N1-Acetyl-5-methoxykynuramine, which decreases in the hippocampus with aging, improves long-term memory via CaMKII/CREB phosphorylation.

Melatonin is a molecule ubiquitous in nature and involved in several physiological functions. In the brain, melatonin is converted to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and then to N1-acetyl-5-methoxykynuramine (AMK), which has been reported to strongly enhance long-term object memory formation. However, the synthesis of AMK in brain tissues and the underlying mechanisms regarding memory formation remain largely unknown. In the present study, young and old individuals from a melatonin-producing strain, C3H/He mice, were employed. The amount of AMK in the pineal gland and plasma was very low compared with those of melatonin at night; conversely, in the hippocampus, the amount of AMK was higher than that of melatonin. Indoleamine 2, 3-dioxygenase (Ido) mRNA was expressed in multiple brain tissues, whereas tryptophan 2,3-dioxygenase (Tdo) mRNA was expressed only in the hippocampus, and its lysate had melatonin to AFMK conversion activity, which was blocked by the TDO inhibitor. The expression levels of phosphorylated cAMP response element binding protein (CREB) and PSD-95 in whole hippocampal tissue were significantly increased with AMK treatment. Before increasing in the whole tissue, CREB phosphorylation was significantly enhanced in the nuclear fraction. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that downregulated genes in hippocampus of old C3H/He mice were more enriched for long-term potentiation (LTP) pathway. Gene set enrichment analysis showed that LTP and neuroactive receptor interaction gene sets were enriched in hippocampus of old mice. In addition, Ido1 and Tdo mRNA expression was significantly decreased in the hippocampus of old mice compared with young mice, and the decrease in Tdo mRNA was more pronounced than Ido1. Furthermore, there was a higher decrease in AMK levels, which was less than 1/10 that of young mice, than in melatonin levels in the hippocampus of old mice. In conclusion, we first demonstrated the Tdo-related melatonin to AMK metabolism in the hippocampus and suggest a novel mechanism of AMK involved in LTP and memory formation. These results support AMK as a potential therapeutic agent to prevent memory decline.

Protective Role of Melatonin and Its Metabolites in Skin Aging.

The skin, being the largest organ in the human body, is exposed to the environment and suffers from both intrinsic and extrinsic aging factors. The skin aging process is characterized by several clinical features such as wrinkling, loss of elasticity, and rough-textured appearance. This complex process is accompanied with phenotypic and functional changes in cutaneous and immune cells, as well as structural and functional disturbances in extracellular matrix components such as collagens and elastin. Because skin health is considered one of the principal factors representing overall "well-being" and the perception of "health" in humans, several anti-aging strategies have recently been developed. Thus, while the fundamental mechanisms regarding skin aging are known, new substances should be considered for introduction into dermatological treatments. Herein, we describe melatonin and its metabolites as potential "aging neutralizers". Melatonin, an evolutionarily ancient derivative of serotonin with hormonal properties, is the main neuroendocrine secretory product of the pineal gland. It regulates circadian rhythmicity and also exerts anti-oxidative, anti-inflammatory, immunomodulatory, and anti-tumor capacities. The intention of this review is to summarize changes within skin aging, research advances on the molecular mechanisms leading to these changes, and the impact of the melatoninergic anti-oxidative system controlled by melatonin and its metabolites, targeting the prevention or reversal of skin aging.

Melatonin and its metabolite N(1)-acetyl-N(1)-formyl-5-methoxykynuramine improve learning and memory impairment related to Alzheimer's disease in rats.

The aim of this study was to investigate the effect of melatonin (MT) and its metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on Alzheimer-like learning and memory impairment in rats intracerebroventricularly injected with streptozotocin (STZ). The results showed that the escape latency of the STZ group was longer than that of the control (CON), MT, and AFMK groups. Increased levels of hyperphosphorylated tau, neurofilament proteins, and malondialdehyde and decreased superoxide dismutase levels were observed in the brains of the rats from the STZ group compared with the brains of the rats from the CON, MT, AFMK high and low group. These results suggest that exogenous MT and AFMK can improve memory impairment and downregulate AD-like hyperphosphorylation induced by STZ, most likely through their antioxidation function. Meanwhile, we found that an equal dose of AFMK had a stronger effect than that of MT. Our results indicate that MT and its metabolite AFMK represent novel treatment strategies for Alzheimer's disease.

Protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin's cascade metabolites C3-OHM and AFMK in RBA1 astrocytes.

Cyclic 3-hydroxymelatonin (C3-OHM) and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) are two major cascade metabolites of melatonin. We previously showed melatonin provides multiple levels of mitochondria-targeted protection beyond as a mitochondrial antioxidant during ionomycin-induced mitochondrial Ca2+ (mCa2+ ) stress in RBA1 astrocytes. Using noninvasive laser scanning fluorescence coupled time-lapse digital imaging microscopy, this study investigated whether C3-OHM and AFMK also provide mitochondrial levels of protection during ionomycin-induced mCa2+ stress in RBA1 astrocytes. Interestingly, precise temporal and spatial dynamic live mitochondrial images revealed that C3-OHM and AFMK prevented specifically mCa2+ -mediated mitochondrial reactive oxygen species (mROS) formation and hence mROS-mediated depolarization of mitochondrial membrane potential (△Ψm ) and permanent lethal opening of the MPT (p-MPT). The antioxidative effects of AFMK, however, were less potent than that of C3-OHM. Whether C3-OHM and AFMK targeted directly the MPT was investigated under a condition of "oxidation free-Ca2+ stress" using a classic antioxidant vitamin E to remove mCa2+ -mediated mROS stress and the potential antioxidative effects of C3-OHM and AFMK. Intriguingly, two compounds still effectively postponed "oxidation free-Ca2+ stress"-mediated depolarization of △Ψm and p-MPT. Measurements using a MPT pore-specific indicator Calcein further identified that C3-OHM and AFMK, rather than inhibiting, stabilized the MPT in its transient protective opening mode (t-MPT), a critical mechanism to reduce overloaded mROS and mCa2+ . These multiple layers of mitochondrial protection provided by C3-OHM and AFMK thus crucially allow melatonin to extend its metabolic cascades of mitochondrial protection during mROS- and mCa2+ -mediated MPT-associated apoptotic stresses and may provide therapeutic benefits against astrocyte-mediated neurodegeneration in the CNS.

Metabolism of melatonin in the skin: Why is it important?

Melatonin is produced in almost all living taxa and is probably 2-3 billion years old. Its pleiotropic activities are related to its local concentration that is secondary to its local synthesis, delivery from distant sites and metabolic or non-enzymatic consumption. This consumption generates metabolites through indolic, kynuric and cytochrome P450 (CYP) mediated hydroxylations and O-demethylation or non-enzymatic processes, with potentially diverse phenotypic effects. While melatonin acts through receptor-dependent and receptor-independent mechanisms, receptors for melatonin metabolites remain to be identified, while their receptor-independent activities are well documented. The human skin with its main cellular components including malignant cells can both produce and rapidly metabolize melatonin in cell-type and context-dependent fashion. The predominant metabolism in human skin occurs through indolic, CYP-mediated and kynuric pathways with main metabolites represented by 6-hydroxymelatonin, N1 -acetyl-N2 -formyl-5-methoxykynuramine (AFMK), N1 -acetyl-5-methoxykynuramine (AMK), 5-methoxytryptamine, 5-methoxytryptophol and 2-hydroxymelatonin. AFMK, 6-hydroxymelatonin, 2-hydroxymelatonin and probably 4-hydroxymelatonin can potentially be produced in epidermis through UVB-induced non-enzymatic melatonin transformation. The skin metabolites are also the same as those produced in lower organisms and plants indicating phylogenetic conservation across diverse species and adaptation by skin of the primordial defense mechanism. As melatonin and its metabolites counteract or buffer environmental stresses to maintain its homeostasis through broad-spectrum activities, both melatoninergic and degradative pathways must be precisely regulated, because the nature of phenotypic regulations will depend on local concentration of melatonin and its metabolites. These can be receptor-mediated or represent non-receptor regulatory mechanisms.

Effects of Melatonin and Its Analogues on Pancreatic Inflammation, Enzyme Secretion, and Tumorigenesis.

Melatonin is an indoleamine produced from the amino acid l-tryptophan, whereas metabolites of melatonin are known as kynuramines. One of the best-known kynuramines is N¹-acetyl-N¹-formyl-5-methoxykynuramine (AFMK). Melatonin has attracted scientific attention as a potent antioxidant and protector of tissue against oxidative stress. l-Tryptophan and kynuramines share common beneficial features with melatonin. Melatonin was originally discovered as a pineal product, has been detected in the gastrointestinal tract, and its receptors have been identified in the pancreas. The role of melatonin in the pancreatic gland is not explained, however several arguments support the opinion that melatonin is probably implicated in the physiology and pathophysiology of the pancreas. (1) Melatonin stimulates pancreatic enzyme secretion through the activation of entero-pancreatic reflex and cholecystokinin (CCK) release. l-Tryptophan and AFMK are less effective than melatonin in the stimulation of pancreatic exocrine function; (2) Melatonin is a successful pancreatic protector, which prevents the pancreas from developing of acute pancreatitis and reduces pancreatic damage. This effect is related to its direct and indirect antioxidant action, to the strengthening of immune defense, and to the modulation of apoptosis. Like melatonin, its precursor and AFMK are able to mimic its protective effect, and it is commonly accepted that all these substances create an antioxidant cascade to intensify the pancreatic protection and acinar cells viability; (3) In pancreatic cancer cells, melatonin and AFMK activated a signal transduction pathway for apoptosis and stimulated heat shock proteins. The role of melatonin and AFMK in pancreatic tumorigenesis remains to be elucidated.

Melatonin and its metabolites as copper chelating agents and their role in inhibiting oxidative stress: a physicochemical analysis.

The copper sequestering ability of melatonin and its metabolites cyclic 3-hydroxymelatonin (3OHM), N(1) -acetyl-N(2) -formyl-5-methoxykynuramine (AFMK), and N(1) -acetyl-5-methoxykynuramine (AMK) was investigated within the frame of the Density Functional Theory. It was demonstrated that these compounds are capable of chelating copper ions, yielding stable complexes. The most likely chelation sites were identified. Two different mechanisms were modeled, the direct-chelation mechanism (DCM) and the coupled-deprotonation-chelation mechanism (CDCM). It is proposed that, under physiological conditions, CDCM would be the main chelation route for Cu(II). It was found that melatonin and its metabolites fully inhibited the oxidative stress induced by Cu(II)-ascorbate mixtures, via Cu(II) chelation. In the same way, melatonin, AFMK, and 3OHM also prevented the first step of the Haber-Weiss reaction, consequently turning off the ˙OH production via the Fenton reaction. Therefore, it is proposed that, in addition to the previously reported free radical scavenging cascade, melatonin is also involved in a concurrent 'chelating cascade', thereby contributing to a reduction in oxidative stress. 3OHM was identified as the most efficient of the studied compounds for that purpose, supporting the important role of this metabolite in the beneficial effects of melatonin against oxidative stress.

Metabolism of melatonin and biological activity of intermediates of melatoninergic pathway in human skin cells.

Indolic and kynuric pathways of skin melatonin metabolism were monitored by liquid chromatography mass spectrometry in human keratinocytes, melanocytes, dermal fibroblasts, and melanoma cells. Production of 6-hydroxymelatonin [6(OH)M], N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5-MT) was detected in a cell type-dependent fashion. The major metabolites, 6(OH)M and AFMK, were produced in all cells. Thus, in immortalized epidermal (HaCaT) keratinocytes, 6(OH)M was the major product with Vmax = 63.7 ng/10(6) cells and Km = 10.2 µM, with lower production of AFMK and 5-MT. Melanocytes, keratinocytes, and fibroblasts transformed melatonin primarily into 6(OH)M and AFMK. In melanoma cells, 6(OH)M and AFMK were produced endogenously, a process accelerated by exogenous melatonin in the case of AFMK. In addition, N-acetylserotonin was endogenously produced by normal and malignant melanocytes. Metabolites showed selective antiproliferative effects on human primary epidermal keratinocytes in vitro. In ex vivo human skin, both melatonin and AFMK-stimulated expression of involucrin and keratins-10 and keratins-14 in the epidermis, indicating their stimulatory role in building and maintaining the epidermal barrier. In summary, the metabolism of melatonin and its endogenous production is cell type-dependent and expressed in all three main cell populations of human skin. Furthermore, melatonin and its metabolite AFMK stimulate differentiation in human epidermis, indicating their key role in building the skin barrier.

Serotonin, melatonin and their precursors and metabolites and vitamin D3 derivatives in honey.

We are commenting recent discoveries on the presence of L-DOPA, dopamine, 5-hydroxytryptophan, tryptamine, serotonin, N-acetylserotonin, melatonin, 2-hydroxymelatonin, AFMK and AMK in honey. Serotonin and melatonin, products of the tryptophan metabolism, are widely produced in nature, serving as hormones, neurotransmitters, biological regulators, neurotransmitters and antioxidants, in a context dependent fashion. Dopamine and tryptamine are important neurotransmitters across different species. Honey is used as one of the most popular healthy food substances. Detection of above molecules in honey accompanied by detection of vitamin D3 and its hydroxyderivatives, is consistent with their detection in insects and plants. Their presence in honey enhances spectrum of its beneficial effects for human health and implicates that these molecules must play important role in social insects physiology, bees development and colony functions.

Stress Response System in the Fish Skin-Welfare Measures Revisited.

The skin of vertebrates acts as a biological barrier defending the organism against many harmful environmental factors. It is well established that the main stress hormone cortisol, together with antioxidants such as melatonin (Mel) and its biologically active metabolites set up a local stress response system in the mammalian skin. Recently, our research group has shown that in fish there are basic conditions for the functioning of a cutaneous stress response system (CSRS) similar to that in mammals, where Mel with its biologically active metabolite AFMK (N1-acetyl-N2-formyl-5-methoxykynuramine) and cortisol act together to protect organism against unfavorable environment. Since aquaculture is making an increasing contribution to the global economy and new laws are demanding people to respect the welfare requirements of animals there has been increasing interest in indicators of fish well-being in aquaculture. This article addresses the problem of on-farm assessment of fish welfare and proposes the CSRS as a new source of information on the welfare status of farmed fish.

Computational study of metal complexes formed with EDTA, melatonin, and its main metabolites: implications in lead intoxication and clues to a plausible alternative treatment.

Melatonin has been proposed as an alternative treatment to the usage of EDTA for lead intoxication. In this computational paper, since previous work has not systematically studied the complexes that may be formed in the existing and proposed treatments, we study 45 possible complexes that we suggest may be formed between Pb and some essential metals with melatonin, melatonin metabolites, and EDTA, analyzing the stability and viability of these through the Gibbs free energy of complexation (ΔΔG), molecular orbitals, and energy decomposition analysis at the DFT level of theory PBE/TZ2P. Our findings show that most complexes present exergonic energies of reaction, and thus spontaneous complex formation. In addition, we show that the AMK and 3OHM melatonin metabolites possess electronic and thermodynamic properties adequate to act as lead trapping molecules due to the lower Pauli repulsion energies involved in the complexes they form and their large negative values of ΔΔG. Therefore, it is shown that both melatonin and some of its metabolites may be employed in a viable treatment for lead intoxication through formation of stable Pb-complexes. Graphical abstract Metal complexes formed with EDTA, melatonin, and its main metabolites.

Binding of Pb-Melatonin and Pb-(Melatonin-metabolites) complexes with DMT1 and ZIP8: implications for lead detoxification.

We have applied the docking methodology to characterize the binding modes of the divalent metal transporter 1 (DMT1) and the zinc transporter 8 (ZIP8) protein channels with: melatonin, some melatonin metabolites, and a few lead complexes of melatonin and its metabolites, in three different coordination modes (mono-coordinated, bi-coordinated and tri-coordinated). Our results show that bi-coordinated and tri-coordinated lead complexes prefer to bind inside the central region of ZIP8. Moreover, the interaction strength is larger compared with that of the free melatonin and melatonin metabolites. On the other hand, the binding modes with DMT1 of such complexes display lower binding energies, compared with the free melatonin and melatonin metabolites. Our results suggest that ZIP8 plays a major role in the translocation of Pb, bi or tri coordinated, when melatonin metabolites are present. Finally, we have characterized the binding modes responsible for the ZIP8 large affinities, found in bi-coordinated and tri-coordinated lead complexes. Our results show that such interactions are greater, because of an increase of the number of hydrogen bonds, the number and intensity of electrostatic interactions, and the interaction overlay degree in each binding mode. Our results give insight into the importance of the ZIP8 channel on lead transport and a possible elimination mechanism in lead detoxification processes. Graphical abstract .

Evaluation of melatonin and AFMK levels in women with breast cancer.

PURPOSE: Changes in the circadian rhythm may contribute to the development of cancer and are correlated with the high risk of breast cancer (BC) in night workers. Melatonin is a hormone synthesized by the pineal gland at night in the absence of light. Levels of melatonin and the metabolite of oxidative metabolism AFMK (acetyl-N-formyl-5-methoxykynurenamine), are suggested as potential biomarkers of BC risk. The aims of this study were to evaluate levels of melatonin and AFMK in women recently diagnosed with BC, women under adjuvant chemotherapy, and night-shift nurses, and compare them with healthy women to evaluate the relation of these compounds with BC risk. METHODS: Blood samples were collected from 47 women with BC, 9 healthy women, 10 healthy night shift nurses, and 6 patients under adjuvant chemotherapy. Compound levels were measured by mass spectrometry. RESULTS AND CONCLUSIONS: Our results showed that women with BC had lower levels of melatonin compared to control group women, and even lower in night-shift nurses and in patients under adjuvant chemotherapy. There was no significant difference of AFMK levels between the groups. In addition to this, high levels of melatonin and AFMK were related to patients with metastasis, and high levels of AFMK were related to the presence of lymph node-positive, tumor > 20 mm and patients who sleep with light at night. Our results showed a reduction of melatonin levels in BC patients, suggesting a relation with the disease, and in addition, point to the importance of melatonin supplementation in women that work at night to reduce the BC risk.

Melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (afmk) enhance chemosensitivity to gemcitabine in pancreatic carcinoma cells (PANC-1).

BACKGROUND: Gemcitabine is a standard chemotherapeutic agent for patients suffering from pancreatic cancer. However, the applied therapy is not effective due to the resistance of tumor cells to cytostatics, caused by inefficiency of the apoptotic mechanisms. Herein, we present the hypothesis that melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) modify the effect of gemcitabine on PANC-1 cells and that this phenomenon is dependent on the modulation of apoptosis. METHODS: PANC-1 cells have been incubated with melatonin, AFMK or gemcitabine alone or in combination to determine the cytotoxity and proliferative effects. In subsequent part of the study, cells were harvested, the proteins were isolated and analyzed employing immunoprecipitation/immunoblotting. RESULTS: Incubation of PANC-1 cells with gemcitabine resulted in upregulation of pro-apoptotic bax and caspases proteins expression, downregulation of anti-apoptotic Bcl-2, heat shock proteins (HSPs) and modulation of cellular inhibitors of apoptosis (IAPs). Both melatonin and AFMK administered to PANC-1 in combination with gemcitabine inhibited the production of HSP70 and cIAP-2 as compared to the results obtained with gemcitabine alone. These changes were accompanied by upregulation of Bax/Bcl-2 ratio and reduction of procaspases-9 and -3 abundance, followed by an increase in the formation of active caspase of PANC-1 cells with combination of gemcitabine plus low doses of melatonin or AFMK led to enhanced cytotoxicity and resulted in the inhibition of PANC-1 cells growth as compared to effects of gemcitabine alone. CONCLUSION: Melatonin and AFMK could improve the anti-tumor effect of gemcitabine in PANC-1 cells presumably through the modulation of apoptotic pathway.

Melatonin pharmacokinetics after transdermal administration changes according to the time of the day.

Melatonin is a neurohormone with multiple and different actions, such as chronobiotic or antioxidant. Melatonin is usually orally administered, but dermal administration is also useful in dermatological diseases or as adjuvant to certain skin treatments. Here, we studied the variability of the pharmacokinetics of melatonin and its metabolite AFMK, when melatonin is transdermally administered to Hairless rat at two different times of day (Zeitgeber Time 4 (ZT4) and ZT16). Moreover, in order to obtain the bioavailability, kinetics after intravenous administration was also studied. In addition, a permeation study was carried out, at both ZTs, to test the amount of melatonin retained in the skin after transdermal administration. Results showed that pharmacokinetic parameters of melatonin administered exogenously depended on the time of the day. When intravenous data were fitted to a compartmental model, the extrapolated plasma concentration at time 0 and the area under the curve were higher at ZT4, while clearance, volumes of central and peripheral compartments and volume of distribution at the steady state were higher at ZT16. Transdermal administration was best fitted to a one-compartment model and tmax, half-life of absorption and area under the curve showed higher values at ZT4, while the absorption rate and constant of absorption were higher at ZT16. AFMK was detected in all cases, but no differences between the two ZTs were observed. Transdermal administration showed better bioavailability also at ZT4. Results indicate that time of day is a variable that should be taken into account when melatonin is transdermally administered.

Melatonin and its metabolites accumulate in the human epidermis in vivo and inhibit proliferation and tyrosinase activity in epidermal melanocytes in vitro.

Melatonin and its metabolites including 6-hydroxymelatonin (6(OH)M), N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5MT) are endogenously produced in human epidermis. This production depends on race, gender and age. The highest melatonin levels are in African-Americans. In each racial group they are highest in young African-Americans [30-50 years old (yo)], old Caucasians (60-90 yo) and Caucasian females. AFMK levels are the highest in African-Americans, while 6(OH)M and 5MT levels are similar in all groups. Testing of their phenotypic effects in normal human melanocytes show that melatonin and its metabolites (10(-5) M) inhibit tyrosinase activity and cell growth, and inhibit DNA synthesis in a dose dependent manner with 10(-9) M being the lowest effective concentration. In melanoma cells, they inhibited cell growth but had no effect on melanogenesis, except for 5MT which enhanced L-tyrosine induced melanogenesis. In conclusion, melatonin and its metabolites [6(OH)M, AFMK and 5MT] are produced endogenously in human epidermis and can affect melanocyte and melanoma behavior.

Network beyond IDO in psychiatric disorders: revisiting neurodegeneration hypothesis.

The involvement of immune system activation in the pathophysiology of certain psychiatric disorders is well documented. Inflammatory molecules such as pro-inflammatory cytokines could enhance the activity of the indoleamine 2,3-dioxygenase (IDO) enzyme which is the first rate-limiting enzyme of the tryptophan degradation pathway, the kynurenine pathway. The increased tryptophan degradation could induce serotonin depletion and depressive mood. On the other hand, the downstream metabolites from this pathway, such as 3-hydroxykynurenine, quinolinic acid and kynurenic acid, are neuroactive metabolites which can modulate several neurotransmissions, such as glutamatergic, GABAergic, dopaminergic and noradrenergic neurotransmissions, which in turn induce changes in neuronal-glial network and neuropsychiatric consequences. In this issue, we have revised the previous 'neurodegeneration hypothesis,' which explained the involvement of cytokines and IDO pathway interaction in depression, with a further extended view related to the network beyond IDO, the network between immune molecules, tryptophan metabolites and different neurotransmitters, in depression and other major psychiatric disorders such as schizophrenia, bipolar disorder and childhood psychiatric disorders.