Showing papers in "Journal of Pineal Research in 2009"

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

Abstract: 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.

Protective effect of melatonin against chlorophyll degradation during the senescence of barley leaves.

Abstract: Melatonin (N-acetyl-5-methoxytryptamine) is a highly conserved molecule whose presence is not exclusive to the animal kingdom. Indeed, numerous studies have demonstrated its presence in plants, where the possible role(s) of this indoleamine is (are) under active investigation. The present work aims to further our knowledge in this respect and presents the results of a study of the effect that melatonin has on foliar senescence. Barley leaves treated with melatonin solutions clearly slowed down the senescence process, as estimated from the chlorophyll lost in leaves. This effect of melatonin was concentration dependent, with an optimal response being obtained at 1 mm melatonin, after 48 hr of incubation in darkness. The already known effects of the phytohormones, kinetin, and abscisic acid, were also assayed. Of the phytohormone and melatonin combinations assayed, 1 mm melatonin presented the best protection against senescence. The levels of endogenous melatonin in control leaves were measured by liquid chromatography with fluorescence detection and in leaves treated with different exogenous melatonin concentrations (to demonstrate the absorption capacity of leaves). The possible physiological implications of this newly revealed action of melatonin in foliar senescence are discussed.

Oxidative stress of the newborn in the pre- and postnatal period and the clinical utility of melatonin.

Abstract: Newborns, and especially those delivered preterm, are probably more prone to oxidative stress than individuals later in life. Also during pregnancy, increased oxygen demand augments the rate of production of reactive oxygen species (ROS) and women, even with normal pregnancies, experience elevated oxidative stress and lipid peroxidation compared with nonpregnant women. Also, there appears to be an increase in ROS generation in the placenta of pre-eclamptic women. In comparison with healthy adults, newborn infants have lower levels of plasma antioxidants such as vitamin E, β-carotene, and sulphydryl groups, lower levels of plasma metal binding proteins including ceruloplasmin and transferrin, and reduced activity of erythrocyte superoxide dismutase. This review summarizes conditions of newborns where there is elevated oxidative stress. Included in this group of conditions is asphyxia, respiratory distress syndrome and sepsis and the review also summarizes the literature related to clinical trials of antioxidant therapies and of melatonin, a highly effective antioxidant and free radical scavenger. The authors document there is general agreement that short-term melatonin therapy may be highly effective and that it has a remarkably benign safety profile, even when neonates are treated with pharmacological doses. Significant complications with long-term melatonin therapy in children and adults also have not been reported. None of the animal studies of maternal melatonin treatment or in postnatal life have shown any treatment-related side effects. The authors conclude that treatment with melatonin might result in a wide range of health benefits, improved quality of life and reduced healthcare costs and may help reduce complications in the neonatal period.

Melatonin protects the mitochondria from oxidative damage reducing oxygen consumption, membrane potential, and superoxide anion production.

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

Protection against cognitive deficits and markers of neurodegeneration by long-term oral administration of melatonin in a transgenic model of Alzheimer disease.

Abstract: The neurohormone melatonin has been reported to exert anti-beta-amyloid aggregation, antioxidant, and anti-inflammatory actions in various in vitro and animal models. To comprehensively determine the potential for long-term melatonin treatment to protect Alzheimer's transgenic mice against cognitive impairment and development of beta-amyloid (Abeta) neuropathology, we administered melatonin (100 mg/L drinking water) to APP + PS1 double transgenic (Tg) mice from 2-2.5 months of age to their killing at age 7.5 months. A comprehensive behavioral battery administered during the final 6 weeks of treatment revealed that Tg mice given melatonin were protected from cognitive impairment in a variety of tasks of working memory, spatial reference learning/memory, and basic mnemonic function; Tg control mice remained impaired in all of these cognitive tasks/domains. Immunoreactive Abeta deposition was significantly reduced in hippocampus (43%) and entorhinal cortex (37%) of melatonin-treated Tg mice. Although soluble and oligomeric forms of Abeta1-40 and 1-42 were unchanged in the hippocampus and cortex of the same melatonin-treated Tg mice, their plasma Abeta levels were elevated. These Abeta results, together with our concurrent demonstration that melatonin suppresses Abeta aggregation in brain homogenates, are consistent with a melatonin-facilitated removal of Abeta from the brain. Inflammatory cytokines such as tumor necrosis factor (TNF)-alpha were decreased in hippocampus (but not plasma) of Tg+ melatonin mice. Finally, the cortical mRNA expression of three antioxidant enzymes (SOD-1, glutathione peroxidase, and catalase) was significantly reduced to non-Tg levels by long-term melatonin treatment in Tg mice. Thus, melatonin's cognitive benefits could involve its anti-Abeta aggregation, anti-inflammatory, and/or antioxidant properties. Our findings provide support for long-term melatonin therapy as a primary or complementary strategy for abating the progression of Alzheimer disease.

Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress

Abstract: The relationship between germination and melatonin applied during osmo- and hydropriming was studied in cucumber seeds. The proportion of nuclei with different DNA contents, the mean ploidy and the (2C + 4C = 8C)/2C ratio in unprimed and primed, dry and imbibed at 10 degrees C seeds were established by flow cytometry. Thiobarbituric acid reactive substances and protein oxidation were also estimated. Melatonin and indole-3-acetic acid (IAA) concentrations in the seeds were determined using high-performance liquid chromatography with electrochemical detection. Being sensitive to chilling stress, seeds that germinated well (99%) at 25 degrees C showed only 30% germination at 15 degrees C, and almost no germination (4%) at 10 degrees C. Hydropriming in water improved seed germination to 50-60% at 15 degrees C and the addition of melatonin (25-100 M) also increased the rate of germination. Osmopriming in polyethylene glycol increased germination at 15 degrees C to 78%, and 98% when combined with 50 M melatonin. Osmoprimed seeds germinated even at 10 degrees C and reached 43%, and 83% when 50 M melatonin was applied. None of the treatments induced DNA synthesis, although during the first 24 hr of imbibition at 10 degrees C the mean ploidy and the (2C + 4C = 8C)/2C ratio increased, which is indicative of the advanced Phase II of germination. Hydro- and osmopriming slightly decreased IAA content in the seeds in most of the cases; only hydropriming with 100 and 500 M melatonin increased it. Melatonin protected membrane structure against peroxidation during chilling, but excessive melatonin levels in cucumber seeds (approximately 4 microg/g fresh weight) provoked oxidative changes in proteins. There is still lack of information explained clearly the role of melatonin in plant physiology. This molecule acts multidirectionally and usually is alliged to other compounds.

Long-term follow-up of melatonin treatment in children with ADHD and chronic sleep onset insomnia.

Abstract: We conducted this study to assess long-term melatonin treatment course, effectiveness and safety in children with attention-deficit/ hyperactivity disorder (ADHD) and chronic sleep onset insomnia (CSOI). This was conducted by means of a structured questionnaire for the parents. The subjects of this study consisted of participants who previously participated in a randomised clinical trial on melatonin efficacy. The response rate was 93% (94/101). The mean time to follow up was 3.7 yr. No serious adverse events or treatment related co-morbidities were reported. Sixty-five percent of the children still used melatonin daily and 12% occasionally. Temporal discontinuation of treatment resulted in a delay of sleep onset in 92% of the children. Nine percent of the children could discontinue melatonin completely because of improvement of sleep onset insomnia. Long-term melatonin treatment was judged to be effective against sleep onset problems in 88% of the cases. Improvement of behaviour and mood was reported in 71% and 61% respectively. We conclude that melatonin remains an effective therapy on the long term for the treatment of CSOI in children with ADHD and has no safety concerns regarding serious adverse events or treatment related co-morbidity. Discontinuation of melatonin treatment usually leads to a relapse of sleep onset insomnia and in resuming melatonin treatment, even after several years of treatment.

Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells

Abstract: Melatonin is a multifunctional molecule that mediates several circadian and seasonal processes in animal reproduction. Melatonin and its metabolites are antioxidants and free radical scavengers. We investigated the effects of melatonin on porcine oocyte maturation and embryo development. We then investigated the local expression of the melatonin receptor 1 (MT1) gene in cumulus cells, granulosa cells, and the oocytes with the reverse transcription-polymerase chain reaction (RT-PCR) method. We further evaluated the antioxidant effects [reactive oxygen species (ROS) levels in cumulus-oocytes complexes] of melatonin supplementation during in vitro maturation (IVM). Compared with control, melatonin supplementation (10 ng/mL) during IVM resulted in a greater proportion of oocytes extruding the polar body (75.6% versus 84.6%). Significantly greater proportion of parthenogenetically activated oocytes developed to blastocysts when the in vitro medium was supplemented with melatonin; however, cleavage frequency and blastocyst cell number were not affected by the treatment. RT-PCR analysis revealed the expression of MT1 gene in cumulus and granulosa cells but not in oocytes. Melatonin-treated oocytes had significantly lower levels of ROS than did control (untreated) oocytes. We conclude that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. Some of the observed effects may be mediated by receptor binding and while others may have been receptor independent, e.g., direct free radical scavenging.

Defining chronodisruption

Abstract: When the International Agency for Research on Cancer (IARC) classified shift-work that involves circadian disruption as probably carcinogenic to humans in 2007, this was the prelude to extensive experimental and epidemiological research in coming years. Indeed, with some 20% of people worldwide being engaged in some type of work at unusual times, including the night, it is a must to investigate, and to clarify as soon as possible, the biologically plausible links via circadian disruption with epidemic cancers such as of the breast or prostate. Surprisingly, neither the IARC information available so far nor the general literature provides a clear definition of what the critical component in the postulated chain of causation, namely circadian disruption, is. Here we offer our definition of chronodisruption (CD), a concept which we proposed in 2003 and which we operationalized recently in research which addressed the putative links between shift-work, time-zone-travel and human cancers independently of the IARC and led to similar causal interpretations. As a basis for further research in this area with possible high relevance for public health, we: (i) elaborate our definition of CD, with melatonin being a key biological intermediary, by putting critical disruptions, and the resulting disorder, of circadian clocks, biological rhythms and circadian organization into thematic and historical context with Colin Pittendrigh’s insights almost half a century ago; (ii) provide material on ‘what are chronodisruptors?’ and (iii) pose a key question which needs to be answered by and for experimental and epidemiological CD research. We hope that defining CD can contribute to studies which may help to find clues to a background incidence of epidemic internal cancers for which – so far in many cases – we lack causal explanations.

The role of the thalamus in sleep, pineal melatonin production, and circadian rhythm sleep disorders

Abstract: The thalamus has a strong nonphotic influence on sleep, circadian rhythmicity, pineal melatonin production, and secretion. The opening of the sleep gate for nonrapid eye movement sleep is a thalamic function but it is assisted by melatonin which acts by promoting spindle formation. Thus, melatonin has a modulatory influence on sleep onset and maintenance. A remarkable similarity exists between spindle behavior, circadian rhythmicity, and pineal melatonin production throughout life. Together, the thalamic and chronobiological control of sleep leads to a new and improved understanding of the pathophysiology of circadian rhythm sleep disorders and also of the principles of sleep hygiene interventions.

Melatonin exists in porcine follicular fluid and improves in vitro maturation and parthenogenetic development of porcine oocytes.

Abstract: This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10(-11) m. Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10(-11), 10(-9), 10(-7), 10(-5) and 10(-3) m) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10(-9) m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 +/- 4.5%, 28 +/- 2.4% and 50 +/- 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10(-7) m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 +/- 8.4%, blastocyst rates 35 +/- 6.7%) were obtained when both the maturation and culture medium were supplemented with 10(-9) m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.

Melatonin improves placental efficiency and birth weight and increases the placental expression of antioxidant enzymes in undernourished pregnancy.

Abstract: Melatonin participates in circadian, seasonal and reproductive physiology. Melatonin also acts as a potent endogenous antioxidant by scavenging free radicals and upregulating antioxidant pathways. The placenta expresses melatonin receptors and melatonin protects against oxidative damage induced in rat placenta by ischemia-reperfusion. One of the most common complications in pregnancy is a reduction in fetal nutrient delivery, which is known to promote oxidative stress. However, whether melatonin protects placental function and fetal development in undernourished pregnancy is unknown. Here, we investigated the effects of maternal treatment with melatonin on placental efficiency, fetal growth, birth weight and protein expression of placental oxidative stress markers in undernourished pregnancy. On day 15 of pregnancy, rats were divided into control and undernourished pregnancy (35% reduction in food intake), with and without melatonin treatment (5 microg/mL drinking water). On day 20 of gestation, fetal biometry was carried out, the placenta was weighed and subsequently analyzed by Western blot for xanthine oxidase, heat shock protein (HSP) 27 and 70, catalase, manganese superoxide dismutase (Mn-SOD) and glutathione peroxidase 1 (GPx-1). A separate cohort was allowed to deliver to assess effects on birth weight. Maternal undernutrition led to a fall in placental efficiency, disproportionate intrauterine growth retardation and a reduction in birth weight. Maternal treatment with melatonin in undernourished pregnancy improved placental efficiency and restored birth weight, and it increased the expression of placental Mn-SOD and catalase. The data show that in pregnancy complicated by undernutrition, melatonin may improve placental efficiency and birth weight by upregulating placental antioxidant enzymes.

Melatonin and serotonin in flowers and fruits of Datura metel L.

Abstract: Datura metel is a plant that contains several different neurologically active phytochemicals which affect human health. On-going research has examined the potential role of the human neuroindoles, melatonin and serotonin, in medicinal plants with neurological efficacy. In this report, we describe the quantification of melatonin and serotonin in flowers and developing fruits of Datura metel and the effects of cold stress on the levels of these neuroindoles in the reproductive tissues of this plant. Melatonin and serotonin were found at the highest levels in the least developed flower buds with decreasing concentrations as the flower buds matured. Cold stress significantly increased the concentration of melatonin in young flower buds. In the developing fruit, melatonin was present at relatively stable, high concentrations for the first 10 days after anthesis. After 10-15 days, the ovule had grown to a sufficient size for excision and analysis and melatonin was found to be at the highest concentrations in the developing ovule with minimal concentrations of the neuroindoles in the fleshy fruit. Together, these data indicate that melatonin may play a role in protecting the reproductive tissues during flower and seed formation in a Datura species.

Chemical stress by different agents affects the melatonin content of barley roots.

Abstract: The presence of melatonin (N-acetyl-5-methoxytryptamine) in plants has been clearly demonstrated. However, while this indoleamine has been intensively studied in animals, especially in mammals, the same is not true in the case of plants, where one of the most interesting aspects is its possible role as antioxidative molecule in physiological processes. Some data reflect the possible protective role that melatonin may exert in some stress situations such as ultraviolet (UV)-radiation, induced senescence and copper stress. The present work was designed to establish how the melatonin content changes in plants as a result of chemically induced stress. For this, barley plants were exposed in different treatments to the chemical-stress agents: sodium chloride, zinc sulphate or hydrogen peroxide. After different times, the content of melatonin in treated roots and control roots were determined using liquid chromatography (LC) with time-of-flight/mass spectrometry and LC with fluorescence detection for identification and quantification, respectively. The data show that the melatonin content in roots increased due to stress, reaching up to six times the melatonin content of control roots. Induction was time dependent, while hydrogen peroxide (10 mm) and zinc sulphate (1 mm) were the most effective inducers. The capacity of roots to absorb melatonin from soil was also studied. The data establish, for first time, that the chemical-stress agents assayed can induce the biosynthesis of melatonin in barley roots and produce a significant increase in their melatonin content. Such an increase in melatonin probably plays an important antioxidative role in the defense against chemically induced stress and other abiotic/biotic stresses.

Profiling of melatonin in the model tomato (Solanum lycopersicum L.) cultivar Micro-Tom.

Abstract: Melatonin exists in a considerable variety of plant species. However, the physiological roles of melatonin in plants are not well understood. In this study, the distribution and accumulation of melatonin during leaf and fruit development were analyzed in Micro-Tom, a model cultivar of tomato (Solanum lycopersicum L.). Melatonin was extracted using an acetone-methanol method and measured by enzyme-linked immunosorbent assay. Melatonin was detected in leaves, stems, roots, flowers, fruits, seedlings and seeds in the range of 1.5-66.6 ng/g fresh weight, with seeds containing the highest concentration of melatonin. In fruits and leaves, melatonin concentrations varied depending on the developmental stage, suggesting that melatonin controls some of the processes involved in plant maturation.

Melatonin induces apoptotic death in LNCaP cells via p38 and JNK pathways: therapeutic implications for prostate cancer

Abstract: Apoptosis, a form of cell death, is a fundamental process for the development and maintenance of multicellular organisms that promotes the removal of damaged, senescent or unwanted cells Induction of cancer cell apoptosis is an important strategy of anticancer therapy In this study, we examined if melatonin, the main secretory product of the pineal gland, inhibited the growth of prostate cancer cells (LNCaP) and promoted apoptosis via mitogen-activated protein kinases (MAPKs), which are closely associated with apoptosis and survival Melatonin treatment significantly inhibited the growth of LNCaP cells in a dose- and time-dependent manner It clearly induced both an early stage of apoptosis (propidium iodide(-), FITC Annexin-V(+)) and a late apoptosis/secondary necrosis (propidium iodide(+) and FITC Annexin-V(+)), which indicated induction of serial stages of apoptosis in cells Moreover, melatonin markedly activated c-JUN N-terminal kinase (JNK) and p38 kinase, whereas extracellular signal-regulated kinase (ERK) was not responsive to melatonin Treatment with MAPK inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor), confirmed that melatonin-induced apoptosis was JNK- and p38-dependent, but ERK-independent In the presence of PD98059, caspase-3 activity increased, while levels of Bax/cytochrome c (Cyt c) and Bcl-2 decreased These effects were opposite to those observed with SP600125 and SB202190 treatments Together, these results strongly suggest that JNK and p38 activation directly participate in apoptosis induced by melatonin Thus, melatonin may be of promise for anti-prostate cancer strategies

Melatonin induces mitochondrial-mediated apoptosis in human myeloid HL-60 cells.

Abstract: The role of melatonin in the mediation of apoptotic events has recently gained attention, especially after recent studies have reported that melatonin exerts antiapoptotic actions in normal cells but may activate proapoptotic pathways in some tumor cells. Here, we have evaluated the effect of melatonin on apoptosis in the human leukemia cell line HL-60. Melatonin treatment (1 mm) induced a significant increase in caspase-3 and -9 activities. The effect of melatonin on the activation of caspases was time dependent, reaching a maximum after 12 hr of stimulation, and then decreasing to a minimum after 72 hr. Treatment with melatonin also evoked mitochondrial membrane depolarization and permeability transition pore induction, which caused loss of mitochondrial staining by calcein, and increased cell death by apoptosis/necrosis as demonstrated by propidium iodide positive-staining of cells after 72 hr of stimulation. In addition, the exposure of cells to melatonin resulted in an activation and association of the proapoptotic proteins Bax and Bid, as well as promoting detectable increases in the expression of both proteins. We conclude that melatonin has proapoptotic and/or oncostatic effects in the human myeloid cell line HL-60.

Melatonin protects kidney grafts from ischemia/reperfusion injury through inhibition of NF-kB and apoptosis after experimental kidney transplantation.

Abstract: Free radicals are involved in pathophysiology of ischemia/reperfusion injury (IRI). Melatonin is a potent scavenger of reactive oxygen and nitrogen species. Thus, this study was designed to elucidate its effects in a model of rat kidney transplantation. Twenty Lewis rats were randomly divided into 2 groups (n = 10 animals each). Melatonin (50 mg/kg BW) dissolved in 5 mL milk was given to one group via gavage 2 hr before left donor nephrectomy. Controls were given the same volume of milk only. Kidney grafts were then transplanted into bilaterally nephrectomized syngeneic recipients after 24 hr of cold storage in Histidine–Tryptophan–Ketoglutarate solution. Both graft function and injury were assessed after transplantation through serum levels of blood urea nitrogen (BUN), creatinine, transaminases, and lactate dehydrogenase (LDH). Biopsies were taken to evaluate tubular damage, the enzymatic activity of superoxide dismutase (SOD) and lipid hydroperoxide (LPO), and the expression of NF-kBp65, inducible nitric oxide synthase (iNOS), caspase-3 as indices of oxidative stress, necrosis, and apoptosis, respectively. Melatonin improved survival (P < 0.01) while decreasing BUN, creatinine, transaminases, and LDH values up to 39–71% (P < 0.05). Melatonin significantly reduced the histological index for tubular damage, induced tissue enzymatic activity of SOD while reducing LPO. At the same time, melatonin down-regulated the expression of NF-kBp65, iNOS, and caspase-3. In conclusion, donor preconditioning with melatonin protected kidney donor grafts from IRI-induced renal dysfunction and tubular injury most likely through its anti-oxidative, anti-apoptotic and NF-kB inhibitory capacity.

Long-term melatonin administration protects brain mitochondria from aging.

Abstract: We tested whether chronic melatonin administration in the drinking water would reduce the brain mitochondrial impairment that accompanies aging. Brain mitochondria from male and female senescent prone (SAMP8) mice at 5 and 10 months of age were studied. Mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation and nitrite, glutathione/glutathione disulfide ratio, and glutathione peroxidase and glutathione reductase activities. Electron transport chain activity and oxidative phosphorylation capability of mitochondria were also determined by measuring the activity of the respiratory chain complexes and the ATP content. The results support a significant age-dependent mitochondrial dysfunction with a diminished efficiency of the electron transport chain and reduced ATP production, accompanied by an increased oxidative/nitrosative stress. Melatonin administration between 1 and 10 months of age completely prevented the mitochondrial impairment, maintaining or even increasing ATP production. There were no major age-dependent differences between males in females, although female mice seemed to be somewhat more sensitive to melatonin treatment than males. Thus, melatonin administration as a single therapy maintained fully functioning brain mitochondria during aging, a finding with important consequences in the pathophysiology of brain aging.

The melatonin metabolite N1‐acetyl‐5‐methoxykynuramine is a potent singlet oxygen scavenger

Abstract: 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.

Melatonin reduces cardiac inflammatory injury induced by acute exercise.

Abstract: Cardiac muscle tissue, when stimulated by acute exercise, presents increased signs of cell damage. This study was designed to investigate whether overexpression of inflammatory mediators induced in the heart by acute exercise could be prevented by melatonin and whether the protective effect of melatonin was related with inhibition of nuclear factor kappa B (NF-kappaB) activation. Male Wistar rats received melatonin i.p. at a dose of 1.0 mg/kg body weight 3 min before being exercised for 60 min on a treadmill at a speed of 25 m/min and a 10% slope. Exercise was associated with a significant increase in myeloperoxidase activity and in TNF-alpha, IL-1 and IL-6 mRNA levels. Both mRNA level and protein concentrations of intercellular adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2 were also significantly elevated. A significant activation of nuclear factor kappa B (NF-kappaB) was observed in exercised rats. These effects were totally or partially prevented by melatonin administration. Data obtained indicate that melatonin protects against heart damage caused by acute exercise. Impaired production of noxious mediators involved in the inflammatory process and down-regulation of the NF-kappaB signal transduction pathway appear to contribute to the beneficial effects of melatonin.

Melatonin downregulates nuclear erythroid 2-related factor 2 and nuclear factor-kappaB during prevention of oxidative liver injury in a dimethylnitrosamine model.

Abstract: Melatonin has potent hepatoprotective effects as an antioxidant. However, the signaling pathway of melatonin in the induction of antioxidant enzymes against acute liver injury is not fully understood. The study aimed to determine whether melatonin could prevent dimethylnitrosamine (DMN)-induced liver injury through nuclear erythroid 2-related factor 2 (Nrf2) and inflammation. Liver injury was induced in rats by a single injection of DMN (30 mg/kg, i.p.). Melatonin treatment (50 mg/kg/daily, i.p.) was initiated 24 hr after DMN injection for 14 days, after which the rats were killed and samples were collected. Serum and antioxidant enzyme activities improved in melatonin-treated rats, compared with DMN-induced liver injury group (P < 0.01). Melatonin reduced the infiltration of inflammatory cells and necrosis in the liver, and increased the expression of NADPH: quinone oxidoreductase-1, heme oxygenase-1, and superoxide dismutase-2, which were decreased by DMN. Melatonin increased expression of novel transcription factor, Nrf2, and decreased expression of inflammatory mediators including tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and inducible nitric oxide synthase. The increased nuclear binding of nuclear factor-kappa B (NF-kappaB) in the DMN-induced liver injury group was inhibited by melatonin. Our results show that melatonin increases antioxidant enzymes and Nrf2 expression in parallel with the decrease of inflammatory mediators in DMN-induced liver injury, suggesting that melatonin may play a role of antioxidant defense via the Nrf2 pathway, by reducing inflammation by NF-kappaB inhibition.

Melatonin ameliorates high fat diet-induced diabetes and stimulates glycogen synthesis via a PKCζ-Akt-GSK3β pathway in hepatic cells.

Abstract: Low levels of melatonin in circulation had been reported to be related to the development of diabetes. Melatonin administration in animals increases hepatic glycogen content to lower blood glucose. However, the signaling pathway for these effects is still unclear. The present study shows that intraperitoneal injection of 10 mg/kg melatonin ameliorated glucose utilization and insulin sensitivity in high fat diet-induced diabetic mice with an increase in hepatic glycogen and improvement in liver steatosis. We used HepG2 cells to investigate the signaling pathways for the melatonin-stimulated hepatic glycogen increment. Treatment of HepG2 cells with 1 nm melatonin markedly increased glycogen synthesis which was blocked by the melatonin receptor antagonist luzindole. In addition, melatonin increased the phosphorylation of subcellular signals at the level of protein kinase C zeta (PKCzeta), Akt, and glycogen synthase kinase 3beta (GSK3beta) while the increase in glycogen synthesis induced by melatonin was inhibited by PKCzeta pseudo-peptide. However, 3',5'-cyclic adenosine monophosphate-activated protein kinase (AMPK) was not influenced by melatonin treatment. Taken together, melatonin improves glucose intolerance and insulin resistance in high fat diet-induced diabetic mice and stimulates glycogen synthesis via a PKCzeta-Akt-GSK3beta pathway in HepG2 cells.

Therapeutic potential of melatonin in traumatic central nervous system injury

Abstract: A vast literature extolling the benefits of melatonin has accumulated during the past four decades. Melatonin was previously considered of importance to seasonal reproduction and circadian rhythmicity. Currently, it appears to be a versatile anti-oxidative and anti-nitrosative agent, a molecule with immunomodulatory actions and profound oncostatic activity, and also to play a role as a potent neuroprotectant. Nowadays, melatonin is sold as a dietary supplement with differential availability as an over-the-counter aid in different countries. There is a widespread agreement that melatonin is nontoxic and safe considering its frequent, long-term usage by humans at both physiological and pharmacological doses with no reported side effects. Endeavors toward a designated drug status for melatonin may be enormously rewarding in clinics for treatment of several forms of neurotrauma where effective pharmacological intervention has not yet been attained. This mini review consolidates the data regarding the efficacy of melatonin as an unique neuroprotective agent in traumatic central nervous system (CNS) injuries. Well-documented actions of melatonin in combating traumatic CNS damage are compiled from various clinical and experimental studies. Research on traumatic brain injury and ischemia/reperfusion are briefly outlined here as they have been recently reviewed elsewhere, whereas the studies on different animal models of the experimental spinal cord injury have been extensively covered in this mini review for the first time.

Melatonin preserves longevity protein (sirtuin 1) expression in the hippocampus of total sleep‐deprived rats

Abstract: Sleep disorders cause cognitive dysfunction in which impaired neuronal plasticity in the hippocampus may underline the molecular mechanisms of this deficiency. As sirtuin 1 (SIRT1) plays an important role in maintaining metabolic homeostasis and neuronal plasticity, this study is aimed to determine whether melatonin exerts beneficial effects on preserving SIRT1 activation following total sleep deprivation (TSD). TSD was performed by disc on water method for five consecutive days. During this period, animals daily received melatonin at doses of 5, 25, 50 or 100 mg/kg. The cytochrome oxidase (COX) histochemistry, SIRT1 immunohistochemistry together with Morris water maze learning test were performed to examine the metabolic, neurochemical, as well as the behavioral changes in neuronal plasticity, respectively. The results indicate that in normal rats, numerous COX and SIRT1 positive-labeled neurons with strong staining intensities were found in hippocampal pyramidal and granular cell layers. Following TSD, both COX and SIRT1 reactivities were drastically decreased as revealed by reduced staining pattern and labeling frequency. Behavioral data corresponded well with morphological findings in which spatial memory test in water maze was significantly impaired after TSD. However, in rats receiving different doses of melatonin, both COX and SIRT1 expressions were successfully preserved. Considerably better performance on behavioral testing further strengthened the beneficial effects of melatonin. These findings suggest that melatonin may serve as a novel therapeutic strategy directed for preventing the memory deficits resulting from TSD, possibly by effectively preserving the metabolic function and neuronal plasticity engaged in maintaining cognitive activity.

Evidence for a biphasic apoptotic pathway induced by melatonin in MCF‐7 breast cancer cells

Abstract: Previous investigations demonstrated that melatonin exerts an oncostatic action on estrogen-responsive breast cancer, both in vitro and in vivo Nevertheless, the pro-apoptotic effect of melatonin is still a matter of debate An experimental study was undertaken to focus on melatonin-related apoptosis and to identify the apoptotic pathways involved Whole cell-count, flow-cytometry analysis and proteins involved in apoptotic pathways [p53, p73, murine double minute 2 (MDM2), caspases-9,-7,-6, cleaved-poly ADP ribose polymerase (PARP), Bcl-2, Bax and apoptotic inducing factor (AIF)] were investigated in human MCF-7 breast cancer cells treated with physiological (1 nM) concentration of melatonin Melatonin exerts a significant growth-inhibitory effect on MCF-7 cells, becoming evident after 72 hr and thereafter increasing linearly up to 144 hr In this model, the growth-inhibition is transforming growth factor beta 1 (TGFbeta1)-dependent and it might be reversed by adding an anti-TGFbeta1 antibody Melatonin induces a significant rise in apoptotic rate, at both 24 and 96 hr The anti-TGFbeta1 antibody almost completely suppresses melatonin-related late apoptosis; however, early apoptosis is unaffected Early programmed cell death is associated with a significant increase in the p53/MDM2 ratio and in AIF release, without modifications in caspase activity or cleaved-PARP levels Activated caspases-9 and -7 and cleaved-PARP increased significantly at 96 hr, concomitantly with a down-regulation of the Bcl-2/Bax ratio These data suggest that two distinct apoptotic processes are triggered by melatonin in MCF-7 cells: an early, TGFbeta1 and caspase-independent response, and a late apoptotic TGFbeta1-dependent process in which activated-caspase-7 is likely to be the terminal effector

Decreased MT1 and MT2 melatonin receptor expression in extrapineal tissues of the rat during physiological aging

Abstract: Aging is a complex process associated with a diminished ability to respond to stress, a progressive increase in free radical generation and a decline in immune function. Melatonin, a molecule with a great functional versatility exerts anti-oxidant, oncostatic, immunomodulatory, and anti-aging properties. Melatonin levels drop during aging and it has been speculated that the loss of melatonin may accelerate aging. This study was designed to elucidate whether aging involves responsiveness to reduced melatonin. Melatonin membrane receptor (MT1 and MT2) expression and MT1 protein expression were analyzed in extrapineal tissues (thymus, spleen, liver, kidney, and heart) of 3- and 12-month-old rats using real time polymerase chain reaction and western blotting analysis. Moreover, melatonin in tissues was measured by high performance liquid chromatography. We report for the first time, an age-related reduction in mRNA MT1 and MT2 expression levels as well as MT1 protein expression in all tissues tested except the thymus, where surprisingly, both melatonin receptor levels were significantly higher in 12-month-old rats and MT1 protein expression maintained unchanged with age. Diminished melatonin concentrations were measured in spleen, liver, and heart during aging. As a conclusion, physiological aging seems to exert responsiveness to melatonin and consequently, the loss of this potent anti-oxidant may contribute to onset of aging.

Anti-aging properties of melatonin in an in vitro murine senescence model: involvement of the sirtuin 1 pathway.

Abstract: Sirtuin 1 is a member of the sirtuin family of protein deacetylases, which have attracted considerable attention as mediators of lifespan extension in several model organisms Induction of sirtuin 1 expression also attenuates neuronal degeneration and death in animal models of Alzheimer's disease and Huntington's disease In this study, an in vitro model of neuronal aging was used to test in several ways whether melatonin acts as a sirtuin 1 inducer and if this effect could be neuroprotective It is shown that melatonin is able to increase the level of this deacetylase in young primary neurons, as well as in aged neurons We also observed an increase in the deacetylation of several substrates of sirtuin 1, such as p53, PGC-1alpha, FoxO1, ADAM10 and NFkappaB In addition, there was a reduction in its nuclear translocation and, subsequently, an improvement in transcriptional activity Sirtinol, a sirtuin 1 inhibitor, was used to correlate these effects with sirtuin It is shown that sirtinol reduces sirtuin 1 expression and impairs the beneficial action of melatonin on cell viability and apoptosis prevention Moreover, some of the sirtuin 1 substrates studied also reversed the melatonin effect when sirtinol is added to the cells, mainly p53 Globally, these results add weight to the findings of previous reports, indicating a new role for melatonin in improving cell function gated to an increased neuroprotective role for the sirtuin 1 pathway

Cranial irradiation‐induced inhibition of neurogenesis in hippocampal dentate gyrus of adult mice: attenuation by melatonin pretreatment

Abstract: Radiation is an important therapeutic tool in the treatment of cancer. The tremendous development in radiotherapeutic techniques and dosimetry has made it possible to augment the patient survival. Therefore, attention has focused on long-range treatment side effects especially in relation to the neurocognitive changes. As cognitive health of an organism is considered to be maintained by the capacity of hippocampal neurogenesis, this study designed to evaluate the delayed effect of cranial irradiation on hippocampal neurogenesis, possible implication of oxidative stress and prophylactic action of melatonin in mice. One month after cranial irradiation (6 Gy, X-ray), changes in the population of immature and proliferating neurons in dentate gyrus were localized through the expression of the microtubule binding protein doublecortin (Dcx) and proliferation marker Ki-67. We found a substantial reduction in the Dcx and Ki-67 positive cells after irradiation. Melatonin pretreatment significantly ameliorated the radiation-induced decline in the Dcx and Ki-67 positive cells. In addition, profound increase in the 4-hydroxynonenal (4-HNE) and 8-hydroxy-2'-deoxyguanosine positive cells were reported in subventricular zone, granular cell layer and hilus after day 30 postirradiation. Immunoreactivity of these oxidative stress markers were significantly inhibited by melatonin pretreatment. To confirm the magnitude of free-radical scavenging potential of melatonin, we measured the in-vitro OH radical scavenging power of melatonin by electron spin resonance. Interestingly, the melatonin was capable of scavenging the OH radicals at very low concentration (IC(50) = 214.46 nm). The findings indicate the possible benefit of melatonin treatment to combat the delayed side effects of cranial radiotherapy.

Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBPβ transcriptional activity

Abstract: Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBPbeta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPARgamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPARgamma (60%), C/EBPalpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBPalpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBPbeta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBPalpha promoter. However, when treated with melatonin, the level of C/EBPalpha promoter activation by C/EBPbeta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3 ) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBPbeta.