Progress in Biophysics and Molecular Biology 87 (2005) 213223

Effects of static magnetic elds at the cellular level

has also been reported that treatment with trace amounts of ferrous ions in the cell culture medium and

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www.elsevier.com/locate/pbiomolbio

0079-6107/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pbiomolbio.2004.08.008

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E-mail address: miyakosh@cc.hirosaki-u.ac.jp (J. Miyakoshi).exposure to a static magnetic eld increases DNA damage, which is detected using the comet assay. Inaddition, many studies have found a strong magnetic eld that can induce orientation phenomena in cellculture.r 2004 Elsevier Ltd. All rights reserved.Junji Miyakoshi

Department of Radiological Technology, School of Health Sciences, Faculty of Medicine, Hirosaki University,

66-1 Hon-Cho, Hirosaki 036-8564, Japan

Available online 19 October 2004

Abstract

There have been few studies on the effects of static magnetic elds at the cellular level, compared to thoseof extremely low frequency magnetic elds. Past studies have shown that a static magnetic eld alone doesnot have a lethal effect on the basic properties of cell growth and survival under normal culture conditions,regardless of the magnetic density. Most but not all studies have also suggested that a static magnetic eldhas no effect on changes in cell growth rate. It has also been shown that cell cycle distribution is notinuenced by extremely strong static magnetic elds (up to a maximum of 10T). A further area of interest iswhether static magnetic elds cause DNA damage, which can be evaluated by determination of thefrequency of micronucleus formation. The presence or absence of such micronuclei can conrm whether aparticular treatment damages cellular DNA. This method has been used to conrm that a static magneticeld alone has no such effect. However, the frequency of micronucleus formation increases signicantlywhen certain treatments (e.g., X-irradiation) are given prior to exposure to a 10T static magnetic eld. It

necrotic morphology compared to the control group. These reports suggest that the effect ofexposure to static magnetic elds varies depending on the cell type.

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J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223214Cell cycle analysis of synchronized and non-synchronized HFLFs cells did not revealstatistically signicant differences between the cells exposed to 0.2, 1.0, or 1.5 T for 1 h/day for 5consecutive days and control cells (Wiskirchen et al., 2000). The population doublings did notindicate any growth modulation during exposure. Proliferation kinetics did not provide any hintof modulating effects of repetitive magnetic eld exposure. HL60 and EA2 cells were exposed tostatic magnetic elds of 1.5 and 7.05T, for periods ranging from 1 to 24 h (Schiffer et al., 2003).Cell cycle analysis did not reveal differences between the exposed and control cells.

2. Genotoxic effects

2.1. Mutation

Ikehata et al. (1999) examined possible mutagenic and co-mutagenic effects of strong staticmagnetic elds using the bacterial mutagenicity test. No mutagenic effect of static magnetic eldsup to 5T (1T=10,000G) was detected using four strains of Salmonella typhimurium (TA98,TA100, TA1535 and TA1537) and Escherichia coli (E. coli) WP2 uvrA. The mutation rate in theexposed group was signicantly higher than in the non-exposed group when cells were treatedwith N-ethyl-N0-nitro-N-nitrosoguanidine, N-methyl-N0-nitro-N-nitrosoguanidine, ethylmethane-sulfonate, 4-nitroquinoline-N-oxide, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline or 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide.

An E. coli mutation assay was used to assess the mutagenic effects of strong static magneticelds (Zhang et al., 2003). Various mutant strains of E. coli were exposed up to 9T for 24 h andthe frequencies of rifampicin-resistant mutations were then determined. The expression of thesoxS::lacZ fusion gene was assessed by measuring b-galactosidase activity. The results for survival1. Cell proliferation and cell cycle distribution

Wiskirchen et al. (1999) reported that no statistically signicant differences could be detected inpopulation doublings and cumulative population doublings between exposed and control cellgroups following exposure to a static magnetic eld of 1.5T for a period of 3 weeks. Clonogenicactivity, DNA synthesis, cell cycle, and proliferation kinetics were not altered by exposure to themagnetic eld and repetitive exposure to a static magnetic eld of 1.5 T exerted no effects onproliferation of human fetal lung broblast (HFLF) cells.Raylman et al. (1996) reported that a 64 h exposure to a 7T magnetic eld produced a reduction

in viable cell numbers in melanoma, (HTB 63), ovarian carcinoma, (HTB 77 IP3) and lymphoma(CCL 86) cell lines. Prolonged exposure to the 7T eld appeared to inhibit growth of the threehuman tumor cell lines in vitro. Alterations in cell growth cycle and gross fragmentation of DNAwere excluded as possible contributory factors. Buemi et al. (2001) examined the effects of a staticmagnetic eld of 0.5mT on the cell proliferation/cell death balance in renal cells (VERO) andcortical astrocyte cultures from rats. After 2, 4 and 6 days of exposure to a magnetic eld, theyobserved a gradual decrease in apoptosis and proliferation and a gradual increase in cells with aor mutation obtained with the wild-type E. coli strain GC4468 and its derivatives defective in

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J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223 2152.2. Sister chromatid exchanges

The effects of static homogeneous magnetic elds of 0.5 and 1.0 T on cells from human bloodwere investigated by examining their inuence on the frequency of gross lesions, sister chromatidexchanges and on the proportion of amodal cells (Cooke and Morris, 1981). Neither treatmenthad a signicant effect on any of the parameters measured.

2.3. Micronucleus formation

Long-term exposure to a 10-T static magnetic eld for up to 4 days did not affect cell growthrate or cell cycle distribution in Chinese hamster ovary CHO-K1 cells (Nakahara et al., 2002).Exposure to the static magnetic eld alone did not affect micronucleus formation. In X-ray-irradiated cells, exposure to a 1-T static magnetic eld also did not affect micronucleus formation,but exposure to a 10-T static magnetic eld resulted in a signicant (po0:05) increase inmicronucleus formation induced after a 4-Gy exposure.The effects of a 4.7 T static eld on the frequency of micronucleated cells in CHL/IU cells

induced by mitomycin C (MMC) were studied in vitro (Okonogi et al., 1996). The cells weresimultaneously exposed to the static eld and MMC for 6 h, and then the cells were cultured innormal condition for the micronucleus expression up to 66 h. Exposure to the static magnetic eldfor 6 h signicantly decreased the frequency of MMC-induced micronucleated cell expression afterculture periods of 18, 42, 54 and 66 h. These results suggested that a 4.7 T static magnetic eldmight have exerted an inuence on the DNA damage stage produced by MMC rather than on theformation of micronuclei during the stage following MMC-induced DNA damage.

2.4. DNA damage

Lymphocyte exposure to a static magnetic eld of 7mT did not affect the number of cells withDNA damage in the comet assay (Zmyslony et al., 2000). Incubation of lymphocytes with FeCl2(10 mg/ml) also did not produce detectable damage of DNA. However, when the FeCl2-incubatedlymphocytes were simultaneously exposed to a 7mT static magnetic eld, the number of damagedcells increased signicantly and reached to approximately 20%. Exposure of the cells to staticmagnetic elds and simultaneous treatment with a known oxidant, ferrous chloride, may thereforeaffect oxidative damage of DNA molecules (Zmyslony et al., 2000).

3. Ca2+ and ion transport

Exposure to a 120mT static magnetic eld resulted in a slight reduction in the peakDNA repair enzymes or redox-regulating enzymes showed no effect of exposure. On the otherhand, the mutation frequency was signicantly increased by exposure to a static magnetic eld of9T in soxR and sodAsodB mutants, which are defective in defense mechanisms against oxidativecalcium current amplitude and shift in the currentvoltage relationship in cultured GH3

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Human lymphocytes were simultaneously exposed to 4.75T for static component and0.7mT for the pulsed component at 500MHz generated by an NMR apparatus for 1 h. Exposure

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J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223216to the static magnetic eld together with a pulsed electromagnetic eld increased the Ca2+ inuxwithout any proliferative, activating or proinammatory effect on either unstimulated orphytohemagglutinin (PHA)-stimulated lymphocytes (Aldinucci et al., 2003a). On the otherhand, exposure of Jurkat cells decreased Ca2+ inux and proliferation signicantly. Theseeffects were consistent with the low levels of IL-2 measured in supernatants of the cells afterexposure. It was also reported that exposure of Jurkat cells signicantly decreased theproliferation indexes, which were 0.770.29 and 0.8770.12 24 and 48 h after exposure,respectively. Moreover, in Jurkat cells, the Ca2+ inux, which was higher than in humanperipheral blood mononuclear cells, was reduced signicantly by about 50% after exposure(Aldinucci et al., 2003b).Exposure to strong homogeneous magnetic elds with various magnetic ux densities of less

than 1.6 T had no signicant effect on either active or passive Rb+ inuxes into HeLa cells(Miyamoto et al., 1996). Exposure to a magnetic eld of 2T at different temperatures (1045 1C)also did not cause any change in active or passive Rb+ inux, and no evidence was obtained forthe presence of a phase transition point of the cell membrane between 10 and 37 1C. The patch-clamp method was used to measure transmembrane Na+ and K+ currents in SH-Sy5Yneuroblastoma cells exposed to static magnetic elds of 0.1, 0.5, and 7.5mT (Sonnier et al., 2003).Application of the magnetic elds did not result in detectable changes in any of the actionpotential parameters chosen for this study.There was a slight shift in the currentvoltage relationship and a less than 5% reduction in peak

current during magnetic eld exposure to 125mT in voltage-activated Na+ channels inproliferating GH3 cells (Rosen, 2003). More pronounced was the increase in the activation timeconstant, tm; during and for at least 100 s following exposure to the eld. The temperaturedependence of this phenomenon was probably due to the greater ease with which the liquid crystalmembrane was deformed. The experimental threshold gradient and the calculated threshold eldintensity for blockade of action potentials by these arrays were estimated to be approximately0.02mT/mm and 0.02mT, respectively. These ndings suggested that spatial variation of themagnetic eld was the principal cause of action potential blockade in dorsal root ganglia (DRG)in vitro (Cavopol et al., 1995).

4. Metabolic activity

Onodera et al. (2003) reported that exposure to a magnetic eld of 10T reduced the viability ofPHA-activated T cells in both the CD4(+) and CD8(+) subclasses. The susceptibility oflymphocytes to magnetic eld exposure differed among activated T-cell subtypes. Thecells using the whole-cell patch clamp technique (Rosen, 1996). Absorbance measure-ments at 660 nm of calmodulin-dependent cyclic nucleotide phosphodiesterase activityunder cell free conditions indicated that 30-min exposure to a weak static magnetic eld(20 mT) intensity altered this activity compared to zero magnetic eld exposure (Liboff et al.,2003).magnetic eld exposure signicantly increased the death of PHA-stimulated lymphocytes by

signicant change in shape that could be detected by scanning electron microscopy (Sato et al.,1992). The growth of HeLa cells was not inuenced by exposure to the magnetic eld. Similarly,exposure for 48 h to the magnetic eld had no effect on growth of normal human gingival

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J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223 217broblasts (Gin-1).Gradient magnetic elds of 6T with 60T/m affected the convection of oating cell aggregations

in the cell culture ask, and reversibly changed the direction of convectional ow (Iwasaka et al.,2003a). HeLa cells exhibited stream-like cell distribution patterns in the direction of the appliedmagnetic eld gradient.Dubey et al. (1999) developed an in vitro assay to study neurite elongation of DRG explants

placed onto one end of magnetically aligned collagen gel rods. The extent of neurite elongationfrom chick embryo DRG neurons into the rods was found to be substantially greater than thatobserved in controls and increased with magnetic eld strength, as did the collagen gel rodbirefringence, indicative of collagen bril alignment along the rod axis. These results maytranslate into an improved method of entubulation repair of transected peripheral nerves bydirecting and stimulating axonal growth through a tube lled with magnetically aligned collagenapoptosis. These results suggested that a 10T magnetic eld had acute effects on immune cellsduring cell division, while the eld exposure had a minimal effect on immune cells in non-dividingphases.Sabo et al. (2002) reported that the metabolic activity was reduced in human leukemic cell line

HL-60 exposed to a 1T static magnetic eld for 72 h. The inhibitory effect was also observed inthe presence of the mixture of the antineoplastic drugs, 5-uorouracil, cisplatin, doxorubicin andvincristine.The rate of cell proliferation of human gingival broblasts, histogram of the nuclear DNA

content, rates of lactate production and glucose consumption and the ATP content weredetermined and cell morphology was investigated by both light- and electron-microscopy in astatic 0.2 T magnetic eld for 6 or 8 months (Yamaguchi et al., 1993). The results showed nosignicant differences between exposed and control cells.

5. Morphology

Pacini et al. (1999) examined morphological changes caused by exposure to a 0.2T magneticresonance tomograph on a normal human neuronal cell culture (FNC-B4). The results showeddramatic changes in morphology: vortexes of cells were formed and exposed branched neuritesfeaturing synaptic buttons. Control (sham-exposed) or non-neuronal cells (mouse leukemia, andhuman breast carcinoma cells) did not show any alteration following exposure. Endothelin-1release from FNC-B4 cells was also dramatically reduced after 5min exposure. They also reportedthat human skin broblast cell morphology was modied with a concomitant decrease in theexpression of some sugar residues of glycoconjugates after 1 h exposure to a 0.2T static magneticeld (Pacini et al., 2003). However, cell viability, assessed by the colony-forming assay, wasunaffected.HeLa cells grew at a normal rate for 96 h in the magnetic eld of 1.5 T and showed nogel.

(AP-1) in immature cultured rat hippocampal neurons with high expression of growth-associatedprotein-43. Exposure to the static magnetic eld increased AP-1 DNA binding through expression

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J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223218of Fra-2, c-Jun and Jun-D proteins in immature cultured hippocampal neurons.Hiraoka et al. (1992) reported that c-fos mRNA in the HeLaS3 cells was undetectable in

untreated cells, but the expression was induced in cells by the magnetic eld exposure of0.180.2T for 224 h. Expression of mRNA changed as a function of time with a peak at a 6 hexposure. c-fos was expressed following heat treatment at 45 1C for 10 and 15min, and itsexpression was further enhanced by treating the cells with heat followed by 4 h of exposure to themagnetic eld.Human peripheral blood mononuclear cells were exposed to a 0.5 T eld generated by a

superconducting MRI unit for 24 h. The exposed and sham-exposed cells were maintained at thesame temperature of 2470.2 1C. The 0.5T eld produced a reduced expression of CD69 fromhuman peripheral blood mononuclear cells in vitro, which was enhanced after PHA stimulation(Salerno et al., 1999). An increased release of IFN-g and IL-4 was also found, which was reducedafter PHA stimulation. The release of TNF-a, IL-6 and IL-10 was not modied.

7. Apoptosis

HL-60 cells were exposed to a static magnetic eld of 6mT with or without DNAtopoisomerase I inhibitor, camptothecin for 5 h. The static magnetic eld alone did not produceany apoptogenic or necrogenic effect in HL-60 cells (Teodori et al., 2002). Static magnetic eldsalone or in combination with camptothecin did not affect overall cell viability, but theyaccelerated the rate of cell transition from apoptosis to secondary necrosis after induction ofapoptosis by camptothecin.Simultaneous exposure of rat lymphocytes to a 7mT static magnetic eld and ferrous chloride

(FeCl2) caused an increase in the number of cells with DNA damage (Jajte et al., 2002). Thedamage of DNA molecules by simultaneous exposure to a 7mT static magnetic eld and iron ionsmay lead to cell death by apoptosis or necrosis. No signicant differences were observed betweenunexposed lymphocytes and lymphocytes exposed to a 7mT static magnetic eld. A 3-h6. Gene expression

Miyakoshi and co-workers used a static magnetic eld exposure system, to expose cells to aspatially inhomogeneous 6T with a strong magnetic eld gradient (41.7T/m) or to a spatiallyhomogeneous 10T (Hirose et al., 2003a). HL-60 cells exposed to either a 6 or 10T static magneticeld for periods of 148 h did not exhibit signicant differences in the levels of c-Myc and c-Fosprotein expression, as compared to sham-exposed cells. In contrast, c-Jun protein expressionincreased in HL-60 cells after exposure to the 6T static magnetic eld for 24, 36, 48, and 72 h.Exposure to a strong magnetic eld gradient induced c-Jun expression, which suggested thatstrong magnetic eld gradients may have signicant biological effects, particularly regardingprocesses related to an elevation of c-jun gene expression.Hirai et al. (2002) reported that brief exposure (15min) to a static magnetic eld of 100mT led

to a marked but transient potentiation of binding of a radiolabeled probe for activator protein-1incubation with FeCl2 (10 mg/ml) did not affect cell viability. However, when lymphocytes were

contribute signicantly to this orientation. The observation of magnetic orientation was directedtoward understanding the fundamental microstructural aspects of the erythrocyte.Bull sperm and paramecium cilium were exposed to uniform static magnetic elds to determine

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J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223 219effects on their orientations and measure their anisotropic diamagnetic susceptibility (Dw) (Emuraet al., 2003). The whole bull sperm and the bull sperm heads became oriented perpendicular to themagnetic elds (1.7 T maximum), while the paramecium cilia became parallel to the magneticelds (8T maximum).It has been reported that exposure to strong static magnetic elds on the order of 1T can result

in the orientation of macromolecules such as collagen (Torbet and Ronziere, 1984) and of animalcells in vitro. Human foreskin rboblasts were also oriented using the magnetic orientation ofcollagen with static magnetic elds of 4.0 and 4.7T (Guido and Tranquillo, 1993). Furthermore,osteoblast cells have been shown to be oriented under exposure to a strong static magnetic eld of8T in the absence of collagen (Kotani et al., 2000).Guido and Tranquillo (1993) showed that the degree of bril orientation, and consequently the

elicited contact guidance, could be controlled by independently varying the magnetic eld strengthor temperature during brillogenesis. They presented the rst quantitative correlation of contactexposed to a 7mT static magnetic eld and simultaneously treated with FeCl2, there was asignicant increase in the percentage of apoptotic and necrotic cells accompanied by signicantalterations in cell viability.Flipo et al. (1998) observed that exposure to a static magnetic eld of 0.0250.15T increased

intracellular Ca2+ levels and decreased mitogenic responses in C57BL/6 murine macrophages,splenic lymphocytes or thymic cells. Exposure to the static magnetic eld produced markedlyincreased apoptosis of thymic cells, as determined by ow cytometry.Static magnetic elds above 600 mT were found to decrease the extent of cell death by apoptosis

induced by several agents in different human cell systems of U937 and CEM cells in an intensity-dependent fashion, reaching a plateau at 6mT (Fanelli et al., 1999). The protective effect wasfound to be mediated by the ability of the elds to enhance Ca2+ inux from the extracellularmedium; accordingly, it was limited to those cell systems where Ca2+ inux was shown to have anantiapoptotic effect.

8. Orientation

Murayama (1965) rst reported that the sickled erythrocytes were oriented perpendicular to themagnetic eld at 0.35T. Higashi et al. (1993) assessed the inuence of a uniform static magneticeld (8T maximum) on normal erythrocytes. The erythrocytes were oriented with their diskplanes parallel to the magnetic eld direction. This effect on erythrocytes was detectable at 1 Tand almost 100% of the cells were oriented when exposed to 4T. They also reported that theintact erythrocytes were oriented with their disk planes parallel to the magnetic eld because ofthe diamagnetism of the cell membrane components, particularly the transmembrane proteins(e.g., Band III, glycopholin) and the lipid bilayer (Higashi et al., 1995). In addition, Higashi et al.(1996) observed that the paramagnetism of membrane-bound hemoglobin was thought toguidance (based on cell orientation) with collagen bril orientation (based on birefringence) for

linearly polarized light (Iwasaka and Ueno, 2003). The change in polarized light intensitythrough the lamellar cell assembly under magnetic elds corresponds to behavioral changes

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J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223220in cell components. They speculated that intracellular macromolecules rotated and showeda displacement due to diamagnetic torque forces during 23 h of magnetic eld exposureat 14T.

9. Conclusions

There have been few studies on the effects of static magnetic elds at the cellular level,compared to those of extremely low frequency electromagnetic elds; therefore, a clear evaluationcannot yet be made. Articles cited here varied with respect to magnetic ux density of staticmagnetic elds and exposure times (from minutes to months) and it is difcult to compare themdirectly. Considering these articles comprehensively, the conclusions are as follows: exposure tostatic magnetic elds alone has no or extremely small effects on cell growth and genetic toxicityregardless of the magnetic density. However, in combination with other external factors such asionizing radiation and some chemicals, there is evidence to strongly suggest that a static magneticeld modies their effects. A static magnetic eld may have effects on intracellular ion control,especially Ca2+. Regarding gene expression, although not a consistent view, static magnetic eldsand strong magnetic eld gradients have an effect on c-Jun expression. Effects of static magneticelds on apoptosis are a potentially interesting phenomenon but the data are denitive. Manystudies report orientation by high magnetic ux densities on cells and collagen bers, which havebeen conrmed as effects of static magnetic elds. However, these effects often depended on a celltype and were not found in various types of cells. MRI has gained widespread use for diagnosisand its magnetic ux densities used are increasing. Studies of the effects of static magnetic elds athuman foreskin broblasts cultured in a collagen gel, by using gels of varying orientation resultingfrom different magnetic eld strengths and temperatures during brillogenesis.Hirose et al. (2003b) reported that human glioblastoma A172 cells embedded in collagen gels

were oriented perpendicular to the direction of the static magnetic eld at 10T. A172 cellscultured in the absence of collagen did not exhibit any specic orientation pattern after 7 days ofexposure to the static magnetic eld. Eguchi et al. (2003) observed that after 60 h of magnetic eldexposure, cultured Schwann cells from dissected sciatic nerves of neonatal rats wereoriented parallel to the magnetic eld at 8T. In contrast, Schwann cells, suspended in a mediumwith collagen, oriented in the direction perpendicular to the magnetic eld after 2 h of magneticeld exposure. In this case, Schwann cells aligned along the collagen ber oriented by magneticelds.The morphological effects of strong static magnetic elds on adherent cells are less well

understood than the effects of magnetic elds on red blood cells. Iwasaka et al. (2003b)reported that a high-intensity magnetic eld of 14T affected the morphology of smoothmuscle cell assemblies and that the cell colonies were extended along the direction of the magneticux. The speculated mechanism was that a diamagnetic torque force acts on cytoskeletonbers, which are dynamically polymerizing and depolymerizing during cell division and cellmigration. They also detected the intracellular macromolecule behavior under 14T eld bythe cellular level should therefore be continued.

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ARTICLE IN PRESS

J. Miyakoshi / Progress in Biophysics and Molecular Biology 87 (2005) 213223 223

Effects of static magnetic fields at the cellular levelCell proliferation and cell cycle distributionGenotoxic effectsMutationSister chromatid exchangesMicronucleus formationDNA damage

Ca2+ and ion transportMetabolic activityMorphologyGene expressionApoptosisOrientationConclusionsReferences