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Transcription in Drosophila melanogaster salivary gland cells is altered following exposure to low frequency electromagnetic fields: Analysis of chromosomes 2R and 2L
39
Bioelectrochemistry and BioenergeGcs, 31(1993)
39-47
Elsevier Sequoia S.A., Lausanne
JEC BB 01578
Transcription in Drosophda melanogaster salivary gland cells is altered following exposure to low frequency electromagnetic fields: analysis of chromosomes 2R and 2L David Weisbrot William Paterson College of NewJersey, 07470 NJ (USA)
Alun Uluc and Ann Henderson Hunter College of the City Uniuersity of New York, New York (USA)
Reba Goodman
l
Columbia Uniuersify Health Sciences, Department of Pathology, 630 West 168 Street, New York,
NY 10032 (USA) (Received 12 August 1992)
Abstract Using the technique of transcription autoradiography, it is possible to identify nascent RNA chains directly on defined regions of Drosophila salivary gland chromosomes. Changes in transcriptional activity have been identified in 17 defined regions of Drosophila melanogasrer salivary gland chromosome arms 2R and 2L following 20 min exposures of salivary glands to five extremely low frequency electric and magnetic fields. Many of the labeled areas of the chromosome have been correlated with regions which contain known gene sites based on chromosome maps of D. melanogaster.
INTRODUCTION
A growing body of evidence, including the results of several epidemiological studies, suggests an association between human exposure to extremely low frequency (ELF) electric and magnetic (EM) fields and the incidence of some forms of cancers [l-3]. As a result of such findings, the effect of ELF EM fields on biological systems has become the focus of considerable research interest.
* To whom correspondence 0302-4598/93/$06.00
should be addressed.
0 1993 - Elsevier Sequoia S.A. All rights reserved
40
EM fields with physical parameters similar to those found in the environment are used in laboratory studies to investigate the interaction between low frequency fields and cells and tissues. Numerous and various effects on cell and tissue function resulting from exposure to EM fields have been reported (reviewed in refs. 4 and 5). Examples of effects include alterations in neurotransmitter release, changes in receptor binding and Con-A induced changes in intracellular Ca2+ levels (reviewed in refs. 4 and 5; see refs. 6-8). We demonstrated previously that significant increases in some transcripts can be measured when human HL-60 cells are exposed for short time periods (up to 40 min) to ELF EM fields (< 300 Hz). The magnitude of the increase is dependent on time, frequency and field strength [9]. Not all genes are affected. Transcript levels for p-2-microglobulin, for example, are unaffected in cells exposed to either EM fields or a.c. current [lo]. The present study is the third in a series that looks at the transcriptional response in Drosophila mefunogusfer salivary gland cells following 20 min exposures to five different ELF EM fields. Drosophila salivary gland cells have proven to be a very useful model for studies of this kind. Their polytene chromosomes, which are in interphase, have 1000-5000 copies of DNA strands side by side on each chromosome which produce identifiable bands. This makes it possible to visualize transcriptional changes directly on the chromosome. Further, these chromosomes have been extensively studied [ll], their bands and interbands mapped and associated specific gene sites have been identified (reviewed in ref. 12). We previously identified regions of chromosome 3R [ll] and 3L and X [12] from salivary gland cells that show differential 3H-uridine uptake into identified chromosome regions in response to exposure to a variety of low frequency EM fields for 20 min. At least two heat shock loci (87AD and 93AD) responded to exposure to each EM field tested without a detectable increase in temperature. Further, the effect of ELF EM fields on protein synthesis was determined by two-dimensional gel electrophoresis [11,13] and, among other changes in protein synthetic patterns, the 70 kD heat shock protein was increased. This report is a continuation of analyses of transcriptional activity on Drosophila chromosomes 2R and 2L in salivary gland cells exposed to ELF EM fields. A total of 17 chromosomal regions have been identified where transcriptional activity has been increased over that observed in chromosomes which were not exposed to ELF EM fields. MATERIALS AND METHODS
Fifteen pairs of salivary glands from third instar larvae were used for each experiment. The glands were dissected partially free from the larval body in Schneider’s Drosophila medium (SDM; GIBCO) in 30 mm glass Petri dishes. Some attachment to the larval body was retained during exposure to the EM fields. After three washes with SDM, 100 &i of 3H-uridine (NEN) was added to each dish (2
41
Waveform Characteristics 380 pSEC , Repetitive Single Pulse (SP) 4.5 mSEC
(Diode Clipped) 72 Hz
Repetitive .-------_ Pulse Burst (PB) 15 Hz
0.8 mV positive amplitude
-/\/\
Sine?
(CW
30 mSEC Equine-33 _____ Repetitive Pulse Burst 1.5 Hz
Fig. 1 The five EM signals used for these experiments with their major physical parameters. The values presented here for the physical parameters were measured using a calibrated inductive search coil (Electra-Biology, Inc.).
ml total volume) and the organ culture was exposed to one of each of the EM fields for 20 min. Three of the fields were pulsing electromagnetic fields (PEMFs; Electra-Biology, Inc., Parsippany, NJ) used clinically for the treatment of nonunion bone fractures, remodeling in osteoporosis or avascular necrosis [121. The PEMFs were compared with two continuous wave sinusoidal fields at frequencies of 60 and 72 Hz. The five EM fields used for these experiments, with their physical parameters, are’given in Fig. 1. For each of the five signals used, at least four separate experiments were performed. As previously described 111,121, the glass Petri dishes (containing the salivary glands) were placed on a Plexiglas form within a pair of 13 cm X 14 cm Helmholtz coils with an 8 cm space. The coils were positioned vertically so that the
42
oscillating magnetic field was generated in the horizontal plane, inducing a relatively uniform electric field in the conductive medium [5,9]. The organ culture containing the glands was exposed to the fields for 20 min at 25°C. Two groups of control cells were used for each experimental series. One group controlled for possible heating effects; these were incubated at 37°C for 20 min. This temperature was selected because it is known to produce specific changes in both transcription and translation. In the experiments reported here, temperature variations during all exposures were +O.S’C, as measured by a thermocouple temperature-sensitive probe (PhysiTemp Instruments, Inc.). The second control group were cells incubated in 3H-uridine for 20 min at 25°C in the-absence of the EM fields. These were shielded in a mu metal box. At the conclusion of each exposure, the dishes containing the organ culture were immediately placed on ice and washed three times with cold SDM, and the salivary glands were dissected free from the larval body. A single pair of glands (containing 70 cells) was placed on individual pre-cleaned glass slides in a drop of 45% acetic : 1% lactic acid. Gland pairs were cover slipped and squashed to release the chromosomes, and the cover slips were removed by freezing them in liquid nitrogen. The slides were dehydrated through a series of alcohols, after which they were air dried. For autoradiography, the slides containing the chromosomes were dipped in a 1: 1 dilution of Ilford K-5 autoradiographic emulsion (Ilford, UK), air dried and stored in light-tight boxes at 4°C. Slide preparations were developed and fixed at 24 h intervals and stained with Giemsa (1: 9). Exposure to the autoradiographic emulsion at 48 h was selected for the analyses presented here because at this exposure time the chromosomes were lightly, but significantly, labeled, which simplified the counting of the silver grains (see Fig. 2). Analysis of silver grains at identified chromosome regions was made from mounted cut-outs of chromosomes 2R and 2L from a minimum of 8 different photographic plates from each experiment or 24 for each determination. RESULTS
The baseline for these experiments was analysis of chromosomes 2R and 2L from control (not subjected to fields) salivary gland cells. Gene activity was deliberately underestimated by limiting the time of autoradiographic exposure. This made both identification of the bands and counting the grains easier. Preparations that were exposed to the autoradiographic emulsion for longer time periods showed no additional labeled regions, but had a heavier grain density with further exposure [ 11,121. In unexposed cells, two chomosome regions on 2L (25BC and 36BD) consistently showed transcriptional activity at this stage of larval development. In addition, chromosome region 25BC was transcriptionally active in cells exposed to any of the five signals. On the other hand, region 36BD was only labeled in controls and cells exposed to the SP signal..No regions on chromosome 2R from unexposed cells showed transcriptional activity at this autoradiographic exposure,
(b)
Fig. 2. Examples of transcription autoradiograms of chromosomes (a) 2R and (b) 2L following exposure to each of five different ELF EM signals, heat shock (37°C) for 20 min in the presence of 3H-uridine. Control preparations are from unexposed cells incubated in the presence of tritiated uridine for 20 min at 25°C. The transcriptively active regions are identified according to published maps [9]. Autoradiographic exposure time was 48 h. In order to identify regions precisely chromosomes were cut to present them in a relatively straight line configuration. (Magnification: 615 x .)
(al
TABLE 1 Grain counts from transcription autoradiographs E33 Chromosome 2R 51BD 55CF 56BC 57AD 58CD 59AF 6OAC
10.5 7.8
CWl2
1.3
10.6
21.1
10.5 10.5
14.6
11.6 9.3 8.5
6.4 4.1
60DF Chromosome 2L 21AB 23AC 25BC 26BC 27EF 28EF 29AC 31AC 33CD 35AB 36BD
CW60
PT
20.2
over chromosome 2R and 2L SP
HS
C
Head specific RNA, maternal restricted transcript Maternal restricted transcript P-tubulin Tudor, c - erbB en-like homeobox
13.8 5.7 9.7 14.8
Maternal restricted transcript, P-tubulin Telomeres; Kruppel
8.2 7.3
10.7
50.6 9.3 8.3
8.7 5.9
12.1 10.3 7.2
10.5 10.6
8.9
8.6
Gene assignment of region
9.6 21.2 21.1
5.7
Telomeres Maternal restricted transcript Collagen-like gene
15.8 src homologous 7.2
6.8 4.7
26.1 10.3
9.3
ADH Myosin-heavy chain
A minimum of eight photographic plates were used for each determination in at least three different experiments. Data are given for salivary gland chromosomes that were exposed to the five EM signals at 2o”C, heat shock (37°C) or unexposed at 25°C for 20 min. Genes known to be associated with specific regions are given. The grain numbers are the average counts within a band or interband region. Grains that were not within this range were not included, though they may have in fact been produced within the same RNAs. All of these are compared with adjacent bands where there are no counts.
nor was transcriptional activity observed at any chromosome region on either 2R or 2L following sudden elevated temperature (heat shock). Chromosomes from cells exposed to the EM fields showed transcriptional activity in 17 regions of 2R and 2L. The results of the average grain counts are given in Table 1. Many chromosome regions responded to exposure to EM fields by increased transcription activity, and some were labeled in a signal-specific fashion. For example, exposure of cells to the sinusoidal fields affects three chromosomal loci in common: 51BD, 55CF and 60DF. Some chromosome regions responded to a single signal only, including five regions (23AC, 27EF, 28EF, 33CD 6OAC) that showed increased incorporation of 3H-uridine following exposure to the sinusoidal 60 Hz signal, but were not labeled following exposure to any of the other signals tested, and two regions specific to sinusoidal 72 Hz exposures (57AD and 58CD). These
45
data suggest that the cells can discriminate within a 12 Hz frequency range, and that response to these low frequency fields is exquisitely sensitive. This observation was also noted in our previous studies on chromosomes 3R, 3L and X [11,121. Differential response to different signals was also observed. For example, on chromosome 2L, some of the signals induced transcription in regions that were not observed in unexposed cells or seen in chromosomes exposed to other EM fields (see Table 1). Another region (55CF) on chr.omosome 2R responded to all signals. Related classes of genes responded to the EM signals. For example, regions which contain maternal restricted transcripts (a group of transcripts found in Drosophila eggs and isolated by cDNA probes) located at regions 23AC, 51BD, 55CF and 6OAC, and those containing genes for p-tubulin (56BD and 6OAC) were among those affected by ELF EM fields. This phenomenon was noted earlier. The’ putative genes for maternal restricted transcripts on chromosome 3 were induced at 83AD, 93DF, 75DE and tubulin at 84AD, 97DF [14,15]. Other regions of interest are those which include genes associated with development such as en-like homeobox (58CD) and Kruppel (6ODF), oncogenes including c-erbB (57BD) and src (29AC) and finally cell structure genes such as myosin (36BD) and collagenlike (35BC). DISCUSSION
The 17 chromosome regions which showed increased transcription contain genes that control and/or regulate cell structure and development. The specific physical component(s) of EM fields that participate in causing alterations in transcription are unknown at present, but it appears that the cell may “see” the EM field as a form of cellular stress. We showed previously that increased transcription, in the absence of temperature elevation, occurs at several heat shock loci in Drosophila salivary gland cells, e.g. chromosome regions 63AC, 93AD and 87AD, following a 20 min exposure to each of the five ELF EM signals used in this present study [11,12]. Many models for EM field interaction with biological systems have been proposed [16-201. There is increasing evidence that specific biological effects resulting from field exposure could be related to mechanisms involved in signal transduction [21]. Such a model would be consistent with observed transcriptional activation resulting from EM field exposure, and could explain how transcriptional activation at specific loci could be realized in the presence of EM fields, including the increase in transcript levels for several heat shock genes in cells exposed to ELF EM fields without an increase in temperature. Heat shock proteins apparently play a number of essential roles in cellular processes, not only under stress conditions but also under non-stress conditions where their synthesis is constitutively or developmentally regulated. They can regulate protein folding and assembly which then allows heat shock proteins to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in DNA repair (reviewed in refs. 22 and
46
23). These events are highly conserved and have been observed in all species studied including E. coli, yeast, murine and human cells. Hsp .90, for example, which is the most abundant constitutively expressed stress protein in the cytosol of eukaryotic cells, participates in the maturation of other proteins, and modulates protein activity in the case of hormone - free receptors and intracellular transport of some newly synthesized kinases [24]. The protective effect of mild heat treatment is eliminated when cycloheximide, an inhibitor of cytoplasmic protein synthesis, is added before the heat shock proteins are produced [25]. We suggest that a search for mechanism(s) of EM field interaction with the cell should include those pathways that have been shown to be implicated when the cells are subjected to stress (chemicals, temperature, pH and alterations in ionic concentration). Alterations in receptor-ligand or receptor-G protein interaction have been reported in cultured mouse bone cells exposed to a 15 Hz PEMF [26]. Alterations in calcium influx and efflux play a significant role in the response of cells to EM fields (reviewed in ref. 26). Time-varying magnetic fields act in combination with static magnetic fields to alter calcium signaling mitogen-activated lymphocytes [6-81 with accompanying increases in transcript levels for c-myc [27]. A recent report shows that calcium plays an essential role in the multistep activation of the heat shock factor in 3T3 cells. Activation of the heat shock factor and transcription of hsp 70 gene requires calcium and, of equal importance in understanding the EM field effect, is sensitive to a protein kinase inhibitor [28]. Finally, we have examined the changes in the biosynthetic responses of dipteran salivary glands cells to two stresses, EM fields and heat shock. We find that there are remarkable similarities in the proteins synthesized by salivary gland cells in response to EM fields and heat shock [29]. ACKNOWLEDGMENTS
We are grateful to Dr. C.F. Blackman for measurements of the local geomagnetic field and Mr. R. Cangialosi (Electra-Biology, Inc.) who made measurements, tested and standardized exposure equipment. This research was supported in part by grants from Electra-Biology, Inc., Parsippany, NJ, the Office of Naval Research (N00014-85-K-0577 (R.G.) and NOOO14-88-K-0105 (A.H.)), the Department of Energy (DE-FGOl-89CE34023) and the Electric Power and Research Institute (RP2965-05). REFERENCES 1 N. Werheimer and E. Leeper, Int. J. Epidemiol., 11 (1982) 345. 2 D. Savitz and E.E. Calle, J. Occup. Med., 29 (1987) 47. 3 Environmental Protection Agency, Evaluation of the Potential Carcinogenic&y of Electromagnetic Fields, Workshop Review Draft EPA/600/0, Washington, DC, 1989. 4 I. Nair, G. Morgan and H. Florig, Power Frequency Electric and Magnetic Field Exposure, Effects, Research and Regulation, US Congressional Office of Technology Assessment, Washington, DC, 1989.
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
R. Goodman and A. Henderson, Bioelectrochem. Bioenerg., 25 (1991) 335. M.G. Yost and R.P. Liburdy, FEBS, 296 (1992) 117. J. Wollazcek and R. Liburdy, FEBS Lett., 271 (1990) 157. R. Liburdy, FEBS Len:, 301 (1992) 53. R. Goodman, L.-X. Wei, J. Bumann and A. Henderson, Electra- and Magnetobiology, 11 (1992) 19. M. Blank, L. Soo, H. Lin, A. Henderson and R. Goodman Bioelectrochem. Bioenerg., 28 (1992) 301. R. Goodman, D. Weisbrot, A. Uluc and A. Henderson, Bioelectromagnetics, 13 (1992) 111. R. Goodman, D. Weisbrot, A. Uluc and A. Henderson, Bioelectrochem. Bioenerg., 28 (1992) 311. R. Goodman and A. Henderson, Proc. Natl. Acad. Sci. USA, 85 (1988) 3928. C. Bridges, J. Hered., 26 (1935) 60. J. Merriam, M. Ashbumer, D.L. Hart1 and F.C. Kafatos, Science, 254 (1991) 221. B.R. McLeod, S. Smith, K. Cooksey and A.R. Liboff, J. Bioelectr., 6 (1987) 1. C.F. Blackman, S.G. Benane, J.R. Rabinowitz, D.E. House and W.T. Joines, Bioelectromagnetics, 6 (1985) 327. W.R. Adey, Physiol. Rev., 61 (1981) 435. S. Andersson, S.T. Hyde, K. Larsson and S. Lidin, Chem. Rev., 88 (1988) 221. V.V. Lednev, Bioelectromagnetics, 12 (1990) 71. R. Luben and H. Duong, Proc. American Society for Cell Biology, 1989, Abstract 348. P.M. Pechan, FEBS, 280 (1991) 1. RI. Morimoto, Cancer Cells, 3 (1991) 295. H. Wiech, J. Buchner, R. Zimmermann and U. Jakob, Nature London, 358 (1992) 169. W.R. Adey and A. Chopart, In M. Blank and E. Find1 (Eds.), Mechanistic Approaches to Interaction of Electromagnetic Fields with Living Systems, Plenum, New York, p. 365. R. Luben, D. Huynh and A. Morgan, The Annual Review of Research of Biological Effects of 50 and 60 Hz Electric and Magnetic Fields, Department of Energy, Milwaukee, WI, p. A-15. R. Liburdy, personal communication, 1992. B.R. Price and SK. Calderwood, Mol. Cell Biol., 11 (1991) 3365. M. Blank, 0. Khorkova and R. Goodman, Proc., 1st World Congr. for Electricity and Magnetism in Biology and Medicine, Orlando, FL, 1992, p. 153.
Bioelectrochemistry and BioenergeGcs, 31(1993)
39-47
Elsevier Sequoia S.A., Lausanne
JEC BB 01578
Transcription in Drosophda melanogaster salivary gland cells is altered following exposure to low frequency electromagnetic fields: analysis of chromosomes 2R and 2L David Weisbrot William Paterson College of NewJersey, 07470 NJ (USA)
Alun Uluc and Ann Henderson Hunter College of the City Uniuersity of New York, New York (USA)
Reba Goodman
l
Columbia Uniuersify Health Sciences, Department of Pathology, 630 West 168 Street, New York,
NY 10032 (USA) (Received 12 August 1992)
Abstract Using the technique of transcription autoradiography, it is possible to identify nascent RNA chains directly on defined regions of Drosophila salivary gland chromosomes. Changes in transcriptional activity have been identified in 17 defined regions of Drosophila melanogasrer salivary gland chromosome arms 2R and 2L following 20 min exposures of salivary glands to five extremely low frequency electric and magnetic fields. Many of the labeled areas of the chromosome have been correlated with regions which contain known gene sites based on chromosome maps of D. melanogaster.
INTRODUCTION
A growing body of evidence, including the results of several epidemiological studies, suggests an association between human exposure to extremely low frequency (ELF) electric and magnetic (EM) fields and the incidence of some forms of cancers [l-3]. As a result of such findings, the effect of ELF EM fields on biological systems has become the focus of considerable research interest.
* To whom correspondence 0302-4598/93/$06.00
should be addressed.
0 1993 - Elsevier Sequoia S.A. All rights reserved
40
EM fields with physical parameters similar to those found in the environment are used in laboratory studies to investigate the interaction between low frequency fields and cells and tissues. Numerous and various effects on cell and tissue function resulting from exposure to EM fields have been reported (reviewed in refs. 4 and 5). Examples of effects include alterations in neurotransmitter release, changes in receptor binding and Con-A induced changes in intracellular Ca2+ levels (reviewed in refs. 4 and 5; see refs. 6-8). We demonstrated previously that significant increases in some transcripts can be measured when human HL-60 cells are exposed for short time periods (up to 40 min) to ELF EM fields (< 300 Hz). The magnitude of the increase is dependent on time, frequency and field strength [9]. Not all genes are affected. Transcript levels for p-2-microglobulin, for example, are unaffected in cells exposed to either EM fields or a.c. current [lo]. The present study is the third in a series that looks at the transcriptional response in Drosophila mefunogusfer salivary gland cells following 20 min exposures to five different ELF EM fields. Drosophila salivary gland cells have proven to be a very useful model for studies of this kind. Their polytene chromosomes, which are in interphase, have 1000-5000 copies of DNA strands side by side on each chromosome which produce identifiable bands. This makes it possible to visualize transcriptional changes directly on the chromosome. Further, these chromosomes have been extensively studied [ll], their bands and interbands mapped and associated specific gene sites have been identified (reviewed in ref. 12). We previously identified regions of chromosome 3R [ll] and 3L and X [12] from salivary gland cells that show differential 3H-uridine uptake into identified chromosome regions in response to exposure to a variety of low frequency EM fields for 20 min. At least two heat shock loci (87AD and 93AD) responded to exposure to each EM field tested without a detectable increase in temperature. Further, the effect of ELF EM fields on protein synthesis was determined by two-dimensional gel electrophoresis [11,13] and, among other changes in protein synthetic patterns, the 70 kD heat shock protein was increased. This report is a continuation of analyses of transcriptional activity on Drosophila chromosomes 2R and 2L in salivary gland cells exposed to ELF EM fields. A total of 17 chromosomal regions have been identified where transcriptional activity has been increased over that observed in chromosomes which were not exposed to ELF EM fields. MATERIALS AND METHODS
Fifteen pairs of salivary glands from third instar larvae were used for each experiment. The glands were dissected partially free from the larval body in Schneider’s Drosophila medium (SDM; GIBCO) in 30 mm glass Petri dishes. Some attachment to the larval body was retained during exposure to the EM fields. After three washes with SDM, 100 &i of 3H-uridine (NEN) was added to each dish (2
41
Waveform Characteristics 380 pSEC , Repetitive Single Pulse (SP) 4.5 mSEC
(Diode Clipped) 72 Hz
Repetitive .-------_ Pulse Burst (PB) 15 Hz
0.8 mV positive amplitude
-/\/\
Sine?
(CW
30 mSEC Equine-33 _____ Repetitive Pulse Burst 1.5 Hz
Fig. 1 The five EM signals used for these experiments with their major physical parameters. The values presented here for the physical parameters were measured using a calibrated inductive search coil (Electra-Biology, Inc.).
ml total volume) and the organ culture was exposed to one of each of the EM fields for 20 min. Three of the fields were pulsing electromagnetic fields (PEMFs; Electra-Biology, Inc., Parsippany, NJ) used clinically for the treatment of nonunion bone fractures, remodeling in osteoporosis or avascular necrosis [121. The PEMFs were compared with two continuous wave sinusoidal fields at frequencies of 60 and 72 Hz. The five EM fields used for these experiments, with their physical parameters, are’given in Fig. 1. For each of the five signals used, at least four separate experiments were performed. As previously described 111,121, the glass Petri dishes (containing the salivary glands) were placed on a Plexiglas form within a pair of 13 cm X 14 cm Helmholtz coils with an 8 cm space. The coils were positioned vertically so that the
42
oscillating magnetic field was generated in the horizontal plane, inducing a relatively uniform electric field in the conductive medium [5,9]. The organ culture containing the glands was exposed to the fields for 20 min at 25°C. Two groups of control cells were used for each experimental series. One group controlled for possible heating effects; these were incubated at 37°C for 20 min. This temperature was selected because it is known to produce specific changes in both transcription and translation. In the experiments reported here, temperature variations during all exposures were +O.S’C, as measured by a thermocouple temperature-sensitive probe (PhysiTemp Instruments, Inc.). The second control group were cells incubated in 3H-uridine for 20 min at 25°C in the-absence of the EM fields. These were shielded in a mu metal box. At the conclusion of each exposure, the dishes containing the organ culture were immediately placed on ice and washed three times with cold SDM, and the salivary glands were dissected free from the larval body. A single pair of glands (containing 70 cells) was placed on individual pre-cleaned glass slides in a drop of 45% acetic : 1% lactic acid. Gland pairs were cover slipped and squashed to release the chromosomes, and the cover slips were removed by freezing them in liquid nitrogen. The slides were dehydrated through a series of alcohols, after which they were air dried. For autoradiography, the slides containing the chromosomes were dipped in a 1: 1 dilution of Ilford K-5 autoradiographic emulsion (Ilford, UK), air dried and stored in light-tight boxes at 4°C. Slide preparations were developed and fixed at 24 h intervals and stained with Giemsa (1: 9). Exposure to the autoradiographic emulsion at 48 h was selected for the analyses presented here because at this exposure time the chromosomes were lightly, but significantly, labeled, which simplified the counting of the silver grains (see Fig. 2). Analysis of silver grains at identified chromosome regions was made from mounted cut-outs of chromosomes 2R and 2L from a minimum of 8 different photographic plates from each experiment or 24 for each determination. RESULTS
The baseline for these experiments was analysis of chromosomes 2R and 2L from control (not subjected to fields) salivary gland cells. Gene activity was deliberately underestimated by limiting the time of autoradiographic exposure. This made both identification of the bands and counting the grains easier. Preparations that were exposed to the autoradiographic emulsion for longer time periods showed no additional labeled regions, but had a heavier grain density with further exposure [ 11,121. In unexposed cells, two chomosome regions on 2L (25BC and 36BD) consistently showed transcriptional activity at this stage of larval development. In addition, chromosome region 25BC was transcriptionally active in cells exposed to any of the five signals. On the other hand, region 36BD was only labeled in controls and cells exposed to the SP signal..No regions on chromosome 2R from unexposed cells showed transcriptional activity at this autoradiographic exposure,
(b)
Fig. 2. Examples of transcription autoradiograms of chromosomes (a) 2R and (b) 2L following exposure to each of five different ELF EM signals, heat shock (37°C) for 20 min in the presence of 3H-uridine. Control preparations are from unexposed cells incubated in the presence of tritiated uridine for 20 min at 25°C. The transcriptively active regions are identified according to published maps [9]. Autoradiographic exposure time was 48 h. In order to identify regions precisely chromosomes were cut to present them in a relatively straight line configuration. (Magnification: 615 x .)
(al
TABLE 1 Grain counts from transcription autoradiographs E33 Chromosome 2R 51BD 55CF 56BC 57AD 58CD 59AF 6OAC
10.5 7.8
CWl2
1.3
10.6
21.1
10.5 10.5
14.6
11.6 9.3 8.5
6.4 4.1
60DF Chromosome 2L 21AB 23AC 25BC 26BC 27EF 28EF 29AC 31AC 33CD 35AB 36BD
CW60
PT
20.2
over chromosome 2R and 2L SP
HS
C
Head specific RNA, maternal restricted transcript Maternal restricted transcript P-tubulin Tudor, c - erbB en-like homeobox
13.8 5.7 9.7 14.8
Maternal restricted transcript, P-tubulin Telomeres; Kruppel
8.2 7.3
10.7
50.6 9.3 8.3
8.7 5.9
12.1 10.3 7.2
10.5 10.6
8.9
8.6
Gene assignment of region
9.6 21.2 21.1
5.7
Telomeres Maternal restricted transcript Collagen-like gene
15.8 src homologous 7.2
6.8 4.7
26.1 10.3
9.3
ADH Myosin-heavy chain
A minimum of eight photographic plates were used for each determination in at least three different experiments. Data are given for salivary gland chromosomes that were exposed to the five EM signals at 2o”C, heat shock (37°C) or unexposed at 25°C for 20 min. Genes known to be associated with specific regions are given. The grain numbers are the average counts within a band or interband region. Grains that were not within this range were not included, though they may have in fact been produced within the same RNAs. All of these are compared with adjacent bands where there are no counts.
nor was transcriptional activity observed at any chromosome region on either 2R or 2L following sudden elevated temperature (heat shock). Chromosomes from cells exposed to the EM fields showed transcriptional activity in 17 regions of 2R and 2L. The results of the average grain counts are given in Table 1. Many chromosome regions responded to exposure to EM fields by increased transcription activity, and some were labeled in a signal-specific fashion. For example, exposure of cells to the sinusoidal fields affects three chromosomal loci in common: 51BD, 55CF and 60DF. Some chromosome regions responded to a single signal only, including five regions (23AC, 27EF, 28EF, 33CD 6OAC) that showed increased incorporation of 3H-uridine following exposure to the sinusoidal 60 Hz signal, but were not labeled following exposure to any of the other signals tested, and two regions specific to sinusoidal 72 Hz exposures (57AD and 58CD). These
45
data suggest that the cells can discriminate within a 12 Hz frequency range, and that response to these low frequency fields is exquisitely sensitive. This observation was also noted in our previous studies on chromosomes 3R, 3L and X [11,121. Differential response to different signals was also observed. For example, on chromosome 2L, some of the signals induced transcription in regions that were not observed in unexposed cells or seen in chromosomes exposed to other EM fields (see Table 1). Another region (55CF) on chr.omosome 2R responded to all signals. Related classes of genes responded to the EM signals. For example, regions which contain maternal restricted transcripts (a group of transcripts found in Drosophila eggs and isolated by cDNA probes) located at regions 23AC, 51BD, 55CF and 6OAC, and those containing genes for p-tubulin (56BD and 6OAC) were among those affected by ELF EM fields. This phenomenon was noted earlier. The’ putative genes for maternal restricted transcripts on chromosome 3 were induced at 83AD, 93DF, 75DE and tubulin at 84AD, 97DF [14,15]. Other regions of interest are those which include genes associated with development such as en-like homeobox (58CD) and Kruppel (6ODF), oncogenes including c-erbB (57BD) and src (29AC) and finally cell structure genes such as myosin (36BD) and collagenlike (35BC). DISCUSSION
The 17 chromosome regions which showed increased transcription contain genes that control and/or regulate cell structure and development. The specific physical component(s) of EM fields that participate in causing alterations in transcription are unknown at present, but it appears that the cell may “see” the EM field as a form of cellular stress. We showed previously that increased transcription, in the absence of temperature elevation, occurs at several heat shock loci in Drosophila salivary gland cells, e.g. chromosome regions 63AC, 93AD and 87AD, following a 20 min exposure to each of the five ELF EM signals used in this present study [11,12]. Many models for EM field interaction with biological systems have been proposed [16-201. There is increasing evidence that specific biological effects resulting from field exposure could be related to mechanisms involved in signal transduction [21]. Such a model would be consistent with observed transcriptional activation resulting from EM field exposure, and could explain how transcriptional activation at specific loci could be realized in the presence of EM fields, including the increase in transcript levels for several heat shock genes in cells exposed to ELF EM fields without an increase in temperature. Heat shock proteins apparently play a number of essential roles in cellular processes, not only under stress conditions but also under non-stress conditions where their synthesis is constitutively or developmentally regulated. They can regulate protein folding and assembly which then allows heat shock proteins to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in DNA repair (reviewed in refs. 22 and
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23). These events are highly conserved and have been observed in all species studied including E. coli, yeast, murine and human cells. Hsp .90, for example, which is the most abundant constitutively expressed stress protein in the cytosol of eukaryotic cells, participates in the maturation of other proteins, and modulates protein activity in the case of hormone - free receptors and intracellular transport of some newly synthesized kinases [24]. The protective effect of mild heat treatment is eliminated when cycloheximide, an inhibitor of cytoplasmic protein synthesis, is added before the heat shock proteins are produced [25]. We suggest that a search for mechanism(s) of EM field interaction with the cell should include those pathways that have been shown to be implicated when the cells are subjected to stress (chemicals, temperature, pH and alterations in ionic concentration). Alterations in receptor-ligand or receptor-G protein interaction have been reported in cultured mouse bone cells exposed to a 15 Hz PEMF [26]. Alterations in calcium influx and efflux play a significant role in the response of cells to EM fields (reviewed in ref. 26). Time-varying magnetic fields act in combination with static magnetic fields to alter calcium signaling mitogen-activated lymphocytes [6-81 with accompanying increases in transcript levels for c-myc [27]. A recent report shows that calcium plays an essential role in the multistep activation of the heat shock factor in 3T3 cells. Activation of the heat shock factor and transcription of hsp 70 gene requires calcium and, of equal importance in understanding the EM field effect, is sensitive to a protein kinase inhibitor [28]. Finally, we have examined the changes in the biosynthetic responses of dipteran salivary glands cells to two stresses, EM fields and heat shock. We find that there are remarkable similarities in the proteins synthesized by salivary gland cells in response to EM fields and heat shock [29]. ACKNOWLEDGMENTS
We are grateful to Dr. C.F. Blackman for measurements of the local geomagnetic field and Mr. R. Cangialosi (Electra-Biology, Inc.) who made measurements, tested and standardized exposure equipment. This research was supported in part by grants from Electra-Biology, Inc., Parsippany, NJ, the Office of Naval Research (N00014-85-K-0577 (R.G.) and NOOO14-88-K-0105 (A.H.)), the Department of Energy (DE-FGOl-89CE34023) and the Electric Power and Research Institute (RP2965-05). REFERENCES 1 N. Werheimer and E. Leeper, Int. J. Epidemiol., 11 (1982) 345. 2 D. Savitz and E.E. Calle, J. Occup. Med., 29 (1987) 47. 3 Environmental Protection Agency, Evaluation of the Potential Carcinogenic&y of Electromagnetic Fields, Workshop Review Draft EPA/600/0, Washington, DC, 1989. 4 I. Nair, G. Morgan and H. Florig, Power Frequency Electric and Magnetic Field Exposure, Effects, Research and Regulation, US Congressional Office of Technology Assessment, Washington, DC, 1989.
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