Showing papers in "Journal of Pineal Research in 2000"

Evidence for melatonin synthesis in mouse and human bone marrow cells.

Abstract: Recently, it was demonstrated that inbred strains of mice have a clearcut circadian rhythm of pineal and serum melatonin. Moreover, it is known that melatonin is involved in many immunoregulatory functions. Among them, hematopoiesis is influenced by the action of melatonin via melatonin-induced opioids on kappa-opioid receptors, which are present on stromal bone marrow cells. Therefore, the present study was carried out to investigate the presence of melatonin in the bone marrow in which immunocompetent cells are generated. Specifically, we aimed at answering the following question: are bone marrow cells involved in melatonin synthesis? In the present study, we demonstrate that (1) bone marrow cells contain high concentrations of melatonin; (2) bone marrow cells have a N-acetyltransferase activity and they express the mRNA encoding hydroxy-O-methyltransferase and (3) bone marrow cells cultured for a prolonged period exhibited high levels of melatonin. Results presented here suggest that mouse and human bone marrow and bone marrow cells are capable of de novo synthesis of melatonin, which may have intracellular and or paracrine functions.

Melatonin‐induced increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo

Abstract: Melatonin displays antioxidant and free radical scavenger properties. Due to its ability with which it enters cells, these protective effects are manifested in all subcellular compartments. Recent studies suggest a role for melatonin in mitochondrial metabolism. To study the effects of melatonin on this organelle we used ruthenium red to induce mitochondrial damage and oxidative stress. The results show that melatonin (10 mg/kg i.p.) can increase the activity of the mitochondrial respiratory complexes I and IV after its administration in vivo in a time-dependent manner; these changes correlate well with the half-life of the indole in plasma. Melatonin administration also prevented the decrease in the activity of complexes I and IV due to ruthenium red (60 microg/kg i.p.) administration. At this dose, ruthenium red did not induce lipid peroxidation but it significantly reduced the activity of the antioxidative enzyme glutathione peroxidase, an effect also counteracted by melatonin. These results suggest that melatonin modulates mitochondrial respiratory activity, an effect that may account for some of the protective properties of the indoleamine. The mitochondria-modulating role of melatonin may be of physiological significance since it seems that the indoleamine is concentrated into normal mitochondria. The data also support a pharmacological use of melatonin in drug-induced mitochondrial damage in vivo.

Randomized, double-blind clinical trial, controlled with placebo, of the toxicology of chronic melatonin treatment.

Abstract: The objective of the present study was to assess the toxicology of melatonin (10 mg), administered for 28 days to 40 volunteers randomly assigned to groups receiving either melatonin (N = 30) or placebo (N = 10) in a double-blind fashion. The following measurements were performed: polysomnography (PSG), laboratory examinations, including complete blood count, urinalysis, sodium, potassium and calcium levels, total protein levels, albumin, blood glucose, triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), urea, creatinine, uric acid, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvate transaminase (GPT), bilirubin, alkaline phosphatase, gama-glutamic transaminase (GGT), T3, T4, TSH, LH/FSH, cortisol, and melatonin serum concentrations. In addition, the Epworth Somnolence Scale (ESS) and a sleep diary (SD) were also applied to the volunteers 1 wk before each PSG. In addition, the volunteers were asked about possible side effects (SE) that appeared during the treatment. The study was carried out according to the following timetable: Visit 0, filling out the term of consent and inclusion criteria; Visit 1, PSG, laboratory examinations, ESS, SD, melatonin serum concentrations; Visit 2, SD, melatonin serum concentrations, SE; Visit 3, melatonin serum concentrations, PSG, ESS, SE; Visit 4, laboratory examinations, SE, melatonin serum concentrations, SD; and Visit 5, PSG, ESS, SE. Analysis of the PSG showed a statistically significant reduction of stage 1 of sleep in the melatonin group. No other differences between the placebo and melatonin groups were obtained. In the present study we did not observe, according to the parameters analyzed, any toxicological effect that might compromise the use of melatonin at a dose of 10 mg for the period of time utilized in this study.

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines.

Abstract: The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.

Nitrosation of melatonin by nitric oxide and peroxynitrite.

Abstract: Peroxynitrite (ONOO-) is an endogenous molecule, formed by rapid coupling between *NO and O2*-. ONOO- is known to be a strong oxidant of thiols and metalloorganic compounds and also a nitrating agent of aromatic compounds such as tyrosine. However, its chemistry is not yet well elucidated under physiological conditions. Melatonin, which is an indole-amine produced by the pineal gland and other organs, has antioxidant properties. We show that melatonin reacts with ONOO- in phosphate-buffered solutions. We provide evidence of nitrosation and oxidation at the pyrrole nitrogen leading to 1-nitrosomelatonin and 1-hydroxymelatonin, these being the major reactions in aqueous phosphate-buffered solutions besides other aromatic hydroxylations and nitration. 4-Nitromelatonin is formed, but in small amounts. The kinetics of all transformations were strictly dependent on ONOO- decay, whereas yields varied with pH and the presence of CO2. The N-oxidation became competitive with nitrosation at pH 7.4, in medium containing a sufficient amount of CO2. A proposed mechanism involves the transient formation of melatonyl radical and ONOO* radical derived from ONOO- decay.

The effect of melatonin on in vitro fertilization and embryo development in mice.

Abstract: To examine the effect of melatonin on in vitro fertilization and embryonic development, mouse embryos after insemination in vitro were cultured in a physiological medium with or without melatonin. Melatonin increased the fertilization rate significantly at a concentration between 10(-6) and 10(-4) M (27.6 vs. 43.9 or 40.4%, P < 0.01). Furthermore, a significant increase in the rate of embryos reaching the four-cell stage (16.0 vs. 26.7%, P < 0.01), the eight-cell stage (12.1 vs. 25.8 or 23.5%, P < 0.01), and blastulation (8.9 vs. 23.5 or 17.5%, P < 0.01) was observed when the embryos were cultured in a medium containing 10(-8) or 10(-6) M melatonin. These results demonstrate that melatonin supports fertilization and early embryo development after in vitro fertilization.

Melatonin increases activities of glutathione peroxidase and superoxide dismutase in fetal rat brain.

Abstract: Melatonin is a powerful scavenger of oxygen free radicals. In humans, melatonin is rapidly transferred from the maternal to the fetal circulation. To investigate whether or not maternal melatonin administration can protect the fetal rat brain from radical-induced damage by increasing the activities of antioxidant enzymes, we administered melatonin to pregnant rats on day 20 of gestation. Melatonin (10 mg/kg) was injected intraperitoneally at daytime (14:00 hr) and, to remove the fetuses, a laparotomy was performed at 1, 2, or 3 hr after its administration. We measured the melatonin concentration in the maternal serum and in fetal brain homogenates and determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in fetal brain homogenates. Melatonin administration markedly increased melatonin concentrations in the maternal serum and fetal brain homogenates, with peak levels achieved 1 hr after melatonin administration (serum: 538.2+/-160.7 pM/mL; brain homogenates: 13.8+/-2.8 pM/mg protein). Between 1 and 3 hr after melatonin administration, GSH-Px activity in fetal brain homogenates increased significantly (P<0.01). Similarly, SOD activity increased significantly between 1 and 2 hr after melatonin administration (P<0.01). These results indicate that melatonin administration to the mother increases antioxidant enzyme activities in the fetal brain and may thereby provide indirect protection against free radical injury. Thus, melatonin may potentially be useful in the treatment of neurodegenerative conditions that may involve excessive free radical production, such as fetal hypoxia and preeclampsia.

Clinical trials of controlled‐release melatonin in children with sleep–wake cycle disorders

Abstract: This is the first study to examine effective doses of controlled-release (CR) melatonin in children with chronic sleep wake cycle disorders. All 42 subjects had severe neurodevelopmental difficulties. Initially, a randomized double-blinded cross-over design was used in 16 children, comparing the effectiveness of fast-release (FR) and CR melatonin. In the remainder of the patients, the CR melatonin was studied on a clinical basis. The effectiveness of treatment was assessed by sleep charts and clinical follow-up. Emphasis was placed on the judgement of the parents, who had guidance from the physicians. The average final CR melatonin dose in the 42 patients was 5.7 mg (2-12 mg). The studies showed that the FR melatonin was most effective when there was only delayed sleep onset, but CR formulations were more useful for sleep maintenance. Children appeared to require higher doses than adults.

Protective effects of melatonin in ischemic brain injury

Abstract: Recent studies have demonstrated that melatonin is a scavenger of oxyradicals and peroxynitrite and an inhibitor of nitric oxide (NO) production. NO, peroxynitrite (formed from NO and superoxide anion), and poly (ADP-Ribose) synthetase (PARS) have been implicated as mediators of neuronal damage following focal ischemia. In the present study, we have investigated the effects of melatonin treatment in Mongolian gerbils subjected to cerebral ischemia. Treatment of gerbils with melatonin (10 mg kg−1, 30 min before reperfusion and 1, 2, and 6 hr after reperfusion) reduced the formation of post-ischemic brain edema, evaluated by water content. Melatonin also attenuated the increase in the brain levels malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischemia. Positive staining for nitrotyrosine was found in the hippocampus of Mongolian gerbils subjected to cerebral ischemia. Hippocampus tissue sections, from Mongolian gerbils subjected to cerebral ischemia, also showed positive staining for PARS. The degrees of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received melatonin. Melatonin treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischemia and reperfusion. Histological observations of the pyramidal layer of CA-1 showed a reduction of neuronal loss in animals that received melatonin. These results show that melatonin improves brain injury induced by transient cerebral ischemia.

Increased levels of oxidatively damaged DNA induced by chromium(III) and H2O2: protection by melatonin and related molecules

Abstract: Chromium (Cr) compounds are known occupational and environmental carcinogens. This trace element is found in the workplace primarily in the valence forms Cr(III) and Cr(VI). Cr(III), which was thought originally to be relatively nontoxic, was recently found to be more reactive toward purified DNA than was chromium(VI). Herein, we examined the ability of Cr(III) to induce oxidative DNA damage by measuring the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in purified calf thymus DNA incubated with CrCl3 plus H2O2. In this system we observed that the Cr(III)-induced formation of 8-OH-dG in isolated DNA was both dose- and time-dependent. When melatonin and related molecules, including 6-methoxy-1,2,3,4-tetrahydro-beta-carboline (pinoline), N-acetylserotonin, 6-hydroxymelatonin and indole-3-propionic acid, were co-incubated with CrCl3 plus H2O2, the accumulations of 8-OH-dG in DNA samples were markedly inhibited in a concentration-dependent manner. The concentrations of each indole required to reduce DNA damage by 50%, i.e. the IC50 values, were 0.48, 0.51, 0.88, 1.00 and 3.08 microM for pinoline, melatonin, N-acetylserotonin, 6-hydroxymelatonin and indole-3-propionic acid, respectively. These results suggest that one of the mechanisms by which Cr(III) may induce cancer is via Fenton-type reactions which generate the hydroxyl radical (*OH). The findings also indicate that the protective effects of melatonin and related molecules against Cr(III)-induced carcinogenesis relate to their direct *OH scavenging ability which thereby reduces the formation of the damaged DNA product, 8-OH-dG.

Therapeutic effect of melatonin on carbon tetrachloride-induced acute liver injury in rats.

Abstract: The therapeutic effect of melatonin on acute liver injury was examined in rats intoxicated with carbon tetrachloride (CCl4). Melatonin (10, 50, or 100 mg/kg body weight [BW]) was intraperitoneally administered to male Wistar rats 6 hr after intraperitoneal injection of CCl4 (1.6 g/kg BW) at which time an apparent liver injury occurred. This post-melatonin administration dose dependently prevented the progression of liver injury at 24 hr after CCl4 injection, judging from the levels of serum transaminases, indices of liver cell damage. Rats injected with CCl4 alone showed an increase in liver lipid peroxide (LPO) content and a decrease in liver reduced glutathione content at 6 and 24 hr after the injection. The post-melatonin administration dose dependently ameliorated both changes found at 24 hr after CCl4 injection. Rats injected with CCl4 alone showed an increase in liver triglyceride (TG) content and decreases in serum TG concentration and liver tryptophan 2,3-dioxygenase (TDO) activity, a marker of the inhibition of liver protein synthesis by CCl4, at 6 and 24 hr after the injection, and also a decrease in serum albumin concentration at 24 hr. The changes in serum TG, albumin concentration, liver TG content, and TDO activity found at 24 hr after CCl4 injection were not ameliorated by the post-administration of melatonin. The same administration of melatonin dose dependently reduced liver LPO content in CCl4-untreated rats. These results indicate that melatonin exerts a therapeutic effect on CCl4-induced acute liver injury in rats, possibly through its antioxidant action.

Melatonin protects against stress‐induced gastric lesions by scavenging the hydroxyl radical

Abstract: The antiulcer effect of melatonin on gastric lesions caused by restraint-cold stress or by indomethacin (IMN) was studied with the intent of determining the mechanism of action of the indole. Melatonin dose-dependently prevents both stress and IMN-induced gastric damage with around 90% inhibition at a dose of 60 mg per kg BW. When compared with already-marketed antiulcer drugs, such as ranitidine and omeprazole, melatonin was found to be more effective than ranitidine but less effective than omeprazole in preventing stress ulcer. When compared with other antioxidants, melatonin was more potent than glutathione and essentially equipotent to alpha-tocopherol in blocking stress-induced ulcer. As stress-induced gastric lesions are mainly caused by oxidative damage due to hydroxyl radicals (*OH), the effect of melatonin in scavenging the *OH generated during stress conditions, as well as in an in vitro model system, was studied. The results indicate that melatonin at the dose of 60 mg per kg BW caused an 88% reduction of endogenous *OH during stress. Melatonin was also highly effective in scavenging *OH generated in vitro by a Cu2+-ascorbate system. In this case, melatonin at 100 microM reduced *OH by 80%. Melatonin was also found to be a more potent radical scavenger than benzoate, a known *OH scavenger. The results indicate that melatonin prevents stress-induced gastric lesions by scavenging the endogenous *OH. As it also protects against IMN-induced gastric damage, it probably also offers gastroprotection by maintaining endogenous prostaglandin levels.

Ototoxicity caused by cisplatin is ameliorated by melatonin and other antioxidants.

Abstract: The mechanism of the ototoxicity caused by cisplatin is based in the generation of reactive oxygen species, which interferes with the antioxidant protection of the organ of Corti. Conversely, the protection of the cochlea with antioxidants ameliorates the ototoxicity by cisplatin. The ototoxicity produced by cisplatin can be reversible or persistent, depending on the age of the patient, cumulative doses, number of chemotherapy cycles, history of noise exposure, and deteriorating renal function. We have obtained in rats an ototoxic chart utilizing cisplatin (10 mg/kg body weight injected intraperitoneally, once only). Together with this treatment, the animals were treated with melatonin in the drinking water (10 mg/L) or injected subcutaneously (250 microg), and with an antioxidant mixture, injected subcutaneously, composed of 0.25 mg alpha-tocopherol acid succinate, 3 mg ascorbic acid, 1 mg glutathione, and 60 mg N-acetylcysteine. The distortion product otoacoustic emissions were determined for a prolonged period of time for each animal. The ototoxicity produced by cisplatin was maximal from days 7 to 10 post-treatment, returning to normal values in a month. When melatonin and the antioxidant mixture were present, the recovery was between days 10 and 15 post-treatment, independent of the means of administration of the pineal product. We conclude that the ototoxicity caused by cisplatin is ameliorated by melatonin and other antioxidants.

Beneficial effects of melatonin in a rat model of splanchnic artery occlusion and reperfusion.

Abstract: The aim of the present study was to investigate the protective effect of the pineal secretary product melatonin in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were sacrificed for tissue histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malondialdehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 hr after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localized in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. Melatonin (applied at 3 mg/kg, 5 min prior to reperfusion, followed by an infusion of 3 mg/kg per hr), significantly reduced ischemia reperfusion injury in the bowel as evaluated by histological examination. This prevented the infiltration of neutrophils into the reperfused intestine, is evidenced by reduced myeloperoxidase activity and reduced lipid peroxidation. This was evaluated by malondialdehyde activity which reduced the production of peroxynitrite during reperfusion, markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats and improved their survival. Taken together, our results clearly demonstrate that melatonin treatment exerts a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Scavenging effect of melatonin on hydroxyl radicals generated by alloxan.

Abstract: Alloxan can act as a generator of reactive oxygen species (ROS) as long as sufficient suitable reducing agents (e.g. reduced glutathione) and oxygen are available. Using electron spin resonance-spectroscopy and the oxygen-centered spin trap DEPMPO, we demonstrate that hydroxyl radicals (OHzrad;) are formed in vitro by alloxan in the presence of glutathione (GSH) and chelated divalent iron. Furthermore, peroxidation of polyunsaturated fatty acids from phosphatidylcholine-containing liposomes with concomitant formation of malondialdehyde (MDA) was used as a further indicator for a preceding OHzrad; formation. Melatonin, the main secretory product of the pineal gland, is an effective scavenger of OHzrad;. The 50%-inhibitor concentration (IC50-value) for melatonin to scavenge OHzrad; generated from the alloxan/GSH-reaction in the presence of ferrous ions was 23 μmol/L. In contrast to the ability to effectively scavenge OHzrad;, the potential of melatonin to prevent lipid peroxidation is considerably less pronounced.

Morning urinary assessment of nocturnal melatonin secretion in older women.

Abstract: We evaluated the feasibility of using morning urine samples in epidemiological studies aimed at clarifying the relationship between nocturnal melatonin levels and breast cancer risk. Initially, a laboratory-based study of 29 women (40- 70 yr old) was performed to examine the correlation between plasma melatonin levels in hourly nocturnal blood samples and both melatonin and its major enzymatic metabolite, 6-hydroxymelatonin-sulfate (6-OHMS) in morning urine samples. In a companion field study, morning urine samples were collected from 203 healthy women to assess similarities and differences in laboratory versus field measures. Taken together, our results indicate: 1) levels of melatonin and of creatinine-corrected 6-OHMS in the first morning void urine are strongly correlated with total nocturnal plasma melatonin output (P < 0.001) and also with peak nocturnal melatonin values (P < 0.001); 2) similar ranges for 6-OHMS were found in the laboratory and the field; and 3) neither menopausal status nor hormonal replacement therapy altered 6-OHMS values in morning void urine. The inclusion of morning urine samples in epidemiological studies of cancer could allow cost-effective, widespread testing of the role played by melatonin in human health and disease.

Comparative effects of melatonin, L-deprenyl, Trolox and ascorbate in the suppression of hydroxyl radical formation during dopamine autoxidation in vitro.

Abstract: Degeneration of nigrostriatal dopaminergic neurons is the major pathogenic substrate of Parkinson's disease (PD). Inhibitors of monoamine oxidase B (MAO-B) have been used in the treatment of PD and at least one of them, i.e., deprenyl, also displays antioxidant activity. Dopamine (DA) autoxidation produces reactive oxygen species implicated in the loss of dopaminergic neurons in the nigrostriatal pathway. In this study we compared the effects of melatonin with those of deprenyl and vitamins E and C in preventing the hydroxyl radical (8OH) generation during DA oxidation. The rate of production of 2,3-dihydroxybenzoate (2,3-DHBA) in the presence of salicylate, an *OH scavenger, was used to detect the in vitro generation of *OH during iron-catalyzed oxidation of DA. The results showed a dose-dependent effect of melatonin, deprenyl and vitamin E in counteracting DA autoxidation, whereas vitamin C had no effect. Comparative analyses between the effect of these antioxidants showed that the protective effect of melatonin against DA autoxidation was significantly higher than that of the other compounds tested. Also, when melatonin plus deprenyl were added to the incubation medium, a potentiation of the antioxidant effect was found. These findings suggest that antioxidants may be useful in brain protection against toxicity of reactive oxygen species produced during DA oxidation, and melatonin, alone or in combination with deprenyl, may be an important component of the brain's antioxidant defenses to protect it from dopaminergic neurodegeneration.

Changes in nocturnal melatonin secretion in perimenopausal women: correlation with endogenous estrogen concentrations.

Abstract: Although age-related decrease in melatonin secretion in humans and animals is well documented, there is a paucity of data on the precise changes in melatonin secretion that occur during the perimenopausal period. The present study was designed to measure changes in nocturnal melatonin and to characterize the role played by estrogen in controlling nocturnal melatonin secretion in perimenopausal women. Nocturnal serum melatonin concentrations were determined every 2 hr in 46 premenopausal women, 44 postmenopausal women, and 11 premenopausal women with uterine leiomyoma scheduled for hysterectomy and bilateral salpingo-oophorectomy. Nocturnal serum melatonin secretion in premenopausal women declined moderately from 17 to 45 years of age, and increased during the period from 46 to 50 years of age. Among postmenopausal women, a steep, age-related decline in nocturnal melatonin secretion was found for up to 15 years postmenopause, followed by an extremely gradual decline thereafter. A significant negative correlation was observed between the peak serum melatonin concentration and the serum 17 β-estradiol concentration in premenopausal women aged 40-50 years (r = - 0.661, P < 0.0005). Daily oral administration of conjugated estrogen (0.625 mg) to postmenopausal women suppressed nocturnal melatonin secretion (P < 0.005). A low estrogen state, induced by oophorectomy of premenopausal women with uterine leiomyoma led to an increase in nocturnal melatonin secretion (P < 0.0001). Our findings suggest that transient elevated nocturnal melatonin secretion during menopause may be related to the existence of a low estrogen environment. The age-related decrease in melatonin secretion observed in other conditions is most likely attributable to other age-related factors.

Low urinary 6‐sulphatoxymelatonin levels in patients with coronary artery disease

Abstract: A decrease in nocturnal serum melatonin levels was reported in patients with clinically uncharacterized coronary artery disease. To assess whether there was a correlation between melatonin production and disease stage, we measured the nocturnal urinary excretion of 6-sulphatoxymelatonin (an index of blood melatonin concentration) in patients with chronic stable or unstable coronary disease and in a group of age-matched controls. Three groups of individuals were studied: a) 24 healthy subjects (mean age: 63 +/- 13 yr); b) 32 patients with chronic, stable, coronary disease (62 +/- 11 yr); and c) 27 patients with unstable angina (62 +/- 12 yr). For 6-sulphatoxymelatonin measurement, urine was collected from 18:00 to 06:00 hr, within 48 hr of hospitalization in the case of unstable angina. 6-Sulphatoxymelatonin was measured by a specific radioimmunoassay. Urinary 6-sulphatoxymelatonin excretion was significantly lower in unstable angina patients than in healthy subjects or in patients with stable angina. 6-Sulphatoxymelatonin correlated negatively with age in healthy subjects, but not in coronary patients. 6-Sulphatoxymelatonin excretion in patients treated with beta-adrenoceptor blockers did not differ significantly from coronary patients not receiving beta-blockers. The results indicate that patients with coronary disease have a low melatonin production rate, with greater decreases in those with higher risk of cardiac infarction and/or death.

Preservation of the antioxidant status in chemically-induced diabetes mellitus by melatonin.

Abstract: Oxidant stress is believed to be enhanced in patients with diabetes mellitus, which may lead to endothelial dysfunction and the development of atherosclerosis. In diabetes, hyperglycemia drives non-enzymatic glycation and oxidation of proteins and lipids which enhances the formation of advanced glycation end products (AGEs), which may be involved in the pathogenesis of diabetic vascular disease. The macrovascular complications of diabetes seem to be due to enhanced cellular oxidant stress by the interaction of AGEs with their receptor. It would be worthwhile to devise methods to reduce this oxidant stress. In alloxan-induced diabetic rats lipid peroxidation products were increased, while levels of nitric oxide glutathione peroxidase and superoxide dismutase were reduced. Melatonin restored these biochemical abnormalities to normalcy independent of hyperglycemia. This model can be used to study the role of oxidant stress in the development of macrovascular complications in diabetes mellitus.

Protective effect of melatonin against the 1-methyl-4-phenylpyridinium-induced inhibition of complex I of the mitochondrial respiratory chain.

Abstract: In the present study, a novel property of melatonin is shown: a protective effect of melatonin on the respiratory chain in isolated rat liver mitochondria and in striatal synaptosomes treated with 1-methyl-4-phenylpyridinium ion (MPP+). The cellular damage caused by MPP+, a compound that produces a Parkinsonian-like syndrome in humans, is the result of the mitochondrial respiration inhibition at the Complex I level and oxidative stress induction. Treatment of mitochondria with MPP+ inhibits the respiration rate. This effect was prevented by the inclusion of melatonin in the incubation mixture. This preventive effect, which is not related to the antioxidative properties of melatonin, seems to be due to the fact that melatonin prevents MPP+ interaction with Complex I. These results suggest that melatonin may protect against the effect of several Parkinsonogenic compounds that are associated with progressive impairment of mitochondrial function and increased oxidative damage.

Differential growth inhibitory effect of melatonin on two endometrial cancer cell lines

Abstract: The effect of melatonin on endometrial cancer cell growth was investigated using two cell lines, SNG-II and Ishikawa, which are different in their estrogen receptor status. A physiological concentration of melatonin (10(-9) M) showed no growth inhibitory effect on SNG-II cells, which are estrogen receptor-negative at all cell densities and incubation times. In contrast, melatonin significantly inhibited Ishikawa cells, which are estrogen receptor-positive at all cell densities tested after 96 hr incubation. The greatest inhibition of Ishikawa cell growth was observed at 10(-9) M melatonin, compared with other supra (10(-6), 10(-8) M) or subphysiological concentrations (10(-10), 10(-12) M). This growth inhibitory effect of melatonin on Ishikawa cells was completely blocked by 10(-10) to 10(-8) M concentrations of 17-beta estradiol administration. Pretreatment with luzindole, which is a selective melatonin receptor antagonist, prior to the addition of melatonin also blocked the inhibitory effect of melatonin on Ishikawa cells. This is the first study to demonstrate an anti-proliferative effect of physiological melatonin on endometrial cancer cells in vitro. The present study revealed that melatonin also inhibits the growth of estrogen receptor positive endometrial cancer cells and that this effect of the pineal indole may be mediated by both steroid and melatonin receptors.

Circadian variation of portal, arterial and venous blood levels of melatonin in pigs and its relationship to food intake and sleep

Abstract: Circadian levels of melatonin were determined in the hepatic portal vein, cranial vena cava, and the lower aorta of ten juvenile pigs. Blood was sampled every hour for a total of 24 hr via temporary cannulas introduced into blood vessels under anesthesia. No peak levels of melatonin were found in the mid-scotophase, but hepatic portal concentrations peaked at 06.00 hr. Overall levels of melatonin were highest in the hepatic portal vein (range 35-65 pg/mL), followed by an artery (range 30-55 pg/mL) and the vena cava (range 25-35 pg/mL). Levels of melatonin exhibit strong variation between individual pigs, but generally the average levels from all three sources follow each other's time course. However, on occasion, melatonin levels in the hepatic portal vein varied independently from the levels in the vena cava. Large portal peaks were usually preceded by a feeding period and were associated with a subsequent period of sleep. The data indicate that: 1) there is no clear circadian rhythm of melatonin in the peripheral blood of pigs, 2) relatively little melatonin is metabolized during the first liver passage, 3) food intake may elevate melatonin levels in the hepatic portal vein, and 4) increased levels of melatonin originated in the gastrointestinal tract may induce sleep.

Influence of melatonin on free radical-induced changes in rat pancreatic beta-cells in vitro.

Abstract: Free radicals may produce cytotoxicity to pancreatic islets under pathophysiological conditions. The aim of our in vitro investigations was to compare functional and morphological changes in pancreatic beta-cells induced by reactive oxygen species (ROS) generated by alloxan or xanthine oxidase/hypoxanthine (XO/HX), respectively. We demonstrate that short-term exposure to alloxan or to XO/HX leads to a temporarily elevated insulin release from isolated pancreatic islets. On application of alloxan, this effect is caused by beta-cell necrosis and can be prevented by administration of melatonin, while in contrast, XO/HX did not lead to long-term morphological changes in the majority of the cells. Among the cells destroyed by alloxan, only necrosis could be detected, while in contrast, some apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction and electron microscopic examinations of cells treated with XO/HX. Melatonin was able to prevent the changes caused by alloxan, but failed to influence the alterations caused by XO/HX. Using electron spin resonance and lipid peroxidation assay, respectively, it was confirmed that melatonin effectively detoxifies hydroxyl radicals. Therefore, we believe that hydroxyl radicals are the toxic principle of alloxan, but not of XO/HX toxicity.

Melatonin inhibits apoptosis during early B-cell development in mouse bone marrow.

Abstract: The pineal secretory product, melatonin, exerts a variety of effects on the immune system. Administration of melatonin stimulates cell-mediated immunity, particularly by inhibiting apoptosis among T lymphocytes in the thymus and inducing production of T-cell-derived cytokines. However, its possible effects on the humoral immune system are unclear. In the present study, we have examined whether melatonin may influence the in vivo development of B lymphocytes in mouse bone marrow, a process in which apoptosis is normally a prominent feature. Double immunofluorescence labeling and flow cytometry were used to quantitate phenotypically defined precursor B-cell and mature B-cell populations and their apoptotic rates in bone marrow of mice fed either melatonin-containing or control diet for 16 days from 9 wk of age. In short-term bone marrow cultures, the incidence of apoptosis among large pre-B cells, including cells expressing the lambda5 component of pre-B-cell receptor, was markedly reduced in melatonin-treated mice, associated with an increase in the absolute number of large pre-B cells in bone marrow. In contrast, apoptosis of earlier precursor B cells and mature B lymphocytes did not differ from control values. The results indicate that orally administered melatonin can substantially promote the survival of precursor B cells in mouse bone marrow. Melatonin treatment may thus boost the survival of newly formed B cells mediating humoral immunity.

Potential involvement of mt1 receptor and attenuated sex steroid-induced calcium influx in the direct anti-proliferative action of melatonin on androgen-responsive LNCaP human prostate cancer cells.

Abstract: Melatonin, a pineal secretory product, has been shown to exert a direct anti-proliferative action on the androgen-sensitive LNCaP prostate cancer cell line through hitherto undefined mechanisms. In this communication, expression of mt1 melatonin receptor protein in human prostate cancer tissues and LNCaP cells was demonstrated by immunohisto(cyto)chemistry and western blotting, hence supporting the use of LNCaP cell line as a model for the study of melatonin signaling in prostate cancer cell growth. Using 3H-thymidine incorporation assay, LNCaP cell proliferation was inhibited by 2-iodomelatonin, a high-affinity melatonin receptor agonist. Furthermore, melatonin inhibited 3H-thymidine incorporation into LNCaP cells and attenuated 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2)-induced stimulation of LNCaP cell proliferation at physiological and pharmacological concentrations. Similar concentration-dependent inhibition of sex steroid-induced stimulation of thymidine incorporation into LNCaP cells by 2-iodomelatonin was also observed. Interestingly, attenuation of sex steroid-stimulated calcium influx into LNCaP cells by pharmacological concentrations of melatonin was recorded, whereas 2-iodomelatonin had no effect on cytosolic calcium changes induced by sex steroids. In addition, proliferative and cytosolic calcium changes were associated with inhibition of total prostate-specific antigen (PSA) production by LNCaP cells at high physiological and pharmacological concentrations of melatonin. Our data suggest that activated mt1 receptor and attenuated sex steroid-induced calcium influx are two important mechanisms mediating the direct anti-proliferative action of melatonin on androgen-responsive human prostate cancer cells.

Nocturnal 6-hydroxymelatonin sulfate excretion in female workers exposed to magnetic fields

Abstract: The objective of this study was to determine whether daytime occupational exposure to extremely low frequency magnetic fields (MFs) suppresses nocturnal melatonin production. Sixty female volunteers were recruited. Thirty-nine worked in a garment factory, and 21 office workers served as a reference group. Exposure assessment was based on the type of sewing machine used and MF measurements around each type of machine. Eye-level MF flux density was used to classify the operators to higher (>1 microT) and lower (0.3-1 microT) exposure categories. A third group of factory workers had diverse MF exposures from other sources. The reference group had average exposure of about 0.15 microT. Urine samples were collected on Friday and Monday for three consecutive weeks. Melatonin production was assessed as urinary 6-hydroxymelatonin sulfate (6-OHMS) excretion. The ratio of Friday morning/Monday morning 6-OHMS was used to test the hypothesis that melatonin production is suppressed after 4 days of occupational MF exposure with significant recovery during the weekend. Possible chronic suppression of melatonin production was evaluated by studying exposure-related differences in the Friday values by multivariate regression analysis. The Monday/Friday ratios were close to 1.0, suggesting that there is no increase in melatonin production over the weekend. The average 6-OHMS excretion on Friday was lower among the factory workers than in the reference group, but no monotonous dose-response was observed. Multivariate regression analysis identified MF exposure, smoking, and age as significant explanatory variables associated with decreased 6-OHMS excretion.

Multi-night exposure to 60 Hz magnetic fields: effects on melatonin and its enzymatic metabolite.

Abstract: Magnetic field-induced suppression of nocturnal melatonin in humans has been reported in occupational and residential studies, but not in laboratory-based exposure studies. The present study examined whether this contrasting pattern of results might be related to associated differences in exposure duration or to field-induced measurement instability over time. Thirty healthy young men were evaluated using a randomized, double-blind test protocol. Statistical analysis indicated that 4 consecutive nights of exposure to power-frequency magnetic fields at occupational intensity (resultant flux density = 28.3 microtesla, muT, [283 milligauss, mG]) had no differential effect on concentrations of melatonin or its major enzymatic metabolite (6-hydroxymelatonin sulfate, 6-OHMS) in daily morning urine samples, compared to equivalent no-exposure sham control conditions. The consistency of intra-individual urinary measurements over the 4 test nights also was quite high (P < 0.01) in the sham control condition. In contrast, repeated nightly exposure to the magnetic field was associated with reduced consistency. Morning urinary measures obtained after exposure on night 4 differed (P < 0.01) from similar measures obtained after the second and third exposure night. Thus, while the overall results of this study do not support the melatonin hypothesis, there is some suggestion of a possible cumulative effect of magnetic field exposure on the stability of individual melatonin measurements over time. Additional research with longer periods of controlled exposure may be warranted.

Melatonin disrupts circadian rhythms of glutamate and GABA in the neostriatum of the awake rat: a microdialysis study

Abstract: The purpose of this study was to investigate possible circadian changes in extracellular concentrations of glutamate (GLU) and gamma-aminobutyric acid (GABA). and the influence of melatonin on the levels of these neurotransmitters in the neostriatum of awake rats using in vivo microdialysis. At the same time, the concentrations of the amino acids taurine (TAU), glutamine (GLN) and arginine (ARG), as well as dopamine (DA) and its metabolites 3, 4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), were measured in the extracellular fluid. When dialysates were collected over a 24-hr period (6 hr dark, 12 hr light, 6 hr dark), both GLU and GABA, without the infusion of melatonin, exhibited statistically significant rhythms, with higher levels of these constituents during the dark and lower levels during the day. Perfusion with melatonin (for 19 consecutive hours) prevented the daytime reductions in both GLU and GABA. Of the amino acids measured in the dialysates collected from the neostriatum of non-perfused rats, only ARG exhibited a significant change during the light:dark cycle; again, lowest concentrations were measured during the day. While melatonin perfusion did not statistically significantly influence neostriatal levels of TAU and ARG, GLN levels continued to drop during the infusion of the indoleamine. Dialysate concentrations of DA, DOPAC and HVA exhibited circadian rhythms which were not influenced by melatonin perfusion. The findings indicate there are differential effects of melatonin on extracellular neurotransmitter concentrations in the neostriatum of the awake rat. The results also suggest that the day:night variations in GLU and GABA may relate to daily changes in endogenous melatonin production, while DA and its metabolites are minimally influenced by this secretory product.

Effect of melatonin on motility pattern of small intestine in rats and its inhibition by melatonin receptor antagonist S 22153.

Abstract: Melatonin is synthesized during the night by the pineal gland. Recently, melatonin binding sites have been identified in the gut. Despite few studies, the physiological role of melatonin in gut function remains unclear. The objective of the present study was to investigate the effects of melatonin in the regulation of intestinal motility by using the melatonin receptor antagonist S 22153 in rats. Twenty-four male Wistar rats (400 +/- 25 g) were equipped with intraparietal electrodes along the small intestine. Rats were subjected to a 12:12 hr light:dark schedule. During the dark phase, intestinal migrating motor complexes (MMCs) frequency increased (P < 0.05) by 20% in the duodenum and in the jejunum compared with daylight. This effect is due to a significant reduction in the irregular spiking activity (ISA) of MMCs. Concurrently, at night, the duration of the postprandial motor response is reduced by 30% in the duodenum and 50% in the jejunum and ileum. The administration of S 22153 (2 mg/kg sc) at night suppressed these nocturnal variations and restored the daylight values. In contrast, S 22153 was ineffective during daylight whatever the digestive state. Administration of melatonin (1 mg/kg iv) during the preprandial state, 3 hr after light onset, decreased (-80%) the duration of the ISA of MMCs at the three intestinal levels. During the satiety phase, melatonin administered 10 min before or 15 min after food onset induced the appearance of a transitory preprandial-like motor profile in the entire small intestine. In contrast, when administered at the end of the meal it was ineffective. Preprandial and postprandial melatonin effects were prevented by S 22153 pretreatment. In conclusion, these findings reveal, first, that endogenous melatonin is physiologically involved in the pre- and postprandial changes of intestinal motility at night. Second, exogenous melatonin produces pharmacological effects on pre- and postprandial intestinal motility. In both cases, the action of melatonin corresponds to an inhibition of ISA and a reinforcement of the cyclic MMC pattern.