Bioelectromagnetics 31:649^655 (2010)
Reduction of the Earth’s Magnetic Field Inhibits
Growth Rates of Model Cancer Cell Lines
Carlos F. Martino,1* Lucas Portelli,1 Kevin McCabe,2 Mark Hernandez,2 and Frank Barnes1
1
Electrical, Computer, & Energy Department, University of Colorado, Boulder, Colorado
2
Civil, Environmental, & Architectural Engineering, University of Colorado, Boulder,
Colorado
Small alterations in static magnetic fields have been shown to affect certain chemical reaction rates ex
vivo. In this manuscript, we present data demonstrating that similar small changes in static magnetic
fields between individual cell culture incubators results in significantly altered cell cycle rates for
multiple cancer-derived cell lines. This change as assessed by cell number is not a result of apoptosis,
necrosis, or cell cycle alterations. While the underlying mechanism is unclear, the implications for
all cell culture experiments are clear; static magnetic field conditions within incubators must be
considered and/or controlled just as one does for temperature, humidity, and carbon dioxide
concentration. Bioelectromagnetics 31:649–655, 2010. 2010 Wiley-Liss, Inc.
Key words: weak static fields; low-level fields; cancer cell; growth rates
INTRODUCTION
The Earth’s natural magnetic field (geomagnetic
field, GMF) is a quasi-static and quasi-uniform
magnetic field that ranges in magnitude from about
25 to 65 mT in unperturbed spaces, although GMF
tends to be significantly decreased and distorted inside
man-made buildings. Variations in GMF tend to be
even more pronounced inside metallic structures,
especially enclosed laboratory devices such as incubators. We have measured the static magnetic fields
between incubators and found them to be quite variable;
differences of 40 mT between two neighboring incubators prompted the current study.
Certain configurations of low-level electric and
electromagnetic fields have consistently been shown
to affect biological systems. Small differences in these
field magnitudes can cause changes in the configuration
of certain proteins, and produce effects at the cell and
organism level [McCaig et al., 2005; Céspedes and
Ueno, 2009]. These effects are reproducible and are
currently being evaluated for therapeutic applications
[Pilla, 2002, 2006; Nuccitelli, 2003; Volpe, 2003;
Balakatounis and Angoules, 2008; Colbert et al.,
2009; Strauch et al., 2009]. However, the mechanism
or mechanisms of action remain an open question
[Eremenko et al., 1997; Harland et al., 1999; Cook et al.,
2002; Barnes, 2006]. More recently, accumulating
proof that some configurations of low-level magnetic
fields have effects on different kinds of cells [Simko
et al., 2001; Del Re et al., 2004; Ravera et al., 2004;
Novikov et al., 2008], tissues [Barnes, 2006; Akdag
7 2010 Wiley-Liss, Inc.
et al., 2007], and organisms of different complexity
[Lednev et al., 1996; Berk et al., 1997; Thomas et al.,
1998; Prato et al., 2005; Barnes, 2006; Martino et al.,
2010] have become hard to ignore.
We have hypothesized that small variations in
static magnetic fields, on the order of differences in
GMF at distant locations as well as those observed
between neighboring incubators, can affect the cellular
behavior of model cancer cell lines in controlled
environments. Although CO2 and temperature levels
are parameters controlled rigorously, the background
static magnetic field has rarely or never been a
parameter under consideration to control. The results
presented in this report are therefore relevant to
virtually all cell culture experiments that do not
currently control for GMF as a variable.
In biological studies, the tight control of as
many variables as possible is critical for the accurate
interpretation of results. In cell cultures, advanced
incubators controlling CO2, humidity, and temperature
have become the norm. However, control of the internal
magnetic field has previously not been considered as a
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*Correspondence to: Carlos F. Martino, Campus Box 425,
Boulder, CO 80309-0425. E-mail: [email protected]
Received for review 22 February 2010; Accepted 14 July 2010
DOI 10.1002/bem.20606
Published online 9 September 2010 in Wiley Online Library
(wileyonlinelibrary.com).
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Martino et al.
critical variable to control. The results here demonstrate
that weak magnetic fields have significant effects on
cellular systems. Standard cell culture incubators are
immersed in the geomagnetic field and are affected by
low-level electromagnetic noise from their integrated
systems (e.g., fans).
MATERIALS AND METHODS
Cell Culture
Fibrosarcoma HT1080 and colorectal HCT116
cancer cells (CCL-121 and CCL-247, respectively,
ATCC, Manassas, VA) were grown and maintained in
Eagle’s Minimum Essential Medium (EMEM) and
McCoy’s 5a Medium (Modified), respectively, supplemented by 10% Fetal Bovine Serum (ATCC). The cells
were cultured in 75 cm2 flasks to expand cell number.
After reaching confluence, the cells were seeded in
35 mm Petri dishes. Medium was then changed every
2 days. The cultures were incubated in a 5% CO2
atmosphere at 37 8C in the same incubator.
Magnetic Stimulating System
The background magnetic field intensity inside
the incubator used in these studies, measured using a
gauss meter (Model FGM 4D2N, Walker Scientific,
Lake Orion, MI), varied from 6 to 13 mT depending on
the relative position in the room and the relative position
of surrounding objects. In order to establish uniform
static magnetic fields, we engineered a pair of tri-axial
square Helmholtz coils driven by a power supply
(HP 6205C Dual, Hewlett Packard, Palo Alto, CA) and
resistive circuitry. These coils established a unidirectional magnetic field perpendicular to the plane of
growth of the cells. The temperature and CO2 levels
were monitored daily and maintained at 37 8C and 5%,
respectively. The difference in temperature between the
upper and lower areas of the incubator was 0.1 8C
(0.003 8C/cm). All experiments were conducted in the
same incubator. To control for location in the incubator
and any associated electromagnetic noise or other
spatial variation, the orientation of experimental and
control cultures were periodically reversed.
Cells were seeded and allowed to rest for
24 h under the same magnetic background conditions,
after which timed magnetic exposure began. This
time is denoted as t0. The colorectal cancer line
HCT116 and fibrosarcoma cancer line HT1080 were
grown under three distinctive static magnetic field
intensities: (1) background static field inside incubator
(6–13 mT); (2) Earth’s average unperturbed static
magnetic field (43–45 mT); (3) reduced magnetic fields
(0.2–0.5 mT).
Bioelectromagnetics
Cell Proliferation Assay
The effects of the magnetic treatment on cellular
proliferation were determined by direct count of cell
numbers after each termination point. For the cell
counting assay, 35 mm Petri dishes were seeded at
a concentration of 8.0 103 cells/cm2 and incubated
in 5% CO2 at 37 8C for 1 day prior to subjecting the
experimental group to weak static magnetic fields. After
magnetic stimulation cycle, the cells in three wells per
termination point were counted three times using a
hemocytometer (VWR, San Francisco, CA).
Flow Cytometry
Flow cyctometric analysis, using a Beckmann
Coulter CyAn Cytometer (Brea, CA) and fluorescentlabeled Annexin-V antibodies and propidium iodide
(PI; Annexin-V-Flour Staining Kit, Roche, Mannheim,
Germany), was performed according to manufacturer’s
specifications to identify any changes in apoptosis and
necrosis. Fixed cell PI analysis examining cell cycle
status was carried out using well-established methods.
Data analysis of cell cycle data was performed using
both FlowJo, where gates were assigned using an
estimated full width half max of the Gaussian curves
for the G1, G2/M and S phases of the cell cycle, with
S being the intervening region; and ModFit which
assigns relative percentages to debris, aggregates,
G1, S, and G2/M based upon calculated Gaussian
distributions.
Statistical Analysis
Statistical analysis was performed using the
ANOVA test with a minimal confidence level of 95%
established as statistically significant (P < 0.05). Each
experiment was performed at least three times with
a minimum of three samples per termination point,
resulting in a total number of nine samples per group
for each experiment. The data shown constitutes a
representative sample of the experiments performed.
RESULTS
Small Variations in Magnetic Fields Change
Growth Rates of Cancer Cells
Small variations in static magnetic fields inside the
same incubator affected growth rates of the fibrosarcoma cancer line, HT1080 (Fig. 1A). The background
static magnetic field was 6 mT, which corresponds to
the resultant field in the x, y plane of the cells and the
z-direction. A tri-axial square Helmholtz coil established a 43 mT static field in the z-direction (perpendicular to the plane of growth of the cells); the fields in the
Reduction of the Earth’s Magnetic Field
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Fig. 2. Reducing the Earth’s magnetic field below background
level inhibits cellular growth (P < 0.05). A: Fibrosarcoma HT1080
cancer cells cultured in the presence of low fields (0.2 ^ 0.5 mT)
and 45 mT fields (errorbars,SD; n ¼ 3 replicates).B:Visualassessment of cells corresponding to cell numbers in (A); magnification
20.
Fig. 1. Small variation in magnetic fields alters growth rates of
model cancer cell lines (P < 0.05). A: Cell population of fibrosarcoma HT1080 increased throughout the culture period. B: Similar
results were obtained for colorectal line HCT116 (error bars, SD;
n ¼ 3 replicates).
x–y plane inside the tri-axial coil were minimized
(0.2–0.5 mT). Similar results were obtained with the
colorectal line, HCT116 (Fig. 1B).
Reduction of the Earth’s Static Magnetic Field
Inhibits Growth Rates of Cancer Cells
Next, we proceeded to annihilate the background
static magnetic field in the incubator. A tri-axial square
Helmholtz coil reduced the background static magnetic
field to the range of 0.2–0.5 mT for the experimental
group for up to 4 days, while another tri-axial square
Helmholtz coil established a 45 mT static field for
the control group. Seeding density was adjusted to
1 103 cells/cm2 to allow for longer culture growth
periods in the log phase. Figure 2A shows the resultant
decrease in cell number at day 4 at a reduced field
of 0.2–0.5 mT with the fibrosarcoma HT1080 line. A
representative photomicrograph of the HT1080 culture
at 45 mT and 0.2–0.5 mT is shown in Figure 2B.
Magnetic Field Effect Disappears as Static Field
Is Lowered Close to Low-Level Fields
We now proceeded to find a threshold for the
magnetic field effect. One group was exposed to 20 mT
while the other group was placed in low-level fields
(0.2–0.7 mT). Cell numbers were similar for both
groups (P > 0.05, n ¼ 3). Effects on cell number with
small variations in static magnetic fields become
negligible (see Table 1).
As a result of the reduction or elimination of
the Earth’s magnetic field below normal (45 mT)
we observed consistent inhibition of cell division in
multiple model cancer cell lines. We examined markers
of apoptosis and necrosis to determine if they were the
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Martino et al.
TABLE 1. Summary of Weak Magnetic Field Effects on Growth Rates of Cancer Cells
Magnetic stimulus
Incubator background fields (6–13 mT) vs. 45 mT control
Low-level fields (0.5–0.7 mT) vs. 45 mT control
Magnetic threshold low-level fields vs. 20 mT
source of reduced cell numbers in the cultures with
decreased magnetic fields. The population of cells
positive for Annexin-V and PI did not differ between
exposure and control groups, suggesting that the lowlevel static magnetic fields do not cause apoptosis or
necrosis.
Since there were no findings of apoptosis or
necrosis, propidium iodide cell cycle analysis by flow
cytometry was carried out. Results from this analysis
Percent change,
Day 2
Percent change,
Day 3
>30%
>30%
Negligible
>30%
>30%
Negligible
indicate no significant difference in relevant proportions of cell cycle stages (Fig. 3) suggesting that there
is no cell cycle checkpoint activation and no major
perturbations of mitotic mechanisms. Along with the
PI/Annexin-V data, we can conclude that the reduced
cell numbers must result from an overall reduction in
cell cycle rate as opposed to apoptosis, necrosis, DNA
damage, mitotic disruptions, or cell cycle checkpoint
activation.
Fig. 3. Cell cycle analysis of HT1080 cells exposed to low-level fields and 45 mT (right and
left, respectively).The raw data were analyzed by two methods: (1) FlowJo (top row) and (2) ModFit
(bottom row).
Bioelectromagnetics
Reduction of the Earth’s Magnetic Field
Time Frame Is Critical for Observation of
Magnetic Field Effects
In the next experiment, HT1080 cells were seeded
at a lower density of 500 cells/cm2. The cell population
grew throughout the culture period. While cell numbers
did not differ between low level and 45 mT magnetic
groups by day 2, a significant change in growth
rates was observed by day 4 (Fig. 4). The absolute cell
numbers and growth rates observed mirror those of
previous experiments, taking into account the relative
doubling time of treated and control cells. While this
test demonstrates a robustness of the static magnetic
field effect under different starting culture conditions,
it also suggests that the time frame in which one
examines this effect is critical; examination of these
cultures at day 2 would have shown no effect, and if
the cultures had been examined later, after contact
inhibition of growth had set in, no effect would have
been seen.
Magnetic Field Effect Is Additive for Longer
Cell Culture Periods
Lastly, the effects of small variations of static
magnetic fields were assessed for longer culture
periods. For this experiment, HT1080 cells were seeded
at 1,000 cells/cm2 and allowed to proliferate under low
level and 45 mT static magnetic fields. On day 4, cells
were counted for both groups: the low-level field,
7.32 105 cells/well; and 45 mT, 9.85 105 cells/well.
This time point corresponds to passage 1 in Figure 5.
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Fig. 5. Small variation in magnetic fields was evaluated for longer
culture periods. HT1080 fibrosarcoma cells were cultured under
low level and 45 mT magnetic fields. Continual exposure to low
levelmagnetic fields significantly (P < 0.05) reduced cellnumbers
compared to exposure to the Earth’s magnetic fieldlevels.
For both cultures, cells were passaged at a 1:9 dilution
and maintained at their previous magnetic field
conditions. After 3 days, cells were again counted:
low-level field, 7.20 105 cells/well; and 45 mT, 1.19 106 cells/well. Figure 5 shows the additive effect of
small changes in static magnetic fields for longer
culture periods indicating that the cells do not adapt to
the altered magnetic field conditions over time.
DISCUSSION
Fig. 4. Magnetic fieldeffectdependson celldensity.Fibrosarcoma
line was seeded at a low concentration of 500 cells/cm2.Decreasing the magnetic field did not change cell numbers by day 2, but
a significant (P < 0.05) change in growth was observed by day 4
(error bars, SD; n ¼ 3 replicates).
Recently, we reported weak magnetic fields
(60 and 120 mT) increased human umbilical vein
endothelial cell (HUVEC) growth by 40% in 48 h,
compared to the group shielded in a m-metal cylinder
(0.2–0.7 mT). Functional parameters that are tied to key
activities in HUVECs, such as endothelial nitric oxide
synthase (eNOS), also increased significantly after the
magnetic exposure [Martino et al., 2010]. In conjunction with our findings herein, we propose a model by
which small changes in static magnetic fields change
the energy required for transitions between electron
spin states (singlet (S) and triplet (T) quantum states),
altering concentrations of free radicals or reaction rates
involving free radicals or free radical intermediates.
Similar field changes have been shown to slightly
alter the rate of some free radical-dependant reactions
ex vivo [Steiner and Ulrich, 1989; Timmel and Henbest,
2004]. If these same effects occur in vivo in even a
modest percentage of biochemical reactions requiring a
free radical or intermediate reaction, they would affect
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Martino et al.
not just the direct production of free radicals, but also a
broad array of metabolic reactions requiring free radical
chemistry. Even a slight change in reaction rate across
such a breadth of metabolic reactions could manifest
in doubling the time alterations observed here, without
the major perturbations of cellular function that
would manifest as apoptosis, necrosis, or gross cell
cycle changes.
An important finding in this report is the static
magnetic field intensity threshold for observing
changes in growth rates. The magnetic field effect on
growth rates disappears when reducing the static field
to 20 mT; low-level field (0.2–0.7 mT) and weak static
magnetic field (10–20 mT) effects on growth rates
become indistinguishable. Low-level magnetic field
effects have also been observed to influence animal
behavior [Choleris et al., 2002; Prato et al., 2005].
In the latter study, effects were observed only when
the ambient fields were shielded by m-metal and not by
canceling the fields with tri-axial coils. This is an
essential distinction that may be implemented in our
future studies. The m-metal shielding attenuates the
ambient static and low frequency magnetic fields, while
the tri-axial coils cancel only the static component. In
our current study, the ambient low frequency magnetic
fields, which are present in both the control and
experimental groups, do not appear to affect growth
rates of cancer cells. Further studies on low-level effects
by shielding or canceling the ambient magnetic fields
are needed and currently under way.
Further exploration of the exact mechanism
underlying these cell cycle alterations is clearly
required. Increased understanding of the underlying
mechanism would also serve to improve the design of
therapeutics utilizing static magnetic fields, increase
their efficacy and provide a sound, scientific underpinning. However, regardless of the underlying mechanism, it is clear that care must be taken in any
experiments requiring growth in devices that may alter
cellular exposure to static magnetic fields to ensure that
variations in these fields are not altering experimental
outcomes. The specific effects of variations in magnetic
fields between incubators, not just fields established
by coils, should be considered. Indeed, a thoughtful
review of relevant previous findings, especially those
demonstrating unexplainable culture variability, must
be considered, and magnetic field effects should be
taken into account in future experimental designs.
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