Frequency sensitivity of nanosecond pulse EMF on regrowth and hsp70 levels in transected planaria

doi:10.4236/jbise.2009.24036

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J. Biomedical Science and Engineering, 2009, 2, 227-238

doi: 10.4236/jbise.2009.24036 Published Online August 2009 (http://www.SciRP.org/journal/jbise/

JBiSE

Published Online August 2009 in SciRes. http://www.scirp.org/journal/jbise

Frequency sensitivity of nanosecond pulse EMF on

regrowth and hsp70 levels in transected planaria

Ash Madkan1*, Avary Lin-Ye1, Spiro P. Pantazatos2, Matthew S. Geddis3, Martin Blank2, Reba Goodman1

1Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, USA;

2Department of Physiology and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, USA;

3Department of Science, Borough of Manhattan Community College-CUNY, New York, USA.

Email: rmg5@columbia.edu

Received 30 June 2009; revised 10 July 2009; accepted 15 July 2009

ABSTRACT

Purpose: To study the effect of time varying/

pulsing electromagnetic fields (PEMF) on bio-

logical systems by measuring regrowth and the

induction of elevated levels of the stress protein

hsp70 in the regenerative model Planaria Duge-

sia dorotocethala. Objective: The outcomes of

studies using electromagnetic fields (EMF) are

dependent on pulse design, field strength (mG),

frequency (Hz), duration and magnetic field/rise

time (dB/ dt). Standardization of effective pulse

design is necessary to avoid continuing confu-

sion in the investigation of pulsing electro-

magnetic field (PEMF) technology. Information

from studies on hsp70 protein induction and

regrowth in transected Planaria provides in-

formation on EMF efficacy for potential clinical

application in the treatment of ischemia reper-

fusion injury and the eventual inclusion of EMF

prophylaxis prior to surgery. Materials and

methods: Planaria were transected equidistant

between the tip of the head and the tip of the tail.

Individual head and tail portions from the same

worm were placed in pond water and exposed to

8, 16 or 72 Hertz PEMF for one hour daily post

transection under carefully controlled exposure

conditions. Regrowth of heads and tails was

measured in PEMF-exposed and sham control.

Protein lysates from PEMF-exposed and sham

control transected heads and tails were ana-

lyzed for hsp70 levels by Western blot analyses.

Conclusion: The degree of regrowth and hsp70

levels in transected heads and tails exposed to

nanosecond PEMF exposures at 8, 16 or 72 Hz

was frequency dependent. There are currently

several views on the interaction mechanism

involved in regrowth. Here we discuss two: in

one [7,8] we propose a direct effect on the DNA

of the PEMF consensus sequence, nCTCTn,

referred to as electromagnetic field response

elements (EMRE) in the promoter region of the

stress response gene HSP70. In the second

mechanism [28] it is proposed that EMF induce

vibrations of proteins through a series of quan-

tized low frequency phonon signals.

Keywords: Planaria; Nanosecond EM Pulse; hsp70

Protein; Regrowth

1. INTRODUCTION

Electromagnetic fields (EMF) of less than 1 Gauss

strength have been shown to induce a variety of specific

effects in cells and tissues over a wide frequency range of

the EM spectrum [7,8]. Nanosecond pulsing electromag-

netic field (PEMF) technology has assumed increasingly

greater importance in clinical treatments [15,29,70]. As a

measure of efficacy, we used the regenerative model Pla-

naria, to test three nanosecond PEMF devices on regrowth

of transected heads and tails and the induction of the stress

response protein hsp70 [42,43,44,45].

Previously we showed that ELF-EMF induce elevated

hsp70 protein levels through the responsiveness of a

specific consensus DNA sequence EMRE (electromag-

netic field response element, nCTCTn) on the heat shock

70 (HSP70) promoter [41,42,43,44,45]. This DNA do-

main is upstream from the heat shock domain. Further-

more, ELF-EMF induction of the hsp70 protein protects

cells and limits the effects of subsequent stress, includ-

ing sudden changes in temperature [12,30].

The degree of electromagnetic field-effects on bio-

logical systems is known to be dependent on a number

of criteria in the waveform pattern of the exposure sys-

tem used; these include frequency, duration, wave shape,

and relative orientation of the fields [6,29,32,33,39,40].

In some cases pulsed fields have demonstrated increased

efficacy over static designs [19,21] in both medical and

experimental settings.

Abbreviations: Extremely low frequency (ELF), electromagnetic fields

(EMF); nanosecond pulsing electromagnetic field (PEMF), heat shoc

rotein (hsp), heat shock gene (HSP), frequency (Hz), Gauss (G), mag-

netic field/rise time (dB/dt); mitogen-activated protein kinase (MAPK).

228 A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238

To examine the effects of PEMF on regrowth follow-

ing transcetion and the induction of hsp70 protein in

transected heads and tails, Planaria, a Platyhelminth,

were used as a model organism because of its recognized

ability to regenerate [55]. Previous studies showed that

EM sinusoidal fields enhance the first three to six days

of regeneration in Planaria and during this time specific

proteins in the MAPKinase cascade are activated [26].

Planarians regenerate through a well defined stem cell

system by restimulating mechanisms that guide the pat-

terning of body structures during embryonic develop-

ment [1,2,3,13,14,56,57,58,59]. Planaria are typically

able to regenerate into a full worm from any body part

within 14-16 days [55,32,33] thus providing a sensitive

model for examining how electromagnetic fields interact

with specific stages of regeneration.

The potential application of the upregulation of the

HSP70 gene by both ELF-EMF and nanosecond PEMF

in clinical practice would include trauma, surgery, pe-

ripheral nerve damage, orthopedic fracture, and vascular

graft support, among others. Regardless of pulse design,

EMF technology has been shown to be effective in bone

healing [5], wound repair [11] and neural regeneration

[31,36,48,49,51,63,64,65,66]. In terms of clinical applica-

tion, EMF-induction of elevated levels of hsp70 protein

also confers protection against hypoxia [61] and aid myo-

cardial function and survival [20,22]. Given these results,

we are particularly interested in the translational signifi-

cance of effect vs. efficacy which is not usually reported

by designers or investigators of EMF devices. More pre-

cise description of EM pulse and sine wave parameters,

including the specific EM output sector, will provide con-

sistency and “scientific basis” in reporting findings.

. MATERIALS AND METHODS

Planaria. Dugesia dorotocethala (Carolina Biological

Supply Company; cat. # 132950) were shipped overnight

and allowed to ‘recover’ for at least 24 hours in fresh

oxygenated pond water (Carolina Biological Supply Co,

NC, USA). Planaria were maintained in near darkness at

22 to 24o C (Precision Scientific incubator; Fisher Scien-

tific, NJ, USA) throughout the experiments.

2.1. Experimental Protocol

Planaria Dugesia dorotocethala were transected equidis-

tant between the tip of the head and the tip of the tail

(Figure 1). The following is the experimental protocol

used in these experiments:

1) Following transection (0 time) head and tail por-

tions were exposed to 8, 16 or 72Hz nanopulse for

one hour twice a day. Measurements of regrowth

were performed from 0 time to at least three day

intervals (n = > 10 experiments).

2) Following transection, heads and tails were ex-

posed to 8, 16 or 72 Hz up to 180 minutes. Samples

were removed at 20 minute intervals for protein ex-

traction and hsp70 analyses (n = >10).

3) Following transection, heads and tails were ex-

posed to 8, 16 or 72 Hz fields for one hour twice

per day over a 12 day time period. Ten head and tail

samples were removed daily and prepared for

hsp70 analyses. Experiments were repeated at least

three times.

Exposure protocol. In the experiments described here,

Planaria Dugesia dorotocethala were transected equidis-

tant between the tip of the tail and the head (Figure 2).

Each head and tail portion was photographed using a

Nikon digital camera mounted on a Wilde dissecting

microscope. Images were stored in a database for sub-

sequent measurements using ImageJ (see section on

Quantitation). Head and tail portions were placed in

Figure 1. Transection of Planaria. The image in the left panel shows an intact sham exposed planaria with head

facing up. The arrow indicates the transection point. The adjacent panels show representative images of heads

and tails from transected Planaria at days 0, 3, and 9. The images have been enlarged 16X to show detail.

A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238 229

250ns width @ 72 Hz

8 V

555 TIMER

Figure 2. EM Probe Device. This device is designed to generate fast magnetic field pulses of low duty cycle in the

near field region. The pulse length is about 250ns driven by a storage capacitor and a MOFSET switch that produces a

10A current in a field generating circuit element. The circuit element is just the single loop of the capacitor and switch

circuit and does not contain any additional coil or turns of a conductor to enhance the magnetic field strength. The

compact single loop design minimizes inductance and increases the speed of the circuit allowing a fast rise pulse pro-

ducing a maximum magnetic field of the order of one Gauss at about 1cm from the device. The field is concentrated in

a region of a few cm3 and falls off rapidly outside that region. The fast rise and fall of the current and magnetic field

pulse implies a broad spectrum of Fourier components contain within the repeated pulse of the waveform. For the

pulse rise time of about 10ns and a pulse frequency of 72Hz, those components extend from the fundamental fre-

quency at 72Hz at the low end to at least 20MHz at the high frequency end. The particular distribution of frequen-

cies is determined by the usual methods of Fourier analysis from the exact pulse shape. This wide range of fre-

quency offer resonant interactions with the biological mechanisms of the organism being treated over a very wide

range and could include molecular, cellular and multicellular level interactions.

separate Petri dishes (Falcon 351007 60 x 15mm; Fisher

Scientific, NJ, USA) containing pond water approxi-

mately 0.6cm deep. Dishes were numbered so that heads

and tails of the same worm could be identified for meas-

urements. Dishes were placed on a firm base and the

PEMF devices were attached to the dishes so that the

entire area of the dish was exposed to a uniform field for

one hour a day. All exposures took place within an incu-

bator with the temperature maintained at 22-24o C and

monitored with a thermocouple probe (sensitivity +/-

0.01oC; Physitemp, model BAT12, Hackensack, NJ,

USA). Growth was assessed at three-day intervals from

day 0 (immediately following transection and the onset

of PEMF exposure) to day 12 post-transection. In a se-

ries of separate experiments, pigmented eye spot devel-

opment was monitored at 12 hour intervals in transected

tails exposed to PEMF and sham control immediately

following transection.

The nanosecond PEMF device (EM-PROBE

Technologies) in Figure 1 generates a near field fast

magnetic pulse of 250 nanosecond (ns) duration, which is

driven by a circuit containing a storage capacitor and a

switch that produces a 10 A current in a field-generating

circuit element. The single loop circuit does not contain

any additional coils of a conductor to enhance the mag-

netic field strength, and allows a rapid dB/dt pulse design

producing a magnetic field of 1 Gauss approximately 1 cm

from the device. The pulse begins with a peak strength of

1.4-1.7 Gauss that deteriorates to zero in about 200ns. At

72Hz this means that there is an active field for 0.00072%

of the time. At 16 Hz there is an active field for 0.00016%

of the time. At 7.8Hz there is an active field for 0.000078%

of the time. The particular distribution of frequencies is

determined by Fourier analysis from the exact pulse shape.

This wide range of frequencies offers resonant interactions

with the biological mechanisms of the organism being

treated. The entire output circuit is optimized for rapid

response by minimizing inductance. The wide range of

frequencies offers resonant interactions with the biological

mechanisms of the organism being treated over a wide

range and might include molecular, cellular and multi-

cellular interactions.

Shielding: Both active (experimental) and sham- ex-

posed (control) samples were enclosed in 30cm high,

15cm diameter cylindrical Mu metal containers (0.040"

thickness) (Amuneal Corp. Philadelphia, PA). Detailed

measurements of background magnetic fields in the in-

cubator, harmonic distortion, DC magnetic fields and

mean static magnetic fields in the incubators were pre-

viously determined [35].

Protein lysates. In a separate series of experiments,

protein was extracted at defined time periods from the

transected heads and tails, both experimental and sham,

to determine effect of PEMF on hsp70 levels. Lysates

were prepared from the heads and the tails of exposed

and sham exposed Planaria for analyses of hsp70 [41, 42,

230 A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238

45]. Protein concentrations were determined by the

Bradford assay (Bio-Rad Redmond, WA, USA).

Western blot. Lysed samples containing 30µg of pro-

tein were subjected to sodium dodecyl sulfate gel elec-

trophoresis (Fisher Scientific, NJ, USA) on 10% poly-

acrylamide gels using appropriate molecular weight

markers (Santa Cruz Biotechnology Inc, Santa Cruz, CA,

USA) and the polypeptides were transferred to polyvi-

nylidene fluoride membrane for immunoblotting. Blots

were probed with anti-hsp70 antibody (1:10,000). The

blots were then stripped using SuperSignal West Pico Sta-

ble Peroxide Solution (Pierce, prod # 1859674, Fisher

Scientific, NJ, USA) and SuperSignal West Pico Luminol

Enhancer Solution (Pierce prod # 1859675, from Fisher

Scientific, NJ, USA) and reprobed with anti-actin (1:1000)

to confirm equivalent loading. Visualization was by the

enhanced chemiluminescent detection system (Fisher

Scientific, NJ, USA) as previously described [43].

Antibodies. The antibody to hsp70 was kindly provided

by Dr. Richard Morimoto, Northwestern University.

Statistical analysis: For determination of regrowth,

multiple high-resolution digital pictures of Planaria

heads and tails were taken at three day intervals and the

rate of regrowth quantified using ImageJ v1.38 (http://

rsb.info.nih.gov/ij). Length measurements were cali-

brated in millimeters and transposed to an Excel spread-

sheet for statistical analysis. Lengths were normalized to

the 0-time. Length differences over each 3-day interval

were then subjected to 2-sample t-tests to assess signifi-

cant differences in mean growth between the control and

exposed conditions for both heads and tails. An addi-

tional analysis was conducted in SAS v9.1 (www.

sas.com) to assess the significance of an interaction ef-

fect, i.e., whether the exposure had greater effect on ei-

ther heads or tails. For this a ‘mixed effects’ analysis was

performed which modeled the average length at 3-day

intervals as a linear sum of the fixed effects of exposure

(experimental vs. control), worm portion (heads vs. tails)

and their interaction.

For quantification of the hsp70 and beta-actin levels

the films from Western blot were scanned and saved as

digital images (Figure 3). The densities of the bands

were quantified using the histogram function in ImageJ

and values transposed to Excel for statistical analysis (2

sample t-tests). A minimum of three replications of each

assay were conducted.

2.2 Western Blots Quantitation of Hsp70

and β-actin Bands

Images from films were scanned into a computer and

analyzed with Image J v1.37 (NIH). The analyze func-

tion for gels was used to plot the spatial signal density

for each lane of hsp70 (see Figure 2). The same was

done for the β-actin controls (not shown). Figure 2 plots

the mean hsp70/-actin ratios for PEMF and Control con-

ditions. These values were imported into a Microsoft

Excel spreadsheet where the signal value of hsp70 was

divided by the value of the-actin signal in the same lane

in order to normalize against variable loading volumes.

-actin is a housekeeping gene and unaffected by EMF or

PEMF. Statistical analyses were also conducted in Excel

using the Data Analysis toolbox.

3. RESULTS

The extraordinary ability of Planaria to regenerate after

injury is attributed to the presence of totipotent neoblasts

capable of differentiating into all of the tissue types [1,

4]. Perhaps most impressive is their ability to regenerate

the nervous system. When transected the tail region

forms a new head complete with bilateral optic nerves

and eyes, a two-lobed brain, and a pair of ventral nerve

cords (VNC) [2,13,14].

3.1. PEMF Accelerates Rate of Regrowth of

Transected Planaria

To assess the efficacy of the nanosecond PEMFs on

Planaria regeneration, the length of ELF-EMF-exposed

and sham control heads and tails were measured at three-

day intervals starting immediately following transection

(Figure 4 A,B,C). The heads and tails of all transected

Planaria (experimental and control) regrew to normal

viable worms. The initial experiments used PEMF at

72Hz.to measure regrowth of transected heads and tails.

As previously demonstrated using ELF-EMF [26], ac-

celerated tail regrowth was significant at days 3 and 6

post transaction (p=0.05). In contrast to the effect of

exposure to72 Hz PEMF, 8Hz PEMF exposure on the

tail portion showed the greatest response to any of the

Figure 3. Images from films were scanned into computer and

analyzed with Image J v1.37 (NIH). The ‘analyze function for

gels’ was used to plot the spatial signal density for each lane of

hsp70. The same was done for the β-actin controls (not shown).

These values were imported into a Microsoft Excel spreadsheet

where the signal value of hsp70 was divided by the value of the

β-actin signal in the same lane in order to normalize against vari-

able loading volumes. β-actin is a so-called ‘housekeeping gene’

and unaffected by EMF or EMP. Statistical analyses were also

conducted in Excel using the Data Analysis toolbox.

A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238 231

frequencies to which the transected heads and tails were

exposed. The sham control samples in general showed

very little change in length over this time period (Figure

3B). A reduced response to the 16Hz PEMF (Figure 3C)

was measured in both transected heads and tails when

compared with 72 and 8Hz (Figure 3A, B).

EM Probe Regeneration - 72 Hz TAILS (normalized)

0.4

0.6

0.8

1.2

1.4

1.6

1.8

0346

Days

Ratio to Day

EM Probe 72 Hz

Control

Figure 4A. Regeneration of heads and tails following transection of Planaria. Histograms show-

ing the mean length of control and PEMF-exposed heads and tails immediately post-transection (0

time), and 3 to 9 days post-transection. (A) Mean length ± 2 standard error of the mean (SEM) of ex-

posed heads (HE, n=48) and sham exposed heads (HC, n=27) at each time. (B) Mean length ± 2SEM

of exposed tails (TE, n=20) and sham exposed tails (TC, n=12) at each time. The asterisks indicate a

significant statistical difference in the rate of regeneration between exposed and sham exposed heads

and tails assessed by 2-sample t-tests of mean difference in length.

A. Exposure to 72Hz accelerated tail regrowth was significant at days 3 and 6. post-transection [data

normalized against day zero (n=3)]. The effect of the 72Hz EM probe is `statistically significant

(p=0.05) at three and six days on the tail portion. The heads and tails of all transected Planaria (ex-

perimental and control) regrew to normal viable worms by 20 days. There was little or no measure-

able effect on regrowth in head portion.

EM Probe Regeneration - 8 Hz TAILS (normalized)

0.4

0.6

0.8

1.2

1.4

1.6

1.8

0234610

Days

Ratio to Day

EM Probe 8 Hz

Control

Figure 4B. In contrast to the effect of exposure to 72 Hz PEMF, transected tails exposed to the 8Hz EM

probe show a steady increase in length. The effect of 8Hz PEMF on the tail portion showed the greatest

response to any of the frequencies to which the transected heads and tails were exposed. The sham con-

trol samples and the head exposed samples showed very little change in length over this time period.

JBiSE

232 A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238

EM Probe Regeneration - 16 Hz HEADS (normalized)

0.4

0.6

0.8

1.2

1.4

1.6

1.8

03467912

Days

Ratio to Day 0

EM Probe 16 Hz

Control

Figure 4C. A reduced response to the 16Hz PEMF was measured in both transected

heads and tails when compared with 72 and 8Hz.

Of particular interest was the general trend of the re-

sponsiveness of the tail to the three frequencies tested as

compared to the head portions (Figure 4 A, B, C). The

third day after transection has been shown to be the time

of maximal ELF-EMF-stimulated growth of the tail [26].

During normal regeneration this is the developmental

stage when the neural networks are actively forming [13,

14] and the ventral nerve cords (VNC) are extending

from the head into the newly formed tail.

3.2. PEMF Activation of Hsp70 Associated

with Injury and Repair

A significant elevation in hsp70 levels was evident in tail

portions that were exposed to the nanosecond PEMF

using all three frequencies at 0, 20, 40, 60 and 80 min-

utes (Figure 5). We also looked at potential differences

in induction of hsp70 protein, comparing all separate

PEMF exposures to the sham control. Tails that were

exposed to 16Hz EM-Probe fields showed the highest

hsp70 production (p= .000007.91).

In terms regrowth, the 8Hz tails shows the quickest

response to PEMF (Figure 4B). The 72Hz also shows a

noted difference between exposed and unexposed worms

(Figure 4A). The 16Hz was too inconsistent in order to

draw any conclusions (data not shown).

We believe the accelerated regrowth above is consis-

tent with the role of hsp70 as a chaperone that monitors

the folding of proteins during repair. PEMF induction of

the HSP70 gene may result from events triggered by the

activation of ERK that utilize a unique consensus se-

quence (electromagnetic field response element; EMRE)

upstream from the heat shock consensus sequence [8,

9,10].

The increase paralleled the accelerated regeneration

noted above and is consistent with the role of hsp70 as a

chaperone that monitors the folding of proteins during

repair. Induction of the HSP70 gene may result from

events triggered by the activation of ERK that utilize a

unique consensus sequence (electromagnetic field re-

sponse element; EMRE) upstream from the heat shock

consensus sequence [42,47]. Stress response protein

hsp70 levels are elevated after exposure to PEMF [43,

44,45]. We next examined the effect of nanosecond

PEMF at 8, 16 and 72 Hz exposures on levels of this

protein. Earlier studied had shown that hsp70 levels

(Figure 5A,B) depicts mean hsp70/-actin ratios and 95%

CI for both 16Hz and 72Hz exposure conditions on

heads and tails respectively vs. their control counterparts.

With both frequencies we observed a spike in hsp70 lev-

els at 40 minutes which concurs with our previous data

[41,45] and as previously noted the hsp70 levels return

to normal levels by 120 minutes. [Mean hsp70/-actin

ratios for 16Hz EMP Heads (n=14), 16Hz EMP Tails

(n=14), 72Hz EMP Tails (n=17) and 72Hz EMP Heads

(n=13).] As expected, measurements of hsp70 during

regrowth showed little to no effect of exposures.

4. DISCUSSION

In the experiments reported here we examined nanosec-

ond PEMF, rapid dB/dt using 8, 16 and 72Hz EM probe

devices on induction of hsp70 levels and Planarian head

and tail regrowth post-transection. Our data show that

the induction of elevated hsp70 levels and regrowth fol-

lowing transection are frequency specific; tail portions

that regenerate nervous system, brain and eyes, are more

responsive to the EM probe device than head portions.

The effectiveness of time varying electromagnetic fields

on biological systems has been shown to be dependent

on pulse design; frequency, duration, magnetic field/rise

time (dB/dt) [29,30]. Measurements of EMF induced

A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238 233

Planaria EM Pulse Solo - 16 Hz and Control

0 20406080

Exposure Time (minutes)

Mean Hsp70 / beta-actin Protein Levels (arbitary units)

Control Heads

Control Tails

Exposed Heads

Exposed Tails

Figure 5A. Histogram showing a quantitative analysis of hsp70 protein levels from regenerating heads,

tails, and sham controls. The relative protein levels are expressed in arbitrary units based on the intensity of

the hsp70 protein band from the Western blots. The PEMF exposed tails show an obvious spike between 20

and 40 minutes. Mean hsp70/β-actin ratios for 16 Hz PEMF heads (n=14), 16 Hz PEMF tails (n=14), 72

Hz PEMF tails (n=17) and 72 Hz PEMF heads (n=13). The control groups for each sample had a sample

size equal to that of its corresponding PEMF group. Error bars represent 95% confidence intervals (

2*Standard Error).]

Planaria TAILS Hsp70 - Days

5000

10000

15000

20000

25000

30000

036

Days

Mean Hsp70 Levels

(arbitrary units)

8 Hz

16 Hz

72 Hz

Control

Figure 5B. Histogram showing a quantitative analysis of hsp70 protein levels from regenerating heads, tails,

and sham controls.[Mean hsp70/β-actin ratios for 16 Hz EMP heads (n=14), 16 Hz PEMF tails (n=14), 72

Hz PEMF tails (n=17) and 72 Hz PEMF heads (n=13)]. The control groups for each sample had a sample

size equal to that of its corresponding EMP group. Error bars represent 95% confidence intervals (

2*Standard Error).]

hsp70 levels and induction of post-transected re-growth

were used as markers for repair efficacy in this study.

Standardization of effective pulse design is necessary to

avoid continuing confusion in the investigation of puls-

234 A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238

ing electromagnetic field (PEMF) technology. The data

from hsp70 protein induction and regrowth in Planaria, a

regeneration model, as a measure of EMF efficacy, sug-

gest excellent opportunity for clinical use in treatment of

ischemia reperfusion injury and the inclusion of EMF

prophylaxis prior to surgery. Our focus in these experi-

ments was on: (a) regrowth of head and tail portions

following transection, (b) immediacy of hsp70 induction

during regeneration of heads and tails, (c) levels of

hsp70 elevation during regeneration of heads and tails

and (d) duration of hsp70 elevation. We have shown that

the tail portion of the transected Planaria is more sensi-

tive to different frequencies both in the induction of

hsp70 levels and in regrowth. Furthermore, these

changes in tail metabolism occur at different times de-

pending upon the frequency used in the exposure.

4.1. Interaction Mechanisms

How weak electromagnetic (EM) fields interact with

DNA to stimulate protein synthesis remains unknown;

EMF interaction with cells and tissues has been exam-

ined in a variety of in vivo and in vitro systems including

enzyme reactions [10], Drosophila [25,27,72], yeast [71]

cultured cells [23,24,53] and on regeneration in Planaria

[26]. Currently no definitive mechanism exists to explain

how EMF interact with DNA and proteins in cells and

tissues, although several theories have been proposed

including cyclotron resonance [40], and a modification

of this idea [6,37].

There is some evidence that EM fields can affect

DNA, both directly and indirectly [9]. We have shown a

unique DNA sequence in a specific domain on the

HSP70 promoter that is sensitive to EM field exposures

and this observation has contributed to the understanding,

in part, of how ELF-EMF affects biological systems.

There are specific regions on the promoters of some

genes, HSP70 and c-myc for example, that contain this

consensus sequence and when this sequence is trans-

fected into promoter of reporter genes, these genes be-

come sensitive to EMF, whereas before they were not.

[41,42,43,44,45]. EM fields initiate up regulation of the

HSP70 gene, increase mRNA transcripts and hsp70 pro-

tein levels. The responsiveness is dependent on the

number of copies of the nCTCTn consensus sequence.

[42]. Electromagnetically, responsive elements act as

‘sensors’. This is an HSF-1 dependent process as shown

by electrophoretic mobility shift assays (EMSA) of pro-

tein extracts from HL60 cells exposed to EMF. HSF1-

DNA binding activity was demonstrated by a super-

shifted band. The magnetic field-inducible heat shock

element-binding activity is HSE sequence-specific and

contains HSF1 [45].

Because of the demonstrated effect of EMF on elec-

tron transfer reactions, it has been proposed that dis-

placement of electrons in the H-bonds that hold DNA

together can lead to DNA chain separation, thus initiat-

ing transcription [7,8]. The resultant charging due to

electron displacement on the dynamics of DNA chain

separation suggests that electron transfer could favor

separation of base pairs e.g. nCTCTn, with the EM field

sensitive DNA sequence acting as the first order iterative

mechanism. In the case of Planaria the DNA in the toti-

potent stem cells created by the injury may be respond-

ing directly to the EM fields. It has been shown that

DNA-mediated charge transport and the oxidative dam-

age that results are extremely sensitive to variations in

the sequence and conformational-adaptive response

leading to stacking of the intervening bases [18,62].

Protein-binding to DNA, for example, through one or

more of the MAPKinase cascades, modulates long-range

charge transport negatively and positively depending

upon the specific protein DNA interactions in play

[52,54,68,69].

A possible mechanism for EMF initiation of protein

synthesis is by acting on the de-localized π electrons in

the base pairs that hold the DNA strands together. This is

one way in which the effect of EMF in ELF range can be

due to the resulting current. The charging of molecular

complexes has been shown to lead to their disaggrega-

tion [10], so local charging would be expected to cause

the DNA strands to disaggregate. A possible sequence of

steps may start with EMF displacing electrons and

charging DNA segments, followed by disaggregation of

DNA strands as a result of the charging.

The properties of the CTCT bases suggest that they

may be involved in the first step in a molecular mecha-

nism for EMF activation of protein synthesis. They

have low electron affinities, so electrons would be more

easily displaced by the EMF. Also, the CTCT are

pyrimidines, and when the H-bonds split between CTCT

and the GAGA (purines) bases on the complementary

chain, the smaller area that results would require less

energy, and make the disaggregation more favorable [7].

Two recent studies of molecular properties of DNA

add support to this proposed mechanism of DNA disag-

gregation. One paper compared the lifetimes of excita-

tion induced by ultraviolet (UV) light in different DNA

structures [60]. Apparently, the induced fluorescence had

significantly longer lifetimes in DNA chains composed

of A and G bases than in chains with C and T bases. The

critical non-thermal stress protein sequences, the CTCT

pyrimidines, were found to have the shortest UV stimu-

lated fluorescence lifetimes. Their conjugate GAGA

purines were found to have lifetimes that are approxi-

mately an order of magnitude longer.

Fluorescence lifetimes are measured in the picosecond

time range, a relevant time scale for molecular rear-

rangements. Since the fluorescence lifetimes relate to the

properties of the excited molecule and not to what

caused the excitation, the measured UV excitation life-

A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238 235

times in the picosecond range also applied to lifetimes of

perturbations due to EMF. Therefore, the studies suggest

that the conjugate segments on the DNA strands will

retain energy for significantly different lengths of time.

The shorter lasting perturbation is evidence of more

rapid dissipation of energy through collisions and a

greater likelihood of reaching a breaking point, while the

rest of the chain is a being held in place by the conjugate

strand.

A second paper [46] on the flexibility of the double

DNA helix found that stretching fluctuations were much

larger than expected and extended over two turns of the

DNA helix (over 10 base pairs). The double helix is ap-

parently much more flexible than would be expected

from the multiple H-bond links between the two strands,

and mechanical stresses are probably more easily trans-

mitted along the strand. The flexibility of the DNA dou-

ble helix would make the local unraveling of the two

strands proceed more easily once it starts.

These recently described properties of the DNA dou-

ble helix support the idea that CTCT sequences are

likely to play a greater role in DNA strand separation

leading to initiation of transcription. Similar responses

can occur with other base sequences, but they would

require greater energy and are less likely to occur at

lower frequencies. At higher frequencies, electron

movements are apt to be induced all along the DNA

strands.

Another approach to mechanism has been proposed

by Gordon [28]. According to this model DNA function

is controlled by acoustic signals (phonons), which can be

enhanced via stochastic summation, i.e use of back-

ground. ‘noise’ if required, to complete the signal at the

necessary strength [28]. This idea proposes that protein

iterative activities account for enzyme activation, chan-

nel completion, and other functions necessary for cell

homeostasis, and have been an integral part of electro-

magnetic responsive elements nCTCTn. Thus the sug-

gestion that paramagnetic/diamagnetic transduction

(damping) is a first order mechanism in this dynamic

centered around the Schrodinger equation [to create a

binary phonon signal series is extant in the literature [28].

According to this theory, beta sub-units direct protein

conformational adaptation in response to acoustic signal

series to complete an EM driven control circuit capable

of directing oxidation-reduction reactions (Ubbink et al.

[67]. An alternate mechanism to variations on cyclotron

resonance has been suggested by [16,17]. Gordon [28]

and Panagopolus [50] suggest that EM fields may act as

classical “forcers” in a resonance system with paramag-

netic/diamagnetic oscillators that “damp” the EM field

via transduction into a normal mode or elementary pho-

non compatible with the intrinsic design and length of

the protein. This hypothesis suggests that displacement

of electrons in the H-bonds that hold DNA together

leading to DNA chain separation and initiating transcrip-

tion are reflections of a phonon resonance/iterative

process.

4.2. Signaling Proteins

How does all this work? In the case of Planaria the DNA

in the totipotent stem cells created by the injury may be

responding directly to the EM fields. DNA-mediated

charge transport and the oxidative damage that results

are extremely sensitive to variations in the sequence and

conformational-adaptive response leading to stacking of

the intervening bases [18]. Protein binding to DNA, for

example, through one or more of the MAPKinase cas-

cades, may modulates long-range charge transport nega-

tively and positively depending upon the specific protein

/DNA interactions in play [52,54].

Increased activation of the MAP-Kinase signaling

pathways and the specific protein binding activities that

normally occurs prior to upregulation of gene expression

are increased by EMF [26,34,38,72]. Protein transcrip-

tion factors in the p38 MAPK pathway are reported to be

involved during both ELF and RF exposures [38]. In-

creased phosphorylation of specific transcription factors

entering the nucleus, bind to specific regions of the rec-

ognition sites on the promoter and are essential for the

initiation of transcription.

4.3. Potential Biomedical Applications

EM field exposures to induce hsp70 expression offer

non-invasive methods to provide cytoprotection before,

during and following surgical procedures or in areas of

highly predictable trauma, e.g. combat, contact sports. In

terms of potential biomedical applications of EM fields,

it is intriguing to consider the data presented here to-

gether with our previous reports on cytoprotection and

potential for gene therapy using a known specific DNA

sequence that is responsive to EM fields, e.g. NGF,

HSP70.

The stimulation of repair is a documented biological

effect of EM fields [5]. However, some of the variability

in results obtained may possibly be related to different

responses to repair in different tissues, the exposure

protocols employed and specific pulse parameters [29].

With modern lightweight designs prophylactic applica-

tions are technologically feasible, and could result in

more rapid repair. Re-exposure at specific intervals

would maintain the increased levels and provide in-

creased protection.

Treatment with EM fields has been shown to protect

and enhance injury repair in ischemia reperfusion injury

[22] and enhanced regeneration of injured sciatic nerve,

in both examples, after EM field exposure was discon-

tinued [36]. Post-EM field exposure (60Hz, 80mG sine

wave) of as little as 40 minutes, induces elevated hsp70

levels that were found to remain elevated for more than

236 A. Madkan et al. / J. Biomedical Science and Engineering 2 (2009) 227-238

3 hours and remained capable of re-stimulation to the

same or higher levels using a higher or lower field

strength [8,23,24]. Gordon [28,29] suggests that para-

magnetic/diamagnetic dynamics in natively responsive

elements generate quantum signal series that control

conformational adaptation of proteins including DNA,

enzymes and membrane proteins. He notes that identifi-

cation of electromagnetic response elements in DNA

was an important first insight and has proposed that

DNA and especially promoter areas are highly respon-

sive to these signal series. Dennis and Goodwin [19]

examined PEMF technologies to define pulse bioeffi-

cacy and reported dB/dt or “rise time” as the critical de-

terminant within the PEMF technologies they examined.

They went on to note, “rectangular pulses with rapid

dB/dt were up to four times more bio-effective in stimu-

lating classes of genes associated with tissue restoration

following trauma compared to DC, sine wave or milli-

second designs”. Our findings tend to corroborate that

assessment, but also note the importance of specific fre-

quencies within the parameters of our study.

4.4. Summary and Conclusions

In the experiments reported here, the flatworm Planaria

Dugesia dorotocethala was used as a model system to

assess whether frequencies that are multiples of eight

induced regrowth and levels of stress response proteins

(hsp70) quicker and with longer duration of effect when

compared to a sinusoidal signal. There is good evidence,

as previously suggested by Dennis and Goodwin [19]

that the critical design determinant in pulsed electro-

magnetic field technologies in terms of bioefficacy is the

dB/dt or “rise time” in such designs and that rise time

bioefficacy could be based upon electromagnetically

responsive elements present in proteins, which would

imbue the protein with highly sophisticated abilities to

select harmonics necessary for homeostasis. The sig-

nificance of effect vs. efficacy is largely avoided among

designers and investigators of PEMF devices. The fail-

ure to address this question has resulted in highly vari-

able results and criticism that PEMF technology lacks

“scientific basis”. This paper is the first in an attempt to

discuss and evaluate specific frequencies in the ELF-

EMF range.

5. ACKNOWLEDGEMENT

Dedicated in loving memory to Abraham J Kremer and Glen A

Gordon. We are grateful to Janet Wilson of Carolina Biologi-

cals and Eve Vagg, Department of Photography and Illustration

for their dedicated help.

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