Although melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) could alleviate salinity stress in plants, the downstream signaling pathway is still not fully characterized. Here, we report that endogenous melatonin and thereafter nitric oxide (<em>N</em>O) accumulation was successively increased in <em>N</em>aCl-stressed rapeseed (Brassica napus L.) seedling roots. Application of melatonin and <em>N</em>O-releasing compound not only counteracted <em>N</em>aCl-induced seedling growth inhibition, but also reestablished redox and ion homeostasis, the latter of which are confirmed by the alleviation of reactive oxygen species overproduction, the decreases in thiobarbituric acid reactive substances production, and <em>N</em>a⁺/K⁺ ratio. Consistently, the related antioxidant defense genes, sodium hydrogen exchanger (<em>N</em>HX1), and salt overly sensitive 2 (SOS2) transcripts are modulated. The involvement S-nitrosylation, a redox-based posttranslational modification triggered by <em>N</em>O, is suggested. Further results show that in response to <em>N</em>aCl stress, the increased <em>N</em>O levels are strengthened by the addition of melatonin in seedling roots. Above responses are abolished by the removal of <em>N</em>O by <em>N</em>O scavenger. We further discover that the removal of <em>N</em>O does not alter endogenous melatonin content in roots supplemented with <em>N</em>aCl alone or together with melatonin, thus excluding the possibility of <em>N</em>O-triggered melatonin production. Genetic evidence reveals that, compared with wild-type Arabidopsis, the hypersensitivity to <em>N</em>aCl in nia1/2 and noa1 mutants (exhibiting null nitrate reductase activity and indirectly reduced endogenous <em>N</em>O level, respectively) cannot be rescued by melatonin supplementation. The reestablishment of redox homeostasis and induction of SOS signaling are not observed. In summary, above pharmacological, molecular, and genetic data conclude that <em>N</em>O operates downstream of melatonin promoting salinity tolerance.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is a hormone synthesized and secreted by the pineal gland mainly during the night, since light exposure suppresses its production. Initially, an implication of this indoleamine in malignant disease was described in endocrine-responsive breast cancer. Data from several clinical trials and multiple experimental studies performed both in vivo and in vitro have documented that the pineal hormone inhibits endocrine-dependent mammary tumors by interfering with the estrogen signaling-mediated transcription, therefore behaving as a selective estrogen receptor modulator (SERM). Additionally, melatonin regulates the production of estradiol through the control of the enzymes involved in its synthesis, acting as a selective estrogen enzyme modulator (SEEM). Many more mechanisms have been proposed during the past few years, including signaling triggered after activation of the membrane melatonin receptors MT-1 and MT-2, or else intracellular actions targeting molecules such as calmodulin, or binding intranuclear receptors. Similar results have been obtained in prostate (regulation of enzymes involved in androgen synthesis and modulation of androgen receptor levels and activity) and ovary cancer. Thus, tumor metabolism, gene expression, or epigenetic modifications are modulated, cell growth is impaired and angiogenesis and metastasis are inhibited. In the last decade, many more reports have demonstrated that melatonin is a promising adjuvant molecule with many potential beneficial consequences when included in chemotherapy or radiotherapy protocols designed to treat endocrine-responsive tumors. Therefore, in this state-of-the-art review, we aim to compile the knowledge about the oncostatic actions of the indoleamine in hormone-dependent tumors, and the latest findings concerning melatonin actions when administered in combination with radio- or chemotherapy in breast, prostate, and ovary cancers. As melatonin has no toxicity, it may be well deserve to be considered as an endogenously generated agent helpful in cancer prevention and treatment.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is synthesized and released by the pineal gland following a circadian rhythm characterized by high levels during the night. It shows several pharmacological effects on diverse cellular and animal models, mainly related to either its antioxidant activity or to its ability to activate specific receptors (MTr). Melatonin is widely used as a self-administered food additive, but its therapeutic potential needs more investigation and is hampered by its poor pharmacokinetics. This review will focus on the medicinal chemistry of agonist ligands of the two human GPCRs MT(1) and MT(2) melatonin receptors. The recent introduction of ramelteon, a non-selective MT(1)/MT(2) agonist for the treatment of insomnia, and the advancement to clinical trials of other MTr agonists have renewed interest for different classes of compounds endowed with this activity. Several chemical classes of MTr agonists are described in the literature, generally characterized by an indole, or an indole bioisostere, carrying an amide side chain and a methoxy group, or substituents with similar stereoelectronic features. Abundant information is available for non-selective MT(1)/MT(2) ligands, and several molecular models, both ligand- and receptor-based, have been proposed to rationalize their structure activity relationships. Fewer classes of selective agonists have been reported in the literature, and they could help clarifying the physiological role of the two receptor subtypes. A brief discussion on the therapeutic potential of this class of compounds is based on the clinical data available for the agonists ramelteon, agomelatine, beta-methyl-6-chloromelatonin (TIK-301) and VEC-162.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is a neurohormone produced in the pineal gland. Its biological properties are related to the circadian rhythm. Recently, the European Food Safety Authority (EFSA) accepted the health claim related to melatonin and the alleviation of subjective feelings of jet lag. This molecule has been detected in some foods. In this work, 13 grape varieties were studied; 7 monovarietal wines were produced in an experimental winery under strictly controlled conditions and were sampled in different steps. The grape varieties used to make the wines were: Cabernet Sauvignon, Merlot, Syrah, Tempranillo, Tintilla de Rota, Palomino Fino and Alpha red. Liquid chromatography tandem mass spectrometry (LC-MS/MS) unequivocally confirmed the presence of melatonin in wines. The main contribution of this paper is the results that clearly show that melatonin is synthesised during the winemaking process, specifically after the alcoholic fermentation. Indeed, melatonin is absent in grapes and musts and is formed during alcoholic fermentation.

The effects on plasma lipids of daily intraperitoneal injections of 4 mg of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) for 10-27-day periods were examined biochemically and morphologically in rats fed regular and high-cholesterol (1% cholesterol, 0.<em>5</em>% cholic acid) diets. Melatonin administration had no significant effect on plasma lipids and lipoproteins in the rats on a normal diet but blunted the effects of a high-cholesterol diet on these parameters. <em>N</em>o effects of melatonin on lipase activity were noted. Melatonin also diminished the fatty infiltration in the liver of animals on the high-cholesterol diet. The high-cholesterol diet produced major increases in VLDL and LDL cholesterol and protein content, and decreases in HDL cholesterol and protein. Melatonin decreased the extent of this plasma lipoprotein increase, although it did not completely prevent the phenomenon. Therefore, the effect is thought to be quantitative and not qualititative in nature.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) plays important roles in regulating both biotic and abiotic stress tolerance, biological rhythms, plant growth and development. Sharing the same substrate (tryptophan) for the biosynthesis, melatonin and auxin also have similar effects in plant development. However, the specific function of melatonin in modulating plant root growth and the relationship between melatonin and auxin as well as underlying mechanisms are still unclear. In this study, we found high concentration of melatonin remarkably inhibited root growth in Arabidopsis by reducing root meristem size. Further studies showed that melatonin negatively regulated auxin biosynthesis, the expression of PI<em>N</em>FORMED (PI<em>N</em>) proteins as well as auxin response in Arabidopsis. Moreover, the root growth of the triple mutant pin1pin3pin7 was more tolerant than that of wild-type in response to melatonin treatment, suggesting the essential role of PI<em>N</em>1/3/7 in melatonin-mediated root growth. Combination treatment of melatonin and <em>5</em>-Triiodobenzoic acid (TIBA) did not enhance melatonin-mediated reduction of root meristem size, indicating that polar auxin transport (PAT) may be necessary for the regulation of root meristem size by melatonin treatment. Taken together, this study indicates that melatonin regulates root growth in Arabidopsis, through auxin synthesis and polar auxin transport, at least partially.

Melatonin (MEL; <em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) plays an important role in plant stress defense. Various plant species rich in this indoleamine have shown a higher capacity for stress tolerance. Moreover, it has great potential for plant biostimulation, is biodegradable and non-toxic for the environment. All this indicates that our concept of seed enrichment with exogenous MEL is justified. This work concerns the effects of corn (Zea mays L.) seed pre-sowing treatments supplemented with MEL. <em>N</em>on-treated seeds (nt), and those hydroprimed with water (H) or with MEL solutions <em>5</em>0 and <em>5</em>00 μM (HMel<em>5</em>0, HMel<em>5</em>00) were compared. Positive effects of seed priming are particularly apparent during germination under suboptimal conditions. The impact of MEL applied by priming on seed protein profiles during imbibition/germination at low temperature has not been investigated to date. In order to identify changes in the corn seed proteome after applying hydropriming techniques, purified protein extracts of chilling stressed seed embryos (14 days, <em>5</em>°C) were separated by two-dimensional electrophoresis. Then proteome maps were graphically and statistically compared and selected protein spots were qualitatively analyzed using mass spectrometry techniques and identified. This study aimed to analyze the priming-induced changes in maize embryo proteome and at identifying priming-associated and MEL-associated proteins in maize seeds subjected to chilling. We attempt to explain how MEL expands plant capacity for stress tolerance.

Plant hormone candidate melatonin has been widely studied in plants under various stress conditions, such as heat, cold, salt, drought, heavy metal, and pathogen attack. Under stress, melatonin usually accumulates sharply by modulating its biosynthesis and metabolic pathways. Beginning from the precursor tryptophan, four consecutive enzymes mediate the biosynthesis of tryptamine or <em>5</em>-hydroxytryptophan, serotonin, <em>N</em>-<em>acetyl</em>serotonin or <em>5</em>-<em>methoxytryptamine</em>, and melatonin. Then, the compound is catabolized into 2-hydroxymelatonin, cyclic-3-hydroxymelatonin, and <em>N</em>¹-<em>acetyl</em>-<em>N</em>²-formyl-<em>5</em>-methoxyknuramine through 2-oxoglutarate-dependent dioxygenase catalysis or reaction with reactive oxygen species. As an ancient and powerful antioxidant, melatonin directly scavenges ROS induced by various stress conditions. Furthermore, it confreres stress tolerance by activating the plant's antioxidant system, alleviating photosynthesis inhibition, modulating transcription factors that are involved with stress resisting, and chelating and promoting the transport of heavy metals. Melatonin is even proven to defense against pathogen attacks for the plant by activating other stress-relevant hormones, like salicylic acid, ethylene, and jasmonic acid. Intriguingly, other precursors and metabolite molecules involved with melatonin also can increase stress tolerance for plant except for unconfirmed <em>5</em>-<em>methoxytryptamine</em>, cyclic-3-hydroxymelatonin, and <em>N</em>¹-<em>acetyl</em>-<em>N</em>²-formyl-<em>5</em>-methoxyknuramine. Therefore, the precursors and metabolites locating at the whole biosynthesis and catabolism pathway of melatonin could contribute to plant stress resistance, thus providing a new perspective for promoting plant stress tolerance.

BACKGROUND

Photoprotective effects of topically applied antioxidants when applied before ultraviolet radiation (UVR) exposure are well known. Their protective effect when applied after UVR exposure is, however, less established.

OBJECTIVE

In a randomized, double-blinded, placebo-controlled human study the short-term photoprotective effects of different antioxidants and of their combinations were evaluated when applied after UVR exposure.

METHODS

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), vitamin E (alpha-tocopherol) and vitamin C (ascorbic acid) were topically administered alone or in combination following UVR exposure as single applications (immediately or 30 min after irradiation, respectively) or as multiple applications (three times: 30 min, 1 h and 2 h after irradiation). The erythemal reaction was evaluated visually and noninvasively with bioengineering methods (skin color and skin blood flow).

RESULTS

No significant protective effect of melatonin or the vitamins when applied alone or in combination were obtained when antioxidants were applied after UVR exposure. No improved photoprotective effect was obtained when multiple applications were done.

CONCLUSIONS

UVR-induced skin damage is a rapid event, and antioxidants possibly prevent such damage only when present in relevant concentration at the site of action beginning and during oxidative stress.

Research on the role of serotonin (<em>5</em>-hydroxytryptamine, <em>5</em>-HT) in the function of the enteric nervous system has been impeded by the lack of specific inhibitors of the enteric neural actions of <em>5</em>-HT. Saturable, reversible, high affinity enteric binding sites for 3H-<em>5</em>-HT have recently been characterized and radioautographically located. Affinity for the 3H-<em>5</em>-HT binding site requires an indole ring substituted with a free hydroxyl group. These 3H-<em>5</em>-HT binding sites have been proposed to be enteric neural <em>5</em>-HT receptors. This hypothesis was tested in the current study by comparing the ability of compounds to inhibit the binding of 3H-<em>5</em>-HT with their electrophysiologically determined actions on myenteric neurons. <em>5</em>-<em>Methoxytryptamine</em> did not inhibit the binding of 3H-<em>5</em>-HT to enteric membranes and neither mimicked nor antagonized the effects of <em>5</em>-HT on the membrane potential of myenteric neurons. Two dipeptides of <em>5</em>-hydroxytryptophan, <em>N</em>-<em>acetyl</em>- and <em>N</em>-hexanoyl-<em>5</em>-hydroxytryptophyl-<em>5</em>-hydroxytryptophan amide (<em>5</em>-HTP-DP and <em>N</em>-hex-<em>5</em>-HTP-DP) inhibited the binding of 3H-<em>5</em>-HT (K1 = 0.2<em>5</em> microM for <em>5</em>-HTP-DP and 1.19 microM for <em>N</em>-hex-<em>5</em>-HTP-DP). <em>5</em>-HTP-DP applied by pressure microejection or superfusion (10 microM) antagonized the slow postsynaptic depolarization of myenteric neurons evoked by microejection of <em>5</em>-HT. <em>5</em>-HTP-DP also blocked the <em>5</em>-HT-induced presynaptic reduction in amplitude of nicotinic fast synaptic potentials; however, <em>5</em>-HTP-DP itself did not affect these responses. Moreover, <em>5</em>-HTP-DP also failed to affect responses of myenteric neurons to microejected substance P, their muscarinic response to <em>acetyl</em>choline, or antidromic action potentials. In contrast, both dipeptides blocked the slow synaptic potentials seen in type II/AH neurons following stimulation of fiber tracts in interganglionic connectives. These data support the hypotheses that enteric 3H-<em>5</em>-HT binding sites are enteric neural <em>5</em>-HT receptors, that dipeptides of <em>5</em>-hydroxytryptophan are specific antagonists at these receptors, and that <em>5</em>-HT is one of the mediators of slow synaptic potentials in the myenteric plexus.

The objectives of this study were to test the effects of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), a natural compound of edible plants on the rooting of certain commercial sweet cherry rootstocks. Shoot tip explants from previous in vitro cultures of the cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus × P. canescens), and M × M 60 (P. avium × P. mahaleb) were included in the experiment. The effect of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) alone or in combination with melatonin was tested concerning their rooting potential. Seven concentrations of melatonin (0, 0.0<em>5</em>, 0.1, 0.<em>5</em>, 1, <em>5</em>, and 10 μM) alone or in combination with <em>5</em>.71 μM of IAA or 4.92 μM of IBA were tested. For each rootstock, 21 treatments were included. The explants were grown in glass tubes containing 10 mL of substrate. The parameters measured include rooting percentage, number of roots per rooted explant, root length, and callus formation. The data presented in this study show that melatonin has a rooting promoting effect at a low concentration but a growth inhibitory effect at high concentrations. In the absence of auxin, 1 μM melatonin had auxinic response concerning the number and length of roots, but 10 μM melatonin was inhibitory to rooting in all the tested rootstocks. The final conclusion of this experiment is that exogenously applied melatonin acted as a rooting promoter and its action was similar to that of IAA.

The Syrian hamster Harderian gland exhibits sexually dimorphic porphyrin biosynthesis, wherein the female glands display an extraordinarily high concentration of porphyrins. Damage derived from this production of porphyrins, mediated by reactive oxygen species, causes the glands to develop autophagic processes, which culminate in detachment-derived cell death; these cells normally play a central role in the secretory activity of the gland. The main aim of this study was to analyze how a change in the redox state impacts autophagy. Female Syrian hamsters were treated daily with melatonin (2<em>5</em> μg, subcutaneously) at ZT 10 for 1-2 months (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), an endogenous antioxidant that ameliorates the deleterious effects of free radicals via a variety of mechanisms. The length of treatment affected the redox balance, the autophagy machinery, and the activation of p<em>5</em>3 and <em>N</em>F-κB. One-month treatment displaces redox balance to the antioxidant side, promotes autophagy through a p<em>5</em>3-mediated mechanism, and increases cell detachment. Meanwhile, 2-month treatment restores redox balance to the oxidant side, activates <em>N</em>F-κB reducing autophagy to basal levels, increases number of type II cells, and reduces number of detached cells. Our results conclude that the redox state can modulate autophagy through redox-sensitive transcriptions factors. Additionally, these findings support a hypothesis that ascribes differences in the autophagic-lysosomal pathway to epithelial cell types, thereby restricting detachment-induced autophagic cell death to epithelial cell type I.

There is some indirect evidence that the pineal hormone melatonin can suppress plasma levels of cholesterol in hypercholesterolemic rats. We have examined the effects of the hormone on cellular cholesterol metabolism in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 20 h in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of [12<em>5</em>I]labeled low density lipoprotein (LDL). Addition of melatonin in increasing concentrations to the incubation medium at zero time inhibited cholesterol synthesis and the specific accumulation and degradation of [12<em>5</em>I]labeled LDL; at a concentration of 100 microM, the inhibitions were 38%, 42%, and 48%, respectively. Similar results were obtained using [14C]mevalonate as precursor. Fatty acid synthesis was not altered under these conditions. In contrast to cholesterol, the synthesis of the first cyclic compound lanosterol was not affected by the pineal hormone. These results implicate that melatonin inhibits this pathway between lanosterol and cholesterol. The action of melatonin on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in binding affinity. Pharmacological characterization of the potential melatonin receptor site using several analogs like tryptamine, <em>5</em>-hydroxytryptamine,<em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptamine, <em>5</em>-<em>methoxytryptamine</em>, and 6-chloromelatonin indicated that the <em>5</em>-methoxy group is indispensible for the hormone action on cholesterol synthesis. The data provide evidence that melatonin can modulate cholesterol metabolism in human cells.

OBJECTIVE

It has been suggested that the vascular endothelial growth factor (VEGF) gene expression plays an important role in radiation-induced injury to the spinal cord. This study assesses the radioprotective effects of <em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em> (melatonin) through its modulation of VEGF expression after localized irradiation of the cervical spinal cord.

METHODS

In this experimental study, we divided 192 male rats into four groups: 1. control (n=48); 2. rats that received an intraperitoneal (IP) injection of melatonin (n=48); 3. rats that received an IP injection of melatonin 30 minutes prior to cervical spinal cord gamma irradiation [dose: 22 Gy; (n=48)]; and 4. rats that received an IP injection of vehicle prior to spinal cord irradiation (n=48). The changes in VEGF expression were assessed using real-time RT-PCR and enzyme-linked immunosorbent assays. Samples for light microscopy were stained with hematoxylin and eosin (H&E). The differences among the groups were analyzed using the analysis of variance (ANOVA) test followed by Tukey's multiple comparisons test.

RESULTS

Up-regulation of VEGF expression was observed from 8 to 22 weeks after irradiation (p<0.0<em>5</em>). Paralysis and other radiation-induced myelopathy manifestations developed within 22 weeks after irradiation. VEGF expression in the melatonin pre-treatment group significantly down-regulated in the 20(th) and 22(nd) weeks after irradiation compared to the radiation-only group.

CONCLUSIONS

The results support the hypothesis that modulation of VEGF expression by melatonin administration may increase the survival rate of irradiated animals.

In most vertebrates and several insects, melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is synthesized enzymatically from serotonin (<em>5</em>-hydroxytryptamine) by the sequential action of arylalkylamine <em>N</em>-<em>acetyl</em>transferase (<em>N</em>AT) and hydroxyindole-O-methyltransferase (HIOMT). In the freshwater planarian Dugesia japonica, which belongs to the most primitive metazoan phylum, activities of <em>N</em>AT and HIOMT, as well as melatonin, were found. The apparent Michaelis constants for substrates of <em>N</em>AT and HIOMT in the planarian were similar to those reported for the mammalian pineal gland and retina. When the planarians were maintained under a 12 h light:12 h dark cycle, the activities of <em>N</em>AT and HIOMT and melatonin levels exhibited a significant diurnal variation, peaking at the mid-dark time. In constant darkness, <em>N</em>AT activity and melatonin levels fluctuated with a circadian (about 24 h) rhythm. These data demonstrate that the planarian synthesizes melatonin through the same pathways as those in most vertebrates and several insects, and that its melatonin synthesis fluctuates in a circadian manner. Thus, it is strongly suggested that the planarian contains a circadian clock controlling melatonin synthesis.

Our previous work showed that melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor-positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2-[12<em>5</em>I]-iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2-[12<em>5</em>I]-iodomelatonin at all concentrations tested (10(-8) to 10(-4) m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.

Melatonin,<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>, is a hormonal product of the pineal gland. Its synthesis is higher at night than during the day in all vertebrates including man. Once melatonin is produced in the pineal gland it is quickly released into the vascular system. The rapid release of melatonin is generally believed to relate to its high lipophilicity which allows it to readily pass through the membrane of the pinealocytes and the endothelial cells which line the capillaries. The result of the nocturnal synthesis and secretion of melatonin is high blood levels at night. Also because of its highly lipophilic nature, melatonin from the blood readily escapes into every other bodily fluid and all cells in the body. Until recently it was generally thought that melatonin's action in the organism depended on its exclusive interaction with specific receptors on cells located in discrete locations. Certainly, the interactions of melatonin with these membrane-bound receptors are believed to mediate the endocrine and circadian rhythm effects of melatonin. It was recently discovered, however, that melatonin's primary action may not depend on the previously described membrane receptors. We have found that melatonin is a very potent hydroxyl radical scavenger; free radicals and the hydroxyl radical in particular, because of its very high reactivity, can be extremely damaging to macromolecules in cells. Compared to glutathione and mannitol, two well known free radical scavengers, melatonin is a more powerful scavenger and affords protection of molecules, especially D<em>N</em>A, from oxidative damage. Melatonin's extremely high diffusibility is important for its scavenging action because this feature allows it to enter all cells and every subcellular compartment. Whereas the free radical quenching activity of melatonin does not require a receptor, we also have evidence that it may be bound in the nucleus thereby providing on-site protection to D<em>N</em>A. Besides scavenging the highly toxic hydroxyl radical, melatonin also stimulates glutathione peroxidase activity which metabolizes the precursor of the hydroxyl radical, hydrogen peroxide, to water. Thus, melatonin has at least two means to protect the cell from oxidative damage, i.e., it breaks down hydrogen peroxide to harmless water and, in the event any hydroxyl radicals are formed, melatonin scavenges them. Melatonin may be the premier molecule to protect the organism from oxidative damage.

Recently, it was suggested that melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is oxidized by activated neutrophils in a reaction most probably involving myeloperoxidase (Biochem. Biophys. Res. Commun. (2000) 279, 6<em>5</em>7-662). Myeloperoxidase (MPO) is the most abundant protein of neutrophils and is involved in killing invading pathogens. To clarify if melatonin is a substrate of MPO, we investigated the oxidation of melatonin by its redox intermediates compounds I and II using transient-state spectral and kinetic measurements at 2<em>5</em> degrees C. Spectral and kinetic analysis revealed that both compound I and compound II oxidize melatonin via one-electron processes. The second-order rate constant measured for compound I reduction at pH 7 and pH <em>5</em> are (6.1 +/- 0.2) x 10(6) M(-1) s(-1) and (1.0 +/- 0.08) x 10(7) M(-1) s(-1), respectively. The rates for the one-electron reduction of compound II back to the ferric enzyme are (9.6 +/- 0.3) x 10(2) M(-1) s(-1) (pH 7) and (2.2 +/- 0.1) x 10(3) M(-1) s(-1) (pH <em>5</em>). Thus, melatonin is a much better electron donor for compound I than for compound II. Steady-state experiments showed that the rate of oxidation of melatonin is dependent on the H(2)O(2) concentration, is not affected by superoxide dismutase, and is quickly terminated by sodium cyanide. Melatonin can markedly inhibit the chlorinating activity of MPO at both pH 7 and pH <em>5</em>. The implication of these findings in the activated neutrophil is discussed.

The administration of melatonin, either peripherally (0.01-10 mg/kg) or intraocularly (0.001-10 mumol/eye), to light-exposed chicks dose-dependently increased serotonin <em>N</em>-<em>acetyl</em>transferase (<em>N</em>AT) activity in retina but not in pineal gland. The effect of melatonin was slightly but significantly reduced by luzindole (2-benzyl-<em>N</em>-<em>acetyl</em>tryptamine), and not affected by two other purported melatonin antagonists, <em>N</em>-<em>acetyl</em>tryptamine and <em>N</em>-(2,4-dinitrophenyl)-<em>5</em>-<em>methoxytryptamine</em> (ML-23). The elevation of the enzyme activity induced by melatonin was substantially stronger than that evoked by <em>5</em>-hydroxytryptamine, <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptamine, or <em>5</em>-<em>methoxytryptamine</em>. The melatonin-evoked rise in the retinal <em>N</em>AT activity was counteracted by two dopamine D2 receptor agonists, quinpirole and apomorphine, and prevented by the dopamine D2 receptor blocker spiroperidol, and by an inhibitor of dopamine synthesis, alpha-methyl-p-tyrosine. Melatonin (0.1-10 mg/kg i.p.) dose-dependently decreased the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), as well as the DOPAC/dopamine ratio, in chick retina but not in forebrain. The results obtained (1) indicate that melatonin in vivo potently inhibits dopamine synthesis selectively in retina, and (2) suggest that the increase in retinal <em>N</em>AT activity evoked by melatonin in light-exposed chicks is an indirect action of the compound, and results from the disinhibition of the <em>N</em>AT induction process from the dopaminergic (inhibitory) signal. The results provide in vivo evidence supporting the idea (derived on the basis of in vitro findings) that a mutually antagonistic interaction between melatonin and dopamine operates in retinas of living animals.

BACKGROUND

Antiepileptic and neuroprotective effects of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) have been shown at higher doses (<em>5</em>0-160 mg/kg). We aimed to investigate the antiepileptic effects of low-dose melatonin (10 mg/kg) on pentylenetetrazol (PTZ)-induced experimental epilepsy model.

METHODS

Twelve male albino guinea pigs weighing <em>5</em>00-800 g were used in our work. Initially, latent period, seizure intensity and mortality parameters were evaluated during the epileptic seizure induced by PTZ. After a recovery period of 7 days, effects of the neuroprotective agent, melatonin (which is dissolved in 2.<em>5</em>% ethanol-saline solution), on epileptic seizures induced by PTZ were evaluated. Effects of 2.<em>5</em>% ethanol, which is an anticonvulsant agent when administered acutely in high concentrations, on PTZ-induced seizures were also evaluated.

RESULTS

Data obtained from the study groups (PTZ, PTZ + melatonin and PTZ + ethanol) were evaluated by paired t-test, and p<0.00<em>5</em> was considered statistically significant. The differences of latent periods between the PTZ and PTZ + melatonin groups were found to be statistically significant (p<0.001).

CONCLUSIONS

Although melatonin does not have a primary anticonvulsant effect at low doses (10 mg/kg), it lowers the mortality rates and attenuates seizure severity while increasing the latent period.

Leaf senescence is a typical symptom in plants exposed to dark and may be regulated by plant growth regulators. The objective of this study was to determine whether exogenous application of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) suppresses dark-induced leaf senescence and the effects of melatonin on reactive oxygen species (ROS) scavenging system and chlorophyll degradation pathway in perennial grass species. Mature perennial ryegrass (Lolium perenne L. cv. 'Pinnacle') leaves were excised and incubated in 3 mM 2-(<em>N</em>-morpholino) ethanesulfonic buffer (pH <em>5</em>.8) supplemented with melatonin or water (control) and exposed to dark treatment for 8 days. Leaves treated with melatonin maintained significantly higher endogenous melatonin level, chlorophyll content, photochemical efficiency, and cell membrane stability expressed by lower electrolyte leakage and malondialdehyde (MDA) content compared to the control. Exogenous melatonin treatment also reduced the transcript level of chlorophyll degradation-associated genes and senescence marker genes (LpSAG12.1, Lph36, and Lpl69) during the dark treatment. The endogenous O2- production rate and H2O2 content were significantly lower in these excised leaves treated with melatonin compared to the water control. Exogenous melatonin treatment caused increases in enzymatic activity and transcript levels of superoxide dismutase and catalase but had no significant effects on ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monohydroascorbate reductase. The content of non-enzymatic antioxidants, such as ascorbate and dehydroascorbate, were decreased by melatonin treatment, while the content of glutathione and oxidized glutathione was not affected by melatonin. These results suggest that the suppression of dark-induced leaf senescence by exogenous melatonin may be associated with its roles in regulating ROS scavenging through activating the superoxide dismutase-catalase enzymatic antioxidant pathway and down-regulating chlorophyll degradation in perennial ryegrass.

Human lymphocytes have recently been described as an important physiological source of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), which could be involved in the regulation of the human immune system. On the other hand, stimulation of IL-2 production by exogenous melatonin has been shown in the Jurkat human lymphocytic cell line. Furthermore, both melatonin membrane and nuclear receptors are present in these cells. In this study, we show that the necessary machinery to synthesize melatonin is present and active in resting and stimulated Jurkat cells. Accordingly, we have found that cells synthesize and release melatonin in both conditions. Therefore, we investigated whether endogenous melatonin produced by Jurkat cells was involved in the regulation of IL-2 production. When melatonin membrane and nuclear receptors were blocked using specific antagonists, luzindole and CGP <em>5</em><em>5</em>644, respectively, we found that IL-2 production decreased, and this drop was reverted by exogenous melatonin. Additionally, PHA activation of Jurkat cells changed the profile of melatonin nuclear receptor mR<em>N</em>A expression. A previous study showed that exogenous melatonin is able to counteract the decrease in IL-2 production caused by prostaglandin E2 (PGE2) in human lymphocytes via its membrane receptor. In our model, when we blocked the melatonin membrane receptor with luzindole, the inhibitory effect of PGE2 on IL-2 production was higher. Therefore, we have demonstrated the physiological role of endogenous melatonin in this cell line. These findings indicate that endogenous melatonin synthesized by human T cells would contribute to regulation of its own IL-2 production, acting as an intracrine, autocrine, and/or paracrine substance.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) and the activities of two melatonin-synthesizing enzymes, serotonin <em>N</em>-<em>acetyl</em>transferase (<em>acetyl</em> coenzyme A: arylalkylamine <em>N</em>-<em>acetyl</em>transferase EC 2.3.1.87; <em>N</em>AT) and hydroxyindole-O-methyltransferase (S-adenosyl-L-methionine: <em>N</em>-<em>acetyl</em>serotonin-O-methyltransferase EC 2.1.1.4; HIOMT), were assayed in extracts of ovaries obtained from virgin Wistar-derived rats (7-9 week-old) during the light period of a 12 h light/12 h dark cycle. Melatonin was detected in the rat ovary using reverse-phase high-performance liquid chromatography (HPLC) coupled with fluorometric detection and radioimmunoassay (RIA). In addition, <em>N</em>AT and HIOMT activities were found in rat ovary. The apparent Michaelis constants (Km) for the substrates of <em>N</em>AT and HIOMT in the rat ovary were similar to those reported for the pineal gland and retina. These data suggest that the rat ovary, like the pineal gland and the retina, may synthesize melatonin from serotonin by the sequential action of <em>N</em>AT and HIOMT.

Globally, gastric cancer (GC) is one of the most common types of cancer and the third leading cause of cancer‑related death. In China, gastric and liver cancers have the highest mortality rates. Melatonin, also known as <em>N</em>-<em>acetyl</em>‑<em>5</em>-<em>methoxytryptamine</em>, is a hormone that is produced by the pineal gland in animals and regulates sleep and wakefulness. Melatonin has been shown to inhibit various carcinomas, including GC. There are many different hypotheses to explain the anticancer effects of melatonin, including stimulation of apoptosis, inhibition of cell growth, regulation of anticancer immunity, induction of free-radical scavenging, and the competitive inhibition of estrogen. However, the underlying mechanism by which these effects are elicited remains elusive. The aim of the present study was to investigate the effects of melatonin on human GC cells and determine the underlying molecular mechanism. We treated SGC-7901 GC cells with melatonin and analyzed the resulting protein changes using protein chip technology. Several proteins related to cell apoptosis and proliferation were identified and further tested in SGC-7901 GC cells. We found that melatonin induced cell cycle arrest and the downregulation of CDC2<em>5</em>A, phospho-CDC2<em>5</em>A (at Ser7<em>5</em>), p21 (p21Cip1/p21Waf1) and phospho-p21 (at Thr14<em>5</em>). Melatonin also induced upregulation of Bax, downregulation of Bcl-xL, an increase in cleaved caspase-9 level and activation of caspase-3, which confirmed the involvement of the mitochondria in melatonin‑induced apoptosis. Upstream regulators of the above proteins, MDM2, phospho-MDM2 (at Ser166) and AKT, phospho-AKT (at Thr308) were all attenuated by melatonin, which led to an increase in p<em>5</em>3. The present study demonstrated that the oncostatic effects of melatonin on SGC-7901 GC cells are mediated via the blockade of the AKT/MDM2 intracellular pathway.