Indian Journal of Biochemistry & Biophysics Vol 45, October 2008, pp. 289-304

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Melatonin: Fifty Years of Scientific Journey from the Discovery in Bovine to Delineation of Functions in Human

Indrajit Chowdhury1, Anamika Sengupta2 and Saumen Kumar Maitra3* 1Department of Obstetrics and Gynecology, 2Department of Physiology, Morehouse School of Medicine, Atlanta, USA 3Department of Zoology, Visva-Bharati University, Santiniketan, 731 235, India Received 24 November 2007; revised 01 July 2008

Melatonin (N-acetyl-5-methoxytryptamine) was first purified and characterized from the bovine pineal gland extract by Aron Lerner and co-workers in 1958. Since then, a plethora of information has piled up on its biosynthesis, metabolism, time-bound periodicity, physiological and patho-physiological functions, as well as its interactions with other endocrine or neuro-endocrine organs and tissues in the body. Melatonin has wide range of applications in physiology and biomedical fields. In recent years, a significant progress has been made in the understanding mechanism of its actions at the cellular and molecular levels. Consistent efforts have uncovered the mystery of this indoleamine, and demonstrated its role in regulation of a large as well as diverse body functions in different groups of animals in general, and in humans in particular. Current review, in commemoration of 50 years of discovery of melatonin, while revisiting the established dogmas, summarizes current information on biosynthesis, secretion, metabolism and molecular mechanism of action of melatonin at cellular level and highlights the recent research on its role in human physiology and clinical biology. Keywords: AA-NAT, Biological rhythm, Cancer, Melatonin, Pineal gland, Reproduction, Seasonal affective disorder , Sleep, Jet-lag.

Until first half of the twentieth century, the pineal gland was considered as an epithalamic appendage of vertebrate brain with enigmatic functions. Discovery of a new biological substance from the bovine pineal gland extract by the dermatologist Aaron B Lerner at the School of Medicine was a major breakthrough in pineal research1. This purified pineal product was chemically identified as N-acetyl-5- methoxy-tryptamine, and named melatonin (Fig. 1), in recognition to McCord and Allen’s2 observations as early as 1917 that bovine pineal extracts added to water caused the larvae to blanch due to aggregation

______Fig. 1—Chemical structure of melatonin (N-acetyl-5-methoxy *Corresponding author tryptamine) Tel: 91-3463-261268; Fax: 91-3463-261268 E-mail: [email protected], [email protected] of melanin granules within the skin cells. Though this Abbreviations: AA-NAT, arylalkylamine N-acetyl transferase; indole hormone was first reported from the pineal AFMK, N1-acetyl-N2-formyl-5-methoxykynuramine; AMK, gland, it was also found in retinae, Harderian gland 1 N -acetyl-5-methoxykynuramine; c3OHM, cyclic3-hydroxy- and gastro-intestinal tracts in vertebrates, as well as in melatonin; camp, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CYp1A2, CYP1A1 or CYP1B1, a wide variety of organisms, ranging from cytochrome P450 mono-oxygenases; ER, estrogen receptor; GnRH, invertebrates to plants. One of the characteristic gonadotropin releasing hormone; HIOMT, hydroxyindole-O- features of this tiny derivative molecule is methyl transferase; 5-HIAA, 5-hydroxy-indole acetic acid, IL, that its synthesis takes place during darkness in a interleukin; LDL, low density lipoprotein, MT, melatonin light-dark cycle, irrespective of the habit of the receptor; NO, nitric oxide; NA, noradreneralin; REM, rapid eye movement; SAD, seasonal affective disorder; MT6s, concerned animals. Therefore, it is often described as 3 6-sulphatoxymelatonin; SCN, suprachiasmatic nucleus. ‘hormone of darkness’ or a ‘chronobiotic molecule’ . 290 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, OCTOBER 2008

Past fifty years, since its discovery from the bovine Biosynthesis, secretion and metabolism pineal gland have witnessed accumulation of Biosynthesis considerable data on different aspects of regulatory In all vertebrates investigated so far, melatonin is mechanism involved in the synthesis and release of primarily synthesized within the pinealocytes of melatonin, its role in the regulation of diverse body 10 pineal gland during the night, regardless of the functions in vertebrates, human in particular. Studies diurnal or nocturnal locomotor activity of the animals. in various animals, especially in mammals, have Biosynthesis of melatonin is a four-step phenomenon. shown the importance of melatonin in regulation of a First, its precursor L-tryptophan is taken up from the wide range of physiological functions, ranging from circulation (blood) into the pinealocyte and converted aging to aggression, hibernation to hypertension, to 5-hydroxytryptophan by tryptophan 5-monooxy sleep to stress, reproduction to tissue regeneration, genase/hydroxylase (L-tryptophan, tetra-hydropterin- scavenging of free radicals to synchronization of body dine: oxygen oxidoreductase, EC 1.14.16.4) and functions with the environmental light-dark cycles. further decarboxylated by L-aromatic amino acid Now, it is clearly known that melatonin acts on its decarboxylase (aromatic L-amino acid carboxylase, target cells/tissues through transmembrane receptors EC 4.1.1.28) to form 5-hydroxytryptamine (5-HT) or MT1, MT2 and MT3 or through orphan nuclear serotonin. Serotonin is acetylated (N-acetylation), to receptors of the retinoic acid receptor family4 Being a form N-acetylserotonin using serotonin-N- lipophilic molecule, it has free access to all cells, acetyltransferase/arylalkylamine N-acetyltransferase tissues, and organs of the body and thereby offers an (AA-NAT; acetyl CoA: aryl-amine N-acetyltrans- additional receptor independent non-hormonal role of ferase, EC 2.3.1.5). Finally, N-acetylserotonin is free radical scavenger in reducing the oxidative stress. methylated by hydroxyindole-o-methyltransferase The physiological and genetical aspects of the (HIOMT; S-adenosyl-L-methionine: N-acetyl- melatonin have been well-documented in recent 3-12 serotonin-o-methyltransferase, EC 2.1.1.4) to form years . As a supplementary to the existing literature 11,19,20 melatonin . AA-NAT is the rate-limiting enzyme and to commemorate the golden jubilee year of the in melatonin synthesis, but serotonin availability is discovery of melatonin, this communication aims at one of the major factors that play an important summarizing the recent progress in the understanding 9 regulatory role in this process . of molecular mechanism of action and physiological role of melatonin with special emphasis on clinical The magnitude and duration of nocturnal increase significance and application in human subjects. in melatonin synthesis is dependent upon the length of the dark phase of photoperiodic cycle or external Melatonin: A ubiquitously distributed biological geophysical cycle and it acts as a “clock” and molecule “calendar” for the entrainment of other biological Melatonin is ubiquitously distributed in living activities21. The rhythm of melatonin synthesis is system and is suggested to represent one of the most generated by interacting networks of circadian clock primitive biological signals appeared on earth13. It has genes located in the suprachiasmatic nucleus (SCN), been identified in all major taxa of organisms circadian oscillator/master pacemaker, of the including bacteria, and unicellular eukaryotes, in in brain, which is considered as the different parts of plants including the roots, stems, major central rhythm-generating system or “clock” in flowers and seeds, and in invertebrate and vertebrate mammals22,23. The pineal gland itself is a self- animals14,15. It is also present in different tissues, sustaining “clock” in some species, except in lower organs such as Harderian gland, extraorbital lacrimal vertebrates24. The SCN clock is set to a 24-h day by gland, retina, bone marrow cells, platelets, lympho- the natural light-dark cycle via retinal light input cytes, skin, enterochromaffin cells of gastrointestinal which then sends circadian signals over a neural (GI) tract and in bile in a variety of animal species10, pathway including sympathetic nerve terminals that 16-18. In fact, its concentration in the GI tract is greater project from the superior cervical ganglia (SCG) to than in the pineal gland or in the circulation and in the the pineal gland and thereby driving rhythmic bile16. The concentration of melatonin in the GI melatonin synthesis. The main photoreceptor pigment tissues surpasses blood levels by 10–100 times and for circadian timing appears to be melanopsin in the there is at least 400× more melatonin in the retinal ganglion cells25. Specifically, SCN is the major gastrointestinal tract than in the pineal gland16. regulatory site of of AA-NAT activity, which is the CHOWDHURY et al.: FIFTY YEARS OF MELATONIN DISCOVERY 291

penultimate and key enzyme in the synthesis of Post-ganglionic sympathetic nerve fibers that end at the melatonin from tryptophan. The mechanism of pineal gland release noradrenalin (NA) or norepinephrine biosynthesis of melatonin is presented in Fig. 2. (NE) which also plays crucial role in the control of

There are notable species differences in the melatonin synthesis. The nocturnal elevation in regulation of AA-NAT synthesis. In human and ovine noradrenergic stimulation via β and α1- adrenergic system, the enzyme is regulated primarily at a post- receptor of pinealocytes increase the intra-cellular transcriptional level, whereas in rodents, the key event concentration of cAMP, which in turn activates AA- appears to be cyclic AMP-dependent phosphorylation NAT20 resulting in increased levels of melatonin at night. of a transcription factor that binds to the AA-NAT Stimulation of α-adrenergic receptors potentiates the β- promoter20. The rapid decline in activity of AA-NAT stimulation and requires participation of other molecules with light treatment at night26 appears to be complex such as calcium ions, phosphatidylinositol, diacyl and associated with the control of catabolism through glycerol, and protein kinase C30. Thus, the synthesis and various phosphorylation of AA-NAT by protein release of melatonin are stimulated by darkness (~80% kinase A (PKA), Rho kinase, checkpoint protein 1 synthesis) and are inhibited by light. The regulation of (CHK1)19 and subsequent association of the melatonin synthesis is also influenced by 5-HT, phosphorylated AA-NAT with the chaperone-like monoamine oxidase, cortisol, corticosterone, aldosterone, 14.3.3 protein27. This dimerization either drives the testosterone and estradiol, but their exact molecular complex to a proteosomal-mediated proteolysis or mechanisms are not yet clearly known31,32. protects the complex from breakdown28. Catabolism of AA-NAT is limited to the pineal gland, whereas Secretion the retinal AA-NAT enzyme appears to be protected The secretion of melatonin is related to the length against breakdown during the day29. of night. A single daily light pulse of suitable

Fig. 2—Diagrammatic presentation of the mechanism of biosynthesis and catabolism of melatonin [The hormone melatonin is synthesized from tryptophan under the control of the enzymes tryptophan 5-hydroxylase, 5-hydroxytryptophan-(5HTP)-decarboxylase, serotonin-N-acetyltransferase (NAT) or AANAT, and hydroxyindole-o-methyl transferase (HIOMT). The formation and secretion of AANAT and HIOMT are influenced by suprachiasmatic nucleus (SCN) activity via seasonal and circadian timing mechanisms. The catabolism of melatonin is through kynuric pathway, including metabolites formed by interaction of N1-acetyl-5-methoxykynuramine

(AMK), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), glucuronides etc.] 292 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, OCTOBER 2008

intensity and duration26 in otherwise constant pineal gland38 suggesting that melatonin is taken up darkness is sufficient to phase shift and to by brain tissues. In the blood, 50-75% of total synchronize the melatonin rhythm in animals to 24 h melatonin is bound reversibly to albumin and via SCN21. There are clear indications that prolonged glycoproteins. The half-life of melatonin is duration of the night leads to longer duration of bi-exponential, with a first distribution half-life of secretion of melatonin in most animal species3. Ocular 2 min and a second of 20 min13. The endogenous half- light serves to entrain/synchronize the rhythm to 24 h life of melatonin in serum is about 30-60 min and and to suppress secretion at the beginning and/or the exogenous melatonin has even shorter half-life, end of the dark phase. The amount of light required to 12-48 min39. suppress melatonin secretion during the night varies The mechanism of melatonin catabolism is less from species to species with the time of night and understood with the exception of conjugation steps with the history of previous light exposure31,32. The that account for ~70% of the ingested dose. Melatonin amplitude of nocturnal melatonin secretion exhibits is metabolized non-enzymatically in all cells, and by considerable inter-individual differences and is free radicals as an oxidant. It is converted into cyclic possibly genetically determined. In humans, serum 3-hydroxymelatonin (c3OHM), when it directly levels of melatonin is low during the day (10-20 scavenges two hydroxyl radicals40. The circulating pg/ml), but significantly higher at night (80-120 melatonin is primarily metabolized through classical pg/ml) with peak between 24:00 h and 03:00 h. The hydroxylation pathway in human and rodent liver by onset of secretion usually takes place during 22.00- microsomal enzymes, cytochrome P450 mono- 02:00 h and the offset between 07:00-09:00 h31,32. oxygenases (isoenzymes CYP1A2, CYP1A1 or Melatonin concentration varies in relation to the CYP1B1) to 6-hydroxymelatonin which further stage of development, puberty, menstrual cycle and conjugates with either sulphate (catalyzed by age of the individuals. Its peak values in blood may sulphotransferase ST1A3) to form 6-sulphatoxy also vary from one individual to the other depending melatonin (MT6s), or glucuronic acid (catalyzed by on their age, sex and disease10. Just after birth, very UDP-glucuronosyltransferase) to form 6-hydroxy little melatonin or 6-sulphatoxymelatonin (aMT6s) is melatonin glucuronide13. Several other metabolites detectable in the body fluids. A robust melatonin (approximately 30% of overall melatonin) are also rhythm appears in 6 to 8 weeks of age33. The serum formed by pyrrole ring cleavage19. This oxidative melatonin level increases rapidly thereafter and catabolism by indoleamine-2, 3-dioxygenase and/or reaches a lifetime peak in between 3-5 yr of age34. myeloperoxidase leads to the formation of unstable The increment is much greater at night (54-75 pg/ml). intermediary kynuramine derivative N1-acetyl-N2- Subsequently, a steady decrease occurs reaching to a formyl-5-methoxy-kynuramine (AFMK), which is mean adult concentration in mid to late teens with the further deformylated to more stable N1-acetyl-5- major decline before puberty, with relatively stable methoxy-kynuramine (AMK) by kynuramine until 35 to 40 yrs, and further reach to low levels formamidase. Thus, the clearance rate of melatonin is (16-40 pg/ml) in old age31,32,35. However, elderly predominantly associated with excretion in the urine women show higher levels of melatonin than in in metabolized form and in small quantities in elderly men. Although melatonin concentration is low unmetabolized form41. in precocious puberty, higher melatonin is found in delayed puberty and hypothalamic amenorrhea, Mechanism of action at cellular level 34 The development of high affinity radioligand compared to age-matched control and abnormal 125 melatonin secretion in patients with pre-menstrual binding assay of 2-[ I] iodomelatonin, autoradio tension36. Low melatonin is associated with graphy and studies with putative melatonin receptor cardiovascular diseases and diabetic autonomic agonists and antagonists have allowed to identify, neuropathology37. characterize and demonstrate distribution patterns of melatonin receptors in various central and peripheral 42,43 Metabolism tissues . The first melatonin receptor was cloned Pineal melatonin is released in the cerebrospinal from Xenopus laevis immortalized melanophore fluid in the third ventricle via pineal recess and attains mRNAs44, but expressed only in non-mammalian up to 20-30 times higher levels than in blood, but species (birds and fish). Subsequently, human rapidly diminishes with increasing distance from the melatonin receptors have also been cloned45. CHOWDHURY et al.: FIFTY YEARS OF MELATONIN DISCOVERY 293

Two forms of high-affinity melatonin receptors and thereby play a role in the initiation of S and M (MT1 and MT2) and a low-affinity receptor (MT3) phases of the cell cycle, cell cycle-related gene have been identified based on pharmacological and expression, and the re-entry of quiescent cells from G0 kinetic differences in 2-[125I] iodomelatonin binding back into the cell cycle55. assay46,47. In human, MT1 receptor consists of 350 Becker-Andre and co-workers56 demonstrated that amino acids, and is mapped to chromosome 4q35.148 beside MT receptors, melatonin has genomic action while gene for MT2 receptor is located in through novel class of orphan nuclear receptors of the chromosome 11q21-22 and cDNA encodes a protein retinoic acid receptor family. These receptors have containing 365 amino acids with 60% homology with been cloned and named as retinoid Z receptor (RZR) MT1 receptor45. These membrane-bound receptors and retinoid acid receptor-related orphan receptor (MT1 and MT2) belong to the superfamily of (ROR)56,57. The RZR/ROR family consists of three guanidine triphosphate binding proteins or G-protein subtypes: α, β and γ. The RORα1, RORα2 and RZRβ coupled receptors containing the typical seven have low affinity in normal and cancer tissues59. 7,46,49,50 transmembrane domains . Studies on RORα1 and RORα2 receptors are involved in 125 immunolocalization, 2-[ I] iodomelatonin binding immune modulation, whereas the receptor RZRβ is assay and cloning of mRNA from different tissues expressed in the CNS, including pineal gland. have shown that these receptors are widely distributed Melatonin also acts in an intracrine, paracrine or in the peripheral tissues, viz. heart, arteries, kidney, autocrine manner in the pineal gland, eye, liver, gall bladder, intestine, adipocytes, granulosa lymphocytes, gut, bone marrow, skin, and gonads for cells of ovarian follicles, uterus, breast cells, prostate the local coordination7,16,17,52,58,60,61. The mechanism of 7 and skin , besides retina, cornea, ciliary body, lens, actions of melatonin at cellular level is schematically 51 choroids and sclera . Even MT1 receptor is expressed presented in Fig. 3. in the SCN, hypophyseal pars tuberalis and other parts of the brain including hypothalamus, thalamus, Functions: A testimony of physiological diversity hippocampus, cerebellum and cerebral cortex52. . The Due to pleiotropic nature of melatonin, a wide density of melatonin receptors not only varies with range of its physiological or patho-physiological species and location, but also with the lighting functions is ascribed to human or other mammalian regime, time of the day, tissues, and developmental or species. Most of the studies have used endocrine status of the concerned animals53. pharmacological doses of melatonin (1 µM and above) and a few studies have confirmed these The SCN is the putative site of circadian action of functions either clinically or experimentally as melatonin, and the hypophyseal pars tuberalis is the opposed to physiological doses (below the nanomolar putative site for its reproductive effects. Melatonin range) of melatonin49,62. Several recent binding with MT1 modulates intracellular signal by publications7,8,11,12,63,64 have reviewed melatonin inhibiting adenylate cyclase and stimulating involvement in multiple physiological functions, but phosphoinositide hydrolysis, while activation of MT2 only a few of them have shown its importance in the receptor inhibits formation of two second messengers 13 context of human health. namely, cAMP and cGMP at cellular levels . The decrease in cAMP production reduces the uptake of Synchronization of rhythmic body functions with the linoelic acid, an essential and major fatty acid by environment 54 specific fatty acid transporters MT1 acts by In all living organisms, circadian periodicity suppressing neuronal firing activity by mediating (for human 24.2 h/cycle) in many of the body vasoconstriction and MT2 acts mainly by inducing functions is an inherited characteristic which appears 46 phase shifts by mediating vasodilation . to be closely related to diurnal preference and the A third mechanism of the biological effects of early or late timing of the circadian system (cBT) in a melatonin is through MT3 receptor, quinine reductase normal entrained situation65. Melatonin acts as an 55 2 (QR2), a known detoxifying enzyme which is endogenous synchronizer either in stabilizing rhythms identified with lower melatonin affinity, very rapid (circadian) of body functions or in reinforcing them, ligand association/dissociation kinetics and widely hence, called a ‘chronobiotic’ molecule66. Melatonin 55 distributed in various tissues of the body . The MT3 is also considered as a “neuroendocrine transducer” or 5 receptor modulates calcium and calmodulin activity , “hormone of darkness” or “biological night” which is 294 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, OCTOBER 2008

Fig. 3—Mechanism of action of melatonin at cellular level: A schematic presentation exclusively involved in signaling the “length of night” cycles kaput), Bmal1 (Brain and Muscle ARNT-like or “time of day” and “time of year” to all tissues. 1, Mop3), the two homologues of Drosophila In mammalian species and humans, exogenous cryptochrome (Cry1 and Cry2) and casein kinase 1ε administration of melatonin is reported to change the (CK1ε). Human clock genes show high similarity to timing of rhythms by increasing sleepiness, wake those of other mammalian clock genes. The Per1, 2, EEG theta activity, REM (rapid eye movement) sleep 3; clock, Bmal1 (Mop3), Cry1 and Cry2 genes are 74,75 propensity, non-REM sleep propensity, endogenous expressed in all peripheral tissues . melatonin and cortisol levels; and decreasing core The clock genes are conserved from flies to human. body temperature that ultimately leads to sleep4,67,68. Nevertheless, the functional roles played by some of This phase shifting effect of melatonin depends upon their corresponding products differ between insects its time of administration. It causes phase-advance of and mammal. In human, the pars tuberalis and SCN the circadian clock when given during the evening clock genes expression pattern show 24 h rhythmicity. and the first half of the night, and phase-delays when Per1 is activated at the beginning of light phase and given during the second half of the night or at early Cry1 at the onset of dark phase. Long or short daytime. The magnitude of phase-advance or phase- photoperiod information is encoded within the SCN. delay depends on the dose of melatonin69. As Thus, synthesis of melatonin is driven by the SCN to melatonin crosses the placenta, it plays an active role convey the photoperiodic information to the pars in synchronizing the fetal biological clock13. tuberalis by virtue of its secretion pattern. This phenomenon, in turn, influences the pattern of The chronobiotic effect of melatonin is caused by expression of the clock genes Per1 and Cry1 within its direct influence on the electrical and metabolic the pars tuberalis, providing a means of translating the activity of the SCN as shown by in vitro as well as in melatonin signal for the control of body rhythm or vivo experiments71-73. Melatonin through various rhythmic synthesis and secretions of hormones42. receptors induces a differential influence on clock However, amplitude modulation is unrelated to clock genes. The currently known mammalian clock genes, gene expression in the SCN42,76. which have been cloned only during the last one- decade include three mammalian homologues of The circadian regulation is also determined by the period (Per1, 2, 3), clock (circadian locomotor output interactions with neurotransmitter functions. The CHOWDHURY et al.: FIFTY YEARS OF MELATONIN DISCOVERY 295

highest concentration of serotonin in the CNS is in the melatonin levels and the quality of sleep, decline at SCN77,78 and its turnover exhibits marked circadian puberty and in elderly82. The period of sleep tends to and seasonal rhythmicity, which is rapidly stimulated become shorter and the quality of sleep poorer with by light exposure77. At the cellular level, circadian decreased amplitude of the and rhythms of other clock genes are driven by the waking in a 12 h light/12 h dark cycle, and sometimes interlocking self-regulatory interaction. The positive with phase advancement of circadian rhythm called arm of the circuit is the heterodimer of the proteins delayed sleep phase syndrome (DSPS)81. Cross CLOCK:BMAL1. This complex binds E-box meridian flights involve disorganization of biological elements (CACGTG/T) at the promoter region of rhythm caused by the rapid change of environment Per1-2 and Cry1-2, including their transcription. The and associated light/dark cues. negative regulators are the translated CRY and PER The clock genes in the SCN gradually adapt to proteins that complex with CK1ε to translocate into phase-shift of the light dark cycle (as found in shift the nucleus, interacting with the CLOCK:BMAL1 work, transmeridian flight). Peripheral clocks in the complex and thus blocking their own transcription. muscle, liver, pancreas, kidney, heart, lung and Phosphorylation of PER and CRY proteins by CK1ε mononuclear leukocytes are entrained directly by the controls their proteosomal degradation, delaying the SCN through some neurohormonal signals, formation of CRY:PER complex and determines the glucocorticoids, retinoic acid, growth factors and by length of the cycle. Additional regulators are negative other Zeitgebers, such as body temperature and loop generated by the transcription of the dec1 feeding time, which resynchronize their clock genes (sharp2/stra13) and dec2 (sharp1) genes, which are at their own rates83,84. Circadian clocks in peripheral also driven by CLOCK: BAML1 via E-boxes in their tissues/cells also exhibit identical features. This promoters. results in “double desynchronisation”-“internal DEC1 and DEC2 proteins may block circadian desynchronisation” between different clocks in the gene expression, in part by forming a non-functional body and brain, and “external desynchronisation” heterodimer with BMAL1, which inhibits the between the timing of body rhythm with respect to the expression of all genes dependent on an E-box as well light-dark cycle. This temporal orchestra of ‘jet lags’ as play a role in light induction of genes in the SCN. (sleep disturbance, mental inefficiency or daytime The circadian oscillation of clock gene expression fatigue) can be corrected by melatonin given at local 85 controls the expression of genes involved in multiple bedtime i.e., between 10 p.m. and mid-night . cellular functions in the 24 h period (clock control Moreover, patient or animal model with primary genes, CCGs) by at least two mechanisms: direct insomnia (wakefulness and inability to fall asleep interaction with E-boxes in the promoters of these before 2.00 to 3.00 a.m.), narcolepsy (a disorder of genes; and through the regulation of other CCGs that disturbed circadian sleep/wake rhythm and REM in turn are influenced by transcription factors like sleep deficit) and sleep disorders in children DBP79. These studies reveal that melatonin is an (hyperactivity disorder) can be successfully corrected 86 effective chronobiotic molecule that synchronizes with pharmacological doses (5-50 mg) of melatonin . rhythmic body functions with the environmental Thus, melatonin can be used at local bedtime to variables and clock genes. resynchronize the circadian oscillator with the new environment for coordination of circadian rhythms and sleep function86,87. However, higher secretion of Regulation of sleep-wakefulness cycle melatonin causes maximum sleep and fatigue at Two-process model of sleep regulation considers night87. These studies indicate that application of the timing and architecture of sleep to be a melatonin in optimum dose and schedule attenuates consequence of a homeostatic process of rising sleep sleep related problems in different human age groups. pressure and the duration of prior wakefulness that is dissipated during the sleep period, and is a function of Regulation of mental state, behaviour and brain functions circadian pacemaker80. Over the last decade, two The pineal gland promotes homeostatic equilibrium important protocols have been developed to through melatonin, acts as a “tranquilizing organ” investigate circadian and sleep homeostatic processes in stabilizing electrical activity of the CNS and in human81. A strong relationship has been found causes rapid synchronization of the EEG88. The between sleep and melatonin levels67. Both, nocturnal classic endogenous or non-seasonal depression is 296 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, OCTOBER 2008

characterized by the insomnia (early morning inhibits cytochrome p450 enzyme CYP1A2, which awakening), appetite suppression, weight loss and metabolizes the melatonin in hepatocytes, leading to advanced onset of nocturnal melatonin release that an increase in endogenous melatonin levels99,100. begins in the spring and persists through the summer Thus, it finally supports serotonergic system to or winter during the period of light-phase change or elevate mood, reduce aggression, increase shortening89. Similarly, seasonal affective disorder the pain threshold, reduce anxiety, relieve insomnia, (SAD) is characterized by late sleep, morning improve impulse control and ameliorate obsessive- hypersomnia, increased appetite, and retarded onset of compulsive syndromes91. However, higher doses or nocturnal melatonin release90 which peaks in the long-term use of TCA has side effects by blocking autumn and spring68. Similar phenomena are muscarinic, histaminergic and adrenergic receptors, associated with individuals with low nocturnal because of its joined benzene ring structure. In melatonin levels and major depressive/panic isolated cases, a long-term use of melatonin has been disorders91. found to cause psychomotor disturbance, increased The epidemiologic and chronobiological evidences seizure risk and blood clotting abnormalities. strengthen the link between melatonin levels, pineal Collectively, these findings suggest that appropriate function and mood disorders. Administration of exogenous melatonin administration can restore different doses (>1 g/day) of melatonin in these human neurological disorders with direct impact on 64 individuals at night prolongs the nocturnal melatonin general health of elderly people . rise and helps in recovering SAD by changing the Scavenger of free-radicals expression of clock gene and by changing the 13 expression of Per2 gene in bipolar or classic Melatonin, because of its lipophilic nature , depression91,92. However, large doses in the morning crosses all morphological, physical and or early afternoon failed to show any clear effect93, hematoencephalic barriers (blood brain barrier, placenta) and reaches all tissues of the body within a though phototherapy as an adjuvant may accelerate 101 response to antidepressants among patients with short period of time . As a result, it exhibits depression94. Melatonin secretion has been shown to antioxidant effect and performs a very important be wavelength-dependent as exposure to mono receptor-independent metabolic function, i.e., multi- chromatic light at 460 nm produces a 2-fold greater faceted scavenger of free radicals. The antioxidant circadian phase-delay95. These results are further effects of melatonin have been well described, and are confirmed by measuring the brain serotonin and known to include both direct and indirect effects with equal efficiency in multiple sites (nucleus, cytosol and tryptophan levels that rise after melatonin 78 administration ,and directly linked with an array of membranes) of the cell . It detoxifies a variety of free neuropsychiatric phenomenon96. The diminished radicals and reactivates oxygen intermediates, central serotonin, as indicated by low levels of including the hydroxyl radical/hydrogen peroxide, serotonin marker 5-hydroxy indole acetic acid peroxy radicals, peroxynitrite anion, singlet oxygen, (5-HIAA) in cerebrospinal fluid is associated with nitric oxide and lipid peroxidation. impulsiveness, aggression and auto-aggregation, Melatonin is a more potent antioxidant than vitamin C alcoholism, compulsive gambling, overeating, and and E102,103. The antioxidant property of melatonin is 97 other obsessive-compulsive behaviours . Moreover, shared by two of its major metabolites, namely N1-acetyl- the requirement of intact β-receptor function for N2-formyl-5-methoxy-kynuramine (AFMK)41 and a melatonin synthesis and stimulatory effect of nor- higher efficient, N1-acetyl-5-methoxykynuramine epinephrine on melatonin synthesis and release (AMK)104. AFMK is produced by both enzymatic and demonstrate a direct relation of melatonin to non-enzymatic mechanisms105 and mainly by 98 depression . myeloperoxidase19. The potent scavenger AMK Administration of tricyclic antidepressants (TCA) consumes additional radicals in primary and secondary at night also exerts sedative effect with increased reactions106. Interestingly, AMK interacts not only with melatonin synthesis through binding with reactive oxygen, but also with reactive nitrogen species107. β-adrenergic receptors of pinealocyte and increased It exerts its effects on electron flux through the respiratory cAMP production, which in turn, activates AA-NAT chain and improves ATP synthesis in conjugation with and enhances melatonin rhythm amplitude. TCA also the rise in complex I and IV activities108. The broad- CHOWDHURY et al.: FIFTY YEARS OF MELATONIN DISCOVERY 297

spectrum antioxidant activity of melatonin also includes system12. Likewise, exogenous administration of indirect effect of upregulating several antioxidative melatonin causes vasodilatation, decreasing internal enzymes and downregulating pro-oxidant enzymes, in artery pulsatile index, increases the cardiac vagal general, and 5- and 12-lipo-oxygenases109, glutathione tone, decreases circulating norepinephrine levels and peroxidase, glutathione reductase, glucose-6-phospahte finally reduces blood pressure in hypertensive dehydrogenase, superoxide dismutase, catalase110 and patients121. Antioxidant properties of melatonin can nitric oxide (NO) synthase40, in particular. also be additive to the treatment of gastric ulcer, Due to strong antioxidant property, melatonin bowel syndrome, ulcerative colitis, diarrhea, glycerol- protects lipid in membranes, proteins in cytosol, DNA in induced renal failure122,123. Thus, melatonin can act as nucleus and mitochondria from free radical damages. a potent free radical scavenger, neutralizing hydroxyl Thus, it can be used in the treatment of several and peroxyl radicals among others, preventing lipid neurodegenerative diseases such as Alzheimer’s disease membrane peroxidation, apoptosis and protecting the (AD), Parkinson’s disease, Huntington’s chorea and DNA from the damage induced by free radicals124. amyotrophic lateral sclerosis, which are caused by inter- Modulation of immune function related processes namely glutamate excitotoxicity, free The role of melatonin as an immunomodulator in the radical-mediated nerve injury and mitochondrial 111-114 regulation of development, differentiation, and functions dysfunction . Studies on disruption of nocturnal of lymphoid tissues is known for nearly past three surge of melatonin in ischaemic stroke patients and 58,125-127 115 decades . In rodents, post-natal pinealectomy patients with acute cerebral haemorrhage and suppresses immunity and thymic atrophy, whereas reduction in the degree of tissue damage against exogenous melatonin treatment increases cell-mediated ischaemic injury through direct free radical scavenging immune function by increasing natural killer cell or by indirect antioxidant activities in these exogenously activity104. Immunomodulatory effects of melatonin are melatonin administered patients suggest the 116 observed in human with bronchial asthma. The neuroprotective role of melatonin in strokes . nocturnal rise in blood melatonin levels in human is The oxidative stress is an important hallmark in associated with increased production of interleukins (IL- disorganizing the cortical actin cytoskeletal assembly 1, IL-2, IL-6, IL-12), thymosin 1a, thymulin and tumor and disruption of accompanied sub cellular intricate necrosis factor α (TNF α). On the other hand, fibrous network composed of microtubules, exogenous melatonin has an adverse effect in patients microfilaments and intermediate filaments (IF) as well with asthma104. The nocturnal asthma is associated with as by associated proteins117. Melatonin prevents elevation and phase-delay of serum melatonin levels128. cytoskeletal structure disruption, followed by cell shape In adjuvant-induced arthritis, both prophylactic and changes and increased lipid peroxidation or apoptosis therapeutic melatonin administration inhibit the induced by okadaic acid of physiological plasma and inflammatory response129. Melatonin implants enhance a cerebrospinal fluid118. In this process, participation of defined T helper 2-based immune response under in vivo PKC as an important signaling molecule has been conditions, suggesting potential role of melatonin as a evident from the studies that a PKC inhibitor novel adjuvant immunomodulatory agent130. (bisindolylmaleimide) abolishes cytoskeletal re- Melatonin acts on immunocompetent cells establishment elicited by melatonin, while the PKC (monocytes, B-lymphocytes, natural killer lymphocytes, agonist (PMA or phorbol 12-myristate 13-acetate) T-helper lymphocytes, cytotoxic T lymphocytes) through reorganizes microtubules and microfilaments119. Thus, MT1 and RZR/RORα orphan nuclear receptor family and melatonin may be used in the treatment of enhances cytokine production/secretions, cell proliferation neurodegenerative diseases as a cytoskeletal modulator or oncostasis131,132. In B-lymphocytes, it binds to the 5,96,120 as well as a free radical scavenger . RZR/RORα receptors to downregulate 5-lipoxy-genase The anti-oxidant properties of melatonin also help expression, which is an important enzyme in allergic and in reducing blood cholesterol (~38%), mainly by inflammatory diseases like asthma and arthritis133. Thus, inhibiting copper-induced oxidation of low-density melatonin and the immune system are linked by complex lipoprotein (LDL) with reduction in blood pressure bidirectional communication. The immune system, in and catecholamine levels via relaxation of smooth turn, reciprocally regulates pineal gland functions, mainly muscles in aortic walls and potentially contributing to via cytokines produced by activated immune cells and an antiatherosclerotic effect on cardiovascular depends on age, sex and species58,125-127. The precise 298 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, OCTOBER 2008

mechanisms involved in autoimmune diseases are yet inhibitory effects on both the release of corticoids and largely unknown and need further elucidation. their peripheral actions by immune depression, hyper- catabolism, thymic involution and adrenal Effects on endocrine network 129 Melatonin affects the synthesis, secretion and suppression , which finally unequivocally delays action of steroid as well as non-steroid hormones. In aging. However, direct experimental evidences are not vitro and in vivo experiments have shown that effects sufficient to demonstrate a relationship between of melatonin on adenohypophysial activity vary in melatonin and hypothalamic-pituitary-adrenal-axis. A different animal species with their age and sex, and in strong relationship is known between the functions of pineal gland and hypothalamo-hypophysial-thyroid axis relation to the concentration of the hormone and 144 experimental conditions. It modifies synthesis and in rodents and several non-mammalian species . secretion of different adenohypophysial hormones Nevertheless, such relationship is yet to be functionally (growth hormone/somatotrophin, GH; thyrotropin, TSH; characterized in humans.

adrenocorticotropin, ACTH) either directly by Effects on reproduction influencing the secretory activity of the cells in the The role of melatonin in regulation of reproduction anterior pituitary, or indirectly by influencing the is one of the major areas to have received serious hypothalamic neurons producing the respective attention for studies in animals145 as well as in neurohormones which stimulate or inhibit the release of human146. Melatonin has a strong influence on the 134,135 adequate adenohypophysial hormones . Melatonin reproductive function in seasonal mammals by its has a positive phase relation with prolactin synthesis, inhibitory action at various levels of the 134-136 and negative with GH ; it is involved in the hypothalamic-pituiatry-gonadal axis147. It down- regulation of calcium and phosphorus metabolism by regulates gonadotropin-releasing hormone (GnRH) stimulating parathyroid gland or inhibiting calcitonin gene expression in a cyclical pattern over a 24 h 137 release and prostaglandin synthesis . A similar change period and exerts an inhibitory effect by acting on in melatonin levels is observed with the nocturnal G-protein coupled melatonin receptors MT1 and MT2 increase and morning decrease in prolactin levels, and nuclear orphan receptors RORα and RZRβ148. In whereas melatonin administration stimulates prolactin neonatal pituitary cells, it inhibits GnRH-induced 138 secretions . The relationship between melatonin and calcium signaling and gonadotropin secretion through GH is poorly understood. MT1 and MT2 receptors. This inhibitory role of Melatonin affects the activity of pituitary-adrenal melatonin on the hypothalamus is responsible for axis by modulating the peripheral action of pubertal maturation. The decline of serum melatonin corticoids134,135. It possibly acts as a corticotrophin- below a threshold value (~115 pg/ml) may constitute releasing factor inhibitor with dis-inhibition of the the activating signal for hypothalamic pulsatile pituitary-adrenal axis in major depression, when secretion of GnRH and subsequent onset of pubertal pineal melatonin levels are low and unable to changes149. The hypothalamic-pituitary-gonadal axis modulate functions of adrenal gland139. Low is active during fetal life, but remains quiescent until melatonin levels are also found in patients with the age of ~10 yr due to high levels of melatonin and Cushing disease (hyperadrenocorticism)140 and is reactivated thereafter with increase in the amplitude hypercortisolemia, which have been linked to several and frequency of GnRH pulses. aspects of aging and age-associated phenomena, Stimulation of pulsatile secretion of luteinizing including glucose intolerance, atherogenesis, impaired hormone (LH) and follicle-stimulating hormone immune function, and cancer141. (FSH) are crucial for pubertal changes and, therefore, The nocturnal melatonin levels decline or almost the decline in melatonin concentration below the completely lost with aging in humans35,142. This close threshold value is very important for the initiation of reciprocal relation of melatonin and corticoids or loss of puberty as a central rhythm generator134,135. The melatonin rhythmicity may be responsible for the inhibitory effects of melatonin on GnRH action pituitary/adrenal axis dis-inhibition, characteristic of gradually decline due to decreased expression of aging. The adrenal of elderly human is apparently functional melatonin receptors150. The low hypersensitive to adrenocorticotropic hormone, and concentration of melatonin would result in premature midnight corticoid levels (low in young) are markedly activation of the hypothalamic GnRH secretion and elevated at old age143. Thus, melatonin has phasic the occurrence of precocious puberty151. These effects CHOWDHURY et al.: FIFTY YEARS OF MELATONIN DISCOVERY 299

are further clinically demonstrated in humans, thereby preventing the formation of its mitogenic showing that acute oral doses of melatonin amplify metabolite 13-hydroxyoctadecadienonic acid55. In LH pulses in early follicular phase, and stimulate addition, melatonin also inhibits the fatty acid growth prolactin and vasopressin secretions152. factor uptake by cancer cells, inhibits telomerase activity Evidences show that the melatonin can also exert by reducing telomere length, which causes apoptosis in effects on reproductive axis by directly binding to cancer cells. It also inhibits endothelin-1 synthesis, an granulosa cells in the ovary153. In human granulosa angiogenic factor that promotes blood vessel growth in cells, both MT1 and MT2 are present and melatonin tumors, and finally modulates the expression of tumor upregulates LH mRNA-receptor too154. LH is suppressor gene TP53 or inhibits transcriptional essential for the initiation of leuteinization. expression of cyclin D1163. The action at different levels Furthermore, melatonin treatment enhances human of signaling pathways in a tumor cell collectively chorionic gonadotropin (hCG) stimulated pro- suggests melatonin to be a supportive anticancer drug in gesterone secretion with an inhibition of GnRH and cancer prevention and treatment. However, exact GnRH receptor expression. GnRH in the ovary is mechanism of melatonin action on cancer remains to be suggested to be an important paracrine and/or understood properly. autocrine regulator, and may be involved in the 134,135,155 Conclusion regression of corpus luteum . These studies The scientific journey initiated 50 years ago with indicate involvement of melatonin in the maintenance the isolation, purification and characterization of of corpus luteum during pregnancy. melatonin from the bovine pineal gland has already Effects on cancer opened up a new vista of understanding on the The study on the effects of melatonin on cancer has a pleiotropic nature of the functions of this small long history. Cohen et al.156 first indicated the possible tryptophan derivative molecule. The information role of pineal gland on the etiology of breast cancer and summarized in this communication by no means suggested that a decrease in pineal function (i.e. reduction describe all actions of melatonin. However, it in melatonin secretion) could induce a state of relative illustrates briefly the versatile functions of melatonin hyper-estrogenism, and the early and prolonged exposure in humans. This physiologically wonder molecule is of the breast tissue to estrogens could be involved in the unique in sharing the properties of receptor-mediated etiology of breast carcinogenesis. Melatonin is known to actions of hormone as well as of the receptor- shift forskolin and estrogen-induced elevation of cAMP independent actions of an antioxidant. In view of the levels by 57% and 45% respectively, thereby affecting data presented herein, one feels compelled to surmise signal transduction mechanisms in human breast cancer that multiple functions of melatonin may be brought cells157. It is proposed that melatonin through the action forth by primary and secondary effects. on neuroendocrine reproductive axis may downregulate Melatonin may interact with its receptors in the expression of estrogen receptor α (ERα), and finally different parts of nervous system and peripheral inhibit the binding of estradiol-ER complex to the organs, and produce its primary effects as a hormone. estrogen response element158. Subsequent studies have Because of its lipophilic nature, it may also interact shown reduced levels of melatonin in patients with certain with numerous receptors for amino acids, biogenic types of cancers, compared to normal healthy people of amines, and peptides and produce its secondary the same age159,160. effects as a co-hormone, or co-transmitter. However, Melatonin has shown an antiproliferative effect its anti-oxidant actions are relatively recent addition through receptors MT1 and MT2 on various types of to its functional repertoire, which because of cancers (breast, lung, metastatic renal cell carcinoma, lipophilic and hydrophilic properties is readily hepatocellular carcinoma, brain metastases from solid accessible to all cells of the body, and thereby does tumors, ovarian carcinoma, human neuroblastoma cells, not require any receptors for carrying out the bladder carcinoma and erythroleukemia) with tumor scavenging actions on free radicals. Collectively, the growth, and the incidence of metastases has shown functions of melatonin would be to orchestrate, physiological to pharmacological effects of synchronize, and refine the multiplicity of biological melatonin161,162. Moreover, melatonin treatment has functions, which make life possible. shown MT1/MT2-dependent inhibition of uptake of fatty The research for and discovery of how melatonin acids, in general, and of linoleic acid in particular, with its apparent omnipotent effects brings forth these 300 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, OCTOBER 2008

multiple functions may raise the exciting prospect of 12 Altun A & Ugur-Altun B (2007) Melatonin: therapeutic providing new avenues of treating numerous diseases, and clinical utilization. Int J Clin Pract, 1-11 13 Claustrat B, Brun J & Chazot G (2005) The basic thus replacing old treatments which sustain life, but 164 physiology and pathophysiology of melatonin. Sleep Med diminish its quality . Nevertheless, there are several Rev 9, 11-24 practical impediments to routine clinical use of 14 Reiter R J & Tan D X (2002) Melatonin: An antioxidant in melatonin. The optimal dose and timing of edible plants. Ann N Y Acad Sci 957, 341-344 administration remain to be clearly defined, particularly 15 Hardeland R & Poeggeler B (2003) Non-vertebrate melatonin. J Pineal Res 34, 233-241 for various human populations in different geographical 16 Bubenik G A (2002) Gastrointestinal melatonin: locations having different photoperiodic conditions. Localization, function and clinical relevance. Dig Dis Sci There is an urgent need of further research for 47, 2336-2348 clarification of its clinical uses and side effects, 17 Carrillo-Vico A, Calvo J R, Abreu P, Lardone P J, Garcia- especially with prolonged use. Obviously, there is a long Maurino S, Reiter R J & Guerrero J M (2004) Evidence of melatonin synthesis by human lymphocytes and its way to go, before we arrive at a firm conclusion on the physiological significance: possible role as intracrine, physiological and clinical significance of melatonin in autocrine and/or paracrine substance. FASEB J 18, 537-539 human. In the context of existing situation, it “may be 18 Konturek P C, Konturek S J, Brzozowski T, Dembinski A, more exciting to travel than to land.” Zembala M, Mytar B & Hahn E G (1997) Gastroprotective activity of melatonin and its precursor, L-tryptophan, Acknowledgement against stress-induced and ischaemia-induced lesions is The authors are thankful to their colleagues, mediated by scavenge of oxygen radicals. 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