Changes in cell proliferation due to environmental non-ionizing radiation

Bioelectrochemistry and Bioenergetics 44 Ž1998. 251–255

Changes in cell proliferation due to environmental non-ionizing radiation 2. Microwave radiation S. Kwee a

a,)

, P. Raskmark

b

Department of Medical Biochemistry, UniÕersity of Aarhus, Building 170, DK-8000 Aarhus C, Denmark b Institute of Communication Technology, Aalborg UniÕersity, DK-9220 Aalborg Ø, Denmark Received 23 August 1997; received in revised form 20 October 1997; accepted 27 October 1997

Abstract Due to the use of mobile telephones, there is an increased exposure of the environment to weak radiofrequency ŽRF. electromagnetic fields, emitted by these devices. This study was undertaken to investigate if the microwave radiation from these fields will have a similar effect on cell proliferation as weak electromagnetic ŽELF. fields. The field was generated by signal simulation of the Global System for Mobile communications ŽGSM. of 960 MHz. Cell cultures, growing in microtiter plates, were exposed in a specially constructed chamber, a Transverse Electromagnetic ŽTEM. cell. The Specific Absorption Rate ŽSAR. values for each cell well were calculated for this exposure system. Experiments were performed on cell cultures of transformed human epithelial amnion cells ŽAMA., which were exposed to 960 MHz microwave fields at three different power levels and three different exposure times, respectively. It was found that cell growth in the exposed cells was decreased in comparison to that in the control and sham exposed cells. Cell proliferation during the period following exposure varied not only with the various SAR levels, but also with the length of exposure time. On the other hand, repeated periods of exposure did not seem to change the effects. There was a general linear correlation between power level and growth change. However, the exposure time required to obtain the maximum effect was not the same for the various power levels. It turned out that at low power level, a maximum effect was first reached after a longer exposure time than at higher power level. A similar phenomenon was registered in the studies on ELF electromagnetic fields. Here, it was found that there was a linear correlation between the length of exposure time to obtain maximum effect and field strength. q 1998 Elsevier Science S.A. Keywords: Electromagnetic fields; Radiofrequency electromagnetic fields; Microwave radiation; Specific absorption rate values; Cell proliferation; Microtiter plate culture

1. Introduction In recent years, the use of mobile telephones has accelerated, resulting in an increasing exposure of the environment to weak radiofrequency ŽRF. electromagnetic fields, generated by these devices. Results of the numerous experimental, epidemiological and theoretical studies are rather controversial, and in all areas so far, no unanimous conclusions have been reached. Besides epidemiological w1x and in vitro w2,3x studies that fail to show any effect from exposure to microwave radiation, there are also quite a number, that show that there is a biological effect from microwave radiation. On cellular level modulated microwave radiation already at low SAR levels, below 10 W kgy1 , changes in cell cycle w4x, cell growth rates w5,6x,

)

Corresponding author.

0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 7 . 0 0 0 9 5 - 0

enzyme activities w7–9x, cell membrane structure w10–12x, cellular transformations w13,14x and the nervous system w15x have been reported, as well as in animal studies w16,17x. At continuous wave radiation effects on cell cycle w4x, cell proliferation w18x, the genome w19x, nervous system w20x were first detected at SAR values over 25 W kgy1 . However, on RNA transcription w21x and membrane structure w22x these effects were also found at lower SAR values. In vivo experiments on young rats showed that the nervous system could be effected in selected spots already by fields at weaker levels than thermal noise w23x. In previous work, we showed that cell growth is affected by exposure to weak ŽELF. electromagnetic fields w24x. Consequently, the next thing to investigate was, if EM fields generated by microwave radiation, would have a similar effect on cell proliferation. Since mobile phones are supposed to affect the head primarily, and consequently the brain, we should have used primary brain

252

S. Kwee, P. Raskmarkr Bioelectrochemistry and Bioenergetics 44 (1998) 251–255

tissue culture for our experiments. However, we wanted to study cell proliferation, so we could not use brain tissue here. Moreover, we decided not to use brain tumor cell cultures in the first place because we wished to compare the effects of microwave radiation under the same conditions as in our previous experiments. Therefore, the following studies were done on the same cell line as the one we used in our previous work.

2. Materials and methods 2.1. Materials and cell lines Celltiter 96 wAQ ueous ŽMTS. non-radioactive cell proliferation assay from Promega was used. Transformed human epithelial amnion cells ŽAMA., grown as monolayer cultures in Dulbecco’s modified Eagle’s medium containing 10% calf serum, penicillin and streptomycin, at 378C in 6.0% CO 2 and kept in a Forma Scientific 3164 incubator. All experiments were done on two different clones.

Fig. 1. Transverse Electromagnetic ŽTEM. cell.

the exposure times at each power level were 20, 30 or 40 min, respectively.

2.2. ELF magnetic field exposure 2.4. Experimental protocol The magnetic fields were generated with the use of Helmholz coils: 12 = 13 cm, 9 cm apart, 2 = 10 turns, using 1 mm diameter copper wire and a copper screen. One pair for the AC magnetic field and three orthogonal pairs to change the static field. The coils were wound around a square plastic container placed inside the incubator. A low-distortion generator and a power amplifier delivered the signal. The magnetic field was oriented in the plane of the cell plate. Fields were measured with a three-axis fluxgate meter, Bartington Inst. model MAG-03 MC. The experimental setup has been described before w24x. 2.3. MicrowaÕe exposure The EM field was generated by signal simulation of the Global System for Mobile communications ŽGSM., a 960 MHz carrier amplitude modulated with a 217 Hz square pulse of duty cycle 12%. The cell cultures, growing in microtiter plates, were exposed in a specially constructed chamber, a Transverse Electromagnetic ŽTEM. cell. Fig. 1 shows the TEM cell and its analysis has been given previously w25x. A schematic diagram of the experimental setup is shown in Fig. 2. The Specific Absorption Rate ŽSAR. values for each cell well were obtained from the FDTD calculations of the EM field distribution in a microtiter suspension well w25,26x. The cell cultures were exposed in the TEM cell to the RF fields at three different power levels, resulting in SAR values of 0.021, 0.21 and 2.1 mW kgy1 , respectively and

The cells were grown in microtiter plates, tissue culture grade, 96 wells, flat bottom. Cells were seeded in the 2 central rows, a6 and a7, A–H in 100 m l culture medium. After 24-h field-free incubation at 378C the cells were growing in their log phase to approximately 40–50% confluency. A plate was then transferred to the exposure chamber, i.e., the Helmholtz coil assembly for ELF magnetic field exposure w24x or the TEM cell for radiofrequency field exposure, in the incubator. Subsequent microtiter plates were exposed to the magnetic field of varying strength and exposure times. During exposure, the field and the temperature in the incubator were monitored continuously. Proliferation of the cell cultures grown in microtiter plates was determined by a colorimetric assay with quantification in an ELISA plate reader, based on the conversion of a tetrazolium salt by mitochondrial dehydrogenases

Fig. 2. Schematic diagram of experimental set-up of microwave exposure system.

S. Kwee, P. Raskmarkr Bioelectrochemistry and Bioenergetics 44 (1998) 251–255

inviable cells, to form a colored formazan product w27x, as follows: 10 m l Celltiter 96 w MTS reagent was added to each well and after 5–6-h incubation at 378C in 6.0% CO 2 , the absorbance was read. The absorbance was measured at the wavelength of 490 nm and the reference wavelength was 655 nm. The number of proliferating cells was proportional to the absorbance. For each series of experiments, cell proliferation was determined prior to exposure to the field, i.e., MTS reagent was added to all the wells in row a6 of each plate. After exposure, cells were allowed to grow for another 24 h in the field-free incubator. Then, cell proliferation was determined in the remaining cells, i.e., MTS reagent was added to all the wells in row a7 of each plate. In the control experiments, the cell cultures were grown and incubated in field-free environment and cell proliferation assayed following the same procedure. In the sham exposure experiments, the cell cultures were kept for 30 min in the exposure chamber with the field turned off. Each series of experiments was duplicated on the same cell culture of a different clone, but which had undergone the same number of passages. To reduce effects due to mutation, all experiments were only run for 4–5 passages of the same cell culture. Then, a new series was run from a freshly thawed culture starting with the same passage number. Proliferation rate was expressed as the ratio between absorbance after 24 h and absorbance at zero time in percent: Percent proliferation rate s

A ts24 h A ts0

P 100.

Change in proliferation rate was expressed as the difference between the percent proliferation rate in the exposed culture and the percent proliferation rate in the control experiments. Change in proliferation rate s proliferation rate exposed y proliferation rate control Measurements of the field in each cell well showed that the field in both outermost horizontal rows, i.e., row A and row H was different from those in the rows B–G. Therefore, only results from measurements in row B–G were included in this analysis. P values were obtained by statistical calculations, based on a paired 2-tailed test.

253

Table 1 Dependence of proliferation rate on exposure time and ELF magnetic field strength in AMA cells Field strengthr mT

Proliferation rater% Exposure timermin

20 30 40 50 60 80

10

20

30

40

45

Control

199 165 163 182 193 165

201 166 166 187 200 170

202 168 174 182 189 164

211 172 166 182 189 164

221

194 163 160 179 181 164

this effect more thoroughly, the experiments in the present study were conducted over a narrower range of field strengths and the interval of the exposure times was more frequent and shorter. The results in Table 1 show proliferation rates after exposure to 50 Hz EM fields of different power levels and at varying exposure times. Each result was based on 8–9 independent experiments. It could now be seen, that there was a maximum effect on proliferation rate at each power level and that the exposure time required to obtain this peak effect was not the same for all field strengths. It showed that at low power level, the maximum effect was first obtained after a longer exposure time than at higher power level. In other words, to each specific field strength a certain exposure time is required to obtain the maximum effect. The correlation existing between the exposure time needed to obtain a maximum effect and that specific field strength is shown in Fig. 3. This may explain why a so-called ‘window’ effect is seen, if experiments are only done at one field strength or exposure time. Our experiments also showed that longer exposure times than those at which the maximum effect was reached did not result in a higher effect. This can be due to adaptation and also explains why no effect will be detected, if cell cultures are exposed for hours, days or weeks to a magnetic field.

3. Results and discussion 3.1. ELF electromagnetic radiation In our previous study w24x, we found that there existed a so-called ‘window’ effect, i.e., the maximum effect on changes in proliferation rate were found at a specific electromagnetic field strength and exposure time. To study

Fig. 3. ELF magnetic field exposure. Relation between exposure time to obtain the maximum change in proliferation rate and field strength.

S. Kwee, P. Raskmarkr Bioelectrochemistry and Bioenergetics 44 (1998) 251–255

254

Table 2 Percent change in proliferation rate in AMA cells after exposure to microwave fields of varying energy levels and different exposure times

Table 3 Dependance of proliferation rate on exposure time and microwave field strength in AMA cells

Field strength SARrmW kgy1

SARrmW kgy1 Proliferation rater%

0.021 0.021 0.021 0.21 0.21 0.21 2.1 2.1 2.1 a

Exposure timermin 20 30 40 20 30 40 20 30 40

Change in proliferation rater% Mean

Exposure timermin

95% Confidence limit a

y2.2 y7.2 y11.7 y1.75a y9.3 y4.8 a 1.3 a y5.5 y10.3

y4.3 y5.5 1.4 y7.6 y0.4 y19.4 y9.9 0.17 5.8

8.7 19.9 21.9 11.1 19.1 28.9 12.6 10.8 14.8

Not significant P ) 0.5 Žpaired test..

3.2. MicrowaÕe radiation In Table 2, the changes in cell growth after exposure to RF electromagnetic fields of varying strength at different exposure times are shown. Each result was based on 12 independent experiments with a 95% confidence interval. It was found that cell growth in the exposed cells differed from that in the control and sham exposed cells and a decrease in cell growth was seen at all three SAR values ŽFig. 4.. Further, it turned out that there was no difference in the changes in cell proliferation for the two clones of cell lines used. Neither did repeated exposures interrupted by a field-free period change the effects. A general linear correlation between exposure time and growth changes was seen for the highest and lowest power intensity. At the intermediate SAR value of 0.21 mW kgy1 , this linear correlation was not clear. The cell proliferation rates resulting from the exposure to microwave radiation were almost equal at all SAR levels ŽTable 3.. We observed that in order to obtain a peak effect the minimum exposure time at all power levels had to be 30 min. The same relation between exposure

Fig. 4. Changes in cell growth after exposure to microwave field. SAR 0.021 mW kgy1 ŽB-.-.; SAR 0.21 mW kgy1 Ž Ø — .; SAR 2.1 mW kgy1 Ž' . . . ..

20 0.021 0.21 2.1

SEM 30

125 6.3 126 7.6 126 7.5

SEM 40

120 8.5 118 9.5 122 5.9

SEM Control SEM

115 8.2 123 13.1 117 5.9

126 126 126

5.0 5.0 5.0

time to reach a peak effect and power level as seen in the case of ELF electromagnetic radiation as described in Section 3.1, was not clear. However, more experiments at a narrower interval of SAR values and exposure times will be necessary to detect if there is the same type of window effect in the case of microwave radiation as in ELF electromagnetic fields. As previously observed by others w5,23x, the effects registered are not linear over the whole radiofrequency field density spectrum. This can be related to the oscillatory nature of cell growth w28x. Apparently, the interaction of microwave radiation with cellular oscillators contributes more to these effects than ELF electromagnetic fields. Contrary to previous assumptions, our results showed that there can be an effect at very low SAR values.

Acknowledgements This research was supported by Novo Nordisk Denmark.

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