The Clastogenic Effects of Cyclophosphamide and a Sixty Hertz Electronagnetic Field on Bone Marrow Cells of CD-I Mice

Western Michigan University ScholarWorks at WMU

Dissertations Graduate College

8-1997

Kevin K. Block Western Michigan University

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Recommended Citation Block, Kevin K., "The Clastogenic Effects of Cyclophosphamide and a Sixty Hertz Electronagnetic Field on Bone Marrow Cells of CD-I Mice" (1997). Dissertations. 1649. https://scholarworks.wmich.edu/dissertations/1649

This Dissertation-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Dissertations by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. THE CLASTOGENIC EFFECTS OF CYCLOPHOSPHAMIDE AND A SIXTY HERTZ ELECTROMAGNETIC FIELD ON BONE MARROW CELLS OF CD-I MICE

Kevin K. Block

A Dissertation Submitted to the Faculty of The Graduate College in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Department of Science Studies

Western Michigan University Kalamazoo, Michigan August 1997

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THE CLASTOGENIC EFFECTS OF CYCLOPHOSPHAMIDE AND A SIXTY HERTZ ELECTROMAGNETIC FIELD ON BONE MARROW CELLS OF CD-I MICE

Kevin K. Block, Ph.D.

Western Michigan University, 1997

Extremely low frequency (ELF) electromagnetic fields (EMF) have

been correlated with the induction of cancer in a number of epidemiological

studies (Feychting and Ahlbom, 1993; T,m and Lee, 1994; London et al. 1991;

Lovely et al., 1994; Tomenius, 1986; Wertheimer and Leeper, 1979, 1982).

Two hypothesis were tested in the present study. The first hypothesis tested

was that exposure to a 7.3 G magnetic field for 24 hours would increase the

frequency of clastogenic and/or mutagenic events in bone marrow cells of

mice. The second hypothesis tested was that exposure to a 7.3 G magnetic

field for 24 hours would increase the firequency of clastogenic and/or

mutagenic events in bone marrow cells of mice also treated with

cyclophosphamide, and hence may e n h an ce the potential for cancer initiation

by a known clastogen, mutagen, and carcinogen (Povirk and Shuker, 1994).

Eight groups of mice, 12 animals per group (6? and 6(f), were

administered a single Lp. injection of cyclophosphamide at a dose of 0, 5, 25,

or 50 mg/kg b.w.. Each group of mice was separated by sex into two plastic

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cages and the two cages placed side-by-side within the center region of an

energized or nonenergized Helmholtz coiL The 60 Hertz alternating current

was regulated to flow through the coil for 19 seconds, followed by a 19 second

interval of no current flow, ad in fin itu m As measured, the energized coil

produced a magnetic field intensity of 7.3 gauss and an electric field

intensity of 17.0 V/m at the center region of the coil.

After 20 hours of ELF EIMF or sham exposure, the mice were injected

with a single i.p. injection of colchicine at a dose of 4 mg/kg b.w., and

returned to their cage within the coil for 4 more hours of ELF EMF or sham

exposure. After 24 hours of ELF EMF or sham exposure, the mice were

sacrificed by cervical dislocation, their femurs removed, and bone marrow

metaphase spreads prepared on glass slides.

The scoring of the slides revealed that a 24 hour exposure to a

magnetic field intensity of 7.3 G did not significantly increase the firequency

of clastogenic and/or mutagenic events in mice that had (or had not) been

treated with cyclophosphamide. The results did not support either of the two

research hypothesis tested in the present study.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Copyright by Kevin K. Block 1997

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

I am deeply indebted to the members of my graduate committee. Dr.

Robert Poel, Dr. Gyula Ficsor, and Dr. Leonard Ginsberg for their patience

and the thoughtful advice they have given me over the last seven years. I

especially thank Dr Robert Poel for keeping me headed in the correct

direction over the last several years, and I thank my mentor and friend. Dr.

Gyula Ficsor for sharing his time and expertise with me.

Secondly I would Hke to acknowledge the patience and support given

to me by my wife Peggy, Thank you Peggy for putting up with me over the

last seven years.

Last I would like to thank Dr. John Crowell for his assistance in

performing the statistical analysis of the data generated in this project. I

also thank two friends of mine, Linda Dine and Roger Acerra, for the

technical support they contributed to this project.

Kevin K. Block

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... ü

LIST OF TABLES...... v

LIST OF FIGURES...... vi

CHAPTER

I. INTRODUCTION...... 1

Historical Background to Problem ...... 1

Statement of Research Problem ...... 19

n. LITERATURE REVIEW ...... 21

Cyclophosphamide ...... 21

Extremely Low Frequency Electromagnetic Fields ...... 31

Replication of Cells ...... 44

Sum m ary ...... 49

m. DESIGN OF STUDY...... 53

Introduction ...... 53

Experimental Design ...... 59

IV. RESULTS...... 70

Body Mass Measurements ...... 70

Chromosome Aberrations ...... 72

Sum m ary ...... 94 hi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents—continued

V. DISCUSSION AND CONCLUSION...... 95

Mutagenicity of Extremely Low Frequency Electromagnetic Fields ...... 95

Uniqueness of Study ...... 96

Conclusion ...... 100

APPENDICES

A. Layout of ELF EMF Exposure Hardware ...... 102

B. Monitoring Positions of Gauss Detector in A nim al Cage 104

C. Institutional Animal Care and Use Committee Form ...... 106

BIBLIOGRAPHY...... I l l

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

1. Summaiy of In \^tro Studies on Cyclophosphamide Mutagenicily. 28

2. Summary of In Vivo Studies on Cyclophosphamide Mutagenicity.. 32

3. Summary of In Vitro Studies on ELF EMF Mutagenicity ...... 39

4. Summary of In \^vo Studies on ELF EMF Mutagenicity ...... 43

5. Summary of Effects of Cyclophosphamide on Cell RepHcation 46

6. Summary of Effects of ELF EMF on Cell Replication ...... 50

7. Eight Different Treatment Groups of Mice Used in This Study 61

8. Body Mass Measurements ...... 71

9. Number of Chromatid Breaks (CB) / Chromatid Fragments (CF)... 74

10. Number of Isochromatid Breaks (IB) / Bichromatid Fragments (BF) 78

11. Number of Exchanges (E) / Rearrangements (R) ...... 81

12. Num ber of Rings ...... 84

13. Number of Highly Damaged Cells (HDC):>15 Breaks/ Fragments. 88

14. Total N um ber of Aberrant M etaphases (TAM) ...... 92

15. Summary of In Vitro Studies on ELF EMF Mutagenicity ...... 97

16. Summary of In Vivo Studies on ELF EMF Mutagenicity ...... 99

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

1. Approximate Wavelengths, Energies, and Frequencies of Various Tÿpes of Electromagnetic Radiations ...... 7

2. Metabolites of Cyclophosphamide ...... 24

3. ELF EM F Exposure A pparatus: Helmholtz Coil ...... 57

4. Normal Chromosome Spread ...... 66

5. Chromatid Break and Chromatid Fragment ...... 66

6. Isochromatid Breaks and Bichromatid Fragments ...... 67

7. Chromosome Exchanges and Chromosome Rings ...... 67

8. Highly Dam aged CeUs:> 15 Breaks/ Fragm ents ...... 68

9. Number of Chromatid Breaks/Chromatid Fragments ...... 75

10. Number of Isochromatid Breaks/Bichromatid Fragments ...... 79

11. Number of Exchanges/Rearrangements ...... 82

12. Num ber of Rings ...... 85

13. Number of Highly Damaged Cells ...... 89

14. Number of Cells With Aberrant Metaphases of Any Type ...... 93

15. Layout of ELF EMF Exposure Hardware ...... 103

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

INTRODUCTION

Historical Background to Problem

Ubiquity of Electromagnetic Fields

In a technological society, such as the United States, the widespread

use of electrical energy is a common fact of life. During the year 1983, the

average household used about 9000 kilowatt-hours (kWh) of electricity, and

this was forecasted to increase to about 10,671 kWh of electricity during

the year 2000. In addition, during the year 1984, both residential and

commercial use of electricity was estimated at 780 billion kWh, and this was

forecasted to increase to 1320 billion kWh during the year 2000 (Aldrich

and Easterly, 1987).

(üoupled with the ever increasing use of of electricity, is the increased

potential for humans to be exposed to electromagnetic fields (EMFs) created

by 50 or 60 Hertz (Hz) electricity flowing within conductors. We ca n n ot see,

hear, or feel the low energy EMFs created by the extremely low firequency

(ELF) alternating electric currents, but the fact remains, most people in a

modem society are immersed in ELF EMFs of varying intensity and for

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 varying durations during each day of their life. During the course of an

average day, a person in the United States may be in close proximity to

several items that are powered by electricity. Such items include electric

powered toothbrushes, radios, hairdryers, lights, razors, small and large

appliances, televisions, stereos, computers, blankets, heaters, waterbeds,

clocks, spas, hand held power tools, machinery, and the list goes on and on.

People are also exposed to EMFs generated &om electricity flowing through

the electrical wiring in their homes and at their work locations, and to EMFs

generated from electricity flowing through overhead (or buried) electric

power transmission lines. In addition, people are also exposed to EMFs

generated by certain types of medical diagnostic tests and medical therapy.

Components of Electromagnetic Fields

There are two physical components of EMFs: (1) an electric field (EF),

and (2) a magnetic field (MF). Both EFs and MFs occur as a result of electric

charges.

Elgçtriç Figlds

Electric fields exert attractive or repulsive forces on electric charges.

There are positive electric charges (protons) and negative electric charges

(electrons) found throughout nature. Each positive or negative electric

charge is surrounded by an electric field (EF) which exerts a force on other

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 positive and negative electric charges. The EF around a positive electric

charge repels other positive electric charges and attracts negative electric

charges. Conversely, an EF around a negative electric charge repels other

negative electric charges and attracts positive electric charges. The intensity

of an EF is measured in units of volts per meter (V/m).

Electric fields are found throughout nature. Static electricity is one

example of an EF that occurs in nature. AH of us are familiar with the build

up of static electric charges fiom walking across a carpeted room and the

associated discharge of those charges when we touch a doorknob or another

person. Lightning is another example of static electric charges that have

accumulated and are then discharged with each ligh tn in g bolt. Other

examples of EFs that occur in nature include the EFs that hold the atoms

and molecules of both living and non-living matter together, and the EFs

found to exist across the plasma membranes of living cells.

Magnetic Fields

An electric current is the movement or flow of electric charges in the

same direction. The greater the current, the greater the strength of the

associated MF that is generated. The strength of an electric current is

measured in units of amperes (A).

When electric charges are in motion in a conductor such as with

alternating electric current moving in transmission hnes, the moving electric

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 charges create a field that exerts a force on other moving electric charges.

The field around the moving electric charges in alternating electric current

is called a magnetic field (MF). The intensity of a MF is measured in units

of gauss(G) or tesla(T), with the equivalence between the two units being:

IT = 10,0000.

Similar to EFs, MFs are also found throughout nature. Examples

include the small magnetic fields originating firom electric currents created

by the electric depolarization of neuron membranes and the depolarization

of cardiac muscle. Other examples include the earth's MF created by electric

charges flowing within the earth's molten center and the MF created by a

bar magnet as a result of a certain alignment of electron spins within the

iron atoms of the magnet. MFs are generated around electric powered items

and around any conductor that has an electric current flowing through it.

Properties of Magnetic and Electric Fields

Magnetic Field

Magnetic fields are force fields generated by moving electric charges

which exert a force upon other moving electric charges. The electric charges

must be in motion in order to generate and maintain a MF. When 60 Hz

electricity is flowing through a conductor, a MF is generated around the

conductor. However, when the electricity is shut off, the electric current

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stops flowing in the conductor and the MF ceases to exist.

Magnetic fields, as they propagate through space, will easily pass

through most objects (including human beings), which is the reason that

overhead or buried residential power lines will contribute to the MFs within

our houses and the buildings we are in while at work. The strongest MFs

are always found closest to the conductor and decrease rapidly in intensity

with increasing distance fiom the conductor.

Electric Field

Electricity must be flowing in a conductor in order for a MF to be

generated. However, an EF may be generated near an electric powered

item merely by having the item connected to a voltage source even when the

item is not in operation.

Unlike the propagation of MFs through space, the propagation of EFs

through space are stopped by most objects around us, such as the walls and

roofs of buildings and houses, or even by the trees so often found in

residential neighborhoods. Like MFs, the strongest electric fields are found

closest to the conductor and decrease rapidly in intensity with increasing

distance fixim the conductor.

Properties of 60 Hertz Alternating Electricitv

The electricity that is generated in the United States is 60 Hz

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 alternating current (AC), which means the electric current alternates back

and forth in direction, sixty times in every second. This alternation of electric

current affects both the MFs and EFs generated by the alternating current.

Unlike the earth's MF which has a fixed direction, the MFs generated firom

60 Hz AC, vary periodically with a period of 1/60 second. In each period, the

MF builds up from zero to a maximum value, then decreases to zero, and

then builds up to a maximum value in the opposite direction, and once again

returns to a zero value.

The wavelengths, firequencies, and energies of the EMFs generated

by 60 Hz AC electricily are part of a spectrum known as the electromagnetic

spectrum (see Figure I). The velocity, in air, of the EMFs generated firom 60

Hz electricity is the same as it is for the velocity of all other types of

electromagnetic energy in air, namely the speed of hght (3 x 10* m/s). The

intensity of EMFs coming firom a point source, such as the EMFs coming

fiom a hand held electric drill, vary inversely with the square of the distance

from the point source. The intensity of EMFs coming firom a line source,

such as the EMFs coming firom electric transmission lines, vary inversely

with the distance fiom the line source.

Public Concern Over EMFs

Since the early 1980's, largely due to epidemiological studies that have

at times been sensationalized by the press, there has been increasing public

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wavelength Energy Frequency Type of Electromagnetic Radiation

(m X 2.9979) (eV X 4.1354) (Hertz)

10*6 10* 1 -*l ELF 60 Hz AC Electricity 10*3 10® 10^ HI

10“ 10'** 10“ H

10- 1 0 ® 10® AM Radio

1 10' ^ 10* Television/radar

10'2 10'® 10^® H | — Microwaves

10'“ 10 '* 10*2 ^ Infrared

10-® 1 0 * 10*“ H | Visible Light

10'* 10 10*® H - Ultraviolet

10-10 10* 10** X-rays

10*2 10® 1Q2® HI

10'*“ 10 ^ 1Q22 H I Gam ma Rays

10'* ® 10® 102“ ■

Wavelength (A.) = c/f = 2.9979 x 10* m s'* / frequency (Hz)

Energy (E) = hf = 4.1354 x 10'*® eV s'* x frequency (Hz)

Figure 1. Approximate Wavelengths, Energies, and Frequencies of Various Types of Electromagnetic Radiations.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 concern over the possiblity that exposure to ELF EMFs may increase the

incidence of certain types of cancer.

One type of epidemiological study that has reported statistically

significant correlations between cancer incidence and exposure to ELF EMFs

is concerned with the population of workers whose occupations have required

them to receive sustained exposures to ELF EMFs at their work locations

(Lin, Dischinger, Conde, and Farrell, 1985; Savitz and Calle, 1987; Pearce,

Reif, and Fraser, 1989; Bastuji-Garin, Richardson, and Zittoun, 1990;

Garland, et al., 1990; Loomis and Savitz, 1990; Demers et al., 1991;

Tornqvist, Knave, Ahlbom, and Persson, 1991; lynes, Anderson, and

Langmark, 1992; Richardson et al., 1992; Floderus et al. 1993; Matanoski,

Elliot, Breysse, and Lynberg, 1993; Theriault et al. 1994; Floderus,

Tornqvist, and Stenlund,1994; Savitz and Loomis, 1995).

A second type of epidemiological study that has reported fin d in g

statistically significant correlations between cancer incidence and exposure

to ELF EMF is concerned with the populations of children and adults who

received sustained exposures to ELF EMFs within their own homes

(Wertheimer and Leeper, 1979; Wertheimer and Leeper, 1982; Tomenius,

1986; Savitz, John, and Kleckner, 1990; London et al., 1991; Feychting and

Ahlbom, 1993; Lovely et al., 1994; Lin and Lee, 1994)

In a review of 53 epidemiological studies that reported fin d in g

statistically significant correlations between the incidence of certain types

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9 of cancer and exposure to ELF EMFs (Bates, 1991), 46 of the studies also

reported the risk odds ratio for acquiring the cancers. A risk odds ratio equal

to 1 means the probability of acquiring a certain type of cancer is the same

as the national incident rate for that type of cancer; a risk odds ratio equal

to 2 means the probability of acquiring a certain type of cancer is 2 times

that of the national incidence rate for that type type of cancer, and so on. Of

the 46 studies reporting nsk odds ratios, 42 studies reported risk odds ratios

between 1 (same as the national incident rate) and 4 (4 times the national

incident rate), 3 studies reported risk odds ratios between 4 to 10, and 1

study reported a risk odds ratio greater than 10. The public is cautioned to

put these reported risk odds ratios in perspective since risk odds ratios in the

1 to 4 range do not demonstrate a strong relationship between a variable (i.e.

ELF EMFs) and cancer incidence. Risk odds ratios of 10 and above show a

much stronger relationship between a variable and cancer incidence such as

in the epidemiological studies which reported statistically significant

correlations between smoking and lung cancer. Such studies reported risk

odds ratios of between 20 to 30 (Hendee and Boteler, 1994).

Another thing that has not always been made clear to the public

about these epidemiological studies, is that a statistical correlation does not

mean the same thing as cause and effect. As Morgan (1992) explains,

statistically significant correlations between two variables such as ELF

EMFs and cancer incidence do not prove that exposure to ELF EMFs caused

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 the higher incidence rate of a cancer. These studies do not (and cannot) look

at all of the possible variables or agents that in fact could actually account

for higher incidence rates of cancer. In real life, we have different lifestyles

and diets, exposing us to a number of different carcinogenic agents. This

makes it very difGcult to propose a cause and effect relationship between

ELF EMFs and higher incidence rates of cancers.

One important finding of the epidemiological studies is that MFs,

generated by 60 Hz electricity, show a higher correlation figure with cancer

incidence than the correlation figure of EFs with cancer incidence. One

possible explanation for this finding may be that MFs are known to

penetrate most materials easily (including the human body), whereas EFs

do not penetrate most materials very easily (including the human body).

Because the energy of the electric field is dissipated very quickly upon

entering the human body, it does not penetrate very deeply into the tissues

of the body. However the energy of a magnetic field is not dissipated quickly

upon entering the human body and as a result, the magnetic field penetrates

deeply into the tissues of the human body. It is for this reason that magnetic

fields may be principally responsible for any biolgical effects induced by ELF

EMFs.

Biological Effects of EMF

Laboratory studies on the biological effects of ELF EMFs have created

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 controversies for a number of reasons. First, a number of the studies have

reported finding biological effects while other studies have reported fin d in g

no biological effects. Second, attem pts to reproduce certain positive studies

that reported finding biological effects, have not always been successful.

Third, there is no generally accepted mechanism for how ELF EMFs can

produce biological effects in organisms. Fourth, certain studies have

reported finding no linear dose response between biological efiects and the

increase of dose rates or total dosages of ELF EMFs. In addition, some

studies have reported thresholds of ELF EMF firequency and intensity

whereby biological effects are seen at a certain firequency and/or intensity

and then disappear at higher or lower firequencies and/or intensities

(Goodman et al., 1995).

The two best understood mechanisms for producing biological damage

in living tissue by EMFs, are those of ionization and thermal effects. At the

firequency of 6 0 Hz electricity, each particle (photon) of the EMF has a very

small energy, 2.5 x 10*‘^ eV (see Figure 1). Unlike the energies of gam m a

rays, X-rays, or even the higher energy ultraviolet electromagnetic

radiations, the ELF EMFs generated by 60 Hz electricity do not have

sufficient energy to ionize atoms, break molecular bonds in biological

molecules, or ionize water molecules in cells resulting in the production of

highly reactive firee radicals. All of these processes would require photons

with energies greater than 1 x 10^ eV.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 It is generally accepted (Morgan, 1992) that the EMFs generated from

60 Hz electricity can move ions within the tissues of our bodies , thereby

producing electric currents in these tissues. However, the electric currents

produced in tissues from ELF EMFs are several times weaker than the

electric currents that naturally occur in the body. The electric currents

produced in the tissues surrounding the heart (as a result of the

depolarization of cardiac tissue) are 100 to 1000 times greater than the

electric currents produced in the body by 60 Hz ELF EMFs. (Pool, 1990).

One result of the weak electric currents produced in tissues by 60

Hz ELF EMFs, is that some heat is produced within the tissues. However,

EFs in the air would have to be between a magnitude 10' and 10® V/m, or

the MFs would have to be between a magnitude of lO'* and 10® G, in order to

produce electric currents sufGcient in magnitude to produce heating effects

comparable to those that exist naturally within the human body. However,

EFs greater than 10® V/m cannot be maintained naturally in air due to spark

discharge of the field, and MFs between 10“* and 10® G are not found to occur

naturally (Aldrich and Easterly, 1987).

Although the ELF EÎMF energies created by 60 Hz AC electricity are

insufficient to ionize atoms, break molecular bonds, or produce thermal

effects of any significance within tissues, several laboratory studies have

shown that ELF EMFs are somehow interacting with organisms and are

affecting the biological processes within them. Exactly how ELF EMFs may

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 produce the biological effects is still not known.

While several of the laboratory studies have reported finding

biological effects associated with 60 Hz EMFs, there have also been a

number of studies that have reported finding no biological effects. Some of

the reported biological affects include the following: (Morgan, 1992)

1. Changes in the amount of ion flow across cell membranes,

especially for the C a^ ions, but also for IT", Li% and Mg^.

2. Changes in the production of melatonin, a hormone important in

regulating the body's daily biological cycle.

3. Changes in the duration of the cell- cycle, whereby the growth rate

of cells and division of the cells is affected.

4. (Changes in the rate that DNA molecules are copied and the rates

and amounts of ribonucleic acid (RNA) copied &om the DNA.

5. Changes in the immune response by certain immune cells.

6 Changes in the activities of certain enzymes.

7. Changes in the response of cells to mitogens.

8. Enhancement in the ability of broken bone to reunite.

9. Enhanced mutagenic effects have been reported in a few studies,

however the majority of studies have reported no such effects.

Carcinogenesis

Moolgavkar and Knudson (1981) have described a two stage model for

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14 carcinogenesis based on two independent events: (1) tumor initiation; and (2)

tumor promotion. Under the two stage model, two separate DNA mutations

must eventually occur before a cell can be transformed into a m alig n a n t cell

capable of transformed clonal expansion. The first DNA mutation occurs

with initiation, whereby the DNA of a cell is changed either by spontaneous

mutation or by the affects of a chemical or physical mutagenic agent. The

initiated cell is now called an intermediate cell. The intermediate cell may

now divide resulting in an intermediate lesion in the DNA that may or may

not be repaired in the cell. The process that increases the proliferation of

the intermediate cells is called promotion, and the agents (usually not

mutagenic themselves) that facilitate the division of intermediate cells, are

called promoters. An intermediate cell, with a mutation, may now undergo

a second mutation resulting in a transformed cell that is malignant and

whose clone of cells wiU result in a malignant tumor. In essence, a promoter

increases the pool of intermediate cells having the required first mutation,

thereby increasing the overall possibility of a second mutation to occur to one

of these cells and for a malignant transformation of the cell to occur

(Severson and Davis, 1991).

In addition to promoting factors, there are also co-promoting factors.

Co-promoters are not direct tumor promoters themselves, but assist in the

carcinogenic process by enhancing the tumor promoting affects of promoters

(Loscher and Mevissen, 1994). For many types of tumors in humans, the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 latent period between the initiating event in a cell and the development of

the malignant tumor is 15 to 45 years depending on the activities of the

tumor promoters and co-promoters (Koihnan, 1993).

ELF EMF as a Cancer Promoter/Co-promoter

There have been a small number of in vitro and in vivo laboratory

studies that have reported finding mutagenic effects in cells as a result of

exposures to ELF EMFs. On the other hand, there have been a far greater

number of laboratory studies that have reported finding no mutagenic effects

due to exposures to ELF EMFs. Taken in total, the preponderance of

negative findings have caused many scientists to conclude that DNA is not

significantly damaged by exposure to ELF EMFs. As a result, many

scientists believe that ELF EMFs may act as cancer promoters or co

promoters rather than as a cancer initiator (Goodman et al., 1995).

Mechanisms proposed to explain how EMFs may act as promoter/co

promoters include the following:

1. Disruption of intercellular communication: EMFs may act

synergistically with promoters at the cell membrane whereby distorted

signals are then sent inward to the nucleus and other organelles. This

may result in a disruption of the normal cell-to-cell communicationthrough

gap junctions, leading to autonomous cell growth (Adey, 1988; 1990).

2. Disruption of intracellular calcium homeostasis: EMFs disruption

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 of a cell's calcium homeoastasis may lead to increased production of &ee

radicals within the cell which in turn may may affect the cell's ability to

protect itself from oxidative attack by toxic chemicals or ionizing radiation

(Stevens, 1993).

3. Reduced levels of melatonin: Melatonin is the principal hormone

released by the pineal gland in the brain. Melatonin acts as the

neuroendocrine controller of the biological clock by synchronizing the release

of hormones fr-om other endocrine glands. Melatonin's action on other

endocrine glands is generally inhibitory. Since ELF EIMFs have been shown

to depress melatonin secretion from the pineal gland, reduced circulating

levels of melatonin could result in increased circulating levels of testosterone

and estrogen. Increased circulating levels of these two steroids could

enhance the carcinogenic process for those types of cancers with positive

receptors for these steroids. Such cancers would include breast and prostate

cancer (Stevens, 1993).

4. Reduced immune functions: There is some evidence that ELF

EMFs can suppress the cytotoxicity of T-lymphocytes (Lyle et al., 1983). The

reduced immune functions could result in reduced elimination of initiated

cells as well as the progression of transformed cells via reduced T-cell

activity (Severson and Davis, 1991).

5. Altered gene expression: Disruption of a cell's calcium homeostasis

by ELF EMFs could affect the activation of certain genes associated with

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 growth processes of the cell, which in turn could be involved in the

carcinogenic process (Goodman, 1990).

Leukem ia

As explained by Kumar, Cotran, and Robbins (1997), "The leukemias

are malignant neoplasms of the hematopoietic stem cells characterized by

diffuse replacement of the bone marrow (cells) by neoplastic cells" (p 373).

The two major classifications of leukemias are (1) acute leukemias, and (2)

chronic leukemias. Both the acute leukemias and the chronic leukemias

may be classified further into (1) lymphocytic, and (2) myelocytic. Thus a

particular leukemia may be placed into one of four different classifications,

(1) acute lymphocytic leukemia (ALL), (2) chronic lymphocytic leukemias

(CLL), (3) Acute myelocytic leukemia (AML), or (4) chronic myeloqrtic

leukem ia (CML) (Kum ar et al., 1997).

Chromosome abnormalities have been found in people who have been

exposed to various physical agents (i.e. gamma radiation or X-rays) or to

various chemical agents (i.e. cyclophosphamide, nitrosourea, benzo(a)pyrene,

7,12-dimethylbenzanthracene). Chromosome abnormalities have also been

found in people suffering firom certain diseases. Cytogenetic examination

of bone marrow cells taken firom people suffering firom leukemia have found

both numerical and morphological chromosome abnormalities in a

percentage of the leukemia patients, although a percentage of the patients

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 have also shown no chromosome abnoinnalities. The numerical

abnormalities found in leukemia patients include both aneuploidy and

euploidy chromosome abnormalities. The morphological abnormalities

include deletions, breaks, gaps, fragments, marker chromosomes, and

dicentrics (Woodliff, 1971).

Certain types of chromosome abnormalities are associated with certain

types of leukemia. As explained by Kumar et al (1997),

Approximately 90% of patients with ALL have nonrandom karyotypic abnormalities. Most common is hyperdiploidy (>50 chromosomes/cell) in early B cell ALL. The Philadelphia chromosome is present in 2% to 5% of children with ALL and in 25% to 30% of adults with ALL.... Several qrtogenetic changes have been noted in AML...Of particular interest is the t(15:17) translocation....In approximately 90% of patients with CML, the Ph (Philadelphia) chromosome, usually representing a reciprocal translocation from the long arm of chromosome 22 to the another chromosome (usually the long arm of chromosome 9) can be identifred.._Approximately 50% of (CLL) patients have karyotypic abnormalities, the most common of which is trisomy 12. (Kumar et al, 1997, pp374-378)

Although chromosome abnormalities are found in only a percentage

of untreated leukemia patients, chromosome abnormalities are more

commonly found in leukemia patients who have undergone therapy with

physical and/or chemotherapeutic agents. The increase in the number and

types of chromosome abnormalities in patients after therapeutic treatments

attest to the ability of the physical and chemical agents to induce

chromosome abnormalities(WeatheraH and Walker, 1965).

Cancer therapy has in many cases been shown to be a two edged

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19 sword. The very agent(s) used to put a cancer into remission may later be

responsible for inducing a secondary cancer. Ionizing radiation (gamma

radiation or X-rays) can cause several different types of chromosomal

aberrations and can also lead to acute leukemia (Woodliff, 1971). Several

chemicals used as a cancer chemotherapeutic can cause chromosome damage

(i.e. cyclophosphamide) and must be considered potentially leukemogenic

(Woodliff, 1971)

Statement of Research Problem

Extremely low frequency (ELF) electromagnetic fields (EMF) have

been correlated with the induction of cancer in a number of epidemiological

studies (Feychting and Ahlbom, 1993; Lin and Lee, 1994; London et al.,

1991; Lovely et al., 1994; Tomenius, 1986; Wertheimer and Leeper, 1979,

1982).

Since damage to chromosomes can initiate the carcinogenic process,

it is important to know if ELF EIMFs are mutagenic to chromosomes. The

first hypothesis tested in the present study was that exposure of mice to a 7.3

G magnetic field, for 24 hours, would increase the firequency of clastogenic

and/or mutagenic events in the bone marrow cells of the mice exposed to the

magnetic field.

It is also important to know if ELF EMFs can enhance the

mutagenicity of known cancer initiators. Cyclophosphamide is a known

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 clastogen, mutagen, and carcinogen (Povirk and Shuker, 1994). The second

hypothesis tested in the present study was that exposure of mice to a 7.3 G

magnetic field for 24 hours would increase the clastogenic and/or mutagenic

efiects of cyclophosphamide in the bone marrow cells of mice. In the present

study, mice were treated with a single intraperitoneal (i.p.) injection of

cyclophosphamide at a dose of either 0, 5, 25, or 50 mg/kg body weight, and

then exposed to a 7.3 G magnetic field intensity for a period of 24 hours.

A significant increase in the firequency of clastogenic and/or mutagenic

events in the bone marrow cells of mice exposed to both qrclophosphamide

and the 7.3 G magnetic field would suggest that ELF EMFs can enhance the

cancer initiation process by cyclophosphamide.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER n

LITERATURE REVIEW

Cyclophosphamide

Introduction

The nitrogen mustard, qrclophosphamide, is a bifunctional alkylating

agent that is used extensively as an anticancer chemotherapeutic in the

treatment of several different types of neoplastic diseases. In additon, due

to cyclophosphamide's immunosuppressive properties, the drug is also

administered as an immunosuppressive agent prior to organ transplants and

in the treatment of such nonneoplastic diseases as rheumatoid arthritis,

systemic lupus erythematosus, multiple sclerosis, psoriatic arthritis, chronic

hepatitis, scleroderma, glomerulonephritis, and chronic interstitial

pneumonia (Anderson, Bishop, Gamer, Ostrosky-Wegman, and Selby, 1995).

In the 1950s, anticancer chemotherapy relied extensively on the use

alkylating agents, including nitrogen mustards. It was not long after the

regular use of alkylating agents for anticancer therapy, that case reports

started appearing in the literature about patients developing secondary

cancers after having received prior treatment with alkylating agents (Povirk

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22 and Shuker, 1994). Of all the alkylating agents used as anticancer

chemotherapeutics, the nitrogen mustards are among the most carcinogenic,

(Kaldor, Day, and Hemminki, 1988). After reviewing the available

information, the International Agency for Research on Cancer (lARC)

designated cyclophosphamide as being carcinogenic to humans (lARC,

1981)(IARC, 1982)(IARC, 1987).

A number of studies have reported a link between the use of

cyclophosphamide as a chemotherapeutic agent and the later onset of

secondary cancers. The onset of secondary bladder cancer in patients who

previously received cyclophosphamide therapy, has been reported in a

number of studies (Fairchild, Spence, Solomon, and Gangai, 1979; Kinlen,

1985; Pederson-Bjergaard, Ersboll, and Hansen, 1988; Travis, Curtis, Boice,

and Fraumeni, 1989; Thrasher, Miller, and Wettlaufer, 1990; Sigal,

Tomaszewski, Brooks, Wein, and LiVasi, 1991; Cannon, Linke, and Cos,

1991; AHvizitos et al., 1991; Ortiz, Gonzales-Parra, Alveraz-Costa, and

Egido, 1992). In addition, there have been a number of studies that have

reported on the onset of leukemia following cyclophosphamide therapy

(Greene, Boice, and Greer, 1982; Pederson-Bjergaard et al., 1985;

Baglin,Galvin, and Pollock, 1987; Geller et al., 1989; Gibbons and

Westerman, 1988; Escalante, Kaufman, and Beardmore, 1989; Henry-Amar

and Dietrich, 1993).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 Metabolism of Cyclophosphamide

The parent cyclophosphamide molecule must first undergo

transformation by hepatic cytochrome P450 enzymes in order to become an

active alkylating agent (see Figure 2). Cyclophosphamide first undergoes an

oxidation in the hepatcxytes firom the parent molecule to 4-

hydroxycydophosphamide. The 4-hydroxycyclophosphamide molecule may

then either nonenzymatically equilibrate with the aldophosphamide

structure, or be metabolized further in the hepatocyes to the biologically

inactive 4-ketcxycdophosphamide molecule. The aldophosphamide molecule

may then be metabolized in the liver to the biologically inactive

carboxyphosphamide molecule or may enter the circulatory system. The

carboxyphosphamide molecule, while itself not being toxic, may then be

metabolized to the nomitrogen molecule which is a very strong alkylating

agent. Inside the cells of the body, the aldophosphamide molecule may then

undergo nonenzymatic cleavage into an acrolein molecule and a

phosphoramide mustard molecule. The phosphoramide mustard molecule

is thought to be the major alkylating agent giving rise to a series of mono-

and crosshnked adducts resembling the nomitrogen molecule.(Anderson et

al., 1995; Stanton and Legendre, 1986; Povirk and Shuker, 1994).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24

C1-CH,-CH2v

CI-CHo-CHT O-CHg

Cyclophosphamide

Cytochrome P450 mixed function oxidases

H 9H H g Cl-CHa-CHA O. N -C H . a-CHa-CHas KT-C. N-X ^CH, . N - X y a , CL-CHo-CHia O-CHg CI-CH2.CH2 O -CHa 4-Hydroxyqrclophosphamide 4-Ketocyclophosphamide > = C > (not biologically active) A

Cl-CHa-CHaV NHa Cl-CHa-CHaS NHa .N-X Cl-CHa-CHa O-CHa-CHa-CHO CI-CHa-CHa O-CHa'CHa-COOH

Aldophosphamide C Carboxyphosphamide (not biologically active)

CI-CHa-CHa, (^,NHa Cl-CHa*CHav .N -P , ,.NH Cl-CHa-CHa OH C I-C H a”CH a Phosphoramide Mustard Nomitrogen Mustard (strong alkylating agent) (strong allqrlating agent) +

CHa= CH-CHO

Acrolein (cytotoxic)

Figure 2. Metabolites of Cyclophosphamide.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 Cyclophosphamide: Mechanism of Action

Alkylating agents act by form ing covalent bonds between an alkyl

group(s) contributed by the all^lating agent and biological molecules in the

body. As explained by Stanton and Legendre (1986),

Alkylating agents act by form in g covalent bonds with organic compoimds,particularly nucleic acids, thereby replacing a hydrogen bond (on the nucleic acid or protein) with an alkyl group (from the alkylating agent). This results in the cross-linking of DNA strands, the breakage of DNA strands, the formation of linkages between different locations on the same DNA strand, and the inhibition of DNA synthesis. Cyclophosphamide's m ain cytotoxic effects result from alkylating reactions with nuclear DNA, cytoplasmic RNA, and various other cytoplasmic and membrane proteins. These reactions result in the prevention of mitosis because of interference with DNA replication, transcription, and RNA translation....Cyclophosphamide, similar to all chemotherapeutic drugs, is most effective on cell populations undergoing rapid division. (Stanton and Legendre, 1986, pp 1319-1320)

In regards to Cyclophosphamide's carcinogenic properties, the drug's

carcinogenic mechanism(s) are still not clearly understood but are thought

to arise from the contribution of one or more mechanisms that include

chromosome point mutations, intragenic deletions, large scale chromosome

aberrations, disturbances in genetic regulation, defects in immunological

surveillance, and/or activation of latent viruses (Povirk and Shuker, 1994;

Stanton and Legendre, 1986).

Cyclophosphamide: Mutagenicity In \^tro

There are a number of in vitro studies that have reported on the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 mutagenicity of cyclophosphamide. Nasr, Goldman, Klein, and Dacre (1988)

reported finding a significant increase in the firequency of sister chromatid

exchanges (SCEs) in cultured Chinese hamster ovary (CHO) cells activated

with rat hver microsomes (S9) and treated with cyclophosphamide at a dose

concentration of 10^ M. Amsdorf-Roubicek and Targa (1990) reported on an

in vivo exposure/in vitro assay to assess the mutagenicity of

cyclophosphamide's metabolites in the blood of rats. Rats were treated with

cyclophosphamide a t doses of 2.5, 5.0,10.0, or 20.0 mg/kg body weight (b.w.)

and then blood plasma was removed firom the treated rats. The authors

reported finding a statistically significant increase in the firequency of SCEs

in cultured human or rat lymphocyes treated with the blood plasma taken

firom the cyclophosphamide treated rats.

Sharma, Sobti, Chaudhry, and Dhar (1987) reported fin d ing a

statisticraUy significant increase in the firequency of chromosome aberrations

(CAs) in the polytene chromosomes firom the salivary gland cells of

Anopheles stephensi mosquito larvae treated with cyclophosphamide. The

mosquito larvae were introduced to 20.7 /ig/ml of cyclophosphamide in

distilled water as a rearing medium. Twenty-four hours post treatment, the

larvae were transferred to ordinary water and allowed to grow. Healthy

larvae were then dissected to obtain the salivary gland cells for chromosome

analysis. Miller (1991) reported fin d in g a statistically significant increase in

the frequency of CAs in mitogen-stimulated human B- and T-lymphocyte

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 populations activated with S9 mix and treated with qrclophosphamide at

concentrations of 10, 15, 20, or 30 /zg/ml. Monteith and Vanstone (1995)

reported finding increased firequencies of CAs in cultured V79 Chinese

hamster cells exposed to cyclophosphamide at doses of 4 or 8 jug/ml.

Oshiro, Piper, Balwierz, and Soelter (1991) reported at least a two-fold

increase in the firequency of micronuclei (MN) found in cultured Chinese

hamster ovary cells exposed to cyclophosphamide at concentrations of 12, 20,

80, or 160 /zg/ml.

Table 1 presents a summary of the in vitro studies reviewed in this

paper on the mutagenicity of cyclophosphamide.

Cvclopbosphamide: Mutagenicitv In Vivo

There are a number of in vivo studies that report on the mutagenicity

of cyclophosphamide. Rabello-Gay, Carvalho, Otto, and Targa (1985)

reported a significant increase in the firequency of CAs in bone marrow cells

taken firom mice treated with cyclophosphamide at a dose of 50 mg/kg b.w..

McFee and Tice (1990) reported dose related increases in the fi’equency of

CAs in first division metaphase bone marrow cells taken fix>m mice treated

with cyclophosphamide at doses of 18.75, 37.5, or 75.0 mg/kg b.w..

Nersessian, Zilfian, and Koumkoumadjian (1992) reported fin d in g a

significant increase in the firequency of CAs in bone marrow cells taken from

Armenian hamsters treated with a single intraperitoneal injection (i.p.) of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 Table 1

Summary of In Vitro Studies on Cyclophosphamide Mutagenicity

Organism/ Reference CehType Dose(CP) End-point Effect

Sharma et al. Anopheles 20.7j£/g/ml CA ++ (1987) mosquito in larvae larvae/ rearing pol]rtene medium chromosomes

Nasr et al. (1988) CHO 10-^ M SCE ++

Rosselli et al. rat/lymphocytes 10 mg/kg CA -H- (1990) to rat.

Amsdorf- rat/ lymphocytes plasma dose SCE -HF Roubicek et al. hum an/ lymphocytes not given SCE +-F (1990)

Miller (1991) human B- and T- 10-30 /zg/ml CA +4- lymphocytes

Oshiro et al. CHO 10/160 jug/ml HGPRT -H- (1991) CHO 5/10/20yug/ml MN > 2 x controls

Monteith et al. Chinese hamster A/79 4/8 M g/m l CA + (1995)

++ = positive results and statistically significant + = positive results but not statistically significant CA = chromosme aberration SCE = sister chromatid exchange MN = micronucleus HGPRT = a specific mutation locus CP = cyclophosphamide CHO = Chinese hamster ovary cells

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 cyclophosphamide at doses of 25, 50, 100, 200, or 300 mg/kg b.w.. Rosselli

et aL (1990) reported finding a significant increase in the firequency of CAs

in the bone marrow cells of rats treated with cyclophosphamide at a dose of

10 mg/kg b.w.. The authors also reported significrant incu^ases in the

frequency of CAs in cultured lymphocytes prepared from rats treated with

a single dose of cyclophosphamide at a dose of 10 mg/kg b.w..

Reimer and Singh (1982) reported finding a significant increase in the

frequency of SCEs in the bone marrow crells of five different strains of mice

treated with a single Lp. injection of cyclophosphamide at a dose of 45 mg/kg

b.w.. Huang, Tan, Sirianni, Pacholec, and Chapman (1990) reported a dose

related increase in the frequency of SCEs in bone marrow cells taken firom

three different strains of miœ treated with cyclophosphamide at doses of 5

or 10 mg/kg b.w..

Jendemy, Walk, Hackenberg, and Rohrman (1988) reported finding

a significant increase in the firequency of SCEs and CAs in bone marrow

cells of miœ afier the mice inhaled cyclophosphamide at doses of 8, 23, or 57

mg/kg b.w. for females, and 21, 49, or 114 mg/kg b.w. for males. The authors

also reported finding a significant increase in the firequency of SCEs and CAs

in bone marrow cells of Chinese hamsters after the am'mal.s inhaled

cyclophosphamide at doses of 30 or 80 mg/kg b.w. by females, and 25 or 40

mg/kg b.w. by males. The authors also reported significant increases in the

frequency of SCEs and CAs in bone marrow cells of mice and Chinese

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30 hamsters that were administered a single i.p. injection of cyclophosphamide

at a dose of 29 or 56 mg/kg b.w. to male mice, 15 or 32 mg/kg b.w. to male

hamsters, 22 or 42 mg/kg b.w. to female mice, and 15 or 32 mg/kg b.w. to

female hamsters.

Krishna , Kropko, Ciaravino, and Theiss (1991) reported finding a

statistically significant increase of micronucleated polychromated

erythrocytes and CAs in bone marrow cells taken firom mice treated with

cyclophosphamide at doses of 20 or 40 mg/kg b.w.. Monteith and Vanstone

(1995)reported an increase in the firequency of MN in bone marrow cells of

mice treated with cyclophosphamide at a dose of 40 mg/kg b.w.. Krishna,

Petrere, Anderson, and Theiss (1995) reported fin d in g a statistically

significant increase in micronucleated polychromated erythrocytes in bone

marrow cells of mice that received cyclophosphamide at doses of 30 or 40

mg/kg b.w..

Krishna et al. (1995b) reported on a dom in a n t lethal (DL) assay in

which male mice were administered cyclophosphamide at a dose of 40 mg/kg

b.w. and then mated with eight different groups of female mice (2 females

per group) over an eight week breeding period. The authors reported that

the total number of mouse embryo implants had been significantly reduced

for matings taking plaœ in weeks 1, 2, 3,6, and 7 of the eight week breeding

period. The authors also reported a significant increase in the number of

mouse embryo implants (dead and resorbed) for breeding weeks 1, 2, and 3.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 Synaptonemal complex (SC) analysis has been used to detect

structural chromosome damage in the germ cells of rodents treated with

cyclophosphamide. Allen, DeWeese, Gibson, Poorman, and Moses, (1987)

reported finding increased frequencies of synaptonemal complex (SC)

damage in the spermatocytes of mice treated with cyclophosphamide at doses

of 30 or 300 mg/kg b.w.. Allen et al. (1988) reported finding a statistically

significant increase in the firequentgr of SC damage in the spermatocytes of

mice treated with cyclophosphamide with a dose as low as 10 mg/kg b.w..

Table 2 presents a summary of the in vivo studies reviewed in this

paper on the mutagenicity of cyclophosphamide.

Extremely Low Frequency Electromagnetic Fields (ELF EMFs)

ELF EMFs: Mutagenicity In Vitro

Nordenson, Mild, Nordstrom, Sweins, and Birke (1984) reported

finding no significant increases in the firequency of CAs in cultured human

lymphocyes exposed to an electric field current density of 1 mA/cm". The

authors also reported finding no significant increases in the firequency of CAs

in cultured lymphocytes exposed to a peak electric field intensity of 2.5 or 3.0

kV/cm created by ten spark discharges, with each spark discharge lasting

3^s in duration. However a significant increase in CAs was found in

cultured lymphocytes exposed to a peak electric field intensity of 3.5 kV/cm

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32 Table 2

Summary of In Vivo Studies on Cyclophosphamide Mutagenicity

Organism / Reference Cell Type Dose(CP) End-point Effect

Reimer et al. 5 strains of mice/ 45mg/kg SCE (1982) bone marrow cells

Rabello-Gay mice/bone marrow 50 mg/kg CA et al. (1985) cells

Allen et al. mice/spermatocytes 30/300 mg/kg SC (1987)

AUen et al. mice/spermatocytes 10 mg/kg SC (1988)

Jendemy et al. mice/bone marrow 8-114 mg/kg SCE (1988) cells CA

Chinese hamster/ 15-80 mg/kg SCE bone marrow cells CA

Huang et al. 3 strains of mice/ 5/10 mg/kg SCE (1990) bone marrow cells

Rosselli et al. rats/bone marrow 10 mg/kg CA (1990) cells

Mcfee et al. mice/bone marrow 18.75/37.5/ CA (1990) cells 75 mg/kg

Krishna et al. mice/bone marrow 20/40 mg/kg MN (1991) cells

Nersessian et Armenian hamsters/ 25/50/100/ CA al. (1992) bone marrow cells 200/300 mg/kg

Monteith et al. mice/bone marrow 40 mg/kg MN (1995) cells

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33 Table 2—Continued

O rganism / Reference Cell Type Dose(CP) End-point Effect

Krishna et al. mice/bone marrow 30/40 MN ++ (1995b) cells mg/kg

mice/embryo 40 mg/kg DL +-F im plants to m ales

= positive results and statistically significant + = positive results but not statistically significant SC = synaptonemal complex DL = dominant lethal CA = chromosome aberration SCE = sister chromatid exchange MN = micronucleus CP = qrclophosphamide

created by ten spark discharges, with each spark discharge lasting 3/us in

duration. d'Ambrosio et al. (1985) reported finding a significant increase in

the fiequenqr of CAs in cultured bovine lymphoqrtes that were exposed to

a 50 Hz electric field with a current density of 2.4 fx A/cm^, for a period of 48

or 72 hours. However, the authors reported finding no significant increase

in the firequency of SCEs in cultured lympho

of the same parameters and for the same period of time.

Rosenthal and Gunter (1989) reported finding no significant increase

in the firequency of CAs or SCEs in cultured human lymphocytes exposed to

a continuous 60-Hz magnetic field intensity of 50 G, for periods of 48 or 72

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34 hours. However, the same authors reported finding a significant increase in

the firequency of SCEs in cultured human lymphocytes that were pretreated

with methylnitrosourea (NMU) or trenimon (TRN) and exposed to a

magnetic field intensity of 75 G, as compared to lymphcxytes pretreated with

NMU or TRN and not exposed to the magnetic field. Khalil and Qassem

(1991) reported finding a statistically significant increase in the firequency

of CAs in cultured h u m an lymphocytes exposed to a 50-Hz pulsed EMF

(PEMF) with a magnetic field intensity of 10.5 G, for a period of 24, 48 or 72

hours. However, the same authors reported that cultured human

lymphocytes under the same exposure conditions, showed no significant

increase in SCEs after 24 or 48 hours of the PEMF exposure, but did show

a significant increase in SCEs after 72 hours of exposure to the PEMF.

Cohen, Kunska, Astemborski, and McCuUoch (1986a) reported on a

study that looked at the firequency of CAs in cultured peripheral

lymphocytes taken firom 10 normal human adults (5 male and 5 females).

The ELF ElMF used in the study had an electric field current density of 30

^cA/cm" with either a 1 or 2 G magnetic field intensity. The ELF EMF

exposure period for the cultured lymphocytes was 69 hours. The authors

reported finding no significant dififerenœ in the number of CAs found in the

exposed cells as compared to the nonexposed œlls. Garcia-Sagredo, Parada,

and Monteagudo (1990) reported finding a significant increase in the

firequency of CAs in cultured human lymphocytes exposed to a PEMF with

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 a magnetic field intensity of 10, 20, or 40 G, for a period of 24 hours, as

compared to the cultured human lymphocytes not exposed to PEMF.

Cohen, Kunska, Astemborski, McCulloch, and Paskewitz (1986b)

reported on a study that looked at the firequenqr of SCEs and CAs in

cultured hum an peripheral lymphoqrtes taken firom 10 norm al adults (5

male and 5 female). The cultures were either exposed, or not exposed, to an

ELF EMF with an electric field current density of 30 txAIcm^ and a magnetic

field intensity of either 1 or 2 G, for a period of 69 hours. The authors

reported finding no significant increase in the firequency of SCEs or CAs in

the exposed cells as compared to the nonexposed cells. The same authors

reported finding no significant increase in CAs in cultured lymphoblastoid

cells taken firom patients having conditions with deleterious genetic

predispositions such as ataxia telangiectasia, Bloom syndrome, Fanconi

anemia, or xeroderma pigmentosum. The cultured lymphoblastoid cells were

exposed to an ELF EMF with an electric field current density of 30 uA/cm~

and a magnetic field intensity of either 1 or 2 G.

Takahashi, Kaneko, Date, and Fukada (1987) reported the results of

two experiments concerned with exposing Chinese hamster V79 cells to

PEM F consisting of 25fx second pulses at a magnetic field intensity of 1.8,

4..4, 6.6, 12, 17, 21, or 25 G. The first experiment exposed V79 cell cultures

to varying magnetic field intensities (1.8-25.0 G) for 24 hours with addition

of 5-bromodeoxy uridine (BrdU) after the PEMF exposure. The second

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 experiment exposed V79 cell cultures to the PEIMF under the same exposure

conditions except the BrdU was added to the cultures prior to the PEMF

exposures. The authors reported fin d in g no significant increase in the

firequency of SCEs of the exposed cells as compared to nonexposed cells in

either of the two experiments. Garcia-Sagredo et al (1990) reported fin d in g

no significant increase in the firequency of SCEs in cultured human

lymphocytes that were exposed to a PEMF with a magnetic field intensity of

10, 20, or 40 G, for a period of 48 hours, as compared to lymphocytes not

exposed to the PEMF.

Livingston, Witt, Gandhi, Cahtterjee, and Roti Roti (1991) reported

finding no significant increase in the firequency of SCEs or MN in cultured

human lymphcxytes or cultured CHO cells exposed to 60-Hz ELF EMF for

72 hours. The ELF EMF consisted of an electric field current density of 3,

30, 300, or 3,000 fxA/cmr, with a constant magnetic field intensity of 2.2 G.

Zwingelberg et al. (1993) reported on an in vivo exposure/in vitro assay in

which female Wistar rats were exposed to a continuous 50-Hz EMF with an

electric field intensity of 50 kV/m and a magnetic field intensity of 300 G, for

24 hours a day over a period of either 7 or 28 days. Cultures were made of

the peripheral lymphcxytes taken firom the rats both prior to ELF EMF

exposure and then again 7 days after exposure to the ELF EMF. The

authors reported finding no significant incnrease in the firequency of SCEs

between the exposed cells and the pre-exposed control cells. Antonopoloulas,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 Yang, Stamm, Heller, and Obe (1995) reported finding no significant

increase in the number of SCEs in cultured human lymphocytes exposed to

50-Hz ELF EMF for periods of 48, 52, 56, 60, 64, or 68 hours as compared to

human lymphocytes not exposed to the ELF EMF for the same periods. The

intensity of the magnetic field component was 5 G and the electric field

induced in the cultures by the magnetic field was determined to be 0.005

V/m.

Reese, Jostes, and Frazier (1988) reported finding no significant

differences in the firequency of DNA single strand breaks (SSB) in cultured

Chinese hamster ovary (CHO) cells that were either exposed or not exposed

to 60-Hz magnetic field intensities of 1 or 20 G, or to electric field intensities

of 1 or 38 V/m, or to a combined magnetic and electric fields of 20 G and 38

V/m respectively, for a period of 1 hour. Fiorani et al. (1992) reported

finding no increase in the firequency of DNA SSB in cultured human tumor

ceU line #562 exposed to 50-Hz electric fields of varying intensities ( 0.2 to

20 KV/m) or to magnetic fields of varying intensities (0.002 to 2 G), or to a

combination of electric and magnetic field of varying intensities, for a period

of 1, 4, 6, 2 or 24 hours. Fairbaim and O'Neill (1994) reported fin d in g no

significant increase in the firequency of DNA SSB in cultured HL-60 cells,

Raji cells, Hela cells, or human lymphocytes that were exposed to a pulsed

(3 msec.) EMF with a magnetic field component of 50 G, for varying periods

of time.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 Fiorio, Morichetti, Vellosi, and Bronzetti (1993) reported finding no

significant increase in the firequency of HGPRT mutants in Chinese hamster

VT9 cell cultures that were exposed to a continuous 50-Hz ELF EMF with a

magnetic field component of 2 G, for periods of 2, 5, 7, or 10 days.

Tabrah, Mower, Batkin, and Greenwood (1994) reported finding no

significant increase in the number of revertant colonies of strain TAIGO cells

( Salmonella typhimurium) that were exposed to ELF EMF for a period of

48 hours. The 60-Hz EJLF EMF had an electric field intensity of 21 mV/cm,

an electric current density of 0.21 ^A/cm~, and a magnetic field intensity of

2 G. However, the same authors did report a significant increase (14%) in

the number of revertant colonies of TAlOO cells that were exposed to the

chemical azide coupled with ELF EMF exposure as compared to TAlOO cells

treated with azide but not exposed to the ELF EMF.

Table 3 presents a summary of the vitro studies reviewed in this

paper on the mutagenicity of ELF EMF.

ELF EMFs: Mutagenicitv In Vivo

The number of in vivo studies that have investigated the mutagenicity

of ELF EMF are relatively few in number compared to the number of in vitro

studies. In regard to in vivo studies, Haider et al. (1994) reported finding a

significant increase in the firequency of MN in mother pollen cells firom the

Tradescantia plant that were exposed to ELF EMF fi-om radio broadcasting

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 Table 3

Summary of In Vitro Studies on ELF EMF Mutagenicity

O rganism / Exp. End- Reference Cell Type Time ELFEMF point Effect

Nordenson et al. hum an/ (10)3 fxs (EQ 1 mA/cm^ CA (1984) lymphocytes sparks OR 11 (EF) 2.5 kV/cm CA tt (EF) 3.0 kV/cm CA rt (EF) 3.5 kV/cm CA -H-

d'Ambrosio et al. bovine/ 48/72 h (EC) 2.4 txAIcvo? CA -H- (1985) lymphocytes SCE

Cohen et al. hum an/ 69 h (EC) 30ixA/cnsr CA - (1986a) lymphocytes (MF) lo r 2 G

Cohen et al. hum an/ 69 h (EC) 30^A/cm" SCE - (1986b) lymphocytes (MF) 1 or 2 G

T akahashi et al. Chinese PEMF (1987) hamster/V79 24 h 25 fÀS puises (MF) 1.8-25 G SCE

Reese et al. CHO I h (EF) 1 or 38 V/m (1988) (MF) 1 or 20 G SSB

Rosenthal et al. hum an/ 48/72 h (MF) 50 G CA (1989) lymphoqrtes OR SCE (MF) 75 G & MNU or TRN SCE -H-

Garcia-Sagredo hum an/ 48 h PEMF et al. (1990) l]nnpho(ytes 26 fxs puises (MF) 10/20/40 G SCE

Garcia-Sagredo hum an/ 24 h PEMF et al. (1991) lymphocytes 26 jUS puises (MF) 10/20/40 G CA ++

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 Table 3—Continued

O rgan ism / Exp. End- Reference Cell Type Time ELFEMF point Effect

Khalil et al. hum an/ 24/48/or PEMF-10 ms (1991) lymphocytes 72 h pulses (MF) 10.5 G SCE

Livingston et al. hum an/ (1991) lymphocytes 72 h (MF) 2.2 G or CHO (EC) 3/30/300/ SCE or 3000 ^A/cm“ MN

Fiorani et al. hum an/ 1/4/6/ (EF) 0.2-20 kV/m (1992) tumor cell or 24 h (MF) 0.002-2 G SSB line #562

Fiorio et al. Chinese 2/5/7/or (MF) 2G HGPRT _ (1993) hamster/ V79 10 days

Fairbaim et al. HL-60 cells, 24 h (MF) 50 G SSB (1994) Raji cells, Hela cells, or human lymphocytes

Tabrah et al. Salmonella 48 h (EF) 21 mV/cm revert. _ (1994) typhimurium/ (EC) 0.21 (xAJcax-‘ colonies TA 100 cells

= positive results and statistically significant = negative results; no difference between treated and controls CA = chromosome aberrations SCE = sister chromatid exchange MN = micronucleus HGPRT = a specific mutation locus SSB = single strand breaks (EF) = electric field intensity (EC) = electric current density (MF) = magnetic field intensity MNU = methyInitrosourea; TRN = trenimon

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 antennae. Tradescantia plant cuttings with flower buds were placed at

various distances away from five different radio broadcasting antennae and

exposed to 10 to 21 MHz ELF E!MF composed of varying electric field

intensities of 1 to 170 V/m and varying magnetic field intensities of <0.01 to

0.11 A/m for a period of 30 hours. The authors reported finding a significant

increase in the firequency of MN in the mother pollen cells of the

Tradescantia plant at aU exposure sites near the antennae. Ma, Chu, and

Zu (1992) reported finding a 3-fold increase in the firequenqr of MN in

mother pollen cells of the Tradescantia plant when plant cuttings with

flower buds were placed inside a Helmholtz coil and exposed to a magnetic

field intensity of 10 G for a period of 6 hours.

Nahas and Oraby (1989) reported finding a significant increase in the

number of MN in polychromatic erythrocytes taken firom male Swiss mice

exposed to a 50-Hz EMF electric field intensity of 100, 170, 200 or 290 kV/m

for a 24 hour period. The electric field intensity of 100 kV/m gave a slightly

elevated but statistically insignificant increase in the number of MN over

that of the controls. However, the electric field intensities of 170, 220, or 290

kV/m caused a significant increase in the firequenty^ of M N in the

polychromated erythrocytes.

Bauchinger, Hauf, Schmid, and Dresp (1981) reported on the

frequency of CAs and SCEs in cultured human peripheral lymphocytes

prepared firom a group of 32 healthy male workers who had been

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42 occupationally exposed to 50-Hz magnetic and electric fields. The control

peripheral human lymphocytes were prepared fix>m a second group of 22

healthy male workers who had not been occupationally exposed to EMFs.

The authors reported finding no significant differences in the firequency of

CAs or SCEs in the lymphocytes of the adult workers occupationally exposed

to the EMFs as compared to the lymphocytes of the nonexposed workers.

Nordenson et aL (1984) reported finding no significant increase in the

frequency of CAs in cultured human lymphocytes prepared fi-om 20 men

exposed to high levels of BIMF while working in a 400-kV electric switchyard

as compared to a group of 17 men who had not been exposed to high levels

of EMF.

Diebolt (1978) reported finding no significant increase in the

firequency of sex-linked recessive lethal mutations in the progeny resulting

fi*om the mating of ELF EMF exposed male firuit flies (Drosophila

melanogaster) to nonexposed female firuit flies. The male firuit flies were

exposed to an electric field intensity of 0.3 kV/cm or to a magnetic field

intensity of 9,226 G for a 24 hour period, and then mated to a nonexposed

female fly.

Table 4 presents a summary of the in vivo studies reviewed in this

paper on the mutagenicity of ELF EMF.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 Table 4

Summary of In \^vo Studies on ELF EMF Mutagenicity

Organism/ Eîxp. End- Reference Cell Type Time ELFEMF point Effect

Diebolt et al. Fruit fiies: 24 h (MF) 9,226 G recessive _ (1978) Drosophila (EF) 0.3 kV/cm lethal melanogaster

Bauchinger et al. human/ > 20 yrs. MF and EF @ SCE — (1981) lymphocytes 380 kV switch CA yard

Nordenson et al. human/ 1-8 wks MF and EF @ CA ++ (1984) lymphocytes 400 kV switch yard

Nahas et al. mice/ 24 h (EF) 170/200/ MN ++ (1989) polychromatic or 290 kV/m erythrcxytes

Ma et al. Tradescantia 6 h (MF) 10 G MN 3 fold (1992) plant/ pollen increase cells

Haider et al. Tradescantia 30 h (EF) 1-170 V/m MN ++ (1994) plant/ pollen (MF) .01.11 A/m. cells

= positive results and statistically significant ~ = no effect seen; no difference between treated and controls (MF) = m agnetic field intensity (EF) = electric field intensity SCE = sister chromatid exchange MN = micronucleus CA = chromosome aberration

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44 Replication of Cells

Effects of CyclophosphaTniHe on Cell Replication

It is now well understood that due to the drastic genotoxic effects that

mutagenic and/or clastogenic agents can have on the chromosomes of cells,

these agents firequently cause cell cycle delay and also frequently cause "cell

death because a great number of mutant cells are apparently incapable of

life" (Novotna et aL, 1990, p342). In support of the above statement, Krishna

et al. (1995b) reported that cyclophosphamide, at doses of 30 or 40 mg/kg

b.w., were cytotoxic to bone marrow tissue of mice as evidenced by

signifrcantly reduced numbers of different bone marrow cell types found in

the bone marrow of the treated mice. Jendemy et al. (1988) reported that

the replication rate of mouse bone marrow cells was significantly reduced

after male and female mice received a single i.p. injection of

cyclophosphamide at a dose of 42 mg/kg b.w. and 56 mg/kg b.w., respectively.

The same authors also reported that the replication rate of Chinese hamster

bone marrow cells was significantly reduced in both sexes after male and

female hamsters received a single i.p. injection of cyclophosphamide at a

dose of 32 mg/kg b.w.. Krishna et al. (1991) reported that the percentage of

rat bone marrow cells found to be in mitosis (mitotic index) was significantly

reduced in male rats that received a single i.p. injection of cyclophosphamide

at a dose of 40 mg/kg b.w., and also in female rats that received a 20 or 40

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 mg/kg b.w. dose of cyclophosphamide.

Novotna et al. (1990) reported that cyclophosphamide administered

to chick embryos at a dose of 3 ^g caused mitotic inhibition in the blood cells

of the embryo, and at a dose of 6jug, caused mitotic inhibition of cells in the

face and Umbs of the embryo. The authors concluded:

This (mitotic) inhibition is probably related, above all, to the mutagenic effect proper. It is assumed that the damage of DNA with S-dependent substances is largely repaired in the course of the Gg phase. This process consists of two steps. The first step is a mitotic delay, and the second one is removal and repair of DNA lesions. The delay of mitosis cam be induced by an arbitrary clastogen. Generally, the greater the clastogenic potential of a substance, the longer the Go phase and consequently the entire cell cycle. (Novotna et al., 1990, p347)

Table 5 presents a summary of the effects of cyclophosphamide on the

replication of cells.

Effects of ELF EMF on CeU Replication

In regards to the effects of ELF EMFs on the rephcaition of cells,

several research studies have reported finding no such effects. Miller et

al.(1976) reported that exposure of Fava bean (Vicia faba) root tips (in

medium) to an electric field intensity of 10 V/m and an electric current

density of 0.1 A/m", or to a magnetic field intensity of 17, 500, or 5000 G for

periods of 1 hour, 1 day, or 2 days, caused no change in the mitotic index of

the rcx)t tip œUs. Inoue et aL (1985) reported that exposure of the Fava bean

(Vicia faba) in medium, to an electric field intensity of 200, 290, or 360 V/m

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46 Table 5

Summary of Effects of Cyclophosphamide on Cell Replication

Organism / Reference Cell Type Dose(CP) End-point Effect

Jendemy et al. mouse/bone 42 or 56 cell replication ++ (1988) marrow cells mg/kg rate

Chinese hamster/ 32 mg/kg cell replication ++ bone m arrow cells rate

Novotna et al. chick embryo/ 3jU gram cell replication + (1990) blood cells rate

chick embryo/ 6a^ gram cell replication + face & limb cells rate

Krishna et al. male rat/bone 40 mg/kg mitotic index ++ (1991) bone marrow cells

female rat/ bone 20 or 40 mitotic index ++ marrow cells mg/kg

Krishna et al. mice/bone 30 or 40 mitotic index ++ (1995) m arrow cells mg/kg

= statistically significant inhibition of cell replication rate/mitotic index + = inhibition of cell replication rate but not statistically significant

caused no change in the mitotic index of the root tip cells. Brulfert et al.

(1985) reported that exposure of pea (Pisum sativum) root tips in medium

to an electric field intensity of 430 V/m and an electric current density of 3

mA/cm^ for a period of 24 or 48 hours, caused no change in the mitotic index

of the root tip cells. Benz et al. (1986) reported that mice exposed to an

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47 electric field intensity of 15 kV/m or 50 kV/m combined with a magnetic field

intensity of 3 or 10 G respectively, for periods of 1 to 28 days, caused no

significant effect on the average length of the cell cycle. Cohen et al. (1986a;

1986b) reported that exposure of cultured human peripheral lymphocytes

(HPLs) to an electric current density of BOfxA/cmr and a magnetic field

intensity of either 1 or 2 G for 69 hours, had no effect on the replication rate

of the exposed cells. Takahashi et al. (1987) reported that exposure of

cultured CHO cells to PEMF with varying magnetic field intensities of 1.8

to 25 G for a 24 hour period, had no significant effect on the mitotic index of

the cultured CHO cells. Livingston et al. (1991) reported that exposure of

cultured HPLs to an electric current density of 3, 30, 300, or 3000 /uA/cmr,

combined with a magnetic field intensity of 2.2 G for a period of 72 hours,

had no effect on the mitotic index of the exposed cells. The same authors also

reported that cultured CHO cells exposed to the same ELF EMF intensities

as the HPLs, showed no significant change in the CHO cell's mitotic index.

Zwingleberg et al. (1993) reported that exposure of rats to an electric field

intensity of 50 kV/m and a magnetic field intensity of 300 G for 24 hours, did

not result in a significant change in either the mitotic index or the

proliferation index in the peripheral lympho(ytes taken firom the a n im a ls.

Fiorani et al. (1992) reported that exposure of cells from the h um an tum or

cell line #562 to varying electric field intensities of 0.2 to 20 kV/m, or to

varying magnetic field intensities of 0.002 to 2.0 G, or to a combination of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 these electric and magnetic intensities, caused no significant change in the

exposed cell's replication rate.

In contrast to the aforementioned studies, there are also a few

research studies that report finding ELF EIMF effects on the mitotic index

and length of the cell cycle of cells exposed to the ELF EMFs. Robertson et

al. (1981) reported that exposure of pea (Pisum sativum) root tips in medium

to electric field intensities of 140 V/m or 490 V/m resulted in a inhibition of

the mitotic index of the root tip cells with the electric field intensity of 490

V/m. Conti et aL (1983) reported that varying magnetic field intensities fi*om

23 to 65 G inhibited the blastogenesis of HPLs stimulated in vitro by the

mitogens PHA and Con A. The authors speculated that since Ca^ are

involved in the control of lymphocyte proliferation, the magnetic fields

somehow alter Ca^ influxes across the membrane of the cells resulting in

inhibition the blastogenesis of HPLs. Brulfert et al. (1985) reported that

exposure of pea (Pisum sativum) root tips in medium to an electric field

intensity of 430 V/m and an electric current density of 3 mA/cm", for 24 or 48

hours, caused the duration of the cell cycle to increase by 10%, which

correlates with a reduction of the normal cell replication rate. Mooney et al.

(1986) reported that the blastogénie response of the mitogen (PHA)

stimulated peripheral blood mononuclear cells, was significantly inhibited

by exposure to a magnetic field intensity of 45 G for a period of 72 hours, but

not for an exposure period of 12 hours. Khalil et al. (1991) reported that

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49 exposure of cultured HPLs to PEMF with, a magnetic field intensity of 10.5

G for periods of 24, 48, or 72 hours, resulted in a significant suppression of

the mitotic index of the cells. In addition, the cell cycle progression was

significantly decreased as a result of the 72 hour exposure period. Conti et

al.(1985) reported that the mitogens PHA and PMA caused an increase in

the influx of Ca*^ into HPLs and the replication rate of the HPLs was

increased. However when the mitogen stimulated HPLs were treated with

verapamil, a well known Ca^ blocker, the influx of Ca^ was reduced and so

was the replication rate of the HPLs. When the mitogen stimulated HPLs

were treated with verapamil and also exposed to a magnetic field intensity

of 60 G, the Ca^ influx and the replication rate of the HPLs were reduced

even further. The authors speculate that the magnetic fields may affect the

movement of CA^ across the HPL plasma membrane resulting in inhibition

of the HPLs replication rate.

Table 6 presents a summary of the effects of ELF EMF on the

replication of cells.

Sum m ary

It is ap parent firom the research studies reviewed in th is p aper th at

cyclophosphamide is highly mutagenic to several different types of cells as

shown in the results of several in vitro and in vivo research studies that

investigated the mutagenicity of the drug. It is also known with certainty

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50 Table 6

Summary of Effects of ELF EMF on Cell Replication

Orga n ism / Reference Cell Type ELFEMF End-point Effect

Miller et al. Fava bean/ (EC^ 0.1 A/m^ mitotic index (1976) root tip cells (EF) 10 V/m OR (MF) 17/500/ mitotic index 500/or 5000 G

Inoue et al. Fava bean/ ^F) 200/290 mitotic index _ (1985) root tip cells or 360 V/m

Brulfert et al. pea/root (EF) 430 V/m mitotic index _ (1985) tip cells (ECÎ) 3 mA/cm"

Benz et al. mice/bone (EF) 15 kV/m cell cycle time _ (Abstract) marrow cells (MF) 3 G (1986) (EF) 50 kV/m œil cycle time _ (MF) 10 G

Cohen et al. human/ (EC) 30yuA/cm" œil replication _ (1986a) lymphocytes (MF) lor 2 G rate (1986b)

T akahashi CHO (MF) 1.8-25 G mitotic index et al. (1987)

Livingston human/ (MF) 2.2 G mitotic index et al.(1991) lymphocytes (EC) 3/30/300/ or CHO or 3000 ixAlcro^

Zwingleberg rat/ (EF) 50 kV/m mitotic index et al.(1993) lymphocytes (MF) 300 G

Fiorani et al. human/tumor (EF)0.2-2 kV/m œil replication (1992) œil line #562 (MF) .002-2.0 G rate

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51 Table 6—Continued

Organism/ Reference Cell Type ELFEMF End-point Effect

Robertson pea/root tips (EF) 140 or mitotic index + et al.(1981) 490 V/m

Conti et al. human/ (MF) 23-65 G blastogenesis + (1983) lymphocytes of lymphcxytes with themitogens PHA or (Don A

Brulfert et al. pea/root tips (EF) 430 kV/m cell cycle time + (1985) (EC) 3 mA/cm"

Conti et al. human/ (MF) 60 G blastogenesis + (1985) lymphocytes of lymphocytes with the mitogens PHA or PMA

Mooney et al. human/ (MF) 45 G blastogenesis of ++ (1986) peripheral PBMC w ith the blood mono mitogen PHA nuclear cells

Khalil et al. human/ (MF) 10.5 G mitotic index -H- (1991) lymphocytes cell cyle time ++

= statistically significant inhibition + = inhibition seen but not statistically significant ” = no effect seen (MF) = magnetic field intensity (EF) = electric field intensity (EC) = electric current density

that qrclophosphamide is carcinogenic to humans and animals (lARC, 1981;

1982; 1987) which is in accord with the latest theories on chromosome

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 damage and the carcinogenic process. It is also known, with some degree of

certainty, that the genotoxicily of cyclophosphamide contributes to an

inhibition of the normal rate of cell replication in the cells exposed to

cyclophosphamide.

In regards to ELF EMF, the vast majority of the in vitro research

studies have reported ELF EMFs not to be mutagenic. However, there are

a small number of in vivo research studies that reported ELF EMFs may be

mutagenic under certain conditions. In addition, as of yet, there are no

controlled research laboratory studies that provide clear, hard evidence that

ELF EMFs are carcinogenic to humans or anim als. In regards to the effects

of ELF EMFs on the replication of cells, while there are a few studies that

report that ELF EMFs are inhibitory to the replication of cells, the largest

number of research studies report that ELF EMFs are not inhibitory to

normal cell replication rates.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER in

DESIGN OF STUDY

Introduction

The present study is concerned with learning more about the

mutagenic and/or clastogenic potential of ELF EMFs. Since carcinogenesis

involves damage to the integrity of chromosomes as a prerequisite to the

formation of transformed cancer cells, it is important to know what

mutagenic and/or clastogenic potential ELF EMFs may have on living cells.

It is also im portant to know if ELF EIMFs can enhance the mutagenic and/or

clastogenic potential of known carcinogens such as cyclophosphamide,

thereby enhancing the carcinogen's potential to initiate carcinogenesis.

Two hypothesis were tested in the present study. The first hypothesis

tested was that mice exposed to a 7.3 G magnetic field would display a

significantly increased firequency of mutagenic and/or clastogenic events in

their bone marrow cells as compared to control animals. The second

hypothesis tested was that mice treated with cyclophosphamide and exposed

to a 7.3 G magnetic field would display a significantly greater number of

mutagenic and/or clastogenic events in their bone marrow cells as œmpared

to mice treated with cyclophosphamide only. 53

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 Experimental Animais

Male and female CD-I strain mice were obtained firom Charles River

Laboratories, Wilmington, Massachuesetts. Upon receipt of the mice, the

animals were housed in plastic cages (11" x 7' x 5") with 6 mice of the same

sex per cage. The animals were acclimated for a period of 16 days prior to

the start of the study. During the acclimatization period, light periodicity

was controlled at 12 hours of artificial light alternated with 12 hours of

darkness. The temperature was thermostatically controlled at 72 degrees

fahrenheit, and thea n im a ls were allowed food and water ad libitum.

During the 24 hour period of exposure to ELF EMF, or during the 24

hour period of sham exposures, six male mice were housed within a single

plastic cage and six female mice were housed within a second plastic cage,

and both plastic cages were positioned, side by side, at the internal center

of the Helmholtz cod. A nim als were allowed food and water ad libitum

during either the ELF EMF or sham exposure periods.

The group of male mice used in the present study had a mean body

mass of 36.0 g, with individual body mass ranging firom 30.0 g to 46.0 g.

The male mice used in the study were 8 weeks of age at the time of

exposures. The group of female an im als used in the study had a mean body

mass of 27.5 g, with individual body mass ranging fi»m 25.0 to 30.0 g. The

female animals used in the study were 10 weeks of age at the time of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 55 exposures. At the time the an im als were ordered from Charles River

Laboratories, the decision was made to use male animals that were two

weeks younger than the female anim als due to the fact that male CD-I mice

will grow more rapidly than the female a n im a ls.

Chemicals Used

Cyclophosphamide was selected for the present study because of it's

known mutagenic, clastogenic, and carcinogenic potential (Povirk and

Shuker, 1994). The cyclophosphamide (No. 21870-7) was obtained firom

Aldrich Chemical Company, Milwaukee, Wisconsin. The drug was dissolved

in isotonic saline (0.9% NaCl) and concentrations of cyclophosphamide

solution were prepared which allowed for doses of the drug to be

adminstered at 5, 25, or 50 mg/kg b.w..

Colchicine was used in the present study because of it's ability to

arrest cells at the metaphase stage of the cell's replication cycle (mitosis).

The cells in the arrested metaphse stage can then be examined under the

light microscope for chromosome damage. The colchicine was obtained firom

the Sigma Chemical Company, St. Louis, Missouri. The drug was dissolved

in isotonic saline (0.9% NaCl) and a concentration of the œlchicine solution

was prepared which allowed for a dose of colchicine to be adminstered at 4

mg /kg b.w..

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56 ELF EMF Exposure Apparatus

The Helmholtz coil used in this study consisted of two horizontal

loops with an equidistant spacing of 13 inches between the two loops. Each

loop measured 26 inches in internal diameter (I.D.) and was constructed

&om a 26 inch (I.D.) a lu m in u m hoop with insulated 14 gauge copper wire

wound cicumferentially around the alu m in u m hoop, 63 times. Each of the

two plastic anim al cages that housed six male or six female mice during the

ELF EMF or sham exposures, had physical dimensions of Il"x7"x5". The

two plastic cages were positioned side-by side within the internal center of

the H elm h o ltz coil and were supported by a wooden stand in which

aluminum nails were used in the construction (see Figure 3).

The electrical circuit that energized the two coil loops consisted of the

following elements: 110 V. alternating current hrom an electrical outlet

flowed into a 145 V. variable-adjust step-up transformer (Staco Variac, No.

GJ901V), and then directly into a step-down transformer (General Electric-

3KVA, No. 9T51B53). From the step-down transformer the current flowed

through a solid state cycle repeat timer(Artisan Controls Company, Model

No. 4610A-6-2-A) and then sequentially through the two coil loops and

returned to the wall outlet. The sohd state <^cle repeat timer pulsed the

electric current through the coil loops in 19 second pulses: Current flowed

through the Helmholtz coil loops for a period of 19 seconds followed by 19

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O t

m :

Figure 3. ELF EMF Exposure Apparatus: Helmholtz Coil.

seconds where there was no flow of electrical current through the coil loops,

with the cycle repeating ad infinitum. An electrical meter (Fluke-45) was

connected in series with the circuit in order to monitor the circuit amperage,

and an electrical meter (Fluke-45) was connected across the circuit to

monitor the circuit voltage (see Appendix A)

The intensity of the magnetic or electric field produced by the

energized Helmholtz coil, was monitored with a meter (Polytek

Manufacturing Incorporated, Scotia, N.Y., Model No. 100-7-8) that had the

capacity to monitor either type of field. During the monitoring of magnetic

field intensities, the meter required the use of a remote monitor probe

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58 attached to the body of the instrument by a cord. The dimensions of the

monitor probe were small enough so as to allow for magnetic field

monitoring at each of the four interior comers as well as the middle region

of each plastic cage. The magnetic field intensity readings at each of the 6

alternative locations within each plastic cage were essentially the same (see

Appendix B). The magnetic field intensities were also found to be essentially

the same inside of either plastic cage, at any single period of time. During

this study, the mice were exposed to a magnetic field intensity of 7.3 G when

the coil was electrically energized, or to an ambient magnetic field intensity

of 0.015 mG when the coil was not energized during sham exposures.

The electric field intensity produced by the energized Helmholtz coil

was monitored using the body of the instrument instead of the monitor

probe. Since the body of the instrument was too large to allow for it to be

placed inside the plastic animal cages, electric field intensity readings were

taken with the instrument resting on top of the plastic cages. For the

present study, when the Helmholtz coil was electrically energized, the meter

recorded an electric field intensity of 17.0 V/m. Through an oversight,

ambient electric field intensities were not recorded when the Helmholtz coil

was not energized. Although not quantified, logic would dictate that the

magnitude of the electric field would be negligible when the Helmholtz cod

is not energized as during sham exposures.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 59 Elxpermiental Design

Ninely-six mice were used in the present study. Mice were assigned

to one of eight different groups and each mouse was assigned a number firom

1 to 96. Each of the 8 groups of mice contained 6 male and 6 female animals.

All an im ais had their body mass measured on a triple beam balance prior to

receiving any intraperitoneal (i.p) .injections and being placed within an

energized or nonenergized Helmholtz coil.

Two groups of mice received a single i.p. injection of vehicle only

(0.9% saline solution without cyclophosphamide); two groups of mice

received a single Lp. injection of cyclophosphamide at a dose of 5 mg/kg b.w.;

two groups of mice received a single i.p. injection of cyclophosphamide at a

dose of 25 mg/kg b.w.; and two groups of mice received a single i.p. injection

of cyclophosphamide at a dose of 50 mg/kg b.w..

One group of mice that received the injection vehicle, one group that

received a 5 mg/kg b.w. dose of cyclophosphamide, one group that received

a 25 mg/kg b.w. dose of cyclophosphamide, and one group that received a 50

mg/kg b.w. dose of qrclophosphamide, were each divided equally by sex and

placed into two Il"x7"x5" plastic cages, with 6 males in one cage and 6

females in the other cage. The two plastic cages containing the animal.*; were

then placed within the energized Helmholtz coil and exposed to a magnetic

field intensity of 7.3 G for a period of 24 hours. This was done sequentially

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 for each, of the four groups of mice (see Table 7).

Each of the remaining four groups of mice that had received either a

single i.p. injection of vehicle or a 5, 25, or 50 mg/kg b.w. dose of

cyclophosphamide, were also divided by sex placing 6 male mice in one

plastic cage and 6 female mice in the other cage. The two plastic cages

containing the animals were then placed within the same nonenergized

Helmholtz coil and the aniniflls were sham exposed for a period of 2 4 hours.

This was done sequentially for each of the four groups of mice (see Table 7).

At 20 hours of ELF EMF or sham exposure, each an im al was briefly

removed from it's plastic cage within the Helmholtz coü, and administered

a single i.p. injection of colchicine at a dose of 4 mg/kg b.w.. The a n im al was

then immediately placed back into it's respective plastic cage within the

the Helmholtz coil and received four more hours of either EMF or sham

exposure.

After spending a total period of 24 hours within either an electrically

energized or nonenergized Helmholtz coil, each anim al was removed from it's

cage, body mass measurements taken on a triple beam balance, and then

sacrificed by cervical dislocation. The mid-torso region of the dead an im al

was then wet down with a 50% methanol solution, and using scissors, an

incision was made through the sk in circumferentially around the mid-torso

region of the animal. The skin of the lower torso was then pulled down the

lower torso until the muscles covering the femur bones were fully exposed.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 61 Table 7

flight Different Treatment Groups of Mice Used in This Study

Numbers Group N um ber Assigned to ELFEMF Num ber of Mice A nim als CP Dose Exposures Sex

1 12 # lto # 1 2 5 mg/ kg b.w. (MF) 7.3 G 6M/6F

2 12 #13 to #24 25 mg/kg b.w. (MF) 7.3 G 6M/6F

3 12 #25 to #36 50 mg/kg b.w. (MF) 7.3 G 6M/6F

4 12 #37 to #48 0 mg/kg b.w. (MF) 7.3 G 6M/6F

5 12 #49 to #60 5 mg/kg b.w. sham exposed 6M/6F

6 12 #61 to #72 25 mg/kg b.w. sham exposed 6M/6F

7 12 #73 to #84 50 mg/kg b.w. sham exposed 6M/6F

8 12 #85 to #96 0 mg/kg b.w. sham exposed 6M/6F

(MF) = magnetic field intensity M= male F = female

With the femurs still attached to the animal, the muscle covering the two

femurs was snipped away with scissors.

Both fem urs were removed firom the an im al carcass by using small

scissors to cut through the cartilage capsule at both articulating ends of the

fem ur. A fter both fem urs were removed firom the anim al, residual muscle

fibers and residual cartilage was carefully removed firom the femur, and the

femur was rubbed clean using a paper tissue. The hard bone articulating

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 62 processes at both ends of the femur were then carefully cut with scissors.

Several, very thin sequential cuts were made through the hardbone

processes until the red colored soft bone would first begin to appear through

the cut end of the bone process.

Using a 26 gauge hypodermic needle attached to a 3 ml syringe filled

with Hanks Buffered Saline Solution (HBSS), both trimmed ends of the

femur were punctured with the needle. Starting at one end of the femur,

pressure was apphed to the syringe until it's needle tip had passed through

the denser region of the soft bone and entered the medulary canal of the

femur. The needle was then retracted fix>m the femm and the same

procedure was performed at the second end of the femur. However, the

needle was not retracted firom second end of the femur until the bone marrow

cells had been flushed out the opposite punctured end of the femur using 3

ml. of HBSS under the pressure of the syringe plunger.

Slide Preparation

The slides used for scoring chromosome/chromatid aberrations were

prepared in the following manner:

1. Cells flushed firom femurs with HBSS, are centrifuged at 1000

revolutions per minute (RPMs) for 10 minutes.

2. Pipette off excess HBSS leaving a small volume of HBSS over the

pelletized cells.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 63 3. Resuspend cells by vigorously tapping bottom of centrifuge tube.

4. Add 7 ml of 0.65% KCl, at 37® Celsius, to tube with cells.

5. Place tube with cells in 37® Celsius water bath for 15 minutes.

6. Centrifuge tube with cells at 1000 RPM for 10 minutes.

7. Pipette off supernatant leaving a small volume of 0.65% KCl

covering the cell pellet. Try not to disturb the cell pellet when pipeting off

the 0.65% KCl.

8. Resuspend cells by vigorously tapping bottom of tube.

9. Add 3 ml of fresh cell fixative (3 parts Absolute Methanol and 1

part Glacial Acetic Acid). Invert tube 3 times.

10. Centrifuge tube and cells at 1000 RPM for 10 minutes.

11. Pippette off supernatant leaving a small amount of fixative

covering the cell pellet.

12. Resuspend cells by vigorously tapping bottom of centrifuge tube.

Cell suspension should be visibly turbid, but not a milky white. If milky

white, add a little more fixative.

13. Pull up turbid cell suspension into a pipette.

14 At arms length distance away from a slide previously chilled in the

refrigerator, drop three drops of cell suspension along the length of the sHde.

BE SURE TO WRITE ASSIGNED ANIMAL NUMBER ON SLIDE WITH

PENCIL.

15. Place slides under flame to bum off alcohol in fixative.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64 16. Dry slides for 48 hours.

17. Immerse slides in Giemsa staining solution for 5 minutes.

18. Rinse slides in tap water tray by 10 up and down movements of the

slide tray.

19. Allow slides to dry a n d coverslip slides w hen dry.

Scoring Chromosome Aberrations

After preparation of the Giemsa stained metaphase chromosome

spreads on glass slides, the a n im al number that had been assigned each

animal and written on each slide, was covered by non-transparent tape,

and each slide was then assigned a 5 digit random number to ensure a

blind scoring of the slide. Records of assigned animal numbers and

corresponding assigned random numbers were kept by an individual who

was not involved in the scoring of the slides for chromosome damage. Before

scoring any slides, all slides were placed in a slide box in increasing order

of the random numbers to further randomize the selection of slides during

the scoring process.

One hundred suitable metaphase chromosome spreads were scored per

anim al In selecting chromosome spreads judged suitable for scoring, it was

required that the chromosomes were sufficiently spread out so as not to be

compacted together with chromosomes overlapping each other. Another

criteria for selecting suitable chromosome spreads was that the chromosomes

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 65 could not be overly condensed making the chromosomes appear very thick

and short thereby not aUowing for definitive scoring of aberrations. Another

criteria for selecting suitable chromosome spreads was that a spread could

not have staining artifact or other types of debris obscurring the structure

of any of the chromosomes. In addition, in a study such as this, many

chromosome spreads wül not display the normal diploid number of

chromosomes (40 for a mouse), since some of the chromosomes will be lost

firom the spread when the cell bursts and the chromosomes are released to

the surface of the sUde. Recognizing this fact, in this study, a chromosome

spread was scored as normal only when no chromosome aberrations were

found in the spread, and there was a total of at least 37 chromosomes

present in the spread (see Figure 4).

Metaphase chromosome spreads were scored for the following four

different types of chromosome aberrations:

1. Chromatid breaks/chromatid fragments in which there is visible

damage to only one chromatid resulting in a break, a gap, or a displaced

section of chromatid (see Figure 5).

2. Isochromatid breaks /bi-chromatid fragments in which there is

visible damage to both attached chromatids resulting in breaks, gaps, or

displaced sections of both chromatids (see Figure 6).

3. Chromatid exchanges /rearrangements (see Figure 7).

4. Chromosomal rings (see Figure 7).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ob

Figure 4. Normal Chromosome Spread.

Figure 5. Chromatid Break and Chromatid Fragment.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 6. Isochromatid Breaks and Bichromatid Fragments.

Figure 7. Chromosome Exchanges and Chromosome Rings.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 68 Statistical Analysis

Six different endpoints were looked at in the present study: (1)

Number of chromatid breaks/chromatid fragments per 100 cells; (2) number

of isochromatid breaks/bichromatid fragments per 100 cells; (3) number of

exchanges/rearrangements per 100 cells; (4) number of rings per 100 cells;

(5) total number of aberrant cells per 100 cells; and (6) the num ber of highly

damaged (shattered) cells per 100 cells. In this study highly damaged cells

were defined as cells displaying chromosome spreads with greater than 15

chromatid and/or isochromatid breaks/fragments (see Figure 8).

Figure 8. Highly Damaged Cells: > 15 Breaks/ Fragments.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 69 Statistical analysis was performed in order to determine if the

difference in the firequency of the chromosome aberration endpoints that

occurred between selected groups of mice was significant. Among the types

of statistical tests used to test for significance were the Chi-square test,

Fisher's exact test. Student's t-test, and a Conditional Poisson test. In

addition, other testing was done in the context of a logistic or Poisson

regression model with appropriate allowances for overdispersion. In each

instance, the statistical test was selected to be consistent with observed

features of the data, such as very small number of counts or overdispersion.

Significance was determined at probability values less than 5 % (P<0.05).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV

RESULTS

Body Mass Measurements

Table 8 displays the mean body mass measurements of the groups of

male and female mice used in this study. Body mass measurements were

taken prior to each animal receiving an i.p. injection of vehicle or

cyclophosphamide and again after 24 hours of ELF EMF or sham exposure.

Each group of female or male mice that received either a 5, 25, or 50

mg/kg dose of cyclophosphamide showed significant loss of body mass

(P<0.05) as compared to groups of mice of the same sex that received the

same ELF EMF or sham exposure, but had not received any

qrclphosphamide. Of the two groups of female mice that received the

injection vehicle only (0 mg/kg dose of cyclophosphamide) with one group

exposed to ELF EMF and the other group sham exposed, the two groups

showed no significant differences in body mass (P>0.05). Of the two groups

of male mice that received the injection vehicle only (0 mg/kg dose of

cyclophosphamide) with one group exposed to ELF EMF and the other group

sham exposed, the two groups showed no significant differences in body mass

(P>0.05) (see Table 8). 70

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 71 Table 8

Body Mass Measurements

Treatm ent Mean Mass ±S.E.fg) (2-tail) CP EMF No. Sex Initial Final t value Probability

0 - 6 M 34.7±1.2 35.3±1.4 -2.00 0.103

0 - 6 F 26.Q±0.7 26.2±0.7 -0.54 0.611

0 + 6 M 35-7±1.6 35.7±1.5 0.00 1.000

0 + 6 F 27.3±0.S 26.2±0.5 2.15 0.084

5 - 6 M 36.2=fc0.9 33.8±0.8 5.53 0.003“

5 - 6 F 26.7±0.2 25.2±0.3 4.39 0.007“

5 + 6 M 32.7±0.4 27.8±0.6 6.87 0.001“

5 + 6 F 27.8±0.2 24.8±0.3 8.22 0.000“

25 - 6 M 38.7±1.9 35-7±2.3 8.22 0.000“

25 - 6 F 27-8±0.5 25.2±0.9 4.78 0.005“

25 + 6 M 35.2±0.8 33.5±1.0 3.95 0.011“

25 + 6 F 28.3±0.7 26.0±0.7 11.07 0.000“

50 - 6 M 37.8±0.5 35-3±0.6 5.00 0.004“

50 - 6 F 28-7±0.9 26.0±0.7 6.32 0.001“

50 + 6 M 36.8±0.3 34.3±0.8 4.04 0.010“

50 + 6 F 27.7±0.4 25.5±0.6 4.54 0.006“

' Paired t-Test: Significant at P<0.05

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 72 The results of the body mass measurements strongly suggest that

EMF exposure at the field parameters used in this study was not a

significant factor in the weight loss of the a n im a ls, and that animal weight

loss was attributable to treatment of an im als with cyclophosphamide.

Chromosome Aberrations

Table 9 / Figure 9, Table 10 / Figure 10, Table 11 / Figure 11, Table 12

/ Figure 12, Table 13 / Figure 13, and Table 14 / Figure 14, respectively

display the firequency of occurrence of the six endpoints look at in this study:

(1) Number of chromatid breaks (CB) and chromatid fi-agments (OF); (2)

number of isochromatid breaks (IB) and bichromatid fi*agments (BF); (3)

number of exchanges (E) and rearrangements (R); (4) number of rings; (5)

number of highly damaged cells (HDQ whose metaphases displayed greater

than 15 breaks and fiagments; and (6) total number of aberrant metaphases

(TAM). Within each table, the firequenqr of occurrence for the selected

endpoint is displayed separately for the groups of male mice and for the

groups of female mice for each of the eight different treatment scenarios used

in this study.

Table 9 shows a nearly equal fi^quenqr of occurrence for CB/CF

between the groups of male and female mice treated with injection vehicle

only and then either exposed to ELF EMF or sham exposed . For the group

of mice that received a 5 mg/kg b.w. dose of cyclophosphamide and then

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 73 sham exposed, the group of female mice showed a greater number of CB/ CF

with a larger firaction of Animals displaying CB/CF than did the group of

male mice. For the groups of mice that received a 5 mg/kg b.w. dose of

cyclophosphamide and then exposed to ELF EMF, the group of male mice

showed a slightly greater number of CB/CF with a slightly larger fraction

of animals displaying CB/CF than did the group of female mice. For the

groups of mice that received a 25 mg/kg b.w. dose of cyclophosphamide and

then sham exposed, the group of male mice showed a greater number of

CB/CF than did the group of female mice, although both the groups of female

and male mice showed equivalent fractions of animals displaying CB/CF.

For the groups of mice that received a 50 mg/kg b.w. dose of

cyclophosphamide and then either sham exposed or exposed to ELF EMF,

both groups of male mice showed a greater number of CB/CF than did the

the corresponding groups of female mice. However, using a conditional

Poisson test for the groups of mice that received vehicle only, and a Poisson

regression model (adjusting for overdispersion) for the groups of mice that

received qrclophosphamide, there were no statistically significant differences

found in the frequency of CB/CF between the male and female groups of

animals for any of the eight possible treatment scenarios.

Figure 9 shows a graph of the combined frequencies of CB/CF for the

combined male and female groups for each of the eight different possible

treatment scenarios. Numerically, the combined fi%quencies of CB/CF for

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 74 Table 9

Number of Chromatid Breaks (CB) / Chromatid Fragments (CF)

Treatm ent Number of Mice No. Cells No. of M ean No. CP EMF Sex Analyzed (+)CB/CF Analyzed CB/CF per cell

0 - M 6 0 600 3 .005

0 - F 6 1 600 2 .003

0 + M 6 2 600 2 .003

0 + F 6 2 599“ 3 .005

5 - M 6 1 600 1 .002

5 - F 6 5 600 21 .035

5 + M 6 3 600 5 .008

5 + F 6 2 600 2 .003

25 - M 6 6 599“ 123 .205

25 - F 5" 5 500 54 .108

25 + M 4"= 4 400 29 .073

25 + F 5" 5 500 28 .056

50 - M 5" 5 427“ 722 1.691

50 - F 6 6 594“ 459 .773

50 + M 6 6 558“ 672 1.204

50 + F 6 6 595“ 239 .402 “ Highly damaged œlls were not included in cells analyzed for CB/CF Slides from one mouse were not scored due to poor readability of slides Slides from two mice were not scored due to poor readability of slides

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 75

NO. OF CHROMATID BREAKS/ CHROMATID FRAGMENTS ^CP DCR&EMF 140

120

100

ü 80 o o

0: ffl 60

5 25 50 DOSE (mg/kg body weight)

Figure 9. Number of Chromatid Breaks/ Chromatid Fragments.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 76 the combined groups of male/female mice that received either a 5, 25, or 50

mg/kg b.w. dose of qrclophosphamide and sham exposed, is greater than the

combined hequendes of the corresponding male/female groups that received

either a 5, 25, or 50 mg/kg b.w dose of cyclophosphamide and exposed

to ELF EMF.

Data were statistically analysed using a conditional Poisson test for

the combined male/female groups of mice that received vehicle only, and a

Poisson regression model (adjusting for overdispersion) for the combined

male/female groups of mice that received either a 5, 25, or 50 mg/kg b.w. dose

of cyclophosphamide. A statistically significant difference in the number

of CB/CF was found only between the two combined male/female groups that

received a 25 mg/kg b.w. dose of cyclophosphamide and then either sham

exposed or exposed to ELF EIMF (P=0.04).

Table 10 shows that for the groups of mice that reœived a 5 mg/kg

b.w. dose of cyclophosphamide and then sham exposed, the group of female

mice showed a greater number of EB/BF with a larger fraction of an im a ls

displaying IB/BF than did the group of male mice from the same treatment

group. For the groups of mice that received a 25 mg/kg b.w. dose of

cyclophosphamide and then sham exposed, the group of female mice showed

a greater number of IB/BF with a larger fraction of a n im als displaying EB/BF

than did the group of male miœ from the same treatment group. For the

groups of mice that received a 50 mg/kg b.w. dose of cyclophosphamide and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 7 then either sham exposed or exposed to EM F, both groups of male mice

showed a greater number of IB/BF than did the corresponding groups of

female mice. However, using a conditional Poisson test for the groups of

mice that received vehicle only, and a Poisson regression model (adjusting

for overdispersion) for the groups of mice that received qrclophosphamide,

there were no statistically significant differences found in the fiequency of

C B/C F between the male and female groups of a n im a is for any of the eight

possible treatment scenarios.

Figure 10 shows a graph of the combined fiequencies of EB/BF for the

combined male and female groups for each of the eight different possible

treatment scenarios. Numerically, the combined firequencies of EB/BF for the

male/female groups that received either vehicle, or a 5, 25, or 50 mg/kg b.w.

dose of cyclophosphamide and sham exposed, is greater than the combined

frequencies of the corresponding male/female groups that received either

vehicle or a 5, 25, or 50 mg/kg b.w dose of cyclophosphamide and exposed

to ELF EMF.

Data were statistically analysed using a conditional Poisson test for

the combined male/female groups of mice that received vehicle only, and a

Poisson regression model (adjusting for overdispersion) for the combined

male/female groups of mice that received a 5, 25, or 50 mg/kg b.w. dose of

cyclophosphamide. No statistically significant difference in the number of

EB/BF was found between any two of the combined male/female groups that

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 78 T able 10

Number of Isochromatid Breaks QIB) / Bichromatid Fragments (BF)

Treatm ent Number of Mice No. Cells No. of M ean No. CP EM F Sex Analyzed (+)EB/BF Analyzed IB/BF per cell

0 - M 6 0 600 0 .000

0 - F 6 2 600 2 .003

0 + M 6 1 600 1 .002

0 + F 6 0 599= 0 .000

5 - M 6 0 600 0 .000

5 - F 6 2 600 8 .013

5 + M 6 1 600 1 .002

5 + F 6 1 600 1 .002

25 - M 6 2 599= 4 .007

25 - F 5" 3 500 14 .028

25 + M 4" 1 400 1 .003

25 + F 5" 2 500 2 .004

50 - M 5" 4 427= 51 .119

50 - F 6 6 594“ 43 .072

50 + M 6 5 558“ 82 .147

50 + F 6 5 595“ 9 .015

® Highly damaged cells were not included in cells scored for EB/BF ^ Slides from one mouse were not scored due to poor readability of slides ' Slides firom two mice were not scored due to poor readability of slides

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 79

NO. ISOCHROMATID BREAKS/BI-CHROMATID FRAGMENTS

0CP □ CP&EMF 12

c/5 8

LU O O o tr LU QÛ

5 25 50 DOSE (mg/kg body weight)

Figure 10. Number of Isochromatid Breaks/ Bichromatid Fragments.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80 had similarly received either vehicle or a specified dose of cyclophosphamide

and then either sham exposed or exposed to ELF EMF.

Table 11 shows that for the groups of mice that received a 25 mg/kg

b.w. dose of cyclophosphamide and then sham exposed, the group of female

mice showed a slightly greater number of E/R with a slightly larger firaction

of mice displaying E/R than did the group of male mice. For the groups of

mice that received a 50 mg/kg b.w. dose of cyclophosphamide and then either

sham exposed or exposed to EMF, the male animals in both treatment

groups showed a greater number of E/R than did the corresponding female

groups. However, using a conditional Poisson test or a Poisson regression

model (adjusting for overdispersion), no statistically significant differences

were found in the firequency of E/R between the male and female groups of

animals for any of the eight possible treatment scenarios.

Figure 11 shows a graph of the combined fi-equencies of E/R for the

combined male/ female groups for each of the eight different treatment

sœnarios used in the study. Numerically, the combined frequencies of E/R

for the male/female groups that received either a 25, or 50 mg/kg b.w. dose

of cyclophosphamide and sham exposed, is greater than the œmbined

fi-equencies of the corresponding male/female groups that reœived either

a 25, or 50 mg/kg b.w dose of cycdophosphamide and exposed to ELF BIMF.

Data were statistically analysed using a conditional Poisson test or

the Poisson regression model (adjusting for overdispersion) for the combined

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 81 T able 11

Number of Elxchanges (E) / Rearrangements (R)

T reatm ent Num ber of Mice No. Cells No. of M ean No. CP EM F Sex Analyzed (+)E/R Analyzed E/R p er cell

0 - M 6 0 600 0 .000

0 - F 6 0 600 0 .000

0 + M 6 0 600 0 .000

0 + F 6 0 600 0 .000

5 - M 6 0 600 0 .000

5 - F 6 0 600 0 .000

5 + M 6 0 600 0 .000

5 + F 6 0 600 0 .000

25 - M 6 0 600 0 .000

25 - F 5“ 1 500 2 .004

25 + M 4'> 0 400 0 .000

25 + F 5“ 0 500 0 .000

50 - M 5“ 3 500 25 .050

50 - F 6 5 600 8 .013

50 + M 6 4 600 9 .015

50 + F 6 2 600 3 .005

“ Slides from one mouse were not scored due to poor readibility of slides

^ Slides from two mice were not scored due to poor readibility of slides

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 82

NO. OF EXCHANGES / REARRANGEMENTS

□ CP&EMF

3.5

2.5

0.5

DOSE (mg/kg body weight)

Figure 11. Number of Exchanges/Rearrangements.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 83 male/female groups of mice that received either vehicle, or a 5, 25, or 50

mg/kg b.w. dose of cyclophosphamide. No statistically significant difference

in the number of E/R was found between any two of the combined

male/female groups that had similarly received either vehicle or a specified

dose of cyclophosphamide and then either sham exposed or exposed to ELF

EMF.

Table 12 shows that for the groups of mice that received a 50 mg/kg

b.w. dose of cyclophosphamide and then either sham exposed or exposed to

ELF EM F exposed, the groups of male mice in both treatment groups showed

a greater number of chromosome rings than did the corresponding groups of

female mice. The groups of male mice in both treatment groups also show

a larger firaction of animals displaying the rings than do the groups of female

mice in each of the respective treatment groups. However, using a

conditional Poisson test or a Poisson regression model (adjusting for

overdispersion), no statistically significant differences were found in the

firequency of E /R between the male and female groups of an im a ls for any of

the eight possible treatment scenarios.

Figure 12 shows a graph of the combined firequencies of chromosome

rings for the combined groups of male and female mice in each of the eight

different treatment scenarios used in the study. Numerically, the combined

firequencies of rings for the male/female groups that received either a , 25, or

50 mg/kg b.w. dose of cyclophosphamide and sham exposed, is greater than

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 84 Table 12

Number of Rings

Treatm ent Number nf Mice No. Cells No. of M ean No. CP EM F Sex Analyzed (+)Rings Analyzed Rings per cell

0 - M 6 0 600 0 .000

0 - F 6 0 600 0 -000

0 + M 6 0 600 0 .000

0 F 6 0 600 0 .000

5 - M 6 0 600 0 .000

5 - F 6 0 600 0 .000

5 M 6 0 600 0 .000

5 + F 6 0 600 0 .000

25 - M 6 2 600 2 .003

25 - F 5“ 1 500 2 .004

25 + M 4^ 0 400 0 .000

25 + F 5“ 0 500 0 .000

50 - M 5“ 4 500 52 .104

50 - F 6 3 600 19 .032

50 + M 6 5 600 19 .032

50 + F 6 2 600 6 .010

® Slides firom one mouse were not scored due to poor readability of slides

Slides firom two mice were not scored due to poor readability of slides

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 85

NO. OF RINGS

mCP □ CP&EMF

o o

DOSE (mg/kg body weight)

Figure 12. Number of Rings

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 86 the combined frequencies of the corresponding male/female groups that

received either a 25 or 50 mg/kg b.w dose of cyclophosphamide and exposed

to ELF EÎMF.

Data were statistically analysed using a conditional Poisson test or

the Poisson regression model (adjusting for overdispersion) for the combined

male/female groups of mice that received either vehicle or a 5, 25, or 50

mg/kg b.w. dose of cyclophosphamide. No statistically significant difference

in the number of chromosome rings was found between any two of the

combined male/female groups that had similarly received either vehicle or

a specified dose of cyclophosphamide and then either sham exposed or

exposed to ELF EMF.

Table 13 shows that for the groups of mice that received a 50 mg/kg

b.w. dose of cyclophosphamide and then either exposed to EMF or sham

exposed, the groups of male mice in both treatment groups show a greater

number of HDC than do the corresponding groups of female mice in each

treatment group. The groups of male mice in both treatment groups also

show a larger fraction of animals displaying the HDC than do the groups of

female mice in each of the respective treatment groups. Using a Fisher's

exact test, no statistically significant differences were found in the frequency

of HDC between the male and female groups of a n im a is treated with vehicle,

5, or 25 mg/kg dose of cyclophosphamide. However, using a logistic

regression test (adjusting for overdispersion), a signficant difference in the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 87 firequency of HDC was found between the male groups of mice that received

a 50 mg/kg b.w. dose of cyclophosphamide and then either sham exposed or

exposed to ELF EMF, and the corresponding groups of female mice.

Figure 13 shows a graph of the combined firequencies of HDC for the

combined groups of male and female mice for each of the eight different

treatment scenarios. Numerically, the combined firequencies of HDC for the

male/female groups that received a 25 or 50 mg/kg b.w. dose of

cyclophosphamide and sham exposed is greater than the combined

fi*equencies of the corresponding male/female groups that received the same

25 or 50 mg/kg b.w.dose of cyclophosphamide but exposed to ELF EMF.

Statistical analysis was performed using a Fisher's exact test or a

logistic regression model (adjusting for overdispersion), for the combined

male/female groups of miœ that reœived either vehicle or a 5, 25, or 50

mg/kg b.w. dose of cyclophosphamide. No statistically significant difference

in the number of HDC was found between any two of the combined

male/female groups that had similarly reœived either vehicle or a specified

dose of cyclophosphamide and then either sham exposed or exposed to ELF

EMF.

Table 14 shows a nearly equal firequency of oœurrenœ for TAM

between the groups of male mice and groups female mice treated with

injection vehicle only and then either sham exposed or exposed to EMF. For

the group of miœ that reœived a 5 mg/kg b.w. dose of cyclophosphamide and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 88 T ab le 13

Number of Highly Damaged Cells (HDC):>15 Breaks/ Fragments

Treatment Number of Mice Number of Cells % of CP EMF Sex Analyzed (+) HDC Analyzed (+) HDC Cells

0 - M 6 0 600 0 .000

0 - F 6 0 600 0 .000

0 + M 6 0 600 0 .000

0 + F 6 1 600 1 .002

5 - M 6 0 600 0 .000

5 - F 6 0 600 0 .000

5 + M 6 0 600 0 .000

5 + F 6 0 600 0 .000

25 - M 6 1 600 1 .002

25 - F 5“ 0 500 0 .000

25 + M 4" 0 400 0 .000

25 + F 5“ 0 500 0 .000

50 - M 5“ 5 500 73 .146

50 - F 6 2 600 6 .010

50 + M 6 6 600 42 .070

50 + F 6 3 600 5 .008

^ Slides from one mouse were not scored due to poor readability of slides

Slides from two mice were not scored due to poor readability of slides

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 89

HIGHLY DAMAGED CELLS: METAPHASES WITH >15 BREAKS/ FRAGMENTS 0CP DCP&EMF

HI o o o cc HI 00

0 4 5 25 50 DOSE (mg/kg body weight)

Figure 13. Number of Highly Damaged Cells.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 90 sham exposed, the group of female mice showed a greater number of TAM

and had a larger firaction of animals displaying TAM than did the group of

male mice of the same treatment. For the group of mice that received a 5

mg/kg b.w. dose of cyclophosphamide and exposed to ELF EMF, the group

of male animals showed a slightly greater number of TAM. For the group of

mice that received a 25 mg/kg b.w. dose of cyclophosphamide and sham

exposed, the group of male mice showed a greater number of TAM than did

the group of female mice, although both the group of female mice and the

group of male mice showed an equivalent firaction of a n im a ls displaying

TAM. For the group of mice that received a 25 mg/kg b.w. dose of

cyclophosphamide and exposed to EMF, the group of female mice showed a

greater number of TAM than did the group of male mice. For groups of mice

that received a 50 mg/kg b.w. dose of cyclophosphamide and then either

sham exposed or exposed to EMF, the male an im als in both treatment

groups showed a greater number of TAM than did the groups of female mice

in the respective treatment groups. However both the group of female mice

and the group of male mice in each of the two treatment groups showed an

equivalent firaction of animals displaying TAM and had a larger firaction of

animals displaying TAM than did the group of male mice of the same

treatment group. Using a standard Chi-square test, no statistically

significant differences were found in the firequenqr of TAM between the male

and female groups of animals treated with vehicle, or a 5 mg/kg dose of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91 cyclophosphamide. Using a logistic regression test (adjusting for

overdispersion), no significant difference in the frequenqr of TAM was found

between the male groups of mice that received a 25 or 50 mg/kg b.w. dose of

cyclophosphamide.

Figure 14 shows a graph of the combined firequencies of TAM for the

combined groups of male and female mice for each of the eight different

treatment scenarios. Numerically, the combined fiiequencies of TAM for the

male/female groups that received a 25 or 50 mg/kg b.w. dose of

cyclophosphamide and sham exposed is greater than the corresponding

male/female groups that received the same 50 mg/kg b.w.dose of

cyclophosphamide but exposed to ELF EMF.

Statistical analysis was performed using a standard Chi-square test

or logistic regression model (adjusting for overdispersion), for the combined

male/female groups of mice that received either vehicle or a 5, 25, or 50

mg/kg b.w. dose of cyclophosphamide. No significant difference was found

in the number of TAM between the male/female groups that had s im ilarly

received either vehicle or a 5 or 50 mg/kg b.w. dose of cyclophosphamide and

then either sham exposed or exposed to ELF EMF. Using a logistic

regression model (adjusting for overdispersion), a statistically significant

difference in the number of TAM was found between the two combined

male/female groups receiving a 25 mg/kg b.w. dose of cyclophosphamide and

then either being sham exposed or exposed to ELF EMF (P=0.016).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92 T able 14

Total Number of Aberrant Metaphases (TAM)

Treatm ent Number of Mice Number of Cells % of CP EMF Sex Analyzed (+) TAM Analyzed (+) TAM Cells

0 - M 6 2 600 3 .005

0 - F 6 2 600 4 .007

0 + M 6 3 600 3 .005

0 F 6 2 600 4 .007

5 - M 6 1 600 1 .002

5 - F 6 5 600 9 .015

5 + M 6 3 600 6 .010

5 + F 6 3 600 3 .005

25 - M 6 6 600 50 .083

25 - F 5“ 5 500 34 .068

25 + M 4^ 3 400 11 .028

25 + F 5“ 4 500 15 .030

50 - M 5“ 5 500 185 .370

50 - F 6 6 600 130 .217

50 + M 6 6 600 190 .317

50 + F 6 6 600 62 .103

® Slides from one mouse were not scored due to poor readability of slides Slides from two mice were not scored due to poor readability of slides

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 93

NO. OF ABERRANT METAPHASES OF ANY TYPE

mCP GCP&EMF

CO 20

HI o o o 15 Od LU CO

I I - h 0 5 25 50 DOSE (mg/kg body weight)

Figure 14. Number of Cells With Aberrant Metaphases of Any Type.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 94 Sum m ary

The present study reports finding no significant increase in the

firequency of CAs in bone marrow cells of male and female CD-I mice that

were administered vehicle only (0.9% NaCl solution) and exposed to a

magnetic field intensity of 7.3 6 for 24 hours, as compared to male and

female CD-I mice administered vehicle only, but sham exposed for 24 hours.

This study also reports fin d in g no significant increase in the fi%quency

of CAs in bone marrow cells of male and female CD-I mice treated with

cyclophosphamide (dosages of either 5, 25, or 50 mg/kg b.w.) and exposed to

a magnetic field intensity of 7.3 G for 24 hours, as compared to male and

female CD-I mice treated with cyclophosphamide (same dosages), but sham

exposed for 24 hours.

In addition, the results show that in regards to the firequency of

CB/CF (Figure 9) and TAM (Figure 14), the firequency of occurrence was

significantly (P<0.G5) lower in m ale and female CD-I mice treated with

cyclophosphamide at a 25 mg/kg b.w. dose and exposed to 7.3 G magnetic

field intensity, as compared to male and female CD-I mice treated with

cyclophosphamide at a 25 mg/kg dose and sham exposed.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V

DISCUSSION AND CONCLUSION

Mutagenicity of Extremely Low Frequency Electromagnetic Fields

There are a number of epidemiological studies that have reported

positive statistical correlations between exposure to ELF EMFs and the

increased incidence of certain types of cancer (Feychting et al., 1993; Lin et

al., 1994; London et al. 1991; Lovely et al., 1994; Tomenius, 1986;

Wertheimer et al., 1979, 1982). Since carcinogenesis involves the damage

of chromosomes (DNA) as a prerequisite to the formation of transformed

cancer cells, it is very important to know if ELF EMF has mutagenic and/or

clastogenic effects on hving cells. It is also important to know if ELF EMFs

can enhance the mutagenic and/or clastogenic effects of physical or chemical

agents that are known to be mutagenic, clastogenic, and carcinogenic. The

problem addressed in the present study was to determine whether or not

ELF EMFs are mutagenic and/or clastogenic to bone marrow cells of CD-I

mice. The present study was also concerned with determining the abüity of

ELF EMFs to enhance the mutagenic and/or clastogenic effects of

cyclophosphamide, a known mutagen, clastogen, and carcinogen (Povirk and

Shuker, 1994)(lARC, 1981, 1982, 1987), in bone marrow cells of mice. 95

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 96 Although the majority of the research studies suggest that electric

and/or magnetic fields do not cause significant DNA damage, there are a few

studies that suggest that ELF EMFs may cause DNA damage. Table 15 and

Table 16 respectively display the results of the in vitro and in vivo studies

investigating the mutagenicity of ELF EMFs.

The results of the present study are in agreement with the majority

of studies cited in the two tables which report finding no mutagenic and/or

clastogenic effects in cells exposed to ELF EMFs. The present study reports

finding no statistically significant increase in the firequency of CAs in bone

marrow cells of CD-I mice that were administered a single i.p. injection of

vehicle only (0.9% NaCl solution) and exposed to a magnetic field intensity

of 7.3 G for 24 hours, as compared to CD-I mice administered a single i.p.

injection of vehicle, but sham exposed for 24 hours.

This study also reports finding no statistically significant increase in

the fiequency of CAs in bone marrow cells of CD-I mice treated with a single

Lp. injection of (yclophosphamide (dosages of either 5, 25, or 50 mg/kg b.w.)

and exposed to a magnetic field intensity of 7.3 G for 24 hours, as compared

to CD-I mice treated with a single Lp. injection of cyclophosphamide (same

dosages), but sham exposed for 24 hours.

Uniqueness of Study

The present study is unique in that it is one of a very few in vivo

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 97 T able 15

Summary of In Vitro Studies on ELF EMF Mutagenicity

Organism/ Exp. End- Reference Cell Type Time ELFEMF point Effect

Nordenson et ai. hum an/ (10)3 ^ s (EC) 1 mA/cm" CA (1984) lymphocytes sparks OR tf (EF) 2.5 kV/cm CA tt (EF) 3.0 kV/cm CA tl (EF) 3.5 kV/cm CA -H-

d'Ambrosio et al. bovine/ 48/72 h (EC) 2.4 jwA/cm" CA -H- (1985) lymphocytes SCE

Cohen et ai. hum an/ 69 h (EC) 30 fjAJcxnr CA — (1986a) lymphocytes (MF) 1 or 2 G

Cohen et al. hum an/ 69 h (EC) SO^tA/cm" SCE — (1986b) lymphocytes (MF) 1 or 2 G

Takahashi et al. Chinese PEM F (1987) hamster/V79 24 h 25 f^s puises (MF) 1.8-25 G SCE

Reese et al. CHO I h (EF) 1 or 38 V/m

(1988) (MF) 1 or 20 G SSB —

Rosenthal et al. h u m an/ 48/72 h (MF) 50 G CA (1989) lymphocytes OR SCE (MF) 75 G & M N U orT R N SCE ++

Garcia-Sagredo hum an/ 48 h PEM F et al. (1990) lymphcxytes 26 fxs puises (MF) 10/20/40 G SCE

Garcia-Sagredo hum an/ 24 h PEMF et al. (1991) lymphocytes 26 fxs puises (MF) 10/20/40 G CA ++

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 98 Table 15—Continued

Organism / Elxp. End Reference Cell Type Time E L F E M F point Effect

Khalil et al. hum an/ 24/48/or PEM F-10 ms (1991) lymphocytes 72 h pulses ^LfF) 10.5 G SCE

Livingston et al. hum an/ (1991) lymphocytes 72 h (MF) 2.2 G or CHO (EC) 3/30/300/ SCE or 3000 ixAlcar MN

Fiorani et al. hum an/ 1/4/6/ (EF) 0.2-20 kV/m (1992) tumor cell or 24 h (MF) 0.002-2 G SSB line #562

Fiorio et al. Chinese 2/5/7/or (M F )2 G HGPRT _ (1993) ham ster/ V79 10 days

Fairbaim et al. HL-60 cells, 24 h (MF) 50 G SSB (1994) Raji cells, Hela cells, or human lymphocytes

Tabrah et al. Salmonella 4 8 h (EF) 21 mV/cm revert. _ (1994) lyphimurium/ (EC) 0.21 fxAlcm-' colonies TA 100 cells

++ = positive results and statistically significant = negative results; no difference between treated and controls CA = chromosome aberrations SCE = sister chromatid exchange MN = micronucleus HGPRT = a specific mutation locus SSB = single strand breaks (EF) = electric field intensity (EC) = electric current density (MF) = magnetic field intensity MNXJ = methylnitrosourea; TRN = trenimon

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 99 T able 16

Summary of In Vivo Studies on ELF EMF Mutagenicity

O rgan ism / Exp. End Reference Cell Type Time E L F E M F point Effect

Diebolt et al. Fruit fiies; 24 h (MF) 9,226 G recessive _ (1978) Drosophila (EF) 0.3 kV/cm lethal melanogaster

Bauchinger et al. human/ > 20 yrs. MF and EF @ SCE — (1981) lymphocytes 380 kV switch CA y ard

Nordenson et al. h u m a n / 1-8 wks MF and EF @ CA -H- (1984) lymphocytes 400 kV switch y ard

Nahas e t al. mice/ 24 h (EF) 170/200/ MN -H- (1989) polychromatic or 290 kV/m erythrocytes

M a et al. Tradescantia 6 h (MF) 10 G MN 3 fold (1992) plant/ pollen increase cells

Haider et al. Tradescantia 30 h (EF) 1-170 V/m MN -H- (1994) plant/ pollen (MF) .01.11 A/m. cells

= positive results and statistically significant “ = no effect seen; no difference between treated and controls (MF) = magnetic field intensity (EF) = electric field intensity SCE = sister chromatid exchange MN = micronucleus CA = chromosome aberration

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 studies (see Table 16) that has looked at the mutagenic and/or clastogenic

effects of ELF EMFs on research animals, as compared to a large number of

in vitro studies that have investigated the mutagenicity of ELF EMFs on

cultured cells.

The present study is also unique in that at the time it was performed,

this investigator found no other study cited in the literature which

attempted to look at the possible synergistic effects of ELF EMFs with a

known clastogen/mutagen/carcinogen, which in the present study was the

clinical drug cyclophosphamide.

Conclusion

The first hypothesis tested in the present study was that exposure of

mice to a 7.3 6 magnetic field for 24 hours, would significantly increase the

firequency of clastogenic and/or mutagenic events in the bone marrow cells

of the mice exposed to the magnetic field. The results show there was not a

statistically significant increase in the firequency of CAs in bone marrow cells

of CD-I mice administered the vehicle and exposed to a 7.3 G magnetic field

for 24 hours, as compared to CD-I mice administered the vehicle but sham

exposed for 24 hours. The results failed to support the hypothesis.

The second hypothesis tested in this study was that exposure of mice

treated with cyclophosphamide and then exposed to a 7.3 G magnetic field

for 24 hours would significantly increase the clastogenic and/or mutagenic

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 101 effects of cyclophosphamide in the bone marrow cells of mice. The results

show there was not a statistically significant increase in the fi-equency of

CAs in bone marrow cells of CD-I mice treated with qrclophosphamide

(dosages of either 5, 25, or 50 mg/kg b.w.) and exposed to a 7.3 G magnetic

field for 24 hours, as compared to CD-I mice treated with a single i.p.

injection of q^clophosphamide (same dosages), but sham exposed for 24

hours. The results failed to support the hypothesis that the ELF EMF would

increase the mutagenicily and clastogenidty of cyclophosphamide and hence

may contribute to the initiation of cancer by cyclophosphamide.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix A

Layout of ELF EMF Exposure Hardware

102

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 103

110V/AC

Q O POWER 0 a SUPPLY

s e STEP DOWN

ISFORMER

TIMER RELAY 110V/AC

VOLT METER

110V/AC

AMP METER

COIL

Figure 15. Layout of ELF EMF Exposure Hardware.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix B

Monitoring Positions of Gauss Detector in Animal Cage

104

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 105

Monitoring Positions of Gauss Detector in Animal Cage.

Gauss Readings at Six Positions in Animal Cage

Voltage Am perage Gauss Readings at Six Positions in A nim al Cag to C oil to Coil Pos.l Pos. 2 Pos. 3 Pos. 4 Pos. 5 Pos. 6

7.35 V 0.85 A 1.6 G 1.6 G 1.6 G 1.6 G 1.6 G 1.6 G

12.23 V 1.52 A 2.6 G 2.6 G 2.6 G 2.6 G 2.6 G 2.6 G

14.72 V 1.82 A 3.2 G 3.2 G 3.2 G 3.2 G 3.2 G 3.2 G

17.07 V 2.13 A 3.7 G 3.7 G 3.7 G 3.7 G 3.7 G 3.7 G

19.52 V 2.42 A 4.2 G 4.2 G 4.2 G 4.2 G 4.2 G 4.2 G

21.67 V 2.67 A 4.7 G 4.7 G 4.7 G 4.7 G 4.7 G 4.7 G

24.11 V 2.98 A 5.2 G 5.2 G 5.3 G 5.2 G 5.2 G 5.2 G

26.52 V 3.27 A 5.8 G 5.8 G 5.8 G 5.8 G 5.8 G 5.8 G

29.10 V 3.60 A 6.4 G 6.4 G 6.4 G 6.4 G 6.4 G 6.4 G

31.40 V 3.88 A 6.9 G 6.9 G 6.9 G 6.9 G 6.9 G 6.9 G

32.88 V 4.05 A 7.3 G 7.3 G 7.3 G 7.3 G 7.3 G 7.3 G (Max.) (Max.)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix C

Institutional Animal Care and Use Committee Form

106

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lA C ü C Number; f y - o - c ( 107 Date of Receipt: y Date of Approval: J . / 7 WESTERN MICHIGAN UNIVERSITY INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (lACUC)

Application to use Vertebrate Animals for Research or Teaching

The use of any vertebrate ««innak in research and/or teaching without prior approval of the Institutional Animal Care and Use Committee (lACUC) is a violation of Western hBchigan Universi^ policies and procedures. This rnmmittee is charged with the institutional responsibility for assuring the appropriate care and treatment of vertebrate animals.

Mail the signed original and five (5) copies of the typed application and any supplements to Research and Sponsored Program, Room A-221 Ellsworth hall, (616) 387-3670.

Any application that includes use of hazardous materials, chemicals, radioisotopes or biohazards must be arffpmpnniiwi with SUPPLEMENT A. (Radiation will be at another organization than WMU. The mice will not contain hazardous material.)

Any application that survival surgery must be accompanied with SUPPLEMENT B.

Principal Investigator/

Signature: Date: ~ / / ~ 9 ^

Department: Binlngical Sciences ______Campus Phone: (6161 387-5630

Responsible Faculty Member: Ovuia Ficaor ______

(if PI not Acuity member)

Signature: ______'' Date:

Department: Biological Sciencm ______Campus Phone: (616) 387- 5630

Title of Project/Course:

Check one: Teaching: Research: JL O ther:__

L ANIMAL USE CATEGORIES (check ONLY one category)

A. ___ Projects that involve little or not discomfort Gnduding iigections).

B. ___ Projects that may result in somAcomlort or pain, but or short duration. Anesthetics, analgesics or tranquilizers will be used.

C. ___ Projects that may result in significant discomfort or pain. Anesthetics, analgesics, or tranquilizers will not be used.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 108 n. ANIMAL USE FACfLITlES

The annnals(s) wfll be housed and maintained in accordance with the WMU Human Gire and Use of Animals Policies and Procedures.

Yes_]L_ N o _____

If no, give explanation.

Please indicate the building and roomfs) where the anhnals(s) will be housed and cared for as well as the location of the experiments and procedures if different from where housed.

The mice will housed in the approved McCiaclcea Hall mu 'im I rooms following the rules and procedures of said animal focility. Same sex mice will be housed in mouse breeding cages not exceeding 6 mice per cage up to one year.

m . ANIMAL USE SUMMARY

In language understandable to a layperson, summarize your primary aims and describe the proposed use of animals as concisely as possible. Bmr in mind that the lACUC is primarily interested in the responsible, necessary, humane use of animals. Include a description of procedures designed to assure that discomfort and pain to anhnals will be minimized, ft should include method of restraint; method of dosing with test compound; and methods of euthanasia or disiinsition of the animal after the experiment.

Groups o f 12 mice (6 6 ^ will be exposed to cyclophosphamide via single i.p. injection at Omg/kg b.wt.. S mg/kg. b.w t., and SOmg/kg b.wt. doses'-^. The animals (housed in their own cage @ 6 mice per cage) will be placed within a Helmholtz coil that generates non-ionizing radiation. The animals will remain within the electrically energized coil for 24 hours, give food and water ad libitum. Excqtt for the single i.p. injection the mice should suffer no discomfort. The non-ionizing radiation will not be folL At 24 hours post treatment, the anim als will be sacrificed by cervical dislocation. Both femurs will be surgically removed feom dead animals and used for preparation of bone-marrow slides for scoring of bone marrow cell aberrations.

' Nersessian, A. K., et al.. Comparative investigation of cyclophosphamide action on bone marrow cells of the Armenian hamster and 4 other species of rodents. Mntatinn Res.. 268;211-21S. 1992. 2 Nazareth Rabello-Gay, M. et al.. The effects of age, sex and diet on the clastogenic action of cyclophosphamide in mouse bone marrow. Mutation Res.. 158:181-188. 1985.

IV. JUSTIFICATION FOR ALL ANIMAL EXPERIMENTS

Please provide a narrative with reference sources which addresses each of the following:

A. What assurance can be provided to indicate that the procedure is not duplicative?

To date, this investigator has found no refistence in the literature that has investigated synergistic effects of cyclophosphamide jqj I non-ionizing radiation.

B. Have non-live animal techniques (e.g. in vitro biological systems, computer simulation, audiovisual demonstration) been considered? Explain why they have not been mHI»»»!-

Humans are exposed to chemotherapeutic dosages of cyclophosphamide, and also to strong magnetic fields at home and work. Since die nature of the reqxmses is unknown in-vivo, it is impossible to design an in-vitro or computer simulation experiment. The only sensible experimental system is an in-vivo animal model which we propose.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 109

C. Why bas this spades been selected for this procedure: For two reasons: 1) mice are traditionally used in genetic experiments, and 2) they will more easily fit into the Helmholtz radiation coil.

D. How many anhnals will he used in this project? How often wfll its procedures he done and over what duration? V» In total M mice will be needed: (12 @ Omg/kg cycio + 8 Gauss (G) EMF) (12 ® Smg/kg cyclo + 8 G EMF) (12 @ 50 mg/kg cyclo + 8 G EMF) (12 ® 0 mg/kg cyclo) (12 ® 5mg/kg cyclo) ( 12 ® SOmg/kg cyclo) for the main experiment, and two mice for each of the six aforementioned groups for a pilot experiment.

E. In light of concern to minimize the number of animals used in experimentation, how will you determine the number of a n im a k to he used?

We chose the minimum of 6 animals of each sex per dose to assure sufficient statistical power for each of the six experimental groups.

NOTE: Items, F, G. H and I require the approval of the Consulting Veterirmrian

F. What is the antidpated pain or distress response of the animals; and what is the duration of discomfort? (Lyections not induded).

Except for the single i.p. injection with a 25 gauge syringe needle, the mice should suffer no discomfort during the 24 hour treatment period (Personal communication, Michael Higgins, The Upjohn Co.). L.C. Becker et al.. (Mutation Res.. 203:317-330.1988) reported on the results of a study where mice were injected i.p. with 100 mg/kg cyclophosphamide and sacrificed the mice 12 days later with no reported ill effects within this time period. This dose is twice as high and the duration of exposure is 12 times longer than in our study.

G. How will the pain in the animal he monitored?

Mice will be monitored within their cage during treatment period of cyclophosphamide and/or non-ionizing radiation. No overt toxicity shown at these doses and time period. Qper mike Higgins ® Upjohn)

H. What sedative, analgesic, or anesthetics will he used, if any? Include dose, route and firequency of administration.

None is necessary

I. What is the justification if pain relieving drugs are not used?

We expect to see no pain experienced by mimmls except for injection

Signature: (Consulting Veterinarian Date

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 110

WESTERN MICHIGAN UNIVERSITY INVESTIGATOR lACUC CERTIFICATE

Title of Project: Induction of Chromosome Aberrations in Bone Marrow Cell? of Mlçe by CvclODhosphflmide and/or TVon-knizingElectromagnerir Rfldiatfnn.

The infomation included In this lACUC application Is accurate to the hest of my knowledge. All personnel listed recognize their responsibility in complying with uniTcrsity policies governing the care and use of anhnals.

I declare that all experiments involving live animals will be performed under by supervision or that of another qualifîed scientist. Technicians or students involved have been trained in proper procedures in animal handling, administration of anesthetics, analgesics, and euthanasia to be used in the project.

If this project is funded by an extramural source, I certify that this application accurately reflects all procedures involving laboratory animal subjects described in the proposal to the funding ageoty noted above.

Any proposed revision to or variations flrom the animal care and use data will be promptly forwarded to the lACUC for approval

Disapproved ^/^proved Approved with the provisions listed below

Provisions or Explanation

______lACUC Chairperson Date: / j L ~ ^ ~

Acceptance FTovisionsy

Signature: Principal Ihvestigator/Listructor Date Date

lACUC Chairperson Final Approval Date

Approved lACUC Nmnber .P- - c l ______

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BIBLIOGRAPHY

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I l l

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