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Alterations of biological parameters in mice chronically exposed to low-frequency (50 HZ) electromagnetic fields
ELSEVIER
Life Sciences, Vol. 62, No. 14, pp. 1271-1280,1998 Copyright Q 1998 Ekvier Scieace Inc. Printed in the USA. All rights msetved 0024-3205/m s19.w t .oo
PII soo24=3205(98)ooo57-5
ALTERATIONS OF BIOLOGICAL P ARAMETERS IN MICE CHRONICALLY EXPOSED TO LOW-FREQUENCY (50 HZ) ELECTROMAGNETIC FIELDS L. Bonhomme-Faivre’, A. Mac&, Y. Bezie’, S. Marion’, G. Bindoula’, A.M. Szekely3, N. FrBnois4, H. Auclair’, S. Orbach-Arbouys’, E. Bizi’ HBpital Paul-Brousse, 14, Avenue P-V-Couturier Villejuif : l-Service de Pharmacie, Laboratoire de Pharmacologic, 2-Laboratoire d’Hkmatologie, 3-Laboratoire d’AnatomoPathologique, 4-Laboratoire de Biochimie. 5-Institut Gustave-Roussy, rue Camille-Desmoulins, Villejuif. (Received in final form January 21,1998)
Summary In an experimental study we measured changes in hematological, biochemical and cortisol parameters in 6-week-old Swiss mice continuously exposed to ELF generated by a transformer station and high current bus bars. Mean daily exposure of 5.0 pT was maintained for 350 days. Hematological parameters were compared to those of control mice (n=12) exposed to a field level lower than 0.1 PT. Serum biochemical parameters (sodium, potassium, chloride, calcium, magnesium, phosphorus, amylase, creatine phosphokinase, and lactate dehydrogenase) were measured after 28 days of exposure and serum cortisol after 90 and 190 days. Granulocyte/macrophage colony-forming cells (GM-CFC) were counted at the end of the 350-day exposure. On day 20, exposed animals showed a significant decrease in leukocyte, erythrocyte, lymphocyte and monocyte counts and in hemoglobin and hematocrit values, while MCV increased. On days 43 and 63 no and erythrocyte significant difference was observed in leukocyte values, as if hemopoiesis had recovered. On day 90, a significant fall in the leukocyte, polynuclear neutrophil and eosinophil counts was observed in the exposed animals. No significant difference was noted in the biochemical parameters studied. On day 190, exposed animals had neutropenia and a decrease in the cortisol value. On day 350, no significant difference in hematological parameters was noted. Individual differences in sensitivity were observed, as 8 mice in the exposed significant decrease in the leukocyte, showed a group polymorphonuclear neutrophil and GM-CFC counts, while in two mice there was a significant increase in these same values compared to those unexposed mice. Key Words: magnetic fields, 50 Hz, neutropenia, leukopenia, anemia, GM-CFC, cortisol, mouse Address for correspondawe : Dr L. Bonhomme-Fake, Laboratoire de phatmacologie, service Phatmacie, Hbpital PaulBrousse, 14 avenue Paul-Vaillant-Couturier, 94800 Villejuif. Phone : 01 45 59 31 10, Fax : 01 45 59 37 28.
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Biological Alterations of ELF-exposed Mice
Vol. 62, No. 14, 1998
Epidemiological studies have shown a correlation between environmental extremely lowfrequency (ELF) magnetic fields and cancer (1,2,3,4). Such ELF fields may affect the immune and hematological systems and therefore play a role in cancer promotion (5). Epidemiological studies have also indicated that chronic occupational exposure to lowfrequency (SO-60 Hz) electromagnetic fields increases the risk of acute myeloid leukemia above a threshold of 0.2 uT (6). The mechanism of action is unknown. This study was undertaken to evaluate the effects of exposure to 50-Hz fields on hematological and biological parameters in mice placed on the laboratory floor above a transformer station and high-current bus bars. GM-CFC (granulocyte-macrophage colony-forming cells) were also measured after 3 50 days of exposure. Materials and Methods Male Swiss mice (Olivet, France) were used in all experiments. They onset of exposure, which started after acclimatization for a week in a magnetic field of less than 0.1 pT. The exposed group (n=12) was cages (four animals per cage), with food and water ad libitum, and laboratory room where the average field strength was 5 uT.
were 6 weeks old at the room with a background housed in three regular placed on the floor of a
The control group (n=12) was placed in three cages in a second room. The temperature (22 f 2°C) lighting 400 lux (Lux meter L x 101 Bioblock. France) and relative humidity (60 * 10%) were identical. The exposure room was located directly above the main electrical service bus bars and transformer station (13 kV). Magnetic field strength was measured every 3 h (Mag Check 50+, USA) and time-averaged over each working day. We measured in all three dimensions and calculated the sum of the squares (7). The square root of the sum was the results of the three partial measurements. The monthly average was approximately 5 pT. The field was spatially homogeneous within the cage. Every day the peak level (6.8 pT) was observed at 10 am and the minimum at 10 pm (3.2 pT). The cycle was fairly reproducible day to day. The natural geomagnetic field in the exposure room was 572 milliGauss (Geo-magnetometer BPM 2001 Bio-physic Mersmann D 5471 Wassenach). Hematological parameters (total leukocytes and differential leukocyte weight were measured every 3 weeks. Blood was taken by retro-orbital always at the same time in the morning.
counts) and body puncture (200 ~1)
Blood samples were analysed with a Sysmex NE 1500- 1OA (Medical Electronics Japan) and included erythrocyte counts (BBC), absolute leukocytes (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and hematocrit. Differential leukocyte counts were carried out after May-GrunwaldGiemsa staining of the slides (200 cells/mouse). GM-CFC were counted atIer 350 days of exposure. GM-CFC were determined in the bone marrow of the femur and tibia according to Worton (8). Briefly an appropriate number of cells was cultured in the alpha modification of Eagle’s medium containing 0.9% methyl cellulose, 20% horse serum, and 10% mouse heart and lung-conditioned medium as the colonystimulating factor. Four replicate cultures were incubated at 37°C in a 5% CO2 atmosphere for 7 days. Colonies consisting of 50 cells were scored as GM-CFC. Biochemical data were
Vol. 62, No. 14, 1998
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obtained on day 28 by using an autoanalyser (Wako 30, Sereno-USA) and included sodium, potassium, chloride, calcium, magnesium, phosphorus, amylase, creatine-phosphokinase, and lactate dehydrogenase. Serum cottisol levels were determined at 8 am by the fluorescence immunopolarization technique (TDX, Abbott Rungis France) on days 90 and 190. All data are given as mean + standard deviation for each group. Analysis of differences between exposed animals and controls on given days was done using the Mann-Whitney test. Within-group changes in blood values were followed by one-factor analysis of variance with repeated measures, P
study
On day 0 there was no statistical difference in hematological parameters or body weight between the groups. At the different study times there was no significant difference in body weight between the exposed and unexposed animals. On day 20, leukocyte (Figure l), erythrocyte, hemoglobin, and hematocrit values were significantly lower and MCV values higher in the exposed mice than in the controls (table 1). Absolute lymphocyte, monocyte and eosinophil counts were significantly diminished in the exposed animals (Figures 2, 3, 4). On day 20, erythrocyte counts had decreased in four control mice and leukocyte counts had decreased in one, but all the exposed animals had lower individual values for these two parameters compared to the control group. On day 43, hematocrit, hemoglobin and MCV values had increased in the exposed mice compared to the control group. No significant difference was found thereafter between the two groups, possibly because of compensation by the hematopoietic system. On day 43 all the control mice have lower leukocyte values than on day 20, while half the exposed mice had an increase in these parameters. Erythrocyte counts increased in 2 controls and 8 exposed ammals. A significant reduction in the eosinophil count occurred between days 20 and 43 in all the exposed mice. On day 63 no significant difference was noted between the two groups as regards blood parameters and the leukocyte formula. On day 90 leukocyte, polymorphonuclear neutrophil (figure S), eosinophil and MCV values were significantly lower in the exposed group. Two mice died after day 90 in the exposed group and 5 mice died in the control group ; all the deaths occurred during or just after blood sampling.
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Biological Alterations of ELF-exposed Mice
--c --c-
0
60
120
180
240
Vol. 62, No. 14, 1998
Exposed Control I
300 360 Exposure (days)
Fig. 1 Influence of the exposure of 6 week-old Swiss mice to a 5 .O pT electromagnetic field on their leukocyte counts : - between successives values of a group = *p
9 .-
8500
r: 6800
1700
I
P 0
L.“,““““~‘.“‘...‘.‘,““‘.““’ 60 120
0
180
240
300 Erposore
360 (days)
Fig. 2 Influence of the exposure of 6 week-old Swiss mice to a 5.0 pT electromagnetic field on their lymphocyte counts : - between successive values of a group = * :p
Vol. 62, No. 14, 1998
Biological Alterations of ELF-exposed Mice
Ok.“‘,‘.““‘.“““...““““““” 0 60 120 180
240
300
360
Exposure
(days)
Fig. 3 Influence of the exposure of 6 week-old Swiss mice to a 5.0 pT electromagnetic field on their monocyte counts : - between successive valuea of a group =* :p
L
ot,,,,,l,,,,,i,,,,,I,,,,,“,,,,‘, 180 0 60 120
I’
240
300
Exposure
’ 360 (days)
Fig. 4 Influence of the exposure of 6 week-old Swiss mice to a 5 .O pT electromagnetic field on their polym~clear eosinophil counts : - betweensuccessives valuesofa group =*p
1275
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Biological Alterations of ELF-exposed Mice
~~___ 90
-- -i
I
180
Vol. 62, No. 14, 1998
I
270 360 Exposure (days)
Fig. 5 Influence of the exposure of 6 week-old Swiss mice to a 5.0 %T electromagnetic field on their polyuuclear neutrophil counts : - between successive values of a group = * :p
On day 190 the polymorphonuclear exposed group
neutrophil
count
was still statistically
lower in the
On day 350 no significant difference was observed between the groups. However, 4 exposed mice had a polymorphonuclear neutrophil count below 800, while the range of counts in the control group was 1424 to 1917. On day 350 the exposed mice fell into two groups on the basis of the peripheral WEIC counts, In 8/10 mice the values were statistically lower than in the controls (p
hemo lymphoid organs. Discrete
Vol. 62,No. 14,1998
Biological Alterations of ELF-exposed Mice
TABLE 1 HEMATOLOGICAL DATA FROM MICE SWISS EXPOSED TO A 5.0 pT ELECTROMAGNETIC FIELD 50 Hz (mean value + SD)
Exposed Day 190 Control
8.1 + 2.3
17.0 f 6.2
33.4 + 12.3
41.0 + 4.7
21.2 f 2.9 52.2 f 8.7”
Exposed
9.2 f0.6 9.1 f 0.9
17.0 & 2 18.0 f 3
41.0+7 44.0 f 7
49.0+10 48.0 f 11
18.7f2.1 19.4f2.1
40.0+10 42.0+ 10
Day 350 Control Exposed
9.1 f 1.1 8.6 z!z0.6
15.2 + 1.5 14.3 f 1.2
48.7 + 5.6 45.7 f 3.0
54.1 + 5.2 53.3 +3.3
16.8 f 0.8 16.6kO.6
31.3 * 2.3 31.2f 1.1
As compared to control : *p
5487 f1420 2326+ 958 3550 + 610 1615 k 239 65798 + 2085
Exposed (n=8) 6455 +1964 2474+715 2590 f 810* 943 * 353* 53646 f 3925**
Exposed (n=2) 6600 +_2036 2985 + 2032 6280f990** 3309 f 657** 88218f3698**
Data expressed as mean + SD *p
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TABLE 3 SERUM BIOLOGICAL VALUES FROM MICE EXPOSED TO 5.0 uT, 50 Hz ELF (mean value k SD)
2.77 k 0.34
2.56 k 0.29
2.37 + 0.39
CPK (U/L)
449 k 230
353 It 189
635 k 517
LDH (mUI/ml)
2623 + 925
2482 + 862
Amylase (mWml)
3736+448
3668k410
Phosphorus
(mmolk)
2.42 f 0.43 416f259
As compared to control : no significant TABLE 4 SERUM CORTISOL VALUES (@ml) OF MICE EXPOSED TO 5.0 pT, 50 Hz ELF (mean values + SD) Length of exposure 90 days Control 16.7 5 5.0
* ~~0.05 as compared to control
Exposed 13.0 + 4.3
Length of exposure 190 days Control
Exposed
19.9 k 9.8
10.4 k 5.0*
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Biological Alterations of ELF-exposed Mice
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Discussion In this experimental study exposure of male Swiss mice to a mean daily electromagnetic field of 5.0 uT generated by electrical transformers and high-tension cabling modified the values of certain hematological parameters and cortisol values. Leukocyte and erythrocyte counts fell after 20 days of exposure. On day 20 the reduction in erythrocyte, hemoglobin and hematocrit values was accompanied by an increase in mean corpuscular volume, suggestive of macrocytic anemia possibly due to folate or vitamin B12 deficiency. Other experimental studies have shown that electromagnetic fields leads to a reduction in iron metabolism, with a knock-on effect on erythrocyte production. Controls animals exhibited a large increase in leukocyte count and red cells when they were 9 weeks of age, as expected for animals in their growth phase (9). On day 43 leukocyte and erythrocyte counts were no longer significantly different from control values. Only some exposed animals developed anemia or/and leukopenia, reflecting interindividual differencesThe average total length of exposure and the cumulative dose must be taken into account when studying the biological effects of low-frequency electromagnetic fields. We have previously observed leukopenia or anemia in a number of growing mice after 3 weeks of a comparable exposure of 1.2 uT ELF of young mice (10) Our hematological findings are in agreement with those from other experimental studies (11,12). The exposed group showed a new reduction in leukocyte counts on day 90, and in polymorphonuclear neutrophil counts on day 190. Our results suggest an action of the 50 Hz ELF field on pluripotent stem cells considering it in view of the hematologic compensation and decompensation phenomena and the fact that on day 350 the variations in leukocytes ran parallel to those of GM-CSF. The alterations observed in peripheral blood cells probably result from an effect of the ELF on pluripotent marrow progenitors (GM-CSF) generating neutrophils and macrophages. Some studies have shown that high-frequency ELFs have an action on hematopoietic stem cells (13, 14). It is possible that ELFs acts on growth factors of hematopoietic stem cells. White cells seemed to be particularly sensitive. ELF might act on GCSF (granulocyte colony-stimulating factor) or GM-CSF (granulocyte-macrophage colonystimulating factor) and on the production of interleukins such as IL6 (15), IL3, IL2 and IL4 involved in the maturation of hematopoietic cells. The iron-binding glycoprotein lactoferrin (LF) has heen described as a negative feedback regulator for GM-CSF (16). It is logical to think that ELF could act on the LF receptor. The aim of this work was to contIrm in mice the leukopenia and neutropenia observed in exposed persons (17). Neurovegetative and immunologic disorders (reduction in CD4, CD3 and CD2 cells) have been observed in persons exposed to ELF (18). The decrease in cortisol we observed here may be explained as follows. Other studies have shown a modification of cortisol levels (19, 20, 21, 22). Withdrawal of exposure to ELF (intermittent exposure) can lead to an abrupt cessation of the increased cortisol levels and lead to adverse effects like those met when steroid treatment is stopped (asthenia, depressive tendency, muscle weakness, mental changes, muscle and joint pain anorexia, nausea). This could explain the subjective symptoms described in humans after chronic exposure to ELF. Another explanation could be an increase in ACTH and cortisol values at high doses and long exposure, and later feed back control with symptoms like acute adrenal insufficency due to an inhibition of hypothalamic pituitary adrenocortical fonction. Epidemiological studies have suggested that ELF are a risk factor for exposed populations. This experimental study in mice provides evidence that hematological alterations can occur.
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References
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D.A. SAVlTZ , D. P. LOOMIS, Am J. Epidemiol. 141 123-124 (1995). P. GUENEL, P. RASKMARK, J. BACH ANDERSEN, E. LYNGE, Br. J. Int Med. 50 8758-64 (1993). B. FLODERUS, T. PERSSON, C. STENLUND, G. LINDER, C. JOHANSSON, J. KIVIRANTIN, H. PARSMAN, M. LINDBLOM, A.WENNBERG, A. OST, B. KNAVE, National Institute of Occupational Health. 1 20 (1992). J.H. OLSEN, A. NIELSEN , G. SCHULGEN, Br. Med. J. 307 89 l-895 (1993). R.J. SMIALOWICS, Bul1N.Y. AcadMed. 55 1094-1119(1979). , P. G.THERIAULT, A GOLDBERG 1 A.B. MILLER , B. ARMSTRONG GUENEL, J DEADMAN. E .IMBERNON ,A. CHEVALIER D. CYR, C. WALL, Am. J. Epidemiol. 139 550-572 (1994). 2”d revised Edition, Magnetic Sciences K. RILEY, Magnetic Field Guide, International, (1993). R. WORTON, JE. TIL , EA. MC CULLOCH, J Cell. Physiol. 74- 17 1 (1969). E.L. GREEN , Biology of the laboratory mouse, MC GRAW. HILL, NEW YORK, (1966). L. BONHOMME-FAIVRE, E. BIZI, G. FREDJ, H. AUCLATR, S. ORBACHARBOUYS, Third International Congress of the EBEA Nancy (France) (1996). D. VALLYO, M.L. PICAZO, P. SANZ, J.L. BARDASANO, Third international Congress of the EBEA Nancy (France) (1996). W.Z FAM, IEEE, Trans Biomed Eng. 27 376-381 (1980). C. PRZEMYSLAW, Ann. NY. Acad. Sci. 247 232-242 (1975). D. ROTKOVSKA, A. VACEK , Ann. N.Y.Acad. Sci. 247 243-250 (1975). S. MEHTA , 0. CHAN, J. PENNUCI, D. CHERLIN, CPOLK, H. WANEBO, Proc ASCO, 12,291 (1993). I. RICH, G. SAWATZKI. Colloque Inserm John Libbey Eurotext LDt. 162 63-66 (1987). L.BONHOMME-FAIVRE, S.MARION,Y.BEZIE, H.AUCLAIR, G. FREDJ, C.HOMMEAU, Archives of environmental Health (in press) (1998). L. BONHOMME-FAIVRE, S. MARION, H. AUCLAIR, S. LEGRAS, C. HOMMEAU, S. ORBACH-ARBOUYS, 17* Annual Meeting Bioelectromagnetics Society. P 175 B (Boston) (1995). E.A. ZAGORSKAYA, G.P. RODINA, Aviakomioeskaja medicina. 24 2 56-60 (1990). E.A. ZAGORSKAYA, Human Physiol. 11 133-139 (1985). M.A. OROZA, L. CALCICEDO, F SANCHEZ FRANCO, L. RIVAS, J. Bioelectr. 6 139-151 (1987). J.M. THOMPSON, F. STORMSHAK, J.R. LEE, D.L. HESS and L. PAINTER, J. Animal Science. 73 3274-3280 (1995).
Life Sciences, Vol. 62, No. 14, pp. 1271-1280,1998 Copyright Q 1998 Ekvier Scieace Inc. Printed in the USA. All rights msetved 0024-3205/m s19.w t .oo
PII soo24=3205(98)ooo57-5
ALTERATIONS OF BIOLOGICAL P ARAMETERS IN MICE CHRONICALLY EXPOSED TO LOW-FREQUENCY (50 HZ) ELECTROMAGNETIC FIELDS L. Bonhomme-Faivre’, A. Mac&, Y. Bezie’, S. Marion’, G. Bindoula’, A.M. Szekely3, N. FrBnois4, H. Auclair’, S. Orbach-Arbouys’, E. Bizi’ HBpital Paul-Brousse, 14, Avenue P-V-Couturier Villejuif : l-Service de Pharmacie, Laboratoire de Pharmacologic, 2-Laboratoire d’Hkmatologie, 3-Laboratoire d’AnatomoPathologique, 4-Laboratoire de Biochimie. 5-Institut Gustave-Roussy, rue Camille-Desmoulins, Villejuif. (Received in final form January 21,1998)
Summary In an experimental study we measured changes in hematological, biochemical and cortisol parameters in 6-week-old Swiss mice continuously exposed to ELF generated by a transformer station and high current bus bars. Mean daily exposure of 5.0 pT was maintained for 350 days. Hematological parameters were compared to those of control mice (n=12) exposed to a field level lower than 0.1 PT. Serum biochemical parameters (sodium, potassium, chloride, calcium, magnesium, phosphorus, amylase, creatine phosphokinase, and lactate dehydrogenase) were measured after 28 days of exposure and serum cortisol after 90 and 190 days. Granulocyte/macrophage colony-forming cells (GM-CFC) were counted at the end of the 350-day exposure. On day 20, exposed animals showed a significant decrease in leukocyte, erythrocyte, lymphocyte and monocyte counts and in hemoglobin and hematocrit values, while MCV increased. On days 43 and 63 no and erythrocyte significant difference was observed in leukocyte values, as if hemopoiesis had recovered. On day 90, a significant fall in the leukocyte, polynuclear neutrophil and eosinophil counts was observed in the exposed animals. No significant difference was noted in the biochemical parameters studied. On day 190, exposed animals had neutropenia and a decrease in the cortisol value. On day 350, no significant difference in hematological parameters was noted. Individual differences in sensitivity were observed, as 8 mice in the exposed significant decrease in the leukocyte, showed a group polymorphonuclear neutrophil and GM-CFC counts, while in two mice there was a significant increase in these same values compared to those unexposed mice. Key Words: magnetic fields, 50 Hz, neutropenia, leukopenia, anemia, GM-CFC, cortisol, mouse Address for correspondawe : Dr L. Bonhomme-Fake, Laboratoire de phatmacologie, service Phatmacie, Hbpital PaulBrousse, 14 avenue Paul-Vaillant-Couturier, 94800 Villejuif. Phone : 01 45 59 31 10, Fax : 01 45 59 37 28.
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Biological Alterations of ELF-exposed Mice
Vol. 62, No. 14, 1998
Epidemiological studies have shown a correlation between environmental extremely lowfrequency (ELF) magnetic fields and cancer (1,2,3,4). Such ELF fields may affect the immune and hematological systems and therefore play a role in cancer promotion (5). Epidemiological studies have also indicated that chronic occupational exposure to lowfrequency (SO-60 Hz) electromagnetic fields increases the risk of acute myeloid leukemia above a threshold of 0.2 uT (6). The mechanism of action is unknown. This study was undertaken to evaluate the effects of exposure to 50-Hz fields on hematological and biological parameters in mice placed on the laboratory floor above a transformer station and high-current bus bars. GM-CFC (granulocyte-macrophage colony-forming cells) were also measured after 3 50 days of exposure. Materials and Methods Male Swiss mice (Olivet, France) were used in all experiments. They onset of exposure, which started after acclimatization for a week in a magnetic field of less than 0.1 pT. The exposed group (n=12) was cages (four animals per cage), with food and water ad libitum, and laboratory room where the average field strength was 5 uT.
were 6 weeks old at the room with a background housed in three regular placed on the floor of a
The control group (n=12) was placed in three cages in a second room. The temperature (22 f 2°C) lighting 400 lux (Lux meter L x 101 Bioblock. France) and relative humidity (60 * 10%) were identical. The exposure room was located directly above the main electrical service bus bars and transformer station (13 kV). Magnetic field strength was measured every 3 h (Mag Check 50+, USA) and time-averaged over each working day. We measured in all three dimensions and calculated the sum of the squares (7). The square root of the sum was the results of the three partial measurements. The monthly average was approximately 5 pT. The field was spatially homogeneous within the cage. Every day the peak level (6.8 pT) was observed at 10 am and the minimum at 10 pm (3.2 pT). The cycle was fairly reproducible day to day. The natural geomagnetic field in the exposure room was 572 milliGauss (Geo-magnetometer BPM 2001 Bio-physic Mersmann D 5471 Wassenach). Hematological parameters (total leukocytes and differential leukocyte weight were measured every 3 weeks. Blood was taken by retro-orbital always at the same time in the morning.
counts) and body puncture (200 ~1)
Blood samples were analysed with a Sysmex NE 1500- 1OA (Medical Electronics Japan) and included erythrocyte counts (BBC), absolute leukocytes (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and hematocrit. Differential leukocyte counts were carried out after May-GrunwaldGiemsa staining of the slides (200 cells/mouse). GM-CFC were counted atIer 350 days of exposure. GM-CFC were determined in the bone marrow of the femur and tibia according to Worton (8). Briefly an appropriate number of cells was cultured in the alpha modification of Eagle’s medium containing 0.9% methyl cellulose, 20% horse serum, and 10% mouse heart and lung-conditioned medium as the colonystimulating factor. Four replicate cultures were incubated at 37°C in a 5% CO2 atmosphere for 7 days. Colonies consisting of 50 cells were scored as GM-CFC. Biochemical data were
Vol. 62, No. 14, 1998
Biological Alterations of ELF-exposed Mice
1273
obtained on day 28 by using an autoanalyser (Wako 30, Sereno-USA) and included sodium, potassium, chloride, calcium, magnesium, phosphorus, amylase, creatine-phosphokinase, and lactate dehydrogenase. Serum cottisol levels were determined at 8 am by the fluorescence immunopolarization technique (TDX, Abbott Rungis France) on days 90 and 190. All data are given as mean + standard deviation for each group. Analysis of differences between exposed animals and controls on given days was done using the Mann-Whitney test. Within-group changes in blood values were followed by one-factor analysis of variance with repeated measures, P
study
On day 0 there was no statistical difference in hematological parameters or body weight between the groups. At the different study times there was no significant difference in body weight between the exposed and unexposed animals. On day 20, leukocyte (Figure l), erythrocyte, hemoglobin, and hematocrit values were significantly lower and MCV values higher in the exposed mice than in the controls (table 1). Absolute lymphocyte, monocyte and eosinophil counts were significantly diminished in the exposed animals (Figures 2, 3, 4). On day 20, erythrocyte counts had decreased in four control mice and leukocyte counts had decreased in one, but all the exposed animals had lower individual values for these two parameters compared to the control group. On day 43, hematocrit, hemoglobin and MCV values had increased in the exposed mice compared to the control group. No significant difference was found thereafter between the two groups, possibly because of compensation by the hematopoietic system. On day 43 all the control mice have lower leukocyte values than on day 20, while half the exposed mice had an increase in these parameters. Erythrocyte counts increased in 2 controls and 8 exposed ammals. A significant reduction in the eosinophil count occurred between days 20 and 43 in all the exposed mice. On day 63 no significant difference was noted between the two groups as regards blood parameters and the leukocyte formula. On day 90 leukocyte, polymorphonuclear neutrophil (figure S), eosinophil and MCV values were significantly lower in the exposed group. Two mice died after day 90 in the exposed group and 5 mice died in the control group ; all the deaths occurred during or just after blood sampling.
1274
Biological Alterations of ELF-exposed Mice
--c --c-
0
60
120
180
240
Vol. 62, No. 14, 1998
Exposed Control I
300 360 Exposure (days)
Fig. 1 Influence of the exposure of 6 week-old Swiss mice to a 5 .O pT electromagnetic field on their leukocyte counts : - between successives values of a group = *p
9 .-
8500
r: 6800
1700
I
P 0
L.“,““““~‘.“‘...‘.‘,““‘.““’ 60 120
0
180
240
300 Erposore
360 (days)
Fig. 2 Influence of the exposure of 6 week-old Swiss mice to a 5.0 pT electromagnetic field on their lymphocyte counts : - between successive values of a group = * :p
Vol. 62, No. 14, 1998
Biological Alterations of ELF-exposed Mice
Ok.“‘,‘.““‘.“““...““““““” 0 60 120 180
240
300
360
Exposure
(days)
Fig. 3 Influence of the exposure of 6 week-old Swiss mice to a 5.0 pT electromagnetic field on their monocyte counts : - between successive valuea of a group =* :p
L
ot,,,,,l,,,,,i,,,,,I,,,,,“,,,,‘, 180 0 60 120
I’
240
300
Exposure
’ 360 (days)
Fig. 4 Influence of the exposure of 6 week-old Swiss mice to a 5 .O pT electromagnetic field on their polym~clear eosinophil counts : - betweensuccessives valuesofa group =*p
1275
1276
Biological Alterations of ELF-exposed Mice
~~___ 90
-- -i
I
180
Vol. 62, No. 14, 1998
I
270 360 Exposure (days)
Fig. 5 Influence of the exposure of 6 week-old Swiss mice to a 5.0 %T electromagnetic field on their polyuuclear neutrophil counts : - between successive values of a group = * :p
On day 190 the polymorphonuclear exposed group
neutrophil
count
was still statistically
lower in the
On day 350 no significant difference was observed between the groups. However, 4 exposed mice had a polymorphonuclear neutrophil count below 800, while the range of counts in the control group was 1424 to 1917. On day 350 the exposed mice fell into two groups on the basis of the peripheral WEIC counts, In 8/10 mice the values were statistically lower than in the controls (p
hemo lymphoid organs. Discrete
Vol. 62,No. 14,1998
Biological Alterations of ELF-exposed Mice
TABLE 1 HEMATOLOGICAL DATA FROM MICE SWISS EXPOSED TO A 5.0 pT ELECTROMAGNETIC FIELD 50 Hz (mean value + SD)
Exposed Day 190 Control
8.1 + 2.3
17.0 f 6.2
33.4 + 12.3
41.0 + 4.7
21.2 f 2.9 52.2 f 8.7”
Exposed
9.2 f0.6 9.1 f 0.9
17.0 & 2 18.0 f 3
41.0+7 44.0 f 7
49.0+10 48.0 f 11
18.7f2.1 19.4f2.1
40.0+10 42.0+ 10
Day 350 Control Exposed
9.1 f 1.1 8.6 z!z0.6
15.2 + 1.5 14.3 f 1.2
48.7 + 5.6 45.7 f 3.0
54.1 + 5.2 53.3 +3.3
16.8 f 0.8 16.6kO.6
31.3 * 2.3 31.2f 1.1
As compared to control : *p
5487 f1420 2326+ 958 3550 + 610 1615 k 239 65798 + 2085
Exposed (n=8) 6455 +1964 2474+715 2590 f 810* 943 * 353* 53646 f 3925**
Exposed (n=2) 6600 +_2036 2985 + 2032 6280f990** 3309 f 657** 88218f3698**
Data expressed as mean + SD *p
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TABLE 3 SERUM BIOLOGICAL VALUES FROM MICE EXPOSED TO 5.0 uT, 50 Hz ELF (mean value k SD)
2.77 k 0.34
2.56 k 0.29
2.37 + 0.39
CPK (U/L)
449 k 230
353 It 189
635 k 517
LDH (mUI/ml)
2623 + 925
2482 + 862
Amylase (mWml)
3736+448
3668k410
Phosphorus
(mmolk)
2.42 f 0.43 416f259
As compared to control : no significant TABLE 4 SERUM CORTISOL VALUES (@ml) OF MICE EXPOSED TO 5.0 pT, 50 Hz ELF (mean values + SD) Length of exposure 90 days Control 16.7 5 5.0
* ~~0.05 as compared to control
Exposed 13.0 + 4.3
Length of exposure 190 days Control
Exposed
19.9 k 9.8
10.4 k 5.0*
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Discussion In this experimental study exposure of male Swiss mice to a mean daily electromagnetic field of 5.0 uT generated by electrical transformers and high-tension cabling modified the values of certain hematological parameters and cortisol values. Leukocyte and erythrocyte counts fell after 20 days of exposure. On day 20 the reduction in erythrocyte, hemoglobin and hematocrit values was accompanied by an increase in mean corpuscular volume, suggestive of macrocytic anemia possibly due to folate or vitamin B12 deficiency. Other experimental studies have shown that electromagnetic fields leads to a reduction in iron metabolism, with a knock-on effect on erythrocyte production. Controls animals exhibited a large increase in leukocyte count and red cells when they were 9 weeks of age, as expected for animals in their growth phase (9). On day 43 leukocyte and erythrocyte counts were no longer significantly different from control values. Only some exposed animals developed anemia or/and leukopenia, reflecting interindividual differencesThe average total length of exposure and the cumulative dose must be taken into account when studying the biological effects of low-frequency electromagnetic fields. We have previously observed leukopenia or anemia in a number of growing mice after 3 weeks of a comparable exposure of 1.2 uT ELF of young mice (10) Our hematological findings are in agreement with those from other experimental studies (11,12). The exposed group showed a new reduction in leukocyte counts on day 90, and in polymorphonuclear neutrophil counts on day 190. Our results suggest an action of the 50 Hz ELF field on pluripotent stem cells considering it in view of the hematologic compensation and decompensation phenomena and the fact that on day 350 the variations in leukocytes ran parallel to those of GM-CSF. The alterations observed in peripheral blood cells probably result from an effect of the ELF on pluripotent marrow progenitors (GM-CSF) generating neutrophils and macrophages. Some studies have shown that high-frequency ELFs have an action on hematopoietic stem cells (13, 14). It is possible that ELFs acts on growth factors of hematopoietic stem cells. White cells seemed to be particularly sensitive. ELF might act on GCSF (granulocyte colony-stimulating factor) or GM-CSF (granulocyte-macrophage colonystimulating factor) and on the production of interleukins such as IL6 (15), IL3, IL2 and IL4 involved in the maturation of hematopoietic cells. The iron-binding glycoprotein lactoferrin (LF) has heen described as a negative feedback regulator for GM-CSF (16). It is logical to think that ELF could act on the LF receptor. The aim of this work was to contIrm in mice the leukopenia and neutropenia observed in exposed persons (17). Neurovegetative and immunologic disorders (reduction in CD4, CD3 and CD2 cells) have been observed in persons exposed to ELF (18). The decrease in cortisol we observed here may be explained as follows. Other studies have shown a modification of cortisol levels (19, 20, 21, 22). Withdrawal of exposure to ELF (intermittent exposure) can lead to an abrupt cessation of the increased cortisol levels and lead to adverse effects like those met when steroid treatment is stopped (asthenia, depressive tendency, muscle weakness, mental changes, muscle and joint pain anorexia, nausea). This could explain the subjective symptoms described in humans after chronic exposure to ELF. Another explanation could be an increase in ACTH and cortisol values at high doses and long exposure, and later feed back control with symptoms like acute adrenal insufficency due to an inhibition of hypothalamic pituitary adrenocortical fonction. Epidemiological studies have suggested that ELF are a risk factor for exposed populations. This experimental study in mice provides evidence that hematological alterations can occur.
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