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.

The Vital Role of Melatonin and Its Metabolites in the Neuroprotection and Retardation of Brain Aging.

While primarily produced in the pineal gland, melatonin's influence goes beyond its well-known role in regulating sleep, nighttime metabolism, and circadian rhythms, in the field of chronobiology. A plethora of new data demonstrates melatonin to be a very powerful molecule, being a potent ROS/RNS scavenger with anti-inflammatory, immunoregulatory, and oncostatic properties. Melatonin and its metabolites exert multiple beneficial effects in cutaneous and systemic aging. This review is focused on the neuroprotective role of melatonin during aging. Melatonin has an anti-aging capacity, retarding the rate of healthy brain aging and the development of age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. Melatonin, as well as its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), can reduce oxidative brain damage by shielding mitochondria from dysfunction during the aging process. Melatonin could also be implicated in the treatment of neurodegenerative conditions, by modifying their characteristic low-grade neuroinflammation. It can either prevent the initiation of inflammatory responses or attenuate the ongoing inflammation. Drawing on the current knowledge, this review discusses the potential benefits of melatonin supplementation in preventing and managing cognitive impairment and neurodegenerative diseases.

Effects of endogenous and exogenous N-acetyl-5-methoxy kynuramine on object recognition memory in male C3H mice.

Prevention of dementia is important, because it is a leading cause of disability in elderly people. We previously reported that acute intraperitoneal treatment with N-acetyl-5-methoxy kynuramine (AMK), a melatonin (MEL) metabolite, enhanced long-term object recognition memory in ICR mice, a MEL deficient strain. Despite the presumable availability of AMK for dementia, its effects on cognitive performance have not been elucidated. It is unclear whether endogenous AMK is responsible for modulating long-term memory performance. To address this question, we assessed the effects of endogenous AMK on learning and memory using an object recognition test. C3H mice, a MEL-proficient strain, showed peak MEL levels at zeitgeber times (ZT) 19 and 22. Object recognition memory at ZT20 was superior to that at ZT8. Norharmane (NHM, 100 mg/kg), an indoleamine-2,3-dioxygenase (IDO) inhibitor, prevented the transformation of MEL to AMK, thereby suppressing AMK synthesis at ZT20. NHM (100 mg/kg) and another IDO inhibitor, 1-methyl-L-tryptophan (1-MT, 100 mg/kg), disrupted elevated cognitive performance at ZT20. These data imply that endogenous AMK may play a physiological role in the modulation of cognitive function. We also investigated the effects of pharmacological doses of MEL and AMK on object recognition memory in young C3H mice. MEL administration of 0.1 mg/kg, but not 0.01 mg/kg, enhanced object recognition memory, whereas 0.01 and 1 mg/kg AMK enhanced object recognition memory. Administration of 0.1 and 1 mg/kg AMK also enhanced object recognition memory in old C3H mice. These findings in MEL-proficient mice should be confirmed in other learning and memory tests before encouraging the clinical use of AMK.

The melatonin metabolite N1-acetyl-5-methoxykynuramine facilitates long-term object memory in young and aging mice.

Melatonin (MEL) has been reported to enhance cognitive processes, making it a potential treatment for cognitive decline. However, the role of MEL's metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in these effects are unknown. The current study directly investigated the acute effects of systemic MEL, AFMK, and AMK on novel object recognition. We also analyzed MEL, AFMK, and AMK levels in hippocampus and temporal lobe containing the perirhinal cortex following systemic MEL and AMK treatment. AMK administered post-training had a more potent effect on object memory than MEL and AFMK. AMK was also able to rescue age-associated declines in memory impairments when object memory was tested up to 4 days following training. Results from administering AMK at varying times around the training trial and the metabolism time course in brain tissue suggest that AMK's memory-enhancing effects reflect memory consolidation. Furthermore, inhibiting the MEL-to-AMK metabolic pathway disrupted object memory at 24 hours post-training, suggesting that endogenous AMK might play an important role in long-term memory formation. This is the first study to report that AMK facilitates long-term object memory performance in mice, and that MEL crosses the blood-brain barrier and is immediately converted to AMK in brain tissue. Overall, these results support AMK as a potential therapeutic agent to improve or prevent memory decline.

Melatonin, Its Metabolites and Their Interference with Reactive Nitrogen Compounds.

Melatonin and several of its metabolites are interfering with reactive nitrogen. With the notion of prevailing melatonin formation in tissues that exceeds by far the quantities in blood, metabolites come into focus that are poorly found in the circulation. Apart from their antioxidant actions, both melatonin and N1-acetyl-5-methoxykynuramine (AMK) downregulate inducible and inhibit neuronal NO synthases, and additionally scavenge NO. However, the NO adduct of melatonin redonates NO, whereas AMK forms with NO a stable product. Many other melatonin metabolites formed in oxidative processes also contain nitrosylatable sites. Moreover, AMK readily scavenges products of the CO2-adduct of peroxynitrite such as carbonate radicals and NO2. Protein AMKylation seems to be involved in protective actions.

Cortisol and melatonin in the cutaneous stress response system of fish.

The stress hormone cortisol, together with antioxidants, melatonin (Mel) and its biologically active metabolites, 5-methoxykynuramines, including AFMK, set up a local stress response system in mammalian skin. Our in vitro study of the European flounder (Platichthys flesus) was designed to examine whether Mel and AFMK would respond to cortisol while a glucocorticoid is added to the incubation medium. The concentrations of cortisol in the incubation medium mimic plasma cortisol levels seen in fish exposed to different types of stresses such as handling, confinement, high density, food-deprivation or air-exposure. We measured Mel and AFMK in skin explants and culture media using high-performance liquid chromatography (HPLC) with fluorescence detection. We also analysed melanosome response (dispersion/aggregation) in the explants subjected to the different treatments. Cortisol stimulated the release of Mel and AFMK from skin explants in a dose-dependent manner. Melanosome dispersion and a darkening of the skin explants were observed after incubation with cortisol. This study is the first to demonstrate the interrelationship between cortisol and Mel/AFMK in fish skin. Our data strongly suggest that the cutaneous stress response system (CSRS) is present in fish. The question remains whether Mel, AFMK or cortisol are synthetized locally in fish skin and/or transported by the bloodstream. The presence of the CSRS should be taken into account during elaboration of new indicators of fish welfare both in aquaculture and in the wild.

Enhanced production of camptothecin and biological preparation of N 1-acetylkynuramine in Camptotheca acuminata cell suspension cultures.

The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a ∼500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N 1-acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM.

Monoamine oxidase inhibitory activities of heterocyclic chalcones.

Studies have shown that natural and synthetic chalcones (1,3-diphenyl-2-propen-1-ones) possess monoamine oxidase (MAO) inhibition activities. Of particular importance to the present study is a report that a series of furanochalcones acts as MAO-B selective inhibitors. Since the effect of heterocyclic substitution, other than furan (and more recently thiophene, piperidine and quinoline) on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesise and evaluate further heterocyclic chalcone analogues as inhibitors of the human MAOs. For this purpose, heterocyclic chalcone analogues that incorporate pyrrole, 5-methylthiophene, 5-chlorothiophene and 6-methoxypyridine substitution were examined. Seven of the nine synthesised compounds exhibited IC50 values <1 µM for the inhibition of MAO-B, with all compounds exhibiting higher affinities for MAO-B compared to the MAO-A isoform. The most potent MAO-B inhibitor (4h) displays an IC50 value of 0.067 µM while the most potent MAO-A inhibitor (4e) exhibits an IC50 value of 3.81 µM. It was further established that selected heterocyclic chalcones are reversible and competitive MAO inhibitors. 4h, however, may exhibit tight-binding to MAO-B, a property linked to its thiophene moiety. We conclude that high potency chalcones such as 4h represent suitable leads for the development of MAO-B inhibitors for the treatment of Parkinson's disease and possibly other neurodegenerative disorders.

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.

Substituents effects on activity of kynureninase from Homo sapiens and Pseudomonas fluorescens.

A series of substituted kynurenines (3-bromo-DL, 3-chloro-DL, 3-fluoro-DL, 3-methyl-DL, 5-bromo-L, 5-chloro-L, 3,5-dibromo-L and 5-bromo-3-chloro-DL) have been synthesized and tested for their substrate activity with human and Pseudomonas fluorescens kynureninase. All of the substituted kynurenines examined have substrate activity with both human as well as P. fluorescens kynureninase. For the human enzyme, 3- and 5-substituted kynurenines have kcat and kcat/Km values higher than L-kynurenine, but less than that of the physiological substrate, 3-hydroxykynurenine. However, 3,5-dibromo- and 5-bromo-3-chlorokynurenine have kcat and kcat/Km values close to that of 3-hydroxykynurenine with human kynureninase. The effects of the 3-halo substituents on the reactivity with human kynureninase may be due to electronic effects and/or halogen bonding. In contrast, for the bacterial enzyme, 3-methyl, 3-halo and 3,5-dihalokynurenines are much poorer substrates, while 3-fluoro, 5-bromo, and 5-chlorokynurenine have kcat and kcat/Km values comparable to that of its physiological substrate, L-kynurenine. Thus, 5-bromo and 5-chloro-L-kynurenine are good substrates for both human as well as bacterial enzyme, indicating that both enzymes have space for substituents in the active site near C-5. The increased activity of the 5-halokynurenines may be due to van der Waals contacts or hydrophobic effects. These results may be useful in the design of potent and/or selective inhibitors of human and bacterial kynureninase.

Oxidation of melatonin by taurine chloramine.

Melatonin is widely known for its antioxidant, immunomodulatory, and anti-inflammatory effects. Hypochlorous acid (HOCl) is one example of an endogenous oxidant that is promptly neutralized by melatonin. Melatonin also inhibits myeloperoxidase, the enzyme that catalyzes the oxidation of chloride to HOCl. Taurine is the most abundant free amino acid in leukocytes. In activated neutrophils, taurine is converted to taurine chloramine (Tau-NHCl) through a reaction with HOCl. In addition, the related compound taurine bromamine (Tau-NHBr) can be released by neutrophils and eosinophils. The aim of this study was to investigate the reactivity of Tau-NHCl and Tau-NHBr with melatonin. We found that melatonin can react with either Tau-NHCl or Tau-NHBr, leading to the production of 2-hydroxymelatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). The reaction was pH-dependent, and it occurs more rapidly at a slightly acidic pH. Tau-NHBr was significantly more reactive than Tau-NHCl. Using Tau-NHBr as the oxidizing agent, 1 mm melatonin was oxidized in less than 1 min. The pH dependence of the reaction with Tau-NHCl and the increased reactivity of Tau-NHBr can be explained by a mechanism based on the initial attack of chloronium (Cl(+)) or bromonium (Br(+)) ions on melatonin. We also found that the addition of iodide to the reaction medium increased the yield of AFMK. These findings could contribute to the establishment of new functions for melatonin in inflammatory and parasitic diseases, where the role of this indoleamine has been extensively investigated.

Analysis of N1-acetyl-N2-formyl-5-methoxykynuramine/N1-acetyl-5-methoxy-kynuramine formation from melatonin in mice.

The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxy-kynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy-AMK and glucuronide-conjugated hydroxy-AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress.

Effect of melatonin in the antioxidant defense system in the locomotor muscles of the estuarine crab Neohelice granulata (Decapoda, Brachyura).

In vertebrates, many studies verified different effects of melatonin in the antioxidant defense system (ADS). In crustaceans, few studies have been conducted to verify this possibility. We verified the melatonin effects in the crab Neohelice granulata using low (0.002 and 0.02 pmol/crab) and high (2.0 and 20.0 pmol/crab) melatonin dosages in short-term (0.5h) and long-term (9.5h) experiments. We analyzed the antioxidant capacity against peroxyl radicals (ACAP), reactive oxygen species (ROS) concentration, levels of by products of lipid peroxidation (LPO), oxygen consumption (VO(2)), the activity of glutamate cysteine ligase (gamma-GCL) and catalase (CAT) and glutathione content (GSH). Finally, the effects of exogenous melatonin were verified in terms of melatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) content in the muscles of N. granulata. In short-term experiment and low dosages, melatonin increased the VO(2), gamma-GCL activity and GSH content (p<0.05) and decreased melatonin content (p<0.05) without effects in ROS, ACAP and LPO (p>0.05). Possibly, melatonin is acting in the ADS increasing its efficiency and/or acting in mitochondrial activity and/or through signaling muscles to increase its consumption. AFMK was only detected in the eyestalk and cerebroid ganglia. In high dosages melatonin effects decreased, possibly by the desensitization of their receptors. In long-term experiment, melatonin decreased ACAP (p<0.05), and CAT activity (p<0.05) in low dosages. In high dosages melatonin reduced VO(2) (p<0.05) and increased ACAP (p<0.05), possibly stimulating others components of the ADS. In conclusion, melatonin in the locomotor muscles of N. granulata affects the antioxidant/pro-oxidant balance in a time and dosage dependent manner.

Kynuramines, metabolites of melatonin and other indoles: the resurrection of an almost forgotten class of biogenic amines.

Kynuramines represent their own class of biogenic amines. They are formed either by decarboxylation of kynurenines or pyrrole ring cleavage of indoleamines. N(2)-formylated compounds formed in this last reaction can be deformylated either enzymatically by arylamine formamidases or hemoperoxidases, or photochemically. The earlier literature mainly focussed on cardiovascular effects of kynuramine, 5-hydroxykynuramine and their N(1),N(1)-dimethylated analogs, including indirect effects via release of catecholamines or acetylcholine and interference with serotonin receptors. After the discovery of N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK) as major brain metabolites of melatonin, these compounds became of particular interest. They were shown to be produced enzymatically, pseudoenzymatically, by various free radical-mediated and via photochemical processes. In recent years, AFMK and AMK were shown to scavenge reactive oxygen and nitrogen species, thereby forming several newly discovered 3-indolinone, cinnolinone and quinazoline compounds, and to protect tissues from damage by reactive intermediates in various models. AMK is of special interest due to its properties as a potent cyclooxygenase inhibitor, NO scavenger forming a stable nitrosation product, inhibitor and/or downregulator of neuronal and inducible NO synthases, and a mitochondrial metabolism modulator. AMK easily interacts with aromates, forms adducts with tyrosyl and tryptophanyl residues, and may modify proteins.

The melatonin metabolite N-acetyl-5-methoxykynuramine is a potent singlet oxygen scavenger.

Singlet oxygen was generated by means of rose bengal under irradiation by visible light. N(1)-acetyl-5-methoxykynuramine (AMK) was rapidly destroyed by this reactive oxygen species, whereas its formylated precursor, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), was remarkably inert. At photon fluence rates of 1400 mumol photons/m(2)s, and using 20 mum rose bengal, most of initially 0.2 mm AMK was destroyed within 2 min, whereas AFMK remained practically unchanged for much longer periods of time. Competition experiments with other scavengers revealed the following order of reactivity towards singlet oxygen: diazabicyclo-[2,2,2]-octane (DABCO) << imidazole < 4-ethylphenol < N(alpha)-acetylhistidine < histidine < melatonin < AMK, the last one being about 150 times more effective than DABCO. Contrary to the oxidation in free radical-generating systems, AMK did not form adducts with the tyrosine side chain fragment, 4-ethylphenol, under the influence of singlet oxygen. In UV-exposed cells (keratinocytes, plant cells) it is likely to be more rapidly destroyed by singlet oxygen than formed from AFMK.

Novel rhythms of N1-acetyl-N2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation.

N1-acetyl-N2-formyl-5-methoxykynuramine (AMFK) is a major metabolite of melatonin in mammals. To investigate whether AFMK exists in plants, an aquatic plant, water hyacinth, was used. To achieve this, LC/MS/MS with a deuterated standard was employed. AFMK was identified in any plant for the first time. Both it and its precursor, melatonin, were rhythmic with peaks during the late light phase. These novel rhythms indicate that these molecules do not serve as the chemical signal of darkness as in animals but may relate to processes of photosynthesis or photoprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10,000-15,000 microW/cm2) compared to those grown under artificial light (400-450 microW/cm2). Melatonin and AFMK, as potent free radical scavengers, may assist plants in coping with harsh environmental insults, including soil and water pollutants. High levels of melatonin and AFMK in water hyacinth may explain why this plant more easily tolerates environmental pollutants, including toxic chemicals and heavy metals and is successfully used in phytoremediation. These novel findings could lead to improvements in the phytoremediative capacity of plants by either stimulating endogenous melatonin synthesis or by adding melatonin to water/soil in which they are grown.

The co-catalytic effect of chlorpromazine on peroxidase-mediated oxidation of melatonin: enhanced production of N1-acetyl-N2-formyl-5-methoxykynuramine.

Accumulating evidence points to relationships between increased production of reactive oxygen or decreased antioxidant protection in schizophrenic patients. Chlorpromazine (CPZ), which remains a benchmark treatment for people with schizophrenia, has been described as a pro-oxidant compound. Because the antioxidant compound melatonin exerts protective effects against CPZ-induced liver disease in rats, in this investigation, our main objective was to study the effect of CPZ as a co-catalyst of peroxidase-mediated oxidation of melatonin. We found that melatonin was an excellent reductor agent of preformed CPZ cation radical (CPZ(*+)). The addition of CPZ during the horseradish peroxidase (HRP)-catalyzed oxidation of melatonin provoked a significant increase in the rate of oxidation and production of N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). Similar results were obtained using myeloperoxidase. The effect of CPZ on melatonin oxidation was rather higher at alkaline pH. At pH 9.0, the efficiency of oxidation of melatonin was 15 times higher and the production of AFMK was 30 times higher as compared with the assays in the absence of CPZ. We suggest that CPZ is able to exacerbate the rate of oxidation of melatonin by an electron transfer mechanism where CPZ(*+), generated during the peroxidase-catalyzed oxidation, is able to efficiently oxidize melatonin.

MT3/QR2 melatonin binding site does not use melatonin as a substrate or a co-substrate.

Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 microM of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 microM).

Metabolism of melatonin by cytochrome P450s in rat liver mitochondria and microsomes.

In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O-demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N(1)-acetyl-N(2)-formyl-5-methoxy-kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N-acetylserotonin, 2-hydroxymelatonin, 6-hydroxymelatonin and the same two unknown products. Eadie-Hofstee plots for 6-hydroxylation and O-demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower K(m) value) and higher V(max) for melatonin 6-hydroxylation and O-demethylation for both high-affinity and low-affinity components as compared with microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high- and low-affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P450s metabolize melatonin principally by 6-hydroxylation, with O-demethylation representing a minor metabolic pathway.

Potential role of tryptophan derivatives in stress responses characterized by the generation of reactive oxygen and nitrogen species.

To face physicochemical and biological stresses, living organisms evolved endogenous chemical responses based on gas exchange with the atmosphere and on formation of nitric oxide (NO(*)) and oxygen derivatives. The combination of these species generates a complex network of variable extension in space and time, characterized by the nature and level of the reactive oxygen (ROS) and nitrogen species (RNS) and of their organic and inorganic scavengers. Among the latter, this review focusses on natural 3-substituted indolic structures. Tryptophan-derived indoles are unsensitive to NO(*), oxygen and superoxide anion (O(2)(*-)), but react directly with other ROS/RNS giving various derivatives, most of which have been characterized. Though the detection of some products like kynurenine and nitroderivatives can be performed in vitro and in vivo, it is more difficult for others, e.g., 1-nitroso-indolic compounds. In vitro chemical studies only reveal the strong likelihood of their in vivo generation and biological effects can be a sign of their transient formation. Knowing that 1-nitrosoindoles are NO donors and nitrosating agents indicating they can thus act both as mutagens and protectors, the necessity for a thorough evaluation of indole-containing drugs in accordance with the level of the oxidative stress in a given pathology is highlighted.