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1 9 IMMUNE SYSTEM and HAEMATOLOGY 1 2 Haematology

1 9 IMMUNE SYSTEM and HAEMATOLOGY 1 2 Haematology

1 9 AND HAEMATOLOGY 2 3 Haematology is the branch of medicine that is concerned with , the blood-forming organs and blood 4 diseases. Studies encompass the growth and development of the leukocyte () populations that 5 form part of the immune system in addition to the erythrocyte (red cell) populations and the non-cellular serum 6 constituents such as serum iron and serum alkaline phosphatase concentrations. Haemopoiesis, the formation of 7 blood cells, occurs primarily in marrow, where there is progressive division and maturation from stem 8 cells through to the formation of mature erythrocytes and leukocytes. Erythrocytes and leukocytes circulate in 9 the bloodstream, from which cell populations and other haematological parameters may be readily sampled. 10 However, there is a continual and active exchange of leukocytes with other body compartments such as the 11 lymphoid system. In the adult , for example, only about 2% of the total pool are present 12 in the blood and lymphocyte subset composition can be varied by a number of different factors including 13 disease. Few studies have examined EMF effects either on immune system function or on heamatology. The 14 tables summarize the results of studies conducted on the immune system and haematology. Only the more 15 significant ones are discussed in the text. 16 17 9.1 Im m une system 18 19 The immune system identifies and responds to invading micro-organisms such as viruses, bacteria, and 20 various single-celled or multicellular organisms, and to ”foreign‘ macromolecules including proteins and 21 polysaccharides. Thus, it serves to protect individuals from infectious diseases and can also act against tumour 22 cells, although these responses are fairly weak. Immunological responses are mediated through intercellular 23 signalling pathways via chemical messengers such as cytokines and interleukins. 24 25 The first line of defence against pathogens is sustained by relatively unspecific (natural or innate) parts of 26 the immune system. These are natural killer (NK)-cells, mononuclear and . The protein 27 —complement system“ mediates many of the cytolytic and inflammatory effects of humoral (non-cell-mediated) 28 immunity. These innate responses are followed by the adaptive (or aquired) antigen-specific responses of the 29 immune system. The cells that mediate the antigen-specific (or acquired) responses are the B-, 30 which secrete antibodies (humoral immunity) which circulate in body fluids, and the T-lymphocytes, which can 31 function as cytotoxic cells (cell-mediated immunity) or as helper T-cells which assist in B- or T-cell activation. 32 Activated cytotoxic T-lymphocytes specifically recognise and kill cells expressing foreign molecules on their 33 surface and are implicated in anti-tumour responses. The acquired immune responses also further involve the 34 recruitment and amplification of the responses of the innate parts of the immune system. 35 36 9.1.1 Hum an studies 37 38 Selmaoui et al. (1996) showed that a one-night (23.00 to 08.00) exposure to either continuous or 39 intermittent (1 hour off and 1 hour with on/off switching every 15 s) 50-Hz, 10-mT magnetic fields did not affect 40 immunological parameters (CD3-, CD4-, CD8-lymphocytes, NK-cells and B-cell populations) in 16 healthy 41 men aged 20œ30 years as compared to 16 healthy sham-exposed men. 42 43 In 2000, Tuschl et al. published some results on immune parameters of ten workers exposed to the 44 magnetic fields associated with induction heaters (50œ600 Hz, up to 2 mT, or 2.8œ21 kHz, 0.13œ2 mT, for at 45 least two years). Overall, there were no differences between exposed and control subjects in the levels of B- and 46 T-cells, cytokines and immunoglobulins. However, the numbers of NK-cells and oxidative bursts of , 47 implicated in cytotoxic responses were significantly increased in the exposed group while monocytes had 48 significantly reduced phagocytic activity compared with those from unexposed personnel. The authors 49 considered that overall the non-specific immunity of the exposed subjects was normal and that the most peculiar 50 finding was the increase in NK-cell population. 51 52 Recently, the Mandeville group has reported effects of 60 Hz magnetic fields on 60 workers of power 53 utilities (Ichinose et al., 2004). They monitored the activity of ornithine decarboxylase (ODC) in white blood 54 cell, the activity of NK-cells, lymphocyte phenotypes, and differential cell counts. They monitored exposure 55 over three consecutive days before collecting peripheral blood. There was no alteration of NK-cell activity nor 56 of the number of circulating , , , or T-lymphocytes. However, there was an 57 association between exposure intensity and a decreased ODC activity and lower NK-cell counts. 58 59 The production of melatonin, which is known to stimulate the immune system, was quantified on the night 60 preceding immune marker determinations. W hile no alteration in melatonin levels could be observed in the

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61 exposed subjects, the decrease in ODC activity, counts of NK- and B-cells, and monocytes were strongest for 62 the workers with lowest melatonin production. According to the authors, the health consequences associated 63 with these changes are not known. 64 65 Using a cross-section approach, Chinese scientists investigated the effects of ELF fields on the immune 66 system. Zhu et al. (2001, 2002) systematically explored its effects on , and white blood 67 cells of peripheral blood taken from people who were working with the electric railway system. They reported 68 that the fields (50 Hz, 0.01œ0.938 mT, or 0œ12 kV m-1) decreased the number of white blood cells and the level 69 of IgA and IgG (Immunoglobulins A and G) antibodies. They also found that the percentage of lymphocytes 70 showing DNA damage was higher in the exposed group than in the control group. The authors concluded that 71 ELF fields might induce DNA damage in lymphocytes, then cause apoptosis of these cells, and further result in 72 the decrease of cell number and immunoglobulin level in the blood. 73 74 Dasdag et al. (2002) compared blood cell counts, and lymphocyte surface antigens of a group 75 of 16 welders with that of a group of 14 healthy male control subjects. Although CD4 and CD8 levels were 76 decreased in the welders and the hematocrit increased, the authors concluded that the differences were not 77 clinically significant and that the results were not suggestive of an ELF effect on immunologic parameters. 78 Table 61. Im m une system responses in hum ans Test Exposure Results Com m ents Authors Numbers of CD3+, CD4+, 50 Hz No effect with either exposure W ell controlled Selmaoui et al., CD8+ lymphocytes, of NK- protocol. study. Low power. 1996 cells and B-cells 10 µT Healthy young men Continuous or intermittent (1 exposed: n=16 h off, 1 h with on/off sham-exposed: n=16 switching every 15 s) Exposure for one night (23:00 to 08:00).

Number of B- and T-cells, 50œ600 Hz No effect on B- and T-cells, Tuschl et al., levels of cytokines and cytokines and 2000 immunoglobulins up to 2 mT immunoglobulins. Numbers of NK cells and or Increase in NK cells and in oxidative bursts of 2.8œ21 kHz bursts of monocytes. monocytes 0.13œ2 mT Decreased phagocytic activity. phagocytic activity Exposure for at least two years W orkers exposed to induction heaters (n=10)

Activity of ornithine 60 Hz Decreased ODC activity. Ichinose et al., decarboxylase (ODC) in 2004 white blood cells Personal magnetic field No alteration of NK activity. Deleted: [field strengths; monitor for 3 consequtive time?] Activity of NK cells working days No change in number of circulating neutrophils, Lymphocyte phenotypes eosinophils, basophils, and T- lymphocytes, lower NK-cell Differential cell counts counts Power-utility workers (n=60) Numbers of blood cells, 50 Hz Increase in red blood cells, Zhu et al., 2001

levels of immunoglobulins, -1 platelets, and haemoglobin. levels of DNA damage in 0-12 kV m , 0.01-0.92 mT lymphocytes (comet assay) Decrease in white blood cells 4.59ê2.64 h / day, and lymphocytes. Daily exposed workers: 9.72ê3.09 year Decrease in IgA and IgG. n=192 Unexposed control workers: Increase in DNA damage of n=106 lymphocytes.

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Numbers of blood cells, 50 Hz Increase in red blood cells and Extension of Zhu Zhu et al., 2002

levels of immunoglobulins, -1 platelets. et al., 2001 levels of DNA damage in 1.69-3.25 kV m , 0.245- lymphocytes (comet assay) 0.938 mT Decrease in white blood cells and lymphocytes. Daily exposed workers: 4.59ê2.64 h / day, 9.4ê3.2 n=33 year Decrease in IgA and IgG. Unexposed control workers: Increase in DNA damage of n=106 lymphocytes. Red blood cells; W elders exposed 3-4 hours CD4, CD8 lower, hematocrit Dasdag et al., Deleted: exposure ; hematocrit; per day per week and for at higher in welders. 2002 details??? platelets; total white blood least 10 years cells; neutrophils; Differences —not clinically lymphocytes; eosinophils; significant“. and CD3, CD4, CD8, and CD4/CD8 Male welders: n=16 Male controls: n=14 79 80 9.1.2 Anim al studies 81 82 Animal studies have been carried out using several approaches: some authors have examined the 83 responsiveness of the whole immune system, while other used blood cell counts and standard in vitro tests on 84 cells taken from the peripheral blood or of exposed animals. This section discusses all experiments done 85 with exposure of the animals even if the tests on their immune cells were done in vitro. Many of these studies 86 have been previously reviewed by ICNIRP (2003) and the general conclusion was that —there is little consistent 87 evidence on any inhibitory effect of power-frequency EM F exposure on various aspects of immune system 88 function“. 89 90 The Löscher group (M evissen et al. 1996) had reported a decreased spleen T-lymphocyte proliferation in 91 rats chronically exposed to 50 Hz magnetic fields. In a follow-up study, the same authors (Mevissen et al. 1998) 92 found that this proliferation was initially increased, after 2 weeks, but then decreased, after 13 weeks, compared 93 to sham-exposed animals. 94 95 Later, the same group (Häussler et al. 1999) reported on two independent experiments on the ex vivo 96 production of interleukins (ILs) by mitogen-stimulated splenic lymphocytes from female Sprague-Dawley rats 97 exposed to 100 µT 50 Hz magnetic fields. In the first experiment, the rats were treated with DMBA and exposed 98 or sham-exposed for 14 weeks. There was no difference between exposed and sham-exposed groups in the level 99 of production of IL-1 by mitogen-activated splenic B-cells. In the second experiment, rats were exposed for 1 100 day, 1 week, or 2 weeks, followed by collection and activation of spleen lymphocytes. There was no difference 101 in IL-1 or IL-2 production from stimulated B- or T-cells. According to the authors, these negative findings 102 suggested that the reported changes in T-cell proliferation in response to magnetic field exposure (Mevissen et 103 al. 1996, Mevissen et al. 1998) was not mediated via alterations in IL production. 104 105 In another experiment, Thun-Battersby et al. (1999) exposed female Sprague-Dawley rats to a 50 Hz, 106 100 µT field for periods of 3 or 14 days or 13 weeks. They performed analyses of T-lymphocyte subsets and 107 other immune cells: NK- cells, B-lymphocytes, , and granulocytes in blood, spleen and mesenteric 108 lymph nodes. They also detected proliferating and apoptotic cells in the compartments of spleen tissue. No 109 effect was found on different types of leukocytes, including lymphocyte subsets for any of the exposure 110 durations. The authors concluded that exposure did not affect lymphocyte homeostasis, but did not exclude that 111 functional alterations in T-cell responses to mitogens and in NK-cell activity, as described in some studies of 112 exposed rodents, may be one of the mechanisms involved in the carcinogenic effects of magnetic field exposure 113 observed in some models of co-carcinogenesis, such as the DMBA model used by this group. 114 115 A number of tests of NK-cell activity have been carried out, mainly on exposed mice. House et al. (1996) 116 reported that the NK-cell activity of young B6C3F(1) female mice was reduced in some experiments after 117 exposure to continuous or intermittent 60 Hz magnetic fields (2œ1000 µT) but not in male mice nor in male or 118 female rats. The authors later did the experiment with older female mice, and observed a similar decrease in 119 NK-cell activity at 1000 µT but not at the lower field intensities (House and McCormick 2000). They concluded 120 that the inhibition of NK-cell activity caused by exposure was consistent across their experiments but had little

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121 biological significance, as it was not associated with an increase in neoplasms in separate investigations with the 122 same type of exposure. 123 124 Arafa et al. (2003) investigated the bioeffects of repeated exposure to 50 Hz high-strength (20 mT) 125 magnetic fields on some immune parameters in mice. The animals were exposed daily for 30 minutes three 126 times per week for 2 weeks. Immune endpoints included total body weight, spleen/body weight ratio, 127 viability, total and differential white blood cell (W BC) counts, as well as lymphocyte proliferation 128 induced by phytohaemagglutinin, concanavalin-A and lipoploysaccharide. Magnetic field exposure decreased 129 viability, W BC count, as well as mitogen-induced lymphocyte proliferation (by approximately 20%). 130 131 The authors also tested the effects of two distinct anti-radical compounds: L-carnitine and Q10. Both drugs 132 were given 1 h prior to each ELF exposure. L-carnitine, but not Q10 attenuated the adverse effects of exposure 133 on the vast majority of the immune parameters tested. It was speculated by the authors that the effect of L- 134 carnitine was due to its anti-ROS properties. 135 136 Ushiyama and Ohkubo (2004a) and Ushiyama et al. (2004b) studied the acute and subchronic effects of 137 whole-body exposure to 50 Hz magnetic field on leukocyte-endothelium interaction using a dorsal skinfold 138 chamber technique in conscious BALB/c mice. They perfomed an acute exposure experiment by exposing for 139 30 min at 0, 3, 30 and 30 mT and a subchronic exposure experiment by continuous exposure for 17 days at 0, 140 0.3, 1 and 3 mT. The intra-microvascular leukocyte adherence to endothelial cells significantly increased at 30 141 mT in the acute exposure and at 3 mT in the subchronic exposure conditions. In a companion study Ushiyama et 142 al. (2004b), however, they failed to find changes in serum tumour necrosis factor (TNF)-M and IL-l N levels 143 under exposure to subchronic exposure to 30 mT. 144 145 The effect of long-term exposure to ELF electric and magnetic fields on the thymocytes of rats was studied 146 by Quaglino et al. (2004). The 2-month-old Sprague-Dawley rats were exposed or sham exposed for 8 months 147 to 50 Hz fields (1 kV m-1, 5 µT or 5 kV m-1, 100 µT). Simultaneous exposure to continuous light and ELF fields 148 did not change significantly the rate of mitoses compared to sham-exposed rats, but the amount of cell death 149 was significantly increased. The conclusion of the authors was that, in vivo, stress, such as that caused by 150 continuous exposure to light and ELF exposure can act in synergy to cause a more rapid involution of the Deleted: , 151 and suggested that this could be responsible for an increased susceptibility to the potentially hazardous Deleted: infection??. 152 effects of ELF-EMF. 153 Table 62. Im m une system responses in anim als Biological endpoint Exposure conditions Results Com m ents Reference T-cell proliferation Spleen lymphocyte 60 Hz No effect. Morris and proliferation Phillips (1982) 100 kV m-1 Swiss-W ebster mice 90œ150 days Spleen T-lymphocyte 50 Hz Decreased T-cell Mevissen et al. proliferation proliferation 1996 50 µT Sprague-Dawley rats 13 weeks Spleen T-lymphocyte 50 Hz Increase in T-cell Mevissen et al., proliferation proliferation after 2 weeks; 1998 100 JT decrease after 13 weeks; no Sprague-Dawley rats 13 weeks effect on B-cells Peripheral blood lymphocyte Pilot study: Reduced B-lymphocyte Considerable Murthy et al. proliferation 60 Hz response in pilot study. heterogeneity in 1995

-1 results of sham Baboons 9 kV m , 20 µT No effect in main study. exposed 5 weeks animals. Main study: 60 Hz 30 kV m-1, 50 µT 5 weeks T-cell function

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Ex vivo production of 50 Hz No effect on production of IL- Häussler et al., interleukins (ILs) by mitogen- 1. 1999 stimulated splenic 100 µT lymphocytes No difference in IL-1 or IL-2- 14 weeks production by stimulated B- Female Sprague-Dawley 1 day, 1 week, 2 weeks or T-cells. rats treated with DMBA T-lymphocyte subsets; NK- 50 Hz No effects. Thun-Battersby cells, B-lymphocytes, et al.,1999 macrophages and 100 µT granulocytes in blood, 3 or 14 days or 13 weeks. spleen and mesenteric lymph nodes; proliferating and apoptotic cells in the compartments of spleen tissue Sprague-Dawley rats Delayed-type 60 Hz No consistent effect. Generally well House et al., hypersensitivity to oxazolone described study. 1996 2, 200, 1000 µT continuous, B6C3F1 mice 1000 µT intermittent (1 h on/off) 13 weeks Resistance to Listeria 60 Hz No effect. Experimental House et al., monocytogenes infection and control data 1996 2, 200, 1000 µT continuous, not shown. Mice (BALB/C) 1000 µT intermittent (1 h on/off) 4 or 13 weeks Long-term effects on IL-1 60 Hz No effect. Hefeneider et and IL-2 activity al., 2001 Deleted: 1991 transmission lines Sheep 1.07, 3.5 µT 12œ27 mo Deleted: ¶ [needs details] NK-cell activity Spleen and blood NK cells 60 Hz No significant effect. McLean et al., 1991 SENCAR mice treated with 2 mT DMBA and TPA 6 h / day, 5 days / week, 21 weeks Spleen natural killer cells 0.8 Hz (pulsed) Enhanced activity at 30 mT De Seze et al., and above. 1993 BALB/C mice 10œ120 mT 10 h / day, 5 days NK-cell activity 60 Hz Reduced NK-cell activity in Generally well House et al., some experiments in female described study, 1996 Young mice and rats 2œ1000 µT, continuous or mice but not in male mice replicate intermittent nor in male or female rats. experiments. NK-cell activity Repeat of above study Reduced NK-cell activity. House and McCormick, Older mice 2000 Spleen NK-cells 60 Hz No consistent effect in males Generally well House et al., or females. described study, 1996 F344 rats 2, 200, 1000 µT continuous, replicate 1000 µT intermittent (1 h experiments. on/off) 6 or 13 weeks Spleen NK-cells 60 Hz Trend for enhanced activity Fully described Tremblay et al., with exposure. study; but 1996 F344 rats 20 µTœ2 mT significant 20 h / day, 6 weeks effects with control rather than sham comparison.

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Macrophage activity Peritoneal macrophages 60 Hz Trend for enhanced Fully described Tremblay et al., hydrogen peroxide release study; but 1996 F344 rats 20 µTœ2 mT with exposure. significant 20 h / day, 6 weeks effects with control rather than sham comparison. Antibody cell activity Circulating antibody levels to 60 Hz No effect. Morris and keyhole limpet haemocyanin Phillips, 1982 100 kV m-1 Immunised Swiss W ebster mice 30 or 60 days Antibody-forming spleen 60 Hz No effect. Putinas and cells Michaelson, 500 µT 1990 Immunised BALB/C mice 5 h on three alternate days Antibody-forming spleen 0.8 Hz (pulsed) No effect. De Seze et al., cells 1993 10œ120 mT Immunised BALB/C mice 10 h / day, 5 days Antibody-forming spleen 60 Hz No effect. Generally well House et al., cells described study; 1996 2, 200, 1000 µT continuous positive Immunised B6C3F1 mice 1000 µT intermittent (1 h controls. on/off) 3 or 13 weeks Body weight, spleen/body 50 Hz Decreased splenocyte Arafa et al., weight ratio, splenocytes viability, W BCs count, and 2003 viability, total and differential 20 mT mitogen-induced lymphocyte W BC counts, lymphocyte 30 min / day, 3 days /week, proliferation. proliferation induced by 2 weeks PHA, Con-A and LPS Only L-carnitine attenuated the effects of exposure. Effect of anti-radical compounds L-carnitine and Q10 Mice Deleted: Animals? Leukocyte-endothelial 50 Hz Increased leukocyte Ushiyama and interaction adherence at 30 mT. Ohkubo, 2004a 0, 3, 10 and 30 mT BALB/c mice 30 min

Leukocyte-endothelial 50 Hz Increased leukocyte Ushiyama et al., interaction; serum TNF- adherence at 3 mT, no 2004b alpha and IL-l beta 0, 0.3, 1 and 3 mT change in serum TNF-alpha and IL-l beta levels. BALB/c mice continuous for 17 days Rate of mitosis in 50 Hz No change in rate of Quaglino et al.,

thymocytes -1 mitoses; cell death 2004 1 kV m , 5 µT significantly increased. 2-month-old Sprague- -1 Dawley rats 5 kV m , 100 µT 8 months 154 155 9.1.3 Cellular studies 156 157 Jandova et al. (1999; Jandova et al. 2001) found that the adherence of leukocytes taken from 158 patients to solid surfaces (such as glass surfaces or plastic materials) was increased after 1 hour of exposure to a 159 50 Hz sinusoidal magnetic field (1 mT and 10 mT), while it was decreased in T-lymphocytes taken from healthy 160 donors. The leukocyte surface properties manifest cell-mediated immunity, since, in the presence of antigens, 161 leucocytes taken from cancer patients exhibit less adherence than leucocytes from healthy humans. The authors

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162 concluded that the response of cell-mediated immunity was altered by external magnetic field exposure and 163 hypothesized about different biophysical mechanisms, among which were the free radical reactions. 164 165 Ikeda et al. (2003) studied the immunological functions of human peripheral blood mononuclear cells 166 (PBMCs) from healthy male volunteers. They assessed the activities of NK and lymphokine activated killer 167 (LAK) cells and the production of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), 168 interleukin-2 (IL-2), and interleukin-10 (IL-10). The PBMCs were exposed for 24 hours to linearly (vertical), or 169 circularly, or elliptically polarised fields, at 50 and 60 Hz (2œ500 µT for the vertical field and 500 µT for the 170 rotating fields). They found no effect of exposure on the cytotoxic activities and the cytokines production of 171 human PBMCs. 172 173 The Simko group in Germany has been very active in recent years studying the effects of 50 Hz, 1 mT 174 magnetic fields on various immune cells. The effects on the production of free radicals was studied by Lupke et 175 al. (2004) in monocytes from the blood of human umbilical cord and in human Mono Mac6 cells. In monocytes 176 a significant increase of superoxide radical anion production was observed (up to 40%) and an increase in ROS 177 release (up to 20%) upon 45-min exposure of monocytes. The increases were even larger in Mono Mac6 cells. 178 179 Rollwitz et al. (2004) gave some evidence of the cell-activating capacity of ELF magnetic fields by 180 reporting a significant increase in free radical production after exposure of mouse -derived (MBM) 181 and macrophages. The superoxide anion radicals were produced in both types of cells. The 182 authors suggested that the NADH-oxidase pathway was stimulated by exposure, but not the NADPH pathway. 183 184 The same research group (Simko and Matsson, 2004) has concluded that some of the effects of ELF 185 magnetic field exposure might be caused by increasing levels of free radicals. They considered four different 186 types of processes: (i) direct activation of macrophages (or other immune cells) by short-term exposure leading 187 to (or other cell specific responses) and consequently, free radical production, (ii) exposure- 188 induced activation including direct stimulation of free radical production, (iii) increase in the 189 lifetime of free radicals under exposure leading to long-term elevation of free radical concentrations, (iv) long- 190 term exposure leading to a durable increase in the level of free radicals, subsequently causing an inhibition of 191 the effects of the hormone melatonin. However, there are no well-established data showing that 192 free radical production is affected by ELF magnetic field exposure. 193 194 Table 63. Im m une system in vitro studies Biological endpoint Exposure conditions Results Com m ents Reference Adherence assay 50 Hz Decreased adherence in Jandova et al., normal leukocytes which 1999, 2001 Leukocytes taken from 1 and 10 mT (measurements normally are adherent. venous blood of normal gave 1.02 and 9.52 mT, donors and cancer patients respectively) Increased adherence in cancer leukocytes that are 1 h usually not adherent to solid Test tubes placed in the surfaces. center of a coil. Exposure Similar effect for longer performed at 37°C. Sham exposure duration (2, 3 and 4 exposure not mentioned. h tested but no data shown). Several CD markers and 50 Hz Slight effect on CD4, CD14 Conti et al., transcription and expression and CD16 receptor 1999 of CD4. 24, 48, 72 h expression, other CD ] receptors not affected. Deleted: [needs details] Peripheral blood 50 Hz, pulsed (2 msec. DNA CD4+ expression Felaco et al,. mononuclear cells impulse duration) generated increased 1999 by a BIOSTIM apparatus CD4 expression mRNA CD4+ expression 1.5 mT increased in resting cells exposed for 24 h, but not 48 Deleted: ¶ 24, 48 and 72 hours or 72 h Deleted: [needs details] Increase in percentage cell cycle progression in S phase

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Activity of NK and LAK cells; 50 and 60 Hz No effects. Ikeda et al., production of IFN-gamma, 2003 TNF-alpha, IL-2, and IL-10 linearly (vertical), circularly, or elliptically polarised PBMCs from healthy male magnetic fields volunteers 2œ500 µT (vertical field) 500 µT (rotating fields) 24 h Monocytes from blood of 50 Hz Increase in superoxide Lupke et al., human umbilical cord and radical anion production in 2004 human Mono Mac6 cells 1 mT monocytes; increase in ROS 45 min release upon 45-min Deleted: [time?] Production of free radicals exposure of monocytes (larger in Mono Mac6 cells). Mouse bone marrow-derived 50 Hz Increase of free radical Rollwitz et al., (MBM) promonocytes and production: superoxide anion 2004 macrophages 1 mT radicals were produced in both types of cells. Deleted: [time?] Production of free radicals 45 min to 24 hours 195 196 9.2 Haem atological system 197 198 Haematological parameters include: leukocyte and erythrocyte counts, haemoglobin concentration, 199 and thrombocyte counts, bone marrow cellularity and prothrombin times, serum iron and serum 200 alkaline phosphatase concentrations and serum triglyceride values. Most studies have included assessments of 201 the differential white blood cell count, that is, the overall concentration of white cells (leukocytes) and their 202 various sub-groups. However, the importance of small alterations of the levels of circulating leukocytes is not 203 clear as there is a continual and active exchange with other body compartments such as the lymphoid system 204 which can be affected by a number of different factors including disease. 205 206 9.2.1 Hum an studies 207 208 Very few studies have been performed on volunteers and none in recent years. 209 210 Selmaoui et al. (1996) exposed or sham exposed 32 male volunteers to 10 µT, 50 Hz horizontally 211 polarised magnetic fields between 23.00 and 08.00 on two separate days. Blood samples were taken from each 212 subject at 3 hourly intervals from 11.00 to 20.00 and hourly from 22.00 to 08.00. One month later, the exposed 213 group was subjected to an intermittent 10 µT, 50 Hz magnetic field between 23.00 and 08.00. In the intermittent 214 regimen, the magnetic field was turned on for one hour and off for the next hour; during the on period, the field 215 was cycled on and off every 15 s. Counts of all cell types showed a strong circadian rhythm with the possible 216 exception of neutrophils and NK-cells; However, values in the group exposed continuously and in those 217 exposed intermittently were always very similar to values in the sham exposed groups. Moreover, inter- and 218 intra- individual variations were so high that small effects due to exposure were unlikely to be detected. 219 220 Bonhomme-Faivre et al. (1998) monitored a few subjects exposed for 8 hours per day for more than 1 year 221 in their hospital laboratory to 50 Hz, 0.2œ6.6 µT magnetic fields. CD3 and CD4 lymphocyte counts were 222 significantly lower than those measured in six control workers, but NK-cell counts were increased. Since 223 exposure levels were measured at ankle level, the whole-body exposure of the individuals was unknown and no 224 health consequences could be attributed to field exposure. 225 226 Table 64. Hum an haem atological studies Biological endpoint Exposure conditions Results Com m ents Reference Counts of all blood cell 50 Hz No effect but strong inter W ell controlled study. Selmaoui et al., 1996 types and intra-individual Low power. 10 µT variations. 23.00 to 08.00 on two separate days

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CD3 and CD4 50 Hz Decrease in CD3 and CD4 Dosimetry not Bonhomme-Faivre et lymphocytes and NK and increase in NK cells. provided. Low number al., 1998 counts 0.2œ6.6 µT at ankle level of subjects (6 exposed, 8 h / day, 1 year 6 controls). 227 228 9.2.2 Anim al studies 229 230 Boorman et al. (1997) exposed Fischer 344/N rats and B6C3F1 mice to 60 Hz magnetic fields (2200 and 231 1000 µT) for 8 weeks (18.5 h per day, 7 days per week). An additional group of rats and mice was exposed 232 intermittently (1 h on and 1 h off) to 1000 µT magnetic fields. There were no haematological alterations that 233 could be attributed to magnetic field exposure. 234 235 Zecca et al. (1998) assessed haematological variables before exposure and at 12-week intervals during 236 exposure up to 32 weeks. Male Sprague-Dawley rats (64 animals per group) were exposed for 8 h per day, 5 237 days per week for 32 weeks at 50 Hz (5 µT and 1 kV m-1, and 100 µT and 5 kV m-1). Blood samples were 238 collected at 0, 12, 24, and 32 weeks. No pathological changes were observed under any exposure conditions in 239 animal growth rate, in morphology and histology of the tissues collected from the , , mesenteric lymph 240 nodes, testes and bone marrow or in serum chemistry. 241 242 Three studies were performed by Korneva et al. (1999) in male CBA mice exposed to 50 Hz, 22 µT 243 magnetic fields for 1 h, at the same time of day, for 5 successive days. In the first study, spleen colony 244 formation was examined and the number of colony-forming units was not higher than in sham-exposed animals. 245 Significant changes were seen in the thymus weight and thymus index of exposed animals when compared to 246 sham-exposed animals. In a second study, mice were given a sublethal dose of X-rays (6 Gy) followed 2 h later 247 with the same magnetic field exposure as above. The number of colonies per spleen showed a consistent, 248 significant increase with exposure and the number of colony forming units per was decreased. In the third 249 study, bone marrow was taken from mice that had been exposed in still the same way, and injected into mice 250 that had been exposed to a lethal dose of X-rays (9 Gy). The number of colony forming units per femur in the 251 recipient mice was significantly reduced at days 1 and 4 after injection. 252 253 Table 65. Anim al haem atological studies Biological endpoint Exposure conditions Results Com m ents Reference Differential white blood cell 60 Hz No consistent effects seen in Replicate studies; Ragan et al.,

count -1 replicate studies. some results 1983 100 kV m variable. Swiss-W ebster mice and Sprague-Dawley rats 15 (rats only), 30, 60 or 120 days Differential white blood cell 50 Hz No effect. Lorimore et al., and bone marrow progenitor 1990 cell count 20 mT CBA/H mice 7 days Splenic lymphocyte subgroup 60 Hz No effect. Generally well House et al., analysis described study. 1996 2, 200, 1000 µT continuous B6C3F1 mice 1000 µT intermittent (1 h on/off) 4 or 13 weeks Differential white blood cell 60 Hz Trend for reduced T-cell Fully described Tremblay et al., count count with exposure; study; significant 1996 20 µTœ2 mT reduced total, cytotoxic and effects with control F344 rats 20 h / day, 6 weeks helper T-cells. rather than sham comparison. Differential white blood cell 50 Hz No effect. Extensive Thun-Battersby count lymphocyte sub-set et al., 1999b 100 µT analysis. Sprague-Dawley rats 3 days, 14 days or 13 weeks

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Differential white blood cell Pilot study: Reduced helper T- Considerable Murthy et al., count lymphocyte count in pilot heterogeneity in 1995 60 Hz study; no effect in main sham exposed Baboons 9 kV m-1, 20 µT study. results. 5 weeks Main study: 60 Hz 30 kV m-1, 50 µT Haematology 60 Hz No effect . Boorman et al., 1997 Fischer rats and B6C3F1 1000 or 2200 JT continuous mice 1000 JT Intermittent (1 h on, 1 h off) 18.5 h / day, 7 days / week, 8 weeks Blood cells count before 50 Hz No effects. Zecca et al.,

exposure, at 12, 24 and 32 -1 1998 weeks of exposure 5 µT, 1 kV m -1 Morphology and histology of 100 JT, 5 kV m different organs (liver, heart, 8 h / day, 5 days / week, 32 mesenteric lymph nodes, weeks testes, bone marrow) Groups of 64 rats sham- exposed Spleen colony formation 50 Hz No effect of EMF alone. Korneva et al., 1999 Bone marrow injected to mice 22 µT Increase in number of exposed to 9 Gy X-rays colonies per spleen; 1 h / day, same time of day, decrease in colony forming Male CBA mice 5 successive days units per femur. 6 Gy X-rays followed after 2 Number of colony forming h by same exposure as units per femur significantly above reduced in the recipient mice Deleted: [not clear which treatment gives what results] Total and differential white 50 Hz Decreased white blood cells Arafa et al., blood cell counts count. 2003 20 mT Mice Deleted: Animals?? 30 min / day, 3 days / week, 2 weeks 254 255 9.2.3 Cellular studies 256 257 Only one paper has been published recently on the effects on cells of the : Van Den 258 Heuvel et al. (2001) studied the effects of 50 Hz, 80 µT magnetic fields on the proliferation of different types of 259 stem cells, including haemopoietic cells. The cytotoxic effects of exposure were investigated on the proliferation 260 of undifferentiated murine 3T3 cells using the neutral red test. Magnetic fields had no cytotoxic effect on this 261 cell line. 262 263 W hen exposed to the same fields, a reduction in the proliferation and differentiation of the - 264 macrophage progenitor (CFU-GM) grown from the bone marrow of male and female mice was shown compared 265 to non-exposed cells. Stromal stem cell proliferation (CFU-f) from female mice showed a reduction while CFU- 266 f from male mice did not decrease. The authors concluded that these effects on CFU-f are equivocal. 267 268 Table 66. Cell proliferation studies Biological endpoint Exposure conditions Results Com m ents Reference

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Cell numbers and colony Nulled fields, 50 Hz No effects. Reipert et al., following efficiency vertical fields, Ca2+ ion 1997 cyclotron resonance Mouse haemopoetic conditions at 50 Hz progenitor cells FDCP mix A4 0.006, 1 and 2 mT 2 hours immediately after seeding 1, 4 or 7 days, one hour after seeding Deleted: [details?] Cell number 50 Hz No effects. Fiorani et al., 1992 K562 myeloid leukaemia 0.2œ200 µT cells up to 24 h Deleted: [time?] 3H-thymidine uptake 72 Hz pulsed No effects. Phillips and McChesney, CCRF-CEM human 3.5 mT 1991 lymphoblastoid cells 0.5œ24 h Proliferation 50 Hz No effects. Van Den Heuvel et al., Stem cells 80 µT 2001 Undifferentiated murine Deleted: [time?] 3T3 cells 4 days Proliferation and 50 Hz Reduction in proliferation and Van Den differentiation of the differentiation. Heuvel et al., granulocyte-macrophage 80 µT 2001 progenitor 7 days Deleted: [time?] Stromal stem cell 50 Hz Decrease in female mice and Van Den proliferation no change in male mice. Heuvel et al., 80 µT 2001 10 days Deleted: [time?] 269 270 9.3 Conclusions 271 272 Evidence for effects of ELF electric of magnetic fields on components of the immune system is generally Deleted: An evaluation 273 inconsistent. Many of the cell populations and functional markers were unaffected by exposure. However, in Deleted: exposure 274 some human studies at fields from10 µT to 2 mT, changes were observed in natural killer cells, showing both 275 increased and decreased cell numbers, and in white blood cell counts, which showed no change or decreased Deleted: effects 276 numbers. In animal studies reduced activity was seen in female, but not male mice or in rats of Deleted: presents a 277 either sex. W hite blood cell counts also showed inconsistency, with decreases or no change reported in different 278 studies. Animal exposures had even a broader range of 2 µT to 30 mT. The difficulty in interpreting the Deleted: lack of 279 potential health impact of these data is due to large variations in exposure and environmental conditions, Deleted: consistency 280 relatively small numbers of subjects tested, and the broad range of endpoints. Deleted: changes 281 282 There have been few studies carried out on the effects of ELF magnetic fields on the haematological Deleted: NK- 283 system. In experiments evaluating differential white blood cell counts, exposures ranging from 2 µT to 2 mT. Deleted: NK- 284 No consistent effects of acute exposure to magnetic fields, or to combined electric and magnetic fields have 285 been found in either human or animal studies. Deleted: and 286 Deleted: Exposure levels in 287 Overall therefore, evidence for effects of ELF electric or magnetic fields on the immune system and human studies varied between 288 haematological system is considered very weak. 10 µT and 2 mT. 289 Deleted: with Deleted: , Deleted: n

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