Mobile phone radiation might alter protein expression in human skin

Abstract

Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people.
Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies.
This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers.

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BMC Genomics
Open Access
Research article
Mobile phone radiation might alter protein expression in human
skin
Anu Karinen, Sirpa Heinävaara, Reetta Nylund and Dariusz Leszczynski*
Address: STUK – Radiation and Nuclear Safety Authority, Laippatie 4, 00880 Helsinki, Finland
Email: Anu Karinen - anu.karinen@stuk.fi; Sirpa Heinävaara - sirpa.heinavaara@stuk.fi; Reetta Nylund - reetta.nylund@stuk.fi;
Dariusz Leszczynski* - dariusz.leszczynski@stuk.fi
* Corresponding author
Abstract
Background: Earlier we have shown that the mobile phone radiation (radiofrequency modulated
electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does
not mean that similar response will take place in human body exposed to this radiation. Therefore,
in this pilot human volunteer study, using proteomics approach, we have examined whether a local
exposure of human skin to RF-EMF will cause changes in protein expression in living people.
Results: Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific
absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-
exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein
expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that
were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were
present in all 10 volunteers. This suggests that protein expression in human skin might be affected
by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected
proteins observed in our earlier in vitro studies.
Conclusion: This is the first study showing that molecular level changes might take place in human
volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening
approach can identify protein targets of RF-EMF in human volunteers.
Background
Physiological functions of human body are regulated by
electric currents. Therefore, is not surprising that placing
human body within electromagnetic field, of sufficient
strength, may affect physiological processes. The possibil-
ity of induction of biological and health effects by low
energy radiation emitted by mobile phones (radiofre-
quency-modulated electromagnetic fields: RF-EMF)
remains a controversial issue. In spite of years of research,
there is still ongoing discussion whether RF-EMF could
induce any physiologically relevant effects [1]. The vast
majority of the so far conducted research has focused on
cancer. However, RF-EMF is also suspected as potential
cause of such ailments as sleep disorders, headaches or
allergy-like symptoms [2].
We have proposed that proteomics screening may be used
to reveal molecular targets of RF-EMF and help to under-
stand the possible biochemical mechanism of the RF-
EMF-induced effects [3]. Our earlier proteomics studies
have shown that changes in protein expression and activ-
ity (phosphorylation) were induced in human endothe-
Published: 11 February 2008
BMC Genomics 2008, 9:77 doi:10.1186/1471-2164-9-77
Received: 13 November 2007
Accepted: 11 February 2008
This article is available from: http://www.biomedcentral.com/1471-2164/9/77
© 2008 Karinen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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lial cell line EA.hy926 that was exposed to RF-EMF [4-7].
These in vitro observed effects, however, do not automat-
ically mean that similar changes would happen in the cells
of mobile phone users. Therefore, the present pilot study
was undertaken to determine whether a local exposure of
human skin to RF-EMF will induce any changes in protein
expression and whether it will be possible to find com-
mon protein(s) that respond to RF-EMF in all volunteers.
Results
Ethical permit to perform this study was obtained from
the Ethics Committee of Department of Surgery of Hospi-
tal District of Helsinki and Uusimaa, Finland. A small skin
area of a forearm of 10 of same sex (female) volunteers
(age 27 – 65 years; mean 51 years) were irradiated for 1
hour with 900 MHz GSM signal at specific absorption rate
(SAR) of 1.3 W/kg, using specially designed exposure
setup [8]. The mobile phone safety limit SAR is 2.0 W/kg,
as recommended by the International Commission on
Non-Ionizing Radiation Protection (ICNIRP).
Immediately after the exposure, a punch biopsy of the
exposed area of skin (experimental sample) was taken by
a physician. Another punch biopsy was taken from the
other, non-exposed, forearm (sham sample). In this
experimental set-up each volunteer acted as its own sham
control. Both exposed and non-exposed skin samples of
all volunteers were immediately snap-frozen in liquid
nitrogen and stored before extraction of proteins.
Proteins from all samples were extracted using TRIzol Rea-
gent (Invitrogen) and separated using 2-dimensional gel
electrophoresis (2-DE) with pH gradient range of 4–7 in
the first dimension and 9% SDS-PAGE gel in the second
dimension (GE Healthcare). Proteins were detected by sil-
ver staining and spot distribution pattern was analyzed
using PDQuest 7.2 software (Bio-Rad).
We have analyzed a fragment of proteome: proteins with
the isoelectric point (pI) 4–7 and the molecular weight
<40 kDa, because the protein spot separation in 2-DE in
this area was clearly distinguishable (Figure 1). Firstly,
using PDQuest software, for each volunteer was generated
an artificial gel, by combining protein expression profiles
from sham and exposed samples. Thereafter, all 10 artifi-
cial gels were combined into single artificial master gel
Artificial master gel for all sham and exposed skin samples of all 10 volunteersFigure 1
Artificial master gel for all sham and exposed skin samples of all 10 volunteers. Statistically significantly affected protein spots
are marked in red color (declined expression) and in green color (increased expression).
3701
8214
6610
4311
3417
7309
4801
4807
47
Isoelectric point (pI)
Molecular weight
< 40 kDa

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and the differentially expressed protein spots, which were
detected in at least 4 volunteers, were statistically ana-
lyzed.
The ratio of exposed and sham sample expression was
analyzed spot by spot, after logarithm transformation,
with variance analysis (anova). Due to small numbers and
potential violations of model assumptions, the ratios
were also studied with the Wilcoxon test. The statistical
analysis has identified 8 differentially expressed proteins
where the change in expression was statistically significant
among the 579 identified proteins spots (Table 1). Two of
the protein spots (#3701, #4801) were present in all 10
volunteers thus showing that it is possible to find com-
mon, responding proteins among the all volunteers. The
p-values are not adjusted for multiple comparisons.
Discussion
Proteomics approach to study effects of mobile phone
radiation on cells has been used so far only by two
research groups, ours in Helsinki and group the Zhejiang
University, Hangzhou, China. Our studies, using human
endothelial cell lines have shown that mobile phone radi-
ation induces statistically significant changes in the
expression of several tens of proteins [6] and that the
response of cell might be proteome-dependent [7]. In one
study, the group in China has not found statistically sig-
nificant differences in protein expression in MCF-7 cells
[9]. The reason for it might be too low number of experi-
ments in MCF-7 study [9]. In our studies statistical analy-
sis was based on 10 different experiments whereas Zeng et
al. [9] based their analysis on only three replicates.
Another reason for the difference might be different sensi-
tivity of MCF-7 cells as compared with ours endothelial
cell lines EA.hy926 and EA.hy926v1. In the other study
from Zhejiang University [10] were found 4 differentially
expressed proteins in lens epithelial cells, among them the
stress response protein Hsp70.
The obtained results, suggesting effect of mobile phone
radiation on protein expression in human cell lines, do
not automatically mean that this exposure will have any
effect on protein expression in humans. The so far con-
ducted human volunteer studies have focused on cogni-
tive responses to RF-EMF [2] and there is no information
available about the proteome, as well as transcriptome,
response to mobile phone radiation in humans. This
study is, to our knowledge, the first one where human
response to RF-EMF was examined on molecular level.
Our results suggest that human skin might respond to RF-
EMF and change protein expression profile. Interestingly,
when adjusting results of our previous cellular study [6]
using the size of proteome analyzed in the present study
(pI 4–7; <40 kDa) the number of the statistically signifi-
cantly affected proteins appears to be similar in this and
in earlier [6] study, 8 spots and 9 spots, respectively. The
number of differentially expressed protein spots in both
studies is below the number of expected false positives.
However, as we have demonstrated experimentally [6]
and discussed previously [11] it is likely that some of the
proteins will be indeed, real positives. However, without
further testing, it is not possible to predict whether these
changes will have impact on skin physiology.
Finally, our study confirms that the proposed by us pro-
teomics approach [3] can identify protein targets of RF-
EMF. This approach to EMF research has been subse-
quently accepted by the EMF scientists [12,13] and has
been included into the 2006 World Health Organization
Research Agenda [14]. However, new and larger study is
urgently needed to strengthen our pilot observations and
to determine what impact mobile phone exposure might
have on human tissues.
Conclusion
é Mobile phone radiation might alter protein expression
in human skin.
Table 1: List of proteins that were present in at least 4 volunteers and which expression has been changed in statistically significant
manner (<0.05) as determined by the variance analysis and the Wilcoxon test. Ratio = exposed sample expression/sham sample
expression.
Protein spot # Prevalence of spot* Average of
ratio
Standard
deviation of ratio
Median of
ratio
Anova p-value Wilcoxon p-value
1. 3417 4 117.35 157.93 1.00 0.0370
+
0.0474
2. 3701 10 0.68 0.39 0.49 0.0139 0.0367
3. 4311 7 68.88 134.67 1.71 0.0369 0.0169
4. 4801 10 1.89 1.01 1.42 0.0162 0.0218
5. 4807 5 223.17 302.78 1.53 0.0307
+
0.0277
6. 6610 4 39.86 52.05 1.00 0.0371
+
0.0474
7. 7309 8 67.38 101.44 12.17 0.0034 0.0076
8. 8214 4 146.60 252.75 1.00 0.0391
+
0.0474
* prevalence shows in how many volunteers, out of 10, was detected given protein spot;
+
variance analysis might be unreliable because of small numbers of cases and potential violations of model assumptions;

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é Physiological significance of this change is not known
and requires further study.
é Larger human volunteer study will be needed to confirm
results of this pilot study.
é Proteomics screening is valid method for search for
molecular targets of mobile phone radiation. Without this
approach the identification of the proteins responding to
mobile phone radiation would not be reasonably possi-
ble.
Methods
Ethical issues
Ethical permit to perform this study was obtained, in
accordance with the Helsinki Declaration, from the Ethics
Committee of the Department of Surgery of the Hospital
District of Helsinki and Uusimaa, Finland (decision
#127/2005 issued on November 23, 2005). Each volun-
teer was informed in detail about all experimental proce-
dures and each of them has signed the informed consent
form (in Finnish language).
Exposure of volunteers to mobile phone radiation
Volunteers were exposed to 900 MHz GSM mobile phone
radiation in an experimental setup described in detail
elsewhere [8]. The source of irradiation was a half-wave
dipole fed with a computer controlled GSM phone. The
specific absorption rate (SAR) induced in the skin was 1.3
W/kg what is below the ICNIRP safety guidelines (2.0 W/
kg). During the exposure small area of the right forearms
was irradiated for one hour. The other, non-irradiated
forearm was used as sham control. Immediately after
exposure skin punch-biopsies were taken from the
exposed and non-exposed skin for protein analysis.
Protein extraction from skin biopsies
Skin punch biopsies, consisting of both dermis and epi-
dermis but without the underlying fat tissue, were frozen
immediately after harvesting in liquid nitrogen and stored
at -80°C. Isolation and separation of proteins were per-
formed in the blinded manner. Proteins were isolated
from frozen skin using TRIzol
®
reagent protocol as
described by the manufacturer (Invitrogen, Carlsbad, CA,
USA) with a few modifications. Briefly, the chopped skin
punch-biopsies were immersed in 0.5 ml of ice-cold TRI-
zol reagent and homogenized on ice with 70 strokes of the
pestle in DUALL 1 ml tissue grinder (Kimble Chase Life
Science and Research Products, Vineland, NJ, USA). After
the phase separation of TRIzol reagent, the organic phase
containing DNA and proteins was collected. DNA was
then precipitated with ethanol and proteins were isolated
from the phenol-ethanol supernatant. The proteins were
then precipitated by isopropyl alcohol and pelleted at
12000 × g for 10 min at +4°C. The protein pellet was
washed 3 times with 0.3 M guanidine hydrochloride solu-
tion in 95% ethanol and once with 99.5% ethanol. Dur-
ing the extraction pellets were grinded with pellet pestle in
order to improve the solubility of the proteins. After each
wash step, proteins were centrifuged 7500 × g for 5 min at
+4°C. The air-dried protein pellet was dissolved in 2-DE
rehydration buffer containing 9 M urea, 2% (w/v) CHAPS,
0.5% (v/v) IPG buffer pH 4–7 and 5 mg/ml DTT (added
as fresh). The protein concentration of sample was meas-
ured using the Bradford method. The samples were stored
at -80°C.
Protein separation with 2-DE
Proteins were separated by standard 2-DE. Briefly, the first
dimension was performed in IPGphor™ (GE Healthcare,
UK) isoelectric focusing (IEF) apparatus. Linear, 24 cm
long, pH 4–7 Immobiline™ DryStrip gels (IPG-strips, GE
Healthcare, UK) were rehydrated in the strip holders for 4
hours in 0.45 ml rehydration buffer containing 9 M urea,
2% (w/v) CHAPS, 0.5% (v/v) IPG-buffer pH 4–7, 1.2%
(v/v) DeStreak™ reagent, a trace of bromophenol blue and
150 μg of total amount of protein. IEF was carried out at
+20°C using following step-and-hold settings: 50 V, 8 h;
100 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 2000 V, 1 h; 8000 V,
until 95000 Vh was achieved. Then, the IPG-strips were
incubated at room temperature in equilibration buffer
(50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol,
2% (w/v) SDS, a trace of bromophenol blue, and 10 mg/
ml DTT) for 15 min and for another 15 min in the same
buffer that contained 25 mg/ml of iodoacetamide instead
of DTT. The second-dimension separation was performed
using 9%SDS-PAGE gels. Electrophoresis was carried out
at +10°C using an Ettan™ DALTsix electrophoresis unit
(GE Healtcare) at a constant power of 3.5 W/gel for 0.5 h
and then 13 W/gel until the dye front reached the bottom
of the gel (about 4 h). The ready gels were silver stained to
visualize protein spots. Stained gels were scanned into
computer using GS-710 Calibrated Imaging Densitometer
(Bio-Rad Laboratories, Hercules, CA, USA). The gels were
analyzed using PDQuest 7.2 software (Bio-Rad).
Abbreviations
CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-
propanesulfonate; 2-DE, 2-dimensional electrophoresis;
DTT, dithiothreitol; EA.hy926, human endothelial cell
line; ICNIRP, International Commission on Non-Ioniz-
ing Radiation Protection; IEF, isoelectric focusing; IPG,
immobilized pH gradient; MCF-7, human breast adeno-
carcinoma cell line; pI, isoelectric point; RF-EMF, radiofre-
quency modulated electromagnetic field; SAR, specific
absorption rate; SDS, sodium dodecyl sulfate; SDS-PAGE,
sodium dodecyl sulfate polyacrylamide gel electrophore-
sis; Tris-HCl, Tris(hydroxymethyl)aminomethane hydro-
chloride;

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Authors' contributions
AK executed the proteomics experiments and performed
analysis of the proteomics data. RN assisted in designing
of the study, participated in writing the grant funding the
study, assisted in analysis of proteomics data. SH per-
formed statistical analysis of the data. DL conceived and
designed the study, obtained grant funding the study,
coordinated execution and analysis of the results and
wrote the draft manuscript. All authors participated in the
writing of the final version of the manuscript, read it and
approved it.
Acknowledgements
We thank Dr. J. Halttunen (Central Hospital of the University of Helsinki)
for taking skin biopsies. Funding was provided by Tekes – Finnish Funding
Agency for Technology and Innovation (HERMO project) and by STUK-
Radiation and Nuclear Safety Authority.
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