INFLUENCE OF NON-THERMAL 900 MHZ MOBILE PHONE RADIATION ON
MORPHOLOGY, METABOLIC ACTIVITY, CELL CYCLE PROGRESSION, APOPTOSIS
INDUCTION AND GLOBAL GENE EXPRESSION IN BOTH BREAST ADENOCARCINOMA
AND NORMAL BREAST EPITHELIAL CELL LINES
Sumari Marais1, Barend A. Stander1, Carin Huyser2, F le R Fourie3, Dariusz Leszczynski4, Annie M. Joubert1
1 Department of Physiology, University of Pretoria, Pretoria, South Africa; 2 Reproductive Biology Laboratory, Department of
Obstetrics and Gynaecology, University of Pretoria, Pretoria, South Africa; 3 South African Bureau of Standards, Pretoria, South
Africa; 4 Functional Proteomics Group, Radiation Biology Laboratory, STUK Radiation and Safety Authority, Helsinki, Finland
INTRODUCTION
Mobile phones and other hand-held type transceivers are widely used in the world and mobile phone utilization currently exceeds landline
communication in Africa. This has raised concerns about the long-term health effects of their ongoing ever-increasing usage. While there is no current
evidence that cell phones pose a significant health risk, there is also no proof that they are risk free. To date, various in vitro models such as cervical
carcinoma (HeLa), Chinese hamster ovary (CHO), immortalized human umbilical vein endothelial cells (EA.hy926), murine lymphoma (L5178Y), human
lung epithelial cell line (L-132) and (mouse embryonic fibroblast) C3H 10T1/2 cells have been used to study the impact of radio frequency (RF) emissions.
Studying the cellular effects as well as identifying the genes differentially expressed in electromagnetic field (EMF)-exposed cells could provide direct
evidence for biological effects of EMF.
METHODS
MCF-7 and MCF-12A cells were seeded and incubated for 24h to allow for attachment. After attachment the cells were incubated in a vertical GSM900
cell exposure chamber and exposed to 2W/kg non-thermal 900 MHz mobile phone radiation for 1h. Cell morphology was assessed employing light
microscopy and fluorescent microscopy by utilizing haematoxylin and eosin (H&E) staining, Hoechst 33342, propidium iodide (PI) nuclear stains and
phalloidin respectively. Mitotic indexes were determined by counting 1000 cells in triplicate of negative control and exposed cells. Viable and
metabolically active cells were determined by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry
analyses were performed utilizing propidium iodide and Annexin V-FITC for cell cycle progression and apoptosis detection respectively. Analysis were
performed with FC-500 and CXP software from Beckman Coulter. Agilent’s Human 1A Oligo Microarray slides with 20,173 known human 60-mer
oligonucleotide probes and the 44K whole human genome microarray slides with 410000+ unique human genes and transcripts represented were
employed. Microanalyses were conducted with GenePix Pro 6 and the Linear Models for Microarray Data (Limma) package from Bioconducter. The
hybridized slides were scanned with the Axon Genepix 4000B Scanner. With Limma, background correction, Global loess normalization within arrays,
Quantile normalization between arrays and the Least Squares linear model fit were performed on each slide with a B-value cut-off of 0.01. Statistically
significantly differentially expressed genes were mapped to metabolic pathways and Gene Ontology (GO) categories by using FATIGO.
RESULTS
MCF-7
MCF-12A
CELL VIABILITY AND MITOTIC INDEX ANALYSIS
CELL MORPHOLOGY
CELL MORPHOLOGY
M CF-12A me tabolic activ ity (M TT) (av e rage of 3
re pe ats)
140
% Cell growth
120
100
80
60
Control
1 Hour exposure
40
20
0
Figure 1b 60 min
2W/kg
non-thermal
900
MHz
mobile
phone
radiationexposed MCF-7 cells
stained with Hoechst
33342 and PI. No
apparent qualitative
changes to nuclear
morphology
were
observed.
Figure 2a 60 min
negative control MCF7 cells stained with
H&E.
Figure 3 Cell viabilty of 2W/kg nonthermal 900 MHz mobile phone
radiation-exposed
MCF-7
cells
compared to negative control cells. A
statistically insignificant increase in
dehydrogenase activity was observed
in exposed cells.
Figure 4 Cell viability of 2W/kg nonthermal 900 MHz mobile phone
radiation-exposed
MCF-12A
cells
compared to negative control cells. A
statistically insignificant decrease in
dehydrogenase activity was observed
in exposed cells.
CELL CYCLE ANALYSIS
14
Figure 5b 60 min
2W/kg
non-thermal
900
MHz
mobile
phone
radiationexposed
MCF-12A
cells stained with
Hoechst 33342 and
PI.No
apparent
qualitative changes to
nuclear morphology
were observed.
Prophase
12
Figure 6a 60 min
negative control MCF12A cells stained with
H&E.
Figure 6b 60 min
2W/kg non-thermal
900
MHz
mobile
phone
radiationexposed
MCF-12A
cells stained with
H&E. No apparent
qualitative changes
were observed.
CELL CYCLE ANALYSIS
Metaphase
10
Anaphase
8
6
Telophase
4
Cell Death
2
0
MCF-7 (1h) Control
MCF-7 (1h) Exp
Figure 8 Mitotic index comparison of negative
control vs 2W/kg non-thermal 900 MHz mobile
phone
radiation-exposed
MCF-7
cells.
Statistically insignificant increases of cells in
anaphase and telophase were observed and
was confirmed with flow cytometry.
Figure 7a Cell cycle histogram
of negative control MCF-7
cells.
Figure 5a 60 min
negative control MCF12A cells stained with
Hoechst 33342 and
PI.
Mitotic Index (G2/M phase)
% of 1000 counted cells
Figure 1a 60 min
negative
control
MCF-7 cells stained
with Hoechst 33342
and PI.
Figure 2b 60 min
2W/kg
non-thermal
900
MHz
mobile
phone
radiationexposed MCF-7 cells
stained with H&E. No
apparent qualitative
changes
were
observed.
Figure 9 Mitotic index comparison of
negative control vs 2W/kg non-thermal
900 MHz mobile phone radiation-exposed
MCF-12A cells. Statistically insignificant
increase of cells in apoptosis were
observed.
APOPTOSIS ANALYSIS
Figure 7b Cell cycle histogram of
2W/kg non-thermal 900 MHz
mobile phone radiation-exposed
MCF-7 cells.
Figure
10a
Cell
cycle
histogram of negative control
MCF-12A cells.
Figure 10b Cell cycle histogram
of 2W/kg non-thermal 900 MHz
mobile phone radiation-exposed
MCF-12A cells.
MICROARRAY ANALYSIS
Table 1. Selected differentially expressed genes (gene name in brackets) of interest in MCF-7 cells
after 1 hour exposure to 2W/kg non-thermal 900 MHz mobile phone radiation exposure revealed by
cDNA microarray and bioinformatics analyses.
Figure 11a PI (FL3 Log) vs
Annexin V (FL1 Log) dot-plot
of negative control MCF-7
cells.
Figure 11b PI (FL3 Log) vs
Annexin V (FL1 Log) dot-plot of
2W/kg non-thermal 900 MHz
mobile phone radiation-exposed
MCF-7 cells.
Figure 12a PI (FL3 Log) vs
Annexin V (FL1 Log) dot-plot
of negative control MCF-12A
cells.
Figure 12b PI (FL3 Log) vs
Annexin V (FL1 Log) dot-plot of
2W/kg non-thermal 900 MHz
mobile phone radiation-exposed
MCF-12A cells.
DISCUSSION AND CONCLUSION
Table 2. Selected differentially expressed
genes (gene name in brackets) of interest in
MCF-12A cells after 1 hour exposure to 2W/kg
non-thermal 900 MHz mobile phone radiation
exposure revealed by cDNA microarray and
bioinformatics analyses.
No statistically significant differences were observed between the 2W/kg non-thermal 900 MHz mobile phone radiationexposed MCF-12A or MCF-12A cells when compared to negative control cells with regard to cell morphology, viability, cell
cycle, mitotic index and apoptotic cells, as a result, more sensitive microarray and bioinformatics analyses were
employed.
Microarray analyses and bioinformatics analyses revealed 31 differentially expressed genes in the MCF-7 and 19 genes in
the MCF-12A cell line. Genes involved in DNA repair in the MCF-7 cells include excision repair cross-complementing rodent
repair deficiency complementation group 4 (ERCC4), DNA cross-link repair 1C (DCLRE1C) and poly (ADP-ribose)
polymerase family member 2 (PARP2) and chromatin assembly factor 1 subunit B (CHAF1B). Genes involved in cell
differentiation, namely epithelial membrane protein 2 (EMP2), germ cell-less homolog 1 (GMCL1) and BarH-like homeobox 1
(BARX1) were down regulated in the MCF-12A cells.
However, the Agilent 22K and 44K slides revealed no gene overlapping between experiment or platforms. Thus, this
preliminary study revealed possible differentially expressed genes in both the MCF-7 and MCF-12A cell lines after 1 hour
exposure of 2W/kg non-thermal 900 MHz mobile phone radiation. Confirmation of these findings with qRT-PCR and
proteomics techniques is needed to verify results.
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