United States
Environmental Protection
Agency
Health Effects Research EPA GOO 1
Laboratory May 1 &8C
Research Triangle Park NC 27711
Research and Development
&EPA
Genetic and Cellular
Effects of Microwave
Radiations
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
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8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-30-027
May 1930
GENETIC AND CELLULAR EFFECTS OF MICROWAVE RADIATIONS
by
S. K. Dutta, Ph.D.
Principal Investigator
Department of Botany
Howard University
Washington, D.C. 20059
EPA Grant #R803561
Project Officer
C. F. Blackman, Ph.D.
Experimental Biology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Labora-
tory, U.S. Environmental Protection Agency, Research Triangle Park,
North Carolina, and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
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FOREWARD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those already in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health
care and surveillance of persons having suffered imminent and substantial
endangerment of their health.
This report is directed at the question: Does radiofrequency
radiation cause mutagenic changes in biological entities? Simple
unicellular organisms are used to examine the mutagenic potential of
several specific frequencies of non-ionizing electromagnetic radiation.
F. Gordon Hueter
Di rector
Health Effects Research Laboratory
ill
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ABSTRACT
This research program was initiated with the overall objective of
determining genetic and cellular effects of CW and pulsed microwave
radiations which are prevalent in our biosphere.
Several tester strains of the bacterium Salmonella typhimurium,
TA-98, TA-10$, TA-1535, and TA-1538; the bacterium Escherichia coli
W3110 (pol A ) and p3438 (pol A , repair deficient); and the yeast Saccharo-
myces cerevisiae D3, D4, and D5 were tested for lethal and mutagenic
events when exposed to microwave radiations. Effects of known elevated
temperatures were studied to distinguish microwave-induced temperature
effects from the direct temperature effects.
Three kinds of microwave exposure systems were used in these studies:
(1) far-field of an antenna (for 2.45 GHz and 8.5-9.5 GHz), (2) waveguide
(for 8-10 GHz), and (3) transmission line (TEM cell) for 915 MHz radiation.
The SAR (specific absorption rate) for various exposures ranged from 0.1
W/kjg to 40 W/kg. Pulse repetition rates were 400 Hz or 1000 Hz for
pulsed microwave radiations.
The studies revealed no increase in mutations or of gene conversions
when cells were exposed to microwave radiations, but yeast and bacterial
strains showed cellular lethality caused by temperature rises at higher
microwave power densities. Results demonstrated that very high elevated
temperatures (above 10?C rise) generated by the microwave exposure could
produce genetic events in microbial assay systems. At a SAR of 12 W/kg,
an indication (0.05 < P < 0.1) of microwave-induced increase in' cell
concentration in terms of increased colony forming units (CPU) was
observed in the E. coli pol A strain at 8.6 GHz when exposed for more
than seven hours. No change in CPU were noticed when E. coli cells were
exposed to 915 MHz, 8.6, 8.8, or 9.0 GHz at very low power densities
(SAR of 1 W/kg). At 10 GHz, E. coli cells were exposed at varying
power levels (SAR of 0.1, 1, or 10 W/kg). No change in CPU was observed.
Mathematical expressions were developed using a SAS-76 computer
program to predict the concentration of bacterial cells (CPU) at any
time within the temperature range 37?C-49t?C and to measure microwave-
induced changes in CPU as a .function of temperature rise. A heat transfer
model was developed for our exposure systems which helps to distinguish
low power density effects when a temperature rise due to microwave
radiations cannot be detected.
This report was submitted in fulfillment of the Grant No. R-803561
by Howard University under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the work period during May 8,
1975, to December 31, 1979.
IV
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CONTENTS
Foreword fltJRARi<
Abstract TV
Figures vi
Tables vii
Acknowledgment ix
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Materials, Methods and Procedures 4
Microbial tester strains 4
Culture media and growth conditions 6
Microwave exposure systems 6
Procedure for estimation of specific absorption
rate (SAR) 9
Exposure of cells to EMR and subsequent
analysis 11
Exposure of cultures to elevated temperatures ... 11
Positive controls and data analysis 12
Calculation of repair index 12
5. Results 14
Lack of microbial genetic response 14
Cellular effects of elevated temperatures and
microwaves 17
Ancillary studies 26
References 31
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FIGURES
Number
1
2
3
4
5
Block diagram for far-field antenna exposure system ....
Block diagram for waveguide exposure system
Block diagram of TEM Crawford cell exposure system
Survival curves of a yeast and a bacterial culture when
exposed to either elevated temperature or microwave
radiation
Composite data showing plot of average N/NQ against time
of exposure
Page
7
8
10
19
30
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TABLES
Number Page
1 Description of microbial strains employed .......... 5
2 Effect of radiation at different power densities on cell
survival and genetic activity of Saccharomyces
cerevisiae D4 (9.0 GHz pulsed EMR) ........... 14
3 Effect of different power levels and EMR frequencies on cell
survival and genetic activity in Saccharomyces
cerevisiae strain D4 .................. 15
4 Effect of pulsed EMR radiation at different frequencies and
power levels on ^. typhimurium strain TA-100 ...... 15
5 Effect of pulsed EMR radiation at different frequencies
on S. typhimurium strain TA-1535 when exposed at
10 mW/cP~TTT" .................... 16
6 Effect of pulsed EMR radiation at different frequencies
on S. typhimurium strain TA-98 when exposed at
10 mW/cmZ . . . .................... 16
7 Effect of elevated temperatures on different strains of
Saccharomyces cerevisiae when exposed for 2 hours
in saline ....................... 17
8 Effect of elevated temperatures on different strains of
S. typhimurium after 2 hours treatment ......... 18
9 Effects of different temperatures and durations of exposure
on repair indices (Aver) of E. coli pol A relative
to o A ....................... 18
10 Composite data_of microwave- induced cell growth of pol A
and pol A in various intervals of time ........ 20
11 Regression analysis of composite data on microwave
radiation . . . .' ................... 21
12 Summary of calculated repair indices at different time
intervals ....................... 21
13 Summary of the cell growth ratios in colony forming (CPU)
of JE. coli pol A strain at 8.6, 8.8, and 9 GHz
using a far-fTeld antenna exposure system at
1 mW/cm2 ........................ 22
14 Summary of cell growth of E. coli pol A at 0.1, 1, and
10 mW/g at 10 GHz pulsed radiation ........... 23
Vll
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TABLES (continued)
Number Page
15 Summary of repair indices at different dose rates 23
16 Summary of cell growth of E. coli pol A strain at varying
dose rates of 915 MHz CW radiations using a TEM
Crawford cell system 24
17 Summary of cell growth of E. coli pol A strain at varying
dose rates of 915 MHz CW radiations using a TEM
Crawford cell system 25
Ancillary Studies
18 Summary of cell growth ratios for E. coli pol A when
exposed to 8.8 GHz pulsed EMR and to 35.2°C 27
19 Summary of cell growth ratios for E. coli pol A when
exposed to 8.8 GHz pulsed EMR anTTo 3572°C 28
20 Summary of_cell growth ratios at 42.2°C of pol A and
pol A strains 29
vm
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ACKNOWLEDGMENTS
On behalf of the Department of Botany and Howard University, Washington,
D.C., the author, S. K. Dutta, wishes to convey our gratefulness to Dr.
W. Ashley, Jr., and Mr. C. Bishop, respective program directors for EPA
Minority Institution Programs, for helping us relentlessly to create this
microwave research capability at Howard University.
Dr. C. F. Blackman, Project Officer, EPA Research Center, Research
Triangle Park, NC, has provided valuable guidance in conducting research
and in publishing several papers. We are grateful to him for his continu-
ing help. Several other scientists, e.g., Mr. 0. S. Ali, Microwave
Engineer; Dr. J. A. Elder, Chief, Cellular Biophysics Branch; and many
members of the Experimental Biology Division of the Health Effects
Research Laboratory, Research Triangle Park, NC, cooperated in successful
conduction of experiments using the far-field antenna microwave exposure
system.
Dr. Henry Ho, Microwave Research Engineer of the Bureau of Radiologi-
cal Health, Food, and Drug Administration, Rockville, MD 20852, has
helped us with his waveguide exposure system. He has also helped us in
standardizing Crawford cells for exposure to electromagnetic radiation.
We are thankful to him.
The expert advice and continuous help of Dr. David J. Brusick,
Director of Genetic Toxicology, Litton Bionetics, Kensington, MD, is
gratefully acknowledged. The hard work and help provided by several
co-workers and graduate students of Howard University like Dr. W. H.
Nelson, Mr. M. A. Hossain, Ms. Carolyn Chambers, Dr. D. K. Mukhopadhyay,
Dr. Johnson Choppala, Mr. L. Douglas Jones, Mr. Emmanuel Samual, Dr.
K. Dey, Mrs. C. Issac, and many other members of the Botany Department
have helped in successful conduction of this project.
Computer Center of the neighboring George Washington University,
Washington, D.C., has provided us with the computer facilities in using
the SAS (Statistical Analysis System) 76 system. This center has been
very helpful in the analysis of a vast amount of data and in developing
mathematical expressions needed for quantitative analysis of bacterial
responses associated with thermal and microwave treatments.
IX
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SECTION 1
INTRODUCTION
Due to their widespread industrial and domestic uses in heating and
drying equipment, spectroscopy, alarm system, diathermy units, radar, TV
transmission, communications, and navigation devices, microwave radiations
have become an environmentally important energy polluter. The effects
of higher intensities of microwave radiation on test systems HI vivo and
i_n vitro have been investigated by various groups (Cleary, 1977). These
studies have revealed effects of microwave radiation on (a) the lens of
the eye, (b) the gonads, (c) embryonic development, (d) chromosome
aberrations, and (e) electrophoretic and immunologic properties and
enzymatic activity of isolated proteins. The results of the i_n vivo
studies generally reflect the physiological responses of the test organism
to the thermal burden imposed by microwaves. In contrast, a number of
Soviet and East European investigators have reported low level microwave
effects at the cellular level (Baranski and Czerski, 1976).
Many of these Soviet works are primarily on effects on cell membrane,
biological macromolecules, tissues, and growth inhibition of unicellular
organisms (Baranski and Czerski, i?76). The potential hazard of such
radiation might escape detection if investigations are not conducted
simultaneously with a battery of highly sensitive genetic and morphological
tests using a spectrum of genetic system—both unicellular and multicellular
haploid and diploid microorganisms. The biological action of many
poorly understood physicochemical phenomena produced by microwave irradia-
tion may conceivably include such long term hazards as (a) recessive
mutations giving rise to genetic diseases and (b) cumulative effects of
deletion mutations that may go undetected for several generations.
Studies on genetic effects of microwaves have been cited as a key
priority area in accordance with the policy report of the Office of
Telecommunications (see report on NEMR U.S. Government Presidents Executive
Office, May, 1974). It is known that a battery of microbial tester
strains are quicker and far more sensitive for detecting minute genetic
differences than are higher animals. In addition, tests using higher
animals are extremely expensive and time consuming, taking several
generations before meaningful results are obtained.
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SECTION 2
CONCLUSIONS
In accordance with the objectives of the project, major thrusts
were two-fold: (1) to detect any minute changes in the genes at the
nucleotide level and (2) to identify any non-genetic (cellular) effects
of microwave radiations. Conclusions arrived for these objectives are
as follows:
The extensive genetic studies made with a wide variety of microbial
tester strains revealed no increase in mutations or in mitotic gene
conversions when the strains were exposed to low or moderate levels of
microwave radiation in a far-field exposure system. However, at higher
power levels, yeast and bacterial strains showed cellular lethality
caused by a temperature rise. These results demonstrated that very high
elevated temperatures (above 10?C rise) generated by microwave exposure
could produce genetic events in microbial assay systems.
Our studies on cellular effects were based on counts of colony
forming units (CPU) of treated and control (sham) experiments. Using a
waveguide exposure system, having precise temperature control, the
colony forming units in an exposed sample of E. coli, pol A strain, was
observed to increase slightly (0.05 < P < O.lJ. In this case, exposures
were conducted for more than seven hours using 8.6 GHz pulsed radiation
at a SAR of 12 W/kg which had a pulse width of 1 usec and a pulse repeti-
tion rate of 1 kHz.
No radiation-induced change in CPU was noticed when E. coli cells
were exposed to 915 MHz, 8.6, 8.8, and 9.0 GHz at very low levels (SAR
of 1 W/kg). At 10 GHz, JE. coli cells were exposed at varying power
levels (SAR of .1, 1, or 10~W7kg). No change in CPU was noticed. Both
waveguide and Crawford cell exposure systems were used.
As a part of our attempt ot obtain some fundamental information on
common problems associated with microwave research, we did extensive
studies on the effects of elevated temperature. The mathematical expres-
sions were developed using a SAS-76 computer program to predict cell
concentrations in terms of colony forming units (CPU) in bacteria at any
time within the temperature range 37
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SECTION 3
RECOMMENDATIONS
There is conclusive evidence for definite cellular effects, but no
genetic damage, from extremely low intensity microwave radiations.
Because of the evidence for cellular effects in prokaryotic and lower
eukaryotic systems, we strongly recommend that studies be performed on
complex eukaryotes, specifically human cells in culture, to investigate
the effects of this radiation on a potentially more labile system which
has direct human relevance.
(1) The existence of power and frequency "windows" which has been
reported to affect ions associated with brain tissue should be studied
more extensively. The well-established human ijie.uroblastoma cell line
AG2202 can be used as a cellular system for Ca efflux studies at
several frequencies and different power levels to rapidly examine these
responses.
(2) Once the frequency and power density "windows" have been
established for neuroblastoma cells in culture, these values should be
used to examine the response of other human cell lines, such as IMR-90,
established from normal tissue. These experiments should be conducted
with very careful attention to the effects of microwave-induced heating
in order to examine other possible causes of microwave-induced biological
changes. Computerized mathematical expressions of cell survivals and/or
concentration increases as a function of temperature rises will be used
to distinguish microwave effect from temperature effects.
(3) After basic information regarding power and frequency "windows"
are obtained, primary explants in culture from skins of human cadavers
should be exposed to see the microwave-induced effect on tissue from the
human body. Studies should involve histological changes like necrosis
or any gross morphological change and/or growth inhibition using 3H-
thymidine incorporation procedure to detect abnormality in rate of
mitosis. Electron microscopic studies can be performed to study any
changes in membrane structure.
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SECTION 4
MATERIALS, METHODS, AND PROCEDURES
MICROBIAL TESTER STRAINS
Table 1 outlines all strains used in our various studies and their
relevant markers and references to their use in genetic screening. The
procedures for maintenance and culturing and the methodology for identifi-
cation and quantisation of changes in genetic and non-genetic events
have been described by Brusick and Mayer (1973), Ames et al. (1975), and
Zimmermann (1975).
Dip!oid strains of Saccharomyces cerevisiae designated D3, D4, and
D5 were obtained from Dr. F. K. Zimmermann, Technische Hoschschule,
Darmstadt, German Federal Republic. These three strains were established
by Zimmermann. Strains D3 and D4 detect mitotic recombination and
mitotic gene conversion, respectively, whereas strain D5 detects mitotic
recombination and probably mitotic gene conversion events simultaneously.
Mitotic events of D3 and DS are identified by the production of red
and/or pink pigment associated with mutant alleles of the ade 2 locus.
Gene conversion is identified in strain D4 heteroalleles at both ade 2
and trp 5 loci which utilize adenine and tryptophan selective media.
The bacterial strains of Salmonella typhimurium designated TA-1535,
TA-1538, TA-98, and TA-100 were obtained from Dr. B. N. Ames, University
of California. All of the ^S. typhimurium strains were scored by numbers
of revertants in histidineless minimal medium. Strains TA-1535 and
TA-100 are basepair substitution mutants whereas TA-1538 and TA-98 are
frameshift mutants.
Escherichia coll strains designated pol A and pol A were obtained
from Dr. H. Rosenkranz, Columbia University, NY. The mutant strain pol
A is deficient in DNA polymerase and is exceedingly sensitive to UV
radiation, radiomimetic agents and agents known to react with cellular
DNA (D'Alisa et al. , 1971; DeLucia and Cairus, 1969; Gross and Gross,
1969). This repair-deficient mutant of E. coli is very sensitive to
nonspecific damange in DNA which is repairable in normal pol A cells but
which results in cell death in repair-deficient pol A cells. The
purity of these two cultures was tested regularly as follows: 0.1 ml
portions of each strain were spread separately on the surface of standard
agar plates. The plates were left at room temperature for two hours.
One tenth ml of methyl methane sulfonate (MMS) was then poured at the
center of each plate which was then placed in the incubator at 37°C for
16 hours. A larger zone of inhibition exhibited by pol A indicated the
purity of the culture.
4
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TABLE 1. DESCRIPTION OF MICROBIAL STRAINS EMPLOYED
Name of the
organism
Salmonella
typhimurium
Escherichia coli
Saccharomyces
cerevisiae
Strai n
designation
TA-1535
TA-98
TA-100
W3110
p3478
D3
D4
D5
Gene
affected
his C
his D
his G
pol A
ade 2, his 8
ade 2, try 5
ade 2
Additional Mutations
Repair LPS R factor
uvr B rfa
uvr B rfa , pKMlOl
uvr B rfa pKMlOl
Normal
No excision
Normal
Normal
Normal
References for methodology
Ames et al . , 1975
Ames et al . , 1975
Ames et al . , 1975
Slater et al. , 1971
Slater et al. , 1971
Zimmermann et al . , 1965
Zimmermann and Schwaier,
Zimmermann, 1975
1967
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CULTURE MEDIA AND GROWTH CONDITIONS
The composition of complete broth medium for yeast has been described
by Brusick (1970), and the composition of minimal medium has been described
by Magni and Von Borstel (1962). Adenine and tryptophan were added to
minimal medium where appropriate at a concentration of 10-20 mg percent.
The composition of complete broth medium for growing bacterial
(Salmonella and E. coli) cultures was Difco Standard Methods Broth
(Difco, Detroit, MI), and the composition of minimal medium was that of
Spizizen (1958).
Each bacterial strain was cultured in complete broth at 37°C for 16
to 20 hours. One hour prior to exposure at a specific temperature, each
culture was diluted to an optical density (OD) of 0.25 at 610 nm in
order to provide between 100 and 300 colonies per plate. All cultures
were maintained for one hour at the appropriate temperature on a shaker
to allow for post-adjustment growth. Just prior to treatment, cell
concentration was determined for each sample by plating on complete
medium.
MICROWAVE EXPOSURE SYSTEMS
Far-field
Exposure to 2.45 GHz microwaves took place in an electrically
anechoic, chamber-type far-field exposure facility that provides linearly
polarized, regulated CW radiation (Elder and Ali, 1975; Blackman et al.,
1975). The X-band exposures also took place in an anechoic chamber-type
system with the following major components: an Applied Microwave Lab
Model MH250 Pulse Modulator, a Raytheon RK2J51 magnetron, a Systron
Donner Model DBH-250 10 dB horn antenna, and a Vista Scientific environ-
mental control system. Samples were exposed to linearly polarized,
rectangularly pulsed RF radiation at frequencies between 8.5 and 9.6 GHz
with a pulse repetition rate of 1000 Hz and a duty cycle of 0.001. Both
facilities have environmental control systems that maintain temperature
and humidity at present levels (50% ± 5% relative humidity and an air
velocity of 5.4 m/min). One chamber, located in the anechoic room,
houses the sample to be exposed while the other chamber houses the
control sample. Temperature of the exposure and control chambers was
maintained as appropriate for the test systems. Figure 1 is a block
diagram of the X-band system.
Waveguide Exposure System
Details of the equipment arrangement for the waveguide exposure
system are depicted in Figure 2 and described in details by Ho (1976).
The pulsed wave had a width of 1 usec and a pulse repetition rate of 1
KHz. One ml of each culture in broth was placed in a metallic holder
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FIGURE 1. Block diagram for far-field antenna system (X-band).
30 db
Coupler
Exposu re
Chamber
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FIGURE 2. Block diagram for waveguide EMR system.
MODULATOR
FREQUENCY
METER
-LJ-1
SOURCEJ
J
FORWARD
POWER METER
REFLECTED
POWER METER
LOW PASS
FILTER
BIDIRECTIONAL
COUPLER
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which was constructed from a piece of waveguide terminated by a shorting
plate. The holder was connected to the system as diagrammed in Figure
2. The holder was immersed in a water bath at 37° ± 0.1°C. Another
holder of the same material containing another 1.0 ml aliquot of the
culture was also immersed in the bath to serve as the sham exposure.
The holders were shaken frequently so that the cells remained in uniform
suspension. This shaking was accomplished without interrupting exposure
of the samples.
Crawford Cell Microwave Exposure System
The Crawford cell originally conceived at the National Bureau of
Standards, now being used at the EPA Laboratory (Instruments for Industry,
Model CC105S), consists of a rectangular TEN (transverse electric and
magnetic) mode transmission line tapered at each end to a transition
which mates with a standard coaxial cable. This test cell offers a new
and very broad-band method of measuring the absorbed power of the sample
placed within the cell. It offers an extremely efficient means of
obtaining broad-band operation up to approximately 1.0 GHz. Figure 3 is
a block diagram for this system.
PROCEDURE FOR- ESTIMATION OF SPECIFIC ABSORPTION RATE (SAR)
The specific absorption rate (SAR) in a far-field system was deter-
mined at selected intensities and frequencies by the procedures of
heating and cooling curve analysis described by All is et al. (1977). In
order to determine the amount of power absorbed in the waveguide exposure
system by bacterial cells and medium, both the incident and reflected
power meters were monitored. The reflected power was minimized by
adjusting the tuner. The power absorbed by the bacterial culture was
maintained at a definite SAR. Details of the procedures are given by Ho
(1976).
The field generated inside the Crawford cell chamber may be calculated
from the expression E = V/D, where E is the field generated in volts per
meter, V is the input voltage to the cell, and D is the septum to top
plate separation in meters. Thus, when the sample is placed inside the
chamber and exposed to microwave, the amount of power absorbed within
the sample will be Pi - (Pr + Pt) where:
Pi, the incident power;
Pr; the reflected power; and
Pt, the transmitted power.
In order to monitor the temperature of the exposure chamber, the Crawford
cell will be placed inside a versatile temperature and humidity controlled
room. The temperature in the chamber can be varied over the range of 18
to 42°C to within ± 0.1°C.
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FIGURE 3. Block diagram of the TEM Crawford cell microwave exposure system.
R.F.
GENERATOR
AMPLIFIER
BIDIRECTIONAL
COUPLIER
r~
SWITCH
1
TUNER
CRAWFORD
CELL
ATTENUATOR
POWER
METER
10
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EXPOSURE OF CELLS TO ELECTROMAGNETIC RADIATION AND SUBSEQUENT ASSAYS
Log-phase of yeast cells cultured in broth were adjusted to a cell
density of IQl cells/ml and were used as stock for subsequent treatments.
Prior to treatment, cultures were shaken for 60 minutes at 30°C. Log-phase
bacteria were grown in complete broth, centrifuged, and then resuspended
in fresh broth to densities of 109 cells/ml; the suspension was then
shaken for 90 minutes at 37°C. This suspension served as the stock
bacterial culture. The yeast suspensions were exposed for 2 hours to
2.45 GHz radiation at 30.0 ± 0.5°C and to X-band radiation at 29.0 ±
0.5?C. Following exposure, yeast cells were plated on complete medium
at a dilution ratio of 10 5 and on selective media either undiluted or
at a dilution ratio of 10 1. Scoring was performed after 72 hours and
the number of convertants per 10-? surviving cells was calculated.
The bacteria were exposed for 90 minutes at 37.0 ± 0.5°C in the
2.45 GHz facility and at 35.0 + 0.5°C in the X-band facility. In both
facilities, the samples were irradiated from above. Zero-hour samples
of bacteria and yeast were plated to determine the initial conditions of
the cultures and to provide a basis for calculating the number of genera-
tions of growth that occurred during treatment. Bacterial cultures were
plated on complete medium at a dilution ratio of 10 1 to determine the
total number of colony forming units per ml and undiluted to determine
the number of revertants per survivor. Scoring was performed after 48
hours of incubation and is expressed as revertant colonies per 10s
survivors.
After exposures, each E. coli sample was plated in 0.1 ml aliquots
on each of five plates (Standard Methods Agar, SMA, Difco, Detroit, MI).
The plates were incubated at 37°C for 24 hours and the total number of
colonies counted. Each such experiment was repeated three times for
every exposure condition. The initial concentration of cells for exposure
and sham was between the range of 2.2 and 3 x 108 cells/ml. Control
experiments were performed by placing cultures in 35 mm circular petri
plates at 37°C.
EXPOSURE OF CULTURES TO ELEVATED TEMPERATURES
Cultures of bacteria and yeast were exposed to various ambient
temperatures up to 15°C above their optimum growth temperature in Thelco
incubators which maintained'the set point temperature within ± 0.3°C.
Three ml cell suspensions from each culture (both in saline and in
broth) were placed in individual 35 mm diameter petri plates and incubated
for either 2 hours for S. typhimurium and Sacch. cerevisiae or for 1, 5,
10, or 15 hours for E. coli.
The preparation of quiescent cultures was different for exposure in
saline (0.9 percent). Overnight cultures of both strains of E. coli
were first washed two times in saline and then resuspended in saline to
an optical density of 0.20 at 610 nm before being placed on the exposure
vessel.
11
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POSITIVE CONTROLS AND DATA ANALYSIS
Genetic Index
Positive control experiments were conducted with all the microbial
tester strains using the known chemical mutagen, ethyl methanesulfonate
(EMS). This was done to ensure that the organisms were responding at
the proper level of sensitivity in these experiments. The results were
consistent with published data (Brusick and Andrews, 1975; Ames et al.,
1975; Slater et al., 1971).
Effects of elevated temperatures and pulsed microwave radiation on
Sacch. cerevisiae and on S. typhimurium were expressed as frequency of
genetic events per surviving cell population. Based on positive control
studies and on historical control data from the literature, the following
method was selected to summarize the data: genetic-activity index =
frequency of genetic events in a treated population divided by the
frequency of genetic events in a control population (Dutta et al.,
1979). If there were no radiation-induced effects, the genetic-activity
index would be 1.0 or less. If there were induction of mutations or
mitotic recombination, then the frequency of either in the treated
population would be higher than that of the control population, and the
genetic-activity index would be greater than 1.0. Based on previous
observations of normal variation in this system, all genetic-activity
index values between 1.0 and 2.0 are considered to be within the range
of normal fluctuation. Values greater than 2.0 are considered "suspect,"
and values greater than 3.0 are considered positive.
CALCULATION OF REPAIR INDEX
The data for £. coli pol A were expressed as a repair-index which
is defined as the ratio of the percent survival of the repair deficient
mutant to the percent survival of the normal strain when both are exposed
under the same conditions. Theoretically, a repair index of less than
one indicates repairable DMA damage caused by the treatment. However,
because of normal fluctuations in growth normally encountered in our
laboratory, we have considered repair index values less than 0.85 to be
positive.
Repair Index (R.I.) is defined as
(1)
N_ N;;
where+N_ and N are the number of irradiated cells of E. coli pol A and
pol A , respectively, and N_ |nd N+ are the number of unexposed cells
(control) of pol A and pol A , respectively.
12
-------
The repair index can also be expressed as a function of time.
Since the growth of the cells at 37°C in nutrient broth is exponential,
the number of cells N at any time t can be expressed as
N = NQekt (2)
where N = the number of cells at time t = 0 and k = a constant represent-
ing growth which is temperature dependent. Substituting Eq. (2) into
Eq. (1)
klt
R.I.1^—S— /—^-f— (3)
ek-t / e^
The superscripts (C,I) on k indicate the k values for control and irradiated
samples and subscripts (+,-) on k values stand for pol A and pol A ,
respectively. The k values are obtained by regression analysis of the
experimental data.
A similar expression for the repair index of sham exposed culture
can be written as
,.sh. / . sh.
where the subscript (sh) stands for sham.
13
-------
SECTION 5
RESULTS
LACK OF MICROBIAL GENETIC RESPONSE
Strain D4 of the yeast Sacch. cerevlslae and strains TA-1535,
TA-100, and TA-98 of the bacterium S. typhimurium were exposed to 2.45
GHz continuous wave or 8.5-9.6 GHz pulsed electromagnetic radiation
(EMR) at various power densities from 1 to 45 mW/cm2. The temperature
during radiation was maintained at 30°C for yeast cultures and at 37°C
for bacterial cultures. The studies revealed no increase in mutations
or of mitotic gene conversions when cells were radiated for two hours or
less. Decreased viability of cells was noted in all cultures tested
after radiation at power densities of 30 mW/cm2 or more; however, no
reliable changes in genetic events occurred. Results are summarized in
Tables 2 through 6.
TABLE 2. EFFECT OF RADIATION AT DIFFERENT POWER DENSITIES
ON CELL SURVIVAL AND GENETIC ACTIVITY OF SACCH. CEREVISIAE
STRAIN D4 (9.0 GHz PULSED EMR)
Genetic
Gene convertants/10§ survivors activity
% ade + tryp + index
mW/cm2 Survival Exposed Sham Exposed Sham ade +/tryp +
1.0
5.0
8.9
10
15
30
35
40
45
96
100
78
95
100
79
88
88
79
5.6
7.1
8.4
4.4
-
2.2
4.0
36.0
48.0
5.7
7.6
8.2
5.5
-
3.6
5.3
42.0
41.0
1.2
2.5
2.7
8.0
3.0
4.4
2.2
2.8
3.4
1.2
2.1
1.9
7.4
1.5
4.3
4.6
2.0
1.9
1.08/1.0
0.93/1.15
1.02/1.37
0.79/1.07
- /0.82
0.61/1.01
0.75/0.49
0.85/1.38
1.17/1.77
14
-------
TABLE 3. EFFECT OF DIFFERENT POWER LEVELS AND EMR FREQUENCIES ON
CELL SURVIVAL AND GENETIC ACTIVITY IN SACCH. CEREVISIAE STRAIN D4
% Survival
Frequency
(GHz)
8.5
8.6
8.8
9.0
9.2
9.4
9.6
Power
ImW
93
88
77
96
-
92
74
densities/cm2
5mW
100
99
78
100
99
100
100
45mW
55
62
59
79
-
82
42
ImW
ade+/trp+
1.41/1.50
1.13/0.92
1.27/1.62
0.92/1.02
1.88/1.42
1.62/1.24
1.14/1.12
Genetic activity index
5mW
ade+/trp+
0.76/0.80
1.20/1.11
1.18/0.95
1.08/1.15
0.91/1.94
0.91/1.79
0.83/0.83
45mW
ade+/trp+
0.60/0.63
1.27/1.37
1.90/1.00
1.16/1.16
1.49/1.24
2.17/0.97
1.00/1.48
TABLE 4. EFFECT OF PULSED EMR RADIATION AT DIFFERENT FREQUENCIES AND
POWER LEVELS ON S. TYPHIMURIUM STRAIN TA-100
Power
mW/cm2
10
10
10
10
10
45
45
45
45
45
45
45
Percent
survival
90
100
100
100
88
82
70
82
84
80
86
79
Frequency
in GHz
8.6
8.8
9.0
9.4
9.6
8.5
8.6
8.8
9.0'
9.2
9.4
9.6
Reversion/108 cells Genetic activity
Irradiated
40
27
31
35
28
42
45
59
47
48
28
41
Sham
33
17
27
25
44
35
34
30
20
47
36
33
i ndex
1.20
1.38
1.16
1.36
0.61
1.20
1.32
1.91
0.43
1.01
0.78
1.23
15
-------
TABLE 5. EFFECT OF PULSED EMR RADIATION AT DIFFERENT FREQUENCIES ON
S. TYPHIMURIUM STRAIN TA-1535 WHEN EXPOSED AT 10 mW/cm2
Frequency
in GHz
8.6
8.8
9.0
9.4
9.6
Percent
survival
96
92
100
100
80
Reversion/108 cell
Irradiated Sham
57 48
27 29
88 34
94 66
23 26
Genetic activity
i ndex
1.19
0.93
2.57
1.43
0.87
TABLE 6. EFFECT OF PULSED EMR RADIATION AT DIFFERENT FREQUENCIES ON
S. TYPHIMURIUM STRAIN TA-98 WHEN EXPOSED AT 10 mW/cm2
Frequency
in GHz
8.6
8.8
9.0
9.4
9.6
Percent
survival
100
95
100
94
100
Reversion/108 cell
Irradiated Sham
324 210
207 150
830 432
122 293
170 420
Genetic activity
i ndex
1.55
1.38
1.91
0.42
0.48
16
-------
CELLULAR EFFECTS OF ELEVATED TEMPERATURES AND MICROWAVES
Effects in Far-field Exposure
The tester strains of ^. typhimurium, E_. coli, and ^. cerevislae
were examined for lethal events when exposed to elevated temperatures or
to X-band, pulsed microwave radiation at various power densities. _When
compared to E. col i pol A under growing conditions, E. col i pol A
exhibited decreased cell growth when exposed to microwave radiation at
power levels at or above 20 mW/cm2 as well as to temperature levels
above 42°C. All yeast and other bacterial strains showed cellular
lethality at similar microwave intensities and elevated temperatures.
When exposed to elevated temperatures in saline, both quiescent yeast
and Salmonella strains exhibited lethal events. However, the Salmonella
strains tested showed comparatively less induction of genetic events in
the quiescent state compared to induction when the cells were actively
growing in both. These results demonstrate that elevated temperatures
generated by microwave exposure could produce genetic events in microbial
assay systems. If such systems are to be of value in examining the
nonthermal genetic potential of microwave radiation, careful control
over exposure conditions will be required to eliminate heat-induced
genetic events. Results are summarized in Tables 7 to 9 and Figure 4.
TABLE 7. EFFECT OF ELEVATED TEMPERATURES ON DIFFERENT
STRAINS OF SACCHAROMYCES CEREVISIAE WHEN EXPOSED
FOR 2 HOURS IN SALINE
Strain
Temp. °C
% Survival
Events /105 Cells
D3 30
33
40
45
D4 30
33
40
45 ,
D5 30
33
40
45
100
74
68
23
100
84
50
21
100
89
65
19
15
20
15
45
5/3
2.5/3
6/5.5
"•
89
91
105
150
The scores of genetic events were done as per the following
criteria:
D3 = Frequency of red+colonies_ or sectors.
D4 = Frequency of ade or try convertants expressed as a
ratio of ade/try.
D5 = Frequency of red/pink, red or pink sectors.
17
-------
TABLE 8. EFFECT OF ELEVATED TEMPERATURES ON DIFFERENT
STRAINS OF SALMONELLA TYPHIMURIUM AFTER 2 HOURS TREATMENT
Broth
Strai n
TA-1535
TA-100
TA-1538
TA-98
Temperature
37
42
47
52
37
42
47
52
37
42
47
52
37
42
47
52
% Surv.
100
83
63
100
92
60
100
63
14
100
60
20
Saline
Events/10*
Cells % Surv.
7
15
10
M_
NO
20
21
30
______ Kin
MO
5
11
37
__ fcl_
NO
78
310
367
______ Mn
NO
100
78
18
Survivals —
100
90
23
Survivals
100
96
21
Survivals
100
75
8
Survivals
Events/108
Cells
1
1
2
7
6
16
5
6
16
4
3
40
The strain TA-98 is extremely sensitive (spontaneous rate being 60/108
cells instead of 5-20/108 cells in other strains) because of an added
episome.
TABLE 9. EFFECTS OF DIFFERENT TEMPERATURES AND DURATIONS OF EXPOSURE
ON REPAIR INDICES (AVER) OF E. COLI POL A RELATIVE TO POL A
Temp. °C
Hours Exposed
10
15
Times
Repeated
37
40
43
46
49
1.00
1.14 ±
1.08 ±
0.95 ±
0.34 ±
.06
.07
.03
.02
1.00
0.98 ± .03
0.96 ± .10
0.82 1 .05
0.11 ± .08
1.00
0.90 ± .07
0.79 ± .09
0.48 ± .11
N.S.
1.00
0.95 ± .07
0.62 ± .05
N.S.
N.S.
Baseline
3
3
3
5
_ j and pol A
to 37°C which was used as the baseTTne (i.e., 100% survival) temperature.
strains were exposed
In each experiment, both E. coli pol A
to 37°C which was used as the baseline
Repair indices (R.I.) were calculated by using the standard formula:
D , _ Survival % of pol A
K.I. - H
Survival % of pol A
N.S. = No Survival
18
-------
100
50
37 40
TEMPERATURE
10 20 45
POWER DENSITY, mW/cm2
Figure 4. Survival curves of yeast and bacterial cultures when exposed
for 1 5 to 2.0 hours to either elevated temperatures or microwave radiation.
The percent cell survivals were calculated by comparing the respective
colonies grown in the sham (control) conducted at 30°C (for yeast) and
37°C (for bacteria) in each test. Figure A represents cell survival
when the yeast, Sacch. cerevisiae strain D4, was exposed to elevated
temperature as weTTas to different power densities of 8.8 GHz pulsed
radiation. Similarly, Figure B shows such survival curves with TA-10U
strain of the bacterium, S. typhimurium.
19
-------
Effects in Waveguide Exposure
The effects of pulsed microwave radiation on the growth of the E.
coli strains were explored. Experiments were performed under controlled
temperature (37° ± 0.1°C) in a waveguide exposure system using 8.6 GHz
pulsed radiation which had a pulse width of 1 usec and a pulse repetition
rate of 1 KHz. The average specific absorption rate of the sample was
12 W/kg. Two parameters were investigated: (1) the effects of+microwave
radiation on actively growing (log phase) JE. coli strains pol A and pol
A and (2) the effect of microwave radiation on cells maintained in a.
non-nutritive salt solution (saline). Exposure of the E. coli pol A
strain for up to 7 hours did not stimulate growth compared to sham-exposed
controls. Similarly, the data did not show microwave-induced growth of
E. coli pol A , although a trend was observed (0.05 < P < 0.1). The E.
coli strains that were irradiated in saline for up to 7 hours showed no
change in the subsequent growth of normal or repair deficient bacteria.
Results are summarized in Tables 10 to 12.
TABLE 10. COMPOSITE DATA OF MICROWAVE INDUCED CELL GROWTH
OF E. COLI POL A AND POL A IN VARIOUS INTERVALS OF TIME
Pol A
Pol A
Time (hrs)
N (sham)
N (sham)
1
1
1
2
2
2
4
4
4
7
7
7
151
130
141
147
131
146
142
149
129
109
111
115
157
132
144
195
179
187
237
238
213
234
284
293
137
128
140
184
177
181
242
259
236
245
286
279
130
140
140
149
129
113
122
141
116
115
137
124
146
169
187
200
177
154
271
286
275
292
395
267
131
174
190
183
183
155
250
281
247
304
342
312
Initial number of colonies in 0.5 ml of diluted culture.
'Number of colonies after exposure in 0.5 ml of diluted culture.
Number of colonies after sham treatment in 0.5 ml of diluted culture.
Sham experiments were conducted under identical conditions except
without microwave exposure.
20
-------
TABLE 11. REGRESSION ANALYSIS OF COMPOSITE DATA
ON MICROWAVE RADIATION
Regression
Strain Treatment coefficient (k) R2
A+
A+
A+
control
sham
irradiated
0.126 ± 0.014
0.124 ± 0.015
0.124 ± 0.011
0.977
0.969
0.984
A
A"
A"
control
sham
irradiated
0.141 ± 0.012
0.144 ± 0.017
0.149 ± 0.022
0.986
0.973
0.947
Regression coefficients (k) are obtained by computer linear
regression analysis of experimental data. The limits repre-
sent the 95 percent confidence, interval._ K value represents
the relative growths of pol A and pol A strains of E. coli.
For best fitting of the curve, the square of the multiple cor-
relation coefficients (R2) should be close to 1.0.
TABLE 12. SUMMARY OF CALCULATED REPAIR INDICES
AT DIFFERENT TIME INTERVALS
Repair index values
treatment
Time (hours
1
2
4
7
Irradiated
1.010
1.020
1.042
1.075
Sham
1.006
1.011
1.023
1.041
Repair index values with respect to control were
calculated using Eqs. (3) and (4) as described in the
text. K values in Eqs. (3) and (4) are obtained by
curve fitting of the experimental data using a SAS
(Statistical Analysis System) computer program in
order to generate the best possible values for the
cell population.
21
-------
Exposure of E. coli Cells to Extremely Low-level Microwave Intensities
Using Various Exposure Systems
Table 13 summarizes results of exposure of E. coli cells to 8.6
GHz, 8.8 GHz, and 9 GHz for varying time of exposures at extremely low
power using a far-field antenna exposure system. No microwave effect
was apparent when compared to sham treatments.
Table 14 summarizes results obtained by exposure of E. coli cells
to 10 GHz pulsed microwave radiation using a waveguide at an SAR of 0.1,
1, or 10 mW/g. No microwave effects were apparent when compared to sham
treatments.
Tables 15 and 16 summarize data when E. coli pol A and pol A strains
were exposed to 0.1, 1.0, or 5.2 mW/g power levels using a Crawford cell
at 915 MHz. Calculated repair-indices did not show any radiation effect
at any of the time intervals (Tables 15, 16, and 17).
TABLE 13. SUMMARY OF CELL GROWTH RATIOS IN TERMS OF COLONY
FORMING UNITS (CFU) OF E. COLI POL A STRAIN AT 8.6,
8.8, AND 8.9 GHz USING A FAR-FIELD ANTENNA EXPOSURE
SYSTEM AT 1 mW/cm2
Hours of
exposure
1
1
1
5
5
5
10
10
10
15
15
15
8.6
N (irr)*
No§
2.19
2.88
2.60
4.03
3.22
3.90
5.58
5.13
5.33
7.56
7.28
7.09
GHz
N (sham)t
No
2.25
3.15
3.06
4.12
2.96
3.23
5.72
5.21
4.98
7.15
7.76
6.84
8.8
N (irr)
No
2.37
2.46
2.47
3.47
3.51
3.66
5.25
5.01
5.16
7.27
7.12
6.84
GHz
N (sham)
No
2.03
2.06
1.89
3.05
3.18
3.15
5.47
4.73
5.31
7.78
6.86
7.15
9
N (irr)
No
2.03
2.32
2.01
4.74
3.68
4.18
5.41
5.28
5.34
6.61
6.75
6.24
GHz
N (sham)
No
2.10
2.05
1.73
4.69
4.26
4.06
4.55
4.86
5.03
7.05
6.37
6.57
*
No. of cells after irradiation.
No. of cells in control tests.
s
No. of cells at initial time (zero time exposure).
22
-------
TABLE 14. SUMMARY OF CELL GROWTH RATIOS OF E. COLI
POL A AT 10 GHz PULSED RADIATION
Hours of
exposure
7
7
7
15
15
15
0.1
N (irr)*
No§
4.91
5.23
4.78
8.00
8.01
7.03
mW/q
N (sham)t
No
4.74
5.13
4.49
7.81
7.14
7.13
1
N (irr)
No
2.66
3.23
3.35
6.33
10.89
8.97
mW/g
N (sham)
No
2.86
3.48
3.23
6.03
10.03
9.45
10
N (irr)
No
5.01
4.69
4.88
7.37
6.82
7.76
mW/g
N (sham)
No
5.17
4.78
4.67
7.56
7.48
7.43
*
No. of cells after irradiation.
No. of cells in control tests.
§
No. of cells at initial time (zero time exposure).
TABLE 15. SUMMARY OF REPAIR INDICES AT DIFFERENT DOSE RATES
Dose rate
(mW/g)
0.1
1.0
5.2
2 hours
1.12
0.96
1.12
Repair
4 hours
1.04
1.01
0.99
Index
7 hours
0.94
0.98
1.11
15 hours
1.02
1.03
1.05
23
-------
TABLE 16. SUMMARY OF CELL GROWTH OF E. COLI POL A STRAIN AT
VARYING DOSE RATES OF 915 MHz CW RADIATIONS USING A
CRAWFORD CELL SYSTEM
Time of
Exposure
(Hours)
2
2
2
4
4
4
7
7
7
15
15
15
0.1 mW/g
N (pol A+)
No (pol A+)
2.66
2.42
2.55
4.09
3.54
4.29
4.67
4.93
4.75
7.92
7.45
7.60
1 mW/g
N (pol A+)
No (pol A+)
3.09
2.18
2.59
3.94
5.41
4.00
4.66
4.93
4.73
7.86
7.07
7.59
5.20 mW/g
N (pol A+)
No (pol A+)
2.18
3.03
2.58
3.60
4.25
4.45
4.09
3.57
4.86
8.76
7.01
7.64
37°C
N (pol A+)
No (pol A+)
2.43
3.30
2.65
4.42
4.04
4.24
4.61
4.16
4.82
9.10
7.02
7.10
No (pol A ) = Number of cells of E. coli pol A strain in 0.5 ml of
culture with 106 times dilution using 0.9% saline at time zero.
N (pol A ) = Number of cells of E. coli pol A strain in 0.5 ml of
culture with 106 times dilution using 0.9° saline following exposure
for any specified time.
24
-------
TABLE 17. SUMMARY OF CELL GROWTH OF E. COLI POL A STRAIN AT
VARYING DOSE RATES OF 915 MHz CW RADIATIONS USING A
CRAWFORD CELL SYSTEM
Time of
Exposure
(Hours)
2
2
2
4
4
4
7
7
7
15
15
15
0.1 raW/g
N (pol A")
No (pol A")
2.61
3.07
2.98
3.75
4.71
4.27
4.75
4.62
5.76
9.32
8.68
8.91
1 mW/g
N (pol A")
No (pol A")
2.37
2.17
3.09
4.38
5.61
3.77
5.55
5.06
4.85
9.22
8.33
9.04
5.20 mW/g
N (pol A")
No (pol A")
3.18
3.29
2.32
3.73
4.58
4.21
5.58
4.88
4.98
11.64
8.06
8.44
37°C
N (pol A")
No (pol A")
2.96
2.78
2.65
4.36
4.76
3.88
4.35
5.50
5.20
8.90
8.86
8.83
No (pol A~) = Number of cells of E. coli pol A strain in 0.5 ml of
culture with 106 times dilution using 0.9% saline at time zero.
N (pol A ) = Number of cells of E. coli pol A strain in 0.5 ml of
culture with 106 times dilution using 0.9% saline following exposure
for any specified time.
25
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ANCILLARY STUDIES
There is a fundamental problem in microwave research which must be
addressed. That is, what is the confunding influence of microwave-induced
temperature rise and how can the influence of this temperature rise be
removed from the overall change in the biological endpoint. One approach
is to observe the response to various temperatures and compare the
predicted response at various temperatures with the response observed
under microwave exposure. With this in view, we performed several
extensive studies. Results of one of those studies are described below.
A Quantitative Comparison of Thermal Versus Microwave-induced Alterations
in Bacterial Growth
An interpretation of microwave-induced biological effects is often
confused due to the inability to discount the thermal response component
from the total response of biological systems. A technique has been
described to quantitatively analyze bacterial growth responses associated
with thermal and microwave treatments using E. coli pol A and pol A .
Data for the thermal response models were obtained by exposing bacterial
cultures in 35 mm diameter petri dishes to a series of temperatures
(37°C to 49?C) for various times (1 hour to 15 hours). Data for the
microwave response were obtained by exposing similarly-prepared cultures
at one temperature (35°C) to 8.8 GHz radiation pulsed at 1000 MH with a
duty cycle of 0.001 and a SAR of 40 W/kg. Knowledge of the temperature
of the microwave-exposed samples allows a comparison of the microwave
growth rate with the thermal growth rate at the same temperature. The
difference between the observed and the thermal growth rates is then
tested for significance by using the two-way analysis of variance. In
this case, the bacterial growth response could be completely described
by the thermal response. The basic approach described here should prove
useful wherever a microwave-induced change is to be evaluated when ,
endpoint is susceptible to temperature perturbation. Results are summarized
in Tables 18 to 20 and Figure 5.
26
-------
TABLE 18. SUMMARY OF CELL GROWTH OF E. COLI POL A
Time of exposure Exposed to 8.8 GHz
(hours) NQ* Nt N/NQ
2
2
2
5
5
5
10
10
10
15
15
15
120
122
157
141
140
143
162
110
114
142
146
126
167
177
221
204
194
207
187
128
141
147
202
130
1.392
1.451
1.408
"1.447
1.386
1.448
1.154
1.164
1.237
1.035
1.062
1.032
Time
(hours)
1
1
1
5
5
5
10
10
10
15
15
15
Exposed at
No Nc
168
168
168
168
168
168
168
168
168
92
122
122
185
179
185
302
309
312
593
551
562
628
701
751
35.2°C
VNo
1.074
1.068
1.098
1.795
1.942
1.857
3.529
3.280
3.348
6.126
5.746
6.205
Number of cells at time zero in 0.5 ml of culture diluted 106 by
0.9% saline.
Number of cells at any specified exposure time in 0.5 ml culture
diluted 10s by 0.9% saline.
27
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TABLE 19. SUMMARY OF CELL GROWTH OF E. COLI POL A
Time of exposure Exposed to 8.8 GHz
(hours) NQ* Nf N/NQ
2
2
2
5
5
5
10
10
10
15
15
15
151
149
144
140
144
142
123
120
127
128
127
140
217
211
196
201
209
199
154
161
159
134
141
171
1.436
1.416
1.361
1.436
1.451
1.401
1.250
1.342
1.252
1.047
1.110
1.221
Time
(hours)
1
1
1
5
5
5
10
10
10
15
15
15
Exposed at
No Nc
149
149
149
149
149
149
149
149
170
110
112
112
155
152
161
271
265
261
481
462
570
530
576
566
35.2?C
Nc/No
1.040
1.017
1.081
1.818
1.781
1.755
3.231
3.101
3.353
4.840
5.129
5.054
t
Number of cells at time zero in 0.5 ml of culture diluted 106 by
0.9% saline.
Number of cells at any specified exposure time in 0.5 ml culture
diluted 106 by 0.9% saline.
28
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TABLE 20. SUMMARY OF THE CELL GROWTH AT 42.2°C OF E. COLI POL A+ AND POL A" STRAINS
Time
(hours)
2
2
2
5
5
5
10
10
10
15
15
15
NQ (pol A+]
232
115
120
128
132
132
115
156
120
156
120
156
) N (pol A+)
279
154
167
183
186
197
151
173
139
166
123
162
N (pol A+)
N0 (PQl A+)
2.356
1.339
1.392
1.426
1.409
1.492
1.313
1.109
1.158
1.064
1.025
1.038
N (pol A )
123
123
109
121
115
115
123
123
160
121
121
160
N (pol A")
177
173
159
171
164
166
149
146
201
135
132
175
N (pol A")
N0 (fi.1 A')
1.439
1.407
1.459
1.412
1.422
1.443
1.211
1.187
1.256
1.116
1.092
1.074
29
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TIME (hours)
Figure 5. Composite data showing plot of average N/N against time of
exposure.
N = Number of cells at specified time
NQ = Number of cells at time zero
Growth of cells N/N treated at 35.2°C in air incubator is shown by A—A
and 0—0 represents survival of cells treated with 8.8 GHz pulsed microwave
radiations in a anechoic exposure chamber at 35.2° ± 0.2°C. The specific
absorption rate (SAR) is 40 W/kg.
30
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REFERENCES
1. Allis, J. W., C. F. Blackman, M. L. Fromme, and S. G. Benane.
Measurement of Microwave Radiation Absorbed by Biological Systems,
1 Analysis of Heating and Cooling Data. Radio Science, 12:1-8,
1977.
2. Ames, B. N., J. McCann, and E. Yamasaki. Methods for Detecting
Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome
Mutagenicity Test. Mutation Res., 31:347-364, 1975.
3. Baranski, S., and P. Czerski. Biological Effects of Microwaves.
Dowden, Hutchinson & Ross, Inc. Library of Congress Catalog #74-7837.
4. Blackman, C. F., S. G. Benane, C. M. Weil, and J. S. Ali. Effects
of Nonionizing Electromagnetic Radiation on Single-Cell Biologic
Systems. Ann. N.Y. Acad. Sci., 247:352-366, 1975.
5. Brusick, D. J. The Mutagenic Activity of ICR-170 in Saccharomyces
cerevisiae. Mutation Res. 10:11-19, 1970.
6. Brusick, D. J., and H. Andrews. Comparisons of the Genetic Activity
of Diethylnitrosamine, EMS, 2-Acetylamino Fluorene and ICR-170 in
Sacch. cerevisiae Strains D3, D4 and D5 Using In Vitro Assays With
and Without Metabolic Activation. Mutation Res., 26:491-500, 1975.
7. Brusick, D. J., and V. W. Mayer. New Developments in Mutagenecity
Screening with Yeast. Environ. Health Perspective, 6:83-96, 1973.
8. Cleary, S. F. Biological Effects of Microwave and Radio-Frequency
Radiation, C.R.C. Critical Reviews in Environmental Control, Vol.
7, pp. 121-166, 1977.
9. D'Alisa, R. M., G. A. Carden, H. S. Carr, and H. S. Rosenkranz.
"Reversion" of DNA Polymerase-deficient Escherichia coli. Mol.
Gen. Genet., 110:23-26, 1971.
10. De Lucia, P., and J. Cairus. Isolation of an E. coli Strain with a
Mutation Affecting DNA Polymerase. Nature, 224:1164-1166, 1969.
11. Dutta, S. K., W. H. Nelson, C. F. Blackman, and D. J. Brusick.
Lack of Genetic Effects of Exposures of Selected Microbial Strains
to 2.45 GHz CW and 8.6-9.6 GHz Pulsed Electromagnetic Radiation.
J. Microwave Power, 14:275-280, 1979.
12. Elder, J. A., and J. S. Ali. The Effect of Microwaves (2450 MHz)
on Isolated Rat Liver Mitochondria. Ann. N.Y. Acad. Sci., 247:251-
262, 1975.
31
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13. Ho, H. S., M. R. Foster, and M. L. Swicord. Microwave Irradiation
Apparatus Design and Dosimetry. Biological Effects of Electromagnetic
Waves, Selected Papers of the USNC/USRI Annual Meeting, Boulder,
Colorado, October 20-23, 1975, Vol. II, pp. 423-434, December,
1976.
14. Report on "Biological Hazards of Non-ionizing Electromagnetic
Radiation" (NEMR), Executive Office of the U.S. President, Office
of Telecommunications Policy, May, 1974.
15. Slater, E. E., M. D. Anderson, and H. S. Rosenkranz. Rapid Detection
of Mutagens and Carcinogens. Cancer Research, 31:970-973, 1971.
16. Symposium on "Biological Effects and Health Implication of Microwave
Radiation," Richmond, Virginia, September 17-19, 1969, S. F. Cleary,
Editor, U.S. Department of Health, Education and Welfare, Bureau of
Radiological Health Publication No. BRA/DBE 70-72.
17. Zimmermann, F. K., and R. Schwaier. Induction of Mitotic Gene
Conversion with Nitrous Acid, l-methyl-3-nitro-l-nitrosoguanidine
and Other Alkylating Agents in Saccharyomyces cerevisiae. Mol.
Gen. Genet., 100:63-76, 1967.
18. Zimmermann, F. K. Detection of Genetically Active Chemicals Using
Various Yeast Systems. In: Chemical Mutagens, Vol. 3, A. Hollaender
(ed.), Plenum Press, N.Y., 1973. pp. 209-237.
19. Zimmermann, F. K. Procedures Used in the Induction of Mitotic
Recombination and Mutation in the Yeast Saccharomyces cerevisiae.
Mutation Res., 31:71-86, 1975.
32
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TECHNICAL REPORT DATA
{Please read Instructions on the reverse before completing}
1. REPORT NO.
EPA-^600/1 -80-027
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
GENETIC AND CELLULAR EFFECTS OF MICROWAVE RADIATIONS
5. REPORT DATE
May 1980
G. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
S.K. Dutta, Ph.D.
8. PERFORMING ORGANIZATION REPC^I
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Botany
Howard University
Washington, DC 20059
10. PROGRAM ELEMENT NO.
1FA628
11. CONTRACT/GRANT NO.
R-803561
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
FINAL, 8 May 1975 - 31 Dec 1979
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
Prepared by Carl Blackman, Project Officer.
16. ABSTRACT
This research program was initiated with the overall objective of determining
genetic and cellular effects from exposure of unicellular organisms to selected
frequencies of CW and pulsed microwave radiation which is prevalent in our biospnere.
Several tester strains of the bacterium Salmonella typimurium, TA-98, TA-100,
TA-1535 and TA-1538; the bacterium Escherichia coli. W311Q [pol Aj and p3438 (pol A",
repair deficient); and the yeast Saccharomyces cerevisiae, D3, D, and D5 were tested for
lethal amd mutagenic events. Effects of known elevated temperatures were studied to
distinguish microwave induced temperature effects from the direct temperature effects.
Three kinds of microwave exposure systems were used in these studies: (1) farfield
antenna (for 2.45 GHz and 8.5 - 9.5 GHz), (2) waveguide (for 8-10 GHz) and (3) TEH
(transverse electric and magnetic mode) transmission lines for 915 MHz radiation. The
SAR (specific absorption rate) for various exposures ranged from 0.1 W/kg to 40 W/kg.
Pulse repetition rates were 400 Hz and 1000 Hz for pulsed microwave radiations.
The studies revealed no increase in mutations or of gene conversions when cells
were exposed to microwave radiations!, but yeast and bacterial strains showed cellular
lethality caused by temperature rises (greater than 10°C) at higher power levels.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Radiation
Microwave
Salmonella typimurium
Escherichia coli
Saccharomyces cerevisiae
06,C,R
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
33
20. SECURITY CLASS I This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
33
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