Showing papers in "Journal of Pineal Research in 2006"

Reduced oxidative damage in ALS by high-dose enteral melatonin treatment.

Abstract: Amyotrophic lateral sclerosis (ALS) is the collective term for a fatal motoneuron disease of different etiologies, with oxidative stress as a common molecular denominator of disease progression. Melatonin is an amphiphilic molecule with a unique spectrum of antioxidative effects not conveyed by classical antioxidants. In preparation of a possible future clinical trial, we explored the potential of melatonin as neuroprotective compound and antioxidant in: (1) cultured motoneuronal cells (NSC-34), (2) a genetic mouse model of ALS (SOD1(G93A)-transgenic mice), and (3) a group of 31 patients with sporadic ALS. We found that melatonin attenuates glutamate-induced cell death of cultured motoneurons. In SOD1(G93A)-transgenic mice, high-dose oral melatonin delayed disease progression and extended survival. In a clinical safety study, chronic high-dose (300 mg/day) rectal melatonin was well tolerated during an observation period of up to 2 yr. Importantly, circulating serum protein carbonyls, which provide a surrogate marker for oxidative stress, were elevated in ALS patients, but were normalized to control values by melatonin treatment. This combination of preclinical effectiveness and proven safety in humans suggests that high-dose melatonin is suitable for clinical trials aimed at neuroprotection through antioxidation in ALS.

Melatonin in Glycyrrhiza uralensis: response of plant roots to spectral quality of light and UV-B radiation.

Abstract: Melatonin (N-acetyl-5-methoxytryptamine) is known to be synthesized and secreted by the pineal gland in vertebrates. Evidence for the occurrence of melatonin in the roots of Glycyrrhiza uralensis plants and the response of this plant to the spectral quality of light including red, blue and white light (control) and UV-B radiation (280-315 nm) for the synthesis of melatonin were investigated. Melatonin was extracted and quantified in seed, root, leaf and stem tissues and results revealed that the root tissues contained the highest concentration of melatonin; melatonin concentrations also increased with plant development. After 3 months of growth under red, blue and white fluorescent lamps, the melatonin concentrations were highest in red light exposed plants and varied depending on the wavelength of light spectrum in the following order red >> blue > or = white light. Interestingly, in a more mature plant (6 months) melatonin concentration was increased considerably; the increments in concentration were X4, X5 and X3 in 6-month-old red, blue and white light exposed (control) plants, respectively. The difference in melatonin concentrations between blue and white light exposed (control) plants was not significant. The concentration of melatonin quantified in the root tissues was highest in the plants exposed to high intensity UV-B radiation for 3 days followed by low intensity UV-B radiation for 15 days. The reduction of melatonin under longer periods of UV-B exposure indicates that melatonin synthesis may be related to the integrated (intensity and duration) value of UV-B irradiation. Melatonin in G. uralensis plant is presumably for protection against oxidative damage caused as a response to UV irradiation.

Melatonin as a cytoskeletal modulator: implications for cell physiology and disease.

Abstract: The cytoskeleton is a phylogenetically well-preserved structure that plays a key role in cell physiology. Dynamic and differential changes in cytoskeletal organization occur in cellular processes according to the cell type and the specific function. In neurons, microtubules, microfilaments and intermediate filament (IF) rearrangements occur during axogenesis, and neurite formation which eventually differentiate into axons and dendrites to constitute synaptic patterns of connectivity. In epithelial cells, dynamic modifications occur in the three main cytoskeletal components and phosphorylation of cytoskeletal associated proteins takes place during the formation of the epithelial cell monolayer that eventually will transport water. In pathological processes such as neurodegenerative and psychiatric diseases an abnormal cytoskeletal organization occurs. Melatonin, the main product secreted by pineal gland during dark phase of the photoperiod, is capable of influencing microfilament, microtubule and IF organization by acting as a cytoskeletal modulator. In this paper we will summarize the evidence which provides the data that melatonin regulates cytoskeletal organization and we describe recent findings, which indicate that melatonin effects on microfilament rearrangements in stress fibers are involved in the mechanism by which the indole synchronizes water transport in kidney-derived epithelial cells. In addition, we review recent data, which indicates that melatonin protects the neuro-cytoskeletal organization from damage caused by free radicals contributing to cell survival, in addition to the already described mechanism elicited by the indole to prevent apoptosis and to scavenge free radicals. Moreover, we discuss the implications of an altered cytoskeletal organization for neurodegenerative and psychiatric illnesses and its re-establishment by melatonin.

Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade

Abstract: The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10-day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co-exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P-PDOT (MT2 receptor selective), inhibited the melatonin-induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2-[125I]-iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2-[125I]-iodomelatonin binding as well as melatonin efficacy to inhibit forskolin-induced cAMP accumulation. Further characterization of the mechanisms underlying melatonin's effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin-mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.

Diabetic Goto Kakizaki rats as well as type 2 diabetic patients show a decreased diurnal serum melatonin level and an increased pancreatic melatonin-receptor status.

Abstract: There are functional inter-relationships between the beta cells of the endocrine pancreas and the pineal gland, where the synchronizing circadian molecule melatonin originates. The aim of this study was to elucidate a putative interaction between insulin and melatonin in diabetic patients and a diabetic rat model. We analyzed glucose, insulin, and melatonin levels of type 2 patients, as well as type 2 diabetic Goto Kakizaki (GK) rats by radioimmunoassay. Expression of pancreatic melatonin and pineal insulin receptors, as well as arylalkylamine-N-acetyltransferase (AANAT), was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The AANAT enzyme activity was measured in pineal homogenates. Diabetic patients showed a decrease in melatonin levels, while in the pancreas of GK rats an upregulation of the melatonin-receptor mRNA was determined. The pancreatic islets of GK rats showed expression of the mRNA for the pancreatic melatonin (MT1) receptor, which had previously been identified in rats and insulinoma (INS1) cells. Besides their presence in animal cells, the MT1-receptor transcript was also detected in human pancreas by RT-PCR. Whereas the rat pancreatic mRNA expression of the MT1-receptor was significantly increased, the activity of the pineal AANAT enzyme was reduced. The latter observation was in accordance with plasma melatonin levels. The insulin-receptor mRNA of the pineal gland was found to be reduced in GK rats. Our observations suggest a functional inter-relationship between melatonin and insulin, and may indicate a reduction of melatonin in the genesis of diabetes.

Interactions of melatonin and its metabolites with the ABTS cation radical: extension of the radical scavenger cascade and formation of a novel class of oxidation products, C2-substituted 3-indolinones.

Abstract: Melatonin had previously been shown to reduce up to four 2,2- azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) cation radicals (ABTS• + ) via a scavenger cascade ending with N 1 -acetyl-N 2 -formyl-5- methoxykynuramine (AFMK). However, when melatonin is added to the reaction system in much lower quantities than ABTS• + , the number of radicals scavenged per melatonin molecule is considerably higher and can attain a value of ten. Under conditions allowing for such a stoichiometry, novel products have been detected which derive from AFMK (1). These were separated by repeated chromatography and the major compounds were characterized by spectroscopic methods, such as mass spectrometry (HPLC- MS, EI-MS and ESI-HRMS), 1 H nuclear magnetic resonance (NMR) and 13 C NMR, heteronuclear multiple bond connectivity (HMBC) correlations. The identified substances are formed by re-cyclization and represent 3- indolinones carrying the side chain at C2; the N-formyl group can be maintained, but deformylated analogs seem to be also generated, according to MS. The primary product from AFMK (1 )i sN-(1-formyl-5-methoxy-3- oxo-2,3-dihydro-1H-indol-2-ylidenemethyl)-acetamide (2), which is obtained after purification as E- and Z-isomers (2a, 2b); a secondary product has been identified as N-(1-formyl-2-hydroxy-5-methoxy-3-oxo-2,3-dihydro-1H-indol- 2-ylmethyl)-acetamide (3). When H2O2 is added to the ABTS• + reaction mixture in quantities not already leading to substantial reduction of this radical, compound 3 is isolated as the major product, whereas 2a and 2b are virtually absent. The substances formed differ from all previously known oxidation products which derive from melatonin and are, among these, the first 3-indolinones. Moreover, the aliphatic side chain at C2 is reminiscent of other substances which have been synthesized in the search for melatonin receptor ligands.

Melatonin attenuates diabetes-induced oxidative stress in rabbits.

Abstract: Oxidative stress is considered to be the main cause of diabetic complications. As the role of antioxidants in diabetes therapy is still underestimated, the aim of the present investigation was to study the antioxidative action of melatonin in comparison with N-acetylcysteine (NAC) under diabetic conditions. Alloxan-diabetic rabbits were treated daily with either melatonin (1 mg/kg, i.p.), NAC (10 mg/kg, i.p.) or saline. Blood glutathione redox state and serum hydroxyl free radicals (HFR), creatinine and urea levels were monitored. After 3 wk of treatment animals were killed and HFR content, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio as well as the activities of glutathione reductase, glutathione peroxidase and gamma-glutamylcysteine synthetase were estimated in both liver and kidney cortex. Diabetes evoked a several-fold increase in HFR levels accompanied by a significant decline in GSH/GSSG ratio in serum and the examined organs. In contrast to NAC, melatonin (at 1/10 the dose of NAC) attenuated diabetes-induced alterations in glutathione redox state and HFR levels, normalized creatinine concentration and diminished urea content in serum. Moreover, the indole resulted in an increase in glutathione reductase activity in both studied organs and in a rise in glutathione peroxidase and gamma-glutamylcysteine synthetase activities in the liver. In contrast to NAC, melatonin seems to be beneficial for diabetes therapy because of its potent antioxidative and nephroprotective action. The indole-induced increase in the activities of the enzymes of glutathione metabolism might be of importance for antioxidative action of melatonin under diabetic conditions.

Protective effects of melatonin in experimental free radical-related ocular diseases.

Abstract: Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine with a range of antioxidative properties. Melatonin is endogenously produced in the eye and in other organs. Current evidence suggests that melatonin may act as a protective agent in ocular conditions such as photo-keratitis, cataract, glaucoma, retinopathy of prematurity and ischemia/reperfusion injury. These diseases are sight-threatening and they currently remain, for the most part, untreatable. The pathogenesis of these conditions is not entirely clear but oxidative stress has been proposed as one of the causative factors. Elevated levels of various reactive oxygen and nitrogen species have been identified in diseased ocular structures. These reactants damage the structure and deplete the eye of natural defense systems, such as the antioxidant, reduced glutathione, and the antioxidant enzyme superoxide dismutase. Oxidative damage in the eye leads to apoptotic degeneration of retinal neurons and fluid accumulation. Retinal degeneration decreases visual sensitivity and even a small change in the fluid content of the cornea and crystalline lens is sufficient to disrupt ocular transparency. In the eye, melatonin is produced in the retina and in the ciliary body. Continuous regeneration of melatonin in the eye offers a frontier antioxidative defense for both the anterior and posterior eye. However, melatonin production is minimal in newborns and its production gradually wanes in aging individuals as indicated by the large drop in circulating blood concentrations of the indoleamine. These individuals are possibly at risk of contracting degenerative eye diseases that are free radical-based. Supplementation with melatonin, a potent antioxidant, in especially the aged population should be considered as a prophylaxis to preserve visual functions. It may benefit many individuals worldwide, especially in countries where access to medical facilities is limited.

Chemoprotective effect of melatonin against cisplatin-induced testicular toxicity in rats.

Abstract: In this study, we investigated the effect of melatonin on cisplatin-induced spermiotoxicity using quantitative, biochemical and histopathological approaches. Cisplatin (CP, 7 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected. The rats were decapitated on 5th (short-term group) or 50th day (long-term group) after CP injection. Traits of reproductive organs, sperm characteristics, testicular histological findings, and the lipid peroxidation in the testicular tissue were determined. Melatonin mitigated CP-induced reductions in testes, epididymis and accessory gland weights in rats decapitated on day 5. Both short- and long-term CP treatment decreased sperm concentration, sperm motility and increased abnormal sperm rates compared with the control. But the reduction of sperm concentration in long-term CP treatment was insignificant. Although treatment with melatonin provided moderately normalization with respect to sperm concentration in short-term treatment group, melatonin caused a marked normalization of sperm motility in both CP + melatonin groups. Both groups treated with the melatonin showed decreases in abnormal sperm rates compared with alone CP. While testicular malondialdehyde levels were elevated after CP treatment, glutathione peroxidase activity decreased significantly in both groups. Glutathione levels reduced after long-term treatment, but not in short-term group by CP administration. Treatment with CP plus melatonin provided significant amelioration of oxidative stress parameters. Histopathological findings of testes in both short- and long-term treatment groups paralleled the biochemical and spermatogenic results. This study clearly indicates that CP-treatment impaired markedly testicular function and combined treatment with melatonin prevented much of the toxicity in rats.

Therapeutic treatments potentially mediated by melatonin receptors: potential clinical uses in the prevention of osteoporosis, cancer and as an adjuvant therapy

Abstract: Melatonin's therapeutic potential is grossly underestimated because its functional roles are diverse and its mechanism(s) of action are complex and varied. Melatonin produces cellular effects via a variety of mechanisms in a receptor independent and dependent manner. In addition, melatonin is a chronobiotic agent secreted from the pineal gland during the hours of darkness. This diurnal release of melatonin impacts the sensitivity of melatonin receptors throughout a 24-hr period. This changing sensitivity probably contributes to the narrow therapeutic window for use of melatonin in treating sleep disorders, that is, at the light-to-dark (dusk) or dark-to-light (dawn) transition states. In addition to the cyclic changes in melatonin receptors, many genes cycle over the 24-hr period, independent or dependent upon the light/dark cycle. Interestingly, many of these genes support a role for melatonin in modulating metabolic and cardiovascular physiology as well as bone metabolism and immune function and detoxification of chemical agents and cancer reduction. Melatonin also enhances the actions of a variety of drugs or hormones; however, the role of melatonin receptors in modulating these processes is not known. The goal of this review is to summarize the evidence related to the utility of melatonin as a therapeutic agent by focusing on its other potential uses besides sleep disorders. In particular, its use in cancer prevention, osteoporosis and, as an adjuvant to other therapies are discussed. Also, the role that melatonin and, particularly, its receptors play in these processes are highlighted.

Melatonin counteracts inducible mitochondrial nitric oxide synthase-dependent mitochondrial dysfunction in skeletal muscle of septic mice

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

Melatonin ameliorates nonalcoholic fatty liver induced by high-fat diet in rats.

Abstract: Nonalcoholic fatty liver disease (NAFLD) is an increasingly recognized condition that may progress to end-stage liver disease, which ranges from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis. Oxidative stress and lipid peroxidation are key pathophysiological mechanisms in NAFLD. We investigate the preventive effects of intraperitoneal administration of melatonin (2.5, 5, 10 mg/kg, daily, respectively) in NAFLD rats induced by high-fat diets for 12 wk. Liver damage was evaluated by serological analysis, serum and hepatic lipid assay as well as hematoxylin-eosin staining in liver sections. Oxidative stress and lipid peroxidation were assessed by measuring malondialdehyde (MDA) levels and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in liver. The results showed that high-fat diet induced oxidative stress with extensive liver steatosis in rats. Melatonin (5 or 10 mg/kg) was effective in reducing hepatic steatosis and inflammation with lowering serum alanine aminotransferase, aspartate aminotransferase, and levels liver total cholesterol and triglycerides in high-fat diet rats. Moreover, melatonin (2.5, 5, 10 mg/kg) increased SOD and GSH-Px activities and the 10 mg/kg dose of melatonin reduced MDA levels in liver. This study shows that melatonin exerts protective effects against fatty liver in rats induced by high-fat diet possibly through its antioxidant actions.

Blue blocker glasses impede the capacity of bright light to suppress melatonin production.

Abstract: Blocking morning light exposure with dark goggles can contribute to the adjustment to night work but these glasses are incompatible with driving. Recently, it was discovered that the biological clock is most sensitive to short wavelengths (blue light). Therefore, we tested the hypothesis that cutting the blue portion of the light spectrum with orange lens glasses (blue blockers) would prevent the light-induced melatonin suppression, a test broadly used as an indirect assessment of the circadian clock sensitivity. Fourteen normal subjects were exposed at night to a 60 min bright light pulse (1300 lx behind filters) between 01:00 and 02:00 hr while wearing orange lens glasses (experimental condition) or grey lens glasses (control condition). The amount of salivary melatonin change observed during the light pulse was compared with a melatonin baseline obtained the night before. Although both glasses transmitted the same illuminance (1300 lx) but at an irradiance 25% higher for the orange lens (408 microW/cm2) compared with the grey lens (327 microW/cm2), a non-significant increase of 6% (95% CI, -20% to 9%) was observed with the orange lens whereas a significant (P < 0.05) reduction of 46% (95% CI, 35-57%) was observed with the grey lens. Blue blockers represent an elegant means to prevent the light-induced melatonin suppression. Further studies are needed to show that these glasses, which are suitable for driving, could facilitate adaptation to night work.

Quantification of melatonin in phototrophic organisms

Abstract: Melatonin, the chief secretory product of the vertebrate pineal gland is also known to occur in numerous photoautotrophic organisms. The indoleamine is suspected to act as a transducer of photoperiodic information and/or to participate in antioxidative protection. In higher plants and other photoautotrophic organisms, contradictory results for melatonin content for samples from the same species show that further improvement of methods for reliable quantification is required. In the present study, melatonin was quantified in tomatoes, ginger and the marine green macroalga, Ulva lactuca, after extraction with three different extraction methods based on ether, acetone or perchloric acid. Melatonin was determined by enzyme-linked immunosorbent assay (ELISA) in high-performance liquid chromatography (HPLC)-purified extracts. The same HPLC system used for purification of extracts was used for parallel quantifications after derivatization of melatonin under alkaline conditions in the presence of hydrogen peroxide (HPLC-PD). Both quantification methods gave similar results with a high correlation [f(x) = 0.99x + 3.01; R(2) = 0.99]. In ginger, the melatonin concentration was below 5 pg/g (fresh weight, f.w.), whereas in tomatoes about 1200 pg/g (f.w.) were found, and in the green alga, U. lactuca, approximately 12 pg/g (f.w.). Taking into account the recovery rates for synthetic melatonin added prior to extraction, no substantial differences were observed in melatonin quantification between different extraction methods. The demonstrated methods based on HPLC purification and subsequent quantification by ELISA and HPLC-PD allow highly sensitive melatonin determinations in diverse photoautotrophic organisms with a low risk of overestimations by false-positive results.

Melatonin stimulates glucose transport via insulin receptor substrate-1/phosphatidylinositol 3-kinase pathway in C2C12 murine skeletal muscle cells.

Abstract: The prevalence of diabetes has exponentially increased in recent decades due to environmental factors such as nocturnal lifestyle and aging, both of which influence the amount of melatonin produced in the pineal gland. The present study investigated the effect of melatonin on signaling pathways of glucose transport in C2C12 mouse skeletal muscle cells. Intriguingly, treatment of C2C12 cells with melatonin (1 nm) stimulated glucose uptake twofold increase. Melatonin-stimulated glucose transport was inhibited with co-treatment with the melatonin receptor antagonist luzindole. Furthermore, treatment of stably over-expressed melatonin receptor type 2B containing C2C12 myotubes with melatonin amplified glucose transport c. 13-fold. Melatonin also increased the phosphorylation level of insulin receptor substrate-1 (IRS-1) and the activity of phosphoinositide 3-kinase (PI-3-kinase). However, 3',5'-cyclic adenosine monophosphate-activated protein kinase (AMPK), another important glucose transport stimulatory mediator via an insulin-independent pathway, was not influenced by melatonin treatment. Activity of p38 mitogen-activated protein kinase (MAPK), a downstream mediator of AMPK, was also not changed by melatonin. In addition, melatonin increased the expression level of forkhead box A2, which was recently discovered to regulate fatty acid oxidation and to be inhibited by insulin. In summary, melatonin stimulates glucose transport to skeletal muscle cells via IRS-1/PI-3-kinase pathway, which implies, at the molecular level, its role in glucose homeostasis and possibly in diabetes. Additionally, exposure to light at night and aging, both of which lower endogenous melatonin levels may contribute to the incidence and/or development of diabetes.

Melatonin induces apoptosis in human neuroblastoma cancer cells

Abstract: Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK-N-MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6-diamidino-2-phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase-3 activation. Cell death was dose and time-dependent; the lowest effective concentration of melatonin was 100 microm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.

Sustained activation of Akt by melatonin contributes to the protection against kainic acid‐induced neuronal death in hippocampus

Abstract: In the present study, the underlying protective mechanism of melatonin on kainic acid (KA)-induced excitotoxicity was examined in the hippocampus of mice. KA, administered intracerebroventricularly (i.c.v.), induced marked neuronal cell death with concurrent microglial activation and subsequent induction of inducible nitric oxide synthase (iNOS) in the hippocampus. Histopathological analysis demonstrated that melatonin (10 mg/kg), administered 1 hr prior to KA, attenuated KA-induced death of pyramidal neurons in the CA3 region. Melatonin obviously suppressed KA-induced microglial activation and consequent iNOS expression that were determined by increased immunoreactivities of microglial marker OX-6 and iNOS, respectively. Increased phosphorylation of Akt in pyramidal neurons was observed as early as 2 hr after administration of melatonin. Further, melatonin resulted in increased expression of astroglial glial cell line-derived neurotrophic factor (GDNF), which started to appear approximately 6 hr after administration of melatonin. The results of the present study demonstrate that melatonin exerts its neuroprotective action against KA-induced excitotoxicity both through the activation of neuronal Akt and via the direct action on hippocampal neurons and through the increased expression of astroglial GDNF, which subsequently activates neuronal PI3K/Akt pathway. Therefore, the present study suggests that melatonin, pineal secretory product, is potentially useful in the treatment of acute brain pathologies associated with excitotoxic neuronal damage such as epilepsy, stroke, and traumatic brain injury.

Urinary 6‐sulfatoxymelatonin levels and their correlations with lifestyle factors and steroid hormone levels

Abstract: Exposure to light at night, as experienced by rotating night shift workers, has been related to lower circulating levels of melatonin, a hormone with recognized cancer protective properties. However, little is known about the relationship of other lifestyle factors or endogenous sex steroid hormones with melatonin levels. We examined cross-sectional associations of age, reproductive and menopausal factors, body mass index (BMI), alcohol consumption, smoking history, night shift work, as well as several other breast cancer risk factors, and circulating sex steroid hormone levels with creatinine-adjusted morning urinary melatonin (6-sulfatoxymelatonin, aMT6s) levels. Participants were 459 healthy, primarily premenopausal (age range 33-50 yr) women from the Nurses' Health Study II (NHS II). Using multiple linear regression, we computed least-square mean hormone levels across categories of lifestyle factors. Age was inversely related to aMT6s levels, particularly before menopause (premenopausal women, or=49 yr; aMT6s, 20.8 ng/mg versus 11.8 ng/mg creatinine; P for trend, 0.02). In multivariate analyses, BMI was significantly and inversely associated with aMT6s levels (P for trend, <0.01). Higher pack-years of smoking were associated with significantly lower aMT6s levels (never smoker versus 15+ pack-years, aMT6s = 17.4 ng/mg versus 12.3 ng/mg creatinine; P for trend, 0.04). We also observed a positive association between parity and aMT6s levels (P for trend, <0.01), but no other reproductive factors nor any of the sex hormones (estradiol, progesterone, estrone, estrone sulfate, dehydroepiandrostenedione, dehydroepiandrostenedione sulfate, testosterone, and androstenedione), as measured either in the luteal or the follicular phase of the menstrual cycle, were significantly associated with aMT6s. In conclusion, higher age, BMI, and heavy smoking were significantly related to lower levels of melatonin, whereas parity was significantly associated with higher aMT6s levels. Melatonin levels may be one mechanism through which these factors influence the development of cancer, but more studies are needed to elucidate these mechanisms definitively.

Effect of TNF-α on the melatonin synthetic pathway in the rat pineal gland: basis for a ‘feedback’ of the immune response on circadian timing

Abstract: A retino-hypothalamic-sympathetic pathway drives the nocturnal surge of pineal melatonin production that determines the synchronization of pineal function with the environmental light/dark cycle. In many studies, melatonin has been implicated in the modulation of the inflammatory response. However, scant information on the feedback action of molecules present in the blood on the pineal gland during the time course of an inflammatory response is available. Here we analyzed the effect of tumor necrosis factor-alpha (TNF-alpha) and corticosterone on the transcription of the Aa-nat, hiomt and 14-3-3 protein genes in denervated pineal glands of rats stimulated for 5 hr with norepinephrine, using real-time reverse transcription-polymerase chain reaction. The transcription of Aa-nat, a gene encoding the key enzyme in melatonin biosynthesis, together with the synthesis of the melatonin precursor N-acetylserotonin, was inhibited by TNF-alpha. This inhibition was transient, and a preincubation of TNF-alpha for more than 24 hr had no detectable effect. In fact, a protein(s) transcribed, later on, as shown by cycloheximide, was responsible for the reversal of the inhibition of Aa-nat transcription. In addition, corticosterone induced a potentiation of norepinephrine-induced Aa-nat transcription even after 48 hr of incubation. These data support the hypothesis that the nocturnal surge in melatonin is impaired at the beginning of an inflammatory response and restored either during the shutdown of an acute response or in a chronic inflammatory pathology. Here, we introduce a new molecular pathway involved in the feedback of an inflammatory response on pineal activity, and provide a molecular basis for understanding the expression of circadian timing in injured organisms.

Melatonin increases survival of HaCaT keratinocytes by suppressing UV-induced apoptosis.

Abstract: Melatonin is a potent antioxidant and direct radical scavenger. As keratinocytes represent the major population in the skin and UV light causes damage to these cells, the possible protective effects of melatonin against UV-induced cell damage in HaCaT keratinocytes were investigated in vitro. Cells were preincubated with melatonin at graded concentrations from 10(-9) to 10(-3) m for 30 min prior to UV irradiation at doses of 25 and 50 mJ/cm2. Biological markers of cellular viability such as DNA synthesis and colony-forming efficiency as well as molecular markers of apoptosis were measured. DNA synthesis was determined by [3H]-thymidine incorporation into insoluble cellular fraction, clonogenicity through plating efficiency experiments and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis experiments showed a strong protective effect by preincubation with melatonin at concentrations of 10(-4) m (P < 0.01) and 10(-3) m (P < 0.001). Additional postirradiation treatment with melatonin showed no increase in the pre-UV incubation protective effect. These results indicate that preincubation is a requirement for melatonin to exert its protective effects. The mechanism of melatonin's protective effect (10(-6) to 10(-3) m) includes inhibition of apoptosis as measured by TUNEL assay. Moreover, the biological significance of these effects is supported by clonogenic studies showing a significantly higher number of colonies in cultures treated with melatonin compared to controls. Thus, pretreatment with melatonin led to strong protection against UVB-induced damage in keratinocytes.

Melatonin decreases neurovascular oxidative/nitrosative damage and protects against early increases in the blood–brain barrier permeability after transient focal cerebral ischemia in mice

Abstract: We have recently shown that melatonin decreases the late (24 hr) increase in blood-brain barrier (BBB) permeability and the risk of tissue plasminogen activator-induced hemorrhagic transformation following ischemic stroke in mice. In the study, we further explored whether melatonin would reduce postischemic neurovascular oxidative/nitrosative damage and, therefore, improve preservation of the early increase in the BBB permeability at 4 hr after transient focal cerebral ischemia for 60 min in mice. Melatonin (5 mg/kg) or vehicle was given intraperitoneally at the beginning of reperfusion. Hydroethidine (HEt) in situ detection and immunohistochemistry for nitrotyrosine were used to evaluate postischemic accumulation in reactive oxygen and nitrogen species, respectively, in the ischemic neurovascular unit. BBB permeability was evaluated by spectrophotometric and microscopic quantitation of Evans Blue leakage. Relative to controls, melatonin-treated animals not only had a significantly reduced superoxide accumulation in neurovascular units in boundary zones of infarction, by reducing 35% and 54% cytosolic oxidized HEt in intensity and cell-expressing percentage, respectively (P < 0.001), but also exhibited a reduction in nitrotyrosine by 52% (P < 0.01). Additionally, melatonin-treated animals had significantly reduced early postischemic disruption in the BBB permeability by 53% (P < 0.001). Thus, melatonin reduced postischemic oxidative/nitrosative damage to the ischemic neurovascular units and improved the preservation of BBB permeability at an early phase following transient focal cerebral ischemia in mice. The findings further highlight the ability of melatonin in anatomical and functional preservation for the ischemic neurovascular units and its relevant potential in the treatment of ischemic stroke.

A comparative ex vivo and in vivo study of day and night perception in teleosts species using the melatonin rhythm.

Abstract: The purpose of this study was to determine and compare the light sensitivity of two commercially important, phylogenetically different teleost species in terms of melatonin production. Three series of experiments were performed on both Atlantic salmon and European sea bass. First, a range of light intensities were tested ex vivo on pineal melatonin production in culture during the dark phase. Then, light transmission through the skull was investigated, and finally short-term in vivo light sensitivity trials were performed. Results showed that sea bass pineal gland ex vivo are at least 10 times more sensitive to light than that of the salmon. Light intensity threshold in sea bass appeared to be between 3.8 × 10 -5 and 3.8 × 10 -6 W/m 2 in contrast to 3.8 × 10 -4 and 3.8 x 10 -5 W/m 2 in salmon. These highlighted species-specific light sensitivities of pineal melatonin production that are likely to be the result of adaptation to particular photic niches. Light transmission results showed that a significantly higher percentage of light penetrates the sea bass pineal window relative to salmon, and confirmed that penetration is directly related to wavelength with higher penetration towards the red end of the visible spectrum. Although results obtained in vivo were comparable, large differences between ex vivo and in vivo were observed in both species. The pineal gland in isolation thus appeared to have different sensitivities as the whole animal, suggesting that retinal and/or deep brain photoreception may contribute, in vivo, to the control of melatonin production.

Melatonin reduces apoptosis and necrosis induced by ischemia/reperfusion injury of the pancreas

Abstract: The pancreas is highly susceptible to the oxidative stress induced by ischemia/reperfusion (IR) injury leading to the generation of acute pancreatitis. Melatonin has been shown to be useful in the prevention of the damage by ischemia-reperfusion in liver, brain, myocardium, gut and kidney. The aim of the study was to evaluate the cytoprotective properties of melatonin against injury induced by IR in pancreas. The obstruction of gastro-duodenal and inferior splenic arteries induced pancreatic IR in male Wistar rats. Melatonin was intraperitoneally administered before or/and after IR injury. The animals were killed at 24 and 48 hr after reperfusion and there were evaluated parameters of oxidative stress (lipoperoxides, superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione), glandular endocrine and exocrine function (lipase, amylase, insulin) and cell injury (apoptosis and necrosis). The IR induced a marked enhancement of oxidative stress and impaired pancreatic function. The histological analysis showed that IR induced acute pancreatitis with the accumulation of inflammatory infiltrate, disruption of tissue structure, cell necrosis and hemorrhage. Melatonin administration before or after pancreatic IR prevented all tissue markers of oxidative stress, biochemical and histological signs of apoptosis and necrosis, and restored glandular function. No histological signs of pancreatitis were observed 48 hr after reperfusion in 80% of the animals treated with melatonin, with only a mild edematous pancreatitis being observed in the remaining rats. Preventive or therapeutic administration of melatonin protected against the induction of oxidative stress and tissue injury, and restored cell function in experimental pancreatic IR in rats.

Effects of melatonin and zinc on lipid profile and renal function in type 2 diabetic patients poorly controlled with metformin

Abstract: Glycemic control and prevention of secondary complications are the most important goals of using pharmacologic treatment of diabetes mellitus (DM). The inadequate responses to oral hypoglycemic agents may be attributed to inadequate postreceptor events even when insulin levels are quite sufficient, and associated with oxidative stress induced by long-term hyperglycemia. The administration of antioxidants such as melatonin and zinc may improve tissue responses to insulin and increase the efficacy of drugs, e.g. metformin, which act through this pathway. This project was designed to evaluate the effects of melatonin and zinc on the lipid profile and renal function in type 2 DM patients poorly controlled with metformin. A placebo-controlled, double-blind clinical trial was performed in which 46 type 2 diabetic patients were selected and allocated into three groups. These groups were treated with single daily oral doses of both 10 mg of melatonin and 50 mg of zinc acetate alone: 10 mg of melatonin and 50 mg of zinc acetate in addition to the regularly used metformin or placebo, given at bedtime for 90 days. Fasting lipid profiles and microalbuminuria (MAU) were measured before initiating the treatments (zero time) and after 30 and 90 days of treatment. Daily administration of melatonin and zinc improved the impaired lipid profile and decreased the level of MAU; the addition of this treatment regimen in combination with metformin improved the tissue responses to this oral hypoglycemic agent. In conclusion, the combination of melatonin and zinc acetate, when used alone or in combination with metformin, improves DM-related complications such as the impaired lipid profile and MAU in type 2 DM patients.

Melatonin inhibits the expression of the inducible isoform of nitric oxide synthase and nuclear factor kappa B activation in rat skeletal muscle

Abstract: This study investigated whether the induction of inducible nitric oxide synthase (iNOS) produced by acute exercise in rat skeletal muscle could be prevented by melatonin and whether iNOS down-regulation was related to inhibition of nuclear factor kappaB (NF-kappaB) activation. Male Wistar rats received melatonin i.p. at a dose of 1.0 mg/kg body weight 30 min before being exercised for 60 min on a treadmill at a speed of 25 m/min and a 10% slope. Exercise caused a significant induction of iNOS protein levels and a marked activation of NF-kappaB that were significantly prevented in rats treated with melatonin. Exercise also resulted in increased IkappaB kinasealpha (IKKalpha) and phosphorylated IkappaBalpha protein levels, whereas IkappaBalpha content decreased. These effects were blocked by melatonin administration. The increase in the muscle concentration of thiobarbituric acid reactive substances and in the oxidized/reduced glutathione ratio induced by exercise was partially prevented by melatonin. Our data indicate that melatonin has potent protective effects against damage caused by acute exercise in rat muscle, preventing oxidative stress, NF-kappaB activation and iNOS over-expression. These findings support the view that melatonin treatment, by abolishing the IKK/NF-kappaB signal transduction pathway, might block the production of noxious mediators involved in the inflammatory process.

Injury switches melatonin production source from endocrine (pineal) to paracrine (phagocytes) - melatonin in human colostrum and colostrum phagocytes.

Abstract: A large number of data show that melatonin has immunomodulatory properties and is produced by immunocompetent cells; also, some evidence suggests a 'feedback' of the activated immune system on the pineal gland. In this paper, we studied immune-pineal interactions in colostrum obtained from healthy puerperae and mothers with mastitis taking into account that, (a) melatonin levels in milk reflects pineal activity and (b) colostrum quiescent mononuclear and polymorphonuclear phagocytes from healthy mothers in culture are adequate for evaluating the ability of immunocompetent cells to produce melatonin. Here we compared the diurnal and nocturnal melatonin levels in colostrum from healthy puerperae and mothers with mastitis; this is a unique noninvasive model for determining pineal activity in the proinflammatory phase of a defense response. In addition, we determined the 'in vitro' production of melatonin by colostrum immunocompetent cells stimulated by enteropathogenic Escherichia coli or zymosan. Suppression of nocturnal melatonin rise in mothers with mastitis was highly correlated with increased tumor necrosis factor-alpha (TNF-alpha) secretion. This result, interpreted taking into account the presence of the transcription factor nuclear factor kappa B in pineal gland, suggest that the proinflammatory cytokine can inhibit nocturnal pineal melatonin production. On the other hand, stimulated, but not quiescent, immunocompetent cells secreted in the colostrum produced melatonin in vitro. In addition, this production ceases after bacteria killing. These results suggest that during the response to an injury the production of melatonin can be transiently shifted from an endocrine (pineal) to a paracrine (immunocompetent cells) source.

Melatonin protects against hydrogen peroxide‐induced cell death signaling in SH‐SY5Y cultured cells: involvement of nuclear factor kappa B, Bax and Bcl‐2

Abstract: Oxidative stress is defined as a disturbance in the prooxidant-antioxidant balance, leading to potential cell damage. Reactive oxygen species such as superoxide radicals, hydroxyl radicals and hydrogen peroxide may act also as secondary intermediaries in intracellular signaling leading to cell death. The neuroprotective effect of melatonin has been observed both in vivo and in vitro. The objective of this research, therefore, was to better understand the cellular mechanisms of neuronal cell degeneration induced via oxidative stress and the protective roles of melatonin on this cell death. In the present study, the effects of melatonin on H(2)O(2)-induced neuronal cell degeneration in human dopaminergic neuroblastoma SH-SY5Y cultured cells were investigated. The results showed that H(2)O(2) significantly decreased cell viability and melatonin reversed the toxic effects of H(2)O(2). An inhibition of caspase enzyme activity by Ac-DEVD-CHO, a caspase-3 inhibitor, significantly increased cell viability in H(2)O(2)-treated cells. The phosphorylation of transcription factors, nuclear factor kappa B (NF-kappaB) was increased in H(2)O(2)-treated cells and this effect was abolished by melatonin. Translocation of phosphorylated NF-kappaB to perinuclear and nuclear sites, estimated using immunofluorescence, occurred to a greater extent in H(2)O(2)-treated cells than in untreated control cells and again this effect was abolished by melatonin. In addition, induction of Bcl-2 and Bax proteins was demonstrated in SH-SY5Y cultured cells treated with H(2)O(2), whereas the induction of Bax but not Bcl-2 was diminished by melatonin. In light of these finding, it is possible that the antioxidative stress effect of melatonin associated with inhibition of Bax expression, may offer a means of treating neuronal degeneration and disease.

Melatonin inhibits both ERα activation and breast cancer cell proliferation induced by a metalloestrogen, cadmium

Abstract: Cadmium (Cd) is a heavy metal affecting human health both through environmental and occupational exposure. There is evidence that Cd accumulates in several organs and is carcinogenic to humans. In vivo, Cd mimics the effect of estrogens in the uterus and mammary gland. In estrogen-responsive breast cancer cell lines, Cd stimulates proliferation and can also activate the estrogen receptor independent of estradiol. The ability of this metalloestrogen to increase gene expression in MCF7 cells is blocked by anti-estrogens suggesting that the activity of these compounds is mediated by ER alpha. The aims of this work were to test whether melatonin inhibits Cd-induced proliferation in MCF7 cells, and also to study whether melatonin specifically inhibits Cd-induced ER alpha transactivation. We show that melatonin prevents the Cd-induced growth of synchronized MCF7 breast cancer cells. In transient transfection experiments, we prove that both ER alpha- and ER beta-mediated transcription are stimulated by Cd. Melatonin is a specific inhibitor of Cd-induced ER alpha-mediated transcription in both estrogen response elements (ERE)- and AP1-containing promoters, whereas ER beta-mediated transcription is not inhibited by the pineal indole. Moreover, the mutant ER alpha-(K302G, K303G), unable to bind calmodulin, is activated by Cd but becomes insensitive to melatonin treatment. These results proved that melatonin inhibits MCF7 cell growth induced by Cd and abolishes the stimulatory effect of the heavy metal in cells expressing ER alpha at both ERE-luc and AP1-luc sites. We can infer from these experiments that melatonin regulates Cd-induced transcription in both ERE- and AP1 pathways. These results also reinforce the hypothesis of the anti-estrogenic properties of melatonin as a valuable tool in breast cancer therapies.

Parallel signaling pathways of melatonin in the pancreatic beta-cell.

Abstract: Previous results demonstrated that melatonin inhibits cAMP production and stimulates IP(3) liberation in rat insulinoma INS1 cells, a model for the pancreatic beta-cell. This study addresses the impact of melatonin on insulin release. Insulin, cAMP and IP(3) levels of INS1 cells in a superfusion system were measured. Initially, forskolin was used to stimulate cAMP and subsequently insulin release. Incubation of forskolin (5 micromol/L)-stimulated cells with melatonin (100 nmol/L) inhibited cAMP and insulin levels (down to 60% of insulin and cAMP release). The G(i)alpha-protein-inhibitor pertussis toxin (PTX) was used to distinguish between the G(i)alpha-dependent cAMP pathway and the G(i)alpha-independent IP(3) pathway. In our experiments we employed a specific stimulation pattern to prove proper inhibition of G(i)alpha-proteins by PTX. In INS1 cells incubated with 250 ng/mL PTX for 24 hr, melatonin was no longer able to inhibit the forskolin-induced cAMP and insulin release. In a study, carbachol was used to stimulate IP(3) and subsequently insulin release. Surprisingly, incubation of carbachol (300 micromol/L)-stimulated cells with melatonin (100 nmol/L) inhibited insulin release (down to 75% of insulin release). Finally, in PTX-incubated INS1 cells, melatonin (100 nmol/L) increased carbachol (300 micromol/L)-induced insulin release (up to 124% of insulin release). In conclusion, we found that the melatonin MT(1)-receptor on pancreatic beta-cells is coupled to parallel signaling pathways, with opposite influences on insulin secretion. The cAMP- and subsequently insulin-inhibiting signaling pathway involves PTX-sensitive G(i)alpha-proteins and is predominant in terms of insulin release.

Melatonin modulates signal transduction pathways and apoptosis in experimental colitis

Abstract: Various evidences have documented that the pineal secretory product melatonin exerts an important anti-inflammatory effect in different experimental models including colitis. The aim of the present study was to evaluate whether melatonin regulates the inflammatory response of experimental colitis in rats at the level of signal transduction pathway. Colitis was induced by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Four days after DNBS administration, a substantial increase of colon TNF-alpha production was associated with the colon damage. In DNBS-treated rats, the colon injury correlated with a significant rise of apoptosis (evaluated by TUNEL coloration) which was associated with a significant increased expression of proapoptotic Bax and decreased colon content of antiapoptotic Bcl-2. This inflammatory response was also related to activation of nuclear factor-kappaB (NF-kappaB) and phosphorylation of c-Jun as well as FAS ligand expression in the colon. Treatment with melatonin (15 mg/kg daily i.p.) was associated with a remarkable amelioration of colonic disrupted architecture as well as a significant reduction of TNF-alpha. Melatonin also reduced the NF-kappaB activation and phosphorylation of c-Jun as well as the Fas ligand expression in the colon. Furthermore, melatonin reduced the expression of Bax and prevented the loss of Bcl-2 proteins as well as the presence of apoptotic cells caused by DNBS. The results of this study show that melatonin administration exerts beneficial effects in inflammatory bowel disease by modulating signal transduction pathways.