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Multiple studies reporting mitochondrial impairment in Parkinson's disease (PD) involve knockout or knockdown models to inhibit the expression of mitochondrial-related genes, including parkin, PINK1, and DJ-1 ones. Melatonin has significant neuroprotective properties, which have been related to its ability to boost mitochondrial bioenergetics. The meaning and molecular targets of melatonin in PD are yet unclear. Zebrafish are an outstanding model of PD because they are vertebrates, their dopaminergic system is comparable to the nigrostriatal system of humans, and their brains express the same genes as mammals. The exposure of 24 hpf zebrafish embryos to MPTP leads to a significant inhibition of the mitochondrial complex I and the induction of sncga gene, responsible for enhancing γ-synuclein accumulation, which is related to mitochondrial dysfunction. Moreover, MPTP inhibited the parkin/PINK1/DJ-1 expression, impeding the normal function of the parkin/PINK1/DJ-1/MUL1 network to remove the damaged mitochondria. This situation remains over time, and removing MPTP from the treatment did not stop the neurodegenerative process. On the contrary, mitochondria become worse during the next 2 days without MPTP, and the embryos developed a severe motor impairment that cannot be rescued because the mitochondrial-related gene expression remained inhibited. Melatonin, added together with MPTP or added once MPTP was removed, prevented and recovered, respectively, the parkinsonian phenotype once it was established, restoring gene expression and normal function of the parkin/PINK1/DJ-1/MUL1 loop and also the normal motor activity of the embryos. The results show, for the first time, that melatonin restores brain function in zebrafish suffering with Parkinson-like disease.

The connection between the innate immune system, clock genes, and mitochondrial bioenergetics was analyzed during aging and sepsis in mouse heart. Our results suggest that the sole NF-κB activation does not explain the inflammatory process underlying aging; the former also triggers the NLRP3 inflammasome that enhances caspase-1-dependent maturation of IL-1β. In this way, aged mice enter into a vicious cycle as IL-1β further activates the NF-κB/NLRP3 inflammasome link. The origin of NF-κB activation was related to the age-dependent Bmal1/Clock/RORα/Rev-Erbα loop disruption, which lowers NAD(+) levels, reducing the SIRT1 deacetylase ability to inactivate NF-κB. Consequently, NF-κB binding to DNA increases, raising the formation of proinflammatory mediators and inducing mitochondrial impairment. The cycle is then closed with the subsequent NLRP3 inflammasome activation. This paired contribution of the innate immune pathways serves as a catalyst to magnify the response to sepsis in aged compared with young mice. Melatonin administration blunted the septic response, reducing inflammation and oxidative stress, and enhancing mitochondrial function at the levels of nonseptic aged mice, but it did not counteract the age-related inflammation. Together, our results suggest that, although with different strengths, chronoinflammaging constitutes the biochemical substrate of aging and sepsis, and identifies the NLRP3 inflammasome as a new molecular target for melatonin, providing a rationale for its use in NLRP3-dependent diseases.

Mucositis is a common and distressing side effect of chemotherapy or radiotherapy that has potentially severe consequences, and no treatment is available. The purpose of this study was to analyze the molecular pathways involved in the development of oral mucositis and to evaluate whether melatonin can prevent this pathology. The tongue of male Wistar rats was subjected to irradiation (X-ray YXLON Y.Tu 320-D03 irradiator; the animals received a dose of 7.5 Gy/day for 5 days). Rats were treated with 45 mg/day melatonin or vehicle for 21 days postirradiation, either by local application into their mouths (melatonin gel) or by subcutaneous injection. A connection between reactive oxygen species, generating mitochondria and the NLRP3 (NLR-related protein 3 nucleotide-binding domain leucine-rich repeat containing receptor-related protein 3) inflammasome, has been reported in mucositis. Here, we show that mitochondrial oxidative stress, bioenergetic impairment and subsequent NLRP3 inflammasome activation are involved in the development of oral mucositis after irradiation and that melatonin synthesized in the rat tongue is depleted after irradiation. The application of melatonin gel restores physiological melatonin levels in the tongue and prevents mucosal disruption and ulcer formation. Melatonin gel protects the mitochondria from radiation damage and blunts the NF-κB/NLRP3 inflammasome signaling activation in the tongue. Our results suggest new molecular pathways involved in radiotherapy-induced mucositis that are inhibited by topical melatonin application, suggesting a potential preventive therapy for mucositis in patients with cancer.

Head and neck squamous cell carcinoma (HNSCC) clearly involves activation of the Akt mammalian target of rapamycin (mTOR) signalling pathway. However, the effectiveness of treatment with the mTOR inhibitor rapamycin is often limited by chemoresistance. Melatonin suppresses neoplastic growth via different mechanisms in a variety of tumours. In this study, we aimed to elucidate the effects of melatonin on rapamycin-induced HNSCC cell death and to identify potential cross-talk pathways. We analysed the dose-dependent effects of melatonin in rapamycin-treated HNSCC cell lines (Cal-27 and SCC-9). These cells were treated with 0.1, 0.5 or 1 mmol/L melatonin combined with 20 nM rapamycin. We further examined the potential synergistic effects of melatonin with rapamycin in Cal-27 xenograft mice. Relationships between inhibition of the mTOR pathway, reactive oxygen species (ROS), and apoptosis and mitophagy reportedly increased the cytotoxic effects of rapamycin in HNSCC. Our results demonstrated that combined treatment with rapamycin and melatonin blocked the negative feedback loop from the specific downstream effector of mTOR activation S6K1 to Akt signalling, which decreased cell viability, proliferation and clonogenic capacity. Interestingly, combined treatment with rapamycin and melatonin-induced changes in mitochondrial function, which were associated with increased ROS production, increasing apoptosis and mitophagy. This led to increase cell death and cellular differentiation. Our data further indicated that melatonin administration reduced rapamycin-associated toxicity to healthy cells. Overall, our findings suggested that melatonin could be used as an adjuvant agent with rapamycin, improving effectiveness while minimizing its side effects.

The NLRP3 inflammasome is involved in the innate immune response during inflammation. Moreover, melatonin blunts the NF-κB/NLRP3 connection during sepsis. Thus, we compared the roles of the NLRP3 inflammasome and/or melatonin treatment in the septic response of wild-type and NLRP3-/- mice. Mouse myocardial tissue was used for this purpose. The nuclear turnover of NF-κB was enhanced during sepsis, with an increase in TNFα, iNOS, and pro-IL-1β. The lack of inflammasome in NLRP3-/- mice significantly reduced that response and blunted IL-1β maturation due to the lack of caspase-1. Clock and Bmal1 did not change in both mouse strains, enhancing Chrono expression in mutants. RORα, which positively regulates Bmal1, was enhanced at a similar extend in both mouse strains, whereas the expression of the Bmal1 repressor, Rev-Erbα, increased in WT but was depressed in NLRP3-/- mice. Nampt, transcriptionally controlled by Bmal1, increased in WT mice together with Sirt1, whereas they remained unchanged in NLRP3-/- mice. Melatonin treatment reduced the septic response in a comparable manner as did the lack of NLRP3, but unlike the latter, it normalized the clock genes turnover through the induction of RORα and repression of Rev-Erbα and Per2, leading to enhanced Nampt and Sirt1. The lack of NLRP3 inflammasome converts sepsis to a moderate inflammatory disease and identifies NLRP3 as a main target for the treatment of sepsis. The efficacy of melatonin in counteracting the NLRP3 inflammasome activation further confirms the indoleamine as a useful therapeutic drug against this serious condition.

Melatonin is part of the evolutionary conserved highly functional network in vertebrates. It plays a central role in the adaptative behavior of the animal to the environment, including entrainment of daily and annual physiological rhythms, reproductive behavior, food intake, locomotor activity, growth, and breeding performance. In zebrafish, apart from its synchronizing capabilities, melatonin seems to have a major role in multiple physiological processes. Extensive knowledge of its genome and the identification of a series of genes with the same functions as those in humans, the relative ease of obtaining mutants, and the similarities between zebrafish and human pathologies make it an excellent experimental model organism of human diseases. Moreover, it is a common experimental species because of easy handling, breeding, and developmental control. Among other pathophysiologies, zebrafish are now used in studies of neurodegeneration and neurological diseases, endocrine diseases, behavior, muscular dystrophies, developmental alterations, circadian rhythms, and drugs screening. The purpose of this review was to update the current knowledge on the synthesis and biological functions of melatonin in zebrafish, keeping in mind its relevance not only in the physiology of the animal, but also in pathophysiological conditions.

While it is accepted that the high production of nitric oxide (NO˙) by the inducible nitric oxide synthase (iNOS) impairs cardiac mitochondrial function during sepsis, the role of neuronal nitric oxide synthase (nNOS) may be protective. During sepsis, there is a significantly increase in the expression and activity of mitochondrial iNOS (i-mtNOS), which parallels the changes in cytosolic iNOS. The existence of a constitutive NOS form (c-mtNOS) in heart mitochondria has been also described, but its role in the heart failure during sepsis remains unclear. Herein, we analyzed the changes in mitochondrial oxidative stress and bioenergetics in wild-type and nNOS-deficient mice during sepsis, and the role of melatonin, a known antioxidant, in these changes. Sepsis was induced by cecal ligation and puncture, and heart mitochondria were analyzed for NOS expression and activity, nitrites, lipid peroxidation, glutathione and glutathione redox enzymes, oxidized proteins, and respiratory chain activity in vehicle- and melatonin-treated mice. Our data show that sepsis produced a similar induction of iNOS/i-mtNOS and comparable inhibition of the respiratory chain activity in wild-type and in nNOS-deficient mice. Sepsis also increased mitochondrial oxidative/nitrosative stress to a similar extent in both mice strains. Melatonin administration inhibited iNOS/i-mtNOS induction, restored mitochondrial homeostasis in septic mice, and preserved the activity of nNOS/c-mtNOS. The effects of melatonin were unrelated to the presence or the absence of nNOS. Our observations show a lack of effect of nNOS on heart bioenergetic impairment during sepsis and further support the beneficial actions of melatonin in sepsis.

We studied the subcellular levels of melatonin in cerebral cortex and liver of rats under several conditions. The results show that melatonin levels in the cell membrane, cytosol, nucleus, and mitochondrion vary over a 24-hr cycle, although these variations do not exhibit circadian rhythms. The cell membrane has the highest concentration of melatonin followed by mitochondria, nucleus, and cytosol. Pinealectomy significantly increased the content of melatonin in all subcellular compartments, whereas luzindole treatment had little effect on melatonin levels. Administration of 10 mg/kg bw melatonin to sham-pinealectomized, pinealectomized, or continuous light-exposed rats increased the content of melatonin in all subcellular compartments. Melatonin in doses ranging from 40 to 200 mg/kg bw increased in a dose-dependent manner the accumulation of melatonin on cell membrane and cytosol, although the accumulations were 10 times greater in the former than in the latter. Melatonin levels in the nucleus and mitochondria reached saturation with a dose of 40 mg/kg bw; higher doses of injected melatonin did not further cause additional accumulation of melatonin in these organelles. The results suggest some control of extrapineal accumulation or extrapineal production of melatonin and support the existence of regulatory mechanisms in cellular organelles, which prevent the intracellular equilibration of the indolamine. Seemingly, different concentrations of melatonin can be maintained in different subcellular compartments. The data also seem to support a requirement of high doses of melatonin to obtain therapeutic effects. Together, these results add information that assists in explaining the physiology and pharmacology of melatonin.

The beneficial effects of atorvastatin are based on both cholesterol-dependent and independent mechanisms. The latter probably include the ability of the estatin to enhance the expression of endothelial nitric oxide synthase (eNOS) and to cause a vasodilatation. In turn, the antioxidant and anti-inflammatory actions of melatonin are related to its vascular protection. In the present study, we investigated the efficacy of the combination of melatonin plus atorvastatin against endothelial cell damage induced by inflammation and oxidative stress injury. Human umbilical vein endothelial cells (HUVEC) were cultured with bacterial lipopolysaccharide (LPS) in the presence or absence of melatonin and/or atorvastatin. LPS inhibited eNOS mRNA and protein expression, which was reversed by atorvastatin and, to a lesser extent, by melatonin. Together, melatonin + atorvastatin induced higher eNOS protein expression than either compound alone. Melatonin, but not atorvastatin, reduced free radical generation, lipid peroxidation, and interleukin-6 levels induced by LPS. In the presence of atorvastatin, the effects of melatonin were maintained or even improved. These data suggest that melatonin improves the beneficial effects of atorvastatin and reduces its side effects in endothelial cells during inflammation and under conditions of oxidative stress.

The antiproliferative and proapoptotic properties of melatonin in human colon cancer cells in culture were recently reported. To address the mechanisms involved in these actions, HT-29 human colon cancer cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37 degrees C. Cell proliferation was assessed by the incorporation of [(3)H]-thymidine into DNA. Cyclic nucleotide levels, nitrite concentration, glutathione peroxidase and reductase activities, and glutathione levels were assessed after the incubation of these cells with the following drugs: melatonin membrane receptor agonists 2-iodo-melatonin, 2-iodo-N-butanoyl-5-methoxytryptamine, 5-methoxycarbonylamino-N-acetyltryptamine (GR-135,531), and the antagonists luzindole, 4-phenyl-2-propionamidotetralin, and prazosin; the melatonin nuclear receptor agonist CGP 52608, and four synthetic kynurenines analogs to melatonin 2-acetamide-4-(3-methoxyphenyl)-4-oxobutyric acid, 2-acetamide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid, 2-butyramide-4-(3-methoxyphenyl)-4-oxobutyric acid and 2-butyramide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid. The results show that the membrane receptors are not necessary for the antiproliferative effect of melatonin and the participation of the nuclear receptor in this effect is suggested. Moreover, the antioxidative and anti-inflammatory actions of melatonin, counteracting the oxidative status and reducing the production of nitric oxide by cultured HT-29 cells seem to be directly involved in the oncostatic properties of melatonin. Some of the synthetic kynurenines exert higher antiproliferative effects than melatonin. The results reinforce the clinical interest of melatonin due to the different mechanisms involved in its oncostatic role, and suggest a new synthetic pathway to obtain melatonin agonists with clinical applications to oncology.

Melatonin membrane (MT1 and MT2) and nuclear (RORα) receptors have been identified in several mammalian tissues, including the liver. The mechanisms regulating hepatic melatonin receptors are yet unknown. This study investigated whether these receptors exhibit daily changes and the effects of melatonin on their levels. Our results show that mRNAs for MT1/MT2 receptors exhibit circadian rhythms that were followed by rhythms in their respective protein levels; the acrophases for the two rhythms were reached at 04:00 and 05:00 hr, respectively. Pinealectomy blunted the rhythms in both mRNAs and protein levels. In contrast, mRNA and protein levels of nuclear receptor RORα increased significantly after pinealectomy. The cycles of the latter receptor also exhibited circadian rhythms which peaked at 03:00 and 03:45 hr, respectively. Melatonin administration (10-200 mg/kg) increased in a dose-dependent manner the protein content of MT1/MT2 receptors, with no effects on RORα. Lunzindole treatment, however, did not affect melatonin receptor expression or content of either the membrane or nuclear receptors. Together with previously published findings which demonstrated the intracellular distribution of melatonin in rat liver, the current results support the conclusion that the circadian rhythms of MT1/MT2 and RORα receptors are under the control of the serum and intracellular melatonin levels. Moreover, the induction of MT1/MT2 receptors after the administration of high doses of melatonin further suggests that the therapeutic value of melatonin may not be restricted to only low doses of the indoleamine.

We studied the subcellular levels of melatonin in cerebral cortex and liver of rats under several conditions. The results show that melatonin levels in the cell membrane, cytosol, nucleus, and mitochondrion vary over a 24-hr cycle, although these variations do not exhibit circadian rhythms. The cell membrane has the highest concentration of melatonin followed by mitochondria, nucleus, and cytosol. Pinealectomy significantly increased the content of melatonin in all subcellular compartments, whereas luzindole treatment had little effect on melatonin levels. Administration of 10 mg/kg bw melatonin to sham-pinealectomized, pinealectomized, or continuous light-exposed rats increased the content of melatonin in all subcellular compartments. Melatonin in doses ranging from 40 to 200 mg/kg bw increased in a dose-dependent manner the accumulation of melatonin on cell membrane and cytosol, although the accumulations were 10 times greater in the former than in the latter. Melatonin levels in the nucleus and mitochondria reached saturation with a dose of 40 mg/kg bw; higher doses of injected melatonin did not further cause additional accumulation of melatonin in these organelles. The results suggest some control of extrapineal accumulation or extrapineal production of melatonin and support the existence of regulatory mechanisms in cellular organelles, which prevent the intracellular equilibration of the indolamine. Seemingly, different concentrations of melatonin can be maintained in different subcellular compartments. The data also seem to support a requirement of high doses of melatonin to obtain therapeutic effects. Together, these results add information that assists in explaining the physiology and pharmacology of melatonin.

The beneficial effects of atorvastatin are based on both cholesterol-dependent and independent mechanisms. The latter probably include the ability of the estatin to enhance the expression of endothelial nitric oxide synthase (eNOS) and to cause a vasodilatation. In turn, the antioxidant and anti-inflammatory actions of melatonin are related to its vascular protection. In the present study, we investigated the efficacy of the combination of melatonin plus atorvastatin against endothelial cell damage induced by inflammation and oxidative stress injury. Human umbilical vein endothelial cells (HUVEC) were cultured with bacterial lipopolysaccharide (LPS) in the presence or absence of melatonin and/or atorvastatin. LPS inhibited eNOS mRNA and protein expression, which was reversed by atorvastatin and, to a lesser extent, by melatonin. Together, melatonin + atorvastatin induced higher eNOS protein expression than either compound alone. Melatonin, but not atorvastatin, reduced free radical generation, lipid peroxidation, and interleukin-6 levels induced by LPS. In the presence of atorvastatin, the effects of melatonin were maintained or even improved. These data suggest that melatonin improves the beneficial effects of atorvastatin and reduces its side effects in endothelial cells during inflammation and under conditions of oxidative stress.

The role of melatonin in improving mitochondrial respiratory chain activity and increasing ATP production in different experimental conditions has been widely reported. To date, however, the mechanism(s) involved are largely unknown. Using high-resolution respirometry, fluorometry and spectrophotometry we studied the effects of melatonin on normal mitochondrial functions. Mitochondria were recovered from mouse liver cells and incubated in vitro with melatonin at concentrations ranging from 1 nm to 1 mm. Melatonin decreased oxygen consumption concomitantly with its concentration, inhibited any increase in oxygen flux in the presence of an excess of ADP, reduced the membrane potential, and consequently inhibited the production of superoxide anion and hydrogen peroxide. At the same time it maintained the efficiency of oxidative phosphorylation and ATP synthesis while increasing the activity of the respiratory complexes (mainly complexes I, III, and IV). The effects of melatonin appeared to be due to its presence within the mitochondria, since kinetic experiments clearly showed its incorporation into these organelles. Our results support the hypothesis that melatonin, together with hormones such as triiodothyronine, participates in the physiological regulation of mitochondrial homeostasis.

It is hypothesized that, besides increased free radical production, aging is a process also related to inflammation. Thus, female and male senescence-accelerated (SAMP8) and senescence-resistant (SAMR1) mice of 5 and 10 months of age were studied to assess this hypothesis. Plasma from these mice was processed to determine nitric oxide (NO), and pro-inflammatory [interleukin (IL)-1beta, IL-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and granulocyte-macrophage colony-stimulating factor] and anti-inflammatory (IL-4, IL-5 and IL-10) cytokines. The results show the presence of an age-dependent increase in IFN-gamma and TNF-alpha and a reduction in IL-2 levels, with minor changes in the remaining cytokines. Moreover, age was associated with a significant increase in NO levels. Chronic melatonin administration between 1 and 10 months of age counteracted the age-dependent production of pro-inflammatory cytokines and NO, reducing them to the levels found at 5 months of age. Melatonin also reduced the levels of the anti-inflammatory cytokines. The results of this study suggest the existence of an inflammatory process during aging and further support that melatonin behaves as an essential molecule against aging, for its anti-inflammatory properties together with its antioxidative role reported elsewhere.

Mitochondrial nitric oxide synthase (mtNOS) produces nitric oxide (NO) to modulate mitochondrial respiration. Besides a constitutive mtNOS isoform it was recently suggested that mitochondria express an inducible isoform of the enzyme during sepsis. Thus, the mitochondrial respiratory inhibition and energy failure underlying skeletal muscle contractility failure observed in sepsis may reflect the high levels of NO produced by inducible mtNOS. The fact that mtNOS is induced during sepsis suggests its relation to inducible nitric oxide synthase (iNOS). Thus, we examined the changes in mtNOS activity and mitochondrial function in skeletal muscle of wild-type (iNOS +/+ ) and iNOS knockout (iNOS -/- ) mice after sepsis. We also studied the effects of melatonin administration on mitochondrial damage in this experimental paradigm. After sepsis, iNOS +/+ but no iNOS -/- mice showed an increase in mtNOS activity and NO production and a reduction in electron transport chain activity. These changes were accompanied by a pronounced oxidative stress reflected in changes in lipid peroxidation levels, oxidized glutathione/reduced glutathione ratio, and glutathione peroxidase and reductase activities. Melatonin treatment counteracted both the changes in mtNOS activity and rises in oxidative stress; the indole also restored mitochondrial respiratory chain in septic iNOS +/+ mice. Mitochondria from iNOS -/- mice were unaffected by either sepsis or melatonin treatment. The data suggest that inducible mtNOS, which is coded by the same gene as that for iNOS, is responsible for mitochondrial dysfunction during sepsis. The results also suggest the use of melatonin for the protection against mtNOS-mediated mitochondrial failure.