Bioelectrochemistry and Bioenergetics, 15 (1986) 39-55

39

A section of J.Electroanal

.Chem., and constituting Vol . 211 (1986)

Elsevier Sequoia S .A., Lausanne -Printed in The Netherlands

828-SOME BIOLOGICAL EFFECTS OF ELECTROMAGNETIC FIELDS

REBA GOODMAN *

Department of Pathology, College ofPhysicians and Surgeons, Columbia University, 630 West 168th Street,

New York, NY 10032 (US.A.)

ANN S. HENDERSON

Biological Sciences, Hunter College and the Graduate Program of the City University ofNew York, 695 Park

Avenue, New York, NY 10021(U.S.A

.)

(Manuscript received February 13th 1985)

SUMMARY

It has been known for some time that specific asymmetric electromagnetic fields can induce union in

non-healing bones . Despite the clinical effectiveness of low frequency electromagnetic(e.m,)

signals,

virtually nothing is known concerning basic mechanisms in cells involved in bone healing, or other cells in

soft tissue, which can also be affected by e .m. signals. It has thus become increasingly important to

understand the molecular basis of a.m. field stimulation in order to evaluate the clinical effectiveness and

the results of inadvertent environmental exposure.

We have developed a test system to examine a fundamental cellular function, that of transcription, in

Sciara polytene chromosomes. Since RNA synthesis is a basic cellular event, alterations arising from

exogenous stimulation of cells by e .m. fields should be reflected in transcriptional patterns . Our results

show this to be the case. e.m_ signals in the 72 Hz frequencyrange, both quasi-rectangular and sinusoidal,

induce new RNA transcription in the 6-10S size class and augment a RNA size class in the 20-25S range .

e .m. signals in the 1.5 to 15 Hz frequency range result in either a similar response as that seen in 72 Hz

signals, or one which is indistinguishable from non-stimulated control cells. Since translational effects

would be expected to be reflected as a result of alterations in transcription, we examined protein

biosynthesis following a.m. field stimulation . Changes in the form of augmentation, deletion, and

appearance of new polypeptides are observed .

Finally, we found that e .m. signals, even at some distance from the non-stimulated control samples,

emit an undefined signal that is detected by the non-stimulated control cells. For this reason, we have

shielded non-stimulated cells .

Our ultimate goal is to correlate a specific parameter of the e.m. signal waveform with cellular

induction of particular classes of RNA and/or molecular weight ranges of protein. To date, the data

suggest that the 72 Hz range isan important frequency component and that waveshape may not be as

crucial to clinical usefulness as previously supposed.

INTRODUCTION

Biological systems ranging from whole organisms to subcellular components have

been exposed to various types of electrical signals as a means of altering or

To whom correspondence should be addressed-

03024598/86/$03.50

0 1986 Elsevier Sequoia S.A .

40

augmenting preexisting endogenous electrical fields [1] . It is possible that the

appropriate distribution of electrical energy in time, amount and/or frequency could

impart informational qualities to a cell, which in turn, would initiate electrical

perturbances and trigger a specific response . An inappropriate distribution of

electrical energy, on the other hand, could dramatically alter normal dependent

cellular activity and interactions . It has been postulated that stimulation of cells by

exogenous electromagnetic (e.m .) fields could initiate, and possibly imitate, normal

electrical interactions within the cell, resulting in a measurable cellular response [2].

Our subsequent studies have demonstrated that exogenously administered e .m. fields

can induce increased transcription in eukaryotic cells [3-71 .

Alterations in endogenous electrical fields within cells may be directly related to

function. It has been reported that d.c. current in bone will produce osteochondro-

genesis [8J and bacteriostatis 19], affect ATP generation, protein synthesis and

membrane transport [10], cause changes in cell shape and actin distribution [11], and

lead to a reduction of the electroretinographic b-wave response in the retina [12] .

Specific commercial applications of electrical field technology include the use of

either non-uniform a .c. fields or d.c. pulses of high intensity to induce cell fusion, as

well as gene transfer [13,14] . Capacitatively coupled currents have been shown to

affect in vitro DNA synthesis [15] . Inductively coupled pulsating currents affect

lysozyme activity [16], lengthen membrane receptor aggregate lifetime in lympho-

cytes [17], increase neurotransmitter release in a clonal nerve cell line [18] and

increase transcription [3] . Inductively coupled pulsating current is characteristic of

fields that have been used clinically in treatment of non-unions and pseudoarthroses

of bone [19-211 . Low frequency e.m. fields also inhibit alkaline phosphatase activity

in osteoblast-like cells in a manner similar to that seen in the presence of parathyroid

hormone [22] . Higher field strengths can cause desickling of erythrocytes [23] . e.m .

fields in the form of sinusoidal waves between 15 and 4000 Hz have been shown to

enhance DNA synthesis in human fibroblasts [241 .

There are many possible consequences to the cell that could arise as the result of

medical or environmental exposures . The initiating events are relatively undefined,

but effects are expected to be reflected in complex interactions between charged cell

membranes and hormones, antibodies, ions and other charged molecular species

125-27] .

THE MODEL SYSTEM

Few studies have concentrated on basic molecular processes in the cells that are

effected by e.m. fields. Since the complexity of the cellular response is such that the

basic phenomena which initiate cellular events following e .m. exposure could be

obscured, a simple model system was selected for our studies . The experimental

design allows us to determine whether transcriptional or translational patterns are

affected by the presence of e .m. fields . Transcription was selected for study because

it was anticipated that this parameter would initiate or signal critical cellular

changes. This approach is based on the rationale that understanding the total

41

cellular response to e .m. fields can be most effectively obtained by measuring

affected components singly. Studying a complex cellular response could result in a

large number of experimental variables . The number of experimental parameters

may be further increased by the paucity of information available concerning the

specific characteristics of a given e .m. field that are effective in stimulating cellular

functional changes . The salivary gland cells of the dipteran, Sciara coprophila, have

been used in our studies as a relatively simple system in which to monitor

transcriptional changes ; i .e ., via transcription autoradiography or sucrose gradient

analysis of changes in RNA transcripts within size classes. The simplicity of this

eukaryotic system is such that varied e .m. fields with different signal characteristics

can be related using identical and precisely defined conditions . One of the ad-

vantages of Sciara salivary gland chromosomes is the presence of DNA puffs which

can be monitored directly for DNA synthesis [28] . These cells, which are synchro-

nized, non-dividing and in constant interphase, provide an extremely useful system

for testing signal characteristics, and assessing clearcut changes in transcriptional

activity of cells following e .m. field stimulation . Since both RNA and DNA puffs are

present in a well-defined and established order, the developmental stage and

stage-dependent genomic activation can be determined. The nuclei contain 4 chro-

mosomes (II, III, IV, and X) which are highly polytenic (4094 strands of DNA/

chromosome) as a result of repeated rounds of DNA replication .

All expected interphase functions are present in salivary gland cells, including

RNA transcription and cyclical DNA replication [28] . The presence of morphologi-

cal chromosome puffs, RNA and DNA signals a high degree of transcriptional

activity at a given chromosomal locus (Fig. 1). Puff transcription, as well as other

transcriptionally active sites can be followed using transcription autoradiography .

This technique identifies nascent radioactive RNA chains on the chromosome via

autoradiography and 3H-uridine incorporation . Modifications in the transcriptional

activity can be induced by the exogenous application of the steroid insect molting

hormone, ecdysone [28], or exposure to environmental stress in the form of sudden

elevated temperature [29]. Since normal transcriptional patterns, as well as induced

alterations in these patterns are well characterized, deviations from normal transcrip-

tional activity can be easily detected .

The presence in Sciaridae (as opposed to Drosophila) of DNA puffs provides

morphological regions on the chromosome where DNA amplification at specific

developmental stages reaches as high as 8000-teny in each puff. The presence or

absence of the DNA puffs serves as one basis for staging cells, and provides another

parameter to test the effects of e .m. fields with respect to incorporation of radioac-

tive precursors into sites of DNA synthetic activity (see Fig . 1). On the molecular

level, adequate quantities of DNA, RNA and protein for analyses can be obtained

from about 30 salivary glands. In our experiments, salivary gland cells from Sciara

are exposed to various e .m. signals for preset time intervals . Transcriptional patterns

of nascent RNA chains are determined by transcription autoradiography [28],

cytological nick-translation, and by analyses of radioactive RNA patterns following

sucrose density gradient centrifugation [3] . Translational patterns are determined on

42

Fig . 1 . Autoradiogram of a squash preparation of salivary gland chromosome 2 from Schwa coprophila .

The chromosomes were incubated in 125 pCi/cm 3 3H-thymidine for 10 minutes prior to fixation . Note

the large DNA puffs and the characteristic pattern of bands and interbands . The autoradiographic grains

indicate chromosomal loci active in DNA synthesis . This synthetic activity is particularly apparent at

DNA puff regions. A similar type of autoradiogram can be obtained by following the incorporation of

3 H-uridine into chromosomal RNA at an earlier larval stage ; RNA puffs become preferentially labelled .

The time of autoradiographic exposure was 24 hours . Lactic-acetic-orecin. X500 (from Zegarelli-Schmidt

and Goodman [281) .

the basis of autoradiographic patterns following gel electrophoresis of 75S-labelled

proteins.

A variety of waveform characteristics can be achieved by the manner in which a

given coil is constructed ; the waveform parameters are directly related to the

electrical properties of the Helmholtz coil . The simplest waveshape is symmetrical

and is generated by a sine wave generator attached to an appropriate amplifier .

Quasi-rectangular signals, on the other hand, are more complex and depend on the

directional winding as well as the physical characteristics of the wire . The perturba-

tions in the rectangular asymmetric waveforms are produced by electronically

modifying the 60 Hz line-current and shaping it into a quasi-rectangular pulse wave,

repeated at selected frequencies . The pulsing e .m. field induces a current in the

electrolytic environment of the extracellular fluid, and the charged molecules inside

the cell . It has been determined that pulsing e.m. fields in clinical use induce an

electric field of about 1 mV/cm in the extracellular fluid, which translates into a

current of a few tsA/cm2 [30,31]. The physical characteristics of the perturbation

can be precisely monitored by exposing a standard coil probe (connected to an

oscilloscope) to pulsing e .m. fields under the same conditions as experimental

biological sample .

Repetition rate, positive and negative widths and amplitude values are defined for

the signals we have tested (Table 1, Fig . 2) . The coil is in an incubator at 20C, with

the generator outside the incubator at room temperature . Control cultures are kept

in a separate incubator in another room under identical conditions . The e.m .

exposures are obtained via specific current waveforms, which are induced in the

solutions by the time variations of a magnetic field . Salivary glands of late 4th instar

TABLE 1

Signal characteristics

380 )u sec

a

Th

X4.5 ms 1

200

N

Sec -S m sec

b

TTThP

2Spsec

0.8 mV

positive

amplitude

Fig. 2. Major waveform characteristics of representative e.m . fields. (a) Single pulse (SP 17) ; (b) pulse

burst (PB 15); (c) sine wave. The characteristics of each of these fields are given in Table 1 . Amplitude is

measured using a Tektronix 2464 (200 MHz) oscilloscope coupledto a coil probe.

Re etitive

singe pulse (SP)

(diode clipped)

72 Hz

Repetitive

pulse burst (PS)

15 Hz

Sine wave (5)

72 Hz

43

Signal B-FielddB/dt

Rate

(Gauss) (10

-2

(Hz)

Gauss/ps)

Positive

amplitude

(mV)

Positive

width

(ps)

Burst

width

(ms)

Negative Negative

space

spike

Relative

transcriptional

induction(s)

(/Is)

SP-112 .4

6,2 15 10 200-

3400 +

SP-2 16 .0 8 .0 15 13 .5 200 4500 +

SP-3 16 .0 8 .0 15 13 .5 200 - 2200 -

SP-4 38 .0 10 .0 72 14.5 380 6000 -

SP-5 38-0 10 .0 72 14.5 380 6000+ +

SP-6 39.0 10 .0 72 14.5 380 - 5800 + + +

SP-17 35 .3 9 .2 72 15 .0 380 4500 + + +

E33(gen) 4.8 1 .9 1 .5 2.5 250 30 10 4 +

E33(bat) 4.8 1 .9 1 .5 2.5 250 30 104

+ +

PB-22.58 1 .0 2 .0

1 .6250 50

10 4-6 +

PB-32.58 1

.04

.01 .6

250 2510 4-6 +

PB-15 19.0 9.5

15 .0 14.5 190 5 28 24 +

Sine72 7.4 0.33 72 - +++

Sine 222 2 .4 0.33 222 - - + +

Sine 44000.12

0.33 4400 - +

44

female larvae are dissected into Schneider's Drosophila medium (GIBCO) containing

'H uridine (either 125 Ci/cm 3 for transcription autoradiography or 250 Ci/cm'

for isolated RNA determinations ; sp. act. 40.8 Ci/mmol New England Nuclear).

Whole glands attached to the larval body are exposed to a selected e .m. field for

varying time points up to an hour. In all cases, non-stimulated (NS) controls are

shielded from the field source. Our experiments have repeatedly shown that NS

controls which are not isolated geographically from active coils exhibit signal effects

which mimic those seen in cells placed in fields, albeit, in a different time frame . All

experiments were performed in incubators at 20C, the optimum growth temper-

ature for Sciara .

Cells are placed in 15 X 60 mm Petri dishes centrally positioned within vertically

oriented 10 x 10 cm Helmholtz-aiding coils which deliver either sinusoidal, single

pulse (SP), or pulse burst (PB) wave types (see Fig. 2). The symmetrical axis is

aligned with the coils . The distribution of current flow within the dish or flask

depends on the geometry of the coil (and the nature of the tissue) . Since the angular

component of the induced fields varies with the radius, the positioning of samples is

placed as close as possible to the three dimensional center of the field, i .e ., the level

of current density (which is also dependent on the height of the medium) is higher

toward the center of the field relative to the peripheral regions . Further, cells are

exposed to a more nearly uniform current density since the magnetic field is known

to be uniform at the center [30,31] .

TRANSCRIPTION STUDIES

Effect of fields on incorporation of 3H uridine into RNA

We have shown that e .m. fields can induce increased RNA transcription in

salivary gland cells as detected by transcription autoradiography in polytene chro-

mosomes and by analyses of uptake of 3 H uridine into RNA of various size classes

[3-7] . This single critical function of the cell was related to exposure of the cell to

e.m. signals of known characteristics . While this approach could represent an

oversimplification of the magnitude of effects anticipated as the result of e .m. field,

it is necessary in light of the inherent complexities in determining how specific signal

characteristics affect cellular processes .

A large series of types of e .m. signals have been tested for their effect on

transcriptional induction on salivary gland cells of Sciara coprophila . We have tested

7 SP signals, 5 PB signals and 3 sinusoidal waves . The first approach for determining

transcriptional activation was at the cytological level via transcriptional autoradi-

ography. Figure 3 gives examples of transcription autoradiograms following ex-

posure of salivary gland chromosomes to asymmetric and symmetric signal shapes,

and demonstrates that overall transcriptional rates are augmented at specific chro-

mosome sites following exposure to e .m. signals .

The second and major means of determining transcriptive changes is by bio-

chemical analysis of discrete changes in the specific activity of 'H RNA . Irrespective

45

Fig. 3 . Transcriptional patterns over salivary gland chromosomes exposed or isolated from e.m . fields.

Following e .m. field exposure, the labelling is heaviest over RNA puff regions or chromosome interbands

(arrows) . Roman numerals indicate the chromosome number. All magnifications are ca. 100x . (A)

Transcription autoradiogram following exposure of the cells to the SP 17 field for 45 minutes in the

presence of 3H-uridine (125 pCi/cm3 ) . Autoradiographic exposure time was 3 days. (B) Transcriptional

autoradiogram of cells which were isolated from e.m. fields. Exposure time was 3 days. (C) Nick-transla-

tion of chromosomes exposed to SP 17 fields for 45 minutes . DNA sites on the chromosome which were

nicked by deoxyribonuclease I were labelled with

tzsldeoxycytidine triphosphate [NEN] by nick

translation repair with DNA polymerise. Active chromosome regions or DNA associated with active

regions are more susceptable to DNAse nicking, and the resultant repair should identify these regions.

Arrows indicate transcriptively active regions (hoispots).Autoradiographic exposure time =18 hours. (D)

Transcriptional autoradiogram following exposure of the cells to the PB 15 e .m . field . The labelling

pattern over the chromosomes is less intense and less specific than that seen following SP 17 exposure (A) .

Specific deviations are noted by arrows . Autoradiographic exposure time was 3 days . (E) Chromosomes

following transcription autoradiography of cells that were isolated from e .m . fields . Exposure was 3 days .

(F) Nick translation of chromosomes following exposure to PB 17 . Othcr conditions are as given in (C).

From Ref . 3 ; used with permission of Science .

of the signal type, effective signals resulted in changes in the overall transcriptional

patterns. Two general RNA size classes are augmented by e .m. fields . Incorporation

of 3H-uridine into RNA was enhanced in RNA size classes of approximately 6-10S

and 20-255, as well as small fragments which probably represent partially tran-

scribed RNA (< 4S) . These size classes are consistent with S values expected for

processed and unprocessed mRNA fractions, although we cannot rule out the

presence of other RNA species at the present time . Experiments in progress, using

poly-dT column separation, will aid in identifying poly-A containing RNA species .

The most consistent assignment of the induced RNA would be mRNA, since our

studies using transcription autoradiography indicate that multiple, widely dis-

tributed genetic sites are activated. Further studies will also allow us to determine

whether the effect is one which augments pre-existing RNA transcription or if new

species are formed.

46

SP signal effect on RNA transcription

The largest number of analyses has been done using the SP # 17 signal (see Table

1 for characteristics) . This signal induces two broad characteristic peaks of RNA

transcription, as seen on sucrose density gradients . The transcriptional induction,

however, is different from other signals in that the time course of 'H uridine uptake

is not linear. The data can be summarized as follows (see Fig. 4) : there is a strong

transcriptional response to the signal at 15 minutes in the field ; a reduction at 30

minutes and a second, more intense induction after 45 minutes . This effect is also

seen in transcription autoradiographs and is probably a characteristic of this

particular field, since the pattern seen with other signals shows incorporation of

'H-uridine as a function of time . A reduction in radioactive RNA transcripts seen

following the use of all signals after 60 minutes may occur simply because the organ

culture is metabolically exhausted .

A pertinent observation was made during the course of experiments using the SP

17 signal. This is of importance both experimentally and clinically, and has environ-

mental implications as well. Cells will react to e.m. signals even when the cells are

20

4-5s

r

18s

V

28s

I

5

10

Fraction Number

Fig . 4 . Effect of 72 Hz repetitive SP 17 signal on the uptake of 3 H-uridine into RNA at 15, 30, 45 and 60

minutes. 100 salivary gland cells (about 2x10 3 cells) were placed in the e .m. field for the time periods

indicated at 20C. The cells were suspended in 0 .5 cm3 Schneider's Drosophila medium (GIBCO)

containing 250 pCi/em3 3 H-uridine. RNA was extracted as described previously [3].Approximately 50

IAg of RNA from exposed cells was layered over 5 to 30% sucrose gradients. The gradients were run for 17

hours at 32000 r .p .m. in an IEC ultracentrifuge. Fractions were collected with automatic monitoring of

the optical density profile. An aliquot was removed from each fraction for specific activity determina-

tions. (A typical profile of RNA from cells which were not exposed to e .m. fields is given in Fig. 6 .)

154

20-

4-5s

18s

26s

r

V

V

154

47

5

10

Fraction Number

Fig . 5 . The effect of the SP 17 signal on transcription in cells placed some distance from the field . In the

present case, the cells were placed five feet from the signal source . The open circles represent incorpora-

tion of 3 H-uridine into RNA at either 15 minutes at 5 feet distance from the field or cells isolated from

the signal for periods up to 1 hour, i .e ., the incorporation is the same . While this incorporation of

3 H-uridine is not equivalent to that seen in RNA of cells placed directly in e .m . fields, it is significantly

greater than background levels . All conditions are as given in Fig . 4 .

some distance from the position of the e .m. coils (Fig. 5) . This has been tested under

conditions where unshielded control cells were in an incubator at about 5 feet from a

signal source in another incubator . Transcriptional induction was noted after 30

minutes, i.e ., non-stimulated control tissue incorporates 3H-uridine into RNA at a

higher rate than geographically isolated cells, but at a significantly slower rate than

that seen in e .m. stimulated controls . Although the response was not of the order

seen for cells placed directly in the signal, this long distance effect could obviously

skew experimental data (see Fig. 4 for comparison) . e.m. signals and coils in

experimental or clinical use should take into account the presence of the experimen-

tal field or other possible sources of e.m. or electrical fields in the general vicinity of

the signals to be tested. The exact distance from the source at which the signal is

effective is not known, but experiments in progress should delineate field strength at

various distances. On the basis of this data, it is important to determine whether or

not there is another, as yet undefined component of e .m. fields, that is emitted other

than that from the primary source ; e.g., radiowave frequencies.

Our preliminary experiments suggest that transcriptional induction can be aug-

mented by placing cells for short time periods in and out of e .m. fields . Presumably

this prevents acclimatization on the part of the cell.. Figure 6 gives the results of a

pilot experiment where cells are placed in the e .m. field for 15 minutes, removed for

48

4-5s

r

45s

r

5

18a

V

18s

V

10

28s

28s

V

154

154

Fraction Number

Fig. 6. Effect of discontinuous exposure of cells to e .m. fields

. Cells were placed in the SP 17 field for 15

minutes, removed for 30 minutes and placed again in the e.m- field for 15 minutes. The incorporation

pattern is shown by closed triangles (A).The pattern should be compared to that seen for 15 minutes in

the field and immediate extraction of the RNA (open circles, 0) or 60 minutes in the field(0). The

pattern of transcription in RNA from cells which were not exposed to e.m. fields is indicated by the open

triangles (a).Note that the effect of removing the cells from the field during the 60 minutes time period

resulting in a higher incorporation of 3H-uridine into RNA than that seen for RNA of cells exposed

continuously over a 60 minutes time period. All conditions are as given in Fig . 4

.

5

10

Fraction Number

Fig . 7. Transcriptional patterns during recovery from exposure to e .m. fields. Cells were

placed in the SP

17 field for 15 minutes, then allowed to recover in the absence of isotope or field stimulation for 0

minutes (0), 25 minutes(0) and 45 minutes (a) . This experiment indicates that the transcripts induced by

the e.m. field are stable for a period of at least 45 minutes

. Other conditions are as given in Fig . 4.

30 minutes and replaced in the e .m. field for an additional 15 minutes . The rate of

incorporation into RNA transcripts is significantly higher under these conditions

than under those of 60 minutes consecutive time in the single pulse field. We have

also examined recovery following various time periods in the SP 17 field . The

experimental data, presented in Fig. 7, demonstrates that the response to the field is

retained by the cell for at least 45 minutes in the absence of further e .m. stimulation .

Comparison of signal types

E #33 is the most effective signal with repetitive pulse burst characteristics that

we have tested (see Table 1). This signal induces a pattern of isotope incorporation

into RNA that is similar to that seen following stimulation with SP signals (Fig . 8) .

The level of 3 H incorporation, however, is much lower. Other PB signals tested

either gave a negligible response to the presence of e .m. fields, or a very slow, but

significant increase in transcriptive activity up to 60 minutes in the signal ; this is in

contrast to the single pulse signal where a dramatic increase in transcriptional

activity is seen at 15 and 45 minutes time points .

The parameters that constitute clinically effective PB signals are not well defined .

A comparison, however, can be made between various parameters of all PS signals

tested with the two PB signals for which we have the most complete data . First, the

magnitude of B-field in E #33 is 4.8, in PB 15, it is 19 . Both these PB signals are

15

49

5

10

Fraction Number

Fig . 8 . Incorporation of 3 H-uridine into RNA following exposure to signal E33 . The open circles are the

pattern seen in cells exposed to the signal for 15 minutes ; solid circles indicate the pattern from cells

treated under identical conditions, but shielded from the signal. Conditions are given in Fig . 4 .

so

effective signals with respect to RNA induction . Since other biologically active

signals were found within this range, the effectiveness of the PB signal cannot be

directly related to gauss values . Positive width, another waveform parameter, varied

from 175 s to 250 s . The most effective signals in our hands, E #33 and PB 15,

respectively, were at the outside minimum and maximum limits of positive width in

the signals tested. With respect to burst width, the most effective signals were also at

the outside limits. Repetition rates varied from 1 .5 Hz to 50 Hz, but no direct

relationship was apparent with respect to effectiveness of a signal . In point, the most

effective PB signal had the lowest repetition rate . Asymmetric characteristics of

negative spike and negative space are also unrelated . We are presently testing signals

with a single variable, that of frequency in order to simplify analysis of the PB

signals. On the basis of transcription autoradiography, the results from exposure to

PB signals indicate that, in contrast to SP signals, the cell is responding to a different

signal parameter in PB signals as compared with SP signals, i .e ., the chromosomes

exhibit different grain densities and distribution depending on which e .m. field is

used. Further experiments should determine if this is the case .

SP and PB are both quasi-rectangular asymmetric e .m. signals . It has been

presumed until now that the asymmetry of the waveshape is a very important factor

in the effectiveness of the clinical response. To address the issue of waveshape, we

have tested the simple symmetrical sinusoidal wave using a frequency component of

18s

28s

T

r

5

10

15$

Fraction Number

Fig . 9 . Incorporation of3H-uridine into RNA of cells exposed to the Sine 72 Hz signal

. The cells

remained in the signal for the time periods indicated . Note that the incorporation is proportional to time

up to 45 minutes. The scale has been expanded from fraction 9-15 in order to illustrate better the second

transcriptional peak at approximately 20-255 .

20

4-5s

r

18s

V

28s

V

45 min

72 Hz-o

222 Hz-o

4400 Hz -A

5

10

Fraction Number

Fig. 10 . Radioactive RNA transcripts from cells exposed to Sine waves of various frequencies for 45

minutes . Patterns from cells exposed to (0) Sine-72 Hz ; (tr) 222 Hz ; (n) 4A kHz. Conditions are as

described in Fig. 4. Graph has been expanded to allow better definition of second transcriptional peak at

approximately 20-25S .

72 Hz (which is the overall repetition rate of the clinically effective single pulse

signal). Figure 9 illustrates that the transcriptional response using the sinusoidal

waveshape at 72 Hz is virtually identical to that observed using asymmetric SP (Fig .

4). Sinusoidal signals with other frequency components have also been investigated

(Fig. 10). When sine waves of 72 Hz, 222 Hz and 4400 Hz are compared, the

frequency component is inversely proportional to signal effectiveness . In general, the

relative rate of induced transcriptional activity is 72 Hz > 222 Hz > 4400 Hz or as

measured magnetic fields, 1 .15, 0.37 and 0.018 mTesla (mT).

Although we have compared e.m. signals with distinct differences in waveform

characteristics, the feature or features which constitute an effective signal are still

unknown, i.e ., although there appears to he some dependence on frequency and

pulse width, no direct correlation between transcriptional induction and a particular

waveform characteristic can be made on the basis of present data . Within the various

single pulse (SP) signals, however, the signals with the highest efficiency with respect

to 3 H incorporation are those with repetition rates of 72 Hz and a positive width of

380 s. These signals also had higher B-field values and increased negative spikes .

Translational patterns following exposure to e, m . signals

The most direct effect of transcriptional induction of RNA is expected to be

translational alterations . Patterns of protein synthesis were compared between

15+

52

normal unstimulated salivary gland cells and those exposed to either SP and PB

fields for varying periods of time up to 60 minutes . The results of analyses of many

one dimensional gel electrophoretic patterns show that the only distinct differences,

seen to date, are quantitative, rather than qualitative. Protein patterns from cells

stimulated by SP and PB and separated by 2-dimensional gel electrophoresis have

been analyzed .

Data has been analyzed from two-dimensional polyacrylamide gel electrophoresis

of polypeptides from Sciara salivary gland cells exposed to three types of pulse

asymmetric signals (SP17, PB-15 and E-33) as well as the symmetrical sine wave at

72 Hz. Both quantitative and qualitative changes occur in translational patterns of

cells exposed to these e .m. signals. Differences in polypeptide profiles are signal-

specific ; e.g., SP 17 induces 22 polypeptides that are specific to this signal, whereas

17 new polypeptides were found in cells exposed to RPB-15 . New and augmented

polypeptide synthesis is seen as well as the suppression and absence of polypeptides

in all cases as compared with unstimulated control samples [291 . Only 3 polypeptides

out of 294 polypeptides found in cells exposed to e .m. fields overlap those seen in

cells that have been heat shocked .

Is there a temperature effect during exposure to e .m. fields?

One facet of the e.m. field stimulation could theoretically be due to localized

temperature changes in the cell or media surrounding the cell . We have been unable

4-5$

Y

185

V

SP 6o Mns=0

.

HS 60 M

i

nsa

SP+ HS 60 MIns = A

28s

Y

5

15+5

10

Fraction Number

Fig. 11 . Effect of heat shock and e .m. fields administered in concert . Incorporation of 3 H-uridine into

RNA of cells exposed to heat shock at 37C while present in the SP 17 field . The open circles indicate the

RNA pattern of cells exposed to SP 17 only ; closed circles are RNA patterns from cells exposed to heat

shock only and the closed Mangles are the patterns from cells exposed to both types of environmental

stress. Time of exposure was 60 minutes . Other conditions are given in Fig 4 .

53

to detect temperature changes in the media using microprobes. In addition, all of our

experiments using Sciara salivary gland chromosomes have been compared to

studies which used heat shock [29]. These experiments demonstrate that transcrip-

tional induction occurs following heat shock, but the response is not detectable until

temperatures greater than 10C above normal growth temperature for Sciara of

20C are reached . (= 32C) . Studies with transcription autoradiography show that a

different subset of RNAs is induced in heat shock as compared with electromagnetic

fields . The most dramatic difference is seen in the induction of ribosomal RNA

synthesis following heat shock. Ribosomal RNA synthesis is noted in Sciara

chromosomes following 45 minutes of heat shock as determined by transcription

autoradiography and the appearance of nucleoli in the region of the rRNA locus . No

synthetic activity at this locus has been observed following exposure to electromag-

netic fields. It should be noted that a transcriptional response to heat shock is not

observed at temperatures below 32C . Since the transcriptional response of the

chromosomal DNA is different between these two forms of environmental stress, we

can tentatively conclude that the effect of electromagnetic fields iss not one which is

due directly to heat (see Fig . 11) .

DISCUSSION

Our research has studied the effects of many e.m. signals on dipteran eukaryotic

cells. Three distinctively different waveshapes were selected for the e.m. field

investigations based on their association with either clinical or environmental

exposures. Since cellular exposure to e.m. fields could result in myriad interactions

within the cell, we have concentrated on a single feature, that of transcriptional

induction, since transcriptional alterations are expected to reflect direct or indirect

response to cellular changes .

Our test for measuring signal capability can be directly correlated with results

obtained from clinical or experimental usage ; that is, signals with proven effec-

tiveness, clinically or experimentally, also give the requisite response with our model

system. Another factor has also been delineated, which relates to experimental, and

also possibly clinical, use of signals . When active e .m. fields are in close proximity to

unshielded nonstimulated controls, these cells will show a time dependent effect that

mimics e.m. stimulated samples after 15 minutes . Active signals should be used only

in areas where other external sources of electrical or electromagnetic fields are

absent (e.g., n.m.r., or heavy equipment), and cells should be shielded in some

fashion from outside influences .

The biological effect that results from a given e.m. signal must be analyzed in

terms of signal characteristics as well as a definable cellular response . We have

determined that transcription and presumably transcriptional regulation can be

affected by specific types of e .m. field stimulation . Analysis of the cellular response

of genomic activity will ultimately provide vital information on both the potential

usefulness and safety of exposure to e.m. fields . On a biological level, an understand-

ing of cellular accomodation to exogenous stimulation will be important in studying

5 4

regulatory mechanisms . It is anticipated that more sophisticated analyses in the

future, e .g., the development of techniques to measure one or a few mRNA species

produced in response to e.m. fields could lead to a correlation between a specific

waveform parameter and signal effect. There are other alternatives, however, which

are supported in part by the present data . These include :

(1) the cell may be responsive to e .m. fields in a generalized fashion, i .e ., any

perturbation or environmental stress will redirect transcription events ; and

(2) the cell may respond to several different factors within signals in an equiv-

alent fashion .

It must also be determined whether there are secondary signals being emitted

which elude detection by standard calibration devices .

A multivariate approach to investigation of various e .m. fields is important for

several reasons. Understanding biological response to the low frequency range of

e.m. fields is critical to the safety of the patient, as well as medical, industrial and

military personnel. Many other occupations involve direct exposure to e .m. fields .

e.m. fields have been implicated in positive clinical outcomes, as well as negative

environmental outcomes. Environmental exposure to e.m. fields has results in

reports as diverse as :

(a) pregnancy problems in video display terminal operators resulting from associ-

ated RF emissions ; these include birth defects and miscarriages [32] ;

(b) significant fetal loss among users of electric blankets [33] ;

(c) malformation in chick embryos with marked effects in fields at 100 Hz, 1 .2

microtesla [34] ;

(d) increased growth of tumor cells in experiments which mimicked exposure to

power lines [35] ; and

(e) danger to underwater flora and fauna resulting from the presence of under-

water communication systems [36] .

In the general environment sine waves in the frequency range of 72 Hz are found

associated with high tension wires and other carriers of electrical current . The fact

that transcription is increased under conditions where cells are exposed to sinusoidal

waves near this frequency does not necessarily imply a negative effect . It does

strongly suggest that the effects of environmental exposure should be more carefully

monitored. In light of increasing human exposure to e .m. fields, it is surprising that

more research has not been oriented towards determining the basic effects of e.m .

fields at the genomic level .

Combining molecular biology and electrochemistry is in its infancy. Much

research remains to be done before the full range of mechanisms behind the

electrical control of cell function can be delineated . Ultimately, it should be possible

to define what the cell sees in its ionic environment, as the result of either

endogenous or exogenous changes in electrical fields, and eventually, to determine

the specific frequency requirements for different biological effects . We stress that

understanding the magnitude and/or specificity of cellular responses to e .m. fields is

dependent on reducing the complexities inherent in experimental determinations .

Our experiments provide one approach ; i.e ., a single feature of the cell is followed

5 5

after exposure to signals of well-defined waveform characteristics. These types of

experiments will set the stage and define conditions for further investigations of

more complicated cellular responses.

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