The effects of extremely low-frequency magnetic fields on melatonin and cortisol, two marker rhythms of the circadian system.

In the past 30 years the concern that daily exposure to extremely low-frequency magnetic fields (ELF-EMF) (1 to 300 Hz) might be harmful to human health (cancer, neurobehavioral disturbances, etc) has been the object of debate, and has become a public health concern. This has resulted in the classification of ELF-EMF into category 2B, ie, agents that are “possibly carcinogenic to humans” by the International Agency for Research on Cancer. Since melatonin, a neurohormone secreted by the pineal gland, has been shown to possess oncostatic properties, a “melatonin hypothesis” has been raised, stating that exposure to EMF might decrease melatonin production and therefore might promote the development of breast cancer in humans. Data from the literature reviewed here are contradictory. In addition, we have demonstrated a lack of effect of ELF-EMF on melatonin secretion in humans exposed to EMF (up to 20 years' exposure) which rebuts the melatonin hypothesis. Currently, the debate concerns the effects of ELF-EMF on the risk of childhood leukemia in children chronically exposed to more than 0.4 μT. Further research is thus needed to obtain more definite answers regarding the potential deleterious effects of ELF-EMF.

range includes the frequencies (50 Hz in Europe, 60 Hz in North America) of the electric power supply and of electric and magnetic fields (EMF) generated by electricity power lines and electric/electronic appliances; intermediate frequency (IF, 300 Hz to <10 MHz) is used in computer monitors, industrial processes, and security systems; and finally, radiofrequency range (RF, 10 MHz to 300 GHz) includes radars, and radio and television broadcasts and telecommunications. Biological effects of ELF-EMF and their consequences on human health have become the subject of important and recurrent public debate. The growth of electric power use in industrialized countries and the parallel increase of environmental exposure to ELF-EMF resulted in a widespread concern that ELF-EMF may have harmful effects in humans, a concern stimulated in the past decades by a number of epidemiologic studies reporting deleterious effects of ELF-EMF on human health. Wertheimer and Leeper 1,2 published the first report, conducted in the Denver area, on the association between childhood cancer and exposure to ELF-EMF, with the conclusion of a higher risk of childhood leukemia at higher residential ELF-EMF exposure. Savitz et al 3 gave support to this assertion with the publication of similar results in the same area (Denver). From then, several epidemiologic papers have reported a possible link, without any experimental evidence, however, between exposure of humans to ELF-EMF and diseases such as leukemia and other cancers, 4-6 depression, and suicide, 7 and neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. [8][9][10][11] All these results, though some of them were conflicting, resulted in a "melatonin hypothesis" as a tentative explanation, with the idea that those potential ELF-EMF deleterious effects might be a consequence of an inhibitory effect of ELF-EMF on the production of melatonin, 12 a hormone whose secretion has been shown to be altered (concentration decline and/or alteration of its circadian rhythm) in some diseases including cancers (review in Hill et al, ref 13), depressive disorders, [14][15][16] and disorders of the circadian time structure. 17,18 The concern regarding public health resulted in reports on this matter of official organizations, the most recent reports being those of the International Agency for Research on Cancer (IARC) in 2002 and the World Health Organization in 2007. 19 Of special interest, the IARC published in 2002 an evaluation of the carcinogenic risks of ELF to humans. 20 The agency classified ELF electric fields into category 3, which in the classification corresponds to "inadequate evidence" of deleterious effects, and classified ELF magnetic fields into category 2B, corresponding to the category of agents that are "possibly carcinogenic to humans." A classification into group 2B is "usually based on evidence in humans which is considered credible, but for which other explanations could not be ruled out." It has to be noted that these extremely-low-frequency electric and magnetic fields are separate entities. Whether or not ELF magnetic field exposure is causally related to increased health risks has led many scientists to examine the potential mechanisms by which ELF magnetic fields might affect human health. It is known that cancer and neurobehavioral alterations may be associated with circadian rhythm disruption and/or effect on melatonin secretion. [21][22][23][24] Theoretically, melatonin could be a good mechanistic candidate to explain potentially deleterious effects of EMF since: i) its secretion is dramatically inhibited by light, 25-28 which is the visible part of EMF; ii) the circadian pattern of the hormone is phase-advanced or -delayed by light according to the time of exposure, which is known as the phase response curve or PRC, 29 and this property might occur with exposure to EMF; iii) the oncostatic properties of melatonin have been described, [30][31][32] which resulted in the hypothesis that a decrease in the secretion of melatonin by the pineal gland might promote the development of breast cancer in humans 12 ; iv) and last, its association with depressive disorders has been put forward. [14][15][16] Since both melatonin and cortisol are major markers of the circadian system, we reviewed data from the literature on these two marker rhythms, in search of deleterious effects of EMF on both their blood levels and abnormalities in their circadian profiles, eg, a phase-advance or a phase-delay which would point out a rhythm desynchronization of the organism, ie, a situation that occurs when the biological clock is no longer in step with its environment. 17,33 component and is physiologically controlled by light. Melatonin is therefore considered as a marker rhythm of the circadian temporal structure. A marker rhythm is a physiological rhythmic variable, whose circadian pattern is highly reproducible on an individual basis and as a group phenomenon, which thus allows characterization of the timing of the endogenous rhythmic time structure and provides information on the synchronization of individuals (Figure 1). 36 Besides melatonin, the most frequent marker rhythms used both in humans and animals are the core body temperature circadian pattern 37 and the cortisol circadian rhythm, since they are also highly reproducible. 36,17 Cortisol also displays a robust and highly reproducible circadian rhythm that does not respond rapidly to minor and transient environmental changes, as they are part of daily life, which also makes it a good candidate as a marker rhythm. 36 Since a relationship between the pineal gland and the adrenal gland has been documented in vitro, 38 and considering the hypothesis of the alteration of melatonin by EMF, it can be useful to look at their potential effects on cortisol, another rhythm marker of the circadian system, and to obtain an additional argument for a circadian desynchronization of the organism.

Animal studies
For the sake of clarity, we present in two different tables the reports on ELF-EMF effects on melatonin. Table Ia displays the reports showing an alteration of melatonin secretion in different animal species, mainly rodents, after exposure to ELF-EMF. Table Ib deals with all of the studies reporting no effect of ELF-EMF on melatonin secretion in the different species under study. The very first data on the topic deal with electric fields (not magnetic fields), and date back to 1981, with the report on the reduction of pineal melatonin and N-acetyltransferase (NAT), the key enzyme for melatonin synthesis, in rats exposed to electric fields 20 h/day for 30 days. 39,40 Other reports, however, failed to find any effect, or were inconclusive or contradictory. 41,42 Then the interest shifted from electric to magnetic fields, with a large number of studies devoted to the effects of ELF-EMF on melatonin levels in different animal species. 43,44 Yellon 45,46 and Wilson et al, 47 documenting the effects of magnetic fields, were the first to report a reduction of both pineal and plasma melatonin in Djungarian hamsters with a short exposure to a sinusoidal 100-µT magnetic field. In addition, Wilson et al 47 also reported an increase in the concentration of norepinephrine in the suprachiasmatic nuclei, the central rhythm-generating system. The majority of laboratory studies were then carried out on rats. Kato et al, 48 in exposing male Wistar-King rats for 6 weeks to a 50-Hz circularly polarized sinusoidal magnetic field using increasing intensities, showed a decrease 14h 17h 20h 22h 23h 00h 01h 02h 03h 04h 05h 06h 07h 08h Decrease in Mel and NAT activity after 100 µT (acute) and 10 and 100 µT (chronic) Decreased and delayed nighttime peak though not replicated in the same paper = inconclusive Decreased and delayed nighttime peak though not replicated in the second part of the paper = inconclusive  T r a n s l a t i o n a l r e s e a r c h in pineal and plasma melatonin concentrations without any dose-response relationship. With the same protocol of exposure and species, but with a horizontal or vertical magnetic field, the same authors failed to find any effect on melatonin levels. 49 Suspecting a possible interference of pigmentation, Kato et al 50,51 then documented in Long-Evans rats the same intensities of a circularly polarized magnetic field and did indeed show a reduction of pineal and plasma melatonin concentrations. Other studies on rats or mice, 52-55 baboons, 56 and hamsters 57,58 also showed a reduction in the nighttime peak of melatonin. The same team reported a phase delay in the nocturnal peak time of melatonin in hamsters, 46,57,58 though they acknowledged in one paper that they were unable to replicate these findings, which make them inconclusive. 58 Some authors have reported an increase in nighttime melatonin levels. [59][60][61] With the aim of comparing short-term and long-term exposure effects, Selmaoui and Touitou 62 used male Wistar rats housed in a 12:12 light:dark schedule and submitted to a 50-Hz sinusoidal magnetic field of 1, 10, or 100 µT intensity, either once for 12 h or repeatedly 18 h per day for 30 days. While a single 12-h exposure to a 1-or 10-µT magnetic field had no effect on plasma melatonin levels or NAT and hydroxyindole-O-methyltransferase (HIOMT) pineal activities, a 100-µT exposure significantly decreased 30% plasma concentrations of melatonin and depressed 23% pineal NAT activity (HIOMT activity unchanged) when compared with sham-exposed rats. In turn, the 30 days' repeated exposure showed that while the 1-µT intensity showed no effects on pineal function, both the 10-and 100-µT intensities resulted in an approximately 42% decrease of plasma melatonin levels. NAT activity was also decreased, and HIOMT activity remained unchanged. This study showed that a sinusoidal magnetic field alters plasma melatonin levels and pineal NAT activity, and that the sensitivity threshold varies with the duration of exposure, thus suggesting that magnetic fields may have a cumulative effect upon pineal function. This melatonin and NAT activity decrease was able to be replicated in adult rats in another study by Selmaoui and Touitou, 63 while they also reported that aged rats were not affected by ELF-EMF. Löscher et al 53 studied the effects of a 24 h/day, 7 days/week, and 3-month exposure to magnetic fields on female rats bearing DMBA-induced mammary tumors; the field intensities were similar to the domestic exposures recorded close to electric power facilities. Whereas a significant decrease of blood melatonin concentrations was observed with 1 µT, no influence on the development of the mammary tumors could be put in evidence. Table Ib presents data on different animal species reporting the lack of effect of ELF-EMF on the concentrations of pineal or blood melatonin and on the urinary concentration of 6-sulphatoxymelatonin, the main metabolite of the hormone. These reports were either inconsistent or failed to show any effect of ELF-EMF in species as different as rats or mice, [64][65][66][67][68][69][70][71][72][73] sheep, 74,75 baboons, 76 Djungarian hamsters, 58,77 cows or heifers, [78][79][80] and kestrels. 81,82 The comparison of Table Ia (effects on melatonin) and Table Ib (lack of effects on melatonin) clearly shows that a number of these studies resulted in inconsistent data, even when the data were replicated by the same team with the same protocol and characteristics of exposure. 48,49,57,58,83,84 Last, some authors studying the effects of exposure to ELF-EMF of various biological systems such as isolated pineal glands [85][86][87][88][89][90] or MCF-7 cells 91-96 were unable to arrive at definite conclusions (Table II).

Human studies
Much of the evidence for the melatonin hypothesis is based on data obtained in rodents with a 25% to 40% reduction in the hormonal concentration, though, as shown above, results on the effects of ELF-EMF in rodents and higher mammals provided controversial results. Since the 1990s several research papers have documented the effects of ELF-EMF on the secretion of melatonin in humans. Most research published has involved an acute exposure (from 30 min to 4 days on average) of healthy volunteers to ELF-EMF with different exposure characteristics (Tables IIIa  and IIIb). The data on humans are controversial, since of the papers published about one third reported a decrease in melatonin secretion [97][98][99][100][101][102][103][104][105][106][107] with, however, some comments to be mentioned such as the lack of evidence for a doseresponse, 97 or a decrease not exclusively related to ELF-EMF and found in some particular subgroups [98][99][100][101][102][103][104][105][106][107] (Table  IIIa). In contrast to the previous ones, two thirds of the reports failed to find any effect of ELF-EMF on melatonin secretion in humans (Table IIIb).  Most work published on humans dealt with short-term exposure for evident ethical reasons. Taking into account the data we have shown on rats of potentially cumulative effects of ELF-EMF, 62 we performed a study in workers chronically exposed daily for 1 to 20 years, both in the workplace and at home, since the workers were housed near the substations. We showed no alteration in their melatonin secretion (plasma level or circadian profiles) which strongly suggests that ELF-EMF do not have cumulative effects on melatonin secretion in humans, and thus clearly rebuts the melatonin hypothesis that a decrease in blood melatonin concentration (or a disruption in its secretory pattern) explains the occurrence of clinical disorders or cancers possibly related to ELF-EMF. 125

ELF-EMF effects on cortisol and corticosterone
In  under study (corticosterone for rats, cortisol for other mammals), exposure characteristics (short-and long-term), and timing and duration of exposure (1 to 6 months) in different animal species are detailed in Table IV. No effect at work. urinary aMT6s decreased at home Decrease in aMT6s excretion in workers exposed to more stable fields during work. Decrease in aMT6s excretion in workers exposed for >2 h aMT6s excretion lower in exposed workers compared with office workers Decrease, primarily in subgroup using medication Decrease in aMT6s associated with mobile phone use Decrease Decrease in aMT6s associated with elevated geomagnetic activity   While the majority of papers failed to find any effect, [131][132][133][134][135][136][137] others have reported either an increase in the hormonal concentrations [138][139][140][141][142][143][144] or a decreased concentration. 145 The results of these studies are thus inconsistent and contradictory. Comparison between studies revealed that the discrepancy in the results might be due in part to the difference in the animal species used (rabbit, ewe lambs, cows, rats, or mice), class of age, and duration and intensity of exposure. Another factor that should be taken into account is that glucorticoids (ie, cortisol or corticosterone) levels are sensitive to many stressors that might affect hormone levels. It is well known that handling or bleeding animals increase corticosterone, a stress marker, and it is thus important to ensure that any external confounding stressor has to be controlled. Overall, these data suggest that no consistent effects have been seen in the stress-related hormones of the pituitary-adrenal axis in a variety of mammalian species. Data on ELF-EMF effects on cortisol in humans are scarce. We have found 7 papers on the matter (Table V). 109,124,[146][147][148][149] All of these papers report only on short exposure of adult volunteers to ELF-EMF, and all failed to find any effect.

Conclusion
We are all exposed to electric and magnetic fields of weak intensity. The levels of exposure of the general population range from 5 to 50 V/m for electric fields and from 0.01 to 0.2 µT for magnetic fields. The possible risk on health with exposure to electromagnetic fields became a concern to the public, which led to numerous studies by scientists on the topic. We have shown in this review that the reported studies are largely contradictory with regard to epidemiologic studies (about half of the studies found a relationship and the other half failed to find any), to the potential biological effects of ELF-EMF, and to the potentially mechanisms put forward; no clear explanations exist for these contradictory results. The relative risk (RR) which establishes the relation between exposure to ELF-EMF and cancer, is approximately 2 to 3. In the absence of clear explanation(s) a number of hypotheses have been raised. The characteristics of the magnetic field (linear or circular polarization, duration, timing), the animal species and, within a species, the strain appears to have a role in determining the biologic response obtained. Therefore, great care   Elevated daytime but no effect on night-time levels Circadian rhythm Altered On day 190, exposed animals showed a decrease in the cortisol Changes in Ser corticosterone Increase of plasma corticosterone must be given when comparing data obtained in different animal species, even within a group as rodents, since differences have been described between rodent species and even between pigmented and albino breeds. A possible change in the spatial structure of the photoreceptor pigment rhodopsin due to the electric field induced by the magnetic field has been proposed. Magnetic fields might also change either the electrical activity of the pinealocytes or their ability to produce melatonin, or both. With regard to the numerous studies performed on the effects of ELF-EMF on melatonin, the differences observed in animals and humans in these effects may be due to the differences in anatomical location and configuration of the pineal gland, and also the difference in the rest-activity cycle between rodents and humans. A different sensitivity to ELF-EMF could also be part of the explanation. Some human subjects may have greater sensitivity to ELF-EMF, but this is difficult to demonstrate because of the important interindividual variability in plasma concentration of melatonin. As far as melatonin is concerned, we have shown a lack of effect of ELF-EMF on melatonin (concentration and circadian rhythm) in workers exposed daily for up to 20 years in their workplace and at home, which strongly suggests that chronic ELF-EMF exposure appears to have no cumulative effects in human adults; this rebuts the "melatonin hypothesis" raised as an explanation for the deleterious sanitary effects of ELF-EMF. 125 In the same way, the application of high-throughput omics technologies to investigate the influences of ELF-EMF is confronted with the heterogeneity among the biological materials investigated, which are as different as blood cells/vessels, tissue cells, nerves, and bacteria, and this makes it difficult to compare data and to arrive at firm conclusions on the potential effects of ELF-EMF on biological systems. 150 As an example, most breast tumors become resistant to tamoxifen, and it has been shown that ELF-EMF reduce the efficacy of tamoxifen in a manner similar to tamoxifen resistance. By exposing cells of the breast cancer line MCF-7 to ELF-EMF, it has been found that ELF-EMF alter the expression of estrogen receptor cofactors, which in the authors' view may contribute to the induction of tamoxifen resistance in vivo. 151 Currently, the debate concerns the effects of ELF-EMF on children, with some data published in the literature pointing out the risk of childhood leukemia in relation to residential exposure, and underlining that this risk (the RR is around 2) can exist when children are chronically exposed to more than 0.4 µT.10 Large-scale collaborative studies are still needed to fill the gaps in our knowledge and provide answers to these numerous questions not yet resolved. Last, the deleterious risk of ELF-EMF on frail populations such as children and aged people may be greater and should be documented, at least for their residential exposure. ❏  This study was carried out in 15 healthy chronically (in the workplace and at home) exposed men (daily and for 1 to 20 years) to a 50-Hz magnetic field in search of any cumulative effect from those chronic conditions of exposure. Fifteen healthy unexposed men served as controls. As shown here, the exposed subjects experienced no change in the hormone levels or circadian patterns of melatonin.