Static and extremely low frequency electromagnetic field exposure: Reported effects on the circadian production of melatonin

The circadian rhythm of melatonin production (high melatonin levels at night and low during the day) in the mammalian pineal gland is modified by visible portions of the electromagnetic spectrum, i.e., light, and reportedly by extremely low frequency (ELF) electromagnetic fields as well as by static magnetic field exposure. Both light and non-visible electromagnetic field exposure at night depress the conversion of serotonin (5HT) to melatonin within the pineal gland. Several reports over the last decade showed that the chronic exposure of rats to a 60 Hz electric field, over a range of field strengths, severely attenuated the nighttime rise in pineal melatonin production; however, more recent studies have not confirmed this initial observation. Sinusoidal magnetic field exposure also has been shown to interfere with the nocturnal melatonin forming ability of the pineal gland although the number of studies using these field exposures is small. On the other hand, static magnetic fields have been repeatedly shown to perturb the circadian melatonin rhythm. The field strengths in these studies were almost always in the geomagnetic range (0.2 to 0.7 Gauss or 20 to 70 μtesla) and most often the experimental animals were subjected either to a partial rotation or to a total inversion of the horizontal component of the geomagnetic field. These experiments showed that several parameters in the indole cascade in the pineal gland are modified by these field exposures; thus, pineal cyclic AMP levels, N-acetyltransferase (NAT) activity (the rate limiting enzyme in pineal melatonin production), hydroxyindole-O-methyltransferase (HIOMT) activity (the melatonin forming enzyme), and pineal and blood melatonin concentrations were depressed in various studies. Likewise, increases in pineal levels of 5HT and 5-hydroxyindole acetic acid (5HIAA) were also seen in these glands; these increases are consistent with a depressed melatonin synthesis. The mechanisms whereby non-visible electromagnetic fields influence the melatonin forming ability of the pineal gland remain unknown; however, the retinas in particular have been theorized to serve as magnetoreceptors with the altered melatonin cycle being a consequence of a disturbance in the neural biological clock, i.e., the suprachiasmatic nuclei (SCN) of the hypothalamus, which generates the circadian melatonin rhythm. The disturbances in pineal melatonin production induced by either light exposure or non-visible electromagnetic field exposure at night appear to be the same but whether the underlying mechanisms are similar remains unknown.     

Electric field exposure alters serum melatonin but not pineal melatonin synthesis in male rats

Sprague-Dawley male rats, maintained in a 14:10 h light:dark cycl were exposed for 30 days (starting at 56 days of age) to a 65 kV/m, 60 Hz electric field or to a sham field for 20 h/day beginning at dark onset. Pineal N-acetyltransferase (NAT), hydroxy-indole-o-methyl transferase (HIOMT), and melatonin as well as serum melatonin were assayed. Preliminary data on unexposed animals indicated that samples obtained 4 h into the dark period would reveal either a phase delay or depression in circadian melatonin synthesis and secretion. Exposure to electric fields for 30 days did not alter the expected nighttime increase in pineal NAT, HIOMT, or melatonin. Serum melatonin levels were also increased at night, but the electric field-exposed animals had lower levels than the sham-exposed animals. Concurrent exposure to red light and the electric field or exposure to the electric field at a different time of the day-night period did not reduce melatonin synthesis. These data do not support the hypothesis that chronic electric field exposure reduces pineal melatonin synthesis in young adult male rats. However, serum melatonin levels were reduced by electric field exposure, suggesting the possibility that degradation or tissue uptake of melatonin is stimulated by exposure to electric fields. © 1994 Wiley-Liss, Inc.     

The organochlorine insecticide 1,2,3,4,5,6-hexachlorocyclohexane (lindane) but not 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) augments the nocturnal increase in pineal N-acetyltransferase activity and pineal and serum melatonin levels

The effect of organochlorine insecticides lindane (1,2,3,4,5,6-hexachlorocyclohexane) and DDT (1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane) were studied in terms of their effects on the rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during the day (2000h) and at night (2300 and 0100h). Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Nocturnal NAT activity was increased after lindane administration; likewise, lindane augmented pineal and serum melatonin levels at 2300h. Conversely, DDT was without a statistically significant effect on either NAT activity or on pineal or serum melatonin levels. Neither lindane nor DDT significantly influenced pineal HIOMT values either during the day or at night. Likewise, neither insecticide consistently influenced pineal levels of either 5-HTP, 5-HT or 5-HIAA. The results indicate that the organochlorine insecticide, lindane, modifies pineal melatonin synthesis in vivo.     

Age-related differences in serum melatonin and pineal NAT activity and in the response of rat pineal to a 50-Hz magnetic field.

In a previous study we have shown that exposure to a 50-Hz sinusoidal magnetic field decreased serum melatonin concentration and pineal enzyme activities in young rats (9 weeks). In the present study we looked for the effect of a magnetic field of 100 microT on serum melatonin and pineal NAT activity in aged rats and compared them to young rats. We hypothesized that aging may change sensitivity of rats to a magnetic field. Two groups of Wistar male rats [aged rats (23 months) and young rats (9 weeks)] were exposed to 50-Hz magnetic fields of 100 microT for one week (18h/day). The animals were kept under a standard 12:12 light: dark cycle with a temperature of 25 degrees C and a relative humidity of 45 to 50%. Control (sham-exposed) animals were kept in a similar environment but without exposure to a magnetic field. The animals were sacrificed under red dim light. Serum melatonin concentration and pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities were studied. Our results showed that sinusoidal magnetic fields altered the production of melatonin (28% decrease; P <0.05) through an inhibition of pineal NAT activity (52% decrease; P <0.05) in the young rats whereas no effect was observed in aged ones. On the other hand, when comparing data from control animals between young and aged rats, we observed that serum melatonin level and NAT activity, but not HIOMT activity, decreased in aged rats (decrease by about 38% and 36% respectively). Our data strongly suggest that old rats are insensitive to the magnetic field.     

Two-phase response of rat pineal melatonin to lethal whole-body irradiation with gamma rays.

Male Wistar rats adapted to artificial light:dark (LD) regimen 12:12 h were whole-body irradiated with a single dose of 9.6 Gy of gamma rays and sham/irradiated in the night in darkness. The rats were examined 60 min, 1, 3 and 5 days after exposure between 22:00 and 01:30 h in the darkness. The results obtained indicate a two-phase reaction of pineal melatonin after the lethal irradiation of rats: the decline of melatonin concentration early after the exposure (at 60 min) with unchanged serotonin N-acetyltransferase (NAT) activity followed by an increase of melatonin synthesis, accompanied by an increase of pineal and serum melatonin on day 5 after the exposure. NAT activity was increased on day 3 after the exposure. Serum corticosterone concentrations in irradiated rats were increased 60 min and 3 days after exposure. With respect to the antioxidant, immunomodulating and stress-diminishing properties of melatonin, we consider the increase in melatonin synthesis during later periods after irradiation as part of adaptation of the organism to overcome radiation stress.     

Elevated daytime rat pineal and serum melatonin levels induced by isoproterenol are depressed by swimming

Isoproterenol (1 mg/kg) was subcutaneously injected into adult male rats during the day to stimulate pineal N-acetyltransferase (NAT) activity and pineal and serum melatonin levels. Two hours after isoproterenol administration when levels of each of these variables had increased significantly, the experimental animals swam for 10 min in 22 degrees C water. At 15 min after swimming onset, pineal and serum melatonin levels were highly significantly depressed compared to those in control animals that did not swim. The high NAT level was not influenced by swimming. In a second study, isoproterenol injected rats swam for either 1, 3, 6 or 10 min and were sampled 15 min after the onset of swimming. The reduction in the elevated pineal melatonin in these animals was correlated with the length of the swim, i.e., as the duration of swim increased the percent reduction in pineal melatonin also increased. Neither pineal NAT nor hydroxyindole-O-methyltransferase (HIOMT) activities were influenced by swimming. The results suggest that elevated pineal and serum melatonin induced by isoproterenol can be depressed with no effect on the activity of the enzymes which convert serotonin to melatonin.     

A comparative study of melatonin production in the retina,pineal gland and Harderian gland of Bufo viridis and Rana esculenta

1. The circadian patterns of melatonin and of its synthesizing enzyme N-acetyltransferase (NAT) were investigated in the serum, retina, pineal gland and Harderian gland (HG) of two amphibian species, Bufo viridis and Rana esculenta.2. Serum melatonin levels showed no diurnal fluctuations in Bufo viridis, whereas, in Rana esculenta, they exhibited a circadian rhythm, with the highest values occurring during the night. Retina melatonin exhibited characteristic circadian patterns in both species, with the highest values occurring during the day, in Bufo, and the highest concentrations occurring at night in Rana.3. In the retina, NAT activity peaked at night in both amphibians, but in Bufo the levels were up to 30 times higher than in Rana. In the HG and in the pineal gland, NAT activity showed different patterns in the two species with no diurnal variations in Bufo, and characteristic circadian rhythms in Rana.4. In the HG and pineal gland of both species, melatonin was only occasionally detectable over the 24-hr period.5. This is the first report exploring melatonin production in Bufo viridis and Rana esculenta. In our experimental conditions, marked differences emerged between the two species.     

Swimming-induced suppression of rat pineal melatonin is prevented by pretreatment with calcium channel blockers

Young adult male rats were treated with isoproterenol during the day to induce high levels of pineal N-acetyltransferase (NAT) activity and melatonin. Roughly 2 hr later when pineal NAT activity and melatonin levels were elevated, animals were given either an injection of a calcium channel blocker, i.e., either nifedipine or verapamil, or diluent. The rats were then forced to swim for 10 min in room temperature (22 degrees C) water. Fifteen minutes after swimming onset, pineal glands were collected for measurement of NAT activity and melatonin. Swimming caused a dramatic reduction in pineal melatonin content without influencing NAT activity. Nifedipine substantially and verapamil completely blocked the drop in pineal melatonin levels due to swimming without influencing NAT activity. The results suggest that calcium may be somehow directly or indirectly involved in melatonin release from the rat pineal gland.     

The influence of different light irradiances on pineal N-acetyltransferase activity and melatonin levels in the cotton rat, Sigmodon hispidus

Wild-captured cotton rats (Sigmodon hispidus) trapped and tested in September and October exhibited a rapid reduction in pineal N-acetyltransferase (NAT) activity and melatonin levels after exposure to a light irradiance of 300 ωW/cm² during the dark period. The half-time for the depression of both NAT and melatonin was on the order of 2 min. The exposure of cotton rats during darkness to much lower irradiances of light, i.e., 5.0, 0.04, 0.03 or 0.01 W/cm², for 32 min also greatly diminished pineal NAT activity and radioimmunoassayable melatonin levels; however, a light irradiance of 0.005 ωW/cm² failed to significantly depress either the acetylating enzyme or the melatonin content of the pineal gland. The results show that the pineal gland of the wild-captured cotton rat, as judged by NAT activity and melatonin levels, is inhibited even by very low irradiances of light.     

Occupational magnetic field exposure and melatonin: interaction with light-at-night

The evidence of magnetic field (MF) effects on melatonin production in humans is limited and inconsistent. Part of the inconsistencies might be explained by findings suggesting interaction with light in pineal responses to MFs. To test this hypothesis, we reanalyzed data from a previously published study on 6-hydroxy melatonin sulfate (6-OHMS) excretion in women occupationally exposed to extremely low-frequency MFs. Based on questionnaire data on exposure to light-at-night (LAN), and measurement-based MF data, the 60 women were classified to four groups: no MF, no LAN; MF, no LAN; no MF, LAN; MF, LAN. The lowest excretion of 6-OHMS was observed in the group of women who were exposed to both MF and LAN, and the differences between the four groups were significant (P < .0001). The result is based on low numbers, but supports the hypothesis that daytime occupational exposure to MF enhances the effects of nighttime light exposure on melatonin production.     

Effects of exposure to a circularly polarized 50-Hz magnetic field on plasma and pineal melatonin levels in rats

We sought to determine whether a 6-week exposure to a 50-Hz rotating magnetic field influences melatonin synthesis by 11–18 week-old Wistar-King male rats. Rats were exposed continuously to a rotating magnetic field at 1, 5, 50, or 250 μT (spatial vector rms) for 6 weeks, except for twice-weekly breaks of about 2 h for cleaning of cages and feeding. The rats were housed in exposure and sham-exposure facilities, which were located in the same room, under a 12:12 light-dark photoperiod (lights on at 06:00 h). The room was constantly illuminated by 4 small, dim red lights (< 0.07 lux in dark period). Levels of plasma and pineal gland melatonin were determined by radioimmunoassay. A significant decrease of melatonin was observed between the control group and groups exposed to a magnetic field at a flux density in excess of l μT during the night time, but no statistical differences were found among the exposed groups. These results indicate that subchronic exposure of albino rats to a 50-Hz rotating magnetic field influences melatonin production and secretion by the pineal gland. © 1993 Wiley-Liss, Inc.     

Day-night differences in the response of the pineal gland to swimming stress

The effect of swimming stress on pineal N-acetyltransferase activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin content was studied during the day and night in adult male rats. At night, elevated pineal activity was suppressed by light exposure before the animals swam. During the day, swimming for 2 hr did not stimulate NAT activity unless the animals were pretreated with desmethylimipramine (DMI), a norepinephrine uptake blocker. Pineal melatonin content after daytime swimming exhibited a weak rise, unless DMI was injected, in which case melatonin levels showed a highly significant increase. Swimming at night caused a greater (compared to daytime levels) increase in NAT activity in both noninjected and DMI-injected rats. Melatonin levels at night were highly significantly stimulated (compared to daytime values) even without pretreatment of the rats with DMI. The greater response of the rat pineal to swimming stress at night may relate either to an increase in the number of beta-adrenergic receptors in the pinealocyte membrane at night or to a reduced capacity of the sympathetic neurons in the pineal to take up excess circulating catecholamines. Pineal HIOMT activity was not influenced by swimming (with or without DMI) either during the day or at night.     

Locally synthesized angiotensin modulates pineal melatonin generation

We aimed to study the mechanisms and the significance of the influence exerted by the renin-angiotensin system (RAS) on the pineal melatonin production. Pineal melatonin and other indoles were determined by HPLC with electrochemical detection after angiotensin AT1-receptor blockade with Losartan in vivo or in cultured glands. N-acetyltransferase (NAT) activity was radiometricaly measured. To test the in vivo relevance of the local RAS, pineal melatonin and its indole precursors were determined in transgenic rats with inhibited production of angiotensinogen exclusively in astrocytes, TGR(ASrAOGEN). Tryptophan hydroxylase (TPH) and NAT mRNA levels were determined by real-time RT-PCR. Pineal melatonin content was significantly decreased by AT1-receptor blockade in vivo, in cultured glands and in TGR(ASrAOGEN) (35%, 32.4% and 17.5% from control, respectively). Losartan produced a significant decrease of pineal 5-hydroxytryptophan, serotonin, 5-hydroxyindole acetic acid and N-acetylserotonin in pineal cultures. Also, the pineal content of the precursor indoles in TGR(ASrAOGEN) rats was significantly lowered. The reduction of 5-hydroxytryptophan levels by 33-75% in both in vivo and in vitro studies suggests a decreased activity of TPH. Moreover, the TPH mRNA levels in TGR(ASrAOGEN) rats were significantly lower than control rats. On the other hand, NAT activity was unaffected by Losartan in pineal culture and its expression was not significantly different from control in TGR(ASrAOGEN) rats. Our results demonstrate that a local pineal RAS exerts a tonic modulation of indole synthesis by influencing the activity of TPH via AT1-receptors.     

Intra-arterially administered vasopressin inhibits nocturnal pineal melatonin synthesis in the rat

1. In order to investigate the possible involvement of arginine-vasopressin (AVP) in the inhibition of nocturnal pineal melatonin synthesis following electrical stimulation of the hypothalamic paraventricular nuclei, adult male rats received injections of 5 micrograms/100 g body weight of the peptide during either day- or night-time. Following survival times of 30 or 120 min, animals were killed and the activity of the melatonin synthesis enzyme N-acetyltransferase (NAT) was determined. 2. At night, NAT activity was significantly decreased 30 and 120 min following AVP injection. 3. During the daytime, NAT activity was unchanged following AVP administration. 4. It is suggested that pineal melatonin synthesis may be affected by PVN stimulation not only via neural pathways but possibly also by PVN-released blood-borne AVP.     

Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 13-lined ground squirrels (Spermophilus Tridecemlineatus)

Pineal melatonin levels were compared in laboratory-raised or wild-captured 13-lined ground squirrels (Spermophilus tridecemlineatus) that were either exposed to 10 h of darkness at night or to light which had an irradiance of 400 μW/cm². In laboratory-born squirrels the period of darkness was associated with a gradual rise in pineal melatonin levels with peak values being reached at 0200 h, 6 h after darkness onset. Thereafter, melatonin levels decreased and were back to low daytime levels by 0800 h, 2 h after light onset. The exposure of laboratory-raised animals to an irradiance of 400 μW/cm² during the night totally prevented the nocturnal rise in pineal melatonin levels in these animals. In wild-captured ground squirrels the period of darkness at night was associated with a rapid rise in pineal melatonin such that by 2200 h, 2 h after lights out, peak melatonin values were already attained; additionally, melatonin levels remained high throughout the period of darkness but returned to daytime values by 0800 h. Exposure of wild-captured squirrels to a light irradiance of 400 μW/cm² during the normal dark period was completely incapable of suppressing pineal melatonin levels. The difference in the sensitivity of the pineal gland of laboratory-raised and wild-captured ground squirrels may relate to their previous lighting history.     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.     

Electrical stimulation of the hypothalamic nucleus paraventricularis mimics the effects of light on pineal melatonin synthesis

In an attempt to clarify further the role of the hypothalamic paraventricular nuclei (PVN) in the control of pineal function, the effects of 2 min electrical stimulation of these nuclei were investigated in acutely blinded, adult, male Sprague-Dawley rats. Pineal serotonin-N-acetyltransferase (NAT) activity, melatonin content and catecholamine levels were measured by means of radio-enzymatic, radioimmunoassay and high-performance liquid-chromatography methods, respectively. All three pineal parameters underwent significant declines following brief PVN stimulation during the night time. These observations lend credence to the view that the neural pathways transmitting light information to the sympathetic innervation controlling pineal melatonin synthesis.     

Pineal-retinal relationships: rhythmic biosynthesis and immunocytochemical localization of melatonin in the retina of the pike (Esox lucius)

Summary The levels of melatonin and the activities of two enzymes of the melatonin biosynthetic pathway, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were measured throughout the light-dark cycle in the retina of a teleost fish, the pike. HIOMT activity did not display significant variations, whereas NAT activity and melatonin content showed a daily rhythm, high levels occurring during the night. The profiles of the latter two rhythms did not closely match one another and differed from those previously described in the pineal organ of the same species. These results are discussed with respect to a possible paracrine role of retinal melatonin. Melatonin-like immunoreactivity was found in the photoreceptor cell layer and in the Müller cells of the inner nuclear layer. The intensity of the melatonin-like immunoreactivity varied throughout the 24 h light-dark cycle, in good correlation with the variations in the melatonin level as measured by radioimmunoassay.This article is dedicated to the memory of Dr. Klaus Hoffmann     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.