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Magnetic resonance imaging (MRI) effects on rat pineal neuroendocrine function
294
Brain Re,~ear~k, 51)f~ (i99t)) 2!)4-29~ Elsevier
BRES 23887
Magnetic resonance imaging (MRi) effects on rat pineal neuroendocrine function Robert LaPorte, Laura Kus, Robert A. Wisniewski, M. Margaret Prechel, Behrooz Azar'Kia and John A. McNulty Departments of Anatomy, Biochemistry and Radiology, Loyola University Stritch School of Medicine Maywood, 1L 60153 (U.S.A.)
(Accepted 12 September 1989) Key words: Pineal gland; Magnetic resonance imaging; Indole; Isoproterenol
This study investigated the effects of MR1 on receptor-mediated activation of pineal gland indole biosynthesis. Exposure of rats to MRI reduced the effects of isoproterenol on pineal serotonin and N-acetylserotonin levels suggesting that strong magnetic fields and/or radio-frequency pulsing used in MRI inhibited beta-adrenergic activation of the gland. There was no effect of MRI on saline controls. There is appropriate interest regarding the biological effects of strong magnetic fields (MF) and radio-frequency (RF) pulsing associated with MRI devices that are being used for clinical imaging. This investigation sought to examine whether exposure to MRI could alter receptor-mediated neuroendocrine functions of the pineal gland. Several studies have shown that electrical activity of the pineal gland and pineal synthesis of the hormone, melatonin, are influenced by weak MF and electric fields 7-1°'13'15-17. The pineal gland exhibits a robust circadian rhythm in the production of melatonin, which is synthesized by a 2-step enzymatic pathway. First, serotonin (5-HT) is converted by N-acetylserotonin (NAS) by the N-acetyltransferase (NAT) enzyme. Then, NAS is O-methylated by the hydroxyindole-O-methyltransferase (HIOMT) enzyme to form melatonin. The nocturnal activation of this pathway is initiated by the release of norepinephrine from sympathetic neurons. This reaction is rapidly interrupted by exposure to light at night 5, and can be activated by the fl-adrenergic agonist isoproterenol 4. Adult, male Sprague-Dawley rats (Sasco/King) were kept for 3 weeks in an accredited animal research facility with a regular 12 h light/12 h dark photoperiod (lights on at 06.00 h) and food and water ad libitum. Animals were transported during the dark phase (22.00-01.00 h) in enclosed boxes to the MRI Center where they were removed from the boxes and anesthetized with chloral hydrate (400 mg/kg i.p.). Preparation and sacrifice of
animals placed in the MRI scanner were carried out in an adjacent room. The MF in this room was approximately 10 Gauss. Animals not exposed to the MRI were kept in a nearby room where the MF was measured at less than 1 Gauss (earth-strength MF ranges between 0.3 and 0.7 Gauss). Half of the animals in each of these two groups were injected with isoproterenol (2.5 mg/kg i.p.); the other half received saline. Animals subjected to the M R I were situated 6 across in a 28.0 cm transmit-and-receive radio-frequency head coil, that was placed in the M R I scanner (General Electric SIGNA). Within the scanner, the MF was unidirectional, static and measured regularly to be 1.5 Tesla (T) (T = 10,000 Gauss). Rats were exposed to radio-frequency (RF) pulsing sequences as would normally be performed in a routine head scan of an infant, which takes about 45 rain. The series of image planes sectioned consisted of sagittals, axials, and coronals. Maximum specific absorption rates (SAR) for each series of scans were calculated assuming that all transmitted power was absorbed in either the rats or the head coil. Series 1 consisted of nine 90 ° and nine t80 ° pulses per period for a whole body SAR of 0.6 W/kg. Series 2 consisted of nineteen 90 ° and thirty-eight 180 ° pulses per period for a whole body average SAR of 0.8 W/kg. Series 3 consisted of fifteen 90 ° and thirty 180° pulses per period for a whole body average SAR of 0.6 W/kg. Because the RF power deposition is primarily due to the RF magnetic field, the spatial peak SAR would be approximately twice
Correspondence: J.A. McNulty, Department of Anatomy, Loyola University Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
295 the whole body SAR 3. An estimate of the temperature rise shows that series 2 might have produced a spatial peak heating as high as 0.2 °C per number of excitations. However, the actual temperature rise in the pineal gland would probably be insignificant due to blood flow 2. Calculations of the gradient magnetic fields gave a peak magnetic field of 6.2 Gauss in any of the gradients. The gradient reached this value in 0.6 ms which gives a time rate of change of 1.0 T/s in the region of interest. -Animals were sacrificed by decapitation alternating between groups with the mean time from injection (isoproterenol vs saline) to sacrifice ranging between 54.5 and 57.5 min for all groups. Pineal glands were rapidly removed, placed on dry-ice and stored at -70 °C for analysis of monoamines by high-performance liquid chromatography (HPLC) as previously described 5. Briefly, glands were homogenized in phosphate buffer (pH 7.0), centrifuged, and aliquots of the supernatant injected into the chromatograph. Separation of compounds was by reverse-phase (Cl8; 10 × 4.6 cm, 3/~m particle size) using a solvent system consisting of 0.1 M sodium phosphate (pH 4.5) mixed with 20% (v/v) methanol. Indoles were measured electrochemically with a glassy-carbon electrode set at +0.80 V relative to the reference electrode (Ag/.AgC1). Identification was based on synthetic standards injected daily. Statistical analysis of the data was by one-way analysis of variance followed by the Tukey-Kramer multiple comparisons test to determine significance among individual groups. For reasons of consistency in statistical treatment of all the data, the statistical analysis of N_AS and melatonin, which were not detectable in individual (unstimulated) glands injected with saline, included substituted values based on pooled daytime glands 6. We justified this based on our findings that pineal 5-HT levels in the saline groups were comparable to those of daytime (unstimulated) glands 6. (Student's t-test applied to the isoproterenol group gave a P < 0.05.) -Animals given isoproterenol exhibited the expected decline in pineal 5-HT levels and elevation in N.AS and melatonin levels ( A N O V A P < 0.05) resulting from fl-adrenergic activation of the rate-limiting NAT enzyme 4. This effect was partially reduced by exposure of rats to the MRI (Fig. 1A,B). Elevated levels of 5-HT and decreased levels of N.AS in glands exposed to the MRI suggest that the effect of the MRI was expressed on the NAT enzyme. Probably because of the short interval between isoproterenol injection and sacrifice, this effect of MRI was not observed on levels of melatonin, which requires a second enzymatic reaction involving hydroxyindole-O-methylation of NAS. Mean (+S,E.M.) levels of melatonin (ng//xg protein) in the isoproterenol stimulated glands were 0.006 (+0.002) and 0.007 (+0.003) for
animals subjected to normal magnetic fields and the MRI, respectively. The present findings indicate that transient receptormediated neuroendocrine effects occur under strong MF combined with RF pulsing currently being applied for clinical imaging using MRI. Our results are consistent with several earlier studies showing a decline in pineal NAT activity and melatonin content following weaker (5-120/~T) MF alterations s-l°'lS. On the other hand, no effect on pineal indole metabolism was observed after exposure of rats to a NMR-strength MF of 0.14 T for 30 min 11. This discrepancy may be related to differences in intensity of the MF (the present study exposed rats to a MF of 1.5 T for 45 min) and to RF pulses used for MRI. RF radiation has been shown to have various effects on tissues including both thermal and non-thermal interactions TM. The effects of MRI and weaker MF alterations on pineal indole metabolism are generally expressed at the level of the rate-limiting NAT enzyme 9"t°'ls (this study).
A
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Fig. 1. Means (+S.E.M.) of pineal levels of: (A) 5-HT and (B) NAS in glands of animals injected with either saline (SAL) or isoproterenol (ISO) and subsequently subjected to normal magnetic fields (clear bars) or the MRI (hatched bars). Tukey-Kramer multiple comparison analysis revealed significant differences (P < 0.05) between individual indole groups that do not have superscripts in common. ND = not detectable. * See text for explanation of statistical analyses.
296 These effects could result from interaction of M F and/or R F pulses with receptor mechanisms at the m e m b r a n e or possibly second messengers as suggested by recent evidence that pineal c A M P levels decline after subjecting rats to M F during the dark phase ~2. The pineal's
cellular mechanisms related to neurotransduction and endocrine functions.
magnetosensitivity makes this organ a potentially important model for studies of the effects of M R I on other
We thank Dr. E.J. Neafsey for assistance with the statistical analysis and Dr. Daniel J. Schaefer (GE) for the calculations of the oscillating components of the electromagnetic fields. This work was partially supported by NSF (BNS-8801726) and a grant from the Department of Radiology.
1 Adey, W.R., Tissue interactions with non-ionizing electromagnetic fields, Physiol. Rev., 61 (1981) 435-583. 2 Athey, T.W., A model of the temperature rise in the head due to magnetic resonance imaging procedures, Magn. Res. Med., 9 (1989) 177-184. 3 Bottomley, P.A. and Andrew, E.R., RF magnetic field penetration, phase shift and power dissipation in biological tissue: implications for NMR imaging, Phys. Med. Biol., 23 (1978) 630. 4 Deguchi, T. and Axelrod, J., Control of circadian changes of serotonin N-acetyltransferase activity in the pineal organ by the fl-adrenergic receptor, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 2547-2550. 5 McNulty, J.A., Prechel, M.M. and Simmons, W.H., Correlations of serotonin and its metabolites in individual rat pineal glands over light:dark cycles and after acute light exposure, Life Sci., 39 (1986) 1-6. 6 Mefford, I.N., Chang, P., Klein, D.C., Namhoodiri, M.A.A., Sugden, D. and Barchas, J., Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal gland, Endocrinology, 113 (1983) 1582-1586. 7 Miline, J., Bajic, M. and Brakus, V., Morphodynamic reactive response of the pineal gland of rats chronically exposed to stable strong magnetic field, Neuroscience, 26 (1988) 1083-1092. 8 Olcese, J. and Reuss, S., Magnetic field effects on pineal gland melatonin synthesis: comparative studies on albino and pigmented rodents, Brain Research, 369 (1986) 365-368. 9 Olcese, J., Reuss, S. and Vollrath, L., Evidence for the involvement of the visual system in mediating magnetic field effects on pineal melatonin synthesis in the rat, Brain Research,
333 (1985) 382-384. 10 Reuss, S. and Olcese, J., Magnetic field effects on the rat pineal gland: role of retinal activation by light, Neurosci. Lett., 64 (1986) 97-101. 11 Reuss, S., Olcese, J., Vollrath, L., Skalej, M. and Meres, M., Lack of effect of NMR-strength magnetic fields on rat pineal melatonin synthesis, IRCS Med. Sci., 13 (1985) 471. 12 Rudolph, K., Wirz-Justice, A., Krauchi, K. and Feer, H., Static magnetic fields decrease nocturnal pineal cAMP in the rat, Brain Research, 446 (1988) 159-160. 13 Semm, P., Schneider, T. and VoUrath, L., Effects of an earth-strength magnetic field on electrical activity of pineal cells, Nature (Lond.), 288 (1980) 607-608. 14 Tenforde, T.S. and Budinger, T.E, Biological effects and physical safety aspects of NMR imaging and in vivo spectroscopy. In S.R. Thomas and R.L. Dixon (Eds.), NMR in Medicine. The Instrumentation and Clinical Applications, Am. Inst. of Physics, 1986, pp. 493-548. 15 Welker, H.A., Semm, P., Willig, R.P., Commentz, J.C., Wiltschko, W. and Vollrath, L., Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland, Exp. Brain Res., 50 (1983) 426-432. 16 Wilson, B.W., Anderson, L.E., Hilton, D.I. and Phillips, R.D., Chronic exposure to 60-Hz electric fields: effects on pineal function in the rat, Bioelectromagnetics, 2 (1981) 371-380. 17 Wilson, B.W., Chess, E.K. and Anderson, L.E., 60-Hz electricfield effects on pineal melatonin rhythms: time course for onset and recovery, Bioelectromagnetics, 7 (1986) 239-242.
Brain Re,~ear~k, 51)f~ (i99t)) 2!)4-29~ Elsevier
BRES 23887
Magnetic resonance imaging (MRi) effects on rat pineal neuroendocrine function Robert LaPorte, Laura Kus, Robert A. Wisniewski, M. Margaret Prechel, Behrooz Azar'Kia and John A. McNulty Departments of Anatomy, Biochemistry and Radiology, Loyola University Stritch School of Medicine Maywood, 1L 60153 (U.S.A.)
(Accepted 12 September 1989) Key words: Pineal gland; Magnetic resonance imaging; Indole; Isoproterenol
This study investigated the effects of MR1 on receptor-mediated activation of pineal gland indole biosynthesis. Exposure of rats to MRI reduced the effects of isoproterenol on pineal serotonin and N-acetylserotonin levels suggesting that strong magnetic fields and/or radio-frequency pulsing used in MRI inhibited beta-adrenergic activation of the gland. There was no effect of MRI on saline controls. There is appropriate interest regarding the biological effects of strong magnetic fields (MF) and radio-frequency (RF) pulsing associated with MRI devices that are being used for clinical imaging. This investigation sought to examine whether exposure to MRI could alter receptor-mediated neuroendocrine functions of the pineal gland. Several studies have shown that electrical activity of the pineal gland and pineal synthesis of the hormone, melatonin, are influenced by weak MF and electric fields 7-1°'13'15-17. The pineal gland exhibits a robust circadian rhythm in the production of melatonin, which is synthesized by a 2-step enzymatic pathway. First, serotonin (5-HT) is converted by N-acetylserotonin (NAS) by the N-acetyltransferase (NAT) enzyme. Then, NAS is O-methylated by the hydroxyindole-O-methyltransferase (HIOMT) enzyme to form melatonin. The nocturnal activation of this pathway is initiated by the release of norepinephrine from sympathetic neurons. This reaction is rapidly interrupted by exposure to light at night 5, and can be activated by the fl-adrenergic agonist isoproterenol 4. Adult, male Sprague-Dawley rats (Sasco/King) were kept for 3 weeks in an accredited animal research facility with a regular 12 h light/12 h dark photoperiod (lights on at 06.00 h) and food and water ad libitum. Animals were transported during the dark phase (22.00-01.00 h) in enclosed boxes to the MRI Center where they were removed from the boxes and anesthetized with chloral hydrate (400 mg/kg i.p.). Preparation and sacrifice of
animals placed in the MRI scanner were carried out in an adjacent room. The MF in this room was approximately 10 Gauss. Animals not exposed to the MRI were kept in a nearby room where the MF was measured at less than 1 Gauss (earth-strength MF ranges between 0.3 and 0.7 Gauss). Half of the animals in each of these two groups were injected with isoproterenol (2.5 mg/kg i.p.); the other half received saline. Animals subjected to the M R I were situated 6 across in a 28.0 cm transmit-and-receive radio-frequency head coil, that was placed in the M R I scanner (General Electric SIGNA). Within the scanner, the MF was unidirectional, static and measured regularly to be 1.5 Tesla (T) (T = 10,000 Gauss). Rats were exposed to radio-frequency (RF) pulsing sequences as would normally be performed in a routine head scan of an infant, which takes about 45 rain. The series of image planes sectioned consisted of sagittals, axials, and coronals. Maximum specific absorption rates (SAR) for each series of scans were calculated assuming that all transmitted power was absorbed in either the rats or the head coil. Series 1 consisted of nine 90 ° and nine t80 ° pulses per period for a whole body SAR of 0.6 W/kg. Series 2 consisted of nineteen 90 ° and thirty-eight 180 ° pulses per period for a whole body average SAR of 0.8 W/kg. Series 3 consisted of fifteen 90 ° and thirty 180° pulses per period for a whole body average SAR of 0.6 W/kg. Because the RF power deposition is primarily due to the RF magnetic field, the spatial peak SAR would be approximately twice
Correspondence: J.A. McNulty, Department of Anatomy, Loyola University Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
295 the whole body SAR 3. An estimate of the temperature rise shows that series 2 might have produced a spatial peak heating as high as 0.2 °C per number of excitations. However, the actual temperature rise in the pineal gland would probably be insignificant due to blood flow 2. Calculations of the gradient magnetic fields gave a peak magnetic field of 6.2 Gauss in any of the gradients. The gradient reached this value in 0.6 ms which gives a time rate of change of 1.0 T/s in the region of interest. -Animals were sacrificed by decapitation alternating between groups with the mean time from injection (isoproterenol vs saline) to sacrifice ranging between 54.5 and 57.5 min for all groups. Pineal glands were rapidly removed, placed on dry-ice and stored at -70 °C for analysis of monoamines by high-performance liquid chromatography (HPLC) as previously described 5. Briefly, glands were homogenized in phosphate buffer (pH 7.0), centrifuged, and aliquots of the supernatant injected into the chromatograph. Separation of compounds was by reverse-phase (Cl8; 10 × 4.6 cm, 3/~m particle size) using a solvent system consisting of 0.1 M sodium phosphate (pH 4.5) mixed with 20% (v/v) methanol. Indoles were measured electrochemically with a glassy-carbon electrode set at +0.80 V relative to the reference electrode (Ag/.AgC1). Identification was based on synthetic standards injected daily. Statistical analysis of the data was by one-way analysis of variance followed by the Tukey-Kramer multiple comparisons test to determine significance among individual groups. For reasons of consistency in statistical treatment of all the data, the statistical analysis of N_AS and melatonin, which were not detectable in individual (unstimulated) glands injected with saline, included substituted values based on pooled daytime glands 6. We justified this based on our findings that pineal 5-HT levels in the saline groups were comparable to those of daytime (unstimulated) glands 6. (Student's t-test applied to the isoproterenol group gave a P < 0.05.) -Animals given isoproterenol exhibited the expected decline in pineal 5-HT levels and elevation in N.AS and melatonin levels ( A N O V A P < 0.05) resulting from fl-adrenergic activation of the rate-limiting NAT enzyme 4. This effect was partially reduced by exposure of rats to the MRI (Fig. 1A,B). Elevated levels of 5-HT and decreased levels of N.AS in glands exposed to the MRI suggest that the effect of the MRI was expressed on the NAT enzyme. Probably because of the short interval between isoproterenol injection and sacrifice, this effect of MRI was not observed on levels of melatonin, which requires a second enzymatic reaction involving hydroxyindole-O-methylation of NAS. Mean (+S,E.M.) levels of melatonin (ng//xg protein) in the isoproterenol stimulated glands were 0.006 (+0.002) and 0.007 (+0.003) for
animals subjected to normal magnetic fields and the MRI, respectively. The present findings indicate that transient receptormediated neuroendocrine effects occur under strong MF combined with RF pulsing currently being applied for clinical imaging using MRI. Our results are consistent with several earlier studies showing a decline in pineal NAT activity and melatonin content following weaker (5-120/~T) MF alterations s-l°'lS. On the other hand, no effect on pineal indole metabolism was observed after exposure of rats to a NMR-strength MF of 0.14 T for 30 min 11. This discrepancy may be related to differences in intensity of the MF (the present study exposed rats to a MF of 1.5 T for 45 min) and to RF pulses used for MRI. RF radiation has been shown to have various effects on tissues including both thermal and non-thermal interactions TM. The effects of MRI and weaker MF alterations on pineal indole metabolism are generally expressed at the level of the rate-limiting NAT enzyme 9"t°'ls (this study).
A
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Q.
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o . •.
-
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B
R,C
o.o..
Y o o6-
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A
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ND~ ND ~o oo,
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ISO
Fig. 1. Means (+S.E.M.) of pineal levels of: (A) 5-HT and (B) NAS in glands of animals injected with either saline (SAL) or isoproterenol (ISO) and subsequently subjected to normal magnetic fields (clear bars) or the MRI (hatched bars). Tukey-Kramer multiple comparison analysis revealed significant differences (P < 0.05) between individual indole groups that do not have superscripts in common. ND = not detectable. * See text for explanation of statistical analyses.
296 These effects could result from interaction of M F and/or R F pulses with receptor mechanisms at the m e m b r a n e or possibly second messengers as suggested by recent evidence that pineal c A M P levels decline after subjecting rats to M F during the dark phase ~2. The pineal's
cellular mechanisms related to neurotransduction and endocrine functions.
magnetosensitivity makes this organ a potentially important model for studies of the effects of M R I on other
We thank Dr. E.J. Neafsey for assistance with the statistical analysis and Dr. Daniel J. Schaefer (GE) for the calculations of the oscillating components of the electromagnetic fields. This work was partially supported by NSF (BNS-8801726) and a grant from the Department of Radiology.
1 Adey, W.R., Tissue interactions with non-ionizing electromagnetic fields, Physiol. Rev., 61 (1981) 435-583. 2 Athey, T.W., A model of the temperature rise in the head due to magnetic resonance imaging procedures, Magn. Res. Med., 9 (1989) 177-184. 3 Bottomley, P.A. and Andrew, E.R., RF magnetic field penetration, phase shift and power dissipation in biological tissue: implications for NMR imaging, Phys. Med. Biol., 23 (1978) 630. 4 Deguchi, T. and Axelrod, J., Control of circadian changes of serotonin N-acetyltransferase activity in the pineal organ by the fl-adrenergic receptor, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 2547-2550. 5 McNulty, J.A., Prechel, M.M. and Simmons, W.H., Correlations of serotonin and its metabolites in individual rat pineal glands over light:dark cycles and after acute light exposure, Life Sci., 39 (1986) 1-6. 6 Mefford, I.N., Chang, P., Klein, D.C., Namhoodiri, M.A.A., Sugden, D. and Barchas, J., Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal gland, Endocrinology, 113 (1983) 1582-1586. 7 Miline, J., Bajic, M. and Brakus, V., Morphodynamic reactive response of the pineal gland of rats chronically exposed to stable strong magnetic field, Neuroscience, 26 (1988) 1083-1092. 8 Olcese, J. and Reuss, S., Magnetic field effects on pineal gland melatonin synthesis: comparative studies on albino and pigmented rodents, Brain Research, 369 (1986) 365-368. 9 Olcese, J., Reuss, S. and Vollrath, L., Evidence for the involvement of the visual system in mediating magnetic field effects on pineal melatonin synthesis in the rat, Brain Research,
333 (1985) 382-384. 10 Reuss, S. and Olcese, J., Magnetic field effects on the rat pineal gland: role of retinal activation by light, Neurosci. Lett., 64 (1986) 97-101. 11 Reuss, S., Olcese, J., Vollrath, L., Skalej, M. and Meres, M., Lack of effect of NMR-strength magnetic fields on rat pineal melatonin synthesis, IRCS Med. Sci., 13 (1985) 471. 12 Rudolph, K., Wirz-Justice, A., Krauchi, K. and Feer, H., Static magnetic fields decrease nocturnal pineal cAMP in the rat, Brain Research, 446 (1988) 159-160. 13 Semm, P., Schneider, T. and VoUrath, L., Effects of an earth-strength magnetic field on electrical activity of pineal cells, Nature (Lond.), 288 (1980) 607-608. 14 Tenforde, T.S. and Budinger, T.E, Biological effects and physical safety aspects of NMR imaging and in vivo spectroscopy. In S.R. Thomas and R.L. Dixon (Eds.), NMR in Medicine. The Instrumentation and Clinical Applications, Am. Inst. of Physics, 1986, pp. 493-548. 15 Welker, H.A., Semm, P., Willig, R.P., Commentz, J.C., Wiltschko, W. and Vollrath, L., Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland, Exp. Brain Res., 50 (1983) 426-432. 16 Wilson, B.W., Anderson, L.E., Hilton, D.I. and Phillips, R.D., Chronic exposure to 60-Hz electric fields: effects on pineal function in the rat, Bioelectromagnetics, 2 (1981) 371-380. 17 Wilson, B.W., Chess, E.K. and Anderson, L.E., 60-Hz electricfield effects on pineal melatonin rhythms: time course for onset and recovery, Bioelectromagnetics, 7 (1986) 239-242.