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60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells
Mutation Research 400 Ž1998. 313–320
60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells Narendra Singh ) , Henry Lai Bioelectromagnetics Research Laboratory, Department of Bioengineering, UniÕersity of Washington, Seattle, WA, USA Received 28 October 1997; revised 13 January 1998; accepted 13 January 1998
Abstract In previous research, we found an increase in DNA strand breaks in brain cells of rats acutely exposed to a 60 Hz magnetic field Žfor 2 h at an intensity of 0.5 mT.. DNA strand breaks were measured with a microgel electrophoresis assay using the length of DNA migration as an index. In the present experiment, we found that most of the magnetic field-induced increase in DNA migration was observed only after proteinase-K treatment, suggesting that the field caused DNA–protein crosslinks. In addition, when brain cells from control rats were exposed to X-rays, an increase in DNA migration was observed, the extent of which was independent of proteinase-K treatment. However, the X-ray-induced increase in DNA migration was retarded in cells from animals exposed to magnetic fields even after proteinase-K treatment, suggesting that DNA–DNA crosslinks were also induced by the magnetic field. The effects of magnetic fields were also compared with those of a known DNA crosslink-inducing agent mitomycin C. The pattern of effects is similar between the two agents. These data suggest that both DNA–protein and DNA–DNA crosslinks are formed in brain cells of rats after acute exposure to a 60 Hz magnetic field. q 1998 Elsevier Science B.V. All rights reserved. Keywords: 60 Hz magnetic field; Mitomycin C; DNA crosslink; Microgel electrophoresis
1. Introduction An increased use of electric energy in the modern society has subjected the general and occupational population to unprecedented levels of exposure to extremely low frequency ŽELF. Ž- 100 Hz. magnetic and electric fields. The frequency of electric power current in the United States is 60 Hz, whereas it is 50 Hz in European and Asian countries. There ) Corresponding author: Department of Bioengineering, Box 357962, University of Washington, Seattle, WA 98195-7962, USA. Tel.: q1-206-685-2060; fax: q1-206-685-2060; e-mail: [email protected].
are speculations that ELF electromagnetic fields can act as co-promoter or promoter of cancer w1,2x. However, both positive and negative data have been reported in epidemiological studies of cancer risk in the general population with respect to exposure to ELF electromagnetic fields Žcf. review by Wrensch et al. w3x.. Exposure to ELF electromagnetic fields can also affect the nervous system. Recent studies have indicated that occupational exposure could increase the risk of development of neurodegenerative diseases w4,5x. In our previous studies, we have found that acute exposure to a 60 Hz magnetic field caused an increase in DNA single and double strand breaks in
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brain cells of the rat w6x. We have also found that the magnetic field-induced DNA strand breaks are caused by free radicals. The effects can be blocked by treating the animals with free radical scavengers: melatonin, and N-t-butyl-a-phenylnitrone ŽPBN. w7x, and the vitamin E analog trolox wunpublished resultsx. Since it is known that free radicals can cause DNA crosslink formation w8,9x, in the present study, we investigated whether exposure to a 60 Hz magnetic field causes DNA – protein and DNA – DNA crosslinks in brain cells of the rat. The microgel electrophoresis assay w10x was used in our studies to measure DNA strand breaks in individual brain cells. This technique involves making a microgel on a microscopic slide consisting of a cell suspension imbedded in agarose in phosphate buffered saline. The cells are then lysed in the microgel in high salt and detergents and electrophoresed in highly alkaline condition for singlestrand break measurements. The DNA is then stained with a fluorescent dye to enable visual measurement of the extent of DNA migration, which is used as an index of DNA damage. The microgel electrophoresis assay has been suggested to be useful in the detection of DNA crosslinking agents w11x. We have modified the assay to study DNA–protein and DNA–DNA crosslinks in the present experiment. To investigate DNA–protein crosslinks, we treated the slides before electrophoresis with proteinase-K, which removes the protein from the DNA, and compared with DNA migration in identical slides without proteinase-K treatment. DNA is negatively charged and moves to the anode during electrophoresis. Most proteins crosslinked to DNA are positively charged and move to the cathode. DNA–protein crosslinks would impede the movement of DNA during electrophoresis. Thus, removal of protein would free the DNA and an increase in migration would be observed. In addition, some slides were irradiated with a dose of X-rays Ž100 rad. that is known to induce random DNA strand breaks and few crosslinks w12x. Presence of crosslinks in DNA after magnetic field exposure are revealed by an impediment of X-ray-induced enhancement of DNA migration. If magnetic field exposure also causes DNA–DNA crosslinks, X-ray-induced migration of brain cell DNA from magnetic field exposed rats would be impeded even after digestion of proteins with proteinase-K. In the
present study, we also compared the effects of magnetic fields with those of a known DNA crosslink inducing agent mitomycin C ŽMMC. w13x.
2. Methods and procedures 2.1. Animals Male Sprague–Dawley rats Ž2–3 months old, 250–300 g., purchased from B & K Laboratory, Bellevue, WA, were used in this research. They were housed in the room in which they would be exposed to magnetic fields for 24 h before an experiment. The rooms were maintained on a 12-h light–dark cycle Žlight on 0700–1900. and at an ambient temperature of 228C and a relative humidity of 65%. Animals were provided with food and water ad libitum. 2.2. Magnetic field exposure system A Helmholtz coil pair system was used to expose rats to a sinusoidal 60 Hz magnetic field. This system has been described in detail previously w14x. Briefly, a computer program was used to design this Helmholtz coil pair system which is capable of producing a magnetic field with minimal heating and field variations over the exposure area. Each Helmholtz coil is made of two sets of 40 turns each of a6 wire wound in rectangular loops, with minimum internal dimensions of 0.86 = 0.543 m2 . During construction, epoxy was layered between loops to glue them together. This minimizes vibration noise when the coils are activated. The coils are wound on frames fabricated from wood and aluminum and, therefore, are completely shielded against emission of electric fields. They are designed with split windings terminated on multi-terminal blocks so that they may be wired in various series or parallel combinations for impedance matching and connecting to multichannel or multifrequency sources. It is wired such that a switch can be used to put the coils ‘in phase’ to generate magnetic fields or in the ‘bucking mode’. In the ‘bucking mode’, the two coils in each set of coils are activated in an anti-parallel direction Žwith the same current as in the ‘in phase’ condition.
N. Singh, H. Lai r Mutation Research 400 (1998) 313–320
to cancel the fields generated by each other. The ‘bucking mode’ was used as a control condition in our research to control for the possible effects of heat and vibration generated by the coils. By varying the input current to the coils, exposure fields could be set anywhere from the ambient level to the maximum coil designed magnetic field strength of 5.6 mT. With an exposure level set at 1 mT, the heat dissipation from each of the Helmholtz coils is less than 8 W of power. The heat generated is efficiently dissipated due to the large surface area of the coils and good ventilation in the exposure room. The magnetic field during exposure was monitored by input current to the Helmholtz coils and measuring the magnetic flux density with an Enertech EMDEX II magnetic field survey meter. The variation of the magnetic fields within the space between the coils as determined by theoretical calculation and actual measurement was "3% of the mean. The ambient magnetic field in our laboratory Ži.e., when the power supply to the coils was turned off. was 0.14 mT. We have two similar exposure systems in two separate rooms in our laboratory. The systems were used alternatively for magnetic field and ‘bucking’ exposure, thus, both conditions could be run simultaneously. During exposure, rats were housed in a plastic cage Žlength 45 cm, width 21 cm, height 22 cm. with a styrofoam cover. The cage was placed in the center of the space between the coils. 2.3. Experimental procedures and assay method for DNA single strand breaks in brain cells of the rat Animals were exposed in the Helmholtz coil system to a sinusoidal 60 Hz magnetic field for 2 h at a flux density of 0.5 mT or with the bucking condition. A maximum of three rats were exposed in a system at one time. Times of exposure between animals were staggered by 10 min to allow time for tissue sample preparation. Exposure was done between 0800–1000 to control for possible variations in responses due to circadian rhythm. Animals were returned to their home cages after exposure. At 4 h after exposure, animals were sacrificed by placing them one at a time in a closed foam box containing dry ice ŽCO 2 . for 65 s. ŽA cardboard was placed on top of the dry ice to prevent its direct contact with
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the animal.. ŽThis 2-h exposure and 4-h postexposure waiting paradigm is the procedure we used in our previous research w6,7x.. Dry ice was chosen in euthanasia because its use minimized red blood cell contamination of tissue samples that could increase baseline DNA strand break. Procedures after this step were carried out in minimal direct light. Rats were then decapitated with a small animal guillotine and their brains were dissected out immediately for DNA strand break assay. Experiments were run blind, i.e., the two experimenters doing the exposurerbrain dissection and DNA strand break assay, respectively, did not know the treatment Žexposure and drug treatment. conditions of the animals. The microgel electrophoresis assay for DNA single strand breaks in rat brain cells was carried out as described previously by us w15x. All chemicals used in the assay were purchased from Sigma ŽSt. Louis, MO. unless otherwise noted. Immediately after dissection, each whole brain was immersed in ice-cold phosphate-buffered saline ŽPBS. ŽNaCl, 8.01 g; KCl, 0.20 g; Na 2 HPO4 , 1.15 g; KH 2 PO4 , 0.20 g, per liter, pH 7.4. containing 200 m M of N-t-butyl-a-phenylnitrone. It was quickly washed four times with the PBS to remove most of the red blood cells then broken into pieces of approximately 1 mm3 in 5 ml of ice-cold PBS using a tissue press ŽBiospec Product, Bartlesville, OK. w16x. Four more washings with cold PBS removed most of the remaining red blood cells. Finally, in 5 ml of PBS, tissue pieces were dispersed into single-cell suspensions using a P-5000 Pipetman. This cell suspension consisted of different types of brain cells. Ten microliters of this cell suspension were mixed with 0.2 ml of 0.5% agarose Žhigh-resolution 3:1 agarose; Amresco, Solon, OH. maintained at 378C, and 50 m1 of this mixture was pipetted onto a fully frosted slide ŽErie Scientific, Portsmouth, NH.. ŽThese slides were precoated with 50 m l of 0.5% agarose using a 24 = 50 mm2 coverglass. Slides were dried and stored at room temperature before use.. After application of the cell sample, slides immediately covered with a 24 = 50 mm2 a1 coverglass ŽCorning Glass Works, Corning, NY. to make a microgel on the slide. Slides were put in an ice-cold steel tray on ice for 1 min to allow the agarose to gel. The coverglass was removed and 200 m l of agarose solution was layered as before. Slides were then immersed in an ice-cold lysing solution
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Ž2.5 M NaCl, 1% sodium N-lauroyl sarcosinate, 100 mM disodium EDTA, 10 mM Tris base, pH 10. containing 1% Triton X-100, mixed thoroughly an hour before use. To measure single strand DNA breaks, after lysing overnight at 48C, slides were treated with DNAasefree proteinase K Ž1 mgrml, Amresco, Solon, OH. in the lysing solution without detergent for 2 h at 378C. For one set of slides, the proteinase-K digestion step was not carried out. In addition, another set of slides were exposed to 100 rad of X-rays before electrophoresis. Therefore, the experiment consists of three main treatment variables: magnetic field exposure of animals, treatment of microgel slides with proteinase-K, and exposure of slides to X-rays. The sample size was eight for each treatment condition. Slides were then put on the horizontal slab of an electrophoretic assembly ŽHoefer Scientific, San Francisco, CA. modified so that both ends of each electrode are connected to the power supply. One liter of an electrophoresis buffer Ž300 mM NaOH, 0.1% of 8-hydroxyquinoline, 2% dimethyl sulfoxide, and 10 mM tetra-sodium EDTA, pH ) 13. was gently poured into the assembly to cover the slides to a height of 6.5 mm above their surface. After allowing 20 min for DNA unwinding, electrophoresis was started Ž0.4 Vrcm, approximately 250 mA, for 60 min. and the buffer was recirculated Ž100 mlrmin.. At the end of the electrophoresis, slides were then removed from the electrophoresis apparatus and immersed in an excess amount of 1 M ammonium acetate in ethanol Ž5 ml of 10 M ammonium acetate in 45 ml of absolute ethanol. for 30 min. Microgels were then dehydrated in absolute ethanol for 2 h and then submerged in 70% ethanol for 5 min to prevent cracking of microgels during the air drying step. Slides were air-dried at room temperature. One slide at a time was pre-stained with 50 m l of 5% DMSO in 10 mM NaH 2 PO4 and 5% sucrose. Slides were then stained with 50 m l of 1 m M solution of YOYO-1 Žstock, 1 mM in DMSO from Molecular Probes, Eugene, OR. and then covered with a 24 = 50 mm2 coverglass. Slides were examined and analyzed with a Reichert vertical fluorescent microscope Žmodel 2071. equipped with a filter combination for fluorescence isothiocyanate Žexcitation at 490 nm, emission filter at 515 nm, and
dichromic filter at 500 nm.. We measured the length of DNA migration Žin microns., using an eyepiece micrometer, from the beginning of the nuclear area to the last three pixels of DNA perpendicular to the direction of migration up to the leading edge. The migration length is used as the index of DNA strand breaks. Fifty cells were randomly chosen and scored from each slide. However, cells that showed extensive damage with DNA totally migrated out from the nuclear region were not included in the measurement. These highly damaged cells probably resulted from the tissue and cell processing procedures and they occurred equally in the samples irrespective of the treatment. 2.4. Effect of mitomycin C on DNA migration in human lymphocytes Whole blood from a finger prick was collected from a healthy adult men. Lymphocytes were isolated by a method described earlier w10x. Cell viablity was determined by dye exclusion test and was found to be ) 99%. Lymphocytes at a concentration of approximately 500,000 per ml were resuspended in RPMI 1640 ŽLife Technologies, Gaithersberg, MD. supplemented with 10% fetal bovine serum ŽHyclone, Logan, UT.. Lymphocytes were treated for 1 h with 5 m grml of freshly prepared mitomycin C ŽSigma, St. Louis, MO. at 378C in a incubator at 5% CO 2 and 100% humidity. Cells were pelleted and microgels were made as described above. After 1 h of lysis in cold, one set of slides from MMC-treated and control samples was exposed to X-rays Ž100 rad. and 1 mgrml of proteinase K ŽAmresco, Solon, OH. for 2 h at 378C. Slides were then processed for alkaline microgel electrophoresis for estimation of DNA single strand breaks. This experiment was run four times. 2.5. Data analysis Data from the brain cell study were analyzed by the analysis of variance for treatment and interaction effects. Difference between two treatment groups was compared by the Newman–Keuls test. Difference at p - 0.05 was considered statistically significant.
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3. Results Data of the brain cell DNA experiment are presented in Figs. 1 and 2. Since there were three treatment variables Žmagnetic field, proteinase-K, and X-ray., eight treatment conditions were involved in this study. Fig. 1 shows the length of DNA migration Žmean q S.E.M.. of brain cells from the eight treatment groups and Fig. 2 shows the percentage distribution of cells as a function of DNA migration length in the eight groups. Analysis of variance of the data in Fig. 1 showed a significant overall treatment effects Ž F w7,56x s 12.8, p - 0.005., and also significant main treatment effects Žmagnetic field, F w1,56x s 13.11, p - 0.005; proteinase-K, F w1,56x s 34.97, p - 0.005; and X-ray, F w1,56x s 23.93, p - 0.005.. A significant magnetic field = proteinaseK interaction was also observed Ž F w1,56x s 8.89, p - 0.005., whereas no significant interaction effect was observed between magnetic field and X-ray Ž F w1,56x s 3.08, p ) 0.05. and proteinase-K and Xray Ž F w1,56x s 0.13, p ) 0.05.. However, the magnetic field = proteinase-K= X-ray interaction effect was statistically significant Ž F w1,56x s 5.48, p 0.025.. Pair comparison of the treatment groups using the Newman–Keuls test showed that in sample without X-ray exposure Žrepresented by the four bars on the left side of Fig. 1., magnetic field exposure significantly increased DNA single strand breaks in brain cells Ž‘no X-rayrPKrmagnetic field’ vs. ‘no X-
Fig. 1. Effects of in vivo exposure to magnetic field ŽMF., and treatment of cell sample slides with proteinase-K ŽPK. or X-ray on the length of migration Žin microns. of DNA from rat brain cells during microgel electrophoresis. N s8 for each treatment.
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rayrPKrbucking’, p - 0.01. as we had reported in our previous studies w6,7x. However, this is only observed after removal of proteins with proteinase-K. There is no significant difference in migration lengths between the ‘no X-rayr no PKrmagnetic field’ and ‘no X-rayrno PKrbucking’ groups. This suggests that DNA–protein crosslinks are formed after magnetic field exposure which hold the DNA and impede its migration during electrophoresis. X-ray treatment significantly increased the migration lengths on bucking-exposed samples and the increase is observed with and without proteinase treatment Ž ‘ X-rayrPKrbucking vs. no XrayrPKrbucking’, p - 0.01; ‘X-rayrno PKrbucking’ vs. ‘no X-rayrno PKrbucking’, p - 0.05.. This indicates that X-rays, at the dose used in the present experiment, did not induce much DNA–protein crosslinks. However, magnetic field exposure impedes the enhanced migration due to X-rays. Particularly, no significant difference was observed between ‘X-rayrPKrmagnetic field’ and ‘X-rayrPKrbucking’ Ž p ) 0.05.. This suggests that DNA–DNA crosslinks are induced by magnetic field exposure. DNA migration length distribution patterns in Fig. 2 basically confirms this conclusion. Data of treatment with mitomycin C on lymphocytes are shown in Fig. 3. The data also have eight treatment groups as in the brain cells study and included: MMC, proteinase-K, and X-ray treatments. Analysis of variance of the data showed a significant overall treatment effects Ž F w7,24x s 72.95, p 0.001., and also significant main treatment effects ŽMMC, F w1,24x s 22.6, p - 0.001; proteinase-K, F w1,24x s 166.8, p - 0.001; and X-ray, F w1,24x s 266.2, p - 0.001.. A significant MMC= proteinaseK interaction Ž F w1,24x s 37.4, p - 0.001., and MMC= X-ray Ž F w1,24x s 12.6, p - 0.001. interaction effect was observed. However, proteinase-K= X-ray Ž F w1,24x s 3.5, p ) 0.05., and MMC = proteinase-K= X-ray Ž F w1,24x s 1.58, p ) 0.05. interaction effects were statistically nonsignificant. The data also show that DNA not treated with proteinase-K had significantly less migration after MMC treatment Ž‘no X-rayrno PKrcontrol’ vs. ‘no X-rayrno PKrMMC’, p - 0.01.. Mitomycin C probably induced a large amount of DNA crosslinks in these cells which severely impeded DNA migration. Treatment with proteinase-K increased the mi-
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Fig. 2. Percent distribution of cells as a function of DNA migration length of the eight treatment groups.
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Fig. 3. Effects of treatment with mitomycin C ŽMMC., and treatment of cell sample slides with proteinase-K ŽPK. and X-ray on the length of migration Žin microns. of DNA from human lymphocytes during microgel electrophoresis. Open bar s control; shaded bar s MMC-treated. N s 4 for each treatment.
gration Ž‘no X-rayrno PKrMMC’ vs. ‘no XrayrPKrMMC’, p - 0.01; and ‘no X-rayrPKrcontrol’ vs. ‘no X-rayrPKrMMC’, p - 0.05., suggesting the presence of DNA–protein crosslinks. Furthermore, X-ray-induced DNA migration was also impeded by MMC treatment. Particularly, there was no significant difference in DNA migration between ‘X-rayrPKr control’ and ‘X-rayrPKrMMC’ treatments. This indicates that MMC also induced DNA– DNA crosslinks.
4. Discussion Data from the present research suggest that acute exposure to a 60 Hz magnetic field causes DNA– protein and DNA–DNA crosslinks in brain cells of the rat. This is shown by the findings that magnetic field-induced DNA migration was only revealed after proteinase-K digestion, magnetic field exposure impeded X-ray-induced migration, and proteinase-K treatment did not further enhance X-ray-induced migration. Some of the strand breaks we observed are probably resulted from the repair of these crosslinks w17x. The conclusion that magnetic field exposure leads to crosslink formation is further supported by the similarity to the effects of MMC on lymphocyte DNA. The mechanism by which magnetic fields interact with biological system is not well understood. How-
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ever, it has been hypothesized that exposure to magnetic fields increases free radical activity w18,19x. The involvement of free radicals in magnetic fieldinduced effects is further supported by our recent research showing that magnetic field-induced DNA strand breaks can be blocked by free radical scavengers w7x. It is well know that free radicals can cause DNA strand breaks w20x. This could be an iron-mediated process Že.g., via the Fenton reaction.. Bertoncini and Meneghini w21x and Meneghini w22x have proposed that the Fenton reaction-mediated OH-radicals attack DNA at the site where iron is bound. Iron induced OH-radicals have been shown to produce DNA–protein w8x and DNA–DNA w9x crosslinks. Evidence that magnetic fields can affect iron metabolismravailability in cells is provided by a study by Phillips w23x reporting that 60 Hz magnetic field exposure increased the number of transferrin receptors on the surface of tumor cells and in phytohaemagglutinin-stimulated human lymphocytes and our unpublished data showing that magnetic field-induced DNA strand breaks in rat brain cells can be blocked by the iron-chelator deferiprone. Further experiment is needed to investigate the role played by iron in the biological effects of magnetic fields. Formation of DNA crosslinks could have serious consequences on the fate of a cell. DNA crosslinks have been associated with cell death w13x. Two recent studies have reported apoptosis in cells after exposure to a 60 Hz magnetic field w24,25x. Related to the present study is that cell death in the central nervous system could lead to neurodegenerative diseases. Increased risk of Alzheimer’s disease w4x and amyotropic lateral sclerosis w5x have been reported after exposure to electromagnetic fields in occupational environment. Results from this study are important in the setting of guidelines for ELF magnetic field exposure in the public and occupational environments. The magnetic field intensity Ž0.5 mT. used in our study is within the limits contained in current occupational and public environment magnetic field exposure guidelines. For example, the guidelines of the International Nonionizing Radiation Committee of the International Radiation Protection Association for maximum levels of magnetic field exposure for occupational situations are 0.5 mT for workday expo-
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sure and 5 mT for short-term exposure, whereas for the general public it is 0.1 mT for 24 hrday exposure and 1 mT for exposure of a few hours per day.
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Acknowledgements w13x
We thank Mrs. Monserrat Carino for technical assistance. Research described in this paper was supported by grants from the National Institute of Environmental Health Sciences ŽES-06290 and ES08865..
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References w16x w1x B. Holmberg, Magnetic fields and cancer: animal and cellular evidence— an overview, Environ. Health Perspect. 103 Žsuppl 2. Ž1995. 63–67. w2x W. Loscher, M. Mevissen, Animal studies on the role of 50r60-hertz magnetic fields in carcinogenesis, Life Sci. 54 Ž1994. 1531–1543. w3x M. Wrensch, M.L. Bondy, J. Wienche, M. Yost, Environmental risk factors for primary malignant brain tumors: a review, J. Neuro-Oncol. 17 Ž1993. 47–64. w4x E. Sobel, Z. Davanipour, R. Sulkava, T. Erkinjuntti, J. Wikstrom, V.W. Henderson, G. Buckwalter, J.D. Bowman, P.-J. Lee, Occupations with exposure to electromagnetic fields: a possible risk factor for Alzheimer’s disease, Am. J. Epidemiol. 142 Ž1995. 515–524. w5x Z. Davanipour, E. Sobel, J.D. Bowman, Z. Qian, A.D. Will, Amyotropic lateral sclerosis and occupational exposure to electromagnetic fields, Bioelectromagnetics 18 Ž1997. 28–35. w6x H. Lai, N.P. Singh, Acute exposure to a 60-Hz magnetic field increases DNA strand breaks in rat brain cells, Bioelectromagnetics 18 Ž1997. 156–165. w7x H. Lai, N.P. Singh, Melatonin and N-tert-butyl-a-phenylnitrone blocked 60-Hz magnetic field-induced DNA single and double strand breaks in rat brain cells, J. Pineal Res. 22 Ž1997. 152–162. w8x S.A. Altman, T.H. Zastawny, L. Randers-Eichhorn, M.A. Cacciuttolo, S.A. Akman, M. Dizdaroglu, G. Rao, Formation of DNA–protein cross-links in cultured mammalian cells upon treatment with iron ions, Free Radic. Biol. Med. 19 Ž1995. 897–902. w9x D.R. Lloyd, D.W. Phillips, P.L. Carmichael, Generation of putative intrastrand cross-links and strand breaks by transition metal ion-mediated oxygen radical attack, Chem. Res. Toxicol. 10 Ž1997. 393–400. w10x N.P. Singh, M.M. Graham, V. Singh, A. Khan, Induction of
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DNA single-strand breaks in human lymphocytes by low doses of g-rays, Int. J. Radiat. Biol. 68 Ž1995. 563–569. S. Pfuhler, H.U. Wolf, Detection of DNA crosslinking agents with the alkaline comet assay, Environ. Mol. Mutagen. 27 Ž1996. 196–201. A.J. Fornace, J.B. Little, DNA crosslinking induced by X-ray and chemical agents, Biochim. Biophys. Acta 477 Ž1977. 343–355. S.R. Keyes, R. Loomis, M.P. DiGiovanna, C.A. Pritsos, S. Rockwell, A.C. Sartorelli, Cytotoxicity and DNA crosslinks produced by mitomycin analogs in aerobic and hypoxic EMT6 cells, Cancer Commun. 3 Ž1991. 351–356. H. Lai, M.A. Carino, A. Horita, A.W. Guy, Effects of a 60 Hz magnetic field on central cholinergic systems of the rat, Bioelectromagnetics 14 Ž1993. 5–15. H. Lai, N.P. Singh, DNA single- and double-strand DNA breaks in rat brain cells after acute exposure to low-level radiofrequency electromagnetic radiation, Int. J. Radiat. Biol. 69 Ž1996. 513–521. N.P. Singh, A rapid method for the preparation of single cells suspension from solid tissue, Cytometry 31 Ž1998. 229–232. G. Speit, A. Hartmann, The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test Žcomet assay., Mutagenesis 10 Ž1995. 555–559. B. Brocklehurst, J.A. McLauchlan, Free radical mechanism for the effects of environmental electromagnetic fields on biological systems, Int. J. Radiat. Biol. 69 Ž1996. 3–24. R.J. Reiter, Melatonin aspects of exposure to low frequency electric and magnetic fields, in: J.C. Lin ŽEd.., Advances in Electromagnetic Fields in Living Systems, Vol. 2, Plenum, New York, 1997, pp. 1–27. J.L. Farber, Mechanisms of cell injury by activated oxygen species, Environ. Health Perspect. 102 Žsuppl 10. Ž1994. 17–24. C.R. Bertoncini, R. Meneghini, DNA strand breaks produced X by oxidative stress in mammalian cells exhibit 3 -phosphogŽ . lycolate termini, Nucleic Acid Res. 23 1995 2995–3002. R. Meneghini, Iron hemeostasis, oxidative stress, and DNA damage, Free Radic. Biol. Med. 23 Ž1997. 783–792. J.L. Phillips, Transferrin receptors and natural killer cell lysis. A study using Colo 205 cells exposed to 60 Hz electromagnetic fields, Immunol. Lett. 13 Ž1986. 295–299. N.C. Blumenthal, J. Ricci, L. Breger, A. Zychlinsky, H. Solomon, G.C. Chen, D. Kuznetsov, R. Dorfman, Effects of low-intensity AC andror DC electromagnetic fields on cell attachment and induction of apoptosis, Bioelectromagnetics 18 Ž1997. 264–272. J.L. Phillips, M. Campbell-Beachler, O. Ivaschuk, T. IshidaJones, W. Haggnen, Exposure of molt-4 lymphoblastoid cells to a 1 g sinusoidal magnetic field at 60-Hz: effects on cellular events related to apoptosis, in: 1997 Annual Review of Research on Biological Effects of Electric and Magnetic Fields from the Generation, Delivery, and Use of Electricity; WrL Associates, Frederick, MD, 1997.
60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells Narendra Singh ) , Henry Lai Bioelectromagnetics Research Laboratory, Department of Bioengineering, UniÕersity of Washington, Seattle, WA, USA Received 28 October 1997; revised 13 January 1998; accepted 13 January 1998
Abstract In previous research, we found an increase in DNA strand breaks in brain cells of rats acutely exposed to a 60 Hz magnetic field Žfor 2 h at an intensity of 0.5 mT.. DNA strand breaks were measured with a microgel electrophoresis assay using the length of DNA migration as an index. In the present experiment, we found that most of the magnetic field-induced increase in DNA migration was observed only after proteinase-K treatment, suggesting that the field caused DNA–protein crosslinks. In addition, when brain cells from control rats were exposed to X-rays, an increase in DNA migration was observed, the extent of which was independent of proteinase-K treatment. However, the X-ray-induced increase in DNA migration was retarded in cells from animals exposed to magnetic fields even after proteinase-K treatment, suggesting that DNA–DNA crosslinks were also induced by the magnetic field. The effects of magnetic fields were also compared with those of a known DNA crosslink-inducing agent mitomycin C. The pattern of effects is similar between the two agents. These data suggest that both DNA–protein and DNA–DNA crosslinks are formed in brain cells of rats after acute exposure to a 60 Hz magnetic field. q 1998 Elsevier Science B.V. All rights reserved. Keywords: 60 Hz magnetic field; Mitomycin C; DNA crosslink; Microgel electrophoresis
1. Introduction An increased use of electric energy in the modern society has subjected the general and occupational population to unprecedented levels of exposure to extremely low frequency ŽELF. Ž- 100 Hz. magnetic and electric fields. The frequency of electric power current in the United States is 60 Hz, whereas it is 50 Hz in European and Asian countries. There ) Corresponding author: Department of Bioengineering, Box 357962, University of Washington, Seattle, WA 98195-7962, USA. Tel.: q1-206-685-2060; fax: q1-206-685-2060; e-mail: [email protected].
are speculations that ELF electromagnetic fields can act as co-promoter or promoter of cancer w1,2x. However, both positive and negative data have been reported in epidemiological studies of cancer risk in the general population with respect to exposure to ELF electromagnetic fields Žcf. review by Wrensch et al. w3x.. Exposure to ELF electromagnetic fields can also affect the nervous system. Recent studies have indicated that occupational exposure could increase the risk of development of neurodegenerative diseases w4,5x. In our previous studies, we have found that acute exposure to a 60 Hz magnetic field caused an increase in DNA single and double strand breaks in
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brain cells of the rat w6x. We have also found that the magnetic field-induced DNA strand breaks are caused by free radicals. The effects can be blocked by treating the animals with free radical scavengers: melatonin, and N-t-butyl-a-phenylnitrone ŽPBN. w7x, and the vitamin E analog trolox wunpublished resultsx. Since it is known that free radicals can cause DNA crosslink formation w8,9x, in the present study, we investigated whether exposure to a 60 Hz magnetic field causes DNA – protein and DNA – DNA crosslinks in brain cells of the rat. The microgel electrophoresis assay w10x was used in our studies to measure DNA strand breaks in individual brain cells. This technique involves making a microgel on a microscopic slide consisting of a cell suspension imbedded in agarose in phosphate buffered saline. The cells are then lysed in the microgel in high salt and detergents and electrophoresed in highly alkaline condition for singlestrand break measurements. The DNA is then stained with a fluorescent dye to enable visual measurement of the extent of DNA migration, which is used as an index of DNA damage. The microgel electrophoresis assay has been suggested to be useful in the detection of DNA crosslinking agents w11x. We have modified the assay to study DNA–protein and DNA–DNA crosslinks in the present experiment. To investigate DNA–protein crosslinks, we treated the slides before electrophoresis with proteinase-K, which removes the protein from the DNA, and compared with DNA migration in identical slides without proteinase-K treatment. DNA is negatively charged and moves to the anode during electrophoresis. Most proteins crosslinked to DNA are positively charged and move to the cathode. DNA–protein crosslinks would impede the movement of DNA during electrophoresis. Thus, removal of protein would free the DNA and an increase in migration would be observed. In addition, some slides were irradiated with a dose of X-rays Ž100 rad. that is known to induce random DNA strand breaks and few crosslinks w12x. Presence of crosslinks in DNA after magnetic field exposure are revealed by an impediment of X-ray-induced enhancement of DNA migration. If magnetic field exposure also causes DNA–DNA crosslinks, X-ray-induced migration of brain cell DNA from magnetic field exposed rats would be impeded even after digestion of proteins with proteinase-K. In the
present study, we also compared the effects of magnetic fields with those of a known DNA crosslink inducing agent mitomycin C ŽMMC. w13x.
2. Methods and procedures 2.1. Animals Male Sprague–Dawley rats Ž2–3 months old, 250–300 g., purchased from B & K Laboratory, Bellevue, WA, were used in this research. They were housed in the room in which they would be exposed to magnetic fields for 24 h before an experiment. The rooms were maintained on a 12-h light–dark cycle Žlight on 0700–1900. and at an ambient temperature of 228C and a relative humidity of 65%. Animals were provided with food and water ad libitum. 2.2. Magnetic field exposure system A Helmholtz coil pair system was used to expose rats to a sinusoidal 60 Hz magnetic field. This system has been described in detail previously w14x. Briefly, a computer program was used to design this Helmholtz coil pair system which is capable of producing a magnetic field with minimal heating and field variations over the exposure area. Each Helmholtz coil is made of two sets of 40 turns each of a6 wire wound in rectangular loops, with minimum internal dimensions of 0.86 = 0.543 m2 . During construction, epoxy was layered between loops to glue them together. This minimizes vibration noise when the coils are activated. The coils are wound on frames fabricated from wood and aluminum and, therefore, are completely shielded against emission of electric fields. They are designed with split windings terminated on multi-terminal blocks so that they may be wired in various series or parallel combinations for impedance matching and connecting to multichannel or multifrequency sources. It is wired such that a switch can be used to put the coils ‘in phase’ to generate magnetic fields or in the ‘bucking mode’. In the ‘bucking mode’, the two coils in each set of coils are activated in an anti-parallel direction Žwith the same current as in the ‘in phase’ condition.
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to cancel the fields generated by each other. The ‘bucking mode’ was used as a control condition in our research to control for the possible effects of heat and vibration generated by the coils. By varying the input current to the coils, exposure fields could be set anywhere from the ambient level to the maximum coil designed magnetic field strength of 5.6 mT. With an exposure level set at 1 mT, the heat dissipation from each of the Helmholtz coils is less than 8 W of power. The heat generated is efficiently dissipated due to the large surface area of the coils and good ventilation in the exposure room. The magnetic field during exposure was monitored by input current to the Helmholtz coils and measuring the magnetic flux density with an Enertech EMDEX II magnetic field survey meter. The variation of the magnetic fields within the space between the coils as determined by theoretical calculation and actual measurement was "3% of the mean. The ambient magnetic field in our laboratory Ži.e., when the power supply to the coils was turned off. was 0.14 mT. We have two similar exposure systems in two separate rooms in our laboratory. The systems were used alternatively for magnetic field and ‘bucking’ exposure, thus, both conditions could be run simultaneously. During exposure, rats were housed in a plastic cage Žlength 45 cm, width 21 cm, height 22 cm. with a styrofoam cover. The cage was placed in the center of the space between the coils. 2.3. Experimental procedures and assay method for DNA single strand breaks in brain cells of the rat Animals were exposed in the Helmholtz coil system to a sinusoidal 60 Hz magnetic field for 2 h at a flux density of 0.5 mT or with the bucking condition. A maximum of three rats were exposed in a system at one time. Times of exposure between animals were staggered by 10 min to allow time for tissue sample preparation. Exposure was done between 0800–1000 to control for possible variations in responses due to circadian rhythm. Animals were returned to their home cages after exposure. At 4 h after exposure, animals were sacrificed by placing them one at a time in a closed foam box containing dry ice ŽCO 2 . for 65 s. ŽA cardboard was placed on top of the dry ice to prevent its direct contact with
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the animal.. ŽThis 2-h exposure and 4-h postexposure waiting paradigm is the procedure we used in our previous research w6,7x.. Dry ice was chosen in euthanasia because its use minimized red blood cell contamination of tissue samples that could increase baseline DNA strand break. Procedures after this step were carried out in minimal direct light. Rats were then decapitated with a small animal guillotine and their brains were dissected out immediately for DNA strand break assay. Experiments were run blind, i.e., the two experimenters doing the exposurerbrain dissection and DNA strand break assay, respectively, did not know the treatment Žexposure and drug treatment. conditions of the animals. The microgel electrophoresis assay for DNA single strand breaks in rat brain cells was carried out as described previously by us w15x. All chemicals used in the assay were purchased from Sigma ŽSt. Louis, MO. unless otherwise noted. Immediately after dissection, each whole brain was immersed in ice-cold phosphate-buffered saline ŽPBS. ŽNaCl, 8.01 g; KCl, 0.20 g; Na 2 HPO4 , 1.15 g; KH 2 PO4 , 0.20 g, per liter, pH 7.4. containing 200 m M of N-t-butyl-a-phenylnitrone. It was quickly washed four times with the PBS to remove most of the red blood cells then broken into pieces of approximately 1 mm3 in 5 ml of ice-cold PBS using a tissue press ŽBiospec Product, Bartlesville, OK. w16x. Four more washings with cold PBS removed most of the remaining red blood cells. Finally, in 5 ml of PBS, tissue pieces were dispersed into single-cell suspensions using a P-5000 Pipetman. This cell suspension consisted of different types of brain cells. Ten microliters of this cell suspension were mixed with 0.2 ml of 0.5% agarose Žhigh-resolution 3:1 agarose; Amresco, Solon, OH. maintained at 378C, and 50 m1 of this mixture was pipetted onto a fully frosted slide ŽErie Scientific, Portsmouth, NH.. ŽThese slides were precoated with 50 m l of 0.5% agarose using a 24 = 50 mm2 coverglass. Slides were dried and stored at room temperature before use.. After application of the cell sample, slides immediately covered with a 24 = 50 mm2 a1 coverglass ŽCorning Glass Works, Corning, NY. to make a microgel on the slide. Slides were put in an ice-cold steel tray on ice for 1 min to allow the agarose to gel. The coverglass was removed and 200 m l of agarose solution was layered as before. Slides were then immersed in an ice-cold lysing solution
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Ž2.5 M NaCl, 1% sodium N-lauroyl sarcosinate, 100 mM disodium EDTA, 10 mM Tris base, pH 10. containing 1% Triton X-100, mixed thoroughly an hour before use. To measure single strand DNA breaks, after lysing overnight at 48C, slides were treated with DNAasefree proteinase K Ž1 mgrml, Amresco, Solon, OH. in the lysing solution without detergent for 2 h at 378C. For one set of slides, the proteinase-K digestion step was not carried out. In addition, another set of slides were exposed to 100 rad of X-rays before electrophoresis. Therefore, the experiment consists of three main treatment variables: magnetic field exposure of animals, treatment of microgel slides with proteinase-K, and exposure of slides to X-rays. The sample size was eight for each treatment condition. Slides were then put on the horizontal slab of an electrophoretic assembly ŽHoefer Scientific, San Francisco, CA. modified so that both ends of each electrode are connected to the power supply. One liter of an electrophoresis buffer Ž300 mM NaOH, 0.1% of 8-hydroxyquinoline, 2% dimethyl sulfoxide, and 10 mM tetra-sodium EDTA, pH ) 13. was gently poured into the assembly to cover the slides to a height of 6.5 mm above their surface. After allowing 20 min for DNA unwinding, electrophoresis was started Ž0.4 Vrcm, approximately 250 mA, for 60 min. and the buffer was recirculated Ž100 mlrmin.. At the end of the electrophoresis, slides were then removed from the electrophoresis apparatus and immersed in an excess amount of 1 M ammonium acetate in ethanol Ž5 ml of 10 M ammonium acetate in 45 ml of absolute ethanol. for 30 min. Microgels were then dehydrated in absolute ethanol for 2 h and then submerged in 70% ethanol for 5 min to prevent cracking of microgels during the air drying step. Slides were air-dried at room temperature. One slide at a time was pre-stained with 50 m l of 5% DMSO in 10 mM NaH 2 PO4 and 5% sucrose. Slides were then stained with 50 m l of 1 m M solution of YOYO-1 Žstock, 1 mM in DMSO from Molecular Probes, Eugene, OR. and then covered with a 24 = 50 mm2 coverglass. Slides were examined and analyzed with a Reichert vertical fluorescent microscope Žmodel 2071. equipped with a filter combination for fluorescence isothiocyanate Žexcitation at 490 nm, emission filter at 515 nm, and
dichromic filter at 500 nm.. We measured the length of DNA migration Žin microns., using an eyepiece micrometer, from the beginning of the nuclear area to the last three pixels of DNA perpendicular to the direction of migration up to the leading edge. The migration length is used as the index of DNA strand breaks. Fifty cells were randomly chosen and scored from each slide. However, cells that showed extensive damage with DNA totally migrated out from the nuclear region were not included in the measurement. These highly damaged cells probably resulted from the tissue and cell processing procedures and they occurred equally in the samples irrespective of the treatment. 2.4. Effect of mitomycin C on DNA migration in human lymphocytes Whole blood from a finger prick was collected from a healthy adult men. Lymphocytes were isolated by a method described earlier w10x. Cell viablity was determined by dye exclusion test and was found to be ) 99%. Lymphocytes at a concentration of approximately 500,000 per ml were resuspended in RPMI 1640 ŽLife Technologies, Gaithersberg, MD. supplemented with 10% fetal bovine serum ŽHyclone, Logan, UT.. Lymphocytes were treated for 1 h with 5 m grml of freshly prepared mitomycin C ŽSigma, St. Louis, MO. at 378C in a incubator at 5% CO 2 and 100% humidity. Cells were pelleted and microgels were made as described above. After 1 h of lysis in cold, one set of slides from MMC-treated and control samples was exposed to X-rays Ž100 rad. and 1 mgrml of proteinase K ŽAmresco, Solon, OH. for 2 h at 378C. Slides were then processed for alkaline microgel electrophoresis for estimation of DNA single strand breaks. This experiment was run four times. 2.5. Data analysis Data from the brain cell study were analyzed by the analysis of variance for treatment and interaction effects. Difference between two treatment groups was compared by the Newman–Keuls test. Difference at p - 0.05 was considered statistically significant.
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3. Results Data of the brain cell DNA experiment are presented in Figs. 1 and 2. Since there were three treatment variables Žmagnetic field, proteinase-K, and X-ray., eight treatment conditions were involved in this study. Fig. 1 shows the length of DNA migration Žmean q S.E.M.. of brain cells from the eight treatment groups and Fig. 2 shows the percentage distribution of cells as a function of DNA migration length in the eight groups. Analysis of variance of the data in Fig. 1 showed a significant overall treatment effects Ž F w7,56x s 12.8, p - 0.005., and also significant main treatment effects Žmagnetic field, F w1,56x s 13.11, p - 0.005; proteinase-K, F w1,56x s 34.97, p - 0.005; and X-ray, F w1,56x s 23.93, p - 0.005.. A significant magnetic field = proteinaseK interaction was also observed Ž F w1,56x s 8.89, p - 0.005., whereas no significant interaction effect was observed between magnetic field and X-ray Ž F w1,56x s 3.08, p ) 0.05. and proteinase-K and Xray Ž F w1,56x s 0.13, p ) 0.05.. However, the magnetic field = proteinase-K= X-ray interaction effect was statistically significant Ž F w1,56x s 5.48, p 0.025.. Pair comparison of the treatment groups using the Newman–Keuls test showed that in sample without X-ray exposure Žrepresented by the four bars on the left side of Fig. 1., magnetic field exposure significantly increased DNA single strand breaks in brain cells Ž‘no X-rayrPKrmagnetic field’ vs. ‘no X-
Fig. 1. Effects of in vivo exposure to magnetic field ŽMF., and treatment of cell sample slides with proteinase-K ŽPK. or X-ray on the length of migration Žin microns. of DNA from rat brain cells during microgel electrophoresis. N s8 for each treatment.
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rayrPKrbucking’, p - 0.01. as we had reported in our previous studies w6,7x. However, this is only observed after removal of proteins with proteinase-K. There is no significant difference in migration lengths between the ‘no X-rayr no PKrmagnetic field’ and ‘no X-rayrno PKrbucking’ groups. This suggests that DNA–protein crosslinks are formed after magnetic field exposure which hold the DNA and impede its migration during electrophoresis. X-ray treatment significantly increased the migration lengths on bucking-exposed samples and the increase is observed with and without proteinase treatment Ž ‘ X-rayrPKrbucking vs. no XrayrPKrbucking’, p - 0.01; ‘X-rayrno PKrbucking’ vs. ‘no X-rayrno PKrbucking’, p - 0.05.. This indicates that X-rays, at the dose used in the present experiment, did not induce much DNA–protein crosslinks. However, magnetic field exposure impedes the enhanced migration due to X-rays. Particularly, no significant difference was observed between ‘X-rayrPKrmagnetic field’ and ‘X-rayrPKrbucking’ Ž p ) 0.05.. This suggests that DNA–DNA crosslinks are induced by magnetic field exposure. DNA migration length distribution patterns in Fig. 2 basically confirms this conclusion. Data of treatment with mitomycin C on lymphocytes are shown in Fig. 3. The data also have eight treatment groups as in the brain cells study and included: MMC, proteinase-K, and X-ray treatments. Analysis of variance of the data showed a significant overall treatment effects Ž F w7,24x s 72.95, p 0.001., and also significant main treatment effects ŽMMC, F w1,24x s 22.6, p - 0.001; proteinase-K, F w1,24x s 166.8, p - 0.001; and X-ray, F w1,24x s 266.2, p - 0.001.. A significant MMC= proteinaseK interaction Ž F w1,24x s 37.4, p - 0.001., and MMC= X-ray Ž F w1,24x s 12.6, p - 0.001. interaction effect was observed. However, proteinase-K= X-ray Ž F w1,24x s 3.5, p ) 0.05., and MMC = proteinase-K= X-ray Ž F w1,24x s 1.58, p ) 0.05. interaction effects were statistically nonsignificant. The data also show that DNA not treated with proteinase-K had significantly less migration after MMC treatment Ž‘no X-rayrno PKrcontrol’ vs. ‘no X-rayrno PKrMMC’, p - 0.01.. Mitomycin C probably induced a large amount of DNA crosslinks in these cells which severely impeded DNA migration. Treatment with proteinase-K increased the mi-
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Fig. 2. Percent distribution of cells as a function of DNA migration length of the eight treatment groups.
N. Singh, H. Lai r Mutation Research 400 (1998) 313–320
Fig. 3. Effects of treatment with mitomycin C ŽMMC., and treatment of cell sample slides with proteinase-K ŽPK. and X-ray on the length of migration Žin microns. of DNA from human lymphocytes during microgel electrophoresis. Open bar s control; shaded bar s MMC-treated. N s 4 for each treatment.
gration Ž‘no X-rayrno PKrMMC’ vs. ‘no XrayrPKrMMC’, p - 0.01; and ‘no X-rayrPKrcontrol’ vs. ‘no X-rayrPKrMMC’, p - 0.05., suggesting the presence of DNA–protein crosslinks. Furthermore, X-ray-induced DNA migration was also impeded by MMC treatment. Particularly, there was no significant difference in DNA migration between ‘X-rayrPKr control’ and ‘X-rayrPKrMMC’ treatments. This indicates that MMC also induced DNA– DNA crosslinks.
4. Discussion Data from the present research suggest that acute exposure to a 60 Hz magnetic field causes DNA– protein and DNA–DNA crosslinks in brain cells of the rat. This is shown by the findings that magnetic field-induced DNA migration was only revealed after proteinase-K digestion, magnetic field exposure impeded X-ray-induced migration, and proteinase-K treatment did not further enhance X-ray-induced migration. Some of the strand breaks we observed are probably resulted from the repair of these crosslinks w17x. The conclusion that magnetic field exposure leads to crosslink formation is further supported by the similarity to the effects of MMC on lymphocyte DNA. The mechanism by which magnetic fields interact with biological system is not well understood. How-
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ever, it has been hypothesized that exposure to magnetic fields increases free radical activity w18,19x. The involvement of free radicals in magnetic fieldinduced effects is further supported by our recent research showing that magnetic field-induced DNA strand breaks can be blocked by free radical scavengers w7x. It is well know that free radicals can cause DNA strand breaks w20x. This could be an iron-mediated process Že.g., via the Fenton reaction.. Bertoncini and Meneghini w21x and Meneghini w22x have proposed that the Fenton reaction-mediated OH-radicals attack DNA at the site where iron is bound. Iron induced OH-radicals have been shown to produce DNA–protein w8x and DNA–DNA w9x crosslinks. Evidence that magnetic fields can affect iron metabolismravailability in cells is provided by a study by Phillips w23x reporting that 60 Hz magnetic field exposure increased the number of transferrin receptors on the surface of tumor cells and in phytohaemagglutinin-stimulated human lymphocytes and our unpublished data showing that magnetic field-induced DNA strand breaks in rat brain cells can be blocked by the iron-chelator deferiprone. Further experiment is needed to investigate the role played by iron in the biological effects of magnetic fields. Formation of DNA crosslinks could have serious consequences on the fate of a cell. DNA crosslinks have been associated with cell death w13x. Two recent studies have reported apoptosis in cells after exposure to a 60 Hz magnetic field w24,25x. Related to the present study is that cell death in the central nervous system could lead to neurodegenerative diseases. Increased risk of Alzheimer’s disease w4x and amyotropic lateral sclerosis w5x have been reported after exposure to electromagnetic fields in occupational environment. Results from this study are important in the setting of guidelines for ELF magnetic field exposure in the public and occupational environments. The magnetic field intensity Ž0.5 mT. used in our study is within the limits contained in current occupational and public environment magnetic field exposure guidelines. For example, the guidelines of the International Nonionizing Radiation Committee of the International Radiation Protection Association for maximum levels of magnetic field exposure for occupational situations are 0.5 mT for workday expo-
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sure and 5 mT for short-term exposure, whereas for the general public it is 0.1 mT for 24 hrday exposure and 1 mT for exposure of a few hours per day.
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Acknowledgements w13x
We thank Mrs. Monserrat Carino for technical assistance. Research described in this paper was supported by grants from the National Institute of Environmental Health Sciences ŽES-06290 and ES08865..
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