8/13/2019 frey-1993

1/10

27 2 N EW S AN D FEATU R ES 0892-6638/9310007-02721 0l .50. FASEB

Special Feature1Electromagnetic field interactions w ith biological system s

ALLAN H . FREY2Random line Inc., Huntingdon Valley , P ennsylvania 19006, USA

ABSTRACT This is a report on Sym posia organized bythe International Society for B ioelectricity and presentedat the 1992 FASEB M eeting. The presentations summ a-rized here were intended to provide a sampling of newan d fruitful lines of research . The theme topics for theSym posia were cancer, neural function , cell signaling,p ineal gland function , and im mune system interactions.L iving organism s ar e com plex electrochem ical system sthat evolved over billions of years in a world with a rela-tively sim ple weak magnetic field and w ith few elec-trom agnetic energy em itters. A s is characteristic of livingorganism s, they interacted w ith and adapted to this en-vironment of electric and m agnetic fields. In recent yearsthere has been a massive introduction of equipm ent thatem its electromagnetic fields in an enorm ous range of ne wfrequencies, m odulations, and intensities. A s liv ing or-ganism s have only recently found themselves imm ersed inthis new and virtually ubiquitous environment, they havenot had the opportunity to adapt to it. This gives us, asbiologists, the opportunity to use these electromagneticfields as probes to study the functioning of liv ing system s.This is a sign ificant opportunity, as ne w approaches tostudying liv ing system s so often provide the means tom ake great leaps in science. In recent years, a d iversity ofbiologists have carried out experiments using electrom ag-netic fields to study the function of liv ing cells and sys-tem s. This approach is now becom ing quite fruitful an dis yielding data that are advancing our know ledge indiverse areas of b iology.- Frey, A . H . E lectrom agneticfield interactions w ith biological systems. F SEB J. 7:272-281; 1993.Key Words: electromagnetic cancer brain pineal . immune

IF ONE USED ELECTROM AGNETIC ENERGY sensors to view theworld from space 100 years ago, the world would have lookedquite dim . Now the world glows w ith electrom agnetic (em )3energy em issions at m ost frequencies of the nonionizing por-tion of the spectrum , such as power line fields, rad io waves,m icrowaves, etc .

Liv ing organ ism s are complex electrochem ical system sthat evolved over billions of years in a world w ith a relativelysimple w eak m agnetic field and w ith few em energy em itters.As is characteristic of living organ ism s, they in teracted w ithand adapted to th is environm ent of electric and magneticfields. O ne example of th is adaptation is the visual system ,which is exquisitely sensitive to em issions in the very narrowportion of the em spectrum that we call ligh t. O rganism s alsoadapted to another portion of the spectrum , the UV , by de-veloping filtering system s in the eye and the skin to protectthemselves from it.

A w ide range of living organism s, including humans,adapted by using em energy to regulate various critical cellu-lar system s; w e see th is in the com plex of circadian rhythms.

Fish , b irds, and even the duckbill platypus developed sys-tems to use electrom agnetic fields to sense prey and tonav igate (1). E lectrom agnetic fields are involved in neuralm embrane function. Even protein conform ation involves thein teractions of electrical fields.

Thus, it is not surprising that the m assive in troduction ofelectromagnetic fields in an enorm ous range of new frequen-cies, m odulations, and in tensities in recent years has affectedliv ing organ ism s (2-5). In fact, it would be incredib le andbeyond belief if these electromagnetic fields did not affect theelectrochem ical system s we call living organism s.

As living organ isms have only so recently found them -selves immersed in this new and virtually ubiquitous en-v ironment, they have not had an opportunity to adapt to it.This gives us, as bio logists, the opportunity to use these emfields as probes to study the functioning of liv ing system s.This is exciting because new approaches to studying liv ingsystem s so often provide the m eans to make great leaps inscience. Energy in the pow er line frequency, rad io, andm icrowave portions of the spectrum , as well as m agneticfields, penetrate the body and can be used as probes; theem itters are now available. The ubiquitousness of elec-tromagnetic fields is also now a m atter of concern to us asbiologists; it is relevant to the question of contro ls needed inexperiments. Look about the lab and consider the em fieldsnow being im posed on test specim ens by all the electricaldevices in use. How do they influence the resu lts o f b io lo g ic alexperiments? One of the Symposium speakers touched onth is q ue stio n.

A report on five sym posia presented on April 7 1992, in A na-h ei m , C ali fo rn ia at the Annual M eeting of the F ed eration of A meri-can Societies for Experim ental B iology by the International Societyfo r B ioelectricity. Symposium titles and their participants are:Cancer and Environm ental Electromagnetic F ields (chaired by R .S te ve ns ); p artic ip an ts : J. Phillips, G . Harrison, and W . 0. Short;Nervous System Function: Interaction w ith E lectromagnetic Fields(A . H . F rey). M . M cGinnis, A . R . Sheppard, and J. Walleczek;Cell S ignaling and E lectrom agnetic Field Interactions (B . R .McLeod). M . Blank, C . C ain , and R . A . Luben; Pineal G landResponse to Electric and M agnetic Fields: Evidence a nd P oss ib leConsequences (L . Anderson). R . Reiter, S. Y ellon, and B . W ilson;Immune System Interaction w ith Environm ental Electrom agneticFields (chaired by D . Lyle). R . P. Liburdy , A . Cossarizza, and W .D . W i nt er s.

2Current address: 11049 Seven H ill Lane, Potomac, MD 20854,U SA. Em ail address is afrey@ uunet.uu .net3A bbre via tio ns: em , e le ctro ma gne tic ; mf, m agnetic field; CREB ,calcium /cyclic AMP binding protein ; SRE , serum response ele-m ent; SRF, serum response factor; mw , m icrow ave; TPA , 12-0-tetradecanoyl-phorbol-13-acetate; dc, d irect current; CN S, centralnervous system ; MRI, magnetic resonance imaging; IBIs, in ter-burst in tervals; em f, electromagnetic field ; DFS , dual-sample (ordifferen tial) fluorescence spectroscopy; F1H , cell parathyroid hor-mone; NAT, N -acety ltransferase; PBMC, peripheral b loodmononuclear cells; PHA , phytohemagglu tin in; NK , natural k iller,MHC, major histocom patib ility com plex ; Con A , concanavalin A .

8/13/2019 frey-1993

2/10

/

-

E LEC TRO M AG N ETIC FIELD S AN D BIO LOG IC AL SY STEM S 27 3

Much of the literature on electromagnetic field in terac-tions published before the m iddle 1980s is irrelevant to bio lo-gists. M ost of it w as generated by the engineering commu-nitys attem pt to find out if their high-power equipm entcreates a hazard . Thus, little attention was paid to the varia-b les that are importan t in bio logy. Instead , efforts werew asted in fru itless controversies such as whether the effectsseen were thermal or nontherm al. As I noted in an earlierpublished paper (2), th is is really a matter of sem antics, notscience. There w as no common definition of thermal - tosome it m eant a change that could be measured w ith a rectalthermometer; to others it m eant unmeasurable vibration ofmolecules at a cell m embrane. Besides, it is a label, not anexplanation.

In recent years, though, the convergence of a num ber oflines of research has led a diversity of b iologists to carry outexperim ents using low -in tensity electromagnetic fields tostudy the function of liv ing cells and system s. This approachis now becom ing quite fru itfu l and is yielding data that areadvancing our know ledge in diverse areas of b iology. Presen-tations at the Symposia summarized here w ere in tended toprovide a sam pling of these new and fru itfu l lines ofresearch . The theme topics for these Symposia were cancer,neural function , cell signaling , p ineal g land function , andim mune system interactions.

CancerIn the first Symposium , chaired by R ichard Stevens, reportsof research were presented by Jerry Phillips, G eorge Harri-son, and W elland Short. Prev ious work by Phillips (6, 7) andothers indicates that exposure of various cell system s to mag-netic fields w ith a variety of signal characteristics producesgeneral increases in gene transcrip tion . The question ad-dressed in Phillips presentation w as what specific genes arealtered at the transcrip tional level. The speaker had previ-

RatioEXP/CTRL

1. 5

1. 0

0. 5

0. 015 30 60

Tim e of Exposure (mm )F igure 1 . Effect of m f exposure on AP-l and CREB DNA-bindingactivity. Results are expressed as the ratio of activity in exposed tocontrol cell cultures, and are presented as the m ean value of 9separate experim ents. Vertical bars represent SEM5. First bar ineach group is AP-1 binding protein 1, second bar is AP-1 bindingprotein 2 and th ird bar is CREB . ***Indicates F < 0.001 andindicates P < 0.01.

ously used the nuclear run-off assay to study the effect of ex-posure of CCRF-CEM cells to a 1 gauss sinuso idal magneticfield (mf) at 60 Hz on the transcrip tion of four genes: c-fos,c-jun, c-myc and protein kinase C (f3 form ). He found a time-dependent, and sometimes a cell density-dependent, changein the transcrip tion of these genes. H e then concentrated onthe m agnetic field-induced increase in c-fos transcrip tion inorder to determ ine the m echanism by which the m f inducedchanges to occur. H is approach w as to use transcrip tion ofth e c-fos gene as an endpoin t, and then m ove upstream onestep at a time to determ ine which steps leading to gene tran-scription are influenced by m f exposure (6 , 7).

Phillips used the mobility sh ift assay to study m f-inducedchanges in the DNA binding activity of specific transcrip tionfactors. Figure 1 summarizes the results of n ine separate ex-periments in which the relative levels of two specific AP-1com plexes w ere m easured, as w as calcium /cyclic AMP(CRE) binding protein , CREB , in exposed compared w ithunexposed control cells. The m f exposure induced asignificant decrease in the levels of both AP-1 com plexes,reaching a nadir after 30 mm of exposure. In contrast, m fex-posure produced no change in the level of CREB capable ofb inding to its recognition sequence. Serum response factor(SRF) binding to the serum response elem ent (SRE) w asmeasured in four separate experiments. In tw o experim entsSRF binding w as unaffected by m f exposure, w hereas in twoother experiments SRF binding was increased substantially,peaking after 30 mm of exposure.

The speaker stated that the data presented indicate thatm f induced changes in the levels of DNA binding activ ity ofspecific transcrip tion factors and are consistent w ith in -creased c-fos transcrip tion and decreased c-jun transcription.He noted that because transcrip tion of the fos gene is down-regulated by the fo s protein, and because AP-1 consists ofeitherfos-jun heterodimers or jun-jun hetero- or hom odim ers,decreased AP-l m ay indicate decreased fos pro tein and thusa rem oval of repression tofos gene transcrip tion . A t the sam etim e, decreased AP-1 signifies decreased levels ofjun protein,and both are consisten t w ith decreased c-jun transcription.

Phillips stated that the resu lts of his em field studies ofSRF binding to the SRE are also consisten t w ith otherreports that detail the mechanism of m itogen-inducedchanges in c-fos transcrip tion . H e noted that it has beenshown that the SRF has two distinct activities: 1 a DNAbinding activity, and 2 a transcriptional-in itia tion stim ula-tory activity . C onsequently, he concluded that depending onthe cell type or the state of a given cell, the DNA binding ac-tivity m ay be constitu tively activated whereas the protein, al-ready bound to the SRE , awaits activation of itstranscrip tional-initiation stim ulato ry function. Conversely ,treatm ent of a cell w ith some agent m ay, through phosphory-lation of the SRF, increase both its DNA binding andtranscrip tional-in itia tion stim ulatory functions. A sim ilarsituation , he said , has been reported for CREB .

Harrison presented data from a series of studies (8 , 9) ofneoplastic transformation induced by 2.45 GHz m icrow ave(mw) energy pulse-modulated at 120 Hz, which w as followedby treatm ent w ith . 0.1 iM of the tumor prom oter 12-0-tetradecanoyl-phorbol-13-acetate (TPA). H e noted that th isenergy has been found to be a promoter by other investiga-tors. In the reported study, m icrow aves w ere tested for theiractivity as an initia tor or a com plete carcinogen. The m etho-dology of these..experim ents fo llow ed the orig inal protocols,yielded dose-response data, and w ere in tended to study themulti-step progression of tum or developm ent culm inating inthe grow th of fatal fibrosarcomas when fully transform edcells are injected in to test anim als. The possible cocarcino-

8/13/2019 frey-1993

3/10

EJ W ITHO UTTP A M W)WI T HT P AW IT HO UT T PAW ITH TPA x -RAYS)

0.0 0.1 1.0 4.4 0 .0 .25 .50 1.0W SA R W/kg) X-RAY DOSE Gy

274 Vol 7 Februa ry 1993 The FA SEB Journal FR E Y

genic potential of m icrowaves combined w ith X rays orbenzo[a]pyrene was also investigated.

The speaker reported that there was no mw transform ingactivity w ithout postcu lturing w ith TPA . But when follow edby TPA treatm ent, m icrowaves were additive w ith X ray-induced transformation . The dose-response curve resultingfrom mw exposure fo llow ed by TPA is shown in F ig. 2 , alongw ith sim ilar X ray-induced responses for com parison . Thespeaker stated that the results were not due to heating .

Short w as in terested in determ ining the effect of high-strength static m agnetic fields on the ability of human malig-nant melanoma cells to increase in number and rem ain via-b le . Normal hum an fibroblasts w ere also studied to deter-m ine whether any alterations were unique to tumorpopulations. H uman cell lines were grown in monolayer cul-ture in vitro and subjected to a static m agnetic field using a4 .7 tesla superconducting magnetic resonance imaging(MRI) m agnet w ith the gradient coils rem oved. The numberof cells w ithin the to tal population w as determ ined by usingan electronic particle counter. C ell viability was estimated bytrypan blue exclusion and the cellu lar m orphology of the at-tached cells was docum ented using m icroscopy. The resultsShort reported show ed that the human m alignant m elanom acells and the norm al hum an cells w ere both unaffected by thepresence of a high-strength m agnetic field in term s of achange in their numbers or v iability . H e found, how ever,that the ab ility of the m alignant m elanom a cells to rem ainattached to the tissue culture surface w as markedly butreversib ly im paired by the presence of the static magneticfields. D isattachm ent was not observed w ith in the hum annormal fibroblasts. The speaker concluded that high fieldstrength static magnetic fields may alter the ability of atum or cell to successfully interact w ith its ex tracellular en-vironment.Neural functionIn the second Symposium , chaired by A llan Frey , researchreports were presen ted by M ichael M cGinnis , A sher Shep-pard, and Jan W alleczek . M cG innis noted that sm all localendogenous direct current (dc) electric fields are associatedw ith a variety of developmental and repair events. In addi-tion , exogenously applied dc fields are able to alter the orien-tation, m igration , and grow th of cells in vitro . This led to thehypothesis that applied fields may be used to influence either

1Ox

>0zwcyLIz0I-

0C )z

0

Figure 2 . The response resulting from mw exposure fo llowed byTPA and sim ilar X -ray-induced responses for comparison.

8

64

2

developm ental or repair events in vivo. H e tested th ishypothesis in the m ammalian central nervous system (CNS)by imposing dc fields across lesioned guinea pig spinal cordsand assayed for the return of function in the simple spinalreflex (10 , 11). H e found a m odest, yet consisten t, effect ofsuch fields on the percentage of animals show ing recovery ofth is reflex months after the original injury.

McG innis extended these findings in a recently com pletedveterinary clinical tria l using dogs w ith com plete paraplegiadue to naturally occurring disk hern iation . Specifically , heused 24 sm all breed dogs presenting w ith com plete paraple-g ia due to in travertebral d isc hern iation, w hich w ere enteredinto the study at the Veterinary Teaching Hospital at PurdueUniversity . Each animal received a neurological exam , a bat-tery of electrodiagnostic tests, and radiographic exam inationbefore surgery . A t surgery, the spinal cords were decom -pressed and an oscilla ting field stimulator was im plantedw ith platinum electrodes placed rostral and caudal to the le-sion. The stim ulator delivered 200 mA of regulated dc cur-ren t that alternated in polarity every 15 mm . This regularreversal of polarity was based on the hypothesis that nervefibers are induced to regenerate only by a distal cathode.Therefore, to affect both ascending and descending fibers,the polarity of the current w as reversed at regular intervals.Evaluations of the neurological and evoked potential w ererepeated at 6 weeks and at 6 m onths after the stim ulator w asimplanted.

There were more instances of recovery of function in the13 electrically treated an imals than in the 11 sham -treatedcontro ls. This difference in the degree of functional recoverywas statistically significant for a com bined neurologicalscore. This incorporated deep pain appreciation , superficialpain appreciation , placing ability , and am bulatory ability .H e noted that the data suggest that applied dc fields may beable to influence the response to traum a in such a way as toallow more functional recovery to occur than would normallyb e ex pec te d.

Sheppard noted that in teractions of the nervous systemwith exogenous electric fields and currents are of significanceto neurophysio logy. H e indicated there is evidence that op-tim al coupling of currents to cells occurs w ith currentsparallel to nerve cell fibers for focal dipole fields and pulsedfields. H owever, it has been shown that very m uch w eakerenvironm ental electric fields (in the m icrovolt range) can besensed by a number of aquatic animals. M agnetic fields ofabout 50 mT can be sensed by organism s as prim itive as bac-teria and as complex as m arine m ammals. He also discussedlaboratory studies using a w ide range of field strengths andfrequencies that indicate effects on hum an card iac rate,evoked brain potentials, anim al behaviors such as learningand time estimation , nerve function, neurite grow th , circa-d ian rhythm s - including brain transm itter and pinealm elatonin rhythm s, EEG rhythm s, brain slice physiology-and on both active and passive properties of indiv idualneurons.The speaker indicated that sh ifts in membrane surfacepotential m ay be more important for situations in whichtransm embrane shifts are insignificantly sm all or observedphenomena are not directly related to membrane potential.A s an exam ple of such indirect effects, he presented datashow ing the influence of a strong 60 Hz m agnetic field on in-tracellularly recorded interburst in tervals (IBIs) of apacemaker neuron of the m arine snail Aplysia. The speakerconcluded that the electric field ( 8 mV/m5) induced inthe experim ental chamber by the magnetic field w as toow eak to produce direct electrical effects on the cell, as therew as a 6 mm delay before effects w ere evident.

8/13/2019 frey-1993

4/10

F 34 0

ELEC TR OM A GN E TIC FIE LD S A ND B IOLO GIC A L S YSTEM S 27 5

Walleczek explained how membrane-m ediated signaltransduction pathways are thought to be an early target ofelectrom agnetic field (em f) interactions w ith cellu lar sys-tem s, including cells of the immune and nervous system s. Inparticu lar, he noted that the early modulation of Ca2 signalsby electrom agnetic fields is a plausible candidate for induc-ing later-stage em f effects on cellular function (12). Thus, onegoal of h is research has been to study the effects of 50/60 Hzem fs on Ca24 regulation in electrically nonexcitab le cells ofthe immune system compared w ith electrically sensitive neu-ronal cells. A s m odel system s for studying em f influences onCa24-m ediated immune system signal transduction events,the hum an leukem ic T cell line JURKAT as well as fresh lyiso lated rat thym ic lymphocytes were used . The speakerstated that a 22 mT, 60 Hz sinusoidal m agnetic field en-hances 45Ca2 uptake in concanavalin A (Con A) -treated ratlym phocytes, but not in resting cells, as determ ined after a60 mm exposure (13). H e also stated that rat pheochromocy-tom a PD-12 cells, a neurosecretory cell line frequently usedas a nerve cell model, were used to study cellular in teractionmechanism s of em fs w ith the nervous system . Neurotrans-m itter secretion in PC -12 cells reported ly is m ediated at leastin part by changes in the free cytosolic calcium concentra-tion , (C a24)1 . H e thus suggested that em f-induced altera-tions in (C a24)m could affect neurotransm itter release, andsubsequently nerve cell function.

To define these effects, W alleczek developed a new fluori-metric technique, termed dual-sample (or d ifferential)fluorescence spectroscopy, or DFS , which he described . Thedesign of the DFS instrum ent was based on the assumptionthat a reliab le and efficien t approach for probing em fphenomena would be to sim ultaneously m onitor real-tim efluorescent changes in an em f-exposed cell sam ple and anidentical isotherm al contro l sam ple. Commercially availablefluorimetry system s (one-sample system s) are lim ited tomeasuring either the experim ental or the control sample,and thus cannot be used in th is manner. W ith DFS , it is nowpossib le to record at the same time real-tim e changes in(C a24)1 in the absence/presence of em fs.

Figure 3. Dual sample FURA-2 fluorescence spectroscopy (DFS)electric and m agnetic field-exposure system . Abbreviations are asfo llow s: CH , nonm etallic , therm ostated cuvette holder; F , in terfer-ence filter (340, 380, or 510 nm bandpass filters); M F, water-cooledbefilar Lee-W hiting magnetic field exposure coil; OC , optical cou-pler; PM T, photom ultiplier tube; SC , nonm etallic sam ple com part-m ent; SM F, sham magnetic field exposure coil (sam e as M F). Solidlines represent electrical pathways; dashed lines represent the exci-tation and em ission light paths.

Figure 3 provides a schematic representation of the keycomponents of the DFS electric and magnetic field exposuresystem . The cells were prevented from sedimenting to thebottom of the cuvette by a custom -m ade, nonm agnetic cellmixing system . This was found to be necessary because themagnet ic flux densities associated w ith magnetic cell stirrers,as used in conventional fluorim eters, are on the order of 1 to10 mT . This has implications for quite a few labs.

C ell sig nalin gIn the th ird Symposium , chaired by Bruce M cLeod, researchreports w ere presented by M artin B lank, Christopher Cain ,and Richard Luben. B lank explored em field effects onN a,K -ATPase activ ity and biosynthesis. The processes he fo-cused on range from relatively sim ple ion transport due tothe Na,K -ATPase to the complex stages of biosynthesis.These system s also represent links in a possible m echanisticchain that couples events outside the cell to processes in the cell.

The speaker reported that ATP splitting by the membraneNa,K -ATPase changes when electric currents are imposedw ith electrodes or induced by m agnetic fields. The effect de-pends on the basal level of enzym e activ ity . Under optim alconditions, electric currents decrease the activity ; when thebasal activity is lowered by inhib itors or by lowering temper-ature, electric currents increase the activity . The speakernoted that both effects have a broad frequency dependencethat is sim ilar (and probably related) to the one calculatedfor ion concentration changes at membrane surfaces.

It was stated that both inhib ition and stimulation , as w ellas the observed dependances on the electric field and level ofenzyme activity , can be explained by increased binding of ac-tivating cations to the N a,K -ATPase. W hen the enzyme is atoptimal activ ity , an increase in ion concentration low ers ac-tiv ity. B elow optim al activ ity , an increase in ion concentra-tion increases the enzym e activ ity . The threshold for inhib i-tion by induced electric currents is about an order ofm agnitude higher than that for currents im posed by elec-trodes. The additional stray m agnetic field in his inducedcurrent experiment appears to have an effect on N a,K -ATPase function which is opposite to that of the electric field .S tudies of transcrip tion suggest sim ilar effects of electric andm agnetic fields. B lank th inks his results indicate that em sig-nal transduction can occur at ion activation sites on the out-side surface of the m embrane Na,K -ATPase. The changesaffect enzyme function and alter the composition of the cell.H e noted that the observed therapeutic effects of em signalson damaged tissues correlate w ith the enhanced activ ity ofthe enzym e, due to electric currents, when it is far from itsoptim um (14, 15).

B lank also noted that changes in biosynthetic response toem fields are complicated , but stated that he found sim ilari-ties in the patterns of the biosynthetic responses of Sciaracoprophila salivary gland cells to two kinds of stress: emfields and heat shock. He converted data from two-dimensional gels into pro tein distribution curves ( mass vs.mw , m ass vs. pI) and found new peaks that w ere charac-teristic for both types of stress. He indicated that changes inthe tw o peaks in the p1 distribution as a function of em fieldstrength define a dose-response curve and the range of effec-tive field in tensities. Em fields and heat shock are quited ifferent in their effects on cells, but sim ilar b iosyntheticresponses indicate that both stimuli affect the sam e finalcommon pathw ay. The cell appeared to respond in anundifferentiated way to what it sees as a stress. A lso , the ap-parent absence of variations in the response resulting fromvariations in em field intensity suggest an all-or-none response.

8/13/2019 frey-1993

5/10

50

40

30

20

10

010 0

80

60

40

20

00cz 40

N

00

0,

0EXP 1 EXP2 EXP3

0 EXP1 EXP2 EXP3

27 6 Vol. 7 February 1993 T he F AS EB J ou rn al FREY

Cain addressed the question of m echanism s for em fieldsand carcinogenesis. H e noted that the effects of em fields onbiological system s are consisten t w ith the concept that thecell m embrane is a primary site of interaction w ith em fields.Thus, he chose a m odel that focuses on m embrane-relatedevents and not on genotoxic and mutagenic events in study-ing the relation between pow er frequency em fields and car-cinogenesis in vitro . H e stated that in multistage carcinogen-esis, m embrane-related events m ediated through receptorm echanism s are involved in tumor promotion that is reversi-b le and environm entally modulated . H e noted that it hasbeen suggested that p lausible bio logical m echanism s bywhich environm ental em fields m ay influence carcinogenesisinclude epigenetic events of tum or promotion such as trans-membrane signal transduction and intercellular communi-cation. However, he felt that there w as a need for d irect ex-perimental ev idence that fields influence tumor promotionper se, although higher-frequency em fields have been shownto influence carcinogenesis in vitro in C3H /1OT1/2 fibrob-lasts. The question he specifically addressed in the reportedexperim entation was whether 60 Hz em field exposure anda chem ical tumor promoter, (TPA), are copromoters.

W ith his in vitro model system , he m easured the focus for-m ation of mutant cells (UV-TDT1Oe) in coculture w ithparental C3H /1OTI/2 fibroblasts. Specifically , he saidC3H /lOTl/2 cells suppress grow th of the transformed pheno-type of their daughter UV -TDTIO e cells by preventing UV-TDT1Oe cells from grow ing in multilayered foci, as occurswhen UV-TDT1Oe cells grow alone. In coculture, TPA pro-m otes focus form ation of mutant UV -TDT1Oe cells cocul-tured w ith parental C3H /1OT1/2 cells.

D isruption of communication betw een transform ed cellsand norm al cells is involved in tumor prom otion. C ain testedthe hypothesis that 60 Hz em field exposure and TPA arecopromoters enhancing focus formation . Em field exposuresalone did not affect the grow th of parental C3H /1OT1/2fibroblasts or daughter mutant cells, UV -TDT1Oe (16). Fur-therm ore, em field exposure alone did not promote focus for-m ation of mutant cells in coculture w ith parental cells underthe conditions tested . H owever, em field exposurecopromoted w ith TPA by increasing focus formation incoculture, as shown in Fig . 4 . V ideo image analysis of threereplicate experim ents show ed that field exposed culturesproduced 1.91-fo ld more foci than sham exposed cultureswhen treated w ith TPA . The to tal area of foci per d ish in-creased 2.22-fold and the num ber of cells in stained foci in-creased 2 .3 4-fo ld.

C ain concluded that his experiments suggest that chronicin term itten t exposures to a 60 Hz em field and a chem icaltum or promoter influence m embrane-related events bycoprom oting focus formation . Thus, a coculture system pro-vides a m ethod to investigate bio logical m echanism s bywhich em fields m ay influence carcinogenesis.

Luben discussed the effects of low -in tensity electromag-netic fields on signal transduction by G protein-linked recep-tors. In previous studies of bone cell parathyro id horm one(PTH) receptors, he noted , pulsed em fields decreased theefficiency of signal transduction by the PTH receptor (i.e., a ta concentration of PTH , which should have been effective ininducing bone cells to decrease their bone form ing activ ities,exposure to pulsed em fields decreased that response by asm uch as 90 ). These changes in response to PT H are con-sisten t w ith the known in vivo effects of pulsed fields, i.e ., anincrease in the rate of bone formation relative to boneresorption. As the observed decreases in responsiveness toP1H were not accompanied by decreases in hormone bind-ing to the receptor, he hypothesized that pulsed fields had

800U ) 600z

400.200

Figure 4. 60 H z em field exposures and TPA co-promoted focusformation. A foci were counted using video analysis softw are forcocultures w ith TPA , 50 ng/m l. B from the same video analysisdata set, the total area of foci per d ish w as calculated . C the to taloptical density of stained foci was calculated . For cocultures w ithTPA: in experim ent i, n w as 10 (sham ) and 13 (field); in experim ent2, n was 10 (sham ) and 11 ( field); in experim ent 3 , n w as 19 (sham )and 16 (field). The m ean values SEM fo r T PA -tre ate d c oc ultu resare depicted for A -C . **Indicates P < 0.001 and indicatesP < 0.05 using the I test.

modified the coupling of receptor to membrane signal trans-duction processes, m ost likely the interaction of m embraneG proteins w ith the receptor. He stated that studies w ithmembrane-specific probes for G proteins and adenyl cyclasesupport th is hypothesis. Further studies w ith specificmonoclonal antibodies to the PTH receptor, he noted , haveconfirm ed that pulsed fields caused change in the exposure

8/13/2019 frey-1993

6/10

ELE C TR O M AG N ETIC FIELD S AN D BIO LOG IC AL SY STEM S 27 7

to the extracellu lar m edium of individual receptor deter-m inants. H e th inks th is indicates that the orientation oraggregation state of the receptor is actively modified by thefields (17 , 18).

Luben reported on experim ents derived from his hypothe-sis that other m embers of the G protein-linked receptor fa-m ily m ight also be susceptib le to em fields effects. He notedthat the hypothesis is based on the observations mentionedabove and the follow ing: 1 the basic structure of the trans-m embrane signaling domains found in all of these receptorsis very highly conserved through a variety of species andreceptors (e.g ., from bacterial rhodopsin through m am-m alian neurotransm itter receptors), 2 the only other clearlydemonstrated receptor for electromagnetic energy is rhodop-sin , a m ember of the G protein-linked fam ily, 3 probablereceptor organs for low -energy electromagnetic fields inm onotremes are m odified retinal/neural cells, which containhigh levels of rhodopsin-like membrane proteins, and 4) emfield effects on neural metabolism and behavior could be ex-plained plausibly by effects on neurotransm itter and/or neu-rohormone receptors, many of which are m embers of the Gprotein-linked class of receptors (e.g ., adrenerg ic, mus-carin ic, sero toninerg ic, and several types of peptidergic neu-rons are known to function by G protein-linked receptors).He stated that he has expanded his studies to include j3-adrenergic receptors because they are w idely distributed ,their properties are w ell characterized , and there is evidencethat they may be involved in som e responses to em fields invarious system s.

In his experimentation, Luben m easured activation ofadenyl cyclase and the binding of hormone analogs to PTHreceptors and /3-adrenerg ic receptors in various cultured celltypes exposed to em fields (using exposure schedules previ-ously demonstrated to be effective in modify ing PTH recep-tor activ ity). H e exam ined 13-adrenergic receptor activities incultured m ouse bone cells, cu ltured m ouse skin fibroblasts,and explanted mouse pineal g lands in organ culture exposedto pulsed em fields. He did not observe significant effects onthe /3-adrenergic receptor w ith a 72 H z field, although thatw as the m ost effective in studies of PTH receptor signaltransduction . A 15 Hz pulse train em field , how ever, w hichalso affects the PTH receptor after 4 h of exposure, inhib itedadenyly l cyclase activation by the 13-adrenerg ic agonistisoproterenol, but not by forskolin, a nonreceptor-dependentactivator of adenyly l cyclase. Som e results are shown inTable 1. Net levels of G i and G s proteins measured by ADP-ribosylation also were not changed by em fields. This, he be-lieves, suggests that the observed effects were specific eitherfor receptor/ligand or receptor/G protein in teractions.

C alculations of the binding constants for the 13-adrenergicligand 1251-labeled cyanopindolol indicate, in his view , thatthe to tal num ber of b inding sites on the cells w as notchanged, i.e., the em field is not inhibiting expression of the

receptor, at least in these short tim e courses. R ather, he con-cluded that the affinity of the receptor for the hormone w asapparently lowered . Exposure to em fields produced no effecton /3-adrenergic receptor activ ities of mouse skin fibroblastsw ith the exposure conditions used for bone cell experim ents.However, he reported that in prelim inary experim ents w ithexplanted m ouse pineal g lands in short-term organ culture,4 h of exposure to em fields at 72 Hz produced significantinhibition of isoproterenol-sensitive adenyly l cyclase andbinding of 1251-labeled cyanopindolol.

Luben suggested that the effects of em fields are specificfor the horm one receptors rather than for o ther parts of themembrane signal transduction system for the fo llow ing rea-sons: 1 there w as no change in the overall level of adenyl cy-clase as m easured by activation w ith forskolin, a reagentspecific for the cyclase independently of o ther m embranecom ponents, and 2 there was no change in the incorpora-tion of [4C]ribose by ADP-ribosylation w ith cholera toxininto membrane proteins in prelim inary experiments. This,he th inks, suggests that m embrane levels of G protein werenot affected. He believes these data indicate that /3-adrenerg ic receptors can respond to em fields in a tissue-specific and signal-specific manner.

Pineal glandIn the fourth Symposium , chaired by Larry Anderson,research reports w ere presented by Russel R eiter, S teven Yel-lon , and Bary W ilson . R eiter d iscussed the importantparam eters of exposure and the potential m echanism s for thein teraction of em fields and the pineal. He noted that thepineal g land produces melatonin almost exclusively at n ight.Extrem ely low -frequency electric fields and pulsed staticmagnetic fields have both been reported to depress pinealmelatonin production. He reported use of weak pulsed mag-netic fields at night to suppress the production of melatoninand associated constituents and to define som e of theparam eters of exposure that are required . He also suggesteda biological mechanism to explain the findings.

In m ost of the studies reported, young adult m ale ratsw ere exposed to a pulsed magnetic field (0 .25-0 .44 gauss) for30-60 mm during the dark phase of the light:dark cycle. Thepineal content of m elatonin and the activ ity of N -acety ltransferase (NAT) activity , w hich is rate-lim iting inm elatonin production , was measured. The initia l study, con-ducted 4 h after the onset of darkness, showed that NAT wasdepressed by approximately 30 after exposure to a repeat-edly (at 1 mm in tervals) inverted m agnetic field . In a subse-quent study, the fields w ere inverted either instan taneouslyby means of a relay sw itch, or more slow ly by using an in-tegrating potentiometer. Only when the fields w ere instan-taneously inverted were both NAT and m elatonin levelsdepressed. He noted that rap id inversions of m agnetic fields

TABLE 1. In hib itio n o f 1 3-a dr en er gic r ece pto r re sp on ses b y p uls ed em fs in m ou se b on e c ell c ultu res

C ell lin e te ste d cA MP accum ulation at 106 M isoproterenol 25 1 -l ab el ed GYP b in d in g a t h al f- ma xim al a cti vi tyB one cells 0.64 0.136 0.52 0.096H EP hepatom a 1.06 0.11 0.93 0.12S ki n f ib ro bl as ts 0.96 0.13 1.06 0.11Pinealcells 0.59 0.146 0.71 0.156

Cells w ere incubated for 6 h with a B i-o ste og en c lin ic al fra ctu re h ea lin g d ev ic e, w hic h p ro du ced a p ulse d 10 gauss field. T he last hour of incubationincluded 0.1 m M is ob uty lm eth ylx an th in e. C ells w ere th en tre ate d with is op ro te re no l a t 10-6 M fo r10 mm and cyclic A MP accum ulation was measuredb y rad io im m un oassay . A ltern ativ ely , b in din g of 1 251 -Iab eled cy an opin dolo l w as m easu red usin g stan dard rad io lig an d b in din g pro ced ures. T he valu esre pr es en t f ie ld -t re ate dl un tr ea te d r ati os . P 0.05 usin g D unn etts m ultip le ran ge test.

8/13/2019 frey-1993

7/10

EM F EXPOSUREN E U R OL O GI C D E T EC 1 1O N

P IN E AL G LA N D M E DIA T EDN E U R OE N D O CR IN E E F FE C T S4

R ED UC TIO N IN M EL AT ON IN R ED UC TIO N IN M EL AT ON INMEDIATED INHIB ITION MEDIATED STIMULATiONII

NEUROMODULATORYIGONADOTROPICTONCOSTATIMUNOMODULATORYEFFECTS EFFECTS EFFECTSJ EFFECTS

II IN E U RO BE HA VIO R R EP R OD U C TION C AN C ER

278 Vol 7 February 1993 T he F AS EB Jo urn al FREY

induce electric currents (eddy currents) in rats; thus, he sug-gested that eddy currents m ay have been the cause of melato-nm suppression. In another study he found that only in them id- and late-dark phase (but not early in the dark phase)is melatonin synthesis suppressed.

By way of explanation, Reiter noted that melatoninproduction in the pineal gland is normally low during theday because light im pinging on the eyes inhib its pinealm elatonin production . A s pulsed magnetic fields, like light,inhib it melatonin production, he suggested that em field ex-posure m ay have sim ilar m echanism s, possibly by causingthe isom erization of 11-cis-retinal to all-trans-retinal in thephotoreceptor of the retina. Retinal is associated w ith the rodphotopigm ent rhodopsin . A nother constituent potentially involved in the coupling of m agnetic fields to the retina/p inealsystem may be dopam ine, a neurotransm itter found at anumber of neural sites and in the retina.

Yellons presentation was concerned w ith his finding thatacute 60 H z magnetic field exposure suppresses the nightmelatonin rise in the pineal and blood of the adult d jzungar-ian hamster. H e noted that in a variety of seasonal breeders,the pineal melatonin rhythm appears to transduce inform a-tion about environm ental photoperiod to contro l reproduc-tion . In the djzungarian hamster, the evidence suggests thatthe duration of the nighttime rise in circulating m elatonin isthe physio logical signal that mediates the effect of long daysto stim ulate and short days to inhib it reproductive function .

To determ ine whether an acute 60 Hz m f exposureinfluences the nocturnal melatonin rhythm in the pineal andblood of the djzungarian ham ster, adult hamsters in longdays l6L:8D, male and female were exposed to a 60 Hz mf(+1 gauss horizontal component) for 15 mm beginning 2 hbefore lights w ere turned off. The tim e of treatment waschosen because it is a w indow during the day when melato-nm is most effective in suppressing gonadal function. Serumobtained from blood collected from the body trunk andpineal g lands w as assayed for melatonin using the Guildhayantisera. The speaker noted that this radio immunoassay formelatonin has been validated for use in this species.

M elatonin concentrations in the pineal and blood in-creased in the contro ls to a peak 3 h after the light w as off.M elatonin concentrations w ere significantly elevated com -pared w ith low day concentrations for the rem ainder of thedark period. The m f exposure significantly reduced the du-ration of increased m elatonin in the pineal at night andblunted the nocturnal rise in serum melatonin. Because theduration of increased m elatonin in blood codes inform ationabout day length , the m f exposed ham sters had an inap-propriate photoperiod cue, i.e ., an extrem ely long day thatis analogous to 20 h of light. The speaker suggested that m fexposure influences the mechanism of photo-periodic timem easurement, which may indicate a disruption of thetimekeeping capabilities of an endogenous biological clock .

W ilson , in his presentation , stated that alterations in neu-ronal and neuroendocrine system function have em erged asthe m ost consisten tly observed in vivo effects of extremelylow-frequency electrom agnetic field exposure in laboratorystudies. E lectrophysio logical, neurochem ical, and behavioralendpoin ts have all been used to study the effect of em f ex-posure. H e noted that em field exposure has been shown toaffect electroencephalogram s, electrocardiograms, and m ag-netoencephalogram s in hum ans. Such exposure alsochanged the concentration of neurotransm itters in both hu-m an and anim al studies (19 , 20).

W ilson noted that a strik ing aspect of the effect of em fieldson the pineal is a reduction or phase shifting of the nightlyrise in m elatonin . He said it has been im plicated as a possi-

b le etiologic factor in cancer, neurobehavioral effects, andreproduction epidem iologic studies. Figure 5 depicts thepossib le importance of em field-induced alterations inm elatonin circadian rhythm s as related to the health out-comes of in terest in em field epidem iologic studies.

He noted that in the case of cancer, there appears to besome correspondence betw een those neoplasm s againstwhich melatonin has been shown to be protective in labora-tory studies and those that appear associated w ith em fieldsin epidem iologic studies. M elatonin has been shown to beprotective against leukem ia, breast cancer, prostate cancer,and m elanom a. A ccording to W ilson , melatonin infusion hasbeen reported to be successfu l in the treatment of lym -phom as and breast cancer, as w ell as leukem ia and severalo ther neoplasm s in humans. Epidem iologic studies haveidentified leukem ia, breast cancer, m elanom a, prostatecancer, brain cancer, and lymphom a as neoplasm s associatedw ith em field exposure.

W ilson said that in 1987 S tevens hypothesized that ifp ineal g land function was affected by em field exposure inhumans, then increased breast cancer risk could be one con-sequence detectable in epidem iologic studies. W ilsonpoin ted out that studies to determ ine the effect of em field ex -posure on breast cancer risk in chem ically in itiated ratsshow ed a sm all, bu t statistically significant, increase in num -ber of tum ors per tum or-bearing animal. Three subsequentepidemiologic studies that assessed breast cancer showed anexcess risk for this d isease in occupationally exposed males.R esults from other epidem iologic studies, which are sugges-tive of increased risk of depressive symptom s and m iscarri-age, are also consisten t w ith the hypothesis that em field ex-posure may influence risk of certain disorders in hum ans viachanges in m elatonin .Imm une systemIn the last Symposium , chaired by D an Lyle, research resultsw ere presented by Robert Liburdy , A ndrea Cossarizza, andW endell W inters. L iburdy reported on tests of twohypotheses. In the first he asked if the cell m embrane,specifically the calcium ion channel, is d irectly involved inem field interactions. He considered th is important becausereceptor sites and ion channels are located in the cell mem -brane and these are the first structures involved in the signaltransduction process. In the second he asked whether altera-tions in calcium ion flux are propagated down the signal

Figure 5. Neuroendocrine effects m odel depicting possible conse-quences of EMF exposure as mediated via the pineal g land .

8/13/2019 frey-1993

8/10

C

+Cl 0C -,

35 0

30 0

25 0

20 0

15 0

10 0

50

ELEC TR OM A GN E TIC FIELD S AN D B IOLO GIC A L SYS TE M S 27 9

transduction cascade to alter events such as gene activation.This question fo llows from his in teraction m odel in whichem fields alter calcium ion flux, and thereby influence subse-quent cellular events in the signal transduction cascade suchas gene activation (21). H e considered his resu lts to indicatethat 1 the cell membrane is a site of em field in teraction , 2t he e ar ly s ig na l t r an sd uc ti on m ar ke rs o f c al ci um i on i nf lu xand in tracellu lar calcium are altered by em fields, and 3these changes in calcium ion second m essenger are linked toinduction of c-myc mRNA, a m id-stage signal transductionm ar ke r. T he la tter, he believes, suggests that field-inducedchanges in calcium ion flux are propagated down the signaltransduction cascade to alter gene activation events.

Some of the data that led to his conclusions are shown inF ig. 6. This figure shows a t yp ic al t im e c ou rs e f or [ Ca 21 jduring Con A activation in the absence or presence of a 1 .7mV/cm 60 Hz electric field . W hen Con A (1 eg/m l) w as ad-ded at 200 s, [Ca24]1 increased during the early phase of cal-cium signaling to a value of approximately 300 nM at 800 s.In the presence of a 60 H z field , when Con A was added at200 s, an increase in [C a24]1 w as observed which w as identi-cal to that shown for unexposed cells during the first 100 s.However, [Ca2]1 increased at a greater rate than for cells inthe absence of the field. By 800 s the plateau phase of[C a24]1 reached approxim ately 380 nM . As he sees it, a ltera-tion of the plateau phase of calcium signaling im plicates thecalcium ion channel of the outer p lasma membrane as a siteof field interaction , in contrast to structures associated w ithcalcium release from intracellu lar stores.

L iburdy separated the early and plateau phase of calciumsignaling in tim e by placing cells in calcium -free media andthen added calcium to the buffer. A t approximately 200 s,Con A was added and [Ca24]1 increased to approximately200 nM at 600 s, corresponding to the early phase, w ithrelease of calcium from intracellular stores. W hen extracellu-lar calcium was added to the cells at approxim ately 670 s, theplateau phase of [Ca24]1 reached 390 nM at 800 s. In the ab-sence of Con A , when a 60 Hz electric field w as applied nochange in [Ca24], was detected . W hen Con A was added tocontrol cells in calcium -free buffer, an increase in [C a24]1 oc-

60Hz Electric F ie ld Enhances Plateau Phaseof Calcium Signaling in Activated Thym ocytes40 0

100 200 300 400 500 600 700 800 9001000Tim e seconds)

Figure 6 . Real tim e measurem ents of in tracellu lar calcium in m ito-gen activated thym ic lym phocytes during 60 H z field exposure.

curred. An essentially identical increase was observed forCon A -treated cells exposed to a 60 H z electric field. H e con-cluded that the electric field does not influence the earlyphase of calcium signaling . H e believes his findings indicatethat an em field influences calcium movement across theouter cell m embrane, w hich implicates the cell m embrane asa site of field interaction .

L iburdy hypothesized that such alterations in early signaltransduction influence subsequent downstream cellu larevents in the signal transduction pathw ay such as gene acti-vation and cell pro liferation . To test th is, he carried out ex-periments to sim ultaneously quantify Ca26 influx (early STm arker) and c-myc mRNA (m id-stage ST marker) in thesam e cell preparation exposed to 60 Hz em fields.

H e carried out three separate Northern analyses of thesam e sam ple that w ere imaged using a cooled CCD cam era.There were three bands for each Northern blo t, w hich cor-respond to: -Con A , +Con A (1 m g/m I), and +Con A plus60 H z. H e performed a m ultivariate analysis of variance form ean pixel in tensity of each band across the three Northernanalyses. Liburdy stated that the results were statisticallysignificant and consistent w ith mRNA findings. He believesthey support a linkage betw een an em field interaction w iththe cell m embrane involv ing Ca24 signaling and down-stream ST pathw ay processes such as gene activation.

Cossarizza, on a somewhat different tack , described hisexperiments in which electromagnetic fields affected cellproliferation and cytokine production in human cells. H isprevious studies indicated that in vitro exposure of hum anperipheral b lood mononuclear cells (PBMC) to pulsed emfields increased in vitro phytohem agglu tinin (PHA ) -inducedlymphocyte pro liferation , as m easured by (3H -TdR) andbrom o-deoxy-uridine incorporation assays (22). M icro titerp lates contain ing the cell cu ltures w ere placed betw een a pairof Helmholtz coils (maintained parallel to the plates) and po-w ered by a pulse generator. The effect was much more evi-dent, he reported , when cells from aged donors or from sub-jects affected by a syndrome of precocious aging such asDowns syndrome were used. H e reported that these fieldsw ere devoid of genotoxic effects as assessed by the cytokinesisb lock m icronucleus method and they did not interfere w ithDNA repair capability of human lymphocytes exposed togenotoxic agents. The exposure system was given to investi-gators at tw o other laboratories and they w ere able toreproduce the results. He reported that in the course ofseveral years he obtained the same increased cell prolifera-tion in pulsed em field-exposed , PHA -stimulated PBMCfrom aged people.

Further experiments were carried out in order to assess 1the reproducib ility of these results, 2 the m echanism (orm echanism s) by which em fields are able to act as com ito-gens in PHA -stimulated PBMC, and 3 the kind of cellsaffected . Cossarizza noted that the pro liferation of hum anPBMC is regulated and m odulated by several cytokines. H estated that previously he dem onstrated that IL -2 is moreefficien tly utilized in pulsed em field exposed cells, as ahigher percentage of such lym phocytes expressed low-affinityIL-2 receptor (CD25 antigen), as shown by cytofluorimetricanalysis. He then presented data suggesting that the produc-tion of other cytokines is also effected by pulsed fields. W henPBMC from healthy donors were exposed to the fields, hereported a dramatic increase in the production of IL-lb andIL-6 compared w ith unexposed contro ls, as shown in Table2 a nd T ab le 3 . H e r ep or te dt ha tt hi sp he no me no n w as n ot ednot only in m itogen-stim ulated cultures, but also in unstimu-lated PEMF exposed cultures. H e th inks these data are in-teresting for two m ain reasons. F irst, they help explain the

8/13/2019 frey-1993

9/10

28 0 Vol. 7 February 1 993 The FAS EB Journal FR E Y

increased cell pro liferation he has reported , as IL -i is criticalin the early events of T cell activation and proliferation . Se-cond, assum ing that such a phenomenon could occur in vivo ,it could explain other effects of pulsed fields, such as thosedescribed in fibroblasts and bone cells, in which beneficialeffects w ith the sam e type of fields have been reported (23).He noted that IL-I an d IL-6 are the most pleiotropiccytokines, and are able to exertprofound effectsn nonim-mune cells including cells of connective tissues and the neu-roendocrine system .

H e also reported studies of the genotoxic potential andcapability of a 50 Hz electric field to affect im portan t im -mune responses, such as the capability of human lympho-cytes to respond to polyclonal stim uli and of hum an naturalk iller (NK ) cells to kill tum or target cell lines. Seventy-fivesubjects w ere studied. H um an PBMC proliferation w as as-sessed by stim ulating cells w ith different doses of PHA orw ith 10 ng/m l anti-CD3 monoclonal antibody (which hestated selectively stimulates T lym phocytes). C ell prolifera-tion was evaluated by measuring the 3H -TdR incorporationd ur in g t he l a st6 h o f a 72 h culture. The data show that themain effects of the electric field w ere on the proliferation ofPBMC from 21 aged subjects and 12 young donors. Hereported that there w as a sligh tdecre aseof 3H-Td R incorpo-ration , w hich w as particu larly evident w ith optimal ands up ra op ti ma lP HA d os es an d i naged subjects. H e c on clu de dthathisdata show thatthe age of the donor is of critical im -portance in the biological effectsf electric fields o n h uma n c ell s.

Winters also spoke about the immune system . He notedthat the biologicalctiviti esf cellsof the immune system ,particu larly those of lymphoid lineage, have repeatedly beenreported to be sensitive to in vitro exposure to a variety oftypes and strengths of environmental e lectromagnetic andm ag ne ti c f ie ld s e mf /m fi . H e s ta te dt ha t a w id e r an ge o fbiocifects, such as significant changes in cellu lar synthesis ofmacrom olecules, receptor functions, release of ions, andregulatoryfactors,have been found in clinicaland basicscien cerese archstudi esto be associated w ith specific popula-tions of immune system cells obtained from norm al donorsand from patients w ith immune cell d isorders, infectious dis-eases, or neoplasia after their exposure to extremely low -frequency em f/mf. He pointed out that the immune cellpopulations studied have included those cells responsible forinitiatingnd maintaining cellularand humoral immuneresponses, immunosurveillance, and resistance againstpathogenicorganisms.

Winters saidthatthe lym phoid cell populations m ost o ftenstudiedin emf/mf exposure investigationsave been mixedsubpopulations of human lymphocytes of the B type plusthoseof regulatorycelltypes,i.e., helpercellsregulatingB and T cellproliferationnd functionsand bearingclustersof differentiation m arker 4 CD4 and class II majorhisto compa tibi lityompl ex (M HC) antigens. Lym phocytes

TABLE 2. Th e pro du ctio n o JIL-1 /3 in the su perna lan ts of 12 -0 letra de-c an oy lp ho rb ol 1 3- ac ela te .s tim u la te d PBMC c ultu re ?

Stimulus Control Exposed PNone 11.3 1 .3 54 ,4 13 .3

8/13/2019 frey-1993

10/10

A s n ot ed i n t he i nt ro du ct io n, e le ct ro ma gn et ic f ie ld sa renot a foreign substance. L iving organism s are electrochem i-calsystemsthatuse emfs in everythingfrom proteinfoldingth rough cellu lar communication to nervous system function .A more appropriate model of how living organisms can beexpected to respond to em fieldswould be to compare themto a radio receiver.

An electromagnetic signal a radio detects letus callit sig -nal x and transduces into the sound of music is a lmost un-measurably weak. Yet the radio is imm ersed in a sea of emsignals from power lines, rad io stations, TV stations, radars,etc. The radio doesnt notice the sea of signals because theyare not the appropriate frequency or m odulation. Thus, theydont d isturb the m usic w e hear. If w e expose the radio to anappropriately tuned em signal or harmonic, how ever, even ifit is very weak com pared to signal x , it w ill in terfere w ith them usic. S im ilarly , if we expose a liv ing system to a very weakem signal, if the signal is appropriately tuned , it couldfacilitate or in terfere w ith normal function (25). This is them odel that m uch bio logical data and theory tell us to use, nota tox icology model. And this is the model that is now startingto be used so fru itfu lly.

REFERENCES1. Frey, A . H ., and Eichert, E . S . (1972) The nature of electrosens-

ing in the fish . B io ph ys . j 12 , 1326-13582. Frey , A . H . (1988) Evolution and results of b iological research

with low -in tensity nonionizing radiation. In Modern B i oe le c tr ic it y(M arino , A . A ., ed) pp . 785-837, M arcel D ekker, Inc., NewYork

3. Frey , A . H . (1967) B rain stem evoked responses associated w ithlow intensity pu lsed UHF energy. j A p pl. P hy sio l. 23 , 984-988

4. Frey , A . H ., and Coren , E . (1979) Holographic assessm ent of ahypothesized m icrow ave hearing mechanism . Science 206 ,232-234

5. Frey , A . H ., and Siefert, E . (1968) Pulse m odulated UHFenergy illum ination of the heart associated w ith change in heartrate. Lfe Sci. 7 , 5 05 -5 12

6 . P h il li ps , J. L ., and McChesney, L . (1991) E ffect of 72 Hz pulsedelectrom agnetic field exposure on m acromolecular synthesis inCCRF-CEM cells. C an ce r B io ch em . B io ph ys . 12, 1-7

7 . Ph il li p s, J. L ., H aggren, W ., Thom as, W . J. ,Ish ida-Jones, T,and Adey, W. R. (1992) M agnetic field-induced changes ins pe cif ic g en e tr an sc rip tio n. B io ch em . B io ph ys . A da 1132, 140-144

8. Balcer-Kubiczek , E . K ., and Harrison, G . H . (1989) Inductionof neoplastic transform ation in C3H /lOTl/2 cells by 2.45 GHzmicrow ave and phorbol ester. R ad ia l. R es. 11 7, 5 31-5 37

9. Balcer-Kubiczek , E. K ., and Harrison, G . H . (1991) Neoplastictransformation of C3H /IOT1/2 cells fo llow ing exposure to 120Hz modulated 2.45 GHz m icrowaves and phorbol ester tum or

promoter . R ad ia t. R es. 125, 65-7210. Borgens, R . B ., Robinson, K . R ., V anable, J. W ., Jr., and

McGinnis, M . E. (1989) El ec tr ic F i el ds i n Ve r te bra t eRepa ir . Alan R.L is s,N ew Y or k11. Borgens, R . B ., B light, A . R ., and M cGinnis, M . E . (1990)Functional recovery after spinal cord hem isection in guineapigs:the effectsf appliedelectricields. C omp . N eu ro l. 296,634-653

1 2. W a ll ec ze k, J. (1992) E lectrom agnetic field effects on cells of theimmune system : the ro le of calcium signaling. FASEB J. 6,3177-3185

1 3. W a ll ec ze k, J. , and L iburdy , R . P . (1990) 60 Hz sinuso idal mag-netic field exposure enhances 45Ca2 uptake in rat thym ocytes:dependence on m itogen activation . FEBS Lett. 271 , 157-160

14. B lank, M . (1992) Na,K -ATPase function in alternating electricfields. FASEBJ 6 , 2 43 4- 24 3815.Blank,M., and Goodman, R . (1988) An electrochem ical modelforthe stimulationf biosynthesisy e xte rn al e le ctr ic f ie ld s. Bio-e l ec t rochem. Bioenerge t. 1 9, 5 69 -5 80

16. C ain, C . D ., Thom as, D . L ., and Adey, W . R . (1993) 60 Hzmagnetic field acts as co-promoter in focus formation ofC3H /1O TI/2 cells. Carcinogenesis In press17 . Cain, D . C ., Adey, W . R ., and Luben, R . A . (1987) Evidencethat pulsed electrom agnetic fields inhibit coupling of adenylatecyclase by parathyro id horm one in bone cells. j B on e M in er . R .e s.2 , 4 37 -4 4118.Luben, R. A. 1991 Effectsf low energy electromagneticfieldson membrane signaltransductionprocessesin biological sys-tems. Heal th Ph ys ic s 61, 15-28

19. W ilson, B . W ., Leung, G ., Buschbom , R ., Stevens, R . G ., A n-derson , L . E ., and Reiter, R . J. (1 98 8) E lectric field s, the p inealgland an d cancer.In T he P in ea l G la nd a nd C an cer (G upta, D ., A t-tan asi o,A., an d Reiter,R. J. ,eds) B rain R esearch P ro motion ,T uebingen, L ondon

20. W ilson , B . W ., S tevens, R . G ., and Anderson , L . E . (1989) Neu-roendocrine m ediated effects of electrom agnetic-field exposure:possible ro le of the pineal g land . Lfe Sci. 4 5, 1 31 9-1 33 2

21. Liburdy , R . P. (1992) Calcium signaling in lymphocytes andE LF fields: evidence for an electric fields m etric and a site of in-teractioninvolvingthe calcium ion channel. FE BS Lett. 301,53-59

22. Cantini, M ., C os sa rizz a, A ., Ber sa ni, F ., C ad os si , R ., C ec ch er elli ,G., Tenconi,R.,Gatti,C.,and Franceschi,C. 1986 Enhancingeffectof low frequencypulsed electromagneticfieldsn lectin-induced human lymphocyte pro liferation. j Bi oe le c tr . 5 91-104

23. Cadossi, R ., B ersani, F ., Cossarizza, A ., Zuccihni, P ., Emilia,G ., Torelli, G ., and Franceschi, C . Lymphocytes and low-frequency electromagneticie lds. FASEB j 6, 2667-2674

24. Parker,J. E. , an d Winters,W. D. 1992 Expressionof gene-specificNA in cultured cells exposed to rotating 60 Hz mag-n et ic f ie ld s. B io c hem is tr y C e ll B io lo g y 70 , 237-241

25. Frey, A . H . (1990) Is a toxicology m odel appropriate as a guidefo r bio log ical research w ith electro magn etic field s?j Bioelectr 9,233-234