Medical/biological study (experimental study)

Effects of the exposure to intermittent 1.8 GHz radio frequency electromagnetic fields on HSP70 expression and MAPK signaling pathways in PC12 cells med./bio.

By: Valbonesi P, Franzellitti S, Bersani F, Contin A, Fabbri E

Published in: Int J Radiat Biol 2014; 90 (5): 382-391

Aim of study (acc. to author)

To investigate the stress responses mediated by Hsp70 and mitogen-activated protein kinases rat PC12 cells exposed to a 1.8 GHz-electromagnetic field.

Background/further details

In a previous study (Franzellitti et al., 2008 ), the authors reported the effects on the gene expression of Hsp70 in human trophoblast cells. In the present work, the same experimental conditions were applied but PC12 cells were used.
Cell cultures were subjected to one of the following conditions: 1.) sham exposure, 2.) exposure to a continuous wave, 3.) exposure to a GSM-217 Hz signal (simulates mobile phone speaking mode) and 4.) exposure to a GSM-217 Hz and GSM-DTX signal (simulates a typical mobile phone conversation with speaking and hearing). Cell cultures were exposed for 4, 16 or 24 hours. For every condition and time period, 6 experiments with 6 petri dishes were performed, respectively. Positive controls were performed (heat shock for Hsp70 gene expression, treatment with H₂O₂ for phosphorylation of mitogen-activated protein kinases).

Endpoint

molecular biosynthesis: gene expression and protein expression of Hsp70 and mitogen-activated protein kinase

Exposure

- 1,800 MHz
- mobile communications
- mobile phone
- GSM

Exposure	Parameters
Exposure 1: 1,800 MHz Modulation type: CW Exposure duration: intermittent, 5 min on/10 min off, for 4, 16, and 24 h continuous wave	SAR: 2 W/kg average over time (± 0.4 W/kg)
Exposure 2: 1,800 MHz Modulation type: pulsed Exposure duration: intermittent, 5 min on/10 min off, for 4, 16, and 24 h GSM-217 Hz signal	SAR: 2 W/kg average over time (± 0.4 W/kg)
Exposure 3: 1,800 MHz Modulation type: pulsed Exposure duration: intermittent, 5 min on/10 min off, for 4, 16, and 24 h GSM-DTX signal + GSM-217 Hz signal	SAR: 2 W/kg average over time (± 0.4 W/kg)

Exposure 1

Main characteristics

Frequency	1,800 MHz
Type	electromagnetic field
Exposure duration	intermittent, 5 min on/10 min off, for 4, 16, and 24 h
Additional info	continuous wave

Modulation

Modulation type	CW

Exposure setup

Exposure source	cavity resonator waveguide
Chamber	The exposure system was developed and built following the specifications in [Schonborn et al., 2000]. Two 128.5 x 65 x 424 mm³ brass single-mode waveguide resonators equipped with ventilators and air temperature probes were placed inside one of the two incubators.
Setup	Six 35-mm Petri dishes per resonator, each containing 3 ml of medium, were arranged in two stacks using a plastic holder and were placed in the H-field maximum of the standing wave (E-polarization).
Sham exposure	A sham exposure was conducted.
Additional info	Within each experiment, six dishes each were RF and sham exposed. Signal characteristics and the exposure/sham condition were assigned to the two waveguides by a computer-controlled signal unit ensuring blinded conditions.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
SAR	2 W/kg	average over time	measured and calculated	-	± 0.4 W/kg

Additional parameter details

The temperature of the monolayer cells was uniformly distributed without localized "hot spots". The increase in temperature due to the exposure was well below 0.1°C per unit SAR. The temperature difference between sham and exposed cells was less than 0.1°C. The absolute uncertainty of the SAR was 20%, and the variation due to the nonuniformity was 28%. The temperature remained at 36.64 ± 0.12°C during the period of measurement, ensuring no thermal effects.

Exposure 2

Main characteristics

Frequency	1,800 MHz
Type	electromagnetic field
Waveform	pulsed
Exposure duration	intermittent, 5 min on/10 min off, for 4, 16, and 24 h
Additional info	GSM-217 Hz signal

Modulation

Modulation type	pulsed
Duty cycle	12.5 %
Repetition frequency	217 Hz
Pulse type	rectangular
Additional info	GSM-217 Hz signal (speaking only) with every 26th frame being idle, adding an 8 Hz modulation component [Pedersen, 1997]

Exposure setup

Exposure source	same setup as exposure 1
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
SAR	2 W/kg	average over time	measured and calculated	-	± 0.4 W/kg

Exposure 3

Main characteristics

Frequency	1,800 MHz
Type	electromagnetic field
Exposure duration	intermittent, 5 min on/10 min off, for 4, 16, and 24 h
Additional info	GSM-DTX signal + GSM-217 Hz signal

Modulation

Modulation type	pulsed
Duty cycle	12.5 %
Repetition frequency	217 Hz
Pulse type	rectangular
Additional info	GSM-Talk signal (33% speaking and 67% hearing) with temporal changes between GSM-217 Hz and GSM-DTX (transmission reduced to 12 active frames per 104 frames) resulting in 2, 8, and 217 Hz modulation components

Exposure setup

Exposure source	same setup as exposure 1
Sham exposure	A sham exposure was conducted.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
SAR	2 W/kg	average over time	measured and calculated	-	± 0.4 W/kg

Reference articles

Valbonesi P et al. (2008): Evaluation of HSP70 expression and DNA damage in cells of a human trophoblast cell line exposed to 1.8 GHz amplitude-modulated radiofrequency fields
Schuderer J et al. (2004): High Peak SAR Exposure Unit With Tight Exposure and Environmental Control for In Vitro Experiments at 1800 MHz
Kuster N et al. (2000): Recommended minimal requirements and development guidelines for exposure setups of bio-experiments addressing the health risk concern of wireless communications
Pedersen GF (1997): Amplitude modulated RF fields stemming from a GSM/DCS-1800 phone

Exposed system:

intact cell/cell culture
PC-12 cells (pheochromocytoma cell line from rat adrenal gland)

Methods Endpoint/measurement parameters/methodology

Investigated system:

isolated bio./chem. substance
DNA/RNA
intact cell/cell culture

Time of investigation:

after exposure

Main outcome of study (acc. to author)

After 16 or 24 hours of exposure to the GSM-217 Hz signal (group 3), the gene expression of Hsp70 was significantly increased compared to the sham exposed cell cultures, while no significant effect was observed in the other exposure groups. Additionally, no differences between the groups were observed regarding protein expression levels and cell viability.
The authors conclude that the observed increase in the Hsp70 gene expression in PC12 cells confirmed the results of the previous study with trophoblasts.

Study character:

medical/biological study
experimental study
full/main study
blind study

Study funded by

Ministero dell' Università e della Ricerca (MIUR (formerly MURST (Ministero dell' Università e della Ricerca Scientifica e Tecnologica); Ministry of University and Research), Italy

Calabro E et al. (2012): Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves
Ding GR et al. (2009): Comparison of Hsps expression after radio-frequency field exposure in three human glioma cell lines
Valbonesi P et al. (2008): Evaluation of HSP70 expression and DNA damage in cells of a human trophoblast cell line exposed to 1.8 GHz amplitude-modulated radiofrequency fields
Franzellitti S et al. (2008): HSP70 expression in human trophoblast cells exposed to different 1.8 Ghz mobile phone signals
Yu Y et al. (2008): Effects of exposure to 1.8 GHz radiofrequency field on the expression of Hsps and phosphorylation of MAPKs in human lens epithelial cells
Sanchez S et al. (2007): In Vitro Study of the Stress Response of Human Skin Cells to GSM-1800 Mobile Phone Signals Compared to UVB Radiation and Heat Shock
Lantow M et al. (2006): Free radical release and HSP70 expression in two human immune-relevant cell lines after exposure to 1800 MHz radiofrequency radiation
Chauhan V et al. (2006): Gene Expression Analysis of a Human Lymphoblastoma Cell Line Exposed In Vitro to an Intermittent 1.9 GHz Pulse-Modulated Radiofrequency Field
Qutob SS et al. (2006): Microarray gene expression profiling of a human glioblastoma cell line exposed in vitro to a 1.9 GHz pulse-modulated radiofrequency field
Nylund R et al. (2006): 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
Lixia S et al. (2006): Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells
Lee S et al. (2005): 2.45 GHz radiofrequency fields alter gene expression in cultured human cells
Capri M et al. (2004): 1800 MHz radiofrequency (mobile phones, different Global System for Mobile communication modulations) does not affect apoptosis and heat shock protein 70 level in peripheral blood mononuclear cells from young and old donors

Effects of the exposure to intermittent 1.8 GHz radio frequency electromagnetic fields on HSP70 expression and MAPK signaling pathways in PC12 cells med./bio.

Aim of study (acc. to author)

Background/further details

Endpoint

Exposure

Exposure 1

Main characteristics

Modulation

Exposure setup

Parameters

Additional parameter details

Exposure 2

Main characteristics

Modulation

Exposure setup

Parameters

Exposure 3

Main characteristics

Modulation

Exposure setup

Parameters

Reference articles

Methods Endpoint/measurement parameters/methodology

Main outcome of study (acc. to author)

Study funded by

Related articles