Juan R. Calvo

Significance of Melatonin in Antioxidative Defense System: Reactions and Products

Melatonin is a potent endogenous free radical scavenger, actions that are independent of its many receptor-mediated effects. In the last several years, hundreds of publications have confirmed that melatonin is a broad-spectrum antioxidant. Melatonin has been reported to scavenge hydrogen peroxide (H2O2), hydroxyl radical (HO·), nitric oxide (NO·), peroxynitrite anion (ONOO–), hypochlorous acid (HOCl), singlet oxygen (1O2), superoxide anion (O2–·) and peroxyl radical (LOO·), although the validity of its ability to scavenge O2–· and LOO· is debatable. Regardless of the radicals scavenged, melatonin prevents oxidative damage at the level of cells, tissues, organs and organisms. The antioxidative mechanisms of melatonin seem different from classical antioxidants such as vitamin C, vitamin E and glutathione. As electron donors, classical antioxidants undergo redox cycling; thus, they have the potential to promote oxidation as well as prevent it. Melatonin, as an electron-rich molecule, may interact with free radicals via an additive reaction to form several stable end-products which are excreted in the urine. Melatonin does not undergo redox cycling and, thus, does not promote oxidation as shown under a variety of experimental conditions. From this point of view, melatonin can be considered a suicidal or terminal antioxidant which distinguishes it from the opportunistic antioxidants. Interestingly, the ability of melatonin to scavenge free radicals is not in a ratio of mole to mole. Indeed, one melatonin molecule scavenges two HO·. Also, its secondary and tertiary metabolites, for example, N1-acetyl-N2-formyl-5-methoxykynuramine, N-acetyl-5-methoxykynuramine and 6-hydroxymelatonin, which are believed to be generated when melatonin interacts with free radicals, are also regarded as effective free radical scavengers. The continuous free radical scavenging potential of the original molecule (melatonin) and its metabolites may be defined as a scavenging cascade reaction. Melatonin also synergizes with vitamin C, vitamin E and glutathione in the scavenging of free radicals. Melatonin has been detected in vegetables, fruits and a variety of herbs. In some plants, especially in flowers and seeds (the reproductive organs which are most vulnerable to oxidative insults), melatonin concentrations are several orders of magnitude higher than measured in the blood of vertebrates. Melatonin in plants not only provides an alternative exogenous source of melatonin for herbivores but also suggests that melatonin may be an important antioxidant in plants which protects them from a hostile environment that includes extreme heat, cold and pollution, all of which generate free radicals.

Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance

It has been historically assumed that the pineal gland is the major source of melatonin (N‐acetyl‐ 5‐methoxytryptamine) in vertebrates. Melatonin plays a central role in fine‐tuning circadian rhythms in vertebrate physiology. In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, antiaging, and immunomodulatory properties. Melatonin has been identified in a wide range of organisms from bacteria to human beings. Its biosynthesis from tryptophan involves four well‐defined intracellular steps catalyzed by tryptophan hydroxylase, aromatic amino acid decarboxylase, serotonin‐N‐acetyltransferase, and hydroxyndole‐O‐methyltransferase. Here, for the first time, we document that both resting and phytohemagglutinin‐stimulated human lymphocytes synthesize and release large amounts of melatonin, with the melatonin concentration in the medium increasing up to five times the nocturnal physiological levels in human serum. Moreover, we show that the necessary machinery to synthesize melatonin is present in human lymphocytes. Furthermore, melatonin released to the culture medium is synthesized in the cells, because blocking the enzymes required for its biosynthesis or inhibiting protein synthesis in general produced a significant reduction in melatonin release. Moreover, this inhibition caused a decrease in IL‐2 production, which was restored by adding exogenous melatonin. These findings indicate that in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that this melatonin could be involved in the regulation of the human immune system, possibly by acting as an intracrine, autocrine, and/or paracrine substance

Melatonin and Its Relation to the Immune System and Inflammation

Abstract: Melatonin (N‐acetyl‐5‐methoxytryptamine) was initially thought to be produced exclusively in the pineal gland. Subsequently its synthesis was demonstrated in other organs, for example, the retinas, and very high concentrations of melatonin are found at other sites, for example, bone marrow cells and bile. The origin of the high level of melatonin in these locations has not been definitively established, but it is likely not exclusively of pineal origin. Melatonin has been shown to possess anti‐inflammatory effects, among a number of actions. Melatonin reduces tissue destruction during inflammatory reactions by a number of means. Thus melatonin, by virtue of its ability to directly scavenge toxic free radicals, reduces macromolecular damage in all organs. The free radicals and reactive oxygen and nitrogen species known to be scavenged by melatonin include the highly toxic hydroxyl radical (·OH), peroxynitrite anion (ONOO−), and hypochlorous acid (HOCl), among others. These agents all contribute to the inflammatory response and associated tissue destruction. Additionally, melatonin has other means to lower the damage resulting from inflammation. Thus, it prevents the translocation of nuclear factor‐kappa B (NF‐κB) to the nucleus and its binding to DNA, thereby reducing the upregulation of a variety of proinflammatory cytokines, for example, interleukins and tumor neurosis factor‐alpha. Finally, there is indirect evidence that melatonin inhibits the production of adhesion molecules that promote the sticking of leukocytes to endothelial cells. By this means melatonin attenuates transendothelial cell migration and edema, which contribute to tissue damage.

Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum.

In the present paper we show the inhibitory effect of melatonin on rat cerebellar nitric oxide synthase (NOS) activity. NO production was monitored by the stoichiometric conversion of L-arginine to L-citrulline. The inhibitory effect of melatonin was dose-dependent, with an IC50 value of about 0.1 mM. However, a significant inhibition of enzyme activity (> 22%) was observed at 1 nM melatonin which is in the range of the physiological serum concentration of the hormone at night. The inhibitory effect of melatonin was observed exclusively in the presence of Ca++. Results suggest a new and important role of the pineal hormone melatonin on central nervous system processes, i.e., by modulating NO production.

Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin

Constitutive rat cerebellar nitric oxide synthase (NOS) activity is shown to be inhibited by physiological concentrations of the pineal hormone melatonin. The inhibition was dose‐dependent and was coupled to an inhibition of the cyclic GMP production activated by L‐arginine. Results also show that calmodulin appears to be involved in this process because its presence in the incubation medium was able to prevent the effect of melatonin on both NOS activity and cyclic GMP production. Moreover, polyacrylamide gel electrophoresis studies suggest that melatonin can interact with calmodulin modifying the binding of the peptide to the synthetic NOS peptide encompassing the calmodulin‐binding domain of constitutive NOS from rat cerebellum, the natural mechanism by which calmodulin activates cerebellar NOS. J. Cell. Biochem. 65:430–442.

Pharmacology and Physiology of Melatonin in the Reduction of Oxidative Stress in vivo

This brief resume summarizes the evidence which shows that melatonin is a significant free radical scavenger and antioxidant at both physiological and pharmacological concentrations in vivo. Surgical removal of the pineal gland, a procedure which lowers endogenous melatonin levels in the blood, exaggerates molecular damage due to free radicals during an oxidative challenge. Likewise, providing supplemental melatonin during periods of massive free radical production greatly lowers the resulting tissue damage and dysfunction. In the current review, these findings are considered in terms of neurodegenerative diseases, cancer, ischemia/reperfusion injury and aging. Besides being a highly effective direct free radical scavenger and indirect antioxidant, melatonin has several features that make it of clinical interest. Thus, melatonin is readily absorbed when it is administered via any route, it crosses all morphophysiological barriers, e.g., blood-brain barrier and placenta, with ease, it seems to enter all parts of every cell where it prevents oxidative damage, it preserves mitochondrial function, and it has low toxicity. While blood melatonin levels are normally low, tissue levels of the indoleamine can be considerably higher and at some sites, e.g., in bone marrow cells and bile, melatonin concentrations exceed those in the blood by several orders of magnitude. What constitutes a physiological level of melatonin must be redefined in terms of the bodily fluid, tissue and subcellular compartment being examined.

The role of melatonin in the cells of the innate immunity: a review.

Melatonin is the major secretory product synthesized and secreted by the pineal gland and shows both a wide distribution within phylogenetically distant organisms from bacteria to humans and a great functional versatility. In recent years, a considerable amount of experimental evidence has accumulated showing a relationship between the nervous, endocrine, and immune systems. The molecular basis of the communication between these systems is the use of a common chemical language. In this framework, currently melatonin is considered one of the members of the neuroendocrine–immunological network. A number of in vivo and in vitro studies have documented that melatonin plays a fundamental role in neuroimmunomodulation. Based on the information published, it is clear that the majority of the present data in the literature relate to lymphocytes; thus, they have been rather thoroughly investigated, and several reviews have been published related to the mechanisms of action and the effects of melatonin on lymphocytes. However, few studies concerning the effects of melatonin on cells belonging to the innate immunity have been reported. Innate immunity provides the early line of defense against microbes and consists of both cellular and biochemical mechanisms. In this review, we have focused on the role of melatonin in the innate immunity. More specifically, we summarize the effects and action mechanisms of melatonin in the different cells that belong to or participate in the innate immunity, such as monocytes–macrophages, dendritic cells, neutrophils, eosinophils, basophils, mast cells, and natural killer cells.

Beneficial pleiotropic actions of melatonin in an experimental model of septic shock in mice: regulation of pro-/anti-inflammatory cytokine network, protection against oxidative damage and anti-apoptotic effects.

Abstract:  Septic shock, the most severe problem of sepsis, is a lethal condition caused by the interaction of a pathogen‐induced long chain of sequential intracellular events in immune cells, epithelium, endothelium, and the neuroendocrine system. The lethal effects of septic shock are associated with the production and release of numerous pro‐inflammatory biochemical mediators including cytokines, nitric oxide and toxic oxygen and nitrogen radicals, together with development of massive apoptosis. As melatonin has remarkable properties as a cytokine modulator, antioxidant and anti‐apoptotic agent, the present study was designed to evaluate the possible protective effect of melatonin against LPS‐induced septic shock in Swiss mice. We observed that intraperitoneally (i.p.) administered‐melatonin (10 mg/kg) 30 min prior, and 1 hr after i.p. LPS injection (0.75 mg/animal) markedly protected mice from the LPS lethal effects with 90% survival rates for melatonin and 20% for LPS‐injected mice after 72 hr. The melatonin effect was mediated by modulating the release of pro‐/anti‐inflammatory cytokine levels, protection from oxidative damage and counteracting apoptotic cell death. Melatonin was able to partially counteract the increase in LPS‐induced pro‐inflammatory cytokine levels such as tumor necrosis factor‐α, IL‐12 and interferon‐γ at the local site of injection, while it increased the production of the anti‐inflammatory cytokine IL‐10 both locally and systemically. Furthermore, melatonin inhibited the LPS‐induced nitrite/nitrate and lipid peroxidation levels in brain and liver and counteracted the sepsis‐associated apoptotic process in spleen. In conclusion, we have demonstrated that melatonin improves the survival of mice with septic shock via its pleiotropic functions as an immunomodulator, antioxidant and anti‐apoptotic mediator.

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide modulate endotoxin-induced IL-6 production by murine peritoneal macrophages.

Vasoactive intestinal peptide (VIP) is a neuropeptide synthesized by immune cells that can modulate several immune aspects, including the function of cells involved in the inflammatory response, such as macrophages and monocytes. Production and release of cytokines by activated mononuclear phagocytes is an important event in the pathogenesis of ischemia‐reperfusion injury. VIP has been shown to attenuate the deleterious consequences of this pathologic phenomenon. We have investigated the effects of VIP and PACAP38 on the production of interleukin‐6 (IL‐6), a proinflammatory cytokine, by endotoxin‐activated murine macrophages. Both neuropeptides exhibit a dual effect on the IL‐6 production by peritoneal macrophages. Whereas VIP and PACAP inhibit with similar dose‐response curves the release of IL‐6 from macrophages stimulated with a LPS dose range from 100 pg/mL to 10 μg/mL, both neuropeptides enhance IL‐6 secretion in unstimulated macrophages and in macrophages stimulated with very low LPS concentrations (1‐10 pg/mL). The inhibition on LPS‐induced IL‐6 production is specific, presumably mediated through a subtype of the PACAP‐R. VIP and PACAP regulate the production of IL‐6 at a transcriptional level. These results were correlated with an inhibition on both IL‐6 expression and release in endotoxemic mice in vivo. These findings support the idea that in the absence of stimulation or in the presence of low doses of LPS, VIP and PACAP could play a role in immune system homeostasis. However, under toxicity conditions associated with high LPS doses, VIP and PACAP could act as protective mediators that regulate the excessive release of IL‐6 in order to reduce inflammation or shock. J. Leukoc. Biol. 63: 591–601; 1998.

Immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells.

Abstract: This paper reviews the evidence that supports the hypothesis of the existence of specific binding sites for melatonin on immune cells. These binding sites have been described in human blood lymphocytes and granulocytes, and thymus, spleen, and bursa of Fabricius from different rodents and birds. The dissociation constant values of these binding sites are in the 0.1 ‐1 nM range, suggesting that melatonin may play a physiological role in lymphocyte regulation. Moreover, melatonin binding sites appear to be modulated by guanine nucleotides. Therefore, in addition to other mechanisms described for the regulation of immune function by melatonin, a direct mechanism of regulation can be involved via binding of melatonin by immunocompetent cells.