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-independent 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.
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 actions and the evidence that melatonin is produced in all plants and animals that have been studied.
Unconventional Aspects of the Pineal and Melatonin
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 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 melatonin-producing 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 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).
Melatonin, Its Potpourri of 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-protein-coupled 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 RORα, RZRα, RORα2, and RZRβ. 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.
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-N2-formyl-5-methoxykynuramine (AFMK), and N1-acetyl-5-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 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 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 γ-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).
Melatonin Doses and Timing of Administration
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 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.
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-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, 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 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), 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 receptor-independent 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
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, mitochondrial-targeted antioxidant. Melatonin may have regulatory actions on mitochondrial uncoupling protein, but these functions remain poorly defined.
Conclusions and Perspectives
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.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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