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Apoptosis in haemopoietic progenitor cells exposed to extremely low-frequency magnetic fields
Life Sciences,Vol. 61, No. 16, pp. l57l-l582,1997 Cqyright 0 1959 ElsevierScienceInc. Printedin the USA. AlI rightsmmwd oL?24-324?5/97 $17.00 t .oo
PII Soo24-3205(97)00736-4
APOPTOSIS
IN HAEMOPOIETIC PROGENITOR CELLS EXPOSED TO EXTREMELY LOW-FREQUENCY MAGNETIC FIELDS
Birgit M. Reipert’, Donald Allan’, Siegfried Reipert’, T. Michael Dexter3 Cancer Research Campaign Departments of Physics & Instrumentation’, Structural Cell Biology’ and Experimental Haematology3, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 9BX, UK (Received in foal form July 7,1!W7)
Epidemiological studies have indicated a modestly increased risk for the development of acute myeloid leukaemia in children who live close to high-voltage power-lines. Recent evidence has suggested that a common property shared by a number of known and suspected tumour promoters is their ability to block the process of apoptosis. Therefore, one possible mechanistic explanation for the apparent leukaemogenic effect of weak, low-frequency magnetic fields, such as emitted by power-lines and electrical appliances, would be their expression of tumour-promoting activity by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. In order to test this hypothesis, we have employed the well-characterized multipotential haemopoietic progenitor cell line These cells are non-leukaemic and undergo apoptosis when FDCPmix(A4). deprived of appropriate growth factors such as Interleukin-3. We have tested a series of different regimes of weak, low-frequency magnetic fields: nulled fields, Ca”-ion cyclotron resonance conditions at 50 Hz, and vertical 50 Hz fields of 6 pTRMs, 1 mT,s and 2 mT,s, exposing the cells for 2 hours, 24 hours, 4 days or 7 days under various culture conditions. We have not seen any significant alteration in apoptosis induced by any of the exposure regimes tested. We therefore conclude that the regulation of viability and apoptosis in FDCP-mix(A4) cells is not disturbed by weak magnetic fields of the magnitude and type indicated. Key words:
tumor promoters, apoptosis, low-frequency electromagnetic fields, multipotent haemopoietic
progenitor cells
A number of epidemiological studies have suggested a link between the exposure to extremely low-frequency magnetic fields (ELFMF) emitted by high-voltage power-lines and electrical appliances and a modestly increased incidence of leukaemia, in particular acute myeloid leukaemia in children (l-6). These suggestions have stimulated a considerable number of experimental studies aimed at providing a mechanistic explanation for a possible tumour promoting activity of weak ELFMFs. However, no consensus on a definite mechanism that might explain the biological activity of such fields has yet emerged (12-15). Furthermore, the cell systems employed by most
Corresponding author: Dr D Allan, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 9BX, UK, Tel: +44 161 446 3120, Fax: +44 161 446 3109, E-mail: [email protected]
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in vitro studies are not necessarily appropriate for elucidating a mechanism by which weak ELFMFs might promote the development of acute myeloid leukaemia (AML), since the evidence suggests that AML originates from an abnormal cell clone developed at an early stage during haemopoiesis, probably at the level of a multipotential progenitor or pluripotential stem cell (16,17). Several lines of evidence support the concept that tumour growth in viva depends on the evasion of normal homeostatic control mechanisms operating via the induction of cell death by apoptosis. Wright et al have recently shown that a common property shared by known or suspected tumour promoters might be the ability to block the process of apoptosis (7). They have tested a total of 10 tumour promoters, and all were found to inhibit cell death. We have therefore tested the hypothesis that weak ELFMFs might act as promoters for the development of AML by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. For this purpose, we have employed the well-defined multipotential haemopoietic progenitor cell line FDCPmix(A4) that can be cultured under well-established conditions which allow either self-renewal of multipotential cells or commitment and differentiation along alternative (granulocyte/macrophage or erythroid) lineages (8,9). These cells are non-leukaemic and, like normal cells, undergo apoptosis when deprived of appropriate growth factors such as Interleukin-3 (IL-3) (10,ll).
Methods Cell line and cell culture The FDCP-mix (A4) cell line was derived from mouse long-term bone marrow cultures (8). These cells have a diploid karyotype, are non-leukaemic and depend upon IL-3 for their survival and selfrenewal. Furthermore, they can be induced to differentiate by either cultivation over stromal layers or by the addition of soluble growth factors (8,10,11). Stocks of the FDCPmix(A4) cell line are held in liquid nitrogen storage. To minimize the possibility of acquisition of a change in phenotype arising from genotypic alteration over time, batches of frozen stock were thawed serially at regular intervals of about 3 months. Cells were maintained in Iscove’s medium (Gibco BRL, Life Technologies, Paisley, UK) supplemented with 20% horse serum (HS) (Sera Lab, Crawley Down, UK) and 1% conditioned medium from X63Ag8-653 cells as a source of mouse IL-3 (mIL3-CM). X63Ag8-653 cells have been transfected with a murine IL-3 cDNA and constitutively secrete large amounts of IL-3 (18). 1% mIL3-CM corresponds to about 100 U/ml mouse IL-3. In all experiments, cells were harvested in exponential growth phase and then washed twice with Iscove’s medium before being resuspended in Iscove’s medium supplemented with 20% HS and the appropriate concentration of mIL3-CM (no IL-3, O.l%, 0.5% or 1.0%). Unless otherwise stated, cells were seeded at a concentration of 16 per ml in 10 ml medium. ELFMF-exposure experiments were carried out using two different protocols. To investigate the possible influence of weak ELFMFs on the kinetics of apoptosis, cells were washed free of IL-3 and then immediately subjected to two hours ELFMF-exposure. For longer-term exposure to ELFMFs over 24 hours, 4 days or 7 days, samples were set up one hour prior to ELFMF exposure. Flow-cytometric analysis Flow-cytometric analysis was carried out using a Coulter EPICS V flow-cytometer cell sorter (Coulter Electronics, Luton, UK). Computer analysis of the data was performed using the Paterson Institute Epics software package (S A Roberts, unpublished).
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For the discrimination and quantification of viable, apoptotic and necrotic cells, a rapid multiparameter assay was used, measuring forward light scatter intensity (FALS) in combination with emission from the DNA-binding fluorophores Hoechst 33342 (Sigma, St Louis, MO, USA) and propidium iodide (Sigma, St Louis, MO, USA) (10,19). Briefly, Hoechst 33342 (10 pM) and propidium iodide (32 pM) were added to the cell suspension (1-2x16 cells in 0.5 ml Iscove’s medium). After 2 min incubation, lo4 cells were analysed, using the argon-ion laser ultraviolet excitation lines (100 mW) centred around 357 nm. FALS, blue fluorescence (Hoechst 33342DNA; log scale, between 420-560 nm) and red fluorescence (propidium iodide-DNA; linear scale, above 630 nm) were recorded for each cell analysed. Cell debris were excluded electronically. The data were processed as two-dimensional dot plots of FALS versus the logarithm of the blue fluorescence intensity, excluding cells exhibiting red fluorescence (necrotic cells). Transmission electron microscopy (TEM) Cell pellets were fixed in 2.5% glutaraldehyde in 0.15 M Sorensen’s buffer (pH 7.4) for 1 hr. After three buffer washes the pellets were postfixed in 1% 0~0, for 1 hr. Subsequently, the pellets were dehydrated in ethanol and embedded in epoxy resin (Agar 100, Agar Scientific Ltd, Stansted, UK). Thin sections were stained in uranyl acetate and Reynolds lead citrate, and viewed at 80 kV in a Philips electron microscope (Philips EM 400). Exposure to electromagnetic fields ELFMF exposures were performed within a 37°C temperature-controlled hot room, as described previously (20). ELFMFs were generated by a custom-designed coil assembly comprising a 170 mm diameter, 300 mm tall, vertical axis solenoid coil wound on a tubular former with a pair of Helmholtz coils 280 mm in diameter set on a horizontal axis and fastened to either side of the solenoid. The whole unit was mounted on a base which allowed rotation about a vertical axis so that, in combination with a dual-channel coil power-supply giving independent control of the currents in the two coil systems, the field environment within the assembly could be adjusted as required for both field strength and direction. The coil assembly base mounting also incorporated a support rack to hold T25 cell culture flasks. The flasks were held within a volume of field calculated to be uniform to better than 1% and sufficiently large to accommodate a batch of three flasks, when both solenoid and Helmholtz coils were used, or five flasks when the field configuration required that only the solenoid be energised. Sham-exposed control samples were placed on a second identical support rack enclosed by a vertical tube matching the solenoid coil former. The coil and sham-exposure assemblies were positively located in fixed jigs spaced about 1.5 m apart, a distance at which stray fields from the coil set were minimal. A precision low-frequency sine-wave generator was designed to supply AC modulation of coil currents in addition to the DC output of the main coil power-supply. This power-supply configuration was confirmed to provide AC and DC coil currents with stabilities of better than 0.1% over three days. Consequently, over the duration of an experiment, it was the variability of the ambient field which limited the stability of the net field at the sample position. In the hot room, the magnitude of the local static field was in the range 25.2 f 0.5 uT (mean f 1 SD) while the RMS alternating field was 0.5 * 0.2 uT (mean * 1 SD). Throughout an experiment, the local ambient magnetic fields and the temperature of both ELFMF-exposed and sham-exposed samples were continuously monitored by a data logging system controlled by a personal computer (PC). A Bartington MAG-03MC triple-axis fluxgate magnetometer, interfaced to the PC, was used for magnetic field measurements. Temperatures were measured using thermocouple probes, positioned immediately beside the sample flasks on the support racks within the tubular formers, with digital recording, again interfaced to the PC. Potential temperature inequalities between ELFMF and sham-exposed samples due to warming of
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the ELFMF-exposed samples by power dissipated in the coil windings were prevented by forced air circulation through the cylindrical formers surrounding the culture flask support racks. Temperature stability over a period of three days was approximately O.l”C and any residual difference between the ELFMF-exposed and sham-exposed samples was established to be within the O.l”C resolution of the recorders.
TABLE I Summary
of ELFMF Exposure Conditions
Sinusoidal Fields (uT, mean f SD) Vertical
Maximum Induced Electric Field @V/m)
Static Fields (uT, mean f SD)
Horizontal
Vertical
Horizontal
I 0.2 (nulled fields)
I 0.2 (nulled fields)
A - nulled fields 0.22 f 0.09 (ambient fields)
0.415~ 0.16 (ambient fields)
0
B - Ca2+-ion cyclotron resonance
0.22 * 0.09 (ambient fields)
30 cir,s (applied fields) C - vertical 50
6 uTrors (applied
field)
0.41 f 0.16 (ambient fields) D - vertical 50
1 mT,,s (applied field)
0.41 i 0.16 (ambient fields) E - vertical 50
2 m’bS
(applied fields)
0.41 f 0.16
(ambient fields)
52
Hzfields 23
Hzfields 3900
Hzfields 7800
conditions at 50 Hz
I 0.2 (nulled fields)
65 VT (applied fields)
of 6 pTRMs
21.6 f 0.8 (ambient fields)
13.1 f 0.9 (ambient fields)
of 1 mTRMs
21.6 f 0.8 (ambient fields)
13.1 f 0.9 (ambient fields)
of 2 mTRMs 21.6 i 0.8
13.1 f 0.9
(ambient fields)
Means and SDS listed describe fields over the entire experimental
(ambient
fields)
programme.
ELFMF exposure conditions The experiments reported were performed under three different field exposure regimes as detailed
in Table I. In all cases, the magnetic field configuration was set using the Bartington fluxgate magnetometer. In the nulled field case (configuration A), both solenoid and Hehnholtz coils were used to counterbalance the ambient static magnetic fields to give near zero field at the ELFMF-exposed sample position. Alternating field components remained at ambient background values. For cyclotron resonance conditions (configuration B), an alternating and static magnetic field combination matching the resonance conditions for Ca” was applied in a horizontal plane using the Helmholtz coil while the solenoid was used to null the vertical component of the ambient static field. Finally, in configurations C, D and E, the solenoid alone was used to generate vertical
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respectively while all other fields alternating field components of 6 pTWs 1 mT,, and 2 mT,, remained at ambient values. Also listed in Table I are calculated values for the maximum alternating electric field induced in the culture fluid by the corresponding alternating magnetic field. These calculations take due account of the dimensions of the culture flasks and the amount of culture fluid used.
22 20 I) '7 3
15
6
12
6 10 *7 5 2
Fig. 1 3-dimensional plot of FDCPmix(A4) cells analysed by flow-cytometry after 24 hr culture in the presence (A) or absence (B) of IL-3 under normal ambient laboratory conditions of ELFMF.
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Fig. 2 TEM micrographs of thin sections of FDCP-mix(A4) cells grown under normal ambient laboratory conditions of ELFMF. Bar = 3 pm. (A) In the presence of IL-3 : Note the characteristic arrangement of heterochromatin at the nuclear periphery in comparison to (B). 24 hours after withdrawal of IL-3 : Apoptotic cells are characterised by (B) margination of electron dense material (arrows) inside their nuclei.
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Results Apoptosis in FDCP-mix(A4) cells under ambient conditions of ELFMF It has previously been shown that withdrawal of IL-3 causes apoptosis in FDCP-mix(A4) cells (10,ll). By 24 hours after the removal of IL-3, the majority of the cells had undergone apoptosis (Fig. 1) and showed electron dense margination inside their nuclei (Fig. 2), supposed to be condensed chromatin (21), which are regarded as morphological hall marks of this process. A significant increase in the percentage of apoptotic cells under normal ambient laboratory conditions of ELFMF was already seen at about 4 to 6 hours after withdrawal of the growth factor (Fig. 3). Thereafter, the proportion of apoptotic cells increased gradually and reached nearly 100 % after 48 hours (Fig. 3). In order to define optimal and different suboptimal conditions for the maintenance of viability in normal ambient conditions of ELFMF, we tested several concentrations of mIL3-CM (O.l%, 0.5%, 1.0%) over different periods of culture. As shown in Fig. 4, there is no significant difference in the maintenance of viability under ambient conditions of ELFMF for concentrations of 0.5% and 1.0% n-&3-CM. However, a significantly higher proportion of apoptotic cells was seen after different periods of culture when only 0.1% mIL3-CM was used.
100 90 80
20 10 0
4 6 8 Culture period (hours)
24
48
Fig. 3 Induction of apoptosis in FJXP-mix(A4) cells under normal ambient laboratory conditions of ELFMF at different time periods after withdrawal of IL-3. The proportion of viable, apoptotic and necrotic cells was analysed by flow cytometry. viable cells fz# apoptotic cells necrotic cells III
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100
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mean + SEM
90 80 (0 2 70 3
80
mlL3-CM ‘i5 50 8 40
0.1%
0.5%
mlL3-CM
mlL3-CM
1.0%
30 20 10
n=lO
1 day culture
4 days culture
n=25
7 days culture
Fig. 4 Proportion of apoptotic cells in FDCP-mix(A4) cells maintained in medium supplemented with different concentrations of mIL3-CM (O.l%, OS%, 1.O%) over different periods of culture under normal ambient conditions of ELFMF. The proportion of apoptotic cells was detected by flow-cytometry.
Induction of apoptosis afrer withdrawal of IL-3 in FDCP-mtx(A4) cells exposed to various regimes of weak EL.FMFs We investigated whether or not the exposure to various regimes of weak ELFMFs affects the kinetics of apoptosis in FDCPmix(A4) cells after withdrawal of IL-3. For this purpose, cells were transferred into the exposure or sham-exposure environment after removal of the growth factor and period, then exposed/sham-exposed for two hours. After the two hour exposure/sham-exposure cells were either analysed immediately, or left for a further 2, 4 or 22 hours in ambient field conditions before being analysed, for apoptosis using flow cytometry. In six separate experiments, all performed in triplicate, no significant alteration in the kinetics of apoptosis due to the exposure to any of the ELFMF regimes tested was evident; the ratios of the proportion of apoptotic cells for exposed (E) and sham-exposed (S) cells, displayed in Table II, were in no case found to be significantly different from unity.
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TABLE II Induction
of Apoptosis
after Withdrawal
of IL-3 in FDCPmix(A4)
E/S (Exposed/Sham-Exposed)
Cells Exposed to ELF&IFS
: mean f SEM (n=6)
ELFMF exposure conditions
(2 hr duration)
Time after withdrawal of IL-3
A
B
C
D
E
2 hours
1.00 f 0.030
1.00 + 0.015
1.00 -c 0.008
1.OO+ 0.011
0.99 f 0.006
4 hours
1.01 zt 0.018
1.00 f 0.016
1.00 f 0.009
1.01 f 0.012
1.00 f 0.017
6 hours
1.01 k 0.030
0.98 * 0.023
1.01 f 0.012
0.98 * 0.020
0.99 f 0.023
24 hours
1.02 f 0.018
1.01 f 0.042
1.00 i 0.014
1.01 f 0.014
0.99 f 0.048
The ratios of the proportion of apoptotic cells for exposed (E) and sham-exposed (S) cells were calculated for each individual experiment (6 experiments, all performed in triplicate). Overall means and SEMs are shown.
TABLE III Maintenance
of Viability
in FDCP-mix(A4)
Cells Exposed to ELFMFs
E/S (Exposed/Sham-Exposed)
: mean f SEM (n=6)
ELFMF exposure conditions Culture conditions
Exposure duration
A
B
C
0.1% mIL3-CM
24 hours 4 days 7 days
1.00 f 0.011 1.00 f 0.017 0.98 f 0.038
1.00 f 0.033 1.02 f 0.012 0.99 f 0.025
1.01 * 0.016 1.02 zt 0.027 1.00 f 0.030
0.5% mIL3-CM
24 hours 4 days 7 days
1.00 f 0.005 0.98 i 0.024 0.99 f 0.046
1.00 -c 0.009 0.98 f 0.019 1.00 f 0.017
1.01 f 0.014 1.01 f 0.015 1.01 + 0.013
1.0% mIL3-CM
24 hours 4 days
1.01 f 0.012 1.01 f 0.021
0.99 f 0.008 1.02 f 0.022
1.00 f 0.042 0.97 f 0.021
The ratios of the proportion of apoptotic cells for exposed (E) and sham-exposed (S) cells were calculated for each individual experiment (6 experiments, all performed in triplicate). Overall means and SEMs are shown.
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Maintenance of viability under optimal and suboptimal culture conditions in FDCP-mix(A4) cells exposed to weak ELFMFs In order to study the influence of various regimes of weak ELFMFs on the maintenance of viability in FDCP-mix(A4) cells maintained in different suboptimal and optimal culture conditions, cells were set up as described in Methods and then exposed/sham-exposed for 24 hours, 4 days or 7 days. Immediately after exposure/sham-exposure, the cells were analysed for viability and apoptosis using flow cytometry. As shown in Table III, the ratios of the proportion of apoptotic cells in exposed (E) and sham-exposed (S) cells were never found to be significantly different from unity. We have therefore not seen any significant alteration in the maintenance of viability due to the exposure to weak ELFMFs.
Discussion The present study was performed to test the hypothesis that weak ELFMFs might promote the development of AML by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. Recent evidence has shown that the ability to block apoptosis might be a common characteristic of known and suspected tumour promoters (7). Therefore, an alteration of apoptosis in the presence of weak ELFMFs might provide an explanation for the increased risk reported for the development of AML in children that have been exposed to weak ELFMFs emitted by high-voltage power-lines. We employed the well-characterised multipotential haemopoietic progenitor cell line FDCPmix(A4) which depends on the permanent presence of IL-3 for survival and self-renewal (10,ll) as a reasonable model system for testing this possibility. We first defined the kinetics of the onset of apoptosis after growth factor withdrawal under ambient laboratory conditions (Fig. l-3) in order to establish the level at which the system would be able to detect any alterations that might be caused by a series of different weak sinusoidal ELFMFs, or were caused by the ambient static geomagnetic field. Differences of more than about 5% would have been significant. However, no modifications in the kinetics of apoptosis even at this level were observed after exposure of FDCP-mix(A4) cells for 2 hours to any of the ELF&IF regimes tested (Table II). Nevertheless, weak ELFMFs might induce more subtle changes in apoptosis that would only be detectable after a longer period of exposure. We have therefore investigated whether or not weak ELFMFs interfere with the maintenance of viability in FDCP-mix(A4) cells cultured under different suboptimal and optimal growth conditions over periods of up to seven days. Significant differences in the proportion of apoptotic and viable cells under ambient laboratory conditions were detected when the culture medium was supplemented with different concentrations of IL-3 (Fig. 4). However, we did not find any significant alteration in the proportion of apoptotic cells due to the exposure to various regimes of weak ELFMFs (Table RI). Clearly, these results do not support the hypothesis that weak ELFMFs express tumour promoting activity by the inhibition of apoptosis in multipotent haemopoietic progenitor cells. In addition, we have reported previously that exposure to similar ELFMFs does not affect growth rate, cellcycle state or clonogenic efficiency of FDCPmix(A4) cells (20). The exposure conditions we have employed are well within the range of typical environmental fields. For example, children exposed to weak ELFMFs emitted by power-lines experience 50 Hz fields of the order of lo-40 pT (for power-lines between 132 and 400 kV) at ground level directly beneath the power-lines, and about 2-8 pT at 25 m lateral displacement from the centre-line, and similar fields exist close to electrical household appliances (22). Also, the exposure conditions we have chosen are very similar to those that have been reported by others to alter cell functions in
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in vitro or in vivo experiments in a variety of different cell systems (23-26). Although some epidemiological studies suggest a possible link between exposure to weak ELFMFs and an increased risk for the development of leukaemia, overall, epidemiological studies of exposure to weak ELFMFs have given somewhat contradictory results (l-6, 27-29). In view of this, it is possible that weak ELFMFs do interact with biological systems, but only under rather specific exposure conditions different from those tested by us, or only in conjunction with additional environmental influences that have yet to be defined.
Acknowledgments We would like to thank M F Hughes and J Barry for their skilled technical assistance with the flow cytometric analysis and P Lucas for instrumentation development. We are particularly grateful to R E Dale for critical comment and discussion. This work was supported by the National Grid Company plc and by the Cancer Campaign [CRC]. TMD is a Gibb Fellow of the Cancer Research Campaign.
Research
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APOPTOSIS
IN HAEMOPOIETIC PROGENITOR CELLS EXPOSED TO EXTREMELY LOW-FREQUENCY MAGNETIC FIELDS
Birgit M. Reipert’, Donald Allan’, Siegfried Reipert’, T. Michael Dexter3 Cancer Research Campaign Departments of Physics & Instrumentation’, Structural Cell Biology’ and Experimental Haematology3, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 9BX, UK (Received in foal form July 7,1!W7)
Epidemiological studies have indicated a modestly increased risk for the development of acute myeloid leukaemia in children who live close to high-voltage power-lines. Recent evidence has suggested that a common property shared by a number of known and suspected tumour promoters is their ability to block the process of apoptosis. Therefore, one possible mechanistic explanation for the apparent leukaemogenic effect of weak, low-frequency magnetic fields, such as emitted by power-lines and electrical appliances, would be their expression of tumour-promoting activity by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. In order to test this hypothesis, we have employed the well-characterized multipotential haemopoietic progenitor cell line These cells are non-leukaemic and undergo apoptosis when FDCPmix(A4). deprived of appropriate growth factors such as Interleukin-3. We have tested a series of different regimes of weak, low-frequency magnetic fields: nulled fields, Ca”-ion cyclotron resonance conditions at 50 Hz, and vertical 50 Hz fields of 6 pTRMs, 1 mT,s and 2 mT,s, exposing the cells for 2 hours, 24 hours, 4 days or 7 days under various culture conditions. We have not seen any significant alteration in apoptosis induced by any of the exposure regimes tested. We therefore conclude that the regulation of viability and apoptosis in FDCP-mix(A4) cells is not disturbed by weak magnetic fields of the magnitude and type indicated. Key words:
tumor promoters, apoptosis, low-frequency electromagnetic fields, multipotent haemopoietic
progenitor cells
A number of epidemiological studies have suggested a link between the exposure to extremely low-frequency magnetic fields (ELFMF) emitted by high-voltage power-lines and electrical appliances and a modestly increased incidence of leukaemia, in particular acute myeloid leukaemia in children (l-6). These suggestions have stimulated a considerable number of experimental studies aimed at providing a mechanistic explanation for a possible tumour promoting activity of weak ELFMFs. However, no consensus on a definite mechanism that might explain the biological activity of such fields has yet emerged (12-15). Furthermore, the cell systems employed by most
Corresponding author: Dr D Allan, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 9BX, UK, Tel: +44 161 446 3120, Fax: +44 161 446 3109, E-mail: [email protected]
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in vitro studies are not necessarily appropriate for elucidating a mechanism by which weak ELFMFs might promote the development of acute myeloid leukaemia (AML), since the evidence suggests that AML originates from an abnormal cell clone developed at an early stage during haemopoiesis, probably at the level of a multipotential progenitor or pluripotential stem cell (16,17). Several lines of evidence support the concept that tumour growth in viva depends on the evasion of normal homeostatic control mechanisms operating via the induction of cell death by apoptosis. Wright et al have recently shown that a common property shared by known or suspected tumour promoters might be the ability to block the process of apoptosis (7). They have tested a total of 10 tumour promoters, and all were found to inhibit cell death. We have therefore tested the hypothesis that weak ELFMFs might act as promoters for the development of AML by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. For this purpose, we have employed the well-defined multipotential haemopoietic progenitor cell line FDCPmix(A4) that can be cultured under well-established conditions which allow either self-renewal of multipotential cells or commitment and differentiation along alternative (granulocyte/macrophage or erythroid) lineages (8,9). These cells are non-leukaemic and, like normal cells, undergo apoptosis when deprived of appropriate growth factors such as Interleukin-3 (IL-3) (10,ll).
Methods Cell line and cell culture The FDCP-mix (A4) cell line was derived from mouse long-term bone marrow cultures (8). These cells have a diploid karyotype, are non-leukaemic and depend upon IL-3 for their survival and selfrenewal. Furthermore, they can be induced to differentiate by either cultivation over stromal layers or by the addition of soluble growth factors (8,10,11). Stocks of the FDCPmix(A4) cell line are held in liquid nitrogen storage. To minimize the possibility of acquisition of a change in phenotype arising from genotypic alteration over time, batches of frozen stock were thawed serially at regular intervals of about 3 months. Cells were maintained in Iscove’s medium (Gibco BRL, Life Technologies, Paisley, UK) supplemented with 20% horse serum (HS) (Sera Lab, Crawley Down, UK) and 1% conditioned medium from X63Ag8-653 cells as a source of mouse IL-3 (mIL3-CM). X63Ag8-653 cells have been transfected with a murine IL-3 cDNA and constitutively secrete large amounts of IL-3 (18). 1% mIL3-CM corresponds to about 100 U/ml mouse IL-3. In all experiments, cells were harvested in exponential growth phase and then washed twice with Iscove’s medium before being resuspended in Iscove’s medium supplemented with 20% HS and the appropriate concentration of mIL3-CM (no IL-3, O.l%, 0.5% or 1.0%). Unless otherwise stated, cells were seeded at a concentration of 16 per ml in 10 ml medium. ELFMF-exposure experiments were carried out using two different protocols. To investigate the possible influence of weak ELFMFs on the kinetics of apoptosis, cells were washed free of IL-3 and then immediately subjected to two hours ELFMF-exposure. For longer-term exposure to ELFMFs over 24 hours, 4 days or 7 days, samples were set up one hour prior to ELFMF exposure. Flow-cytometric analysis Flow-cytometric analysis was carried out using a Coulter EPICS V flow-cytometer cell sorter (Coulter Electronics, Luton, UK). Computer analysis of the data was performed using the Paterson Institute Epics software package (S A Roberts, unpublished).
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For the discrimination and quantification of viable, apoptotic and necrotic cells, a rapid multiparameter assay was used, measuring forward light scatter intensity (FALS) in combination with emission from the DNA-binding fluorophores Hoechst 33342 (Sigma, St Louis, MO, USA) and propidium iodide (Sigma, St Louis, MO, USA) (10,19). Briefly, Hoechst 33342 (10 pM) and propidium iodide (32 pM) were added to the cell suspension (1-2x16 cells in 0.5 ml Iscove’s medium). After 2 min incubation, lo4 cells were analysed, using the argon-ion laser ultraviolet excitation lines (100 mW) centred around 357 nm. FALS, blue fluorescence (Hoechst 33342DNA; log scale, between 420-560 nm) and red fluorescence (propidium iodide-DNA; linear scale, above 630 nm) were recorded for each cell analysed. Cell debris were excluded electronically. The data were processed as two-dimensional dot plots of FALS versus the logarithm of the blue fluorescence intensity, excluding cells exhibiting red fluorescence (necrotic cells). Transmission electron microscopy (TEM) Cell pellets were fixed in 2.5% glutaraldehyde in 0.15 M Sorensen’s buffer (pH 7.4) for 1 hr. After three buffer washes the pellets were postfixed in 1% 0~0, for 1 hr. Subsequently, the pellets were dehydrated in ethanol and embedded in epoxy resin (Agar 100, Agar Scientific Ltd, Stansted, UK). Thin sections were stained in uranyl acetate and Reynolds lead citrate, and viewed at 80 kV in a Philips electron microscope (Philips EM 400). Exposure to electromagnetic fields ELFMF exposures were performed within a 37°C temperature-controlled hot room, as described previously (20). ELFMFs were generated by a custom-designed coil assembly comprising a 170 mm diameter, 300 mm tall, vertical axis solenoid coil wound on a tubular former with a pair of Helmholtz coils 280 mm in diameter set on a horizontal axis and fastened to either side of the solenoid. The whole unit was mounted on a base which allowed rotation about a vertical axis so that, in combination with a dual-channel coil power-supply giving independent control of the currents in the two coil systems, the field environment within the assembly could be adjusted as required for both field strength and direction. The coil assembly base mounting also incorporated a support rack to hold T25 cell culture flasks. The flasks were held within a volume of field calculated to be uniform to better than 1% and sufficiently large to accommodate a batch of three flasks, when both solenoid and Helmholtz coils were used, or five flasks when the field configuration required that only the solenoid be energised. Sham-exposed control samples were placed on a second identical support rack enclosed by a vertical tube matching the solenoid coil former. The coil and sham-exposure assemblies were positively located in fixed jigs spaced about 1.5 m apart, a distance at which stray fields from the coil set were minimal. A precision low-frequency sine-wave generator was designed to supply AC modulation of coil currents in addition to the DC output of the main coil power-supply. This power-supply configuration was confirmed to provide AC and DC coil currents with stabilities of better than 0.1% over three days. Consequently, over the duration of an experiment, it was the variability of the ambient field which limited the stability of the net field at the sample position. In the hot room, the magnitude of the local static field was in the range 25.2 f 0.5 uT (mean f 1 SD) while the RMS alternating field was 0.5 * 0.2 uT (mean * 1 SD). Throughout an experiment, the local ambient magnetic fields and the temperature of both ELFMF-exposed and sham-exposed samples were continuously monitored by a data logging system controlled by a personal computer (PC). A Bartington MAG-03MC triple-axis fluxgate magnetometer, interfaced to the PC, was used for magnetic field measurements. Temperatures were measured using thermocouple probes, positioned immediately beside the sample flasks on the support racks within the tubular formers, with digital recording, again interfaced to the PC. Potential temperature inequalities between ELFMF and sham-exposed samples due to warming of
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the ELFMF-exposed samples by power dissipated in the coil windings were prevented by forced air circulation through the cylindrical formers surrounding the culture flask support racks. Temperature stability over a period of three days was approximately O.l”C and any residual difference between the ELFMF-exposed and sham-exposed samples was established to be within the O.l”C resolution of the recorders.
TABLE I Summary
of ELFMF Exposure Conditions
Sinusoidal Fields (uT, mean f SD) Vertical
Maximum Induced Electric Field @V/m)
Static Fields (uT, mean f SD)
Horizontal
Vertical
Horizontal
I 0.2 (nulled fields)
I 0.2 (nulled fields)
A - nulled fields 0.22 f 0.09 (ambient fields)
0.415~ 0.16 (ambient fields)
0
B - Ca2+-ion cyclotron resonance
0.22 * 0.09 (ambient fields)
30 cir,s (applied fields) C - vertical 50
6 uTrors (applied
field)
0.41 f 0.16 (ambient fields) D - vertical 50
1 mT,,s (applied field)
0.41 i 0.16 (ambient fields) E - vertical 50
2 m’bS
(applied fields)
0.41 f 0.16
(ambient fields)
52
Hzfields 23
Hzfields 3900
Hzfields 7800
conditions at 50 Hz
I 0.2 (nulled fields)
65 VT (applied fields)
of 6 pTRMs
21.6 f 0.8 (ambient fields)
13.1 f 0.9 (ambient fields)
of 1 mTRMs
21.6 f 0.8 (ambient fields)
13.1 f 0.9 (ambient fields)
of 2 mTRMs 21.6 i 0.8
13.1 f 0.9
(ambient fields)
Means and SDS listed describe fields over the entire experimental
(ambient
fields)
programme.
ELFMF exposure conditions The experiments reported were performed under three different field exposure regimes as detailed
in Table I. In all cases, the magnetic field configuration was set using the Bartington fluxgate magnetometer. In the nulled field case (configuration A), both solenoid and Hehnholtz coils were used to counterbalance the ambient static magnetic fields to give near zero field at the ELFMF-exposed sample position. Alternating field components remained at ambient background values. For cyclotron resonance conditions (configuration B), an alternating and static magnetic field combination matching the resonance conditions for Ca” was applied in a horizontal plane using the Helmholtz coil while the solenoid was used to null the vertical component of the ambient static field. Finally, in configurations C, D and E, the solenoid alone was used to generate vertical
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respectively while all other fields alternating field components of 6 pTWs 1 mT,, and 2 mT,, remained at ambient values. Also listed in Table I are calculated values for the maximum alternating electric field induced in the culture fluid by the corresponding alternating magnetic field. These calculations take due account of the dimensions of the culture flasks and the amount of culture fluid used.
22 20 I) '7 3
15
6
12
6 10 *7 5 2
Fig. 1 3-dimensional plot of FDCPmix(A4) cells analysed by flow-cytometry after 24 hr culture in the presence (A) or absence (B) of IL-3 under normal ambient laboratory conditions of ELFMF.
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Fig. 2 TEM micrographs of thin sections of FDCP-mix(A4) cells grown under normal ambient laboratory conditions of ELFMF. Bar = 3 pm. (A) In the presence of IL-3 : Note the characteristic arrangement of heterochromatin at the nuclear periphery in comparison to (B). 24 hours after withdrawal of IL-3 : Apoptotic cells are characterised by (B) margination of electron dense material (arrows) inside their nuclei.
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Results Apoptosis in FDCP-mix(A4) cells under ambient conditions of ELFMF It has previously been shown that withdrawal of IL-3 causes apoptosis in FDCP-mix(A4) cells (10,ll). By 24 hours after the removal of IL-3, the majority of the cells had undergone apoptosis (Fig. 1) and showed electron dense margination inside their nuclei (Fig. 2), supposed to be condensed chromatin (21), which are regarded as morphological hall marks of this process. A significant increase in the percentage of apoptotic cells under normal ambient laboratory conditions of ELFMF was already seen at about 4 to 6 hours after withdrawal of the growth factor (Fig. 3). Thereafter, the proportion of apoptotic cells increased gradually and reached nearly 100 % after 48 hours (Fig. 3). In order to define optimal and different suboptimal conditions for the maintenance of viability in normal ambient conditions of ELFMF, we tested several concentrations of mIL3-CM (O.l%, 0.5%, 1.0%) over different periods of culture. As shown in Fig. 4, there is no significant difference in the maintenance of viability under ambient conditions of ELFMF for concentrations of 0.5% and 1.0% n-&3-CM. However, a significantly higher proportion of apoptotic cells was seen after different periods of culture when only 0.1% mIL3-CM was used.
100 90 80
20 10 0
4 6 8 Culture period (hours)
24
48
Fig. 3 Induction of apoptosis in FJXP-mix(A4) cells under normal ambient laboratory conditions of ELFMF at different time periods after withdrawal of IL-3. The proportion of viable, apoptotic and necrotic cells was analysed by flow cytometry. viable cells fz# apoptotic cells necrotic cells III
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mean + SEM
90 80 (0 2 70 3
80
mlL3-CM ‘i5 50 8 40
0.1%
0.5%
mlL3-CM
mlL3-CM
1.0%
30 20 10
n=lO
1 day culture
4 days culture
n=25
7 days culture
Fig. 4 Proportion of apoptotic cells in FDCP-mix(A4) cells maintained in medium supplemented with different concentrations of mIL3-CM (O.l%, OS%, 1.O%) over different periods of culture under normal ambient conditions of ELFMF. The proportion of apoptotic cells was detected by flow-cytometry.
Induction of apoptosis afrer withdrawal of IL-3 in FDCP-mtx(A4) cells exposed to various regimes of weak EL.FMFs We investigated whether or not the exposure to various regimes of weak ELFMFs affects the kinetics of apoptosis in FDCPmix(A4) cells after withdrawal of IL-3. For this purpose, cells were transferred into the exposure or sham-exposure environment after removal of the growth factor and period, then exposed/sham-exposed for two hours. After the two hour exposure/sham-exposure cells were either analysed immediately, or left for a further 2, 4 or 22 hours in ambient field conditions before being analysed, for apoptosis using flow cytometry. In six separate experiments, all performed in triplicate, no significant alteration in the kinetics of apoptosis due to the exposure to any of the ELFMF regimes tested was evident; the ratios of the proportion of apoptotic cells for exposed (E) and sham-exposed (S) cells, displayed in Table II, were in no case found to be significantly different from unity.
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TABLE II Induction
of Apoptosis
after Withdrawal
of IL-3 in FDCPmix(A4)
E/S (Exposed/Sham-Exposed)
Cells Exposed to ELF&IFS
: mean f SEM (n=6)
ELFMF exposure conditions
(2 hr duration)
Time after withdrawal of IL-3
A
B
C
D
E
2 hours
1.00 f 0.030
1.00 + 0.015
1.00 -c 0.008
1.OO+ 0.011
0.99 f 0.006
4 hours
1.01 zt 0.018
1.00 f 0.016
1.00 f 0.009
1.01 f 0.012
1.00 f 0.017
6 hours
1.01 k 0.030
0.98 * 0.023
1.01 f 0.012
0.98 * 0.020
0.99 f 0.023
24 hours
1.02 f 0.018
1.01 f 0.042
1.00 i 0.014
1.01 f 0.014
0.99 f 0.048
The ratios of the proportion of apoptotic cells for exposed (E) and sham-exposed (S) cells were calculated for each individual experiment (6 experiments, all performed in triplicate). Overall means and SEMs are shown.
TABLE III Maintenance
of Viability
in FDCP-mix(A4)
Cells Exposed to ELFMFs
E/S (Exposed/Sham-Exposed)
: mean f SEM (n=6)
ELFMF exposure conditions Culture conditions
Exposure duration
A
B
C
0.1% mIL3-CM
24 hours 4 days 7 days
1.00 f 0.011 1.00 f 0.017 0.98 f 0.038
1.00 f 0.033 1.02 f 0.012 0.99 f 0.025
1.01 * 0.016 1.02 zt 0.027 1.00 f 0.030
0.5% mIL3-CM
24 hours 4 days 7 days
1.00 f 0.005 0.98 i 0.024 0.99 f 0.046
1.00 -c 0.009 0.98 f 0.019 1.00 f 0.017
1.01 f 0.014 1.01 f 0.015 1.01 + 0.013
1.0% mIL3-CM
24 hours 4 days
1.01 f 0.012 1.01 f 0.021
0.99 f 0.008 1.02 f 0.022
1.00 f 0.042 0.97 f 0.021
The ratios of the proportion of apoptotic cells for exposed (E) and sham-exposed (S) cells were calculated for each individual experiment (6 experiments, all performed in triplicate). Overall means and SEMs are shown.
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Maintenance of viability under optimal and suboptimal culture conditions in FDCP-mix(A4) cells exposed to weak ELFMFs In order to study the influence of various regimes of weak ELFMFs on the maintenance of viability in FDCP-mix(A4) cells maintained in different suboptimal and optimal culture conditions, cells were set up as described in Methods and then exposed/sham-exposed for 24 hours, 4 days or 7 days. Immediately after exposure/sham-exposure, the cells were analysed for viability and apoptosis using flow cytometry. As shown in Table III, the ratios of the proportion of apoptotic cells in exposed (E) and sham-exposed (S) cells were never found to be significantly different from unity. We have therefore not seen any significant alteration in the maintenance of viability due to the exposure to weak ELFMFs.
Discussion The present study was performed to test the hypothesis that weak ELFMFs might promote the development of AML by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. Recent evidence has shown that the ability to block apoptosis might be a common characteristic of known and suspected tumour promoters (7). Therefore, an alteration of apoptosis in the presence of weak ELFMFs might provide an explanation for the increased risk reported for the development of AML in children that have been exposed to weak ELFMFs emitted by high-voltage power-lines. We employed the well-characterised multipotential haemopoietic progenitor cell line FDCPmix(A4) which depends on the permanent presence of IL-3 for survival and self-renewal (10,ll) as a reasonable model system for testing this possibility. We first defined the kinetics of the onset of apoptosis after growth factor withdrawal under ambient laboratory conditions (Fig. l-3) in order to establish the level at which the system would be able to detect any alterations that might be caused by a series of different weak sinusoidal ELFMFs, or were caused by the ambient static geomagnetic field. Differences of more than about 5% would have been significant. However, no modifications in the kinetics of apoptosis even at this level were observed after exposure of FDCP-mix(A4) cells for 2 hours to any of the ELF&IF regimes tested (Table II). Nevertheless, weak ELFMFs might induce more subtle changes in apoptosis that would only be detectable after a longer period of exposure. We have therefore investigated whether or not weak ELFMFs interfere with the maintenance of viability in FDCP-mix(A4) cells cultured under different suboptimal and optimal growth conditions over periods of up to seven days. Significant differences in the proportion of apoptotic and viable cells under ambient laboratory conditions were detected when the culture medium was supplemented with different concentrations of IL-3 (Fig. 4). However, we did not find any significant alteration in the proportion of apoptotic cells due to the exposure to various regimes of weak ELFMFs (Table RI). Clearly, these results do not support the hypothesis that weak ELFMFs express tumour promoting activity by the inhibition of apoptosis in multipotent haemopoietic progenitor cells. In addition, we have reported previously that exposure to similar ELFMFs does not affect growth rate, cellcycle state or clonogenic efficiency of FDCPmix(A4) cells (20). The exposure conditions we have employed are well within the range of typical environmental fields. For example, children exposed to weak ELFMFs emitted by power-lines experience 50 Hz fields of the order of lo-40 pT (for power-lines between 132 and 400 kV) at ground level directly beneath the power-lines, and about 2-8 pT at 25 m lateral displacement from the centre-line, and similar fields exist close to electrical household appliances (22). Also, the exposure conditions we have chosen are very similar to those that have been reported by others to alter cell functions in
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in vitro or in vivo experiments in a variety of different cell systems (23-26). Although some epidemiological studies suggest a possible link between exposure to weak ELFMFs and an increased risk for the development of leukaemia, overall, epidemiological studies of exposure to weak ELFMFs have given somewhat contradictory results (l-6, 27-29). In view of this, it is possible that weak ELFMFs do interact with biological systems, but only under rather specific exposure conditions different from those tested by us, or only in conjunction with additional environmental influences that have yet to be defined.
Acknowledgments We would like to thank M F Hughes and J Barry for their skilled technical assistance with the flow cytometric analysis and P Lucas for instrumentation development. We are particularly grateful to R E Dale for critical comment and discussion. This work was supported by the National Grid Company plc and by the Cancer Campaign [CRC]. TMD is a Gibb Fellow of the Cancer Research Campaign.
Research
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