SOMATIC AND GENETIC EFFECTS OF LOW SAR
2.45 GHz MICROWAVE RADIATION ON WISTAR
RATS
A Ph.D THESIS
BY
USIKALU, MOJISOLA RACHAEL
MATRIC NO: CUGP050144
August, 2010
i
SOMATIC AND GENETIC EFFECTS OF LOW SAR
2.45 GHz MICROWAVE RADIATION ON WISTAR
RATS
A Ph.D THESIS
BY
USIKALU MOJISOLA RACHAEL
(CUGP050144)
Submitted to the School of Post Graduate Studies of Covenant University, Ota
In partial fulfillment of the requirements for the award of degree of
Doctor of Philosophy (Ph.D.) in Radiation and Health Physics
AUGUST, 2010
ii
CERTIFICATION
This is to certify that Mrs. USIKALU, Mojisola Rachael (Matric No: CUGP050144) carried out
this research work in partial fulfillment of the requirements for the award of Doctor of
Philosophy (Ph.D.) degree in Physics (Radiation and Health Physics) of Covenant University,
Ota, under our supervision.
Dr. M. A. Aweda
Signature
Date …………………….
Signature
Date …………………….
Signature
Date …………………….
(Main Supervisor)
Professor Guangming Zhou
(Co-Supervisor)
Professor E.B. Babatunde
(Co-Supervisor)
iii
DECLARATION
I hereby declare that this thesis entitled “SOMATIC AND GENETIC EFFECTS OF LOW SAR
2.45 GHz MICROWAVE RADIATION ON WISTAR RATS” was compiled and written by me
and that it is a record of my research work. It has not been presented in any previous application
for a higher degree. All quotations and the sources of information are specifically acknowledged
by means of references.
USIKALU, Mojisola R.
iv
DEDICATION
This work is dedicated to the Almighty God the giver and source of all knowledge. Also to my
dearly beloved husband, Oladipo Victor and God’s perfect gift Ayomikun
v
ACKNOWLEDGEMENTS
I am grateful to God Almighty the giver of power, wisdom, and strength, from whom all mercies
flow for seeing me through the research work and this dissertation.
I wish to express my profound gratitude to Dr. M.A. Aweda and Professor Guangming Zhou for
their words of encouragement and for thorough supervision of this work. I appreciate very much
their guidance, making their libraries available to me and for their support. I appreciate them also
for their readiness to attend to my questions on this work at any time.
The support of Third World Organization for Women in Science (TWOWS) Trieste, Italy is
gratefully acknowledged for the TWOWS fellowship granted me to complete this research work
in the Institute of Modern Physics, Chinese Academy of Sciences, China.
The support of Institute of Modern Physics, Chinese Academy of Sciences, China, is also
gratefully acknowledged for granting me access to their laboratories and equipment. I am
particularly grateful to all staff of the institute most especially radiobiological group for their
care and affection throughout my stay in Lanzhou, China.
I wish to show my gratitude to the management of Covenant University for the staff
development program and for granting me study leave to conclude the research in Institute of
Modern Physics, Chinese Academy of Sciences, China under TWOWS fellowship.
I acknowledge with thanks the encouragement received from members of staff of Department of
Physics, Covenant University Ota under the leadership of Dr. Uhuegbu C.C. I also appreciate
Professor E.B. Babatunde, my co-supervisor for his support and fatherly advice.
Finally, I wish to express my deep gratitude to my husband, Dipo, perfect gift of God, Ayomikun
and my family members for their love, prayers, understanding, word of encouragement and for
standing by me during the period of this study.
vi
TABLE OF CONTENTS
Title page
i
Certification
ii
Declaration
iii
Dedication
iv
Acknowledgements
v
Table of contents
vi
List of Figures
xi
List of Tables
xiii
List of Plates
xiv
Abstract
xvi
CHAPTER ONE
INTRODUCTION
1.1
Preamble
1
1.2
Statement of the Problem
4
1.3
Rationale for the study
4
1.4.1 Aim and Objectives
4
1.4.2 Specific Objectives
5
CHAPTER TWO
LITERATURE REVIEW
2.1
Microwave Origin and Spectrum
6
2.2
Generation and Uses of Microwaves
7
2.2.1 Telecommunication
7
2.2.2 Remote Sensing
8
2.2.3 Health
9
2.2.4 Navigation
9
2.2.5 Power
9
2.3
10
Interactions of Microwave with Biological Materials
2.3.1 Thermal Mechanisms of Interaction
10
2.3.2 Athermal (Non-thermal) Mechanisms of Interaction
11
vii
2.4
Macroscopic Interactions
13
2.5
Microscopic Interaction -- Molecular Mechanisms
18
2.5.1 Cellular Interactions
22
2.5.2 Other Interaction Mechanisms
23
2.6
23
Temperature Effects
2.6.1 Effects: High – Frequency Fields Force
26
2.7
Radiometry of Non-ionizing Radiation
28
2.8
Free Radicals
29
2.8.1 Hydroxyl Radical
29
2.8.2 Nitric Oxide
30
2.8.3 Superoxide (O2-)
30
2.8.4 Hydrogen Peroxide
30
2.8.5 Trichloromethyl (CCI3)
31
2.8.6 Free Radicals and Lesion in DNA
31
2.9
Plausible Biological Mechanism
31
2.10
Non-human Studies
32
2.10.1 Thermoregulatory Responses to RF radiation
32
2.10.2 Reproductive Effects
32
2.10.3 Effects on the Blood-forming and Immune Systems
33
2.10.4 Nervous System
35
2.10.5 Behavioural Effects
36
2.10.6 Molecular, Subcellular and Cellular Effects
37
2.11
Genetic Toxicology
37
2.12
Epidemiological Investigations
40
CHAPTER THREE
MATERIALS AND METHODS
3.1
Calibration of Microwave (MW) Source
42
3.2
Determination of Specific Absorption Rate (SAR)
42
3.3
Animal and Sample Preparations
42
viii
3.4
Behavioural Studies
43
3.4.1 Exploratory Behavioural Activity Study
43
3.4.2 Anxiolytic Activity Study
43
3.4.2.1 Elevated Plus Maze Study
43
3.4.2.2 Y- maze Study
44
3.5
46
Fertility Studies
3.5.1 The Weight of the Specimens
46
3.5.2 Semen Collection
46
3.5.3 Sperm Motility Study
46
3.5.4 Sperm Morphology Study
46
3.5.5 Sperm Count
47
3.5.6 Histopathological Studies on the Reproductive Organs
47
3.6
Chromosomal Aberration Study from Rats Bone Marrow
47
3.7
Deoxyribonucleic Acid (DNA) Assay
48
3.7.1 DNA Extraction and Washing of the Blood Cells
48
3.7.2 Lysing of WBC and other Organs
48
3.7.3 Phenol Extraction and Precipitation of DNA
49
3.8
Quantification of DNA Sample
49
3.9
Primers
49
3.10
Amplification and Electrophoresis
49
3.11
Single Cell Gel Electrophoresis (Comet Assay)
50
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1
Calibration and Determination of Specific Absorption Rate (SAR)
53
4.2
Effects of Microwave Radiation on the Behavioural Study
53
4.2.1 Effects of Microwave on the Exploratory Behaviour
53
4.2.2 Effects of Microwave Radiation on the Elevated Plus Maze and Y-Maze Study
54
4.3
66
Effects of Microwave Exposure on the Animal Reproductive Organs
4.3.1 Effects on the Body and Organ Weights of the Wistar Rats after Exposure to MW
Radiation
66
ix
4.3.2 Effects of Microwave Radiation on the Spermatozoa Parameters
67
4.3.3 Effects MW Radiation on the Histopathology Study
76
4.4
Effects of Microwave Radiation on the Chromosomal Study
84
4.5
Genotoxic Effects of Microwave
86
4.5.1 Microwave Effects on DNA Direct Amplification of Length Polymorphisms
(DALP) Analysis
86
4.5.2 Microwave radiation Effects on Comet Assay
87
4.6
105
Conclusion and Recommendation
REFERENCES
108
Appendix
132
x
LIST OF FIGURES
Figure
Title
Page
1.1
Electromagnetic Spectrum
2
4.1
Variation of Thermistor Resistance with Temperature (Calibration Curve)
57
4.2
Variation of Rectal Temperature with Time
58
4.3
Variation of SAR with Mass
58
4.4
Microwave Effects on the Exploratory Behaviour in Male Rats
59
4.5
Microwave Effects on the Exploratory Behaviour in Female Rats
59
4.6
% Time Spent in Open Arms of EPM by Male Rats
62
4.7
% Time Spent in Open Arms of EPM by Female Rats
62
4.8
% Time Spent in Open Arms of Y-Maze by Male Rats
65
4.9
% Time Spent in Open Arms of Y-Maze by Female Rats
65
4.10
Variation in Body weights over a period of 4 weeks (Male)
69
4.11
Variation in Body weights over a period of 4 weeks (Female)
69
4.12
Relative weight of vital organs four weeks post exposure (Male)
72
4.13
Relative weight of vital organs four weeks post exposure (Female)
72
4.14
Variations in the semen cells morphology four weeks post exposure
74
4.15
Variations in the gross sperm motility four weeks post exposure
74
4.16
Variations in the % Life/dead Sperm four weeks post exposure
75
4.17
Variations in the Sperm Counts four weeks post exposure
75
4.18
Variations of the Chromosomal Aberrations in Male Rats
85
4.19
Variations of the Chromosomal Aberrations in Female Rats
85
4.20
Densitometric track analysis of male and female blood DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals
4.21
Densitometric track analysis of male and female brain DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals
4.22
95
Densitometric track analysis of male and female lung DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals
4.23
94
96
Densitometric track analysis of male and female heart DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals.
97
xi
4.24
Densitometric track analysis of male and female liver DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals.
98
4.25A Densitometric track analysis of Ovary DNA. Lane a is the DNA of the
control; b-j are DNA from exposed animals
99
4.25B Densitometric track analysis of Testis DNA. Lane a is the DNA of the
control; b-j are DNA from exposed animals
4.26
99
Densitometric track analysis of male and female kidney DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals
100
4.27A Densitometric track analysis of male and female Tails DNA before
exposure having peak 1 only.
101
4.27B Densitometric track analysis of Prostate DNA. Lane a is the DNA of the
control; b-j are DNA from exposed animals.
4.28
Densitometric track analysis of male and female Thyroid DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals
4.29
101
102
Densitometric track analysis of male and female Spleen DNA. Lane a is the
DNA of the control; b-j are DNA from exposed animals
103
4.30
The mean + SD of % DNA in tail after exposure to 2.45 GHz microwave radiation 104
4.31
The mean + SD of Olive moment after exposure to 2.45 GHz microwave radiation 104
xii
LIST OF TABLES
Table
Title
Page
4.1
Cumulative time spent by the male rats in the Elevated Plus Maze
60
4.2
Cumulative time spent by the female rats in the Elevated plus Maze
61
4.3
Cumulative time spent by the male rats in the Elevated Y-Maze
63
4.4
Cumulative time spent by the female rats in the in Elevated Y-Maze
64
4.5
Effects of microwave radiation exposure on the average weight of the male rats
68
4.6
Effects of microwave radiation exposure on the average weight of the female rats
68
4.7
Effect of exposure on matured male Sprague Dawley to 2.45 GHz microwave radiation
after four weeks post-exposure period on reproductive organ and other visceral vital
organs weight
4.8
70
Effect of exposure on matured female Sprague Dawley to 2.45 GHz microwave
radiation after four weeks post-exposure period on reproductive organ and other
vital organs weight
4.9
71
Effect of exposure of matured male Sprague Dawley to 2.45 GHz microwave
radiation after four weeks post-exposure period on semen parameters
73
xiii
LIST OF PLATES
Plate
Title
Page
4.1A
Micrograph of kidney for control group (female)
78
4.1B
Micrograph of kidney for SAR 0.48 W/kg (female)
78
4.1C
Micrograph of kidney for SAR 1.43 W/kg (female)
78
4.1D
Micrograph of kidney for SAR 1.91 W/kg (female)
78
4.1E
Micrograph of kidney for SAR 2.39 W/kg (female)
79
4.1F
Micrograph of liver for control group (female)
79
4.1G
Micrograph of liver for SAR 0.95 W/kg (female)
79
4.1H
Micrograph of liver for SAR 1.43 W/kg (female)
79
4.1I
Micrograph of liver for SAR 1.91 W/kg (female)
80
4.1J
Micrograph of liver for SAR 2.39 W/kg (female)
80
4.1K
Micrograph of ovary for control group (female)
80
4.1L
Micrograph of ovary for SAR 0.48 W/kg (female)
80
4.1M Micrograph of ovary for SAR 0.95 W/kg (female)
81
4.1N
Micrograph of ovary for SAR 1.43 W/kg (female)
81
4.1O
Micrograph of ovary for SAR 1.91 W/kg (female)
81
4.1P
Micrograph of ovary for SAR 2.39 W/kg (female)
81
4.1Q
Micrograph of testis for control group (male)
82
4.1R
Micrograph of testis for SAR 0.48 W/kg (male)
82
4.1S
Micrograph of testis for SAR 0.95 W/kg (male)
82
4.1T
Micrograph of testis for SAR 1.43 W/kg (male)
82
4.1U
Micrograph of testis for SAR 1.91 W/kg (male)
83
4.1V
Micrograph of liver for control group (male)
83
4.1W Micrograph of liver for SAR 0.48, 0.95 and 1.43 W/kg (male)
83
4.1X
Micrograph of liver for SAR 1.91 and 2.39 W/kg (male)
83
4.2
Hybridization of the blood DNA in male and female rats. Note the
appearance of other bands in lane b –j (exposed animals).
4.3
89
Hybridization of the brain DNA in male and female rats. Note the
appearance of other bands in lane b –j (exposed animals).
89
xiv
4.4
Hybridization of the lung DNA in male and female rats. Note the
appearance of other bands in lane b –j (exposed animals).
4.5
Hybridization of the heart DNA in male and female rats. Note the
appearance of other bands in lane b –j (exposed animals).
4.6
92
Hybridization of the Thyroid DNA in male and female rats. Note the
appearance of other bands in lane d –j (exposed animals).
4.11
92
Hybridization of the prostate and control tails DNA in male and female rats.
Note the appearance of other bands in lane b –j (exposed animals).
4.10
91
Hybridization of the kidney DNA in male and female rats. Note the
appearance of other bands in lane b –j (exposed animals).
4.9
91
Hybridization of the ovary DNA. B. Hybridization of the testis DNA.
Note the appearance of other bands in lane b –j (exposed animals).
4.8
90
Hybridization of the liver DNA in male and female rats. Note the
appearance of other bands in lane b –j (exposed animals).
4.7
90
93
Hybridization of the spleen DNA in male and female rats. Note the
appearance of other bands in lane b –j (exposed animals).
93
xv
ABSTRACT
The somatic and genetic effects of 2.45 GHz Microwave radiation (MWR) on male and female
Sprague Dawley rats were studied. Two hundred rats were used for this study. They were
grouped into control and exposed according to the parameters under consideration. The animals
were exposed to various levels of specific absorption rate (SARs) using the microwave
generator, model ER660E, Serial No MX704CCR from Toshiba UK Ltd. All animals were kept
in healthy, and radiation free environment with water and feed provided ad libitum. The study is
divided into three parts; firstly, the effects of MWR on the anxiolytic and exploratory behaviour
of the animals. MWR produced dose-dependent significant decrease in the cumulative time spent
in the open arms of maze in the first twelve days after exposure and gradually increased towards
the control value before the end of two weeks. It also affects the exploratory behavoiur of the
animals, for the first two weeks after exposure but by the third week there was no significant
difference between the control and the exposed animals. The modifications in the behaviour were
not sex dependent as there were no significant differences between male and female rats.
Secondly, the effects of MWR on the vital organs of male and female most especially, male
reproductive functions were studied. The sperm count, gross motility and sperm morphology
were determined using standard methods. The results showed that 2.45 GHz MWR reduced the
sperm concentration, gross motility while increasing the percentage abnormal sperm cells. There
was a decrease in reproductive organ weights and increased dead sperm cells as large numbers of
the sperm cells membranes took up the eosin-nigrosin vital stain in the exposed group. Lastly,
the genotoxic effects of MWR were studied using Chromosomal aberrations, single cell gel
electrophoresis (comet assay) and direct amplification of length polymorphisms (DALP). There
was a significant increase in the frequency of chromosomal aberrations between control and
xvi
exposed groups. The exposure of vital mammalian cells to 2.45 GHz radiation altered the band
patterns of the exposed animals, especially in the range 40 – 120 bp. Starting with appearance of
additional peaks compared with the control which was not seen in their tail DNA before
exposure to MW and these were confirmed through the densitometric gel analysis. There was a
statistically significant difference in the Olive moment and % DNA in tail of the exposed
animals compared with control (p < 0.05). Thus, these findings support the hypothesis that
exposure to 2.45 GHz MWR at studied SARs may have potential somatic and genetic effects.
xvii