Sumari MaraisSumari Marais11, Barend A. Stander, Barend A. Stander11, Carin Huyser, Carin Huyser22, F le R Fourie, F le R Fourie33, , Dariusz LeszczynskiDariusz Leszczynski44, Annie M. Joubert, Annie M. Joubert11

1 1 Department of Physiology, University of Pretoria, Pretoria, South Africa; Department of Physiology, University of Pretoria, Pretoria, South Africa; 22 Reproductive Biology Laboratory, Department of Reproductive Biology Laboratory, Department of Obstetrics and Gynaecology, Obstetrics and Gynaecology, University of Pretoria, Pretoria, South Africa; University of Pretoria, Pretoria, South Africa; 33 South African Bureau of StandardsSouth African Bureau of Standards, Pretoria, South , Pretoria, South Africa; Africa; 44 Functional Proteomics Group, Radiation Biology Laboratory, STUK Radiation and Safety Authority, Helsinki, Finland Functional Proteomics Group, Radiation Biology Laboratory, STUK Radiation and Safety Authority, Helsinki, Finland

INFLUENCE OF NON-THERMAL 900 MHZ MOBILE PHONE RADIATION ON INFLUENCE OF NON-THERMAL 900 MHZ MOBILE PHONE RADIATION ON MORPHOLOGY, METABOLIC ACTIVITY, CELL CYCLE PROGRESSION, APOPTOSIS MORPHOLOGY, METABOLIC ACTIVITY, CELL CYCLE PROGRESSION, APOPTOSIS INDUCTION AND GLOBAL GENE EXPRESSION IN BOTH BREAST ADENOCARCINOMA INDUCTION AND GLOBAL GENE EXPRESSION IN BOTH BREAST ADENOCARCINOMA AND NORMAL BREAST EPITHELIAL CELL LINESAND NORMAL BREAST EPITHELIAL CELL LINES

INTRODUCTION

METHODS

CELL MORPHOLOGY

CELL CYCLE ANALYSIS

MICROARRAY ANALYSIS

MCF-7 MCF-12A

Mobile phones and other hand-held type transceivers are widely used in the world and mobile phone utilization currently exceeds landline 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 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 evidence that cell phones pose a significant health risk, there is also no proof that they are risk free. To date, various in vitroin vitro models such as cervical models such as cervical carcinoma (HeLa), Chinese hamster ovary (CHO), immortalized human umbilical vein endothelial cells (EA.hy926), murine lymphoma (L5178Y), human 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. 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 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. evidence for biological effects of EMF.

MCF-7 and MCF-12A cells were seeded and incubated for 24h to allow for attachment. After attachment the cells were incubated in a 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 vertical GSM900 cell exposure chamber cell exposure chamber and exposed to 2W/kg non-thermal 900 MHz mobile phone radiation for 1h. Cell morphology was assessed employing light 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 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 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 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 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 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 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 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, 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 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. significantly differentially expressed genes were mapped to metabolic pathways and Gene Ontology (GO) categories by using FATIGO.

Figure 1a 60 min Figure 1a 60 min negative control negative control MCF-7 cells stained MCF-7 cells stained with Hoechst 33342 with Hoechst 33342 and PI. and PI.

Figure 1b 60 min Figure 1b 60 min 2W/kg non-thermal 2W/kg non-thermal 900 MHz mobile 900 MHz mobile phone radiation- phone radiation- exposed MCF-7 cells exposed MCF-7 cells stained with Hoechst stained with Hoechst 33342 and PI. No 33342 and PI. No apparent qualitative apparent qualitative changes to nuclear changes to nuclear morphology were morphology were observed. observed.

Figure 2b 60 min Figure 2b 60 min 2W/kg non-thermal 2W/kg non-thermal 900 MHz mobile 900 MHz mobile phone radiation-phone radiation-exposed MCF-7 cells exposed MCF-7 cells stained with H&E. No stained with H&E. No apparent qualitative apparent qualitative changes were changes were

observed.observed.

Figure 2a 60 min Figure 2a 60 min negative control MCF-negative control MCF-7 cells stained with 7 cells stained with H&E.H&E.

Figure 5a 60 min Figure 5a 60 min negative control MCF-negative control MCF-12A cells stained with 12A cells stained with Hoechst 33342 and Hoechst 33342 and PI. PI.

Figure 5b 60 min Figure 5b 60 min 2W/kg non-thermal 2W/kg non-thermal 900 MHz mobile 900 MHz mobile phone radiation-phone radiation-exposed MCF-12A exposed MCF-12A cells stained with cells stained with Hoechst 33342 and Hoechst 33342 and PI.No apparent PI.No apparent qualitative changes to qualitative changes to nuclear morphology nuclear morphology were observed. were observed.

Figure 6b 60 min Figure 6b 60 min 2W/kg non-thermal 2W/kg non-thermal 900 MHz mobile 900 MHz mobile phone radiation-phone radiation-exposed MCF-12A exposed MCF-12A cells stained with cells stained with H&E. No apparent H&E. No apparent qualitative changes qualitative changes

were observed.were observed.

Figure 6a 60 min Figure 6a 60 min negative control MCF-negative control MCF-12A cells stained with 12A cells stained with H&E.H&E.

CELL VIABILITY AND MITOTIC INDEX ANALYSIS

APOPTOSIS ANALYSIS

MTT (average of 3 repeats)

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Figure 3 Cell viabilty of 2W/kg non-Figure 3 Cell viabilty of 2W/kg non-thermal 900 MHz mobile phone thermal 900 MHz mobile phone radiation-exposed MCF-7 cells radiation-exposed MCF-7 cells compared to negative control cells. A compared to negative control cells. A statistically insignificant increase in statistically insignificant increase in dehydrogenase activity was observed dehydrogenase activity was observed in exposed cells.in exposed cells.

Figure 4 Cell viability of 2W/kg non-Figure 4 Cell viability of 2W/kg non-thermal 900 MHz mobile phone thermal 900 MHz mobile phone radiation-exposed MCF-12A cells radiation-exposed MCF-12A cells compared to negative control cells. A compared to negative control cells. A statistically insignificant decrease in statistically insignificant decrease in dehydrogenase activity was observed dehydrogenase activity was observed in exposed cells.in exposed cells.

MCF-12A metabolic activity (MTT) (average of 3 repeats)

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Figure 8 Mitotic index comparison of negative Figure 8 Mitotic index comparison of negative control vs 2W/kg non-thermal 900 MHz mobile control vs 2W/kg non-thermal 900 MHz mobile phone radiation-exposed MCF-7 cells. phone radiation-exposed MCF-7 cells. Statistically insignificant increases of cells in Statistically insignificant increases of cells in anaphase and telophase were observed and anaphase and telophase were observed and was confirmed with flow cytometry.was confirmed with flow cytometry.

Figure 9 Mitotic index comparison of Figure 9 Mitotic index comparison of negative control vs 2W/kg non-thermal negative control vs 2W/kg non-thermal 900 MHz mobile phone radiation-exposed 900 MHz mobile phone radiation-exposed MCF-12A cells. Statistically insignificant MCF-12A cells. Statistically insignificant increase of cells in apoptosis were increase of cells in apoptosis were observed.observed.

Mitotic Index (Interphase)

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Figure 10a Cell cycle Figure 10a Cell cycle histogram of negative control histogram of negative control MCF-12A cells.MCF-12A cells.

Figure 10b Cell cycle histogram Figure 10b Cell cycle histogram of 2W/kg non-thermal 900 MHz of 2W/kg non-thermal 900 MHz mobile phone radiation-exposed mobile phone radiation-exposed MCF-12A cells.MCF-12A cells.

Figure 7a Cell cycle histogram Figure 7a Cell cycle histogram of negative control MCF-7 of negative control MCF-7 cells.cells.

Figure 7b Cell cycle histogram Figure 7b Cell cycle histogram of 2W/kg non-thermal 900 MHz of 2W/kg non-thermal 900 MHz mobile phone radiation-exposed mobile phone radiation-exposed MCF-7 cells.MCF-7 cells.

CELL MORPHOLOGY

CELL CYCLE ANALYSIS

RESULTS

Figure 12a PI (FL3 Log) vs Figure 12a PI (FL3 Log) vs Annexin V (FL1 Log) dot-plot Annexin V (FL1 Log) dot-plot of negative control MCF-12A of negative control MCF-12A cells.cells.

Figure 12b PI (FL3 Log) vs Figure 12b PI (FL3 Log) vs Annexin V (FL1 Log) dot-plot of Annexin V (FL1 Log) dot-plot of 2W/kg non-thermal 900 MHz 2W/kg non-thermal 900 MHz mobile phone radiation-exposed mobile phone radiation-exposed MCF-12A cells.MCF-12A cells.

Figure 11a PI (FL3 Log) vs Figure 11a PI (FL3 Log) vs Annexin V (FL1 Log) dot-plot Annexin V (FL1 Log) dot-plot of negative control MCF-7 of negative control MCF-7 cells.cells.

Figure 11b PI (FL3 Log) vs Figure 11b PI (FL3 Log) vs Annexin V (FL1 Log) dot-plot of Annexin V (FL1 Log) dot-plot of 2W/kg non-thermal 900 MHz 2W/kg non-thermal 900 MHz mobile phone radiation-exposed mobile phone radiation-exposed MCF-7 cells.MCF-7 cells.

Table 1. Selected differentially expressed genes (gene name in brackets) of interest in MCF-7 cells 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 exposureafter 1 hour exposure to 2W/kg non-thermal 900 MHz mobile phone radiation exposure revealed by revealed by cDNA microarray and bioinformatics analyses.cDNA microarray and bioinformatics analyses.

Table 2. Selected differentially expressed Table 2. Selected differentially expressed genes (gene name in brackets) of interest in genes (gene name in brackets) of interest in MCF-12A cells after 1 hour exposure to 2W/kg MCF-12A cells after 1 hour exposure to 2W/kg non-thermal 900 MHz mobile phone radiation non-thermal 900 MHz mobile phone radiation exposureexposure revealed by cDNA microarray and revealed by cDNA microarray and bioinformatics analyses.bioinformatics analyses.

DISCUSSION AND CONCLUSION No statistically significant differences were observed between the 2W/kg non-thermal 900 MHz mobile phone radiation-No statistically significant differences were observed between the 2W/kg non-thermal 900 MHz mobile phone radiation-exposed MCF-12A or MCF-12A cells when compared to negative control cells with regard to cell morphology, viability, cell exposed 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, cycle, mitotic index and apoptotic cells, as a result, more sensitive microarray and bioinformatics analyses were more sensitive microarray and bioinformatics analyses were employed. employed.

Microarray analyses and bioinformatics analyses revealed 31 differentially expressed genes in the MCF-7 and 19 genes in 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 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) 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 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 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.(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 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 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 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. proteomics techniques is needed to verify results.

REFERENCES 1.1. Cotgreave IA. Biological stress responses to radio frequency electromagnetic radiation: are mobile phones really so (heat) shocking? Archives of Biochemistry and Biophysics. 2005; 435(1): 227-240.Cotgreave IA. Biological stress responses to radio frequency electromagnetic radiation: are mobile phones really so (heat) shocking? Archives of Biochemistry and Biophysics. 2005; 435(1): 227-240.2.2. Leszczynski D. The need for a new approach in studies of the biological effects of electromagnetic fields. Proteomics. 2006; 6(17):4671-4673.Leszczynski D. The need for a new approach in studies of the biological effects of electromagnetic fields. Proteomics. 2006; 6(17):4671-4673.3.3. Nylund R, Leszczynski D. Mobile phone radiation causes changes in gene and protein expression in human endothelial cell lines and the response seems to be genome- and proteome-dependent. Proteomics. 2006; 6(17):4769-4780.Nylund R, Leszczynski D. Mobile phone radiation causes changes in gene and protein expression in human endothelial cell lines and the response seems to be genome- and proteome-dependent. Proteomics. 2006; 6(17):4769-4780.4.4. Remondini D, Nylund R, Reivinen J, Poulletier de Gannes F, Veyret B, Lagroye I, Haro E, Trillo MA, Capri M, Franceschi C, Schlatterer K, Gminski R, Fitzner R, Tauber R, Schuderer J, Kuster N, Leszczynski D, Bersani F, Maercker C. Gene expression changes in Remondini D, Nylund R, Reivinen J, Poulletier de Gannes F, Veyret B, Lagroye I, Haro E, Trillo MA, Capri M, Franceschi C, Schlatterer K, Gminski R, Fitzner R, Tauber R, Schuderer J, Kuster N, Leszczynski D, Bersani F, Maercker C. Gene expression changes in

human cells after exposure to mobile phone microwaves. Proteomics. 2006; 6(17):4745-4754.human cells after exposure to mobile phone microwaves. Proteomics. 2006; 6(17):4745-4754.