17 Elevated Sister Chromatid Exchange Frequencies in Dividing Human Peripheral Blood Lymphocytes Exp
Bioelectromagnetics28:281^288(2007) Elevated Sister Chromatid Exchange
Frequencies in Dividing Human
Peripheral Blood Lymphocytes Exposed
to50Hz Magnetic Fields
M.A.Wahab,1J.V.Podd,2B.I.Rapley,3and R.E.Rowland1*
1Institute of Molecular BioSciences,Massey University,Palmerston North,New Zealand 2School of Psychology,Massey University,Palmerston North,New Zealand
3Atkinson&Rapley Consulting Ltd.,Palmerston North,New Zealand
The in vitro cytomolecular technique,sister chromatid exchange(SCE),was applied to test the
clastogenic potentiality of extremely low frequency(ELF)electromagnetic?elds(EMFs)on human
peripheral blood lymphocytes(HPBLs).SCE frequencies were scored in dividing peripheral blood
lymphocytes(PBLs)from six healthy male blood donors in two rounds of experiments,R1and R2,to
determine reproducibility.Lymphocyte cultures in the eight experiments conducted in each round
were exposed to50Hz sinusoidal(continuous or pulsed)or square(continuous or pulsed)MFs at?eld
strengths of1m T or1mT for72h.A signi?cant increase in the number of SCEs/cell in the grouped
experimental conditions compared to the controls was observed in both rounds.The highest SCE
frequency in R1was10.03for a square continuous?eld,and10.39for a square continuous?eld was
the second highest frequency in R2.DNA crosslinking at the replication fork is proposed as a model
which could explain the mechanistic link between ELF EMF exposure and increased SCE frequency. Bioelectromagnetics28:281–288,2007.?2006Wiley-Liss,Inc.
Key words:ELF EMF;human peripheral blood lymphocytes;sine and square waveform;
SCE;chromosome;DNA crosslinking
INTRODUCTION
Electricity has become an essential commodity in our technology-based society.The widespread use of electric power for domestic and industrial appliances at home and in the work place results in exposure to extremely low frequency electromagnetic?elds(ELF EMFs).These are set at50Hz in New Zealand and 60Hz in the USA.Growing public awareness of EMFs in the environment has stimulated the need for sound scienti?c information concerning possible biological effects.The present study was conducted to evaluate possible genetic effects via the sister chromatid exchange(SCE)assay in human peripheral blood lymphocytes(HPBLs)exposed to50Hz ELF EMFs.
SCE is widely recognized as a sensitive and reliable test for clastogenicity[Sarto et al.,1985; Tucker et al.,1993;Albertini et al.,2000],a clastogen being de?ned as any environmental agent which is harmful to DNA and chromosomes.The technique exploits the instability of the DNA helix during replication at S phase in a dividing population of cells, and evidence of genetic damage is accepted if the number of SCEs in an experimental group is more signi?cant statistically than a selected control group. Many clastogenic factors are known to interfere with DNA replication and cause the helix to twist.This exerts a tension on the newly replicating half-strand which switches across to the opposite backbone.In the presence of bromodeoxyuridine,a halogenic analog of uridine,this can be detected in c-metaphase complements as an SCE.It is well documented that any increase in the frequency of SCE in an individual is linked to ill health[Sandberg et al.,1984].
?2006Wiley-Liss,Inc.——————
Grant sponsor:Palmerston North Medical Research Foundation, New Zealand.
*Correspondence to:R.E.Rowland,Institute of Molecular BioSciences,Massey University,P.O.Box11-222,Palmerston North,New Zealand.E-mail:R.E.Rowland@https://www.360docs.net/doc/4017674114.html,
Received for review3November2005;Final revision received18 July2006
DOI10.1002/bem.20289
Published online1November2006in Wiley InterScience (https://www.360docs.net/doc/4017674114.html,).
SCE frequencies from two sets of experiments were scored in HPBLs of six(3t3)healthy,non-smoking male blood donors matched for age,lifestyle, occupational history,medical history,and diet.The experiments were duplicated with a different set of donors to determine whether the results could be repeated or not.Cultured lymphocytes were exposed to all combinations of two different?ux densities,one strong(1mT)and one weak(1m T),two different waves (sine or square),and two different durations(continu-ous or pulsed).A reductionist approach was followed in order to clarify the parameters involved should genetic effects be observed.
MATERIALS AND METHODS
Subjects
Human peripheral blood samples were obtained with informed consent from six(3t3)non-smoking healthy male donors.Donors were matched and selected based on information gathered from a ques-tionnaire that detailed information on age,occupation, diet,lifestyle,and medical history.The study was approved by Massey University Human Ethics Com-mittee PN99/64.
A set of eight experiments was conducted in duplicate with each duplicate experiment using the same EMF conditions(Table1).These two sets of eight experiments each were called R1and R2.The eight experiments in R1were conducted using blood samples from the?rst three donors and blood samples from the remaining three donors were used in the R2 experiments.
Lymphocyte Cultures
Ten milliliters of blood was taken via venipunc-ture from each donor.Each culture tube contained5ml of Medium-199,1ml of fetal bovine serum,and0.1ml of phytohemagglutinin(PHA)M form(all from GibcoBRL).When conducting the SCE technique it is imperative that the white blood cell(WBC)count is constant[Bender et al.,1992].The WBC count was determined and then adjusted to produce a solution of3.25million cells/culture tube by adding between 0.3and0.6ml of blood to each sample.?0.05ml of 10à2M5-bromodeoxyuridine(BrdU)was added to each culture tube,adjusted to give a?nal concentration of20m M.The culture tubes were incubated at378C for72h,then0.05m l of colchicine,giving a?nal concentration of0.05%,was added1h immediately prior to harvest.Lymphocytes were harvested by centrifugation,treated with0.075M KCl and?xed in fresh methanol:acetic acid(3:1).Ten slides per donor were stained using the modi?ed Fluorescence-Plus-Giemsa(FPG)method of Perry and Wolff[1974]. ELF MF Exposure Conditions
A specially designed EMF?eld incubator,built by members of our research team,was equipped with a Helmholtz coil pair system.Each coil was180mm in diameter.The coils were made of enameled0.7mm diameter copper wire and wound in100turns.A high power ampli?er and a programmable time base generator were used to produce MFs of selected strength,waveform,and duration.To determine the exact nature of the?eld produced by the coil,the system was modeled using the EMF simulation program, MagneSim,developed by Foster and Page[Rapley et al.,1998].Figure1,Tables2and3illustrate the magnetic?eld parameters of the Helmholtz coils.The Helmholtz coils were housed in a purpose-built incubator situated in an electrically shielded room away from any MF sources.
For R1and R2,culture tubes were situated within the pair of Helmholtz coils and subjected to50Hz1m T (sine wave or square wave)or1mT(sine wave or square wave)MFs,in either a pulsed(4s on/4s off)or continuous duration for72h,inclusive of the1h colchicine pretreatment period.The incubator was heated by a?ow of warm air from a distant source(2m) and stabilized at a constant378C by a thermostat,the temperature at which the PBLs were cultured.Control tubes were similarly cultured over a separate72h incubation period,but received no exposure(sham)to MFs.To eliminate bias,an independent person set the MF parameters that were unknown to the researcher. Scoring Criteria
The ten slides of each donor were randomly coded(a–j)by the researcher and examined serially. Approximately200consecutive second mitotic meta-phase cells were selected and scored per donor. Each selected cell showed a full complement of
TABLE1.Peripheral Blood Lymphocytes From Each of Three Participants Were Exposed to the Eight Combinations of Waveform,Duration,and Strength(R1)and Repeated
on Another Three Participants(R2)
Waveform Duration Strength(T) Sine Continuous1m Sine Continuous1m Sine Pulsed1m Sine Pulsed1m Square Continuous1m Square Continuous1m Square Pulsed1m Square Pulsed1m 282Wahab et al.
46chromosomes,good chromosome morphology,differential staining for SCE and no chromosome overlapping.The images were captured by a JVC 3-CCD Color Video Camera using Silicon Graphics and Image Capture software.SCEs were scored according to guidelines of Sweirenga et al.[1991].The researcher was blind to whether he was scoring an experimental or control slide.RESULTS
A total of 5145and 6141c-metaphase cells with clear exchanges were analyzed in R1and R2,
respectively.Figure 2is an illustration of a dividing HPBL at c-metaphase showing a number of SCEs.Analysis of Round-1
Initially,the data of R1were examined by collapsing across all experimental conditions so that the mean number of SCEs could be compared with the control group.Table 4summarizes the data for the number of cells analyzed (N),the mean SCE and standard deviations of the control group and that of the experimental group.A statistically signi?cant difference was observed between the mean SCE of the control group and that of the entire set of experimental groups,t (5143)?4.09,P <.001.
Table 5summarizes the data in R1(from all donors combined)on the number of cells observed for SCE (N),mean SCE and standard deviations for all eight experiments and controls.The mean SCE value of the three control experiments in R1is 8.92.The mean SCE values range from 8.08for sine continuous at 1m T to 10.03for square continuous at 1mT .The low N values for sine continuous 1m T and square pulsed 1m T are attributed to a low mitotic index for these ?elds in R1.
Analysis of Round-2
As in R1,the data of R2(from all donors combined)were also examined by collapsing across all experimental conditions so that the mean number of SCEs could be compared with the control group.Table 6summarizes the data for the SCE means and standard deviations of the control group and that of the experimental group.A t -test showed a statistically signi?cant difference between the mean SCE of the control group and that of the entire set of experimental groups,t (6139)?4.57,P <.001.
Table 7summarizes the data in R2on the number of cells observed for SCE (N),mean SCEs and standard deviations for all eight experiments.The overall mean SCE value for the controls in R2is higher than that in R1(9.66and 8.92,respectively).The mean SCE
values
Fig.1.Lollipopplotoftheresultant vectorsinalongitudinalsection through the‘‘Z-axis’’of the Helmholtz coil pair.The vectors’angles represent thedirectionoftheresultant magneticfieldsin Cartesian co-ordinates with the length of the vector representing the relative magnetic field strength.The bold outlines represent the position of the culture tubes within the three-dimensional coil.The dottedverticallinesrepresent the coilwindings.
TABLE 2.Angles in Cartesian Co-Ordinates of the Resultant Magnetic Field Vectors in the Helmholtz Coil Pair inc.75614216344318299285inc.3561117145355346343349435503313593573573605357359360003603600133593593603600360001111003600360360359359310360360003603593575360357357359133035543493433463555141711356inc.
285
299
318
344
16
42
61
75
inc.
The bold text corresponds to the approximate position of the culture tubes.inc.,represents a value which falls within a physical turn of the coil and is thus incalculable.
Elevated Sister Chromatid Exchange Frequencies 283
range from9.66for square pulsed1mT to10.61for sine pulsed1m T.
Interactions
Parameter interactions that reached signi?cance or near signi?cance are reported.A detailed analysis showed that a square continuous waveform produced a higher number of SCEs per cell compared to the square pulsed waveform,t(1172)?1.89with a P-value of near signi?cance(0.06)in R1.In R2,the same square continuous waveform compared to square pulsed produced a signi?cantly higher number of SCEs per cell,t(2398)?4.45,P<.001.Going a step further,a square waveform for R1produced a signi?cantly higher number of SCEs per cell with increasing?eld strength,t(1772)?3.57,P<.001.An exposure of continuous duration irrespective of waveform(in R1)produced a signi?cantly higher number of SCEs per cell with increasing strength,t(1885)?5.51,P<.001. DISCUSSION
The results of this study show that ELF EMFs appear to exert a weak but measurable effect on human chromosomes.In R1and R2the data showed an elevation in SCE frequency which reached signi?cance in both rounds,although the elevation was small: 0.42and0.51of an SCE in each respective round.Thus, it is equivocal as to whether the statistical signi?cance observed is biologically meaningful.On the other hand, if the effect is naturally weak,it is equally plausible that the increase in SCE observed in the exposed cells is what one could expect.
Interactions
A key element in this body of work was the adoption of a reductionist approach to try and establish whether any particular factor or combination of factors could affect the genetic machinery.A detailed analysis of parameter interactions showed the most interesting results.A square continuous waveform produces a near signi?cant higher number of SCEs per cell in R1 (P?.06).Similarly,we can see in R2that the same square continuous waveform indeed produces a sig-ni?cantly higher number of SCEs per cell.Going a step further,a square waveform produces a signi?cantly higher number of SCEs per cell with increasing?eld strength,and exposure with continuous duration,
TABLE3.Relative Magnetic Field Strengths in the Helmholtz Coil Pair Where‘‘1.000’’Represents the Strongest Field inc. 1.0000.4610.2830.2110.2110.2830.461 1.000inc.
0.8640.7620.5930.4730.4140.4140.4730.5930.7620.864 0.6040.5980.5660.5300.5070.5070.5300.5660.5980.604 0.5310.5430.5420.5360.5300.5300.5360.5420.5430.531 0.5070.5230.5310.5330.5330.5330.5330.5310.5230.507 0.5070.5230.5310.5330.5330.5330.5330.5310.5230.507 0.5310.5430.5420.5360.5300.5300.5360.5420.5430.531 0.6040.5980.5660.5300.5070.5070.5300.5660.5980.604 0.8640.7620.5930.4730.4140.4140.4730.5930.7620.864
inc. 1.0000.4610.2830.2110.2110.2830.461 1.000inc. The bold text corresponds to the approximate position of the culture tubes.The chosen?eld strength was set at the position of the culture
tubes.inc.,represents a value which falls within a physical turn of the coil and is thus
incalculable.
Fig.2.A c-metaphase chromosome spread of a dividing periph-eral blood lymphocyte from a sham exposed control that has undergone two rounds of DNAreplicationin the presence of BrdU, showing11SCEs(arrowheads).[The color figure for this article is available online at https://www.360docs.net/doc/4017674114.html,.]TABLE4.Overall Mean SCE and Standard Deviations of the Control Group and That of the Entire Set of Experimental Groups for R1
Group N Mean SCE Standard Control15548.92 3.19 Experimental35919.34 3.49
284Wahab et al.
irrespective of waveform,produces a signi?cantly higher number of SCEs per cell with increasing strength.Thus,it appears that a square continuous waveform with increasing strength may be of impor-tance here in producing a higher number of SCEs per cell.
The authors note that the SCE frequency of 10.61scored for a sine pulsed waveform at1m T in R2 was the highest recorded,which theoretically could mean that this?eld has the greatest clastogenic effect of all parameters,but caution is warranted here;the highest SCE frequency in R1was10.03for a square continuous?eld,and the SCE frequency of10.39for a square continuous waveform in R2(albeit a different strength)was the second highest in this latter round. Waveform
The results outlined in this paper are indicative of an increased biological effect(increased incidence of SCEs)with the application of a square waveform in contrast to a sine waveform.To explain this observation, it is necessary to understand the physics of these waveforms and how they are obtained as a magnetic ?eld in the time domain in three-dimensional space.
In order to generate a close approximation to a square wave in terms of the magnetic?eld produced within the coils the authors used a transconductance (current feedback)ampli?er.This ampli?er is able to deliver a high voltage spike at the beginning of each half cycle in order to drive suf?cient current through the coil(overcoming the coils inherent back EMF)to approximate the fast rise time of a square wave.The result is a good approximation of a square wave that is characterized by its rich harmonic envelope,consisting largely of odd-numbered harmonics.
A perfect example of this rich harmonic envelope is the‘‘ultimate’’square wave,a single spike,such as is produced by the spark plug of an internal combustion engine.Such a waveform is capable of generating radio interference over a wide range of frequencies.This explains why a lawn mower,for example(without radio suppression circuitry),can produce radio interference across a wide band of both radio and television broadcast frequencies.
The fast rise time of a square wave may be the critical factor for understanding the interaction mech-anism of alternating magnetic?elds with biological systems.A fast rise time,expressed mathematically as d b/d t where the product is a large number,is known to induce greater electrical current within a system.This electrical current translates to an ionic current in a biological cell and may be the causal factor in exerting a biological effect.
In contrast,a sine waveform is relatively poor in its harmonic envelope with a relatively small number of harmonics produced.Accordingly,the analysis of the harmonic spectra of a standard sine wave reveals the majority of the power is expressed at the fundamental frequency.For this reason sine waves are generally used in bioelectromagnetic research in order to focus the exposure on a‘‘single’’frequency.In general terms,a sine wave is considered to be a single,pure frequency.
With respect to possible differences in power between a square and sine waveform,the authors went to considerable lengths to ensure that the total power for the two exposure conditions,1mT and1m T, were equivalent for both waveforms.Therefore,any difference observed in the results could not be attributed to dissimilarities in power levels created by virtue of the
TABLE5.Mean SCE and Standard Deviations of All Experiments in R1Based on all Three Donors
Waveform Duration Strength
(T)
Mean
SCE
Standard
deviation N
Sine Continuous1m8.08 2.7387 Sine Continuous1m9.62 3.22600 Sine Pulsed1m9.55 3.07443 Sine Pulsed1m8.82 3.12600 Square Continuous1m9.09 2.98600 Square Continuous1m10.03 3.67600 Square Pulsed1m8.70 3.2879 Square Pulsed1m9.28 4.59495 Control(?3)8.92 3.191554 Total5145 TABLE6.Overall Mean SCE and Standard Deviations of the
Control Group and That of the Entire Set of Experimental
Groups for R2
Group N Mean SCE Standard deviation Control15359.66 3.72 Experimental460610.17 3.79
TABLE7.Mean SCEs and Standard Deviations of All Experiments in R2,Based on All Three Donors
Waveform Duration
Strength
(T)
Mean
SCE
Standard
deviation N Sine Continuous1m9.98 3.58508 Sine Continuous1m10.21 3.85521 Sine Pulsed1m10.61 4.09516 Sine Pulsed1m9.98 3.62473 Square Continuous1m10.39 3.86600 Square Continuous1m9.89 3.83600 Square Pulsed1m9.83 3.66600 Square Pulsed1m9.66 3.74600 Control(?3)9.66 3.721535 Total6141 Elevated Sister Chromatid Exchange Frequencies285
different waveform.This was achieved by utilizing root mean square(RMS)current measurements in the Helmholtz coil pair for each waveform during calibration.
In terms of the biological experiments conducted here,the square waveform spreads its power across a wider spectrum than is the case for a sine wave. Therefore,it is reasonable to assume that if there is a biological window of susceptibility that is frequency dependent,in this case with respect to the generation of SCEs,it could be expected that the wider frequency spectrum of a square waveform would have a greater chance of hitting the target frequency(or frequencies) thus generating a measurable biological response, compared to the narrow harmonic bandwidth of a sine wave.Indeed,the results presented here accord with this view.
MFs and SCE Mechanism
If MFs can exert an effect on the genetic machinery,a central question then arises:what is the mechanism by which this effect occurs?SCEs are known to arise consequent to events at the DNA replication fork.The replication bypass model for SCE was originally proposed in1975to explain DNA crosslink-induced SCEs,since it appeared that(i) crosslinking agents were the most potent inducers of SCEs[Latt,1974;Perry and Evans,1975];(ii)cross-links would inhibit or prevent the normal separation of DNA strands as required for bidirectional replication [Geiduschek,1961];and(iii)that crosslink repair required an undamaged parallel DNA duplex in order to complete a recombinational exchange[Cole,1973, 1974].
On this basis,Shafer[1977]proposed a model in which SCEs could result from the completion of DNA replication at the site of an unrepaired DNA crosslink. The most important characteristic of this proposed model(amongst several advanced)was that SCEs could result from a series of sequential events that might occur as bidirectional replication encounters a crosslink.It is interesting to mention here that the induction of DNA crosslinks was observed in rat brain cells when exposed to a60Hz MF[Singh and Lai,1998].The effects of these MFs were also compared with those of a known DNA crosslink-inducing agent,mitomycin C. The pattern of effects was similar between these two agents.Therefore,the presence of a signi?cant increase in the frequency of SCEs in the present study could be the ultimate product of ELF EMF exposure via DNA-crosslinks.Of all the models proposed linking SCEs with ELF EMF effects,this one appears to be the soundest.MF Exposures and SCE Frequency
The overall increase in number of SCE/cell as a consequence of MF exposure that was observed in the current study is in good agreement with the results reported previously by Khalil and Qassem[1991].They similarly showed that in vitro exposure of human cells to MFs resulted in a signi?cant increase in frequency of SCE.In their study,PHA-stimulated lymphocytes were exposed to a pulsed?eld(50Hz,1.05mT peak;pulse duration,10ms)for24,48,and72h.No signi?cant alterations in the baseline frequency of SCE were observed in cultures exposed to MFs for24or48h.In contrast,however,a statistically signi?cant twofold increase in the frequency of SCE was observed in cultures continuously exposed for72h.Apart from the current study,this is the only report,as far as we are aware,which shows a positive result using SCE analysis on extremely weak MFs.
It is interesting to note,however,that Yaguchi et al.[1999]also observed an increase in SCE in mouse m5S chromosomes when they applied much stronger MFs(5–400mT).Furthermore,several other studies of human[Garcia-Sagredo and Monteagudo,1991;Nor-denson et al.,1994]as well as animal[Lai and Singh, 1997]cells exposed to ELF EMFs have also shown clastogenic effects via different methods.On the other hand,these?ndings must be balanced against those of other researchers who have found no effects. For example,Rosenthal and Obe[1989]exposed mitogen-stimulated adult peripheral lymphocytes to a sinusoidal50Hz?eld from0.1to7.5mT and reported no effect of MFs on the frequency of SCEs or chromosome breaks.A similar study using a pulsed MF at 1.0, 2.0,and 4.0mT,applied as a quasirectangular pulse of26m s,in5ms trains,repeated at14Hz,also showed no effect on SCE[Garcia-Sagredo et al.,1990].Livingston et al.[1991]exposed Chinese hamster ovary?broblasts and PHA-stimulated adult and newborn lymphocytes to a?xed MF of 0.22mT,with60Hz electric?elds(current densities of 3,30,300,and3000m A/cm2).No signi?cant effects on SCE were observed in any cell type or?eld examined. Similarly,exposures of Chinese hamster cells to pulsed MFs from0.18to2.5mT did not show any increase in the baseline frequency of SCE[Takahashi et al.,1987]. The data from several other studies have indicated an absence of signi?cant differences in the incidence of CA,SCE as well as micronucleus frequency between RFR-exposed and sham-exposed cells[Vijayalaxmi et al.,1997,2001a,b;Bisht et al.,2002;d’Ambrosio et al.,2002].
The con?icting data above may not be antago-nistic but merely indicate the complexity of the?eld of
286Wahab et al.
enquiry.It is more productive to focus on the parameters involved as highlighted in the current study. Habituation
In Garcia-Sagredo and Monteagudo’s[1991] study,HPBLs were exposed to pulsed MFs at4mT and a statistically signi?cant increase in frequency of CAs was observed.Nordenson et al.[1994]exposed human amniotic cells to a sinusoidal50Hz,30m T pulsed MF(15s on/15s off)for72h and similarly observed a signi?cant increase in the frequency of CAs. Among exposed cells,the aberration frequency was4% compared to2%in sham-exposed cells.The same authors observed a similar increase in CA in another series of eight experiments in a2s on/2s off pulsed ?eld.The rationale used to explain MF effects observed with pulsed?elds but not continuous?elds is sometimes attributed to a phenomenon called‘‘habituation,’’or in this case a lack of habituation.According to this theory, cells can become habituated to a constant continuous ?eld and thus do not show a response.With pulsed ?elds,the opposite prevails:the view expressed is that cells do not become habituated and therefore do show a response.No mechanism as to how habituation could be achieved has been documented that the current authors are aware of,and whilst agreeing that the idea is attractive,no evidence was found in the present study to support the theory.
CONCLUSION
Our?ndings show that a square continuous waveform increases the number of SCEs in dividing HPBLs.Our?ndings were con?rmed in two identical sets of experiments.These results may in large part be due to the rich harmonic envelope which is character-istic of a square waveform in the time domain.The ?ndings endorse the DNA crosslinking model for the generation of SCEs with ELF magnetic?elds. REFERENCES
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