Q 1991 MUNKSGAARD
Pineal gland “magnetosensitivity” to static magnetic fields is a consequence of induced electric currents (eddy currents) Lerchl A, Honaka KO, Reiter RJ. Pineal gland “magnetosensitivity” to datic magnetic fields is a consequence of induced electric currents (eddy currents). J Pineal Res 1991:10:109- 1 16. Abstract: During the past decade, a number of reports indicated that the mammalian pineal gland is magnetosensitive in terms of spatial orientation. This indication is based on observations that artificial alterations of the direction of the earth’s magnetic field (MF) markedly decreased the gland’s capability to synthesize melatonin. These findings, however, seem paradoxical since animals as well as humans experience such alterations whenever they turn their heads. Therefore, the potential of the pineal for sensing magnetic fields was re-investigated. During the dark phase, rats were exposed to repeatedly inverted MFs, generated by two identical pairs of Helmholtz coils; one pair connected to a power supply automatically, the other pair manually using an integrated potentiometer. Only the pineals of animals exposed to the automatically activated field responded with a reduced activity of the rate-limiting enzyme serotonin-N-acetyltransferase, lower melatonin levels and increases in serotonin and 5-hydroxyindole acetic acid. Hence, MF exposure itself did not affect the pineal. Rather, induced eddy currents in the animals, resulting from rapid On/Off transients of the artificially applied MF, are most likely the explanation.
The pineal gland, and its chief hormone, melatonin, are involved in a variety of physiological processes including seasonal reproduction [Reiter, 19801, diurnal activity rhythms [Armstrong, 19891, and the immune system [Maestroni et al., 1988; Withyachumnarnkul et al., 19901. Melatonin suppresses the incidence rate of certain cancers and prolongs the life span in mice [Maestroni et al., 19881 while in vitro, melatonin, at physiological concentrations, is capable of inhibiting the proliferation of human MCF-7 breast cancer cells [Hill and Blask, 19881. Moreover, patients with estrogen receptor-positive breast cancer have lower nocturnal plasma melatonin levels [Tamarkin et al., 19821. The possible involvement of the pineal in reproduction of nonhuman primates [Lerchl and Kiiderling, 19891 and man [Reiter, 19861 is another example of the gland’s multiple properties. In the pineal, the enzyme N-acetyltransferase (NAT; EC 2.3.1.5.) catalyzes the N-acetylation of serotonin to N-acetylserotonin which, in turn, is further 0-methylated to melatonin by the enzyme hydroxyindole-0-methyltransferase(HIOMT) [Deguchi and Axelrod, 1972a; Klein and Weller,
Alexander Lerchl, Keico 0. Nonaka, and Russel J. Reiter From the Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA (K.O.N., R.J.R.); Institute for Reproductive Medicine, University of Munster, FRG (A.L.)
Key words: electromagnetic fields-melatoninserotonin-5-hydroxyindole acetic acidserotonin-N-acetyltransferase
Dr. Russel J. Reiter, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7762, U.S.A. Accepted for publication October 30, 1990.
19701. Both pineal and serum melatonin levels exhibit a pronounced circadian rhythm, with highest levels occurring during the night [Quay, 1984; Reiter, 19801. Pineal serotonin, the substrate for NAT, and 5-hydroxyindole acetic acid (SHIAA), a serotonin metabolite, show an inverse concentration pattern with highest levels during the day [Mefford et al., 1983; Champney et al., 19841. During the winter (long nights), the nocturnal synthesis of melatonin is reportedly more prolonged than during spring or summer [Hoffmann et al., 1985; Illnerova et al., 1985; Rollag and Niswender, 19761. These patterns are caused by changes in the NAT activity [Klein and Weller, 19701 which is suppressed by light above a certain, species-specific intensity threshold [Reiter, 19851. Even 1 sec light pulses at night inhibit pineal NAT and melatonin [Reiter et al., 19861. In mammals, NAT activity is controlled indirectly by the environmental lightldark cycle [Wurtman et al., 19631, as perceived by the eyes, and also involving the suprachiasmatic nucleus and the superior cervical ganglia. The nocturnal release of norepinephrine from nerves within the pineal activates NAT [Deguchi and Axelrod, 109
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1972aI. Seasonal affective disorder (SAD), a form of depression which occurs almost exclusively during winter, may be related to an alteration of melatonin rhythmicity; in fact, exposure of SAD patients to bright light during early morning hours significantly improves their condition [Lewy et al., 19871. A number of investigations showed an apparent sensitivity of the pineal gland to other environmental factors such as electric [for review see Wilson et al., 19891 or weak, static magnetic fields [Olcese et al., 1985; Olcese et al., 1988a,b; Reuss et al., 1983; Semm et al., 1980; Semm, 1988; Stehle et al., 1988, Welker et al., 19831. Electric and magnetic fields are vector quantities, having both magnitude and direction. Changes in those fields result from alterations in either, or both, parameters. Usually Helmholtz coils are used to rotate, or invert, respectively, the vertical or the horizontal component of the earth’s magnetic field (MF). These coils provide a uniform MF while its strength is regulated by the applied current. The effects of rotated, or inverted, MFs on the pineal during nighttime hours are sometimes dramatic, with the glands exhibiting a suppression of NAT and reduced melatonin levels. In rats and mice, pineal serotonin and 5-HIAA levels are increased after 1 h exposure to an earth’s strength, intermittent MF [Lerchl et al., 19901. Interestingly, a local compensation of the natural MF by Helmholtz coils has no effect on the pineal [Khoory, 19871. Since pineal concentrations of CAMP are reduced due to MF exposure as well [Rudolph et al., 19881, it is assumed that its innervation is involved in the response to an altered MF. The findings are significant when seen in the context of growing public concerns about possible health risks caused by electromagnetic fields (EMF). There may be a causal relationship between EMFs and diseases such as cancer and leukemia [Calle and Savitz, 1985; Coleman et al., 1983; McDowall, 1983; Savitz and Calle, 1987; Wilson et al., 19891. Since every electrical current is accompanied by a MF, the exposure of animals to artificial MFs is, in principal, a good method to investigate possible effects of EMFs. However, both the published methods described and the discussions of the obtained results are highly confusing and have produced more questions than answers. Some have designated the pineal a “magnetosensitive” organ since even a slight horizontal rotation of the earth’s MF (by 5”) reportedly affected the pineal [Welker et al., 19831. However, these findings appear paradoxical because, for the animals investigated, and, of course, also for humans, a’rotation, an inversion or any change of the direction of the ambient MF occurs 110
whenever the head is moved. That these movements do not interfere with the nighttime rise of melatonin is obvious, especially in species which are active during the night when melatonin levels are high. Moreover, the pineal reportedly always responded with a reduction in melatonin synthesis due to MF exposure, never with an increase; if the pineal would be in fact involved in spatial orientation, one would expect at least sometimes an increased melatonin production. Hence, it seems illogical to assume that the observed effects are merely a consequence of the fact that the MF has been rotated or inverted, as presumed earlier [Lerchl et al., 19901. In an attempt to clarify the mechanism(s) by which the pineal is affected by an artificially manipulated MF, we sought an answer to the following question: is the fact that the MF is inverted responsible for the effects on the pineal or is it a consequence of a related process? Materials and Methods Artificial Magnetic Field
Two identical pairs of Helmholtz coils were used. Radius = clearance = 0.5 m; each coil = 300 turns of copper wire; time constant approximately 7.25 msec; total inductivity approximately 0.5 Henry. The strength of the generated field (BH in Gauss) was calculated by the equation BH = c n I r-’, with c = 0.899. VsA-lm-’, n = turns, I = current in amperes, and r = radius in meters [see Semm et al., 19801. At a current of 150 mA, coils produced a MF of 0.8 Gauss. When oriented in a magnetic North-South direction, the horizontal component of the earth’s MF (at approximately 0.4 Gauss in the animal rooms) was inverted (Fig. 1). Field strengths were measured with a Gaussmeter (Model MG-7D, Walker Sci., Worcester, MA). The coils were attached to two identical power supplies in different ways. One pair was connected by a relay which, in turn, was activated by a timing circuit. The voltage of the other power supply was regulated manually by means of an integrating potentiometer. While the latter procedure took about 1 sec for both activating and deactivating the artificial MF, the relay instantaneously switched the full voltage to the coils. Although in both cases the artificially generated MFs inverted the horizontal component of the earth’s MF between the coils, the inductivity of the Helmholtz coils prevented the current (and, consequently, the generated MF) from immediately following the applied voltage. Rather, the current in the coils followed an e-function both when the voltage was connected and disconnected. For that *
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“Magnetosensitivity” of the pineal gland Helmholtz Coils
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FIR. 1 . Top: Two identical pairs of Helmholtz coils (one pair shown) were oriented with their common axis in a magnetic North-South direction. The animals were placed between the coils in macrolon cages which were supported by cardboard boxes, both magnetically permeable materials. To prevent imhomogeneously applied MFs, the metal grid-covers of the cages were removed. Middle: The direction and strength of the earth’s MF depends on the location at the planet’s surface and is a result of a horizontal component (HC) and a vertical component (VC). The length and direction of the resulting vector are equivalent to the total strength and the direction of the MF. Bottom: When an artificial MF with a 2-fold greater intensity than the HC of the earth’s MF is generated, but oriented in the opposite direction (dotted arrows), the resulting HC has the same strength as the “natural” HC, but the opposite direction; hence, the HC is inverted. Consequently, the resulting vector “flips” as indicated by the curved arrow; the VC remains unchanged.
reason, a search coil [Watson and Downes, 19791 was placed between the Helmholtz coils to monitor the rate of change of the generated MF (dB/dt) by measuring the currents (eddy currents) induced in the search coil. Manual activation (or deactivation) with the integrating potentiometer did not produce measurable eddy currents in the search coil although the MF was inverted; however, the automatic activation produced eddy currents when the power supply was switched both on and off with maximal dB/dt values occurring at the moments of connecting, and disconnecting, the voltage, respectively.
After roughly 25 msec, the automatically activated coils completely inverted the MF within their active area [for additional details see Lerchl et al. , 19901. The principal effects of the MF-inversion as well as the aforementioned paradox are presented in Figure 2. Animals
Four groups of male Sprague Dawley rats (body weight 100-120 g, Harlan, Indianapolis, IN) were used. They were kept at 22” 5 2°C and constant 111
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Fig. 2 . When no artificial field is applied, only the earth's MF is present as indicated by the arrow (top). Note that the real earth's MF, as a resultant of a horizontal and a vertical component, is a vector, facing downward at most locations in the northern hemisphere. When a field is applied (bottom), which inverts the horizontal component of the earth's MF, the vector flips (see also Fig. 1). However, for the animal the very same situation exists (namely, a rostra1 oriented vector in this example) when it turns its head by 180" (right).
humidity with unlimited access to tap water and food for 10 days prior to the experiment. The lighvdark cycle was regulated automatically (14Ll 10D; lights on at 7:OO AM CST). Treatments and housing conditions were in accordance with institutional guidelines. One group (8 animals) was killed by decapitation during the light phase (6:OO PM CST); the remaining three groups (10 each) were killed during the following night (2:OO AM CST) under dim red light in alternating order. Pineals were quickly removed, frozen on solid CO, and stored at -60°C until assayed. While the daytime and one nighttime group was not exposed, one group was subjected to the manually activated field, and the third group to the automatically activated field. Before exposure, all animals were transferred to plastic cages (macrolon; 5 animals per cage) that were covered with identical upside-down cages, leaving wide slits to provide an air supply. Control animals were handled in the same way. Two cages each were placed between the coils (see Fig. 1) or at a remote comer in the same room. No changes in the ambient MF due to the exposure facilities were detectable at the locations of the control cages. The Helmholtz coils were intermittently activated for 1 h, beginning at 1:OO AM CST (4 h after darkness onset) by turning them on and off at 1 min intervals. 112
Hence, the MFs were inverted 60 times during the exposure period. After exposure, but prior to the sacrifice, both power supplies were slowly regulated to zero voltage in order to prevent off transients (see Discussion for explanation). Chemical and Statistical Analyses
Pineal NAT activity was estimated by a radioenzymatic method as described previously [Deguchi and Axelrod, 1972bl. Melatonin levels were determined by radioimmunoassay, using [3H]-melatonin (TRK 798, Amersham) as tracer, and a highly specific melatonin antiserum (Batch G/S/704-8483, Stockgrand Ltd., Guildford, UK). For antiserum characteristics see Fraser et al. [ 19831. This assay has been validated extensively by Webley et a]. [1985]. In our determinations, the assay sensitivity was better than 2 pg/ml; intra- and inter-assay variations were 7.8% and 12.8%, respectively. Pineal serotonin and 5-HIAA were measured by means of high performance liquid chromatography with electrochemical detection (HPLC-EC) as described by Martin and Aldegunde [ 19891. Statistical comparisons were performed by ANOVA followed by the StudentNewman-Keuls test. Means standard errors are shown. +_
“Magnetosensitivity” of the pineal gland
tissue’s conductivity [Geddes and Baker. 1967: Lunt, 19821. Hen&, if an animal is exposed to a rapidly changing MF, an induced eddy current occurs that may affect the nervous system. This conclusion is supported by the observation that the synaptic transmission is affected by exposure to electric fields [Jaffe et al., 19801. Presumably, the eyes are involved in the effects caused by the presence of an inverted (or rotated), weak static magnetic field, as shown in albino rats [Olcese et al., 19851. Magnetophosphenes (visual sensations generated by magnetic fields), however, only occur at much higher field strengths (e.g., 100-fold greater than the earth’s MF) [Lovsund et al., 19801. Moreover, when a MF is generated by a direct current power supply, magnetophosphenes only occur when the circuit is closed or opened; no such effects are observed when the current reaches a constant value. The fact that only albino gerbils, but not pigmented members of the same species, show a “magnetosensitivity” of the pineal in terms of reduced melatonin production [Stehle et al., 19881 suggests that a loss of pigmentation increases sensitivity not only to light but also to rapidly changing MFs. This possibility needs further investigation. Studies on the effects of artificially altered, static magnetic fields on the pineal usually neither describe important physical parameters of the Helmholtz coils (e.g., inductivity, time constant) nor give precise information about how the power supplies
Results
Levels of pineal melatonin and NAT activity were higher during the dark phase than during the day (Fig. 3), as expected. Likewise, nighttime levels of serotonin and 5-HIAA were lower than the daytime ~alues. Exposure to an artificially inverted MF depressed nighttime NAT activity and reduced nelatonin levels (Fig. 3), and increased both serotonin and 5-HIAA (Fig. 4) only in the pineals of those animals where the power supply was switched mtomatically. The increased levels of both serotonin and 5-HIAA are presumably the consequence of a reduced NAT activity, thus resulting in an accumulation of its substrate, serotonin, and, subsequently, in its increased conversion to 5-HIAA. Discussion
Based on our findings, OniOff-effects with respect to artificially generated static MFs are very likely the reason for the changes in pineal indole metabolism. Hence, the explanation for the effects is that induced currents (eddy currents), caused by the rapid rises and the decays of the MF affect the pineal gland either directly, or, more likely, indirectly, via an action on the neural input to the gland. The mere presence of an altered MF has no influence. Every change of a MF produces an electric field. Depending on the tissue exposed to such a field, an appropriate eddy current occurs, depending on the
N AT A c t ivi ty
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Fig. 3. The effects of magnetic field (MF) exposure on pineal NAT activity and melatonin content.
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Fig. 4 . Pineal content of serotonin and 5-HIAA. Labels as in Figure 3.
were connected to the coils. However, the results described herein clearly prove that these parameters are equal to or even more important than statements about the field strength or the coil diameter. For example, if one pair of Helmholtz coils is made with wire of 0.1 mm diameter, and another one, otherwise identical, is made with a 0.3 mm diameter wire, and an identical current is applied, both pairs will invert the horizontal component of the Earth’s MF. Since the electrical resistance of the coils also determines their inductivity, the 0.3 mm coil will have a lower time constant, resulting in an increased induced electrical field when the coils are activated or deactivated. Thus, different effects on the pineal gland would be expected. It has not been routine to describe exactly how (or if) the current was turned off after the exposure of the animals to a static MF. If, for instance, the coils were deactivated just prior to pineal collection, an off-transient induced eddy current rather than the MF exposure itself may have caused the effects. Without this information, we are unable to compare our observations quantitatively with other findings. Nevertheless, our results are, qualitatively at least, well in accordance with previous reports with respect to the suppression of pineal NAT activity and melatonin [Olcese et al., 1985; Olcese et al., 1988a; Semm et al., 1980; Semm, 1983; Reuss et al., 1983; Welker et al., 19831. Furthermore, our previous findings [Lerchl et al., 19901 were found to be reproducible with respect to the increase in 114
pineal serotonin and 5-HIAA. In the aforementioned study, however, we did not observe a drop in pineal melatonin. In the present study we report diminished levels of pineal melatonin as a consequence of MF exposure. This difference may be explained by the fact that in the present study we activated the coils more frequently than in our previous investigation, thus further supporting the assumption that the effets are most likely a consequence of On/Off-transients rather than a result of the inversion of the horizontal component of the earth’s MF. In future investigations dealing with physiological effects of magnetic fields, these methodological parameters as well as an exact description of the physical characteristics of the Helmholtz coils are indispensible if comparisons between reports are to be made. In conclusion, we feel that artificial, earth’s strength static magnetic fields do not affect the pineal gland of mammals due to their presence, either directly or indirectly. Rather, eddy currents, produced by rapid (in the low msec range) rises and decays of MFs are the likely explanation for the response of the gland. The findings reported herein should be considered in the context of studies showing effects of pulsed, weak magnetic fields on intracellular transcription [Goodman et al., 19831 and DNA synthesis [Takahashi et al., 19861, respectively. The authors of both papers assume that certain characteristics of the applied MF, such as pulse width and frequency, determine the observed
“Magnetosensitivity” of the pineal gland
effects. Hence, future investigations dealing with MF-induced alterations in the pineal gland have to focus on those parameters as well. A recent report by Matanoski et al. [ 19891 proposed that “spikey” (high dB/dt) magnetic fields present in certain telephone company offices may be an important factor in increased risk of prostate and male breast cmcer found in telephone workers. Whether these findings, however, are related to malfunctions of the pineal gland due to these artificial fields remains to be seen. Acknowledgments
This work was supported by NSF grant # DCB 87- 1 1241. We acknowledge the support provided by Dr. W.W. Morgan, this institution. A.L. was supported by a fellowship from the NATO scientific committee through the German Academic Exchange Service, K.O.N. by the CNPq from Brazil. Literature Cited AKMSTRONG, S.M. (1989) Melatonin and circadian control in mammals. Experientia 45:932-938. CALLE,E.E., D.A. SAVITZ(1985) Leukemia in occupational groups with presumed exposure to electrical and magnetic fields. N. Engl. J. Med. 313:147&1477. CHAMPNEY, T.H., A.P. HOLTORF,R.W. STEGER,R.J. REITER (1984) Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland. J. Neurosci. Res. 11:5946. COLEMAN, M., J. BELL,R. SKEET(1983) Leukemia incidence in electrical workers. Lancet 1:982-983. DEGUCHI, T., J. AXELROD (1972a) Control of circadian change of serotonin N-acetyltransferase in the pineal organ by the P-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 69:2547-2550. DEGUCHI,T., J. AXELROD(1972b) Sensitive assay for serotonin N-acetyltransferase activity in rat pineal gland. Anal. Biochem. 50:174-179. FRASER, S., P. COWEN,M. FRANKLIN, C. FRANEY, J. ARENDT (1983) Direct radioimmunoassay for melatonin in plasma. Clin. Chem. 29:396397. GEDDES, L.A., L.E. BAKER(1967) The specific resistance of biological material-a compendium of data for the biomedical engineer and physiologist. Med. Biol. Eng. 5:271-293. GOODMAN, R., C.A. BASSET,A.S. HENDERSON (1983) Pulsing electromagnetic fields induce cellular transcription. Science 220: 1283-1285. HILL,S.M., D.E. BLASK(1988) Effects of the pineal hormone melatonin on the proliferation and morphological characteristics of human breast cancer cells (MCF-7) in culture. Cancer Res. 48:612 1-6 126. HOFFMANN, K., H. ILLNEROVA, J. VANECEK (1985) Comparison of pineal melatonin rhythms in young, adult and old Djungarian hamsters (Phodopus sungorus) under long and short photoperiods. Neurosci. Lett. 56:39-53. H., P. ZVOLSKY, J. VANECEK (1985) The circadian ILLNEROVA, rhythm in plasma melatonin concentration of the urbanized man: The effet of summer and winter time. Brain Res. 328: 186-1 89.
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L., D. DANFORTH,A. LICHTER,E. DEMOSS,M. TAMARKIN, COHEN,B. CHABNER, M. LIPPMAN(1982) Decreased nocturnal plasma melatonin peak in patients with estrogen receptorpositive breast cancer. Science 216:1003-1005. WATSON,J., E.M. DOWNES(1979) Clinical aspects of the stimulation of bone healing using electrical phenomena. Med. Biol. Eng. Comput. 17:161-169. WEBLEY,G.E., H. MEHL,K.P. WILLEY(1985) Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species. J. Endocrinol. 106:387-394. WELKER,H . A . , P. SEMM,R.P. WILLIG,J.C. COMMENTZ, W. WILTSCHKO,L. VOLLRATH(1983) Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content in the rat pineal gland. Exp. Brain Res. 50:426-432. WILSON,B.W., R.G. STEVENS,L.E. ANDERSON (1989) Neuroendocrine mediated effects of electromagnetic-field exposure: Possible role of the pineal gland. Life Sci. 4.513191332. WITHYACHNUMNARNKUL, B., K.O. NONAKA,C. SANTANA, M.A. ATTIA,R.J. REITER(1990) Interferon-gamma modulates melatonin production in rat pineal glands in organ culture. J. Interferon Res., 10:403411. WURTMAN, R.J., J. AXELROD,L.S. PHILLIPS(1963) Melatonin synthesis in the rat pineal gland: Control by light. Science 142:1071-1073.
Pineal gland “magnetosensitivity” to static magnetic fields is a consequence of induced electric currents (eddy currents) Lerchl A, Honaka KO, Reiter RJ. Pineal gland “magnetosensitivity” to datic magnetic fields is a consequence of induced electric currents (eddy currents). J Pineal Res 1991:10:109- 1 16. Abstract: During the past decade, a number of reports indicated that the mammalian pineal gland is magnetosensitive in terms of spatial orientation. This indication is based on observations that artificial alterations of the direction of the earth’s magnetic field (MF) markedly decreased the gland’s capability to synthesize melatonin. These findings, however, seem paradoxical since animals as well as humans experience such alterations whenever they turn their heads. Therefore, the potential of the pineal for sensing magnetic fields was re-investigated. During the dark phase, rats were exposed to repeatedly inverted MFs, generated by two identical pairs of Helmholtz coils; one pair connected to a power supply automatically, the other pair manually using an integrated potentiometer. Only the pineals of animals exposed to the automatically activated field responded with a reduced activity of the rate-limiting enzyme serotonin-N-acetyltransferase, lower melatonin levels and increases in serotonin and 5-hydroxyindole acetic acid. Hence, MF exposure itself did not affect the pineal. Rather, induced eddy currents in the animals, resulting from rapid On/Off transients of the artificially applied MF, are most likely the explanation.
The pineal gland, and its chief hormone, melatonin, are involved in a variety of physiological processes including seasonal reproduction [Reiter, 19801, diurnal activity rhythms [Armstrong, 19891, and the immune system [Maestroni et al., 1988; Withyachumnarnkul et al., 19901. Melatonin suppresses the incidence rate of certain cancers and prolongs the life span in mice [Maestroni et al., 19881 while in vitro, melatonin, at physiological concentrations, is capable of inhibiting the proliferation of human MCF-7 breast cancer cells [Hill and Blask, 19881. Moreover, patients with estrogen receptor-positive breast cancer have lower nocturnal plasma melatonin levels [Tamarkin et al., 19821. The possible involvement of the pineal in reproduction of nonhuman primates [Lerchl and Kiiderling, 19891 and man [Reiter, 19861 is another example of the gland’s multiple properties. In the pineal, the enzyme N-acetyltransferase (NAT; EC 2.3.1.5.) catalyzes the N-acetylation of serotonin to N-acetylserotonin which, in turn, is further 0-methylated to melatonin by the enzyme hydroxyindole-0-methyltransferase(HIOMT) [Deguchi and Axelrod, 1972a; Klein and Weller,
Alexander Lerchl, Keico 0. Nonaka, and Russel J. Reiter From the Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA (K.O.N., R.J.R.); Institute for Reproductive Medicine, University of Munster, FRG (A.L.)
Key words: electromagnetic fields-melatoninserotonin-5-hydroxyindole acetic acidserotonin-N-acetyltransferase
Dr. Russel J. Reiter, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7762, U.S.A. Accepted for publication October 30, 1990.
19701. Both pineal and serum melatonin levels exhibit a pronounced circadian rhythm, with highest levels occurring during the night [Quay, 1984; Reiter, 19801. Pineal serotonin, the substrate for NAT, and 5-hydroxyindole acetic acid (SHIAA), a serotonin metabolite, show an inverse concentration pattern with highest levels during the day [Mefford et al., 1983; Champney et al., 19841. During the winter (long nights), the nocturnal synthesis of melatonin is reportedly more prolonged than during spring or summer [Hoffmann et al., 1985; Illnerova et al., 1985; Rollag and Niswender, 19761. These patterns are caused by changes in the NAT activity [Klein and Weller, 19701 which is suppressed by light above a certain, species-specific intensity threshold [Reiter, 19851. Even 1 sec light pulses at night inhibit pineal NAT and melatonin [Reiter et al., 19861. In mammals, NAT activity is controlled indirectly by the environmental lightldark cycle [Wurtman et al., 19631, as perceived by the eyes, and also involving the suprachiasmatic nucleus and the superior cervical ganglia. The nocturnal release of norepinephrine from nerves within the pineal activates NAT [Deguchi and Axelrod, 109
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1972aI. Seasonal affective disorder (SAD), a form of depression which occurs almost exclusively during winter, may be related to an alteration of melatonin rhythmicity; in fact, exposure of SAD patients to bright light during early morning hours significantly improves their condition [Lewy et al., 19871. A number of investigations showed an apparent sensitivity of the pineal gland to other environmental factors such as electric [for review see Wilson et al., 19891 or weak, static magnetic fields [Olcese et al., 1985; Olcese et al., 1988a,b; Reuss et al., 1983; Semm et al., 1980; Semm, 1988; Stehle et al., 1988, Welker et al., 19831. Electric and magnetic fields are vector quantities, having both magnitude and direction. Changes in those fields result from alterations in either, or both, parameters. Usually Helmholtz coils are used to rotate, or invert, respectively, the vertical or the horizontal component of the earth’s magnetic field (MF). These coils provide a uniform MF while its strength is regulated by the applied current. The effects of rotated, or inverted, MFs on the pineal during nighttime hours are sometimes dramatic, with the glands exhibiting a suppression of NAT and reduced melatonin levels. In rats and mice, pineal serotonin and 5-HIAA levels are increased after 1 h exposure to an earth’s strength, intermittent MF [Lerchl et al., 19901. Interestingly, a local compensation of the natural MF by Helmholtz coils has no effect on the pineal [Khoory, 19871. Since pineal concentrations of CAMP are reduced due to MF exposure as well [Rudolph et al., 19881, it is assumed that its innervation is involved in the response to an altered MF. The findings are significant when seen in the context of growing public concerns about possible health risks caused by electromagnetic fields (EMF). There may be a causal relationship between EMFs and diseases such as cancer and leukemia [Calle and Savitz, 1985; Coleman et al., 1983; McDowall, 1983; Savitz and Calle, 1987; Wilson et al., 19891. Since every electrical current is accompanied by a MF, the exposure of animals to artificial MFs is, in principal, a good method to investigate possible effects of EMFs. However, both the published methods described and the discussions of the obtained results are highly confusing and have produced more questions than answers. Some have designated the pineal a “magnetosensitive” organ since even a slight horizontal rotation of the earth’s MF (by 5”) reportedly affected the pineal [Welker et al., 19831. However, these findings appear paradoxical because, for the animals investigated, and, of course, also for humans, a’rotation, an inversion or any change of the direction of the ambient MF occurs 110
whenever the head is moved. That these movements do not interfere with the nighttime rise of melatonin is obvious, especially in species which are active during the night when melatonin levels are high. Moreover, the pineal reportedly always responded with a reduction in melatonin synthesis due to MF exposure, never with an increase; if the pineal would be in fact involved in spatial orientation, one would expect at least sometimes an increased melatonin production. Hence, it seems illogical to assume that the observed effects are merely a consequence of the fact that the MF has been rotated or inverted, as presumed earlier [Lerchl et al., 19901. In an attempt to clarify the mechanism(s) by which the pineal is affected by an artificially manipulated MF, we sought an answer to the following question: is the fact that the MF is inverted responsible for the effects on the pineal or is it a consequence of a related process? Materials and Methods Artificial Magnetic Field
Two identical pairs of Helmholtz coils were used. Radius = clearance = 0.5 m; each coil = 300 turns of copper wire; time constant approximately 7.25 msec; total inductivity approximately 0.5 Henry. The strength of the generated field (BH in Gauss) was calculated by the equation BH = c n I r-’, with c = 0.899. VsA-lm-’, n = turns, I = current in amperes, and r = radius in meters [see Semm et al., 19801. At a current of 150 mA, coils produced a MF of 0.8 Gauss. When oriented in a magnetic North-South direction, the horizontal component of the earth’s MF (at approximately 0.4 Gauss in the animal rooms) was inverted (Fig. 1). Field strengths were measured with a Gaussmeter (Model MG-7D, Walker Sci., Worcester, MA). The coils were attached to two identical power supplies in different ways. One pair was connected by a relay which, in turn, was activated by a timing circuit. The voltage of the other power supply was regulated manually by means of an integrating potentiometer. While the latter procedure took about 1 sec for both activating and deactivating the artificial MF, the relay instantaneously switched the full voltage to the coils. Although in both cases the artificially generated MFs inverted the horizontal component of the earth’s MF between the coils, the inductivity of the Helmholtz coils prevented the current (and, consequently, the generated MF) from immediately following the applied voltage. Rather, the current in the coils followed an e-function both when the voltage was connected and disconnected. For that *
*
“Magnetosensitivity” of the pineal gland Helmholtz Coils
0 0
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FIR. 1 . Top: Two identical pairs of Helmholtz coils (one pair shown) were oriented with their common axis in a magnetic North-South direction. The animals were placed between the coils in macrolon cages which were supported by cardboard boxes, both magnetically permeable materials. To prevent imhomogeneously applied MFs, the metal grid-covers of the cages were removed. Middle: The direction and strength of the earth’s MF depends on the location at the planet’s surface and is a result of a horizontal component (HC) and a vertical component (VC). The length and direction of the resulting vector are equivalent to the total strength and the direction of the MF. Bottom: When an artificial MF with a 2-fold greater intensity than the HC of the earth’s MF is generated, but oriented in the opposite direction (dotted arrows), the resulting HC has the same strength as the “natural” HC, but the opposite direction; hence, the HC is inverted. Consequently, the resulting vector “flips” as indicated by the curved arrow; the VC remains unchanged.
reason, a search coil [Watson and Downes, 19791 was placed between the Helmholtz coils to monitor the rate of change of the generated MF (dB/dt) by measuring the currents (eddy currents) induced in the search coil. Manual activation (or deactivation) with the integrating potentiometer did not produce measurable eddy currents in the search coil although the MF was inverted; however, the automatic activation produced eddy currents when the power supply was switched both on and off with maximal dB/dt values occurring at the moments of connecting, and disconnecting, the voltage, respectively.
After roughly 25 msec, the automatically activated coils completely inverted the MF within their active area [for additional details see Lerchl et al. , 19901. The principal effects of the MF-inversion as well as the aforementioned paradox are presented in Figure 2. Animals
Four groups of male Sprague Dawley rats (body weight 100-120 g, Harlan, Indianapolis, IN) were used. They were kept at 22” 5 2°C and constant 111
Lerchl et al.
Fig. 2 . When no artificial field is applied, only the earth's MF is present as indicated by the arrow (top). Note that the real earth's MF, as a resultant of a horizontal and a vertical component, is a vector, facing downward at most locations in the northern hemisphere. When a field is applied (bottom), which inverts the horizontal component of the earth's MF, the vector flips (see also Fig. 1). However, for the animal the very same situation exists (namely, a rostra1 oriented vector in this example) when it turns its head by 180" (right).
humidity with unlimited access to tap water and food for 10 days prior to the experiment. The lighvdark cycle was regulated automatically (14Ll 10D; lights on at 7:OO AM CST). Treatments and housing conditions were in accordance with institutional guidelines. One group (8 animals) was killed by decapitation during the light phase (6:OO PM CST); the remaining three groups (10 each) were killed during the following night (2:OO AM CST) under dim red light in alternating order. Pineals were quickly removed, frozen on solid CO, and stored at -60°C until assayed. While the daytime and one nighttime group was not exposed, one group was subjected to the manually activated field, and the third group to the automatically activated field. Before exposure, all animals were transferred to plastic cages (macrolon; 5 animals per cage) that were covered with identical upside-down cages, leaving wide slits to provide an air supply. Control animals were handled in the same way. Two cages each were placed between the coils (see Fig. 1) or at a remote comer in the same room. No changes in the ambient MF due to the exposure facilities were detectable at the locations of the control cages. The Helmholtz coils were intermittently activated for 1 h, beginning at 1:OO AM CST (4 h after darkness onset) by turning them on and off at 1 min intervals. 112
Hence, the MFs were inverted 60 times during the exposure period. After exposure, but prior to the sacrifice, both power supplies were slowly regulated to zero voltage in order to prevent off transients (see Discussion for explanation). Chemical and Statistical Analyses
Pineal NAT activity was estimated by a radioenzymatic method as described previously [Deguchi and Axelrod, 1972bl. Melatonin levels were determined by radioimmunoassay, using [3H]-melatonin (TRK 798, Amersham) as tracer, and a highly specific melatonin antiserum (Batch G/S/704-8483, Stockgrand Ltd., Guildford, UK). For antiserum characteristics see Fraser et al. [ 19831. This assay has been validated extensively by Webley et a]. [1985]. In our determinations, the assay sensitivity was better than 2 pg/ml; intra- and inter-assay variations were 7.8% and 12.8%, respectively. Pineal serotonin and 5-HIAA were measured by means of high performance liquid chromatography with electrochemical detection (HPLC-EC) as described by Martin and Aldegunde [ 19891. Statistical comparisons were performed by ANOVA followed by the StudentNewman-Keuls test. Means standard errors are shown. +_
“Magnetosensitivity” of the pineal gland
tissue’s conductivity [Geddes and Baker. 1967: Lunt, 19821. Hen&, if an animal is exposed to a rapidly changing MF, an induced eddy current occurs that may affect the nervous system. This conclusion is supported by the observation that the synaptic transmission is affected by exposure to electric fields [Jaffe et al., 19801. Presumably, the eyes are involved in the effects caused by the presence of an inverted (or rotated), weak static magnetic field, as shown in albino rats [Olcese et al., 19851. Magnetophosphenes (visual sensations generated by magnetic fields), however, only occur at much higher field strengths (e.g., 100-fold greater than the earth’s MF) [Lovsund et al., 19801. Moreover, when a MF is generated by a direct current power supply, magnetophosphenes only occur when the circuit is closed or opened; no such effects are observed when the current reaches a constant value. The fact that only albino gerbils, but not pigmented members of the same species, show a “magnetosensitivity” of the pineal in terms of reduced melatonin production [Stehle et al., 19881 suggests that a loss of pigmentation increases sensitivity not only to light but also to rapidly changing MFs. This possibility needs further investigation. Studies on the effects of artificially altered, static magnetic fields on the pineal usually neither describe important physical parameters of the Helmholtz coils (e.g., inductivity, time constant) nor give precise information about how the power supplies
Results
Levels of pineal melatonin and NAT activity were higher during the dark phase than during the day (Fig. 3), as expected. Likewise, nighttime levels of serotonin and 5-HIAA were lower than the daytime ~alues. Exposure to an artificially inverted MF depressed nighttime NAT activity and reduced nelatonin levels (Fig. 3), and increased both serotonin and 5-HIAA (Fig. 4) only in the pineals of those animals where the power supply was switched mtomatically. The increased levels of both serotonin and 5-HIAA are presumably the consequence of a reduced NAT activity, thus resulting in an accumulation of its substrate, serotonin, and, subsequently, in its increased conversion to 5-HIAA. Discussion
Based on our findings, OniOff-effects with respect to artificially generated static MFs are very likely the reason for the changes in pineal indole metabolism. Hence, the explanation for the effects is that induced currents (eddy currents), caused by the rapid rises and the decays of the MF affect the pineal gland either directly, or, more likely, indirectly, via an action on the neural input to the gland. The mere presence of an altered MF has no influence. Every change of a MF produces an electric field. Depending on the tissue exposed to such a field, an appropriate eddy current occurs, depending on the
N AT A c t ivi ty
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Fig. 3. The effects of magnetic field (MF) exposure on pineal NAT activity and melatonin content.
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.;
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Fig. 4 . Pineal content of serotonin and 5-HIAA. Labels as in Figure 3.
were connected to the coils. However, the results described herein clearly prove that these parameters are equal to or even more important than statements about the field strength or the coil diameter. For example, if one pair of Helmholtz coils is made with wire of 0.1 mm diameter, and another one, otherwise identical, is made with a 0.3 mm diameter wire, and an identical current is applied, both pairs will invert the horizontal component of the Earth’s MF. Since the electrical resistance of the coils also determines their inductivity, the 0.3 mm coil will have a lower time constant, resulting in an increased induced electrical field when the coils are activated or deactivated. Thus, different effects on the pineal gland would be expected. It has not been routine to describe exactly how (or if) the current was turned off after the exposure of the animals to a static MF. If, for instance, the coils were deactivated just prior to pineal collection, an off-transient induced eddy current rather than the MF exposure itself may have caused the effects. Without this information, we are unable to compare our observations quantitatively with other findings. Nevertheless, our results are, qualitatively at least, well in accordance with previous reports with respect to the suppression of pineal NAT activity and melatonin [Olcese et al., 1985; Olcese et al., 1988a; Semm et al., 1980; Semm, 1983; Reuss et al., 1983; Welker et al., 19831. Furthermore, our previous findings [Lerchl et al., 19901 were found to be reproducible with respect to the increase in 114
pineal serotonin and 5-HIAA. In the aforementioned study, however, we did not observe a drop in pineal melatonin. In the present study we report diminished levels of pineal melatonin as a consequence of MF exposure. This difference may be explained by the fact that in the present study we activated the coils more frequently than in our previous investigation, thus further supporting the assumption that the effets are most likely a consequence of On/Off-transients rather than a result of the inversion of the horizontal component of the earth’s MF. In future investigations dealing with physiological effects of magnetic fields, these methodological parameters as well as an exact description of the physical characteristics of the Helmholtz coils are indispensible if comparisons between reports are to be made. In conclusion, we feel that artificial, earth’s strength static magnetic fields do not affect the pineal gland of mammals due to their presence, either directly or indirectly. Rather, eddy currents, produced by rapid (in the low msec range) rises and decays of MFs are the likely explanation for the response of the gland. The findings reported herein should be considered in the context of studies showing effects of pulsed, weak magnetic fields on intracellular transcription [Goodman et al., 19831 and DNA synthesis [Takahashi et al., 19861, respectively. The authors of both papers assume that certain characteristics of the applied MF, such as pulse width and frequency, determine the observed
“Magnetosensitivity” of the pineal gland
effects. Hence, future investigations dealing with MF-induced alterations in the pineal gland have to focus on those parameters as well. A recent report by Matanoski et al. [ 19891 proposed that “spikey” (high dB/dt) magnetic fields present in certain telephone company offices may be an important factor in increased risk of prostate and male breast cmcer found in telephone workers. Whether these findings, however, are related to malfunctions of the pineal gland due to these artificial fields remains to be seen. Acknowledgments
This work was supported by NSF grant # DCB 87- 1 1241. We acknowledge the support provided by Dr. W.W. Morgan, this institution. A.L. was supported by a fellowship from the NATO scientific committee through the German Academic Exchange Service, K.O.N. by the CNPq from Brazil. Literature Cited AKMSTRONG, S.M. (1989) Melatonin and circadian control in mammals. Experientia 45:932-938. CALLE,E.E., D.A. SAVITZ(1985) Leukemia in occupational groups with presumed exposure to electrical and magnetic fields. N. Engl. J. Med. 313:147&1477. CHAMPNEY, T.H., A.P. HOLTORF,R.W. STEGER,R.J. REITER (1984) Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland. J. Neurosci. Res. 11:5946. COLEMAN, M., J. BELL,R. SKEET(1983) Leukemia incidence in electrical workers. Lancet 1:982-983. DEGUCHI, T., J. AXELROD (1972a) Control of circadian change of serotonin N-acetyltransferase in the pineal organ by the P-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 69:2547-2550. DEGUCHI,T., J. AXELROD(1972b) Sensitive assay for serotonin N-acetyltransferase activity in rat pineal gland. Anal. Biochem. 50:174-179. FRASER, S., P. COWEN,M. FRANKLIN, C. FRANEY, J. ARENDT (1983) Direct radioimmunoassay for melatonin in plasma. Clin. Chem. 29:396397. GEDDES, L.A., L.E. BAKER(1967) The specific resistance of biological material-a compendium of data for the biomedical engineer and physiologist. Med. Biol. Eng. 5:271-293. GOODMAN, R., C.A. BASSET,A.S. HENDERSON (1983) Pulsing electromagnetic fields induce cellular transcription. Science 220: 1283-1285. HILL,S.M., D.E. BLASK(1988) Effects of the pineal hormone melatonin on the proliferation and morphological characteristics of human breast cancer cells (MCF-7) in culture. Cancer Res. 48:612 1-6 126. HOFFMANN, K., H. ILLNEROVA, J. VANECEK (1985) Comparison of pineal melatonin rhythms in young, adult and old Djungarian hamsters (Phodopus sungorus) under long and short photoperiods. Neurosci. Lett. 56:39-53. H., P. ZVOLSKY, J. VANECEK (1985) The circadian ILLNEROVA, rhythm in plasma melatonin concentration of the urbanized man: The effet of summer and winter time. Brain Res. 328: 186-1 89.
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L., D. DANFORTH,A. LICHTER,E. DEMOSS,M. TAMARKIN, COHEN,B. CHABNER, M. LIPPMAN(1982) Decreased nocturnal plasma melatonin peak in patients with estrogen receptorpositive breast cancer. Science 216:1003-1005. WATSON,J., E.M. DOWNES(1979) Clinical aspects of the stimulation of bone healing using electrical phenomena. Med. Biol. Eng. Comput. 17:161-169. WEBLEY,G.E., H. MEHL,K.P. WILLEY(1985) Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species. J. Endocrinol. 106:387-394. WELKER,H . A . , P. SEMM,R.P. WILLIG,J.C. COMMENTZ, W. WILTSCHKO,L. VOLLRATH(1983) Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content in the rat pineal gland. Exp. Brain Res. 50:426-432. WILSON,B.W., R.G. STEVENS,L.E. ANDERSON (1989) Neuroendocrine mediated effects of electromagnetic-field exposure: Possible role of the pineal gland. Life Sci. 4.513191332. WITHYACHNUMNARNKUL, B., K.O. NONAKA,C. SANTANA, M.A. ATTIA,R.J. REITER(1990) Interferon-gamma modulates melatonin production in rat pineal glands in organ culture. J. Interferon Res., 10:403411. WURTMAN, R.J., J. AXELROD,L.S. PHILLIPS(1963) Melatonin synthesis in the rat pineal gland: Control by light. Science 142:1071-1073.
