The influence of homogeneous magnetic fields up to 5.5 T on contractile frequency and mortality in the ciliate protozoan spirostomum ambiguum stimulated by 2,2′ PDS is reported. Magnetic fields are observed to decrease contractile frequency and to significantly increase mortality

Ettienne, E. M.

Frankel, Richard B.

Ripamonti, Angela

English

DigitalCommons@CalPoly

EFFECT OF MAGNETIC FIELDS ON DRUG INDUCED CONTRACTILITY AND MORTALITY IN SPIROSTOMUM Angela RIPAMONTI, R.B. FRANKEL Francis Bitter National Magnet Laboratory *, Massachusetts Institute of Technology, Cambridge, MA 02139, USA and E.M. ETTIENNE ** University ofMassachusetts Medical School, Worcester, MA 01600, USA The influence of homogeneous magnetic fields up to 5.5 T on contractile frequency and mortality in the ciliate proto­zoan spirostomum ambiguum stimulated by 2,2' PDS is reported. Magnetic fields are observed to decrease contractile frequency and to significantly increase mortality. Possibilities for installation oflarge scale magnetic ~vices for energy production have stimulated inquiries Blo the biological effects of magnetic fields [1,2]. 'ecently magnetic fields have been reported by .. eking et al. [3] to influence the force developed ring contraction of isolated frog sartorius muscle. ey observed that in homogeneous magnetic fields to 5 T, the amplitude of isometric force develop­ent in isolated skeletal muscle is reduced by about o. The authors speculate, following Dudoladov and incher [4], that the s"tructural state of intracellular ter adjacent to protein or membrane surfaces can altered by an external magnetic field. We have investigated the effects of homogeneous agnetic fields on the mechanism of cellular contrac­n by studying the contraction frequency in cells Uowing chemical stimulation with and without an plied magnetic field. In this study we used the ntractile ciliate spirostomum ambiguum (Ward's ological Supply, Rochester, Ny), inducing contrac­fiS with 10 ,uM of the thiol reagent 2,2'-dipyridyl-Sponsored by the National Science Foundation Work partially sponsored by the Muscular Dystrophy Association of America. disulfide (PDS) (Sigma Chemical Co., S1. Louis, MO) in a medium consisting of 2,uM NaCl, 0.5 ,uM KCI; 0.05,uM CaCI2 ; 0.1 ,uM KH2 P04 ; and 0.1 ,uM KOH (pH 6.3). In all experiments, the temperature was monitored and held constant at 20 or 22°C during application of the field. PDS is a sulfhydryl oxidizing agent which, through action on the microsomal respiratory chain of cells [5] decreases cytoplasmic ratios of NADPH/NADP and GSH/GSSG (glutathione). These ratios have been implicated in the oscillatory regulation of ionized free calcium concentration in the cytoplasm over ranges which include thres­hold levels for contraction in spirostomum [6,7]. It has been demonstrated that contractility in spirostomum is fundamentally like that of striated muscle and that there is Ca2+ dependent regulation of contractile elements [8] with Ca2+ levels con­trolled through intracellular membranous compart­ments. Because its physiology has been elucidated in detail, we feel that spirostomum is an excellent organism for the study of the effects of magnetic fields on mechano-chemical activity. Magnetic fields of 0.5 and 0.92 T were produced by a water-cooled electromagnet with flat, iron pole pieces and at 3.0 and 5.5 T by a water-cooled Bitter ----~ o CONTROL .0.5 T:=' 9	 I ,) 0.92 T=> z	 ! ~ 6 3.0 T o 5.5 T~	 I -' <t 80 0:: w	 I > a. 6	 '>I 0::(/) => 0z	 (/) 60 ~	 z u 0 ti 3 t 40.... Z -' 0 U 0 a. 20 0 'J a. => 0	 15 20 0 10 20 30Fig. 1. Time course and magnitude of contractile response of TIME (min)Spirostomum to treatment with 10-5 M 2,2' PDS in zero field (e) and 0.92 T (0). Significant suppression of contrac­ Fig. 2. Percentage survival as a function of time for cells stimulated with 2,2' PDS in homogeneous magnetic fields tions are seen to occur at 7, 10, 13, 15 and 16 min. of 0 T (control), 0.5 T, 0.92 T, 3.0 T and 5.5 T. The data are also tabula ted in table 1. solenoid. PDS induced contractions per minute were monitored over an interval of 20 min in populations is shown in fig. 1 for magnetic field H o = 0.92 T and of 15 or more cells exhibiting synchronous contrac­ H o =0 controls. At 7, 10, 13 and 16 min following tions. The results per time point of 10 to 30 experi­ incubation, peaks are observed in the contraction ments were analyzed statistically according to the frequency for Ho =0 (see fig. 1) [6,7]. In the external Student '1' test [9]. The frequency of contractions magnetic field, the values for the peak contraction as a function of time following stimulation with PDS frequencies are depressed by 50% (P < 0.001), 37% Table 1 Percentage survival as a function of time following simultaneous stimulation with PDS and exposure to 0.5, 0.92, 3 and 5.5 T magnetic fields Field	 Control (0 T) 0.5 T 0.92 T n * =15 n * =15 n* =15 Time x± sd x± sd S+ X ± sd S+ (min) 10 99 ± 3.22 91.6 ± 9.96 P < 0.01 89.6 ± 12.2 P < 0.001 20 93.7 ± 9.6 76.12 ± 5.92 P < 0.01 66.3 ± 13.6 P < 0.001 30 74.1 ± 10.2 44.9 ± 15.3 P < 0.0005 33.1 ± 16 P < 0.0005 40 44.2 ± 27.8 7.56 ± 8.5 P < 0.0005 4.02 ± 6.3 P < 0.0005 Field	 Control (0 T) 3.0T 5.5 T n * =15 n * =10 n* =10 Time x± sd x± sd S+ x± sd S+ (min) 10 99. ± 3.22 74.7 ± 4.5 P< 0.001 52.2 ± 7.4 P < 0.001 20 93.7 ± 9.6 41.09 ± 8.2 P< 0.0005 26.8 ± 9.3 P < 0.0005 30 74.1 ± 10.2 44.3 ± 9.5 P< 0.0005 3.3 ± 5.9 P < 0.0005 40 44.2 ± 27.8 2.8 ± 5.2 P < 0.0005 O±O P < 0.0005 n* : Number of experiments with at least 15 cells per sample. S+ : Significance compared to control. (P < 0.001),33% (P < 0.001), and 33% (P < 0.001), respectively. The duration of the relaxation phase in magnetic fields was qualitatively observed to be extended over controls. In addition to the observed effects on contraction frequency, there are also very marked effects on cell mortality. In fig. 2, the percentages of survivors in 12 experiments with >15 cells each at 10,20,30 and 40 min after incubation are plotted as a function of time for magnetic fields Ho =a(control), 0.50 T, 0.92 T, 3.0 T and 5.5 T. In table I we present per­centage survival and statistical significance compared to controls over the given time intervals for the four magnetic field values. At 30 min, the effects of 0.50 T, 0.92 T, 3.0 T and 5.5 T on survival were all significant to P < 0.0005. We note that in the absence of PDS, there were no observed effects of exposure to magne­tic fields up to 5.5 T for 30 min on cell viability. Experiments were also conducted in which samples of spirostomum (IS cells per sample) were exposed to 5.5 T for 30 min in the absence of the drug, and then removed from the field. Immediately following field exposure, PDS was administered and the mortal­ity in zero field was monitored as a function of time and compared with controls which had not been exposed to the external field. The results, presented in table 2 and fig. 3, show that at 10,20,30 and 40 min experimental values are 83% (P < 0.001), 55% (P < 0.0005), 33% (P < 0.0005) and 15% (P < 0.0005) of controls, respectively. These results imply that the effect of the magnetic field on mor­tality cannot be attributed solely to the direct effect Table 2 Percentage survival as a function of time after PDS stimulation, following 30 min exposure to 5.5 T magnetic field Time x ± sd (min) Control (0 T) 10 98.03 ± 3.2 * = 10n 20 94.1 ± 5.9 30 65.6 ± 7.8 40 46.2 ± 22.5 5.5 T 10 81.4 ± 10.5 P < 0.001 * = 10n 20 5\.8 ± 16.1 P< 0.0005 30 22.2 ± 15.2 P < 0.0005 40 7.1 ± 8.3 P < 0.0005 n* : number of experiments with at least 15 cells per sample. P : significance compared to control. 100K:"--_~ o CONTROL • 5.5 T ~ 80 > a: ~ 60 z o ~ 40 -' ::> ll. o ll. 20 o L----;"rIoc-----~20~--~3'ocO----4:";:Oc----' TIME (min) Fig. 3. Percentage survival as a function of time for cells exposed for 30 min to 0 T (controls) and 5.5 T, followed by removal of the field and stimulation with 2,2' PDS. The abscissa correspond to time following stimulation with.2,2' PDS. The data are also tabulated in table 2. of the field on PDS, and indicate significant long last­ing effects of the field on the organism. We have seen that the magnetic field acts to decrease contraction frequency and increase mortal­ity in response to PDS. Based on the known physiol­ogy of spirostomum [5,8] the observations can be interpreted as reflecting a decrease in the enzymatic transport of Ca2+ out of the cytoplasm following contraction in the magnetic field. Reduced Ca2+ transport might occur as a result of an interaction between the magnetic field and the intracellular membranous compartments. This hypothesis could be verified by observation of prolongation of Ca2+ transients in vivo. Alternatively, Ca2+ in transport vesicles isolated from cells exposed to magnetic fields could be measured in vitro with 45Ca. These experiments are presently being pursued. Informa­tion from these experiments will enable us to a3cer­tain whether the locus of the magnetic field effect is associated with the Ca2+ transport membranes. Acknowledgements We are pleased to acknowledge the continuing interest and support of D.B. Montgomery and P. Marston, and thank Y. Shapira for a careful reading of the manuscript. References [1] H. Aceto, C. Tobias and 1.1. Silver, IEEE Trans. Magn. MAG-6 (1970) 368. [2) A. Sheppard and M. Eisenbud, Biological Effects of Electric and Magnetic Fields of Extremely Low Frequency (University Press, New York, 1977). (3) J. Biicking, M. Herbst and P. Pointer, Rad. Environ. Biophys. 11 (1974) 79. [4] A.G. Dudoladov and K.S. Trincher, Biofiz. 16, (1971) 547. (5) M.A. Levine and E. Ettienne, Microbios Lett. in press. [6) E. Ettienne and S. Dikstein, Nature, 250 (1974) 782. [7] S. Dikstein and R.B. Hawkes, Experientia 32 (1976) 1029. (8) E.M. Ettienne, J. Gen. Physiol. 56 (1970) I68. [9) W.C. Scheffler, Statistics for Biological Sciences (Addison Wesley, New York, 1973). 

Effect of Magnetic Fields on Drug Induced Contractility and Mortality in Spirostomum

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Effect of Magnetic Fields on Drug Induced Contractility and Mortality in Spirostomum

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