effect of a 7-tesla homogeneous magnetic field on mammalian cells

7
Ž . Bioelectrochemistry and Bioenergetics 49 1999 57–63 www.elsevier.comrlocaterbioecechem Effect of a 7-tesla homogeneous magnetic field on mammalian cells Hideki Sakurai a , Kazumasa Okuno a , Atsushi Kubo b , Kayoko Nakamura b , Makoto Shoda a, ) a Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226 8503, Japan b Department of Radiology, School of Medicine, Keio UniÕersity, 35 Shinanomachi, Shinjyuku-ku, Tokyo, Japan Received 11 June 1999; received in revised form 20 September 1999; accepted 27 September 1999 Abstract When two types of mammalian cells, mouse leukemia cells, P388, and Chinese hamster fibroblast cells, V79, were grown under a Ž. Ž . 7-tesla T homogeneous magnetic field which was produced by a newly constructed superconducting magnet biosystem SBS , the growth pattern of cells under 7 T magnetic field and the geomagnetic field control showed no differences. The DNA distribution of the two cells was compared by flow cytometry after exposure to 7 T for 3 and 24 h, but there was no significant differences between magnet-exposed cells and unexposed cells. When the two types of cells were exposed to different concentrations of the antitumor agent, Ž . bleomycin BLM , for 3 h under 7 T, their viable cell numbers were almost the same as that of the control although sensitivity to BLM was different between the two cells. These results suggest that the 7 T homogeneous magnetic field exerts no adverse effects on mammalian cells. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Mouse leukemia cells; Chinese hamster fibroblast cells; High magnetic field 1. Introduction With the recent progress in the development of super- conducting materials, superconducting magnet systems will accelerate the application of strong magnetic fields. Conse- quently, the chances of humans being exposed to strong magnetic fields will increase in the future. Although sev- eral studies have dealt with effect of strong magnetic fields wx on the function and morphology of various cells 1 , the results are often conflicting. No sufficient scientific con- clusions have been drawn yet concerning the effects of magnetic fields on living organisms, partly due to the lack of appropriate measuring instruments. In order to conduct detailed experiments, we developed a superconducting Ž . magnet biosystem SBS , and bacterial growth under ho- mogeneous and inhomogeneous magnetic fields was mainly w x investigated 2–4 . In this study, we investigated growth, DNA distribution during cell cycle and drug sensitivity of two types of Ž. mammalian cells under 7 tesla T homogeneous magnetic field in SBS which was modified for cultivation of mam- malian cells. ) Corresponding author. Tel.: q81-45-9245274; fax: q81-45-9245276; e-mail: [email protected] 2. Experimental method 2.1. Magnet system The SBS was described in detail in previous papers w x 5,6 and only a brief outline of its structure and character- istics is given here. The SBS consists of a superconducting magnet, a control for geomagnetic field, an incubator unit, and a temperature-controlled water supply unit, as shown Ž in Fig. 1. The superconducting magnet has a 16-cm in . diameter bore in the horizontal direction which can pro- duce a magnetic strength of 0.5 to 7 T. The distribution of the axial magnetic field strength of the SBS, measured by a flux meter in situ in the magnetic bore when a magnetic w x field of 7 T was shown in previous papers 3,4 . An area, 16 cm in diameter = 20 cm long in the bore is the homoge- neous magnetic field region, outside of which the magnetic field gradually decreases and the maximum gradient of the magnetic field is formed with a gradient of 23 Trm. As the growth rate of mammalian cells is significantly lower than that of bacteria, the precise temperature control, aseptic CO supply and 100% humidity are essential for 2 maintenance of stable growth of mammalian cells. There- fore, the following modification of SBS was conducted. The incubator unit designed for mammalian cells is shown Ž . in Fig. 2. The incubator unit 180 = 119 = 26 mm which 0302-4598r99r$ - see front matter q 1999 Elsevier Science S.A. All rights reserved. Ž . PII: S0302-4598 99 00066-5

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Page 1: Effect of a 7-tesla homogeneous magnetic field on mammalian cells

Ž .Bioelectrochemistry and Bioenergetics 49 1999 57–63www.elsevier.comrlocaterbioecechem

Effect of a 7-tesla homogeneous magnetic field on mammalian cells

Hideki Sakurai a, Kazumasa Okuno a, Atsushi Kubo b, Kayoko Nakamura b, Makoto Shoda a,)

a Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226 8503, Japanb Department of Radiology, School of Medicine, Keio UniÕersity, 35 Shinanomachi, Shinjyuku-ku, Tokyo, Japan

Received 11 June 1999; received in revised form 20 September 1999; accepted 27 September 1999

Abstract

When two types of mammalian cells, mouse leukemia cells, P388, and Chinese hamster fibroblast cells, V79, were grown under aŽ . Ž .7-tesla T homogeneous magnetic field which was produced by a newly constructed superconducting magnet biosystem SBS , the

growth pattern of cells under 7 T magnetic field and the geomagnetic field control showed no differences. The DNA distribution of thetwo cells was compared by flow cytometry after exposure to 7 T for 3 and 24 h, but there was no significant differences betweenmagnet-exposed cells and unexposed cells. When the two types of cells were exposed to different concentrations of the antitumor agent,

Ž .bleomycin BLM , for 3 h under 7 T, their viable cell numbers were almost the same as that of the control although sensitivity to BLMwas different between the two cells. These results suggest that the 7 T homogeneous magnetic field exerts no adverse effects onmammalian cells. q 1999 Elsevier Science S.A. All rights reserved.

Keywords: Mouse leukemia cells; Chinese hamster fibroblast cells; High magnetic field

1. Introduction

With the recent progress in the development of super-conducting materials, superconducting magnet systems willaccelerate the application of strong magnetic fields. Conse-quently, the chances of humans being exposed to strongmagnetic fields will increase in the future. Although sev-eral studies have dealt with effect of strong magnetic fields

w xon the function and morphology of various cells 1 , theresults are often conflicting. No sufficient scientific con-clusions have been drawn yet concerning the effects ofmagnetic fields on living organisms, partly due to the lackof appropriate measuring instruments. In order to conductdetailed experiments, we developed a superconducting

Ž .magnet biosystem SBS , and bacterial growth under ho-mogeneous and inhomogeneous magnetic fields was mainly

w xinvestigated 2–4 .In this study, we investigated growth, DNA distribution

during cell cycle and drug sensitivity of two types ofŽ .mammalian cells under 7 tesla T homogeneous magnetic

field in SBS which was modified for cultivation of mam-malian cells.

) Corresponding author. Tel.: q81-45-9245274; fax: q81-45-9245276;e-mail: [email protected]

2. Experimental method

2.1. Magnet system

The SBS was described in detail in previous papersw x5,6 and only a brief outline of its structure and character-istics is given here. The SBS consists of a superconductingmagnet, a control for geomagnetic field, an incubator unit,and a temperature-controlled water supply unit, as shown

Žin Fig. 1. The superconducting magnet has a 16-cm in.diameter bore in the horizontal direction which can pro-

duce a magnetic strength of 0.5 to 7 T. The distribution ofthe axial magnetic field strength of the SBS, measured bya flux meter in situ in the magnetic bore when a magnetic

w xfield of 7 T was shown in previous papers 3,4 . An area,16 cm in diameter=20 cm long in the bore is the homoge-neous magnetic field region, outside of which the magneticfield gradually decreases and the maximum gradient of themagnetic field is formed with a gradient of 23 Trm.

As the growth rate of mammalian cells is significantlylower than that of bacteria, the precise temperature control,aseptic CO supply and 100% humidity are essential for2

maintenance of stable growth of mammalian cells. There-fore, the following modification of SBS was conducted.The incubator unit designed for mammalian cells is shown

Ž .in Fig. 2. The incubator unit 180=119=26 mm which

0302-4598r99r$ - see front matter q 1999 Elsevier Science S.A. All rights reserved.Ž .PII: S0302-4598 99 00066-5

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( )H. Sakurai et al.rBioelectrochemistry and Bioenergetics 49 1999 57–6358

Fig. 1. Schematic diagram of the SBS used for mammalian cells.

is made of acrylic resin was designed to accommodate two60 ml tissue culture flasks or one 24-well plate for staticcultivation of mammalian cells. Inside the incubator, asilicone tube was placed along the walls. Two acrylic resin

Žwater bath units 169=85=20 mm and 136=75=10.mm were placed on the top and the bottom of the

incubator unit, respectively. Air containing 5% CO gas in2

a cylinder was supplied at 10 mlrmin through a 0.45-mm

Fig. 2. Structure of incubation unit prepared for growth of mammalian cells, which consists of two water baths and a silicone tube to control thetemperature.

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sterile membrane filter to the incubator unit. A waterŽ .container 70=27=10 mm filled with sterilized water

was placed to maintain 100% humidity inside the incuba-tor. Temperature control by the water supply unit is real-ized by supplying water to the two water bath units and asilicone tube inside the incubator to maintain the tempera-ture of flasks or 24-well plate at 37"0.48C. The sameincubator unit was also prepared for the control.

2.2. Growth of two types of mammalian cells used

Mouse leukemia cells, P388, which grow in suspensionculture were used. The medium is RPMI medium supple-

Ž . Ž .mented with 10% fetal bovine serum FBS Gibco . TheRPMI medium consists of 10.4 g RPMI medium 1640Ž .Gibco and 2 g NaHCO in 1 l water. The cell suspension3

of P388 in the growth phase was centrifuged at 1000 rpmfor 5 min and the supernatant was removed by a sterilepipette. Five milliliter of fresh medium was added to theprecipitated cells to resuspend them. An aliquot of 0.8 mlof cell suspension was transferred into two 24-well platesŽ .Corning and one plate was placed in an incubator unitunder 7 T homogeneous magnetic field and the other undergeomagnetic field as a control. The two plates were culti-vated statically at 378C at 100% humidity in air containingof 5% CO for 8 days. Cells from 2–3 wells were sampled2

daily from the two plates, 0.8 ml of trypan blue solutionŽ .Wako, Osaka, Japan was added and unstained cells werecounted as viable cells using a hemocytometer. The result-ing growth curves were compared.

Chinese hamster fibroblast cells, V79, which grow inmonolayer culture were also used. The medium is F-10medium supplemented with 10% FBS. The F-10 medium

Ž .Fig. 3. Growth of P388 cells under 7 T magnetic field ` and geomag-Ž .netic field ^ at 378C. The error of each datum was less than "5%.

Ž .Fig. 4. Growth of V79 cells under 7 T magnetic field ` and geomag-Ž .netic field ^ at 378C. The error of each datum was less than "5%.

Ž .consists of 9.8 g F-10 nutrient mixture Gibco and 1.2 gNaHCO in 1 l water. V79 cells grown on the surface of a3

Žtissue culture flask 60 ml, Asahi Techno glass, Tokyo,. Ž .Japan were treated with 0.25 ml of 0.25% trypsin Gibco

Žsolution in phosphate-buffered saline Na HPO P12H O2 4 2. Ž .2.9 g, KH PO 0.2 g, NaCl 8 g, KCl 0.2 g in 1 l PBS2 4

and the suspended cells were mixed with 5 ml freshmedium. An aliquot of 0.8 ml of the cell suspension wasadded to two 24-well plates and the plates were placedunder magnetic field and under geomagnetic field as acontrol. Cultivation was carried out under the same condi-tions as those of P388. Every day, cells in 2–3 wells weredispersed with 0.25% trypsin in PBS and the viable cellnumber was counted using a hemocytometer after trypanblue exclusion treatment.

2.3. DNA distribution during cell cycle

P388 cells were precultured in the tissue culture flaskseach of which contained 5 ml culture medium and then,1% of the preculture was inoculated into two tissue cultureflasks and cultivated for 1 day. Then, one flask wasexposed to 7 T homogeneous magnetic field for 3 or 24 hat 378C and the other was exposed to geomagnetic field ascontrol. The cell suspension of P388 was washed with PBS

Žand then treated with 1 ml of PI solution 50 mgrmlŽ . Ž .propidium iodide Sigma , 0.1% sodium citrate Wako ,Ž .0.2% Nonidet p-40 Nakarai Chem., Tokyo , 0.25 mgrml

Ž ..RNase Sigma for more than 10 min at room temperatureto stain DNA. Then, the cell suspension was subjected to a

Ž .flow cytometer EPICS PROFILE II, Coulter for theanalysis of the distribution of DNA at different phases ofthe cell cycle and the resulting histograms were compared

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between the magnet-exposed and unexposed cells. Then,the cells of V79 were trypsin-treated and subjected to aflow cytometer in a manner similar to P388.

2.4. Cytotoxicity

A 0.6-ml aliquot of P388 cells in the growth phase waspoured into each well of a 24-well plate to make aconcentration of about 4=104 cellsrml and cultivated for

Ž .12 h. Then, 0.6 ml of medium RPMI mediumq10%FBSŽ .containing different concentrations of bleomycin BLM

Ž .Nihon Kayaku, Tokyo was added to each well and theplate was exposed to 7 T homogenous magnetic field for 3h at 378C. After exposure, the cells were recovered fromthe wells and centrifuged at 10000 rpm for 10 min. Theprecipitated cells were resuspended in fresh medium andcultivated for 2 days under geomagnetic field and theviable cell number was counted using a hemocytometer.The trypan blue dye exclusion method was applied toassay for cell viability, and relative cell viability wascompared between samples exposed to the magnetic fieldand the geomagnetic field.

The V79 cells in a monolayer culture were treated with3 Žtrypsin and 2=10 cells in 0.6 ml medium F-10 medium

.q10%FBS were added to each well of a 24-well plateand cultivated for 12 h. Then, 0.2 ml of the mediumcontaining different concentrations of BLM was added intoeach well and the plate was exposed to 7 T homogenousmagnetic field for 3 h at 378C. After exposure, the culturecontaining BLM was removed by a pipette and the cellswere washed in fresh medium. Then, 1 ml of fresh mediumwas added, and the plate was cultivated for 3 days undergeomagnetic field. The cells were trypsin-treated and thecell suspension was subjected to trypan blue treatment.Then the viable cell number was counted using a hemocy-tometer. Relative cell viability was compared in the samemanner as that for P388.

3. Result

Fig. 3 shows the growth curves of viable P388 cellsunder 7 T homogeneous magnetic field and under geomag-netic field which were measured using a hemocytometer.The experiment was repeated at least five times, and thederivation of each datum was within "5%. No significantdifferences in the growth patterns between the magneticfield-exposed cells and control cells were observed.

Fig. 4 shows the growth curves of viable V79 cellsunder 7 T homogeneous magnetic field and under geomag-netic field. The 7 T homogeneous magnetic field gave nospecific effect on the rate of growth of V79.

Ž . Ž .Fig. 6. Ratio of viable cell number of P388 cells A and V79 cells Bgrown under geomagnetic field for 2 and 3 days, respectively, after thetwo types of cells were exposed to BLM at various concentrations for 3 hunder 7 T homogeneous magnetic field. The ratio of viable cell numberwithout BLM was set at 100%. The bars show the deviation of the data.`: cells of P388 or V79 exposed to BLM. ^: cells of P388 or V79unexposed to BLM.

Fig. 5 shows the histogram of the cell number againstthe DNA content as measured by flow cytometry of thetwo types of cells. The ordinate is DNA content per unitcell and the abscissa is cell number for each DNA content.The big peak corresponded to cells of G0 and G1 phasesof the mammalian cell cycle. A small peak on the right

Fig. 5. Distribution of number of cells containing DNA in different phases as measured by flow cytometry after V79 or P338 cells were grown under 7 Tmagnetic field for 3 and 24 h. The control for each experiment is also shown.

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side shows that the cells of G2 and M phases existedbetween the two peaks. Although the histograms are differ-ent between P388 and V79, no significant differences wereobserved between unexposed and exposed cells at anyexposure time for each cell. When the measurement wasrepeated for each cell, the histogram patterns were notexactly the same at each time, but there was no significantdifference of the pattern between 7 T exposed cells andgeomagnetic field exposed cells.

Fig. 6 shows the relative ratio of viable cell numbers ofP388 and V79 cells when the viability at zero BLMconcentration was set at 100%. When they were exposedto BLM for 3 h under 7 T magnetic field and thenincubated for 2 days or 3 days under geomagnetic field,the sensitivity to BLM differed between them, but nosignificant differences were detectable in the magneticfield-exposed cells and control cells.

4. Discussion

As there are many cellular components that are suscep-tible to magnetic field, such as water, proteins, nucleic

w xacids and lipid bilayers 7–10 , the effect of a strongmagnetic field on cells is anticipated. The effects of mag-netic field on mammalian cells have been reported fromdifferent aspects of biological and biochemical phenom-ena. They include inhibition of poly-ADP ribosylation of

w x w xproteins 11 , induction of c-fos gene expression 12 ,w xorientation of transmembrane protein or lipid bilayer 13 ,

change in Ca concentration such as in neurotransmissionw x w x14,15 and perfusion change in neurons 16 . However,these changes are not necessarily reflected by the changein growth or morphology especially in suspension cultures.However, in monolayer culture, the effect of magneticfield is related to the ability of attachment. Even thoughthe exposure to magnetic field gave no apparent change incell number or viability of the cells which exhibited mono-

w xlayer growth, cell adhesion ability was impaired 17 and adecrease in the ability of attachment and growth occurredw x w x18 . Impaired cell adhesion induced apoptosis 19 , whichmight eventually lead to the death of the cells. Therefore,we selected two typical mammalian cells; P388 whichgrows in suspension culture and V79 which exhibits mono-layer growth; and SBS was used for the different stages ofexperiments. Under 7 T homogeneous magnetic field, theapparent growth of the two cells was almost the same asthat in the control. Using flow cytometry, the distributionof DNA content was compared between the cells exposedto 7 T and those in control, but no significant differencewas observed. As the exposure to inhomogeneous mag-netic field led to a significant change in morphology and

w xviability of cells in microbial experiments 2–4 , the inho-mogeneous magnetic field in SBS may exhibit significantchange even in mammalian cells.

As the effects of magnetic field are related to experi-mental conditions, such as the quality of magnetic field or

the period and sequence of the exposure, magnetic fieldw xsensitivity depends on cell type 20,21 or relative amount

w xof cultured cells in the G1 phase 22 .The fact that the concentration and lifetime of free

radicals are influenced by magnetic field is scientificallyw xestablished 6 . An increases in the yield or lifetime of

radicals by magnetic field will cause membrane damage,w xcell lysis or membrane permeability 23 . The increase in

the death rate of human erythrocytes due to free radicalsw x24 . There are many cell components that can react with

Ž .radicals e.g., lipids, proteins , so the local radical concen-trations generated intracellularly may be influenced bymagnetic field. Therefore, if exposure to magnetic fieldenhances the probability of radical reactions with cellular

w xcomponents 25 , the effect of magnetic field will berevealed. The antitumor function of BLM is based oncleavage of double-stranded DNA with radical reactionsw x26 and its cytotoxicity was potentiated by additives in

w xV79 cells 27 . Furthermore, combined exposure to mag-netic field and drugs or X-rays was investigated and theresults are as follows: no change in growth or sensitivity to

w x w xX-rays 28,29 , enhanced cytotoxicity of drugs 30 andw xdecrease in viability after X-ray irradiation 31 were ob-

served. Therefore, if a strong magnetic field enhances theradical pair lifetime, the sensitivity of the cells to BLMshould be increased. However, experiments have shownthat these effects cannot be expressed in this system.Radical generation and attack by radicals in cells aresupposed to be nonspecific, and this indicates that theintracellular radical extinguishers prevail over the activityof radicals. An in vitro test using a mixture of plasmidpBR322 and BLM under 7 T magnetic field revealed that

Ž .DNA cleavage of pBR322 was stimulated data not shown .This indicates the clear difference in the magnetic effectbetween in vitro test and in vivo test.

Higher field strength MRI is now under considerationand the magnetically levitated train in Japan is at thetesting stage. We conclude that the 7 T homogeneousmagnetic field would cause the least adverse effect onmammalian cells because it did not cause any significantdifferences in cell proliferation, cell cycle and sensitivityto drug in the two types of mammalian cells. Similarexperiments are under progress under an inhomogeneousmagnetic field in consideration of spatial and transientdistributions of magnetic field in some facilities.

Acknowledgements

We thank Dr. J. Miyakoshi, Kyoto University for pro-viding valuable information on the design of an incubator.

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