- Email: [email protected]
The minimizing effect of electromagnetic noise on the changes in cell proliferation caused by ELF magnetic fields
ELSEVIER
Bioelectrochemistxy and Bioenergetics 40 (1996) 193- 196
The minimizing effect of electromagnetic noise on the changesin cell proliferation causedby ELF magnetic fields P. Raskmark a, S. Kwee bT* a Institute of Communication Technology, b Institute ofMedical Biochemistry, University
Aalborg University, of Aurhus, Building
DK-9220 Aalborg 0, Denmark 170, DK-8000 Aarhus C, Denmark
Received 26 January 19%; accepted 15 February I996
Abstract A significant increase in cell growth was registered in human epithelial amnion (AMA)
cells, when exposed to a sinusoidal 50 Hz, 50
FT electromagnetic field (S. Kwee and P. Raskmark, Bioelectrochem. Bioenerg., 36 (199.5) 109). To study the effects of incoherent magnetic fields on the biological changes caused by the electromagnetic fields, varying levels of noise were superimposed on the above-mentioned field. No inhibition of the coherent magnetic field effects were seen at low noise magnetic field densities. However, when the noise reached a level of 7040% of the magnetic field, a significant inhibition of the increased cell proliferation was registered. The imposed inhibition remained constant up to a noise level equal to the the magnetic field. No significant change in proliferation rate was seen when the cell culture was exposed to a noise field only. Keywords:
Electromagnetic noise; Cell proliferation;
AMA cells; Microtiter plate cultivation; Respons inhibition
1. Introduction In spite of numerous studies on the biological effects of exposure to weak electromagnetic fields, the issue of whether continuous exposure to these fields involves health risks, is still a subject of considerable debate. Epidemiological studies have linked the incidence of cancer, especially childhood leukemia, to exposure to ELF EM fields [l-4]. On a cellular level, effects on cell proliferation, enzyme activities, calcium transport, transcription and chromosome aberations have been reported [5- lo]. However it cannot be denied that there are also several studies reporting negative results and no effects of exposure to EM fields [ 11- 121. A true mechanism for these observations has not yet been found. Litovitz et al. found that a magnetic field will only induce biological effects in the exposed system if the field is coherent for at least 5- 10 s [ 131. They therefore suggested that electromagnetic field effects could be inhibited by making the field incoherent, e.g. by superimposing electromagnetic noise. In two separate systems, (the ornithine decarboxylase (ODC) enhancement in L929 murine cells [13] and abnormalities in chick embryos [ 141) both * Corresponding author. 0302-4598/96/$15.00 PII
SO302-4598(96)05060-X
0 1996 Elsevier Science S.A. All rights reserved
induced by exposure to a 60 Hz magnetic field, they could show that these effects could be blocked by superimposing noise on an ELF EM field [151. A related study showed that electromagnetic noise could also inhibit the enhancement of steady state c-myc transcript levels in human leukemia cells induced by exposure to a 60 Hz magnetic field [ 161. We have previously shown that exposure to a 50 Hz pT EM field results in a significant increase in cell growth in human epithelial amnion (AMA) cells [ 171. This change in proliferation rate was dependent on exposure times and field densities. A maximum increase in cell proliferation was only found at a specific field density and exposure time i.e. there were “window” and adaptation effects. The purpose of this study is to investigate whether a superposition of a noise EM field, can inhibit the changes in proliferation rate in AMA cells induced by exposure to ELF magnetic fields.
2. Materials 2.1. Materials
and methods and cell lines
Cell proliferation Reagent WST- 1 from Boehringer Mannheim Biochemica was used.
194
P. Raskmark, S. Knee/ Bioelectrochemistry
and Bioenergetics 40 11996) 193-196
Fig. 1. Diagram showing experimental set-up for noise added ELF magnetic field exposure. Magnetic fields are measured before exposure using the fluxgate. The current in the coils are monitored during exposure using the rms voltmeter.
Transformed human epithelial amnion cells (AMA) were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium containing 10% calf serum, penicillin and streptomycin, at 37 “C in 6.0% CO, and kept on a Forma Scientific 3 164 incubator. 2.2. EM exposure and noise generation The exposure system was designed to generate ELF magnetic fields in accordance to the principles outlined in [ 18,191e.g. we measured static and stray fields and checked temperature rise during exposure. The apparatus setup is basically the same as used in [20], see Fig. 1 i.e. a signal generator and filter followed by a power amplifier delivering current to Helmholz coils and using a fluxgate to measure the magnetic fields. As an exposure measure we used the magnetic field measured before exposure in the same position as the exposed cells. We used the current supplied to the coils to monitor the fields during exposure. The exposure levels are traceable through the Bartington fluxgate and the Hewlett Packard multimeter. The Helmholz coils had the following attributes: dimensions of 12 cm x 13 cm, 9 cm apart, 2 times 10 turns using 1 mm diameter copper wire and a copper screen. The magnetic fields were oriented in the plane of the cell plates and the variation in the magnetic fields was not more than 5% as a function of cell well position (see field distribution in Ref. [20]). If more homogeneous fields are required the method of Kirschvink [21] can be applied, in which a double wrapped system is recommended. In our experiments the sensitivity to field strength was weak and since the temperature is controlled, a simple Helmholz coil system was considered sufficient. A multifunction synthesizer followed by a band pass filter generated the required voltage, see Fig. 1. The synthesizer was programmed to deliver a constant 50 Hz signal and 5 different levels of Gaussian white noise were added. The composite signal is bandpass filtered using first a lowpass- and then a highpass-maximally flat four pole Butterworth filter (24 dB per octave response, 50 Hz cutoff frequency). The excitation voltage was amplified using a voltage-to-current power amplifier (high impedance out-
put) to secure a frequency independent voltage to magnetic field transfer. Interference signal pickup was minimized having all instruments signal ground except the floating signal generator. The generated magnetic fields were measured using a fluxgate with the frequency range O-l kHz and analyzed with two different instruments: a rms voltmeter and a dynamic signal analyzer. In contrast to sinusoidal signals the low frequency noise signals are somewhat difficult to measure. Determining the noise signal by using an oscilloscope [15] is not reproducible, a better criterion is rms together with information on bandwidth. Since standard so called “true rms” voltmeters will display varying results due to limited crest factors, we used the hp3458A multimeter (with the longest possible integration time, approx. 20 s). In addition we used the hp35660A Dynamic Signal Analyzer to measure the power spectra, as shown in the example in Fig. 2. 2.3. Assay Proliferation of the cell cultures grown in microtiter plates was determined by a calorimetric assay with quan-
10 5 I
-351
/
I lo2
10'
Frequency/
HZ
Fig. 2. Measured magnetic fields for 50 +T 50 Hz with added 20 FT (rms) bandpass filtered white noise. The voltage output from the fluxgates are plotted in dB relative to 1 V rms in the frequency range lo-300 HZ; 14 dB corresponds to 5 V rms or 50 FT.
P. Raskmurk, S. Kwee/Bioelecrrochemis?ry
tification in an ELISA plate reader, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases i viable cells, in the following way: 10 ~1 WST-1 reagent was added at zero time to each well and after 5-6 h incubation at 37 “C in 6.0% CO,, the absorbance was read. The absorbance was measured at the wavelength of 490 nm and the reference wavelength was 655 nm. For maximum absorbance a second reading was taken after 24 h. The number of proliferating cells was proportional to the absorbance.
and Bioenergefics 40 (1996) 193-196
195
Table 1 Percent change in proliferation rate in AMA cells after 30 min exposure to EM field of 50 Hz and 50 *T with varying noise levels % change in profileration rate
Field density/kT
Noise level/ pT
mean
95% confidence limits
50 50 50 50 50 50
0 10 20 30 40 50
0 + 0.0 a - 2.25 a - 6.3 a - 16.0 -20.1
- 16.4 - 14.6 - 23.0 - 25.7 -31.2
16.4 10.15 10.5 - 6.2 -9.1
a Not significant P value > 0.5 (paired t-test).
2.4. Experimental protocol The cells were seeded in 100 p,l culture medium into tissue culture grade, microtiter plates with 96 wells and a flat bottom. Cells were seeded in the 2 central rows, 6 and 7, A-H. After 24 h field-free incubation at 37 “C the cells were growing in their log phase to approximately 40-50% confluency. A .plate was then transferred to the Hehnholtz coil assembly in the incubator and exposed to a 50 Hz, 50 p.T magnetic field. Subsequent microtiter plates were exposed to this magnetic field with superimposed noise fields of varying strength. Exposure time 30 min. Other microtiter plates were exposed to noise fields only. During exposure the field and temperature were monitored continuously in the incubator. For each series of experiments cell proliferation was determined prior to exposure to the field i.e. WST-1 reagent was added to all the wells in row 6 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. WST-1 reagent was added to all the wells in row 7 of each plate. In the control experiments similar cell cultures were grown and incubated in a field-free environment and cell proliferation was 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. During exposure the temperature in the cultures was measured, but temperature rise was less than O.l”C.
3. Results and discussion Proliferation rate was expressed as the ratio between absorbance after 24 h and absorbance at zero time in percent. *,=
Percent proliferation rate = -
24h
. 100
A,=0 Change in proliferation rate was expressed as the difference between the percent proliferation rate in the exposed
culture and the percent proliferation experiments.
rate in the control
change in proliferation rate = proliferation rateexposed - proliferation ratecontrol Change in proliferation rate due to the superimposed noise field was expressed as the difference between proliferation rate in the culture exposed to both a magnetic and a noise field and the proliferation rate in the culture only exposed to the magnetic field. effect of noise field = proliferation ratenoise+nMfield- proliferation rateEMfield The changes in proliferation rate after 30 min exposure to a 50 Hz, 50 ~.LT electromagnetic field with varying superimposed noise levels are reported in Table 1. Each figure was based on the average mean of 8-9 independent experimental results with a 95% confidence interval. From Table 2 it can be seen that if only a noise field was applied without an EM field, no significant effect on cell growth was detected as compared to exposure to the ELF field or to the control. Table 1 shows that the superimposed noise field causes an inhibition of the increased cell growth caused by the ELF field. However, the inhibiting effect is only significant at a noise field from 40 PT on and higher. At noise levels below 30 p.T there is hardly any inhibiting effect. This means that the total effective field reaches a level of 64 p,T (i.e. 50 FT signal + 40 p,T noise) before significant growth inhibition is detected. These results are comparable to those found by Litovitz et al. Their experiments showed a steady decrease [15] in ODC activity of L929 cultures, starting at a noise to signal Table 2 Percent change in proliferation rate in AMA cells after 30 min exposure to EM field of 50 Hz or to noise only % change in proliferation rate
Field density/kT
Noise level/ pT
mean
95% confidence limits
0 50 0
0 0 50
0 + 15.1 + 9.3 a
5.1 - 1.7
a Not significant P value > 0.5 (paired t-test).
24.5 20.4
196
P. Raskmurk, S. Kwee /Eioelectrochemisrty
ratio of approx. 0.05 and reaching almost complete inhibition at at noise to signal ratio of 1. In our experiments a significant inhibiting effect is first obtained at a noise to signal ratio of 0.8, i.e. appproximately two times more noise is required than found by Litovitz et al. [15]. It should be taken into account that our experimental conditions differed with respect to cell cultures, measurement of biological data and the ELF em and noise fields. Moreover it must be pointed out that it is premature to conclude from our results that the the fields mentioned will affect cellular functions under in vivo conditions in a similar way and induce the same biological effects. This would require additional experiments on whole organisms, such as animals and human beings under normal living conditions.
Acknowledgements This research was supported by Danfoss A/S, Denmark.
References fi] D.A. Savitz et al., Am. J. Epidemiol., 128 (1988) 21. [2] M. Feychting and A. Ahlbom, Am. J. Epidemiol., 138 (1993) 467. [3] P.GuCnel, P. Raskmark, J. Bach Andersen and E. Lynge, Br. J. Ind. Med., 50 (1993) 758 [4] J.H. Ohlsen et al., Br. Med. J., 307 (1993) 891.
und Bioenergetics 40 (19961 193-196 [5] S. Johann, T. Ledeter, S. Mikotey, W. Kraus and G. Bliimel, Bioelectrochem. Bioenerg., 30 (1993) 127. [6] M.S. Markov, J.T. Ryaby, J.J. Kaufman and A.A. Pilla in M.J. Allen, S.F. Cleary. A.E. Sowers and D.D. Shillady (eds.), Charge and Field Effects in Biosystems 3, Birkhaiiser, Boston 1992, p. 151. [7] M.S. Markov, D.J. Muehsam and A.A. Pilla in M.J. Allen, S.F. Cleary and A.E. Sowers (eds.), Charge and Field Effects in Biosysterns 4. World Scientific, Singapore 1994, p. 274. [8] R. Goodman, M. Blank, H. Lin, R. Dai, 0. Khorkova, L. Soo, D. Weisbrot and A. Henderson, Bioelectrochem. Bioenerg.. 33 (1994) 115. [9] I. Nordenson, K. Hansson Mild, G. Andersson and M. Sandstrom, Bioelectromagnetics. 15 (1994) 293. [lo] S. Tofani, A. Ferrara, L. Anglesio and G. Gilli, Bioelectrochem. Bioenerg., 36 (1995) 9. [ 111 P. Verkasalo et al., Br. Med. J., 307 (1993) 895. [12] S. Galt, J. Wahlstrbm, Y. Hamnerius, D. Holmqvist and T. Joharnesson, Bioelectrochem. Bioenerg.. 36 (1995) 1. [ 131 T.A. Litovitz, D. Krause and J.M. Mullins, B&hem. Biophys. Res. Commun., 178 (1991) 862. [14] T.A. Litovitz, C.J. Montrose, P.Doinov, K.M. Brown and M. Barber, Bioelectromagnetics, 15 (1994) 105. [15] T.A. Litovitz, D. Krause, C.J. Montrose and J.M. Mullins, Bioelectromagnetics (I 995) in press. [16] H. Lin and R. Goodman, Bioelectrochem. Bioenerg., 36 (1995) 33. [17] S. Kwee and P. Raskmark in M.J. Allen, SF. Cleary and A.E. Sowers (eds.), Charge and Field Effects in Biosystems 4, World Scientific, Singapore 1994, p. 255. [ 181 M. Misakian et.al. Bioelectromagnetics Suppl. 2 (1993) 1. [ 191 M. Chiabrera et al. COST 244 temporary document no. 244TDc95m9. [20] S. Kwee and P. Raskmark, Bioelectrochem. Bioenerg., 36 (1995) 109. [21] J.L. Kirschvink, Bioelectromagnetics, 13 (1992) 401.
Bioelectrochemistxy and Bioenergetics 40 (1996) 193- 196
The minimizing effect of electromagnetic noise on the changesin cell proliferation causedby ELF magnetic fields P. Raskmark a, S. Kwee bT* a Institute of Communication Technology, b Institute ofMedical Biochemistry, University
Aalborg University, of Aurhus, Building
DK-9220 Aalborg 0, Denmark 170, DK-8000 Aarhus C, Denmark
Received 26 January 19%; accepted 15 February I996
Abstract A significant increase in cell growth was registered in human epithelial amnion (AMA)
cells, when exposed to a sinusoidal 50 Hz, 50
FT electromagnetic field (S. Kwee and P. Raskmark, Bioelectrochem. Bioenerg., 36 (199.5) 109). To study the effects of incoherent magnetic fields on the biological changes caused by the electromagnetic fields, varying levels of noise were superimposed on the above-mentioned field. No inhibition of the coherent magnetic field effects were seen at low noise magnetic field densities. However, when the noise reached a level of 7040% of the magnetic field, a significant inhibition of the increased cell proliferation was registered. The imposed inhibition remained constant up to a noise level equal to the the magnetic field. No significant change in proliferation rate was seen when the cell culture was exposed to a noise field only. Keywords:
Electromagnetic noise; Cell proliferation;
AMA cells; Microtiter plate cultivation; Respons inhibition
1. Introduction In spite of numerous studies on the biological effects of exposure to weak electromagnetic fields, the issue of whether continuous exposure to these fields involves health risks, is still a subject of considerable debate. Epidemiological studies have linked the incidence of cancer, especially childhood leukemia, to exposure to ELF EM fields [l-4]. On a cellular level, effects on cell proliferation, enzyme activities, calcium transport, transcription and chromosome aberations have been reported [5- lo]. However it cannot be denied that there are also several studies reporting negative results and no effects of exposure to EM fields [ 11- 121. A true mechanism for these observations has not yet been found. Litovitz et al. found that a magnetic field will only induce biological effects in the exposed system if the field is coherent for at least 5- 10 s [ 131. They therefore suggested that electromagnetic field effects could be inhibited by making the field incoherent, e.g. by superimposing electromagnetic noise. In two separate systems, (the ornithine decarboxylase (ODC) enhancement in L929 murine cells [13] and abnormalities in chick embryos [ 141) both * Corresponding author. 0302-4598/96/$15.00 PII
SO302-4598(96)05060-X
0 1996 Elsevier Science S.A. All rights reserved
induced by exposure to a 60 Hz magnetic field, they could show that these effects could be blocked by superimposing noise on an ELF EM field [151. A related study showed that electromagnetic noise could also inhibit the enhancement of steady state c-myc transcript levels in human leukemia cells induced by exposure to a 60 Hz magnetic field [ 161. We have previously shown that exposure to a 50 Hz pT EM field results in a significant increase in cell growth in human epithelial amnion (AMA) cells [ 171. This change in proliferation rate was dependent on exposure times and field densities. A maximum increase in cell proliferation was only found at a specific field density and exposure time i.e. there were “window” and adaptation effects. The purpose of this study is to investigate whether a superposition of a noise EM field, can inhibit the changes in proliferation rate in AMA cells induced by exposure to ELF magnetic fields.
2. Materials 2.1. Materials
and methods and cell lines
Cell proliferation Reagent WST- 1 from Boehringer Mannheim Biochemica was used.
194
P. Raskmark, S. Knee/ Bioelectrochemistry
and Bioenergetics 40 11996) 193-196
Fig. 1. Diagram showing experimental set-up for noise added ELF magnetic field exposure. Magnetic fields are measured before exposure using the fluxgate. The current in the coils are monitored during exposure using the rms voltmeter.
Transformed human epithelial amnion cells (AMA) were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium containing 10% calf serum, penicillin and streptomycin, at 37 “C in 6.0% CO, and kept on a Forma Scientific 3 164 incubator. 2.2. EM exposure and noise generation The exposure system was designed to generate ELF magnetic fields in accordance to the principles outlined in [ 18,191e.g. we measured static and stray fields and checked temperature rise during exposure. The apparatus setup is basically the same as used in [20], see Fig. 1 i.e. a signal generator and filter followed by a power amplifier delivering current to Helmholz coils and using a fluxgate to measure the magnetic fields. As an exposure measure we used the magnetic field measured before exposure in the same position as the exposed cells. We used the current supplied to the coils to monitor the fields during exposure. The exposure levels are traceable through the Bartington fluxgate and the Hewlett Packard multimeter. The Helmholz coils had the following attributes: dimensions of 12 cm x 13 cm, 9 cm apart, 2 times 10 turns using 1 mm diameter copper wire and a copper screen. The magnetic fields were oriented in the plane of the cell plates and the variation in the magnetic fields was not more than 5% as a function of cell well position (see field distribution in Ref. [20]). If more homogeneous fields are required the method of Kirschvink [21] can be applied, in which a double wrapped system is recommended. In our experiments the sensitivity to field strength was weak and since the temperature is controlled, a simple Helmholz coil system was considered sufficient. A multifunction synthesizer followed by a band pass filter generated the required voltage, see Fig. 1. The synthesizer was programmed to deliver a constant 50 Hz signal and 5 different levels of Gaussian white noise were added. The composite signal is bandpass filtered using first a lowpass- and then a highpass-maximally flat four pole Butterworth filter (24 dB per octave response, 50 Hz cutoff frequency). The excitation voltage was amplified using a voltage-to-current power amplifier (high impedance out-
put) to secure a frequency independent voltage to magnetic field transfer. Interference signal pickup was minimized having all instruments signal ground except the floating signal generator. The generated magnetic fields were measured using a fluxgate with the frequency range O-l kHz and analyzed with two different instruments: a rms voltmeter and a dynamic signal analyzer. In contrast to sinusoidal signals the low frequency noise signals are somewhat difficult to measure. Determining the noise signal by using an oscilloscope [15] is not reproducible, a better criterion is rms together with information on bandwidth. Since standard so called “true rms” voltmeters will display varying results due to limited crest factors, we used the hp3458A multimeter (with the longest possible integration time, approx. 20 s). In addition we used the hp35660A Dynamic Signal Analyzer to measure the power spectra, as shown in the example in Fig. 2. 2.3. Assay Proliferation of the cell cultures grown in microtiter plates was determined by a calorimetric assay with quan-
10 5 I
-351
/
I lo2
10'
Frequency/
HZ
Fig. 2. Measured magnetic fields for 50 +T 50 Hz with added 20 FT (rms) bandpass filtered white noise. The voltage output from the fluxgates are plotted in dB relative to 1 V rms in the frequency range lo-300 HZ; 14 dB corresponds to 5 V rms or 50 FT.
P. Raskmurk, S. Kwee/Bioelecrrochemis?ry
tification in an ELISA plate reader, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases i viable cells, in the following way: 10 ~1 WST-1 reagent was added at zero time to each well and after 5-6 h incubation at 37 “C in 6.0% CO,, the absorbance was read. The absorbance was measured at the wavelength of 490 nm and the reference wavelength was 655 nm. For maximum absorbance a second reading was taken after 24 h. The number of proliferating cells was proportional to the absorbance.
and Bioenergefics 40 (1996) 193-196
195
Table 1 Percent change in proliferation rate in AMA cells after 30 min exposure to EM field of 50 Hz and 50 *T with varying noise levels % change in profileration rate
Field density/kT
Noise level/ pT
mean
95% confidence limits
50 50 50 50 50 50
0 10 20 30 40 50
0 + 0.0 a - 2.25 a - 6.3 a - 16.0 -20.1
- 16.4 - 14.6 - 23.0 - 25.7 -31.2
16.4 10.15 10.5 - 6.2 -9.1
a Not significant P value > 0.5 (paired t-test).
2.4. Experimental protocol The cells were seeded in 100 p,l culture medium into tissue culture grade, microtiter plates with 96 wells and a flat bottom. Cells were seeded in the 2 central rows, 6 and 7, A-H. After 24 h field-free incubation at 37 “C the cells were growing in their log phase to approximately 40-50% confluency. A .plate was then transferred to the Hehnholtz coil assembly in the incubator and exposed to a 50 Hz, 50 p.T magnetic field. Subsequent microtiter plates were exposed to this magnetic field with superimposed noise fields of varying strength. Exposure time 30 min. Other microtiter plates were exposed to noise fields only. During exposure the field and temperature were monitored continuously in the incubator. For each series of experiments cell proliferation was determined prior to exposure to the field i.e. WST-1 reagent was added to all the wells in row 6 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. WST-1 reagent was added to all the wells in row 7 of each plate. In the control experiments similar cell cultures were grown and incubated in a field-free environment and cell proliferation was 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. During exposure the temperature in the cultures was measured, but temperature rise was less than O.l”C.
3. Results and discussion Proliferation rate was expressed as the ratio between absorbance after 24 h and absorbance at zero time in percent. *,=
Percent proliferation rate = -
24h
. 100
A,=0 Change in proliferation rate was expressed as the difference between the percent proliferation rate in the exposed
culture and the percent proliferation experiments.
rate in the control
change in proliferation rate = proliferation rateexposed - proliferation ratecontrol Change in proliferation rate due to the superimposed noise field was expressed as the difference between proliferation rate in the culture exposed to both a magnetic and a noise field and the proliferation rate in the culture only exposed to the magnetic field. effect of noise field = proliferation ratenoise+nMfield- proliferation rateEMfield The changes in proliferation rate after 30 min exposure to a 50 Hz, 50 ~.LT electromagnetic field with varying superimposed noise levels are reported in Table 1. Each figure was based on the average mean of 8-9 independent experimental results with a 95% confidence interval. From Table 2 it can be seen that if only a noise field was applied without an EM field, no significant effect on cell growth was detected as compared to exposure to the ELF field or to the control. Table 1 shows that the superimposed noise field causes an inhibition of the increased cell growth caused by the ELF field. However, the inhibiting effect is only significant at a noise field from 40 PT on and higher. At noise levels below 30 p.T there is hardly any inhibiting effect. This means that the total effective field reaches a level of 64 p,T (i.e. 50 FT signal + 40 p,T noise) before significant growth inhibition is detected. These results are comparable to those found by Litovitz et al. Their experiments showed a steady decrease [15] in ODC activity of L929 cultures, starting at a noise to signal Table 2 Percent change in proliferation rate in AMA cells after 30 min exposure to EM field of 50 Hz or to noise only % change in proliferation rate
Field density/kT
Noise level/ pT
mean
95% confidence limits
0 50 0
0 0 50
0 + 15.1 + 9.3 a
5.1 - 1.7
a Not significant P value > 0.5 (paired t-test).
24.5 20.4
196
P. Raskmurk, S. Kwee /Eioelectrochemisrty
ratio of approx. 0.05 and reaching almost complete inhibition at at noise to signal ratio of 1. In our experiments a significant inhibiting effect is first obtained at a noise to signal ratio of 0.8, i.e. appproximately two times more noise is required than found by Litovitz et al. [15]. It should be taken into account that our experimental conditions differed with respect to cell cultures, measurement of biological data and the ELF em and noise fields. Moreover it must be pointed out that it is premature to conclude from our results that the the fields mentioned will affect cellular functions under in vivo conditions in a similar way and induce the same biological effects. This would require additional experiments on whole organisms, such as animals and human beings under normal living conditions.
Acknowledgements This research was supported by Danfoss A/S, Denmark.
References fi] D.A. Savitz et al., Am. J. Epidemiol., 128 (1988) 21. [2] M. Feychting and A. Ahlbom, Am. J. Epidemiol., 138 (1993) 467. [3] P.GuCnel, P. Raskmark, J. Bach Andersen and E. Lynge, Br. J. Ind. Med., 50 (1993) 758 [4] J.H. Ohlsen et al., Br. Med. J., 307 (1993) 891.
und Bioenergetics 40 (19961 193-196 [5] S. Johann, T. Ledeter, S. Mikotey, W. Kraus and G. Bliimel, Bioelectrochem. Bioenerg., 30 (1993) 127. [6] M.S. Markov, J.T. Ryaby, J.J. Kaufman and A.A. Pilla in M.J. Allen, S.F. Cleary. A.E. Sowers and D.D. Shillady (eds.), Charge and Field Effects in Biosystems 3, Birkhaiiser, Boston 1992, p. 151. [7] M.S. Markov, D.J. Muehsam and A.A. Pilla in M.J. Allen, S.F. Cleary and A.E. Sowers (eds.), Charge and Field Effects in Biosysterns 4. World Scientific, Singapore 1994, p. 274. [8] R. Goodman, M. Blank, H. Lin, R. Dai, 0. Khorkova, L. Soo, D. Weisbrot and A. Henderson, Bioelectrochem. Bioenerg.. 33 (1994) 115. [9] I. Nordenson, K. Hansson Mild, G. Andersson and M. Sandstrom, Bioelectromagnetics. 15 (1994) 293. [lo] S. Tofani, A. Ferrara, L. Anglesio and G. Gilli, Bioelectrochem. Bioenerg., 36 (1995) 9. [ 111 P. Verkasalo et al., Br. Med. J., 307 (1993) 895. [12] S. Galt, J. Wahlstrbm, Y. Hamnerius, D. Holmqvist and T. Joharnesson, Bioelectrochem. Bioenerg.. 36 (1995) 1. [ 131 T.A. Litovitz, D. Krause and J.M. Mullins, B&hem. Biophys. Res. Commun., 178 (1991) 862. [14] T.A. Litovitz, C.J. Montrose, P.Doinov, K.M. Brown and M. Barber, Bioelectromagnetics, 15 (1994) 105. [15] T.A. Litovitz, D. Krause, C.J. Montrose and J.M. Mullins, Bioelectromagnetics (I 995) in press. [16] H. Lin and R. Goodman, Bioelectrochem. Bioenerg., 36 (1995) 33. [17] S. Kwee and P. Raskmark in M.J. Allen, SF. Cleary and A.E. Sowers (eds.), Charge and Field Effects in Biosystems 4, World Scientific, Singapore 1994, p. 255. [ 181 M. Misakian et.al. Bioelectromagnetics Suppl. 2 (1993) 1. [ 191 M. Chiabrera et al. COST 244 temporary document no. 244TDc95m9. [20] S. Kwee and P. Raskmark, Bioelectrochem. Bioenerg., 36 (1995) 109. [21] J.L. Kirschvink, Bioelectromagnetics, 13 (1992) 401.