Electromagnetic fields and DNA damage

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Pathophysiology 16 (2009) 79–88
Electromagnetic fields and DNA damage
J.L. Phillips a,∗, N.P. Singh b,H.Lai
b
aDepartment of Chemistry, University of Colorado at Colorado Springs, Colorado Springs, CO 80918, USA
bDepartment of Bioengineering, University of Washington, Seattle, WA 98195, USA
Received 24 October 2008; received in revised form 16 November 2008; accepted 16 November 2008
Abstract
A major concern of the adverse effects of exposure to non-ionizing electromagnetic field (EMF) is cancer induction. Since the majority of
cancers are initiated by damage to a cell’s genome, studies have been carried out to investigate the effects of electromagnetic fields on DNA and
chromosomal structure. Additionally, DNA damage can lead to changes in cellular functions and cell death. Single cell gel electrophoresis, also
known as the ‘comet assay’, has been widely used in EMF research to determine DNA damage, reflected as single-strand breaks, double-strand
breaks, and crosslinks. Studies have also been carried out to investigate chromosomal conformational changes and micronucleus formation
in cells after exposure to EMF. This review describes the comet assay and its utility to qualitatively and quantitatively assess DNA damage,
reviews studies that have investigated DNA strand breaks and other changes in DNA structure, and then discusses important lessons learned
from our work in this area.
© 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Electromagnetic field; DNA damage; Comet assay; Radiofrequency radiation; Cellular telephone
1. The comet assay for measurement of DNA strand
breaks
DNA is continuously damaged by endogenous and exoge-
nous factors and then repaired by DNA repair enzymes. Any
imbalance in damage and repair and mistakes in repair result
in accumulation of DNA damage. Eventually, this will lead
to cell death, aging, or cancer. There are several types of
DNA lesions. The common ones that can be detected easily
are DNA strand breaks and DNA crosslinks. Strand breaks in
DNA are produced by endogenous factors, such as free radi-
cals generated by mitochondrial respiration and metabolism,
and by exogenous agents, including UV, ionizing and non-
ionizing radiation, and chemicals.
There are two types of DNA strand breaks: single- and
double-strand breaks. DNA single-strand breaks include
frank breaks and alkali labile sites, such as base modifica-
tion, deamination, depurination, and alkylation. These are
the most commonly assessed lesions of DNA. DNA double-
strand breaks are very critical for cells and usually they are
∗Corresponding author.
E-mail address: jphillip@mail.uccs.edu (J.L. Phillips).
lethal. DNA strand breaks have been correlated with cell
death [1–5], aging [6–8] and cancer [9–13].
Several techniques have been developed to analyze single-
and double-strand breaks. Most commonly used is micro-
gel electrophoresis, also called the ‘comet assay’ or ‘single
cell gel electrophoresis’. This technique involves mixing
cells with agarose, making microgels on a microscope slide,
lysing cells in the microgels with salts and detergents,
removing proteins from DNA by using proteinase K, unwind-
ing/equilibrating and electrophoresing DNA (under highly
alkaline condition for assessment of single-strand breaks or
under neutral condition for assessment of DNA double-strand
breaks), fixing the DNA, visualizing the DNA with a fluores-
cent dye, and then analyzing migration patterns of DNA from
individual cells with an image analysis system.
The comet assay is a very sensitive method of detect-
ing single- and double-strand breaks if specific criteria are
met. Critical criteria include the following. Cells from tis-
sue culture or laboratory animals should be handled with
care to minimize DNA damage, for instance, by avoiding
light and high temperature. When working with animals
exposed to EMF in vivo, it is better to anesthetize the animals
with CO2before harvesting tissues for assay. Antioxidants
0928-4680/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.pathophys.2008.11.005

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80 J.L. Phillips et al. / Pathophysiology 16 (2009) 79–88
such as albumin and sucrose, or spin-trap molecules such
as ␣-phenyl-tert-butyl nitrone (PBN), should be added dur-
ing dispersion of tissues into single cells. Cells should be
lysed at 0–4 ◦C to minimize DNA damage by endonucle-
ases. Additionally, antioxidants such as tris and glutathione,
and chelators such as EDTA, should be used in the lysing
solution. High concentrations of dimethylsulfoxide (DMSO)
should be avoided due to its chromatin condensing effect.
Treatment with proteinase K (PK; lyophilized DNAse-free
proteinase-K from Amresco is ideal) at a concentration of
0.5–1 mg/ml (depending upon cell type and number of cells
in the microgel) should be used for 1–2 h at 37 ◦C to reveal all
possible strand breaks which otherwise may go undetected
due to DNA–protein crosslinks. Longer times in PK will lead
to loss of smaller pieces of DNA by diffusion. Glass slides
should be chosen based on which high resolution agarose
(3:1 high resolution agarose from Amresco is ideal) will stick
well to the slide and on the ability of the specimen to be visu-
alized without excessive fluorescence background. Choice
of an electrophoresis unit is important to minimize slide-to-
slide variation in DNA migration pattern. A unit with uniform
electric field and buffer recirculation should be used. Elec-
trophoresis buffers should have antioxidants and chelators
such as DMSO and EDTA. DNA diffusion should be mini-
mized during the neutralization step by rapidly precipitating
the DNA. Staining should employ a sensitive fluorescent dye,
such as the intercalating fluorescent labeling dye YOYO-1.
A cell-selection criteria for analysis should be set before the
experiment, such as not analyzing cells with too much dam-
age, although, the number of such cells should be recorded.
There are different versions of the comet assay that have
been modified to meet the needs of specific applications and
to improve sensitivity. Using the most basic form of the
assay, one should be able to detect DNA strand breaks in
human lymphocytes that were induced by 5 rad of gamma-ray
[14,15].
2. Radiofrequency radiation (RFR) and DNA
damage
In a series of publications, Lai and Singh [16–19] reported
increases in single- and double-strand DNA breaks, as mea-
sured by the comet assay, in brain cells of rats exposed for 2 h
to a 2450-MHz RFR at whole body specific absorption rate
(SAR) between 0.6 and 1.2 W/kg. The effects were blocked
by antioxidants, which suggested involvement of free radi-
cals. At the same time, Sarkar et al. [20] exposed mice to
2450-MHz microwaves at a power density of 1 mW/cm2for
2 h/day over a period of 120, 150, and 200 days. Rearrange-
ment of DNA segments were observed in testis and brain
of exposed animals. Their data also suggested breakage of
DNA strands after RFR exposure. Phillips et al. [21] were
the first to study the effects of two forms of cell cellular
phone signals, known as TDMA and iDEN, on DNA dam-
age in Molt-4 human lymphoblastoid cells using the comet
assay. These cells were exposed to relatively low intensities
of the fields (2.4–26 ␮W/g) for 2–21 h. They reported both
increased and decreased DNA damage, depending on the type
of signal studied, as well as the intensity and duration of expo-
sure. They speculated that the fields may affect DNA repair in
cells. Subsequently, different groups of researchers have also
reported DNA damage in various types of cells after expo-
sure to cell phone frequency fields. Diem et al. [22] exposed
human fibroblasts and rat granulosa cells to cell phone signal
(1800 MHz; SAR 1.2 or 2 W/kg; different modulations; for
4, 16 and 24 h; intermittent 5 min on/10 min off or continu-
ous). RFR exposure induced DNA single- and double-strand
breaks as measured by the comet assay. Effects occurred after
16 h of exposure to different cell phone modulations in both
cell types. The intermittent exposure schedule caused a sig-
nificantly stronger effect than continuous exposure. Gandhi
and Anita [23] reported increases in DNA strand breaks and
micronucleation in lymphocytes obtained from cell phone
users. Markova et al. [24] reported that GSM signals affected
chromatin conformation and ␥-H2AX foci that co-localized
in distinct foci with DNA double-strand breaks in human
lymphocytes. The effect was found to be dependent on carrier
frequency. Nikolova et al. [25] reported a low and transient
increase in DNA double-strand breaks in mouse embryonic
stem cells after acute exposure to a 1.7-GHz field. Lixia et
al. [26] reported an increase in DNA damage in human lens
epithelial cells at 0 and 30 min after 2 h of exposure to a
1.8-GHz field at 3 W/kg. Sun et al. [27] reported an increase
in DNA single-strand breaks in human lens epithelial cells
after 2 h of exposure to a 1.8-GHz field at SARs of 3 and
4 W/kg. DNA damage caused by the field at 4W/kg was irre-
versible. Zhang et al. [28] reported that an 1800-MHz field at
3.0 W/kg induced DNA damage in Chinese hamster lung cells
after 24 h of exposure. Aitken et al. [29] exposed mice to a
900-MHz RFR at a SAR of 0.09 W/kg for 7 days at 12 h per
day. DNA damage in caudal epididymal spermatozoa was
assessed by quantitative PCR (QPCR) as well as by alka-
line and pulsed-field gel electrophoresis. Gel electrophoresis
revealed no significant change in single- or double-strand
breaks in spermatozoa. However, QPCR revealed statistically
significant damage to both the mitochondrial genome and the
nuclear ␤-globin locus. Changes in sperm cell genome after
exposure to 2450-MHz microwaves have also been reported
previously by Sarkar et al. [20]. Related to this are sev-
eral publications that have reported decreased motility and
changes in morphology in isolated sperm cells exposed to
cell phone radiation [30], sperm cells from animals exposed
to cell phone radiation [31], and cell phone users [32–34].
Some of these in vivo effects could be caused by hormonal
changes [35,36].
There also are studies reporting no significant effect of cell
phone RFR exposure on DNA damage. After RFR-induced
DNA damage was reported by Lai and Singh [16] using
2450-MHz microwaves and after the report of Phillips et
al. [21] on cell phone radiation was published, Motorola
funded a series of studies by Roti Roti and colleagues [37] at

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J.L. Phillips et al. / Pathophysiology 16 (2009) 79–88 81
Washington University to investigate DNA strand breaks
in cells and animals exposed to RFR. None of the stud-
ies reported by this group found significant effects of RFR
exposure on DNA damage [38–40]. However, a different ver-
sion of the comet assay was used in these studies. More
recently, four additional studies from the Roti-Roti labora-
tories also reported no significant effects on DNA damage
in cells exposed to RFR. Li et al. [41] reported no signif-
icant change in DNA strand breaks in murine C3H10T1/2
fibroblasts after 2 h of exposure to 847.74- and 835.02-
MHz fields at 3–5 W/kg. Hook et al. [42] showed that a
24-h exposure of Molt-4 cells to CDMA, FDMA, iDEN or
TDMA-modulated RFR did not significantly alter the level of
DNA damage. Lagroye et al. [43,44] also reported no signifi-
cant change in DNA strand breaks, protein–DNA crosslinks,
and DNA–DNA crosslinks in cells exposed to 2450-MHz
RFR.
From other laboratories, Vijayalaxmi et al. [45] reported
no increase in DNA stand breaks in human lymphocytes
exposed in vitro to 2450-MHz RFR at 2.135W/kg for 2h.
Tice et al. [46] measured DNA single-strand breaks in human
leukocytes using the comet assay after exposure to various
forms of cell phone signals. Cells were exposed for 3 or 24 h at
average SARs of 1.0–10.0 W/kg. Exposure for either 3 or 24 h
did not induce a significant increase in DNA damage in leuko-
cytes. McNamee et al. [47–49] found no significant increase
in DNA breaks and micronucleus formation in human leuko-
cytes exposed for 2 h to a 1.9-GHz field at SAR up to 10 W/kg.
Zeni et al. [50] reported that a 2-h exposure to 900-MHz GSM
signal at 0.3 and 1 W/kg did not significantly affect levels of
DNA strand breaks in human leukocytes. Sakuma et al. [51]
exposed human glioblastoma A172 cells and normal human
IMR-90 fibroblasts from fetal lungs to cell phone radiation
for 2 and 24 h. No significant changes in DNA strand breaks
were observed up to a SAR of 800 mW/kg. Stronati et al. [52]
showed that 24 h of exposure to 935-MHz GSM basic signal
at 1 or 2 W/Kg did not cause DNA strand breaks in human
blood cells. Verschaeve et al. [53] reported that long-term
exposure (2 h/day, 5 days/week for 2 years) of rats to 900-
MHz GSM signal at 0.3 and 0.9 W/kg did not significantly
affect levels of DNA strand breaks in cells.
3. Extremely low frequency electromagnetic fields
(ELF EMF) and DNA damage
To complete the picture, a few words on the effects of ELF
EMF are required, since cell phones also emit these fields and
they are another common form of non-ionizing EMF in our
environment. Quite a number of studies have indicated that
exposure to ELF EMF could lead to DNA damage [54–69].
In addition, two studies [70,71] have reported effects of ELF
fields on DNA repair mechanisms. Free radicals and interac-
tion with transitional metals (e.g., iron) [60,62,63,69] have
also been implicated to play a role in the genotoxic effects
observed after exposure to these fields.
4. Some considerations on the effects of EMF on
DNA
From this brief literature survey, no consistent pattern of
RFR exposure inducing changes in or damage to DNA in
cells and organisms emerges. However, one can conclude that
under certain conditions of exposure, RFR is genotoxic. Data
available are mainly applicable only to radiation exposure
that would be typical during cell phone use. Other than the
study of Phillips et al. [21], there is no indication that RFR at
levels that one can experience in the vicinity of base stations
and RF-transmission towers could cause DNA damage.
Differences in experimental outcomes are expected since
many factors could influence the outcome of experiments
in EMF research. Any effect of EMF has to depend on the
energy absorbed by a biological organism and on how the
energy is delivered in space and time. Frequency, intensity,
exposure duration, and the number of exposure episodes can
affect the response, and these factors can interact with each
other to produce different effects. In addition, in order to
understand the biological consequence of EMF exposure, one
must know whether the effect is cumulative, whether com-
pensatory responses result, and when homeostasis will break
down. The contributions of these factors have been discussed
in a talk given by one us (HL) in Vienna, Austria in 1998
[72].
Radiation from cell phone transmission has very com-
plex patterns, and signals vary with the type of transmission.
Moreover, the technology is constantly changing. Research
results from one types of transmission pattern may not be
applicable to other types. Thus, differences in outcomes of
the research on genotoxic effects of RFR could be explained
by the many different exposure conditions used in the studies.
An example is the study of Phillips et al. [21], which demon-
strated that different cell phone signals could cause different
effects on DNA (i.e., an increase in strand breaks after expo-
sure to one type of signal and a decrease with another). This is
further complicated by the fact that some of the studies listed
above used poor exposure procedures with very limited doc-
umentation of exposure parameters, e.g., using an actual cell
phone to expose cells and animals, thus rendering the data
from these experiments as questionable.
Another source of influence on experimental outcome is
the cell or organism studied. Many different biological sys-
tems were used in the genotoxicity studies. Different cell
types [73] and organisms [74,75] may not all respond simi-
larly to EMF.
Comment about the comet assay also is required, since
it was used in many of the EMF studies to determine DNA
damage. Different versions of the assay have been developed.
These versions have different detection sensitivities and can
be used to measure different aspects of DNA strand breaks. A
comparison of data from experiments using different versions
of the assay could be misleading. Another concern is that most
of the comet assay studies were carried out by experimenters
who had no prior experience with this technique and mistakes

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82 J.L. Phillips et al. / Pathophysiology 16 (2009) 79–88
Fig. 1. A representation of the Fenton reaction and its role as a mediator in
EMF-induced bioeffects.
were made. For example, in the study by Lagroye et al. [43]
to investigate the effect of PK digestion on DNA migration
after RFR exposure, PK was added to a lysing solution con-
taining the detergent Triton X-100, which would inactivate
the enzyme. Our experience indicates that the comet assay
is a very sensitive and requires great care to perform. Thus,
different detection sensitivities could result in different labo-
ratories, even if the same procedures are followed. One way
to solve this problem of experimental variation is for each
research team to report the sensitivity of their comet assay,
e.g., the threshold of detecting strand breaks in human lym-
phocytes exposed to X-rays. This information has generally
not been provided for EMF-genotoxicity studies. Interest-
ingly, when such information was provided, a large range of
sensitivities have been reported. Malyapa et al. [40] reported a
detection level of 0.6 cGy of gamma radiation in human lym-
phocytes, whereas McNamee et al. [76] reported 10–50 cGy
of X-irradiation in lymphocytes, which is much higher than
the generally acceptable detection level of the comet assay
[15].
A drawback in the interpretation and understanding of
experimental data from bioelectromagnetics research is that
there is no general acceptable mechanism on how EMF
affects biological systems. The mechanism by which EMF
produces changes in DNA is unknown. Since the energy level
associated with EMF exposure is not sufficient to cause direct
breakage of chemical bonds within molecules, the effects are
probably indirect and secondary to other induced biochemical
changes in cells.
One possibility is that DNA is damaged by free radicals
that are formed inside cells. Free radicals affect cells by dam-
aging macromolecules, such as DNA, protein, and membrane
lipids. Several reports have indicated that EMF enhances free
radical activity in cells [18,19,61,62,77,78], particularly via
the Fenton reaction [62]. The Fenton reaction is a process
catalyzed by iron in which hydrogen peroxide, a product of
oxidative respiration in the mitochondria, is converted into
hydroxyl free radicals, which are very potent and cytotoxic
molecules (Fig. 1).
It is interesting that ELF EMF has also been shown to
cause DNA damage. Furthermore, free radicals have been
implicated in this effect of ELF EMF. This further supports
the view that EMF affects DNA via an indirect secondary
process, since the energy content of ELF EMF is much lower
than that of RFR. Effects via the Fenton reaction predict how
a cell would respond to EMF. For instance:
(1) Cells that are metabolically active would be more sus-
ceptible to EMF, because more hydrogen peroxide is
generated by mitochondria to fuel the reaction.
(2) Cells that have high level of intracellular free iron would
be more vulnerable to EMF. Cancer cells and cells under-
going abnormal proliferation have higher concentrations
of free iron because they uptake more iron and have less
efficient iron storage regulation. Thus, these cells could
be selectively damaged by EMF. Consequently, this sug-
gests that EMF could potentially be used for the treatment
of cancer and hyperplastic diseases. The effect could be
further enhanced if one could shift anaerobic glycoly-
sis of cancer cells to oxidative glycolysis. There is quite
a large database of information on the effects of EMF
(mostly in the ELF range) on cancer cells and tumors.
The data tend to indicate that EMF could retard tumor
growth and kill cancer cells. One consequence of this
consideration is that epidemiological studies of cancer
incidence in cell phone users may not show a risk at all
or even a protection effect.
(3) Since the brain is exposed to rather high levels of
EMF during cell phone use, the consequences of EMF-
induced genetic damage in brain cells are of particular
importance. Brain cells have high levels of iron. Spe-
cial molecular pumps are present on nerve cell nuclear
membranes to pump iron into the nucleus. Iron atoms
have been found to intercalate within DNA molecules. In
addition, nerve cells have a low capacity for DNA repair,
and DNA breaks could easily accumulate. Another con-
cern is the presence of superparamagnetic iron-particles
(magnetites) in body tissues, particularly in the brain.
These particles could enhance free radical activity in cells
and thus increase the cellular-damaging effects of EMF.
These factors make nerve cells more vulnerable to EMF.
Thus, the effect of EMF on DNA could conceivably be
more significant on nerve cells than on other cell types of
the body. Since nerve cells do not divide and are not likely
to become cancerous, the more likely consequences of
DNA damage in nerve cells include changes in cellular
functions and in cell death, which could either lead to
or accelerate the development of neurodegenerative dis-
eases. Double-strand breaks, if not properly repaired, are
known to lead to cell death. Cumulative DNA damage in
nerve cells of the brain has been associated with neurode-
generative diseases, such as Alzheimer’s, Huntington’s,
and Parkinson’s diseases. However, another type of brain
cell, the glial cell, can become cancerous as a result of
DNA damage. The question is whether the damaged cells

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J.L. Phillips et al. / Pathophysiology 16 (2009) 79–88 83
would develop into tumors before they are killed by EMF
due to over accumulation of genetic damages. The out-
come depends on the interplay of these different physical
and biological factors—an increase, decrease, or no sig-
nificant change in cancer risk could result from EMF
exposure.
(4) On the other hand, cells with high amounts of
antioxidants and antioxidative enzymes would be less
susceptible to EMF. Furthermore, the effect of free
radicals could depend on the nutritional status of an
individual, e.g., availability of dietary antioxidants, con-
sumption of alcohol, and amount of food consumption.
Various life conditions, such as psychological stress and
strenuous physical exercise, have been shown to increase
oxidative stress and enhance the effect of free radicals in
the body. Thus, one can also speculate that some indi-
viduals may be more susceptible to the effects of EMF
exposure.
Additionally, the work of Blank and Soo [79] and Blank
and Goodman [80] support the possibility that EMF exposure
at low levels has a direct effect on electron transfer processes.
Although the authors do not discuss their work in the con-
text of EMF-induced DNA damage, the possibility exists that
EMF exposure could produce oxidative damage to DNA.
5. Lessons learned
Whether or not EMF causes biological effects, let alone
effects that are detrimental to human health and development,
is a contentious issue. The literature in this area abounds
with apparently contradictory studies, and as presented in this
review, the literature specific to the effects of RFR exposure
on DNA damage and repair in various biological systems is
no exception. As a consequence of this controversy, there
are several key issues that must be addressed—contrary data,
weight of evidence, and data interpretation consistent with
known science.
Consider that EMF does not share the familiar and com-
forting physical properties of chemical agents. EMF cannot
be seen, tasted, smelled, or felt (except at high intensities).
It is relevant, therefore, to ask, in what ways do scientists
respond to data, especially if that data are contrary to their
scientific beliefs or inconsistent with long-held hypotheses?
Often such data are ignored, simply because it contradict what
is accepted as conventional wisdom. Careful evaluation and
interpretation of data may be difficult, because technologies
used to expose biological systems to EMF and methodologies
used to assess dosimetry generally are outside the experience
of most biomedical scientists. Additionally, it is often diffi-
cult to assess differences in methodologies between studies,
one or more of which were intended to replicate an origi-
nal investigation. For instance, Malyapa et al. [40] reported
what they claimed to be a replication of the work of Lai
and Singh [16]. There were, however, significant differences
in the comet analyses used by each group. Lai and Singh
precipitated DNA in agarose so that low levels of DNA dam-
age could be detected. Malyapa et al. did not. Lai and Singh
treated their samples with PK to digest proteins bound to
DNA, thus allowing DNA to move toward the positive pole
during electrophoresis (unlike DNA, most proteins are nega-
tively charged, and if they are not removed they will drag the
DNA toward the negative pole). The Malyapa et al. study did
not use PK. There were other methodological differences as
well. Such is also the case in the study of Hook et al. [42],
which attempted to replicate the work of Phillips et al. [21].
The latter group used a PK treatment in their comet assay,
while the former group did not.
While credibility is enhanced when one can relate data
to personal knowledge and scientific beliefs, it has not yet
been determined how RFR couples with biological systems
or by what mechanisms effects are produced. Even carefully
designed and well executed RFR exposure studies may be
summarily dismissed as methodologically unsound, or the
data may be interpreted as invalid because of inconsisten-
cies with what one believes to be correct. The quintessential
example is the belief that exposure to RFR can produce no
effects that are not related to the ability of RFR to produce
heat, that is, to raise the temperature of biological systems
[81,82]. Nonetheless, there are many examples of biologi-
cal effects resulting from low-level (athermal) RFR exposure
[83,84]. Consider here the work of Mashevich et al. [85]. This
group exposed human peripheral blood lymphocytes to an
830-MHz signal for 72 h and at different average SARs (SAR,
1.6–8.8 W/kg). Temperatures ranged from 34.5 to 38.5◦C.
This group observed an increase in chromosome 17 aneu-
ploidy that varied linearly with SAR. Temperature elevation
alone in the range of 34.5–38.5 ◦C did not produce this geno-
toxic effect, although significant aneuploidy was observed
at higher temperatures of 40–41 ◦C. The authors conclude
that the genotoxic effect of the radiofrequency signal used is
elicited through a non-thermal pathway.
Also consider one aspect of the work of Phillips et al. [21].
In that study, DNA damage was found to vary in direction;
that is, under some conditions of signal characteristics, signal
intensity, and time of exposure, DNA damage increased as
compared with concurrent unexposed controls, while under
other conditions DNA damage decreased as compared with
controls. The dual nature of Phillips et al.’s [21] results
will be discussed later. For now consider the relationship of
these results to other investigations. Adey et al. [86] per-
formed an in vivo study to determine if rats treated in utero
with the carcinogen ethylnitrosourea (ENU) and exposed to
an 836.55-MHz field with North American Digital Cellular
modulation (referred to as a TDMA field) would develop
increased numbers of central system tumors. This group
reported that rather than seeing an increase in tumor inci-
dence in RFR-exposed rats, there was instead a decrease in
tumor incidence. Moreover, rats that received no ENU but
which were exposed to the TDMA signal also showed a
decrease in the number of spontaneous tumors as compared

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84 J.L. Phillips et al. / Pathophysiology 16 (2009) 79–88
with animals exposed to neither ENU nor the TDMA signal.
This group postulated that their results may be mechanis-
tically similar to the work of another group. Stammberger
et al. [87] had previously reported that rats treated in utero
with ENU and then exposed to low doses of X-irradiation
exhibited significantly reduced incidences of brain tumors
in adult life. Stammberger and colleagues [87] hypothe-
sized that low-level X-irradiation produced DNA damage that
then induced the repair enzyme 06-alkylguanine-DNA alkyl-
transferase (AT). Numerous groups have since reported that
X-irradiation does indeed induce AT activity (e.g., [88,89]).
In this context, it is significant that Phillips et al. [21] found
that cells exposed in vitro to a TDMA signal identical to that
used in the study of Adey et al. [86] produced a decrease in
DNA damage under specific conditions of intensity and time
of exposure (lower intensity, longer time; higher intensity,
shorter time). These results raise the intriguing possibility
that the decrease in tumor incidence in the study of Adey et al.
[86] and the decrease in DNA damage in the study of Phillips
et al. [21] both may have been the result of induction of AT
activity resulting from DNA damage produced by exposure
to the TDMA signal. This remains to be investigated.
Because the issue of RFR-induced bioeffects is con-
tentious, and because the issue is tried in courtrooms and
various public forums, a term heard frequently is weight of
evidence. This term generally is used to describe a method
by which all scientific evidence related to a causal hypothesis
is considered and evaluated. This process is used extensively
in matters of regulation, policy, and the law, and it provides
a means of weighing results across different modalities of
evidence. When considering the effects of RFR exposure
on DNA damage and repair, modalities of evidence include
studies of cells and tissues from laboratory animals exposed
in vivo to RFR, studies of cells from humans exposed to
RFR in vivo, and studies of cells exposed in vitro to RFR.
While weight of evidence is gaining favor with regulators
[90], its application by scientists to decide matters of science
is often of questionable value. One of the reasons for this
is that there generally is no discussion or characterization
of what weight of evidence actually means in the context
in which it is used. Additionally, the distinction between
weight of evidence and strength of evidence often is lack-
ing or not defined, and differences in methodologies between
investigators are not considered. Consequently, weight of evi-
dence generally amounts to what Krimsky [90] refers to as
a “seat-of-the-pants qualitative assessment.” Krimsky points
out that according to this view, weight of evidence is “a vague
term that scientists use when they apply implicit, qualitative,
and/or subjective criteria to evaluate a body of evidence.”
Such is the case in the reviews by Juutilainen and Lang [91]
and Verschaeve and Maes [92]. There is little emphasis on
a critical analysis of similarities and differences in biolog-
ical systems used, exposure regimens, data produced, and
investigator’s interpretations and conclusions. Rather, there is
greater emphasis on the number of publications either finding
or not finding an effect of RFR exposure on some endpoint.
To some investigators, weight of evidence does indeed refer
to the balance (or imbalance) between the number of stud-
ies producing apparently opposing results, without regard to
critical experimental variables. While understanding the role
these variables play in determining experimental outcome
could provide remarkable insights into defining mechanisms
by which RFR produced biological effects, few seem inter-
ested in or willing to delve deeply into the science.
A final lesson can be derived from a statement made by
Gos et al. [93] referring to the work of Phillips et al. [21]. Gos
and colleagues state, “The results in the latter study (Phillips
et al., 1998) are puzzling and difficult to interpret, as no con-
sistent increase or decrease in signal in the comet assay at
various SARs or times of exposure was identified.” This state-
ment is pointed out because studies of the biological effects of
exposure to electromagnetic fields at any frequency are often
viewed as outside of or distinct from what many refer to as
mainstream science. However, what has been perceived as an
inconsistent effect is indeed consistent with the observations
of bimodal effects reported in hundreds of peer-reviewed
publications. These bimodal effects may be dependent on
concentration of an agent, time of incubation with an agent,
or some other parameter relating to the state of the system
under investigation. For instance, treatment of B cells for
a short time (30 min) with the protein kinase C activator
phorbol 12,13-dibutyrate increased proliferative responses
to anti-immunoglobulin antibody, whereas treatment for a
longer period of time (≥3 h) suppressed proliferation [94].
In a study of ␬-opioid agonists on locomotor activity in
mice, Kuzmin et al. [95] reported that higher, analgesic doses
of ␬-agonists reduced rearing, motility, and locomotion in
non-habituated mice. In contrast, lower, subanalgesic doses
increased motor activity in a time-dependent manner. Dierov
et al. [96] observed a bimodal effect of all-trans-retinoic acid
(RA) on cell cycle progression in lymphoid cells that was
temporally related to the length of exposure to RA. A final
example is found in the work of Rosenstein et al. [97]. This
group found that the activity of melatonin on depolarization-
induced calcium influx by hypothalamic synaptosomes from
rats sacrificed late evening (2000 h) depended on melatonin
preincubation time. A short preincubation time (10 min) stim-
ulated uptake, while a longer preincubation (30 min) inhibited
calcium uptake. These effects were also dependent on the
time of day when the rats were sacrificed. Effects were max-
imal at 2000 h, minimal at 2400h, and intermediate at 400h.
At 1000 h, only inhibitory effects of melatonin on calcium
uptake were observed. These examples point out that what
appears to be inconsistency may instead be real events related
to and determined by the agents involved and the state of the
biological system under investigation. The results of Phillips
et al. [21] may be the result of signal modulation, signal
intensity, time of exposure, or state of the cells. The results
may indicate a bimodal effect, or they may, as the investiga-
tors suggest, represent time- and signal-dependant changes
in the balance between damage and repair because of direct
or indirect effects of RFR exposure on repair mechanisms.

Author's personal copy
J.L. Phillips et al. / Pathophysiology 16 (2009) 79–88 85
6. Summary
Exposure of laboratory animals in vivo and of cultured
cells in vitro to various radiofrequency signals has produced
changes in DNA damage in some investigations and not in
others. That many of the studies on both sides of this issue
have been done well is encouraging from a scientific perspec-
tive. RFR exposure does indeed appear to affect DNA damage
and repair, and the total body of available data contains
clues as to conditions producing effects and methodologies
to detect them. This view is in contrast to that of those who
believe that studies unable to replicate the work of others are
more credible than the original studies, that studies showing
no effects cancel studies showing an effect, or that stud-
ies showing effects are not credible simply because we do
not understand how those effects might occur. Some may
be tempted to apply incorrectly the teachings of Sir Karl
Popper, one of the great science philosophers of the 20th
century. Popper proposed that many examples may lend sup-
port to an hypothesis, while only one negative instance is
required to refute it [98]. While this holds most strongly for
logical subjects, such as mathematics, it does not hold well
for more complex biological phenomena that are influenced
by stochastic factors. Each study to investigate RFR-induced
DNA damage must be evaluated on its own merits, and then
studies that both show effects and do not show effects must be
carefully evaluated to define the relationship of experimental
variables to experimental outcomes and to assess the value
of experimental methodologies to detect and measure these
outcomes (see Section 2).
The lack of a causal or proven mechanism(s) to explain
RFR-induced effects on DNA damage and repair does not
decrease the credibility of studies in the scientific literature
that report effects of RFR exposure, because there are sev-
eral plausible mechanisms of action that can account for the
observed effects. The relationship between cigarette smok-
ing and lung cancer was accepted long before a mechanism
was established. This, however, occurred on the strength of
epidemiologic data [99]. Fortunately, relevant epidemiologic
data relating long-term cell phone use (>10 years) to central
nervous system tumors are beginning to appear [84,100–102],
and these data point to an increased risk of acoustic neuroma,
glioma and parotid gland tumors.
One plausible mechanism for RFR-induced DNA damage
is free radical damage. After finding that two free radi-
cal scavengers (melatonin and N-tert-butyl-␣-phenylnitrone)
prevent RFR-induced DNA damage in rat brain cells, Lai
and Singh [62] hypothesized that this damage resulted from
free radical generation. Subsequently, other reports appeared
that also suggested free radical formation as a result of RFR
exposure [103–105]. Additionally, some investigators have
reported that non-thermal exposure to RFR alters protein
structure and function [106–109]. Scientists are familiar with
molecules interacting with proteins through lock-and-key or
induced-fit mechanisms. It is accepted that such interactions
provide energy to change protein conformation and protein
function. Indeed, discussions of these principles are presented
in introductory biology and biochemistry courses. Perhaps
then it is possible that RFR exposure, in a manner similar to
that of chemical agents, provides sufficient energy to alter the
structure of proteins involved in DNA repair mechanisms to
the extent that their function also is changed. This has not yet
been investigated.
When scientists maintain their beliefs in the face of con-
trary data, two diametrically opposed situations may result.
On the one hand, data are seen as either right or wrong and
there is no discussion to resolve disparities. On the other
hand, and as Francis Crick [110] has pointed out, scientists
who hold theoretically opposed positions may engage in fruit-
ful debate to enhance understanding of underlying principles
and advance science in general. While the latter certainly is
preferable, there are external factors involving economics and
politics that keep this from happening. It is time to acknowl-
edge this and embark on the path of fruitful discussion. Great
scientific discoveries await.
Acknowledgment
We thank Khushbu Komal and Ji-Sun Park for assistance
in the preparation of the manuscript.
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