Monitoring intracellular melatonin levels in human prostate normal and cancer cells by HPLC.

Melatonin (N-acetyl-5-methoxytryptamine) is a potent endogenous antioxidant and free radical scavenger that has attracted much attention as a consequence of its multiple biological functions. In addition to other physiological properties, it has clear antiproliferative activity in several types of cancer cell. The concentration of melatonin necessary to inhibit cell growth is much higher than its blood physiological concentrations in some tumor types. For years its indolic nature has impeded proper monitoring, by molecular or immunological techniques, of its uptake by cancer cells. In this work we developed a simple, rapid, and validated analytical method for detection and quantification of MEL inside normal and cancer cells. For this purpose we performed high-performance liquid chromatographic analysis after liquid-liquid extraction of the indole from biological samples. The method was validated, and the correlation coefficient for amounts from 0.125 to 1.25 microg was higher than 0.999, with a range of recovery near 100%. Precision was evaluated as repeatability, and for intermediate precision, the relative standard deviation was less than 5%. The method was used to study the stability of the indole in solution and to determine intracellular melatonin concentrations in normal (PNT1A) and several cancer (LNCaP, DU-145, PC-3) prostate cell lines. Intracellular LOQ/LOD were 7.23/2.83, 23.17/9.07, 4.03/1.83, and 6.51/2.53 nmol L(-1), or 1.82/4.66, 0.56/1.45, 3.26/8.34, and 2.02/5.17 attogram in each cell in PNT1A, LNCaP, DU145, and PC-3 cells, respectively. Because there was no information about intracellular levels of melatonin inside normal or tumor prostate cells after treatment with the indole, nor a relationship between its antiproliferative activity and its intracellular concentration, this is the first time that, by using an analytical method combined with measurement of cellular volume by flow cytometry, the intracellular concentration of MEL has been estimated. Also, data obtained here explain why the antiproliferative properties of MEL vary in different cell types. This is, moreover, the first time that by increasing the intracellular concentration of melatonin, its antitumor properties have been promoted in prostate cancer cells. This process can be monitored by the method developed here.

Antioxidants do not prevent acrylonitrile-induced toxicity.

Several reports have recently described that acrylonitrile (ACN) toxicity resides in its capacity for inducing oxidative stress. ACN can be conjugated with glutathione (GSH), diminishing its cellular content, or being metabolized to cyanide. In the present report, we determine the effect of ACN on the viability of primary-cultured astrocytes as well as the oxidative damage generated by ACN by measuring GSH levels in primary cultured astrocytes. We also analyzed whether the ACN (2.5mM) toxicity could be avoided by using antioxidants such as taurine (5mM), N-acetylcysteine (20 mM), trolox (100 microM), estradiol (10 microM) and melatonin (100 nM-1mM). In this cell culture model, antioxidants were not able to prevent ACN-induced cell damage, with the exception of NAC, confirming that only GSH seems to play a key role in ACN-derived toxicity. Additionally, we measured different parameters of oxidative stress such as catalase activity, lipid peroxidation and GSH concentration, as indicators of the potential oxidative stress mediated by the toxicity of ACN, after exposure of Wistar rats to a concentration of 200 ppm ACN for 14 days. At the concentration assayed, we did not find any evidence of oxidative damage in the brain of ACN-treated rats.

Protection against oxidative protein damage induced by metal-catalyzed reaction or alkylperoxyl radicals: comparative effects of melatonin and other antioxidants.

Melatonin is a well-known hydroxyl radical (*OH) scavenger that protects DNA and lipids from free radical attack. In this paper, we studied the ability of melatonin to prevent oxidative damage to bovine serum albumin (BSA) induced by two different paradigms: the metal-catalyzed oxidation (MCO) induced by Cu(2+)/H(2)O(2) and the alkoxyl and alkylperoxyl radicals formed by the azo initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH, 40 mM). The protective effects of melatonin were compared with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), glutathione (GSH), ascorbate, 3,4',5-trihydroxy-trans-stilbene (resveratrol, 0.1 microM-4 mM) and mannitol (50 microM-100 mM). Melatonin efficiently prevented protein modification induced by both models, as assayed by polyacrylamide gel electrophoresis and carbonyl content. Both trolox and ascorbate had an obvious pro-oxidant effect in the Cu(2+)/H(2)O(2) model, whereas both prevented BSA damage induced by AAPH. In the MCO model, the efficacy of GSH in terms of protein protection was higher than melatonin at relatively high concentrations (250 microM-4 mM); however, at lower concentrations (50-250 microM), the efficacy of melatonin was superior to GSH. D-Mannitol (50 microM-100 mM) and resveratrol did not protect BSA from the site-specific damage induced by Cu(2+)/H(2)O(2). On the other hand, the relative protective efficiency in the AAPH model was melatonin approximately trolox>GSH>ascorbate.

Identification of highly elevated levels of melatonin in bone marrow: its origin and significance.

Bone marrow is an important tissue in generation of immunocompetent and peripheral blood cells. The progenitors of hematopoietic cells in bone marrow exhibit continuous proliferation and differentiation and they are highly vulnerable to acute or chronic oxidative stress. In this investigation, highly elevated levels of the antioxidant melatonin were identified in rat bone marrow using immunocytochemistry, radioimmunoassay, high performance liquid chromatography with electrochemical detection and mass spectrometry. Night-time melatonin concentrations (expressed as pg melatonin/mg protein) in the bone marrow of rats were roughly two orders of magnitude higher than those in peripheral blood. Measurement of the activities of the two enzymes (N-acetyltransferase (NAT) and hydroxyindole-O-methoxyltransferase (HIOMT)) which synthesize melatonin from serotonin showed that bone marrow cells have measurable NAT activity, but they have very low levels of HIOMT activity (at the one time they were measured). From these studies we could not definitively determine whether melatonin was produced in bone marrow cells or elsewhere. To investigate the potential pineal origin of bone marrow melatonin, long-term (8-month) pinealectomized rats were used to ascertain if the pineal gland is the primary source of this antioxidant. The bone marrow of pinealectomized rats, however, still exhibited high levels of melatonin. These results indicate that a major portion of the bone marrow's melatonin is of extrapineal origin. Immunocytochemistry clearly showed a positive melatonin reaction intracellularly in bone marrow cells. A melatonin concentrating mechanism in these cells is suggested by these findings and this may involve a specific melatonin binding protein. Since melatonin is an endogenous free radical scavenger and an immune-enhancing agent, the high levels of melatonin in bone marrow cells may provide on-site protection to reduce oxidative damage to these highly vulnerable hematopoietic cells and may enhance the immune capacity of cells such as lymphocytes.

N1-acetyl-N2-formyl-5-methoxykynuramine, a biogenic amine and melatonin metabolite, functions as a potent antioxidant.

The biogenic amine The biogenic amine N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) was investigated for its potential antioxidative capacity. AFMK is a metabolite generated through either an enzymatic or a chemical reaction pathway from melatonin. The physiological function of AFMK remains unknown. To our knowledge, this report is the first to document the potent antioxidant action of this biogenic amine. Cyclic voltammetry (CV) shows that AFMK donates two electrons at potentials of 456 mV and 668 mV, and therefore it functions as a reductive force. This function contrasts with all other physiological antioxidants that donate a single electron only when they neutralize free radicals. AFMK reduced 8-hydroxydeoxyguanosine formation induced by the incubation of DNA with oxidants significantly. Lipid peroxidation resulting from free radical damage to rat liver homogenates was also prevented by the addition of AFMK. The inhibitory effects of AFMK on both DNA and lipid damage appear to be dose-response related. In cell culture, AFMK efficiently reduced hippocampal neuronal death induced by either hydrogen peroxide, glutamate, or amyloid b25-35 peptide. AFMK is a naturally occurring molecule with potent free radical scavenging capacity (donating two electrons/molecule) and thus may be a valuable new antioxidant for preventing and treating free radical-related disorders.

Melatonin reduces oxidative neurotoxicity due to quinolinic acid: in vitro and in vivo findings.

The in vivo and in vitro effects of melatonin on quinolinic acid-induced oxidative damage in rat brain were determined. The concentrations of malonaldehyde and 4-hydroxyalkenals were assayed as an index of oxidatively damaged lipid. In in vitro experiments, the increase in malonaldehyde and 4-hydroxyalkenals concentrations induced by quinolinic acid were concentration-dependent and time-dependent. The accumulation of products of lipid peroxidation induced by quinolinic acid were very significantly reduced by melatonin in a concentration-dependent manner. Additionally, at the highest concentrations of melatonin used in quinolinic acid treated homogenates, it reduced the levels of oxidatively damaged lipid products below those measured in control homogenates (no quinolinic acid or melatonin). When quinolinic acid (200 mg/kg) was intraperitonally injected into 11-day-old rats, lipid peroxidation in the brain was significantly increased 24 hours later compared to levels in control rats. When melatonin (10 mg/kg) was injected i.p. 30 min before and 4 and 20 hours after the administration of quinolinic acid, the increased lipid peroxidation induced by quinolinic acid was significantly reduced. Likewise, neurobehavioral signs associated with quinolinate administration were attenuated by melatonin. These results show that both in vitro and in vivo pharmacological levels of melatonin confer protection against quinolinic acid-induced oxidative toxicity in the brain. The findings also indicate that melatonin may be pharmacologically useful in combatting quinolinic neurotoxicity which is associated with several acute and chronic neurodegenerative neurological diseases.

Mast cells in the Harderian gland of female Syrian hamsters during the estrous cycle and pregnancy: effects of the light/dark cycle.

The number of identifiable mast cells and the intraluminal area occupied by porphyrin deposits was studied on semithin sections from female hamster Harderian glands during the estrous cycle and pregnancy. Although the serum levels of estradiol, progesterone, luteinizing hormone and follicle stimulating hormone exhibited significant changes throughout the cycle, no correlation between these changes and the variations in the number of recognizable mast cells was observed. However both during diestrous 1 and proestrous cycles, the number of identifiable mast cells was higher at midnight than at noon (in 14 h light:10 h dark photoperiod with lights on at 07:00 h). A more exhaustive study revealed the presence of 'degranulated mast cells' which were not stained with toluidine blue. Thus, a diurnal cycle in degranulation might occur in the Harderian glands from female hamsters. No significant variations were observed in the area occupied by intraluminal porphyrin deposits during the estrous cycle. However, both the relative number of mast cells and the area occupied by intraluminal porphyrins decreased from day 4 of pregnancy to day 14 showing a strong correlation. The Harderian glands from female Syrian hamsters might provide a useful model for the study of mast cell degranulation during porphyria.

Melatonin as a pharmacological agent against neuronal loss in experimental models of Huntington's disease, Alzheimer's disease and parkinsonism.

This review summarizes the experimental findings related to the neuroprotective role of melatonin. In particular, it focuses on research directed at models of Huntington's disease, Alzheimer's disease and Parkinsonism. Melatonin has been shown to be highly effective in reducing oxidative damage in the central nervous system; this efficacy derives from its ability to directly scavenge a number of free radicals and to function as an indirect antioxidant. In particular, melatonin detoxifies the highly toxic hydroxyl radical as well as the peroxyl radical, peroxynitrite anion, nitric oxide, and singlet oxygen, all of which can damage macromolecules in brain cells. Additionally, melatonin stimulates a variety of antioxidative enzymes including superoxide dismutase, glutathione peroxidase and glutathione reductase. One additional advantage melatonin has in reducing oxidative damage in the central nervous system is the ease with which to crosses the blood-brain barrier. This combination of actions makes melatonin a highly effective pharmacological agent against free radical damage. The role of physiological levels of melatonin in forestalling oxidative damage in the brain is currently being tested.

Regulation of the aminolevulinate synthase gene in the Syrian hamster Harderian gland: changes during development and circadian rhythm and role of some hormones.

The Syrian hamster Harderian gland has been advocated as a model to study the porphyrin biosynthetic pathway, since it shows by far the highest porphyrin concentration known to date. Another particular characteristic is the sexual dimorphism at both the morphological and the biochemical levels. We found a variation in the ALV-S (aminolevulinate synthase) gene expression according to sex, with females exhibiting much higher mRNA levels than do males. After castration, ALV-S mRNA rose considerably in males, this increase being inhibited by darkness or treatment with melatonin. Treatment with hCG or progesterone did not vary the ALV-S mRNA levels in females. Castrated males, however, showed a much larger increase when they were treated with hCG. No variations have been found in the expression of the ALV-S gene in female HG throughout the estrous cycle. During development, males and females showed similar ALV-S mRNA levels until they were 20 days old. Afterwards, they started showing gender-associated differences. In females, ALV-S mRNA levels rose during the first 3 months of life, and thereafter they decreased progressively with aging. A circadian rhythm has been found in the gene expression of ALV-S mRNA in females, showing very low levels in the morning and reaching a peak during the first hours of darkness. It was an endogenous rhythm, probably regulated at the transcriptional level. It is proposed that the light-dark period duration modulates this rhythm through the suprachiasmatic nucleus which in turn acts on the pineal secretion of melatonin that regulates ALV-S gene expression.

Ultrastructural confirmation of neuronal protection by melatonin against the neurotoxin 6-hydroxydopamine cell damage.

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and cultured neuronal cells. Biochemical and molecular approaches showed previously that low doses of 6-OHDA induced apoptosis in PC12 cells, while high doses of this neurotoxin induced necrosis. Melatonin has been shown to protect against the neuronal programmed cell death induced by 6-OHDA, although it was not able to prevent the massive necrotic cellular death occurring after the addition of high doses of the neurotoxin. In the present work, we demonstrate by ultrastructural analysis that although low doses of 6-OHDA induced apoptosis in PC12 cells, it also damaged the non-apoptotic cells, morphologically corresponding this damage to incipient and reversible necrotic lesions. When the doses of the neurotoxin increase, there are still apoptotic cells, although most of the cells show necrotic irreversible lesions. We also found that melatonin partially prevents the incipient necrotic lesions caused by low doses of 6-OHDA. The fact that melatonin was shown in previous work to prevent apoptosis caused by low doses of 6-OHDA, but not necrosis induced by high doses of the neurotoxin, seemed to indicate that this agent is only able to protect against apoptosis. However, our present results, melatonin preventing also the incipient necrotic neuronal lesions, suggest that this hormone may provide a general protection against cell death, suggesting that higher doses should be tried in order to prevent the necrotic cell death induced by high doses of the neurotoxin.

Melatonin decreases mRNA for histone H4 in thymus of young rats.

The antiproliferative properties of melatonin have been previously demonstrated for several normal and tumoral tissues. In a recent report we have shown that melatonin is able to inhibit programmed cell death in thymus both, in vivo and in vitro. Given that other authors have related programmed cell death and cell proliferation and that no previous reports on melatonin and cell division exist on thymus, we decide to study the possible antiproliferative effect of melatonin in this organ measured as the levels of mRNA for the histone H4. We found that melatonin inhibits cell division on thymus when administered chronically both, at high (500 microg/body weight) and low (50 microg/body weight) dose. We also found a circadian rhythm of the mRNA for histone H4, opposed to the one previously described for melatonin, supporting the negative regulation by this hormone of cell division on thymus. A single dose of melatonin (50 microg/body weight) was not able to decrease the levels of mRNA for H4 in the time-points studied but after two hours of its administration. Finally, we report the inhibitory effect of melatonin in the cell proliferation of Harderian gland, brain, lung and kidney.

Androgen-dependent mast cell degranulation in the Harderian gland of female Syrian hamsters: in vivo and organ culture evidence.

In previous articles we have reported the "disappearance" of Harderian gland mast cells (HGMC) after treatment with testosterone. In the present work we study: (a) if the apparent decrease in the number of mast cells caused by this androgen is real or is due to the fact that testosterone induces mast cell degranulation that avoids its recognition by toluidine blue staining; (b) if testosterone acts through its receptor directly on the Harderian gland (HG). In order to give an answer to the first question, we observed HG of female Syrian hamsters treated with testosterone under the electron microscope to find the possible degranulated mast cells not recognizable with the aid of the toluidine blue staining. We also studied in vivo and in vitro the effects of the beta-agonists isoproterenol and salbutamol, given that they increase cAMP and can therefore prevent degranulation of mast cells. Finally we have used cytocalasin B, which inhibits degranulation by blocking actin depolimerization. Both the beta-agonists and cytochalasin B were able to prevent the decrease of mast cells, as recognized by staining with toluidine blue after treatment with testosterone. Indeed, when observed under the electron microscope, abundant degranulated mast cells were found after treatment with testosterone. For solving the second issue we analyzed the effect of the antiandrogen cyproterone acetate in vivo and in vitro. Our results demonstrate that testosterone is able to induce degranulation of HGMC in the Syrian hamster Mesocricetus auratus and that this effect is achieved directly through its receptor on the Harderian gland.

Development and hormonal regulation of mast cells in the Harderian gland of Syrian hamsters.

The morphological features and relative number of mast cells per mm2 were studied in the Harderian glands of male and female Syrian hamsters (Mesocricetus auratus) under different experimental conditions. The structural and ultrastructural characteristics of Harderian mast cells corresponded to those of connective tissue mast cells. The Harderian glands from female hamsters contained more mast cells than those of male hamsters. A subcutaneous implant of testosterone (2 mg/24 mg beeswax) resulted in a rapid decrease in the number of recognizable mast cells 6 h after the implantation. Neither orchidectomy nor ovariectomy significantly altered the relative number of mast cells. However, the daily subcutaneous injection of 20 IU of human chorionic gonadotropin during 20 days resulted in a significant decrease of identifiable mast cells. The administration of another steroid such as progesterone or the induction of states of hypo- and hyperthyroidism did not alter the distribution of mast cells in the Harderian glands of female Syrian hamsters.

Evaluation of the biological effect of Ti generated debris from metal implants: ions and nanoparticles.

Metallic implants placed in humans exhibit wear and corrosion that result in the liberation of metal-containing by-products. In the case of titanium (Ti) containing implants, the metal containing debris may exist in a number of states, including metallic particles produced by mechanical wear and the products of metal corrosion in biological environments, such as the joints and surrounding fluids and tissues. In addition, these constituents may dissolve in both intracellular and extracellular solutions generating Ti ions. Both species, ions and nanoparticles, show different cellular toxicities. In this work we have evaluated the possible evolution of TiO2 nanoparticles (NPs) into soluble Ti metal ions by contact with biological fluids. For this aim, an in vitro study to address quantitative Ti solubilisation from TiO2 nanoparticles (with a diameter of 21 nm) after incubation with human serum at different concentrations has been conducted. Total Ti determination revealed low solubilisation rates ranging from 0.53 to 0.82% after just one week of incubation in the serum. The incubated serum was then subjected to speciation analysis by anion exchange liquid chromatography using an inductively coupled plasma mass spectrometer (ICP-MS) as an elemental detector for Ti monitoring. The obtained results revealed a significant increase in the Ti signal associated with the fraction of the protein transferrin and preferentially with one of the metal binding sites of the protein, the N-lobe. Thus, the effect of Ti at the cellular level has been evaluated by considering that it can be present either as ions or as nanoparticles using two different cells lines: human enterocytes HT29 and murine osteoblasts MC3T3. Significant toxicity was found at the highest concentration assayed (50 µg mL(-1)) for both Ti species (ions and NPs) and slightly higher for the ionic species at lower concentrations (1 and 10 µg mL(-1)).

Development and validation of new methods for the determination of melatonin and its oxidative metabolites by high performance liquid chromatography and capillary electrophoresis, using multivariate optimization.

Melatonin (N-acetyl-5-metoxytriptamine, MEL) has focused a lot of attention as consequence of its multiple functions. MEL is a potent endogenous antioxidant and a free radical scavenger that reacts with several sort of radicals generating various metabolites. Two of them are N1-acetyl-N2-formyl-5-methoxykynurenine (AFMK) and N1-acetyl-5-methoxykynurenine (AMK). These compounds are important because they have also antioxidant actions as well as other important biological properties. In the present work, we develop two methods to detect and quantify these compounds (MEL, AFMK and AMK) in the same sample. For this purpose we used an experimental design, and utilized high performance liquid chromatography (HPLC-DAD) and micellar electrokinetic chromatography (MEKC) techniques with diode array detector in both of them. The limit of detection/quantification for MEL, AFMK and AMK were respectively 44/94, 18/38 and 23/51 ng mL(-1) by using HPLC and 13/44, 37/124 and 47/156 ng mL(-1) by using MEKC. This is the first time that these compounds have been separated in the same chromatogram or electroferogram. The time of analysis was faster using MEKC. Furthermore, this technique showed better resolution but HPLC offered better limit of detection and quantification for metabolites. Both methods were validated and correlation coefficients were higher than 0.999 and the range of recovery of those methods were 99.6-103.7%. Precision was evaluated as repeatability and intermediate precision with relative standard derivation <5%. When a 5 microg mL(-1) solution of these compounds were analyzed with both methods we do not observed any statistically significance differences. Moreover, we analyzed 3COHM (cyclic-3-hydroximelatonin), another known metabolite of melatonin, by using the same methods. The employment of these methods will offer a useful tool to contribute to answer the role of MEL, AFMK and AMK in biological system and both methods can be used in routine analysis for these compounds.

Glutamate induces oxidative stress not mediated by glutamate receptors or cystine transporters: protective effect of melatonin and other antioxidants.

Glutamate is responsible for most of the excitatory synaptic activity and oxidative stress induction in the mammalian brain. This amino acid is increased in the substantia nigra in parkinsonism due to the lack of dopamine restraint to the subthalamic nucleus. Parkinson's disease also shows an increase of iron levels in the substantia nigra and a decrease of glutathione, the antioxidant responsible for the ascorbate radical recycling. Considered together, these facts could make the antioxidant ascorbate behave as a pro-oxidant in parkinsonism. Since both glutamate and ascorbate are present in the synaptosomes and neurons of substantia nigra, we tested 1) if glutamate is able to induce oxidative stress independently of its excitatory activity, and 2) if ascorbate may have synergistic effects with glutamate when these two molecules co-exist. Brains were homogenized in order to disrupt membranes and render membrane receptors and intracellular signaling pathways non-functional. In these homogenates glutamate induced lipid peroxidation, indicating that this amino acid also may cause oxidative stress not mediated by its binding to glutamate receptors or cystine transporters. Ascorbate also induced lipid peroxidation thus behaving as a pro-oxidant. Both substances together produced an additive effect but they did not synergize. Given that melatonin is a potent physiological antioxidant with protective effects in models of neurotoxicity, we tested the role of this secretory product on the pro-oxidant effect of both compounds given separately or in combination. We also checked the protective ability of several other antioxidants. Pharmacological doses of melatonin (millimolar), estrogens, pinoline and trolox (micromolar) prevented the oxidant effect of glutamate, ascorbate, and the combination of both substances. Potential therapeutic application of these results is discussed.

Melatonin regulation of antioxidant enzyme gene expression.

Antioxidant enzymes (AOEs) are part of the primary cellular defense against free radicals induced by toxins and/or spontaneously formed in cells. Melatonin (MLT) has received much attention in recent years due to its direct free radical scavenging and antioxidant properties. In the present work we report that MLT, at physiological serum concentrations (1 nM), increases the mRNA of both superoxide dismutases (SODs) and glutathione peroxidase (GPx) in two neuronal cell lines. The MLT effect on both SODs and GPx mRNA was mediated by a de novo synthesized protein. MLT alters mRNA stability for Cu-Zn SOD and GPx. Experiments with a short time treatment (pulse action) of MLT suggest that the regulation of AOE gene expression is likely to be receptor mediated, because 1-h treatment with MLT results in the same response as a 24-h treatment.

Melatonin increases gene expression for antioxidant enzymes in rat brain cortex.

During the last years several reports have demonstrated that melatonin is a efficient free radical scavenger and general antioxidant. In addition, it has been shown that this neurohormone is able to increase the activity of glutathione peroxidase in rat brain cortex as well as the gene expression for some antioxidant enzymes in the Harderian gland of female Syrian hamster. Also, it is well known that brain cells are particularly exposed to free radicals, with antioxidant enzymes as the major defense mechanism that the brain uses to neutralize reactive oxygen species. The aim of the present study was to examine the influence of melatonin on gene expression for antioxidant enzymes in rat brain cortex. Our results clearly demonstrate that exogenously administered melatonin increases the levels of mRNA for glutathione peroxidase, copper-zinc superoxide dismutase, and manganese superoxide dismutase in this tissue. These stimulatory effects are observed after both acute and chronic treatment with this hormone, producing in the latter case the more marked increase. We therefore conclude that melatonin exerts an important role in providing indirect protection against free radical injury by stimulating gene expression for antioxidant enzymes. Consequently, melatonin could be considered as a potential therapeutic agent in some age-related neurodegenerative diseases where excessive free radical production has been implicated.

Inhibition of cell proliferation: a mechanism likely to mediate the prevention of neuronal cell death by melatonin.

In a previous work we demonstrated that melatonin is able to prevent apoptosis induced by low doses of 6-hydroxydopamine (6-OHDA) in undifferentiated and neuronal PC12 cells. We also reported how this neurohormone was able to prevent the decrease in the mRNA for antioxidant enzymes caused by 6-OHDA. Although the antioxidant capability of melatonin seems to be clearly implicated in its antiapoptotic activity, literature suggests that its antiproliferative property could also be involved in its prevention of apoptosis. In the present work we demonstrated that melatonin is able to inhibit cell proliferation in undifferentiated PC12 cells, decreasing cell number and the total amount of DNA, and the mRNA for the histone H4, which are known to increase during DNA synthesis. Melatonin does not decrease the number of cells in nonproliferating PC12 cells, indicating that it does not cause cell death. Additionally, we demonstrate that other inhibitors of cell proliferation, as well as other antioxidants, are able to mimic the antiapoptotic effect of melatonin. This is interpreted to mean that melatonin acts by both mechanisms to inhibit apoptosis caused by 6-OHDA and the findings support the hypothesis of a relationship between oxidative stress and regulation of the cell cycle.

Neurally-mediated and neurally-independent beneficial actions of melatonin in the gastrointestinal tract.

Melatonin (N-acetyl-5-methoxytryptamine), originally discovered in the pineal gland, is now known also to be present in the gastrointestinal tract from the stomach to the colon. It is localized and likely synthesized in the enterochromaffin cells of the mucosal lining. Its functions in the gut generally seem to be protective of the mucosa from erosion and ulcer formation and to possibly influence movement of the gastrointestinal contents through the digestive system. In this brief review, we summarize the work documenting the function of melatonin in influencing bicarbonate secretion in the stomach and its role in preventing and repairing ulcers in the stomach and duodenum. Melatonin's actions in the control of bicarbonate secretion involve the central and peripheral sympathetic nervous systems and the actions are receptor mediated. Conversely, melatonin's actions in reducing ulcer formation also seemingly involve the ability of the indole to directly scavenge toxic oxygen-based reactants, e.g., the hydroxyl radical, and possibly to promote antioxidative enzyme activities. These same processes may be involved in the mechanisms by which melatonin promotes ulcer healing. Additionally, however, melatonin's effects on the healing of ulcers includes actions of blood flow in the margins of the ulcer and also on the sensory nerves. All indications are that melatonin has a variety of beneficial effects in the gastrointestinal tract. It is likely, however, that additional actions of melatonin on the digestive system will be uncovered.