Medical/biological study (experimental study)

Effects of 2.45 GHz electromagnetic fields with a wide range of SARs on bacterial and HPRT gene mutations med./bio.

By: Koyama S, Takashima Y, Sakurai T, Suzuki Y, Taki M, Miyakoshi J

Published in: J Radiat Res 2007; 48 (1): 69-75

Aim of study (acc. to author)

This in vitro study was performed to investigate the effects of radiofrequency electromagnetic field on bacterial mutations and hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation in mammalian cells.

Background/further details

The hypoxanthine-guanine phosphoribosyl transferase (HPRT) is an enzyme in the purine (for example guanine) synthesis pathway. HPRT-negative cells are not able to use guanine for GTP synthesis und thus, they have to synthesize the base de novo. Using the guanine analog 6-thioguanine HPRT-positive cells use 6-thioguanine for nucleotide synthesis. However, integration of 6-thioguanine leads to DNA and RNA damage and it is lethal for these cells. In contrast, HPRT-negative cells are not able to utilize 6-thioguanine and survive.
The HPRT gene mutation assay is a well-established mutagenicity assay based on the selection of clones resistant to the purine analog 6-thioguanine. I.e. for determination of the induction of 6-thioguanine resistant mutation in the HPRT gene exposed cells are plated in a medium containing 6-thioguanine.
Experiments were performed with different specific absorption rates.
The frequency of HPRT gene mutations were compared between: 1.) sham exposed, 2.) radiofrequency electromagnetic field exposed, 3.) co-exposed (radiofrequency electromagnetic field exposure plus bleomycin treatment) and 4.) heat (39°, 41°, 44° with or without bleomycin) treated cells.

Endpoint

genotoxicity/mutation: mutation frequency

Exposure

- 2.45 GHz
- microwaves

Exposure	Parameters
Exposure 1: 2.45 GHz Exposure duration: continuous for 30 min bacterial cells	SAR: 200 W/kg (5, 50, 100, 200 W/kg)
Exposure 2: 2.45 GHz Exposure duration: continuous for 2 h CHO-K1 cells	SAR: 200 W/kg (5, 10, 20, 50, 100, 200 W/kg)

Exposure 1

Main characteristics

Frequency	2.45 GHz
Type	electromagnetic field
Exposure duration	continuous for 30 min
Additional info	bacterial cells
Additional info	Ames, B. N., McCann, J., and Yamazaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31: 347-363.

Exposure setup

Exposure source	waveguide rectangular, TE₁₀ mode
Chamber	Details of the exposure system have been described previously [Koyama et al., 2004]. To generate standing waves, one end of the waveguide was terminated with a short-circuiting plate. Exposure was performed in an acrylic incubator with an atmosphere of humidified 95% air and 5% CO₂ that was "installed into inner space of the core".
Setup	Aliquots of 20.1 ml of the cell suspensions were seeded into a specially designed culture dish that was placed on two slits bored on the waveguide through which cells were exposed.
Sham exposure	A sham exposure was conducted.
Additional info	Bacterial cells were exposed according to the protocol of the pre-incubation method of the Ames test [Ames et al., 1975]. Three chemical mutagens were used as positive control.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
SAR	200 W/kg	-	measured	-	5, 50, 100, 200 W/kg

Measurement and calculation details

SAR distribution of the exposure setup was measured.

Exposure 2

Main characteristics

Frequency	2.45 GHz
Type	electromagnetic field
Exposure duration	continuous for 2 h
Additional info	CHO-K1 cells

Exposure setup

Exposure source	same setup as exposure 1
Sham exposure	A sham exposure was conducted.
Additional info	As a positive control or co-mutagenic treatment, CHO-K1 cells were exposed to bleomycin for 1 h before EMF exposure. During heat treatment, cells were incubated for 2 h at 39, 41, and 44°C corresponding to the heat induction of about 50, 100, and 200 W/kg. Co-mutagenic treatment with bleomycin was also performed.

Parameters

Measurand	Value	Type	Method	Mass	Remarks
SAR	200 W/kg	-	measured	-	5, 10, 20, 50, 100, 200 W/kg

Reference articles

Koyama S et al. (2004): Effects of 2.45-GHz electromagnetic fields with a wide range of SARs on micronucleus formation in CHO-K1 cells

Exposed system:

intact cell/cell culture
bacterium
Salmonella typhimurium (TA98, TA100, TA1535, TA1537), Escherichia coli (WP2 uvrA) and CHO cells K1 (chinese hamster ovary K1)

Methods Endpoint/measurement parameters/methodology

genotoxicity/mutation: bacterial mutation (number of revertant colonies, Ames test), frequency of the hypoxanthine-guanine phosphoribosyl transferase gene mutations (number of 6-thioguanine resistent colonies)

Investigated system:

intact cell/cell culture
bacterium

Time of investigation:

after exposure

Main outcome of study (acc. to author)

There was no significant difference in the number of revertant colonies between sham exposed and radiofrequency electromagnetic field exposed bacterial strains.
A significant difference in the HPRT gene mutation frequency between sham exposed and electromagnetic field exposed cells was only observed at the highest specific absorption rate (200 W/kg), but not at lower ones.
The mutation frequency of co-exposed cells at 5, 10 and 20 W/kg were the same as bleomycin alone treated controls. However, co-exposure at 50, 100 and 200 W/kg resulted in a dose-dependent increase of mutation frequency.
Heat treatment revealed a significant increase in HPRT gene mutation at 44° (corresponding to the 200 W/Kg radiofrequency electromagnetic field). Combined treatment of heat (41° and 44°) and bleomycin induced significant difference in gene mutation compared to bleomycin alone treated cells. Therefore the increase of mutation frequency might be a result of a thermal effect.

Study character:

medical/biological study
experimental study
full/main study

Study funded by

Ministry of Internal Affairs and Communications, Japan

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Effects of 2.45 GHz electromagnetic fields with a wide range of SARs on bacterial and HPRT gene mutations med./bio.

Aim of study (acc. to author)

Background/further details

Endpoint

Exposure

Exposure 1

Main characteristics

Exposure setup

Parameters

Measurement and calculation details

Exposure 2

Main characteristics

Exposure setup

Parameters

Reference articles

Methods Endpoint/measurement parameters/methodology

Main outcome of study (acc. to author)

Study funded by

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