Melatonin: Exceeding Expectations Russel J. Reiter, Dun Xian Tan and Annia Galano Physiology 29:325-333, 2014. doi:10.1152/physiol.00011.2014 You might find this additional info useful... This article cites 67 articles, 9 of which can be accessed free at: /content/29/5/325.full.html#ref-list-1 This article has been cited by 1 other HighWire hosted articles Living a Healthier Lifestyle Physiology, September , 2014; 29 (5): 302-303. [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: /content/29/5/325.full.html Additional material and information about Physiology can be found at: http://www.the-aps.org/publications/physiol
Physiology (formerly published as News in Physiological Science) publishes brief review articles on major physiological developments. It is published bimonthly in January, March, May, July, September, and November by the American Physiological Society, 9650 Rockville Pike, Bethesda, MD 20814-3991. Copyright © 2014 by the American Physiological Society. ISSN: 1548-9213, ESSN: 1548-9221. Visit our website at http://www.the-aps.org/.
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PHYSIOLOGY 29: 325–333, 2014; doi:10.1152/physiol.00011.2014
Melatonin: Exceeding Expectations Melatonin is a small, highly conserved indole with numerous receptor-mediated and receptor-independent actions. Receptor-dependent functions include circadian rhythm regulation, sleep, and cancer inhibition. The receptor-inde-
Russel J. Reiter,1 Dun Xian Tan,1 and Annia Galano2 1 Department of Cellular and Structural Biology, UT Health Science Center, San Antonio, Texas; and 2Departamento de Quimica, Universidad Autonoma Metropolitana-Iztapalapa, Mexico D.F., Mexico [email protected]
pendent actions relate to melatonin’s ability to function in the detoxification of free radicals, thereby protecting critical molecules from the destructive effects of oxidative stress under conditions of ischemia/reperfusion injury (stroke, heart attack), ionizing radiation, and drug toxicity, among others. Melatonin has numerous applications in physiology and medicine.
1548-9213/14 ©2014 Int. Union Physiol. Sci./Am. Physiol. Soc.
actions and the evidence that melatonin is produced in all plants and animals that have been studied.
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When Aaron B. Lerner and his associates (29) isolated and characterized the methoxy derivative of serotonin from bovine pineal tissue near the middle of the last century, in their most optimistic outlook they would never have envisioned the multiple functions this molecule would eventually display. As dermatologists, they had engaged in this effort because, based on a publication that appeared 50 years earlier (35), they knew there was something in the pineal gland that blanched cutaneous chromatophores and thereby lightened the skin of amphibians. Indeed, the name they selected for their newly discovered molecule, i.e., melatonin, is in part based on its effect on skin pigmentation (“mela” from melanin and “tonin” from serotonin). After melatonin’s discovery in pineal tissue, the image of the gland quickly changed from that of a functionless vestige to one of an active organ of internal secretion. Biochemists identified the steps in the synthetic pathway of melatonin (4, 41, 67) (FIGURE 1), and the physiologists described a now well known function of the pineal, i.e., the regulation of seasonal reproduction in photoperiod-sensitive mammals (22, 24). By the end of what has been referred to as the Decade of Transformation (mid-1950s to mid-1960s) (7), the pineal gland and melatonin were both recognized as legitimate components of the endocrine system: the pineal was known to synthesize melatonin (67), the gland influenced seasonal reproduction in photosensitive species (23, 24), and it required its sympathetic innervation to remain functional (43, 66). Research within the last 60 years, however, has revealed that melatonin is functionally much more diverse than being only a regulator of the neuroendocrine-reproductive axis in photoperiod-dependent, seasonally breeding mammals. Also, the conflating of melatonin exclusively with the pineal gland has been shown to be untenable (47). Herein, we briefly summarize the data documenting the heterogeneity of melatonin’s multiple
In vertebrates, the pineal gland synthesizes melatonin in a circadian fashion, with nighttime darkness being a requirement for maximal production (57); because of this, melatonin is referred to as the chemical expression of darkness (42). The influence of the prevailing photoperiod on circadian melatonin production is accomplished by a restricted bandwidth of visible light, i.e., wavelengths in the range of 460 – 480 nm (blue light) (FIGURE 1). These wavelengths account for the inhibition of melatonin synthesis in the pineal gland during the day or, if they occur, at night. For the retinal processing of blue wavelengths, the mammalian retinas have up to five highly specialized subtypes of intrinsically photosensitive retinal ganglion cells (ipRGCs), which use a specialized photopigment, melanopsin, to respond to light (31, 68). The message related to the light environment is then transferred to the central biological clock, the suprachiasmatic nuclei (SCN), in the hypothalamus via the retinohypothalamic tract, which is embedded in the optic nerve. The SCN is a critical relay center that conveys a neural signal to the pineal gland (39) (FIGURE 1). The neural message arrives at the pinealocytes from the SCN via the central and peripheral sympathetic nervous system. This is one of several features that distinguishes the pineal from classic endocrine organs, i.e., its output is governed by a neural input, which, if destroyed, renders the gland totally inept (43); likewise, feedback effects from peripherally derived hormonal signals are usually not considered to be major factors in changing the quantity of melatonin produced or altering the phasing of its rhythm (11). Hence, the melatonin rhythm is more 325
REVIEWS or less independent of endogenous influences, and melatonin synthesis is determined almost exclusively by the prevailing photoperiod, thereby making the pineal an unconventional endocrine gland. There is also nothing phylogenetically that ties melatonin production to the pineal gland. Melatonin is not unique to vertebrates but is also produced in invertebrates, unicells, and plants, none of which possess a pineal gland and some of which, i.e., unicells, have no organs whatsoever (37, 50, 51). These nonvertebrates need melatonin for some of the same reasons vertebrates do, e.g., as an antioxidant (37). Some plants already have been genetically engineered to produce elevated quantities of melatonin, which protects them from free
radical-producing environmental stressors (temperature extremes, drought, disease) (8, 9). Melatonin probably evolved more than 2.5 billon years ago, likely in purple nonsulfur bacteria, presumably in Rhodospirillum rubrum (61), to protect them from an oxidizing environment that was a consequence of increasing atmospheric oxygen concentrations associated with the Great Oxygen Event (the Oxygen Catastrophe). These melatoninproducing bacteria were subsequently phagocytized by eukaryotes, a process referred to as endosymbiosis, where they eventually evolved into mitochondria. Because of this, we have speculated that mitochondria in all eukaryotic cells may have retained the ability to produce melatonin (61); if
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FIGURE 1. A diagrammatic representation of the means by which light wavelengths regulate the central circadian pacemaker A diagrammatic representation of the means by which 460- to 480-nm light wavelengths (blue light), detected by the intrinsically photoreceptive retinal ganglion cells (ipRGC), regulate the central circadian pacemaker, the suprachiasmatic nucleus (SCN), and the transfer of photoperiodic information to the pineal gland for the regulation of melatonin synthesis from tryptophan. This vertebrate pathway involves synapses in the paraventricular nucleus (PVN) of the hypothalamus, the intermediolateral cell column (ILCC) at thoracic cord levels 1 and 2, and the superior cervical ganglia (SCG). Melatonin released into the cerebrospinal fluid (CSF) and blood act on the SCN to modulate its circadian activity and on circadian oscillations in peripheral tissues. Besides influencing circadian rhythms, melatonin has many other functions, as summarized in the text. In the pinealocyte depicted at the top right, melatonin synthesis as it occurs in the mammalian pineal gland is shown. Based on currently available data, the synthetic pathway for melatonin production in plant cells differs slightly from that in animals. In plants, tryptophan is initially decarboxylated to tryptamine, which is then hydroxylated to form serotonin. The final two steps are similar to those in the mammalian pineal gland. AANAT, arylalkylamine N-acetyltransferase; AC, adenylate cyclase; ASMT, acetylserotonin methyl transferase; CaMK, calcium/calmodulin protein kinase; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; CSF, cerebrospinal fluid; NE, norepinephrine; NMDA, N-methyl-D-aspartate receptor; NO, nitric oxide; PACAP, pituitary adenylate cyclase-activating peptide; PKA, protein kinase A; RHT, retinohypothalamic tract. 326
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Melatonin, Its Potpourri of Actions Receptor-Mediated Actions Melatonin has an uncommonly large skill set, and, considering that melatonin is billions of years old, it has had ample time to hone its functions. For several decades after its discovery, melatonin was assumed to mediate all of its actions via receptors that were bound to membranes of a limited number of cells, e.g., neurons of the SCN (56). Two membrane receptors were pharmacologically characterized, subsequently cloned, and are now identified as the MT1 (Mel1a) and MT2 (Mel1b) receptors. These are both members of the superfamily of transmembrane, G-proteincoupled receptors. The MT1 receptor is 350 amino acids in length, whereas MT2 consists of 363 amino acids; the receptors share 60% homology (14). There is also an orphan molecule referred to as a melatonin-related receptor (MRR; also called GPR50) that shares 45% homology to MT1 and MT2; little is known of its function. In contrast to the original assumption that the membrane melatonin receptors were located on only a few cells, subsequent investigations have in fact localized them on many cells (55), and they may exist on the membranes of all cells. There is also what is referred to as an MT3 receptor located in the cytosol of a few cells. It is not coupled to a G protein and exhibits low affinity for
iodo-melatonin; it may be equivalent to quinone reductase 2, a detoxification enzyme (21). Finally, melatonin may carry out some of its activities by an interaction with a group of nuclear receptors referred to as retinoid orphan receptors (ROR) or retinoid Z receptors (RZR). The superfamily members that reportedly bind melatonin include the ROR␣, RZR␣, ROR␣2, and RZR. These nuclear receptors contain an NH2-terminal domain, a DNA binding domain, a ligand binding domain (in the COOH terminal), a zinc double finger, and a hinge region. The nuclear receptors may be differentially distributed among tissues (1) and perhaps are best functionally described in the immune system (12, 27). In most cases, their specific functions are under debate.
Receptor-Independent Actions In addition to its abundant actions mediated by its multiple receptors described above, melatonin also directly detoxifies reactive oxygen species (ROS) and reactive nitrogen species (RNS) by nonreceptor-mediated means (45). Like some other classic radical scavengers, major chemical mechanisms by which melatonin ensnares radicals include single electron transfer and hydrogen transfer, but other yet to be defined processes may be involved (15). Consistent with its presumed high intramitochondrial concentrations, melatonin improves the activities of several respiratory chain complexes, thereby reducing electron leakage and free-radical generation (34), a process referred to as radical avoidance (20). Moreover, metabolites of melatonin [cyclic-3-hydroxymelatonin (c3OHM), N1-acetyl-N2formyl-5-methoxykynuramine (AFMK), and N1-acetyl5-methoxykynuramine (AMK)] (FIGURE 2) provide additional protection against oxidative damage by the same mechanisms described above for melatonin and perhaps also by radical adduct formation (16, 17, 48, 59, 62). The relative efficacies of melatonin, c3OHM, AFMK, and AMK as scavengers of the hydroxyl radical (·OH) and the hydroperoxyl radical (·OOH) have been examined in several settings. These results were generated using a variety of techniques, including functional density theory. Although there are slight differences in their relative efficiencies, each of the four molecules scavenge the highly reactive ·OH at diffusion-controlled rates, regardless of the polarity of the environment. Thus they are all highly effective scavengers of the devastatingly reactive ·OH (17, 59, 62). With regard to the ·OOH, neither melatonin, AFMK, nor AMK are particularly effective in neutralizing this agent. This is consistent with melatonin per se being an inefficient chain-breaking antioxidant (33) but inconsistent with the data showing that both in vitro and in vivo melatonin
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so, all tissues in multicellular organisms generate melatonin for their own use (64). Of interest in this regard is that melatonin concentrations in the mitochondria of rat hepatocytes greatly exceed blood levels, and they do not drop after pinealectomy, which depletes melatonin from the blood (64). Many extrapineal organs, as sites of melatonin production, have been identified in vertebrates. Unlike the pineal gland, most of these organs (exception, the retinas) may not synthesize melatonin in a circadian manner nor do they release it into the blood in any significant amount. Rather, in these organs, melatonin functions as an antioxidant, as an autocoid, or as a paracoid to regulate intracellular events. Another misconception that should be dispelled is that blood melatonin levels are representative of its concentrations throughout the body. This is clearly not the case given that the bile (58) and the cerebrospinal fluid (54) have melatonin concentrations that exceed those in the blood by several orders of magnitude, and, as already noted, intracellular levels are also higher than those in the blood. Thus, because of its unequal distribution, blood melatonin concentrations should not be used to judge melatonin levels in other body fluids or within subcellular compartments (47).
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REVIEWS conclusion of these studies is that melatonin both limits the initiation of lipid breakdown and reduces its propagation when the metabolite c3OHM scavenges the ·OOH. Melatonin also reportedly detoxifies the strong oxidizing agent, the peroxynitrite anion (ONOO⫺) and hypochlorous acid (20). Whether melatonin’s metabolites likewise function in these capacities remains untested. To illustrate the importance of melatonin as an antioxidant, throughout evolution, no organism that has been investigated has lost the ability to synthesize this indoleamine, as is the case with some other radical scavengers, e.g., vitamin C. Additionally, besides being endogenously produced, melatonin is ingested in the diet of all animal species given that it is produced in plants (28). These dual routes ensure that melatonin is always available, again unlike some other radical scavengers. Finally, at least in some animal and plant species, melatonin synthesis is upregulated under circumstances where free-radical generation is exaggerated (3). This greatly increases its protective activities. Related to melatonin’s capability of reducing molecular damage due to free radicals are its effects on anti- and pro-oxidative enzymes (45). These actions are prominent and highly reproducible but, mechanistically, not well investigated, although preliminary evidence suggests the involvement of the membrane receptors MT1 and MT2 (49). The major antioxidative enzymes that are stimulated by melatonin under basal conditions include the intracellular superoxide dismutases (CuZnSOD and MnSOD), the selenium-containing glutathione peroxidases (GPX1, GPX2 and GPX3), and catalase (CAT) (FIGURE 3). Conversely, under toxic conditions of high oxidative stress, these proteins are protected from free-radical damage, and their enzymatic activity is preserved. Melatonin also maintains the activities of enzymes that enhance intracellular levels of reduced glutathione (GSH), an important intracellular antioxidant. Thus melatonin promotes glutathione reductase (GRd) activity, which reduces glutathione disulfide (GSSG) to the sulfhydryl form of GSH and ␥-glutamylcysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis (63). The pro-oxidative enzymes inhibited by melatonin include nitric oxide synthase, myeloperoxidase, and eosinophil peroxidase (20).
FIGURE 2. Melatonin and its metabolites that are formed when the parent molecule scavenges toxic radicals
Melatonin Doses and Timing of Administration
This series of reactions is referred to as melatonin’s antioxidant cascade, and it permits melatonin to directly and indirectly detoxify multiple radicals. These molecules differ somewhat in terms of their efficacies toward the radicals they scavenge. 328
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When melatonin is to be used, the dose and timing of when it is given may be critical in determining its efficacy as a treatment. The doses typically used in animal/human studies for
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markedly reduces lipid peroxidation (45). This action was then assumed to be a consequence of melatonin’s ability to prevent the onset of lipid peroxidation by neutralizing the initiating agents, ·OH and the peroxynitrite anion. Subsequently, it was found, however, that c3OHM, in contrast to the other three molecules, is highly effective in scavenging the ·OOH (17, 59). Moreover, c3OHM was found to detoxify the ·OOH almost 100 times faster than trolox (water soluble vitamin E). This is remarkable since vitamin E is considered the primer peroxyl radical scavenging agent and inhibitor of lipid peroxidation. The
REVIEWS promoting activity (10). Each of these functions relies on the circadian message provided by the pineal-derived blood and cerebrospinal fluid melatonin rhythms that are transferred to cells that “have a need to know.” Melatonin is not impeded by any of the known morphophysiological barriers, e.g., blood-brain barrier, blood-testis barrier, etc., and it readily crosses the placenta in an unaltered form to impact the fetus (46). Melatonin’s concentration in some bodily fluids, e.g., CSF, bile, etc., exceeds that in the serum by several orders of magnitude. Likewise, at least in those cells where measurements have been made, subcellular organelle melatonin levels are greater than in the blood and may not rely on the blood as a source of their melatonin (36, 64). We have speculated that all cells have the capability of synthesizing melatonin, particularly in their mitochondria. This locally produced melatonin is for protection from free radicals and from cells in the neighborhood via autocoid and paracoid actions and is not normally released into the blood. Receptor-independent actions of melatonin and its metabolites relate to their ability to directly quench free radicals and nonradical, but toxic,
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membrane receptor-mediated circadian rhythm regulation are usually lower than those used for defeating free radicals, an action that is membrane receptor independent. Moreover, when melatonin is used in situations where an influence on circadian processes is a factor, e.g., sleep in humans, melatonin should be taken in the evening before sleep. Conversely, if the purpose of melatonin use is to harness free radicals, melatonin would be most effective if given when the free radical event, e.g., stroke, occurs, regardless of the time of day. The statement has been made that melatonin functions as a free-radical scavenger only when given in amounts that are considered pharmacological. This is incorrect. For any scavenger, even a single molecule is capable of neutralizing a radical. To combat the massive number of free radicals generated under conditions of severe oxidative stress, however, pharmacological amounts of any antioxidant must be given to neutralize the overwhelming numbers of these toxic brigands that are produced. Vitamins C and E, for example, are often taken in gram quantities, even under conditions where severe oxidative damage is not a consideration. Thus, when melatonin is used to reduce free-radical destruction, it is given in pharmacological doses since the free radicals are also being produced at “pharmacological” levels.
Melatonin Isomers Isomers of melatonin have been identified in plant products (19, 65), and we have predicted they will eventually be found throughout the plant and animal kingdoms (60). Isomers are molecules with the identical molecular size as melatonin; they also possess the same methoxy residue and the aliphatic side chain as melatonin itself, but they are in different locations on the indole nucleus. Presently published reports show that the isomers bind to classic membrane melatonin receptors and function in free-radical detoxification (60).
Melatonin: Functionally, a Swiss Army Knife Like the multi-tooled device bearing the Swiss coat of arms, melatonin has a bewildering array of functions and employs a variety of means to carry them out (FIGURE 4). These actions likely impact every cell in every organism throughout the plant and animal kingdoms. As a consequence, the physiological and pathophysiological actions of melatonin are numerous. Melatonin is best known for its mediation of circannual variations in metabolism and reproductive competence in photosensitive species (5), its ability to influence circadian processes that are ubiquitous in organisms and in cells, and its sleep-
FIGURE 3. The relationship of molecular oxygen to the formation of reactive oxygen species, reactive nitrogen species, and the production of hypochlorous acid The relationship of molecular oxygen (O2) to the formation of reactive oxygen species (O2·⫺; 1O2; H2O2; ·OH), reactive nitrogen species (NO·; ONOO⫺), and the production of hypochlorous acid (HOCl), a toxic chloride species. Also shown are the enzymes that are either stimulated (1) or inhibited (2) by melatonin. CAT, catalase; GCL, glutamylcysteine ligase; GPx, glutathione peroxidase; GRd, glutathione reductase; MPO, myeloperoxidase; NOS, nitric oxide synthase; SOD, superoxide dismutase.
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REVIEWS physiological collapse, further exaggerating the production of free radicals. Melatonin, because of its ability to neutralize radicals, defers age-related dysfunction of several organs (22). Increased age leads to a gradual diminished melatonin production such that, in the elderly, the nocturnal melatonin rise in the circulation is either greatly attenuated or it no longer exists. The consequences of this reduction may be highly significant in terms of health (44). Since the cycle of melatonin strengthens circadian rhythms by acting at the SCN and also on peripheral slave oscillators, normal circadian rhythms deteriorate, and, considering their importance for optimal health, the dysregulation of these rhythms, e.g., the sleep/wake cycle, negatively impacts organisms. Additionally, the loss of melatonin during aging contributes to the accelerated accumulation of oxidative stress due to the reduced availability of this important antioxidant. This presumably contributes to the progression of diseases that have a free-radical component, e.g., neurodegenerative diseases (52), cardiovascular disease (13), skin deterioration (25), and metabolic syndrome (38),
FIGURE 4. Some of the numerous actions of melatonin in mammals Melatonin has both receptor-dependent and receptor-independent actions. The indole binds to well known membrane receptors (MT1 and MT2) and, via several signal transduction pathways, influences a host of physiological effects. MT1 and MT2 may homo- and/or heterodimerize in some cases, and they may interact with nuclear receptors (binding sites). There is considerable debate regarding the existence/function of the orphan nuclear melatonin receptors ROR and RZR. The cytosolic receptor, MT3, is a detoxifying enzyme, quinone reductase 2. Receptor-independent actions are mediated by the ability of melatonin and its metabolites to scavenge reactive oxygen (ROS) and reactive nitrogen species (RNS). These actions allow melatonin to protect against a wide variety of toxins and processes that generate highly toxic reactants. Any cell can simultaneously respond to melatonin by both its receptor-mediated and receptor-independent actions. Many of the documented physiological and molecular actions of melatonin are not listed in this figure. Additionally, the figure does not include melatonin functions in nonvertebrates or in plants. 330
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species. Excessive free-radical generation is notoriously destructive and kills cells secondary to massive oxidative damage, which induces cellular apoptosis or necrosis. The loss of cells due to programmed cell death is a consequence and/or contributes to many diseases as well as to age-related deterioration. Melatonin’s ability to prevent molecular damage meted out by free radicals and the cellular mutilation becomes manifested when the indole protects against that destruction. Such damage can be produced by ultraviolet and ionizing radiation–ingestion of toxins, heavy metals, alcohol, and prescription drugs–smoking, ischemia/ reperfusion injury, which occurs during a heart attack or stroke, severe inflammation, neurodegenerative diseases, and many other pathophysiological situations (45). Similarly, aging is associated with the progressive accumulation of oxidative debris, which contributes to functional inefficiency of cellular processes, thereby inducing additional free radicals to be produced. Thus aging becomes a vicious cycle. As molecular processes fail, oxidative damage accumulates, which leads to additional
REVIEWS among others. In experimental studies, supplementing aging animals with melatonin defers some of these debilitating changes (22). Some functions of melatonin may depend on both receptor-mediated events and on receptorindependent processes. As an example, melatonin inhibits the proliferation of many cancer cells (40) as well as limiting tumor metastases (32). The ability of melatonin to forestall cancer cell division and the molecular processes associated with the relocation of cancer cells to a secondary site may involve the classic membrane receptors that these cells possess as well as its receptor-independent antioxidative functions; the latter also may account for melatonin’s ability to promote apoptosis of cancer cells (6). This pro-apoptotic action of melatonin in cancer cells is diametrically opposite to its anti-apoptotic function in normal cells, a differential action that has been difficult to explain (6).
Melatonin, Its Special Association With Mitochondria
Melatonin’s flummoxing array of physiological actions as well as the numerous means by which these actions are mediated supports the idea that melatonin is a multitasking molecule. Regarding both these features, melatonin has certainly exceeded the expectations of the most ardent melatonin devotees. Yet, there is likely more to come. Stable circadian rhythms, which melatonin aids in regulating, are crucial for the optimal generalized physiology of organisms and for the microphysiology of molecular functions. Likewise, melatonin’s ability to modulate oxidative processes and protect against free-radical mutilation of essential molecules is indispensible for flawless cellular function. Research on melatonin seems to be at the “end of the beginning.” Obviously, extensive research has been performed regarding the actions of melatonin in experimental cells, plants, and animals. Its uncovered actions are uniformly beneficial, although not all the specific mechanisms have been described. In addition to its multiple positive physiological actions, melatonin has an uncommonly high safety profile. Thus it is time to advance this research to the next level, i.e., to more extensively test its use in clinical trials (26). Although small steps are being taken in this direction (53), it is essential that multi-centered, blinded, well controlled studies be performed using adequate amounts of melatonin and for appropriate durations to determine melatonin’s ability to defer or prevent certain diseases, as suggested by the numerous experimental studies that have been performed. This is especially important since prevention always trumps treatment. 䡲
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Mitochondria are a major site of free-radical generation since electrons that are fumbled during their transfer through the electron transport chain reduce molecular oxygen to the superoxide anion radical (O2·⫺). This oxidizing agent is quickly dismutated to form hydrogen peroxide (H2O2), which is transformed, in the presence of transition metals, to the ·OH (FIGURE 3). Alternatively, O2·⫺ may couple with nitric oxide to produce the highly toxic ONOO⫺. If the prediction that melatonin is produced in mitochondria is valid, its generation in these organelles would be a major advantage for reducing mitochondrial damage and preserving energy production. Melatonin is highly effective as an antioxidant at the mitochondrial level (2). When compared with synthetically produced, mitochondrial-targeted antioxidants Mito Q and Mito E, which are concentrated up to 500-fold in mitochondria (18), melatonin still performed better in reducing every aspect of damage to this organelle (30). The implication is that melatonin, when administered via any route, is absorbed and makes its way to mitochondria in sufficiently high amounts where it is more effective than the synthetic antioxidants, which are known to accumulate in the matrix of mitochondria, in preserving the function of these critically important organelles. Thus, besides being produced in mitochondria, when it is taken as a supplement or consumed in the diet it is readily available to mitochondria. Thus melatonin may be classified as a naturally occurring, mitochondrialtargeted antioxidant. Melatonin may have regulatory actions on mitochondrial uncoupling protein, but these functions remain poorly defined.
Conclusions and Perspectives
No conflicts of interest, financial or otherwise, are declared by the author(s). Author contributions: R.J.R. and D.-X.T. analyzed data; R.J.R., D.-X.T., and A.G. interpreted results of experiments; R.J.R. drafted manuscript; R.J.R. and D.-X.T. edited and revised manuscript; R.J.R., D.-X.T., and A.G. approved final version of manuscript; A.G. prepared figures.
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12. Carrillo-Vico A, Guerrero JM, Lardone PL, Reiter RJ. A review of the multiple actions of melatonin on the immune system. Endocrine 27: 189 –200, 2005. 13. Dominguez-Rodriguez A, Abreu-Gonzalez P, Avanzas P. The role of melatonin in acute myocardial infarction. Front Biosci 17: 2433–2441, 2012. 14. Dubocovich ML, Markowska M. Functional MT1 and MT2 melatonin receptors in mammals. Endocrine 27: 101–110, 2005. 15. Galano A. On the direct scavenging activity of melatonin towards hydroxyl and series of peroxyl radicals. Phys Chem Chem Phys 13: 7178 –7188, 2011. 16. Galano A, Tan DX, Reiter RJ. On the free radical scavenging activities of melatonin’s metabolites, AFMK and AMK. J Pineal Res 54: 245–257, 2013.
25. Kim TK, Kleszczynski K, Janjetoric Z, Sweatman T, Lin Z, Li W, Reiter RJ, Fischer TW, Slominski AT. Metabolism of melatonin and biological activity of intermediates of melatoninergic pathway in human skin cells. FASEB J 27: 2742–2755, 2013. 26. Korkmaz A, Reiter RJ, Topal T, Manchester LC, Oter S, Tan DX. Melatonin: an established antioxidant worthy of use in clinical trials. Mol Med 15: 43–50, 2009. 27. Lardone PJ, Guerrero JM, Fernandez-Santos JM, Rubio A, Martin-Lacave I, Carrillo-Vico A. Melatonin synthesized by T-lymphocytes as a ligand of the retinoic acid-related orphan receptor. J Pineal Res 51: 454 – 462, 2011. 28. Lei Q, Wang L, Tan DX, Zhao Y, Zheng XD, Chen H, Li QT, Zuo BX, Kong J. Identification of genes for melatonin synthetic enzymes in ‘Red Fuji’ apple (Malus domestica Borkh. cv Red) and their expression and melatonin production during fruit development. J Pineal Res 55: 443– 451, 2013. 29. Lerner AB, Case JD, Takahashi Y, Lee TH, Mori W. Isolation of melatonin, the pineal factor that lightens melanocytes. J Am Chem Soc 80: 2587– 2592, 1958. 30. Lowes DA, Webster NR, Murphy MP, Galley HF. Antioxidants that protect mitochondria reduce interleukin-6 and oxidative stress, improve mitochondrial function, and reduce biochemical markers or organ dysfunction in a rat model of acute sepsis. Br J Anaesth 110: 472– 480, 2013. 31. Lucas RJ, Peirson SN, Berson DM, Brown TM, Cooper HM, Czeisler CA, Figuero MG, Gamlin PD, Lockley SW, O’Hagan JB, Price LL, Provencio I, Skene DJ, Brainard GC. Measuring and using light in the melanopsin age. Trends Neurosci 37: 1–9, 2014. 32. Mao L, Dauchy RT, Blask DE, Slakey LM, Xiang S, Yuan L, Dauchy EM, Shan B, Brainard GC, Hanifin JP, Frasch T, Duplessis TT, Hill SM. Circadian gating of epithelial-to-mesenchymal transition in breast cancer cells via melatonin-regulation of GSK3. Mol Endocrinol 26: 1808 –1820, 2012. 33. Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI, Halliwell B. Evaluation of the antioxidant activity of melatonin in vitro. Free Radic Biol Med 21: 307–315, 1996.
17. Galano A, Tan DX, Reiter RJ. Cyclic 3-hydroxymelatonin, a key metabolite enhancing the peroxyl radical scavenging activity of melatonin. Roy Soc Chem Adv 4: 5220 –5227, 2014.
34. Martin M, Macias M, Escames G, Reiter RJ, Agapito MT, Ortiz GG, Acuna-Castroviejo D. Melatonin-induced increased activity of respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo. J Pineal Res 28: 242–248, 2000.
18. Galley HF. Bench-to-beside review: targeting antioxidants to mitochondria in sepsis. Crit Care 14: 230 –238, 2010.
35. McCord CP, Allen FP. Evidences associating pineal gland function with alterations in pigmentation. J Exp Zool 23: 207–224, 1917.
19. Gomez FJV, Raba J, Cerutti S, Silva MF. Monitoring melatonin and its isomers in Vitis vinifera cv. Malbec by UHPLC-MS/MS from grape to bottle. J Pineal Res 52: 349 –355, 2012.
36. Menendez-Pelaez A, Reiter RJ. Distribution of melatonin in mammalian tissues: the relative importance of nuclear versus cytosolic localization. J Pineal Res 15: 59 – 69, 1993.
20. Hardeland R. Antioxidative protection by melatonin: multiplicity of mechanisms from radical detoxification to radical avoidance. Endocrine 27: 119 –130, 2005.
37. Mercolini L, Mandrioli R, Raggi MA. Content of melatonin and other antioxidants in grape-related foodstuffs: measurement using a MEPSHPLC-F method. J Pineal Res 53: 21–28, 2012.
21. Hardeland R. Melatonin: signaling mechanisms of a pleiotropic agent. Biofactors 35: 183–192, 2009.
38. Nduhirabandi F, du Toit EF, Lochner A. Melatonin and the metabolic syndrome: a tool for effective therapy in obesity-associated abnormalities? Acta Physiol (Oxf) 205: 209 –223, 2012.
22. Hardeland R. Melatonin and the theories of aging: a critical appraisal of melatonin’s role in antiaging mechanisms. J Pineal Res 55: 325–356, 2013. 23. Hoffman RA, Reiter RJ. Pineal gland: influence on gonads of male hamsters. Science 148: 1609 – 1611, 1965.
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39. Owens L, Buhr E, Tu DC, Lamprecht TL, Lee J, Van Gelder RN. Effect of circadian clock gene mutations on nonvisual photoreception in the mouse. Investig Ophthalmol 53: 454 – 460, 2012. 40. Proietti S, Cucina A, Reiter RJ, Bizzarri M. Molecular mechanisms of melatonin’s inhibitory actions on breast cancer. Cell Mol Life Sci 70: 2139 – 2157, 2013.
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41. Quay WB. Circadian rhythms in rat pineal serotonin and its modifications by the estrous cycle and photoperiod. Gen Comp Endocrinol 3: 473– 479, 1963. 42. Reiter RJ. Melatonin: the chemical expression of darkness. Mol Cell Endocrinol 79: C153–C158, 1991. 43. Reiter RJ, Hester RJ. Interrelationships of the pineal gland, the superior cervical ganglia and the photoperiod in the regulation of the endocrine systems of hamsters. Endocrinology 79: 1168 –1170, 1966. 44. Reiter RJ, Guerrero JM, Garcia JJ, AcunaCastroviejo D. Reactive oxygen intermediates, molecular damage, and aging: relation to melatonin. Ann NY Acad Sci 854: 410 – 424, 1998. 45. Reiter RJ, Paredes SD, Manchester LC, Tan DX. Reducing oxidative/nitrosative stress: a newlydiscovered genre for melatonin. Crit Rev Biochem Mol Biol 44: 175–200, 2009. 46. Reiter RJ, Tan DX, Korkmaz A, Rosales-Corral SA. Melatonin and stable circadian rhythms optimize maternal, placental and fetal physiology. Human Reprod Update 20: 293–307, 2014. 47. Reiter RJ, Tan DX, Rosales-Corral SA, Manchester LC. The universal nature, unequal distribution and antioxidant functions of melatonin and its derivatives. Mini Rev Med Chem 13: 373– 384, 2013. 48. Ressmeyer AR, Majo JC, Zelosko V, Sainz RM, Tan DX, Poeggeler B, Antolin I, Zsizsik BK, Reiter RJ, Hardeland R. Antioxidant properties of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction. Redox Rep 8: 205–213, 2003. 49. Rodriguez C, Mayo JC, Sainz RM, Antolin I, Herrera F, Martin V, Reiter RJ. Regulation of antioxidant enzymes: a significant role for melatonin. J Pineal Res 36: 1–9, 2004. 50. Rodriguez-Naranjo MI, Torija MJ, Mas A, CantosVillar E, Garcia-Parilla MC. Production of melatonin by Saccharomyces strains under growth and fermentation conditions. J Pineal Res 53: 219 –224, 2012. 51. Roopin M, Levy O. Temporal and histological evaluation of melatonin patterns in a ‘basal’ metazoan. J Pineal Res 53: 259 –269, 2012. 52. Rosales-Corral SA, Acuna-Castroviejo D, CotoMontes A, Boga JA, Manchester LC, FuentesBroto L, Korkmaz A, Ma S, Tan DX, Reiter RJ. Alzheimer’s disease: pathological mechanisms and the beneficial role of melatonin. J Pineal Res 52: 167–202, 2012. 53. Sanchez-Barcelo E, Mediavilla MD, Tan DX, Reiter RJ. Clinical uses of melatonin: evaluation of human trials. Curr Med Chem 17: 2070 –2095, 2010. 54. Skinner DC, Malpaux B. High melatonin concentrations in third ventricular cerebrospinal fluid are not due to Galen vein blood circulating through the choroid plexus. Endocrinology 140: 4399 – 4405, 1999. 55. Slominski RM, Reiter RJ, Schlobritz-Loutsevitch N, Ostrom RS, Slominski AT. Melatonin membrane receptors in peripheral tissues: distribution and functions. Mol Cell Endocrinol 35: 152–166, 2012. 56. Stankov B, Reiter RJ. Melatonin receptors: current status, facts and hypothesis. Life Sci 46: 971–982, 1990. 57. Stehle JH, Saade A, Rowashdeh O, Ackermann K, Jilg A, Sebesteny T, Maronde E. A survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases. J Pineal Res 51: 17– 43, 2011.
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11. Cano P, Jimenez-Ortega V, Larrad A, Reyes Toso CF, Cardinali DP, Esquifino AI. Effect of high-fat diet on 24-h pattern of circulating levels of prolactin, luteinizing hormone, testosterone, corticosterone, thyroid-stimulating hormone and glucose and pineal melatonin content, in rats. Endocrine 33: 118 –125, 2008.
24. Hoffman RA, Reiter RJ. Responses of some endocrine organs of female hamsters to pinealectomy and light. Life Sci 5: 1147–1151, 1966.
REVIEWS 58. Tan DX, Manchester LC, Reiter RJ, Qi W, Hanes MA, Farley NJ. High physiological levels of melatonin in the bile of mammals. Life Sci 65: 2523– 2529, 1999. 59. Tan DX, Manchester LC, Reiter RJ, Plummer BF. Cyclic 3-hydroxymelatonin: a melatonin metabolite generated as a result of hydroxyl radical scavenging. Biol Signals Recpt 8: 70 –74, 1999. 60. Tan DX, Hardeland R, Manchester LC, RosalesCorral SA, Coto-Montes A, Boga JA, Reiter RJ. Emergence of naturally occurring melatonin isomers and their proposed nomenclature. J Pineal Res 53: 113–121, 2012. 61. Tan DX, Manchester LC, Liu X, Rosales-Corral SA, Acuna-Castroviejo D, Reiter RJ. Mitochondria and chloroplasts as the original sites of melatonin synthesis: a hypothesis related to melatonin’s primary function and evolution in eukaryotes. J Pineal Res 54: 127–138, 2013.
62. Tan DX, Hardeland R, Manchester LC, Galano A, Reiter RJ. Cyclic-3-hydroxymelatonin (c3HOM), a potent antioxidant, scavenges free radicals and suppresses oxidative reactions. Curr Med Chem 21: 1557–1565, 2014. 63. Urata Y, Honma S, Goto S, Todoroki S, Iida T, Cho S, Honma K, Kondo T. Melatonin induces gamma-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells. Free Radic Biol Med 27: 838 – 847, 1999. 64. Venegas C, Garcia JA, Escames G, Ortiz F, Lopez A, Doerrier C, Garcia-Corzo L, Lopez LC, Reiter RJ, Acuna-Castroviejo D. Extrapineal melatonin: analysis of its subcellular distribution and daily fluctuations. J Pineal Res 52: 217–227, 2012.
65. Vitalini S, Gardana C, Simonetti P, Fico G, Iriti M. Melatonin, melatonin isomers and stilbenes in Italian traditional grape products and their antiradical activity. J Pineal Res 54: 322–333, 2013. 66. Wurtman RJ, Axelrod J, Fischer JE. Melatonin synthesis in the pineal gland: effect of light mediated by the sympathetic nervous system. Science 143: 1328 –1330, 1964. 67. Wurtman RJ, Axelrod J, Phillips LS. Melatonin synthesis in the pineal gland control by light. Science 142: 1071–1073, 1963. 68. Zhoa X, Stafford BK, Godin AL, King WM, Wong KY. Photoresponse diversity among the five types of intrinsically photosensitive retinal ganglion cells. J Physiol 592: 1619 –1636, 2014.
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Physiology (formerly published as News in Physiological Science) publishes brief review articles on major physiological developments. It is published bimonthly in January, March, May, July, September, and November by the American Physiological Society, 9650 Rockville Pike, Bethesda, MD 20814-3991. Copyright © 2014 by the American Physiological Society. ISSN: 1548-9213, ESSN: 1548-9221. Visit our website at http://www.the-aps.org/.
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This information is current as of November 21, 2014.
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PHYSIOLOGY 29: 325–333, 2014; doi:10.1152/physiol.00011.2014
Melatonin: Exceeding Expectations Melatonin is a small, highly conserved indole with numerous receptor-mediated and receptor-independent actions. Receptor-dependent functions include circadian rhythm regulation, sleep, and cancer inhibition. The receptor-inde-
Russel J. Reiter,1 Dun Xian Tan,1 and Annia Galano2 1 Department of Cellular and Structural Biology, UT Health Science Center, San Antonio, Texas; and 2Departamento de Quimica, Universidad Autonoma Metropolitana-Iztapalapa, Mexico D.F., Mexico [email protected]
pendent actions relate to melatonin’s ability to function in the detoxification of free radicals, thereby protecting critical molecules from the destructive effects of oxidative stress under conditions of ischemia/reperfusion injury (stroke, heart attack), ionizing radiation, and drug toxicity, among others. Melatonin has numerous applications in physiology and medicine.
1548-9213/14 ©2014 Int. Union Physiol. Sci./Am. Physiol. Soc.
actions and the evidence that melatonin is produced in all plants and animals that have been studied.
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When Aaron B. Lerner and his associates (29) isolated and characterized the methoxy derivative of serotonin from bovine pineal tissue near the middle of the last century, in their most optimistic outlook they would never have envisioned the multiple functions this molecule would eventually display. As dermatologists, they had engaged in this effort because, based on a publication that appeared 50 years earlier (35), they knew there was something in the pineal gland that blanched cutaneous chromatophores and thereby lightened the skin of amphibians. Indeed, the name they selected for their newly discovered molecule, i.e., melatonin, is in part based on its effect on skin pigmentation (“mela” from melanin and “tonin” from serotonin). After melatonin’s discovery in pineal tissue, the image of the gland quickly changed from that of a functionless vestige to one of an active organ of internal secretion. Biochemists identified the steps in the synthetic pathway of melatonin (4, 41, 67) (FIGURE 1), and the physiologists described a now well known function of the pineal, i.e., the regulation of seasonal reproduction in photoperiod-sensitive mammals (22, 24). By the end of what has been referred to as the Decade of Transformation (mid-1950s to mid-1960s) (7), the pineal gland and melatonin were both recognized as legitimate components of the endocrine system: the pineal was known to synthesize melatonin (67), the gland influenced seasonal reproduction in photosensitive species (23, 24), and it required its sympathetic innervation to remain functional (43, 66). Research within the last 60 years, however, has revealed that melatonin is functionally much more diverse than being only a regulator of the neuroendocrine-reproductive axis in photoperiod-dependent, seasonally breeding mammals. Also, the conflating of melatonin exclusively with the pineal gland has been shown to be untenable (47). Herein, we briefly summarize the data documenting the heterogeneity of melatonin’s multiple
In vertebrates, the pineal gland synthesizes melatonin in a circadian fashion, with nighttime darkness being a requirement for maximal production (57); because of this, melatonin is referred to as the chemical expression of darkness (42). The influence of the prevailing photoperiod on circadian melatonin production is accomplished by a restricted bandwidth of visible light, i.e., wavelengths in the range of 460 – 480 nm (blue light) (FIGURE 1). These wavelengths account for the inhibition of melatonin synthesis in the pineal gland during the day or, if they occur, at night. For the retinal processing of blue wavelengths, the mammalian retinas have up to five highly specialized subtypes of intrinsically photosensitive retinal ganglion cells (ipRGCs), which use a specialized photopigment, melanopsin, to respond to light (31, 68). The message related to the light environment is then transferred to the central biological clock, the suprachiasmatic nuclei (SCN), in the hypothalamus via the retinohypothalamic tract, which is embedded in the optic nerve. The SCN is a critical relay center that conveys a neural signal to the pineal gland (39) (FIGURE 1). The neural message arrives at the pinealocytes from the SCN via the central and peripheral sympathetic nervous system. This is one of several features that distinguishes the pineal from classic endocrine organs, i.e., its output is governed by a neural input, which, if destroyed, renders the gland totally inept (43); likewise, feedback effects from peripherally derived hormonal signals are usually not considered to be major factors in changing the quantity of melatonin produced or altering the phasing of its rhythm (11). Hence, the melatonin rhythm is more 325
REVIEWS or less independent of endogenous influences, and melatonin synthesis is determined almost exclusively by the prevailing photoperiod, thereby making the pineal an unconventional endocrine gland. There is also nothing phylogenetically that ties melatonin production to the pineal gland. Melatonin is not unique to vertebrates but is also produced in invertebrates, unicells, and plants, none of which possess a pineal gland and some of which, i.e., unicells, have no organs whatsoever (37, 50, 51). These nonvertebrates need melatonin for some of the same reasons vertebrates do, e.g., as an antioxidant (37). Some plants already have been genetically engineered to produce elevated quantities of melatonin, which protects them from free
radical-producing environmental stressors (temperature extremes, drought, disease) (8, 9). Melatonin probably evolved more than 2.5 billon years ago, likely in purple nonsulfur bacteria, presumably in Rhodospirillum rubrum (61), to protect them from an oxidizing environment that was a consequence of increasing atmospheric oxygen concentrations associated with the Great Oxygen Event (the Oxygen Catastrophe). These melatoninproducing bacteria were subsequently phagocytized by eukaryotes, a process referred to as endosymbiosis, where they eventually evolved into mitochondria. Because of this, we have speculated that mitochondria in all eukaryotic cells may have retained the ability to produce melatonin (61); if
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FIGURE 1. A diagrammatic representation of the means by which light wavelengths regulate the central circadian pacemaker A diagrammatic representation of the means by which 460- to 480-nm light wavelengths (blue light), detected by the intrinsically photoreceptive retinal ganglion cells (ipRGC), regulate the central circadian pacemaker, the suprachiasmatic nucleus (SCN), and the transfer of photoperiodic information to the pineal gland for the regulation of melatonin synthesis from tryptophan. This vertebrate pathway involves synapses in the paraventricular nucleus (PVN) of the hypothalamus, the intermediolateral cell column (ILCC) at thoracic cord levels 1 and 2, and the superior cervical ganglia (SCG). Melatonin released into the cerebrospinal fluid (CSF) and blood act on the SCN to modulate its circadian activity and on circadian oscillations in peripheral tissues. Besides influencing circadian rhythms, melatonin has many other functions, as summarized in the text. In the pinealocyte depicted at the top right, melatonin synthesis as it occurs in the mammalian pineal gland is shown. Based on currently available data, the synthetic pathway for melatonin production in plant cells differs slightly from that in animals. In plants, tryptophan is initially decarboxylated to tryptamine, which is then hydroxylated to form serotonin. The final two steps are similar to those in the mammalian pineal gland. AANAT, arylalkylamine N-acetyltransferase; AC, adenylate cyclase; ASMT, acetylserotonin methyl transferase; CaMK, calcium/calmodulin protein kinase; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; CSF, cerebrospinal fluid; NE, norepinephrine; NMDA, N-methyl-D-aspartate receptor; NO, nitric oxide; PACAP, pituitary adenylate cyclase-activating peptide; PKA, protein kinase A; RHT, retinohypothalamic tract. 326
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Melatonin, Its Potpourri of Actions Receptor-Mediated Actions Melatonin has an uncommonly large skill set, and, considering that melatonin is billions of years old, it has had ample time to hone its functions. For several decades after its discovery, melatonin was assumed to mediate all of its actions via receptors that were bound to membranes of a limited number of cells, e.g., neurons of the SCN (56). Two membrane receptors were pharmacologically characterized, subsequently cloned, and are now identified as the MT1 (Mel1a) and MT2 (Mel1b) receptors. These are both members of the superfamily of transmembrane, G-proteincoupled receptors. The MT1 receptor is 350 amino acids in length, whereas MT2 consists of 363 amino acids; the receptors share 60% homology (14). There is also an orphan molecule referred to as a melatonin-related receptor (MRR; also called GPR50) that shares 45% homology to MT1 and MT2; little is known of its function. In contrast to the original assumption that the membrane melatonin receptors were located on only a few cells, subsequent investigations have in fact localized them on many cells (55), and they may exist on the membranes of all cells. There is also what is referred to as an MT3 receptor located in the cytosol of a few cells. It is not coupled to a G protein and exhibits low affinity for
iodo-melatonin; it may be equivalent to quinone reductase 2, a detoxification enzyme (21). Finally, melatonin may carry out some of its activities by an interaction with a group of nuclear receptors referred to as retinoid orphan receptors (ROR) or retinoid Z receptors (RZR). The superfamily members that reportedly bind melatonin include the ROR␣, RZR␣, ROR␣2, and RZR. These nuclear receptors contain an NH2-terminal domain, a DNA binding domain, a ligand binding domain (in the COOH terminal), a zinc double finger, and a hinge region. The nuclear receptors may be differentially distributed among tissues (1) and perhaps are best functionally described in the immune system (12, 27). In most cases, their specific functions are under debate.
Receptor-Independent Actions In addition to its abundant actions mediated by its multiple receptors described above, melatonin also directly detoxifies reactive oxygen species (ROS) and reactive nitrogen species (RNS) by nonreceptor-mediated means (45). Like some other classic radical scavengers, major chemical mechanisms by which melatonin ensnares radicals include single electron transfer and hydrogen transfer, but other yet to be defined processes may be involved (15). Consistent with its presumed high intramitochondrial concentrations, melatonin improves the activities of several respiratory chain complexes, thereby reducing electron leakage and free-radical generation (34), a process referred to as radical avoidance (20). Moreover, metabolites of melatonin [cyclic-3-hydroxymelatonin (c3OHM), N1-acetyl-N2formyl-5-methoxykynuramine (AFMK), and N1-acetyl5-methoxykynuramine (AMK)] (FIGURE 2) provide additional protection against oxidative damage by the same mechanisms described above for melatonin and perhaps also by radical adduct formation (16, 17, 48, 59, 62). The relative efficacies of melatonin, c3OHM, AFMK, and AMK as scavengers of the hydroxyl radical (·OH) and the hydroperoxyl radical (·OOH) have been examined in several settings. These results were generated using a variety of techniques, including functional density theory. Although there are slight differences in their relative efficiencies, each of the four molecules scavenge the highly reactive ·OH at diffusion-controlled rates, regardless of the polarity of the environment. Thus they are all highly effective scavengers of the devastatingly reactive ·OH (17, 59, 62). With regard to the ·OOH, neither melatonin, AFMK, nor AMK are particularly effective in neutralizing this agent. This is consistent with melatonin per se being an inefficient chain-breaking antioxidant (33) but inconsistent with the data showing that both in vitro and in vivo melatonin
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so, all tissues in multicellular organisms generate melatonin for their own use (64). Of interest in this regard is that melatonin concentrations in the mitochondria of rat hepatocytes greatly exceed blood levels, and they do not drop after pinealectomy, which depletes melatonin from the blood (64). Many extrapineal organs, as sites of melatonin production, have been identified in vertebrates. Unlike the pineal gland, most of these organs (exception, the retinas) may not synthesize melatonin in a circadian manner nor do they release it into the blood in any significant amount. Rather, in these organs, melatonin functions as an antioxidant, as an autocoid, or as a paracoid to regulate intracellular events. Another misconception that should be dispelled is that blood melatonin levels are representative of its concentrations throughout the body. This is clearly not the case given that the bile (58) and the cerebrospinal fluid (54) have melatonin concentrations that exceed those in the blood by several orders of magnitude, and, as already noted, intracellular levels are also higher than those in the blood. Thus, because of its unequal distribution, blood melatonin concentrations should not be used to judge melatonin levels in other body fluids or within subcellular compartments (47).
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REVIEWS conclusion of these studies is that melatonin both limits the initiation of lipid breakdown and reduces its propagation when the metabolite c3OHM scavenges the ·OOH. Melatonin also reportedly detoxifies the strong oxidizing agent, the peroxynitrite anion (ONOO⫺) and hypochlorous acid (20). Whether melatonin’s metabolites likewise function in these capacities remains untested. To illustrate the importance of melatonin as an antioxidant, throughout evolution, no organism that has been investigated has lost the ability to synthesize this indoleamine, as is the case with some other radical scavengers, e.g., vitamin C. Additionally, besides being endogenously produced, melatonin is ingested in the diet of all animal species given that it is produced in plants (28). These dual routes ensure that melatonin is always available, again unlike some other radical scavengers. Finally, at least in some animal and plant species, melatonin synthesis is upregulated under circumstances where free-radical generation is exaggerated (3). This greatly increases its protective activities. Related to melatonin’s capability of reducing molecular damage due to free radicals are its effects on anti- and pro-oxidative enzymes (45). These actions are prominent and highly reproducible but, mechanistically, not well investigated, although preliminary evidence suggests the involvement of the membrane receptors MT1 and MT2 (49). The major antioxidative enzymes that are stimulated by melatonin under basal conditions include the intracellular superoxide dismutases (CuZnSOD and MnSOD), the selenium-containing glutathione peroxidases (GPX1, GPX2 and GPX3), and catalase (CAT) (FIGURE 3). Conversely, under toxic conditions of high oxidative stress, these proteins are protected from free-radical damage, and their enzymatic activity is preserved. Melatonin also maintains the activities of enzymes that enhance intracellular levels of reduced glutathione (GSH), an important intracellular antioxidant. Thus melatonin promotes glutathione reductase (GRd) activity, which reduces glutathione disulfide (GSSG) to the sulfhydryl form of GSH and ␥-glutamylcysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis (63). The pro-oxidative enzymes inhibited by melatonin include nitric oxide synthase, myeloperoxidase, and eosinophil peroxidase (20).
FIGURE 2. Melatonin and its metabolites that are formed when the parent molecule scavenges toxic radicals
Melatonin Doses and Timing of Administration
This series of reactions is referred to as melatonin’s antioxidant cascade, and it permits melatonin to directly and indirectly detoxify multiple radicals. These molecules differ somewhat in terms of their efficacies toward the radicals they scavenge. 328
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When melatonin is to be used, the dose and timing of when it is given may be critical in determining its efficacy as a treatment. The doses typically used in animal/human studies for
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markedly reduces lipid peroxidation (45). This action was then assumed to be a consequence of melatonin’s ability to prevent the onset of lipid peroxidation by neutralizing the initiating agents, ·OH and the peroxynitrite anion. Subsequently, it was found, however, that c3OHM, in contrast to the other three molecules, is highly effective in scavenging the ·OOH (17, 59). Moreover, c3OHM was found to detoxify the ·OOH almost 100 times faster than trolox (water soluble vitamin E). This is remarkable since vitamin E is considered the primer peroxyl radical scavenging agent and inhibitor of lipid peroxidation. The
REVIEWS promoting activity (10). Each of these functions relies on the circadian message provided by the pineal-derived blood and cerebrospinal fluid melatonin rhythms that are transferred to cells that “have a need to know.” Melatonin is not impeded by any of the known morphophysiological barriers, e.g., blood-brain barrier, blood-testis barrier, etc., and it readily crosses the placenta in an unaltered form to impact the fetus (46). Melatonin’s concentration in some bodily fluids, e.g., CSF, bile, etc., exceeds that in the serum by several orders of magnitude. Likewise, at least in those cells where measurements have been made, subcellular organelle melatonin levels are greater than in the blood and may not rely on the blood as a source of their melatonin (36, 64). We have speculated that all cells have the capability of synthesizing melatonin, particularly in their mitochondria. This locally produced melatonin is for protection from free radicals and from cells in the neighborhood via autocoid and paracoid actions and is not normally released into the blood. Receptor-independent actions of melatonin and its metabolites relate to their ability to directly quench free radicals and nonradical, but toxic,
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membrane receptor-mediated circadian rhythm regulation are usually lower than those used for defeating free radicals, an action that is membrane receptor independent. Moreover, when melatonin is used in situations where an influence on circadian processes is a factor, e.g., sleep in humans, melatonin should be taken in the evening before sleep. Conversely, if the purpose of melatonin use is to harness free radicals, melatonin would be most effective if given when the free radical event, e.g., stroke, occurs, regardless of the time of day. The statement has been made that melatonin functions as a free-radical scavenger only when given in amounts that are considered pharmacological. This is incorrect. For any scavenger, even a single molecule is capable of neutralizing a radical. To combat the massive number of free radicals generated under conditions of severe oxidative stress, however, pharmacological amounts of any antioxidant must be given to neutralize the overwhelming numbers of these toxic brigands that are produced. Vitamins C and E, for example, are often taken in gram quantities, even under conditions where severe oxidative damage is not a consideration. Thus, when melatonin is used to reduce free-radical destruction, it is given in pharmacological doses since the free radicals are also being produced at “pharmacological” levels.
Melatonin Isomers Isomers of melatonin have been identified in plant products (19, 65), and we have predicted they will eventually be found throughout the plant and animal kingdoms (60). Isomers are molecules with the identical molecular size as melatonin; they also possess the same methoxy residue and the aliphatic side chain as melatonin itself, but they are in different locations on the indole nucleus. Presently published reports show that the isomers bind to classic membrane melatonin receptors and function in free-radical detoxification (60).
Melatonin: Functionally, a Swiss Army Knife Like the multi-tooled device bearing the Swiss coat of arms, melatonin has a bewildering array of functions and employs a variety of means to carry them out (FIGURE 4). These actions likely impact every cell in every organism throughout the plant and animal kingdoms. As a consequence, the physiological and pathophysiological actions of melatonin are numerous. Melatonin is best known for its mediation of circannual variations in metabolism and reproductive competence in photosensitive species (5), its ability to influence circadian processes that are ubiquitous in organisms and in cells, and its sleep-
FIGURE 3. The relationship of molecular oxygen to the formation of reactive oxygen species, reactive nitrogen species, and the production of hypochlorous acid The relationship of molecular oxygen (O2) to the formation of reactive oxygen species (O2·⫺; 1O2; H2O2; ·OH), reactive nitrogen species (NO·; ONOO⫺), and the production of hypochlorous acid (HOCl), a toxic chloride species. Also shown are the enzymes that are either stimulated (1) or inhibited (2) by melatonin. CAT, catalase; GCL, glutamylcysteine ligase; GPx, glutathione peroxidase; GRd, glutathione reductase; MPO, myeloperoxidase; NOS, nitric oxide synthase; SOD, superoxide dismutase.
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REVIEWS physiological collapse, further exaggerating the production of free radicals. Melatonin, because of its ability to neutralize radicals, defers age-related dysfunction of several organs (22). Increased age leads to a gradual diminished melatonin production such that, in the elderly, the nocturnal melatonin rise in the circulation is either greatly attenuated or it no longer exists. The consequences of this reduction may be highly significant in terms of health (44). Since the cycle of melatonin strengthens circadian rhythms by acting at the SCN and also on peripheral slave oscillators, normal circadian rhythms deteriorate, and, considering their importance for optimal health, the dysregulation of these rhythms, e.g., the sleep/wake cycle, negatively impacts organisms. Additionally, the loss of melatonin during aging contributes to the accelerated accumulation of oxidative stress due to the reduced availability of this important antioxidant. This presumably contributes to the progression of diseases that have a free-radical component, e.g., neurodegenerative diseases (52), cardiovascular disease (13), skin deterioration (25), and metabolic syndrome (38),
FIGURE 4. Some of the numerous actions of melatonin in mammals Melatonin has both receptor-dependent and receptor-independent actions. The indole binds to well known membrane receptors (MT1 and MT2) and, via several signal transduction pathways, influences a host of physiological effects. MT1 and MT2 may homo- and/or heterodimerize in some cases, and they may interact with nuclear receptors (binding sites). There is considerable debate regarding the existence/function of the orphan nuclear melatonin receptors ROR and RZR. The cytosolic receptor, MT3, is a detoxifying enzyme, quinone reductase 2. Receptor-independent actions are mediated by the ability of melatonin and its metabolites to scavenge reactive oxygen (ROS) and reactive nitrogen species (RNS). These actions allow melatonin to protect against a wide variety of toxins and processes that generate highly toxic reactants. Any cell can simultaneously respond to melatonin by both its receptor-mediated and receptor-independent actions. Many of the documented physiological and molecular actions of melatonin are not listed in this figure. Additionally, the figure does not include melatonin functions in nonvertebrates or in plants. 330
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species. Excessive free-radical generation is notoriously destructive and kills cells secondary to massive oxidative damage, which induces cellular apoptosis or necrosis. The loss of cells due to programmed cell death is a consequence and/or contributes to many diseases as well as to age-related deterioration. Melatonin’s ability to prevent molecular damage meted out by free radicals and the cellular mutilation becomes manifested when the indole protects against that destruction. Such damage can be produced by ultraviolet and ionizing radiation–ingestion of toxins, heavy metals, alcohol, and prescription drugs–smoking, ischemia/ reperfusion injury, which occurs during a heart attack or stroke, severe inflammation, neurodegenerative diseases, and many other pathophysiological situations (45). Similarly, aging is associated with the progressive accumulation of oxidative debris, which contributes to functional inefficiency of cellular processes, thereby inducing additional free radicals to be produced. Thus aging becomes a vicious cycle. As molecular processes fail, oxidative damage accumulates, which leads to additional
REVIEWS among others. In experimental studies, supplementing aging animals with melatonin defers some of these debilitating changes (22). Some functions of melatonin may depend on both receptor-mediated events and on receptorindependent processes. As an example, melatonin inhibits the proliferation of many cancer cells (40) as well as limiting tumor metastases (32). The ability of melatonin to forestall cancer cell division and the molecular processes associated with the relocation of cancer cells to a secondary site may involve the classic membrane receptors that these cells possess as well as its receptor-independent antioxidative functions; the latter also may account for melatonin’s ability to promote apoptosis of cancer cells (6). This pro-apoptotic action of melatonin in cancer cells is diametrically opposite to its anti-apoptotic function in normal cells, a differential action that has been difficult to explain (6).
Melatonin, Its Special Association With Mitochondria
Melatonin’s flummoxing array of physiological actions as well as the numerous means by which these actions are mediated supports the idea that melatonin is a multitasking molecule. Regarding both these features, melatonin has certainly exceeded the expectations of the most ardent melatonin devotees. Yet, there is likely more to come. Stable circadian rhythms, which melatonin aids in regulating, are crucial for the optimal generalized physiology of organisms and for the microphysiology of molecular functions. Likewise, melatonin’s ability to modulate oxidative processes and protect against free-radical mutilation of essential molecules is indispensible for flawless cellular function. Research on melatonin seems to be at the “end of the beginning.” Obviously, extensive research has been performed regarding the actions of melatonin in experimental cells, plants, and animals. Its uncovered actions are uniformly beneficial, although not all the specific mechanisms have been described. In addition to its multiple positive physiological actions, melatonin has an uncommonly high safety profile. Thus it is time to advance this research to the next level, i.e., to more extensively test its use in clinical trials (26). Although small steps are being taken in this direction (53), it is essential that multi-centered, blinded, well controlled studies be performed using adequate amounts of melatonin and for appropriate durations to determine melatonin’s ability to defer or prevent certain diseases, as suggested by the numerous experimental studies that have been performed. This is especially important since prevention always trumps treatment. 䡲
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Mitochondria are a major site of free-radical generation since electrons that are fumbled during their transfer through the electron transport chain reduce molecular oxygen to the superoxide anion radical (O2·⫺). This oxidizing agent is quickly dismutated to form hydrogen peroxide (H2O2), which is transformed, in the presence of transition metals, to the ·OH (FIGURE 3). Alternatively, O2·⫺ may couple with nitric oxide to produce the highly toxic ONOO⫺. If the prediction that melatonin is produced in mitochondria is valid, its generation in these organelles would be a major advantage for reducing mitochondrial damage and preserving energy production. Melatonin is highly effective as an antioxidant at the mitochondrial level (2). When compared with synthetically produced, mitochondrial-targeted antioxidants Mito Q and Mito E, which are concentrated up to 500-fold in mitochondria (18), melatonin still performed better in reducing every aspect of damage to this organelle (30). The implication is that melatonin, when administered via any route, is absorbed and makes its way to mitochondria in sufficiently high amounts where it is more effective than the synthetic antioxidants, which are known to accumulate in the matrix of mitochondria, in preserving the function of these critically important organelles. Thus, besides being produced in mitochondria, when it is taken as a supplement or consumed in the diet it is readily available to mitochondria. Thus melatonin may be classified as a naturally occurring, mitochondrialtargeted antioxidant. Melatonin may have regulatory actions on mitochondrial uncoupling protein, but these functions remain poorly defined.
Conclusions and Perspectives
No conflicts of interest, financial or otherwise, are declared by the author(s). Author contributions: R.J.R. and D.-X.T. analyzed data; R.J.R., D.-X.T., and A.G. interpreted results of experiments; R.J.R. drafted manuscript; R.J.R. and D.-X.T. edited and revised manuscript; R.J.R., D.-X.T., and A.G. approved final version of manuscript; A.G. prepared figures.
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