Vol.
174,
January
No.
2, 1991
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Pages 742-749
31, 1991
DELINEATION EFFECTS
OF ELECTRIC
OF EXTREMELY
LOW
RADIATION James J. Greene,
William
Department
Received
November
FIELD
ELECTROMAGNETIC
ON TRANSCRIPTION
J. Skowronski,
J. Michael
of Biology and Institute The Catholic University
Miguel
MAGNETIC
FREQUENCY
Washington,
Department
AND
Penafiel
Mullins,
and Roland
for Biomolecular of America
M. Nardone
Studies
DC 20064
and Robert
Meister
of Electrical Engineering and Vitreous State Laboratory The Catholic University of America Washington, DC 20064 19,
1990
The relative effects of the electric and magnetic field components of extremely low frequency electromagnetic radiation (ELF) on transcription were examined in human leukemia HL-60 cells. Delineation of the individual field contributions was achieved by irradiating cells in separate concentric compartments of a culture dish within a solenoid chamber. This exposure system produced a homogeneous magnetic field with a coincident electric field whose strength varied directly with distance from the center of the culture dish. Irradiation of HL-60 cells with sine wave ELF at 60 Hz and a field strength of 10 Gauss produced a transient increase in the transcriptional rates which reached a maximum of 50-60% enhancement at 30-120 minutes of irradiation and declined to near basal levels by 18 hours. Comparison of transcription responses to ELF of cells in different concentric compartments revealed that the transcriptional effects were primarily the result of the electric field component with little or no contribution from the magnetic field. 0 1991Acadenlc mess,1°C.
Extremely
low frequency electromagnetic radiation (ELF),
encompassing the
frequency range from 0 to 1000 Hz, is the most common form of nonionizing radiation (1). It is pervasive in environments where electricity is used, being radiated by
transmission lines as well
as by virtually
all
electric
appliances. Several
epidemiological studies have suggested a correlation between ELF and some forms of cancer, although no consensushas been reached (2-6). Moreover, acceleration of bone healing and other whole-body effects have been attributed to ELF
(7-9). Cellular
studies directed to identifying the underlying basis for these demonstrable or perceived 0006-293X/91 $1.50 Copyright Q 1991 by Academic Press, Inc. All rights of’ reproduction in atzy form reserved.
742
Vol.
174,
No.
effects
2, 1991
have
transcription
revealed
the relative
roles
cellular
since
individual
prior
leukemia
studies
responses.
did
exposure
Distinguishing
biological
address
systems
which
in
of this coupling.
(12, 13). Nevertheless,
not
of ELF
pulse shape, frequency
fields in modifying either
basis for these
field components
strength,
including
this
and
the separate
responses question
complicated
remain or
were
isolation
of
present
it is generally not possible to vary one component
without
property
cells were
a solenoid
of the radiation
delineated
such as its frequency.
chamber, of ELF
by irradiation
an exposure independent
at 60 Hz caused a significant
rate that could be attributed from the magnetic
the underlying
a better understanding field
processes
fields are always
of each component ELF
of
COMMUNICATIONS
study, the electric and magnetic field effects on transcription
HL-60
within
that sine wave
regarding
Since both the electric and magnetic
or another
In the present
assessment
a diversity
and magnetic
provide
in their
radiation,
the other
RESEARCH
energy is coupled to cellular
would
by limitations
compartments
little is known
and magnetic
these
in electromagnetic
human
affect
of electromagnetic
field effects.
affecting
can
BIOPHYSICAL
on cells have all been examined
of the electric
constrained
AND
of the electric
responses
influences
of ELF
obscure
ELF
how the ELF
contributions
The timing
that
(10, 11). However,
effects, expecially
inducing
BIOCHEMICAL
in
of cells in concentric
system which
allowed
for the
of the other. This study shows transient
to the electric field with
increase
in transcription
little or no direct contribution
field.
MATERIALS
AND
METHODS
Solenoid Irradiator. The solenoid coil system used for irradiation is illustrated in Figure 1A. The irradiator consisted of a solenoid coil encased in a mu metal shield to insulate the interior from exogenous fields. The solenoid itself was 11 cm in diameter and 50 cm in length and together with its casing measured 17 cm x 17 cm x 50 cm. Magnetic fields within the solenoid were generated by a Carver magnetic field power amplifier modulated by a Tenma generator. The irradiator was placed within a water-jacketed CO, incubator with a thermostated water circulation system incorporated within the solenoid to regulate the temperature of the cell cultures at 37°C 2 O.l”C. A lo-stage plexiglass culture dish holder with holes to allow for gas exchange was placed within the cavity of the solenoid for irradiation. Cell Culture. HL-60 cells were grown in Eagle’s minimum essential medium (Sigma Chemical Co., St. Louis MO) supplemented with 10% fetal bovine serum (Biofluids Inc., Rockville, MD). The cells were kept in the log phase of growth by passaging three times per week. One hour prior to irradiation, cells were inoculated into 60 mm culture dishes at a density of 1.0 x lo6 cells/ml, except for overnight exposures which were inoculated at a density of 0.5 x lo6 cells/ml to allow for one doubling of the cells. Dishes. Three types of culture dishes were used: standard 60 mm tissue culture 60 mm organ culture dishes, and custom-made 60 mm dishes with concentric 743
dishes, Lucite
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
rings. Organ culture dishes contained two concentric compartments. The of the inner compartment was 0.90 cm while the average radius compartment was 2.03 cm. The concentric ring dishes contained two inner and outer annular compartments with average radii of 0.87 cm respectively.
average radius of the outer rings, defining and 2.49 cm,
3H-Uridine Pulse Labeling and TCA Precipitation. Cells were inoculated as described above into conventional, organ, or custom 60 mm culture dishes one hour prior to ELF radiation exposure. Irradiated cultures were placed randomly into various slots of the plexiglass culture holder then lowered into the solenoid cavity for exposure for various times. Matched control cultures were placed onto a plastic holder situated outside of the solenoid within the same incubator as the irradiator and incubated for the same periods as the irradiated cultures. Both the irradiated cultures and their corresponding controls in duplicate were pulse-labeled for the final 15 minutes of incubation by the addition of 3H-uridine (27.1 Ci/mmol - DuPont/NEN, Boston, MA) to a final concentration of 20 &i/ml. After labeling, cells were harvested by centrifugation and lysed in a buffer of 1% SDS, 10mM Tris-Cl, 5mM EDTA, pH 7.5. The lysates were vortexed and an equal volume of 20% TCA was added. Acid precipitable material was collected on Whatman GFB glass microfibre filters by vacuum filtration, washed 3X with 5% TCA, and 1X with 95% ethanol. After drying, incorporation of uridine into newly synthesized RNA was quantified by scintillation counting.
RESULTS The solenoid field with
configuration
its attendant
wave was determined
Irradiation
in the exposure
dish and increased
in the culture
center
(Figure
of cells to a uniform field produced
the cross-section
1B). The induced to the outer
fields were
constant
dishes
to an electric
by a 60 Hz sine
of the solenoid
electric
cavity
field strength
edge of the cavity (Figure
was 1C).
along the length of the solenoid.
to ELF
radiation
within
the solenoid
field that was 0 at the center
of the
although
environment
the geometry
and magnitude
was independent
of the electric field
of the dish position
from
the
of the cavity (14). To examine
dishes were
the effect of ELF on transcription,
exposed
in the solenoid
for various
HL-60
at a field strength
of 10 Gauss. These conditions
radiation
emitted
transmission
was assessed of irradiation.
by electric
by the incorporation Results
30 minutes
of this pulse-label
of exposure
lines and appliances.
of 3H-uridine kinetics
into RNA
are within
60 mm 60 Hz
the range of
The transcription
rate
during the final 15 minutes
experiment
to ELF, there was a marked 744
cells in standard
lengths of time to sine wave
radiation
Within
magnetic
radially from the center. The culture dishes were aligned along the
axis of the solenoid
induced
throughout
linearly
and magnetic
exposure
The magnetic
of cells in 60 mm circular
cavity resulted
central
periphery
and increased
Both the electric
field.
to be uniform
except at the extreme 0 at the center
electric
allowed
are shown
in Figure
30-50% enhancement
2. in
Vol.
174,
No.
mu metal
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
shield casing
solenoid
coils
cooling plexiglass dish
coils culture holder
60 mm culture
dish
-2.0
-1.0 Distance
0 From
1.0 Center
2.0 km)
1. A) The solenoid ELF exposure system.The exposure systemconsistedof a solenoid encased in a mu-metal shield placed inside a CO, equilibrated waterjacketed incubator. Cell cultures were irradiated in 60 mm dishesthat were placed onto one of 10 stages of a plexiglassculture carrier. The culture carrier was constructed so that the 10 stageswere positionedin the homogeneousregion of the ELF field. Ventillation holes in the carrier allowed for gas exchange between the cultures and the incubator environment. Control cultures were placed on a separate carrier in the same incubator as the solenoid.B) Magnetic field strength determined across the culture dish diameter. A Bell 620 Gaussmeterwith an axial Hall effect probe was used to measurethe magneticfield distribution at various points acrossthe solenoid. The average field strength for these measurementsis midway in the range of l-10 Gauss used in the transcription studies.Throughout this range, the magnetic field was uniform within 2 0.05 Gauss. C) Induced electric field strength within the culture medium.The electric field inducedby a 10 Gaussmagneticfield was measured with a 0.9 mm platinum element dipole that was inserted into the medium contained within a 60 mm tissue culture dish. The accuracy of the field measurementswas within r 0.1 mv. Figure
the uridine incorporation rate. This enhanced level of transcription was sustained for 5 hours after which the levels declined to near control levels by 15-20 hours. Fractionation and nuclear transcription studies using specific gene probes showed that the increased transcription could be largely accounted for by an acceleration in the transcription of ribosomal 45 S precursor RNA. The contributions of the magnetic and electric fields to the enhancement in transcription
were studied by exploiting the field characteristics of the solenoid
irradiation system. Given the radially varying electric field produced in the culture environment within the solenoid, cells could be exposed to the same magnetic, but different electric field strength when irradiated at different radii from the center in the same dish. Therefore, HL-60 cells were exposed to ELF in culture dishes that 745
Vol.
174,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
SameE DiffsrentB
Different E Same 6
80
-0 \
2
60
i 8 c
40
B
2
0
I=
“\\./
02
-10’
0
5
Time
10
lrradioted
15
20
I 25
-I 1
(hrs)
2
3
4
Time
0
Irradiated
1
2
3
(hrs)
Fkure 2. Effect of ELF exposure on the incorporation of 3H-uridine. A total of 3 x lo6 HL-60 cells was inoculated in Eagle’s minimum essential medium supplemented with 10 % fetal bovine serum at a density of 1 x lo6 cells/ml into each of duplicate 60mm culture dishes 1 hour prior to irradiation. Irradiated cultures were exposed to 10 Gauss 60 Hz sine wave radiation at staggered times so that all cultures in the kinetics experiments were pulse-labeled at the same time. Control cultures in duplicate dishes were loaded into carriers placed outside of the solenoid coil but within the same incubator as the irradiated cultures. Pulse-labeling of the irradiated and control cultures are as described in Materials and Methods. Data shown are expressed as the % enhancement of incorporation over that of controls. Each point represents an average of five experiments except for points for 20 and 24 hours which are an average of two experiments. The standard error for these points is & 515% except for data corresponding to 20 and 24 kinetic time points for which the error is < & 5%. Much of this error is attributed to small variations in the kinetics of the transcription response from experiment to experiment rather than the magnitude of the response. Figure 3. Delineation of electric and magnetic field components on the incorporation of H3-uridine. A) Comparison of the kinetics of H3-uridine incorporation during exposure to ELF under conditions of different electric field but same magnetic field strengths. HL-60 cells were inoculated at a density of 1 x lo6 cells/ml into the inner and outer compartments of duplicate 60 mm organ culture dishes and exposed to sine wave 60 Hz radiation at a field strength of 10 Gauss. The effective radius (radius from the center of the dish encompassing 50% of cells in the compartment) for cells in the outer compartment was 2.03 cm at which the cells experienced an electric field of 3.40 mV/m. The effective radius for cells in the inner compartment was 0.2 cm at which the electric field was 0.34 mv/m. ELF exposure and pulse-labeling of cells is as described in Materials and Methods. Data is presented as % enhancement in H3-uridine incorporation over that of corresponding control cultures. Each point is the average of three experiments. Standard error was k5-10%. The relative difference in the kinetics between the cells in the inner and outer compartments was the same in each of the three experiments. Open circles - kinetics for cells in inner compartment; closed circles - kinetics for cells in the outer compartment. B) Comparison of the kinetics of H3-uridine incorporation during exposure to ELF under conditions of same electric field but different magnetic field strengths. HL-60 cells in the outer compartment of duplicate 60 mm organ culture dishes were exposed to ELF for the indicated times at a field strength of 1 Gauss. Under these conditions, the average electric field experienced by these cells was 0.34 mV/m. The H3-uridine incorporation was determined as for Figure 2 and is compared to that of cells in the inner compartment exposed to 10 Gauss which experienced the same electric field but a magnetic field an order of magnitude higher. Open circles - kinetics for cells exposed to 1 Gauss radiation; closed circles - kinetics for cells exposed to 10 Gauss radiation. Standard error for this experiment was also 2 5-10%.
746
4
5
Vol.
174,
were
No.
2, 1991
divided
concentric
into
Both
BIOPHYSICAL
chambers.
RESEARCH
of either
by gluing acrylic rings onto 60 mm culture
dishes, or
allowed
dishes
inner well surrounded for
homogeneous
These
COMMUNICATIONS
consisted
a central
configurations
to the same
AND
annular
dishes containing dish
populations electric
concentric
ring dishes fabricated
organ culture well.
BIOCHEMICAL
by an outer
the simultaneous
magnetic
field
exposure
and different,
of two but
cell
defined,
fields. The 3H-uridine
and 10 Gauss
pulse-label
kinetics
of HL-60
cells exposed
to ELF
field in the center and outer wells of organ culture
mV/m,
exhibited
greater
incorporation
in the inner well which average electric in the
exposed
field of 0.34 mV/m.
inner
represented
were
of 3H-uridine
and
outer
wells
This relative
was
observed
the inner
and outer
However,
in these dishes, the difference
in the inner dishes,
and outer wells
reflecting
concentric
in each
annular
of
the
four
cells were
ring dishes
in the uridine
than did cells field, but an
in the response
obtained when
of concentric
(data
incorporation
of cells
experiments irradiated
in
not shown).
rate of the cells
was not as great as for the cells in the organ culture
the smaller
separation
affect the transcriptional
The
magnetic
field
by comparing
of the organ culture exposed
experienced
to
between
the inner and outer
wells
in the
wells
process.
contribution
the kinetics
to the
of uridine
enhancement
incorporation
dishes exposed to 1 Gauss radiation
10 Gauss
radiation.
by these cells were
the enhancement and outer
difference
field of 3.40
ring dishes. These results strongly indicate that the electric field component
can directly
examined
to controls
to the same 10 Gauss magnetic
in Figure 3A. Similar results were chambers
relative
at 60 Hz
dishes are shown
in Figure 3A. The cells in the outer well, exposed to an average electric
well
annular
in uridine
Under
comparable
incorporation
even though
these
in
to that of cells in the inner
conditions,
strength
the electric
As shown
was nearly identical field
was
of cells in the outer well
at 0.34 mV/m.
the magnetic
transcription
fields
in Figure
3B,
for cells in the inner
differed
by an order
of
magnitude.
DISCUSSION Collectively,
these results
elicited by the interaction of the magnetic
indicate
of the electric field with
field. Predominance
is consistent
magnetic
field is capable of directly altering membranes,
its dielectric the orientation
in transcription
field in producing
effects on cells. Indeed, the charge distribution
of charged biomolecules, 747
can be
cells, with little or no contribution
of the electric
response
and organelle
with
that an enhancement
this biologic
the electric
but not
on the cell’s outer and the counterion
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
polarization
(15). These effects become particularly
the range
of 60 Hz
conceivable
that
transcription
dielectric
field strength cells were
the permittivity dielectric
generated
within
appear
changes
surprising
to overcome
and electric
which
8, and a radius field strength
considers
field-induced
relationship
in view
high.
in
It
in modifying
is the
underlying
of the relatively
low electric
the randomization however,
the periodicity
changes
of cellular
is reconciled of electric
in membrane
for the spherical
the HL-60
%ininlnnl-
HL-60
cell with
I 1 ”
1 r2
generally
processes by the model
(18).
Using
a membrane
estimate
the
believed
produced
fields together
potential
cell can respond
-~2 kTd 3 4xeoKm
electric field to which
a field strength
of 15 x 10e6m, the conservative
to which
frequencies
and the possible
the solenoid. The maximum
noise (17). This discrepancy,
Astumian
can participate
exposed was on the order of 10 mV/cm,
and Astumian
at lower
of the cell is very
effects
of the transcriptional
may initially
to be insufficient thermal
manifested
COMMUNICATIONS
(16).
occurrence effects
which
each of these
process
The
for
RESEARCH
by
of Weaver
with
thermal
the Weaver-
thickness
for the minimum
of 75 electric
is given by:
=
10 mvlcm
where k is the Boltzmann constant, T the absolute temperature (310°K), d the thickness of the cell membrane (5 x 10e7cm), K,,, the membrane’s dielectric constant (approximated as 2.5), and E, the permittivity
of free space. If the cells are exposed
to a periodic field, as in this case, and this periodic@ accounted for by signal averaging, then the Eminimum is reduced by a factor of (ft).” frequency of radiation and t the exposure time”
where f is the
to lower the Eminimum one to two
orders of magnitude. These considerations clearly indicate that the minimum detectable field imposed on the HL-60 cells by competition between the applied field and thermal noise is within the range generated within the solenoid coils. The transcriptional changes observed in this study can potentially be mediated by a number of mechanisms. While the proximal causual event has not been identified, the correlation of the initiation of this cellular response solely to the electric field component of ELF narrows the possibilities by which ELF is coupled to cellular effects. ACKNOWLEDGMENTS The assistance of Julie Brent in the culturing and preparation of HL-60 cells is greatly appreciated. The authors thank Howard Bassen, Edward Elson, David Krause, 748
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Theodore Litovitz, Robert Mohr and Charles Montrose for many helpful and advice. The support of the Department of the Army is appreciated.
discussions
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Lee, J., M., Jr. et al. (1989). Electrical and Siological Effects of Transmission Lines: A Review. U.S. Dept. of Energy, Portland, OR. Wertheimer, N., and Leeper, E. (1979) Am. J. Epidemol. 109, 273-284. Wertheimer, N., and Leeper, E. (1982) Znt. .I. Epidemiol. 11, 345-355. Coleman, M., Bell, J., and Skeet, R. (1983) Lancet 1, 982-983. Milham Jr., S. (1982) N. En@. J. Med. 307, 249. Pearce, N. E. et aZ.,(1985) Lancet 1, 811-812. Bassett, C. A. L., Mitchell, S. N., and Gaston, S. R. (1982) J. Am. Med. Assoc. 247, 623-628. Bassett, C. A. L., Schink, M. M., and Gaston, S. R. (1981) Trans. First Annu. Meet. Bioelec. Repair and Growth Sot. 1, 38-47. Brighton C. et aZ., (1981) .I. Bone and Joiflt Sq. 63-A, 2-21. Clear-y, S. F. (1987) ZEEE Eng. Med. and Biol. 6, 26-30. Polk, C., and E. Postow, E. (1985) CRC Handbook of Biological Effects of Electromagnetic Field, CRC Press, Boca Raton, FL. Goodman, R., Bassett, C. A. L., and Henderson, A. (1983) Science 22, 12831285. Goodman, R., and Henderson, A. S. (1986) Bioelectromagnetics 7, 23-29. Bassen, H. et al., Manuscript submitted. Foster, K. R., and Schwan, H. P. (1989) CRC Critical Reviews in Biomed. Eng. 17, 25 104. Blank, M., and Goodman, R. (1988) Bioelectrochem. Bioenerget. 19, 565-571. Schwan, H. P., and Foster, K. R. (1980) IEEE Trans. Biomed. Eng. 68, 104-113. Weaver, J. C., and Astumian, R. D. (1990) Science 247, 459-462.
749
174,
January
No.
2, 1991
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Pages 742-749
31, 1991
DELINEATION EFFECTS
OF ELECTRIC
OF EXTREMELY
LOW
RADIATION James J. Greene,
William
Department
Received
November
FIELD
ELECTROMAGNETIC
ON TRANSCRIPTION
J. Skowronski,
J. Michael
of Biology and Institute The Catholic University
Miguel
MAGNETIC
FREQUENCY
Washington,
Department
AND
Penafiel
Mullins,
and Roland
for Biomolecular of America
M. Nardone
Studies
DC 20064
and Robert
Meister
of Electrical Engineering and Vitreous State Laboratory The Catholic University of America Washington, DC 20064 19,
1990
The relative effects of the electric and magnetic field components of extremely low frequency electromagnetic radiation (ELF) on transcription were examined in human leukemia HL-60 cells. Delineation of the individual field contributions was achieved by irradiating cells in separate concentric compartments of a culture dish within a solenoid chamber. This exposure system produced a homogeneous magnetic field with a coincident electric field whose strength varied directly with distance from the center of the culture dish. Irradiation of HL-60 cells with sine wave ELF at 60 Hz and a field strength of 10 Gauss produced a transient increase in the transcriptional rates which reached a maximum of 50-60% enhancement at 30-120 minutes of irradiation and declined to near basal levels by 18 hours. Comparison of transcription responses to ELF of cells in different concentric compartments revealed that the transcriptional effects were primarily the result of the electric field component with little or no contribution from the magnetic field. 0 1991Acadenlc mess,1°C.
Extremely
low frequency electromagnetic radiation (ELF),
encompassing the
frequency range from 0 to 1000 Hz, is the most common form of nonionizing radiation (1). It is pervasive in environments where electricity is used, being radiated by
transmission lines as well
as by virtually
all
electric
appliances. Several
epidemiological studies have suggested a correlation between ELF and some forms of cancer, although no consensushas been reached (2-6). Moreover, acceleration of bone healing and other whole-body effects have been attributed to ELF
(7-9). Cellular
studies directed to identifying the underlying basis for these demonstrable or perceived 0006-293X/91 $1.50 Copyright Q 1991 by Academic Press, Inc. All rights of’ reproduction in atzy form reserved.
742
Vol.
174,
No.
effects
2, 1991
have
transcription
revealed
the relative
roles
cellular
since
individual
prior
leukemia
studies
responses.
did
exposure
Distinguishing
biological
address
systems
which
in
of this coupling.
(12, 13). Nevertheless,
not
of ELF
pulse shape, frequency
fields in modifying either
basis for these
field components
strength,
including
this
and
the separate
responses question
complicated
remain or
were
isolation
of
present
it is generally not possible to vary one component
without
property
cells were
a solenoid
of the radiation
delineated
such as its frequency.
chamber, of ELF
by irradiation
an exposure independent
at 60 Hz caused a significant
rate that could be attributed from the magnetic
the underlying
a better understanding field
processes
fields are always
of each component ELF
of
COMMUNICATIONS
study, the electric and magnetic field effects on transcription
HL-60
within
that sine wave
regarding
Since both the electric and magnetic
or another
In the present
assessment
a diversity
and magnetic
provide
in their
radiation,
the other
RESEARCH
energy is coupled to cellular
would
by limitations
compartments
little is known
and magnetic
these
in electromagnetic
human
affect
of electromagnetic
field effects.
affecting
can
BIOPHYSICAL
on cells have all been examined
of the electric
constrained
AND
of the electric
responses
influences
of ELF
obscure
ELF
how the ELF
contributions
The timing
that
(10, 11). However,
effects, expecially
inducing
BIOCHEMICAL
in
of cells in concentric
system which
allowed
for the
of the other. This study shows transient
to the electric field with
increase
in transcription
little or no direct contribution
field.
MATERIALS
AND
METHODS
Solenoid Irradiator. The solenoid coil system used for irradiation is illustrated in Figure 1A. The irradiator consisted of a solenoid coil encased in a mu metal shield to insulate the interior from exogenous fields. The solenoid itself was 11 cm in diameter and 50 cm in length and together with its casing measured 17 cm x 17 cm x 50 cm. Magnetic fields within the solenoid were generated by a Carver magnetic field power amplifier modulated by a Tenma generator. The irradiator was placed within a water-jacketed CO, incubator with a thermostated water circulation system incorporated within the solenoid to regulate the temperature of the cell cultures at 37°C 2 O.l”C. A lo-stage plexiglass culture dish holder with holes to allow for gas exchange was placed within the cavity of the solenoid for irradiation. Cell Culture. HL-60 cells were grown in Eagle’s minimum essential medium (Sigma Chemical Co., St. Louis MO) supplemented with 10% fetal bovine serum (Biofluids Inc., Rockville, MD). The cells were kept in the log phase of growth by passaging three times per week. One hour prior to irradiation, cells were inoculated into 60 mm culture dishes at a density of 1.0 x lo6 cells/ml, except for overnight exposures which were inoculated at a density of 0.5 x lo6 cells/ml to allow for one doubling of the cells. Dishes. Three types of culture dishes were used: standard 60 mm tissue culture 60 mm organ culture dishes, and custom-made 60 mm dishes with concentric 743
dishes, Lucite
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
rings. Organ culture dishes contained two concentric compartments. The of the inner compartment was 0.90 cm while the average radius compartment was 2.03 cm. The concentric ring dishes contained two inner and outer annular compartments with average radii of 0.87 cm respectively.
average radius of the outer rings, defining and 2.49 cm,
3H-Uridine Pulse Labeling and TCA Precipitation. Cells were inoculated as described above into conventional, organ, or custom 60 mm culture dishes one hour prior to ELF radiation exposure. Irradiated cultures were placed randomly into various slots of the plexiglass culture holder then lowered into the solenoid cavity for exposure for various times. Matched control cultures were placed onto a plastic holder situated outside of the solenoid within the same incubator as the irradiator and incubated for the same periods as the irradiated cultures. Both the irradiated cultures and their corresponding controls in duplicate were pulse-labeled for the final 15 minutes of incubation by the addition of 3H-uridine (27.1 Ci/mmol - DuPont/NEN, Boston, MA) to a final concentration of 20 &i/ml. After labeling, cells were harvested by centrifugation and lysed in a buffer of 1% SDS, 10mM Tris-Cl, 5mM EDTA, pH 7.5. The lysates were vortexed and an equal volume of 20% TCA was added. Acid precipitable material was collected on Whatman GFB glass microfibre filters by vacuum filtration, washed 3X with 5% TCA, and 1X with 95% ethanol. After drying, incorporation of uridine into newly synthesized RNA was quantified by scintillation counting.
RESULTS The solenoid field with
configuration
its attendant
wave was determined
Irradiation
in the exposure
dish and increased
in the culture
center
(Figure
of cells to a uniform field produced
the cross-section
1B). The induced to the outer
fields were
constant
dishes
to an electric
by a 60 Hz sine
of the solenoid
electric
cavity
field strength
edge of the cavity (Figure
was 1C).
along the length of the solenoid.
to ELF
radiation
within
the solenoid
field that was 0 at the center
of the
although
environment
the geometry
and magnitude
was independent
of the electric field
of the dish position
from
the
of the cavity (14). To examine
dishes were
the effect of ELF on transcription,
exposed
in the solenoid
for various
HL-60
at a field strength
of 10 Gauss. These conditions
radiation
emitted
transmission
was assessed of irradiation.
by electric
by the incorporation Results
30 minutes
of this pulse-label
of exposure
lines and appliances.
of 3H-uridine kinetics
into RNA
are within
60 mm 60 Hz
the range of
The transcription
rate
during the final 15 minutes
experiment
to ELF, there was a marked 744
cells in standard
lengths of time to sine wave
radiation
Within
magnetic
radially from the center. The culture dishes were aligned along the
axis of the solenoid
induced
throughout
linearly
and magnetic
exposure
The magnetic
of cells in 60 mm circular
cavity resulted
central
periphery
and increased
Both the electric
field.
to be uniform
except at the extreme 0 at the center
electric
allowed
are shown
in Figure
30-50% enhancement
2. in
Vol.
174,
No.
mu metal
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
shield casing
solenoid
coils
cooling plexiglass dish
coils culture holder
60 mm culture
dish
-2.0
-1.0 Distance
0 From
1.0 Center
2.0 km)
1. A) The solenoid ELF exposure system.The exposure systemconsistedof a solenoid encased in a mu-metal shield placed inside a CO, equilibrated waterjacketed incubator. Cell cultures were irradiated in 60 mm dishesthat were placed onto one of 10 stages of a plexiglassculture carrier. The culture carrier was constructed so that the 10 stageswere positionedin the homogeneousregion of the ELF field. Ventillation holes in the carrier allowed for gas exchange between the cultures and the incubator environment. Control cultures were placed on a separate carrier in the same incubator as the solenoid.B) Magnetic field strength determined across the culture dish diameter. A Bell 620 Gaussmeterwith an axial Hall effect probe was used to measurethe magneticfield distribution at various points acrossthe solenoid. The average field strength for these measurementsis midway in the range of l-10 Gauss used in the transcription studies.Throughout this range, the magnetic field was uniform within 2 0.05 Gauss. C) Induced electric field strength within the culture medium.The electric field inducedby a 10 Gaussmagneticfield was measured with a 0.9 mm platinum element dipole that was inserted into the medium contained within a 60 mm tissue culture dish. The accuracy of the field measurementswas within r 0.1 mv. Figure
the uridine incorporation rate. This enhanced level of transcription was sustained for 5 hours after which the levels declined to near control levels by 15-20 hours. Fractionation and nuclear transcription studies using specific gene probes showed that the increased transcription could be largely accounted for by an acceleration in the transcription of ribosomal 45 S precursor RNA. The contributions of the magnetic and electric fields to the enhancement in transcription
were studied by exploiting the field characteristics of the solenoid
irradiation system. Given the radially varying electric field produced in the culture environment within the solenoid, cells could be exposed to the same magnetic, but different electric field strength when irradiated at different radii from the center in the same dish. Therefore, HL-60 cells were exposed to ELF in culture dishes that 745
Vol.
174,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
SameE DiffsrentB
Different E Same 6
80
-0 \
2
60
i 8 c
40
B
2
0
I=
“\\./
02
-10’
0
5
Time
10
lrradioted
15
20
I 25
-I 1
(hrs)
2
3
4
Time
0
Irradiated
1
2
3
(hrs)
Fkure 2. Effect of ELF exposure on the incorporation of 3H-uridine. A total of 3 x lo6 HL-60 cells was inoculated in Eagle’s minimum essential medium supplemented with 10 % fetal bovine serum at a density of 1 x lo6 cells/ml into each of duplicate 60mm culture dishes 1 hour prior to irradiation. Irradiated cultures were exposed to 10 Gauss 60 Hz sine wave radiation at staggered times so that all cultures in the kinetics experiments were pulse-labeled at the same time. Control cultures in duplicate dishes were loaded into carriers placed outside of the solenoid coil but within the same incubator as the irradiated cultures. Pulse-labeling of the irradiated and control cultures are as described in Materials and Methods. Data shown are expressed as the % enhancement of incorporation over that of controls. Each point represents an average of five experiments except for points for 20 and 24 hours which are an average of two experiments. The standard error for these points is & 515% except for data corresponding to 20 and 24 kinetic time points for which the error is < & 5%. Much of this error is attributed to small variations in the kinetics of the transcription response from experiment to experiment rather than the magnitude of the response. Figure 3. Delineation of electric and magnetic field components on the incorporation of H3-uridine. A) Comparison of the kinetics of H3-uridine incorporation during exposure to ELF under conditions of different electric field but same magnetic field strengths. HL-60 cells were inoculated at a density of 1 x lo6 cells/ml into the inner and outer compartments of duplicate 60 mm organ culture dishes and exposed to sine wave 60 Hz radiation at a field strength of 10 Gauss. The effective radius (radius from the center of the dish encompassing 50% of cells in the compartment) for cells in the outer compartment was 2.03 cm at which the cells experienced an electric field of 3.40 mV/m. The effective radius for cells in the inner compartment was 0.2 cm at which the electric field was 0.34 mv/m. ELF exposure and pulse-labeling of cells is as described in Materials and Methods. Data is presented as % enhancement in H3-uridine incorporation over that of corresponding control cultures. Each point is the average of three experiments. Standard error was k5-10%. The relative difference in the kinetics between the cells in the inner and outer compartments was the same in each of the three experiments. Open circles - kinetics for cells in inner compartment; closed circles - kinetics for cells in the outer compartment. B) Comparison of the kinetics of H3-uridine incorporation during exposure to ELF under conditions of same electric field but different magnetic field strengths. HL-60 cells in the outer compartment of duplicate 60 mm organ culture dishes were exposed to ELF for the indicated times at a field strength of 1 Gauss. Under these conditions, the average electric field experienced by these cells was 0.34 mV/m. The H3-uridine incorporation was determined as for Figure 2 and is compared to that of cells in the inner compartment exposed to 10 Gauss which experienced the same electric field but a magnetic field an order of magnitude higher. Open circles - kinetics for cells exposed to 1 Gauss radiation; closed circles - kinetics for cells exposed to 10 Gauss radiation. Standard error for this experiment was also 2 5-10%.
746
4
5
Vol.
174,
were
No.
2, 1991
divided
concentric
into
Both
BIOPHYSICAL
chambers.
RESEARCH
of either
by gluing acrylic rings onto 60 mm culture
dishes, or
allowed
dishes
inner well surrounded for
homogeneous
These
COMMUNICATIONS
consisted
a central
configurations
to the same
AND
annular
dishes containing dish
populations electric
concentric
ring dishes fabricated
organ culture well.
BIOCHEMICAL
by an outer
the simultaneous
magnetic
field
exposure
and different,
of two but
cell
defined,
fields. The 3H-uridine
and 10 Gauss
pulse-label
kinetics
of HL-60
cells exposed
to ELF
field in the center and outer wells of organ culture
mV/m,
exhibited
greater
incorporation
in the inner well which average electric in the
exposed
field of 0.34 mV/m.
inner
represented
were
of 3H-uridine
and
outer
wells
This relative
was
observed
the inner
and outer
However,
in these dishes, the difference
in the inner dishes,
and outer wells
reflecting
concentric
in each
annular
of
the
four
cells were
ring dishes
in the uridine
than did cells field, but an
in the response
obtained when
of concentric
(data
incorporation
of cells
experiments irradiated
in
not shown).
rate of the cells
was not as great as for the cells in the organ culture
the smaller
separation
affect the transcriptional
The
magnetic
field
by comparing
of the organ culture exposed
experienced
to
between
the inner and outer
wells
in the
wells
process.
contribution
the kinetics
to the
of uridine
enhancement
incorporation
dishes exposed to 1 Gauss radiation
10 Gauss
radiation.
by these cells were
the enhancement and outer
difference
field of 3.40
ring dishes. These results strongly indicate that the electric field component
can directly
examined
to controls
to the same 10 Gauss magnetic
in Figure 3A. Similar results were chambers
relative
at 60 Hz
dishes are shown
in Figure 3A. The cells in the outer well, exposed to an average electric
well
annular
in uridine
Under
comparable
incorporation
even though
these
in
to that of cells in the inner
conditions,
strength
the electric
As shown
was nearly identical field
was
of cells in the outer well
at 0.34 mV/m.
the magnetic
transcription
fields
in Figure
3B,
for cells in the inner
differed
by an order
of
magnitude.
DISCUSSION Collectively,
these results
elicited by the interaction of the magnetic
indicate
of the electric field with
field. Predominance
is consistent
magnetic
field is capable of directly altering membranes,
its dielectric the orientation
in transcription
field in producing
effects on cells. Indeed, the charge distribution
of charged biomolecules, 747
can be
cells, with little or no contribution
of the electric
response
and organelle
with
that an enhancement
this biologic
the electric
but not
on the cell’s outer and the counterion
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
polarization
(15). These effects become particularly
the range
of 60 Hz
conceivable
that
transcription
dielectric
field strength cells were
the permittivity dielectric
generated
within
appear
changes
surprising
to overcome
and electric
which
8, and a radius field strength
considers
field-induced
relationship
in view
high.
in
It
in modifying
is the
underlying
of the relatively
low electric
the randomization however,
the periodicity
changes
of cellular
is reconciled of electric
in membrane
for the spherical
the HL-60
%ininlnnl-
HL-60
cell with
I 1 ”
1 r2
generally
processes by the model
(18).
Using
a membrane
estimate
the
believed
produced
fields together
potential
cell can respond
-~2 kTd 3 4xeoKm
electric field to which
a field strength
of 15 x 10e6m, the conservative
to which
frequencies
and the possible
the solenoid. The maximum
noise (17). This discrepancy,
Astumian
can participate
exposed was on the order of 10 mV/cm,
and Astumian
at lower
of the cell is very
effects
of the transcriptional
may initially
to be insufficient thermal
manifested
COMMUNICATIONS
(16).
occurrence effects
which
each of these
process
The
for
RESEARCH
by
of Weaver
with
thermal
the Weaver-
thickness
for the minimum
of 75 electric
is given by:
=
10 mvlcm
where k is the Boltzmann constant, T the absolute temperature (310°K), d the thickness of the cell membrane (5 x 10e7cm), K,,, the membrane’s dielectric constant (approximated as 2.5), and E, the permittivity
of free space. If the cells are exposed
to a periodic field, as in this case, and this periodic@ accounted for by signal averaging, then the Eminimum is reduced by a factor of (ft).” frequency of radiation and t the exposure time”
where f is the
to lower the Eminimum one to two
orders of magnitude. These considerations clearly indicate that the minimum detectable field imposed on the HL-60 cells by competition between the applied field and thermal noise is within the range generated within the solenoid coils. The transcriptional changes observed in this study can potentially be mediated by a number of mechanisms. While the proximal causual event has not been identified, the correlation of the initiation of this cellular response solely to the electric field component of ELF narrows the possibilities by which ELF is coupled to cellular effects. ACKNOWLEDGMENTS The assistance of Julie Brent in the culturing and preparation of HL-60 cells is greatly appreciated. The authors thank Howard Bassen, Edward Elson, David Krause, 748
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Theodore Litovitz, Robert Mohr and Charles Montrose for many helpful and advice. The support of the Department of the Army is appreciated.
discussions
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Lee, J., M., Jr. et al. (1989). Electrical and Siological Effects of Transmission Lines: A Review. U.S. Dept. of Energy, Portland, OR. Wertheimer, N., and Leeper, E. (1979) Am. J. Epidemol. 109, 273-284. Wertheimer, N., and Leeper, E. (1982) Znt. .I. Epidemiol. 11, 345-355. Coleman, M., Bell, J., and Skeet, R. (1983) Lancet 1, 982-983. Milham Jr., S. (1982) N. En@. J. Med. 307, 249. Pearce, N. E. et aZ.,(1985) Lancet 1, 811-812. Bassett, C. A. L., Mitchell, S. N., and Gaston, S. R. (1982) J. Am. Med. Assoc. 247, 623-628. Bassett, C. A. L., Schink, M. M., and Gaston, S. R. (1981) Trans. First Annu. Meet. Bioelec. Repair and Growth Sot. 1, 38-47. Brighton C. et aZ., (1981) .I. Bone and Joiflt Sq. 63-A, 2-21. Clear-y, S. F. (1987) ZEEE Eng. Med. and Biol. 6, 26-30. Polk, C., and E. Postow, E. (1985) CRC Handbook of Biological Effects of Electromagnetic Field, CRC Press, Boca Raton, FL. Goodman, R., Bassett, C. A. L., and Henderson, A. (1983) Science 22, 12831285. Goodman, R., and Henderson, A. S. (1986) Bioelectromagnetics 7, 23-29. Bassen, H. et al., Manuscript submitted. Foster, K. R., and Schwan, H. P. (1989) CRC Critical Reviews in Biomed. Eng. 17, 25 104. Blank, M., and Goodman, R. (1988) Bioelectrochem. Bioenerget. 19, 565-571. Schwan, H. P., and Foster, K. R. (1980) IEEE Trans. Biomed. Eng. 68, 104-113. Weaver, J. C., and Astumian, R. D. (1990) Science 247, 459-462.
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