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SOMATIC AND GENETIC EFFECTS OF LOW SAR 2.45 GHz MICROWAVE RADIATION ON WISTAR RATS

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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  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. 
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 Interactions of Microwave with Biological Materials 10
 2.3.1 Thermal Mechanisms of Interaction 10
2.3.2 Athermal (Non-thermal) Mechanisms of Interaction 11 
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 Temperature Effects 23
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
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 Fertility Studies 46
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 Effects of Microwave Exposure on the Animal Reproductive Organs 66
4.3.1 Effects on the Body and Organ Weights of the Wistar Rats after Exposure to MW Radiation.
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 Conclusion and Recommendation 105
REFERENCES 108
Appendix 132 
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 94
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 95
4.22 Densitometric track analysis of male and female lung DNA. Lane a is the
 DNA of the control; b-j are DNA from exposed animals 96
4.23 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 
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 99
4.26 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. 101
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 102
4.29 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 
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 70
4.8 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 71
4.9 Effect of exposure of matured male Sprague Dawley to 2.45 GHz microwave
 radiation after four weeks post-exposure period on semen parameters 73 
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). 89
4.3 Hybridization of the brain DNA in male and female rats. Note the
 appearance of other bands in lane b "j (exposed animals). 89
4.4 Hybridization of the lung DNA in male and female rats. Note the
 appearance of other bands in lane b "j (exposed animals). 90
4.5 Hybridization of the heart DNA in male and female rats. Note the
 appearance of other bands in lane b "j (exposed animals). 90
4.6 Hybridization of the liver DNA in male and female rats. Note the
 appearance of other bands in lane b "j (exposed animals). 91
4.7 Hybridization of the ovary DNA. B. Hybridization of the testis DNA.
 Note the appearance of other bands in lane b "j (exposed animals). 91
4.8 Hybridization of the kidney DNA in male and female rats. Note the
 appearance of other bands in lane b "j (exposed animals). 92
4.9 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). 92
4.10 Hybridization of the Thyroid DNA in male and female rats. Note the
 appearance of other bands in lane d "j (exposed animals). 93
4.11 Hybridization of the spleen DNA in male and female rats. Note the
 appearance of other bands in lane b "j (exposed animals). 93 

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