Copyright is owned by the Author of the thesis.  Permission is given for 
a copy to be downloaded by an individual for the purpose of research and 
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EFFECTS OF 
EXTREMELY LOW FREQUENCY 
ELECTROMAGNETIC FIELDS 
ON 
HUMAN CHROMOSOMES. 
A thesis presented in partial fulfilment of the 
requirements for the degree of 
Doctor of Philosophy 
in 
Genetics 
at the 
Institute of Molecular BioSciences 
Massey University, Palmerston North, 
New Zealand 
by 
MOHAMMED ABDUL WAHAB 
2005 
11 
III 
Abstract. 
Electromagnetic fields (EMFs) have been associated with increased incidences of 
cancer as suggested by epidemiological studies. The in vitro sister chromatid exchange 
(SCE) technique, radiation-induced micronucleus assay (MN assay), COMET assay, 
and fluorescence in situ hybridization (FISH) were used in the present study to test the 
carcinogenic potentiality of extremely low frequency (ELF) EMFs on human peripheral 
blood lymphocytes. All experiments were performed single blind and used lymphocytes 
taken from 6 age-matched donors. The SCE experiments were conducted twice: round I 
(RI) and round 2 (R2), in order to determine whether or not the results obtained could 
be duplicated. 
Detailed analysis of the SCE results showed that there was a significant increase in the 
number of SCEs/cell in the grouped experimental conditions compared to the controls 
in both rounds. Similarly, in the MN assay, a significant increase of mean number of 
micronucleated CB cells/lOO CB cells (Ma) and mean number of micronuclei/l 00 CB 
cells (Mb) was observed in the grouped experimental conditions compared to the 
controls. Moreover, the highest SCE frequency in RI was 1 0 .03 for a square continuous 
field, and the SCE frequency of 10 .39 for a square continuous field in R2 (albeit a 
different strength) was the second highest in this latter round. But in the MN assay a 
square pulsed field with increasing EMF strength showed the greatest effect on the 
DNA repair system. The COMET assay also showed that both a l mT  square field 
(continuous or pulsed) resulted in significant fragmentation of the DNA. On the other 
hand, a FISH analysis failed to show any translocations. 
In the field of EMF research, perhaps the most outstanding question that remains to be 
answered with certainty is how weak EMFs exert their effects at the molecular level. 
Various mechanisms are reviewed and evaluated in this thesis. From the results of the 
research performed in the current study which concentrated on testing and discovering 
genetic effects, a model is postulated that weak EMFs stimulate the production of free 
radicals which result in genetic damage. Further extensive research should be conducted 
to test this hypothesis. 
IV 
Acknowledgments. 
I wish to extend my sincere gratitude to my supervisors, Dr. R.E. Rowland, and Dr. J.V. Podd, without 
whom I would not have had the wonderful opportunity to tackle this piece of work. Their constant 
invaluable guidance, advice, encouragement and supervision kept me on track and from both of them I 
have learnt a lot. 
I would like to express my sincere thanks to: 
• Elizabeth Nickless-who was an endless help in providing the invaluable advice throughout my 
research on methods and equipment. 
• Charlotte James - who was my friend in need, and endless helper during my study. 
• Bruce Rapley - for his invaluable contribution in building the special incubator to generate accurate 
ELF EMFs and assistance during the study. 
• Chris Kendrik - his assistance with blood collection was invaluable and greatly appreciated. Chris s 
smiling face continually inspired me. 
• Chad Johnson - who taught me how to perform the COMET assay. 
• Ted Drawneek - is a nice person, who assisted me with all the statistical analysis. 
• Paul Hauquard, Michael Wilson - who were two of my blood donors and gave me endless 
assistance and made my IMBS life much easier. 
• Amira Pearson - Auckland University Medical School, who is a person with infinite patience from 
whom I learned the MN assay technique. 
• Colin Dicks and his oncology staff in the Palmerston North Hospital - for their endless friendship 
and co-operation during this study. 
• Rissa Ota, Matthew Phillips, Leon Perrie, Kerryn Slack, Tim White, and Andrew Clarke - who 
generously offered their computer expertise. 
To my family, especially my wife Kamrunnahar Begum, and my many friends and colleagues who 
have given me endless support and space to complete this work, I give my hearfelt thanks. Their help and 
encouragement are sincerely appreciated. 
Finally, I would like to express my sincere thanks to Massey University for providing me with the 
Massey Doctoral Scholarship and Palmerston North Medical Research Foundation for providing me with 
a research grant. 
DEDICATED 
TO 
MY BELOVED WIFE 
KAMRUNNAHAR BEGUM (Hira) 
IN APPRECIATION OF HER 
CONTINUOUS HELP, 
SUPPORT AND 
LOVE. 
v 
Table of Contents. 
Opening page 
Title page 
Abstract 
Acknowledgments 
Dedication 
Table of Contents 
List of Tables 
Lists of Figures 
Chapter One: Introduction. 
1. INTRODUCTION 
1.1 Sister Chromatid Exchange (SCE) 
1.2 Micronucleus Assay (MN Assay) 
1.3 The COMET Assay 
1.4 Fluorescent In Situ Hybridization (FISH) 
1 .5 Aim, Objective and Prediction 
Chapter Two: Review of Literature. 
I 
Il 
III 
IV 
V 
VI 
XI 
XII 
1 
4 
5 
5 
6 
7 
2. REVIEW OF LITERATURE 8 
2.1 ELF EMFs and Living Organisms 8 
2.1.1 Human Exposure to ELF EMFs 9 
2.1.2 Electromagnetic Field Mechanism( s) 10 
2.1.3 EMFs and Cancer 17 
2.1.4 EMFs and Transcription 22 
2.1.5 EMF and Enhancement of the Intracellular Ca2+ Response 25 
VI 
VII 
2.1.6 EMF and Plasma Membrane 26 
2.1.7 Melatonin and ELF EMFs 27 
2.1.8 Geomagnetic Field (GMF) 34 
2.1.9 DNA Damage, Chromosomal Aberration and ELF EMFs 42 
2.2 Sister Chromatid Exchange (SCE) 46 
2.2.1 Introduction 46 
2.2.2 Genetic Basis of SCE 51 
2.2.3 SCE Models 55 
2.2.4 SCE Induction 77 
2.2.5 SCE and Disease 80 
2.2.6 Human Reproductive Hormones and SCE 86 
2.2.7 Lymphocyte Concentration and SCE 87 
2.2.8 Baseline SCE and the Influence of Genetic, Chemical, 
and Environmental Agents 87 
2.3 Micronucleus Assay (MN Assay) 99 
2.3.1 Introduction 99 
2.3.2 Radiation Dose and Micronuclei 100 
2.3.3 Radiosensitivity and Micronuclei 102 
2.3.4 Genetic Studies Using the MN Assay 103 
2.3.5 Micronucleus Frequency as a Biomarker of Cancer Risk 106 
2.3.6 Effects of Age and Gender on Micronucleus Frequency 108 
2.3.7 MN Assay and the Influence of Chemical and 
Environmental Agents 112 
2.3.8 Concentration of Cytochalasin B 129 
2.4 The COMET Assay 130 
2.4.1 Introduction 130 
2.4.2 Mechanism of Cornet Tail Formation 132 
2.4.3 The COMET Assay in Genotoxicity Testing 133 
2.4.4 EMF Exposure and COMET Assay 135 
2.4.5 Evaluation and Interpretation of Results 135 
2.4.6 DNA Damage and Use of COMET Assay 136 
2.5 Fluorescent In Situ Hybridization (FISH) 138 
2.5.1 Introduction 138 
2.5.2 Advantage, and Limitations of FISH Analysis 139 
2 . 5 . 3  Translocation Study and the Use of  FISH 
Chapter Three: Materials and Methods. 
3. MATERIALS AND METHODS 
3.1  Sister Chromatid Exchange (SCE) 
3 . 1 . 1  Collection of blood samples 
3 . 1 .2 SCE protocol 
3.2 Micronuclei Assay (MN) 
3 .2. 1 Collection of blood samples 
3 .2 .2 MN assay protocol 
3.3 COMET Assay 
3 .3 . 1  Materials 
3 . 3 .2 Generation of EMFs and Field Exposure 
3 . 3.3 Collection of Blood Samples 
3 . 3 .4 Lymphocyte Cultures 
3 . 3 . 5  Lymphocyte Preparation 
3 .3 .6 Lymphocyte Concentration and Cell Viability 
3 .3 .7 Pre-treatment Method 
3 .3 . 8  The COMET Assay Methodology 
3 .3 .9 Slide Analysis (Quantitative) 
3.4 Fluorescent in situ Hybridization (FISH) 
3 .4 . 1 Collection of Blood Samples 
3 .4.2 FISH Protocol 
Chapter Four: Results. 
4. RESULTS 
4.1 Sister Chromatid Exchange (SCE) 
4 . 1 . 1  Analysis of Round- l (SCE) 
4. 1 .2 Analysis of Round-2 (SCE) 
4. 1 . 3  Complex MF 
1 40 
1 44 
1 44 
1 44 
1 45 
1 56 
1 56 
1 56 
1 6 1  
1 6 1  
1 66 
1 66 
1 66 
1 66 
1 67 
1 69 
1 7 1  
1 73 
1 76 
1 77 
1 77 
1 8 1  
1 8 1  
1 82 
1 89 
1 93 
VIII 
4.2 Micronucleus Assay (MN Assay) 
4.2. 1 Complex MF 
4.3 COMET Assay 
4.4 Fluorescent in situ Hybridization (FISH) 
4.5 Chromosomal Anomalies 
Chapter Five: Discussion. 
5. DISCUSSION 
5.1 SCE Analysis 
5.2 MN Assay 
5.3 COMET Assay 
5.4 FISH 
5.5 FUTURE RESEARCH 
Chapter Six: Summary and Limitations. 
6. SUMMARY AND LIMITATIONS 
6.1 Summary 
6.2 Limitations of Present Study 
References. 
Appendix One (a): A simplified comparison between electrical pressure 
and water pressure. 
Appendix One (b): A simplified comparison between electrical fields 
and magnetic fields. 
Appendix One (c) : Magnetic field decreases with increasing distance 
1 94 
205 
205 
209 
209 
2 1 1 
2 1 1 
224 
230 
233  
234 
237 
237 
239 
242 
296 
297 
from the source and measured in Tesla (T). 298 
Appendix Two : Personal Questionnaire 299 
Appendix Three : Consent Form Appendix Two: Consent Form 3 1 1  
Appendix Four : Showing 50  ( 1 -50) random microscope coordinates 
at (X and Y positions) for each of the 2 gels per slide 3 1 2 
Appendix Five : Figure 1 a & 1 b 3 1 4  
IX 
Appendix Five 
Appendix Five 
Appendix Five 
Appendix Six 
Appendix Seven 
: Figure 2a & 2b 
: Figure 3a & 3b 
: Figure 4a & 4b 
: Sinusoidal wave & Square wave 
: Table l a  - Recorded Temperatures 
(Control condition: coil off & no MF) 
: Table 1 b- Recorded Temperatures 
(Coil on @ 1mT MF) 
: Figure l a  & 1 b - Graph of recorded temperatures 
(Control condition: coil off & no MF) 
: Figure 1 c & 1 d - Graph of recorded temperatures 
(Coil on @ 1mT MF) 
3 1 5  
3 1 6  
3 1 7  
3 1 8  
3 1 9  
320 
32 1 -322 
323-324 
x 
List of Tables. 
Table 4. 1 .  Overall mean SCE and standard deviations of the control group 
XI 
and that of the entire set of experimental groups for RI. 1 82 
Table 4.2. Mean SCE and standard deviations of all experiments in RI based 
on all three donors. 1 85 
Table 4 .3 .  Overall ANOVA for RI. 1 86 
Table 4.4. Overall mean SCE and standard deviations of the control group 
and that of the entire set of experimental groups for R2. 1 89 
Table 4 .5 .  Mean SCE and standard deviations of all experiments in R2 based 
on all three donors. 1 9 1  
Table 4.6. Overall ANOV A for R2. 1 92 
Table 4 .7 .  Overall SCE mean of control and complex experiments in RI. 1 93 
Table 4 .8 .  Overall SCE mean of control and complex experiments in R2. 1 94 
Table 4.9.  Ma' s and standard deviations of control group and that of 
the entire set of experimental groups. 195  
Table 4 . 1  O. Ma's and standard deviations of  all experiments based on all 
three donors. 1 96 
Table 4. 1 1 .  Overall ANOV A for Ma. 1 97 
Table 4. 1 2 . Mb' s  and standard deviations of control groups and that of the 
entire set of experimental groups. 200 
Table 4. 1 3 .  Mb' s and standard deviations of all experiments based on all three 
donors. 201  
Table 4. 1 4. Overall ANOVA for Mb. 202 
Table 4. 1 5 . Overall Ma of control and complex experiments ofMN assay. 205 
Table 4 . 1 6. Overall Mb of control and complex experiments of MN. 205 
Table 4 . 1 7. Summary results of three parameters (Tail length, Tail moment 
and Olive tail moment) of the COMET assay experiments. 207 
Table 4. 1 8 . {-tests for all three parameters (Tail length, Tail moment and Olive 
tail moment) between the three COMET assay experiments. 208 
Table 4. 1 9 . Summary results of control and one FISH experiment. 209 
XII 
List of Figures. 
Figure 1 .l . Showing the flow of current and magnetic field (Giancoli, 2000) . 1 
Figure 2 .0. Pathways of reactive oxygen species (ROS) involvement in cellular 
reactions subjected to short- and long-term EMF-exposure. 1 4b 
Figure 2 . l .  Biological signal transduction pathways involving first- and 
second-messenger systems.  1 6  
Figure 2.2. Showing geographic and geomagnetic poles. 35  
Figure 2 .3 . A c-metaphase chromosome spread of a dividing peripheral blood 
Lymphocyte showing 1 0  SCEs. 46 
Figure 2.4. Showing how SCE staining method produces differentially stained 
chromatids by the incorporation of BrdU. 48 
Figure 2.5. Showing "single" and "twin" sister chromatid exchanges .  50  
Figure 2.6. Showing the single stranded (unineme) nature of chromosomes. 5 1  
Figure 2.7.  SCE model of Kato. 57 
Figure 2 .8 .  Multiple lesion pathways leading to a small number of 
SCE-inducing lesion states. 59 
Figure 2.9. Replication crosslink bypass model of SCE. 60 
Figure 2 . 1 0. Alternate rejoining processes for the replication bypass of SCE. 62 
Figure 2. 1 1 . Model for possible effects of DNA-damaging agents on cluster 
replication and segregation. 64 
Figure 2 . 1 2. Double-strand recombination at the junction between replicon 
clusters. 65 
Figure 2 . 1 3 .  Showing the Cleaver's SCE model .  67 
Figure 2 . 14 .  Topoisomerase II subunit exchange model of SCE. 69 
Figure 2. 1 5 .  Replication detour SCE model of Ishii and Bender. 70 
Figure 2 . 1 6. Strand switching by homologous displacement at blocked 
replication forks. 72 
Figure 2. 1 7. Strand switching in the model of Shafer. 75 
Figure 2 . 1 8 . SCE between replicated strands by two topoisomerase . 
I I-mediated strand switches. 76 
Figure 3 . 1 .  A.  Incubator apparatus. 1 46 
Figure 3 .2 .  Helmholtz coils . 148 
XIII 
Figure 3 . 3 .  Lollipop-plot of the resultant vectors i n  a longitudinal section 
through the' Z-axis' of a Helmholtz coil .  1 50 
Figure 3 .4 .  Magnitude of resultant vectors in 'mT' for the pair of 
Helmholtz coils. 1 50 
Figure 3 . 5 .  Magnitude of  resultant vectors i n  'J.lT' for the pair of 
Helmholtz coils. 1 5 1  
Figure 3 .6 .  Angles in Cartesian co-ordinates of resultant magnetic field vectors 
relating to the pair of Helmholtz coils. 1 5 1  
Figure 3 .7 .  A Giemsa-stained c-metaphase chromosome complement. 
(2n = 46.) 1 53 
Figure 3 .8. Diagrammatic representation of various differential staining patterns 
observed after incorporation of BrdU into replicating DNA. 1 55 
Figure 3 .9. Cytokinesis-blocked (CB) binucleated cells (a-h) with varying 
numbers (0-7) of micronuclei. 1 59 
Figure 3 . 1 0 . Binucleate cells which can be scored for micronuclei. 1 60 
Figure 3 . 1 l . Typical appearance and relative size of micronuclei in 
binucleated cells that meet the scoring criteria. 1 60 
Figure 3 . 12 .  Cellular structures which resemble micronuclei but were 
not scored. 1 6 1  
Figure 3 . 1 3 . I l lustrative diagram of what was observed after centrifugation of 
whole blood during isolation using Ficol, concentration gradient. 1 67 
Figure 3 . 1 4. Diagram representing the dimensions of a haemocytometer 
counting chamber. 1 68 
Figure 3 . 1 5 . I llustrative image showing how the gel layers were formed 
using the pre-treatment method. Image not to scale. 1 70 
Figure 3 . 1 6 .  Image indicating the placement of microscope slides in 
electrophoresis tank. 1 72 
Figure 3 . 1 7. Image capturing hardware used in the current study. 1 73 
Figure 3 . 1 8 . Orientation of cell (directed left to right) for analysis by 
the CASP software. 1 74 
Figure 3 . 1 9. Image outlining 7 of the 1 3  different variables that are measured 
by the CASpTM software. 1 76 
XIV 
Figure 4. 1 .  Graph of mean SCE of control groups and EMF exposed groups 
of three donors in Rl. 1 83 
Figure 4.2 .  A c-metaphase chromosome spread from a sham exposed 
control showing 1 1  SCEs. 1 84 
Figure 4.3 . A c-metaphase chromosome spread from an EMF exposed 
experiment showing 20 SCEs. 1 84 
Figure 4.4. A c-metaphase chromosome spread of a dividing peripheral blood 
lymphocyte that has undergone three rounds of DNA replication. 1 85 
Figure 4 . 5 .  Graph of mean SCE of  different Wave and Form in  RI. 1 87 
Figure 4.6. Graph of mean SCE of different Wave and Strength in RI. 1 87 
Figure 4 .7 .  Graph of mean SCE of different Form and Strength in RI. 1 87 
Figure 4 .8 .  Graph of mean SCE of different Wave and three Donors. 1 88 
Figure 4.9 .  Graph of mean SCE of different Form and three Donors. 1 88 
Figure 4. 1 0 . Graph of mean SCE of different Strength and three Donors. 1 88 
Figure 4. 1 1 .  Graph of mean SCE of control groups and EMF exposed groups 
of three donors in R2. 1 90 
Figure 4. 1 2 . Graph of mean SCE of different Wave and Form in R2. 1 92 
Figure 4. 1 3 .  Graph of mean SCE of different Form and Strength in R2. 1 93 
Figure 4. 1 4. Graph of Ma of control groups and EMF -exposed groups for three 
donors. 1 95 
Figure 4. 1 5 . Graph of Ma of different Wave and Strength. 1 98 
Figure 4. 1 6 . Graph of Ma of different Form and Strength. 1 98 
Figure 4. 1 7 . Graph of Ma of different Form and Wave. 1 98 
Figure 4. 1 8 . Graph of Ma of different Wave and three Donors. 1 99 
Figure 4 . 1 9 . Graph of Ma of different Form and three Donors. 1 99 
Figure 4.20. Graph of Ma of different Strength and three Donors. 1 99 
Figure 4.2 1 .  Graph ofMb of control groups and EMF-exposed groups for 
three donors. 200 
Figure 4.22. Graph of Mb of different Wave and Strength. 203 
Figure 4.23. Graph ofMb of different Form and Strength. 203 
Figure 4 .24. Graph ofMb of different Form and Wave. 203 
Figure 4.25 . Graph of Mb of different Wave and three Donors. 204 
Figure 4.26. Graph ofMb of different Form and three Donors. 204 
Figure 4.27. Graph of Mb of different Strength and three Donors. 204 
xv 
Figure 4.28.  Photographs showing single-strand DNA migration pattern 
of individual peripheral blood lymphocytes .  206 
Figure 4.29. Mean values for Tail length, Tail moment, Olive tail moment for 
control, Square continuous 1 m T  and Square pulsed 1 mT. 208 
Figure 4.30 .  Photographs showing a human peripheral blood lymphocyte cell 
with six whole chromosome (2, 3 and 5) labelled with FITC (green). 2 1 0  
Chapter One: Introduction 1 
Chapter One 
Introd uction. 
The use of electrical energy has been one of the most important conditions for the 
development of our present-day society. Electrical energy provides benefits and 
conveniences that society now deems necessities. As a consequence this has meant that 
technological development in the modem industrial world has lead to a steady increase 
in the production of artificial alternating current (AC) electromagnetic fields (EMFs). 
Almost all living organisms, including man, are exposed to a potpourri of these EMFs, 
but little is known about the biological effects of this kind of exposure, especially in the 
extremely low frequency (ELF) range as emitted by power appliances. This was the 
rationale for pursuing the current study in an attempt to answer the question: do weak 
EMFs exert an effect on human genetic material? 
EMFs are invisible lines of force, which consist of an electric and magnetic component 
(Figure 1 . 1 ). The movement of current creates magnetic fields (MFs) and their strength 
Magnetic 
field 
Figure 1 . 1 : Showing the electric charge and magnetic 
field (Giancoli, 2000). 
I = Electric 
charge 
Chapter One: Introduction 2 
increases with the strength of current. NIOSH (National Institute for occupational safety 
and health) ( 1 995) has drawn a comparison with water pressure in order to illustrate in 
simple terms the flow of electricity and the generation of MF s (Appendix 1 a and 1 b). 
Appendix 1 c shows approximate MF strengths recorded from around a photocopier by 
EPA (Environmental Protection Agency, U .S.A.)  ( 1 992), and illustrates that the 
strength of the field decreases with increasing distance from the source. The harmful 
effects of short wavelengths in the electromagnetic spectrum, such as gamma rays, X­
rays and ultraviolet (UV) light, are well known. These short wavelengths possess 
sufficient energy to break a chemical bond and can thereby destabilize an atom or 
molecule. Hence the term ionizing radiation. Longer wavelengths in the microwave 
range ( 1 09_ 1 011 Hz), however, possess insufficient energy to disrupt chemical bonds 
(non-ionizing radiation), but do cause rapid heating of tissue as energy is absorbed. 
Even longer frequencies such as those used in the conductance of electricity (50-60 Hz) 
exert negligible thermal effects (Weaver and Astumian, 1 990; Adair 1 99 1 ;  Tenforde, 
1 996). Nevertheless, evidence that these weak fields can exert a biological effect is  
steadily growing. 
In recent years, interest has focused on the 50-60 Hz power-frequency range, largely 
because epidemiological studies have shown an increased risk of leukemia in children 
living close to electric power distribution lines (Blank, 1 995).  A great deal of attention 
has been generated by epidemiological studies associating weak EMFs with small 
increases in the odds ratios for various types of cancers (Werheimer and Leeper, 1 979; 
Savitz et al. 1 988; London et al. 1 99 1 ;  Feychting and Ahlbom, 1 993) .  Washbum et al. 
( 1 994), in a meta-analysis of 1 3  published studies, also found statistically significant 
elevation in the risk of childhood leukemia and nervous tissue tumours among subjects 
living in close proximity to electricity transmission and distribution equipment. 
Trans Power , the company which controls the distribution of electricity in New 
Zealand, has acknowledged some research findings which show a possible link between 
adverse health effects and EMFs (Power Line, 1 994). Elevated risk of breast cancer 
among electrical workers was also reported in some studies (Matanoski et al. 1 99 1 ;  
Demers et al. 1 99 1 ;  Tynes et al. 1 992; Loomis et al. 1 994). But no association between 
residential exposure and cancer risk among adults has been observed in several 
independent studies (National Radiological Protection Board, NRPB, 1 992). Blackman 
Chapter One: Introduction 3 
et al. ( l 985a, b) reported that, in some experiments, 'windows' of frequency or intensity 
have been observed, that is, effects were visible at one frequency and/or intensity that 
disappeared for both higher and lower values of the parameters. 
On the other hand, electric devices based on ELF pulsed electromagnetic fields 
(PEMFs) have been approved for therapeutic use to accelerate healing of bone fractures 
(Andrew and Bassett, 1 995 ; Fontanesi et af. 1 986). Low and high frequency time­
varying MFs are used in medical diagnostic procedures such as magnetic resonance 
imaging (MRI) (Brandon et af. 1 996). High frequency EMFs also have a number of 
well-known medical uses ranging from diathermic warming of painful tissues to 
inducing hyperthermia in tumors. 
Obviously, the need for sound scientific information has increased with growing public 
awareness of EMFs in the environment. In view of the above, the present study was 
conducted to evaluate the possible genetic effects in human peripheral blood 
lymphocytes (HPBLs) of ELF EMFs (50 Hz). Using a specially designed incubator that 
was built by members of our research team, cultured lymphocytes were exposed to all 
combinations of two different flux densities, one strong (lmTtand one weak ( 1 �T)b, 
two different Waves (sine or square) and two different Forms (continuous or pulsed). In 
addition, a separate one-off experiment was conducted whereby a peripheral blood 
lymphocyte (PBL) culture was subjected to a complex field as emitted from the back of 
a computer, in order to detect whether or not this type of exposure can result in genetic 
effects. 
The fol lowing four methodologies were applied in this study to investigate possible 
genetic effects on PBLs which were cultured for 3 days in the presence of certain ELF 
EMFs: 
1 )  Sister Chromatid Exchange (SCE), 
2) The Micronucleus Assay (MN assay), 
3) The COMET Assay, and 
4) Fluorescence in situ Hybridization (FISH). 
(a) lmT: one milli-Tesla, (b) lilT: one micro-Tesla. 
Chapter One: Introduction 4 
1.1 Sister Chromatid Exchange (SCE) 
SCE is a widely acknowledged procedure for detecting genetic damage. The usefulness 
of this technique in determining DNA damage exploits the instability of the DNA helix 
during replication at S phase in a dividing population of cells. Many c1astogenicC factors 
are known to interfere with DNA replication and cause the helix to twist. This exerts a 
tension on the newly replicating half-strand which switches across to the opposite 
backbone. In the presence of bromodeoxyuridine, a halogenic analogue of uridine, this 
can be detected in c-metaphase complements as an SCE (Figure 2.3) .  It is well 
documented that any increase in the frequency of SCE in an individual is linked to ill 
health (Sandberg et al. 1 984). 
In a normal healthy human, the background SCE frequency per cell varies with the 
conditions of the experiment, for example, lymphocyte concentration. Nevertheless, it is 
widely accepted that in a controlled experiment, any increase in the frequency of SCE is 
interpreted as evidence of c1astogenic activity (Perry and Evans, 1 975; Popescu et al. 
1 977). 
In the present study, the frequency of SCE from two sets of experiments was analyzed 
in peripheral blood lymphocytes of six (3+3) healthy, non-smoking male blood donors. 
Participants were selected with similar age, lifestyles, occupational history, medical 
history and diet history in each round in order to identify any of the factors that may 
confound the analysis of SCEs. The background information of each individual in this 
study was obtained through a questionnaire (Appendix 2). All  experiments were 
performed blind in order to eliminate bias in the analysis of data which have the 
potential to be highly controversial. It is important to mention here that the aim of this 
study was to compare cellular effects not to compare individual effects. The whole 
study was repeated blind under the same strict conditions. 
(c) Clastogen: any substance or environmental agent that causes chromosomal 
abnormalities .  
Chapter One: Introduction 5 
1.2 Micronucleus Assay (MN assay) 
In our daily life we are unknowingly exposed to different environmental agents that 
damage our genetic material. But fortunately our body possesses molecular mechanisms 
that repair this damage. If these biological processes start to malfunction due to old age, 
ill health or as a consequence of clastogenic activity, then it is not unusual to find 
fragments or damaged chromosomes in dividing lymphocytes, which fail to attach to 
the mitotic spindle during cytokinesis. Chromosomes, which have lost their 
centromeres, or small acentric fragments, are excluded from nuclei and form what are 
called micronuclei (Figure 3.9). 
The MN assay is a sophisticated cytomolecular technique, developed over recent years, 
that measures the degree of malfunctioning of a person's DNA repair system in the G1 
phase of the cell cycle. The technique has been extensively used for the biological 
monitoring of populations exposed to mutagenic and carcinogenicd agents in various 
environmental and occupational settings. Many cancer laboratories apply the technique 
when following the effects of radiotherapy and chemotherapy on patients (Catena et al. 
1993). Some researchers speculate that in a few years the method may even be used as 
an alert signal in individuals prior to the clinical manifestation of cancer (Scott et al. 
1996). 
In essence, the MN assay involves counting numbers of micronuclei per hundred 
binucleated cells. An increase in the frequency of micronuclei in a targeted group 
compared to the controls indicates a breakdown of DNA repair. This technique was 
employed in the current study to determine whether or not ELF EMFs affect the DNA 
repair system in humans to any degree. 
1.3 The COMET Assay 
The COMET assay is also known as micro gel electrophoresis. The COMET assay takes 
its name from the fact that highly fractionated lymphocytes look like comets under a 
microscope. They have a head and a tail, with the head being the stained remains of the 
(d) Carcinogenic: A cancer-causing substance or agent. 
Chapter One: Introduction 6 
lymphocyte nucleus and the tail being fractionated DNA, which has tailed out along the 
gel when electrophoresis is conducted. When DNA is  broken, it breaks into little 
fractions, thus fractionation. The more fractionation, the more damage. The COMET 
assay measures the amount of DNA fractionation in any particular person studied. 
Caution is  necessary when applying this assay: it i s  very sensitive and thus open to 
various interpretations as to what may be causing any fractionation observed .  
Nevertheless, i t  is a test that is widely conducted when searching for any genetic 
damage. 
In the COMET assay, fresh lymphocytes are mixed on an agarose gel and mounted on a 
special microscope slide. The lymphocytes are lysed so that the nuclear membrane is  
disrupted. With the aid of various chemicals all of the non-DNA materials are removed. 
The slide is then placed into a buffer solution and electrophoresis performed. After a 
certain length of time the lymphocytes are stained with a fluorescent dye and observed 
under a fluorescent microscope. The background is naturally  dark and the residual 
lymphocytes look like bright yellow comets with short tail s  in a sky (Figure 4.28b). In 
the current study a computer software package was used to analyse the length and 
morphology of these tails, but in essence, the longer the tail, the greater the degree of 
fractionation and thus the greater the genetic damage. 
1.4 Fluorescence in situ Hybridization (FISH) 
It is well known that one of the effects of radiation damage in humans is an increase in 
chromosome translocations (Bauchinger et al. 200 1 ;  Kodama et al. 200 1 ;  Lambert et al. 
200 1 ;  Nakano et al. 200 1 ;  and Vorobtsova et al. 200 1 )  which have the potential to lead 
to ill health, particularly a range of cancers, or inherited defects which may be passed on 
to offspring. To search for possible translocations as a consequence of EMF exposure, 
another technique was employed in this current study (FISH : fluorescent in situ 
hybridization) to selectively paint the chromosomes. By annealing a DNA probe from 
whole chromosomes 2, 3 and 5 ,  these chromosomes were selectively stained with a 
green fluorescent dye (FITC) and the rest of the chromosomes were stained with a blue 
fluorescent dye (DAPI) (Figure 4 .30). With this technique it is possible to detect any 
translocations (shift of any green across to a blue chromosome) that may occur as a 
consequence of exposure to a certain EMF during the 3-day period of peripheral 
Chapter One: Introduction 7 
lymphocyte culture. In addition, hybridization of all the chromosomes with a pan­
centromeric DNA probe labelled with a pink fluorescent dye was done in order to 
search for dicentric or multi centromeric chromosomes ,  which is common in radiation 
studies (Figure 4 .30) .  
1.5 Aim, Objective and Prediction 
The aim of this thesis was to test the null hypothesis that 50 Hz, ELF EMFs, as used in 
electric appliances, do not exert an effect on the human genetic material and thereby 
will not manifest clastogenic or mutagenic potentiality. 
The objective of this thesis was to test the hypothesis using four cytomolecular 
techniques: 
1 )  Sister Chromatid Exchange (SCE), 
2) The Micronucleus Assay (MN assay), 
3) The COMET Assay, and 
4) Fluorescent in situ Hybridization (FISH). 
Following the empirical method for scientific investigation, a prediction was made for 
each technique that if the null hypothesis were true, then no significant statistical 
difference should be observed between lymphocytes exposed to a weak EMF and sham­
exposed controls. 
Chapter Two: Review of Literature. 8 
Chapter Two 
Review of Literature 
2.1 ELF EMFs and Living Organisms 
Electric power is a modern convenience that is irreplaceable. It  benefits our daily lives 
and fuels appliances and machinery that we use frequently . Conversely, electric power 
lines, household wiring and electronic devices can cause serious injury from electric 
shock if handled improperly. To avoid danger, however, electric power comes with 
certain precautions that are well known to the user. 
Appliances that operate either with batteries or which are connected to a household 
electric outlet are usually equipped with an AC/DC switch. If switched to alternating 
current (AC), the appliance uses electric power that flows back and forth or "alternates" 
at a (NZ) rate of 50 cycles per second (50 hertz, or Hz). If direct current (DC) is chosen, 
current flows in one direction from the battery to the appliance. AC fields induce weak 
electric currents in conducting objects, including humans; DC fields do not, unless the 
DC field changes in space or time relative to the person in the field. Induced currents 
from AC fields have been a focus for research on how EMFs could affect human health. 
Power lines, electrical wiring and appliances all produce electric fields and MFs. EMFs 
are invisible l ines of force that surround any electrical device while operating. Electric 
fields and MFs have different properties and possibly different ways of causing 
biological effects. It is worth mentioning here that while electric fields are easily 
shielded or weakened by conducting objects (e.g. ,  trees, buildings, and human skin), 
MFs are not. However, both electric fields and MFs weaken with increasing distance 
from the source (Appendix lc). 
There is increasing concern that the household and environmental exposures to power 
frequency (50-60 Hz) EMFs may cause adverse health effects in humans. Interest in the 
possible health effects of EMFs grew with the publication of epidemiological studies 
that have shown an increased risk of cancer in children living close to electric power 
distribution l ines (Wertheimer and Leeper, 1 979). Two similar studies (Savitz et al. 
Chapter Two: Review of Literature. 9 
1 988 ;  London et at. 1 99 1 )  also found associations between residential wmng 
configuration and the incidence of childhood cancer. The study by Savitz et al. ( 1 988) 
measured fields within the home in addition to classification by wiring configuration, 
and found an increase in risk correlated with MFs. However, the study by London et al. 
( 1 99 1 )  observed no correlation between measured MFs within the home and the 
incidence of cancer. 
The first evidence of  co-carcinogenic effects of ELF EMF exposure came from 
epidemiological studies by Savitz and Ahlbom ( 1 994). DNA damage that accumulates 
in cells over a period of time could be the cause of slow-onset diseases, such as cancer. 
DNA strand breaks may affect cellular functions, which could lead to carcinogenesis 
and cell death and possibly to the onset of neuro-degenerative diseases. 
2. 1 .1  Human Exposure to ELF EMFs 
Most human exposure to ELF EMFs comes from electrical appliances, household 
wiring, AC transmission, and distribution l ines. Below overhead transmission lines, 
electric field strengths may be as high as 12 kilovolts per metre (k Vim), depending on 
the line voltage and the distance between the conductor and the ground. Around 
electricity generating stations, electric fields up to 1 6  kV Im may be encountered 
(Simon, 1 992). Electric fields around appliances can be of the order of 0 .5  kV Im. 
Naturally occurring 50-60 Hz electric fields are of the order of 0. 1 millivolts per metre 
(mV/m), and accompanying MFs are approximately 0 .0 1 nano-Tesla (nT). In residences 
and most work places, the average 50-60 Hz MFs are between 0 . 1  and 0.3 IlT (National 
Research Council, NRC, USA, 1 996). Many household appliances produce MF, which 
range from 50- 1 50 /IT; the fields decrease rapidly with increased distance from the 
source. Below overhead transmission lines, the average magnetic flux density can be up 
to 30  /IT for multi-conductor 765 kV lines and 1 0  /IT for 380 kV l ines .  Around power 
generating stations, average fields may be as high as 40 /IT, and peak fields of 270 /IT 
may be encountered. Industrial processes can be responsible for occupational exposures 
in welders as high as the 1 30 mT peak fields (Simon, 1 992). 
Chapter Two: Review of Literature. 1 0  
2.1 .2 Electromagnetic Field Mechanism(s) 
Embarking on any investigation into whether ELF EMFs  have an effect on living 
systems undoubtedly draws controversy and sometimes outright skepticism. Despite 
these sometimes strongly held opinions, the researcher of the present thesis has 
followed strict scientific procedures in formulating a null hypothesis (that ELF EMFs 
have no effect on human genetic material), followed by the construction of an empirical 
experiment to test the hypothesis (namely the application of established cytomolecular 
techniques that are known to detect clastogenicity), with the intention of attempting to 
determine with the technology available whether or not any effect can be observed. 
Many researchers have published in reputable international journals claiming to have 
observed cellular effects (Nordenson et al. 1 984; 1 988; 1 994; Rosenthal and Obe, 1 989; 
Khalil and Qassem, 1 99 1 ;  Garcia-Sagredo and Monteagudo, 1 99 1 ;  Cohen et al. 1986a 
and 1 986b; Lai and Singh, 1 997a, 1 997b and 1 997c) and indeed whole organism effects 
with ELF EMFs (Milham, 1 982; NRPB, UK. 1 992; Theriault et al. 1 994), which 
inevitably gives rise to the question, "what is the underlying mechanism whereby ELF 
EMFs exert their effect?" This is where many biologists become "unstuck" because 
many of them do not possess sufficient depth of knowledge in physics to offer a 
plausible mechanism. The present author is one such researcher; he readily admits that 
his knowledge of physics is not strong; he i s  first and foremost a cytogeneticist, but this 
does not invalidate the observations made either by him or any other non-physicist in 
the stated area of study. Nevertheless, it is beholden upon any person studying EMFs to 
possess at least a basic understanding of the various ideas and models that have been 
advanced to explain those effects that have been observed. The author will attempt here 
to present his understanding of the literature as follows. 
One of the main areas of controversy involves "thermal" vs "non-thermal" effects. All 
living systems possess thermal kinetic energy (in joules, .1), known as kT, (where k is 
the Boltzmann constant and T is the absolute temperature in kelvin, k), which is the 
background energy from the movement of molecules inside cells that are involved in 
life processes. It  is  universally accepted that the kT inside a cell is  below that of ELF 
EMFs, which, if an effect were observed, would probably rule out any thermal model as 
an explanation for any biological effect. This means one has to search for a "non-
Chapter Two: Review of Literature. 1 1  
thermal" model, which is where problems and considerable controversies arise. Most of 
the models are untestable empirically, which contravenes accepted scientific practice. 
Nevertheless, Copemicus faced the same difficulty in his day, which shows that science 
does not always progress with full understanding when an idea is in its infancy. 
The models most frequently advanced to explain observed biological effects of ELF 
EMFs embrace a variety of views as outlined below. 
A number of innovative models have been proposed over the course of the last few 
decades. One class of models that has received particular attention is resonance models. 
Ion cyclotron resonance (feR) model of Lib off 
The term 'cyclotron' itself derives from the so-called cyclotron accelerator - a familiar 
tool in atomic physics since the 1 930s - in which charged particles move inside an 
evacuated chamber mounted between the poles of a magnet. Under the influence of the 
MF, the particles spiral round at a fixed frequency (the cyclotron frequency) while they 
receive regular accelerating pulses from an electric field applied at the same point in 
each orbit so that their energy increases in a resonant fashion (Male, 1 992). 
The resonance model involves the combined action of an ELF MF and the static 
geomagnetic fields (GMFs). Liboff ( 1 985)  first proposed the ICR model as a 
mechanism that could facilitate the movement of ions such as Ca ++ through membrane 
channels in the presence of the GMF and a weak ELF MF tuned to ICR frequency. The 
resonance condition is formally expressed by the relationship!c = QB/2nm. where!c = 
ICR frequency, Q = ion charge, B = a DC MF, and m = ion mass. For the typical value 
of the GMF over the surface of the earth (50 IlT), the resonant frequencies of many 
biologically important ions such as Na +, K+, and Ca ++ fall within the ELF range. This 
has given the basis to assume that a cyclotron resonance could underlie the observed 
phenomena. Many authors developed the theme of such resonance in magnetobiology 
(Liboff et. al. 1 987; Smith et al. 1 987; Parkinson and Hanks, 1 989; Liboff and 
Parkinson, 1 99 1 ;  Durney et al. 1 992 ; Gait et al. 1 993 ; and Coulton et al. 1 993 ) .  
Nonetheless, i t  has not received recognition due to  the lack of correct physical 
Chapter Two: Review of Literature. 1 2  
substantiation.  At the same time, these experiments showed the essential role o f  ions, 
especially Ca ++, in magnetobiology. 
Quantum beats or parametric resonance model 
Lednev ( 1 99 1 )  proposed this model, in which combined static and ELF fields affect 
vibrational energy levels and transition probabilities of bound ions (e.g., Ca ++ ions 
bound to calmodulin). Shuvalova et al. ( 1 99 1 )  have obtained data on the effect of 
combined fields on the rate of calmodulin-dependent phosphorylation of myosin that 
appear to support the predictions of the Lednev model .  Another study, however, in 
which an optical technique was used to study Ca ++ binding to calmodulin and to 
metallochromic dyes, failed to find any effects of combined static and time-varying 
fields under the resonance conditions predicted by Lednev' s  model (Bruckner-Lea et al. 
1 992). In a separate analysis Blanchard & Blackman ( 1 994) claimed that the original 
Lednev model contained mathematical errors that led to an incorrect prediction for the 
ion transition probability between energy levels. Using their revision of the Lednev 
model, these investigators obtained a reasonably good fit to data on combined static and 
EMF field inhibitory effects on neurite outgrowth in cultured PC- 12  cells stimulated by 
nerve growth factor (Blackman et al. 1 994). In view of the significant differences in the 
original and revised Lednev's model, there is a clear need for further investigations on 
both a theoretical basis and biological implications of this resonance model for the 
action of combined static and ELF MFs. However, regardless of the details of the 
model, Adair ( 1 992a) has pointed out that the Lednev model of resonant field effects is 
improbable because of the long lifetime of the excited vibrational states (approximately 
8 sec). 
Ionic cyclotron resonance model oJZhadin & Fesenko 
Zhadin & Fesenko ( 1 990) proposed this model in which the rotational energy levels of 
bound ions are pumped by combined static and ELF MFs. Similar arguments, which 
have been presented against the Lednev model, might be raised against the Zhadin & 
Fesenko ( 1 990) model. In addition, the rate of transition of a bound ion to an excited 
state that is predicted by Zhadin & Fesenko' s  model is so low that a transition would 
occur only once in several months at typical environmental MF levels. 
Chapter Two: Review of Literature. 1 3  
Stochastic resonance model 
The concept of stochastic resonance first put forward in the seminal paper by Benzi et 
al. ( 1 98 1 )  has been proposed as a possible mechanism through which weak EMFs could 
be detected in biological systems (Kruglikov and Dertinger, 1 994). Stochastic resonance 
is the phenomenon whereby the addition of an optimal level of noise to a weak 
information-carrying input to certain non-linear systems can enhance the information 
content at their outputs. For example, in some systems, such as radio receivers, turning 
up the volume in order to hear a faint signal amidst much noise usually only results in 
turning up the noise as well. However, in other systems, increasing the amount of 
ambient noise actually enhances (up to a certain point) the signal-to-noise ratio through 
a complicated non-linear cooperation between the system and detector. This effect, 
known to operate in neurons, and lasers, is called stochastic resonance. The noise 
fluctuations might be stochastic (meaning totally random) but the detection of a desired 
signal can be maximised by tuning the noise. 
Preliminary experiments with mechanoreceptor hair cells from crayfish have given 
evidence for the enhanced detection of weak stimuli in the presence of random noise 
(Wiesenfeld and Moss, 1 995). It has been suggested that stochastic resonance is the 
basis for this effect, and perhaps underlies many other examples of weak signal 
detection by sensory systems (Douglas et al. 1 993).  Arguments have also been made 
that stochastic resonance is a plausible mechanism for the detection of periodic EMFs 
under conditions in which the signal-to-noise ratio is less than one (Kruglikov and 
Dertinger, 1 994). This hypothesis clearly merits further evaluation and experimental 
testing in biological systems that exhibit reproducible responses to weak ELF fields. 
Biogenic magnetite 
Biogenic magnetites are natural microscopic crystals of magnetite, within the body of 
some animals and bacteria, which are capable of being magnetized. An external MF 
influences such crystals so that they tend to change their orientation. Consequently, the 
crystals exert pressure on the adjacent tissue and cause a biological response. In a 
number of cases, it is possible to explain by this manner the biological reception of a 
weak static MF. A series of chains of uniformly sized single domain magnetite particles 
Chapter Two: Review of Literature. 1 4  
embedded in the ethmoid tissue in the front of the cranium of Chinook Salmon fish 
(Oncorhynchus tshawytscha) is used for accurate navigation (Kirschvink et at. 1 99 2 ) .  
Microscopic crystals of magnetite or greigite (Fe304) were found in the brain of some 
birds, which are known to navigate well in the GMF (Kirschvink et al. 200 1 ). Traces of 
magnetite are also found in magnetotactic bacteria (Blakemore, 1 982 and Torres de 
Araujo, 1 986) . 
Magnetic contamination 
Ferromagnetic contamination is one of the ideas in the area of magnetobiology 
proposed by Kobayashi et al. ( 1 995) . The authors suggested that small polluting 
magnetic particles are present not only in the air dust but also deposited on the surface 
of laboratory devices, and penetrate into plastics, glasses, chemicals and even into 
purified water .  Their mean size is about 1 0-5 cm. The particles consist of ferro- and 
ferrimagnetics, which possess spontaneous magnetization. The authors have shown that 
routine laboratory procedures like pouring or/and rinsing give rise to enrichment of the 
cell cultures by pollutants. The number of particles in cultures could be 1 0  times more 
than that of cells . The energy of such a particle is approximately three orders of 
magnitude greater than kT. In this research the authors postulated that the magnetic 
particle absorbed by a cell membrane can transfer its energy to the adjacent biophysical 
structures, for example, mechanically activated ion channels. 
These mechanisms of magnetobiological effects stand apart and do not solve the basic 
problem of magnetobiology. Indeed, many unicellular organisms, wherein magnetite is 
absent, are capable of reacting to the MF. The reaction in many cases is of a complex 
non- l inear, character depending on the MF parameters . The basic problem of 
magnetobiology i s  exactly in the explanation of this  phenomenon, which is a 
paradoxical one from the viewpoint of orthodox physics . 
Chapter Two: Review of Literature. 14a 
Free radicals 
Very recently, Simko et al. 2004 have also proposed a mechanism that genotoxicity after 
exposure to EMF is due to secondary induced mechanisms, such as the production of free 
radicals, which must be induced by different signal transduction pathways.  Free radicals are 
important intermediates in natural processes and are released during natural cell 
metabolism. These intermediates arise in mitochondrial respiration and are also a key 
feature of the phagocytic process. These authors speculate on the basis of experiments they 
have conducted in rats that EMFs might be a stimulus to induce an "activated state" of the 
cell such as phagocytosis, which then enhances the release of free radicals, in turn leading 
to genotoxic events. They envisage that EMF exposure increases free radical levels which 
can cause both acute and chronic effects on different cellular processes. The pathways they 
propose are as fol lows: ( 1 )  Direct activation of macrophages (or other cells) by short-term 
exposure to EMF leads to phagocytosis (or other cell specific responses) and consequently, 
free radical production. (2) EMF-induced macrophage (cell) activation which includes 
direct stimulation of free radical production. (3) An increase in the lifetime of free radicals 
by EMF which leads to persistently elevated free  radical concentrations. (4) Long-term 
EMF exposure leads to a chronically increased level of free radicals, subsequently causing 
an inhibition of the effects of the pineal gland hormone melatonin. Taken together, they 
advance the hypothesis that these EMF-induced reactions could lead to a higher incidence 
of DNA damage and therefore, to an increased risk of tumour development (see Figure 
2.0). 
Chapter Two: Review of Literature. 
short-term exposure EMF long-term exposure 
activation of macrophages 
(or other cel l s) 
(� 
lifetime extension 
of radicals 
phagocytosis -----I.� 
free radicals 
(cel l sneci fic resnonse) 
l 
activation of 
( immune) resnonse 
DNA-damage 
reduction of 
melatonin svnthesi s 
Figure 2.0:  Pathways of reactive oxygen species (ROS) involvement in cel lular 
reactions subjected to short- and long-term EMF-exposure. Stimulation of the 
immune system through macrophage activation is a favourable response to short­
term EMF- exposure (a) .  Free radical production may arise directly from 
macrophage or other cell specific activation (b) or radical lifetime extension (c) . 
Similarly, increase of ROS has been attributed to the inhibiting capacity of EMF on 
the availability of the pineal gland hormone melatonin which is a known scavenger 
for free radicals (d) after long-term EMF-exposure (Simko et al. 2004). 
1 4b 
Chapter Two: Review of Literature. 1 5  
Other proposed interaction mechanisms 
Current evidence suggests that cell processes can be influenced by weak EMFs (Nair et 
at. 1 989; Adey, 1 990; Anderson, 1 99 1 ; Tenforde, 1 992). It would also be true to say 
that the effects represent a weak perturbation of the homeostatic state, that EMFs appear 
to be a stress that a cell can adapt to in most cases without catastrophic consequences. 
But there is no question that a change in the physiological state of the plasma membrane 
in particular, representing the boundary between the external environment and the 
organism, is measurable and significant in response to certain weak EMFs (Azanza and 
Del Moral, 1 99 3 ;  Goodman et al. 1 995). Lawrence and Adey ( 1 982) have demonstrated 
this in their cell membrane studies. Strands of protein are strategically located on the 
surface of cel ls  in tissue, where they act as detectors of electrical and chemical 
messages arriving at cell surfaces, transducing them and transmitting them to the cell 
interior. Through the protein strands, cell membranes perform a triple role in signal 
detection, signal amplification, and signal transduction to the cell interior. 
The most important issue in understanding the pathways by which ELF EMF signals 
might influence membrane and cellular functions is the elucidation of mechanisms by 
which these fields are transduced within the cell membrane. One approach that has been 
taken in addressing this question is to consider the intricate biochemical pathways that 
have evolved as a mechanism by which a living cell communicates with its extracellular 
environment. As illustrated in Figure 2. l A  by Tenforde ( 1 993), the binding of a single 
molecule of a messenger substance to a specific receptor within the membrane triggers a 
cascade of events that involve conformational shifts in membrane-associated proteins. 
These events in turn lead to signal transduction and amplify the production of 
cytoplasmic second messengers and internal effectors such as free Ca ++ and protein 
phosphorylases (kinases) that regulate DNA transcription and protein biosynthesis 
(A Ikon & Rasmussen, 1 988; Luben 1 99 1 ) .  The end result of a single messenger binding 
event at the membrane surface is thus a cytoplasmic signal that is amplified to a level 
that can produce robust effects on macromolecular synthesis and cellular responses 
involving significant changes in functional states of the cell. 
Similarly, the interaction of EMF fields with biological membranes might in principle 
lead to alterations in each component of the signaling process that occurs in living cells .  
Chapter Two: Review of Literature. 1 6  
A useful working hypothesis (Figure 2 . 1 B), which was advanced by Tenforde ( 1 993), is 
that the particular fields and currents induced by an applied EMF initiate 
A 
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C'¥toplnmlc 
M ... sengers 
{cAMP, cGMP, I P , •  
dtaeylgl)'clllml) 
E«ec:!s on ONA 
TranllCflpllon ana 
Hallslallon 
"'eIIl\lll\lcnt-lioUl\d Effector 
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C o  o 0 0 Internal Effectors 
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" 
Cellular Responses 
(growth. differentiation, etc.) 
I ELF rId I B 
I Induced Electn<;; CU\"f\!'l'lta I in Pericellular Fluids t 
EIltctJochemlcal E.vents at Cell Surfact! 
--- Transmembrane Interactions 
--- Signal Transduction and Amplltication 
I Cytoplumic AallponH. (eaH'. cAMP, klnases, etc.} I - Altered Macromolecule, Syntlluls I 
Etle<:ts on Cell Glowti'l, Dlfferentiat'Jon and 
Func\lc>nal Properties 
Figure 2 . 1 : (A) Biological signal transduction pathways involving first- and second­
messenger systems. Abbreviations- cAMP: cyclic adenosine monophosphate; cGMP: 
cyclic guanosine monophosphate; IP3 : triphosphoinositol (which releases Ca ++ from 
intracellular stores). (B) Hypothesized sequence of membrane-mediated events leading to 
cellular and tissue responses to applied ELF fields (Tenforde, 1 993).  
electrochemical events within the cell  membrane that are important elements of the 
primary signal transduction and amplification process. These biochemically-mediated 
events then produce cytoplasmic second messenger responses that trigger changes in the 
biosynthesis  of macromolecules and perhaps alterations i n  cellular growth, 
differentiation, and functional properties. 
In examining the in vivo and in vitro response of cells  to low frequency EMFs there is a 
growing consensus among investigators that the sequence of events leading to EMF 
signal transduction and amplification at the cellular level does occur. For example, 
Luben et al. ( 1 982) have demonstrated that pulsed e lectromagnetic fields (PEMF) with 
EMF repetition frequencies inhibit the production of cyclic adenosine monophosphate 
(cAMP) by bone cells in response to the binding of parathyroid hormone (PTH) to 
Chapter Two: Review of Literature. 1 7  
surface receptors. Moreover, studies by Cain et al. ( 1 987) have shown that PEMF action 
can lead to the inability of the PTH-receptor complex to activate the alpha subunit of 
the G protein, thereby interfering with the sequence of events that activates adenylate 
cyclase at the cytoplasmic membrane interface. Byus et al. ( 1 984) in a separate study 
using human lymphocytes has shown that exposure to microwave fields with amplitude 
modulation at ELF frequencies leads to the inhibition of non-cAMP-dependant histone 
kinases. 
Some other interaction models has been advanced to explain a possible interaction 
between EMFs and biological systems, but none have gained prominence as an 
acceptable working model and are thus not highlighted in this treatise. They include: ( 1 )  
Eddy electric currents, induced by alternating MF (Lerchl et al. 1 990; Schimmelpfeng 
et al. 1 997); (2) Interference of quantum states of bound ions and electrons (Binhi, 
1 997, 1 997a) ;  (3) Coherent quantum excitations (Frohlich & Kremer, 1 983);  and 4) 
Magnetosensitive free-radical reactions and other "spin" mechanisms (Grundler et al. 
1 992). 
2.1 .3 EMFs and Cancer 
The debate over a possible link between cancer and ELF EMFs began first with a study 
by Wertheimer and Leeper ( 1 979) that showed an increased risk of cancer in children 
l iving close to electric power distribution lines . Subsequently, a number of other 
epidemiological studies have also reported that residential and occupational exposure of 
ELF (50-60 Hz) EMFs is related to different types of cancers (Davies  et al. 1 992 ; 
Sagan, 1 992; Stevens et al. 1 992; Savitz and Ahlbom, 1 994). However, the National 
Research Council, National Academy of Sciences, Washington, D .C .  ( 1 996) reported 
that the results from such epidemiological studies have been inconsistent and 
contradictory. In reference to the ambiguous results of these epidemiological studies on 
association between ELF EMFs exposure and risk of cancer, laboratory studies are 
necessary to determine a link, if any, between such fields and carcinogenesis. 
The development of cancer is a multi stage process.  During normal development, 
immature cells undergo a process called differentiation in which they become highly 
Chapter Two: Review of Literature. 1 8  
specialized and are less able to continue proliferating. In the first stage of cancer, 
termed initiation, a cell 's  DNA is damaged through mutation, causing a differentiated 
cell to resemble an immature one, in effect reversing the process of differentiation. In 
the second stage, promotion, normal cellular controls  are disrupted and the mutated cell 
multiplies. 
The majority of in vitro studies have indicated that ELF-EMFs have no mutagenic 
effects and do not initiate cancer. It has been suggested, however, that they could play a 
role as a cancer promoter (McCann et al. 1 993 and Murphy et al. 1 993) through 
epigenetic mechanisms and may induce cancer in cells that have already mutated. Many 
cellular studies support this view as ELF EMFs are observed to influence enzyme 
action, signal transduction, calcium ions (Ca2+),  protein synthesis, gene expression, 
ornithine decarboxylase (ODC), cell proliferation, and melatonin. 
Tumour promotion is often accompanied by an increase in the activity of the enzyme 
ODC. ODC is a key enzyme in the biosynthesis of polyamines, which have roles in cell 
proliferation and control of gene expression. Changes in ODC have been reported from 
50  Hz exposures in vivo (Mevissen et al. 1 995). In this study, female rats were exposed 
to a 50 /IT EMF to observe ODC activity in different tissues, including mammary 
tissue. Rats were exposed for a period of six weeks either with or without oral 
administration of the chemical carcinogen 7- 1 2-dimethylbenz(a)anthracene (DMBA) 
and all data were compared with those from sham-exposed controls .  MF exposure 
resulted in an approximate doubling of ODC in mammary tissue. A significant ODC 
increase was also seen in the spleen, but not in the liver, small intestine, bone marrow, 
and ear skin. The ODC increase produced by EMF exposure in the mammary tissues 
was of a similar magnitude as that observed after treatment with DMBA. Combined 
treatment with EMF and DMBA was not as effective in increasing ODC as treatment 
with DMBA alone, except in liver tissue. These results on in vivo increases of ODC by 
EMF exposure strengthen the hypothesis that weak 50-Hz EMFs affect ODC activity 
and may thus function as a tumor-promoting or co-promoting agent. 
A tumour promoting effect of the EMFs was also observed in three different studies of 
mammary cancer development in rodents exposed to ELF MFs fol lowing tumour 
initiation with a chemical initiator. In the first study, rats were inj ected intravenously 
Chapter Two: Review of Literature. 1 9  
with a 50  mg/kg dose of nitrosomethylurea (NMU), which produced mammary tumours 
in 54% of the rats (Beniashvili et al. 1 99 1 ) . An increased percentage (86%) of rats with 
mammary tumours were observed when a group of rats were injected with NMU 
followed by an exposure of 0.2 mT MF (50 Hz), 3 h / day for 5 weeks. In the second 
study, mammary tumours were initiated through the administration of oral doses of 
DMBA (20 mg total) followed by 23 weeks exposure to a 30  mT MF (50 Hz) 
(Mevissen et al. 1 993). A significantly increased number of tumors were observed per 
tumor-bearing rat. A third study, which was conducted by Loscher et al. ( 1 993), used 
the same protocol as the second study described above, except that a considerably 
smaller MF level of 0 . 1 mT was used. In this study, 50% higher mammary tumour 
incidence was observed in the MF exposed rats than the sham-exposed controls. 
In a separate study, Kumlin et al. ( 1 998) investigated the possible influence of MFs and 
simulated solar radiation (SSR) on ODC and polyamines in mouse epidermis. Chronic 
exposure to combined EMF and SSR, however, did not cause persistent effects on ODC 
activity or polyamines compared to the animals exposed only to SSR, although the same 
EMF treatment was previously found to accelerate skin tumor development. 
S ignal transduction (ST) is one of the major, fundamental pathways for cell 
communication and it is a general process in multi-cellular organisms in which a ligand 
molecule binds to its receptor site on the cell surface, which in turn triggers a cascade of 
biochemical events associated with the cell membrane . Liburdy et al. ( 1 993) 
investigated receptor-l igand binding in human lymphocytes exposed to EMF. Human 
peripheral blood lymphocytes pre-Iabelled with fluorescently modified antibody 
markers against CD20 (pan B-cell surface marker) or against the CD3 (T-cell receptor) 
determinant were exposed to 22 �T EMF (60-Hz sinusoidal) at 3 7C for an hour. 
Following field exposures, T-Iymphocytes were reported to show more anti-CD3 
antibody released from cells compared to sham exposed controls, whereas EMF-treated 
B-Iymphocytes showed only a slight increase in CD20 release compared to controls. 
This suggests that EMFs have the potential to alter receptor binding involving the CD3 
receptor in T -lymphocytes. The CD3 molecule is physically integrated with the T -cell 
receptor in the lymphocyte cell membrane and is the major complex responsible for T­
cell activation and triggering of the ST cascade in this cell. 
Chapter Two: Review of Literature. 20 
The series of events in the ST cascade that follows receptor-ligand binding is the 
activation of membrane bound enzymes. Many researchers have studied different 
membrane bound enzymes during EMF exposures. Protein kinase C (PKC) is a key 
enzyme in the ST cascade and is believed to be the membrane receptor for phorbol 
esters, which are a potent class of tumour promoters (Kikkawa et al. 1 989). Therefore, 
if the binding of this class of tumour promoters to PKC is influenced by EMF then 
altered cancer promotion would be expected. Monti et al. ( 1 99 1 )  showed that HL-60 
lymphocytes exposed to a 50 Hz, 8 mT EMF have increased binding of the PKC­
specific phorbol ester, suggesting that EMFs may modify the cellular response to 
tumour promoters. PKC activity was also seen to elevate in human pre-B leukemia cells 
exposed to 60 Hz, 0 . 1 mT EMFs. Moreover, activation of PKC was dependent on the 
activation of lyn kinase, a tyrosine kinase of the src family which is known to be 
involved in proliferation of leukemia clones (Uckun et al. 1 995).  
The final endpoint in the process of carcinogenesis is  an obvious loss of control of cell 
proliferation. This is a complex process, which is under the control of cellular signal 
transduction (ST) pathways .  In one study, Rosenthal and Obe ( 1 989) exposed human 
peripheral lymphocytes to a 5 mT (50 Hz) EMF and showed a 1 0- 1 5% increase in cell­
cycle progression. Antonopoulos et al. ( 1 995) independently confirmed the effects of a 
5 mT (50 Hz) field reported by Rosenthal and Obe ( 1 989). West ( 1 994) demonstrated 
increased colony growth in anchorage-independent JB6 cells after 1 0- 14  days exposure 
to a 1 . 1  mT (60 Hz) EMF. On the other hand, Hiroaki et al. (2002), whilst not refuting 
that EMFs have an effect on cell proliferation, reported no effect of MFs specifically 
upon the rate of DNA synthesis.  This was measured by determining the rate of 
eH]thymidine incorporation into DNA when cell lines derived from human tumours 
were exposed to either a 50 Hz or 60 Hz field for 3 days. 
Cridland et al. ( 1 999a) used human fibroblasts to investigate the effects of power 
frequency magnetic fields on the kinetics of cell cycle progression using human 
fibroblasts. In this study, normal human fibroblasts were synchronised in the Go phase 
of the cell cycle and exposed to 50  Hz magnetic fields at a range of flux densities. 
Progression through the cycle was monitored by examining the timing of entry into S 
phase, as characterised by the onset of DNA synthesis. Simultaneous positive controls 
were exposed to human recombinant fibroblast growth factor to demonstrate that the 
Chapter Two: Review of Literature. 2 1  
system was responsive t o  external stimuli .  Exposure to magnetic fields at 2 0  and 200 
�T induced a small but significant increase in the l ength of the G1 phase of the cell 
cycle. Exposure at higher flux densities of 2 and 20 mT had no significant effect. 
Chen et at. (2000) used a Friend erythroleukemia cell l ine, which can be chemically 
induced to differentiate, to determine whether ELF EMF could alter proliferation and 
differentiation in these cells in a manner similar to that of a chemical tumor promoter. 
Exposure of this cell line to 60 Hz ELF EMF resulted in  a dose-dependent inhibition of 
differentiation, with maximal inhibition peaking at 40% at 4 �T. ELF EMF at l �T and 
2 . 5  �T inhibited differentiation at 0 and 20%, respectively. ELF EMF at 1 00 �T and 
ImT stimulated cell proliferation 50% above the sham-treated cells. The activity of 
telomerase, a marker of undifferentiated cells, decreased l OO-fold when the cells were 
induced to differentiate under sham conditions, but when the cells were exposed to 5 0  
�T  there was only a I D-fo ld decrease. They concluded that ELF EMF can partially 
block the differentiation of Friend erythroleukemia cells, and this results in a larger 
population of cells remaining in the undifferentiated, proliferative state, which is similar 
to the published results of Friend erythroleukemia cell s  treated with chemical-tumor 
promoters. 
In a wel l-designed study, Mattei et at. ( 1 999) exposed cultured bone cells to a pulsed 
EMF (PEMF) for different times to find the minimal exposure time necessary to 
stimulate an increase of DNA synthesis. These researchers used human osteosarcoma 
cell  l ines and normal human osteoblast cells and observed that the proliferative 
responses of normal human osteoblast cell (NHOC) and human osteosarcoma cell lines 
to PEMF exposure are quite different. Moreover, NHOC required minimal exposure 
times to PEMF to increase their cell proliferation, similar to that needed to stimulate 
bone formation in vivo. 
In summarizing this section, it is evident from all the studies described above that ELF 
EMF whilst perhaps not directly initiating cancer, do appear to play an important role in 
carcinogenesis by influencing enzyme action, signal transduction, cell-cycle 
progression, protein synthesis, ODC, and cell proliferation. However, further research is 
indeed warranted to substantiate this far-reaching claim. 
Chapter Two: Review of Literature. 22 
2. 1 .4 EMFs and Transcription 
Several key studies seem to indicate that ELF-EMFs can influence the rate of mRNA 
transcription. In an elegant study, Goodman and Henderson (1991) observed an increase 
in mRNA transcription levels for selected chromosomes of salivary gland cells of 
Drosophila and Sciara after brief exposures of up to 60 min at 37C, to 0.5-1.1 mT 
EMFs (sinusoidal, 72 and 60 Hz). Some chromosomes showed loci with an increase in 
transcription from 10 to 1000-fold over sham exposed control cells. Exposure of human 
HL-60 cell line to 0.57-570 �T EMFs (sinusoidal, 60 Hz) also induced an increase in 
mRNA transcription (Goodman and Henderson, 1992). 
In an earlier study, Goodman et al. (1983) worked with two types of pulsed fields : 
repetitive single pulse and repetitive pulse train. These were used to test the hypothesis 
that weak pulsed EMFs can modify biological processes. They observed that a single 
pulse increased the specific activity of messenger RNA after 15 min and 45 min of 
exposure . Pulse train, however, increased specific activity only after 45 min of 
exposure. 
In a similar study, Litovitz et al. (1990) exposed cells to relatively low-intensity, pulsed, 
ELF EMFs and observed transient augmentation of RNA synthesis. They also observed 
that under certain conditions of irradiation, the augmentation of RNA synthesis 
enhanced with the increase in EMF strength. 
In a separate study, transcriptional rates in HL-60 cel ls exposed to sinusoidal 60 Hz 
EMFs were investigated by Greene et al. (1991). In their study, transcriptional rates 
were measured by labeling the cells with eH]uridine. A maximum of 50-60% increase 
in overall transcriptional rates was reported in HL-60 cells exposed to a ImT, sinusoidal 
60 Hz EMF. This increase was time dependent and transient with maximum 
enhancement occurring at 30-120 min of exposure with a decline to basal levels by 18 
h. The increase in transcription activity in HL-60 cel ls and the time-dependence are in 
general agreement with the findings of Goodman and Henderson (1992). 
Human HL-60 cells were also exposed for 20 minutes to an EMF at frequencies ranging 
from 0.2-2.3 mT in a similar study by Lin-Xiang et al. (1990). Following each 
Chapter Two: Review of Literature. 23 
exposure, quantitative levels of c-myc and h istone H2B transcription were detennined 
by dot-blot hybridization analysis and compared with sham exposed control samples. A 
four-fold increase in transcription was observed after each exposure at 50  Hz compared 
with sham exposed controls. 
Similarly, with the help of transcription autoradiographic methods, Goodman et al. 
( 1 992) also observed changes in transcriptional activity at 1 3  defined regions of the 
right arm of chromosome 3 (3R) in Drosophila following 20 min exposures of salivary 
glands to five different intensities of ELF EMFs. Other observations that were made 
included changes in translational patterns in ELF EMFs exposed cells, and an increase 
in over-all polypeptide synthesis. 
All these above studies are milestone papers linking weak EMF exposure to gene 
activity . S ince these pioneer studies were performed, several papers have been 
published supporting these data to varying degrees .  Noteworthy research, relevant to the 
current study, is  reviewed as follows: 
Ding et al. (2002) investigated the distribution and expression of growth associated 
protein-43 (GAP-43) in human glioma cells (MO 54) after exposure to a 60 Hz, 5 mT 
MF with or without initial X-ionizing radiation (2 Gy). Immunocytochemistry and the 
reverse transcription polymerase chain reaction (RT-peR) were used to determine the 
EMF -effect. GAP-43 was present in the cytoplasm, accumulating in the perinuclear 
area. An increase in GAP-43 expression was observed with a peak at 1 0  h in mRNA 
levels and at 1 2  h in protein levels, after exposure to the EMF. The increased level of 
GAP-43 protein returned to a nonnal level within 24 h of exposure to a 5 mT EMF. The 
kinetic pattern of GAP-43 expression induced by X-ionizing radiation was very similar 
to that induced by the EMF. The investigators suggest that the stimulation of GAP-43 
expression could occur by a similar mechanism following exposure to X-rays or EMFs. 
The effects of a 50  Hz sinusoidal ELF EMF on the expression of genes relating to 
cytokine receptors were studied in HL60 cells by Zhou et al. (2002). Transcription 
levels of tumor necrosis factor receptor (TNFR) p55 and p75,  interleukin-6 receptor 
(IL-6R) and transforming growth factor-receptor 1 (TGFR1 )  were quantified in cells 
exposed to an intensity of 0 . 1  or 0.8 mT for periods ranging from 30 min to 72 h. The 
Chapter Two: Review of Literature. 24 
results showed that MF exposure at 0 . 1  and 0 .8  mT for 72 h increased TNFR p75 and 
IL-6R mRNA expression in HL60 cells. No significant change in gene expression levels 
ofTNFR p55 and TGFR1 was observed under any of the exposure conditions. 
Ghadire & Mahnaz (2002) studied the biological effect of alternating EMFs on 
developmental stages of Drosophila melanogaster. In this study, D. melanogaster eggs, 
first, second and third instar larvae were exposed to an 1 1  mT 50 Hz field produced 
from a pair of Helmholtz coils. Eggs and each of the larval stages were exposed to 
EMFs for 2, 4, 6 and 8 h. A significant number of adult flies derived from larvae with 
varying degrees of exposure was observed with morphological changes in head thorax 
and abdomen. Whereas, no significant increase in abnormal morphology was observed 
in the group ari sing from EMF -exposed eggs. They also observed a dose-dependent 
increase in the number of abnormal flies compared to the control .  
In a very recent in vitro study that used pluripotent embryonic stem (ES) cells, Czyz et 
al. (2004) studied the non-thermal effects of 50 Hz, ELF EMF. In their study, wild-type 
(wt) and p53-deficient ES cells were exposed either continuously or pulsed (various 
on/off cycles) to power line frequency MF with different magnetic flux densities (0 . 1  
mT, 1 .0 mT or 2 . 3  mT) for 6 h or 48 h. Transcript levels of regulatory genes, such as 
egr- l ,  p2 1 ,  c-jun, c-myc, hsp70 and bcl-2, were analysed by semi-quantitative RT-PCR 
immediately after exposure or after a recovery time of 1 8  h. Pulsed EMF exposure to 5 
min on/30 min off cycles at a flux density of 2 .3  mT for 6 h resulted in a significant up­
regulation of c-jun, p2 1 and egr- l mRNA levels in p53-deficient, but not in wild-type 
cells. No significant EMF effects were observed in both cell systems at lower flux 
densities, longer exposure time or after 1 8  h recovery time. They also reported that 5 
min on/30 min off pulsed MF exposure is capable of causing non-thermal responses in 
ES cells. 
The effect of ELF EMFs on biochemical properties of human oral keratinocytes (HOK) 
was studied by Manni et al. (2004). Cells exposed to a 2 mT, 50 Hz EMF showed no 
changes in gene expression but also modification in shape and morphology by scanning 
electron microscopy (SEM). These modifications were also associated with different 
actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells 
had a smaller c lonogenic capacity , and decreased cellular growth. Indirect 
Chapter Two: Review of Literature. 25 
immunofluorescence with fluorescent antibodies against involucrin and �-catenin, both 
differentiation and adhesion markers, revealed an increase in involucrin and �-catenin 
expression. The advance in differentiation was confirmed by a decrease of expression of 
epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that 
exposure to EMF carries keratinocytes to higher differentiation level .  These 
observations support the hypothesis  that 50  Hz EMFs may not only affect gene 
expression, but also modify cell morphology and interfere in differentiation and cellular 
adhesion of normal keratinocytes. 
Shin-ichi et al. (200 1 )  exposed HeLa cells to 60 Hz, AC, 0.25 to 0 .5  T MFs to 
investigate the biological effects of EMFs on DNA synthesis, transcription or repair, 
using in vitro model systems with defined sequences. In contrast to most studies, these 
researchers observed no statistically significant change in the rate and fidelity of DNA 
polymerase catalyzed DNA synthesis, nor of RNA polymerase catalyzed RNA 
synthesis. Similarly, Cridland et al. ( 1 999b) also showed a null effect of EMFs on RNA 
synthesis in human fibroblasts when exposed to a 50 Hz field at a range of flux densities 
between 2 IlT and 20 mT. 
Most of the research to date on EMFs and gene expression appears to have established a 
link between weak EMF exposure and gene activity. The biological ramifications of 
such a conclusion are enormous when one considers the level of exposure living 
organisms are now subjected to in the modern world. These are fields to which living 
organisms have never been exposed in their long evolutionary history prior to the 
generation of man-made electricity. Research is only just beginning to discover the 
possible genetic consequences of human technological progress. 
2. 1 .5 EMF and Enhancement of the Intracellular Ca2+ Response 
Calcium plays an important role for many cellular functions, especially for transmission 
of extracellular signals, the regulation of intracellular transport of compounds, the 
release of secretion products, bone metabolism, and muscle contraction. Therefore, 
maintaining an optimal cellular calcium concentration is of key importance. This is 
obtained by a number of very precise active ion-transport mechanisms through cell 
Chapter Two: Review of Literature. 26 
membranes .  Many investigations, mostly in vitro, have shown that ELF EMFs influence 
the release of calcium from the cell (Blackman et at. 1 988). 
Persuing this line of study, Lindstrom et al. ( 1 993) exposed Jurkat cells to 50 Hz, 0 . 1 
mT EMFs and examined free intracellular Ca2+ in individual cells, using Fura-2. Within 
1 5-200 sec after the Helmholtz coils were energized, intracellular Ca2+ increased to 200-
400 nM from baseline levels of 50- 1 00 nM. When similar experiments were performed 
with mitogen-activated peripheral lymphocytes,  a similar enhancement in intracellular 
Ca2+ response was observed. However, only 1 0% of the lymphocytes tested responded 
to the EMFs, whereas about 85% of the Jurkat cells showed enhanced intracellular Ca2+ 
in response to an applied EMF. EMFs failed to enhance intracellular Ca2+ levels in non­
dividing endocrine pancreatic cells from mice or rats or when Ca2+ was absent from the 
cell perfusion medium. These studies have an additional unique feature in that single 
cells were observed continuously before and after EMFs were activated and the change 
in Ca2+ level was perfectly correlated with EMFs. 
2. 1 .6 EMF and Plasma Membrane 
The majority of theories addressing the pathways of interaction between biological 
systems and EMFs suggest that the primary site of interaction is the plasma membrane. 
However, direct experimental data showing EMF -induced membrane perturbations are 
sparse. Grandolfo et al. ( 1 99 1 )  exposed chick myoblasts to a 50  Hz sinusoidal MF at 
intensities between 1 and 1 0  mT. No field effects were observed on the fusion of 
myoblasts although decreases in the conductivity and permittivity of the cells were 
observed. The conductivity changes were interpreted as evidence for a decrease in 
transport of various ions, but the site of inhibition was not determined. The decrease in 
permittivity was interpreted as a change in surface charge owing to alteration in lipids, 
proteins or both. An interesting aspect of this study was the observation that the 
maximum response of the cells was found at 5 mT rather than at the highest applied 
intensity of 1 0  mT, suggesting that a l inear dose response might not be involved. It is 
noteworthy that Goodman and Henderson ( 1 99 1 )  also reported that the lower intensity 
fields in the flT range are more effective in inducing bioeffects than more intense fields. 
Chapter Two: Review of Literature. 27 
Fisher et al. ( 1 986) examined the effect of PEMFs on eH]glucosamine incorporation in 
plasma membranes of human fibroblasts and rat sarcoma cells.  About a 1 00% increase 
in labeled glucosamine incorporation into existing cell surface glycoproteins following 
a 48 h exposure to PEMFs was observed. Chromatographic data indicated that anionic 
residues (N -acety 19lucosamine, N-acetylgalactosamine) were also being synthesized 
and incorporated on the cell surface.  
The effects of ELF EMFs ( 5 0-60 Hz) in the form of alteration of membrane transport 
processes were observed by Stange et al. (2002) in Vicia faba root tips. In their study, 
Vicia faba seedlings were exposed to a 1 0  11 T MF (50 Hz, square wave) for 40 min 
together with a radioactive pulse, and they observed a marked increase in amino acid 
uptake into intact roots. A more modest increase was observed with a 1 00 IlT (50 Hz, 
square wave) . Moreover, an increase in media conductivity at low field intensities from 
1 0  IlT ( 5 0  Hz square wave), 1 00 IlT (50 Hz sine wave), and 1 00 IlT (60 Hz square 
wave) fields, indicated an alteration in the movement of ions across the plasma 
membrane, most l ikely due to an increase in net outflow of ions from the root cells. 
S imilarly, a marked elevation in media pH, indicating increased alkalinity, was 
observed at 1 0  and 1 00 IlT for both square and sine waves at both 5 0  and 60 Hz. 
2.1 .7 Melatonin and ELF EMFs 
Recent studies have indicated that certain ELF EMFs can influence melatonin levels in 
humans, and that this in turn may have a bearing on genetic events (Stevens, 1 987;  
Pfluger and Minder, 1 996; Griefahn et al. 2002 ; Wood et al. 1 998). Thus it  is  important 
that the current status of research conducted in this area should be addressed. 
Circadian rhythms in humans and animals are driven by the day/night cycle and are 
synchronised with natural GMFs. The major control gland over this natural cycle is the 
pea-sized pineal gland (Cherry, 1 997), weighing approximately 5 0- 1 5 0 mg in man and 
1 mg in the rat. It is  located in the centre of the human brain. In recent years, the pineal 
gland has been recognized as a highly important endocrine organ. It has been found to 
be sensitive to l ight levels and i s  located on the top of the brain of many early 
vertebrates. For most animals, including humans, it is located in one of the innermost, 
and protected, areas of the head. The pineal gland is a very active organ, having the 
Chapter Two: Review of L iterature. 28 
second highest blood flow after the kidneys, and is equal in volume to the pituitary 
gland. Although it is located in the centre of the brain, it is actually outside the blood­
brain barrier. 
A main function of the pineal gland is the synthesis of the hormone, melatonin. The 
ability of the pineal gland to produce melatonin i s  directly related to the amount of 
visible electromagnetic radiation (i .e. ,  light) that is detected by the eyes. At night, the 
absence of light due to sleep stimulates the pineal gland to produce melatonin, which is 
derived from the amino acid, tryptophan. After its uptake by the endocrine cells of the 
pineal g land, known as pinealocytes, tryptophan is converted to serotonin (5-
hydroxytryptamine) and eventually converted to melatonin (N-acetyl-5 -methoxytrypta 
mine), which is an indole (Jan et at. 1 994) . During the day, light, which falls on the 
eye ' s  retina, produces signals that are biochemically amplified to stimulate the pineal 
gland to reduce its melatonin production (Omura et at. 1 993). 
Wilson et at. ( 1 98 1 ) first reported the in vitro evidence for EMF -dependent depression 
of melatonin in animals. They reported that 60 Hz electric field exposure depressed 
nocturnal melatonin levels in rats. It has also been reported that 50 Hz circular polarized 
MFs, but not linear 50 Hz MFs (Kato et at. 1 994), depress melatonin in rats (Kato et at. 
1 993) .  Yellon ( 1 994) reported that 60 Hz MFs depress melatonin in hamsters, and 
Selmaoui and Touitou ( 1 995) reported depression of pineal serotonin N-acetyl 
transferase and serum melatonin in rats. 
It is important to mention here that melatonin is a most efficient natural cell protection 
and anti-carcinogenic agent in our bodies because of its ability to eliminate free radicals 
(Reiter et at. 1 993 & 200 1 ;  Omura et at. 1 993) .  Production of melatonin floods our 
bodies particularly at night, eliminating the build up of free radicals that are being 
produced, allowing DNA synthesis and cell division to occur with a much lower chance 
of damage and hence producing more healthy cells. Melatonin also dampens the release 
of estrogen, prolonged exposure to which may increase the risk of breast cancer 
(Cherry, 1 997). 
Based on a number of studies it has been reported in the field of chemistry that ELF 
EMFs increase the yields of some types of free radicals (Scaiano et at. 1 994a, b; 
Chapter Two: Review of Literature. 29 
Canfield et al. 1 994; Grissom, 1 995). Free radicals are known to be an important factor 
in cell proliferation and hence to be known as cancer promoters. The production of free 
radicals (.OH) is a consequence of the utilization of oxygen by all organisms. About 1 -
2% of inspired oxygen ends up as toxic free radicals which can damage macromolecules 
such as DNA, proteins and lipids. This damage is referred to as oxidative stress. 
Melatonin is an efficient neutraliser of these free radicals. To i llustrate this ,  safrole, 
which is a known chemical carcinogen, damages DNA by inducing the production of 
large numbers of oxygen free radicals. But Tan et al. ( 1 993) showed that the hormone 
melatonin inhibits DNA damage induced by safrole in vivo. When they injected rats 
with safrole alone, they observed extensive DNA damage after only 24 hours. However, 
when melatonin was injected along with safrole the DNA damage was reduced by 99%. 
Since melatonin is a known natural cell protector and possesses anti-carcinogenic 
properties (Reiter, 2000; and Liburdy et al. 1 993),  it is believed that any factors that 
suppress its secretion may contribute to an increased cancer risk. There are a number of 
reports on human and animals suggesting that synthesis of melatonin and its precursors 
are altered in the presence of an EMF. For example, Stevens ( 1 987) first put forward the 
hypothesis that the use of electric power may increase the risk of breast cancer. This 
hypothesis was based on a number of experimental reports indicating an effect of light 
at night (LAN) and powerline-frequency (50 or 60 Hz) EMFs on pineal melatonin 
production, and on the relationship of melatonin to mammary (breast) carcinogenesis. 
Referring to the Stevens ' s  hypothesis, EMF and LAN are said to reduce circulating 
levels of melatonin. This in turn would allow estrogen levels to rise and stimulate the 
turnover of breast epithelial stem cells and increase the risk for malignant 
transformation. A follow-up study of the Stevens' hypothesis was performed by 
Graham et al. (200 1 b) . In their three laboratory-based studies a total of 53 healthy 
young women were exposed at night to EMF or to LAN under controlled exposure 
conditions to determine whether such exposures reduced melatonin and altered estrogen 
levels. But their experiment failed to show any effect of industrial-strength EMF­
exposure (28 . 3  IlT, 60 Hz,) on the blood levels of melatonin or estradiol. However, 
nocturnal melatonin levels were profoundly suppressed, and the time of peak 
concentration was significantly delayed in women exposed to LAN, regardless of 
whether they were in the follicular or luteal phase of the menstrual cycle. 
Chapter Two: Review of Literature. 30  
In a study of  seven healthy men ( 1 6-22 years), Griefahn et al. (2002) reported that light 
suppressed melatonin production and delayed night-time rectal temperature and heart 
rate . During the control session, subjects were kept at complete bed rest for 24 h at less 
than 30 lux at 1 8C. Experimental subjects were exposed to either light (at 1 500 lux), 0.2 
mT MF ( 1 6 .7 Hz), or infrared radiation at 65C from 5 pm to 1 am. Salivary melatonin 
level was determined hourly, but rectal temperature and heart rate were recorded 
continuously . Melatonin synthesis was completely suppressed by light but resumed 
thereafter with darkness. The night-time rectal temperature and heart rate were delayed. 
The MF had no effect. Infrared radiation elevated rectal temperature and heart rate. 
Only bright l ight affected the circadian rhythms of melatonin synthesis, rectal 
temperature, and heart rate . 
In another study, Wood et al. ( 1 998) studied the effects of power-frequency MFs on 
night-time plasma melatonin in a group of 30 adult male human subjects. The time of 
onset of rise in melatonin concentration for each sham-exposed subject was predicted 
from a previous screening night. Then the subjects were exposed to 20 IlT (50 Hz) at 
certain times and the time of onset of rise in melatonin concentration for a particular 
individual was recorded. Response to this exposure was compared to sham-exposure (in 
random order). When exposure preceded onset of rise, a significant delay in onset time 
relative to sham-exposure of approximately half an hour was observed, with indications 
of a reduction in maximum melatonin level .  MFs generated by square-wave currents 
produce more marked reductions in the maximum level when compared to sinusoidal 
waveforms, but there was no significant difference in onset time. 
Levallois et al. (200 1 )  studied the effect of residential exposure to electric fields and 
MFs from high-power lines on female urinary excretion of 6-hydroxymelatonin sulfate 
(6-0HMS). A sample of 22 1 women living near a 735-kV line was compared with 1 95 
women the same age living away from any power lines. Participants provided morning 
urine samples on two consecutive days and wore a magnetic dosimeter for 36 
consecutive hours to measure personal magnetic exposure. The indoor electric field was 
assessed by spot measurements. After adjustment for other factors associated with low 
melatonin secretion, such as medication use or light exposure, night-time concentration 
of 6-0HMS was similar in the two groups .  When either 24 h or sleep-time exposure to 
MF or electric field measurements was used, no exposure-effect relation was evident. 
Chapter Two: Review of Literature. 3 1  
However, the trend o f  decreasing 6-0HMS concentration with age was more 
pronounced for women living near the lines, as was a lower 6-0HMS concentration in 
women with high body mass index. Chronic residential exposure to MFs from high­
power lines may accentuate the decrease in melatonin secretion observed in some 
vulnerable subgroups of the population. 
In a similar study, Pfluger and Minder ( 1 996) investigated the effects of EMF exposure 
( 1 6 .7  Hz) on pineal melatonin production in healthy humans. The study was based on 
comparing urinary 6-0HMS levels of 1 08 male railway workers. A repeated measure 
design was used, which means each volunteer served as his own control . The exposure 
ranged between 1 1-1T to 20  !IT. Melatonin level was measured by sampling urinary 6-
OHMS both in the morning and the early evening. Their results suggested that MFs do 
alter 6-0HMS excretion in humans exposed to 1 6 .7 Hz MFs. 
In another study, Davis et al. (200 1 )  also studied the reduction of melatonin levels by 
night-time residential EMF exposure (60 Hz) in humans. The researchers investigated 
the association between night-time residential EMF exposure with lower nocturnal 
urinary concentration of 6-0HMS in 203 women (20-74y) with no history of breast 
cancer. They observed reduction in nocturnal melatonin production. 
In a similar study, Burch et al. (2000) investigated the melatonin metabolite in electric 
utility workers. In this study, personal exposure to MFs and ambient light, and excretion 
of the melatonin metabolite, 6-0HMS, were measured over 3 consecutive workdays in 
electric util ity workers. There was a MF-dependent reduction in adjusted mean 
nocturnal and post-work 6-0HMS levels among men working more than 2 hours per 
day in substation and 3-phase environments and no effect among those working 2 hours 
or less. No changes were observed among men working in I -phase environments. The 
results suggest that circular or elliptical MF polarization, or another factor linked to 
substations and 3 -phase e lectricity, i s  associated with MF-induced melatonin 
suppression in humans. 
To study the secretory activity of the pineal gland, Welker et al. ( 1 983) investigated the 
effects of artificial MFs on pineal serotonin-N-acetyltransferase (NAT) activity and 
melatonin content in male Sprague-Dawley rats .  Experimental inversion of the 
Chapter Two: Review of Literature. 32 
horizontal component of the natural MF, performed at night-time, led to a significant 
decrease of both parameters investigated. During day-time, this effect was less 
conspicuous. During night-time, inversion of the horizontal component was followed by 
a reduced pineal secretory activity for about 2 h. After a 24 h exposure to the inverted 
horizontal component, return to the natural condition was followed by a renewed clear 
depression of pineal N AT activity and melatonin content, indicating that the main 
stimulus is not the inverted MF itself but rather its change. Changing the inclination of 
the local MF from 63 degrees to 58 degrees, 68 degrees or 78 degrees, respectively also 
decreased the secretory activity of the rat pineal gland. 
In an interesting study of rats, Olcese et al. ( 1 985) investigated whether MF effects on 
mammalian pineal function are direct or indirect via retinal disturbances. In this study, 
acutely blinded and intact male rats were exposed to a single nocturnal magnetic 
stimulus. Then pineal N-acetyltransferase activity and melatonin content were assayed. 
Only in intact animals did the magnetic stimulus significantly reduce pineal activity, 
i . e . ,  no effects were detected in bl inded animals .  These data point to a retinal 
magnetosensitivity, which may serve to modulate pineal gland function. 
In persons with intact vision, pineal secretion is highest early in the morning and 
reduces fol lowing exposures to l ight. Therefore, increased exposures to LAN may 
decrease nocturnal secretion and thus contribute to the observed increases in breast 
cancer incidence rates. It has been reasoned that women who are profoundly blind, and 
therefore not susceptible to LAN, should have a reduced risk of breast cancer compared 
with those with intact vision. In one study, Verkasalo et al. ( 1 999) studied the cancer 
risk patterns in persons with visual impairments to explore the hypothesis that women 
with visual impairment might have decreased breast cancer risk. A total of 1 0,935 
women with visual impairment were identified from the Finnish register of visual 
impairment and followed up for cancer through the Finnish cancer registry for the years 
1 983- 1 996. The authors observed a statistically significant decreased breast cancer risk 
with increased visual impairment, which suggests a dose-response relationship between 
visible light and breast cancer risk. 
Chapter Two: Review of Literature. 3 3  
A series of  studies has shown that low exposure to EMFs can inhibit melatonin's cancer­
preventive activity. For example, Harland and Liburdy ( 1 996) reported that a 1 .2 �T 
EMF could significantly reduce the growth-inhibitory action of melatonin and 
tamoxifen on breast cancer cell MCF -7 growth. Blackman ( 1 996) found that melatonin 
can inhibit the growth of MCF-7 cells in culture, and that a 1 .2 �T, 60 Hz MF can 
completely block this growth-inhibitory action. Harland and Liburdy ( 1 996) observed 
that 1 .2 �T computer monitor MFs also inhibit the abi lity of melatonin to suppress 
breast cancer cells in vitro . Finally, Liburdy and Afzal ( 1 996) suggested that 1 .2 �T 60 
Hz exposure of a Video Display Terminal (VDT) MF inhibits the cancer-preventive 
action of melatonin in vitro. 
A number of other studies in animals where responses have been observed suggest that 
specific aspects of the exposure might be critical (Rogers et al. 1 997). For example, 
Yellon ( 1 996) suggested that repeated MF exposures result in a loss of the detection of 
the melatonin effect in hamsters, i .e . ,  where the melatonin depression is seen only after 
a single exposure but not with continued 3-week exposures. 
Yellon ( 1 99 1 )  had previously shown that the nocturnal production of pineal melatonin 
in Djungarian hamsters is extremely sensitive to a brief 1 5  min exposure to a sinusoidal 
60-Hz, 0 . 1  mT MF. When the field was applied 2 h before the onset of darkness, the 
night-time rise in pineal melatonin was heavily suppressed and the peak production of 
this hormone was phase shifted by approximately 2 h to a later part of the dark period. 
Acute exposures of short-day animals produced similar results (Yellon, 1 994). By 
contrast, daily exposures of the same type for as long as 3 -weeks had no effect. 
Reiter ( 1 994) proposes that melatonin is more rapidly taken up by tissues during the 
ELF EMF exposure. He noted that if EMFs result in higher levels of free radicals, then 
an antioxidant like melatonin would disappear from the blood more quickly than is 
normal because it would be required for the scavenging of free radicals. Reduction of 
melatonin at night, by any means, increases the cell's vulnerability to alteration by 
carcinogenic agents. Thus, if in fact artificial EMF exposure increases the incidence of 
cancer in humans, a plausible mechanism could involve a reduction in melatonin that is 
a consequence of such exposures. 
Chapter Two: Review of L iterature. 34 
Referring to the above discussion, i t  is the view of the current author that although there 
have been a number of negative experimental findings concerning the biological effects 
of EMF on melatonin production, there exists a persistent pattern of evidence, both in 
vitro and in vivo, showing that fields can produce significant biological responses in 
humans even at low levels. The responses are as follows: 1) suppression of melatonin 
production; (2) increase of estrogen levels; (3) reduction in the growth-inhibitory action 
of melatonin and tamoxifen on breast cancer cell MCF -7 growth; (4) inhibition of 
cancer-preventive action of melatonin; and (5) MFs generated by square-wave currents 
produce more marked reductions in the maximum level night-time plasma melatonin 
when compared to sinusoidal waveforms. 
2.1 .8 Geomagnetic Field (GMF) 
The current study was performed to determine the possible effects of l !-lT or ImT (50 
Hz,  AC) ELF EMFs on human chromosomes .  Although the earth' s  geomagnetic field 
(GMF) is DC and is quite different to artificially produced AC fields, it is nevertheless a 
weak MF to which living organisms respond in a wide variety of circumstances. It i s  
therefore appropriate to review the discoveries that have been made during the last few 
decades concerning the ability of living organisms, including humans, to respond to the 
GMF. 
The Earth acts like a great spherical magnet, and is therefore surrounded by a MF. The 
Earth's MF resembles, in general, the field generated by a dipole magnet (i .e, a straight 
magnet with a North and South Pole) located at the centre of the Earth. The axis of the 
dipole is offset from the axis of the Earth's rotation by approximately 1 1 .5 degrees. This 
means that the north and south geographic poles and the north and south magnetic poles 
are not located in the same place (Figure 2.2). The GMF originates from electric current 
flow in tIre Earth' s  core and Dubrov ( 1 978) published the first extensive review of the 
biological effects of the GMF. The Eearth' s  MF is irregular, and at the surface of the 
earth has a steady component of approximately 5 0  �T. 
Sensitivity of living organisms to the Earth' s  MF has been one of the most controversial 
topics in the behavioural and natural sciences for many years. Biologists criticized the 
Chapter Two: Review of Literature. 35  
Geomagnetic Axis 
Axis of Earth's  Rotation 
Geomagnetic North Pole Geographic North Pole 
Plane of Magnetic 
Equator 
Figure 2.2: Showing geographic and geomagnetic poles. 
earlier reports of magnetic effects on animals because they were unable to reproduce the 
effects, and physicists could not determine a genuine biophysical mechanism by which 
animals could detect the weak MF of the Earth (Griffin, 1 944, 1 952). 
Radical changes occurred, however, during the last two decades in three distinct areas: 
1 )  Discovery of many highly reproducible magnetic effects on behaviour, which 
includes the magnetotactic bacteria and protozoans, and magnetic effects on homing 
and navigational behaviour by migrating animals .  It has been revealed that apparently 
two separate magnetic sensory systems exist in higher animals :  a directional compass 
and a magneto intensity sense. 2) At least two plausible biophysical mechanisms are 
now known through which the Earth' s  MF can be transduced to the nervous system. 
These mechanisms include use of the ferromagnetic mineral magnetite (Fe304), which 
is a biochemical precipitate in virtually all groups of higher organisms, including 
humans. It forms chainlike structures in several groups of vertebrates ideally suited for 
responding to MFs (Kirschvink et al. 1 985 ;  Mann et al. 1 988;  Beason and Brennan, 
1 986). Another potential transduction mechanism is "Optical pumping" (Leask, 1 977; 
Chapter Two: Review of Literature. 36 
Schulten, 1 982; Schulten and Windemeuth, 1 986) by which Earth MPs could influence 
charge-transfer reactions in organic molecules. 3) A group of researchers obtained clear 
records of magnetically influenced signals in single nerve units connecting magnetite­
bearing tissues with the brain, and these signals suggest strongly that the magnetite­
bearing tissues of the ethmoid sinus contain a magnetoreceptor. 
The next section is a brief review of these three developments discussed above. 
Magnetoreception 
Kirschvink et al. (200 1 )  reported that orientation, navigation, and homing were critical 
traits expressed by organisms ranging from bacteria through to higher vertebrates .  
Sensory systems that aid such behavior have provided key selective advantages to these 
groups over the past 4 billion years, and are highly evolved; magnetoreception is no 
exception. Across many species and groups of organisms, compelling evidence exists 
that the physical basis of this response is tiny crystals of single-domain magnetite . 
Kirschvink postulated that all MP sensitivity in l iving organisms, including 
elasmobranch fishes, is  the result of a highly evolved, finely-tuned sensory system 
based on single-domain, ferromagnetic crystals.  
Blakemore ( 1 982) and Torres de Araujo et al. ( 1 986) reported a good example of 
directional compass response in magnetotactic bacteria. Microscopic observation shows 
that living cells swim in straight lines parallel to a MP, and their swimming direction 
can be changed instantly by using a small hand magnet. The cells of these organisms 
contain 0 . 5-2.0  % magnetite or greigite by weight, and the arrangement of crystals into 
chains provides each cell with magnetic moments large enough to align the organism 
passively like a compass needle in the GMF. 
Soon after the discovery of magnetotactic bacteria, Blakemore ( 1 975) proposed the 
theory that all magnetotactic bacteria are microaerophilic and indigenous in sediments. 
Frankel et al. ( 1 997) clearly showed that these bacteria passively align and actively 
swim along the inclined GMF -lines as a result of their magnetic dipole moment. 
Blakemore called this behavior magnetotaxis, which helps to guide the cells down to 
less oxygenated regions of aquatic habitats at the surface of sediments. Once cells have 
Chapter Two: Review of Literature. 37 
reached their preferred microhabitat they would presumably stop swimming and adhere 
to sediment particles until conditions changed, as for example, when additional oxygen 
was introduced. This theory is supported by the predominant occurrence of magne­
totactic bacteria that are north-seeking (i .e . ,  swim in the direction indicated by the 
north-seeking pole of a magnetic compass needle) under oxic conditions in the northern 
hemisphere whereas bacteria are predominately south-seeking in the Southern 
Hemisphere . Due to the negative and positive sign of the GMF-inc1ination in the 
Northern and Southern Hemispheres,  respectively, magnetotacti c  bacteria in both 
Hemispheres therefore swim downward toward the sediments (Blakemore, 1 982). 
A possible connection between magnetic material and MF effects was found in 
honeybee behavior, and these behavioural effects have been reviewed by von Frisch 
( 1 967), Martin and Lindauer ( 1 977), Martin et al. ( 1 989), Gould ( 1 980), Hsu and Chia­
wei ( 1 994). Worker honeybees communicate the location of a food source by means of 
a "waggle dance" to the other worker bees in a hive on a vertical honeycomb. The angle 
between the direction of the dance and the vertical direction indicates the angle between 
the food source and the Sun. Consistent errors in the dance angle occur when the MF in 
the hive is eliminated by means of external coils. In anomalous situations where bees 
are made to dance on horizontal surfaces, after an initial period of disorientation they 
dance eight magnetic, compass directions (N, NE, E, SE, etc .) .  If  the GMF is eliminated 
the dances become disoriented. Martin and Lindauer ( 1 977) suggested that bees could 
also use the diurnal variations in the GMF to set their circadian rhythms.  
Phillips and Sayeed ( 1 993) reported a directional compass response in male fruit flies, 
(Drosophila melanogaster). This discovery is an exciting development, as numerous 
genetic mutants exist for Drosophila that could be used as an aid in the localization of 
the receptor cells, as well  as aid in the understanding of the transduction mechanism. 
The experimental protocol followed appears to be straightforward and simple. It has 
been suggested that the fruit fly could be used to replace the honeybee as the organism 
of choice in magnetic studies, if the results prove to be reproducible. 
Lohmann e t  al. ( 1 99 1 )  also observed a directional compass response in manne 
mollusks, ( Tritonia diomeda) . Lohmann et al. ( 1 99 1 )  used intracel lular electro­
physiological recording techniques and observed that the large cells of the left and right 
Chapter Two: Review of L iterature. 38  
pedal ( IPe5 and rPe5) alter their firing patterns slowly but consistently in response to a 
rotation of the MF direction. The ability to identify these distinct, magnetically sensitive 
cells offers hope that neurological staining techniques may eventually locate the actual 
receptor cells and help to clarify the transduction mechanism. 
A directional compass response was also found in sockeye salmon (fish) 0. nerka by 
Quinn & Brannon ( 1 982), male eastern red-spotted newts (amphibians) Notophthalmus 
viridescens by Phillips ( 1 986), and in loggerhead sea turtle (reptiles) Caretta caretta L.  
by Lohmann ( 1 99 1 ) .  
Magnetointensity perception 
A study by Walcott et at. ( 1 979) on pigeon homing suggested that MF information 
could be used for orientation. The ability of pigeons to sense MFs may be associated 
with a small, unilateral structure between the brain and the skull that contains a magnet, 
and appears to be a single domain. 
In a separate study, however, Wa1cott ( 1 99 1 )  concluded that the primary navigation of 
homing pigeons is by the position of the sun but when the sun is obscured by cloud then 
they use the GMF for navigation. Similarly, migratory birds are also able to use the 
GMF as a compass to find and to maintain direction. Migratory sparrows (Passerculus 
sandwichensis), for example, navigate by the GMF (Able and Able, 1 995). 
Kirschvink et at. ( 1 986) tested the hypothesis that cetaceans use weak anomalies in the 
GMFs as cues for orientation, navigation and/or piloting. Using the positions of 2 1 2  
stranding events of live animals i n  the Smithsonian compilation which fall  within the 
boundaries of the East-Coast Aeromagnetic Survey (USGS), they found that there are 
highly significant tendencies for cetaceans to beach themselves near coastal locations 
with local magnetic minima. Monte-Carlo simulations confirm the significance of these 
effects . These results suggest that cetaceans have a magnetic sensory system 
comparable  to that in other migratory and homing animals,  and predict that the 
magnetic topography and in particular the marine magnetic lineations may play an 
important role in guiding long-distance migration. The 'map' sense of migratory animals 
may therefore be largely based on a simple strategy of following paths of local magnetic 
Chapter Two: Review of Literature. 3 9  
minima and avoiding magnetic gradients. A central problem, however, in the study of 
magnetic sensitivity in animals has been the lack of behavioural techniques sufficiently 
powerful for the systematic psychophysical work required for an understanding of 
magnetosensory capacity and of the transduction mechanism. 
In the past, it has been shown repeatedly that free-flying honeybees can be trained in 
discrimination experiments to respond to local magnetic anomalies (Walker and 
Bitterman, 1 989, 1989a, b; Kirschvink and Kobayashi-Kirschvink, 1 99 1 ,  Kirschvink et 
at. 1 992a). Therefore, honeybees are one of the best experimental animals available for 
testing biophysical hypotheses concerning the sensory transduction of the GMF in 
animals .  Walker and Bitterman ( 1 989b) developed an experimental protocol in which 
individual honeybees were trained to discriminate between the presence or absence of a 
smal l static (DC) magnetic anomaly in one of two targets mounted vertically. 
Kirschvink et at. ( 1 992a) reported the replication of this model and used it to measure 
the range of frequencies, of constant amplitude, to which the honeybee magnetoreceptor 
system was capable of responding. Honeybees demonstrated an ability to detect AC 
fields of 2.2 mT peak amplitude from (DC) frequencies up to 60 Hz. Above 60 Hz the 
behaviour approached randomness. This result was broadly consistent with the 
biophysical prediction for a magnetite-based magnetoreceptor (Kirschvink et at. 1 992a). 
Walker and Bitterman ( 1 989) reported a remarkable measurement of the threshold level 
of static magnetic intensity perception in the honeybee. Such was the ingenuity of their 
experimental design, it is worth detailing their procedure. In their two-choice threshold 
conditioning experiments, free-flying honeybees were conditioned to discriminate the 
presence of localized magnetic dipole anomalies superimposed on the uniform 
background field of the earth. In the beginning they developed a two-choice training 
paradigm using two sucrose-water feeder assemblies mounted on a vertical window 
frame. Each assembly had a pair of double-wrapped coils that could either produce a 
sharply focused magnetic anomaly or a matching null-field anomaly, but with the same 
thermal effects . Individual foraging bees were trained via a reward-punishment scheme 
to feed preferentially from the feeder paired with the magnetic anomaly. Usually within 
1 0-20 repeat visits they would learn to land at the feeder with the magnetic anomaly and 
avoid the non-magnetic one. By starting with a moderately strong anomaly (3 mT) and 
by reducing the amplitude of the anomaly in small exponential steps, the point at which 
Chapter Two: Review of Literature. 40 
the bees were no longer able to discriminate correctly could determine the threshold 
sensitivity. Of nine bees run through the procedure, the median was 250 nT in the 
presence of the Earth's field. The best bee lost the ability to discriminate in fields below 
25 nT (0.06% of background). 
Kirschvink et al. ( 1 997) used a modified Walker and Bitterman ( 1 989b) protocol to 
place relative constraints on the threshold sensitivity of honeybees to AC MFs at 
frequencies of 1 0  and 60 Hz to complement their results for static magnetic anomalies. 
Their results indicate that the sensitivity of the honeybee magnetoreception system 
decreases rapidly with increasing frequency. At 60 Hz, alternating field strengths above 
1 00 Il T are required to show discrimination. In other words, the magnetic sense of the 
honeybee has its maximum sensitivity below 1 0 Hz, which is consistent with the 
evolution of this system within the known geomagnetic frequency spectrum. 
Biophysics of magnetoreception 
The materials found in living organisms are normally either nonmagnetic (e .g . ,  water, 
fatty substances) or paramagnetic (e.g . ,  deoxyhaemoglobin in red blood cells). The 
direct physical influence of the Earth' s  MF is very weak on these types of materials, 
with the energy of magnetic interaction being many orders of magnitude below that of 
the background thermal energy, kT (where, k = Boltzmann constant and T = absolute 
temperature) . However, another category of materials, known as ferromagnetic, 
interacts very strongly with the Earth's MF. Magnetotactic bacteria are guided by the 
direct interaction of the field with particles of ferromagnetic material secreted within 
their cytoplasm (Tenforde, 1 989). In the marine diatom, Soffeaeformis, movement is 
enhanced by calcium uptake in MFs (Male, 1 992) . 
As mentioned previously, many higher organisms including mammals, birds and insects 
are thought to be able to detect and use the GMF for navigation and homing purposes, 
although the nature of the primary interaction mechanisms that may be involved is not 
well understood. In mammals, the pineal gland is a light-sensitive time-keeping organ in 
which the cell activity is affected by MF pulses of the order of the strength of the GMF 
(Semm et al. 1 980). 
Chapter Two: Review of Literature. 4 1  
Natural magnetoreception 
Kisliuk and I slay ( 1 977) have observed foetal effects on pupae of hornets when a 
horizontal MF was laid down over a hive. Upon emerging from pupae, young hornets 
were sluggish for a few days and then they built abnormally oriented combs. 
A series of chains of uniformly sized single domain magnetite particles embedded in the 
ethmoid tissue in the front of the cranium of Chinook Salmon fish (Oncorhynchus 
tshawytscha) is used for accurate navigation (Kirschvink et al. 1 985).  Salamanders 
(Eurycea lucifuga) were trained to choose which corridors to move along in response to 
the angle of the EMF surrounding the corridor (Phillips, 1 977). 
The apparently synchronous turning and wheeling of flocks of several bird species, 
particularly starlings (Sternus vulgaris) has long been a phenomenon of curiosity and 
interest (Frank and Haffner, 1 974). Based on several reports the authors suggested that 
birds may be able to detect the earth' s  MF and the turning movements in bird flocks 
may be coordinated by geomagnetic means. Such a signal would be instantaneous, 
could reach all birds in the flock regardless of their position relative to the signal source, 
and could carry sufficient information for turning movements. Analysis of films of 
turning flocks of birds and experimental studies of startle reaction times in birds have 
suggested that the geomagnetic radiation model could provide a parsimonious 
explanation for the observed behaviour of the birds. 
In a seminal paper, Walker et al. ( 1 997) described the key components of a magnetic 
sense underpinning this navigational ability in the rainbow trout fish (Oncorhynchus 
mykiss) .  The authors identified an area in the nose of the trout fish where candidate 
magnetoreceptor cells are located and reported the behavioural and electrophysiological 
responses to MFs. They have tracked the sensory pathway from these newly identified 
candidate magnetoreceptor cells to the brain and associated the system with a learned 
response to MFs. 
They have identified single neurons in the superficial ophthalmic ramus (ros V) of the 
trigeminal nerve which do respond to varying magnetic intensity but do not respond to 
the direction of an imposed MF. They have also identified the candidate magnetite-
Chapter Two: Review of Literature. 42 
based magnetoreceptor cells in the nose of the trout with the aid of new imaging and 
confocal microscopic techniques. The cells contain crystals that are iron-rich and almost 
identical in form to single-domain magnetite crystals previously extracted from sockeye 
salmon (Mann et al. 1 988).  They have also traced fine branches of the ros V nerve to 
the same cellular layer (the lamina propria within the olfactory lamellae) as the 
candidate magnetoreceptor cells . Identification of the critical components of the 
magnetic sense brings a new perspective to the study of long-distance orientation in a 
variety of vertebrate groups. 
It  appears from the above discussion that living organisms do respond to the minute 
static GMFs. These responses of living organisms to GMFs are in the form of 1 )  a 
directional compass - used by magnetotactic bacteria, honey bee, drosophila, marine 
mollusks, sockeye salmon, male eastern red-spotted newts, and loggerhead sea turtle ;  2) 
magneto intensity perception - used in homing, navigation and orientation by pigeon, 
migratory sparrows, and cetaceans; 3) In mammals, the pineal gland is a l ight-sensitive 
time-keeping organ in which the cell activity is affected by MF pulses of the order of 
the strength of the GMF. 
2.1.9 DNA Damage, Chromosomal Aberration and ELF EMFs 
It is generally accepted that ELF EMFs do not transfer energy to cells in sufficient 
amounts to directly damage DNA. It is possible, however, that certain cellular processes 
altered by exposure to ELF EMF, such as free radicals, indirectly affect the structure of 
DNA. Most researchers have looked for strand breaks and other chromosomal 
aberrations (CAs), including SCE, formation of micronuclei, and/or effects on DNA 
repair. In a number of studies conducted to assess the effects of ELF EMFs, human 
peripheral blood lymphocytes (HPBLs) have been exposed to different magnetic flux 
densities. 
In one study, EMFs were found to induce CAs in human lymphocytes (Khalil and 
Qassem, 1 99 1 ). Exposure of human lymphocytes to a pulsed field with a peak magnetic 
flux density of 1 .05 mT for 24-72 h induced a statistically significant, 2-fold increase in 
the frequency of CAs and simultaneous lowering of the mitotic index. 
Chapter Two: Review of Literature. 43 
Garcia-Sagredo et al. ( 1 99 1 )  studied the effects of ELF EMFs on HPBLs in vitro. An 
increase of CAs was observed after exposure to PEMFs of amplitudes ranging from 
1mT to 4 mT. The treatment effect at 4 mT was statistically significant. This result led 
them to conclude that PEMFs may have a clastogenic effect on humans. 
In a PEMF study by Vijaylaxmi et al. ( 1 997), HPBLs were exposed to radio-frequency 
radiation (RFR) at 2.45 gigahertz (GHz) for 30  min on-30 min off over 90 min. 
Lymphocytes were cultured immediately after exposure for 48 h and 72 h. An analysis 
showed no s ignificant difference between RFR-exposed and sham-exposed 
lymphocytes with respect to i) mitotic index, ii) chromosome damage, iii) exchange 
aberrations, and iv) number of micronuclei. 
Scientific studies have also focus sed on the question of whether radiation emitted from 
mobile phones causes any damage to human health. With this aim, Mashevich et al. 
(2003) exposed HPBLs to continuous 830 MHz EMFs and observed losses and gains of 
chromosomes (aneuploidy), a major "somatic mutation" leading to genomic instability 
and thereby to cancer. 
A linear increase in chromosome 1 7  aneuploidy was observed as a function of the 
specific absorption rate (SAR) value, demonstrating that this radiation has a genotoxic 
effect. The SAR-dependent aneuploidy was accompanied by an abnormal mode of 
replication of the chromosome 1 7  region engaged in segregation (repetitive DNA arrays 
associated with the centromere), suggesting that epigenetic alterations are involved in 
SAR-dependent genetic toxicity. Control experiments (i .e . ,  without any RF radiation) 
carried out in the temperature range of 34.5-38 .5C showed that elevated temperature is 
not associated with either the genetic or epigenetic alterations observed following RFR, 
the increased levels of aneuploidy, and the modification in replication of the 
centromeric DNA arrays. These findings indicate that the genotoxic effect of the 
electromagnetic radiation is elicited via a non-thermal pathway. One important aspect of 
the above study not noted by the authors, but observed in passing by the writer of this 
thesis is that chromosome 1 7  houses one of the most important tumour suppressor genes 
in humans :  p53 .  Loss of this chromosome in a cell line by aneuploidy could have far­
reaching effects on the proliferation of that line. 
Chapter Two: Review of L iterature. 44 
Cytogenetic investigations were performed by Maes et at. (2000) in HPBLs following 
exposure to 50 Hz MFs alone or in combination with the chemical mutagen, mitomycin 
C, or with X-rays. No significant effects of EMFs up to 2,500 /IT on the chromosome 
aberration and SCE frequency were observed. 
Nordenson et at. ( 1 994) observed a significant increase in the frequency of 
chromosomal aberrations in human amniotic cells after exposure to a sinusoidal 50 Hz, 
30 /IT, pulsed MF. Amniotic cells from eight different foetuses were exposed to a 50  
Hz, 30  /IT MF in a 1 5  sec on and 15  sec off fashion for 72  h .  Among exposed cells, 
aberration frequency was 4% compared to 2% in sham-exposed cells. A similar increase 
in CA was observed in another series of eight experiments in a 2 sec on and 2 sec off 
fashion. On the other hand, continuous exposure of cells to 50  Hz, 300 /IT for 72 h did 
not increase the frequency of chromosomal aberrations. 
In a study by Rapley et al. ( 1 998) seedlings of Viciafaba were exposed to different MFs 
continuously for 3 days :  0 Hz (DC) at 5 mT, 50 Hz at 1 .5 mT, 60 Hz at 1 . 5 mT, and 75 
Hz at 1 . 5 mT.  The length of all the mitotic phases differed from the controls in all 
treatments using alternating MFs and for prophase and metaphase in the DC condition. 
In all treatments, the length of the prophase increased significantly in meristematic root­
tip cells compared with the controls. The length of the prophase, however, did not vary 
significantly between the treatments. Moreover, exposed seedlings had a lower 
frequency of chromosome breakage than the controls .  
The effects of acute exposure to 60 Hz MFs on DNA strand breaks, a common form of 
DNA damage, in the brain cells of rat were observed by Lai and Singh ( 1 997a) . The 
'Comet-assay '  (DNA microgel electrophoresis) was used in their study to detect DNA 
fragmentation in single cells .  Rats were exposed for 2 h to a 60 Hz MF (flux densities 
0. 1 , 0 .25 and 0 . 5  mT) and a dose-dependent increase in DNA strand breaks in the brain 
cells at 4 h post-exposure was observed. An increase in single-strand DNA breaks were 
also observed in all three parameters of exposure (0. 1 ,  0.25 and 0 .5  mT) but double­
strand DNA breaks were observed only in higher parameters of exposure (0.25 and 0 .5  
mT). 
Chapter Two: Review of Literature. 45 
Setlow and Setlow (1 972) reported that several types of electromagnetic radiation might 
cause damage to a cel l ' s  DNA. Strong energy radiation is effective at breaking DNA 
and promoting chemical modification in the form of gamma-endonuclease sites 
(Patterson et al. 1 976) or protein-DNA crosslinks (Fomace et al. 1 980). It is well known 
that ultraviolet l ight produces pyrimidine dimers, as well as causing numerous other 
alterations in DNA (Setlow and Setlow, 1 972), while irradiation of BrdU substituted 
DNA with near-ultraviolet light produces a different set of changes, most notably 
single-strand DNA breaks (Hutchinson, 1 973).  
In a study by Fiorani et al. ( 1 992), a human tumor cell l ine (k562) was grown in 
suspension culture and exposed to 50  Hz, 0.2, 20, 1 00, or 200 IlT MFs for 4, 6, 1 2, or 
24 h in an alkaline elution assay and analysed for DNA strand breaks. The induced 
electric field calculated from the geometry of the exposure system reported by Dacha et 
al. ( 1 993) was 0-004, 0-4, 0-40, 0-80, or 0-200 Il Vim, respectively. No increase in strand 
breaks was observed in cultures exposed to the MF as compared to controls. 
An overall summary of all the studies reviewed above shows a mix of positive and 
negative findings with respect to the possible effects of ELF EMFs on the genetic 
apparatus . On balance, however, it is the current author' s  opinion that the weight of 
evidence at this stage would favour the view that some genetic effects can occur under 
certain circumstances, the nature of which remains to be clarified. 
Chapter Two: Review of Literature. 46 
2.2 Sister Chromatid Exchange (SCE) 
The SCE technique is one of the most widely used specialized techniques that has been 
applied to investigate clastogenicity. It is a highly sensitive technique that can be used 
to determine the levels of genetic damage and was applied in the current study in two 
complete rounds of experiments. It is therefore important to review the SCE technique 
and its utilization in detail . 
2.2.1 Introduction 
A sister chromatid exchange (SCE) may be defined as a reciprocal exchange between 
sister chromatids of the same replicating chromosome. This interchange is the product 
of DNA breakage and rejoining at apparently homologous loci and is evidenced by 
differences in chromatin staining intensity in metaphase chromosome preparations 
(Figure 2.3). 
Figure 2.3: A c-metaphase chromosome spread of a 
dividing peripheral blood lymphocyte that has undergone 
two rounds of DNA replication in the presence of BrdU, 
showing 1 0  SCEs (arrows). 
Chapter Two: Review of Literature. 47 
Taylor et al. ( 1 957), who used 3H-thymidine to differentially label the DNA of 
replicating cells and autoradiography to distinguish the silver grain pattern on the two 
sister chromatids, first visualized SCEs. This method was eventually replaced by 
cytomolecular methods for differentiating sister chromatids. Latt ( 1 973) demonstrated 
that when the halogenated thymidine analogue, bromodeoxyuridine (BrdU) was 
incorporated into DNA, it could quench the fluorescence of the fluorochrome Hoechst 
3 3258 .  Perry and Wolff ( 1 974) found that incorporated BrdU also diminished the 
uptake of Giemsa stain into the chromatin. 
DNA replicates semi conservatively and each chromosome acts as if it i s  composed of a 
single DNA duplex. Chromosomes that have replicated in the presence of BrdU for two 
cell cycles each contain one unifilarly substituted and one bifil arly substituted 
chromatid (Figure 2.4) and one chromatid will stain more intensely than the other in 
second-generation metaphase cells. If cells are allowed three consecutive generations in 
the presence of Br dU, only one out of four chromatids will stain more intensely. 
I f  it is agreed that these exchanges arise by a breakage and reumon between 
corresponding sites of a duplex DNA in each chromatid, then some predictions can be 
made about the structure of the chromatid and the nature of recombination. The SCE 
might take place after the first round of replication in BrdU or after the second round. If  
cells are treated with colchicine, chromosome segregation after the first round is 
prevented and both pairs of sister chromatids are retained in the same cell . When an 
exchange occurs after the second round of replication it will affect only one of the two 
original sister chromatids (Figure 2.4). However, if it occurs after the first round, it will 
be perpetuated by replication, so that it is  seen in both pairs in tetraploid cells (Figure 
2 . 5). S ince there are twice as many chromosomes in the second round, there should be 
twice as many single exchanges as twin exchanges. 
Unlabelled DNA strand 
• • •  • • •  BrdU-Iabelled DNA strand 
-+ 
DNA double helix 1 st. round rep. in BrdU 
Unifilar substitution 
~ 
Seg. 
at 
Ana. 
-+ 
-+ 
2nd. 
round 
rep. m 
BrdU 
Bifilar substitution 
No exchange D 
_
_
_
_
_
 
.... � a 
r 
k 
L • 
• 
• 
• g • 
• 
h • 
Exchange t 
• 
D 
a 
r 
k 
Figure 2.4: Showing how SCE staining method produces differentially stained chromatids by the incorporation ofBrdU. 
L 
g 
h 
t 
D 
a 
r 
k 
L 
g 
h 
t. 
(") 
t:r � 
'9. Cl> .... 
..., 
� 
� <: 
� . 
0 -, 
t: ,.;-Cl> .... 
� Cl> 
� 00 
Chapter Two: Review of Literature. 49 
This assumes that the frequency of exchange is the same in both division cycles. This 
cannot be accepted a priori, as bifilarly substituted DNA might be more active in 
exchange than unifilarly substituted DNA. However, a study by Mazrimas and Stetka 
( 1 978) indicates that there is no difference in the frequency of SCE between the two 
cycles. Attempts to measure the ratio between single and twin exchanges with 
autoradiography were not successful but with the BrdU technique a ratio of very close 
to 2: 1 has been measured (Wolff and Perry, 1 975).  This suggests that a single unineme 
duplex of DNA constitute the only unit that can be exchanged between chromatids. Any 
exchange of a half strand of DNA would generate different patterns (Kihlman, 1 975;  
Shafer et al. 1 976 and Taylor, 1 958). If this were so, after the first round of replication, 
totally substituted and totally unsubstituted DNA could be produced (Figure 2 .6) . 
SCEs have been found in all organisms in which they have been sought: in a variety of 
mammals, including humans (Khalil and Qassem 1 99 1 ;  Bender et al. 1 992a; Atalay et 
al. 2000), in birds (Bloom, 1 978; Bloom and Hsu, 1 975), in fishes (Kligerman, 1 979; 
Kligerman and Bloom, 1 976; Kligerman et al. 1 975), in insects (Gatti et al. 1 979), in 
plants (Schubert et al. 1 980) and in marine worms (Pesch and Pesch, 1 979). Most SCE 
analyses have been performed on cultured cells, but in vivo studies have been performed 
on mammalian bone marrow, spleen, regenerating liver, and spermatogonia (Kanda, 
1 982). In principle, SCEs can be observed in any cell that has completed two replication 
cycles in the presence ofBrdU. The biological significance of SCEs is unclear, since the 
genetic makeup of a cell is not altered if a SCE takes place at identical sites on the sister 
chromatids. However, if the breakpoints in the two chromatids are different, the 
resultant unequal (ectopic) SCE leads to a duplication of the intervening segment on 
one chromatid and its deletion from the other chromatid. For instance, the variation of 
the heterochromatic segment on the Y chromosome obviously has come about through 
unequal SCE (Eeva and Millard, 1 992). 
D = Dark, L = Light, D **  = Twin exchange, D* = Single exchange 
--- Unlabelled DNA strand 
.. . . . . . . . . . . .  BrdU-Iabelled DNA Strand 
-+ 
DNA double 
helix 1 st. round rep. 
in BrdU 
I 
• 
• 
• 
-+ 
I 
• 
• 
• 
• 
1'"'1 
'-J 
Exchange 
I 
• 
/. = 
! e 
g. 
a 
t 
A 
n 
a 
p I 
h • • 
a • 
s 
� I . 2"", 
round 
rep. in 
BrdU 
I 
• 
• 
• 
• 
I 
• 
• 
• 
D 
No 
Exchange L 
D 
D 
L 
Exchange 
- D 
L 
D 
I 
• 
• 
• 
• 
I 
• 
• 
• 
• 
I 
• 
• 
• 
I 
h 
f'---I 
Figure 2.5: Showing "single" and "twin" sister chromatid exchanges. Sister chromatid exchange might take place after the first 
round of replication in BrdU or after the second round. If an exchange occurs after the first round, it will be perpetuated in both 
pairs of cells, such exchanges are called twin exchanges (D**).When an exchange occurs after the second round of replication, it 
will affect only one of the two original sister chromatids, these are called single exchanges (D*). 
L 
D** 
L 
L 
D** 
L 
D* 
L 
() ::r 
{l ..... (1) ... 
� 
� 
� < 
;. 
� 
0 ...., 
t"'" ;:-.' (1) ... � 
� 
VI o 
Chapter Two: Review of Literature. 
-- + 
(a) 
-� + 
1 1 
Unlabelled DNA strand 
BrdU-Iabelled DNA strand 
t 
All 
dark 
Dark 
Normal 
dark 
5 1  
Figure 2.6: Showing the single stranded (unineme) nature of chromosomes, i .e. ,  a 
single unineme duplex of DNA constitutes the only unit that can be exchanged 
between chromatids. Any exchange of a half strand of DNA would generate different 
patterns (a or b). If this were so, after the flrst round of replication, totally substituted 
(normal dark) and totally un substituted ( light) DNA could be produced. It is 
important to know that this pattern has not been observed after the flrst round of 
replication in BrdU. 
2.2.2 Genetic Basis of SCE 
If the present study shows elevated SCE frequencies as a consequence of exposure to 
ELF EMFs, then it is imperative that one should have a sound understanding of how 
SCEs are thought to be generated, and how EMFs might exert their effect.  Therefore, 
the following section covers the various models that have been advanced to explain how 
SCEs arise at the molecular level. 
Chapter Two: Review of Literature. 52 
The measurement of SCE, first described by Taylor ( 1 958) using autoradiography, has 
become much more accurate since the introduction of dye-photometric techniques (Latt, 
1 973) and in particular, the Fluorescence-Plus-Giemsa (FPG) technique of Wolff and 
Perry ( 1 974). Once it became possible to rapidly assess SCE frequencies with this new 
technique by substitution of 5-bromodeoxyuridine (BrdU) in thymidine sites in DNA, 
the SCE assay has become extensively used as a sensitive test for agents with mutagenic 
or carcinogenic potential (Chaganti et al. 1 974; Dutrillaux et al. 1 974; German et al. 
1 965, 1 974; Kato, 1 974a; Latt, 1 974a, b; Perry and Evans, 1 975;  Perry and Wolff, 
1 974). 
In humans, the most common system for ascertaining the possible mutagenic or 
carcinogenic potential of substances, either chemical or physical , is  based on 
observations of peripheral blood lymphocytes. Human blood cultures can be treated 
with a particular agent and lymphocytes can be grown in the presence of BrdU. The 
number of SCEs is then scored. An increase in SCE rate is widely considered to be an 
indication of the mutagenic and carcinogenic potential of an agent (Perry and Evans, 
1 975 ;  Nichols et al. 1 978;  Wolff, 1 979; Carrano et al. 1 979; Husum et al. 1 98 1 ;  
Sandberg, 1 982;  Popescu et al. 1 977; Popescu and Dipaolo, 1 982; Kram, 1 982; Wolff, 
1 983;  Sandberg et al. 1 984; Latt et al. 1 984; Perera et al. 1 992). 
Whether SCEs are actually associated with or causally linked to mutagenic or 
carcinogenic alterations is still unclear. To resolve this issue, an understanding of SCE 
mechanisms at the molecular level is necessary. Taylor s ( 1 958) early SCE studies 
suggested two major assumptions about eukaryotic chromosome structure which are 
fundamental to describing the model: i) that eukaryotic chromosomes have a unineme 
organization with one DNA duplex molecule in each chromatid, and ii) that the 
rejoining of DNA strands is restricted by the structural polarity of each duplex strand. 
While only autoradiographic methods were available, these conclusions remained in 
doubt primarily because the observation of small isolabeling segments suggested the 
possibility of a bineme or polyneme organization (Gatti et al. 1 974) . These issues have 
been clarified by the new high resolution BrdU/staining methods which show that 
isolabeling is an artifact of grain scatter (Wolff and Perry, 1 974; Kato, 1 974c) and that 
an equal exchange of chromatid material occurs even with an extremely high SCE 
frequency. Other studies also confirm structural polarity (Wolff and Perry, 1 975; Tice et 
Chapter Two: Review of Literature. 53 
al. 1 975) and establish that polarity extends through the centromere (Lin and Davidson, 
1 974). Therefore, chromosome organization may be simply described as a single DNA 
duplex in the G ]  stage before replication, and as a pair of daughter duplexes in the G2 
and metaphase stages after replication. A unineme organization would, of course, make 
cells highly vulnerable to genetic damage particularly in the G] stage. 
Formation of SCE is tightly coupled to DNA synthesis; i .e . ,  cells need to pass through 
S-phase before SCE induction can be observed (Wolff et al. 1 974) . Kato ( I 974a) 
observed maximum SCE induction during the DNA synthetic period while DNA 
synthetic activity of Chinese hamster cells was determined autoradiographically in 
BrdU-substituted duplicate cultures by labeling the cells with 3H-thymidine during the 
period of visible illumination. DNA synthetic activity was expressed as the % labeled 
metaphases. Observations were also made in synchronized cells that maximum 
induction occurs at the start of S-phase and decreases progressively throughout the rest 
of S-phase (Latt and Loveday, 1 978). 
The phenomenon of SCEs is now widely accepted as intrachromosomal exchanges of 
DNA replication products, presumably at homologous sites on each chromatid and 
involve DNA breakage and reunion (Latt et al. 1 98 1 ). Although the precise nature and 
mechanism(s) involved in the formation of SCE are not clear, SCE analysis has come 
into use as a most sensitive means of detecting DNA damage (Perry and Evans, 1 975;  
Abe and Sasaki, 1 977, 1 982; Wol ff and Carrano, 1 979; Tucker et al. 1 986) .  
Nevertheless, i t  is widely accepted that in a controlled experiment, any increase in the 
frequency of SCE is interpreted as evidence of clastogenic activity. 
Although SCEs are readily observed, the molecular mechanism(s) responsible for the 
exchange are not fully known. Several mechanisms have been proposed to explain SCE 
formation, which are consistent with uninemic chromosome structure . These 
mechanisms belong to two basic groups .  One group is assumes that the replicating point 
is the site of SCE formation (Kato, 1 974a; Shafer, 1 977; Schvartzman and Gutierrez, 
1 980; Ishii and Bender, 1 980; Kato, 1 9 80). The other group assumes that the SCE may 
be a post-replicational event closely linked to S-phase (Kato, 1 977; Schvartzman et al. 
1 979; Painter, 1 980; Maria et al. 1 989). 
Chapter Two: Review of Literature. 54 
In the former category, Shafer ( 1 977) proposed a model in which SCE results from 
completion of DNA replication at the site of an unrepaired DNA crosslink. The model 
was subsequently generalized to apply to other lesions (Shafer, 1 982) and he discussed 
the possible mechanism of the alternative replication bypass model . There are different 
alternative replication bypass mechanisms, all of which are based on the hypothesis that 
different SCE mechanisms may be required for different DNA lesion states and 
conditions, the principle being that SCEs occur as ongoing replication processes that 
encounter intact or partially repaired DNA lesions . Kato ( 1 974a) postulated that in 
BrdU-substituted chromosomes, fluorescent light induces the formation of SCEs but 
only if irradiation coincides with the time of DNA replication. The replication detour 
model of Ishii and Bender, ( 1 980), based on Kato s work (Kato, 1 974a), suggests that 
SCE occurs at the replicating fork of DNA. 
Schvartzman and Gutierrez ( 1 980) studied the effectiveness of a given dosage of visible 
light in inducing increased yields of SCE in Allium cepa L .  meristems. Cells were 
grown for 1 cycle time in the presence of BrdU and then irradiated at different times 
throughout the second cell cycle. The effectiveness of this treatment in provoking the 
formation of SCE increases the closer the irradiation time is to the beginning of the S 
phase, and then decreases rapidly as cells progress through the S period. The largest 
increase in SCE is obtained when irradiation coincides with early S phase. SCE may 
arise at the time of DNA replication due to the presence of unrepaired lesions. Since 
repair appears to be a time-dependent process, the shorter the interval between damage 
induction and DNA replication, the greater the number of lesions that remain un­
repaired, and as a consequence, the higher the effectiveness of the damaging treatment 
in provoking the formation of SCE. 
In contrast, homologous displacement involving topoisomerase II alone provides a 
mechanism for the strand switching required in the models of Kato ( 1 977) and Cleaver 
( 1 98 1 )  in which SCEs occur between replicated double strands. Pommier et al. ( 1 985) 
suggested that SCE results from exchange of topoisomerase II subunits. Homologous 
displacement , an alternative mechanism, is proposed in which strand switching occurs 
during removal of parental helical turns by topoisomerases. 
Chapter Two: Review of Literature. 55  
Painter s ( 1 980) SCE model i s  based on the idea that double-strand breaks are generated 
at junctions between a completely duplicated replicon cluster. Most models generally 
provide a rationale for strand switches relative to the replication fork, but vary in exact 
details.  
2.2.3 SCE Models 
If low frequency EMFs can affect SCE frequency, then it is essential to understand the 
current models advanced to explain how SCEs arise. Only by examining the different 
models in detail at the molecular level, is it possible to interpret ELF EMF effects 
should they occur. The following sections, which examine SCE formation are rather 
long for a Literature Review, but inclusion of these models, and criticisms thereof, was 
thought necessary by the author towards providing a meaningful mechanism if an effect 
is observed. 
SCE model of Kalo 
Ben-Hur and Elkind ( 1 972) reported that fluorescent light (FL) induces strand breaks 
only in bromodeoxyuridine (BrdU)-substituted DNA, and that these breaks eventually 
lead to the formation of SCEs (Ikushima and Wolff, 1 974; Kato, 1 974a; Wolff and 
Perry, 1 974). Kato ( 1 977) studied the response of SCEs to Fluorescent light (FL) in 
Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution 
had occurred either in one or three strands. The FL-induced SCE frequency did not 
differ greatly between these two types of chromosomes. However, when they were 
submitted to caffeine treatment, a drastic increase in the frequency was detected in the 
trifilarly-substituted (sic) chromosomes while a significant decrease occurred in the 
unifilarly-substituted (sic) chromosomes. 
Based on these above results, Kato ( 1 977) hypothesized that at least two different 
mechanisms are involved in the induction of SCEs (Figure 2 .7) .  One operates at 
replication forks probably utilizing the machinery of DNA replication, and the other is 
considered to act only in the post-replicational DNA portion. 
Chapter Two: Review of Literature. 56 
To test the above hypothesis, Kato ( 1 977) in his experimental system controlled the 
labeling conditions and obtained chromatids with a known number of BrdU-Iabeled 
DNA strands and thus a known number of break-bearing strands following photolysis 
by FL. The labeling patterns of DNA in the chromosome at the second S phase are 
shown in Figure 2.7 A and B ,  respectively. Chromosome A is referred to as a pulse­
labeled (PL) which has undergone one round of replication in the presence of BrdU 
and followed by a subsequent round in its absence. On the other hand, chromosome B is 
referred to as a continuous-Iabeled (CL),  which has undergone two rounds of 
replication in the presence of BrdU. 
According to the author, the first pathway (Figure 2 .7 :  I, Il) needs only one FL-induced 
single-strand break for its initiation. One of the possibilities may be that a free end of a 
single-strand break could somehow accomplish exchange with the discontinuously 
replicating DNA strand, probably with the aid of the machinery of DNA replication. 
Temporal detachment of histones from the replicating point may facilitate such an 
interaction. Thus, strictly, this pathway would require two breaks for its initiation, one 
being induced by FL and the other occurring spontaneously, both residing in co-parallel 
strands. Therefore, if the exchange site is restricted to the replicating point as shown in 
Figure 2 .7 :  I, Il, A & B, the exchange frequency would be the same for both types of 
chromosomes. 
The second type of pathway (Figure 2 .7 :  III a or b) would become available only when 
normal repair of FL-induced DNA damage is disturbed somehow, and function only in 
the post-replicational DNA regions, probably in a similar fashion to that assumed in 
models of meiosis or gene conversion (Holliday, 1 964; Whitehouse, 1 963). It requires 
the presence of two single-strand breaks at juxtaposition in two sister chromatids, and 
eventual displacement of a free end of a single strand at the break point, thereby 
enabling the interaction of two DNA molecules. If the exchange site were located in the 
duplicated DNA region, the exchange frequency in the CL-chromosomes would be 
higher than in the PL-chromosomes. 
Chapter Two: Review of Literature. 
A 
/----------------� -�--'" �-----------------
,/ J -�,.,  ; �-- --
/ ... �IIII:">---- ---.. --l..../'/' \ 1l - , � I , ,' ..... I '\'''''' ."�". ,_e'-_-_-:..-_-=--=--=--=--=--=--=--=--=-:.:.:: 
III 
--..... -,- .. 
57 
B 
,r-------- ------­
/ ""- ------
.... - <' 
�"------. .. 
Figure 2.7: A and B .  Schematic illustration of the labeling of chromosomes at the 
second post-labeling S phase and possible steps (I, 11 and Ill) of the initiation of the 
formation of s ister chromatid exchange fol lowing introduction of strand breaks by 
photolysis in BrdU-substituted DNA. A and B are tentatively designated as pulse­
labeled (PL) and continuous-labeled (CL) chromosomes, respectively. Thick lines 
indicate BrdU-substituted DNA strands and thin lines unsubstituted strands. 
Discontinuities in thick lines show the sites of photolysis. In IlIa, the site of a break in 
the BrdU-substituted parental strand is shown, for convenience sake, at juxtaposition 
with another break in the co-parallel nascent strand. Encircled regions in step 11 are 
considered to be the site of the exchange initiation, though the exact process remains 
to be elucidated (Kato 1 977). 
The replication bypass model of SCE 
There were only a few important clues to an SCE mechanism before the rapid influx of 
BrdU-incorporation SCE-studies. Taylor ( 1 958) reported that a double-strand exchange 
was involved in SCEs. Wolff et al. ( 1 974) pointed out that lesions induced in DNA had 
to pass through an S phase before they could result in SCEs, and Kato ( 1 974a) likewise 
noted that SCE induction was maximized during the period of DNA synthesis.  
Considering the above findings, the replication bypass model for SCE was originally 
proposed in 1 975 to explain DNA crosslink-induced SCEs, since it appeared that i) 
crosslinking agents were the most potent inducers of SCEs (Perry and Evans, 1 975;  
Chapter Two: Review of Literature. 58  
Latt, 1 974a); ii) Crosslinks would inhibit or prevent the normal separation of DNA 
strands as required for bidirectional replication (Geiduschek, 1 96 1 ) ; and iii) that 
crosslink repair required an undamaged parallel DNA duplex in order to complete a 
re combinational exchange (Cole, 1 973 and 1 974) .  
On the basis of the above clues, Shafer ( 1 977) proposed a model in which SCEs result 
from completion of DNA replication at the site of an unrepaired DNA crosslink. The 
model was subsequently generalized to apply to other lesions (Figure 2 .8)  (Shafer, 
1 982), and he discussed the possible mechanism of the alternative replication bypass 
model. The most important characteristic of this proposed model was that SCEs could 
result from a series of sequential events, which might occur as bidirectional replication 
encountered a crosslink. These events may be summarized as follows: a) the advance of 
a leading strand of nascent DNA toward a crosslink site would create distortional stress 
near the crosslink resulting in a parental-strand break or incision; b) the leading nascent 
strands would approach the crosslink along opposite parental strands; c) two-resultant 
breaks or incisions would occur contralaterally on opposite parental strands above and 
below the crosslink (Figure 2.9, D).  Due to convergent unwinding processes, the free 
ends produced in the first stage become terminally aligned irrespective of their opposite 
polarity. Completion of an SCE could then be accomplished by either of two rejoining 
processes-terminal ligation of the free ends via nascent Okazaki pieces or heteroduplex 
complementation by overlapping of the free ends (Figure 2.9 & 2 . 1 0). 
This bypass mechanism would allow replication to continue past the crosslink, leaving 
it intact, but would also result in a switching of parental strands and their attached 
incomplete nascent strands above and below the crosslink site, producing an apparent 
exchange between sister chromatids. 
Evidence and criticism of Shafer s replication bypass model. 
The replication bypass model for SCE proposed by Shafer is  examined in detail by 
Stetka ( 1 979). Although it was acknowledged that alternate replication bypass 
mechanisms for single-strand lesions were suggested, this evaluation criticized the 
model for its limitations solely as a crosslink bypass mechanism and, on this basis, 
found it inadequate for explaining SCEs induced by other lesions. Secondly, an 
assumption was made, without justification, that the model predicted that crosslinks 
Chapter Two: Review of Literature. 59 
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c> z >- 0 
< 0 ...J Z 
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z 
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..-. u (/') 
� i t ! � z c> 0 z u :  � u : : 0 !;S  = :  e l  :z: : �  'u : 41 (/') in � : �  : .,  : !I  
. ..  
: .. t: '" UJ 0.- ... : .. . ... E �  LU ., :.! g :J.!  S.! ., i; ...J ::E E a  ., : u ,;:: � . ... ., : u >- en- " : " �:i Q.:� N fO : C  .... : e ;;:� :z: E :� 0· 0 E :  W w ."!! ; -g E :  � : c  :0 =  i ... : ...:- "0 - "  ,,- ., "0 - .. 
V") c: 
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V') " E � c c: UJ .!:! :! 0 "" .. tJ ... -' c .. :> .. := tV  "Q � .. " cD  " 
.. <0: '" '" � � z .. -' .. 
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< ... c: � 0 .. c c: 0 co .. ::E -= '0 
..-. 
.. 
.. 
.. "" 0 '" .. " 
-;;; � � .. 
:; 
; � ., c; E ... Z tU '" ... .. U ...J co cc VI ..... :x: U 
Figure 2.8: Multiple lesion pathways leading to a small number of SCE-inducing lesion 
states. Some lesions such as crosslinks and strand breaks may cause SCE directly, 
though the latter lesion type has only a short time span. Most other lesions are 
converted by a variety of repair processes to a similar strand incision stage and 
subsequent repair intermediate stages that may induce SCE by a single or several SCE 
mechanisms. Prior to completion of repair any one or more of these open-strand stages 
may induce SCEs. Alternatively, SCE induction with such lesions may be caused if 
replication encounters a lesion in the process of endonuclease binding (Shafer, 1 982). 
Chapter Two: Review of Literature. 60 
A 8 C D E 
" 
" 
" 
:' 
" 
- ' 
: 
� 
� 
: 
� 
� 
::. 
:: 
• :: ::. : 
.. :; 0- ; � , 
, :: -:: --::. -
:: 
:: 
::: :: 
:: 
: --
: 
::: 
0 d 0 b 0 c b . b 
:: 
C-l :: 
et � � � ci I) cl :: I:i 0" : C C 
:: --
::: 
::: 
:: . 
:: : 
::: 
" 
:: 
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, :: 
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::: 
" 
': 
::: 
::: 
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Il 
': ::: � ::: 
Figure 2.9 of Shafer, 1977: (For caption, see next page). 
Chapter Two: Review of Literature. 61 
Figure 2.9 from Shafer, 1977: Replication crosslink bypass model of the SCE 
mechanism. A) The interphase G1 chromosome is depicted as a single DNA duplex 
with structural polarity . Based on the BrdU/ SCE staining methods, the shaded 
strand b-b' indicates the distinctly staining thymidine-retaining strand and the 
unshaded strand a-a' indicates the BrdU substituted strand. An interstrand crosslink 
is indicated at C-L. The crosslink bonding angle is based specially on the covalent 
l inkages which occur between alternate bases on their 5' side with agents such as 
mitomycin-C, nitrogen mustard, and psoralen, plus l ight. For graphic simplicity 
only two replicons and their origins 0 and 0' are represented. B) Bidirectional 
replication is initiated and the nascent strands d and c' which advance towards the 
crosslink are on opposite parental strands b and a'. C) As the advancing strands d 
and c' proceed along the growing fork, the complementary regions on the opposite 
parental strands are back-filled with Okazaki pieces represented by the nascent 
strands c and d'. When the advancing strands d and c' approach the crosslink, they 
will come into parallel competition with the parental strands a and b' at the stress 
points (arrows) .  At this stage normal replication should be prevented by the 
crosslink. The proposed SCE mechanism would occur by a two step 
displacement/rejoining event which may involve unique incision and/or l igation 
enzyme systems.  The stress points (arrows) indicate where the displacement will 
occur when the advancing strands d and c' reach the crosslink. The SCE 
mechanism is completed in D. After the chain breaks are affected, the advancing 
strands d and c' terminate replication at the crosslink; and new Okazaki pieces c 
and d' are initiated which backfill from the break point. Visualized three­
dimensionally, the break sites are in contralateral proximity such that they may line 
up in correct polarity across the break via the new Okazaki pieces. L igation of the 
breaks may occur by chemical processes similar to the normal means for linking 
completed Okazaki pieces during replication. E) After replication the daughter 
duplexes form the two chromatids . Above and below the crosslink the shaded 
strand segments b and b' containing Thymidine are now separated into different 
chromatids and results in the SCE staining effect. This SCE mechanism occurs 
during replication and does not involve recombination as the incomplete nascent 
complementary strands remain bound to their original parental strand during the 
exchange.  The effect observed at metaphase appears equivalent to a post­
replication double strand exchange. The crosslink remaining in one chromatid may , now be removed by crosslink repair. These diagrams only depict a hypothetically 
perfect SCE mechanism in which no bases are lost in the exchange (see Fig. 2 . 1 0) .  
Enzymatic incision may not, in fact, be possible nearer than two bases from the 
crosslink (Shafer, 1977). 
Chapter Two: Review of Literature. 
A 
,­
I 
J 
1 
I 
1 
B or c 
-\ 
I \ \ I 
\ I 
\ : \-(> I 
I 
I 
I 
I¥. 
� 
... 
D 
Figure 2 .10:  Alternate rejoining processes for the replication bypass SCE 
mechanism. A represents the structural detail of stage D in Figure 2.9. In 
this hypothetically perfect process, opposite parental strands may be 
rejoined by terminal l igation (at open arrow) via the formation of nascent 
Okazaki pieces. The shaded bases represent the normal thymidine bases 
retained by semi conservative replication. These bases allow the chromatid 
portions carrying them to stain darkly at metaphase (B). C represents the 
structural details of rejoining by aberrant complementation (between open 
arrows). Overlapping parental strands may rejoin in regions of similar or 
repetitive sequences producing heteroduplex segments. While this process 
may occur quite readily, the resulting metaphase structure (D) would lead to 
base loss andlor mutational error in the daughter cells (Shafer, 1 977). 
62 
could not be repaired prior to a second cell cycle. Stetka concluded that, according to 
Shafer s model, the retention of crosslinks through two S phases would cancel the 
observation of twin SCEs in induced tetraploid cells. Finally, secondary speculations 
about Bloom syndrome (BS) and Fanconi anemia (FA) were evaluated as if they could 
be considered experimental tests of the model. Stetka also suggested that Shafer s 
model was unsupported because one such hypothesis stimulated by the model, that high 
SCEs in BS may result from deficient crosslink repair, was in conflict with evidence 
that BS cells had nonnal repair capacity. 
Chapter Two: Review of Literature. 63 
Involvement of replication and repair in SCE. 
SCE studies of other types have also confinned a close relation between SCE induction 
and DNA replication. By comparing SCE effects to chromosome replication banding 
patterns,  Latt et al. ( 1 977) demonstrated that the location of PUVA-induced 
[8Methoxypsoralen (8MOP) plus near UV=PUVA] SCEs correlated with those regions 
undergoing DNA replication at the time of induction. Hoo and Parslow ( 1 979) reported 
similar replication-related differences in the location of SCEs using a method allowing 
simultaneous observation of SCEs and chromosome replication bands. Kato s ( 1 980) 
study clearly pinpointed a precise relation of SCE induction to the replication fork, 
where cells were grown in fluorodeoxyuridine (FudR) to preferentially accumulate 
replication forks and then SCE induction was evaluated with blue fluorescent light. 
Using equipotent light treatments, this study demonstrated that the relative frequency of 
SCEs increased as the number of growing points increased and, thus, implied that the 
site of exchange was confined to the replication forks. 
The post replication SCE model of Painter 
This model is based on the idea that double-strand breaks occur frequently at the 
junctions of adjacent replicon clusters during their replication. Painter ( 1 980) 
hypothesized that the specific supercoiled subunits of the chromosome somehow allow 
DNA at a junction to be susceptible to double-strand break fonnation during replication 
of adjoining clusters. 
DNA damage in replicon clusters that are at various stages of completion may present a 
condition where a delay in the progression of the replication fork in a replicating cluster 
may extend the time when replicated clusters adjoin unreplicated ones. Painter ( 1 980) 
suggests that DNA double strand breakage at these junctions (mediated perhaps by 
topoisomerase-II) and subsequent joining of the daughter strands of the replicated 
replicon c luster to the strands of the unreplicated cluster may be responsible for the 
fonnation of an SCE (Figure 2. 1 1 ) .  
Chapter Two: Review of Literature. 
Figure 2 .1 1 :  Model for possible effects of DNA-damaging agents on cluster 
replication and segregation. An array of these clusters is depicted; the one on the 
left is completely repl icated and the one on the right is unreplicated. In the 
center, damage has blocked fork progression so that the cluster is only partially 
replicated. Lines represent the super-supercoiled Watson-Crick DNA double 
helices. Junctions where RNA and/or proteins maintain the separate domains of 
clusters are indicated by circles. In the lower half of the diagram, segregation in 
the completely replicated cluster is accompanied by a double strand-break at the 
junction between this cluster and the partially replicated cluster (Painter, 1 980). 
64 
If this happens, both a daughter and parental strand of each polarity would be available 
for ligation with the DNA of the unreplicated adjacent cluster (Figure 2. 1 2), and there 
would be a definite probability that the daughter strands, rather than the parental ones, 
will join with the unreplicated strands. When replication of the other strand finishes, the 
normal ligation process at the junction will complete the exchange. 
This exchange requires only one double-strand break in the parental strands, which is 
consistent with data showing that the production of SeEs is a linear function of dose 
(Carrano et at. , 1 978; Perry and Evans, 1 975). That is, SCE is a product of a single hit, 
even though two double-stranded molecules participate in each exchange. Agents that 
block chain elongation will often cause DNA in clusters to remain partially replicated 
for long times. 
Chapter Two: Review of Literature. 
Figure 2.12:  Double-strand recombination at the junction between replicon 
clusters. The exchange shown is between the newly formed strands of the 
replicated cluster and the parental strands of the unreplicated cluster. The 
system responsible for joining DNA at the junction will later complete the SCE 
(Painter, 1 980). 
65 
This hypothesis may also explain the high number of spontaneous SCEs observed in 
cells from individuals with Bloom s syndrome (Chaganti et al. , 1 974). Hand and 
German ( 1 975) have shown that the average rate of DNA-fork displacement is about 
30% slower in these cells than in normal cells. An examination of their data shows that 
this average decrease is due to a large fraction of replicons whose fork-displacement 
rate is about two-thirds that of normal cells and a smaller fraction whose average fork­
displacement rate is the same as in normal cells. In Bloom s syndrome patient s cells, a 
partially defective polymerase or other component of DNA replication may exist in a 
fraction of the clusters that are in operation at any one time; the rate of chain elongation 
in these clusters would be slower than in nearby c lusters containing normal 
components. The slowly replicating clusters would not finish their duplication at the 
same time as adjacent clusters, resulting in the same effects seen in mutagen-damaged 
cells. 
Chapter Two: Review of Literature. 66 
The proposed hypothesis of Painter does not implicate any repair process in the 
formation of SCEs; the only role for repair is to reduce the number of lesions before the 
damaged DNA replicates and thereby to lower the frequency of SCEs. ELF EMFs 
might be one of the agents which blocks the chain elongation which cause the DNA in 
clusters to remain partially replicated for a long time and as a result double strand 
breaks take place in replicating cluster junctions. Then both newly synthesized strands 
may join with the parental strands and when synthesis resumes from these free ends of 
the two remaining parental strands, SCE is completed. 
Cleaver s SCE model 
Baseline SCE increases as a function of average replicon size in a variety of human and 
Chinese hamster cell lines (Wolff et al. 1 975 ,  1 977 and Takehisa and Wolff, 1 978) .  
This observation i s  the basis for this model in which SCEs are generated by errors in the 
unraveling of daughter double helices by topoisomerases. According to Cleaver ( 1 98 1 ), 
these errors cause exchanges behind the replication fork, not at the fork as several 
current models assume. This model also provides an explanation for SCEs induced 
when residual DNA damage that was replicated interferes with the normal processes of 
unraveling the daughter strands. 
In Cleaver s model the newly replicated DNA (Figure 2. 1 3 , C) is considered to be 
initially in an entwined form because of the Watson-Crick helical structure of the 
parental DNA strands before replication, and because of the multiple orders of coiling 
involved in the packaging of eukaryotic DNA (Cook and Brazell, 1 976). The two 
entwined replicated DNA double helices may be unwound and unknotted by the action 
of enzymes, such as topoisomerases I and 11 to produce separate daughter helices (Baldi 
et al. 1 980; Hsieh and Brutlag, 1 980; Kreuzer and Cozzarelli, 1 980) .  Cells with small 
replicons would presumably have less demand for topoisomerases to unravel the 
replicated DNA than would cells with large replicons. Therefore, there would be less 
opportunity for errors to generate SCE in cells that replicate DNA in small units as 
compared to large. This principle is consistent with the report of Blumenthal et al. 
( 1 974) that the DNA replicon size in Drosophila cells is very small and which may lead 
to low frequency baseline SCE (Gatti et al. 1 979) .  Similarly, unrepaired damage 
remaining in double stranded, replicated daughter DNA could interfere with the 
Chapter Two: Review of Literature. 67 
recognition and function of topoisomerases such that homologous chromatids are 
rejoined incorrectly and generate SCEs. 
Origin 
A 1�:_: ______ �t_: ________ .I ________ � ________ �j
B 
C 
D 
ACCURATE 
, Initiation 
, -
;::===�: "'-
- -
ONA DoualE � UNRAVElUNG 
STRAND BREAKS  - {TOPO ISOMERASE III �-- �NACCURAn: 
(Damaged DNA or 
o.fectiy. TOPO 11) �,...:-�------- ---------,-- ---/',-- --.... ":;<,..C-------:;y:"" ----'--
�."'-'---- - ------ - ... � �-----"A -- -"A"'-- --'f" 
E F SISTER CHROMATIC EXCHANGES 
Figure 2.13:  Showing the possible mechanism of SCE formation. Replication past 
damaged sites is presumed to leave damage in parental strands in intact daughter 
double helices without gaps or other abnormal structures. The normal entwined 
configuration of newly replicated DNA is postulated and unravelled by double strand 
breakage and reunion through the action ofDNA-topoisomerases. Errors introduced by 
DNA damage or a low frequency of spontaneous errors will cause faulty recognition, 
crossing and rejoining by topoisomerases and ultimately cause SCEs. Large origin-to­
origin distances, i .e. ,  large replicons, will require more action of topoisomerases for 
unravelling and consequently a higher possibility for error. The length of DNA is 
indicated as having multiple origins (A & B). An individual replicon is in an entwined 
configuration soon after initiation (C), and subsequent action of topoisomerases allows 
accurate or inaccurate unravelling of the strand (D). SCEs are produced by inaccurate 
rejoining (F) (Cleaver, 1 98 1 ). 
According to this model for SCE formation (Figure 2 . 1 3) ,  excision-defective 
Xeroderma pigmentosum (XP) cells show high UV-induced SCE formation because of 
the high frequency of damage sites remaining in replicated DNA (de Weerd-Kastelein 
et al. 1 977). This model can also be used to explain the high baseline SCE frequency in 
Bloom s syndrome (Chaganti et al. 1 974) in terms of a possible defect in the enzymes 
involved in unravelling newly replicated DNA. 
Chapter Two: Review of Literature. 68 
Topoisomerase 11 subunit exchange model of SCE 
That SCEs occur at or near the replication fork is generally accepted, based on the 
observation that in synchronized cells the ability of a DNA-damaging agent to induce 
SCEs decreases with the amount of the genome remaining to be replicated (Latt and 
Loveday, 1 978) .  Schvartzman and Gutierrez ( 1 980) also suggested that the SCEs arise 
at the time of DNA replication due to the presence of unrepaired lesions. They argued 
that the DNA repair system is a time-dependent process, the shorter the interval 
between damage induction and DNA replication, the greater the number of lesions that 
remain unrepaired, and as a consequence, the higher the effectiveness of the damaging 
treatment in provoking the formation of SCEs. The exact locations at which the strand 
switches occur relative to a replication fork differ among other proposed models. In the 
topoisomerase II subunit exchange model of Pommier et al. ( 1 985) all 4 strand switches 
occur in a concerted action. The property of topoisomerases of forming DNA covalent 
linkages after DNA incision is a central factor in this model. 
DNA -topoisomerase complexes and homologous displacement 
The Homologous displacement model (Pommier et al. 1 985) proposes that strand 
switching occurs during removal of parental helical turns by topoisomerases. It has been 
shown (Hsiang et al. 1 985 and Chen et al. 1 984) that both topoisomerase I and II form 
covalent linkages to DNA in the process of DNA incision (Figure 2 . 1 4, A and B). These 
covalent linkages are broken in the process of topoisomerase-mediated ligation. A 
single-strand incision is created by topoisomerase I and the enzyme covalently binds to 
the 3 '  end of the incision. The strand on the other side of the incision with free 5 '  may 
rotate around the intact complemetary strand prior to ligation. Topoisomerase n, which 
is a dim er, causes double strand incision. On each side of the double-strand incision, the 
5 '  strand is covalently linked to one of the topoisomerase II monomers while the 3 '  
strand remains free. Another intact DNA double-strand duplex can pass through the 
double-strand incision. The covalently-linked complexes between topoisomerases and 
DNA have been called cleavable complexes . 
Chapter Two: Review of Literature. 
A 
8 
3' S' 
3' S' /'�""""�'" ---,""'4' • • --'--------�- i • �/�}----
" ""�: ,/" 
c 3'�-"''''''''\-­S' 
.. �. 
5' 
3' 
5' 
3' 
Figure 2.14: (A) Topoisomerase I cleavable complex; single-strand 
incision with topoisomerase I covalently bound to the 5 '  end. (B) 
Topoisomerase II  cleavable complex; double-strand incision with 
each topoisomerase II monomer covalently bound to a 5 '  end. (C) 
Homologous displacement; a homologous strand has displaced the 
non-covalently bound strand in a topoisomerase I cleavable complex 
(Dillehay et al. 1 989). 
69 
A strand switch could conceivably occur if a nearby homologous strand of DNA not 
involved in a cleavable complex displaced, through complementary binding, a non­
covalentIy-bound strand from a cleavable complex. A topoisomerase I cleavable 
complex is illustrated in (Figure 2 . 14, C). True matching of the displaced and displacing 
strands could result in ligation of the latter to the covalently-bound strand. The authors 
speculate that such a homologous displacement strand-switching mechanism may be 
involved in SCE. Therefore, it is necessary to fulfill two conditions, the means and the 
opportunity, to make homologous displacement occur. First a cleavable complex must 
form at the proper location and then a homologous strand with a true-matching free end 
must come in close proximity. 
Replication detour model of Ishii and Bender 
Ishii and Bender s ( 1980) replication detour model of SCE is shown in Figure 2. 1 5 . In 
this model it has been assumed that replication on the template strand containing a 
lesion has stopped, but that synthesis on the undamaged template strand continues. This 
Chapter Two: Review of Literature. 70 
A B 
,� 'L1 --;/ � 
C D 
, L2SX � ,/ fN2 
E F 
� • ;/ 
Figure 2 . 1 5 :  Replication detour SCE model of Ishii and Bender. Newly 
synthesized DNA strands are represented by the heavier lines. (A) A lesion (square) 
has blocked replication of the upper template strand. Replication of the other 
template strand has progressed farther downstream. N I  marks the site at which a 
nick (incision) occurs in the template strand downstream from the lesion. (B) 
Improper ligation (L 1) has joined the downstream end of the nick in the template 
strand to the end of the newly synthesized strand. (C) N2 marks the site at which a 
nick occurs in the other template strand. (D) The downstream end of this nick has 
ligated (L2) to the end of the blocked newly replicated strand. (E) Replication 
resumes from the new free ends. (F) SCE following replication completion 
(Dillehay et al. 1 989). 
model states that the first step towards the formation of SCE is nicking (incision) of the 
lesion-containing template strand downstream from the lesion (Figure 2 . 1 5 , A). After 
incision, the end of one of the newly replicated strands displaces the template strand 
(the two strands being homologous) on the upstream side of the incision and is then 
ligated to the template strand on the downstream side of the incision (Figure 2 . 1 5 , B) .  
Following this event, the model then requires a second incision to be made in the other 
template strand at the position opposite the point at which synthesis has stopped on the 
lesion-containing strand (Figure 2. 1 5 , C). The blocked strand then displaces its 
homologue on one side of the incision and is ligated to the other side (Figure 2 . 1 5, D).  
Two of the four strand switches required for an SCE accomplished by the above steps. 
Following these steps, there remain two free ends of two original template s DNA 
Chapter Two: Review of Literature. 7 1  
strands at the replication fork. Ishii and Bender postulated that the replication fork could 
reorient (Figure 2. 1 5 ,  E) and synthesis resume from these free ends, accomplishing the 
last two joinings of template to daughter strands to complete the SCE (Figure 2. 1 5 ,  F). 
To prove whether homologous displacement accomplishes any of the steps in this 
model, they first consider the case in which the lesion is blocking synthesis of the new 
strand, which grows in the 5 '  to 3 '  direction. The model assumes that atopoisomerase 1-
cleavable complex has arisen downstream from the replication fork, with the incision in 
the template strand containing the lesion. If replication continued off the other template 
strand, as suggested in the model, then the configuration shown in Figure 2. 1 6, A would 
result when the replication fork reached the topoisomerase I complex. Consequently, if 
homologous displacement and ligation then occurred, the first two steps of the model 
would be accomplished. 
On completion of the first two steps, could homologous displacement involving 
topoisomerase I accomplish the next two steps? The formation of a topoisomerase 1-
cleavable complex exactly opposite the blocked newly replicated strand is shown in 
Figure 2. 1 6, C. Homologous displacement would first require that the newly replicated 
strand come sufficiently close to displace the template strand. Such a displacement 
appears impossible since the strand, which would have to be displaced, is covalently 
linked to the topoisomerase I. However, homologous displacement at a topoisomerase 
II-cleavable complex appears possible. I f  a topoisomerase II-cleavable complex were 
present at the site shown in Figure 2. 1 6, D, the template strand to be displaced would 
not be covalently linked. The necessity for proximity of the homologous strand might 
be achieved if the DNA containing the lesion were being passed through the double­
strand break at the topoisomerase II-cleavable complex. 
The first two steps for the case of blocking the lesion in the other template strand is 
considered in Figure 2. 1 6, B. Homologous displacement at a topoisomerase I-cleavable 
complex could not occur, again because of protein-DNA covalent attachment at the 
strand to be displaced. By the same reasoning used above, homologous displacement at 
a topoisomerase II-cleavable complex would be required for the first two steps and at a 
Chapter Two: Review of Literature. 72 
Figure 2.16:  Strand switching by homologous displacement at blocked replication 
forks. (A) Replication fork near a lesion (square) on the template strand whose 
daughter strand replicates continuously (5 ' to 3 ' ) .  Topoisomerase I (large ope} 
circle) has nicked this strand and is covalently bound (dark circle within the open 
circle) to the 3 '  (downstream) end of the nick. This corresponds to step A in Fig. 
2. 1 5 . Homologous displacement of the strand on the 5' end of the nick by the newly 
synthesized strand of the same polarity would accomplish step B in Fig. 2. 1 5 .  In 
this figure an arrow indicates the movement of the displaced strand. (B) Same 
situation as A except the lesion is on the template strand which is replicated 
discontinuously (3 ' to 5 ') . When topoisomerase I nicks this strand it is bound 
covalently to the 3 '  (upstream) end of the incision, which cannot be displaced by 
the 3 '  end of the newly synthesized strand. (C ) Situation if topoisomerase I 
performs steps A and B in Fig. 2. 1 5  and topoisomerase I nicks the other template 
strand (Step C in Fig. 2. 1 5) .  Since topoisomerase I is bound to the 3 '  end of the 
incision, homologous displacement could not occur. (D) If  topoisomerase II 
performed the second incision (Step C in Fig. 2. 1 5), displacement of the free 3 '  end 
and ligation (Step D in Fig. 2. 1 5) would be possible (Dillehay et al. 1 989). 
topoisomerase I cleavable complex for the next two steps. However, in this case, some 
additional mechanisms would be necessary to bring the blocked free end in to proximity 
with the topoisomerase I cleavable complex. Thus, homologous displacement appears to 
be a plausible mechanism by which SCEs could occur within the Ishii and Bender s 
model; both topisomerase I and II would be active in each SCE and only lesions on the 
continuously-replicated strand would lead to SCEs. 
Chapter Two: Review of Literature. 73  
Criticism of Ishii and Bender s model. 
Ishii and Bender in their model (Figure 2 . 1 5 ,  F) claimed the completion of SCE when 
replication resumes from the new free ends, but it is very clear from the figure that in 
the upper DNA duplex there is a dark-pale-dark region and only dark on the lower one. 
This is quite difficult for the SCE model to explain. 
Dark Pale Dark 
................................... 'f -'-'-'-'-'-'-' ......................................... .. 
D 
• 
Lesion 
..... -. .  -. .  -..  - .. -.. -. .  - .. -. .  -.. -. .  -. .  -. .  -. .  - .. - .. � Dark 
From figure 2.15, F 
Does homologous displacement occur in the Replication Cluster Junction model of 
Painter? 
Painter ( 1 980) proposed that an SCE could occur when a replication fork reaches a 
replicon cluster junction before the oppositely-moving replication fork of the adjacent 
replicon. Although Painter suggested that topoisomerases could be involved, no detailed 
description of the strand switching was given and there is no evidence that 
topoisomerases are located at these sites. However, if SCEs occurred at these sites by 
homologous displacement, both a topoisomerase I and topoisomerase II-mediated strand 
switch would be required. 
Chapter Two: Review of Literature. 74 
Does homologous displacement appear to be a plausible mechanism for the Shafer s 
Converging Replication Forks model? 
In Shafer s ( 1 977) proposed model, an SCE results from completion of DNA replication 
at the site of an unrepaired DNA crosslink. The model was eventually generalized to 
apply to other lesions (Shafer, 1 982) .  The model states that SCEs occur when 
bidirectionally-moving replication forks meet a crosslink that prevents them from 
merging. As in the two formerly discussed models, this model also needs an incision in 
each of the two template strands and ligation of one side of the break to a newly­
synthesized strand. 
The two strand switches involve DNA of the same polarity, so if performed by 
topoisomerases,  then both switches would have to be done by topoisomerase I or both 
by topoisomerase 11. A topoisomease I-cleavable complex formed at the site of one of 
the template incisions needed in the model is shown in (Figure 2. 1 7, A). The structure, 
which results after the first ligation step in the model, is shown in (Figure 2. 1 7, B).  To 
get from the structure in Fig. 2 . 1 7 ,  A to that in F igure 2 . 1 7 ,  B ,  the newly-synthesized 
strand that must do the displacing from the topoisomerase is not homologous to the 
strand to be displaced. Thus, homologous displacement does not appear to be a 
plausible mechanism for the strand switches required in this model. 
Does SCE occur by homologous displacement between replicated strands? 
Cleaver ( 1 98 1 ) proposed that SCE occurred during the removal of parental turns (Figure 
2 . 1 3) like other replication fork model discussed before. However, he suggested that the 
exchange occurred between replicated double strands and resulted from an unknown 
topoisomerase action. Only topoisomerase II would be  expected to be able to remove 
parental turns in replicated DNA as described in Figure 2 . 1 8 .  Kato ( 1 977) also 
suggested a model (Figure 2 .7) in which SCEs occur between replicated strands .  He 
postulated that a free end of a single-strand break in one of the chromatids would pair 
with the complimentary sequence on the sister chromatid and induce a break in the 
displaced strand. Two such nearby events would be required to produce an SCE. How 
homologous displacement could produce these events is shown in Figure 2. 1 8 , A-D. 
Each strand switch would be initiated by a single-strand break in one of the two double 
strands. Homologous displacement could then occur when this double strand was being 
passed through a topoisomerase II-cleavable complex on the opposite strand. The 
Chapter Two: Review of Literature. 75 
structure shown in Figure 2 . 1 8 , C would be the result of two homologous displacements 
at nearby sites. Melting and reannealing could then produce the structure shown in 
Figure 2 . 1 8, D, and the ligation ofthe two remaining strands breaks would complete the 
SCE. 
A 
3' 5' 
5'�3' 
. '---- / 
3'� � 5' 5' 3' 
3' 5' 
5' 
3' 
5' 3' 
Figure 2.17: Strand switching in the model of Shafer. In (A), two replication 
forks have converged at an interstrand crosslink (two linked squares) which 
prevents them from merging. Shafer proposed that a break occurs in a 
template strand on one side of the crosslink, followed by ligation of one end 
of the break to a newly synthesized strand on the opposite side of the 
crosslink. (B) If the process is repeated, DNA synthesis could be completed 
and an SCE would have occurred. In A, the incision is shown as a 
topoisomerase I-cleavable complex . However, since the displacing and 
displaced strands are not homologous, homologous displacement does not 
appear a plausible mechanism for the required strand switching (Dillehay et 
al . 1 989). 
Cleaver postulated that DNA-damaging agents increase SCE because unrepaired 
damage remaining in the replicated DNA stimulates faulty topoisomerase action. For 
the homologous displacement mechanism, the strand break needed in the template 
strand (Figure 2 . 1 8, A) might be generated by repair incision at the site of unrepaired 
damage. However, this mechanism also needed a strand break in the newly replicated 
Chapter Two: Review of Literature. 
A 
d 
l' 
��tl __________ __ 
-.:.- ;  .... 1- ----­\ ... 9 
B 
;z::�-----' !-
c J 
D 
-� 
76 
E 
Figure 2.18:  SCE between replicated strands by two topoisomerase II-mediated 
strand switches .  (A) The first strand switch would be initiated by a single-strand 
break in the upper double strand opposite a topoisomerase II-cleavable complex on 
the lower double strand. This strand switch would result in the configuration on the 
left side of B. The second strand switch would be initiated by a second strand break 
opposite topoisomerase II shown on the right side of B .  The configuration that 
results after the second strand switch is shown in C. Reannealing of the displaced 
strands results in D. Ligation of the two remaining strand breaks would complete the 
SCE. Alternatively, as shown in E, the second strand switch might be initiated by a 
free end at a replication fork, as in Fig. 3 . 1 6, D (Dillehay et al. 1 989). 
(intact) strand for the second strand switch. By exposing cells to light Kato ( 1 977) 
produced breaks in BrdU-substituted DNA. Based on the SCEs induced with different 
substitution patterns, he concluded that not all SCEs could be occurring completely in 
the replicated DNA; for some of the SCEs one or more strand switches would have to 
be initiated at a nearby replication fork. In Figure 2 . 1 8 , E it has been shown how the 
free end required for a displacing strand in the second switch could be supplied at a 
blocked replication fork. The second switch would then be exactly the same process as 
the second switch in the Ishii and Bender model (Figure 2. 1 6, D). 
Chapter Two: Review of Literature. 77 
Thus it appears that this model can reasonably account for the increased induction of 
SCEs by DNA-damaging agents. However, this model is different from that of Ishii and 
Bender, and it is not clear what mechanism would ensure that both strand switches take 
place and complete the SCE. 
2.2.4 SCE Induction 
SCE analysis has been used most extensively to assess the impact of clastogens on 
chromosomes. A wide variety of agents that cause chromosome breaks have also been 
found to induce SCE s. These exchanges are now known to occur as a normal event of 
cell division in mammalian tissues (Tice et al. 1 976; Latt et al. 1 98 1 ;  Schubert and 
Rieger, 1 98 1 ;  Sandberg, 1 982; Tucker et al. 1 986) . SCEs are efficiently induced by 
substances that form covalent adducts to the DNA or that otherwise interfere with DNA 
metabolism or repair (Perry and Evans, 1 975;  Wolff, 1 977;  Carrano and Thompson, 
1 982). The four-strand DNA exchange that results in the SCE occurs during DNA 
replication (Wolff et al. 1 974) and it has been demonstrated that the polarity of DNA is 
maintained in this process (Taylor, 1 958 ;  Wolff and Perry, 1 975). Many of the agents 
initially used to induce SCE are also well known mutagens (Carrano et al. 1978; Perry 
and Evans, 1975); hence, it has been suggested that SCE analysis could be used to assay 
and monitor mutagens and carcinogens. 
Attempts to correlate SCE induction with known DNA repair have failed. de Weerd­
Kastelein et al. ( 1 977) measured the induction of SCEs by ultraviolet light in normal 
and Xeroderma pigmentosum cells and found no systematic correlation between the 
ability of the cell to perform excision repair and the production of SCEs. They also 
observed similar SCE frequencies in both normal and Xeroderma pigmentosum variant 
cells, which presumably have a defect in post replication repair . Thus, although there 
is obviously a role for DNA damage in the production of SCEs, the known mammalian 
DNA repair processes do not seem to be active participants. 
One of the more striking features of SCEs is that their yield is many times lower than 
the number of lesions in DNA; for instance, the number of SCEs is about 1 120,000 the 
number of ultraviolet-induced dimers in the DNA (Reynolds et al. 1979). 
Chapter Two: Review of Literature. 78  
Another feature of SCE is the relatively high frequency found after damage by agents 
which cause DNA lesions that retard the progression of the replication fork during DNA 
replication, and the relatively low frequency induced by agents that cause damage 
resulting in inhibition of the initiation of replicons. In the former category are agents 
such as Mitomycin C (MMC) that cause crosslinks. By their nature, crosslinks must be 
strong blocks to chain elongation. Another example is ultraviolet light, which forms 
dimers that act as either temporary or permanent blocks to fork progression but causes 
very few strand breaks. In contrast, X-radiation, which is the best example of a DNA­
strand-breaking agent, causes either very few or no SCEs (Littlefield et al. 1 979; Wolff 
et al. 1 974). Strand breaks seem to be the most important DNA lesions involved in 
inhibition ofreplicon initiation (Povirk and Painter, 1 976). 
Chemicals capable of alkylating DNA are among the most effective and extensively 
studied agents that induce SCEs. A clear relationship has not yet emerged between the 
specificity with which particular agents react with DNA, or the type of DNA repair 
stimulated by these reactions, and the relative potential with which SCEs are induced 
(Latt, 1 98 1  b). SCE formation can be increased both by alkylating agents that are very 
sensitive to target nucleophilicity and by those that are not. The former, which include 
methylmethanesulfonate (MMS) and dimethylsulfate (DMS), react relatively cleanly 
with DNA, forming primarily N7 guanine or N3 adenine adducts, while the latter, which 
include methylnitrosourea (MNUA) and N-methyl-N -nitro-N-nitrosoguanidine 
(MNNG), react at many different sites, forming among other products, highly 
mutagenic 06 guanine substituents (Lawley, 1 974). 
After three rounds of DNA replication in the presence of BrdU third-division metaphase 
cells can be scored for the frequencies of SCE that occurred during cycles one and two, 
and also for the frequency of SCE that occurred during cycle three. This procedure was 
used to resolve the issue of SCE induction by replication of BrdU-substituted DNA 
templates vs. induction by BrdU incorporation into nascent DNA (Stetka and Spahn, 
1 984) . Third-cycle SCE frequencies in Chinese hamster ovary (CHO) cells are 
dependent upon the amount of BrdU that was present during cycles one and two and are 
not dependent on the BrdU concentration during the third cycle. BrdU serving as a 
template, rather than BrdU being incorporated in the nascent DNA will initiate the SCE 
event. 
Chapter Two: Review of Literature. 79 
Induction of SCEs in human chromosomes is greatly increased by alkylating agents, but 
ionizing radiation is not very effective. A dose-dependent increase in SCEs in CHO 
cells with X-irradiation was reported by Perry and Evans ( 1 975), but only if the 
irradiation was done during the 01 or S phase prior to harvest. Such cells had already 
undergone at least one cycle of BrdU incorporation, and damage produced at sites of 
BrdU incorporation might well have caused the SCE induction. X-irradiated human 
lymphocytes during the preharvest 01  phase induce SCEs (Solomon and Bobrow 1 975, 
Abramovsky et al. 1978) is subject to a similar explanation. On the other hand, ionizing 
radiation breaks chromosomes effectively, but increases SCE only slightly. A dose of 
X-rays that increases chromosome breaks 20-fold only doubles the SCE rate (Kato, 
1 977) .  Cells from patients with the disease Xeroderma pigmentosum are hypersensitive 
to the induction of SCEs by ultraviolet light and alkylating agents (Wolff et al. 1 975, 
1 977). 
In a comparative study by Carrano et al. ( 1 979), CHO cells were treated with DNA­
crosslinking chemicals, MMC and porfiromycin (POR), and their monofunctional 
derivative, decarbamoyl MMC. After exposure, the cells were studied for the induction 
of SCEs and mutations at the hypoxanthine phosphoribosyltransferase and adenine 
phosphoribosyltransferase loci. All three compounds were potent inducers of SCEs, but 
only weakly mutagenic .  All three chemicals by concentration were approximately 
equally effective in inducing SCEs or mutations. The results gained in this study 
indicate that the DNA interstrand crosslink is not the major lesion responsible for the 
induction of SCE or mutation by these compounds. 
The high�st incidence of SCEs has been achieved with bifunctional alkylating agents. 
The most effective SCE inducers include methylmethanesulfonate, MMC, 
dimethylsulfate, ethylmethane-sulfonate, and the mustards (Latt, 1 98 1  a) . Another 
powerful inducer of SCE is ultraviolet light and is well known to produce pyrimidine 
dimers, as well as numerous other alterations in DNA (Setiow and Setlow, 1 972). 
Chapter Two: Review of Literature. 80 
2.2.5 SCE and Disease 
The frequency of spontaneous, and MMC-induced SCE was investigated by Yokota et 
al. ( 1 987) in PBLs of 3 1  untreated patients with cervical cancer, as well as of seven 
untreated patients with myoma uteri and 1 6  healthy women. Factors that might have 
affected SCE frequency were also investigated, including age and smoking, and were 
not found to influence the SCE frequency. However, a family history of cancer 
significantly increased MMC-induced SCE frequency in the cancer group. The MMC­
induced SCE frequency was also higher in the cancer group. Spontaneous and MMC­
induced SCE frequency gradually increased with the progression of cervical cancer. All 
chromosome groups equally contributed to the increased SCE frequency in the cancer 
group. These results suggested that DNA of all chromosomes were damaged in the 
patients with cervical cancer. 
The frequencies of spontaneous and MMC-induced SCEs were investigated by Sou et 
al. ( 1 986) in PBLs of women in different stages (O-IlI) of cervical cancer. The 
spontaneous SCE frequency was found to be significantly higher in the cancer group 
than that of the control group, and the SCE frequency gradually increased with the 
progression of the cervical cancer. The SCE frequency with carcinoma in situ (CIS), the 
lowest stage of cervical cancer, was significantly higher than that of the controls. The 
frequency of MMC-induced SCE was higher than that of spontaneous SCE in all groups 
and the difference in frequency between MMC-induced and spontaneous SCEs of 
cervical cancer group of the stages I-Ill was significantly different from that of the 
control group. The average generation time of the cervical cancer group was shorter 
than that of the control group, but the difference was not statistically significant. 
Cortes-Gutierrez et al. (2000) reported that the frequency of SCE in peripheral 
lymphocytes is significantly higher in cervical cancer patients than those in normal 
individuals. The mean number of SCEs per metaphase in women with carcinoma of the 
cervix uteri (7.80 - 1 .05) was higher than the control group (6.98 - 1 . 1 3) .  
SCEs were studied in 20 patients with breast cancer (stage Il)  before surgery, one 
month after surgery, and after three years as a follow-up study. Data from 50 age­
matched, normal healthy females, preferably from the affected families, served as 
Chapter Two: Review of Literature. 8 1  
controls. I n  each patient, 50 metaphase spreads were scored for SCEs. The mean values 
of SCEs per metaphase were 5 .80, 4.69, and 5 .98 in breast cancer patients before 
surgery, one month after surgery, and after a gap of three years as a follow-up, 
respectively. A one-way analysis of variance showed that there was a highly significant 
difference in the frequency of SCE in these patients before surgery, one month after 
surgical removal of cancerous tissue, and after three years as a follow�up study. 
Galloway and Evans ( 1 975) applied the SCE technique to human cells to examine the 
distribution of SCE between different people and within different chromosomes. The 
results show: ( 1 )  That there were no large differences in the incidence of SCE between 
blood leukocyte chromosomes from male and female adults and newborn, and that 
similar frequencies were found in cells from two patients with Ataxia telangiectasia 
which, nevertheless, showed the typical increases in chromosomal aberrations. (2) The 
distribution of SCE between chromosomes in the complement was found to be 
proportional to chromosome length, although the smaller chromosomes were under­
represented, but not significantly so. (3)  The distribution of SCE within chromosomes 
was non-random, with a deficiency in the centromeric and excess in the mid-arm 
regions. There was no evidence for an excess of SCE in chromosome regions rich in AT 
DNA sequences. (4) The frequency of SCE is to some extent dependent on BrdU 
concentration, but the influence of concentration is minimal in the range of 1 - 1 60 IlM. 
Human cells exposed over two cell cycles at which higher BrdU levels have around 14  
SCE per cell - a frequency virtually identical with that observed in  cultured cells from 
the Chinese hamster, wallaby, rat and kangaroo. 
Information on a possible association between SCE and cancer is controversial . Studies 
of SCE frequency in lymphocytes have proven to be useful to evaluate the exposure of 
living organisms to mutagens or carcinogens. Husum et al. ( 1 98 1 )  examined SCE 
frequency in peripheral blood lymphocytes of 1 3 1  women patients affected with breast 
tumour and observed an average 9.39 ± 0. 1 7  SCE/cell in 52 patients with carcinoma of 
the breast and 9 .88  ± 0 . 1 8  SCE/cel l  in 79 patients with non-malignant 
fibroadenomatosis. This is significantly different to healthy controls. 
Phenytoin (PHT) is a widely prescribed anti-epileptic drug. Kaul and Goyle ( 1 999) used 
SCE frequency assay to investigate its potential to interact with genetic material in a set 
Chapter Two: Review of Literature. 82 
of 30 epileptic patients aged 1 0  to 30 years prior to and following the administration of 
PHT over a period of nine months. In this study 40 control subjects were used in 
relation to age, sex, duration of drug therapy, and plasma concentration of PHT. Plasma 
levels of the PHT were measured by biochemical assay in epileptic patients before and 
after the PHT therapy. The peripheral blood lymphocytes were cultured and harvested at 
72 h .  The frequency of SCE was significantly higher in both age groups ( 1 0  to 20 and 
2 1  to 30 years) for PHT -treated epileptics compared to PHT -untreated and control 
subjects. However, there were no considerable variations in SCE findings between the 
control and PHT -untreated patients. Between the two age groups, a significantly higher 
SCE frequency was observed in PHT-treated patients in the older age group (2 1 to 30 
years). Mean SCE frequency did not differ between the male and female in the controls, 
PHT -untreated, or treated epileptics. Correlation between the plasma concentration of 
PHT and the incidence of SCE among 30 patients was not significant. PHT 
monotherapy appears to have a genotoxic effect as expressed by the induction of 
increased SCE rates in treated epileptics, while disease does not play any role in 
inducing genetic damage as shown by no difference in SCE frequencies between control 
subjects and PHT -untreated epileptic patients. 
Determination of SCE frequency, CA and micronucleus rate of cultured peripheral 
lymphocytes in 32 patients with lung cancer, 33  miners and 40 non-mining workers in 
Yunnan Tin Mine was carried out by Hu et al. ( 1 987).  The results showed that the 
cancer patients had the highest SCE incidence,  CA and micronucleus rate, the non­
mining workers had the least and miners on an intermediate level.  There was a 
significant difference of SCE, CAs and micronucleus rate between patients and non­
mining workers. They also found that miners had a significantly higher SCE and CA 
rate but not micronucleus rate as  compared with the non-mining workers. They 
proposed that some carcinogens present in the Yunnan Tin Mine might be responsible 
for the genetic damages in the miners. Chemical drugs may be considered to contribute 
to the genetic damage in cancer patients who had a tendency toward an increased SCE, 
CA and micronucleus rate as compared to the miners, though without reaching 
statistical significance. It has been suggested that a combination of assays of SCE with 
CA or MN assay may be a useful index for screening of the high risk population and 
monitoring the chemical drug prevention of lung cancers in Yunnan Tin Mine. 
Chapter Two: Review of Literature. 83  
The spontaneous and MMC-induced SCEs and CAs in cultured lymphocytes from 
members of "high risk" and " low risk" oesophageal cancer families in Linxian County, 
China were studied by Wu ( 1 986). The results from this study showed that the 
frequencies of the spontaneous SCE of "high risk" and "low risk" oesophageal cancer 
groups were 7.8 - 0.25 and 8 .3  - 0.25/per cell. They observed no difference between 
these two groups. The frequencies of the SCE induced by MMC in "high risk" and " low 
risk" oesophageal cancer groups were 43 .8  - 2.4 and 2 1 .6 - 1 . l Iper cell. This is a 
noteworthy difference. Also, the frequencies of the spontaneous and MMC-induced 
CAs in "high risk" cancer families were higher than those of the " low risk" group. 
Mertens et al. ( 1 995) investigated the SCE and lymphocyte subsets of children with 
acute lymphoblastic leukemia (ALL) during chemotherapy. The treatment followed a 
protocol called ALL-BFM-90. At the time of diagnosis children with ALL showed 
statistically significant higher SCE frequencies (4.9 - 0.77) than healthy controls (3 .6  -
0.93).  The in vivo effects of cyclophosphamide (CP) resulted in a dramatic increase of 
the SCE frequency (20.5-3 .76). This increased SCE level of lymphocytes might reflect 
an instability of DNA or a deficiency of DNA repair. One could suggest that 
1ymphocytes of children with ALL might have a higher susceptibility to harmful 
influences; and this could be a co-factor towards the development of the malignant 
disease. However, one week after the administration of CP, the SCE rate decreased. 
This decline of SCE frequency correlated with a severe reduction of the absolute 
number of T lymphocytes .  The observed reduction of SCE frequency may be due to a 
loss ofT lymphocytes, or SCE became repaired during one week. 
A study by Baltaci et at. (2002) used SCE assays and the COMET assay to study DNA 
damage in patients with different types of ovarian malignancies and in healthy 
volunteers. They found that the SCE frequencies of cancer were significantly greater 
than that of normal controls and that the frequency of exchange in chromosomal groups 
A, B, and C ,  which include chromosomes 1 - 1 2, was higher than that of the other 
chromosomal groups in both groups of patients. This is perhaps not surprising 
considering the larger size of group A, B ,  and C chromosomes compared to groups D,  
E, and G. A comparison of the results of  the alkaline COMET assay in patients and 
control subjects showed a significant difference in the number of DNA-damaged cells. 
And when samples were assayed using the COMET test it was found that the DNA 
Chapter Two: Review of Literature. 84 
migration of cells in the women with ovarian malignancies was higher than that of 
control women. It was therefore concluded that both the SCE and COMET assays could 
be used successfully to monitor DNA damage in women with ovarian cancer. 
The increased risk of neoplasia following cytotoxic therapy for both malignant and non­
malignant disease is  well known. SCE frequencies in patients receiving such 
chemotherapy are often elevated, and the persistence of high levels after treatment may 
provide an indicator for susceptibility to secondary neoplasia. The cytostatic drug 
razoxane has been used for the treatment of psoriasis, acute myeloid leukemia (AML), 
and colorectal carcinoma. Prolonged use of this drug, however, has been associated 
with the subsequent development of AML. Price et al. ( 1 992) studied the SCE 
frequencies of 34  patients with colorectal carcinoma who were receiving or had 
previously been treated with razoxane. They claimed that no significant increase in SCE 
levels were observed in the razoxane group compared with either normal controls or 
untreated patients. 
Maurya et al. ( 1 987) reported that changes in SCE frequency, the patient s response to 
anticonvulsant therapy and seizure severity are interrelated. In their study, SCE 
frequency was scored from PBLs of epileptic patients (untreated and treated) and 
controls. Significantly higher number of SCEs were observed in samples taken from 
both treated (recipient of anticonvulsant therapy) and untreated (seizure) epileptic 
patients than was seen in the normal healthy control group. However, those who 
received the anticonvulsant therapy had a decreased number of SCEs in comparison to 
the untreated patients. 
Melanomas are highly clonogenic. Genetic variability and polymorphism of tumour cell 
populations have been reported. However, no direct evidence of mutator activity as a 
source of genetic polymorphism for melanoma cells has been described. Some 
intermediates of melanin synthesis are cytotoxic and genotoxic and their mutagenic 
powers have been described. Miranda et al. ( 1 997) observed that the rate of SCE of the 
line of human melanoma cells varies with the concentration of the melanin precursor L­
tyrosine, in the culture medium. An increase of melanin synthesis results in increased 
SCE rates. The highest values of SCEs are found in melanotic melanoma cells 
Chapter Two: Review of Literature. 85 
compared with the amelanotic ones. Observation shows that melanoma cell has higher 
levels of SCE compared with normal human lymphocytes. 
Atalay et al. (2000) assessed the impact of malignant mesothelioma on the frequencies 
of SCE in the pleural effusion cells. Ten patients with mesothelioma and 20 control 
subjects were included in the study. The SCE frequencies of malignant pleural 
mesotheliomas were significantly higher than that ofthe control subjects. 
Spontaneous SCE frequencies were measured by Parkes et al. ( 1 985) in cells 
undergoing their second mitosis at 54, 68 ,  72, 78 or 90 h after PHA stimulation of 
whole blood cultures from seven normal donors. A consistent pattern of fluctuation of 
SCE levels was observed between 54 and 78 h. The magnitude of change in SCE 
frequency between consecutive fixation times was as high as 80% for some donors. 
These observations support the hypothesis of Snope and Rary ( 1 979) that there are 
subpopulations of lymphocytes with different spontaneous SCE levels and different 
proliferation rates .  In untreated cancer patients the patterns of change in SCE frequency 
with time were different from that seen in normal donors. This may be because of 
quantitative changes in T -lymphocyte sUb-populations that have been observed in 
cancer patients. Changes in SCE frequency of less than about a factor of two observed 
at a single sampling time may not be indicative of genotoxic events or genetic 
instability but simply represent changes  in the composition of lymphocyte sub­
populations and/or in their rates of proliferation in vitro. 
SCE frequencies were used to compare two types of osteoporosis  treatment for 
genotoxicity by Sahin et al. (2002). This study included 57 women, aged between 40 
and 64 years. SCE values of patients under estrogen replacement therapy (ERT) or 
alendronate therapy were compared to control s  who never used any drugs for 
osteoporosis. The difference between the SCE values of women taking ERT and control 
women was found to be statistically significant. The difference between women taking 
alendronate and untreated controls was not statistically significant. The author 
concluded that alendronate does not have genotoxic effects based on SCE frequency, 
while ERT increases SCE frequencies. 
Chapter Two: Review of Literature. 86 
Kayikcioglu et al. (2000) evaluated the frequency of SCE during hormone replacement 
therapy in postmenopausal women. Thirty-four asymptomatic postmenopausal women 
with a minimum 12 months since their last menstrual period and surgical menopausal 
women were included in the study. Seventeen patients who were in spontaneous 
menopause were administered conjugated estrogen and medroxypro-gesterone acetate 
(group A), and the others who were in surgical menopause were given 1 7-beta-estradiol 
only (group B). Peripheral lymphocytes were obtained at the beginning and at the end 
of the third month of therapy. The mean age of the patients was 50.67 - 4.79 y. There 
were statistically significant differences in terms of SCE frequencies between pre- and 
post-treatment levels of both groups. It is likely that estrogens with or without 
progesterone increase SCE frequency and this issue may be evidence for the increased 
potential for malignancies. 
2.2.6 Human Reproductive Hormones and SCE 
Joseph-Lemer et al. ( 1 993) tested the hypothesis that sex hormones, particularly 
steroids, influence the frequency of SCEs in women undergoing ovulation induction for 
in vitro fertilization treatment. These women undergo extreme hormonal changes and 
therefore serve as a good model for testing the rate of genetic damage due to these 
changes. The controls were fertile women with regular menstrual cycles who received 
no hormonal treatment. Peripheral lymphocytes were obtained during different stages of 
the normal and treated cycles. They found an increased SCE frequency during ovulation 
time in the controls and during the time of human chorionic gonadotropin 
administration in the group undergoing ovulation induction. However, in the later 
group, SCE frequency was significantly higher. SCE frequency was positively 
correlated with the level of testosterone and follicle stimulating hormone (FSH) in the 
ovulation induction group and positively correlated with the estradiol level in both 
groups. 
D'Souza et al. ( 1 988) observed a higher rate of cellular genetic damage in terms of 
increased frequencies of SCEs and chromosome aberrations during "ovulatory" and 
"estrogenic" stages as compared with those of "progestogenic" stages of the menstrual 
cycle in women and the reported fluctuations in SCEs as a function of time in men. This 
indicates that chronobiologic considerations are essential in the design of studies of 
Chapter Two: Review of Literature.  87 
human and animal experiments. It seems that female subjects are more influenced by 
biological rhythms because of specific hormonal cycles and are sensitive to genetic 
damage during ovulatory/estrogenic stages of the menstrual cycle. Sharma and Das 
( 1 986) have reported an increased frequency of SCEs and CAs in women during the 
advanced stages of pregnancy. This has been attributed to the high levels of sex steroids 
present during the last trimester of pregnancy. 
Oral contraceptive use has been associated with increased SCEs (Bala Kishna Murthy 
and Prema, 1 979, 1 983) .  However, Husum et at. ( 1 982) observed no effect of 
contraceptives on the frequency of SCE. 
2.2.7 Lymphocyte Concentration and SCE 
Bender et at. ( 1 992b) observed that the lymphocyte concentration in the culture tube 
leads to variation in SCE frequency. The number of SCEs decreased with the increased 
number of lymphocytes . Therefore, to conduct SCE experiments it is necessary to 
standardize the lymphocyte concentration. 
2.2.8 Baseline SCE and the Influence of Genetic, Chemical, and Environmental 
Agents 
BrdU itself, like eH]thymidine (Brewen and Peacock, 1 969; Gibson and Prescott, 
1 972), induces SCEs (Gatti et at. 1 979; Kato, 1 974b; Latt, 1 974a; Wolf and Perry, 
1 974) and may be responsible for most of the baseline SCEs observed in the absence of 
additional clastogens. However, Stetka and Spahn ( 1 984) demonstrated that replication 
of BrdU-substituted DNA results in SCE induction, while incorporation of BrdU into 
nascent DNA does not. The latter phenomenon was reported by Mazrimas and Stetka 
( 1 978) and Davidson et at. ( 1 980), and was interpreted as an indication that BrdU may 
induce SCEs by two different mechanisms, one involving incorporation and the other 
not. 
Baseline SCE frequency appears to be directly correlated with genome size, not 
chromosome number (Kato, 1 977). The SCE number within a cell or within a given 
chromosome is randomly distributed with a few exceptions (Wolf and Perry, 1 974; 
Chapter Two: Review of Literature. 88  
Chaganti e t  al. 1 974;  Latt, 1 974a; Galloway and Evans, 1 975 ,  and Dutrillaux e t  al. 
1 974). The number of exchanges per chromosome is positively correlated with 
chromosome length. In man, among small (E, F and G groups) chromosomes there are 
fewer exchanges than expected (Hsu and Pathak, 1 976; Latt, 1 974a; Galloway and 
Evans, 1 975 ; Dutrillaux et al. 1974, and Latt et al. 1 975). 
Increased baseline SCE frequencies were observed in some human diseases such as 
Bloom s Syndrome (Chaganti et al. 1 974; Shiraishi et al. 1 976), multiple sclerosis 
(Sutherland et al. 1 980;  Vijayalaxmi et al. 1 983 ;  Senecal-Quevillon et al. 1 986), 
Down s syndrome (Shubber et al. 1 99 1 ), Schistosoma hematobium (Shubber, 1 987) ,  
Crohn s disease (Myung-Hee et al. 1 997) and systemic Lupus erythematosus (Palmer et 
al. 1 987). In a recent cytogenetic study with prostate cancer patients (Dhillon and 
Dhillon, 1 998) observed that SCE frequency is significantly higher in prostate cancer 
patients than in the controls. 
Technical factors that influence baseline SCE included the tissue culture medium used, 
the amount of BrdU in the medium, and the method of differential staining. Cigarette 
smoking, vaccination, viral infections and therapeutic drugs may also affect baseline 
SCE, and hence, it is important to check an individual s medical and occupational 
history in evaluating the SCE levels. Two other factors also have been reported to 
influence the baseline SCE frequency: the serum used in the culture medium and the 
culture temperature (Carrano and Natarajan 1 988) .  Das and Sharma ( 1 984) observed 
that the frequency of SCEs in human lymphocytes increased as a function of culture 
temperature with a maximum at 40C. 
EMFs and SCE 
Zwingelberg et al. ( 1 993) exposed nine female Wistar rats for seven days or 28 days, 24 
h per day to a homogeneous 50 Hz, 30 mT (9.4 T/s) horizontal MF. The calculated 
internal electric field was 0- 1 70 m Vim assuming an elliptical torso cross-section 5 cm 
high and 1 2.5  cm long. Peripheral blood was sampled one day prior to and immediately 
following exposure. Cyclophosphamide, a potent inducer of SCEs, was used as a 
positive control. Investigators reported that neither exposure period resulted in a 
statistically significant increase in SCEs in PBLs in any of the exposed animals when 
Chapter Two: Review of Literature. 89 
compared to rates determined prior to exposure . Six additional animals, which were 
sham-exposed controls, produced similar results. 
Antonopoulos et al. ( 1 995) incubated human peripheral blood cultures in the presence 
of an EMF of 50 Hz and 5 mT which lead to a stimulation of the cell cycle of dividing 
lymphocytes but had no influence on the frequencies of the SCE. Comparative studies 
with two different exposure systems and with different culture temperatures indicated 
that the effect on the cell cycle results from the EMF and is not a thermal effect. These 
data support the assumption that with respect to their suspected carcinogenic effects 
EMFs have no initiating effects, although they may have promoting effects. 
Human lymphocytes culture was exposed by Khalil and Qassem ( 1 99 1 )  to a PEMF (50 
Hz, 1 .05 mT) for 24, 48 and 72 h. They observed a statistically significant suppression 
of mitotic activity and a higher incidence of CAs. Shorter exposure time (24 and 48 h) 
had no effect on the cell proliferation index (CPI) or SCE but continuous 72 h exposure 
exhibited a significant reduction of CPI and an elevation of SCE rate. 
Yaguchi et al. ( 1 999) evaluated the induction of SCE in cultured mouse m5S cells, 
which were exposed to 5,  50 and 400 mT. Exposure to 5 mT and 50 mT MF led to a 
very small increase in the frequency of SCEs, but no significant difference was 
observed between exposed and unexposed control cells. The cells exposed to 400 mT 
exhibited a significant elevation of the SCE frequencies suggesting that a 400 mT MF 
may induce DNA damage. 
According to a number of in vitro studies, ELF EMFs are unable to induce CAs in white 
blood cells as measured by the analysis of SCEs (Cohen et al. 1 986a; Rosenthal and 
Obe, 1 989). SCEs are good indicators of genetic effects induced by chemicals but not 
by ionizing (Evans, 1 977) or non-ionizing radiation (Maes et al. 1 993). A significant 
increase in DNA synthesis was sometimes observed (Lester and Moore, 1 982) as well 
as an increase in cell division and transcriptional activity (Liboff et al. 1 984; Goodman 
et al. 1 989b). 
Chapter Two: Review of Literature. 90 
Rosenthal and Obe ( 1 989) cultured HPBL in the presence of 50 Hz EMFs and observed 
no alteration of the spontaneous frequencies of SCEs and CAs, but observed an 
enhancement of the cell cycle progression of HPLs in vitro. 
Gamma-irradiation and SCE 
Shah and Mittal ( 1 987) studied the effects of different doses of gamma (y)-irradiation 
on SCE induction in unifiliarly BrdU-substituted DNA in various phases of cell cycle. 
y-irradiation-induced SCE frequency was measured by post-irradiation treatment with 
antimutagen L-cysteine. Perturbation in cellular proliferation kinetics due to y­
irradiation and y-irradiation plus L-cysteine was also studied. It was observed that y­
irradiation is an efficient inducer of SCE and is most effective in S phase. L-cysteine 
also causes SCE induction, which is slightly higher than the spontaneous level of SCEs 
found in HeLa cells. However, post-irradiation addition of L-cysteine reduces SCE 
frequency in y-irradiated cultures and this reduction is maximum in G1 phase irradiated 
cells. y-irradiation delayed the mitosis considerably and this delay continued to increase 
with increasing doses. L-cysteine reduced the delay in cell cycle caused by y-irradiation. 
Influence of Age, Gender, Smoking on SCE 
Gender is an important factor for the frequency of the SCE. However, several studies 
have observed no significant differences between male and female SCE frequencies 
(Galloway and Evans, 1 975;  Alhadeff and Cohen, 1 976; Crossen et at. 1 977; Latt and 
Juergens, 1 977;  Morgan and Crossen, 1 977;  Cheng et al. 1 979; De Arce, 1 98 1 ;  
Waksvik et al. 1 98 1 ;  Carrano and Moore, 1 982; and Livingston et at. 1 983) .  Other 
studies have observed baseline SCE frequencies at approximately 0.5 exchanges per cell 
higher in females than males (Soper et at. 1 984; Margolin and Shelby 1 985 and Bender 
et al. 1 992a). Bender et at. ( l 992a) believed that it might be a consequence of the 
increased chromosome length (DNA content) represented by the second X-chromosome 
in females versus the smaller Y -chromosome in males. 
Milosevic and Marinkovic ( 1 999) reported the variability in the frequency of SCE in 
lymphocytes of peripheral blood in relation to age and gender of individuals in a 
Chapter Two: Review of Literature. 9 1  
Yugoslavian population. William et al. ( 1 977) studied the incidence of SCE in cultured 
human lymphocytes from 50 normal individuals using the BrdU-Giemsa technique. The 
mean SCE frequency per metaphase was 7 .9  with a standard deviation of 1 . 36 and a 
range of 1 -2 1  and the incidence of exchanges was influenced neither by gender nor by 
age. 
Bukvic et al. (200 1 )  studied the possible effect of age, gender and smoking using the 
micronuclei and SCE frequencies. Peripheral blood obtained from 38  subjects ranging 
in age from 1 6  to 63 years and 1 6  centenarians was used in this study. The mean 
number of binucleated cells with micronuclei varied as a function of age and gender, 
particularly in women. Smoking habits had no effect on micronuclei frequency. Gender 
and smoking habits significantly influenced SCE per cell frequencies, but age had no 
effect on them. 
Rates of SCE in dividing RPBLs were determined and compared between smoking and 
non-smoking young women between the ages of 1 6  and 25 by Rowland and Rarding 
( 1 999) . Chromosomes block-stained with Giemsa were also examined for CAs. A 
striking difference in the frequency of SCE was found between young women who 
smoked and those who did not. Smokers scored a significantly higher rate and range of 
SCE than those observed in non-smokers. No statistical difference in the frequency of 
CAs was found between smokers and non-smokers. The significantly higher frequency 
of exchange in young smoking women may indicate that initial damage to the DNA in 
many of these women has probably already occurred, thus causing an increased risk of 
developing cancer later in life. 
Lazutka et al. ( 1 992) assessed the effects of alcohol consumption, cigarette smoking 
and age on SCE frequency in human lymphocytes by means of multiple linear 
regression. They observed an increase in the rate of SCE, which was associated with 
alcohol consumption, smoking, and to a small extent age . They concluded that these 
three confounding factors explain 48% of the inter-personal variation in SCE rates 
among subjects studied. 
The effects of age, gender and smoking on SCE frequency and distribution in human 
lymphocytes were assessed by means of multiple linear regression (Lazutka et al. 
Chapter Two: Review of Literature. 92 
1 994). Differences in SCE scores were associated with all above variables: SCE 
increased with age and c igarette smoking intensity, and higher SCE frequencies were 
observed in females. 
Elio (2000) examined a l arge sample of 4-day old chick embryos from a commercial 
strain to determine the baseline SCE frequency and the nature of the distribution. A 
mean frequencies of 1 . 1 5  SCE/cell were observed in a total of 6,400 cells from 1 28 
male and female embryos : the SCE frequencies for males and females were similar. 
Diet and SCE 
The role of diet has not been taken in to consideration when SCE frequencies are 
studied. However, Bala Kishna Murthy et al. ( 1 980) reported that severe protein calorie 
malnutrition in children is associated with increased lymphocyte SCE frequencies. SCE 
frequencies were observed to decrease following nutritional rehabilitation. 
Occupational Hazards and SCE 
Chromosomal aberrations and SCEs in lymphocytes from coke oven workers, an 
occupational group known to be at increased cancer risk, were studied by Bender et al. 
( 1 988) .  It was observed that coke oven workers as a group have a statistically 
significant increase in the frequency of CAs and SCEs. 
SCE frequencies were measured in the peripheral lymphocytes of 1 04 greenhouse 
farmers exposed to pesticides and 44 unexposed workers in a study by Shaham et al. 
(2001 ) .  The frequency of SCE was significantly higher among the farmers who were 
exposed compared with the unexposed group. 
SCE in blood lymphocytes was determined in 32 male workers occupationally exposed 
to lead (Pb) and zinc (Zn) and in 20 unexposed controls  matched for age and smoking 
habits by Donmez et al. ( 1 998). Exposed workers had higher SCE mean values than 
control workers . In exposed persons, blood Pb concentrations were also significantly 
higher than was found in the controls, but the difference between Zn levels in the blood 
of these groups was not significant. The results indicate that Pb may be genotoxic and 
Chapter Two: Review of Literature. 93 
harmful to humans; however, exposure to zinc seem to have no harmful  effect on 
humans. 
Lazutka et al. ( 1 999) performed cytogenetical analysis of CAs in 1 75 ,229 cells from 
1 , 1 1 3  individuals, both unexposed and occupationally or environmentally exposed to 
heavy metals (mercury and lead), organic (styrene, formaldehyde, phenol and 
benzo[a]pyrene) and inorganic (sulfur and nitrogen oxides, hydrogen and ammonium 
fluorides) volatile substances and/or ionizing radiation. In addition, 1 1  ,250 cel ls  from 
225 individuals were scored for the frequency of SCE. Increased frequencies of CAs 
were observed in all occupationally exposed groups. A principal difference between the 
exposure to heavy metals and organic substances was found. An increase in the CA 
frequency was dependent on the duration of exposure to mercury, but not dependent on 
duration of exposure to styrene, formaldehyde, and phenol .  Higher CA incidences were 
found in lymphocytes of  children living in the vicinity of  a plant manufacturing 
phosphate fertilizers. This suggests that children are a sensitive study group for the 
assessment of environmental exposure. 
The genotoxic effects of occupational exposure of 20 nurses who handled cytostatic 
drugs in medical oncology and haematology units were investigated by Kasuba et al. 
( 1 999) . The effects were evaluated by SCE test. The duration of employment in the 
units and of exposure to cytostatic compounds ranged from I to 3 1  years. They 
observed no statistically significant differences in the frequency of SCEs. 
Exposure to ionizing radiation was found to cause chromosome breaks and SCEs in 
Chemobyl clean-up workers. Chromatid breaks, SCEs, dicentric chromosomes, and 
chromosome translocations were found in workers from Ignalina Nuclear Power Plant. 
The increased frequency of SCEs in these individuals exposed to ionizing radiation was 
quite unexpected, as ionizing radiation is not known to cause SCEs. This increase may 
be attributed to the action of some unrecognized lifestyle or occupational factors, or to 
be a result of radiation-induced genomic instability. 
Tsun-Jen et al. (2000) used lymphocyte SCE frequencies as a parameter to investigate 
the genotoxicity of low-level ethylene dichloride (EDC) and vinyl chloride monomer 
(VCM) exposure in VCM-manufacturing plant workers. The SCE frequency was 
Chapter Two: Review of Literature. 94 
determined for 4 1  male workers with exposure to VCM and/or EDC and for 20 male 
workers who were unexposed. Moderate EDC exposure of around one part per million 
(ppm) corresponded to a significantly greater SCE frequency than was found in the low 
EDC exposure group. However, VCM exposure of similar level was not associated with 
increased SCE and concluded that EDC may cause genotoxicity at a relatively low level 
of exposure. 
Shaham et al. (2002) studied the SCEs in peripheral lymphocytes of 90 pathology 
department workers from 1 4  different hospitals in I srael who were occupationally 
exposed to formaldehyde (FA) and of 42 unexposed workers from the administrative 
section of the same hospitals. The mean exposure period to FA was 1 5 .4  years (range 1 -
39). The results of SCEs are expressed in two variables: (a) the mean number of SCEs 
per chromosome, and (b) the proportion of high frequency cells (cell s  with more than 
eight SCEs). A high correlation was observed between these two variables.  The 
adjusted means of both SCE variables were significantly higher among the exposed 
group compared with that of the unexposed group. 
Chronic exposure to low frequency (LF) noise and whole-body vibration (WBV) 
induces both physiological and psychological alterations in humans. Silva et al. ( 1 999) 
have shown that long-term occupational exposure to LF noise and WBV produces 
genotoxic effects in humans expressed as an increase in SCE levels in lymphocytes. 
SCEs were also analyzed by Silva et al. (2002) in spleen lymphocytes of mice exposed 
to LF noise alone and in combination with WBV for 300 and 600 h. An effect at the cell 
cycle kinetics level was also investigated. The results revealed significant increases in 
the mean SCE number per cell and in the proportion of cells with a high frequency of 
SCEs (HFCs) in lymphocytes of mice submitted to combined noise and WBV over 
controls. No significant differences were found between single noise-exposed and 
control mice. A cell cycle delay was observed exclusively in the noise and WBV 
exposure groups. They concluded that prolonged exposure to LF noise in combination 
with WBV determines an increase in SCE level in mice while LF noise alone was not 
effective in SCE induction. 
The evaluation of SCE is used to establish the cytogenic damage in subjects exposed to 
toxic substances. The test is considered to be one of the most sensitive and accurate 
Chapter Two: Review of Literature. 95 
indicators of genetic damage and responds to toxic chemicals at low doses. Stea et al. 
(2000) evaluated the incidence of SCE in peripheral lymphocytes of patients with 
artificial limbs. Subjects with artificial limbs made of titanium-aluminium-vanadium 
alloys presented a significantly higher SCE number than that of the control population, 
whereas subjects with artificial limbs made of chrome-cobalt alloy or mixed artificial 
limbs presented a higher SCE value than the controls, but not significantly so. 
Duydu et al. (200 1 )  studied inorganic lead exposure in 3 1  volunteers employed in a 
storage battery plant. The genotoxicity of lead was measured in terms of SCE. 
Erythrocyte delta-aminolevulinic acid dehydrogenase (ALA D) activity, urinary delta­
aminolevulinic acid (V-ALA), and blood lead levels (PbBs) were also determined to 
evaluate some possible relations between these lead exposure indicators and the 
observed SCE frequencies. Blood lead concentration of 36.3 1 g/decilitre (decilitre 
= 
1 00 ml) was determined as an average level in the workers. Consequently, decreased 
ALAD activity in erythrocytes and increased U-ALA excretion was observed in 
statistically higher PbBs when compared with the control group. A statistically 
significant correlation was observed between the PbBs and SCE frequencies. Moreover, 
the correlation between V-ALA excretion and SCE frequencies was relatively higher 
than the correlation between PbBs and SCE frequencies. They concluded that these 
results might indicate a possible mechanism of ALA mediation in the genotoxic effects 
of lead. 
Influence of Drugs and Alcohol on SCE 
Cocaine is a widely abused drug. Chang-Tze et al. ( 1 999) reported that it induces 
teratogenesis in both humans and animals .  Cocaine-induced teratogenesis has been 
associated with reactive oxygen species (ROS), which are generated by cytochrome 
P450 during cocaine biotransformation. Since ROS have been reported to induce 
genotoxicity, it is of interest to determine whether cocaine and/or its metabolites are 
also genotoxic. In this study, CRO K 1  cells were employed as a model system to 
investigate the genetic toxicity of cocaine in the presence or absence of rat liver S9 
fraction. Cocaine-induced cytotoxicity was enhanced when S9 was present, indicating 
the cytochrome P450 metabolism p lays a role in cocaine-mediated cytotoxicity. 
Cocaine treatments alone induced few chromosome aberrations, while treatments of 
Chapter Two: Review of L iterature. 96 
cocaine plus S9 caused a significant increase in chromosome aberrations. In contrast, 
cocaine induced micronuclei formation and hypoxanthine-guanine phosphoribosyltrans­
ferase mutation only in the presence of S9. Therefore, cocaine itself is at best a weak 
clastogen, whereas metabolites of cocaine are truly inducers of clastogenesis and 
mutagenesis. Cocaine treatments alone also induced a significant increase in SCE 
frequency, but the addition of S9 did not affect the results. Free radical scavengers, 
including superoxide dismutase and catalase, efficiently decreased the frequency of 
cocaine plus S9- induced micronuclei, implying that ROS are indeed important 
components in cocaine-induced genotoxicity. The observation that non-toxic doses of 
cocaine can inhibit intercel lular metabolic cooperation suggests that cocaine may also 
be a tumour promoter. The data support the idea that cocaine could possess genotoxicity 
in addition to its well-known neurotoxicity and teratogenicity. 
The induction of SCE by caffeine is very minimal (Kato, 1 973 ;  Waksvik et al. 1 977; 
Vogel and Bauknecht, 1 978; Basler et al. 1 979; Mourelatos, 1 979; and Zamansky et al. 
1 980). However, chronic exposure of human peripheral blood lymphocytes to caffeine 
has been shown to double the SCE frequency (Guglielmi and Tice, 1 980) and retard cell 
proliferation. In another study caffeine was shown to reduce the inhibitory effect of 
alkylating agents on replicon progression (Mumane et al. 1 980). Increased SCEs have 
also have been reported in alcoholics (Butler and Sanger, 1 98 1 ). 
Hirsch et al. ( 1 992) reported that SCE frequencies are significantly affected by coffee 
drinking habits.  A stepwise multiple regression analysis showed that together, smoking 
and coffee habits entered at the first step accounted for 2 1  % of the observed variance in 
SCE. Their twin analysis showed that after adjustment of the data set for smoking and 
other significant predictors, genetic factors accounted for approximately 30% of the 
variation in SCE rates. 
Mycotoxins and SCE 
lia-Sheng and Groopman ( 1 999) reported that mycotoxins are toxic fungal metabolites, 
which are structurally diverse, common contaminants of the ingredients of animal feed 
and human food. To date, mycotoxins with carcinogenic potency in experimental 
animal mode l s  include aflatoxins, sterigmatocystin, ochratoxin, fumonisins, 
Chapter Two: Review of Literature. 97 
zearalenone, and some Penicillium toxins. Most of these carcinogenic mycotoxins are 
genotoxic agents with the exception of fumonisins, which is currently believed to act by 
disrupting the signal transduction pathways of the target cells. Aflatoxin B 1 (AFB 1 ), a 
type I known human carcinogen and the most potent genotoxic agent, is mutagenic in 
many model systems.  I t  has been shown to produce CAs, micronuclei, SCE, 
unscheduled DNA synthesis, and chromosomal strand breaks, as well as form adducts 
in rodent and human cells. 
SCE Frequency as a pollutant indicator 
Etroplus suratensis, a brackish water fish species, has been examined by Paramananda 
et al. ( 1 996) for its applicability as a test organism in screening genotoxic pollutants in 
aquatic environments. A detailed karyotype study has been carried out and methods 
developed for demonstrating sister chromatid differentiation (SCD) and SCE. The 
spontaneous SCE value was found to be 1 .79 per metaphase. This study showed that the 
fish appeared to be a suitable model for in vivo cytogenetic assays in pollution 
monitoring. 
Arsenic is one of the few human carcinogens for which there is not yet a reliable animal 
model for studying cancer. As such, the classification of arsenic as a carcinogen is 
based upon data derived from human epidemiological studies. Although the 
mechanisms of action of arsenic as a toxic agent have been known for many years, the 
inability to cause cancer by arsenic in laboratory animals has confounded the 
operational characterisation of arsenic as an initiator, promoter, complete carcinogen, or 
co-carcinogen for humans. Mass ( 1 992) reported that arsenic is a genotoxic agent and it 
induces CAs, micronuclei and SCE in mammalian cells, as well as neoplastically 
transforming Syrian Hamster embryo cells. However, it is not a classical point mutagen 
that causes the exchange in DNA of one nitrogenous base for another. 
SCEs were analysed in lymphocytes from 1 2  control persons and 33  Chemobyl clean­
up workers by Lazutka and Dedonyte ( 1 995). The group ofChernobyl clean-up workers 
consisted of civilians, who were forced to go to Chernobyl to clean up environmental 
contamination caused by the Chemobyl disaster. On average, they received 0. 1 3  Gy of 
Chapter Two: Review of Literature. 98 
external irradiation before returning home. Cytogenetic analyses were performed 6-8 
years after the irradiation. Standard cytogenetic techniques were used. Mean SCE 
frequency was 7.45 SCE/cell in controls and 1 0.30  SCE/cell in clean-up workers. In 
addition, increased frequencies of CAs due to exposure at Chernobyl and alcohol 
consumption were observed. However, there was no correlation between external dose 
of irradiation and the frequency of CAs. Thus, even 6-8 years after the irradiation, 
cytogenetic effects in lymphocytes of Chernobyl clean-up workers are still significant. 
Ozturk et al. (2002) conducted a study to investigate the genotoxic effect of acute 
overexposure to combustion products originating from coal or wood stoves in patients 
presented with acute carbon monoxide exposure. The frequency of SCEs and the 
carboxyhemoglobin concentration was observed in 20 patients without a history of 
smoking or drug use who had been treated in the Emergency Care Unit of the Istanbul 
Medical Facility due to acute carbon monoxide exposure. All of these cases were 
domestic accidents due to malfunctioning coal or wood stoves. The results were 
compared with a control group of 20 non-smoking, non-drug using healthy individuals 
matched for age, sex, and absence of other chemical exposure. The mean SCE 
frequency per metaphase was significantly higher in the study group compared to the 
control group. No positive correlation was found between the blood carboxyhemoglobin 
concentration and SCE frequency. They concluded that acute exposure to combustion 
products of wood or coal is genotoxic to DNA. 
Petrochemical Exposure and SCE 
SCE frequencies were investigated by Bukvic et al. ( 1 998) in the PBLs from 22 men 
with low average exposure to benzene and 1 9  control men who had not been exposed. 
The majority of these men (2 1 exposed, 1 9  controls) were also investigated using the 
micronuclei. The exposed subjects were employed at ten different petrol stations in or 
near the city (Bari/South Italy). SCE frequencies were significantly related with age and 
smoking habits, but no relation was observed between SCE and length of employment. 
Micronuclei frequencies were significantly increased in relation to the length of 
employment; but no relation was observed when age and smoking habits were taken 
into consideration. 
Chapter Two: Review of Literature. 99 
Summary of SCE Review 
In conclusion, it is important to note that the replication fork in S-phase is believed to 
be the site of SCE formation and SCEs are good indicators of genetic damage. The SCE 
technique has been used comprehensively and successfully in many studies discussed 
above to determine the levels of genetic damage caused by several classes of clastogens. 
This review shows the importance of including the SCE technique in the current study. 
2.3 Micronucleus (MN) Assay 
In this section, the MN assay that was employed in the current study to measure the 
degree of malfunctioning of a person's DNA repair system in the G[ phase of the cell 
cycle is reviewed. This section includes the definition, o rigin and genetic basis of 
micronuclei, and the use of the MN assay as a biomarker. In order to isolate the effect of 
EMF exposure from the exposure of other environmental agents and lifestyle factors, 
which might contribute to DNA repair mechanisms, each donor was required to 
complete a detailed personal questionnaire covering the foHowing : present and past 
occupational history, exposure history, medical history, smoking history, diet history, 
and genetic history. Thus, it was considered necessary by the author to include in this 
review a survey of related studies where MN assay has been performed to monitor the 
effects of various environmental agents and lifestyle factors on DNA repair. 
2.3 . 1  Introduction 
Many endogenous and exogenous agents are known to cause spontaneous cytogenetical 
or physiological damage in living cells. Chromosomal damage has been linked to severe 
clinical disorders (Frank and Riccardi, 1 977). Therefore, it i s  important to determine if 
these agents are capable of inducing such harmful effects in human cells. 
The impact of exogenous agents on chromosomal damage has been studied extensively 
for many years (Raj et al. 1 983;  Yager et al. 1 988; Surralles & Natarajan, 1 997; Fenech 
et al. 1 999; Kryscio et al. 200 1 ) . Cytological examination of metaphase preparations 
treated with a test agent either in vivo or in vitro has been the traditional tool used in 
these studies (Evans, 1 976). However, this technique is time consuming and yields 
Chapter Two: Review of Literature. 1 00 
results that are statistically weak due to the relatively low numbers of affected cells that 
can be scored. 
In contrast, techniques such as the MN assay are faster and easy to perform. The 
existence of micronuclei has been recognized for many years. The linkage between 
micronuclei and DNA damage was well  known to early workers in the radiation field 
and their occurrence has been frequently mentioned in the early literature (Brenneke 
and Mather 1 937, Heddle et al. 1 983;  Russell and Russell, 1 954). Evans et al. ( 1 959) 
made the first attempt to use micronuclei as a monitor of cytogenetic damage by using 
the frequency of micronuclei to measure the chromosomal damage that had been 
induced in root-tips by fast neutrons and gamma rays in the presence and absence of 
oxygen. It was observed that all chromosomal breaks, as well as asymmetrical and 
incomplete symmetrical exchanges, had produced acentric fragments at mitosis, and 
that these fragments were frequently excluded from the daughter nuclei. The fragments 
appeared in the fol lowing interphase as micronuclei. 
Micronuclei are small extra-nuclear bodies that arise in mitotic cells from acentric 
chromosomal fragments or whole chromosomes that lag behind in anaphase and are not 
incorporated into the daughter nucleus (Rieger et al. 1 968;  Evans, 1 976 and Fenech, et 
al. 1 999). Hence, micronuclei provide a measure of both chromosomal breakage and 
chromosomal loss. It is worth mentioning here that the mechanistic origin of individual 
micronuclei can be determined. Micronuclei arising from lagging chromosomes can be 
identified by the presence of a kinetochore using anti kinetochore antibodies or by the 
presence of centromeric DNA sequences using FISH (Thomson and Perry, 1 988 ;  
Norppa et al. 1 993). Micronuclei that do not contain kinetochore/centromeric DNA 
sequences are interpreted to harbour acentric chromosomal fragments. 
2.3.2 Radiation Dose and Micronuclei 
Fenech et al. ( 1 990) applied the cytokinesis-block MN assay to measure chromosome 
damage in lymphocytes of 1 1  cancer patients undergoing fractionated partial-body 
irradiation. Measurements performed before, during, and after cessation of radiotherapy 
showed a dose-related increase in micronucleus frequency in each of the patients 
studied. A general decline in micronuclei frequency was observed during the post-
Chapter Two: Review of Literature. 1 0 1  
treatment period down to 5 7% after 1 2  months. However, there was considerable 
variation among individuals. 
In another study, the micronuclei of PBLs from radiation-exposed people were 
monitored by He et al. (2000), using the binucleated lymphocyte. Micronuclei 
frequencies in people with radiation-disease were radiation exposed, and a control 
group were found to be 1 .26%, 0.42% and 0 .33%, respectively. The micronuclei 
frequencies of patients with radiation-disease were significantly higher than other 
groups. The difference between the radiation-exposed group and the control group was 
not significant. Meanwhile, CA of the three groups was determined. The results were 
similar to those previously seen, while the micronuclei frequencies were 2.06%, 0.93% 
and 0.69%, respectively. CA rate of the radiation-disease group was significantly higher 
than other groups. The difference between the radiation-exposed group and the control 
group was not significant. The study indicates that the binucleated lymphocyte is a 
rapid, sensitive and accurate method, which can be used to monitor a large popUlation 
exposed to radiation. 
Fenech and Morley ( 1 985a) observed no effect of cytochalasin B on micronuclei 
production and the cytochalasin B method is simple to perform. The in vivo 
micronucleus frequency in normal human individuals was 4.4 - 2.6 micronuclei/500 
cytokinesis-blocked binucleated (CB) cells, and for lymphocytes irradiated in vitro 
there was a linear relationship between dose of radiation and number of induced 
micronuclei. 
Kryscio et al. (200 1 )  studied the long-term genotoxic effect of occupational radiation 
exposure of uranium miners. In this study the frequency of micronuclei with and 
without a centromere were scored using the cytochalasin B technique. It  is known that 
micronuclei can be comprised of acentric fragments and/or whole chromosomes. 
Micronuclei containing whole chromosomes were identified by means of FISH with a 
centromere-specific probe. The frequency and percentage of micronuclei with 
centromeres were analysed in lymphocytes taken from controls and from uranium 
miners who were exposed to large doses of radiation several decades ago. The miners 
were subdivided into those with and those without bronchial carcinoma. 
Chapter Two: Review of Literature. 1 02 
It had been shown previously (Bakou et al. 2002) that the relative frequency of 
micronuclei with centromeres decreased with dose, which meant that the number of 
micronuclei originating from acentric fragments increases . No statistically significant 
difference in the overall micronuclei frequency was seen between the analysed groups .  
The fraction of micronuclei with centromeres, however, was highest in  lymphocytes of 
controls (74 .6%) followed by healthy miners (62. 1 %) and those suffering from cancer 
(55 .8%). These results indicated the occurrence of a genomic instability in lymphocytes 
of miners, especially those with cancer, and it appeared that the low percentage of 
micronuclei with centromeres might be a marker of genomic instability and cancer 
predisposition. 
It is evident from the discussion above that there is a linear relationship between 
micronucleus frequency and radiation dose, but the relative frequency of micronuclei 
with centromeres decreased with higher dose. 
2.3.3 Radiosensitivity and Micronuclei 
In a study of breast cancer patients, Burrill et al. (2000) found that 30% of the patients 
were sensitive in a lymphocyte assay of radiation-induced chromosome damage 
(micronucleus induction) compared with 1 0% of healthy controls. Twenty-two first­
degree relatives of 1 1  sensitive patients had an average micronucleus yield significantly 
higher than that of 68 controls. In this assay, the authors suggested that radiosensitivity 
might be an inherited characteristic associated with predisposition to breast cancer. 
Erexson et al. ( 1 99 1 )  compared the radiosensitivity of human, rat and mouse PBLs by 
analysing frequencies of micronuclei in CB cells. For each species and dose, 4 ml  
aliquots of  whole blood were X-irradiated to obtain doses of  38, 75 ,  1 50 or 300 centi­
Grey (cGy). Controls were sham-irradiated. After exposure to X-rays, mononuclear 
leukocytes were isolated using density gradients and cultured in RPMI 1 640 medium 
containing phytohemagglutinin (PHA) to stimulate mitogenesis. At 2 1  h cytochalasin B 
was added to produce CB cells, and all cultures were harvested at 52  h post-initiation by 
cyto-centrifugation. Significant dose-dependent increases in the percentage of 
Chapter Two: Review of L iterature. 1 03 
micro nucleated CB cells and the number of micronuclei per CB cell were observed in 
all three species. 
2.3.4 Genetic Studies Using the MN Assay 
The genetic integrity of human populations is increasingly under threat due to certain 
industrial activities, which result in exposure to chemical and physical genotoxins. 
Other factors which can influence genetic damage include l ife-style factors (diet, 
smoking, alcohol), various medical therapies, and climatic changes (increased exposure 
to ultraviolet radiation due to depletion of atmospheric ozone) (Fenech & Neville, 
1 992) .  Fortunately, cells of human beings have evolved sophisticated mechanisms to 
repair any DNA breakages (Braithwaite et al. 1 998). If this did not happen, one would 
quickly become ill . If one's  DNA repair system is impaired it can be linked to a number 
of cancers and perhaps a range of il lnesses. Indeed some researchers view this as a 
causal link in tumorigenesis (Wei et al. 1 996 & Mohrenweiser, 1 998). 
The increased micronucleus frequencies in human subj ects i s  now under intense 
evaluation for its association to cancer risk (Chang & Mon-hsiung, 200 1 ;  Fenech et al. 
1 999). Many medical laboratories throughout the world conduct tests to monitor the 
efficiency of a cancer patient ' s  DNA repair system whilst undergoing radiotherapy 
(Scott, 2000) . The technique most commonly applied to measure the efficiency of an 
individuals DNA repair system is the MN Assay. 
Countryman and Heddle ( 1 976) first proposed the use of micronuclei as a measure of 
chromosomal damage in HPBLs.  In recent years this method has been improved with 
the development of the cytokinesis-blocked binucleated (CB) cell technique, which 
prevents cytokinesis without inhibiting nuclear division (Fenech and Morley, 1 985a and 
1 985b). This has enabled such cells to be recognized by their binucleate appearance. 
Cytochalasin B is an inhibitor of actin polymerisation which is required for the 
formation of the micro filament ring that constricts the cytoplasm between the daughter 
nuclei during cytokinesis (Carter, 1 967). Scott et al. improved this method in 1 998 and 
a slightly modified version of it was used in the present study . In this method Go 
lymphocyte cultures were irradiated with a high dose rate (HDR) of X-irradiation, 3 . 5  
Chapter Two: Review of Literature. 1 04 
Gray (Gy) at 1 Gy min- I . This pulverises the cel l ' s  DNA and caused breakages. The 
irradiated lymphocyte cultures were incubated at 3 7C under different experimental 
EMF conditions for a period of six hours to allow for the expression of differential 
DNA repair (Jones et al. 1 995), followed by mitogenic stimulation with the mitogen 
phytohaemagglutinin (PHA). After 24 h of mitogenic stimulation Cytochalasin B was 
added. In order to achieve the additional advantage of a higher yield of binucleate cells, 
lymphocytes were harvested at 90 h in lieu of the usual 72 h from the time of mitogenic 
stimulation. 
The MN assay has been used to measure the frequency of micronuclei that have 
occurred in peripheral blood lymphocytes (PBLs), which acts as a marker for the levels 
of DNA damage that have been induced in a cell (Fenech and Morley, 1 985a; Tucker 
and Preston, 1 996; Miller at al. 1 997; Kirsch-Volders, 1 997; Evans, 1 997). The 
technique has also been used to a lesser extent to study epithelial cells and fibroblasts 
(Fenech et al. 1 999). 
Using the MN assay it is a relatively simple task to score a large number of cells,  
compared to the classical metaphase analysis, which gives the MN assay greater 
statistical power (Fenech et al. 1 999) . Metaphase analysis can be highly tedious with 
only a small number of cells that can be scored. However, the MN assay allows 
thousands of cells to be scored in a relatively short time, which will give a statistically 
stronger result than results gained from metaphase analysis. Most laboratories using the 
MN assay usually score from 500 up to 2000 CB cells (Bonassi et al. 200 1) .  This is a 
highly recognized assay that provides reliable data when the scoring of micronuclei is 
limited to those cells that have completed one nuclear division (Fenech and Morley, 
1 986; Evans, 1 997; Fenech, 1 997; Fenech and Morley, 1 985a) .  
BRCA J or BRCA2 Protein 
The BRCA1 and BRCA2 gene products are believed to play an important part in the 
onset and/or development of many sporadic mammary cancers. It has been reported by 
Welcsh et al. (2000) that these two proteins contribute to a centrosome function, which 
is believed to help maintain the integrity of the chromosome segregation process. This 
Chapter Two: Review of L iterature. 1 05 
may mean that a reduced level of the BRCAl or BRCA2 protein in mammary cells will 
occasionally lead to non-disjunctional chromosomal loss or gain. Ban et al. (200 1 )  
reported that spontaneous micronuclei arising from chromosome( s) which fai l  to be 
incorporated into the relevant daughter nuclei during mitosis tend to occur more 
frequently in BRCAl- or BRCA2-defective human cancer cells than in BRCA-positive 
cancer cells. According to the author, some cases of mammary carcinogenesis might 
stem from the loss of integrity of chromosome segregation in cells, which have a 
reduced capacity to express either BRCAl or BRCA2. 
Turner's and Down 's Syndrome 
In a micronucleus induction study Scarfi et al. ( 1 996) studied the PBLs from 1 5  
subjects affected by Turner's syndrome (TS) and between 2 and 24 years of age to 
evaluate the spontaneous and Mitomycin-C (MMC) induced micronucleus frequency. A 
group of 1 5  healthy subj ects from the same age group were used as control .  As 
expected, statistically significant differences between spontaneous and MMC-induced 
micronuclei were found either in TS or in healthy subjects. Unexpectedly, when the two 
groups of donors were compared, TS subjects showed lower spontaneous and MMC­
induced micronucleus frequencies when compared with healthy subjects. Cell 
proliferation kinetic and cytotoxicity were also measured applying the cytokinesis-block 
proliferation index (CB PI) and the results showed that the MMC, at the employed 
concentration, does not induce cell cycle delay both in healthy and in TS donors .  When 
CBPI from TS and healthy donors were compared, a faster proliferation was observed 
in TS patients in both untreated and MMC-treated cultures. 
Similarly, Scarfi et al. ( 1 990) studied the PBLs from seven patients with Down's  
syndrome (DS; trisomy 2 1 )  and 14  healthy age-matched controls by the cytokinesis­
block method. The spontaneous incidence of micronucleus in lymphocytes from DS 
subjects was lower than that of control cultures. When lymphocytes were treated with 
MMC at the beginning of the culture period, an increase in micronucleus formation was 
found in cells from both DS and control subjects. In DS subjects this increase was much 
more marked than in control donors. This effect had to be ascribed to cells from older 
DS subjects (37-55 years old), which showed an MMC-induced micronucleus formation 
Chapter Two: Review of Literature. 1 06 
that was markedly and significantly higher than that observed in cells from younger (9-
16 years old) DS subjects. These data indicate that age has to be considered as a major 
variable when studies on the genetic instability of DS subjects are performed. 
2.3.5 Micronucleus Frequency as a Biomarker of Cancer Risk 
It is evident from epidemiological studies that long-term risks are associated with 
human exposure to mutagenic or carcinogenic agents. During the last few decades, 
many progressive changes have occurred in workplaces, in the environment and in 
lifestyles. Ultimately, this has resulted in different exposure patterns and exposures to 
new substances, and therefore new and more sensitive tools should be utilised to 
investigate cancer risk from these substances.  Moreover, there are aspects of 
metabolism and susceptibility previously unknown or poorly understood, such as 
metabolic polymorphisms, which can dramatically modify individual responses to toxic 
and carcinogenic agents. These features have made the identification of etiological 
factors more difficult, especially for cancers with a long induction period, and the use of 
traditional epidemiologic outcomes in many cases, such as cancer incidence or 
mortality, no longer seem sufficient for the evaluation of cancer risk in human 
populations. 
Biomarkers that have been validated for their predictive value may be used for the 
timely identification of increased cancer risk, which can be used in the prevention or 
control of disease. The assumption strengthening the use of a biomarker as a surrogate 
of genetic disease is that the observed relationship between exposure and the marker 
wil l  translate into a similar relationship between exposure and disease (Schatzkin et al. 
1 990). Confirmation of this assumption is crucial to the validation of the MN assay as a 
predictor of cancer. However, micronucleus frequency measured by the MN assay may 
not identify all chromosome damage events; e .g . ,  aberrations such as symmetrical 
reciprocal translocations are not expressed as micronuclei,  but asymmetrical 
translocations, such as dicentric chromosomes, and their associated acentric fragments 
may be observed as neoplasmic bridges and micronuclei, respectively. 
Chapter Two: Review of Literature. 1 07 
A causal relationship between micronucleus frequency and cancer risk could be inferred 
from studies of structural chromosomal aberrations (CA). A huge body of data has been 
published on the association between CA and neoplastic transformation, which supports 
the hypothesis that somatic chromosome damage is involved in cancer etiology (Tucker 
et al. 1 997) . Such damage can occur via chromosome breakage and the relative 
frequencies of each vary with the inducing chemical. 
A direct association between the frequency of micronuclei and cancer development is 
supported by the following findings: ( 1 )  Cheng et al. ( 1 996) and Duffaud et al. ( 1 997) 
observed an increased frequency of micronuclei in PBLs in cancer patients. (2) Rudd et 
al. ( 1 988) and Rosin and German ( 1 985) suggested that the subjects affected by 
congenital diseases such as Bloom syndrome or Ataxia telangiectasia have both 
abnormally high micronucleus frequencies and an increased risk of cancer. (3) Clinical 
chemo-prevention trials on oral pre-malignancies have used micronuclei in oral mucosa 
as a surrogate endpoint of cancer (Benner et al. 1 994; Desai et al. 1 996). (4) A 
suggestive correlation exists between carcinogenicity and genotoxicity for some agents 
able to increase micronucleus frequencies in humans and animals, e .g. ,  ionizing 
radiation, ethylene oxide, benzene, tobacco smoke (Sorsa et al. 1 992) . (5) Micronucleus 
frequency is strongly associated with the blood concentration of vitamins and folates, 
whose deficiencies are associated with increased risks for some cancers (Fenech and 
Rinaldi, 1 995 ;  Fenech et at. 1 997 and 1 998;  Blount et at. 1 997). All the findings 
mentioned above clearly suggest a causal link between an increase in micronucleus 
frequency and cancer. 
Enhanced sensitivity to the chromosome-damaging effects of ionizing radiation is a 
feature of many cancer-predisposing conditions. Scott et al. ( 1 994) reported that 42% of 
an unselected series of breast cancer patients and 9% of healthy control subjects with 
elevated chromosomal radio-sensitivity of lymphocytes irradiated in the G2 phase of the 
cell cycle. They suggested that in addition to the highly penetrant genes BRCAl and 
BRCA2, which confer a very high risk of breast cancer and are carried by about 5% of 
all breast cancer patients, there are also low-penetrance predisposing genes carried by a 
much higher proportion of breast cancer patients, a view supported by epidemiological 
studies (Teare et al. 1 994; Chen et at. 1 995). Ideally, testing for the presence of these 
Chapter Two: Review of L iterature. 1 08 
putative genes should involve the use of simpler methods than the G2 assay, which 
requires metaphase analysis of chromosome damage. Scott et al. ( 1 998) reported on the 
use of a simple, rapid MN assay in GO lymphocytes exposed to high dose rate (HDR) or 
low dose rate y-irradiation, with delayed mitogenic stimulation. In this assay, they 
obtained a good assay reproducibility, particularly with the HDR protocol, which 
identified 3 1  % ( 1 2  out of 39) of breast cancer patients compared with 5% (2 out of 42) 
of healthy controls as having elevated radiation sensitivity. According to the authors, in 
the long term, such cytogenetic assays may have the potential for selecting women for 
intensive screening for breast cancer. This HDR method was also employed during this 
study to determine whether or not weak EMFs affect the DNA repair system in humans 
to any degree. 
2.3.6 Effects of Age and Gender on Micronucleus Frequency 
Several studies (Fenech and Morley, 1 986; Bolognesi et al. 1 997) have investigated the 
role of age as a confounding factor in cytogenetic bio-monitoring. Research by these 
groups has found that there is an age-related increase in the frequency of micronuclei. 
Fenech and Morley ( 1 986), to overcome the kinetic problems that are found when they 
have used the human lymphocytes for MN assays, developed the cytokinesis-block 
micronucleus technique. Using this technique the number of spontaneous micronuclei in 
lymphocytes from 42 individuals aged between 20 and 85 years was studied and was 
found to increase at a rate of 4.3% per year. Comparison with the results obtained with 
the conventional MN assay confirmed that the conventional method markedly 
underestimates this age effect. The sensitivity of the cytokinesis-block (CB) method 
was determined by studying the effect of low-dose (less than 50  rad*) X-irradiation. 
The results indicated that the dose-response was linear and a single in vitro exposure to 
5 rad of X-rays could be unequivocally detected. They concluded that the CB method 
was more sensitive and precise than the conventional MN assay and classical metaphase 
*Rad (rd) : A unit of energy absorbed from ionising radiation, equal to 1 00 ergs per 
gram or 0.0 1 joules per kilogram of irradiated material. The Gray (Gy) has replaced it 
as a standard scientific unit. 
Chapter Two: Review of Literature. 1 09 
analysis, and predicted that the MN assay would be of value for detecting chromosome 
damage induced in vivo by genotoxic agents. 
Various authors have reported intra- and inter-individual variations of baseline 
frequencies of cytogenetic end points in lymphocytes of human populations. Personal 
characteristics seem to account for a significant proportion of this variability (Fenech, 
1 999; Bolognesi et at. 1 997a). For this reason Fenech (personal communication) has 
established certain criteria for the application of the MN assay. All studies should be 
designed to allow for the detection of five micronuleated cells per 1 ,000 binucleated 
cells between an experimental group and their controls. 
Bolognesi et al. ( 1 997a) quantitatively evaluated the effect of age on SCE, CA, and 
micronuclei through the analysis of a population sample that included data from several 
bio-monitoring studies performed over the last few decades in 1 2  Italian laboratories .  
The large size of the data set, more than 2 ,000 tests for each end point, allowed them to 
estimate the independent effect of age, gender, smoking habits, occupational exposure, 
and inter- and intra-laboratory variability. A greater frequency of the mean standardised 
values with increasing age was observed for all of the end points. A levelling off was 
evident in the last age classes in the trend of micronuclei frequencies. Frequency ratios 
(FRs), which express the increase of the cytogenetic damage with respect to the first age 
classes, 1 - 1 9  years, were estimated using Poisson regression analysis after adjustment 
for potential confounding factors and confirmed the increasing trend by age class for all 
three end points . The most dramatic increase was observed for micronuclei, with a FR 
that approaches the values of 2 at the age class 50-59 and remains mainly unchanged 
thereafter. The trend of FRs for CA is more homogeneous, with a constant rise even in 
the older classes, whereas the frequency of SCE increases with age to a lesser extent, 
reaching a plateau in the age class 40-49 and the maximum value of FR in the age class 
over 70. Their results pointed to an age-related increase of the chromosome damage in 
lymphocytes and emphasise the need to take into account the potential confounding 
effect of this variable in the design of bio-monitoring studies based on chromosome 
damage. 
Chapter Two: Review of Literature. 1 1 0 
In a biomonitoring study, Bolognesi et al. (2002) investigated whether exposure to 
complex pesticide mixtures in ornamental crop production represents a potential 
genotoxic risk. Exposed and control subjects were selected in western Liguria (Italy) .  
The area was chosen for its intensive use of pesticides. The main crops produced were 
roses, mimosas, carnations and chrysanthemums, as ornamental non-edible plants, and 
tomato, lettuce and basil, as edible ones. The levels of micronuclei were analysed in 
PBLs of 1 07 floriculturists (92 men and 15  women) and 61  control subjects (42 men 
and 1 9  women). A statistically significant increase in binucleated cells with micronuclei 
(BNMN) was detected in floriculturists with respect to the control population. The 
mean number of BNMN varied as a function of gender and age. Smoking habit had no 
effect on micronuclei frequency. A positive correlation between years of farming and 
micronuclei frequency in peripheral blood lymphocytes was also observed. The 
conditions of exposure were also associated with an increase in cytogenetic damage, 
with a 28% higher micronuclei frequency in greenhouse workers compared with 
subjects working only outdoors in fields. Workers not using protective measures during 
high exposure activities showed an increase in micronuclei frequency. Their findings 
suggest a potential genotoxic risk due to pesticide exposure. 
In another study, Bakou et al. (2002) used FISH to evaluate spontaneous and 
aneuploidogen-induced micronucleus frequencies and non-disjunction of chromosomes 
X and 8 in cultured binucleated lymphocytes of women of two age groups .  
Demecolcine and vincristine were used as model aneuploidogens to induce micronuclei 
and chromosome mal segregation. Four of the women were aged between 22-26 (mean 
24.3)  years and the other four were 47-50 (mean 49.0) years. Pancentromeric FISH was 
applied to micronuclei to identify chromosomes and double-colour centromeric FISH 
performed in binucleates of two young and two older women to assess the involvement 
of chromosomes X and 8 in micronuclei and non-disjunction. This confirmed that age 
increases the frequencies of micronuclei .  Micronuclei that contained entire 
chromosomes were found predominantly in o lder females .  Age also enhanced 
micronuclei containing acentric chromosome fragments. The inclusion of chromosomes 
X and 8 in micronuclei was enhanced by age, and chromosome X was generally over­
represented. Non-disjunction of chromosomes X and 8 also increased with age,  
chromosome X being the more sensitive. Treatment of lymphocytes with vincristine and 
Chapter Two: Review of Literature.  1 1 1  
demecolcine increased micronucleus frequency and mal segregation of chromosomes X 
and 8 in both age groups. Comparison of the estimated frequencies of micronucleation 
and non-disjunction for all human chromosomes showed that non-disjunction is the 
main type of chromosome malsegregation. 
Chromosomal damage as measured by frequency of translocations, acentric fragments, 
telomere shortening, nondisjunction, chromosome loss, aneuploidy, and micronucleus 
formation has been shown to increase progressively with age. Fenech et at. ( 1 998) used 
the cytokinesis-block micronucleus technique, which provides an efficient measure of 
chromosomal breakage and loss, to show that aging can explain at least 25% of the 
variation in chromosomal damage rate in lymphocytes from both males and females. He 
also performed cross-sectional and placebo-controlled intervention studies to determine 
the relationship between the micronucleus frequency in lymphocytes and diet, and 
blood status for vitamins C, E, B 1 2, and folic acid. Their studies have shown that 
micronucleus frequency in  the 4 1 -60 year age group i s  significantly lower in 
vegetarians when compared to non-vegetarians, however the reverse was found to be 
true in males aged between 20 and 40 years. This was accounted for by a deficient/low 
B 1 2  status in vegetarian males; there was no difference in the micronucleus frequency 
of vegetarian and nonvegetarian subjects aged between 6 1  and 90 years. Results from 
this study also showed significant negative correlations of micronucleus frequency with 
folic acid and vitamin B 1 2  but not with vitamin C or vitamin E. 
Furthermore, the spontaneous micronucleus yield in lymphocyte cultures (Fenech and 
Morley, 1 985a) from healthy donors aged 0-82 years was estimated at 72 h and 96 h of 
culture . At both 72 and 96 h there was a positive correlation of micronucleus expression 
with increasing age, with an approximately four-fold increase in micronuclei in cultures 
from 80-year-old donors when compared to cultures from newborn donors. Since there 
is some evidence that the effect of DNA-damaging agents may increase with age, 
lymphocytes from individuals of various ages were exposed to X-rays and mitomycin C 
and micronuclei were scored after 72 and 96 h of culture . The total number of 
micronuclei formed after exposure to these agents were, however, decreased in cells 
from elderly individuals,  most l ikely due to kinetic differences between the 
lymphocytes of old and young individuals. 
Chapter Two: Review of Literature. 1 1 2 
To conclude the above section, the important point to note i s  that there is a clear 
indication of age related increase in micronucleus frequency. 
2.3.7 MN Assay and the Influence of Chemical and Environmental Agents 
EMF Exposure 
In an elegant study, Simk6 et al. ( 1 998) studied the effects of ELF EMFs exposure in 
humans. In their study, human squamous cell carcinoma cell line (SCL II) and an 
amniotic fluid cell line (AFC) was used. Cells were exposed to 50 Hz, 0. 1 - 1 .0 mT, MFs 
for durations of 24, 48 and 72 h. Micronucleus frequency and apoptic cells were scored 
from this study. A statistically significant increase of frequency of micronuclei and the 
induction of apoptosis in SCL I I  cells after both 48 h and 72 h of continuous exposure 
to 50 Hz MFs (0.8 and 1 .0 mT) were found. However, exposure of AFC cells to MFs of 
different intensities and for different exposure times showed no statistically significant 
differences when compared to controls. Dose-dependent induction of apoptosis and 
genotoxic effects, resulting in increased micronucleus formation, could be demonstrated 
in the transformed cell l ine, whereas the non-transformed cell l ine did not show 
statistically significant effects. 
Several epidemiological studies (Wertheimer and Leeper, 1 979; Savitz et al. 1 988; and 
London et al. 1 99 1 )  have suggested that EMFs are associated with increased incidence 
of cancer. To test the carcinogenic potency of EMFs, the in vitro MN assay with Syrian 
Hamster Embryo (SHE) cells was used as a screening method for genotoxicity in a 
study by S imk6 et al. (2001 ). A 50  Hz, 1 mT strength MF was applied either with or 
without the tumour initiator benzo(a)pyrene (BP) or the tumour promoter 1 2-0-
tetradecanoylphorbol- 1 3-acetate (TPA) . All three treatments were applied in single, 
double or triple treatment regimes. MF or TP A (1 nM) alone did not affect the number 
of micronuclei in initiated and non-initiated SHE cells. Changing the schedule of the 
typical initiation protocol, namely by applying the initiator BP during exposure to MF, 
results in a 1 .8-fold increase in micronuclei formation compared to BP treatment alone. 
A combined experiment with BP,  TP A and MF exposure did not cause further 
Chapter Two: Review of Literature. 1 1 3 
micronuclei formation. S ince initiation during MF exposure caused a significant 
increase in micronuclei formation, their findings suggest that MFs enhance the initiation 
process of BP. It was concluded that this MF -enhanced co-carcinogenic effect is caused 
by an indirect "cell activation" process. The resulting genomic instability is proposed to 
be due to free radicals and/or to the unscheduled "switching-on" of signal transduction 
pathways. 
Scarfi et al. ( 1 999) studied the micronucleus induction and cell proliferation in HPBLs 
cultured in vitro and exposed to a 50 Hz sinusoidal MF for 72 h at different intensities 
( 1 .0,  0 .75,  0 .5 ,  0 .25, and 0.05 mT). A total of 42 healthy human donors, aged between 
26 and 54 were used. The cytokinesis-block MN assay results showed no genotoxic 
effects for the field intensities tested. However, cell proliferation was slightly elevated. 
In a separate genotoxic evaluation study, Scarfi et al. ( 1 994) exposed HPBL cultures to 
50 Hz pulsed MFs in order to evaluate a possible genotoxic effect of such non-ionizing 
radiation. The genotoxic effect was evaluated in terms of both micronucleus induction 
and classical CAs; the mitotic index (MI) was also calculated. In this study lymphocytes 
from five healthy human donors were examined using the tests mentioned above. No 
genotoxic effects were found, but an increased MI was found in exposed samples 
compared to the controls .  
CAs, micronuclei, and SCEs were analysed by Heimers (2000) in HPBLs of 1 8  
Concord pilots who were exposed to cosmic radiation and 1 0  controls. It was found that 
there was an 8-fold significant increase of dicentric chromosomes in the Concord group. 
The yield of micronuclei was also significantly elevated. However, SCEs in the 
Concord group did not differ from the controls. 
Abramsson-Zetterberg and Grawe (200 1 )  used the flow cytometer-based MN assay to 
study the effects on the chromosomes of erythroid cells of CBA/Ca mice after an 
extended exposure to a 5 0  Hz, 1 4  JlT MF. The study included two different 
experiments: (a) mice exposed in utero during 1 8  days of their prenatal stage, and (b) 
adult mice exposed for 1 8  days. In experiment (a) the mice were exposed 35 days in 
total and then peripheral blood was drawn from the exposed mice to determine whether 
Chapter Two: Review of L iterature. 1 1 4 
the exposure had a genotoxic effect on the pluripotent erythroid stem cells .  About 
200,000 polychromatic erythrocytes (PCE) and 200,000 normochromatic erythrocytes 
(NCE) were analysed from each of 20 exposed mice. The EMF exposure did not 
significantly change the frequency of micronucleated PCE or NCE in comparison with 
20 sham-irradiated mice. There was no difference in the proportion of PCE between 
exposed and unexposed animals .  Similarly, in experiment (b) no differences were seen 
between EMF -exposed and unexposed adult mice when samples of peripheral blood 
were taken at the end of exposure and analysed for micronuclei in PCE and NCE. The 
proportion of PCE was similar in both groups .  The results indicate that exposure to 
EMF does not induce direct or indirect effects on chromosomes in erythroid cells 
expressed as increased levels of micronucleated erythrocytes of mice. No indications of 
delayed genetic effects were found. 
Lagroye and Poncy ( 1 997) used the cytokinesis-blocked MN assay to investigate a 
possible amplification of the genotoxic effects of ionizing radiations in cells exposed to 
combined static or power-frequency EMFs. In this study, rat tracheal epithelial cell lines 
were used. First, the cells were exposed in vitro to 60CO y-radiation (either 0, 2 or 6 Oy) 
and then cultured for 24 h in a homogeneous sinusoidal 1 00 J.!T EMF (50 Hz) combined 
with an artificial geomagnetic-like field created by the use of horizontal and vertical 
pairs of Helmholtz coils .  Control cells were cultured in an adjacent incubator where the 
background EMF was approximately 0 . 1 J.!T. Under the in vitro experimental 
conditions, the EMF showed no significant direct effect on micronucleus induction in 
rat tracheal cell lines. However, an increased frequency of EMF-exposed binucleated 
cells with micronuclei was observed in cells exposed to 6 Oy of y-rays compared with 
controls. 
Livingston et al. ( 1 99 1 )  studied human lymphocytes and Chinese Hamster Ovary 
(CHO) fibroblasts for cytogenetic and cytotoxic endpoints to determine whether 
exposure to power frequency (60 Hz) EMFs interfered with normal cell growth and 
reproduction. An exposure chamber was built in order to apply variable electric current 
densities of 3 ,  3 0, 3 00,  and 3 ,000 microampere per square centimetre (J.!A/cm2) 
simultaneously with a fixed MF of 0 .22 mT to proliferating cells. The current densities 
were chosen to bracket those that may be induced in the human body by fields 
Chapter Two: Review of Literature. 1 1 5 
measured beneath high voltage (765 kV) power transmission lines. The electric current 
was applied through the media of a cell culture chamber positioned between two 
stainless steel e lectrodes but separated from direct contact with the culture media by a 
salt bridge composed of a 1 % agarose gel . The MF was generated using two pairs of 
Helmholtz coils driven 73 degrees out of phase producing an elliptically polarised MF 
36 degrees out of phase with the electric field. The EMFs were measured and mapped 
inside the cell culture chamber to ensure their uniformity. CHO cells were exposed 
continuously for 24-96 h (depending on the experiment) and human lymphocytes were 
exposed continuously for 72 h. The EMFs were monitored throughout the entire 
treatment period using a multi channel chart recorder to verify continuous application of 
the desired fields. SCE and micronuclei were monitored to evaluate the potential for 
genotoxicity. In addition, standard growth curves, clonogenicity, and cell cycle kinetics 
were analysed to evaluate possible cytotoxic effects. The results showed no effects on 
both cell types exposed to the EMFs. 
The micronucleus can serve as a suitable indicator for the assessment of exposure to 
potentially genotoxic agents such as radio-frequency electromagnetic radiation (RFR) 
and the analysis  of mitotic activity as an additional parameter for efficient bio­
monitoring. In a study by Garaj -Vrhovac ( 1 999), the effects of RFR on the cell kinetics 
and genome damages in PBLs were determined in lymphocytes of 1 2  subjects 
occupationally exposed to RFR. Their results showed an increase in the frequency of 
micronuclei, as well as disturbances in the distribution of cells over the first, second and 
third mitotic division in exposed subjects compared to controls. 
Haider et al. ( 1 994) used the Tradescantia-micronucleus bioassay to determine the 
genotoxic effects of short-wave EMFs ( l 0-2 1 MHz) which is used for broadcasting. In 
their experiment, plant cuttings bearing young flower buds were exposed to this short­
wave EMFs for 30  h. A statistically higher micronucleus frequency than controls were 
observed at all exposure sites except one in the immediate vicinity of the antenna. 
Hence, they concluded that short-wave radiation from the broadcasting antennae had a 
clastogenic effect. 
Chapter Two: Review of Literature. 1 16 
Tice et al. (2002) studied the potential genotoxicity of radio frequency signals emitted 
by cellular telephones. The in vitro studies evaluated the induction of DNA and 
chromosomal damage in human b lood leukocytes and lymphocytes,  respectively . The 
signals were voice-modulated 837 MHz signals and produced by an analog signal 
generator or by a time division multiple access cellular telephone. 837 MHz was 
generated by a code division multiple access cellular telephone (not voice modulated), 
and voice modulated 1 909.8 MHz was generated by a global system of mobile 
communication-type personal communication systems cellular telephone. 
In this study, DNA damage was assessed in leukocytes using the alkaline (pH> 1 3) 
single cell gel electrophoresis assay, which i s  also known as the CO MET assay. 
Chromosomal damage was evaluated in lymphocytes mitogenically stimulated to divide 
post-exposure using the cytochalasin B-binucleate cell .  Cells were exposed at 37  ± 1 C, 
for 3 or 24 h at average specific absorption rates (SARs) of 1 .0- 1 0.0 W/kg. Exposure for 
either 3 or 24 h did not induce a significant increase in DNA damage in leukocytes, nor 
did exposure for 3 h induce a significant increase in micronuc1eated cells among 
lymphocytes. However, exposure to each of the four RP signal technologies for 24 h at 
an average SAR of 5.0 or 1 0.0 W/kg resulted in a significant and reproducible increase 
in the frequency of micronucleated lymphocytes. The magnitude of the response 
(approximately 4-fold) was independent of the technology, the presence or absence of 
voice modulation, and the frequency (837 vs. 1 909.8 MHz). This research demonstrates 
that under extended exposure conditions, RF signals at an average SAR of at least 5 .0 
W /kg are capable of inducing chromosomal damage in human lymphocytes. 
In another study,  Vijayalaxmi et al. (200 1 )  studied the chromosomal damage and 
micronucleus formation in HPBLs exposed in vitro to RFR at a cellular telephone 
frequency (847.74 MHz). In this study, peripheral blood samples collected from four 
healthy nonsmoking human volunteers were diluted with tissue culture medium and 
exposed in vitro for 24 h to 847.74 MHz RPR (continuous wave), a frequency employed 
for cellular telephone communications. 
A code division multiple access technology was used with a nominal net forward power 
of 75 W and a nominal power density of 950 W/m2 (95 mW/cm2) .  The mean SAR was 
Chapter Two: Review of L iterature. 1 1 7 
A code division multiple access technology was used with a nominal net forward power 
of 75 W and a nominal power density of 950 W/m2 (95 mW/cm2) .  The mean SAR was 
4.9 or 5 . 5  W/kg. Blood alliquots that were sham-exposed or exposed in vitro to an acute 
dose of 1 .5 Gy of y-radiation were included in the study as controls. The temperatures 
of the medium during RFR and sham exposures in the Radial Transmission Line facility 
were controlled at 37  ± 0 . 3C .  Immediately after the exposures, lymphocytes were 
cultured at 37 ± 0.3C for 48 or 72 h. 
The extent of genetic damage in this study was assessed from the incidence of CAs and 
micronuclei. The kinetics of cell proliferation was determined from the mitotic indices 
in 48 h cultures and from the incidence of binucleate cells in 72 h cultures. The data 
indicated no significant differences between RFR-exposed and sham-exposed 
lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, 
excess fragments, binucleate cells, and micronucle i .  The response of y-irradiated 
lymphocytes was significantly different from that of both RFR-exposed and sham­
exposed cells for all of these indices. Thus, the authors concluded that there was no 
evidence for induction of CAs and micronuclei in HPBLs exposed in vitro for 24 h to 
847.74 MHz RFR (CDMA) at SARs of 4.9 or 5 .5  W/kg. 
In a similar study, Vijayalaxmi et al. (2001 )  carried out cytogenetic investigations using 
HPBL exposed in vitro to RFR at a cellular telephone frequency of 835 .62 MHz. 
Freshly collected peripheral blood samples from four healthy human volunteers were 
diluted with RPMI 1 640 tissue culture medium and exposed to 83 5 .62 MHz RFR in 
vitro in T -7 5 tissue culture flasks for 24 h. This frequency was employed for customer­
to-base station transmission of cellular telephone communications. An analog signal 
was used, and the access technology was frequency division multiple access 
(continuous wave) . A nominal net forward power of 68 W was used, and the nominal 
power density at the centre of the exposure flask was 860 W 1m2 . The mean SAR in the 
exposure flask was 4.4 or 5 .0 W/kg. 
Aliquots of diluted blood that were sham-exposed or exposed in vitro to an acute dose 
of 1 . 50  Gy of y-radiation were used as negative or positive controls, respectively. 
Immediately after the exposures, the lymphocytes were stimulated with PHA, a 
Chapter Two: Review of L iterature. 1 1 8 
mitogen, and cultured for 48 or 72 h to determine the extent of genetic damage, as 
assessed from the frequencies of CAs and micronuclei. 
The extent of alteration in the kinetics of cell proliferation was determined from the 
mitotic indices in 48 h cultures and from the incidence of binucleate cells in 72 h 
cultures. The data, however, indicated no significant differences between RFR-exposed 
and sham-exposed lymphocytes with respect to mitotic indices, incidence of exchange 
aberrations, excess fragments, binucleate cells, and micronuclei .  In contrast, the 
response of the lymphocytes exposed to y-radiation was significantly different from 
both RFR- and sham-exposed cells for all of these indices. 
In a study by Hook et al. ( 1 999) the possible effect of wireless phone signals (analog, 
TDMA, CDMA and PCS) on micronuclei formation in human lymphocytes was 
explored. No effects were found after three hours of exposure at SARs of 0, 1 , 2 .5,  5 or 
1 0  W/kg for any of the signals tested. At 5 W/kg, increases of lesser magnitude were 
observed from analog and TDMA exposure. Finite Difference Time Domain (FDTD) 
calculations indicated peak SAR values may have reached as much as twice the average 
SAR values in parts of the cell pellet and repeat studies were planned to attempt to 
clarify the findings and determine whether sample heating was a confounding factor. In 
the following repeat studies the authors observed clear evidence of DNA disruption and 
micronuclei formation, which means that the cellular DNA had divided unequally . 
Vijayalaxmi et al. ( 1 997) exposed aliquots of peripheral blood samples collected from 
two healthy human volunteers to continuous wave 2450 MHz RFR. The exposures were 
either continuously for a period of 90 min or intermittently for a total period of 90 min 
(30 min on and 30 min off, repeated three times) . B lood aliquots that were sham­
exposed or exposed in vitro to 1 50 cGy y-radiation served as controls .  
The continuous wave 2450  MHz RFR was generated with a net forward power of 34 .5  
W and transmitted from a standard gain rectangular antenna horn in a vertical ly 
downward direction. The mean power density at the position of the cells was 5 .0 
mW/cm2. The mean SAR calculated by FDTD analysis was 1 2 .46 W/kg. Lymphocytes 
Chapter Two: Review of Literature. 1 1 9 
There were no significant differences between RFR-exposed and sham-exposed 
lymphocytes with respect to: (a) mitotic indices; (b) incidence of cells showing 
chromosome damage; (c) SCE; (d) acentric fragments; (e) binucleate cell, and (t) 
micronuclei, for either the continuous or intermittent RFR-exposures. In contrast, the 
response of positive control cells exposed to 1 50 cOy y-radiation was significantly 
different from RFR-exposed and sham-exposed lymphocytes. Thus, there is no evidence 
for an effect on mitogen-stimulated proliferation kinetics or for excess genotoxicity 
within 72 h in human blood lymphocytes exposed in vitro to 2450 MHz RFR. 
Diener and Eberle ( 1 996) investigated the possible effects of RF exposure (400 MHz, 
900 MHz, and 1 . 8 OHz) on gene and chromosome mutations and cell growth. 
Exposures ranged from 39 to 70 h resulted in no effects on CA rate, SCE, micronucleus 
frequency or cell proliferation. 
Several in vitro studies have also been conducted to detect effects of microwave 
exposure on the genetic material. In one study, Zotti-Martell i  et al. (2000) investigated 
the in vitro genotoxic effect of microwave exposure in HPBL. Lymphocytes were 
exposed in 00 to EMFs with different microwave frequencies (2.45 and 7.70Hz) and 
power density ( 1 0, 20 and 30  mW/cm2) for three times ( 1 5 , 3 0  and 60 min). The results 
showed that microwaves are able to cause cytogenetic damage in human lymphocytes, 
but only at high power density (30 mW/cm2) and long exposure times (30 and 60 min). 
Fucic et al. ( 1 992) chose a new mathematical approach to separate clastogenic from 
aneugenic activity of three well-known mutagens (vinyl chloride monomer, X-rays and 
microwaves) on the genome of human somatic cells .  The comparison of frequencies of 
size distribution of micronuclei in the lymphocytes of humans exposed to each of these 
three mutagens showed that X-rays and microwaves were preferentially clastogens 
while vinyl chloride monomer showed aneugenic activity as well .  Microwaves possess 
some mutagenic characteristics typical of chemical mutagens. 
Oaraj -Vrhovac et al. ( 1 992) exposed human whole-blood samples to continuous 
microwave radiation (frequency 7.7 OHz, power density 0 .5 ,  1 0  and 30 mW/cm2 for 1 0, 
30  and 60 min) . A correlation between specific CAs and the incidence of micronuclei 
Chapter Two: Review of Literature. 1 20 
Garaj -Vrhovac et al. ( 1 992) exposed human whole-blood samples to continuous 
microwave radiation (frequency 7.7 GHz, power density 0 .5 , 1 0  and 3 0  mW/cm2 for 1 0, 
3 0  and 60 min) . A correlation between specific CAs and the incidence of micronuclei 
after in vitro exposure was observed. In all experimental conditions, the frequency of all 
types of CAs was significantly higher than in the control samples . In the irradiated 
samples the presence of dicentric and ring chromosomes was established. The incidence 
of micronuclei was also higher in the exposed samples. The results of the structural CA 
test and of the micronucleus test were comparatively analysed. The values obtained 
showed a positive correlation between micronuclei  and specific CAs (acentric 
fragments and dicentric chromosomes).  The results of this  study indicated that 
microwave radiation causes changes in the genome of somatic human cells and that the 
applied tests are equally sensitive for the detection of the genotoxicity of microwaves .  
Cossarizza et  al. ( 1 989) examined the question of whether EMFs interfere with DNA 
repair following y-irradiation. In these experiments lymphocytes were stimulated with 
PHA for 66 h, SUbjected to 100 Gy of 60CO y-radiation, pulsed with [ 3H]thymidine, and 
subjected to pulsed EMFs for 6 h. The later 6 h period corresponds to the normal period 
of radiation damage DNA repair. Their data indicate that post-exposure to EMFs did not 
interfere with the normal repair processes induced by y-irradiation. 
Micronuclei in lymphocytes have been examined (Livingston et al. 1 99 1 ;  Scarfi et al. 
1 99 1 ,  and 1 994) to determine if their electric or pulsed MFs affect chromosomal 
breakage or induce mis-segregation. The micronuclear test is a sensitive genotoxic 
marker that "evaluates the frequency of small round bodies derived from chromosome 
fragments or whole chromosomes whose centromeres have lost their affinity for the 
mitotic spindle" (Scarfi et al. 1 99 1 ) . Again, no effects were found in these studies. 
Maes et al. ( 1 993) and Vijayalaxmi et al. ( 1 998) observed an increase in micronucleus 
in HPBLs and peripheral blood and bone marrow of cancer-prone mice, respectively 
when they applied much stronger MFs 2.45 GHz microwave RFR. In Maes et al. ( 1 993) 
study, HPBLs were exposed to microwaves at 2.45 GHz. They observed a marked 
increase in the frequency of CAs (including dicentric chromosomes and acentric 
fragments) and micronuclei. On the other hand the microwave exposure did not 
Chapter Two: Review of Literature. 1 2 1  
influence the cell kinetics or the SCE frequency. Formation of  dicentric chromosomes is 
considered to be the "hallmark" of ionizing radiation exposure. However, Vijayalaxmi 
et af. ( 1 998) exposed peripheral blood and bone marrow of cancer-prone mice 
chronically to 2450 MHz microwave RFR and observed a significant increase in 
micronuclei. 
It is  evident from the review of different studie s  mentioned above that EMFs do 
influence micronucleus frequency, and this can serve as a suitable indicator for the 
assessment of exposure to potentially genotoxic agents. Thus, the MN assay was 
included in the present study. 
Exposure to Cytostatic Drugs 
Kasuba et af. ( 1 999) used the MN assay to study the genotoxic effect of occupational 
exposure of 20 nurses who handled cytostatic drugs in medical oncology and 
haematology units . The duration of employment in the units and of exposure to 
cytostatic drugs ranged from 1 to 3 1  years. The exposed nurses showed an increased 
proportion of cells with micronuclei as compared to the control group. Nurses exposed 
to cytostatic drugs for 20-3 1 years showed a higher frequency of micronuclei, whereas 
there was no difference in frequencies between the control group and the group exposed 
for 1 - 1 4  years.  The influence of the exposure period proved to be a significant 
parameter for the MN assay. 
To evaluate genotoxicity of cytostatic drugs, 1 00 employees who were occupationally 
exposed to cytostatic drugs in hospitals and/or pharmacies, were studied using the MN 
assay by Hessel et al. (2001 ) . A total of 247 peripheral blood samples were collected 
from different exposure levels. The levels of exposure were "baseline level" ,  i .e . ,  
without any cytostatic drugs exposure before recruiting or after at least 3 weeks without 
cytostatic drugs contact and at three times (cycle 1 -3) post-exposure. Blood samples 
from 60 office employees served as controls. 
Statistical analyses were performed under consideration of age,  gender and X-ray 
exposure . The frequency of micronuclei was significantly related to the age of the 
Chapter Two: Review of Literature. 1 22 
subjects . However, there were no significant differences in the frequencies of 
micronuclei between controls and exposed hospital workers. Similarly, micronucleus 
rates were not significantly different at the various sampling time points and there was 
no correlation between duration of employment and micronucleus rates. 
In another study, Telez et al. (200 1 )  investigated the genotoxic potential of chronic 
long-term therapy with the antihypertensive drug nimodipine, using SCEs and 
micronuclei in HPBLs of patients with long-term exposure to this drug. HPBLs of 
control individuals exposed in vitro to nimodipine were also studied to assess the effect 
of the drug itself. FISH using a centromeric probe was performed to determine the 
origin of the induced micronuclei. The in vivo study was carried out on five patients 
under antihypertensive treatment with nimodipine. The in vitro study was performed on 
five control individuals by adding the drug to the culture medium at a final 
concentration similar to the levels found in plasma (controls/medium). The in vivo study 
showed no genotoxic effects of long-term therapy with nimodipine because the 
frequencies of SCE and micronuclei in exposed patients did not show significant 
differences as compared with control individuals. A statistically significant increase in 
the frequency of micronuclei was detected in controls/medium as compared with 
control individuals without the drug. FISH analysis revealed statistically significant 
differences with respect to the frequency of centromeric signals in nimodipine-induced 
micronuclei in vitro. With regard to the in vivo results, chronic long-term therapy with 
nimodipine is not associated with increased genotoxicity . The differing results in vivo 
and in vitro could be due to extensive metabolism of nimodipine, indicating that the 
cytogenetic effect observed was due to the drug itself rather than its metabolites or to an 
adaptive response to nimodipine in vivo. 
Exposure to Pesticide 
The potential cytogenetic damage associated with pesticide use in Greek agricultural 
workers was evaluated by Pastor et al. (200 1 )  using the frequency of micronuclei as 
bio-markers in lymphocytes of peripheral blood and exfoliated cells of the buccal 
mucosa. In addition, the effects of pesticide exposure and other variables on the 
cytokinesis block proliferation index (CB PI)  in lymphocytes were also evaluated. 
Chapter Two: Review of Literature. 1 23 
Micronuclei were analysed in 50 agricultural workers exposed to pesticides (30 men 
and 20 women) and in 66 non-exposed individuals that constituted the control group (4 1 
men and 25 women). The comparison between workers and controls did not reveal any 
statistical significant difference in the micronuclei frequency for either lymphocytes or 
buccal cells .  Regarding CBPI, the value found in the exposed group was lower than in 
controls, the difference being statistically significant. 
Exposure to Genotoxic Chemicals 
Micronucleus frequency from PBLs of two groups of workers from hospital and 
cyclophosphamide (CP) processing industries who were potentially exposed to CP were 
analysed using the MN assay by Yager et al. ( 1 988). Increased numbers of micronuclei 
were observed in both exposed groups compared to controls .  The finding was 
independent of the age of the subjects, which was also correlated with micronuclei 
formation. 
Exposure to certain chemical agents in occupational settings has been identified as 
carcinogenic to the human bladder. Micronucleus analysis in exfoliated urothelial cells 
is an interesting method for biomonitoring genetic damage in human populations. In 
one of the few studies to have been performed in an occupational context, Fontana et al. 
(2001 )  performed the MN assay using exfoliated urothelial cell s  to examine whether the 
occupational use of a mineral jelly induced a genotoxic risk for workers employed at a 
factory producing bearings. The prevalence of micronucleated exfoliated urothelial cells 
(MNC) was determined in 35 female workers with dermal exposure to the jelly and 4 1  
female controls. They observed a significant occupational effect on the prevalence of 
MNC.  This may be due to the presence of mutagens/carcinogens in the jelly: an 
aromatic amine, N-phenyl- 1 -naphthylamine, which is carcinogenic in mice, or sodium 
nitrite, which is genotoxic in human cell systems. These results suggest that the use of 
the mineral j elly could present a genotoxic risk for workers and the MN assay on 
exfoliated cells could be valuable for biological monitoring purposes in occupational 
contexts as a marker of significant exposure to bladder mutagenic/carcinogenic agents. 
Chapter Two: Review of Literature. 1 24 
Oliveira et al. (200 1 )  report on the genotoxicity of the boron neutron capture (BNC) 
reaction in human metastatic melanoma cells assessed by the MN assay using p-borono­
L-pheny lalanine (BP A) as the boron delivery agent. Different concentrations of BP A 
(0.48, 1 .2 and 2 .4  mM) and different influences of thermal neutrons were studied. 
Substantial genotoxic potential of alpha and lithium particles generated inside or near 
the malignant cell by the BNC reaction was observed in a dose-response manner as 
measured by the frequency of micronucleated binucleated melanoma cells and by the 
number of micronuclei per binucleated cell .  The distribution of the number of 
micronuclei per micro nucleated binucleated cell was also studied. The BNC reaction 
clearly modifies this distribution by increasing the frequency of micronucleated 
binucleated cells with two or three micronuclei and consequently decreasing the 
frequency of micronucleated cel l s  with one micronucleus. A decrease in cell 
proliferation was also observed which correlated with micronuclei formation. 
In a study by Nesti et al. (2000), primary liver fibroblasts were studied in a MN assay in 
combination with FISH using two protocols. In protocol-A (Prot. A), cytochalasin B 
was added at the end of the treatment time directly to the medium containing the 
standard compounds, whereas, in protocol-B (Prot. B) the chemical-containing medium 
was removed and fresh medium with cytochalasin B was added. The study was 
performed using a fungicidal antibiotic griseofulvin (GF) and the clastogen mitomycin 
C (MMC) as standard compounds. With both protocols GF induced a significant 
increase in micronuclei frequency over controls in a dose-related manner at the lower 
concentrations tested (7 .5  and 1 5  !lg/ml). At the highest dose (30 !lg/ml) the antibiotic 
effect was substantially reduced. Micronuclei induction obtained with Prot. A was 
significantly higher (approximately 3 -fold) than with Prot. B .  Micronuclei induced by 
MMC showed a dose- and time-dependent increase in both protocols. In contrast to GF, 
the greater clastogenic response induced by MMC in human l iver fibroblasts was 
obtained with Prot. B, approximately 3 -fold higher than Prot. A and approximately 2-
fold with 24 h treatment at 0 . 1 7  !lg/ml MMC. FISH analysis showed that MMC induced 
mainly micronuclei containing acentric fragments rather than whole chromosomes. 
These researchers demonstrated that chemically induced genetic effects are strongly 
dependent on the cell culture employed, treatment schedule and intra- and post­
treatment experimental conditions. 
Chapter Two: Review of L iterature. 1 25 
The in vivo rodent MN assay is widely used as a cytogenetic assay to detect the 
clastogenic activity of  a chemical in vivo. The MN assay is one of three tests 
recommended by the fourth International Conference on Harmonisation (ICH4) of 
Genotoxicity Guidelines and as such, many regulatory authorities have accepted it. 
However, the determination of a positive result in a genotoxicity test, including MN 
assay, has been an issue of debate among toxicologists and biometricians. Kim et at. 
(2000) compared several statistical procedures that have been suggested for the analysis 
of MN assay data and indicate which one is the most powerful. The standard protocol of 
MN assay has at least three dose levels plus the control dose and uses at least four 
animals per group. For each animal, two thousand polychromatic erythrocytes (PCE) 
were counted. 
In a genotoxicity study, female mice of hybrid strain B6C3F l , 8- 1 0  weeks old were fed 
on powdered food with or without 2% caffeic acid (Raj et al. 1 983) .  Caffeic acid (3 ,4-
dihydroxy cinnamic acid) is widely distributed in plant materials in both free and 
combined forms and, as such, is a component of the human diet. After one week on 
these diets, some of  each group of mice were injected with 7, 1 2-dimethyl 
benz[a]anthracene (25 mg/kg) dissolved in dimethyl disulfoxide (DMSO). In the course 
of separate experiments, bone-marrow samples were taken at various intervals after 
injection for analysi s  in the MN assay .  From each mouse, 500 polychromatic 
erythrocytes were scored to determine the frequency of micronuclei. At the time at 
which the maximum response was observed, which differed between experiments, the 
frequency of micronuclei induced by 7, 1 2-dimethyl benz[a]anthracene (DMBA) was 
reduced by 50% by the presence of caffeic acid. The results suggest that caffeic acid 
provides significant protection against the genotoxicity of DMBA. 
Meng and Zhang ( 1997) studied the frequencies of CA and micronuclei in PBLs of 40 
workers at a phosphate fertiliser factory in North China. Hydrogen fluoride (HF) and 
SiF 4 are the main air pollutants and small amounts of dust containing fluoride, NH3, and 
S02 were also present in the factory . The mean observed frequencies per 1 00 
metaphases of major CA type (chromosome rings, translocations, and dicentrics) of the 
workers and the non-exposed controls were 0 .9 1  and 0 .24, respectively. The average 
percentages of lymphocytes with micronuclei of the workers and the controls were 1 .55 
Chapter Two: Review of Literature. 1 26 
± 0 .7 1  and 0 .62 ± 0.54, respectively. The authors concluded that both the CA and 
micronuclei frequency of the workers increased with the length of the chemical 
exposure period. 
Anaesthetic Gases 
To minimise the possible health risks posed by waste anaesthetic gases the National 
Institute of Occupational Safety and Health (NIOSH, USA, 1 994, publication 94- 1 00) 
& Health and Safety Executive (HSE, London, 1 994, Guidance note EH 40/94) 
recommends certain exposure limits. Wiesner et al. (200 1 )  investigated the genotoxicity 
of a previously established occupational exposure exceeding these l imits (high-level 
exposure) and of one within these limits (low-level exposure) .  Genotoxicity was 
assessed by the formation of micronucleated lymphocytes in 25 anaesthetists and 
anaesthetic nurses of an Eastern European (High-Level Exposure Group) and a German 
(Low-Level Exposure Group) university hospital. Each exposed group was compared 
with a group of non-exposed personnel of the same hospital . Compared with the control 
group, there was an increased fraction of micronucleated lymphocytes per 1 ,000 
binucleated cells in the High-Level Exposure Group but not in the Low-Level Exposure 
Group. They conclude that a high-level exposure to inhaled anaesthetics is associated 
with an increase in chromosome damage, and measures were recommended to decrease 
exposure levels. 
Occupational exposure to anaesthetic gases is associated with various adverse health 
effects. Genetic material has been shown to be a sensitive target of numerous harmful 
agents. Rozgaj et al. (200 1 )  examined a group of 43 hospital workers of three 
professions (anesthesiologists, technicians and operating room nurses) and 26 control 
subj ects for CAs, SCEs, and micronucleus frequency. The exposed groups were 
matched in duration of exposure to anaesthetics, but not in age. An equal ratio between 
women and men was possible in all groups except nurses. L ikewise, the ratio between 
smokers and non-smokers was also not comparable. An increase in chromosome 
damage was found in all exposed groups. While the increase in SCE frequency was not 
significant, CAs and micronucleus frequency increased significantly, showing higher 
Chapter Two: Review of Literature. 127 
rates in women. Their results suggest that the micronucleus test i s  the most sensitive 
indicator of changes caused by anaesthetic gases. 
Air pollution and Micronuclei Frequency 
Atmospheric pollution represents a relevant environmental hazard, which has been 
associated with considerable excess mortality, morbidity, and increased rates of 
respiratory diseases in humans. To date, more than 3 ,000 environmental chemical 
compounds have been identified in the ambient atmosphere, including a variety of 
mutagenic and/or carcinogenic agents, such as polycyclic aromatic hydrocarbons 
(PAHs), aromatic amines, and heterocyclic compounds (Bolognesi et al. 1 997b; Waiter 
& Crinnion, 2000). Positive associations between cytogenetic markers and airborne 
levels of P AHs have been reported by experimental and human studies. Traffic has been 
implicated as the major determinant for the concentration of PAHs and, therefore, for 
the genotoxic activity of urban air. 
The Tradescantialmicronuclei test (TRAD/MCN) is a well-validated test for monitoring 
environmental genotoxicants. These pollutants induce at the early meiotic stage of 
pollen mother cells chromosome fragments, which become micronuclei at the tetrad 
stage. The standard test protocol requires several hours of exposure of the 
inflorescences and a recovery time of about 24 h to reach the early tetrad stage. Since 
the recovery period represents a critical step of the TRAD/MCN, experiments were 
performed by Falistocco et al. (2000)  to establish the appropriate length of time in 
plants for clone 4430 of the hybrid T hirsutiflora X T subacaulis, which is widely used 
in environmental monitoring. The aim of the research was to acertain the exact duration 
of recovery time in order to improve the sensitivity of the TRAD/MCN test. First, 
studies were performed to select the flowers at the beginning of meiosis, and then 
anthers were sampled and studied for a period of 48 to 86 h. To study complete meiosis 
in the plants required approximately 80 h. Second, exposure to genotoxic substances 
followed by different recovery times was performed in order to demonstrate that the 
effectiveness of the TRAD/MCN test is closely related to the duration of the recovery 
time. In order to conduct the tests, the inflorescences were exposed to known mutagens 
(sodium azide and maleic hydrazide) for 6 h followed by different recovery times (24-
Chapter Two: Review of Literature. 1 28 
72 h) . The results from these experiments showed that the frequency of micronuclei in 
the pollen mother cells increased with the length of the recovery time. 
S imilarly, Batalha et al. ( 1 999) designed a study to determine the clastogenicity of 
particulate matter (with an aerodynamic diameter smaller than 1 0 �m) in the urban 
polluted air in the city of Sao Paulo. The TRAD/MCN assay was used throughout this 
study to evaluate the clastogenicity of the extracts of the particulate matter. 
Tradescantia pallida (Rose) Hunt. cv. purpurea, an indigenous cultivar, was used in the 
TRAD/MCN assay. The efficacy of this plant material for the TRAD/MCN assay was 
validated with dose-response studies using formaldehyde and beta radiation. Dose­
response curves were established with these known mutagens. The extracts of the PMI O  
particles at concentrations between 5 and 5 0  ppm induced a dose-related increase in 
MCN frequencies. The results indicate that T. pallida is equally sensitive to mutagens 
as the standard Tradescantia clone 4430 or 03 and the particulate matter in the urban air 
are clastogenic to the chromosomes of this plant. Inhalation of these particles by urban 
dwellers may affect their health by inducing similar genetic damage. 
Alcohol 
In a genotoxic exposition study, Ramirez and Saldanha (2002) used the MN assay as an 
indicator of genotoxic exposition, as it is associated with CAs. An increased mutation 
rate in oral squamous cells, indicated by an increased micronucleus frequency, is also 
related to the development of oral carcinomas. The frequencies of micronuclei and other 
metanucleated anomalies in the buccal squamous cells of 30  alcoholics with oral or 
oropharyngeal carcinomas were evaluated and compared them to a control group of 
abstinent healthy individuals. Microscopic examination was made of 2,000 cells per 
individual from each of three distinct areas of the mouth: around the lesion (A), 
opposite to the lesion (B) and in the upper gingival-Iabial gutter (C). Area C was used 
as a control region because of low tumour frequency. A 7-fold increase in micronucleus 
frequency in region B, 3-fold increases in region A, and a 2-fold increase in C, was 
observed. The authors concluded that the MN assay could be used for monitoring 
clinical evaluation, by means of intra- and inter-individual cellular comparisons. 
Chapter Two: Review of Literature. 1 29 
Diet 
Fenech & Neville ( 1 992) investigated whether the consumption of cooked meat could 
induce chromosome damage. In their study, the bone-marrow MN assay was used. They 
observed no difference in the micronucleus frequency of mice on normal diet (cereal­
based, non-purified diet) and mice on nonnal diet supplemented with microwaved meat. 
However, supplementing the normal diet with well-done pan-fried meat, or rare 
charcoal-barbecued meat, or well-done charcoal-barbecued meat produced significant 
increments in  the micronucleus frequency of polychromatic erythrocytes .  The 
increments were of the order of 73%, 90%, and 1 36%, respectively and they were 
observed after a 2 1 -day feeding trial. These results suggest that ingestion of well-done 
pan-fried or barbecued meat may increase genetic damage; however, the accompanying 
decreased intake of vegetable constituents may have also contributed to the observed 
changes. 
2.3.8 Concentration of Cytochalasin B 
Scarfi et al. ( 1 993) carried out a study in order to set up a standardised quantitative 
assay for spontaneous micronuclei in bovine lymphocytes .  For this purpose the 
cytochalasin B micronucleus method, originally proposed by Fenech and Morley 
( 1 985a) for human lymphocytes, and was applied to PBLs of 20 healthy cows of the 
Italian Friesian breed.  The results demonstrate that the optimal concentration of 
cytochalasin B to obtain the highest frequency of binucleated cells was 6 Jlg/ml. The 
baseline frequency of spontaneous micronucleus formation in 500 binucleated cells was 
1 2 . 3  ± 4 . 1 ,  3 -times higher than that reported in human lymphocytes (Fenech and 
Morley, 1 985a, and Scarfi et al. 1 99 1 ) .  
As  a comparative system, Erexson e t  al. ( 1 987) developed a procedure to test mouse 
PBLs using the modified technique for assessing micronuclei frequency in human PBLs 
described by Fenech and Morley ( 1 985a) .  In this assay, male mice (C57B 1 I6) were 
injected with five different doses (0, 2 .5 ,  5 .0, 7 .5,  or 1 0 .0 mg) of diaziquone (AZQ)/kg. 
After 24 h the mice were bled by cardiac puncture, PBLs were isolated on a Ficoll­
density gradient and then cultured in RPMI 1 640 medium using 8 Jlg of PHA/ml .  In 
Chapter Two: Review of Literature. 1 3 0  
some cultures cytochalasin B was added at 2 1  h during the medium change to block 
cytokinesis. In other cultures, cytochalasin B was omitted to compare the sensitivity of 
analysing micronuclei in binucleate versus unblocked mononucleate cells.  All doses of 
AZQ yielded significant increases in micronuclei-containing binucleated PBLs. The use 
of cytochalasin B in the mouse PBL MN assay increased the sensitivity by 
approximately 3 -fold. The authors concluded that the MN assay in mouse PBLs should 
be useful in comparative cytogenetic studies of mice and humans. 
The review above clearly indicates that environmental agents and lifestyle factors do 
influence the frequency of micronucleus and thus affect the DNA repair mechanism. In 
the following section (Section 2.4), the COMET assay which has been employed to 
assess any possible damage to the DNA as a consequence of ELF EMF exposure is 
discussed. Section 2.4 includes definitions of comet and COMET assay, mechanism of 
comet tail formation, use of COMET assay as a tool in the studies of testing 
genotoxicity, DNA damage, and effects of EMF exposure. It is concluded that the 
COMET assay should be included in the present study. 
2.4 The COMET Assay 
2.4.1 Introduction 
The COMET assay is one of several methods developed over the last two decades to 
measure DNA strand breaks produced in individual cells .  This assay, which is also 
known as the single-cell gel assay (SCG) or microgel electrophoresis (MGE), is an 
effective test for measuring genotoxicity, investigating DNA repair and apoptosis, or 
monitoring populations for exposure against an environmental agent with clastogenic or 
genotoxic potentiality. 
In 1 978, Rydberg and lohanson first estimated DNA damage in individual cells by a 
novel technique. In their experiment, human lymphocytes were mixed in agarose to 
make microgels on microscopic slides. They then lysed the cells and unwound DNA 
using sodium hydroxide. The number of pieces of single stranded DNA that were 
generated under these alkaline conditions depended upon the number of breaks in the 
Chapter Two: Review of Literature. 1 3 1  
double stranded DNA. Using acridine orange (AO), an intercalating metachromatic dye 
(AO fluoresces green when bound to double stranded DNA and red when bound to 
single stranded DNA at optimum concentrations), they quantified greenness and redness 
for estimating DNA damage. 
In 1 984, Ostling and 10hanson introduced a microgel electrophoresis technique under 
neutral conditions for detecting DNA damage at the level of a single cell after y­
irradiation. In their technique, cells embedded in agarose were placed on a microscope 
slide, lysed by detergents and high salt, and the l iberated DNA electrophoresed under 
neutral conditions. Ethidium bromide was used to stain the DNA.  The image obtained 
looked like a "comet" with a distinct head containing intact DNA, and a tail comprising 
damaged or broken pieces of DNA. Hence the name "COMET" assay was given. The 
extent of DNA liberated from the head of the comet was a function of the dose of 
irradiation. However, thi s  procedure is able to detect only DNA double-strand 
breakages (DSBs). A short time later, the alkaline version of the COMET assay was 
introduced by Singh et al. ( 1 988). In this version of the COMET assay, electrophoresis 
was performed under high alkaline (pH > 1 3) conditions for detecting DNA damage in 
single cells .  Unlike the former method, this assay could detect DNA single-strand 
breakages (SSBs) and DSBs, alkaline-labile sites that are expressed as SSBs, and 
transient DNA strand breaks arising due to DNA repair processes. Under certain 
conditions, the assay can also detect DNA-DNA and DNA-protein crosslinking, which 
appears as a relative decrease in DNA migration compared with concurrent controls 
(Hartmann et al. 2003) .  Due to its high pH, which denatures the DNA, fragments 
caused by SSBs are released so they can migrate through a gel when exposed to an 
electric current. This has made the assay more sensitive than the neutral version and it 
has become the assay of choice for DNA damage investigators. This assay was used in 
the current study. 
Evidence of DNA damage is gained by observing the cell under an epifluorescent (or 
similar) microscope and information can be gained using quantitative or qualitative 
methods. The comet tail can be analysed qualitatively according to its DNA content 
(indicated by the intensity of the stain) and by comparing this value to control (healthy) 
Chapter Two: Review of Literature. 1 32 
cells .  Scoring can be made according to low, medium, or high intensity tail DNA 
content. 
Quantitative methods involve recording values such as tail migration distance, image 
length, nuclear size, and using these values to calculate the tail moment, which is a good 
indicator of DNA damage. Several software packages (such as Komet) are available and 
with the aid of a fluorescent microscope and a digital camera these quantitative values 
can be quickly analysed. 
The COMET assay has widespread applications in genotoxicity testing compared with 
other assays. This is because: 1 )  it is highly sensitive for detecting low level of DNA 
damage; 2) it has widespread applications in genotoxicity testing (Tice, 1 995;  Anderson 
et al. 1 998; Rojas et al. 1 999) ;  3 )  it requires a small number of cells per sample; 4) it is 
relatively inexpensive and studies can be performed even with a low budget; 5) it is 
easy to apply; and 6) samples can be prepared and evaluated very quickly by visual 
scoring or by an image analytical system (Collins et al. 1 993).  
2.4.2 Mechanism of Comet Tail Formation 
The application of the alkaline COMET assay in different genotoxicity studies has 
increased exponentially although the underlying mechanism of comet tail formation has 
not been entirely resolved. Ostling and Johanson ( 1 984) first proposed that strand 
breaks might be responsible to stretch out DNA loops during electrophoresis, and hence 
form the comet tail. Relaxation of loops was also proposed to be the primary underlying 
basis for comet formation under alkaline conditions (Collins et al. 1 997). On the other 
hand, Klaude et al. ( 1 996) presented an excellent approach to demonstrate that the 
comet tail under neutral conditions consists of relaxed loops, whereas, comet tails under 
alkaline conditions consist of DNA fragments .  
Chapter Two: Review of Literature. 1 33  
2.4.3 The COMET Assay in Genotoxicity Testing 
DNA strand break assays have been used to test the genotoxicity of various chemical 
and physical agents i n  a variety of cells .  Peripheral blood is the usual source for 
screening a large number of subjects exposed to genotoxic agents . DNA single strand 
breaks and alkali labile sites are easiest to detect and are by far the largest number of 
lesions in DNA in general. They are relevant to human health as they have been 
correlated with mutagenicity, teratogenicity, cancer, aging (Newton et al. 1 989; Elia et 
al. 1 99 1 ;  Chicca et al. 1 996 and Ames, 1 998) and cell death (Sorensen et al. 1 998). 
The majority of X-ray-induced lesions are SSBs or alkali labile sites, which are 
converted to SSBs under alkaline conditions (Moran and Wallace, 1 985) and can be 
detected by alkaline micro gel electrophoresis. Similarly, other carcinogenic chemical or 
physical agents produce oxidative lesions in DNA and most of these lesions are either 
DNA SSBs or alkali labile sites (A verbeck et al. 1 993 and Melvin et al. 1 998). DNA 
DSBs are more important but less frequent lesions in DNA. Their repair is more 
complicated and even a few unrepaired breaks lead to cell death (Tounekti et al. 1 995;  
Ahmad e t  al. 1 998) . DNA DSBs also have been correlated to mutagenicity, 
carcinogenicity, teratogenicity and aging (Chicca et at. 1 996; Mayer et at. 1 989 and 
Lieber et at. 1 998). 
Many types of cells are suitable for detection of SSBs / alkali labile sites using the 
alkaline microgel electrophoresis assay. For example, single cells from invertebrates 
(Salagovic et at. 1 996) and plants (Koppen and Verschaeve 1 996) can be analysed for 
DNA damage using the COMET assay. The neutral comet method has been used to 
identify DNA damage in irradiated meats, fish and seeds (Creda et al. 1 997). The 
COMET assay is such a sensitive tool to detect DNA damage that SSBs caused by as 
low as 5 cOy irradiation is possible without the necessity of radiolabeling DNA (Singh 
et al. 1 994; Malyapa et al. 1 998). Sperm cells, however, naturally carry abundant alkali­
labile sites (Singh et al. 1 989). Therefore, it was not possible to see the effects of even 
very high doses of radiation in well lysed and proteinase-K-treated cells in microgels. In 
1 997, Singh and Stephens introduced a neutral version of the micro gel electrophoresis 
assay to detect X-ray-induced DNA damage in human lymphocytes (Singh and 
Chapter Two: Review of Literature. 1 34 
Stephens, 1 997).  Using this neutral version they were able to detect DNA DSBs in 
sperm cells (Singh and Stephens, 1 998). 
Cell viability is an important factor in COMET assay procedures and greater than 95% 
viability should be considered for analysis if only 50  cells per sample are analysed 
(Singh, 2000). It is not possible to assess cell viability on cells from solid tissues (Singh 
& Lai, 1 998). This is due to the fact that most of the techniques which involve physical 
separation of cells from tissues, are associated with the disruption of cell membranes at 
desmosomal (cell to cell junctions) level. In the present study, in each slide, 1 0  III of 
cell suspension having approximately 1 0,000 cells was mixed with 90 III of Low 
Melting Temperature Agarose (LMA 1 % per volume, Life Technologies Inc.). 
Comparative investigations were performed between the COMET assay and the SCE 
test, which is known to be very sensitive in detecting chemical mutagens (Hartmann et 
at. 1 994; Hartmann and Speit, 1 995) .  For most of the chemicals these researchers 
observed similar sensitivity for the two assays. The only exceptions were chemicals 
inducing DNA-crosslinks, which were not sensitively detected with the standard version 
of the COMET assay. Meanwhile, modifications were introduced which can not only 
detect DNA-DNA or DNA-protein crosslinks, but even enable the investigator to 
distinguish between the different types of crosslinks (Pfuhler et at. 1 996; Merk and 
Speit, 1 999). 
In another comparative study, Betti et al. ( 1 994) compared the COMET assay and SCE 
to determine the better method for detecting DNA damage. They used these methods to 
establish if damage had occurred in the DNA in individuals as a result of smoking. 
They took samples from one smoker and one non-smoker of the same ages every 3 
months for a year and found that the comet tail of lymphocytes taken from the smoker 
was significantly longer than that of the non-smoker. This meant that the smoker' s  
DNA had been damaged more than that of  the non-smoker. When comparing the 
COMET assay and SCE they were able to detect the effects of smoking in a small 
sample using the COMET assay where SCE analysis had failed. 
Chapter Two: Review of Literature. 1 3 5  
2.4.4 EMF Exposure and COMET Assay 
Lai and Singh ( 1 997b) exposed live rats to a 60 Hz MF (flux densities 0 . 1 ,  0.25 and 0.5 
mT) for 2 h .  With the aid of the COMET assay method at 4 h post-exposure they 
observed a dose-dependent increase in DNA strand breaks in brain cells. An increase in 
single-strand DNA breaks was observed after exposure to MFs of 0. 1 ,  0.25, and 0.5 mT, 
whereas an increase in double-strand DNA breaks was observed at 0 .25 and 0.5 mT. 
Similarly, exposure to RFR, 2450 MHz, at a whole body specific absorption rate (SAR) 
of 0 .6 and 1 .2 W/kg for 2 h caused an increase in both SSBs and DSBs in DNA of brain 
cells in the rat (Lai and Singh 1 995,  1 996). 
In another EMF study, Ahuja et al. ( 1 999) exposed HPBLs to 5 doses (2, 3 , 4, 5 and 1 0  
mT, 50  Hz) of MF and they observed significant increase i n  DNA strand breaks 
compared to sham-exposed controls in each magnetic flux density except one. In this 
study comet tail lengths were used as a measure of DNA damage. Using the COMET 
assay, Svedenstal et al. ( 1 999a) also observed an increase in DNA strand breaks in brain 
cells of mice after 32 days of exposure to MFs at a low intensity of about 8.0 JlT. 
Similarly, an increase in DNA strand breaks in brain cells of mice after 1 4  days of 
exposure to a low intensity of 0.5 mT MFs were observed by Svedenstal et al. ( 1 999b). 
Human diploid fibroblasts were used in a study by Ivancsits et al. (2002) in order to 
evaluate the genotoxic effects of ELF EMFs in the form of DNA SSBs and DSBs. In 
their study, human diploid fibroblasts were exposed continuously or intermittently (5 
min field-on/ 1 0  min field-off) to EMFs ( l mT, 50 Hz, sinusoidal) for 24 h and then 
subj ected to both the alkaline and the neutral COMET assays .  They observed a 
significant increase in DNA DSBs only for intermittent fields compared to sham­
exposed controls. There was no observed difference between the continuous field­
exposed and the sham-exposed cells .  
2.4.5 Evaluation and Interpretation of Results 
The widespread applicability of the COMET assay in genotoxicity testing is reflected 
by the wealth of data published in the last few years. However, Collins et al. ( 1 997) 
proposed a precaution with the interpretation of increased effects in the COMET assay. 
Chapter Two: Review of Literature. 136  
According to them, the COMET assay detects primary DNA lesions, such as  DNA 
strand breaks. These lesions may not be of much relevance since they may be repaired 
error-free or represent transient repair sites. In order to study comet-effects in more 
detail ,  Collins et at. ( 1 997) proposed to use lesion-specific enzymes in human 
monitoring studies. Furthermore, COMET assay data from biomonitoring studies should 
always be discussed in conjunction with results of other tests run in parallel .  
Interestingly, in a study by Hartmann et al. ( 1 998) with more than 90 individuals they 
observed increased comet values in leukocytes of waste disposal workers and also 
detected increased CAs, but observed no correlation between these two parameters. 
2.4.6 DNA Damage and Use of COMET Assay 
The use of pesticides has been increasing in recent years, resulting in the need for 
increased production of pesticides. However, some pesticides may represent a hazard to 
human health, especially by causing cancer. In one study, Grover et al. (2003) evaluated 
the genetic damage in workers employed in pesticide production using the COMET 
assay. Blood leukocytes of a group of 54 pesticide workers and an equal number of 
control subj ects were used. The two groups had similar mean ages and smoking 
prevalence .  The mean comet tail length was used to measure DNA damage. The 
exposed workers had significantly greater mean comet tail lengths than those of 
controls. Smokers had statistically significant larger mean tail lengths than non-smokers 
did. Further analysis of covariance showed that occupational exposure and smoking had 
significant effects on mean tail length, whereas age and gender had no effect on DNA 
damage. 
In another study, higher DNA damage was observed by Baltaci et al. ( 1 998) in a group 
of women who suffered from habitual abortion compared to normal controls. A 
statistically significant difference in the tail lengths was observed using the COMET 
assay on the blood samples obtained from these groups. Although these women did not 
suffer from any systematic disorder, other than their abortion history, it seems that 
chromosomal abnormalities and DNA damage detected by the COMET assay plays an 
important role in women with a history of recurring abortions. However, it was not 
determined if emotional stress is a factor in these cases. 
Chapter Two: Review of Literature. 1 37 
One of the most important aspects of cancer i s  the loss of genomic stability. To assess 
genomic instability Colleu-Durel et al. (200 1 )  studied 1 9  patients who suffered from 
sporadic breast cancer. The COMET assay was used to study the lymphocytes of 
patients before radiotherapy and/or chemotherapy. The study was conducted on cells 
which were or were not exposed to various levels of irradiation. The results show that 
the patients have higher baseline values than controls.  At an exposure of 2 .0  Gy, 
damage score, the mean tail moment, and the percentage of DNA in the tail increased 
for both groups but these values are much higher for patients. It is clear from their 
results that the lymphocyte DNA of cancer patients is more easily damaged than that of 
controls of the same group, and subsequently, a hypothesi s  was put forward that the 
baseline DNA damage reflects a genomic instability in sporadic breast cancer. This 
instability seems to increase following in vitro irradiation. 
An interesting study on the use of the COMET assay in studying the relationship 
between EMF exposure and apoptosis was conducted by Robison et al. (2002). They 
found that EMF exposure protects heat induced apoptosis in human cancer cell lines in 
a time dependent manner. In their study, HL-60, HL-60R, and Raj i cell lines were 
grown in a 0 . 1 5  mT 60 Hz sinusoidal EMF for 4 to 24 h. After induction of apoptosis, 
cells were analyzed by the neutral comet assay to determine the percentage of apoptotic 
cells . To discover the duration of this protection, cells were grown in the EMF for 24 h 
and then removed for 24 to 48 h before heat shock and neutral comet assays were 
performed. Their results showed that EMF exposure offers significant protection from 
apoptosis after 1 2  h of exposure and that protection can last up to 48 h after removal 
from the EMF. They also demonstrated the effect of the EMF on DNA repair rates .  
DNA repair data were gathered by exposing the same cell  l ines to the EMF for 24 h 
before damaging the exposed cells and non-exposed cells with H202• Cells were 
allowed to repair for time periods between 0 and 1 5  min before analysis using the 
alkaline comet assay. Their results showed that EMF exposure significantly decreased 
DNA repair rates in HL-60 and HL-60R cell lines, but not in the Raji cell line. 
In a similar study using HL-60 cells, Ding et al. (2004) found that although the 
magnetic field itself (60 Hz, 5mT) could not induce apoptosis, it did exert a promoting 
effect on H202-induced cell death. 
Chapter Two: Review of Literature. 1 38  
2 .5  Fluorescence In Situ Hybridization (FISH) 
2.5. 1 Introduction 
Increased chromosomal translocation is one of the known features of radiation effects in 
humans, which has the potential to lead to ill health, particularly a variety of cancers or 
inherited defects which may be passed on to the next generation. In the present study, to 
investigate the incidence of translocation frequency in PBLs induced by EMF exposure, 
the fluorescent in situ hybridization (FISH) technique was employed. In situ 
hybridization was first introduced in 1 969 simultaneously by two groups of scientists 
(Pardue and Gall, 1 969; John et al. 1 969) . The technique has multiple uses in molecular 
morphology due to its unique capability of visualizing nucleic acid sequences without 
altering the cell ' s  cytological or chromosomal integrity. F ISH uses fluorescent 
molecules to vividly paint genes or chromosomes. This technique i s  particularly useful 
for gene mapping and for identifying chromosomal abnormalities .  The usefulness of 
FISH is also growing rapidly in genomics, cytogenetics, prenatal research, tumour 
biology, radiation labels,  gene mapping, gene amplification, and basic biomedical 
research. 
In principle, the technique is quite straightforward. A stretch of DNA, which matches 
the area of interest in the genome, is labeled with nucleotides linked to a fluorochrome 
and this labeled DNA is called the probe. When double stranded DNA in the probe or in 
the cell is heated, the hydrogen bonds holding the strands together will open up. When 
the heated probe mixture is put onto heated cellular DNA that has been fixed on a slide, 
the probe wil l  align with the matching cellular DNA. As the slide cools slowly, new 
hydrogen bonds will form between the probe strands and the cellular strands. When 
stimulated with the correct wavelength, the fluorochrome linked to the probe will l ight 
up the region of the cellular DNA of interest. 
Although a number of different hybridization protocols exist, the variations among them 
are relatively minor. The basic steps are outlined below: (a) tissue i s  denatured to create 
single-stranded DNA; (b) denatured probe is applied to the slide or tissue; (c) slide or 
tissue is incubated overnight to allow hybridization I reannealing; (d) the hybridization 
reaction identifies, or labels, target genomic sequences. After washing and possible 
Chapter Two: Review of L iterature. 1 39 
signal amplification, their location and size is identified for the cellular DNA of interest 
and studied by fluorescent microscopy. 
2.5.2 Advantage, and Limitations of FISH Analysis 
FISH is appl icable to several types of tissue preparations : fresh or frozen tissue, 
cytological preparations, previously Wright-stained smears (or fresh smears stored at -
70C), frozen tissue sections, formalin-fixed paraffin-embedded tissue and previously 
immunostained cells (Krahl, 1 999 and Christine et al. 2002) . The main advantage of 
FISH lies in its relative rapidity with which it can be performed as compared to 
conventional metaphase cytogenetic evaluation. FISH also allows analysis of terminally 
differentiated tumour cells, which never enter mitosis, or tumours with low mitotic rate. 
Conventional cytogenetic evaluation of such tumours is difficult or impossible. Another 
advantage lies in the increased number of cells studied when utilizing FISH, as opposed 
to metaphase cytogenetics. This abi lity to study large numbers of cells allows detection 
of low frequency abnormalities, such as detecting minimal residual disease or early 
relapse. On the other hand, FISH has limitations too. One of the main limitations of 
FISH as a molecular tool is  signal fading. Furthermore, FISH requires significant 
technical expertise to obtain consistent results and a comprehensive set up with a good 
fluorescent microscope, computer, and a digital camera. 
In terms of sensitivity and resolution, FISH is better than karyotyping and comparative 
genomic hybridization (CGH), but worse than polymerase chain reaction (PCR)-based 
assays for detecting small nucleotide alterations. CGH is a new approach of FISH 
(Kallioniemi et al. 1 992) that allows one to screen losses and gains in DNA copy 
number along the entire genome in a single hybridization (Du Manoir et al. 1 993) .  
Conventional FISH procedure is limited to alterations of several Mb in size, whereas 
CGH can be designed to detect single-base mutations. Since FISH probes are typically 
at least 30  Kb in size, alterations need to be equally large for reliable detection. On the 
other hand, PCR is  more sensitive than FISH for the detection of abnormal fusion 
transcripts resulting from translocation; it picks up as few as one per million cells. 
Chapter Two: Review of L iterature. 140 
2.5.3 Translocation Study and the Use of FISH 
So far no studies have been reported on the application of FISH in cultured cells 
exposed to ELF EMFs. Therefore, the survey presented here i llustrates the application 
of the technique in radiation studies using the ionizing section of the electromagnetic 
spectrum only. 
In a radiation study, Lambert et al. (200 1 )  used tricolour FISH with whole 
chromosome-specific probes for chromosomes 2 ,  4 and 8 for scoring translocations 
induced by iodine- I 3 I  therapy in thyrotoxicosis patients one year after the 
administration of the radio label led compound. The study was carried out on nine 
patients (one male and eight females). From the genomic translocation frequencies, 
derived using the Lucas formula, equivalent whole-body doses were calculated, based 
on the in vitro 60CO y-ray dose-response curve. The average radiation dose obtained for 
this group of nine patients was 0.79 ± 0.22 Gy. 
A total of 1 0 1  translocations were observed in 4,864 metaphases in the patient group, 
63% being of the two-way type. In the control group used for obtaining dose-response 
data, nine translocations were observed in 5 ,278 metaphases, 5 5% being two-way 
translocations. Although they observed 1 2  times higher translocation frequencies in the 
iodine-therapy patients groups than in the control group involving chromosomes 2, 4 
and 8, no correlation was reported between the observed frequency of translocation and 
administered radioactivity. Furthermore, they observed that chromosome 4 contributed 
more in the formation of two-way translocation, which was detectable one year after the 
administration of the radiolabelled compound. 
In another study, using FISH, Lucas et al. ( 1 999a) studied the relationship between 
background chromosome translocation frequency and age with translocation frequency 
measured to a high statistical precision. They also determined the amount of 
background ionizing radiation which can cause the production of chromosome 
translocations in a control population. In their study, lymphocytes from 3 5  healthy 
control individuals ( 1 5  females and 20 males) were used to measure the frequency of 
Chapter Two: Review of Literature. 141 
chromosomal translocation. These control subjects were of varying ages, ranging from 0 
(cord blood) to 98 years. 
They reported that the background translocation frequency in control individuals 
follows a curvilinear relationship with age. No significant variation was observed 
between individuals of the same age .  Clastogenic processes of normal aging and 
physiological factors in additional to ionizing radiation play a major role in the 
production of chromosome translocations in a control population. Background radiation, 
however, appears to play a minor role in chromosome translocation production in 
control individuals living near sea level . 
Furthermore, a study to detect complete and incomplete chromosomal exchanges 
(translocations) in human lymphocytes (Deng & Lucas, 1 999) combined FISH with 
pan-telomeric peptide nucleic acid (PNA) and whole chromosome-specific DNA 
probes. In their study, human lymphocytes were irradiated in vitro with 0.9 Gy low 
dose-rate (0 .0 1 9  Gy/h) tritium beta-rays. 
After hybridization, all three pairs of labelled chromosomes together with 92 telomeres 
were readily visible. The whole chromosomes 1 ,  2 and 4 were painted orange, and all 
telomeres were painted green. Unpainted chromosomes were counterstained with blue. 
In the observed 680 CAs induced by tritium beta-rays in human lymphocytes after 52 h 
of culture, no evidence of telomere addition was detected. Incomplete and hidden 
complete exchanges and terminal deletions were definitively discriminated. According 
to the authors, this was the pioneer study in the simultaneous detection of telomeres and 
specific whole chromosomes to analyse accurately the complete and incomplete 
chromosome exchanges involving painted chromosomes in human lymphocytes. 
In a similar study using FISH, Lucas et al. ( 1 999) measured the effects of incubation 
temperature during irradiation, and of donor age, on the in vitro induction of 
chromosomal translocations in human lymphocytes. In their study, six human males 
were used as lymphocyte donors. Lymphocytes were exposed over 48 h continuously at 
37  or 20C to tritium beta-rays or 60CO gamma-rays. 
Chapter Two: Review of Literature. 142 
They observed no age-related difference in the alpha coefficients of the fitted induction 
curves for gamma-ray-exposed lymphocytes obtained from four donors whose ages 
ranged from 24 to 79 years, or for tritium beta-ray-exposed lymphocytes from two 
donors aged 36 and 62 years. The conclusion of the study is that the S-ratio (the ratio of 
induced complete to incomplete translocations) was found to be independent of 
radiation dose, donor age and exposure temperature. 
Natarajan et al. ( 1 992) used in situ hybridization with chromosome-specific DNA 
libraries to analyse radiation-induced stable translocations in  HPBLs. These data were 
compared with radiation-induced unstable-type aberrations (dicentrics) in the same 
samples. The results indicate that far more stable aberrations are induced by radiation in 
comparison to unstable aberrations. 
In another study, Stephan and Pressl ( 1 997) used FISH to determine the yield of 
symmetric and asymmetric exchange aberrations in metaphase chromosomes after in 
vitro exposure of PBLs to 250  kV X-rays (0-3 . 0  Gy) . For the aberration analyses, 
chromosomes 2, 4 and 8 and all centromeres were painted. Centric rings amounted to 
about 8% of the dicentric yield. The proportion of inversions and insertions was about 
5% of the total translocations. Regarding the spontaneous levels, the frequency of total 
induced translocations was higher by a factor of 1 . 1 3  than that of dicentrics. The 
involvement of chromosomes 2, 4 and 8 in translocations is significantly different from 
the expected ratio concerning physical length. Furthermore, the frequency of 
translocations was evaluated in three radiation workers who received an accidental 
radiation exposure 1 1  years previously. About 75% of the translocations were identified 
as complete in comparison with 79% in the in vitro experiments. In the radiation 
workers chromosome 2 showed an under-representation in translocations, whereas 
chromosome 4 was over-represented as in the in vitro experiments. The summarized 
results for the radiation workers showed a mean genomic translocation frequency of 
1 3 .4 per 1 000 cells. This frequency is not significantly different from the mean 
frequency of dicentrics which were determined by conventional Fluorescent Plus 
Giemsa (FPG) staining, after detection of the accidental radiation exposure about 1 1  
years previously (8.6 dic/ l OOO cells). There were, however, some differences between 
Chapter Two: Review of L iterature. 143 
individuals affecting this comparison. The distribution patterns of dicentrics showed an 
over-dispersion, whereas the translocations occurred single in cells. 
Hsieh et al. ( 1 999) used FISH to measure the alpha coefficient, the initial slope of the 
translocation dose-response curve, for chromosome translocations in  human 
lymphocytes exposed to chronic 60Co y-rays at body temperature. In their study, human 
lymphocytes from two 24-year old male blood donors were exposed to 0, 0 .32, 0 .62 and 
0.92 Gy of chronic 60 Co y-rays under conditions that reduce the metabolic stress to the 
cells. Chromosome translocation frequencies were measured using FISH with a whole­
chromosome probe cocktail specific for chromosomes 1 ,  2, 4 (orange) and 3, 5 ,  6 
(green) . A total of 72,383 metaphases were analysed (3 3 ,429 in exposed cells) in two 
donors. The shape of the dose-response curves for translocations was linear, and alpha 
coefficient was measured as 0 .024 ± 0.002 translocations per cell per Gy for the 
combined data. The authors concluded that the alpha coefficients measured after 
chronic exposure were in good agreement with that reported in the literature for acute, 
low-dose exposure of human lymphocytes to 60Co y-rays. 
In the above review, the utilization of the FISH technique is seen to be comprehensively 
successful in detecting different types of translocation as a consequence of ionizing 
radiation exposure, which thus justifies its application in the current study. 
Chapter Three: Materials and Methods. 144 
Chapter Three 
Materials and Methods 
3.1 Sister Chromatid Exchange (SCE) 
In this study, the frequency of SCEs from two rounds, round- I (RI )  and round-2 (R2), of 
experiments was studied in peripheral blood lymphocytes from six healthy, non-smoking 
male blood donors. Donors were selected with similar age, lifestyles, medical history, and 
dietary history. Ages of the first three donors ranged from 50-56 years and the remaining 
three donors ranged from 3 1 -44 years . A set of eight experiments was conducted in 
duplicate with each duplicate experiment using the same strict EMF conditions. These two 
sets of eight experiments each were called RI and R2. The eight experiments in RI were 
conducted using blood samples from the first three donors and blood samples from the 
remaining three donors were used in the R2 experiments. 
3.1 . 1  Collection of Blood Samples 
Blood samples were obtained (with ethical approval) by venipuncture from the six healthy 
human male adult donors. Each donor was required to complete a detailed questionnaire 
(Appendix 2) .  Donors were also required to complete a consent form (Appendix 3 )  
allowing blood samples (about 10  ml) to be  drawn by a qualified haematologist (Mr. Chris 
Kendrick of the Institute of Molecular BioSciences at Massey University) for analysis .  Two 
heparinised tubes were used to collect blood samples from each donor: one tube was used 
to perform computerized b lood analysis to obtain the white blood count (WBC), and the 
second tube was used for blood culture. The blood specimens were then gently mixed to 
prevent clotting. 
Chapter Three: Materials and Methods. 145 
3.1.2 SCE P rotocol 
Generation of EMFs 
The incubator, which was used to deliver EMFs (Figure 3 . 1 -A), were constructed, 
calibrated and designed by Mr. B. I. Rapley of the Institute of Technology and Engineering 
(Massey University, Palmerston North, New Zealand). To generate low power, a laboratory 
power amplifier, 0 .22 V I AMP (Figure 3 . 1 - i) was used, whereas an AC/DC power 
amplifier was used to generate high power (Figure 3 . 1 -iv) . To generate sine and square 
waves a programmable time base generator was used (Figure 3 . l -i i) .  The oscilloscope 
(Figure 3 . 1 -iii) was used to check the wave form (sine, square) accuracy. To generate the 
'pulsing wave form' a programmable pulse-width modulator (Figure 3 . 1 -v) was used.  
Culture tubes were housed in a compartment within a pair of Helmholtz coils (Figure 3 . 1 -
vi, and 3 . 1 C) .  A computer (Figure 3 . 1 -vii) was used to generate the 'complex' wave. The 
incubator temperature (37C) was generated by six 1 50 W bulbs (Figure 3 . 1 -xi) and was 
regulated by a thermostat (Figure 3 . 1 - viii). The incubator was placed in an electrically 
shielded room, and well away from any MF sources. 
Field exposure 
The culture tubes of controls were placed between a pair of parallel Helmholtz coils (Figure 
3 . 1 -B & C) placed at a 20° angle to the horizontal to facilitate cell growth in the incubator. 
These cells received no exposure (sham) to EMFs. However, the culture tubes of EMF­
exposed experiments were placed between the same pair of parallel Helmholtz coil s  
identically to  the controls with the exception that during the whole culture time, the culture 
tubes were exposed to alternating 50 Hz EMFs with the following characteristics: 
Wave type - Sine or Square or Complex 
Wave form - Continuous or Pulsed (4 sec on and 4 sec off) 
EMF strengths - 1 � T or 1 m T 
Chapter Three: Materials and Methods. 1 46 
For the complex experiment, lymphocytes were grown in the presence of a complex MF 
produced from the back of a computer (Figure 3 . I -vi). There was no ambient AC EMFs in 
the chamber of the incubator where the Helmholtz coils were placed. 
V III IX 
B 
--. . t"  
. " , 
c 
Figure 3. 1 :  A. Incubator apparatus (i) Laboratory power amplifier-low power, 0.22 
V/AMP, (ii) Programmable time base generator, (iii) Oscilloscope, (iv) Power 
amplifier ACIDC-high power, (v) Programmable Pulse-width modulator, (vi) 
Culture tube compartment with thermostat, and (vii) Computer. B & C. Side view 
and front view of culture tube placement between a pair of Helmholtz coils. (viii) 
Temperature control of thermostat, and (ix) Bulbs. 
Chapter Three: Materials and Methods. 
Helmholtz coil and EMF 
Description of the coil 
Wire type 
Wire diameter 
Total turns 
Coil diameter 
Coil modeling 
: enameled copper 
: 0.7 mm 
: 1 00 
: 1 80 mm 
147 
The MF produced by the induction coil (described above) was modeled using the EMF 
simulation programme, MagneSim, developed by Brent Foster and Wyatt Page for 
Resonance Research. MagneSim models the MFs produced by circular form: single turn; 
solenoid; Maxwell  and Helmholtz pairs. The numeric solution is accurate to within ± 1 % of 
the actual values as determined by measurement (in the ' Z'  -axis). 
The plane of calculation 
The MF produced by a circular form induction coil exists as a three dimensional 
phenomenon in space surrounding the coil .  Circular symmetry exists about the Z (or long) 
axis, which means that it is only necessary to determine the field in a two dimensional 
plane. This plane may then be rotated about the Z-axis to model the field in three­
dimensional space. The calculation plane is shown in Figure 3 .2. 
Chapter Three: Materials and Methods. 148 
Calculation Plane 
Figure 3.2: Helmholtz coils: Two coils of wire, each with 1 00 turns and separated by 
a distance equal to the radius (R), 90 mm. 
The calculation matrix 
MagneSim models the MF of a coil using a 1 Ox 1 0  matrix of points on a two-dimensional 
plane about the Z-axis, thus bisecting the coil along the long axis. All points are equally 
spaced along their axis .  However, the x and y axes may be of arbitrary length. The 
calculation matrix may exist on any part of the calculation plane and is not restricted to 
having the coil in the centre. Indeed it is possible to choose an area anywhere either within 
or outside the coil. The only constraint is that the points are automatically equally spaced in 
the x and y direction. If a calculation point of the MF in the calculation plane falls directly 
on the geometric x-y position of a turn of wire, MagneSim returns a "?", as the magnitude 
is, by definition, incalculable. 
Chapter Three: Materials and Methods. 149 
The EMF vector 
To visualize the MF, MagneSim draws a ' lollipop' plot of the MF matrix points on the 
calculation plane of the coil .  The field at each point is represented by a ' lollipop' ,  that is, a 
point with a stick attached. The 'point' of the lollipop defines the exact geometric position 
on the calculation plane where the field is calculated, whereas the ' stick' represents the 
field magnitude and the direction in vector form. The length of the stick represents the 
magnitude of the field so that the largest value represents two matrix units against which all 
other field values are auto-scaled. For example, a line drawn from a dot directly parallel to 
the Z-axis pointing to the right represents a vector at zero degrees Cartesian. The lollipop 
plot of the coil is shown in Figure 3 . 3 .  
Magnitude of the EMF resultant vector 
The magnitudes of the MF vectors for the Helmholtz coils used in this research are shown 
below. The magnitude of resultant vectors inside the two culture tubes (shaded in green 
colour) from a ImT and a l ilT exposed experiment are shown in mT (0.95 1 6- 1 . 1 326) 
(Figure 3 .4) and in I1T (0.95 1 6- 1 . 1 326) (Figure 3 .5) for the pair of Helmholtz coils.  
Direction of the MF resultant vectors 
Figure 3 . 6  shows the directions of the resultant magnetic field vectors in Cartesian 
coordinates, which are the same for all exposure levels within the Helmholtz coils. 
Chapter Three: Materials and Methods. 
? 
1 .6205 
1 . 1 326 
0.9960 
0.9500 
0.9500 
0.9960 
1 . 1 326 
1 .6205 
? 
r 
Culture 
tubes 
� 
Helmholtz 
Coil (a) 
t-"<=------�--�""""T-+-+_+-Helmholtz 
Coil (b) 
Resultant vectors 
Figure 3.3: Lollipop-plot of the resultant vectors in a longitudinal 
section through the 'Z-axis' of a Helmholtz coil. The shaded area 
(green) shows the relative position of the culture tubes with the 
three-dimensional coil, while the vector arrows display the angle, 
in the plane of the coil, at the resultant magnetic fields generated. 
Arrow lengths are auto-scaled to represent the relative field 
strength in gauss. 
1 .8755 0.8647 0.5308 0.3960 0.3960 0.5308 0.8647 1 .8755 
1 .4282 1 . 1 1 30 0.8864 0.7764 0.7764 0.8864 1 . 1 1 30 1 .4282 
1 . 1 2 19 1 .061 4  0.9935 0.95 1 6  0.95 1 6  0.9935 1 .06 14  1 . 1 2 1 9  
1 .0 1 83 1 .0 169 1 .0047 0.9460 0.9460 1 .0047 1 .0 1 69 1 .0 1 83 
0.98 1 1 0.9954 0.9993 0.9991 0.9991 0.9993 0.9954 0.98 1 1  
0.98 1 1  0.9954 0.9993 0.999 1 0.9991 0.9993 0.9954 0.98 1 1  
1 .0 1 83 1 . 1 069 1 .0047 0.9460 0.9460 1 .0047 l . l 069 1 .0 1 83 
1 . 1 2 1 9  1 .061 4  0.9935 0.95 1 6  0.95 16  0.9935 1 .06 14  1 . 1 2 1 9  
1 .4282 1 . 1 1 30 0.8864 0.7764 0.7764 0.8864 1 . 1 1 30 1 .4282 
1 .8755 0.8647 0.5308 0.3960 0.3960 0.5308 0.8647 1 .8755 
Figure 3.4: Magnitude of resultant vectors in 'mT' for the pair of Helmholtz coils. 
1 50 
? 
1 .6205 
1 . 1 326 
0.9960 
0.9500 
0.9500 
0.9960 
1 . 1 326 
1 .6205 
? 
Chapter Three: Materials and Methods. 1 5 1  
? 1 .8755 0.8647 0.5308 0.3960 0.3960 0.5308 0.8647 1 .8755 ? 
1 .6205 1 .4282 1 . 1 130 0.8864 0.7764 0.7764 0.8864 1 . 1 1 30 1 .4282 1 .6205 
1 . 1 326 1 . 1 2 19  1 .061 4  0.9935 0.95 16  0.95 1 6  0.9935 1 .061 4  1 . 1 2 1 9  1 . 1 326 
0.9960 1 .0 1 83 1 .0 1 69 1 .0047 0.9460 0.9460 1 .0047 1 .0 1 69 1 .0 1 83 0.9960 
0.9500 0.98 1 1 0.9954 0.9993 0.9991 0.9991 0.9993 0.9954 0.98 1 1 0.9500 
0.9500 0.98 1 1 0.9954 0.9993 0.999 1 0.9991 0.9993 0.9954 0.98 1 1 0.9500 
0.9960 1 .0 1 83 1 . 1 069 1 .0047 0.9460 0.9460 1 .0047 1 . 1 069 1 .0 1 83 0.9960 
1 . 1 326 1 .1 2 19  1 .0614  0.9935 0.95 1 6  0.95 1 6  0.9935 1 .0614  1 . 1 2 1 9  1 . 1 326 
1 .6205 1 .4282 1 . 1 1 30  0.8864 0.7764 0.7764 0.8864 1 . 1 1 30 1 .4282 1 .6205 
? 1 . 8755 0.8647 0.5308 0.3960 0.3960 0.5308 0.8647 1 .8755 ? 
Figure 3.5: Magnitude of resultant vectors in ' )IT' for the pair of He lmholtz coils. 
? 75 6 1  42 1 6  344 3 1 8 299 285 ? 
D 356 1 1  1 7  1 4  5 355 346 343 349 4 355 0 3 3 1 359 357 357 360 5 357 359 360 0 o 360 360 0 1 3 
359 359 360 360 o 360 0 0 1 1 
1 1 0 o 360 o 360 360 359 359 
D 3 1 o 360 360 0 o 360 359 357 5 360 357 357 359 1 3 3 o 355 4 349 343 346 355 5 1 4  1 7  1 1 356 
? 285 299 3 1 8 344 1 6  42 6 1  75 ? 
Figure 3.6: Angles in Cartesian co-ordinates of resultant magnetic field vectors relating to the 
pair of Helmholtz coils. 
Chapter Three: Materials and Methods. 1 5 1 a  
Warming effect of the Helmholtz coil 
One of the most important variables that must be considered when conducting 
experiments on weak EMFs i s  temperature. Experiments conducted by the 
Bioelectromagnetics Research Team at Massey University have previously investigated 
possible warming effects and possible temperature fluctuations in the design of the 
Helmholtz coil used in the current series of experiments which were conducted at 37C. 
The data obtained showed no measurable increase in temperature around the coil at 50 Hz 
ImT, the highest strength used in this study. Nevertheless, duplicate experiments have 
recently been conducted to confirm this finding. Two electronic probes were placed 
inside the culture chamber: one in  the culture tube and one around the coil. The culture 
chamber was heated by a flow of warm air from a distant source and stabilized at 37C by 
a thermostat, the temperature at which the PBLs are cultured. Temperature readings were 
taken over a 24 h period with the coil turned off (control) (Table l a, Appendix 7) and 
over another 24 h period with the coil turned on (50 Hz, AC) (Table I b, Appendix 7). 
The graphs (Figures I a, I b, I c & I d of Appendix 7) show no detectable differences in 
temperature fluctuations or warming effects within the chamber with the coil turned off 
or on. Any warming effect of the coil would indeed be very small (calculations of the 
heating capacity of the coil show that it contributes less than I % of the heat to the 
chamber), and irrespective of this point, an independent heat source is needed to elevate 
the temperature inside the culture chamber to 3 7C, which is the temperature required for 
lymphocyte growth. 
Some researchers use a double-wound bi-fi lar coil in their EMF experiments, the 
rationale being that such a coil nullifies the magnetic field generated by the current. In 
our laboratory we designed and tested a simple double-wound, bi-filar coil and could 
detect a weak magnetic field some distance from the coil. The magnetic field does not 
disappear entirely. For the experiments conducted in this thesis the decision was made to 
turn off the current completely for our control experiments, the rationale being that one 
could then be confident in knowing that all magnetic fields generated by the coil were 
eradicated entirely. 
Chapter Three: Materials and Methods. 1 52 
Lymphocyte cultures 
F or each experimental series, blood samples from three of the six donors were used. Two 
culture tubes were set up for each donor. Each tube contained 5 ml of Medium- 1 99 
(GibcoBRL, Cat. No. 3 1 1 00-035), 1 ml of fetal bovine serum, (GibcoBRL, Cat. No. 1 0093-
1 36), and 0 . 1 ml of phytohaemagglutinin (PHA) M form (GibcoBRL, Cat. No. 1 0576-0 1 5). 
Using the WBC count, calculations were made to obtain 3 .25-million cells/per culture tube 
by adding approximately 0 .3-0.6 ml of blood from the second samples. When conducting 
the SCE technique it is imperative that the WBC is constant (Bender et ai, 1 992b). 0.05 ml 
of 1 0.2 M 5 -Bromodeoxyuridine (BrdU) (Sigma, B-9285) was added to each culture tube 
(Falcon, 8 ml polystyrene, round-bottom tube, 1 3  x 1 00 mm style). The culture tubes were 
incubated at 37C for 72 h, which included a treatment with colchicine (0.05%, BDH, Prod. 
27805FM) for 1 h. 
Harvesting 
Harvesting of cells and slide preparation were accomplished usmg the modified 
Fluorescence-Plus-Giemsa (FPG) method (Perry and Wolf, 1 975) .  Culture tubes were 
removed from the incubator after 72 h, mixed gently then centrifuged for 1 0  min at 1 000 
rpm. Supernatant was gently removed from the top leaving approximately 1 cm above the 
pellet. Deposits were mixed thoroughly with a vortex stirrer for 5 sec, to avoid clotting, 
then re suspended in 5 ml of warm KCI (0.075 M) hypotonic solution at 37C. The tubes 
were then mixed gently by inversion 6-8 times. The tubes were incubated at 37C in a water 
bath for 1 0  min to activate the water transport system (osmosis), which ultimately swells 
the lymphocytes .  The tubes were centrifuged again for 1 0  min and the supernatant was 
removed from the top leaving approximately 1 cm above the pellet. Deposits were mixed 
well again with the vortex stirrer for 3 sec. They were then re suspended in 5 ml of acetic 
acid (6%) under constant agitation and kept for 5 min at room temperature. Cultures were 
once again centrifuged and the supernatant removed. Cultures were resuspended in 8 ml of 
ice cold fixative (Methanol : Acetic Acid = 3 : 1 ), centrifuged immediately and the 
supernatant removed. This last step was then repeated. Finally, after removing the 
supernatant to 5 mm without disturbing the pellet, 2-3 drops of fixative was added to give a 
cell suspension of light turbidity. 
Chapter Three: Materials and Methods. 153  
Giemsa block staining 
Air-dried, freshly prepared slides were stained with 1 0% Giemsa (Gurr, BDH, Prod. 
3 50864X) in Sorensen's buffer (PH 6.8) for 7-9 min, then rinsed in Sorensen's buffer for 2-
3 min and air-dried. Slides were mounted using DPX. Figure 3 .7, shows a Giemsa block 
stained c-metaphase chromosome spread from a control experiment showing a normal 
complement of2n = 46. 
Figure 3.7: A Giemsa-stained c-metaphase chromosome spread of a 
dividing peripheral blood lymphocyte from a control experiment 
showing a normal complement of2n = 46. 
Chapter Three: Materials and Methods. 1 54 
Fluorescence-plus-Giemsa (FPG) staining 
Fluorescence-plus-Giemsa staining protocol was a modification of methods devised by 
Wolf and Perry ( 1 975), and Sandberg and AnneMarie ( 1 987). This procedure is based on 
UV sensitivity of heavily BrdU-Iabeled DNA. The one week old prepared slides were 
soaked in Sorensen's buffer solution (pH 6.8) for 5 min, rinsed in distilled water. The air­
dried slides were mounted using fluorochrome Hoechst 3 3258 (bisbenzimide) solution (5 
flg mr ' ) and covered with coverslips for 30  min (in dark). Hoechst 33258 staining 
photosensitizes degradation of BrdU-stimulated DNA, creating single-strand nicks. After 
30 min coverslips were removed by rinsing with Sorensen' s buffer and washed with 
distilled water (in dark) . To avoid chromosome damage, slides were mounted in 
Macllvaine' s  buffer (pH 7.0), covered with coverslips, and i lluminated to 356 nm UV l ight 
for 2 .5  h (at a distance of approximately 1 0  cm). Coverslips were removed by rinsing with 
Sorensen's buffer and washed with distilled water (in dark). The slides were immediately 
incubated for 20 min in salt-sodium-citrate buffer (2 x SSC, pH 8 .0) at 65C to elute small 
DNA fragments. Slides were stained in Giemsa ( 1 0%) (Gurr; BDH, Prod. 3 50864X) in 
Sorensen 's  buffer (pH 6 .8) for 7- 1 0  minutes then rinsed in Sorensen's buffer for 2-3 
minutes. Air-dried slides were mounted in DPX. 
Scoring criteria 
The 1 0  microscope slides of every donor were randomly coded (a - j) by the researcher to 
ensure anonymity of samples and examined 'blind' (without knowledge of controls or 
experimental source). To eliminate bias, an independent person was involved in setting the 
EMF parameters, which were thus blind to the researcher. The slides were investigated 
serially. Two hundred consecutive second mitotic metaphase cell s  per donor, which 
showed good chromosome morphology, differential staining for SCE and no chromosome 
overlapping, were selected. The images were captured by a lVC 3-CCD Colour Video 
Camera using Silicon Graphics and Image Capture software and scored for SCE from the 
computer screen at 1 000X magnification. SCEs were expressed per cell, so it was 
necessary for a full complement of 46 chromosomes to be present. SCEs were analysed 
according to guidelines of Swierenga et al. ( 1 99 1 )  (see Figure 3 .8). 
Chapter Three: Materials and Methods. 
Sister chromatids 
VJ ----. Q) I-< Q) 
S a b c d e 0 
� s:: Q) a U 
f g h 
Figure 3.8: Diagrammatic representation of various differential 
staining patterns observed after incorporation of BrdU into 
replicating DNA: (a) No SCE, (b) a single SCE (arrow), (c) two 
SCEs, (d) three SCEs, (e) SCE at the centromere, (f) not 
counted as SCE, (g & b) incorporation of BrdU for more than 
two complete cycles of DNA synthesis, (i) reciprocal pattern of 
staining less than one chromatid in width and not counted as 
SCE (Swierenga et al. 1 99 1 ) .  
1 55 
Chapter Three: Materials and Methods. 1 56 
3.2 Micronuclei Assay (MN assay) 
In the present study, the frequency of micronuclei (MN) from one round of experiments 
was studied in peripheral blood lymphocytes from the same three blood donors used in RI  
of  the SCE experiments. Only one set of  eight experiments was conducted with the same 
strict EMF conditions as followed in RI and R2 of the SCE experiments. For the MN assay 
experiments, the incubator that was used to conduct SCE experiments was used again. 
Lymphocyte cultures for all experiments conducted for MN were exposed to 50  Hz 
sinusoidal (continuous or pulsed) or square (continuous or pulsed) MFs at field strengths of 
l ilT or I mT for 90 h. Lymphocyte cultures of three controls were sham exposed. In the 
twelfth experiment of MN, lymphocyte cultures were exposed to a complex MF produced 
from the back of a computer, for 90 h. All experiments were performed ' single blind' as 
followed in the SCE experiments. 
3.2.1 Collection of Blood Samples 
Fresh blood samples (5 ml) were obtained by venipuncture by the same liematologist, Mr. 
Chris Kendrick. Only one heparinized tube was used to collect blood samples from each 
donor. The blood specimen was then gently mixed to prevent clotting. 
3.2.2 MN Assay Protocol 
Generation of EMFs 
The same incubator (Figure 3 . 1 ) which was used to deliver EMF intensities for the SCE 
experiments was also used to conduct MN experiments. 
Field exposure 
The culture tubes of controls were placed between a pair of parallel Helmholtz coils (Figure 
3 . 1 ,  B and C) placed at a 20° angle to the horizontal in the incubator and received no 
exposure (sham) to EMFs. 
Chapter Three: Materials and Methods. 1 57 
The culture tubes of EMF -exposed experiments were placed between the same pair of 
parallel Helmholtz coils, identically to the controls, with the exception that during the 
whole culture time (90 h) the culture tubes were exposed to alternating 50 Hz EMFs with 
the following characteristics :  
Wave type - Sine or Square or Complex 
Wave form - Continuous or Pulsed (4 sec on and 4 sec off) 
EMF intensities - l ilT or ImT 
Lymphocytes were grown (90 h) in the presence of a complex MF produced from the back 
of a computer (Figure 3 . 1 -vi) . There were no ambient AC EMFs in the chamber of the 
incubator where the Helmholtz coils were placed. 
Preparation of cytochalasin-B stock solution 
Cytochalasin-B (Sigma, C-6762) was made up as a stock solution in dimethyl sulphoxide 
(DMSO) at a concentration of 2 mg/ml and stored at below -20C. The stock solution was 
thawed and added 24 h after the mitotic stimulation of the lymphocytes at a final 
concentration of 6/lg mr' . 
Lymphocyte cultures 
For the MN experiments two culture tubes were set up for each donor from fresh blood 
samples which were obtained by venipuncture from the donors. 0 .5  ml of fresh whole blood 
was added to culture tubes containing 5 ml of culture medium (4 ml GibcoBRL Medium-
1 99, Cat. No. 3 1 1 00-035 and 1 ml fetal bovine serum, Cat. No. 1 0093 - 1 36) pre-warmed to 
3 7C .  The six culture tubes were placed in a water bath for one h at 37C before being 
irradiated with X-rays at 3 . 5  Gy (0.4375 Gy min- ' , with a total 8 min exposure time). After 
irradiation the culture tubes were incubated (in dark) at 3 7C under different experimental 
EMF conditions. The controls were sham exposed. All experiments were performed single 
blind. 
Six hours from the point of irradiation the lymphocytes were stimulated to divide using 
mitogen phytohaemagglutinin (PHA) (M form, GibcoBRL, Cat. No. 1 0576-0 1 5) at a 
Chapter Three: Materials and Methods. 1 58 
concentration of 1 0llg mr l . Twenty-four h after PHA stimulation, the cytokinesis-blocking 
agent, cytochalasin-B (Sigma, C-6762) was added to the cell cultures at a concentration of 
61lg mrl to arrest the dividing cells in cytokinesis. First generation post-mitotic cells could 
subsequently be identified as binucleated cells (Fenech and Morley, 1 985a and Scott et al. 
1 998). 
Harvesting of lymphocytes, slide preparation 
Harvesting of cells and slide preparation was accomplished by the modified method of 
Fenech and Morley ( 1 985a), and Scott et al. ( 1 998). Culture tubes were incubated for 90 h, 
mixed gently, and centrifuged for 1 0  min at 1 000 rpm. Supernatant was gently removed 
from the top leaving approximately 1 cm above the pellet. Deposits were mixed thoroughly 
using a vortex stirrer for 3 sec and then resuspended in 5 ml of a warm hypotonic solution 
(KCI, 0 .075M) at 37C and mixed gently by inverting 6-8 times. Tubes were incubated at 
37C in a water bath for 4 min in order to swell the binucleated cells to allow more accurate 
scoring. Tubes were centrifuged immediately and the supernatant was drawn off to 1 cm 
above the pel let. Deposits were again mixed using a vortex machine for 3 sec. 
Lymphocytes were re suspended in 5 ml of fresh ice-cold fixative (Methanol : Acetic Acid 
= 3 : 1 )  in three steps, mixed gently by inversion, and centrifuged immediately. The 
supernatant was then removed and 5 ml of the fixative was added directly, mixed gently, 
and centrifuged. After centrifugation the supernatant was removed to 5 mm above the 
bottom of the tube without disturbing the pellet. Two to three drops of fixative were added 
to give a cell suspension of light turbidity . The cell suspension (3-4 drops) was dropped 
gently along an acid-alcohol washed ( 1ml of 1M  HCI and 50 ml of 95% ethanol) air-dried 
slide. Twelve slides were prepared per donor. All harvesting procedures and reagents were 
at ambient temperature unless otherwise stated. 
The slides were stained with 1 0% Giemsa (Gurr, BDH, Prod. 3 50864X) in Sorensen' s  
buffer (pH 6 .8) for 8 min, then rinsed in Sorensen's  buffer for 2-3 min and air-dried. The 
slides were mounted in DPX. 
Chapter Three: Materials and Methods. 1 59 
Coding of slides and scoring of micronuleus 
The slides of every donor were randomly coded by the researcher to ensure anonymity of 
samples and analysed at 400X magnification under a light microscope. In the cytokinesis­
blocked method, nuclei are scored only in cells that have been inhibited from undergoing 
cytokinesis (Figure 3.9). Cytokinesis-blocked (CB) cells are easily recognisable by their 
binucleate appearance, as they must be dividing cells, which have completed nuclear 
division but not cytoplasmic division. Approximately 2,000 cells were scored from each of 
the 3 donors. Figures 3. 1 0, 3.1 1 ,  and 3. 1 2  illustrate the method used to score micronulei • 
• 
• 
a b c d 
• 
e f g 
• 
h 
Figure 3.9: Cytokinesis-blocked (CB) binucleated cel ls (a-h) with varying 
numbers (0-7) of micronuclei. 
Chapter Three: Materials and Methods. 1 60 
Criteria for choosing binucleate cells 
A B c D 
Figure 3.10:  Binucleate cells which can be scored for micronuclei, A .  Ideal 
binucleate cell; B. Binucleate cell with touching nuclei; C. Binucleate cell with 
narrow nucleoplasmic bridge between nuclei; D. Binucleate cell with relatively 
wide nucleplasmic bridge. 
A B c D 
Figure 3.1 1 :  Typical appearance and relative size of micronuclei in binucleated 
cells that meet the scoring criteria: A. Cell with 2-micronuclei . The first with one 
third and second with one ninth the diameter of one of the main nuclei within the 
cell .  B. Micronuclei  are touching but are not overlapping the main nuclei .  C .  
Binucleated cell with nucleoplasmic bridge between the two main micronuclei 
and, D. Binucleated cell with six micronuclei of various size. This type of cell is 
rarely seen. 
Chapter Three: Materials and Methods. 
A B c D 
Figure 3. 12 :  Cellular structures which resemble micronuclei but were not scored: A. 
micronuclei with a diameter greater than one third the diameter of the main nuclei, 
B. Dense stippl ing in a specific region of the cytoplasm, C. Extruded nuclear 
material that appears as a micronucleus with a narrow nucleoplasmic connection to 
the main nucleus, and D. Nuclear blebs which overlap with the main nucleus. 
3.3 COMET Assay 
1 6 1  
In  the present study, two COMET assay experiments were carried out in peripheral blood 
lymphocytes of donor- l only, which was used in RI of the SCE study. A total of two 
experiments were conducted with the same strict EMF conditions that were followed in the 
SCE and MN experiments. 
Lymphocyte cultures were exposed to either a 50 Hz square continuous field or a 50 Hz 
square pulsed field (4 sec on and 4 sec off) at a field strength of ImT for 72 h. The control 
lymphocyte cultures were sham exposed. Both the COMET assay experiments were 
conducted single blind. 
3.3. 1 Materials 
The materials used during this COMET assay were stored at room temperature, unless 
otherwise stated. Sterilization of these materials was not required, unless stated, due to the 
nature of the assay. If sterilization was an issue, an aseptic technique was used. 
Chapter Three: Materials and Methods. 
Agarose gels 
1 % Low melting-point agarose (LMA) 
LMAgarose (Life Technologies Inc.) 
IX PBS (Bio-Rad Laboratories) 
1 g 
1 00 ml 
1 62 
The agarose powder was mixed well with PBS and heated in a microwave with the cap 
loosened for �30 sec, or until the solution turned clear. The agarose was prepared as stock 
and aliquots were melted using a hotplate and transferred into a smaller vial as required for 
experimental work. 
0.5 % Normal melting point agarose (NMA) 
NMA Powder (Life Technologies Inc.) 
IX PBS (Bio-Rad Laboratories) 
0.5 g 
1 00 ml 
The agarose powder was mixed well with PBS and heated in a microwave with the cap 
loosened for 60 sec, or until the solution turned clear. The agarose was prepared as stock 
and aliquots were melted using a hotplate and transferred into a smaller vial as required for 
experimental work. 
Cell preparation media 
Ficoll-Paque media 
Ficoll-Paque Plus media was obtained (Amersham Bioscience) and used undiluted. An 
equal volume of Ficoll was layered beneath a lymphocyte :PBS 1 :  1 mixture before 
centrifugation. Ficoll-Paque media was stored at 4C. 
I x  Phosphate buffered saline (PBS) 
l aX PBS (No Ca2+ or Mg2+) (Bio-Rad Laboratories) 
Milli-Q Water (MqH20) 
1 0  ml 
90 ml 
Chapter Three: Materials and Methods. 1 63 
1 0X concentrated PBS was mixed well with an appropriate volume of mqH20 and 
refrigerated. 
Trypan blue stain 
Trypan blue stain (BDH Chemicals) 
DH20 
The stain was mixed well until all powder had dissolved. 
DNA stains 
4', 6-Diamidino-2-phenylindole (OAPI) stain 
0 . 1  g 
1 0  ml 
Vectashield™ with DAPI was obtained (In Vitro Life Science Technologies Inc.) and used 
undiluted. OAPI was stored at 4C in the dark to maintain viability. 
Molecular biology buffers and solutions 
Dimethyl sulfoxide (OMSO) 
OM SO was obtained (Life Technologies Inc.) and used undiluted. DMSO was stored at 
room temperature in the dark. 
O.2M EDT A, pH 8 
0 . 5  M EDTA (BDH Laboratories) 
MqH20 
6 ml 
9 ml 
0.5 M EOTA was diluted with mqH20 to reach the desired concentration 
Chapter Three: Materials and Methods. 
0 .5M EDTA, pH 8 
EDTA (BDH Laboratories) 
MqH20 
1 0  M NaOH (BOH Laboratories) 
14.6 1 g 
50 ml 
1 64 
The solution was mixed well and NaOH was added drop-wise until the desired pH was 
reached and all powder was dissolved. 
70 % Ethanol solution 
Absolute ethanol (BDH Laboratories) 
DH20 
COMET assay materials 
3 50 ml 
1 50 ml 
All solutions for the COMET assay in this section were made fresh for each sampling day. 
Alkaline electrophoresis buffer, �pH 1 3  
NaOH (BOH Laboratories) 
0 .5  M EDTA (BDH Laboratories) 
MqH20 
1 2  g 
2 ml 
1 000 ml 
The solution was mixed well until fully dissolved and left to chill at 4C for at least 60 min 
before use. 
Alkaline solution, pH 1 3  
NaOH powder (BDH Laboratories) 
0.2 M EDTA (BOH Laboratories) 
MqH20 
0 .6 g 
250  ).11 
5 0  ml 
Chapter Three: Materials and Methods. 165 
The solution was mixed well until fully dissolved and left to cool at room temperature for 
at least 60 min before use 
Lysis solution 
Per 1 0  slides: 
Lysis solution (Trevigen) 
DMSO (Life Technologies Inc.) 
40 ml 
400 ml 
The solution was mixed well by gentle agitation and stored at 4C for at least 30  min before 
use. The lysis solution contains the following components (Singh et aI, 1 988). 
NaCI 
EDTA 
NaOH 
Trizma base 
1 46. 1 g 
37.2 g 
8.0 g 
1 .2 g 
The components were added to approximately 700 ml of dH20, and mixed well. NaOH was 
added to allow powders to dissolve for about 20 min. The pH was adjusted to 1 0 .0 using 
concentrated NaOH or HCI, before filling to 890 ml with mqH20. The solution was stored 
at room temperature. Before each assay, 3 5 .6 ml of the above solution was measured and 
400 III of Triton X- lOO and 4 ml ofDMSO was added fresh to a final volume of 40 m!. The 
solution was then stored at 4C for at least 30  min before use. This protocol is included only 
for researchers who wish to make their own lysis solution. The lysis solution used in this 
thesis was obtained commercially. 
Cell culture media 
Wellcome media (media 1 99) (GIB CO) 
AB serum (GIBCOBRL, Cat No. 1 0093- 1 36) 
Phytohaemagglutinin (PHA) (GIBCOBRL, Cat No. 1 0576-0 1 5) 
5 ml 
1 ml 
0. 1 ml 
Chapter Three: Materials and Methods. 1 66 
The cell culture media was prepared fresh when required. Aseptic technique was used 
during the use of this material to prevent bacterial contamination. The total volume of 
media prepared depended on what was required. 
3.3.2 Generation of EMFs and Field Exposure 
The same incubator (Figure 3 . 1 ) which was used to deliver EMF intensities for the SCE 
and MN assay experiments was also used to conduct COMET assay experiments. The field 
exposure conditions and parameter combinations were identical to these used in the SCE 
study and MN assay. 
3.3.3 Collection of Blood Samples 
Fresh blood samples (5 ml) were obtained by venipuncture. Only one heparinized tube was 
used to collect blood samples from the donor. The blood specimen was then gently mixed 
to prevent clotting. 
3.3.4 Lymphocyte Cultures 
For the COMET assay experiments, two culture tubes per experiment were established. 
Each tube contained 5 ml of Medium- 1 99, 1 ml of fetal bovine serum, and 0. 1 ml of PHA, 
M form. Approximately 0.5 ml blood from the fresh sample was added per culture tube. 
Then the culture tubes were incubated (under sham exposure or EMF exposure) at 37C for 
72 h. 
3.3.5 Lymphocyte Preparation 
After 72 h the lymphocyte cultures were removed from the incubator, the cells were 
resuspended by gently inverting the tube followed by vortex. In a new 8 ml Falcon ™ tube, 
3 ml of this lymphocyte culture was carefully layered on to an equal volume of a 
Ficol-Paque Plus lymphocyte separation media (Amersham Bioscience). This was then 
centrifuged for 20 min at 800 x g, or until the lymphocytes were completely separated from 
the erythrocytes and plasma. After centrifugation the sample resembled Figure 3 . 1 3 .  The 
top layer was a yellowish colour that consisted of plasma, platelets, and a small amount of 
Chapter Three: Materials and Methods. 1 67 
erythrocytes and peripheral blood mononulceate cells (pBMes). The large, clear layer 
contained the Ficol gradient, and the dark red mass at the bottom of the tube consisted 
mainly of erythrocytes .  A wispy, white "buffy" layer was observed between the p lasma 
layer and the Ficol layer; this layer contained the majority of the lymphocytes .  A Pasteur 
pipette was used to collect the layer in a swirling motion, taking care to collect as little of 
the upper and lower layers as possible. The lymphocytes were transferred to an 8 ml Falcon 
tube containing 1 ml of chilled PBS buffer (PH 7), mixed slowly by vortex, and stored on 
ice. The PBS contained no Ca2+ or Mg2+ to ensure minimal in vivo DNA repair during the 
assay procedure. 
.- Screw cap 
.- Layer of Plasma 
�=� .- Layer of Lymphocytes 
+-- Layer ofFicol 
+-- Layer of Erythrocytes 
Figure 3.13: I l lustrative diagram of what was observed after centrifugation 
of whole blood during isolation using Ficol, concentration gradient. 
3.3.6 Lympbocyte Concentration and Cell Viability 
To determine the lymphocyte concentration of the isolated sample, an "Improved 
Neubauer" haemocytometer was used (Weber Scientific International Ltd). A 
haemocytometer is a specialised slide with 2 chambers, each with grid patterns that are 
used to determine the concentration of cells in a medium. Each grid is made up of 9 large 
squares with sides that are 1 mm in length as illustrated in Figure 3 . 1 4 . 
When the coverslip was placed on the clean haemocytometer there was a slight rainbow 
appearance over the edges of the coverslip. This indicated that the distance between the 
Chapter Three: Materials and Methods. 1 68 
slide and the coverslip is 0 . 1 mm and therefore able to give an accurate cell count. The 
large square circled in Figure 3 . 1 4  was used for all counts. This square is made up of 25 
smaller squares, each with sides that are 0.2 mm in length. The volume contained in the 
large centre square is 0 . 1  mm3 ( 1  mm x 1 mm x 0. 1 mm). There is 1 ml in 1 cm3, and 1 000 
mm3 in 1 cm3 , and 1 x 1 04 ( 1 000 mm3 x 1 x 1 0) volumes of the counting square in 1 ml. 
Therefore, by counting the number of cells in the large centre square and multiplying this 
by 1 x 1 04, it will give the concentration of the cell sample in cells/ml. 
V 
� {  f \. 
• 
-
lInIn  
Figure 3.14: Diagram representing the dimensions of a 
haemocytometer counting chamber. 
F or this study, the calculation of cell viability was required. Cell concentration and cell 
viability was determined using trypan blue exclusion and a haemocytometer. One drop of 
trypan blue stain was added to 1 drop of cell suspension in an Eppendorf tube, mixed well, 
and allowed to incubate for 1 min. After 1 min approximately 20 III of the stained cell 
solution was taken up using a Pasteur pipette and slowly added to the edge of the 
haemocytometer' s  coverslip, which is spread across the slide by capillary action .  The 
sample was added until the chamber was filled, taking care not to overfill. The slide was 
observed under an Olympus® CHA light microscope using 400X magnification to 
determine the viability of the sample. 
Chapter Three: Materials and Methods. 1 69 
Non-viable cells are visible as dark blue structures due to the dye permeating the 
compromised cell membrane, while viable cells appear to be relatively clear against a 
darker background. Only cell samples with a cell viability of > 90 % were used for the 
COMET assay (Visvardis et aI, 1 994) . A dilution factor of 2 was taken into consideration 
when determining lymphocyte concentrations due to addition of trypan blue stain. 
Lymphocytes were distinguished from erythrocytes by visual morphology only, although 
identification by other methods can be used, e .g . ,  Turk' s  stain or using a haemolytic 
solution. 
The total number of cells in the middle counting grid were counted, as well as the number 
of viable cells. The total cell number was multiplied by 2 x 1 04 ( 1  x 1 04 multiplied by a 
dilution factor of 2) to give the approximate concentration of cells/ml. For example, if the 
number of cells in the large centre square was found to be 1 0, then the concentration of the 
cell solution is approximately 200,000 or 2 x 1 05 cells/ml. Cell viability was determined by 
the number of viable cells divided by the total number of cells counted, all multiplied by 
100. 
The cell concentration required for the COMET assay is approximately 1 x 1 05 cells/ml. 
Samples were diluted with PBS as required, or if the concentration was too low then they 
were concentrated by centrifugation and resuspended in a lower volume of PBS. Unless 
otherwise stated, a vortex mixer was used throughout this research for the resuspension of a 
pellet. 
3.3.7 Pre-treatment Method 
The current COMET assay slides were prepared from conventional microscope slides using 
a modified version of the pre-treatment method outlined by Singh et al ( 1 988). 26 mm x 76 
mm microscope slides (Lomb Scientific Ltd) were first marked at the centre with an 
electronic engraver to identify the sample number that was to be added to each slide. The 
slides were then cleaned using a lint-free tissue to remove grease, moisture, and dust, all of 
which can cause the gel to detach during the assay. An aliquot of electrophoresis-grade, 
normal melting-temperature agarose (NMA, 0 .5  % per volume, Life Technologies Inc.) 
was melted using a microwave, and 2 aliquots of 75 III of the molten agarose was pipetted 
onto each of the slides, 1 at each end. A 22 mm x 22 mm glass microscope coverslip 
Chapter Three: Materials and Methods. 1 70 
(Biolab Scientific) was placed on top of each aliquot to act as a type of mould. The slides 
were stored at 4C for at least 60 min to allow this base layer of agarose to solidify. This 
layer served as an attachment layer for later agarose gel layers. During seasons of low 
humidity, such as winter, these gels are prone to drying too fast before a coverslip can be 
added. To overcome this, slides were placed on top of a slide warmer that had been covered 
with a damp tissue to create artificial humidity during application of the base gel layer. This 
change would not affect results, as no cells were present in this layer. 
An aliquot of electrophoresis-grade, low melting-temperature Agarose (LMA, 1 % per 
volume, L ife Technologies Inc.) was melted using a boil ing water bath. After 1 0  min the 
LMA was transferred to a 42C water bath to cool for at least 1 0  min. Cells were not added 
until the gel had cooled in order to prevent temperature-associated damage (Speit et al. 
1 999). The coverslip was carefully removed from the base layer by sliding, taking care not 
to tear or disturb the base agarose layer. Ten III of the cell solution from the cell 
suspension, as prepared in Section 3 . 3 . 5 ,  was mixed well with 90 III of the molten LMA by 
gentle agitation in an Eppendorf tube. Seventy III of this celllagarose mixture was added to 
each of the base gel layers and a fresh coverslip was again placed on top of each of the new 
layers. The slide was stored at 4C for 20 min to allow the agarose to solidify. The coverslip 
was again removed and another 70 III of LMA 42C layer was added. This layer contained 
no cells and functioned as a protective layer to prevent detachment of the cells from the gel 
while in solution. A fresh coverslip was then placed on top of this new layer and the slide 
was stored at 4C for 30  min to allow the agarose to solidify. A diagrammatic representation 
of the gel layers is seen in Figure 3 . 1 5 . 
I 
" " 
Gel- l t Gel-2 
Microscope Slide 
.. 
..... 
I 
Coverslips 
LMAgarose Layer 
LMAgarose + Cell 
Sample Layer 
Normal Agarose 
Layer 
Figure 3.15:  Il lustrative image showing how the gel layers were 
formed using the pre-treatment method. Image not to scale. 
Chapter Three: Materials and Methods. 1 7 1  
Two replicate slides were prepared for each sample, each with 2 gels (designated Gel 1 and 
Gel 2 as illustrated in Figure 3 . 1 5) at either end. Each slide was processed 1 step apart from 
its counterpart to take into account any variability that may occur between sample runs, i .e . ,  
while the first slide was immersed in alkaline solution (the second step of the COMET 
assay) the second slide was immersed in the lysis solution (the first step of the COMET 
assay). Two gels per slide were prepared as a precaution as the gels would sometimes 
become detached from the slide while in solution, and hence become unusable. After the 
gels had solidified the coversl ips were carefully removed so that the slides could be 
subjected to the COMET assay. 
3.3.8 The COMET Assay Methodology 
The COMET assay was performed using a modified version of the methodology described 
by S ingh et al. ( 1 988). All solutions for the COMET assay were prepared at least I h before 
the assay was commenced. Unless otherwise stated, solutions were prepared using mqH20. 
The lysis solution and electrophoresis solution were prepared and stored in a 4C 
refrigerated room, while the alkaline solution was cooled and stored at room temperature. 
Samples were kept in the dark during all stages of the assay .  
Lysis solution 
After the coverslips were removed, the slides were immediately immersed in 40 ml of lysis 
solution (Trevigen Inc.), which had been chilled at 4C or on ice for at least 60 min before 
use . The slides were immersed until levels just covered the gels .  Immersion was conducted 
at 4C for 40 min in the dark. The slides were then carefully removed from the solution, 
drained, and then immediately transferred to the alkaline solution. 
Alkaline solution 
The slides were immersed in 50 ml of alkaline solution and incubated for 40 min at room 
temperature, in the dark. At no point during this stage was the container of alkaline solution 
disturbed or moved, as gels tend to detach from the sl ides at this point due to the 
conditions. The slides were then carefully removed and any excess solution was drained 
off. Slides were immediately transferred to the electrophoresis apparatus .  
Chapter Three: Materials and Methods. 1 72 
Alkaline electrophoresis 
An electrophoresis apparatus was filled with 1 Litre CL) of chilled, alkaline electrophoresis 
buffer at least 1 h before use. The volume used will depend upon the apparatus used. Work 
involving the apparatus was conducted at 4C in the dark to reduce any temperature-related 
damage due to heating of the solution by the electric current. Heat may affect the results of 
the assay, or cause the gel to detach from the slide. A maximum of five slides was placed in 
the centre of the apparatus in each run. The placement of slides in the apparatus is 
illustrated in Figure 3 . 1 6. All slides were placed in the apparatus so that all comet tails were 
directed from left to right in order to accommodate the COMET assay analysis software 
that was used to quantify the genetic damage. The settings for electrophoresis were applied 
at 1 V fcm in length of the electrophoresis apparatus at 3 00 mA. The apparatus was 
approximately 30 cm in length and so the voltage applied was 30 V .  A current of 300 mA 
was applied to normalise the volume of electrophoresis buffer used between gel runs. This 
current is applied regardless of the size of the apparatus. The amount of buffer will affect 
the electrophoresis current, and so buffer was added or removed until this current was 
achieved. The voltage was applied for 30  min. 
-... �� Direction of DNA Migration 
II 
III 
IV 
Figure 3.16:  Image indicating the placement of microscopic 
slides in electrophoresis tank. 
The slides were then removed from the apparatus and immersed in 70 % ethanol for 5 min. 
The slides were removed from the ethanol and allowed to dehydrate overnight in a slide 
Chapter Three: Materials and Methods. 1 73 
box that contained desiccant (sil ica, AnalaR). The drying step is important as it brings the 
cells into the same field of view to allow observation by fluorescent microscopy. 
3.3.9 Slide Analysis (Quantitative) 
Fluorescent microscopy was utilised to visualise the slides after applying the COMET 
assay. Various fluorescent DNA stains were trialed to detect comets in this study, such as 
SYBR Green™, and propidium iodide. Although these stains give clear images, they were 
very photosensitive and faded quickly, making them unreliable for digital imaging. 
However, DAPI is a semi-permanent stain and during trials for a suitable stain was found to 
be very effective in regards to both clarity of image and stability. 
Fifteen III of DAPI was added to the centre of each gel and a coverslip was place on top to 
allow the stain to spread. The slides were then refrigerated overnight at 4 °c and observed 
the following day. All microscope work and digital imaging was conducted using an 
Olympus® BX5 1 fluorescent microscope, under 400X magnification. The wavelengths for 
excitation and emission of DAPI occur at 358 nm and 461 nm, respectively. Therefore, the 
stain was detected using a U excitation (wide band) filter (abbreviated WU). Digital 
imaging was conducted using an Optronics MagnaFire S99802 digital camera with 
MagnaFire™ frame-grabbing software on a 2 GHz Pentium 4 ™ computer with 256 MB of 
RAM and a 1 28 MB video card. Cell images were captured using an exposure time of 200 
ms throughout the sampling period. Figure 3 . 1 7  shows the hardware used during the 
current study. 
Figure 3. 1 7: Image capturing hardware used in the current study. 
Chapter Three: Materials and Methods. 1 74 
In the current study 1 03 ,  99 and 70 cells were photographed for the control and two 
experiments (square continuous ImT, and square pulsed ImT), respectively. 
Cell images were captured so that they would accommodate the COMET assay analysis 
software (The CASP, © Krzysztof Konca). The main requirement is that COMET tails 
were to be directed from left to right as illustrated in Figure 3 . 1 8 . 
Each cell that was to be captured was required to be free of debris and other cellular 
material, and be completely separated from other comets (both head and tails) around the 
perimeter of the cell of interest. A set of 50 random microscope coordinates (X and Y 
positions) for each of the 2 gels per slide were generated using Microsoft Excel™ 
(Appendix 4) by determining the perimeter of the 2 gels per slide and the corresponding 
area of the microscope stage. These coordinates were used in the current study. Once the 
microscope had been directed to the coordinate, separate images were taken of all comets 
that could be seen in the field of view that matched the prerequisites outlined in this 
paragraph. 
Figure 3.18: Orientation of the cell (directed left to right) 
for analysis by the CASP software. 
Images of comet cells were analysed using The CASP version 1 .0 1  (The COMET Assay 
Software Project, Krzysztof Konca ©) software package with a 902 MHz Intel Celeron ™ 
computer, with 256 MB of RAM and a 64 MB video card. The CASP software captures 1 3  
variables from each comet cell, which can be used to determine the extent of the DNA 
damage. 
Chapter Three: Materials and Methods. 1 75 
An explanation of these 1 3  variables is outlined below: 
Head Area - Area of the comet head in pixels (sum of pixels in the head) 
Tail Area - Area of the comet tail in pixels (sum ofpixels in the tail) 
Head DNA - Amount of DNA in the comet head (derived from the sum of intensities of 
pixels in the head) 
Tail DNA - Amount of DNA in the comet tail (derived from the sum of intensities of pixels 
in the tail) 
Head DNA % - The Percentage of the cell ' s  total DNA that is located in the comet head 
Tail DNA % - The Percentage of the cell' s  total DNA that is located in the comet tail 
Head Radius - Radius of the comet head (in pixels) 
Tail Length - Length of the comet tail measured from right border of head area to end of 
tail (in pixels) 
Comet Length - Length of the entire comet from the left border of head area to the end of 
tail (in pixels) 
Head MeanX - Centre of gravity of DNA in the head (x-coordinate) 
Tail MeanX - Centre of gravity of DNA in the tail (x-coordinate) 
Tail Moment - TailDNA% x TailLength ( [percent of DNA in the tail] x [tail length]) 
Olive Tail Moment (OTM) - TailDNA% x (TaiIMeanX-HeadMeanX) ([percent of DNA 
in the tail] x [distance between the centre of gravity of DNA in the tail and the of centre of 
gravity of DNA in the head in x-direction]) 
A pictorial explanation of some of these variables i s  illustrated in Figure 3 . 19 .  
Chapter Three: Materials and Methods. 1 76 
Of the 1 3  variables, only 3 have been found to be the most indicative of genetic 
damage. The comet's Tail Length (TL) was used for many years due to its ease of 
measurement via an eyepiece micrometer. Tail moment (TM) and Olive Tail Moment 
(OTM) (Olive et al. 1 990) have become more prominent in recent literature. These 3 
variables were used as a measure of genetic damage in the current study. 
Figure 3.19:  Image outlining 7 of the 1 3  different variables that are measured by 
the CASpTM software. 
3.4 Fluorescent in situ Hybridization (FISH) 
In the present study, FISH experiments were carried out in peripheral blood 
lymphocytes of donor- l only, who was studied in RI of the SCE study. One experiment 
was conducted with the same strict EMF conditions as followed in the SCE, MN assay 
and COMET assay experiments . 
Lymphocyte cultures of the experiment conducted for FISH were exposed to a 50  Hz 
square continuous MF at the field strength of 1 mT for 72 h. The lymphocyte cultures 
of the control were sham exposed. 
Chapter Three: Materials and Methods. 1 77 
3.4.1 Collection of Blood Samples 
Fresh blood samples (5 ml) were obtained by venipuncture . Only one heparinized tube was 
used to collect blood samples from the donor. The blood specimen was then gently mixed 
to prevent clotting. 
3.4.2 FISH Protocol 
Generation of EMFs and Field Exposure 
The same incubator (Figure 3 . 1 )  which was used to deliver EMF intensities for the SCE, 
MN and COMET assay experiments was also used to conduct FISH experiments. All field 
parameters and exposure conditions were identical as followed in the SCE, MN assay and 
COMET assay experiments. 
Lymphocyte cultures 
Two culture tubes for both control and experiment were established from a fresh blood 
sample. Each tube contained 5 ml of Medium-1 99 (GibcoBRL, Cat. No. 3 1 1 00-035), 1 ml 
of fetal bovine serum, (GibcoBRL, Cat. No . 1 0093 - 1 36) ,  and 0 . 1  ml of  
phytohaemagglutinin (PHA) M form (GibcoBRL, Cat. No. 1 0576-0 1 5) .  Using the WBC 
count, calculations were made to obtain 3 .25-million cells per culture tube by adding 
approximately 0 . 3-0.6 ml of blood from the sample. The culture tubes were incubated 
(under sham exposure or EMF exposure) at 37C for 72 h, which included a 1 h treatment 
with colchicine (0.05%, BDH, Prod. 2780SFM). 
Lymphocyte harvesting 
Harvesting of lymphocytes was accomplished by the same method as used for the SCE 
experiments with a little modification. After final centrifugation the supernatant was 
removed to 5 mm above the bottom of the tubes without disturbing the pellet. Then the 
pellets of the two tubes were transferred to one Eppendorf ( 1 .5  ml) tube, filled with fresh 
fixative and stored in the freezer (at -20C) until sl ides were required for chromosome 
painting. 
Chapter Three: Materials and Methods. 1 78 
Slide preparation and fluorescence in situ hybridization 
Day-one : 
Lymphocytes dropping on pre-cleaned slide 
Prior to dropping of lymphocytes on slides, Eppendorf tubes containing dividing 
lymphocytes were thawed at ambient temperature for approximately 1 0- 1 5  mins. Each tube 
was sUbjected to brief vortex and then spun for 5 - 1 0  sec using a microcentrifuge (Model 
No. C- 1 200, 220V/50 Hz, National Labnet Co) . After centrifugation, the supernatant was 
removed to approximately 5 mm above the bottom of the tubes without disturbing the 
pellet. A single drop of the lymphocyte was then placed on a pre-cleaned slide and the area 
of the droplet was marked with an electronic engraver on the specimen slide. The slide was 
then kept at ambient temperature in the dark for 24 h. 
Day-two : 
Pre-hybridisation slide washing and drying 
Nine clean Coplin jars (1) with lids were placed in the bio-hood. Each of the jars (from J- l 
to j-5) were filled with 70 ml of freshly prepared solution of 70%, 85%, 95%, 1 00%, 1 00% 
alcohol, respectively. J-6 was filled with 70 ml of Roticlear solution, and the final three Jars 
(1-7 to J-9) were filled with 1 00% alcohol. The slides were immersed in each solution 
(from J- l to J-5) then for 1 0, 1 0, 1 0, 1 0  and 30  min. The slides were then immersed in 
Roticlear solution (J-6) for 30 min followed by 1 0  min in each of final three jars (1-7 to J-
9). Slides were then transferred to a hot plate at 3 7C for 30 min. 
Preparation of probe mixture and in situ hybridisation 
For the FISH analysis, chromosomes 2, 3 and 5 were painted green by in situ hybridisation 
with whole chromosome probes (Vysis) labelled with fluoresce in isothiocyanate (FITC). 
The centromeres were labelled red with Cy3-pancentromeric probe (Cambio) . The rest of 
the chromosomes were stained with the blue fluorescent dye, DAPI . The co-denaturation 
Chapter Three: Materials and Methods. 1 79 
process was used in this study; this simplifies FISH by combining denaturation of probe 
mixture and specimen into a single step. 
In a sterile Eppendorf tube, a cocktail of the probes and hybridization buffer was prepared 
to a total of 1 0 �l in the following ratios: 
Pancentromeric probe (PCP) 
Chromosome 2 probe 
Chromosome 3 probe 
Chromosome 5 probe 
Hybridization buffer 
Total 
The probe cocktail was then mixed thoroughly by centrifuging the Eppendorf tubes for 3-5  
sec with a micro-centrifuge, followed by vortex for 3-5  sec ,  and then centrifugation again. 
Ten �l of the probe mixture was placed on the targeted area of the sample slide, covered 
with a 22 mm x 22 mm coverslip (Biolab Scientific), and then sealed with rubber cement 
(vulcanizing rubber solution). The slides were then kept in the dark at an ambient 
temperature for 1 5-30 mins to dry the rubber cement. The slides were then placed onto a 
micro-heating system (Vysis HYBrite ™ hybridization system) at 75C for 5 min to allow 
denaturation of the probes and DNA. The slides were then placed on moist tissue in a 
closed plastic container overnight at 37C in a thermostat control room to allow 
hybridisation. 
Day-three: 
Rapid post hybridisation wash, drying and counterstain 
Post hybridisation wash for probes was carried out by immersing the slides in 0.4 x SSC / 
3% NP 40 ( 1  x SSC = 1 50 mM NaCI, 1 5  mM sodium citrate) for 2 min at 7 1 C  and 1 min in 
2 x SSC / 0. 1 % NP 40 at ambient temperature. After draining off excess liquid and drying 
for 30  min, the slides were stained with 1 5  �l of OAP!.  Slides were covered with glass 
Chapter Three: Materials and Methods. 1 80 
coverslips (22 X 22 mm), sealed with nail polish, and kept in the dark for 1 0- 1 5  min. The 
slides were stored at 4C until analysis (between 1 and 24 h). 
Cytogenetic evaluation 
For the analysis of translocation (shift of any green across to a blue chromosome) 545 cells 
from control and 506 cells from EMF -exposed experiments were scored. The formulas, 
proposed by Lucas et al. ( 1 992) and Straume and Lucas ( 1 993),  could be used for 
comparison of data obtained with different chromosome cocktails .  These formulas are 
based on the assumption that the probabi lity of a chromosome being involved in a 
particular exchange aberration is proportional to its DNA content. 
Chapter Four: Results. 1 8 1  
Chapter Four 
Results 
4.1 Sister Chromatid Exchange (SCE) 
In the present study, the frequency of SeEs from two rounds, round- l (RI )  and round-2 
(R2), of experiments were studied in peripheral blood lymphocytes (PBLs) from six 
healthy, non-smoking male blood donors . Donors were selected with similar lifestyles, 
medical history, and dietary history. Ages of the first three donors were 50-56 years and 
the remaining three donors were 3 1 -44 years. The background information of each 
donor was obtained through a detailed questionnaire but a careful analysis of these data 
showed no record of any confounding factor(s), which could influence the results. None 
of the participants was a smoker or substantial consumer of alcohol or caffeine, which 
are the main factors that could influence the results. 
A set of eight experiments was conducted in duplicate, with each duplicate experiment 
using the same strict EMF conditions. The eight experiments in R I  were conducted 
using blood samples from the first three donors, and blood samples from the remaining 
three donors were used in the R2 experiments. A specially designed and built EMF field 
generator (incubator) was used to generate different EMF waves (sine and square) ,  
different EMF flux densities, one weak ( l ilT), and one strong ( 1  mT), and continuous or 
pulsed fields (4 sec on and 4 sec off). 
Lymphocyte cultures in eight experiments conducted in each round were exposed to 50  
Hz sinusoidal (continuous or pulsed) or square (continuous or  pulsed) MFs at field 
strengths of l ilT or ImT for 72 h. Lymphocyte cultures of three controls were sham 
exposed in each round. In the twelfth experiment of each round, lymphocyte cultures 
were exposed to a complex MF, produced from the back of a computer, for 72 h. 
To eliminate bias, an independent person was involved in setting the EMF parameters, 
which were unknown to the researcher. A total of 5 145 and 6 1 4 1  c-metaphase cells with 
clear exchanges were analysed in RI  and R2. 
Chapter Four: Results. 1 82 
4.1 . 1  Analysis of Round-l (SCE) 
Initially, the data ofR l  were examined by collapsing across all experimental conditions 
so that the mean number of SCEs in all experimental conditions could be compared 
with the control condition. Table 4. 1 summarises the data for the number of cells 
analysed (N), the mean SCE and standard deviations of the control group and that of the 
experimental group. The data were analysed by t-test to see whether there was any 
difference between the mean SCE for the control group and that of the combined 
experimental groups. 
A statistically significant difference was observed between the mean SCE of the control 
group and that of the entire set of experimental groups, t (5 1 43)  = 4 .09, P < .00 1 . 
Table 4.1 :  Overall mean SCE and standard deviations of the control 
group and that of the entire set of experimental groups for RI . 
Expt. N Mean Std. Deviation 
Control 1 554 8 .92 3 . 1 9  
Experimental 3 59 1  9.34 3 .49 
Does the result shown in Table 4. 1 apply to all three donors? Figure 4. 1 shows the 
overall results but for each donor separately . D - l  (donor- I )  and D-2 showed a 
significant difference t ( 1 608) = 4.09, P < .00 1 ,  and t ( 1 947) = 4 .33 ,  P < .00 1 ,  
respectively, while 0-3 showed only a small increase in SCE per/cell for the exposed 
conditions. This difference was not significant (t < 1 ) .  
Chapter Four: Results. 1 83 
1 1 .0 -+- Contro\ 
1 0.5 
_ EMF exposed 
1 0.0 t ( 1 608) = 4.09 ! .... � t ( \ 584) = 0.67 
P < .OOl P = .5 � 9.5 t u --� 9.0 U L. .... { rZJ 
a 8.5 Cl) • :::E 8.0 t ( 1 947) = 4.33 
7.5 P < .00 1 
7.0 
0- 1 0-2 0-3 
Figure 4.1 :  Graph of mean SCE of control groups and EMF exposed groups of 
three donors in RI . 
Figure 4.2 shows an example of a Giemsa stained complement from a sham-exposed 
control experiment showing 1 1  SCEs (arrowheads) . Figure 4.3 is a similar complement 
but from an EMF-exposed experiment which shows 20 SCE (arrows). However, in 
some spreads both the chromatids in some chromosomes show light or pale colour (Fig. 
4.4). Figure 4.4 is an example of a cell that has undergone three rounds of DNA 
replication in the presence ofBrdU. 
Table 4.2 summarises the data in RI  on the number of cells observed for SCE (N), 
mean SCE and standard deviations for all eight experiments, controls, and complex. 
The mean SCE values of the three controls and complex experiment for RI are 8 .92 and 
9.34, respectively. The mean SCE values range from 8.08 for sine continuous IpT to 
1 0.03 for square continuous ImT It is also notable that the mean SCE value increases 
with an increase of field strength in all parameter combinations except for a downward 
trend in sine pulsed IpT and ImT (9.55 and 8.82, respectively). The lowest standard 
deviation (2.73) was observed in the experiment sine continuous IpT, and the highest 
(4.59) for the experiment square pulsed ImT, but a comparatively uniform standard 
deviation was observed for all other experiments (2.98-3 .67). 
Chapter Four: Results. 
Figure 4.2: A c-metaphase chromosome spread of a 
dividing peripheral blood lymphocyte from a sham 
exposed control that has undergone two rounds of 
DNA replication in the presence of BrdU, showing 
1 1  SCEs (arrowheads). 
Figure 4.3: A c-metaphase chromosome spread of a 
dividing peripheral blood lymphocyte from an EMF 
exposed experiment that has undergone two rounds of 
DNA replication in the presence of BrdU, showing 20 
SCEs (arrows). 
1 84 
Chapter Four: Results. 
Figure 4.4: A c-metaphase chromosome spread of a 
dividing peripheral blood lymphocyte that has 
undergone three rounds of DNA replication in the 
presence of BrdU, showing both the chromatids light 
or pale (arrows). 
Table 4.2: Mean SCE and standard deviations of all experiments in RI based 
on all three donors. 
Wave Form Strength Mean Std. N 
(T) (SCE) Deviation 
Sine Continuous YA 8.08 2.73 87 
Sine Continuous lm  9.62 3 .22 600 
Sine Pulsed lp 9.55 3 .07 443 
Sine Pulsed fm 8.82 3 . 1 2  600 
Square Continuous JP 9.09 2.98 600 
Square Continuous lm 1 0.03 3.67 600 
Square Pulsed l)A 8 .70 3.28 79 
Square Pulsed l m  9.28 4.59 495 
Complex 9.34 3.0 1 87 
Control (x3) 8 .92 3 . 1 9  1 554 
Total 5 1 45 
Note: T = Tesla. 
1 85 
Chapter Four: Results. 1 86 
These mean differences were subjected to a 4-way ANOVA (analysis of variance) test 
(Table 4 .3) .  To reiterate for clarity, Wave (sinusoidal, square), Form (continuous, 
pulsed), Strength ( l flT, ImT) and Donor (D- I ,  D-2, D-3) were the factors. There was a 
significant main effect for Strength, F ( 1 ,  24) = 1 6 .64, P = . 0 1  and a near significant 
main effect for Wave, F ( 1 , 24) = 7.04, P = .09. However, the main effect for Strength 
was qualified by a near significant Form x Strength interaction, F ( 1 , 24) = 1 0.29, P = 
.08 .  This interaction is shown graphically in Figure 4 .7 .  Follow up {-tests showed that 
for the continuous wave form the SCE/cell count was significantly greater for the 1 mT 
field strength compared to the 1  flT field strength, { ( 1 885) = 5 .5 1 ,  P < .00 1 ,  whereas the 
1 flT field produced a higher SCE count than the 1 mT field for the pulsed wave, { 
( 1 6 1 5) = 2.0 1 ,  P = .04 (see Figure 4.7). 
Table 4.3 : Overall ANOV A for RI . 
Source df F P 
Intercept 1 692.83 0 .001  
Wave 1 7 .04 0 .09** 
Form 1 1 . 1 5  0 .38 
Strength 1 1 6.64 0 .0 1  * 
Donor 2 - - * * *  
Wave x Form 1 2 . 5 5  0.24 
Wave x Strength 1 0 . 30  0.88 
Form x Strength 1 1 0.29 0.08* *  
Wave x Form x Strength 1 1 . 3 3  0 .36 
Wave x Donor 2 0 . 1 3  0 .89 
Form x Donor 2 2 .89 0.98 
Wave x Form x Donor 2 0 .38 0.72 
Strength x Donor 2 0 .03 0.98 
Wave x Strength x Donor 2 1 .89 0 .35  
Form x Strength x Donor 2 0.69 0 .59 
Wave x Form x Strength x Donor 2 3 .29 0 .04* 
Note: ( *  = significant, * *  = Near significant, and * * *  = The SPSS could not 
calculate because it cannot compute the error degrees of freedom using 
Satterthwaite's  method. 
Chapter Four: Results. 
10.0 
9.8 
9.6 
9.4 
9.2 
9.0 
8.8 
t ( 1 728) = 0.55 
P = .59 
t r;_------: ..,··  iI 
1-'--Continuous 1 !- Pulsed 1 
t ( 1 1 72) = 1 .89 
P =.06 
8.6 +---------r-----------. 
Sine Square 
Figure 4.5: Graph of mean SCE of different Wave and Form in R I  
1 0.0 
9.8 
- 9.6 a:i t ( 1 728) = 0.55 () -... P = .59 � U 9.4 t.., m 
§ 9.2 v 
� 
9.0 
8.8 
8.6 
Sine Square 
1 87 
Figure 4.6: Graph of mean SCE of different Wave and Strength in R I  
10.0 I : I �T I _ lmT 
9.8 
- 9.6 a:i () -... 
� 9.4 t ( 1 885) = 5 .5 1  U m P <  .001 
§ 9.2 v 
� 
9.0 
8.8 
8.6 
Continuous Pulsed 
Figure 4.7: Graph of mean SCE of different Form and Strength in R I  
Chapter Four: Results. 
1 0.5 
1 0.0 
- 9.5 "0 Q 
iil 
u 9.0 
r/) 
§ IU 8.5 � 
8.0 
7.5 
0-1 0-2 0-3 
Sine 
Square 
1 88 
Figure 4.8: Graph of mean SCE of different Wave and three Donors. 
1 0.5 
1 0.0 
9.5 
9.0 
8.5 
8.0 
-+--Continuou 
_Pulsed 
7.5 +------...---------r-------. 
0- 1 0-2 D-3 
Figure 4.9: Graph of mean SCE of different Form and three Donors. 
-+-- lflT 
1 0.5 -.- lmT 
1 0.0 
-
� 
"0 9.5 
� 
� 
u 
: r/) 9.0 § <1) 
� 8.5 
8.0 
7.5 
0-1 0-2 D-3 
Figure 4.10:  Graph of mean SCE of different Strength and three Donors. 
Chapter Four: Results. 189 
There was also a 4-way interaction across Wave, Form, Strength, and Donor, F (2, 24) 
= 3 .29, P = .04, suggesting that the EMF effect is somewhat dependant on all four 
factors. 
The pair-wise interactions involved can be seen in Figures 4 . 5  - 4. 1 0. By far the biggest 
component of this interaction was the contribution made by the Form x Strength 
interaction (Figure 4 .7) and the Wave x Strength interaction (Figure 4 .6). Concerning 
the donor' s  contribution, D-3 always had the highest number of SCE/cell  compared 
with the other two donors . 
4.1 .2 Analysis of Round-2 (SCE) 
As in RI , the data of R2 were also examined by collapsing across  all experimental 
conditions so that the mean number of SCEs in all experimental conditions could be 
compared with the control condition. Table 4 .4 summarises the data for the SCE means 
and standard deviations of the control group and that of the experimental group. The 
data were again analysed by Hest to see whether there was any major difference 
between the mean SCE for the control group and that of the combined experimental 
groups. 
Table 4.4: Overall mean SCE and standard deviations of the control 
group and that of the entire set of experimental groups for R2. 
Expt. N Mean Std. Deviation 
Control 1 53 5  9.66 3 .72 
Experimental 4606 1 0. 1 7  3 .79 
A statistically significant difference was observed between the mean SCE of the control 
group and that of the entire set of experimental groups, t (6 1 39) = 4.57, P < .00 1 .  
Similar to RI , Figure 4 . 1 1  shows that the difference i s  mainly due to donors D-4 and D-
6, D-5  making a minor contribution. D-6 and D-4 showed a significant difference, t 
( 1 686) = 4.45,  P < .00 1 ,  and t (2280) = 2.8 1 ,  P < . 0 1 ,  respectively, while D-5 showed 
Chapter Four: Results. 1 90 
small increase in SCE / cell for the exposed conditions. This difference was not 
significant (t = 1 . 1 2). 
1 1 .0 
1 0.5 
1 0.0 
- 9.5 � u 
P:l 9.0 U 
r/J 
� 8.5 11) 
::E 8.0 
7.5 
7.0 
r·······: 
... 
• 
t (2280) = 2.8 1 
P < .01  
D-4 
r·······1 
-+- Control 
_ EMF exposed 
... . -=:!E::.:+-----....... t ( 1 686) = 4.45 
P < .00 1 
D-5 D-6 
Figure 4. 1 1 :  Graph of mean SCE of control groups and EMF -exposed groups of 
three donors in R2. 
In summary, looking at Figures 4. 1 and 4 . 1 1  it can be seen that four of six donors 
showed statistically significant changes between the control and exposed conditions. 
However, even though the remaining two donors did not yield significance, the data 
showed a trend towards an effect from EMF exposure. 
Table 4.5 summarises the data in R2 on the number of cells observed for SCE (N), 
mean SCEs and standard deviations for all 1 2  experiments. It is worth mentioning here 
that the overall mean SCE value in R2 is higher than that in RI . The mean SCE value of 
the three control experiments and complex experiment for R2 are 9.66 and 9.46, 
respectively. The mean SCE values range from 9.66 for square pulsed Im T to 1 0.6 1 for 
sine pulsed I p T. It is also notable, in contrast to R I ,  that the mean SCE value decreases 
with an increase of field strength in all parameter combinations except for an upward 
trend between sine continuous I p T  and I m T  (9.98 and 1 0.2 1 ,  respectively). The 
standard deviations differ little across all the experiments ( 3 . 5 2-4.09), with the standard 
deviation value of the experiment sine pulsed 1 p T  being the highest. 
Chapter Four: Results. 1 9 1  
Table 4.5: Mean SCEs and standard deviations of all experiments in R2, 
based on all three donors. 
Wave Form Strength Mean Std. N 
(T) (SCE) Deviation 
Sine Continuous 1 �  9.98 3 .58 508 
Sine Continuous 1 m  1 0.2 1 3 . 85 52 1 
Sine Pulsed 111 1 0.6 1 4.09 5 1 6  
Sine Pulsed 1 m  9.98 3 .62 473 
Square Continuous 111 1 0. 39  3 .86 600 
Square Continuous 1 m  9.89 3 . 83 600 
Square Pulsed 1 Jl  9.83 3 .66 600 
Square Pulsed 1m  9.66 3 .74 600 
Complex 9.46 3 . 52 1 88 
Control (x3) 9.66 3 . 72 1 53 5  
Total 6 14 1  
These mean differences were subjected to  a 4-way ANOV A test (Table 4.6) as in R I .  
No significant difference for any of the main effects, Wave, Form, Strength or Donor 
was observed. However, there was a significant Wave x Form interaction, F ( 1 , 24)= 
90.62, P < . 0 1 .  This interaction is shown visually in Figure 4 . 1 2 . Follow up {-tests 
showed that there was a significant difference in SCE count between Continuous and 
Pulsed for the Square wave, { (2398) = 4 .45 ,  P < .00 1 ,  but no difference for the Sine 
wave, { (20 1 6) = 1 .28, P = . 1 9. 
Comparing R I  with R2, it will be recalled that there was a near significant interaction 
between Form and Strength (P = .08) in R I ,  Table 4.3 . Table 4.6 shows that while this 
interaction did not reach significance in R2 (P = 0 . 1 6) there was a trend towards an 
interaction. This can be seen in Figure 4. 1 3 , which can be compared with Figure 4 .7 .  
The main feature to note i s  that in both cases the trend i s  exactly the same. For a 
Continuous wave form, there was a greater SCE/cell for the 1mT field compared to the 
1 � T field, whereas the reverse was true for the Pulsed wave form. 
The largest difference between R I  and R2 involved the two factors, Wave and Form. 
There was a significant Wave x Form interaction in R2, whereas in RI this interaction 
showed only a small trend, if anything. 
Chapter Four: Results. 
Table 4.6: Overall ANOV A for R2 .  
Source df F P 
Intercept 1 1 1 43 .82 0.00 1 
Wave 1 3 .63 0. 1 7  
Form 1 2.77 0.24 
Strength 1 0.01 0.95 
Donor 2 8 .58 0.23 
Wave x Form 1 90.62 0.0 1 * 
Wave x Strength 1 0.69 0.49 
Form x Strength 1 4.66 0. 1 6  
Wave x Form x Strength 1 0.35 0 .61  
Wave x Donor 2 3 .42 0.99 
Form x Donor 2 - - * *  
Wave x Form x Donor 2 0.06 0.94 
Strength x Donor 2 2 .22 0.67 
Wave x Strength x Donor 2 0 .96 0.5 1 
Form x Strength x Donor 2 0.6 1 0.62 
Wave x Form x Strength x Donor 2 2.07 0. 1 3  
Note: * = significant, **  = SPSS could not calculate because it cannot 
compute the error degrees of freedom using Satterthwaite 's  method. 
-+--Continuous 
10.6 _Pulsed 
1 0.4 
- t (2398) = 4.45 Q) () 1 0.2 P < .00 1 ---
U.l 
U 
rn 
� 1 0.0 t (20 1 6) = 1 .28 
� 
P = . 1 9  
9.8 
9.6 
Sine Square 
Figure 4. 1 2 :  Graph of mean SCE of different Wave and Form in R2 .  
1 92 
Chapter Four: Results. 1 93 
1 0.6 
--+- I �T 
-+- lmT 
10.4 t (2 1 87) = 2.02 
r o :  P = .04 - t � � 1 0.2 � U I ........ ·) r.n (2227) = 2.02 1a 1 0.0 t Q) P = .09 ::E 
9.8 
9.6 
Continuous Pulsed 
Figure 4. 13:  Graph of mean SCE of different Form and Strength in R2. 
4.1.3 Complex MF 
Table 4.7 summarises the number of cells analysed (N), the mean SCEs and standard 
deviations of the mean of the control and complex experiment in RI . No statistically 
significant difference, t ( 1 639) = 1 .20, P = 0.23, was found between the control and 
complex experiment in the mean frequency of SCE of R I .  This experiment was 
duplicated in R2. As Table 4 . 8  shows, R2 produces the same result for this complex 
fi eld, t ( 1 72 1 )  = 0. 70, P = 0.48. 
Table 4.7: Overall SCE means of control and complex experiment in RI . 
Expt. N Mean Std. Deviation 
Complex 87 9.34 3 .0 1  
Control (x3) 1 5 54 8 .92 3 . 1 9  
Chapter Four: Results. 194 
Table 4.8: Overall SCE mean of control and complex experiments in R2. 
Expt. N Mean Std. Deviation 
Complex 1 88 9.46 3 .52 
Control (x3) 1 53 5  9.66 3 .72 
It is particularly notable, however, that the number of dividing cells cultured in a 
complex field in both R I  and R2 was considerably reduced (complex N = 87 and 
control N = 5 1 8  in RI ; Complex N = 1 88 and control N = 5 1 1  in R2). Unfortunately, no 
reliable data to compare mitotic indexes are available. Nevertheless, the smaller number 
of dividing cells in s lides prepared from lymphocytes cultured in a complex field is 
interesting, a matter returned to in the Discussion. 
4.2 Micronucleus Assay (MN Assay) 
In the MN assay, the frequency of micronuclei from one set of eight experiments was 
studied using PBLs from the same three blood donors used in RI  of the SCE 
experiments . The same strict EMF conditions used in RI and R2 of the SCE 
experiments were followed. For the MN experiments, the same incubator that was used 
to conduct the SCE experiments was used again (See section 3 .2) . 
Initially, the data of MN experiments were examined by collapsing across all 
experimental conditions so that the mean number of micronucleated cytokinesis-blocked 
(CB) cells per 100 CB cells (Ma) in all experimental conditions could be compared with 
those of the control condition. Table 4.9 summarises the data for the number of CB 
cells analysed (N), Ma, and standard deviations of the control group and that of the 
experimental groups. The data were analysed by t-test to see whether there was any 
major difference between the means for the control group and that of the combined 
experimental groups. 
Chapter Four: Results. 1 95 
A statistically significant difference was observed between the mean, Ma, of the control 
group and that of the entire set of experimental groups, t (34) = 2 . 37, P = . 0 3 .  
Table 4.9:  Mas and standard deviations o f  control group 
and that of the entire set of experimental groups. 
Expt. N Ma 
Std. 
Deviation 
Control 1 8466 1 9. 3 9  5 . 2 8  
Experimental 502 1 6  2 5 . 06 8.43 
Figure 4. 1 4  shows the overall results for each donor separately. Interestingly, none of 
the differences between control and experimental conditions for any donor reached 
significance, t ( 1 0) = 1 . 36, P = .20, t ( 1 0) = 0. 2 8, P = 0 . 78 and t ( 1 0) = 1 . 74, P = . 1 1 , for 
D I ,  D2 and D3, respectively. Even though there were no significant differences here, it 
can be noted (Fi gure 4. 1 4) that the differences between control and experimental 
conditions for all three donors were in the direction of an EMF effect. However, as for 
the SCE analysis (R I ), one donor (D-2 ) had little effect on the outcome. Analysing 
more cells, or using more subj ects, would have increased the level of statistical power. 
The clear trends seen in Figure 4. 1 4  may then have reached significance. 
30.0 
2 5 . 0  
20.0 
1 5 .0 
1 0. 0  
5 . 0  
f ( 1 0) = 1 . 36 
p =  .20 
... 
I ( 1 0) = 0.28 
p =  0.78 
-+- Control 
_ Experimental 
I ........ � 
f ( l 0) = 1 . 74 
p =  . 1 1  
0 . 0  +-----..------.-------., 
D-1 D-2 D-3 
Figure 4. 14 :  Graph of Ma of control groups and EMF-exposed groups 
for three donors. 
Chapter Four: Results. 1 96 
Table 4 . 1 0  summarises the MN data for complex, three controls, and eight experiments. 
The average Ma value of the three controls and complex experiment for MN are 1 9.39 
and 1 8 . 84, respectively. The Ma value differs considerably among all the experiments 
( 1 6 . 70-3 5 . 1 8), with the Ma value of the experiment Square pulsed ImT being the 
highest. It is also notable that the Ma value increases with an increase of field strength in 
two parameter combinations Sine continuousI pT and 1 mT ( 1 6.70 to 24.02), and Square 
pulsed IpT  and ImT (33 .57  to 3 5 . 1 8), while the other two parameter combinations 
showed downward trends, sine pulsed IpT and ImT (27 . 1 5  to 1 7 .69), and Square 
continuous 1 pT  and ImT (24.98 to 22.57) .  The standard deviation (SD) of Ma for the 
experiment sine pulsed ImTwas lowest (3 .08), and highest ( 1 2 .29) for the experiment 
Sine continuous 1 mT, but a fairly uniform deviation was observed among all other 
experiments (4.05-7.40). The reason for the high SD in Sine continuous ImT ( 12 .29) 
was due to an unusually high number of CB cells with micronuclei found in D-3 . 
Table 4 . 10: Ma' s  and standard deviations of all experiments based on all three donors. 
N= Total number of CB cells observed. 
Wave Fonn Strength Ma 
Std. Deviation N 
(T) 
Sine Continuous I II 1 6.70 5 .90 6 129 
Sine Continuous 1 m  24.02 1 2 .29 6095 
Sine Pulsed I II 27. 1 5  7.40 6 1 06 
Sine Pulsed 1m  1 7.69 3 .08 606 1 
Square Continuous I II 24.98 4 .93 6322 
Square Continuous 1m  22.5 7  5 . 34  6345 
Square Pulsed I II 33 . 57  4 . 52  6086 
Square Pulsed 1m  35 . 1 8  4.05 7072 
Complex 1 8 .84 4.27 6 1 00 
Control (x3)  1 9. 39  6.67 1 8,466 
Total 74,782 
Chapter Four: Results. 1 97 
Table 4.1 1 :  Overall ANOVA for Ma. 
Source df F P 
Intercept 1 202.74 0.005 
Wave 1 3 . 74 0. 1 9  
Form 1 3 .28 0 .2 1 
Strength 1 0 . 1 7  0 .7 1 
Donor 2 0 . 38  0 .7 1  
Wave x Form 1 3 .20 0.22 
Wave x Strength 1 0 .06 0.82 
Form x Strength 1 5 .00 0 . 1 5* 
Wave x Form x Strength 1 1 .99 0.29 
Wave x Donor 2 - * * *  -
Form x Donor 2 - * * *  -
Wave x Form x Donor 2 0.28 0.78 
Strength x Donor 2 - * * *  -
Wave x Strength x Donor 2 0 .06 0 .94 
F arm x Strength x Donor 2 - * * *  -
Wave x Form x Strength x Donor 2 1 2.59 0.00 1 * *  
* = Near significant, * *  = Highly significant, and * * *  = SPSS could not calculate 
because it cannot compute the error degrees of freedom using Satterthwaite' s  
method. 
The only statistically significant outcome was a 4-way interaction involving Wave, 
Form, Strength and Donors F (2, 24) = 12. 59, P < .00 1 .  
The pair wise interactions can b e  seen in Figures 4. 1 5  through 4.20. Figure 4. 1 7  shows 
a significant difference in Ma, t ( 1 0) = 3 .25,  P < .0 1 ,  in Pulsed wave form between the 
Square wave and Sine wave. Figures 4. 1 8  to 4.20 show data for each donor separately, 
and it can be seen that D-3 is not contributing at all to Ma. Figures 4. 1 8  and 4. 1 9  show 
substantial differences between Sine and Square wave and Continuous and Pulsed wave 
forms. However, Strength had no effect on any donor. 
Chapter Four: Results. 
3 5 .0 
30.0 
25.0 
20.0 
1 5 .0 
1 0.0 
5.0 
0.0 
t ( 1 0) = .03 
P =  .97 
Sine 
t ( 1 0) = .08 
P = .94 
Square 
-+- I �T 
-+- l mT 
Figure 4. 15: Graph of Ma of different Wave and Strength. 
-+- I �T 
35.0 -+- l mT 
30.0 
25.0 • 
t ( 1 0) = .62 
20.0 • P =  .55 
t ( 1 0) = .59 
1 5 .0 p = .57 
1 0.0 
5.0 
0.0 
Continuous Pulsed 
Figure 4.16: Graph of Ma of different Form and Strength. 
40.0 
35.0 
30.0 
25.0 
20.0 
r· ... ··� 
• 
1 5 .0 t ( 1 0) = .93 
10.0 
p = .37 
5.0 
0.0 
Continuous 
-+- Sine 
-+- Square 
i .... .. � t ( 1 0) = 3.25 
� P < .0 1 
... 
Pulsed 
Figure 4.1 7: Graph of Ma of different Form and Wave. 
1 98 
Chapter Four: Results. 
35.0 
30.0 
25.0 
os 
� 20.0 
1 5 .0 
1 0.0 
5.0 
0.0 
0- 1 0-2 0-3 
Figure 4. 18: Graph of Ma of different Wave and three Donors. 
35.0 
30.0 
25.0 
os 
� 20.0 
1 5.0 
1 0.0 
5.0 
0.0 
D- I 0-2 D-3 
-+- Continuous 
_Pulsed 
Figure 4.19: Graph of Ma of different Form and three Donors. 
35.0 
30.0 
25.0 
os 
� 20.0 
1 5 .0 
10.0 
5.0 
0.0 
D- I 0-2 0-3 
1-+- I IlT I 1_ lmT I 
Figure 4.20: Graph of Ma of different Strength and three Donors. 
1 99 
Chapter Four: Results. 200 
The data of MN experiments were also examined by collapsing across all experimental 
conditions so that the mean number of micronuclei per 100 CB cells (Mb) in all 
experimental condition could be compared with those of the control condition. Table 
4. 1 2  summarises the data for the number of CB cells analysed (N), Mb, and the standard 
deviations of the control group and the experimental groups. The data were analysed by 
t-test to see whether there was any major difference between the means for the control 
group and that of the combined experimental groups. 
Table 4.1 2 :  Mb'S and standard deviations of control groups 
and that of the entire set of experimental groups. 
Expt. N Mb Std. Deviation 
Control 1 8466 23.80 6.97 
Experimental 502 1 6  32.02 1 3 .06 
A statistically significant difference was observed between Mb of the control group and 
that of the entire set of experimental groups, t (34) = 2.40, P < .0 1 .  
Does the result shown in Table 4. 1 2  apply to all three donors? Figure 4.2 1 shows the 
results for each donor separately. 
40.0 
35.0 
30.0 
25.0 .0 
::E 20.0 
1 5 .0 • 
t ( 10) = 1 .23 
1 0.0 p =  .25 
5.0 
0.0 
D-I 
• 
t ( 1 0) = 0.43 
p =  .67 
0-2 D-3 
�Control 
__ Experimental 
• 
t ( 1 0) = 1 . 5 1  
P = . 1 6  
Figure 4.2 1 :  Graph of Mb of control groups and EMF-exposed groups 
for three donors. 
Chapter Four: Results. 20 1 
Again, none of the differences between control and experimental conditions for the 
donors reached significance, t ( 1 0) = 1 .23,  P = .25,  t ( 1 0) = 0.43,  P = .67 and t ( 1 0) = 
1 .5 1 ,  P = . 1 6, for D- 1 ,  D-2 and D-3, respectively. 
Table 4. 1 3  summarises the data for complex, three controls and nine experiments. 
Average Mb values of the three controls  and complex experiment for MN is 23.80 and 
2 1 .66, respectively. The Mb value differs considerably across all  the experiments 
( 1 9.70- 48 .98),  with the Mb value of the experiment, Square pulsed lmT being the 
highest. It is also notable that the Mb value decreases with an increase of field strength 
in two parameter combinations, Sine pulsed If.JT and lmT (34.96-20.44), and Square 
continuous lilT and lmT (3 1 .39-27.66). The other two-parameter combinations showed 
upward trends, sine continuous 1 f.JT and lrnT ( 1 9 .70-29 .27), and Square pulsed lilT and 
lmT (45 .3 8-48.98) .  The standard deviations of Mb for the two experiments were the 
highest ( 1 5 . 1 0  and 1 1 .76, respectively) for Sine continuous lrnT, Sine pulsed lilT, and 
lowest for Sine pulsed lmT (3 .99), but a uniform deviation was observed among all 
other experiments (5 . 1 5-9.27) .  The reason for the high standard deviation in Sine 
continuous lmT ( 1 5 . 1 0) was due to an unusually high number of CB cells with varying 
number of micronuclei observed in D-3 . 
Table 4.13: MbS and standard deviations of all experiments based on all three donors . 
N= Total number of CB cells observed. 
Wave Form Strength Mb Std. Deviation N (T) 
Sine Continuous I II 1 9 .70 7.87 6 1 29 
Sine Continuous 1m  29.27 1 5 . 1 0  6095 
Sine Pulsed I II 34.96 1 1 .76 6 1 06 
Sine Pulsed 1m  20.44 3 .99 606 1 
Square Continuous III 3 1 .3 9  7.2 1 6322 
Square Continuous lm  27.66 7 .97 6345 
Square Pulsed III 45.38  8 .20 6086 
Square Pulsed 1m  48.98 7 .94 7072 
Complex 2 1 .66 5 . 1 5  6 1 00 
Control (x3) 23 .80 9.27 1 8,466 
Total 74,782 
Chapter Four: Results. 202 
Table 4. 1 4  shows that the only statistically significant outcome was a 4-way interaction 
involving Wave, Form, Strength and Donors F (2, 24) = 1 2.47, P < .00 1 ,  the same result 
that was obtained using Ma. Figure 4.24 shows that one main source of this interaction 
was the Form x Wave interaction. It can be seen that a significant difference, t ( 1 0) = 
3 . 33 ,  P < .0 1 ,  in Pulsed wave form between the Square wave and Sine wave (Figure 
4.24) occurred. Figures 4.25 to 4 .27 show data for each donor separately, and it can be 
seen that D-3 makes l ittle contribution, as for Ma. Thus, the factor, Donor, plays an 
important part in the observed 4-way interaction. 
Table 4.14 :  Overall ANOV A for Mb• 
Source df F P 
Intercept 1 1 76.77 0.006 
Wave 1 5 .64 0. 1 4* 
Form 1 3 . 82 0. 1 9  
Strength 1 0.3 6  0.6 1 
Donor 2 0 .32 0 .74 
Wave x Form 1 6. 1 8  0. 1 3 *  
Wave x Strength 1 1 .3 3  0 .37 
Form x Strength 1 2.02 0.29 
Wave x Form x Strength 1 2 . 1 3  0 .28 
Wave x Donor 2 - -* * *  
Form x Donor 2 - -* * *  
Wave x Form x Donor 2 0. 1 7  0.85 
Strength x Donor 2 - - * * *  
Wave x Strength x Donor 2 0.04 0.96 
Form x Strength x Donor 2 - -* * *  
Wave x Form x Strength x Donor 2 1 2.47 0.001 * *  
* = Near significant, * *  = Highly significant, and * * *  = SPSS could not 
calculate because it cannot compute the error degrees  of freedom using 
Satterthwaite's method. 
Chapter Four: Results. 
50.0 
40.0 
� 30.0 
� 
20.0 
1 0.0 
0.0 
t ( 1 0) = . 1 7  
P =  .87 
Sine 
t ( 1 0) = .06 
P =  .95 
Square 
-+- l flT 
_ l mT 
Figure 4.22: Graph of Mb of different Wave and Strength. 
50.0 
40.0 t ( 1 0) = .5 1 P =  62 
� 30.0 t ....... 
� 
20.0 
1 0.0 
0.0 
Continuous Pulsed 
Figure 4.23: Graph of Mb of different Form and Strength. 
50.0 
40.0 
30.0 � 
� 
20.0 
1 0.0 
0.0 
t ( 1 0) = .98 
P =  .35 
t . : ..... ...... ( 
• 
Continuous 
-+- Sine 
_ Square 
� .... � t ( 1 0) = 3.33 
i P < .01  
.. 
Pulsed 
Figure 4.24: Graph of Mb of different Form and Wave. 
203 
Chapter Four: Results. 
-+- Sine 
50.0 _ Square 
40.0 • • • 
30. 0 
� 
20.0 
1 0.0 
0.0 
D- l D-2 D-3 
Figure 4.25: Graph of Mb of different Wave and three Donors. 
-+- Continuous 
50.0 _ Pulsed 
40.0 
30.0 
� 
20.0 
1 0.0 
0.0 
D- l D-2 D-3 
Figure 4.26: Graph of Mb of different Form and three Donors. 
� 
50.0 
40.0 
30.0 
20.0 
1 0.0 
l-+- l ll
.
T !- l mT 
0.0 +-------.-----.....,....-------, 
D- l D-2 D-3 
Figure 4.27: Graph of Mb of different Strength and three Donors. 
204 
Chapter Four: Results. 205 
4.2. 1 Complex MF 
Table 4. 1 5  summarises the data for the number of cells analysed (N), Ma, and standard 
deviation of the mean of the control and complex experiment. No statistically 
significant difference was observed between the mean, Ma, of control and complex 
experiment, t ( 1 0) = 0. 1 6, P = 0.88 .  A simlar result was found for Mb (Table 4. 1 6), t 
( l 0) = 0.48, P = 0 .64. 
Table 4.15:  Overall Ma of control and complex experiment ofMN assay.  
Expt. N Ma 
Std. Deviation 
Complex 6, 1 00 1 8 .84 4 .53  
Control (x3) 1 8 ,466 1 9.39 5 .28 
Table 4.16:  Overall Mb of control and complex experiment ofMN. 
Expt. N Mb 
Std. Deviation 
Complex 6, 1 00 2 1 .66 5 . 59  
Control (x3) 1 8 ,466 23 .80 6.97 
4.3 COMET Assay 
The COMET assay experiments in the current study were performed in peripheral blood 
lymphocytes of one donor (D- l )  only, who took part in R I  of the SCE study. Two 
lymphocyte culture tubes were established for each of the two experiments and control .  
Lymphocyte cultures of the two experiments conducted for the COMET assay were 
exposed to 50 Hz square continuous or pulsed (4 sec on and 4 sec off) MFs at the field 
strength ImT for 72 h. The lymphocyte cultures of the control were sham exposed. Both 
the COMET assay experiments and control were conducted single blind. A total of 272 
comets were analysed for the control and those two experiments. Two cell images are 
shown in Figure 4.28, which are typical of their respective conditions : (a) shows an 
Chapter Four: Results. 206 
undamaged cell that was typical of the control conditions, whereas (b) shows a damaged 
cell that was typical of the EMF exposed conditions. 
(a) (b) 
Figure 4.28: Photographs showing single-strand DNA migration pattern of 
individual peripheral blood lymphocytes from D- l .  (a) Control Sample - This cell 
is typical of the control sample. It is undamaged and will most likely be found in a 
negative control; (b) Experimental - This cell is typical of the experimental 
samples. This cell has DNA that is extremely damaged. It is most likely apoptotic. 
Table 4. 1 7  summarises the data of the COMET assay experiments on the number of 
comets observed (N), mean Tail length, Tail moment and Olive tail moment for control, 
Square continuous lmT and Square pulsed lmT. The mean Tail length values range 
from 3 3 .99 for Square pulsed ImT to 44.02 for Square continuous ImT. The Tail 
moment values range from 4. 1 4  for control to 14.96 for Square continuous ImT, and 
similarly, Olive tail moment values range from 3 .76 for control to 1 0. 1 2  for Square 
continuous ImT. The standard deviation for Tail length values range from 24.39 for 
control to 3 8.87 for Square continuous ImT. The standard deviation for Tail moment 
values range from 1 0.33 to 28.07 for Square continuous ImT, and similarly, the Olive 
tail moment values standard deviation values range from 6.5 1 for Square pulsed ImT to 
1 5 .83 for Square continuous ImT. It  is important to note here that Square continuous 
1 m T  has the highest mean values for all three parameters (Tail length, Tail moment and 
Olive tail moment). 
Chapter Four: Results. 
Table 4.17: Summary results of three parameters (Tail length, Tail moment and 
Olive tail moment) of the COMET assay experiments. 
Parameters Experiments Mean Std. 
Deviation 
Control 34.45 24.39 
Tail length Square continuous 1 mT 44.02 38 .87 
Square pulsed 1 mT 33 .99 30.23 
Control 4 . 1 4  1 0 .33 
Tail moment Square continuous 1 mT 14 .96 28.07 
Square pulsed 1 mT 9.03 1 4 .92 
Control 3 .76 6.87 
Olive tail moment Square continuous 1 mT 1 0. 1 2  1 5 .83 
Square pulsed 1 mT 5 .67 6 .5 1 
207 
N 
1 03 
99 
70 
1 03 
99 
70 
1 03 
99 
70 
The mean differences between each pair of conditions were analysed using t-tests (see 
Table 4 . 1 8) .  Statistically significant differences were observed for the mean Tail length, 
mean Tail moment, mean Olive tail moment between control and Square continuous 
ImT, P < . 04, P < .00 1 ,  and P < .00 1 ,  respectively. It should be noted that all these 
outcomes involve the continuous waveform. However, there was also statistically 
significant difference for the mean Tail moment between control and square pulsed 
1 m T, P < .02 .  Furthermore, there was a significant difference between the two 
waveforms, P < . 0 1 . These differences are also shown graphically in Figure 4.29. 
Chapter Four: Results. 208 
Table 4.18: t-tests for all three parameters (Tail length, Tail moment and Olive tail 
moment) between the three COMET assay experiments. 
Parameters 
Tail length 
Tail 
moment 
Olive tail 
moment 
50  
45 
40 
35  
30 
25 
20 
1 5  
1 0  
5 
Experiments t 
Control vs Sq. cont. 1 mT 2.09 
Control vs Sq. pulsed 1 mT 0. 1 1  
Sq. cont. 1 mT vs Sq. pulsed 1 mT 1 . 8 1  
Control vs S q .  cont. 1 mT 3 . 6 1  
Control v s  Sq. pulsed I mT 2.38 
S q .  cont. 1 mT v s  S q .  pulsed 1 mT 1 .78  
Control vs S q .  cont. 1 mT 3 .68  
Control v s  S q .  pulsed I mT 1 .84 
Sq. cont. 1 mT vs Sq. pulsed I mT 2.5 1 
df Sig. Mean (2-tailed) difference 
200 .04 9.57 
1 7 1 .92 0.46 
1 67 .07 1 0.03 
200 .00 1  1 0.82 
1 7 1  .02 4.89 
167 .08 5 .94 
200 .00 1 6 .36 
1 7 1  .07 1 .9 1  
1 67 .01  4.45 
• Control 
Sq. cont. 1mT 
• Sq. pulsed 1mT 
O ���--�--�����--�� 
Tail 
length 
Tail Olive tail 
moment moment 
Figure 4.29: Mean values for Tail length, Tail moment, O l ive tail moment for control ,  Square 
continuous I m T and Square pulsed I m T.  
Chapter Four: Results. 209 
4.4 Fluorescent in situ Hybridization (FISH) 
The FISH experiments were performed on peripheral blood lymphocytes for only one 
donor (D- 1 )  in the current study. Only one FISH experiment was conducted because of 
time and resource constraints. 
Two lymphocyte cultures for both the experiment and the control were established. 
Lymphocyte cultures of the FISH experiment were exposed to a 50  Hz square 
continuous 1 mT  MF for 72 h. Lymphocyte cultures of the control experiment were 
sham exposed. A total of 1 05 1  metaphase cells were analysed in the experiment and the 
control conditions (Table 4. 1 9). No translocations or dicentric chromosomes were 
observed. Figure 4.30 shows a cell image typical of the control conditions. 
Table 4. 19:  Summary results of control and one FISH experiment. 
Experiments No. of cells observed No. of translocations observed 
Control 545 0 
Square continuous ImT 506 0 
4.5 Chromosomal Anomalies 
In the current study, the main aim was to investigate the biological effects of EMFs on 
the HPBL. Although it affects the frequency of SCE, frequency of micronucleus, and 
causes single DNA strand breakage (SSB), it is the chromosomal anomalies that are of 
particular interest. Of these, endoreduplication is the strangest phenomenon observed in 
PBL cultures of all donors studied. Figures 1 (a & b) and 2 (a & b) of Appendix 5 ,  
shows the result o f  endoreduplication i n  cultured lymphocytes at metaphase. The 
chromosomes have replicated but were unable to separate correctly at anaphase, thus 
resulting in this abnormal configuration. 
Chapter Four: Results. 
Figure 4.30: Photographs showing a human peripheral blood 
lymphocyte cell with six whole chromosome (2, 3 and 5) 
labelled with FITC (green), centromeres labelled red with Pan 
Centromeric Probe (PCP, Cambio) and rest of the chromosome 
labelled with DAPI in the analysis of metaphase cells. 
2 1 0  
Among other anomalies; multiple SCEs (3 & 4) are shown in Figure 3 (a & b), and 
chromosome complexes formed by 2-pairs of chromosomes are shown in Figure 4 (a & 
b) of Appendix 5,  respectively. 
Chapter Five: Discussion. 2 1 1  
Chapter Five 
Discussion 
In this section the author explores the significance of the findings in this study as they 
relate to the current body of knowledge about EMF effects on cells, in particular on the 
genetic machinery. Because this area is highly contentious, the researcher was cognisant 
of the fact that the study had to be conducted under strict conditions, i .e. ,  sufficient 
power to detect any effects if they existed, ensure that the fields to which the 
lymphocytes were exposed in all experiments were clearly defined and, most 
importantly, demonstrated to be so, and that the study be conducted blind. In addition, 
the researcher wanted to determine whether the results obtained could be repeated and 
in different subjects. For this reason, the study was conducted twice (Round 1 and 
Round 2) for the initial main focus on SCE. Limited time and limited resources did not 
permit the whole study to be repeated for the MN assay, COMET assay and F I SH. 
Thousands of cells were examined and scored which involved a considerable effort, but 
this meant that any conclusions drawn were supported by reasonably secure data. 
5.1 SCE Analysis 
The SCE experiments for both rounds (Rl and R2) were conducted to evaluate whether 
or not weak EMFs affect the frequency of SCE, which is used as an indicator of 
clastogenicity, or genotoxicity, in blood lymphocytes of humans. Six donors took part in 
this study (identified as D-l  to D-6). The first three donors (D- l  to D-3) were included 
in R l , whilst D4-D6 were included in the replicate set of experiments (R2). For all 
experiments, BrdU-treated GO lymphocyte cultures that were subjected to both control 
and experimental conditions were placed in a specially designed incubator at 3 7C .  The 
incubator was placed in an electrically quiet room where stray magnetic and electric 
fields as measured with a standard Gaussmeter, were negligible. Lymphocyte cultures 
of experimental conditions were exposed each to a certain MF continuously for 3 days 
while the lymphocyte cultures of the control experiments were sham-exposed (no EMF 
fi eld). In an additional experiment, lymphocytes from all donors were grown in the 
presence of a complex MF produced from the back of a computer. Lymphocytes were 
harvested at the end of a 72 h incubation period, which included 1 h of treatment with 
Chapter Five: Discussion. 212  
colchicine. Approximately 200 second mitotic metaphase cells for SCE were scored per 
donor, per experiment. 
The SCE results show that ELF EMFs do have an effect on human chromosomes. These 
results, however, are sometimes conflicting and difficult to interpret. While a significant 
difference between the experimental and sham control conditions was observed, there 
were mixed results throughout the different parameter combinations as well as 
inconsistencies in and across both rounds of experiments. 
The overall means of SCE/cell of the controls and that of the entire set of experimentals 
for RI and R2 are shown in Tables 4 . 1  and 4.4. The overall mean of SCE/cell for the 
controls in RI and R2 were 8.92 and 9.66, respectively, whereas the overall mean of 
SCE/cell of the entire set of experimentals in R I  and R2 were 9.34 and 1 0. 1 7, 
respectively. A (-test showed that there was a significant increase (P < .00 1 )  in the 
number of SCEs/cell in the grouped experimental conditions compared to the controls 
in both RI  and R2. The important point to note here is the repetition of an overall 
increase in the number of SCE/cell as a consequence of EMF exposure in R2 that was 
previously observed in RI . 
SCE results were analysed further to determine whether all six donors contributed 
equally to the results of RI and R2. The results showed significant increases in the 
formation of SCE/cell under grouped experimental conditions compared to the sham 
exposed controls for four donors out of six, while the other two donors showed only a 
small increase in the effects for the experimental conditions. However, even though 
these remaining two donors did not yield significance results, the data showed a trend 
towards an effect from EMF exposure (Figures 4. 1 and 4. 1 1 ). 
A key element in this body of work was the adoption of a reductionist approach to try 
and establish whether any particular factor or combination of factors could affect the 
genetic machinery. Unfortunately the results of the SCE study are complex and 
interpreting the results is not easy. Nevertheless, a close examination shows some 
interesting patterns. First, in RI the mean SCE values increased with increasing EMF 
field strength in all parameter combinations except for a downward trend in sine p ulsed 
1 pT to 1 mT (Table 4.2), while the SCE values for R2 are largely the reverse. 
Chapter Five: Discussion. 213  
Paradoxically, the SCE values decreased in R2 with an increase of  EMF field strength 
in all parameter combinations except for an upward trend between sine continuous 1 p T  
and I m T  (Table 4 .5) . Thus, the mean SCE values increased with increasing EMF field 
strength (JpT to Im1) in both RI and R2 but only for sine continuous. The trend of 
increasing SCE frequency for sine continuous with increasing field strength was exactly 
the same in both rounds. But for sine pulsed the mean SCE value was downward for 
both RI  and R2 with increasing field strength. For both square continuous and square 
pulsed the SCE value was upward in RI and downward in R2 with increasing field 
strength. It would appear from these data that there are no straightforward conclusions 
that can be drawn concerning a linear increase in EMF field strength and genetic effect, 
although a closer examination may indicate that some subtle effects may be occurring. 
Interactions 
A detailed analysis of parameter interactions showed the most interesting results. Figure 
4.5 shows that a square continuous field produces a near significant higher number of 
SCEs per cell in RI (P = 0.06). Similarly, we can see from Figure 4 . 1 2  in R2 that the 
same square continuous field indeed produces a significantly higher number of SCEs 
per cell. Going a step further, Figure 4.6 shows that a square wave produces a 
significantly higher number of SCEs per cell with increasing field strength, and Figure 
4.7 shows that a continuous form produces a significantly higher number of SCEs per 
cell with increasing strength. Thus, it appears that a square continuous field with 
increasing strength may be of importance here in producing a higher number of SCEs 
per cell. 
The author notes that the SCE frequency of 1 0.61  scored for a sine pulsed field at I J.lT 
in R2 was the highest recorded, which theoretically could mean that this field has the 
greatest c1astogenic effect of all parameters, but caution is warranted here; the highest 
SCE frequency in RI was 1 0.03 for a square continuous field, and the SCE frequency 
of 1 0.39 for a square continuous field in R2 (albeit a different strength) was the second 
highest in this latter round. The possible importance of a square field generating a 
c1astogenic effect became even more apparent with subsequent experimentation using 
the Micronucleus assay and COMET assay. 
Chapter Five: Discussion. 214 
Complex field 
The mean SCE value of the three control experiments and complex experiment for RI 
and R2 are shown in Tables 4.7 and 4.8, respectively. No statistically significant 
difference was observed in either RI or R2 between the mean SCE/cell of the control 
groups and that of the complex field. The author notes, however, that despite attempting 
to culture lymphocytes on three separate occasions in a complex field, difficulty was 
always encountered in obtaining a sufficient number of dividing metaphase cells. Only 
87 cells could be scored in RI , and only 1 88 cells in R2, in all 3 subjects. A similar 
reduction has also been noted by Nagy and Fischl (2004) in the sporulation of fungi. 
The low mitotic index observed in the present study severely limits the statistical power 
of the observations and consequently restricts the conclusions that can be drawn. 
Nevertheless, a repeatedly low mitotic index must be a cause for concern, despite the 
lack of significant differences in SCE frequencies between the complex-field-exposed 
cultures and the controls. This raises questions concerning health issues and could be 
the focus of a more intensive study in the future. 
EMF exposures and SCE 
The overall increase in number of SCE/cell as a consequence of EMF exposure that was 
observed in the current study is in good agreement with the results reported previously 
by Khalil and Qassem ( 1 99 1 ). They similarly showed that in vitro exposure of human 
cells to EMFs resulted in a significant increase in frequency of SCE. In their study, 
PHA-stimulated lymphocytes were exposed to a pulsed field (50 Hz, 1 .05 mT peak; 
pulse duration, 1 0  msec) for 24 h, 48 h, and 72 h. No significant alterations in the 
baseline frequency of SCE were observed in cultures exposed to EMFs for 24 h or 48 h. 
In contrast, however, a statistically significant 2-fold increase in the frequency of SCE 
was observed in cultures continuously exposed for 72 h. This is the only report so far 
available which shows this positive result using SCE analysis on extremely weak MFs. 
It is interesting to note, however, that Yaguchi et at. ( 1 999) also observed an increase in 
SCE in mouse m5S chromosomes when they applied much stronger MFs (5-400 mT). 
Furthermore, several other studies of human (Garcia-Sagredo and Monteagudo, 1 99 1 ;  
Nordenson et al. 1 994) as well as animal (Lai and Singh 1 997a) cells exposed to ELF 
EMFs have also shown c1astogenic effects via different methods. 
Chapter Five: Discussion. 2 15  
In  Garcia-Sagredo and Monteagudo' s ( 1 99 1 )  study, HPBLs were exposed to  pulsed 
EMFs at 4 mT and a statistically significant increase in frequency of CAs was observed. 
More recently, Nordenson et al. ( 1 994) exposed human amniotic cells to a sinusoidal 50 
Hz, 30  J.lT pulsed MF ( 1 5  sec on / 1 5  sec oft) for 72 h, and similarly observed a 
significant increase in the frequency of CAs. Among exposed cells, the aberration 
frequency was 4% compared to 2% in sham-exposed cells. The same authors observed a 
similar increase in CA in another series of eight experiments in a 2 sec on / 2 sec off 
pulsed field (Nordenson et al. 1 994). The rationale used to explain EMF effects 
observed with pulsed fields but not continuous fields is sometimes attributed to a 
phenomenon called "habituation", or in this case a lack of habituation. According to this 
theory, cells can become habituated to a constant continuous field and thus do not show 
a response. With pulsed fields, the opposite prevails: the view expressed is that cells do 
not become habituated and therefore do show a response. No mechanism as to how 
habituation could be achieved has been documented that the current author is aware of, 
and whilst agreeing that the idea is attractive, no evidence was found in the present 
study to support the theory. 
Conflicting results 
In the current study conflicting results were observed in some parameters between the 
two successive rounds of identical experimentation. Sometimes a significant effect was 
observed in RI but not in R2 and vice versa. Such contradictions are not unusual in this 
field of study as is illustrated by the following examples. DNA strand breaks which are 
a common form of DNA damage were observed in brain cells of rats in a study 
conducted by Lai and Singh ( 1 997a). In their study, rats were exposed for 2 h to a 60 Hz 
MF (flux densities 0. 1 ,  0.25 and 0.5 mT) and they observed a dose-dependent increase 
in DNA strand breaks in the brain cells at 4 h post-exposure. An increase in single­
strand DNA breaks was also observed in all three flux densities, but double-strand DNA 
breaks were observed only at the higher parameters of exposure (0.25 and 0.5 mT). On 
the other hand, other authors have reported that ELF EMFs affect neither the frequency 
of SCE nor CA in HPBLs. Rosenthal and Obe ( 1989) exposed mitogen-stimulated adult 
peripheral lymphocytes to a sinusoidal 50 Hz field from 0. 1  mT to 7.5 mT and reported 
no effect of EMF on the frequency of both chromosome breaks and SCEs. A similar 
study using a pulsed MF at 1 .0, 2 .0, and 4.0 mT, applied as a quasirectangular pulse of 
Chapter Five: Discussion. 2 1 6  
26 J.lsec, in 5 msec trains, repeated at 1 4  Hz, also showed no effect on SCE (Garcia­
Sagredo et al. 1 990). 
Similar findings of null EMF effects were also reported by Antonopoulos et al. ( 1995) 
and Cohen et al. ( 1 986a, b). HPBLs were exposed to 5 mT, 5 0  Hz MF, in the former 
study, but in the latter, the same were exposed to 60 Hz, circulatory polarized fields at 
an electric current density of 30 IlNcm2 andlor MFs at 0. 1 and 0.2 mT. In both studies 
no effect of magnetic, electric, or a combined EMF was observed on the frequency of 
SCE, mitotic rate or chromosome breakage. 
In a continuation of null EMF effects, an animal genotoxic study performed by 
Livingston et al. ( 199 1 )  on Chinese hamster ovary fibroblasts and PHA-stimulated adult 
and newborn lymphocytes were exposed to a fixed MF of 0.22 mT, with 60 Hz electric 
fields (current densities of 3, 30, 3 00, and 3 000 IlNcm2). No significant effects on SCE 
were observed in any cell type or field examined. In a similar study, exposures of 
Chinese hamster cells to pulsed EMFs from 0 . 1 8-2.5 mT did not show any increase in 
the baseline frequency of SCE (Takahashi et al. 1 987). The data from several other 
studies have indicated an absence of significant differences in the incidence of CA, SCE 
as well as micronucleus frequency between RFR-exposed and sham-exposed cells 
(Vijayalaxmi et al. 1 997, 200 1 a, b; d' Ambrosio et al. 2002; Bisht et al. 2002). 
EMF effects non-reproducible 
As noted previously, although an overall effect was observed in the present SCE study, 
different parameter combinations in RI and R2 did not yield exactly duplicate results. 
This is perplexing. Other workers have encountered similar difficulties in reproducing 
their own positive findings. Maes et al. ( 1 996, 1 997) reported a significant increase in 
CA and a weak effect on SCEs in RFR-exposed human blood lymphocytes but this was 
not confirmed in their own subsequent investigation (Maes et al. 200 1 ) .  Similarly, 
subsequent studies by other investigators have sometimes not confirmed previous 
observations. For example, Lai and Singh ( 1 995, 1 996, and 1 997a) observed an in vivo 
significant increase in DNA single and double strand breaks in brain cells of rats, when 
they were exposed to 2.45 GHz, but this was not confirmed in several subsequent 
Chapter Five: Discussion. 2 17  
studies using rodent and human cells (Malayapa et al. 1 997a, b, 1 998; Maes et al. 1 997; 
Vijayalaxmi et al. 2000; Li et al. 200 1 ;  Tice et al. 2002). 
Donor 
Two groups of three healthy donors were used in the present SCE study. The intention 
of performing the entire experiments twice with a different set of donors in each round 
was to determine whether observed effects could be replicated in different people. A 
key point to note is that the results did show significant differences between control and 
combined experimental conditions in both rounds. The frequency of SCE was 
significantly higher in the exposed group in both rounds (P < .001 ) .  From these data the 
conclusion can be drawn that certain EMFs can exert a biological effect, but the effects 
vary. This could be due to a chance event or may be attributed to variability of response 
in the different donors. It is recognized that the sample size of the two groups is too 
small to draw any firm conclusion, but then, it was not the aim of the overall study to 
compare individual effects. The aim of the current study was to observe cellular effects. 
Subtle response of cells to EMFs 
The controversy relating to variability of response to EMFs could be due to the subtle 
response of cells to EMFs and this could explain the conflicting results sometimes 
obtained when experimental replications are attempted. Some of the problems affecting 
reproducibility have been addressed by Delgado et al. ( 1 982). Their study is one of the 
best examples of a concerted attempt to reproduce an EMF bio-effect involving six 
independent laboratories trying to replicate the developmental alterations on chicks. In 
the Delgado experiments, chicken eggs that were exposed to pulsed MFs ( la, 1 00, and 
1 000 Hz, 0. 1 2  to 1 .2 llT) exhibited significant developmental malformations. In the 
replicate studies all investigators used identical equipment constructed in one laboratory 
and followed similar but not identical protocols. Fertilized eggs were evaluated for 
fertility, development, morphology, and stage of maturity. Ten eggs were sham-exposed 
and ten eggs were exposed to a unipolar pulsed MF (500 msec pulse, 1 00 pulses / sec, 
l 11T peak). Because the fertilized eggs were obtained from a local market supplier, the 
genetic characteristics of the eggs were not controlled. In five of six laboratories more 
embryos exhibited structural anomalies than did the sham-exposed controls. Statistically 
Chapter Five: Discussion. 218  
significant differences, however, were observed only in  two laboratories; a third 
laboratory had marginally significant differences (Berman et al. 1 990). When the data 
were pooled from all laboratories, approximately 25% of exposed embryos showed 
abnormalities compared to 1 9% for sham-exposed controls. 
The disconcerting lesson from the study described above is that subtle factors yet to be 
identified may play a dominant role in bio-effect studies of EMFs. In Delgado et al. 's 
study an effort was made by all of the research groups to reproduce the original 
experiments as closely as possible and yet the results were still ambiguous. As 
mentioned above, one possible explanation for the overall lack of agreement might be 
the genetic variability in the eggs. Support for this suggestion was obtained by Litovitz 
et al. ( 1 993), who, using conditions and end points similar to those in Berman's study, 
report evidence that changes in the breeding flock may be a strong determinant as to 
whether or not an EMF-related defect is observed. In some experiments involving 
multiple replications with eggs from one breeding flock, a strong effect was obtained 
each time the experiment was performed. In other experiments, using eggs from the 
same supplier but a different breeding flock, no defects were ever obtained. These data 
strongly suggest that a yet to be defined factor in a supposedly homogeneously bred 
stock may be involved in susceptibility to EMFs. 
SCE is a sensitive marker for genotoxicity 
Although the relationship between SCE, DNA damage and carcinogenesis is not well 
understood, the SCE technique is still recognised as a sensitive marker for genotoxicity. 
It is generally believed that induction of seEs represents the interchange of DNA 
replication products at apparently homologous loci involving DNA breakage and 
reunion (Latt et al. 198 1 ). In the present study, acute exposure to a 50 Hz MF resulted 
in a significant overall increase in the frequencies of SeEs in both rounds. This result 
suggests that DNA breakage might be enhanced by ELF EMFs, as shown by Lai and 
Singh ( 1 997a). As noted previously, they found by using the COMET assay that acute 
exposure to a 60 Hz ELF EMF could significantly increase the amounts of single- and 
double-stranded DNA breakages in the brain cells of rats. Therefore, the presence of 
both an increase in the frequency of SeEs and the increase in the levels of DNA 
Chapter Five: Discussion. 2 1 9  
damage detected by the COMET assay may indicate that both tests are mechanistically 
related to effects of exposure to ELF EMFs. 
SCE mechanism 
If EMFs can exert an effect on the genetic machinery, a central question then arises :  
what is the mechanism by which this effect occurs? There were only a few important 
clues to an SCE mechanism before the rapid influx of BrdU-incorporation SCE-studies. 
From his autoradiographic studies, Taylor ( 1 958) reported that a double-strand 
exchange was involved in SCEs. Wolff et al. ( 1 974) subsequently showed that lesions 
induced in DNA had to pass through an S phase before they could result in SCEs, and 
Kato ( 1 974a) likewise noted that SCE induction maximized during the period of DNA 
synthesis. Considering the above findings the replication bypass model for SCE was 
originally proposed in 1 975 to explain DNA crosslink-induced SCEs, since it appeared 
that: i) crosslinking agents were the most potent inducers of SCEs (Perry and Evans, 
1 975 ;  Latt, 1 974a); ii) crosslinks would inhibit or prevent the normal separation of 
DNA strands as required for bidirectional replication (Geiduschek, 1 961) ;  and iii) that 
crosslink repair required an undamaged parallel DNA duplex in order to complete a 
recombinational exchange (Cole, 1 973 and 1 974). 
On this basis, Shafer ( 1 977) proposed a model in which SCEs could result from the 
completion of DNA replication at the site of an unrepaired DNA crosslink. The most 
important characteristic of this proposed model was that SCEs could result from a series 
of sequential events that might occur as bidirectional replication encounters a crosslink. 
It is interesting to mention here that the induction of DNA crosslinks was observed in 
rat brain cells when exposed to a 60 Hz MF (Singh and Lai, 1 998). The effects of these 
MFs were also compared with those of a known DNA crosslink.-inducing agent, 
mitomycin C .  The pattern of effects was similar between these two agents. Therefore, 
the presence of a significant increase in the frequency of SCEs in the present study 
could be the ultimate product of ELF EMF exposure via DNA-crosslinks. Of all the 
models proposed linking SCEs with ELF EMF effects (as covered in the Literature 
Review), this one appears to be the soundest. 
Chapter Five: Discussion. 220 
Free radicals and melatonin 
Free radicals are known to be an important factor in cell proliferation and hence to be 
cancer promoters (Burdon and Rice-Evans, 1 989; Rose and Bode, 1 993) .  The 
production of free radicals (.OH) is a consequence of the utilization of oxygen by all 
organisms. About 1 -2% of inspired oxygen ends up as toxic free radicals, which can 
damage macromolecules such as DNA, proteins and lipids. This damage is referred to 
as oxidative stress. On the other hand, the pineal hormone, melatonin, eliminates free 
radicals through its most efficient natural cell protection and anti-oxidative stress ability 
in our bodies (Reiter et al. 1 994, and 200 1 ;  Hardeland et al. 1 993; Vijayalaxmi et al. 
1 995; Omura et af. 1993). 
Melatonin's DNA damage inhibitory activity by eliminating free radicals can be seen in 
the in vivo study using safrole that was conducted by Tan et al. ( 1 993). Safrole is a 
known chemical carcinogen, and damages DNA by inducing the production of large 
numbers of oxygen free radicals. However, melatonin inhibits DNA damage induced by 
safrole in vivo. Tan et al. ( 1 993) injected rats with safrole alone and observed extensive 
DNA damage after 24 h. However, when melatonin was injected along with safrole, the 
DNA damage was reduced by 99%. Lai and Singh ( 1 997b, c) have also observed 
similar in vivo effects of melatonin. In their studies, rats were treated with melatonin 
before exposure to EMFs (ELF EMF and RFR) and damaging effects ofEMFs on DNA 
previously observed were now blocked. Vijayalaxmi et al. (1 995) similarly observed 
that melatonin protects human blood lymphocytes from radiation-induced chromosome 
damage. In their study, cells of human peripheral blood were treated in vitro with 
increasing concentrations of melatonin (0.5  or 1 .0 or 2.0 mM) for 20 min at 37  ± 1 C  and 
then exposed to 1 50 cGy gamma-radiation from a 137 Cs source. The lymphocytes which 
were pre-treated with melatonin exhibited a significant concentration-dependent 
decrease in the frequency of radiation-induced chromosome damage as compared with 
the irradiated cells which did not receive the pre-treatment. 
On the other hand, it has been reported from the results of many previous studies that 
exposure of human cells (Pflunger and Minder, 1 996; Ametz et al. 1 996; Wilson et al. 
1 990; Graham et al. 1 994; Davis, 1 997; Wood et al. 1 998; Burch et al. 1 997, 1 998, 
1 999, 2000; and Griefahn et al. 2002) as well as animal cells (Rosen et al. 1 998; Olcese 
Chapter Five: Discussion. 221 
et al. 1 985 ;  Kato et al. 1 993; Yellon, 1 99 1 ,  1 994) to ELF EMFs can reduce the amount 
of melatonin. Wood et al. ( 1 998) also reported that MF generated by a square wave 
significantly reduced melatonin level compared to MF generated by sinusoidal 
waveforms. This suggests that ELF EMF exposure enhances free radical activity in cells 
through the suppression of melatonin production, which in turn leads to DNA damage 
and ultimate production of a higher number of SCEs per cell. 
MF exposure limits 
In the present research, fields of l ilT and 1mT were deliberately selected for study in 
order to cover the range of exposure normally encountered by humans. The ELF 
magnetic flux density in the environment varies over a wide range. For example, 
household and office levels can vary from 0.0 1 - 1 IlT. Intermittently, levels can reach 
more than 1 0  IlT. Levels near a power transmission line can be 1 0-30 IlT, whereas the 
magnetic flux density can vary between 0. 1 and 1mT near some electrical appliances 
(e.g. ,  electric blankets, hair dryers) . Much higher levels are expected in occupational 
exposures (Bernhardt, 1 985; Gauger, 1 984; Tenforde and Kaune, 1 987). Recommended 
maximum levels also vary. For example, the interim guidelines of the International 
Nonionizing Radiation Committee of the International Radiation Protection Association 
(INIRCIIRPA, 1 990) for occupational situations are 0.5 mT for workday exposure and 5 
mT for short-term (2 h) exposure, whereas for the general public they are 0. 1 mT for 24 
h/day exposure and lmT for exposure of a few h per day. The National Radiological 
Protection Board (NRPB) of England recommends a limit of 2 mT for both 
occupational and general public exposure to ELF EMFs (NRPB, 1 989). In the current 
study, exposure to ELF EMFs with a magnetic flux density ranging from l ilT to 1mT 
produced significantly higher SCEs/cell than the sham-exposed controls. These flux 
densities are much higher than the majority of people encounter in daily life. However, 
they are still within the limits contained in current MF exposure guidelines of the 
INIRC/IRP A and NRPB and can be encountered in both public and occupational 
situations. 
Chapter Five: Discussion. 222 
kT- problem 
One of the criticisms sometimes directed towards suggestions of ELF EMFs being able 
to exert a biological effect is that the fields are far too weak. The kT of the fields is 
much lower than the kT inside the cells; this is an indisputable fact. However, over the 
last 20 years, biologists have continued to conduct research in this area, and the 
evidence is compelling that weak EMFs can exert a biological effect. The number of 
studies which confirm this are too many to ignore. As Hill ( 1 965) stated long ago, it is 
not necessary to identify a biological mechanism for classifying a causal effect of ELF 
EMFs. Nevertheless, this point has been the main bone of contention by skeptics who 
dismiss claims of a biological effect on the grounds that a mechanism is  not evident. 
The author of the current treatise acknowledges this point and recognizes that it is 
helpful to strengthen the argument (of an effect) beyond doubt if a plausible biological 
mechanism of EMF interaction can be identified and supported by sound scientific 
research. 
EMF mechanism 
Mechanisms of interaction between EMFs and biological systems have been studied for 
decades and a number of innovative models have been proposed (see earlier review 
Section 2 . 1 .2). Although there is no generally accepted mechanism of EMF interaction 
to explain how EMFs can affect biological systems, there is a general agreement that the 
primary site of interaction is the cell membrane. Signal transduction is the basis for cell 
communication. It is a general process in which a ligand molecule binds to its receptor 
site on the cell surface triggering a cascade of molecular interactions in the cell 
membrane and inside the cell leading to enzyme activation, gene induction, protein 
synthesis which eventually leads to growth, differentiation and cell proliferation. 
Liburdy (1 995) hypothesised that EMFs interact with the membrane, triggering an early 
event in the signal transduction cascade, such as calcium ion influx. The calcium ion 
influx process has been confirmed experimentally (Liburdy, 1 992a, b; and Zimmerman 
et al. 1 990) in the absence of a release of calcium from intracellular stores. These results 
implicated the cell membrane as being involved in the EMF interaction. 
Chapter Five: Discussion. 223 
Adair ( 199 1 )  has attempted to address the question of ELF EMFs being too weak to 
influence cell behaviour that they are too weak to be recognized and are thus lost in the 
general thermal and environmental noise. Adair advanced the opinion that ELF signals 
need amplification if they are to influence the action of messengers, antibody and 
cancer promoter molecules at their membrane receptor sites. There have been several 
hypotheses as to how these frequencies can be amplified in biological systems. 
Kruglikov and Dertinger (1 994) proposed that the signals are amplified via the 
phenomenon of stochastic resonance, a view that is gaining currency (Astumian et al. 
1 995), and that this amplification occurs at the cell membrane in ion channels. The ion 
channels can form a network, which is capable of amplifying the signal by several 
orders of magnitude. 
The concept of stochastic resonance (SR) first put forward in the seminal paper by 
Benzi et al. ( 198 1 )  has ultimately been proposed as a possible mechanism through 
which weak EMF fields could be detected in biological systems (Kruglikov and 
Dertinger, 1994). Stochastic resonance is the phenomenon whereby the addition of an 
optimal level of noise to a weak information-carrying input to certain non-linear 
systems can enhance the information content at their outputs. 
In some systems, for example, such as radio receivers, turning up the volume in order to 
hear a faint signal amidst much noise usually only results in turning up the noise as 
well.  However, in other systems increasing the amount of ambient noise actually 
enhances the signal-to-noise ratio through a complicated non-linear cooperation 
between the system and detector. This phenomenon has been shown to exist in various 
physical systems, but has only recently received support in biology (Bezrukov & 
Vodyanoy, 1 997). The noise fluctuations might be stochastic but the detection of a 
desired signal can be maximised by tuning the noise. Of all the theories advanced, this 
appears to be the most plausible mechanism to explain how weak forces can be 
enhanced to a level where an increase in SCE can be observed. 
In the following section the findings of the MN assay are discussed and an attempt is 
made to establish any common EMF effects between the SCE technique and MN assay. 
Chapter Five: Discussion. 224 
5.2 MN Assay 
MN assay experiments were conducted in the present study to ascertain whether or not 
weak EMFs affect the DNA repair system in humans to any degree. In this study, both 
control and experimental GO lymphocyte cultures, established from fresh blood, were 
irradiated with 3 .5  Gy X-rays and placed immediately in an incubator at 37C. Following 
irradiation, a rest period of 6 h was allowed to repair the damaged DNA at GO. After 
this 6 h period, the lymphocytes were stimulated with the mitogen PHA. Twenty-four 
hours after this stimulation, the cytokinesis-blocking agent Cytochalasin-B was then 
added in order to capture binucleate cells with any micronuclei arising from MF 
exposure. Experimental lymphocyte cultures were exposed to different MFs for the 
entire culture period (90 h after addition of PHA), whereas the control lymphocyte 
cultures were sham-exposed. 
The results of the micronucleus study showed ELF EMF effects similar to that seen in 
the SCE study. There was a significant difference between the pooled experimental 
groups and the control, but mixed results throughout the different parameter 
combinations for both the mean number of micronucleated CB cells/} 00 CB cells (Ma) 
and the mean number of micronuclei/} 00 CB cells (Mb). 
Ma and Mb results for all experimental conditions and the control are summarised in 
Tables 4.9 and 4 . 12 .  The overall means of the controls and experimentals for Ma were 
1 9.39 and 25 .06, respectively, whereas the overall means of the controls and 
experimentals for Mb were 23 .80 and 32.02, respectively. As with the SCE study, a t­
test showed that there was a significant increase in Ma and Mb in the grouped 
experimental conditions compared to the controls. The most important point to reiterate 
is the observation of an overall increase in mean value of both Ma and Mb in the 
experimentals compared to the controls as a consequence of EMF exposure. These 
results strongly support the view that ELF EMFs affect the efficiency of DNA repair in 
cultured PBLs. 
Comparing the effects of the different types of fields is not straightforward, but 
nevertheless interesting in its complexity. First, let us examine those parameters with 
the greatest effects. A Square pulsed field at strengths of both l /lT and ImT produced 
Chapter Five: Discussion. 225 
the highest frequency of Ma (33 .57 for l ilT and to 35 . 1 8  for lmT) and Mb (45.38 for 
l ilT and 48.98 for ImT). These figures are convincingly higher than the average of the 
3 controls (1 9.33 for Ma and 23 .74 for Mb). This could be interpreted as a Square pulsed 
field affecting DNA repair in HPBLs greater than any other parameter combination. 
Interestingly, it was noted previously that the highest SCE frequency in RI was 10.03 
for a square continuous field, and the SCE frequency of 1 0.39 for a square continuous 
field in R2 (albeit a different strength) was the second highest in this latter round. It is 
tempting here to draw the conclusion that a square wave has a more general damaging 
effect on DNA, bearing in mind that one must proceed with caution in interpreting these 
data, not least in acknowledging that the SCE test and the MN assay are measuring two 
quite different phenomena. Nevertheless, one could argue that the commonality here 
between the SCE and MN assays is a square wave and that a square pulsed field in 
particular has the greatest effect on the DNA repair system. Having made this point, 
however, it should also be noted (which also reinforces the perplexity one encounters in 
this field of scientific endeavour) that a sine pulsed field at l ilT produced a relatively 
high Mb frequency (34.96) and interestingly, a sine pulsed field at l ilT produced the 
highest SCE frequency ( 10.6 1)  of any parameter. 
Interactions 
A comparison between the results observed in the SCE study with that of the MN assay 
shows other interesting parallel features. Table 4. 1 1  shows a near significant interaction 
between Form and Strength, the same as that seen in Table 4.3 in RI of the SCE 
experiments. Also, as noted previously, a Square pulsed field showed an increase in Ma 
and Mb values with an increase in field strength from l ilT to ImT, the same trend as 
that observed in RI of the SCE study. (SCE means in R2 for square pulsed 1 pT and 
square pulsed ImTwere 9.83 and 9.66, respectively, which is a negligible difference.) 
What can one infer from the above parallel observations? Although the SCE test and the 
MN assay measure quite different cellular phenomena, the similar trends in effects 
could indicate that a common mechanism is operating at the molecular level .  
Furthermore, the importance of these parallel observations become even more 
interesting when we compare the results of the COMET assay in a later section. 
Chapter Five: Discussion. 226 
Energy of square waves 
The rationale for a square wave being the strongest in its biological effect may be due to 
its higher power output than a sine wave. The higher power-generating capacity of 
square waves was reported by both Loss (200 1 )  and Islam (2003). The root mean square 
(RMS) average, which describes the energy within the sine and square waves, was 
calculated using instantaneous voltages measured over a complete cycle (Islam, 2003 
and Loss, 200 1 ) .  They observed that a sine wave has a RMS value 0.707 times that of 
its peak value but the same RMS average value for a square wave is equal to its peak 
value which is 1 .0 (see appendix 6). 
In sine waves,  the voltage increases smoothly from the negative maximum to the 
positive maximum and back again. Square waves, on the other hand, do not increase in 
voltage smoothly, but shift suddenly from negative to positive, stay there for half a 
cycle, then jump to full negative and stay there for half a cycle, repetitively. Three 
important points can be drawn from the above discussion: 1 )  the RMS voltage output of 
a square wave is higher than a sine wave, which might contribute to a higher magnetic 
flux density; 2) a square wave changes its voltage abruptly which might cause energy 
splatter; 3) while a sine wave has a single frequency in it (50 Hz in New Zealand), a 
square wave contains many higher frequencies in it as well, called harmonics, which 
can cause problems; and moreover 4) pulsing also causes extra energy splatter. It is a 
plausible conjecture that these four factors of square waves (50 Hz) might influence 
enzymatic processes involved in DNA repair, leading to an accumulation of DNA 
strand breaks and thereby inducing a higher number of micronuclei. On the other hand, 
these might also influence the enzymatic processes to induce more cross-links, which 
might lead to a higher number of SCE per cell. 
Donor 
In the current MN assay study only one round of eight experiments was performed 
(because of time constraints) using the same first group of three donors (D-l to D-3) 
which was used in RI of the SCE study. The rationale for conducting the MN assay was 
to establish whether or not weak EMFs affect DNA repair mechanisms in a similar 
fashion to the SCE study, which yielded inconsistent results among the different 
Chapter Five: Discussion. 227 
parameter combinations, both within and between rounds. Indeed, the MN assay 
experiments produced similar inconsistent results among different parameter 
combination as seen in the SCE study, even though the overall frequency of micronuclei 
was significantly higher in the exposed group. Further analysis on the donor 
contribution towards these results showed that none of the differences between control 
and experimental conditions for any one donor reached significance. Even though there 
were no significant differences between control and experimental conditions for each of 
the three donors, all observations were in the direction of an EMF effect. Only when the 
results were pooled was an effect observed. From these data the conclusion was drawn 
that certain EMFs can exert a biological effect on DNA repair mechanisms, but the 
effects vary. This could be due to a chance event or may be attributed to variability of 
response in the different donors. 
Complex field 
Attempts were also taken to see whether there was any difference between the Ma and 
Mb of the control and that of a complex field (Tables 4. 1 3  and 4 . 14) .  No statistically 
significant difference was observed between the means of each of Ma and Mb of the 
controls compared to the complex field. From these observations one can tentatively 
deduce that a complex field, at least the field produced from the rear of the particular 
computer used in this experiment, causes no damage to DNA repair mechanisms in 
dividing PBLs. However, as previously noted with the SCE experiments, difficulty was 
always encountered in obtaining a sufficient number of dividing cells. 
Positive and null EMF effects 
In the current study, the results show that acute exposure to a 50 Hz MF causes an 
overall increase in micronucleus frequency as a consequence of EMF exposure which is 
in agreement with results reported previously by Simko et al. ( 1 998). They similarly 
showed that in vitro exposure of human squamous carcinoma cell line (SCL II) to a 
sinusoidal 50 Hz MF (0.8 and ImT) resulted in a significant increase in the frequency of 
micronuclei. In their study, they examined the effects of ELF-EMFs in a human 
squamous cell carcinoma cell line (SCL 11) and in a human amniotic fluid cell line 
(AFC) for different durations (24, 48, and 72 h) and different field intensities (O. I - lmT) 
Chapter Five: Discussion. 228 
on micronucleus formation and induction of apoptosis. A statistically significant 
increase in micronucleus frequency and induction of apoptosis in SCL 11 cells after 48-h 
and 72-h continuous exposure to 50 Hz MF (0.8 and ImT) was found. On the other 
hand, at lower (0. 1 and 0.5 mT) field intensities, no effects could be detected. Exposure 
of AFC cells to EMF of different intensities and for different exposure times showed no 
statistically significant differences when compared with the controls.  This study by 
Simko is the only report so far available at 50 Hz which shows a significant increase in 
micronucleus frequency using the MN assay as a marker for effects of extremely weak 
MFs, although Scarfi et al. ( l997a) observed an increase in micronucleus frequency 
when cultured human lymphocytes were exposed to MFs at 100 Hz. 
It is interesting to note, however, that Maes et al. ( 1 993) and Vijayalaxmi et al. ( 1 998) 
also observed an increase in micronucleus frequency in HPBLs and peripheral blood 
and bone marrow of cancer-prone mice, respectively when they applied much stronger 
MFs (2450 MHz microwave RFR). In Maes et al. ( 1 993) study, HPBLs were exposed to 
microwaves at 2450 MHz. They observed a marked increase in the frequency of CAs 
(including dicentric chromosomes and acentric fragments) and micronuclei. On the 
other hand, the microwave exposure did not influence either the cell kinetics or the SCE 
frequency. Similarly, Vijayalaxmi et al. ( 1 998) exposed peripheral blood and bone 
marrow of cancer-prone mice chronically to 2450 MHz microwave RFR and observed a 
significant increase in micronuclei. 
Intriguingly, Tofani et al. ( 1 995) claimed that a DC field can induce the formation of 
micronuclei. In their in vitro study, when human peripheral lymphocytes were exposed 
to 32 or 50 Hz (75 or 1 00 �T) with no DC component, no micronuclei were induced. 
However, when the lymphocytes were exposed to the 32 Hz field with a 42 �T DC 
component, micronuclei were induced. 
The results of most other studies on micronucleus formation due to EMF exposure show 
a null effect in micronucleus frequency. For example, Scarfi et al. ( 1 99 1 ,  1 993, 1 994) 
were consistently unable to detect an increase in micronucleus frequency in normal 
cultured human lymphocytes exposed to a 50 Hz pulsed MF or a sinusoidal electrical 
field. Similarly, Paile et al. ( 1 995) exposed human peripheral lymphocytes to 50 Hz, 30 
�T, 300 �T, and I mT MF for 65 h.  They observed no statistically significant effect on 
Chapter Five: Discussion. 229 
the frequency of micronuclei .  Interestingly, however, cells from patients with Turner's 
syndrome showed an 80-100% increase in micronuclei after similar exposure (Scarfi et 
al. 1 997). 
Hypothesis involving DNA repair 
ELF MFs do not have enough energy to break chemical bonds directly in DNA 
molecules. A possible explanation of the present observations is that 50  Hz MFs, in 
particular a square pulsed field, might affect the enzymatic processes involved in DNA 
repair, leading to an accumulation of DNA strand breaks. This hypothesis is supported 
by a recent report by Phillips et al. ( 1995) that acute exposure to a 60 Hz MF 
significantly affected the activity of poly-ADP-ribose polymerization, an enzymatic 
activity involved in DNA repair. A similar effect on poly-ADP-ribose polymerization 
has also been observed by Sarker et al. (1 994) in brain cells of rats after chronic 
exposure to a 50 Hz MF. 
Furthermore, the results gained in the current study suggest that either the cell' s  DNA 
repair mechanism is affected in such a way that it was unable to repair any DNA 
damage that was induced by the ELF EMF, or the damage was so severe that although 
the cell was able to repair some of the strand breakages, not all damage induced could 
be repaired. Although the true cause behind the increased numbers of micronuclei 
observed in the current study cannot be elucidated, the results of the present study 
support the view that weak EMFs can affect DNA repair. 
In the following section (5.3 COMET assay) the author discusses the findings of the 
COMET assay and endeavours to establish any common EMF effects between the SCE 
technique, MN assay and COMET assay. Furthermore, the author explores whether the 
EMF effects observed with the SCE technique, MN assay and COMET assay are 
ultimately controlled by the same mechanism. 
Chapter Five: Discussion. 230 
5.3 COMET Assay 
The application of the COMET assay in both in vitro and in vivo studies has been 
shown to be a reliable measure of genetic damage induced by genotoxic agents. 
Damage induced by physical, chemical or environmental agents in different cell 
systems can be accurately and sensitively assessed by this assay (Ostling and Johanson, 
1 984; Singh et al. 1988, 1 990, 1 99 1 , 1 994, Singh, 2000; Vijayalaxmi et a!' 1 992, 1 993 
and Betti et al. 1 994). Moreover, the COMET assay seems highly reproducible in the 
same subject and is an effective tool for the evaluation of DNA damage induced by 
chemotherapy (Ostling et al. 1 987; Tice et a!. 1 992) or by syndromes involving defects 
in excision repair, such as Xeroderma pigmentosum (Green et al. 1 992). This assay is 
also highly sensitive in revealing DNA-damaging lifestyle factors such as smoking 
(Anderson et al. 1 994). Damage by UV radiation has been examined extensively using 
the alkaline COMET assay (Green et al. 1 992; Gedik et al. 1 992; Arlett et al. 1 993). 
There is also some indication that the alkaline COMET assay can detect DNA repair 
deficiency after irradiation of lymphocytes from patients with systemic lupus 
erythematosus and rheumatoid arthritis (McCurdy et al. 1 997). For these reasons, a total 
of two COMET assay experiments (square continuous ImT and square pulsed I m 1)  
were conducted to evaluate whether or not weak EMFs damage the DNA to any degree 
in humans.  Scarcity of time and resources are the reasons why only two COMET assay 
experiments were conducted. However, the results of the SCE study and MN assay 
showed that a square continuous and square pulsed I mT MF produced significant 
effects. This was the rationale for choosing these fields to conduct the same experiments 
with the COMET assay. 
In the current study, both control and experimental lymphocytes were cultured in an 
incubator at 37C. Lymphocyte cultures of the experimentals were exposed to each of 
the two MFs while the lymphocyte cultures of the controls were unexposed (sham) for 
72 h. Cell suspensions were prepared from these cultures and the lymphocytes were 
resuspended in low melting agarose then layered onto slides precoated with a 0.5% 
normal melting-temperature agarose. The cells were lysed with a concentrated salt 
solution in order to remove cellular proteins and liberate the damaged DNA. The 
liberated DNA was subjected to unwinding under alkaline conditions to allow DNA 
supercoils to relax and express DNA single strand breaks and alkali labile sites .  
Chapter Five: Discussion. 23 1 
Electrophoresis was then performed also under highly alkaline conditions (pH > 1 3), 
which allowed the broken ends to migrate under the effect of an electric field, towards 
the anode. After neutralization, the nuclei were stained with a fluorescent DNA stain, 
DAPI. The slides were then kept in a humidified slide chamber until they were scored. 
A total of 70- 1 03 comets were captured. Only three parameters: Tail Length (in pixels), 
Tail moment and Olive Tail Moment (arbitrary units) were analysed and used to assess 
DNA damage. 
Similar EMF effects 
In the current study, the results showed that exposure for 72 h to a 50  Hz, square 
continuous lmT or a square pulsed l m T  MF caused a significant increase in single­
strand DNA breaks in HPBLs. This is in good agreement with the results reported 
previously by Lai and Singh ( 1995, 1 996 and 1 997a) and Ivancsits et al. (2002). While 
Lai and Singh observed effects of 60 Hz MF exposure on DNA in brain cells of the rat, 
Ivancsits et al. (2002) observed effects of intermittent ELF EMFs, 50 Hz, sinusoidal, on 
human diploid fibroblasts. In Lai and Singh's study, when rats were exposed for 2 h to 
an EMF at intensities of 0. 1 ,  0.25, or 0.5 mT, they observed single strand breaks, 
whereas an increase in double strand breaks was observed at 0 .25 and 0.5 mT, but not 
0. 1 mT. The effect was proportional to the intensity of the MF (Lai and Singh, 1 997a). 
In Ivancsits et al. (2002) study, human diploid fibroblasts were exposed to continuous 
or pulsed (5 min-on / 1 0  min-off) for 24 h to a 1 mT EMF intensity. They observed a 
significant increase in DNA strand breaks due to pulsed exposure, mainly DSBs as 
compared to non-exposed controls. Similarly, exposure to RFR, 2450 MHz, at a whole 
body specific absorption rate (SAR) of 0.6 and 1 .2 W/kg for 2 h caused an increase in 
both single and double strand breaks in DNA of brain cells in the rat (Lai and Singh 
1 995, 1 996). 
In addition to the above example, Ahuja et al. ( 1999) and Svedenstal et al. ( l999a, b) 
also reported an increase in DNA strand breaks in cells after MF exposure. In Ahuja et 
al. ' s  ( 1999) study, HPBLs were exposed to five different field strengths (2, 3 , 4, 5  and 
1 0  mT, all at 50 Hz) and they observed a significant increase in DNA strand breaks in 
each magnetic flux density except one. Interestingly, Svedenstal e t  al. ( 1999a) observed 
an increase in DNA strand breaks in brain cells of mice after 32 days of exposure to MF 
Chapter Five: Discussion. 232 
at a low intensity of about 8 � T. Similarly, Svedenstal et al. (1 999b) also observed an 
increase in DNA strand breaks in brain cells of mice after 14  days of exposure to a low 
intensity of 0.5 mT MFs. 
A square wave in particular appears to exert multiple effects across all three tests (SCE, 
MN and COMET) which ostensibly measure different molecular phenomena. It is 
interesting to recall that in an earlier discussion the current author noted the widely­
accepted damaging effect of free radicals on macromolecules such as DNA, proteins 
and lipids (see p220 Free radicals and melatonin). The findings of Wood et al. ( 1998) 
were also noted (p221 ), who reported that MFs generated by a square wave significantly 
reduced melatonin levels, which are thought to inhibit the damaging effect of free 
radicals. The author also noted (P2 19) the possible link between MF exposure, DNA 
cross-links and SCE induction. One can only conjecture about the possibility of a 
common molecular mechanism operating here, but the current author offers the 
speculative view in the following section that free radicals, formed as a consequence of 
MF exposure, may have a wide range of effects on the genetic machinery. This may 
conceivably involve the formation of DNA cross-links, accumulation of DNA strand 
breaks, interference with DNA repair, resulting ultimately in genetic damage. 
EMF effects: a possible explanation 
It is an accepted fact that the energy from ELF EMFs is incapable of breaking chemical 
bonds directly in DNA molecules. A possible explanation of the present observations in 
this thesis is  that a 50 Hz MF might initiate an iron-mediated process, known as the 
Fenton reaction, which increases hydroxyl free radical formation in cells, leading to 
DNA strand breaks (Christophersen et af. 1 991 ), and perhaps even widespread effects 
on the genetic apparatus as seen with the SCE, MN and COMET assays.  To diverge 
briefly, the Fenton reaction is an iron-mediated process. Cells with high iron intake such 
as proliferating cells, brain cells, peripheral blood cells, and cells infected by DNA virus 
may be more susceptible to the effects of EMF because a high metabolic rate generates 
high amounts of hydrogen peroxide via the mitochondrial electron transport pathway 
(oxidative phosphorylation). For proliferating cells, the most vulnerable time may be 
during the /S phases of the cell cycle when transferrin receptors are expressed and iron 
influx is high. This coincides with when DNA repair occurs with the MN assay and 
Chapter Five: Discussion. 233 
when SCEs are initiated. Hydroxyl radicals are generated from hydrogen peroxide via 
the Fenton reaction, which is the iron-saIt-dependent decomposition of di-hydrogen 
peroxide, generating the highly reactive hydroxyl radical. The equation that illustrates 
the Fenton reaction is, Fe2+ + H202 � Fe3+ + OH + OR (Wedrychowski et al. 1 986). 
Returning to the central argument, the above hypothesis is supported by recent reports 
(Lai and Singh, 1 997b, c and Lai, 200 1),  which state that treatment of rats before 
exposure with melatonin (a free radical scavenger) blocked the effects of EMF (ELF­
EMF and RFR) on DNA. This suggests that EMFs enhance free radical activity in cells, 
which in turn leads to DNA damage. They also observed that EMF exposure caused 
DNA-protein and DNA-DNA crosslinks (Singh and Lai, 1 998) and increased rates of 
apoptosis and necrosis in brain cells of rats. Furthermore, they observed that pre­
treating rats with an iron-chelator could block the effects of EMF exposure on DNA. 
This means, in the presence of an iron chelator, the Fenton reaction can no longer 
activate and produce free radicals and consequently yield no DNA damage. Further 
detailed investigation is undoubtedly required to substantiate the conjecture that the 
effects of free radicals represent the common theme in the results observed in the 
present study, which is discussed in the Future Research section on page. 
5.4 FISH 
Translocations are often referred to as stable CAs as they may remain reasonably 
constant for years (Buckton et al. 1 967, 1 978 ; Buckton, 1 983; Lucas, 1 997; Lloyd et al. 
1 998). Increased CA frequency is a well-known feature of ionising radiation in humans. 
The scoring of chromosomal translocations in HPBLs has long been established as a 
method for personal dosimetry in radiological protection (IAEA, 1986). Exciting 
progress, however, has been made in this area over the last decade. The recent 
development of FISH methods has brought the objective scoring of translocations to a 
new level (Pinkel et al. 1 988; Lucas et al. 1989), ushering in a new era in the detection 
of stable aberrations. In the current study, of course, the effects of non-ionising 
radiation exposure over 3 days were investigated. Nevertheless, in view of the above 
developments, FISH was employed to investigate the incidence of translocation 
frequency in PBLs, should they be induced by ELF EMF exposure. The aim was 
scientifically sound even if the results were somewhat predictable. 
Chapter Five: Discussion. 234 
An extensive literature search showed no reports of translocations occurring in humans 
or any other model system as a result of ELF EMF exposure. It seems surprising to this 
author that no such studies have been performed. Perhaps the absence of publications is 
a consequence of investigators not finding any evidence of major CAs arising from 
exposure to weak EMFs. This would not be totally unexpected as the forces involved to 
break a chromosome and thereby give rise to a translocation are much greater than the 
forces required to disrupt molecular events at the replication fork, for instance, and thus 
affect the frequency of SCE. Certainly the results obtained in the current study support 
the view that ELF EMFs are too weak to affect whole chromosome structure. Although 
only one field (square continuous Im1) was studied, a count of 506 cells showed no 
evidence of translocations occurring in PBLs subjected to this exposure. 
5.5 Future Research 
In the field of EMF research, perhaps the most outstanding question that remains to be 
answered with certainty is how weak EMFs exert their effects at the molecular level. In 
other words, "What is the mechanism by which weak EMFs exert their biological 
effect?" One of the exciting ideas advanced by the author of the current thesis is that 
free radicals, formed as a consequence of MF exposure, may have a wide range of 
effects on the genetic machinery. The possible role of free radicals may conceivably 
involve the formation of DNA cross-links, accumulation of DNA strand breaks, 
interference with DNA repair, resulting ultimately in genetic damage. Simko et al. 
(2004) have also recently proposed a mechanism where EMFs indirectly affect the 
DNA by way of free radical production, the speculation being on the basis of 
experiments they conducted in rats. Free radicals are widely known to cause genetic 
damage (Tan et al. 1 993). Simko's group, however, have not tested this mechanism. 
That EMFs may induce the production of an increased number of free radicals, could 
herald a breakthrough in providing a long sought-after model by which very weak 
EMFs affect genetic material as observed in this thesis. Future research should be 
conducted to test this hypothesis, especially as it relates to assays such as sister 
chromatid exchange, micronucleus assay and COMET assay, all of which are known to 
detect genotoxicl clastogenic effects. 
Chapter Five: Discussion. 235 
According to my proposed model, exposure to certain 50 Hz MFs increases the number 
of free radicals, which are known genotoxic elements. My hypothesis is that when 
dividing PBLs are cultured in a suspected clastogenic EMF field, this results in a higher 
frequency of sister chromatid exchange, a higher number of micronuclei and an increase 
in DNA degradation as a consequence of exposure to free radicals. If this is true, then 
exposure of PBLs to a free radical scavenger (e.g. melatonin) when growing in the 
presence of a selected EMF field, should reduce the amount of genetic damage. 
To test this hypothesis, I propose that three assays be applied: sister chromatid 
exchange, the radiation-induced MN assay and the COMET assay. The aim of these 
investigations is to investigate whether the application of melatonin (a known free 
radical scavenger) to an PBL culture can reduce the effect of free radicals allegedly 
produced by 50 Hz MF exposure. 
First, however, we should confirm by way of a surrogate experiment that free radicals 
can cause an increase in genotoxic damage as observed by the above three assays. 
Safrole is a well-known chemical carcinogen which is known to damage DNA by 
inducing the production of large numbers of free radicals (Tan et al. 1 993). In my 
proposed study, a PBL culture should be grown in the presence of safrole which would 
thus expose dividing blood lymphocytes to a large number of free radicals. I predict 
that this would result in a higher frequency of sister chromatid exchange, a higher 
number of micronuclei and increased DNA degradation as compared to an unexposed 
control culture. 
As mentioned, melatonin is a free radical scavenger and is thereby a known protector of 
genetic damage. If melatonin is added to the PBL culture along with safrole, the result 
should show a decrease in genetic damage frequencies compared to the safrole­
administered culture. If this predicted result from these experiments is observed, then 
this would support the view that free radical damage of DNA may be mitigated by 
melatonin. 
The next step in this investigation would be to culture PBLs in the presence of 
melatonin and certain EMFs which have been shown to give rise to genetic damage. 
The assumption here is that the observed relationship between exposure to safrole and 
Chapter Five: Discussion. 236 
melatonin will translate into a similar relationship between exposure to EMF and 
melatonin. 
In the first instance a square pulsed 50 Hz ImT field should be tested. If my 
assumption is correct, then exposure of lymphocytes to a combined melatoninlEMF flux 
density should result in a decrease in frequency of sister chromatid exchange, a decrease 
in frequency of micronuclei and less DNA degradation as compared to the results 
already obtained from exposing the lymphocytes to the EMF alone. Even then, 
however, one would have to be cautious in drawing conclusions from this investigation, 
as cause and effect would still not have been established. Nevertheless, experimentation 
with different concentrations of safrole and melatonin could be a fruitful avenue to 
pursue in depth. 
Chapter S ix :  Summary and Limitations 237 
Chapter Six 
Summary and Limitations 
6.1 Summary 
Current evidence suggests that 50  Hz weak EMFs can influence cell processes. A 
comparison between the results observed in the SCE study with that of the MN assay 
show interesting parallel features .  In the SCE study, an overall significant increase in 
the number of SCEs/cell in the grouped experimental conditions compared to the 
controls in both rounds was observed. Similarly, the MN assay also showed an overall 
significant increase of mean number 0/ micronucleated CB cellslI 00 CB cells (Ma) and 
mean number o/micronuclei/lOO CB cells (M b) in the grouped experimental conditions 
compared to the controls. Although results from both the studies (SCE study and MN 
assay) showed an overall EMF effect, the relation between effect and applied field 
intensities is neither simple nor linear. 
From a detailed analysis of SCE data, it appears that a square continuous field with 
increasing field strength produces a higher number of SCEs per cel l ,  although the 
highest SCE frequency ( 1 0.6 1 )  was scored for a sine pulsed field at 1 �T in R2. 
In the MN assay, a square pulsed field showed an increase in Ma and Mb values with an 
increase in field strength, the same trend as that observed in R I  of the SCE study. 
Moreover, the highest SCE frequency in R I  was 1 0.03 for a square continuous field, 
and the SCE frequency of 1 0 .39 for a square continuous field in R2 (albeit a different 
strength) was the second highest in this latter round. But in the MN assay a square 
pulsed field with increasing EMF strength showed the greatest effect on the DNA repair 
system. 
It is tempting here to draw the conclusion that a square wave has a more general 
damaging effect on DNA, bearing in mind that one must proceed with caution in 
interpreting these data, not least in acknowledging that the SCE test and the MN assay 
are measuring two quite different phenomena. Nevertheless, one could argue that the 
Chapter Six: Summary and Limitations 238 
commonality in effect here between the SCE and MN assays is a square wave and that a 
square pulsed field in particular has the greatest effect on the DNA repair system. 
Furthermore, the importance of these parallel observations becomes even more 
interesting when we compare them with the results of the COMET assay. The COMET 
assay also showed that both a 1 mT square field (continuous or pulsed) resulted in 
significant fragmentation of the DNA. On the other hand, a FISH analysis failed to 
show any translocations. 
The results obtained in this study lead one to the conclusion that a square wave may 
exert multiple effects across all three tests (SCE, MN and COMET) which presumably 
measure different molecular phenomena. From the literature it appears that free radicals 
have the potential to damage DNA, protein and lipids. Square waves significantly 
reduced melatonin levels, which are thought to inhibit the damaging effect of free 
radicals. With reference to these facts one can speculate upon the involvement of a 
common molecular mechanism. Formation of free radicals as a consequence of MF 
exposure seems to be a plausible common mechanism, which may have a wide range of 
effects on the genetic machinery. This may conceivably involve the formation of DNA 
cross-links, accumulation of DNA strand breaks, interference with DNA repair, 
resulting ultimately in genetic damage. 
With regard to the null hypothesis, 3 of the 4 tests applied (SCE, MN and COMET), 
during this research do not support the null hypothesis originally stated, which is thus 
rejected. From the data gathered in this study, the author concludes that ELF EMFs do 
have a clastogenic effect on human chromosomes. 
One of the most exciting developments in this thesis that should be followed up in 
subsequent research is a thorough investigation into the possible production of free 
radicals in the presence of certain EMFs and whether this is the cause of the genetic 
effects observed in the current study. 
Chapter Six: Summary and Limitations 239 
6.2 Limitations of Present Study 
A number of limitations became apparent III the present study, which could be 
overcome in further research. 
I .  In the current SCE study two different groups of three healthy blood donors were 
used when replicating the EMF experiments. The rationale for conducting the entire 
experiments twice with a different set of donors in each round was to determine 
whether observed effects could be replicated, as well as in different people. The 
limitation in doing this is the risk of detecting possible variability of response 
between donors, which could impact on interpreting apparently conflicting results. 
It i s  very important to note that the results did show significant differences between 
control and exposed conditions in both rounds. From these results the conclusion was 
drawn that certain EMFs can exert a biological effect, but the effects vary. This could 
be due to a chance event or may be attributed to variability of response in the 
different donors. It is obvious that the sample size of the two groups is too small to 
draw any firm conclusion, but then, it was not the aim of the overall study to 
compare individual effects. The aim of the current study was to observe cellular 
effects. 
In further EMF studies, in order to observe individual effects a concerted attempt 
using a larger number of donors and involving more than two independent 
laboratories could be useful . Certainly this would be a task beyond that of one person 
if all the current tests were conducted. In the replicate studies all investigators should 
use identical equipment constructed in one laboratory and they should follow 
identical protocols. 
2. The author notes that the overall SCE reading is higher in R2 than RI , which might 
be due to individual susceptibility to the marker used, BrdU. This was also noted by 
Morgan and Crossen ( 1 977) . Replacement of BrdU with a different marker could be 
helpful in solving possible differences in individual susceptibilities towards the 
marker. But at present there is no other marker available to replace BrdU in this type 
of experiment. 
Chapter Six: Summary and Limitations 240 
3 .  No statistically significant difference was observed i n  either R I  or R2 between the 
mean SCE/cell of the control groups and that of the complex field. The author notes, 
however, that despite attempting to culture lymphocytes on three separate occasions 
in a complex field, difficulty was always encountered in obtaining a sufficient 
number of dividing metaphase cells. Only 87 cells could be scored in RI , and only 
1 88 cells in R2, in all 3 donors. This low mitotic index severely limits the statistical 
power of the observations and consequently restricts the conclusions that can be 
drawn. Nevertheless, a repeatedly low mitotic index must be a cause for concern, 
despite the lack of significant differences in SCE frequencies between the complex­
field-exposed cultures and the controls .  This raises questions concerning health 
issues and could be the focus of a more intensive study in the future. 
4 .  In the MN assay, steps were also taken to determine whether there was any 
difference between the Ma and Mb of the control and that of a complex field. No 
statistically significant difference was observed between the mean of both Ma and 
M b of the control and complex experiment. From these observations one can 
tentatively deduce that a complex field, at least the field produced from the rear of 
the particular computer used in this experiment, causes no damage to DNA repair 
mechanisms in dividing peripheral blood lymphocytes. Appropriately designed 
multi-laboratory international validation studies are necessary to gather information 
on additional potential sources of variability and on the intra- or inter laboratory 
reproducibility of MN assay results. 
5 .  In  the current MN assay study, only one round of eight experiments was performed. 
The current author scored micronuclei from a large number of binucleate cells per 
experiment, which was time consuming without having access to an automated 
system. Insufficient time l imited the current author in the extent of his study, 
although according to Fenech et al. ( 1 999), other end-points such as apoptosis, 
necrosis, and cytostasis should also be considered when interpreting controversial 
data. 
6 .  There seem to be problems associated with using cytochalasin B to produce 
binucleate cells. Shimizu et al. ( 1 998) observed the formation of micronuclei as a 
Chapter Six: Summary and Limitations 241 
result of gene amplification in which the cell eliminates excess amplified DNA 
directly from the nucleus by nuclear budding during S phase. This is known as 
mitotic slippage. Elhajouj i  et al. ( 1 997) also observed that the application of specific 
mitotic spindle inhibitors might cause mitotic slippage leading to polyploid nuclei 
and micronuclei in mononucleated cells. The concern here is that the frequency of 
micronuclei observed in the present EMF study may be artificially elevated by 
cytochalasin B,  even given the use of controls. Therefore, it would be useful to 
study further the possible effects of cytochalasin B .  
7 .  I n  view of  the current findings and the contradictory published reports, it i s  
important that more investigations be  conducted using various types of  cells from 
both humans and animals under different experimental conditions to resolve the 
controversy of ELF EMF bio-effects. 
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Anderson LE. 1 990. Evidence of an effect of ELF electromagnetic fields on human pineal gland function. 
J Pineal Res. 9(4):259-269. 
Wolff S. 1 983. Sister chromatid exchange as a test for mutagenic carcinogens. Ann. NY. Aead. Sei. 
407: 1 42- 1 53 .  
WolffS.  1 977. Sister-chromatid exchange. Ann. Rev. Genet. 1 1 :  1 83-201 .  
Wolff S.  1 979. Sister chromatid exchange: the most sensitive mammalian system for determining the 
agents. Academic Press, New York, pp. 229-246. 
Wolff S, & Carrano AV. 1 979. Report on the workshop on the util ity of sister chromatid exchange. 
Mutation Res. 64:53-56. 
Wolff S, & Perry P. 1 974 . Differential Giemsa staining of sister chromatid and the study of sister 
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Wolff S, Bodycote J ,  & Painter RB. 1 974. Sister-chromatid exchanges induced in Chinese hamster cells 
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Wood A W, Armstrong SM, Sait ML, Devine L, & Martin MJ. 1 998.  Changes in human plasma melatonin 
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Yager JW, Sorsa M, & Selvin S. 1 988.  Micronuclei in cytokinesis-blocked lymphocytes as an index of 
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Zimmerman AM, Winters WD, & Cameron IL. 1 990. Influence of 60 Hz magnetic fields on sea urchin 
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Zotti-MarteII i  L, Peccatori M, Scarpato R, & Migliore L. 2000. Induction of micronuclei in human 
Iymphocytes exposed in vitro to microwave radiation. Mutation Res. 472(1 -2):5 1 -58.  
Zwingelberg R,  Obe G, Rosenthal M, Mevissen M, Buntenkotter S, & Locher W. 1 993. Exposure of rats 
to a 50-Hz, 30-mT magnetic field influences neither the frequencies of sister chromatid exchanges nor 
proliferation characteristics of cultured peripheral lymphocytes. Mutation Res. 302:39-44. 
Appendix One. 296 
Appendix One (a) : A simplified comparison between 
electrical pressure and water pressure. 
Electrical pressure 
Voltage: Electrical pressure-the 
potential to do work. Measured in 
volts (V) or in kilovolts (kV). 
1 kV=1 000 volts. 
Lamp plugged in 
and turned off 
220 V 
Current: The movement of electric 
charge (e.g., electrons). Measured 
in amperes (A). 
Lamp plugged in 
and turned on 
220 V 
l A  
Water Pressure 
Hose connected to an open faucet 
but with the nozzle turned off 
Nozzle closed 
Hose connected to an open faucet 
and with the nozzle turned on. 
___ ;;::=s' . 
Nozzle open 
Source: NIOSH Facts: EMFs in the workplace, 1 995.  
Appendix One. 297 
Appendix One (b) : A simplified comparison between 
electrical fields and magnetic fields. 
Electric fields 
• Produced by voltage. 
t t 
+ 
Lamp plugged in but turned off 
voltage produces an electric field. 
• Measured in volts per meter 
(V Im) or in kilovolts per meter 
(kV/m). 
• Easily shielded (weakened) by 
conducting objects like trees 
and buildings 
• Strength decreases with 
increasing distance from the 
source. 
Magnetic fields 
Produced by current. 
Lamp plugged in but turned on voltage 
produces a magnetic field also. 
Measured in tesla (T) or gauss (G). 
1 T = 1 0,000 G. 
Not easily shielded (weakened) by 
most material. 
Strength decreases with increasing 
distance from the source. 
Source: NIOSH Facts: EMFs in the workplace, 1 995. 
Appendix One. 298 
Appendix One (c) : Magnetic field decreases with 
increasing distance from the source and measured in tesla 
(T) . This magnetic field is a 60 Hz power frequency field. 
61 cm, 0.7 �T 
r----�IIE__-
30 cm, 2 �T 
,---�IIIr-----.lIIk- 15 cm, 9 � T 
Source: EMF in your environment, EPA, 1992. 
Appendix Two. 299 
Appendix 2: PERSONAL QUESTIONNAIRE 
Please read the following questions carefully and answer them as thoroughly and 
accurately as possible. The information you give will not be associated with your name 
in any public document and will be known only to the principal investigator of this 
study . The answers you provide may have a direct bearing on the interpretation of our 
results. Therefore, we ask that you kindly cooperate fully in providing correct 
information. Thank you for your interest. 
Name: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
Address:  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
Contact Phone: . . . . . . . . . . . . . . . . . . . . . . . . . . . .  (Home) . . . . . . . . . . . . . . . . . . . . . . . . . . . . (Work) 
To be filled in by principal investigator: 
Code Number: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
Date: . . . . . . . . . . .  .1 . . . . . . . . . . .  . 1  . . . . . . . . . . .  . 
This sheet is to be detached from the remainder of the questionnaire and filed by the 
principal investigator. Only the code number will be used as an identifier in subsequent 
pages. If additional space is needed for the completion of an answer, please write on the 
back of the page and identify the remaining part of the answer with the question' s 
number. 
Appendix Two. 
Personal History 
1 .  Date: . . . . . . . . . . . . . .1 . . . . . . . . . . . . . . .  .1 . . . . . . . . . . . . . . . . . .  . 
Code No . . . . . . . . . . .  . . 
2.  What is your birth date? . . . . . . . . . . .  .1 . . . . . . . . . . .  ./ . . . . . . . . . . . . . . . 
(Month) (Day) (Year) 
Present and Past Occupational History 
3 .  Are you currently employed? D YES O NO 
4. What is the name of the Company/Institution for which you now work or 
if unemployed, last worked? 
5 .  For how long have you worked for this Company or Institution? 
6. What type of work do / did you do? 
300 
Appendix Two. 301 
Code No . . . . . . . . . . . . . . .  . 
Exposure History (work and non-work related) 
7. Have you ever been exposed to any of the following in your job? 
Asbestos 
Radiation 
Coal 
Products 
Dust (such 
as wood, 
leather) 
Pesticides, 
Herbicides 
Petroleum 
Products 
Dyes 
Solvents 
Other 
Chemicals 
(specify in 
question 
No. 8) 
When were you 
first exposed? 
(month , year) 
When were you 
last exposed? 
(Month, Y ear) 
How long in terms 
of days, months, or 
years in total were 
you exposed? 
o YES---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
O NO 
D YES ---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
O NO 
o YES---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ' "  
O NO 
D YES ---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ' "  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
O NO 
D YES ---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
O NO 
D YES ---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
O NO 
D YES ---7 . ,  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ' "  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
O NO 
D YES ---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
O NO 
D YES ---7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
O NO 
Appendix Two. 302 
Code No . . . . . . . . . . .  . . 
8. List the names of any specific substances to which you know you were exposed by 
either breathing or direct skin contact at work either in the last year or within the 
past ten years. 
In the last year 
( 1 2  months) 
How frequently 
exposed on a 
monthly average 
Within the How frequently 
past 1 0  years exposed on a 
monthly average 
9. Please list any chemical or physical exposure you experienced in the last year while 
practicing a hobby or other activities either at home or in other non-occupational 
settings.  Refer back to the list in question No. 7 but do not limit your answers to 
only these substances. 
In the last year 
( 1 2  months) 
How frequently 
exposed on a 
monthly average 
Within the How frequently 
past 1 0  years exposed on a 
monthly average 
Appendix Two. 303 
Code No . . . . . . . . . . . .  . 
Medical History 
1 0. Have you taken any medication prescribed by a doctor in the past lyear (for 
example, blood pressure pills, antibiotics, insulin, tranquilizers, muscle relaxants, 
etc.)? 
If yes, please indicate below: 
Type of medication Dose 
D YES O NO 
Frequency? Time period: 
Began (month) Ended (month) 
1 1 . Have you taken any non-prescription medication in the past 1 year (for 
example, aspirin, antacid, anti-histamines, sedatives, or other drugs)? 
D YES D NO 
If yes, please indicate below: 
Type of medication Dose Frequency? Time period: 
Began (month) Ended (month) 
Appendix Two. 304 
Code No . . . . . . . . . . . .  . 
1 2. Do you take any vitamins currently or have you in the past 6 months? 
D YES O NO 
If yes, please indicate below: 
What kind of vitamins: Dose Frequency 
1 3 .  Have you ever had any of the following illnesses? 
Cancer D YES O NO 
Hepatitis D YES O NO 
Mononucleosis D YES O NO 
Herpes D YES O NO 
AIDS D YES O NO 
Meningitis D YES O NO 
Bacterial or viral infection D YES O NO 
Cardiovascular disease D YES O NO 
Diabetes D YES O NO 
Other major illness D YES O NO 
If yes, please specifY what illnesses, when you were ill, and indicate treatment. 
Illness Period of illness 
(month, year to month, year) 
Treatment 
Appendix Two. 305 
Code No . . . . . . . . . . . .  . 
1 4. List any other illnesses and their treatments you have experienced in the past 1 2  
months (these should include colds, flu, etc.) 
Illness Period of illness 
(month, year to month, year) 
Treatment 
1 5 . List any vaccination which you have received in the past 1 2  months . 
Type of vaccination Date administered 
1 6. List any diagnostic or therapeutic X -rays other than dental you have received in 
the past 1 0  years . 
Reason for X-rays year received 
Appendix Two. 
1 7. Have you ever had any dental X-ray? 0 YES 
If yes, within: o the last month 
o the last 6 months 
o the last 6- 1 2  months 
o over one year 
1 8 . Have you had any surgery during the past year? 
Date Reason 
O NO 
1 9. Give dates you have had any high fevers during the past year. 
306 
Code No . . . . . . . . . . . .  . 
Date Associated illness Medication taken 
Appendix Two. 307 
Code No . . . . . . . . . . . .  . 
Smoking History 
20. If  you previously smoked cigarettes, cigars or a pipe, but have now given 
up, when did you last have a smoke? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
2 1 .  Do you currently smoke cigarettes? 
If yes, how many packs do you smoke each day? 
o less than half a pack 
o half- l  pack 
o more than 1 pack 
D YES 
If you smoke more than one pack a day please state how many: 
22. Do you currently smoke cigars? 
If yes, how many cigars do you smoke each day? 
o I cigar 0 2-3 cigars 0 4  or more cigars 
23 . Do you currently smoke a pipe? 
If yes, how many pipesful do you smoke each day? 
o 1 pipeful 
o 2-3 pipesful 
o 4 or more pipesful 
24. Do you smoke any substance other than tobacco ? 
D YES 
D YES 
D YES 
O NO 
O NO 
O NO 
O NO 
Appendix Two. 308 
Code No . . . . . . . . . . .  . 
Diet History (should only reflect current habits) 
25 .  Do you eat vegetables? 
26. Do you eat meat? 
27. Do you use diet sweeteners? 
D YES 
D YES 
D YES 
O NO 
O NO 
O NO 
How much per day or week? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
28.  Do you use diet drinks? D YES O NO 
How many per day or week? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
29. Pertinent comments concerning diet not covered by above questions, 
e .g . ,  special diet such as high protein, low carbohydrate, etc. 
30 .  Do you drink coffee? D YES O NO 
If yes, how much / day? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . .  
Decaffeinated? 
3 1 .  Do you drink tea? 
D YES 
D YES 
O NO 
O NO 
If yes, how much/day? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
Appendix Two. 309 
Code No . . . . . . . . . . .  . .  
32. Do you drink beer? O YES O NO 
If yes, please indicate your average weekly beer consumption: 
o 1 -6 cans (375 ml) a week or less 
o 7- 1 2  cans a week 
o 1 3-24 cans a week 
o more than 24 bottles a week. 
3 3 .  Do  you drink wine? O YES O NO 
If yes, please indicate your average weekly wine consumption :  
o 1 -4 glasses a week or  less 
o 5-8 glasses a week 
o 9- 1 6  glasses a week 
o more than 1 6  glasses a week. 
34. Do you drink other alcoholic beverages (excluding beers and wines)? 
o YES O NO 
If yes, please indicate type and average weekly consumption: 
o 1 -4 glasses ( 1 5  ml liquor) a week or less. 
o 5-8 glasses a week 
o 9- 16  glasses a week 
o more than 1 6  glasses a week. 
Appendix Two. 3 1 0  
Code No  . . . . . . . . . . . .  . 
Genetic History 
35 .  Are you aware of any birth defects or other genetic disorders or inherited diseases 
which affect your parents, brothers, sisters, or their children? 
O YES O NO 
If yes, please specify 
Thank you for your time. 
Appendix Three. 3 1 1  
Appendix 3 :  Consent Form 
I,  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ' "  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (print name), agree to 
have approximately 1 Oml of blood withdrawn from my body by a qualified person for 
the sole purpose of culturing my blood lymphocytes for chromosome examination. 
I have read the accompanying Invitation Letter and Personal Questionnaire and fully 
understand the issues outlined in them. I further acknowledge that the withdrawal of my 
blood and the subsequent examination of my chromosomes by Mr. Wahab at Massey 
University will be for the express purpose of research into the effects of low frequency 
electromagnetic fields (EMFs) on human chromosomes .  
I agree to participate III this experiment on the understanding that anonymity IS 
preserved and that any results obtained from the analysis of my chromosomes will 
remain confidential except for public disclosure of possible EMF effects on my genetic 
material. I further understand that I will be able to have access to all results concerning 
me at any time. 
I am also aware that I can withdraw from this research programme at any time. 
Signature: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . 
Date: 
Appendix Four. 3 12 
Appendix 4: Showing 50 ( 1 -50) random microscope coordinates at (X and Y positions) 
for each of the 2 gels per slide. 
Slide Area 1 Slide Area 2 
Cell # X Direction Y Direction Cell # X Direction Y Direction 
0 1  1 70 1 3  01 1 1 7 1 3  
02 1 69 1 0  02 1 1 7 1 7  
03 1 7 1  06 03 1 20 1 3  
04 1 7 1 1 3  04 1 1 6 1 0  
05 1 64 1 0  05 1 06 1 6  
06 1 62 1 4  06 1 1 4 1 0  
07 1 70 1 8  07 1 1 7 1 4  
08 1 7 1  1 0  08 1 1 3 2 1  
09 1 58 2 1  09 1 1 8 07 
10 1 56 08 1 0  1 1 0 1 9  
1 1  1 64 07 1 1  1 09 1 0  
1 2  1 54 08 12 1 1 0 2 1  
13 1 67 05 13 1 07 1 0  
1 4  1 65 07 14 1 1 9 1 6  
1 5  1 56 09 1 5  1 1 1  1 8  
1 6  1 65 1 0  1 6  1 09 1 8  
1 7  1 65 2 1  1 7  1 1 9 23 
18 1 67 14  1 8  1 04 2 1  
1 9  1 59 1 0  19 1 08 2 1  
20 1 60 1 3  20 1 1 2 08 
2 1  1 58 20 2 1  1 08 1 6  
22 1 55 1 6  22 1 04 09 
23 1 63 1 7  23 1 1 8 03 
24 1 56 22 24 1 20 1 1  
25 1 7 1  1 9  25 1 05 2 1  
Appendix Four. 3 1 3  
26 1 54 1 7  26 1 1 3 1 1  
27 1 53 1 5  27 1 2 1  05 
28 165 08 28 1 07 09 
29 1 54 08 29 1 1 4 07 
30 1 69 1 4  30 I I I  1 5  
3 1  1 69 22 31 1 22 03 
32 166 1 1  32 1 1 6 06 
33 1 58 1 4  33 1 1 9 08 
34 1 58 23 34 1 1 5 12  
35 165 09 35 1 1 5 03 
36 1 7 1  2 1  36 1 02 1 5  
37 1 72 1 4  37 1 1 1  07 
38 1 57 03 38 1 1 8 1 9  
39 1 6 1  1 9  39 1 1 7 03 
40 1 64 1 3  40 1 02 08 
41 1 55 07 41 1 05 1 2  
42 1 67 08 42 1 1 6 1 7  
43 1 52 1 0  43 1 09 1 8  
44 1 57 03 44 1 1 0 1 3  
45 1 54 02 45 1 09 08 
46 1 72 1 7  46 1 07 10  
47 167 2 1  47 1 1 2 1 0  
48 167 09 48 1 03 04 
49 1 58 14  49 1 1 1  12  
50 1 7 1 22 50 1 03 14  
Appendix Five. 
Appendix 5: 
." 
(a) � ' 
I1 � 
(b) 
DNA Rep 1 
double In 
helix BrdU 
, 
� 
",.:- ), 
, 
No 
seg. 
at 
Ana. 
,. 
;: . 
. 
� 
Rep 2 in BrdU 
· · 
· · 
· · 
· · 
· · 
· · 
� � 
� � 
Figure 1 :  (a) A c-metaphase chromosome spread of a dividing 
peripheral blood lymphocyte that has undergone two rounds of DNA 
replication in the presence BrdU, showing failure of chromosome to 
segregate after the first round of replication. Arrows = sister chromatid 
exchange. (b) A diagrammatic representation of DNA replication and 
sister chromatid exchange as has occurred in the arrowed chromosome 
pair. Solid line = unlabelled; dotted line = DNA labelled with BrdU. 
3 14 
Appendix Five. 
Appendix 5:  
(a) 
(b) 
DNA Rep 1 
double in .
� 
helix BrdU � 
f( : 
� --+ I � i ( ...... ��g .  : 
� � 
.
.
..
. 
�a � 
: : +. • : = = ••• : 
" '� : 
Rep 2 in 
BrdU 
No 
seg. 
at 
Ana. 
Rep 3 in 
BrdU 
Figure 2: (a) A c-metaphase chromosome spread of a dividing peripheral 
blood lymphocyte that has undergone three round of DNA replication in 
the presence of BrdU, showing failure of chromosomes to segregate after 
the second round of replication (arrows ) . (b) A diagrammatic 
representation of DNA replication as has occurred in the arrowed 
chromosome pairs. Solid line = unlabelled DNA; dotted line = DNA 
labelled with BrdU. 
3 1 5  
Appendix Five. 
Appendix 5:  
(a) 
(b) 
) 
/ 
� 
( 
'( 
/' 
) 
� 
,I' 
I' 
Figure 3: (a & b) Two c-metaphase spreads of a dividing 
peripheral blood lymphocyte subjected to EMF exposure 
which has undergone two rounds of DNA replication in 
the presence of BrdU, showing 3 and 4 SeEs (arrows), 
respectively. 
3 1 6  
Appendix Five. 
Appendix 5 :  
(a) 
• 
1 
(b) 
Figure 4: (a & b) Two c-metaphase spreads of a dividing peripheral 
blood lymphocyte subjected to EMF exposure which has undergone two 
rounds of DNA replication in the presence of BrdU, arrows showing 
complexes formed by 2-pairs of chromosomes. 
3 1 7  
Appendix Six. 
Appendix 6 :  
Sinusoidal wave 
1 
1 
1 cycle 
• • . . . . . . . . . . . . . . . • . • • . • . . . . • • • • • . . . • . . . • .
• 
. 
· 
· 
. . . . . . . .  i . . . . . . . . . . � . . . . . . . . . . . . . . . . .  . 
· 
� • • . . . . . . • . . . . . . . • . • . . . . . . . . . . . . . . . . . .  � 
Period 
3 1 8  
1 = peak 
O.707= Avg 
Rectified sinusoidal wave 
I Square wave I 
Period 
. 
: .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... : 
1 = Peak =A vg. 
o 
Rectified Square wave 
Appendix Seven. 3 1 9  
Appendix 7 :  
Table la:  Showing the raw data of recorded temperatures (Control condition: coil off & 
no MF) inside the culture tube, near the coil, inside the cabinet, and the room where the 
incubator was placed over a 24 h period. 
Time (b) Culture Tube Coil Cabinet Room Real Time 
0 37.0 37.0 3 7.0  22.0 5 
1 37.0 37.0 37 .0 2 l .7 6 
2 37.0 37.0 3 7.0  2 1 .5 7 
3 37.0 3 7.0 3 7. 0  2 1 .2 8 
4 37.0 37.0 37 .0 20.8 9 
5 37.0 37.0 37 .0 20.7 1 0  
6 37.0 37 .0 37 .0  20 .3  1 1  
7 37.0 37.0 37 .0 20. 1  mid night 
8 37.0 37.0 37 .0 20.0 1 
9 37.0 37.0 37.0 1 9 .9  2 
1 0  37.0 37.0 37 .0 20.0 3 
1 1  37.0 37.0 3 7.0 20.0 4 
1 2  37. 1 37.0 37.0 1 9.9  5 
1 3  37.0 37 .0 3 7.0  20.6 6 
1 4  37.0 37.0 3 7.0 20. 5  7 
1 5  37.0 37.0 37 .0 2 l .2 8 
1 6  37.0 37.0 37 .0 2 1 .6 9 
1 7  37.0 37.0 3 7.0 2 1 .9 1 0  
1 8  37.0 37.0 37.0 22.0 1 1  
1 9  37.0 37.0 37 .0 22.0 noon 
20 37.0  37 .0 3 7.0 22.0 1 
2 1  37.0 37.0 37 .0 22.0 2 
22 37.0 37.0 37 .0 22.0 3 
23 37.0 37 .0 37 .0  22.0 4 
24 36.9 37 .0  37 .0  2 1 .2 5 
23 37.0 37.0 37 .0 22.0 4 
24 36.9 37 .0 37.0 2 1 .2 5 
Appendix Seven. 320 
Table Ib: Showing the raw data of recorded temperatures (Coil on @ ImT MF) inside 
the culture tube, near the coil, inside the cabinet, and the room where the incubator was 
placed over a 24 h period. 
Time (h) Culture Tube Coil Cabinet Room Real Time 
0 37 .0 37.0 37.0 22.0 5 
1 37.0 37.0 37 .0 2 1 .5 6 
2 37 .0  37 .0  3 7.0 2 1 . 1  7 
3 37.0 37.0 37.0 20.8 8 
4 37 .0 37.0 37.0 20.4 9 
5 37 .0 37 .0  37 .0 20. 1 1 0  
6 37 .0 37.0 37.0 20. 1 1 1  
7 37 .0 37 .0 37 .0 1 9.8  mid night 
8 37.0 37.0 37 .0 1 9. 8  1 
9 37.0 37.0 37.0 1 9.6 2 
1 0  37.0 37.0 37 .0 1 9.7  3 
1 1  37.0 37.0 37.0 1 9. 5  4 
1 2  37.0 37.0 37.0 1 9.0  5 
1 3  37.0 37.0 37.0 1 9 . 1  6 
1 4  37.0 37 .0 37 .0 20.0 7 
1 5  37.0 37 .0  37 .0 20.5  8 
1 6  37.0 37 .0 37.0 2 1 .2 9 
1 7  37.0 37.0 37 .0 2 1 .9 1 0  
1 8  37.0 37.0 37.0 2 1 .9 1 1  
1 9  37.0 37 .0  37 .0  2 1 .9 noon 
20 37.0 37 .0  37 .0  2 1 . 8 1 
2 1  37.0 37 .0 37 .0 2 1 .9 2 
22 37.0 37.0 37 .0 2 1 .9 3 
23 37.0 37 .0 37 .0 2 1 .9 4 
24 37.0 37 .0 37 .0 2 1 .5 5 
Appendix Seven 
Appendix 7: 
Recorded Temperatures: Control Condition - No Field 
40.0�----------------------------------------------------�
r n r n n n n n n  
35.0 
30.0 
25 .0 
20.0 
1 5 .0 +---------,r---------r-------r---------f 
o 6 1 2  
Time - hours 
1 8  24 
--+-Culture Tube 
____ Coil 
Cabinet 
� Room 
Figure la: Graph of recorded temperatures (Control condition: coil off & no MF) inside the culture tube, near the coil, 
inside the cabinet, and the room where the incubator was placed over a 24 h period. 
32 1 
Appendix Seven 
38.0 
37.8 
37.6 
37.4 
U 
� 37.2 � � a.. 
ell � 
Q 
36.8 
36.6 
36.4 
36.2 
36.0 
0 
Recorded Temperatures: Control Condition - No Field 
6 1 2  
Time - hours 
1 8  24 
322 
--+-Culture Tube 
____ Coil 
Cabinet 
--*- Room 
Figure Ib: Graph of recorded temperatures (Control condition: coil off & no MF) inside the culture tube, near the coil, 
and inside the cabinet over a 24 h period. 
Appendix Seven 
U 
VJ � � I. I:)J) � 
� 
323 
Recorded Temperatures : Coil On @ 1 mT 
40.0 �---------------------------------------------------------, 
n n n 
35 .0 
30.0 • Culture Tube 
25 .0 
20.0 
____ Coil 
Cabinet 
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -M--Room 
l 5 .0+-------------r-----------��----------_,------------� 
o 6 12  
Time - hours 
1 8  24 
Figure le: Graph of recorded temperatures (Coil on @ ImT MP) inside the culture tube, near the coil, inside the cabinet, 
and the room where the incubator was placed over a 24 h period. 
Appendix Seven 
324 
Recorded Temperatures: Coil On @ 1 mT 
38.0 
37.8 
37.6 
37.4 
--+-Culture Tube 
U 37.2 
rIl � � 37.0 '"' � � 
36.8 Q 
36.6 
36.4 
36.2 
36.0 
0 6 1 2  
Time - bours 
1 8  24 
--4--Coil 
Cabinet 
�Room 
Figure Id:  Graph of recorded temperatures (Coil on @ ImT MP) inside the culture tube, near the coil, and inside the 
cabinet over a 24 h period.