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Increased dexamethasone-induced apoptosis of thymocytes from mice exposed to long-term extremely low frequency magnetic fields

Increased dexamethasone-induced apoptosis of thymocytes from mice exposed to long-term extremely low frequency magnetic fields
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  Bioelectromagnetics 19:131–135 (1998) Brief Communication Increased Dexamethasone-Induced Apoptosis of Thymocytes FromMice Exposed to Long-Term Extremely Low Frequency Magnetic Fields Sebastia ˜ o J. Ismael, 1 Fernando Callera, 1  Aglair B. Garcia, 1 Oswaldo Baffa, 2 and Roberto P. Falca ˜ o 1 * 1 Department of Clinical Medicine, School of Medicine, Ribeira˜ o Preto, Sa˜ o Paulo, Brazil   2 Department of Physics and Mathematics, FFCLRP-USP, Ribeira˜ o Preto, Sa˜ o Paulo, Brazil  To address the effect of extremely low frequency electromagnetic fields on programmed cell deathwe assessed both the spontaneous and dexamethasone (Dex)-induced apoptosis of thymocytes andspleen cells from mice submitted to a long-term continuous exposure of a 0.4–1.0  m T 60 Hz magneticfield or an 8–20  m T direct current (DC) magnetic field. Dex-induced apoptosis but not spontaneousapoptosis was substantially increased in thymocytes from 0.4 to 1.0  m T 60 Hz field-exposed animals.Spontaneous apoptosis and Dex-induced apoptosis of spleen cells were not affected by the0.4–1.0  m T 60 Hz field exposure. In addition, spontaneous apoptosis and Dex-induced apoptosis of thymocytes and spleen cells from mice exposed to an 8–20  m T DC field were similar to the controls.These findings represent the first demonstration that thymocytes from mice exposed to a long-term0.4–1.0  m T 60 Hz field may show abnormal response to Dex apoptotic stimuli.  Bioelectromagnetics19:131–135, 1998.    1998 Wiley-Liss, Inc. Key words: magnetic fields; apoptosis; dexamethasone-induced apoptosis; thymocytes INTRODUCTION  Apoptosis is a morphologically distinct form of programmed cell death that occurs through the activa-Electromagnetic fields (EMF) are characterized tion of a cell-intrinsic suicide mechanism which playsas non-ionizing radiation emitted from devices that an important role in the development and homeostasisproduce, transmit, or use electric power. Increasingly, of many tissues [Steller, 1995]. To address the possiblepublic attention has focused on whether exposure to effect of ELF-EMF on programmed cell death we haveEMF could result in adverse human health effects studied spontaneous apoptosis and dexamethasone[Shulman, 1990; Savitz et al., 1989; Sagan, 1992; (Dex)-induced apoptosis of thymocytes and spleenBates, 1991]. Experimental exposures in vivo to differ- cells from mice exposed to an ELF-EMF (60 Hz) RMSent extremely low frequency (ELF-EMF) flux densities[Ahlbom and Feychting, 1993; Lo¨scher and Mevissen,1994] have been associated with the development [Be- Contract grant sponsor: Conselho Nacional de Desenvolvimento Cien- niashvili et al., 1991; Rannug et al., 1993], implantation  tifico e Tecnolo´gico (CNPq); Contract grant sponsor: Financiadora deEstudos e Projetos (FINEP). [Thomson et al., 1988], and chemical induction of tu-mor cells [Rannug et al., 1993; McLean et al., 1991; *Correspondence to: Roberto P. Falca˜o, M.D., Ph.D., Department of  Stuchly et al., 1992; Lo¨scher et al., 1993]. However,  Clinical Medicine, School of Medicine, Av. Bandeirantes 3900, Ribeira˜oPreto, Sa˜o Paulo, 14049-900, Brazil. E-mail: rpfalcao@fmrp.usp.br there are few data concerning the effects of EMF onthe mechanisms of cell death or apoptosis [Bellossi et  Received for review 11 February 1997; Revision received 18 September1997 al., 1996].   1998 Wiley-Liss, Inc.  132 Ismael et al. value of 0.4–1.0  m T [Mevissen et al., 1993] or an evaluation at necropsy). The level of fighting amonganimals was assessed by the clinical observation of the8–20  m T direct current (DC) magnetic field.skin lesions and was similar in all cages.After 12 months, animals from the 3 groups (con-trols weighing 47.6–58.6 g, group A weighing MATERIALS AND METHODS 45.4–54.9 g, and group B weighing 44.5–53.7 g) weresacrificed by cervical dislocation by the same personMice were coded and randomized to minimizeat the same time of the day. The thymus (controls  n Å systematic error and observer bias. Thirty 22-day-old19, group A  n  Å  23, and group B  n  Å  18) and spleenSwiss male mice weighing 13.7–14.8 g were housed(controls  n  Å  21, group A  n  Å  21, and group B  n  Å in 2 cages (15 mice per cage) of 42 cm length, 32 cm18) were collected and studied separately. The naturewidth, and 18 cm height, and placed in the bottom of of the thymus tissue was confirmed by histologicala cylindrical coil generating a 60 Hz alternating currentanalysis.(AC) magnetic field which varied from 0.4 to 1.0  m TThe thymus and spleen were teased in RPMI 1640(group A). A second group of 30 animals of the samemedium (not phenol red free) and separated in Ficoll-age weighing 14.0–15.1 g was exposed to a DC fieldHypaque (density 1.077). The cell suspensions werewhich varied from 8 to 20  m T (group B), and a thirdadjusted to a concentration of 1.5  1  10 6 cells/ml ingroup of 30 animals (the control group) of the sameRPMI 1640 supplemented with 5% fetal calf serumage weighing 13.8–15.0 g was placed in the coil with(FCS). Aliquots of 2 ml from each cell suspensionno power connected to it. The cylindrical coils were(thymocytes and spleen cells) were placed in 12  1 placed more than 3 m apart with their axis pointing in75 mm polystyrene tubes and incubated for 8 h atthe vertical orientation, producing a magnetic field in37   C, 5% CO 2 , without (spontaneous apoptosis) andthis direction. The cylindrical coils were made withwith (induced apoptosis) Dex 10 0 7 M. Spontaneoustransparent acrylic sheets 2.5 mm thick and wereapoptosis and Dex-induced apoptosis were assessed by88 cm in diameter and 43 cm long.the detection of DNA fragmentation after lysing cellsThe coils were connected to a power supply capa-in a hypotonic solution (0.1% sodium citrate, 0.1%ble of producing AC and DC voltages; 94.5 mV peak  Triton  1 100) containing 50  m g/ml propidium iodideto peak was used for the coil employed to produce the (PI) and analyzed by flow cytometry (FACScan, Bec-60 Hz AC field (480 turns of copper wire, resistance ton Dickinson, San Jose, CA). At least 10 4 cells fromR  Å  43  V , and inductance L  Å  199 mH) and 8 V for each sample were analyzed. All measurements werethe coil (49 copper wire turns) used to produce the DC performed under the conditions described by Nicolettifield. The magnetic field was measured in the midplane et al. [1993] (Fig. 1A,B).(21.5 cm distant from each extremity) at the center and Apoptosis was also determined by the in situthe periphery of the coil. The local earth’s field is terminal deoxytransferase-catalyzed DNA nick endapproximately 20  m T. The laboratory where the experi- labeling (TUNEL, ONCOR ApopTag TM Plus peroxi-ments were carried out had no magnetic fields other dase, Gaithersburg, MD) staining method [Gavrielethan the ones produced by the low power lines and the et al., 1992] on cytocentrifuge-prepared slides. Dupli-general purpose appliances such as bulbs. The mag- cate slides of thymocyte suspensions were obtainednetic field was checked once a month with a fluxgate from 6 of the 19 control samples and from 6 of the 23magnetometer model 428C (Applied Physic Systems, group A samples. In addition, spleen cell suspensionsUSA) and variations from the above reported values were obtained from 6 of the 21 control samples andwere in the range of   { 5%. No efforts were made to from 6 of the 21 group A samples. These samplesmonitor short-term variations that might have occurred, were chosen completely at random. The percentagesince the power line in the laboratory is very stable. of positive cells (peroxidase-stained nuclei) was deter-The mice were continuously exposed (24 h/day) mined by counting at least 500 cells from each slideduring the 12 month period of the experiment. How- (Fig. 1C,D).ever, the cages were changed daily, leaving the animals As the apoptosis data were not normally distrib-with about 15–30 min without exposure. No heat was uted, the Kruskal-Wallis (KW) analysis of varianceproduced by the coils that could change the temperature (ANOVA) with Dunn’s multiple comparisons test, theof the cages. Housing conditions were controlled in Wilcoxon test (paired samples), and the Mann-Whitneyorder to avoid noise and vibration of the cages. Animals test (non-paired samples) were used for statistical anal-received food and water ad libitum and were free of  ysis. The level of significance for each type of test was P  õ  .05.infection during the experiment (data obtained by tissue  Dex-Induced Apoptosis of Thymocytes and EMF 133 Fig.1.DNAfluorescencehistogramsofpropiumiodide-stainedthymocytesafter8hoursincuba-tion in medium alone (   A   ) or in medium plus dexamethasone 10 0 7 M (  B  ) showing the percentageof apoptotic cells (1.5% and 21.4% respectively). TUNEL staining of dexamethasone-inducedapoptosis in thymocytes after 8 hours incubation in medium alone (  C  ) or in medium plusdexamethasone 10 0 7 M (  D  ). Characteristic apoptotic cells are marked by arrows. RESULTS AND DISCUSSION  spleen cells from exposed and non-exposed mice wassimilar. Group A (0.4–1.0  m T 60 Hz Field Exposure)Group B (8–20  m T DC Field Exposure) The spontaneous apoptosis of thymocytes andspleen cells from this group was similar to the controls. To confirm that EMFs were responsible for theincreased Dex-induced apoptosis we exposed mice toThymocytes and spleen cells from exposed and non-exposed controls showed an increased Dex-induced a vertical constant magnetic field of the same magni-tude as the local earth’s field (20  m T) and superposedapoptosis (Wilcoxon test,  P õ .05, related to respectivespontaneous apoptosis). In addition, we observed that to it. Interestingly, the flow cytometric analysis of bothspontaneous apoptosis and Dex-induced apoptosis of Dex-induced apoptosis measured by the FACScan orTUNEL method was significantly higher in thymocytes thymocytes and spleen cells from this group showedvalues similar to those observed in non-exposed mice.from exposed mice (KW ANOVA with Dunn’s multi-ple comparisons test,  P õ .01, and Mann-Whitney test, The percentage of the spontaneous apoptosis and Dex-induced apoptosis of thymocytes and spleen cells from P  õ  .01, respectively). Dex-induced apoptosis of   134 Ismael et al. TABLE 1.PercentofSpontaneousApoptosisandDex-InducedApoptosisofThymocytesandSpleenCellsDetectedbytheFACScanor TUNEL Method From Mice Exposed to Different Magnetic Fields † % Apoptotic cellsSpontaneous Dex-inducedFACScan TUNEL FACScan TUNELThymocytesControls 0.9  {  0.1 1.3  {  0.5 12.6  {  1.5 7.5  {  1.0(n  Å  19) (n  Å  6) (n  Å  19) (n  Å  6)Group A 1.7  {  0.3 1.4  {  0.4 25.7  {  3.2* 17.5  {  1.9(n  Å  23) (n  Å  6) (n  Å  23) (n  Å  6)Group B 2.2  {  0.8 — 9.8  {  1.3** —(n  Å  18) (n  Å  18)KW ANOVA test  P  Å  .07  P  õ  .001Mann-Whitney test  P  Å  .87  P  õ  .01Spleen cellsControls 2.1  {  0.2 1.9  {  0.5 5.0  {  0.6 7.0  {  1.6(n  Å  21) (n  Å  6) (n  Å  21) (n  Å  6)Group A 3.0  {  0.2 1.2  {  0.3 8.1  {  0.6 4.2  {  0.8(n  Å  21) (n  Å  6) (n  Å  21) (n  Å  6)Group B 2.6  {  0.3 — 6.1  {  0.9 —(n  Å  18) (n  Å  18)KW ANOVA test  P  Å  .06  P  Å  .07Mann-Whitney test  P  Å  .23  P  Å  .12 † Group A  Å  0.4–1.0  m T 60 Hz field and group B  Å  8–20  m T DC field. Values are expressed as mean  {  SEM.* P  õ  .01 and ** P  ú  .05 in comparison with Dex-induced control (Dunn’s multiple comparisons test). mice exposed to different magnetic fields are shown in (CNPq) and by the Financiadora de Estudos e Projetos(FINEP).Table 1.Histological comparison of the thymus and thespleen from control and group A mice after 1 year of  REFERENCES exposure using 5  m m hematoxylin-eosin-stained paraf- Ahlbom A, Feychting M (1993): EMF and Cancer. Science 260:13– fin sections did not reveal any morphological differ- 16. ences. In addition, the relative weights (g) of the thy- Bates MN (1991): Extremely low frequency electromagnetic fields and mus and the spleen (organ weight/body weight) from cancer: the epidemiologic evidence. 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Callera contributed equally Cancer promotion in a mouse-skin model by a 60-Hz magnetic to this work. This study was supported by the Conselho  field: II. Tumor development and immune response. Bioelectro-magnetics 12:273–287. Nacional de Desenvolvimento Cientifico e Tecnolo´gico  Dex-Induced Apoptosis of Thymocytes and EMF 135 Mevissen M, Stamm A, Buntenko¨tter R, Zwingelberg R, Wahnschaffe frequency electric and magnetic fields. J Amer Med Assoc268:625–629.U, Lo¨scher W (1993): Effects of magnetic fields on mammarytumor development induced by 7,12-dimethylbenz(a)anthracene Savitz DA, Pearce NE, Poole C (1989): Methodological issues in theepidemiology of electromagnetic fields and cancer. Epidemiolin rats. Bioelectromagnetics 14:131–143.Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1993): Rev 11:59–78.Shulman S (1990): Cancer risks seen in electro-magnetic fields. 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