Libro_Biological Effects of Magnetic and Electromagnetic Fields(the Language of Science)

7/28/2019 Libro_Biological Effects of Magnetic and Electromagnetic Fields(the Language of Science)

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MAG NETIC NERVE STIMULATION ANDEFFECTS OF MAGNETIC FIELDS ONBIOLOGICAL, PHYSICAL AND CHEMICALPROCESSES

Shoogo Ueno and Masakazu IwasakaInstitute of Medical ElectronicsFaculty of Medicine, U niversity of TokyoTokyo 113, Japan

INTRODUCTIONBiological effects of magnetic fields are classified into three categories; the effectsof (1) time-varying magnetic fields, (2) DC or static magnetic fields, and (3) multiplicationof both static fields and other energy such as light and radiation. For each category, a differentstrategic approach is required to shed light on the biomagnetic effects.Time-varying magnetic fields produce eddy currents which stimulate excitabletissues at low frequencies. Magnetic brain stimulation can be realized by this effect. The firstpart of this paper focuses on m agnetic nerve stimulation.Biological effects of static magnetic fields have been poorly understood. Recognitionof the role of diamagnetic, paramagnetic and ferrimagnetic materials in the body may helpin unraveling the underlying mechanisms. The latter part of this paper focuses on the effectsof magnetic fields on the behavior of diamagnetic water and paramagnetic oxygen. Theembryonic development of frogs, blood coagulation, fibrinolytic processes and other biochemical processes are also observed under strong magnetic fields up to 8 T

BIOMAGNETIC PHENOMENABiomagnetic phenomena in different intensities of magnetic fields and their frequencies are shown in Fig. 1. Effects of magnetic fields on living system s and biological ma terials

have been observed mostly in the range of magnetic fields higher than the earth's magneticfield. Recently, superconducting magnets are being used in MRI (magnetic resonanceimaging) systems, where human subjects are exposed to static magnetic fields of 1.5 T 2.0T. Mo re intense mag netic fields (4 T order) are used in advanced M RI sy stems. We observedthe phenomenon that surface of water is parted by static magnetic fields of up to 8 T FibrinBiological Effects of Magnetic an d Electromagnetic Fields, Ediled by S. UenoPlenum Press, New York, 1996 I


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S. L'eno and M. Iwasaka

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Magnetic Fields on Biological, Pliysical and Chem ical Processes 3and lung (Magnetopulmogram, MPG) can be measured . This chapter , however , does notinc lude these b iomagnet ic measu reme n ts .

MAGNETIC NERVE STIMULATIONMagnetic stimulation of excitable tissues has been studied by several investigators

(D'Arsonval, 1896, Bickford et al, 1965, Irwin et al, 1970, Maass et al, 1970, Oberg. 1973. ).Among them, Maass and Asa proposed a t ransformer type of s t imulat ion in which a nervebund le was threaded through a core as the secondary wind ing (M aass et al , 1970) . T heydemonstrated that the f lux change in the core could be used to excite nerves. Oberg proposedan airgap type of s timulat ion , in which a nerve bundle w as exposed to a l ternat ing ma gnet icfields (Oberg, 1973). Induced eddy currents in the membrane tissues could be expected tos t imulate nerves . Although these types of magnet ic nerve s t imulat ion were exper imental lydemonstrated , the under ly ing nerve exci ta t ion processes fo l lowing magnet ic s t imulat ionwere not yet understood (Ueno et al, 1978).

We carried out an experiment to measure the action potentials of lobster giant axonsunder t ime-va ry ing m agnet ic f ie lds (Ueno et a l , 1981, 1986) . The axon me mb rane wa sexcited by galvanic stimulation, and the action potential was recorded intercellularly withmicro-electrodes . Dur ing the propagat ion of the act ion potent ia l a long the axon, a l ternat ingor pulse d ma gne tic f ields were applied acro ss the mi ddle of the axon in order to study wh eth eror not the magnetic f ields had any effect on parameters such as conduction velocity andrefractory period of the nerve f iber and am plitu de, duration and shape of the action pot ent ials.

The results obtained from the lobster experiments suggest that nerve excitation byma gne tic f ield influen ce is me diate d via the induction of eddy curre nt in the tissue s urrou nding the nerve. The current density induced depends on geometrical factors as well as theresistivity of the tissue in which the current f lows. In other words, for nerve excitation, themicroscopic eddy currents that f low ins ide the microstructures of the axon membrane arenot very important. What is important are the macroscopic eddy currents that f low along thenerve axon and in the tissues surrounding the nerve, as they contribute to the depolarizationo f the membrane .

After the study of single nerve axons, we proposed a new type of magnetic nervestimulation (Ueno et al, 1984). An insulated magnetic core is implanted into the body witha nerve bundle positioned on the core aperture. The nerve can be stimulated by the eddycurrents that flow in the body fluids around the core when the magnetic flux in the core ischanged. There is no in ter l inkage between the core and the nerves . Therefore , th is methodis a good ex am ple for ver ify ing that the nervous sys tem responds to t ime-vary in g mag net icfields through eddy currents induced in the body.

Magnetic stimulation of the human brain was f irst reported by Barker et al. (Barkeret al, 1985, 1986, 1987). Parameters of muscular potentials, such as conduction velocity,la tency and ampli tudes were s tudied by a number of researchers by record ing the electromyographic (EMG) responses to s t imulat ion of the motor cor tex (Day et a l , 1987, Hesset al, 1987a. 1987b, Mills et all , 1987, Ro thwe ll et al, 1987). Single coils were used for thesestudies. Current pulses were passed through a single coil placed outside the head, and eddycurrents induced into the head by the pulsed magnetic f ields stimulated the brain. With thismethod, however , broad areas of the brain are s t imulated s imultaneously . We developed amethod of local ized magnet ic s t imulat ion of the human cor tex (Ueno, Tashiro , and Harada,1988).

The basic idea is to concentrate induced eddy currents locally in the vicinity of atarget by a pair of opp osin g pulsed ma gne tic f ields which can be produ ced by a f igure-eightcoi l . U sin gth ism et ho d, w e were ab le to s t imulate the mo tor cortex of the hum an brain w ith in


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4 S. Leno and M. Iwasakaa 5 mm resolution (Ueno, Matsuda, Fujiki, and Hori, 1989, Ueno, Matsuda, and Fujiki,1989a). Since the concentrated eddy currents beneath the intersection of the figure - eightcoil flow in a direction parallel to the tangent of both circular co ils, vectorial stim ulation canbe achieved. The neural fibers can be excited easily when the fibers are stimulated by theeddy currents that flow parallel to the nerve fibers.Based upon this principle, we obtained functional maps related to the hand and footareas (Ueno, Matsuda, and Fujiki, 1989a, Ueno, Matsuda, and Fujiki, 1989b, Ueno , M atsuda.and Hiwaki, 1990). We have observed that an optimal direction of stimulating currents forneural excitation exists in each functional area in the cortex. We have also observed that thefunctional map s of the cortex vary with the orientation of the stimulating curren t. To explainthe mechanism that is responsible for producing this anisotropic response to brain stimulation, we developed a model of neural excitation elicited by magnetic stimulation (Ueno,Matsuda, and Hiwaki, 1991).

The m odel explains our observation that the directions of the induced cu rrent ve ctorsreflect both the functional and the anatomical organization of neural fibers in the brain. Weneed to emphasize that the point of excitation is not at a point under the intersection withthe coil. Instead it is a few ten mm away from the point of intersection; in the case in whichthe coil diameter used is 50 mm, the distance between the coil and the nerve fiber is 10 mm.

This phenomenon was experimentally studied by Nilsson et al in magnetic stimulation of a peripheral nerve (Nilsson et al, 1992). Magnetic stimulations of the human brainand peripheral nerve were also studied by other groups (Amassian et al, 1989, 1992, Basseret al, 1991, Maccabee et al, 1990, Olney, et al,1990, Esselle and Stuchly, 1992, Rothwell,1994, Cohen et al, 1990, Reilly et al, 1989).Magnetic stimulation of the spinal cord and heart were also studied (Ue no, Matsuda,and H iwaki, 1990, Ueno and Hiw aki, 1991, Bourland et al. 1990, 1992. Ny enhu is, 1994.Hosono, 1992),

PARTING OF WATER BY MAGNETIC FIELD: MOSE'S EFFECTSWater, which is a diamagnetic material, is one of the most general and abundantdiamagnetic fluid. It is an interesting problem for humans as to whether or not magneticfields have any influence on water. Diamagnetic water is often used for calibration and

correction in determining magnetic susceptibilities of materials with the magnetic balancesystem. However, little has been observed about the hydrodynamic behavior of water ordiam agne tic fluids, in horizontal m agnetic fields of 100 - 1000 T- / m order.We investigated the dynamic behavior of water in high gradient magnetic fields. Weused a horizontal type of superconducting magnet 700 mm long with a 100 mm diameterwide bore. The superconducting magnet has a distribution of high gradient magnetic fieldsnear the center of the magnet, as show in Fig. 2. When the magnet produced 8 T at its center,the maximum product of the magnetic field and the gradient was 400 T" / m at z= 5 mm,where the z-axis was directed to the bore axis. A water chamber, 50 mm wide, 60 mm high,and 700 mm long was filled with distilled water.When the water chamber was inserted into the bore of the magnet, we observed aphenom enon in which the surface of the water was pushed back by magne tic fields of highergradients. Two "frozen" cascades were formed; the surface of the water near the center ofthe magnet was parted, and the bottom of the water chamber could be seen. The water levelat both ends of the chamber was raised (Ueno and Iwasaka, 1994 a). We call this phenom enonthe Moses' effects. This phenomenon reminds us of the Biblical story from Exodus, i.e..Moses parting the Red Sea.


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Ma|net i Fie lds on Biological , P i iyskal and Chemieal Processes

Water chamber

Figure 2. Distribution of magnetic fields along the horizonla! orz-axis in a superconducting magnet.

100 mm

-200 0 200Distance z [mm]

We also found the Mo se s' effect w ith agarose gels. The agarose so lution was allom'cdto gel in the superconducting magnet. Fig. 3 shows that the surface of the agarose gel wasfortBed in the same manner as in the water experiment.To measure changes in the water level in the magnetic fields, one of the edges of thewater chamber was positioned at the center of the magnet, and the water level wa.s observedby a video camera. M agnetic fields in the center of the magnet w ere changed from 0 to 8 T,and a decrease in the water level was observed. The chan ges in the lowest level of water was-22 mm when magnetic fields were changed from 0 T to 8 T as shown in Fig.4. The decreasein water level was proportionate to B^ at the center of the magnet. This result shows that theenergy used for the formation of water-walls in a steady state is proportiona te to the inag.neticenergy of the water (Ueno and Iwasaka, 1994 b).

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Fi gu re 3 . Mo ses' effect. A visible ink was addedinto the agarose solution, and the solution was allowed to gel in the magnetic fields. 5 cm


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S. Ucno and M. bvasaka

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80 Figure 4. Change in water surfaee level inmagnetic fields.

A Stress analysis was carried out to explain the mechanism of the Moses" effect. Thestress tensor of a diamagnetic f luid ( in indicial notation) is given by

T"' n, = (p"' + (loH '. ' 2 + poMH / 2) n, , 1p"' = - | i , |M H 2 + |a {" M d H - p g z + c o n st , 2

wh ere n = (n,,nj,n |j) is a unit vector that is perpen dicu lar to the wate r surface, p '" is the staticfluid p ress ure, i ,, is m agne tic perm eability, p is ma ss per unit vo lum e, g is gravita tionalconstant, M is the magnetization of diamagnetic f luid and H is a magnetic f ield (Ueno andIwa saka , 1994 a) .

On the other hand, the stress tensor that is outside of the diamagnetic f luid is uivenby

T " , j n j = - - ( p " + H 0 H 2 / 2 ) n i . 3where p " is the environm ental pressure.

On the sur fac e of the w ate r, T"",!, n equ al to T",j Uj. E qu at ion s ( 1), (2) a nd (3) can becombined to g ive

z = h = (Mo / rg) lO" M dH = z, Mo H- / 2rg . 4Decreases in water levels calculated by equation (4) are shown in Fig. 4 and Fig. 5.

The hydro dyna mic s of d iam agnet ic f lu id in ~40() T ' ' m is com parab le with that offerromagnetic f luid in weak magnetic f ields. Remarkable effects of high gradient magneticfields are expected in blood circulation, cerebral spinal fluid system and other intra and extracel lu lar so lu t ions .

Th e ma gnetic force ac ting on water mo lecule s is given by

F = i B ^Mo d z 5where x is the magnetic susceptibility of water molecules, B is the magnetic f lux density.and Mo is the mag netic p erm eab ility of the vacuum . M olec ular sus ceptib ility of water is- 1 2 . 9 7 X 10"'' [cgs] at 29 3 K. Th e ma gne tic forces act ing on 100 ml of water is sho wn in Fig.


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Magnetic Fields on Biological, Physical and Chemical Processes

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Figure 5. Surface profile of diamagnetic water inmagnetic fields of up to 8 T.- 3 0-300 -200 -100 0 100 200 300

Distance [mm]

6. Amaxiniiim magactic force of 0.288 [N], m'hich is about one third of the force of gravity,is obtained wliere BxclB/dz = 400 T^/DI.Magnetic levitations of diamagoetic materials such as water, etlianol and acetone i0magnetic fields of more than 20 T produced by a vertical type of superconducting magnetwere reported by Beaugnoo et al (Beaugnon et al, 1991). Distortion in the surface of liquidhelium by magnetic fields of 6.5 T was also studied (Frost et a!).Studying the role of diamagnetic fluids in gradient magnetic fields is important inunderstanding the mechanism of biological effects of magnetic fields. The phenomenaobserved in the present experiments will provide a new aspect to applied pliysics andbiomagnctics.

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Figure 6. Magnetic force acting on water. IFimax = 0.28 8 {H / 1 0 0 m i] a t C


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Magnetic Fields on Biological, Physical and Chemical Processes

Figure 8. Redistribution of dissolved oxygen concentrationby magnetic fields of up to 8 T. The concentration of dissolvedoxygen before magnetic field exposure was controlled byintroducing oxygen gases into the water. 1 2 3 4 5 6 7Sect ion number

The experimental set-up is shown in Fig. 7. We used a horizontal type of superconduct ing m agnet 700 mm long with a bore 100 mm in d iameter . When the magnet produc ed8 T at i ts center , the maximum product of the magnetic f ield and the gradient wa.s 400 T- mat 5 mm , w he re the z-axis is the b ore a xis .

An acrylic water chamber, 700 mm long, 70 mm high, 50 mm wide was 100 "o f illedwith distil led water , and the water chamber was sealed with an acrylic cover to separate thewater from the air atmosphere. The water chamber consisted of 7 sections, and the length ofeach section was 100 mm. Partit ions, i .e. , dividers, were removable.

The d isso lved oxygen concentrat ion w as contro l led by in troducing oxygen gases in tothe distil led water in the range from 8 mg/1 to 26 mg/1. The w ater ch am ber w ithou t divi derswas placed in the magnet 's bore, and the water was exposed to magnetic f ields of up to 8 Tfor 60 min. After the water chamber was taken out of the magnet, the water in the chamberwas divided by six acrylic dividers into seven sections. The dissolved oxygen concentrationat each section was measured with a Clark type DO (dissolved oxygen) meter .

To obtain dynamic behaviors of dissolved oxygen during and after magnetic f ieldexposures , t ime-courses of changes in d isso lved oxygen concentrat ion were measured in the4th sect ion , changing the exposure per iods .

Fig. 8 show s the redistr ibution of dissolve d oxygen co ncen tration by mag neti c f ieldsof up to 8 T for 60 min. The dissolve d oxyge n conce ntration be fore ma gnetic f ield e xpo sur es.i .e. , an initial con cen tratio n, w as controlled from 11 mg/1 to 22 mg/1. Th e horizon tal axisshow s the section num ber of each sect ion d iv ided by acry l ic d iv iders . The d isso lved oxygenconcentration in sections 3 5 at the central part of the magnet was increased.

Fig. 9 shows the normalized data of the redistr ibution of dissolved oxygen concentration. The data were averaged over 8 tr ials where the dissolved oxygen concentration at

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Figure 9. Effect of an 8 T magnetic fie ld exposure for 60min on distribution of dissolved oxygen concentration. Thedata were averaged over 8 trials where the dissolved oxygen concentration in section 1 and 7 fell in the range 14mg'lto 16 mg/1.

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10 S. L'eno and M. Iwasaka

Initial concentration [mg/l]

Figure 10. Effect of magnetic fields of up to 8 T on ch ange sin concentration of dissolved oxygen in water. The concentration of dissolved oxygen before magnetic Held exposurewas changed from 8 mg.l to 26 mg. 1,

both edges of the cham ber fell in the range of 14 mg/l 16 mg/l after magnetic field exposuresof 60 min. The results show that the increase in dissolved oxyge n con centration was 5 %.Fig. 10 shows the effect of initial concentration on the redistribution of dissolvedoxygen concentration. When the initial concentration changed from 8 mg/i to 26 mg/l. thechange of dissolved oxygen was 0% to 13 %.The magnetic force acting on a group of oxygen molecules is described as follows.

X n dB ,. .. . . B 3, (in z-direction)dzHowhere x is the magnetic susceptibility of a group of oxygen molecules, B is the magneticflux density, and \1Q is the magnetic permeability of the vacuum. For oxygen mo lecules, x =3449 X 10^ [cgs] at 300 K. The magnetic force acting on 1 molar of oxygen is shown in Fig.11.

High gradient magnetic separation (HG MS) has made it possible to separate m icron-sized paramagnetic particles from solution. For instance, Melville et al succeeded inseparating red cells from whole blood by HGM S when the hemoglobin was in the comp letelydeoxygenated state (Melville et al, 1975) . Friedlaender et al captured small paramagneticparticles such as Mn203 and CriOj by HGMS (Friedlaender et al, 1978).The magnetic energy of an oxygen molecu le is 3 x 10 '-'' [J] at I T and 2 x 10'-'' [.I]at 8 r, respectively. Thermal energy at 300 K is 4 x 10 -' [J] . It is estimated that oxygenmolecules drift in a particular direction in magnetic fields of more than 8 T when at least2000 oxygen molecules aggregate.Oxygen molecules are attracted by a maximum magnetic force where BxdB/dz islargest in magnetic fields. When there are two maximum peaks in a magnetic force as shownin Fig. II, dissolved oxygen molecules are trapped in the area between the two peaks

-200 20 0Distance [mm]

Figure 11. Magnetic force acting on dissoK ed oxygen molecules per mol.


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Magnetic Fieldi on Biological, Physical and Chemical Processes 11resulting in the redistribution of dissolved oxygen concentration. In other words, spatialdistribution of dissolved oxygen concentration conforms to a single peak distribution in asteady state, similar to the distribution of magnetic fields, but not to the distribution ofmagnetic force. In contrast, in the case with captures of paramagnetic particles by HGMStechniques, trapped particles accumulate in the vicinity of wires where the magnetic forceis the strongest.

liffects of high gradient magiicric fields on biological systems are expected 'Alien aliving tissue contains a high concentrarioii of dissolved oxygeii. For instance, the oxygeneffect is a piienomeaon when radiation iniury of tissues with a higli concen tration of oxygenbecom es serious, compared with the case of a low concentration ofoxygen^ If the beha\iorsof dissolved oxygen could be controlled by magnetic fields, a new type of cancer ilierapymight be contrived (IJeno and Ffarada, 1986).

EFF ECT S OF MAG NETIC FIELDS ON GAS-FLOW ANDCOMBUSTIONIn relation to oxygen dynamics in water, we observed oxygen dynamics in air.Combustion is an oxidation reaction which involves both a burning phenomena in the airand cell respiration in the living body. The effects of magnetic fields on combustion ofhydrocarbons and alcohol with the aid of platinum catalysis have been studied to simulatein part the oxidation of organic matter in the living body, and we observed that thecombustion velocities of gasoline and alcohol were influenced by magnetic fields (Ueno etal , 1985, 1986). In order to explain this pheomenon, we examine the effect of gradient

magn etic fields on burning flames. A candle was burned in the airgap between mag neticpoles, and the candle flames were exposed to gradient magnetic fields. D uring m agnetic fieldexposures, the flames were pressed down, and the shape of the flames changed like amushroom (Ueno and Harada, 1987). The field intensity in the center of the airgap was 1.2T.Apart from the combustion experiments, we have observed that the flow of gasessuch as carbon dioxide, nitrogen, oxygen and argon are blocked or disturbed by magneticfields. A model called a "magnetic curtain" has been introduced to explain these pheno me na.It is assumed tiiat the magn etic curtain is a wall of air caused by magnetic fields as show nin Fig. 12.To clarify the mechanism of the magnetic curtain, trajectories of gas flow near andinside the magnetic curtain are simulated on the basis of molecular dynamics (Ueno andIwasaka, 1990, Lfeno et al, 1993). It is assumed that gas molecules such as nitrogen andoxygen molecules are particles, and these particles move in two-dimensional space. TheBrownian movement of particles in air atmosphere is caused by molecular collisions. We

Gas flow

Figure 12. Formation of the magnetic curtain. Magnetic field off(a)

M-^net i&vcurtai,, \flEgnetic field on

(b)


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12 S. Ueno and \1 . Iwasaka

Gas flow | o i r

G a s f l o w I 0 1 mm

Figure 13. Trajectories of nitrogen molecules in gradient magnetic fields, (a) without magnetic field, (b) with magnetic fields.

assumed that the dynamic movement of paramagnet ic molecules such as oxygen is constrained by a magnetic force.

Let us con sider the cond ition in whic h 10 particle s start toget her at an uppe r sta rtingline and go down with a velocity of mean free path. Each particle receives randomacceleration through the collision. When a magnetic force is applied to the particle f low, thevelocity component of the z axis is changed by the collision with oxygen particles. Fig. 13(a) sho ws the trajectories of gas flow with 10 million collis ions. W hen the region is expo sedto a gradient magnetic f ield of 225 T-/m, the gas f low is blocked, as shown in Fig. 13 (b) .We have des igned an electroma gnet w ith a pair of co lum nar m agnet ic p oles in whichinner s idepieces were h ol lowed out . A candle was burned in the hol lowed space betw een themagnet ic poles , and candle f lames were exposed to magnet ic f ie lds . The f lames were

,.r - Magnetic Curtain^^^^^mFigure 14. A mouse in a space surrounded byMagnetic Curtain magnetic curtains.


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14 S. Ueno and M. luasakaexperiment was carried out in gradient magnetic fields (gradient fields: B x dB/dz = 350 -400 T ' m). The mean level of FDPs released in a gradient mag netic field of up to 400 T ' /m was more than 190 % of the control samples. In this case, there are large gradients alongthe horizontal line. Solutions containing w ater, plasmin and fibrin could diffuse to horizontaldirections to promote the reactions of fibrinolysis. The results show that fibrin gels and watercontaining plasmin dilTused to less intense magnetic fields, and fibrin dissolved in specificdirections. It is possible to vectorially control the dissolution of a fibrin clot by magneticforces.

We also carried out an experiment in order to understand how the fibrin oriented ina magnetic field dissolves (Ueno et al, 1993). FDPs in the dissolved holes in the fibrinprepared in either the presence or the absence of an 8 T magnetic field w ere assayed. Fibringels formed with a magnetic field were more soluble than those formed without a magneticfield. It was observed that the shapes of holes in dissolved fibrin changed to ellipsoidalpatterns when fibrin plates were formed with a magnetic field at 8 T. The transversal axis ofthe ellipse was parallel to the magnetic fields.As a possible mechanism of these effects, changes in concentrations of diama gneticmacrom olecules in a solution in gradient m agnetic fields w ere examined. We carried out anexperiment to measure changes in the concentration of macromo lecules (Iwasaka et al, 1994b). A solution of fibrinogen and throm bin in a plastic tube made of vinyl chloride, 10 mm indiameter and 310 min long, was coagulated w ithin 12 hours in magnetic fields up to 8 T (B-dB/d2


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Ma gnetic Fields on Biological, Physical and Chemical Processes 15When a solution, containing water and diamagnetic macromolecuies is exposed togradient fields, magnetic forces act on the water and diamagnetic macromolecuies. Thedistance of drifts of a macromolecule in the water in a unit time is described as follows:

/ = ll(Xv-Xw)V nA(H =)-dt-2m 7where t is time, V is volume of fibrin polymer, Xv is magnetic susceptibility of fibrin pervolume, Xw 's a magnetic susceptibility of water per volume, and m is inass of fibrin polym erper unit volume.

Magneto-phoresis of fibrin polymers in gradient magnetic fields occurred whenmagnetic energy -(/(v^Xw)VnnH' II was larger than thennal energy kT. We conclude thatdiam agne tic m acrom olecuies in water drift in a specific direction due to the difference in thediamagnetic susceptibility of fibrin and water.It is possible for large macromolecuies, such as fibrins and fibrin polymers, to beaffected by gradient magnetic fields. To investigate the effect of homogeneous magneticfields on enzymatic activity of plasmin, we measured a synthetic substrate hydrolysis withplasmin (Iwasaka et al, 1994 c). As the synthetic substrate, D-Val-Leu-Lys-pNA (S-2251;molecular weight is 551.5) is a small molecule compared to fibrin, it is expected that theeffects of gradient magnetic fields can be ignored. In order to reduce the effect of gradientmagn etic fields, the center part of the superconducting m agne t's bore, where dB/dy < 4 T/mand B = 8 T was used. The concentration of S-2251 and that of plasmin was 0.1 mM and0.023 casein units/ml, respectively. After incubation at 37C 200 ul of 99.9% acetic acidwas added, and the absorbance was measured at 405nm with a spectrophotometer. An 8 Tmagnetic field did not have a distinct effect on ether the maximum velocity (Vmax) or theMichaelis constant (Km ). However, when the absorbance changed non-linearly and reached6090 % of the maxim um absorbance, absorbance of the mixture exposed to 4 - 8 T for 60min at 37C was lower than control sam ples (maximum 11 % ). The results show that along-term exposure to magnetic fields inhibited the synthetic substrate hydrolysis withplasmin.

EMBRYONIC DEVELOPMENT OF XENOPUS LAEVIS UNDERMAGNETIC FIELDSWe have studied a possible influence of intense magnetic fields on the early embryonic development of frogs (Ueno et al, 1984, 1990). Some of the most serious hazardouseffects that could be induced by intense m agnetic fields are teratogenic etTects on developin gembryos. Embryos ofXenopus laevis were exposed to magnetic fields of up to 8 T for theperiod from the pre-cleavage stage to neurula stage. Embryos were then cultured in Brown-Caston's medium until the feeding-tadpole stage (Ueno, Iwasaka and Shiokawa, 1994).We carried out an experiment, in which 100 embryos were cultured for 20 hours inmagne tic fields. No app arent teratogenic effects were observed when e mbryo s were culturedfor 20 h from the stage of uncleaved fertilized egg to the neurula stage under magnetic fieldsof 8 T We conclude that static magnetic fields of up to 8 T do not appreciab ly affect the rapid

cleavage and the following cell multiplication and differentiation in Xenopits laevis.We have also studied the early em bryonic developm ent oiXenopus laevis in a 40 nTmagnetic field, or one-thousandths of the earth's magnetic field, and obtained negativeresults. Thus, again under this very low magnetic field, fertilized eggs developed normallyand formed tadpoles with no appreciable abnormality.It is a miracle or paradox that animals such as Xenopus laevis develop normally understrong magnetic fields, although we have observed very clear biomagnetic effects such as


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16 S. L'cno and M. Iwa sakaparting water by magnetic fields, magnetic orientation of diamagneic polymers, and redistribution of dissolved oxygen by magnetic fields. It seems that biological cells arc robustagainst magnetic fields.

GENETIC EFFECTS OF MAGNETIC FIELDS ON DROSOPHILAMELANOGASTERThe genetic effects of magnetic fields on somatic reversion and somatic recombination in Dmsophila melanogaster have been reported (Levengoo d, 1966, Mittler, 1971, Koanaet al. 1994, Yoshikawa et al, 1994).We examined the somatic reversion of the white locus and the som atic recomb inationofmwh and //v genes in Drusophila melanogaster after they were exposed to a static m agne ticfield at 8 T for 8 hours or exposed to an alternating magnetic field (3.3 mT) at 20 Hz for 8hours (Yoshikawa, Iwasaka and Ueno, 1994). We have developed a mutation assay systemin which both types of mutations are genetically combined so as to detect mosaic spots oneye facets and on wing blades in single individuals. The reverse eye color mutation fromwhite-ivoiy (w') to wild type fw*) is associated with the loss of a 2.9-kilobase DNA fragmentduplicated in the white locus on the X chromosome. The formation of mutant spots on thewing blades results from recombination at wing anlage cells in larvae, trans-heterozygousfor the mutations multiple w ing hairs (mwh) an d/lare (fir) on the 3rd chromosom e. The staticmagnetic fields with 8 T were effective in producing the eye spots caused by intragenicmutation and the wing hair spots resulting from somatic recombination. In the exposuregroups, eye spots occurred 1.7 times more frequently than in the control groups, and winghair spots 1.4 times more. In contrast, the alternating magnetic fields with 3.3 mT at 20 Hzwere not more efficient for induction of both somatic spots. Mutant clones induced at thebeginning of larval life will be large in size while those produced in the second or third instarwill be successively smaller Hence, classification of mosaic spots into size classes andsubsequent analysis of clone size distribution can be used to estimate the point in time of theinduction of the mutational events. The increased number of mosaic spots in size classes 1,2, and 3 ^ of the groups that exposed to an 8 T magnetic field were characteristic whencompared to the control groups. It is possible that the period of these increments maycorrespond to the third instar of the larval stage. It seems reasonable to conclude that an 8T magnetic field induced reverse mutation of eye color.A wing spot test in Drosophila melanogaster was also carried out by Koana et al(Koana et al, 1994). It was reported that static magnetic fields of 5 T increased chromosomalrecombination 50 %.

MAG NETIC F IELD EFFECT S IN SPIN CHEMISTRYPossible biomagnetic and chemical effects can be expected wh en biological systemsare exposed to both static magnetic fields and other energy such as light and radiation (Ueno

and Harada, 1986). Photochemical reactions produced by a radical-pair intermediate insolution can be exposed to show magnetic field effects that arise from an electron Zeemaninteraction, electron-nuclear hyperfine interaction, or hyperfine interaction mechanismincluding an electron-exchange interaction in a radical-pair interme diate. That is, a comm onmechanism of magnetic field effects on photochemical p rocesses is that a chemical yield ofthe cage- or escape-product com es to show a magnetic field dependence when a singlet-triplet intersystem crossing can be subject to magnetic perturbations. The magnetic field effects


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Magnetic Fields on Biological, Physical and Chemical Processes 17on photochemical reactions were verified (Hata, 1976, Tanimoto et al. 1976, Shulten. 1976.Nagakura and Molin, 1992).Chemical reactions in the skin and eyes may have potential biomagnetic effects whenthe tissues are irradiated by both laser and magnetic fields (Ueno and Harada, 1986). If aprocess of enzymatic reactions involve a radical-pair, there is a possibility that magneticfields affect the singlet-triplet conversion rate of radical pairs. Some enzymatic reactionswhich involve photochemical processes are the candidates for the magnetically-sensitiveenzymatic reactions.

The question of whether magnetic fields affect enzymatic activities or not is ofconsiderable interest in biochemistry. To clarify the effect of magnetic fields on enzymaticreactions in living systems, it is important to study the enzymatic reactions that involveradicals which are generated from non-photochemical processes. We examined the possibleeffects of static magnetic fields on biochemical reactions catalyzed by xanthine oxidase andby catalase.Haberditzl reported that magn etic fields of up to 6 T enhanced the activity of catalase4.9 ~ 52 % (Harberditzl, 1967). Recently, it has been reported that magnetic fields of 0.10 -0.15 T affected B,, ethanolamine ammonia lyase (Harkins and Grissom, 1994).Xan thine ox idase, contained in the liver, lungs, intestine and other organs, catalyzesthe degradation of hypoxanthine to xanthine, and xanthine to uric acid, which is the terminalwaste of purine nucleotides in mammals. During the oxidation of xanthine, the enzymereleases superoxide anion radicals as intermediates which reduce ferricytochrom e c (Fe-'*).Superoxide anion, as well as any type of free radical, is also paramagnetic. Reducedcytochrome c (Fe^*) has an absorbance maximum at 550 nm which can be detected by aspectrophotometer. Superoxide dismutase inhibits the reduction of cytochrome c by accel

erating the dismutation of superoxide anions to hydrogen peroxide and oxygen, and thecatalase converts hydrogen peroxide into water and molecular oxygen.We observed that magnetic fields of up to 1.0 T did not alter the reduction rate ofcytochrome c by superoxide anion which was produced by the reaction catalyzed by xan thineoxidase (Ueno and Harada, 1986). It indicates that neither the electron transfer from xan thineto molecular oxygen nor the transfer from superoxide anion to cytochrome c was affectedby the magnetic fields of this range.Catalase, which is an important heme protein in the body used to decompose toxichydrogen peroxide, contains a ferric state (Fe^*) iron atom, therefore its magnetic propertyshows paramagnetism. The activity of catalase can be inhibited by hydrogen cyanide,

potassium cyanide and others.In the case of catalase, the influence of mag netic fields on the activity was de terminedby the catalytic decom position of hydrogen peroxide. The rate of disappearance of hydrogenperoxide was followed by observing the rate of decrease in absOrbance at 240 nm using aspectrophotometer. Magnetic fields of up to 1.0 T did not alter the rate of decomposition ofhydrogen peroxide catalyzed by catalase (Ueno and Harada, 1986). These biochemicalreactions were also exposed to gradient mag netic fields of the same intensities as used in thecombustion experiments. However, no clear positive effects were observed.

ACKNOWLEDGMENTSCollaborators in this work include Dr. T. Matsuda, Dr. O. Hiwaki and Dr. M. Fujikion magnetic brain stimulation, Dr. H. Tsuda on the hematological experiment. Dr. M.Nakamura on the enzymatic activity in magnetic fields, and Prof K. Shiokawa for theembryogenetic experiment. The work was supported in part by grants from the Ministry ofEducation, Science and Culture in Japan.


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BIOLOGICAL INTERACTION OFEXTREMELY-LOW-FREQUENCYELECTROMAGNETIC FIELDS

T. S. TenfordeHealth DivisionPacific Noriiiwest LaboratoryRichland, Washincton

INTRODUCTIONConsiderable controversy has arisen during the past decade over the question of

whether public and occupational exposures to electromagnetic fields in the extremely-low-frequency (ELF) range may be linked to adverse health effects, particularly an elevation inthe risk of cancer. Since the original publication by Wertheimer and Leeper (1979), whichreported an apparent association between childhood cancer risk and proximity of homes topower distribution lines with a high-current configuration, there have been nearly 20additional reports on this topic. The basic hypothesis resulting from the original study wasthat the contribution of power-line fields to the ambient 50 60 Hz tleld level in the homemay be linked to cancer risk by some unknown mechanism(s). Although there werenumerous technical deficiencies in the methods used in the original Wertheimer and Leeperstudy, more carefully designed and executed studies in recent years have added some supportto the original hypothe sis. For example , in a meta-analysis of 13 published studies Washburne! al. (1994) found a statistically significant elevation in the risk of childhood leukemia andnervous tissue tumors among subjects living in close proximity to electricity transmissionand distribution equipment. A similar association between residential exposures and elevatedcancer risk among adults has not been observed in several independent studies (NationalRadiological Protection Board [NRPB], 1992).

In addition to the residential cancer studies, more than 50 publications have appearedduring the past decade on the subject of occupational exposure to ELF fields and cancer risk.Although the results of these studies exhibit a number of inconsistencies, there does appearto be an elevation in the risk of leukemia and nervous tissue tumors am ong electrical workers(Theriau lt, 1990; NR PB, 1992). Limited evidence also suggests an elevated risk of breastcancer among electrical workers (Matanoski et al., 1991; Demers a a!.. 1991; Tyncs ei al..1992; Loomis , 1994).A major problem in the interpretation of these epidemiological findings is the lackof convincing evidence that the underlying cause of the elevated cancer risk is exposure toBioloiiiciil Effecfs of Xfaf^nt'tlc and lilcctionhii^iwlic Field.'.. F:dited by S. IJenoPlenum Press. New York. 1996 2.1


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24 T. S. Tcn fordeELF fields from power lines or in the workplace, rather than exposure to some confoundingvariable or set of variables that have not as yet been identified. In general, ELF fieldexposures based on short-term measurements have not correlated strongly with cancer riskamong the exposed subjects, although a few exceptions have been reported. The issue ofconfounding variables has also been closely examined in several studies, but even whenpotential confounders such as chemical exposures have been identified, they have had littleimpact on the cancer risk estimates based on living in homes close to power lines or workingin electrical occupations.

Another problematic issue is the fact that a biologically plausible mechanism has notbeen identified through which ELF fields at low exposure levels could significantly influencethe development or progression of malignant tumors. As discussed below, the strength ofELF fields encountered in occupational or public settings is far too low to directly affectDNA or chroinatin structure. For that reason the emphasis in laboratory studies during thepast several years has been on determining whether 50/60 Hz field exposures can operatethrough epigenetic mechanisms to promote the development of tumors from clones of cellspreviously transformed either by spontaneous mutation or by exposure to carcinogenicagents such as chemicals, ionizing radiation, or oncoviruses.

I'he results of several recent studies that address the issue of tumor promotion byELF fields are discussed in this article, along with other factors such as changes in geneexpression and endocrine regulation that could influence carcinogenic processes. One aspectof these laboratory-based studies that merits attention is the fact that field levels required toelicit reproducible biological responses are typically larger than, and frequently orders ofmagnitude greater than, the weak 50.60 Eiz fields to which humans are commonly exposedin the home or workplace. These exposure levels are generally in the range of 10 to 50 V/mfor the electric field component and 0.1 to 0.3 jiT for the magnetic field component, whichinduce electric fields within the body on the order of 5 |.iV/m or less. It is a major challengefor both laboratory research and biophysical modeling to elucidate plausible mechanisms bywhich weak ELF fields could significantly affect biological processes when the signal levelsare comparable to, or less than, the intrinsic physical and biological noise present in livingsystems. Several contemporary hypotheses on the physical and chemical mechanismsthrough which weak ELF fields could perturb the functioning of living systems are describedand critically evaluated in this article.

P H Y S I C A L P R O P E R T I E S O F E L F F I E L D SELF fields in tissue have a long wavelength (-1000 m at 60 Hz) and skin depth(~150 m at 60 Hz), as a result of which these fields behave as though they are composed ofindepe nden t, quasistatic electric and magnetic field com ponents (Tenforde, 1991). As aconsequence, the radiating properties of ELF fields can be neglected in their interactionswith tissue. Another important property of ELF fields is their extremely small energy. Forexample, the energy of a 60-Hz photon is 2.5 x 10""' eV, which is 11 orders of magnitudesmaller than the Boltzmann thermal energy, kT (= 2.7 x 10'- eV at 310 K), and 14 orders ofmagnitude less than the energy required to break a chemical bond. Substantial laboratory

evidence supports the physical expectation that ELF fields do not disrupt the chemical bondsin DNA, proteins or other biological molecules. Direct genotoxic effects of ELF fieldsleading to cell death, gene mutation, or neoplastic transformation would therefore not beexpected, and in fact, have not been observed.A third important feature of ELF fields applied to living organisms through air is thenonthermal nature of their interactions. The highest field in tissue that can be induced by anELF field applied through air is about 1 V/m, which leads to a specific energy absorption


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Biological Interaction of Kxtrcmcly-Low-Frcquency Electromagnetic Fields 25rate of 10"* W /kg. T his rate of energy d epo sition is four orde rs of m ag nit ud e les s than th ebody's basal metabolic rate and produces a negligible rate of temperature r ise (about 3 x 10 '^ C s ) . The interactions of ELF fields applied to the body through air are therefore of anonthermal nature .

B I O L O G I C A L E F F E C T S A N D M E M B R A N E I N T E R A C T I O N SDespite the minimal perturbations of molecular structure by environmental levels of

FLF fields, there is abundant evidence for responses to these f ields at the tissue and cellularlevels (Na irt ' r a/. , 1989; Adey, 1990a; An derso n, 199 1; Tenforde, 1992). For exa m ple , smallfunctional changes in excitable tissues and neuroendocrine alterations have been reported mresponse to ELF fields that induce tissue voltage gradients < 10 mV/m. These alterationsinclude changes in evoked brain potentials (Graham ct al.. 1990). heart rate (Graham ci ai.1990), and nocturnal synthesis of pineal melatonin (Wilson ci al, 1981; Lerchl ci al.. 1990;Kato L'l al.. 1993). In addition, a large number of cellular phenomena, including alterationsin growth rate, gene expression and macromolecular synthesis, have been reported to occurin respons e to fields of mo dera te to weak intensity (Tenforde, 1992). Th ese effects includealterations in biosynthesis of specif ic messenger RNA and proteins at f ield levels below 1mV in (Go odm an and He nders on. 1991), Finally, there is a rapidly grow ing body ofinformation that implicates the cell m em bran e as a primary site of FL F field in teractio ns(Adey, 1990a; Adey. 1990b; Tenforde, 1992; Tenforde and Ka une, 1987). A wid e variety ofcell membrane structural and functional properties have been reported to be altered inresponse to ELF fields, including changes in Ca** binding to anionic fixed charges at thecell surface {e.g.. sialic acid residues of membrane glycoproteins) , changes in the transportof ions such as Ca*^ and the secretion of small solutes such as insulin, and alterations inligand-receptor interactions that tr igger changes in the biosynthetic and functional states ofcells. The threshold tissue field levels that lead to such effects appear to vary widelyde pe nd ing upon the end point stud ied, but in nea rly all cas es arc < 0.1 V 'm . In the specificcas e of field-induced Ca*" des orptio n from fixe d-cha rge s ites on the mem br an e surfac e, theeffective field level has been reported to be as low as 1-10 |.iV m (Bawin and .Adey, 1976;B la c k m a n tv a / . , 1 9 8 5 ) .

The phospholipid bilayerthat forms the structural matrix in membranes of living cellsis an electr ical insulator , and the membrane electr ical conductivity is about f ive orders ofmagnitude less than that of the extracellular medium or the cytoplasm. As a result , themembrane of a living cell forms an excellent electr ical barrier , as well as a superb chemicalbarrier , that mediates cellular interactions with the external environment. For this reason, i tis generally believed that cellular responses to weak ELF fields arc initiated by membraneinteractions that serve as the primary mechanism of f ield transduction. Under typicalexp osure co ndit ions with induced ELF fields in the extrace llular m edium of < 1 V'm . the"ie ak ag e" f ield in the cell cytoplasm is less than 1 |J V m. This conclusio n also holds for thecircu lating electr ic f ields induced directly in the cell cyto plasm by m agne tic indu ction. Forexample, a sinusoidal 60-Hz, 0.1-mT field induces a maximum electr ic f ield of 0.2 pV/m inthe cytoplas m of a cell with a 10 jim radius. The se consid eratio ns reinforce the i mp ortan ceof the cell membrane in ELF signal reception and transduction.Another point to be made regarding the role of cell membranes in ELF signaltransduc tion is the amplif ication of the extrace llular f ield that occu rs across the me m bra ne .By solving M axw ell 's equ ations for the specif ic case of a dielectr ic shell (m em bra ne )surrounding a spherical conductor (cytoplasm), i t can be easily shown that the electr ic f ieldacross the me m bran e is greater than that in the extrace llular m ediu m by a factor 1.5 R d.where R is the cell radius and d is the membrane thickness. For a spherical cell with a radius


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26 I. S. I enfo rdeof 10 [xm and a membrane thickness of 5 nm. the field across the membrane is thereforepredicted to be 3000 times greater than that in the extracellular medium. A weak environmental field that induces a voltage gradient of 5 nV/m in the extracellular fluid thus producesa field of about 15 mV m across the cell inembrane.

ELECTRICAL NOISE IN BIOMEMBRANESSeveral physical and biological sources of electrical noise within the cell membranemay im pose a lower limit on the strength of an ELF field that can be recognized as a coherentsignal (Fishman and Leuchtag. 1990; Weaver and Astumian. 1990; Adair, 1991). The fourmajor sources of electrical noise in biological membranes include: (1) Johnson-Nyquistthermally-generated electrical noise, which produces a 3 nV transmem brane voltage shift atphysiological temperatures; (2) l/f noise associated with ion current flows through mem

brane channels, which typically produces a 10 )iV transmembrane voltage shift: (3) "shot"noise, which results from the discrete nature of ionic charge carriers and is a minor sourceof mem brane electrical no ise; and (4) endogenous biological background fields produced byelectrically active organs such as the heart, muscles and the nervous system, which canexceed the contribution of physical noise sources by an order of magnitude or more.Because of these various sources of membrane noise, it is of interest to explore theminimum strength of induced electric fields in tissue that can achieve a signal-to-noise ratiogreater than one within the cell membrane. For example. Weaver and Astumian (1990)arrived at an estimate of approximately 0.1 V/m as the smallest applied electric field thatexceeds the Johnson -Nyquist noise signal in the mem brane of a single cell with an elongated

cylindrical geometry (such as a fibroblast, neuron, or muscle cell). Larger threshold fieldlevels, greater than 1 V/m, were predicted for small spherical cells such as lym phocytes.Further increases in the threshold field level are expected if other sources of electrical noisewithin the cell membrane are taken into account.These calculations of signal-to-noise ratio consider only the transmem brane electricpotential shift introduced by an extracellular field. It has been argued rather convincinglythat the field and noise sources that are most relevant to biological signal transduction arethose that reside within the highly charged electrical double layer that exists at the cellsurface. This double layer, frequently referred to as the Helmholtz-Stern layer, is comprisedof fixed anionic charges on the outer membrane surface and diffusible cations in the

surrounding medium. As described originally by Debye and Hiickel, the average thicknessof the diffuse electrical dou ble layer at cell surfaces is approximately 0.8 nm in a physio logical medium (Tenforde, 1970). By considering Johnson-Nyq uist noise and other sources ofelectrical noise within the double layer, it can be concluded that the minimum electric fieldrequired in the extracellular medium to achieve a signal-to-noise ratio greater than one isabout 10 mV m.Another important factor to be considered in calculating the threshold field level thatexceeds intrinsic electrical noise in biological m embranes is the junction al coupling thatoccurs between cells in organized tissues. For an aggregate of N cells with electricallycoupled membranes, the threshold field level required to achieve a signal-to-noise ratio of

one is lower than the threshold for single cells by approximately N'-''^ as a result of twofactors: (I) field amplification due to the larger size of the aggregate, and (2) reduction ofmembrane voltage noise as a result of the larger capacitance of the aggregate (Weaver andAstum ian, 1992). For example, if an aggregate of 10*' electrically coupled cells is considered,the threshold field level to achieve a signal-to-noise ratio of one is predicted to be lower by100,000 than the threshold for a single cell. Because of the junctional coupling exhibited bymost biological tissues, the threshold field to achieve membrane and cellular respo nses may


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Biological Interaction of K\tr em clv-l.o -F req ucn cy Klectromagnt'tic Fields 27there fore be on the order of I i iV/m as con traste d to the valu e of abou t 0.1 V i n or hig herpredic ted for single cells . A similar conclusion has been reached by Pilla (1993) from anelectr ical netw ork mod el of cells that com mu nica te electr ically via Junctiona l coup ling . Theelectr ical c ondu ctivity of gap jun ctio ns in cell m em bran es is , how ever, lower than that ofthe extracellu lar m edium . As a result , the electr ical couplin g and the gain in sig nal- to -noiseratio for cell aggregates in tissue is probably less than that predicted from relatively simplemodels .

B I O L O G I C A L S I G N A L T R A N S D U C T I O N P A T H W A Y S AN DP O S S I B L E C A R C I N O G E N I C E F F E C T S O F E L F F I E L D S

The most singly important issue in understanding the pathways by which weak HI^Fsignals coidd influence me mb rane and cellular functions is the elucida tion of me cha nism sby which these f ields arc transduced within the cell membrane. One approach that has beentaken in addressing this question is to consider (he intr icate biochemical pathways that haveevoKed as a mechanism by which a living cell communicates with its extracellular en\ iron-ment. The binding of a single molecule of a mitogenic substance to a specif ic receptor w iihinthe membrane tr iggers a cascade of events that involve conformational shif ts in membrane-assoc iated protei ns. These ev ents, in turn, lead to signal transd uction and am plif ication \ lathe production of cytoplasmic second messengers and internal efTectors such as free C'a*^and protein phosphorylascs (kinases) that regulate DNA transcription and protein biosynthesis ( .Alkon and Rasmussen, 1988; Luben, 1991). The end result of a single mitogenbindin g event at the mem bra ne surface is thus a cytop lasm ic signal that is amplif ied lo alevel that can produce robust effects on macromolecnlar synthesis and cellular responsesinvolving signif icant changes in functional and proliferative states.

The interaction of FI.F f ields with biological membranes could, in principle, lead toaltera tions in each com pone nt of this elegant signaling p rocess that occurs in living cells . Auseful wo rking hypo thesis is that the pericellular f ields and curren ts induced by an appliedbLI- field initiate electrochemical events within the cell membrane that are importantelem ents of the prima ry signal transduction and amplif ication process ( len fo rd e, I99.i) .These b iochemical ly-mediated events then produce cytoplasmic second messenger responses that tr igger changes in the biosynthesis of macromolecules and alterations in cellulargrow th, differentiation, and functional p ropertie s. During the past dec ade , a grov\ing bodyof experimental evidence has been acquired that supports this general picture of the sequenceof events leading to HLF signal transduction and amplification at the cellular level. It hasbeen demonstrated, for example, that pulsed electromagnetic f ields (PKMK) with HLFrepetition trequ enei es inhibit the production of cA M P by bone cells in response to the bind ingof parathyroid hormone (PTH) to sinTace receptors (Luben ci al.. 1982). Further studies haveshown that the PEMF action leads to inability of the PTIi-receptor complex to activate thealpha subunit of G protein, thereby interfering with the sequence of events that activatesadenylate cyclase at the cy toplasmic membrane in ter face (Cain ei


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28 I. S. I enforde

Outer Surface

PhosphorylationFigure 1. Phosphokinase C pathway leading to an elevated intracellular calciinn concentration and increasedkinase activity. The binding of a mitogen to the membrane receptor triggers a meinbrane-associatcd phospho li-pasc C (PLC) hydrolysis of phosphotidylinositol 4,5-bisphosphatc (PIPT). thereby producing diacylglycerol(DAG), which activates protein kinase C, and inositol 1,4,5-triphosphate (IP)), which mobilizes intracellularCa**. There is also evidence that the conversion by a kinase of IP, to IP4 may lead to an influx of free Ca**and other ions through membrane channels. These ions, together with calmodulin (CAL). lead to furtheractivation of kinases.

human HL-60 cells exposed to a 50-Hz pulsed magnetic field (Monti ei al.. 1991). The fieldeffect was con siderab ly dainped by adding EGTA, a Ca" ^ chelator, to the me dium . T hisobservation is consistent with previous findings that kinase-C activation relies on themobilization of Ca** ions. Another relevant study is the recent finding that stimulation ofCa** uptake into rat thymocytes by the plant lectin, Concan avalin A (Co n-A ), is significantlyaugmented by exposure to a sinusoidal 60-Hz magnetic field (Liburdy, 1992). In lymphoidceils the binding of Con-A to surface receptors triggers cytoplasmic signaling eventsinvolved in the kinase-C pathway, including the activation of kinase-C and an elevation ofthe cytosolic Ca** concentration. Enhancement of the Con-A effect by a 60-Hz inagneticfield was shown to be dependent upon the strength of the electric field induced in theextracellular medium.

Several in vivo studies have been conducted to test the hypothesis that ELF fieldsmay act as tumor-promoting agents, possibly through the stimulation of proliferation intransformed cell popu lations via activation of the kinase-C signaling pathway. T hese studieshave been conducted primarily with skin, mammary, liver, and brain tumor models inrodents. In studies on skin tuinor promotion, three studies in mice have shown no prom oting


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Biological Interaction of Extremely-Low-Frequency Electromagnetic Fields 29effect of chronic exposure to power-frequency magnetic fields with flux densities rangingfrom 0.05 to 2 mT on the development of skin papillomas induced by topical application of10 nanomole of the carcinogen, 7-12-dimethylbenz(a)anthracene (DMBA) (McLean et al..1991; Ra nnu ger a/., 1993a; Ran nu ge ra/ ., 1994). In positive control groups of anima ls, skintumor development was observed when the DMBA-initiated m ice were treated tw ice weeklywith topical application of the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA).In one experiment the effect of an intermittently applied 50-Hz magnetic field (15 sec on/15sec off) was tested for tumor-promoting activity in comparison with a continuously appliedfield (Rannug et al.. 1994). A small increase in the number of skin tumors was observed inthe mice exposed to intermittent versus continuous fields, but neither group of animalsshowed a statistically significant increase relative to control mice treated with DMBA alone.Hence there was no indication of a tumor-promoting effect with either type of magnetic fieldexposure.

Another in vivo study of particular interest in the context of possib le EL F field effectson the kinase-C signaling pathway and tumor promotion is the recent finding by Stuchly etal . (1992) that a 60-Hz, 2-mT magnetic field exerts a copromoting effect on mouse skincarcinogen esis. In these experiments skin tumors were initiated by the topical application of10 nanomole of DMBA, and were then promoted by the application of 4.9 nanomole of thephorbol ester TPA, once per week for a total of 23 weeks. In the group of mice that receivedDMBA and TPA plus exposure to the 60-Hz field for 6 h/d, 5 d/week througho ut the tumorpromotion phase, both the percentage of mice with tumors and the number of tumors permouse increased more rapidly with time than in the group of mice that received DMB.A plusTPA alone. For exam ple, at week 18 the percentage of mice with tum ors in these two grou pswere, respectively, 2 5% and 8% and the mean number of tumors per mouse were 1.90 x 0.69(SEM) and 0.65 0.46. By week 23 the differences between the mice exposed to the mag neticfield and the nonexposed group were no longer statistically significant. Other studies by thesame group of investigators had previously shown that a 60-Hz, 2-mT m agnetic field actingalone on D MB A-initiated skin cells does not have a tumor-promoting effect (McLean et al.,1991). It would be of considerable interest to conduct further experiments in which comparative measurements are made of kinase-C activity in the affected skin cells throughoutthe tumor promotion phase in the field-exposed and nonexposed groups of mice. Suchexperiments would p rovide a test of the hypothesis that ELF fields may exert a coprom otingeffect on tumor development through an influence on the kinase-C signaling pathwayactivated by phorbol esters.

Three studies of mammary cancer development in rodents exposed to ELF magneticfields following tumor initiation with a chemical initiator have provided some evidence fora tumor-promoting effect of the fields (Beniashvili et al., 1991; Loscher et al., 1993;Mevissen et al., 1993). In the first of these studies, rats were injected intravenously with a50 mg/kg dose of nitrosomethylurea (NMU), which produced mammary tumors in 54% ofthe rats (Ben iashvili et al., 1991). This percentage increased to 86 % in a group of rats initiatedwith NMU and exposed to a 50-Hz, 0.2-mT magnetic field for 3 h/d for 5 weeks. The latencytime to tumor developm ent w as also found to decrease significantly in the rats expo sed toboth NMU and the magnetic field relative to rats that received only NMU. In a second study,rats that were initiated by oral doses of DMBA (20 mg total dose) were exposed for 23 w eeksto a 30-mT, 50-Hz magnetic field (Mevissen et al., 1993). Although the overall tumorincidence did not differ significantly between the field-exposed and control groups, therewas a statistically significant increase in the number of tumors per rat in the group exposedto the 30-mT field. A third study was conducted with the same protocol as the second studydescribed above, except that a considerably smaller field level of 0.1 mT was used (Loscheret al., 1993). In this experiment the overall mammary tumor incidence was observed to be50% higher in the field-exposed rats relative to the control group of animals.


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30 1. S. TenfordeAlthough most of the studies on chemically-initiated mammary tumor developmentin rats exposed to 50-Hz magnetic fields provided evidence consistent with a tumor-promoting effect of the fields, the findings of increased tumor incidence could also be related to afield-induced suppression of pineal melatonin and a resultant elevation in breast cancer risk

(Tamarkin et al., 1981; Stevens et al., 1992). Further research on the effects of ELF fieldson the development of chemically-induced mammary tumors in rodents, including parallelmeasurements of field effects on the level of pineal melatonin in the exposed animals, wouldprovide useful insights into the pathways by which these fields may exert carcinogeniceffects.Two series of experimen ts have been designed to study the effects of chronic expo sureto 50-Hz m agnetic fields on the development of chem ically-induced liver tumors in partiallyhepalectomizedrats(Rannugt';i//., 1993b; Rannugef a/., 1993c). In both studies liver tumordevelopment was initiated by a 30-mg dose of diethylnitrosamine (DENA) administeredintraperitoneally 24 h following partial removal of liver tissue. Tumor development was

promoted with phenobarbital as a positive control. The development of transformed foci ofliver cells was assayed by histochemical staining for the enzymes y-glutamyl transpeptidaseand glutathione S-transferase. The development of tumor foci in the livers of the chemically-initiated rats was not significantly influenced by a 3-month exposure to SO-Hz magneticfields with flux d ensities ranging from 0.5 MT to 500 |aT, thus indicating a lack oftumor-promoting effect by the fields. Similarly, no evidence was obtained for a copromotingeffect in rats exposed to both phenobarbital and 50-Hz fields following tumor initiation withDENA.A recently com pleted study provided evidence that chemically-induced brain tumorsin rats are not promoted by exposure to 50-Hz magnetic fields (Brugere et a/., submitted for

publication). Brain tumors of various histological types, including gliomas and astrocytomas, were induced by intravenous injection of 50 mg/kg ethylnitrosourea (ENU) intofemale rats on the 19th day of pregnancy. After weaning the offspring were chronicallyexposed to fields of 1, 10 and 100 |iT and studied for survival time. Neither male or femaleoffspring showed a statistically significant difference in mean survival time relative tocontrol rats that were exposed to ENU but not to 50-Hz magnetic fields.Overall, with the possible exception of mammary tumors, the available evidence isnot strong for a promoting or copromoting effect of ELF fields on tumor development inrodents. However, further studies are needed to clarify the possible influence of ELF fieldson second messenger signaling and endocrine regulation in exposed an imals, both of which

are factors that could influence the development of tumors by promoting the proliferationof initiated cells.

PHYSICAL MECHANISMS OF ELF FIELD INTERACTIONSIn the search for mechanisms by which extremely weak ELF fields could exert.significant biological effects, a number of innovative models have been proposed over thecourse of the last two decades. One class of models that has received particular attentionduring the past several years are resonance models that involve the combined action of an

ELF field and the static geomagnetic field. For examp le, ion cyclotron resonan ce (ICR ) wasproposed by Liboff (1985) as a possible mechanism that could facilitate the transport of ionssuch as Ca** through membrane channels in the presence of the geomagnetic field and aweak ELF field tuned to the ICR frequency. Although some data on Ca** uptake bylymphoc ytes (Liboff e al., 1987) and diatoms (Sm ith el ai, 1987) have been cited as lendingsupport to this model, there are also negative experimental findings (Parkinson and Hanks,1989; Liboff and Parkinso n, 1991 ; Parkinson and Su lik, 1992). In addition, a number of


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Biological Interaction of Extrem ely-Low -Frequcncy Electrom agnetic Fields 31physica l argum ents can be raised against the ICR model (Tenforde, 1992), the most seriousof which is the collisional damping of resonant ion motion that is expected to occur in acondensed phase (Halle, 1988).

Two other resonance models that have been proposed recently are the "quantumbeats" model of Lednev (1991), in which combined static and ELF fields affect vibrationalenergy levels and transition probabilities of bound ions (e

Libro_Biological Effects of Magnetic and Electromagnetic Fields(the Language of Science)

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