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Carcinogenesis |
University of California, Riverside, California 92521 [W. R. A., C. V. B.]; University of California, Davis, California 95616 [R. J. H.]; Pettis Veterans Affairs Medical Center, Loma Linda, California 92357 [C. D. C., R. A. J., C. J. K., A. M., R. B. S.]; Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland [N. K.]; and Loma Linda University School of Allied Health Professions, Loma Linda, California 92350 [G. Z.]
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ABSTRACT |
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INTRODUCTION |
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No direct comparisons have been made in an animal bioassay of possible differences in their bioeffects, particularly with respect to brain tumorigenesis. FM signals remain constant in amplitude, whereas signals generated in accordance with the North American Digital Code create a train of pulses (packets) at 50/s, cycling on for one-third of an epoch and remaining silent for two-thirds (TDMA amplitude or pulse modulation). As will be discussed (1, 2, 3, 4, 5, 6, 7, 8) , a spectrum of amplitude modulation-dependent physiological responses have been reported. Most mobile phones operate in close proximity to the head. Portable FM phones, with maximum output powers of 0.6 W in the 800-MHz band, induce fields in the most exposed tissues at energy absorption rates equivalent to 1 W/kg ± 6 db, depending on the device’s position and design (9) .
There have been previous studies of tumor incidence in rats and mice after long-term microwave exposure (10, 11, 12, 13) . However, in none of these studies was detection of brain tumors a prime goal but was incidental in the course of whole-body analysis, both gross and histopathological. To achieve high accuracy in tumor numbers, we have examined a sequence of 20–25 sections/brain, as compared with three standard sections approved in protocols of the NTP.
In this study with Fischer 344 rats exposed to simulated FM phone fields, we have addressed four major questions: (a) Did lifetime exposure to the FM fields alter the spontaneous incidence of primary CNS tumors? (b) Did sham exposure of the rats, involving their immobilization, particularly in the near-field exposure system, cause levels of stress that might markedly alter primary CNS tumor incidence in these animals? (c) Did the short-lived neurocarcinogen ENU, given as a single low dose in utero, cause an expected increase in brain tumor incidence as a positive control? and (d) Did lifetime intermittent exposure to the FM fields for 24 months alter the incidence of low-level carcinogen-induced brain tumors, i.e., did the FM field act as a tumor promoter or as a tumor progression agent?
This experiment was designed as a 2-year bioassay to test the hypothesis that FM field exposure might alter either survival or primary CNS tumor incidence. We aimed to simulate a life-long exposure, beginning with exposures of fetal and preweanling rats. A low ENU dose was selected to give maximum sensitivity for detection of a modification of tumor incidence by the FM fields as a lifetime response.
Our choice of the Fischer 344 rat was based on: (a) its comprehensive pathology databases in the NTP; (b) an overall incidence and subtypes of CNS tumors resembling those of humans (14) ; (c) reduced variations in pathological responses as an inbred strain when compared with most outbred strains (15) ; and (d) greater longevity at 24 months (80%), mainly because of a lowered spontaneous tumor incidence, a vitally important factor in long-term studies (16) .
Single-strand breaks in rat brain DNA have been reported after acute low-intensity microwave exposure (17 , 18) and after prolonged exposure in mice (19) , but single strand breaks were not confirmed in a similar study (20) . Their reported increased incidence after microwave exposure is unlikely to result from a direct genotoxic microwave action but rather to interference with enzymatic DNA repair mechanisms (21, 22, 23) . In a mouse model, life-long intermittent microwave exposure has been related to an increased lymphoma incidence late in life (24) .
As in our counterpart study with digital phone field exposures in this rat model (25) , the major objective was to evaluate whether FM phone fields met some of the defining criteria for known neurocarcinogenic agents (26) . They include: (a) a consistent capability to increase tumor incidence; and (b) a reduction in latency period of tumors (reduced rat survival time).
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MATERIALS AND METHODS |
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ENU Administration.
A single dose (4 mg/kg body weight) of ENU (Sigma Chemical, St. Louis,
MO) was administered on GD 18 to dams in groups ES, EC, and EF
(n = 52) via the lateral tail vein as a
freshly prepared solution in a phosphate/citrate/saline buffer (pH 6.0,
adjusted with citric acid). The remaining 52 dams were injected with an
equal volume of buffer only (Sham-ENU injection). Because no data are
available for a dose-response relationship between transplacental ENU
and brain tumor incidence for the Fischer 344 rat, the dosage selected
was based on published data on the incidence of brain tumors in
offspring of Sprague Dawley rats (20
, 21)
and other rat
strains, such as the BD-IX (22)
. These strains have a
brain tumor incidence ranging from 12% to as high as 40% in some
experiments for ENU doses between 1 and 5 mg/kg administered on GD 20.
Experimental Groups.
All pups (n = 778) were born on GD 22 (84%)
or GD 23 (16%). Two of the 104 dams were not pregnant. Experimental
groups were then set up from weanlings of litters from the assigned
groups of dams. Gender distribution remained unknown until sexing at
weaning. In group EF (see alphabetic group designations below), two
litters (19 pups) were discarded because of unsatisfactory ENU
injections and consequent dose uncertainty. There were 750 pups for
distribution into the final experimental groups, 329 males and 421
females. We aimed at gender balance, based on an initial random
selection of three males and three females from each litter. However,
this selection method failed in group EF, where there was an aggregate
of only 38 males, leading to a ratio 38:52, male:female. These progeny
then became treatment cohorts in six groups, comprised of: Sham
ENU/Sham Field (SS) n = 90, 45 males and 45
females; Sham ENU/Field Exposed (SF), n = 90,
45 males and 45 females; ENU/Sham Field (ES), n = 90, 45 males and 45 females; ENU/Field Exposed (EF),
n = 90, 38 males and 52 females; ENU/Cage
Control (EC), n = 90, 45 males and 45
females; and Sham ENU/Cage Control (SC), n = 90, 45 males and 45 females (total, n = 540).
The cage control groups EC and SC were established to test for effects
of reported stresses associated with tube restraint of the type used in
the near-field FM field exposures (groups SS and ES).
FM Far Field (FF) and Near Field (NF) Exposures.
Daily (7 days/week) FF exposures of the pregnant females in groups SF
and EF began on GD 19 and continued until parturition 3 or 4 days
later. Thereafter, FF exposure of dams and offspring continued until
the pups were aged 21 days (25 FF exposures). All FF and sham exposures
were performed on rats in their home cages. To accommodate all dams
with their litters, three exposure time periods were used: 1600–1800,
1900–2100, and 2200–2400 h. NF exposures began at age 31 days. In the
10-day interim, the offspring were weaned and individually identified
by numbered tail tattooing but received no field exposures. NF
exposures were resumed on 4 consecutive days weekly until experiment
termination at 731–734 days (total, 384 NF exposures). NF and sham
exposures occurred at the same time of day for each group. Weekly
weighings and colony inspections were carried out on the fifth day.
Animal Handling and Veterinary Surveillance.
Rats were weighed weekly and housed by sex, exposure group, and
injection group, with two males or three females per cage. No
substitutions were made after tail tattooing. Rats were observed daily.
With the appearance of one or more clinical signs, such as progressive
weight loss, head tilt, ocular discharge, and others, they were
evaluated by the project veterinarian (C. J. K.), who determined
whether immediate euthanasia was required. All procedures (a.m.) were
in compliance with a protocol approved by the Animal Care Committee of
the Loma Linda Veterans Affairs Medical Center. Animal facilities at
this Center are fully accredited by the American Association for
Advancement of Laboratory Animal Care. The experiment was performed in
as blind a manner as possible in that at no time prior to the
conclusion of the experiment did the veterinarian or the pathologist
know to which groups the animals were assigned.
Necropsy Procedures and Pathological Examination.
Termination of the experiment took place over 4 days when the rats were
731–734 days of age. To the extent possible, the same numbers of
animals from each experimental group were euthanized on each of the 4
days. All animals that were terminated (n = 372) were lightly anesthetized in a CO2 chamber
and then received 1 ml heparin i.p. (1000 IU/ml), followed by deep
pentobarbital anesthesia (55 mg/kg i.p.). They were then perfused with
a peristaltic pump via the left ventricle with 240 ml of 10%
phosphate-buffered formol saline at a sustained intracardiac pressure
of 120 mm Hg. A complete gross necropsy of extraneural organs was
performed, and each carcass was then stored in fresh perfusion fixative
until the brain and spinal cord were removed for histopathological
examination. Using a rat brain matrix (Harvard Apparatus, Holliston,
MA), transverse sections of the brain 1.0-mm thick were cut and
processed sequentially (approximately 25 and 20 sections for males and
females, respectively). Transverse spinal cord sections were cut every
1 cm. Additional sections were taken at extraneural sites with gross
lesions. All sections were cut at 5-µm thickness and stained with H&E
and evaluated microscopically (R. J. H.). Thus, a greater number of
brain sections/brain was examined in this study as compared with
standard procedures in the carcinogenesis studies in Fischer 344 rats
in the NTP, which limited examinations to three coronal sections of the
brain at the levels of: (a) the frontal cortex and the basal
ganglia; (b) the parietal cortex and the diencephalon; and
(c) the cerebellum and pons.
Primary tumors of the CNS were classified into histological subtypes using guidelines reported previously (14 , 27 , 28) . Tumors grossly visible on external examination or after transverse sectioning of brain or spinal cord were classified by size as Ts or as MTs if only detected microscopically (29) . The number of primary brain tumors in each rat was evaluated by sequential arrangement of histological brain sections. Causes of death in rats not surviving to the end of the experiment were based on gross autopsy findings, supplemented where appropriate by histological evaluation of gross lesions. Additional independent peer review of tumor numbers and classification of CNS tumor subtypes was done by a panel organized by Experimental Pathology Laboratories, Inc. (Research Triangle Park, NC).
Data Analysis Methods.
Experimental design parameters and all statistical analyses were
contributed by our statistician (G. Z.). In planning this study, a
calculation of experimental power was made, based on tumor incidence
observed in our preceding Fischer rat study, where we tested
spontaneous and ENU-induced brain tumor incidence in exposures to
digital phone fields (25)
. Here, with an assumed
spontaneous brain tumor incidence of 8.3% from the previous study and
sample sizes of 90 rats in each group, we would be able to detect an
increase of 13% with a power of 80%. Similarly, with an ENU-induced
tumor incidence of 15% (25)
, we would be able to detect
an increase of 15% with a power of 80%. Using two-tailed tests, the
corresponding increases are 15 and 18% for a sample size of 90 rats
and a power of 80%.
Kaplan-Meier survival analysis (30)
was used to compare
all six groups and selected data subsets. As a product limit,
nonparametric estimate of the probability of survival, it avoids
assumptions of normality in follow-up studies where survival time is
known only for individuals dying during the study period. This estimate
is based on constructing time intervals containing only one death in
each interval. Here, the test’s usefulness is limited by severe
censoring of data. Incidence rates were compared using Z-statistics and
2 analysis. Both one-tailed and two-tailed
tests of the null hypothesis were calculated, with the initial premise
that the one-tailed test would be appropriate for our hypothesized
increase in tumor incidence.
Microwave Exposure Systems.
These have been described in detail elsewhere (18)
and
will be summarized here. An ideal simulation of exposure conditions for
the human cellular phone user would be restricted to a NF model.
However, because fetal tissue is believed to be maximally sensitive to
other known neurocarcinogens and because some groups received a
transplacental dose of the carcinogen ENU on GD 18, exposure of
pregnant animals was required. We therefore developed two complete
exposure systems. System I approximated FF conditions. It provided
initial exposures for pregnant dams, and later, for offspring up to
weaning at 21 days of age. After weaning, System II produced a NF
exposure from weaning to the end of the experiment, with each rat in
individual restraints.
System I: Large Horn Radiator for Simulated FF Exposures.
FF conditions were simulated with an approximately plane wave in a
large tapered horn. Rat cages were positioned in a vertically oriented
3 x 3 matrix at the square aperture of the horn (2.0 m
on a side). Sham exposures were made in a square chamber of identical
dimensions and materials. A power input of 37.2 W produced a mean field
power density at the horn aperture of 2.6 ± 0.50
mW/cm2 (mean ± SD). The horn was
excited in circular polarization to reduce possible
orientation-dependent coupling to the animals, because dams and pups
were free to move about their cages. Wavefront circularity (axial
ratio) was within 2.3 dB, and power density was within 1.6 dB across
the cage exposure area.
System II: Carousel NF Exposure System.
Long-term, intermittent exposures (2 h/day, 4 days/week) to the NF
began at age 33 days, using exposure platforms with 10 rats oriented
radially around a central antenna (the carousel system). The antenna is
a standard production half-wave sleeved dipole (Motorola). To
accommodate 120 rats simultaneously (60 field exposed, 60 sham), 12
exposure platforms were used. A plastic tubular restraint confined each
rat for the duration of the exposure. They faced the antenna at a fixed
distance from the tip of the nose (30 mm from weaning to 120 days, 45
mm thereafter). After a short training period (1 week) they would
freely enter the tubes and often slept during exposure. To accommodate
the 360 exposed/sham exposed rats in this study, exposures were
conducted in three shifts.
SARs in the NF.
We have tested a FM 836.55 MHz signal, with ±12.5 KHz maximum
deviation. Modulation was by a recorded pattern of "balanced
speech" that generated all major speech components in a 2-min epoch
that recycled continuously. The average antenna power of 2.5 ± 0.1 W was selected to produce the same average SARs in the
exposed brains as the slot average SAR in our previous study with
digital cell phone fields (25)
. Energy absorption levels
were tested by two different techniques, each of which was verified by
an independent method: numerical modeling verified by electric probe
measurements, and thermography verified by thermometric probes.
Numerical dosimetry (31
, 32)
was based on magnetic
resonance imaging data sets of a rat cadaver with a resolution of 0.125
mm3 in the brain and 1.0
mm3 in the rest of the body. The results have
been validated at 30 specific points within a cadaver brain, using an
electric field probe. Thermography exposed hemisected rat cadavers to a
235-W field at 836 MHz for 
90 s and acquisition of a series of
infrared images of the cut surfaces for 2 min. Thermography suffered
from "smearing" of the thermal image from effects of thermal
diffusion. Thermometry was based on a Vitek thermistor probe (BSD
Medical Devices, Salt Lake City, UT) placed on the cut surface of the
brain and readings compared with the thermographic images. These
measurements were complicated by imperfect contact between the sensor
and the tissue and by radiative cooling from thermistor leads. However,
within the described limitations, the dosimetric assessments agree well
(Table 1)
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RESULTS |
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80% of the natural life span of the Fischer 344 rat. Of the
original 540 rats, 168 (31%) either died spontaneously
(n = 45; 8%) or were euthanized
(n = 123; 23%) prior to the end of the
experiment because of severe clinical impairment. Deaths in the course
of the experiment are summarized in Fig. 1
. With the use of Kaplan-Meier statistics, the survival curves for all
six groups were compared. Because most rats in each group survived to
be sacrificed at the end of the study, each Kaplan-Meier analysis has
considerable censored data.
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There were the expected rates of a large granulocyte lymphocyte leukemia and pituitary adenomas; both are endogenous diseases in the Fischer 344 rat strain. Large granulocyte lymphocytes were detected in 137 of 540 rats (25%), based on hematocrits, differential WBC counts, and necropsy findings. There was no evidence of a differential incidence attributable to separate or combined exposures to ENU or FM fields. Incidence of pituitary tumors was assessed on necropsy inspection of the sella region and the adjoining hypothalamus and confirmed histologically, but histopathology was not done in normal-appearing pituitary glands. In 243 (45%) of 540 rats, no pituitary tumors were detected. In 194 rats (36%), macroscopic tumors were observed, and in 103 rats (19%), the lesions were large enough to produce hypothalamic compression. No significant difference in gender incidence was detected.
Incidence of Primary CNS Tumors.
There was a total of 60 primary tumors of the CNS in all groups. We
detected a total of 54 glial tumors (48 in the brain and 6 in the
spinal cord; Table 2
). These 54 tumors occurred in 52 rats. With the exception of two rats
each having two tumors (one rat with two astrocytomas in group EC, and
one rat with two mixed gliomas in group EF), all other rats had only a
single tumor. No FM field-mediated increase in number or incidence of
either spontaneous brain tumors (SS versus SF) nor of
ENU-induced brain tumors (ES versus EF) was observed (Table 2)
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By comparison, CNS tumor incidence increased sharply in rats receiving
ENU (ES, 22.2%; EF, 17.8%; EC, 14.4%). This increase was highly
significant in comparison with the spontaneous rates
(P < 0.0001), but as in the spontaneous
tumor groups, there were no statistically significant effects
attributable to FM field exposures. Sham exposure, where rats were
placed in an unenergized NF exposure apparatus, did not alter tumor
incidence when cage controls (EC and SC) were compared with their
counterpart groups (ES and SS). Analysis of overall brain tumor
incidence alone, with exclusion of spinal cord tumors, did not differ
from the combined tumor analysis for either spontaneous or ENU-induced
tumors. Here also, the presence of the FM field produced no measurable
effect. No significant gender differences were detected in incidence of
any of the six types of primary nervous system tumors listed in Table 2
.
In comparing the relative size of CNS tumors in rats that survived to full term, there were 2 Ts and 2 MTs in the SC group, 10 Ts and 6 MTs in the EC group, 20 Ts and 1 MT in the ES group, and 16 Ts and 1 MT in the EF group.
In comparing the incidence of tumor subtypes, comparison of sham and control rats with those receiving ENU indicated increased numbers of mixed gliomas in the ENU rats, but there were no effects on any of these characteristics attributable to FM field exposure.
Findings in Rats Dying before Experiment Termination (Unscheduled
Sacrifice, Preterm Rats).
In our previous research with animal tumor bioassays of 60-Hz magnetic
field effects, we noted that some magnetic field effects were most
apparent early during the copromotion phase, but their statistical
significance declined later in the carcinogenic process
(33)
. In the present study, rats dying prior to experiment
termination or found dead (unscheduled sacrifice, referred to hereafter
as "preterm" rats) have therefore been grouped separately in
analysis of tumor incidence and histopathology.
Six major categories of probable causes of death were identified in
rats dying before experiment termination at 731–734 days (Table 3)
. Of the 168 rats, 17 died in the SS group, 19 in the SF group, 34 in
the ES group, 35 in the EF group, 39 in the EC group, and 24 in the SC
group. There were no obvious differences in death rates within the
three groups not receiving ENU nor within the three groups that had
received ENU, nor did the additional factor of FM field exposure modify
death rates in either of these two clusters. Where primary neural
tumors were determined to be the cause of death (Table 3
, category 1),
no significant differences were detected in tumor incidence or tumor
histopathology attributable to FM field exposure (Table 4)
. Rats in category 7 (Table 3)
had no gross lesions at necropsy, and
confirmation of cause of death awaits complete histopathological
examination of all extraneural tissues.
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DISCUSSION |
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No previous long-term studies of microwave exposures at athermal levels in rats and mice have evaluated brain tumor incidence as a primary goal, reporting only findings incidental to whole-body gross and histopathological examinations. We have taken special care to minimize failures in recognizing small CNS tumors by examining a sequence of 20–25 sections/brain. This contrasts with the three standard sections used in the NTP. Other studies have used carrier frequencies from 435 MHz to 2.45 GHz with both continuous wave and pulsed fields. They tested effects on general health and longevity, immune status, and mammary tumor incidence, with negative findings. No primary brain tumors were reported in 200 exposed and sham-exposed Sprague Dawley rats exposed to pulsed and ELF-modulated (8 Hz) 2.45 GHz fields for 25 months (10) . In a series of studies in mice prone to mammary cancer, exposure to circularly polarized 2.45-GHz fields (SARs, 0.3–1.0 W/kg) for 18–21 months had no effects on tumor incidence, tumor growth rate, latency to tumor onset, or longevity (11, 12, 13) . In Eµ-Pim1 transgenic mice, exposures simulating Global System for Mobile Communications (GSM) digital cell phone fields were associated with a significant increase in follicular lymphomas appearing after the age of 10 months (24) . Autopsy on an adult squirrel monkey exposed to 2.45 GHz fields for 90 min each week for 3 years revealed a frontoparietal primitive neuroectodermal tumor (41) .
A review of published data on the genetic toxicology of RF radiation with a focus on mobile phone carrier frequencies concluded that they are not genotoxic; they do not induce genetic effects in vitro or in vivo, at least under athermal conditions, and do not seem to be teratogenic nor to induce cancer (42) . RF/microwave fields lack necessary photon energies to disrupt chemical bonds. The threshold for disruption of chemical bonds by electromagnetic fields occurs at photon energies around 7–10 electron V, first seen in UV spectral regions and at progressively shorter wavelengths, extending into X-ray spectral zones.
Single strand breaks in rat brain DNA reported after acute low-intensity microwave exposure suggest a role for defective DNA repair mechanisms in carcinogenesis. Both animal models and clinical disorders suggest a role for defective DNA repair mechanisms in carcinogenesis. There was a 90% reduction in ENU-induced CNS gliomas if exposure to ionizing radiation (2 Gy) occurred 24 h before ENU dosage (21) . The reduction correlated with activation by the radiation of the DNA repair enzyme alkylguanine-DNA-6-alkyltransferase. Defects in cloned repair genes have been associated with a predisposition to certain cancers (22) . Moreover, free radicals of the oxygen and nitrogen species may act as complete carcinogens, the outcome depending on interactions between DNA damage, antioxidant levels, and DNA repair systems (6 , 23) .
In a life-term rat model, we have assessed possible brain tumor promotion by simulated FM mobile phone microwave fields and compared the findings with a comparable exposure to digitally encoded (TDMA) phone fields (18) . Taken jointly, a summary of the experimental evidence points to a perturbation in brain tumor incidence by the digitally encoded signal not detected in exposure to FM fields. Although consistent but not attaining significance in the experiment overall (spontaneous CNS tumors, P < 0.08 one-tailed, P < 0.16 two-tailed; ENU-induced tumors, P < 0.08 one-tailed, P < 0.16 two-tailed), the trend was significant (P < 0.015 one-tailed, P < 0.03 two-tailed) in rats that received ENU and died prior to experiment termination, with a primary brain tumor as the cause of death. The effect was seen among glial-derived but not in nonglial histological subtypes. There were no effects on the incidence of primary nervous system tumors attributable to FM field exposure in spontaneous CNS tumors nor in the higher incidence in cohorts that received the carcinogen ENU. In a similar Fischer 344 rat study testing the TDMA digital field, we observed trends toward reduced incidence of both spontaneous and drug-induced CNS tumors (25) . However, nonglial CNS tumors in that study numbered only four.
As already noted, there are fundamental physical differences between a digital microwave signal transmitted as a series of packets or pulses and the unvarying intensity of analogue (FM) fields. There may also be fundamental differences in their modes of eliciting bioeffects. Resonant interactions with molecules or portions of molecules may be expected in the millimeter wave/far infrared spectral regions. But for typical microwave fields with carrier frequencies far below these spectral regions, theoretical considerations and experimental data support the concept of collision-broadened, rather than resonant, spectra in their biomolecular interactions (43) . Heating is thus their prime mode of interaction. As reviewed elsewhere (6) , in cell culture preparations with SARs less than 5 W/kg, cellular responses have been reported primarily from exposures to microwaves that were amplitude or pulse modulated at ELF frequencies (i.e., typically <300 Hz), where heating does not appear to be the mediator of these bioeffects. In contrast, effects on cell cultures have been reported with microwave fields lacking amplitude or pulse modulation (continuous wave or FM), all using SARs equal to or greater than 10 W/kg.
From initial observations of modulation frequency-dependent responses of calcium binding in cerebral tissue (1, 2, 3) , there are now many reports that RF fields, when either pulse or amplitude modulated at ELF, may regulate a wide range of in vitro responses, including cell membrane ion transfer, enzyme activation, neurotransmitter release, and cell growth regulation, even where these fields are below levels where tissue heating may mediate the responses (so-called "athermal exposures"; Ref. 4, 5, 6 ). Exposures here and in the comparable digital field study (25) were in this range (brain SAR, 1 W/kg). There is the implication that some form of envelope demodulation occurs in tissue recognition of ELF components. A suggested basis for envelope demodulation at cell surfaces may reside in the spatial anisotropy of the intensely anionic charge distribution on strands of glycoprotein protruding from the cell interior (4, 5, 6 , 44) .
Thus, the aggregate evidence suggests a biological role for ELF amplitude- or pulse-modulated microwave fields with absorbed energies below 5 W/kg, where heating is not the mediator of these bioeffects. If the concept of amplitude or pulse modulation frequency dependence gains further experimental support, elucidation of the tissue detection mechanisms for these responses becomes important (6) . Low-level microwave fields with 16-Hz amplitude modulation (SAR, 0.08 W/kg) increased ODC activity in CHO hamster ovary cells by 50%, but responses decreased to control levels at 6 and at 100 Hz in a modulation frequency-dependent manner (45) . ODC and its polyamine products are responsive to both 60-Hz ELF fields and to radiofrequency fields with 60-Hz amplitude modulation (7 , 8 , 46 , 47) .
A direct comparison of ODC activity in murine fibroblasts (L929) exposed to either a simulated TDMA digital phone field producing 50 pulses (packets)/s or to an FM phone field at athermal levels (SAR, 2.5 W/kg, 60-KHz deviation) showed a 90% increase with digital exposures but no response to the FM fields (47) . This study also reported the ODC response to be a function of modulation frequency, significantly increased at 16, 55, 60, and 65 Hz but not at 6 or 600 Hz. Iridium cell phone fields (11 packets/s) modified activity of ODC and levels of its polyamine product putrescine in fetal rat brains (48) . These electromagnetic sensitivities of ODC and its key functions in regulation of cell growth suggest possible links between overexpression of ODC in initiated cells and tumorigenesis in the absence of any other promoter (49) .
The observed range of spontaneous CNS tumor incidence of 1.1–4.4% and brain tumor incidence of 1.1–3.3% approximated the range of 0.77–3.3% (CI upper bound, 6.5%) for brain tumors for the Fischer 344 rat in 2-year carcinogenicity studies of the NTP (50 , 51) . These studies report widely differing incidences, with the highest incidence in a single study of 4% (52 , 53) . In our study, tumor incidence was sharply increased to 15–22% over the lifetime of rats receiving a single dose of ENU in utero. They thus became a positive control in seeking possible promotional effects of FM field in tumorigenesis. Accuracy of tumor counts in the present study may have benefited from use of 20–25 sections/brain, permitting detection of MTs possibly missed in the NTP standard procedure with a maximum of three sections/brain. Other studies have reported trends toward a higher tumor incidence when at least five to eight sections/brain are used (54) .
In summary, there were no effects on incidence of either spontaneous or ENU-induced primary tumors of the CNS associated with lifetime exposure of rats to FM microwave fields simulating electrical characteristics of hand-held mobile telephone transmissions at the head of the user.
ACKNOWLEDGMENTS
We are indebted to Dr. A. R. Sheppard for support in
experiment design and exposure system development. Dr. G. Stoica, Texas
A and M University, College Station, Texas, provided an
invaluable baseline on the carcinogen ENU. Dr. B. Zook, George
Washington University, Washington DC, pioneered the NF exposure system.
M. Burkhardt, K. Popovic, and F. Schonborn at the Swiss National
Technological Institute (ETH) Zurich conceived and refined many
microwave dosimetry models. Dr. Q. Balzano, Motorola Corporation, has
been unfailing in his innovative leadership throughout the study.
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FOOTNOTES |
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1 This study was performed with the support of the
Motorola Corporation. 
2 To whom requests for reprints should be
addressed, at Department of Biochemistry, Boyce Hall, Room 1407,
University of California, Riverside, CA 92521. Phone and Fax:
(909) 787-4623; E-mail: RossAdey{at}citrus.ucr.edu
3 The abbreviations used are: FM, frequency
modulated; RF, radiofrequency; TDMA, time division multiple access;
NTP, National Toxicology Program; ENU,
N-ethyl-N-nitrosourea; GD, gestation day;
ES, ENU/Sham Field; EF, ENU/Field Exposed; EC, ENU/Cage Control; SS,
Sham ENU/Sham Field; SF, Sham ENU/Field Exposed; SC, Sham ENU/Cage
Control; FF, far field; NF, near field; CNS, central nervous system; T
and MT, tumor and microtumor, respectively; SAR, specific energy
absorption rate; ELF, extremely low frequency; CI, confidence interval;
ODC, ornithine decarboxylase.
Received 6/ 8/99. Accepted 2/ 2/00.
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 S. Takahashi, S. Inaguma, Y.-M. Cho, K. Imaida, J. Wang, O. Fujiwara, and T. Shirai Lack of Mutation Induction with Exposure to 1.5 GHz Electromagnetic Near Fields Used for Cellular Phones in Brains of Big Blue Mice Cancer Res., April 1, 2002; 62(7): 1956 - 1960. [Abstract] [Full Text] [PDF]
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 P. A. Mason, T. J. Walters, J. DiGiovanni, C. W. Beason, J. R. Jauchem, E. J. Dick Jr, K. Mahajan, S. J. Dusch, B. A. Shields, J. H. Merritt, et al. Lack of effect of 94 GHz radio frequency radiation exposure in an animal model of skin carcinogenesis Carcinogenesis, October 1, 2001; 22(10): 1701 - 1708. [Abstract] [Full Text] [PDF]
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 H. Frumkin, A. Jacobson, T. Gansler, and M. J. Thun Cellular Phones and Risk of Brain Tumors CA Cancer J Clin, March 1, 2001; 51(2): 137 - 141. [Full Text] [PDF]
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 J. E. Muscat, M. G. Malkin, S. Thompson, R. E. Shore, S. D. Stellman, D. McRee, A. I. Neugut, and E. L. Wynder Handheld Cellular Telephone Use and Risk of Brain Cancer JAMA, December 20, 2000; 284(23): 3001 - 3007. [Abstract] [Full Text] [PDF]
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