Yamanashi Med. J. 8(I), 9--l3, 1993
Effects ofStatic Magnetic Fields on the Induction ofSister Chromatid
Exchange in Human Lymphocytes in vitro
Sumio IuiMA, Zentaro YAMAGATA, Toshimi OoMA, Tatsuya TAKEsmTAi), Makoto HiGuRAsm2), aRd Akio AsAKA
Dopartment ofHealth Sciences, Yamanashi Medical Universdy, Tamaho, Nakahoma, Yamanashi 409-38, i)Dopartment ofHlygtene and Preventive Medicine, School ofMedicine, Osaka Universdy, Kita-ku, Osaka 53 0, and 2)Dopartment ofMaternal and Child Health, Facudy ofMedicine, The Universdy ofTodyo, Bttndyo-hu, Toltyo I13,JaPan
Abstract: The effects of static magRetic fields (MF) on the induction of sister chromatid exchange (SCE) were examined in human lymphocytes using whole blood culture. The SCE frequency in the group unexposed to MF was 9.23±O.08. IR the groups exposed to O.4 and O.8 T of MF, the SCE frequency was 9.88±e.38, 8.93±O.64, respectively. There were no significant differences between unexposed and exposed groups. Analysis ofthe cell cycle kinetics revealed no
marked differeltces between unexposed and exposed groups.
The SCE frequency increased dose-dependently by the treatment with the known rnutagen ethylmethanesulfonate (EMS)･, there being Ro significant difference between MF exposed and unexposed groups. There was alse no difference between MF-exposed and MF-unexposed groups in the SCE frequency in the control group without EMS. These findings were in good accordance with the prevlous reports and suggest that static MF could not induce SCE.
Key words: Magnetic fields, Sister chromatid exchange (SCE), Hurnan lymphocyte, methanesulfonate (EMS), Cell cycle klRetics
INTRODUCTION
There is increasing public concern about the biological effects of electric fields andlor magnetic fields (EMF). EMF has been reported te be carcinogenic in several reportsimii) but not carcinogenic in other repertsi2-i4); the results of epidemiological studies on EMF and cancer are still controversial. Sister chromatid exchange (SCE) is regarded as a sensitive indicator of mutagenic carcinegensi5-i9).
There are several reports efi the induction of SCE using either static magnetic fields (MF) or time-varying EMF. Static MF did not iRduce SCE in spite of very high intensities of MF: up
to several teslas20-23). IR several studies,
time-varying EMF did not iRduce SCE24-29) and
recently in a few studies it has been reported to induce SCESO･3i).
Rosenthal and Obe32) reported the induc-tion of a significantly higher frequency of SCE in human peripheral lymphocytes pretreated
with known chemical mutagens in the presence of EMF when compared to cells cultivated in
the absence of EMF.
In the present study, we examined the
dose-response relationship of the effects of static MF on SCE induction in human
lympho-cy￡es (Experiment 1.) and their effect in
combination with a known mutagen,
ethyl-methanesulfonate (EMS) (Experiment 2.). ReceivedAccepted
February 26, 1993 March l6, 1993
MATERIALS AND METHODS
Euperiment 1
Heparinized peripheral blood was drawn
from three healthy donors. Whole blood fromeach donor (O.3 ml) was exposed to O.4 and O.8
T of MF for 7 hours using aR electromagnet
(Tohoku Kinzeku Co Ltd; SEE-9 model).
Immediately after the exposure, each blood
specimen was added to 5 ml RPMI1640
medium (GIBCO) containing 15% fetal bovine
serum (GIBCO) and S% phytohemagglutinin
M (GIBCO). The medium also contained 40
paM bromodeoxyuridine (Sigma) for the entire culture period. The cultures were incubated at
370C for 72 hours in complete darkness. Six
hours before fixation colcemid (fina} concen-tration, 2×1Oww7 M; WAKO) was added to each culture. The cells were collected by centrifuga-tien, then exposed to O.075 M KCI hypotonic solution for 8 min and fixed 3 times in an
'
ethanol;acetic acid (3:1) solution. Air-dried
chremosome preparations were made, and a
modification of the fluerescence-plus-Giemsa
method was applied to obtain harlequin
chro [}osemes33). Cells dividing for the first, second, and third or more times in culture can be determined in such preparations34)35).
One hundred metaphase cells were scored for cell cycle kinetics, and 8e consecutive second-division metaphases were scored for SCEs per point per person.
Esperiment 2
Heparinized peripheral blood was drawn
from a healthy donor. The whele blood wasadded to RPMI1640 medium (GIBCO)
con-taining 15% fetal bovine serum (GIBCO) and
3% phytohemagglutinin M (GIBCO). The
medium also contained 40 paM
bromodeoxy-uridine (Sigma) for the entire culture period. The medium was poured into a silicen culture dish with four wells (O.7 ml) and ethyimethane-sulfonate (EMS) was added at the concentra-tion of O, 25, 50 and 10e uglml. This dish was
put in the middle of the magnetic field of
O.985T permanent magnet and the magnet
was introduced into a COg incubator. Then the cultures were incubated at 370C for 72 hours in complete darkness. Six hours before fixation,
celcemid (final concentration, 2xlOM7 M;
WAKO) was added to each culture.
Chromo-some preparations were made and stained bythe same technique as described for
Experi-ment l.
REsuLTs
Esperiment 1
The SCE frequency in the unexposed group
was 9.23±O.08. In groups treated with O.4 and
e.8T of MF, the SCE frequency was 9.88±
O.38, 8.93±O.64, respectively. There were ito significant differences between unexposed and exposed groups (Student's t test, Table 1). No
dose-response relationships were observed,
Table 1. The frequencies of SCEs in human lymphocytes exposed to magnetic
fields SCEs1cell") o lntensMes of magnetic
OA
fields O.8 (T) 9.10 ± O.55 9.23 ± O.57 9.36 ± O.60 10.14 ± O.65 9.l4 ± l.29 10.36 ± O.53 7.66 9.6} 9.52 ± ± ± O.50 O.74 O.60Average 9.23±O.08
") Data are expressed as mean±S.E.9.88 ± with data
e.38
shown for each 8.93 ± donor.
Effect of magnetic fields on SCE ll
Table 2. Cell cycle kinetics in human lymphocytes exposed to magnetic
fields
cell cycle kiRetics") (%)
e
intensi￡ies of magnetic fields
e.4 O.8 (T) 1 2 3 X}
X2
X3+
XlX2
X3+
X}X2
X8+
l8 32 se l6 25 59 ll 35 54 8 so 62 22 29 49 16 32 52 19 34 47 16 26 58 16 22 62 a)Data second are expressed as (X2), and thirdpercentages of cells dividing for the or more (X3+) time in culture.
first (Xl),
Table 3. The frequencies of SCEs in human lym-phocy{es exposed to EMS with or without magnetic fields
in frequency of SCE, there were no significant
differences between the MF-exposed groups
and unexposed groups.
SCEs/cella)
EMS
(ptglml) magnetlc (-) fields (O.985T) (+)e
25 50 1eo 7.74 10.43 I5.40 l8.75 ± O.59 ± O.69 ± O.94 ! 2.i6 8.82 14.25 16.oe 20.80 ± 1.02 ± 2.ll ± 1.49 ± o.ge DIscuSSIoNa)Data are expressed as mean ± S.E.
either. Analysis of the cell cycle kinetics by the sister chromatid differential staiRing method
revealed no notable differences between the
cell cycle kinetics of unexposed and exposed groups (Table 2).
Euperiment 2
MF was applied with the mutagen EMS. The SCE frequency in the control group without EMS was 8.82±1.02 in the MF-exposed group,
and 7.74±O.59 in the unexposed group (Table 3), there being no significant difference
be-tween the two groups. Although the EMS
treatmeRded to a clear dose-related increase
In Experiment l, SCE was not induced by
MF treatment. This finding was in good
accordance with the previous reports20m23). However, recently Khalil and Quassem30) re-ported a sigRificant increase in the SCE in the cultures continuously exposed to a pulsing EMF (50 Hz, l.05 mT) for 72 hours but not
with a shorter exposure time (24 and 48
hours). Garcia-Sagredo and Monteagudo3i)
also reported that low-level pulsed EMF
caused a significant increase in SCE.
There-fore, under certain conditions EMF may
in-duce SCE.
Analysis of the cell cycle kinetics by the sister chromatid differential staining method has
been shown to be a very good iRdicator of the cytotoxicity35). However, iR the present experi-ment, there were no notable differences in the cell cycle kinetics between the MF-exposed and
unexposed groups. This suggests that MF
under the present condition does not affect the cell cycle kinetics.
In Experiment 2, SCE was not significantly
enhanced when MF was applied with EMS.
Rosenthal and Obe32) reported that cultivation
of human lymphocytes in the presence of
50-Hz EMF does not alter the spolttaneousfrequeflcy of SCE but in some cases the
frequency of SCE in humalt lymphocytes
pretreated with mutagens was higher when the
cells were cultured iR the preseRce of EMF.
This was always less than the doubling of the
SCE frequencies obtained when pretreated
cells were cultured in the absence of EMF.Therefore EMF might cause SCE after
pre-treatment with a mutagen. The differences between the above report and our findings may be due to the duration of pretreatmentand the form of EMF.
IR the present study, the SCE frequency in
{he groeps exposed to static MF was not
significantly different from that in the
unex-posed groups even when the MF was applied
with EMS. Further studies using various fire-quencies,intensities and wave forms are clearly required.
ACKNOWLEDGMENT
This work was supported in part by a
Grant-in-Aid for Scientific Research from the Ministry of Education, ScieRce and Culture of
Japan.
REFERENCES
l) Wer{heimer N, Leeper E. Electrical wiring configurations and childhood cancer. Am J Epidemiol 1979; le9: 273-284.
2) WertheimerN,LeeperE.Adultcancerrelated
to electricaj wires near the home. Int J Epidemiol 1982; 11: 345-355.
3) Tomenius L. 50-Hz elec￡romagBetic ments and the incidence of childhood tumors in Stockholm County. Bioelectromagnetics 19861 7: 191-207.
4) Milham S. Mor￡ality from leukemia in workers exposed to electrical and magRetic fields. New Eng J Med l982; 3e7: 249.
5) McDowall ME. Mortality of resident in the
vicinity of electricity traRsmission facilities.
Lancet l983' 77: 246.
,
6) Coleman MP, Bell CMJ, Taylor HL,
Zakeji. Leukemia and residence near electricity transmission equipmen￡: a case-control study. Br J Cancer l989; 6e: 793-798.
7) Lin RS, Dischinger PC, Conde J, Farrell KP. Occupa￡ional exposure to electromagnetic fields aRd the occurrence of brain tumors. J Occup Med l985: 27: 413-419.
8) Thomas TL, Stolley PD, Stemhagen A, et al. Brain tumor mortality risk among men with
electrical and electronics jobs: A case-control study. J Natl Cancer Inst 1987; 79: 233-236. 9) Savitz DA,John EM, Kleckner RC. Magnetic field exposure from electric applicances and childhood cancer. Am J Epidemiol l990; 131: 763-773.
Ie) Bastuiji-Garin S, Richardson S, Zittoun R. Acute leukemia in workers exposed to magnetic fields. Eur J Cancer I990; 26: 1119-l120.
Il) Tomqvist S, Knave B, Ahlborn A, Persson T. Incidence ef leukemia and braiB tumours in some "electrical occupations". Br J Ind Med 1991; 48: 597-603.
}2) Fulton JP, Cobb S, Preble L, et al. Electrical wiring configurations and childhood leukemia in Rhode Island. Am J Epidemiol 1980; 111: 292-296.
I3) London Sll, Thomas DC, Bowman JD, et al. Exposure to residential electric and magne￡ic fields aRd risk of childhood leukemia. Am J Epidemiol 199I; 134: 923-937.
I4) Verreault R, Weiss NS, HolleRbach KA. et al. Use of electric blankets and risk of ￡esticular
cancer. Am J Epidemiol l990; 131: 759-762.
15)
IerJL. Sister chromatid exchange as an tor of mutagenesis. Nature (Lond) I978; 271: 55l-553,
16) Kato H, Shirnada H. Sister chromatid
chaRges induced by mitomycin C: a new
method of detecting DNA damage at somal level, Mutat Res l975; 28: 459--464. 17) Perry P, Evans HJ. Cytolegical detection of mutagen-carcinogen exposure by sister matid exchange. Nature (Lond) 1975; 258: 121-125.
I8) StetkaDG,WolffS.Sisterchromatidexchange as an assay for genetic damage induced by mu￡agen-carcinogeRs. Mutat Res 1976; 41: 833-S42.
Effiect of magnetic fields on SCE 13
exchangers induced by mutageRic carcinogens in Rormal and xeroderma pigmentosum cells. Nature (Lond) l977; 265: 347-348. 20) Wolff S, Crooks LE, Brown P, et aL Tests for DNA and chromosomal damage induced by nuclear magnetic resonance imaging. gy I980; 136: 707-710.
21) Wolff S,James TL, Young GB, et al. Magnetic Resonance Imaging: AbseRce of in vitro cytogenetic damage. Radiology 1985; 55: l65.
22) Cooke P, Morris PG. The effects of NMR exposure on liviBg organisms. II. A geRetic study of human lymphocytes. BrJ Radiol 1981; 54: 622-625.
23) Peteiro-Cartelle EJ, Cabezas--Cerrato J. sence of kiRetics and cytogenetic effects on human lymphocytes exposed to s￡atic magnetic fields. J Bioelectricity 1989; 8: ll-19. 24) Bauchinger M, Hauf R, Schmid E, Drisp J. Analysis of structural chromosome changes
aRd SCE after occupational longterm posure to electric and magRetic fie}ds from 380 kV systems. Radiat Environ Biophys 1981; 19: 235-238.
25) NordeRsonI,Hansson-MildK,NordstromS,et al. Clastogenic effects in human lymphocytes of power frequency electric fields: In vivo and in vitro studies. Radiat Environ Biopkysics l984; 23: 191-201.
26) Cohen MM, Kunska A, Asterr}borskiJA, et al. Effect of low-level 60-Hz electrorr}agnetic fields on humaR lymphoid cells. I. Mitotic rate and chromosome breakage in human peripheral
lymphocytes. Bioelectromagnetics 1986; 7: 415-423.
27) Cohen MM, Kunska A, AstemborskiJA, et al. The effect of low-level 60-Hz electromagnetic fields oR human lymphoid cells. II.
chromatid-exchanges in peripheral cytes aRd lymphoblastoid cells lines. Mutat Res 1986; 172: 177-l84.
28) Takahashi K, Kaneko I, Date M, Fukuda E. Influence of pulsing electromagne￡ic field olt the frequency of sister-chromatid exchanges iR cultured mammaliaR ce}ls. ExperieRtia 1987; 43: 33F332.
29) Garcia-Sagredo JM, Parada LA, Monteagudo JL. Effect on SCE in human chromosomes in
vitro oflow--level pulsed magnetic field. EnviroR
Mol Mut 199e; l6: 185-l88.
80) KhalilAM,QuassemW.Cytogeneticeffectsof pulsing electremagnetic field on human phocytes in vitro: chromosome aberratioRs, sis￡er-chromatid exchaRges and cell kinetics. Mgta￡ Res }991; 247: 141-146.
31) Garcia-SagredoJM,MonteagudoJL.Effectof low-level pulsed electromagRetic fields on man chromosomes in vitro: analysis somal aberrations. Hereditas l991; l15: 9-Il. 32> Rosenthal M, Obe G. Effects of50-hertz tromagnetic fields on proliferation and on chromosomal alterations in human peripheral lymphocytes untreated or pretreated with
mical mutagens. Mutat Res l989; 210:
329-335.
38) GotoK,MaedaS,KanoY,Sugiyama[{".Factors involved iR differential Gier{}sa staining of sister chromatids. Chromosoma 1978; 66: 351-359.
34) Tice RR, Schneider EL, RaryJM. The tion of bromodeoxyuridlne incorporation into DNA for the analysis of cellular kiltetics. Exp Cell Res 1976; 102: 232-236.
35) Morimoto K, Wolff S. Increase of sister matid exchanges and pertubations of cell sion kinetics in humaB lyrnphocytes by benzene me￡abolites. Cancer Res l980; 40: 1189-1193.
