Absence of synergistic effects on micronucleus formation after exposure to electromagnetic fields and asbestos fibers in vitro

Exposure of human amniotic fluid (AFC) cells to horizontally applied magnetic fields (hMF) of 50 Hz and 1 mT generated in a Helmholtz-coil system leads to a significant increase in micronucleus frequency (MN), without affecting cell proliferation. To
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  Toxicology Letters 108 (1999) 47–53 Absence of synergistic effects on micronucleus formationafter exposure to electromagnetic fields and asbestos fibersin vitro M. Simko´  a, *, E. Dopp  b , R. Kriehuber  a a Di   ision of En  ironmental Physiology ,  Institute of Cellular Physiology and Biosystems Technology ,  Uni   ersity of Rostock  , Uni   ersita¨tsplatz  2  ,  D - 18051 ,  Rostock  ,  Germany b Di   ision of Cellular Pathophysiology ,  Institute of Cellular Physiology and Biosystems Technology ,  Uni   ersity of Rostock  ,  18055  , Rostock  ,  Germany Received 28 January 1999; received in revised form 27 April 1999; accepted 4 May 1999 Abstract Exposure of human amniotic fluid (AFC) cells to horizontally applied magnetic fields (hMF) of 50 Hz and 1 mTgenerated in a Helmholtz-coil system leads to a significant increase in micronucleus frequency (MN), without affectingcell proliferation. To investigate whether hMF-exposure has an additive or synergistic effect on the genotoxic capacityof asbestos fibers, MN induction was investigated in hMF pre-exposed cells, treated before or after with asbestos (1  g / cm 2 ). Neither synergistic nor additive effects on MN induction were observed. The results indicate, that under ourexperimental conditions, exposure to hMF and treatment with asbestos fibers possess genotoxic capability, but nointeractive effects, in AFC cells. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords :   Electromagnetic fields (ELF-EMF); Micronuclei (MN); Asbestos fibers; Amniotic cells; Tumour promoter; Growth curve;Mitotic / locate / toxlet 1. Introduction The correlation between exposure to environ-mental electromagnetic fields (EMF) and in-creased cancer risk is controversial.Epidemiological studies have indicated a correla-tion between EMF-exposure and an increasedincidence of childhood leukaemia, cancer of thenervous system, and lymphomas (Wertheimer andLeeper, 1979; Savitz et al., 1988; Washburn et al.,1994). On the other hand, a recent publicationreported no clear correlation between EMF andgenotoxicity (Moulder and Foster, 1995). How-ever, animal studies have showed a possible co-promoter capacity of EMF on tumour growth(Lo¨scher and Mevissen, 1994). In vitro studiesshowed biological effects after exposure to EMF,such as increased colony growth in anchorage-in- * Corresponding author. Tel.:  + 49-381-4981935; fax:  + 49-381-4981918. E  - mail address : (M.Simko´)0378-4274 / 99 / $ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.PII: S0378-4274(99)00115-0  M  .  Simko´ et al  .  /   Toxicology Letters  108 (1999) 47–53  48 dependent JB6 cells (West et al., 1994). Cain et al.(1993) showed that exposure to EMF combinedwith treatment of a chemical tumour promoter,phorbol ester (TPA), enhances cell transformationin vitro. These data suggest that EMF and chem-ical tumour promoter are able to act as co-pro-moters, which may lead to tumour enhancementin initiated cell population.Epidemiological results prove syncarcinogenesisfor asbestos exposure and smoking, radon expo-sure and smoking, and exposure to aromaticamines and smoking (Popp, 1996). Animal experi-ments point to additive effects in carcinogenesisfor different nitrosamines and substances like ben-zo(a)pyrene and ionising radiation. Popp (1996)concluded that syncarcino genetic mechanismsmay not only result from genotoxicity but alsofrom influences on cell proliferation and mitoticactivity.The ability of asbestos to promote tumourgrowth in initiated cells is well known (Jaurand etal., 1988). Asbestos fibers are carcinogenic wheninhaled by both humans and animals; however,the mechanisms by which the fibers exert theireffect are unknown. Aneuploidy is a commoncharacteristic of asbestos-induced tumours, and ithas been hypothesised that a shift in chromosomecomplement plays a major role in the early stagesof neoplastic development (Barrett et al., 1990). Ithas also been shown that free radicals play animportant role in asbestos-induced diseases(Kamp et al., 1992). A number of investigationshave evaluated the co-carcinogenic effects of fibers and chemical carcinogens. Selikoff et al.(1968) was the first to demonstrate that a syner-gistic relationship exists between asbestos expo-sure and cigarette smoking for the production of bronchial carcinomas. Co-carcinogenic effectswere also found after exposure of animals toasbestos and benzo[a]pyrene (Pylev and Shabad,1973) or  N  -nitrosoheptamethyleneimine (Harrisonand Heath, 1991). Synergistic effect between as-bestos and radiation in tumour production issupported by experimental evidence as well (War-ren et al., 1981; Hei et al., 1984). EMF, as acommon environmental ‘possible carcinogen’(NIEH, 1998) might therefore also interact withasbestos fibers in a synergistic or even in a co-car-cinogenic manner.In this study, we investigated the micronucleus(MN) frequency in human amniotic fluid cells(AFC) following exposure to alternating EMFwith an amplitude of 1 mT in the presence orabsence of asbestos fibers. Different experimentswere used to perceive possible co-carcinogenicinteractions between EMF and asbestos fibers. 2. Material and methods 2  . 1 .  Cell culture Human amniotic fluid cells (AFC cells,GM00472, Coriell Cell Repositories, MD, USA)were used. AFC cells were maintained in RPMI1640 medium supplemented with 15% foetal calf serum (Gibco BRL, FRG) and were incubated at37°C in a humidified atmosphere of 5% CO 2  and95% air. Cells (0.5–1 × 10 5 ) were plated on coverslips (21 × 26 mm) in six well plastic plates 12 hprior exposure to EMF or to asbestos. 2  . 2  .  Electromagnetic field exposure system For exposure to EMF, we used a Helmholtz-coil system with a pair of perpendicular coils(Phywe Systeme, GmbH, Go¨ttingen, FRG) each400 mm in diameter and with a distance betweenthe coils of 200 mm, which was placed in astandard incubator. The coils were wound with amagnetic wire 154 times around an approximatesquare frame with 2.1   . No significant tempera-ture changes could be detected within the experi-ments (37  0.2°C). The uniformity of themagnetic flux density was measured using a preci-sion F.W. Bell Model 4048 Gauss / Tesla Meterand was found to be 1  0.01 mT. In the orienta-tion used, the exposure system generated horizon-tally magnetic fields in respect to the culturemedium. 2  . 3  .  Growth control  To determine the influence of 1 mT EMF expo-sure and asbestos fibers on the growth character-istics of AFC cells, conventional growth curveswere determined for exposed and control cell cul-  M  .  Simko´ et al  .  /   Toxicology Letters  108 (1999) 47–53   49 tures over a period of 6 or 12 days. For each dayof growth, two 6-well culture plates were preparedas follows: AFC cells (2 × 10 5 ) were plated intoeach of six replicate culture wells and placed intothe exposure system and the control plates in asecond incubator, respectively. For 6 or 12 days,the number of cells were determined daily in eachof the six replicate culture wells. Every 3–4 dayscells were fed with fresh culture medium. Exposedand control cultures were treated similarly. Exper-iments were repeated six times. 2  . 4  .  Exposure of human amniotic fluid cells toelectromagnetic fields and asbestos fibers Exponentially growing cells were continuouslyexposed to asbestos and / or to 50 Hz EMF for 24,48, and 72 h to 1 mT by placing the culture platesin the centre of the exposure system contained ina standard tissue culture incubator. Cells weretreated with crocidolite asbestos fibers (UICCstandard) at a concentration of 1   g / cm 2 . Cro-cidolite asbestos was obtained from Dr Linnain-maa (University of Helsinki, Finland). Theaverage dimension for crocidolite was 1.71   m inlength and 0.25   m in diameter (Dopp et al.,1995). Fibers were sterilised by autoclaving(120°C for 20 min) and were suspended in PBS. Acrocidolite concentration of 1   g / cm 2 was used,because the best capability of MN induction wasshown at the concentration of 1–5   g / cm 2 inAFC cells (Dopp et al., 1997).Different exposure schedules for the studieswere used: 1. AFC cells were exposed to horizon-tal magnetic fields (hMF) for 24 h and thenexposed to crocidolite asbestos fibers (1   g / cm 2 )and hMF for 48 h; 2. AFC cells exposed to hMFfor 48 h were exposed to crocidolite and hMF for24 h; 3. AFC cells were simultaneously exposed tohMF and crocidolite asbestos fibers for 72 h; 4.cells were exposed to crocidolite asbestos fibersfor 24 h and then simultaneously exposed tocrocidolite asbestos fibers and hMF for 48 h.Untreated control samples, as well as samplesexposed to asbestos fibers alone and to EMFalone were run at the same time and were used asconcurrent controls. 2  . 5  .  In   itro micronucleus assay After EMF-exposure and / or asbestos treat-ment, cover slips were washed in PBS and fixedwith  − 20°C methanol (100%) for 2 h. Air-driedslides were washed in PBS and stained for 1 minwith the fluorescent DNA dye bisbenzimide(Hoechst 33258; 1   g / ml). After washing in dis-tilled water, slides were mounted for fluorescencemicroscopy and evaluated for MN formation andfor mitotic cells (MC). For identification of MNthe criteria of Countryman and Heddle (1976)were applied as followed: the MN has to be atleast one third smaller than the main nucleus andnot touching the main nucleus. Cells containingone or more micronuclei were scored as MN-pos-itive-cells. The number of MN were counted onthree simultaneously exposed slides in at least1000 cells for each time point of measurement.For evaluation slides were coded. Experimentswere repeated 3–5 times. Data of exposed cellswere analysed with reference to their own controldata for every time point. Differences betweenMN rates / number of MC in control and in ex-posed cells were tested for significance using theone-tailed Student’s  t -test. 3. Results Standard growth curves were determined todetect the influence of EMF- and asbestos fibersexposure on the cell growth after continuous ex-posure of AFC cell for 12 days to hMFs (1 mT / 50Hz) and 6 days to crocidolite asbestos fibers (1  g / cm 2 ), respectively. Fig. 1 illustrates the increas-ing cell number as a function of time for the EMFexposed and control cultures. Data represent themean of the pooled data from six independentexperiments, which include six replicate cultureseach. No statistical significant differences could beobserved between EMF-exposed and unexposedcells in regard to the cell growth. This is incontrast to the results obtained for asbestos. Thenumber of cells continuously decreases after 24 hof exposure to 1   g / cm 2 crocidolite asbestos fibers(Fig. 1) and, after 6 days of treatment no livingcells were observed in the cell cultures. No signifi-  M  .  Simko´ et al  .  /   Toxicology Letters  108 (1999) 47–53  50Fig. 1. Standard growth curve of AFC cells representing control (  ), hMF-exposed (1 mT) (  ) and crocidolite asbestos fibers(1   g / cm 2 ) (  ) treated cells. Control and hMF exposed cells show no differences in growth characteristics, whereas in asbestos fiberstreated cells no cell proliferation could be detected. After 6 days of asbestos-treatment no living cells could be observed in the cellcultures. Error bars represent standard deviation (S.D.). cant differences could be detected in the mitoticactivity of control and hMF exposed cells after24, 48 and 72 h exposure time.MN induction was detected in AFC cells aftercontinuous exposure to hMF (1 mT) and expo-sure times of 24, 48 and 72 h (Table 1). MNfrequency was significantly increased from 20 / 1000 in controls to 28 / 1000 after 24 h of hMFexposure up to 34 / 1000 after 48 h of exposure( P  0.05). A further increase in MN frequencyafter 72 h of exposure was not detectable. Similardata were found after treatment of AFC cells withasbestos fibers (Table 1). The highest increase inMN induction was found to be after 48 h of asbestos-treatment (36 / 1000). After 72 h asbestos-treatment, the number of MN was significantlyelevated in comparison to control data; however,the MN frequency was smaller (30 / 1000) thanafter 48 h treatment (37 / 1000).Simultaneously performed experiments with co-exposed cultures to hMF and crocidolite asbestosfibers showed neither an increase nor a furtherelevation in MN frequency as well (Table 2). Cellsexposed to hMF (24 or 48 h), which were thenadditionally exposed to asbestos fibers for 24 or48 h, respectively, showed no further increase inMN frequency. Treatment with asbestos fibers for24 h followed by hMF exposure for 48 h also didnot enhance MN induction. Co-exposure to hMFand asbestos fibers for 72 h resulted in a signifi-cant suppressive effect ( P  0.05) on MN fre- Table 1Micronucleus (MN) induction in AFC cells after exposure to1 mT / 50 Hz horizontally oriented magnetic fields (hMF) andafter treatment with asbestos fibers (1   g / cm 2 ) a MN / 1000 cells  S.D.48 h 72 h24 h24.42  6.420.29  6.9 27.44  6.2Control34.00  8.8* 33.8028.20  6.3*hMF  7.5**Asbestos 23.38  4.2 36.63  8.5** 30.22  6.1* a Each exposure time represents the mean of triplicate ex-posed cultures from three experiments.* Represents statistical analysis of exposed and control datadetermined by the Student’s  t -test on the significance level of  P  0.05.** Represents statistical analysis of exposed and controldata determined by the Student’s  t -test on the significancelevel of   P  0.01.  M  .  Simko´ et al  .  /   Toxicology Letters  108 (1999) 47–53   51Table 2Micronucleus (MN) induction in AFC cells after co-exposureto 1 mT / 50 Hz horizontally oriented magnetic fields (hMF)and asbestos fibers (Asb. 1   g / cm 2 ) for different time periodsand for different exposure conditions a MN / 1000  P -value c t -test vs. b Exposure con-cells  S.D.ditionshMF (72 h)33.5  6.5 n.s. d 24 h hMF + 48 hhMF / Asb Asb (48 h) n.s.hMF (72 h)30.75  6.9 n.s.48 h hMF + 24 hhMF / Asb Asb (24 h) n.s.hMF (72 h)72 h hMF / Asb 0.0428  6.2Asb (72 h) n.s.35.8  9.524 h Asb + 48 h hMF (48 h) n.s.Asb / hMF Asb (72 h) n.s. a Each exposure time represents the mean of triplicate ex-posed cultures from three experiments. b Data of co-exposed cells were compared to correspondingdata for significance (Student’s  t -test on the significance levelof   P  0.05). c P -values represents statistical analysis of exposed and con-trol data. d n.s., non-significant. as, in an altered mitotic cell rate. Since no effectwas observed on the cell growth (Fig. 1), and inthe mitotic rate, a clastogenic mechanism of MNformation after hMF-exposure is highly improba-ble. Livingston et al. (1991) reported no changesafter EMF exposure in the cell doubling time andin the duration of G2 / M-phase as well, but theyalso reported no effects on MN induction. How-ever there is a discrepancy in the literature aboutthe genotoxic effectiveness of EMF in vitro. Whilea number of studies reported no genotoxic effects,others found genotoxic effects, such as sister chro-matid exchanges (SCE) and chromosome aberra-tions in human cells in vitro [for review seeLacy-Hulbert et al. (1998)]. Chromatid breaks inbovine lymphocyte cultures after EMF exposurewere reported by D’Ambrosio et al. (1985, 1988).In human lymphocytes no chromosomal effectswere found, whereas an enhanced cell cycle pro-gression and a dose-dependent increase of SCEfrequency could be shown in chemically pre-treated cells followed by EMF exposure (Rosen-thal and Obe, 1989). Antonopoulos et al. (1995)described a stimulation of the cell cycle in humanlymphocytes after exposure to EMF (5 mT), butdetected no influence on the rate of SCE. Norden-son et al. (1994) reported chromosomal aberra-tions in human amniotic cells after exposure to300   T EMF, whereas no genotoxic effects couldbe observed after continuous exposure for 72 h.Summarising, the different results reported in theliterature and the discrepancy between the ob-served data seems to be due to differences in thecell types used, exposure duration and exposureconditions, as well as exposure systems.In contrast to the results obtained after expo-sure to EMF, an altered growth curve and adecreased mitotic cell rate were observed aftertreatment of cells with crocidolite asbestos fibers(Fig. 1). It is known that asbestos induces chro-mosomal breakage and aneuploidy (Barrett et al.,1989). Similar results were obtained by Hesterberget al. (1987) in rat tracheal epithelial cells, by Hartet al. (1992) in Chinese hamster ovary cells and byDong et al. (1994) in rat pleural mesothelial cells.Wydler et al. (1988) observed a decrease in mitoticindex in rat primary fibroblasts exposed to cro-cidolite fibers. Induction of micronuclei in as-quency in hMF / asbestos-treated samples (28 / 1000) when compared to hMF-exposure alone(34 / 1000). However, this significant differencecould not be confirmed when data of co-exposedcells were compared with data of asbestos fibersalone. 4. Discussion These data show that exposure to EMF (1 mT)induces micronuclei in AFC, when EMF is gener-ated in a Helmholtz-coil system and the magneticfield component is oriented horizontally, with re-spect to the cell culture medium (Table 1). How-ever, vertically applied magnetic fields do notcause a significant increase in MN frequency(Simko´ et al., 1998a,b). Disturbances in the G2 / M-phase of the cell cycle, would be expected whenDNA-strand breaks and / or chromosome breaksare involved in MN formation, and such damagewould result in an altered growth curve, as well
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