EXTREMELY LOW FREQUENCY MAGNETIC FIELDS

Effects of weak (less than 100 microT) extremely low frequency (ELF, 3 - 300 Hz) magnetic fields' (MF) on living systems have been frequently demonstrated in scientific studies. The motivation for researching was a possible health risk these fields represent. ELF magnetic fields can influence biological systems in a number of ways. Reported results show very heterogeneous (stimulatory, inhibitory) and nonlinear effects (power and frequency windows etc.) and are mostly very hard to reproduce. Very variable exposure conditions among different laboratories represent an additional difficulty which makes it hard to infer on any mechanisms involved. Despite this, many physical theories were proposed trying to reveal the mystery of the MF biological influence mechanism and, based on their assumptions, predict the MF exposure conditions that would produce biological effects. At that point another problem had to be dealt with - lack of suitable detection systems, that would be sensitive and at the same time reliable enough (in the sense of reproducibility of effects) for testing the theories. At the same time the importance of geomagnetic field intensity and orientation for difficulties in reproducing successful experiments in different laboratory has been emphasized.

Considering all this, we have developed a new detection system for weak, ELF MF in order to test some of the proposed theories about the mechanisms involved in the influence of ELF MF on biological systems, where the conditions of exposure are precisely determined. With this system we could also monitor possible effects simultaneously. The system is based on bioluminescence of dinoflagellates, which has already been thoroughly investigated.
 

BIOLUMINESCENCE

Bioluminescence is the emission of light from living organisms without generating heat (1 ). The light is produced when a high energy substrate, luciferin, is oxidized in the presence of enzyme luciferase. It can be emitted continuously (i.e. bacteria) or occurs as flashes, typically of 0,1-1 s duration. This phenomenon is found in many organisms like fish, fireflies, worms, cnidarians, ctenophores, molluscs, fungus, dinoflagellates, bacteria and others.

   Two cells of Gonyaulax scrippsae.

Bioluminescent dinoflagellates are often used for biological research due to their sensitivity to external influences. Dinoflagellates emit light in the blue region. Emission occurs in two forms: as a continuous low-level glow and as a rapid flashing (app. 100 ms). Numerous parameters can trigger these emissions, including pH, temperature, electric currents, osmotic shock, mechanical stress, and various chemicals. Bioluminescence in dinoflagellates reflects the state of the cell and associates with many cell functions - enzyme (luciferase) activity, electron transport, translation of proteins, proton translocation, iron uptake, oxidative metabolism etc. It is sensitive to different wavelengths of light as well as to different irradiation time intervals, temperature, chemicals that influence calcium ions in any way (bind, displace or influence its transport) and also some other chemical substances (creatine, anisomycin, cycloheximide). Calcium ion proved to be a very important factor in biological response to weak, ELF MF.

In autotrophic dinoflagellates like Gonyaulax sp. (probably G.scrippsae), the intensity of bioluminescent emission varies on a circadian basis (as well as cell division, photosynthesis, geotaxis and swarming behavior, activities of some enzymes i.e. nitrate reductase) - it is much more intense during their night phase then during their light phase. Light is emitted from many organelles (app. 400 per cell), called scintillons, which contain luciferin-binding protein (LBP) and luciferase. Scintillons are spherical evaginations of cytoplasm into the cell vacuole, which preserves the continuity of the vacuolar membrane conducting the triggering action potential. The opening of membrane proton channels causes a transient pH change in the scintillones, which activates the reaction, and a flash. At the end of the dark phase there is a peak of emission intensity, when both proteins are destroyed and later resynthesized  in the next cycle. Their synthesis is regulated on the translation level. Remarkably, also the scintillons are broken down and reformed each day. The sensitivity to external factors mostly reflects in changes of this diurnal rhythm of emission intensity. Biorhythms also proved to be very sensitive to weak electromagnetic fields.
 

EXPERIMENTAL RESEARCH - TESTING OF LEDNEV'S PRM (1991)

First we studied gonyaulax bioluminescence properties. Its bioluminescence follows circadian rhythm which is influenced by the cell size, activity, age, density and temperature. It was the most sensitive to 50 Hz 11,2 mT magnetic field when exposed during D3 and D5 of the dark phase. 50 Hz 1,2 mT and 35 Hz 0,7, 1 and 1,2 mT were not effective (2).
After experimentally establishing sensitivity of our biological system to weak, ELF MF we tested Lednev's parametric resonance model (PRM) proposed in 1991 (3). The model predicts that the probability of biological effects appearance due to weak alternating magnetic fields (B_AC) in presence of static geomagnetic field or some other static field (B_DC) depends on the relationship between the magnitudes of B_AC and B_DC and the angular frequency. The target is an ion bound to oxygen ligands in calcium-binding proteins (e.g. calmodulin). This model has already been successfully tested for Ca²⁺ and K⁺ ions by Frank S. Prato and co-workers (4) and some other scientists (5), as well as Lednev himself and his co-workers. We tested PRM for Ca²⁺ ion.
Dinoflagellates were exposed to the combination of a parallel 46 microT static magnetic field and a 35 Hz alternating magnetic field of 115 microT, 243 microT, 340 microT in 745 microT. The model predicts maximum effects of around 115 microT and 340 microT, opposite maximum effects at B_AC 243 microT and no effects at B_AC 745 microT. With exposure to these fields in the dark phase there were no obvious effects. On the other hand, exposure in the day phase showed influence of magnetic fields according to the PRM predictions. Exposure to 50 Hz 30 mT (magnetophosphenes) alternating magnetic field did not influence bioluminescence.

Cirkadian rhythm of Gonyaulax scrippsae.

ELF magnetic field influence on G.scrippsae bioluminescence in the light phase. The bioluminescence is triggered around 15 minutes after onset of magnetic field when effects are predicted by PRM (1991).

ELF magnetic field influence on G.scrippsae bioluminescence in the light phase. When environmental temperature is higher then 25.5 deg.C, a slight peak in emission intensity is observed around 1 hour after the begining of the measurement. The exposure to magnetic field which should produce oposite effects acording to PRM (1991) reduces and delayes the peak.
 

CONCLUSIONS
The results indicate calcium ions involvement in the cell response to weak ELF magnetic fields, possibly through the mechanism proposed by PRM. Other mechanisms are not excluded, i.e. the influence through the heat shock pathway (M.Blank and R.Goodman). Magnetic fields interaction with gonyaulax bioluminescence was non-linear and dependent on physiological state of the cell.
The bioluminescence of Gonyaulax scrippsae proved to be very useful for researching weak ELF magnetic fields influence on biological systems. Its great advantage is a real-time monitoring of magnetic fields' effects. In addition, bioluminescence measurement is a non-invasive and easily implemented technique, which provides various responses, critical for testing the theories about influence mechanisms.
 
 

INTERESTING LINKS
The Bioluminescence Webpage
Bioluminescence websites
Dinoflagellate websites
J.Woodland Hastings Homepage
International Society for Bioluminescence and Chemiluminescence (ISBC)
 
 

Application of bioluminescence measurements
Soil and water toxicity monitoring with bacterial bioluminescence (Vibrio fischeri) according to International standard ISO 11348.

References
1. Wilson, T. and J.W. Hastings (1998): Bioluminescence. Annu Rev Cell Dev Biol 14: 197.
2. Berden, M., Zrimec, A., Jerman, I. (2001): New biological detection system for weak ELF magnetic fields and testing of the parametric resonance model (Lednev 1991). Electro- and Magnetobiology 20(1): 27.
3. Lednev, V.V. (1991): Possible mechanism for the influence of weak magnetic fields on biological systems. Bioelectromagnetics 12: 71.
4. Prato, F.S., Kavaliers, M., Thomas, A.W. (2000): Extremly low frequency magnetic fields can either increase or decrease analgaesia in the land snail depending on field and light conditions. Bioelectromagnetics 21(4): 287.
5. Yost, M.G., Liburdy, R.P. (1992): Time-varying and static magnetic fields act in combination to alter calcium signal transduction in the lymphocyte. FEBS Letters 296: 117.
 

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