0 1992 MUNKSGAARD
An oral melatonin replacement regimen that re-establishes the normal circadian levels of urinary 6-sulphatoxymelatonin in functionally pinealectomized rats John TM, Brown MC, Brown GM. An oral melatonin replacement regimen that re-establishes the normal circadian levels of urinary 6-sulphatoxymelatonin in functionally pinealectomized rats. J. Pineal Res. 1992: 13: 145-150.
I
Abstract: Wistar rats maintained on a 12-hr daily photoperiod (LD 12:12 cycle) exhibited a diurnal rhythm in urinary 6-sulphatoxymelatonin (aMT6s) concentrations with peak levels in the scotophase. Light-induced functional pinealectomy (FPX) abolished the nocturnal rise in aMT6s, lowering it to photophase levels. The objective of the study was to formulate an oral melatonin replacement regimen that would restore a normal rhythmic output of urinary aMT6s in functionally pinealectomised rats. Three regimens of sequential doses of melatonin were tested. Of these, the regimen with melatonin concentrations of 4 ng, 12 ng, 65 ng, and 4 ng per ml of drinking water given to rats during the lst, 2nd, 3rd, and 4th 3-hr periods, respectively, of the 12-hr FPX phase, was found to generate a urinary aMT6s level that 'losely resembled the natural level and rhythm exhibited under an LD 12:12 cycle. This dose is considered appropriate to restore certain melatonin-mediated physiological functions in Wistar rats subjected to FPX.
Introduction
In most mammals, circulating as well as pineal levels of melatonin exhibit a circadian rhythm with a peak in the night and a trough in the day [Reiter, 19861. It is also known that light has a suppressive effect on melatonin levels [Lewy et al., 1980; Rollag et al., 1980; Reiter, 19831, a feature presumably pivotal in transmitting seasonal and daily photoperiodic information into a neuroendocrine signal. Furthermore, melatonin is believed to be involved in mediating many rhythmic physiological events [Wetterberg, 1978; Arendt and Broadway, 19871, and is also considered to impose synchronicity on a multitude of daily rhythms in cells, tissues, and organs of the internal milieu [Armstrong, 19891. A,major strategy for investigating the role of melatonin has been to administer melatonin to animals in which the endogenous melatonin production has been blocked or curtailed by pinealectomy or other methods, and observe whether this would antagonize the effects produced by the deficiency in
T. Mathew John,' M. Catherine Brown,' and Gregory M. Brown' 'Department of Biomedical Sciences, McMaster University, Hamilton, Canada; *Clarke Institute of Psychiatry, Toronto, Canada
Key words melatonin-6-sulphatoxymelatonin Address reprint requests to Dr Gregory M Brown, The Clarke Institute of Psychiatry, 250 College Street, Toronto, Ontario, Canada M5T 1R8 Received March 2, 1992, accepted July 29, 1992
the endogenous melatonin. Due to the existence of a rhythmic pattern of the circulating melatonin levels in animals living under a normal day-night cycle, it is desirable that in experiments involving melatonin replacement, the concentrations resulting from exogenous melatonin administered should conform to the normal endogenous levels in the same rhythmic pattern as would occur in normal day-night cycle. Most methods of melatonin administration, however, do not comply with these criteria, and the use of such methods often elicits inconsistent and contradictory experimental results. In the present study, we have formulated a regimen of oral dosage of melatonin that would simulate in functionally pinealectomized rats a normal level and rhythm of urinary 6-sulphatoxymelatonin (aMT6s), the principal metabolite of melatonin. The concentration of urinary aMT6s is considered to be a reliable index of melatonin secretion [Arendt et al., 1985; Bojkowski et al., 1987; Fellenberg et al., 1981; Young et al., 1988; Bojkowski and Arendt, 1990; Kennedy et al., 19901 and pineal activity
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John et al.
[Brown et al., 19911. Measurement of urinary aMT6s has definite advantages over serum melatonin measurement in that it circumvents the need for blood sampling in circumstances where blood sampling is difficult [Bojkowski and Arendt, 19901 and could provide a more integrated estimate of total melatonin output during a fixed time period [Kennedy et al., 19901. Materials and methods Animals
Male Wistar rats (200-220 g) were obtained from a local supplier and were individually housed in metabolic stainless steel cages suspended inside a controlled environment cabinet. The floors of the cages were made of stainless steel with mesh, under which a detachable stainless steel funnel was attached when urine collection was necessary. The rats were given drinking water and commercial rat-feed ad libitum, and were acclimated to a 12-hr daily photoperiod and an ambient temperature of 22 2 0.5”C for at least 3 weeks prior to the commencement of the experiment. The rats weighed over 290 g when the experiments were conducted. Lighting was provided by “Cool White” fluorescent bulbs which supplied an illumination of approximately 110pW/cm2 (as per reading on an Opikon radiometer with broad spectrum head and infra-red filter). Red photosafe bulbs were used to allow visualization while carrying out testing procedures in the dark. In rats maintained under similar conditions, distinct circadian rhythms of pineal and serum melatonin levels [Ho et al., 1984; 19851 as well as of urinary aMT6s [Brown et al., 19911 have been observed previously in this laboratory. Experimental procedure
Experiment 1: This experiment, using 12 rats maintained on LD 12:12 cycle (lights on at 0930 and off at 2130), was intended to verify the 24 hr pattern of baseline urinary aMT6s output. Levels of urinary aMT6s have previously been reported to be low during the photophase, showing no fluctuation between 3 to 12 hr after lights were on, and to be relatively high during the scotophase, peaking between 6 and 12 hr after the onset of darkness [Brown et al., 19911. In the present experiment, the total daytime (12 hr) urine excreted from each rat was collected at the end of the photophase, whereas the night time urine was collected in 3-hr blocks over the 12-hr scotophase. At the end of each collection period, the funnel from beneath the cage bottom was removed and rinsed with 25 ml of distilled water which was consequently added to the urine. 146
After recording the volume of each collected sample, it was centrifuged at 5000g for 20 min. An aliquot of the supernatant was separated and frozen at -20°C until assays were performed. Experiment 2: Twenty three rats were randomly divided into 3 groups comprising seven or eight rats each, and were subjected to light-induced functional pinealectomy (FPX). During FPX, groups 1 and 2 received melatonin through their drinking water, while group 3 served as control, receiving only the vehicfe solution through the drink. Melatonin was given only during the 12-hr FPX period (2130 to 0930), with the concentration of melatonin in the drink being changed after every 3-hr block. Rats in group 1 received drinking water with melatonin concentrations of 100 ng, 300 ng, 1000 ng, and 0 ng per ml during the 1st, 2nd, 3rd, and 4th 3-hr periods of the FPX phase, respectively. Rats in group 2 were given a lesser dose of melatonin, with concentrations of 10, 30, 100, and 0 ng per ml during the 4 periods, respectively. Controls also received fresh drinks every 3 hr. All drink bottles contained 200 ml of fresh drink when replaced at the beginning of each 3-hr period. The urine was harvested at every 3-hr interval during the FPX period and stored for aMT6s assay, as described in experiment 1. Experiment 3: Since the urinary aMT6s values obtained following melatonin replacement using both the regimens in experiment 2 appeared much higher than the normal baseline values, it was considered necessary to repeat the experiment with melatonin doses further lowered. A group of nine rats received melatonin at concentrations of 4, 12, 65, and 4 ng per ml of drink during the four consecutive 3-hr blocks of the FPX phase respectively. A control group of nine rats received vehicle drink. The drink consumed (including spillage if any) by each rat was recorded at the end of each 3hr block. All other procedures including sequential urine collection remained the same as described in experiment 2. Functional pinealectomy (FPX)
In the context of the present study, FPX refers to light-induced suppression of pineal activity during one 12-hr dark period. In rats maintained regularly on an LD 12:12 cycle (lights on at 0930 and off at 2130), the FPX was effected by imposing “daytime” lighting conditions during what would normally be the dark phase (2130 to 0930) of the LD cycle. Assay
The aMT6s radioimmunoassay materials supplied by CIDtech Research Inc. (Mississauga, Ontario,
Physiological melatonin replacement
Canada) were used for the measurement of urinary aMT6s. The assay was based on a modified version [Brown et al., 19911 of the method used by Aldhous and Arendt [ 19881.
3800. 0boselme
3400 - I FPX only (n=8)
3000 - Lz9
2600 -
2200 - E Z
Melatonin
Crystalline melatonin (Lot no. 108F 0646, Sigma Chemical Co., St. Louis, Missouri) was initially dissolved in absolute ethanol and subsequently diluted with water to a final alcohol concentration of 1% (v/v). Melatonin solutions were freshly prepared less than an hour prior to the beginning of the 12-hr functional pinealectomy period.
Data analysis Repeated measures analysis of variance was employed for the statistical evaluation of the results. Newman-Keuls test was used for multiple comparisons, when warranted. P < 0.05 was considered acceptable as significant.
Values under
12 1 2 LD cycle ( n = 1 2 )
,-.
1800-
.c 1?
a v
FPX and MEL replacement (100, 300, 1000. Onq/ml)
(n=7) FPX and MEL replacement ( l o ; 30; 100, Ong/ml)
(n=8)
1 4 0 0 - EZJ FPX and MEL replocement (4; 12;6 5 ; 4flg/ml) (n=9) 1000-
600 -
2
180
d c
1601
5>r
140
=
1201 100
:ji 20
0 Period 1 21 30-OO3Oh
Results
Experiment 1: The existence of a diurnal variation in urinary aMT6s output was confirmed. The lowest level of aMT6s output (3.11 ? 0.79 ng/3 hr) was registered in samples collected during the photophase. The level gradually rose during scotophase, peaking between 6 and 9 hr after the onset of darkness and then falling. The levels at both the 3rd and the 4th quarters of the scotophase were significantly higher than those of the rest of the periods. Data from scotophase are incorporated in Figure 1 . ANOVA results: F = 23.6051; df = 4, 44; P < 0.0001. Experiment 2 (Fig. 1): Rats subjected to FPX showed no scotophase-induced normal rise in urinary aMT6s level under an LD 12:12 cycle (Fig. 1). The functionally pinealectomized rats receiving melatonin doses of 100, 300, 1000, and 0 ng per ml drink or 10, 30, 100, 0 ng per ml drink showed an aMT6s rhythm similar to that exhibited by rats maintained on LD 12:12 cycle, but both regimens of melatonin doses showed a trend to elicit higher urinary aMT6s values than in normal rats maintained under LD 12:12 cycle (Fig. 1). Rats on the higher melatonin dose regimen exhibited higher aMT6s output, showing a peak value of 3366 ng/3 hr (6-9 hr into functional pinealectomy period), which was over 50 times greater than that in normal rats during the corresponding period. ANOVA results: treatment, P < 0.0001; time, P < 0.0001; interaction, P < 0.0001. Experiment 3: As in experiment 2, functional pinealectomy abolished the scotophase-related rise
Period 2
Period 3
Period 4
0030-0330h
0330-0630h
0630-0930h
Fig. I . Effects of functional pinealectomy (FPX) and oral melatonin (MEL) replacement on urinary 6-sulphatoxymelatonin (aMT6s) levels in Wistar rats. MEL was given in drinking water in sequential doses of either 10 ng, 30 ng, 100 ng, and 0 ng/ml (Exp. 2 ) or 100 ng, 300 ng, 1000 ng, and 0 ng/ml (Exp. 2), or 4 ng, 12 ng, 65 ng, and 4 ng/ml (Exp. 3), during the lst, 2nd, 3rd, and 4th 3-hr periods of the FPX phase, respectively. Under normal LD cycle (Exp. l), lights were turned on at 0930 and off at 2130. Under FPX (Exp. 2 and 3), lights were not turned off at 2130. Since aMT6s values under FPX in Experiment 3 were not significantly different from corresponding values in Experiment 2, only those from Experiment 2 are represented on the graph.
in urinary aMT6s observed under LD 12:12 cycle. The urinary aMT6s output in functionally pinealectomized rats receiving a melatonin dose regimen of 4, 12, 65, and 4 ng per ml drink was not significantly different from the baseline values observed under an LD 12:12 cycle (Fig. 1). ANOVA results: treatment, P < 0.0001; time, P < 0.0001; interaction, P < 0.0001. Although the consumption of melatonin drink, in general, showed a trend to be lower than that of vehicle drink (Fig. 2), the difference was not significant (treatment, P > 0.1; time, P < 0.05; interaction, P > 0.01). A trend towards a lower urine output, overall, was also apparent (treatment, P = 0.058; time, P < 0.01; interaction, P < 0.01) in the melatonin-treated rats in comparison to that in the control rats (Fig. 2).
147
John et al. 0-0
.-*
Vehicle control (n=9) Melotonin-treoted (4 , 12. 65, 4 ng/ml) (n=9)
I
2c Period 1
Period 2
Period 3
Period 4
2130-0030h
0030-0330h
0330-0630h
0630-0930h
F ig . 2. Drink consumption (A) and urine output (B) in Wistar rats under functional pinealectomy . Lighting conditions are the same as explained in Fig. 1 . Melatonin sequential dose: 4 ng, 12 ng, 65 ng, and 4 ngiml drink. Values are mean i SEM.
Discussion
The urinary aMT6s output in Wistar rats used in the present study showed a circadian pattern with lower values in the photophase and higher in the latter half of the scotophase. These observations are consistent with an earlier report by Brown et al. [1991] on aMT6s profile in this species. The present study also corroborated their observation that light-induced functional pinealectomy abolished the nocturnal rise in urinary aMT6s, reducing the levels to that seen during the photophase. Over the years, investigators have used several methods for melatonin replacement, including single or multiple injections, subcutaneous implantation of melatonin in beeswax pellets or silastic capsules, and oral feeding. However, the exogenous melatonin thus administered often does not provide a rhythmic circulating level that would mimic the natural rhythm and level of the endogenous melatonin. In order to make proper assessment of the effects of melatonin replacement, it is important that the concentration of exogenous melatonin administered conforms to the normal endogenous basal levels in the same rhythmic pattern as would occur in normal day-night cycle. Failure to achieve such a condition may well be the reason for many of 148
the inconsistencies and contradictory results often reported in the literature following melatonin treatment. It has been proposed that continuous availability of melatonin from implants provides a releasable pool which saturates melatonin receptors, making them insensitive to an acute signal or pulse of melatonin, and subsequently resulting in a perpetual state of “down regulation” [Reiter et al., 198 1 ; Vaughan, 19811. The injection method, on the other hand, provides melatonin for only a limited period following the injection. Oral administrations appear to have an advantage over the other methods in that the dosage can be regulated without the use of any painful injections or surgeries. With oral administration, since the melatonin infusion is somewhat stretched over time, an appropriately timed alteration in oral dosage can bring about a gradual, rather than an abrupt change in the circulating melatonin level, better simulating the natural rhythm. It may be mentioned in this connection that both the absolute duration of the melatonin peak and the circadian timing of the peak have been proposed as crucial parameters in transmitting photoperiodic information [Goldman and Darrow, 1983; Stetson and Watson-Whitmyre, 1986; Goldman and Elliot, 19881. Among the three regimens of sequential dosage tested in the present study, we have found that the regimen with melatonin concentrations of 4 ng, 12 ng, 65 ng, and 4 ng per ml of drink, given to Wistar rats during the lst, 2nd, 3rd, and 4th 3-hr periods, respectively, of the functional pinealectomy phase, would generate an urinary aMT6s level and rhythm that would closely resemble the natural level and rhythm exhibited under an LD 12:12 cycle. Since the concentration of urinary aMT6s is considered to be a reliable index of melatonin secretion [Arendt et al., 1985; Bojkowski et al., 1987; Fellenberg et al., 1981; Young et al., 1988; Bojkowski and Arendt, 1990; Kennedy et al., 19901, a rhythmic pattern in circulating melatonin levels identical to that of urinary aMT6s is also to be expected to occur with those dosage. However, whether the rhythms in both melatonin and aMT6s occur concurrently or with a time-lag is not certain, although in humans it has been reported that changes in urinary aMT6s levels followed that of plasma melatonin levels by 12 to 108 min., depending on the time of the year [Matthews et al., 19911. It may be pointed out here that, although the normal pattern of urinary aMT6s secretion over any 3-hr period may be reproduced by the present oral replacement of melatonin, it is not clear whether the treatment would produce patterns of circulating melatonin precisely like those in normal rats. It has recently been demonstrated in rats that blood melatonin exhibits an episodic pattern with pulses
Physiological melatonin replacement
superimposed on the basal level [Chan et al., 19911. Since rats do not drink continuously, it is possible that there could be episodic fluctuations/short-term pulses in blood melatonin levels, but such fluctuations/pulses need not necessarily synchronize with natural fluctuations/pulses. Since only a urinary metabolite of melatonin is measured in the present study, some concern could be raised as to whether the metabolism of orally administered melatonin is identical to that of melatonin secreted directly into the blood and whether any similarities exhibited in resulting aMT6s levels indeed reflect similarities in blood melatonin levels. However, a major pathway for the metabolism of melatonin being the hydroxylation of melatonin by hepatic microsomal enzymes [Quay, 19741, one could presume that the metabolism of both oral and endogenous melatonin takes place in the liver prior to excretion via the kidney. In a separate experiment (to be reported separately) involving FPX, we have found that melatonin replacement with a sequential dose of 4, 12, 65, and 4 ng per ml drink, when administered as in the present study, nullified the effect of light-induced FPX on the analgesic rhythm in Wistar rats [Bar-Or and Brown, 19891, providing proof that the dose is appropriate to restore this melatonin-mediated function. It may also be mentioned here that oral administration of melatonin has previously been reported to influence reproductive activity in sheep [Kennaway et al., 1982; Arendt et al., 1983; English et al., 1986; Stellflug et al., 1988; Kusakari et al., 19911 and in mice [Hamed et al., 19911. The trend towards a lower urine output observed in melatonin-treated rats than in control rats may be considered as a consequence of the reduced drink consumption apparent in these rats. It is not clear, however, why the consumption of melatonin drink showed a trend to be lower than that of vehicle drink. An aversion to the taste and/or smell of the melatonin drink or an inhibitory effect of melatonin on thirst are some of the possible reasons that may be probed. In recent years, melatonin has been proposed as a powerful chronobiotic, having great potential as a prophylactic and therapeutic agent for stabilizing or re-entraining disturbed internal rhythms, thereby averting or remedying disorders arising from disturbed circadian physiology [Armstrong, 19891. Melatonin administered in inappropriate doses or at an incorrect time of the day may aggravate the desynchrony of internal rhythms and consequently the disease. The melatonin administration regimen that simulated the normal circadian rhythm of urinary aMT6s in rats used in the present study could serve as a model for formulating therapeutic melatonin replacement schedules, but the precise
dosage and the timing of administration may have to be individually designed for each animal species. Acknowledgments These studies were supported in part by the Medical Research Council of Canada. G. M. Brown is an Ontario Mental Health Foundation Research Associate. Thanks are expressed to S. Kashur for assistance with the aMT6s assay.
Literature cited ALDHOUS, M.E., J. ARENDT(1988) Radioimmunoassay for 6-sulphatoxymelatonin in urine, using an iodinated tracer. Ann. Clin. Biochem. 25:298-303. ARENDT,J., C. BOJKOWSKI, C. FRANEY,J. WRIGHT, V. MARKS ( 1985) Immunoassay of 6-hydroxymelatonin sulfate in human plasma and urine: abolition of the 24-hour rhythm with atenolol. J. Clin. Endocrinol. Metab. 60:1166-1173. ARENDT,J., J. BROADWAY (1987) Light and melatonin as zeitgebers in man. Chronobiol. Int. 4:273-283. ARENDT, J., A.M. SYMONS,C.A. LAUD,S.J. PRYDE(1983) Melatonin can induce the early onset of the breeding season in ewes. J. Endocrinol. 97:395400. ARMSTRONG, S.M. (1989) Melatonin: the internal zeitgeber of mammals? Pineal Res. Rev. 7: 157-202. BAR-OR,A,, G.M. BROWN(1989) Pineal involvement in the diurnal rhythm of nociception in the rat. Life Sci., 44:10671075. BOJKOWSKI, C.J., J. ARENDT(1990) Factors influencing urinary 6-sulphatoxymelatonin, a major melatonin metabolite, in normal human subjects. Clin. Endocrinol. 33:435-444. BOJKOWSKI, C.J., J. ARENDT,M.C. SHIH, S.P. MARKEY (1987) Melatonin secretion in humans assessed by measuring its metabolite, 6-sulfatoxymelatonin. Clinical Chem. 33: 1343-1 348. BROWN,G.M., A. BAR-OR, D. GROSSl, S . KASHUR,E. JOHANSSON, S.M. YIE (1991) Urinary 6-sulphatoxymelatonin, an index of pineal function in the rat. J. Pineal Res., 10:141-147. CHAN,Y.S., Y.M. CHEUNG, S . F . PANG(1991) Rhythmic release pattern of pineal melatonin in rodents. Neuroendocrinology, 53 (supp. 1):6&67. ENGLISH, J., A.L. POULTON, J. ARENDT,A.M. SYMONS (1986) A comparison of the efficiency of melatonin treatment in advancing oestrus in ewes. J. Reprod. Fertil. 77:321-327. FELLENBERG, A.J., G. PHILLIPOU, R.F. SEAMARK(1981) Urinary 6-sulphatoxymelatonin excretion and melatonin production rate: studies in sheep and man. In: Pineal Function. C.D. Matthews, R.F. Seamark, eds. Elsevier, Oxford, pp. 143-149. GOLDMAN, B.D., J.M. DARROW (1983) The pineal gland and mammalian photoperiodism. Neuroendocrinology , 37:38& 396. GOLDMAN,B.D., J.A. ELLIOTT(1988) Photoperiodism and seasonality in hamsters: Role of the pineal gland. In: Processing of Environmental Information in Vertebrates. M.H. Stetson, ed. Springer, New York, pp. 203-218. HAMED,M.Y., E.M. MOSTAFA,S.S. TOUS(1991) Antifertility effect of orally formulated melatonin tablets in mice. Int. J. Pharm. (Amst.) 69:93-102. Ho, A . K . , T.G. BURNS,L.J. GROTA,G.M. BROWN(1985) Scheduled feeding and 24-hour rhythms of N-acetylserotonin and melatonin in rats. Endocrinology 116: 1858-1862. Ho, A.K., L.J. GROTA,G.M. BROWN(1984) Relationship between pineal N-acetyltransferase activity, pineal melatonin
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John et a]. and serum melatonin in rats under different lighting conditions. Neuroendocrinology 39:465-470. KENNAWAY, D.J., R.A. GILMORE,R.F. SEAMARK (1982) Effect of melatonin feeding on serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicity in sheep. Endocrinology 110:1766-1772. KENNEDY, S.H., G.M. BROWN,P.E. GARFINKEL, G. MCVEY, D. COSTA,V. PARIENTI (1990) Sulphatoxy melatonin: An index of depression in anorexia nervosa and bulimia nervosa. Psychiatry Res. 32:221-227. KUSAKARI, N., Y. TAJIMA, S. ITOH,K . SEN-NA,S. SERLKAWA, T. HAITA,M. OHARA,Y. MORI(1991) Diurnal changes in plasma melatonin and the timing of reproductive onset in anestrous sheep fed melatonin. J. Vet. Med. Sci. 53:457-462. LEWY,A.J., T.A. WEHR,F.K. GOODWIN, D.A. NEWSOME, S.P. MARKEY (1980) Light suppresses melatonin secretion in humans. Science 210:1267-1269. MATTHEWS, C.D., M.V. GUERIN,X. WANG(1991) Human plasma melatonin and urinary 6-sulphatoxymelatonin: studies in natural annual photoperiod and in extended darkness. Clin. Endocrinol. 35:21-27. QUAY,W.B. (1974). Pineal Chemistry. Charles C. Thomas Publishers, Illinois. REITER,R.J. (1983) The role of light and age in determining melatonin production in the pineal gland. In: The Pineal and Its Endocrine Role. J Axelrod, F. Fraschini, G.P. Velo, eds. Plenum, New York, pp. 227-241.
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REITER,R.J. (1986) Normal patterns of melatonin levels in the pineal gland and body fluids of humans and experimental animals. J. Neural Transm. Suppl. 21:35-54. B.A. RICHREITER,R.J., L.Y. JOHNSON,M.K. VAUGHAN, ARDSON (198 1) Pineal constituents and reproductive physiology. In: Physiopathology of Endocrine Diseases and Mechanisms of Hormone Action. Liss, New York, pp. 163-178. C. TRAROLLAG,M.D., E.S. PANKE,W.K. TRAKULRUNGSI, KULRUNGSI,R.J. REITER(1980) Quantification of daily melatonin synthesis in the hamster pineal gland. Endocrinology 1061231-236. STELLFLUG, J.N., T.M. NETT,C.F. PARKER (1988) Melatonin advances June breeding and fall lambing in range and farmflock type ewes. Theriogenology 29543-655. (1986) Effects of STETSON,M.H., M. WATSON-WHITMYRE exogenous and endogenous melatonin on gonadal function in hamsters. J. Neural Transm. Suppl. 21:55-80. VAUGHAN, M.K. (1981) The pineal gland-a survey of its antigonadotrophic substances and their actions. In: International Review of Physiology, Vol. 24: Endocrine Physiology 111. S.M. McCann, ed. University Park Press, Baltimore. WETTERBERG, L. (1978) Melatonin in humans: physiological and clinical studies. J. Neural Transm. Suppl. 13:289-310. YOUNG,I.M., P.L. FRANCIS, A.M. LEONE,P. SLOVELL, R.E. SILMAN(1988) Constant pineal output and increasing body mass account for declining melatonin levels during human growth and sexual maturation. J. Pineal Res. 5:71-86.
An oral melatonin replacement regimen that re-establishes the normal circadian levels of urinary 6-sulphatoxymelatonin in functionally pinealectomized rats John TM, Brown MC, Brown GM. An oral melatonin replacement regimen that re-establishes the normal circadian levels of urinary 6-sulphatoxymelatonin in functionally pinealectomized rats. J. Pineal Res. 1992: 13: 145-150.
I
Abstract: Wistar rats maintained on a 12-hr daily photoperiod (LD 12:12 cycle) exhibited a diurnal rhythm in urinary 6-sulphatoxymelatonin (aMT6s) concentrations with peak levels in the scotophase. Light-induced functional pinealectomy (FPX) abolished the nocturnal rise in aMT6s, lowering it to photophase levels. The objective of the study was to formulate an oral melatonin replacement regimen that would restore a normal rhythmic output of urinary aMT6s in functionally pinealectomised rats. Three regimens of sequential doses of melatonin were tested. Of these, the regimen with melatonin concentrations of 4 ng, 12 ng, 65 ng, and 4 ng per ml of drinking water given to rats during the lst, 2nd, 3rd, and 4th 3-hr periods, respectively, of the 12-hr FPX phase, was found to generate a urinary aMT6s level that 'losely resembled the natural level and rhythm exhibited under an LD 12:12 cycle. This dose is considered appropriate to restore certain melatonin-mediated physiological functions in Wistar rats subjected to FPX.
Introduction
In most mammals, circulating as well as pineal levels of melatonin exhibit a circadian rhythm with a peak in the night and a trough in the day [Reiter, 19861. It is also known that light has a suppressive effect on melatonin levels [Lewy et al., 1980; Rollag et al., 1980; Reiter, 19831, a feature presumably pivotal in transmitting seasonal and daily photoperiodic information into a neuroendocrine signal. Furthermore, melatonin is believed to be involved in mediating many rhythmic physiological events [Wetterberg, 1978; Arendt and Broadway, 19871, and is also considered to impose synchronicity on a multitude of daily rhythms in cells, tissues, and organs of the internal milieu [Armstrong, 19891. A,major strategy for investigating the role of melatonin has been to administer melatonin to animals in which the endogenous melatonin production has been blocked or curtailed by pinealectomy or other methods, and observe whether this would antagonize the effects produced by the deficiency in
T. Mathew John,' M. Catherine Brown,' and Gregory M. Brown' 'Department of Biomedical Sciences, McMaster University, Hamilton, Canada; *Clarke Institute of Psychiatry, Toronto, Canada
Key words melatonin-6-sulphatoxymelatonin Address reprint requests to Dr Gregory M Brown, The Clarke Institute of Psychiatry, 250 College Street, Toronto, Ontario, Canada M5T 1R8 Received March 2, 1992, accepted July 29, 1992
the endogenous melatonin. Due to the existence of a rhythmic pattern of the circulating melatonin levels in animals living under a normal day-night cycle, it is desirable that in experiments involving melatonin replacement, the concentrations resulting from exogenous melatonin administered should conform to the normal endogenous levels in the same rhythmic pattern as would occur in normal day-night cycle. Most methods of melatonin administration, however, do not comply with these criteria, and the use of such methods often elicits inconsistent and contradictory experimental results. In the present study, we have formulated a regimen of oral dosage of melatonin that would simulate in functionally pinealectomized rats a normal level and rhythm of urinary 6-sulphatoxymelatonin (aMT6s), the principal metabolite of melatonin. The concentration of urinary aMT6s is considered to be a reliable index of melatonin secretion [Arendt et al., 1985; Bojkowski et al., 1987; Fellenberg et al., 1981; Young et al., 1988; Bojkowski and Arendt, 1990; Kennedy et al., 19901 and pineal activity
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[Brown et al., 19911. Measurement of urinary aMT6s has definite advantages over serum melatonin measurement in that it circumvents the need for blood sampling in circumstances where blood sampling is difficult [Bojkowski and Arendt, 19901 and could provide a more integrated estimate of total melatonin output during a fixed time period [Kennedy et al., 19901. Materials and methods Animals
Male Wistar rats (200-220 g) were obtained from a local supplier and were individually housed in metabolic stainless steel cages suspended inside a controlled environment cabinet. The floors of the cages were made of stainless steel with mesh, under which a detachable stainless steel funnel was attached when urine collection was necessary. The rats were given drinking water and commercial rat-feed ad libitum, and were acclimated to a 12-hr daily photoperiod and an ambient temperature of 22 2 0.5”C for at least 3 weeks prior to the commencement of the experiment. The rats weighed over 290 g when the experiments were conducted. Lighting was provided by “Cool White” fluorescent bulbs which supplied an illumination of approximately 110pW/cm2 (as per reading on an Opikon radiometer with broad spectrum head and infra-red filter). Red photosafe bulbs were used to allow visualization while carrying out testing procedures in the dark. In rats maintained under similar conditions, distinct circadian rhythms of pineal and serum melatonin levels [Ho et al., 1984; 19851 as well as of urinary aMT6s [Brown et al., 19911 have been observed previously in this laboratory. Experimental procedure
Experiment 1: This experiment, using 12 rats maintained on LD 12:12 cycle (lights on at 0930 and off at 2130), was intended to verify the 24 hr pattern of baseline urinary aMT6s output. Levels of urinary aMT6s have previously been reported to be low during the photophase, showing no fluctuation between 3 to 12 hr after lights were on, and to be relatively high during the scotophase, peaking between 6 and 12 hr after the onset of darkness [Brown et al., 19911. In the present experiment, the total daytime (12 hr) urine excreted from each rat was collected at the end of the photophase, whereas the night time urine was collected in 3-hr blocks over the 12-hr scotophase. At the end of each collection period, the funnel from beneath the cage bottom was removed and rinsed with 25 ml of distilled water which was consequently added to the urine. 146
After recording the volume of each collected sample, it was centrifuged at 5000g for 20 min. An aliquot of the supernatant was separated and frozen at -20°C until assays were performed. Experiment 2: Twenty three rats were randomly divided into 3 groups comprising seven or eight rats each, and were subjected to light-induced functional pinealectomy (FPX). During FPX, groups 1 and 2 received melatonin through their drinking water, while group 3 served as control, receiving only the vehicfe solution through the drink. Melatonin was given only during the 12-hr FPX period (2130 to 0930), with the concentration of melatonin in the drink being changed after every 3-hr block. Rats in group 1 received drinking water with melatonin concentrations of 100 ng, 300 ng, 1000 ng, and 0 ng per ml during the 1st, 2nd, 3rd, and 4th 3-hr periods of the FPX phase, respectively. Rats in group 2 were given a lesser dose of melatonin, with concentrations of 10, 30, 100, and 0 ng per ml during the 4 periods, respectively. Controls also received fresh drinks every 3 hr. All drink bottles contained 200 ml of fresh drink when replaced at the beginning of each 3-hr period. The urine was harvested at every 3-hr interval during the FPX period and stored for aMT6s assay, as described in experiment 1. Experiment 3: Since the urinary aMT6s values obtained following melatonin replacement using both the regimens in experiment 2 appeared much higher than the normal baseline values, it was considered necessary to repeat the experiment with melatonin doses further lowered. A group of nine rats received melatonin at concentrations of 4, 12, 65, and 4 ng per ml of drink during the four consecutive 3-hr blocks of the FPX phase respectively. A control group of nine rats received vehicle drink. The drink consumed (including spillage if any) by each rat was recorded at the end of each 3hr block. All other procedures including sequential urine collection remained the same as described in experiment 2. Functional pinealectomy (FPX)
In the context of the present study, FPX refers to light-induced suppression of pineal activity during one 12-hr dark period. In rats maintained regularly on an LD 12:12 cycle (lights on at 0930 and off at 2130), the FPX was effected by imposing “daytime” lighting conditions during what would normally be the dark phase (2130 to 0930) of the LD cycle. Assay
The aMT6s radioimmunoassay materials supplied by CIDtech Research Inc. (Mississauga, Ontario,
Physiological melatonin replacement
Canada) were used for the measurement of urinary aMT6s. The assay was based on a modified version [Brown et al., 19911 of the method used by Aldhous and Arendt [ 19881.
3800. 0boselme
3400 - I FPX only (n=8)
3000 - Lz9
2600 -
2200 - E Z
Melatonin
Crystalline melatonin (Lot no. 108F 0646, Sigma Chemical Co., St. Louis, Missouri) was initially dissolved in absolute ethanol and subsequently diluted with water to a final alcohol concentration of 1% (v/v). Melatonin solutions were freshly prepared less than an hour prior to the beginning of the 12-hr functional pinealectomy period.
Data analysis Repeated measures analysis of variance was employed for the statistical evaluation of the results. Newman-Keuls test was used for multiple comparisons, when warranted. P < 0.05 was considered acceptable as significant.
Values under
12 1 2 LD cycle ( n = 1 2 )
,-.
1800-
.c 1?
a v
FPX and MEL replacement (100, 300, 1000. Onq/ml)
(n=7) FPX and MEL replacement ( l o ; 30; 100, Ong/ml)
(n=8)
1 4 0 0 - EZJ FPX and MEL replocement (4; 12;6 5 ; 4flg/ml) (n=9) 1000-
600 -
2
180
d c
1601
5>r
140
=
1201 100
:ji 20
0 Period 1 21 30-OO3Oh
Results
Experiment 1: The existence of a diurnal variation in urinary aMT6s output was confirmed. The lowest level of aMT6s output (3.11 ? 0.79 ng/3 hr) was registered in samples collected during the photophase. The level gradually rose during scotophase, peaking between 6 and 9 hr after the onset of darkness and then falling. The levels at both the 3rd and the 4th quarters of the scotophase were significantly higher than those of the rest of the periods. Data from scotophase are incorporated in Figure 1 . ANOVA results: F = 23.6051; df = 4, 44; P < 0.0001. Experiment 2 (Fig. 1): Rats subjected to FPX showed no scotophase-induced normal rise in urinary aMT6s level under an LD 12:12 cycle (Fig. 1). The functionally pinealectomized rats receiving melatonin doses of 100, 300, 1000, and 0 ng per ml drink or 10, 30, 100, 0 ng per ml drink showed an aMT6s rhythm similar to that exhibited by rats maintained on LD 12:12 cycle, but both regimens of melatonin doses showed a trend to elicit higher urinary aMT6s values than in normal rats maintained under LD 12:12 cycle (Fig. 1). Rats on the higher melatonin dose regimen exhibited higher aMT6s output, showing a peak value of 3366 ng/3 hr (6-9 hr into functional pinealectomy period), which was over 50 times greater than that in normal rats during the corresponding period. ANOVA results: treatment, P < 0.0001; time, P < 0.0001; interaction, P < 0.0001. Experiment 3: As in experiment 2, functional pinealectomy abolished the scotophase-related rise
Period 2
Period 3
Period 4
0030-0330h
0330-0630h
0630-0930h
Fig. I . Effects of functional pinealectomy (FPX) and oral melatonin (MEL) replacement on urinary 6-sulphatoxymelatonin (aMT6s) levels in Wistar rats. MEL was given in drinking water in sequential doses of either 10 ng, 30 ng, 100 ng, and 0 ng/ml (Exp. 2 ) or 100 ng, 300 ng, 1000 ng, and 0 ng/ml (Exp. 2), or 4 ng, 12 ng, 65 ng, and 4 ng/ml (Exp. 3), during the lst, 2nd, 3rd, and 4th 3-hr periods of the FPX phase, respectively. Under normal LD cycle (Exp. l), lights were turned on at 0930 and off at 2130. Under FPX (Exp. 2 and 3), lights were not turned off at 2130. Since aMT6s values under FPX in Experiment 3 were not significantly different from corresponding values in Experiment 2, only those from Experiment 2 are represented on the graph.
in urinary aMT6s observed under LD 12:12 cycle. The urinary aMT6s output in functionally pinealectomized rats receiving a melatonin dose regimen of 4, 12, 65, and 4 ng per ml drink was not significantly different from the baseline values observed under an LD 12:12 cycle (Fig. 1). ANOVA results: treatment, P < 0.0001; time, P < 0.0001; interaction, P < 0.0001. Although the consumption of melatonin drink, in general, showed a trend to be lower than that of vehicle drink (Fig. 2), the difference was not significant (treatment, P > 0.1; time, P < 0.05; interaction, P > 0.01). A trend towards a lower urine output, overall, was also apparent (treatment, P = 0.058; time, P < 0.01; interaction, P < 0.01) in the melatonin-treated rats in comparison to that in the control rats (Fig. 2).
147
John et al. 0-0
.-*
Vehicle control (n=9) Melotonin-treoted (4 , 12. 65, 4 ng/ml) (n=9)
I
2c Period 1
Period 2
Period 3
Period 4
2130-0030h
0030-0330h
0330-0630h
0630-0930h
F ig . 2. Drink consumption (A) and urine output (B) in Wistar rats under functional pinealectomy . Lighting conditions are the same as explained in Fig. 1 . Melatonin sequential dose: 4 ng, 12 ng, 65 ng, and 4 ngiml drink. Values are mean i SEM.
Discussion
The urinary aMT6s output in Wistar rats used in the present study showed a circadian pattern with lower values in the photophase and higher in the latter half of the scotophase. These observations are consistent with an earlier report by Brown et al. [1991] on aMT6s profile in this species. The present study also corroborated their observation that light-induced functional pinealectomy abolished the nocturnal rise in urinary aMT6s, reducing the levels to that seen during the photophase. Over the years, investigators have used several methods for melatonin replacement, including single or multiple injections, subcutaneous implantation of melatonin in beeswax pellets or silastic capsules, and oral feeding. However, the exogenous melatonin thus administered often does not provide a rhythmic circulating level that would mimic the natural rhythm and level of the endogenous melatonin. In order to make proper assessment of the effects of melatonin replacement, it is important that the concentration of exogenous melatonin administered conforms to the normal endogenous basal levels in the same rhythmic pattern as would occur in normal day-night cycle. Failure to achieve such a condition may well be the reason for many of 148
the inconsistencies and contradictory results often reported in the literature following melatonin treatment. It has been proposed that continuous availability of melatonin from implants provides a releasable pool which saturates melatonin receptors, making them insensitive to an acute signal or pulse of melatonin, and subsequently resulting in a perpetual state of “down regulation” [Reiter et al., 198 1 ; Vaughan, 19811. The injection method, on the other hand, provides melatonin for only a limited period following the injection. Oral administrations appear to have an advantage over the other methods in that the dosage can be regulated without the use of any painful injections or surgeries. With oral administration, since the melatonin infusion is somewhat stretched over time, an appropriately timed alteration in oral dosage can bring about a gradual, rather than an abrupt change in the circulating melatonin level, better simulating the natural rhythm. It may be mentioned in this connection that both the absolute duration of the melatonin peak and the circadian timing of the peak have been proposed as crucial parameters in transmitting photoperiodic information [Goldman and Darrow, 1983; Stetson and Watson-Whitmyre, 1986; Goldman and Elliot, 19881. Among the three regimens of sequential dosage tested in the present study, we have found that the regimen with melatonin concentrations of 4 ng, 12 ng, 65 ng, and 4 ng per ml of drink, given to Wistar rats during the lst, 2nd, 3rd, and 4th 3-hr periods, respectively, of the functional pinealectomy phase, would generate an urinary aMT6s level and rhythm that would closely resemble the natural level and rhythm exhibited under an LD 12:12 cycle. Since the concentration of urinary aMT6s is considered to be a reliable index of melatonin secretion [Arendt et al., 1985; Bojkowski et al., 1987; Fellenberg et al., 1981; Young et al., 1988; Bojkowski and Arendt, 1990; Kennedy et al., 19901, a rhythmic pattern in circulating melatonin levels identical to that of urinary aMT6s is also to be expected to occur with those dosage. However, whether the rhythms in both melatonin and aMT6s occur concurrently or with a time-lag is not certain, although in humans it has been reported that changes in urinary aMT6s levels followed that of plasma melatonin levels by 12 to 108 min., depending on the time of the year [Matthews et al., 19911. It may be pointed out here that, although the normal pattern of urinary aMT6s secretion over any 3-hr period may be reproduced by the present oral replacement of melatonin, it is not clear whether the treatment would produce patterns of circulating melatonin precisely like those in normal rats. It has recently been demonstrated in rats that blood melatonin exhibits an episodic pattern with pulses
Physiological melatonin replacement
superimposed on the basal level [Chan et al., 19911. Since rats do not drink continuously, it is possible that there could be episodic fluctuations/short-term pulses in blood melatonin levels, but such fluctuations/pulses need not necessarily synchronize with natural fluctuations/pulses. Since only a urinary metabolite of melatonin is measured in the present study, some concern could be raised as to whether the metabolism of orally administered melatonin is identical to that of melatonin secreted directly into the blood and whether any similarities exhibited in resulting aMT6s levels indeed reflect similarities in blood melatonin levels. However, a major pathway for the metabolism of melatonin being the hydroxylation of melatonin by hepatic microsomal enzymes [Quay, 19741, one could presume that the metabolism of both oral and endogenous melatonin takes place in the liver prior to excretion via the kidney. In a separate experiment (to be reported separately) involving FPX, we have found that melatonin replacement with a sequential dose of 4, 12, 65, and 4 ng per ml drink, when administered as in the present study, nullified the effect of light-induced FPX on the analgesic rhythm in Wistar rats [Bar-Or and Brown, 19891, providing proof that the dose is appropriate to restore this melatonin-mediated function. It may also be mentioned here that oral administration of melatonin has previously been reported to influence reproductive activity in sheep [Kennaway et al., 1982; Arendt et al., 1983; English et al., 1986; Stellflug et al., 1988; Kusakari et al., 19911 and in mice [Hamed et al., 19911. The trend towards a lower urine output observed in melatonin-treated rats than in control rats may be considered as a consequence of the reduced drink consumption apparent in these rats. It is not clear, however, why the consumption of melatonin drink showed a trend to be lower than that of vehicle drink. An aversion to the taste and/or smell of the melatonin drink or an inhibitory effect of melatonin on thirst are some of the possible reasons that may be probed. In recent years, melatonin has been proposed as a powerful chronobiotic, having great potential as a prophylactic and therapeutic agent for stabilizing or re-entraining disturbed internal rhythms, thereby averting or remedying disorders arising from disturbed circadian physiology [Armstrong, 19891. Melatonin administered in inappropriate doses or at an incorrect time of the day may aggravate the desynchrony of internal rhythms and consequently the disease. The melatonin administration regimen that simulated the normal circadian rhythm of urinary aMT6s in rats used in the present study could serve as a model for formulating therapeutic melatonin replacement schedules, but the precise
dosage and the timing of administration may have to be individually designed for each animal species. Acknowledgments These studies were supported in part by the Medical Research Council of Canada. G. M. Brown is an Ontario Mental Health Foundation Research Associate. Thanks are expressed to S. Kashur for assistance with the aMT6s assay.
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REITER,R.J. (1986) Normal patterns of melatonin levels in the pineal gland and body fluids of humans and experimental animals. J. Neural Transm. Suppl. 21:35-54. B.A. RICHREITER,R.J., L.Y. JOHNSON,M.K. VAUGHAN, ARDSON (198 1) Pineal constituents and reproductive physiology. In: Physiopathology of Endocrine Diseases and Mechanisms of Hormone Action. Liss, New York, pp. 163-178. C. TRAROLLAG,M.D., E.S. PANKE,W.K. TRAKULRUNGSI, KULRUNGSI,R.J. REITER(1980) Quantification of daily melatonin synthesis in the hamster pineal gland. Endocrinology 1061231-236. STELLFLUG, J.N., T.M. NETT,C.F. PARKER (1988) Melatonin advances June breeding and fall lambing in range and farmflock type ewes. Theriogenology 29543-655. (1986) Effects of STETSON,M.H., M. WATSON-WHITMYRE exogenous and endogenous melatonin on gonadal function in hamsters. J. Neural Transm. Suppl. 21:55-80. VAUGHAN, M.K. (1981) The pineal gland-a survey of its antigonadotrophic substances and their actions. In: International Review of Physiology, Vol. 24: Endocrine Physiology 111. S.M. McCann, ed. University Park Press, Baltimore. WETTERBERG, L. (1978) Melatonin in humans: physiological and clinical studies. J. Neural Transm. Suppl. 13:289-310. YOUNG,I.M., P.L. FRANCIS, A.M. LEONE,P. SLOVELL, R.E. SILMAN(1988) Constant pineal output and increasing body mass account for declining melatonin levels during human growth and sexual maturation. J. Pineal Res. 5:71-86.
