 0.552, 95% CI ?3.630
The present study indicates that in well-con-
trolled experiments, ELF-MF alone did not induce
NIH3T3 cell transformation. Furthermore, ELF-MF
had no influence on the transformation activities of
stress factors such as IR, reactive oxygen species,
and oncogenic activation.
Many laboratory studies designed to detect
DNA and chromosomal damage reported mutational
events and increased transformation frequency in re-
sponse to ELF-MF exposure [National Radiological
Protection Board (NRPB), 1992; Tenforde, 1996;
Villarini et al., 2006; Ruiz-Go ´mez and Martı ´nez-
Morillo, 2009; Jime ´nez-Garcı ´a et al., 2010]. More-
over, recent papers also suggested that repetitive
ELF-MF exposure with 6 mT decreased cell viability
[Kim et al., 2010], and intermittent exposure of hu-
man primary fibroblasts to a 50 Hz ELF-MF at a flux
density of 1 mT induced a slight but significant in-
crease of DNA fragmentation in the Comet assay
[Focke et al., 2010]. However, there are some nega-
tive results of ELF-MF alone without initiators [Erdal
et al., 2008; Di Loreto et al., 2009; Akdag et al.,
2010] and the lack of an effect on the chromosome
structure suggests that if ELF-MF were to influence
carcinogenesis, they would be more likely to act as a
promoter to enhance the proliferation of genetically
altered cells rather than as an initiator to cause
Fig. 4. a:Imunoblottingofc-Mycproteinafter transfectionofempty vector (V5) orc-Mycwasper-
formed in NIH3T3 cells. b: Cellular transformation assay in control and V5 or c-Myc transfected
NIH3T3cells.c:Morphologyandd:numberofcolonieswithexposureto 4 hELF(1 mT),2 GyIR,or
ELF þ IRinc-Myctransfectedand V5 transfected cells.Scalebar ¼ 50 mm.?Significantlydiffer-
ent fromcorrespondinguntreatedcontrolcellsatP < 0.05(mean ? SD).CON,unexposedcontrol
ELF, ELF-exposed cells; IR, ionizing radiation-exposed cells; ELF þ IR, combined exposure of
the original lesion in DNA or chromatin. Indeed,
exposure to an ELF-MF (5 mT) in combination with
the genotoxic agents, methyl methane sulfonate
(MMS) or H2O2, increased the number of apurinic/
apyrimidinic (AP) sites compared with the genotoxic
agents alone [Koyama et al., 2008].
Therefore, further studies are needed to investi-
gate the possible effects of ELF-MF exposure on the
promotion and progression of tumor development
following initiation by a chemical or environmental
carcinogen; the WHO Agenda also recommended the
priority of these types of studies [WHO, 2007]. The
present results, in combination with IR, H2O2, and
c-Myc oncogenic activation, did not suggest a trans-
formation effect of ELF-MF exposure at 1 mT, a
level below Korean exposure limits. Exposure levels
of ELF-MF higher than 1 mT may affect transforma-
tion activity in combination with stress factors; how-
ever, this does not reflect the real exposure life.
Therefore, these results might be more related to res-
idential or occupational exposure situations. Al-
though the negative results obtained in the present
study are specific for NIH3T3 cells under the condi-
tions tested, the failure of ELF-MF exposure to
induce transformation in a relatively sensitive cell
culture system argues against an increased cancer
risk in those exposed to ELF-MF.
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