Journal of Pineal Research 7:205-209 (1989)

Antigonadal Activity of the Melatonin Analogs 2-Iodomelatonin and

2-Chloromelatonin in the Juvenile Djungarian Hamster, Phodopus

sungorus campbelli

David Sugden Department of Physiology, King's College London, London

The antigonadal effects of daily (20 pg, s.c.) injection of melatonin and two analogs, 2-iodomelatonin and 2-chloromelatonin, were compared in juvenile Djungarian hamsters housed under long photoperiod (L:D 16:s). Melatonin, 2-iodomelatonin, and 2-chloromelatonin injected 3 h before lights off for 16 days ( 17-34 days of age) significantly inhibited testis growth compared to vehicle-injected hamsters. In addition, melatonin and both analogs significantly reduced body weight gain. These 2-substituted analogs appear to be melatonin agonists with a potency in vivo similar to the parent compound, melatonin.

Key words: melatonin, antigonadal, hamster

INTRODUCTION

The pineal hormone melatonin is thought to mediate the effects of photoperiod on the reproductive system in seasonally breeding mammals [Tamarkin et al., 19851. It has been suggested that the duration of the nocturnal melatonin signal is the critical parameter conveying daylength information [Carter and Goldman, 1983; Bittman et al., 19831. Implant studies have indicated that the major target sites for melatonin are within the hypothalamus [Glass and Lynch, 1981 1. Recent in vitro autoradiographic studies using 2-[ '251]iodo- melatonin [Vakkuri et al., 19841 support this suggestion in that high-affinity binding sites selective for melatonin were identified in the median eminence/ arcuate nucleus area and suprachiasmatic nucleus in rat and hamster brain [Vanecek et al., 1987; Weaver et al., 19881. Other studies have reported lower affinity binding sites distributed throughout the brain [Laudon and Zisapel, ' .'

Received October 4, 1988; accepted February 14, 1989.

Address reprint requests to Dr. David Sugden, Department of Physiology, Kensington Campus, Campden Hill Road, London W8 7AH, England.

0 1989 Alan R. Liss, Inc.

206 Sugden

1986; Duncan et al., 19881. Despite its use in these binding studies little is known of the biological activity of 24odomelatonin. Recent work suggests that it is a melatonin agonist. It shares melatonin’s ability to inhibit [3H]dopamine release from chicken retina [ Dubocovich and Takahashi, 19871. Furthermore, infusion of 2-iodomelatonin into pinealectomized pregnant Djungarian hamsters mimicked the action of melatonin and stimulated the reproductive development of the fetus [Weaver et al., 19881, The present study compared the effects of melatonin, 2-iodomelatonin, and another novel analog, 2-chloromelatonin, on body weight and testicular development in juvenile Djungarian hamsters housed in a long photoperiod. Antigonadal responses to exogenous melatonin occur much more rapidly in juvenile male Djungarian hamsters than in adults [Carter and Goldman, 19831, thus offering a good model system in which to test the effects of putative melatonin agonists. The results indicate that both of these 2-substituted analogs are potent melatonin agonists in vivo.

MATERIALS AND METHODS

Male Djungarian hamsters, Phodopus sungorus campbelli, obtained from Wrights, Latchingdon, U.K. were used in this study. Hamsters were housed six per cage in a well-ventilated, temperature-controlled (2 l°C) room with food and water available ad libitum. All hamsters (and their mothers) were housed from birth in a photoperiod of 16 h of light per day (16LBD; lights on,

Melatonin and its analogs (20 pg in 1 : l O ethanolic saline) were adminis- tered S.C. daily at 1800 h from 17 days of age. Control hamsters received daily vehicle injections ( 1 : l O ethanolic saline, 0.1 ml s.c.). Each animal was weighed on day 17 (2 days after weaning) and 24 h after the last injection just prior to sacrifice on day 34 using an Oertling OB152 top-loading digital balance. Reproductive development was assessed by determining paired testicular weight on day 34 using a Mettler model H l 6 balance. Data were analyzed using analysis of variance followed by Student’s t-test.

2-lodomelatonin was obtained from Research Biochemicals Inc., Natick, MA, and melatonin from Sigma Chemical Co., Poole, U.K. 2-Chloromelatonin was synthesised by Dr. C. Smithen, Roche Products Ltd, Welwyn Garden City, U.K.

0500-2100 h).

RESULTS

Average testis weight in control hamsters increased from 169 k 9 mg in 17-day-old hamsters to 702 * 48 mg in 34-day-old hamsters. Daily injections of melatonin, 2-iodomelatonin, and 2-chloromelatonin significantly reduced the increase in testis weight (Table 1). Figure 1 shows the average testis weight of individual male hamsters in each of the groups. Treatment with melatonin and these two analogs also significantly reduced the gain in body weight (Table 1 ).

DISCUSSION

These experiments suggest that in addition to melatonin, 2-iodomelatonin and 2-chloromelatonin are capable of inhibiting testicular growth in juvenile

Antigonadal Effect of Melatonin Analogs 207

TABLE 1. Effect of Melatonin, 2-Iodomelatonin, and 2-Chloromelatonin on Body Weight and Testis Weight of Male Djungarian Hamsters'

~

Average testis Body weight weight

n (g) (mg) Control 8 34.6 2 1.2 702 t 411 Melatonin 12 30.9 ? 0.9' 224 ? 49; 2-Iodomelatonin 12 30.3 1.0* 313 * 65* 2-Chloromelatonin 12 28.4 t_ 1.4* 353 t 91'

'Hamsters were injected S.C. daily with 20 bg of melatonin, 2-iodomelatonin, or 2-chloromelatonin from 17 to 34 days of age. Initial body weights at 17 days of age were not significantly different; 17.8 * 1.6, 18.0 2 2.0, 17.7 t 1.5, and 17.8 & 1.5 g for control, melatonin, 2-iodomelatonin, and 2-chloromelatonin groups, respectively. Average testis weight at 17 days of age was 169 2 9 mg (n = 5). Values given in the table are the mean k S.E.M. body and testis weight at 34 days of age. Significantly different from the control group. *P < .05 using Student's t-test.

Djungarian hamsters and are melatonin agonists in vivo. As has been previously noted the inhibition of testes development was accompanied by a reduction in body weight gain. 2-lodomelatonin and 2-chloromelatonin injected daily 3 h before lights off at a dose of 20 pg produced clear suppression of testis growth in the majority of the treated hamsters (Fig. 1). Not all hamsters treated with 2-iodomelatonin and 2-chloromelatonin, or even melatonin itself, showed this response. This may have been because the animals were not treated for a long enough period of time, although previous work indicates that significant inhibition of testes growth can be observed after just a few days of treatment of juvenile Pbodopus sungorus sungorus [ Goldman et al., 19841. Alternatively, the daily period of melatonin sensitivity may differ slightly between l? sungorus sungorus [Stetson et al., 19861 and l? sungorus campbelli, the strain that was used in these experiments.

It appeared that 2-chloromelatonin and 2-iodomelatonin at the dose used were slightly less effective than melatonin itself in reducing testicular growth, although this did not seem to be the case for body weight. Clearly a comparison of the in vivo potency of these compounds must await a full dose-response study. Recent data indicate that 2-chloromelatonin and 2-iodomelatonin, like melatonin itself, produce condensation of the pigment granules of cultured Xenopus leuvis melanophores at subpicomolar concentrations [ Sugden, 19891. These results indicate that the halogenated analogs are full agonists with a potency comparable to melatonin. As the daily period of melatonin sensitivity in Pbodopus is relatively brief, differences in the absorption, distribution or metabolism of these analogs following injection may be particularly important in determining the in vivo potency of the analogs. No information is yet

Previous studies have established that both 5-methoxy and N-acetyl groups are essential for the biological activity of melatonin [Messenger and Warner, 1977; Rollag, 19821. Surprisingly, the substitution of a large atom (-I,-Cl) at the 2-position of the indole ring does not seem to interfere appreciably with the biological activity of melatonin [Sugden, 19891. Future studies on additional melatonin analogs should help define the structural features of the

available on the metabolic disposition of these melatonin analogs. , *

208 Sugden

-

P W 3 6oo 400 t P 200 7 0

* .

t . : . I

0 . i :. :

C M IM CM

Fig. 1. Average testis weight of individual hamsters in each group. Melatonin, 2-iodomelatonin, and 2-chloromelatonin were injected S.C. (20 kg) daily from 17 days to 34 days of age at 1800 h, 3 h before lights off. Controls received daily vehicle (1:10 ethanolic saline, 0.1 ml s.c.) injections. All animals were killed 24 h after the last injection.

melatonin molecule required for interaction with its receptor and may lead to the rational design of potent melatonin agonists and antagonists.

LITERATURE CITED

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Dubocovich, M.L., J.S. Takahashi ( 1987) Use of 24 '"I]iodomelatonin to characterise melatonin binding sites in chicken retina. Proc. Natl. Acad. Sci. U S A . 84:3916-3920.

Duncan, M.J., J.S. Takahashi, M.L. Dubocovich (1988) 2-[ '251] lodomelatonin binding sites in hamster brain membranes: Pharmacological characteristics and regional distribution. Endo- crinology 122: 1825-1 8 3 3 .

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Antigonadal Effect of Melatonin Analogs 209

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