rf-induced calcium ion efflux enhancement from neuroblastoma

8/9/2019 RF-Induced Calcium Ion Efflux Enhancement From Neuroblastoma

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Bioelectromagnetics 10:197-202 (1989)

Rad of requency Radiat on-InducedCalcium Ion Efflux EnhancementFrom Human and OtherNeuroblastoma Cells in CultureS.K. Dutta, B. Ghosh, and C.F. BlackmanDepartments of Botany and Radiotherapy and the Cancer Research Center, HowardUniversity, Washington, DC (S.K. D., 6. .); Health Effects Research Laboratory,Environmental Protection Agency, Research Triangle Park, North Carolina (C.F.B.)

To test the generality of radiofrequency radiation-induced changes in 4sCa2+ efflux fromavian and feline brain tissues, human neuroblastoma cells were exposed to electromagn eticradiation at 147 MHz, am plitude-modulated (A M ) at 16 Hz, at specific absorption rates(SAR) of 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0005 W/kg. Significant 4s Ca 2+efflux wasobtained at SAR values of 0.05 and 0.005 Wikg. Enhanced efflux at 0.05 W/kg peaked atthe 13-16 Hz and at the 57 .5-60 Hz modulation ranges. A Chinese hamster-mouse hybridneuroblastoina was also shown to exhibit enhanced radiation-induced Ca2+ e f f l u x at anSAR of 0.05 Wikg, using 147 MH z, AM at 16 Hz. Th ese results confirm thatamplitude-modulated radiofrequency radiation can induce responses in cells of nervoustissue origin from widely different animal species, including humans. The results are alsoconsistent with the repoits of similar findings in avian and feline brain tissues and indicatethe general nature of the phenomenon.

Key words: frequency windows, intensity windows, power line frequency

INTRODUCTIONCalcium ions are important in maintaining normal physiologic function in braintissues. Therefore, radiation-enhanced calcium efflux may affect nervous systemfunction. Baw in et al. [1975] and Blackman e t al. [197 9, 198 0a, 19811 have studiedradiofrequency (RF)-induced efflux of radioactive calcium ions with low-power 147MHz carrier waves, sinusoidally amplitude-modulated (AM) at 16 Hz. All thesestudies at extremely low modulation frequencies were performed with freshlyremoved avian and feline brain tissues. We hav e tested the generality of the responseby using cultured neural cells derived from human and another vertebrate sources. Ina previous study [D utta et al ., 19841, we reported enhanced efflux of calcium ions

Received for review March 18, 1988; revision received September 19, 1988.Address reprint requests to Dr. S . K . Dutta, Department of Botany, Howard University, Washington, DC20059.

0 989 Alan R. Liss, Inc.


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198 Dutta et al.from human neuroblastoma cells in culture exposed to RF radiation at 915 MH z, AMat 16 Hz. This is a report of the effects of AM 147 MHz radiation, similar to theexposure used in the study by Bawin et al. [1975] and Blackman et al. [1979, 1980a,19811, on neuroblastom a ce lls derived from h uman s and other vertebrates. O urstudies confirm and extend the generality of R F-induced enhanced 45 Ca 2f efflux tomammalian neural cells.MATERIALSAND METHODS

Human neuroblastoma cells (IMR 32) were obtained from the American TypeCulture Collection (ATCC). This cell line was established from a neuroblastoma in a13-month-old white male. In our routine microscopic examinations, this culture is amorphologically distinct cell type. The other cell line was the Chinese hamster-mousehybrid neuroblastoma cell line NG-108 obtained from the U. S . National Institute ofHealth.The cultures were grown in minimum essential medium (MEM-Eagle-Earlesbalanced salt solution, with nonessential amino acids and sodium pyruvate) supple-mented with 10% fetal bovine se rum , glutamine (0.02 mM ), and gentamycin ( 1% ) .All media components were obtained from M.A. Bioproducts (Walkersville, MD).Each cell culture was seeded in T-flasks (2 5 tin' growth surface) with identical 5 mlaliquots of medium and was grown to a confluent m onolayer containing about 2.0 x107cells.Th e exposure system is the same as that described earlier by Dutta et al. [19841.The exposure chamber, a Crawford cell (Instrument for Industry, M odel BC-1 lo ),consisted of a transmission line tapered at each end to mate with a standard coaxialcable. A signal generator (Hewlett Pack ard, M odel 86 40) provided the 147 M Hzcarrier frequency, which was sinusoidally AM (80%) at low frequencies with afunction generator (Krohn -Hite, Model 5200). T he modulated signals were amplifiedwith a linear amplifier (Amplifier Research, Model IW 1000). Two identicalthermoelectric power meters (Hewlett-Packard, Model 432B) and sensors (HewIett-Packard Model 478A), attached to 20 dB bidirectional couplers (Narda, Model3020A), were used in conjunction with a switch (Sage Laboratories, ModelSTN 2180A , Type 1P2T) to measure the forward, reflected, and transmitted powers.Specific absorption rates (SARs) were determined from incident, reflected, andtransmitted powers measured with 1) an empty C rawford cell, 2) a Crawford cell withan empty flask, and 3) a Crawford cell and flask with culture and 5 ml of growthmedium. The reflected power was adjusted to zero with a tuner (WeinschelEngineering, model D S-109L L) for each m easurement. SAR for a given incidentpow er was determined as described previously [Du tta et al., 19841. No attempt wasmade to estimate the local SAR in the monolayer of cells; i t was assumed that thecomposition of the cells was sufficiently close to the medium so that the measuredSAR was an adequate approximation for the cells as well. The measured SAR wasthen varied by increasing or decreasing the forward power once the percentage ofabsorption w as determined. T he static, local geomagnetic field (LGF) in the exposuresystem as measured by a gauss meter (Bell model 640) was 16 pT ( 0 . 1 6 gauss) at aninclination of 53".

Procedures for cell labeling, exposures, and assay have been describedpreviously [Dutta et al ., 19841. Four iden tical confluent flasks containing the cells of


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Neuroblastoma Cell Responses 199TABLE 1. Calcium Ion Efflux in T w o Cell Lines*

Difference in release,Release of calcium during exposure paired samples P value, for

Cells Control ? SEM(n)a Exposedf SEM(n)" mean * SEM(n)" paired samplesIMR-32b 2,192 k 228 (14) 3,011 2 28 2 (14) 818 2 1 4 0 ( 1 4 )


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200 Dutta et al.TABLE 2. Calcium Ion Eftlux From Human Neuroblastoma Cells* Exposed to Different SARsof 147 MHz Radiation Amplitude Modulated at 16 Hz

Difference in release,Release of calcium durin g exposure paired samples P value,

SA R (W/kg) Control t SE M (n)3 Exposed 2 SE M (n). mean * SE M (n ) for paired samples0.0001 1,314 -+ 141 (9) 1,324 2 88 (9) 10 2 73 (9) 0.897O.OOO5 1,2YY & 132 (5 ) 1.282 ? 136 (5) - 17 * 25 (5) 0.5370.0007 2,391 2 529 (4) 2,384 2 459 (4) - 7 t 143 (4) 0.9640.001 1.8 I I 2 206 (9) 1,795 2 205 (9) ~ 16 * 92 (9) 0.8610.002 1,948 f 153 ( 11 ) 1,941 t 128(11) - 6 ? 77 ( 1 1 ) 0.9380.005 2,684 2 141 (9 ) 3,402 2 193 (9) 719 2 170 (9)


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NeuroblastomaCell Responses 201TABLE 3. Calcium Ion Efflux From Human Neuroblastoma Cells* Exposed to 147 M HzRadiation** Amplitude Modulated at Different FrequenciesAM freq. paired samples for(Hz) Control 2 SEM (n)" Exposed -+ SEM (n)" mean 2 SE M (n)" paired samples9 2,750 2 333 (7 ) 2,711 t 343 (7) - 38 2 187 (7) 0.84413 2,696 t 200 (7 ) 2,932 2 243 (7) 236 * 88 (7) 0.03718 1,673 2 129 (7) 1,969 2 172 (7) 296 2 176 (7) 0.14320 1,953 2 192 (7) 1,609 k 226 ( 7 ) - 343 2 155 (7 ) 0.06855 1,608 2 127 (7) 1,713 2 92 (7) 105 2 128 (7) 0.44557.5 2,510 2 520 (7 ) 3,140 t 485 (7) 630 2 94 (7)


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202 Dutta et al.information on the role the LGF might play in modulated RF radiation results,because the demodulation site and process are not understood.The final conclusion from this study is that cell lines derived from tumors of theCNS, neuroblastomas, respond to modulated RF fields in a manner identical to thenormal, forebrain-tissue preparations from newborn chicks and from felines. Thisindicates that the response is not due exclusively to the transformed state of the cells.Furthermore, cells of non-CNS origin (e.g., human nonneuronal cancer cells andfibroblasts) did not show a similar response to the fields (unpublished pilot data).Thus cells of CNS origin may respond to the modulated RF fields in a very specificmanner. However, this result cannot be generalized without qualification because ofthe small number of cell types tested.ACKNOWLEDGMENTS

The authors wish to acknowledge support from the U . S . EnvironmentalProtection Agency (Cooperative Agreement No. CR-8 12100) and from the U.S.Department of Energy (Interagency Agreement No. DE-A I06-87RLI 1374 to U . S .EPA).The research described in this article has been reviewed by the Health EffectsResearch Laboratory, U .S . Environmental Protection Agency, and approved forpublication. Approval d oes not signify that the contents necessarily reflect the viewsand policies of the Agency, nor does mention of trade names or commercial productsconstitute endorsement or recommendation for use.REFERENCESBawin SM , Adey W R, Sabbot IM (19 78 ): onic factors in release of %a2+ from chicken cerebral tissueby electromagnetic fields. Proc Natl Acad Sci USA 75:6314-6318.Bawin SM, Kaczmarek LK, Adey WR (1975): Effects of modulated VHF fields on the central nervoussystem. Ann N Y Acad Sci 247:74-8 I .Blackman C F, Benane SG , Elder JA, House D E, Lam pe JA , Faulk JM (19 80a): Induction of calcium-ionefflux from brain tissue by radiofrequency radiation: Effect of sample number and modulationfrequency on the pow er-density window. Bioelectromagnetics I :35 -43.Blackman C F, Benane SG , House D E, Joines WT (1985a): Effects of ELF (1-120 Hz) and modulated (50Hz) R F fields on the efflux of calcium ions from brain tissue in vitro. Bioelectromagnetics 6: -1 1 .Blackman CF, Benane SG , Joines WT, Hollis M A, House D E (1980b): Calcium-ion efflux from braintissue: Power-density versus internal field-intensity depend encies at 50-M Hz R F radiation.Bioelectromagnetics 1 277-283.Blackman CF , Benane SG, Rabinowitz J R , House D E, Joines W T (1985b): A role for the magnetic fieldin the radiation-induced efflux of calcium ions from brain tissue in vitro. BioelectromagneticsBlackman C F, Elder JA , Well CM , Benane S G , Eichinger DC (1979): Induction of calcium-ion effluxfrom brain tissue by radiofrequency radiation: Effects of modulation frequen cy and field strength .Radio Sci 14(68):93-98.Blackman CF, Joines WT, Elder JA (1981): Calcium-ion efflux in brain tissue by radiofrequencyradiation. In Illinger KH (ed): Biological Effects of N onionizing Radiation. Washington, DC:American C hemical Society, pp 299-314.Dutta SK , Subramoniam A, Ghosh B, Parshad R (1984 ): Microwav e radiation-induced calcium ion effluxfrom hu man neuro blastoma cells in culture. B ioelectrom agnetics 5:7 1-78.

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rf-induced calcium ion efflux enhancement from neuroblastoma

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