Lawson Innovation Prize

Real-time methodologies for the measurement of

biological effects of static and pulsed magnetic fields
on cell suspensions

Jeffrey J.L. Carson1,2, Michelle Belton1 and Frank S. Prato1,2

1ImagingProgram, Lawson Health Research Institute, London Ontario
Canada, N6A 4V2
2Department of Medical Biophysics, Schulich School of Medicine and
Dentistry, University of Western Ontario, London Ontario Canada

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 Lawson Innovation Prize

Background

In 1979, an epidemiological report by Wertheimer and

Leeper suggested an association between childhood
leukaemia and residential power distribution lines.
In 1993, Feychting and Ahlbom observed that this
association may be related to the historical pattern of
magnetic field exposure
This apparent link between cancer in humans and
exposure to magnetic fields resulted in much debate
within the scientific community.

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 Lawson Innovation Prize

Motivation

To establish a biological basis for the epidemiological

results, many scientists investigated the short-term
effects of higher intensity (up to 200 mT) magnetic fields
on cells in vitro (e.g. Aarholt et al., 1981; Dixey and
Rein, 1982; Goodman et al., 1983; Goodman and
Henderson, 1988; and Liboff et al., 1984).
These and many other reports suggested that Ca2+-
dependent biological processes and gene expression
are affected by magnetic fields.

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 Lawson Innovation Prize

[Ca2+]c
Early work examined the effect of magnetic fields on cytosolic
free calcium in HL-60 cells (Carson et al., AJP, 1990).

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 Lawson Innovation Prize

Real-time [Ca2+]c measurements

Motivated by this effect we

constructed a system to
measure cytosolic free
calcium in real-time
(Carson & Prato, RSI,
1996).

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 Lawson Innovation Prize

Outcome
However, using this real-time system, robust and reproducible
effects of magnetic fields were not observed.
• Cytosolic free Ca2+ in HL-60 cells during exposure to extremely low frequency gradient
magnetic fields. Carson JJL, PhD Thesis (Chapter 5; 1995):
• Dependence of cytosolic free Ca2+ in a human cell line on frequency and field strength
during exposure to a sinusoidal magnetic field. Carson JJL, PhD Thesis (Chapter 6;
1995).
• Factors confounding cytosolic calcium measurements in Jurkat E6.1 cells during
exposure to ELF magnetic fields. McCreary CR, Thomas AW, Prato FS.
Bioelectromagnetics. 23(4):315-28 (2002).
• Real-time measurement of cytosolic free calcium concentration in Jurkat cells during
ELF magnetic field exposure and evaluation of the role of cell cycle. McCreary CR,
Dixon SJ, Fraher LJ, Carson JJL, Prato FS. Bioelectromagnetics. 27(5):354-64 (2006).

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 Lawson Innovation Prize

Latest Research

Fast-forward to present day...

The availability of miniature high-intensity

magnetic field generators and genetic
reporters has provided an opportunity to
re-examine basic questions in
bioelectromagnetics.

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 Lawson Innovation Prize

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 Lawson Innovation Prize

Outline

Study I Real-time cytosolic free Ca2+ measurements at

high flux density, i.e. > 50 X the previous work (100
mT).
Study II Real-time monitoring of heat shock protein
(HSP70) during magnetic field exposure using
bioluminescent reporter technology.

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 Lawson Innovation Prize

Study I
Effect of Static a Magnetic Field on
Cytosolic Free Calcium Concentration
Real-time fluorescence measurements

1. Real-time measurement of cytosolic free calcium concentration in HL-60 cells during

static magnetic field exposure and activation by ATP. Belton M, Commerford K, Hall J,
Prato FS, Carson JJ. Bioelectromagnetics. 29(6):439-46 (2008).
2. Real-time measurement of cytosolic free calcium concentration in DEM-treated HL-60
cells during static magnetic field exposure and activation by ATP. Rozanski C, Belton
M, Prato FS, Carson JJ. Bioelectromagnetics. 30(3):213-21 (2009).
3. Effect of 100 mT homogeneous static magnetic field on [Ca2+]c response to ATP in
HL-60 cells following GSH depletion. Belton M, Prato FS, Rozanski C, Carson JJ.
Bioelectromagnetics. 30(4):322-9 (2009).

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 Lawson Innovation Prize

Background - Calcium

• Calcium is involved in a number of signal

transduction pathways
– Often involving ATP
• Cellular signaling results in changes in
cytosolic free calcium concentration
([Ca2+]C)

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 Lawson Innovation Prize

Background – Magnetic Field

• SMF have been shown to affect [Ca2+]C

• Rosen AD. 1996. Biochim Biophys Acta 1282;149-55
• Radical pair mechanism offers a possible
explanation for SMF effects
• Manipulation of antioxidant levels (Glutathione
(GSH)) may increase probability of observing a
biological effect of SMF

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 Lawson Innovation Prize

Objective

• To determine if [Ca2+]C (resting and ATP) is

affected by depletion of GSH by
diethylmaleate (DEM) in the absence or
presence of a 100mT SMF.

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 Lawson Innovation Prize

Methods – SMF exposure system

5x106 HL-60 cells loaded
with Fura-2 AM and placed
in a disposable cuvette

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 Lawson Innovation Prize

Methods – Real-time Data Trace

•DEM: 1mM-15mM
•ATP: 1µM
100mT
Counts[1/s]

DEM

ATP
EGTA

BR-A23187

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 Lawson Innovation Prize

Results – Effect of DEM on [Ca2+]C

Representative Time Trace n = 8, p<1x10-6

(nM)

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 Lawson Innovation Prize
Results – Effect of DEM/SMF on [Ca2+]C response
to ATP

Measurement

n=8

Effect of [DEM] – p<1x10-6

Representative time traces.
Effect of 100mT SMF p>0.05
No SMF

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 Lawson Innovation Prize

Results – Effect of SMF on FWHM

N=7–8

Effect of [DEM] – p< 8e-8

Effect of 100 mT SMF – p>0.05

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 Lawson Innovation Prize

Conclusions – Exposure to DEM

• A 1500s exposure to DEM significantly

increases the [Ca2+]C in a dose-dependent
manner.
• Exposure to DEM significantly decreases the
size of the Ca2+ response following addition
of 1µM ATP.
• Observations most likely resulted from
emptying of intracellular stores due to GSH
depletion.

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 Lawson Innovation Prize

Conclusions – DEM + SMF

• There was no significant effect of a 100mT

SMF and DEM on the peak [Ca2+]C response
to ATP
• There was no significant effect of a 100mT
SMF and DEM on the FWHM of the [Ca2+]C
response to ATP
• Therefore, exposure to a SMF in
combination with an increase in free radicals
did not influence resting or activated [Ca2+]c

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 Lawson Innovation Prize

Outline

Study I Real-time cytosolic free Ca2+ measurements at

high flux density, i.e. > 50 X the previous work (100
mT).
Study II Real-time monitoring of heat shock protein
(HSP70) during magnetic field exposure using
bioluminescent reporter technology.

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 Lawson Innovation Prize

Study II

Effect of a Magnetic Field on Heat Shock Proteins

Real-time Bioluminescence measurements

The effect of 100 mT SMF on activation of the hsp70

promoter in a heat shock/luciferase reporter system.
Belton M, Rozanski C, Prato FS, Carson JJ.
J Cell Biochem. 108(4):956-62 (2009).

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 Lawson Innovation Prize

Heat Shock Proteins

• Act as chaperones and to promote proper protein

folding
• cytoprotective
• Some heat shock proteins (HSPs), including Hsp70,
are produced in response to a number of cellular
stresses.
• Heat
• Oxidative stress

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 Lawson Innovation Prize

Oxidative Stress

• Use DEM to deplete GSH as in Study I

• A number of groups have found that modulating the

concentration of GSH in the cell alters Hsp70 levels.

•S. Guo, et al. Cell Stress Chaperones 12 (2007) 245-54.

•W. Hassen, et al. Toxicology 232 (2007) 294.
•J.W. Park, et al. Neurosci Lett 413 (2007) 260.

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 Lawson Innovation Prize

Magnetic Field Effects

• Exposure to magnetic fields (MF) can alter levels of HSP701.

• Most evidence in power line frequency EM fields
• Effect depends on a number of variables
• Synergistic effects observed when application of MFs are
combined with a primary stress such as heat exposure2,3.
• Evidence that MFs influence chemistry involving free radicals
• This influence most commonly observed with static magnetic
fields (SMF)

1. Jung et al. (2007)

2. Junkersdorf et al. (2000)
3. Tokalov and Gutzeit (2004)

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 Lawson Innovation Prize

Objective

To analyze the amount of bioluminescence produced by a

Hsp70/luciferase reporter system in order to evaluate the
possibility of a synergistic effect of exposure to a 100 mT
static magnetic field (SMF) following A) cellular GSH
depletion, B) exposure to heat or C) a combination of GSH
depletion and heat.

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 Lawson Innovation Prize

Methods
• Stably transfected NIH3T3 cells (C.Contag, Stanford)
• Hsp70/luciferase
• Cells trypsinized, suspended in DMEM
• 0.75 mg D-luciferin
• Placed in exposure system

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 Lawson Innovation Prize

Timing - Exposure

Heat SMF 42°C

DEM

37°C

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 Lawson Innovation Prize

Timing - Measurements

Average Slope
Heat SMF
Measurement times
i ii iii iv
i = Pre-field
DEM ii = Early field
iii = Late field
iv = Post field

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 Lawson Innovation Prize

Slope of the Curve – No Heat

0.25

Before Field
0.2 Early Field
Photon counts / s2

Late field

0.15 Post Field

0.1

0.05

0
C D FD F
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 Lawson Innovation Prize

Slope of the Curve – Heat

70
Before Field
60
Early Field
50
Photon counts / s2

Late Field
Post Field
40

30

20

10

0
H HD HDF HF
-10

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 Lawson Innovation Prize

Statistical Results
No Heat Heat
Av. Slope (p - value) Av. Slope (p - value)
100 mT SMF 0.42 0.75
Pre-Field 1 mM DEM *0.02 * < 0.01
Interaction 0.44 0.95
100 mT SMF 0.54 0.66
Early Field 1 mM DEM 0.16 * < 0.01
Interaction 0.58 0.83
100 mT SMF 0.14 0.62
Late Field 1 mM DEM * 0.03 *< 0.01
Interaction 0.53 0.77
100 mT SMF 0.12 0.57
Post Field 1 mM DEM * 0.02 *< 0.01
Interaction 0.78 0.71
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 Lawson Innovation Prize

Summary
• No effect of a 100 mT SMF at any time point with or
without heat
• Significant reduction in BL for 1 mM DEM at all time
points except no-heat early field.
• Depleting GSH lowers hsp70 levels
• No interaction between SMF and DEM under any
conditions

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 Lawson Innovation Prize

General Conclusions

Studies I and II
• GSH depletion significantly alters [Ca2+]C and
HSP70 levels
• A 100 mT SMF does not alter [Ca2+]C or HSP70
levels

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 Lawson Innovation Prize

Quality of Results

 Positive controls
 For calcium measurements: DEM, ATP
 For bioluminescence measurements: heat

 Performance of measurement systems is as expected

 Fura-2 signals had good dynamic range (calibration)
 Luciferase bioluminescence had large dynamic range

 Intrasample (pre-, exposure, post-) Vs. Intersample groups

(sham and magnetic field) provided confidence in results

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 Lawson Innovation Prize

Potential Pitfalls of Real-Time

 Ever more precise measurement techniques lead to

sensitivity to seemingly smaller and smaller effects
 Real effects
 Effects due to sample handling
 Effects due to sample ordering
 Effects due to subtle protocol differences
 Effects due to other unidentifiable systematic errors

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 Lawson Innovation Prize
Future Approach:
Dual Real-time Measurements

• Originally championed by Dr. Jan Walleczek in the mid

1990’s
• Construct two exposure systems capable of
simultaneous readout of a biologically relevant metric
• Enables usage of identical starting material in each
setup
• Improved sham Vs exposure pairing
• Combined with real-time measurements could provide
a powerful way to observe biological effects from
EMFs.

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 Lawson Innovation Prize

Thank you

jcarson@lawsonimaging.ca
www.lawsonimaging.ca

BEMS 2011 – Halifax, Nova Scotia, Canada

Dalhousie University
June 12-17, 2011
www.bioelectromagnetics.org
www.bems.org

Student travel awards

Best student paper awards
Best journal Paper Award
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