United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 2771 1
Research and Development
EPA-600/S1-83-008 Sept. 1983
oEPA Project Summary
Microwaves, Hyperthermia, and
Human Leukocyte Function
Norbert J. Roberts, Jr., Shin-Tsu Lu, and Sol M. Michaelson
Studies were performed to deter-
mine whether human leukocytes are
affected by exposure to microwave
energies that equal or even exceed
current safety standard recommenda-
tions. There were no detectable effects
on viability or function of human mono-
nuclear leukocytes resulting from ex-
posure to microwave energy at specific
absorption rates up to 4 mW/ml. In
contrast to studies in other laboratories,
results were highly reproducible and
provided no evidence that current
safety standard recommendations are
inappropriate insofar as leukocyte func-
tion is concerned.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory, Research Triangle Park, NC, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
All individuals are exposed to radio-
frequency/microwave energies to variable
degrees. Studies by several investigators
have raised the possibility that the immuno-
competent cells of humans are particularly
susceptible to microwaves. These studies
were admitted to be poorly reproducible
and nonquantitative. Nonetheless, they
are frequently cited, and have provided the
limited data available, on exposure of
human leukocytes, to be used by those
individuals and agencies that develop en-
vironmental health standards. Many ani-
mal systems have been studied, but the
species, microwave power intensities,
environmental conditions and other fac-
tors have been so varied that extrapolation
to humans would be exceedingly difficult,
even if appropriate.
The studies reported here were per-
formed to determine whether human
leukocytes are affected by exposure to
microwave energies that equal or even
exceed current safety standard recom-
mendations. Exposure to microwave ener-
gy at specific absorption rates up to 4
mW/ml resulted in no detectable effects
on viability, or unstimulated or stimulated
DNA, RNA, total protein or mterferon
synthesis by human mononuclear leuko-
cytes. In contrast to the earlier studies
cited above, results were highly repro-
ducible and provided no evidence that
current safety standard recommendations
are inappropriate.
Materials and Methods
Human mononuclear leukocytes were
exposed in a waveguide system to 2450
MHz (CW) microwaves for 2 hours at
specific absorption rates (SARs) from 0.5
to 4 mW/ml. The safety standard limit
proposed by Committee C-95.4 of the
American National Standards Institute is
0.4 mW/g, which is equivalent in these
cultures to 0.4 mW/ml. This standard
incorporates a ten-fold safety factor rela-
tive to bioeffects reported using animal
models. The waveguide exposure system
and methods of preventing temperature
mhomogeneity, and methods of determin-
ing specific absorption rates (SARs) have
been described previously (EPA-600/S1-
81-041). In addition to leukocyte cultures
enclosed within waveguides for exposure
or sham-exposure, we included control
cultures located within the same incubator
but external to the waveguides. No at-
tempt was made to counteract microwave-
induced heating of the leukocyte cultures
since we wished to observe any potential
microwave-induced effects, thermal or
otherwise.
-------
Viability was determined by total cell
counts and percent of cells able to exclude
trypan blue dye and ethidium bromide.
DNA, RNA and total protein synthesis
were measured by cellular incorporation
of tritiated thymidine, urinde, or leucine,
respectively, using established methods.
Unstimulated and mitogen-stimulated pre-
cursor incorporation were assayed from
immediately to 5 days after exposure. The
mitogen phytohemagglutmin (PHA) was
added at a concentration shown to yield
optimum responses with normal mono-
nuclear leukocytes, as well as at several
suboptimal concentrations. Responses
(mean cpm ± S.E.) are shown in Results
for unstimulated and optimal PHA-stimu-
lated leukocytes.
Results
Exposure of the leukocytes at SAR=4
mW/ml produced no significant changes
in cell viability for up to one week after
exposure (Table 1). Results were similar
with exposures at lower SARs.
Unstimulated and mitogen-sttmulated
DNA, RNA and total protein synthesis
were examined after exposure of the
mononuclear leukocytes to microwaves at
SARs of 4 mW/ml or less. There were no
significant differences between micro-
wave (4mW/ml)-exposed, sham-exposed,
and control leukocytes in unstimulated
DNA synthesis, or in responses of the
leukocytes to an optimal concentration of
mitogen (Figure 1). Results were similar
using suboptimal concentrations of mito-
gen, and using lower SARs (0.5 and 1.0
mW/ml) for the microwave-exposed cul-
tures (data not shown). Results were
similar with measurements of RNA and
total protein synthesis. Microscopic in-
spection of Wnght-Giemsa-stained cyto-
spin preparations did not reveal any dis-
crepancies between morphologic lymph-
ocyte blastogenesis (used in some of the
studies cited earlier) and determinations
using incorporation of the radiolabelled
precursors.
In addition to determinations of total
protein synthesis, we measured spon-
taneous production of interferon (none
detected in any cultures), and production
of influenza virus-induced interferon-a
and PHA-induced interferon-y, at 1 and 3
days after induction. Virtually all detect-
able virus-induced mterferon-a was present
by 24 h, with equivalent amounts produced
by microwave-exposed (SAR=4mW/ml),
sham-exposed, and control leukocytes
(Figure 2A). PHA-mduced interferon-y,
usually produced by 48-72 h, was not
detected in any culture supernatant fluid
at 24 h. By 72 h, interferon-y was detected
Table 1.
Exposure
Total Viable Mononuclear Leukocytes after Exposure to Microwave Energy at
SAH=4 mW/ml
Days After Exposure8
Microwave
Sham
Control
58±7b
65+11
54±6
60+9
63±15
56±6
41+4
39±2
41±8
47±16
72+36
46+13
39±10
4J±9
36±7
40±W
37+8
38+ Tl
aInsufficient numbers of observations (<5) were available 3 days after exposure. Viability was
assessed by the ability of the cells to exclude trypan blue dye and ethidium bromide.
bData represent mean total number of viable cells (total cells x percent viable) x TO'4, + S.E.
Unstimulated
0.6
04
°2
I
36
D
PHA -Stimulated
Microwave-Exposed
Sham-Exposed
Control
I
o
s-
S 32
.c
28
24
| 20
s
> 16
C:
?2
01 2345
Days After Exposure
Figure 1. DNA Synthesis by microwave (mW)-exposed, sham-exposed, and unexposed
/control) human mononuclear leukocytes SAR = 4 mW/ml DNA synthesis by
unstimulated cells and by cells stimulated by an optima/ concentration of
PHA (160 g/ml) are shown. Columns indicate mean cpm tritiated thymidine
incorporated + S.E., from immediately to 5 days after exposure.
in all PHA-mduced cultures, with no signif-
icant differences between the microwave-
exposed (SAR=4mW/ml), sham-exposed,
and control leukocytes (Figure 2B). Re-
sults were similar with exposures to micro-
waves at lower SARs.
Discussion
The current studies provide the first
clear, reproducible data regarding exposure
of human leukocytes to microwave ener-
gies relevant to current public safety
recommendations. Direct extrapolation to
the in vivo setting with many physiological,
homeostatic, integrated systems is not
appropriate. However, these data do sug-
gest that earlier reports of possible micro-
wave effects on human leukocytes, at
such energy levels, remain poorly repro-
ducible and should not form a basis for the
resetting of safety standards. Most studies
of environmental physical factors examine
effects on resting cell populations even
though, under normal conditions, man is
commonly exposed to more than one
environmental stress at a time. Thus,
-------
SA) Virus-Induced
12 h
10
L
(B)PHA -Induced
Microwave-Exposed
K/ Sham-Exposed
I I Control
Figure 2.
Cell Cultures
Interferon-a and mterferon-y synthesis by microwave (mW)-exposed, sham-
exposed, andunexposed(control)humanmononuclearleukocytes. SAR = 4mW/ml
(A) Interferon-a tilers present 24 hours after induction with influenza virus, and (Bj
Interferon- y liters present 72 hours after induction with PHA are shown Columns
indicate mean log? units/ml ± S.E
these results are notable further for indi-
cating that human leukocytes exposed to
microwaves, as a potential physical stress
factor, can respond normally to a second
biological factor, such as the commonly
encountered infectious agent, influenza
virus.
The current studies do not exclude the
existence of microwave-induced effects
on human leukocytes resulting from ex-
posures at greater SARs. Such exposures
commonly produce effects that can be
related to the degree and/or the rate of
heating of the cell cultures or tissues in
vivo. Furthermore, the current studies do
not completely exclude potential microwave-
induced effects resulting from exposure at
similar SARs, but applied by almost in-
numerable different possible wave forms
(frequencies, modulations, etc.).
Recommendations
The ubiquitous distribution of micro-
wave energy and the potential differences
between animal models and humans sug-
gest that further investigations with human
leukocytes and other cells may be war-
ranted. The literature regarding micro-
waves includes animal studies reporting
deleterious effects of exposure and animal
studies reporting beneficial effects, over a
broad range of SARs. Potential health
hazards for humans should be further
defined and limited, and potential health
benefits, such as the use of microwave-
induced hyperthermia in the treatment of
cancer, should be further defined and
expanded.
NorbertJ. Roberts, Jr., Shin- TsuLu, and Sol M. Michaelson are with the University
of Rochester School of Medicine, Rochester, NY 14642
Ralph J. Smialowicz is the EPA Project Officer (see below).
The complete report, entitled "Microwaves, Hyperthermia, and Human Leukocyte
Function," (Order No. PB 83-225 375; Cost: $7.00, subject to change} will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
------- |